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INTRODUCTION {#SEC1}
============
Although many processes, which occur in the archaeal cell are exclusively present in this unique third domain of life, other processes are often a mosaic of bacterial and eukaryotic properties. This is especially the case for information processing, like the regulation of transcription. While transcriptional regulators resemble their bacterial counterparts, the basal transcription apparatus is considered a simplified version of the more complex system found in eukaryotes ([@B1],[@B2]). In contrast to eukaryotes, archaea exhibit a much smaller set of general transcription factors (GTFs). The basal archaeal transcription machinery comprises a multi-subunit RNA-polymerase (RNAP) and the GTFs representing homologs of the eukaryotic TATA box binding protein (TBP) and the transcription factor TFIIB (TFB). Most of the archaeal promoters show structural similarity to the eukaryotic RNA polymerase II system ([@B3]), as they contain the typical sequences for TATA box, TFB responsive element (BRE) and initiator element (Inr).
The archaeal transcription machinery, including the initiation of transcription, has been extensively studied ([@B4]). Briefly, TBP recognizes and binds to the TATA box. Subsequently, TFB associates with the TBP--DNA complex and forms sequence specific interactions with the BRE site followed by a conformational change, i.e. bending, of the DNA, which determines the direction of transcription. Finally, the RNAP is recruited by the N-terminal part of TFB ([@B7]). *In vitro* analyses showed that only TBP, TFB and the RNAP are required and sufficient to initiate transcription ([@B11],[@B12]). Furthermore, archaea possess a homolog of the eukaryotic GTF TFIIE (TFE). TFE is involved in the stimulation of transcription processivity, since it is able to increase the transcription of certain promoters, especially those, which show suboptimal interaction with TBP ([@B13],[@B14]). Additionally, it has been shown that TFE plays an important role in promoter escape as it competes with the elongation factor Spt4/5 for RNAP binding ([@B15],[@B16]).
Most of the sequenced archaeal genomes encode multiple homologs of the GTFs (i.e. TBP and TFB) and the numbers vary depending on the species. Recent structural comparisons revealed similarity between eukaryotic TFIIB, archaeal TFB and bacterial σ factors ([@B17]). The multiplicity of GTFs is extensively studied in euryarchaea and a role in the adaptation to changing environmental conditions has been proposed for several organisms. The function of multiple GTFs has best been characterized in the halophilic euryarchaeon *Halobacterium salinarum* NRC. *H. salinarum* NRC encodes seven TFBs and six TBPs, which are used in different combinations to regulate the transcription of genes essential during the heat shock response ([@B18]), oxidative stress ([@B19]) and adaptation to low temperatures ([@B20]). The euryarchaeon *Pyrococcus furiosus* contains two TFBs and one TBP. Transcript levels of *tfb2* increased dramatically upon heat shock, while *tfb1* levels remained stable, suggesting a role of TFB2 in the response to higher temperatures ([@B21]). In contrast to the detailed studies, which are available for euryarchaea, information about the role of multiple GTFs in crenarchaea is still limited. The thermoacidophilic crenarchaeal model organisms *Sulfolobus solfataricus* and *Sulfolobus acidocaldarius* both possess three *tfb* genes and one *tbp* gene ([@B22],[@B23]). In general, TFBs have a conserved structure with two major domains ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}). The N-terminal domain harbors a zinc ribbon motif and a conserved B-finger and is required for the recruitment of RNAP ([@B11],[@B24]). The C-terminal domain, which represents two third of the protein, is essential for the interaction with the TBP--DNA complex and contains a helix-turn-helix (HTH) motif, which is used to form sequence specific contacts with the BRE site ([@B25]). In *Sulfolobus* species, TFB3 is significantly smaller compared to TFB1 and TFB2. It comprises only a short C-terminal domain and lacks the B-finger and the HTH motif ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}). While TFB1, which supports transcription initiation *in vitro* ([@B26]), functions as a classical TFIIB homolog fulfilling widespread roles in the regulation of housekeeping genes ([@B11],[@B27]), the alternative transcription factors TFB2 and TFB3 show much lower transcript levels and are therefore proposed to have specialized functions ([@B28]). Genome wide microarray experiments in which transcription levels were studied during the cell cycle of *S. acidocaldarius*, showed that *tfb2* is induced during the transition from the G~1~ to the S phase, indicating that expression is cell cycle dependent and that TFB2 plays a role in cell-cycle regulation ([@B29]).
Two independent transcriptome studies using microarrays of *S. acidocaldarius* and *S. solfataricus* revealed that *tfb3* is one of the most upregulated genes following UV irradiation ([@B30],[@B31]). To further explore the role of TFB3 in UV stress response, Paytubi and White performed *in vitro* transcription assays with purified proteins from *S. solfataricus* ([@B32]). In this study, DNA fragments containing promotors that were previously shown to be either unresponsive to UV stress (T6, *ssb*) or to be activated (*dps, thsB*) or repressed (*sta1*) after UV stress were used. The experiments revealed that TFB3 had a stimulating effect on the transcription of all analyzed promoters and that transcription required TFB1 bound at the promoter. Therefore, TFB3 was proposed to interact with the TBP--TFB1--DNA complex and to stimulate RNAP recruitment and thus to function as a transcriptional activator ([@B32]).
In this study, the role of TFB3 in early UV stress response in *S. acidocaldarius* is addressed by *in vivo* tagging of the protein for immunodetection and co-immunoprecipitation as well as promoter activity studies, creation of a *tfb3* insertion mutant and a comprehensive transcriptome analysis.
MATERIALS AND METHODS {#SEC2}
=====================
Growth conditions {#SEC2-1}
-----------------
*Sulfolobus acidocaldarius* strains MW001, MW001*^pyrEF+^*, MW1104, *tfb3::pyrEF, tfb3*~C~SF and MW001\_*tfb3p* were grown aerobically at 75°C in basal Brock medium ([@B33]) pH 3.5, supplemented with 0.1% (w/v) NZ- amine, 0.2% (w/v) dextrin and for strains MW001, MW004 and *tfb3*~C~SF with 0.2% (w/v) uracil. The growth of the cells was monitored by measuring the optical density at 600 nm (OD~600~).
Creation of the genomic *HA*-tagged and Strep-/FLAG-tagged versions of *tfb3* {#SEC2-2}
-----------------------------------------------------------------------------
The *tfb3-C terminal HA* strain and the *tfb3-C terminal* Strep-/FLAG strain were obtained using the 'pop in-pop out' method described previously ([@B34]). To generate the HA-tagged strain, specific overlapping primer pairs (7518/7519, [Supplementary Table S1](#sup1){ref-type="supplementary-material"}) were used to amplify the upstream and downstream regions of the stop codon of *tfb3*. These regions were connected by overlap polymerase chain reaction (PCR) using the external primers 7516/7517 ([Supplementary Table S1](#sup1){ref-type="supplementary-material"}). The PCR product was purified and cloned into plasmid pSVA407 by ApaI and BamHI resulting in the plasmid pSVA3640. For the construction of the Strep-/FLAG-tagged strain, upstream and downstream regions of the *tfb3* gene were amplified using *S. acidocaldarius* DSM639 genomic DNA as a template and Strep-/FLAG tag sequences were introduced via PCR using the primer pairs SF01/SF03 and SF02/SF04 ([Supplementary Table S1](#sup1){ref-type="supplementary-material"}). Overlap extension PCR was performed to fuse the DNA fragments using primer pair SF01/SF04. The PCR product was inserted into pSVA406 via BamHI and NcoI restrictions sites resulting in the plasmid pSVA406-*tfb3*~C~SF. After verification by DNA sequencing, *Escherichia coli* ER1821 was used for the methylation of the plasmids. *S. acidocaldarius* MW001 cells were transformed with the respective methylated insertion-construct. Mutant screening was performed as described previously ([@B34]). The resulting strains MW1104 and *tfb3*~C~SF comprise the HA tag and the Strep-/FLAG tag, respectively, at the C-terminus of TFB3.
Generation of the *tfb3* insertion mutant {#SEC2-3}
-----------------------------------------
The *tfb3* mutant strain *tfb3::pyrEF* was created via double homologous recombination and insertion of the *Sso-pyrEF* cassette in the *tfb3* gene. A total of 86 bp of the *tfb3* gene and 106 bp of the *tfb3* gene were fused upstream and downstream to the *pyrEF* cassette, which was amplified via PCR using pSVA406 as a template*. S. acidocaldarius* MW001 cells were transformed with 300 ng of the PCR product via electroporation. The resulting cells were selected on plates and liquid media lacking uracil but containing 0.1% (w/v) NZ-amine. Clones were tested for the presence of the *pyrEF* cassette via colony PCR and positive clones were confirmed by sequencing. Attempts to create a marker-less deletion mutant via the 'pop in-pop out' method ([@B34]) failed. Whereas the recombination step in which the *Sso-pyrEF* cassette containing plasmid was integrated in the chromosome was successful, all colonies (over 200 colonies were screened) that lost the plasmid in the second recombination step still contained the complete *tfb3* gene despite approximately equally sized regions for the two possible recombination steps. This phenomenon has been observed previously, e.g. ([@B35]) and suggests that either the resulting marker-less mutant is harmful to the cell, or that secondary or tertiary DNA structures strongly favor the recombination resulting in the original genomic arrangement.
Generation of the *tfb3* reporter gene construct {#SEC2-4}
------------------------------------------------
In order to determine the activity of the *tfb3* promoter after UV stress, the maltose promoter in the shuttle vector pSVA1450 ([@B34]) was replaced with the *tfb3* promoter including 200 bp of its upstream promoter region. The DNA fragment was obtained via PCR using *S. acidocaldarius* DSM639 genomic DNA as a template and the primer pair RGA01/RGA02 ([Supplementary Table S1](#sup1){ref-type="supplementary-material"}). The resulting PCR product was inserted into pSVA1450 via the NcoI and SacII restriction sites, yielding the reporter gene construct pSVA1450-*tfb3*p.
UV treatment and aggregation assays {#SEC2-5}
-----------------------------------
Exposure of *S. acidocaldarius* cells to UV light was performed as described previously ([@B36]). Briefly, 10 ml of a culture (OD~600~ = 0.2--0.3) was induced with 100 J/m² UV light (254 nm, Spectroline UV-crosslinker) in a Petri dish. After that, cultures were incubated at 75°C for 3 h. Subsequently, 5 μl of each culture (diluted to OD~600~ = 0.2) was spotted on a microscope slide coated with 1% (w/v) agar. Single and aggregated cells (*n* ≥ 3) were analyzed by the ImageJ cell counter plug-in (NIH, Bethesda, MD, USA) from at least three independent experiments. The percentage of cells found in aggregates was subsequently calculated.
Quantitative real-time-polymerase chain reaction (qRT-PCR) {#SEC2-6}
----------------------------------------------------------
To compare the expression of genes of the *ups* operon and genes regulated by UV before and 3 h after UV induction, total RNA was isolated from a 10 ml culture grown to an OD~600~ of 0.4 using Trizol (Sigma) followed by DNAse I treatment. cDNAs were synthesized with the First Strand cDNA synthesis Kit (Thermo Scientific) following the instructions of the manufacturer. Quantitative real-time-PCR (qRT-PCR) was performed using the Maxima SYBR green master mix in a Rotor-Gene Q qPCR cycler (Qiagen). Gene-specific primer sets were designed for the following genes: *UpsX* (Saci_1493), *upsE* (Saci_1494), *upsA* (Saci_1496), *cedB* (Saci_0748) (for details, see [Supplementary Table S1](#sup1){ref-type="supplementary-material"}). The housekeeping gene *secY* served as a control (primer set 1480/1481). The threshold cycle (CT) values obtained were determined to compare the non-UV-induced expression with the UV-induced expression of the tested genes. Furthermore, expression levels of tested genes of MW001*^pyrEF+^* and *tfb3::pyrEF* were compared. Differences in expression are displayed as log2 changes.
ONPG assay {#SEC2-7}
----------
The o-nitrophenyl-β\--D-galactopyranoside (ONPG) assay was performed as described previously with few modifications ([@B37]). Briefly, 50 and 350 ml Brock medium containing 0.1% (w/v) NZ-amine and 0.2% (w/v) dextrin were used for the pre-cultures and the main cultures, respectively, and the main cultures were grown until an OD~600~ of 0.2--0.4 was reached. UV treatment was performed as described above. Samples before treatment (control) as well as after treatment with UV irradiation (+UV) and without UV irradiation (−UV) were collected by centrifugation at 5000 × *g* for 20 min (4°C). Cell disruption was performed using a Precellys cell homogenizer (Peqlab) and the lysate was centrifuged (16 000 × *g*, 45 min, 4°C) to remove cell debris. The protein concentration in the soluble fraction was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The ONPG assay was performed in a total volume of 500 μl containing 5 μg protein of the lysate in the Z-buffer. In all steps, 2-mercaptoethanol and Triton X-100 were omitted from the Z-buffer. The measurements were performed at 75°C using a spectrophotometer with a 1 cm cuvet (Specord). Specific activities were determined using the following equation: $$\documentclass[12pt]{minimal}
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}{}\begin{equation*}{\rm{Specific \, activity [U}}/{\rm{mg]}} = \frac{{\Delta {\rm{E}}/{\rm{min}}*{{\rm{V}}_{{\rm{tot}}}}}}{{{\rm{\varepsilon }}*{\rm{c}}*{\rm{d}}*{{\rm{V}}_{{\rm{Protein}}}}}}\end{equation*}\end{document}$$with ΔE/min, extinction change per minute; V~tot~, total volume of the assay \[ml\]; V~Protein~, volume of the protein solution applied in the assay \[ml\]; ϵ, extinction coefficient; c, concentration of the protein solution applied in the assay \[mg/ml\]; d, thickness of the cuvette \[cm\].
Generation and purification of polyclonal antibodies {#SEC2-8}
----------------------------------------------------
Antibodies were generated against purified recombinant 6x-His-TFB1. 1 mg of protein was used for the immunization of one rabbit (28 days Speedy protocol, Eurogentec, Belgium). The antibodies were purified from the final bleed antiserum using protein A agarose (Roche) following the manufacturer's instructions.
Western blotting and immunodetection {#SEC2-9}
------------------------------------
Cell pellets from UV treated and control cultures were resuspended in modified N-buffer (25 mM Tris--HCl pH 7.5, 10 mM MgCl~2~, 100 μM ZnSO~4~, 1 mM Tris(2-carboxyethyl)phosphin supplemented with 500 mM NaCl and protease inhibitor cocktail and subsequently disrupted using a Precellys cell homogenizer (Peqlab). To remove unbroken cells, the lysate was centrifuged at 16 000 × *g* and 4°C for 45 min. The protein concentration was determined according to the method by Bradford ([@B38]) using the Bio Rad protein assay following the manufacturer\'s instructions. Following separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ([@B39]), the proteins were analyzed via western blotting and immunodetection using an anti-FLAG-tag antibody (NEB, dilution 1:1000) and a polyclonal anti-TFB1 antibody (Eurogentec, dilution 1:10 000) as primary antibodies. As a secondary antibody, a horseradish peroxidase (HRP)-conjugated anti-rabbit-IgG raised in mouse (NEB) was used with a dilution of 1:10 000. Signals were detected using the SignalFire™ Elite ECL Reagent (NEB) and visualized by autoradiography.
Co-immunoprecipitation {#SEC2-10}
----------------------
The strains MW001 and MW1104 were grown in Brock medium to OD~600~ = 0.4. Cells were exposed to 100 J/m² UV light (254 nm, Spectroline UV-crosslinker) in 10 ml aliquots and then incubated at 75°C for 3 h. Formaldehyde crosslinking (1% (v/v) final concentration) was carried out in a 35 ml culture for 10 min. The reaction was quenched by addition of 125 mM glycine. After that, cells were harvested by centrifugation at 5000 × *g* for 20 min (4°C) and stored at −20°C. The cell pellets were resuspended in Pierce IP Lysis/Wash Buffer (0.025 M Tris--HCL pH 7.4, 0.15 M NaCl, 0.001 M ethylenediaminetetraacetic acid, 1% (v/v) NP40, 5% (v/v) glycerol) and then lysed by sonication (40% amplitude, 15-s pulse, 15-s pause) using a KE76 tip (Bandelin). Soluble proteins were obtained by ultracentrifugation at 100 000 × *g* for 45 min at 4°C. The pulldown assay was performed using the Pierce Anti-HA Magnetic Beads kit (Thermo Scientific) following the manufacturer\'s instructions. Protein identification was performed by the Mass spectrometry facility of the Center for Biological Systems Analysis (ZBSA) Freiburg.
RNA isolation for RNA sequencing {#SEC2-11}
--------------------------------
RNA was isolated for all samples and replicates with Trizol (ThermoFisher, Waltham, USA) as described previously ([@B40]). The obtained RNA samples were treated with RNase-free DNase (Qiagen) according to the protocol and afterward purified by ethanol precipitation. Ribosomal RNA was depleted using a RiboZero magnetic kit for bacteria (Illumina, San Diego, USA) with a modified protocol. Only 90 μl instead of 225 μl magnetic beads were used and for the rRNA removal reaction and only 1 μg RNA was mixed with 4 μl removal solution in a total volume of 20 μl instead of 40 μl.
RNA sequencing and data analysis {#SEC2-12}
--------------------------------
Sequencing libraries for all samples and replicates were prepared with the TruSeq^®^ Stranded mRNA HT kit (Illumina) starting with the RNA fragmentation step after elution of precipitated RNA in 19 μl of the Fragment-Prime-Mix. Sequencing libraries were quantified with a High-Sensitivity Chip on a Bioanalyzer (Agilent, Böblingen, Germany) and a measurement with a Quant-iT PicoGreen^®^ dsDNA Assay Kit (Invitrogen, Carlsbad, USA) on a Microplate Reader Tecan Infinite 200 (Tecan, Männedorf, Switzerland). Sequencing was performed on a HiSeq1500 instrument (Illumina) in rapid mode with a read length of 2 × 25 nt. Sequencing reads were mapped with Bowtie2 ([@B41]) against the reference genome. Since *S. acidocaldarius* MW001 is a deletion mutant of *S. acidocaldarius* DSM639, the latter one was chosen as a reference (*S. acidocaldarius* DSM639, genome size: 2 225 959 nt, RefSeq ID: NC_007181.1). The data were visualized in the software ReadXplorer and mapped reads were counted for each gene ([@B42]). Subsequently RPKM values ([@B43]) were manually calculated for each gene. In contrast to the original value, only reads mapping to coding sequences were included in order to get the total number of reads. For determination of regulated genes, a statistical analysis was performed using DESeq2 ([@B44]). Sequences relating to *tfb3* found in the *tfb3::pyrEF* strain are related to the small 5′ and 3′ regions of *tfb3* that remained intact after insertion of the *pyrEF* cassette, but do not represent expression of the complete gene.
Identification of a non-palindromic hexanucleotide motif {#SEC2-13}
--------------------------------------------------------
All possible sequence patterns for the desired motif were searched with Bowtie2 (Version: 2.3.0) \[([@B41]); 38 in draft\] in *S. acidocaldarius* DSM639. No mismatches were allowed for the alignment. The results were filtered by a custom Python script to only contain features where the complete motif was between 30 and 80 bp upstream of a gene start. All steps were automated via Snakemake (Version: 3.13.3) ([@B45]). The logo plot was created with WebLogo (Version: 2.8.2) ([@B46]).
RESULTS {#SEC3}
=======
TFB3 protein levels are increased upon UV irradiation {#SEC3-1}
-----------------------------------------------------
In order to follow the expression of TFB3 *in vivo*, the MW001 derived strain *tfb3*~C~SF in which a C-terminally Strep-/FLAG-tag tagged TFB3 is expressed from the native promoter was constructed. To study the expression levels of TFB3 after UV treatment *in vivo, tfb3*~C~SF was cultured in shaking flasks (350 ml) and grown until an OD~600~ of 0.2--0.4 was reached. The experimental setup is shown in Figure [1](#F1){ref-type="fig"}. First, 30 ml of the culture were removed as untreated control sample. The remaining culture was split (10 ml portions each) and transferred to petri dishes. Ten petri dishes were either exposed to UV light (100 J/m^2^) or were handled in the same manner but without UV treatment. Subsequently, the cells were transferred to fresh Erlenmeyer flasks with complex medium and growth was continued at 75°C. In contrast to previous studies focusing on DNA repair mechanisms ([@B30],[@B31]) cells were not maintained in the dark after UV treatment to prevent photolyase activity. Samples for total protein extraction were taken before (control) as well as 45, 90 and 180 min after the treatment with UV irradiation (+UV) or without UV irradiation (−UV). The total protein extracts were analyzed via SDS-PAGE (Figure [2A](#F2){ref-type="fig"}) and immunodetection using an anti-FLAG-tag antibody (Figure [2B](#F2){ref-type="fig"}). As shown in Figure [2B](#F2){ref-type="fig"}, no signal for TFB3 was detected before UV treatment nor in any of the samples that were not treated with UV. TFB3 was also not detected at 45 min after UV irradiation (Figure [2B](#F2){ref-type="fig"}, lane 4), but could be detected 90 min after UV irradiation (Figure [2B](#F2){ref-type="fig"}, compare lanes 5 and 6). TFB3 levels increased further until 180 min (Figure [2B](#F2){ref-type="fig"}, compare lanes 7 and 8). Our data confirm two previous studies ([@B30],[@B31]) and show that TFB expression is induced by UV irradiation.
{#F1}
{#F2}
The activity of the *tfb3* promoter is induced by UV irradiation {#SEC3-2}
----------------------------------------------------------------
To test the activity of the *tfb3* promoter under the influence of UV stress, a 200 bp fragment of the *tfb3* promoter was fused before the *lacS* gene in the shuttle vector pSV1450. MW001 transformants were grown in shaking flasks under standard growth conditions until an OD~600~ of 0.2--0.4 was reached and the UV treatment was performed as described above. Samples were taken at 45, 90 and 180 min after the treatment. The β-galactosidase activity in the total protein extracts was determined using the ONPG assay.
As shown in Figure [3](#F3){ref-type="fig"}, a basal activity of 3.7 U/mg was determined in the sample before the treatment (control). While no significant increase of the activity was observed in all samples without UV irradiation, higher β-galactosidase activities were observed in the UV irradiated samples increasing from 4.1 U/mg to 6.7 U/mg and 8.7 U/mg after 45, 90 and 180 min, respectively. These results were in agreement with the observed expression of the TFB3 protein after 90 min in UV treated cells (Figure [2B](#F2){ref-type="fig"}). The β-galactosidase activity in the sample before treatment indicates a basal expression from the *tfb3* promoter. Indeed analysis of increased amounts of crude extracts (100 μg protein compared to 30 μg protein extract loaded on SDS-PAGE previously (Figure [2](#F2){ref-type="fig"})) allowed for detection of TFB3 in non UV treated cells ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}).
{#F3}
TFB3 *in vivo* protein--protein interaction {#SEC3-3}
-------------------------------------------
In order to identify proteins that interact with TFB3 *in vivo, S. acidocaldarius* strain MW1104, which carries the endogenously HA-tagged *tfb3* gene was constructed. Soluble proteins were extracted from MW001 and MW1104, were immune-precipitated using an anti-HA antibody and these fractions were analyzed on a SDS-PAGE followed by silver staining. Mass spectrometry was further used to identify proteins (indicated by black rectangles, Figure [4](#F4){ref-type="fig"}) that were observed in MW1104 but not in MW001. As shown in Figure [4](#F4){ref-type="fig"}, candidates specifically identified in the TFB3-HA immunoprecipitation fractions could be identified ([Supplementary Table S2](#sup1){ref-type="supplementary-material"}). Notably, TFB1 and the RNAP subunits RpoA2, RpoA1, RpoB and the TBP-binding protein (Tip49) were detected. TFB3 was previously reported to interact with the RNAP and probably TFB1 in the pre-initiation complex *in vitro* ([@B32]). Remarkably, also *Sa*-Lrp (Saci_1588) ([@B47]) was specifically detected in MW1104. *Sa*-Lrp was previously shown to bind to the promoters of the *ups* genes and the deletion mutant of *Sa*-Lrp showed abnormal sizes of the UV-induced aggregates ([@B47]).
{ref-type="supplementary-material"}: Tip49, TBP interacting protein 49kDa; TUF, Elongation factor 1-α; UpsE, ATPase of the ups pilus; RopA(1,2,B), RNAP subunits; Rrp42, Exosome complex component; Rps19, 30S ribosomal protein S19; Rsps8e, 30S ribosomal protein S8e; M, marker in kDa.](gky527fig4){#F4}
Cellular aggregation after UV treatment {#SEC3-4}
---------------------------------------
Upon UV stress, *S. acidocaldarius* cells express *ups* pili (UV-inducible pili operon of *Sulfolobus*), which result in the formation of aggregates in which subsequently DNA exchange can occur ([@B36],[@B48]). This DNA exchange is an important mechanism to repair DNA damage via homologous recombination. Deletion of e.g. *upsE* that encodes the secretion adenosinetriphosphatase (ATPase) involved in *ups* pili formation results in a strain (MW109), which does no longer aggregate after UV treatment ([@B49]). In order to further investigate the involvement of TFB3 in this UV stress response, the *tfb3* insertion mutant strain *tfb3::pyrEF* was generated. In this strain, a cassette containing the *S. solfataricus pyrEF* genes is inserted into the *tfb3* gene resulting in disruption of the *tfb3* gene. As a control the *S. acidocaldarius* strain MW001*^pyrEF+^* (MW001 complemented with the *pyrEF* cassette ([@B35])), was used. Aggregation assays performed with MW001*^pyrEF+^* showed that 180 min after UV treatment, 45--85% of the cells were present in aggregates, whereas in the *ΔupsE* strain MW109 \<10% of the cells were present in aggregates (Figure [5](#F5){ref-type="fig"}). Interestingly, the *tfb3* disruption mutant *tfb3::pyrEF* also showed almost no aggregates after UV treatment (5%) (Figure [5B](#F5){ref-type="fig"}) suggesting that TFB3 might be involved in the regulation of *ups* pili formation. To study this possibility, qRT-PCR was performed on selected genes involved in cellular aggregation and DNA exchange ([@B36],[@B49],[@B51]). This showed that the *upsX, upsE* and *upsA* genes, contrary to the reference strain MW001^pyrEF+^, were not induced in the *tfb3* mutant 180 min after UV stress. Moreover, when expression of these three genes was compared to that in MW001*^pyrEF+^* (Figure [6A](#F6){ref-type="fig"}, on the right side), their expression was also significantly lower in non-UV-treated samples. Beside the *ups* genes, also the transcription of the *cedB* gene, which encodes the membrane protein involved in DNA transport in aggregates after UV treatment ([@B51]) was dramatically decreased in the *tfb3* mutant compared to that of MW001*^pyrEF+^* (Figure [6B](#F6){ref-type="fig"}) ([@B30],[@B31]). Therefore, it is concluded that TFB3 is involved in the regulation of the *ups* and *ced* genes.
{#F5}
{#F6}
Early transcriptional response to UV irradiation {#SEC3-5}
------------------------------------------------
To investigate the role of TFB3 in the early response to UV treatment, gene expression in the MW001*^pyrEF+^* and the *tfb3::pyrEF* strain were studied by a genome wide transcriptome analysis. To exclude an effect of the *pyrEF* cassette on the differential transcription of genes, strain MW001*^pyrEF+^* was used as reference. Cells (see [Supplementary Figure S3](#sup1){ref-type="supplementary-material"} for detailed transcript levels) were cultivated until an OD~600~ of 0.2--0.4 was reached and were subsequently UV-treated with 100 J/m^2^ as shown in Figure [1](#F1){ref-type="fig"}. RNA was extracted from samples taken before as well as 45 and 90 min after UV treatment. A detailed overview of the transcription of the *S. acidocaldarius* genes in the MW001*^pyrEF+^* strain and the *tfb3::pyrEF* strain at the different timepoints is given in [Supplementary Table S3](#sup1){ref-type="supplementary-material"}. [Supplementary Tables S4--6](#sup1){ref-type="supplementary-material"} show a detailed presentation of all significant differences in gene expression (*P*-value ≤ 0.01). In MW001*^pyrEF+^*, 45 min after UV irradiation, 12 genes were, when compared to the not UV treated cells, regulated more than 4-fold and 100 genes more than 2-fold. Ninety minutes after UV irradiation, 45 genes were regulated more than 4-fold and 191 genes more than 2-fold (for details see [Supplementary Figures S4, 5 and 8](#sup1){ref-type="supplementary-material"}). In the *tfb3::pyrEF* strain, after 45 min, 8 genes were regulated more than 4-fold and 122 genes more than 2-fold and after 90 min 24 genes were regulated more than 4-fold and 190 genes more than 2-fold, when compared to non UV treated cells (for details see [Supplementary Figures S4, 5 and 8](#sup1){ref-type="supplementary-material"}).
When the *tfb3::pyrEF* strain was compared with strain MW001*^pyrEF+^*, 7, 11 and 15 genes were regulated more than 4-fold (46, 30 and 45 genes more than 2-fold) at the timepoints before UV treatment and at 45 and 90 min after UV treatment, respectively (for details see [Supplementary Figures S6 and 7](#sup1){ref-type="supplementary-material"}). The regulated genes are distributed evenly over the whole genome ([Supplementary Figure S11](#sup1){ref-type="supplementary-material"}). To further analyze the identified genes, they were classified according their arCOG (Archaeal Clusters of Orthologous Genes) classes. This identified major changes in the classes describing genes involved in transcription (K), replication (L), cell-cycle control and division (D), as well as nucleotide metabolism and transport (F), indicating that the organism adapts to UV stress by regulating a variety of biological processes ([Supplementary Figures S10 and 11](#sup1){ref-type="supplementary-material"}).
The comparison of the UV response in both strains allows distinguishing between UV-responsive genes whose expression depends on the presence of TFB3 and thus is lower in the *tfb3::pyrEF* strain compared to the MW001^+^*^pyrEF^* strain and genes whose transcription is independent of TFB3 and therefore shows no significant difference in the *tfb3::pyrEF* and MW001^+^*^pyrEF^* strains. The data also enables to differentiate between genes that show an early response (45 min) and a delayed response (90 min). Tables [1](#tbl1){ref-type="table"} and [2](#tbl2){ref-type="table"} show a selection of genes, which are affected by the UV treatment but are either dependent or independent on the presence of TFB3.
######
TFB3-dependent regulation of selected genes following UV irradiation in *S. acidocaldarius* MW001*^pyrEF+^* (WT) and *tfb3::pyrEF* (Mut)
---------------------
---------------------
######
TFB3-independent regulation of selected genes following UV irradiation in *S. acidocaldarius* MW001*^pyrEF+^* (WT) and *tfb3::pyrEF* (Mut)
---------------------
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TFB3-dependent UV stress response {#SEC3-6}
---------------------------------
As expected, *tfb3* is one of the five genes that are more than 4-fold upregulated 45 min after UV treatment (WT0 versus WT1), while upregulation of all other genes, which are expressed differentially in a TFB3-dependent manner, follows after 90 min (WT0 versus WT2) (Table [1](#tbl1){ref-type="table"}). We found that *tfb3* is slightly upregulated as a later response to UV stress in the *tfb3* mutant strain (Mut0 versus Mut2). Furthermore, a slight down regulation could be observed in the irradiated *tfb3* mutant strain compared to the reference strain (WT1 versus Mut1, WT2 versus Mut2). This is due to the fact that in the insertion mutant transcription of the gene occurs and short reads of the disrupted gene might be mapped during sequencing despite the *pyrEF* insertion. Five and 22 genes are differentially regulated more than 4-fold 45 and 90 min after UV treatment in the reference strain MW001^+^*^pyrEF^*, while no response was observed in the *tfb3* insertion mutant *tfb3::pyrEF* (see [Supplementary Figure S4C and D](#sup1){ref-type="supplementary-material"} for details). Accordingly, the expression of a subset of these genes was significantly different in the untreated and/or irradiated reference compared to the *tfb3::pyrEF* mutant strain (WT0 versus Mut0, WT1 versus Mut1, WT2 versus Mut2, see [Supplementary Figure S6](#sup1){ref-type="supplementary-material"} for details).
Comparing the expression in the reference and the mutant strain almost all genes show a strong downregulation in the mutant strain and only one gene is slightly upregulated (2-fold) under all conditions ([Supplementary Figure S7](#sup1){ref-type="supplementary-material"}), Saci_1770 annotated as 1,2-phenylacetyl-CoA epoxidase, catalytic subunit (arCOG Q). The enzyme shows significant structural similarity to the recently characterized *E. coli* phenylacetic acid degradation protein (probability 99.96; *E*-value 2e-29; (HHPRED, ([@B52])); 3pvt_A, ([@B52])) that is involved in the aerobic and anaerobic hybrid degradation pathway for aromatic compounds. More distant structural similarity is found to ribonucleotide reductases (probability 99,79; *E*-value 3.1e-20, 4M1H_A (HHPRED), ([@B53])), which catalyze the formation of deoxyribonucleotides from ribonucleotides suggesting a possible role of Saci_1770 in the regulation of DNA synthesis in *S. acidocaldarius*. All other genes showed an upregulation in the MW001*^pyrEF+^* strain when compared to the *tfb3::pyrEF* strain. Most regulation was observed 90 min after UV irradiation, suggesting that this is the result of the expression of TFB3. Indeed upregulation of *tfb3* was observed after 45 min and the TFB3 protein was detected after 90 min.
Among the TFB3-dependent UV responsive genes identified, the *ups* gene cluster (*upsX*, Saci_1493; *upsE*, Saci_1494; *upsF*, Saci_1495; *upsA/B*, Saci_1496) was most highly upregulated (more than 4-fold). In addition, also the *cedA1, cedA2* and *cedB* genes encoding the membrane and the ATPase components of the Ced (crenarchaeal system for exchange of DNA) DNA importer were more than 4-fold upregulated. Remarkably, also the membrane-bound VirB4/HerA/CedB homolog Saci_0667, which is encoded in close vicinity of the Ced system is highly upregulated. It has been proposed that Saci_0667 either encodes an additional ATPase involved in the Ced system or might function as a part of a putative export machinery on the donor site of DNA transfer events ([@B51]).
Furthermore, Saci_0950 shows more than 2-fold TFB3 dependent upregulation at 90 min after UV treatment. Saci_0950 shows 29% sequence identity (*e*-value = 8e^−18^) to the characterized pantoate kinase from *Thermococcus kodakarensis* (TK2141) involved in coenzyme A biosynthesis ([@B54]). Other genes, which show a TFB3-dependent UV stress response are the putative transporter protein encoding Saci_1270 (annotated as a MFS permease) and two genes of unknown function, Saci_0951 and Saci_1225, which were also previously observed to be upregulated upon UV irradiation ([@B31]).
TFB3-independent UV stress response {#SEC3-7}
-----------------------------------
To find genes that showed a TFB3 independent response to UV treatment, genes that were similarly regulated in the MW001*^pyrEF+^* strain and the *tfb3::pyrEF* strain were identified. Seven genes were identified that were at least 4-fold up- or downregulated at 45 min after UV treatment and 23 genes were identified after 90 min ([Supplementary Figure S4C and D](#sup1){ref-type="supplementary-material"}).
Among the genes that show an upregulation is one of the three Cdc6 (cell division cycle 6) proteins, which are key players in the initiation step of DNA replication. Archaeal homologs of proteins involved in the eukaryotic origin recognition complex have been studied in *Methanothermobacter thermautotrophicus* and it was proposed that Cdc6 proteins directly interact with the replicative minichromosome maintenance helicase MCM and bind to replication origins ([@B55]). *Cdc6-2* (Saci_0903) was one of the most highly induced genes upon UV stress, whereas *cdc6-3* (Saci_0001) remained unaffected *and cdc6-1* (Saci_0722) was slightly downregulated. This is in accordance with previous studies ([@B30],[@B31]). For *S. solfataricus* a putative role of Cdc6-2 as a repressor of DNA replication was suggested based on the presence of Cdc6-2 only in the post-replicative G2 phase of the cell cycle and the binding of Cdc6-2 to DNA sequences that overlap with Cdc6-1 or Cdc6-3 binding site ([@B56]). In contrast, Cdc6-1 and Cdc6-3 have been discussed to act on the origins in a positive manner to facilitate replication initiation.
Also the Saci_0478 and Saci_0483 genes, which are annotated as conserved conjugative plasmid proteins possessing HTH DNA-binding motifs similar to that of the transcriptional regulator XRE and MarR families, respectively, show upregulation after UV irradiation. The XRE (xenobiotic response element) family of transcriptional regulators is present in all three domains of life ([@B57]) and for MarR like regulators a role in antibiotic resistance, oxidative stress response, virulence or catabolism of aromatic compounds is reported ([@B58]). A new member of this family, BldR2, involved in aromatic stress response was characterized in *S. solfataricus* ([@B59]). One can speculate that the regulators have a possible role in conjugative processes induced in the course of oxidative stress response upon formation of pyrimidine and purine photoproducts. This is in accordance with the observed 2-fold upregulation of the predicted superoxide dismutase (Saci_0195) in the delayed UV response of both strains.
At last, also the ArnR encoding gene Saci_1180 is highly induced upon UV irradiation. ArnR is together with ArnR1 (Saci_1181) one of the two transcriptional regulators (i.e. activators) of the archaellum regulatory network (Arn), which induce the expression of the filament forming archaeallins encoded by *flaB*. The archaellum operon of *S. acidocaldarius* is divided into two loci, *flaB* and *flaX-J*, which encode essential genes for the biosynthesis of the archaellum, a rotating type IV pilus-like structure, which acts as the motility structure of this organism ([@B60],[@B61]). No upregulation of FlaB expression was observed in our experiments. Indeed a delay between ArnR induction and the expression of *FlaB* was observed under starvation condition where the assembly of the archaellum is strongly induced after 4 h of starvation ([@B62]).
Among the genes, which were downregulated, predicted transcriptional regulators are found. Saci_1012, which is strongly downregulated upon UV stress, encodes a putative Mn-dependent transcriptional regulator of the DtxR family and might be involved in metal homeostasis. Manganese-responsive DtxR family regulators were studied in many bacteria, including the MntR protein of *E. coli* ([@B63]) and *Bacillus subtilis* ([@B64]). An MntR mediated repression of a manganese transporter has been reported for *Mycobacterium tuberculosis* ([@B65]). Manganese plays an important role in free radical-detoxifying enzymes, including superoxide dismutase and catalase ([@B66]). Therefore, the downregulation of a possible repressor of manganese transport (Saci_1012) might also aid oxidative stress response mediated by the upregulated predicted superoxide dismutase (Saci_0195).
Remarkably, the gene encoding ArnC (Saci_1193*)*, one of the Hanks-type protein kinases (eSTKs) (besides ArnD, Saci_1694) that have been shown to be involved in the regulation of the archaellum ([@B61],[@B67],[@B68]), is downregulated 2-fold after 45 min and more than 4-fold after 90 min after UV treatment in both investigated strains. Deletion mutants of ArnC were only slightly affected in their motility but no direct effect on FlaB expression was observed, the filament protein of the archaellum, which indicated that they might be involved in other regulatory pathways possibly interconnected with the archaellum regulatory network ([@B68]).
Several genes involved in purine biosynthesis (e.g. Saci_0214, formate-dependent phosphoribosylglycinamide formyl-transferase (*purT*)); Saci_1607, *purC*, phosphoribosylaminoimidazolesuccinocarboxamide synthase and Saci_1608 and Saci_1610, which encode the PurS and the PurL domains of the phosphoribosylformylglycinamidine synthase complex) were also strongly downregulated. Also Saci_1701, which encodes a conserved protein annotated as a xanthine/uracil/vitamin C permease of the AzgA family is downregulated after UV stress in a TFB3 independent manner. Its amino acid sequence shows 30% identity (*e*-value: 2e^−43^) with the AzgA protein of *Aspergillus nidulans*, which has been characterized as a purine transporter with high affinity to hypoxanthine, guanine and adenine ([@B69]). Saci_0789 and Saci_1672, which both encode predicted deoxycytidine deaminases were also found to be repressed 90 min after UV irradiation. However, whereas Saci_0789 was downregulated 4- and 2-fold in the WT and *tfb3::pyrEF* strain, respectively, for Saci_1672 only a 2-fold change in the WT strain was observed. Saci_0789 possesses similarity to the characterized bifunctional dCTP deaminase-dUTPase (DCD-DUT) from *Methanocaldococcus jannaschii* MJ0430 (28% protein sequence identity, *e*-value = 6e^−10^) ([@B70],[@B71]) and is therefore assumed to play an essential role in pyrimidine metabolism.
Further genes that show a downregulation at 90 min after UV irradiation are Saci_0203 and Saci_0204, which are annotated as an uncharacterized protein and as a ParA ATPase involved in chromosome partitioning, respectively. The two genes show high similarity to SSO0035/SegB (sequence identity = 53%, *e*-value = 4e^−38^) and SSO0034/SegA (sequence identity = 71%, *e*-value = 1e^−111^), which were shown to play an essential role in chromosome partitioning ([@B72]). While SegA is an ortholog of bacterial Walker-type ParA proteins, SegB is considered an archaea specific factor ([@B72]). In addition, also the annotated Holliday junction (HJ) resolvase Hjc, encoded by Saci_1741 was strongly downregulated. The corresponding gene product shows high similarity to several other archaeal HJ resolvases, e.g. Hjc (SSO0575, sequence identity = 42%, residues 8 to 79, *e*-value = 2e^−14^) and Hje (SSO1176, sequence identity = 73%, *e*-value = 4e^−66^) from *S. solfataricus*. For the genes Saci_1228 annotated as a predicted P-loop ATPase superfamily protein and the adjacent gene Saci_1229, which is annotated as a transcriptional regulator of the GntR family, a 4- and 2-fold downregulation, was observed. Saci_1228 shows distant homology to ATP-dependent HrpA-like helicases, which are absent in archaeal genomes. Hence, the Saci_1228 gene product might represent a HrpA counterpart involved in cell cycle progression in archaea. ([@B29],[@B73]). Thus our data suggest that the TFB3-independent UV response includes the repression of nucleotide biosynthesis and transport, replication and cell cycle progression in order to allow DNA repair.
Identification of a non-palindromic hexanucleotide motif {#SEC3-8}
--------------------------------------------------------
Previous motif searches identified a non-palindromic hexanucleotide motif in *S. acidocaldarius* (5′-A(N)TTTC-3′) that was supposed to be important in UV-inducible promoters ([@B74]). The motif is located 30 to 80 bp upstream of the transcription start, and a so far unknown regulator was supposed to bind to the motif and to interact with TFB3, which lacks a DNA binding domain. It is speculated that TFB3 then interacts with the RNAP and thus activates transcription via bridging the RNAP and TFB1--TBP--DNA complex ([@B32],[@B74]). In our study, we performed an automated motif search and identified in total 299 genes in *S. acidocaldarius*. Among these genes were 31 and 24 (log~2~ fold change ≥ 2: 11 and 2) significantly upregulated genes and 9 and 15 (log~2~ fold change ≤ −2: 3 and 3) down regulated genes after UV treatment in the reference strain and tfb3 mutant strain, respectively (for details see [Supplementary Table S7 and Figure S12](#sup1){ref-type="supplementary-material"}). Two of the 11 highly upregulated genes (more than 4-fold) in the reference strain, with the searched motif in its upstream region, were also highly upregulated in the *tfb3* insertion mutant indicating that the presence of TFB3 is not required for the observed regulation. One of these genes was *cdc6-2* (Saci_0903) (Table [2](#tbl2){ref-type="table"}). From the highly downregulated genes (more than 4-fold) all three were found in the reference strain and the *tfb3* insertion mutant. We, therefore, speculate that the motif is important for binding of a so far unknown factor or factors that regulate TFB3-independent transcription probably also under other growth or stress conditions.
DISCUSSION {#SEC4}
==========
In *Sulfolobus*, UV-induced DNA damage leads to the formation of photoproducts like cyclobutane pyrimidine dimers (CPD) and 6,4-pyrimidine-pyrimidones (6-4 PP) as well as single and double strand breaks ([@B30]). Interestingly, contrary to what has been previously observed in eukaryotes and bacteria, no significant transcriptional induction of DNA repair genes was observed in *Sulfolobales* ([@B30],[@B31]). Instead, upregulated genes were mainly involved in cell cycle control (*cdc6-2*), oxidative stress (*dps*) and transcription regulation (*tfb3*). In contrast, DNA replication proteins are repressed during UV stress ([@B31]).
Two independent DNA microarray studies in *Sulfolobus* demonstrated that the gene encoding the alternative TFIIB homolog TFB3 is one of the highest upregulated genes upon UV irradiation ([@B30],[@B31]). Our studies with chromosomally tagged TFB3 and promoter activity assays (Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}) showed that the previously observed changes in transcript levels ([@B31]), as expected, result in a significant increase in the cellular TFB3 concentration after UV irradiation, while the concentrations of TFB1 remained stable. Remarkably, changes in transcript level are observed already at 45 min, whereas an increase in the TFB3 protein concentration is only observed at 90 min after UV treatment indicating that changes in *tfb3* transcript numbers are manifested only after about 45 min in changes in the TFB3 protein concentration (Figure [2](#F2){ref-type="fig"}).
This study also provided first evidence of the *in vivo* function of TFB3. The *tfb3::pyrEF* strain was impaired in functional Ups pili formation and UV induced cell aggregation (Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}), a process, which normally leads to DNA exchange. This demonstrated that TFB3 is required for Ups pili expression after UV treatment.
Furthermore, several interaction partners of TFB3 were identified. Using the *tfb3*-HA-tagged strain MW1104, we were able to show the interaction of TFB3 with TFB1 and the RNA polymerase (represented by the large subunit RpoA2) *in vivo* by co-immunoprecipitation (Figure [4](#F4){ref-type="fig"}). This was consistent with previous *in vitro* studies where co-immunoprecipitation of TFB3 with the ternary complex of DNA--TBP--TFB1 and an interaction of TFB3 with the completely assembled pre-initiation complex was observed ([@B32]). Co-immunoprecipitation with TFB3-HA also identified *Sa*-Lrp (Saci_1588). Sa-Lrp is a member of the leucine-responsive regulatory protein (Lrp)-like family of transcriptional regulators in *S. acidocaldarius* ([@B47]). *Sa*-Lrp binds to promoter regions of genes with a variety of functions including ammonia assimilation, transcriptional control and UV-induced pili synthesis. *In vivo* studies with the *Sa-lrp* deletion mutant showed that the absence of *Sa*-Lrp lead to reduced UV-induced cell aggregation. A differential effect of *Sa*-Lrp on the expression of the *upsA* gene before and after UV exposure was observed and it was suggested that *Sa*-Lrp acts in conjunction with other transcriptional regulators in driving the transcription of the *ups* operon ([@B47]). Our pulldown experiments provided first evidence that TFB3 might be the interacting factor of *Sa*-Lrp.
The genome wide transcriptome analysis using the MW001^+^*^pyrEF^* and the *tfb3::pyrEF* strains revealed two separate UV stress response pathways. While the regulation of several genes depends on the presence of TFB3, other genes are differentially transcribed in a TFB3-independent manner. The TFB3-dependent response showed a strict chronological order. Only after expression of TFB3 (45 min), an effect on target gene expression (90 min) is observed, demonstrating that the presence of high levels of TFB3 is a prerequisite for the enhanced transcription of the TFB3-dependent target genes. The gene encoding the classical housekeeping GTF *tfb1* was found not to be regulated upon UV stress, confirming the stable expression that was observed in all samples during the immunodetection experiment with the chromosomally tagged strain *tfb3*~C~SF (Figure [2B](#F2){ref-type="fig"}).
Among the processes that are under control of TFB3 (Table [1](#tbl1){ref-type="table"}) is the exchange of DNA via the Ups and Ced systems after UV treatment ([@B49],[@B51]). DNA exchange between the cells occurs in order to repair UV-induced DNA damages via homologous recombination. The observed TFB3-dependent regulation is in accordance with the results of two previous studies on the response of *S. solfataricus* and *S. acidocaldarius* to UV damage. In both microarray studies, the *ups* genes as well as the *ced* genes were upregulated upon UV stress ([@B30],[@B31]). The fact that all genes, which exhibit a differential expression depending on the presence of TFB3, are downregulated in the *tfb3::pyrEF* strain compared to MW001^+^*^pyrEF^* strain and no upregulation was observed in this comparison, confirms that TFB3 acts as an activator of transcription ([@B32]).
Analysis of the TFB3-independent response, which is observed in both the MW001^+^*^pyrEF^* strain and the *tfb3::pyrEF* strain(Table [2](#tbl2){ref-type="table"}) showed that especially the transcription of genes, which drive replication, genome segregation and cell division, is significantly reduced upon UV stress. Consistently, a large number of these genes shows a cell cycle-dependent transcriptional induction during the respective cell cycle stage under standard growth conditions as demonstrated by studies with cell cycle-synchronized *S. acidocaldarius* cultures ([@B29],[@B73]). For example, the predicted chromosome partitioning ATPase (*parA*, SegA, Saci_0204) and its preceding gene Saci_0203 (SegB) ([@B72]), which in our transcriptome study both are downregulated more than 4-fold upon UV treatment, were among the very first genes to be induced in the synchronized cultures under standard growth conditions. This confirms their function in genome segregation, the first cell-cycle process to be initiated after release from G2 arrest ([@B29]). In addition, the annotated HJ resolvase (Saci_1741) shows a significant downregulation. The biochemical and structural properties and function of these endonucleases, which catalyze the cleavage/resolution of HJs and are of fundamental importance in chromosome segregation, have been described in detail ([@B75],[@B76]). One might speculate that the accumulation of HJs, because of the lower abundance of HJ resolvases, may intentionally impair cell-cycle progression to allow DNA repair. The predicted HrpA counterpart Saci_1228 and the neighboring gene Saci_1229, which are strongly repressed upon UV stress in both strains have also been reported to be highly induced at the mitosis/cytokinesis stage in cell cycle-synchronized cultures and a function in cell-cycle progression was proposed ([@B29],[@B73]).
Furthermore *cdc6-2*, which encodes one of the three identified Cdc6s in *S. acidocaldarius*, was among the early, highly upregulated genes, whereas *cdc6-1* was slightly downregulated at 90 min. For cdc6-2, a function as repressor of DNA replication initiation was proposed, whereas Cdc6-1 and Cdc6-3 seem to promote initiation of DNA replication ([@B56]). However, our studies indicate that Cdc6-2 might also operate as an inducible transcription factor, like TFB3. Thus, our data show that upon UV stress, mechanisms that inhibit DNA replication and cell division are initiated to allow DNA repair. Notably all these mechanisms are part of the TFB3-independent UV response in *S. acidocaldarius* and except *cdc6-2* all genes are downregulated as part of the delayed UV response. The inhibition of DNA replication following UV-induced DNA damage has been reported previously both for bacteria and eukaryotes ([@B77],[@B78]).
Exposure to UV light is accompanied by DNA damage, e.g. misincorporation of bases during the replication process, deamination of bases, depurination and depyrimidination as well as oxidative stress caused by UV radiation-induced free radicals or reactive oxygen species ([@B79],[@B80]). Although the most observed consequences of UV radiation are pyrimidine photoproducts, e.g. CPDs, characterized by covalent linkage between adjacent pyrimidines and (6-4) pyrimidone photoproducts (6--4 PPs), several purine photoproducts have also been reported. These include the adenine dimer, the Porschke photoproduct and the T-A photoadduct ([@B79],[@B81],[@B82]). Accordingly, we found that processes, which are involved in synthesis and uptake of nucleotides, are strongly affected upon UV treatment in the MW001^+^*^pyrEF^* strain and the *tfb3::pyrEF* strain. The genes involved in purine biosynthesis and uptake are highly downregulated in the early UV response and two predicted deoxycytidine deaminases for pyrimidine biosynthesis in the delayed UV response. In previous studies the amount of transcripts of the pyrimidine synthesis gene cluster (Saci_1607-Saci_1613) and the uptake system (Saci_0214) have been shown to be coordinately increased in the chromosome replication stage of the cell cycle (S-phase), where high amounts of DNA building blocks are required ([@B29]).
Our data show that multiple predicted proteins, which share structural relationships to well-known families of transcriptional regulators (Saci_0478 (XRE family) and Saci_0483 (MarR family) (both annotated conserved conjugative plasmid proteins with HTH motif); Saci_1012 (DtxR family), Saci_1180 (ArnR)), exhibit a strong UV stress response in both investigated MW001 strains. In addition, the expression of the gene encoding the Hanks-type protein kinase ArnC (Saci_1193) is drastically reduced upon UV treatment in both investigated strains suggesting a general regulatory role in cellular UV response.
In summary, our results confirm the UV-dependent expression of TFB3 on both protein and gene level. Using the *tfb3::pyrEF* strain, we identified genes, whose differential gene expression upon UV irradiation depends on the presence of TFB3 and demonstrated the importance of TFB3 in UV-mediated cellular aggregation and DNA exchange (i.e. *ups* pili operon and the Ced DNA importer) in order to allow for DNA repair via homologous recombination. Our findings confirm TFB3 as a key player and activator of transcription in early UV stress response, since all genes are downregulated in the *tfb3::pyrEF* strain compared to the MW001^+^*^pyrEF^* strain. However, we observed an additional clear TFB3-independent response that targets major cellular processes resulting in repression of DNA replication, cell-cycle control as well as nucleotide biosynthesis in order to allow for DNA repair (Figure [7](#F7){ref-type="fig"}).
{#F7}
Supplementary Material
======================
######
Click here for additional data file.
F.S. and B.R. were supported by the German Research Foundation, DFG Graduate Training Program 1431 'Transcription, chromatin structure and DNA repair in development and differentiation'. BS, A.A., J.K. A.G. and P.B. acknowledge funding by the Ferderal Ministery of Education and Research (BMBF) within the e:Bio initiative HotSysAPP. A.G. and P.B. acknowledge technical assistance by the Bioinformatics Core Facility/Professorship of Systems Biology at JLU Giessen and access to resources financially supported by the BMBF grant FKZ 031A533 to the BiGi center within the de.NBI network. T.N.L. acknowledges funding by the Minstry of Education and Training, Vietman. S.V.A. acknowledges funding by the European Research councli (ERC starting grant, ARCHAELLUM). We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.
*Author Contributions*': B.S. conceived the study. F.S., B.S., S.V.A., A.G. and J.K. designed the experiments. F.S., B.R., T.L., P.B. and A.A. performed the experiments. Computational motif Search P.B., A.G.. F.S. and B.S. wrote the manuscript with input from A.A., T.L., C.D., S.V.A. All authors approved the final manuscript.
SUPPLEMENTARY DATA {#SEC5}
==================
[Supplementary Data](https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gky527#supplementary-data) are available at NAR Online.
FUNDING {#SEC6}
=======
German Research Foundation \[DFG Graduate Training Program 1431 to F.S., B.R.\]; Federal Ministry of Education and Research (BMBF) \[e:Bio initiative, HotSysAPP, 03120078A to B.S., 031L0078C to A.A., J.K., 031L0078D to A.G., P.B., de.NBI network FKZ 031A533 to A.G., P.B.\]; Ministry of Education and Training, Vietnam \[911 grant to T.N.L.\]; European Research council \[ERC starting grant, ARCHAELLUM, 311523 to S.V.A.\]. Funding for open access charge: University Duisburg-Essen (Bettina Siebers).
*Conflict of interest statement*. None declared.
[^1]: Present address: Bettina Siebers. Molecular Enzyme Technology and Biochemistry (MEB), Biofilm Centre, Centre for Water and Environmental Research (CWE), Faculty of Chemistry, University of Duisburg-Essen, Universitätsstraße 5, 45141 Essen, Germany.
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When we started the process of creating this special issue of *Public Health,* we were seeking to respond to an increasing call to recognise gambling harms as a public health problem and to address concerns about a potential growing epidemic of harms that threatened the health and well-being of populations across the globe. That this is increasingly being accepted as a public health issue, albeit among a relatively small field of gambling researchers and practitioners, is not in question. However, the question that faced us was what such a response, for this Journal, at this point in time, should look like.
We began to realise that what was needed was a kind of 'next step'. An evolution of discussion that goes beyond the simple repetition that 'gambling is a public health problem' brings the issue to a wider audience of academics, public health and healthcare professionals. And for this to include what public health policy and practice might be in the future.
To facilitate mature debate, we needed to help public health, primary care and healthcare professionals see that gambling is not necessarily a harmless pastime, and to understand that gambling harms contribute to many of the social and economic inequalities that are determinants of health and well-being for individuals, their families and the communities in which they live. In short, we needed to bring what was known from the evidence base to the readers of this Journal. As with any issue of *Public Health,* we wanted to do that in a way which helped readers see both the reason for the concerns relating to gambling harms across the globe and the potential for translating such evidence into public health action. This issue is the result of those discussions.
What we did not discuss was how you present a special issue of *Public Health* on what some are coming to see as an epidemic of gambling-related harms, when the world is experiencing a global pandemic. At the time of writing, COVID-19 is still an emerging disease. Whilst we await population surveillance based on antibody testing, containment measures will continue to focus on the isolation of symptomatic cases and social lockdowns. Across the world, public health attention has, naturally, been directed towards the pandemic response, whether locally or as advisors for national and local politicians.
Yet, even in the midst of this pandemic, we need to be aware that gambling harms are still occurring. The pandemic has not interrupted gambling, merely changed how it is happening. We can but speculate on how keeping people in their own homes, many without their usual occupations, activities and social networks, might impact their use of online gambling platforms. Obviously, sporting events have been cancelled and land-based venues closed during lockdowns, vastly reducing the availability of gambling, but other options online have sprung up. As competitive sports and horse racing restart, albeit behind 'closed doors', and social distancing continues to affect the footfall in gambling venues, in the US at least, one company has created 'drive-thru' gambling centres.[@bib1]
Debate has already started about the impact the COVID-19 pandemic has had on gambling behaviour. Industry commentators point to lost revenues, which are likely to be significant. The industry will regroup and a critical business consideration for them will be how to future-proof itself against such shocks of this nature. A greater push to online gambling seems inevitable. It may also be tempting for governments to use gambling expansion and its subsequent revenues to recover resources which will be a priority with the inevitable economic depression looming. However, from a public health perspective, we need to focus on people, not purses. We need to consider the impact on individuals and communities and to assess how gambling harms are changing in the context of our vastly altered post--COVID-19 world.
In Britain, as elsewhere, one of the aims of gambling legislation is stated to be the protection of the vulnerable. Importantly, vulnerability is not a static state but something that may vary for individuals based on their life experiences and conditions. In the context of great economic and social uncertainty, it is vital to understand who are the new 'vulnerable' and what measures should be put in place to protect them. This may mean taking and maintaining a more precautionary approach to gambling regulation whilst the knowledge about impact is generated. COVID-19 is likely to create many more vulnerable people and to exacerbate existing inequalities: inequalities that are already expressed in the distribution of gambling harms. It is vital that politicians, regulators and public health officials are sighted of these changes and able to respond rapidly.
What this collection of special articles shows is how varied and diverse the experience of gambling harms can be, how far reaching for individuals, families and communities, and how global the potential impact could be. Vulnerability to harms is manifest through socio-economic or cultural status, through the areas in which people live and through the practices engaged in by the industry. These things are not simply the preserve of the individual but of individuals embedded within their broader social and environmental contexts.
It is particularly heartening to see the local actions being undertaken that are sensitive to these contexts. At the same time, COVID-19 aptly reminds us of the global reach of gambling and the need to embed local responses within global strategies to reduce harm. That the World Health Organization (WHO) is starting to take interest in championing strategy in this area is positive. Yet, there is much still to do. Research and policy nearly always plays catch-up to industry developments. The industry, especially the online industry, is sophisticated in its use of data and technology to promote, protect and expand its services. Increasingly, researchers interested in gambling will have to also be interested in data science and technological infrastructure if they are to truly understand the nature of gambling products, the industries responsible for generating them and their impacts.
We agree a new framing is necessary: we need to acknowledge that the context of people\'s lives, broader social, economic and environmental circumstances and the actions of politicians, regulators and corporations, along with individual characteristics, can all influence behaviours. This is a public health framing, one where responsibility lies with multiple actors and agencies for preventing gambling harms. In Britain, as politicians begin the process of reviewing the 2005 Gambling Act and updating it for the digital world, there is the potential for such change to occur. But it requires bold thinking and -- crucially -- action to overcome challenges in how to deliver such a joined-up, global and systems-based approach to reducing gambling harms. The articles in this special issue offer hope that with sustained effort and action, this may be possible.
We all recognise that the world into which we will return will be very different, and within that new world, we will have an opportunity to do things differently. Once the immediate pandemic is past its peak and lockdown is slowly released, the public health community will refocus on what recovery will be needed and begin planning for the new normal. With this pandemic, we are already seeing questions being asked about how we can 'reset' rather than 'recover'.
Paine[@bib2] (1737--1809), the British-born American political theorist and revolutionary, wrote: 'We have it in our power to begin the world over again'. As we reset from the COVID-19 pandemic, should we not see this as a real opportunity to rebuild the world in a way that also addresses and reduces gambling harms? If we are to have the sort of mature discussion around building the public health response to gambling harms, this is an important time to start.
Author statements {#sec1}
=================
Competing interests {#sec1.1}
-------------------
P.M. reports serving as an advisor to the Scottish Chief Medical Officer and the Scottish Government on the public health response to gambling harms. P.M. reports being invited to speak at the launch of the Gambling Commission\'s National Strategy to Reduce Gambling Harms in Scotland. He reports not being remunerated for this. He reports receiving funding from the Gambling Commission to undertake a project developing a public health strategy to prevent gambling harms in Glasgow for his employing agency, Public Health Scotland. This funding was provided as part of a regulatory settlement to the Gambling Commission.
Between May 2015 and March 2020, H.W. reports serving as the Deputy Chair of the Advisory Board for Safer Gambling, an independent group that provides advice on gambling policy and research to the Gambling Commission and was remunerated by them. She reports working on one project funded by 10.13039/501100008625GambleAware on gambling and suicidality in the last three years. H.W. reports running a research consultancy, Heather Wardle Research Ltd. She reports not providing consultancy or any other services for the industry. She reports providing evidence at the House of Lords Select Committee enquiry into the social and economic impact of gambling as an unpaid expert witness in summer 2019. She reports serving as a member of WHO panel on gambling.
S.G. reports serving as an Emeritus Professor at the Chinese University of Hong Kong which has institutionally received charitable and research support from the Hong Kong Jockey Club. She reports being the Deputy Chair and Trustee of GambleAware, which is unremunerated, Chair of the Safer Gambling Board which is responsible for the BetRegret Campaign. In addition, S.G. reports being an Associate Non-Executive Board Member of the Public Health England Board Global Health Committee.
G.R. reports currently being a member of the World Health Organization\'s panel on gambling and the Howard League\'s Commission on Problem Gambling and Crime. Neither position is remunerated. She reports receiving research funds from the 10.13039/501100000272National Institute for Health Research, the 10.13039/501100000269Economic and Social Research Council, the 10.13039/501100000265Medical Research Council and the 10.13039/501100000286British Academy in the past three years. Between 2009 and 2014, she reports being a remunerated member of the Responsible Gambling Strategy Board (now the Advisory Board for Safer Gambling): an independent body that advised the Gambling Commission on research and strategy.
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Introduction {#Sec1}
============
Type II REases recognize defined DNA sequences and cleave within or in close, defined vicinity of this particular sequence. These enzymes, in the form of monomers, dimers or tetramers, typically act independently of the associated methyltransferase. They cut DNA generating either 'blunt' double-stranded DNA ends or cohesive ss and almost all of them require Mg^2+^ ions as cofactors (Loenen et al. [@CR3]). There are both enzymes which recognize symmetric and asymmetric sequences, whereas interrupted palindromes such as GC\|TNAGC recognized by EspI (Calléja et al. [@CR1]) are also considered symmetric.
Type II REases are ubiquitous tools in recombinant DNA technology; numerous applications of these enzymes are exemplified by: restriction analysis of DNA fragments and entire genomes, molecular cloning, 'Southern blot', constructing protein production systems, DNA amplification methods, studies on DNA-protein interactions, genetic disease analysis (RFLP and other methods), taxonomy, crime detection, molecular archaeology, creating transgenic plants and animals (Roberts [@CR4]). To meet the demands of biotechnology and expanding research techniques, new specificities are needed for scientific and commercial usage. As of 06/05/2015, only 363 different prototype specificities have been described biochemically or genetically (REBASE; Roberts [@CR5]), out of tens of thousands statistically possible combinations of 4--8 bp DNA sequences. Novel REases are being located by both sequencing of entire genomes followed by bioinformatics analyses and candidate coding genes' cloning, and a more traditional approach---biochemical screening of bacterial cell lysates.
Type II RM have also been used to identify pathogenic bacteria, such as *Helicobacter pylori*, since they are highly diversified between strains. It has been shown that a total of 22 RM systems with 18 specificities are found in six *Helicobacter pylori* strains and it has been concluded that RM-based methylation patterns of chromosomal DNA may serve as a new typing system to discriminate *Helicobacter pylori* isolates for clinical purposes (Xu et al. [@CR10]; Vale and Vitor [@CR9]). Thus, this paper has two aims: to serve as an aid in the identification of insect-infecting *M. morganii* and to extend the availability of REases for DNA manipulation purposes.
Results and discussion {#Sec2}
======================
Identification of the restriction endonuclease MmoSTI {#Sec3}
-----------------------------------------------------
A *M. morganii* strain has been isolated from a tropical *A. selene* moth's larva, exhibiting 100 % deadly infection symptoms. In a recent work we have shown that the disease is caused by a mixed baculovirus-*M. morganii* infection (Skowron et al. [@CR8]). As part of the bacterial strain's characterization, a lysate was prepared and tested for DNA restriction activity.
Crude lysate was subjected to phosphocellulose chromatography and the restriction activity of effluents was determined. The highest REase activity was observed in fractions with a NaCl concentration of 400--500 mM and just one chromatographic step was sufficient to obtain high quality DNA digests. Therefore, clear, relatively unobscured by non-specific nucleases restriction activity and accompanying REases displaying different specificities was detected in the crude *M. morganii* extract. The detected site-specific REase has been named MmoSTI, according to current nomenclature (Roberts et al. [@CR6]). The overall production of MmoSTI was estimated by serial dilutions as exceeding app. 2000 units/g cells (not shown). Prolonged incubations resulted in clear digestion patterns, indicating low non-specific nucleases content (not shown).
A series of DNA cleavage reactions by the MmoSTI revealed an optimum reaction temperature of 30 °C, activity within a wide range of salt concentration from low through medium to high buffer, with optimum at medium salt buffer (not shown). Determination of the recognition sequence and cleavage site was carried out by three different methods: (*i*) cleavage pattern analysis of short PCR substrates with a few or isolated putative site(s), (*ii*) run-off sequencing of digestion products and (*iii*) shotgun cloning. An additional, general methodological advantage of this combined approach is that it enables the determination of REases' recognition/cleavage sites even when analyzing partial digests. This is beneficial when analyzing low-concentration enzyme preparations, fast screening for REases or those REases which inherently yield partial digests, regardless of whether an excess of the enzyme is used, e.g., some sub-type IIG REases ( Zylicz-Stachula et al. [@CR11]).
Recognition sequence estimation by restriction pattern analysis {#Sec4}
---------------------------------------------------------------
Since MmoSTI is a frequent cutter, short PCR substrates were used for restriction pattern analysis instead of standard longer DNAs, such as plasmids or bacteriophage genomes. Digestion of a 390 bp PCR substrate ( Zylicz-Stachula et al. [@CR12]) by MmoSTI gave two distinct DNA bands in PAGE analysis (Fig. [1a](#Fig1){ref-type="fig"}). Basing on the length of the fragments (between 170 and 220 bp), over ten candidate palindromes and interrupted palindromes were located around MmoSTI cleavage points within the 390 bp sequence. Comparing the run-off sequencing results of pUC19 digestion products, the sites 5′-CCTGG-3′ and 5′-CCCGG-3′ were selected for further analysis. Cleavage of the 390 bp PCR substrate cleavage should generate three fragments: 42, 173, and 175 bp (Fig. [1b](#Fig1){ref-type="fig"}). Fragments 173 and 175 bp would be visible as one DNA band, as the length difference is below PAGE resolution. The presence of only two restriction fragments after PAGE was due to partial cleavage, as the enzyme obtained was not concentrated. In this case, 173 and 217 as well as 175 and 215 bp fragments were generated, with 173 and 175 bp fragments visible as one and 215 and 217 bp as another band, which collaborates with the pattern obtained (Fig. [1](#Fig1){ref-type="fig"}). To evaluate all potential variants of the MmoSTI cognate site with respect to the 3rd variable base, four 54 bp PCR substrates, differing only in a single bp within asymmetrically located 5′-CCNGG-3′ sequences, were generated. The digestion products would be 33 and 16 bp in each case, if all variants are cleaved. As shown in Fig. [2a, b](#Fig2){ref-type="fig"}, all 5′-CCNGG-3′ are cleaved both by MmoSTI and the prototype ScrFI. To finally validate this analysis, a comparative digestion of the pUC19 plasmid, as well as a long 1789 bp PCR substrate (Krefft et al. [@CR2]), with MmoSTI and ScrFI was conducted and resulted in the same digestion patterns. As both enzymes cleave DNA frequently, the obtained multiple bands are grouped within a small fragment size range (Fig. [2c](#Fig2){ref-type="fig"}).Fig. 1Digestion pattern of a 390 bp PCR substrate cleaved by MmoSTI. **a** 12 % polyacrylamide/TBE PAGE. Lane M, molecular weight marker---O'RangeRuler 20 bp DNA Ladder, Ready-to-Use, Thermo Scientific; lane 1, MmoSTI-digested 390 bp PCR substrate. *White arrows* indicate the generated DNA bands and their corresponding length in bp. **b** DNA sequence of the 390 bp PCR substrate. Three restriction fragments generated by MmoSTI are marked: *italics*---175 bp, *bold*---42 bp, and *regular*---173 bp. *Arrows* indicate cleavage sites of MmoSTIFig. 2Comparative digestions by MmoSTI and ScrFI. **a** 15 % polyacrylamide/TBE PAGE: Digestions of 54 bp PCR substrate variants containing the sites 5′-CCAGG-3′ or 5′-CCTGG-3′. Lane M, O'RangeRuler 20 bp DNA Ladder, Ready-to-Use, Thermo Scientific; lanes A, uncut 5′-CCAGG-3′ substrate, ScrFI cut 5′-CCAGG-3′ substrate, MmoSTI cut 5′-CCAGG-3′ substrate; lanes T, uncut 5′-CCTGG-3′ substrate, ScrFI cut 5′-CCTGG-3′ substrate, MmoSTI cut 5′-CCTGG-3′ substrate. **b** 15 % polyacrylamide/TBE PAGE: digestions of 54 bp PCR substrate variants containing the sites 5′-CCGGG-3′ or 5′-CCCGG-3′. Lane M, O'RangeRuler 20 bp DNA Ladder, Ready-to-Use, Thermo Scientific; lanes G, uncut 5′-CCGGG-3′ substrate, ScrFI cut 5′-CCGGG-3′ substrate, MmoSTI cut 5′-CCGGG-3′ substrate; lanes C, uncut 5′-CCCGG-3′ substrate, ScrFI cut 5′-CCCGG-3′ substrate, MmoSTI cut 5′-CCCGG-3′ substrate. **c** Agarose gel electrophoresis (1.5 %/TBE) of digested pUC19 and pACYC184-derived 1789 bp PCR substrate. Lane M, O'RangeRuler 20 bp DNA Ladder, Ready-to-Use, Thermo Scientific. Lanes 1, uncut pUC19 DNA, MmoSTI cut pUC19, ScrFI-cut pUC19; lanes 2, uncut 1789 bp PCR substrate, MmoSTI cut 1789 bp PCR, ScrFI-cut 1789 bp PCR
Determining the MmoSTI recognition sequence and cleavage site by run-off sequencing of pUC19/MmoSTI digestion products {#Sec5}
----------------------------------------------------------------------------------------------------------------------
In run-off sequencing, DNA polymerase extends DNA strands to the point where it falls off the end of the template. The sudden stop in sequencing peaks obtained from the forward primer shows the recognition site of MmoSTI: 5′-CCGGG-3′ and the cleavage point being before the first C (Fig. [3](#Fig3){ref-type="fig"}). In the case of the sequencing read generated by the reverse primer, the last peak representing G is smaller than previous peaks, but clearly stands out from the background. Such artefacts at the ends of DNA sequencing are frequently observed, as polymerases have difficulties in the last base incorporation efficiency, whereas capillary electrophoresis is also of lower resolution with a terminal stretch of identical bases. The sequencing results indicate that the last five nt of the cut fragment are 5′-CCGGG-3′ or 5′-CCCGG-3′, thus the MmoSTI recognition site is an interrupted palindrome, with a variable internal 3rd base.Fig. 3Run-off sequencing read showing MmoSTI cleavage site in pUC19. *Top*: forward primer, *bottom*: reverse primer; *box*: recognition site of MmoSTI; *arrows* indicate cleavage site
MmoSTI recognition and cleavage sequence determination by shotgun cloning and sequencing {#Sec6}
----------------------------------------------------------------------------------------
Fragments of λ and *E. coli* genomes digested with MmoSTI were blunted with T4 DNA polymerase/dNTPs, ligated into the SmaI site of pUC19 and insert junctions were sequenced. The clones' sequencing results provided independent confirmation of the MmoSTI putative recognition site. Further analysis considered both the potential MmoSTI recognition sites present in the inserts and DNA forming the clipped off flanking regions. Table [1](#Tab1){ref-type="table"} shows λ or *E. coli* genome fragments with MmoSTI recognition sequences marked.Table 1Determination of the MmoSTI recognition sequence and cleavage site by shotgun cloning and sequencing of MmoSTI restriction fragmentsBase numbering refers to the 5′ → 3′ orientation of λ or *E. coli* genome. Grey and emboldened, terminal part of restriction fragment generated by MmoSTI, blunted with T4 DNA polymerase/dNTPs and cloned into pUC19. Black, λ or *E. coli* genome sequence adjacent to the cloned restriction fragment. Grey box, MmoSTI recognition sequence
These results show that the recognition site of MmoSTI is 5′-CCNGG-3′. The fact that the entire recognition sequence is within the filled-in inserts indicates that MmoSTI cleaves DNA leaving 5-nt long, ss, 5′- cohesive ends. Thus, MmoSTI, recognizing an interrupted palindrome, is a subtype IIP REase (Loenen et al. [@CR3]) exhibiting high cleavage frequency.
Current REBASE data (04/06/2015) indicates that the ScrFI prototype enzyme from *Streptococcus cremoris* F recognizes a 5′-CC\|NGG-3′ sequence and cleaves after the second C of the recognition site, leaving 1-nt protruding 5′ ss DNA termini. The REase has 129 isoschizomers and neoschizomers, found either through biochemical analyses of bacterial cellular extracts or as putative genes, detected using genomic bioinformatics. Most originate from bacteria with a relatively narrow range of optimal temperature---mezophiles growing between 26 and 37 °C, however, there are some present in psychrophiles (0 °C) and thermophiles (55 °C). MmoSTI was isolated from *M. morganii* ST with an optimal growth temperature of 30--37 °C. Out of all known ScrFI isoschizomers, 97 comprise putative ORFs, predicted to code for REases, recognizing 5′-CCNGG-3′ sites, without experimental validations and with no estimates of cleavage positions. Four isoschizomers cleave recognition sites like the prototype, ScrFI, whereas 12 neoschizomers cleave before the first C, leaving long 5-nt protruding 5′ ss DNA termini and no isoschizomer cleaving before the second C was found (REBASE 04/06/2015). Thus far, no REases have been found and characterized biochemically in *M. morganii*. However, several putative MTase-coding genes, with predicted 5′-GATC-3′ and two with predicted 5′-CCNGG-3′ specificities of coded MTases, (M.MmoKTORF102P and M.MmoSC01ORF3004P, REBASE 04/06/2015) were found in genomes of *M. morganii*.
To conclude, the discovered REase can serve for two main purposes: *i)* aid in pathogenic bacterial strain identification and *ii)* provide a high frequency cutting REase for DNA manipulations, which can be extracted from an easily cultured bacterium, *M. morganii*, using a simple purification protocol.
Materials and methods {#Sec7}
=====================
Bacterial strains, DNAs, reagents {#Sec8}
---------------------------------
*Escherichia coli* (*E. coli*) DH11S \[*mcr*A ∆(*mrr*-hsd RMS-*mcr* BC) ∆(*lac*-*pro*AB) ∆(*rec*A 1398) *deo*R, *rps*L *srl-thi*, *sup*E/F', *pro*AB-*lac*I^q^ Z ∆M15\] was from Novagen / Merck KGaA (Darmstadt, Germany). *M. morganii* was isolated from *A. selene* larva cultivated in Stratford Butterfly Farm (Stratford-Upon-Avon, UK). Plasmid pUC19, *E. coli* DNA and λ cI857ts were isolated according to Sambrook et al. [@CR7]. Marathon Taq DNA Polymerase was from A&A Biotechnologies (Gdansk, Poland), Oligonucleotide synthesis was performed at Genomed (Warsaw, Poland). DNA, protein markers and the Miniprep DNA purification kit were from Thermo Scientific (Fermentas, Vilnus, Lithuania). All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).
Screening for DNA restriction activity {#Sec9}
--------------------------------------
Cuticle surfaces of infected *A. selene* larvae were imprinted and streaked on agar plates with LB medium (Sambrook et al. [@CR7]) and incubated for 48 h. The dominant strain, as a potential etiological agent, was further studied, which included colony morphology description, Gram staining, antibiotic resistance determination, growth temperature optimization and identification through mass spectrometry (Skowron et al. [@CR8]). Bacterial lysate was prepared using a single bacterial colony grown at 30 °C on LB plates, which was introduced into 20 ml of LB and incubated overnight at 30 °C in a shaker. Subsequently, 1000 ml of LB was inoculated, grown with vigorous aeration until the culture's entrance into the stationary phase, chilled down and centrifuged. The bacterial pellet was suspended in phosphocellulose chromatography buffer (50 mM K/PO4 pH 7.2, 0.5 mM EDTA, 50 mM NaCl, 0.05 % Triton X-100, 5 mM β-mercaptoethanol, 0.5 mM PMSF, 1/4 Sigma tablet protease inhibitors, containing AEBSF, aprotinin, bestatin, EDTA, E-64, leupeptin). Lysozyme was added to 0.5 mg/ml and the suspension was incubated in ice for 1 h, sonicated, and centrifuged. The obtained crude lysate was subjected to phosphocellulose chromatography. Crude lysate and chromatography fractions were assayed for restriction activity by incubation with 500 ng of substrate DNA from 1 h to overnight at 30 °C in 25 μl reactions in medium salt buffer (50 mM NaCl, pH 7.5, 10 mM MgCl~2~; crude lysate additionally supplemented with 50 μg/ml of RNase A). Reactions determining the recognition/cleavage specificity of MmoSTI were carried out in the same conditions. The non-specific nuclease content was estimated through long incubations (4 h to overnight) of serial dilutions of the purified enzyme with pUC19 DNA in medium salt buffer and assessment of the digestion patterns' clarity.
Determination of MmoSTI recognition and cleavage sites {#Sec10}
------------------------------------------------------
### Run-off sequencing of digestion products {#Sec11}
Plasmid pUC19 was digested overnight at 30 °C with MmoSTI. Proteins were removed by proteinase K digestion, phenol/chloroform extracted and ethanol precipitated. Purified cleaved products were subjected to Sanger automated sequencing at Genomed (Warsaw, Poland) using standard pUC19 primers.
### Cleavage pattern analysis of PCR substrates {#Sec12}
A set of custom PCR substrates was prepared (Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}). The 390 bp substrate was amplified with sequence-modifying primers from a pBR322 template ( Zylicz-Stachula et al. [@CR12]) and the 1789 bp substrate was amplified from the pACYC184 plasmid (Krefft et al. [@CR2]). Four variants of the 54 bp substrate, differing in a single bp within isolated 5′-CCNGG-3′ sites, were prepared as follows: four 54 nt ssDNAs were synthesized and used as a PCR template (MmoSTI putative recognition sites in bold, underlined): 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT**[CCAGG]{.ul}**GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT**[CCTGG]{.ul}**GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT**[CCCGG]{.ul}**GCCGCGCTGCGGACCC-3′, 5′-GGGCGCCATCTCGACCGAAGAGAGGCCAAGCAT**[CCGGG]{.ul}**GCCGCGCTGCGGACCC-3′. The amplification was conducted with primers: 5′-GGGCGCCATCTCGACC-3′ and 5′-GGGTCCGCAGCGCGGCC-3′ in 100 μl reactions, containing 1x Marathon PCR buffer, 0.4 mM of each dNTP, 0.5 μM of each primer, 100 ng template, and 1 unit of Marathon DNA polymerase. The hot-start PCR profile included 32 cycles of 95, 50, and 68 °C. Substrates were purified on 1.8 % agarose/TBE gels, electroeluted, phenol/chloroform extracted and ethanol precipitated. MmoSTI-digested PCR substrates were treated with proteinase K, phenol/chloroform extracted, ethanol precipitated and subjected to PAGE analysis.
### Shotgun cloning {#Sec13}
1.5 μg of λ and *E. coli* DNA were digested by MmoSTI at 30 °C for 3 h. Cohesive DNA ends were filled by T4 DNA polymerase/dNTPs. DNA was purified through phenol/chloroform extraction and ethanol precipitated. Resulting DNA fragments were cloned into the SmaI site of pUC19 vector and electrotransformed into *E. coli*. Bacteria were plated onto X-Gal/IPTG plates (α-complementation screening). Nineteen selected clones were used directly as a template in PCR with the standard primers: 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′ and 5′- AGCGGATAACAATTTCACACAGG-3′. Recombinant bacteria were cultured in LB and plasmids were isolated through alkaline lysis, phenol/chloroform extracted, ethanol precipitated, and subjected to Sanger sequencing.
This work was supported by University of Gdansk task funds no. 530-8640-D509-14 and 530-L140-D242-14.
[^1]: Communicated by: Agnieszka Szalewska-Palasz
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1. Introduction {#sec0001}
===============
1.1. PTSD heterogeneity {#sec0002}
-----------------------
Modern medicine has demonstrated a substantial interest in exploring heterogenous subtypes of psychiatric illness, corresponding to the characterization of unique psychopathology and associated aberrant neural circuitry ([@bib0138] [@bib0037]). Critically, a dissociative subtype of posttraumatic stress disorder (PTSD+DS) has been formalized recently, which corresponds to a specific group of individuals that exhibit additional symptoms of depersonalization/derealization and related emotion *over*modulation ([@bib0004]). In contrast to the typical presentation of PTSD, PTSD+DS is often associated with more severe PTSD symptoms, childhood trauma, increased comorbidity, and increased suicidality, pointing towards a patient group with heightened psychopathology and additional treatment needs ([@bib0073]; [@bib0128]; [@bib0084]). It has been shown repeatedly that posttraumatic stress disorder (PTSD) is associated with decreased regulation and inhibition from the medial prefrontal cortex (mPFC), where increased activation of the amygdala/limbic system and midbrain is thought to precipitate associated symptoms of hyperarousal, vivid re-experiencing, and emotion *under*modulation ([@bib0035]; [@bib0100]; [@bib0137]; [@bib0091]; [@bib0090]; [@bib0037]; [@bib0039]; [@bib0056]). By contrast, PTSD+DS is uniquely characterized by increased regulatory activation of the mPFC, resulting in hypoactivation of the amygdala/limbic system, with associated symptoms of depersonalization, derealization, and emotion *over*modulation ([@bib0059]; [@bib0074]; [@bib0085]; [@bib0091]; [@bib0093]; [@bib0037]; [@bib0081]). Indeed, PTSD and PTSD+DS show directed connectivity patterns that are consistent with their emotion under- and over-modulation symptom profiles, respectively. For example, [@bib0091] showed that PTSD patients demonstrate bottom-up directed connectivity from the amygdala and periaqueductal gray, brain regions involved in emotion generation and threat processing, to the vmPFC. By contrast, PTSD+DS patients displayed top-down directed connectivity from the vmPFC, an area involved in emotion regulation, to the amygdala and periaqueductal gray, which may facilitate emotion *over*modulation symptoms observed in these patients. Interestingly, unique neural correlates have also been documented within central hubs of intrinsic connectivity networks (ICNs) when comparing PTSD, PTSD+DS, and healthy individuals during both symptom provocation and the resting state ([@bib0060]; [@bib0089], [@bib0094]; [@bib0091]; [@bib0054]; [@bib0104], [@bib0105]; [@bib0129]).
1.2. Intrinsic connectivity networks {#sec0003}
------------------------------------
Neuroscientists have increasingly begun to emphasize the characterization of psychiatric disorders as conditions reflecting altered distributed neural networks ([@bib0061]; [@bib0077]). The default mode network (DMN), which consists of the posterior cingulate cortex (PCC), ventromedial prefrontal cortex (vmPFC), hippocampus, and cortical midline/parietal structures, is active predominantly at rest and is critical to autobiographical self-referential processing, future-oriented thinking, and continuous experience of the self across time and into the future ([@bib0048]; [@bib0019]; [@bib0125]; [@bib0103]; [@bib0042]). The central executive network (CEN) is a frontoparietal and cerebellar network centered around the dorsolateral prefrontal cortex (dlPFC) and is involved in higher order executive functioning, including the cognitive control of thought, emotion regulation, and working memory ([@bib0088]; [@bib0099]; [@bib0069]; [@bib0117]; [@bib0049]; [@bib0001]). The salience network (SN) encompasses the insula, the dorsal anterior cingulate cortex (ACC), and the amygdala, where this network is involved in interoceptive processing, environmental monitoring and the subsequent apprehension of personally salient stimuli ([@bib0032]; [@bib0117]; [@bib0126]). Indeed, central hubs within the DMN, CEN, and SN display aberrant activation and functional connectivity patterns in PTSD and its dissociative subtype during both symptom provocation and during the resting-state ([@bib0060]; [@bib0089], [@bib0094], [@bib0093], [@bib0051]; [@bib0104], [@bib0105]; [@bib0095]; [@bib0129]).
1.3. Intrinsic connectivity networks in PTSD {#sec0004}
--------------------------------------------
Neuropathological disruptions within ICNs have been shown to be related to specific symptoms of PTSD and PTSD+DS, which are closely tied to the unique function of each network ([@bib0077]; [@bib0037]). DMN functional disruptions in PTSD patients are hypothesized to be related to negative self-referential thoughts as well as alterations in social cognition and autobiographical memory, where PTSD+DS patients additionally experience alterations in somatic self-referential processing, including symptoms of depersonalization (i.e., out-of-body experiences) ([@bib0016]; [@bib0030]; [@bib0131]; [@bib0037]; [@bib0042]). CEN functional disruptions are associated with PTSD symptoms of decreased cognitive functioning across multiple domains, as well as emotion *under*modulation associated with impaired regulation of limbic structures ([@bib0006]; [@bib0101]; [@bib0063]; [@bib0027]; [@bib0077]; [@bib0015]). Interestingly, PTSD+DS is uniquely characterized by emotion *over*modulation from executive functioning areas, and dissociative symptoms are transdiagnostically related to exacerbated CEN impairments of attention, executive functioning, memory, and social cognition ([@bib0079]; [@bib0017]; [@bib0037]; [@bib0076]). Finally, alterations within the SN have been linked to PTSD symptoms of hyperarousal and bodily reactivity, as well as PTSD+DS symptoms of emotion *over*modulation associated with emotional detachment and lack of interoceptive awareness related to symptoms of depersonalization ([@bib0127]; [@bib0130]; [@bib0137]; [@bib0094]; [@bib0001]; [@bib0052]).
Neuroimaging studies examining functional connectivity within the DMN among subtype non-differentiated PTSD patients at rest reveal decreased coupling between the PCC, vmPFC and other DMN structures ([@bib0030]; [@bib0102]; [@bib0127]a; [@bib0026]; [@bib0120]; [@bib0066]; [@bib0068]; [@bib0087]). Here, recent studies have found decreased DMN functional connectivity to be associated with increased PTSD symptom severity, with both decreased functional integration (less efficiency of overall communication across the network) ([@bib0002]; [@bib0058]), and increased segregation (the capacity of specialized processing) in patients with PTSD ([@bib0002]). With regard to PTSD+DS, depersonalization/derealization symptoms have been associated with reduced connectivity of regions linked to self-referential processing within the DMN ([@bib0130]). Moreover, during tasks that require executive functioning, previous studies report decreased functional connectivity within the CEN in PTSD and increased connectivity within the DMN ([@bib0030]). This suggests aberrant ICN functioning in PTSD, as DMN recruitment would typically be decreased during tasks that require executive functioning ([@bib0030]). Indeed, this may underlie aforementioned disruptions in cognition functioning among PTSD patients ([@bib0079]). Additionally, during the resting-state, subtype non-differentiated PTSD patients demonstrate reduced functional integration of the CEN, which is associated with reduced orbitofrontal-amygdala connectivity, indicative of reduced regulation of the limbic system as compared to healthy controls ([@bib0139]). Supporting models of PTSD emotion *under*modulation, a recent study has also shown weaker connectivity within nodes of the CEN (right and left dlPFC) in patients with PTSD ([@bib0058]). By contrast, it is hypothesized that PTSD+DS patients exhibit over-recruitment of CEN areas relating to emotion *over*modulation ([@bib0137]; [@bib0037]). Finally, it is well documented that subtype non-differentiated PTSD patients often display elevated SN connectivity during the resting-state indicative of hyperarousal, hypervigilance and threat processing symptoms ([@bib0108]; [@bib0127]; [@bib0068]).
Although disruptions in these aforementioned neural networks have already been associated with subtype nonspecific PTSD populations ([@bib0001]; [@bib0070]; [@bib0077]; [@bib0082]; [@bib0106] [@bib0119]; [@bib0120]; [@bib0137]), functional connectivity of the DMN, CEN and SN has not been compared between PTSD, PTSD+DS and healthy controls, nor has activation within these networks been used to classify diagnoses via machine learning. Hence, it is unclear how alterations within the DMN, CEN and SN may contribute to heterogeneous symptom presentation in PTSD versus PTSD+DS patients, an effort which may guide/inform treatment interventions aimed at restoring these brain networks in trauma-related illness.
1.4. Multivariate pattern analysis: machine learning applications in neuroimaging {#sec0005}
---------------------------------------------------------------------------------
In contrast to univariate neuroimaging analyses, multivariate machine learning applications for fMRI constitute a more powerful method by which to evaluate subtle and spatially distributed signal patterns within the brain ([@bib0116]). Multivariate machine learning algorithms for neuroimaging data provide a means by which to classify patients, identify illness subtypes, and predict response to treatment based on highly complex sources of neural information, and due to their multivariate properties, these machine learning methods can achieve relatively greater sensitivity ([@bib0116]). Of particular relevance to psychiatry, these analyses are sensitive enough to facilitate inference/classification at the single-subject level ([@bib0097]; [@bib0044]; [@bib0136]), hence offering the capacity to predict individual diagnoses. Indeed, identifying objective neural network classifiers that can categorize PTSD heterogeneity may offer valuable clinical insight into guiding treatments for PTSD versus PTSD+DS by matching specific individuals to a personalized treatment. Recently, a growing number of studies have applied multivariate machine learning methods to neuroimaging datasets to predict and characterize psychiatric disease ([@bib0014]; [@bib0086]; [@bib0111]; [@bib0109]), including PTSD ([@bib0046]; [@bib0065]; [@bib0078]; [@bib0096]; [@bib0045]; [@bib0047]; [@bib0064]; [@bib0113]). Indeed, we have shown recently that machine learning algorithms were able to accurately classify PTSD, PTSD+DS, and healthy controls based on neural activation (91.63% accuracy), amygdala complex functional connectivity (85.00% accuracy) ([@bib0090]) and insula subregion functional connectivity (80.40% accuracy) ([@bib0052]). To date, however, no studies have utilized fMRI machine learning to classify PTSD and its dissociative subtype from healthy individuals based on ICN activation with the DMN, CEN, and SN.
1.5. Study objective {#sec0006}
--------------------
The purpose of the current study was to a) compare DMN, CEN, and SN functional connectivity between PTSD, PTSD+DS and healthy individuals; and b) to examine the predictive validity of machine learning algorithms in classifying PTSD, PTSD+DS, and healthy individuals based on DMN, CEN and SN activation. Given the distinct neurobiological underpinnings of PTSD and PTSD+DS with respect to both activation and functional connectivity within central hubs of ICNs, we hypothesized unique group differences between the two PTSD groups and healthy controls in terms of functional connectivity within the DMN (PCC and mPFC), CEN (dlPFC), and the SN (insula and amygdala). Specifically, with regard to the CEN and SN, we predicted increased integration of prefrontal cortex emotion regulation regions in PTSD+DS as compared to PTSD, indicative of emotion overmodulation. In addition, we predicted increased DMN connectivity in PTSD+DS as compared to PTSD and healthy controls, a pattern reflective of the altered self-referential processing and bodily self-consciousness associated with depersonalization (i.e., out-of-body experiences, emotional detachment). By contrast, in the PTSD group as compared to PTSD+DS and healthy controls, we predicted decreased CEN and SN connectivity to prefrontal cortex emotion regulation regions, consistent with emotion undermodulation. Moreover, in the PTSD group as compared to PTSD+DS and healthy controls, we predicted increased SN connectivity to the insula, consistent with hyper-monitoring of salient stimuli and hyperarousal at rest seen in this group. Finally, we hypothesized that these distinct ICN dynamics may contribute to high predictive accuracy when classifying individuals using machine learning computations based on whole-network activation.
2. Methods {#sec0007}
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2.1. Participants {#sec0008}
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Our sample consisted of 186 participants \[PTSD (*n* = 81); PTSD+DS (*n* = 49); healthy controls (*n* = 56); [Table 1](#tbl0001){ref-type="table"}\]. Although there were no statistically significant associations between group and biological sex, the majority of the sample consisted of female participants. There were also no statistically significant differences in terms of age between groups (see Supplemental Material for details). Machine learning analyses on a portion of this sample have been reported in previous manuscripts ([@bib0090]; [@bib0052]). Participants were recruited from 2009--2018 through referrals from family physicians, mental health professionals, psychology/psychiatric clinics, community programs for traumatic stress, and posters/advertisements within the London, Ontario community. The inclusion criteria for either PTSD group included a primary diagnosis of PTSD as determined using the Clinician-Administered PTSD Scale \[CAPS; versions IV (for 85 participants) and 5 (for 45 participants\] ([@bib0012]; [@bib0140]) and the DSM-IV Structured Clinical Interview (SCID) ([@bib0038]). Additionally, PTSD+DS patients were identified by scoring ≥ 2 for both frequency and intensity on either depersonalization or derealization CAPS-IV symptoms, or at least two in symptom severity on the CAPS-5 scale for depersonalization or derealization symptoms, as per standard methods ([@bib0051]; [@bib0090]). Exclusion criteria for patients included alcohol or substance abuse/dependence not in sustained full remission and diagnosis of bipolar disorder or schizophrenia. Exclusion criteria for the control group included lifetime Axis-I psychiatric disorders, evaluated using the SCID and CAPS. Ideally, all patients would be evaluated with the same version of the CAPS; however, this large dataset of almost 200 participants has been collected since 2010, and participants were evaluated with the best diagnostic criteria at the time. Exclusion criteria for all participants included non-compliance with 3 Tesla fMRI safety standards, significant untreated medical illness, pregnancy, a history of neurological or pervasive developmental disorders, and previous head injury with loss of consciousness. The study was approved by the Research Ethics Board at Western University, Canada, and written informed consent was obtained from all participants.Table 1Demographic and Clinical Information.Table 1PTSDPTSD+DSHealthy ControlsN**814956**Sex**46 female38 female36 female**MeasureValueSDValueSDValueSDAge3911.794013.523411.98CAPS-IV Total\*66.60 ^a^14.9181.60 ^a,b^12.890.602.59CAPS-5 Total36.589.2141.377.76n/an/aCTQ-Total\*56.06 ^a^23.0069.74 ^a,b^19.4132.108.80BDI\*23.21 ^a^8.3335.13 ^a,b^11.700.961.91MDI-Total\*53.64 ^a^14.8380.89 ^a,b^22.2033.963.82MDI-Dep/Dereal\*7.72 ^a^2.7312.97 ^a,b^4.595.200.51STAI5.6 ^a^2.16.2 ^a^2.53.30.6RSDI-Dissociation\*3.6 ^a^1.44.9 ^a,b^2.02.70.4RSDI-Reliving Experiences3.0 ^a^1.33.3 ^a^1.52.10.3nPastnPastnPastMDD\*12 ^a^2423 ^a,b^9----Panic Disorder/Agoraphobia10696----Social Phobia2260----OCD3202----GAD1000----Medication2919----[^1]
A battery of questionnaires was administered consisting of the Childhood Trauma Questionnaire (CTQ) ([@bib0010]), Beck\'s Depression Inventory (BDI) ([@bib0007]), and the Multiscale Dissociation Inventory (MDI) ([@bib0018]). Additionally, scores on the Responses to Script-Driven Imagery Scale (RSDI; [@bib0059]), and the State-Trait Anxiety Inventory (STAI; [@bib0124]) were collected during the fMRI scan to evaluate state clinical PTSD symptoms. Ninety percent of PTSD patients had early aversive experiences (confirmed if the patient scored above the 'none/minimal' threshold for any trauma category according to the CTQ scoring manual). Among PTSD participants, 48 (PTSD, *n* = 29; PTSD+DS, *n* = 19) were receiving psychotropic treatment at the time of the study. Medications included antidepressants, atypical antipsychotics, sedatives, and anticonvulsants (see Supplemental Material for details). Although the healthy control group was free of Axis-I disorders, a small portion of this group reported elevated levels on the CTQ. Critically, medication use was not found to significantly affect results when used as a covariate.
2.2. fMRI image acquisition, protocol and preprocessing {#sec0009}
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We acquired and preprocessed the resting-state neuroimaging data according to standard procedures in several of our manuscripts ([@bib0089], [@bib0091], [@bib0090]). We utilized a 3 Tesla MRI Scanner (Trio, Siemens Medical Solutions, Erlangen, Germany) with a 32-channel phased array head coil for brain imaging. During the resting-state scan, 120 volumes of whole brain BOLD (blood oxygen level dependent) images were acquired with the manufacturer\'s standard T2\* gradient-echo planar imaging (EPI) pulse sequence (single-shot, blipped-EPI, interleaved slice acquisition order and tridimensional prospective acquisition correction) with the following parameters: TR = 3000 ms, TE = 20 ms, isotropic resolution 2 mm, FOV = 192 × 192 × 128 mm^3^ (94 × 94 matrix, 64 slices), flip angle = 90° High-resolution T1-weighted anatomical images were acquired with a Magnetization-Prepared Rapid Acquisition Gradient Echo sequence (192 slices, 1 mm isotropic resolution). For the resting-state procedure, participants were instructed to close their eyes and let their minds wander while trying not to focus on anything in particular for 6 minutes ([@bib0016]; [@bib0040]; [@bib0054]), after which we assessed state-based clinical symptoms experienced during the scans (see below).
Preprocessing of the functional images was performed with SPM12 (Wellcome Department of Cognitive Neurology, London, UK) within Matlab 2017a. After discarding the 4 initial volumes, the standard preprocessing routine included spatial alignment to the mean image using a rigid body transformation, reslicing, and co-registration of the functional mean image to the anatomical image. The co-registered images were segmented using the "New Segment" method implemented in SPM12. The functional images were normalized to MNI space (Montréal Neurological Institute) and were subsequently smoothed with a FWHM (full-width at half-maximum) Gaussian kernel of 6 mm. Additional correction for motion was implemented using the ART software package (Gabrieli Lab, McGovern Institute for Brain Research, Cambridge, MA), which computes regressors that account for outlier volumes, in addition to the six movement regressors computed during standard realignment in general linear modeling. The smoothed functional images were subsequently bandpass filtered (high-pass 0.012 Hz, low-pass 0.1 Hz) (software by co-author Jean Théberge).
3. Data analyses {#sec0010}
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3.1. Part A) analysis of intrinsic connectivity networks {#sec0011}
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This section of the methods describes the analysis pertaining to objective one of the study, which was to compare DMN, CEN, and SN functional connectivity between PTSD, PTSD+DS and healthy individuals.
### 3.1.1. Independent component analysis {#sec0012}
Group spatial independent component analysis (ICA) was performed on the resting-state fMRI data with all subjects in order to identify spatially independent networks using the Group ICA of fMRI Toolbox (GIFT v4.0b) ([@bib0022], [@bib0023]; [@bib0003]). The Infomax algorithm was used to identify 20 independent components (ICs) within the data set, following minimum description length (MDL) criteria ([@bib0003]; [@bib0112]; [@bib0063]; [@bib0067]). In order to ensure reliability of the components, the ICA estimation was repeated 20 times through ICASSO ([@bib0057]). This procedure resulted in a set of group aggregate spatial maps (which included brain regions that represent a network/component) and corresponding time courses of the BOLD signal change across time. For each component, single-subject spatial maps and time courses were then back-reconstructed to individual subject space and converted to Z-scores, which denote the strength of each voxel\'s correlation (i.e., connectivity) with the aggregate component\'s time course. This data-driven approach was used to identify ICNs instead of masking the data with standard ICN templates, as previous studies suggest that ICNs within patients with PTSD networks may be aberrant ([@bib0030]; [@bib0120]; [@bib0107]; [@bib0130]; [@bib0093]). Of importance, ICA also identifies motion-related sources, along with vascular, ventricular, and susceptibility artifacts, which are incorporated into "noise" components.
### 3.1.2. Component identification: spatial sorting analysis {#sec0013}
We first visually inspected the obtained components for the presence of artifacts (ensuring peak activations in gray matter, low spatial overlap with known vascular, ventricular, motion, and susceptibility artifacts, and investigated signal time course frequency fluctuations) ([@bib0003]). Subsequently, the spatial sorting function within the GIFT toolbox was used to identify components that shared features with reference network templates in the literature. Here, we utilized reference ICN masks derived from the GIFT toolbox (GIFT v4.0b), and from <https://findlab.stanford.edu/functional_ROIs.html> ([@bib0121]; [@bib0106]; [@bib0093]). Rationale for examining the left and right CENs separately was derived in part from our recent ICA study, which suggests differential hemisphere recruitment within the CEN among PTSD patients during emotion regulation tasks ([@bib0093]). Furthermore, recent findings suggest greater involvement of the left CEN in explicit cognitive emotion regulation and language paradigms, while the right CEN is associated with implicit perceptual, somesthetic, and nociceptive processing ([@bib0123]; [@bib0072]; [@bib0055]). We also examined the anterior and posterior SN separately, as we have recently shown differential connectivity patterns of the anterior salience network (anterior insula) and posterior salience network (posterior insula) among PTSD, PTSD+DS, and healthy controls ([@bib0094]; [@bib0052]). Furthermore, it has been shown that the anterior insula is associated more with arousal/interoceptive awareness, cognitive emotional processing ([@bib0029]; [@bib0083]), heightened alertness, and autobiographical memory ([@bib0071]), representing a major hub within the anterior SN along with the dorsal ACC ([@bib0121]). By contrast, the posterior insula has been identified as a multimodal convergence zone for sensory information, including pain processing ([@bib0141]; [@bib0142]), and is the major hub of the posterior SN ([@bib0121]). Interestingly, the CEN and SN naturally separated into left/right and anterior/posterior networks, respectively, when conducting our ICA protocol. In summary, we carried forward 1 DMN component, 2 CEN components (left/right), and 2 SN components (anterior/posterior).
### 3.1.3. Group spatial comparisons for intrinsic networks {#sec0014}
The resulting component spatial maps of networks of interest were entered into second-level analyses within SPM12, with the central aim to examine network differences in the strength of regional functional connectivity between participant groups. For each network, a mask was created by entering the single-subject spatial maps into a voxel-wise one-sample *t*-test. This was thresholded at *q* \< 0.05 with false discovery rate (FDR) correction and saved as a mask for use in subsequent paired *t*-tests on the single-subject spatial maps. This ensured that all findings would be restricted to brain regions actually contributing to the respective component. We first conducted a 3 (group) × 5 (network) full-factorial ANOVA that principally focused on the interaction between participant group (PTSD, PTSD+DS, healthy controls) and network (DMN, left/right CEN, anterior/posterior SN). Next, we evaluated between group differences in functional connectivity for each network, with separate two-sample *t*-tests. All analyses were evaluated at the conservative threshold of *p-*FDR *\<* 0.05 *k* = 10 observed at the cluster-corrected level in order to control for multiple comparisons (see [@bib0033]) where we set the initial uncorrected cluster-forming threshold in SPM at *p* \< .001, *k* = 20.
3.2. Part B) multivariate machine learning classification analysis on network activation {#sec0015}
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This section of the methods describes the analysis pertaining to the second objective of the study, which was to observe the predictive validity of machine learning algorithms with regard to classifying PTSD, PTSD+DS, and healthy individuals based neural activation within ICNs.
3.2.1. Extraction of resting-state data for the classification machine learning analysis {#sec0016}
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We first extracted individual subject activation maps that would serve as inputs for the multivariate classification machine learning analyses after being masked with network maps. Specifically, we computed the mean amplitude of low-frequency fluctuations (mALFF) of the BOLD signal, denoting spontaneous resting-state brain activation across the whole-brain. The mALFF has been used previously as an input for machine learning analyses to predict accurately PTSD symptoms ([@bib0046]; [@bib0078]; [@bib0090]), which was found to be more accurate than functional connectivity features ([@bib0090]). Here, we used the REST toolbox (http:// [www.restfmri.net/forum](http://www.restfmri.net/forum){#interref0002}) within Matlab2012a and SPM8 in order to de-trend and extract individual ALFF maps from the preprocessed fMRI data for each participant across the frequency band 0.01 Hz to 0.08 Hz. Individual subject mALFF maps were then obtained by normalizing ALFF spatial maps (where each voxel was divided by the whole-brain ALFF mean).
3.2.2. Multivariate machine learning classification analysis {#sec0017}
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To assess the predictive value of neural activation within the DMN, CEN, and SN as a means to classify PTSD, PTSD+DS, and healthy controls, we implemented Multiclass Gaussian Process Classification algorithms within PRoNTo toolbox (http:// [www.mlnl.cs.ucl.ac.uk/pronto/](http://www.mlnl.cs.ucl.ac.uk/pronto/){#interref0003}) ([@bib0116]) running under Matlab2017a . Using standard template ICN masks from the GIFT toolbox (GIFT v4.0b), mALFF maps denoting activation within either the DMN, left CEN, right CEN, anterior SN or posterior SN were inputted into separate machine learning analyses. For each network, a resting-state design was modelled with no conditions. A feature set was prepared on mALFF data for voxels within each network of interest. Features were mean-centered, and a Multiclass Gaussian Process Classifier (MGPC) ([@bib0143]; [@bib0116]) was used to test if network activation could accurately predict the three groups (PTSD, PTSD+DS, and healthy controls). Critically, PRoNTo software implements kernel methods as a result of the high dimensionality of pattern vectors in neuroimaging data relative to the number of subjects (for more information see [@bib0116]). We implemented a supervised machine learning approach such that MGPC could inform multiclass membership with predictive probabilities (i.e., PTSD, PTSD+DS and healthy control group classification) ([@bib0143]; [@bib0116]; [@bib0134]). We then used a leave-one-subject-out (LOSO) cross-validation procedure to estimate the generalizability of our classifiers. Consistent with our previous machine learning publication, we also computed a leave-one-subject-out-per-group (LOSOPG) cross-validation procedure in order to provide an exhaustive approach to this analysis (see Supplemental Results) ([@bib0090]). The MGPC analysis provided probabilistic predictions for each diagnostic category in which balanced accuracy measures were computed to account for unequal group sizes. Statistical significance of these accuracy measures was determined by permutation testing (1000 permutations). For visualization purposes, anatomical atlas weights were computed to illustrate the regional pattern of weights used by the decision function of the machine to classify each group ([@bib0115]). Finally, we evaluated the predictive accuracy of activation features from all networks (DMN, left/right CEN and anterior/posterior SN) within the same MGPC analysis.
Of importance, utilizing reference template network masks of the DMN, CEN, and SN in Part B (as opposed to corresponding network components/masks identified in Part A) ensured that the features inputted into the classification machine learning analysis were not biased by the previous independent component analysis. In Part A, the independent component analysis has indeed \'learnt\' from the BOLD signal in all of the subjects, where the cross-validation procedure used in the machine learning analysis for Part B assumes that the hold-out set has not informed the decision function of the machine. As PRoNTo software does not allow for a feature extraction in which an ICA can be run on the inner cross validation loop of the MGPC (i.e., generating the ICA based on the training data by leaving-one-subject-out, then training the MGPC on these features, and finally, classifying the hold-out subject), we elected to utilize non-bias template ICN masks and not components identified in Part A.
3.3. Clinical data and motion statistical analyses {#sec0018}
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We computed Kruskal-Wallis H tests and Games-Howell post-hoc analyses in order to test for potential group differences with regard to the following clinical measures: CAPS-total, CTQ, BDI, MDI-total, and MDI depersonalization/derealization average scores. Additionally, scores on the RSDI scale (used to assess dissociation and reliving experiences subscales at the time of the scan) and the STAI (assessing state anxiety at the time of the scan) were compared among groups to assess for differences on state-based clinical symptoms. Finally, we computed separate chi-squared statistics on the number of motion outlier parameters generated by ART across each group, MDD diagnosis between patient groups, and frequency of medication use between patient groups.
4. Results {#sec0019}
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4.1. Part A) analysis of intrinsic connectivity networks {#sec0020}
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### 4.1.1. Spatial sorting analysis: component identification {#sec0021}
We identified five artifact-free components corresponding to the DMN, left CEN, right CEN, anterior SN, and posterior SN (see [Fig. 1](#fig0001){ref-type="fig"}). The DMN component consisted mainly of a large bilateral cluster in the posterior cingulate cortex (PCC), precuneus, and superior parietal lobe (SPL), as well bilateral clusters in medial and lateral PFC regions, the temporal pole and ACC. The left CEN component primarily covered a large prefrontal area in the left hemisphere, including the left dlPFC, the dmPFC, and the orbitofrontal cortex, as well as clusters in the left SPL. The right CEN component primarily covered a large prefrontal area in the right hemisphere, including the vmPFC, the dlPFC, the dmPFC, and the orbitofrontal cortex, as well as clusters in the right SPL. The anterior SN component comprised mainly a large bilateral anterior cluster spanning the dorsal ACC, as well as the bilateral anterior insula. The posterior SN mainly comprised clusters in the bilateral posterior insula, supramarginal gyrus, precuneus and cerebellum (lobules V, VI).Fig. 1Mean components generated by the group-level independent component analysis (ICA), pertaining to the default mode network, left and right central executive networks, and the anterior and posterior salience networks.Fig. 1:
### 4.1.2. Group spatial comparison for intrinsic networks: Summary {#sec0022}
In summary, when comparing functional connectivity within ICNs between PTSD, PTSD+DS and healthy controls, we found differential patterns of connectivity to brain regions involved in emotion regulation, in addition to limbic structures and areas involved in self-referential processing, interoception, consciousness, attention, orienting responses, and depersonalization/derealization. Specifically, the omnibus full-factorial ANOVA revealed a significant interaction effect between group and network involving the cuneus/precuneus and the middle dorsal PFC (see [Table 2](#tbl0002){ref-type="table"}). Below we present in detail follow-up comparison results for each ICN.Table 2Intrinsic Network Spatial Comparison: Full-Factorial ANOVA.Table 2Intrinsic NetworkBrain RegionHBACluster SizeMNI CoordinateF Stat.Z score*p FDR* ClusterxyzNetwork by Group InteractionCuneus/precuneus1794−2−92205.634.880.036Middle dorsal PFCR9932836404.414.010.037[^2]
### 4.1.3. Default mode network {#sec0023}
PTSD+DS patients showed more DMN functional connectivity to the left middle dorsal PFC/middle frontal gyrus as compared to PTSD patients (see [Table 3](#tbl0003){ref-type="table"} and [Fig. 2](#fig0002){ref-type="fig"}). Additionally, the healthy control group displayed increased DMN functional connectivity to the left precuneus/SPL, as compared to PTSD+DS patients.Table 3Follow-up Between Group Spatial Comparisons for Intrinsic Networks.Table 3Intrinsic NetworkGroup ComparisonBrain RegionHBACluster SizeMNI Coordinatet Stat.Z score*p FDR* ClusterxyzDMNPTSD+DS \> PTSDMiddle dorsal PFC/MFGL8/985−320644.774.740.016Healthy \> PTSD+DSPrecuneus/ superior parietal lobeL775−6−68544.714.680.032Left CENHealthy \> PTSDSuperior/middle temporal gyrusL21129−64−54104.334.310.008Right CENPTSD+DS \> PTSDLateral orbitofrontal cortexR11914830−103.923.900.012Anterior SNHealthy \> PTSD+DSPrimary visual cortexR171139−8415.085.040.005Posterior SNPTSD+DS \> PTSDMiddle dorsal PFCR8/92012830464.504.480.003PTSD \> PTSD+DSAnterior insulaL79−2818104.584.550.029PTSD+DS \> HealthyCuneus/precuneus17860−92204.954.920.020Precentral gyrusL455−3−22704.644.610.039R45442−8544.094.070.039PTSD \> HealthyPosterior insulaL88−36−20175.155.110.007Cuneus/precuneus17124−2−90204.924.890.002Healthy \> PTSDSupramarginal gyrusR4020564−46345.115.080.001dlPFCR9686014275.115.070.040[^3]Fig. 2Between group comparisons of default mode, central executive, and salience network connectivity (FDR-cluster level *p* \< .05, *k* = 10) comparing PTSD patients, PTSD+DS patients, and healthy controls, as indicated by contrast notation (\< or \>). Abbreviations: PTSD = Posttraumatic stress disorder group, PTSD+DS = Dissociative subtype posttraumatic stress disorder group, FDR = False discovery rate cluster corrected, dlPFC = Dorsolateral prefrontal cortex, SPL = Superior parietal lobe, MFG = Middle frontal gyrus, STG = Superior temporal gyrus, MTG = Middle temporal gyrus. Coordinates are given in MNI space and images were produced using MRIcron.Fig. 2:
### 4.1.4. Left central executive network {#sec0024}
As compared to patients with PTSD, the healthy control group showed increased left CEN functional connectivity to the left superior/middle temporal gyrus (see [Table 3](#tbl0003){ref-type="table"} and [Fig. 2](#fig0002){ref-type="fig"}).
### 4.1.5. Right central executive network {#sec0025}
PTSD+DS patients displayed increased right CEN functional connectivity to the right lateral orbitofrontal cortex, as compared to PTSD patients (see [Table 3](#tbl0003){ref-type="table"} and [Fig. 2](#fig0002){ref-type="fig"}).
### 4.1.6. Anterior salience network {#sec0026}
The healthy control group showed more anterior SN functional connectivity to the right primary visual cortex as compared to PTSD+DS patients (see [Table 3](#tbl0003){ref-type="table"} and [Fig. 2](#fig0002){ref-type="fig"}).
### 4.1.7. Posterior salience network {#sec0027}
PTSD+DS patients displayed more posterior SN functional connectivity to the right middle dorsal PFC as compared to PTSD patients (see [Table 3](#tbl0003){ref-type="table"} and [Fig. 2](#fig0002){ref-type="fig"}). By contrast, we found that the PTSD group displayed increased posterior SN connectivity to the left anterior insula, as compared to the PTSD+DS group. The PTSD+DS group also showed more posterior SN functional connectivity to the cuneus/precuneus, and the bilateral precentral gyrus, as compared to the healthy control group. Additionally, PTSD patients displayed increased posterior SN functional connectivity to the left posterior insula and the cuneus/precuneus, as compared to the healthy control group. Finally, healthy controls showed more posterior SN functional connectivity to the right dlPFC and the right supramarginal gyrus as compared to PTSD patients.
4.2. Part B) multivariate machine learning classification analysis on network activation {#sec0028}
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### 4.2.1. Default mode network {#sec0029}
Activation within the DMN resulted in the highest accuracy measures across all ICNs with regard to classifying PTSD diagnoses with machine learning computations. Indeed, we found that activation within the DMN was able to predict group classification of PTSD, PTSD+DS, and healthy individuals with 80% balanced accuracy (*p* \< .005 during permutation testing) ([Fig. 3](#fig0003){ref-type="fig"}). Here, the class accuracy was 86% for controls, 78% for PTSD, and for 74% for PTSD+DS. Moreover, the class predictive value was 79% for controls, 84% for PTSD, and 72% for PTSD+DS. Network regions with the highest decision function weights included multiple clusters in the mPFC, as well as midline parietal areas in the SPL and the PCC.Fig. 3Results for the Multiclass Gaussian Process Classification machine learning analysis, examining the predictive validity of activation within intrinsic connectivity networks: a) default mode network, b) left central executive network, c) right central executive network, d) anterior salience network, e) posterior salience network. On the left side of each section is the balanced accuracy of group classification for each network, with regional decision function weight vectors displayed below (visual representation of the weights that the machine learning algorithm uses to classify each group, based on features inputted from each network). Displayed in the middle are the individual class predictive values for each group. Finally, the graphs on the right side of each section displays the respective confusion matrix, where an ideal confusion matrix is diagonal and all predicted class labels correspond to the truth. Here, no classes are sacrificed in order to gain accuracy in other classes. For the confusion matrix, Group 1 = healthy control group, Group 2 = PTSD+DS patients, Group 3 = PTSD patients. Numbers on top of the bars in each graph correspond to the number of correctly classified individuals in each group. Abbreviations: PTSD = posttraumatic stress disorder group, PTSD+DS = dissociative subtype posttraumatic stress disorder group.Fig. 3:
### 4.2.2. Left central executive network {#sec0030}
Activation within the left CEN was able to predict group classification of PTSD, PTSD+DS, and healthy individuals with 75% balanced accuracy (*p* \< .005 during permutation testing) ([Fig. 3](#fig0003){ref-type="fig"}). The class accuracy was 84% for controls, 74% for PTSD, and 68% for PTSD+DS. Moreover, the class predictive value was 76% for controls, 78% for PTSD, and 70% for PTSD+DS. Network regions with the highest weights included areas in the left hemisphere pertaining to the dlPFC, the SPL, and the cerebellum crus 1.
### 4.2.3. Right central executive network {#sec0031}
We found that activation within the right CEN was able to predict group classification of PTSD, PTSD+DS, and healthy individuals with 76% balanced accuracy (*p* \< .005 during permutation testing) ([Fig. 3](#fig0003){ref-type="fig"}). The class accuracy was 81% for controls, 75% for PTSD, and 72% for PTSD+DS. Moreover, the class predictive value was 72% for controls, 80% for PTSD, and 76% for PTSD+DS. Network regions with the highest weights included areas in the right hemisphere pertaining to the inferior orbitofrontal cortex, the dlPFC, the SPL, the supramarginal gyrus, and the cerebellum crus 1.
### 4.2.4. Anterior salience network {#sec0032}
Activation within the anterior SN was able to predict group classification of PTSD, PTSD+DS, and healthy individuals with 74% balanced accuracy (*p* \< .005 during permutation testing) ([Fig. 3](#fig0003){ref-type="fig"}). The class accuracy was 79% for controls, 68% for PTSD, and 74% for PTSD+DS. Moreover, the class predictive value was 70% for controls, 74% for PTSD, and 72% for PTSD+DS. Network regions with the highest decision function weights included the anterior dorsal ACC, the bilateral middle dorsal PFC, and the right anterior insula.
### 4.2.5. Posterior salience network {#sec0033}
Activation within the posterior SN was able to predict group classification of PTSD, PTSD+DS, and healthy individuals with 70% balanced accuracy (*p* \< .005 during permutation testing) ([Fig. 3](#fig0003){ref-type="fig"}). The class accuracy was 82% for controls, 72% for PTSD, and 56% for PTSD+DS. Moreover, the class predictive value was 71% for healthy individuals, 75% for PTSD, and 63% for PTSD+DS. Network regions with the highest weights included the right posterior insula, the left superior temporal gyrus, the right postcentral gyrus, and the bilateral supramarginal gyrus.
A noteworthy concept is that all voxels within each of the specified ICN activation maps will contribute to the decision function of the machine. Indeed, the weight maps produced by the machine learning analysis are a spatial representation of the predictive function and show the relative contribution of all voxels for the model. Hence, we present regional contributions above and in [Fig. 3](#fig0003){ref-type="fig"} for illustrative purposes only. This is in contrast to many common neuroimaging analyses which utilize a univariate approach. In this multivariate analysis, as all voxels inputted into the algorithm will contribute to the machine\'s prediction, it is not possible to arbitrarily threshold or to single out whether any one region is predictive in isolation. Finally, similar results were obtained for all machine learning analyses when using a LOSOPG cross-validation procedure. Here, accuracy measures were within +/- 1% of original balanced accuracy percent values reported via the LOSO cross-validation (see Supplemental Results). Moreover, all anatomical weights retained expected ranking positions, indicating stable ranking of the regions across folds. Of importance, confusion matrices in [Fig. 3](#fig0003){ref-type="fig"} show an optimal and diagonal pattern of classification, where no classes are sacrificed in order to gain accuracy in other classes. Finally, including activation features from all networks in the same MGPC analysis yielded a balanced accuracy measure of 88.99% (*p* \< .005 during permutation testing) (class accuracy 98.21% for healthy individuals, 81.63% for PTSD+DS, and 90.12% for PTSD; class predictive value 88.71% for healthy individuals, 90.91% for PTSD+DS, and 91.25% for PTSD). Collectively, these results highlight the importance of examining ICNs in PTSD; below we discuss each network in turn, an approach consistent with these findings demonstrating the unique contributions of each network to the accuracy of classification.
4.3. Clinical data and motion artefacts {#sec0034}
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We found that all three groups (PTSD, PTSD+DS and controls) differed significantly in terms of CAPS-IV, CTQ, BDI, MDI-total, and MDI depersonalization/derealization average scores. The PTSD+DS group exhibited the highest scores among these clinical variables as compared to the PTSD and control groups (see Supplemental Material Table s1 and [Table 1](#tbl0001){ref-type="table"}), where the PTSD group had higher scores on these clinical variables as compared to the control group only.
With regard to state-based clinical measures assessed at the time of the fMRI scan, the PTSD+DS group scored significantly higher on state dissociation scores as measured by the RSDI-dissociation subscale as compared to the PTSD group (see Supplemental Material Table s1 and [Table 1](#tbl0001){ref-type="table"}). By contrast, scores for state anxiety (STAI) and reliving symptoms (RSDI scale) did not differ significantly between the PTSD and PTSD+DS groups. However, all state-based clinical measures were significantly higher in the PTSD and PTSD+DS groups as compared to the healthy control group, which is a common finding in the PTSD literature and suggests active psychopathology even at rest among PTSD patients (also see [@bib0054]; [@bib0091]; [@bib0076]). Dissociative symptoms collected prior to the scan (MDI depersonalization/derealization averages) and during the scan (RSDI depersonalization/derealization averages) were highly correlated (*r* = 0.70, *p* \<0.001).
The relation between medication use frequency and group was non-significant. MDD diagnosis was significantly more frequent in the PTSD+DS group as compared to the PTSD group (*p* \< .001), where it has been suggested that higher scores of depression and PTSD symptom severity are associated with the dissociative subtype of PTSD ([@bib0128]; [@bib0050]). Finally, when MDD diagnosis was used as a covariate in Part A, regional clusters did not change, albeit the magnitude of statistical significance decreased marginally. Similarly, medication use was not found to significantly affect results when used as a covariate. Our analysis regarding motion outliers yielded non-significant results when comparing observed outlier frequencies across groups X^2^ (2, *N* = 186) = 0.90, *ns*.
5. Discussion {#sec0035}
=============
In comparison to patients with PTSD, PTSD+DS is characterized by additional psychopathology, more severe PTSD symptoms, increased comorbidity, and suicidality, which together translates into unique treatment needs ([@bib0073]; [@bib0128]; [@bib0084]). Critically, the functional connectivity of intrinsic networks has not been compared among heterogeneous PTSD subtypes (PTSD versus PTSD+DS) and healthy controls, nor has activation within these networks been used to predict PTSD subtype diagnosis using machine learning. Indeed, it was previously unclear how alterations within the DMN, CEN and SN may contribute to heterogeneous symptom presentation in PTSD versus PTSD+DS patients, which may inform treatment interventions aimed at restoring large scale brain networks. Given the distinct neurobiological correlates of PTSD and PTSD+DS at the activation and functional connectivity level within central hubs of ICNs, we hypothesized unique group differences in terms of functional connectivity within the DMN (PCC and mPFC), CEN (dlPFC), and the SN (insula and amygdala). In Part A, we observed differential group connectivity of the DMN, CEN, and SN among PTSD, PTSD+DS and healthy individuals to brain regions involved in emotion regulation, in addition to limbic structures and areas involved in self-referential processing, interoception, bodily self-consciousness, attention, orienting responses, and depersonalization/derealization. In support of our hypotheses, our results generally support *increased* connectivity with PFC emotion regulation areas among all intrinsic networks in patients with PTSD+DS as compared to *decreased* PFC emotion regulation connectivity among patients with PTSD. Importantly, unique ICN functional connectivity within each PTSD group may underlie differential symptom presentations among PTSD subtypes. In Part B, using machine learning classification algorithms, we were also able to show that activation within each of the aforementioned networks accurately predicts the classification of PTSD, PTSD+DS, and healthy individuals. Here, the DMN was the most accurate in classifying PTSD patient diagnoses --- an expected finding given that feature selection was performed on resting-state data and that the DMN has been shown to be highly implicated in PTSD psychopathology ([@bib0077]; [@bib0137]; [@bib0001]). The second-best predictor of PTSD patient diagnoses using machine learning classification algorithms was activation within the CEN (the ICN most implicated in emotion regulation and executive functioning), which is a finding consistent with emotion-modulation models of PTSD (see below; [@bib0074]; [@bib0037]).
5.1. Default mode network {#sec0036}
-------------------------
The DMN is thought to mediate altered self-referential processing, autobiographical memory, and social cognition ([@bib0077]), where trauma has been shown to have lasting effects on the sense of self manifested at both the cognitive and somatic level ([@bib0042]). When comparing PTSD patient groups, we hypothesized unique connectivity differences as PTSD+DS patients experience additional alterations in self-referential processing and bodily self-consciousness associated with depersonalization (i.e., out-of-body experiences, emotional numbing) ([@bib0076]; [@bib0042]). Studies investigating DMN intrinsic functional connectivity among subtype non-differentiated PTSD at **rest** generally report decreased coupling between the PCC, vmPFC, and other DMN structures where aberrant DMN functioning has been associated with PTSD symptoms ([@bib0063]; [@bib0077]; [@bib0137]; [@bib0001]; [@bib0037]). Importantly, during tasks that require executive functioning, previous studies report increased connectivity within the DMN and decreased connectivity within the CEN among patients with PTSD as compared to healthy individuals ([@bib0030]).
While previous studies typically examine undifferentiated PTSD patient samples as compared to healthy controls, results from the current study demonstrate unique DMN connectivity between patients with PTSD and its dissociative subtype. Here, increased DMN connectivity to the middle dorsal PFC in the PTSD+DS as compared to the PTSD group likely represents emotion *over*modulation (over regulation of the hyperactive limbic system in PTSD) related to increased symptoms of depersonalization/derealization in this group ([@bib0074], [@bib0073]; [@bib0106]; [@bib0091]; [@bib0090]; [@bib0037]). Interestingly, and in direct support of our results, the frequency of dissociative experiences has been positively correlated to DMN connectivity with the dorsal PFC, a region involved in the CEN and emotion *over*modulation ([@bib0016]). It is noteworthy that dissociative responses have also been shown to involve key areas of the DMN, including the medial PFC, medial parietal lobe, and the temporoparietal junction ([@bib0144]), further suggesting that alterations within the DMN may be a potential mechanism underlying depersonalization-related disturbances in self-referential processing ([@bib0106]; [@bib0042]). Critically, hyperconnectivity of the DMN with prefrontal CEN areas in PTSD+DS may also reduce the availability of the CEN for use in other cognitively demanding tasks, hence underlying symptoms of poor cognitive performance in this group ([@bib0079]). Moreover, PTSD+DS patients may be uniquely characterized by alterations in connectivity between the DMN and CEN regions, which may also relate to aforementioned difficulties in switching between DMN and CEN modalities ([@bib0077]; [@bib0076]; [@bib0079]). Mirroring our results and further supporting models of emotion *under*modulation in PTSD patients without the dissociative subtype, a recent study found that PFC dysconnectivity in the DMN was linked to PTSD symptom severity ([@bib0002]).
The precuneus is an additional node within the DMN and is critical for relating the self to socially relevant emotional stimuli, bodily self-consciousness, first-person perspective taking, visuo-spatial imagery and episodic memory retrieval ([@bib0048]; [@bib0025]; [@bib0021]). Here, decreased precuneus connectivity in PTSD+DS as compared to healthy controls may point to alterations in these functions related to depersonalization (i.e., out-of-body experiences) and dissociation driven changes in social-cognition (disruption in the ability to use, encode, and store information about others that we gain from social interactions) ([@bib0079]). The PTSD+DS group also displayed decreased connectivity to the SPL involved in attention ([@bib0008]; [@bib0133]), as compared to healthy controls. This may be a mechanism underlying dissociation driven changes in attention and cognitive dysfunction that occur in PTSD patients and in other psychiatric disorders transdiagnostically ([@bib0009]; [@bib0079]; [@bib0076]).
Additionally, results from the multivariate machine learning analysis in Part B suggest that aberrant DMN activation may be a useful predictor of PTSD group classification, where the DMN yielded the most accurate classification results when predicting individual diagnoses among PTSD, PTSD+DS and healthy controls. Taken together, this provides further evidence that the DMN may support unique clinical presentations of self-referential processing, including depersonalization and related identity disturbance.
5.2. Central executive network {#sec0037}
------------------------------
Alterations within the CEN are thought to underlie both emotion regulatory and cognitive dysfunctions observed in PTSD ([@bib0030]; [@bib0043]; [@bib0077]; [@bib0079]; [@bib0001]; [@bib0119]). A recent study found that during the **resting-state**, subtype non-differentiated PTSD patients demonstrated reduced CEN convergence which was associated with decreased orbitofrontal-amygdala connectivity in PTSD, indicative of reduced prefrontal regulation on the resting limbic system ([@bib0139]). This is consistent with previous literature that suggests decreased recruitment and functional connectivity within the CEN among subtype non-differentiated PTSD patients ([@bib0028]; [@bib0063]; [@bib0077]; [@bib0058]). Critically, these studies suggest that altered CEN functional connectivity may have cascading negative effects on emotion regulation, and thus may be critical to the neural underpinnings of emotion *under*modulation symptoms observed in PTSD. By contrast, PTSD+DS patients are known to exhibit emotion *over*modulation patterns of neural connectivity, paralleling extreme top-down regulation from CEN areas, including the prefrontal cortex, which may promote emotional detachment symptoms including depersonalization and derealization ([@bib0074]; [@bib0091]; [@bib0037]). Furthermore, PTSD is associated with disrupted cognitive functioning across multiple domains (declarative memory, short-term memory, attention, and executive functioning) ([@bib0006]; [@bib0101]; [@bib0079]).
In support of our hypotheses, we found increased right CEN connectivity with the right lateral orbitofrontal cortex in PTSD+DS as compared to PTSD. Increased CEN connectivity with the orbitofrontal cortex may contribute to the neurobiological basis of emotion *over*modulation among PTSD+DS patients. Indeed, exacerbated recruitment of emotion regulation areas, including the orbitofrontal cortex among PTSD+DS patients has repeatedly been shown to be related to over-regulation of the limbic system with associated depersonalization, derealization and emotional detachment ([@bib0060]; [@bib0036]; [@bib0074]; [@bib0135]; [@bib0091]; [@bib0090]; [@bib0037]; [@bib0081]; [@bib0105]). Furthermore, dissociative symptoms have been shown to impair CEN functions related to attention, executive functioning, memory, and social cognition ([@bib0006]; [@bib0101]; [@bib0028]; [@bib0063]; [@bib0079]; [@bib0015]).
Additionally, the PTSD group displayed decreased left CEN connectivity to the left superior/middle temporal gyri as compared to the healthy control group. Interestingly, alterations in superior/middle temporal gyri processing have been associated with trauma-related symptoms, such as reexperiencing and avoidance, where decreased CEN connectivity to this area my correspond to decreased control over these symptoms ([@bib0075]; [@bib0060]; [@bib0122]; [@bib0114]). Interestingly, the temporoparietal junction, which includes the posterior superior temporal gyrus and the supramarginal gyrus, is critical for multisensory integration, bodily self-consciousness and embodiment (which is the sense of being localized within one\'s physical body and therefore constitutes a fundamental aspect of the self) ([@bib0005]; [@bib0013]; [@bib0062]). Furthermore, the left superior/middle temporal gyrus is also part of the dorsal attention network critical for multisensory integration and executive functioning ([@bib0031]). Critically, these functions related to bodily self-consciousness, multisensory integration, embodiment, and executive functioning are hypothesised to be disrupted in patients with PTSD ([@bib0077]; [@bib0051]).
Finally, results from the multivariate machine learning analysis in Part B suggest that activation within the left and right CEN may be useful biomarkers for predicting PTSD subtype classification. Indeed, our CEN results from Part A & B strongly support the hypothesis that unique symptom presentation among PTSD subtypes may emanate from emotion *under*- and *over*- modulation within the CEN in PTSD and PTSD+DS patients, respectively.
5.3. Salience network {#sec0038}
---------------------
Alterations in SN functioning in patients with PTSD may contribute to the hypervigilance and hyperarousal symptoms commonly observed in patients with PTSD ([@bib0077]; [@bib0137]; [@bib0037]), where subtype non-differentiated PTSD patients have been shown to display elevated SN connectivity during the resting state ([@bib0108]; [@bib0127]; [@bib0068]). Additionally, the SN and anterior insula are thought to mediate "dynamic switching" between DMN and the CEN, in order to bring online higher-order cognitive processing ([@bib0117]; [@bib0126]; [@bib0083]).
The anterior insula is associated with arousal/interoceptive awareness, cognitive emotional processing ([@bib0029]; [@bib0083]), heightened alertness, and auto- biographical memory ([@bib0071]) and represents a major hub within the **anterior SN** along with the dorsal ACC ([@bib0121]). In the current study, our results showed decreased anterior SN connectivity to the primary visual cortex in PTSD+DS as compared to healthy controls. Critically, the anterior SN facilitates orienting responses to salient stimuli via connections with dorsal visual stream regions, which includes the primary visual cortex ([@bib0083]; [@bib0132]). Here, we hypothesize that PTSD+DS patients may exhibit disrupted SN processing with regard to orienting to salient visual stimuli in the environment, which may be mediated by depersonalization and derealization symptoms ([@bib0077]; [@bib0052]).
By contrast, the posterior insula region has been identified as a multimodal convergence zone for sensory information, including pain and body condition ([@bib0141]; [@bib0142]) and is the major hub of the **posterior SN** ([@bib0121]). In the current study, PTSD patients displayed increased posterior SN connectivity to the anterior insula as compared to PTSD+DS, and additionally displayed increased posterior SN connectivity with the posterior insula as compared to healthy controls. In the PTSD group, increased posterior SN connectivity with the anterior insula may represent exacerbated sensory processing related to arousal/interoceptive awareness, emotional state processing and heightened alertness ([@bib0029]; [@bib0083]; [@bib0077]; [@bib0094]), as compared to PTSD+DS. Indeed, PTSD+DS and accompanying emotional detachment, depersonalization and derealization, has been associated with anterior insula under-engagement ([@bib0060]; [@bib0041]; [@bib0074]; [@bib0037]). These findings support exacerbated interoceptive/arousal processing in the anterior insula among PTSD patients as compared to PTSD+DS ([@bib0092]), which may precipitate hypervigilance and avoidance symptoms in PTSD patients ([@bib0098]; [@bib0137]). Notably, hyperactivation in the anterior insula has been correlated *positively* with state re-experiencing scores and *negatively* with state dissociation scores ([@bib0060]). By contrast, increased posterior SN connectivity to the posterior insula in PTSD as compared to healthy controls may reflect exacerbated multisensory processing related to salient threat detection, pain and body condition, hypervigilance and hyperarousal PTSD symptoms ([@bib0060]; [@bib0074]; [@bib0100]; [@bib0135]; [@bib0080]; [@bib0037]).
Finally, both the PTSD and PTSD+DS groups showed increased posterior SN connectivity to areas within attention/memory networks and areas related to episodic memory processing (precuneus, cuneus, precentral gyrus), as well as areas within the posterior insula network related to consciousness (precuneus) and motor processing (precentral gyrus) ([@bib0025]; [@bib0024]; [@bib0020]) as compared to healthy controls. Additionally, when compared to healthy individuals, the PTSD group evidenced decreased posterior SN connectivity with the supramarginal gyrus, a temporoparietal junction area heavily implicated in self-referential processing, bodily self-consciousness and embodiment, where these results collectively support disruptions among these neural processes within patients with PTSD ([@bib0013]; [@bib0118]; [@bib0062]; [@bib0051]; [@bib0129]).
The emotion modulation model also appears to be a relevant framework for characterizing PTSD subtypes with regard to SN connectivity. In support of our hypotheses, we found increased posterior SN connectivity among PTSD+DS patients to the middle dorsal PFC as compared to PTSD patients. This finding suggests that the middle dorsal PFC may be attenuating/ overmodulating salient threat processing and multisensory integration of the posterior SN within PTSD+DS patients ([@bib0034]; [@bib0091]). Similarly, PTSD patients also displayed decreased posterior SN connectivity to the dlPFC as compared to healthy controls. This further supports models of emotion *under*modulation in PTSD patients, consistent with attenuated connectivity between emotion regulation areas and limbic structures ([@bib0074]; [@bib0137]; [@bib0091]; [@bib0037]). Finally, we were able to predict the diagnosis PTSD, PTSD+DS and healthy controls based on SN activation using machine learning computations. The anterior salience network had greater classification accuracy as compared to the posterior SN during our resting-state scans. Notably, SN functioning related to threat detection may predict classification with higher accuracy during symptom provocation/emotion induction as opposed to resting-state paradigms. Indeed, functional connectivity patterns of ICNs may further polarize during emotion induction paradigms when comparing PTSD versus PTSD+DS patients, where these patient groups are known to exhibit unique symptoms in response to such paradigms ([@bib0060]; [@bib0074]).
5.4. Future directions and limitations {#sec0039}
--------------------------------------
It will be critical for future studies to investigate additional subtypes of PTSD illness, as well as investigate accuracy of prediction during emotion induction paradigms. Future studies may benefit from a longitudinal experimental design that would allow for prospective examination of the effects of treatment response and medication type/dosage on neural network integrity in PTSD. Additional studies are also required to elucidate further the effects of different classes of medication on patterns of neural response, and to determine how specific biomarkers of psychiatric illness may predict response to specific treatments. Future studies should also examine the predictive accuracy of data driven networks from PTSD populations and explore influences of sex on network classification in PTSD. It will also be important to examine changes in network connectivity in relation to patterns of patient comorbidity. Additionally, PTSD and PTSD+DS differed significantly on CAPS-IV total severity scores; no such finding emerged for the CAPS-5. The absence of an effect in participants assessed by the CAPS-5 may stem, in part, from the smaller sample size of these participants, thus limiting power to detect a difference. Future studies that include a larger sample size will be needed to confirm this hypothesis and to additionally compare the sensitivity of such diagnostic measures. Finally, we implemented a very conservative data driven approach, where additional group differences were found at a more liberal threshold for the DMN, SN, and CEN; hence, replication and increased power are critical to fully elucidating these dynamics.
6. Conclusion {#sec0040}
=============
Alterations within intrinsic connectivity networks have long been shown to be associated with psychiatric illnesses, including PTSD. Of importance, we present the first study to compare directly intrinsic connectivity network (DMN, CEN, and SN) functional connectivity among PTSD, dissociative subtype PTSD and healthy individuals. Specifically, we show unique group differences in terms of ICN functional connectivity with emotion regulation areas, limbic structures, somatosensory and interoception brain areas, as well as regions involved in self-referential processing, consciousness, and depersonalization/derealization. Using multivariate machine learning classification algorithms, we were also able to show that activation within these networks predicts accurately the diagnosis of PTSD, its dissociative subtype, and healthy individuals with high accuracy. Critically, identifying objective neural network classifiers that can categorize PTSD heterogeneity may prove valuable by pointing towards targeted treatments for PTSD and its dissociative subtype that address specific alterations in the neural networks associated with each and thus match specific individuals to personalized treatment approaches. The present results also suggest strongly that the specific patterns of alterations within intrinsic connectivity networks associated uniquely with PTSD and with PTSD+DS may contribute, in part, to the contrasting clinical presentation observed across these subtypes.
CRediT authorship contribution statement {#sec0040a}
========================================
**Andrew A. Nicholson:** Conceptualization, Data curation, Funding acquisition, Formal analysis, Writing - original draft. **Sherain Harricharan:** Conceptualization, Data curation, Writing - review & editing, Visualization. **Maria Densmore:** Formal analysis, Methodology. **Richard W.J. Neufeld:** Conceptualization, Writing - review & editing. **Tomas Ros:** Conceptualization, Writing - review & editing. **Margaret C. McKinnon:** Conceptualization, Writing - review & editing. **Paul A. Frewen:** Conceptualization, Writing - review & editing. **Jean Théberge:** Conceptualization, Writing - review & editing. **Rakesh Jetly:** Conceptualization, Writing - review & editing. **David Pedlar:** Conceptualization, Writing - review & editing. **Ruth A. Lanius:** Conceptualization, Supervision, Data curation, Funding acquisition, Writing - original draft.
Declaration of Competing Interest
=================================
All authors declare no financial interests or potential conflicts of interest with regard to the current study.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Appendix. Supplementary materials {#sec0043}
=================================
Image, application 1
Dr. Andrew A. Nicholson has received funding support from the European Union\'s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Individual Fellowship (grant agreement No. 897709). Our research group would also like to acknowledge the following funding sources in which we are extremely grateful for their support: the Canadian Institutes of Health Research (CIHR), General Dynamics Land Systems, Mitacs, the Canadian Institute for Veteran Health Research (CIMVHR), IBM and the Homewood Research Institute. We also thank Suzy Southwell and Stephanie Nevill for their contributions with data collection, and Dr. David Steyrl for providing a high level of machine learning expertise. Open access funding provided by the University of Vienna.
Supplementary material associated with this article can be found, in the online version, at [doi:10.1016/j.nicl.2020.102262](https://doi.org/10.1016/j.nicl.2020.102262){#interref0004}.
[^1]: Abbreviations: PTSD= Posttraumatic Stress Disorder, PTSD+DS= Dissociative Subtype Posttraumatic Stress Disorder Patients, CAPS = Clinician-Administered PTSD Scale, CTQ = Childhood Trauma Questionnaire (*none or minimal childhood trauma = 25--36, moderate = 56--68, extreme trauma \> 72*), BDI = Beck\'s Depression Inventory, MDI = Multiscale Dissociation Inventory, Dep/Dereal = Depersonalization and Derealization Average, MDD = Major Depressive Disorder, OCD = Obsessive Compulsive Disorder, GAD = Generalized Anxiety Disorder. State clinical measures taken during the scan: STAI = State Trait Anxiety Inventory, RSDI = Responses to Script-Driven Imagery Scale.\* indicates the clinical variables on which all groups differed significantly from one another (*p* \< .05). a. indicates significantly higher clinical measures within a group as compared to the control group, b. indicates significantly higher clinical measures as compared to the PTSD group.
[^2]: Results from the full-factorial 3 (group) by 5 (network) ANOVA (*p-FDR* cluster-corrected *\<* 0.05, *k* = 10). Abbreviations: PFC = Prefrontal cortex, *H* = Hemisphere, BA = Brodmann area, *p* FDR = False discovery rate correction for multiple comparisons.
[^3]: Follow-up group comparisons of network functional connectivity evaluated via 2-sample *t*-tests (*p*-FDR cluster-corrected \< 0.05, *k* = 10). Data represents between group differences in terms of network functional connectivityfor the DMN, CEN and SN. Comparisons were computed between PTSD patients, PTSD+DS patients, and healthy controls, as indicated by contrast notation (\< or \>). Abbreviations: DMN = Default mode network, CEN = central executive network, SN = salience network, dlPFC = Dorsolateral prefrontal cortex, PFC = Prefrontal cortex, MFG = Middle frontal gyrus, PTSD = Posttraumatic stress disorder patient group, PTSD+DS = Dissociative subtype posttraumatic stress disorder group, Healthy= age-matched healthy control group, BA = Brodmann Area, MNI = Montreal Neurological Institute, FDR = False discovery rate cluster corrected, H = Hemisphere.
| {
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Humans and their closest evolutionary relatives, the chimpanzees and bonobos, differ from one another in morphological, cognitive, behavioural and physiological traits \[[@pone.0134548.ref001]--[@pone.0134548.ref003]\]. The availability of genome sequences for human, chimpanzee, bonobo and rhesus macaque allows for the identification of lineage-specific sequence changes. However, since most of the sequence differences are likely to be neutral \[[@pone.0134548.ref004]\], it remains a challenging task to identify those that have important biological consequences \[[@pone.0134548.ref005]\]. For a relative small number of loci, human lineage-specific genomic changes have been linked to phenotypic differences to the other great apes \[[@pone.0134548.ref006]\]. One example is a deletion within the human gene *CMAH* which leads to the loss of the sialic acid Neu5Gc and to altered pathogen susceptibility \[[@pone.0134548.ref007]\].
Using the great apes to identify connections between genotype and phenotype in humans is hampered by the comparatively limited biomedical and physiological information about great apes \[[@pone.0134548.ref001], [@pone.0134548.ref005], [@pone.0134548.ref008]\]. Blood provides an opportunity to quantitatively analyze metabolic products that can serve as markers of metabolic and hormonal states \[[@pone.0134548.ref009]\]. The genes and metabolic pathways underlying these metabolic products are often well-characterized and it is therefore sometimes possible to identify the potential genetic basis for differences in metabolite levels.
While blood biomarkers are extensively studied in humans, there have been only limited comparative studies of humans and great apes. These that exist have been carried out in small numbers of captive animals \[[@pone.0134548.ref010], [@pone.0134548.ref011]\] or involve a single species \[[@pone.0134548.ref012]--[@pone.0134548.ref015]\].
The aim of this study was to identify biomarkers that show lineage-specific changes in their levels or activity in the serum of humans, chimpanzees and bonobos. We analyzed serum samples from 277 wild-born and captive-born great apes (121 Central African chimpanzees, 95 West African chimpanzees, 61 bonobos), and from 312 healthy human volunteers from Germany. For each sample we measured up to 33 biomarkers that are routinely used in human and veterinary medicine to quantify heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We assigned lineage-specific changes based on comparison to serum levels of the same biomarkers in 20 rhesus macaques from Germany ([Fig 1](#pone.0134548.g001){ref-type="fig"}). We identified biomarkers that are likely to have changed on one lineage and divide these into changes that are strongly influenced by environmental factors and changes that are unlikely to be explained by environmental effects alone.
![Flow chart of the analytic approach to identify lineage-specific biomarker levels.\
**A:** differences in biomarker levels were sorted as specific to (i) humans, (ii) great apes, (iii) bonobos, (iv) chimpanzees, (v) Central African chimpanzees, (vi) West African chimpanzees and as (vii) non-lineage specific. **B:** Human-specific changes were defined as significant differences to chimpanzees and bonobos taken together (but not between the latter two species) as well as to rhesus macaques (shown); and as significant differences between humans and the individual great ape species (not shown), regardless of significant differences between species born and living under different environments. Relations of species as shown in cladograms derived from \[[@pone.0134548.ref103], [@pone.0134548.ref104]\].](pone.0134548.g001){#pone.0134548.g001}
Methodology {#sec002}
===========
Ethics statement {#sec003}
----------------
All animal work was conducted according to relevant national, EU and international guidelines. In all cases, the animals were not subjected to any experimental procedures, and the blood samples used were left-over aliquots collected by veterinarians carrying out routine medical examinations. Authorization for use of the samples was obtained from the respective Ministries of Environment as well as by the Ministère de la Recherche Scientifique (DRC) to "Les Amis des Bonobos du Congo", the Uganda Wildlife Authority and the Uganda National Council for Science and Technology, and the Ministère de l\'Enseignement Supérieur et de la Recherche Scientifique from Republic of Congo. The international transport of samples was approved (CITES numbers: Uganda E-3520/05, Kenya E-1259/05, DRC E-0908/07, Republic of Congo E-1274/07). The proposal that in part covers this research (233297, TWOPAN) was reviewed and approved by the European Commission.
Samples {#sec004}
-------
Sera from 121 (65 male and 56 female) wild-born Central African chimpanzees (*Pan troglodytes troglodytes*), 76 (34 male and 42 female) wild-born and 19 (6 male and 13 female) captive-born West African chimpanzees (*Pan troglodytes verus*), 50 (28 male and 22 female) wild-born and 11 (7 male and 4 female) captive-born bonobos (*Pan paniscus*), 20 (3 male and 17 female) captive-born rhesus macaques (*Macaca mulatta*) and from 312 (156 male and 156 female) humans was used for this study.
All samples of wild-born great apes were collected in 2007 and 2009 during annually planned health checks. The samples of wild-born Central African chimpanzees were collected at Tchimpounga Sanctuary (Pointe Noire, Republic of Congo), the samples of wild-born West African chimpanzees were collected at Tacugama Sanctuary (Freetown, Sierra Leone) and the samples of wild-born bonobos were collected at Lola Ya Bonobo Sanctuary (Kinshasa, Democratic Republic of Congo).
The wild-born great apes were fasting from the evening before the anaesthesia and were then anaesthetized with a combination of medetomidine (Domitor, Pfizer, NY, USA, 0.03 to 0.05 mg/kg) and ketamine (Imalgen, Merial, GA, USA, 3 to 4 mg/kg), injected intramuscularly with either a dart (Telinject, France) or a syringe (Terumo, Japan). Blood was collected from the femoral vein with the Vacutainer (Becton Dickinson, NJ, USA) or with the Monovette-system (Sarstedt, Germany). Then blood was processed according to a standardized protocol in order to minimize preanalytic influences. Blood was allowed to clot for 30 minutes at room temperature, then centrifuged at 4° C for 10 minutes at 1600 g. The supernatant serum was transferred into a freezing tube, stored on dry ice, protected from light and transferred to our laboratory for subsequent biomarker analysis. Two bonobos were excluded from analysis since they died shortly after the sampling of the blood. One Central African chimpanzee was excluded since he was an outlier in a principle component analysis. Median ages for wild-born bonobos, Central and West African chimpanzees were 6 years (range: 3 to 20 years), 8 years (range: 2 to 42 years) and 4 years (range: 1 to 23 years), respectively ([Table 1](#pone.0134548.t001){ref-type="table"}). Median weights were 18.8 kg (2.5^th^ to 97.5^th^ percentiles: 4.9 and 45 kg), 38.8 kg (2.5^th^ to 97.5^th^ percentiles: 10 and 67 kg) and 27 kg (2.5^th^ to 97.5^th^ percentiles: 9.9 and 53.2 kg).
10.1371/journal.pone.0134548.t001
###### Results for clinical chemical biomarkers in serum samples from wild- and captive-born great apes (wild-born Central African chimpanzees from the Republic of Congo (Ch---CG), wild-born West African chimpanzees from Sierra Leone (Ch---SL) and captive-born chimpanzees from Germany (Ch---DE), wild-born bonobos from the Democratic Republic of the Congo (B---CD) and captive-born bonobos from Germany (B---DE)), captive-born rhesus macaques from Germany (Rh---DE), and from healthy human volunteers (H---DE).
{#pone.0134548.t001g}
Species
-------------------------------------------------------------- ----------------- --------------- ---------------- --------------- ------------------ --------------- ---------------- ---------
Age \[years\] *median* 3.5 6 14 8 4 20 27
*range* 1--22 3--20 3--37 2--42 1--23 8--46 18--65
Alanine transaminase activity \[μkat/L\] *median* 0.10 0.27 0.54 0.48 0.48 0.65 0.18
*percentiles/range* 0.07--0.15 0.09--0.66 0.43--0.83 0.21--0.94 0.27--0.94 0.41--1.93 0.08--0.40
Albumin \[g/L\] *median* 42.8 33.3 40.0 36.9 37.6 39.3 43.8
*percentiles/range* 34.4--47.6 24.8--40.2 35.5--42.6 29.1--41.1 32.4--42.2 28.8--43.5 38.7--48.2
Apolipoprotein A \[g/L\]---bs *median* 1.40 2.15 2.51 1.63 1.85 1.71 1.58
*percentiles/range* 0.80--2.08 1.47--2.83 2.06--3.52 1.15--2.28 1.25--2.41 1.08--3.10 1.11--2.26
Apolipoprotein B \[g/L\] *median* 0.37 0.79 0.76 0.59 0.70 0.73 0.72
*percentiles/range* 0.20--0.47 0.50--1.33 0.48--0.97 0.35--0.89 0.46--0.92 0.29--1.24 0.42--1.31
Aspartate transaminase \[μkat/L\] *median* 1.20 0.53 0.40 0.79 0.39 0.48 0.42
*percentiles/range* 0.87--1.66 0.21--2.91 0.27--0.80 0.39--1.80 0.22--0.56 0.26--1.08 0.26--0.69
Total bilirubin^a^ \[μmol/L\]---hs *median* \< 1.71 \< 1.71 \< 1.71 \< 1.71 \< 1.71 \< 1.71 3.40
*percentiles/range* \< 1.71 \<1.71--2.70 \< 1.71--2.00 \< 1.71--2.40 \< 1.71--2.40 \< 1.71--2.10 \< 1.71--13.34
Cholinesterase \[μkat/L\]---hs *median* 201.2 156.9 214.0 199.7 208.0 209.3 126.0
*percentiles/range* 136.9--448.9 98.0--282.0 159.60--339.90 105.6--345.2 124.8--280.1 154.8--364.9 72.0--191.2
Total cholesterol \[mmol/L\] *median* 3.42 5.63 5.50 4.63 5.07 4.58 4.72
*percentiles/range* 1.84--4.39 3.90--8.54 4.61--6.45 3.00--7.06 3.78--6.78 2.90--8.07 3.24--6.95
Colloid osmotic pressure \[mmHg\] *median* 26.1 24.9 n\. a. 22.4 22.2 n\. a. 28.3
*range* 22.3--29.2 20.5--29.3 19.0--25.4 18.7--24.1 24.2--32.5
C-reactive protein \[mg/L\] *median* 1.11 23.90 0.84 2.32 0.53 1.00 0.72
*percentiles/range* \< 0.30--27.09 0.71--239.24 \< 0.30--3.39 1.00--45.80 \< 0.30--50.82 \< 0.30--3.57 \< 0.30--10.28
Creatine kinase \[μkat/L\] *median* 42.28 2.14 4.10 2.97 1.80 3.68 1.19
*percentiles/range* 18.97--86.40 0.56--12.11 2.57--6.91 1.31--10.40 0.92--6.23 1.17--13.32 0.50--4.39
Creatinine \[μmol/L\] *median* 76.0 46.5 82.0 62.0 58.5 75.00 70.0
*percentiles/range* 52.0--118.0 14.5--93.9 34.0--123.0 33.1--121.9 35.6--103.1 50--253 48.5--95.0
Ferritin \[ng/ml\] *median* n\. a. 21.3 71.5 105.4 69.1 203.2 27.0
*percentiles* 4.0--157.2 31.0--171.9 15.5--388.2 18.0--268.8 17.5--883.3 5.5--144.6
Folate \[nmol/L\] *median* n\. a. 31.50 21.18 28.31 18.69 36.16 20.56
*percentiles* 15.43--45.40 12.84--45.40 17.51--45.40 11.02--29.84 28.50--45.00 9.27--39.36
Gamma-glutamyl transpeptidase \[μkat/L\] *median* 1.59 0.10 0.15 0.26 0.32 0.44 0.28
*percentiles/range* 0.72--3.40 \< 0.05--0.32 0.09--0.34 0.11--0.99 0.18--0.58 0.18--1.13 0.12--1.75
Glucose \[mmol/L\] *median* n\. a. 4.37 6.31 5.17 5.18 6.46 4.49
*percentiles* 2.53--7.96 4.46--9.67 2.19--10.17 2.62--7.65 4.34--9.89 2.85--7.84
Glutamate dehydrogenase \[μkat/L\] *median* 0.27 0.11 0.04 0.12 0.10 0.07 0.04
*percentiles/range* 0.19--0.32 0.03--0.52 0.02--0.75 0.06--0.43 0.05--0.28 0.03--0.29 0.02--0.19
High-density lipoprotein cholesterol \[mmol/L\] *median* 0.83 1.67 2.35 1.58 1.68 1.60 1.45
*percentiles/range* 0.46--1.29 0.56--2.84 2.06--4.16 0.84--2.66 0.87--2.40 0.38--3.08 0.90--2.41
Lactate dehydrogenase \[μkat/L\]---hs *median* 9.55 6.56 4.59 12.33 5.95 6.18 2.07
*percentiles/range* 6.14--13.28 3.55--32.93 3.96--9.07 6.07--48.73 4.15--8.34 2.96--9.18 1.43--2.98
Lipase \[μkat/L\] *median* 0.22 0.18 0.13 0.16 0.18 0.19 0.53
*percentiles/range* 0.00--0.71 0.13--0.32 0.09--0.24 0.13--0.24 0.14--0.25 0.12--0.27 0.25--1.30
Lipoprotein (a) \[mg/dl\] *median* 37.3 102.8 74.1 43.6 181.0 97.4 37.3
*percentiles/range* 9.9--116.1 3.5--269.8 31.9--120.3 2.1--166.2 65.9--296.90 14.6--163.0 \< 5.0--154.4
Low-density lipoprotein cholesterol \[mmol/L\] *median* 1.89 3.03 2.90 2.48 2.66 2.40 2.72
*percentiles/range* 0.42--2.50 1.71--5.94 1.79--3.47 1.29--4.06 1.67--4.03 1.71--5.64 1.41--4.60
N-terminal prohormone of brain natriuretic peptide \[pg/mL\] *median* \<5.0 204.4 73.1 195.0 154.5 194.3 33.2
*percentiles* \<5.0--9.9 26.9--2563.6 35.7--401.6 17.9--775.7 59.0--502.7 \< 5.0--836.7 6.9--144.6
Phosphate \[mmol/L\] *median* n\. a. 1.58 1.13 1.43 1.57 1.32 1.29
*percentiles* 0.86--2.16 0.47--2.00 0.57--1.93 0.82--2.31 0.36--1.93 0.96--1.65
Total protein \[g/L\] *median* 66.7 83.1 71.8 76.9 68.9 69.6 72.0
*percentiles/range* 58.4--74.0 64.9--98.5 65.9--75.7 65.7--87.6 61.5--78.5 64.7--81.0 64.0--79.6
Thyroid-stimulating hormone \[mU/L\] *median* \< 0.005 3.73 4.61 1.71 3.06 3.56 1.34
*percentiles* \< 0.005--0.007 0.33--19.88 2.43--18.10 0.51--5.38 0.74--11.09 0.005--8.50 0.39--3.61
Free thyroxine \[pmol/L\] *median* 14.14 11.33 14.41 12.37 12.14 16.61 16.53
*percentiles/range* 7.92--24.37 6.53--18.37 10.26--25.19 7.19--20.87 8.15--21.03 11.40--24.21 13.11--20.76
Triglycerides \[mmol/L\] *median* 0.69 1.15 0.56 1.02 1.12 1.08 1.28
*percentiles/range* 0.44--1.01 0.58--3.11 0.28--1.70 0.56--2.30 0.63--1.94 0.73--3.06 0.55--3.95
Free triiodothyronine \[pmol/L\] *median* 5.71 6.47 6.57 6.59 5.01 6.63 5.15
*percentiles/range* 1.71--17.37 1.28--11.22 4.30--8.83 4.04--11.09 3.05--10.44 3.69--10.00 3.92--6.66
Troponin T \[μg/L\] *median* n\. a. \< 0.01 \< 0.01 \< 0.01 \< 0.01 \< 0.01 \< 0.01
*percentiles/range* \< 0.01--0.18 \< 0.01--0.04 \< 0.01--0.05 \- \< 0.01--0.25 \< 0.01
Urea \[mmol/L\] *median* 7.60 1.50 1.95 1.60 1.45 2.60 4.43
*percentiles/range* 4.30--13.20 \< 0.83--6.74 0.80--3.60 \< 0.83--4.99 \< 0.83--3.25 1.30--7.80 2.56--7.30
Uric acid \[μmol/L\] *median* \< 11.9 203.0 128.0 145.5 145.5 124.0 265.5
*percentiles* \< 11.9--12.0 106.4--371.6 93.0--192.0 75.0--226.8 64.5--221.8 81--224 142.1--407.5
Vitamin B12 \[pmol/L\] *median* n\. a. 272.7 974.3 233.3 593.9 1476 278.9
*percentiles/range* 126.8--1459.6 173.3 - \>1476 82.4 - \>1476 215.2 - \>1476.0 1475 - \>1476 147.5--576.6
ISO 3166 codes for the representation of countries of origin of serum samples; median, 2.5th---97.5th percentiles for wild-born great apes and humans and range for captive born great apes and rhesus macaques; ranges for age, colloid osmotic pressure and for lipoprotein (a) in wild-born Central African chimpanzees; "n. a." = not analyzed; "bs" indicates a bonobo-specific biomarker and "hs" indicates human-specific biomarkers, green border denotes bonobo-specific biomarkers; blue border denotes human-specific biomarkers; "\<"/"\>" indicate below lower limit/above upper limit of quantification; percent of individuals below lower level of quantification (LLOQ) for bilirubin: wild- and captive-born bonobos: 82 and 55 percent, Central African chimpanzees: 75 percent; wild- and captive-born West African chimpanzees: 82 and 63 percent; rhesus macaques 100 percent; humans: 12.3 percent; see "[Methodology](#sec002){ref-type="sec"}" for details on the origin of the samples. The underlying data can be downloaded as [S4 Dataset](#pone.0134548.s004){ref-type="supplementary-material"}.
Samples from captive-born West African chimpanzees and bonobos were obtained from the Leipzig Zoo during routine health checks between 2005 and 2013. Animals fasted for 12 hours prior to anaesthesia with a combination of xylazine (2 to 3 mg/kg for bonobos and 2.4 to 4 mg/kg for chimpanzees) and ketamine (4 to 6 mg/kg for bonobos and 2.5 to 4 mg/kg for chimpanzees), injected intramuscularly with a dart. Blood was taken either from the vena saphena parva superficialis or from the vena mediana cubiti. Sera were stored at -80°C prior to analysis. No weights were available for captive-born great apes. Median ages for captive-born bonobos, and West African chimpanzees were 14 years (range: 3 to 17 years) and 20 years (range: 8 to 46 years), respectively ([Table 1](#pone.0134548.t001){ref-type="table"}).
Sera from rhesus macaques were purchased from the German primate center (Göttingen/Germany). The sera were from healthy individuals kept for breeding purposes, and were collected during routine physical exams in May 2007 from the vena saphena of the proximal forth of the thigh. Animals had been anaesthetized with ketamine after a 12 hour fast. All sera were stored at -20° C prior to analysis. Although individual weights were not recorded for each rhesus macaque, males were between 2 and 3 kg and females between 7 and 8 kg. The median age for rhesus macaques was 3.5 years (range: 1 to 22 years; [Table 1](#pone.0134548.t001){ref-type="table"}).
As the human reference group we used serum of a group of 312 young healthy blood donors previously described \[[@pone.0134548.ref016]\]. Median age was 27 years (range: 18 to 65 years; [Table 1](#pone.0134548.t001){ref-type="table"}); median weight was 70 kg (2.5^th^ to 97.5^th^ percentiles: 52 and 98 kg).
Biochemical analyses {#sec005}
--------------------
All biochemical analyses were performed at the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics of the University Clinic Leipzig/Germany except for parts of the analysis of lipoprotein(a) (Lp(a)) which was performed at the Department of Medical Genetics, Molecular and Clinical Pharmacology, Divisions of Genetic Epidemiology and Human Genetics of the Innsbruck Medical University (Innsbruck/Austria).
Total cholesterol, HDL and LDL cholesterol, triglycerides, albumin, total protein, creatinine, urea and uric acid, bilirubin, glucose and phosphate were measured by using commercial photometric tests on an automated Modular P analyzer (Roche/Hitachi; [S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}) \[[@pone.0134548.ref017]--[@pone.0134548.ref031]\]. Concentrations of apolipoprotein A-I and apolipoprotein B-100, C-reactive protein and Lipoprotein (a) were measured by using immunoturbidimetric assays (Tina-quant apo AI version 2 and Tina-quant apo B-100 version 2, C-reactive protein Gen.3; Tina-quant Lipoprotein (a), Roche; [S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}) on Modular P \[[@pone.0134548.ref032]--[@pone.0134548.ref035]\]. The catalytic activities of alanine transaminase, aspartate transaminase, cholinesterase, creatine kinase, gamma-glutamyl transpeptidase, glutamate dehydrogenase, lactate dehydrogenase and lipase were determined using a photometric test on the same automated analyzer ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}) \[[@pone.0134548.ref036]--[@pone.0134548.ref040]\]. Ferritin, free and total triiodothyronine and thyroxine, amino-terminal prohormone of brain natriuretic peptide (NTproBNP), troponin T and vitamin B12 were analyzed by a heterogeneous electrochemiluminescence immunoassay on an automated Modular E analyzer (Roche/Hitachi, [S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}) \[[@pone.0134548.ref041], [@pone.0134548.ref042]\]. Lp(a) quantification in samples from the Republic of Congo, Sierra Leone and the Democratic Republic of Congo was performed as described in detail \[[@pone.0134548.ref043], [@pone.0134548.ref044]\] with a double-antibody enzyme-linked immunosorbent assay (ELISA), using an affinity-purified polyclonal apo(a) antibody for coating and the horseradish peroxidase-conjugated monoclonal antibody for detection. Colloid osmotic pressure was determined via direct hydrostatic pressure measurement on a colloid osmometer (Osmomat 50, Gonotec, Berlin, Germany) \[[@pone.0134548.ref045], [@pone.0134548.ref046]\] in triplicates.
A list of all biomarkers measured in this study with the corresponding method of detection, associated gene name (where possible) and pairwise alignment score for the comparison of proteins between humans and both West African chimpanzees and rhesus macaques is provided in [S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}. A protein search was done using the HomoloGene function on NCBI's webpage to obtain homologous protein sequences of human, West African chimpanzee and rhesus macaque proteins. When a homologous protein was not available for any of the non-human primate species using the HomoloGene function, a protein blast with the human protein was performed using UniProt to find the homologous protein in the remaining species.
The expression level of UGT1A1 transcripts in humans, chimpanzees, and rhesus macaques ([Fig 2](#pone.0134548.g002){ref-type="fig"}) was determined by transcript sequencing (RNA-Seq on an Illumina GA IIx) of liver samples from 3 males and 3 females from each species \[[@pone.0134548.ref047]\]. Significant differences in expression levels between species were determined using the negative binomial model implemented in DESeq \[[@pone.0134548.ref048]\].
![Liver UDP-glucuronosyltransferase 1A1 (UGT1A1) promoter transcript expression in rhesus macaques, chimpanzees and humans \[[@pone.0134548.ref047]\] and respective TATAA-box length \[[@pone.0134548.ref056], [@pone.0134548.ref057], [@pone.0134548.ref105]\]\
- UGT1A1 transcript expression was determined from RNA-Seq of human, chimpanzee and rhesus macaque liver RNA samples from 3 males and 3 females of each species \[[@pone.0134548.ref047]\]. Relative expression levels were calculated from the original dataset setting human expression levels at 100 percent (also see [S1 Table](#pone.0134548.s007){ref-type="supplementary-material"} for the variability of TA repeats in TATA box of UGT1A1 promoter in archaic hominins, humans and non-human primates).](pone.0134548.g002){#pone.0134548.g002}
The expression levels of UGT1A1 transcripts in mice fed either a raw or a cooked diet composed of meat or of tuber were measured by RNA-Seq ([Fig 3](#pone.0134548.g003){ref-type="fig"})\[[@pone.0134548.ref049]\]. Total RNA was prepared from 17 individuals and sequenced as a pool on two lanes of an Illumina HiSeq 2500. Significant differences in expression between mice fed raw diets and mice fed cooked diets were quantified using DESeq \[[@pone.0134548.ref048]\].
![Liver *UGT1A1*-mRNA expression in mice on raw and cooked diets:\
Liver mRNA expression of UGT1A1 transcripts in mice fed either a raw or cooked meat or raw or cooked or tuber diets was measured by RNA-Seq \[[@pone.0134548.ref049]\]. Total RNA was prepared from 17 individuals and sequenced as a pool on two lanes of an Illumina HiSeq 2500. Significant differences in expression between mice fed raw diets and mice fed cooked diets were quantified using DESeq \[[@pone.0134548.ref048]\].](pone.0134548.g003){#pone.0134548.g003}
Statistical analysis {#sec006}
--------------------
Calculations were performed with IBM SPSS Statistics software (version 20.0.0) and R (version 2.12.1; <http://cran.r-project.org/>).
The study population was split into species groups. Percentages of change are given as medians (2.5th to 97.5th percentiles and ranges). The statistical significance of the differences between groups was assessed by using the Wilcoxon rank-sum test with the significance threshold set at 0.01.
Results {#sec007}
=======
Sex-specific differences in serum biomarker levels {#sec008}
--------------------------------------------------
Our study included both male and female individuals and we therefore expect some differences in biomarker levels to reflect sex-specific traits. Using average timing of sexual maturity in each species \[[@pone.0134548.ref050]\] we expect that approximately 50 percent of the rhesus macaques, 30 and 73 percent of the wild- and captive-born bonobos, 49 percent of the wild-born Central African chimpanzees, 35 and 100 percent of the wild- and captive-born West African chimpanzees, and 100 percent of the humans in this study were sexually mature. We tested to what extent biomarker levels differ between males and females in each of the species. Sex-specific differences in serum biomarker levels are shown in [S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}. Humans showed the largest number of significantly different biomarkers between females and males (67 percent of total number of biomarkers differed compared to between 0 and 12 percent in the non-human primates; [S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}). In wild bonobos mean levels of low-density lipoprotein cholesterol, apolipoprotein A, apolipoprotein B and total cholesterol were significantly higher in females than in males. However, this difference was not seen in captive-born bonobos where levels of these same biomarkers were not significantly different between males and females ([S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}).
Wild-born male Central African chimpanzees showed a significantly higher catalytic activity of creatine kinase, and higher levels of uric acid and ferritin than females. Cholinesterase was found to be significantly increased in wild-born male West African chimpanzees, while lactate dehydrogenase was significantly higher in captive-born West African male chimpanzees ([S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}). In rhesus macaques no significant differences between the sexes were found for any of the biomarkers. In humans, all biomarkers with the exception of total cholesterol, colloid-osmotic pressure, folic acid, glucose, lactate dehydrogenase, lipoprotein (a), Thyroid-stimulating hormone, troponin T and vitamin B12 showed significant differences between the sexes ([S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}).
Lineage-specific biomarker levels {#sec009}
---------------------------------
We assigned differences in biomarker levels as: (i) human-specific, (ii) great ape-specific, (iii) bonobo-specific, (iv) chimpanzee-specific, (v) Central African chimpanzee-specific, (vi) West African chimpanzee-specific and (vii) as uncategorized changes using the rhesus macaques as an outgroup ([Fig 1](#pone.0134548.g001){ref-type="fig"}). We did not identify rhesus macaque-specific changes since this would require an appropriate outgroup. Human-specific changes were defined as significant differences to all chimpanzees and bonobos taken together (but not between the two apes) as well as to rhesus macaques; and as significant differences between humans and the individual great ape species (Wilcoxon rank-sum test, p \< 0.01). Using this approach, we identified human-specific differences in three biomarkers (bilirubin, cholinesterase, lactate dehydrogenase), and a bonobo-specific difference in one biomarker (apolipoprotein A). There was no biomarker difference specific to either the great apes as a group, to chimpanzees as a group or to West or Central African chimpanzees ([Fig 1](#pone.0134548.g001){ref-type="fig"}). Twenty-nine biomarkers showed differences that could not be assigned uniquely to one lineage because they were either not statistically significant or because they have changed on multiple lineages. These were classified as "non-lineage-specific".
Amino acid differences in protein sequences may affect test results by altering antibody affinity for an analyte or the catalytic activities of enzymes. For three of the four biomarkers with species-specific differences, the genes underlying the biomarker can be identified. We compared the protein sequences of these genes between human, chimpanzee and rhesus macaque ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}). The bonobo sequence of apolipoprotein A was 99.6, 99.6 and 94.8 percent identical to those from West African chimpanzees, humans and rhesus macaques, respectively. Human cholinesterase was 99.2, and 95.8 percent identical to those from West African chimpanzees, and rhesus macaques ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}; a bonobo sequence was not available). Lactate dehydrogenase consists of three subunits (LDHA, LDHB and LDHC) with a median identity of 99.7 percent between humans and West African chimpanzees and a median identity of 98.9 percent between humans and rhesus macaques ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}; a bonobo sequence was not available). As a metabolite, the fourth biomarker, bilirubin, is structurally identical in all species of our study \[[@pone.0134548.ref051]\].
Biomarker level differences between wild- and captive-born individuals {#sec010}
----------------------------------------------------------------------
Differences in biomarker levels may represent organismal responses to short- or long-term environmental factors, genetic differences between species, or both. We assume that biomarkers that show large differences between members of the same species living in different environments are those most affected by short-term environmental factors. We therefore compared each biomarker between wild- and captive-born individuals of the same species. We were only able to compare 32 biomarkers since colloid osmotic pressure was neither determined in captive-born bonobos nor in captive-born chimpanzees. Of these, 17 biomarkers differed between wild- and captive-born bonobos and 14 differed between wild- and captive-born chimpanzees; Wilcoxon rank-sum test, p \< 0.01; [S3 Dataset](#pone.0134548.s003){ref-type="supplementary-material"}). When excluding the 25 biomarkers showing an environmental effect, only seven biomarkers remained (apolipoprotein B, bilirubin, total cholesterol, low-density lipoprotein cholesterol, N-terminal prohormone of brain natriuretic peptide, Thyroid-stimulating hormone and troponin T).
Biomarker levels that were significantly higher in both captive-born chimpanzees and captive-born bonobos were alanine transaminase, creatine kinase, ferritin, glucose, and vitamin B12 ([S3 Dataset](#pone.0134548.s003){ref-type="supplementary-material"}). The levels or catalytic activity of aspartate transaminase, gamma-glutamyl transpeptidase, the free thyroid hormones triiodothyronine and thyroxine, urea and folic acid were only significantly higher in captive-born chimpanzees but not in captive-born bonobos. The levels or catalytic activity of albumin, apolipoprotein A, cholinesterase and high-density lipoprotein cholesterol were only significantly higher in captive-born bonobos.
No biomarker was consistently higher in both wild-born great apes, whereas C-reactive protein, lactate dehydrogenase, lipase, total protein, triglycerides and uric acid were higher in wild-born and lipoprotein A and phosphate in wild-born chimpanzees ([S3 Dataset](#pone.0134548.s003){ref-type="supplementary-material"}).
Quantification of influence of environment and species on biomarkers {#sec011}
--------------------------------------------------------------------
To assess the relative influence of genetic and environmental factors in our dataset, we tested whether chimpanzees and bonobos from similar environments showed more similar biomarker levels than those from different environments. More specifically, we compared biomarker levels between captive- and wild-born bonobos and captive- and wild-born West African chimpanzees (see description of [S1 Fig](#pone.0134548.s005){ref-type="supplementary-material"} for details). We observe that wild-born bonobos and wild-born West African chimpanzees show the smallest differences. In contrast, comparisons between different environments (wild-born bonobos to captive-born West African chimpanzees and captive-born bonobos to wild-born West African chimpanzees) show significantly larger differences than the comparison between wild-born bonobos and wild-born chimpanzees (Wilcoxon rank-sum test, p \< 0.001 in both comparisons; [S1 Fig](#pone.0134548.s005){ref-type="supplementary-material"}). Larger differences are also observed in the comparison of captive-born bonobos and chimpanzees. We conclude that short-term environmental influences may account for many of the observed differences in biomarker levels.
Exclusion of biomarkers that are significantly different between individuals of the same species in different environments {#sec012}
--------------------------------------------------------------------------------------------------------------------------
To identify potential genetic differences for biomarker concentrations we eliminated from consideration biomarkers that differed between wild- and captive-born great apes since we reasoned that these are likely to be influenced by short-term environmental factors. Of the four lineage-specific biomarkers in our study, only bilirubin levels were equivalent among members of the same species from distinct habitats.
A human-specific increase in serum bilirubin {#sec013}
--------------------------------------------
Humans have significantly higher serum levels of bilirubin than great apes and rhesus macaques (Median: 3.4 μmol/L in humans vs. \< 1.71 μmol/L in the remaining species). While 12.3 percent of human samples were below the lower limit of quantification for bilirubin, the fraction in the other species was higher (between 55 and 82 percent in the great apes and 100 percent in the rhesus macaques) ([Table 1](#pone.0134548.t001){ref-type="table"}; [Fig 4](#pone.0134548.g004){ref-type="fig"}).
{#pone.0134548.g004}
Genome-wide association studies have shown that the major gene associated with serum bilirubin levels is *uridine diphosphoglucuronosyltransferase 1* (UGT1A1) \[[@pone.0134548.ref052]--[@pone.0134548.ref054]\]. The expression of *UGT1A1* largely depends on a microsatellite in the promoter of the *UGT1A1* gene, with an inverse relationship between the number of TA repeats and the activity of the gene \[[@pone.0134548.ref055]\]. The genome sequences show differences in TA repeat length with 3 repeats in rhesus macaques (reference assembly rheMac2), 3 to 4 repeats in chimpanzees (chimpanzee resequencing data from \[[@pone.0134548.ref056]\]) and 5--8 repeats in humans (13 human genomes from \[[@pone.0134548.ref057]\]; [Fig 2](#pone.0134548.g002){ref-type="fig"}, [S1 Table](#pone.0134548.s007){ref-type="supplementary-material"}). Furthermore, we found that the transcript expression levels of *UGT1A1* in rhesus macaque and chimpanzee livers were 46 and 35 percent higher than in humans (p = 0.15 and 0.27, respectively; [Fig 2](#pone.0134548.g002){ref-type="fig"}, [S2 Table](#pone.0134548.s008){ref-type="supplementary-material"}). The microsatellite length in two Neandertal individuals \[[@pone.0134548.ref057]\] and one Denisovan \[[@pone.0134548.ref058]\] are similar to present-day humans outside of Africa in having a TA repeat of length 6 in the promoter of *UGT1A1* ([S2 Table](#pone.0134548.s008){ref-type="supplementary-material"}).
By protein sequence alignment of *UGT1A1*, we found one human-specific amino acid substitution F518L ([S2 Fig](#pone.0134548.s006){ref-type="supplementary-material"}). The substitution lies between a transmembrane domain and di-lysine motifs in the cytosolic tail that confer retention of UGT1A-proteins to the endoplasmic reticulum (ER) and alter the half-life of UGT1A1 protein \[[@pone.0134548.ref059]\].
UGT1A1 is the main isozyme responsible for the glucuronidation of bilirubin. However, it is also involved in the glucuronidation that aides excretion of phytoalexins which are toxic plant-derived compounds \[[@pone.0134548.ref060]\]. To assess the effect of a plant- or animal-derived diet on *UGT1A1*-expression, we determined *UGT1A1* expression in liver samples of mice fed tuber or meat diets that were either cooked or raw (Carmody et al. in preparation). Mice on a raw tuber diet showed a tendency of higher *UGT1A1* expression than mice on either meat or cooked tuber diets ([Fig 3](#pone.0134548.g003){ref-type="fig"}).
Discussion {#sec014}
==========
The dataset presented here is, to our knowledge, the largest screen of biomarkers in wild- and captive-born great apes. While this dataset can be used for a variety of analyses, we have chosen to concentrate on the identification of lineage-specific changes in biomarker levels.
Large effort has been made to minimize preanalytic influences. All samples, including those from Africa, were frozen immediately after serum extraction. Studies have shown that long-term storage (up to 10 years) does not affect the results for any of the biomarkers we have tested \[[@pone.0134548.ref061]\]. All analyses were carried out using the same automated analyser.
We note that all great apes had to be sedated before sampling. Ketamine anaesthesia has been shown to influence blood levels of some of the biomarkers we measured (alanine and aspartate aminotransferase and creatine kinase \[[@pone.0134548.ref062], [@pone.0134548.ref063]\], phosphate \[[@pone.0134548.ref064]\]) whereas bilirubin is not changed \[[@pone.0134548.ref065], [@pone.0134548.ref066]\]. However, the differences we observe between captive-born and wild-born apes indicate that anaesthesia is not a major factor determining differences in blood-chemistry between groups.
All animals included in this study were deemed healthy by a trained veterinarian. However, we cannot exclude that subclinical infection could explain high values of C-reactive protein in the bonobos from Lola Ya Bonobo.
Our laboratory analyses were performed using established automated assays optimized for use in human clinical medicine. We are aware that this may affect results obtained in other species \[[@pone.0134548.ref010], [@pone.0134548.ref011], [@pone.0134548.ref067]\]. The sequence identity of proteins measured in our study lies between 95.1 and 100 percent compared to humans for West African chimpanzees and between 87.2 and 100 percent for rhesus macaques ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}). Results obtained for some antibody-based assays (e.g. immunoassays) may therefore be species-dependent. Furthermore, serum catalytic activity as the product of enzymatic concentration and activity of each enzyme may be influenced by differences in protein sequence and/or structure.
Our study included serum samples from both male and female individuals. As sex-specific biomarker traits may affect overall biomarker level distributions, we analyzed our data for gender-specific differences. The number of significantly different biomarkers between sexes was greatest in humans compared to all other species ([S2 Dataset](#pone.0134548.s002){ref-type="supplementary-material"}). The reason for this finding is not clear. The finding is not consistent with the extent of sexual dimorphism \[[@pone.0134548.ref068]\]. Possible explanations include differences in proportions of male and female individuals in our study groups (female excess among rhesus macaques and captive-born chimpanzees, [Table 1](#pone.0134548.t001){ref-type="table"}) and the proportion of mature individuals (highest in our human study group).
We find evidence for only few species-specific biomarker differences. Lactate dehydrogenase, cholinesterase and bilirubin showed human-specific differences and apolipoprotein A showed a bonobo-specific difference.
We found many differences in biomarker levels between members of the same species born and living under different environments. The biomarker levels that showed significant differences between captive- and wild-born individuals include those that are known to be influenced by dietary factors (ferritin, folic acid, glucose, the free thyroid hormones triiodothyronine and thyroxine, vitamin B12) and enzymes whose release into blood is dependent upon muscle mass or activity (aspartate transaminase, creatine kinase; [S3 Dataset](#pone.0134548.s003){ref-type="supplementary-material"} and [Table 1](#pone.0134548.t001){ref-type="table"}) \[[@pone.0134548.ref026]\]. Except for bilirubin the comparison between captive-born and wild-born apes indicates that environmental differences may explain these lineage-specific differences.
In our study levels of apolipoprotein A-I were specifically higher in bonobos. In agreement with this result, levels of apolipoprotein A-I and high-density lipoprotein cholesterol have been found to be similar in captive-born West African chimpanzees and humans \[[@pone.0134548.ref010]\]. However, a study comparing lipid status in free-ranging and captive macaque species and humans found that levels varied more between the same species in different environments than between the different macaque species \[[@pone.0134548.ref067]\]. Further, apolipoprotein A-I was found to be elevated in female wild-born bonobos, female captive-born chimpanzees, and in female humans whereas this pattern was not seen in the remaining species. High-density lipoprotein cholesterol levels, which are highly correlated with apolipoprotein A-I, are known to be influenced by both diet and exercise in both humans and macaques \[[@pone.0134548.ref019], [@pone.0134548.ref026], [@pone.0134548.ref069]--[@pone.0134548.ref073]\]. We therefore cannot exclude that the lineage-specific change in bonobos is due to some environmental factor.
Lactate dehydrogenase showed a human-specific decrease compared to the apes and rhesus macaques. However, we detected significant variation in serum LDH activity between wild- and captive-born great apes, suggesting that LDH activity is likely to be environmentally influenced. As a cytoplasmic enzyme and constituent of all tissues, lactate dehydrogenase is liberated into the blood in response to tissue damage, which may also include muscle activity and alarm reactions before blood taking \[[@pone.0134548.ref026]\]. It is therefore conceivable that the non-human primates in our study group have higher lactate dehydrogenase activity due to a difference in physical activity.
Serum cholinesterase activity showed a human-specific decrease. The exact function of serum cholinesterase is unclear \[[@pone.0134548.ref074]\]. It is abundant in plasma, liver, pancreas, spleen and the white matter of the brain and is commonly used in clinical medicine to quantify liver synthetic capacity \[[@pone.0134548.ref026]\]. In humans genetic variants with catalytic activities that differ by a factor of ten exist \[[@pone.0134548.ref026]\]. Cholinesterase activity is furthermore known to be associated with weight, body mass index (BMI) and muscle mass \[[@pone.0134548.ref075]\]. Possible explanations for the observed differences may include a combination of the above mentioned factors.
The protein sequences of apolipoprotein A-I, serum cholinesterase and lactate dehydrogenase differ between species ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}). It is not known whether these differences change their interaction with binding antibodies or their catalytic activity and therefore alter the measurement of these biomarkers with the standard assays applied here. For example, in our study apolipoprotein A-I was determined with an immunoturbidimetric assay that may be sensitive to protein differences. The human and chimpanzee apolipoprotein A-I protein sequence is identical while bonobos differ from both by one amino acid. We can therefore not distinguish whether the lineage-specific changes in apolipoprotein A-I in bonobos, and serum cholinesterase and lactate dehydrogenase in humans are due to differences in their protein sequence ([S1 Dataset](#pone.0134548.s001){ref-type="supplementary-material"}).
Bilirubin levels were consistently higher in humans than in West and Central African chimpanzees and bonobos (both captive and wild-born), as well as in rhesus macaques. This is in agreement with a small study that showed that bilirubin levels are twice as high in humans as in captive chimpanzees \[[@pone.0134548.ref010]\].
Serum concentration of bilirubin is a function of extrahepatic formation and intrahepatic excretion of bilirubin. The magnitude of bilirubin formation depends mainly on erythrocyte half-life \[[@pone.0134548.ref076], [@pone.0134548.ref077]\]. Since the erythrocytes of chimpanzees and rhesus macaques have a half-life which is about 50 percent shorter than that of human erythrocytes, bilirubin production should be higher in the great apes and rhesus macaques \[[@pone.0134548.ref078], [@pone.0134548.ref079]\]. However, we observe serum bilirubin levels that are lower in all the non-human primates, suggesting that bilirubin depletion and not bilirubin formation may cause the difference in levels.
To explore whether genetic changes might explain this difference we focused on the gene encoding UDP-glycosyltransferase 1 (*UGT1A1*) which is the major enzyme involved in the glucuronidation of bilirubin (99.5 percent of total turnover) and in the excretion of phytoalexins which are toxic plant-derived compounds \[[@pone.0134548.ref060], [@pone.0134548.ref080], [@pone.0134548.ref081]\]. Levels of bilirubin are highly heritable and association studies have shown that *UGT1A1* is the main contributor to these levels \[[@pone.0134548.ref052], [@pone.0134548.ref053]\]. The gene has not been found to vary in copy number \[[@pone.0134548.ref082]\]. However, Gilbert's syndrome (GS, MIM\*143500), an inherited form of mild hyperbilirubinemia, is typically caused by a homozygous TA insertion in the TATAA element of the 5' promoter region in Europeans (normal A(TA)~6~TAA) (*UGT1A1*\*28) \[[@pone.0134548.ref083]\]. Homozygotes for the A(TA)~7~TAA sequence have higher levels of bilirubin whereas bilirubin levels of heterozygotes are between homozygotes and subjects carrying the wild type \[[@pone.0134548.ref083], [@pone.0134548.ref084]\].
While the promoter repeat length varies from 5 to 8 in humans, rhesus macaques and chimpanzees show a reduced repeat length of 3 and 3 to 4, respectively. Based on the shorter promoter repeat length we would predict that UGT1A1 expression is reduced in humans compared with non-human primates. Indeed we find RNA expression to be 46 and 35 percent higher in rhesus macaques and chimpanzees than in humans ([Fig 3](#pone.0134548.g003){ref-type="fig"}. And [S2 Table](#pone.0134548.s008){ref-type="supplementary-material"}). Notably, the *UGT1A1* promoter in the Neandertal and Denisovan genomes ([S1 Table](#pone.0134548.s007){ref-type="supplementary-material"}) carries the sequence A(TA)~6~TAA, common in humans today. Since the Michaelis constant K~m~ of UDP-glycosyltransferase (the substrate concentration at which the reaction rate is half of the maximum rate) lies at bilirubin levels of 0.26 μmol/l \[[@pone.0134548.ref085]\], well below the median bilirubin of 3.4 μmol/l in humans, lower expression of *UGT1A1* may well increase serum bilirubin levels.
Protein sequence alignment of *UGT1A1* revealed one human-specific amino acid substitution at position F518L ([S2 Fig](#pone.0134548.s006){ref-type="supplementary-material"}). Interestingly, this substitution is situated between di-lysine motifs in the cytosolic tail and a transmembrane domain that confer retention of UGT1A-proteins to the endoplasmic reticulum (ER) \[[@pone.0134548.ref059]\]. This amino acid substitution between two ER-retention signals may alter the percentage of protein that stays in the ER and thus the turnover of UGT1A1 independently of expression differences. Further work is necessary to elucidate if and to what extent this protein difference and the promoter difference contribute to the altered bilirubin concentrations in humans.
In the process of heme degradation, mammals reduce water-soluble biliverdin to the potentially toxic \[[@pone.0134548.ref086]--[@pone.0134548.ref091]\] bilirubin in an energy-dependant step \[[@pone.0134548.ref051]\]. Because bilirubin is poorly soluble in water, it is further conjugated with glucuronic acid by UGT1A1 and transported into the bile, both steps requiring additional energy. Whether this particular mode of heme catabolism serves further purposes remains elusive \[[@pone.0134548.ref092]\]. Both *in vitro* \[[@pone.0134548.ref051], [@pone.0134548.ref093]\] and *in vivo* \[[@pone.0134548.ref094], [@pone.0134548.ref095]\] anti-oxidative and neuroprotective \[[@pone.0134548.ref096]\] properties of bilirubin have been known for many years. Clinical studies have shown associations between elevated bilirubin levels and lower incidence of cardiovascular disease \[[@pone.0134548.ref097]\], respiratory disease \[[@pone.0134548.ref098]\], cancer \[[@pone.0134548.ref099]\] and stroke \[[@pone.0134548.ref100]\]. However, it is not clear if this association is due to the inherently higher bilirubin levels or whether more bilirubin is degraded due to the disease \[[@pone.0134548.ref092]\].
Modern human populations derive more of their dietary energy from meat than the great apes (Leonard, Snodgrass et al. 2007). Improved diet quality due to cooking and consumption of animal protein and fat has been proposed as one among other factors allowing the increased brain size in humans \[[@pone.0134548.ref101], [@pone.0134548.ref102]\]. Our results indicate that expression of *UGT1A1* is higher in mice fed a raw plant diet than in mice fed either a meat or cooked-plant diet ([Fig 3](#pone.0134548.g003){ref-type="fig"}), consistent with the role UGT1A1 plays in the detoxification and excretion of plant toxins \[[@pone.0134548.ref060]\]. We hypothesise that the consumption of cooked food in the last million years of hominin history may either have reduced the selective constraint on sufficiently high levels of UGT1A1 expression or even driven reduction in the levels of UGT1A1 expression. The higher level of bilirubin may be a side-effect of the change in UGT1A1 expression, but may also have come under selection on its own right due to the beneficial effects associated with higher levels of bilirubin. The change in bilirubin may thus constitute an example of the wide-ranging consequences of cooking in the evolution of humans.
Supporting Information {#sec015}
======================
###### Alignment scores.
Alignment scores were derived from the NCBI and UniProt websites.
(XLSX)
######
Click here for additional data file.
###### Influence of sex on biomarker measurements.
(XLSX)
######
Click here for additional data file.
###### Influence of environment on biomarker measurements using wild- vs. captive-born great apes.
(XLSX)
######
Click here for additional data file.
###### Supplementary dataset to lineage-specific changes in biomarkers in great apes and humans.
(XLSX)
######
Click here for additional data file.
###### Divergence of biomarker levels in various great ape species.
Distributions of p-values in all pairwise comparisons of 8 individuals to 8 individuals between two groups. Brackets on top and bottom show the results of a two-sided Wilcoxon rank test between distributions that are informative for environmental effect (zoo vs. wild compared to either zoo vs. zoo or wild vs. wild).
(DOCX)
######
Click here for additional data file.
###### Alignment of UDP-glucuronosyltransferase (UGT) 1A1 protein in humans and non-human primates.
Red circle indicating human-specific amino acid substitution (F518L); sequences derived from \[[@pone.0134548.ref056], [@pone.0134548.ref106]--[@pone.0134548.ref108]\]; descriptions of protein folding and functional domains \[[@pone.0134548.ref059], [@pone.0134548.ref109]--[@pone.0134548.ref111]\].
(DOCX)
######
Click here for additional data file.
###### Variability of TA repeats in TATA box of UDP-glucuronosyltransferase (UGT) 1A1 promoter in archaic hominins, humans and non-human primates.
Sequences derived from \[[@pone.0134548.ref056]--[@pone.0134548.ref058], [@pone.0134548.ref082], [@pone.0134548.ref084], [@pone.0134548.ref105], [@pone.0134548.ref112], [@pone.0134548.ref113]\].
(DOCX)
######
Click here for additional data file.
###### Expression data of genes analyzed in this study.
Data derived from \[[@pone.0134548.ref047], [@pone.0134548.ref114], [@pone.0134548.ref115]\].
(DOCX)
######
Click here for additional data file.
We thank W. Enard for helpful discussions; A. Bernhard and the staff of Zoo Leipzig (Leipzig, Germany), the German Primate Center (Göttingen, Germany), the Tacugama Chimpanzee Sanctuary (Freetown, Sierra Leone), Ngamba Island Chimpanzee Sanctuary (Uganda), the Tchimpounga Chimpanzee Rehabilitation Center (Pointe-Noire, Republic of Congo) and the Lola Ya Bonobo sanctuary (Kinshasa, Democratic Republic of Congo) for providing samples and Konrad Schmidt at the Department of Medical Genetics, Molecular and Clinical Pharmacology, Divisions of Genetic Epidemiology and Human Genetics of the Innsbruck Medical University (Innsbruck/Austria) for the analysis of lipoprotein(a).
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: CR JT SP KP JK. Performed the experiments: CR MD MS CH UC JT SP KP JK. Analyzed the data: CR MD MS CH UC JT SP KP JK. Contributed reagents/materials/analysis tools: MH AF MB CA RA LM. Wrote the paper: CR SP KP JK.
| {
"pile_set_name": "PubMed Central"
} |
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Introduction {#s1}
============
Ca^2+^-activated K^+^ channels (SK and BK channels, collectively K~Ca~) are known to play various roles that involve repolarisation of cell membranes, including the regulation of firing rates in central neurons, of smooth muscle tone, and of synaptic transmission [@pone.0107073-Berkefeld1]. They have been described in a variety of other cell types, including dorsal-root ganglion cells [@pone.0107073-Scholz1], [@pone.0107073-Mongan1], though there are conflicting reports about the possible occurrence of K~Ca~ channels in sensory terminals of low-threshold mechanoreceptors, in particular those of the mammalian muscle spindle [@pone.0107073-Hunt1], [@pone.0107073-Kruse1]. Our own interest in this possibility arose from our work on the small (50 nm), clear vesicles present in mammalian mechanosensory terminals [@pone.0107073-Bewick1], [@pone.0107073-Banks1]. Despite wide variation in form, associated accessory cells, and function of the terminals, all of them seem to possess a population of the vesicles [@pone.0107073-Zelen1], indicating the existence of an important common underlying mechanism. The vesicles share many properties with those of presynaptic terminals, but as the sensory terminals are emphatically not synaptic we refer to the vesicles as synaptic-like (SLV). Using sensory endings of rat muscle spindles as a model of the role of SLVs we have presented evidence that they are involved in autogenic modulation of sensory-ending excitability, mediated by glutamate released from SLVs during their recycling [@pone.0107073-Bewick1]. This presynaptic similarity of mechanosensory endings prompted us to investigate Ca-dependent mechanisms that might regulate SLV recycling, and/or afferent firing.
As with the similar vesicles in presynaptic terminals, fusion of SLVs with the sensory terminal membrane is Ca^2+^-dependent, and blocking Ca^2+^ influx with inorganic ions (Co^2+^ or Ni^2+^/Cd^2+^) severely inhibits or abolishes the sensory response in muscle spindles [@pone.0107073-Bewick1]. More specific blocking of P/Q-type channels with ω-agatoxin IVA or ω-conotoxin MVIIC powerfully increased firing rates (2--3 fold approximately) in response to stretch. A similar effect was produced if either BK or SK channels were blocked with charybdotoxin, iberiotoxin or apamin [@pone.0107073-Simon1], [@pone.0107073-Simon2].
Here we investigate the expression of SK1-3 in sensory terminals of muscle spindles and in lanceolate endings of hair follicles using immunocytochemistry. The synaptic vesicle protein synaptophysin (SYN) was used as a marker of sensory terminals, which show strong immunoreactivity to SYN presumably because of their SLV content. In addition, as a further marker of the sensory terminals, we also examined the location of immunoreactivity to the candidate mechanotransducer channel component ASIC2, also known as BNaC1. We have previously found immunoreactivity to ASIC2 in the sensory endings of spindles, where it colocalises with that to SYN [@pone.0107073-Simon3]; and immunoreactivity to the BNaC1α isoform has been reported in cutaneous mechanoreceptor endings, including the lanceolate (or palisade) ending of the hair follicle [@pone.0107073-GarcaAoveros1]. In the lanceolate ending, unlike the spindle primary ending, individual terminals are closely invested by satellite glial cells (SGCs) in an association so intimate that the SGCs may usefully be considered to be a non-neural component of the ending. We used immunoreactivity to the Ca^2+^-binding protein S100, often regarded as a marker of glial cells [@pone.0107073-Rice1], to identify SGCs.
SK1 expression has recently been reported in the dorsal-root ganglion cells and sensory receptor cells of the special sense organs in zebrafish (*Danio*), though post-cranial sensory endings were not described [@pone.0107073-Cabo1]. We did not detect SK1 in either muscle-spindle or lanceolate endings, apart from some unidentified axon-like structures, whereas immunoreactivity to SK2 was present in both spindle terminals and lanceolate endings. Immunoreactivity to SK3 was found in the lanceolate endings in a pattern consistent with a predominantly, or exclusively, SGC expression.
Materials and Methods {#s2}
=====================
Ethics Statement {#s2a}
----------------
All procedures were carried out in accordance with UK legislation: Animals (Scientific Procedures) Act, 1986 and all possible care was taken to ameliorate suffering. The study was carried out under UK Home Office Project Licence no. PPL 60/3963, with the approval of Durham University's Life Sciences Ethical Review Process Committee, granted 2010.
Tissue Preparation and Imaging {#s2b}
------------------------------
Adult rats (2) were deeply anaesthetized with sodium pentobarbitone (45 mg kg^−1^, I.P.) and fixed by transcardial perfusion with 4% (w/v) formaldehyde (from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.4. Tissue samples, taken from the pinnae and from a spindle-rich region of the deep masseter muscles of each animal, were cryoprotected by immersion in 30% sucrose in 0.1 M phosphate buffer overnight, before being placed in moulds containing Tissue-Tek and rapidly frozen in isopentane cooled to −160°C with liquid N~2~. 10 µm thick cryosections were cut using a Leica CM 1850 UV cryostat. Sections of skin from the inner surfaces of the pinnae were double labelled with one of four groups of antibody (Ab) combinations: 1) anti-SK channel (1.25--5 µg/ml, goat or rabbit polyclonals, Santa Cruz Biotechnology/Alomone Laboratories) + anti-SYN (1 µg/ml, mouse monoclonal, Millipore); 2) anti-SK + anti-S100 (1∶400, mouse monoclonal, Santa Cruz Biotechnology); 3) anti-ASIC2 (5 µg/ml, goat polyclonal, Santa Cruz Biotechnology) + anti-SYN; 4) anti-ASIC2+ anti-S100. Sections were incubated with primary antibodies for 48 hrs at 4°C. Secondary antibodies were Alexa Fluor (AF) conjugated antibodies (all at 1∶250 dilution, AF 594 donkey anti-goat and AF 488 donkey anti-mouse or AF 594 goat anti-rabbit and AF 488 goat anti-mouse, Invitrogen). Incubations with secondary antibodies were for 1 hr at ambient temperature (circa 20°C). Sections of muscle were incubated only with combinations 1) and 3) as there are no SGCs associated with spindle sensory endings. Channel antibodies pre-incubated with the peptides to which they had been raised were used as controls. Sections were viewed and optically sectioned with a Leica SP5 Confocal Laser Scanning Microscope, using a x63 NA 1.4 objective. Serial confocal planes (Z-stacks) used for movies, animations and reconstructions were 0.5 µm apart. A diagrammatic survey of the key structural components of the primary ending and the lanceolate ending in relation to planes of imaging is given in [Figure 1](#pone-0107073-g001){ref-type="fig"}.
{#pone-0107073-g001}
Colocalisation Statistical Analysis {#s2c}
-----------------------------------
To assess the colocalisation of SK and ASIC2 immunoreactivity with either anti-S100 or anti-SYN labelling, we calculated Manders' correlation coefficients (M~x~, M~y~) [@pone.0107073-Manders1], with automatic thresholding, for pairs of images of single confocal planes, using Volocity software (Perkin-Elmer, Cambridge, UK; automatic thresholding is implemented according to the protocol of Costes et al. [@pone.0107073-Costes1], in Volocity). Regions of interest (ROIs) were drawn freehand to include sensory endings or, where appropriate, SGC bodies and to exclude most of the unlabelled background so that thresholds were not unduly biased by pixels with 0, or close to 0, intensity. In the present data, M~x~ is always the summed intensities in the green channel (AF 488) for pixels above threshold in both channels, as a proportion of the total intensities for all pixels in the green channel; and M~y~ is the equivalent for the red channel (AF 594). To clarify the presentation of the quantitative data in the subsequent results, M~x~ and M~y~ are further specified according to the immunoreactivities being compared, e.g. M~x~ = M~SK2/SYN~ signifies the proportion of total intensities of thresholded pixels immunopositive for SYN that are also immunopositive for SK2, and the complementary M~y~ = M~SYN/SK2~ signifies the proportion of total intensities of thresholded pixels immunopositive for SK2 that are also immunopositive for SYN. Merged images were created using ImageJ (<http://imagej.nih.gov/ij> National Institutes of Health, Bethesda, MD). Calculation of Manders' coefficients effectively normalises fluorescent intensities, so reducing variability arising from differences in absolute intensities from ending to ending and preparation to preparation. The coefficients can take values between 0 and 1, and are usually not distributed normally. We therefore made statistical comparisons using Mann-Whitney *U*. Sample sizes (n values) refer to numbers of sensory endings.
Colocalisation Visualisation and 3D Surface Rendering {#s2d}
-----------------------------------------------------
Interpretation of the results of immunolabelling in the lanceolate endings of hair follicles is more complicated than in the sensory endings of muscle spindles, due to the smaller size of the lanceolate's sensory terminals and their close, parallel association with thin processes of SGCs, the distance between the opposed membranes being well below the resolution of the light microscope [@pone.0107073-AndresK1]. Care was needed, therefore, to avoid the possibility of misinterpreting proximity for colocalisation, which is a particular problem with longitudinal sections ([Fig. 1F](#pone-0107073-g001){ref-type="fig"}). In order to minimise this problem, data used in the quantitative analysis of [Table 1](#pone-0107073-t001){ref-type="table"} were taken only from image planes close to transverse with respect to the sensory terminals. Longitudinal and oblique sections were used exclusively for qualitative observations. Qualitative interpretation was further facilitated in more complex sections by viewing Z-stacks of images sequentially in a movie, making it much easier to follow particular structures in 3 dimensions. Some of these were subsequently converted to surface-rendered 3D objects using Imaris software (Imaris 7.7 with colocalisation; Bitplane, Belfast, UK) to investigate spatial relationships between labels further and visualise the colocalisation. Examples of both these treatments are appended as supplementary files. The validity of our interpretation of colocalisation is illustrated by the lack of colocalisation volume when sections were double labelled with anti-SYN (terminals) and anti-S100 (glia), and 3D surface-rendering found no colocalisation volumes present ([Movies S1](#pone.0107073.s001){ref-type="supplementary-material"} and [S2](#pone.0107073.s002){ref-type="supplementary-material"}).
10.1371/journal.pone.0107073.t001
###### Colocalisation analysis using Manders' coefficients for double-labelled immunofluorescence in muscle spindles and lanceolate endings.
{#pone-0107073-t001-1}
muscle spindles
------------------------ ---------- ----------------------- ------- -------- ----------------- ---------------------- ------ --------- -------------- ------------------- ------ --------
green SYN M~X~ = M~SK2/SYN~ 0.74 8
red SK2 M~Y~ = M~SYN/SK2~ 0.88
green SYN M~X~ = M~SK3/SYN~ 0.01
red SK3 M~Y~ = M~SYN/SK3~ 0.47 2
green SYN M~X~ = M~ASIC2/SYN~ 0.64
red ASIC2 M~Y~ = M~SYN/ASIC2~ 0.78 10
**lanceolate endings**
**data** **statistical tests**
**channel** **Ab** **coloc coeff** **n** **Ab** **coloc coeff** **n** ***U*** ***P***
green SYN M~X~ = M~SK2/SYN~ 0.90 S100 M~X~ = M~SK2/S100~ 0.81 M~SK2/SYN~ *v* M~SK2/S100~ 61 \<0.05
red SK2 M~Y~ = M~SYN/SK2~ 0.81 15 SK2 M~Y~ = M~S100/SK2~ 0.96 16 M~SYN/SK2~ *v* M~S100/SK2~ 27.5 \<0.01
green SYN M~X~ = M~SK3/SYN~ 0.33 S100 M~X~ = M~SK3/S100~ 0.42 M~SK3/SYN~ *v* M~SK3/S100~ 63.5 ns
red SK3 M~Y~ = M~SYN/SK3~ 0.63 11 SK3 M~Y~ = M~S100/SK3~ 0.88 15 M~SYN/SK3~ *v* M~S100/SK3~ 12 \<0.01
green SYN M~X~ = M~ASIC2/SYN~ 0.66 S100 M~X~ = M~ASIC2/S100~ 0.48 M~ASIC2/SYN~ *v* M~ASIC2/S100~ 98 ns
red ASIC2 M~Y~ = M~SYN/ASIC2~ 0.67 20 ASIC2 M~Y~ = M~S100/ASIC2~ 0.79 15 M~SYN/ASIC2~ *v* M~S100/ASIC2~ 95 ns
Abbreviations: Ab, antibody; coloc coeff, colocalisation coefficient; M~X~, M~Y~, Manders' coefficients; SYN, synaptophysin.
Results {#s3}
=======
SK2 {#s3a}
---
We begin with SK2 as we found anti-SK2 immunoreactivity in the terminals both of sensory endings of muscle spindles and lanceolate endings of hair follicles. In addition, anti-SK2 immunoreactivity was present in lanceolate-ending SGCs. Control sections incubated with anti-channel antibodies pretreated with the respective antigenic peptides showed no immunofluorescence.
### SK2 immunoreactivity in muscle spindles {#s3a1}
SK2 immunoreactivity was detected in sensory terminals and other, less well defined structures, probably inner capsule cells ([Fig. 2A--C](#pone-0107073-g002){ref-type="fig"}). Pre-terminal axons are rarely included in 10 µm thick sections of muscle spindles, owing to their very restricted distribution. Nevertheless, in one section, SK2 immunoreactivity was present in what had the structural characteristics of a pre-terminal axon ([Fig. 2D](#pone-0107073-g002){ref-type="fig"}). Sensory terminals were identified by their size, shape and positive immunoreactivity for SYN. Manders' coefficients (SYN, green channel; SK2, red channel) were both high (mean M~x~ = M~SK2/SYN~ = 0.74; mean M~y~ = M~SYN/SK2~ = 0.88, n = 8) for ROIs drawn as single envelopes enclosing all recognisable sensory terminals in each section, indicating strong colocalisation of SK2 and SYN in the terminals. For comparison, we also carried out double labelling with antibodies against SYN (green channel) and ASIC2 (red channel). We have previously described ASIC2 immunoreactivity in the spindle sensory terminals, as a putative component of the mechanotransduction channel [@pone.0107073-Simon3]. The SYN/SK2 immunoreactivity colocalisation of ROIs enclosing sensory terminals was at least as strong as that for SYN/ASIC2 (mean M~x~ = M~ASIC2/SYN~ = 0.64; mean M~y~ = M~SYN/ASIC2~ = 0.78, n = 10).
{#pone-0107073-g002}
### SK2 immunoreactivity associated with hair follicle sensory innervation {#s3a2}
Examination of pairs of images taken in the red (SK2) and green (SYN or S100) channels, as well as corresponding merged images, showed at least partial colocalisation of SK2 immunoreactivity with both SYN and S100 immunoreactivity in sections passing through the palisade-like ring of sensory terminals and SGC processes of lanceolate endings ([Fig. 3](#pone-0107073-g003){ref-type="fig"}). Manders' coefficients were: for SYN/SK2 (mean M~x~ = M~SK2/SYN~ = 0.90; mean M~y~ = M~SYN/SK2~ = 0.81, n = 15); and for S100/SK2 (mean M~x~ = M~SK2/S100~ = 0.81; mean M~y~ = M~S100/SK2~ = 0.96, n = 16). Corresponding pairs of coefficients differed significantly (M~x~) or highly significantly (M~y~) ([Table 1](#pone-0107073-t001){ref-type="table"}). Collectively, these results indicate that SK2 is expressed in both the sensory terminals and the SGCs. Confirmation was provided by sections passing through the bulb regions of hair follicles, where SGCs and their initial processes could be seen, prior to their association with sensory terminals or axons. Thus, SK2 immunoreactivity was present in the cell bodies (cytoplasm) and processes of SGCs as well as in axons and their terminals, whereas SYN immunoreactivity was present only in the axons and terminals ([Fig. 4](#pone-0107073-g004){ref-type="fig"} and [Movies S3](#pone.0107073.s003){ref-type="supplementary-material"},[S4](#pone.0107073.s004){ref-type="supplementary-material"}).
{#pone-0107073-g003}
{#pone-0107073-g004}
In addition we sometimes found SK2 immunoreactivity associated with hair follicles, but outside the lanceolate endings themselves (as defined by the sensory terminals and SGCs). In individual sections it was usually not possible to tell whether this was associated with pre-terminal axons of the lanceolate endings, though in some cases the structures involved appeared to be portions of circumferential endings ([Fig. 5](#pone-0107073-g005){ref-type="fig"}). SK2 immunoreactivity was also shown by cells of the sebaceous glands and non-neural cells at the base of the follicles, whereas the inner and outer sheath cells did not show reactivity.
{#pone-0107073-g005}
SK3 {#s3b}
---
### SK3 in muscle spindles {#s3b1}
We did not detect immunoreactivity to SK3 in the terminals of muscle spindle sensory endings. However, we cannot rule out the possibility that SK3 is present in the pre-terminal axonal branches, in view of the difficulty of finding them in our sections, as described above.
### SK3 associated with hair follicle sensory innervation {#s3b2}
Although we found no evidence for SK3 in spindles, immunoreactivity to SK3 was found in restricted locations in lanceolate endings. Double labelling demonstrated that SK3 immunoreactivity occurred in association with both SYN and S100 immunoreactivities, but that it was only partiallycolocalised with either. Rather, it seemed to be localised at or near the interface between terminals (represented by SYN) and SGC processes (represented by S100) ([Fig. 6](#pone-0107073-g006){ref-type="fig"} and [Movie S5](#pone.0107073.s005){ref-type="supplementary-material"}). Manders' coefficients were: for SYN/SK3 (mean M~x~ = M~SK3/SYN~ = 0.33; mean M~y~ = M~SYN/SK3~ = 0.63, n = 11); and for S100/SK3 (mean M~x~ = M~SK3/S100~ = 0.42; mean M~y~ = M~S100/SK3~ = 0.88, n = 15). There was no significant difference in a statistical comparison of M~SK3/SYN~ and M~SK3/S100~, whereas M~SYN/SK3~ and M~S100/SK3~ differed highly significantly ([Table 1](#pone-0107073-t001){ref-type="table"}). SK3 immunoreactivity was detectable in the cell bodies and processes of some SGCs; however, not all S100 immunoreactive profiles also showed SK3 reactivity, even when closely adjacent in the same section ([Fig. 7](#pone-0107073-g007){ref-type="fig"} and [Movie S6](#pone.0107073.s006){ref-type="supplementary-material"}). These results are consistent with a restricted expression of SK3 in SGCs, especially where their processes are closely adjacent to sensory terminals. It is also possible that not all SGCs express SK3, and we cannot exclude the possibility, on the present evidence, of limited expression in the sensory terminals.
{#pone-0107073-g006}
{#pone-0107073-g007}
### ASIC2 colocalisation pattern for comparison with SK channels {#s3b3}
ASIC2 immunoreactivity showed a similarly very restricted distribution in lanceolate endings to that of SK3, and also relatively low colocalisation with both SYN and S100 immunoreactivities ([Fig. 8](#pone-0107073-g008){ref-type="fig"} and [Movie S7](#pone.0107073.s007){ref-type="supplementary-material"}). Manders' coefficients were: for SYN/ASIC2 (mean M~x~ = M~ASIC2/SYN~ = 0.66; mean M~y~ = M~SYN/ASIC2~ = 0.67, n = 20); and for S100/ASIC2 (mean M~x~ = M~ASIC2/S100~ = 0.48; mean M~y~ = M~S100/ASIC2~ = 0.79, n = 15). Neither corresponding pair of coefficients differed significantly (M~x~ nor M~y~) ([Table 1](#pone-0107073-t001){ref-type="table"}). In this case, however, there was no evidence of ASIC2 expression in SGC cell bodies or their processes, except where sensory terminals were also likely to be present. Conversely, there is clear evidence that ASIC2 (BNaC1α) is expressed in the sensory axons and terminals [@pone.0107073-GarcaAoveros1] and the restricted distribution in relation to SYN immunoreactivity in our own results is consistent with ASIC2 being localised especially at the sensory terminal membrane, in particular, the regions where it is known to be exposed. That is, at the edges of the long axis of the lanceolate terminal blade closest to, and furthest from, the hair shaft where there are gaps between the SGC processes [@pone.0107073-AndresK1].
{#pone-0107073-g008}
SK1 {#s3c}
---
We did not detect immunoreactivity to SK1 in the terminals either of sensory endings in muscle spindles or of lanceolate endings; however, there was some specific labelling with anti-SK1 antibody in unidentified axon-like structures, apparently located immediately beneath the lanceolate endings around some hair follicles ([Fig. 9](#pone-0107073-g009){ref-type="fig"}).
{#pone-0107073-g009}
Discussion {#s4}
==========
Opening the black box -- low-threshold sensory mechanotransduction in mammals {#s4a}
-----------------------------------------------------------------------------
Despite their different locations, structural organisations and associations with very different accessory cells, the sensory endings of muscle spindles and the lanceolate endings of hair follicles share some fundamentally important properties in common, together with other cutaneous, joint and muscle afferents, and some visceral afferents such as baroreceptors [@pone.0107073-Delmas1]. Thus, all of these are low-threshold mechanoreceptors, responding to low force stimuli, often of minute amplitude; and all are formed of the peripheral sensory terminals of primary afferent axons whose cell bodies are located in the dorsal-root or cranial nerve ganglia. They also share in common the fact that the molecular basis of their mechanosensitivity is unknown. In some cases, such as the muscle spindle, classical neurophysiology has provided us with very detailed input:output (I/O) properties where the inputs are well-defined and precisely controlled mechanical stimuli, and the outputs are the resulting spike trains in the afferent axons (see, for example, [@pone.0107073-Hulliger1]). In such experiments the overall process of mechanosensory transduction is treated as a black box, whose transfer function can, at least in principle, be determined from the I/O properties alone (see, for example, [@pone.0107073-Poppele1]). This treatment is very useful in bioengineering, but it is clearly unsatisfactory for our understanding of a fundamentally important biophysical process. For that we need to see inside the 'black box'.
Much of our recent work has centred on the role of a glutamatergic system mediated by SLVs in mechanosensory terminals [@pone.0107073-Bewick1], [@pone.0107073-Banks1]. The rate of SLV turnover is activity dependent, and experimental manipulation of the system alters the sensory ending's I/O properties, at least of the muscle spindle, so it is feasible that the system is part of an automatic gain control of the mechanosensory black box, operating with a time course of seconds to minutes. We encountered the importance of Ca^2+^-activated K^+^ channels, both SK and BK, in the course of investigating the possible role of voltage-gated Ca^2+^ channels in SLV recycling in muscle spindles [@pone.0107073-Simon1]. SLV recycling is a Ca^2+^-dependent process, so we were surprised initially to find that blocking P/Q Ca^2+^ channels enhanced rather than inhibited muscle-spindle sensory responses to stretch; however, as P/Q channels are frequently associated with K~Ca~ channels, we also tried blocking SK and BK channels and found that blocking either or both types produced similar effects to P/Q blockage [@pone.0107073-Simon2]. It might be thought likely that these channels would be localised especially at sites in the sensory endings where action potentials are generated and where they could be particularly effective in regulating the firing rate. Heminodes, of which there may be several in any one sensory ending and which are located in preterminal branches, are thought to be the most important such sites, but they were not amenable to study in the present work where we have concentrated on the sensory terminals and associated accessory cells. We have carried out preliminary observations on the immunohistochemistry of BK, which indicate that it is not present in the sensory terminals of either muscle spindles or lanceolate endings (FCS,RWB), but we have yet to investigate whether immunoreactivity to BK or SK can be detected at heminodes.
SK2 in sensory terminals {#s4b}
------------------------
In the light of the previous consideration, our clear evidence for SK2 expression throughout the sensory terminals of both muscle spindles and lanceolate endings is all the more remarkable. In the very large terminals of muscle spindles much of the immunoreactivity was found within the terminals, though it was not completely colocalised with SYN, the SLV marker. Any SK2 localised internally is presumably sequestered in a reserve pool, as its functional site must surely be the sensory terminal membrane. The much smaller terminals of the lanceolate endings do not allow us to decide whether or not SK2 is similarly localised in part within the terminals as well as in their membranes, but the higher value of Manders' coefficient M~x~ = M~SK2/SYN~ in lanceolate (0.90) as compared to muscle spindle endings (0.74) is at least consistent with this. Conversely, M~y~ = M~SYN/SK2~ is somewhat higher for muscle spindles (0.88) than for lanceolate endings (0.81), which might reflect the additional expression of SK2 in the SGCs that are a prominent feature of lanceolate endings, but are absent from muscle spindles.
The sensory terminals are, of course, thought to be the sites where receptor potentials are produced in response to the mechanical deformation of the terminals resulting in gating of stretch-activated channels in their membranes. Hunt et al. (1978) [@pone.0107073-Hunt1] succeeded in recording muscle-spindle receptor potentials from the parent axons, rather than the inaccessible terminals, by blocking action potentials with tetrodotoxin. They were able to attribute certain features of the receptor potential to K^+^ influx, but they also showed that removal of external Ca^2+^ had no effect on these features and presumably, therefore, the K^+^ currents responsible were not due to Ca^2+^-activated channels. This is a very important observation in the context of our results, as it suggests that the SK2 channels of the sensory terminals may require much greater, transient, depolarisations to invade the terminals by backfiring action potentials in order to activate P/Q channels associated with them, and that the role of the P/Q-SK2 channel complex within the terminals may therefore concern registration of the fact of action potential firing rather than modulation of the receptor potential itself. In contrast to the results of Hunt et al. (1978), Kruse and Poppele (1991) [@pone.0107073-Kruse1] found very clear evidence for a role for K~Ca~ channels in the mid-frequency dynamics of the muscle-spindle primary ending's response to small-amplitude sinusoidal stretch, which they modelled as a negative feedback pathway within the process of receptor potential generation. What is perhaps particularly significant is that Kruse and Poppele (1991) had not blocked impulse activity and were, in fact, recording the responses as impulse activity from the parent afferent nerve fibre. Our finding of SK2 immunoreactivity in the sensory terminals may therefore be seen as providing further support to the idea of such a feedback pathway.
SK2 and SK3 in satellite glial cells {#s4c}
------------------------------------
In lanceolate endings spatial resolution with immunofluorescence was rarely sufficient to separate clearly the immunoreactivity of the sensory terminals and their associated SGC processes, even in single confocal planes. The quantitative data analysis must therefore be interpreted using additional qualitative information, provided by examination of SGC bodies and sensory axons in the region of the hair follicles deep to the palisade endings. At this level, at least, we could be sure that immunoreactivity to both SK2 and SK3 was present in SGCs, but in sensory axons only that to SK2 was present. Consistent with such a pattern of expression, at the level of the sensory terminals, we found Manders' coefficients to be generally high for SK2 colocalisation with both SYN and S100 (all 4 values of M~x~ and M~y~ ranged between 0.81 and 0.96), but only M~y~ = M~S100/SK3~ was high (0.88) for SK3 colocalising with S100, implying that almost all the SK3 immunoreactivity colocalised with that of S100. The converse, however, was not true (M~x~ = M~SK3/S100~ = 0.42). Together with the low, quantitative, colocalisation of SK3 with SYN (M~x~ = M~SK3/SYN~ = 0.33, M~y~ = M~SYN/SK3~ = 0.63), this reflects the very restricted expression of SK3 apparently close to the interface between the sensory terminals and the SGC processes.
Individual sensory terminals of the lanceolate ending are very closely invested by processes of SGCs, and are otherwise exposed only on their inner and outer edges at narrow gaps between pairs of SGC processes. The SGCs are therefore well placed to influence the flow of any receptor current generated by the sensory terminals, in addition to perhaps playing a role in transmission of mechanical stimuli to the terminals. Our demonstration of the presence of SK2 and SK3 in SGCs, in particular their processes, suggests that one possible contribution they may make is the regulation of \[K^+^\] in the extraterminal space.
Our findings concerning the expression of SK3 in SGCs are consistent with previous reports of its presence in other types of glia, both centrally in astrocytes of the rat supraoptic nucleus [@pone.0107073-Armstrong1], and peripherally in SGCs of the trigeminal ganglion [@pone.0107073-Vit1]. Increases in intracellular \[Ca^2+^\] in other types of glia have been shown to regulate neuronal activity [@pone.0107073-Rousse1], but the signal for activation of SK channels in SGCs of the hair follicle are unknown. In lanceolate endings of rat vibrissae, however, Takahashi-Iwanaga et al. (2008) [@pone.0107073-TakahashiIwanaga1] have described a dual system of intercellular Ca^2+^ signalling in SGCs that could conceivably activate SK channels, in response to direct mechanical stimulation of the SGC processes themselves. Mechanical stimulation results in local elevation of intracellular \[Ca^2+^\], the signal then being propagated intercellularly by purine- and gap-junction-mediated pathways, effectively coupling the SGCs into a functional network [@pone.0107073-TakahashiIwanaga1].
Supporting Information {#s5}
======================
Mpg-format movies created using Imaris software (Bitplane , Zurich, Swizerland) from z-stacks of confocal images.
######
**3D surface rendering of image stacks showing the distinct cellular localisations of anti-SYN and anti-S100 labelling.** The original *en face* raw anti-SYN image stack (green) of the hair follicle palisade is followed by addition of the anti-S100 (red) of the glial cell, then any colocalisation volume is rendered in yellow. This composite is then rotated to alternative viewing aspects. Note the absence of any colocalisation volume (yellow), indicating complete cellular segregation, with no synaptophysin labelling in the S100-positive glial cell, or *vice versa*.
(ZIP)
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Click here for additional data file.
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**3D surface rendering of image stacks showing the distinct cellular localisations of anti-SYN and anti-S100 labelling.** The original *en face* raw anti-SYN image stack (green) of the hair follicle palisade is followed by addition of the anti-S100 (red) of the glial cell, then any colocalisation volume is rendered in yellow. This composite is then rotated to alternative viewing aspects. Note the absence of any colocalisation volume (yellow), indicating complete cellular segregation, with no synaptophysin labelling in the S100-positive glial cell, or *vice versa*.
(ZIP)
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Click here for additional data file.
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**3D surface rendering of image stack showing the relationship between anti-SK2 and anti-SYN labelling.** The original *en face* raw SK2 image stack (red) of the almost complete circle of the hair follicle palisade is followed by addition of the lanceolate terminal (synaptophysin, green) and colocalisation volumes (yellow), which is then rotated to alternative viewing aspects. Note the extensive fragment of glial cell body (bottom centre) labelled for SK2, and the circumferential and longitudinal labelling (in the z-axis) in the rest of the follicle. The white framework indicates the total volume of the section that was imaged. Solid surface rendering is then applied to the SYN, SK2 then colocalisation volumes. The original image volume and the frame are then removed. Zooming in on the lanceolate terminals (green) shows they are predominantly enclosed by a larger (glial) volume labelled for SK2. However, transparency of the red and green channels allows the extensive labelling of SK2 within the terminals (yellow) to be seen. A lower power view then confirms the SK2 labelling is within the terminals, not just overlapping labels directly in line sight from a single view point, as the non-colocalised SK2 and SYN labels are progressively peeled away, leaving only the surface rendered colocalisation volume, before being reinstated.
(ZIP)
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Click here for additional data file.
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**3D surface rendering of image stack showing another example of colocalisation of anti-SK2 with anti-SYN labelling in lanceolate terminals.** As for [movie S1](#pone.0107073.s001){ref-type="supplementary-material"}, but in an oblique section deeper within the follicle that contains much more extensive glial cell, and less nerve terminal, material.
(ZIP)
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Click here for additional data file.
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**3D surface rendering of image stack showing anti-SK3 (red) labelling is very predominantly in the glial cell processes, and only colocalises (yellow) with the terminal anti-SYN (green) labelling at the interface between the lanceolate endings and their enclosing glial cell processes.** Note in this follicle, sectioned at a more superficial level of the skin, there are no cell bodies but the glial cell processes are clearly interpolated between the lanceolate terminals.
(ZIP)
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Click here for additional data file.
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**Confocal optical section stack close to the base of a lanceolate ending, double-labelled with anti-SK3 (red channel) and anti-S100 (green channel) antibodies.** Colocalisation of the immunoreactivities may be seen in some SGCs and their processes, but not others, and may be followed in the 3 dimensions of the image stack. Compare with the endings shown in [Figure 7D--F](#pone-0107073-g007){ref-type="fig"} and [Figure 8](#pone-0107073-g008){ref-type="fig"}, labelled in the same way. (Avi-format movie created using Windows Moviemaker from z-stack of confocal images.).
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Click here for additional data file.
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**3D surface rendering of an image stack to contrast the relationship of anti-ASIC2 and anti-SYN labelling with that for the SK channels above.** Note the extensive ASIC2 labelling (red) in both longitudinal and circumferential elements. While there is substantial anti-ASIC2 labelling in the glial cell processes, it is also found extensively within most of the lanceolate terminals, colocalising with anti-SYN labeling.
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Click here for additional data file.
We thank Mr Gytis Markevicius for help in preparing the 3D surface rendered movies, and Mr Kevin McKenzie for expert technical assistance in image processing.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: FCS GSB RWB. Performed the experiments: FCS. Analyzed the data: FCS GSB RWB. Contributed reagents/materials/analysis tools: GSB RWB. Contributed to the writing of the manuscript: FCS GSB RWB.
[^3]: Current address: Institute for Ageing and Health, Campus for Ageing and Vitality, Newcastle University, United Kingdom
| {
"pile_set_name": "PubMed Central"
} |
Immunotherapy, including anticancer vaccination and adoptive transfer of tumour-specific cytotoxic T lymphocytes, has been considered as a promising treatment for head and neck squamous cell carcinoma (HNSCC), although its effects on tumour regression remain limited ([@bib40]; [@bib24]). The inefficacy of such therapies implies that head and neck carcinoma has developed diverse strategies to evade tumour-specific immunity ([@bib12]).
Studies show that regulatory T (Treg) cells and M2 macrophages are crucial players in immune tolerance ([@bib28]; [@bib19]; [@bib50]; [@bib1]; [@bib29]). Recently, to evoke antitumour immunity *in vivo*, we blocked tumour trafficking of CD45RA^−^Foxp3^high^ activated Treg (aTreg) cells using a CCR4 antagonist, which led to inhibition of tumour growth and prolonged survival ([@bib45], [@bib43], [@bib44]). However, the effect of targeting a single immunosuppressive population is not satisfactory. This prompted us to employ a combined strategy targeting both aTreg cells and M2 macrophages and further investigate their interactions.
In this study, we evaluated the clinical importance of tumour-infiltrating aTreg cells and M2 macrophages in laryngeal squamous cell carcinoma (LSCC) patients and confirmed their positive correlation. We demonstrated that cancer cell-activated M2-like macrophages induced aTreg cells from CD4^+^CD25^−^ T cells *in vitro*. Reciprocally, the generated aTreg cells skewed the differentiation of monocytes towards the M2-like phenotype, forming a positive-feedback loop. Blockade of aTreg cell trafficking combined with depletion of tissue macrophages reduced both intratumoural aTreg cells and M2 macrophages to the maximum extent and significantly enhanced antitumour immunity in experimental mice.
Materials and methods
=====================
Immunohistochemical analysis
----------------------------
The main clinical and pathological characteristics of LSCC patients are presented in [Supplementary Table S1](#sup1){ref-type="supplementary-material"}. Clinical staging and the anatomical site of tumours were assessed according to the sixth edition of the Union for International Cancer Control (UICC 2008) tumour-node-metastasis classification of malignant tumours. Paraffin-embedded, formalin-fixed 4 *μ*m-thick tissue sections were processed for immunohistochemical staining with anti-FoxP3 (Ab22510, IgG1, 1 : 300 dilution, Abcam, Cambridge, MA, USA), anti-CD163 (Ab87099, IgG, 1 : 80 dilution, Abcam) and anti-p16/INK4a (Clone EPR1473, 1 : 250 dilution, Epitomics Inc., Burlingame, CA, USA) antibodies. The densities of FoxP3^+^ or CD163^+^ cells was evaluated quantitatively by the mean count of four representative fields at 400 × magnification by two independent observers who were blinded to the clinical outcome. Nuclear and cytoplasmic staining of \>70% tumour cells was considered p16 positive and believed to correlate with Human papilloma virus (HPV) ([@bib5]).
Cell lines and animals
----------------------
Human LSCC cell lines (SNU46 and SNU899) and a nasopharyngeal squamous cell carcinoma (NPSCC) cell line (HONE1) were kindly provided by Professor Ja-Lok Ku (Seoul National University College of Medicine, South Korea). Tumour culture supernatants (TSNs) were prepared as described previously ([@bib22]). The mouse squamous cell carcinoma (SCC VII) cell line was kindly provided by Professor Si-Xi Liu (West China Hospital, China). Cells were cultured under standard conditions.
Eight-week old male C3H-HeN mice were purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, China). All mice were housed under specific pathogen-free conditions. Experimental procedures were approved by the Institutional Animal Studies Committee and conducted in accordance with Institutional Animal Care and Use Committee guidelines.
Cell preparation
----------------
The study protocol was approved by The Ethics Committee of The First Affiliated Hospital of Sun Yat-Sen University and used for research purposes only (Approval No. 2012-349). Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by Ficoll density gradient centrifugation. CD14^+^ cells were isolated from PBMCs using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with or without 20% TSN for 6 days to obtain macrophages.
To isolate CD4^+^CD25^−^ T cells and Treg subsets, cells were stained with anti-hCD4-FITC, anti-hCD25-APC, and anti-hCD45RA-eFluor 450 (eBioscience, San Diego, CA, USA) antibodies, and sorted using a BD Influx cell sorter (BD Biosciences). Three Treg subsets were prepared as live cells as described previously ([@bib31]). Foxp3^low^CD45RA^+^ cells, which were CD25^++^ (I), Foxp3^high^CD45RA^−^ cells, which were CD25^+++^ (II), and Foxp3^low^CD45RA^−^ cells, which were CD25^++^ (III), were prepared by sorting CD25^++^CD45RA^+^, CD25^+++^CD45RA^−^, and CD25^++^CD45RA^−^ cells, respectively.
Cell co-culture
---------------
Cells were co-cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin, and 10% heat-inactivated foetal calf serum. For Treg cell generation, 1 × 10^6^ autologous macrophages were co-cultured with 2 × 10^6^ CD14^+^-depleted PBMCs in the presence of 0.5 *μ*g ml^−l^ anti-CD3 mAb or with 1 × 10^6^ CD4^+^CD25^−^ T cells in the presence of 2 *μ*g ml^−l^ anti-CD3 mAb plus 1 *μ*g ml^−l^ anti-CD28 mAb for 4 days. For macrophage differentiation, CD14^+^ cells were seeded in a round bottom 96-well plate at 2 × 10^5^ cells per well. CD4^+^CD25^−^ T cells and the three Treg subsets were added to autologous CD14^+^ cells at a ratio of 1 : 2 and co-cultured for 40 h in the presence of 50 ng ml^−l^ anti-CD3 mAb with or without 20% TSN from SNU899 cells.
For neutralisation experiments, neutralising antibodies against TGF-β1 (5 *μ*g ml^−l^, mouse IgG1, clone 9016), IL-10 (5 *μ*g ml^−l^, goat IgG), IL-4 (5 *μ*g ml^−l^, mouse IgG1, clone 3007), or IL-13 (5 *μ*g ml^−l^, mouse IgG1, clone 32116) and isotype controls (mouse IgG1 or goat IgG) were added at the start of the co-culture. All from R&D Systems (Minneapolis, MN, USA).
Flow cytometric analysis
------------------------
To determine the phenotype of macrophages, the following antibodies were used: anti-hCD163-APC, anti-hCD206-Alexa Fluor 488, anti-hHLA-DR-eFluor 450, anti-hCD86-PE, and anti-hCD80-PE-Cy5. To determine the frequency of the three distinct Treg subsets, the following antibodies were used: anti-hCD4-FITC, anti-hCD25-APC, and anti-hCD45RA-eFluor 450 for surface staining; anti-hFoxp3-APC for intracellular staining.
For the intracellular cytokine assay, the sorted CD4^+^CD25^−^ T cells and each Treg subset were stimulated with a cocktail of phorbol 12-myristate 13-acetate (PMA), ionomycin, and Golgi stop (brefeldin A and monensin; eBioscience) for 5 h. Then, we performed intracellular staining using anti-hIL-4-PE-Cy7, anti-hIL-10-PerCP-eFluor 710, anti-hIL-13-PE (Sony Biotechnology, Inc, San Jose, CA, USA), and anti-hIFN-*γ*-APC-eFluor 780 antibodies. All antibodies were purchased from eBioscience unless specified otherwise.
Cytokine detection
------------------
The concentrations of IL-10, IL-6, IL-12p70, TNF-*α*, IL-4, IL-5, IL-13, IL-2, IFN-*γ*, and TGF-β1 in culture supernatants was determined using ELISA kits according to the manufacturer's instructions (IL-2, IFN-*γ*, and TNF-*α* from R&D Systems; others from RayBiotech, Inc, Norcross, GA, USA).
Combined targeting of aTreg cells and M2 macrophages *in vivo*
--------------------------------------------------------------
Twenty mice were randomly and equally divided into four groups and ear tagged prior to treatment. On day 0, 1 × 10^5^ tumour cells were injected subcutaneously into the right flank. A CCR4 antagonist (87.5 *μ*g; TOCRIS, Minneapolis, MN, USA; [@bib44]), GdCl~3~ (0.2 mg; Sigma-Aldrich, Saint Louis, MO, USA; [@bib16]), or CCR4 antagonist+GdCl~3~ were injected intraperitoneally three times per week for 3 weeks from day 5 after tumour cell injection. A group of five mice for the saline control was set in parallel. Tumour size was measured every 4 days using fine calipers. Tumour volumes were calculated as length × (width)^2^ × 0.5. All mice were killed at 24 h after the last injection.
After killing the mice, tumours and spleens were isolated and prepared as single cell suspensions. Mononuclear cells were isolated using a Ficoll density gradient and analysed for aTreg cells, M2 macrophages, CD4^+^ and CD8^+^ T cells, and Th1 cytokines by flow cytometry using anti-mouse CD4-eFluor 450, anti-mouse CD8-Alexa Fluor 700, anti-mouse CD25-APC, anti-mouse CD45RA-FITC (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-mouse CD206-PE-Cy7 (Sony Biotechnology, Inc), anti-mouse F4/80-PE-Cy5.5, anti-mouse FoxP3-PE, anti-mouse IL-2-PE, anti-mouse IFN-*γ*-PE-Cy5.5, and anti-mouse TNF-*α*-PE-Cy7 antibodies, and a fixation/permeabilisation kit. Antibodies were purchased from eBioscience unless specified otherwise. Immunohistochemical staining of CD3 (Ab16669, IgG, 1 : 100 dilution, Abcam) was also performed to assess intratumoural CD3^+^ T cells. Another randomly divided four groups of mice were used for survival analysis. Observation was aborted on day 50.
Statistical analysis
--------------------
All statistical analysws were performed with SPSS for Windows version 13.0 (IBM, Chicago, IL, USA). For Foxp3 and CD163, the cutoff to define the subgroups was the median value. Analysis of the correlation between variables was conducted using Spearman's rank coefficient tests. Survival variables were estimated using the Kaplan--Meier method and compared using log-rank tests. Differences between groups were assessed using the Mann--Whitney *U*-test, Student's *t*-test, or Kruskal--Wallis test. Multivariate analysis using the Cox proportional hazard model was used to determine the influence of each variable, when adjusted to the others, on overall survival. *P*-values of \<0.05 were considered as statistically significant.
Results
=======
Accumulation of aTreg cells and M2 macrophages is positively associated with each other in LSCC tissue and predicts poor survival
---------------------------------------------------------------------------------------------------------------------------------
We first examined the prevalence of tumour-infiltrating aTreg cells and M2 macrophages in 65 patients with LSCC, the common type of HNSCC ([Supplementary Table S1](#sup1){ref-type="supplementary-material"}). Immunohistochemical staining of Foxp3 and CD163 was performed in paraffin-embedded tissues. Both Foxp3^+^ aTreg cells and CD163^+^ macrophages had substantially infiltrated into the stroma, but scarcely into tumour sites ([Figure 1A](#fig1){ref-type="fig"}). To determine the clinical importance of Foxp3+ aTreg cells and CD163+ macrophages in stromal of tumours, we analysed their associations with clinicopathological characteristics and overall survival. The levels of Foxp3^+^ aTreg cell and CD163^+^ macrophage infiltration in patients at late clinical stages (III and IV) were significantly higher than those at early clinical stages (I and II; *P*\<0.01 and *P*\<0.05, respectively; [Figure 1B and C](#fig1){ref-type="fig"}) ([Supplementary Table S2](#sup1){ref-type="supplementary-material"}). Expression of Foxp3 and CD163 increased with the pathological stage, although no significant difference was found in Foxp3 expression ([Figure 1D and E](#fig1){ref-type="fig"}; [Supplementary Table S2](#sup1){ref-type="supplementary-material"}). Patients with high numbers of intratumoural Foxp3^+^ aTreg cells/CD163^+^ macrophages had shorter overall survival (medians: 74/78 months) than those with low numbers of Foxp3^+^ aTreg cells/CD163^+^ macrophages (medians: 94/90 months; [Figure 1F and G](#fig1){ref-type="fig"}). Furthermore, there was a significant difference between the group with high expression of both Foxp3 and CD163 and the group with high expression of either Foxp3 or CD163 and the group with low expression of both Foxp3 and CD163 (medians: 71, 87, and 94 months, respectively; [Figure 1H](#fig1){ref-type="fig"}). These data supported important roles of both aTreg cells and M2 macrophages in accelerating tumour progression and predicting poor survival. In a Cox Multivariate analysis ([Supplementary Table S3](#sup1){ref-type="supplementary-material"}), clinical stage, nodal status, tobacco and alcohol consumption were associated with overall survival. The presence of high numbers of intratumoural Foxp3^+^ aTreg cells in combination with high numbers of intratumoural CD163^+^ macrophages was an independent prognostic marker for overall survival, while the HPV status had no independent prognostic value ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}).
Moreover, Spearman's rank correlation coefficient was calculated to assess the relationship between Foxp3^+^ aTreg cells and CD163^+^ macrophages. The densities of the two markers were positively correlated with each other in LSCC specimens ([Figure 1I](#fig1){ref-type="fig"}). This quantitative relationship encouraged us to further investigate their biological interactions.
Macrophages exposed to tumour culture supernatant from LSCC-derived cell lines exhibit an M2-like phenotype and augment the Foxp3^+^ Treg population in peripheral blood mononuclear cells
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
To investigate whether cancer cells with a distinct epithelial phenotype have a different ability to activate macrophages, we cultured freshly isolated human monocytes in medium with 25% tumour culture supernatant (TSN) from a nasopharyngeal carcinoma cell line (HONE1) or laryngeal carcinoma cell lines (SNU46 or SNU899) for 6 days to obtain macrophages. [Figure 2A](#fig2){ref-type="fig"} shows that, relative to untreated macrophages, TSN-treated macrophages displayed strong up-regulation of typical M2 macrophage markers CD163 and CD206 (data not shown) and reduced expression of HLA-DR, CD86, and CD80, which are associated with immunosuppression. They also produced higher amounts of an M2 macrophage cytokine (interleukin (IL)-10) and lower levels of M1 macrophage cytokines (tumour necrosis factor-*α* (TNF-*α*), IL-6, and IL-12p70) ([Figure 2B](#fig2){ref-type="fig"}), indicating that macrophages were activated to an 'M2-like state' by TSN. Moreover, the level of activation by TSN from SNU899 cells, as measured by cytokine production and surface marker expression of M2 macrophages, was the highest among all groups, suggesting that SNU899-derived TSN was a strong macrophage activator.
To determine whether these activated macrophages induced Foxp3^+^ Treg cells, they were co-cultured with autologous peripheral blood mononuclear cells (PBMCs). Consistent with the changes in surface markers and cytokines, more Foxp3^+^ Treg cells were derived from macrophages treated with TSN from SNU899 (Mφ~SNU899~: 82.0±4.7%) than untreated macrophages (Mφ: 36.5±2.9%) or those treated with TSNs from other HNSCC cell lines (Mφ~SNU46~: 68.2±3.2% Mφ~HNOE1~: 64.0±3.4% [Figure 2C](#fig2){ref-type="fig"}). In addition, to investigate the overall effect of macrophages on the immune response, we examined cytokines in the co-cultures by immunosorbent assays (ELISAs). The levels of Th2 cytokines (IL-5 and IL-13) were correspondingly increased in the culture supernatants ([Figure 2D](#fig2){ref-type="fig"}), while the production of Th1 cytokines (interferon-*γ* (IFN-*γ*), IL-2, and TNF-*α*) was suppressed to undetectable levels (data not shown), implying that the TSN-exposed macrophages mediated a tumour-promoting effect.
Taken together, these results indicate that macrophages activated by TSN from LSCC cell lines have an M2-like phenotype and may serve as an important initiator of Treg cell induction.
TSN-exposed M2-like macrophages induce an aTreg cell subset from CD4^+^CD25^−^ T cells
--------------------------------------------------------------------------------------
To eliminate the possibility that TSN-exposed macrophages were merely expanding the pre-existing naturally occurring Treg cells present at the initiation of culture, we sorted autologous CD4^+^CD25^−^ T cells as responder cells before adding macrophages (*n*=3). A clear conversion from CD4^+^CD25^−^ Foxp3^−^ T cells to CD4^+^CD25^+^ Foxp3^+^ Treg cells was observed by flow cytometric analysis ([Figure 3Aa and Ab](#fig3){ref-type="fig"}). We further identified the functionally distinct subsets of these newly generated Treg cells ([Figure 3Ac](#fig3){ref-type="fig"}), because Treg cells with a phenotype of CD45RA^+^Foxp3^low^ or CD45RA^−^Foxp3^high^ can exhibit immunosuppressive functions ([@bib31]). We found a dominant increase in CD45RA^−^Foxp3^high^ aTreg cells of the suppressive Treg subsets ([Figure 3B and C](#fig3){ref-type="fig"}), indicating that the Treg cells induced by M2-like macrophages may directly mediate an inhibitory effect on tumour immunity.
To determine the nature of the soluble factors involved in the induction of aTreg cells, neutralising antibodies against transforming growth factor (TGF)-β1, IL-10, or TGF-β1/ IL-10 were added to the cocultures. Our results showed that neutralising IL-10 partially attenuated the typical increase in aTreg cell frequency resulting from co-culture of Mφ~SNU899~ with CD4^+^CD25^−^ T cells, whereas blocking TGF-β1 had no significant effect ([Figure 3D and E](#fig3){ref-type="fig"}). Therefore, IL-10 attributed to Mφ~SNU899~ was involved in the induction of aTreg cells from CD4^+^CD25^−^ T cells.
aTreg cells skew the differentiation of monocytes toward M2 macrophages in the TSN
----------------------------------------------------------------------------------
To investigate whether the Mφ~SNU899~-induced Treg subsets skew the differentiation of monocytes to M2-like macrophages, CD14^+^ monocytes were cultured alone, with CD4^+^CD25^−^ T cells, or with each sorted Treg subset (*n*=5). An anti-CD3 monoclonal antibody (mAb) was present in all conditions to stimulate the T cells. After 40 h, cells were collected for flow cytometry. Interestingly, compared with control monocytes, monocytes co-cultured with aTreg cells, but not CD45RA^+^Foxp3^low^CD4^+^ resting Treg (rTreg) cells or CD45RA^−^Foxp3^low^CD4^+^ T cells, displayed strong up-regulation of the typical M2 macrophage marker CD163. In addition, reduced expression of HLA-DR, CD86, and CD80 was observed ([Figure 4A](#fig4){ref-type="fig"}). The differences in the modulation of monocyte/macrophage functions may lie in the distinct cytokine patterns of these Treg subsets. Of note is that the aTreg subset was the highest producer of IL-10, CD45RA^−^Foxp3^low^CD4^+^ T cells produced high amounts of IL-4, IL-13, and IFN-*γ*, while the rTreg subset was a poor producer of these cytokines ([Figure 4B and C](#fig4){ref-type="fig"}). To validate the cytokine effect on the aTreg cell-M2 macrophage induction, neutralising antibodies against IL-10, IL-4/IL-13, or IL-10/IL-4/IL-13 were added to the co-cultures. Unsurprisingly, neutralising IL-10 was sufficient to reverse the upregulation of CD163 and the down-regulation of HLA-DR (data not shown), whereas neutralising IL-4/IL-13 had no effect ([Figure 4D](#fig4){ref-type="fig"}).
Next, to examine this aTreg-mediated effect on monocyte/macrophage functions in a tumour context, we added SNU899 cell-derived TSNs to the co-cultures. Interestingly, CD163 expression was increased further and accompanied by a further decline in the HLA-DR level. As a control, TSN alone could only induce a very small increase in the expression of CD163 and a marginal decrease in the HLA-DR level ([Figure 4E](#fig4){ref-type="fig"}). These data imply that the tumour cell induced soluble microenvironment may enhance the ability of aTreg cells to convert monocytes into M2 macrophages.
The positive-feedback loop between tumour-infiltrating aTreg cells and M2 macrophages contributes to immunosuppression
----------------------------------------------------------------------------------------------------------------------
Because TSN-exposed M2-like macrophages induced aTreg cells that in turn promoted induction of M2 macrophages *in vitro*, we investigated the feedback relationship between aTreg cells and M2 macrophages *in vivo*. After administration of a C--C chemokine receptor type 4 (CCR4) antagonist to specifically block tumour recruitment of aTreg cells, we observed that the percentage of tumour-infiltrating M2 macrophages was decreased by 27.17% ([Figures 5Ab and Bb](#fig5){ref-type="fig"}; 61.1±4.10% in control *vs* 44.50±3.54% in CCR~4~ antagonist, *n*=5, paired *t*-test, *P*\<0.05). In turn, treatment with gadolinium chloride (GdCl~3~) to deplete tissue macrophages attenuated the proportion of aTreg cells by 28.20% ([Figures 5Aa and Ba](#fig5){ref-type="fig"}; 30.85±1.91% in control *vs* 22.15±2.05% in GdCl~3~, *n*=5, paired *t*-test, *P*\<0.05), suggesting mutually dependent decreases or increases of aTreg cells and M2 macrophages in tumours.
Both aTreg cells and M2 macrophages have a key role in subversion of anti-tumour immunity. We found that targeting aTreg cells or M2 macrophages resulted in expansion of CD4^+^ and CD8^+^ T cells in tumours ([Figures 5Ac, Bc and Bd](#fig5){ref-type="fig"}). Moreover, T cells in CCR~4~ antagonist- and GdCl~3~-treated groups secreted higher amounts of IFN-*γ*, IL-2, and TNF-*α* than T cells in the control group ([Figure 5C and D](#fig5){ref-type="fig"}), which is consistent with previous reports ([@bib32]; [@bib44]). Therefore, the positive-feedback loop between aTreg cells and M2 macrophages by promoting induction of each cell type may significantly contribute to immunosuppression in the TME.
Combined targeting aTreg cells and M2 macrophages hampers their positive-feedback loop and evokes a potent antitumour immune response *in vivo*
-----------------------------------------------------------------------------------------------------------------------------------------------
This aTreg cell-M2 macrophage loop by enhancing tumour-induced immunosuppression represents a barrier to successful immunotherapy. Therefore, combined targeting of the generation of the two suppressive cell populations is a desirable goal in chemo- and immuno-therapeutic approaches. We first observed that tumour growth was significantly inhibited and the survival of mice was significantly prolonged in the CCR4 antagonist+GdCl~3~-treated group compared with the PBS control group and single-treated groups ([Figure 6A--C](#fig6){ref-type="fig"}; [Supplementary Table S4](#sup1){ref-type="supplementary-material"}). The weight of tumours, which were excised at 28 days after tumour cell injection, was significantly lower in the combined treatment group than in other groups ([Figure 6D and E](#fig6){ref-type="fig"}; [Supplementary Table S4](#sup1){ref-type="supplementary-material"}), whereas spleen weights were higher in this group ([Figure 6F](#fig6){ref-type="fig"}; [Supplementary Table S4](#sup1){ref-type="supplementary-material"}). These results suggest the effectiveness and practicability of this combined strategy targeting both aTreg cells and M2 macrophages.
To investigate whether the inhibition of tumour growth in the combined treatment group was associated with T-cell-mediated antitumour immunity, we analysed the composition of T cells that had infiltrated into the tumour tissues of mice. Immunohistochemical staining and flow cytometric analysis showed that the frequencies of CD3^+^, CD4^+^, and CD8^+^ T cells and Th1 cells were significantly higher in tumour tissues of the combined treatment group than in those of the other groups ([Figure 6G and H](#fig6){ref-type="fig"} and [Figure 5Ac, Bc--d, C and D](#fig5){ref-type="fig"}). These results indicated that the CCR4 antagonist+GdCl~3~ treatment evoked the most potent T-cell responses in the TME. Notably, this treatment had no obvious effect on T cells in the spleen ([Figure 6I](#fig6){ref-type="fig"}), which supports its potential therapeutic implications.
We hypothesised that the combined treatment targeting aTreg cells and M2 macrophages hampers their positive-feedback loop inducing each other, leading to enhanced T-cell populations ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}). Our results showed that, after the combined treatment, the frequency of tumour-infiltrating aTreg cells was lower than that induced by blockage of aTreg cells alone ([Figures 5Aa and Ba](#fig5){ref-type="fig"}; 9.85±2.05% *vs* 17.30±3.54%), while the M2 macrophage content was also decreased to 21.5±2.12% from 37.80±2.55% of single M2 macrophage depletion ([Figures 5Ab and Bb](#fig5){ref-type="fig"}), indicating an effect on the feedback loop. Therefore, this combined strategy reduces suppressor cells to the least extent by targeting them directly and interfering with their interactions in the TME.
Discussion
==========
In this study, we show that M2-polarised macrophages in the tumour environment promote the differentiation of CD4^+^CD25^−^T cells into aTreg cells. In turn, these generated aTreg cells skew the differentiation of monocytes toward M2 macrophages, forming a positive-feedback loop. This M2 macrophage-aTreg cell loop contributes to immunosuppression and is associated to advanced clinical stage and poor survival in LSCC patients.
Tumour-infiltrating Treg cells promote local tumour growth by exerting immunosuppressive effects against tumour-associated antigen (TAA) T-cell responses ([@bib9]; [@bib38]; [@bib19]; [@bib50]; [@bib25]). Yet Treg cells have been reported to be both negative and positive prognostic factors for cancers ([@bib9]; [@bib48]; [@bib2]; [@bib15]; [@bib14]; [@bib13]; [@bib7]; [@bib17]; [@bib21]). This contradiction regarding the role of Treg cells in cancer prognosis might because Treg cells in humans are functionally heterogeneous, and include suppressive and non-suppressive cell subsets ([@bib31]; [@bib10]). Tumour-infiltrating suppressive aTreg cells were proved to be the real Treg subset controlling the poor prognosis of cancers ([@bib45], [@bib43]; [@bib36]). Recently, to evoke antitumour immunity *in vivo*, we blocked tumour trafficking of aTreg cells using a CCR4 antagonist, which led to inhibition of tumour growth and prolonged survival ([@bib44]). However, the effect was not satisfactory, indicating that a single target in the treatment of HNSCC has a limitation.
Macrophages are a major component of the leukocyte infiltrate in tumours and considered to be another crucial mediator of tumour immunity ([@bib4]; [@bib33]; [@bib32]). Different types of macrophages, including M1 and M2, are of high scientific importance for subtyping. M1 macrophages are immunostimulatory and anti-tumoural, while M2 macrophages are immunosuppressive and pro-tumoural. Thus far, studies have been mainly based on whole intratumoural macrophages and have not reached a consensus on their effects on tumours. In the thyroid, lungs, oral cavity, and hepatocellular cancers, an increased macrophage density is associated with poor survival ([@bib30]; [@bib8]; [@bib35]; [@bib49]). However, in pancreatic and gastric cancers, high intratumoural macrophages correlate with increased survival ([@bib20]; [@bib46]). The causes of such conflicting results may lie in the fact that whole macrophages were taken into account and not the subsets of macrophages, especially M2, because the phenotype of macrophages can vary in different tumour types. In our study, the tumour-infiltrating macrophages in LSCC tissues were a distinct M2-polarised population and negatively associated with overall survival.
Different types of malignant tumours, including HNSCC, have various abilities to polarise macrophages, which is characterised by the expression of M2 macrophage surface markers and the ability to secrete M2 cytokines. Our study showed that, compared with HNSCC cell lines SNU46 and HONE1, laryngeal carcinoma cell line SNU899-derived TSNs had the most significant influence on the differentiation of monocytes toward M2 macrophages, resulting in the highest expression of M2-specific marker and cytokine levels. The varied degree of polarisation may lead to a difference in the function of macrophages, such as their interaction with lymphocytes ([@bib6]).
Our study demonstrated that the high intratumoural M2 macrophage density was positively correlated with the elevated intratumoural Foxp3^+^ Treg population. Accumulation of M2 macrophages and/or Foxp3^+^ Treg cells in LSCC tissue is associated to advanced T stage and poor survival. We hypothesised that these cells cooperate with each other to enhance immunosuppression in tumours. Our *in vitro* results revealed that TSN-exposed macrophages with the most typical M2 features had the strongest ability to induce Foxp3^+^ Treg cells by acting on responder PBMCs. To identify whether the induced Tregs were converted from CD4^+^CD25^−^T cells, rather than expansion of previous Tregs in the culture system, we replaced the responder cells with CD4^+^CD25^−^T cells and observed clear upregulation of Treg cell surface antigens by flow cytometry, thereby confirming their interactions. Previous study has demonstrated that Foxp3^+^ Treg cells are composed of three phenotypically and functionally distinct subpopulations: CD45RA^+^Foxp3^low^ rTreg cells and CD45RA^−^Foxp3^high^ aTreg cells, both of which are suppressive *in vitro*, and cytokine-secreting CD45RA^−^Foxp3 ^low^ nonsuppressive T cells ([@bib31]). Based on this classification of human Tregs, our study provided evidence to support the notion of heterogeneous Treg subsets in HNSCC patients ([@bib45], [@bib43], [@bib44]). We showed that aTreg cells, as the predominant cell population among tumour-infiltrating Foxp3^+^ Treg cells, accelerate disease progression by suppressing TAA immunity in patients with HNSCC ([@bib44]; [@bib47]). To determine whether TSN-exposed M2-like macrophages contribute to an increase in aTreg cells, we analysed the subsets of induced Treg cells. Interestingly, the aTreg cell frequency was dominantly increased in the suppressive Treg-cell population, indicating that the Treg cells induced by M2-like macrophages can directly mediate an inhibitory effect on tumour immunity.
Conversely, to examine the effect of aTreg cells on macrophage functions in the context of tumour-induced immunosuppression, we added TSNs to the co-culture system to mimic the tumour microenvironment (TME). Interestingly, aTreg cell-affected monocytes expressed significantly higher levels of an M2-specific marker in the TSNs compared with the medium alone, wheresas single TSNs had only a marginal effect on M2 marker induction within the specific time. These results indicated that TSNs have a good promotion effect on M2 macrophage induction by aTreg cells. The reverse process connected the formation of M2 macrophages and aTreg cells to a positive-feedback loop that may contribute to an increase of their number in tumours. Therefore, this biological mechanism can serve as an explanation for the positive correlation between aTreg cells and M2 macrophages in LSCC tissue.
To validate the positive-feedback relationship between M2 macrophages and aTreg cells and its importance *in vivo*, we targeted the two populations individually in a tumour-bearing mouse model. As a result, depletion of intratumoural M2 macrophages simultaneously attenuated the intratumoural aTreg cell frequency, whereas blockage of aTreg cell tumour trafficking indirectly decreased the percentage of intratumoural M2 macrophages. These data indicate that it is imperative to further investigate combined strategies to interfere with both M2 macrophages and aTreg cells in the TME.
Indeed, targeting M2 macrophages and aTreg cells simultaneously had a superior effect on regulating the two cell populations. Both aTreg cells and M2 macrophages have a key role in subversion of anti-tumour immunity. The former can suppress TAA-T-cell responses by cell contact, while the latter have a poor antigen-presenting capacity and suppress T-cell activation and proliferation by releasing prostaglandins, IL-10, and TGF-β ([@bib18]; [@bib27]; [@bib26]; [@bib37]). In mice, we found that the combined strategy was more efficient than single targeting for inhibition of tumour growth and prolongation of survival. The efficiency is derived from deregulation of antitumour immunity because the proportions of CD4^+^ and CD8^+^ T cells and Th1 cells were increased significantly. Furthermore, we found that this strategy had no significant influence on the peripheral immune status, thus indicating greater potential therapeutic implications.
This combined targeting strategy, based on the theory of the M2 macrophage-aTreg cell positive-feedback loop, may offer new approaches to overcome the barriers of immunosuppression in the TME. Various options can be provided for the specific therapeutic program, including blocking tumour trafficking of macrophages/Treg cells using CSF1R/CCR4 antagonists, respectively ([@bib42]; [@bib34]; [@bib23]), ligation of GITR to down-regulate Foxp3 expression and reduce production of IL-10 by Treg cells ([@bib39]), and re-educating M2 macrophages to an M1 phenotype using Toll-like receptor 3 ligands or STING agonists ([@bib41]; [@bib11]; [@bib3]). A rational combination of the above methods may exert an unexpected antitumour effect in LSCC treatment.
In summary, our study suggests an important role of the positive-feedback loop between tumour-infiltrating M2 macrophages and aTreg cells in tumour progression. This positive-feedback loop is essential to maintain or promote immunosuppression in the TME and could be a potential therapeutic target against tumour progression.
This work was supported by grants from the Natural Science Foundation of Guangdong Province (2016A030310153, 2014A030313031, and 2016A030313257), the Science and Technology Planning Project of Guangdong Province (2014A020212141), the Natural Science Foundation of China (81602365, 81670902, and 81470674), the Medical Scientific Research Foundation of Guangdong Province (A2017216), and the Guangzhou Key Laboratory of Otorhinolaryngology Head and Neck Surgery (201605030003).
[Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License.
The authors declare no conflict of interest.
Supplementary Material {#sup1}
======================
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[^1]: These authors contributed equally to this work.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S0001}
============
The results of many epidemiological and clinical studies have shown that osteoporosis is a multifactorial disease and develops mainly in women during menopause. The most important modifiable risk factors for osteoporosis include the ones connected with lifestyle, among which the most important are dietary factors (including in particular the customary intake of calcium and vitamin D), factors associated with physical activity and the non-food source of vitamin D, which is skin synthesis through exposure to sunlight \[[@CIT0001], [@CIT0002]\].
Nutrition is the primary determinant of reconfigurable chronic diseases. Change of diet eliminates errors in eating habits and has a significantly impact on improving the health and quality of life of premenopausal women. It also gives a chance to reduce the risk of osteoporosis after menopause.
In an osteoporosis preventive therapy healthy lifestyle plays a huge role. This includes proper nutrition, balanced in energy, providing the necessary nutrients in the right amounts. Among these, calcium and vitamin D are especially important.
Vitamin D is essential to maintain proper calcium and phosphorus homeostasis of the organism and to ensure the proper functioning of many tissues, organs and cells independent of the mineral market. Vitamin D deficiency increases the risk of fractures in osteoporosis and can lead to osteomalacia. An important endogenous source of vitamin D is regular exposure to the sun. However, it should be noted that commonly used sun blockers can reduce the yield of synthesis of skin under the influence of UVB radiation by up to 90% \[[@CIT0003], [@CIT0004]\].
Polish recommendations for the prevention of vitamin D deficiency developed by a group of experts carefully lay down the optimal vitamin D photo conversion in the skin. In our geographical area skin synthesis takes place from April to September, ensuring a minimum of 15 minutes sun exposure of 18% of body surface area (exposed arms and legs partially) from 10 am to 3 pm without the use of protective filters. However, from October to March skin synthesis of vitamin D practically does not occur at all \[[@CIT0005]\].
An essential element in determining the importance of nutrition in the path physiology and prevention of osteoporosis is insufficient calcium intake. Bone tissue is a reservoir of calcium and calcium homeostasis has a significant influence on it when the achievement of peak bone mass is reached and also later throughout life. As the body ages the process of increased release of calcium from the bones occurs. The hormonal changes of menopause also disrupt the economy of calcium \[[@CIT0006], [@CIT0007]\].
Forecasts for epidemiology of osteoporosis are worrying. Therefore, the consequences of low-energy fractures will remain a growing health problem in the world. Therefore, it is necessary to undertake studies leading to the definition of the type and number of factors responsible for the occurrence of underestimation of bone mineral density and to determine for which of them comes to the biggest and most common failures in the population of females.
The aim of this study was to evaluate the diet with particular reference to calcium and vitamin D and to sun exposure in premenopausal women in terms of osteoporosis risk.
Material and methods {#S0002}
====================
The study involved 300 women aged 45-55 years from three districts of Warsaw: Bielany, Bemowo and Wola. This sample was selected randomly from a set of women living in the area covered by the study.
Hormonal status of women was rated on the basis of a self-assessment questionnaire with the classification of the World Health Organization \[[@CIT0008]\]:
premenopause -- period before menopause characterized by rhythmic menstrual cycles,perimenopause -- period immediately preceding menopause, in which menstrual cycles are longer, become irregular or the menstrual bleeding came to an end; however, at the time of the study 12 consecutive months without bleeding have not passed from a woman\'s last period (amenorrhea).
The method used was the dietary interview (standardized questionnaire for the assessment of food intake of products most important for calcium and vitamin D intake) assessing the frequency of consumption of the last three months, developed in the Department of Epidemiology and Nutrition Standards of Food and Nutrition Institute in Warsaw.
The questionnaire assessing the frequency of consumption is an advanced tool that allows one to assess the frequency and quantity (g/person/day) of food intake usually in the past three months. Moreover, it allows one to assess the amount of calcium and vitamin D intake in the daily food ration (mg or µg/person/day). The main part of the questionnaire contains closed questions on the usual consumption frequency of products containing calcium and vitamin D as well as their quantity. The list of products mainly included milk, dairy products divided into fermented products, dairy desserts, cheeses, white cottage cheese, blue cheese, meat and sausages, bread, cereal and cereal products, spreads (margarine, butter) and the types of water consumed. Moreover, women were also asked about the use of supplements, the dose and frequency of intake. Help in determining the type and amount of food eaten and drinks was provided by the "Photo Album of products and dishes in different sizes", incorporating 201 colour photos, prepared and published by the Institute of Food and Nutrition \[[@CIT0009]\].
Statistical analysis of selected nutrient intake was performed with the computer program Diet 5.0 extended version. The interpretation of the results was made according to the guidelines for the assessment of group intake created by the Standards of Human Nutrition for the Polish population 2012 developed by the Food and Nutrition Institute in Warsaw \[[@CIT0010]\]. In the intake assessment a standard average demand group, EAR (Estimated Average Requirement), was used, in the assessment of calcium and vitamin D in the diet, and taking into account the supplementation of women surveyed.
Exposure to the sun evaluated using a questionnaire prepared in cooperation with the Institute of Food and Nutrition in Warsaw.
The examination concerned the period from April to September, where due to the geographical zone skin synthesis takes place. In the winter period, due to high zenith angles of the sun, above the area of Poland a deficiency of radiating D~3~ previtamin for effectiveness is observed in Poland. In relation to the daily dose recommended for the maintenance of health of 0.25 MED \[[@CIT0011]\], months in which sun exposure enables the skin synthesis of vitamin D were analysed.
The questionnaire consisted of three sections. The first section was related to sun exposure. The second part included questions about the sun exposure frequency. And finally, the third part concerned clothes worn during sun exposure and the assessment of body surface exposure to UV radiation.
For the purpose of the project 8 profiles describing attitudes towards prevention of osteoporosis were created:
PROFILE I -- high exposure to sunlight, recommended intake of calcium and vitamin D,PROFILE II -- high exposure to sunlight, recommended intake of calcium and deficiency of vitamin D,PROFILE III -- high exposure to sunlight, calcium deficiency, recommended intake of vitamin D,PROFILE IV -- high exposure to sunlight, deficiency of calcium and vitamin D,PROFILE V -- insufficient exposure to sunlight, recommended intake of calcium and vitamin D,PROFILE VI -- insufficient exposure to sunlight, recommended intake of calcium, vitamin D deficiency,PROFILE VII -- insufficient exposure to sunlight, calcium deficiency, recommended intake of vitamin D,PROFILE VIII -- insufficient exposure to sunlight, deficiency of calcium and vitamin D.
Categories of high and insufficient exposure were developed by summing points from the data obtained from the questionnaire: the sum of the two categories frequency of exposure to the sun and the actual exposure to sunlight.
High exposure to sunlight was adopted in accordance with the guidelines and recommendations for the prevention of vitamin D deficiency produced by a group of experts: in the period from April to September to ensure a minimum of 15 minutes of sun exposure for at least 18% of the body surface area (exposed arms and legs in part), and, moreover, without the use of protective filters \[[@CIT0005]\].
Statistical analysis of the data obtained was performed using STATISTICA. Arithmetic means and standard deviations were calculated in the data set.
The project (its goals, objectives, methods, and selection of the sample) received the consent of the Senate Ethics Committee for Scientific Research, Jozef Pilsudski University of Physical Education in Warsaw.
Results {#S0003}
=======
Computer analysis of the data gathered from the interview among surveyed women showed insufficient dietary intake of calcium and vitamin D. Moreover, the average content of females' nutrients in daily food rations was located well below the normal average demand of a group EAR (Estimated Average Requirement) ([Table I](#T0001){ref-type="table"}).
######
Calcium and vitamin D in the daily diet of examined women
------------------------------------------------------------------------------------------------------------
Mean ± SD EAR for female adults[\*](#TF0001){ref-type="table-fn"}
-------------------- --------------- --------------------------------------------------------- -------------
Calcium (mg/day) 444.0 ± 472.0 19-50 years\ 800 mg/day\
\> 50 years 1000 mg/day
Vitamin D (µg/day) 2.8 ± 2.9 10 µg/day
------------------------------------------------------------------------------------------------------------
EAR (Estimated Average Requirement), Jarosz M (ed.): The standards of nutrition for the population of Polish-amendment. Food and Nutrition Institute, Warsaw 2012
There have been a significant proportion of women who have had deficiency of both calcium and vitamin D. Intake of vitamin D recommended in the standard EAR occurred only in 3.7% of all surveyed women ([Fig. 1](#F0001){ref-type="fig"}).
{#F0001}
The highest percentage of women surveyed reported exposure to the sun at a frequency of 1-2 times a week, or even higher than two days. Half of the respondents reported only occasional exposure to the sun ([Table II](#T0002){ref-type="table"}).
######
Weekly exposure to the sun
Frequency of exposure to the sun Exposure to the sun
----------- ---------------------------------- --------------------- ------ ----- ------ ------ ------
Women (%) 9.3 44.0 43.3 3.4 17.0 50.0 33.0
As a result, the highest percentage of women represented profile number VIII, which describes attitudes towards prevention of osteoporosis as insufficient exposure to sunlight and dietary deficiency of both calcium and vitamin D ([Fig. 2](#F0002){ref-type="fig"}).
{#F0002}
Discussion {#S0004}
==========
Vitamin D is essential for bone health. It promotes the efficient utilization of calcium by the body. Calcium is essential for the proper functioning of the nervous system, bone growth and maintenance of normal bone density \[[@CIT0008]\].
Our study reported inadequate intake of calcium and vitamin D in the diets of women surveyed. The average content of these nutrients in daily food rations was well below the normal average demand for the group recommended by EAR. This result confirms previous reports of deficiency of these nutrients in the diet of both adults and children. Significant deficiency is even more disturbing because of the pleiotropic effects of vitamin D (influencing more than one thing at the same time), which can protect the body against many chronic diseases including osteoporosis.
Hollis *et al*. found in their research that many people had insufficient levels of vitamin D, even if one takes a few thousand units per day. Thus, adult deficit of vitamin D in industrialized countries is the rule rather than the exception \[[@CIT0009]\].
Many authors draw attention to the fact of very common subclinical vitamin D deficiency \[[@CIT0010]\]. A report of the International Osteoporosis Foundation indicates the broad global prevalence of hypovitaminosis D in almost every region of the world which was covered by the study. Although we still lack the complete data from parts of Asia and most part of Africa, vitamin D deficiency is seen as a clinical problem and significantly increases the risk of many diseases \[[@CIT0012]\].
What may be worrying is the high percentage of vitamin D deficiency found in a variety of age groups in the Polish population, where the most common nutrient deficiencies in the diet (in more than 95% of the surveyed women) were found in the levels of vitamin D and calcium \[[@CIT0011]\].
The human body is capable of endogenous synthesis of cholecalciferol of 7-dehydrocholesterol in the skin cells by ultraviolet radiation. Therefore, the efficiency of this process is dependent on skin pigmentation, age, time of exposure to sun and the latitude in which we live. Deficiency of vitamin D in the body can be caused by too low endogenous synthesis or by insufficient supply in the diet \[[@CIT0013], [@CIT0014]\].
The vast majority of surveyed women from Warsaw presented the least favourable profile of attitudes towards prevention of osteoporosis. Insufficient exposure to sunlight and at the same time deficiency of calcium and vitamin D from their diet show a lack of health-related behaviours regarding bone protection against osteoporosis.
This is a negative conclusion for the group study, because the shortage of food calcium must be rebalanced at the expense of bone tissue. This increases the pace of decline in bone density. It is estimated that approximately 80-100% of the daily requirement for vitamin D~3~ is derived from biosynthesis in the skin, and only to a small extent is supported by dietary sources. Among the examined groups of women it was found that the acquisition of vitamin D from diet as well as from outside sources is not sufficient. As a result this leads to higher risk of osteoporosis in elderly life.
Insufficient exposure to sunlight may be associated with fear of skin diseases including melanoma. It has been proven that excessive exposure to sunlight increases the risk of skin cancer, but there is no evidence that reasonable exposure to the sun increases this risk. Therefore, reasonable and recommended skin exposure to the sun should be perceived as the best source of vitamin D. Exposure to the sun as a result of which we observe the minimum erythema is equivalent to about 20,000 IU vitamin D from food and supplements. Thus, the exposure of arms and legs to ensure a minimum dose can be compared to the consumption of approximately 3000 IU of vitamin D \[[@CIT0015]\].
Nutritional support (adequate intake of calcium and vitamin D in diet, use of products enriched with calcium and vitamin D, right supplementation) and appropriate exposure to the sun as the main external source of vitamin D are important aspects of physiological slowing the loss of BMD in premenopausal women. Moreover, they can be considered as the main means of prevention of osteoporosis in postmenopausal women.
Conclusions {#S0005}
===========
Proper diet according to the recommendations determines an adequate supply of calcium and vitamin D which is important to maintaining the health and well-being of the skeletal system.Equally important is adequate exposure to the sun as the main non-nutrition source of vitamin D both to allow achieving peak bone mass, and as the main factor in the prevention of osteoporosis.An important element of the prevention of diseases of the skeletal system is provided by nutritional education and modification of women\'s behaviour toward a healthy lifestyle.
I would like to give my special thanks to Professor Jadwiga Charzewska for her kindness, support and extra effort in directing me in my work.
The project is financed by the Jozef Pilsudski University of Physical Education in Warsaw as part of the activities of young scientists (DM-14).
Disclosure {#S0006}
==========
Author reports no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Oral cancer is a major devastating head and neck cancer subtype and is any cancerous tissue growth located in the oral cavity or oropharynx \[[@CR1]\]. There are several types of oral cancers, but around 90% of oral cancers are squamous cell carcinomas. Oral cancer is the sixth most common malignancy in humans. Its incidence and mortality have also increased over the past decades. In 2013, oral cancer resulted in 135,000 deaths, up from 84,000 deaths in 1990 \[[@CR2]\]. Oral cancer is characterized by poor prognosis and a low survival rate despite sophisticated surgical and radiotherapeutic modalities. The five-year survival rate is only 63% in the United States.
Metastasis is one of the main causes of oral cancer patients' death. The process of oral cancer metastasis is a series of sequential and interdependent events involving detachment of cells from tumor tissue, increased cell motility and local invasion, angiogenesis, intravasation of invading cells into the vasculature or lymphatic systems, extravasation and subsequent deposition and proliferation at a second site. Oral cancer tends to spread primarily to the regional lymph nodes of the neck first before it spreads to remote sites. Lymph node metastasis is called locoregional metastasis and lung (or other organs) metastasis is called distant metastasis \[[@CR3]\]. Early diagnosis of lymph node metastasis is important for improving clinical outcomes of oral cancer patients \[[@CR4]\].
The clinical diagnosis of lymph node metastasis of oral cancer is currently based on imaging techniques and sentinel lymph node biopsy (SLNB) \[[@CR5]\]. However, current imaging tests have been proven to be unreliable, especially in the detection of early nodal diseases. Localization of SLNB in patients with certain cancers such as floor-of-mouth carcinoma is difficult. The difficulties of detecting micrometastasis in frozen sections also limit SLNB as an adequate guide to clinical decision-making \[[@CR6]\]. Therefore, better understanding of the lymphatic metastasis of oral cancer and developing new diagnostic strategies to predict the clinical behavior of the disease are desired \[[@CR4]\].
Identifying reliable gene signatures or molecular biomarkers for oral cancer lymph node metastasis is highly valuable to use as a potential diagnostic or prognostic tool for routine clinical practice of oral cancer. Microarray analysis has been extensively used to examine the global gene expression changes in non-metastatic versus metastatic human tumor samples. Comparing the gene expression profiles of primary tumors from non-metastatic and metastatic tumor generated a list of genes whose expression was significantly different in these two groups. The list of genes is called "metastasis signatures". Several "metastasis signatures" have been generated in oral cancer aimed at predicting lymph node metastasis \[[@CR7]--[@CR17]\]. However, defined "metastasis signatures" from different studies are highly variable, which hinders their translation to clinical applications. To date, none of the identified signatures or molecular biomarkers has been successfully implemented as a diagnostic or prognostic tool applicable to routine clinical practice of oral cancer.
In this study, in order to identify differentially expressed genes (gene signature) and pathways that contribute to oral cancer metastasis to lymph nodes, we designed a new bioinformatic analysis strategy. The GSE70604-associated study is aimed at identifying lymph node metastasis-associated genes and pathways in oral cancer by comparing gene profiles in lymph nodes with metastasis to those of normal lymph nodes. The GSE2280-associated study tried to achieve this goal by comparing gene profiles in squamous cell carcinomas of the oral cavity with lymph node metastasis to those in tumors without lymph node metastasis. Then overlapping analysis was performed to identify common DEGs (gene signature) and pathways in these two studies. We focused on those 28 common DEGs that show consistent changes in both datasets and defined a "metastasis signature": CCND1, JUN and SPP1 for oral cancer lymph node metastasis. Both Jun and SPP1 are known molecules involved in cancer cell invasiveness and tumor metastasis. CCND1 (cyclinD1) is a known critical factor in cell cycle progression, but the function of CCND1 in human cancer cell migration/invasion is not sufficiently understood despite a recent study showing that it directly regulates the focal adhesion pathway and promotes R3327 rat prostatic tumor cell migration/invasion and tumor metastasis \[[@CR18]\] and affects cell migration and invasion in breast cancer \[[@CR18]--[@CR21]\]. Our study identified a novel "focal adhesion"-related gene signature (CCND1, JUN and SPP1) that might be applicable for diagnosis of oral cancer metastasis to lymph nodes.
Materials and methods {#Sec2}
=====================
Cell lines {#Sec3}
----------
OECM-1 is a human oral cancer cell line derived from gingival epidermoid carcinoma of a patient \[[@CR22]\]. The cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 2 mmol/ l glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO~2~ at 37 °C.
siRNA interference {#Sec4}
------------------
SPP1, JUN and CCND1 siRNA smart pool (Cat. L-009152-00-0005) was purchased from Dharmacon/Thermo Fisher Scientific. Transfection of the siRNA oligonucleotide duplexes was performed in a 6-well plate (1 × 10 ^5^ cells per well) with Lipofectamine 2000 (Invitrogen, Inc.), using the methods recommended by the manufacturer. Knockdown of SPP1, JUN and CCND1 with siRNA was examined 72 h after siRNA transfection through western blotting.
Western blot {#Sec5}
------------
Cells were lysed in a lysis buffer containing 50 mmol/L TRIS-HCl, pH 7.4, 150 mmol/L NaCl, 0.5% NP40, 50 mmol/L NaF, 1 mmol/L Na~3~VO~4~, 1 mmol/L phenyl-methylsulfonyl fluoride, 25 μg/mL leupeptin, and 25 μg/mL aprotinin and clarified by centrifugation (14,000 g for 30 min at 4 °C). The protein concentration of the cell lysates was determined using the Bradford Coomassie blue method (Pierce Chemical Corp.). Whole-cell lysates were separated by sodium dodecyl sulfate (SDS)-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked with PBS containing 5% (*w*/*v*) skim milk at 4 °C for 2 h, washed with PBST (PBS with 0.05% Tween-20), and then incubated overnight with primary antibody. After washing with PBST, the membrane was incubated with a second antibody at room temperature for 2 h, washed with PBST and then developed with the ECL system. The results of Western blot were analyzed with Odyssey software version 3.0.
Cell invasion assay {#Sec6}
-------------------
Cell invasion potential was measured with a Boyden transwell chamber consisting of upper inserts with 8-μm-pore-size filter membranes at the bottom of the inserts and lower wells in 24-well cell culture plates (Corning Life Sciences). 20 μl of 1:6 diluted Matrigel (2--3 mg/ml protein) was added to the center of each cell well insert. Coated inserts were placed in an incubator to allow the Matrigel to solidify for 20--30 min. Cells (3.5 × 10^5^ cells in 0.2 mL) suspended in serum-free medium with 0.1% bovine serum albumin were seeded into the inserts of the chambers. The inserts were then placed over the wells filled with 0.5 mL of 10% FBS culture medium and incubated in a 37 °C incubator for 24 h. Cells that had not penetrated the filter membrane in the inserts were wiped off with cotton swabs, and the cells on the underside of the filter membrane were fixed and stained with the HEMA-3 kit (Fisher Diagnostics). Invaded cells were counted in a total of 10 fields for each sample under a microscope with the 10X objective and the stained cell number per field was calculated.
Differentially expressed genes (DEG) analysis {#Sec7}
---------------------------------------------
Differentially expressed genes of the GSE2280-associated study and GSE2280-associated study were analyzed. The expression abundance (FPKM) value of each gene was estimated by running cufflinks \[[@CR23]\] and the differentially expressed genes were assessed by cuffdiff. Statistically differentially expressed genes between two groups were those genes with logFC (fold change) \> 0.45 and adjusted *p* value \< 0.05. The adjusted p value was obtained through applying Benjamini and Hochberg's (BH) false discovery rate correction on the original p value, and the fold change threshold was selected based on our purpose of focusing on significantly differentially expressed genes.
Hierarchical clustering {#Sec8}
-----------------------
Hierarchical clustering was conducted \[[@CR24]\] to classify analyzed samples based on gene expression profiles. Hierarchical clustering using differentially expressed genes (DEGs) demonstrated the global gene expression patterns in the samples. In addition, the DEGs were further extracted and classified in specific biological processes (Gene Ontology terms) and KEGG pathways. The expression pattern of those DEGs was characterized and heat maps of the DEGs were classified in targeted biological processes or KEGG pathways using the R package.
GO and KEGG pathway analysis {#Sec9}
----------------------------
We used the R packages GO.db, KEGG.db and KEGGREST to detect Gene Ontology categories and KEGG pathways with significant enrichment in DEGs for comparison across all measured genes. The significantly enriched biological processes were identified by *p* value less than the threshold value 0.05. For the KEGG pathway, the p value was also set to less than 0.05.
Results {#Sec10}
=======
Identification of potential genes related to oral cancer metastasis to lymph nodes through screening GEO database {#Sec11}
-----------------------------------------------------------------------------------------------------------------
In order to find the key genes regulating oral cancer metastasis to lymph nodes, we screened the GEO (gene expression omnibus) database for GSE70604 (<https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70604>) \[[@CR25]\] and GSE2280 (<https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2280>) \[[@CR12]\] as shown in Additional file [1](#MOESM1){ref-type="media"}: Table S1 and Additional file [2](#MOESM2){ref-type="media"}: Table S3.
In GSE70604, the comparisons of gene expression profiles were made between lymph nodes with metastasis of oral squamous cell carcinoma (OSCC) and normal lymph nodes (comparison 1).
In GSE2280, the comparisons of gene expression profiles were made between primary tumors of OSCC which has lymph node metastasis and nonmetastatic primary OSCC without lymph node metastasis (comparison 2).
Differentially expressed genes (DEGs) of both comparisons were obtained where lymph nodes with OSCC metastasis were compared to normal lymph nodes in comparison 1 and metastatic OSCC primary tumors were compared to non-metastatic OSCC primary tumors in comparison 2. Both comparisons had the \|log(fold change)\|(logFC) \> 0.45 and *p* value \< 0.05, indicating the overall changes as statistically significant. In comparison 1, gene expression of 7 lymph nodes with metastasis of OSCC was compared to that of a normal lymph node. Figure [1](#Fig1){ref-type="fig"} shows the distribution of DEGs in comparison 1; we found that 1392 genes had expression changes (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Among those genes, we identified 723 down-regulated genes (Additional file [3](#MOESM3){ref-type="media"}: Table S2) and 699 up-regulated genes (Additional file [2](#MOESM2){ref-type="media"}: Table S3). In comparison 2, gene expression of 5 primary tumors of OSCC with lymph node metastasis was compared to that of 8 non-metastatic primary tumors of OSCC. Figure [2](#Fig2){ref-type="fig"} shows the distribution of DEGs in comparison 2; we found 890 genes that had expression changes (Additional file [4](#MOESM4){ref-type="media"}: Table S4). Among those genes, we identified 477 down-regulated genes (Additional file [5](#MOESM5){ref-type="media"}: Table S5) and 413 up-regulated genes (Additional file [6](#MOESM6){ref-type="media"}: Table S6). To identify potential genes related to oral cancer metastasis to lymph nodes, we then analyzed the overlapping DEGs in comparison 1 and comparison 2 and found 114 overlapping DEGs. Among those DEGs, 28 of 114 DEGs had a consistent change trend (consistent increase or decrease in both comparisons) (Additional file [7](#MOESM7){ref-type="media"}: Table S7) and the remaining 86 had an opposite change trend (Additional file [8](#MOESM8){ref-type="media"}: Table S8). The 28 genes with consistent changes in comparison 1 and comparison 2 are the potential genes related to oral cancer lymph node metastasis.Fig. 1Heat map of distribution of DEGs in GSE70604. The comparisons of gene expression profiles were made between lymph nodes with metastasis of oral squamous cell carcinoma (OSCC) and normal lymph nodesFig. 2Heat map of distribution of DEGs in GSE2280. The comparisons of gene expression profiles were made between primary tumors of squamous cell carcinomas of the oral cavity (OSCC) which has lymph node metastasis and nonmetastatic primary OSCC without lymph node metastasis
GO-KEGG analysis of consistent overlapping DEGs {#Sec12}
-----------------------------------------------
We further analyzed the 28 consistent overlapping DEGs by GO-KEGG analysis. GO biological process analysis indicated that these 28 genes were involved in 448 biological processes; Additional file [9](#MOESM9){ref-type="media"}: Table S9 shows the top 10 biological processes. KEGG pathway analysis showed that those genes mainly participated in 12 signaling pathways (Additional file [10](#MOESM10){ref-type="media"}: Table S10) and the top 3 KEGG signaling pathways are colorectal cancer, Toll-like receptor signaling pathway and Chagas disease. We also identified 7 interacting gene pairs among these 28 overlapping genes (Additional file [11](#MOESM11){ref-type="media"}: Table S11). Among those genes, JUN, ATF3 and FOS were presented as the key connecting nodes in the network of signaling pathways according to connectivity. JUN and FOS cooperatively participated in 9 common KEGG signaling pathway among the 12 KEGG pathways we identified. JUN, ATF3 and FOS were involved in 257, 115 and 183 GO-biological processes respectively.
Definition of key regulatory genes involved in oral cancer metastasis to lymph nodes {#Sec13}
------------------------------------------------------------------------------------
Among 12 KEGG pathways we identified by analyzing the 28 consistent overlapping DEGs (Additional file [10](#MOESM10){ref-type="media"}: Table S10), we mainly focus on the focal adhesion pathway, which is important for cancer cell migration/invasion and tumor metastasis.
Three DEGs associated with the focal adhesion pathway in the 28 consistent overlapping DEGs are CCND1, JUN and SPP1. Both JUN and SPP1 are genes known to promote cancer cell invasiveness and tumor metastasis. CCND1 (cyclinD1), a critical gene regulating cell cycle progress, recently has been reported to directly regulate the focal adhesion pathway and promote R3327 rat prostatic tumor cell migration/invasion and tumor metastasis \[[@CR18]\]. The direct involvement of CCND1 in human cancer cell migration/invasion is virtually unknown. Our study identified a novel focal adhesion-related gene signature (CCND1, JUN and SPP1) that might be important for oral cancer metastasis to lymph nodes.
Gene signature CCND1, JUN and SPP1 are required for oral cancer cell invasion {#Sec14}
-----------------------------------------------------------------------------
To confirm the function of CCND1, JUN and SPP1 in oral cancer cell invasion, we knocked down expression of CCND1, JUN and SPP1 through siRNA in the human oral cancer cell line OECM-1 and examined the alteration of cell invasion ability. Deletion of CCND1, JUN and SPP1 expression in OECM-1 via siRNA interference significantly impaired cell invasion although in varying degrees (Fig. [3a-c](#Fig3){ref-type="fig"}). The result further supports the hypothesis that the focal adhesion-related gene signature (CCND1, JUN and SPP1) is important for oral cancer invasion.Fig. 3CCND1, JUN and SPP1 are involved in oral cancer cell invasion. **a** Human oral cancer cell line OECM-1 was transfected with SPP1 siRNA, SPP1 protein expression was examined through western blot, and alteration of cell invasion ability was examined and quantified as a histogram. **b** Human oral cancer cell line OECM-1 was transfected with JUN siRNA, JUN protein expression was examined through western blot, and alteration of cell invasion ability was examined and quantified as a histogram. **c** Human oral cancer cell line OECM-1 was transfected with CCND1 siRNA, CCND1 protein expression was examined through western blot, and alteration of cell invasion ability was examined and quantified as a histogram
C- JUN expression is correlated with oral cancer metastasis in clinical practice {#Sec15}
--------------------------------------------------------------------------------
We further tried to explore the correlation of expression of CCND1, JUN and SPP1 with oral cancer metastasis through statistically analyzing the clinic data in the literature. Based on the original data of Rickman's work published in 2008 in Oncogene \[[@CR26]\], we compared JUN expression in oropharyngeal squamous cell carcinoma in patients without metastatic events at 5 years with that of patients with metastatic events at 5 years \[[@CR26]\]. Metastatic oropharyngeal squamous cell carcinoma had 1.69-fold higher JUN gene expression than non-metastatic oropharyngeal squamous cell carcinoma (*p* = 0.025). Figure [4](#Fig4){ref-type="fig"} compares JUN expression in oral cavity squamous cell carcinoma in patients without metastatic events at 3 years with that of patients with metastatic events at 3 years \[[@CR26]\]. Metastatic oral cavity squamous cell carcinoma had 2.69-fold higher JUN gene expression than non-metastatic oral cavity squamous cell carcinoma (*p* = 0.012) (Fig. [4](#Fig4){ref-type="fig"}). Figure [5](#Fig5){ref-type="fig"} compared JUN expression in oral cavity squamous cell carcinoma in patients without metastatic events at 5 years than that of patients with metastatic events at 5 years \[[@CR26]\]. Metastatic oral cavity squamous cell carcinoma had 2.39-fold higher JUN gene expression than non-metastatic oral cavity squamous cell carcinoma (*p* = 0.025) (Fig. [5](#Fig5){ref-type="fig"}). Figure [6](#Fig6){ref-type="fig"} compares JUN expression in oropharyngeal squamous cell carcinoma in patients without metastatic events at 5 years with that of patients with metastatic events at 5 years \[[@CR26]\]. Metastatic oropharyngeal squamous cell carcinoma had 1.69-fold higher JUN gene expression than non-metastatic oropharyngeal squamous cell carcinoma (p = 0.025) (Fig. [6](#Fig6){ref-type="fig"}). The results suggest that c-JUN is strongly correlated with oral cancer metastasis. We have not found similar gene expression data of CCND1 and SPP1 in oral cancer metastasis. However, we observed that higher expression of Jun or CCND1 or SPP1 was associated with short survival of oral squamous cell carcinoma patients (Figs. [7](#Fig7){ref-type="fig"}, [8](#Fig8){ref-type="fig"} and [9](#Fig9){ref-type="fig"}).Fig. 4Comparison of JUN expression in oral cavity squamous cell carcinoma without metastatic events at 3 years and with metastatic events at 3 yearsFig. 5Comparison of JUN expression in oral cavity squamous cell carcinoma without metastatic events at 5 years and with metastatic events at 5 yearsFig. 6Comparison of JUN expression in oropharyngeal squamous cell carcinoma without metastatic events at 5 years and with metastatic events at 5 yearsFig. 7Comparison of JUN expression in oropharyngeal squamous cell carcinomaFig. 8Comparison of CCND1 expression in hypopharyngeal squamous cell carcinomaFig. 9Comparison of SPP1 expression in oropharyngeal squamous cell carcinoma
Discussion {#Sec16}
==========
Through bioinformatics analysis of the gene expression profile of oral cancer metastatic and non-metastatic lymph nodes and primary tumors, we identified a new oral cancer metastatic gene signature: CCND1, JUN and SPP1. The gene knockdown experiment on the oral cancer cell line and clinical data correlation analysis indicated strong association of this gene signature expression with oral cancer invasion and metastasis.
c-Jun is a subunit of transcription factor AP-1. Activated AP-1 increases the transcription of target genes and plays roles in cell division, proliferation, differentiation, apoptosis and so on. Although overexpression of c-Jun promotes invasion and metastasis of various tumors \[[@CR27]--[@CR29]\], it has not been reported in oral cancer. c-Jun plays a role during the initiation and progression of OSCC. High expression of c-Jun is associated with poor prognosis of OSCC \[[@CR30]\]. Through a gene knockdown experiment in oral cancer cell line and clinical data analysis, our study confirmed that it is also required for oral cancer invasion and metastasis.
SPP1 (secreted phosphoprotein 1) encodes osteopontin (OPN). Osteopontin is a secreted non-collagenous, sialic acid rich, chemokine-like, matricellular phosphoglycoprotein that facilitates cell--matrix interactions and promotes tumor progression \[[@CR31]\]. Osteopontin is a multifunctional cytokine regulating cell proliferation, survival, drug resistance, invasion, and stem-like behavior. Its aberrant expression and/or splicing is functionally responsible for many disease pathologies including cancer \[[@CR31]\]. The expression of OPN was elevated in 95% of OSCC and can be used as a diagnostic marker for oral cancer \[[@CR32]\]. It is also a poor prognostic factor in OSCC treated with cisplatin-based IC followed by CCRT \[[@CR33]\]. However, its correlation with OSCC invasion and metastasis has not been studied anywhere else. The gene knockdown experiment in an oral cancer cell line and clinical data analysis showed that SPP1 is important for oral cancer invasion and metastasis in this study. Besides aberrant expression, the pattern of isoform expression (gene splicing) and post-translational modification are other SPP1 regulation methods. This regulation is cell-type specific and may influence the potential role of OPN in malignancy as a cancer biomarker \[[@CR34]\] For example, invasive breast tumor cells generate three splice variants of OPN, while non-invasive breast cells express only the unspliced form or no OPN at all \[[@CR35]\]. We do not know the splice variants of SPP1 in non-metastatic oral cancer and metastatic oral cancer with lymph node metastasis beside the expression difference, which deserves further investigation.
A recent study showed that CCND1 (cyclin D1) together with its binding partner CDK4 does not only simply act as a transcriptional regulator to control cell proliferation, but also controls cell adhesion, migration and metastasis under normal and pathological conditions. The focal adhesion component paxillin is a cytoplasmic substrate of CCND1·CDK4. This complex phosphorylates a fraction of paxillin specifically associated with the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumor cells \[[@CR18]\]. Cytoplasmic CCND1 controls the migration and invasiveness of mantle lymphoma cells \[[@CR36]\]. However, more evidence is needed to support the function of CCND1 in cell invasion and metastasis, especially in clinic samples. CCND1 is one of the DEGS with elevated expression in both metastatic lymph nodes and metastatic primary tumor of oral cancer in our analysis. The results suggested the important function of CCND1 in promoting oral cancer lymph node metastasis although the detained mechanism still needs to be explored.
In sum, this study identifies a unique gene signature -- CCND1, JUN and SPP1 -- which could be a new early biomarker for diagnosing oral cancer lymph node invasion and metastasis.
Additional files
================
{#Sec17}
Additional file 1:**Table S1.** DEGs of GSE70604 Associated Study. (XLSX 106 kb) Additional file 2:**Table S3.** Upregulated DEGs of GSE70604 Associated Study. (XLSX 57 kb) Additional file 3:**Table S2.** Downregulated DEGs of GSE70604 Associated Study. (XLSX 59 kb) Additional file 4:**Table S4.** DEGs of GSE2280 Associated Study. (XLSX 70 kb) Additional file 5:**Table S5.** Downregulated DEGs of GSE2280 Associated Study. (XLSX 42 kb) Additional file 6:**Table S6.** Upregulated DEGs of GSE2280 Associated Study. (XLSX 39 kb) Additional file 7:**Table S7.** Consistent Overlapping DEGs. (XLSX 11 kb) Additional file 8:**Table S8.** Opposite Overlapping DEGs. (XLSX 13 kb) Additional file 9:**Table S9.** Top 10 GO Biological Processes of Consistent Overlapping DEGs. (XLSX 12 kb) Additional file 10:**Table S10.** KEGG Pathways of Consistent Overlapping DEGs. (XLSX 10 kb) Additional file 11:**Table S11.** Interacting Gene Pairs of Consistent Overlapping DEGs. (XLSX 9 kb)
CCND1
: Cyclin D1
DEGs
: Differentially expressed genes
GEO
: Gene expression omnibus
OPN
: Osteopontin
OSCC
: Oral squamous cell carcinoma
SLNB
: Sentinel lymph node biopsy
SPP1
: Secreted phosphoprotein 1
This work was supported by Bureau of Science & Technology and Intellectual Property Nanchong City, China (16YFZJ0026).
Funding {#FPar1}
=======
This work was supported by the Bureau of Science & Technology and Intellectual Property Nanchong City, China (16YFZJ0026).
Availability of data and materials {#FPar2}
==================================
Not applicable.
LY and LQ carried out the molecular genetic studies, participated in the sequence alignment. LK and ZX drafted the manuscript. HX and WH carried out the immunoassays. ZL and TX participated in the sequence alignment. ZX and MH participated in the design of the study and performed the statistical analysis. LK and FG conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
Not applicable.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
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AFTER CATASTROPHE, WE SEEK INDIVIDUALS TO BLAME {#sec1}
===============================================
After a disaster such as the COVID-19 pandemic, there will be an irresistible desire to blame others. People will attribute intentionality to actions that were purely accidental and deem people careless for failing to prevent unforeseeable harms.[^1^](#fn1){ref-type="fn"} Even assessments of causation will be influenced by whether we think someone acted selfishly.[^2^](#fn2){ref-type="fn"} We will blame our neighbors for going to the grocery store without wearing masks. We will blame those who hoarded pallets of toilet paper. We will blame the Chinese. We will blame the broken health care system. We will blame the physician who ignored our appeals for a ventilator for our dad. After so much death, we may even blame God. The post-mortems will frustratingly attempt to identify *the* cause of this catastrophe, as if there were that simple.
THE PRIMARY TARGET OF OUR BLAME SHOULD BE THE PRESIDENT AND HIS ADMINISTRATION {#sec2}
==============================================================================
Hopefully, in the midst of this powerful need to blame, we will not forget who the primary target of our outrage should be---our federal executive branch. We are only midway through the crisis, and it is already quite clear that the president's response has been nothing short of reckless.[^3^](#fn3){ref-type="fn"} He has made a number of notable missteps that may have exacerbated the death toll; to make this plain I will canvass just a few of them here. Rather than immediately using the Defense Production Act to order companies to produce needed tests and medical supplies, the president downplayed the seriousness of the virus.[^4^](#fn4){ref-type="fn"} The failure to immediately develop testing capacity led directly to an inability to conduct effective contact tracing. When tests were initially developed by the Centers for Disease Control (CDC), they were determined to be contaminated due to sloppy laboratory procedures.[^5^](#fn5){ref-type="fn"} Other laboratories began developing reliable tests for the virus, but the CDC wanted to be the sole source, and Federal Drug Administration (FDA) regulations that required emergency use authorization were slow to be approved.[^6^](#fn6){ref-type="fn"} These initial testing failures allowed the virus to quietly spread among the many who are asymptomatic.[^7^](#fn7){ref-type="fn"} Due to the termination of the senior director for global health security and biodefense in 2018, there has not been a coordinated, authoritative federal response.[^8^](#fn8){ref-type="fn"} Doctors have complained that they are not looking to the CDC for trustworthy data, and are instead relying on more informal networks and non-governmental publications.[^9^](#fn9){ref-type="fn"}
The president also frequently misstated the availability of testing, leading people to bombard their local health care facilities for tests that they did not have.[^10^](#fn10){ref-type="fn"} Regarding treatment, the President later claimed that the FDA had approved a 'game-changer' drug (hydroxychloroquine), despite the FDA later correcting that the drug was still being tested for safety and efficacy.[^11^](#fn11){ref-type="fn"} His frequent false statements led many to call for the networks to stop broadcasting his press-briefings.[^12^](#fn12){ref-type="fn"}
Even worse, he created a bidding war between states for much needed supplies like masks and ventilators,[^13^](#fn13){ref-type="fn"} with his administration apparently dolling them out preferentially to the states who had given him political support.[^14^](#fn14){ref-type="fn"} While private businesses stepped into the void to begin producing hand sanitizer and ventilators, White House officials "doubled down on proposed cuts to health services and the Centers for Disease Control and Prevention (CDC)".[^15^](#fn15){ref-type="fn"} Then, perhaps to deflect from these gross errors, he puzzled everyone by announcing he would be defunding the World Health Organization in the midst of the largest pandemic of our lifetimes, while he investigated its mismanagement of the coronavirus.[^16^](#fn16){ref-type="fn"} This move was described by experts as 'counterproductive' and "leav\[ing\] the U.S. and the world less safe." [^17^](#fn17){ref-type="fn"}
He then claimed he had 'total' authority to mandate a premature reopening of the state economies, which went against his health expert's advice, and notably the US Constitution.[^18^](#fn18){ref-type="fn"} When armed individuals in Michigan, Minnesota, and Virginia protested the continued social distancing orders and closure of non-essential businesses, he appeared to encourage insurrection, tweeting "LIBERATE VIRGINIA, and save your great 2nd Amendment. It is under siege!"[^19^](#fn19){ref-type="fn"}
GOVERNMENT IMMUNITY INSULATES WRONGDOING {#sec3}
========================================
Lamentably, despite being the primary mistake-maker here, the federal government itself will enjoy broad immunity, and will be essentially protected from any tort liability related to its COVID-19 response. The concept of sovereign immunity derives from English law, where it was assumed that "the King can do no wrong."[^20^](#fn20){ref-type="fn"} Since the 13th century, the English monarchy could not be sued unless it agreed to waive immunity. The concept was somewhat clumsily borrowed and applied to our federal and state governments in the USA, despite our emphatic rejection of an unaccountable Crown.
There are principled justifications to federal sovereign immunity. Namely, the separation of powers supports executive agency policy decisions being protected from being second-guessed by the courts.[^21^](#fn21){ref-type="fn"} Immunity may also be defended on instrumental grounds, as the government's motive is to protect the general welfare, and not to maximize profits and avoid liability.[^22^](#fn22){ref-type="fn"} While these claims have merit, there are even more powerful counterarguments, which go to the very structure and purpose of our democratic government. Erwin Chemerinsky has effectively argued that the doctrine is an 'anachronistic relic' and that "\[n\]o government - federal, state, or local - should be accorded sovereign immunity in any court."[^23^](#fn23){ref-type="fn"} He bases his claim on both functional and historical accounts of the Constitution. According to this view, democratic principles require maximizing governmental accountability, not reducing it, and the courts can provide an important check on the power and mismanagement of the federal government.[^24^](#fn24){ref-type="fn"} To be sure, the presence of broad immunity may encourage defendants to "push the doctrine to its limits."[^25^](#fn25){ref-type="fn"} That is, knowing that immunity exists may incentivize reckless and authoritarian behavior.[^26^](#fn26){ref-type="fn"} For this reason, immunity should be as narrow as possible to effect its policy rational. It works best when it insulates individuals from even the prospect of a lawsuit for actions that are objectively reasonable under the circumstances. It works much less well when it is shielding individuals from accountability for gross negligence or abuse of discretion.[^27^](#fn27){ref-type="fn"}
Recognizing that fairness dictates that the federal government should waive immunity in some instances, Congress passed the Federal Tort Claims Act (FTCA) in 1946. [^28^](#fn28){ref-type="fn"} In theory, the FTCA allowed private citizens to sue the federal government for civil damages when it acted like a private party.[^29^](#fn29){ref-type="fn"} However, the statute retains immunity in so many cases of intentional wrongdoing that in practice it continues to be quite difficult to obtain damages from the federal government.[^30^](#fn30){ref-type="fn"}
Of relevance here, when the federal government or its agencies perform a discretionary function or duty, there is no waiver of immunity under the FTCA even if that discretion is found to be abused.[^31^](#fn31){ref-type="fn"} Discretionary acts are those that "involv\[e\] an element of judgment or choice," rather than an official merely following a directive.[^32^](#fn32){ref-type="fn"} They thus cover a wide range of government activity. Most of the federal government's response to COVID-19 would be described as 'discretionary,' as there was no rulebook dictating particular moves. Thus, immunity would not be waived, and those injured by the government's failures cannot sue for tort damages.[^33^](#fn33){ref-type="fn"}
While the chief mismanagement appears to be on the federal level, there are even greater barriers to holding state governments accountable should one desire to do so. States have government immunity statutes similar to the FTCA that typically retain immunity for discretionary functions. However, even if a plaintiff was able to describe a specific, non-discretionary duty that was breached, she would likely still lose due to the common law 'public duty' doctrine.
The public duty doctrine holds that because governments owe a duty of care to every citizen, they do not owe a duty to any one particular citizen. Both state and federal courts have interpreted this to mean that government officials such as emergency responders cannot be sued in tort when they fail to perform their duties, such as sending aid in response to a 911 call. If there was no direct contact or assurance of aid to a particular individual, then there was no duty for the dispatcher, for example, to perform her functions. The public duty doctrine has astonishing consequences, and often insulates the police, emergency responders, and many state agencies from being accountable for their negligence or wrongdoing. There is nothing to suspend this doctrine during public health emergencies, and in fact, the emergency situation is likely to expand its application.
1. Political Consequences Are Insufficient {#sec4}
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To summarize, as a result of both the discretionary function exception and the public duty doctrine, we can expect that injured individuals will not be able to seek adequate legal recourse from our federal or state governments for their failure to respond appropriately to the pandemic. Thus, one of the only ways the federal government will be accountable for wrongdoing will be if the president suffers political consequences in the next election. Relying solely on political accountability is worrisome, however.
For one, political accountability has been significantly watered down in the last decade, in part due to almost unlimited corporate money in political campaigns,[^34^](#fn34){ref-type="fn"} and the distortions of representation in the electoral college[^35^](#fn35){ref-type="fn"}. This is also fueled by information asymmetry; we cannot assume that the electorate has the bandwidth to pay attention to the many conflicting news stories and evidence of political wrongdoing. Political accountability requires a great deal of focus and time that many voters simply do not have. This may be in part why it has taken considerable presidential mismanagement to move the needle at all. The president's approval ratings were at an all-time high despite some major mistakes in his response to COVID-19. They only very recently have started to decline.[^36^](#fn36){ref-type="fn"} Importantly, however, even if the president suffers significant political consequences from his mishandling of the pandemic, almost none of this will trickle down to the agency executives or career bureaucrats who did his bidding. While political accountability in theory provides a check on shoddy political decision-making, it provides a very weak check on agencies.[^37^](#fn37){ref-type="fn"} This is sometimes by design[^38^](#fn38){ref-type="fn"}.
Perhaps most important of all, political accountability, even if perfect, does absolutely nothing to compensate those who were individually injured by executive recklessness. The need to compensate injured parties provides the strongest basis for removing immunity. If government is responsibly conducting their risk benefit analyses, there will not be many plaintiffs seeking compensation. But the complete lack of a remedy for carelessly inflicting personal harm flies in the face of our democratic principles of government.
TARGETED IMMUNITY FOR PARTICULAR GROUPS {#sec5}
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In addition to governmental immunity, there are a patchwork of immunities that attach to various groups for their undertakings in response to COVID-19. On March 17th, the Secretary of Health and Human Services issued an emergency declaration that triggered broad immunity protections contained in the Public Readiness and Emergency Preparedness Act (PREP Act) of 2005.[^39^](#fn39){ref-type="fn"} This Act immunizes, from all federal and state lawsuits, health care professionals who administer or use specified countermeasures as antiviral medications during declared public health emergencies such as COVID-19.[^40^](#fn40){ref-type="fn"} So long as health care providers administer the approved treatments during the window of the declared emergency, they can only be liable under the PREP Act if they engaged in willful misconduct.[^41^](#fn41){ref-type="fn"}
Some states have passed similar immunity for providers who prescribe unapproved drugs, such as hydroxychloroquine.[^42^](#fn42){ref-type="fn"} This is concerning. Immunity should be used to remove the threat of a lawsuit when providers act in ways that are objectively reasonable, given the circumstances. Prescribing treatments that are not yet proved to be safe or effective, even in a pandemic, could violate the standard of care. Therefore, this is not the kind of conduct that should be categorically protected from tort liability.[^43^](#fn43){ref-type="fn"}
On March 27th, the president signed the "Coronavirus Aid, Relief, and Economic Security Act" (CARES Act) into law.[^44^](#fn44){ref-type="fn"} The Act provides broad liability immunity for companies that manufacture respiratory protective devices. It also preempts state law to immunize volunteer health care workers from tort liability for negligence in actions or omissions in the course of providing volunteer health care services related to COVID-19.[^45^](#fn45){ref-type="fn"} The constitutionality of the latter preemption will no doubt be challenged, as the states typically regulate intrastate tort liability. Indeed, most states already immunize volunteer health care workers for carelessness when they respond to an emergency.[^46^](#fn46){ref-type="fn"} Even so, the immunity provisions in the PREP and CARES Acts speak to Congressional recognition that the fear of liability might stymie our pandemic response, both at the state and federal levels.
2. Paid Health Care Providers Are Left Out from These Targeted Immunity Provisions {#sec6}
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Of all of the predictable defendants to be sued after a pandemic, there is one group that is glaringly absent from this list: non-volunteer health care workers.[^47^](#fn47){ref-type="fn"} Even when they work for a state hospital, nurses, and physicians are typically not protected under the state's governmental immunity, as they "exercised medical judgment, regardless of whether it related to policy decision."[^48^](#fn48){ref-type="fn"} As parties will be looking for someone to hold accountable for the terrible outcomes they have experienced, physicians are the only group that is out in front, with enormous bullseyes on their backs.
This situation is particularly distressing as there is a huge asymmetry between the risks health care workers are undertaking, and the legal protections afforded them. Due to multiple government failures in the early days of SARS-cov-2 virus, physicians and nurses have been rendered particularly vulnerable. In addition to not being able to be tested themselves before reporting to be overworked in the ICUs, shortages in personal protective equipment have exposed many of them to the deadly virus.[^49^](#fn49){ref-type="fn"} Nearly 20 percent of all confirmed COVID-19 cases in the USA are in health care workers,[^50^](#fn50){ref-type="fn"} and worldwide more than a hundred have died so far from the infection.[^51^](#fn51){ref-type="fn"}
What's more, again due in part to government failures, some hospital's resources may soon be overwhelmed---with insufficient ventilators, blood products, and personnel to meet the demand. Physicians may have to decide whom to admit, whom to ventilate, and whom to treat with extracorporeal membrane oxygenation (ECMO). Ad hoc decision-making leads to discrimination against people of color, people with disabilities, and people who simply lack the entitlement to question their denial of care.[^52^](#fn52){ref-type="fn"} It also places significant moral responsibility on the individual physician, who may struggle to shoulder this weight.[^53^](#fn53){ref-type="fn"}
3. Rationing Policies, and Lack of Institutional Policies, Expose Physicians to Liability {#sec7}
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To encourage more ethical decision-making and relieve the pressure on individual physicians, several working groups, states, and institutions have developed triage protocols, or Crisis Standards of Care plans (CSCs) based on different notions of distributive justice. For example, the American College of Chest Physicians (CHEST) Task Force for Mass Critical Care has published suggestions for rationing ICU beds and treatments that have been adopted by some institutions[^54^](#fn54){ref-type="fn"}. According to this consensus statement, patient groups with a life expectancy of less than 1 year or with a predicted mortality rate of over 90 per cent should not be admitted to an ICU when resources are overwhelmed during a pandemic.[^55^](#fn55){ref-type="fn"} Other protocols prioritize short-term clinical factors, but then suggest using life-cycle considerations as a tiebreaker, with priority going to younger patients.[^56^](#fn56){ref-type="fn"} There is a robust debate occurring about the ethics and potential illegal discriminatory impacts of these policies.[^57^](#fn57){ref-type="fn"} The purpose of this article is not to advocate for a particular framework, but rather to provide legal immunity for physicians who make decisions in compliance with a documented policy.[^58^](#fn58){ref-type="fn"} If an institution has no documented policy, and leaves rationing up to the individual physician, they expose both the physician and the institution to legal liability from discriminatory or substandard decisions. Implementing a policy that complies with anti-discrimination law is prudent in these situations to reduce this risk.
Despite strong recommendations to do so, many institutions have failed to develop CSCs, or if they have their specific requirements are unclear.[^59^](#fn59){ref-type="fn"} Even hospitals that have developed plans are struggling with how to incorporate emerging data regarding COVID-19 risk factors and urgent requests for deviation from the protocol. While often not part of any CSC, there is also the problem with a kind of 'soft' rationing, where patients are not even offered transfers from a nursing home to an ICU. The lack of institutional or legal guidance leaves a lot of unwanted discretion within the hands of individual doctors. For example, if an institution supports a 'lottery' system once near-term survival is taken into account, it is not clear how that would actually be operationalized in practice.
There are only so many physicians and nurses, and only so many beds. The possibility of a medical malpractice lawsuit will do nothing to prevent the need to ration. If there is only one ICU bed available, and 10 patients vying for it, the possibility of *ex post* tort liability will not instantly create nine more beds. Perhaps if the government could be accountable for this failure, more beds and ventilators could be produced or reallocated from regions that are not as hard hit by COVID-19. But the physicians on the frontlines cannot be expected to create additional resources while fighting this pandemic. The normal incentives of deterrence will thus not work to create better rationing or to remove the need for rationing. Indeed, the possibility of liability will only make the necessary rationing more discriminatory and unfair, as physicians cater to the loudest, wealthiest, or most educated patients who fight the hardest, and who seem most likely to sue.[^60^](#fn60){ref-type="fn"}
4. COVID-19 Specific Protocols Expose Health Care Workers to Liability {#sec8}
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There will be many institutional policies related to COVID-19 that will expose individual physicians to liability, and these instances may be even more common than the need to ration scarce resources. Physicians are being instructed by their states, professional associations, and institutions to do things differently, and in ways that may violate the standard of care if it were not for COVID-19.[^61^](#fn61){ref-type="fn"} For example, during cardiac resuscitation some physicians are being told that all patients must be intubated, rather than using manual ventilation, like a bag. Intubation reduces the risk of transmitting COVID-19 to the medical team through the patient's coughing, but it creates other risks for the patient, and imposes delays. I have received reports of physicians in some areas being instructed not to use cardiac catheterization on heart attack patients, due to risks of physician exposure to COVID-19.[^62^](#fn62){ref-type="fn"} Other policies include mandatory emergency intubation during thrombectomies for people experiencing stroke. These procedures would ordinarily not involve intubation, but for the risk of COVID-19 being aerosolized during the procedure. If there is an injury or bad outcome from the intubation, a patient who did not have COVID-19 may question the wisdom of intubating them.
In addition to taking extra precautions during procedures, physicians are also being instructed by their employers to reschedule or cancel cancer, heart, and lung interventions that they think can wait several weeks. This is done both to reduce the risk of the immune-compromised from being infected at the hospital, as well as to reserve ventilators and beds for COVID-19 patients who might later need them. Of course, prognosticating is difficult, and physicians have no crystal ball to assess whether an individual patient really can wait several weeks before having a tumor resected or fluid drained. It is possible that patients who had a delayed diagnosis or treatment might sue the physicians who made this call, if it turns out not to have been clinically appropriate for them. While these decisions might all be perfectly reasonable during a pandemic, the fear (or reality) of having to defend these utilitarian decisions in court might add undue stress on the physicians who are doing their best to follow state or institutional policy.[^63^](#fn63){ref-type="fn"} The value of immunity comes not just from rejecting the second-guessing of emergency decisions that were thrust upon an overwhelmed industry. There is also considerable value in giving physicians peace of mind that the forced choice between two terrible possibilities will not later be penalized in court.
During normal times, emotions run high when a loved one is denied medical treatment or injured during a procedure. The resulting break-down of communication may lead to a lawsuit, as many people will be unhappy with how resources were rationed or decisions made. Under ordinary circumstances, a physician may be liable for a clinical judgment that could foreseeably cause a patient's death. However, these are not ordinary circumstances, and health care providers are being asked to make impossible decisions that may aid our ability to respond to COVID-19, but which might increase the risk to individual patients. There is often no third option that avoids putting a particular patient at risk to help others. Immunity is appropriate in these situations, where the providers' decisions are objectively reasonable and defensible *ex ante*, and for which there would be great psychological value in removing the possibility of suit.
While it might seem unlikely that many families will sue, as they should appreciate that the extenuating circumstances of the pandemic, not all families will be so understanding or reasonable.[^64^](#fn64){ref-type="fn"} To be sure, there may be greater distrust of physician decision-making and increased incentives to sue in the wake of this pandemic, as people are more isolated and anxious, and may be suffering from severe economic instability. Additionally, as the social distancing measures cities and states have adopted to "flatten the curve" are working, the lack of a big surge in infections as was seen in New York might anger those patients who assume that the precautionary measures were not necessary.[^65^](#fn65){ref-type="fn"}
Given this, we can expect that some number of families will claim that a physician who denied their family member treatment, or performed a procedure differently due to COVID-19 precautions, caused them a compensable injury. Depending on the circumstances, a judge and jury may agree. Causation and breach are not discoverable facts of nature, but rather are morally laden and susceptible to hindsight bias.[^66^](#fn66){ref-type="fn"} Hindsight bias could be particularly severe here. Cases likely will not be litigated until after the chaos of the pandemic has subsided, which might permit juries to forget just how extenuating the circumstances appeared to be *ex ante*. Therefore, even if physicians were doing their best at the time to fairly allocate resources and follow institutional policies, a jury could find them liable for making an intolerable choice that caused harm. Indeed, even if the possibility of an award is remote, the very prospect of this possibility may create unfair and paralyzing fear for physicians, who as a group already overestimate the risk of being sued.[^67^](#fn67){ref-type="fn"}
HEALTH CARE PROVIDERS NEED STATE-CONFERRED TORT IMMUNITY WHEN FOLLOWING PROFESSIONAL, INSTITUTIONAL OR STATE POLICIES RELATED TO COVID-19 {#sec9}
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This article advocates for removing the possibility of a medical malpractice claim for individual physicians and independent health care providers, when they are complying with published state, professional, or institutional COVID-19 policies in good faith. This would include deviations from normal practice to reduce the risk of SARS-cov-2 transmission, as well as rationing decisions that comply with pre-existing policy directives. Ideally, as part of any emergency response packages that are passed, legislatures should either develop, or require hospitals to develop, protocols for rationing ventilators, ECMO machines, ICU beds, and personnel. It would be best if the policies were mandatory at the state level, to encourage cooperation between health care facilities.[^68^](#fn68){ref-type="fn"} Further, and more to the point, when enacting these statutes, legislatures should incorporate tort immunity for health care workers who comply with COVID-19 treatment protocols in good faith. In the absence of a state directive, this article also advocates for immunity for individual physicians when they comply with a recognized professional organization's guidance related to COVID-19, or institutional policies related to their COVID-19 response.
This is not merely to limit the liability of a powerful group. It is to recognize the unbearable situation that health care workers face during a pandemic, which is not at all of their own creation. It is also to recognize that certain decisions might appear unreasonable *ex post*, but they were not unreasonable *ex ante*. Health care providers are under an inordinate amount of stress as they expose themselves to a serious or deadly disease, often while working incredibly long hours. The extenuating circumstances of a pandemic necessitate immunity for physicians who are doing their best to bravely make critical decisions, with imperfect information, institutional and professional directives that run against the normal standard of care, and with highly constrained resources. We should not put physicians in the position of having to choose between the pre-COVID-19 standard of care, and violating their institutional policies. Importantly, as the hospitals and institutions are the ones making the cost-benefit analysis regarding triage and mandatory intubations, immunity should not attach to them.[^69^](#fn69){ref-type="fn"} Patients could still sue institutions and hospitals for policies that, under the circumstances of the pandemic, were not state-mandated and were objectively unreasonable.
There is precedent for this sort of immunity. Many state statutes extend immunity to volunteer health care workers under so-called Good Samaritan laws, as the CARES Act does. However, these volunteers often have to be responding to an emergency clinical situation, and must be unpaid, so these laws would not immunize ordinary health care workers during a pandemic.[^70^](#fn70){ref-type="fn"}
Medical futility statutes provide some guidance, as they have been enacted in many states to shield physicians from negligence claims if they refuse to offer ineffective life-sustaining treatment.[^71^](#fn71){ref-type="fn"} However, medical futility statutes should not be used during the COVID-19 pandemic to confuse medically ineffective care with care that would be appropriate if there were not resource constraints. Indeed, physicians who put pressure on particular groups to reconsider their end-of-life plans, to discourage only certain groups from refusing life-sustaining treatment, should emphatically not be immunized from tort liability.[^72^](#fn72){ref-type="fn"} Further, given our experience with medical futility statutes, it would be prudent to afford clearer protection from suit than what the medical futility statutes typically provide.[^73^](#fn73){ref-type="fn"} For examples targeted at providing physicians immunity in response to a pandemic, we can also look to laws in effect in Maryland and New York.
IMPROVING UPON THE MARYLAND AND NEW YORK PROVIDE EXAMPLES {#sec10}
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At least two states have passed targeted immunity laws, which would protect paid health care providers during a pandemic, for different kinds of activities. The Maryland statute immunizes health care providers from civil or criminal liability when "acting in good faith and in accordance with a catastrophic health emergency disease surveillance and response program." [^74^](#fn74){ref-type="fn"} This is part of a law focused on penalizing non-compliance with public health directives. This immunity focuses on protecting clinicians who follow the state-approved ventilator rationing protocols, "regardless of the negative consequences arising from the withdrawal of a patient's ventilator."[^75^](#fn75){ref-type="fn"} It does not provide immunity for institutional or personal medical decisions that are not prescribed by the state's public health emergency response program. In this sense, the immunity is fairly limited. It also does not attach in cases of willful misconduct, or when the physician acts in bad faith.[^76^](#fn76){ref-type="fn"}
New York's immunity law came by way of executive order. On March 23, 2020, Governor Cuomo issued an order revising an education law to provide.
> "that all physicians, physician assistants, specialist assistants, nurse practitioners, licensed registered professional nurses and licensed practical nurses shall be immune from civil liability for any injury or death alleged to have been sustained directly as a result of an act or omission by such medical professional in the course of providing medical services in support of the State's response to the COVID-19 outbreak."[^77^](#fn77){ref-type="fn"}
The immunity does not attach if the injury was caused by gross negligence, but it is otherwise much broader than the Maryland statute. For one, it provides immunity for actions that are not performed in accordance with any state public health directive, but are rather just 'in support' of the state's response. It also provides for immunity in non-emergency situations. A reasonable interpretation could include immunizing providers who have personally decided to delay treating their non-COVID-19 patients, in order to prioritize COVID-19 patients. This would be an act or omission in the course of providing medical services to support the state's response, but it may not be the kind of immunity intended. Thus, if a health care provider decided to cancel all non-COVID-19 procedures, in order to keep ventilators available for COVID-19 patients, this order may prohibit the non-COVID-19 patient from suing in tort, even if that decision was unreasonable under the circumstances. Of course, the order says nothing about immunizing health care institutions who make and enforce similar policies.
The Maryland statute is both too narrow and too broad. It is too narrow as it focuses only on immunizing rationing decisions that are part of the state's public health directive, and not care that is provided in conformity with institutional or professional recommendations. It is too broad in that it immunizes physicians from criminal charges and intentional wrongs. In general, immunity from criminal charges, or even anything above gross negligence, is both unnecessary and unwise. Prosecutors are exceedingly unlikely to bring charges against physicians who are making these kinds of pandemic-related decisions, and in any event if a prosecutor could show that a physician acted with an intent to kill, then a criminal action seems completely appropriate. A civil action also seems appropriate, if unlikely, where a plaintiff can make out a prima facie claim that the physician acted with gross negligence or worse. Immunity should only cover negligence claims to strike the right balance between protecting physicians and compensating plaintiffs who were clearly wronged. Denying actions for criminal, or grossly negligent actions, provides too much immunity and has the potential to absolve, or even encourage, bad actors.
The New York order is also overly broad, but for different reasons. It immunizes everything related to the state's response to COVID-19, which might include too many clinical decisions that are not within the standard of care, and which are not required by a physician's institution or even recommended by her professional organization. Providing physicians negligence immunity for good faith decisions that are made either in response to professional guidance, state law, or institutional policy related to COVID-19 best balances the need to recognize (a) the extenuating circumstances of the pandemic, (b) the conflicting obligations to the individual patient, themselves, other patients, and institutional obligations, (c) the heavy toll of hindsight bias in negligence actions, and (d) the need for liability for decisions that are grossly negligent or not in good faith.
In summary, states should pass statutes now that immunize individual physicians and other health care providers from negligence liability when, in good faith, they comply with an institutional or state directive, or clear professional guidance, in response to the COVID-19 pandemic, or future pandemics.[^78^](#fn78){ref-type="fn"} This recognizes that in many cases physicians are not responding to state-level legal directives, but are rather complying with institutional or professional policies that either mandate or strongly encourage certain kinds of action. While plaintiffs could ordinarily sue both the nurses, institution, and physicians together, these laws would limit negligence suits to the institutions that are making and enforcing COVID-19 specific policies. Physicians would not be immunized for every personal clinical decision related to COVID-19. That level of immunity is too broad, encourages too much carelessness, and denies appropriate compensation to injured patients. While under my proposal, institutions would remain liable for policy decisions they implement, this might be unsatisfactory as it is harder for a plaintiff to prove that a policy, as opposed to an individual physician's decision, violated the standard of care.
CONCLUSION {#sec11}
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Ideally, the cost of making difficult decisions during a pandemic should not fall on individual patients. Those who are injured by others' carelessness should have some legal resource when they are wronged. But our powerful desire to correct a potential injustice, and to shift the cost elsewhere, should not come at the expense of holding individual health care workers responsible for decisions that were reasonable *ex ante*. There is great risk of hindsight bias, where juries might forget the very real and conflicting pressures physicians were facing, especially if communities do not experience the overwhelming surges that were predicted, but do experience significant financial hardship.
The summary judgment standard of "no reasonable juror could find breach" provides a good guide for tailoring immunity to those situations where it can do the most good and the least harm. Immunity laws recognize that even if a plaintiff is properly denied recovery at the summary judgment stage, getting to this point imposes considerable emotional and financial costs on the individual physicians. In addition to having to review case files, be deposed, and respond to interrogatories, health care providers often have to report all medical malpractice complaints, regardless of the outcome, every time they seek new credentials. Providing negligence immunity for decisions in good faith conformity with state, professional or institutional guidelines, should significantly curb the number of meritless cases that are filed.[^79^](#fn79){ref-type="fn"} Physicians did not create this pandemic situation. While they are intimately involved in its mitigation, these heroic efforts should not expose them to unnecessary malpractice liability, merely because government immunity makes them the last target standing.
Our state and federal governments should be accountable, as public health agencies and government figures have a moral and legal duty to protect their citizens and prepare for health emergencies.[^80^](#fn80){ref-type="fn"} There is a possibility that the federal government could waive immunity for personal injury related to its response to the pandemic. Following the threat of a previous H1N1 pandemic, Congress passed the Swine Flu Act, which created a private right of action against the USA from injuries resulting from swine flu inoculation.[^81^](#fn81){ref-type="fn"} Or, the government could set up a compensation fund for families adversely affected by rationing decisions. While removing immunity---where the government acted recklessly or in ways that abused its discretion---would be a sensible way to provide accountability, it also seems politically quite unlikely.
We *can* hope for some political recourse, as the executives and their agencies that failed to prepare or respond may be removed from office. However, for the reasons described above, this is an enormously imperfect check. Crucially, it provides no remedy for the parties who are injured by carelessness or wrongdoing. If a corporation develops a defective product that injures thousands, removing the executives in charge does nothing to compensate those who were harmed.
We are learning many lessons from this pandemic, some welcome and some not. One lesson we might take away from this is the need for better remedies against government officials when they completely fail to perform their public duties. The time has come to pass legislation that more significantly waives immunity in line with our democratic ideals. Now, more than ever, we must fight the dangerous notion that our leaders are kings.
Mark Alicke, *Blaming Badly*, 8 J. of Cognition and Culture 179 (2008).
Joshua Knobe and Scott Shapiro, *Proximate Cause Explained: An Essay in Experimental Jurisprudence*, forthcoming in the University of Chicago Law Review, https://ssrn.com/abstract=3544982 or http://dx.doi.org/10.2139/ssrn.3544982, at 16--17; see also Fiery Cushman, Joshua Knobe, & Walter Sinnott-Armstrong. *Moral Appraisals Affect Doing/Allowing Judgments,* 108 COGNITION 281 (2008).
Ed Yong, How the Pandemic Will End, The Atlantic, Mar. 25, 2020, <https://www.theatlantic.com/health/archive/2020/03/how-will-coronavirus-end/608719/>.
Zolan Kanno-Youngs and Ana Swanson, *Wartime Production Law Has Been Used Routinely, but Not With Coronavirus,* NY Times, Mar. 31, 2020, <https://www.nytimes.com/2020/03/31/us/politics/coronavirus-defense-production-act.html>.
Sheila Kaplan, *C.D.C. Labs Were Contaminated, Delaying Coronavirus Testing, Officials Say,* NY Times, Apr. 18, 2020, <https://www.nytimes.com/2020/04/18/health/cdc-coronavirus-lab-contamination-testing.html>.
Bob Ortega, Scott Bronstein, Curt Devine, and Drew Griffin, *How the government delayed coronavirus testing*, CNN, Apr. 9, 2020, <https://www.cnn.com/2020/04/09/politics/coronavirus-testing-cdc-fda-red-tape-invs/index.html>.
David Frum, *This is Trump's Fault*, Apr. 7, 2020, The Atlantic, <https://www.theatlantic.com/ideas/archive/2020/04/americans-are-paying-the-price-for-trumps-failures/609532/>.
Liz Alesse, *Did Trump try to cut the CDC's budget as Democrats claim?: ANALYSIS*, ABC News, Feb. 28, 2020, <https://abcnews.go.com/Politics/trump-cut-cdcs-budget-democrats-claim-analysis/story?id=69233170> ("The president fired the pandemic specialist in this country 2 years ago," former NYC mayor Michael Bloomberg said. "So, there's nobody here to figure out what the hell we should be doing.")
Robert Baird, *How Doctors on the Front Lines Are Confronting the Uncertainties of COVID-19,* The New Yorker, Apr. 5, 2020, <https://www.newyorker.com/science/medical-dispatch/how-doctors-on-the-front-lines-are-confronting-the-uncertainties-of-covid-19?itm_content=footer-recirc>.
Meg Kelly, Sarah Cahlan and Elyse Samuels, *What went wrong with coronavirus testing in the U.S.*, Washington Post, Mar. 30, 2020, <https://www.washingtonpost.com/politics/2020/03/30/11-100000-what-went-wrong-with-coronavirus-testing-us/> (On March 6th, President Trump: "A*nybody that needs a test, gets a test. They're there. They have the tests. And the tests are beautiful."*).
Shannon Pettypiece, *Trump's coronavirus claims have not matched response reality*, NBC News, Mar. 20, 2020, <https://www.nbcnews.com/politics/white-house/trump-s-coronavirus-claims-haven-t-matched-response-reality-n1164041>. During an official briefing, the President also infamously suggested that ingesting disinfectants or ultra-violet rays might be a promising treatment, despite this being a dangerous and unapproved treatment. He later said that these remarks were meant to be "sarcastic." See, Sheera Frenkel and Davey Alba, *Trump's Disinfectant Talk Trips Up Sites' Vows Against Misinformation*, NY Times, Apr. 30, 2020, <https://www.nytimes.com/2020/04/30/technology/trump-coronavirus-social-media.html>.
Chantal da Silva, Over 200,000 Sign Petition Calling for End to Live Coverage of Trump's Coronavirus Briefings
Newsweek, Mar. 31, 2020, <https://www.newsweek.com/nearly-100000-sign-petition-calling-end-live-coverage-trumps-coronavirus-briefings-1495195>.
Jeanne Whelan et al., Scramble for medical equipment descends into chaos as U.S. states and hospitals compete for rare supplies, Washington Post, Mar. 24, 2020, <https://www.washingtonpost.com/business/2020/03/24/scramble-medical-equipment-descends-into-chaos-us-states-hospitals-compete-rare-supplies/>.
Manu Raju, *Colorado Democrat believes Trump awarded ventilators as political favor to vulnerable GOP senator*, CNN, Apr. 9, 2020, <https://www.cnn.com/2020/04/08/politics/degette-gardner-trump-ventilators-favor/index.html> ("Process employed by the White House shows that the President appears to be doling out the ventilators to his allies at a time when the virus is affecting people of all political persuasions.")
Niv Elis, Trump budget chief holds firm on CDC cuts amid virus outbreak, The Hill, Mar. 10, 2020, <https://thehill.com/policy/finance/486817-trump-budget-chief-holds-firm-on-cdc-cuts-amid-virus-outbreak>.
[Kai Kupferschmidt](https://www.sciencemag.org/author/kai-kupferschmidt), [Jon Cohen](https://www.sciencemag.org/author/jon-cohen), *'Short-sighted.' Health experts decry Trump's freeze on U.S. funding for WHO as world fights pandemic*, Science Magazine, Apr. 14, 2020, [https://www.sciencemag.org/news/2020/04/trump-freezes-us-funding-who-world-fights-pandemic\#](https://www.sciencemag.org/news/2020/04/trump-freezes-us-funding-who-world-fights-pandemic).
[Kai Kupferschmidt](https://www.sciencemag.org/author/kai-kupferschmidt), [Jon Cohen](https://www.sciencemag.org/author/jon-cohen), *'Short-sighted.' Health experts decry Trump's freeze on U.S. funding for WHO as world fights pandemic*, Science Magazine, Apr. 14, 2020, [https://www.sciencemag.org/news/2020/04/trump-freezes-us-funding-who-world-fights-pandemic\#](https://www.sciencemag.org/news/2020/04/trump-freezes-us-funding-who-world-fights-pandemic).
Adam Taylor et al., *Trump endorses ending coronavirus social distancing soon, against health experts' advice*, Washington Post, Mar. 23, 2020, <https://www.washingtonpost.com/world/2020/03/23/coronavirus-latest-news/>.
Ben Collins and Brandy Zadrozny, *In Trump's 'LIBERATE' Tweets, Extremists See a Call to Arms*, NBC News, Apr. 17, 2020, <https://www.nbcnews.com/tech/security/trump-s-liberate-tweets-extremists-see-call-arms-n1186561>.
Erwin Chemerinsky, *Shifting the Balance of Power? The Supreme Court, Federalism, and State Sovereign Immunity*: *Against Sovereign Immunity,* 53 Stan. L. Rev. 1201, 1201 (2001).
Jameson B. Bilsborrow, *Keeping the Arms in Touch: Taking Political Accountability Seriously in the Eleventh Amendment Arm-of-the-state doctrine*, 64 Emory L.J. 819, 819 (2015).
Lawrence Rosenthal, *A Theory of Governmental Damages Liability: Torts, Constitutional Torts, and Takings*, 9 U. Pa. J. Const. L. 797, 798--799 (2007). This author also supports immunity on corrective justice grounds, as "the government passes its legal costs along to the taxpayers, who bear little meaningful culpability for the underlying tortious conduct..." However, this assumes that corrective justice is all about punishment, as opposed to also being about compensation for plaintiffs. Immunity cannot be justified on grounds of compensating individual victims of government wrongs.
Erwin Chemerinsky, *Shifting the Balance of Power? The Supreme Court, Federalism, and State Sovereign Immunity*, *Against Sovereign Immunity*, 53 Stan. L. Rev. 1201, 1201 (2001).
See, Erwin Chemerinsky, *Shifting the Balance of Power? The Supreme Court, Federalism, and State Sovereign Immunity*, *Against Sovereign Immunity*, 53 Stan. L. Rev. 1201, 1206 (2001); see also Paul Gowder, *The Rule of Law Against Sovereign Immunity in a Democratic State*, 93 Tex. L. Rev. Online 247, 255 (2015); and Eric Berger, *The Collision of the Takings and State Sovereign Immunity Doctrines*, 63 Wash & Lee L. Rev. 493, 548--549 (2006).
Katherine Florey, *Sovereign Immunity's Penumbras: common law, accident, and the policy in the development of sovereign immunity doctrine*, 43 Wake Forest L. Rev. 765, 769 (2008).
This is particularly concerning given that the president has been making more statements in recent weeks indicating he believes the executive to be beyond reproach and to have complete authority: (President Trump: "when somebody is the president of the United States, the authority is total.") See, Meghan Flynn & Allyson Chiu, Trump says his 'authority is total.' Constitutional experts have 'no idea' where he got that, Washington Post, Apr. 14, 2020, available online at \[x\].
Thus, there is no principled reason to immunize private businesses from tort liability for behavior that might be considered objectively careless under the circumstances. See, Natalie Andrews, *Mitch McConnell Wants to Shield Companies From Liability in Coronavirus-Related Suits*, Washington Post, Apr. 29, 2020, <https://www.wsj.com/articles/house-delays-return-to-capitol-amid-uncertainty-over-next-round-of-coronavirus-stimulus-11588091849>.
28 USCS § 2671 (West 2020).
*United States v. Olson*, 546 U.S. 43, 47, 126 S. Ct. 510, 513, 163 L. Ed. 2d 306 (2005).
Katherine Florey, *Sovereign Immunity's Penumbras: common law, accident, and policy in the development of sovereign immunity doctrine*, 43 Wake Forest L. Rev. 765, 765 (2008); For example, the FTCA exempts from waiver any claims "for damages caused by the imposition or establishment of a quarantine by the United States." See, 28 U.S.C.A. § 2680 (West) It also exempts actions for intentional wrongdoing like false imprisonment and battery. Even if the federal government or agencies intentionally misrepresented facts or deceived people, there would be no waiver of immunity. See, 28 U.S.C.A. § 2680(h) (2020).
28 U.S.C. § 2680(a).
*Dalehite v. United States*, 346 U.S. 15, 33 (1953); see also *United States v. Gaubert*, 499 U.S. 315, 322 (1991).
Where the discretionary function exception applies, individuals would need to show that a government official violated their "clearly established" civil rights, which is a tall order, in order to pursue a remedy under 42 U.S. Section 1983. See, *Bivens v. Six Unknown Named Agents of Fed. Bureau of Narcotics*, 403 U.S. 388(1971) See also, *Howlett v. Rose*, 496 U.S. 356, 358, 110 S. Ct. 2430, 2433 (1990) (holding that a state's invocation of sovereign immunity when the petitioner alleges colorable Fourth Amendment claims violated the supremacy clause of the Constitution). Section 233(a) of the Public Health Service Act "grants absolute immunity to PHS officers and employees for actions arising out of the performance of medical or related functions within the scope of their employment by barring all actions against them for such conduct." See, *Hui v. Castaneda*, 559 U.S. 799, 806 (2010); In some cases, even a *Bivens* remedy is precluded, such as when public health service officers or employers are sued under the Public Service Act.
Deborah J. Brooks, and Michael Murov, *Assessing Accountability in a Post-Citizens United Era: The Effects of Attack Ad Sponsorship by Unknown Independent Groups*, 40 American Politics Research, 383--418 (2012).
Katherine Florey, *Losing Bargain: Why Winner-Take-All Vote Assignment Is the Electoral College's Least Defensible Feature*, 68 Case W. Res. L. Rev. 317, 328 (2017).
Despite the documented failures on the part of the federal government to respond to COVID-19, as of April 16th, 43 per cent of American adults still approve of the way Trump is handling his job as president, while 54 per cent say they disapprove. See, Justin Wise, Trump job approval slips 6 points in past month: poll, The Hill, available online at <https://thehill.com/homenews/administration/493165-trump-job-approval-slips-6-points-in-past-month-poll>.
"\[S\]ome policy decisions must be insulated from politics ---and, conversely, that insulating decisions too much creates accountability problems." Miriam Seifter, Understanding State Agency Independence, 117 Mich. L. Rev. 1537, 1541 (2019).
Lisa Schultz Bressman & Robert B. Thompson, *The Future of Agency Independence*, 63 Vand. L. Rev. 599, 600 (describing the desired insulation of independent agencies, as compared to executive agencies).
Public Readiness and Emergency Preparedness Act, 42 U.S.C.A. § 247d-6d (West 2020)
<https://www.govinfo.gov/content/pkg/FR-2020-03-17/pdf/2020-05484.pdf>, <https://www.phe.gov/Preparedness/legal/prepact/Pages/default.aspx>.
42 U.S.C.A. § 247d-6d (West).
Utah Emergency Health Care Access and Immunity Amendments,
<https://legiscan.com/UT/text/SB3002/2020/X3>; see also Associated Press, Utah Buys Malaria Drugs Touted by Trump, Panned by Doctors, Apr. 21, 2020, <https://www.usnews.com/news/best-states/utah/articles/2020-04-21/utah-buys-malaria-drugs-touted-by-trump-panned-by-doctors>.
The Utah statute places the burden on the individual patients, by suggesting that so long as they provide informed consent, immunity is appropriate for prescribing unapproved medications. This appears to be linked with the "Right to Try" movement. However, there are sound reasons why we do not permit individuals to be treated with medications that are not proved as safe or effective, even if an individual's cost-benefit analysis skews toward welcoming greater risk, and we certainly should not absolve providers from liability when their off-label use is negligent. See, *Abigail All. for Better Access to Developmental Drugs v. Von Eschenbach*, 373 U.S. App. D.C. 386, 389 (2006).
S.3548---CARES Act116th Congress (2019--2020), <https://www.congress.gov/bill/116th-congress/senate-bill/3548/text>.
See, Section. 4216. *Limitation on liability for volunteer health care professionals during covid-19 emergency response*, CARES Act, <https://www.congress.gov/bill/116th-congress/senate-bill/3548/text#toc-id5B4AC6639BF64A26916E41980A9BC7EB>.
"Many jurisdictions extend immunity to all persons administering emergency care; others limit coverage to specified medical personnel, or to physicians alone." See, Danny R. Veilleux, *Construction and application of "good Samaritan" statutes,* 68 A.L.R.4th 294 (Originally published in 1989).
Immunity is often discussed in terms of physicians, because nurses and physician assistants are typically indemnified by their employer, and their negligence is imputed to the attending physician or institution. However, where health care providers are independently overseeing clinical care, they too could be sued for medical malpractice; thus, the targeted immunity should cover them as well.
See, *Lather v. Beadle County*, 879 F.2d 365 (8^th^ Cir. 1989).
Somini Sengupta, *A N.Y. Nurse Dies. Angry Co-Workers Blame a Lack of Protective Gear*, NY Times, Mar. 26, 2020, <https://www.nytimes.com/2020/03/26/nyregion/nurse-dies-coronavirus-mount-sinai.html>; Michael Rothfeld et al., *13 Deaths in a Day: An 'Apocalyptic' Coronavirus Surge at an N.Y.C. Hospital*, New York Times, Mar. 25, 2020, <https://www.nytimes.com/2020/03/25/nyregion/nyc-coronavirus-hospitals.html>; see also, Hannah Rappleye, Andrew W. Lehren, Laura Strickler and Sarah Fitzpatrick, *'This system is doomed': Doctors, nurses sound off in NBC News coronavirus survey*, NBC News, Mar. 20, 2020, <https://www.nbcnews.com/news/us-news/system-doomed-doctors-nurses-sound-nbc-news-coronavirus-survey-n1164841>.
Centers for Disease Control and Prevention, *Characteristics of Health Care Personnel with COVID-19---United States*, *February 12--April 9, 2020*, Apr. 17, 2020, <https://www.cdc.gov/mmwr/volumes/69/wr/mm6915e6.htm?s_cid=mm6915e6_x> "Among 315,531 U.S. COVID-19 cases reported to CDC during February 12--April 9, data on HCP occupational status were available for 49,370 (16 per cent), among whom 9282 (19 per cent) were identified as \[health care providers.\]
Medscape, *In Memoriam: Healthcare Workers Who Have Died of COVID-19*, Apr. 1, 2020, <https://www.medscape.com/viewarticle/927976>.
Necia Hobbes, Out of the Frying Pan Into the Fire: heightened discrimination and reduced legal safeguards when pandemic strikes, 72 U. Pitt. L. Rev. 779, 784 (2011).
The psychological impacts of treating COVID-19 patients have proved to be devastatingly real. See, Eric Levenson, *Stress on health care workers is creating 'second victims' in the coronavirus pandemic*, CNN, Apr. 29, 2020, <https://www.cnn.com/2020/04/29/us/coronavirus-health-care-mental-health/index.html>.
While the initial emphasis of these protocols was on rationing ventilators, the propriety of mechanical ventilation for COVID-19 patients is being questioned, and rationing for future outbreaks might focus instead on nursing resources, beds, and other medications such as sedation or antivirals.
Michael D. Christian et al., Care of the Critically Ill and Injured During Pandemics and Disasters: CHEST Consensus Statement, 146 CHEST e61S--74S (2014) Policies vary, and could advocate for prioritization based on different grounds, such as need, equity, or protecting the most vulnerable. See, James Tabery et al., Ethics of Triage in the Event of an Influenza Pandemic, 2 Disaster Med Public Health Preparedness, 114--118 (2008).
Douglas White and Bernard Lo, *A Framework for Rationing Ventilators and Critical Care Beds During the COVID-19 Pandemic*, J. Am. Med. Assoc. Mar. 27, 2020. DOI: 10.1001/jama.2020.5046.
Colorado Department of Public Health and Environment. CDPHE All Hazards Internal Emergency Response and Recovery Plan: Annex B: Colorado Crisis Standards of Care Plan. Published 2018 (accessed Mar. 21,
2020).
This proposal does nothing to alter liability under federal statutes such as 42 US Section 1983, or anti-discrimination law, such as that contained in Section 1557 of the Affordable Care Act. See, 42 U.S.C. §18116 (2020).
"\[A\]uthorities should ensure that there is a legislative framework and structure to support critical care triage. The legislative and legal frameworks to address issues, especially rationing, during a disaster or public health emergency are highly complex and in many jurisdictions, are unclear or nonexistent." See Michael D. Christian et al., *Care of the Critically Ill and Injured During Pandemics and Disasters: CHEST Consensus Statement,* 146 CHEST e61S-74S (2014).
For an argument against providing immunity for health care providers in these situations (though focusing on volunteers) see George Annas, *Standard of Care---In Sickness and in Health and in Emergencies*, 362v N Eng. J. Med 2126 (2010).
"\[B\]y definition, the standard of care in an emergency would take into account the exigent conditions in which providers were working." See, Sharona Hoffman, *Responders' Responsibility: Liability and Immunity in Public Health Emergencies*, 96 Geo. L.J. 1913, 1926 (2008).
Cath Lab Digest, *Data Shows Reduction in U.S. Heart Attack Activations During COVID-19 Pandemic*, Apr. 10, 2020, <https://www.cathlabdigest.com/content/data-shows-reduction-us-heart-attack-activations-during-covid-19-pandemic>.
Valerie Gutmann Koch, & Beth E. Roxland, *Unique Proposals for Limiting Legal Liability and Encouraging Adherence to Ventilator Allocation Guidelines in an Influenza Pandemic*, 14 DePaul J. Health Care L. 467, 469 (2013).
Lawrence Gostin et al., *Standard of Care---In Sickness and in Health and in Emergencies,* response to George Annas, 363 N Engl J Med 1378 (2010) ("Liability claims can and do flow from emergencies, illustrated by ongoing civil litigation against Dr. Anna Pou and others after Hurricane Katrina."
Kaylee DeWitt, *Salt Lake County Mayor Jenny Wilson says social distancing measures are working in flattening the curve*, ABC4 News, Apr. 2, 2020, <https://www.abc4.com/coronavirus/salt-lake-county-mayor-jenny-wilson-says-social-distancing-measures-are-working-in-flattening-the-curve/>; Kristen Schorsch, *Chicago Has 'Flattened The Curve' Of COVID-19 Cases, Mayor Lori Lightfoot Says*, WBEZ News, Apr. 15, 2020, <https://www.wbez.org/stories/chicago-has-flattened-the-curve-of-covid-19-cases-mayor-lori-lightfoot-says/cee77a42-b01f-4138-bf79-d1e681928eed>.
Joshua Knobe and Scott Shapiro, *Proximate Cause Explained: An Essay in Experimental Jurisprudence*, forthcoming in the University of Chicago Law Review, https://ssrn.com/abstract=3544982 or http://dx.doi.org/10.2139/ssrn.3544982 , at 16--17; see also Fiery Cushman, Joshua Knobe, & Walter Sinnott-Armstrong. *Moral Appraisals Affect Doing/Allowing Judgments,* 108 COGNITION 281 (2008).
Ann Lawthers et al., *Physicians' Perceptions of the Risk of Being Sued*, 17 J Health Polit Policy Law 463 (1992).
Brooke Courtney et al., *Legal Preparedness: Care of the Critically Ill and Injured During Pandemics and Disasters: CHEST Consensus Statement*, 146 CHEST e134S (2014).
If the practice is physician-owned this means that practicing physicians may still be liable, albeit through their partnership rather than individually, and this would sound not in medical malpractice but in corporate negligence. See, With respect to entities---including hospitals, clinics, and health care organizations---claims are likely to sound in corporate negligence and vicarious liability. See, Valerie Gutmann Koch & Beth E. Roxland, *Unique Proposals for Limiting Legal Liability and Encouraging Adherence to Ventilator Allocation Guidelines in an Influenza Pandemic*, 14 DePaul J. Health Care L. 467, 474--75 (2013).
See Evan D. Anderson & James G. Hodge, *Emergency Legal Preparedness Among Select US Local Governments*, 3 (Suppl. 2) Disaster Med. & Pub. Health Preparedness S1, S5 (2009); Valerie Guttman Koch & Beth Roxland, *Unique Proposals For Limiting Legal Liability and Encouraging Adherence to Ventilator Guidelines in an Influenza Pandemic*, 14 DePaul J. Health Care L. 467, 483 (2011).
Teneille R. Brown, *Medical Futility and Religious Free Exercise*, 15 First Amend. L. Rev. 43, 44 (2016). One way to do this would be to require gross negligence or more before a physician could be liable for refusing someone hospital resources, rather than immunizing them from negligence only when their conduct was reviewed as "reasonable." The reasonability review embedded in medical futility statutes guts them of their immunity protection and removes the desired peace of mind they are intended to bestow.
A policy that permitted categorical age discrimination would violate Section 1557 of the Affordable Care Act.
Thaddeus Pope, *Medical Futility States: no safe harbor to unilaterally refuse life-sustaining treatment*, 75 Tenn. L. Rev. 1, 58 (2007).
Md. Code Ann., Health-Gen. § 18--907 (West 2020).
I. Glenn Cohen, Andrew Crespo, and Doug White, *Protect the Doctors and Nurses Who Are Protecting Us*, N.Y. Times, Apr. 1, 2020, <https://www.nytimes.com/2020/04/01/opinion/coronavirus-ventilators-doctors.html>.
Md. Code Ann., Health-Gen. § 18--907 (West 2020).
Executive Order of the Governor of New York, Mar. 23, 2020, No. 202.10: Continuing Temporary Suspension and Modification of Laws Relating to the Disaster Emergency, <https://www.governor.ny.gov/news/no-20210-continuing-temporary-suspension-and-modification-laws-relating-disaster-emergency>.
There may be more than one professional organization, and each might provide guidance that is inconsistent with another organization. Rather than permitting liability in these circumstances, this is a perfect example of how difficult these clinical decisions are. So long as a physician can show that a recognized professional society issued guidance that she followed in good faith, this should trigger the negligence immunity.
However, importantly, immunity statutes cannot reduce these meritless lawsuits to zero. While immunity statutes provide a clear signal to plaintiffs' attorneys, especially the majority of them working on a contingency fee basis, that they are quite likely to lose and not be paid, there may still be some who fail to internalize that risk and sue. There does not seem to be a principled way to remove this risk, without over-correcting and unconstitutionally eliminating access to the courts.
Brooke Courtney et al., *Legal Preparedness: Care of the Critically Ill and Injured During Pandemics and Disasters: CHEST Consensus Statement*, 146 CHEST e134S, e136S (2014).
Swine Flu Act, 42 U.S.C. Section 247b(k)(2)(A) (Lexis, 2020).
[^1]: Teneille R. Brown is a law professor at the University of Utah, S.J. Quinney College of Law and an adjunct professor of General Internal Medicine, in the Program on Medical Ethics and Humanities. She is also married to a critical care intensivist, who is treating patients with COVID-19. However, due to adequate planning by both the Utah Department of Health and his employer, the University of Utah, a detailed crisis plan has been put in place that would not require him to personally make any rationing decisions.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
The National Institute of Statistics and Geography (INEGI) reported in 2018 that cardiovascular diseases are the primary cause of death in Mexico.^\[[@R1]\]^ The most important risk factors for the onset of these diseases are high levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides, and low levels of high-density lipoprotein cholesterol (HDL-C). Genetic studies of the loci associated with lipid levels have enabled the identification of polymorphisms that may serve to develop new approaches for the prevention and treatment of cardiovascular diseases. In particular, our research group has collaborated with other research institutions with the objective of characterizing the loci associated with dyslipidemia in the Mexican and Hispanic populations. In a genome-wide association study (GWAS), we found that the genes, *CELSR2*, *ZNF259/APOA5*, *KANK2/DOCK6* and *NCAN/MAU2,* were associated with TC levels; *CELSR2, APOB,* and *NCAN/MAU2* with LDL-C levels^\[[@R2]\]^; and *DAGLB* with HDL-C levels.^\[[@R3]\]^ The same GWAS study failed to find an association between the haptoglobin-encoding (*HP*) gene (located at chromosome 16q22.2) with lipid levels. However, previous studies have showed that the A allele of the rs2000999 polymorphism in *HP* is associated with high levels of TC and LDL-C. It has been proposed that the rs2000999 A allele, which is associated with reduced *HP* expression, has decreased antioxidant protection for APOE, contributing to elevated cholesterol levels.^\[[@R4],[@R5]\]^
Hp is an alpha-glucoprotein commonly found in plasma; it is composed of 2 light chains (α) and 2 heavy chains (β) that are covalently bound. It is mainly produced in the liver and has been reported to play a role in cholesterol esterification, particularly with respect to the binding of apolipoprotein A--I to HDL-C, which promotes cholesterol efflux from cells and stimulates the enzyme lecithincholesterol acyltransferase to esterify cholesterol.^\[[@R6]\]^ Hp has been proposed to target and protect the ApoA--I effector domain of lecithin-cholesterol acyltransferase from oxidative stress; Hp can also bind to ApoE. ApoA-I and ApoE contain similar sequences, which are able to stimulate LCAT to achieve cholesterol esterification in reverse cholesterol transport.^\[[@R7]--[@R10]\]^ Recently, Boettger et al. (2016) identified a strong association between the rs2000999 (G/A) polymorphism in *HP* with TC and LDL-C levels. The rs2000999 A allele decreases Hp expression, consequently reducing its antioxidant capacity, which in turn leads to higher cholesterol levels.^\[[@R4]\]^ The human *HP* gene has 2 common alleles, Hp1 and Hp2, which are the result of a small intragenic duplication of the *HP* gene.^\[[@R11]\]^ The Hp1 allele exhibits enhanced antioxidant activity compared to Hp2. The frequency of the alleles Hp1 and Hp2 is quite variable in world populations. In general, the Hp1 allele is less frequent in East Asian populations than in other population groups, including European, African, and Native American groups.^\[[@R12]--[@R17]\]^
Studies in patients with diabetes revealed that the Hp2-1 or Hp2-2 genotypes are associated with increased risk of vascular injury compared to the Hp1-1 genotype.^\[[@R18],[@R19]\]^ In addition, the rs2000999 A allele is almost exclusively associated with the Hp2 isoform and decrease in expression of Hp; on the other hand, rs2000999 (G) is associated with the Hp1 isoform.^\[[@R4]\]^ A study performed in Chinese diabetic patients reported that the variant rs2000999 is not associated with diabetic macrovascular diseases, although, the A allele is associated with higher levels of LDL-C.^\[[@R20]\]^
To date, the association of rs2000999 of *HP* with serum lipids in the Latin American diabetic population is uncharacterized. Therefore, the aim of this study was to analyze the association of rs2000999 with TC, HDL-C, and LDL-C and in the Mexican population with type 2 diabetes (T2D).
2. Material and methods
=======================
2.1. Study population
---------------------
We investigated 1,200 Mexican adults of both sexes (546 adults with T2D, the T2D group; and 654 controls without T2D, the No T2D group) from 4 centers of social security in Mexico City from 2012 to 2015. The T2D group was selected based on fasting glucose levels, in accordance with American Diabetes Association (ADA) guidelines.^\[[@R21]\]^ The present work was designed as an observational case-control study with a convenience sample size.
The study protocol complies with the ethical guidelines of the 1975 Declaration of Helsinki. The study was authorized by the Instituto Mexicano del Seguro Social ethics committee and informed consent was obtained from all participants.
2.2. Procedure
--------------
All participants were weighed using a digital scale (Seca, Hamburg, Germany). Height was measured with a portable stadiometer (Seca 225, Hamburg, Germany). Body mass index (BMI), calculated as weight (kg)/height (m)^2^. Waist circumference was measured taken after expiration at the midpoint between the low rib margin and iliac crest. Systolic and diastolic blood pressure (SBP and DBP) were measured using a mercurial sphygmomanometer (ALPK2, Tokyo, Japan). Data regarding age and smoking status (current smokers/non-smokers) was determined by self-report.
Two blood samples were collected from each participant to estimate the biochemical parameters and for DNA extraction. The IL-650 equipment (Instrument Laboratory, Bedford, MA, USA) was used to quantify TC, HDL-C, LDL-C, triglycerides (TG), and glucose levels measured by enzymatic colorimetric method (kit numbers 0018250540, 0018255740, 0018256040, 0018480500 and 0018250740 of Werfen Czech s.r.o, respectively). According with Grundy et al,^\[[@R22]\]^ metabolic syndrome was defined with the presence of 3 or more risk factors (waist circumference ≥102 cm in men or ≥88 cm in women, triglycerides ≥150 mg/dl, HDL-C \< 40 mg/dl in men or \<50 in women, systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg and fasting glucose ≥ 100 mg/dl).
DNA isolation was performed in the AutoGen Flex Star (AutoGen, MA, USA) following the manufacturer\'s recommendations. DNA quantity (260 nm) and purity (260/280 nm ratio) were evaluated in the Epoch Microplate Spectrophotometer using Gen5 Microplate Data Analysis (BioCell, VT, USA). DNA integrity was also assessed by electrophoresis in agarose gels (.8%).
Genotyping of rs2000999 was performed by real time polymerase chain reaction using the TaqMan allelic discrimination assay C\_\_11439054_10 on the 7900HT Fast Real-Time PCR system (Applied Biosystems, CA, USA), following standard protocols. Duplicates were performed in 10% of the samples. Genotype discrimination was evaluated using the SDS software (Applied Biosystems, CA, USA).
Estimation of individual ancestral proportions: Given that the individuals in this sample are of mixed ancestry, the Axiom LAT microarray (Affymetrix, CA, USA) was used to determine the Native-American (NAM), European and African ancestry proportions of the participants using the program ADMIXTURE.^\[[@R23]\]^ We added individual ancestral proportions as a covariate in the association analyses.
2.3. Statistical analysis
-------------------------
The normal distribution of continuous variables was tested using the Kolmogorov-Smirnov test. For the traits that significantly deviate from normality, rank based inverse normal transformations were applied. Differences between the cases and controls for continuous and categorical traits were evaluated using the Student *t* test and Chi-Squared test, respectively. We tested the association of rs200099 with individual ancestral proportions, using an additive model, with adjustments for age, sex and T2D diagnosis. The association of rs2000999 with lipid concentrations was assessed using linear regression under different genetic models (codominant, dominant and recessive). All statistical analyses were performed using SPSS software (version 22.0, IBM, Armonk, NY, USA). Two-sided *P* values \< .05 were considered significant.
3. Results
==========
3.1. Characteristics of the study population
--------------------------------------------
The characteristics of the 1200 participants included in the sample are presented in Table [1](#T1){ref-type="table"}. Compared with non-diabetic individuals, the T2D group exhibited significantly higher frequency of women, age, waist circumference, BMI, TG, ratios of TC/HDL-C, NonHDL-C/HDL and TG/HDL-C, blood pressure, glucose, and frequency of current smokers and individuals with metabolic syndrome. However, the non-diabetic individuals showed significantly higher levels of TC, HDL-C and LDL-C than the T2D group. LDL-C/HDL-C ratio and NonHDL-C did not display significant difference between the non-diabetic and T2D groups. Similarly, the non-diabetic and T2D groups had similar frequencies of the rs2000999 A. The polymorphism was in Hardy--Weinberg equilibrium in both groups (*P*~NoT2D~ = .530 and *P*~T2D~ = .991).
######
General characteristics of cases and controls of type 2 diabetes Mexican adults.
3.2. Association of rs2000999 A allele with individual ancestry proportions
---------------------------------------------------------------------------
The rs2000999 A allele had a strong positive association with the percentage of European ancestry (β ± SE \[standard error\] = 0.028 ± 0.011, *P* = .013) and to a lesser extent African ancestry (β ± SE = 0.003 ± 0.002, *P* = .041). On the other hand, the percentage of Native-American ancestry showed a strong negative association with the A allele of rs2000999 of *HP* (β ± SE = −0.031 ± 0.012, *P* = .012).
3.3. Association of rs2000999 with serum lipids
-----------------------------------------------
We tested the association of the variant rs2000999 with serum lipids under different genetic models, using the A allele as the effect allele. Under the codominant model (AA vs AG vs GG) the rs200099 A allele was associated with higher TC and LDL-C levels in the No T2D group (TC: β ± SE = 9.102 ± 3.677, *P* = .014; LDL-C: β ± SE = 6.425 ± 3.144, *P* = .042) and the T2D group (TC: β ± SE = 8.918 ± 3.955, *P* = .025; LDL-C: β ± SE = 8.222 ± 3.240, *P* = .012) (Table [2](#T2){ref-type="table"}). Under the recessive model (AA vs AG + GG), AA homozygotes are significantly associated with higher TC and LDL-C levels in the No T2D group (TC: β ± SE = 10.102 ± 4.038, *P* = .013; LDL-C: β ± SE = 7.287 ± 3.453, *P* = .035) and T2D group (TC: β ± SE = 10.196 ± 5.555, *P* = .026; LDL-C: β ± SE = 8.662 ± 3.736, *P* = .021) (Table [2](#T2){ref-type="table"}). For HDL-C, there are also significant associations under the codominant and recessive models in the T2D group (codominant: β ± SE = 2.402 ± 1.126, *P* = .034; recessive: β ± SE = 3.283 ± 1.295, *P* = .012), but not in the non-diabetic group (Table [2](#T2){ref-type="table"}). Under the dominant model, rs20009999 was not significantly associated with lipid levels, although we saw similar trends indicating higher lipid levels in carriers of the A allele, particularly for TC and LDL-C (Table [2](#T2){ref-type="table"}).
######
Association of variant rs2000999 of *haptoglobin* gene with serum lipids in 654 non diabetic and 546 diabetic Mexican adults.
4. Discussion
=============
This is the first study to describe an association of the rs2000999 variant of *HP* with serum lipids in a Latin American diabetic population. The allelic frequency of the variant was quite similar in the No T2D and T2D groups. In this work, we show that the percentage of Native-American ancestry is negatively associated with the rs2000999 A allele. In contrast, the rs2000999 A allele has a strong positive association with European ancestry, and to a lesser extent, with African ancestry (it is important to note that the average African ancestry in the Mexican sample is quite low, around 3.5%). Our results are quite consistent with data from different world populations. In the allele frequency database ALFRED (<https://alfred.med.yale.edu/alfred/index.asp>), rs2000999 A allele frequencies in Southern European populations are higher than 20%, and in Native American groups from Mexico (Pima, Maya) do not surpass 10%. The frequencies of the A allele in African populations range from 0% to 12.8%. Given that the rs2000999 A allele is present almost exclusively on Hp2 haplotypes,^\[[@R4]\]^ our results indicate that that frequency of the Hp2 allele is lower in the Native American ancestral population than in the European or African ancestral populations. This is consistent with studies that have reported that the frequencies of the Hp1 allele are quite high in some Native American groups, including indigenous groups from Mexico.^\[[@R16],[@R17]\]^ Further studies are needed to describe in detail the linkage disequilibrium patterns between rs200099 and the Hp1 and Hp2 alleles in different population groups, including indigenous groups from the Americas.
We observed that under codominant and recessive models, rs2000999 is associated with TC and LDL-C levels in both the No T2D and T2D groups. Interestingly, under codominant and recessive models, rs2000999 was also significantly associated with HDL-C levels in the T2D group, but not in the non-diabetic group. Our results indicate that carriers of the A allele have higher lipid concentrations than non-carriers, although dominant models did not reach significance. These findings are in agreement with previous studies showing significant positive associations of the A allele with lipid concentrations in other population groups.^\[[@R20],[@R24]\]^ Given the associations reported between the rs2000999 A allele and the Hp2 allele, our results are also consistent with previous studies that have reported that Hp2 is associated with 5-times higher risk of developing cardiovascular diseases, when compared to the Hp1 allele, in the T2D population.^\[[@R25]\]^
The present study also has some limitations. Patients with T2D included in the study are undergoing a healthy eating program and physical activity. These interventions may improve HDL-C levels of the T2D group. The sample size is relatively small, and we did not determine the Hp1 and Hp2 alleles of *HP,* so we could not evaluate the extent to which the effect of rs2000999 and Hp1/Hp2 on lipid levels is independent or not. Of note, a recent meta-analysis have indicated that the effect of rs2000999 on Haptoglobin levels is independent from the effect of the Hp1/Hp2 alleles.^\[[@R26]\]^ This suggests that these 2 polymorphisms may have independent effects on lipid levels. Our results suggest that rs2000999 could be important in evaluating the genetic predisposition to high levels of lipids in patients with T2D and people without T2D. However, as future directions, longitudinal studies in Latin American population, through the evaluation of the response of carriers of rs2000999 A allele to pharmacological intervention, could offer evidence to improve the treatment lipids and the programs to prevent or delay the onset of cardiovascular diseases.
In conclusion, our results evidence that in the Mexican population, the rs2000999 A allele is positively associated with the percentage of European (and to a lesser extent African ancestry), and negatively associated with Native American ancestry. We also show that carriers of the A allele have increased levels of TC and LDL-C, independently of T2D diagnosis, and also increased concentrations of HDL-C in the T2D sample, but not in the non-diabetic group.
Acknowledgments
===============
We thank all the individuals who volunteered to participate in the study.
Author contributions
====================
**Conceptualization:** Adan Valladares-Salgado.
**Data curation:** José J Peralta-Romero.
**Formal analysis:** Fernando Suarez-Sanchez.
**Funding acquisition:** Adan Valladares-Salgado.
**Methodology:** Miguel Vazquez-Moreno, Ema Herrera-Lopez, Jaime H Gomez-Zamudio, José J Peralta-Romero, Osvaldo D Castelan-Martinez.
**Project administration:** Adan Valladares-Salgado.
**Supervision:** Osvaldo D Castelan-Martinez, Miguel Cruz, Esteban J Parra, Adan Valladares-Salgado.
**Writing -- original draft:** Fernando Suarez-Sanchez, Miguel Vazquez-Moreno, Ema Herrera-Lopez.
**Writing -- review & editing:** Jaime H Gomez-Zamudio, Miguel Cruz, Esteban J Parra, Adan Valladares-Salgado.
Abbreviations: GWAS = genome-wide association study, HDL-C = high-density lipoprotein cholesterol, *HP* = haptoglobin-encoding gene, LDL-C = low-density lipoprotein cholesterol, SE = standard error, T2D = type 2 diabetes, TC = total cholesterol, TG = triglycerides.
How to cite this article: Suarez-Sanchez F, Vazquez-Moreno M, Herrera-Lopez E, Gomez-Zamudio JH, Peralta-Romero JJ, Castelan-Martinez OD, Cruz M, Parra EJ, Valladares-Salgado A. Association of rs2000999 in the haptoglobin gene with total cholesterol, HDL-C and LDL-C levels in Mexican type 2 diabetes patients. *Medicine*. 2019;98:39(e17298).
Fernando Suarez-Sanchez and Miguel Vazquez-Moreno Equal contribution.
Financial support: This work was supported by the Fondo Sectorial de Investigación en Salud y Seguridad Social (SSA/IMSS/ISSSTE-CONACYT) project 150352.
The authors have no conflicts of interests to disclose.
| {
"pile_set_name": "PubMed Central"
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1. Introduction {#sec1}
===============
Cloud storage provides an elastic, highly available, easily accessible, and cheap data repository to users who do not want to maintain their own storage or just for convenience, and such a way of storing data becomes more and more popular. In many cases, especially when the users want to store their sensitive data such as business documents, it requires the security guarantees against the cloud provider since an internal staff may access to the data maliciously. Directly encrypting the sensitive documents using traditional encryption techniques such as AES is not an ideal solution since the user will lose the ability to effectively search for the desired documents.
One solution for effectively searching over encrypted data is *searchable encryption* technique. It enables a user to securely outsource his private documents to a third party while maintaining the ability to search the documents by keywords. The scenario is simple: the user submits some encrypted keywords to the server, and then the server performs the search and returns the encrypted documents which contain the queried keywords. However, current searchable encryption techniques either directly return the matched documents or return in the first round some limited information (guided mode) which is prestored in metadata, such as the name and a short static abstract for each matched document. The more documents stored, the more possible matched results will be, and finding the desired documents also becomes a problem. Moreover, the bandwidth cost must be taken into consideration such that returning a large amount of matched documents seemed to be impractical.
Another solution for effectively searching for the desired data is through content preview, which is the main topic of this paper. In modern search engine, if a user searches for a web page by keywords, the search engine will return the name, URI, and a small *query-biased snippet* for each matched page. The snippet explains why such page is matched. Then the user could make a final choice and selectively browse the needed pages without opening all matched links. The same way could be used for searching the desired encrypted documents since the scenario is the same. It could also be combined with searchable encryption to improve the user experience.
However, obtaining a query-biased snippet from an encrypted data is quite challenging. For a general search engine, in order to get a query-biased snippet from a plaintext, it must scan each matched document dynamically, extract the snippets where the keywords occur, then rank the results and finally return the *top-ranking snippet*. While data is encrypted, dynamic scanning becomes quite impossible. Precomputing a snippet file for preview is also impossible because there is no way to know in advance what the queried keywords are, and building all static (keyword, snippet) pairs for each document costs too much storage space even far more than the document itself. Thus, we consider dividing a document to many equal-size encrypted snippets and preconstruct an index to address each snippet. The index stores the information about the keyword frequency in each snippet, which enables the server to dynamically calculate the best snippet for the user when queried by multiple keywords.
There are two major security problems. First, the snippet is the part of a document; therefore the encryption scheme used may affect the snippet retrieval. We use a pad-and-divide scheme to preprocess the document to make it compatible with any cryptosystem such as DES and RSA. Second, the information in the index is private, and no partial information about the document should be leaked to the server. Therefore, we encrypt the index based on the core method of searchable encryption. Since each keyword maps an entry in the index, if queried by some keywords, directly returning the related score information without calculating leaks the information about the number of queried keywords (equals to the number of returned entries) to an eavesdropper, and it also costs multiple communication bandwidth as the number of requested keywords increases. A *homomorphic encryption* scheme could be adopted such that the server could directly operate over the encrypted data and produce a single result, while keeping the ciphertext still secure. However, homomorphic encryption scheme is often costly when dealing with a large amount of data. Observing that all the data are very small, we propose a novel lightweight substitution for homomorphic encryption to construct such secure index.
In this paper, our contributions are the following. (1) To the best of our knowledge, we formalize the problem of securely retrieving query-biased snippet over encrypted data for the first time. We generalize the notion of *secure query-biased preview* (SecQBP) and its security model. (2) We propose a lightweight solution to deal with matrix data with partial homomorphic property, named *matrix additive coding* (Matrix-AC), which could efficiently add two rows of small numbers while keeping the data still encrypted. (3) Based on Matrix-AC and private information retrieval protocol, we construct a *secure additive ranking index* (SecARI) that enables the server to efficiently compute the top-ranking snippet over encrypted data while no partial information about the document is leaked, and then we propose the complete construction to realize SecQBP and prove that it is secure under our security model. (4) We propose a high level solution to combine the preview scheme with searchable encryption technique, which greatly improves the user experience.
The rest of the paper is organized as follows. [Section 2](#sec2){ref-type="sec"} presents the related work. [Section 3](#sec3){ref-type="sec"} presents the notations and preliminaries. [Section 4](#sec4){ref-type="sec"} presents our proposed additive coding scheme. In [Section 5](#sec5){ref-type="sec"} we formally define the preview scheme and its security model and present the construction in detail. We present the application in searchable encryption and analyze the performance in [Section 6](#sec6){ref-type="sec"}. [Section 7](#sec7){ref-type="sec"} concludes this paper.
2. Related Work {#sec2}
===============
We categorize the related work into four topics, and each topic is summarized separately.
2.1. Query-Biased Snippet {#sec2.1}
-------------------------
Query-biased snippet refers to a piece of the content in a document that contains the queried keywords. Query-biased snippet generation schemes are widely used in modern search engine. It is also named *dynamic summary* or *keyword-in-context* (KWIC) snippet generation. The term was used firstly in \[[@B39]\]. The improvements were introduced in \[[@B11]--[@B42]\]. However, as far as we know, all query-biased schemes are focused on dynamically retrieving snippets from the plaintext. If the document is encrypted, dynamic scanning becomes impossible. Static preview refers to a snippet summarizing the content in advance, which is always the same regardless of the query. It is generally composed of either a subset of the content or metadata associated with the document. A lightweight static preview scheme over the encrypted data was introduced in \[[@B30]\]. For more details, please refer to \[[@B31]\] for a survey of the recent preview schemes.
2.2. Searchable Encryption {#sec2.2}
--------------------------
Our proposed scheme and security model are based on searchable encryption technique. The basic goal of searchable encryption is to enable a user to privately search over encrypted data by keywords. The first scheme was introduced in \[[@B37]\]. Later on, many index-based symmetric searchable encryption schemes were proposed. The first secure index was introduced in \[[@B20]\], and the security model of adaptive chosen keyword attack (IND-CKA) was also introduced. Reference \[[@B14]\] introduced two constructions to realize symmetric searchable encryption: the first is SSE-1 which is nonadaptive and the second is SSE-2 which is adaptive. A generalization for symmetric searchable encryption was introduced in \[[@B9]\]. Another type of searchable encryption schemes is public-key based. The first scheme was introduced in \[[@B2]\], the improved definition was introduced in \[[@B1]\], and the strongest security model was introduced in \[[@B3]\].
There are many functional extensions for the basic searchable encryption schemes. Reference \[[@B24]\] introduced a scheme supporting conjunctive keyword search. References \[[@B6]--[@B38]\] introduced ranked keyword search over encrypted data. References \[[@B4]--[@B28]\] introduced fuzzy keyword search over encrypted data. Similar to fuzzy keyword search but different, \[[@B12], [@B27]\] introduced similarity search over encrypted data.
2.3. Homomorphic Encryption {#sec2.3}
---------------------------
Our proposed additive coding method is based on the core concept of homomorphic encryption. The classical homomorphic encryption schemes are based on group operation such as the unpadded RSA in \[[@B34]\], the variant of ElGamal introduced in \[[@B36]\], Goldwasser and Micali\'s bit homomorphic encryption scheme introduced in \[[@B22], [@B23]\], and Paillier\'s encryption scheme introduced in \[[@B32]\]. Many improvements have been proposed based on these classical series of schemes. The referred schemes are public-key based, and few symmetric homomorphic schemes have been proposed. The series of symmetric homomorphic schemes which is based on one-time pad was introduced in \[[@B8]\]. Some ring-based homomorphic schemes have been proposed recently, which are also referred to as full homomorphic encryption, such as the one in \[[@B18]\] that is based on ideal lattices and the one in \[[@B41]\] that does not require ideal lattices.
2.4. Private Information Retrieval {#sec2.4}
----------------------------------
We encapsulate a private information retrieval (PIR) protocol and extend the use of it in our scheme. PIR schemes allow a user to privately retrieve the *i*th bit of an *n*-bit database. The notion was fist introduced in \[[@B10]\] by Chor et al., and the notion of private block retrieval (PBR) was also introduced. Kushilevitz and Ostrovsky introduced a single-server and single-round computational PIR scheme in \[[@B26]\], which achieves communication complexity of $O(\sqrt{n})$ for the basic scheme and could achieve *O*(*n* ^*ϵ*^) with arbitrary small *ϵ* theoretically ($2^{O(\sqrt{\log n\text{loglog}n})}$ is achieved assuming security parameter is polylogarithmic in *n*). In \[[@B7]\], Cashin et al. introduced a single-database PIR scheme with polylogarithmic communication complexity for the first time, about *O*(log^8^ *n*) as suggested. Gentry and Ramzan introduced a PBP scheme with *O*(*k* + *d*) communication cost in \[[@B19]\], where *k* ≥ log*n* is a security parameter that depends on *n*, which is nearly optimal.
3. Notations and Preliminaries {#sec3}
==============================
3.1. Basic Notations {#sec3.1}
--------------------
We write *x*←~*U*~ *X* to represent sampling element *x* uniformly random from a set *X* and write *x* ← *𝒜* to represent the output of an algorithm *𝒜*. We write *a*\|\|*b* to refer to the concatenation of two strings *a* and *b*. We write \|*A*\| to represent its cardinality when *A* is a set and write \|*a*\| to represent its bit length if *a* is a string. We write ⊕ to represent bitwise exclusive OR (XOR) and "≪*n*" to represent bitwise shift left for *n* bits. We write ⌈*x*⌉ to represent the least integer less than or equal to *x*. We write ${\underset{\_}{s}}_{b}$ to represent a bit string that contains either 0 or 1 (e.g., ${\underset{\_}{001101}}_{b}$). A function *μ*(*k*) : *ℕ* → ℝ is negligible if for every positive polynomial *p*(·) there exists an inter *N* \> 0 such that for all *k* \> *N*, \|*μ*(*k*)\|\<1/*p*(*k*). We write poly(*k*) and negl(*k*) to denote polynomial and negligible functions in *k*, respectively.
We write Δ = (*w* ~1~,..., *w* ~*n*~) to present a dictionary of *n* words in lexicographic order. We assume that all words are of length polynomial in *k*. We write *d* to refer to a document that contains poly(*k*) words. We write $\overset{¯}{d}$ to represent the identifier of *d* that uniquely identifies the document, such as a memory location. We write *s* to refer to a snippet (50 characters in general) extracted from the document and write $\overset{¯}{s}$ to represent the identifier of *s*, such as the position in the document.
3.2. Cryptographic Primitives {#sec3.2}
-----------------------------
A function *f* : {0,1}^*k*^ × {0,1}^*n*^ → {0,1}^*m*^ is pseudorandom if it is computable in polynomial time in *k* and for all polynomial size adversaries *𝒜*, it cannot be distinguished from random functions. If *f* is bijective then it is a pseudorandom permutation. We write the abbreviation PRF for pseudorandom functions and PRP for pseudorandom permutations.
Let `ES` represent an encryption scheme. Let `ES.Gen`(1^*k*^) represent the key generation algorithm (*k* is the secure parameter). Let `ES.Enc` ~K~(*d*) represent the encryption algorithm that encrypts data *d* using key *K*, and let `ES.Dec` ~K~(*c*) represent the decryption algorithm that decrypts data *c* to gain the plaintext *d*. In our scheme, a lot of data will be encrypted using the same key; therefore the encryption scheme must be at least CPA (chosen plaintext attack) and CCA (chosen ciphertext attack) secure. For example, ECB (electronic codebook) mode in DES or RSA without OAEP (optimal asymmetric encryption padding) should not be used.
3.3. Homomorphism {#sec3.3}
-----------------
Let *ℳ* denote the set of the plaintexts, let *𝒞* denote the set of the ciphertexts, let ⊙ denote the operation between the plaintexts and ⊗ the operation between the ciphertexts, and let "←" denote "directly compute" without any intermediate decryption. An encryption scheme is said to be homomorphic if for any given encryption key *k*, the encryption function *E* or the decryption function *D* satisfies $$\begin{matrix}
\left. {}\forall m_{1},m_{2} \in \mathcal{M},\quad E\left( {m_{1} \odot m_{2}} \right)\longleftarrow E\left( m_{1} \right) \otimes E\left( m_{2} \right), \right. \\
\end{matrix}$$ $$\begin{matrix}
\left. {}\forall c_{1},c_{2} \in \mathcal{C},\quad D\left( {c_{1} \otimes c_{2}} \right)\longleftarrow D\left( c_{1} \right) \odot D\left( c_{2} \right). \right. \\
\end{matrix}$$
Sometimes, property ([2](#EEq2){ref-type="disp-formula"}) is also referred to as homomorphic decryption. If the operation is upon a group, we say it is a group homomorphism. If the operation is upon a ring, we say it is a ring homomorphism and is also referred to as full homomorphism. If the operator is addition, we say it is additively homomorphic, and if the operator is multiplication, we say it is multiplicatively homomorphic.
3.4. Private Block Retrieval Protocol {#sec3.4}
-------------------------------------
Let *B* = (*B* ~1~,..., *B* ~*n*~) represent a database of *n* blocks; all blocks have equal size *d*. The user wants to privately retrieve the *i*th block from the server; therefore he runs a private block retrieval protocol. At a high level, we define the single database and single round computational PBR as follows.
Definition (computational PBR protocol)A computational PBR protocol scheme is a collection of four polynomial-time algorithms `CPBR` = (`Setup`, `Query`, `Response`, `Decode`) such that we have the following. *P*←`Setup`(*B*) is a probabilistic algorithm that takes as input the database *B* and outputs a parameter set *P*. It is run by the database owner, and *P* is known to all users. *t*←`Query`(*i*) is a probabilistic algorithm that takes as input a block index *i* and outputs a token *t*. It is run by the user. *t* is sent to the server. *r*←`Response`(*t*) is a deterministic algorithm that takes as input the requested token *t* and outputs a result *r*. It is run by the server. *r* is sent to the user. *B* ~*i*~←`Decode`(*r*) is a deterministic algorithm that takes as input the response *r* from the server and outputs the requested data block *B* ~*i*~. It is run by the user.
In our preview scheme, we adopt the computational PBP scheme as a primitive introduced in \[[@B19]\]. In the setup algorithm, we set the database size as the maximal possible document size (e.g., 10 MB) and reuse prime number set and prime power set in all documents. The communication complexity is *O*(log\|*d* \| +\|*s*\|) where \|*d*\| is the document length and \|*s*\| is the snippet length.
4. Secure Additive Coding {#sec4}
=========================
Before introducing the preview scheme, we first introduce a novel coding method called matrix additive coding (Matrix-AC) that enables addition of two rows in a matrix in a homomorphic fashion, which is very fast and suitable for dealing with small numbers (the integer is coded to a specific bit string) and is especially useful for computing statistical table in encrypted form. Since all operated integers are correlative, it is not a homomorphic encryption scheme which could encrypt data independently.
Matrix-AC is used in the preview scheme to construct the secure additive ranking index (SecARI). Becouse a large number of small numbers will be calculated in the preview scheme, using homomorphic encryption schemes is costly. Therefore, we use Matrix-AC scheme as a substitution for homomorphic encryption scheme to achieve optimal performance.
We note that, for all the schemes (including the preview scheme in the next section), we only consider the confidentiality of the data. Mechanism about protecting data integrity is out of the scope of this paper.
4.1. Basic Idea {#sec4.1}
---------------
The basic idea of coding small integers *𝕊* ~*N*~ = (0,1, 2,..., *N*) with homomorphic property is simple: we consider an integer vector **m** = (*m* ~1~,..., *m* ~*n*~), where *m* ~*i*~ ∈ *𝕊* ~*N*~ and ∑~*i*=1~ ^*n*^ *m* ~*i*~ ≤ *N*. We define a "vernier" that has *N* bits, and each integer *m* ~*i*~ is mapped to such vernier for *m* ~*i*~ bits in different position. A global cursor *g* is autoincreased to process the mapping. To code a message, a random string as a one-time-pad key is used and XORed with the mapped data. The decoding is simple: just operate ⊕ with the key and count the number of bit-1 to make reverse mapping.
For example (as shown in [Figure 1](#fig1){ref-type="fig"}), consider a vernier that has 8 bits, and we map three integers (2,1, 3) as a vector to three pairs ($2,{\underset{\_}{00000011}}_{b}$), ($1,{\underset{\_}{00000100}}_{b}$), and ($3,{\underset{\_}{00111000}}_{b}$). It is easy to see that the homomorphic property holds as 2 + 3 = 5, and ${\underset{\_}{00000011}}_{b} \oplus {\underset{\_}{00111000}}_{b} = {\underset{\_}{00111011}}_{b}$ which has exactly 5 bit-1. Thus, let the vector be (*m* ~1~ = 2, *m* ~2~ = 1, *m* ~3~ = 3), and let the keys be (*K* ~1~, *K* ~2~, *K* ~3~) that each key is a random string; then the ciphertext vector will be $(c_{1} = {\underset{\_}{00000011}}_{b} \oplus K_{1}$, $c_{2} = {\underset{\_}{00000100}}_{b} \oplus K_{1}$, $c_{3} = {\underset{\_}{00111000}}_{b} \oplus K_{1})$. To perform addition for any two plaintexts *m* = *m* ~1~ + *m* ~2~, the server could directly compute the corresponding ciphertexts *c* = *c* ~1~ ⊕ *c* ~2~ and the decryption key becomes *K* = *K* ~1~ ⊕ *K* ~2~. Using the new key, it is easy to decrypt the ciphertext and count the number of bit-1 to restore the result.
The problem of the basic scheme is that the vernier may be used up. That is why we set the restriction that a vernier is just used in a single vector. Another drawback is that, as the max *N* increases, the length of the vernier also increases in linear *O*(*N*). Thus, the targeted data must be small enough to save storage space. A good application is not for dealing with few such integers but for computing a large number of small data in parallel.
4.2. Coding a Matrix {#sec4.2}
--------------------
We extend the basic idea to code the data matrix. Let *A* ~*m*×*n*~ represent a matrix with *m* rows and *n* columns, let *a* ~*ij*~ represent the element in row *i* and column *j*, and *a* ~*i*~ represent the *i*th row. Matrix-AC scheme is described in [Algorithm 1](#alg1){ref-type="fig"}. Note that there are *n* cursors that control the mapping for each column.
Let us check the homomorphism for decoding: let *D* represent the decryption algorithm, for arbitrary two ciphertext rows *c* ~1~ and *c* ~2~, *D*(*c* ~1~ ⊕ *c* ~2~) = *D*(*c* ~1~) + *D*(*c* ~2~), where the decryption key for *c* ~1~ ⊕ *c* ~2~ is *K* ~1~ ⊕ *K* ~2~.
There is a problem if the scheme is directly used in the application. In the real world, there is no way to directly represent, for example, data of 5 bits (there is an extended "bitset" class in C++, but it treats the bits as a set, and all operations are performed over set, and it is very slow). In computer, the data is represented by "byte" that a valid number is stored in such a byte. Thus, 5-bit data is stored in one byte (8 bits) as a "character," 12-bit data is stored in two bytes (16 bits) as a "short integer," and a 20-bit data is stored in four bytes (32 bits) as an "integer." Thus, the XOR operation is performed over byte, and the data should be extended to such standard length. However, since all data in Matrix-AC are in fact a bit string, sometimes the data in the same row could be "chained" together. For example, suppose *N* = 5 and there are 6 data in a row; the row could be chained to a 30-bit string and stored in a 32-bit integer. Another problem is that the "bit-counting" algorithm is realized indirectly by "mod 2" operation or setting *N* masks to see if the masked bit is 1. Therefore, the performance would be improved if some dedicated hardware directly dealing with bits is used.
4.3. Proof of Security {#sec4.3}
----------------------
Intuitively, the scheme is secure if any two matrices (the numbers of elements are the same) prepared by the adversary are indistinguishable, which also implies that any two elements from the same matrix are indistinguishable. We define the security of Matrix-AC as follows.
LemmaIf *K* ~1~,..., *K* ~*n*~ are random strings, then Matrix-AC is CPA secure.
ProofWe briefly prove the scheme since the mechanism is simple. We describe a PPT simulator *𝒮* for all PPT adversaries *𝒜*. *𝒮* generates matrix **X** ~*m*×*n*~\* with *m* random strings of length *n* · *N*. For any row *x* ~*i*~ in the original matrix **X** ~*m*×*n*~, no matter how it maps, the last computation is a string XORed with a one-time-pad random string *K* ~*i*~; thus the result is indistinguishable from random. For any two rows *x* ~*i*~ and *x* ~*j*~ from the same matrix, each row is XORed with different random strings such that the results are indistinguishable from each other. For any two rows *x* ~*i*~ and *x* ~*i*~\* from two matrices, as discussed previously, *x* ~*i*~ is indistinguishable from the random string *x* ~*i*~\*.
5. Secure Query Biased Preview Scheme {#sec5}
=====================================
The preview scheme contains two steps: (1) storage at which the data owner prepares the previewable document and a searchable index; (2) retrieval at which the user privately retrieves the snippet from the server.
The basic idea of constructing a query-biased previewable document is as follows: divide the document into *n* snippets with equal size, extract keywords from each piece to form a keyword set which records the snippet information as (keyword, frequency) pairs, and build an index to address the snippets according to the distinct keywords. The index *R* is a *m* × *n* two-dimensional matrix of the form *R*(keyword, snippet index), and the value is the keyword frequency in the corresponding snippet. An example is shown in [Table 1](#tab1){ref-type="table"}. The keyword is represented by *w* ~*i*~, and the snippet index is represented by *s* ~*i*~.
The main process of retrieving the best snippet by multi keywords follows the following steps. The user submits multikeywords to the server. The server retrieves the multirows in the index according to the submitted keywords and adds the rows together. The result is a single entry that contains the information about the best matched snippet. The user decrypts the entry, selects the snippet identifier (index number) with the highest score (for simplicity, the score equals the frequency), and privately retrieves the snippet from the server by running a PBR protocol. In order for the server to perform the "addition" operation over the encrypted data, a homomorphic encryption scheme could be used to encrypt the index. We adopt Matrix-AC as the encryption scheme instead of a standard homomorphic encryption scheme as discussed previously.
Now we begin to introduce the definition and the security model of the preview scheme. Note that we assume the server is honest but curious. Additional methods could be added to make those solutions robust against malicious attack; however, we restrict our discussion on honest-but-curious fashion. We also note that all documents are treated as text files the same way as search engine does. For example, if a document is a web page, the style tags will be pruned.
5.1. Scheme Definition {#sec5.1}
----------------------
The secure-query biased preview (SecQBP) scheme contains two parties: a user *U* and a remote server *S*. *U* encrypts his private document *d* to *D*, generates a secure additive ranking index (SecARI) *H*, and then outsources them to *S*. *S* stores the document, performs the computation for the scores when queried by multiple keywords, and returns the result to *U*. *U* then selects the best snippet indexed by *i* and privately retrieves it from *S*.
Without loss of generality, we consider the construction for a single document. The scheme could be extended to a document collection with ease. Now we define the SecQBP scheme as follows.
Definition (secure query-biased preview scheme)SecQBP scheme is a collection of six polynomial-time algorithms `SecQBP` = (`Gen`, `Setup`, `Query`, `ComputeScore`, `DecScore`, `DecSnip`) as follows. *K*←`Gen`(1^*k*^) is a probabilistic algorithm that takes as input a security parameter *k* and outputs the secret key collection *K*. It is run by the user, and the keys are kept secret. (*D*, *H*)←`Setup` ~K~(*d*) is a probabilistic algorithm that takes as input a document *d* and outputs a encrypted document *D* (using any cryptosystem) and an index *H*. It is run by the user, and *D*, *H* are outsourced to the server. *q*←`Query` ~K~(**w**) is a deterministic algorithm that takes as input the queried multiple keywords **w** = (*w* ~1~,..., *w* ~*n*~) and outputs a secret query token *q*. It is run by the user, and *q* is sent to the server. *r*←`ComputeScore`(*q*, *H*) is a deterministic algorithm that takes as input the secret query *q* and the index *H* and outputs the result *r* that contains the final score information about each snippet. It is run by the server. *i*←`DecScore` ~K~ $(\mathbf{w},\overset{-}{d},r)$ is a deterministic algorithm that takes as input the queried keywords **w**, the document identifier $\overset{¯}{d}$, and the query result *r* and outputs the snippet index number *i*. It is run by the user. *s* ~*i*~←`DecSnip` ~K~(*D* ~*i*~) is a deterministic algorithm that takes as input the ciphertext *D* ~*i*~ and outputs the recovered plaintext snippet *s* ~*i*~. It is run by the user. Note that, if the user retrieves the entire encrypted document, he could decrypt the document by decrypting each snippet.
5.2. Security Model {#sec5.2}
-------------------
Informally speaking, SecQBP must guarantee that, first, given the encrypted document *c* and the index *H*, the adversary cannot learn any partial information about the document; second, given a sequence of queries *q* = (*q* ~1~,..., *q* ~*n*~), the adversary cannot learn any partial information about the queried keywords and the matched snippet (including the index number and the content). We now present the security definition for adaptive adversaries.
Definition (semantic security against adaptive chosen keyword attack, CKA2-security)Let ∑ = (SecQBP algorithm + SecQBP protocol) be the preview scheme. Let *k* ∈ *ℕ* be the security parameter. one considers the following probabilistic experiments, where *𝒜* is an adversary and *𝒮* is a simulator. Real~Σ,*𝒜*~(*k*): the challenger runs `Gen`(1^*k*^) to generate the key *K*. *𝒜* generates a document *d* and receives (*D*, *H*)←`Setup` ~K~(*d*) from the challenger. *𝒜* makes a polynomial number of adaptive queries **w** ~1~,..., **w** ~*n*~ (each set **w** ~*i*~ contains multiple keywords in *d*), and for each queried keyword set **w** ~*i*~, *𝒜* receives a query token *q* ~*i*~←`Query` ~K~(**w** ~*i*~) from the challenger. Finally, *𝒜* returns a bit *b* that is output by the experiment. Sim~Σ,*𝒜*,*𝒮*~(*k*): *𝒜* generates a document *d*. Given only the size \|*d*\|, *𝒮* generates and sends (*D*\*, *H*\*) to *𝒜*. *𝒜* makes a polynomial number of adaptive queries **w** ~1~,..., **w** ~*n*~ (each set **w** ~*i*~ contains multiple keywords in *d*), and for each queried keyword set **w** ~*i*~, *𝒜* receives a query token *q* ~*i*~\* from *𝒮*. Finally, *𝒜* returns a bit *b* that is output by the experiment.We say that SecQBP is semantic secure against adaptive chosen keyword attack if, for all PPT adversaries *𝒜*, there exists a PPT simulator *𝒮* such that $$\begin{matrix}
{\left| {\text{Pr}\left\lbrack {\text{Rea}\text{l}_{\sum,\mathcal{A}}\left( k \right) = 1} \right\rbrack - \text{Pr}\left\lbrack {\text{Si}\text{m}_{\sum,\mathcal{A},\mathcal{S}}\left( k \right) = 1} \right\rbrack} \right| \leq \text{negl}\left( k \right),} \\
\end{matrix}$$ where the probabilities are over the coins of `Gen` and `Setup` (related to the underlying cryptosystem).
Note that, with *q* ~*i*~ or *q* ~*i*~\*, *𝒜* could run `ComputeScore`(*q*, *H*) to get the result *r*, and any internal state is also captured by *𝒜*. *𝒜* could also send query according to the previous result.
5.3. Concrete Construction {#sec5.3}
--------------------------
Now we describe the concrete construction for SecQBP. We describe the constructions for some core components, and then represent the complete construction.
### 5.3.1. Encrypting a Document {#sec5.3.1}
We consider the problem of extracting keywords from a document. In general, a keyword is followed by a separator. Thus, in a general snippet of 50 characters, no more that 25 keywords are contained. Another problem is that not all words are keywords, and such words do not need indexing, for instance, the words "a," "the," and "and." This kind of words can be found in most of the sentences such that it is useless as a key to index a file. They are called stop-word and firstly researched in \[[@B29]\]. The most classical stop word list used abroad is a list of 425 words suggested in \[[@B16]\].
There is a problem that the last word in a snippet may be cut off. In other words, the last word of a snippet may be not short enough to fit the space, and it cannot be split into two words because neither of them is a valid keyword. In a general search engine, such overflowed word is omitted. However, in the scenario of precomputing snippets, if the word is omitted, a keyword may be lost. It means that, when querying the omitted keyword, there will be no matched snippet returned, where actually there is a match for the document. Thus, we add the full word to both the keyword sets of the snippets which contain part of the keyword.
The basic idea for encrypting a document is dividing the document with equal size; therefore, a padding scheme is needed when the last piece of the document is not long enough. We modify the CBC plaintext padding scheme introduced in \[[@B33]\] to meet our goal. Let \|*s*\| represent the length of the snippet; the snippet is treated as a sequence of bytes. If the last snippet is *a* bytes, then pad the snippet with \|*s* \| −*a* bytes with value \|*s* \| −*a*. After decryption, the padding will be deleted to recover the original plaintext. For instance, suppose \|*s* \| = 50; if the final snippet has 15 plaintext bytes, then pad the snippet as $$\begin{matrix}
\left. {}\text{byt}\text{e}_{1}||\text{byt}\text{e}_{2}||\cdots||\text{byt}\text{e}_{15}||35||35||\cdots||35, \right. \\
\end{matrix}$$ where there are 35 bytes that have the number 35. If the snippet is divisible by \|*s*\|, here is 50, then add a new snippet with all bytes being 50: $$\begin{matrix}
\left. {}50||50||\cdots||50. \right. \\
\end{matrix}$$
Let *d* represent a document, and *D* is the encrypted form of *d*. We introduce the scheme for encrypting a document, shown in [Algorithm 2](#alg2){ref-type="fig"}. In the algorithm, "a valid keyword" means the token is not a separator, not a stop word, and not a random-looking string. A word dictionary could be used to check its validity.
### 5.3.2. Constructing the Secure Index {#sec5.3.2}
The secure additive ranking index (SecARI) is the encryption form of the snippet index, as shown in [Table 1](#tab1){ref-type="table"} (PAD denotes the padding with a random string), and each row is an encrypted entry. For security reason, the number of entries of SecARI must be padded to a certain amount which is independent of the actual number of keywords in the content, or it will leak the information about the number of distinct keywords in the document (it equals the number of rows). An example of a SecARI is shown in [Table 2](#tab2){ref-type="table"}. In the table, *π* is a pseudorandom permutation which randomizes the order of the keywords, and the value *c* ~*ij*~ is the encrypted score.
Let us consider the secure amount of the entries. If the document *d* is small, let a keyword occupy only one byte; then the maximum possible number of keywords is \|*d* \| /2 (as discussed, a valid keyword is at least 2 bytes); thus, the number of entries must be set to \|*d* \| /2 (the fractional part is ignored). If the document *d* is large, the maximum possible number of keywords equals the total number of words in the dictionary. Reference \[[@B15]\] made a detailed word statistical analysis based on 450 million words on Corpus of Contemporary American English (1990--2012). The statistics show that the total words used are about 60000. We set the dictionary used as Δ and define the maximum keyword amount as \|Δ \| = 60000. Thus, we define the number of entries as follows.
Definition (number of entries)To guarantee security, the number of entries *N* ~ent~ for a SecARI is $$\begin{matrix}
{N_{\text{ent}} = \begin{cases}
{\frac{\left| d \right|}{2},} & {\frac{\left| d \right|}{2} < \left| \Delta \right|,} \\
{\left| \Delta \right|,} & {\frac{\left| d \right|}{2} \geq \left| \Delta \right|.} \\
\end{cases}} \\
\end{matrix}$$
SecARI is in fact a sparse look-up table, and we use indirect addressing method to manage it. Indirect addressing method is also called *FKS dictionary* introduced in \[[@B17]\], which is also adopted in symmetric searchable encryption scheme in \[[@B14]\]. It manages sparse table of the form (address, value). The address is a *virtual address* that could locate the value field. Given the address, the algorithm will return the associated value in constant look-up time and return *Ø* otherwise.
In addition, we make use of a pseudorandom permutation *π* to index an entry and a pseudorandom function to generate the one-time-pad keys for Matrix-AC: $$\begin{matrix}
\left. {}\pi:\left\{ 0,1 \right\}^{k} \times \left\{ 0,1 \right\}^{|w|}\rightarrow\left\{ 0,1 \right\}^{|w|}, \right. \\
\left. {}f:\left\{ 0,1 \right\}^{k} \times \left\{ 0,1 \right\}^{|w| + |\overset{¯}{d}|}\rightarrow\left\{ 0,1 \right\}^{N \cdot n}, \right. \\
\end{matrix}$$ where \|*w*\| is the keyword length and $\left| \overset{¯}{d} \right|$ is the length of the document identifier. *N* is the upper bound discussed in Matrix-AC and *n* is the number of snippets that is calculated from the document size. The submitted keyword is encrypted by *π* such that the server cannot figure out what the keyword the user queries.
Let *H* be a {0,1}^\|*w*\|^ × {0,1}^*N*·*n*^ × *N* ~ent~ data matrix managed by indirect addressing technique as discussed previously. Now we describe SecARI in [Algorithm 3](#alg3){ref-type="fig"}.
### 5.3.3. The Complete Scheme {#sec5.3.3}
In order to hide the information about the number of queried keywords, a SecARI is not enough. When the user submits the queried multiple keywords, each query should be of the same length so that an eavesdropper cannot learn the information about the number of keywords in a query. Let the maximum number of keywords allowed in a single query be *W* ~max~; the remaining space must be padded. The user and the server should initiate a secure channel such as SSL to transport such message, or the padding may be discovered by an eavesdropper. Since the size of a keyword is small, the bandwidth waste of the padding is rather negligible.
We also determine the upper bound *N* for Matrix-AC. As discussed, a general snippet contains at most 25 keywords; thus we set *N* = 32 (stored as a standard integer).
Let *f* be the pseudorandom function, and *π* is the pseudorandom permutation as described previously. Now we describe the complete scheme in [Algorithm 4](#alg4){ref-type="fig"}, and describe the storage and retrieval protocol in [Protocol 1](#protocol1){ref-type="other"}. The retrieval protocol describes the retrieval of a query-biased snippet from document *d* by submitting a multikeyword query **w** = (*w* ~1~,..., *w* ~*n*~).
{#protocol1}
Note that it is a scenario for a single document. The protocol also works for a document collection. Thus, the user could retrieve multiple snippets for multiple documents in the same round.
5.4. Proof of Security {#sec5.4}
----------------------
The server stores the SecARI, performs homomorphic computation for a query, and returns to the user the score information as a single entry. We prove the security by introducing a theorem as follows.
TheoremIf *f* is a pseudorandom function, if *π* is a pseudorandom permutation, and if `ES` is CPA and CCA secure, then SecQBP is CKA2 secure.
ProofWe describe a polynomial-size simulator *𝒮*, for all polynomial-size adversaries *𝒜*, Real~∑,*𝒜*~(*k*) and Sim~∑,*𝒜*,*𝒮*~(*k*) are indistinguishable. Consider the simulator that given the size of the document \|*d*\|, *𝒮* generates the data as follows. (Simulating *H*\*) *𝒮* computes *m* = *N* ~ent~, *n* = ⌈\|*d* \| /\|*s*\|⌉. For 1 ≤ *i* ≤ *m*, *𝒮* generates a string *a* ~*i*~\*\|\|*c* ~*i*~\* such that each *a* ~*i*~\* is a distinct string of length \|*w*\| chosen uniformly at random, and each *c* ~*i*~\* is a string of length *N* · *n* bits chosen uniformly at random. All strings form *H*\*.(Simulating *q* ~*i*~\*) *𝒮* prepares a query list *L* that stores the query history. The value in *L* is of the form (*w*, *a*\*). When queried by a keyword set **w** ~*i*~, for each keyword *w* ~*k*~ in **w** ~*i*~, *𝒮* first scans *L* to see if there is a match. If not, *𝒮* randomly chooses a distinct *a* ~*k*~\* which is not in *L* and stores the pair (*w* ~*k*~, *a* ~*k*~\*) into *L*. *𝒮* gets (*a* ~1~\*,..., *a* ~\|**w**~*i*~\|~\*) according to **w** ~*i*~ and sets *q* ~*i*~\* = (*a* ~1~\*,..., *a* ~\|**w**~*i*~\|~\*).(Simulating *D* ~*i*~\*) *𝒮* sets *D* ~*i*~\* to a \|*D* ~*i*~\|-bit string chosen uniformly at random. Note that \|*D* ~*i*~\| is a global parameter known by the user and the server.We claim that no polynomial-size distinguisher *𝒟* could distinguish the following pairs. (*H* and *H*\*) recall that *H* consists of *N* ~ent~ values. Each value consists of either a string of the form (*π* ~*K*~*p*~~(*w* ~*i*~)\|\|*c* ~*i*~) or a random string. In any case, with all but negligible probability, the PRP key *K* ~*p*~ is not included; therefore the pseudorandomness of *π* guarantees that *π* ~*K*~*p*~~(*w* ~*i*~) is indistinguishable from random. The PRF key *K* ~*m*~ is also not included; therefore the pseudorandomness of *f* guarantees that the derived key *k* ~*i*~ for each data row is indistinguishable from random, and then the underlying Matrix-AC is CPA-secure, which means that *c* ~*i*~ is indistinguishable from random. *H*\* contains *N* ~ent~ random values. Therefore, as discussed, *H* and *H*\* are indistinguishable.(*q* ~*i*~ and *q* ~*i*~\*) recall that *q* ~*i*~ is the evaluation of the PRP *π*. In any case, with all but negligible probability, the PRP key *K* ~*p*~ is not included; therefore the pseudorandomness of *π* guarantees that all *π* ~*K*~*p*~~(*w* ~*i*~) in *q* ~*i*~ are indistinguishable from random, and *q* ~*i*~\* is a random string of the same length of *q* ~*i*~.(*D* ~*i*~ and *D* ~*i*~\*) recall that *D* ~*i*~ is encrypted by a CPA and CCA secure encryption scheme. Since the encryption key *K* ~*d*~ is not known by the adversary, the security of the encryption scheme guarantees that *D* ~*i*~ and *D* ~*i*~\* are indistinguishable.
6. Comparison, Application, and Performance Analysis {#sec6}
====================================================
First, we compare the functionalities and performance of our work with previous works. Then, as a significant example, we discuss how to combine the preview scheme with symmetric searchable encryption to improve the user experience. We also discuss the performance of the preview scheme in the concrete application example.
6.1. Scheme Comparison {#sec6.1}
----------------------
Let *s* denote the snippet length and *d* the document size; the comparisons of our work with other representative works are shown in [Table 3](#tab3){ref-type="table"}.
The query-biased preview mode is widely used in general search engine, as introduced in \[[@B40]\]. In the scheme, the search engine dynamically scans the document line by line to find the top-ranking snippet. Therefore, the computation complexity is *O*(*d*). In \[[@B30]\], Mithal and Tayebi proposed a static preview scheme over encrypted data based on content mask technique. In the scheme, some segments of the plaintext are extracted in advance and are masked with noise in such a way that the so called "masked preview content" could be sent to the user as a preview when queried. The static scheme is fast and informative but does not explain why a document is matched by a query. Note that our scheme costs one extra round of communication since the score results have to be returned to the user in the first round.
6.2. Symmetric Searchable Encryption Extension {#sec6.2}
----------------------------------------------
We review the generalized definition of symmetric searchable encryption (SSE) introduced in \[[@B9]\]. We assume that the searchable encryption scheme is in guided mode. In other words, the server will first return to the user the identifiers of the matched documents, and the user makes a final choice to select some document identifiers and sends them to the server to retrieve the selected ones.
Definition (extended symmetric searchable encryption)In guided mode, a symmetric searchable encryption scheme is a collection of six polynomial-time algorithms `SSE` = (`Gen`, `Enc`, `Token`, `Search`, `Retrieve`, `Dec`) such that we have the following. *K* ← Gen(1^*k*^) is a probabilistic algorithm that takes as input a security parameter *k* and outputs a secret key *K*. It is run by the user, and the output key is kept secret by the user. (*γ*, *C*) ← Enc~*K*~(*D*) is an algorithm that takes as input a secret key *K* and a document collection *D* = (*D* ~1~,..., *D* ~*n*~) and outputs a searchable structure *γ* and a sequence of encrypted documents *C* = (*C* ~1~,..., *C* ~*n*~). It enables a user to query some keywords, and the server returns the matched documents. For instance, in an index-based searchable symmetric encryption scheme, *γ* is the secure index. It is run by the user, and (*γ*, *C*) is sent to the storage server. *t* ← Token~*K*~(**w**) is a deterministic (possibly probabilistic) algorithm that takes as input a secret *K* and a set of some keywords **w** = (*w* ~1~,..., *w* ~*n*~) and outputs a search token *t* (also named trapdoor or capacity). It is run by the user. *I* ← Search(*γ*, *t*) (guided mode) is a deterministic algorithm that takes as input the query token *t* and the searchable structure *γ* and outputs the matched document identifiers *I* = (*I* ~1~,..., *I* ~*m*~). It is run by the server, and the result *I* is sent to the user. Note that, if not in guided mode, this algorithm returns the matched documents directly. It is run by the server. *C*′ ← Retrieve(*C*, *I*′) is a deterministic algorithm that takes as input the encrypted documents and the selected document identifiers *I*′⊆*I* and outputs the selected documents corresponding to the identifiers. It is run by the server. *D* ~*i*~ ← Dec~*K*~(*C* ~*i*~) is a deterministic algorithm that takes as input a secret key *K* and the returned encrypted document *C* ~*i*~ and outputs the recovered plaintext *D* ~*i*~. It is run by the user.
The preview scheme is applied in SSE as follows. The user runs `SSE.Gen`, `SecQBP.Gen`, `SSE.Enc, and SecQBP.Setup,` respectively. The server stores the outsourced structure generated by SSE and the encrypted documents generated by SecQBP scheme. To search for some documents, the user runs `SSE.Token` and `SecQBP.Query`, respectively, and sends them to the server. The server produces the identifiers of the matched documents, runs `SecQBP.ComputeScore` for the corresponding documents one by one, and returns the document identifiers and the score results together. The user decodes the score, retrieves the preview snippets from the server, then makes the choice, and sends the selected document identifiers to the server to retrieve the interested documents.
6.3. Performance Analysis {#sec6.3}
-------------------------
We adopt SSE-2 introduced in \[[@B14]\] as an instance of a SSE scheme. [Table 4](#tab4){ref-type="table"} shows the time complexity and storage complexity for single SSE-2 scheme and SSE-2 plus SecQBP in detail.
Let *C* represent the encrypted document collection, so the total size is \|*C*\| bytes. Other than the returned encrypted documents, the extrastorage cost for SSE-2 is \|*C* \| /8 bytes; thus the storage cost is *O*(*n*). The extrastorage cost for SecQBP is *H* for each document. By definition, the storage cost is *O*(*n*). For SSE, the server searches the matched documents and decrypts the identifier list. For SecQBP, the server searches the indices for all matched documents, returns score results for all matched documents, and finally returns the snippets. They are both in time complexity of *O*(1). The number of rounds for SSE is two (guided mode). First, the server returns the identifiers of the matched documents and next returns the selected documents. SecQBP adds extra round for retrieving snippets from the snippet server. Moreover, for each matched document, the size of the messages for SEE is *O*(1). SecQBP is *O*(log(*d*/*s*) + *s* + *d*/*s*), where *d* is the document length, and *s* is the snippet length, since the user will receive a score result of size *d*/*s* and a snippet of size *s*.
The detailed performance of SSE is analyzed in \[[@B25]\]; therefore we just analyze the performance of the SecQBP part. The content of a document *d* is varied in the real world. By observation in \[[@B15]\], the number of keywords in a document increases along with the document size which satisfies log model, and the worst case satisfies linear model (each word in the document is keyword, such as a dictionary). However, the design for security in our scheme guarantees that the encrypted indices generated from any document are indistinguishable. Therefore, the computation for the server is independent from the models (i.e., the computations for all documents are the same). To simulate the reality, we design the data generator that simulates documents using log model.
In order to demonstrate the optimization for the server, we compare our suggested Matrix-AC scheme with the simplest and, as far as we know, the fastest symmetric homomorphic encryption scheme \[[@B8]\] denoted by SHE and a well-known homomorphic cryptosystem \[[@B32]\] denoted by Paillier cryptosystem. We consider that 100 users submit queries simultaneously. Each query contains 5 keywords, and the score computation is over 100 matched documents (SSE generates the identifiers of the matched documents). The size of each document increases from 50 KB to 1 MB (the sizes for all stored documents are the same), and the computation cost is described by millisecond.
The algorithms are coded in C++ programming language and the server is a Pentium Dual-Core E5300 PC with 2.6 GHz CPU. The result is shown in [Figure 2](#fig2){ref-type="fig"}. It demonstrates that:the following. (1) The scheme is secure. The figure shows a linear computation cost, which means the computation is independent of the document content. In other words, the server does not see any differences for all documents while performing the search. (2) In cloud environment, computation for 100 users simultaneously on a single server becomes a burden as the size of the document increases. In other words, the number of servers run as services is determined by the size of the stored documents and the accepted queries. (3) The performance is improved as we adopt Matrix-AC to substitute the homomorphic encryption schemes. From the data, Matrix-AC is about 30% faster than using SHE or Paillier cryptosystem. We assume that the user does not modify the document frequently, and the main operation is just searching for some documents. Therefore, the performance improvement is significant since it could save about 30% virtual machines in the cloud.
7. Conclusions {#sec7}
==============
In this paper, we propose a generalized method of securely retrieving query-biased snippet over outsourced and encrypted data, which allows the users to take a sneak preview over their encrypted data. The preview scheme has strong security and privacy guarantees with relatively low overhead, and it greatly improves the user experience.
Part of this work is supported by the Fundamental Research Funds for New Century Excellent Talents in Chinese Universities (Grant no. NCET-10-0298) and Ministry of Science and Technology of Sichuan province (no. 2012HH0003).
{#fig1}
{#fig2}
{#alg1}
{#alg2}
{#alg3}
{#alg4}
######
Example of a snippet index.
Keyword *s* ~1~ *s* ~2~ *⋯* *s* ~*n*~
----------- --------- --------- ----- -----------
*w* ~1~ 2 1 *⋯* 2
*w* ~2~ 1 2 *⋯* 1
*⋯* *⋯* ... *⋯* *⋯*
*w* ~*i*~ 2 5 *⋯* 3
*⋯* *⋯* ... *⋯* *⋯*
*w* ~*m*~ 3 2 *⋯* 2
######
Example of a SecARI.
Index *s* ~1~ *s* ~2~ *⋯* *s* ~*n*~
---------------------- ------------ ------------ ----- ------------
*π* ~*K*~(*w* ~2~) *c* ~21~ *c* ~22~ *⋯* *c* ~2*n*~
*⋯* *⋯* *⋯* *⋯* *⋯*
PAD PAD PAD PAD PAD
*π* ~*K*~(*w* ~1~) *c* ~11~ *c* ~12~ *⋯* *c* ~1*n*~
*⋯* *⋯* *⋯* *⋯* *⋯*
*π* ~*K*~(*w* ~*m*~) *c* ~*m*1~ *c* ~*m*2~ *⋯* *c* ~*mn*~
PAD PAD PAD PAD PAD
*π* ~*K*~(*w* ~3~) *c* ~31~ *c* ~32~ *⋯* *c* ~3*n*~
*⋯* *⋯* *⋯* *⋯* *⋯*
######
Comparisons of preview schemes.
Data type Preview mode Round Communication Storage Computation
---------------------------------- ------------------------- -------------- ------- ------------------------------------- ---------- -------------
General search engine \[[@B40]\] Plaintext Query biased 1 *O*(*s*) *d* *O*(*d*)
Content mask \[[@B30]\] Plaintext or ciphertext Static 1 *O*(*s*) *O*(*d*) *O*(1)
Our scheme Ciphertext Query biased 2 *O*(log(*d*/*s*) + *s* + *d*/*s*) *O*(*d*) *O*(1)
######
Properties of SSE-2 + SecQBP.
Properties SSE-2 SSE-2 + SecQBP
---------------------- ---------- -------------------------------------
Adaptive adversaries Y Y
Number of servers 1 1
Server storage *O*(*n*) *O*(*n*)
Server computation *O*(1) *O*(1)
Number of rounds 2 3
Extracommunication *O*(1) *O*(log(*d*/*s*) + *s* + *d*/*s*)
[^1]: Academic Editors: T.-Y. Chang and C. L. Hsu
| {
"pile_set_name": "PubMed Central"
} |
###### Strengths and limitations of this study
- This is the first review that seeks to identify the greatest amount of information related to mechanical ventilation settings and monitoring during physical therapy interventions.
- This study follows a recommended method to answer research questions through a scoping review.
- A biomedical librarian will perform systematic search of the databases and two independent physiotherapists will carry out the study selection.
- The databases will be selected according to the availability of our institution. To minimise the possibility of missing relevant articles, we will carry out a hand search of the reference lists of the relevant articles and a search in a grey literature database.
- The search is limited to documents in English and Spanish.
Introduction {#s1}
============
Mechanical ventilation (MV) is one of the most common interventions in the intensive care unit (ICU).[@R1] Goals of MV include alleviation of the work of breathing and gas exchange enhancement while avoiding additional lung or respiratory muscle injury.[@R2] Positive pressure ventilatory support must synchronically interact with the negative pressure generated by the respiratory muscles to achieve the objectives of MV.[@R3] There are several ventilatory strategies to improve patient-ventilator synchrony during stationary conditions (eg, assisted or proportional modes),[@R4] but little is known about ventilatory support during physical therapy.
Physical therapy is frequently restricted in mechanically ventilated patients,[@R5] though early mobilisation has been extensively advocated to improve functional outcomes.[@R7] Physical therapy interventions include passive movements of the extremities for deeply sedated patients,[@R8] in-bed and out-of-bed mobility,[@R9] active or passive cycling,[@R10] neuromuscular electrical stimulation[@R11] and ambulation.[@R12] Clinical trials of these physical therapy interventions have reported inconsistent effects on functional outcomes in mechanically ventilated patients.[@R9] These results could be explained by a lack of knowledge on the optimal dosage of physical therapy for ICU patients (in terms of intensity, duration and frequency).[@R18] Alternatively, during physical therapy a mismatch between ventilatory support and exercise-induced ventilatory demand may excessively increase work of breathing, limiting physical therapy performance and eventually leading to fatigue.[@R22]
Despite expert consensus and recommendations on active mobilisation of mechanically ventilated patients,[@R23] ventilator settings and safety parameters to monitor during mobilisation have not yet been defined. Some authors have suggested strategies to adjust ventilatory support during physical therapy; for example, increasing support level during pressure support ventilation,[@R24] using proportional ventilation modes,[@R22] employing assist-control ventilation before and after physical therapy[@R25] or increasing the fraction of inspired oxygen.[@R25] In addition, mechanical ventilators allow real-time monitoring of respiratory (eg, respiratory rate or minute ventilation) and metabolic variables (eg, carbon dioxide production)[@R22] that could provide useful information to individualise the dosage of physical therapy or ventilatory support. There is therefore a need to summarise the existing knowledge on the use of MV as a potential tool to provide safe and efficient physical therapy for ICU patients.
A first step for professionals to improve the decision-making during physical therapy intervention is to identify the MV monitoring and setting. Scoping review is a solid method to map research areas, providing a rigorous and systematic approach to the synthesis of knowledge, particularly useful when a body of literature has not yet been thoroughly reviewed.[@R27] The aim of this review is to comprehensively map and summarise current knowledge on adjustments of respiratory support and respiratory or metabolic monitoring during physical therapy in adult critically ill mechanically ventilated patients.
Methods {#s2}
=======
This scoping review will be based on the Joanna Briggs Institute (JBI) method[@R27] as initially conceived by Arksey and O'Malley.[@R30] This project was reviewed, approved and registered by the Research and Clinical Trials Unit of the *Departamento Científico Docente* of *Clínica Alemana de Santiago* (N° 2019--752).
This scoping review will be based on the six steps recommended by the JBI: (1) identifying the research question, (2) identifying relevant studies, (3) study selection, (4) data extraction, (5) collating, summarising and reporting the results, and (6) consultation.[@R30] To control the quality of this review, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for Scoping Reviews Checklist will be used.[@R31]
Stage 1: identifying the research questions {#s2-1}
-------------------------------------------
The research questions of this scoping review emerged from knowledge gaps identified by the authors when trying to adjust ventilator settings in order to maintain synchrony and avoid respiratory distress during physical therapy interventions. Additionally, modern mechanical ventilators provide a wealth of respiratory and metabolic data that could be used to monitor the physiological response to physical therapy. This information, in turn, could be used to further adjust ventilator settings or the dose of physical therapy in an individualised manner. The following research questions were structured based on the *Population, Concept and Context* method (PCC)[@R27]:
1. What MV settings have been reported or studied during physical therapy interventions in mechanically ventilated adult critically ill patients?
2. What respiratory and metabolic variables have been reported or studied to monitor the physiological response to physical therapy interventions in adult mechanically ventilated critically ill patients?
Stage 2: identifying relevant studies {#s2-2}
-------------------------------------
To identify relevant studies, a biomedical librarian (AJS) and two critical care physiotherapist (AC-M and FG-S) will search the literature based on the three stages recommended by the JBI.[@R27] First, an iterative search will be conducted in PubMed (FG-S and AC-M) using keywords and MeSH terms associated to 'mechanical ventilation', 'exercise', 'intensive care unit' and 'assisted mechanical ventilation'. Second, the keywords identified in the relevant articles of the first search will be reviewed to include them in a second search in the electronic databases selected for relevance of content and accessibility (limited to the databases available in our institution). The following relevant biomedical databases will be consulted: PubMed (NCBI), CINAHL plus with full text (EBSCO), Rehabilitation & Sport Medicine (EBSCO), Scielo Citation Index (Clarivate), Epistemónikos, Clinical Trials, Cochrane Library (free access by Ministerio de Salud de Chile) and PEDro (see eg, of the full search strategy in [online supplementary file table S1](#SP1){ref-type="supplementary-material"}). It will be filtered by language (English and Spanish) and all types articles will be considered, including research articles, conference abstracts, clinical practice guidelines and expert recommendations. In addition, the grey literature search will be conducted using Opengrey. Third, a hand search of the reference lists of the relevant articles identified in the final search will be performed. The authors of primary studies or reviews will be contacted if additional information is deemed necessary. Bibliographical management software will be used to download and manage the reference database (EndNote Clarivate Analytics, Boston, Massachusetts, USA). The results for the initial search using relevant keywords and MeSH terms developed for each database are presented in [table 1](#T1){ref-type="table"}. The final search will be made within 2 weeks when this protocol is approved and the search cut-off date will be 30 June 2019.
10.1136/bmjopen-2019-030692.supp1
######
Initial database search results
------------------------------------------------------------------ -----
PubMed (NCBI) 675
CINAHL plus with full text (EBSCO) 264
Epistemónikos 105
Clinical Trials 41
Rehabilitation & Sport Medicine (EBSCO) 21
Scielo Citation Index (Clarivate) 11
PEDro 13
Cochrane Library (free access by *Ministerio de Salud de Chile*) 3
------------------------------------------------------------------ -----
Stage 3: study selection {#s2-3}
------------------------
Two researchers (FG-S and AC-M) will independently carry out the article selection in three stages: filter by title, abstract and full text according to the selection criteria. Potential disagreements about the inclusion or exclusion of articles will be discussed and, if the discrepancy is not resolved, a third one (RPA) will judge. The 'include rather than exclude' methodology will be used to select by title, abstract and full text, to avoid potential loss of relevant information.[@R32] This stage will be reported using the PRISMA flow chart.[@R33]
The following inclusion criteria will be used, based on the components of the PCC method[@R27] ([figure 1](#F1){ref-type="fig"}): *Population---*adult critically ill patients on MV for at least 48 hours, defined as any ventilation through an endotracheal tube, tracheostomy tube or non-invasive ventilation[@R34]; *Concept (intervention*)---physical therapy interventions during ICU stay[@R35] performed by a physiotherapy, nurse or other allied-health professional, including (but is not limited to): passive, active or active-assisted range of motion, progressive mobility, neuromuscular facilitation, therapeutic exercises, resistant exercises, manual skills, neuromuscular electrical stimulation, cycling and dangle sitting; *Concept (Outcomes*)---description of the MV settings used before, during and after of any physical therapy interventions. In addition, articles describing any respiratory or metabolic variables used to monitor before, during and after any physical therapy interventions will be selected; *Context (studies*)---all types of scientific articles, including (but not limited to) original studies, conference abstracts, reviews, clinical practice guidelines, expert recommendations and protocols or ongoing studies. The following article exclusion criteria will be used: those concerning interventions initiated outside the ICU, paediatric or neonatal population, thoracic or respiratory physiotherapy interventions (eg, inspiratory muscle threshold and airway clearance techniques), animal models, in vitro studies and articles published in languages other than English or Spanish.
![Inclusion criteria used in this scoping review based on the population, concept and context method.[@R27] AROM, active range of motion; AAROM, active-assisted range of motion; ICU, intensive care unit, PROM, passive range of motion.](bmjopen-2019-030692f01){#F1}
Stage 4: data extraction {#s2-4}
------------------------
Relevant data extraction from the selected studies will be performed using Peters *et al* recommendations[@R27] and compiled using the PCC nomenclature in a custom-made Excel 2011 (Microsoft, V.14.6.6) spreadsheet ([online supplementary material table S2](#SP1){ref-type="supplementary-material"}). Four reviewers (FG-S, AC-M, JMB and RPA) will extract the data from the qualified articles, including information from the supplementary materials and appendices. The four reviewers will complete the same data extraction form and will meet to determine if the information extracted is consistent among them to answer the questions and the purpose of the study.
The least information to be extracted from the articles will be: authors, year of publication, country, sample size, study design, objective of the study, type of publication, study population, type of ventilation used, MV settings used during physical therapy intervention (eg, ventilatory mode, mandatory respiratory rate, fraction of inspired oxygen, positive end expiratory pressure, targeted pressure or volume prescription), respiratory or metabolic variables used to monitor physical therapy (eg, respiratory rate, tidal volume, minute ventilation, oesophageal pressure or diaphragm electrical activity derived variables, pulse oximetry, breathing pattern, oxygen consumption and carbon dioxide production) and type of physical therapy intervention. Moreover, some clinically relevant causes of complications (eg, vomiting, agitation, airway trouble) will be extracted.
Physical therapy interventions will be the following[@R21]: passive, active or active-assisted range of motion, progressive mobility, neuromuscular facilitation, therapeutic exercises, resistant exercises, manual skills, neuromuscular electrical stimulation, cycling and dangle sitting.
Outcomes will be classified using the following definitions: MV adjustments during physical therapy session and respiratory/metabolic real-time variables used to monitor physical therapy interventions.
Stage 5: collating, summarising and reporting results {#s2-5}
-----------------------------------------------------
Compiled data from the selected studies will be synthesised by four researchers (FG-S, AC-M, JMB and RPA) in a logical and descriptive order aligned with the study questions. According to the methodology described by Arksey and O'Malley,[@R30] we anticipate that the extraction and synthesis of the data will be an iterative process that will depend on the literature found. The results will be presented based on four categories: (1) bibliometric variables of the included articles, (2) physical therapy interventions, (3) MV settings and (4) respiratory and metabolic variables used to monitor physical therapy interventions. A descriptive summary and synthesis of the study findings will be presented in results tables.
Potential discrepancies or uncertainties in the synthesis of information between the four reviewers will be discussed with a senior critical care physician (JGS).
Stage 6: consultation {#s2-6}
---------------------
If necessary, other experts on MV and physical therapy or the corresponding authors of selected articles will be consulted. A focused search of exercise physiology articles may be carried out if the findings extracted in the scoping review require physiological explanations not found in the original sources.
Patient and public involvement {#s2-7}
------------------------------
The scoping review to follow on this protocol will not require the participation of any patients or the general public.
Ethics and dissemination {#s3}
========================
This scoping review does not require the ethics committee approval. This study seeks an integrated research approach including physiotherapists, a respiratory therapist, a critical care physician and a biomedical librarian. This will facilitate knowledge translation at the end of the study, integrating physical therapy and respiratory therapy in the ICU setting.
The knowledge translation of the scoping review results will be carried out based on the End-of-Grant objectives.[@R36] These include three broad stages: diffusion (let it happen), dissemination (help to make it happen) and application (make it happen).[@R36] Diffusion will take place in a peer-reviewed scientific journal covering as many users of intensive care knowledge as possible. Dissemination of the findings will take place through the presentation of the results and clinical recommendations in relevant international critical care congresses. Training to MV stakeholders in the use of the MV during physical therapy interventions will facilitate its application. The results of this scoping review could provide the basis for future studies on patient--ventilator interaction during physical therapy.
Supplementary Material
======================
###### Reviewer comments
###### Author\'s manuscript
**Contributors:** FG-S conceived the study. FG-S, AC-M, AJS and JGS contributed to the protocol design and plan. FG-S, AC-M and AJS developed and conducted the initial literature search. FG-S, AC-M, JMB, RPA and JGS worked collaboratively to several draft and revise the manuscript. All the authors made substantive intellectual contributions to the development of this protocol and approved the final version.
**Funding:** The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
**Competing interests:** None declared.
**Patient consent for publication:** Not required.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
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Introduction
============
Although it is generally accepted that type 1 diabetes is associated with ten percent less bone mineral density (BMD) value compared with no diabetic adults,[@B1],[@B2] there is no consensus on osteoporosis risk in people with type 2 diabetes. Nicodemus and Folsom[@B3] found that women with type 2 diabetes had 1.70-fold higher risk of incident hip fracture than women without diabetes. Two other studies revealed no difference in bone density between type 2 diabetes patients and control subjects.[@B4],[@B5] Some have reported higher bone mass in type 2 diabetic patients relative to no diabetic control subjects.[@B6],[@B7],[@B8]
The aim of this study to compare the BMD values of postmenopausal women with diabetic, hypertensive and hyperlipidemic postmenopausal women.
Materials and Methods
=====================
The medical records and BMD measurements of 50 diabetic hypertensive postmenopausal women followed-up at our institution for 4 years were evaluated retrospectively. A cohort of women with known diabetes mellitus (DM) was identified by retrospectively reviewing our patient database. Approval (Date: 1.08.2014, Number: 115) of the Bozok University Medical School\'s local ethics committee has been obtained. Participants were naturally postmenopausal women under 70 years of age. Exclusion criteria included surgical menopause, smoking, chronic medical illness (Chronic Obstructive Lung Disease). Essential hypertension and diabetes diagnosis was obtained from hospital records. The diagnosis is made in our hospital according to \"the Seventh Report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure\" and \"World Health Organization (WHO) report on the diagnosis and classification of DM. Women treated with diet or drugs or both; or either a fasting serum glucose value more than 126 mg/dL describe as known DM. Patients who had essential hypertension previously or patients whose blood pressure measurements had been more than 140/90 were included in the study. Those women with and hypertension who had undergone screening BMD in our institution were identified; this group of women comprised our study group. Increase of triglyceride (\> 160 mg/dL) or cholesterol levels (\> 200 mg/dL) are described as hyperlipidemia. Fifty-one healthy postmenopausal women were also included in the study as the control group. Our institution as a large tertiary referral center, offers a comprehensive menopause health program to our patients with screening BMD, mammogram and Pap-test. Our control group was selected from these patients. Data included the age, gravidity, parity, body mass index (BMI), BMD measurements, serum alkaline phosphatase (ALP), calcium and phosphorous levels were collected. BMD was measured using dual energy X-ray absorptiometry (DXA). BMD measurements of the hip (femoral neck) and lumbar spine (L1-L4) were performed in the anterior posterior (AP) view for the lumbar spine DXA scanner (Hologic QDR 4500 Elite, Bedford, MA, USA). The BMD value for each region was calculated as the ratio of bone mineral content to the area of the interested region (g/cm^2^). According to WHO criteria, a T-score of ≥ -1 denotes normal bone, a T-score between -1 and -2.5 denotes osteopenia, and a T-score of ≤ -2.5 denotes osteoporosis. BMI wascalculatedbydividingthe weighttothesquare of height (kg/m^2^).
The statistical analyses were performed using the SPSS statistical package (SSPS Inc., Chicago, IL, USA). The normality of variables were assessed using the Kolmogorov-Smirnov test. The data were reported as mean ± standard deviation (for normally distributed data) or as median and range (for non-normally distributed data). For data with normal distribution, the Student t test was applied. Otherwise, the Mann-Whitney U test was used as a nonparametric test. Correlations between BMD and biochemical marker levels were made using Pearson\'s correlation coefficient and a partial correlation analysis after adjustments for age and menopausal time. A *P* value of less than 0.05 was considered significant.
Results
=======
There were no significant differences between two groups in respect to lumbar and vertebral BMD values, age, BMI, gravidity, parity ([Table 1](#T1){ref-type="table"}). Serum cholesterol and fasting glucose levels were significantly different between each groups (*P* = 0.0001, *P* = 0.002). There were no significant differences in serum ALP, calcium and phosphorous levels between the diabetic patients and the controls. There was a negative correlation between lumbar spine BMD and duration of menopause. But we didn\'t find a correlation between blood glucose levels and BMD ([Table 2](#T2){ref-type="table"}). After adjusting for age and menopausal time, the partial correlation of BMD with biochemical marker levels were shown in [Table 3](#T3){ref-type="table"}.
Discussion
==========
Data regarding the impact of DM on the BMD are controversial. Studies have found that people with type 2 diabetes have increased,[@B6],[@B7],[@B8] similar[@B4],[@B5] or decreased bone mass in comparison to healthy control subjects.[@B3] Although BMD measurements of femur neck and lumbar (L2-L4) spines were lower in study group. We couldn\'t find significant differences between study and control group. Regarding the effect of glycaemia on BMD, there are some data that depict the relation between glycaemia and BMD. Hyperglycemia induces decreased bone turnover with osteoblast dysfunction and suppresses serum osteocalcin levels.[@B9],[@B10] According to the previous studies that defend a relation between DM and osteoporosis, osteoporosis is more frequent among chronic DM patients, who have retinopathies, neuropathies or vascular disease.[@B11] Kanazawa et al.[@B12] claimed a negative correlation between serum osteocalcin and hemoglobin A1c (HbA1c) levels. Viegas et al.[@B13] found remarkable osteoporosis and vertebral fracture ratios among chronic diabetic postmenopausal women who have retinopathy and renal dysfunction. But there are also some studies that claim no relation between DM and osteoporosis. For example Anaforoglu et al.[@B14] found no relation between type 2 diabetes and osteoporosis among postmenopausal women but a negative correlation between femoral neck BMD and micro albuminuria due to DM. Other studies showed that BMD values of type 2 DM patients were higher than non-diabetic patient\'s values.[@B15]
Although there is no consensus about the effects of hypercholesterolemia on BMD, many authors noticed a negative correlation between hypercholesterolemia and BMD. In vitro studies, it was shown that lipids and lipoprotein oxidation products hampered the functions and differentiation of osteoblasts.[@B16] Low-density lipoproteins (LDL) cholesterol has got direct negative effect on bone metabolism. This negative effect of LDL cholesterol particles is explained by increased catabolism rather than bone production.[@B17],[@B18] Similarly, Poli et al.[@B19] found low BMD among 1303 postmenopausal patients who have high plasma LDL levels. Cui et al.[@B20] reported a negative correlation between LDL levels and lumber BMD values in postmenopausal patients. In another study lumber BMD values found lesser among postmenopausal women who have high serum cholesterol and LDL levels.[@B21] On the other hand Demir et al.[@B22] couldn\'t find any correlation between cholesterol levels and BMD. In another study it was reported that lipid profile wasn\'t a unique factor on BMD besides lipids duration of menopause and level of estrogen were also effective on BMD.[@B23]
In our study we couldn\'t find significant differences between diabetic, hyperlipidemic and hypertensive women and control group. There is expediency with literature. In our study we didn\'t inquire medication among hypertensive and hyperlipidemic patients. In the literature positive effects of antihypertensive and antihyperlipidemic drugs on BMD were reported. Some previous studies reported positive effects of anti-lipid drugs on BMD.[@B24],[@B25],[@B26] In another study Olmos et al.[@B27] reported that thiazide group drugs were effective in increasing BMD in hypertensive patients. The relation between BMD and Cardiovascular Disease risk factors such as hypertension and hyperlipidemia couldn\'t be clearly defined. In another study, no relation was found between hypertension and BMD.[@B28],[@B29]
There are no significant differences between study group and control group in terms of BMD measurements. We found that, accompanying chronic diseases such as diabetes, hypertension and hyperlipidemia don\'t affect the BMD measurements at postmenopausal period. So these postmenopausal women don\'t have excess risk regarding osteoporosis.
**Conflict of Interest:** No potential conflict of interest relevant to this article was reported.
###### Demographic and clinical characteristics of the two groups
Values are expressed as mean ± standard deviation (SD)
^\*^Values are median (minimum-maximum)
ALP: alkaline phosphatase, BMI: body mass index, Ca: calcium, FSH: follicle stimulating hormone, HDL: high-density lipoprotein, LDL: low-density lipoprotein, LH: luteinizing hormone, P: phosphorus, TG: triglycerides, TSKF: T score femoral neck, TSKV: T score lumbar vertebra spine
###### Analysis of correlation of T score femoral neck, T score lumbar spine
FSH: follicle stimulating hormone, LH: luteinizing hormone, Ca: calcium, P: phosphorus, TSKF: T score femoral neck, TSKV: T score lumbar vertebra spine
###### Analysis of partial correlation of T score femoral neck, T score lumbar spine (after adjust for age and menopause time)
FSH: follicle stimulating hormone, LH: luteinizing hormone, Ca: calcium, P: phosphorus, TSKF: T score femoral neck, TSKV: T score lumbar vertebra spine
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Background
==========
Marine environments have become an easily reasonable exposure site for pollutants such as polycyclic aromatic hydrocarbon (PAH) compounds. PAHs are a group of environmental contaminants formed during the incomplete combustion of organic materials such as coal, fossil xfuel, and wood, as well as from forest fires, volcanic activity and petroleum seeps \[[@B1]-[@B5]\]. PAHs are well known environmental pollutants and are included in priority pollutant list of the European Union and US Environmental Protection Agency (EPA) due to their mutagenic and carcinogenic properties \[[@B6],[@B7]\].
Source determination of PAH in the marine environment seems to be difficult, because they are produced by three major processes: Pyrogenic PAH, Petrogenic PAH and Diagenic PAH. Pyrogenic PAHs result from an incomplete but high temperature and short-duration combustions of organic matters including fossil fuels and biomass \[[@B3],[@B8]\]. Petrogenic PAHs are formed by biogenic processes at a relatively low temperature over geologic time scale, leading to the formation of petroleum and other fossil fuels containing PAHs \[[@B3],[@B9]\]. Diagenic PAHs refer to PAHs from biogenic precursors, like plant terpenes \[[@B10],[@B11]\]. The first two types of sources may be both natural and anthropogenic sources. Petrogenic PAH dominates oil-polluted samples, while Pyrogenic PAH tends to dominate the samples from industrial areas. The toxicity, environmental persistence, bioaccumulation, and trophic transfer of PAHs in aquatic ecosystem are vital to assess the ecological risk of contaminants \[[@B12]-[@B14]\].
All kinds of Fish definitely respond to pollutants by altering/adapting their metabolic functions. Hence, Fish have been often used as appropriate bioindicators of the chemical contaminants \[[@B15]-[@B17]\]. Mudskippers (Gobiidae: Oxudercinae) live in intertidal mudflats and in mangrove ecosystem \[[@B18]\]. They spend much of their life buried, being in close contact with the sediments \[[@B12]\]. The susceptibility of using mudskippers to monitor marine pollution, like PAHs, points to the fact that mudskippers are able to accumulate and record the PAHs presented in the coastal environments. Therefore, the analysis of mudskipper tissues may give an indication of the bioavailable portion of environmental PAH contamination \[[@B12]\].
The Persian Gulf is considered as one of the most polluted ecosystems in the world. It is an exposed and stressed area as a result of oil pollution \[[@B19]\]. The environmental condition of the Persian Gulf became more critical because of the war related activities \[[@B20]\]. A large number of studies have addressed PAHs in the Persian Gulf, mainly to determine concentration, composition and degradation \[[@B15],[@B19],[@B21]-[@B29]\]. In line with these trends of research, this study was conducted: the objective of the present paper was to investigate the levels of PAHs in *B. dussumieri*, water and sediment samples collected from this area.
Materials and methods
=====================
Study species
-------------
Mudskippers (i.e., *B. dussumieri*) were selected as the main organism for this research. A Mudskipper has a preferential uptake of PAHs from the sediments due to its benthic characteristics. This is considered important because the most carcinogenic PAHs are not usually present in the dissolved phase in a marine environment. These compounds are bound to the sediments or the suspended matters. Accordingly, Mudskippers (*B. dussumieri*) have been used in this research as a sentinel organism for the common chemical analysis in the coastal areas of the Persian Gulf.
Study sites
-----------
Five different stations were chosen along the north western coast of the Persian Gulf (see Table [1](#T1){ref-type="table"} and Figure [1](#F1){ref-type="fig"}). Most of the locations were near the mouths of discharges of commercial and domestic waste. Furthermore, they were selected along a doubtful pollution gradient based on the earlier information available in literature about the local contaminant levels \[[@B30]\].
######
Background information on the sampling stations in the North West of Persian gulf
**Sampling sites** **Coordinates** **Description**
-------------------- ----------------- ----------------- ----------------------------------------------------------------
**Arvand(St1)** 48º 40′58″E 29º54′35″N Abadan refineries
**Jafari(St2)** 49º06′52″E 30º26′52″N Special economic and petroleum zone, urban and industrial dump
**Zangi(St3)** 49º03′52″E 30º28′52″N landing port for vessels, petroleum industries
**Samayeli(St4)** 49º10″51″E 30º27″45″N landing port for fishing vessels, petroleum saving tank
**Bahrakan(St5)** 49º50′54′ E 30º08′14″N Reference site
St: Station.
{#F1}
Sampling and preparation for chemical analysis
----------------------------------------------
The sediments, the coastal water and the biota (i.e., B. dussumieri) were collected in October 2011. The mudskippers (n = 30) were collected from each of the five sampling sites by using hand nets. For the transportation of the fish to the laboratory, they were stored inside the aquaria with a volume of 60 L (semi-static to be aerated under ambient conditions: a water temperature of 25.2 ± 0.7°C, a salinity of 38 g/l).
Immediately after transportation of the fish to the laboratory, mudskippers were anesthetized by pouring dry extracts of pink cloves into water column. Subsequently, the fish were removed and were all measured in length and weight: a mean length of 17 ± 0.1 cm and a mean weight of 16 ± 0.2 g. The fish were immediately dissected and the liver was quickly removed at the ice. The Liver tissues were wrapped in the foils and were stored at -20°C for the following PAHs pending analyses.
Superficial sediment samples (top 8 cm) were taken from five different locations on each of the five study sites. The sediment samples were placed in a Teflon-lined amber vial on an ice pack until it could be stored at -20°C to be ready for the following PAHs analysis. These steps were taken following the standards of ICES ACME Report 1997 \[[@B31]\].
Coastal sea water was collected in acid-washed amber glass bottles fitted with Teflon screw caps from three different locations at each five study sites. To remove suspended matter, samples were filtered through glass fiber filters (Whatman GF/C). Filtered samples were stored in pre-cleaned (with distilled water) glass bottles at 4°C prior to the PAHs analysis.
Extraction and purification of PAHs in tissues
----------------------------------------------
In order to extract and purify PAHs, the same method proposed earlier by Perugini et al., in 2007 was followed \[[@B32]\]. This determination was carried out in composite pools of tissues dissected from 30 fish (five samples, each constituted by tissues of six specimens). After being thawed, the assay sample (2 g dw) was inserted into a l00mL round-bottomed flask with a 10 mL of 1 M KOH in an ethanolic solution. The mixture was placed in a reflex system in a temperature of 80°C for 3 hours. The liquid phases were transferred to a separation funnel and were extracted with a 10 mL of cyclohexane. They were then shaked rigorously for 30 minutes. The cyclohexane KOH phase was drained and discarded. Next, the liquid phases were rinsed with 10 mL cyclohexane once more. The samples were allowed to pass through the anhydrous sodium sulphate column. After that, the organic phase was concentrated in a rotary evaporator (Model Buchi B-490) to a volume of 5 mL under a reduced pressure. The samples passed through a column filled with florisil and concentrated in rotary evaporator (30°C, 19--21 kPa) to volume of 1 mL. Finally, the extracts were evaporated with a gentle stream of nitrogen at room temperature and were then reconstituted in 1 mL of acetonitrile.
Extraction and purification of PAHs in sediment
-----------------------------------------------
For the extraction and purification of PAHs the method proposed by Moopam in 1999 was adopted \[[@B33]\]; however, this was done by implementing some slight modifications based on the context of the present research. PAHs in the sediments (N = 25) obtained from five different locations on each of the five study sites were measured. Sediment samples (each 200grams) were freeze dried (freeze-drier Model: OPR-FDB-5503, Korea) at -40ºC to a constant weight. Subsequently, a 10 g of each sample were mixed with a 250 mL of n-hexane and dichloromethane mixture (1:1 ratio). Following that, the mixture was placed for 8 hours in Soxhelet. The combined extracts were concentrated to about 1 mL by vacuum rotary evaporation. To eliminate sulphur and the related compounds, a 3 g of active copper was added to the mixture and was then allowed to pass through a folded filter paper (Whatman GF/C, 24micron) for 24 hours. The mixture was concentrated in a rotary (Buchi B-490) to a volume of 5 mL. The Samples were passed through a column which contained a 10 g of silica gel in n-hexane, 1-2 g of anhydrous sodium sulphate, and a 10 mL of alumina. Then, a 30 mL of n-hexane and dichloromethane mixture (9:1 ratio) was added to the column. The samples were concentrated once more in a rotary evaporator to a volume of 5 mL. After that, the concentrated extract was dried under the nitrogen steam (N-E VAP 112, USA). The residue was finally dissolved in a 1 mL of acetonitrile.
Extraction and purification of PAHs in water
--------------------------------------------
Extraction and purification of PAHs in water was conducted as the following method. The experimental procedure is based on the USEPA Method 550.1 of Bashe and Baker in 1990 \[[@B34]\]. Solid phase partitioning was carried out by processing 500 ml of the sample through a 25 mm C18 Empore TM extraction disk mounted in a side arm vacuum flask for approximately 30 min. The disks were dried and desorption was performed by eluting with 4 × 5 ml of acetonitrile. Samples were concentrated to 0.5 ml under a gentle stream of nitrogen. PAHs in the particulate fraction were isolated from the glass fiber filters with 4 \_ 5 ml of acetonitrile by ultrasonication during 30 min. Samples were then centrifuged and concentrated to 0.5 ml under nitrogen steam.
Chemical analysis
-----------------
The sixteen most toxic PAHs introduced by the Environmental Protection Agency (EPA) were assessed in the coastal waters, the sediments and the biota (mudskipper: B. dussumieri). The abbreviations used for the investigated PAHs are: naphthalene: N; acenaphthylene: AC; acenaphthene: ACE; Fluorene: F; phenanthrene: Ph; anthracene: A; fluoranthene: Fl; pyrene: Py; benz(a)anthracene: B(a)A; chrysene: Ch; benzo(b)fluoranthene: B(b)Fl; benzo(k)fluoranthene: B(k)Fl; benzo(a)pyrene: B(a)Py; indeno(1,2,3-c,d)pyrene: IPy; dibenz(a,h)anthracene: DB(ah)A; benzo(ghi)perylene: B(ghi)Pe. Hewlett-Packard 1100 HPLC equipped with an Agilent-1100 fluorescence detector was used. Injection volume was 10 μL. The initial mobile phase was a 60% of acetonitrile and a 40% of HPLC water for 40 minutes with a flow rate of 0.7 mL min^-1^, which was then gradiently changed to 100% acetonitrile.
Gross morphometric indices
--------------------------
Gross morphometric indices were computed for each individual Mudskipper consenting to the following formulas:
$$\begin{matrix}
{{Condition}\ {factor}\mspace{2mu}\left( {CF} \right):\left( {WT}/3 \right) \times 100} \\
{{Hepatosomatic}\ {index}\mspace{2mu}\left( {HSI} \right):\left( {WH}/{WT} \right) \times 100} \\
\end{matrix}$$
Where WT: total wet weight (g); L: total length (cm); WH: liver weight (g).
Quality control
---------------
Replicate samples, certified reference materials IAEA-417 and procedural blanks were used as quality control procedures. All the obtained values for PAHs in CRM were in the reported range. Reproducibility and recovery were high (85--110%) with relative standard deviation (RSD) 4--11%. To measure the quality control, the procedural blanks were periodically analyzed for each batch of 10 samples. Quantitative analysis was conducted on a five-point linear calibration of PAH solution, obtained by dilution of the certified standard mixture of 16-PAH (kit 610-N-Supelco4-7351). Blank samples were processed together with samples, and limits of detection (LODs) were estimated as the average signal of the blanks plus five times the standard deviation of the signal of the blanks. LODs for biota, sediment and water measurements ranged from 0.18 to 0.4 ng g^\_1^ dw and 0.5--1.4 ng g^\_1^ dw, 0.1-0.12 μg/l respectively. Satisfactory linearity was obtained with values of the correlation coefficient R above 0.99.
Statistical analyses
--------------------
Statistical analysis was done using the SPSS software (version 19). The data were tested to check for the normality using the Kolmogorov--Smirnov test and indicated a normal distribution. To assess changes in PAHs levels at different sampling sites, One way analysis of variance (ANOVA), followed by Tukey's multiple range test for mean comparisons was applied (p \< 0.05).
Results
=======
PAH pollution of the sediments
------------------------------
Poly aromatic hydrocarbons contents determined in the sediment from five sampling sites in the Persian Gulf as well as the results of the related statistical analyses are displayed in Table [2](#T2){ref-type="table"}. Among 16 PAHs, levels of B(a)A represented the highest concentration among all sampling sites. The highest concentration of total PAHs (Σ16PAH) was measured at Jafari followed by Zangi \> Arvand \> Samayeli \> and Bahrakan. For total PAHs levels (Σ16PAH), significant variations in all of the sampling sites were also found (p \< 0.05). The composition patterns of PAHs in all the sites were dominated by the presence of four-ring PAH except for the PAH in Bahrakan where three-ring structures followed by four-ring ones prevailed (Figure [2](#F2){ref-type="fig"}).
######
**Mean and standard deviation + 16PAHspercent per T-PAHs values of sediment (ng g**^**-1**^**dry weight) and water (μg/l) collected from different sites**
**PAHs** **Samples** **Arvand** **Jafari** **Zangi** **Samayeli** **Bahrakan**
---------- ------------------- ---------------------- ---------------------- --------------------- --------------------- -------------------
N S nd nd nd nd nd
W nd nd nd nd nd
AC S 13.297 ± 1.451^a^ 132.112 ± 9.90^b^ 74.903 ± 6.015^c^ 1.979 ± 0.461^d^ nd
W 1.584 ± 0.331^a^ 1.121 ± 0.155^a^ 0.810 ± 0.110^a^ 2.634 ± 0.522^b^ nd
ACE S nd nd nd nd nd
W nd nd nd nd nd
F S 44.005 ± 1.590^a^ 17.353 ± 1.480^b^ 3.323 ± .792^c^ 4.319 ± .690^c^ 4.698 ± .690^c^
W 1.123 ± 0.223^a^ 0.566 ± 0.026^b^ 0.286 ± 0.065^c^ 0.341 ± 0.031^c^ 0.300 ± 0.026^c^
Ph S 20.989 ± 2.769^a^ 632.682 ± 27.72^b^ 33.429 ± 2.690^a^ 36.861 ± 3.003^a^ 15.438 ± 1.64^a^
W 0.406 ± 0.028^a^ 11.104 ± 1.391^b^ 0.449 ± 0.051^a^ 0.455 ± 0.040^a^ 0.373 ± 0.014^a^
A S nd nd nd nd nd
W nd nd nd nd nd
Fl S 5.164 ± 0.237^a^ 178.975 ± 9.38^b^ 29.546 ± 3.001^c^ 17.445 ± 2.432^d^ 14.289 ± 1.74^d^
W 0.734 ± 0.091^a^ 2.818 ± 0.632^b^ 2.162 ± 0.467^bc^ 1.472 ± 0.371^ac^ nd
Py S 114.260 ± 4.645^a^ 126.051 ± 7.979^a^ 426.015 ± 14.55^b^ 71.298 ± 6.074^c^ 28.476 ± 3.46^d^
W 0.167 ± 0.080^de^ 0.174 ± 0.015^de^ 0.348 ± 0.054^c^ 0.208 ± 0.023^d^ 0.125 ± 0.040^e^
B(a)A S 623.201 ± 18.16^a^ 1132.562 ± 59.923^b^ 649.151 ± 20.06^a^ 188.623 ± 8.388^cd^ 33.220 ± 3379^d^
W 0.453 ± 0.015^a^ 0.565 ± 0.083^a^ 0.460 ± 0.064^a^ 0.308 ± 0.072^b^ nd
Ch S 330.579 ± 16.05^a^ 760.706 ± 7.28^b^ 307.695 ± 14.09^a^ 40.299 ± 3.245^cd^ 17.379 ± 1.491^d^
W nd 1.150 ± 0.190^a^ 0.791 ± 0.089^a^ 0.645 ± 0.121^a^ nd
B(b)Fl S 26.360 ± 3.203^a^ 59.813 ± 4.120^b^ 111.277 ± 7.901^c^ Nd nd
W nd nd nd nd nd
B(k)Fl S 27.968 ± 3.721^a^ 41.532 ± 4.198^b^ 72.833 ± 4.854^c^ nd nd
W nd nd nd nd nd
B(a)Py S 240.500 ± 12.494^a^ 302.557 ± 14.891^b^ 199.636 ± 7.206^c^ nd nd
W 0.763 ± 0.025^a^ 0.839 ± 0.033^a^ 0.741 ± 0.109^a^ nd nd
IPy S nd nd nd nd nd
W nd nd nd nd nd
DB(ah)A S nd nd nd nd nd
W nd nd nd nd nd
B(ghi)Pe S nd nd nd nd nd
W nd nd nd nd nd
Ʃ PAH S 1446.323 ± 200.950\* 3384.34 ± 376.946\* 1907.81 ± 210.486\* 360.82 ± 65.028 113.50 ± 10.334\*
W 5\. 23 ± 0.479^a^ 18.34 ± 3.648^b^ 6.12 ± 0.603^a^ 6.06 ± 0.887^a^ 0.80 ± 0.127^d^
Values followed by the same letters (a, b, c, d) are not statistically different between the 16PAHs in each row (P \> 0.05).
\*Indicates significant difference between the Ʃ PAH in each row (P \> 0.05); nd = not detected; S = sediment; W = water.
{#F2}
PAH pollution of the water
--------------------------
The results of statistical analysis of 16 PAH levels in the coastal water samples are presented in Table [2](#T2){ref-type="table"}. For the total PAHs levels (Σ16PAH), the highest concentrations were detected at Jafari followed by Zangi \> Samayeli \> Arvand \> and Bahrakan. Significant differences were found between Jafari and Bahrakan and with other sites (p \< 0.05); however no significant difference was revealed among Arvand, Zangi and Samayeli sampling sites (p \> 0.05). The composition patterns of PAHs was prevailed by two-ring structures at Arvand and Samayeli, whiles three-ring structures dominated in three other sites followed by those with two-ring (Figure [2](#F2){ref-type="fig"}).
PAHs concentrations in liver tissues
------------------------------------
The results of the determination of 16 PAH accumulated in liver tissue of mudskipper together with the results of the statistical analysis are shown in Table [3](#T3){ref-type="table"}. The levels of PAHs in the liver from the five sites showed that only nine PAHs could be identified at detectable levels. For the total PAHs levels (Σ16PAH) in the liver, the highest concentration was detected at Jafari followed by Zangi \> Arvand \> Samayeli \> and Bahrakan. When comparing concentration of total PAHs among sites, there were only significant difference between Jafari and Bahrakan (p \< 0.05). However, there was no significant difference between the other sites compared two by two (p \> 0.05). Samayeli and Bahrakan sites were prevailed with three-ring structures. Moreover, a three -and four-ring PAH structures prevailed in Arvand section (Figure [2](#F2){ref-type="fig"}).
######
**Mean and standard deviation + 16PAHspercent per T-PAHs values of liver and gill tissues (ng g**^**-1**^**dry weight) collected from different sites**
**PAHs** **Tissues** **Arvand** **Jafari** **Zangi** **Samayeli** **Bahrakan**
----------- ------------------- -------------------------- -------------------------- -------------------------- -------------------------- -------------------------
N L nd nd nd nd nd
G nd nd nd nd nd
AC L 3.532 ± 0.49^a^ 4.381 ± 0.54^a^ 1\. 829 ± 0.177^b^ 2.233 ± 0.55^b^ nd
G 1.956 ± 0.580^bc^ 1.934 ± 0.694^bc^ 1.432 ± 0.382^c^ 3.009 ± 0.984^d^ nd
ACE L nd nd nd nd nd
G nd nd nd nd nd
F L 2.471 ± 0.55^a^ 1.051 ± 0.132^b^ 0.558 ± 0.083^b^ 0.660 ± 0.095^c^ nd
G 2.137 ± 0.570^a^ 0.993 ± 0.187^b^ 0.980 ± 0.191^cb^ 1.450 ± 0.344^ab^ 1.963 ± 0.621^a^
Ph L 3.576 ± 0.676^a^ 9.265 ± 0.826^b^ 2.619 ± 0.449^a^ 3.073 ± 0.625^a^ 1.053 ± 0.330^e^
G 1.218 ± 0.287^ce^ 11.388 ± 1.630^b^ 1.761 ± 0.501^c^ 1.342 ± 0.430^ce^ 0.177 ± 0.048^e^
A L nd nd nd nd nd
G nd nd nd nd nd
Fl L 1.019 ± 0.435^a^ 3.222 ± 0.763^b^ 1.036 ± 0.431^a^ 4.119 ± 0.337^db^ nd
G 1.623 ± 0.316^a^ 3.118 ± 0.402^b^ 3.190 ± 0.847^b^ 1.562 ± 0.388a nd
Py L 5.634 ± 0.830^a^ 2.168 ± 0.468^b^ 9.586 ± 1.030^c^ 13.296 ± 0.976^b^ 2.932 ± 0.792^b^
G 0.860 ± 0.140^a^ 0.326 ± 0.0.40^b^ 0.736 ± 0.180^a^ 0.665 ± 0.090^a^ 0.973 ± 0.126^a^
B(a)A L 5.275 ± 1.110^a^ 14.460 ± 2.931^b^ 7.153 ± 1.451^c^ nd nd
G nd nd nd nd nd
Ch L 4\. 110 ± 0.627^a^ 9.828 ± 1.270^b^ 4.097 ± 0.897^a^ nd nd
G nd nd nd nd nd
B(b)Fl L 0.750 ± 0.183^a^ nd 3.036 ± 0.847^b^ nd nd
G nd nd nd nd nd
B(k)Fl L nd nd nd nd nd
G nd nd nd nd nd
B(a)Py L 3.307 ± 0.827^a^ 2.263 ± 0.687^b^ 2.303 ± 0.524^b^ nd nd
G nd nd nd nd nd
IPy L nd nd nd nd nd
G nd nd nd nd nd
DB(ah)A L nd nd nd nd nd
G nd nd nd nd nd
B(ghi)Pe L nd nd nd nd nd
G nd nd nd nd nd
**Ʃ PAH** L **29.67 ± 1.678**^**a**^ **46.64 ± 4.782**^**b**^ **32.22 ± 2.971**^**a**^ **23.38 ± 4.981**^**a**^ **3.99 ± 1.328**^**d**^
**G** **7.79 ± 0.524**^**a**^ **17.76 ± 4.504**^**b**^ **8.10 ± 0.963**^**a**^ **8.03 ± 0.858**^**a**^ **3.11 ± 0.894**^**c**^
Values followed by the same letters (a, b, c, d) are not statistically different between the 16PAHs in each row (P \> 0.05) nd = not detected; L = Liver; G = Gill.
PAHs concentrations in gill tissues
-----------------------------------
The results of the determination of 16 PAH accumulated in gill tissue of mudskipper together with the results of the statistical analysis are shown in Table [3](#T3){ref-type="table"}. As clearly observable form the table, only five PAHs (Py, Fl, Ph, F, and AC) can be identified at the detectable levels, Nevertheless, among the five PAHs pollutant detected in the gill samples, only three ones (Py, Fl, Ph) were found at Bahrakan site. The highest concentrations of the total PAHs levels (Σ16PAH) in the gill were found at Jafari site followed by Zangi \> Samayeli \> Arvand \> and Bahrakan. As Figure [3](#F3){ref-type="fig"} displayed, PAHs were accumulated more significantly in the liver than in the gill tissues in all stations except at Bahrakan (p \< 0.05). The composition pattern of PAHs in the gill tissues was prevailed by two-ring structures at Arvand, Samayeli and Bahrakan, On the other hand PAHs were dominated with three-ring structures at two other sites, that is, Jafari and Zangi (Figure [2](#F2){ref-type="fig"}).
{#F3}
Biological indices
------------------
The HSI value results and the related statistical analysis are illustrated in Figure [4](#F4){ref-type="fig"}. A strong correlation (r =0.90) was found between HSI and total PAHs (Σ16PAH) in the liver samples (Figure [5](#F5){ref-type="fig"}). In this study, CFs were found in the fish collected from all five sites. As Figure [6](#F6){ref-type="fig"} showed, the CFs of the fish living in the stations were not significantly different (p \> 0.05).
{#F4}
{#F5}
{#F6}
Origin of the contaminants
--------------------------
To characterize origin of PAH contamination more appropriately, according to the processes from which they were generated, molecular indices were developed based on the thermodynamic stability of various isomeric compounds. However, because of the complexity of the parameters governing PAH distributions in the environment, simultaneous consideration of these molecular indices is necessary to be able to distinguish PAHs originating from various sources \[[@B35]-[@B38]\] Accordingly, three PAH ratios (phenanthrene/anthracene, fluoranthene/pyrene and chrysene/benzo(a)anthracene) were selected, These ratios were also suggested by other researchers \[[@B12],[@B35],[@B36]\] for the identification of pyrolytic or petrogenic origin of PAHs. The former refers to combustion emissions, and the latter addresses oil petroleum spillages, industrial effluents, and urban discharges. However, we could not use all of the ratios because levels of anthracene concentrations in some stations were below the detection limit of the instrument applied.
In the case of the sediment and the liver samples, Fluoranthene/Pyrene ratios were found to be lower than 1 except for those at Jafari site. The value of Fluoranthene/Pyrene ratios were more than 1 for the water and the gill samples except at Bahrakan site. Chrysene/benzo(a)anthracene ratios were more than 1 for the sediment and the liver samples at Arvand, Jafari and Zangi sites, but not the same result was found at two other sites. Chrysene/benzo(a)anthracene ratios for the water samples were found to be higher than 1 at Arvand, Jafari; nevertheless, it was lower than 1 at Zangi sites.
Discussion
==========
Persian Gulf is considered as one of the most pollutant marine ecosystems in the world. Almost, two-thirds of the world's proven oil reserves are located in the Persian Gulf \[[@B39]\]. This region has undergone considerable development, urbanization and industrialization; port areas have become the major sources of pollution in marine environments.
Khuzestan coast in the north western of the Persian Gulf was selected, since the problems associated with oil pollution have been perceived to be of crucial importance. Ports, refineries and other petroleum industrial wastes have had significant influence on this area, suggesting that the coastal animals are probably exposed to PAHs pollution. Mudskipper (*B. dussumieri*) was selected since they were abundant in the area, and they had also the potentiality to function as regional bioindicator.
The total amount of 16 PAH concentration did not exceed the NOAA sediment quality guideline value for the effects range low (ERL) of 4022 ng/g dry weight at five sampling sites. The composition patterns of the PAHs in the sediment samples were dominated by three-and four-ring structures. Different orders of the PAHs in sampling sites seemed to be related to different sources of PAHs. The large amount of low condensate ring structures in the sediments indicated oil pollution as the major source of PAHs pollution as it was found by Sericano et al., 2001 \[[@B40]\].
To assess the potential eco-risk of PAHs in Khuzestan coast sediments, the PAH concentrations which were already measured were compared with the effect-based sediment guideline values as suggested earlier in Long et al., 1995 \[[@B41]\] and Ningjing et al., 2010 \[[@B42]\] studies. The concentrations of B(a)A, F, AC, Ph and Ch at eight cases were higher than effect range-low (ERL) values, but they were lower than effect range-medium (ERM) values (Table [4](#T4){ref-type="table"}). These results indicated that negative eco-risk are likely to occur, although with a likelihood below 50% effects in this area. However, other individual PAH concentrations were below ERL values which indicated a low risk of biological effect caused by PAHs (Table [4](#T4){ref-type="table"}). Similar to this study, Imma Tolosa et al., 2005 \[[@B15]\] came to the same conclusions about the sediments in the southern part of the Persian Gulf (i.e., Bahrain, Qatar, UEA and Oman coast).
######
Sediment PAHs concentration compared with ERL and ERM values
**PAHs** **Guidelines** **Sites**
---------- ---------------- ----------- ------------------- ------------------- ------------------- -------- --------
B(a)A 261 1600 ERL \< ... \< ERM ERL \< ... \< ERM ERL \< ... \< ERM \< ERL \< ERL
F 19 540 ERL \< ... \< ERM \< ERL \< ERL \< ERL \< ERL
AC 44 640 ERL\> ERL \< ... \< ERM ERL \< ... \< ERM \< ERL nd
Ph 24 1500 \< ERL ERL \< ... \< ERM \< ERL \< ERL \< ERL
Ch 384 2800 \< ERL ERL \< ... \< ERM \< ERL \< ERL \< ERL
B(a)Py 430 1600 \< ERL \< ERL \< ERL nd nd
Fl 600 5100 \< ERL \< ERL \< ERL \< ERL \< ERL
Py 665 2600 \< ERL \< ERL \< ERL \< ERL \< ERL
TPAHs 4022 44792 \< ERL \< ERL \< ERL \< ERL \< ERL
nd: non detected.
The different patterns of PAHs found in the liver of mudskipper showed that there was large amount of the lower molecular weight pollution that is three-and four-ring structures. Low molecular weight PAHs found in the liver tissues might be the result of metabolic transformations of higher molecular weight PAHs occurring in fish liver \[[@B43]\]. Based on the findings of this re, It could be concluded that the PAH composition in fish liver was a sign of predominant derivation of PAH from oil pollution rather than from pyrolitic sources. A similar pattern was also found in the liver of *Epinephelus coioides* which exist in the southern part of the Persian Gulf \[[@B15]\].
Lower PAH concentrations in mudskipper liver tissues compare to the sediment might be the result of metabolic transformations of higher molecular weight PAHs occurring in fish liver \[[@B43],[@B44]\]. Fish can convert up to 99% of the PAHs to metabolites within 24 hours of uptake, changing the pattern and the concentrations of PAHs in several tissues \[[@B32],[@B45],[@B46]\]. Moreover, half-life of PAHs is generally very short in all kinds of fish \[[@B43],[@B47]\].
The Joint FAO/WHO Expert Committee on Food Additives has adopted a specification, which requires that the concentration of benzo\[a\]pyrene should not exceed a limit of 10 μg/kg \[[@B6],[@B48]-[@B50]\]. In the current study, B(a)Py could be identified at detectable levels in the liver tissues of mudskipper from three sites (i.e., Arvand, Jafari and Zangi), while their values were much below the limit.
Eight and five PAH compounds were detected in water and gill samples, respectively, where two-and three-ring PAHs were dominated. This could be due to the fact that PAHs with lower solubility are attracted by particulate matters, depositing more in bottom sediments. Furthermore, PAHs are not concussively adsorbed by the gill systems of marine organisms although more soluble compounds have more persistency in seawater. They are more bioavailable and could be easily absorbed through gill membrane or food ingestion. Similar to this finding, Bummard,1998 \[[@B35]\] found that low molecular weight (LMW) compounds, rather than high molecular weight (HMW), are more abundant in marine organisms. The results of this study were in agreement with those of Law et al., 1997 \[[@B51]\] who reported high concentration of two- and three-ring PAHs in sea water around England. The concentrations of PAHs in sea water of Marmara Sea \[[@B52]\] were also comparable with the ones found in the present study.
There were wide variations in the total parent PAH concentration in the water, the sediment and the biota samples. The PAH compositions at each site were found to be different even when the discharges were almost similar. Obviously, each composition is a characteristic of the particular waste being discharged from. There was a river near the Arvand station introducing some terrestrial organic matters and waste discharge of the refineries which could therefore introduce high PAHs to the sediment of this site. Jafari, Zangi and Samayeli sample areas are located in Khoure-Mousa bay which is very shallow and stagnant having limited water circulation. Unfortunately, without receiving any pre-treatments, the local industries, the refineries and the ports discharge their wastes to this bay. Regarding PAH concentration resulted from the pollutions, in the sample areas, Jafari showed higher total parent PAH concentration. Presence of Mahshahr Special economic and petroleum zone as a major refineries zone in North Western part of Persian Gulf which introduce high PAHs to Jafari site may be responsible for the higher contamination level observed at this station.
The Hepatosomatic index (HSI) values showed that there were significant differences among the sampling stations. However, no significant differences were observed among the sites in case of condition factors (CFs). A high correlation was found between HSI and total parent PAH concentrations in the liver. This indeed indicates that accumulation of PAHs in mudskipper can influence its liver tissue as a target organ of the fish to detoxify PAH. HSI are potentially indicative of toxicant effects. They provide useful information about energy reserves and the ability of individuals to tolerate chemical pollution challenge or other kinds of environmental stress \[[@B53]-[@B56]\].
In order to clarify potential sources or origins of PAHs in marine sediments, several PAH rations have been recommended \[[@B12],[@B57],[@B58]\]. The Fl/Py ratios of the sediment and the liver tissues pointed to petrogenic origin for Arvand, Zangi, Samayeli, and Bahrakan, Nonetheless, pyrolytic source was established at Jafari site. In case of the water and the gill samples, the values of Fl/Py ratios strongly implied a pyrolytic origin except for Bahrakan site. Although Ch/B(a)A ratio suggested a petrogenic origin for the sediment and the liver tissues at all sampling sites, it indicated a pyrolytic origin for water at Jafari and Arvand and petrogenic source at Zangi. In addition, PAH fingerprints in all tissues together with the adjacent sediments and water showed an overall predominance of LMW PAHs. The high presence of low condensate ring structures in all samples indicated oil pollution as the major source of PAHs contamination. Most importantly, different origins of PAHs were identified by the diagnostic ratios used in this study. This finding might be owing to the complexity of the parameters which determine PAHs distributions in the environment.
Conclusion
==========
This study provides important information on PAH concentrations in surface sediments, water and *B. dussumieri* from north western part of the Persian Gulf. The Persian Gulf is generally considered as an extremely polluted ecosystem with respect to oil and refineries. This is particularly true about Khuzestan coast which is located in the north west of the Persian Gulf. This study, however, showed that PAH concentrations in this region did not exceed the NOAA sediment quality guideline value for the effects range low (ERL). Our results also revealed a negative eco-risk effects occasionally occur in this area. The higher presence of low condensate ring structures reflected a predominant origin of petrogenic and some cases of pyrolitic sources in North West of Persian Gulf. These findings suggest that mudskipper *(B. dussumieri*) is not robust as a bioindicator of PAHs pollution in marine ecosystems, further research is needed. Regular monitoring of the Persian Gulf area is suggested in order to determine if any dumping activities have occurred in this particular area.
Competing interests
===================
We have received funding from national institute of oceanography that has applied for PAHs analysis relating to the content of the manuscript.
Authors' contributions
======================
AM: participated in the design of the study and performed the statistical analysis. Both authors read and approved the final manuscript.
Acknowledgments
===============
Thanks are due to Mr Shahabi, Mr Edgi pour, Mr Hashemi, Mr Ghadamgahi, Mr Sahraiyan for supporting this work.
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The third author's name is spelled incorrectly. The correct name is: Dirk Spitzer. The correct citation is: Zhong H, Sanchez C, Spitzer D, Plambeck-Suess S, Gibbs J, Hawkins WG, et al. (2013) Synergistic Effects of Concurrent Blockade of PI3K and MEK Pathways in Pancreatic Cancer Preclinical Models. PLoS ONE 8(10): e77243. doi:[10.1371/journal.pone.0077243](http://dx.doi.org/10.1371/journal.pone.0077243)
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{#sp1 .187}
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INTRODUCTION {#s1}
============
Pregnancy causes major changes in all systems of the body. Physiological changes protect the fetus, support development, and also prepare the mother for birth. These changes affect the cardiovascular, renal, pulmonary, endocrine, metabolic, hematologic and visual systems.^[@ref1]^ For example, in early pregnancy cardiac output and blood volume increase by 30-50%. Decreased fibrinolytic activity and increases in plasminogen, fibrinogen and factors I, V, VII, IX and X result in a predisposition toward coagulation. Toward the end of pregnancy, extracellular fluid increases by up to two liters. Cellular immunity decreases, but there are no changes in immunoglobins.^[@ref2]^
Ocular changes during pregnancy are categorized as physiological or pathological. Pregnancy-related pathological changes may present as new ocular developments, changes in existing ocular pathology, and ocular complications of systemic diseases.^[@ref3],[@ref4]^
This article examines pregnancy-related physiological and pathological changes in the eye and visual system; the diagnosis, monitoring and treatment of these changes; and issues to be considered.
PHYSIOLOGICAL OCULAR CHANGES {#s2}
============================
The most frequent pregnancy-related physiological change is an increase in pigmentation around the eyes. Darkening of the face during pregnancy is referred to as pregnancy mask, cloasma or melasma and develops through increased estrogen, progesterone and melanocyte-stimulating hormone.^[@ref5]^ Unilateral ptosis has been reported during pregnancy and following normal delivery. Ptosis is believed to develop as a result of fluid and hormonal effects on the levator aponeurosis, and it resolves postpartum.^[@ref6]^
A decrease in conjunctival capillaries and an increase in granularity in conjunctival venules may occur, and they also resolve in the postpartum period.^[@ref3]^
Pregnancy can also affect tear film physiology and lead to dry eye. This may be attributable to increased immune reaction in the lacrimal duct cells and the direct destruction of acinar cells by prolactin, transforming growth factor beta-1 and epidermal growth factor. Dryness can be further increased by dehydration resulting from nausea and vomiting and the use of anti-nausea medications.^[@ref7]^
During pregnancy there may be a decrease in the sensitivity of the cornea which becomes more evident toward the end of pregnancy.^[@ref8]^ The cornea thickens in response to corneal edema. Alterations in corneal curvature may occur, increasing in late pregnancy and resolving after the conclusion of the birth and breastfeeding period.^[@ref9]^ Edema-related changes in corneal thickness and refractive index may occur, which therefore affects refraction.^[@ref10]^ Changes in the cornea and lacrimal system during pregnancy may lead to contact lens intolerance.^[@ref11]^
Increase in lens curvature may cause myopic shift. Temporary accommodation loss and insufficiency during pregnancy and the postnatal breastfeeding period have been reported. Therefore, new eyeglass and contact lens prescriptions should be avoided during pregnancy, and are best postponed until several months after delivery. Refractive surgery is contraindicated during pregnancy.^[@ref3]^
Krukenberg's spindle may appear in the first two trimesters; with increasing ease of outflow in the last trimester and postpartum, the spindle shrinks and disappears.
Intraocular pressure (IOP) decreases during pregnancy. A 19.6% reduction for individuals with normal IOP and a 24.4% reduction for ocular hypertension patients have been reported. Various mechanisms have been proposed to explain pregnancy-related IOP reduction, including increased aqueous outflow, lower episcleral venous pressure due to decreased systemic vascular resistance, lower scleral rigidity as a result of increased tissue elasticity, and general acidosis during pregnancy.^[@ref12],[@ref13],[@ref14]^ Existing glaucoma typically improves during pregnancy, although there are reports of cases in which IOP was difficult to regulate.^[@ref15],[@ref16],[@ref17]^ Pregnant patients may not want to use glaucoma medication because of their teratogenic effects; in that situation, the need for medication can be lessened with laser trabeculoplasty before the patient plans to become pregnant.^[@ref16],[@ref17]^ During normal pregnancy, no physiological changes in the retinal arterioles, venules and capillary bed have been observed.
Changes in the visual field may occur. The pituitary gland grows physiologically during pregnancy; this can lead to changes such as bitemporal concentric visual field defects in cases of an abnormal anatomical relationship between the pituitary gland and the optic chiasma.^[@ref18]^
PATHOLOGICAL OCULAR CHANGES {#s3}
===========================
A. Existing Ocular Pathology Affected by Pregnancy {#s3a}
--------------------------------------------------
### Diabetic Retinopathy {#s3aa}
Diabetes is the one of the most common diseases. During pregnancy, diabetic retinopathy (DR) can progress quickly. Aggravation of the condition is dependent on several factors such as the degree of retinopathy at the beginning of pregnancy, how long a patient has been diabetic, glycemic control and comorbid hypertension.^[@ref19]^
As gestational diabetes carries a very small risk of developing retinopathy, ophthalmologic examination is not necessary. Studies have shown that 10% of patients without DR at the beginning of pregnancy developed nonproliferative changes, and a very small proportion of those patients developed proliferation. Therefore, a baseline examination in the first trimester is sufficient in the absence of visual symptoms.
In patients with nonproliferative DR (NPDR), retinopathy findings during pregnancy showed 50% progression. These findings generally regressed in the third trimester and in the postpartum period. Among patients with severe NPDR, 5-20% transition to proliferative diabetic retinopathy (PDR). Progression of up to 45% can be seen in cases of PDR. However, with pre-pregnancy laser treatment, the risk of progression is reduced by 50%. Pre-pregnancy laser therapy is recommended for patients with PDR or severe NPDR. PDR may regress in the third trimester and postpartum period; these patients require monthly examinations.^[@ref20]^
Hematologic, hormonal, metabolic, cardiovascular and immunologic factors have pathophysiological roles. Increased retinal capillary blood flow has been shown in diabetic women during pregnancy. It has been posited that this increase may cause endothelial cell damage at the capillary level. During pregnancy, the release of several angiopoietic factors also increases. Progesterone may elevate the production of vascular endothelial growth factor (VEGF) and other angiopoietic factors.^[@ref21]^
Diabetic macular edema (DME) may develop or worsen during pregnancy. DME is generally observed in pregnant patients with proteinuria or hypertension associated with diabetes.^[@ref20]^ Laser therapy is recommended for clinically significant macular edema. There are no studies in which treatment was started during pregnancy, and as there have been many cases of spontaneous correction in the postpartum period, observation is sufficient.^[@ref22]^
### Tumors {#s3ab}
#### Pituitary Adenomas {#s3aba}
Previously asymptomatic pituitary adenoma or microadenomas may grow during pregnancy and cause various ophthalmological symptoms. Headache, visual field changes (most frequently bitemporal defect), lowered visual acuity and rarely diplopia may occur. Following pregnancy, adenomas shrink and leave no visual sequelae. In patients with known adenoma, monthly ophthalmologic examination and visual field monitoring is necessary to monitor for tumor growth.^[@ref23]^
### Meningioma {#s3ac}
Pre-exising meningiomas may vascularize and grow in the second half of pregnancy. It is believed that estrogen and progesterone may be mediators.^[@ref24]^
### Uveal Melanoma {#s3ad}
Higher ocular melanoma incidence and reactivation rates have been reported in pregnant women compared with nonpregnant women of the same age.^[@ref25]^ In a later study, a hormonal correlation with uveal melanoma could not be established.^[@ref26]^
### Graves'Disease {#s3ae}
Graves' disease is the most common cause of hyperthyroidism during pregnancy. Exacerbation may occur in the first trimester. The condition may subside during the rest of the pregnancy, but become exacerbated again in the postpartum period. Patients require multi-disciplinary monitoring. The preferred medical treatment is propylthiouracil.^[@ref3]^
### Toxoplasmosis {#s3af}
Toxoplasmosis is a common infection worldwide, including in Turkey. A primary infection during pregnancy (through transplacental transmission from mother to baby) can result in congenital infection. The fetus is severely effected when the infection occurs in the first trimester; however, transmission to the fetus is more frequently seen in the third trimester, when maternal and fetal circulation are in greatest contact. Latent infections in the mother may become active. With the typical retinochoroiditis presentation, toxoplasmosis is easily diagnosed; however, atypic presentations such as neuroretinitis, papillitis, scleritis and acute retinal necrosis have also been reported.^[@ref27]^ Oral treatment with the macrolide antibiotic spiramycin is recommended. In the final stage of pregnancy, use of the sulfamethoxazole/trimethoprim combination should be avoided due to the risk of neonatal kernicterus. As current treatment options, use of intravitreal klindamycin (1.0 mg/0.1 ml) and dexamethasone (400 µg/0.1 ml) injections to avoid systemic toxicity has been reported.^[@ref28]^
### Multiple Sclerosis {#s3ag}
As with other inflammatory conditions, the rate of multiple sclerosis attacks decreases during pregnancy, although it may increase in the first three months postpartum. Optic neuritis attacks may occur as a result of immune-mediated changes during this period. Multiple sclerosis presenting for the first time during pregnancy has also been reported.^[@ref29]^
### Pituitary Apoplexy-Sheehan's Syndrome {#s3ah}
Pituitary apoplexy is pituitary gland enlargement due to sudden infarct or hemorrhage in pituitary adenomas. Pregnancy is one of the risk factors for this condition, and occurs as a result of serious postpartum hemorrhage. The condition is a vision-threatening complication and is characterized by sudden headache, vision loss (52%) and visual field loss (64%) and/or ophthalmoplegia. Typical vision field loss is bitemporal superior quadrant defect. Cavernous sinus compression most frequently affects the third cranial nerve, followed by the fourth and less often the sixth. Ptosis, diplopia, anisocoria (midriasis) and lateral-inferior deviation of the globe is seen in cases of third cranial nerve involvement. Horner's syndrome may also arise as a result of damage to sympathetic fibers.^[@ref30],[@ref31]^
### Idiopathic Intracranial Hypertension {#s3ai}
During pregnancy, idiopathic intracranial hypertension is known to progress with weight gain. The condition occurs with obesity and is characterized by an increase in intracranial pressure of unknown cause. Headache is the most common symptom, and may be accompanied by nausea and vomitting. Ocular findings include blurred vision, scotomas, photopsia, diplopia and retrobulbar pain. Papillary edema is apparent during fundus examination. Keeping weight gain under control is emphasized in treatment.^[@ref32],[@ref33]^
### Uveitis {#s3aj}
During pregnancy, it has been reported that increased endogenic steroids along with multifactorial and complex mechanisms cause both ocular and systemic signs of noninfectious uveitis to subside and attack frequency to decrease.34 Improvements in both the ocular and systemic symptoms of sarcoidosis, spondiloartropathy and rheumatoid arthritis have been observed. However, six months after delivery there may be recurrences.
These pregnancy-related improvements may serve as an advantage for pregnant women with chronic sight-threatening uveitis. It may be possible to reduce or suspend the use of immunosuppressive drugs used to treat uveitis for which data regarding safety is lacking or for which there are known teratogenic effects. Potential attacks can be treated with local corticosteroids.^[@ref34]^
Vogt-Koyanagi-Harada syndrome, which is characterized by bilateral granulomatous uveitis, exudative retinal detachment, meningeal symptoms, hearing loss and pigment loss has been reported to regress and in some cases complete resolve during pregnancy and the postpartum period.^[@ref35]^
### Posterior Scleritis {#s3ak}
Posterior scleritis worsens and recurrence increases during pregnancy.^[@ref36]^ Although the standard treatment is oral steroids, posterior sub-Tenon's triamcinolone injection is recommended for pregnant patients.^[@ref12]^
### Choroidal Neovascularization {#s3al}
There have been case reports of choroidal neovascularization (CNV) during pregnancy. These cases were diagnosed with myopia, punctate inner choroidopathy (PIC), presumed ocular histoplasmosis syndrome (POHS) and idiopathic CNV.^[@ref37],[@ref38],[@ref39]^
B. Ocular Pathology Emerging During Pregnancy {#s3b}
---------------------------------------------
### Preeclampsia and Eclampsia {#s3ba}
In a normotensive pregnant woman, the triad of systemic blood pressure of over 140/90 mmHg, edema and proteinuria after week 20 of pregnancy is defined as preeclampsia. With the addition of contractions without any other cause, the condition is called eclampsia. The incidence of preeclampsia is approximately 5%, and ocular sequelae have been reported in one in three of these patients.36 Though patients frequently complain of blurred vision, they may also experience photopsia, scotoma and diplopia.
Changes in preeclampsia-related retinopathy resemble those in hypertensive retinopathy. The most common finding is retinal arteriolar narrowing, which is usually focal but may also be generalized. Other changes include retinal hemorrhage, edema, exudate, nerve fiber layer infarcts and intravitreal hemorrhage secondary to neovascularization. There is a positive correlation between the severity of preeclampsia and the degree of retinopathy. Most of these findings return to normal following the resolution of preeclampsia. Cases of preeclampsia-related retinopathy with underlying diabetes, chronic hypertension and kidney disease may be more severe.
Optic nerve findings in preeclampsia are papillary edema, ischemic optic neuropathy and optic atrophy. Exudative retinal detachment is seen in 1% of preeclamptic patients and 10% of eclamptic patients.[@ref36] In a Turkish study by one of the authors of this manuscript (RBK) including 47 patients with preeclampsia, 3 patients exhibited exudative retinal detachment; two of the cases resolved spontaneously following birth, while one required postpartum systemic steroid treatment due to very low visual acuity.^[@ref40]^
### Central Serous Chorioretinopathy {#s3bb}
Pregnancy is believed to be a risk factor for the development of central serous chorioretinopathy (CSCR). It is most frequently seen in the third trimester, though it may also appear in the first and second trimesters. In one study, 90% of pregnant CSCR patients had fibrinous subretinal exudate, while this rate was 20% in nonpregnant CSCR patients.41 Diagnosis is easily made with posterior segment optical coherence tomography. Spontaneous regression is observed at the end of pregnancy or after birth; however, there may be a tendency for recurrence in the same eye in subsequent pregnancies.^[@ref41],[@ref42]^
### Vascular Occlusive Diseases {#s3bc}
This group of conditions includes retinal artery occlusions, retinal vein occlusions, disseminated intravascular coagulopathy (DIC), thrombotic thrombocytopenic purpura (TTP), antiphospholipid antibody syndrome (APS), amniotic fluid embolism, and cerebral venous thrombosis. As indicated previously, hypercoagulability occurs in pregnancy. Pregnancy-related central and branch retinal artery occlusion has rarely been reported. Retinal vein occlusions are rarer than arterial occlusions.
DIC may develop in pregnancies with complications such as abruptio placenta, preeclampsia/eclampsia, complicated birth, amniotic fluid embolism, intrauterine infection and intrauterine death. DIC is a serious condition characterized by diffuse small vessel thrombosis and subsequent hemorrhage and tissue necrosis.^[@ref43],[@ref44]^ In the eye, the choroid layer is most affected; thrombosis in the choriocapillaris disrupting the retinal pigment epithelium may cause serous retinal detachment. Ocular symptoms improve with DIC treatment, though mild pigmentary changes may persist.^[@ref45]^
HELLP syndrome is a condition characterized by hemolysis, elevated liver enzymes and lowered thrombocyte count; it is typically seen in preeclamptic patients and generally appears with DIC. Serous retinal detachment, vitreous hemorrhage, central retinal vein occlusion and Purtscher-like retinopathy have been reported in these patients.^[@ref46],[@ref47],[@ref48]^
TTP is a rare disease, and ocular changes are observed in 10% of TTP patients. Fundus changes occur in the form of serous retinal detachment, retinal hemorrhage, exudates and narrowing of the arterioles. Involvement of the vessels supplying the optic nerve may lead to optic atrophy. Anisocoria, subconjunctival hemorrhage, scintillating scotoma, extraocular muscle paresis and homonymous hemianopia may occur.[@ref49]
APS is a thrombophilic condition; patients are predisposed to arterial and venous thrombosis and antiphospholipid antibody-related pregnancy morbidity is observed. Anterior segment findings accompanying this syndrome may include conjunctival telangiectasia and microaneurysms, episcleritis, limbal or filamentous keratitis, and iritis. Posterior segment findings may include vitreitis, retinal detachment, posterior scleritis, central retinal vein occlusion, retinal vein branch occlusion, cilioretinal artery occlusion, increased venous tortuosity, retinal hemorrhage, and soft exudates. Furthermore, vascular thrombosis may develop in the choroid, optic nerve, visual pathways and ocular motor nerves.^[@ref50],[@ref51]^
Amniotic fluid embolism, though rare, is a serious condition with fatal complications; 85% of cases end in mortality. Symptoms include chills, cyanosis, convulsions and shock. The optic nerve, visual pathways and occipital cortex may be affected and central retinal artery occlusion may develop.^[@ref52]^
Risk of venous and sinus thrombosis increases during pregnancy due to hormonal changes. Between 5-20% of cerebral venous thrombosis cases are women who are pregnant or in the postpartum period and papilledema is found in 35% of these cases.^[@ref53]^
### Medication Use During Pregnancy {#s3bd}
Regardless of attempts to avoid or postpone the use of medication during pregnancy, in some cases it cannot be avoided. It is therefore essential that physicians are well informed of the potential teratogenic effects of the medications being prescribed. Based on available clinical studies and experience, the U.S. Food and Drug Administration (FDA) has classified drugs into five categories (A, B, C, D and X) according to the severity of potential teratogenic effects.^[@ref54]^
### Food and Drug Administration Categories: {#s3be}
#### - Category A: {#s3bea}
Adequate and well-controlled studies did not show a risk to the fetus in the first trimester of pregnancy, and there are no data suggesting a risk in the second and third trimesters. Category A is the safest category.
#### - Category B: {#s3beb}
Animal studies did not show a risk to the fetus, but there are not adequate and well-controlled studies in pregnant women. Alternatively, adverse effects appeared in animal studies, but adequate and well-controlled studies with pregnant women did not reveal a risk to the fetus during any trimester. Category B drugs can be used if necessary.
#### - Category C: {#s3bec}
Adverse effects were observed in animal studies, and adequate and well-controlled studies in humans have not been conducted. However, the potential benefits may justify the use of the drug in pregnant women despite the potential risks.
#### - Category D: {#s3bed}
There is evidence indicating risk to the human fetus based on adverse reaction data from investigational or marketing experience, but the potential benefits may justify the use of the drug during pregnancy despite the potential risks. The drug may be used with caution if the mother and fetus face greater risks from not using it.
#### - Category X: {#s3bee}
Fetal abnormalities were seen in animal or human studies and/or there is evidence indicating risk to the human fetus based on adverse reaction data from investigational or marketing experience. The drug's risks outweigh the potential benefits of its use during pregnancy. Use is not recommended.
### Use of Ophthalmic Drugs During Pregnancy {#s3bf}
As a general principle, to limit systemic absorbtion and avoid toxicity, the lowest dose possible should be given and after administering eye drops the patient should perform punctal occlusion by applying pressure to the nasolacrimal canal and wipe away excess to prevent systemic absorption. There is a lack of in-depth research regarding the effects of ophthalmologic drugs on pregnancy and lactation.^[@ref55]^
### Drugs Used in Diagnostic Tests {#s3bg}
Fluorescein crosses the placenta and is a category C drug. Indocyanine green does not cross the placenta, but is also in category C.
Tropicamide, cyclopentolate and epinephrine, used for dilatation and cycloplegic purposes, are category C drugs. Although there are no reports of teratogenic effects from topical use, minor fetal malformations have been reported from systemic use of phenylephrine, atropine and homatropine, and there is relative contraindication for their ophthalmologic use.^[@ref56]^ In the first three months of pregnancy, even their application for the purpose of examination is not recommended.
### Antibiotics {#s3bh}
Chloramphenicol, neomycin and tetracycline must not used during pregnancy. Erythromycin, ophthalmic tobramycin, ophthalmic gentamicin, polymyxin B, acyclovir and quinolones are reported to be safe.^[@ref56],[@ref57]^ Systemic tobramycin is a category D drug, topical tobramycin is category B, netilmicin and tetracycline are category D, and all other antibiotics used locally are category C. Among antiviral drugs, trifluridine is category C and acyclovir is category B.
### Anti-inflammatory and Anti-allergy Drugs {#s3bi}
In general, the potential risks and benefits must be considered when prescribing these drugs. Systemic corticosteroids are contraindicated due to their teratogenic effects and their role in CSCR; topical steroids have no known teratogenic effects. All topical steroid and nonsteroid anti-inflammatory drugs are in category C. Topical cyclosporine is in category C. Among topical antihistamines and anti-allergy drugs, ketotifen, olopatadine and epinastine are category C drugs; sodium cromoglycate and emedastine are in category B.^[@ref54]^
### Glaucoma Medications {#s3bj}
Beta-blockers, prostaglandin analogues, carbonic anhydrase inhibitors and miotics are category C drugs. Beta-blockers should be used with caution in the first trimester and be discontinued a few days before birth to prevent neonatal beta blockade. As beta-blockers are concentrated in breast milk, they should not be used while lactating.^[@ref57]^
Systemic and topical carbonic anhydrase inhibitors have teratogenic and hepatorenal effects and are therefore contraindicated both in pregnancy and during lactation.^[@ref58]^ The use of miotic drugs during pregnancy appears to be safe. Prostaglandins may cause early delivery or miscarriage. In a study including 11 pregnant women using latanoprost, 1 patient miscarried, 1 patient did not continue follow-up, and 9 patients completed their pregnancies without complications.^[@ref59]^ Brimonidine, an alpha-2 agonist, is in category B. However, maternal use during the lactation period carries the risk of apnea and bradycardia in the newborn^[@ref60]^
### Anti-Vascular Endothelial Growth Factor Agents {#s3bk}
Anti-VEGF agents are used to treat PIC, POHS, miopic CNV, diabetic and uveitic macular edema, and retina and iris neovascularization in PDR in patients of child-bearing age. Although systemic absorption is very low and bevacizumab cannot cross the placenta due to its high molecular weight (149 kD), it should be kept in mind that it can theoretically affect placental vasculature. Among anti-VEGF agents, bevacizumab and ranibizumab are category C and pegaptanib is category B.
There are case reports in the literature regarding the use of anti-VEGF intravitreal injection during pregnancy.37,39 One case underwent photodynamic therapy (PDT) in the first two weeks of pregnancy and received 1.25 mg intravitreal bevacizumab in the third month; the pregnancy was completed with no problems.^[@ref37]^ Another case received intravitreal bevacizumab in the third trimester and encountered no problems. A patient with idiopathic choroidal neovascularization received ranibizumab in the third trimester and also experienced no problems.39 In one study, four pregnant patients received an average of 2.5 intravitreal injections (range, 1-6) of bevacizumab with no adverse effects, while in another report, two patients with unknown pregnancies miscarried following bevacizumab treatment in the first trimester.^[@ref38]^
### Verteporfin {#s3bl}
Verteporfin is a category C drug. Anophthalmia and microphthalmia have been reported in rats exposed during organogenesis to verteporfin at 40 times the dose used in humans.^[@ref37]^ However, two patients with unknown pregnancies underwent PDT at 3 and 12 weeks post-conception and later gave birth to healthy babies.^[@ref61],[@ref62]^
### Anesthetic Agents {#s3bm}
For pregnant patients for whom ocular surgery is unavoidable, the safest anesthetics may be lidocaine and citanest, which are category B drugs.54 They may be used as sub-Tenon's and peribulbar anesthesia. Bupivacaine, a category C drug, is not recommended due to the risk of bradycardia in the fetus. Proparacaine hydrochloride, which is used as a topical anesthetic, is also in category C.^[@ref54]^
CONCLUSION {#s4}
==========
Visual complaints are a common occurrence in pregnant women. It is extremely important that ophthalmologists are aware of the various physiological and pathological conditions that may arise or be altered during pregnancy, and are knowledgeable about their differential diagnosis, treatment and monitoring. Especially rare and serious complications may include visual symptoms and pregnant patients may initially be seen by an ophthalmologist. Responding carefully, rapidly and strategically to patients with these complaints can prevent possible risks to mother and baby.
**Concept:** Nursal Melda Yenerel, Raciha Beril Küçümen, **Design:** Nursal Melda Yenerel, **Data Collection or Processing:** Nursal Melda Yenerel, **Analysis or Interpretation:** Nursal Melda Yenerel, Raciha Beril Küçümen, **Literature Search:** Nursal Melda Yenerel, **Writing:** Nursal Melda Yenerel, Raciha Beril Küçümen, **Peer-review:** Externally peer-reviewed.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Molecular phylogenetic studies of maples, *Acer* L. (Sapindaceae), have relied mainly on the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) ([@b10-ebo-02-07]; [@b1-ebo-02-07]; [@b60-ebo-02-07]; [@b53-ebo-02-07]; [@b64-ebo-02-07]). To the extent that they overlapped in species sampling, these studies have yielded contradictory relationships, although usually without statistical support. The ITS region used in these studies is part of the rDNA cistron coding for the 35S pre-RNA, which consists of 5′ ETS (external transcribed spacer), 18S, ITS1, 5.8S, ITS2, 25S, and 3′ ETS, with ITS1 having evolved from an intergenic spacer and ITS2 from an expansion segment in the rDNA large subunit ([@b25-ebo-02-07]; [@b27-ebo-02-07]). Plant genomes have thousands of copies ([@b24-ebo-02-07]) located in one or several loci, distributed on one or several chromosomes, and hybrid or allopolyploid individuals can contain different parental rDNA repeats ([@b71-ebo-02-07]; [@b69-ebo-02-07]). Functional and pseudogenic copies can recombine, further increasing sequence variation within individuals. For all these reasons, signal contained in ITS sequences cannot always be mapped onto bifurcating trees, although concerted evolution constantly homogenizes new variation among the numerous rDNA repeats of an individual ([@b58-ebo-02-07]; [@b61-ebo-02-07]; [@b3-ebo-02-07]; [@b73-ebo-02-07]; [@b9-ebo-02-07]; [@b68-ebo-02-07]; [@b45-ebo-02-07]; [@b2-ebo-02-07]; [@b55-ebo-02-07]; [@b21-ebo-02-07]; [@b50-ebo-02-07]; [@b56-ebo-02-07]; [@b75-ebo-02-07]). Direct sequencing of this region has therefore long been the accepted practice, and indeed the ITS sequences used in studies of *Acer* ([@b10-ebo-02-07]; [@b1-ebo-02-07]; [@b60-ebo-02-07]; [@b64-ebo-02-07]) were obtained from direct sequencing.
An initial survey that involved 160 cloned ITS sequences from 84 individuals of *Acer*, representing 45 species and 20 subspecies, revealed non-identical copies in most individuals ([@b20-ebo-02-07]). This required sampling multiple accessions of as many species as possible. *Acer* includes many polyploids, and species are known to hybridize, although the frequency and extent of hybridization in nature appear to be limited ([@b67-ebo-02-07]; [@b53-ebo-02-07]). The genus contains at least 124 species ([@b67-ebo-02-07]) of which almost half occur in China. The closest relative of *Acer, Dipteronia*, with two species, also is endemic to China. As sampling increased to currently more than 600 clones, including clones from all known polyploid species, it became clear that species were characterized not by single nucleotide (nt) substitutions, but by sequence motifs. Sequence motifs or elements are series of linked substitutions and/or insertions and deletions (indels) within a given region ([@b25-ebo-02-07]; [@b25-ebo-02-07]; [@b20-ebo-02-07]; [@b70-ebo-02-07]; [@b71-ebo-02-07]; [@b15-ebo-02-07]). ITS motifs in length-polymorphic regions (LPRs, comprising up to 50 nt) have been found to be phylogenetically informative ([@b20-ebo-02-07]). For the present study, we greatly increased sampling density to test whether ITS motifs might clarify contradictory relationships found in the previous studies of *Acer* and whether they can be traced within Sapindaceae or other Sapindales (to which *Acer* belongs; [@b6-ebo-02-07]; [@b65-ebo-02-07]; [@b22-ebo-02-07]).
Several approaches are available to analyze contradictory signal within and between phylogenetic data sets. We here employ phylogenetic networks as well as comparison of bootstrap values on branches obtained with different inference methods. Networks graphically represent signal from mutations in sequences, sets of trees, or inferred genetic distances. Competing signal may arise from the stochastic substitution process, poorly fitting evolutionary models, or the heuristic nature of many tree search algorithms. Or it may result from hybridization, lineage sorting, or lateral gene transfer. Whatever the causes of contradictory signals, a rapid method to visualize their extent is a splits-based graph. Splits graphs comprise median networks from sequences, consensus networks from trees, and split decomposition and neighbor-nets from genetic distances ([@b4-ebo-02-07]; [@b32-ebo-02-07]; [@b30-ebo-02-07]; [@b7-ebo-02-07]; [@b29-ebo-02-07]; [@b8-ebo-02-07]; [@b28-ebo-02-07]; [@b36-ebo-02-07]; [@b44-ebo-02-07]; [@b74-ebo-02-07]; [@b33-ebo-02-07]). In splits graphs, each set of parallel edges represents a split or bipartition of the data into non-overlapping groups, with edge lengths being proportional to the number of mutations supporting a particular split. Tree-like parts of the diagrams represent un-contradicted patterns, whereas box-like structures represent conflict. Compared to a visual screening of alternative bifurcating trees, splits-based graphs allow a more thorough and qualitatively different assessment of alternative relationships supported by the data, especially if different types of splits graphs are employed.
Running full maximum likelihood (ML) analyses (e.g. computing 200 ML trees and 1,000 ML bootstrap replicates) under a parameter-rich model on matrices of some 600 sequences and 460 characters required a high-performance computational approach, such as implemented in RAxML ([@b59-ebo-02-07]). RAxML-VI-HPC allows for ML-based computation of phylogenetic trees for datasets of up to 25,000 taxa with 1500 base pairs or 2,100 taxa with up to 50,000 base pairs ([@b59-ebo-02-07]). We also used RAxML-based bootstrap analyses to explore the effects of using the several hundred original ITS sequences as opposed to using smaller matrices of consensus sequences that used ambiguity coding to account for within-species and subspecies variation.
Our multi-tiered approach to extracting phylogenetic signal from the multicopy ITS region thus combined (i) maximum likelihood tree inference from matrices of different composition, (ii) networks for exploring contradictory signal, and (iii) motif analyses at different hierarchical levels. This study also presents the most thorough attempt so far to identify major clades in *Acer*, the largest tree genus of the northern hemisphere besides *Quercus*.
Material and Methods
====================
Taxon sampling
--------------
A list of the 83 species and subspecies of *Acer* and *Dipteronia* included in the analysis with their sources and gene bank accession numbers given in Appendix 1. Species were chosen to represent all sections and series of [@b67-ebo-02-07]; Table 1). Where possible, species identifications were confirmed by comparison with herbarium specimens. Taxonomic resources (e.g. [@b18-ebo-02-07]; ITIS 2006; [@b66-ebo-02-07]) disagree markedly in recognized species and subspecies; we decided to follow in principal the most recent monograph of *Acer* ([@b67-ebo-02-07]). A total of 606 ITS sequences (579 clones plus 22 clones with missing data for either ITS1 or ITS2 and 5 directly sequenced PCR products obtained by S.-F. Huang, Department of Biology, University of Missouri-St Louis, personal communication, 2003) was generated and uploaded to GenBank and the EMBL database.
Outgroup selection relied on [@b22-ebo-02-07], who demonstrated that *Acer* and *Dipteronia* together are sister to a clade comprising *Aesculus* (13--19 species in China, Europe, and North America), *Billia* (two species in the neotropics), and *Handeliodendron* (one species in China). All belong in Sapindaceae and hence Sapindales ([@b6-ebo-02-07]; [@b65-ebo-02-07]; [@b22-ebo-02-07]). ITS sequences of *Aesculus, Handeliodendron*, and more distant Sapindaceae cannot unambiguously be aligned with *Acer* and *Dipteronia* to a sufficient degree. Hence, only *Dipteronia* could be included for alignment-based analyses. For motif analysis, we downloaded all ITS sequences of other families in the Sapindales from GenBank (December 2005): Anacardiaceae, 62 accessions, Burseraceae, 119 accessions, Rutaceae, 16 accessions, Meliaceae, 72 accessions, and Sapindaceae, 15 accessions. Sequences of *Handeliodendron* were generated for this study.
DNA isolation, amplification, sequencing, and alignment
-------------------------------------------------------
Isolation of total DNA followed a modified cethyltrimethylammonium bromide (CTAB) protocol ([@b19-ebo-02-07]). DNA amplification by the polymerase chain reaction (PCR) was carried out with Vent®-polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and the plant specific primers ITS-A and ITS-D ([@b15-ebo-02-07]; modified after [@b35-ebo-02-07]), which amplify the 3′ end of 18S rDNA, ITS1, the 5.8S rDNA, ITS2, and the 5′ end of 25S rDNA. Amplified fragments were purified with QIAquick gel extraction kits (QIAGEN, Hilden, Germany). Ligation and transformation were performed using a pUC18 vector *E. coli* strain DH5α system. Up to 15 positive clones per sample were cultivated overnight, mixed 1:1 with glycerin, and stored at −70°C for plasmid isolation and data documentation. Cultures are available upon request. The DNA of up to ten clones per sample was isolated with High Pure Plasmid Isolation Kit® (Roche) and prepared for sequencing. Sequencing was done on an ABI Prism® automated sequencer with the universal primer M13 forward and reverse primers, and sequences were then edited and subsequently aligned with CHROMAS® V.1.45 (Technelysium Pty, Tewantin, Australia) and SeqMan II® plus MegAlign® (DNAStar, Madison, U.S.A.). Alignments were optimized manually, and for regions with length polymorphisms, we followed a protocol developed by [@b20-ebo-02-07], p 11), taking into consideration results from independently computed alignments for each subspecies, species, or well supported clades to optimize the placements of gaps.
Phylogenetic analyses
---------------------
Parsimony analyses relied on PAUP version 4.0b.10 ([@b62-ebo-02-07]), and Bayesian analyses on MrBayes version 3.1 ([@b31-ebo-02-07]; [@b57-ebo-02-07]). Maximum likelihood analyses used RAxML-VI-HPC ([@b59-ebo-02-07]; software available at ic[www.epfl.ch/\~stamatak](www.epfl.ch/~stamatak)); with computations performed on the computer cluster of the Cyber-Infrastructure for Phylogenetic Research project (CIPRES, [www.phylo.org](www.phylo.org)) at the San Diego Supercomputer Center. Distance analyses were performed with neighbor-joining (NJ) and neighbor-net (NN) algorithms implemented in PAUP and SplitsTree version 4.3 ([@b33-ebo-02-07]; available at [www.splitstree.org](www.splitstree.org)).
One matrix consisted of 584 original ITS sequences. Reduced matrices were built as follows: (1) A matrix of 101 original sequences was used for computation of some NN splits graphs. (2) A matrix of 105 sequences included one consensus sequence per species or subspecies as long as these taxa exhibited minor (≤1 nt long) nucleotide polymorphism among ITS clones. Taxa that exhibited length polymorphisms or distinct ITS variants were represented by several consensus sequences. Arboretum samples that deviated from the wild type of the respective species were represented by additional consensus sequences. (3) A matrix of 40 sequences included one to three semi-strict (as defined below) consensus sequences for each of the species groups that had diagnostic ITS motifs high and (\>75%) ML bootstrap support. To create semi-strict consensus sequences, intraspecific variability was ignored as long as it was restricted to a single species/subspecies. For consensus sequences that represented more than one species, mutational divergence was retained by using ambiguity coding.
Parsimony analyses were performed using the parsimony ratchet analysis for PAUP (PRAP) command block ([@b46-ebo-02-07]), with ten random taxon-addition replicates. Bayesian analyses used one cold and three incrementally heated Monte Carlo Markov chains (MCMC) in two simultaneous runs. Chains were run for 1 million cycles, with trees sampled every 100th generation, each using a random tree as a starting point and a temperature parameter value of 0.2 (the default in MrBayes). The first 298 trees of each run were discarded as burn-in; converging log-likelihoods, potential scale reduction factors for each parameter, and inspection of tabulated model parameters suggested that stationarity had been reached thereafter. The remaining trees were used to compute posterior probabilities of nodes.
Models for minimum evolution (distance) and Bayesian analyses were selected from the 24 models implemented in MrModeltest 2.1 ([@b48-ebo-02-07]) employing the Akaike information criterion (AIC). Best model decisions from MrModeltest were compared to best models found for the same data via simultaneous evaluation of the 56 models implemented in DT-ModSel ([@b42-ebo-02-07]). The latter uses a Bayesian information criterion based on decision theory to gauge the different models' performance in terms of branch-length error and degree of over-fitting. For the 105-taxon data set (84 species and subspecies), the general time-reversible (GTR) model plus a gamma shape parameter (Γ) and a proportion of invariable sites (I) received the best AIC score in MrModeltest, while the less parameter-rich transition model (TiM) plus Γ scored best in DT-ModSel. TiM + Γ cannot be specified in MrBayes, so we opted for the next parameter-rich model (GTR + Γ). Parameter estimation in MrBayes ran for the duration of specified MCMC runs. For the RAxML analyses, we also used the GTR + Γ model, with model parameters estimated over the duration of specified runs. Distance analyses (NJ) were performed under the GTR + Γ model and the HKY + Γ + I model ([@b23-ebo-02-07]), using the parameter values found with MrModeltest.
Clade support was assessed with posterior probabilities (PP) computed with MrBayes and non-parametric bootstrapping ([@b17-ebo-02-07]) as implemented in PAUP and RAxML. Bootstrap support (BS) under parsimony (BS~P~) and neighbor joining (BS~NJ~) is based on 10,000 replicates. Parsimony bootstrap replicates used a simple taxon addition tree as the starting point, tree-bisection-reconnection swapping, and one tree held in memory; more computation-intensive heuristic approaches have been shown not to increase the reliability of bootstrapping ([@b47-ebo-02-07]). Bootstrap support under ML (BS~ML~) is based on 1,000 replicates computed with the parallel message-passing-interface-based version of RAxML-VI-HPC on a LINUX cluster.
Alternative splits in bootstrap replicates and in the Bayesian partitions table were visualized as split networks with SplitsTree. In addition, we computed 200 ML trees on the original 584-taxon alignment in parallel, using 200 distinct randomized MP starting trees produced by RAxML. The advantage of using randomized MP starting trees is that the ML-based search starts from different points in the vast search space and it is thus less likely to get stuck in local ML maxima.
Neighbor-net (NN) splits graph analysis ([@b7-ebo-02-07]), implemented in SplitsTree, was used to infer the distribution of incompatible splits. Neighbor-net starts with genetic distances. In addition, bipartition networks were obtained by coding the partitions tables from RAxML, PAUP, and MrBayes as split matrices for SplitsTree. The frequency of each split becomes an edge length in the bipartition network, and contradictory splits can thereby be visualized. Splits graphs used either uncorrected p-distances or distances computed under HKY + Γ + I, with the model parameters found by MrModeltest (above).
Mutational patterns within length polymorphic regions (LPR1 and LPR2, see below) were treated as logically dependent (linked) characters and as one sequence motif. The ITS1 and ITS2 regions of *Acer* and *Dipteronia* were also screened for sequence elements conserved among major clades (conserved motifs). For motif analysis, variants were placed next to each other in a network that minimized the number of mutational changes between adjacent motifs. Motif variants were then mapped onto a constrained topology to visualize mutational trends and differentiation levels. The obtained motif differentiation pattern for *Acer* and *Dipteronia* was compared to homologous DNA stretches in other Sapindales.
Results
=======
Phylogenetic relationships in *Acer*
------------------------------------
We generated 606 ITS sequences from 231 leaf samples, representing 81 species and subspecies of *Acer* and both species of *Dipteronia*. A table summarizing sequence length, GC content, and sequence divergence for the amplified regions ITS1, 5.8S rDNA, and ITS2 is available from the first author. Data matrices have been uploaded to TreeBase (accessions SN2898--11606 to 11608; fully annotated NEXUS files for SplitsTree and PAUP/MrBayes can be obtained upon request). The 18S, 5.8S, and 25S rDNA gene portions were excluded from all phylogenetic analyses, as were ten ITS clones judged to be pseudogenes based on increased AT content and deletions within their 5.8S sequences (not shown). We also excluded two highly divergent and length polymorphic regions (LPRs), one of 49 bp in the ITS1 region and one of 53 bp in the ITS2 region (subsequently referred to as LPR1 and LPR2); these polymorphic regions were only included in some NN analyses (as specified below). In total, 584 ITS sequences (mostly cloned) were included as original taxonomic units.
All methods recovered three supraspecific groups: the aceroid, the palmatoid, and the platanoid cluster ([Figs. 1](#f1-ebo-02-07){ref-type="fig"}--[3](#f3-ebo-02-07){ref-type="fig"}), and many of the traditional sections and series of *Acer* (Table 1) were also recovered under ML ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}; TreeBase accession SN2898-11610), in the neighbor-net ([Fig. 2](#f2-ebo-02-07){ref-type="fig"}), and in the bipartition network ([Fig. 3](#f3-ebo-02-07){ref-type="fig"}). Traditional groups that have low statistical support (with all matrices and methods) were the *Acer* core clade and the *Caudata* clade ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}). Nevertheless, both were recovered in the best-known ML tree obtained from the 584 original sequences included in the largest matrix.
Molecularly well-supported groupings ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}) not previously recognized based on morphology were (i) the placement of *A. wardii* inside the *Palmata* clade, (ii) that of sections *Pentaphylla* and *Trifoliata*, and (iii) that of *A. campbellii* subsp. *campbellii* (sect *Palmata* ser *Sinensia* sensu [@b12-ebo-02-07]) with *A. tschonoskii* (sect. *Macrantha*; the clade is labeled *Macrantha 2* in all our figures).
The geographic distribution of the major clades seen in the ITS data is shown in [Fig. 1](#f1-ebo-02-07){ref-type="fig"}. Six clades of two or more species/subspecies are endemic in East Asia, four occur in East Asia and North America, two occur in Western Eurasia and East Asia, and one is widespread in the northern hemisphere.
The two species of *Dipteronia*, the sister genus to *Acer*, did not group together in the ML tree ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}) or any of the bipartition networks ([Fig. 3](#f3-ebo-02-07){ref-type="fig"} and Appendix 2). To investigate this unexpected result, we computed NN splits graphs from the 584-sequence matrix and from a 101-sequence matrix (of original sequences, not consensus sequences) and excluded/included the six clones representing *D. dyeriana* and *D. sinensis* as well as the LPR1 and LPR2 regions. In none of these experimental analyses did *D. dyeriana* and *D. sinensis* group together, although the previously seen aceroid, palmatoid and platanoid clusters were nearly always recovered (Table 2; [Fig. 2](#f2-ebo-02-07){ref-type="fig"}, additional graphs not shown). The main effect of excluding the LPR1 and LPR2 regions was that species of the *Macrantha 2* clade separated from the platanoid cluster (Table 2). With genetically distant taxa excluded, the LPR1 and LPR2 could be aligned unambiguously (as explained above, these polymorphic regions were excluded from other analyses). Resultant NN splits graphs ([Fig. 2](#f2-ebo-02-07){ref-type="fig"}) agreed with those from the 584 sequences: The inclusion of *Dipteronia* species mainly affected relationships among basal *Acer* lineages (compare length of central edges and position of non-clustered *Acer* species in [Fig. 2A and Fig. 2B](#f2-ebo-02-07){ref-type="fig"}).
Method-dependent topological differences (evaluated for the 105-consensus-sequence matrix) were restricted to nodes that received moderate to low support (PP \<0.95, BS \<75%), and there were no model-dependent topological differences between GTR + Γ and HKY + Γ + I NJ trees (not shown). Topological differences among the 683 most parsimonious trees obtained from the 105-consensus-sequence matrix were restricted to nodes near the leaves, as was the case for the 200 ML trees inferred from the 584-original-sequence matrix without bootstrapping. Nodes with posterior probabilities of \>0.95 (and BS \>75%) were consistently recovered in all trees, irrespective of optimality criterion, while all nodes that varied with method had low or moderate probabilities. On the other hand, several nodes found in all parsimony and ML trees had low posterior probabilities and bootstrap support (see Node Support).
Effects of using consensus sequences
------------------------------------
To explore the effect of using consensus sequences that "masked" within-species and among clone variation via ambiguity coding, we compared bootstrap support (using 1,000 RAxML-computed replicates) obtained with the 584 original sequences, the 105 consensus sequences, and the 40 consensus sequences. Generally, corresponding nodes, i.e. nodes defining the same groups of taxa, had higher bootstrap support with the 105-consensus- than the 584-original-sequence matrix (see Appendix 3). The weakly supported *Acer* core clade had a PP of 0.85 and a BS~ML~ of 57% with the 105-consensus-sequence matrix, while with the 584-original sequence matrix it had a BS~ML~ of only 41%. Using the 40- instead of the 105-consensus-sequence matrix increased bootstrap support only slightly and support for a few terminal nodes actually decreased. An exception from these general trends was the sister taxon relationship between *A. distylum* and *A. nipponicum* ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}), which received low ML bootstrap support (41%) with the 584-original-sequence matrix, 33% with the 105-consensus-sequence matrix, and 37% with the 40-consensus-sequence matrix.
Node support
------------
Many backbone nodes of the preferred ML tree ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}) have PP \< 0.95 and BS \< 75% (Appendix 3). Such low support can reflect absence of phylogenetic signal or contradictory signal. The bipartition networks (one of which is shown as [Fig. 3](#f3-ebo-02-07){ref-type="fig"}) show that backbone nodes in the ML tree ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}) generally correspond to the longest edges. Where placements have poor support, as is the case for *A. caesium, A. carpinifolium*, the *Acer* core clade, and the *Ginnala* clade, alternative splits are almost equally probable ([Fig. 3](#f3-ebo-02-07){ref-type="fig"}), but basically there is too little signal in the data. Low support for the *Macrantha 2* clade, however, appears due to alternative bipartitions, some of which indicate a closer relationship of *Macrantha 2* taxa to the palmatoid cluster, *A. distylum, A. nipponicum*, and *D. dyeriana*, while others pull *Macrantha 2* taxa to the platanoid cluster ([Figs. 2](#f2-ebo-02-07){ref-type="fig"} and [3](#f3-ebo-02-07){ref-type="fig"}, Appendices 2 and 3). In the case of *A. negundo*, the low support for the placement of *A. negundo* as sister to the *Cissifolia* clade ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}) is mainly due to a single alternative split involving *A. caudatum* subsp. *ukurunduense* of the *Caudata* clade ([Fig. 3](#f3-ebo-02-07){ref-type="fig"} and Appendices 2 and 3).
Motif analysis
--------------
We screened all sequences for motifs, including the LPR1 and LPR2 regions (the location of which is shown in [Fig. 4A](#f4-ebo-02-07){ref-type="fig"}). Examples of motifs found in *Acer-Dipteronia* and Sapindales are illustrated in [Figures 4](#f4-ebo-02-07){ref-type="fig"} (3′ part of LPR2) and 5 (3′ end of ITS1). Transitions are the most common substitutions, but appear restricted to specific motif positions.
The LPR1 of *Acer* and *Dipteronia* is 21 nt (clones of *A. buergerianum*) to 49 nt long (several *A. ibericum* clones) and is characterized by an initial motif of 6 to 7 G, followed by AG, a 20 nt long central motif, and an 8 nt long terminal pyrimidine motif. The initial GGGGGG-AG motif is conserved across the two genera. A generalized *Acer* LPR1 sequence is found in *A. caesium* (aceroid cluster), *A. negundo*, and all species of the *Arguta*, *Caudata* (except *A. caudatum* subsp. *ukurunduense*), and *Cissifolia* clades (palmatoid cluster), *A. macrophyllum*, all species of *Macrantha 2*, and all but one species of the *Platanoidea* clade (platanoid cluster). The remaining palmatoid and platanoid species, as well as *D. dyeriana* and *D. sinensis*, have LPR1 motifs that differ in 1 to 2 nt from the consensus sequence. Increased motif divergence, including prominent (≥3 nt long) length polymorphism, is restricted to the aceroid cluster. The LPR1 of *Acer* and *Dipteronia* cannot unambiguously be aligned with the remaining Sapindales. Sapindaceae other than *Acer* and *Dipteronia* all lack the initial GGGGGG-AG motif and the 8 nt long terminal pyrimidine motif. The 5′ end of the LPR1 central motif of Burseraceae (119 accessions, representing 7 genera) sometimes differs only in 1 nt from the 5′ end of the central motif of *Acer* and *Dipteronia*, and, as in *Acer* and *Dipteronia*, the LPR1 region of Anacardiaceae (62 ITS accessions, representing 12 genera), Burseraceae, and Rutaceae (16 accessions from 5 genera) is less than 30 nt long and has a 5′ G-dominated motif and a 3′ C-dominated motif. Unlike in Sapindaceae, however, major (≥3 nt long) intra- and intergeneric length polymorphism in these families is uncommon. In the Meliaceae (72 accessions from 10 genera) and *Murraya* (Rutaceae; 2 species sequenced), the LPR1 equivalents have increased length (55 to 65 nt) and point mutational variability.
The LPR2 ([Fig. 4](#f4-ebo-02-07){ref-type="fig"}) of *Acer* and *Dipteronia* starts with a C-dominated motif (≤14 nt long) that is followed by three to five purines (predominantly 3 A) and a motif with pyrimidines (C and T), which is followed downstream by a purine-dominated motif (A and G; [Fig. 4B](#f4-ebo-02-07){ref-type="fig"}). The 3′ end of LPR2 is defined by ACTTGGCC (with some taxon-specific modifications) downstream of the LPR2. Further LPR2 motifs characterize subspecies, species, or species groups: For example, (i) most species have an 11 to 20 nt long pyrimidine motif, \[CCCCT\]~2~CTC or CCCCT\[CTC\]~2~, usually accompanied by a 15 to 20 nt long purine motif that can be generalized as GAAA-\[GAGA\]~1--2~-CGA-GGGG ([Fig. 4B](#f4-ebo-02-07){ref-type="fig"}) (ii) Other species differ in two to five point mutations and indels from the basic motif (shown in the center of [Fig. 4B](#f4-ebo-02-07){ref-type="fig"}). (iii) In the *Pentaphylla-Trifoliata* clade ([Figs. 1](#f1-ebo-02-07){ref-type="fig"}, [4B](#f4-ebo-02-07){ref-type="fig"}) and in several clones of *A. palmatum* subsp. *amoenum*, the downstream pyrimidine-purine motifs are partially deleted.
The LPR2 regions of *Dipteronia*, while differing from each other and from *Acer*, can still be aligned with each other, and LPR2-homologous DNA stretches are detectable in the remaining Sapindales. In Sapindaceae, they are 23 to 41 nt long and start with a C-dominated motif that ends with two or three purines (AA, GAA, AAG), followed by up to 15 downstream pyrimidines and a purine-dominated terminal part ([Fig. 4C](#f4-ebo-02-07){ref-type="fig"}). In Burseraceae, the LPR2 starts with two C-dominated motifs (each with 6 or more Cs); Anacardiaceae and Meliaceae have three 5′ purines after the initial C-dominated motif, often three A, and then a 14 nt long motif comprising all four nucleotides in more or less equal frequencies; in Rutaceae (for which we screened 21 ITS2 accessions from five genera), the LPR2 is mainly composed of 5′ Cs and 3′ Gs ([Fig. 4C](#f4-ebo-02-07){ref-type="fig"}).
In addition to the LPR1 and LPR2 ITS regions, we screened the remainder of ITS1 and ITS2 for clade-conserved motifs (approx. 12 to 20 nt long; [Fig. 5](#f5-ebo-02-07){ref-type="fig"} and Appendix 4). Motif variants of sister taxa usually involve the fixation of a single mutation (mostly transitions), and the detected variants can be ordered parsimoniously within species clusters. Examples are the permutations of the T-dominated ITS1 motif illustrated in [Figure 5](#f5-ebo-02-07){ref-type="fig"}. Different clades of *Acer* have fixed substitutions of a C at specific positions in this motif. The last two nucleotides of the motif, TT or CT in *Acer* and *Dipteronia* and predominantly CT, TC, CC in other Sapindales, are relatively conserved. For example, 190 accessions of Burseraceae, representing seven genera, all had the CC ending.
More 'ancestral' and more 'derived' motif variants can co-exist within *Acer* individuals, subspecies, or species, or may be confined to taxon clusters, as indicated in [Figure 5A](#f5-ebo-02-07){ref-type="fig"}.
The above-described motifs in the LPR1 and LPR2 regions plus eight motifs in the remainder of ITS1 and ITS2 (Appendix 4) support relationships found in the bipartitions network and the NN splits graphs ([Figs. 2](#f2-ebo-02-07){ref-type="fig"} and [3](#f3-ebo-02-07){ref-type="fig"}). Motifs also weakly support the monophyly of *Dipteronia*, but the two species have different mutational trends, with the ITS of *D. sinensis* exhibiting a bias towards C/G substitutions compared to *D. dyeriana* ([Fig. 4B](#f4-ebo-02-07){ref-type="fig"}).
Polyploidy and ITS divergence in *Acer*
---------------------------------------
To assess whether polyploid maples have increased ITS divergence, we compared as many diploid/polyploid pairs (with similar cloning efforts) as possible. Uncorrected p-distances in four pairs were:
i. *A. monspessulanum* subsp. *monspessulanum* (*Acer* core clade, diploid), 56 clones from 14 individuals: 0--0.013; 38 clones from 10 individuals: 0--0.013; 12 clones from three individuals: 0--0.009; *A. opalus* (*Acer* core clade, diploid), 32 clones from 12 individuals: 0--0.009; *A. pseudoplatanus* (*Acer* core clade, tetraploid), 38 clones from 13 individuals: 0--0.015.
ii. *A. sempervirens* (*Acer* core clade, diploid), 19 clones from 4 individuals: 0--0.009; *A. velutinum* (*Acer* core clade, tetraploid), 19 clones from 3 individuals: 0--0.014.
iii. *A. rubrum* (*Rubra* clade, hexa- to octopolyploid), 9 clones from 3 individuals: 0--0.007; *A. saccharinum* (*Rubra* clade, tetraploid), 12 clones from 3 individuals: 0--0.013.
iv. *A. buergerianum* (*Pentaphylla*-*Trifoliata* clade, diploid): 4 clones from an arboretum: 0.003--0.008, 6 clones from the wild: 0--0.015; *A. griseum* (*Pentaphylla-Trifoliata* clade, diploid): 4 clones from an arboretum: 0.004--0.01, 4 clones from the wild: 0--0.005; *A. laurinum* (diploid), 4 clones from the wild: 0.001--0.007, *A. carpinifolium* (tetraploid), 4 clones from an arboretum: 0--0.008.
In *Acer ibericum* (erroneously synonymized under *A. monspessulanum* by [@b77-ebo-02-07]), we found two co-existing ITS haplotypes in all four individuals (11 clones). Both variants share the *A. ibericum*-typical mutations and are undergoing concerted evolution. The ploidy level of *A. ibericum* is not known. Similar cases of co-existing haplotypes were discovered in *A. campestre* and *A. mono* subsp. *mono*.
Discussion
==========
The main goal of this study was to assess whether ITS data can be used for inferring the phylogeny of *Acer* in spite of intra-individual and intra-specific sequence divergence. Earlier phylogenetic studies of the *Acer* have all relied on directly sequenced ITS data ([@b10-ebo-02-07]; [@b1-ebo-02-07]; [@b60-ebo-02-07]; [@b64-ebo-02-07]), and obtained largely unsupported trees. Results obtained here with a ML analysis of 584 ITS sequences from multiple accessions of most species show that sequences largely group by species, putting to rest suspicions that ITS might be a dubious phylogenetic marker in *Acer*. We found no evidence of increased ITS divergence in wild material as opposed to trees from botanical gardens or in polyploid as compared to diploid species. Polyploidy can be accompanied by the coexistence of several nucleolus organization regions (NORs; [@b38-ebo-02-07]), and several NORs can also coexist in experimental hybrids ([@b37-ebo-02-07]). Assuming that most polyploids are allopolyploids ([@b38-ebo-02-07]), one might have expected that coexisting NORs from different parents would result in sets of divergent ITS variants (homoeologs). Polyploid species of *Acer* are highly concentrated in the aceroid cluster, but there was no difference in within-species ITS divergence among polyploid and diploid species. We also found little evidence of within-individual co-occurrence of different parental ITS haplotypes. For *Nicotiana* it has been shown that in stabilized allopolyploids, one parental rDNA lineage can (but need not) be completely eliminated, while the other undergoes significant restructuring ([@b68-ebo-02-07]; [@b39-ebo-02-07]; [@b71-ebo-02-07]; [@b69-ebo-02-07]). The scarcity of divergent ITS variants in diploid and polyploid species of *Acer* cannot be fully understood without the investigation of experimental hybrids, but probably indicates rapidly acting concerted evolution.
While our results justify the use of ITS as a phylogenetic marker in *Acer* ([@b10-ebo-02-07]; [@b1-ebo-02-07]; [@b60-ebo-02-07]; [@b64-ebo-02-07]), they also show that intra-specific ITS divergence is sufficiently high to require inclusion of multiple sequences per species. Construction of consensus sequence matrices, however, is not obsolete because such matrices allow more complete searches as well as being visually more easily understood. Using 105 or 40 consensus sequences rather than the 584 original sequences resulted in slightly improved bootstrap support, probably because of slightly reduced contradictory signal and the more complete searches of a reduced tree space. The search space given by the number of all possible trees is 1.07 · 10^1338^-times larger for 584 taxa compared to 105 taxa and 4.55 · 10^1476^- times compared to 40 taxa. Reduction from 105 to 40 taxa reduces the number of possible trees by the factor 4.25 · 10^138^. Despite these huge differences in the size of the search space, matrix size had no significant effect on topology, indicating the efficiency of heuristic search algorithms.
The unsatisfactory (contradictory) results in some of the earlier ITS-based phylogenetic studies of *Acer* to some extent may be due to paralogy problems, but likely also to sparse taxon sampling and direct sequencing, resulting in numerous ambiguous base calls (a tabulation of GenBank *Acer* sequence quality is included in [@b20-ebo-02-07]). Just as found here, *Dipteronia sinensis* in these earlier studies was nested inside *Acer*, and *D. dyeriana* and *D. sinensis* did not group together ([@b53-ebo-02-07]; [@b64-ebo-02-07]). Using multiple newly generated sequences for these species, we found that their ITS contains very little signal. Moreover, there are identical mutational fixation trends in *Acer* and one of the two species of *Dipteronia*. Chloroplast sequences from four loci (*trnL* region, *rpl16* intron, *psbA*-*trnH* spacer, *rbcL* gene) generated for 62 species of *Acer* plus five Sapindaceae outgroups, including both *Dipteronia* species, strongly support the mutual monophyly of *Acer* and *Dipteronia* (S.S. Renner, L. Beenken, G.W. Grimm, A. Kocyan, R.E. Ricklefs, unpublished data).
Based on the fossil record, the initial radiation of *Acer* took place at the end of the Cretaceous (\>65 Ma; [@b76-ebo-02-07]; [@b5-ebo-02-07]; [@b41-ebo-02-07]), and much of the phylogenetic signal in the ITS region seems to have been overwritten since then. Nevertheless and as noted in a benchmark study of seed plant ITS ([@b25-ebo-02-07]), there are highly conserved sections, which correlate with phylogenetic divergence events as old as 350 Ma (also [@b25-ebo-02-07]). Analogously, basic ITS motifs could have survived in clades of *Acer* and other Sapindales since the Eocene.
A study of fossil *Acer* leaves and fruits from North America ([@b76-ebo-02-07]) distinguished seven morphotypes from the Early and Middle Eocene (≥47Ma) and assigned them to extinct and extant sections. We sequenced several species from their extant sections, namely *A. caudatum* (two subspecies), *A. nipponicum*, *A. distylum*, and *A. spicatum*. Assuming that the fossils are correctly assigned, the earliest record of the *Distyla* lineage would be from the Late Eocene of East Asia ([@b63-ebo-02-07]) and the earliest record of the *Spicata* lineage would come from the Middle Eocene of East Asia and the Late Eocene of Central Europe ([@b72-ebo-02-07]; [@b63-ebo-02-07]). *Acer nipponicum* and *A. caudatum* belong to the geologically younger section *Parviflora* (Late Oligocene/Early Miocene, N America; [@b76-ebo-02-07]). These species all place relatively basal in the ML tree or near the centers of networks ([Figs. 1](#f1-ebo-02-07){ref-type="fig"}, [2](#f2-ebo-02-07){ref-type="fig"}, [3](#f3-ebo-02-07){ref-type="fig"}), and their sequence motifs are close to the inferred basic *Acer* ITS motifs ([Fig. 4B](#f4-ebo-02-07){ref-type="fig"} and Appendix 4). In a broad sense, then, early appearance in the fossil record correlates with possession of basic ITS sequence motifs and relatively early divergence in the phylogenetic tree. More critical placements of fossils, however, are necessary to test this further.
The species of *Acer* have been grouped into many subgenera, sections, and series ([@b51-ebo-02-07]; [@b52-ebo-02-07]; [@b54-ebo-02-07]; [@b43-ebo-02-07]; [@b49-ebo-02-07]; [@b11-ebo-02-07]; [@b13-ebo-02-07]; [@b14-ebo-02-07]; [@b40-ebo-02-07]; [@b12-ebo-02-07]; our Table 1). While these authors' higher-ranked groups are not recovered by ITS data, ten of de Jong's 16 sections are supported, either in the trees and networks, or in the cases of sections *Negundo* and *Macrantha* by characteristic motifs. Of de Jong's 19 series, 14 are supported by trees, networks, or motifs. In addition, six species that have been difficult to place based on morphology are clearly assigned by the ITS data: *Acer carpinifolium* is closest to *A. caesium*, *A. tataricum* (Ginnala clade), and to species of the *Acer* core clade; *A. laurinum* is sister to the *Rubra* clade; *A. mandshuricum* and *A. pentaphyllum* belong in the *Pentaphylla-Trifoliata* clade; *A. pilosum* is nested within the aceroid cluster, where it is basal to either *A. laurinum* + *Rubra* or the *Pentaphylla-Trifoliata* clade; and *A. wardii* belongs to the *Palmata* clade ([Fig. 1](#f1-ebo-02-07){ref-type="fig"}).
The results of this study show that the ITS region in *Acer*, when analyzed such that within-species variation is taken into account, contains clear phylogenetic signal, and the geographic clustering and overall agreement with morphologically defined taxa (species, series, sections) suggest that the relationships seen indeed mirror the evolutionary unfolding of maples.
For plant material we thank the Missouri Botanical Garden, the Morris Arboretum of the University of Pennsylvania, especially its curator, Anthony S Aiello, the Prague Botanical Garden, the University of Tübingen Botanical Garden, the United States National Arboretum in Washington, DC, Washington Park, Seattle, and the local staffs of the US National Park service. Karin Stögerer is thanked for lab assistance; Thomas Denk and Charles Sheviak for samples and taxonomic expertise; M. Blessing, U. Edelmann, M. Langer, M. Schlee, S.-F. Huang, and R.E. Ricklefs for samples from arboreta and natural stands; S.-F. Huang for five sequences from direct sequencing; and G. Schneeweiss, T. Stuessy, M. Chase, and M. Hasebe for DNA aliquots. For critical comments on an earlier version we thank R.E. Ricklefs. GWG and K. Stögerer were supported by German Science Foundation grants (He549/14-1, 2 to VH and V. Mosbrugger; Gr3123/1-1 to GG). SSR was supported by the National Evolutionary Synthesis Center (NESCent), NSF \#EF-0423641.
{#f1-ebo-02-07}
{#f2-ebo-02-07}
{#f3-ebo-02-07}
{#f4-ebo-02-07}
{ref-type="fig"}. **A.** Within *Acer* lineages, single substitution events suffice to derive motif variants from one another. **B.** Evolution of the same motif among Sapindaceae genera. Motif variants in *Acer, Dipteronia, Aesculus wangii,* and *Handeliodendron (Hd.) bodinieri* are more similar to *Hamamelis,* a basal eudicot, than to other Sapindaceae. Roman numerals in parentheses refer to major Sapindales clades sensu [@b22-ebo-02-07]. **C.** Evolution of the same motif among the Sapindales families Anacardiaceae, Burseraceae, Meliaceae, and Rutaceae. The same point mutations that account for motif variability within *Acer* are found among these families. Abbreviations as in [Fig. 4](#f4-ebo-02-07){ref-type="fig"}.](EBO-02-07-g005){#f5-ebo-02-07}
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Cancer is the leading cause of human deaths worldwide. Cancer therapeutics are intensively studied, and immunotherapy represents one of the novel promising therapeutic approaches. In this type of therapy, the immune system is recruited to fight against tumor development and expansion, and the most successful immunotherapeutics to date have been immune checkpoint inhibitors, such as anti-programmed cell death protein 1 (PD-1), anti-PD-L1, and anti-CTLA-4 antibodies.^[@CR1]^ Under normal conditions, T-cells can identify and kill tumor cells by recognizing the antigens on tumor cells. However, one tumor cell mechanism, which allows them to avoid killing by taking advantage of the tightly regulated nature of T-cells, has evolved. Specifically, PD-1, the surface receptor on T-cells, is an immune checkpoint molecule responsible for avoiding autoimmunity. Upon the binding of PD-1 to its ligand, PD-L1, the T-cells are deactivated. Therefore, tumor cells can present PD-L1 on their surfaces and escape death by deactivating T-cells.^[@CR2]^ Immune checkpoint inhibitors have been developed to block the interaction between PD-1 and PD-L1, allowing the immune system to act against tumor.^[@CR3]^ US Food and Drug Administration (FDA) have approved anti-PD-1 (nivolumab, pembrolizumab), anti-PD-L1 (atezolizumab), and anti-CTLA-4 (ipilimumab) drugs for the treatment of different kinds of cancers, such as melanoma, non-small-cell lung cancer, bladder cancer, head and neck cancer, and renal cell carcinoma.^[@CR4]--[@CR6]^ Clinical trials, examining the anti-tumor activity of PD-1/PD-L1 blocking antibodies against other solid and hematological malignancies are in progress, demonstrating that the PD-1 pathway represents a promising target for anti-cancer therapy.^[@CR7]^
Although the efficacy of immunotherapy has been demonstrated, treatment response is only observed in a subset of patients.^[@CR8]--[@CR10]^ Therefore, the identification of patients that can potentially respond to drugs and the understanding of the underlying mechanisms are necessary. Rizvi et al.^[@CR10]^ demonstrated that the mutation load, the number of nonsynonymous point mutations, may be a useful predictive biomarker for treatment response. An increased number of nonsynonymous point mutations is associated with improved objective response, durable clinical benefit (DCB), and progression-free survival (PFS). However, whole-exome sequencing, necessary for the determination of mutation load is not sufficiently cost and time-effective to be applied as a standard clinical test. In contrast, cancer gene panels composed of about 300--600 cancer-related genes are used in clinical practice to investigate the genetic profile of tumors.^[@CR11],[@CR12]^ Therefore, the application of the next-generation sequencing (NGS) gene panels for the precise estimation of the mutation load and treatment response prediction was investigated. Johnson et al.^[@CR13]^ showed that the mutation counts detected in the 315-gene NGS panel for melanoma are highly correlated with those assessed by whole-exome sequencing (Spearman correlation coefficient = 0.995). Additionally, patients with high mutation counts detected by NGS gene panels were demonstrated to have a significantly higher PFS than those with the low gene panel mutation counts.^[@CR12]^ Further, Roszik et al.^[@CR14]^ developed a novel algorithmic method to accurately predict total mutation load within tumors using approximately 170 genes in the NGS panels. These results indicate that the NGS gene panels with hundreds of genes can be used to estimate the mutation load of tumors and to predict the efficacy of immunotherapy. However, Campesato et al.^[@CR12]^ further demonstrated that the predictive accuracy is apparently lost when the number of genes in the NGS panel is lower than 150, suggesting that the comprehensive gene panels, comprising more than 300 cancer-related genes, should be employed. Unfortunately, the cost of the NGS gene panels with more than 300 genes is still high, and this may be unattainable for the routine clinical tests in most hospitals worldwide.
Here, based on the publicly available cancer genomics information, we proposed a computational framework for the construction of a mutation load estimation model for lung adenocarcinoma, the most common type of lung cancer, and we analyzed the effectiveness of this model for the prediction of cancer immunotherapy response. Furthermore, the computational framework was applied to construct the mutation load estimation models for melanoma and colorectal cancer, respectively. These cancer-specific models may allow the design of customized panels for the targeted sequencing of selected genes to estimate mutation load, instead of whole-exome sequencing, decreasing the cost and time required for the assessment of mutation load.
Results {#Sec2}
=======
Computational framework overview {#Sec3}
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The flowchart of the computational framework used during the mutation load estimation model construction for lung adenocarcinoma is shown in Fig. [1](#Fig1){ref-type="fig"}. We generated the mutation matrix with the somatic mutation data downloaded from The Cancer Genome Atlas (TCGA)^[@CR15]^ as the training data. Subsequently, the candidate genes were selected based on a set of defined criteria. Afterward, a simple linear model was used for the construction of mutation load estimation model. Least squares parameter estimation method was employed for parameter identification and Bayesian information criterion (BIC) was used for model selection. After the selection of the most appropriate model, the performance of the mutation load estimation model was evaluated and verified using the mutation information obtained from the independent validation data. Details of this procedure are presented in Materials and methods.Fig. 1Computational framework used during the construction of the lung adenocarcinoma mutation load estimation model
Mutation load estimation model for lung adenocarcinoma was constructed using only 24 genes {#Sec4}
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With the lung adenocarcinoma somatic mutation data downloaded from TCGA database, a computational framework was developed to construct the mutation load estimation model. After selecting nonsynonymous point mutations, the mutation matrix with 13,526 genes and 230 patients was generated. Subsequently, based on the defined selection criteria (mutation frequency ≥ 10%, coding DNA sequence (CDS) length ≤ 15,000, and Bonferroni corrected *p*-value \< 0.05 in Wilcoxon test), 62 candidate genes were selected ([Materials and methods](#MOESM1){ref-type="media"}, Supplementary Fig. [1](#MOESM1){ref-type="media"}, and Supplementary Table [1](#MOESM1){ref-type="media"}).
For the 62 candidate genes selected, there are 2^62^−1 combinations of gene sets, resulting in 2^62^−1 possible models. Based on the least squares parameter estimation and BIC for model selection ([Materials and methods](#MOESM1){ref-type="media"}, [Supplementary Methods](#MOESM1){ref-type="media"}), the most appropriate mutation load estimation model for lung adenocarcinoma was shown to contain only 24 genes, selected as follows:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{array}{l} \hat y{{ = }}68.72 \cdot {\mathrm{{\it PXDNL} + }}64.27 \cdot {\mathrm{{\it NOTCH4} + }} \cdots {{ + }}27.1 \cdot {\mathrm{\it PAPPA2}} \\ + 22.57 \cdot {\mathrm{{\it ZFHX4} + }}47.24, \end{array}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$\hat y$$\end{document}$ is the estimated mutation load using the 24-gene model. The complete list of genes and their corresponding parameters in the constructed estimation model are shown in Table [1](#Tab1){ref-type="table"}. With the model constructed as shown by equation ([1](#Equ1){ref-type=""}), the mutation counts in these 24 genes of a patient allow the estimation of the mutation load.Table 1Genes and the corresponding parameters used in the constructed lung adenocarcinoma mutation load estimation modelGene symbolEntrez IDParameterGene symbolEntrez IDParameter*PXDNL*13790268.72*ASXL3*8081639.52*NOTCH4*485564.27*ERICH3*12725437.88*CSMD2*11478458.51*HRNR*38869737.14*PLPPR4*989054.54*LRP2*403636.12*NRXN1*937850.57*ASTN1*46035.65*KMT2C*5850849.19*RYR3*626335.57*ADAMTS12*8179246.68*MXRA5*2587834.54*COL6A3*129345.95*ADGRG4*13937831.22*ZNF831*12861141.88*NALCN*25923229.79*FAM135B*5105941.01*LRP1B*5335328.99*FLG*231240.99*PAPPA2*6067627.10*FAM47C*44244440.62*ZFHX4*7977622.57Constant term47.24
The constructed model for lung adenocarcinoma can be used for the precise estimation of the mutation load and accurate prediction of the immunotherapy treatment response {#Sec5}
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For the performance evaluation of the constructed model for lung adenocarcinoma, the mutation load for all patients in the training data from TCGA (*n* *=* 230) was estimated using this model. *R*^*2*^ between the estimated and actual mutation load was shown to be 0.9336 (Supplementary Fig. [2](#MOESM1){ref-type="media"}), indicating that the estimated mutation loads highly correlate with the actual mutation loads. Additionally, in order to validate the constructed mutation load estimation model, two independent validation datasets (*n* *=* 211) were applied as well, to test the performance (Materials and methods),^[@CR10],[@CR16]^ and *R*^2^ between the estimated and actual mutation load was shown to be 0.7626 for the independent validation cohort (Fig. [2a](#Fig2){ref-type="fig"}).Fig. 2Performance evaluation of the mutation load estimation model. **a** Estimated mutation load vs. actual mutation load using the independent validation data (*n* *=* 211). **b** Survival analysis comparing PFS in patients with the high estimated mutation loads (*n* = 15) with those with the low estimated mutation loads (*n* = 15). The log-rank test results indicate that the higher estimated mutation load correlates with improved PFS (*p* = 0.0003). **c** ROC curve for the classification of DCB/NDB patients using the estimated mutation load. The red point indicates the optimal discrimination threshold 141. AUC = 0.8744. **d** Immunotherapy response prediction using the estimated mutation load. Gold horizontal line represents the optimal discrimination threshold, 141
We analyzed the performance of the mutation load estimation model for lung adenocarcinoma, in the prediction of the immunotherapy treatment response, using information from an independent validation cohort.^[@CR10]^ The actual and estimated mutation loads of the patient subgroups with different clinical characteristics are presented in Supplementary Figs. [3](#MOESM1){ref-type="media"} and [4](#MOESM1){ref-type="media"}, respectively. Survival analysis was applied for the comparison of the PFS between the patients (*n* *=* 30) with high/low estimated mutation loads, and we demonstrated that a high mutation load, as estimated using our constructed model, was significantly associated with the improved PFS (*p* = 0.0003, log-rank test) (Fig. [2b](#Fig2){ref-type="fig"}). Univariate analysis showed that strong PD-L1 expression and high mutation load (either actual or estimated mutation load) are significantly associated with the improved PFS. In multivariate analysis, after adjusting for the PD-L1 expression, high estimated mutation load remained significantly associated with improved PFS (Supplementary Table [2](#MOESM1){ref-type="media"}). The estimated mutation loads were also employed to predict whether the patients have DCBs or no durable benefits (NDBs) following the immunotherapy. To this end, we determined a discrimination threshold first. Because the higher estimated mutation load correlates with the improved PFS, if the estimated mutation load of a patient is higher than or equal to the discrimination threshold, that patient is more likely to have DCB, and vice versa. Therefore, the receiver operating characteristic (ROC) curve was used to determine the optimal discrimination threshold, and the estimated mutation load ≥ 141 was identified as the threshold combining the maximal sensitivity and specificity. The area under the curve (AUC) for DCB/NDB classification using our constructed model was shown to be 0.8744, demonstrating that the estimated mutation load can predict the immunotherapy treatment response quite well (Fig. [2c](#Fig2){ref-type="fig"}). According to the estimated mutation load of each lung adenocarcinoma patient and the identified optimal threshold, the sensitivity and specificity of DCB/NDB classification using our constructed model were shown to be 0.8462 and 0.9333, respectively (Fig. [2d](#Fig2){ref-type="fig"}). Furthermore, the accuracy of the cancer immunotherapy response prediction was 0.8929, obtained using our estimation model, which is comparable to that obtained using whole-exome sequencing.^[@CR10]^
Performance verification by random models {#Sec6}
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Although we demonstrated that our estimation model for lung adenocarcinoma can be used for the precise estimation of the mutation load of a patient, and the estimated mutation load is useful for the prediction of cancer immunotherapy treatment response, we further verified the results, comparing them with the results of a model constructed using 24 randomly selected genes. Therefore, 24 genes were randomly selected from the generated mutation matrix, and a random model was constructed with the help of the least squares parameter estimation method. The procedure was repeated 10,000 times, resulting in 10,000 random models. Subsequently, the performances of these random models were evaluated. The empirical distribution of *R*^2^ between the estimated and actual mutation loads in the independent validation cohort for 10,000 random models is presented in Fig. [3a](#Fig3){ref-type="fig"}. The *R*^2^ of our constructed model (0.7626) was shown to be higher than all *R*^2^ calculated by random models, and the empirical *p*-value of *R*^2^ was *p* \< 0.0001. Further, based on the random models and the immunotherapy treatment response data, the ROC curves for all 10,000 random models were plotted (Fig. [3b](#Fig3){ref-type="fig"}) and the empirical distribution of AUC is shown in Fig. [3c](#Fig3){ref-type="fig"} (empirical *p-*value = 0.0002). For each random model, the optimal discrimination threshold can also be identified using the ROC curve, allowing the determination of the classification accuracy. The empirical distribution of classification accuracy for 10,000 random models is displayed in Fig. [3d](#Fig3){ref-type="fig"} and the empirical *p-*value of our constructed model was 0.0001.Fig. 3Performance verification using 10,000 random models. **a** Empirical distribution of *R*^2^ between the estimated and actual mutation load for 10,000 random models. **b** ROC curves for the constructed model and 10,000 random models. Blue line, the ROC curve of classifier based on the mutation load estimation model. **c** Empirical distribution of AUC statistic for 10,000 random models. **d** Empirical distribution of the classification accuracy for 10,000 random models
Cancer-type-specific mutation load estimation model is necessary for clinical application {#Sec7}
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In addition to lung adenocarcinoma, previous studies showed that the tumor mutation load is associated with the degree of clinical benefit of immunotherapy in melanomas.^[@CR17],[@CR18]^ Therefore, we investigated whether the mutation load estimation model developed using lung adenocarcinoma data can also be employed to estimate the mutation load and predict immunotherapy treatment response for melanoma patients. Mutation data of the melanoma patients^[@CR17]--[@CR20]^ and the clinical outcomes for patients treated with anti-CTLA-4^[@CR17],[@CR18]^ or anti-PD-1^[@CR20]^ agents were retrieved. The constructed 24-gene estimation model of lung adenocarcinoma was applied to estimate the mutation load of these melanoma patients. *R*^2^ between the estimated mutation load and actual mutation load was shown to be 0.6574, and the accuracy of classification with the optimal discrimination threshold was shown to be 0.6437 and 0.6579 for anti-CTLA-4 and anti-PD-1 treatments, respectively. These results demonstrate that the mutation load estimation model trained for lung adenocarcinoma can be used for the estimation of the mutation load and the immunotherapy treatment response prediction in melanoma patients to a certain extent. However, the performance of the constructed model was not as good as that for lung adenocarcinoma patients. To test whether a melanoma mutation estimation model can yield better results, we utilized the somatic mutation data of melanoma patients obtained from TCGA database (*n* = 333)^[@CR21]^ to train a melanoma mutation load estimation model, using the same approaches as the one used for the lung adenocarcinoma patients. The constructed melanoma model contained 22 genes (Table [2](#Tab2){ref-type="table"}). *R*^2^ between the estimated mutation load and actual mutation load in an independent validation cohort collected from four studies (*n* = 333)^[@CR17]--[@CR20]^ was shown to be 0.8124 (Fig. [4](#Fig4){ref-type="fig"}), which is superior to that calculated using the lung adenocarcinoma model, indicating that cancer-type-specific mutation load estimation models are necessary. Additionally, clinical responses in the melanoma patients treated with anti-CTLA-4^[@CR17],[@CR18]^ or anti-PD-1^[@CR20]^ agents were acquired to assess the performance of the immunotherapy response prediction. The actual and estimated mutation loads of patient subgroups with different clinical characteristics are presented in Supplementary Figs. [5](#MOESM1){ref-type="media"} and [6](#MOESM1){ref-type="media"}, respectively. Overall survival (OS) for both anti-CTLA-4 and anti-PD-1 treatments were shown to have no significant correlation with the estimated mutation load (Supplementary Fig. [7](#MOESM1){ref-type="media"}). For the anti-CTLA-4 treatment patients, AUC for the classification of clinical benefit using estimated mutation load was 0.6270 (Fig. [5a](#Fig5){ref-type="fig"}), and the accuracy of classification with the optimal discrimination threshold was shown to be 0.6494 (Fig. [5c](#Fig5){ref-type="fig"}). Univariate analysis showed that M category, serum lactate dehydrogenase (LDH) level, prior courses of systemic therapy, and mutation load (either actual or estimated mutation load) are significantly associated with clinical benefit. In multivariate analysis, after adjusting for M category, LDH level and prior systemic therapy, estimated mutation load was shown to remain significantly associated with the clinical benefit (Supplementary Table [3](#MOESM1){ref-type="media"}). Furthermore, AUC for the classification of clinical benefits using estimated mutation load for the anti-PD-1 treatment patients was 0.5812 (Fig. [5b](#Fig5){ref-type="fig"}), and the accuracy of classification was shown to be 0.6053 (Fig. [5d](#Fig5){ref-type="fig"}). However, there were no significant differences in the mutation load between the treatment responders and non-responders (Supplementary Table [4](#MOESM1){ref-type="media"}). These results indicate that the melanoma model can estimate the mutation load of the melanoma patients more precisely than the lung adenocarcinoma model. Unexpectedly, however, the prediction accuracy of the treatment response of melanoma model was similar to that of the lung adenocarcinoma model. In addition to the lung adenocarcinoma and melanoma, the proposed computational framework was also applied for colorectal cancer, where mutation load estimation is currently not available. Somatic mutation information downloaded from TCGA was used as the training data (*n* = 536)^[@CR22]^ and the constructed colorectal mutation load estimation model contained 22 genes (Supplementary Table [5](#MOESM1){ref-type="media"}). The mutation data for colorectal cancer patients from two independent studies (*n* = 691)^[@CR23],[@CR24]^ were employed as the validation data. *R*^2^ between the estimated mutation load and actual mutation load was shown to be 0.8794 (Supplementary Fig. [8](#MOESM1){ref-type="media"}). The actual and estimated mutation loads of patient subgroups with different clinical characteristics are presented in Supplementary Figs. [9](#MOESM1){ref-type="media"} and [10](#MOESM1){ref-type="media"}, respectively. Since no immunotherapy response data for these colorectal cancer samples exist, the treatment response prediction accuracy of the constructed colorectal model cannot be evaluated.Table 2Genes and the corresponding parameters used in the constructed melanoma mutation load estimation modelGene symbolEntrez IDParameterGene symbolEntrez IDParameter*TNXB*714893.24*RYR2*626250.95*NPAP1*2374280.88*LRP2*403649.34*DNAH10*19638575.59*COL4A4*128641.92*ADGRG4*13937869.31*RP1*610140.24*SCN10A*633658.65*APOB*33838.24*CMYA5*20233355.98*UNC13C*44027936.52*FAT3*12011454.53*XIRP2*12944635.84*ZNF831*12861153.47*MXRA5*2587833.81*CSMD3*11478853.14*DNAH11*870130.16*MYH4*462252.94*MUC17*14045328.28*PKHD1L1*9303551.74*DNAH9*177027.21Constant term18.17Fig. 4Performance evaluation comparing the actual mutation load with the estimated mutation load using the melanoma model in an independent validation cohort (*n* *=* 333)Fig. 5Performance evaluation of the immunotherapy treatment response prediction using the melanoma mutation load estimation model. **a** ROC curve for the classification of clinical benefits using the estimated mutation load in the anti-CTLA-4 treatment patients. Red point, the optimal discrimination threshold 264. AUC = 0.6270. **b** ROC curve for the anti-PD-1 treatment patients. Red point, the optimal discrimination threshold 206. AUC = 0.5812. **c** Immunotherapy response prediction using the estimated mutation load for the anti-CTLA-4 treatment patients. Gold horizontal line represents the optimal discrimination threshold 264. The accuracy of the classification is 0.6494. **d** Immunotherapy response prediction for the anti-PD-1 treatment patients. Gold horizontal line represents the optimal discrimination threshold 206. The accuracy of the classification is 0.6053
Discussion {#Sec8}
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Immunotherapy using immune checkpoint inhibitors has emerged as a promising new therapeutic approach to cancer treatment in recent years. However, there are still patients who do not respond to this type of therapy, and the potential predictive biomarkers that can be used to identify the potential responders of immunotherapy are intensively studied, since this information can support the medical decision-making. Previous studies demonstrated that the mutation load measured by whole-exome sequencing may predict the sensitivity to cancer immunotherapy. However, due to the high costs and technical threshold, the routine use of whole-exome sequencing is generally not feasible in medical institutions, which hinders the application of this method as a standard clinical test. In this study, we developed a computational framework for the construction of the mathematical model that can be used for the estimation of the patient mutation load using the genetic information on a small number of genes. The constructed mutation load estimation model for lung adenocarcinoma using only 24 genes was shown to allow the precise estimation of the mutation load and the highly accurate prediction of the immunotherapy response in the lung adenocarcinoma patients, and this accuracy was shown to be similar to that of the whole-exome sequencing. Furthermore, all performance indices demonstrated that our mutation load estimation model outperforms the random models, which shows the effectiveness of the computational framework proposed in this study.
Previous studies showed that the commercial or institutional gene panels that consist of genes known or suspected to be relevant to cancer can be used to estimate the mutation load.^[@CR25]^ However, the number of genes in these panels is considerably higher than that in our model, including as many as 170, 315, and 641 genes.^[@CR12],[@CR14]^ Additionally, only four genes used in our lung adenocarcinoma model are currently included in other cancer gene panels, and only one of them is included in all three panels (Supplementary Table [6](#MOESM1){ref-type="media"}). This suggests that the majority of genes used in our model is not well-recognized as cancer-associated genes. Since the mutational profile of these 24 lung adenocarcinoma model genes was shown to be highly associated with the responses to cancer immunotherapy and mutation load, the role of these genes in cancer development and progression should be elucidated in future studies.
The genes used in our lung adenocarcinoma mutation load estimation model have a total CDS length of 187,188, which is much shorter than that in the commercial or institutional gene panels.^[@CR11],[@CR25],[@CR26]^ Therefore, this represents an additional advantage of a gene panel developed based on our mutation load estimation model, since panel cost depends on the total lengths of the genes selected. Our model should help decrease the cost and time required for panel analysis, which will further accelerate the establishment of diagnosis and medical decisions. Additionally, since there are many gene transcripts, and the CDS length information retrieved from the Ensembl BioMart represents the length of the transcripts, the CDS length of the longest transcript was used when selecting the candidate genes. Therefore, if the panels are developed using the most common transcript of each gene, the total CDS length and cost can be further reduced. Moreover, mutational hotspots can be considered as well when developing a gene panel to minimize the cost.
Although the cost can vary across different platforms, panel designs, analysis pipelines, and practices, we believe a customized targeted gene panel based on our 24-gene lung adenocarcinoma model may be a cost-effective solution for the mutation load estimation and prediction of responses to cancer immunotherapy in lung adenocarcinoma patients. A previous study directly compared the costs of a targeted sequencing panel (Einstein_v1, with a targeted region of 4.98 Mb) to whole-exome sequencing, using the same sequencing platform.^[@CR27]^ The cost of Einstein_v1 was shown to be approximately one-fourth lower than that of the whole-exome sequencing (USD \$281.25 vs. \$1266). The targeted region in our 24-gene model (approximately 0.2 Mb) is much smaller than that in Einstein_v1, and the cost can be anticipated to reduce further. Additionally, targeted gene panel approach shortens the turnaround time. A previous study estimated that the data processing CPU time for a 90-gene panel is one-twentieth of that needed for the whole-exome sequencing (5 vs. 100 h).^[@CR28]^ Furthermore, targeted gene panel approach can substantially increase the throughput, because of its high multiplexing capabilities. For example, in the aforementioned study comparing Einstein_v1 and whole-exome sequencing, whole-exome sequencing allowed only three samples per lane, whereas the targeted sequencing panel Einstein_v1 can analyze 16 samples per lane.^[@CR27]^ These are all important issues determining clinical applicability of a test.
When we applied the mutation estimation model trained using lung adenocarcinoma data on the melanoma patients, its performance was not as good as that observed for the lung adenocarcinoma patients. Since there are considerable differences in the mutational landscapes between different types of cancer, this was not surprising, and cancer-type-specific mutation load estimation model was shown to be necessary to estimate precisely the mutation load in different types of cancer. We demonstrated that the *R*^2^ between the estimated mutation load and actual mutation load in melanoma patients is higher when using the melanoma model compared with that obtained when using the lung adenocarcinoma model. Additionally, we showed that the colorectal cancer model can precisely estimate the mutation load in colorectal cancer patients, where mutation load estimation is currently not available. However, we noted that the prediction accuracy of the treatment response in melanoma patients with the melanoma model is not superior to that of the lung adenocarcinoma model. This may be due to the relatively weaker association between the mutation load and treatment response in melanoma patients compared with that in the lung adenocarcinoma patients, and mutation load alone may not be sufficient to predict clinical benefits in the melanoma patients, which agrees with the previously obtained results.^[@CR17],[@CR20]^ To elucidate this issue further, we plotted the ROC for clinical benefit prediction using the actual mutation loads of patients treated with anti-CTLA-4/anti-PD-1 immunotherapy (Supplementary Fig. [11](#MOESM1){ref-type="media"}), and the AUCs for this classification were shown to be 0.6587 and 0.6092, respectively. Furthermore, the accuracy of clinical benefit prediction using actual mutation load in melanoma patients was shown to be 0.6149 and 0.6842 for anti-CTLA-4 and anti-PD-1 treatments, respectively. These moderate performance indices indicate that the predictive value of mutation load for the treatment response in melanoma patients is not as high as that in the lung adenocarcinoma patients. Consequently, although the melanoma model can be used for the estimation of the mutation load as precisely as the lung adenocarcinoma model, their accuracy in predicting the treatment response is not comparable. This suggests that the ability to predict treatment response for a mutation estimation model depends upon its precision in estimating the mutation load and the nature of the disease as well. The development of different approaches may be necessary to predict immunotherapy treatment response in different types of cancer in future.
The limitation of our study is a relatively small number of cases, since the immunotherapy treatment response data for lung adenocarcinoma patients included only 28 cases,^[@CR10]^ and therefore, a larger number of cases is required for the validation of the performance of the treatment response prediction. Furthermore, the datasets in this study were mostly obtained in the Caucasian population, and the performance of our model in other ethnicities should be tested. Recently, in addition to the mutation load, other features such as microsatellite instability and neoantigen burden emerged as potential predictive biomarkers for cancer immunotherapy treatment response as well.^[@CR29]--[@CR31]^ Therefore, the strategies that integrate different features may be more effective biomarkers for the accurate prediction of cancer immunotherapy response in future.^[@CR32]^
In summary, we have proposed a computational framework and successfully constructed a mathematical model using only 24 genes that can be used to estimate the mutation load in lung adenocarcinoma precisely. The estimated mutation load can be used to predict the clinical outcome of cancer immunotherapy with high accuracy. Therefore, a customized panel for the targeted sequencing of these selected genes can be designed, instead of whole-exome sequencing. Consequently, by using our mutation load estimation model, the cost and time needed for the assessment of the mutation load should considerably decrease and the cancer immunotherapy response prediction should be more obtainable in the standard clinical setting.
Materials and methods {#Sec9}
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Data used for model construction {#Sec10}
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Genomics data, specifically somatic mutation information, were used for the construction of the mutation load estimation model. As the training data for the construction of the lung adenocarcinoma model, the somatic mutation data were downloaded from TCGA database (*n* *=* 230).^[@CR15]^ As the validation data, the somatic mutation data from two independent studies were retrieved (*n* *=* 181 for Imielinski et al.;^[@CR16]^ *n* *=* 30 for Rizvi et al.,^[@CR10]^ excluding four patients with squamous cell carcinoma). Additionally, we retrieved the data showing the treatment responses to anti-PD-1 immunotherapy.^[@CR10]^ For the melanoma model, the somatic mutation data was obtained from TCGA database (*n* = 333)^[@CR21]^ as the training data. The somatic mutation information from four independent studies (*n* = 333)^[@CR17]--[@CR20]^ and clinical outcomes of melanoma patients treated with anti-CTLA-4 (Snyder et al. (*n* = 64)^[@CR17]^ and Van Allen et al. (*n* = 110)^[@CR18]^) or anti-PD-1 therapy (Hugo et al. (*n* = 38)^[@CR20]^) were used as the validation data for the melanoma model. For the colorectal model, as the training data, the somatic mutation data obtained from TCGA database (*n* = 536)^[@CR22]^ were used, while the validation data were the mutation data retrieved from two independent studies (*n* *=* 619 for Giannakis et al.^[@CR23]^; *n* *=* 72 for Seshagiri et al.^[@CR24]^).
Selection of nonsynonymous point mutations and the construction of mutation matrix {#Sec11}
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Since the number of nonsynonymous point mutations has been demonstrated to be associated with the clinical benefits of immunotherapy,^[@CR10]^ the first step was selecting nonsynonymous point mutations from the training data downloaded from TCGA. Here, the column "Variant_Classification" indicates the translational effect of a variant. There are 11 different types of variant classification in TCGA lung adenocarcinoma somatic mutation data and three of them, including nonsense mutation, nonstop mutation, and missense mutation, are considered nonsynonymous point mutations. The mutations of these three types were selected and used for mutation matrix construction. Mutation matrix is an *m* × *n* matrix where *m* indicates the number of genes and *n* represents the number of patients. Each element in the mutation matrix specifies the number of nonsynonymous point mutations in a gene in one patient. Following the selection of the nonsynonymous point mutations, the "Variant_Type" information in TCGA somatic mutation raw data, showing variant types, was used for the calculation of mutation count. The types of variants used here were single-nucleotide polymorphism (SNP), double-nucleotide polymorphism (DNP), and tri-nucleotide polymorphism (TNP), indicating the mutations in one, two, or three consecutive nucleotides, respectively. Therefore, the mutation count calculation was one, two, and three for SNP, DNP, and TNP, respectively. The summation of all mutation counts of a gene in a patient represented the total number of nonsynonymous point mutations. For example, three SNPs, two DNPs, and one TNP in a gene A of a patient gave ten nonsynonymous point mutations in gene A. In this way, the number of nonsynonymous point mutations in each gene for each patient was calculated, generating the mutation matrix.
Candidate gene selection {#Sec12}
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There are about 20,000 genes in human genome,^[@CR33]^ and it is impractical to consider all genes with nonsynonymous point mutations for the model construction. Therefore, candidate genes, which may help estimate the mutation load precisely were selected based on the following three characteristics: mutation frequency, CDS length, and the association between mutation status and mutation load (Fig. [1](#Fig1){ref-type="fig"}). For each gene in the mutation matrix, the mutation frequency, i.e., the percentage of patients with mutation in one gene, can be calculated. If the constructed model comprises genes with low mutation frequency, more genes are required for the precise estimation of the mutation load, and, to avoid this, we selected the genes with mutation frequency higher than or equal to 10%. Since we aimed to reduce the cost of mutation load estimation, and the cost of the customized panel is proportional to the number of selected genes and their corresponding CDS lengths, genes with the large CDS lengths were avoided when constructing the model. Here, the CDS lengths for each gene were obtained from the Ensembl BioMart database,^[@CR34]^ and genes with the CDS lengths larger than 15,000 nucleotides were excluded from further analysis. Furthermore, we aimed to select the mutation load-associated genes that can be used to precisely estimate the mutation load of the patients, and for those where the mutation load was shown to be significantly different between the patients with mutations in a particular gene and the patients with the wild-type gene, these genes were identified as the mutation load-associated gene and selected as potential candidate genes. For example, based on the mutation information of the gene A in the mutation matrix, the patients can be separated into two groups: the mutated group, in which the patients carry the mutation in gene A, and the wild-type group, where the patients do not carry gene A mutations. Wilcoxon rank sum test was employed to test the difference in the mutation loads between these two groups. The genes with Bonferroni corrected *p*-values lower than 0.05 were identified as the mutation load-associated genes and selected as potential candidate genes. The genes that met all three criteria were selected as the candidate genes for further model construction.
Construction of the mutation load estimation model {#Sec13}
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Based on the selected candidate genes, a linear mathematical model was used to estimate the mutation load:$$\documentclass[12pt]{minimal}
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\begin{document}$$y_m{\mathrm{ = }}c{\mathrm{ + }}\mathop {\sum}\nolimits_{i = 1}^n {a_i \cdot x_{mi}} {\mathrm{ + }}e_m$$\end{document}$$where *y*~*m*~ is the mutation load of the *m*-th patient, *x*~*mi*~, *i* = 1, ..., *n*, indicates the mutation count of the selected model gene *i* in the *m*-th patient, *a*~*i*~, *i* = 1, ..., *n*, represents the weighting of each selected model gene *i* on the mutation load, *c* specifies the constant term, and *e*~*m*~ is the model uncertainty for the *m*-th patient. The equation shows that the mutation load of a patient can be calculated using the mutation counts of the selected model genes multiplied by the corresponding weightings and adding the constant term and the model uncertainty.
In the mutation load estimation model shown in equation ([2](#Equ2){ref-type=""}), the mutation load *y*~*m*~ and the mutation counts of the selected genes *x*~*mi*~ can be obtained from the generated mutation matrix. On the other hands, the weighting of each selected gene *a*~*i*~ and the constant term *c* represent the model parameters that had to be identified. Subsequently, least squares parameter estimation method was employed for parameter identification and BIC was used for model selection. BIC is a model selection criterion widely used in the field of system identification.^[@CR35]^ It measures the trade-off between the estimated error and model complexity. The model with the lower value of BIC can estimate the mutation load more precisely without including too many genes in the model. Therefore, the model with the minimal BIC statistics was selected as the most appropriate mutation load estimation model. Details are presented in [Supplementary Methods](#MOESM1){ref-type="media"}.
Performance evaluation and validation {#Sec14}
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We have selected the most appropriate model containing *p* genes with the minimal BIC value. Afterward, we evaluated the performance of the mutation load estimation model by calculating *R*^2^ between the estimated and actual mutation load using the independent validation data. Furthermore, based on the PFS/OS information and the estimated mutation load for each patient, a survival analysis comparing patients with high/low estimated mutation loads was used to determine if the estimated mutation load correlates with the clinical outcome of immunotherapy. Since the immunotherapy response data for lung adenocarcinoma were obtained from Rizvi et al.,^[@CR10]^ the estimated mutation loads were employed to discriminate between the patients with DCB or NDB as well. The ROC curve was plotted to determine the optimal discrimination threshold and the AUC was calculated. Subsequently, the patients with the estimated mutation load higher than the optimal discrimination threshold were predicted to have DCBs. In contrast, the patients with the estimated mutation load below the optimal discrimination threshold were predicted to have NDBs. In this way, the sensitivity, specificity, and accuracy of the classification were evaluated. Furthermore, we compared the performance of our model with the performances of random models composed of *p* randomly selected genes. Therefore, 10,000 random models with *p* genes were constructed and their performance were evaluated. We generated the empirical distributions of *R*^2^ between the estimated and actual mutation load, AUC statistic of classifier, accuracy of classification for 10,000 random models, and the empirical *p-*values showing the performance of our mutation load estimation model were determined.
Statistical analysis {#Sec15}
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Differences in mutation loads were examined by using the Mann--Whitney *U*-test or the Kruskal--Wallis exact test. The log-rank test was used to compare Kaplan--Meier survival curves. Cox proportional-hazards regression model was used to estimate hazard ratios and their associated 95% confidence intervals.
Data availability {#Sec16}
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All data used in this study were publicly available prior to analysis (Materials and methods).
Code availability {#Sec17}
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The code for mutation load estimation model construction is available upon request.
Electronic supplementary material
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{#Sec19}
Supplementary Information
**Electronic supplementary material**
**Supplementary Information** accompanies the paper on the *npj Genomic Medicine* website (10.1038/s41525-018-0051-x).
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This work was supported by Ministry of Science and Technology, Taiwan (MOST 104-2221-E-010-008-MY2, MOST 106-2221-E-010-019-MY3).
Y.-C.Y. and Y.-C.W. conceived of the study. G.-Y.L. and Y.-C.W. developed the method. G.-Y.L., Y.-H.Y., Y.-C.Y., and Y.-C.W. analyzed the data. G.-Y.L., Y.-C.Y., and Y.-C.W. wrote the manuscript.
Competing interests {#FPar1}
===================
The authors declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
Background {#s1}
==========
A sophisticated olfactory system is a key physiological trait for insect survival and reproduction. Olfaction is crucial to many behaviors, including feeding, mating, toxin avoidance, and negative taxis [@pone.0067151-Zhou1]. Previous studies have shown that the major olfactory-related proteins in insects are odorant-binding proteins (OBPs), odorant receptors (ORs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs) and odorant-degrading enzyme (ODEs) [@pone.0067151-Leal1]. The process of insect olfaction can be generalized as having several main steps. First, odorants reaching the pore tubules enter a sensillum and are bound and solubilized by OBPs; then, the OBPs are transported through the sensillar lymph that fills the cavity around the dendrites, where they finally activate membrane-bound ORs [@pone.0067151-Leal1]. After OR activation, the odorant will be rapidly removed by ODEs to restore the sensitivity of the sensory neuron [@pone.0067151-Vogt1], [@pone.0067151-Vogt2].
In addition, some other proteins are also thought to be involved in insect olfaction. Soluble chemosensory proteins (CSPs) are also found in abundance within the sensillar lymph. Similar to OBPs, CSPs are thought to affect insect chemoreception by enhancing the solubility of semiochemicals and delivering them to the chemosensory receptors [@pone.0067151-JacquinJoly1]. SNMPs, located in the dendritic membranes of pheromone sensitive neurons [@pone.0067151-Vogt2], [@pone.0067151-Rogers1], appear to trigger ligand delivery to the olfactory receptor [@pone.0067151-Benton1]. To maintain olfactory system sensitivity, ODEs then remove the odorant. Carboxylesterases (CXEs) and aldehyde oxidases (AOXs), which degrade ester and aldehyde pheromones [@pone.0067151-Ishida1], [@pone.0067151-Durand1], were the first identified ODEs in insects [@pone.0067151-Vogt3], [@pone.0067151-Rybczynski1]. Additionally, P450s play important roles in many physiological functions in insects, such as signal molecule metabolism, adaptation to host plants, and insecticide resistance. However, some reports have revealed that P450s may be involved in odorant processing in antennae [@pone.0067151-Wojtasek1]--[@pone.0067151-Ai1].
Most Neuroptera are natural enemies of agroecosystem pests. The lacewing *Chrysopa pallens* (Rambur) (Neuroptera: Chrysopidae) are the most important natural predators of various pests such as aphids, coccids, thrips, mites, and caterpillars [@pone.0067151-Winterton1]. The larvae of *C. pallens* are called aphid-lions because each can eat more than 800 aphids during its larval stage. *C. pallens* are voracious predators that adapt well to different agroecosystems [@pone.0067151-Athhan1]--[@pone.0067151-New1], so they are very useful auxiliaries in integrated pest management (IPM). Mass production and release of *C. pallens* in fields is economically unfeasible compared with recruitment of existing individuals in the environment. Based on the feeding habits of adult lacewings, several plant-derived compounds were suggested as attractants [@pone.0067151-JunweiZhu1]--[@pone.0067151-Toth1]. Recently, individual aphid sex pheromone compounds (enantiomers of nepetalactol and nepetalactone) were shown to attract predatory males of *Chrysopa* spp. and can potentially be used to enhance biological control of aphids [@pone.0067151-Koczor1]. Furthermore, field attraction of *Chrysopa* lacewings to (1*R*, 4a*S*, 7*S*, 7a*R*)-nepetalactol has been reported [@pone.0067151-Boo1], [@pone.0067151-Zhang1].
To enhance the effectiveness of *C. pallens* in IPM via tools such as the "push-pull" strategy, in which combinations of repellent and attractive stimuli are used to alter the populations of insect pests and natural enemies [@pone.0067151-Cook1], research about olfactory behavior, including hunting, mating, and oviposition, is essential. Discovery of chemoreception genes is the primary step for exploring the mechanism underlying insect olfactory behavior. Unfortunately, sequence data from *C. pallens* is scarce. Therefore, more sequence information and transcriptome analyses could serve as valuable molecular resources leading ultimately to more effective use of *C. pallens* in pest control and contributing to the understanding of co-evolution between *C. pallens* and its prey.
Deep sequencing data can provide extensive information about genomes and gene expression profiling via next-generation high-throughput techniques. For example, Illumina technology have proven to be an efficient and low cost method to find genes in many insects, such as *Spodoptera littoralis*, *Manduca sexta*, *Helicoverpa armigera*, *Apis cerana cerana*, *Cnaphalocrosis medinalis*, and *Nilaparvata lugens* [@pone.0067151-Wang1]--[@pone.0067151-David1]. High-throughput sequencing has been successfully used to identify olfactory genes in Lepidoptera [@pone.0067151-Legeai1], [@pone.0067151-Liu1]--[@pone.0067151-Jordan1], but no transcriptome information has been reported from Neuroptera.
Given the lack of sequence information for *C. pallens*, we constructed a whole-body cDNA library and obtained 50,396 distinct unigenes by Illumina RNA sequencing and sequence assembly. In total, 14 *OBPs*, *22 CSPs*, 60 *P450s*, 16 *IRs*, 14 *ORs*, 2 *SNMPs*, 54 *CXEs* and 21 *AOXs* were obtained. Furthermore, 10 digital gene expression (DGE) libraries were constructed and the gene expression profiles were compared among different tissues.
Results {#s2}
=======
Illumina Sequencing and Sequence Assembly {#s2a}
-----------------------------------------
A library of *C. pallens* was constructed by Illumina sequencing. A total of 58,580,430 raw reads were obtained from the sample. After removing low-quality, adaptor, and contaminating sequences, 54,331,274 clean reads (Q~20~ = 98.87%) with an accumulated length of 4,889,814,660 nucleotides (nt) were generated and assembled into 104,603 contigs with an N~50~ length of 567 nt. Using paired-ends reads, these contigs were further assembled into 50,396 distinct unigenes, including 10,662 clusters and 39,734 singletons, with a mean length of 722 nt and N~50~ of 1142 bp ([Table 1](#pone-0067151-t001){ref-type="table"}). The size distribution indicated that the 10,107 unigenes were longer than 1000 bp ([Fig. 1](#pone-0067151-g001){ref-type="fig"}).
{#pone-0067151-g001}
10.1371/journal.pone.0067151.t001
###### Summary of *Chrysopa pallens* transcriptome.
{#pone-0067151-t001-1}
Number
----------------------------------- ---------------
**Total number of raw reads** 58,580,430
**Total number of clean reads** 54,331,274
**Total nucleotides** 4,889,814,660
**Q~20~ percentage** 98.87%
**GC percentage** 37.95%
**Total number of contigs** 104,603
**Mean length of contigs** 340
**N~50~ of contigs (nt)** 567
**Total number of unigenes** 50,396
**Mean length of unigenes** 722
**N~50~ of unigenes (nt)** 1,142
**Number of distinct clusters** 10,662
**Number of distinct singletons** 39,734
Gene Identification and Functional Annotation {#s2b}
---------------------------------------------
For annotation, all of 50,396 distinct unigenes longer than 200 bp were analysised by searching against NR, Swiss-Prot, KEGG, COG, and GO databases using BLASTX with a cut-off e-value of 10^−5^. A total of 21,644 unigenes (42.96%) returned a BLAST result; 20,524, 16,967, 14,938, 8,154, and 6,209 were annotated by NR, Swiss-Prot, KEGG, COG, and GO, respectively. A much higher cut-off e-value of 10^−20^ were also used to search against NR, Swiss-Prot, KEGG and COG databases by BLASTX, and 15584, 12051, 10109 and 3774 unigenes were annotated, respectively.
GO annotation was used to classify the function of the *C. pallens* unigenes. Among the distinct unigenes, 6,209 (12.33%) corresponded to at least one GO term. A total of 14,790, 8,609, and 5,706 unigenes were classified as being involved in the categories of biological process, cellular component, and molecular function, respectively. Within biological process, cellular process and metabolic process represented the most abundant GO terms. Most unigenes that corresponded to cellular component were involved in cell and cell part. Binding and catalytic activity were most prevalent in of molecular function ([Fig. 2](#pone-0067151-g002){ref-type="fig"}).
{#pone-0067151-g002}
To further evaluate the functions of the unigenes, COG annotation was used. A total of 8,154 of the unigenes were distributed into 25 COG classifications. Among these, the cluster "general function prediction only" was the biggest group. The clusters "replication, recombination and repair", "transcription" and "translation, ribosomal structure and biogenesis" were also enriched ([Fig. S1](#pone.0067151.s001){ref-type="supplementary-material"}). The species distribution of unigenes was annotated with the NR protein database. The *C. pallens* sequences revealed substantial (35.34%) matches with *Tribolium castaneum* ([Fig. 3](#pone-0067151-g003){ref-type="fig"}).
{#pone-0067151-g003}
We identified 14 candidate *OBPs*, 22 candidate *CSPs*, 60 candidate *P450s*, 16 candidate *IRs*, 14 candidate *ORs*, 2 candidate *SNMPs*, 54 candidate *CXEs*, and 21 candidate *AOXs* with BLASTX and BLASTN ([Table S1](#pone.0067151.s005){ref-type="supplementary-material"}).
Additionally, 36 putative chemoreception genes, including 14 *OBPs* and 22 *CSPs*, were used to confirm transcriptome assemblies by sequencing their PCR products. The sequences obtained from positive clones had ≥99% identities at the nucleic acid level with corresponding sequences from the transcriptome, indicating that unigene assemblies were adequate.
Sequence Alignment and Phylogenetic Analyses {#s2c}
--------------------------------------------
### *OBP* genes {#s2c1}
All unigenes were searched by BLASTX, 14 distinct unigenes encoding OBPs were identified from the *C. pallens* transcriptome. Based on sequence analysis, 10 sequences contained a full length open reading frame with a predicted signal peptide sequence ([Fig. 4A](#pone-0067151-g004){ref-type="fig"}). These 14 genes were used to construct a phylogenetic tree with other OBPs from five species ([Fig. 5](#pone-0067151-g005){ref-type="fig"}). The tree revealed that all the candidate OBP sequences were distributed among orthologous groups. Three of them showed higher homology to ApisOBPs.
{#pone-0067151-g004}
{#pone-0067151-g005}
### *CSP* genes {#s2c2}
Sequence annotation led to the identification of 22 different candidate CSPs. Fifteen were predicted to have full lengths with signal peptides by sequence analysis ([Fig. 4B](#pone-0067151-g004){ref-type="fig"}). These 22 candidate CSPs were phylogenetically analyzed with data from five other species. Most CSPs from the same species formed monophyletic groups, unlike the OBPs. Nevertheless, eight CSPs representing five of the six species clustered together with a bootstrap percentage \>50 ([Fig. 6](#pone-0067151-g006){ref-type="fig"}).
{#pone-0067151-g006}
### *P450* genes {#s2c3}
The 134 putative P450 sequences in the *C. pallens* transcriptome assembly were identified based on similarity to known insect P450s. Forty-seven P450s (containing 28 full-length sequences) more than 220 amino acids in length were used to construct a phylogenetic tree with 82 *B. mori* P450s ([Fig. 7](#pone-0067151-g007){ref-type="fig"}). The 47 predicted P450 belonged to four major clades of P450s [@pone.0067151-Feyereisen1], namely the CYP2, CYP3, CYP4, and mitochondrial clades. Most belonged to CYP3 (22 candidates) and CYP4 (26).
{#pone-0067151-g007}
DGE Library Sequencing {#s2d}
----------------------
Ten DGE libraries of *C. pallens* including, female antennae (FA), female heads without antennae (FH), female legs (FL), female wings (FW), female thoraxes and abdomens (FB), male antennae (MA), male heads without antennae (MH), male legs (ML), male wings (MW), and male thoraxes and abdomen (MB), were constructed to investigate the expression profiles of the unigenes in these tissues. In total, 5.76, 5.65, 6.25, 5.82, 5.80, 5.61, 5.28, 5.27, 5.80, and 5.63 million raw tags were generated in each library. The percentage of clean tags among the raw tags was more than 95% in each library ([Fig. S2](#pone.0067151.s002){ref-type="supplementary-material"}), and the percentages of clean tags that could be mapped to unigenes ranged from 69.05--79.15% ([Table 2](#pone-0067151-t002){ref-type="table"}).
10.1371/journal.pone.0067151.t002
###### Tag analysis statistics of *Chrysopa pallens* transcriptome.
{#pone-0067151-t002-2}
Summary Tissue
---------------------------------------------------------------------------- -------- --------- --------- --------- --------- ---------
Total raw data Female 5759655 5797561 5652300 6247713 5815595
Male 5614292 5625370 5278529 5265384 5797437
Distinct raw data Female 201804 260245 213145 184043 199779
Male 189924 179199 184821 169576 195394
Total clean tags Female 5578201 5552846 5379205 6065829 5604779
Male 5422631 5376574 5096997 5076253 5605875
Distinct clean tag Female 83838 108157 87545 77603 82132
Male 79548 72274 78626 68830 80616
All tags mapping to genes Female 3852874 3934109 4009853 4720243 3869649
Male 3854895 4123339 3797634 4017871 3920390
All tags mapping to genes[1](#nt101){ref-type="table-fn"} Female 69.07 70.85 74.54 77.82 69.04
Male 71.09 76.69 74.51 79.15 69.93
Distinct tags mapping to Genes Female 38853 51146 43389 39611 34238
Male 36804 36143 39242 35134 38068
Distinct tags mapping to Genes[1](#nt101){ref-type="table-fn"} Female 46.34 47.29 49.56 51.04 41.69
Male 46.27 50.01 49.91 51.04 47.22
Unambiguous tags mapping to genes Female 3475421 3414969 3429608 3900638 3433164
Male 3443210 3583915 3280687 3375695 3409000
Unambiguous tags mapping to genes[1](#nt101){ref-type="table-fn"} Female 62.30 61.50 63.76 64.31 61.25
Male 63.50 66.66 64.37 66.50 60.81
Distinct unambiguous tags mapping to genes Female 33152 43132 36658 33421 29460
Male 31539 30621 33143 29734 32542
Distinct unambiguous tags mapping to genes[1](#nt101){ref-type="table-fn"} Female 39.54 39.88 41.87 43.07 35.87
Male 39.65 42.37 42.15 43.20 40.37
All tag-mapped genes Female 18369 22715 20058 18196 16619
Male 18056 17612 18836 17016 17888
All tag-mapped genes[2](#nt102){ref-type="table-fn"} Female 36.45 45.07 39.80 36.11 32.98
Male 35.83 34.95 37.38 33.76 35.49
Unambiguous tag-mapped genes Female 13833 17581 15173 13650 12558
Male 13636 13228 14142 12747 13552
Unambiguous tag-mapped genes[2](#nt102){ref-type="table-fn"} Female 27.45 34.89 30.11 27.09 24.92
Male 27.06 26.25 28.06 25.29 26.89
Unknown tags Female 1725327 1618737 1369352 1345586 1735130
Male 1567736 1253235 1299363 1058382 1685485
Unknown tags[1](#nt101){ref-type="table-fn"} Female 30.93 29.15 25.46 22.18 30.96
Male 28.91 23.31 25.49 20.85 30.07
Distinct unknown tags Female 44985 57011 44156 37992 47894
Male 42744 36131 39384 33696 42548
Distinct unknown tags[1](#nt101){ref-type="table-fn"} Female 53.66 52.71 50.44 48.96 58.31
Male 53.73 49.99 50.09 48.96 52.78
percentage of clean tags (%).
percentage of reference unigenes (%).
thoraxes and abdomens.
To evaluate the DGE data, we analyzed the distribution of clean tags in each library. Genes with more than 100 copies constituted more than 77.94% of the clean tags but less than 7.04% of the genes ([Figs. S3](#pone.0067151.s003){ref-type="supplementary-material"} and [S4](#pone.0067151.s004){ref-type="supplementary-material"}). In contrast, genes with low expression levels (fewer than five copies) represented many distinct clean tags in each library.
Differentially Expressed Genes between Tissues {#s2e}
----------------------------------------------
In this study, pair-wise comparison of each tissue within each sex against all the other tissues, respectively, was used to determine the gene expression. Compared with other tissues of each sex, between 439 and 1030 unigenes (females, 439--1030; males, 552--1026) were up-regulated in antennae ([Table 3](#pone-0067151-t003){ref-type="table"}, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). Among the 10 most up-regulated unigenes in these eight comparisons (after duplicates were removed), only 11 genes were up-regulated: seven olfactory-related genes (*OBPs* and *CSPs*), a *luciferase*, a *P450*, an *alpha*-*amylase*, and an *extradiol ring-cleavage dioxygenase* ([Table S2](#pone.0067151.s006){ref-type="supplementary-material"}). According to the GO classification, most of the gene sets that were up-regulated were related to transport. Most of the olfactory and circadian genes were up-regulated in antennae, the center of olfaction.
{#pone-0067151-g008}
10.1371/journal.pone.0067151.t003
###### Differentially-expressed unigenes between different tissues of *Chrysopa pallens.*
{#pone-0067151-t003-3}
Tissues FA FB FH FL FW MA MB MH ML MW
--------- ------ ------ ------ ----- ----- ------ ------ ------ ------ ------
**FA** ― 3191 953 568 720 104 N N N N
**FB** 928 ― 794 571 673 N 676 N N N
**FH** 439 2152 ― 208 608 N N 91 N N
**FL** 748 3143 726 ― 859 N N N 68 N
**FW** 1030 3367 1532 775 ― N N N N 331
**MA** 120 N N N N ― 1086 852 597 910
**MB** N 3004 N N N 1026 ― 882 637 1442
**MH** N N 284 N N 552 547 ― 321 967
**ML** N N N 101 N 718 774 658 ― 664
**MW** N N N N 98 715 1177 1163 1005 ―
The number of up-regulated unigenes is listed in the upper right of the table, and the number of down-regulated genes is listed in the bottom left for each tissue comparison.
N: not compared. FA: female antennae, FH: female heads without antennae, FL: female legs, FW: female wings, FB: female thoraxes and abdomens, MA: male antennae, MH: male heads without antennae, ML: male legs, MW: male wings, MB: male thoraxes and abdomen.
When body (thorax and abdomen) tissues were compared with other tissues of each sex, between 547 and 3367 (females, 2152--3367; males, 547--1177) unigenes were up-regulated ([Table 3](#pone-0067151-t003){ref-type="table"}, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). Among the top 10 up-regulated unigenes in the comparisons, 11 genes, including a *eupolytin*, a *vitellogenin receptor*, two *enteropeptidases*, two *hemolymph lipopolysaccharide*-*binding proteins*, two *chymotrypsins*, a *lysozyme*, and three *cellular FABPs* were identified ([Table S3](#pone.0067151.s007){ref-type="supplementary-material"}). Most of the up-regulated genes were involved in the GO metabolic process. Most of the genes related to metabolic process and digestion were more abundant in the body.
In the comparative analysis between the heads and other tissues of each sex, a range from 658 to 1532 (females, 726--1532; males, 658--1163) unigenes were up-regulated ([Table 3](#pone-0067151-t003){ref-type="table"}, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). Fifteen genes were identified from the top 10 up-regulated unigenes in the comparisons: a *serine proteinase inhibitor*, three *glucose dehydrogenases*, a *flagelliform silk protein*, a *calmodulin-binding protein*, two *lysozymes*, a *chymotrypsin*, a *venom allergen*, an *ultraviolet-sensitive opsin*, two *cuticular proteins*, a *P450*, and a *transient receptor potential channel* ([Table S4](#pone.0067151.s008){ref-type="supplementary-material"}). Base on the GO functional classifications, most gene sets were correlated to gene expression and transport.
In legs, as compared with other tissues of each sex, as few as 208 and as many as 1005 (females, 208--775; males, 321--1005) unigenes had significantly higher expressions ([Table 3](#pone-0067151-t003){ref-type="table"}, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). Among the top 10 up-regulated unigenes in the comparisons, 25 unigenes were identified: three *glucose dehydrogenases*, a *4-hydroxyphenylpyruvate dioxygenase*, a *serine proteinase inhibitor*, a *trimeric intracellular cation channel*, a *CSP*, two *cuticular proteins*, a *facilitated trehalose transporter*, two *pro-phenol oxidases*, a *long chain fatty acids protein*, a *calcium-independent phospholipase*, an *ionotropic glutamate receptor*, a *P450*, a *vitellogenin*, a *allantoicase*, a *ultraviolet-sensitive opsin*, a 1,*6-bisphosphate aldolase*, a *progestin and adipo Q receptor*, a *chitin deacetylase*, a *cellular FABP-like protein*, and a *glutamate receptor interacting protein* ([Table S5](#pone.0067151.s009){ref-type="supplementary-material"}). Most of the unigene sets enriched in GO process were involved in metabolic processes. Some of these up-regulated genes were related to the respiratory electron transport chain, which may be involved in providing energy for movement.
Differences in the gene-expression profiles between wings and other tissues of each sex, from 608 to 1442 (females, 608--859; males, 664--1442) unigenes were notably up-regulated ([Table 3](#pone-0067151-t003){ref-type="table"}, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). An analysis of the top 10 up-regulated unigenes in the comparisons found that nine unigenes had defined functions, including two *P450s*, one *nose resistant to fluoxetine protein*, two *cuticular proteins*, a *lysozyme*, a *polyprotein*, a *glucose dehydrogenase*, and an *extradiol ring-cleavage dioxygenase* ([Table S6](#pone.0067151.s010){ref-type="supplementary-material"}). Most of up-regulated unigenes were related to gene expression and metabolic process in females and to transport and metabolic process in males.
Equivalent tissues were also compared between females and males (Table. 3, [Fig. 8](#pone-0067151-g008){ref-type="fig"}). Comparing MA and FA, two signal transduction genes, *calmodulin-binding protein* and *G-protein coupled receptor*, were up-regulated in the FA library, and an *OBP gene* was up-regulated in MA. Two *CSPs* gene were up-regulated in FW relative to MW ([Table S7](#pone.0067151.s011){ref-type="supplementary-material"} and [S8](#pone.0067151.s012){ref-type="supplementary-material"}).
Quantitative Real-time PCR Validation {#s2f}
-------------------------------------
According to the DGE data, most of the *OBP*s and all of *OR* were abundant mainly in antennae. Among the 14 *OBP* genes, all had more than 10 TPMs (transcripts per million clean tags). We examined the relative expressions of these 14 *OBP* genes. The qRT-PCR results for these genes were consistent with the DGE results ([Fig. 9](#pone-0067151-g009){ref-type="fig"}).
{#pone-0067151-g009}
By comparing the tissues between females and males, three (*CL961-1*, *U6888*, and *U81231*) had antennal specific expressions and seven (*CL979-2*, *U15134*, *U15188*, *U21061*, *U66755*, and *U79506*) were enriched in antennae. *U29002* and *U29658*showed broad expression profiles with higher expression levels in legs. Interesting, two *OBP*s displayed head enrichment.
Discussion {#s3}
==========
*C. pallens* is one of the most important natural enemies of insect pests in China. Until now, no transcriptome information has been available for Neuroptera and sequence data for *C. pallens* were scarce, so more data was a research priority for investigating gene function in this species. In this study, a reference transcriptome was completed, yielding 4.89 Gb of transcriptome data and 56.85 Mb of gene expression tags, using next-generation sequencing technology. To our knowledge, this is the first report of whole transcriptome and DGE profile information for *C. pallens* and could serve as a valuable resource leading ultimately to more effective application of this insect in pest control.
Transcriptome *de novo* assembly was carried out with short reads because of the lack of *C. pallens* genome sequences. In this study, the N~50~ of the unigenes was 1,142 bp, much higher than in other studies [@pone.0067151-Wang1], [@pone.0067151-Xue1], suggesting high quality sequencing and assembly. In the transcriptome annotation, only 40.73% of 50,396 unigenes had matches in the NR database, and 12.32% could be annotated to one or more GO terms, indicating that large numbers of the unigenes were either non-coding or specific to *C. pallens*.
Using BLASTX annotation of the *C. pallens* transcriptome, we found that *C. pallens* shared more similarity with *T. castaneum* than with the other species examined, with a significant percentage (35.34%) of highest-similarity sequences. There were 28,690 protein sequences for *Anopheles gambiae* and 27,406 for *T. castaneum*, much more than for the other species. Nevertheless, only 4.37% of the *A. gambiae* were most homologous, much lower than the value for *T. castaneum*. The similarity between *C. pallens* and *T. castaneum* transcriptomes reflected the relatively close phylogenetic relationship between their orders (Neuroptera and Coleoptera).
To analyze the gene expression information, 10 DGE libraries of different tissues were constructed. Most of the differentially-expressed genes were up-regulated in FB. The majority of up-regulated genes were involved in metabolic processes, suggesting that the thorax and abdomen have enhanced metabolic activities, perhaps because these tagmata are the most important sites of digestion, circulation, and reproduction. Compared with other tissues, the *period*, *timeless*, and *clock* genes, which are involved in circadian rhythms, were more up-regulated in heads, antennae and, particularly, in wings, which are the most important organs for circadian rhythms in *C. pallens* [@pone.0067151-Merlin1]--[@pone.0067151-Plautz1].
The insect olfactory system is a highly specific and sensitive chemical detector essential for feeding, mating, and finding oviposition sites. To better understand the sophisticated olfactory system of *C. pallens*, 66 putative chemoreception genes(including *OBPs*, *CSPs*, *ORs*, *IRs*, and *SNMPs*) were identified from our transcriptome. This number is more than the numbers found in *Cnaphalocrosis medinalis* (22) [@pone.0067151-Li1] and *Cotesia vestalis* (28) [@pone.0067151-Nishimura1], but less than that of *Spodoptera littoralis* (82) [@pone.0067151-Legeai1], *Manduca sexta* (94) [@pone.0067151-GrosseWilde1] and *Helicoverpa armigera* (99) [@pone.0067151-Liu1].
OBPs are thought to aid in capture and transport of odorants and pheromones to chemoreceptors [@pone.0067151-Pelosi1], [@pone.0067151-Wanner1], which were discovered in the early 1980s in the giant moth *Antheraea polyphemus* [@pone.0067151-Vogt3]. On the basis of BLASTX results, all 14 candidate *OBPs* were classified into two groups: 12 *OBPs* and 2 *PBPs*. Base on the *OBP* sequences, where only the cysteine motif defines the relatedness, insect OBPs have been further grouped into: Classic OBPs (with 6 conserved cysteines), Plus-C OBPs (with more than six conserved cysteines) and Minus-C (with only four conserved cysteines) [@pone.0067151-Zhou1], [@pone.0067151-Gong1]--[@pone.0067151-McKenna1]. According to DGE and qRT-PCR results ([Fig 9](#pone-0067151-g009){ref-type="fig"} and [Table S1](#pone.0067151.s005){ref-type="supplementary-material"}), *OBPs* and *ORs* were abundant mainly in antennae, consistent with studies on *OBPs* and *ORs* in other insects [@pone.0067151-Gong1],[@pone.0067151-Fort1]. Three of 14 *OBPs*, which had antennae-specific expressions, may play important roles in pheromone detection. Interestingly, two putative *OBP*s (*U29002* and *U29658*) were expressed at very high levels in legs, and two *OBP*s showed head enrichment. This high expression might suggest the unique functions in chemoreception.
As other soluble secreted proteins, CSPs are also found in the sensillum lymph, but their roles in olfaction remain elusive. Insect CSPs were also known as OS-D-like proteins [@pone.0067151-McKenna1] or sensory appendage proteins (SAPs) [@pone.0067151-Robertson1] before being named CSPs [@pone.0067151-Angeli1]. In our study, most of the *CSPs* showed broad expression profiles which were consistent with previous studies on these genes in other insects [@pone.0067151-Gong2]--[@pone.0067151-Gu1]. In brief, antennae-enriched OBPs and CSPs may be involved in *C. pallens* identifying and binding volatile from pests, compatriots or pest-damaged plants.
Antennae-restricted expression is a useful criterion to identify genes involved in specific olfactory functions, including ODEs. Because the sequences of these transcripts were so short in the transcriptome, most have not been detected by DGE. Interestingly, 12 of the 60 *P450s* were clearly enriched in or restricted to antennae, suggesting an ODE function. In addition, *CL147-6* and *U30217* were related to the CYP4A subfamily of P450s ([Fig. 7](#pone-0067151-g007){ref-type="fig"}). Several genes in this family are olfaction specific and also expressed in olfactory sensilla [@pone.0067151-MaibecheCoisne1].
To better understand the OBPs and CSPs, phylogenetic trees of each of these genes were constructed. Three antennae enriched candidate OBPs showed higher homology to *A. pisum* OBPs ([Fig. 5](#pone-0067151-g005){ref-type="fig"} and [Fig. 9](#pone-0067151-g009){ref-type="fig"}). Because aphids are the major prey of *C. pallens*, the higher homology of candidate OBPs between *C. pallens* and *A. pisum* OBPs may play an important role in hunting. In the CSPs phylogenetic tree, most of the CSPs from the same species formed a clade. Nevertheless, eight CSPs from five of the six species clustered together ([Fig. 6](#pone-0067151-g006){ref-type="fig"}). This high conservation of CSPs may indicate the multiple copies formed after these insect orders diverged from one another and that these proteins may have crucial functions. Competitive fluorescence binding assays, fluorescent *in situ* hybridization, RNAi, and behavioral bioassays may be used to characterize these olfaction-related genes in further studies [@pone.0067151-He1], [@pone.0067151-Sun1].
The DGE results were consistent with the major functions of different tissues, most of the olfaction genes were up-regulated in male and female antennae, the center of olfaction. The antennae-enriched P450s may play roles in digesting poisonous odors to protect the sensitivity of sensory neurons [@pone.0067151-MaibecheCoisne1], [@pone.0067151-Pottier1]. Additionally, three insecticide-resistance-related genes, including two *P450s*, were enriched in wings, indicating that wings may play an important role in insecticide resistance. Some genes related to energy generation and transmission, three *glucose dehydrogenases*, an *ionotropic glutamate receptor*, and a *facilitated trehalose transporter*, were up-regulated in legs, consistent with their important function in movement.
Conclusions {#s3a}
-----------
Using next-generation sequencing technology, we provided large-scale sequence information for *C. pallens* and identified 14 *OBPs*, 22*CSPs*, 60 *P450s*, 16 *IRs*, 14 *ORs*, 2 *SNMPs*, 54 *CXEs,* and 21 *AOXs*. This large number of insect chemosensory genes will provide the basis for functional studies. Together with the evolutionary analysis, the results provide new insights into the differentiation and evolution of insect OBPs and CSPs, as well as data for further studies on non-olfaction genes in *C. pallens* and other Neuropterans. Understanding how structure, function, and gene expression interact in the olfactory systems of predatory insects can inform the development of new semiochemical tools that will improve biological control in sustainable agriculture.
Methods {#s4}
=======
Insects {#s4a}
-------
The *C. pallens* used in this experiment were provided by the Plant Protect & Environment Protect Research Institute, Beijing Academy of Agriculture and Forestry Sciences, China. Experimental insects were the offspring of a single female and reared in the laboratory on *Acyrthosiphon pisum*. Rearing conditions were 25±1°C, a 14∶10 h light/dark (L:D) photoperiod, and 65±5% relative humidity (RH). Pupae were kept in separate cages for eclosion, after which adults were supplied with *A. pisum*. Thirty 3-day-old virgin females and males, respectively, were used to collect female antennae (FA), heads (FH), wings (FW), legs (FL) and thoraxes and abdomens (FB) and male antennae (MA), heads (MH), wings (MW), legs (ML) and thoraxes and abdomens (MB). Tissue samples were kept at −80°C until RNA isolation.
cDNA Library Preparation and Illumina Sequencing for Transcriptomes {#s4b}
-------------------------------------------------------------------
Total RNA was extracted by SV Total Isolation System (Promega, Madison, WI, USA) following the manufacturer's instructions. cDNA library construction and Illumina sequencing of the samples were performed at Beijing Genomics Institute -- Shenzhen, Shenzhen, China [@pone.0067151-Zhang2]. Briefly, oligo(dT) beads were used to isolate poly (A) mRNA from 20 µg of pooled total RNA(FA : FH : FW : FL : FB : MA : MH : MW : ML : MB at 5∶1:1∶1:1∶5:1∶1:1∶1 ratio). To interrupt mRNA into short fragments, fragmentation buffer and divalent cations were used at 94°C for 5 min. Using these short fragments as templates, random hexamer-primers were used to synthesize first-strand cDNA. Second-strand cDNA was generated using buffer, dNTPs, RNAseH, and DNA polymerase I. After end-repair and ligation of adaptors, the products were amplified by PCR and purified with QIAquick PCR extraction kit (Qiagen, Venlo, Netherlands) and resolved with EB buffer for end reparation and adding poly (A). Then, the short fragments connected to sequencing adapters and detected by agarose gel electrophoresis were selected as templates for PCR amplification and sequencing using Illumina HiSeq™ 2000 (San Diego, CA, USA).
Assembly and Function Annotation {#s4c}
--------------------------------
Transcriptome *de novo* assembly was carried out with the short-read assembly program Trinity [@pone.0067151-Grabherr1], which generated two classes of unigenes: clusters (prefix CL) and singletons (prefix unigene). Finally, unigenes larger than 150 bp were first aligned by BLASTX to protein databases including Nr, Swiss-Prot, KEGG and COG (e-value\<10^−5^) and by BLASTN to the NCBI nucleotide databases (Nt; e-value \<10^−5^) to retrieve proteins with the highest sequence similarity with the given unigenes along with their protein functional annotations.
To validate the assembly results, 14 OBPs and 22 CSPs were confirmed by end-to-end RT-PCR using specific primers designed using Primer Premier 5.0 ([Table S9](#pone.0067151.s013){ref-type="supplementary-material"}). The PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega) and then subcloned into a T/A plasmid using the pEASY-T3 cloning vector system (TransGene, Beijing, China) following manufacturer's instructions. In this study, the clean read and computationally assembled sequences were submitted and available from the NCBI/SRA data base and NCBI/TSA repository (SRA experiment accession number: SRX219870, TSA accession number: GAGF01000001 - GAGF01050315, BioProject accession number: PRJNA186574).
Sequence Alignment and Phylogenetic Analysis {#s4d}
--------------------------------------------
The amino acid sequence alignment of the candidate OBPs and CSPs were performed using CLUSTALX 2.0 [@pone.0067151-Larkin1] and then arranged by Jalview 2.4.0 b2 [@pone.0067151-Waterhouse1]. The 14 OBP, 22 CSP, and 47 P450 conceptually-translated sequences from the *C. pallens* transcriptome, along with OBPs, CSPs, and P450s from other insect species, were used to construct three phylogenetic trees based on the amino sequences. The OBP data set contained OBPs from five other insect species (46 from *T. castaneum*, 46 from *Drosophila melanogaster*, 43 from *Bombyx mori*, 13 from *Acyrthosiphon pisum*, and 27 from *Apis mellifera*) ([Table S10](#pone.0067151.s014){ref-type="supplementary-material"}). The CSP data set contained CSPs from five other insect species (20 from *T. castaneum*, 19 from *D. melanogaster*, 16 from *B. mori*, 6 from *A. pisum*, and 6 from *A. mellifera*) ([Table S11](#pone.0067151.s015){ref-type="supplementary-material"}). The signal peptide predicted by SignalIP 4.0 (<http://www.cbs.dtu.dk/services/SignalP/>) was deleted from the amino acid sequences of OBPs and CSPs before phylogenetic analyses. The P450 data set contained 82 P450 sequences from *B. mori* [@pone.0067151-Ai1]. Amino acid sequences for each protein were aligned using ClustalX 1.83 [@pone.0067151-Thompson1]. Neighbor-joining trees were produced using MEGA5 [@pone.0067151-Tamura1] with Poisson correction of distances, and 1000 neighbor-joining bootstrap replicates were performed.
DGE Library Preparation and Sequencing {#s4e}
--------------------------------------
Six µg total RNA were extracted from FA, FH, FL, FW, FB, MA, MH, ML, MW, and MB as described above, and DGE library preparation and sequencing followed previously-described protocols [@pone.0067151-Xue1]. mRNA were purified by using Oligo (dT) magnetic beads adsorption, 5′ and 3′ ends of tags were generated by endonuclease *MmeI*, *NlaIII* and *DpnII* ([Table S12](#pone.0067151.s016){ref-type="supplementary-material"}). Illumina adaptors(sense: 5′ACACTCTTTCCCTACACGACGCTCTTCCGATC3′ and 5′GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG3′) were ligated to the sticky 5′ and 3′ ends of tags, respectively. At last, the short fragments connected to sequencing adapters were selected as templates for PCR amplification and sequencing using Illumina HiSeq™ 2000.
Analysis and Gene Expression Annotation of DGE Tags {#s4f}
---------------------------------------------------
DGE tag mapping followed previously-described protocols [@pone.0067151-Xue1]. The raw sequences were filtered to clean tags, and were mapped to the transcriptome, used as reference sequences containing all the possible clean tags containing CATG and 17 bases length sequences of the reference gene sequences. The number of unambiguous clean tags for each gene was calculated and then normalized to TPM (number of transcripts per million clean tags) [@pone.0067151-Hoen1], [@pone.0067151-Morrissy1].
Screening of Differentially Expressed Genes {#s4g}
-------------------------------------------
A rigorous algorithm was developed to identify differentially expressed genes (DEGs) between the different tissues of *C. pallens*, referring to the method described previously [@pone.0067151-Audic1]. *P* Value corresponds to differential gene expression test. False discovery rate (FDR) was used to determine the threshold of *P* value in multiple tests and analysis through manipulating the FDR value. We use FDR ≤0.001 and the absolute value of log~2~ ratio ≥1 as the threshold to judge the significance of gene expression difference [@pone.0067151-Benjamini1].
GO Functional Enrichment Analysis for DGEs {#s4h}
------------------------------------------
In gene expression profiling analysis, GO enrichment analysis of functional significance followed previously-described protocols [@pone.0067151-Xue1]: applies hypergeometric test to map all differentially expressed genes to terms in GO database, looking for significantly enriched GO terms in DEGs comparing to the genome background. The calculating formula is:
In this equation, *N* is the number of all genes with GO annotation; *n* is the number of DEGs in *N*; *M* is the number of all genes that are annotated to the certain GO terms; *m* is the number of DEGs in *M*. For GO enrichment analysis, all of the *P* values were calculated with Bonferroni correction. We selected a corrected *P* value ≤0.05 as a threshold to determine significant enrichment of the gene sets.
Quantitative Real-time PCR Validation {#s4i}
-------------------------------------
Total RNA was extracted as described above. The concentration of each RNA sample was standardized to 0.5 µg/µL for reverse transcription. cDNAs were synthesized using Reverse Transcription System (Promega) according to the manufacturer's protocol. The results were normalized by the internal controls *GTP-binding protein* (*U15543*) and *ribosomal protein* (*U29931*). qRT-PCR primers were designed based on the nucleotide sequences of the chosen unigenes using Beacon Designer 7.7. Primers and unigenes used in qRT-PCR are listed [Table S13](#pone.0067151.s017){ref-type="supplementary-material"}. qRT-PCR reactions were run in triplicate (technical repeats) with three independent biological replicates. The quantitative validation was analyzed by a relative quantitative method (2^--△△CT^) [@pone.0067151-Pfaffl1].
Supporting Information {#s5}
======================
######
COG classification of *Chrysopa pallens* unigenes. This figure shows the COG classifications of the unigene BLASTX results against the COG database. The X-axis shows the function class of unigenes. The Y-axis shows the number of unigenes.
(TIF)
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Different components of the raw tags in each *Chrysopa pallens* tissue sample. The percentages of tags containing Ns, only adaptors, a tag copy number \<2, and clean tags are shown. FA: female antennae, FH: female heads without antennae, FL: female legs, FW: female wings, FB: female thoraxes and abdomens, MA: male antennae, MH: male heads without antennae, ML: male legs, MW: male wings, MB: male thoraxes and abdomen.
(TIF)
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Distribution of total and distinct clean tags in each female *Chrysopa pallens* sample. Numbers in square brackets indicate the copy number ranges for each tag category. Data in parentheses indicate the numbers and percentages of each category of tags. (A) Distribution of total clean tags. (B) Distribution of distinct clean tags.
(TIF)
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Distribution of total and distinct clean tags in each male *Chrysopa pallens* sample. Numbers in square brackets indicate copy number ranges for each tag category. Data in parentheses indicate the numbers and percentages of each category of tags. (A) Distribution of total clean tags. (B) Distribution of distinct clean tags. FA: female antennae, FH: female heads without antennae, FL: female legs, FW: female wings, FB: female thoraxes and abdomens, MA: male antennae, MH: male heads without antennae, ML: male legs, MW: male wings, MB: male thoraxes and abdomen.
(TIF)
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BLASTX results and digital gene expression profiles of candidate *OBPs*, *CSPs*, *P450s*, *IRs*, *ORs*, *CXEs* and *AOXs* from *Chrysopa pallens.* Information includes gene ID in this transcriptome, open reading frame length, gene name, accession number, species, E-value, identity to other proteins, and number of transcripts per million clean tags (TPM).
(XLSX)
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Top 10 most up-regulated unigenes in antennae compared with other tissues, including gene name, expression ratio, GO process, and annotation result.
(XLSX)
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Top 10 most up-regulated unigenes in the thorax and abdomen compared with other tissues, including gene name, expression ratio, GO process, and annotation result.
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Top 10 most up-regulated unigenes in heads compared with other tissues, including gene name, expression ratio, GO process, and annotation result.
(XLSX)
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Top 10 most up-regulated unigenes in legs compared with other tissues, including gene name, expression ratio, GO process, and annotation result.
(XLSX)
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Top 10 most up-regulated unigenes in wings compared with other tissues, including gene name, expression ratio, GO process, and annotation result.
(XLSX)
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Top 10 most up-regulated unigenes in male compared with female tissues, including gene name, expression ratio, GO process and annotation result.
(XLSX)
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Top 10 most up-regulated unigenes in female compared with male tissues, including gene name, expression ratio, GO process and annotation result.
(XLSX)
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Primers and unigenes used in end-to-end RT-PCR validation, including unigene names and primer sequences.
(XLSX)
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Odorant-binding proteins used in phylogenetic tree construction, including protein name and GenBank accession number.
(DOCX)
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Chemosensory proteins used in phylogenetic tree construction, including protein name and GenBank accession number.
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Recognition sites of several endonucleases on cDNA in the sample prepared for the construction of DGE libraries.
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Primers and unigenes used in quantitative real-time PCR validation, including unigene names and primer sequences.
(XLSX)
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Click here for additional data file.
We thank Yan-Nan Zhang (Nanjing Agricultural University, China) for useful discussion; Na Li (Institute of Cotton Research, Chinese Academy of Agricultural Sciences) for help with experiments; and Fan Zhang (Plant Protect & Environment Protect Research Institute, Beijing Academy of Agriculture and Forestry Sciences, China) for providing insects.
[^1]: **Competing Interests:**The authors declare that they have no competing interests.
[^2]: Conceived and designed the experiments: ZL SD JC. Performed the experiments: ZL. Analyzed the data: SZ YM JL LL CW. Contributed reagents/materials/analysis tools: ZL SZ SD JC. Wrote the paper: ZL.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
As more and more protein sequences become available with their structures undetermined, recognizing functional signatures directly from sequences is particularly desirable in functional proteomics ([@b1]--[@b3]). Automatic discovery of patterns in unaligned biological sequences is an important problem in molecular biology ([@b5]--[@b9]). For a good review on the mining algorithms, the readers can refer to Refs ([@b10]--[@b12]). When compared with the approaches based on multiple sequence alignment in identifying functional regions, pattern mining algorithms have the advantage of automatically determining the subset of sequences involved in the final mining results ([@b13]). The derived patterns are useful in many research issues in Bioinformatics, including automatic functional annotation of sequences, database search of homologues, detection of functional sites and prediction of hot regions in protein--protein interactions ([@b2],[@b14]--[@b16]).
Pattern mining algorithms can be categorized by the description models they employ. Frequently used models include regular expressions, profiles and hidden Markov models (HMMs) ([@b11]). This paper focuses on discovery of patterns expressed in regular expression and considers only exact components in a pattern. An exact component permits only one specific amino acid in one position, such as the capital letters in the pattern N-R-x(5,19)-Y-x-G-x(3)-D. In this example, 'x' stands for a wildcard that matches any amino acid. Both 'x' and 'x(3)' are called rigid gaps, a gap of fixed length, which are composed of one and three wildcards, respectively. On the other hand, x(5,19) is a flexible gap, a gap of flexible length, which admits at least five successive wildcards and at most 19 successive wildcards in between the exact components 'R' and 'Y'. A flexible gap handles the don\'t-care regions where large insertions and deletions might happen during evolution, while a rigid gap deals with the conservative substitutions allowed in biological sequences.
When only exact elements are considered in mining process, the derived patterns are usually very sparse, in which the pattern elements are interleaved with a large amount of gaps. Patterns of this type are hard to detect but are greatly appreciated because they concisely highlight the important residues associated with protein functional sites. In proteins, the conserved residues usually appear as clusters (it is called a block in this paper), and multiple clusters together constitute an important substructure. The conserved regions that strongly correlate with each other and conserved simultaneously are usually interleaved with large irregular gaps ([@b16],[@b17]). In other words, the residues associated with a functional motif are not necessarily found in one region of the sequence ([@b2],[@b16]--[@b20]). This complicates the mining process and often confuses the approaches based on multiple sequence alignment.
Regular expression is considered as a deterministic model contrary to the probabilistic models such as profiles and HMMs ([@b11]). A deterministic pattern can be matched or not matched by a sequence. In the mining process, a pattern will be reported as long as it matches more than a user-specified percentage of the input sequence set. This is the so-called minimum support constraint ([@b21]). A pattern is said to be diagnostic for a family if it matches all the known sequences in the family, and no other known sequence ([@b10]). However, a diagnostic pattern does not always correspond to a functional signature. By setting the minimum support constraint as a lower value, MAGIIC-PRO can discover patterns that really present as functional signatures but are only conserved in a subset of input sequences. Such patterns are more informative and useful in predicting ligand binding or protein interaction.
Discovering sparse and flexible patterns which are conserved in only a subset of input sequences is a time-consuming task due to the large search space of solutions. So many related studies employ other constraints in addition to the minimum support constraint to expedite the mining process. Mining algorithms that consider only short conserved words ([@b5],[@b6],[@b18],[@b19]) or rigid gaps ([@b7]--[@b9],[@b13]), such as web service Teiresias ([@b13]), are efficient and effective in identifying short motifs. On the other hand, the Pratt ([@b22]) algorithm introduced the concept of gap flexibility to enlarge the search space. However, allowing large flexible gaps might derive patterns with the conserved residues scattered. Furthermore, it has been shown by experiments in our recent work that considering large flexibilities causes the failure of Pratt to deliver satisfied results within an acceptable time ([@b17]). Different from the previous works, our approach considers two types of gaps to improve the mining efficiency, where the gaps within a conserved region are called an intra-block gap and the gaps in between two adjacent conserved regions are called inter-block gaps ([@b17]). Using two types of gap constraints for different purposes improves the efficiency of mining process while keeping high accuracy of mining results. The server MAGIIC-PRO further employs rigid intra-block gaps instead of the flexible ones proposed in Ref. ([@b17]) since it has been observed in protein sequences that insertions and deletions are seldom present in highly conserved regions ([@b2],[@b13]). Our experimental results also reveal that considering only rigid gaps within a block is useful in eliminating noisy patterns.
MAGIIC-PRO provides many useful tools for examining and visualizing the derived patterns, which will be described in detail later. After that, we will show by experiments that MAGIIC-PRO is efficient and effective in identifying functional sites and predicting hot regions in protein--protein interactions.
METHOD
======
The web service MAGIIC-PRO is in particular designed for mining protein sequences, where the kernel algorithm executing sequential pattern mining is based on our previously developed algorithm MAGIIC ([@b17]) incorporated with several state of the art data mining techniques. MAGIIC-PRO first quickly identifies rigid gapped blocks by bounded-prefix growth technique of MAGIIC. After that, the candidate blocks are concatenated into patterns with large irregular gaps by exploiting the antimonotone characteristic of this problem ([@b20],[@b21]). Finally, a newly proposed bounded-gap closure checking scheme developed based on Ref. (23) is executed to eliminate patterns that can be covered by other super patterns with the same occurrences.
After the mining process terminates, MAGIIC-PRO generates a pattern snapshot that shows all the derived patterns in alignment with the query protein. The residues present in different patterns are combined together to create a conservation plot, where the conservation level of each residue is determined by the percentage of total number of supporting proteins merged from different patterns. The conservation plot provides a whole picture about the conserved residues of a query protein.
Input
-----
We assume that every user of MAGIIC-PRO has a protein sequence of interest at hand. MAGIIC-PRO takes a protein sequence as input, and helps the users to prepare the training data for pattern mining. The task of collecting relative sequences of the query protein can be achieved by using Swiss-Prot annotations or executing the PSI-BLAST program. Once the query protein and the training data have been determined, the mining process is executed using the parameters described in the following subsection.
Parameters
----------
The most important parameter of MAGIIC-PRO is the minimum support constraint. A pattern will be reported as long as it matches at least a certain number of sequences. The support constraint is critical to the mining results, but it might not be possible to know in advance by what percentage level a satisfied pattern can be discovered. Since lower values bring more patterns, the users are suggested to start with a large support constraint, e.g. 90%, and MAGIIC-PRO will decrease it gradually until a desired number of patterns have been found.
In addition to the minimum support constraint, MAGIIC-PRO has some other parameters for advanced users. Before going into the details, we first give a formal definition of a pattern block. Assume that a pattern is consisted of pattern elements as a sequence, and each successive pair of elements is either interleaved with a gap or not. In this work, small and rigid gaps are considered as intra-block gaps, while large and flexible gaps are treated as inter-block gaps. This thus defines the boundaries of the blocks. The notation x(*a*,*b*), *a* \< *b*, is used for a flexible gap with minimum length of *a* and maximum length of *b*, and x(*a*) stands for a rigid gap with a fixed length of *a*. The wildcard x(*a*) is omitted if *a* = 0, and is written as x if *a* = 1, i.e. *x* = *x*(1). The first group of the advanced parameters specifies the gap constraints. The maximum length of an intra-block gap (default value = 3);The maximum relative flexibility of an inter-block gap with respect to the length of the inter-block gap present in the query protein (default value = 30%);The second group of the advanced parameters specifies the size or length constraints.The minimum number of elements in a block (default value = 3);The minimum number of blocks in a pattern (default value = 2);
We argue that a pattern should have at least two blocks to be meaningful, because an important region is seldom to be conserved singly either from structural or functional aspects. In this way, the users can be directly guided to the important discoveries.
Output
------
After the mining process finishes, the users can first take a look on the conservation plot and pattern snapshot. As shown in the [Figure 1a](#fig1){ref-type="fig"}, the locations of the conserved regions are summarized in the complete conservation plot derived from all the patterns. It can be observed in [Figure 1a](#fig1){ref-type="fig"} that there are nine conserved regions in the query protein. In the same web page, the users are provided with an interactive interface to collect patterns of interest in a pattern snapshot. Different from the conservation plot, a pattern snapshot in addition tells which pattern blocks are simultaneously conserved during evolution. The users are suggested to browse the lists of the top 10 high-support and top 10 large-size patterns. The size of a pattern is defined as the number of exact components it contains. A pattern with a high support usually highlights the most highly conserved residues that are related to a functional region, while a longer pattern with a lower support in general provides a complete signature with respect to a functional site.
Here we use the same example from [Figure 1a](#fig1){ref-type="fig"} to explain how the interactive snapshot can facilitate examining the mining results. In [Figure 1b](#fig1){ref-type="fig"}, we first examine the top 10 high-support patterns with 3 or more blocks. Similar patterns can be considered as being associated with the same functional site, but each of them is distinct from the others because the sets of supporting sequences are different. The top one pattern in [Figure 1b](#fig1){ref-type="fig"} identifies the most three important regions of this query protein, which are related to the binding sites of the ligands FAD and NAP, denoted as the blocks 1, 2 and 3 in both [Figures 1a](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}. Next, we can request the top ten large-size patterns with three or more blocks. It is observed in [Figure 1c](#fig1){ref-type="fig"} that blocks 4 and 5 are the next most conserved blocks that are simultaneously conserved with blocks 1, 2 and 3, and the further next is the block 6. The top one large-size pattern in [Figure 1c](#fig1){ref-type="fig"} is plotted with an available PDB structure in [Figure 2](#fig2){ref-type="fig"}, showing that these six blocks together constitute a complete signature regarding the binding sites of ligands FAD and NAP.
To facilitate studying the patterns of interest, we provide five useful links for each pattern. First, the web page highlights the locations of the pattern in its supporting sequences. Second, the derived pattern can be plotted with a protein structure if there are PDB entries available for any of the supporting sequences. Third, the derived pattern can be fed to the ScanProsite web service to check its selectivity, the ability to reject false positive matches. Fourth, the users can perform a multiple sequence alignment on the segments of supporting sequences that are associated with the selected pattern. This helps the user to construct a more generalized pattern with amino acid substitutions considered. Fifth, MAGIIC-PRO aligns each excluded sequence with the segment of the query protein. This helps to tell why a particular sequence does not match the pattern.
RESULTS AND DISCUSSIONS
=======================
In this section, we first demonstrate the efficiency of MAGIIC-PRO in identifying long patterns based on the 13 datasets with different levels of similarities listed in [Table 1](#tbl1){ref-type="table"}. With the default settings of the advanced parameters, MAGIIC-PRO starts the search by setting the minimum support constraint as 90%, and decreases this constraint step by step until at least one pattern have been found. At this stage, we observed that most of the patterns with the maximum support are related to a functional site of the query protein but do not serve as a complete signature of a functional site. In order to find patterns with more conserved blocks involved, we continued decreasing the minimum support constraint and stopped the process when the calculation time of a single mining task is longer than 60 s. [Table 1](#tbl1){ref-type="table"} reports the minimum support where we stopped for each dataset, as well as the searching time used in the latest search. [Table 1](#tbl1){ref-type="table"} also shows the number of blocks generated in the first stage of the mining process and the number of derived patterns with at least two blocks. It is observed in [Table 1](#tbl1){ref-type="table"} that a large amount of single blocks do not collaborate with other blocks to form a longer pattern. The number of patterns converges rapidly when the number of blocks in a pattern increases. The patterns found in the top 10 high-support and the top 10 large-size lists of each dataset demonstrate the potential of MAGIIC-PRO in identifying functional sites and hot regions in protein--protein interactions. Owing to the limited space, we only show one experimental result in the following paragraph, while the others are provided on the web page of MAGIIC-PRO.
Here we use the case of query protein P00730 to illustrate that the long patterns found by MAGIIC-PRO are biologically meaningful. The pattern in [Figure 3a](#fig3){ref-type="fig"} constitutes the pocket for INF (N-(Hydroxyaminocarbonyl) Phenylalanine) and the zinc ions. This pattern matches 42 sequences in the training data. A longer pattern with a lower support (28 sequences) is plotted in [Figure 3b](#fig3){ref-type="fig"}. It is of interest that this pattern constitutes the substructure which presents its importance from another protein (P04852) in the complex with the LCI protein. This small example shows the necessity of finding motifs with different conservation levels that match different subsets of sequences in the training data. On the other hand, the diagnostic patterns provided in the PROSITE database simply capture the signature regarding the zinc-binding site.
Limitation of MAGIIC-PRO
------------------------
Since the minimum number of the elements in a block is suggested to be set as 3, it might happen that some residue or two of the residues are conserved but cannot be found by MAGIIC-PRO in its primitive results. In this case, the users are suggested to perform a multiple sequence alignment for an interested pattern on the matched segments of supporting sequences through the link provided by MAGIIC-PRO. By this way, the derived patterns can be enhanced with multiple sequence alignment to have both singly conserved residues and conservative substitutions well considered.
CONCLUSION
==========
Detecting functional signatures directly from primary information is a challenging task. The mining process is tedious especially when the users have no prior knowledge about the query protein that can be used to judge how the mining results are. MAGIIC-PRO quickly guides the biologists directly to the most highly conserved regions, and after that the users can extend the derived patterns by using the advanced parameters to refine the mining results. The derived patterns are useful in prediction of protein functions and structures, protein--ligand interactions and protein--protein interactions.
The authors would like to thank National Science Council of Republic of China, Taiwan, for the financial support under the contract NSC 94-2213-E-002-125. Funding to pay the Open Access publication charges for this article was provided by Yuan Ze University and National Taiwan University.
*Conflict of interest statement*. None declared
Figures and Tables
==================
{#fig1}
{#fig2}
{#fig3}
######
Analysis of the efficiency of MAGIIC-PRO
Query protein Size of training data Setting of minimum support (%) No. of blocks derived in the first stage No. of the derived patterns with different number of blocks Time used (in s)
-------------------------------------------------------- ----------------------- -------------------------------- ------------------------------------------ ------------------------------------------------------------- ------------------ ----- ----- ----- -----
O14965 (STK6_HUMAN) Serine/threonine-protein kinase 6 1910 60 23 4 --- --- --- --- 27
P51656 (DHB1_MOUSE) Estradiol 17-beta-dehydrogenase 1 494 20 211 462 34 --- --- --- 9
P19120 (HSP7C_BOVIN) Heat shock cognate 71 kDa protein 473 90 115 3 --- --- --- --- 12
P00962 (SYQ_ECOLI) Glutaminyl-tRNA synthetase 346 70 75 12 4 --- --- --- 4
P10933 (FENR1_PEA ) Ferredoxin---NADP reductase 280 20 1490 1112 948 618 192 7 23
P08622 (DNAJ_ECOLI) Chaperone protein dnaJ 275 80 79 86 78 66 5 --- 5
P25910 (BLAB_BACFR) Beta-lactamase type II precursor 267 5 2712 860 139 26 --- --- 24
P27142 (KAD_BACST) Adenylate kinase 243 80 146 178 33 --- --- --- 2
P22887 (NDKC_DICDI) Nucleoside diphosphate kinase 233 70 78 187 169 37 --- --- 1
P09372 (GRPE_ECOLI) Protein grpE 195 30 479 1533 1836 599 23 --- 9
P00730 (CBPA1_BOVIN) Carboxypeptidase A1 precursor 57 50 141 49 72 9 3 --- 1
P08692 (ARSC1_ECOLI) Arsenate reductase 51 70 18 7 --- --- --- --- \<1
P35568 (IRS1_HUMAN) Insulin receptor substrate 1 25 80 20 2 --- --- --- --- \<1
The symbol hyphen stands for 'no patterns found.'
| {
"pile_set_name": "PubMed Central"
} |
***Background.*** Transmission efficacy of Influenza in aerosols is dependent on environmental factors such as temperature, relative humidity (RH), air flow, and UV-light. We studied the impact of these factors on the recovery of an aerosolized wild-type Influenza virus.
***Methods.*** Influenza A virus (A/WS/33 H1N1) was aerosolized via nebulizer (droplet nuclei) and airbrush (droplet nuclei/droplet mix). Virus was collected through a mannequin head into a breathing bag of an artificial lung system filled with virus transport media. During emission virus was exposed to varying environmental conditions: (1) indoor (20^o^C; 40%RH); (2) winter (6^o^C; 35%RH); and (3) summer (34^o^C; 77%RH). Within each condition, directed air flow (low: 137 feet/second, high: 154 feet/second) and exposure to UV-C light (low dose: 18W; high dose: 36W) was applied. Quantitative virus detection was performed via plaque assay in MDCK cell cultures.
***Results.*** With indoor set as standard, winter reduced virus recovery by 44% (p = 0.01) in droplet nuclei and increased recovery, though not significantly (12%, p = 0.58) in droplet nuclei/droplet mix. Summer decreased recovery by 64% in droplet nuclei (p = 0.0001) and 11% in droplet nuclei/droplet mix (p = 0.67). Within each condition, increased air flow led to a reduction in droplet nuclei (p \< 0.0001). There was indication that increased air flow may improve recovery across all conditions for droplet nuclei/droplet mix (p = 0.09). Application of low dose UV-C resulted in a \>98% reduction for droplet nuclei across all conditions (p \< 0.0001). For droplet nuclei/droplet mix, low dose UV-C had no effect across all conditions (p = 0.35), but high dose UV-C decreased droplet nuclei/droplet mix (p = 0.0019).
***Conclusion.*** Indoor conditions most effectively supported the potential for airborne Influenza transmission by small particles (droplet nuclei). However, winter shifted virus recovery to large particles making droplet transmission more likely. Summer decreased both, small and large particles. Within each condition, directed airflow reduced small particle collection by dilution, while increasing larger particle recovery by acceleration towards the collection point. UV-C light reduced virus recovery with the smallest impact observed in large particles.
***Disclosures.*** **All authors:** No reported disclosures.
[^1]: **Session:** 106. Clinical Respiratory Infections
[^2]: Friday, October 10, 2014: 12:30 PM
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s01}
============
The ATR kinase plays a crucial role in normal physiology by functioning as an apical organizer of the cellular response to DNA replication stress, a deleterious condition caused by the slowing or stalling of replication forks ([@bib22]). Replication stress is a recognized driver of genome instability that can fuel the development of cancer and other severe pathologies ([@bib6]; [@bib10]; [@bib29]). ATR-dependent phosphorylation of a range of downstream effector proteins collectively enables cells to prevent the collapse of stalled replication forks and suppress the firing of new replication origins until the replicative stress has been resolved ([@bib22]).
Stimulation of ATR kinase activity entails the recruitment of ATR and its partner protein ATRIP to replication protein A (RPA)--coated stretches of single-stranded DNA, which accumulate upon functional uncoupling of replicative DNA helicase and polymerase activities after replication fork stalling ([@bib31]; [@bib22]). This brings ATR-ATRIP into close proximity with TopBP1, which is independently recruited to replication stress sites via the Rad9-Rad1-Hus1 complex and directly stimulates ATR kinase activity by means of an ATR-activating domain (AAD; [@bib14]; [@bib11]; [@bib16]). While the requirement of this pathway for promoting the essential ATR--CHK1 checkpoint signaling axis has long been recognized, recent findings revealed that the ETAA1 protein independently promotes ATR signaling by means of dual RPA-binding motifs and an AAD that stimulates ATR kinase activity via a mechanism analogous to that of the TopBP1 AAD ([@bib3]; [@bib12]; [@bib17]; [@bib26]). Loss of ETAA1 sensitizes cells to replication stress and is synthetic lethal with TopBP1 depletion, resulting from quantitative suppression of ATR signaling, defective G2/M checkpoint control, and ensuing gross chromosomal instability ([@bib12]). Thus, full ATR activation in vertebrate cells relies on independent TopBP1- and ETAA1-mediated pathways, although the precise division of labor in promoting ATR-mediated signaling responses remains unclear. For instance, while ablation of TopBP1 or the functionality of its AAD leads to early embryonic lethality in mice, ETAA1 knockout (KO) gives rise to a comparatively mild phenotype characterized by incompletely penetrant embryonic lethality, reduced body size, and defective clonal expansion of T lymphocytes ([@bib30]; [@bib20]). Recent studies suggested roles of ETAA1 in promoting an ATR-mediated S/G2 checkpoint suppressing mitotic entry before completion of DNA replication and ATR activation during mitosis, where ATR has been shown to facilitate Aurora B activation at centromeres and accurate chromosome segregation ([@bib13]; [@bib23]; [@bib2]). However, it remains unclear which, if any, of these activities represents the key cellular functions of ETAA1 in maintaining chromosome stability. In addition, whether and how the ATR-activating capacity of ETAA1 is modulated by DNA replication and genome integrity status are not known.
Here, we used an unbiased genome-scale CRISPR-Cas9 screening approach to reveal a specific and critical function of ETAA1 in promoting cell fitness and chromosome stability upon genetic or chemical disruption of DNA replication efficiency. We established a central role in this process of cell cycle-- and replication stress--regulated, phosphorylation-dependent control of ETAA1-mediated ATR activation.
Results and discussion {#s02}
======================
ETAA1 becomes critical for cell fitness upon disruption of the DNA replication machinery {#s03}
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To define the key cellular functions of human ETAA1, we performed an unbiased, genome-scale, CRISPR-Cas9--based screen to identify genes whose ablation is synthetic lethal with loss of ETAA1 expression. To this end, we transduced WT and ETAA1-deficient (*ETAA1Δ*) HeLa cells ([@bib12]) with a lentiviral CRISPR-Cas9 KO single guide RNA (sgRNA) library (GeCKO v2) targeting 19,050 human genes ([@bib24]). We then used next-generation sequencing to identify sgRNAs that were selectively depleted in the *ETAA1Δ* background upon long-term proliferation ([Fig. 1 A](#fig1){ref-type="fig"} and Fig. S1 A). Among genes whose KO selectively impaired proliferation of *ETAA1Δ* cells, we identified several factors with known roles in promoting DNA synthesis, including MCM10, POLE3, POLE4, and CDC25A ([@bib1]; [@bib7]; [@bib5]), while sgRNAs targeting essential DNA replication factors including proliferating cell nuclear antigen and CMG helicase subunits were depleted in both WT and *ETAA1Δ* cells as expected ([Fig. 1, B and C](#fig1){ref-type="fig"}; Fig. S1 B; and Table S1). Gene ontology analysis indicated notable enrichment of terms associated with DNA replication and cell cycle progression among gene KOs displaying synthetic lethality with ETAA1 loss ([Fig. 1 D](#fig1){ref-type="fig"}), suggesting that ETAA1 function becomes critical when DNA replication efficiency is compromised. Knockdown of MCM10 or POLE3 by siRNAs impaired clonogenic survival to a much greater extent in HCT116 *ETAA1Δ* cells than in their WT counterparts, validating these screen hits and demonstrating that their genetic interaction with ETAA1 is not cell type--specific ([Fig. 1, E and F](#fig1){ref-type="fig"}; and Fig. S1, C and D). Interestingly, combined loss of ETAA1 and MCM10 led to a markedly elevated proportion of cells undergoing mitosis in the presence of DNA damage relative to MCM10 depletion alone ([Fig. 1, G and H](#fig1){ref-type="fig"}). Moreover, upon low-dose treatment with the DNA polymerase inhibitor aphidicolin (APH) to chemically slow down DNA replication, *ETAA1Δ* cells displayed a faster progression into mitosis than did WT cells ([Fig. 1 I](#fig1){ref-type="fig"}). Together, these data suggest that ETAA1 function becomes critical for cell fitness and genome stability by restraining mitotic entry when DNA replication integrity is compromised.
![**ETAA1 becomes critical for cell fitness upon disruption of the DNA replication machinery. (A)** Schematic overview of CRISPR-Cas9 KO screen workflow. **(B)** Volcano plot of CRISPR-Cas9 screen results, showing gene KOs that selectively impair fitness of HeLa WT (blue; P \< 0.01) or *ETAA1Δ* (red; P \< 0.01) cells. Point sizes (q value) represent the corrected P value for growth effect (−log~10~). Fold change depicts the median difference in sgRNA representation between the two cell lines at t21. **(C)** Boxplot depicting normalized counts for selected screen hits. **(D)** Gene ontology (GO) analysis of sensitizing hits specific to HeLa *ETAA1Δ* cells. **(E)** Clonogenic survival (relative to nontargeting control siRNA \[siCTRL\]) of HCT116 cell lines transfected with MCM10 siRNA (mean ± SEM; *n* = 3 independent experiments; unpaired *t* test). **(F)** As in E, but using POLE3 siRNA (mean ± SEM; *n* = 3 independent experiments; unpaired *t* test). **(G)** Representative images of siRNA-transfected cells coimmunostained with γ-H2AX and H3-pS10 antibodies. Scale bar, 10 µm. **(H)** Flow cytometry analysis of γ-H2AX positivity in mitotic cells in G, gated based on DNA content and H3-pS10 positivity (mean ± SEM; *n* = 3 or 4 independent experiments; multiple *t* test). **(I)** Cells synchronized by double thymidine block were released into medium containing nocodazole in the presence or absence of APH (0.2 µM), collected 12 h later, and processed for flow cytometry to determine the relative mitotic index (APH/untreated) by H3-pS10 staining (mean ± SEM; *n* = 3 experiments; unpaired *t* test). \*\*\*, P \< 0.0005; \*\*, P \< 0.005; \*, P \< 0.05; ns, not significant.](JCB_201905064_Fig1){#fig1}
ETAA1 prevents mitotic chromosome abnormalities associated with under-replicated DNA {#s04}
------------------------------------------------------------------------------------
To explore the potential role of ETAA1 in preventing genetic alterations arising from incompletely replicated DNA, we analyzed the impact of ETAA1 loss in HCT116 and HeLa cells on mitotic chromosome abnormalities. For this, we quantified bulky and ultrafine anaphase DNA bridges (UFBs) that are hallmarks of unresolved replication intermediates ([@bib18]), during both an unperturbed cell cycle and following replication stress induced by low-dose treatment with APH or hydroxyurea (HU). Consistent with a role of ETAA1 in linking DNA replication integrity and chromosome stability, *ETAA1Δ* cells showed elevated frequencies of anaphase chromatin bridges and lagging chromatin that were exacerbated upon replicative stress ([Fig. 2, A--C](#fig2){ref-type="fig"}; and Fig. S1, E--H). Likewise, *ETAA1Δ* cells displayed a pronounced increase in UFBs marked by PICH and associated with FANCD2 twin foci ([Fig. 2, A and D--F](#fig2){ref-type="fig"}; and Fig. S1, I and J), indicative of elevated common fragile site (CFS) expression ([@bib19]). In late G2 cells, ETAA1 colocalized with FANCD2 foci, which demarcate CFSs, but not with centromeric or telomeric markers (Fig. S1 K), suggesting a specific association of ETAA1 with UFBs arising from under-replicated DNA. We also observed a subset of UFBs that were coated by RPA (Fig. S1, L and M; [@bib18]; [@bib8]), although we failed to detect ETAA1 associated with these structures (data not shown), suggesting that it suppresses UFB formation by restricting mitotic entry of cells containing incompletely duplicated loci. In agreement with this possibility, cells lacking ETAA1 displayed an increased level of metaphase chromosome breaks that was further enhanced following APH treatment ([Fig. 2, A, G, and H](#fig2){ref-type="fig"}). Moreover, ETAA1 KO led to a markedly elevated proportion of APH-treated and MCM10-depleted cells undergoing mitotic DNA synthesis (MiDAS; [Fig. 2, A, I, and J](#fig2){ref-type="fig"}; and Fig. S1 N), a process that resolves under-replicated DNA at CFSs in early mitosis ([@bib19]). We conclude that ETAA1 is important for preventing mitotic chromosome abnormalities arising from incompletely replicated loci.
{#fig2}
Cell cycle-- and replication stress--regulated phosphorylation of the ETAA1 AAD promotes its ATR-activating potential {#s05}
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In line with a role for ETAA1 in protecting against chromosome instability arising from under-replicated DNA even during unperturbed cell proliferation, previous work by others and us demonstrated that ETAA1 promotes ATR signaling in a normal S phase ([@bib12]; [@bib23]). Because ETAA1 harbors both RPA-binding and ATR-activating determinants and might therefore be capable of promoting ATR activation irrespective of cell cycle position, we reasoned that regulatory control of this function may be important for its integration with genome duplication status. The ATR-activating ability of TopBP1 can be stimulated by direct ATM-mediated phosphorylation within its AAD ([@bib28]). Correspondingly, we noted that the ETAA1 AAD contains highly conserved, putative CDK phosphorylation sites (S95 and S111 in human ETAA1) in close proximity to aromatic amino acids (F106 and W107) that are essential for stimulation of ATR kinase activity ([Fig. 3 A](#fig3){ref-type="fig"}; [@bib3]; [@bib12]). Phospho-specific antibodies to S95 and S111 in human ETAA1 showed that both residues are phosphorylated in cells ([Fig. 3, B and C](#fig3){ref-type="fig"}; and Fig. S2 A). Interestingly, whereas the levels of S95 and S111 phosphorylation were low in asynchronous cells, these modifications were robustly up-regulated during S and G2 phases but declined as cells entered mitosis ([Fig. 3 C](#fig3){ref-type="fig"}). Using an established high-content imaging-based assay for monitoring ETAA1 AAD-mediated ATR activation in cells ([@bib12]; [@bib26]), we found that phospho-mimicking substitutions at S95 or S111 (S95D or S111D) but not other potential CDK sites within the ETAA1 AAD (T79D and T165D) potentiated the ATR-activating capacity of ectopic ETAA1 AAD expressed at low levels ([Fig. 3 D](#fig3){ref-type="fig"} and Fig. S2 B). Combining the S95D and S111D substitutions synergistically augmented this activity ([Fig. 3 D](#fig3){ref-type="fig"} and Fig. S2 B). Moreover, unlike WT ETAA1, whose AAD we have previously shown is a direct target of ATR-mediated phosphorylation ([@bib12]), an S95A+S111A AAD phosphorylation-deficient ETAA1 allele was refractory to phosphorylation by ATR upon replication stress ([Fig. 3 E](#fig3){ref-type="fig"} and Fig. S2, A and C). These observations suggested that phosphorylation of the ETAA1 AAD stimulates its ATR-activating potential and interplay with ATR. Interestingly, phosphorylation of S95 was strongly up-regulated upon replication stress induced by camptothecin or HU ([Fig. 3 F](#fig3){ref-type="fig"} and Fig. S2 D) in an ATR-dependent manner ([Fig. 3 G](#fig3){ref-type="fig"} and Fig. S2, E and F), while S111 phosphorylation showed a mild increase, indicating a potential positive feedback loop underlying ETAA1-mediated ATR activation.
![**Cell cycle-- and replication stress--regulated phosphorylation of the ETAA1 AAD promotes its ATR-activating potential. (A)** Sequence alignment of the ETAA1 AAD core region. Highly conserved residues (blue) and aromatic residues required for ATR activation (asterisks) are indicated. **(B)** Extracts of HCT116 cells transfected with WT or mutant versions of GFP-ETAA1 AAD were subjected to GFP IP under denaturing conditions and immunoblotting (IB). **(C)** Cells released from a G1/S arrest by double thymidine block and collected 4 h (mid-S phase), 8 h (late S), 12 h (G2-M), or 21 h (prometaphase arrested with nocodazole) later were processed for IP of endogenous ETAA1 under denaturing conditions followed by IB. **(D)** U2OS cells transfected with GFP-ETAA1 AAD-3xNLS constructs were immunostained with γ-H2AX antibody and analyzed by quantitative imaging to reveal single-cell correlations between GFP expression and pan-nuclear γ-H2AX signal intensity as readout for ETAA1-induced ATR activation ([@bib12]; [@bib26]). Plot shows mean γ-H2AX signal intensity in cells expressing GFP-ETAA1 AAD-3xNLS at a low level (Fig. S2 B) from a representative experiment (red bars, median; \*\*\*\*, P \< 0.0001, Kruskal--Wallis test; \>800 cells quantified per condition). **(E)** HCT116 *ETAA1Δ* cells stably expressing GFP-ETAA1 WT or S95A+S111A mutant were exposed or not to CPT for 90 min and subjected to GFP IP under denaturing conditions followed by IB. **(F)** Cells were exposed or not to CPT for 90 min and processed as in C. **(G)** Cells incubated with CPT or HU for 90 min in the presence or absence of ATR inhibitor (ATRi) were processed as in F. **(H)** As in C, except that cells in late S phase (S) were treated or not with CDK inhibitor (CDKi) for 90 min before harvesting. Asyn, asynchronous cells.](JCB_201905064_Fig3){#fig3}
Next, we investigated whether S95 and S111 were phosphorylated by CDKs. In contrast to ATR inhibition, suppressing interphase, but not mitotic, CDK activity strongly reduced the basal cell cycle--dependent phosphorylation of S95 and S111, but had no impact on the replication stress--induced hyper-phosphorylation of S95 ([Fig. 3 H](#fig3){ref-type="fig"} and Fig. S2, F and G). Moreover, both S95 and S111 could be directly phosphorylated by cyclin A--CDK2, but not ATR, in vitro (Fig. S2, H and I). Collectively, these findings suggest that phosphorylation of S95 and S111 during unperturbed S phase is directly catalyzed by CDKs, while replication stress--induced hyperphosphorylation of S95 is mediated by an ATR-dependent pathway involving an as yet unknown non-CDK effector kinase.
AAD phosphorylation is critical for ETAA1-dependent suppression of replication stress-induced chromosome instability {#s06}
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We next asked whether the ETAA1 AAD and its phospho-dependent regulation are required for suppressing chromosome instability following impaired DNA replication progression, using a panel of ETAA1 KO cell lines stably reconstituted with inducible WT or mutant forms of GFP-tagged ETAA1 (Fig. S3 A). As expected, the mitotic chromosome abnormalities and increased MiDAS resulting from ETAA1 loss could be rescued by ectopic WT ETAA1, but not mutants lacking the AAD or RPA-binding motifs ([Fig. 4, A--E](#fig4){ref-type="fig"}; and Fig. S3 B), providing a framework for probing the importance of ETAA1 AAD phosphorylation in protecting against chromosomal aberrations. Notably, similar to the ETAA1 ΔAAD and ΔRPA1+2 mutants, the ETAA1 S95A+S111A mutant failed to efficiently reverse mitotic chromosome aberrations and DNA synthesis in an *ETAA1Δ* background, supporting a key role for AAD phosphorylation in promoting ETAA1-mediated suppression of chromosome instability ([Fig. 4, D--H](#fig4){ref-type="fig"}; and Fig. S3 C). We previously demonstrated that ETAA1 promotes cell survival following replication stress in a manner dependent on both its AAD and dual RPA-binding motifs ([@bib12]). Similarly, we found that stably reconstituted ETAA1 S95A+S111A protein was unable to correct the survival defect of *ETAA1Δ* cells exposed to replication stress ([Fig. 4 I](#fig4){ref-type="fig"}). These data further support an important role of phosphorylation-mediated regulation of ATR activation by ETAA1 in promoting genome stability and cell fitness when DNA replication integrity is challenged.
{ref-type="fig"}. DAPI-positive chromatin bridges were quantified (mean ± SEM; *n* = 3 independent experiments; 120 anaphases/condition; unpaired *t* test). **(B)** Lagging chromatin in cells in A (mean ± SEM; *n* = 3 independent experiments; 120 anaphases/condition; unpaired *t* test). **(C)** Cells in A were immunostained with PICH antibody and analyzed for PICH-coated UFBs (mean ± SEM; *n* = 3 independent experiments; 150 anaphases/condition; unpaired *t* test). **(D)** Chromosome breaks in metaphase spreads of cells treated as in A were scored. Box plot shows median, upper and lower quartiles (boxes), and 10th and 90th percentiles (whiskers; 40 metaphases/condition; Kruskal--Wallis test). **(E)** As in D, but EdU foci in prometaphase were quantified (red bars, median; 120--125 prometaphases/condition; Kruskal--Wallis test). **(F)** As in A, using indicated cell lines (mean ± SEM; *n* = 3 independent experiments; 120 anaphases/condition; unpaired *t* test). **(G)** Lagging chromatin in cells in F (mean ± SEM; *n* = 3 independent experiments; 120 anaphases/condition; unpaired *t* test). **(H)** As in C, using indicated cell lines (mean ± SEM; *n* = 3 independent experiments; 150 anaphases/condition; unpaired *t* test). **(I)** Clonogenic survival of CPT-treated HCT116 WT and *ETAA1Δ* cell lines stably expressing GFP-ETAA1 WT or S95A+S111A mutant. Results were normalized to mock treatment (mean ± SEM; *n* = 3 independent experiments; unpaired *t* test). \*\*\*\*, P \< 0.0001; \*\*, P \< 0.005; \*, P \< 0.05; ns, not significant.](JCB_201905064_Fig4){#fig4}
Our studies demonstrate that ETAA1 becomes critical for chromosome stability and cell fitness under conditions of compromised DNA replication efficiency, and that phosphorylation-mediated control of its ATR-stimulating potential is central to this function. The notion that ETAA1 suppresses a range of mitotic chromosome abnormalities associated with incompletely replicated DNA via its ATR-activating ability is well aligned with recent work by others and us showing that ETAA1, but not TopBP1, promotes ATR signaling during a normal S phase ([@bib12]; [@bib23]). Moreover, in contrast to ETAA1, TopBP1 has been shown to localize to UFBs and promote MiDAS ([@bib21]). This apparent division of labor between ETAA1 and TopBP1 has a notable analogy to the distinct roles played by AAD-containing proteins in budding yeast, where Dna2 promotes activation of the ATR homologue Mec1 at RPA-coated single-stranded DNA on the lagging strand during normal DNA replication, whereas the TopBP1 homologue, Dpb11, drives Mec1-Rad53 activation upon replication stress ([@bib4]). We established that cell cycle-- and replication stress--responsive phosphorylation of residues within the AAD core are critical for integrating the ATR-activating potential of ETAA1 with DNA replication status and for its ability to suppress chromosome instability. Recent insights into the structure of Mec1-Ddc2, the yeast homologue of ATR-ATRIP, suggested that acidic patches within AADs may interact with the basic C-terminal PRD (PIKK regulatory domain) domain in Mec1/ATR to promote conformational changes associated with kinase activation ([@bib27]). This offers a possible mechanistic rationale for how negatively charged, phosphorylated residues within the ETAA1 AAD domain might enhance its ATR-activating potential, a notion awaiting validation by biochemical or structural approaches.
While an intra-mitotic function of ETAA1 was recently reported ([@bib2]), the role of ETAA1 in suppressing mitotic chromosome aberrations described here seems unlikely to reflect such an involvement for three reasons. First, the spectrum of mitotic chromosome abnormalities accumulating in *ETAA1Δ* cells exposed to mild replication stress is characteristic of defects arising due to the persistence of unresolved replication intermediates ([@bib18]). Second, the stimulatory phosphorylations within the ETAA1 AAD, which we show are important for its ability to suppress chromosome abnormalities associated with under-replicated DNA, are largely confined to S/G2-phase cells and present at only low levels in mitosis. Third, we did not observe significant defects in Aurora B--associated mitotic phosphorylation events in our *ETAA1Δ* cell lines (Fig. S3, D and E).
Collectively, our data suggest that whereas TopBP1 may be the principal activator of the essential ATR-CHK1 checkpoint pathway, the role of ETAA1 in promoting cell fitness becomes particularly important following impediments to the DNA replication machinery that increase the risk posed by mitotic entry in the presence of unresolved DNA replication intermediates. This could help to explain why ETAA1 deficiency in mice manifests with partially penetrant lethality during embryogenesis and defective clonal expansion of T lymphocytes ([@bib20]), processes entailing rapid cell proliferation where efficient ETAA1-mediated G2/M checkpoint control may be instrumental in suppressing gross chromosomal instability. Targeted inhibition of ETAA1 functionality might thus unmask a selective vulnerability in highly proliferative malignant cells that generally experience deregulated control of cell cycle progression and elevated replicative stress, providing potential opportunities for the continued evolution of promising therapeutic strategies targeting ATR signaling in cancer ([@bib15]).
Materials and methods {#s07}
=====================
Cell culture {#s08}
------------
Human U2OS, HCT116, HeLa, and HEK293FT cell lines obtained from American Type Culture Collection were cultured in DMEM containing 10% FBS and regularly tested negative for mycoplasma infection. HeLa and HCT116 cell lines with targeted KO of ETAA1 (*ETAA1*Δ**), generated by transfecting parental cells with pX459-sgETAA1 construct and selected with puromycin, were described previously ([@bib12]). To generate derivative cell lines inducibly expressing WT or mutant forms (S95A+S111A, ΔRBM1+2 \[Δ603-618+Δ892-926\], and ΔAAD \[Δ56-220\]) of GFP-tagged human ETAA1, HCT116 *ETAA1*Δ** cells were cotransfected with pcDNA4/TO-GFP-ETAA1 and pcDNA6/TR (Invitrogen) plasmids, and positive clones were selected by incubation in medium containing Blasticidin S (Invitrogen) and Zeocin (Invitrogen) for 14 d.
Unless otherwise stated, the following drug concentrations were used: thymidine (2 mM; Sigma-Aldrich), camptothecin (1 µM; Sigma-Aldrich), HU (2 mM; Sigma-Aldrich), ATR inhibitor (AZ20; 1 µM; Sigma-Aldrich), APH (0.4 µM; Sigma-Aldrich), pan-CDK inhibitor (R547; 5 µM; Sigma-Aldrich), CDK1 inhibitor (RO-3306; 7 µM; Millipore), nocodazole (40 ng/ml), and colcemid (0.1 µg/ml).
Plasmids and siRNA {#s09}
------------------
For doxycycline-inducible expression of GFP-ETAA1, cDNA encoding human *ETAA1* was cloned into the destination vector pcDNA4/TO-GFP using Gateway LR Clonase (Invitrogen). For nuclear expression of the ETAA1 AAD, cDNA encoding residues 56--220 of human ETAA1 was inserted between the GFP and 3xNLS (nuclear localization signal) tags in the pAcGFP-Nuc vector (Clontech), as described previously ([@bib12]). To ensure optimal fluorescence detection, ETAA1 AAD(56--220)-3xNLS was subcloned into pEGFP-C1 (Clontech). Introduction of point mutations into pcDNA4/TO-GFP-ETAA1 (S95A+S111A) and pEGFPC1-ETAA1-AAD-3xNLS (FW/AA \[F106A+W107A\], S95D, S111D, S95D+S111D, T79D, and T165D) was performed using a Q5 Site-directed mutagenesis kit (NEB), according to the manufacturer's protocol.
Plasmid DNA transfections were performed with FuGENE 6 (Promega) or GeneJuice (Merck Millipore), according to the manufacturer's instructions. For siRNA transfections (typically 48--72 h), Lipofectamine RNAiMAX (Invitrogen) was used according to the manufacturer's protocol. All siRNAs were used at a final concentration of 50 nM. The following siRNA oligonucleotides were used: non-targeting control: 5′-GGGAUACCUAGACGUUCUA-3′; MCM10, 5′-GACGAUUUCUCGGAACAAA-3′; and POLE3, ON-TARGET Plus POLE3 siRNA pool (LQ-008460-01-0002; Dharmacon; [@bib5]).
Genome-scale CRISPR-Cas9 screen {#s10}
-------------------------------
For preparation of lentivirus, HEK293FT cells were transfected with a human CRISPR KO pooled library (GeCKO v2; 1000000048; Addgene) and the lentiviral packaging plasmids pMDLg/pRRE, pRS-Rev, and pMD2.G using Lipofectamine 3000 according to the manufacturer's protocol. After 3 d, the supernatant was passed through a 0.45-µm syringe filter unit, and the multiplicity of infection was determined. HeLa WT and *ETAA1*Δ** cells were infected with the pooled lentiviral library at a multiplicity of infection of 0.3 and selected with puromycin 24 h after transduction. After 2 d of puromycin selection, a fraction of the cells was harvested ("t0" time point), and the remaining cells were cultured for 21 d ("t21" time point). The experiment was performed in duplicates, and library coverage was kept at a minimum of 200× per replicate throughout the entire screen. Genomic DNA was extracted from 3 × 10^7^ cells as described previously ([@bib9]).
From 130 µg genomic DNA, sgRNA sequences were amplified by PCR using Herculase II Fusion DNA polymerase kit with the following primers: CRISPR F1, 5′-AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3′, and CRISPR R1, 5′-TCTACTATTCTTTCCCCTGCACTGTTGTGGGCGATGTGCGCTCTG-3′. A second PCR was then performed to attach Illumina adaptors and barcodes with primer sequences described previously ([@bib25]).
The amplicons from the second PCR were gel-extracted and subjected to next-generation DNA sequencing. Samples from WT and *ETAA1Δ* cells were multiplexed and subjected to single-end 75-bp-read high-throughput DNA sequencing on an Illumina NextSeq instrument with 30% PhiX spike-in, generating more than 400 million reads. Analysis of sgRNA count data was conducted using MEMcrispR (<https://github.com/grimbough/MEMcrispR>). MEMcrispR uses linear mixed-effect models for each gene to assess the fold change and significance between two conditions. The use of mixed effect allows addition of "fixed" and "random" terms to the model. Days of cell growth (t0 and t21) and the cell lines correspond to "fixed" effects, whereas the sgRNAs and the replicates correspond to "random" effects. The data were normalized by the median distribution of the internal nontargeting sgRNAs. Two models were calculated, one with the treatment effect (t0 versus t21) and a null model without treatment. An ANOVA likelihood ratio test was calculated between the two models to estimate significant differences. The data were corrected for multiple testing using the Benjamini--Hochberg procedure. The β scores were converted to fold changes and used for the directionality of the effect of each gene (positive or negative) as well as for the magnitude of the effect. Fold changes represent sgRNA read counts between treatment and control conditions. MEMcrispR identifies differentially represented genes between t0 versus t21 and combined, and the advanced models use the cell line as an additional fixed parameter. Data from MEMcrispR analysis using the cell line effect are shown, and hits are specific to HeLa *ETAA1Δ* or WT cells (q values for growth are depicted). Gene ontology enrichment analysis was performed on the individual analyses of each cell line. Terms were calculated by ToppGene and reduced by Revigo.
Immunoblotting, immunoprecipitation (IP), and dot blot analysis {#s11}
---------------------------------------------------------------
For immunoblotting experiments, cells were lysed in EBC buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1 mM DTT) containing protease and phosphatase inhibitors and sonicated, and the lysate was clarified by centrifugation. For IP of endogenous ETAA1, cells were lysed in denaturing buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.5% Igepal, 0.5% sodium deoxycholate, and 0.5% SDS) supplemented with protease and phosphatase inhibitors. Cleared lysates were incubated with ETAA1 antibody (1.5--2 µg/sample) coupled to Protein G agarose beads (20398; Thermo Fisher Scientific) overnight at 4°C on a rotary wheel. Beads with bound proteins were washed four times with denaturing buffer and eluted by boiling in 2× Laemmli sample buffer. GFP IPs were performed using similar conditions, except that cell lysates were incubated with GFP-Trap agarose (Chromotek) for 3 h at 4°C with rotation. For dot blots, N-terminally biotin-labeled unmodified or S95-/S111-phosphorylated peptides spanning amino acids 91--115 of human ETAA1 (SSFSSPNDPDGQNDIFWDQNSPLTK and SSFS(pS)PNDPDGQNDIFWDQN(pS)PLTK; synthesized by Peptide 2.0) were spotted onto a 0.22-µm nitrocellulose membrane (Whatman Protran BA83). The membrane was dried for 30 min and processed for immunoblotting.
Antibodies used in this study included mouse monoclonals to actin (MAB1501; Millipore), cyclin A (611268; BD Biosciences), cyclin B (610220; BD Biosciences), GFP (11814460001; Roche), γ-H2AX (2577, Cell Signaling; and 05-636, Millipore), PICH (04-1540; Millipore), and vinculin (V9131; Sigma-Aldrich); rabbit polyclonals to Aurora B pT232 (600-401-677; Rockland), Chk1 pS317 (2344; Cell Signaling), FANCD2 (NB100-182; Novus Biologicals), GFP (sc-8334; Santa Cruz), histone H3-pS10 (06-570; Millipore), MCM10 (12251-1-AP; Proteintech Europe), phospho-(Ser) CDK substrate (pSP; 2324; Cell Signaling), and phospho-ATM/ATR substrate (pSQ; 9607; Cell Signaling). Polyclonal phospho-specific antibodies to S95 and S111 in human ETAA1 were produced in rabbit (GenScript) using internal ETAA1 peptides as antigens. Polyclonal sheep ETAA1 antibody raised against full-length human ETAA1 was described previously ([@bib12]).
Immunofluorescence microscopy {#s12}
-----------------------------
Cells were washed twice in PBS before fixation with 4% formaldehyde for 15 min. Cells were then subjected to a permeabilization step with PBS containing 0.2% Triton X-100 for 5 min and incubated with primary antibodies diluted in PBS containing 2% BSA for 1 h at room temperature. Following incubation with secondary antibodies (Alexa Fluor; Life Technologies) diluted in PBS containing 2% BSA for 1 h at room temperature, coverslips were mounted in Vectashield mounting medium (Vector Laboratories) containing DAPI nuclear stain (Molecular Probes). Images were acquired with a Leica AF6000 wide-field microscope (Leica Microsystems) equipped with HC Plan-Apochromat 63×/1.4 oil immersion objective, using standard settings. Image acquisition and analysis were performed with LAS X software (Leica Microsystems). Raw images were exported as TIFF files, and if adjustments in image contrast and brightness were applied, identical settings were used on all images of a given experiment.
For high-content imaging and analysis of EGFP-ETAA1-AAD-3xNLS expression, cells were fixed, permeabilized, and stained as described above. Nuclear DNA was counterstained with DAPI alongside incubation with secondary antibodies. Cells were then mounted onto glass slides using ProLong Gold Antifade (Invitrogen, Molecular Probes). Images were acquired with an Olympus IX-81 wide-field microscope equipped with an MT20 Illumination system and a digital monochrome Hamamatsu C9100 CCD camera. Olympus UPLSAPO 10×/0.4 NA and 20×/0.75 NA objectives were used. Automated and unbiased image analysis was performed with the ScanR analysis software. Data were exported and processed using Spotfire (Tibco) software.
Analysis of mitotic chromosome abnormalities {#s13}
--------------------------------------------
Asynchronously growing cells were mock-treated or incubated with APH (0.4 µM) or HU (100 µM) for 14 h, and subsequently treated with the CDK1 inhibitor (CDK1i) RO-3306 (7 µM) for 4 h. For analysis of chromatin bridges, lagging chromatin, and UFBs, G2-arrested cells were washed and released into fresh prewarmed medium for 45 min. Cells were fixed with 4% paraformaldehyde containing 0.2% Triton X-100 in PBS for 20 min. For MiDAS analysis, G2-arrested cells were released into fresh medium containing EdU (20 µM) for 30 min. Prometaphase cells were gently shaken off and spotted on poly-L-lysine slides. Cells were fixed with 4% paraformaldehyde, and the Click-iT reaction was performed as described previously ([@bib19]). For metaphase spreads, G2-arrested cells were released into fresh medium containing colcemid (0.1 µg/ml) for 1 h. Mitotic cells were shaken off, treated with hypotonic buffer (35 mM KCl) for 15 min, resuspended in methanol/acetic acid (3:1) solution, and spread onto glass slides.
Flow cytometry {#s14}
--------------
Cells collected by trypsinization were fixed in 70% ethanol, permeabilized in 0.25% Triton X-100 for 10 min, and washed in PBS containing 2% BSA. Cells were incubated with primary antibodies diluted in PBS containing 2% BSA for 1 h at room temperature, followed by a washing step in PBS containing 2% BSA. Cells were then stained for 1 h with secondary antibody (Alexa Fluor; Life Technologies) and washed, and DNA was counterstained in PBS containing 0.1 mg/ml propidium iodide containing RNase (19101; 20 µg/ml; Qiagen) for 30 min at 37°C or, alternatively, with DAPI. Flow cytometry analysis was performed using a FACS Calibur (BD Biosciences) and CellQuest Pro software, or using a LSR Fortessa (BD Biosciences) and FACSDiva software. Exported data were analyzed using FlowJo software (v.10.1).
Clonogenic survival assays {#s15}
--------------------------
For colony formation assays, cells were counted and, where indicated, reverse-transfected with siRNAs for 48 h. Cells were then seeded in dishes in triplicates and incubated for 7--10 d to form visible colonies. To assay camptothecin (CPT) sensitivity, cells were allowed to adhere for a minimum of 16 h following seeding and then treated with 10 nM CPT for 24 h, washed, and released into fresh medium. After 7--10 d of colony outgrowth, plates were washed once in PBS, left to dry, and stained with cell staining solution (0.5% wt/vol Crystal Violet; 25% vol/vol methanol). Colonies were counted using an automated colony counter and its associated software (GelCount; Oxford Optronix). The surviving fraction was calculated as no. colonies/(no. seeded cells × plating efficiency) and normalized to mock control.
In vitro kinase assays {#s16}
----------------------
GST-tagged ETAA1(56--220) was expressed in Rosetta 2 (DE3) pLysS bacteria and purified using glutathione-based affinity purification. For radioactive kinase assays, 100 ng of purified His-Cyclin A/CDK2 (PV3267; Thermo Fisher Scientific) was incubated with 2 µg recombinant GST-ETAA1(56--220) in kinase assay buffer (10 mM MnCl~2~, 1 mM DTT, and 50 mM β-glycerophosphate). Reactions were started by addition of 100 µM ATP spiked with \[γ-^32^P\]ATP (1 µCi; Perkin Elmer), incubated at 30°C with gentle shaking, and terminated by addition of Laemmli sample buffer and boiling for 10 min. Samples were then resolved by SDS-PAGE and stained with Colloidal Blue (Life Technologies). Relative phosphorylation was assayed by autoradiography. For kinase assays comparing cyclin A/CDK2 and ATR/ATRIP kinase activity toward the ETAA1 AAD, 100 ng of purified His--cyclin A/CDK2 or FLAG-ATR/Myc-ATRIP (14--953; Eurofins) was incubated with 2 µg recombinant GST-ETAA1(56--220) and kinase assay buffer. Samples were processed as above and immunoblotted with phospho-specific antibodies to ETAA1 S95 and S111.
Quantification and statistical analysis {#s17}
---------------------------------------
Statistical analysis of data was performed using GraphPad Prism (version 7). Information about statistical tests is provided in the figure legends. No samples were excluded from the analysis, and no statistical method was used to predetermine sample size. For all experiments, samples were not randomized, and the investigators were not blinded to the group allocation during experiments and outcome assessment.
Online supplemental material {#s18}
----------------------------
Fig. S1 shows the impact of ETAA1 deficiency on clonogenic survival and mitotic chromosome abnormalities. Fig. S2 shows the characterization of ETAA1 S95 and S111 phosphorylation. Fig. S3 shows the impact of altered ETAA1 functional status on mitotic chromosome abnormalities and Aurora B-dependent phosphorylation events. Table S1 shows full results and analysis of genome-scale CRISPR-Cas9 KO screen in HeLa WT and *ETAA1Δ* cells.
Supplementary Material
======================
###### Supplemental Materials (PDF)
###### Table S1 (Excel)
We thank Francisco G. Rodriguez Gonzalez for assistance with next-generation sequencing, and Michal Zimmermann and Daniel Durocher for technical advice on CRISPR screens.
This work was supported by the Novo Nordisk Foundation (grant no. NNF14CC0001), Independent Research Fund Denmark (grant no. 7016-00055), European Research Council (grant agreement no. 616236 \[DDRegulation\]), Danish National Research Foundation (grant no. DNRF115), and Danish Cancer Society (grant no. A10769).
The authors declare no competing financial interests.
Author contributions: D. Achuthankutty, R.S. Thakur, P. Haahr, and S. Hoffmann carried out experiments. D. Achuthankutty, R.S. Thakur, P. Haahr, S. Hoffmann, A.P. Drainas, A.H. Bizard, J. Weischenfeldt, I.D. Hickson, and N. Mailand designed experiments and analyzed the data. N. Mailand wrote the manuscript, and all authors edited it.
[^1]: D. Achuthankutty, R.S. Thakur, and P. Haahr contributed equally to this paper.
| {
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1. Introduction {#sec1}
===============
Some 'good-genes' models of sexual selection predict that attractive males or males that are superior competitors in intrasexual selection are of high genetic quality and hence offer indirect genetic benefits to females ([@bib47]). Empirical tests of such models need to control for potentially confounding effects of, for example, differential female investment into embryos and juveniles ([@bib51]). Therefore, the ideal species to test for genetic effects of sexual selection are those with no parental care, external fertilization and large family size, i.e. some frogs ([@bib74]) and some fish species (e.g. Wedekind *et al*. [@bib72], [@bib73]; [@bib62]; [@bib55]). However, it is still unclear whether colours can be linked to genetic quality in such species.
Animals often use colours to advertise their quality and to attract mates. Two main groups have been discussed as candidates in this context ([@bib43]; [@bib27]): carotenoid-based yellow or red colours and melanin-based brown, black and grey colours. The reliability of the signals they produce is assumed to be given by the costs of colour production and/or maintenance ([@bib23]). Various non-exclusive types of costs have been discussed ([@bib28]) as follows: (i) the pigments are costly to acquire, e.g. because they are rare or owing to costly physiological handling (metabolism or transport), (ii) the pigments are required by other physiological processes, e.g. for immune functioning, or (iii) the pigments are toxic or the colours are dangerous, e.g. because they enhance conspicuousness to predators or they signal a social status that may need to be defended.
In vertebrates, carotenoids that are responsible for yellow and red colours have to be obtained through the diet. They may therefore reliably signal foraging quality ([@bib50]) or foraging strategy ([@bib5]). Such colours have often been investigated in sexual selection studies because they are usually conspicuous and based on pigments that also have antioxidant and immunoregulatory properties ([@bib43]; [@bib53]). Hence, by using these pigments, the signaller may reveal its superior health and vigour to potential mates ([@bib45]). In the three-spined stickleback (*Gasterosteus aculeatus*), for example, males use conspicuous red colours to attract females ([@bib46]; [@bib4]). These colours are based on at least three different carotenoids ([@bib71]) that are known to be important in immune function ([@bib39]). Accordingly, red colours in these fish reveal good condition ([@bib21]; [@bib7]) and high resistance against parasite infection ([@bib46]; [@bib6]), and females benefit from preferring red males especially when males are energy constrained during their paternal care ([@bib11]).
In species without paternal care, i.e. where males only provide genes to their offspring, the role of carotenoid-based colours in signalling and mate choice is less clear. In Arctic charr (*Salvelinus alpinus*), for example, first observations found a link between red colours and immune function ([@bib64]), but these colours do not seem to be useful indicators of health and vigour or resistance against infection: [@bib65] even found a positive correlation between red coloration and parasite intensity, and [@bib42] reported a positive link between red coloration and milt characteristics that indicate low dominance in male--male interaction at the spawning place ([@bib63]). In guppies (*Poecilia reticulata*), another fish with no paternal care, variation in carotenoid-based colour traits can be linked to infections and reactions of the immune system ([@bib25]; [@bib38]), and females have been found to prefer males with larger amounts of carotenoids in their orange spots ([@bib24]). However, female responsiveness to male colours varies with carotenoid availability ([@bib26]) and age ([@bib37]), and females are also reported to prefer larger males to brightly coloured ones ([@bib31]).
In contrast to carotenoid-based colours, melanin-based colours can be synthesized by the animal itself (e.g. in specialized organelles called melanosomes; [@bib69]) and the synthesis is under strong genetic control ([@bib40]). Melanin-based colours may have therefore been less obvious candidates for reliable signals of genetic quality, but it has recently been recognized that significant relationships exist between melanin-based pigmentation and overall health and vigour ([@bib58]; [@bib43]). In birds, melanin-based traits are frequently linked to fitness traits (reviewed by [@bib59]; recent examples include [@bib9], [@bib16] and [@bib60]). These links could be due to the antioxidant activity of melanin ([@bib43]) or its involvement in calcium, zinc and iron metabolism and in the maintenance of body structures ([@bib48]; [@bib61]; [@bib44]). In addition, melanin plays important roles in the developmental pathways of various functions and may therefore be an indicator of homeostasis ([@bib3]; [@bib58]). Finally, although melanin-based colours often function as camouflage, some melanin-based signals may make the signaller more conspicuous in some environments ([@bib66]), and only animals in good health and vigour may be able to pay the costs of being conspicuous ([@bib36]).
The brown trout is a salmonid with external fertilization and no paternal care, and with migratory and resident forms. The resident form (*Salmo trutta* resident morph) develops brown, black and red spots on the body sides and the adipose fin. The red spots on the skin and the adipose fin contain large quantities of different carotenoids (xanthophylls and astacene esters) and much more so than any other tissue studied ([@bib67]). The migratory forms of brown trout are usually larger and therefore expected to be more dominant at the spawning place ([@bib34]); they have dark spots but usually no red spots when they arrive at the spawning place. We chose a resident population that has not been supplemented by hatchery fish since at least 10 years. We tested whether males differ in their genetic quality, determined as their offspring viability, and if so, whether male colour traits reveal genetic quality. We also tested for heritabilities of colour traits by comparing 2-year-old juveniles with their fathers.
2. Material and methods {#sec2}
=======================
Brown trout were collected from their natural spawning place in River Enziwigger (near Willisau, Switzerland) in November by electrofishing. Fifteen mature males were narcotized, measured for size and weight and their milt stripped for the *in vitro* fertilization of the eggs of one large female of the same population. The eggs of this one female were about equally distributed to 15 Petri dishes and 20 μl of milt were added each and activated with sand-filtered lake water (8°C), following the methods of [@bib70]. The resulting embryos were reared in four separate Petri dishes per sibship in 50 ml sand-filtered lake water at 4.7°C (mean number of eggs per Petri dish: 22.3±12.0 s.d.; three unfertilized eggs were earlier discarded).
The males were individually marked and kept together in a large tank at approximately 8°C for another breeding experiment 14 days later ([@bib34]). They were then killed with a sharp blow on their head and photographed from both sides under standardized conditions with a digital camera (Nikon E995, 2048×1536 pixels; [figure 1](#fig1){ref-type="fig"}*a*,*b*). Scale samples were taken from below the adipose fin near the lateral line to estimate the age of the fish from yearly grow rings ([@bib57]).
From day 60 after fertilization onwards, non-viable embryos and hatched larvae were recorded and carefully removed from the Petri dishes with a plastic spoon (in regular intervals of approx. 10 days each). Alevins were kept in darkness in running water at 7--8°C until all embryos had hatched and most alevins had nearly used up their yolk sac, i.e. until day 131 after fertilization. We then released all fish plus some additional ones from two other studies ([@bib34]; [@bib15]) into a 600 m long semi-natural, structured streamlet near Willisau that is typical for the streamlets in the region and that is confined by physical barriers. The streamlet has a width of up to 0.5 m and an average depth of approximately 10 cm. We removed all present trout by electrofishing and released our fish by carefully distributing them over the full length of the streamlet during a period when water discharge was low and not obviously affecting the larvae. We caught the fish back 20 months later again by electrofishing, measured their weight and length and took standard photos from both sides (Olympus C77OUZ, 2288×1712 pixels; [figure 1](#fig1){ref-type="fig"}*c*). DNA was extracted from fin clips, and eight microsatellite markers (Mst85, Mst543AE, BS131, T3-13, AETG1, Sssosl417, Ssa171 and Str58) were used for parental assignment following the procedure described by [@bib34]. Paternity was established by exclusion using the [Cervus]{.smallcaps} program ([@bib41]). All inferred parent--offspring pairs were checked for the absence of mismatching loci.
The photos of the parental and juvenile fish were used to count the number of red and brown or black spots. Further colour analyses were done on TIFF files in the open-access software [ImageJ]{.smallcaps} (<http://rsb.info.nih.gov/ij/>). We used a mode that defines hue, saturation and brightness for every pixel of the image (the 'HSB mode'). Hue is given as an angle on a continuous circular scale from 0° to 360°, with 0° for red, 60° for yellow, 120° for green, 180° for cyan, 240° for blue and 300° for magenta. Saturation is the purity of the colour from 0% (grey) to 100% (fully saturated colour), and brightness is the relative lightness or darkness of the colour from 0% (black) to 100% (white). In [ImageJ]{.smallcaps}, all three values are converted to a scale from 0 to 255, and thus every pixel is defined by three values within this range. To obtain the measurements of the red coloration of our fish, we defined *a priori* a range of reddish hue values (225°--255° and 0°--20°). We then counted all the pixels within this range to determine a fish\'s overall redness as the total red area on the body sides and the adipose fin divided by the total area of the body sides and the adipose fin. The average modal hue and saturation of the red coloration, i.e. the most frequent hue and saturation values within the area that was perceived as red, were used as a qualitative measure of the red pigmentation. We also determined the average modal grey value on both body sides, i.e. the most frequent grey value as a measure of overall dark pigmentation. Our methods did not, however, allow us to measure colours in the ultraviolet spectrum. See [@bib68] for a discussion of colour measurements from digital photos. [Figure 1](#fig1){ref-type="fig"} indicates the range of observed male phenotypes. The analogous measures were taken from the standard photos of the 2-year-old offspring.
Statistical analyses were done in JMP v. 6.0 ([www.jmp.com](http://www.jmp.com)) or the open-access software R ([www.r-project.org](http://www.r-project.org); see also [@bib8]). Pearson\'s correlation coefficients *r* are used when graphical inspection of the data suggested that the model assumptions of this statistics were not notably violated, otherwise Spearman\'s rank-order correlation coefficients *r*~s~ are used. Mixed-effects logistic models were used to test for sire effects on offspring viability. To avoid potentially confounding Petri-dish effects, we excluded all Petri dishes that could be considered as outliers, i.e. they were linked to embryo mortalities that were 1.5 times larger than the interquartile of the respective half-sib family (4 out of 60 Petri dishes). The sire effect was then assessed in two tests: first, a log-likelihood ratio test between a reference model (including both Petri dish and sire effect) and a reduced model (with only one of the two parameters); second, the generation of a log-likelihood distribution of the model under the null hypothesis by random permutation (*n*=1000) and a rank comparison of the observed likelihood with this distribution.
3. Results
==========
Fertilization success was close to 100% for all males (only three out of in total 1454 eggs did not seem to contain an embryo). Embryos were clearly visible on day 60 after fertilization, with no apparent mortality until then. Total embryo survival until hatching was 86.9% (median 90%, range 20--100% per Petri dish). Males differed in the viability of their embryos (permutation test, male effect on embryo mortality: *p*=0.002; [table 1](#tbl1){ref-type="table"}).
All red coloration measures seemed to be independent of male size and age (the sires\' overall redness, the number of red spots or red hue and saturation versus age or size measures: −0.39\<*r*~s~\<0.11, *n*=15, always *p*\>0.05). Embryo viability was negatively correlated with the sires\' overall redness and the number of red spots ([figure 2](#fig2){ref-type="fig"}). Embryo survival increased with increasing dark pigmentation of their fathers as revealed by low modal grey values ([figure 3](#fig3){ref-type="fig"}). Increasing dark pigmentation was also linked to male age (*r*~s~=−0.53, *p*=0.04) and male weight (*r*~s~=−0.51, *p*=0.05), but embryo survival could not significantly be predicted by male age or size (0.002\<*r*~s~\<0.18, always *p*\>0.05).
Out of 1261 hatchlings that were released into the wild, 10 juveniles (0.8%) could be caught back 20 months later. They were sired by eight males that did not differ significantly from the other seven males in age or in any of the colour or size measurements (Kruskal--Wallis, always *p*\>0.05). There was also no significant correlation between juveniles\' size or condition factor and the sires\' age, size or colour traits (Spearman\'s rank-order correlations, always *p*\>0.05). The low recapture rate remains unexplained but could be linked to an extraordinary high water that occurred few months before recapture and that caused strong currents at the study site.
Spottiness, i.e. the total number of black, brown and red spots on both body sides, appeared to be heritable ([figure 4](#fig4){ref-type="fig"}*a*), while no evidence for a heritability of other pigmentation traits was found (correlation between sire and offspring, overall dark pigmentation: *r*=−0.28, *p*\>0.05; overall redness: *r*=−0.23, *n*=8, *p*\>0.05; red spots, [figure 4](#fig4){ref-type="fig"}*b*). We found no variation in the hue value of the red colour patches among the juveniles that could be recaptured.
4. Discussion
=============
In fishes, nuptial coloration can function both in inter- and intrasexual selection ([@bib36]). In the case of the brown trout, the relative importance of agonistic interactions between males and courtship to females is still unclear. It is even unclear whether there is any female mate choice in the brown trout ([@bib54]; [@bib34]). However, generalizations about a species\' breeding system are difficult to draw because fishes often respond opportunistically to temporal or spatial variation in sex ratio, population density, habitat structure and other ecological factors ([@bib36]). Our results suggest that if females choose and base their mate choice on colours, they should prefer darker males in order to increase the viability of their embryos. Contrary to what could be found in many other species (see [§1](#sec1){ref-type="sec"} and below), they should not prefer red males. In fact, they should even avoid mating with bright and red males owing to the low viability of the embryos these males sire. However, we could not detect any positive or negative links between sire colour characteristics and the viability of their juvenile offspring. This could be due to either low statistical power or the different nature of embryo and juvenile mortality ([@bib29]).
In salmonids, females are usually restricted in their choice of mates. They often defend spawning territories where they spawn their eggs in several batches, and males compete for access to these females ([@bib17]; [@bib56]). Large males are more successful in such interactions, and females seem to prefer larger males as they deposit more eggs with larger males ([@bib18]), and they are more aggressive towards small males than towards large males ([@bib2]) or may delay spawning when courted by non-preferred males ([@bib14]). Some *Oncorhynchus* species, especially the anadromous and non-anadromous forms of *Oncorhynchus nerka* (sockeye salmon and kokanee, respectively) or *Oncorhynchus mykiss* (steelhead and rainbow trout, respectively), develop intense red colours during the spawning season. These colours are carotenoid based ([@bib10]; [@bib13]), and, as in the three-spined stickleback (see [§1](#sec1){ref-type="sec"}), the red breeding colour of *O. nerka* has been found to positively influence mate choice ([@bib12]; [@bib19]).
Animals often announce their aggressive stage and their fighting ability in colours and other characteristics ([@bib33]; [@bib59]), and fighting ability could be correlated with overall health and vigour and hence with good genes. However, it is not yet clear whether darker skin colours signal dominance or subordination in brown trout. Among juvenile fishes, social subordination has been linked to darker skin colours in rainbow trout (*O. mykiss*; [@bib1]), Atlantic salmon (*Salmo salar*; [@bib49]) and Arctic charr (*S. alpinus*; [@bib30]). Among spawners, however, the situation could be very different ([@bib17]; [@bib14]). A change to dark colours that may signal dominance or attractiveness during the spawning season is often seen in freshwater fishes ([@bib36]).
There are different types of fish chromatophores, including melanophores that contain melanized organelles and that produce dark colours on the skin ([@bib22]; [@bib69]). Some chromatophores show very motile responses, for example, in response to their surroundings (some fishes are dark on a black background and pale on a white background; [@bib69]). The variation in colours that we observed is, however, unlikely to reflect responses of the fish to their light environment. We controlled for such environmental variation by keeping the males for 14 days in one large unstructured tank before they were photographed.
Carotenoid-based colours can have a genetic basis ([@bib13]), but the within-population variation is often due to diet and factors that influence carotenoid uptake and storage ([@bib13]; [@bib28]). Darker skin patterns are usually due to higher melanin content ([@bib30]; [@bib52]). Compared with carotenoid-based colours, melanin-based colours seem to have a stronger genetic basis ([@bib32]; [@bib28]), but environmental influences are nevertheless possible ([@bib30]; [@bib16]). We found a significant father--offspring correlation in the total number of spots, i.e. this aspect of the skin coloration appears to be heritable. An analogous heritability has been observed in rainbow trout ([@bib35]). We found no significant evidence for any heritability of red colour traits, but since our heritability analyses are based on a rather small sample size, such non-significant findings cannot be taken as evidence for a lack of heritability.
In a recent study on brown trout, Forsberg *et al*. ([@bib20]) found '... no apparent reproductive skew that would have indicated a strong 'good genes' effect'. However, they also found that their most successful males had a higher 'microsatellite score', i.e. these males were more different in their average genetics from the rest of the sample than males with lower reproductive success. It remains unclear whether this microsatellite score is linked to variation in genetic load or to the origin of the breeders (the authors used a mixture of hatchery- and wild-born males). In the former case, within-population variation in mating success may be caused by variation in genetic load. Our findings would then suggest that the genetic load is revealed by melanin-based colour traits. However, contrary to other species, a mate preference for red colours would not promote indirect genetic benefits in the case of the brown trout.
The study was done with permission of the *Fischerei- und Jagdverwaltung des Kanton Luzern* and conforms to Swiss laws.
We thank P. Amrein for providing the fish; P. Amrein, B. von Siebenthal, D. Urbach and K. von Wattenwyl for their discussion and assistance in the field; and S. Cotton, A. Roulin and the anonymous reviewers for their constructive comments on the manuscript. The project was supported by the Swiss National Science Foundation.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
######
Sire effects on embryo survival. (Three mixed-effects logistic models to test whether sire effects explain a significant part of the variance in embryo survival (a binary response variable; egg number *n*=1290). Model parameters are random effects. The goodness of fit is given by the logarithm of the approximated likelihood (log *L*) and the Akaike information criterion (AIC). A measure to compare the quality of fit between two models is the difference in AICs between two models. Likelihood ratio tests between the reference model and the other models are used to test which parameter significantly improves the goodness of fit.)
likelihood ratio test against the reference model
--------------------- ------------ ------------------ --------- -------- --------------------------------------------------- --- --------
reference model Petri dish, sire −383.79 773.58
environmental model sire Petri dish −387.15 778.31 6.73 1 0.0095
sire model Petri dish sire −383.79 771.58 \<0.01 1 1
[^1]: Present address: INRA, UMR985, Ecology and Health of Ecosystems, 35000 Rennes, France.
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} |
D. Yang, F. C. Löhrer, V. Körstgens, A. Schreiber, B. Cao, S. Bernstorff, P. Müller‐Buschbaum, In Operando GISAXS and GIWAXS Stability Study of Organic Solar Cells Based on PffBT4T‐2OD:PC~71~BM with and without Solvent Additive. Adv. Sci. 2020, 7, 2001117 10.1002/advs.202001117
Solar energy harvested with organic solar cells (OSCs) is considered as a promising potential alternative renewable and green energy sources due to the low costs, flexibility, solution processing, and large‐scale fabrication.^\[^ [^1^](#advs1879-bib-0001){ref-type="ref"}, [^2^](#advs1879-bib-0002){ref-type="ref"} ^\]^ Since many years, increasing the power conversion efficiency (PCE) of champion devices is in the main research direction of organic photovoltaics (OPVs).^\[^ [^3^](#advs1879-bib-0003){ref-type="ref"}, [^4^](#advs1879-bib-0004){ref-type="ref"} ^\]^ Now the champion PCE for single‐junction OSCs is around 16%,^\[^ [^5^](#advs1879-bib-0005){ref-type="ref"}, [^6^](#advs1879-bib-0006){ref-type="ref"}, [^7^](#advs1879-bib-0007){ref-type="ref"}, [^8^](#advs1879-bib-0008){ref-type="ref"} ^\]^ which is quite close to the value of theoretical predictions.^\[^ [^9^](#advs1879-bib-0009){ref-type="ref"} ^\]^ However, despite high PCE values reached so far, a poor device stability is seriously blocking the real application of OSCs today.^\[^ [^10^](#advs1879-bib-0010){ref-type="ref"}, [^11^](#advs1879-bib-0011){ref-type="ref"} ^\]^ As a consequence, a better understanding of the degradation mechanism of OSCs is attracting more and more attention.^\[^ [^12^](#advs1879-bib-0012){ref-type="ref"}, [^13^](#advs1879-bib-0013){ref-type="ref"}, [^14^](#advs1879-bib-0014){ref-type="ref"} ^\]^ In general, the various degradation processes occurring in OSCs can be roughly divided into two branches, namely chemical and physical degradation processes.^\[^ [^10^](#advs1879-bib-0010){ref-type="ref"}, [^15^](#advs1879-bib-0015){ref-type="ref"} ^\]^ The chemical degradation is mainly attributed to the reaction between water and oxygen with the materials in the device.^\[^ [^16^](#advs1879-bib-0016){ref-type="ref"}, [^17^](#advs1879-bib-0017){ref-type="ref"} ^\]^ Hence, encapsulation techniques and more stable materials are being developed to avoid these problems. In contrast, physical degradation is considered to arise mainly from the morphology deterioration of bulk heterojunction (BHJ) films.^\[^ [^10^](#advs1879-bib-0010){ref-type="ref"} ^\]^ So far, two major morphological degradation pathways have been determined: a demixing‐driven coarsening of inner structures and a mixing‐driven loss of connectivity due to shrinkage of domains.^\[^ [^18^](#advs1879-bib-0018){ref-type="ref"}, [^19^](#advs1879-bib-0019){ref-type="ref"} ^\]^ Recent experiments indicated more complexity in the morphological degradation^\[^ [^20^](#advs1879-bib-0020){ref-type="ref"} ^\]^ and thus the operation‐driven morphology changes in BHJ devices still need to be further explored.
Solvent additives are widely used to modify the morphology of BHJ films to achieve a good interpenetrating network of donor/acceptor (D/A) materials for an improved exciton dissociation and charge transport.^\[^ [^21^](#advs1879-bib-0021){ref-type="ref"}, [^22^](#advs1879-bib-0022){ref-type="ref"}, [^23^](#advs1879-bib-0023){ref-type="ref"}, [^24^](#advs1879-bib-0024){ref-type="ref"}, [^25^](#advs1879-bib-0025){ref-type="ref"}, [^26^](#advs1879-bib-0026){ref-type="ref"} ^\]^ A solvent additive usually has two features, namely that the boiling point is higher than that of the host solvents, and one component of the donor and acceptor blend is selectively dissolved in the solvent additive.^\[^ [^27^](#advs1879-bib-0027){ref-type="ref"}, [^28^](#advs1879-bib-0028){ref-type="ref"} ^\]^ It has been reported in our previous work that the residual solvent additive in the final device leads to an obvious decay of the fill factor (FF) during the operation when the leftover solvent is escaping from the device under vacuum conditions.^\[^ [^18^](#advs1879-bib-0018){ref-type="ref"}, [^20^](#advs1879-bib-0020){ref-type="ref"} ^\]^ Therefore, avoiding the presence of residual solvent in the final devices should be taken into consideration to avoid such degradation pathway.
In earlier work, PTB7‐Th:PC~71~BM films were fabricated at room temperature and no post treatments were involved in the device fabrication process.^\[^ [^20^](#advs1879-bib-0020){ref-type="ref"} ^\]^ In the present work, poly\[(5,6‐difluoro‐2,1,3‐benzothiadiazol‐4,7‐diyl)‐alt‐(3,3′′′‐di(2‐octyldodecyl)‐2,2′,5′,2′′,5′′,2′′′‐quaterthiophen‐5,5′′′‐diyl)\] (PffBT4T‐2OD) and \[6,6\]‐phenyl‐C71‐butyric acid methyl ester (PC~71~BM) are chosen as the donor and acceptor materials, respectively. The PffBT4T‐2OD:PC~71~BM films are spin‐coated at 110 °C, followed by thermal annealing at 85 °C for 5 min under nitrogen atmosphere. The annealing step is selected to remove the solvent additives to a large extent. Moreover, PffBT4T‐2OD is a highly crystalline polymer,^\[^ [^29^](#advs1879-bib-0029){ref-type="ref"}, [^30^](#advs1879-bib-0030){ref-type="ref"}, [^31^](#advs1879-bib-0031){ref-type="ref"} ^\]^ which is beneficial for a high charge carrier mobility.^\[^ [^32^](#advs1879-bib-0032){ref-type="ref"}, [^33^](#advs1879-bib-0033){ref-type="ref"}, [^34^](#advs1879-bib-0034){ref-type="ref"} ^\]^ We fabricate solar cells without solvent additive, with 1,8‐diiodoctane (DIO, boiling point at 332 °C) and with *o*‐chlorobenzaldehyde (CBA, boiling point at 212 °C), respectively. From the comparison of the degradation behavior, we learn about the impact of the type of solvent additive.
In operando grazing‐incidence small/wide angle X‐ray scattering (GISAXS/GIWAXS) characterizations are successfully used to determine the correlation between the evolution of BHJ morphology and the photovoltaic device performance.^\[^ [^18^](#advs1879-bib-0018){ref-type="ref"}, [^19^](#advs1879-bib-0019){ref-type="ref"}, [^35^](#advs1879-bib-0035){ref-type="ref"}, [^36^](#advs1879-bib-0036){ref-type="ref"}, [^37^](#advs1879-bib-0037){ref-type="ref"} ^\]^ In the present study, in operando GISAXS and GIWAXS are sequentially performed on a series of PffBT4T‐2OD:PC~71~BM‐based devices, fabricated without and with solvent additives, to reveal the operation‐induced changes of the nanostructures and crystallinity in the BHJ layers during the aging process. The measurement chamber, the experimental protocol, and other experimental details are described in the Supporting Information. Notably, all devices are made without any special encapsulation and the experiment chamber is evacuated to avoid degradation caused by the presence of oxygen and moisture in the air during the measurements.
The temporal evolution of the normalized photovoltaic parameters of the devices detected during the in situ GIWAXS measurements is displayed in **Figure** [**1**](#advs1879-fig-0001){ref-type="fig"}. Since the device performance has been tracked during the in situ GISAXS and in situ GIWAXS measurements individually, two sets of temporal evolution of the photovoltaic parameters are obtained. Figure S1, Supporting Information, shows the respective temporal evolution of the normalized photovoltaic parameters of the devices recorded during the in situ GISAXS experiments. Small individual differences in the decrease of the parameters are seen in particular in the initial burn‐in phase. At later stages of the device degradation, the overall temporal characteristics agree well for the in operando GISAXS and GIWAXS experiments. The individual differences arise from the well‐known spread in the photovoltaic parameters among different devices of the same type. The average photovoltaic performance of all devices is shown in Table S1, Supporting Information, as measured immediately after fabrication in ambient in the laboratory. The PffBT4T‐2OD:PC~71~BM solar cells fabricated with DIO have higher PCE values than the other devices. Doping with CBA cannot further enhance the device performance, which is attributed to the small difference of the boiling points between 1,2‐dichlorobenzene (DCB, 180 °C) and CBA (212 °C). However, comparing the temporal evolution of the normalized PCE values of all devices (Figure [1](#advs1879-fig-0001){ref-type="fig"}), it can be seen that the devices with CBA are the most stable and the devices with DIO show the strongest decay of the device performance. To make the comparison clearer, we model the PCE decay curves via exponential functions (Figure S2, Supporting Information) and find that 85% of the PCE maintain even after 2000 min in the device with CBA treatment, but only 66% of the PCE preserves in the device with DIO additive, which directly indicates that a solvent additive can affect the device stability. The 10% loss of the short circuit current (*J* ~sc~) is the main degradation factor in the device fabricated without a solvent additive.
{#advs1879-fig-0001}
An efficient charge carrier separation and transport highly depends on the sizes of the nano‐structured domains in the BHJ layer.^\[^ [^38^](#advs1879-bib-0038){ref-type="ref"}, [^39^](#advs1879-bib-0039){ref-type="ref"} ^\]^ As the exciton diffusion length is typically in the range of 10--20 nm for conjugated polymers, the domain sizes determine if excitons can reach the D/A interface to dissociate. Thus, the inner nanostructure of the active layer directly impacts on the device performance.^\[^ [^19^](#advs1879-bib-0019){ref-type="ref"}, [^40^](#advs1879-bib-0040){ref-type="ref"} ^\]^
Changes of the morphology of the BHJ layer are tracked on a nanometer scale during the device degradation process via in operando GISAXS. Figure S3, Supporting Information, displays the 2D GISAXS data of each device at selected times in the operation process. Detailed observations of morphological changes are determined via extracting the scattering signals of PffBT4T‐2OD at the Yoneda^\[^ [^41^](#advs1879-bib-0041){ref-type="ref"} ^\]^ region via horizontal line cuts of the 2D GISAXS data. The horizontal line cuts are shown in Figure S4, Supporting Information. To evaluate the nanostructures of the polymer, these horizontal line cuts are modeled based on the effective interface approximation and the local monodisperse approximation (details are given in the Supporting Information).^\[^ [^42^](#advs1879-bib-0042){ref-type="ref"}, [^43^](#advs1879-bib-0043){ref-type="ref"} ^\]^ In our modeling (red curves in Figure S4, Supporting Information), three characteristic cylinder nanostructures are applied for the samples with and without solvent additive. The average radii of the polymer domains at the respective times are plotted for each device in **Figure** [**2**](#advs1879-fig-0002){ref-type="fig"}. It can be seen that the initial radii of the largest polymer domains in the sample without solvent additive is around 11.5 ± 0.3 nm, which is smaller than that in the devices with DIO (15.5 ± 0.3 nm) and CBA (15.0 ± 0.4 nm) additives. The slight increase in the polymer domain sizes in the devices with solvent additives is due to polymer aggregation in solution. Thus, the addition of DIO and CBA leads to a morphology modification in the active layers, which agrees well with previous reports.^\[^ [^21^](#advs1879-bib-0021){ref-type="ref"}, [^44^](#advs1879-bib-0044){ref-type="ref"}, [^45^](#advs1879-bib-0045){ref-type="ref"} ^\]^ During operation, the average radii of the largest polymer domains increase with time in the device fabricated without solvent additive (Figure [2a](#advs1879-fig-0002){ref-type="fig"}). Contrary no obvious changes of the polymer domain radii are visible in the devices with DIO (Figure [2b](#advs1879-fig-0002){ref-type="fig"}) and CBA (Figure [2c](#advs1879-fig-0002){ref-type="fig"}) additives. It has been claimed that solvent additives, having a high boiling point and selectively dissolving PC~71~BM, provide a better integration of the PC~71~BM molecules into the donor polymer aggregates.^\[^ [^44^](#advs1879-bib-0044){ref-type="ref"}, [^46^](#advs1879-bib-0046){ref-type="ref"} ^\]^ The more pronounced interpenetration between donor and acceptor molecules in the system with an additive gives less opportunity for the migration of molecules, which can explain the more stable BHJ morphology for devices with DIO and CBA additive. We conclude that the continuous decay of *J~sc~* in the device without an additive is induced by the increase of polymer domains with time, probed in the GISAXS measurements, which leads to a decrease of the chances for charge carrier separation. The differences in the decay of *J* ~sc~ between the devices with DIO and CBA solvent additive illustrate the complexity of the morphology impact on individual device parameters. Besides the simple size of the polymer domains also other morphology parameters such as their connectivity and crystallinity are of importance.
{#advs1879-fig-0002}
In earlier work, the loss of residual solvent additives from the BHJ layer was identified to cause a shrinkage of the polymer domains, which reduced the connectivity of domains and caused a decrease of the FF.^\[^ [^18^](#advs1879-bib-0018){ref-type="ref"}, [^20^](#advs1879-bib-0020){ref-type="ref"} ^\]^ In contrast in the present work, the FF is observed to be the most stable photovoltaic parameter in the devices fabricated with solvent additive (DIO or CBA, Figure [1b](#advs1879-fig-0001){ref-type="fig"},[c](#advs1879-fig-0001){ref-type="fig"}), which we attribute to having almost no more additive left inside the active layers to be removed during the device operation. Actually, we detect no solvent additive losses from the vertical line cuts of the 2D GISAXS data (Figure S5, Supporting Information), because the distance and amplitude of the resonant diffuse scattering along the *q~z~* direction are almost stable during the entire measurement time. In contrast, the decrease of the open circuit voltage (*V* ~oc~) dominates the device degradation in the devices with solvent additive (DIO or CBA), as shown in Figure [1](#advs1879-fig-0001){ref-type="fig"}. *V* ~oc~ is impacted by many factors, such as recombination rates, density‐of‐state (DOS) shape, charge carrier, and exciton mobility in organic photovoltaics.^\[^ [^47^](#advs1879-bib-0047){ref-type="ref"}, [^48^](#advs1879-bib-0048){ref-type="ref"} ^\]^ All these impact factors are closely related with the crystalline state of the materials in the BHJ layer.^\[^ [^35^](#advs1879-bib-0035){ref-type="ref"}, [^49^](#advs1879-bib-0049){ref-type="ref"}, [^50^](#advs1879-bib-0050){ref-type="ref"} ^\]^ Thus, in operando GIWAXS measurements are applied to reveal changes of the crystalline parts of the active layers during the in operando measurements.
Figure S6, Supporting Information, shows the collected 2D GIWAXS data of each device at selected times during operation. Cake cuts of the 2D GIWAXS data are performed to extract the respective crystalline information (see Supporting Information). The pronounced (100) Bragg peak located at *q~z~* = 0.27 Å^−1^ is ascribed to the PffBT4T‐2OD crystallites, whereas the polymer (010) Bragg peak (1.72 Å^−1^) and the fullerene peak (1.30 Å^−1^) are both weak in intensity and broad (Figure S7, Supporting Information). Thus, an edge‐on orientation of the polymer crystallites is dominant in these films while the face‐on orientation is strongly suppressed. Changes in crystallinity caused by device operation are seen from a comparison of the initial and the final vertical cake cuts of the device with DIO as example, since the polymer (010) Bragg peak is only observed for this device (Figure S8, Supporting Information). We find that the PC~71~BM peak and the polymer (010) Bragg peak are almost constant during the measurements, whereas the polymer (100) Bragg peak changes. Consequently, we determine changes of the polymer crystalline state during the operation process by tracking the PffBT4T‐2OD (100) Bragg peak. By fitting with Gaussian functions, we obtain the q‐position, the crystalline grain size (estimated via Scherrer equation) and the peak intensity for all devices (a fitting example is shown in Figure S9, Supporting Information, for the device made with CBA). The respective results are summarized in **Figure** [**3**](#advs1879-fig-0003){ref-type="fig"}. It should be noted that the initial stage, marked in Figure [3](#advs1879-fig-0003){ref-type="fig"} with grey dashed lines, is considered as a "burn‐in" phase, since the peak intensity shows a fast increase in the beginning of the operation for all samples.^\[^ [^51^](#advs1879-bib-0051){ref-type="ref"} ^\]^ A "burn‐in" stage was reported in the literature before, while its origin is still debated. Oxygen trapped within the films, the broad polydispersity of the polymers used in the active layers, and organic or inorganic impurities in the polymers (such as palladium catalysts) were suggested as possible origin of the "burn‐in" phenomena.^\[^ [^52^](#advs1879-bib-0052){ref-type="ref"}, [^53^](#advs1879-bib-0053){ref-type="ref"}, [^54^](#advs1879-bib-0054){ref-type="ref"}, [^55^](#advs1879-bib-0055){ref-type="ref"}, [^56^](#advs1879-bib-0056){ref-type="ref"} ^\]^ Since the "burn‐in" is followed by the device degradation, we will restrict to the aging in the present work. In Figure [3](#advs1879-fig-0003){ref-type="fig"}, it can be observed that the q‐position of the (100) Bragg peak stays almost unchanged during the operation process in all devices after the initial "burn‐in" stage. Thus, the polymer crystallites undergo no changes of the crystal lattice during operation after having passed the burn‐in.
{#advs1879-fig-0003}
The crystalline grain sizes of PffBT4T‐2OD remain nearly constant in the devices without an additive and with CBA additive, whereas they decrease in the device with DIO additive. To further determine the influence of the applied bias voltage and the used vacuum conditions on the crystallinity of the BHJ film, a dark in operando measurement is carried out on another fresh DIO device. The bias voltage is periodically applied on the device without illumination and an in‐situ GIWAXS measurement is performed like for the illuminated device. The results of the dark in operando measurements are shown in Figure S10, Supporting Information. The crystalline grain sizes almost stay constant within the error bars after the burn‐in stage in the dark control experiment, which indicates that the morphological changes observed in the illuminated sample require the presence of voltage and illumination. It was reported in a previous study that a codependence exists between crystalline states of the BHJ layer and the *V* ~oc~ value probed during the aging process.^\[^ [^35^](#advs1879-bib-0035){ref-type="ref"} ^\]^ In **Figure** [**4**](#advs1879-fig-0004){ref-type="fig"}, the crystalline grain sizes and *V* ~oc~ values are plotted together for all three devices. We can see that the changes of *V* ~OC~ show a reasonable correlation with the evolution of the crystalline grain sizes after the burn‐in stage. The crystal grain sizes and *V* ~oc~ values in the device without an additive and with CBA additive are almost stable after the burn‐in stage, while the crystalline grain sizes and *V* ~oc~ value show a correlated decrease in the device with DIO additive. Thus, the shrinkage of the crystalline grain sizes causes the observed decrease of the *V* ~oc~ values in the device with DIO additive, as seen before in case of P3HT based devices.^\[^ [^35^](#advs1879-bib-0035){ref-type="ref"} ^\]^ Moreover, the initial *V* ~oc~ value of the device with DIO additive is 0.72 V, which is lower than that of the devices without an additive and with CBA additive (0.76 V, as shown in Table S1, Supporting Information). Based on the GIWAXS observations, we know that the crystallite sizes in the device with DIO additive are slightly larger than those present in the samples with CBA additive and without any additive. It was demonstrated that the donor crystallites with extended *π*‐conjugation could cause a slight increase of the highest occupied molecular orbital (HOMO) level of the donors and therefore result in a decrease of the *V* ~oc~ value.^\[^ [^57^](#advs1879-bib-0057){ref-type="ref"}, [^58^](#advs1879-bib-0058){ref-type="ref"} ^\]^ Furthermore, the lower degree of polymer crystallinity in the device with DIO additive, obtained by comparing the initial Bragg peak intensities of all devices (Figure [3](#advs1879-fig-0003){ref-type="fig"}), might lead to a lower *V* ~OC~ value as well.^\[^ [^59^](#advs1879-bib-0059){ref-type="ref"} ^\]^ In detail, we can also observe that the Bragg peak intensities almost remain constant in the devices without an additive and with DIO additive during operation. However, a slight decrease of Bragg peak intensity is recorded in the device with CBA additive, suggesting a decrease of the polymer crystallinity during the operation time and resulting in a loss of *J* ~sc~. Because high crystallinity facilitates charge transport, the mobility of charge carriers in polymer crystallites is higher than that in amorphous domains.^\[^ [^60^](#advs1879-bib-0060){ref-type="ref"}, [^61^](#advs1879-bib-0061){ref-type="ref"} ^\]^
{#advs1879-fig-0004}
The absolute peak intensities of the device with CBA additive are significantly higher than those of the devices without additive and with DIO additive (Figure [3](#advs1879-fig-0003){ref-type="fig"}). We propose that a competition of polymer molecules between forming interpenetrating D/A networks and growing crystallites is triggered by an additive in the BHJ films. Combining the results of GISAXS and GIWAXS measurements, we know that the polymer morphology experiences a phase separation (shown in **Figure** [**5**a](#advs1879-fig-0005){ref-type="fig"}) during the device aging in the device without solvent additives, which causes a motion of polymer molecules.^\[^ [^62^](#advs1879-bib-0062){ref-type="ref"} ^\]^ Therefore, the polymer domains become larger during the in operando measurements, resulting in the decay of the *J* ~SC~. However, from the GIWAXS measurements we observe that the polymer crystallites are more stable in this device, which means that the growth of the domains is not caused by growing polymer crystals but by the addition of amorphous polymers. In contrast, from the use of a high boiling point additive like DIO, an interpenetrating D/A network is promoted (shown in Figure [5b](#advs1879-fig-0005){ref-type="fig"}), resulting in a better *J* ~sc~ and FF (Table S1, Supporting Information).^\[^ [^44^](#advs1879-bib-0044){ref-type="ref"}, [^63^](#advs1879-bib-0063){ref-type="ref"} ^\]^ A low boiling point solvent additive like CBA is beneficial for polymer molecule aggregation as well, but more favorable for polymer crystallite growth (shown in Figure [5c](#advs1879-fig-0005){ref-type="fig"}).^\[^ [^46^](#advs1879-bib-0046){ref-type="ref"} ^\]^ Moreover, the extended film drying time in case of doping with solvent additives is also beneficial for forming face‐on orientated crystallites.^\[^ [^64^](#advs1879-bib-0064){ref-type="ref"} ^\]^ The out‐of‐plane (010) and (100) Bragg peaks correspond to the face‐on and edge‐on crystallites of the polymer phase.^\[^ [^65^](#advs1879-bib-0065){ref-type="ref"} ^\]^ Figure S7, Supporting Information, shows the initial vertical cake cuts of all devices. There are almost no face‐on crystallites in the BHJ film prepared without solvent additives, whereas, for the devices fabricated with solvent additives, the (010) Bragg peak becomes observable, especially in the sample with DIO additive.
{#advs1879-fig-0005}
In summary, we investigate the impact of solvent additives on the stability of PffBT4T‐2OD:PC~71~BM solar cells with in operando GISAXS and GIWAXS measurements. Due to post‐production annealing we ensure that no substantial residual solvents are released during the device operation. In case of PffBT4T‐2OD:PC~71~BM solar cells, doping with solvent additives helps to obtain more donor and acceptor interpenetrating networks in the BHJ layers to form a stable morphology, which is reflected by a stable FF. However, solvent additives also can cause competition between forming a D/A interpenetrating network and crystallite growth in the donor polymer molecules. DIO enhances the polymer\'s ability to form interpenetrating networks in the BHJ film, but it lowers the polymer crystallinity, and results in a lower *V* ~oc~. Thus, the DIO additive turns out to be most beneficial for the performance of PffBT4T‐2OD:PC~71~BM solar cells. However, the best stability is present in devices with CBA additive, because these devices have a higher polymer crystallinity in the active layer. Thereby, our work gives a further understanding of the degradation mechanism in high‐efficiency OSCs based on solvent additives. The findings can help for choosing a suitable solvent additive, which can well balance the interpenetrating network character and the crystallinity in a BHJ film for next generation solar cells.
Experimental Section {#advs1879-sec-0020}
====================
{#advs1879-sec-0030}
### {#advs1879-sec-0040}
#### Materials {#advs1879-sec-0050}
Poly\[(5,6‐difluoro‐2,1,3‐benzothiadiazol‐4,7‐diyl)‐alt‐(3,3′′′‐di(2‐octyldodecyl)‐2,2′,5′,2′′,5′′,2′′′‐quaterthiophen‐5,5′′′‐diyl)\] (PffBT4T‐2OD) was purchased from California organic semiconductors Inc., the molecular weight (*M* ~w~) was around 131 kDa, and \[6,6\]‐phenyl‐C71‐butyric acid methyl ester (PC~71~BM) was purchased from 1‐materials Inc. All solvents, which were used in this work, including the solvent additives DIO and CBA were obtained from Sigma‐Aldrich and all chemicals were used as received without any further purification.
#### Device Fabrication {#advs1879-sec-0060}
For device fabrication, indium‐doped tin oxide (ITO) coated glass substrates (8--12 Ω per sq) were sequentially cleaned via ultra‐sonication in Alconox, ethanol, acetone, and isopropyl alcohol, each for 10 min. Afterward, the substrates were dried with N~2~ and treated in an O~2~‐plasma cleaning step (Plasma‐System‐Nano, Diener Electronic GmbH). The ZnO precursor solution, which was prepared following an earlier report,^\[^ [^66^](#advs1879-bib-0066){ref-type="ref"} ^\]^ was spin‐cast at 3000 rpm on top of the cleaned ITO substrates, and subsequently annealed at 150 °C for 30 min in air, resulting in a transparent ZnO thin film with a thickness of around 30 nm. A mixture of PffBT4T‐2OD: PC~71~BM (1:1.2, 20 mg mL^−1^ in total) was dissolved in a combination of 1,2‐dichlorobenzene (DCB) and chlorobenzene (CB) (1:1, 3% DIO or 5% CBA) while stirring and heating (100 °C) overnight. Prior to spin‐coating a BHJ layer, the ITO substrates with ZnO film were heated for 1 min at 120 °C, then the hot PffBT4T‐2OD: PC~71~BM solution was dropped onto the ZnO layer, the time interval between placing a substrate and starting spin‐coating was around 10 s. Afterward, the samples were annealed under nitrogen atmosphere at 85 °C for 5 min. Finally, a thin layer of MoO~3~ (about 10 nm) and a layer of Al (60 nm) were deposited successively via thermal evaporation under vacuum conditions. The active area of each pixel of the devices was about 12 mm^2^.
#### Film Characterization {#advs1879-sec-0070}
In operando GISAXS and GIWAXS measurements were performed at the Austrian SAXS beamline of the Elettra Sincrotrone Trieste, at an X‐ray energy of 8 keV. For the GISAXS measurements, the incidence angle was set to 0.35° and the sample‐detector distance to 1365.5 mm. The DPDAK software^\[^ [^67^](#advs1879-bib-0067){ref-type="ref"} ^\]^ was applied to extract the polymer scattering signals. For the GIWAXS measurements, the incidence angle was around 0.3° with a sample‐detector distance changed to 296 mm. The obtained 2D scattering patterns were evaluated using the GIXSGUI software.^\[^ [^68^](#advs1879-bib-0068){ref-type="ref"} ^\]^
Conflict of Interest {#advs1879-sec-0090}
====================
The authors declare no conflict of interest.
Supporting information
======================
######
Supporting Information
######
Click here for additional data file.
The authors acknowledge funding from Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via International Research Training Group 2022 Alberta/Technical University of Munich International Graduate School for Environmentally Responsible Functional Materials (ATUMS) and under Germany\'s Excellence Strategy---EXC 2089/1---390776260 (e‐conversion) and as well as from TUM.solar in the context of the Bavarian Collaborative Research Project "Solar Technologies Go Hybrid" (SolTech) and the Center for NanoScience (CeNS) and the Future Energy Systems of the University of Alberta. D.Y. acknowledges the China Scholarship Council (CSC) funding and the support within TUM IGSSE. The authors thank Dr. Johannes Schlipf and Bernhard Kalis for building the in operando chamber. The authors acknowledge the CERIC−ERIC Consortium for the access to the SAXS beamline.
| {
"pile_set_name": "PubMed Central"
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1.. Introduction {#S0001}
================
Angiogenesis is the process of new blood vessel formation \[[@CIT0001]\]. It occurs throughout life in both health and disease and relies on migration, proliferation, and differentiation of endothelial cells (ECs), which line the inside wall of blood vessels. In established vessels, ECs have a cobblestone-like appearance and are often referred to as phalanx cells \[[@CIT0002]\]. However, in the presence of pro-angiogenic signals such as growth factors and hypoxia, ECs can rapidly switch to an angiogenic state and become motile and invasive \[[@CIT0003],[@CIT0004]\]. Upon detection of pro-angiogenic stimuli, such as VEGF (vascular endothelial growth factor), ECs lose their adherence junctions, and matrix metalloproteases degrade the basement membrane, thereby creating a scaffold for EC migration \[[@CIT0003],[@CIT0004]\]. In response to such stimuli, a vessel sprout emerges in which individual ECs adopt distinct and functionally specialized phenotypes \[[@CIT0003]\].
Vessel sprouting is initiated by the differentiation of ECs into specialized tip and stalk cells. Tip cells extend long filopodia and guide the new sprout toward an angiogenic stimulus but proliferate rarely, whereas stalk cells have fewer filopodia, proliferate to elongate the branch and form a vascular lumen \[[@CIT0003],[@CIT0004]\]. When the new branch is formed and perfused, ECs regain their quiescent phalanx cell phenotype. Recruitment of pericytes and vascular smooth muscle cells, together referred to as mural cells, provides stability, maintains vessel integrity and regulates perfusion \[[@CIT0003],[@CIT0004]\]. Furthermore, extracellular matrix proteins, laid down by both phalanx cells and pericytes, establish a basement membrane at the basal side of the endothelium \[[@CIT0003]--[@CIT0005]\].
Blood vessels not only deliver oxygen and nutrients to the body's tissues but also support diseases such as cancer \[[@CIT0006]\]. Rapidly growing tumors are in continuous demand for oxygen and nutrients. Therefore, they have an excessive production of angiogenic stimuli, which creates an imbalance in pro- versus antiangiogenic signaling. This results in an abnormal, leaky, and hypoperfused vascular network, characterized by hypoxia, acidosis, and high interstitial fluid pressure. This hostile tumor microenvironment stimulates the production of pro-angiogenic factors even more and fuels an endless self-reinforcing loop of nonproductive angiogenesis \[[@CIT0007],[@CIT0008]\]. Paradoxically, however, this nonproductive angiogenesis leads to the formation of less functional vessels. This results in hypoxic sites within the tumor, deprived of nutrients and growth factors, creating a hostile microenvironment, from where cancer cells attempt to escape, thereby favoring cancer cell invasion and dissemination, further aided by the leaky endothelium, through which cancer cells can escape \[[@CIT0007],[@CIT0008]\]. Moreover, the abnormal tumor vasculature impairs perfusion and thus drug delivery and distribution. Together, these features stimulate overall malignancy \[[@CIT0007],[@CIT0009],[@CIT0010]\].
Therapeutic approaches for inhibition of angiogenesis have been developed to treat cancer and have led to the approval of several antiangiogenic drugs. So far, innumerable patients have benefited from these therapies but limited efficacy and resistance pose clinical challenges \[[@CIT0008],[@CIT0011]--[@CIT0013]\]. A novel paradigm is to heal the abnormal tumor vasculature in a process called tumor vessel normalization \[[@CIT0010]\]. Tumor vascular normalization (TVN) strategies reduce metastasis and improve the response to conventional anticancer therapies \[[@CIT0010],[@CIT0014]\].
2.. Role of abnormal vasculature in tumor progression and therapeutic strategies {#S0002}
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2.1.. Tumor vessels are highly abnormal {#S0002-S2001}
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Tumor blood vessels are structurally and functionally highly abnormal. They are tortuous, leaky, irregular and form a chaotic network. Moreover, they are heterogeneous in size and shape, ranging from capillaries to big, thin-walled vessels \[[@CIT0007],[@CIT0009],[@CIT0010]\] ([Figure 1](#F0001){ref-type="fig"}). ECs lining tumor vessels have an irregular shape and are disorganized. They establish weak junctions, which results in a loose association, promoting EC trans-migration from their resident site. In certain regions, ECs are stacked upon each other and extend multiple protrusions within the lumen, thus obstructing the blood flow, while at other sites, ECs move away from their position, leaving gaps behind. In addition, mural cells show abnormal structural features. They have an abnormal shape and are often loosely associated with ECs or absent. Finally, the basement membrane of tumor vessels is also abnormal. In some tumors, it is unusually tick, while very thin, discontinuous, or absent in others \[[@CIT0007],[@CIT0009],[@CIT0010]\]. Figure 1.Tumor vessels are structurally and functionally abnormal.Endothelial cells lining tumor vessels demonstrate aberrations in shape, they are hyperproliferative and hypermigrative and are often separated by wide and irregular inter-endothelial junctions. In addition, tumor vessels are covered by fewer pericytes, which are often detached from endothelial cells. These structural abnormalities lead to hypoperfusion and hypoxia, which stimulate cancer cells to escape and metastasize in distant organs. Moreover, the accompanying functional aberrations limit delivery and distribution of chemotherapeutics to and into the tumors. BM: basement membrane. Adapted from \[[@CIT0009]\].
Abnormalities in tumor vessel morphology impair perfusion. Blood flow often changes direction, flowing rapidly in certain vessels and being stagnant in others. In addition, the structural abnormalities lead to increased vessel permeability. Hence, intravascular fluids and plasma proteins can easily extravasate and increase interstitial fluid pressure, further impeding blood flow. Moreover, these leaky vessels facilitate intravasation of cancer cells and metastasis \[[@CIT0008]--[@CIT0010]\]. The combination of poor perfusion and increased interstitial fluid pressure creates hypoxic and acidic sites. In such conditions, invasive cancer cells are selected. Overall, the aforementioned tumor vessel abnormalities increase metastasis and impair the delivery and efficacy of systemically administered drugs, therefore reducing the efficacy of anticancer therapies \[[@CIT0007]--[@CIT0010]\].
2.2.. Vascular normalization as anticancer strategy: an emerging paradigm {#S0002-S2002}
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Anti-VEGF therapies were originally designed to restore the balance between pro- and antiangiogenic molecules and the vascular architecture of tumors by pruning immature vessels \[[@CIT0015]--[@CIT0017]\]. Early preclinical studies were promising and demonstrated a significant tumor growth delay and reduced metastasis \[[@CIT0016],[@CIT0018]\]. Unfortunately, the effects of such anti-VEGF agents in cancer patients during clinical trials have not fulfilled the expected hopes as the survival improvement by antiangiogenic therapy is rather modest \[[@CIT0019]\]. Monotherapy with anti-VEGF monoclonal antibody often failed to attain substantial response rates or survival benefits \[[@CIT0020]--[@CIT0022]\]. These data suggest that anti-VEGF therapy alone cannot efficiently induce sustained tumor shrinkage or complete tumor eradication in most cancer patients.
However, anti-VEGF therapy in combination with systemic chemotherapy has often shown improvements in progression-free survival when compared with chemotherapy \[[@CIT0021],[@CIT0023]--[@CIT0028]\]. This has been interpreted to imply that anti-VEGF therapy may enhance the efficacy of systemic chemotherapy. These findings are counterintuitive since anti-VEGF therapies were designed to promote vascular pruning; yet, the efficacy of chemotherapy relies on adequate tumor blood supply to ensure drug delivery. Thus, tumor vessel pruning by anti-VEGF treatment should theoretically decrease rather than enhance the chemotherapy efficacy \[[@CIT0010]\]. The hypothesis of vascular normalization may resolve this paradox \[[@CIT0029]\]. This hypothesis posits that rather than destroying vessels, antiangiogenic therapy might restore the normal structure and function of tumor vessels by 'healing' abnormal disorganized and dysfunctional tumor vessels. These changes would improve tumor oxygenation, thereby reducing metastasis and improving therapy responses, in part through more efficient drug delivery and lessened tumor hypoxia (which can improve the efficacy of several chemo- or radiotherapeutics, given that they rely on the conversion of oxygen to its radicals, and of immunotherapy \[see below\]). Thus, anticancer therapies given upon normalization might achieve greater efficacy \[[@CIT0029]\].
Initial evidence for vascular normalization stems from preclinical studies with anti-VEGF, which show that blockade of VEGF signaling remodels the abnormal tumor vasculature into a more normal vasculature \[[@CIT0014]\]. Of relevance, the kinetics of vascular normalization determined the overall outcome of combined antiangiogenic and conventional therapy. Preclinical and clinical tumor studies with anti-VEGF agents identified a 'normalization window,' typically occurring within a few days after therapy onset followed by a closure coinciding with the loss of normalization features \[[@CIT0010]\]. The transient nature of the normalization of tumor vessels may relate either to excessively high or continued dosing of antiangiogenic therapy or to the development of resistance by activation of alternative pro-angiogenic pathways \[[@CIT0010]\]. High doses of antiangiogenic drugs prune the immature tumor vessels, causing a rapid reduction in blood perfusion and consequent increase in hypoxia, which is a strong stimulus for cancer cells to invade, intravasate, and metastasize \[[@CIT0008]\]. On the contrary, properly timed low dose of anti-VEGF therapy promotes the formation of less tortuous vessels, normal basement membrane, and greater vessel coverage by pericytes. Indeed, low doses of anti-VEGFR2 antibody normalized the breast cancer vasculature and improved tissue perfusion, reprogramming the tumor microenvironment from immunosuppression toward enhanced cancer vaccine therapy \[[@CIT0030]\].
Other mechanisms might contribute to the vascular normalization effect of anti-VEGF agents. For instance, upon anti-VEGFR2 inhibition, mural cells upregulate angiopoietin-1 (ANG-1), promoting vessel maturation, stability, and integrity \[[@CIT0031]\]. The ANG/TIE-2 system represents another target for antiangiogenic therapies to induce tumor vessel normalization \[[@CIT0001],[@CIT0008],[@CIT0010]\]. In contrast to ANG-1, ANG-2 is primarily synthesized by ECs and antagonizes the effects of ANG-1. ANG-2 destabilizes blood vessels and promotes vascular permeability. Hence, preclinical reports demonstrated that blockade of ANG-2 induces vessel normalization by recruitment of pericytes and tightening of endothelial junctions \[[@CIT0032],[@CIT0033]\]. Moreover, combined targeting of VEGF and ANG-2 showed a greater degree of vessel normalization \[[@CIT0034]\].
2.3.. Emerging successful tumor vessel normalization strategies {#S0002-S2003}
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In addition to targeting the VEGF/VEGFR and ANG/TIE-2 axes, a number of strategies that alter other molecules in stromal and cancer cells can induce vascular normalization. The oxygen sensor prolyl hydroxylase domain protein 2 (PHD2) plays an important role in mediating abnormalities in tumor vessels. In the tumor microenvironment, lack of oxygen reduces the enzymatic activity of PHD2 on one hand, and possibly as a feedback, also upregulates the expression levels of this enzyme. Nonetheless, hypoxia results in the activation of hypoxia-inducible transcription factors, key mediators of transcriptional response to hypoxia \[[@CIT0035]\]. In turn, VEGF is upregulated, thereby promoting abnormal angiogenesis \[[@CIT0036]\]. Studies in genetically modified mice have shown that global (stromal) or endothelial-specific PHD2 haplodeficiency does not impair physiological angiogenesis but induces sustained normalization of tumor vessels \[[@CIT0002],[@CIT0037]\]. There are currently no pharmacological agents available to inhibit PHD2 specifically in ECs. In addition, global PHD2 haplodeficiency also induces tumor vessel normalization in part by decreasing the activation and contraction of cancer-associated fibroblasts (CAFs) \[[@CIT0038]\]. Mechanistically, PHD2 haplodeficient cancer cells inhibit CAF-induced matrix deposition that cancer cells use as a migration scaffold for dissemination, thereby reducing metastasis \[[@CIT0038]\]. This suggests that administration of a pharmacological blocker inhibiting PHD2 in both stromal and cancer cell compartments might offer therapeutic benefit by reducing metastatic disease.
Specific targeting of the transmembrane glycoprotein L1 on tumor vasculature also promotes tumor vessel normalization, resulting in reduced tumor growth and metastasis \[[@CIT0039]\]. The regulator of G-protein signaling 5 (RGS5) is a marker of mural cells that has been identified as a key mediator of the abnormal tumor vasculature \[[@CIT0040]\]. Tumor vessels of RGS5-deficient mice were characterized by reduced vessel permeability and leakage, increased structural homogeneity, improved oxygenation, and coverage by more mature pericytes. This enhanced the influx of immune effector cells into the tumor and prolonged the survival of the tumor-bearing mice \[[@CIT0040]\], offering alternative therapeutic opportunities for immunotherapy and anticancer therapy \[[@CIT0040]\]. The modulation of tumor-associated macrophages (TAMs) may be another way to control tumor vessel abnormalities. For instance, histidine-rich glycoprotein suppressed the expression of placental growth factor and induced the polarization of TAMs, promoting TVN and enhancement of immunity \[[@CIT0041]\].
The anti-malaric drug chloroquine also yielded promising results in cancer patients \[[@CIT0042]\]. While its anticancer cell activity relies on the blockade of cancer cell autophagy at a high dose, a low dose of chloroquine targets ECs via an autophagy-independent increase in Notch signaling, which promotes tumor EC quiescence and thereby tumor vessel normalization \[[@CIT0043],[@CIT0044]\]. A number of FDA-approved drugs also target molecules that indirectly contribute to the tumor vessel abnormalities, namely by targeting cancer cells that subsequently affect tumor vessels. Examples include the human epidermal growth factor receptor 2, the phosphoinositide-3-kinase/AKT serine/threonine kinase 1/mammalian target of rapamycin (mTOR) axis, Ras, and the EGF receptor (reviewed in Ref. \[[@CIT0010]\]).
Taken together, the aforementioned studies suggest that targeting stromal, cancer, and other cells in the tumor milieu may improve the efficacy of tumor vessel normalization. The challenge for the future is to explore whether these approaches also promote sustained tumor vessel normalization in patients in clinical settings, as observed in preclinical studies.
2.4.. Vessel normalization as strategy to improve cancer immunotherapy {#S0002-S2004}
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In addition to the examples mentioned above, a growing body of evidence suggests that tumor vessel normalization may also enhance the efficacy of immunotherapy \[[@CIT0030],[@CIT0040],[@CIT0041],[@CIT0045]\]. During tumor progression, cancer cells co-opt immune checkpoint pathways to promote immune evasion, particularly by cytotoxic T cells \[[@CIT0046]\]. Different strategies to interfere with ligand--receptor interactions involved in immune checkpoint pathways have been developed and have entered clinical practice \[[@CIT0047]\]. However, several hurdles need to be overcome to further improve such treatments.
Indeed, the efficacy of anticancer immunotherapy by blocking immune checkpoints is hampered by hypoxia and poor infiltration of T cells inside the tumor, as a result of the poor perfusion in the disorganized tumor vessels \[[@CIT0048]\]. Abnormal tumor vessels also limit the adhesion and extravasation of leukocytes and impair their infiltration inside the tumor core \[[@CIT0007],[@CIT0009]\]. Hypoxia increases the immunosuppressive nature of the stromal tumor environment, by impairing T-cell effector functions (T-cell receptor signaling, proliferation, and cytokine production by T cells) \[[@CIT0048],[@CIT0049]\]. In contrast, hyperoxia increases cytotoxic T-cell performance, which correlates with better clinical responses to blockade of the immune checkpoint molecule programed death 1 (PD-1) \[[@CIT0050]\]. In addition, nutrient deprivation (such as glucose) impedes T-cell proliferation and activation into CD8^+^ effector cells \[[@CIT0051]\].
Hence, tumor vessel normalization increases tumor perfusion, and thereby oxygen and nutrient supply, and thus can be expected to improve the overall anticancer immunotherapy response. This hypothesis is supported by findings that anti-VEGF therapy promotes antitumor immunity, in part by improving vessel function and reducing hypoxia \[[@CIT0030]\]. Restoring vessel integrity improves tumor perfusion and decreases interstitial fluid pressure, processes that would be expected to improve the influx of immune cells into the tumor \[[@CIT0040],[@CIT0052]\]. Moreover, the fewer gaps between ECs in the normalized vasculature might create a more continuous vascular surface to support leucocyte rolling and diapedesis \[[@CIT0053]\]. However, the exact mechanism by which tumor vessel normalization strategies increase the influx of immune cells into the tumor, while reducing cancer cell escape, remains to be defined. The increased antitumor immunity responses observed upon anti-VEGF therapy might be also related to the inhibition of the immunosuppressive functions exerted by VEGF on effector T cells \[[@CIT0054]\]. Thus, blocking VEGF signaling enhances effector T-cell function by increasing their activation and delivery to the tumor (via tumor vessel normalization) on one hand and by inhibiting the VEGF-induced upregulation of inhibitory immune checkpoints, on the other hand \[[@CIT0030],[@CIT0055]\].
2.5.. Vessel normalization: a clinical perspective {#S0002-S2005}
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Unfortunately, the high expectations of antiangiogenic therapies could not be fulfilled, as the high therapy efficacy observed in preclinical models could not be fully reproduced in clinical trials \[[@CIT0056]\]. Therapy effects in cancer patients are mostly short lived due to intrinsic refractoriness or development of acquired resistance upon antiangiogenic therapy. Several modes of resistance have been identified preclinically, but they are less well characterized in the clinical setting \[[@CIT0057],[@CIT0058]\].
Limitations to better clinically characterize the mode of action of antiangiogenic therapies are related to multiple reasons. Amongst those, one reason may relate to the fact that most antiangiogenic drugs are used for the treatment of advanced stage disease (mostly metastatic), where tissue sampling is only possible from accessible tumors (for instance, rectal cancer via rectoscopy); and therefore, translational investigations are often limited to blood sampling or imaging studies \[[@CIT0019]\]. Nevertheless, in a subset of patients, tumor vessel normalization has been observed upon administration of antiangiogenic therapy \[[@CIT0059]\] ([Table 1](#T0001){ref-type="table"}). Table 1.Clinical studies investigating vascular normalization in humans.Tumor typeTherapeutic strategyMeasurement of vascular normalizationClinical finding and translational observationRectal cancer \[[@CIT0060]\]Bev in combination with radio-chemotherapyTissue biopsies, tumor imaging by FDG-PET and functional dynamic CTTVN upon bev monotherapy (radiological and histological evaluation), local control rate and DFS in phase II studyGlioblastoma multiforme \[[@CIT0061]\]CediranibTumor perfusion by DCE-MRIPerfusion response (increase) correlated with prolonged OSNSCLC \[[@CIT0062]\]Bev in combination with doublet chemotherapyTumor perfusion by perfusion CT (MTT)Prolongation of MTT correlated with prolonged OSNSCLC \[[@CIT0063]\]Bev in combination with doublet chemotherapyTumor uptake of radiolabeled chemotherapyReduced chemo uptake after bev applicationBreast cancer \[[@CIT0064]\]Bev with combinational chemotherapyTissue biopsies after bev monotherapy (single dose)Predictive pre-therapy MVDBreast cancer \[[@CIT0065]\]Sunitinib with chemotherapyTissue biopsies, DCE-MRIIncreased VNI and perfusion in the combinational treatment armColorectal cancer \[[@CIT0066]\]Bev in combination with 5-FU^18^F-5-FU PET/CT scanningReduced 5-FU trace uptake short term after bev application[^1]
The first studies investigating the influence of the VEGF neutralizing antibody bevacizumab on tumor vessel normalization were performed in rectal cancer patients. Bevacizumab monotherapy decreased the interstitial fluid pressure, while tumor blood vessels were covered with more pericytes as compared to pre-therapy investigations \[[@CIT0067]\]. Long-term follow-up of the subsequent phase II study showed high local tumor control rate and promising disease-free and overall survival upon neoadjuvant bevacizumab treatment in combination with standard chemoradiotherapy \[[@CIT0060]\], suggesting that vessel normalization features might induce a better therapeutic outcome. In support of this hypothesis, treatment with cediranib (a VEGFR tyrosine kinase inhibitor) resulted in increased tumor perfusion and prolonged survival in a subgroup of patients with recurrent glioblastoma multiforme \[[@CIT0061]\]. Accordingly, improved tumor vascularization after bevacizumab combination therapy with carboplatin and nab-paclitaxel was associated with longer survival in patients with advanced non-small cell lung cancer \[[@CIT0062]\]. Overall, the abovementioned studies may imply that antiangiogenic therapy regimens that promote tumor vessel normalization offer benefit to cancer patients. Clearly, the number of clinical studies showing beneficial outcome of tumor vessel normalization is still limited, and additional studies are needed to corroborate these initial clinical findings. In addition, it is of utmost importance to identify predictive biomarkers for vascular normalization and therapy stratification in future clinical trials \[[@CIT0019]\].
A recent clinical trial, including a small number of patients with metastatic renal cell carcinoma treated with bevacizumab in combination with an anti-PD-ligand 1 antibody showed that the combination treatment increases the intra-tumoral infiltration of CD8^+^ T cells, thus enhancing the anticancer immune-specific responses \[[@CIT0068]\]. The ongoing early phase I/II clinical trials will further reveal whether the anti-VEGF therapies in combination with checkpoint inhibitors increase the anticancer effects \[[@CIT0069]\].
Mechanistically, it has been hypothesized (and supported by preclinical evidence) that upon tumor vessel normalization, the improved vessel functionality leads to enhanced tumor delivery of chemotherapeutics \[[@CIT0070]\]. However, some clinical trials investigating the effect of antiangiogenic drugs on tumor delivery showed that antiangiogenic drugs reduced (rather than increased) the delivery of chemotherapy or biological drugs \[[@CIT0071],[@CIT0066]\]. A reduction in tumor perfusion and vessel permeability has been proposed to explain the decreased tumor uptake of chemotherapeutics upon bevacizumab therapy \[[@CIT0072]\]. Further, drug penetration in tumors was enhanced only when the chemotherapeutic agent was administered within a defined interval after anti-VEGF therapy \[[@CIT0073],[@CIT0074]\]. Together, while tumor vessel normalization is an attractive strategy to impede metastasis and improve chemo-, radio-, and immunotherapy in preclinical models, and initial signs have been recognized in clinical settings, other studies also highlight the importance of testing different treatment regimen schedules and dosing of bevacizumab in order to examine their effect on perfusion and permeability and to identify the normalization window in which the antiangiogenic agent exerts beneficial effects. Nonetheless, it will remain challenging to clinically translate the preclinical efficacy of VEGF-signaling blockers to induce long-lasting tumor vessel normalization required for optimal therapeutic benefit. Hence, we will need additional agents with a completely different mechanism, which are capable of inducing persistent tumor vessel normalization (as for instance documented in mice for chloroquine, PHD2 gene haplodeficiency, etc. -- see above).
2.6.. Tumor vessel normalization by targeting EC metabolism {#S0002-S2006}
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An entirely different and new antiangiogenic approach to promote tumor vessel normalization is to target EC metabolism. This strategy is based on the postulate that EC metabolism is the engine onto which pro-angiogenic signals like VEGF and others converge and that 'cooling down the overheated metabolism' of ECs can paralyze angiogenic ECs and reduce pathological angiogenesis, regardless of how many angiogenic signals are still present upon neutralization of VEGF \[[@CIT0003],[@CIT0075],[@CIT0076]\]. Since ECs are highly glycolytic \[[@CIT0077]\], targeting glycolysis might provide an alternative new therapeutic opportunity for reducing pathological angiogenesis. Indeed, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a key regulator of glycolysis in ECs. Inhibition of PFKFB3, either by genetic loss in ECs or by global pharmacological blockade with the small molecule 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), reduces vessel sprouting by inhibiting EC proliferation and migration, not only in physiological but also in pathological conditions of inflammation and tissue injury \[[@CIT0077],[@CIT0078]\]. Notably, however, PFKFB3 silencing or blockade reduces EC glycolysis only partially by no more than 35%, but still sufficient to normalize the hyper-glycolysis of sprouting ECs to maintenance levels found in quiescent ECs \[[@CIT0077],[@CIT0078]\]. Thus, even a modest decrease in glycolysis was sufficient to impair vessel sprouting by promoting quiescence \[[@CIT0078]\]. In addition, the effect of 3PO *in vivo* was transient because of its short half-life (30 min) and rapid clearance. As a result of the partial, transient reduction (not elimination) of glycolysis, and the fact that ECs are more glycolysis addicted than other cell types, the effect of 3PO was well tolerated \[[@CIT0078],[@CIT0079]\].
This type of anti-glycolytic approach differs substantially from previous anti-glycolytic anticancer therapies, which were not always successful \[[@CIT0080],[@CIT0081]\], mainly because they attempted to eliminate glycolysis completely and permanently, which causes adverse effects. Indeed, the non-metabolizable glucose analog 2-deoxy-[d]{.smallcaps}-glucose (2DG), which reduces glycolysis by 80%, causes ATP depletion and EC death \[[@CIT0078]\]. Since high 2DG doses are needed to compete with the high levels of glucose in the blood, its effects are toxic.
Deletion of both PFKFB3 alleles in ECs decreases angiogenesis and perfusion in tumors \[[@CIT0082]\]. A more recent study showed however that endothelial haplodeficiency of PFKFB3 does not inhibit tumor growth but reduces metastasis and improves the delivery and response to chemotherapy, by normalizing tumor vessels \[[@CIT0083]\]. Treatment with a low dose of 3PO, which reduces glycolysis by only 15% in ECs, induces similar effects \[[@CIT0083]\]. Since ECs' lining tumor blood vessels have a much higher glycolytic rate than healthy ECs, they are more sensitive to PFKFB3 blockade \[[@CIT0083]\]. This can explain why even the deletion of one allele of PFKFB3 or the use of a low dose of 3PO already induces tumor vessel normalization \[[@CIT0083]\]. Unlike traditional antiangiogenic agents \[[@CIT0084]--[@CIT0086]\], PFKFB3 haplodeficiency or blockade does not reduce tumor vessel density or total vascular area. Instead, PFKFB3 inhibition enlarges the vessel lumen and stabilizes tumor vessels by increasing vessel maturation through pericyte recruitment. These morphological changes improve tumor perfusion and thereby lower tumor hypoxia \[[@CIT0083]\]. Overall, PFKFB3 blockade reduces metastasis and increases chemotherapy delivery and efficacy ([Figure 2](#F0002){ref-type="fig"}). Figure 2.Targeting endothelial cell metabolism induces tumor vessel normalization.a. Schematic representation of the glycolytic pathway converting glucose into pyruvate. PFKFB3 is a key regulator of glycolysis by producing fructose-2,6-bisphosphate (F2,6P~2~), the most potent allosteric activator of phosphofructokinase-1 (PFK-1). G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F2,6P~2~, fructose-2,6-bisphosphate; PFK, phospho-fructokinase; 3PG, 3-phospho glyceraldehyde; TCA, tricarboxylic acid cycle; ATP, cellular adenosine 5ʹ-trisphosphate.b. Upon inhibition of PFKFB3 in hyperglycolytic tumor endothelial cells, tumor vessels show smoother endothelial surface, reduced intercellular gaps, more prominent basement membrane and increased pericyte coverage. All these changes improve tumor vessel perfusion and thereby lower hypoxia, contributing to reduced invasion, intravasation and metastasis. Adapted from \[[@CIT0008]\].
3.. Conclusions {#S0003}
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Since the concept of tumor vessel normalization for anticancer therapy was proposed in 2001, promising progress has been made in preclinical studies and clinical trials. By restoring the balance between pro- and antiangiogenic factors in the tumor microenvironment, antiangiogenic therapy using adequate antiangiogenic agents and delivery regimens is able to 'heal' the perturbed tumor vessels and to restore a more normal tumor vasculature, capable of more efficiently delivery of cytotoxic drugs and other therapies such as immunotherapies to tumors. While preclinical studies have amply demonstrated the therapeutic benefit of tumor vessel normalization strategies, translation to the clinic is now required to provide proof of evidence for similar benefit in cancer patients. However, this requires clinical development of new alternative strategies, capable of inducing more long-lasting and more efficient tumor vessel normalization. Targeting endothelial metabolism by lowering glycolysis in tumor ECs is emerging as a novel anticancer therapeutic approach, capable of inducing tumor vessel normalization, and hence reducing metastasis while improving chemo-, radio-, and immunotherapy.
4.. Expert opinion {#S0004}
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More than a decade of clinical experience reveals that we have yet to realize the full potential of antiangiogenic therapy. Most antiangiogenic agents received market authorization after providing proof of efficacy in a phase III trial that PFS or overall survival is prolonged compared to the most efficacious available therapy. However, these studies did not sufficiently investigate the fundamental mechanisms of how antiangiogenic drugs in combination with chemotherapy produce clinical benefit, and the exact time window in which the antiangiogenic agents exert the greatest benefit remains largely undefined.
Cancer patients receive disrupting scheduling of anti-VEGF regimens, which are often interrupted because of toxicity, resistance, or high costs. Discontinuation of antiangiogenic therapy might cause a rebound effect, leading to disease progression and metastasis (as observed at least in preclinical studies \[[@CIT0011],[@CIT0012],[@CIT0085],[@CIT0087]\]). One possible alternative, which has been poorly explored to date, might be to administer low (i.e. lower than maximally tolerated) doses of antiangiogenic drugs with the objective to promote tumor vessel normalization. In this case, the increased anticancer activity would not result from vessel pruning upon administration of high doses of antiangiogenic drugs, but rather from the restoration of a more functional tumor microenvironment that not only facilitates the delivery of chemotherapy and immune effector cells to the tumor but also impairs metastasis. Since targeting VEGF signaling often normalizes tumor vessels transiently and may ultimately provoke vessel regression, the development of alternative antiangiogenic strategies with a fundamentally distinct mechanism is mandated.
The recent data that the glycolytic regulator PFKFB3 controls vessel sprouting and its blockade promotes tumor vessel normalization \[[@CIT0083]\] raise the question if strategies targeting EC metabolism could increase the response of cancer patients to current anticancer treatment and represent a complementary or alternative antiangiogenic approach. A clinical trial using a small chemical PFKFB3 blocker has been initiated to target, in the first instance, cancer cells by using maximally tolerated doses \[[@CIT0088]\], based on preclinical studies designed to inhibit cancer cell proliferation \[[@CIT0089]\]. Since ECs are very sensitive to even small changes in glycolysis levels and, in fact, more responsive to PFKFB3 blockade than various cancer cell lines *in vitro* \[[@CIT0083]\], a clinical trial with another design using lower doses of the PFKFB3 blocker will be required to test whether PFKFB3 blockade can impair metastasis, while improving standard care therapy by promoting tumor vessel normalization. It will remain to be determined whether a high, or rather a low, dose of the PFKFB3 blocker is capable of inducing tumor vessel normalization, and its associated therapeutic benefits of reduced metastasis and improved response to chemotherapy.
Article highlights {#S0005}
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Anti-VEGF therapy promotes vascular pruningExcessive vascular pruning increases the risk of metastasis and impairs drug delivery and efficacyTumor vessel normalization represents an alternative strategy for antiangiogenic cancer treatmentTargeting endothelial cell glycolysis normalizes tumor vesselsPFKFB3 blockade reduces metastasis and promotes chemotherapy efficacy
This box summarizes key points contained in the article.
Declaration of interest {#S0006}
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P. Carmeliet declares to be named as inventor on patent applications, claiming subject matter related to the results described in this paper. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
[^1]: bev: bevacizumab; FDG-PET: 18-fluorodeoxyglucose positron emission tomography; CT: computed tomography; MVD: microvessel density; NSCLC: non-small cell lung cancer; VN: vessel normalization; DFS: disease-free survival; OS: overall survival; DCE-MRI: dynamic contrast enhance magnetic resonance imaging; MTT: mean transient time; VNI: vascular normalization index; 5-FU: 5-fluorouracil.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
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Most cases of end stage renal disease (ESRD) are caused by diabetes, and the number of cases has increased numerically and proportionally during the last few decades. Diabetic nephropathy, which causes the adverse outcomes of kidney failure, cardiovascular disease and premature death, is now the single most common cause of ESRD in both Western and Eastern countries \[[@B1]\]. However, our ability to assess early renal impairment in diabetic nephropathy, when active management is important, is still poor in comparison to biomarker-based approaches for other diseases, such as serum troponin concentration in acute myocardial infarct \[[@B2]\].
Serum creatinine level is the most commonly used surrogate measurement of glomerular filtration rate (GFR) in a clinical setting. In 2002, the Kidney Disease Outcomes Quality Initiative (K/DOQI) organization published guidelines for chronic kidney disease \[[@B3]\]. This classification, based on the modification of diet in renal disease (MDRD) study estimation equation for GFR using serum creatinine concentration, was immediately widely embraced \[[@B4]\]. However, serum creatinine concentration does not increase until renal function decreases to less than 50% of the normal value \[[@B5]\]. The variances according to age, sex, physical exercise, and lean body mass are also important limitations \[[@B6]\]. Gold standard tests using radioisotopes such as ^51^Cr-labeled EDTA, ^99m^Tc-labeled DTPA, and ^125^I-labeled iothalamate or iohexol \[[@B7],[@B8]\] are most reliable for measurement of GFR but are too difficult to use in the clinic setting \[[@B9]\]. Therefore, an accurate and available marker of GFR for patients with diabetic nephropathy is needed.
In the past three decades, urinary albumin excretion has assumed a central role in the diagnosis and management of kidney disease among people with diabetes mellitus \[[@B2]\]. This led investigators during the early 1980s to search for early predictors of diabetic nephropathy through the measurement of low concentrations of albumin in the urine, especially in type 1 diabetic patients \[[@B10],[@B11]\]. Despite a great deal of controversy \[[@B4],[@B12]\], microalbuminuria is now widely accepted as a predictive marker of overt proteinuria \[[@B13]\] and increased cardiovascular disease and mortality \[[@B14]\] not only in type 1 diabetes mellitus \[[@B15]\], but also in type 2 diabetic mellitus \[[@B13]\]. The importance of albuminuria has increased in practice as well as in laboratory research.
However, urinary albumin excretion can be affected by several factors including plasma concentrations of atrial natriuretic peptide, arginine vasopressin, angiotensin II, aldosterone and fasting blood glucose, glycated hemoglobin, and mean arterial blood pressure \[[@B16]\] and albumin can be degraded in a manner consistent with the activity of endogenous urinary proteases \[[@B17]\]. Furthermore, the intraindividual variation is as high as 47%. Because of these problems, all results that are initially positive for albuminuria need to be confirmed with a second sample collected on a different day, and in cases of discrepancies between the first and second sample, a third sample is necessary. Despite the convenience of urinary albumin/creatinine ratio (uACR) measurement in random urine samples compared to 24-hour urine collection, it incommodes clinicians who provide primary care to diabetic patients and causes a delay in the recognition and initial treatment for diabetic nephropathy. It is a fundamental problem because its main purpose is the detection of diabetic nephropathy at an early stage. Therefore, while continuing to encourage the use of the K/DOQI classification, which provides a well-established clinical guideline, the diabetology community should eagerly seek other methods to measure or better estimate early kidney damage of diabetic nephropathy, while maintaining the virtue of uACR. Further, changes in urinary albumin excretion are dynamic, whereas changes in GFR are usually progressive. Although increases in urinary albumin excretion generally precede a decline in GFR, some patients follow a non-albuminuric pathway to renal impairment. Therefore, in the assessment of diabetic nephropathy, the roles of albuminuria and GFR are complementary rather than competitive \[[@B18]\].
Recently, cystatin C was introduced as a good marker of early renal function decline \[[@B19],[@B20]\], particularly in diabetic \[[@B21]\] or elderly \[[@B22]\] patients. Cystatin C is being considered as a potential replacement for serum creatinine because it appears to be less affected by muscle mass \[[@B23]\], and most studies have shown that it is more highly correlated with GFR than is serum creatinine \[[@B20],[@B24]\]. Furthermore, there is no interference from metabolites, like bilirubin, ketones, and various drugs like antidiabetic medications \[[@B25]\]. Cystatin C is a non-glycosylated protein belonging to the super family of cysteine protease inhibitors, and it is produced by all nucleated cells at a constant rate \[[@B26]\]. Serum cystatin C fulfills a number of the criteria that would make it a suitable marker of GFR. It can be freely filtered at the glomerular membrane because of a low molecular weight (M*r*=13359) \[[@B9]\] and reabsorbed and catabolized by renal tubular cells \[[@B27]\]. Furthermore, its concentration is stable in stored plasma, unlike urinary albumin, probably due to the high concentration of other proteinase inhibitors \[[@B28]\]. Perkins et al. \[[@B29]\] reported longitudinal data demonstrating that the change in 100/cystatin C was more accurately correlated with the change in iothalamate-GFR than with the change in estimated GFR (eGFR) as calculated from serum creatinine.
Some studies have investigated the correlation between albuminuria and serum cystatin C level, but they evaluated serum cystatin C and albuminuria in cross-sectional datasets \[[@B30],[@B31]\]. Therefore, we analyzed the longitudinal correlation between cystatin C and albuminuria in Korean patients with type 2 diabetes mellitus.
METHODS
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Subjects
--------
We collected and retrospectively reviewed the medical records of 1,058 subjects with type 2 diabetes mellitus who visited the out-patient department of the Endocrinology and Metabolism Division at Yonsei University, Gangnam Severance Hospital, Seoul, South Korea from March 2006 to March 2010 ([Fig. 1](#F1){ref-type="fig"}). Inclusion criteria were as follows: 1) previously known type 2 diabetic mellitus; 2) more than 3 years of follow-up for serum creatinine, cystatin C, and uACR; and 3) age between 18 and 85 years. Subjects were excluded if they had 1) significant hepatic disease (alanine aminotransferase or aspartate aminotransferase \>2.5-fold the normal value); 2) thyroid disease or medication due to thyroid disease during the previous six months; 3) any other medical condition that could alter the level of serum cystatin C or uACR; or 4) if they were pregnant. The subjects were classified into three groups, according to uACR: 1) normoalbuminuria group (uACR \<30 µg/mg); 2) microalbuminuria group (30 µg/mg≤uACR\<300 µg/mg); and 3) overt albuminuria group (uACR≥300 µg/mg Cr). Obesity was defined according to 2000 WHO criteria, the Asia-Pacific perspective, as follows; 1) normal weight (body mass index \[BMI\] \<23 kg/m^2^); 2) overweight (23 kg/m^2^≤BMI\<25 kg/m^2^); 3) obese (BMI≥25 kg/m^2^). This study was approved by our human research ethics committee.
Study design
------------
BMI was calculated as body weight in kilograms divided by height in meters squared (kg/m^2^). Hypertension was defined as systolic blood pressure (SBP) ≥140 mm Hg or diastolic blood pressure (DBP) ≥90 mm Hg and/or reported treatment with antihypertensive medications in the previous 2 weeks. SBP and DB*P* values were classified by the Joint National Committee (JNC) 7 report in 2003.
Blood was sampled from all of the subjects after fasting for more than 10 hours, and random urine was collected. Serum cystatin C was measured by latex particle-enhanced turbidimetric immunoassays (Cobas 6000 analyzer; Roche Diagnostics, West Sussex, UK). Serum and urine creatinine levels were measured by automatic picric colorimetry (Hitachi 7600-110 automatic analyzer; Hitachi Co., Tokyo, Japan). Hemoglobin A1c (HbA1c) was determined by means of high-performance liquid chromatography (Variant II; Bio-Rad, Hercules, CA, USA). Total cholesterol (TC), triacylglyceride (TG), and high density lipoprotein cholesterol (HDL-C) levels were measured by an enzymatic method using an enzymatic color test (747 automatic analyzer; Hitachi Co., Tokyo, Japan), and low density lipoprotein cholesterol (LDL-C) was calculated according to Friedewald\'s formula.
The estimated GFR level was calculated using the MDRD formula (MDRD-eGFR) \[[@B32]\]:
MDRD-eGFR=186×(serum creatinine \[mg/dL\])^-1.154^×age^-0.203^×(0.742 if female)
The cystatin C-based estimated GFR (CysC-eGFR) equation \[[@B33]\]:
CysC-eGFR=78/(serum cystatin C in mg/L)+4
To compare the change in variables over three years, we calculated the initial and last value of cysC-eGFR and MDRD-eGFR using two different approaches, as follows:
1\) Simple subtraction
ΔcysC-eGFR=last cysC-eGFR-initial cysC-eGFRΔMDRD-eGFR=last MDRD-eGFR-initial MDRD-eGFR
2\) Declining slope
cysC-eGFR change rate=ΔcysC-eGFR/initial cys-eGFRMDRD-eGFR change rate=ΔMDRD-eGFR/initial MDRD-eGFR
Statistical analysis
--------------------
All statistical analyses were performed using SPSS version 18.0 (IBM, Chicago, IL, USA), except for the generalized linear mixed model for longitudinal analysis, which was performed using SAS version 9.0 (SAS Institute Inc., Cary, NC, USA). Serum cystatin C and creatinine concentration were transformed to the reciprocal by multiplying by 100 for analysis. This transformation was performed to assure that changes over time had the same direction as the changes in estimated GFR calculated using the MDRD formula. Descriptive data are expressed as mean±standard deviation, but in the generalized linear mixed model, we used standard error instead of standard deviation. *P* values less than 0.05 were considered statistically significant.
RESULTS
=======
Clinical and biochemical characteristics of subjects
----------------------------------------------------
The clinical characteristics of the study subjects are shown in [Table 1](#T1){ref-type="table"}. The group of 264 subjects was male-dominant, with an average age of 61.8±10.6 years in the baseline period. The subjects were followed for three years. The proportion of elderly subjects, defined as older than 65 years, constituted almost half of the study group. The mean BMI of these subjects, 24.2±3.0 kg/m^2^, was in the overweight range, although fewer than 10% of the subjects were obese. Two-thirds of the subjects were prescribed angiotensin-converting enzyme inhibitors and/or angiotension receptor blocker as antihypertensive drugs, and their blood pressures were under control. The mean fasting glucose level of the subjects, 131.1±65.7 mg/dL, was relatively high, but only one-sixth of the patients had an HbA1c greater than 9%, indicating that, overall, the blood sugar levels were under control in this study group.
The mean uACR of subjects was in the microalbuminuria range, and all stages of albuminuria were included. Patients in the nephritic range of proteinuria greater than 2 g/day were excluded because the possibility of other kidney disease could not be eliminated. The portion of patients with renal failure, defined as an eGFR lower than 60 mL/min/1.73 m^2^, was almost half of the study group. However, if eGFR was calculated using a cystatin C-based equation rather than the MDRD formula, the portion decreased to 34.3%. Patients with end stage renal failure were not included because their status of albuminuria could not be interpreted properly since their urinary albumin was not only glomerular in origin, but also tubular, and some of them had an anuric status.
Longitudinal trends in uACR and renal function marker
-----------------------------------------------------
As a result of longitudinal analysis using a general linear mixed model, only 100/cystatin C significantly affected uACR (*P*=0.0407), while 100/serum creatinine and MDRD-eGFR did not. Serum cystatin C and creatinine values were transformed to the reciprocal multiplied by 100 to assure that changes over time had the same direction as changes in MDRD-eGFR. CysC-eGFR was not involved in this analysis because, unlike MDRD-eGFR, its equation has no other variables except serum cystatin C. uACR decreased to 4.8321 mg/g when 100/cystatin C increased to 1 L/mg ([Table 2](#T2){ref-type="table"}). Even after adjustment for age, sex, serum uric acid, and HbA1c, these results did not change (*P*=0.0106); however, the other two renal function markers did not show any effect at all ([Table 3](#T3){ref-type="table"}). This result was consistent, regardless of the stage of chronic kidney disease stage.
Additionally, age and serum uric acid concentration showed a significant correlation with uACR in serial measurement (*P* value=0.0023 and 0.027, respectively), although gender and HbA1c did not (*P*=0.1137, 0.4063, and 0.7372, respectively).
Accordance between uACR and cysC-eGFR or MDRD-eGFR
--------------------------------------------------
CysC-eGFR showed better direction accordance (39.4%) with uACR in comparison to MDRD-eGFR (23.8%) ([Fig. 2](#F2){ref-type="fig"}) when analyzing declining slope. When we used the simple subtraction value ([Fig. 3](#F3){ref-type="fig"}), the accordance with uACR increased in both cases (52.3% and 36.8%, respectively), although that of cysC-eGFR was superior. When we categorized the linear variables according to the K/DOQI classification, the accordance rate with uACR was 46.5% in the case of cys-eGFR and 36.1% in the case of MDRD-eGFR. A close look at the discordance portion revealed that cysC-eGFR was worse than MDRD-eGFR at detecting aggravation of albuminuria, and that MDRD-eGFR was worse than cysC-eGFRat detecting improvement in albuminuria or preservation status.
DISCUSSION
==========
In our 3-year follow-up of a hospital cohort of type 2 diabetic patients, neither serum creatinine nor MDRD-eGFR corresponded with the trend in uACR change ([Tables 1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). This result indicates that the underlying pathophysiology of albuminuria and GFR loss are different in the early stages of diabetic kidney disease \[[@B27]\], and overt albuminuria does not always precede a significant loss of GFR in the setting of diabetes. Therefore, measuring albuminuria alone does not fully capture the scope of early diabetic kidney disease \[[@B10],[@B12]\]. Instead, albuminuria and GFR loss may represent complementary, if overlapping, manifestations of kidney damage \[[@B2]\].
Most recent studies that have assessed serum cystatin C as a measure of GFR have been cross-sectional in nature, but longitudinal studies are needed to further define the potential role of cystatin C in clinical care \[[@B29]\]. Serum cystatin C has great advantages compared to inconvenient uACR measurements, as mentioned above. Furthermore, there is evidence that cysC-eGFR is more valuable than MDRD-eGFR in the prediction of microalbuminuric stage in a cross-sectional clinical study of Korean type 2 diabetic patients \[[@B31]\]. To our knowledge, this is the first study to suggest a correlation between serum cystatin C and uACR in longitudinal studies for Korean type 2 diabetic patients. In contrast to serum creatinine and MDRD-eGFR, 100/serum cystatin C showed a statistically significant correlation with the trend of albuminuria (estimate -4.8321±2.2339, *P*=0.0407) ([Table 2](#T2){ref-type="table"}). Rather, the effect of 100/serum cystatin C on albuminuria increased after adjustments for age, sex, serum uric acid concentration and glycated hemoglobin (estimate -7.6667±2.8110, *P*=0.0106). However, the effects of 100/serum creatinine (*P*=0.3857) and MDRD-eGRF (*P*=0.7229) on uACR remained insignificant ([Table 3](#T3){ref-type="table"}).
In our study, we demonstrated the superiority of cysC-eGFR in reflecting the trend of albuminuria. But, the discordance portion was nearly half of all subjects ([Table 4](#T4){ref-type="table"}), so serum cystatin C alone might have limited value as a replacement for uACR measurement.
There are some controversies about the usefulness of serum cystatin C. Many studies have shown that serum cystatin C is a better predictor of cardiovascular disease, renal function \[[@B31]\] and mortality than is the serum creatinine-based eGFR formula \[[@B34]\], although some researchers disagree with this conclusion \[[@B21]\]. The discordance comes from the difference in subject characteristics, mostly with regard to the mean age of the population or proportion of renal failure patients. Our study population was evenly distributed in terms of age and chronic kidney disease stage, thus reinforcing the reliability of our data. It is critical that the ideal marker of diabetic nephropathy not be limited to a certain stage of kidney disease or specific age group because diabetic nephropathy progresses throughout the entire life of diabetic patients and has a broad spectrum from incipient nephropathy to end stage renal disease, which requires dialysis.
The limitations of our study are its retrospective design, the relatively small number of subjects and the fact that the data were collected from a single institute. Additional well-designed, prospective, and multicenter studies with a large population are required in the future.
No potential conflict of interest relevant to this article was reported.
{#F1}
{#F2}
{#F3}
######
Baseline clinical and biochemical characteristics of the 264 subjects
Values are presented as mean±standard deviation or percentage.
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; ACEi, angiotension-converting enzyme inhibitors; ARB, angiotension receptor blocker; uACR, urine albumin/creatinine ratio; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; MDRD-eGFR, estimated glomerular filtration rate calculated using the modification of diet in renal disease formula; CysC-eGFR, estimated glomerular filtration rate calculated using serum cystatin C.
######
The effect of each variable on urinary albumin/creatinine ratio change analyzed by generalized linear mixed model
Unadjusted for confounding variables.
SE, standard errors; MDRD-eGFR, estimated glomerular filtration rate calculated by the modification of diet in renal disease formula.
######
The effect of each variable on urinary albumin/creatinine ratio change analyzed by generalized linear mixed model
Adjusted for age, sex, serum uric acid, HbA1c.
SE, standard errors; MDRD-eGFR, estimated glomerular filtration rate calculated by the modification of diet in renal disease formula; CKD, chronic kidney disease.
######
Accordance of stage change between uACR and cysC-eGFR or MDRD-eGFR using simple subtraction
uACR, urine albumin/creatinine ratio; eGFR, estimated glomerular filtration rate; MDRD, modification of diet in renal disease; cysC-eGFR, eGFR calculated using serum cystatin C; MDRD-eGFR, eGFR calculated using MDRD formula.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
The Internet and social media provide a powerful avenue for information exchange and the development of opinions on a diversity of issues. The wide variety of sources makes it difficult for a reader to navigate among different trustworthy or/and misleading stories, suggestions and recommendations freely accessible through social media, often amplifying peoples\' fears and hopes. Many of the subjects involved in viral stories existed in pre-internet times. For example, the anti-vaccination movement started long before the widespread use of the Internet (see [@bib8] for analysis of its impact), so the later intensive anti-vax attack of Russian troll farms ([@bib2]) sowed the seed of distrust on fertile ground. As with the anti-vax movement, the influencing of the 2006 Israeli election through fake political news ([@bib1]) reflects the effects of fake news on potentially life-changing decisions. Yet, misleading stories can provoke not only decisions, but also practices, and therefore there is a need for an interdisciplinary approach in order to reduce their spread and "to address the underlying pathologies it has revealed" ([@bib24]). There is a growing body of research on the mechanisms of the spread of information and methods to analyze the information itself ([@bib4]; [@bib50]; [@bib14]; [@bib48] and references therein), yet more research is still needed. To understand if and how social media can help to invent tradition (sensu [@bib11]) we need to analyze something not noticed earlier that has recently become popular.
A good candidate for such an exploration is Ivan-chaj, a beverage (hereafter referred to as Ivan-chaj) prepared from the fermented leaves of rosebay willowherb *Epilobium angustifolium* L. (hereafter referred to as willowherb). It is important to note that Ivan-chaj is also the name of the plant in Russian, along with other names like kiprej and koporskij chaj, but we will use this term only to refer to the beverage. Making tea is very practical work, demanding several skills. One needs to be able to recognize the plant, and know its habitat, time of collection, method of collection, processing and preservation techniques, etc. While in the last six to seven years, sporadic use of dried (not fermented) leaves and flowers of willowherb for making tea has been documented in some Slavic-speaking regions (e.g. [@bib40]; [@bib43]; [@bib39]; [@bib41]; [@bib27]), the people mentioning their use have been very few. Fieldwork carried out in summer 2018, however, showed a comparably wide use of Ivan-chaj in several of the researched sites, especially in the Russian Federation, where around a quarter of the interviewees reported the current use of Ivan-chaj as a recreational tea ([@bib16]). Significantly, [@bib16] demonstrated that while the specifically processed leaves of willowherb were initially sold as fake black tea in the 18th century and later sporadically promoted and used as a tea substitute in the European part of the current Russian Federation, the fermented version of Ivan-chaj have been used mainly within the last five years.
A basic Internet search revealed that social media is overflowing with information on Ivan-chaj and its "glorious" history which uses elements of the past in order to build a believable narrative. Most of them were published in the last six to seven years and have presented Ivan-chaj as a drink that has been used in Russia for centuries ([@bib47]; [@bib44]; and many others). Yet, the "history" represented on social media is far from reality, forming *in corpore* a perfect example of a false narrative (*sensu* [@bib14]). The rapid rise of interest in Ivan-chaj has happened within the last decade, which coincides with the expansion of the social network itself; however, it is not solely a social media-based phenomenon.
The aim of this study was to understand the role of fake news and the involvement of social media in the construction of the symbolic identity narrative and, through that, the invention of "tradition". To this end, we 1) recorded and analyzed current practices in selected regions potentially affected by social media, 2) recorded and analyzed current reflections on Ivan-chaj on various social media, and 3) evaluated the mechanisms supporting the current popularity of Ivan-chaj.
2. Data and methods {#sec2}
===================
Our data consist of a variety of sources: videos, texts and visuals circulating on the Internet, and robust empirical field material collected through face-to-face conversations, without involvement of the Internet. The complexity of the subject required the use of mixed methods for analysis.
2.1. Field study and data analysis {#sec2.1}
----------------------------------
In order to understand if and how Internet-based narratives affect practice, we relied on qualitative analysis of the responses of 240 people interviewed in summer 2018 in three regions: the Republic of Karelia, Russian Federation (37 of the 70 people interviewed mentioned Ivan-chaj), Setumaa and Võrumaa in Estonia (16/95), and the border region between Lithuania and Belarus (6/141). The proportion of the people that mentioned the use of Ivan-chaj was higher in the region closer to the "historical center" of the narrative, decreasing with distance. The general framework of the interviews, which lasted from 30 min to 3 h, was about personal experience using wild food and medicinal plants in each specific region. In each location, we interviewed the major linguistic and/or ethnic groups: Karelians, Russians and a few newcomers from other parts of the former Soviet Union in the Russian Federation; Setu, Estonians and Russians in Estonia; and Lithuanians and Poles in the Lithuanian-Belarusian borderland. We used pseudo-random and snowball methods to identify the interview subjects. As this was a part of a larger cross-border ethnobotanical research project investigating the effect of different socio-political scenarios on Local Ecological Practice, Ivan-chaj was named among many other food and medicinal plants. The initial information on the use of willowherb was mainly provided spontaneously, with brief ad hoc questions asked afterward. The structure of the questionnaire was built on the basis of dishes and diseases, so the plant was rarely asked about explicitly. There were two exceptional cases in which the interviewer did explicitly ask if the participant used Ivan-chaj, as their neighbors suggested that they did -- the answer, in both cases, was negative.
The study was approved by the Ethics Committee of Università Ca\' Foscari and strictly followed the ethical guidelines outlined by the International Society of Ethnobiology ([@bib15]).
The interview transcripts were entered into RQDA software ([@bib12]) and a keyword list was developed on the basis of the content analysis of subjects that arose during the interviews. The emic keywords from the transcribed interviews were grouped according to their similarity in meaning, using the exact wording of interviewees as much as possible. The subsequent analysis was guided by the logic of the results.
2.2. Media as a resource {#sec2.2}
------------------------
For understanding the representation of Ivan-chaj in the media we extracted several datasets from online sources like Google trends, websites (e.g. blogs, micro-blogs, and forums), and social networks (e.g. Twitter, YouTube, and Instagram) ([Table 1](#tbl1){ref-type="table"}, Supplement 1). These platforms are quite popular channels in the study area for sharing and accessing media, including pictures, videos, and texts.Table 1The various media sources analyzed.Table 1What is analyzedSourceNumberTime of extractionType of analysisSample analysisNarrativesblogs, homepages, forums370Oct-18timeline, contentMemes17contentSystematic analysisTweetsTwitter446Oct-19timeline, contentPhotosInstagram1713VideosYouTube513**Total sum**3062
We used only publicly available data (users with privacy restrictions were not included in our dataset) by using the API (application programming interface). The data for the sample analysis was collected using snowball and subject saturation methods, while for extracting data for systematic analysis R software (Version 3.6.1) was used ([@bib5]). After data scraping we conducted data cleaning, deleted duplicates, removed spaces and non-printing characters, transformed and rearranged columns, fixed dates, etc.
A total of 3062 samples, posted by active users, were accessed. The content was manually classified and double-checked for consistencies. We stored a variety of metadata for each of these posts. The most important of which were data about social activity ("likes", "dislikes", "shares" and comments).
The keywords used to detect the data in the different Internet environments and online social media were *"иван-чай"* and its Latinized equivalent *"ivanchai"* for all but written narratives, for which the searches were conducted in the languages of the areas under investigation and their immediate surrounding territories: Russian *"иван-чай", "копорский чай"*; Belarusian *"іван-чай", "скрыпень"*; Ukrainian *"скрипень"*; Lithuanian *"gaurometis", "ivan arbata", "ivan čaj"*; Latvian *"šaurlapu ugunspuku"*; Estonian *"ivanatee", "ivan tsai", "ivan(i) tee", "põdrakanepi tee"*; Finnish *"maitohorsma", "Ivan ceai"*; Polish *"iwan czaj, "wierzbówka kiprzyca"*; and Romanian "*ceaiul lui Ivan*".
For making timelines, we used Excel and RAW Graphs, while for the content analysis we used different approaches depending on the type of media. For YouTube and Instagram we identified the most used words presented in the title of the video or description of the photo, respectively. The visuals were chosen for analysis on the basis of diversity, in an attempt to form a thematic overview of the parodies. The written narratives were thoroughly studied and qualitatively categorized in different plots according to the ad hoc sub-themes covered. We applied qualitative content analysis to explore the narrative structure. Frequently appearing words with regard to plots about drinks and plants were visualized using word clouds.
3. Results and discussion {#sec3}
=========================
3.1. Ivan-chaj in practice {#sec3.1}
--------------------------
### 3.1.1. Republic of Karelia {#sec3.1.1}
The high percentage of interviewees from the Republic of Karelia (Russian Federation) familiar with Ivan-chaj is not surprising as the region is quite close to the claimed place of origin of Ivan-chaj production (Koporye, Leningrad Oblast). Nevertheless, the subjects addressed by those people in relation to Ivan-chaj are diverse and the history of use not straightforward. A Karelian woman born in 1955 narrated: "*Now Ivan-chaj is widely sold. It is fashionable now. I once tasted it but did not like it; it is necessary to do it the right way, to catch the phase, and to have a dryer.*" Only one Russian woman (b. 1966) claimed to have made Ivan-chaj throughout her life: "*We have always had herbal teas. Even now I drink koporskij chaj more often than usual tea. I make it myself, I ferment it. It becomes very fragrant, pure honey. \[I use\] only leaves. If I want -- drink it pure and clean, if I want -- add some currants.*"
#### 3.1.1.1. Current use {#sec3.1.1.1}
The content regarding current use is often controversial. Many of our interviewees had fermented or dried Ivan-chaj at home, either done themselves, bought or received as a present ([Figure 1](#fig1){ref-type="fig"}). Ten people claimed to make Ivan-chaj themselves, and eight described the technology of fermentation (each with different wording). Three people also described experimenting after seeing teachings on the Internet and two people claimed to have learned from others. Five people described the technology as complicated. Eight people asserted that others make Ivan-chaj and three of them suggested that it has now become a fashionable thing. One man (b. 1946) said that he wants to try it, but had not yet had the chance: "*I wanted Ivan-chaj, it is promoted on TV, but I have not yet tried it. On TV they told us that in Russia Ivan-chaj was drunk before; I don\'t remember what broadcast it was; it was about the tea, various countries and that now we had forgotten Ivan-chaj*". One Russian woman, born in 1978, who makes rag dolls and teaches this in workshops, learned from medicinal books that willowherb was poisonous: "*I drank Ivan-chaj until I realized that I have \[health\] problems because of it. I dry it simply for the dolls. It goes into dolls as stuffing material*". A woman of Belarusian origin, born in 1955, narrated: "*As the Chinese say, if Ivan-chaj were to grow in China, every Chinese would be a millionaire. Here it grows, but we don\'t really use it\...*" A Russian woman, born in 1986, who produces Ivan-chaj narrated:""A few years ago, when we started making tea, we had several blends. And only we made complex blends with black tea. That is, people made all kinds of herbs, mixed them, but it wasn\'t with black tea. And we were thinking - why not smoke it? And somehow we smoked, and so far no one else does it. We first ferment it. There were classic recipes on the Internet, there are one million one hundred thousand of them, but in the end we developed our own. Now you can see it - it will be yellow or red, it is not yet ready. Fermentation has not yet been completed, but in winter it will be just right, it needs to stay for another six months. When we realized that our product was starting to be in demand, we realized that this could generate income. We are constantly studying the market in order to survive, because a small Ivan-chaj industry has appeared in Karelia and this is sad for all manufacturers. Not only in terms of income, but in terms of quality.""Figure 1Examples of various homemade Ivan-chaj preparations produced in the Republic of Karelia by interviewees for their own consumption. Upper row: different stages of the fermentation of leaves; lower row: mixtures of dried leaves, flowers and other plants. Photos by Valeria Kolosova and Olga Belichenko 2018.Figure 1
Those who indicated the start of the practice spontaneously mentioned one to four years ago. There was one exception: a Russian woman born in 1935 mentioned 1989 as the starting year (a time when various recipes of fermented Ivan-chaj were published; [@bib19]): "*No, it wasn\'t done here earlier. I think Ivan-chaj is used now particularly a lot, I know that for sure. And they always ferment it, someone came and taught us in 1989. From then Ivan-chaj has been used until now*".
#### 3.1.1.2. Past uses {#sec3.1.1.2}
Eleven people claimed to have used willowherb tea in the past, but only four of them stated that they personally drank it in their childhood as a tea, while five asserted that past use was restricted to flowers. One Karelian woman (b. 1972) told us that her mother recalled drying Ivan-chaj, while another woman born in 1966 mentioned that her grandmother considered it a famine food that she never used afterward. Four people claimed that they think that it may have been used in the past. At the same time, three people asserted that their family buys black tea, seven people were convinced that Ivan-chaj was not used in their childhood, and one person recalled that only a few individuals used Ivan-chaj in the past. Three people described its past use for animal fodder: "*willowherb was collected in childhood to sell to sovkhoz as food for polar foxes and minks. All children of my generation did it. We did not drink it ourselves*" (Karelian woman, b. 1954).
### 3.1.2. Outside the Russian Federation {#sec3.1.2}
In Estonia, a woman with higher education (b. 1954) referred directly to pseudo-history: "*Ivan-tšai\'s history is such that in Russia there was a factory where Ivan-chaj was made. The first thing that the Germans destroyed was the factory, because then the Russian soldiers had nowhere from which to get their strength".* As she was selling medicinal plants, she also copied teachings from the Internet, which she gave with the tea pack. Another Estonian woman (b. 1948) created her own story: "*Now it is called ivan-tšai, this name came from Russia. I always say to the men that this is the plant that Peter I always gave his soldiers as a compulsory drink because it takes care of men\'s potency. He was the first to call it ivan-tšai*". However, on the Estonian-language Internet, there is only a single statement that Peter I was the first to begin drinking black Chinese tea in Russia. In addition, two people knew that this was the drink of wealthy men in the past, stating: "*I read that the Russian boyar had used this tea and that it was also traded. It was very profitable in earlier times but I do not know where it was sold \..."* (woman, b. 1957). Also, "*Russians make the tea, call it ivan-tšai or tsaar-tšai. I\'ve picked it myself; it should be good for men, for the prostate*" (woman, b. 1938).
In Lithuania, narratives revealed the very recent origin of the practice and the described confusion introduced through social media. A Polish-speaking woman (b. 1941) referred to *Hypericum perforatum* L. as "Ivan-chaj". She associated the Russian name of Ivan with the Catholic Saint John and said: "*Świętojańskie ziele - it is in our language. And easier for you* \[to understand\] *- Ivan-chai*". The most enthusiastic users of Ivan-chaj in the study region were an Old Believer (b. 1968) and his wife of Polish origin (b. 1972). They collected flowering tops of willowherb, having learned about this plant on the Internet. They were waiting for the start of winter to drink it. The couple remarked that local Polish and Lithuanian people are not familiar with the tradition of using Ivan-chaj. They experimented with tea fermentation: the husband thinks that longer fermentation yields a greater taste, but his wife said that longer fermentation kills the aroma. As a compromise, they fermented willowherb for 4 days. A man of mixed Lithuanian-Polish ancestry (b. 1963) also experimented with tea fermentation. He made different teas from willowherb by altering the fermentation and drying process. He knows many recipes from books and the Internet and tries to choose the best ones.
The difference in manufacturing technology is the clear marker identifying current uses prompted by social media. The way to ferment the leaves of willowherb into proper Ivan-chaj is mainly learned from the Internet or through mediators, often experimenting with different suggested recipes. People re-narrate stories found on the Internet or other media, which they are eager to alter according to their understanding and preferences, often taking earlier use for granted and stories being true without questioning their provenance.
3.2. Ivan-chaj story in the media {#sec3.2}
---------------------------------
### 3.2.1. The start of the campaign {#sec3.2.1}
Google trends showed a sudden global increase in the interest in Ivan-chaj starting July 2013. The search rate increase repeats itself yearly and accelerates during the summer months with a peak in July when the plant is collected ([Figure 2](#fig2){ref-type="fig"}).Figure 2Google trends for proportional global interest in Ivan-chaj and its other two most widespread names (source: [trends.google.com](http://trends.google.com){#intref0095}). January 2004--October 2018.Figure 2
Television might have given the first push: in March 2009 tea made of willowherb was mentioned on the program *Dobroye utro, Rossiya!* \[Good morning, Russia\] on the Rossiya-2 TV channel ([@bib36]).
The first great rise in interest coincided with the date (March 2013) when the Russian channel RenTV aired a TV program dedicated to Ivan-chaj ([@bib28]). This channel, blacklisted for spreading fake news, colorfully illustrated its "glorious history".
#### 3.2.1.1. Pseudo-history narratives {#sec3.2.1.1}
We found 160 original narrative segments representing all languages except for Finnish. Analyzing the content of the narrative lines ([Figure 3](#fig3){ref-type="fig"}), we divided the resulting 43 plots into two separate categories: narratives about the **plant** and narratives about the **drink**. The narratives about the plant (willowherb) cover its origin and properties of the plant and the origin of the name. The narratives about Ivan-chaj as a fermented drink cover its origin, knowledge, medicinal properties, relation to identity and economic importance. None of the narratives have an actual historical basis and the diversity of the covered subjects is impressively wide.Figure 3Timelines of publishing Ivan-chaj related content on [twitter.com](http://twitter.com){#intref0100}, [youtube.com](http://youtube.com){#intref0105} and [instagram.com](http://instagram.com){#intref0110}.Figure 3
A large number of storylines refer to historical figures and events (not necessary factual) familiar in the specific region which they are addressing, building on the audience\'s existing knowledge. It is clearly evident that the Russian idea of cultural identity established through Ivan-chaj was mirrored in other countries, local origin of the tradition of drinking Ivan-chaj. If in one case there was a reference to common roots or it referred to Slavic (Russian) unity, there were several cases pinpointing a specific ethnic origin (Finno-Ugric, Baltic, Belarusian, Kievan Rus\'). A narrative line of ancestral roots (found only outside Russia) highlights the construction of cultural identity and strategies of ethnic survival through Ivan-chaj.
As shown in [Figure 4](#fig4){ref-type="fig"}, the narratives originating from Russia clearly dominate the data and were produced in all years and cover all subjects. Some narratives did not leave the borders of Russia, while many others are spread across almost all studied languages and a small proportion is dispersed in specific countries. Remarkably, until 2013 no narrative was translated into other languages and only since 2016 have the pseudo-stories gone viral across borders and moved from the Russian-speaking center to many of the countries covered in this study through translation into national languages. The two narratives encountered in Romanian are related only to the origin of the plant name, while the modest representation of narratives in Latvian, Belarusian and Ukrainian may be due to the Russian language still serving as the *lingua franca* in those countries. We might consider this broadening of narrative frames through which Ivan-chai tea is reported as reflecting what [@bib10] refer to as "domestication", since a key feature of the domestication process is the way in which globally significant events, and here we would include Ivan-chaj, are rendered "comprehensible, appealing and "relevant" through reference to a "narrative framework that is already familiar to and recognizable by" domestic audiences ([@bib10]: 206--7).Figure 4Alluvial diagram of Ivan-chaj narratives by language, year of publication and subjects covered based on 370 narratives.Figure 4
Finally, we have an image of Ivan-chaj, spreading on blogs, micro-blogs and local forums, as a healthy, trendy, and delicious high-class beverage for high-class people. The studied narratives have very detailed storylines, which are typical for newly created ones ([@bib30]). There is also a strong folklorization ([@bib23]) of the plant and the drink in contemporary discourse, and the mythologization of this phenomenon ([@bib38]). We can also trace the adoration and exclusivity of this drink, as well as the creation and dissemination of new drinking rituals (for example, Ivan-chaj drinking ceremonies). The ritualization of consumption through social media exposure adds to what Grasseni called the virtualization of local foods ([@bib9]). As a result, in social media, TV programs, festivals and advertisements, Ivan-chaj is ever more depicted as a symbol of specific territories, through reference to national and cultural identity. It is consequently treated as a political and economic resource. To claim "local" status for a drink not only means re-inventing (just as "traditions" are invented or constructed): it can mobilize strategies of self-rediscovery of the patrimonialization of local histories, places and landscapes.
### 3.2.2. The potential impact of social media {#sec3.2.2}
Social media is highly visual: images can be sent and received as messages ([@bib26]), occupying a key place in communication and information sharing on online platforms. Ivan-chaj has inspired an impressive number of images, tweets and videos ([Table 2](#tbl2){ref-type="table"}). The emergence of social media is providing an alternative avenue for information exchange and opinion formation on related issues. Collective online discourse in such media leads to the formation of a complex narrative - reaction, conveying public views and perceptions. Ultimately, we argue that collected data has potential value in helping us understand the social experience of the practical uses of Ivan-chaj, but studying these types of data presents theoretical and methodological challenges ([@bib25]).Table 2Impact of selected social media channels in spreading information on Ivan-chaj.Table 2CharacteristicsInstagramTwitterYouTubeNumber of reposts-2871-Number of creators676398421Maximum number of posts per creator14113322Proportion of creators with one post31%49%60%Number of likes102225525741395Number of dislikes\--2718Number of comments-312-Number of views\--15807281
As depicted in [Figure 5](#fig5){ref-type="fig"}, "ЗОЖ (здоровый образ жизни)" ("healthy lifestyle") and "Russian" were the most frequent words in the Instagram and Youtube datasets. Among users of Instagram, **health-related** terms such as "herbaltea," "healthyfood", "health" "healingherbs", "полезный" ("helpful"), "ппперекус" ("proper nutrition snack"), "plantmedicine" and "wellness" were observed, indicating that users mostly shared information about the health properties of the studied plant. We also found identity-specific words such as "russiantea", "Russia", "russiancuisine", "ru", and "сибирский чай" ("Siberian tea"). Among users of Youtube, **practice-related** terms such as "готовим" ("cook"), "сбор (собрать)" ("collecting (collect)"), "сушить" ("dry"), "выращивание" ("growing"), "произодство" ("producing") were frequently noted, indicating that these were important words for characterizing new practices. Also, frequently encountered terms in the Youtube data corpus about life hacks ("правильно" ("right"), "настоящий ("real") "секрет", ("secret")) are about how to better collect the plant and prepare the drink.Figure 5Word clouds of the most often repeated words in descriptions of Instagram pictures and titles of YouTube videos.Figure 5
Another important trend is that 72% of the videos were posted by men, which is not characteristic of traditional knowledge transfer mechanisms in Slavic countries, where the domain of plants is dominated by women.
### 3.2.3. Jokes as a statement {#sec3.2.3}
Collective discourse in online social media leads to the formation of a complex narrative, conveying public views and perceptions. Some humorous posts start 'trending', becoming the most circulated ([@bib25]). In a digital economy where attention is scarce, images are a quick and efficient way to communicate thoughts and feelings ([@bib29]). We found that the production and consumption of humorous texts and images highlighted changes in the representation of the studied plant -- a potential turn from top-down understanding of the plant to bottom-up, citizen informed views. While many such images contain references to Russia dominance, they also ridicule this new symbol: the theme of powerful and healthy beverages in these images and texts provide a counter-narrative to mainstream media accounts.
The range of humorous reactions covering the subject of Ivan-chaj can be divided into five groups. The first group can be labeled as *"Drink Russian!"* ([Figure 6](#fig6){ref-type="fig"}). For instance, one of the memes referred to one of the most changed Soviet propaganda posters combating alcoholism -- "Нет!" ("No!"). In the modern context, one of the memetic replacements takes place with Ivan-chaj. Accommodation via references to local culture creates a feeling of recognition and commonality and renders the process of entextualization smoother. One of the vernacular reactions to international sanctions against Russia over the Ukraine crisis was memes that offered Ivan-chaj as an alternative to importing English tea.Figure 6a) *"No! I drink only Ivan tea"* (meme-arsenal.ru); b) *"No! Only Russian Ivan-tea"* ([vk.com](http://vk.com){#intref0115}); c) *"And which tea do you drink?"* (demoviators.ru); d) *"English tea vs Russian tea. Sanctions made available to everyone"* ([vk.com](http://vk.com){#intref0120}); e) *"English tea vs Russian Ivan tea. Sanctions made available to everyone"* ([vk.com](http://vk.com){#intref0125}); f) *"Ivan tea. Epic. Our brand. Russian highest quality. Young leaves of fireweed. Alcohol 0%. The best choice of Russian boyars. Sale in St. Petersburg. Representatives in all cities"* ([vk.com](http://vk.com){#intref0130}).Figure 6
Memes and texts circulating on Twitter about Ivan-chaj are mostly stereotypical: *"- Здравствуйте, сударь! - Мне блин с картошкой и салат "русский цезарь". - Пить что будете? - Иван-чай. - Сударь, не хотите ли к заказу еще взять брошюрку о спасителе белой расы Адольфе Гитлере? - Что? - Что?*" ("- Hello, sir! -- For me, please, pancake with potatoes and "Russian Caesar" salad. - What will you drink? -- Ivan-chaj. - Sir, would you like to order a booklet about the savior of the white race Adolf Hitler? - What? - What?") (Twitter, user \@Zh-d, 30-Aug-19). Ivan-chaj equally became an object of ridicule. Many tweets consider drinks as the main actor in life: "*Мужчины в моей жизни: - Max Factor, - Дымов. -- Рентген. - Mr. Proper. - Б.Ю. Александров. - Иван-чай. Ни о чом не жалею*" ("Men in my life: - Max Factor, - Dymov \[*brand of sausages and meat delicacies*\] - Röntgen. - Mr. Proper. - B.Yu. Alexandrov \[*chocolate glazed curd cheese brand*\]. -- Ivan-chaj. I have no regrets") (Twitter, user \@n-a, 1-Sep-19).
In contrast to the "drink national beverages" narrative, the joke is a reference to the label of the top-selling American whiskey Jack Daniel\'s, converted into Ivan-chaj. Or on Twitter "*срочная новость. кока-колу переименовали в иван-чай*" ("Breaking news. Coca-Cola was renamed Ivan-chaj") (Twitter, user \@ni-os, 13-May-19). *"- Что это? - Иван чай. - А можно мне чай без Ивана?" (\"- What is it? -- Ivan-chaj. "Can I take tea without Ivan?")* (Twitter, user \@P-4, 12-Jun-16).
Another group of humorous reactions ([Figure 7](#fig7){ref-type="fig"}) refers to a value set that includes the power of Ivan-chaj (a reference to Star Wars Episode III) and it\'s magical health.Figure 7a) *"You underestimate the power of Ivan tea!"* (meme-arsenal.ru) b) *"When the cat didn\'t drink Ivan tea. - Where is my TEA?"* ([vk.com](http://vk.com){#intref0135}).Figure 7
In contrast, the next group of Ivan tea jokes references delirium tremens after drinking/smoking Ivan-tea (popular Soviet film "Kidnapping, Caucasian Style": "*We will cure you - alcoholics are our profile*"). In the next case, the authors of the visual narratives emphasize that it is not so easy to abandon the use of Ivan tea ("*One does not simply walk into Mordor*" from a scene in the film adaption of J.R.R. Tolkien\'s Lord of the Rings) ([Figure 7](#fig7){ref-type="fig"}).
This motive of the following joke is also quite popular among twitter users: *"- Колян, мне кажется чай пахнет коноплей. -- Нет. - Да пахнет, принюхайся. -- Нет, Иван-чаем пахнет. - Чо хочешь сказать, что вчера Иван-чай курили"* ("- Kolyan, it seems to me that tea smells like hemp. - Not. - Smells, sniff it. - No, Ivan-chaj smells. - What do you want to say, that yesterday we smoked Ivan-chaj?") (Twitter, user @ na-a, 1-Sep-19).
Local motives are successfully combined with modern global forms and elements ([Figure 8](#fig8){ref-type="fig"}). The popular Snorp meme was photoshopped into the famous Russian work of art "Girl with peaches"; in a scene from Martin Scorsese\'s movie "Shutter Island", the taste of Ivan-chaj was questioned; the Philosoraptor dinosaur (part of a series on advice animals) deeply immersed in metaphysical inquiries) (see [Figure 9](#fig9){ref-type="fig"}).Figure 8a) *"Ivan-tea! Make your mind easy!"* (meme-arsenal.ru); b) *"Was it definitely Ivan-tea? -- I got stoned!"* ([memesmix.net](http://memesmix.net){#intref0140}); c) *"You just need to smoke Ivan-tea"* (risovach.ru).Figure 8Figure 9a) *"When you are waiting for Ivan-tea to brew"* ([vk.com](http://vk.com){#intref0145}); b) *"Have you tasted Ivan-tea? - Once. - How did you like it? - ..."* (risovach.ru); c) *"If Ivan-chaj heals, and microwaves disable, what will happen if Ivan-chaj is heated in a microwave - drug or poison?"* ([memesmix.net](http://memesmix.net){#intref0150}).Figure 9
We can also note a group of linguistic jokes about Ivan-chaj using wordplay, such as homography (words identically spelled in different languages), which involves exploiting the ambiguous meanings of words for humorous or rhetorical effect ([Figure 10](#fig10){ref-type="fig"}): "*пью иван-чай в надежде, что стану сегодня николаем"* ("I drink Ivan chaj in the hope that I will become Nicholas today") (Twitter, user \@T-na, 11-Jul-16); "*иван-чай, катя-водка"* (ivan-tea, katya-vodka) (Twitter, user \@wh-y, 1-Nov-16); "*Иван чай ака джонни ти*" ("Ivan tea aka Johnny tea") (Twitter, user \@R--S, 18-Jan-15). Also, "*Что-то у них много Джеков: джек-пот, джек юнион, блэк джек, джек рассел терьер, джек потрошитель. А у нас только Иван-чай*" ("Somehow they have too many Jacks: jackpot, Union jack, Blackjack, Jack Russell terrier, Jack the Ripper. And we have only Ivan chaj") (Twitter, user \@h-om, 19-Mar-14).Figure 10a) "*Ivan-tea. Gosha -- cappuccino*" (risovach.ru); b) "*Ivan -- tea. Andrei -- kefir*" (pikabu.ru); c) "*Ivan-tea. Ivan tea -- Russian tea. Vova-tea. Boba tea -- Thai tea*" ([vk.com](http://vk.com){#intref0155}).Figure 10
### 3.2.4. Virality of the grand narrative of "Russian tea" and its economic impact {#sec3.2.4}
Winner of the Nobel Prize in Economics Robert J. Shiller defined the term "viral narrative" as "a simple story or easily expressed explanation of events that many people want to bring up in conversation, on the news or on social media because it can be used to stimulate the concerns or emotions of others and/or because it appears to advance self-interest". [@bib37] suggested that it is hard to predict which narratives, their elements or their mutations suddenly become popular or "go viral", but once they do so, they "spread far, even worldwide, with economic impact". He also stressed that "narratives are major vectors of rapid change in culture, in zeigeist, and ultimately in economic behavior" if the story can establish a "reference point, which has influence on decisions". The "reference point" of Ivan-Chaj was certainly established, as by 2018 nine patents concerning willowherb had been issued; four of them involve tea production ([@bib35]). There are more than 70 producers of Ivan-chaj in the Russian Federation, and the business was profitable ([@bib51]).
The first documented Ivan-chai festival (*Праздник Иван-чая*) was celebrated in Nizhny Novgorod Oblast in 2010, and it subsequently became a yearly event claiming to be a revival of traditional holiday celebrations ([@bib13]). Now, numerous Ivan-chaj festivals are organized throughout the Russian Federation which have a similar structure and are combined with fairs. The key events of Ivan-chaj holidays include picking the plant and fermentation workshops and demonstrations. At these events, a Russian tea ceremony with Ivan-chaj has recently been gaining popularity ([@bib7]). The holiday usually is scheduled on the Ivan Kupala (St. John\'s night) (from 6 to 7 July in Russia), which is correlated with the legendary origin of the plant in modern Internet narratives ([@bib33]). According to popular belief, it is the time of the summer solstice -- the best time for all plant gathering including willowherb. The Ivan-chaj holiday organizers are usually private individuals -- representatives of agro-tourism, ecovillages and other cultural institutions. There are suggestions to include Ivan-chaj on the list of local dishes within the framework of gastronomic tourism, for example, in Yamal ([@bib3]).
In 2015, Ivan-chaj seemed to have been promised more official support by the state in the Russian Federation. On the wave of import substitution, the Public Chamber of the Russian Federation conducted public hearings on the issue: "Developing a legislative framework for the development of the Ivan-tea industry in the Russian Federation and the support of domestic Ivan-chaj producers". The members believed it necessary to develop a National Standard for producing Ivan-chaj products from willowherb, to create several different kinds of Ivan-chaj, and even to established a professional holiday of Russian herbalists (Ivan-chaj Day), as well as to recommend the government to consider reducing tea imports and finding ways to promote Ivan-chaj products in foreign markets ([@bib34]). However, "The National Union of Producers of "Russian Tea", which was created in 2015 and organized these hearings, quickly collapsed, they did not even have time to register it (Ivan-chaj, 2018).
The growing cohort of Ivan-chaj producers in the Russian Federation tends to abstain from pseudoscience or even taking a critical stance and investing their time in dispelling the myths created by their predecessors (see, for example, an exhaustive explanation in ([@bib49])). Nevertheless, the value of Ivan-chaj itself is not questioned as it is still claimed to have proven quality and health-improving abilities. The latest major appearance of Ivan-chaj on Russian TV happened in 2017 ([@bib6]). The stance has changed from myth-propagating to robust 'science': the viewers learned that fermentation technology was brought from China. The episode was concluded by tea tasting and dégustation of sophisticated dishes made with willowherb leaves and flowers. The matter of zero cost is less and less discussed in the media for obvious reasons, even though this is an important issue for a majority of the audience.
The fake history initially supporting the grand narrative of Ivan-chaj was also backed by food conspiracy narratives (cf. the narrative about yeast in bread ([@bib18])) thanks to their presence in a majority of media outlets (newspapers, TV, social media). Together they could play a promoting role in consumer groups of various backgrounds, from younger educated urban dwellers to older inhabitants of rural areas. At the same time, Ivan-chaj sold in shops and local markets cannot be called cheap ([Figure 11](#fig11){ref-type="fig"}); for example, in a Belarusian market a pack of tea is sold for ca 4--10 Belarusian rubles, which is very expensive for people earning the minimum monthly salary of 325 rubles (as of 1 January 2019), a common salary in the countryside. Ivan-chaj is often presented as elite and a bioproduct, accessible only to wealthier individuals. However, there also seems to be some signs of over-production of Ivan-chaj, as, for example, a pack of it was offered as the final bonus to kitchenware sold on a TV shop commercial broadcast on the RenTV Baltic Channel, 11 July 2019.Figure 11One of many examples of the highlighting of Ivan-chaj in local markets. Minsk, Kamaroŭski Market. February 2019. Photo by Julia Prakofjewa.Figure 11
Ivan-chaj has also recently been introduced as a regional food culture brand in the area around Lake Peipsi, the historic site of Russian Old Believers in Estonia. In 2015--2016, a village society in Lohusuu started to advertise itself to tourists through Ivan-chaj culture, offering trainings and presentations ([@bib17]) and publishing a leaflet ([@bib45]), a regional recipe book, including instruction on producing Ivan-chaj ([@bib46]), and an exhibition of regional food culture (including Ivan-chaj) in the Estonian National Museum ([@bib21], [@bib22]); supported by media publications ([@bib32]). However, the in-depth inventory of culinary traditions of Old Believers in Estonia did not mention the use of Ivan-chaj and the local plants were perceived mainly as taste additives to black tea ([@bib20]).
Mati Rebane, the natural therapist, translated legends about Ivan-chaj into Estonian and then published them on his website ([@bib31]). In a few months, these stories were published on the homepage of one of Estonia\'s most popular natural and ecological product distributors, looduspere.ee, despite the fact that this online store had already been selling Ivan-chaj with various additives for almost a year. The success of Ivan-chaj in Estonia has been supported by the long time promotion of its use against prostatitis, well publicized by popular herbalists and official medicine ([@bib42]). By the end of 2018, several local small-scale companies produced, sold and introduced Ivan-chaj, supported by agricultural, entrepreneurship and regional programs in Estonia.
There are also numerous producers of Ivan-chaj in Belarus, Ukraine, Latvia, Lithuania, and other countries. It should be noted, though, that the Ivan-chaj holiday has not yet spread beyond the Russian Federation.
4. Conclusions {#sec4}
==============
Following on from the idea of Shiller and our field results, we suggest that the wider a base the narrative has, the more people it addresses, and the greater chance it has to go viral and cause an economic impact. An overall positive image of Ivan-chaj was presented through the sum of all the storylines and other means of modern marketing formed into a general narrative, changing the way it was perceived. The diversity of narrative lines and their distribution created a situation where there was literally "something important" for everyone, and this most likely engaged a critical mass of people. Even the criticism of memes and jokes seems not to be able to ruin the positive image of Ivan-chaj. Currently, in the Russian Federation Ivan-chaj is drifting toward becoming a national culinary practice in which the taste, properties and relation to identity are regarded as the most important features of the drink. It also entered other countries through narratives on its medicinal usefulness and cultural importance, adding some more colorful narrative lines, best understood in the context of a given country. Plausible narratives were easily adapted for the potential audience and quickly spread by media. Having appeared in Russia, mostly as a Russian cultural marker, the narrative went viral and spread beyond its borders where neighbors tried in turn to embrace Ivan-chaj as their own cultural marker by proclaiming it a local tradition. Indeed, narratives regarding Ivan-chaj spread easily in countries sharing some linguistic, historical and/or cultural elements with Russia (via the nexus of the Soviet Union).
While the influence of the Internet and social media on the spread of invented tradition is not easily detectable and results may often not be univocally interpretable, we recommend future research in this direction in order to understand the differences between claimed and factual practice and the mechanisms of information transmission.
Declarations {#sec5}
============
Author contribution statement {#sec5.1}
-----------------------------
Julia Prakofjewa, Raivo Kalle, Renata Sõukand: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Olga Belichenko, Valeria B. Kolosova: Performed the experiments; Conceived and designed the experiments; Wrote the paper.
Funding statement {#sec5.2}
-----------------
This work was supported by the European Research Council (10.13039/100010663ERC) under the European Union\'s Horizon 2020 research and innovation programme (grant agreement No 714874).
Competing interest statement {#sec5.3}
----------------------------
The authors declare no conflict of interest.
Additional information {#sec5.4}
----------------------
No additional information is available for this paper.
Appendix A. Supplementary data {#appsec1}
==============================
The following is the supplementary data related to this article:Supplement 1Supplement 1
We are grateful to the local people who shared with us their knowledge and practice on willowherb use. We thank Povilas Šarka, Baiba Prūse, Ieva Mežaka and Andra Simanova for assisting in the fieldwork, and also Giulia Mattalia for insightful comments on initial manuscript.
| {
"pile_set_name": "PubMed Central"
} |
Soresina A, Moratto D, Chiarini M, et al. Two X‐linked agammaglobulinemia patients develop pneumonia as COVID‐19 manifestation but recover. Pediatr Allergy Immunol. 2020;00:1--5. 10.1111/pai.13263
The peer review history for this article is available at https://publons.com/publon/10.1111/pai.13263
Key MessagePatients with agammaglobulinemia can recover from SARS‐Cov2 infection despite lack of B cells.
1. INTRODUCTION {#pai13263-sec-0006}
===============
Since February 21, 2020, several cases of coronavirus disease 2019 (COVID‐19), caused by the β‐coronavirus associated with human severe acute respiratory syndrome (SARS) officially named SARS‐CoV‐2, were identified in northern Italy.[^1^](#pai13263-bib-0001){ref-type="ref"} The disease has a broad spectrum of clinical phenotypes ranging from mild symptomatic cases characterized by influenza‐like manifestations, which occur in the majority of subjects (about 80%), to severe pneumonia requiring admission to intensive care units in 5% or invasive mechanical ventilation, or even death.[^2^](#pai13263-bib-0002){ref-type="ref"} Patients with severe disease usually present with comorbidities and are older than those with non‐severe disease. Most of the COVID‐19 patients exhibit lymphopenia, which is more pronounced in severe cases; thrombocytopenia; and elevation of C‐reactive protein. The disease has an incubation period of 5‐14 days and manifests with fever, cough, vomiting, and diarrhea lasting up to 2‐3 weeks.
Little is known about the mechanism of immune response against the SARS‐CoV‐2, but the first reports suggest that active immunity requires the formation of antigen‐specific cytotoxic T cells and synthesis of neutralizing antibodies directed against the virus.[^3^](#pai13263-bib-0003){ref-type="ref"}, [^4^](#pai13263-bib-0004){ref-type="ref"} There is no specific antiviral treatment for patients with this infection, but many antiviral or immunomodulatory drugs are under evaluation.[^5^](#pai13263-bib-0005){ref-type="ref"}, [^6^](#pai13263-bib-0006){ref-type="ref"} Indeed, patients with severe COVID‐19 have been successfully treated with immune plasma derived from convalescent patients, suggesting that virus‐specific neutralizing antibodies are important for disease recovery.[^7^](#pai13263-bib-0007){ref-type="ref"}, [^8^](#pai13263-bib-0008){ref-type="ref"}
2. METHODS {#pai13263-sec-0007}
==========
After the identification of patients with SARS‐CoV‐2 infection in Italy, we started a survey protocol on patients with chronic diseases who presented with COVID‐19 to identify conditions that could constitute a risk factor for an unfavorable outcome. SARS‐CoV‐2 infection was confirmed in all patients by the detection of viral RNA by real‐time reverse transcription polymerase chain reaction (RT‐PCR) assay in nasopharyngeal swab specimens obtained during the clinical manifestations. Patient care and research were conducted in compliance with the Case Report guidelines and the Declaration of Helsinki. This study was performed after ethics approval (Ethics Committee of Brescia, protocols NP4000 and NP4047).
3. RESULTS {#pai13263-sec-0008}
==========
Herein we describe two patients with agammaglobulinemia and absence of circulating B cells due to *BTK* mutations who live in northern Italy and have developed COVID‐19 during the 2020 outbreak.
Patient 1 is a 34‐year‐old man who was diagnosed with agammaglobulinemia at 4 months of age because of perianal abscess associated with low levels of immunoglobulins (IgG 161 mg/dL, IgA 6.5 mg/dL, IgM 10 mg/dL as compared to IgG 222‐846 mg/dL, IgA 6‐60 mg/dL, IgM 28‐39 mg/dL in age‐matched children) and lack of B cells in peripheral blood (Figure [1](#pai13263-fig-0001){ref-type="fig"}). He started intravenous immunoglobulin treatment at the time of diagnosis and switched to subcutaneous immunoglobulins in March 2013. *BTK* genetic analysis revealed a non‐sense mutation in exon 2 (K19X) which will cause termination of protein synthesis prematurely. Chest computed tomography (CT) scan performed in January 2010 did not reveal bronchiectasis. On January 14, 2020, he performed outpatient blood tests (IgG 860 mg/dL) and received immunoglobulin treatment; then, he continued immunoglobulin therapy at home every 28 days.
{#pai13263-fig-0001}
On March 5, he presented with fever and cough, which required medical evaluation and following treatment with oral antibiotics. Because patient general conditions were maintained with normal breathing and unremarkable chest X‐ray, he was advised to remain at home in self‐isolation to reduce contacts with potential sources of SARS‐CoV‐2 infection. However, on March 13, fever and cough reappeared and he was admitted to the hospital suspecting SARS‐CoV‐2 infection, which was confirmed by nasopharyngeal swab. Because chest radiography revealed interstitial pneumonia with bilateral infiltrates (Figure [2A](#pai13263-fig-0002){ref-type="fig"}), we started empirical treatment with lopinavir/ritonavir‐associated hydroxychloroquine, and subcutaneous infusion of immunoglobulins (400 mg/kg), but he never needed oxygen ventilation. During his stay in the hospital, he developed leukopenia (WB cells: 2910 cells/ul) and an increase in CRP and ferritin (Table [1](#pai13263-tbl-0001){ref-type="table"}), which gradually improved in the following days. He was always able to breathe spontaneously and was discharged on March 27 in good health. Analysis of lymphocyte subpopulation during the infection revealed normal T‐lymphocyte subsets and low levels expression of the activation marker HLA‐DR on both CD4 and CD8 subsets (Table [2](#pai13263-tbl-0002){ref-type="table"}), while B cells were about 0.03% of total lymphocytes (Figure [1](#pai13263-fig-0001){ref-type="fig"}).
{#pai13263-fig-0002}
######
Clinical chemistry values of two XLA patients with COVID‐19
Patient 1 Patient 2 Normal values
------------------------------ ----------- ----------- --------------- ----- -----------
C‐reactive protein (mg/L) 26 78 3.6 1.5 (\<5)
Lactate dehydrogenase (U/L) 170 194 235 248 (135‐225)
Fibrinogen (mg/dL) 598 737 424 517 (170‐410)
Ferritin (µg/L) 362 469 603 774 (30‐400)
Aspartate transaminase (U/L) 24 22 30 65 (18‐54)
Alanine transaminase (U/L) 17 19 26 230 (10‐50)
John Wiley & Sons, Ltd
This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.
######
Lymphocyte subsets of XLA patients
Patient 1 Patient 2 Normal values
-------------------------------------------- ----------- ----------- ---------------
CD3^+^ T lymphocytes (%) 88.4 94.7 (57.1‐87.6)
CD3^+^ (cells/µL) 1040 1791 (721‐2562)
CD4^+^ T cells (%) 42.1 44.9 (28.5‐65.6)
CD4+ (cells/µL) 495 849 (273‐1882)
CD8+ T cells (%) 43.3 30.6 (10.5‐37.7)
CD8+ (cells/µL) 509 578 (177‐783)
γδ^+^ T cells (%) 3.8 32.4 (0.9‐11.2)
B cells CD19^+^ (%) **‐** **‐** (5.8‐22.1)
CD19^+^ (cells/µL) **‐** **‐** (86‐684)
NK cells (CD3^‐^CD56^+^. CD3^‐^CD16^+^, %) 11.6 5.3 (3.4‐28.4)
CD3^+^CD4^+^ (%)
HLADR^+^ 3.1 2.4 (1.6‐12.2)
Naïve 68 63.7 (20.4‐63.6)
RTE 41.2 46.9 (11.4‐48.1)
RTE abs 204 398 (115‐913)
Central memory 10.4 22.1 (18.7‐46.2)
Effector memory 17.2 12.2 (7.1‐38.0)
Terminally differentiated 4.4 2 (0.3‐9.1)
CD3^+^CD8^+^ (%)
HLADR^+^ 7.2 7.2 (2.7‐31.7)
Naïve 29.7 30.6 (13.1‐66.5)
Central memory 0.5 4.2 (2.6‐24.5)
Effector memory 15.5 14 (10.1‐47.4)
Terminally differentiated 54.3 51.2 (5.2‐63.5)
Abbreviation: RTE, recent thymic emigrants.
John Wiley & Sons, Ltd
This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.
Patient 2 is a 26‐year‐old man who was diagnosed with agammaglobulinemia at 16 months of age because of respiratory tract infections and low immunoglobulins (IgG 63 mg/dL, IgA 7 mg/dL, IgM 12 mg/dL as compared to IgG 264‐1509 mg/dL, IgA 17‐178 mg/dL, IgM 48‐337 mg/dL in age‐matched children), virtual absence of B cells in the blood, and *BTK* mutation (S578Y). Since then, he received regular intravenous immunoglobulin infusions every 28 days and stayed in good health as chest CT scan in June 2010 did not reveal bronchiectasis. In the following years, he moved to London for work reasons and returned occasionally to see his family in northern Italy. In February 2020, he visited his family living in Milan just a few weeks before the SARS‐CoV‐2 epidemics. On March 23, he performed blood test to determine IgG levels, which were normal (IgG 923 mg/dL), before receiving immunoglobulin infusion. On April 1, he presented with anorexia, asthenia, and vomiting, without fever or diarrhea. Execution of nasopharyngeal swab at a local hospital in Milan area revealed SARS‐CoV‐2 infection; chest X‐ray showed an interstitial pneumonia (Figure [2B](#pai13263-fig-0002){ref-type="fig"}), while IgG levels were 863 mg/dL. In the following days, the patient presented with fever, but he never required oxygen supplementation. We started treatment with hydroxychloroquine, azithromycin, and ceftriaxone and administered immunoglobulin infusion at 400 mg/kg. In the following days, his conditions have been improving. Flow cytometry analysis of lymphocyte subpopulations, which was performed 7 days before the appearance of the first symptom, showed normal distribution of T and NK subsets (Table [2](#pai13263-tbl-0002){ref-type="table"}).
4. DISCUSSION {#pai13263-sec-0009}
=============
We describe two patients with XLA who have been exposed to SARS‐CoV‐2 and developed interstitial pneumonia and lymphopenia. Despite the absence of B cells, both subjects could recover without the need for intensive care or oxygen ventilation. This observation suggests that T‐cell response is probably important for immune protection against the virus, while B‐cell response might be unessential. This is in agreement with preliminary studies that have shown that in normal subjects infected by SARS‐CoV‐2, the number of cytotoxic T cells expressing activation markers such as HLA‐DR and CD38 increases during infection.[^4^](#pai13263-bib-0004){ref-type="ref"} However, the risk to develop pneumonia after SARS‐CoV‐2 infection is quite low in young adults such as the two XLA patients, suggesting that the lack of antibody production is probably contributing to disease severity. Moreover, there is also evidence that passive antibody administration through transfusion of convalescent plasma constitutes a feasible strategy to confer immunization to patients with severe COVID‐19 and contributes to improve the clinical manifestations and the outcome of these subjects, suggesting that production of neutralizing antibodies might also be an important step for disease recovery.[^7^](#pai13263-bib-0007){ref-type="ref"}, [^8^](#pai13263-bib-0008){ref-type="ref"} Therefore, the lack of neutralizing antibodies in XLA patients might place these subjects at higher risk of disease relapse, suggesting the need for careful monitoring during convalescence.
The observation that patients with X‐linked agammaglobulinemia can recover from SARS‐CoV‐2 infection suggests that human immune system could use multiple paths to counteract viral infection and that a normal T‐cell immune response can be sufficient to defeat the virus in subjects who cannot synthesize antigen‐specific immunoglobulins.
However, BTK is expressed not only in B cells, but also in myeloid cells. In these cell types, BTK is involved in Toll‐like receptor--mediated production of pro‐inflammatory cytokines, such as IL‐6 and TNF‐α, which are produced in large amount in patients with SARS‐CoV‐2 infection.[^9^](#pai13263-bib-0009){ref-type="ref"} Therefore, the lack of BTK in myeloid cells of XLA patients might provide a subordinate advantage for these patients by preventing the development of the inflammatory stage of the disease which has been associated with the possible fatal outcome of COVID‐19.
However, we cannot draw final conclusions from the description of these two cases of XLA, because the clinical and immunologic features of patients with this disease can be very heterogeneous. Indeed, Bruton\'s tyrosine kinase is required for many checkpoints during B‐cell differentiation and *BTK* mutations might also have variable effects on B‐cell precursor survival.[^10^](#pai13263-bib-0010){ref-type="ref"} In one of the two agammaglobulinemia patients, we detected an extremely low number of B cells at various stages of differentiation, suggesting that these cells might play a role in the immune response against the virus. Moreover, both subjects have received immunoglobulin infusions before the infection and have been further supplemented with immunoglobulins at the time of infection. Infusion of polyclonal immunoglobulins derived from pools of normal donors has been used to treat COVID‐19 patients,[^11^](#pai13263-bib-0011){ref-type="ref"} but there is limited evidence of efficacy. One might speculate that immunoglobulin pools might contain antibodies that could cross‐react with SARS‐CoV‐2 proteins by exerting a priming effect on host immune response. Alternatively, immunoglobulins could provide an immunomodulatory action on monocytes and tissue‐resident macrophages that are involved in the so‐called "cytokine storm" in the advanced phases of the infection in patients with severe COVID‐19.[^12^](#pai13263-bib-0012){ref-type="ref"}
Overall, our report provides evidence that B‐cell response can be dispensable in the immune response against SARS‐CoV‐2 infection, but clinical decisions should be based on data derived from a more broad survey of patients with IEI.
AUTHOR CONTRIBUTION {#pai13263-sec-0010}
===================
**Annarosa Soresina:** Conceptualization (lead); Investigation (lead); Writing‐review & editing (supporting). **Daniele Moratto:** Data curation (equal); Formal analysis (equal); Investigation (equal); Visualization (equal). **Marco Chiarini:** Data curation (equal); Investigation (equal). **Ciro Paolillo:** Investigation (equal); Validation (equal). **Giulia Baresi:** Data curation (equal); Formal analysis (equal); Investigation (supporting). **Emanuele Foca\':** Formal analysis (supporting); Investigation (equal). **Michela Bezzi:** Data curation (equal); Formal analysis (equal); Investigation (supporting). **Barbara Baronio:** Data curation (supporting); Formal analysis (equal). **Mauro Giacomelli:** Data curation (equal); Formal analysis (equal); Investigation (equal). **Raffaele Badolato:** Conceptualization (lead); Supervision (lead); Writing‐original draft (lead).
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Has the time come to put to bed the concept of a harm threshold when discussing the ethics of reproductive decision making and the legal limits that should be placed upon it? This is the question asked by Anna Smajdor in her article in this issue. For her, the answer is a resounding yes. She claims---after outlining the concept of the harm threshold in reproductive decision making, noting its many proponents in the philosophical community, and observing its inclusion in legal and policy documents related to reproduction---that no entity can be harmed by being brought into existence, regardless of the extent to which they will suffer once born.
Smajdor comes to this conclusion by appealing to a well-known literature that supports the notion that the act of creation cannot be identical with the acts of harming and benefiting. For if, in accordance with this literature, she suggests, existence should not be considered to be a 'real' predicate---that is, if existence cannot be considered an attribute of an object and thus fails to add to the concept of a thing---no entity can be harmed or benefited by being born. In short, her argument is based on the following simple claim: there does not exist a 'logical connection between the assertion that some lives are not worth living, and the claim that such people are harmed by being conceived' \[[@CR1]\]. Thus, her article, although she does not choose to characterize it in this way, actually constitutes the suggestion that those who subscribe to a harm based and person affecting account of morality (or of the limits of law) must acknowledge that, in holding this view, they are also required[1](#Fn1){ref-type="fn"} to accept the conclusions of a remarkably *strong version* of a problem in philosophy, known as the 'non-identity problem'.
In this commentary, we defend the claim that despite the conclusions of the non-identity problem, there are good moral reasons, based upon consideration of the suffering that would be experienced by the individual concerned, not to decide to bring him or her to birth. We have in mind cases in which a foetus or embryo is likely to become an individual whose life is variously described in the philosophical literature as 'empty of all the things that make life worth living' \[[@CR2]\], 'dominated by pain and suffering' \[[@CR3]\], 'intractably miserable' \[[@CR4]\], 'not worth living' \[[@CR5]\], or 'worse than no life at all' \[[@CR6]\].
Our arguments are, however, more than mere commentary. We aim to settle some tenacious misunderstandings of the logic of this corner of moral discourse by exploring and explaining the difference between the use of comparative and non-comparative accounts of harm in non-identity cases and the problems that occur when such accounts are conflated. We thus begin our commentary by providing a reconstruction of the major components of the arguments contained within Smajdor's article. We then question whether Smajdor's use of Derek Parfit's arguments in her own is a charitable one that truly captures the spirit in which they were made. After this, we note that although a threshold account of prenatal harm may not be compatible with comparative accounts of harm, the logical inconsistencies Smajdor associates with this account do not occur on non-comparative accounts of harm, such as the one championed by one of us, John Harris, in his book *Wonderwoman and Superman*.
Doing away with the harm threshold: reconstructing, summarising, explaining, and situating Smajdor's position in relation to our own {#Sec2}
====================================================================================================================================
Smajdor begins her attack on the harm threshold in reproductive decision making by noting the benefits that are often associated with harm-based, person-affecting accounts of morality and the proper limits of legislation. She notes, for example, that although she does not subscribe to the belief 'that harm to offspring is the sole focus of moral concern in reproductive decisions' \[[@CR1]\], there is something to be said for a harm-based approach to legislation and morality. This can be found, she suggests, in the fact that a focus on the harms our choices may impose on others are more identifiable and less subjective than a focus on deontological principles or impersonal and free-floating harms. She thus suggests that because of this more empirical focus, harm based approaches 'may be a useful interface between morality and legislation' \[[@CR1]\]. She then observes, however---in recognising the conclusions of the non-identity problem, as have many before her, including ourselves (see, for example, \[[@CR7], [@CR8]\])---that this approach seems to offer very few of the benefits it provides in cases of harms to extant persons in the context of reproduction.
This is so because if we limit moral and legal criticism only to those acts which can be said to negatively affect the interests of some particular entity, it turns out that our reproductive decisions will, in many cases, have little or no moral content, despite our intuitions to the contrary. That is, if we also accept the relatively uncontroversial proposition that our coming into existence is highly precarious---'dependent on the conditions under which we and our descendants procreate, with the slightest difference in the conditions of conception sufficient, in a particular case, to \[ensure\] the creation of a different future person' \[[@CR9]\].
There are a number of versions of what might be called 'the precariousness proposition', which produce slight differences in the kinds of circumstances in which non-identity cases are generated. This is so as the particular version to which one subscribes depends greatly on one's views concerning what it is that makes one numerical person the same numerical person over time and change. However, the version to which Smajdor appeals in her article seems to be based on an acceptance of the precariousness proposition as it is formulated in the work of Parfit in his two versions of the 'time dependence claim' (TDC). As she does not make it clear to which version she subscribes---and this actually makes little difference in the cases she considers---we will assume her allegiance is to the slightly weaker form, which, as Parfit notes, is *in fact true*, although not necessarily so:"TDC 2: 'If any particular person had not been conceived within a month of the time when he was in fact conceived, he would in fact never have existed'. \[[@CR10], p. 352\]"
This version of the precariousness proposition may be interpreted in three different ways. It can be seen, firstly, as a causal claim regarding the importance of our genome for the development of personal identity (understood as psychological connectedness and continuity) by noting, for example, that 'differences in \[genetic\] material make for later differences in virtually all aspects of a person: change the sperm, and there will be substantial changes (of both a physical and psychological kind) in the later human being' \[[@CR11]\]. Secondly, it can be viewed as a weak version of Kripke's origins claim regarding the importance of our material origins for numerical identity on biological accounts of personal identity over time. This interpretation suggests that just as all material things must have their origin in at least some of the matter from which they are constituted, so too must all numerical persons have their origins in, *inter alia*, the genetic material from which they are constituted in order to be considered the same numerical entity \[[@CR8]\]. Thirdly, however, one might view it as a temporal or environmental claim regarding the importance of possible epigenetic factors and others flowing from the time, place, and manner of our conception. Time, place, and manner may thus embrace factors such as differences in the method of conception, gestational environment, maternal nutrition, the general external environment, and exposure to potentially teratogenic substances during pregnancy.
Depending on one's interpretation, it can therefore be noted that non-identity is not simply related to conception and factors tied to the timing of conception, nor, as the Parfit of *Reasons and Persons* perhaps believed, simply to genetic identity \[[@CR8], [@CR11], [@CR12]\]. However, Smajdor's interpretation of the claim does seem to fall into either the causal or Kripkean camp, as she notes numerous times in her article, the importance for personal identity of our being conceived from the particular egg or sperm from which we were conceived. This means that, whilst non-identity may not occur in cases of straightforward prenatal harm, such as when a pregnant woman ingests a teratogen or a foetus is injured in some other way during pregnancy, it will be commonplace in cases of genetic decisions regarding, for example, with whom we choose to procreate and when we wish to do so. For this understanding of the TDC2, when paired with a person affecting account of morality that rests on a comparative account of harm, yields a particular prescription. This is that many reproductive decisions turn out to affect not the interests of persons created as a result of such decisions but their identities, and thus cause no harm to those created as a result.
Indeed, the trap of non-identity is not only evidenced in cases of seemingly harmful conceptions. Many people at the time of World War II, for example, including the parents of one of the present authors, decided to await the result of the war and the defeat of Nazism before conceiving or conceiving again. Such people believed they were somehow acting in the interests of the child that would be born to them, despite the fact that they seemed to be 'guilty' of falling into the trap of non-identity. It should be noted too that non-identity poses an 'intriguing theoretical obstacle' \[[@CR13]\] to questions of intergenerational justice and, as has been most recently noted, to cases of affirmative action and of apology or reparation for historic injustices. For, ironically, were it not for the wrong that was done to the ancestors of those now seeking apology or compensation, the latter would almost certainly never have existed \[[@CR14]\].
Smajdor illustrates this point in the reproductive case by providing an example of a woman who is receiving treatment for syphilis and must make a choice between conceiving now and giving birth to a child with congenital syphilis, or waiting until after she has been cured and giving birth to a 'healthy' child (free from congenital syphilis). Like Parfit, Smajdor suggests that despite many people's intuitions to the contrary, the woman has little reason based on the interests of the children she would create to choose to wait. For, whilst many of us tend to believe that it is better to be born without a disability than with one, as disability is, by definition, disadvantageous, person-affecting morality actually gives us little reason based on the interests of the child created to wait to conceive.
If one accepts the TDC2 and holds too that our actions only have moral status when they affect the interests of distinct numerical persons, one can see that regardless of the decision she makes, her child will not be harmed as different children will come to exist depending upon her choice. For, whilst it is true that if she conceives now, her child will be born with congenital syphilis---an undoubtedly harmful condition which may result in cerebral palsy, hearing loss, and musculoskeletal deformities---should she wait to conceive a child free of syphilis, a different child will be born as 'a different egg and sperm will be involved, resulting in a genetically different individual' \[[@CR1]\]. The child the woman could conceive now could not exist without suffering from congenital syphilis. Being brought into existence suffering from such a condition cannot harm him. For, whilst he might rationally prefer a life without his affliction, the alternative for him is not a life without the effects of congenital syphilis, but no life at all, as syphilis is a condition of his very existence.
Here, however, is where Smajdor takes her position to depart from Parfit's. For, whilst she agrees that in the case of congenital syphilis, the woman would not harm her child by bringing him into existence with this affliction, non-identity statements are not always so simple.
For although this is not always the case, such statements often come with a qualifier that warns against the creation of lives that are 'empty of all the things that make life worth living' \[[@CR2]\], 'dominated by pain and suffering' \[[@CR3]\], 'intractably miserable' \[[@CR4]\], 'not worth living' \[[@CR5]\], 'worse than no life at all' \[[@CR6]\]. This qualifier can be found in the works of many authors who write on the non-identity problem. Smajdor, for example, notes that Robertson suggests that although it is normally the the case that 'a child's interests are hardly protected by preventing that child's existence..., this objection would not hold if the ... conditions of his life would be so harmful to him that from his perspective he would prefer not to live' \[[@CR15], p. 75\].
It is this qualifier with which Smajdor takes issue. For, she holds that its addition entails a commitment to a questionable view: the view that 'a child born with a worse disease than congenital syphilis *could* have been harmed---if the disease is so terrible as to mean that she does not have a minimally acceptable quality of life' \[[@CR1]\]. In other words, she claims that to add this qualifying phrase entails a commitment to the view that conception can constitute a harm to the child in the latter but not the former case because 'a threshold has been passed ... which was not breached in the case of the child with congenital syphilis' \[[@CR1]\]. Despite the fact that Smajdor fails to truly unpack the threshold account of harm as it is said to apply to non-identity cases, her characterisation of the account is accurate albeit bare. For, those who subscribe to a threshold account of harm in such cases should be seen to hold that some procreative choice made at T~1~ harms a person if and only if it causes into existence a person who falls below some normatively defined threshold of wellbeing, interest satisfaction, etc.
For Smajdor, such accounts are deeply problematic as she is sceptical about the existence of a 'logical connection between the assertion that some lives are not worth living, and the claim that such people are harmed by being conceived' \[[@CR1]\]. After a rather long section in which she notes the seeming arbitrariness of a threshold account of harm and a number of troubling conclusions associated with this \[[@CR1]\], she provides support for her scepticism by asking the reader to consider Kant's refutation of Anselm's ontological argument for the existence of God.
In his *Proslogion,* Anselm claims to derive the existence of God from definition: the concept of God as 'something than which nothing greater can be thought' \[[@CR16]\]. He held that if such a definition is true, God must exist in reality, since if He does not, a greater being can be conceived of: one than which nothing greater can be thought, and which, in fact, exists. Kant however, argued that Anselm committed a grave error in his argument, suggesting that although Anselm treats existence as a property that things may either possess or fail to possess, to say that some entity exists is not to confer existence on it. Instead, it is to say that the concept of the thing is exemplified in the world, just as to say that some entity does not exist is not to state that a thing lacks the property of existence but to say that the concept of that thing is not exemplified in the real world. In other words, Kant argued that a God that exists is identical to a God that does not. For, whether something exists or does not exist does not add to or alter the properties it possesses, it is the mere positing of a thing: 'a hundred real thalers do not contain the least coin more than a hundred possible thalers' \[[@CR17], pp. B626-7\] and 'the real God is not a few degrees more perfect than a conceptual one' \[[@CR1]\].
With this done, Smajdor suggests that we may straightforwardly apply Kant's argument to the act of creation and the question of whether an entity can be harmed by creation itself. For, she notes, 'the act of creation can be construed uncontroversially as the act of bringing something into existence,' and thus, 'whatever is logically true of existence in general should be true of creation insofar as it is the conferring of existence.... If existence itself cannot entail any other property, then it follows that the mere act of giving existence cannot encompass the act of conferring any property---other than existence---on the entity which is created' \[[@CR1]\]. With this in mind, Smajdor concludes that creating something, or bringing it into existence, cannot be the same as harming it. For, although harm is not a predicate in the usual sense, the fact of having a harmful genetic constitution is. It qualifies an entity in the same way as other predicates. Thus, she suggests that 'if we cannot make X greater, more perfect, or more valuable by bringing it into existence, neither can we harm X by bringing it into existence however greatly X must suffer' \[[@CR1]\]. She therefore concludes:"There is no specific act that can be construed as harming a future child when the child's condition is directly linked with the circumstances surrounding its conception. Nor can these questions be a matter of degree, since the logical and metaphysical constraints that prevent us from concluding that a child born with a moderate amount of suffering has been harmed apply equally to all cases of creation. \[[@CR1]\]"
Is Smajdor's characterisation of Parfit's position fair? {#Sec3}
========================================================
With the major components of Smajdor's argument explained and laid out above, we now return to the point at which she takes her position on the possibility of harming by the act of creation to differ from Parfit's. For, we contend that the characterisation she offers of Parfit's view on the matter of harms to individuals with 'lives that are less than worth living' is, at best, uncharitable, and at worst, based on a misunderstanding of the claims made by Parfit on this matter.
The point at which she holds her position to diverge from Parfit's occurs just after an acceptance of the conclusions of the non-identity problem in the case of the child with congenital syphilis. For, as is noted above, she and Parfit agree that in this case there is little reason for the woman to choose to wait based upon the interests of the child she creates. Her choice is a choice between lives as opposed to a choice of whether to harm or benefit her child. However, whilst this is so, Smajdor argues that Parfit does not discount the possibility that there is a point at which the woman's child could be harmed by his conception: the point at which this child's life would be less than worth living, dominated by suffering, or where the quantity and quality of whatever it is that makes life worth living falls below some threshold level, such that the harms he would suffer throughout his life outweigh its pleasures.
Smajdor, however, denies that this could be the case, holding that even if the woman's choice was between a worthwhile and a less than worthwhile life, no harm would be done to the child created should she choose to bring to birth a child whose life falls below this threshold level. She characterizes Parfit's view regarding lives that are dominated by suffering as follows: 'that the child could not have existed without that condition does *not* prevent us from concluding that \[he\] has been harmed. A threshold has been passed in the latter case, which was not breached in the \[former\] case' \[[@CR1]\].
She then explores what Parfit might mean by this: asking *why* Parfit should hold that in the case of congenital syphilis the child created cannot be harmed by his mother's choice, but would be harmed should he suffer more seriously. What, she asks, is it that makes the suffering of the child bear on questions of the morality of the mother's actions in the latter but not the former case? Why does Parfit seem to divorce the concept of harm from its normal relationship with suffering, turning it into a mercurial entity that flashes into existence only in certain very specific circumstances? \[[@CR1]\]
After all, Parfit claims, as do many others who accept the conclusions of the non-identity problem, that non-identity is a logical and metaphysical problem as opposed to one relating to the degree of suffering an individual must endure before he can be deemed harmed, all things considered. The mother is---according to its logic---not morally responsible 'because there is no causal mechanism by which we can understand him to be harmed' \[[@CR1]\]. Her blamelessness has nothing to do with the fact that her child does not suffer enough or because the harms of his existence fail to outweigh the pleasures his life contains. Her child is not harmed and she may not be criticized morally because he could not have existed absent his condition and she could have done nothing to alleviate his suffering without causing some other child to exist instead of him.
Smajdor thus notes that due to this there is no reason to assume that the conclusions of the non-identity problem should not apply equally in the more serious case. The relevant facts are, she contends, the same in both, regardless of the extent to which the created child suffers once brought into existence. Thus, she holds that Parfit and others are guilty of contradicting themselves when they state that in cases of 'lives less than worth living', the child created could be harmed as they are essentially asserting that despite their logical position, 'existence cannot harm someone ... \[but\] it is, after all, a question of degree' \[[@CR1]\].
Yet, whilst Smajdor's point is well made---the non-identity problem does seem to apply equally to cases of worthwhile and less than worthwhile lives as the facts relevant to its generation are the same---at no point within *Reasons and Persons* does Parfit suggest that this is not so. He does not claim, despite Smajdor's assertion, that the child created in the more serious case can be harmed by the act of his creation because his life is less than worth living. He leaves this open to interpretation just as he leaves open to interpretation the question of whether causing some person to exist can be said to cause them some peculiar benefit. He does this in virtue of the fact that he views both a negative and positive answer to the question of whether existence can constitute a predicate as being defensible \[[@CR10], p. 358\].
It should be noted too, in fairness to Smajdor, that because of this, Parfit does not make clear that the conclusions of the non-identity problem may still hold in cases of less than worthwhile lives dependent on the views one holds regarding this. All of his non-identity cases, for example, regard unquestionably worthwhile lives and thus say nothing about the conclusions of the non-identity problem in cases of lives which are less than worth living. Parfit considers, for example, a child whose mother had him too young but whose life, despite its 'bad start..., \[is\] predictably worth living' \[[@CR10], p. 358\]; a choice between conserving our resources for future generations or depleting them such that future generations will live lives of much lower but still acceptable (although barely so) quality \[[@CR10], pp. 361-4\]; and a woman who must choose between waiting to conceive or having a child with a painful but not terrible disability \[[@CR18]\]. In other words, although Smajdor asserts that Parfit's claim is based on a mistake---the formation of an untenable connection between the assertion that some lives are not worth living and the claim that such people are harmed by being conceived---the mistake here seems to be her own. The claim she attributes to Parfit can, on a close reading, be found nowhere in the work she references although, as can be seen in appendix G of *Reasons and Persons*, he is sympathetic to this view.
Can a child be harmed by his own conception? On comparative and non-comparative accounts of harm in genesis cases {#Sec4}
=================================================================================================================
Smajdor seems to suggest that the only plausible accounts of harming are those that are comparative in nature. That is, she seems to recognise as proper only accounts of harming that compare some particular numerical entity's current state of welfare, happiness, interest satisfaction, etc. to the state that he would have been in had some particular action not occurred. Examples of such accounts are that of the diachronic account, according to which some particular action (or inaction) done at time t~1~ is harmful *iff* it causes some person (p) to be worse off at some later time t~2~ than they were at t~1~, and the subjunctive historical account made famous by Joel Feinberg, which suggests that some particular action (or inaction) done at t~1~ is harmful for p *iff* it causes p to be worse off at t~2~ than he would have been at t~2~ had it not occurred \[[@CR5]\].
Such accounts necessarily exclude the possibility that present persons may harm future and merely possible persons by acts of creation in cases of both worthwhile and less than worthwhile lives. This is so because in order to make a harm claim on comparative accounts, one is required to compare the state of p's interests, welfare, happiness, etc. with the state he would have been in had the act not been performed (subjunctive historical) or was in prior to the performance (diachronic). Thus, as the alternative in non-identity cases is non-existence and non-existence is no state at all, there can be no way to make such a comparison. The writing is, on such accounts, already on the wall as it is nonsensical to discuss a harm threshold when its existence is precluded by the nature of the account in question.
Yet, that this is so does not preclude the possibility that comparative accounts may be compatible with the following argument: although a child who will have a less than worthwhile life may not be harmed by his conception, he may be harmed if his suffering does not end as soon as it begins (whether before or after birth), and this may constitute a good reason not to conceive such a child in the first place. This is not the question addressed by Smajdor in her article, and as such, it has been understandably glossed over. However, it does seem important. For, it means that on comparative accounts, although we may not say that a child can be harmed by his conception even if his life is one that is less than worth living, we can provide good person-affecting reasons to support a moral requirement to prevent the suffering of a child whose life will be dominated by suffering.
For, if there exists a moral duty not only to refrain from causing persons to suffer but also to actively seek to ameliorate suffering where it occurs, we may find that in cases of lives dominated by suffering, there is a moral duty to end the lives of such individuals as soon as they come to suffer. We need not claim that the child created is harmed by his own conception or that those responsible for his conception are to blame for his poor prospects. He is not and they are not, as nothing could have been done to avoid this unfortunate stacking of the deck. Yet, whilst the prospective child may not be harmed by his conception, we may hold that those responsible for his conception are morally blameworthy for failing to stop his experience of such severe and uncompensated suffering in the face of full knowledge of his devastatingly poor life prospects.
Consideration of the nature of different accounts of harm seems to uncover another major problem inherent in Smajdor's article. This can be found in the fact that it is poor philosophical practice to attempt to criticise one account by merely showing that it is different from another. That such is the case is already transparent. The utilitarian may not fairly criticise the Kantian by stating only that he is not a utilitarian. He must instead uncover some fatal flaw in the theory of his foe or appeal to good reasons suggesting his account is more plausible. With this in mind, we can note that those who subscribe to comparative accounts of harm cannot refer to the inadequacy of non-comparative theories by merely noting that they are not the same as their own. The threshold account is a non-comparative theory. As such, what is needed on Smajdor's part is an explanation of *why* the particular account of harm she seeks to criticise should be seen to be lacking in some sense, and this can only be done via explication *or* the provision of an explanation of *why* one's own account overcomes such shortcomings by demonstrating both its superiority and its avoidance of other equally troubling conclusions.
Comparative accounts are, however, not the only plausible accounts of harm to which one may appeal. To be sure, they are intuitive, since generally, when we think of harm, we think of things going worse for someone than they otherwise might have. Yet, despite this, such accounts are not a panacea. They face a number of problems that are not limited only to the fact that they fail to account for our intuitions in non-identity cases. Thus, although we might be willing, as Smajdor is, to bite the bullet in such cases and accept the counterintuitive conclusions comparative accounts engender in such cases, these are not the only counterintuitive conclusions such accounts are often argued as entailing. Comparative accounts have been said, for example, to fail to account for circumstances in which we wish to say that an individual has been both significantly harmed and greatly benefited by an act which causes an on-balance benefit, such as in Seana Shiffrin's example of the generous but dangerous millionaire who drops large blocks of gold bullion from the sky as gifts to citizens of a neighbouring town where the million dollar manna ends up falling on and injuring one of the recipients \[[@CR19]\]. They are charged with multiplying harms excessively due to the fact that, on such accounts, harms are grounded in comparisons, leading to confusing and questionable determinations. For when one is harmed, one does not undergo a separate harm relative to each earlier moment in one's life at which one fared better \[[@CR20]\], as would be the case on the diachronic comparative account, and neither does one undergo a number of different harms in cases where one is shot just because one would have been better off had one's assailant not pulled the trigger, had the gun not gone off, or had the bullet missed one's body, as would be the case on the subjunctive historical account \[[@CR20]\]. Comparative accounts, it has been said, fail to account for the harm in beneficial cases of self-harming, as in the case of the 'Blighty' wound \[[@CR7], p. 92\],[2](#Fn2){ref-type="fn"} and for pre-emptive harms \[[@CR20]\]. Indeed, whilst this is not the place for a lengthy discussion of these shortcomings, they are numerous and have been widely discussed within the literature.
One account of harm often presented as a credible alternative to comparative accounts, and which seems to overcome a great number of the problems often associated with them, has been championed by one of the authors of this commentary, Harris. It is set out in great detail in his book *Wonderwoman and Superman* and is often termed the 'harmed state account'. On this account, and on other similar accounts of harm such as that proposed by Shiffrin \[[@CR19]\], the notions of harming and wronging set out in comparative accounts of harm seem to be turned upside down, and a particular numerical entity can be said to be harmed when it is simply the case that he has been put into a condition that is harmful. As Harris explains:"A condition that is harmful ... is one in which the individual is disabled or suffering in some way or in which his interests or rights are frustrated. The disability or suffering may be slight, just as harms are trivial.... I would want to claim that a harmed condition obtains wherever someone is in a disabling or hurtful condition, even \[if\] that condition is only marginally disabling and even \[if\] it is not possible for that particular individual to avoid the condition in question. \[[@CR7]\] p. 88\]"On this account of harm, therefore:"To suffer harm is to come to be in---or perhaps better, is simply to be in---a certain sort of non-comparatively bad state. It is to come to be in ... a state in which one fares, not worse than one fared, or would have fared, in some alternative state of affairs, but simply badly. The seriousness of a given harm, according to this way of thinking, is proportionate to the (non-comparative) badness of this state. \[[@CR20]\]"
Such accounts therefore leave open the possibility of all sorts of harms befalling future and present individuals even if they cannot be said to have been made worse off. Quite often, such individuals will, of course, be made worse off, but this is not a central question. As such, the possibility is left open that individuals can, and quite often *will*, be deemed harmed in non-identity cases, because although they would never have come into existence had the act not been done, in order to determine harm, one needs only to point to the fact that the individual suffers.
Returning to the congenital syphilis case, then, we note that should the woman choose to conceive now, she will conceive a child who suffers from the effects of congenital syphilis, and thus, her child will be born harmed by her action. Despite the fact that had she made a different choice he would not exist, she is still to be held responsible for the harm that has occurred as 'where B is in a condition that is harmed and A and/or C is responsible for B's being in that condition, then A and/or C have harmed B' \[[@CR7], p. 89\]. Non-identity does, however, still represent a problem on such accounts if we are unwilling to appeal to impersonal harms and wrongs[3](#Fn3){ref-type="fn"} in cases such as the above, resulting in harmed but still worthwhile lives, but it occurs for the reason that no wrong can be determined. It remains a problem:"Not because the life in question has not been impaired, not because the individuals are not suffering, not because they have not been harmed: it has, they are, and they have: rather because it is not possible to regard them as having been wronged. You might harm someone in order to benefit them, but if so, you do not wrong them unless you violate their will in order to do so or breach some other obligation to them. The mother giving a life with some measure of disability to a child who will find such a life worth having does not wrong her child. She is like the doctor giving a drug \[that\] has damaging side effects but side effects \[that\] are worth enduring for the sake of staying alive. \[[@CR7], p. 95\]"
In the congenital syphilis case, the child *should* be seen as harmed by his mother's act, but the mother is saved from blameworthiness *because* her child has a life that is worth living. He will not be wronged by her because, 'like those with Blighty wounds or those who have to endure the harmful side-effects of beneficial drugs, \[he\] has received a net benefit from what has happened to \[him\] and none of \[his\] rights have been violated' \[[@CR7], pp. 95-6\]. It would be irrational for him to condemn his mother for her choice as he would not have existed without his condition and his existence is, although not perfectly so, pleasurable.
Yet, returning to the more serious case of the choice to bring to birth a child whose life will be less than worth living, we note that non-comparative accounts can state that to make such a choice, in full knowledge of this fact, constitutes both a harm and a wrong.[4](#Fn4){ref-type="fn"} In other words, non-comparative accounts are, unlike comparative accounts, compatible with a subscription to a harm threshold. This, of course, requires an answer to the question of what it actually means for an individual to be in a worse off condition than non-existence, and to make a determination of where this threshold lies. However, despite Smajdor's protestations, an answer can be easily found. For, although non-existence is not a state of being and thus cannot be said to be preferable *for* an individual, it can be in an individual's interests to end her life or to have her life ended for her. If it were not, we would not view it to be 'better' to painlessly end the life of a suffering animal than to allow it to continue existing in a state that is both terrible and terrifying for it, and neither would we seriously consider questions relating to rational suicide or voluntary euthanasia. As Parfit notes:"A certain kind of life may be judged to be either good or bad---either worth living or not worth living. If a certain kind of life is good, it is better than nothing. If it is bad, it is worse than nothing.... Consider someone dying painfully who has already made his farewells. This person may decide that lingering on would be worse than dying. To make this judgement, he need not *compare* what it would be like to linger on with *what it would be like to have died* ... he might consider what lay before him, and decide whether he did or did not want to undergo it. \[[@CR10], p. 487\]"
With this in mind, it is held that for existing individuals, the question of what constitutes a less than worthwhile life can only be answered subjectively, for in such cases we ask it in regards to 'individuals who can have a view about the desirability of their own existence'. In this sense, 'a condition is worse than non-existence if and only if the subject would rather not exist than exist in such a condition' \[[@CR7], p. 93\]. Where the threshold lies for conception to be considered wrongful is trickier to determine, however. For in such cases, we cannot ask the child to be created whether he would consider his life worth living, and nor may we appeal to the conditions under which we would consider our own lives to be less than worth living. Instead, we must attempt to determine, in light of the information available to us, whether the child to be created is likely to consider his life to be worth having, and this is to be done by assessing 'whether or not such a life has a favourable balance of satisfactions over miseries' \[[@CR7], p. 93\].
If we turn, then, to Smajdor's concerns regarding whether existence can constitute a predicate, we can note that it is not at all clear how such considerations bear on the question of harmful conceptions and wrongful lives on threshold accounts. For on such accounts, whether we do or do not choose to bring to birth a child with a harmful genetic constitution has little bearing on the properties with which he would enter the world. To be sure, non-existent things lack all properties, as existence is a precursor for having properties, and thus, to state that a child can be harmed or benefited by not being brought into existence is nonsensical; however, existent things can, on non-comparative accounts of harm, possess as a necessary property the property of harm itself. For example, we can note that in the congenital syphilis case, whether or not the mother decides to birth the child/children she could have before the cure, it will be deemed that his/their necessary properties are harmful as such properties result in suffering. The harm is inherent in that child's necessary properties, and thus, the concept of the child with congenital syphilis already contains harm, which is not altered in the least bit by our choice of whether or not to bring him to birth or by whether we view existence to constitute a predicate.
Indeed, the corollary on such accounts seems to confirm this: for, non-comparative accounts such as Harris's also hold that if an effective treatment were discovered after the child's birth it is the harm that the child experiences which provides the motive for administering the treatment. If the child were not in a harmed condition, if the condition is not harmful, then why attempt to treat the congenital syphilis or seek to discover a cure?
The same, incidentally, goes for understanding the moral imperatives engaged by the possibility of human enhancement, the possibility of improving on normal species functioning or species typical functioning. The moral motive for human enhancement is generated by the possibility of ameliorating the human condition, of seeing the harmfulness of things as they are, the imperfections of human nature. If there is such a thing as human nature, this 'fact' does not prevent us from seeing its limitations and harmful effects, even when we have no different extant states of being with which to compare. The possibility of enhancing evolution, of improving on human nature, seems, on such accounts, to create a new conception of what it is to be in a harmed condition, not relative to existing alternative states, but to possible enhancements \[[@CR21], pp. 86-109\].
Conclusion {#Sec5}
==========
We have considered a number of arguments forwarded by Anna Smajdor in her article How Useful is the Concept of the "Harm Threshold" in Reproductive Ethics and Law?' where she sought to uncover a number of problems inherent in the concept of the harm threshold by appealing to Kantian arguments concerning the question of whether existence can constitute a predicate. After exploring and explaining Smajdor's arguments, we acknowledged that the question of whether existence can constitute a predicate is relevant for questions of the ethics of reproductive decision making on comparative accounts of harm, and thus, that the concept of the harm threshold lies on shaky ground on such accounts. However, while such is the case, we have also shown that Smajdor's characterisation of Parfit's position on the possibility of threshold harms in non-identity cases rests on a mistake and that the arguments to which Smajdor appeals do not seem to apply on non-comparative accounts of harm, which, incidentally, tend to be the accounts of harm to which those who actually appeal to the concept of a harm threshold subscribe.
Smajdor, therefore, fails to provide compelling reasons for those who subscribe to non-comparative accounts to abandon their claim that in cases of lives less than worth living, persons can be both harmed and wronged by being brought into existence. With this in mind, we suggest that should Smajdor wish to continue her work in this area, she must try to engage more fully with non-comparative accounts of harm and provide arguments giving those who subscribe to such accounts reasons to abandon their view. For, if she believes, as her article seems to suggest, that we may only appeal to comparative harms when making ethical and legal decisions, such a position cannot be defended along the lines she proposes.
Provided they accept some version of the claim that our existence is dependent on our being conceived from the gametes from which we are actually conceived.
The term 'Blighty wound' is often used to refer to a wound (often self inflicted) sustained by a soldier during wartime that is serious enough to get him sent home from the battlefield but not so serious as to kill or reduce his quality of life in any significant way, thus, benefiting him overall.
Broadly defined as those resulting from the bringing of avoidable suffering into the world.
Note that in cases where the decision to bring to birth lives that are highly likely to be less than worth living for reasons other than the child's own limitations, such as for reasons of social justice, there may be other proper subjects of blame than the mother. Consider, for example, the choice to bring to birth a black child in a highly racist society or a case in which an individual would have a life that is worth living but for the denial of the resources that should, in accordance with one's particular theory of justice, be provided to him.
We would like to express our gratitude to the Arts and Humanities Research Council and the Wellcome Trust who provided us with both the funding that facilitated the undertaking of this project and the ability to publish it in an open access format.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Schizophrenia is a chronic mental illness. Some studies show up to 90% of individuals with mental disorders live with relatives who provide long-term practical and emotional support for them ([@B1], [@B2]) and caregivers' burden increases with more contact with patient and the time patients live with their families ([@B3]). Family caregivers engaged in providing care for a patient member may experience grief for loss of patient member's former personality and family lifestyle ([@B4]).
Family burden in caring patient members with long-term disorder may lead to objective and subjective difficulties ([@B5]) and need educational and treatment interventions. Caregivers may experience objective burden such as facing disruption of family relationships, constrained levels of social, work and leisure activities and financial problems. They may experience subjective burden such as feeling of loss, anxiety, sadness and pressure in confronting with disturbing behaviors of their patient family members and frustration that change in relationships impose on them ([@B6]).
In Iran, caregivers play a significant role in supporting family members with schizophrenia. Some studies show Iranian caregivers of patients with schizophrenia endure significant burden in maintaining their patient members ([@B7]), especially when psychopathology of the patients and deficiencies in basic life skills are manifested among patient members ([@B8]). A recent study showed Iranian caregivers experience a significant level of anxiety, depression, disturbance in sleep, and psychosomatic problems and 76% of the parents reported disturbance in their mental health status ([@B9]) but despite all these problems, Iranian caregivers prefer to maintain their patient members and they reported need for hospital beds at the time of relapse and prefer rehabilitation and educational programs face schizophrenia ([@B10]). In recent years, some sporadic studies have shown case management ([@B11]) and family interventions ([@B12]) have been effective in both reducing burden of care giving among Iranian caregivers and some symptoms of the patients but such interventions can be more effective if they are implemented based on characteristics and differences of main groups of consumers and their caregivers.
In regard with critical limitations in financial resources for treating Iranian consumers, the necessity for implementing treatment services for consumers and educational services for their caregivers with optimal effectiveness is a health priority in Iran. This trend is budget saving and may contribute to maximum treatment outcome but this goal is achieved when characteristics and differences of the two mentioned groups of consumers are identified and treatment and educational programs are implemented based on these identified characteristics and differences. This issue may also contribute to promote the efficacy and effectiveness of the implemented services.
To partly contribute to this aim, the current study was designed to describe and compare demographics, clinical and symptom-related characteristics among the two mentioned groups of consumers and knowledge on schizophrenia, family function, burden, and availability of social services and support among their caregivers referring to the Iranian Society Supporting Individuals with Schizophrenia (ISSIS) as one of major centers for consumers and their caregivers in Tehran, Iran.
Materials and Methods
=====================
***Participants***
Two hundred and thirty one consumers with their main caregivers from (ISSIS) were eligible to enter into the study and agreed to be interviewed and completed all the measurements. Of the 231 consumers, 106 consumers were current chronically hospitalized (group 1) and 125 of them were current household maintained (group 2).
Those consumers who were diagnosed as mentally retarded, or were diagnosed with comorbid psychiatric and/or severe physical disorder and current use of substance and alcohol were excluded.
Current chronically hospitalized consumers were characterized by at least one time of hospitalization in psychiatric hospitals in each year of the past 5 years because of severe presence of psychiatric symptoms and more than 5 years of psychiatric diagnosis with schizophrenia while current household maintained consumers were characterized by constant residence with families with or without history of psychiatric hospitalization in the past 5 years and less than 5 years of psychiatric diagnosis with schizophrenia.
The majority of the consumers were elderly single and unemployed males but we also recruited female consumers while the majority of caregivers were elderly mothers who were homemakers with limited education and financial resources but we also interviewed spouses, fathers, brothers and other family members of the consumers. All the participants were provided with oral and written informed consent forms and the protocol of the study was approved by the institutional review board of Shahid Beheshti University in Tehran.
***Assessment procedure***
*Patient-related assessment:*In the first phase, diagnosis of schizophrenia in participants was confirmed by using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders 4^th^ edition (DSM-IV), Psychotic Disorders Version, and severe combined immune deficiency (SCID-I) ([@B13]). Demographic and clinical characteristics of the participants were completed based on a questionnaire designed to do so. The severity of positive symptoms of schizophrenia was measured by the Scale of Assessment of Positive Symptoms (SAPS) ([@B14]) and the severity of negative symptoms was measured by the Scale of Assessment of Negative Symptoms (SANS) ([@B15]). In this study, internal reliabilities via Cronbach's alpha coefficients for positive and negative symptoms were 0.83% and 0.87%, respectively.
Kohlman Evaluation of Living Skills (KELS) ([@B16]) was administered to measure abilities of participants in basic life skills. In this study, the internal reliability via Cronbach's alpha coefficient was obtained 0.72%.
*Caregiver-related assessment:*After completing socio-demographics for caregivers, Knowledge Questionnaire for Caregivers was administered to evaluate the family knowledge about schizophrenia. A brief version of this questionnaire developed by Khazailie with 31 questions was administered covering the symptoms, treatment and family's awareness and behavior toward the patient ([@B17]). The reliability of this questionnaire within a week was 0.82%. Cronbach's alpha coefficient in the present study was 0.75%.
Family Experience Interview Schedule (FEIS) was administered to evaluate family characteristics and burden ([@B18]). In this study, a short version of FEIS including 41 questions was administered and internal reliability via Cronbach's alpha coefficient within a week was 0.89% ([@B11]). In this study, Cronbach's alpha coefficient was 0.90%.
To evaluate the perceived social support, Social Support Scale (SSS) ([@B19]) was administered. Internal reliability via Cronbach's alpha coefficient of this scale in the present study was 0.70%.
To evaluate family function, we administered Family Assessment Device (FAD) devised by Epstein et al. In the original study, internal Cronbach's alpha ranged from 0.72-0.92% between the subscales ([@B20]). In this study, Cronbach's alpha ranged between 0.61-0.86%, respectively.
***Data Analysis***
A series of independent sample t-test and Mann-Whitney U analysis tests in SPSS for Windows 16.0 (SPSS Inc., Chicago, IL, USA) performed to explore the differences between the two groups.
Results
=======
***General characteristics of the patients***
The age range of the study subjects was 18 to 64 years and the majority of them were males. Mean age of the group 1 (68.1 ± 40.9 years) was similar to the mean age of group 2 (67.9 ± 35.8 years). Moreover, the number of lifetime psychiatric hospitalization in group1 was (5.5 ± 9.7) times that was dramatically more than the similar number (2.3 ± 3.2) for group 2. Length of illness in the group 1 was 9.4 ± 18.2 years that was considerably more than length of illness e.g. 3.8 years in group 2 ([Table 1](#T1){ref-type="table"}).
The caregivers' age range was 19 to 84 years. The majority of the caregivers were elderly women. A considerable number of caregivers in group 1 were mothers (32.1%), sisters (19.8%), fathers and brothers with the same proportion (17.9%); while the proportion of mothers (52.8%), fathers (18.4%) and spouses (16.0%) in caring household maintained subjects were more reported while the remaining care givers were other family members([Table 2](#T2){ref-type="table"}).
*Characteristics related to the consumers*
The hospitalized subjects were older (t = 3.72; p *\<*0.002), had longer length of illness (t = 3.69; p *\<*0.003) ([Table 3](#T3){ref-type="table"}), greater severity of negative symptoms (t = 8.12; p *\<* 0.001), and positive symptoms (t = 2.045; p *\<* 0.042) and lower efficacy in basic life skills (t = 8.48; p *\<* 0.001) compared with household maintained consumers ([Table 4](#T4){ref-type="table"}).
*Characteristics related to the caregivers*
Caregivers of the hospitalized subjects reported experiencing greater objective burden (t = 4.24; p \< 0.001), subjective burden (t = 4.70; p \< 0.003) and less knowledge on schizophrenia (t= -5.78; p \< 0.001) compared with caregivers of household maintained consumers ([Table 5](#T5){ref-type="table"}).
*Availability of supportive and health-related services*
Caregivers of the household maintained subjects reported availability of more social support (t = -8.98; p \< 0.01) ([Table 6](#T6){ref-type="table"}), more availability of medical insurance (t = -2.07; p *\<*0.038), more access to supportive organization (t = -3.76; p *\<*0.001) and availability of assistant caregiver (t = -3.60; p *\<*0.001) and substitute caregiver (t = -2.33; p *\<*0.020) compared with caregivers of the hospitalized subjects ([Table 7](#T7){ref-type="table"}).
######
Demographics of the consumers
**Variable** **Characteristics** **Group 1 (n = 106)** **Group 2 (n = 125)**
------------------------------------------------------- --------------------- ----------------------- -----------------------
**Gender** Male 81 (76.4%) 102 (81.6%)
Female 25 (23.6%) 23 (18.4%)
**Mean age (Years)** 68.1 ± 40.9 67.9 ± 35.8
**Age range (Years)** 18-64 22-60
**Marital status** Never married 65 (61.3%) 90 (72%)
Currently married 13 (12.3%) 24 (19.2%)
Divorced 26 (24.5%) 9 (7.2%)
Widower / widow 2 (1.9%) 2 (1.6%)
**Education (Years)** Less than 12 years 63 (59.4%) 46 (36.8%)
12 years 31 (29.2%) 64 (51.2%)
More than 12 years 12 (11.2%) 15 (12.0%)
**Job status** Yes 11 (10.4%) 27 (21.6%)
No 95 (89.6%) 98 (78.4%)
**Medical insurance** Yes 33 (31.1%) 108 (86.4%)
No 73 (58.9%) 17 (13.6%)
**Mean of lifetime psychiatric hospitalization** 05.5 ± 9.7 02.3 ± 3.2
**Frequency of lifetime psychiatric hospitalization** 01-25 00-10
**Length of illness (Years)** 9.4 ± 18.20 3.8 ± 14.7000
######
Demographic characteristics of the caregivers
**Variable** **Characteristics** **Group 1 (n = 106)** **Group 2 (n = 125)**
------------------------------------- --------------------- ----------------------- -----------------------
**Mean age (Years)** 54.1 ± 14.3 55.0 ± 11.7
**Age range (Years)** 19-80 25-84
**Marital status** Currently married 61 (57.5%) 93 (74.4%)
Widower / widow 21 (19.8%) 22 (17.6%)
Divorced 15 (14.2%) 4 (3.2%)
Never married 9 (8.5%) 6 (4.8%)
**Education (Years)** Less than 12 59 (34.2%) 56 (46.5%)
12 29 (27.3%) 58 (46.4%)
More than 12 18 (17%) 11 (8.8%)
**Income (Per month)** Less than \$300 48 (45.3%) 54 (43.2%)
USD 300-750 53 (50%) 66 (54.5%)
More than \$750 5 (4.7%) 5 (4%)
**Type of relation with consumers** Mother 34 (32.1%) 66 (52.8%)
Father 19 (17.9%) 23 (18.4%)
Spouse 8 (7.5%) 20 (16.0%)
Sister 21 (19.8%) 12 (9.6%)
Brother 19 (17.9%) 2 (1.6%)
Child 3 (2.8%) 1 (8%)
Grandmother 2 (1.9%) 1 (8%)
######
Subject differences in related demographic and clinical variables
**Variable** **Group** **Mean (SD)** **t-test** **df** **P-value**
---------------------------------------- --------------- --------------- ------------ -------- ---------------------------------------
**Age** 1 41.03 (11.02) -3.72 229 0.002[\*](#TFN1){ref-type="table-fn"}
2 35.93 (9.81)
**Length of illness (Years)** 1 18.29 (9.04) -3.69 229 0.003[\*](#TFN1){ref-type="table-fn"}
2 14.07 (8.31)
**Number of lifetime Hospitalization** 1 09.76 (5.85) -0.210 225 0.834\*
2 11.61 (90.53)
p \< 0.01
######
Subjects differences in symptom-related and basic life skills variables
----------------------- --------------- ---------------- ------------ -------- -----------------------------------------
**Variable** **Group** **Mean (±SD)** **t-test** **df** **P-value**
**Negative symptoms** 1 66.13 (±12.35) 8.120 192 0.001[\*\*](#TFN3){ref-type="table-fn"}
2 53.17 (±9.74)
**Positive symptoms** 1 63.06 (±12.54) 2.045 192 0.042[\*](#TFN2){ref-type="table-fn"}\*
2 59.75 (±9.83)
**Basic life skills** 1 8.06 (±2.00) 8.480 192 0.001[\*\*](#TFN3){ref-type="table-fn"}
2 5.72 (±1.83)
----------------------- --------------- ---------------- ------------ -------- -----------------------------------------
p \< 0.05;
p \< 0.01
######
Caregivers' differences in family characteristics and burden variables
**Variable** **Group** **Mean (±SD)** **t-test** **df** **P-value**
------------------------------ ---------------- ---------------- ------------ -------- ---------------------------------------
**Problem solving** 1 02.08 (±0.533) -0.407 203 0.194\*
2 02.11 (±0.546)
**Communications** 1 02.26 (±0.147) 1.210 203 0.680\*
2 02.19 (±0.408)
**Roles** 1 02.61 (±0.462) 0.610 203 0.540\*
2 02.57 (±0.421)
**Affective responsiveness** 1 02.31 (±0.505) 0.136 203 0.890\*
2 02.30 (±0.577)
**Affective involvement** 1 02.39 (±0.503) 1.180 203 0.236\*
2 02.31 (±0.518)
**Behavior control** 1 02.46 (±0.457) 1.510 203 0.249\*
2 02.39 (±0.428)
**General functioning** 1 02.42 (±0.480) 0.160 203 0.870\*
2 02.41 (±0.533)
**Objective burden** 1 26.97 (±5.85) 4.240 203 0.001[\*](#TFN4){ref-type="table-fn"}
2 23.01 (±7.46)
**Subjective burden** 1 71.47 (±13.63) 4.700 203 0.003[\*](#TFN4){ref-type="table-fn"}
2 60.73 (±18.78)
**Family knowledge** 1 20.33 (±3.18) -5.780 203 0.001[\*](#TFN4){ref-type="table-fn"}
2 22.77 (±2.84)
p \< 0.01
######
Caregivers' differences in the variable of perceived social support
**Variable** **Group** **Mean (±SD)** **t-test** **df** **P-value**
-------------------- -------------- ---------------- ------------ -------- ---------------------------------------
**Social support** 1 5.24 (±2.59) 8.98 192 0.001[\*](#TFN5){ref-type="table-fn"}
2 8.43 (±2.53)
p \< 0.01
######
Subjects and caregivers' difference in access to supportive and health-related services
**Variable** **Group** **Mean** **Z score** **P-value**
----------------------------- ----------- ---------- ------------- -----------------------------------------
**Medical insurance** 1 106.20 -2.07 0.038[\*](#TFN6){ref-type="table-fn"}
2 123.30
**Supportive organization** 1 103.53 -3.76 0.001[\*\*](#TFN7){ref-type="table-fn"}
2 130.70
**Assistant caregiver** 1 098.89 -3.60 0.001[\*\*](#TFN7){ref-type="table-fn"}
2 125.95
**Caregiver substitute** 1 105.43 -2.33 0.020[\*](#TFN6){ref-type="table-fn"}
2 123.10
p \< 0.05;
p \< 0.01
Discussion
==========
In this study, we revealed some characteristics and differences between chronic hospitalized and household maintained consumers and their caregivers which met different importance. As a developing country with limited financial resources for treating consumers, identifying such characteristics and differences among the two main groups of consumers and their caregivers could reflect their treatment and educational needs and priorities which can be used in designing and implementing effective interventions.
We observed most of the consumers were male and the majority of their caregivers were females especially homemaker mothers. Our study findings are consistent with what we are observing in the developed countries that show patients with schizophrenia are typically male and their caregivers are female especially mothers ([@B21], [@B22]).
We found a greater proportion of elderly consumers with a longer length of illness in group 1 compared with group 2. Although these observations do not state causation but older age in group 1 could reflect longer years of challenge with schizophrenia disorder and long length of living of these patients with their disorder and its chronic severity. Elderly consumers were also likely not to be able to take care of themselves, and old age and prolonged duration of schizophrenia were likely to impose more subjective and objective burden on caregivers, and it was likely to result in greater inpatient admission of these patients.
This study finding was in consistent with studies in developed countries emphasizing older age of psychiatric patients as a burden ([@B23]) and was in contrast with some studies in developed countries that suggest shorter duration of illness is a burden for caregivers ([@B24]) not longer duration of illness which results in longer hospitalization.
We found that group 1 had lower efficacy in performing basic life skills and greater symptoms than group 2. This issue could reflect the chronic and disabling nature of schizophrenia disorder which disabled these patients in doing their daily routines. It may be also partly due to this issue that in family atmosphere, caregivers allocated more time and care to the basic needs of their disable members and showed more sympathy toward their affective needs which were likely reduce deficiency in basic life skills and severity of symptoms compared with crowded inpatient psychiatric wards of hospitals but whether chronic inpatient admission results in increasing deficiency in basic life skills and more severity of symptoms or these issues result in chronic psychiatric hospitalization among Iranian consumers is an avenue for future research. A study showed deficiency in carrying out life skills among consumers is associated with feeling burden among caregivers, and heavy costs ([@B25]). Moreover, some studies show clinical symptoms ([@B26]), the severity of patient symptoms ([@B26]) result in feeling burden by caregivers and patient's dependence on caregiver.
Caregivers of group 1 had more objective and subjective burden and lower level of knowledge on schizophrenia compared to the caregivers of group 2. Feeling burden and lower knowledge on schizophrenia were likely to result in feeling distress and burden and referring them to inpatient service. This study finding emphasizes family psycho-educational and supportive interventions that are considered a best practice in the treatment of schizophrenia ([@B27]).
We found a greater proportion of the caregivers of group 1 had more perceived social support and availability of medical insurance for their patients, more access to supportive organization, assistant caregiver and substitute caregiver during critical periods due to patient compared with caregivers of group 2. These issues were likely to reduce emotional and financial burden of care giving and encouraged the caregivers to continue caring of their patients at home and not to refer them to psychiatric hospitals.
These findings support other studies that show perceived social support by caregivers, availability of help from professional agencies, a larger number of social contacts, more emotional supporters and more social interaction an reduce burden and distress ([@B26]).
Our study had also several limitations. We limited our sample to ISSIS with mainly male consumers and a limited number of female consumers and elderly caregivers with limited education and income who agreed to cooperate with us which are subjects to further studies with larger sample populations and other caregivers such as employed caregivers and relatives but our sample was representative of the group we studied. In fact, we referred to many centers in Tehran for sampling; but they disagreed to cooperate with us. Survey studies may give a better picture of differences between chronic hospitalized and household maintained consumers which might be helpful in designing treatment programs based on their needs and differences. To sum up, our findings revealed a wide range of differences in domains of patient characteristics and family characteristics as well as availability of social and supportive services in a developing country like Iran. Interventions to support and develop the consumer's ability to contribute to the household and management of their own care, such as skills training or wellness recovery, as well as promoting and developing educational programs for caregivers and access to social and supportive services are most likely to meet the needs and enhance the quality of life of people with mental illness and their families rather than inpatient service utilization.
The current study was the results of a MA thesis in psychology which was carried out in Family Research Institute of Shahid Beheshti University in Tehran, Iran. No grant was received for either conducting the project or writing the paper. The authors would like to thank the Iranian Society Supporting Individuals with Schizophrenia for their collaboration.
Authors\' contributions
=======================
NM designed and conducted the study with data collection. SKM participated in designing and evaluating the study and helped to draft the manuscript. NC participated in statistical analysis and re-evaluating the manuscript. SR performed parts of the statistical analysis and re-evaluated the whole statistical analyses. MM and EM helped in reviewing literature and participated in statistical analysis. All authors read and approved the final manuscript.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Fast and accurate discrimination of where another person is looking at, especially the judgment of whether another individual is making eye contact or not, is an important skill supporting social interaction. Not only humans use their eyes to capture visual information, but also to signal their social intentions (for a review, see [@B34]), and specialized neural systems subserve visual and social aspects of gaze processing ([@B48]).
Emotions influence how people think about and understand others and themselves in social settings ([@B19]). Face perception and particularly perception of facial expressions is modulated by concurrent affective information emanating, for example, from the sender themselves as well as from the contextual information related to the physical environment and other surrounding people (for a review, see [@B64]). Perceivers' own emotions also influence their perception of others' facial expressions. Negative emotional state facilitates recognition of negative facial expressions and positive emotional state facilitates recognition of positive facial expressions ([@B52]; [@B60]; [@B7]; [@B47]; [@B36]; [@B18]; [@B67]; [@B53]). Altogether these findings indicate that recognition of facial expressions is facilitated by affectively congruent contexts and perceivers' emotions.
As eyes are the most salient feature in the face and gaze direction is a rich source of socially relevant information, a surprisingly limited number of studies have investigated the impact of emotion on gaze perception. Some previous studies have investigated gaze direction and eye contact perception in the context of emotional facial expressions. These studies have reported that averted gaze is identified faster when the face is fearful rather than angry, while direct gaze is identified faster when the face is angry rather than fearful ([@B1]). [@B41], [@B39]) and [@B40] conducted a series of studies investigating the effects of facial expressions on perceived gaze direction. In these studies, they presented face pictures with different facial expressions and gaze angles to participants. Participants were required to judge whether the face was looking at them or not, or to indicate the perceived gaze direction by moving a slider. The results indicated that participants were more likely to interpret happy faces as looking at them as compared to angry, fearful, or neutral faces. Taken together, these studies suggested that gaze direction perception was modulated by contextual affective information provided by the gaze sender's facial expression.
However, it remains unresolved whether gaze perception would be influenced by an observer's *own* affective state, and the present study was designed to answer this question. As a communicative social signal, gaze direction both signals a sender's approach-avoidance tendencies and activates corresponding motivational tendencies in the observer, thus, strongly regulating social connectedness ([@B5]; [@B2], [@B3]; [@B28]; [@B65]). Positive affect is associated with approach behavior and, thus, enhances a tendency of being cooperative and socializing, while a negative affect is associated with avoidance and leads to the opposite ([@B31]; [@B15]; [@B14]). Therefore, it could be expected that a person in a positive affective state would seek and perceive more social communication signals, i.e., more eye contact, as compared to a person in neutral and negative affective states.
To the best of our knowledge, there are no previous studies investigating the perceiver's affective state on gaze perception. However, some affect-related traits, such as social anxiety, have been demonstrated to modulate individuals' gaze perception. Individuals with social anxiety are prone to overestimate direct gaze from others ([@B22]; [@B54]; [@B6]). Additionally, there are some studies investigating the influence of perceivers' affect on their own gaze behavior. One study demonstrated that compared with the controls, individuals with induced positive affect established eye contact more often, whereas participants with induced negative affect had less frequent and shorter periods of eye contact with a confederate ([@B46]). Similar findings were reported in studies with clinically diagnosed patients. Depressed patients have been found to maintain shorter periods of eye contact and show more gaze aversion compared to control participants ([@B29]; [@B63]).
In two experiments of the present study, we investigated the effects of a perceiver's affective state on eye contact judgments. Additionally, we measured participants' social anxiety in Experiment 2 to assess the potential interplay between affective state and trait variables. Pleasant, neutral, and unpleasant odors were used to induce corresponding affective states. Previous studies have demonstrated that olfactory stimuli are effective in influencing mood, reflected on both physiological and self-reported measures ([@B11]; [@B27]; [@B49]). Importantly, manipulating affect by odors is unobtrusive and can be done simultaneously when participants are performing different tasks. Thus it is well suited for laboratory studies on emotion--cognition interactions. Odor-induced affects also modulate individuals' cognitive processes and behavior (for a review, see [@B26]). Recognition of facial expressions has been shown to be facilitated when the odor context is affectively congruent with the expressions ([@B36]; [@B35]), and odors can also influence the likability ratings of neutral faces ([@B38]). Furthermore, studies have reported effects of odors on people's approach-avoidance tendencies (for a brief review, see [@B30]; [@B66]). For example, a study on consumer behavior indicated that inoffensive scents (e.g., certain floral scents) compared to no scent in the environment, increased customers' intentions to visit the store ([@B59]). Pleasant ambient scents have also been shown to increase social interaction behaviors, e.g., eye contact, physical contact, and conversation, compared to a no scent condition ([@B66]). Thus, this evidence suggested that odors are well suited affect-inducers for the purpose of the present study.
Experiment 1
============
In Experiment 1, participants viewed computer-generated faces with varying gaze angles and were required to judge whether the face was looking at them or not. Participants' affective state was manipulated with pleasant, neutral, and unpleasant odors. To investigate the effect of odor-induced affects on eye contact judgments, we analyzed the width of gaze cone. Gaze cone refers to a width of gaze direction range perceived as eye contact and it has been used as a dependent variable in many previous studies investigating eye contact perception (e.g., [@B21]; [@B16]; [@B22]; [@B61]; [@B42]). We expected that participants induced to have a positive affective state, as compared to neutral and negative affective states, would be prone to perceive a wider range of gaze directions as eye contact, i.e., would have wider width of gaze cone.
Materials and Methods {#s1}
---------------------
### Participants
Twenty-four participants (19 females, age range 19--34 years, mean 23 years) with self-reported normal or corrected-to-normal vision, normal sense of smell, and without any neurological or psychiatric diagnosis were recruited. All participants were informed about the general procedure of the experiment and they signed a consent form. They were requested not to wear any perfume or other products with strong smell. After the experiment, participants were given a movie ticket for their participation. The research protocol was approved by the Ethics Committee of the Tampere region.
### Stimuli
For odor stimuli, pyridine (Merck, 0.1% dilution), lemon essential oil (1% dilution), and water were used as an unpleasant, pleasant, and neutral odor, respectively. Pyridine and lemon essential oil have been effectively used as unpleasant and pleasant odors in prior studies ([@B36]). For gaze direction stimuli, eight characters (four males and four females) with nine different gaze angles (direct gaze and gaze averted 2, 4, 6, and 8° toward left and right) were created using a 3D animation software \[Digital Art Zone (Daz) 3D Studio^[1](#fn01){ref-type="fn"}^\] (**Figure [1A](#F1){ref-type="fig"}**). To avoid potential influence of facial asymmetry, all original stimuli were also presented horizontally flipped.
{#F1}
### Procedure
The experiment was run using a fully within-subjects design. It consisted of three odor conditions, and the trials in each condition were presented in two blocks. The procedure in each condition was identical except that the odor was either unpleasant, pleasant, or neutral. The order of the odor conditions was counterbalanced between the participants. Before each condition, the experimenter prepared the odor apparatus. A container with a clean cotton swab inside was attached on a chinrest so that the swab was 7--8 cm away from the participant's nose. Two milliliter of odor solution was dropped on the cotton at the beginning of each odor condition and 1 ml of liquid was added on the cotton between the blocks in order to keep the level of smell constant. A small air pump was connected to the bottom of the container to blow air into the container continuously during the experiment. Participants wore earmuffs to block low-frequency noise coming from the air pump. After each odor condition, participants took a 5-min break outside the testing room. The experimenter opened the windows until there was no detectable odor left in the room and then prepared the next odor condition. The air conditioning was always on in the testing room.
Each condition consisted of 80 trials with an equal number of 0, 2, 4, 6, and 8° averted gaze (half left/half right) trials. On each trial, a fixation cross was presented first in the center of the screen for 800 ms. This was followed by the presentation of the gaze stimulus for 150 ms. After the stimulus disappeared, participants were asked to make two judgments: first they judged whether the face was looking at them or not by pressing "1" or "2," and immediately after this they evaluated the strength of their feeling of whether the face made eye contact or not on a 3-point scale (strong, intermediate, and weak) by pressing "1," "2," or "3." The task was not paced, but response time (RT) was limited to 7 s. Participants gave responses with the numeric keypad on the right side of the keyboard. Response key mapping for the 'looks at me' task was counterbalanced across participants. After the participant's second response, there was a 500-ms interval before the next trial. After each condition, participants rated the pleasantness of the odor using a scale ranging from 1 (unpleasant) to 9 (pleasant) presented on a computer screen.
### Data Analysis
A one-way analysis of variance (ANOVA) was conducted on the odor ratings. For the eye contact judgment, five participants were excluded from the analysis because the manipulation check showed no differences in pleasantness ratings between odors for these participants. For computing the width of gaze cone, the proportion of looking-at-me responses was first calculated for each gaze angle separately in each odor condition. By using a binary logistic regression model for the proportion of looking-at-me responses data, the point at which a gaze stimulus had equal probabilities to be subjectively judged as eye contact or gaze aversion was calculated. This angle can be interpreted as the width of gaze deviation angle that an individual accepts as eye contact, i.e., gaze cone ([@B16]; [@B61]). Even though previous studies have suggested a symmetrical horizontal gaze cone ([@B62]), it is still possible that left and right gaze cones are asymmetrically influenced by affective state. Thus, the width of gaze cone was calculated separately for gaze averted to the left and right. Because of the positive skew of the distribution of the angles, the gaze cone data were first normalized with a log10 transformation and then entered into a repeated-measures ANOVA with odor (pleasant, neutral, and unpleasant) and gaze direction (left and right) as within-subject factors. Responses to looking at me responses and the eye contact strength data were combined to range from 1 to 6 (1 = not looking at me, strong impression; 2 = not looking at me, intermediate impression; 3 = not looking at me, weak impression; 4 = looking at me, weak impression; 5 = looking at me, intermediate impression; 6 = looking at me, strong impression) and analyzed with a 3 (odor) × 5 (gaze angle) × 2 (gaze direction) ANOVA.
For violations of sphericity, a Huynh-Feldt correction procedure was applied. Least significant difference (LSD) test was performed for all multiple comparisons. For the sake of brevity, uncorrected degrees of freedom were reported. All statistical analyses were performed using the SPSS package.
Result and Discussion
---------------------
For the subjective pleasantness ratings, a one-way ANOVA showed a significant effect of odor, *F*(2,46) = 72.22, *p* \< 0.001, $\eta_{p}^{2}$ = 0.758. Pairwise comparisons revealed that the smell of lemon (*M* = 7.08, *SE* = 0.27) was rated as significantly more pleasant than water (*M* = 4.75, *SE* = 0.16, *p* \< 0.001) and water, in turn, was rated more pleasant than pyridine (*M* = 2.92, *SE* = 0.26, *p* \< 0.001).
The ANOVA on gaze cone width showed that the main effect of odor condition was not significant, *F*(2,36) = 0.76, *p* = 0.473, $\eta_{p}^{2}$ = 0.041, and there was no main effect of gaze direction (*p* = 0.339) or interaction between odor condition and gaze direction (*p* = 0.281) either (see **Figure [2A](#F2){ref-type="fig"}**). The proportions of looking-at-me responses for the nine gaze angles as a function of odor condition are presented in the Supplementary Table [S1](#SM1){ref-type="supplementary-material"}. For the eye contact strength rating data (**Figure [2B](#F2){ref-type="fig"}**), the analysis expectedly showed a main effect of gaze angle, *F*(4,72) = 188.50, *p* \< 0.001, $\eta_{p}^{2}$ = 0.913. The strength of the eye contact feeling decreased with larger deviations of gaze angle from the direct gaze. Again, there was no main effect of odor condition or gaze direction (*p* = 0.754 and *p* = 0.284, respectively) or interactions involving odor condition, gaze angle, and gaze direction (*p* = 0.551, *p* = 0.640, *p* = 0.689, and *p* = 0.611).
{#F2}
Experiment 1 showed no effect of affective state on eye contact judgments. This was true both for the analyses based on the width of gaze cone and on the eye-contact strength ratings. The odor ratings indicated that our odor manipulation was, however, successful and a large number of previous studies have demonstrated the effect of odors on emotion, cognition, and behavior (for a review, see [@B26]). Particularly, a study using an identical odor manipulation procedure with the present experiment showed that participants recognized happy faces faster than faces expressing disgust in a pleasant odor context, whereas a reversed pattern of results was observed in an unpleasant odor context ([@B36]). One possibility for the lack of the odor effect could be that, although the gaze stimuli were briefly presented (150 ms), the participants were nevertheless given quite a long time to give their responses after the stimuli (7 s). Thus, the participants had ample time to evaluate the looking direction of the presented gaze stimulus and it is possible that this diminished the effect of odor context on eye contact judgments. To deal with this problem, in Experiment 2, we measured RTs and asked participants to respond as fast as possible.
It is also possible that the present results were affected by a confounding variable, i.e., by participants' level of social anxiousness. [@B22] investigated gaze cone width in clinical samples with social anxiety. The results indicated that, compared to controls, participants with social anxiety showed a wider gaze cone, i.e., they were more likely to perceive averted gazes as being directed at them ([@B22]). Through a web-based approach, another study showed a positive relationship between social anxiety and the direct gaze judgment: individuals with higher social anxiety scores had a stronger feeling to be looked at by others ([@B54]). There is even evidence that, in participants with social anxiety disorder, the reduction of social anxiety symptoms as a result of Cognitive Behavioral Therapy is accompanied by decrease of the width of gaze cone ([@B24]). Thus, social anxiety may have played an important, modulatory role in gaze perception in Experiment 1. Therefore, we took social anxiety into account in Experiment 2.
Experiment 2
============
In Experiment 2, participants were required to judge five different gaze directions as either looking-at-me or not looking-at-me as fast as possible. Like in Experiment 1, the task was performed in three different affective contexts. We expected that such a setting would pressure participants to exhibit less controlled responses and, therefore, the task would be more sensitive to the influence of the odor context. Additionally, real faces instead of animated faces were used in Experiment 2 to increase realism of the stimuli. We also included the Social Phobia Scale (SPS, [@B43]) to control the potential influence of social anxiety.
Materials and Methods {#s2}
---------------------
### Participants
The recruitment procedures and inclusion criteria of participants were identical with those in Experiment 1. Twenty-eight participants (22 females, age range 20--39 years, mean 25 years) were enrolled in the present experiment.
### Stimuli
Considering that, in Experiment 1, 5 participants (out of 24) did not show differences in their pleasantness ratings between odors, we increased the intensity of unpleasant odor (to 0.6% dilution) and replaced lemon with orange essential oil (1% dilution). Like in Experiment 1, water was used as a neutral odor. For gaze direction stimuli, grayscale photographic images of six Finnish persons (three males and three females) with five different gaze angles (direct gaze, 4 and 8° averted toward left and right) were selected from a set of stimuli prepared for a study by [@B61] (**Figure [1B](#F1){ref-type="fig"}**). All original stimuli were also presented horizontally flipped to avoid any potential influence of facial asymmetry.
### Procedure
The procedures regarding the odor manipulation and block order were identical with those in Experiment 1. In each odor condition, the trials were presented in two blocks. Each block consisted of 48 trials with an equal number of direct, 4 and 8° averted gaze faces. On each experimental trial, first a fixation cross was presented in the center of the screen for 500 ms. This was followed by the presentation of the gaze stimulus for 500 ms. Participants were required to respond whether they felt that the person was "looking at me" or not as fast and accurate as possible by pressing "D" or "K" on the keyboard. They were allowed to give their response within a time-window of 3500 ms starting from the stimulus onset. Response key location was counterbalanced across participants. The inter-trial interval was 1000 ms. After each condition, participants were required to rate the pleasantness of the odor from 1 to 9 (1 = unpleasant, 9 = pleasant).
After the computer task, all participants completed the Social Phobia Scale (SPS) which consists of 20 items and each item is rated on a five-point Likert scale ranging from 0 (not at all) to 4 (extremely) ([@B43]).
### Data Analysis
A one-way ANOVA was conducted on the odor ratings. For the eye contact judgment, four participants were excluded from the analysis because the manipulation check showed no differences in pleasantness ratings between odors for these participants. Two more participants were excluded due to low response accuracy for direct and 8° averted gaze (69 and 64%, respectively). For each participant, we calculated both the gaze cone width and the RT for each gaze angle (separately for the gaze averted to the left and right) in each odor condition. A repeated-measures ANOVA with odor condition and gaze direction as within-subject factors was performed on the gaze cone data. For the RT data, the data from trials with incorrect responses to the gaze directions (16%) and trials with response latencies shorter than 2.5 standard deviations (SDs) below or longer than 2.5 SDs above each participant's mean (1.7%) were excluded. The averaged RT data were first normalized with a log10 transformation and then entered into a 3 (odor condition: pleasant, neutral, and unpleasant) × 3 (gaze angle: 0, 4, and 8°) × 2 (gaze direction: left, right) ANOVA.
The possible modulating influence of social anxiety on gaze cone width in different odor conditions was investigated using an analysis of covariance (ANCOVA) with odor condition and gaze direction as repeated-measures factors and SPS score as a covariate.
Results and Discussion
----------------------
For the pleasantness ratings of odors, there was a significant effect of odor, *F*(2,54) = 65.62, *p* \< 0.001, $\eta_{p}^{2}$ = 0.709. Pairwise comparisons revealed that the smell of orange (*M* = 6.07, *SE* = 0.35) was rated as significantly more pleasant than water (*M* = 4.71, *SE* = 0.18, *p* = 0.001) and water, in turn, was rated more pleasant than pyridine (*M* = 2.04, *SE* = 0.21, *p* \< 0.001).
The ANOVA on gaze cone width showed no main effect of odor condition, *F*(2,42) = 0.78, *p* = 0.467, $\eta_{p}^{2}$ = 0.036. Additionally, there was no main effect of gaze direction (*p* = 0.389) or interaction between odor condition and gaze direction (*p* = 0.773) (**Figure [3A](#F3){ref-type="fig"}**). The proportions of looking-at-me responses for the five gaze angles as a function of odor condition are presented in the Supplementary Table [S2](#SM2){ref-type="supplementary-material"}. For the RT data (**Figure [3B](#F3){ref-type="fig"}**), there was a main effect of gaze angle, *F*(2,42) = 20.03, *p* \< 0.001, $\eta_{p}^{2}$ = 0.488. Overall, participants were faster to respond to 8° averted gaze (*M* = 696 ms, *SE* = 38.21) than to direct gaze (*M* = 741 ms, *SE* = 36.63, *p* = 0.006) and to 4° averted gaze (*M* = 762 ms, *SE* = 41.32, *p* \< 0.001). Importantly, there was no main effect of odor condition or gaze direction (*p* = 0.639 and *p* = 0.845, respectively) or interactions involving odor condition, gaze angle, and gaze direction (*p* = 0.168, *p* = 0.283, *p* = 0.964, and *p* = 0.596).
{#F3}
The ANCOVA on gaze cone width showed that there was no main effect of odor condition or gaze direction (*p* = 0.797 and *p* = 0.333, respectively) or interaction involving odor condition, gaze direction, and social anxiety score (*p* = 0.291, *p* = 0.756, *p* = 0.213, and *p* = 0.303). Importantly, the results revealed a significant main effect of social anxiety, *F*(1,20) = 4.34, *p* = 0.050, $\eta_{p}^{2}$ = 0.178. A correlation analysis between each participant's gaze cone width (left and right gaze cone combined and averaged across odor conditions) and SPS scores showed a positive correlation between gaze cone width and SPS scores (*r* = 0.422, *p* = 0.025, one-tailed) (**Figure [4](#F4){ref-type="fig"}**). Thus, participants with higher levels of social anxiety tended to have a wider gaze cone, that is to say, they were more likely to perceive a gaze as being directed at them. The correlation between each participant's average RT and SPS score was not significant (*r* = 0.287, *p* = 0.196).
{#F4}
Finally, in order to increase the power of statistical testing, we pooled the left and right gaze cone width data from Experiments 1 and 2 and performed a repeated-measures ANOVA. Again, the results showed no main effect of odor condition (*p* = 0.235), main effect of gaze direction (*p* = 0.494) or interaction between odor condition and gaze direction (*p* = 0.458). From the pooled data, we also analyzed the 95% confidence intervals (CIs) for the differences between the odor conditions. These were: -0.7 ≤ CI (μ~pleasant~ - μ~neutral~) ≤ 2.5; -0.8 ≤ CI (μ~pleasant~ - μ~unpleasant~) ≤ 1.7; -1.2 ≤ CI (μ~neutral~ - μ~unpleasant~) ≤ 0.4. These CIs are rather small indicating that with a high probability there were no differences in gaze cone width between different odor conditions.
In Experiment 2, we analyzed both gaze cone width and the RTs to eye contact judgment in pleasant, neutral, and unpleasant odor conditions. The results replicated the findings of Experiment 1: eye contact judgments were not modulated by perceivers' affective state, even after controlling for social anxiety scores. In addition, the present experiment replicated the results of previous studies showing that participants with higher levels of social anxiety interpreted wider gaze deviations from a true direct gaze as an eye contact ([@B22]; [@B54]).
General Discussion
==================
Our main finding was that across two experiments, neither positive nor negative affective state influenced eye contact judgment. Instead, individual differences in social anxiety were associated with eye contact judgments, with high anxiety leading to more liberal criterion in the gaze contact detection.
We start discussing these findings by asking first how affective states come to influence our social judgments. It has been suggested that individuals' own affective states may be used as diagnostic information when making social judgments regarding other people, because it provides information about the elicited action tendencies toward others ([@B55]; [@B17]; [@B23]). For example, in judging the likability of another person, individuals with a positive affect mistakenly interpret their own positive affect as their feeling about the judged person, and evaluate the target person more positively when in a happy rather than a sad mood ([@B23]; [@B56]). Another possibility is that an affective state may activate associated concepts, words, themes, and inference rules, and these activated representations will become more likely to be accessed in subsequent judgments ([@B10]). Consequently, "these mental sets then act as interpretive filters of reality" and bias individuals' social judgments ([@B10], p. 395). Via these mechanisms, affective states lead to affect-congruent judgments. Thus, affective states may influence, for example, predictions regarding the weather (sunny-rainy), one's own future (optimistic-pessimistic), the likability ratings of a neutral face (likable--dislikable), and the recognition of facial expressions (happy--sad) ([@B32]; [@B9]; [@B44]; [@B7]; [@B38]). However, if the judgment to-be-made as such is not strongly associated with a positive or negative affect, the affective state may not exert its influence on the judgment. Indeed, this may be the case in the present study regarding the eye contact judgment: Evaluating whether another person is looking at me or not is not an affective judgment as such. Therefore, the present results suggest that the affective congruency effects, whether based on affect as information type of explanations ([@B57]) or spreading activation theories ([@B8]), exert strong influences in instances where the social judgment as such is affect-related, but less so in situations where the social decision is devoid of affective contents.
The present results also suggest that, in contrast to a relatively robust congruency effects in affective judgments ([@B8]; [@B44]), corresponding congruency effect is absent for approach-avoidance motivation. We hypothesized that by inducing positive (approach) and negative (avoidance) affective states in individuals, we could influence participants' evaluations of others' approach/avoidance tendencies inferred from their eye gaze. The present results, however, imply that approach and avoidance motivation does not sensitize an observer to perceive congruent social signals in their environment similarly as affects do. This accords with recent data showing that when recognizing morphed expressions of anger, an unpleasant (versus pleasant or neutral) odor significantly lowered the morphing threshold required for accurate recognition ([@B35]). Thus, even if the unpleasant odor context had induced avoidance motivation in the participants, they, nevertheless, were more sensitive to valence-congruent, but motivation-incongruent facial expression of anger. Anger is considered to be an affectively negative emotion, but associated with an approach motivation (for a brief review, see [@B25]).
Affect-related states, such as stress, social anxiety, and feelings of ostracism, increase the likelihood of interpreting a gaze as looking toward the self ([@B22]; [@B51]; [@B54]; [@B42]). If affective states do not influence judgments of gaze direction, as postulated above, how then these studies have shown the effect? The reason likely relates to that although all these states involve (negative) affects, the affective state *per se* was not the cause for the biases in the gaze direction judgments. For example, stress occurs when individuals perceive that they do not have sufficient sources to cope with a threatening or a demanding situation ([@B13]). In such situations, it is adaptive to become more alert and self-centered and, consequently, individuals are more likely to interpret another person's gaze as directed toward themselves ([@B51]). Social anxiety is characterized by an intense fear and avoidance of social situations in which an individual may be scrutinized by others ([@B4]) and, at the same time, individuals with social anxiety also show biases in information processing which drives them more likely to interpret social situations in a negative way ([@B12]). Indeed, compared to controls, individuals with social anxiety experience eye contact as aversive ([@B45]), and are also prone to interpret a gaze direction as being directed at them ([@B22]; [@B54]). Being ostracized does not only evoke negative affect, but also lowers one's experience of fulfillment of the basic needs (e.g., belongingness) ([@B65]). [@B42] suggested that socially excluded individuals are biased toward gaze contact detection due to their need for reaffiliation. Thus, even though previous research has shown increased eye contact perception caused by stress, social anxiety, and feelings of ostracism, these studies cannot be considered as having provided evidence that affective states as such would influence the gaze direction judgments.
Although the present results did not show the influence of affective state on eye-contact judgments, we found a statistically significant correlation between the width of gaze cone and self-reported social anxiety. In accordance with prior studies, participants with higher levels of social anxiety accepted a wider range of gaze directions as eye contact ([@B22]; [@B33]; [@B54]).
[@B20] proposed a good-effort criterion for accepting the null hypothesis. He suggested that once a good effort has been made to find the effect but none has been found, the null hypothesis should be accepted. An objective criterion for a good effort is demonstrating a related effect ([@B20]). In our study, we demonstrated this by finding a significant positive correlation between gaze cone width and social anxiety. This related effect indicated that the present study was methodologically sound, it had a sufficient number of participants and trials, and the variances were well-controlled. Furthermore, the CIs analyzed from the data pooled across Experiments 1 and 2 were small; small CIs have also been considered as one of the criteria of good effort to find an effect ([@B20]).
A limitation of the present study is that although we used a valence based classification in our odor manipulation conditions (positive, neutral, and negative), it is a fact that distinct emotions with the same valence differentially influence thoughts and judgments ([@B50]; [@B37]). For example, people's assessments of risk probabilities have been shown to be positively related to their level of dispositional fear but negatively related to their level of dispositional anger ([@B37]). The authors explained their findings by suggesting that each emotion activated a corresponding appraisal pattern. This appraisal pattern automatically guided subsequent perceptions and judgments. For example, fear activated an appraisal tendency to perceive negative events as uncertain and outside of personal control, and consequently led to pessimistic risk perception. On the contrary, anger activated an appraisal tendency to perceive negative events as certain and under personal control, consequently leading to optimistic perceptions ([@B58]; [@B37]). In the present studies, we manipulated the affective context by using olfactory stimuli of lemon/orange, water, and pyridine. The smell of pyridine was unpleasant and negative to the participants, as shown by their self-ratings, and, for most people, the smell was specifically disgusting. Thus, it is likely that it automatically activated a different appraisal tendency as compared to other emotions with negative valence, e.g., sadness, anger, and fear. Future studies should investigate the eye contact perception with a broader variety of induced emotions.
Conclusion
==========
We conclude that observers' affective state does not influence their eye contact judgments. The results were consistent when considering both the width of gaze cone and RTs, and across two experiments using both animated and real face pictures as stimuli. Consistent with previous studies, our study, however, showed a positive relationship between gaze cone width and social anxiety. We suggest that affective states are not likely to influence social judgments if the judgments *per se* are not related to evaluations involving the dimension of affective valence.
Author Contributions
====================
TC and JH were involved in the experimental design, data collection and analysis. All the three authors were involved in manuscript writing and data interpretation work.
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.
**Funding.** This work was supported by the GATE Erasmus Mundus Project (to TC), the Finnish Cultural Foundation (to JH), the Academy of Finland (MIND program grants \#266187 to JH and \#265915 to LN), and ERC Starting Grant (\#313000 to LN).
We thank Helena Kiilavuori for her help in data collecting.
<http://www.daz3d.com>
Supplementary Material
======================
The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fpsyg.2017.00373/full#supplementary-material>
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Click here for additional data file.
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Click here for additional data file.
[^1]: Edited by: *Wenfeng Chen, Institute of Psychology, Chinese Academy of Sciences, China*
[^2]: Reviewed by: *Jan Van Den Stock, Katholieke Universiteit Leuven, Belgium; Sebastian Korb, University of Vienna, Austria*
[^3]: This article was submitted to Emotion Science, a section of the journal Frontiers in Psychology
| {
"pile_set_name": "PubMed Central"
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**Core tip:** B cells lymphoma is one of the most challenging extra-hepatic manifestations of hepatitis C virus. Recently, a new kind of B-cell lymphoma, named Double-hit B, was characterized with an aggressive clinical course. This is the first case described in literature of double hit lymphoma with co-existing chronic hepatitis C, successfully treated by new direct antiviral therapies. This case suggests a potential favorable effect of the new antiviral therapies on double hit lymphoma regression.
INTRODUCTION
============
Hepatitis C virus (HCV) is a major global public health problem, although the newest and most effective antiviral therapies (AVTs), free from severe side effects related to interferon (IFN), are spreading in developed countries. This could lead in the next future to a lower prevalence of chronic hepatitis C (CHC) and of its extra-hepatic manifestations. Among these, the hematologic manifestations are the most challenging. Indeed, HCV could induce B cell proliferation and cause mixed cryoglobulinaemia and non-Hodgkin lymphoma (NHL)\[[@B1]\]. The typical HCV-related lymphomas are marginal zone lymphoma (MZL) and diffuse large B cell lymphoma (DLBCL)\[[@B2]\]. The first could benefit from AVTs, whereas the second group needs more aggressive chemotherapies and the only AVTs seem to be ineffective. More recently, a new kind of lymphoma named double-hit B cell (DHL) was described, characterized by chromosomal rearrangements, specifically of *Myc* oncogene and either B cell lymphoma 2 and B cell lymphoma 6 oncogenes (*Bcl2*-*Bcl6*) or gene for Cyclin D1 (*Ccnd1*)\[[@B3],[@B4]\]. DHL represents about 5% of all cases of DLBCL and affected patients generally have an aggressive clinical course with poor prognosis, despite combination chemotherapy, with a median overall survival of less than 1-2 years. To date, due to the limited literature concerning this type of lymphoma, the specific treatment is still not clear. A potential correlation between HCV and DHL has never been investigated.
We describe the first case, to our knowledge, of a patient affected by DHL and CHC who underwent a successful antiviral treatment with IFN-free therapy (ombitasvir/paritaprevir/ritonavir and dasabuvir).
CASE REPORT
===========
The patient was a 39-year-old Caucasian woman, who tested HCV positive in 2013 during a routine medical evaluation. She had not history of blood transfusions or other risk factors for hepatitis virus transmission. The CHC was sustained by genotype 1b virus, with low necro-inflammatory activity and mild liver fibrosis according to non-invasive evaluation performed by Fibroscan^®^ (4.5 kPa), whereas HCV showed a high replication rate (4500000 UI/mL, Cobas AmpliPrep/Cobas TaqMan^®^-Roche, Rotkreuz, Switzerland). Ultrasound scans of the liver did not demonstrate signs of fibrosis or spleen enlargement secondary to portal hypertension. Of interest, the patient reported a previous episode of major depression occurred in 2006 and, since then, she had been taking venlafazine 75 mg/d.
In July 2013, due to the enlargement of neck lymph nodes, the patient underwent a hematological evaluation and a lymph node biopsy, and she was finally diagnosed with DLBCL (CD20+, CD30-, *Bcl2*+, Ki67 50%, *Bcl6* + 25%, negativity for CD10 and *Mum1*), with involvement of nodes on both sides of the diaphragm, bone marrow and spleen (Figure [1](#F1){ref-type="fig"}). Treatment with cyclophosphamide, doxorubicin, vincristine, prednisolone plus Rituximab (R) (*R-CHOP* regimen) plus prophylactic intrathecal injection of methotrexate and prednisone, was started. In December 2013, *i.e*., after six courses of *R-CHOP*, the patient showed a complete remission of lymphoma according both to Contrast Tomography plus Positron Emission Tomography scans and to bone marrow biopsy. Two additional doses of R were prescribed and, at that time, blood tests showed normal levels of transaminases, while HCV replication did not show any substantial changes (Figure [2](#F2){ref-type="fig"}). In the following months, the patient was referred to our Liver Unit and she was evaluated for AVT. However, since "all-oral" AVTs for CHC was at that time unavailable in Italy, the patient was evaluated for IFN-based AVT, but she presented relative and absolute contraindications to IFN use, such as a previous episode of major depression, mild anemia, leucopenia (*i.e*., Haemoglobin 10.7 g/dL, White cells counts 1.980 cells/mm^3^), as well as to ribavirin (RBV) use (anemia). We therefore asked our case to be evaluated for the expanded access program (EAP) which was ongoing with Paritaprevir (PTV), a NS3/4A protease inhibitor co-formulated with NS5A inhibitor Ombitasvir (OBV) and the pharmacokinetic enhancer ritonavir (r), plus non-nucleoside polymerase inhibitor Dasabuvir (DSV). Since lymphoma is a possible extra-hepatic manifestation of HCV, the pharmaceutical industry committee accepted our patient for participation to the EAP. Unfortunately, in October 2014, the patient experienced the first relapse of lymphoma at the right maxillary sinus (Figure [3](#F3){ref-type="fig"}), whereas bilateral bone biopsies were negative. Since chemotherapy was urgent, cisplatin, cytarabin, dexamethasone plus R (*R-DHAP* regimen) were started. After the second cycle of *R-DHAP*, liver tests worsened, with a rapid evolution to liver failure, *i.e*., jaundice and ascites (Figure [2](#F2){ref-type="fig"}). She was admitted to our Unit and the liver decompensation was successfully treated with diuretics and anti-oxidant infusion (glutathione 2 g/d until recovery).
{#F1}
{#F2}
{#F3}
From February 2015 to March 2015 she underwent local radiotherapy on the right maxillary sinus (30 Gray). After the liver tests returned to normal levels, she started the triple DAAs regimen in the EAP, for a duration of 12 wk (Figure [2](#F2){ref-type="fig"}). The HCV-RNA showed a fast drop since after two weeks of AVT (42 UI/mL), while viremia became undetectable (limit of quantification according to our virological test was 15 UI/mL) after 8 wk of therapy, and persisted undetectable both at the end of AVT and 24 wk after. However, in May 2015, *i.e*., after 4 wk from the end of AVT, the patient experienced a new relapse of lymphoma (Figure [4](#F4){ref-type="fig"}). Due to the appearance of multiple cutaneous nodules on the middle third of the left arm, on the posterior region of the chest wall and on the right and left flanks, a biopsy of one of them was indicated, showing a dense lymphoid infiltrate with atypical growth pattern and cells of large size (CD20+, CD79a+, CD5+, Bcl2+, CD3-, CD30-, *Bcl6*-, *Mum1*-, Ki67 80%). A new PET scan showed multiple pathological uptake of fluorodeoxyglucose in inguinal regions and at the level of the subcutaneous nodules, while the bone marrow biopsy did not show any infiltration. Consistent with the history of two previous relapses and to the high expression of *Bcl2*, a histological reassessment was required on both the lymphoid tissue available at diagnosis and that from the biopsy of the subcutaneous nodule. An analysis with fluorescence *in situ* hybridization for the detection of lymphoma-associated chromosomal abnormalities was performed (Vysis LSI *Bcl2* break apart probe, Vysis LSI Myc break apart probe), showing additive copies of *Bcl2* (18q21) in 82% and rearrangement of *Myc* in 89% of the nuclei analyzed (8q24), respectively. Therefore, the final diagnosis was that of DHL, a more aggressive subtype of DBCL. In July 2015, the patient was firstly prescribed a cycle of ifosfamide, etoposide, epirubicin plus R (*R-IEV* regimen), followed by stem cell collection for a possible autologous stem cell transplantation; subsequently, she underwent a second cycle of *R/IEV/R* (regimen with an additive administration of R), which was burdened by infective complications requiring hospitalization and prolonged antibiotic therapies. At the last hepatological follow-up visit in November 2015, *i.e*., 24 wk after the end of AVT, HCV-RNA was permanently undetectable despite the immunosuppressive therapy. Afterwards, the patient was evaluated for an allogenic stem cells transplantation, which was made in another Hospital in January 2016, and she is free from lymphoma recurrence according to recent clinical and radiological evaluations.
{#F4}
DISCUSSION
==========
The role of HCV in pathogenesis of NHL is well established. The regression of HCV-associated indolent lymphomas after a successful AVT adds the demonstration that there is a relationship between HCV and lymphomagenesis, but there are no data about the response of DHL to AVTs. Because HCV is not integrated into the host genome, there should be indirect mechanisms to induce malignancy ("hit and run theory"). The main hypothesis links the antigenic stimulation caused by CHC to the chronic proliferation of B cells, to produce firstly a polyclonal and after a monoclonal expansion of these cells resulting, in conjunction with further occurrence of additional genetic mutations, in an overt NHL\[[@B5]\]. Notably, several years have to pass in order to accumulate mutational changes, whereas a genetic predisposition or additional mutagenic effects in genes *Bcl2*, *Bcl6* or *Ccnd1* could explain the DHL mutation occurrence. Indeed, HCV could induce a mutator phenotype, which involves enhanced mutations of many somatic genes. In the management of HCV-related DLBCL, anthracycline-based chemotherapy, usually *CHOP*, associated with R (chimeric anti-CD20 monoclonal antibody) is the standard of care\[[@B6]\]. Unlike indolent B-cell lymphomas\[[@B7]\], AVT does not play a significant role in HCV-positive DLBCL. Sequential immune-chemotherapy followed by ATV has been used in two studies with promising results leading to improved clinical outcome and prolonged disease free survival\[[@B8],[@B9]\]. On the other hand, a French study reported a higher overall survival and progression free survival in patients treated firstly by AVT, followed in some cases by chemotherapy\[[@B10]\]. All these studies are developed in the "IFN era", both alone or in combination with RBV and, more recently, with protease inhibitors like telaprevir and boceprevir. An IFN-based immunomodulatory and anti-angiogenetic mechanism could be supposed, although IFN-based AVTs cannot be started in most cases, because of contraindications and heavy side effects.
A few reports demonstrated lymphoma regression after HCV clearance with new DAAs. Rossotti et al\[[@B11]\] reported a rapid virological and hematological response with the combination of a NS3/NS4A inhibitor (faldaprevir) and a non-nucleoside NS5B inhibitor (deleobuvir) in a patient with HCV associated splenic marginal zone lymphoma. In another report, Sultanik et al\[[@B12]\] showed complete regression of MZL after 12 wk of therapy with sofosbuvir (an NS5B RNA-dependent RNA polymerase inhibitor) and RBV. In a case series from France of five patients with HCV-related lymphoma, two DLBCL were successfully treated with sofosbuvir and daclatasvir (an NS5A protease inhibitor), with complete hematological response after 6 mo\[[@B13]\]. There are no reported cases of DHL and CHC treated by new DAAs.
According to clinical data, we could exclude an advanced liver fibrosis in our patient and we estimated a minimal risk of liver decompensation. Therefore, the patient was firstly successfully treated with *R-CHOP*, but after the first hematological relapse, a new immunosuppressive therapy with *R-DHAP* schedule caused an overt liver failure. R has been described to enhance viral replication due to the immune system imbalance, but the rate of severe hepatic complications remains low, with some exceptions, such as in presence of hepatitis B virus or HIV infection, cirrhosis or hepatocellular carcinoma\[[@B7],[@B14]\]. There are controversial studies about the impairment of liver function, which could be caused both by a toxicity of immune-chemotherapy treatment and by HCV reactivation. Indeed, it is not clear if the anecdotal episodes of liver failure in this setting are due to higher HCV replication and enhanced necro-inflammatory activity, or to a direct chemo-toxicity. Chemotherapy-induced HCV reactivation in DLBCL is rarely reported, whereas there are no data about HCV and DHL\[[@B15]-[@B17]\]. In our case the liver failure may have been caused by an add-on effect of HCV reactivation and liver chemo-toxicity, as well suggested by a higher increase of Gamma-Glutamyl Transpeptidase, marker of liver toxicity, instead of a pure elevation of Alanine Aminotransferase, a possible expression of an inflammatory response HCV-mediated. Interestingly, during the liver failure HCV-RNA slightly dropped, so we can suppose that the event was caused by immune re-activation rather than by HCV direct effect on the necro-inflammatory activity. Following the second immune-chemotherapy and after recovering from the acute liver failure, the patient started antiviral therapy with second generation DAAs: OBV/PTV/r and DSV. This multi-targeted 3-DAAs regimen in combination with or without RBV is approved in many countries to treat HCV genotype 1-4 infection. Approval for the treatment of HCV genotype 1 patients with compensated cirrhosis was based on the evidence of a phase 3 trial of 380 patients in which OBV/PTV/r and DSV plus RBV achieved SVR rates at post-treatment week 12 (SVR12) of 91.8% and 95.9% after 12 or 24 wk of therapy, respectively\[[@B18]\]. In the past, the AVTs for HCV were conceived with IFN-based regimens, so an immunomodulatory effect due to interactions with both the adaptive and innate immune response of the host, further than an anti-inflammatory and antiviral effect by inhibiting the synthesis of various cytokines, were possibly responsible for the remission of HCV-related lymphomas. In this context, at the moment, there are no definitive data about IFN free therapies, and it remains to be solved whether IFN is crucial for its direct antitumor and anti-proliferative effect against NHL further than for its antiviral effect.
In conclusion, this is the first case, to our knowledge, of DHL and CHC successfully treated by new DAAs. According to our experience, DHL must be suspected in case of HCV-related lymphoma, and an early diagnosis could direct towards a different hematological management, because a poor prognosis should be expected. Moreover, DAAs triple regimen with OBV/PTV/r and DSV achieves a rapid virological response, and the result is sustained during immunosuppressive therapy, without evidence of HCV reactivation. Finally, we can suggest that in cases of aggressive HCV-related lymphoma, it is mandatory to treat the HCV infection with the new IFN-free regimens at least after the first chemotherapy cycle. This choice could avoid a liver failure in case of re-treatment with hepatotoxic drugs, and it allows a better and safer management of hematological disease. A potential favorable effect of the AVTs on DHL regression should be investigated.
ACKNOWLEDGMENTS
===============
We thank Cristina Madaudo and Lucia Lo Scalzo for preparing figures and graphics.
COMMENTS
========
Case characteristics
--------------------
A 39-year-old Caucasian woman presented with neck lymph nodes enlargement and hepatitis C virus (HCV)-infection.
Clinical diagnosis
------------------
Painless swelling in the right side of the neck with increased thickness.
Differential diagnosis
----------------------
Inflammatory process of the throat, metastatic cancer of head-neck sites, lymphoproliferative disease.
Laboratory diagnosis
--------------------
All blood tests were within normal limits except for high HCV-viremia.
Imaging diagnosis
-----------------
Computed tomography showed multiple lymph nodes on both sides of the diaphragm.
Pathological diagnosis
----------------------
Double-hit B cell lymphoma.
Treatments
----------
Chemotherapy, radiotherapy and antiviral therapy for HCV.
Related reports
---------------
Double-hit B cell lymphoma is a rare entity with no standardized chemotherapeutic strategies and its association with HCV-infection is not ever been investigated. There are no reports about the efficacy of antiviral therapy for HCV plus chemotherapies, in order to cure this pathology in presence of HCV.
Terms explanation
-----------------
HCV could induce B cell proliferation and cause non-Hodgkin lymphoma. The typical HCV-related lymphomas are marginal zone lymphoma and diffuse large B cell lymphoma. Double-hit B cell (DHL) is a new kind of lymphoma with more aggressive outcome, and it is characterized by chromosomal rearrangements, specifically of *Myc* oncogene and either B cell lymphoma 2 and B cell lymphoma 6 oncogenes or gene for Cyclin D1.
Experiences and lessons
-----------------------
DHL must be suspected in case of HCV-related lymphoma, and an early diagnosis could direct towards a different hematological management, because a poor prognosis should be expected. A potential favorable effect of new antiviral therapies on DHL regression should be investigated.
Peer-review
-----------
This manuscript has reported the first case of DHL and CHC successfully treated by new DAA.
Manuscript source: Unsolicited manuscript
Specialty type: Gastroenterology and hepatology
Country of origin: Italy
Peer-review report classification
Grade A (Excellent): A
Grade B (Very good): 0
Grade C (Good): C, C
Grade D (Fair): 0
Grade E (Poor): 0
Institutional review board statement: This case report was exempt from the Institutional Review Board standards at University of Campus Bio Medico of Rome.
Informed consent statement: The patient involved in this clinical case gave her written informed consent authorizing use and disclosure of her protected health information.
Conflict-of-interest statement: All the authors have no conflicts of interests to declare.
Peer-review started: March 24, 2016
First decision: June 12, 2016
Article in press: August 16, 2016
P- Reviewer: Einberg AP, El-Shabrawi MH, Ji FP S- Editor: Qiu S L- Editor: A E- Editor: Li D
[^1]: Author contributions: All the authors contributed equally to the conception and design of the article.
Correspondence to: Giovanni Galati, MD, Unit of Internal Medicine and Hepatology, Department of Medicine, Università Campus Bio-Medico di Roma, via Álvaro del Portillo 21, 00128 Rome, Italy. <[email protected]>
Telephone: +39-062-25411446 Fax: +39-062-25411944
| {
"pile_set_name": "PubMed Central"
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Introduction
============
Lung cancer (LC) is the most deadly type of cancer and represents a major public health problem worldwide. It is the leading cause of cancer-related death in Brazil and in the world, with 1.3 million deaths annually.( [@B01] ) Another major cause of morbidity and mortality, especially in developing countries, is tuberculosis.( [@B02] )
It has been suggested that inflammation and pulmonary fibrosis caused by tuberculosis can induce genetic damage, which can increase LC risk.( [@B03] - [@B05] ) The increased occurrence of LC in patients with tuberculosis can also be related to infection-induced immunosuppression.( [@B06] ) In contrast, cancer-induced or chemotherapy-induced immunosuppression can also lead to increased rates of tuberculosis reactivation in patients with solid tumors.( [@B07] )
The simultaneous or sequential occurrence of pulmonary tuberculosis and LC in the same patient has been reported in various case series and case-control studies.( [@B08] - [@B13] ) The association between these two diseases is important, since both are quite prevalent and have a major impact on public health. In addition, considering that one study( [@B10] ) demonstrated that the association between tuberculosis and LC varies in different ethnic groups and also in different regions, it is important to know the characteristics of these cases in Brazil. Therefore, the objective of this study was to describe the clinical characteristics of patients with pulmonary tuberculosis and LC.
Methods
=======
This was a cross-sectional study, involving retrospective data collection, conducted in order to determine the characteristics of patients with tuberculosis and LC who were treated at the *Hospital de Clínicas de Porto Alegre* (HCPA, Porto Alegre *Hospital de Clínicas*), located in the city of Porto Alegre, southern Brazil. The HCPA is a general tertiary university hospital with 750 beds and approximately 30,000 hospitalizations annually. The HCPA Research Ethics Committee granted permission for access to patient records. A waiver of informed consent was obtained, and the researchers signed a confidentiality agreement.
The study involved patients diagnosed with tuberculosis and LC, either simultaneously or sequentially. We performed a search of the files of the pulmonology outpatient clinics for patients with tuberculosis and LC, and the electronic medical records of these patients were reviewed. A standardized form was completed for each patient included in the study. Demographic data and data relating to the diagnoses of tuberculosis and LC were collected, as were pulmonary function test results.
The diagnosis of pulmonary tuberculosis was based on consensus criteria( [@B14] ): positive Ziehl-Neelsen staining for AFB (two positive smears); positive Ziehl-Neelsen staining for AFB (a positive smear and a positive culture for *Mycobacterium tuberculosis*); positive Ziehl-Neelsen staining for AFB and radiological findings consistent with pulmonary tuberculosis; a single positive culture for *M. tuberculosis*; or epidemiological, clinical, and radiological findings consistent with pulmonary tuberculosis, associated with a favorable response to treatment with antituberculosis drugs. The diagnosis of LC was based on anatomopathological findings.
The diagnoses of LC and tuberculosis were classified by timing as follows: simultaneous--when the diagnoses of tuberculosis and LC occurred simultaneously or when the time between the two diagnoses was \< 2 months; sequential (LC first)--when tuberculosis was diagnosed ≥ 2 after LC diagnosis and within 12 months of completion of LC treatment; sequential (tuberculosis first)--when LC is diagnosed ≥ 2 months after tuberculosis diagnosis, indefinitely, because of the possibility of scar cancer.( [@B10] , [@B15] , [@B16] )
The data were entered into Microsoft Excel spreadsheets, after which they were processed and analyzed with the Statistical Package for the Social Sciences, version 18.0 (SPSS Inc., Chicago, IL, USA). We carried out a descriptive analysis of the study variables. Quantitative data are presented as mean ± SD. Qualitative data are expressed as n (%).
Results
=======
The study involved 24 patients diagnosed with tuberculosis and LC between 2009 and 2012. The principal characteristics of the patients are shown in [Table 1](#t01){ref-type="table"}. In 10 of the 24 patients, the diagnoses of tuberculosis and LC occurred simultaneously, whereas, in 14, tuberculosis was diagnosed before LC. The median time between the two diagnoses was 5 years (interquartile range: 1-30 years).
Table 1Principal characteristics of the patients.a
Three patients (12.5%) reported having had tuberculosis twice. The diagnosis of tuberculosis was established by the following methods: smear microscopy of spontaneous sputum, in 3 patients; culture of spontaneous sputum, in 2; smear microscopy of induced sputum, in 2; bronchoalveolar lavage (BAL) culture, in 2; PCR positivity for *M. tuberculosis* in BAL samples, in 4; and clinical and radiological findings consistent with tuberculosis, in 11.
Tuberculosis treatment was as follows: a regimen consisting of rifampin, isoniazid, and pyrazinamide, in 11 patients (45.8%); and a regimen consisting of rifampin, isoniazid, pyrazinamide, and ethambutol, in 7 (29.2%). In 6 patients (25.0%), data on treatment regimen were either not provided or unavailable. Only 1 patient (4.2%) reported noncompliance with tuberculosis treatment, 3 (12.5%) died, and 16 (66.7%) were discharged as cured. For 5 (20.8%) of the patients, no treatment outcome data were available.
The mean age at LC diagnosis was 62.8 ± 10.6 years. Of the 24 patients, 7 (29.2%) presented with distant metastases at diagnosis; of those 7 patients, 71% were diagnosed with LC and tuberculosis simultaneously. The following LC treatments were performed: radiotherapy alone, in 6 patients (25.0%); chemotherapy and radiotherapy, in 4 (16.7%); surgery alone, in 3 (12.5%); chemotherapy alone, 2 (8.3%); surgery and radiotherapy, in 2 (8.3%); surgery and chemotherapy, in 2 (8.3%); and supportive treatment alone, in 5 (20.8%).
Discussion
==========
In this study, we described the characteristics of 24 patients developing tuberculosis and LC, either simultaneously or sequentially. Tuberculosis was diagnosed before LC in most of the patients, and in none of the cases was LC diagnosed before tuberculosis. Non-small cell lung cancer, especially adenocarcinoma, was the most common histological type.
The coexistence of pulmonary tuberculosis and LC was first described in 1810, and it was demonstrated histologically a few years later.( [@B09] ) Since then, several studies on this association have been published, most of which are case series and case-control studies.( [@B08] - [@B13] ) However, it has always been questioned whether this association was casual or whether it could be explained by a plausible biological mechanism. One hypothesis would be that inflammation associated with infections can contribute to carcinogenesis.( [@B04] ) Reactive oxygen or nitrogen species produced by activated neutrophils can bind to the DNA, inducing genetic damage and neoplastic transformation.( [@B17] , [@B18] ) In fact, it has been shown that alterations of the fragile histidine triad gene might be involved in lung carcinogenesis in patients with chronic pulmonary tuberculosis.( [@B19] - [@B21] ) In addition, during tissue repair, there is increased cell proliferation and angiogenesis, and the epithelium is more prone to metaplasia. Furthermore, carcinogens concentrate preferentially in hyperactive areas to induce neoplastic changes.( [@B22] , [@B23] )
The cases described here are similar to those reported in previous studies.( [@B08] - [@B13] ) Adenocarcinoma was the most common histological type in our case series. In a meta-analysis( [@B10] ) of 37 case-control studies and 4 cohort studies, the association between tuberculosis and LC was significant for adenocarcinoma (relative risk = 1.6), but not for epidermoid carcinoma or small cell carcinoma. In a case series of LC in Japan,( [@B24] ) adenocarcinoma was also the most common cancer. Another study( [@B09] ) demonstrated that all scar carcinomas were adenocarcinomas, and that, even in cases in which there was no proximity between the scar and the neoplasm, adenocarcinoma was the most common histological type.
The vast majority of the patients described here were smokers or former smokers. Despite the cumulative effects of tobacco as a carcinogen, the relationship between pulmonary tuberculosis and LC persists even after control for smoking, with cancer risk being 2.5-fold higher among patients with tuberculosis.( [@B06] , [@B08] , [@B25] ) A meta-analysis( [@B10] ) corroborated this evidence, showing that the association between tuberculosis and LC was not due to the effects of smoking, because, when considering only nonsmoking patients, there was a 1.78-fold increase in LC risk among patients with tuberculosis. That same meta-analysis demonstrated that the association between tuberculosis and LC was not due to the time since diagnosis of tuberculosis. Because the initial symptoms of LC can be mistaken for the symptoms of pulmonary tuberculosis, that analysis was restricted to studies in which tuberculosis was diagnosed more than 1 year before cancer, in order to minimize this bias. The studies were grouped by time between the two diagnoses (1-5, 6-10, 11-20, and more than 20 years). The increase in cancer risk was higher in the first 5 years after tuberculosis; diagnosis; however, the risk remained 1.99-fold higher among those for whom the time between the diagnoses was more than 20 years. This was true in our sample as well, given that the median time between the diagnoses of tuberculosis and LC was 5 years (interquartile range: 1-30 years). Another, more recent study( [@B11] ) showed that LC risk was highest in the 2-year window after tuberculosis diagnosis (OR = 5.01) but remained elevated even 2 years after diagnosis (OR = 1.53).
Approximately 30% of the patients presented with distant metastases at LC diagnosis. Of those, 71% were diagnosed with LC and tuberculosis simultaneously. Another case series found that 50% of the patients had stage IV LC.( [@B24] ) Because the initial symptoms of these two diseases are similar, it should be considered that there can be a delay in the diagnosis of either condition and, consequently, patients can present with either LC or tuberculosis at a more advanced stage.( [@B26] )
In none of our reported cases was LC diagnosed before pulmonary tuberculosis. In contrast, in a recent, retrospective case-control study( [@B15] ) involving 36 patients with LC, 10 (27.8%) were diagnosed with tuberculosis and cancer concomitantly, whereas 26 (72.2%) were diagnosed with tuberculosis after being diagnosed with cancer. In a case series conducted in Japan,( [@B24] ) the diagnoses were concomitant in 6 patients, tuberculosis was diagnosed before LC in 5 cases, and LC was diagnosed before tuberculosis in the remaining 5. It is possible that tuberculosis was diagnosed before LC more frequently because of a reverse causality bias, i.e., an occult cancer can reduce immunity and lead to reactivation of latent tuberculosis. Therefore, tuberculosis can present clinically before LC.( [@B16] , [@B19] )
Our study has limitations that need to be considered. The major one is that the cases were identified retrospectively on the basis of a search of the files of the specialized outpatient clinics at our hospital. Retrospective studies are at risk of selection bias (cases lost to follow-up) and measurement bias (data obtained from medical records). We should also consider the Berkson bias, in which patients with an index diagnosis are more likely to be diagnosed with another disease than are those without an index diagnosis. For instance, patients with tuberculosis, at follow-up chest X-ray, are more prone to be diagnosed with cancer than are those not diagnosed with tuberculosis.( [@B27] ) Nevertheless, because the occurrence of tuberculosis and LC, either simultaneously or not, may have different characteristics according to ethnic group and region,( [@B10] ) it is relevant that cases identified locally be described. In addition, the importance of these cases lies in the fact that patients diagnosed with tuberculosis should be advised to avoid lung carcinogens, such as tobacco smoking, as much as possible, since these agents contribute to a substantial increase in LC risk.
In conclusion, the present study demonstrated that most of the patients with tuberculosis and LC were smokers, and that tuberculosis was diagnosed either before or simultaneously with LC. Non-small cell lung cancer, especially adenocarcinoma, was the most common histological type.
Study carried out at the Federal University of Rio Grande do Sul School of Medicine, Porto Alegre, Brazil.
Financial support: Dirceu Felipe Valentini Junior is the recipient of a Young Investigator Grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development).
Artigos Originais
Tuberculose pulmonar e câncer de pulmão: ocorrência simultânea ou sequencial
\*
Silva
Denise Rossato
Professora Adjunta de Pneumologia
Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil
Valentini
Dirceu Felipe
Junior
Acadêmico de Medicina
Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil
Müller
Alice Mânica
Doutoranda
Programa de Pós-Graduação em Ciências Pneumológicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil
Almeida
Carlos Podalirio Borges de
Mestrando
Programa de Pós-Graduação em Ciências Pneumológicas, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil
Dalcin
Paulo de Tarso Roth
Professor Associado de Pneumologia
Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil
Endereço para correspondência
: Denise Rossato Silva. Rua Ramiro Barcelos, 2350, Santa Cecília, CEP 90035-903, Porto Alegre, RS, Brasil. Tel. 55 51 3359-8241. E-mail:
denise.rossato\@terra.com.br
OBJETIVO:
=========
O câncer de pulmão (CP) é o tipo mais letal de câncer na população mundial e representa um importante problema de saúde pública. A tuberculose é outra causa significativa de morbidade e mortalidade, especialmente em países em desenvolvimento. A ocorrência de tuberculose pulmonar e CP no mesmo paciente, simultaneamente ou não, tem sido descrita em diversas séries de casos e estudos de caso-controle. O objetivo deste estudo foi descrever as características de pacientes com tuberculose e CP, com apresentação simultânea ou sequencial.
MÉTODOS:
========
Estudo transversal, realizado através de revisão de prontuários.
RESULTADOS:
===========
Foram incluídos no estudo 24 pacientes com diagnósticos de tuberculose e CP no período entre 2009 e 2012. Os diagnósticos de tuberculose e CP ocorreram simultaneamente em 10 pacientes, enquanto a tuberculose foi diagnosticada antes do CP em 14. A mediana do tempo entre os diagnósticos foi de 5 anos (variação interquartil: 1-30 anos). Quatorze pacientes (58,3%) eram do sexo masculino, 20 (83,3%) eram brancos, e 22 (91,7%) eram tabagistas ativos ou ex-tabagistas. O tipo histológico mais comum foi o adenocarcinoma, em 14 casos (58,3%), seguido de carcinoma epidermoide, em 6 (25,0%). Sete pacientes (29,2%) já apresentavam metástases à distância no momento do diagnóstico; desses 7, 5 (71%) tiveram o diagnóstico de CP e TB simultaneamente.
CONCLUSÕES:
===========
Neste estudo, a maioria dos pacientes com tuberculose e CP eram tabagistas ou ex-fumantes e a tuberculose foi diagnosticada tanto antes quanto simultaneamente ao diagnóstico de CP. O carcinoma brônquico não pequenas células, em especial o adenocarcinoma, foi o tipo histológico mais comum.
Tuberculose/diagnóstico
Neoplasias Pulmonares/diagnóstico
Comorbidade
Introdução
==========
O câncer de pulmão (CP) é o tipo mais letal de câncer na população mundial e representa um importante problema de saúde pública. É a principal causa de morte por câncer no Brasil e no mundo, com 1,3 milhões de óbitos ao ano atribuídos ao CP mundialmente.( [@B01] ) A tuberculose é outra causa significativa de morbidade e mortalidade, especialmente em países em desenvolvimento.( [@B02] )
Tem sido sugerido que a inflamação e a fibrose pulmonar decorrentes da tuberculose podem induzir dano genético, podendo aumentar o risco de CP.( [@B03] - [@B05] ) A ocorrência aumentada de CP em pacientes com tuberculose também pode estar ligada à imunodepressão causada pela infecção.( [@B06] ) Por outro lado, a imunodepressão causada pelo câncer ou pela quimioterapia também pode aumentar a reativação de tuberculose em pacientes com neoplasias sólidas.( [@B07] )
A ocorrência de tuberculose pulmonar e CP no mesmo paciente, simultaneamente ou não, tem sido descrita em diversas séries de casos e estudos de caso-controle.( [@B08] - [@B13] ) A associação entre essas duas doenças é importante, já que ambas são bastante prevalentes e acarretam grande impacto na saúde pública. Além disso, considerando que um estudo( [@B10] ) demonstrou que a associação entre tuberculose e CP varia em diferentes grupos étnicos e também em diferentes regiões, seria importante conhecer as características desses casos em nosso meio. Portanto, o objetivo do presente estudo foi descrever as características clínicas de pacientes com tuberculose pulmonar e CP.
Métodos
=======
Trata-se de um estudo transversal com coleta de dados de forma retrospectiva, realizado com o objetivo de determinar as características de pacientes com tuberculose e CP atendidos no Hospital de Clínicas de Porto Alegre (HCPA), na cidade de Porto Alegre, sul do Brasil. O HCPA é um hospital geral, terciário e universitário com 750 leitos e aproximadamente 30.000 hospitalizações/ano. O Comitê de Ética do HCPA aprovou o acesso aos registros dos pacientes. Foi aprovada a dispensa do consentimento livre e informado, e os investigadores assinaram um termo de confidencialidade.
Foram incluídos no estudo pacientes com diagnóstico de tuberculose e CP, seja concomitante, seja com um dos diagnósticos antecedendo o outro. Foi realizada uma busca nos ambulatórios de pneumologia para localizar os pacientes com tuberculose e CP, e o prontuário eletrônico desses pacientes foi revisado. Um formulário padronizado foi preenchido para cada paciente incluído no estudo. Foram coletados dados demográficos, dados referentes ao diagnóstico de tuberculose e de CP, assim como resultados de provas de função pulmonar.
O diagnóstico de tuberculose pulmonar seguiu os critérios estabelecidos em um consenso( [@B14] ): detecção de BAAR pela coloração de Ziehl-Neelsen (duas amostras positivas); detecção de BAAR pela coloração de Ziehl-Neelsen (uma amostra positiva e uma cultura positiva para *Mycobacterium tuberculosis*); detecção de BAAR pela coloração de Ziehl-Neelsen e achados radiológicos compatíveis com tuberculose pulmonar; somente uma cultura positiva para *M. tuberculosis*; ou presença de achados epidemiológicos, clínicos e radiológicos compatíveis com tuberculose pulmonar, associados com uma resposta favorável ao tratamento com tuberculostáticos. O diagnóstico de CP foi baseado em resultados de exames anatomopatológicos.
Os diagnósticos de CP e tuberculose foram considerados da seguinte maneira: simultâneos -- quando o diagnóstico de tuberculose e CP foi simultâneo ou com uma diferença \< 2 meses entre os dois diagnósticos; sequencial (CP primeiro) -- quando a tuberculose foi diagnosticada ≥ 2 meses após o diagnóstico de CP e dentro de 12 meses do término do tratamento de CP; e sequencial (tuberculose primeiro): quando o CP foi diagnosticado ≥ 2 meses após o diagnóstico de tuberculose, indefinidamente, pela possibilidade de câncer de cicatriz.( [@B10] , [@B15] , [@B16] )
Os dados foram digitados em planilhas do programa Microsoft Office *Excel*, sendo processados e analisados com auxílio do programa *Statistical Package for the Social Sciences*, versão 18.0 (SPSS Inc., Chicago, IL, EUA). Foi realizada uma análise descritiva das variáveis em estudo. Os dados quantitativos são apresentados como média ± dp. Os dados qualitativos estão expressos em n (%).
Resultados
==========
Foram incluídos no estudo 24 pacientes com diagnósticos de tuberculose e CP no período entre 2009 e 2012. As principais características dos pacientes estão demonstradas na [Tabela 1](#t02){ref-type="table"}. Dos 24 pacientes, 10 tiveram o diagnóstico simultâneo de tuberculose e CP e 14 foram primeiramente diagnosticados com tuberculose. A mediana do tempo entre os diagnósticos foi de 5 anos (variação interquartil:1-30 anos).
Tabela 1Principais características dos pacientes.a
Três pacientes (12,5%) referiram história de tuberculose duas vezes. O diagnóstico da tuberculose foi realizado através de baciloscopia de escarro espontâneo, em 3 pacientes; cultura de escarro espontâneo, em 2; baciloscopia de escarro induzido, em 2; cultura de lavado broncoalveolar (LBA), em 2; PCR positiva para *M. tuberculosis* no LBA, em 4; e achados clínicos e radiológicos compatíveis com tuberculose, em 11.
O tratamento da tuberculose foi realizado com o esquema rifampicina, isoniazida e pirazinamida, em 11 pacientes (45,8%); e esquema rifampicina, isoniazida, pirazinamida e etambutol, em 7 (29,2%), enquanto 6 pacientes (25,0%) não souberam informar ou não havia informações no prontuário sobre o esquema de tratamento utilizado. Apenas 1 paciente (4,2%) relatou abandono de tratamento da tuberculose, 3 (12,5%) evoluíram a óbito, e 16 (66,7%) tiveram alta por cura. Em 5 (20,8%) dos pacientes, não se obteve o desfecho do tratamento.
A média de idade dos pacientes no momento do diagnóstico de CP foi de 62,8 ± 10,6 anos. Dos 24 pacientes, 7 (29,2%) já apresentavam metástases à distância no momento do diagnóstico; desses, 71% tiveram o diagnóstico de CP e tuberculose simultaneamente. Os tratamentos realizados para CP foram radioterapia exclusiva, em 6 (25,0%); quimioterapia e radioterapia, em 4 (16,7%); cirurgia exclusiva, em 3 (12,5%); quimioterapia exclusiva, em 2 (8,3%); cirurgia e radioterapia, em 2 (8,3%); cirurgia e quimioterapia, em 2 (8,3%); e tratamento de suporte exclusivo, em 5 (20,8%).
Discussão
=========
No presente estudo, relatamos as características de 24 pacientes que apresentaram, simultaneamente ou não, tuberculose pulmonar e CP. O diagnóstico de tuberculose ocorreu antes do de CP na maioria dos pacientes e, em nenhum caso, o diagnóstico de CP ocorreu antes do de tuberculose. O carcinoma brônquico não pequenas células, em especial o adenocarcinoma, foi o tipo histológico mais comum.
A coexistência de tuberculose pulmonar e CP foi primeiramente descrita em 1810, sendo demonstrada histologicamente alguns anos mais tarde.( [@B09] ) Desde então, foram publicados diversos estudos sobre essa associação, em sua maioria séries de casos e estudos de caso-controle.( [@B08] - [@B13] ) Entretanto, os autores sempre questionaram se essa associação seria casual ou se poderia ser explicada por algum mecanismo biológico plausível. Uma hipótese seria que a inflamação associada com infecções pode contribuir para a carcinogênese.( [@B04] ) Espécies reativas de oxigênio ou nitrogênio produzidas por neutrófilos ativados podem se ligar ao DNA, induzindo dano genético e transformação neoplásica.( [@B17] , [@B18] ) De fato, já foi evidenciado que alterações no gene *fragile histidine triad* podem estar envolvidas na carcinogênese pulmonar em pacientes com tuberculose pulmonar crônica.( [@B19] - [@B21] ) Além disso, durante o reparo tecidual, há uma maior proliferação celular, angiogênese, e o epitélio é mais propenso a sofrer metaplasia. Ainda, os carcinógenos se concentram preferencialmente em locais de hiperatividade para induzir alterações neoplásicas.( [@B22] , [@B23] )
Os casos que descrevemos são semelhantes aos relatados em estudos prévios.( [@B08] - [@B13] ) O adenocarcinoma foi o tipo histológico mais frequente em nossa série de casos. Em uma meta-análise( [@B10] ) de 37 estudos de caso-controle e 4 estudos de coorte, a associação entre tuberculose e CP foi significativa com adenocarcinoma (risco relativo = 1,6), mas não com carcinoma epidermoide ou de pequenas células. Em uma série de casos de CP no Japão,( [@B24] ) o adenocarcinoma também foi o câncer mais comum. Outro estudo( [@B09] ) demonstrou que todos os carcinomas de cicatriz eram adenocarcinomas e, mesmo nos casos em que não havia proximidade entre a cicatriz e a neoplasia, esse ainda era o tipo histológico mais comum.
A grande maioria dos pacientes que descrevemos tinha história de tabagismo (ativo ou no passado). Apesar dos efeitos aditivos do tabaco como carcinógeno, a relação entre tuberculose pulmonar e CP persiste mesmo após controle para o tabagismo, com um risco até 2,5 vezes maior de câncer entre os pacientes com tuberculose.( [@B06] , [@B08] , [@B25] ) Uma meta-análise( [@B10] ) corroborou essa evidência, mostrando que a associação entre tuberculose e CP não era devida aos efeitos do tabagismo, pois, analisando apenas os pacientes não tabagistas, houve um aumento de 1,78 vezes no risco de CP entre os pacientes com tuberculose. Naquela mesma meta-análise, foi demonstrado que a associação entre tuberculose e CP não era decorrente do tempo de diagnóstico da tuberculose. Como os sintomas iniciais de CP podem ser confundidos com os sintomas de tuberculose pulmonar, os estudos incluídos na análise foram restritos àqueles em que a tuberculose foi diagnosticada há mais de 1 ano antes do câncer, para minimizar esse viés. Os estudos foram combinados de acordo com o tempo entre os dois diagnósticos (1-5, 6-10, 11-20 e mais de 20 anos). O aumento do risco de câncer foi maior nos primeiros 5 anos após o diagnóstico de tuberculose; entretanto, o risco permaneceu 1,99 vezes maior no tempo entre diagnósticos por mais de 20 anos. Isso foi observado em nossa amostra, visto que a mediana do tempo entre o diagnóstico de tuberculose e o de CP foi de 5 anos, com um intervalo interquartil de 1-30 anos. Outro estudo mais recente( [@B11] ) mostrou que o maior risco de CP era no período de 2 anos após o diagnóstico de tuberculose (OR = 5,01), mas permanecia elevado mesmo após 2 anos de diagnóstico (OR = 1,53).
Aproximadamente 30% dos pacientes apresentavam metástases à distância no momento do diagnóstico de CP. Desses, 71% tiveram o diagnóstico de CP e tuberculose simultaneamente. Outros autores também demonstraram em uma série de casos que 50% dos pacientes apresentaram CP em estádio IV.( [@B24] ) Como os sintomas iniciais dessas duas patologias se assemelham, deve-se considerar que pode haver atraso no diagnóstico de uma das duas e, consequentemente, com a apresentação de CP ou tuberculose em uma fase mais avançada.( [@B26] )
Não relatamos nenhum caso de CP diagnosticado antes da tuberculose pulmonar. Contrariamente, em um recente estudo de caso-controle retrospectivo,( [@B15] ) dos 36 pacientes com CP, 10 (27,8%) tiveram o diagnóstico de tuberculose concomitantemente com o de câncer, enquanto 26 (72,2%) tiveram o diagnóstico de tuberculose após o de câncer. Em uma série de casos japonesa,( [@B24] ) em 6 pacientes, o diagnóstico foi concomitante; em 5 casos, a tuberculose precedeu o CP; e, nos 5 restantes, o CP antecedeu a tuberculose. É possível que a tuberculose tenha sido mais frequentemente diagnosticada antes do CP devido ao viés de causalidade reversa, ou seja, um CP oculto pode reduzir a imunidade e levar à reativação da tuberculose latente. Sendo assim, a tuberculose pode se apresentar clinicamente antes do CP.( [@B16] , [@B19] )
Nosso estudo apresenta limitações que precisam ser consideradas. A principal é que os casos foram identificados retrospectivamente a partir de uma busca ativa no grupo de pacientes atendidos nos ambulatórios especializados em nosso hospital. Estudos retrospectivos correm risco de vieses de seleção (por perda de casos), bem como de aferição (dados obtidos de prontuários médicos). Há ainda que se considerar o viés de Berkson, em que é mais provável que os pacientes com um diagnóstico índice tenham a outra doença diagnosticada do que aqueles pacientes sem o diagnóstico índice. Por exemplo, pacientes com tuberculose, ao fazer radiografias de tórax para acompanhamento, podem ter o diagnóstico de câncer mais facilmente realizado do que pacientes sem o diagnóstico de tuberculose.( [@B27] ) Apesar disso, como a ocorrência simultânea ou não de tuberculose e CP pode ter características próprias conforme o grupo étnico e a região,( [@B10] ) torna-se relevante a descrição dos casos identificados localmente. Adicionalmente, a importância desses casos reside no fato de que pacientes com diagnóstico de tuberculose devem ser orientados a evitar ao máximo a exposição a carcinógenos pulmonares, como o tabagismo, que contribuiriam para um aumento substancial no risco de CP.
Em conclusão, no presente estudo demonstramos que a maioria dos pacientes com tuberculose e CP são tabagistas e que a tuberculose ocorreu tanto antes quanto simultaneamente ao diagnóstico de CP. O carcinoma brônquico não pequenas células, em especial o adenocarcinoma, foi o tipo histológico mais comum.
Trabalho realizado na Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre (RS) Brasil.
Apoio financeiro: Dirceu Felipe Valentini Junior é bolsista de iniciação científica do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-cells-06-00048}
===============
The liver is one of the largest organs in the body, weighing up to 1.5 kg (3.3 lb.) in adults \[[@B1-cells-06-00048]\]. Interestingly, although this can constitute only \~2.5% of body weight, the liver receives around 25% of the cardiac output \[[@B2-cells-06-00048]\]. The liver is located in the upper right portion of the abdominal cavity, beneath the diaphragm and on top of the stomach, right kidney, and intestines. The human liver has of two main lobes, which are divided in eight segments (four each lobe). The segments are then microscopically divided in hepatic lobules, which may have anatomic variations between different species. Blood enters into the liver to circulate within the lobules through liver microvessels, while bile, produced and secreted by hepatocytes, flows in the opposite direction. Bile drains via several bile canaliculi that connect with larger ducts to ultimately form the common hepatic duct. The common hepatic duct transports bile to the gallbladder, and finally to its final destination: the duodenum.
The liver has a unique hemodynamic scheme. Blood from the spleen, pancreas, and gastrointestinal circulation reaches the liver via the portal vein together with arterial input from the hepatic artery. It is estimated that \~80% of the blood volume arises from the portal circulation, while the remaining \~20% originates from arterial flow \[[@B3-cells-06-00048],[@B4-cells-06-00048]\]. Once they enter the liver, both portal vein and hepatic artery will branch into smaller segments to irrigate several liver lobules through the hepatic portal spaces. Blood will then slowly flow within the sinusoids, being later drained out of the liver by the centrilobular vein via the hepatic vein. This single vascular architecture together with slow blood flow creates an intimate relationship between the systemic circulation and liver cells. The high permeability of capillary endothelium to water, macromolecules, and solutes is explained by the presence of special transporting systems, including channels, vesicles, diaphragms, and fenestrae. In fact, liver sinusoids are one of the most permeable microvessels in the body, and millions of large fenestrae (\>100 nm) can be found along the sinusoidal surface \[[@B5-cells-06-00048]\]. Lining their lumen, there is a specialized subtype of endothelial cell named LSECs (liver sinusoidal endothelial cells). LSECs comprise around \~20% of liver cells \[[@B6-cells-06-00048]\] and are located at the interface between hepatic microcirculation and hepatocytes. On the luminal side, LSECs continuously survey blood from the gastrointestinal tract, exerting a close relationship with resident liver macrophages (Kupffer cells) and all leukocytes that are in the circulation or those that constantly patrol liver vessels (including monocytes, NK, and NKT cells). On the other side (facing the Disse Space), LSECs interact with hepatic stellate cells (or Ito cells) and hepatocytes. This is crucial for liver metabolism since LSECs are a permeable barrier that mediates the exchange, active uptake, and degradation of circulating molecules \[[@B7-cells-06-00048]\]. LSECs also possess a high endocytic capacity, and numerous endocytic vesicles can be found under homeostatic conditions in their cytoplasm. It is well accepted that LSECs may perform effective uptake of a wide variety of substances from the blood by receptor-mediated endocytosis \[[@B8-cells-06-00048]\]. Therefore, considering the absence of a regular basal lamina together with the presence of fenestrae, LSECs are different and unique from any other type of endothelial cell in the body.
Considering that liver hemodynamic features and vascular architecture create a niche for blood surveillance, it is not surprising that \[[@B9-cells-06-00048]\] the hepatic environment harbors one of the largest populations of immune cells in the body. Virtually, subsets of all leukocytes and resident phagocytes can be found within the liver even under homeostatic conditions. Strikingly, these cells are not randomly distributed throughout the tissues; instead, they are strategically located within the different hepatic compartments (intravascular and subcapsular niches; discussed below), while a large population of these immune cells can be also found patrolling the sinusoidal lumen ([Figure 1](#cells-06-00048-f001){ref-type="fig"}). In addition, liver immune cell population can be rapidly and dramatically changed during inflammation, and this can be associated with the pathogenesis of several diseases. In this review, we will discuss how the differential tissular location of liver immune cells may influence their function during homeostasis and disease.
2. Immune System Ontogeny and the Correlation with the Fetal Liver {#sec2-cells-06-00048}
==================================================================
The liver harbors different immune cell populations that are established during the embryonic period of life. The ontogeny of fetal macrophages occurs in successive and overlapping waves that arise from both extra- and intra-embryonic sites, leading to the sequential acquisition of myeloid, erythroid, and lymphoid lineage potentials ([Figure 2](#cells-06-00048-f002){ref-type="fig"}). The yolk sac is the first hematopoietic organ where macrophages develop in mammals \[[@B8-cells-06-00048],[@B9-cells-06-00048],[@B10-cells-06-00048],[@B11-cells-06-00048],[@B12-cells-06-00048]\] and several studies have reported that macrophages arise in embryos before the generation of the first hematopoietic stem cells (HSCs). In mouse embryos, the first wave is termed primitive hematopoiesis and develops from the posterior plate mesoderm in the blood islands of the extra-embryonic yolk sac around E7.5, giving rise to primitive erythroblasts, megakaryocytes, and primitive macrophages \[[@B9-cells-06-00048],[@B13-cells-06-00048],[@B14-cells-06-00048],[@B15-cells-06-00048]\]. These cells are derived from c-Kit^lo^ CD41^lo^ precursors, which are also dependent on the expression of the transcription factor PU.1 \[[@B13-cells-06-00048],[@B16-cells-06-00048]\].
Between E8.0 and E8.5, the heart is formed and the fetal cardiovascular system is established and connected with the vitelline and umbilical vessels \[[@B17-cells-06-00048]\]. At this time, a second wave of hematopoietic progenitors occurs, called erythro-myeloid precursors (EMPs). They arise from the yolk sac hemogenic endothelium \[[@B13-cells-06-00048],[@B16-cells-06-00048]\]. These progenitors (EMPs) are phenotypically defined as c-Kit^+^ AA4.1^+^ (CD93^+^) CD41^+^ VE-cadherin^+^ CD16/32^+^ (FCgII and FCgIII receptors) CD45^lo^ \[[@B18-cells-06-00048],[@B19-cells-06-00048]\] and exhibit erythroid and broad myeloid---but not lymphoid---potential \[[@B20-cells-06-00048]\]. EMP-derived hematopoiesis is sufficient to support survival of HSC deficient embryos until birth \[[@B21-cells-06-00048]\]. In addition, EMPs emerge in a Runx1-dependant endothelial-to-hematopoietic transition \[[@B22-cells-06-00048]\]. The number of EMPs in the yolk sac peaks between E9.5 and E10.5, and they seed the fetal liver as soon as E9 \[[@B23-cells-06-00048],[@B24-cells-06-00048]\]. These cells will expand and differentiate into multiple lineages, including fetal macrophages, which will colonize the liver giving rise to Kupffer cells (KCs) and colonize to other organs, including the fetal lung \[[@B25-cells-06-00048]\]. Yolk-sac EMPs express the gene encoding the transcription factor c-Myb (Myb) \[[@B26-cells-06-00048]\], but their commitment and differentiation into the myeloid fate is unaltered in c-Myb-deficient embryos, although their erythroid potential is blocked \[[@B25-cells-06-00048],[@B27-cells-06-00048],[@B28-cells-06-00048]\]. Therefore, c-Myb is required for the commitment and differentiation of EMPs into the erythroid fate \[[@B29-cells-06-00048]\] but is dispensable for myeloid differentiation.
Almost concomitant with the emergence of the late EMPs, a new wave of hematopoietic progenitors arises from the intraembryonic hemogenic endothelium, which begins with the generation of immature HSCs in the para-aortic splanchnopleura region and proceeds to give rise to fetal HSCs in the aorta, gonads, and mesonephros regions at E10.5 \[[@B30-cells-06-00048]\], as well as the umbilical and vitelline arteries \[[@B31-cells-06-00048]\]. These precursors migrate to the fetal liver, where they expand and differentiate from E12.5 until definitive hematopoiesis begins to shift to the bone marrow. HSCs colonize the embryonic bone marrow at E15, and active hematopoiesis starts at E17 \[[@B24-cells-06-00048],[@B32-cells-06-00048]\]. Fetal and adult HSCs require c-Myb for their self-renewal and maintenance, and loss of c-Myb expression leads to rapid HSC-derived hematopoiesis failure \[[@B27-cells-06-00048],[@B33-cells-06-00048],[@B34-cells-06-00048]\]. In addition, HSCs also require the transmembrane receptor Notch1 for their emergence, in contrast to EMPs, as Notch1^−/−^ embryos have normal numbers of hematopoietic progenitor cells in the yolk sac but very few in the body of the embryo \[[@B35-cells-06-00048]\].
At 12 days of gestation, the number of liver macrophages with the ability to engulf blood cells rapidly increases and most of them are attached to the endothelial cells in the sinusoid. From E12.5, the fetal liver becomes the major hematopoietic organ within the embryo and contains progenitors of different origins and varied potentials, which together will give rise to the emergent immune system. The hepatic hematopoiesis becomes most prominent from 16 to 18 days of gestation, whereas it decreases in the perinatal period and disappears within a week after birth.
3. Differential Location of Immune Cells throughout the Liver {#sec3-cells-06-00048}
=============================================================
3.1. Phagocytes {#sec3dot1-cells-06-00048}
---------------
### 3.1.1. Macrophages and Monocytes {#sec3dot1dot1-cells-06-00048}
Hepatic macrophages were first observed in 1876 by Karl Wilhelm von Kupffer, who described them as an integral part of the sinusoid endothelium and were initially called "Sternzellen" (star cells) \[[@B7-cells-06-00048]\]. In 1898, after several years of research, Tadeusz Browicz correctly identified them as macrophages, and they received the name of Kupffer-Browicz cells, also known as Kupffer cells (KCs). The liver comprises the largest population of resident macrophages in the body representing \~80--90% of total fixed macrophages and 35% of the liver non-parenchymal cells in normal adult mice \[[@B36-cells-06-00048]\]. Different from other organs where the resident macrophages are located in the tissue parenchyma, in the liver these cells are inside the sinusoids in direct contact with blood circulation \[[@B7-cells-06-00048],[@B17-cells-06-00048],[@B37-cells-06-00048],[@B38-cells-06-00048]\]. KCs are adhered to the endothelium and emit extensions into the extravascular space (space of *Dissé*) acting as a bridge between the blood and components of the liver parenchyma. Moreover, the continuous structure formed by KCs and endothelial cells forms the reticuloendothelial system (RES), which serves as the first line of defense against particles and immunorreactive material passing from the gastrointestinal tract via the portal circulation and may be considered the final component in gut barrier function \[[@B39-cells-06-00048]\].
Kupffer cells have the ability to migrate through the tissue against or in favor of blood flow by locomotion at 4 μm/min on average \[[@B40-cells-06-00048]\], half the speed observed in neutrophils that patrol the liver \[[@B41-cells-06-00048]\]. However, their speed greatly reduces when these cells perform phagocytosis and many of them lose their ability to move in these conditions \[[@B40-cells-06-00048]\]. Despite this locomotion ability described under homeostasis conditions, KCs maintain a constant pattern of tissue distribution. Most of them are in the sinusoidal zones, close to the portal spaces, and in smaller numbers in the centrilobular zones \[[@B38-cells-06-00048]\]. These patterns of location also affect morphology, phagocytic capacity, and the metabolic functions exerted by the cells in these different regions of the hepatic lobules \[[@B42-cells-06-00048],[@B43-cells-06-00048]\]. In addition to morphological, functional, and tissue location variations, hepatic macrophages are also a heterogeneous phenotype population. Under homeostatic conditions, two F4/80^+^ Kupffer cell subsets may exist: a CD68^+^ subset with phagocytic activity and a CD11b^+^ subset with cytokine producing capacity \[[@B44-cells-06-00048]\]. Furthermore, subpopulations of KCs have differential expression of CD11c and major histocompatibility complex class I and class II (MHCI and MHCII) \[[@B38-cells-06-00048]\]. Interestingly, unlike other organs and tissues such as brain, intestine, lung, kidneys, spleen and skin, hepatic macrophages do not express the fractalkine receptor CX3CR1 in homeostatic conditions ([Figure 3](#cells-06-00048-f003){ref-type="fig"}) \[[@B38-cells-06-00048],[@B45-cells-06-00048]\]. More recently, an important common feature between KC subsets that allows their separation from other resident macrophages is the expression of the gene *Clec4f*, a KC-specific marker \[[@B46-cells-06-00048],[@B47-cells-06-00048],[@B48-cells-06-00048]\].
For many years, it was believed that tissue-resident macrophages are originated and continuously maintained by blood-circulating monocytes, which arose from progenitors in the adult bone marrow (BM). This cellular hierarchy was a foundational concept in the definition of the "mononuclear phagocyte system" (MPS) by Van Furth and colleagues in the 1970s that grouped together promonocytes and their precursors in the BM, monocytes in the peripheral blood, and macrophages in the tissues \[[@B49-cells-06-00048],[@B50-cells-06-00048]\]. After the discovery of a common myeloid precursor (macrophage DC precursor-MDPs) of monocytes, macrophages, and dendritic cells, the knowledge of the ontogeny of the cell types within the MPS has dramatically changed \[[@B51-cells-06-00048]\]. Since then, efforts have been dedicated in deciphering the developmental lineages of monocytes, DCs, and macrophages \[[@B52-cells-06-00048],[@B53-cells-06-00048],[@B54-cells-06-00048],[@B55-cells-06-00048],[@B56-cells-06-00048]\]. Currently, the MPS grouped together monocytes, macrophages, dendritic cells, and all their precursors based on morphology, expression of surface markers, functional specialization, and ontogeny \[[@B57-cells-06-00048]\]. Although monocytes and macrophages belong to the same group, recent ontogeny studies have shown that the emergence, establishment, and maintenance of resident macrophages populations---such as KCs---are independent of circulating monocytes \[[@B25-cells-06-00048],[@B56-cells-06-00048],[@B58-cells-06-00048],[@B59-cells-06-00048],[@B60-cells-06-00048]\].
In the fetal liver, macrophages play a scavenger function and support hematopoiesis. These macrophages begin to show peroxidase activity in the nuclear envelope and endoplasmic reticulum after 17 days of gestation \[[@B15-cells-06-00048],[@B61-cells-06-00048],[@B62-cells-06-00048]\], corresponding to KCs in the adult liver \[[@B63-cells-06-00048],[@B64-cells-06-00048]\]. Fetal macrophages rapidly expand to transform into KCs in the late stage of ontogeny and after birth. Fetal tissue macrophages also have a high proliferative capacity that is important for their survival in loco and for their colonization from the fetal liver to the other fetal tissues via blood stream. From this point of view, the fetal liver is a central organ for producing and supplying macrophages and their precursors to the other tissues. Although myeloid precursors are found in fetal hematopoiesis, the production of monocytes during the hematopoiesis of the yolk sac is poorly developed. Compared to monocytes originating from bone marrow precursors, the number of peroxidase-positive granules in monocytes originating from yolk sac precursors is significantly lower \[[@B9-cells-06-00048]\]. In the fetal liver, monocytes increase in number and show an increase in the number of peroxidase-positive granules \[[@B15-cells-06-00048]\]. In the middle stages of hepatic hematopoiesis, their ultra-structural features resembled those seen during bone marrow hematopoiesis.
Further studies have revealed that the contribution of HSCs to tissue-resident macrophages differs among organs and frequently increases with age. The contribution of HSCs to adult tissue-resident macrophages is minor (\<5%) in the brain, liver, and epidermis \[[@B26-cells-06-00048],[@B27-cells-06-00048],[@B58-cells-06-00048]\]. Although small, their contribution increases with age in the lungs, heart and spleen \[[@B25-cells-06-00048],[@B65-cells-06-00048]\], and might predominate in gut lamina propria after weaning \[[@B66-cells-06-00048],[@B67-cells-06-00048]\]. Partial replacement of tissue-resident macrophages is also observed following γ-irradiation, bone marrow transplantation, or adoptive-transfer experiments \[[@B51-cells-06-00048],[@B68-cells-06-00048],[@B69-cells-06-00048]\] and in macrophage-depletion studies, such as intravenous injection of clodronate-loaded liposomes (CLL) \[[@B38-cells-06-00048]\]. KCs are also reported to be replaced by bone marrow--derived progenitor cells following, for example, massive death of Kupffer cells in severe experimental infection with *Listeria monocytogenes* \[[@B70-cells-06-00048]\] and drug induced liver injury. However, different resident macrophages---including KCs, microglia, alveolar macrophages, peritoneal macrophages, and splenic macrophages---have the potential to proliferate and self-renewing \[[@B37-cells-06-00048],[@B59-cells-06-00048],[@B71-cells-06-00048],[@B72-cells-06-00048]\]. In some cases, tissue-resident macrophages can immediately self-replenish following severe depletion \[[@B59-cells-06-00048],[@B71-cells-06-00048],[@B73-cells-06-00048]\] and exert their functions in the tissue.
Macrophages play a central role in both tissue homeostasis and inflammation, accomplishing essential tissue-specific functions as well as protecting the organism from infection. It is currently believed that the characteristic functions exerted by the different populations of resident macrophages are attributed to three main factors: their exposure to specialized tissue environments \[[@B46-cells-06-00048],[@B74-cells-06-00048],[@B75-cells-06-00048]\], the contribution of distinct embryonic or fetal progenitors to distinct subsets \[[@B25-cells-06-00048],[@B58-cells-06-00048],[@B76-cells-06-00048]\] and the preferential expression of transcription factors \[[@B46-cells-06-00048]\]. The rapid recognition and bacterial clearance from the blood is a crucial step in the first-line innate immune defense against systemic infection. In liver, the efficient phagocytosis of pathogens is ensured by the strategic location of the KCs and by their different phagocytic mechanisms. One of them is via the complement receptor of the superfamily Ig, named CRIg \[[@B77-cells-06-00048]\]. In addition to phagocytosis mediated by Fc receptors, KCs recognize bacteria opsonized by the C3b and iC3b complement component via CRIg, which enables the removal of pathogens from circulation \[[@B77-cells-06-00048]\]. CRIg is also important in the detection and uptake of viral vectors through recognition of C3 complementary components present in viruses \[[@B78-cells-06-00048]\]. However, the internalization of viral particles is associated with higher rates of KC depletion, compromising host innate immune response and increasing the susceptible to systemic infections \[[@B78-cells-06-00048]\].
Highlighting the relevance of a rapid removal of bacteria from the circulation in the prevention of systemic infections, recent studies have identified new mechanisms of phagocytosis performed by resident macrophages in the liver. It has been shown that bacteria that reach the liver through arterial blood (fast flow) are rapidly phagocytosed via scavenger receptors when they remained non-opsonized and not bound to platelets \[[@B79-cells-06-00048]\]. However, bacteria flowing through the venous blood (slow flow) are rapidly opsonized, binding to platelets and being phagocytosed via CRIg \[[@B79-cells-06-00048]\], elucidating two distinct bacterial clearance pathways. Moreover, scavenger receptors are the main receptor family that mediates a fast-track clearance of bacteria, and phagocytosis of Gram-positive bacteria by KCs may occur even when opsonization with complement is not present \[[@B80-cells-06-00048]\]. In this case, CRIg on KCs directly binds lipoteichoic acid (LTA) on Gram-positive bacteria, such as *Staphylococcus aureus* and *Listeria monocytogenes*. However, it is not clear if CRIg is relevant in the capture of Gram-negative bacteria \[[@B80-cells-06-00048]\].
The close proximity of KCs to parenchymal and nonparenchymal cells supports their ability to regulate hepatic function, both in health and disease. In a healthy liver, KCs exhibit a tolerogenic phenotype promoting and maintaining what has been termed "immunological tolerance": an anti-inflammatory mechanism to limit deleterious tissue injury in infections \[[@B81-cells-06-00048]\]. This tolerance is necessary to prevent overt immune responses against immunoreactive molecules from the hepatic sinusoids, including gut-derived antigens, and also damage-associated molecular patterns (DAMPs) from dead or dying cells as they are cleared from the circulation in the liver \[[@B81-cells-06-00048],[@B82-cells-06-00048]\]. Mechanistically, tolerance in liver can be established by either direct deletion or tolerogenic priming of CD8 T cells \[[@B83-cells-06-00048],[@B84-cells-06-00048]\] or by induction of regulatory T-cell responses \[[@B85-cells-06-00048],[@B86-cells-06-00048]\]. This function of ensuring immunological tolerance is also related to phagocytosis. Particles removed from circulation can induce tolerogenic T-cell responses in homeostatic conditions, preventing immune diseases in other organs \[[@B87-cells-06-00048]\]. It is important to emphasize that this induced liver tolerance is directly related to the original KCs with tolerogenic profile (M2-like), which are different from infiltrated monocytes (M1-like) with immunogenic profiles. This means that upon tissue injury, tolerance might be broken \[[@B87-cells-06-00048]\].
Another important feature of macrophages is the plasticity that allows the adaptation and phenotypic alteration according to environmental changes, which lead to the activation of KCs and their consequent differentiation in M1-like macrophages (classical) and M2-like macrophages (alternative) \[[@B88-cells-06-00048]\]. Despite the actual value of segregating the diverse macrophage polarization phenotypes under the "M1/M2 category" still being under debate and might be excessively simplistic, in this review, we will still referee to these populations in this way due to didactic reasons. Inflammatory cytokines and microbial products, such as LPS, can induce differentiation of KCs in an M1-like profile \[[@B89-cells-06-00048]\]. M2-like profile can be induced by IL-4, IL-10, IL-13, IL-33, transforming growth factor (TGF-β), and granulocyte colony-stimulating factor (G-CSF). M1-like macrophages are key effector cells for the elimination of pathogens, virally infected, and cancer cells and produce large amounts of IL-12, IL-23 \[[@B90-cells-06-00048]\], nitric oxide (NO), and production of ROS \[[@B91-cells-06-00048]\]. M2-like macrophages, in turn, are usually associated with resolution and tissue repair, being responsible for the production of IL-10, TGF-β, and extracellular matrix components \[[@B91-cells-06-00048]\]. In fact, the dysregulation of the inflammatory (M1)/tolerogenic (M2) phenotypic balance is an important mechanism governing the pathogenesis of chronic inflammatory diseases, suggesting that strategies restraining macrophage polarization may protect against exacerbated inflammation and thus limit tissue injury. Moreover, activation of M1 KCs to secrete pro-inflammatory mediators is a key event in the initiation of fatty liver diseases. However, tolerogenic M2 KCs are able to induce apoptosis of activated M1 KCs by inhibiting pro-inflammatory signaling and reducing tissue damage \[[@B92-cells-06-00048]\].
During liver injury, the dynamics of monocytes and macrophages varies according to tissue damage. In mild injuries that lead to moderate or no loss of tissue-resident macrophages, or when few or no blood monocytes are recruited, macrophage repopulation occurs exclusively from the initial endogenous tissue-resident population. The remaining embryonic-derived macrophages have the potential to repopulate themselves locally \[[@B59-cells-06-00048]\]. In infection models, macrophage repopulation occurs from both local and blood-derived precursors, which ultimately leads to a mosaic 'macrophage chimera' situation with mixed macrophage compartments that are of both embryonic and adult origin \[[@B78-cells-06-00048],[@B93-cells-06-00048]\]. However, we do not yet fully understand whether the blood-derived cells persist and become fully integrated into the macrophage network, or they are a temporary addition to the endogenous population. Macrophage repopulation occurs from blood monocytes or blood-derived precursors following severe inflammatory injuries that lead to major tissue-resident macrophage loss or the partial suppression of their self-renewal capacities \[[@B48-cells-06-00048],[@B59-cells-06-00048],[@B71-cells-06-00048]\]. In situations of hepatocyte necrosis, KCs may help in recruiting circulating monocytes into the damaged tissue and these infiltrating monocytes are responsible for an increase in tumor necrosis factor-α, and the subsequent proliferation of liver progenitor cells (LPCs) \[[@B94-cells-06-00048]\]. Recent data on ontogeny and different origins of resident macrophages has raised questions about the possible consequences of the substitution of original macrophages of embryonic origin by macrophages derived from monocytes throughout life and by various tissue lesions. Different groups have demonstrated that the emergency replacement of liver macrophages has acute \[[@B38-cells-06-00048]\] and long-term \[[@B48-cells-06-00048]\] consequences. Therefore, understanding the origins, the developmental pathways and the homeostatic processes that regulate tissue-resident macrophages is fundamental to enable the design of future intervention strategies to modulate macrophage functions at specific sites.
### 3.1.2. Dendritic Cells {#sec3dot1dot2-cells-06-00048}
Paul Langerhans was the first to describe the dendritic cells (DCs). He characterized the Langerhans cells in the skin, and the term 'dendritic cell' was coined by Steinman, Cohn, and Banchereau due to their morphology: they have large nucleus, abundant cytoplasm and dendrites \[[@B95-cells-06-00048],[@B96-cells-06-00048]\]. DCs are antigen-presenting cells (APCs) capable of inducing immune and tolerogenic responses in lymphoid and non-lymphoid organs, including the liver. Their location within the liver has been disputed over the years, although it is now well established that hepatic DCs represent a heterogeneous and large population within the liver immune milieu \[[@B97-cells-06-00048]\].
Hepatic DCs have been described as an interstitial and nonphagocytic cell population residing periportally, around central veins and in the liver capsule \[[@B98-cells-06-00048],[@B99-cells-06-00048],[@B100-cells-06-00048]\]. The DCs located underneath the liver capsule are morphologically different from the ones found around large vessels: capsular DCs are larger and have more dendrites \[[@B38-cells-06-00048]\]. Unlike KCs, DCs are rarely distributed within the parenchyma and immunohistochemical staining of normal adult livers shows that hepatic DCs express MHC class II. Recent studies have indicated that there is a distinct and large hepatic resident cell population inhabiting the subcapsular space, the CX3CR1^+^ cells. Gene expression analysis between classical splenic DCs and CX3CR1^+^ cells isolated from mice liver classifies them as potential hepatic DCs \[[@B38-cells-06-00048]\], once they have a high ability to present antigens and lower phagocytic behavior. Nevertheless, it is not clear whether these cells are DCs or a distinct non-KC macrophage population \[[@B101-cells-06-00048]\]. The presence of a widespread and distinct cell population underneath the mesothelium suggests that the location of these cells may be strategic for sealing and preventing liver exposure to bacteria and hazard substances from the peritoneal cavity into the liver in certain injury contexts, since the liver capsule is in contact with the peritoneal cavity. Therefore, these cells may play an important role not by directly killing pathogens, but presenting antigens and recruiting other phagocytic cell types to the injury site. Once they represent a numerous cell population in the liver, it is essential to study their origin and their role in different contexts ([Figure 4](#cells-06-00048-f004){ref-type="fig"}).
Dendritic cells are essential to capture, process, and present antigens by interacting with T cells, playing an important role to initiate immune responses. DCs have a distinct role in the liver to maintain a tolerogenic condition due to liver contact to products of digestion, from drug metabolism, microorganism products and intact bacteria \[[@B102-cells-06-00048],[@B103-cells-06-00048]\]. The heterogeneous population of DCs in the liver is described to be fully derived from the bone marrow, mainly from common-myeloid precursors (CMPs). Three subsets of hepatic murine DCs (CD19^−^CD11c^+^) are now characterized: lymphoid (CD8α^+^B220^−^CD11b^−^), myeloid (CD8α^−^B220^−^CD11b^+^), and plasmacytoid (B220^+^CD11b^−^). DCs may be also classified into two main subsets that include classical/conventional DCs (cDCs) and plasmacytoid DCs (pDCs) \[[@B76-cells-06-00048]\]. Murine cDCs express typical myeloid antigens and are typically distinguished as CD11c^+^MHCII^+^. They consist of two types of cells: cDC1s and cDC2s. The cDC1 cells resemble CD8^+^ lymphoid DCs, have migratory capacity and are efficient in presenting cell-associated antigens \[[@B55-cells-06-00048],[@B104-cells-06-00048]\]. The cDC2s development in most non-lymphoid organs depends on the presence of FMS-like tyrosine kinase 3 ligand (FLT3L), a factor that enhances global T cell and humoral immunity, and macrophage colony-stimulating factor (M-CSFR). The cDCs also bestow two subsets: CD103^+^CD11b^+^ and CD103^+^CD11b^−^. Another hepatic DCs population, the plasmacytoid DCs, expresses lower levels of MHCII and functions as major producer of type I interferons (IFNs) in response to viral infections. pDCs can be characterized as CD11c^low^ or CD11c^+^CD11b^−^B220^+^Gr-1^+^ in mice \[[@B105-cells-06-00048],[@B106-cells-06-00048],[@B107-cells-06-00048],[@B108-cells-06-00048]\].
Different subsets of DCs are identified in fetal tissues and are related to adult populations, and they mediate immune responses during gestation \[[@B109-cells-06-00048]\]. However, neither of the molecules here presented exclusively identify DCs. Many of the surface markers can be used to study other myeloid cell types, such as neutrophils and lymphocytes, especially during inflammation. For example, there are mice KCs that express CD11c in steady state conditions and during hepatic replenishment after clodronate injection \[[@B38-cells-06-00048]\]. Despite CD11c is considered a classic marker for DCs, it is not very reliable when studying hepatic DCs. Therefore, it is important to effectively excluding these double-cell populations in certain analysis. These immunophenotype strategies are relevant to study the functions of DCs in different types of hepatic injury.
During homeostasis, DCs are tolerogenic and immature. The immature dendritic cells (IDCs) interact with antigens by capturing: they phagocyte particles and direct them to compartments rich in MHCII to form MHCII-peptide complexes \[[@B110-cells-06-00048]\]. In a context of chronic inflammation, mature DCs have a proinflammatory profile. For example, in a model of murine fibrosis induced by nonalcoholic steatohepatitis (NASH), hepatic CD11c^+^ DCs are able to limit CD8^+^ T cells expansion, produce elevated immune-modulatory cytokines---such as IL-6 and TNFα, but interestingly not IL-10---and activate CD4^+^ T cells \[[@B111-cells-06-00048]\], hence modulating hepatitis and fibrosis in NASH. In addition, it is well known that hepatic DCs are pivotal in the generation of both innate and adaptive immunity in response to lipopolysaccharide (LPS). In a murine experiment model, Chen et al. isolated hepatic DCs and characterized the expression of toll-like receptor 4 (TLR4) in response to LPS stimulation, cytokine productions and ability to ablate T cells after mice had been injected with plasmid-GM-CSF \[[@B112-cells-06-00048]\]. Liver DCs had a role in stimulating a regulatory response as expanding CD4^+^Foxp3^+^ T regulatory cells and promoting secretion of IL-27. These data suggest an immunoregulatory role of these cells. Nevertheless, in response to viral infections, hepatic CD103^+^ DCs induce and sustain CD8^+^ T cells activity against hepatotropic antigens in situ \[[@B113-cells-06-00048]\], opening possibilities to target these cells for strategies like treatments and vaccination.
3.2. Granulocytes {#sec3dot2-cells-06-00048}
-----------------
### 3.2.1. Neutrophils {#sec3dot2dot1-cells-06-00048}
Neutrophils, a subset of polymorphonuclear leukocyte, are the predominant immune cell population in human blood, being crucial for controlling bacterial and fungal infections. In healthy individuals, more than 10^9^ neutrophils per kg body weight are released from the bone marrow every 24 h \[[@B114-cells-06-00048]\]. Neutrophils develop from hematopoietic stem cells in bone marrow, a process called 'granulopoiesis', and granulocyte colony-stimulating factor (G-CSF) is the major factor regulating the neutrophil life cycle by increasing cell proliferation, survival, differentiation, and mobilization to blood circulation. In the liver, neutrophils migrate from the blood to the inflammatory focus driven by chemokynes and chemotactic agents. These mediators are released to establish an efficient chemotactic gradient within the liver intravascular compartment \[[@B115-cells-06-00048]\]. Once attracted, neutrophils accumulate within the hepatic microvasculature, which includes sinusoids and postsinusoidal venules, before transmigration process ([Figure 5](#cells-06-00048-f005){ref-type="fig"}). Neutrophil transmigration involves the upregulation of adhesion molecules, such as selectins and transient interactions between selectins and their ligands result in neutrophil adhesion and rolling, the first step of the leukocyte recruitment cascade \[[@B116-cells-06-00048],[@B117-cells-06-00048]\]. Neutrophil extravasation from the sinusoids into the parenchyma is mediated by β2 integrin, intercellular adhesion molecule-1 (ICAM-1), or β1 integrin/vascular adhesion molecule-1 (VCAM-1) interactions \[[@B118-cells-06-00048]\]. On the other hand, in situations of extensive endothelial cell damage, neutrophils may have direct access to the parenchyma without a CAM-dependent transmigration process \[[@B119-cells-06-00048]\]. Once at the inflammation site, neutrophils initiate clearance process, including phagocytosis, release of DNA extracellular traps. In addition, neutrophils can secrete a large amount of granules containing proteolytic enzymes (elastase, cathepsins and proteinase-3), bactericidal proteins (presenilin 1, defensins, bactericidal/permeability increasing protein), matrix metalloproteinases, and lysozymes \[[@B120-cells-06-00048],[@B121-cells-06-00048]\].
Although neutrophils are known for their excellence in capturing and killing bacteria, these cells also play a key role in sterile liver damage \[[@B122-cells-06-00048]\]. Using a model of thermal hepatic injury, Wang and colleagues \[[@B123-cells-06-00048]\] demonstrated that neutrophils penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. In contrast to what is seen in cases of clearance of pathogens, they neither die at the injury site nor are phagocytized by macrophages. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey passing through the lungs before entering the bone marrow, where they undergo apoptosis. There is a long time debate regarding the role of neutrophil-induced cell injury in the liver. During hepatic inflammation, neutrophils are recruited to the damage, and in high number, these cells can generate sufficient oxidative stress to kill hepatocytes \[[@B124-cells-06-00048],[@B125-cells-06-00048],[@B126-cells-06-00048]\]. Neutrophils produce ROS through the NADPH oxidase system, initiating toxicity. Furthermore, myeloperoxidase, an enzyme present in neutrophil granules and released upon activation, causes significant oxidative stress and protein dysfunction \[[@B124-cells-06-00048],[@B127-cells-06-00048]\]. Despite the damages caused by neutrophil infiltration and activity, the role of these cells in tissue repair is indispensable. In sterile liver lesion, 12 h after injury, the neutrophils fill the areas that had been occupied by the collapsed sinusoids helping in clearance the area. In contrast, antibody-mediated depletion of neutrophils results in far more debris remaining in the injury site and a delay on tissue repair \[[@B123-cells-06-00048]\]. Therefore, despite neutrophils' role in exacerbating tissue damage in acute inflammatory responses, these cells may be crucial in the resolution and repairing phase.
### 3.2.2. Eosinophils {#sec3dot2dot2-cells-06-00048}
The role for eosinophils and their actual location within the liver is still poorly understood. Eosinophils are granulocytes characterized by cytoplasmic granules with an affinity for acid aniline dyes, such as eosin. The origin of the eosinophils is also in the bone marrow, from pluripotent stem cells differentiated after stimuli of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and more particularly interleukin 5 (IL-5) \[[@B128-cells-06-00048]\]. Eosinophils are predominantly tissue cells that migrate from the blood into tissues as a result of several correlated events which involve adhesion pathways and chemoattractants \[[@B129-cells-06-00048]\]. The recruitment of eosinophils to the damaged liver is regulated by numerous events involving cytokines and chemokines released by another eosinophils and T lymphocytes \[[@B130-cells-06-00048]\]. Once at the damaged tissue, eosinophils may be activated by numerous mediators that can drive variable profiles of cell activation \[[@B131-cells-06-00048]\]. The location and the consequences of eosinophilic infiltration of the liver depends on the inflammation focus or disease condition. In biopsies from individuals with chronic hepatitis C the number of these cells was greater in the larger portal tracts and strongly associated with liver steatosis and fibrosis \[[@B132-cells-06-00048]\]. In a drug-induced liver injury (DILI), the role for eosinophil is controversial. Bjornsson and colleagues related a favorable outcome to the occurrence of liver eosinophilia after evaluation of 570 case reports of DILI \[[@B133-cells-06-00048]\]. In contrast, Pham et al. suggest that the release of cationic proteins by eosinophils may contribute to liver cell damage in patients with DILI based on immunohistochemical assays \[[@B131-cells-06-00048]\].
3.3. Lymphocytes {#sec3dot3-cells-06-00048}
----------------
In humans, up to 65% of all hepatic lymphocytes consist of NK cells, NKT cells, and unconventional T cells (γδ) \[[@B134-cells-06-00048]\]. These cell populations can proliferate under certain experimental or pathological conditions. The dominating presence of these populations in the liver and in early defense against pathogens places these cells in a key position among effector lymphocytes in liver immune surveillance.
### 3.3.1. Liver Natural Killer (NK) {#sec3dot3dot1-cells-06-00048}
NK cells are classically known as a subset of the innate immune system, but can play an important role in shaping the adaptive immune response \[[@B135-cells-06-00048]\]. NK cells are also derived from the bone marrow and are distributed throughout the body in both lymphoid and non-lymphoid tissues \[[@B136-cells-06-00048],[@B137-cells-06-00048]\]. However, a growing body of evidence has indicated that the presence of hematopoietic progenitors and immature NK cells at extra-medullary sites \[[@B138-cells-06-00048]\]. Interestingly, the liver is an NK cells enriched organ. Hepatic NK cells are not only a vast population in the liver, but are also naturally activated as they show higher cytotoxicity against tumor cells than other NK cells in rodents and in humans, including splenic or peripheral blood NK cells \[[@B139-cells-06-00048],[@B140-cells-06-00048]\]. Over the last decade, data have suggested the involvement of NK cells in the pathogenesis of liver diseases, mainly tumors and viral infections \[[@B140-cells-06-00048],[@B141-cells-06-00048],[@B142-cells-06-00048],[@B143-cells-06-00048]\].
In 1976, Wisse and colleagues described a new type of liver resident NK cells in rats, named "Pit cells" \[[@B144-cells-06-00048]\]. Further studies revealed that Pit cells are attached to the endothelial lining, which they might penetrate with the microvilli of parenchymal cells. These cells are often closely attached to KCs, suggesting some type of physical relationship \[[@B145-cells-06-00048]\]. The lobular distribution of Pit cells in the liver was found to be predominantly periportal (\~60%). The human liver does not harbor a morphological equivalent of the rat Pit cell.
In mice, liver NK cells are present at significantly higher frequencies than NK cells in the bone marrow, peripheral blood, and spleen, accounting for approximately 5--10% of the total lymphocytes present in this tissue. Although liver-resident NK cells resemble immature circulating NK cells in phenotype, adoptive transfer studies showed that these cells preferentially home to the liver and do not convert to circulating phenotype, suggesting that liver-resident NK cells are stable under steady-state circumstances \[[@B138-cells-06-00048]\]. In humans, the liver also contains a unique CD49a^+^ NK cell subset that resembles murine liver-resident NK cells \[[@B146-cells-06-00048]\]. Similar to their counterparts in the mouse liver, human CD49a+ NK cells are T-bet^+^ Eomes^−^ and are not detectable in afferent or efferent hepatic venous or peripheral blood \[[@B145-cells-06-00048]\].
### 3.3.2. Liver NKT Cells {#sec3dot3dot2-cells-06-00048}
NKT cells are a subset of lymphocytes that express both αβ TCR (T cell marker) and cell surface receptors characteristic of NK cells (NK1.1 in C57BL/6 mice) \[[@B147-cells-06-00048]\]. Mouse liver lymphocytes contain about 20% to 30% NKT cells, which are further elevated in pathological conditions. NKT cells play an important role in induction of liver injury in models of liver injury induced by concanavalin A, α-galactosylceramide, alcohol, and drugs \[[@B148-cells-06-00048]\]. To better understand the role of hepatic NKT cells, Geissman and colleagues observed in vivo that NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance \[[@B149-cells-06-00048]\]. Besides, they observed that CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. Therefore, it is believed that NKT cells are predominantly a population of intravascular cells.
### 3.3.3. Liver γδ T Cells {#sec3dot3dot3-cells-06-00048}
Unconventional T cells that do not express NK cell markers include the major group of TCR γδ cells (also called γδ T cells). This group represents 15% to 25% of all intrahepatic T cells, thereby rendering the liver one of the richest sources of γδ T cells in the body \[[@B150-cells-06-00048]\]. γδ T cells have oligoclonal or invariant TCRs that recognize a limited range of antigens such as stress proteins and nonprotein antigens. In the liver, γδ T cells were predominantly found in portal infiltrates and areas of bile duct proliferation or fibrogenesis, but the exact contribution of these cells to liver immunopathology remained elusive \[[@B151-cells-06-00048]\]. However, the results obtained in human liver disease as well as murine models about the role of these cells are not fully conclusive at present, and the effects of γδ T cells on the outcome of liver disease might vary, depending on etiology and stage of disease.
The normal liver contains a large number of lymphocytes that include not only specialized NK and NKT cells, but also CD4 and CD8 T cells. In inflammatory conditions, the number of lymphocytes in the liver increases and the type and distribution of these infiltrating cells will determine the nature of the inflammation. Under healthy conditions, human liver contains significant numbers of T lymphocytes in the portal tracts and scattered through the parenchyma \[[@B152-cells-06-00048]\]. At homeostasis conditions of the liver, both CD4 and CD8 T cells are found in portal tracts albeit at low numbers and a population of cells with the characteristics of intraepithelial lymphocytes are found in association with biliary epithelium \[[@B153-cells-06-00048]\].
### 3.3.4. Liver B Cells {#sec3dot3dot4-cells-06-00048}
B-lymphocytes perform various immunological functions, including production of antibodies, antigen presentation, secretion of multiple cytokines, and regulation of immune responses. However, little is known about the functional biology of liver B cells. The main reasons for the relative lack of knowledge in this regard may be due to the small number of B cells residing in the healthy liver and the experimental difficulty in isolating and analyzing specifically B cells \[[@B154-cells-06-00048]\]. Hepatic B cells comprise only \~5% of intrahepatic lymphocytes. During infection, intraportal lymphoid follicles display a germinal center-like structure in which activated B cells are surrounded by a follicular DC network. The distribution of IgM-, IgD-, and IgG-positive B cells and the gene expression patterns in intrahepatic germinal centers resemble those in lymph nodes, suggesting that intrahepatic germinal centers function as functional follicular structures \[[@B154-cells-06-00048],[@B155-cells-06-00048]\].
4. Concluding Remarks {#sec4-cells-06-00048}
=====================
It is becoming increasingly clear that the liver is not only an 'accessory organ' for the digestive system. Despite the vital role as a metabolic organ, the liver has emerged as one of the main immune and lymphoid organs of the body. In this context, the liver also harbors one of the most complex and active populations of immune cells in the body. New imaging and immunophenotyping techniques have allowed the identification of these different populations using both in vivo and in vitro assays ([Table 1](#cells-06-00048-t001){ref-type="table"}). Interestingly, these cells are extremely organized in the different liver compartments, and the hepatic hemodynamic scheme favors an intimate contact of blood contents with these cells. Therefore, expanding our knowledge on the frequency, activation status, and the changes in cell compartmentalization throughout the liver during diseases may hold interesting venues for investigation not only for basic science, but also for the ethiopathogenesis of different hepatic diseases.
We would like to thank Maxillofacialtips, BD Biosciences, and Nikon for providing reagents, and financial and technical support.
The authors declare no conflict of interest.
This work was supported by FAPEMIG (Rede Mineira de Imunobiológicos--RED-00140-16), CAPES (Biocomputacional) and CNPq (Brazil).
{#cells-06-00048-f001}
{#cells-06-00048-f002}
{#cells-06-00048-f003}
{#cells-06-00048-f004}
{#cells-06-00048-f005}
cells-06-00048-t001_Table 1
######
Liver immune cells.
Liver Immune Cells Location in Homeostasis Main Surface Markers Putative Role in Diseases
---------------------- -------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------
Kupffer Cells Inside the sinusoids; adhered to the endothelium F4/80, CD11b Controlling inflammation; Kupffer cell depletion is associated with worse prognosis
Dendritic Cells Underneath hepatic capsule; around large vessels CD19^−^CD11c^+^; CD8α^+^B220^−^ CD11b^−^ (lymphoid); CD8α^−^ B220^−^CD11b^+^ (myeloid); B220^+^ CD11b^−^ (plasmacytoid) Enhanced response to viral infections, controlling viral spread and T cell activation
Monocytes Inside the sinusoids as patrolling cells CD11b^hi^CD115^hi^Gr1^lo^ Infiltrating monocytes control pathogen spread and heal tissue injury
Neutrophils Ly6G^+^CD11b^+^F4/80^−^ Overt infiltration is associated with enhanced liver injury in several models
Eosinophils CD11b^+^CD193^+^Siglec F^+^ Role in pathogenesis of liver diseases through release of granules containing TNF-α, highly cytotoxic proteins such as major basic protein and eosinophilic cationic protein
Natural Killer Cells CD3^−^NK1.1^+^ Involved in the pathogenesis of liver diseases, mainly tumors and viral infections; higher cytotoxicity than other NK cells
NKT Cells CD3^+^NK1.1^+^ Patrolling liver sinusoids to provide intravascular immune surveillance
T lymphocytes CD3^+^CD4^+^ (T CD4 cells); CD3^+^CD8^+^ (T CD8 cells) Clearance of virus and in virus-induced immunopathology
B lymphocytes CD19^+^ Antibody-secreting cells within germinal centers of intraportal lymphoid follicles, during viral hepatitis
γδ T cells CD24^+^ CD25^−^ CD27^+^ Controlling early viral infections; expressing perforin, lysing virus-infected targets, and releasing IFN-γ
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
The hormone receptor status in breast cancer (BC) is crucial for deciding treatment regimen for BC patients. The presence of estrogen receptor (ER) predicts treatment response to endocrine therapy, primarily due to its role in driving ER positive breast cancer cells to proliferate^[@CR1]^. However, it has been observed that at least 50% of ER positive tumors display de novo resistance to endocrine therapies such as tamoxifen, and many of those initially sensitive acquire resistance despite expressing non-mutated ER^[@CR2]^. Earlier studies suggest activation of mTOR potentially plays a role in endocrine resistance^[@CR3],[@CR4]^. Recently we demonstrated that activated mTOR (as measured by phosphorylation at serine (S) 2448 residue) in treatment naive breast tumors is positively associated with overall survival (OS) and recurrence free survival (RFS) in ER positive breast cancer patients who were later treated with tamoxifen^[@CR5]^. Also, we demonstrated that ER is a substrate of mTOR and interacts with it further supporting the crosstalk between ER and mTOR. Therefore, we concluded that in breast tumors where there is an intact estrogen regulated mTOR signaling, mTOR is associated with an increased likelihood of responsiveness to endocrine therapy^[@CR5]^. Furthermore, very recently, we observed that N-myristoyltransferase (NMT1) is a downstream target of mTOR (Jaksic *et al*. 2018 unpublished data).
mTOR is a serine/threonine kinase that regulates cell growth, proliferation, motility and survival^[@CR6]^. It is a component of the phosphatidylinositol 3-kinase (PI3K) cell survival pathway and operates at a key junction in the PI3K pathway as it acts both upstream as well as downstream of protein kinase B (PKB or Akt)^[@CR6]^. As a signaling hub, mTOR exists in two different multiprotein complexes: mTORC1 and mTORC2 that are involved in cell growth^[@CR7],[@CR8]^. mTORC1 is mainly responsible for the regulation of protein synthesis necessary for cell growth and proliferation^[@CR9],[@CR10]^, whereas mTORC2 in a feedback loop manner is responsible for the phosphorylation of Akt at S473^[@CR11]^. mTORC1 is mainly activated by PI3K/Akt pathway whereas inhibited by the tuberous sclerosis 1/2 (TSC1/TSC2) complex^[@CR12]^. Once the mTORC1 is active it can exert numerous biological effects by phosphorylating the downstream targets.
Rapamycin, produced by the bacterium *Streptomyces hygroscopicus*, was identified as an anti-fungal agent^[@CR13]^. Rapamycin and its derivatives have been used as therapeutic agents with immunosuppressant and anti-tumor properties. The action of rapamycin is mediated by the specific inhibition of mTOR protein kinase. Rapamycin and its analogues are first generation mTOR inhibitors and have been used in human tumors as monotherapy or as a component of combination therapy. Activation of various growth factor receptors such as human epidermal growth factor receptor 2 (HER-2) and insulin like growth factor receptor (IGFR) results in dysregulation of PI3K/Akt signaling. Dysregulation of various components of mTOR signaling pathway has been reported in various cancers such as breast, ovarian, renal, colon and head and neck cancers. Activation of Akt-mTOR pathway is also associated with the initiation of melanocyte tumors^[@CR14]^. In conclusion, mTOR has been suggested to play a key role in the oncogenic process. mTOR is a downstream target of EGFR signaling and therefore considered as an important therapeutically attractive target for the treatment of various types of cancer.
Myristoylation is a co-translational lipid modification that involves the attachment of a 14-carbon saturated fatty acid or myristate to the N-terminal glycine residue of a subset of eukaryotic proteins^[@CR15],[@CR16]^. NMT belongs to GCN5-related N-acetyltransferases superfamily of proteins and catalyzes myrisate transfer^[@CR17],[@CR18]^. NMT has been isolated and characterized in yeast, fungi, protozoan parasites, plants and mammals including mouse, rat and human^[@CR19]--[@CR22]^. Studies have shown that most of the mammalian species including humans possess two NMT enzymes that are encoded by distinct genes^[@CR23]^. NMT1 and NMT2 in humans share 77% amino acid sequence identity and have similar substrate affinities^[@CR23],[@CR24]^. Human NMT1 and NMT2 are homologous to mouse and rat. However, human and mouse versions of NMT1 and NMT2 are highly homologous and share more than 95% amino acid sequence identity^[@CR23]^. Myristoylation is an integral part of apoptosis and the myristoylated proteins have been reported to be involved in various cellular processes including cellular proliferation and oncogenesis^[@CR25]--[@CR29]^. Some examples of myristoylated proteins are the catalytic subunit of cAMP-dependent protein kinase, *β*-subunit of calcineurin, *α*-subunit of several G-proteins, several tyrosine kinases, etc^[@CR30]^. Evidence from various studies have suggested the involvement of NMT1 in cancer^[@CR26]^. The altered expressions of NMT1 are observed in different types of cancer such as colon, breast, gallbladder and brain^[@CR26]^. NMT activity and expression has been reported to be up-regulated during the progression of colorectal cancer^[@CR31]^. Strong expression of NMT has been reported in malignant breast tissues compared with normal breast cells suggesting that NMT could be an important player in breast cancer^[@CR29]^.
Very recently, we observed that NMT1 is a downstream target of mTOR (Jaksic *et al*., 2018 unpublished data). In this work, we investigated the regulation of NMT1 by mTOR and further determined the impact of perturbations such as the effect of drugs by combining *in vitro* experiments and mathematical modelling approaches. We treated ER positive breast cancer cells with rapamycin and determined the effect of mTOR inhibition on NMT1 in a time dependent manner. Signaling pathways involving mTOR have not been extensively studied mathematically or computationally^[@CR32]--[@CR35]^. Most models of mTOR pathway computationally investigate the signaling upstream of mTOR, in particular, the relationship between insulin signaling and mTOR. The complexity of these models is variable, from a few molecules to dozens, allowing to investigate the outcome of potential signaling events, in order to have a better knowledge of the pathway and/or to determine the impact of perturbations such as the effect of drugs^[@CR32],[@CR33],[@CR36]--[@CR38]^. To the best of our knowledge, the regulation of NMT1 by mTOR has never been mathematically modelled. In this study, we propose a collection of models of this regulation, including the inhibition of mTOR by rapamycin. The use of a collection of models allowed us to consider a variety of assumptions on the endogenous level of mTOR, the feedback regulation of mTOR by NMT1 and characteristics of the pathway when perturbed by rapamycin. All models were calibrated and validated by fitting their responses to experimental data; then, the best models were identified. Confronting models' predictions to experimental data will help us determine key characteristics that are difficult to obtain experimentally, such as the relevance of the negative feedback of NMT1 on mTOR and the reversibility of the inhibition of mTOR by rapamycin.
Results {#Sec2}
=======
Rapamycin acts as an inhibitor of mTOR and inhibits phosphorylation at S2448 residue of mTOR. In this study, we investigated the effects of rapamycin treatment on the expression of total NMT1 over time.
Rapamycin augments NMT1 expression {#Sec3}
----------------------------------
The MCF7 cells were treated with either 100 nM rapamycin or an equivalent volume of DMSO (10 *μ*L) for 5′, 10′, 30′, 60′, 180′, 360′, 720′and 1440′ following which cell lysates were collected and subjected to Western blot analyses as indicated in the Materials and Methods section. Rapamycin treated MCF7 cells showed a decrease in the p-mTOR (S2448) with a maximum decrease at 60′, whereas there was no significant change in the total mTOR levels under all experimental conditions (Fig. [1A and B](#Fig1){ref-type="fig"}) in comparison with either the control (no treatment) or the cells treated with the vehicle, DMSO. Rapamycin treatment resulted in an increase in the expression of total NMT1 levels over time, with a maximum increase of 6-fold at 360′ compared to control cells whereas, no significant change in NMT1 levels in cells treated with DMSO was observed (Fig. [1A and C](#Fig1){ref-type="fig"}). The total NMT1 expression was normalized against *β*-actin. Rapamycin treatment showed a correlation between the levels of p-mTOR (S2448)/total mTOR levels and total NMT1/*β*-actin levels, whereas, there was no significant change when the cells were treated with DMSO alone (Fig. [2](#Fig2){ref-type="fig"}). Four datasets were produced and were used for model calibration and selection.Figure 1Rapamycin treatment decreased the phosphorylation of mTOR (S2448) and augmented NMT1 levels in a time-dependent manner. MCF7 cells at 70--80% confluence were starved and treated with rapamycin (100 nM) or DMSO for indicated time points and protein lysates were prepared as mentioned in the materials and methods section. (**A**) A representative Western blot analysis displaying the expression levels of p-mTOR (S2448), mTOR total, NMT1 and *β*-actin. The depiction of relative band intensities of p-mTOR (S2448)/total mTOR (**B**) and NMT1/*β*-actin (**C**) when MCF7 cells were treated with rapamycin or DMSO for indicated time points. All the experimental data were normalized against control 0′ (no treatment) which is 1-fold. The data point is an average of four independent experiments and the bars represent the standard deviation between them.Figure 2Correlation between active mTOR and NMT-1 in rapamycin treated MCF7 cells. Relative band intensities of p-mTOR (S2448), total mTOR, NMT1 and *β*-actin were obtained from Western blot analyses. Active mTOR is the ratio of relative band intensities of p-mTOR(S2448)/mTOR Total and NMT1 is the ratio of relative band intensities of NMT1/*β*-actin.
Modelling mTOR-NMT1 without rapamycin {#Sec4}
-------------------------------------
The three models, NT, NTt and NTf are considered to investigate the dynamics of the mTOR-NMT1 system without perturbations due to rapamycin treatment (see Fig. [3](#Fig3){ref-type="fig"}). Table [1](#Tab1){ref-type="table"} provides a summary of the characteristics and assumptions of the models; the core assumption of these models is the regulation of NMT1 phosphorylation by p-mTOR. All models have been studied analytically, as detailed in the Supplementary material. Using the four control datasets, parameter values for each model have been optimized to fit the experimental data. All these models are able to reproduce the trends in the proportion of phosphorylated mTOR and total NMT1 observed for all experimental datasets without rapamycin (see Figs [4](#Fig4){ref-type="fig"} and [S4](#MOESM1){ref-type="media"}). Experimental observations combined with the modelling approach validate the regulation of NMT1 by mTOR.Figure 3Derivation of the models studied, starting from model NT and applying multiple assumptions (constant total mTOR, feedback regulation of mTOR by NMT1 and rapamycin binding); corresponding graphs of interactions are in the background. For models with constant total mTOR, only the dynamics of *T* and *R*~*c*~ are considered with rapamycin, thus *T*~*p*~ = *T*~*t*~ − *T* − *R*~*c*~ while without rapamycin only *T*~*p*~ is explicitly described then *T* = *T*~*t*~ − *T*~*p*~. One may note that there is no model with constant total mTOR and irreversible rapamycin binding or feedback, to ensure biologically realistic dynamics. (Inset) Schematic relations between the model variables, corresponding to System ([1](#Equ1){ref-type=""}). The dashed arrow indicates that p-mTOR is activating the phosphorylation of NMT1. The bar-headed line represents an inhibition and the arrows indicate a transfer or an activation.Table 1Collection of models studied with the number of parameters *p*, assumptions on rapamycin binding, feedback regulation of mTOR by NMT1 and mTOR dynamics (s/d meaning an explicit synthesis and degradation of mTOR).Model*p*rapamycinfeedbackmTORNT10nonos/dNTt9nonoconstantNTf11noyess/dNTRr12reversiblenos/dNTtRr11reversiblenoconstantNTRi12irreversiblenos/dNTRrf13reversibleyess/dNTRif13irreversibleyess/dFigure 4Best fit for models NTt (without rapamycin, on the left) and NTtRr (with rapamycin, on the right) for dataset 2 (solid line), with the envelope (dotted lines) corresponding to a variation of ±10% of the nominal parameter values (best set of parameters).
Then the effects of explicit synthesis and degradation of mTOR components and inhibition of mTOR by NMT1 on the dynamics are investigated. For datasets 1 to 3, model NTt, characterized by a constant endogenous level of total mTOR (no explicit synthesis and degradation) and no feedback regulation of mTOR by NMT1, is selected as the best model with a strong level of evidence compared to models NT and NTf. Model NTt has the lowest AIC~*c*~ among the three models considered and its Akaike weight is over 0.9 for these datasets (see Table [2](#Tab2){ref-type="table"}). Moreover, the evidence ratio for model NTt compared to the second best model NT is higher than 15 for each dataset. For dataset 4, model NTt is also selected as the best model, however with a lower Akaike weight of 0.6. Furthermore, a feedback regulation of mTOR by NMT1 is less likely to occur; the Akaike weight of the feedback assumption is lower than 0.01 for each dataset.Table 2Corrected Akaike Information criteria AICc~*i*~ and Akaike weights *w*~*i*~ for models with and without rapamycin for datasets 1 to 4, with *k*~*i*~ = *p* + 1 the number of parameters considered to compute the AICc~*i*~, with *i* denoting the model considered. The weights corresponding to each assumption are obtained by summing the weights of the models verifying the assumption (see Table [1](#Tab1){ref-type="table"}).ModelsDataset 1Dataset 2Dataset 3Dataset 4*k* ~i~AICc~*i*~*w* ~*i*~AICc~*i*~*w* ~i~AICc~*i*~*w* ~*i*~AICc~*i*~*w* ~*i*~(a) Without rapamycin NT11−6.90.062−101.90.033−67.20.045−109.20.379 NTt10−12.4**0**.**936**−108.6**0**.**967**−73.5**0**.**957**−110.2**0**.**609** NTf12−0.70.003−92.910^−4^−61.20.002−102.40.012**Assumptions** Feedback0.0030.00040.0020.01 No feedback**0**.**9970**.**99960**.**9980**.**99**(b) With rapamycin NTRr13−2.30.393−107.110^−4^−75.90.025−58.80.407 NTtRr12−1.40.248−124.9**0**.**997**−83.1**0**.**929**−58.20.305 NTRi13−2.00.351−113.50.003−76.70.039−58.10.282 NTRrf147.00.004−91.610^−8^−73.20.007−48.00.002 NTRif146.60.005−102.410^−5^−66.610^−4^−49.40.004**Assumptions** Reversible**0**.**640**.**99670**.**9610**.**714** Irreversible0.360.00330.0390.286 Feedback0.00810^−5^0.0070.006 No feedback**0**.**9920**.**999990**.**9930**.**994**
In the following, the sensitivity and robustness of the best model NTt to parameters were investigated in details. The LHS-PRCC global sensitivity analysis of model NTt indicates that the proportion of p-mTOR and total NMT1 are significantly sensitive to at least one parameter (see Fig. [5](#Fig5){ref-type="fig"}) and that no parameter is negligible. One may note that total NMT1 is directly sensitive to all parameters, in particular parameters governing the dynamics of mTOR components.Figure 5Global sensitivity analysis of the proportion of p-mTOR *T*~*p*~/*T*~*t*~ and total NMT1 *N* + *N*~*p*~, using LHS-PRCC for Models NTt (top) and NTtRr (bottom) at time *t* = 2880 min. The red area determines the area of significance (levels 0.05, 0.01 and 0.001).
Figure [4](#Fig4){ref-type="fig"} displays trajectories of the model NTt with dataset 2; the dotted lines indicate the minimum and maximum trajectories corresponding to a ±10% variation of the nominal parameter values obtained for dataset 2. Model responses for the proportion of p-mTOR and total NMT1 appear to be robust to small changes in parameter values. The estimated parameter values are of the same order of magnitude for all 4 datasets, with some small differences resulting from variations in the experimental levels of NMT1 and p-mTOR (see Figure [S6](#MOESM1){ref-type="media"}).
Modelling mTOR-NMT1 with rapamycin {#Sec5}
----------------------------------
The dynamics of mTOR and NMT1 is now studied with rapamycin treatment by considering five models, NTRr, NTtRr, NTRi, NTRrf and NTRif (see Fig. [3](#Fig3){ref-type="fig"}). Table [1](#Tab1){ref-type="table"} provides a summary of the assumptions of these models. These five models are calibrated using four datasets treated with rapamycin. All models are able to reproduce the trends observed in experimental data with rapamycin, in particular a decrease in the proportion of p-mTOR and an increase in total NMT1 (see Figs [4](#Fig4){ref-type="fig"} and [S5](#MOESM1){ref-type="media"}). Similarly to the control, model responses support the regulation of NMT1 by p-mTOR in the presence of rapamycin.
Considering these five models allowed us to test the assumptions previously investigated in the control case, as well as the reversibility of rapamycin effect (see Fig. [3](#Fig3){ref-type="fig"} and Table [1](#Tab1){ref-type="table"}).
For datasets 2 and 3, model NTtRr, characterized by a reversible effect of rapamycin, a constant total mTOR and an absence of feedback, is the most likely to be the best model; it has the lowest AIC~*c*~ and a weight above 0.9 (see Table [2](#Tab2){ref-type="table"} and Fig. [4](#Fig4){ref-type="fig"}). The best models with and without rapamycin are based on the same assumptions. Moreover, the conclusions of the global sensitivity analysis of model NTtRr are the same as for model NTt (see Fig. [5](#Fig5){ref-type="fig"}); parameters common to both models have similar impacts.
However, for datasets 1 and 4, multiple models have similar AIC~*c*~ and Akaike weights, not allowing us to determine the best model with a sufficient support. For instance, the best model selected for datasets 2 and 3, NTtRr, overestimates the initial value of p-mTOR on dataset 1, leading to a higher error (see Supplementary material). Even if the best model cannot be selected for datasets 1 and 4, a reversible effect of rapamycin and an absence of feedback are the most probable assumptions, similarly to the other datasets (see Table [2](#Tab2){ref-type="table"}).
Estimated parameter values are of the same order of magnitude for all four datasets for model NTtRr (see Figure [S6](#MOESM1){ref-type="media"}); parameters common to models NTt and NTtRr have similar values.
Discussion {#Sec6}
==========
In our earlier studies using *in-vitro* kinase assay, we have demonstrated that Akt phosphorylates NMT1, which causes a decrease in its activity^[@CR29]^. Since mTORC1 is a downstream target of Akt we were interested in determining the effect of mTOR inhibition on NMT1. Rapamycin binds to the intracellular receptor, FKBP12. The FKBP12-rapamycin complex binds to mTOR and inhibits its autophosphorylation at S2448^[@CR39]^. Rapamycin has also been proposed to inhibit mTOR by destabilizing the mTOR-raptor complex^[@CR40]^. The regulation of NMT1 by mTOR was previously not known. Upon inhibiting mTOR with rapamycin we observed an increased expression of NMT1 in a time dependent manner whereas, DMSO, the vehicle failed to show any significant changes in the total NMT1 levels (see Fig. [1](#Fig1){ref-type="fig"}).
Furthermore, we performed similar experiments in another ER positive T47D breast cancer cell line. The ER positive T47D breast cancer cells were treated with rapamycin for the same concentration and time points as that of MCF7 cells. The expression pattern of NMT1 upon rapamycin treatment in T47D was found to be similar to that of MCF7 cells (see Figure [S3](#MOESM1){ref-type="media"}). The dynamic expression of p-mTOR and NMT1 when treated with DMSO (vehicle control) or rapamycin in T47D was similar to that of MCF7 cells. This data confirms that the regulation of NMT1 by mTOR is not a cell line dependent phenomenon. We used the best model obtained from MCF7 data fitting, NTt and NTtRr, to fit the data obtained by treating T47D cells with or without rapamycin. Our results indicate that the model outputs are a good representation of experimentally obtained data (see Figures [S9](#MOESM1){ref-type="media"} to [S11](#MOESM1){ref-type="media"}). Moreover, estimates of parameter values for both the cell lines, MCF7 and T47D are in similar ranges.
It is possible that activated mTOR acts on NMT1 and primes it for a proteasome-mediated degradation. Ubiquitin-mediated proteasome degradation of proteins is involved during stress-response, signal transduction, antigen processing, DNA-repair, transcriptional regulation, apoptosis, lytic degradation of transcription factors, misfolded proteins, parasitic proteins, etc^[@CR41]--[@CR46]^. Zhao *et al*. reported that inhibition of mTORC1 by rapamycin for as long as 16 h caused a reduction in proteolysis due to decreased proteasome expression^[@CR47]^. The study by Zhang *et al*. supports our observation that the inhibition of mTOR by rapamycin could possibly prevent proteasome-mediated degradation of NMT1^[@CR48]^.
There are a number of proteins, which are degraded due to phosphorylation that makes them susceptible to ubiquitination and proteasome-mediated degradation such as glycogen synthase kinase-3 (GSK-3), which phosphorylates *β*-catenin thus tagging it with ubiquitin for proteasome mediated degradation. Many cell cycle regulated proteins such as cyclins are subjected to proteasomal-mediated degradation for the transition from one phase of cell cycle to another.
Studies in yeast clearly demonstrate the role of NMT in the cell morphogenesis through the regulation of proteasomal activity. A temperature sensitive mutant swoF1 with mutation in NMT encoding gene in *Aspergillus nidulans* could not maintain growth during cell elongation. It was further demonstrated that swoF1 NMT mutants had an increased 26S proteosomal activity^[@CR49]^. In fact, the defect of NMT gene in swoF1 mutants could be partially reversed by the introduction of a second mutation, which mutated 20S proteasomal subunit. All these studies strongly support the loss of NMT activity in mutant fungus is associated with an increased proteasome activity. Research led by Khandelwal have demonstrated that Akt, a protein upstream of mTOR localizes into the nucleus, where it could act as a transcription factor, so the other alternative could be that as a transcription factor Akt could be a down regulator of transcription of NMT1 gene, thus blocking the expression of NMT1 mRNA. Although, the phosphorylation of NMT1 by Akt has been demonstrated. It is unknown whether Akt regulates the transcription of NMT1 in the nucleus^[@CR50]^.
Considering the collection of mathematical models and data, we can validate the assumption of a regulation of NMT1 phosphorylation downstream of mTOR. Under control and perturbed conditions, a probable scenario for the mTOR-NMT1 regulation is characterized by a constant endogenous level of total mTOR and an absence of feedback regulation of mTOR by NMT1. Moreover, rapamycin appears to have a reversible effect on mTOR. The assumption of a constant endogenous level of total mTOR, shared by the best models with and without rapamycin, is in agreement with previously published models of mTOR pathways^[@CR32]--[@CR34]^. These previous models described explicitly signalling upstream of mTOR by using mass-action law. As our models are driven and depend on experimental data for their calibration, only a phenomenological representation of mTOR upstream regulation (described as its phosphorylation) is used in the current work. Despite this phenomenological representation of the upstream regulation of mTOR some of our estimates (maximal rates of phosphorylation and dephosphorylation of mTOR) can be compared to those from previously published work and are of the same order of magnitude after rescaling^[@CR32]^. However, to the best of our knowledge, as this work introduces the first models coupling the dynamics of both mTOR and NMT1, estimates for other parameters are not presently available in the literature for comparison.
When considering control experimental data and the corresponding three models, the model selection is conclusive and select the same best model. However, when including the effect of rapamycin, several models are similarly good for some datasets and it is thus difficult to select one of these models as the best. In the models, we assumed that rapamycin concentration in the cell remained constant over time and that rapamycin only interacts with mTOR. As rapamycin was used to perturb mTOR and to keep the models simple, the interaction between mTOR and rapamycin was modelled as reversible or irreversible using mass action type terms. However, it is possible that rapamycin dynamics is more complex than actually modelled with respect to its action on mTOR and the consequent effect on NMT1. Furthermore, rapamycin could induce further intracellular changes because of slight variations in the expression data due to differences in passages of MCF7 cells used for the trials, despite this the biological trend remained consistent in four trials. The modelling work reported in this study is the first step in the construction of a general model of this cellular pathway for future studies, which can aide in determining regulations upstream of mTOR and downstream of NMT1.
Methods {#Sec7}
=======
Cell culture and treatment {#Sec8}
--------------------------
Human breast adenomacarcinoma, MCF7 cells were a kind gift from Dr. Leigh Murphy (University of Manitoba, Canada). The cells were maintained in Dubecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, glucose, 100 U/ml of penicillin and 100 *μ*g/ml streptomycin at 37 °C with 5% CO2 in a humidified incubator. MCF7 cells were grown in 100 mm tissue culture dishes.
Western Blot Analyses {#Sec9}
---------------------
MCF7 cells of \~75--85% confluence were starved in the culture medium depleted of FBS for 6 h and treated with either 100 nM rapamycin or the vehicle, DMSO for a period of 5′, 10′, 30′, 60′, 180′, 360′, 720′ and 1440′. At indicated times the cells were washed with cold phosphate buffered saline (PBS). Following the treatment total protein was extracted by lysing the cells in a lysis buffer containing HEPES (pH 7.4, 50 mM), sucrose (150 mM), sodium orthovanadate (2 mM), *β*-glycerophosphate (80 mM), sodium fluoride (10 mM), sodium pyrophosphate (10 mM), sodium EGTA (2 mM), sodium EDTA (2 mM), triton X-100 (1%), SDS (0.1%), phenyl methyl sulphonyl fluoride (PMSF; 1 mM) and protease inhibitor cocktail (1%; Sigma Aldrich). The cells were scraped, collected in an eppendorf and allowed to stand on ice for 10 min. The lysates were spun at 13,000 rpm for 10′ at 4 °C, the pellet was discarded and the supernatant was collected and stored at −20 °C for future use. The protein lysates (30 *μ*g) were resolved in 8% and 10% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Quebec, Canada) and blocked with 5% milk dissolved in PBS with tween-20 (PBST) for 1 hour at room temperature. The membranes were then probed with rabbit polyclonal antibody against p-mTOR S2448 (Sigma, Canada) and mouse monoclonal anti-NMT1 antibody (a kind gift from VastCon Inc, Canada). The excess antibodies were removed by washing the membranes three times with PBST, 10′ each and incubated with HRP-conjugated secondary antibody for an hour at room temperature. The membranes were washed three times in PBST, 10′ each and visualization was done using ClarityWestern ECL substrate (Bio-Rad) reagent and a Molecular Imager ChemiDoc XRS System (Bio-Rad) and Image Lab software Version 3.0. The membranes were stripped and re-probed with antibodies against total mTOR and *β*-actin. *β*-actin is a housekeeping protein and was used as a loading control. Densitometric analysis was performed using Image lab software version 3.0 and relative band intensities were presented as a ratio of phosphorylated mTOR (S2448) over total mTOR or NMT1 over *β*-actin, compared with control (no treatment, 0′). Four independant experiments were performed and the representative blots are shown in Figs [1](#Fig1){ref-type="fig"} and [S1](#MOESM1){ref-type="media"}.
Mathematical models {#Sec10}
-------------------
We designed a collection of eight models describing the dynamics of mTOR and NMT1. These eight models result from alternative hypotheses, which are difficult to test experimentally,Does the regulation of endogenous levels of mTOR components impact the dynamics?Does NMT1 have a negative feedback effect on mTOR?Is the effect of rapamycin on mTOR reversible or irreversible?
The models are based on biological assumptions and designed to accommodate the available data: intracellular p-mTOR, total mTOR and total NMT1 over time, with or without perturbations due to rapamycin treatment. The upstream regulation of mTOR is not explicitly included in the models but phenomenologically described in the phosphorylation of mTOR. The core of all these models is the regulation of NMT1 phosphorylation by p-mTOR.
Hence, we consider the dynamics of mTOR *T*, p-mTOR *T*~*p*~, NMT1 *N*, p-NMT1 *N*~*p*~ and the complex rapamycin-mTOR *R*~*c*~, as follows,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{array}{rcl}\frac{{\rm{d}}T}{{\rm{d}}t} & = & \mathop{\overbrace{\frac{-{\alpha }_{T}T}{{K}_{T}+T}}}\limits^{{\rm{phosphorylation}}}+\mathop{\overbrace{\frac{{\alpha }_{{T}_{p}}{T}_{p}}{{K}_{{T}_{p}}+{T}_{p}}}}\limits^{{\rm{dephosphorylation}}}+\mathop{\overbrace{{{\rm{\Pi }}}_{T}}}\limits^{{\rm{synthesis}}}+\mathop{\overbrace{f(T,N)}}\limits^{{\rm{feedback}}}+\mathop{\overbrace{g(T,{R}_{c}),}}\limits^{{\rm{rapamycin}}\,{\rm{effect}}}\\ \frac{{\rm{d}}{T}_{p}}{{\rm{d}}t} & = & \mathop{\overbrace{\frac{{\alpha }_{T}T}{{K}_{T}+T}}}\limits^{{\rm{phosphorylation}}}-\mathop{\overbrace{\frac{{\alpha }_{{T}_{p}}{T}_{p}}{{K}_{{T}_{p}}+{T}_{p}}}}\limits^{{\rm{dephosphorylation}}}-\mathop{\overbrace{{\delta }_{{T}_{p}}{T}_{p},}}\limits^{{\rm{degradation}}}\\ \frac{{\rm{d}}N}{{\rm{d}}t} & = & \mathop{\overbrace{-\frac{{\alpha }_{N}{T}_{p}N}{{K}_{N}+N}}}\limits^{{\rm{phosphorylation}}}+\mathop{\overbrace{{{\rm{\Pi }}}_{N},}}\limits^{{\rm{synthesis}}}\\ \frac{{\rm{d}}{N}_{p}}{{\rm{d}}t} & = & \mathop{\overbrace{\frac{{\alpha }_{N}{T}_{p}N}{{K}_{N}+N}}}\limits^{{\rm{phosphorylation}}}-\mathop{\overbrace{{\delta }_{{N}_{p}}{N}_{p},}}\limits^{{\rm{degradation}}}\\ \frac{{\rm{d}}{R}_{c}}{{\rm{d}}t} & = & \mathop{\overbrace{h(T,{R}_{c}\mathrm{).}}}\limits^{{\rm{rapamycin}}\,{\rm{effect}}}\end{array}$$\end{document}$$
Figure [3](#Fig3){ref-type="fig"} displays a schematic representation of the interactions between the variables corresponding to the system defined in ([1](#Equ1){ref-type=""}), as well as a graphical representation of the relationship between models in the collection. Model assumptions are summarized in Table [1](#Tab1){ref-type="table"}.
The regulation of the endogenous level of mTOR is modelled by the synthesis of mTOR and the degradation of p-mTOR. When the synthesis of mTOR and the degradation of p-mTOR are not explicitly described in the model, the total amount of mTOR *T*~*t*~ is assumed to be constant over time and the corresponding models are labelled constant total mTOR. We do not explicitly model the concentration of rapamycin *R* in the cell, assuming it is abundant and available throughout the experiment, and consider that it remains constant for the timescale considered. The absorption of rapamycin into the cell is not the focus of the study and is thus not described. When needed, enzymatic reactions are assumed to follow Michaelis-Menten dynamics^[@CR51],[@CR52]^. To reduce the number of parameters and the complexity of (1) and without experimental data to quantify p-NMT1, the dephosphorylation of p-NMT1 is not considered; however, we proved mathematically that it does not change the dynamics of the models. For all models there exists at least a positive stable equilibrium under some conditions on parameters. Mathematical analyses of all models are detailed in Supplementary material, see [S2.1](#MOESM1){ref-type="media"}.
### Modelling mTOR-NMT1 without rapamycin {#Sec11}
Models NT, NTt and NTf consider only mTOR and NMT1 without rapamycin (see Table [1](#Tab1){ref-type="table"} and Fig. [3](#Fig3){ref-type="fig"}), this implies that *g*(*T*, *R*~*c*~) = *h*(*T*, *R*~*c*~) = 0. Model NT describes the dynamics of mTOR, p-mTOR, NMT1 and p-NMT1 with synthesis and degradation of mTOR components and without feedback *f*(*T*, *N*) = 0. Model NTt is derived from model NT by setting a constant total mTOR *T*~*t*~ = *T* + *T*~*p*~, which allowed us to reduce the number of equations and describe only the dynamics of p-mTOR *T*~*p*~. Model NTf allowed us to test the assumption of a feedback regulation of mTOR by NMT1 using *f*(*T*, *N*) = −*βNT* (Table [1](#Tab1){ref-type="table"} and Fig. [3](#Fig3){ref-type="fig"}). We did not consider a negative feedback and constant total mTOR at the same time, as the resulting dynamics is biologically unrealistic (no positive steady state). Model equations are provided in Supplementary material, see [S2.1](#MOESM1){ref-type="media"}.
### Modelling the effect of rapamycin {#Sec12}
Models NTRr, NTtRr, NTRi, NTRrf and NTRif, additionally describe the interaction between mTOR and rapamycin (see Table [1](#Tab1){ref-type="table"} and Fig. [3](#Fig3){ref-type="fig"}). In models with an irreversible binding of rapamycin, *g*(*T*, *R*~*c*~) = −*γRT* and $\documentclass[12pt]{minimal}
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\begin{document}$$h(T,{R}_{c})=\gamma RT-{\delta }_{{R}_{c}}{R}_{c}$$\end{document}$. A reversible binding is described by *g*(*T*, *R*~*c*~) = −*γRT* + *κR*~*c*~ and *h*(*T*, *R*~*c*~) = *γRT* − *κR*~*c*~.
From model NT are derived two models by adding a reversible (NTRr) or irreversible (NTRi) binding to rapamycin. Models NTRrf and NTRif were obtained by adding the effect of rapamycin to model NTf or, alternatively, by adding the description of the feedback to models NTRr or NTRi (see Fig. [3](#Fig3){ref-type="fig"}). Finally, model NTtRr was derived from model NTRr and characterized by a constant total mTOR, when the rapamycin binding is reversible. In this case, *T*~*t*~ = *T* + *T*~*p*~ + *R*~*c*~ and we consider only the dynamics of mTOR *T* and the complex rapamycin-mTOR *R*~*c*~ (p-mTOR *T*~*p*~ is thus deduced from *T*~*t*~, *T* and *R*~*c*~). Similarly to the control, the constant total mTOR assumption is not considered with irreversible binding and/or feedback. Model equations are provided in Supplementary material, see [S2.1](#MOESM1){ref-type="media"}.
### Model calibration and selection {#Sec13}
In order to calibre the outputs of the models to the experimental data, we first estimated parameter values for each model; the number of estimated parameters for each model is listed in Table [1](#Tab1){ref-type="table"}. To reduce the number of estimated parameters, we set *R* = 1, as we assumed that the concentration of rapamycin inside the cell remains constant during the course of the experiment. For each of the four datasets, the following experimental data are measured at *m* = 9 time points: total mTOR $\documentclass[12pt]{minimal}
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\begin{document}$${T}_{p}^{exp}$$\end{document}$ and total NMT1 $\documentclass[12pt]{minimal}
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\begin{document}$${N}_{total}^{exp}$$\end{document}$, all normalized by *β*-actin at the time point considered. For the four datasets, the residual sum of squares between experimental and simulated data for model *i* is defined as follows$$\documentclass[12pt]{minimal}
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\begin{document}$${{\rm{RSS}}}_{i}=\sum _{j=1}^{m}\,[{({T}_{p}^{exp}({t}_{j})-{T}_{p}^{i}({t}_{j}))}^{2}+{({T}_{total}^{exp}({t}_{j})-{T}_{total}^{i}({t}_{j}))}^{2}+{({N}_{total}^{exp}({t}_{j})-{N}_{total}^{i}({t}_{j}))}^{2}],$$\end{document}$$where *t*~*j*~ are the *m* time points, $\documentclass[12pt]{minimal}
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\begin{document}$${T}_{total}^{i}({t}_{j})={T}^{i}({t}_{j})+{T}_{p}^{i}({t}_{j})$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$${N}_{total}^{i}({t}_{j})={N}^{i}({t}_{j})+{N}_{p}^{i}({t}_{j})$$\end{document}$ account for the responses in model *i*. To obtain the best fit and thus the best set of parameters, we minimized the residual sum of squares between the experimental data and the model output with a genetic algorithm, and we repeated this procedure multiple times (all the fitting procedure was performed using Matlab R2016b). For a given dataset, a model will thus be associated with a nominal set of parameters.
Then, model selection was used to identify the best model and characterize the required mechanisms. In order to select the best model, given the collection of models and experimental data, we computed the Akaike Information Criterion (AIC~*i*~) for each model *i*, which takes into account the number of estimated parameters and the goodness of the fit^[@CR53],[@CR54]^ (see Table [2](#Tab2){ref-type="table"}), as follows:$$\documentclass[12pt]{minimal}
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\begin{document}$${{\rm{AIC}}}_{i}=n\,\mathrm{ln}\,(\frac{{{\rm{RSS}}}_{i}}{n})+2{k}_{i},$$\end{document}$$where *n* = 3*m* is the number of data points used for parameters estimation and *k*~*i*~ is the number of estimated parameters for model *i*, including the estimation of RSS~*i*~/*n*. As the number of data points was small (*n* = 27 for each dataset) in comparison to the number of parameters we used the AIC corrected for small sample sizes instead of the AIC, as follows:$$\documentclass[12pt]{minimal}
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\begin{document}$${{\rm{AICc}}}_{i}={{\rm{AIC}}}_{i}+\frac{2{k}_{i}({k}_{i}+\mathrm{1)}}{n-{k}_{i}-1}=n\,\mathrm{ln}\,(\frac{{{\rm{RSS}}}_{i}}{n})+2{k}_{i}\frac{n}{n-{k}_{i}-1}\mathrm{.}$$\end{document}$$
Recall the best model is the model with the lowest AICc^[@CR53]^.
For the collection of models, we can then calculate the Akaike weight *w*~*i*~, which can be interpreted as the probability that the model *i* is the best model given the experimental data and the set of models considered:$$\documentclass[12pt]{minimal}
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\begin{document}$${w}_{i}=\frac{\exp (\,-\,{{\rm{\Delta }}}_{i}\mathrm{/2)}}{{\sum }_{j=1}^{N}\,\exp (\,-\,{{\rm{\Delta }}}_{j}\mathrm{/2)}},$$\end{document}$$with *N* the total number of models in the collection and $\documentclass[12pt]{minimal}
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\begin{document}$${{\rm{\Delta }}}_{i}={{\rm{AICc}}}_{i}-\mathop{{\rm{\min }}}\limits_{j=\mathrm{1..}N}\,({{\rm{AICc}}}_{j})$$\end{document}$.
To quantify the probability of each assumption, we computed their respective weights by summing the Akaike weights of the models verifying the assumption. Thus, the weight of models with reversible (resp. irreversible) rapamycin effect will be obtained by summing the weights of models including this assumption namely NTRr, NTtRr and NTRrf (resp. NTRi and NTRif). The weight of the feedback assumption corresponds to the weight of model NTf without rapamycin or models NTRrf and NTRif with rapamycin.
### Robustness and sensitivity analysis {#Sec14}
For the best models, we evaluated the global sensitivity to parameters, using the LHS-PRCC (Partial Rank Correlation Coefficient with Latin Hypercube Sampling) method^[@CR55],[@CR56]^ (see Fig. [5](#Fig5){ref-type="fig"}). Sets of parameter values were sampled uniformly using LHS (the sampling interval includes nominal parameter values). We then eliminated the parameters not satisfying the existence condition for a positive equilibrium (see Supplementary materials Section [S2.1](#MOESM1){ref-type="media"}). These constraints on parameter values were imposed to ensure a biologically relevant behavior for the models. Then models were simulated until time 2880 minutes (twice the experimental time) to ensure equilibrium is reached. The proportion of p-mTOR and total NMT1 at steady state were used to compute the PRCC (see Fig. [5](#Fig5){ref-type="fig"}). The sensitivities of all non aggregated variables are shown in Figure [S13](#MOESM1){ref-type="media"}.
In order to determine the impact of small variations of parameters on the best model outputs, we sampled random sets of parameters, with each parameter following a uniform distribution between 0.9 and 1.1 times the nominal value obtained by fitting. This allowed us to study the effect of parameter fluctuations around the nominal value, assess the robustness of models and define minimal and maximal values of the outputs over time (see Fig. [4](#Fig4){ref-type="fig"} and Supplementary materials Section [S2.2.2](#MOESM1){ref-type="media"}).
Electronic supplementary material
=================================
{#Sec15}
Supplementary information
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Marine Jacquier and Shiby Kuriakose contributed equally.
Stéphanie Portet and Shailly Varma Shrivastav jointly supervised this work.
Electronic supplementary material
=================================
**Supplementary information** accompanies this paper at 10.1038/s41598-018-30447-0.
S.V.S. would like to thank Manitoba Medical Service Foundation (grant number 8-2017-08) operation grant for the financial support. A.S. would like to thank Prostate Cancer Fight Foundation and CFI. A.B. is recipient of Queen Elizabeth-II scholarship and The University of Winnipeg President's scholarship. S.P. would like to thank NSERC for financial support.
M.J. and S.K. have contributed equally to the work. S.K. has planned, executed all the experiments and contributed in manuscript writing. M.J. has performed modelling, mathematical and simulation studies and contributed towards writing of the manuscript. A.B. has executed the experiments and contributed towards manuscript writing. Y.Z. has performed modeling and mathematical studies. A.S. has analyzed the experimental results and contributed towards writing the manuscript. S.P. has conceptualized the mathematical and simulation work and supervised M.J. and Y.Z., analyzed and contributed towards writing of the manuscript. S.V.S. has conceptualized, supervised S.K. and A.B. while conducting the experiments, analyzed the experimental data and contributed towards writing the manuscript.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
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ERRATUM {#h0.0}
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Volume 7, no 3, doi:10.1128/mBio.00281-16, 2016. On PDF page 5, first column, the GenBank accession number "1910160" should be updated to "[KX174310](http://www.ncbi.nlm.nih.gov/nuccore/KX174310)." The correct URL is [http://www.ncbi.nlm.nih.gov/nuccore/KX174310](http://www.ncbi.nlm.nih.gov/nuccore/kx174310).
On PDF page 11, second column, "RD1^mon^, +700/− less than 5,062 bp but greater than 700 bp)" should be updated to "RD1^mon^, +309 bp/−513 bp)."
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Introduction
============
Epidural injections are one of the most commonly utilized treatment modalities for managing chronic low back pain with or without lower extremity pain.[@b1-jpr-5-301]--[@b12-jpr-5-301] Despite increasing utilization of lumbar epidural injections, significant debate continues regarding their effectiveness, specifically any conditions other than disc herniation and compressive radiculitis. The pathophysiology of low back pain and radicular pain is the subject of ongoing research and controversy, with discogenic pain assuming a major role as a cause of non-specific low back pain, beyond disc herniation.[@b13-jpr-5-301]--[@b16-jpr-5-301] In fact, soon after the description of intervertebral disc herniation by Mixter and Barr[@b17-jpr-5-301] in American medical literature in 1934 with their landmark description of the herniated nucleus pulposus, Mixter and Ayers[@b18-jpr-5-301] showed that radicular pain can occur without disc herniation. Further, non-specific low back pain constitutes 80% or 90% of low back pain without identifiable causes with a large proportion having chronic axial low back pain secondary to progressive degenerative disc disease.[@b2-jpr-5-301],[@b19-jpr-5-301]--[@b22-jpr-5-301] It has been shown that discs have innervation with deep ingrowth into degenerated intervertebral discs.[@b23-jpr-5-301]--[@b26-jpr-5-301] Animal models have identified upregulation of various molecules such as calcitonin gene-related peptide and substance P in dorsal root ganglion neurons innervating degenerated intervertebral discs.[@b27-jpr-5-301],[@b28-jpr-5-301] Research also has detected high levels of inflammatory mediators in degenerated discs. While the majority of patients with axial low back pain improve with conservative management, various types of interventions have been described for chronic patients, but most interventions are highly variable and are associated with poor outcomes.[@b29-jpr-5-301]--[@b40-jpr-5-301]
In the past, all axial pain was attributed to disc degeneration. However, the development of controlled diagnostic blocks, and interventional techniques including discography, facet joint blocks, and sacroiliac joint blocks, have provided evidence that axial pain can also be caused by facet joints and sacroiliac joints. Utilizing provocation discography, the prevalence of pain due to internal disc disruption was reported to be 39% in patients suffering with chronic low back pain,[@b41-jpr-5-301] whereas primary discogenic pain was reported in 26%[@b42-jpr-5-301] when no other cause was suspected. In addition, facet joint pain has been shown to be present in 21%--41% of patients,[@b43-jpr-5-301] whereas sacroiliac joint pain has been established in 10%--38% of a selected population.[@b44-jpr-5-301]
The underlying mechanism of action for epidurally administered local anesthetic and steroids has been described, though not well understood. However, historically it has been believed that epidural steroids function by reducing inflammation, thus limiting the indications to compressive radiculopathy or, at best, radiculitis secondary to chemical irritation. Much of the literature on lumbar interlaminar epidurals has been negative except in recent years when fluoroscopic guidance was utilized.[@b39-jpr-5-301],[@b45-jpr-5-301]--[@b53-jpr-5-301] A variation of lumbar interlaminar injections, caudal epidural injections, have also been proven to be effective in multiple causes of low back pain with or without lower extremity pain.[@b38-jpr-5-301],[@b48-jpr-5-301],[@b54-jpr-5-301]--[@b57-jpr-5-301] Recent evidence also has demonstrated effectiveness for fluoroscopically administered epidural injections in the cervical spine[@b58-jpr-5-301]--[@b61-jpr-5-301] as well as the thoracic spine.[@b62-jpr-5-301] These evaluations have illustrated the effectiveness of epidural injections not only for disc herniation, but also for axial or discogenic pain after eliminating facet and sacroiliac joint pain, spinal stenosis, and post-surgery syndrome. In fact, in the published preliminary results of the current study, lumbar interlaminar epidural injections provided improvement in 74% of patients who received local anesthetic only, and 63% in the group who received local anesthetic and steroids.[@b39-jpr-5-301]
The current report evaluates the role of lumbar interlaminar epidural injections for patients with chronic axial or discogenic low back pain in 120 patients with a 1-year follow-up.[@b39-jpr-5-301]
Methods
=======
This active control, randomized, double-blind trial was conducted in an interventional pain management practice, in a specialty referral center, with approval of the Institutional Review Board (IRB). It follows Consolidated Standards of Reporting Trials (CONSORT) guidelines.[@b63-jpr-5-301] The study is registered with the US Clinical Trial Registry with an assigned number of NCT00681447.
The internal resources of the practice were used to conduct the study. There was no external funding, either from industry or from elsewhere.
Interventions
-------------
Patients were assigned into one of two groups. Group I patients received lumbar interlaminar epidural injections with 6 mL of lidocaine 0.5% preservative free; Group II patients received lumbar interlaminar epidural injections with 5 mL of lidocaine 0.5% preservative-free mixed with 6 mg or 1 mL of non-particulate betamethasone.
Participants
------------
All patients were recruited from new patients presenting to the center who met the inclusion criteria. The IRB-approved protocol and informed consent, which described in detail all aspects of the study and its process, were provided to all participating patients.
Pre-enrollment data collection
------------------------------
The data collected included Numeric Rating Scale (NRS) for pain, Oswestry Disability Index 2.0 (ODI) for functional status, medical and surgical history of any co-existing disease(s), radiologic investigations, physical examination, work status, and opioid intake.
Inclusion criteria
------------------
Inclusion criteria included only the patients with a diagnosis of lumbar axial or discogenic pain; over the age of 18 years; a history of chronic function-limiting low back pain of at least 6 months duration; and the ability to understand the study protocol and provide voluntary, written informed consent, and participate in outcome measurements.
Additional criteria were a failure to improve with conservative management, including, but not limited to, physical therapy, chiropractic manipulation, exercises, drug therapy, and bedrest.
Exclusion criteria were a positive response for lumbar facet joint or sacroiliac joint pain by means of controlled, comparative local anesthetic blocks; previous lumbar surgery; uncontrollable or unstable opioid use; uncontrolled psychiatric disorders; uncontrolled medical illness, either acute or chronic; any conditions that could interfere with the interpretation of the outcome assessments; pregnant or lactating women; and a history or potential for adverse reaction(s) to local anesthetics or steroids.
Description of interventions
----------------------------
All participating patients were evaluated with controlled, comparative local anesthetic lumbar facet joint nerve blocks or sacroiliac joint injections. The process started with diagnostic facet joint nerve blocks with 0.5 mL of 1% lidocaine, followed by the blockade of facet joint nerves with 0.25% bupivacaine on separate occasions. A positive response was 80% pain relief.[@b42-jpr-5-301]--[@b44-jpr-5-301],[@b64-jpr-5-301] Controlled, comparative local anesthetic blocks were also performed for suspected sacroiliac joint pain, with 2 mL of 1% lidocaine and 0.25% bupivacaine.[@b42-jpr-5-301],[@b44-jpr-5-301]
Lumbar interlaminar epidural procedures were performed by one physician (LM) in an ambulatory surgery setting, in a sterile operating room, utilizing fluoroscopy. Patients were in the prone position with intravenous access and sedation as indicated. The epidural space entry was confirmed by an injection of non-ionic contrast medium. All procedures were performed either between L5 and S1 or at a higher level based on the patient's pain complaints. Following this, an injection of 6 mL of lidocaine hydrochloride 0.5% preservative-free, or 5 mL of lidocaine mixed with 6 mg of non-particulate betamethasone was given.
Additional interventions
------------------------
If a patient required additional lumbar interlaminar epidural injections, these were provided based on the response to the previous injection, with deterioration of pain relief to less than 50%. Patients who were non-responsive and continued with conservative management were followed without further epidural injections with medical management, unless they requested unblinding.
Co-interventions
----------------
There was no specific physical therapy, occupational therapy, bracing, or other interventions offered other than the study intervention. In addition, if patients were improving significantly and the medical necessity for drugs was lacking, medications were stopped or dosages were decreased. For some patients, based on medical necessity, dosages were increased. However, all patients continued previously directed exercise programs, as well as their employment.
Objective
---------
The study was designed to assess the effectiveness of lumbar interlaminar epidural injections containing local anesthetic with or without steroids in managing chronic axial low back pain of discogenic origin.
Outcomes
--------
Primary outcome measures included the NRS on a scale of 0--10, and the ODI on a 0--50 scale. Secondary outcome measures included employment status, and opioid intake in terms of morphine equivalents. The value and validity of the NRS and ODI have been reported.[@b65-jpr-5-301],[@b66-jpr-5-301] Recently, previously established thresholds were questioned.[@b67-jpr-5-301],[@b68-jpr-5-301] Thus, significant pain relief or improvement and function were considered to be at least a 50% reduction in NRS and the ODI, which is similar to the measurements in other trials.[@b38-jpr-5-301],[@b46-jpr-5-301],[@b47-jpr-5-301],[@b54-jpr-5-301]--[@b62-jpr-5-301],[@b69-jpr-5-301]--[@b71-jpr-5-301]
The opioid intake was converted into morphine equivalents.[@b72-jpr-5-301]
Assessment of employment and work status were determined based on employability at the time of enrollment. Thus, they were classified into multiple categories such as employable, housewife with no desire to work outside the home, retired, or over the age of 65. Patients who were unemployed due to pain, employed but on sick leave, or made redundant were considered as employable.
The epidurals were considered to be successful if a patient obtained significant improvement for at least 3 weeks with the first and second procedures. All others were considered to be failures.
Sample size
-----------
The sample size was calculated based on significant pain relief. Considering a 0.05 two-sided significance level, a power of 80%, and an allocation ratio of 1:1, 55 patients in each group were estimated.[@b73-jpr-5-301] Allowing for a 10% attrition/ non-compliance rate, 60 patients were required.
Randomization
-------------
Sixty patients were randomly assigned into each group from a total of 120 patients who met inclusion criteria.
Sequence generation
-------------------
Simple randomization was utilized to allocate patients into groups.
Allocation concealment
----------------------
Patients were randomized into two groups by one of the three operating room nurses who were also study coordinators. The same person also prepared the drugs.
Blinding (masking)
------------------
The patients and physician were blinded to group assignment and both injectates were clear. In addition, the blinding was ensured by mixing the study patients with other patients receiving routine treatment. All patients chosen for 1-year follow-up were selected by a statistician not participating in provision of the patients' care and the unblinding results were not disclosed to either the treating physician, other participants, or patients. Thus, the nature of blinding was not interrupted.
Statistical methods
-------------------
Data analyses were carried out using SPSS software (v 9.01; SPSS Inc, Chicago, IL). For categorical and continuous data comparison, Chi-square (Fisher test where necessary) and *t*-tests were used, respectively. Because the outcome measures of the participants were measured at four points in time, repeated measures analysis of variance were performed with the post hoc analysis.
Intent-to-treat analysis
------------------------
Either the last follow-up data or initial data were utilized in the patients who dropped out of the study and no other data were available for the intent-to-treat analysis.
Best case, worst case, and last follow-up score scenarios were used for sensitivity analysis.
Results
=======
Participant flow
----------------
[Figure 1](#f1-jpr-5-301){ref-type="fig"} illustrates the participant flow. The recruitment period lasted from January 2008 through May 2010.
Baseline data
-------------
[Table 1](#t1-jpr-5-301){ref-type="table"} shows the basic demographic characteristics, pain distribution, onset of the pain, numeric rating scale of pain, and ODI for functional status summary scores. There were no statistically significant differences between the two groups in terms of these baseline data (all *P* values \> 0.05), except for weight (*P* = 0.000).
Pain relief and functional assessment
-------------------------------------
[Table 2](#t2-jpr-5-301){ref-type="table"} presents the results of repeated measures analysis. Regarding pain scores, there were significant differences within groups by time (*P* = 0.001). In the ODI for functional status, there were significant differences in summary scores within group by time (*P* = 0.001).
A post hoc analysis indicated that all the mean differences between baseline and with the scores at other time points were significant at the 0.05 level.
Pain relief and functional status improvement
---------------------------------------------
The percentage of patients with significant improvement is presented in [Figure 2](#f2-jpr-5-301){ref-type="fig"}. In Group I and II, 77% and 67% showed significant improvement, respectively. In the successful groups, significant improvement was seen in 84% in Group I and 71% in Group II.
Therapeutic procedural characteristics
--------------------------------------
[Table 3](#t3-jpr-5-301){ref-type="table"} lists therapeutic procedural characteristics. Lumbar interlaminar procedures were performed in 91% of cases at L5/S1 and 9% of cases at L4/5.
Employment characteristics
--------------------------
[Table 4](#t4-jpr-5-301){ref-type="table"} lists employment characteristics in both groups. Among the patients eligible for employment, the total employed changed from 12 at baseline to 13 at the end of 12 months in Group I; it changed from 14 to 18 in Group II, a nonsignificant increase of 6% in Group I and 21% in Group II.
Opioid intake
-------------
[Table 5](#t5-jpr-5-301){ref-type="table"} presents the results of repeated measures analysis for opioid intake. There were significant differences in opioid intake within group by time (*P* \< 0.001). A post hoc analysis indicated that all the mean differences in scores between baseline and other time points were significant at the 0.05 level.
Changes in weight
-----------------
[Table 6](#t6-jpr-5-301){ref-type="table"} shows changes in weight, with no significant differences in changes among the groups.
Adverse events
--------------
Of the 454 lumbar epidural procedures performed, there were two subarachnoid punctures that did not result in headache. One patient experienced weight gain due to a high dose of steroid from an unrelated medical problem.
Discussion
==========
This randomized, active control trial shows that carefully selected patients with axial or discogenic chronic low back pain can receive significant pain relief and functional status improvement with lumbar interlaminar epidural injections. Their pain was not caused by disc herniation, facet joints, or the sacroiliac joints. Significant pain relief and functional status improvement of ≥50% were seen in 77% of Group I and 67% of Group II. A better picture emerges when each group was divided into failed and successful outcomes. Significant pain relief and functional status improvement was seen in 84% of the successful outcomes in Group I; 71% in the successful outcomes in Group II. The average procedures per year and average weeks of total relief for the successful outcome patients were: Group I, 3.9 procedures and 40.0 ± 15.6 weeks; Group II, 4.0 procedures and 39.6 ± 12.4 weeks.
The results of this evaluation essentially illustrate that if patients are selected appropriately, lumbar epidural injections provide significant improvement. These results are in line with other studies separating the patients into failed and successful groups.[@b38-jpr-5-301],[@b46-jpr-5-301],[@b47-jpr-5-301],[@b54-jpr-5-301],[@b55-jpr-5-301],[@b58-jpr-5-301]--[@b62-jpr-5-301] The results illustrate that both pain relief and improvement in functional status are clinically and statistically significant. Strict criteria were incorporated into the study and only participating patients judged not to have facet joint or sacroiliac joint pain were included, thus avoiding the criticism that including those with facet joint or sacroiliac joint pain in a study contributes to negative results. As confirmed in this report, epidural injections do not provide long-term relief. But, properly selected patients and appropriate procedures under fluoroscopy can provide long-term relief with judicious use.
Despite multiple publications, there is still significant debate regarding the medical necessity and indications for lumbar epidural injections, either by interlaminar approach, caudal approach, or transforaminal approach. Multiple systematic reviews, guidelines, and other reviews have identified weak indications for epidural injections, namely radicular pain from herniated lumbar intervertebral discs. However, there is a lack of evidence or recommended indications for other conditions. The preliminary report of the current manuscript showed positive results with interlaminar epidural injections.[@b39-jpr-5-301] Similarly, the previous results with caudal epidurals in appropriately selected patients were positive.[@b38-jpr-5-301]
The results of this evaluation are similar to caudal epidural injections for axial or discogenic pain.[@b38-jpr-5-301] However, overall relief was superior in the present study, compared to the caudal study.[@b38-jpr-5-301] Steroids did not have any superiority over local anesthetic alone in either study.
There is a paucity of literature evaluating epidural injections for axial or discogenic pain. The role of caudal epidural injections for axial or discogenic pain, after ruling out facet joint pain or diagnosing it with provocation discography, was evaluated in three studies.[@b38-jpr-5-301],[@b74-jpr-5-301],[@b75-jpr-5-301] Only one study[@b51-jpr-5-301] looked at lumbar interlaminar epidural injections other than the preliminary version of this study.[@b39-jpr-5-301] Butterman[@b51-jpr-5-301] evaluated the role of interlaminar epidural steroids showing improvement only at the 3-month follow-up. All of the studies reported modest results.
The mechanism of action of steroids and local anesthetics continues to be debated. Multiple hypothesis have been emerging.[@b76-jpr-5-301]--[@b84-jpr-5-301] The evidence shows that steroids, as well as local anesthetics, have significant effects on the modulation of noxious stimulation by various mechanisms. Long-term effects are provided by both local anesthetics and steroids or when in combination, in experimental as well as clinical studies.[@b38-jpr-5-301],[@b46-jpr-5-301],[@b47-jpr-5-301],[@b54-jpr-5-301]--[@b56-jpr-5-301],[@b58-jpr-5-301]--[@b61-jpr-5-301],[@b76-jpr-5-301]--[@b84-jpr-5-301]
Comparative effectiveness research and evidence-based medicine have been considered as pivotal to health care policy not only in the US, but across the world.[@b10-jpr-5-301],[@b11-jpr-5-301],[@b85-jpr-5-301]--[@b89-jpr-5-301] In general, practical studies conducted in a generally applicable environment are considered more valuable than pragmatic or practical clinical trials with an active control group instead of a placebo group. Practical studies measure effectiveness, which is considered more appropriate than explanatory trials which measure efficacy.[@b90-jpr-5-301]--[@b93-jpr-5-301] Thus, this study meets the criteria for a practical clinical trial, specifically in contemporary interventional pain management practices; it meets the appropriate selection criteria and repeats the procedures based upon the return of pain, rather than a predetermined schedule. The procedures were performed under fluoroscopy, only after conservative management had failed. The study also confirms the long-held belief that if the first two procedures do not provide at least a minimum of 3 weeks of relief, the procedures may not provide relief on a long-term basis. This was observed in the failed patients, suggesting that it may be futile to continue to repeat these procedures in these patients, unless there are compelling reasons to do so.
The present study may be criticized for not focusing on a placebo group. However, most studies have utilized inappropriate methodology with placebo groups with reference to interventional techniques.[@b88-jpr-5-301],[@b89-jpr-5-301],[@b94-jpr-5-301]--[@b99-jpr-5-301] The only appropriately designed placebo trial by Ghahreman et al[@b99-jpr-5-301] showed a lack of significant effect when sodium chloride solution was injected into an inactive structure. Consequently, when sodium chloride solution or other agents such as local anesthetics, which are considered as placebo by some do not yield the same results, this leads to inaccurate methodology and conclusions.[@b100-jpr-5-301]--[@b103-jpr-5-301]
Some of the other weaknesses include differences in baseline demographic characteristics with respect to weight and sex; however, these differences were not considered to have caused any significant effect on the final results.
The implications of this trial are enormous in the health care arena. Studies with proper methodology in practical settings are not only crucial, but mandatory. Proper application of interventions will improve not only patients' pain and function and reduce drug use, it may also return them to the workforce; however, by the same token, inappropriate provision of any type of intervention, specifically interventions with substantial expenses, will not provide any benefit. Instead, it can harm the patient, thus depleting resources and reducing access. Similarly, inappropriately performed evaluations in the name of methodology, leading to inaccurate conclusions, may reduce health care expenditures, and will also increase patient suffering and reduce function by impeding access to much needed medical care.
Conclusion
==========
This study illustrates that overall significant improvement was seen in 77% of the patients in Group I and 67% of the patients in Group II. In the successful outcome groups, in those who received at least 3 weeks of relief with the first two procedures, the improvement was 84% in Group I and 71% in Group II. Pain relief and functional status improvement was achieved with an average procedures per year in the successful outcome groups of 3.9 in Group I and 4.0 in Group II, and an average total relief per year of 40.0 ± 15.6 weeks in Group I and 39.6 ± 12.4 in Group II.
Dr Manchikanti and Dr Benyamin designed the study; Mr Pampati performed the statistical analysis, and Ms Cash and Ms McManus were two of the three coordinators. The study was approved by the IRB and was registered in the US Clinical Trial Registry. All ethical guidelines were followed.
**Disclosure**
The authors report no conflicts of interest in this work.
{#f1-jpr-5-301}
{#f2-jpr-5-301}
######
Baseline demographic and clinical data
Group 1 (60) Group II (60) *P*-value
-------------------------------- --------------- --------------- -----------
Sex
Male 23% (14) 40% (24) 0.077
Female 77% (46) 60% (36)
Age
Mean ± SD 41.2 ± 11.9 42.7 ± 11.4 0.477
Weight
Mean ± SD 211.2 ± 60.9 168.6 ± 40.6 0.000
Height
Mean ± SD 65.8 ± 3.7 66.4 ± 4.1 0.430
Duration of pain (months)
Mean ± SD 104.2 ± 106.5 129.0 ± 90.9 0.173
Onset of pain
Gradual 67% (40) 70% (42) 0.845
Injury 33% (20) 30% (18)
Pain distribution
Unilateral 20% (12) 25% (15) 0.662
Bilateral 80% (48) 75% (45)
Back pain distribution
Back pain only 15% (9) 20% (12) 0.849
Back pain worse than leg pain 65% (39) 60% (36)
Leg pain worse than back pain 5% (3) 3% (2)
Both equal 15% (9) 17% (10)
Numeric rating score
Mean ± SD 8.0 ± 1.0 7.7 ± 0.9 0.082
Oswestry disability index
Mean ± SD 30.7 ± 4.5 29.2 ± 5.2 0.096
######
Comparison of numeric rating scale for pain and Oswestry disability index score summaries at four time points
Time points Numeric pain rating scale Oswestry disability index
--------------------------- ----------------------------------------------------------- ----------------------------------------------------------- ------------------------------------------------------------ ------------------------------------------------------------
Baseline 8.0 ± 1.0 7.7 ± 0.9 30.7 ± 4.5 29.2 ± 5.2
3 months 3.6[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 0.9 (88%) 3.5[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 1.2 (83%) 14.9[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 4.3 (83%) 14.6[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 5.1 (78%)
6 months 3.9[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 1.1 (77%) 3.6[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 1.2 (82%) 15.4[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 4.8 (73%) 14.4[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 5.2 (77%)
12 months 3.7[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 1.2 (78%) 3.7[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 1.3 (72%) 14.9[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 5.0 (77%) 15.0[\*](#tfn2-jpr-5-301){ref-type="table-fn"} ± 6.4 (70%)
Group difference 0.208 0.395
Time difference 0.001 0.001
Group by time interaction 0.448 0.210
**Notes:** Lower the value, the better the condition;
Significant difference with baseline values within the group (*P* \< 0.05); ( ) illustrates proportion with significant pain relief (≥50%) from baseline.
######
Therapeutic procedural characteristics with procedural frequency, average relief per procedure, and average total relief in weeks over a period of 1 year
Average relief Successful patients Failed patients Combined
----------------------------------- --------------------- ------------------ --------------- ---------------- ----------------- -----------------
1st procedure relief 6.1 ± 3.8 (55) 6.5 ± 4.3 (54) 0.9 ± 1.0 (5) 0.5 ± 0.8 (6) 5.7 ± 4.0 (60) 5.9 ± 4.5 (60)
2nd procedure relief 10.2 ± 6.8 (55) 10.0 ± 6.7 (54) 1.0 ± 1.4 (2) 0.8 ± 1.1 (3) 9.9 ± 6.9 (57) 9.5 ± 6.8 (57)
3rd procedure relief 11.9 ± 4.1 (51) 11.0 ± 3.5 (50) 2.0 (1) 5.0 ± 5.7 (2) 11.7 ± 4.3 (52) 10.8 ± 3.7 (52)
4th procedure relief 12.2 ± 4.5 (38) 12.3 ± 2.3 (41) -- 2.0 ± 1.4 (2) 12.2 ± 4.5 (38) 11.8 ± 3.1 (43)
5th procedure relief 12.6 ± 1.1 (16) 13.3 ± 2.7 (18) -- -- 12.6 ± 1.1 (16) 13.3 ± 2.7 (18)
Number of procedures per year 3.9 ± 0.9 4.0 ± 0.9 1.6 ± 0.9 2.2 ± 1.5 3.7 ± 1.1 3.8 ± 1.1
For initial 2 procedures in weeks 8.6 ± 10.0 8.2 ± 5.9 0.9 ± 1.0 0.6 ± 0.8 8.2 ± 9.9 7.6 ± 6.0
After initial 2 procedures 12.1 ± 3.9 11.9 ± 3.1 2.0 3.5 ± 3.8 12.0 ± 4.0 11.6 ± 3.4
All procedures 10.1 ± 5.4 (215) 10.1 ± 5.0 (218) 1.1 ± 1.0 (8) 1.5 ± 2.5 (13) 9.8 ± 5.6 (223) 9.7 ± 5.3 (231)
Total relief per year (weeks) 40.0 ± 15.6 39.6 ± 12.4 1.6 ± 1.7 3.2 ± 5.4 36.8 ± 18.4 36.0 ± 16.2
**Note:** Successful groups had at least 3 weeks of relief with first two procedures.
######
Employment characteristics
Employment status Group I Group II
-------------------------- --------- ---------- ---- ----
Employed part-time 7 5 3 5
Employed full-time 5 8 11 13
Unemployed (due to pain) 2 1 2 0
Not working 3 4 3 1
Eligible for employment 17 17 19 19
Total employed 12 13 14 18
Housewife 3 3 7 7
Disabled 39 38 32 32
Retired/over 65 1 1 2 2
Total number of patients 60 60 60 60
######
Comparison of opioid intake (morphine equivalents in mg) summaries at four time points
Time Group I (60) Group II (60)
--------------------------- ------------------------------------------------------- -------------------------------------------------------
Baseline 57.2 ± 61.4 53.4 ± 53.8
3 months 35.5[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 24.2 40.3[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 35.7
6 months 36.1[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 27.0 41.8[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 37.3
12 months 36.3[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 27.0 41.8[\#](#tfn5-jpr-5-301){ref-type="table-fn"} ± 37.3
Group difference 0.366
Time difference 0.001
Group by time interaction 0.629
**Note:**
Significant difference from their baseline values (*P* \< 0.05).
######
Characteristics of weight monitoring
Weight (lbs) Group I (60) Group II (60) *P*-value
--------------------- -------------- --------------- -----------
Weight at beginning 211.2 ± 60.9 168.6 ± 40.6 0.000
Weight at 1 year 211.4 ± 64.0 166.1 ± 40.5 0.000
Change 0.2 ± 13.3 −2.5 ± 10.8 0.227
Lost weight 37% (22) 57% (34)
No change 23% (14) 13% (8) 0.078
Gained weight 40% (24) 30% (18)
| {
"pile_set_name": "PubMed Central"
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Iliopsoas impingement (IPI) is an uncommon cause of persistent pain after total hip arthroplasty (THA). Previous studies have demonstrated an incidence as high as 4.3% after THA.[@bib1] IPI after THA often occurs because of hardware irritation. Most commonly, it is due to anterior overhang of the acetabular component, due to either a retroverted or oversized component. Other causes of IPI that have been described after THA include anterior acetabular screw protrusion,[@bib2] oversized femoral head (typically in metal on metal prosthesis), and acetabular component cement extrusion.[@bib3] When prominent hardware causes IPI, conservative management is less likely to provide long-term relief, but should still be pursued initially.
Clinically, patients describe groin pain, especially when standing from a seated position. This pain can be reproduced by moving the hip from a flexed and externally rotated position to an extended and internally rotated position.[@bib4] A snapping sensation may also accompany pain during this physical examination maneuver.[@bib5] Conservative management of IPI includes activity modification, physical therapy for stretching of the iliopsoas, and an injection of cortisone and local anesthetic into the tendon sheath. This injection can be very helpful from both a therapeutic standpoint and a diagnostic standpoint.[@bib6]
When conservative management fails, operative release of the iliopsoas tendon is an option for definitive management. Multiple open techniques have been described to release the iliopsoas tendon with good results.[@bib7], [@bib8] However, repeat open exposure of the hip after THA can be challenging for the surgeon, and temporarily debilitating and possibly harmful to the patient. Consideration of anatomic variations of the iliopsoas tendon is also critical during surgical release, because incomplete release may result in residual pain. In a previous anatomic study, the prevalence of a single-, double-, and triple-banded iliopsoas tendon was 28.3%, 64.2%, and 7.5%, respectively.[@bib9] Arthroscopic techniques to release the iliopsoas tendon have been described in the central compartment, peripheral compartment, and at the level of the lesser trochanter. Anatomic studies have demonstrated that at the level of the labrum (central compartment), the iliopsoas consists of 40% tendon and 60% muscle, compared with 60% tendon and 40% muscle at the level of the lesser trochanter.[@bib10] Because of this discrepancy, hypothetically a more thorough release could be performed at the level of the lesser trochanter.
The purpose of this Technical Note is to demonstrate a technique for arthroscopic release of the iliopsoas tendon at the lesser trochanter after THA.
Technique {#sec1}
=========
Preoperative Workup {#sec1.1}
-------------------
Radiographs of the affected hip should be obtained (anteroposterior and lateral) looking closely at femoral head size and position, screw prominence, anterior overhang of the acetabular component, and version of the acetabular component. Physical examination for suspected iliopsoas impingement should include an evaluation of passive and active range of motion of the hip, pain with resisted hip flexion, and significant pain with bringing the hip from neutral into hip flexion, abduction, and external rotation and then back to neutral.[@bib11] A diagnostic and therapeutic injection of the iliopsoas tendon sheath should be performed with local anesthetic and corticosteroid and the response monitored.[@bib12], [@bib13] The patient should be re-examined and questioned after injection to assess pain relief.
Operating Room Setup {#sec1.2}
--------------------
The patient is positioned supine on the surgeon\'s preferred hip arthroscopy table with traction boots applied to the bilateral legs. Large C-arm should be draped and accessible, coming in from the contralateral side, to localize the lesser trochanter ([Fig 1](#fig1){ref-type="fig"}). No peroneal post is required for this operation. Traction does not need to be applied. For a list of recommended equipment, see [Table 1](#tbl1){ref-type="table"}.Fig 1Demonstration of an operating room setup. Note that the patient is supine, in traction boots, with the right hip prepped and draped. Also, the large C-arm is draped and entering from the contralateral side. Note the neutral position of the ipsilateral foot.Table 1Recommended EquipmentEquipment • Smith and Nephew 4.5- and 5.0-mm access cannulas • Smith and Nephew spinal access needles • Nitinol guidewires • Smith and Nephew E-flex ablater • 70° arthroscope
Portal Placement {#sec1.3}
----------------
The greater trochanter should be palpated and drawn out along with the anterior superior iliac spine (ASIS). A line should be drawn from the ASIS toward the patella and a perpendicular line to this at the tip of the greater trochanter ([Fig 2](#fig2){ref-type="fig"}). The proximal portal is marked out just distal to the level of the greater trochanter and 2 to 3 cm lateral to the line drawn from the ASIS. The distal portal is in the same medial-lateral plane and approximately 5 cm distal to the proximal portal. Access needles (Smith & Nephew, Andover, MA) are used with the femur in neutral rotation to localize the lesser trochanter. Fluoroscopic imaging is used to access the iliopsoas tendon at the level of the lesser trochanter ([Fig 3](#fig3){ref-type="fig"}). Once the proper trajectory of the needle is confirmed with fluoroscopy and tactile feedback, a guidewire is placed through the access needle. The 5.0-mm access cannula is then twisted into the area overlying the lesser trochanter. A second portal is now created approximately 5 cm distal to the first portal using the access needle, then a guidewire, and finally the 4.5-mm access cannula.Fig 2Right hip, supine position, traction boots with no traction, neutral rotation of the operative leg. The greater trochanter and anterior superior iliac spine (ASIS) are palpated and marked. A line is drawn from the ASIS to top of the patella, with a perpendicular line at the level of the greater trochanter. The proximal portal is marked out just distal to the level of the greater trochanter and 2 to 3 cm lateral to the line drawn from the ASIS. The distal portal is in the same medial-lateral plane and approximately 5 cm distal to the proximal portal.Fig 3Right hip, supine position, traction boots with no traction, neutral rotation of the operative leg. Anterior posterior radiograph of the appropriate trajectory of Smith and Nephew access needles from both proximal and distal portals. Note that the leg is in neutral rotation at this time to prevent injury to the neurovascular structures.
Once both cannulas are in place safely beneath the neurovascular structures, the hip is flexed to 30° and the foot is externally rotated to bring the lesser trochanter into view ([Fig 4](#fig4){ref-type="fig"}). Some blunt dissection with the cannula is performed up and down to clear some of the soft tissues around the iliopsoas tendon. Blunt dissection in the medial-lateral plane should be avoided to minimize risk to the neurovascular structures.Fig 4Right hip, supine position, traction boots with no traction, external rotation of the operative leg. Once cannulas are placed and soft tissue planes have been established beneath the neurovascular structures, the hip can be flexed to 30° and maximally externally rotated bringing the lesser trochanter into view.
Psoas Tenotomy {#sec1.4}
--------------
A 70° arthroscope is then placed through the proximal 5.0-mm cannula and the pressure is set to 40 mm Hg. Before the initiation of inflow, the belly is palpated so a baseline of gross abdominal pressure is obtained. A 60° ablater (Smith & Nephew) is inserted through the distal 4.5-mm cannula and the iliopsoas bursa is identified and cleared off the tendon ([Fig 5](#fig5){ref-type="fig"}).Fig 5Right hip, supine position, traction boots with no traction, external rotation of the operative leg. Viewed from the proximal portal, shaver from the distal portal. The bursa overlying the iliopsoas tendon is removed to achieve adequate visualization of the tendon. Care is taken to shave toward the femur.
Once excellent visualization of the iliopsoas tendon is obtained, release of the iliopsoas tendon is performed while taking care to cut toward the femur ([Fig 6](#fig6){ref-type="fig"}). Previous anatomic studies have demonstrated that the average tendon diameter is 2.7 cm at the level of the lesser trochanter.[@bib10] Because of the trajectory of instrumentation and rounded nature of the lesser trochanter, the most posterior bands can be difficult to release with the rigid ArthroCare device. The Smith and Nephew E-flex device may facilitate complete release of the more posterior tendon.Fig 6Right hip, supine position, traction boots with no traction, external rotation of the operative leg. Viewed from the proximal portal, shaver from the distal portal. A 60° ablater (A) can be used to perform the tenotomy (B) taking care to cut toward the femur. Probe should be used to assess complete tenotomy and measure the distance retracted.
Hemostasis is achieved using the E-flex electrocautery or ArthroCare device and all fluid is evacuated using suction. Abdominal pressure is again assessed with palpation of the abdomen. If any significant increase in pressure is noted, then communication with anesthesia is critical to ensure that there are no hemodynamic signs of abdominal compartment syndrome. See [Table 2](#tbl2){ref-type="table"} for a list of technical pearls and pitfalls for this procedure.Table 2Pearls and PitfallsTechnical PearlsPitfalls• When creating and instrumenting portals keep the hip in neutral rotation, after soft tissue tracts are developed then externally rotate to minimize neurovascular risk\
• First portal (proximal) should be aimed approximately 1 cm above the lesser trochanter, and the second distal portal right at the top of the lesser trochanter\
• When portals are created, the leg is externally rotated and the hip flexed\
• Use the cannula to gently sweep away some of the bursal tissues for easier identification of the tendon\
• Use a cautery device and always cut toward the femur to avoid iatrogenic damage to the surrounding structure; if the most posterior area is difficult to access, use a flexible cautery\
• Assess belly pressures before turning on inflow and at the end of the case to monitor for abdominal compartment syndrome• While creating portals, blunt dissection in the medial-lateral plane with the cannula should be avoided to minimize risk to the neurovascular structures\
• Avoid external rotation of the femur before creating the proper soft tissue tracts to minimize risk to the neurovascular structures\
• If the posterior iliopsoas is left intact, symptoms can continue. Ensure entire release by maximal external rotation and use of a flexible cautery
Rehabilitation {#sec1.5}
--------------
Postoperatively, the patient is made weight bearing as tolerated, and range of motion as tolerated. Physical therapy is used for gait training and iliopsoas stretching to ensure minimal adhesion formation.
Please see the accompanying [Video 1](#appsec1){ref-type="sec"}.
Discussion {#sec2}
==========
Although more technically challenging compared with open releases, arthroscopic iliopsoas release has been shown to achieve improvement in patients\' symptoms after THA. Filanti et al.[@bib14] reported a case series of 35 patients with IPI after THA with improvement in Harris hip scores from 44.1 preoperatively, to 83.2 at 24 months\' follow-up from arthroscopic iliopsoas release. However, in this series of patients, the release was performed in the central compartment requiring access to the hip joint. Ilizaliturri et al.[@bib4] reported a randomized control trial of 19 patients who underwent iliopsoas release at either the level of the lesser trochanter, or through the peripheral compartment, and found no difference in patient outcomes, although patients in their study were not THA patients. There have been other case series that have reported improvement in symptoms after arthroscopic iliopsoas release in the central compartment after THA.[@bib15], [@bib16]
Concerns have been raised about an increased risk of complications when a release is performed at the level of the lesser trochanter due to its proximity to the femoral neurovascular structures. Gédouin and Huten[@bib17] reported a case series of 10 patients who underwent arthroscopic iliopsoas release at the lesser trochanter after THA. No complications were reported and 90% of patients were satisfied with their results. To minimize risk to neurovascular structures, all portals are created with the foot in relative neutral to slight internal rotation, which keeps the neurovascular bundle more medial. Once instruments are in place and the proper soft tissue tracts have been created safely, the foot is externally rotated for better visualization of the lesser trochanter and iliopsoas tendon. All portals are also started lateral to the level of the anterior superior iliac spine.
After release of the iliopsoas tendon, some postoperative weakness can be expected. Previous studies have demonstrated that with central compartment release the volume of the iliopsoas is reduced by 25% and seated hip flexion strength is decreased by 20%; however, supine hip flexion strength remains intact.[@bib18] Previous randomized control trials have noted no difference in return of hip flexion strength and patient reported outcomes in those with release of the iliopsoas at the level of the lesser trochanter compared with the central compartment.[@bib19] The patient can be expected to climb stairs without support approximately 8 weeks after release, regardless of the technique.[@bib19]
The advantages of performing a release at the level of the lesser trochanter are multiple. First, there is minimal disruption and scarring of the native anatomy at this level, because most surgeons do not formally expose the lesser trochanter for routine THA. Second, entrance into the hip joint is not required. Theoretically, this decreases the risk of periprosthetic joint infection and minimizes any possible iatrogenic instability from capsular penetration and release. Finally, there is a higher proportion of tendon as opposed to muscle at this level (60% tendon, 40% muscle), compared with the central compartment (40% tendon, 60% muscle), therefore allowing for a more complete release. Conversely, the central compartment may not require as much of a release because the central compartment is where the tendon is being impinged on in most cases, and so this release may be all that is required. There are possible disadvantages of this procedure: (1) it is less familiar to arthroscopists than traditional central and peripheral compartment arthroscopy, (2) a higher percentage of tendon is released, which theoretically could lead to more postoperative weakness, and (3) the risk of neurovascular compromise is theoretically higher. For a list of advantages and disadvantages of this technique of release, see [Table 3](#tbl3){ref-type="table"}.Table 3Advantages and Disadvantages for Release Off the Lesser TrochanterAdvantagesDisadvantages• More complete release. At the level of the lesser trochanter, the iliopsoas is 60% tendon, compared with 40% in the central compartment. Release of a larger portion of the tendon will allow more lengthening of the tendon and result in a lower chance of continued tendinitis• Approach is less familiar to hip arthroscopists as most lengthen the psoas in non-THA patients in the central or peripheral compartment• Surgically unaltered area, minimal scar tissue, so tendon identification and lengthening is simple• Instrumentation heading toward the neurovascular bundle, which if unfamiliar may lead to damage• No disruption of static stabilizers to the hip joint, which may decrease the risk of subsequent hip instability• More complete release, theoretically will lead to more postoperative weakness• Does not violate the hip joint, which may decrease the risk of subsequent hip joint infection due to direct inoculation[^1]
Arthroscopic release of the iliopsoas tendon at the lesser trochanter is a safe, reproducible procedure that provides advantages compared with a release at the central compartment.
Supplementary Data {#appsec1}
==================
Video 1Step-by-step guide to performing arthroscopic release of the iliopsoas tendon at the level of the lesser trochanter. Patient characteristics: Right hip, supine position, traction boots with no traction, external rotation of the operative leg.ICMJE Author Disclosure Forms
The authors report that they have no conflicts of interest in the authorship and publication of this article. Full ICMJE author disclosure forms are available for this article online, as [supplementary material](#appsec1){ref-type="sec"}.
[^1]: THA, total hip arthroplasty.
| {
"pile_set_name": "PubMed Central"
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Related literature {#sec1}
==================
For applications of the 3,5-dichlorosalicylaldehydate ligand in the preparation of Schiff base--metal complexes, see: Akitsu *et al.* (2009[@bb7]); Akitsu & Einaga (2005*a* [@bb4],*b* [@bb5]); Akitsu (2007[@bb1]). For *trans* and *cis* forms of complexes, see: Akitsu & Einaga (2004*a* [@bb2],*b* [@bb3]); Akitsu *et al.* (2005[@bb6]). For related complexes, see: Chen (2006[@bb9]); Chen *et al.* (2007[@bb10]); Xiong & Liu (2005[@bb14]).
Experimental {#sec2}
============
{#sec2.1}
### Crystal data {#sec2.1.1}
\[Zn(C~7~H~3~Cl~2~O~2~)~2~(H~2~O)~2~\]~2~·\[Zn~2~(C~7~H~3~Cl~2~O~2~)~4~(H~2~O)~2~\]*M* *~r~* = 1889.61Triclinic,*a* = 8.7532 (9) Å*b* = 13.6973 (15) Å*c* = 14.2833 (15) Åα = 96.244 (2)°β = 91.700 (1)°γ = 106.096 (1)°*V* = 1632.4 (3) Å^3^*Z* = 1Mo *K*α radiationμ = 2.19 mm^−1^*T* = 100 K0.15 × 0.15 × 0.08 mm
### Data collection {#sec2.1.2}
Bruker APEXII CCD area-detector diffractometerAbsorption correction: multi-scan (*SADABS*; Sheldrick, 1996[@bb12]) *T* ~min~ = 0.735, *T* ~max~ = 0.8459504 measured reflections7275 independent reflections5671 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.021
### Refinement {#sec2.1.3}
*R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.038*wR*(*F* ^2^) = 0.118*S* = 0.757275 reflections445 parametersH-atom parameters constrainedΔρ~max~ = 0.59 e Å^−3^Δρ~min~ = −0.46 e Å^−3^
{#d5e731}
Data collection: *APEX2* (Bruker, 2005[@bb8]); cell refinement: *SAINT* (Bruker, 2005[@bb8]); data reduction: *SAINT*; program(s) used to solve structure: *SHELXS97* (Sheldrick, 2008[@bb13]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008[@bb13]); molecular graphics: *ORTEP-3 for Windows* (Farrugia, 1997[@bb11]); software used to prepare material for publication: *SHELXL97*.
Supplementary Material
======================
Crystal structure: contains datablocks global, I. DOI: [10.1107/S160053680904896X/xu2645sup1.cif](http://dx.doi.org/10.1107/S160053680904896X/xu2645sup1.cif)
Structure factors: contains datablocks I. DOI: [10.1107/S160053680904896X/xu2645Isup2.hkl](http://dx.doi.org/10.1107/S160053680904896X/xu2645Isup2.hkl)
Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?xu2645&file=xu2645sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?xu2645sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?xu2645&checkcif=yes)
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [XU2645](http://scripts.iucr.org/cgi-bin/sendsup?xu2645)).
This work was supported by the Kato Foundation for the Promotion of Science.
Comment
=======
3,5-Dichlorosalcylaldehydato plays an important role in preparation of Schiff base metal complexes because of electronic properties due to Cl-groups for example supramolecular interactions between metallodendrimers (Akitsu *et al.*, 2009) or photochromic compounds (Akitsu & Einaga, 2005*a* & 2005*b*; Akitsu, 2007). Depending on amine reagents and their steric requirement, *trans* (Akitsu & Einaga, 2004*a* & 2004*b*) or *cis* (Akitsu *et al.*, 2005) forms of complexes can be formed. However, we focused on only 3,5-dichloroaldehyde moiety to elucidate structural features without amine moiety.
The title compound (I) is composed of a co-crystal of monomeric \[Zn(C~7~H~3~Cl~2~O~2~)~2~(H~2~O)~2~\] and dimeric \[Zn(C~7~H~3~Cl~2~O~2~)~2~(H~2~O)\]. Both complexes afford a six-coordinated coordination environment exhibiting significant distortion. In contrast to known zinc(II) complexes incorporating saldehyde-derivertive ligands (Chen, 2006; Chen *et al.*, 2007; Xiong & Liu, 2005), both ligands bind to Zn(II) ions in a *cis*-configuration for (I).
Experimental {#experimental}
============
Crystals were obtained accidentally as a byproduct of the treatment of 3,5-dichlorosalcylaldehyde (0.95 g, 5.00 mmol) in methanol (30 ml), *L*-alanine (0.44 g, 5.00 mmol) in water (5 ml), zinc(II) acetate dihydrate (0.55 g, 2.50 mmol) and several drops of triethylamine at c.a. 350 K for 2 hr.
Refinement {#refinement}
==========
Water-H atoms were located based on D-map and refined in riding mode. Other H atoms were placed at the calculated positions with C---H = 0.95 Å and refined in riding mode. U~iso~(H) = 1.2U~eq~(O,C).
Figures
=======
![The molecular structure of (I), showing the atom labeling scheme \[symmetry code: (i) 2 - x, 1 - y, 2 - z\]. Displacement ellipsoids for non-H atoms are drawn at the 50% probability level.](e-65-m1640-fig1){#Fap1}
Crystal data {#tablewrapcrystaldatalong}
============
------------------------------------------------------------------------------------- ---------------------------------------
\[Zn(C~7~H~3~Cl~2~O~2~)~2~(H~2~O)~2~\]~2~·\[Zn~2~(C~7~H~3~Cl~2~O~2~)~4~(H~2~O)~2~\] *Z* = 1
*M~r~* = 1889.61 *F*(000) = 940.0
Triclinic, *P*1 *D*~x~ = 1.922 Mg m^−3^
Hall symbol: -P 1 Mo *K*α radiation, λ = 0.71073 Å
*a* = 8.7532 (9) Å Cell parameters from 2792 reflections
*b* = 13.6973 (15) Å θ = 2.4--27.8°
*c* = 14.2833 (15) Å µ = 2.19 mm^−1^
α = 96.244 (2)° *T* = 100 K
β = 91.700 (1)° Prismatic, yellow
γ = 106.096 (1)° 0.15 × 0.15 × 0.08 mm
*V* = 1632.4 (3) Å^3^
------------------------------------------------------------------------------------- ---------------------------------------
Data collection {#tablewrapdatacollectionlong}
===============
--------------------------------------------------------------- --------------------------------------
Bruker APEXII CCD area-detector diffractometer 7275 independent reflections
Radiation source: fine-focus sealed tube 5671 reflections with *I* \> 2σ(*I*)
graphite *R*~int~ = 0.021
Detector resolution: 8.333 pixels mm^-1^ θ~max~ = 27.9°, θ~min~ = 1.4°
φ and ω scans *h* = −11→11
Absorption correction: multi-scan (*SADABS*; Sheldrick, 1996) *k* = −16→17
*T*~min~ = 0.735, *T*~max~ = 0.845 *l* = −12→18
9504 measured reflections
--------------------------------------------------------------- --------------------------------------
Refinement {#tablewraprefinementdatalong}
==========
------------------------------------- ----------------------------------------------------------------------------------------------
Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
*R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.038 Hydrogen site location: inferred from neighbouring sites
*wR*(*F*^2^) = 0.118 H-atom parameters constrained
*S* = 0.75 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.1*P*)^2^ + 1.2111*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3
7275 reflections (Δ/σ)~max~ = 0.001
445 parameters Δρ~max~ = 0.59 e Å^−3^
0 restraints Δρ~min~ = −0.46 e Å^−3^
------------------------------------- ----------------------------------------------------------------------------------------------
Special details {#specialdetails}
===============
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Experimental. Refinement of F^2^ against ALL reflections. The weighted *R*-factor *wR* and goodness of fit S are based on F^2^, conventional *R*-factors *R* are based on F, with F set to zero for negative F^2^. The threshold expression of F^2^ \> 2sigma(F^2^) is used only for calculating *R*-factors(gt) *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on F^2^ are statistically about twice as large as those based on F, and R-- factors based on ALL data will be even larger. The water-H atoms wre located in a D-map and refined in riding mode with *U*~iso~(H) = 1.2*U*~eq~(O).\"
Geometry. All e.s.d.\'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.\'s are taken into account individually in the estimation of e.s.d.\'s in distances, angles and torsion angles; correlations between e.s.d.\'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.\'s is used for estimating e.s.d.\'s involving l.s. planes.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords}
==================================================================================================
------ -------------- -------------- -------------- -------------------- --
*x* *y* *z* *U*~iso~\*/*U*~eq~
Zn1 0.80461 (4) 0.83299 (3) 0.96067 (3) 0.00981 (11)
Zn2 0.92258 (4) 0.39423 (3) 0.93218 (3) 0.00861 (10)
C1 1.0506 (4) 0.8558 (2) 0.8153 (2) 0.0115 (7)
C2 1.2112 (4) 0.8851 (2) 0.7897 (2) 0.0106 (7)
C3 1.2547 (4) 0.8968 (2) 0.6987 (2) 0.0114 (7)
H3 1.3640 0.9165 0.6855 0.014\*
C4 1.1363 (4) 0.8794 (3) 0.6263 (2) 0.0123 (7)
C5 0.9784 (4) 0.8516 (3) 0.6459 (2) 0.0144 (7)
H5 0.8984 0.8400 0.5963 0.017\*
C6 0.9340 (4) 0.8400 (2) 0.7388 (2) 0.0109 (7)
C7 0.7636 (4) 0.8084 (2) 0.7507 (2) 0.0122 (7)
H7 0.6966 0.7972 0.6946 0.015\*
C8 0.8264 (4) 0.8649 (2) 1.1745 (2) 0.0089 (6)
C9 0.9119 (4) 0.8798 (2) 1.2634 (2) 0.0111 (7)
C10 0.8403 (4) 0.8716 (3) 1.3478 (2) 0.0152 (7)
H10C 0.9029 0.8815 1.4053 0.018\*
C11 0.6749 (4) 0.8485 (3) 1.3479 (2) 0.0140 (7)
C12 0.5844 (4) 0.8358 (3) 1.2644 (2) 0.0134 (7)
H12 0.4719 0.8215 1.2653 0.016\*
C13 0.6573 (4) 0.8437 (2) 1.1784 (2) 0.0105 (6)
C14 0.5495 (4) 0.8274 (2) 1.0955 (2) 0.0130 (7)
H14 0.4403 0.8178 1.1070 0.016\*
C15 1.1453 (4) 0.3785 (2) 0.7795 (2) 0.0105 (6)
C16 1.3048 (4) 0.3981 (2) 0.7511 (2) 0.0107 (7)
C17 1.3436 (4) 0.4026 (2) 0.6585 (2) 0.0119 (7)
H17 1.4517 0.4171 0.6429 0.014\*
C18 1.2213 (4) 0.3855 (3) 0.5875 (2) 0.0127 (7)
C19 1.0649 (4) 0.3623 (3) 0.6098 (2) 0.0133 (7)
H19 0.9827 0.3478 0.5611 0.016\*
C20 1.0254 (4) 0.3598 (2) 0.7050 (2) 0.0109 (6)
C21 0.8572 (4) 0.3365 (3) 0.7224 (2) 0.0130 (7)
H21 0.7847 0.3167 0.6683 0.016\*
C22 0.9237 (4) 0.4081 (2) 1.1431 (2) 0.0089 (6)
C23 0.9985 (4) 0.4002 (2) 1.2300 (2) 0.0103 (6)
C24 0.9167 (4) 0.3743 (2) 1.3089 (2) 0.0125 (7)
H24 0.9724 0.3710 1.3660 0.015\*
C25 0.7501 (4) 0.3529 (3) 1.3036 (2) 0.0120 (7)
C26 0.6708 (4) 0.3553 (2) 1.2201 (2) 0.0125 (7)
H26 0.5578 0.3389 1.2166 0.015\*
C27 0.7539 (4) 0.3816 (2) 1.1396 (2) 0.0097 (6)
C28 0.6570 (4) 0.3854 (2) 1.0564 (2) 0.0100 (6)
H28 0.5461 0.3742 1.0636 0.012\*
O1 1.0177 (3) 0.84419 (18) 0.90218 (16) 0.0114 (5)
O2 0.6955 (3) 0.79453 (18) 0.82386 (17) 0.0129 (5)
O3 0.9021 (3) 0.87169 (18) 1.09647 (16) 0.0120 (5)
O4 0.5816 (3) 0.82436 (18) 1.01226 (16) 0.0128 (5)
O5 1.1173 (3) 0.37770 (17) 0.86854 (16) 0.0107 (5)
O6 0.7986 (3) 0.33961 (18) 0.79914 (16) 0.0118 (5)
O7 1.0093 (3) 0.44105 (17) 1.07188 (16) 0.0090 (5)
O8 0.7034 (3) 0.40163 (17) 0.97769 (16) 0.0116 (5)
Cl1 1.36080 (9) 0.90539 (6) 0.87857 (6) 0.01455 (18)
Cl2 1.19192 (11) 0.89319 (7) 0.51136 (6) 0.0199 (2)
Cl3 1.11849 (9) 0.90686 (6) 1.26347 (6) 0.01406 (18)
Cl4 0.58270 (11) 0.83281 (7) 1.45386 (6) 0.0221 (2)
Cl5 1.45564 (9) 0.41888 (6) 0.83825 (6) 0.01241 (17)
Cl6 1.27119 (11) 0.39485 (7) 0.47120 (6) 0.0197 (2)
Cl7 1.20508 (9) 0.43012 (6) 1.23861 (6) 0.01256 (17)
Cl8 0.64683 (11) 0.32313 (7) 1.40347 (6) 0.0193 (2)
O9 0.8187 (3) 0.98658 (18) 0.94441 (17) 0.0133 (5)
H9A 0.9028 1.0128 0.9186 0.016\*
H9B 0.8160 1.0363 0.9696 0.016\*
O10 0.7513 (3) 0.67389 (17) 0.97417 (17) 0.0118 (5)
H10A 0.8109 0.6479 0.9410 0.014\*
H10B 0.7730 0.6619 1.0275 0.014\*
O11 0.8612 (3) 0.23683 (17) 0.95443 (17) 0.0120 (5)
H11A 0.9147 0.2065 0.9205 0.014\*
H11B 0.8921 0.2170 1.0031 0.014\*
------ -------------- -------------- -------------- -------------------- --
Atomic displacement parameters (Å^2^) {#tablewrapadps}
=====================================
----- -------------- ------------- -------------- -------------- --------------- --------------
*U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^
Zn1 0.00838 (19) 0.0114 (2) 0.0092 (2) 0.00159 (15) −0.00065 (14) 0.00263 (14)
Zn2 0.00720 (19) 0.0103 (2) 0.00819 (19) 0.00178 (14) −0.00008 (14) 0.00230 (14)
C1 0.0112 (16) 0.0069 (15) 0.0166 (17) 0.0020 (12) 0.0003 (13) 0.0044 (12)
C2 0.0133 (16) 0.0074 (15) 0.0109 (16) 0.0028 (12) −0.0022 (13) 0.0019 (12)
C3 0.0106 (15) 0.0103 (16) 0.0142 (17) 0.0030 (13) 0.0036 (13) 0.0046 (12)
C4 0.0169 (17) 0.0127 (16) 0.0072 (15) 0.0031 (13) 0.0053 (13) 0.0025 (12)
C5 0.0153 (17) 0.0101 (16) 0.0164 (18) 0.0016 (13) −0.0022 (14) 0.0021 (13)
C6 0.0109 (16) 0.0094 (16) 0.0104 (16) −0.0005 (13) 0.0010 (12) 0.0019 (12)
C7 0.0117 (16) 0.0118 (16) 0.0123 (16) 0.0022 (13) −0.0021 (13) 0.0021 (12)
C8 0.0136 (16) 0.0064 (15) 0.0076 (15) 0.0044 (12) 0.0010 (12) 0.0007 (11)
C9 0.0113 (16) 0.0074 (15) 0.0152 (17) 0.0033 (12) 0.0016 (13) 0.0029 (12)
C10 0.0176 (18) 0.0165 (18) 0.0106 (17) 0.0037 (14) −0.0013 (13) 0.0011 (13)
C11 0.0158 (17) 0.0128 (17) 0.0137 (17) 0.0034 (14) 0.0057 (13) 0.0027 (13)
C12 0.0098 (16) 0.0130 (17) 0.0156 (17) −0.0006 (13) 0.0025 (13) 0.0039 (13)
C13 0.0110 (16) 0.0089 (15) 0.0120 (16) 0.0029 (12) −0.0001 (13) 0.0024 (12)
C14 0.0080 (15) 0.0112 (16) 0.0184 (18) 0.0001 (13) −0.0010 (13) 0.0029 (13)
C15 0.0124 (16) 0.0087 (15) 0.0097 (16) 0.0018 (13) −0.0003 (13) 0.0012 (12)
C16 0.0103 (15) 0.0084 (15) 0.0108 (16) −0.0012 (12) −0.0027 (12) 0.0009 (12)
C17 0.0084 (15) 0.0118 (16) 0.0134 (17) 0.0006 (13) 0.0002 (13) −0.0007 (12)
C18 0.0176 (17) 0.0127 (16) 0.0075 (15) 0.0033 (13) 0.0047 (13) 0.0013 (12)
C19 0.0111 (16) 0.0173 (18) 0.0113 (16) 0.0044 (14) −0.0025 (13) 0.0017 (13)
C20 0.0104 (15) 0.0113 (16) 0.0128 (16) 0.0051 (13) 0.0028 (13) 0.0025 (12)
C21 0.0097 (16) 0.0137 (17) 0.0138 (17) 0.0009 (13) −0.0028 (13) 0.0015 (13)
C22 0.0107 (15) 0.0043 (14) 0.0090 (15) −0.0026 (12) 0.0003 (12) 0.0017 (11)
C23 0.0091 (15) 0.0090 (15) 0.0111 (16) −0.0005 (12) −0.0031 (12) 0.0023 (12)
C24 0.0157 (17) 0.0088 (16) 0.0130 (17) 0.0030 (13) −0.0010 (13) 0.0031 (12)
C25 0.0136 (16) 0.0101 (16) 0.0118 (16) 0.0007 (13) 0.0044 (13) 0.0052 (12)
C26 0.0092 (15) 0.0098 (16) 0.0174 (17) 0.0014 (13) 0.0033 (13) −0.0003 (13)
C27 0.0103 (15) 0.0061 (15) 0.0103 (16) −0.0009 (12) −0.0013 (12) −0.0006 (12)
C28 0.0057 (14) 0.0124 (16) 0.0100 (16) 0.0006 (12) 0.0007 (12) −0.0014 (12)
O1 0.0089 (11) 0.0191 (13) 0.0080 (11) 0.0054 (9) 0.0018 (9) 0.0048 (9)
O2 0.0097 (11) 0.0143 (12) 0.0140 (12) 0.0018 (9) 0.0000 (9) 0.0032 (9)
O3 0.0105 (11) 0.0133 (12) 0.0108 (12) 0.0006 (9) 0.0001 (9) 0.0030 (9)
O4 0.0106 (11) 0.0154 (12) 0.0124 (12) 0.0039 (10) −0.0012 (9) 0.0015 (9)
O5 0.0103 (11) 0.0127 (12) 0.0103 (11) 0.0039 (9) 0.0001 (9) 0.0045 (9)
O6 0.0068 (11) 0.0156 (12) 0.0123 (12) 0.0012 (9) −0.0003 (9) 0.0036 (9)
O7 0.0069 (10) 0.0107 (11) 0.0083 (11) 0.0006 (9) 0.0000 (9) 0.0023 (9)
O8 0.0091 (11) 0.0133 (12) 0.0129 (12) 0.0038 (9) −0.0004 (9) 0.0018 (9)
Cl1 0.0096 (4) 0.0199 (4) 0.0140 (4) 0.0040 (3) −0.0021 (3) 0.0026 (3)
Cl2 0.0194 (4) 0.0286 (5) 0.0104 (4) 0.0033 (4) 0.0041 (3) 0.0053 (3)
Cl3 0.0105 (4) 0.0191 (4) 0.0124 (4) 0.0037 (3) −0.0011 (3) 0.0028 (3)
Cl4 0.0225 (5) 0.0287 (5) 0.0146 (4) 0.0045 (4) 0.0097 (3) 0.0060 (4)
Cl5 0.0082 (4) 0.0159 (4) 0.0121 (4) 0.0018 (3) −0.0015 (3) 0.0022 (3)
Cl6 0.0197 (4) 0.0279 (5) 0.0089 (4) 0.0024 (4) 0.0032 (3) 0.0020 (3)
Cl7 0.0089 (4) 0.0177 (4) 0.0105 (4) 0.0020 (3) −0.0008 (3) 0.0043 (3)
Cl8 0.0196 (4) 0.0249 (5) 0.0147 (4) 0.0052 (4) 0.0092 (3) 0.0084 (3)
O9 0.0118 (11) 0.0118 (12) 0.0164 (13) 0.0028 (10) 0.0007 (9) 0.0038 (9)
O10 0.0104 (11) 0.0145 (12) 0.0112 (12) 0.0046 (9) −0.0006 (9) 0.0026 (9)
O11 0.0118 (11) 0.0128 (12) 0.0133 (12) 0.0057 (9) 0.0012 (9) 0.0042 (9)
----- -------------- ------------- -------------- -------------- --------------- --------------
Geometric parameters (Å, °) {#tablewrapgeomlong}
===========================
----------------------- ------------- --------------------------- -------------
Zn1---O1 2.040 (2) C14---O4 1.229 (4)
Zn1---O2 2.096 (2) C14---H14 0.9500
Zn1---O3 2.049 (2) C15---O5 1.303 (4)
Zn1---O4 2.084 (2) C15---C20 1.422 (4)
Zn1---O9 2.112 (2) C15---C16 1.426 (5)
Zn1---O10 2.130 (2) C16---C17 1.379 (5)
Zn2---O5 2.011 (2) C16---Cl5 1.730 (3)
Zn2---O6 2.114 (2) C17---C18 1.405 (4)
Zn2---O7 2.081 (2) C17---H17 0.9500
Zn2---O7^i^ 2.176 (2) C18---C19 1.373 (5)
Zn2---O8 2.069 (2) C18---Cl6 1.739 (3)
Zn2---O11 2.134 (2) C19---C20 1.415 (5)
C1---O1 1.298 (4) C19---H19 0.9500
C1---C2 1.422 (5) C20---C21 1.452 (5)
C1---C6 1.429 (4) C21---O6 1.226 (4)
C2---C3 1.378 (5) C21---H21 0.9500
C2---Cl1 1.739 (3) C22---O7 1.327 (4)
C3---C4 1.395 (5) C22---C23 1.415 (4)
C3---H3 0.9500 C22---C27 1.427 (4)
C4---C5 1.374 (5) C23---C24 1.378 (5)
C4---Cl2 1.741 (3) C23---Cl7 1.737 (3)
C5---C6 1.407 (5) C24---C25 1.404 (5)
C5---H5 0.9500 C24---H24 0.9500
C6---C7 1.454 (5) C25---C26 1.370 (5)
C7---O2 1.225 (4) C25---Cl8 1.736 (3)
C7---H7 0.9500 C26---C27 1.406 (5)
C8---O3 1.313 (4) C26---H26 0.9500
C8---C9 1.419 (4) C27---C28 1.453 (4)
C8---C13 1.431 (4) C28---O8 1.228 (4)
C9---C10 1.376 (5) C28---H28 0.9500
C9---Cl3 1.743 (3) O7---Zn2^i^ 2.176 (2)
C10---C11 1.393 (5) O9---H9A 0.8400
C10---H10C 0.9500 O9---H9B 0.7416
C11---C12 1.379 (5) O10---H10A 0.8400
C11---Cl4 1.743 (4) O10---H10B 0.8234
C12---C13 1.403 (5) O11---H11A 0.8400
C12---H12 0.9500 O11---H11B 0.8371
C13---C14 1.449 (4)
O1---Zn1---O3 94.50 (9) C12---C13---C14 115.3 (3)
O1---Zn1---O4 176.22 (8) C8---C13---C14 123.4 (3)
O3---Zn1---O4 88.77 (9) O4---C14---C13 128.2 (3)
O1---Zn1---O2 87.95 (9) O4---C14---H14 115.9
O3---Zn1---O2 177.47 (10) C13---C14---H14 115.9
O4---Zn1---O2 88.76 (9) O5---C15---C20 124.4 (3)
O1---Zn1---O9 91.79 (10) O5---C15---C16 120.1 (3)
O3---Zn1---O9 92.35 (9) C20---C15---C16 115.5 (3)
O4---Zn1---O9 86.17 (9) C17---C16---C15 123.2 (3)
O2---Zn1---O9 86.93 (9) C17---C16---Cl5 119.1 (3)
O1---Zn1---O10 95.85 (9) C15---C16---Cl5 117.7 (2)
O3---Zn1---O10 91.64 (9) C16---C17---C18 119.2 (3)
O4---Zn1---O10 85.94 (9) C16---C17---H17 120.4
O2---Zn1---O10 88.74 (9) C18---C17---H17 120.4
O9---Zn1---O10 171.08 (9) C19---C18---C17 120.4 (3)
O5---Zn2---O8 171.11 (9) C19---C18---Cl6 120.6 (3)
O5---Zn2---O7 102.25 (9) C17---C18---Cl6 119.0 (3)
O8---Zn2---O7 86.59 (9) C18---C19---C20 120.3 (3)
O5---Zn2---O6 86.25 (9) C18---C19---H19 119.8
O8---Zn2---O6 84.87 (9) C20---C19---H19 119.8
O7---Zn2---O6 170.91 (9) C19---C20---C15 121.2 (3)
O5---Zn2---O11 89.56 (9) C19---C20---C21 116.7 (3)
O8---Zn2---O11 89.17 (9) C15---C20---C21 122.1 (3)
O7---Zn2---O11 91.76 (9) O6---C21---C20 127.1 (3)
O6---Zn2---O11 85.01 (9) O6---C21---H21 116.4
O5---Zn2---O7^i^ 93.02 (9) C20---C21---H21 116.4
O8---Zn2---O7^i^ 89.27 (9) O7---C22---C23 120.8 (3)
O7---Zn2---O7^i^ 81.32 (9) O7---C22---C27 123.6 (3)
O6---Zn2---O7^i^ 101.65 (8) C23---C22---C27 115.6 (3)
O11---Zn2---O7^i^ 172.99 (9) C24---C23---C22 123.5 (3)
O1---C1---C2 120.8 (3) C24---C23---Cl7 118.6 (2)
O1---C1---C6 124.5 (3) C22---C23---Cl7 117.9 (3)
C2---C1---C6 114.7 (3) C23---C24---C25 119.2 (3)
C3---C2---C1 123.8 (3) C23---C24---H24 120.4
C3---C2---Cl1 118.4 (3) C25---C24---H24 120.4
C1---C2---Cl1 117.7 (2) C26---C25---C24 119.9 (3)
C2---C3---C4 119.3 (3) C26---C25---Cl8 120.8 (3)
C2---C3---H3 120.4 C24---C25---Cl8 119.4 (3)
C4---C3---H3 120.4 C25---C26---C27 121.1 (3)
C5---C4---C3 120.1 (3) C25---C26---H26 119.4
C5---C4---Cl2 120.9 (3) C27---C26---H26 119.4
C3---C4---Cl2 119.0 (3) C26---C27---C22 120.7 (3)
C4---C5---C6 120.7 (3) C26---C27---C28 115.9 (3)
C4---C5---H5 119.7 C22---C27---C28 123.3 (3)
C6---C5---H5 119.7 O8---C28---C27 126.8 (3)
C5---C6---C1 121.4 (3) O8---C28---H28 116.6
C5---C6---C7 115.7 (3) C27---C28---H28 116.6
C1---C6---C7 122.9 (3) C1---O1---Zn1 127.5 (2)
O2---C7---C6 128.1 (3) C7---O2---Zn1 125.6 (2)
O2---C7---H7 116.0 C8---O3---Zn1 127.4 (2)
C6---C7---H7 116.0 C14---O4---Zn1 126.4 (2)
O3---C8---C9 120.5 (3) C15---O5---Zn2 128.2 (2)
O3---C8---C13 124.5 (3) C21---O6---Zn2 126.7 (2)
C9---C8---C13 115.0 (3) C22---O7---Zn2 121.66 (19)
C10---C9---C8 123.7 (3) C22---O7---Zn2^i^ 115.7 (2)
C10---C9---Cl3 119.1 (3) Zn2---O7---Zn2^i^ 98.68 (9)
C8---C9---Cl3 117.2 (3) C28---O8---Zn2 124.9 (2)
C9---C10---C11 119.2 (3) Zn1---O9---H9A 109.5
C9---C10---H10C 120.4 Zn1---O9---H9B 144.2
C11---C10---H10C 120.4 H9A---O9---H9B 93.4
C12---C11---C10 120.3 (3) Zn1---O10---H10A 109.5
C12---C11---Cl4 120.1 (3) Zn1---O10---H10B 113.6
C10---C11---Cl4 119.6 (3) H10A---O10---H10B 102.5
C11---C12---C13 120.4 (3) Zn2---O11---H11A 109.5
C11---C12---H12 119.8 Zn2---O11---H11B 122.7
C13---C12---H12 119.8 H11A---O11---H11B 91.6
C12---C13---C8 121.3 (3)
O1---C1---C2---C3 178.7 (3) C23---C24---C25---Cl8 −178.5 (3)
C6---C1---C2---C3 −0.8 (5) C24---C25---C26---C27 −1.7 (5)
O1---C1---C2---Cl1 −0.3 (4) Cl8---C25---C26---C27 178.5 (3)
C6---C1---C2---Cl1 −179.8 (2) C25---C26---C27---C22 −1.0 (5)
C1---C2---C3---C4 0.1 (5) C25---C26---C27---C28 −177.6 (3)
Cl1---C2---C3---C4 179.0 (3) O7---C22---C27---C26 −174.7 (3)
C2---C3---C4---C5 0.4 (5) C23---C22---C27---C26 3.4 (5)
C2---C3---C4---Cl2 −179.4 (3) O7---C22---C27---C28 1.7 (5)
C3---C4---C5---C6 −0.2 (5) C23---C22---C27---C28 179.8 (3)
Cl2---C4---C5---C6 179.7 (3) C26---C27---C28---O8 −176.2 (3)
C4---C5---C6---C1 −0.6 (5) C22---C27---C28---O8 7.3 (5)
C4---C5---C6---C7 −178.9 (3) C2---C1---O1---Zn1 165.8 (2)
O1---C1---C6---C5 −178.4 (3) C6---C1---O1---Zn1 −14.8 (5)
C2---C1---C6---C5 1.0 (5) O3---Zn1---O1---C1 −160.0 (3)
O1---C1---C6---C7 −0.3 (5) O2---Zn1---O1---C1 19.3 (3)
C2---C1---C6---C7 179.2 (3) O9---Zn1---O1---C1 −67.5 (3)
C5---C6---C7---O2 −179.0 (3) O10---Zn1---O1---C1 107.9 (3)
C1---C6---C7---O2 2.7 (6) C6---C7---O2---Zn1 9.7 (5)
O3---C8---C9---C10 −178.5 (3) O1---Zn1---O2---C7 −16.7 (3)
C13---C8---C9---C10 2.1 (5) O4---Zn1---O2---C7 161.5 (3)
O3---C8---C9---Cl3 −0.1 (4) O9---Zn1---O2---C7 75.2 (3)
C13---C8---C9---Cl3 −179.5 (2) O10---Zn1---O2---C7 −112.6 (3)
C8---C9---C10---C11 −0.7 (5) C9---C8---O3---Zn1 170.2 (2)
Cl3---C9---C10---C11 −179.1 (3) C13---C8---O3---Zn1 −10.4 (4)
C9---C10---C11---C12 −1.1 (5) O1---Zn1---O3---C8 −169.0 (3)
C9---C10---C11---Cl4 177.4 (3) O4---Zn1---O3---C8 12.9 (3)
C10---C11---C12---C13 1.4 (5) O9---Zn1---O3---C8 99.0 (3)
Cl4---C11---C12---C13 −177.1 (3) O10---Zn1---O3---C8 −73.0 (3)
C11---C12---C13---C8 0.2 (5) C13---C14---O4---Zn1 4.1 (5)
C11---C12---C13---C14 179.1 (3) O3---Zn1---O4---C14 −9.8 (3)
O3---C8---C13---C12 178.8 (3) O2---Zn1---O4---C14 170.7 (3)
C9---C8---C13---C12 −1.8 (4) O9---Zn1---O4---C14 −102.2 (3)
O3---C8---C13---C14 −0.1 (5) O10---Zn1---O4---C14 81.9 (3)
C9---C8---C13---C14 179.3 (3) C20---C15---O5---Zn2 −20.6 (5)
C12---C13---C14---O4 −175.5 (3) C16---C15---O5---Zn2 160.1 (2)
C8---C13---C14---O4 3.4 (5) O7---Zn2---O5---C15 −158.3 (3)
O5---C15---C16---C17 −178.4 (3) O6---Zn2---O5---C15 24.9 (3)
C20---C15---C16---C17 2.2 (5) O11---Zn2---O5---C15 110.0 (3)
O5---C15---C16---Cl5 0.7 (4) O7^i^---Zn2---O5---C15 −76.5 (3)
C20---C15---C16---Cl5 −178.7 (2) C20---C21---O6---Zn2 6.7 (5)
C15---C16---C17---C18 −1.1 (5) O5---Zn2---O6---C21 −18.1 (3)
Cl5---C16---C17---C18 179.8 (3) O8---Zn2---O6---C21 162.4 (3)
C16---C17---C18---C19 −1.5 (5) O11---Zn2---O6---C21 −108.0 (3)
C16---C17---C18---Cl6 178.2 (3) O7^i^---Zn2---O6---C21 74.2 (3)
C17---C18---C19---C20 2.8 (5) C23---C22---O7---Zn2 151.4 (2)
Cl6---C18---C19---C20 −176.8 (3) C27---C22---O7---Zn2 −30.6 (4)
C18---C19---C20---C15 −1.6 (5) C23---C22---O7---Zn2^i^ −89.0 (3)
C18---C19---C20---C21 178.8 (3) C27---C22---O7---Zn2^i^ 89.0 (3)
O5---C15---C20---C19 179.8 (3) O5---Zn2---O7---C22 −141.2 (2)
C16---C15---C20---C19 −0.8 (5) O8---Zn2---O7---C22 37.8 (2)
O5---C15---C20---C21 −0.7 (5) O11---Zn2---O7---C22 −51.2 (2)
C16---C15---C20---C21 178.7 (3) O7^i^---Zn2---O7---C22 127.6 (3)
C19---C20---C21---O6 −172.9 (3) O5---Zn2---O7---Zn2^i^ 91.22 (10)
C15---C20---C21---O6 7.6 (5) O8---Zn2---O7---Zn2^i^ −89.78 (9)
O7---C22---C23---C24 174.7 (3) O11---Zn2---O7---Zn2^i^ −178.85 (9)
C27---C22---C23---C24 −3.4 (5) O7^i^---Zn2---O7---Zn2^i^ 0.0
O7---C22---C23---Cl7 −2.5 (4) C27---C28---O8---Zn2 15.4 (5)
C27---C22---C23---Cl7 179.4 (2) O7---Zn2---O8---C28 −31.0 (3)
C22---C23---C24---C25 1.0 (5) O6---Zn2---O8---C28 145.8 (3)
Cl7---C23---C24---C25 178.1 (3) O11---Zn2---O8---C28 60.8 (3)
C23---C24---C25---C26 1.7 (5) O7^i^---Zn2---O8---C28 −112.4 (3)
----------------------- ------------- --------------------------- -------------
Symmetry codes: (i) −*x*+2, −*y*+1, −*z*+2.
Hydrogen-bond geometry (Å, °) {#tablewraphbondslong}
=============================
-------------------- --------- --------- ----------- ---------------
*D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A*
O9---H9A···O3^ii^ 0.84 2.02 2.790 (3) 153
O9---H9B···O1^ii^ 0.74 2.45 3.015 (3) 134
O10---H10A···O7^i^ 0.84 2.24 2.998 (3) 150
O10---H10B···O5^i^ 0.82 1.95 2.741 (3) 161
O11---H11A···O3^i^ 0.84 2.17 2.931 (3) 151
O11---H11B···O1^i^ 0.84 1.93 2.751 (3) 169
-------------------- --------- --------- ----------- ---------------
Symmetry codes: (ii) −*x*+2, −*y*+2, −*z*+2; (i) −*x*+2, −*y*+1, −*z*+2.
###### Selected bond lengths (Å)
------------- -----------
Zn1---O1 2.040 (2)
Zn1---O2 2.096 (2)
Zn1---O3 2.049 (2)
Zn1---O4 2.084 (2)
Zn1---O9 2.112 (2)
Zn1---O10 2.130 (2)
Zn2---O5 2.011 (2)
Zn2---O6 2.114 (2)
Zn2---O7 2.081 (2)
Zn2---O7^i^ 2.176 (2)
Zn2---O8 2.069 (2)
Zn2---O11 2.134 (2)
------------- -----------
Symmetry code: (i) .
###### Hydrogen-bond geometry (Å, °)
*D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A*
-------------------- --------- ------- ----------- -------------
O9---H9*A*⋯O3^ii^ 0.84 2.02 2.790 (3) 153
O9---H9*B*⋯O1^ii^ 0.74 2.45 3.015 (3) 134
O10---H10*A*⋯O7^i^ 0.84 2.24 2.998 (3) 150
O10---H10*B*⋯O5^i^ 0.82 1.95 2.741 (3) 161
O11---H11*A*⋯O3^i^ 0.84 2.17 2.931 (3) 151
O11---H11*B*⋯O1^i^ 0.84 1.93 2.751 (3) 169
Symmetry codes: (i) ; (ii) .
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Ageing is a multifactorial process developing at different rates depending on individual, environmental, and life style factors.^([@B1])^ Ageing is among others a substantial risk factor for most human diseases. The underlying mechanism of the aging process is still unclear. Ageing is characterized by impaired responsiveness to stress and by reduced efficacy of endogenous protective mechanisms.^([@B2])^ It is postulated that the imbalance between pro-oxidant generation and antioxidant defences increases during ageing. Mitochondria constitute the major source of reactive oxygen species (ROS) within the cell. The mitochondrial theory of ageing holds that the accumulation of ROS damage over time leads to age-associated mitochondrial impairment.^([@B3],[@B4])^ Another age-related alteration is the decline in mitochondrial ability to synthesize ATP.^([@B5],[@B6])^
Coenzyme Q10 (CoQ10) is ubiquitously synthesized in almost all cells and membranes of human tissues. CoQ10 is suspected to be involved in age-related alterations of cells and membranes. Due to its two main functions as an electron carrier in mitochondrial bioenergetics and as a lipophilic antioxidant, deficiency in CoQ10 may impair mitochondrial energy production and increase production of ROS, or susceptibility toward them. CoQ10 is associated with several age-related diseases like diabetes, hypercholesterolemia, cardiac insufficiency, and neurodegenerative diseases, and the beneficial effects of CoQ10 supplementation are discussed.^([@B7],[@B8])^ For instance, in Parkinson's disease, there is a marked decrease in mitochondrial CoQ10 levels,^([@B9])^ while the exogenous uptake of CoQ10 slows the progressive disability.^([@B10])^
The ageing process is thought to influence CoQ concentration in tissue and plasma samples in several species. Beyer *et al.*^([@B11])^ found that CoQ content of several tissues in rats increased after birth, reaching a maximum level at 18 months, after which CoQ content decreased with advancing age. In pigs and mice the CoQ level decreases with age, while the oxidized proportion increases.^([@B12])^ Kamzalov *et al.*^([@B13])^ have postulated that age-related changes of CoQ are more obvious in mitochondria, where CoQ biosynthesis takes place. They found no significant differences in CoQ content in plasma or several tissue homogenates from rats at different ages, whereas the mitochondrial level of CoQ in tissues decreased with age. Likewise, the mitochondrial CoQ content in mouse skeletal muscle decreased with age.^([@B14])^ In rat brains, there are no changes in CoQ levels after an initial increase during the first month of life,^([@B15])^ and in human brain,^([@B16],[@B17])^ heart and pancreas,^([@B18])^ lower concentrations of CoQ in elderly people are reported. CoQ10 measurement in tissues is considered to be the most reliable standard for diagnosis of CoQ10 deficiency.
While tissue CoQ10 levels mainly depend on *de novo* synthesis,^([@B19])^ plasma CoQ10 concentrations are significantly influenced by dietary uptake.^([@B20])^ However, serum or plasma CoQ10 concentrations are frequently used clinically as a raw proxy for the functional CoQ10 status in humans. CoQ10 is considered the main antioxidant in low-density lipoproteins (LDLs). Lower CoQ10 levels in plasma are related to higher lipid peroxidation.^([@B21])^ It is thought that CoQ10 levels decrease in ageing individuals.^([@B22])^ On the other hand, there are studies^([@B1],[@B23],[@B24])^ that indicate that plasma CoQ10 levels in older people are higher than in young people. Besides ageing, sex may influence the blood level of CoQ10; given that the antioxidative defences in LDLs tend to decrease in a sex-specific manner in ageing men.^([@B25])^ As the findings on CoQ10 plasma levels during ageing are inconsistent and often not associated with lipoprotein concentration, and furthermore, information on changes in the redox status is missing, the present study was conducted to examine the impact of aging and sex on total and cholesterol-adjusted CoQ10 concentration and redox-status in a large cohort of 860 subjects aged 18--82 years.
Materials and Methods
=====================
Basic study population
----------------------
Sample characteristics of subjects and study design have been recently described.^([@B26])^ In short: we determined the CoQ10 status in a large European study collective (*n* = 1,911), which was based on the PopGen control cohort^([@B27])^ and the FoCus cohort.^([@B28])^ The participants in this European study collective were recruited in cooperation with the University Hospital Schleswig-Holstein (UKSH), Kiel, Germany as healthy blood donors, or in the adiposity ambulance of UKSH. In the present study, we used sub-groups 1, 3 and 4 for analysis.^([@B26])^ Out of this pool, we used 860 subjects (age range 18--82 years) who fulfilled the inclusion criteria based on questionnaires regarding prevalent diseases (diagnosed by a physician). Exclusion criteria for participation were diabetes, or hepatic, renal or gastrointestinal diseases (chronic diarrhea and inflammatory bowel diseases). Subjects with a history of apoplexy or suffering from neurological disorders (Parkinson's disease, epilepsy, essential tremor, and restless legs syndrome), cardiac insufficiency or coronary heart diseases were excluded. The upper limit for total cholesterol was set at \<6.2 mmol/L (high-risk group), and for body mass index at \<40 kg/m^2^ (adiposity grade III, extreme). The participants had an average BMI of 26.3 ± 4.7 kg/m^2^ and cholesterol concentration of 4.6 mmol/L. A total of 58% were female. The study was approved by the Ethics Committee of the Medical Faculty and was consistent with the Declaration of Helsinki. All volunteers gave written consent.
Intervention study
------------------
Subject characteristics and study design for the intervention study have been described previously.^([@B29])^ Fifty-three healthy male volunteers aged between 21 and 48 years received 150 mg/day of the reduced form of CoQ10 (Q~10~H~2~, ubiquinol; Kaneka Corporation, Japan) with each principal meal for 14 days. Fasting blood samples were taken before (T~0~) and after (T~14~) supplementation. Heparin-plasma was frozen at −84°C until analysis.
Sample preparation and analysis
-------------------------------
Blood samples were taken after an overnight fast and immediately centrifuged. Serum samples were stored at −84°C. The simultaneous analysis of both the oxidized (ubiquinone-10) and reduced forms (ubiquinol-10) of CoQ10 was based on the method of high-performance liquid chromatography (HPLC) with electrochemical detection, as described previously.^([@B30])^ As internal standards, 56 pmol ubiquinol-9 plus 9 pmol ubiquinone-9 (Sigma--Aldrich, Taufkirchen, Germany) in 50 µl ethanol were added to a 50-µl serum aliquot. After hexane extraction and centrifugation (5 min, 1,000 *g*, 4°C), the separated hexane phase was evaporated to dryness under a stream of argon and the dry residue was re-dissolved in 50 µl ethanol for injection into the HPLC system. The analytical column was a Prontosil 120-3-C18-SH PEEK column (Bischoff, Leonberg, Germany). The detection system consisted of a Coulochem II electrochemical detector (ESA, Bedford, MA) connected with a Model 5021A conditioning cell and a Model 5011A analytical cell. A 10-µl sample was stored at −84°C for analysis of cholesterol (CHOD-PAP enzymatic photometric test, Human Diganostics, Wiesbaden, Germany).
Analysis of CoQ10 was performed during 2011--2014. On each day the co-analysis of pool samples verified that redox status and concentration were maintained within control limits (3 sigma).
Statistics
----------
Statistical analysis was performed using the Winstat software package (R. Fitch Software, Bad Krozingen, Germany). Data are expressed as the mean ± SDM. To test for significant differences regarding sex, the Mann--Whitney *U* test was used. To test for significant differences regarding CoQ10 status and cholesterol concentration between age groups, one-way-analysis of variance (Scheffe post hoc test) was used. The correlation of parameters was tested by Spearman's rank correlation. The significance level was set at *p*\<0.05 for all tests.
Results and Discussion
======================
Sex-specific changes in CoQ10 status in different age groups
------------------------------------------------------------
The study subjects (*n* = 860) were divided into three age groups: young (18--40 years, *n* = 326), middle aged (41--60 years, *n* = 388), and old (61--82 years, *n* = 146) (Table [1](#T1){ref-type="table"}). We found an inverse U-shaped relationship between CoQ10 concentration and age, with the highest concentrations in middle-aged subjects. The cholesterol level increased during ageing in women; whereas in men, cholesterol concentration was lower in old than middle-aged subjects. Cholesterol-related CoQ10 concentration exhibited an inverse U-shaped course during ageing, with the highest concentration in the middle-aged group without reaching statistical significance. The distinctly elevated cholesterol-related CoQ10 concentration in men compared to women was the most striking sex-related difference and found for all age groups. Compared to the young and middle-aged groups, the proportion of the oxidized form of CoQ10 was higher in older individuals in both sexes. In young subjects (18--40 years) the proportion of oxidized CoQ10 was higher in women than men. The oxidized proportion of CoQ10 was high in the present study (mean 12.9%). Our own previous studies yielded mean oxidized proportions of CoQ10 in healthy children and adults of \~9%,^([@B31],[@B32])^ however, the redox status of CoQ10 was analysed in heparin plasma in these studies. A comparative analysis of intra-day variations of serum versus heparin plasma pool revealed significantly increased oxidized proportions in serum, while the total concentration was unaffected (data not shown). However, this did not influence sex differences or the general age course of the redox status.
The redox status of CoQ10 is suggested to be a useful biomarker of oxidative stress.^([@B33])^ In its reduced form, CoQ10 is one of the most potent endogenously synthesized lipophilic antioxidants.^([@B34])^ A shift towards oxidized CoQ10 is likely a sign of increased oxidative stress.^([@B35]--[@B37])^ High levels of CoQ10 in lipoproteins are directly related to high resistance to initiation of lipid peroxidation.^([@B38])^ Since men had increased cholesterol-related concentrations combined with increased total ubiquinol concentrations, the antioxidative capacity and demand seemed to be sex specific.
Sex-related differences were also found by Del Pozo-Cruz *et al.*,^([@B24])^ who showed that elderly men had greater CoQ10/cholesterol ratios than elderly women; however, men also exhibited higher lipid peroxidation, which suggests an increased demand in antioxidant capacity in old men. That study group also found that BMI did not influence the lipid profile but showed a negative correlation with total CoQ10 level, which contradicts our own findings. We found a positive association between BMI and cholesterol concentration, as well as between BMI and both CoQ10 concentration and oxidized proportion. However, the upper inclusion limits for BMI and cholesterol concentration were set at higher level in our study.
Kaikkonen *et al.*^([@B23])^ found a positive association between total plasma CoQ10 and age, while men had higher levels than women (*n* = 518; age range 45--70 years). Lu and Frank^([@B39])^ found no significant differences in CoQ10 concentration with respect to sex but an age-dependent increase in total CoQ10 concentration (*n* = 1,317; age range 1--94 years). Unfortunately, in both studies, no information was given about lipid-adjusted CoQ10 or redox status. Miles *et al.*^([@B40])^ found in the plasma of self-reported healthy adults (*n* = 148; age range 28--80 years) significantly increased CoQ10 concentration in men compared to women. However, this sex-related difference equalized, when CoQ10 concentration was related to cholesterol. They also reported that the redox status of CoQ10 in plasma was independent of sex. This contradicts our own findings in which sex-related differences in CoQ10 concentration were strengthened after lipid adjustment, and the oxidized proportion of CoQ10 was higher in young women compared to young men. Differences in the analysis of CoQ10 in plasma between the study groups may be explained by factors like race differences or divergent life habits.
In the present study, the cholesterol-related CoQ10 concentrations of adults showed distinctly lower values than those previously found for healthy children,^([@B31])^ in which an age-related decrease during childhood was evident: while infants (*n* = 85, 0--1 year) had mean values of 303 µmol CoQ10/mol cholesterol, there was a significant decrease to 260 in preschool children (*n* = 60, 2--6 years) and to 226 in school-aged children (*n* = 54, 6--15 years). This decrease in lipid-adjusted CoQ10 concentrations during childhood was confirmed by others.^([@B41])^ Likewise, there was a decrease in total CoQ10 during childhood, reaching the level of young adults during school age (mean 0.807 µmol CoQ10/l).^([@B31])^ However, during adulthood the total CoQ10 concentration in blood again increases with age with a peak at 41--60 years, and decreases in old age, especially in men.
Relationships between CoQ10 concentration and total cholesterol, LDL cholesterol, HDL cholesterol, and BMI
----------------------------------------------------------------------------------------------------------
As shown in Table [2](#T2){ref-type="table"}, there was a strong positive correlation between CoQ10 and cholesterol concentrations. Concentrations of both parameters showed similar and parallel changes with age (Fig. [1](#F1){ref-type="fig"}). CoQ10 and LDL-cholesterol concentrations also showed a strong positive correlation (Table [2](#T2){ref-type="table"}). In contrast, HDL-cholesterol level was not significantly related to CoQ10 concentration. The strong relationship between the total cholesterol or the LDL-cholesterol and CoQ10 concentration respectively in serum was anticipated as virtually all CoQ10 in the circulation is associated with lipoproteins.^([@B42])^ Furthermore, CoQ10 and cholesterol share in parts a common synthetic pathway.^([@B22])^ However, the present findings emphasize the necessity for adjustment of CoQ10 concentration to lipid concentration while diagnosing the functional status of CoQ10 in human blood.
Both CoQ10 and cholesterol concentrations were positively correlated with BMI (Table [2](#T2){ref-type="table"}). BMI and the proportion of the oxidized form of CoQ10 were also positively correlated (Table [2](#T2){ref-type="table"}), which indicates increased oxidative stress in obese people.
Correlation between CoQ10 concentration and redox state
-------------------------------------------------------
We found a strong negative correlation (Spearman *p*≤1E-07) between the concentration of CoQ10 and the proportion of the oxidized form of CoQ10 (Table [2](#T2){ref-type="table"}). This correlation was even stronger after correction of CoQ10 concentration for cholesterol concentration. The resulting negative correlation between CoQ10/cholesterol and redox state of CoQ10 reached Spearman values of *p*≤1E-14 (Table [2](#T2){ref-type="table"}). This correlation was present in all age groups. Namely, subjects with a low concentration of CoQ10 had a concomitant reduced redox capacity of CoQ10, meaning less ubiquinol and more ubiquinone. Based on these results, all data points were stratified into four groups, and compartmented into quadrants corresponding to mean values of both cholesterol-adjusted CoQ10 concentration and redox state of CoQ10 (Fig. [2](#F2){ref-type="fig"}). Of special interest are group I (low concentration, high oxidized form) and group IV (high concentration, low oxidized form), representing a low (group I) and high (group IV) CoQ10 status, respectively. Forty-one percent of all subjects in the old-age group (61--82 years) comprised group I, but only 19% group IV. Subjects with low total CoQ10 in the circulation are suspected of having impaired antioxidative capacity, an increased consumption of their antioxidative resources, or to be more susceptible towards oxidative stress. This situation is intensified when the amount of the antioxidant CoQ10 is not able to assure the protection of the lipoproteins. Since subjects in the old age group (61--82 years) showed increased oxidized proportions of CoQ10 combined with decreasing levels in total CoQ10, elderly people may be more susceptible towards oxidative stress. However, future studies should also consider the association between CoQ10 status, age and other biomarkers for oxidative stress, such as glutathione S-transferase or malondialdehyde levels. It was shown by Del Pozo-Cruz *et al.*^([@B24])^ that physical activity in individuals of advanced age increased the levels of total and lipid-adjusted CoQ10, and lowered the levels of lipid peroxidation in human plasma. However, a reliable improvement in antioxidant capacity may be achieved by oral supplementation of CoQ10. Re-analysis of our intervention study^([@B29])^ revealed that supplementation of 150 mg/day ubiquinol for 14 days attenuated the negative correlation between CoQ10 concentration and redox state of CoQ10: the cholesterol-related CoQ10 concentration significantly increased (Mann--Whitney *U* test *p*≤0), and the oxidized proportion of CoQ10 significantly decreased (Mann--Whitney *U* test *p*≤1E-10) while the negative correlation between both parameters flattened (Fig. [3](#F3){ref-type="fig"}).
Conclusions
-----------
In the current study, it was shown that CoQ10 blood concentrations showed an inverse U-shaped course during adulthood. In old people, the antioxidative properties of CoQ10 in the circulation may be impaired, as reflected by a decrease in CoQ10 concentrations, accompanied by a shift in redox status in favour of the oxidized proportion. This shift in redox status was seen in both sexes; however, women irrespective of age had lower cholesterol-adjusted CoQ10 levels in comparison to men. Therefore, women may either be more susceptible to oxidative damage or men may have an increased demand in antioxidant capacity. There is a clear negative correlation between the cholesterol-adjusted CoQ10 level and its oxidized proportion of CoQ10. Thus, the decrease of CoQ10 levels in old age associated with a loss of antioxidant capacity is not limited to human tissues or cell organelles, but applies also to lipoprotein protection in the blood. The concurrent occurrence of low cholesterol-adjusted concentrations and high oxidized proportions of CoQ10 increase the susceptibility to oxidative stress in old age, which may be overcome by oral supplementation.
This work was supported by the patients' self-help group "Elterninitiative Tumorkranker Kinder e.V. der Vestischen Kinderklinik Datteln", Germany, and by the foundation "Peter und Ruth Wirts Stiftung", Switzerland. We thank all participants of the study cohort for their invaluable contribution to the study. The popgen 2.0 network is supported by a grant from the German Ministry for Education and Research (01EY1103). The FoCus cohort is supported by a grant of the German Ministry for Education and Research (0315540A). Determining and analyzing the CoQ10 status in the large study population is supported by Kaneka Corporation, Japan.
CoQ
: coenzyme Q
HDL
: high-density lipoprotein
HPLC
: high-performance liquid chromatography
LDL
: low-density lipoprotein
ROS
: reactive oxygen species
Conflict of Interest
====================
No potential conflicts of interest were disclosed.
{#F1}
{#F2}
{#F3}
######
CoQ10 status and cholesterol level in 860 study subjects divided into age groups stratified by sex
Age-range number (K/J) 18--40 326 (129/197) 41--60 388 (177/211) 61--82 146 (55/91)
------------------------------------ ------------------------ ---------------------- ---------------------- --------------------
CoQ10 (µmol/L) Total 0.759 ± 0.268^A^ 0.885 ± 0.302^C^ 0.830 ± 0.235^B^
Male 0.796 ± 0.293^A^ 0.939 ± 0.335^B^ 0.835 ± 0.235^AB^
Female 0.734 ± 0.248^A^ 0.839 ± 0.265^B^ 0.827 ± 0.236^B^
*p*≤ (K vs J) 0.01
Cholesterol (mmol/L) Total 4.33 ± 0.79^A^ 4.78 ± 0.73^B^ 4.80 ± 0.68^B^
Male 4.27 ± 0.88^A^ 4.83 ± 0.74^B^ 4.53 ± 0.60^A^
Female 4.37 ± 0.72^A^ 4.74 ± 0.71^B^ 4.97 ± 0.67^C^
*p*≤ (K vs J) 0.001
CoQ10/cholesterol (µmol/mol) Total 175 ± 53^A^ 184 ± 53^A^ 173 ± 44^A^
Male 185 ± 51^A^ 192 ± 53^A^ 184 ± 46^A^
Female 169 ± 54^A^ 177 ± 53^A^ 166 ± 42^A^
*p*≤ (K vs J) 0.005 0.005 0.01
Redox-status (% oxidized in total) Total 12.7 ± 2.4^A^ 12.8 ± 2.2^A^ 13.7 ± 2.0^B^
Male 12.4 ± 2.7^A^ 12.5 ± 2.0^A^ 13.7 ± 2.3^B^
Female 12.9 ± 2.1^A^ 13.0 ± 2.4^A^ 13.6 ± 1.9^B^
*p*≤ (K vs J) 0.005
Data are shown as mean ± SDM; data with different superscripts are statistically different in regard to age groups as calculated by ANOVA (Scheffe post-hoc test) *p*≤0.05; sex-related differences within age groups were calculated by Mann--Whitney *U* test.
######
Correlation (Spearman's rank correlation) of parameters of CoQ10 status, cholesterol concentration, and BMI in 860 study subjects ranging in age from 18 to 82 years
Parameter 1 Parameter 2 *p*≤ Correlation
--------------------------------- -------------------- ---------- -------------
Total CoQ10 Total cholesterol 1.00E-74 0.57
Total CoQ10 HDL-cholesterol^§^ ns
Total CoQ10 LDL-cholesterol^§^ 1.00E-30 0.54
Total CoQ10 BMI 1.00E-04 0.13
Total cholesterol BMI 0.001 0.12
Redox-status (% oxidized CoQ10) BMI 0.002 0.10
Redox-status (% oxidized CoQ10) Total CoQ10 1.00E-07 --0.18
Redox-status (% oxidized CoQ10) CoQ10/cholesterol 1.00E-14 --0.27
^§^HDL and LDL data in *n* = 383 subjects
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Several reports on the chemistry of 4-thiazolidinones have been published \[[@B1-molecules-06-00510],[@B2-molecules-06-00510],[@B3-molecules-06-00510],[@B4-molecules-06-00510],[@B5-molecules-06-00510],[@B6-molecules-06-00510]\], but literature reports on reactions of 4-oxo-2-thioxo-thiazolidines with dipolar species are rather limited and treat the problem of alkylation of the 3-unsubstituted derivatives with diazomethane \[[@B7-molecules-06-00510],[@B8-molecules-06-00510]\] and cycloaddition of nitrilimines to the thiono function of 5-aroyl-methylene-3-phenyl-4-oxo-2-thioxo-1,3-thiazolidine \[[@B9-molecules-06-00510]\]. The intention of the present work was to study the reactivity of thiono as well as exocylic double bond functions in E,Z-5-aroylmethylene-3-benzyl-4-oxo-2-thioxo-1,3-thiazolidines (**3a-c**) towards 4-methoxyphenyl- and 4-chlorophenylnitrile oxides (**4a,b**). Thus a series of E,Z-5-aroylmethylene-3-benzyl-4-oxo-2-thioxo-1,3- thiazolidines (**3a-c*)*** has been synthesised and subsequently reacted with 4-methoxyphenyl- and 4- chlorophenylnitrile oxides (**4a,b**).
Results and Discussion
======================
Compounds **E,Z-3a-c** were synthesised by a method similar to that of Nagase \[[@B10-molecules-06-00510]\] by treating 5-(2- aryl-2-oxoethyl)-4-oxo-2-thioxo-1,3-thiazolidines (**2a-c**) (obtained in turn by reaction of 3-aroylacrylic acids (**1a-c**) with ammonium benzyldithiocarbamate), with bromine as shown in **[Scheme 1](#molecules-06-00510-f001){ref-type="scheme"}**. Omar *et al.* \[[@B2-molecules-06-00510]\] studied the stereochemistry of compounds **3** and reported the formation of E- and Z- stereoisomers with the predominance of the former one.
{#molecules-06-00510-f001}
Our intent was to perform the reactions in pyridine as, on the one hand, it is able to dissolve the starting thiazolidinones which are sparingly soluble in most organic solvents, and, on the other hand, it can be used to liberate the required nitrile oxide *in situ* from stable α-hydroximinobenzyl chlorides precursors.
Nitrile oxides **4a** and **4b,** produced *in situ* upon addition of α-hydroximino-4-methoxy- and α- hydroximino-4-chlorobenzyl chlorides to pyridine reacted with **E,Z-3a-c** to afford the Z-isomers of either of the starting 4-oxo-2-thioxo- compounds **Z-3** or the Z-2,4-dioxo- analogues **5**, or a mixture of them, along with the corresponding head to tail dimerised product, namely 3,6-di-(4-methoxyphenyl)- 1,4,2,5-dioxadiazine (**6a**), in the former case and the 4-chlorophenyl counterpart (**6b**), in the latter case, as outlined in **[Scheme 2](#molecules-06-00510-f002){ref-type="scheme"}**. Small amounts of the unreacted starting materials somewhat enriched with Z- isomers were isolated in all the studied examples. (cf. [Table 1](#molecules-06-00510-t001){ref-type="table"}).
The structures of the isomerized 4-oxo-2-thioxo-1,3-thiazolidines (**Z-3b** and **c**) and the respective 2,4-dioxo counterparts (**Z-5a-c**) were deduced from microanalytical, IR, ^1^H-NMR and MS spectral data (cf. [Table 2](#molecules-06-00510-t002){ref-type="table"} and [Table 3](#molecules-06-00510-t003){ref-type="table"}). All the IR spectra of compounds **5** show the complex carbonyl pattern extending to 1750 cm^-1^ consistent with the stretching vibrations of coupled carbonyl groups, whereas those of **3** show the carbonyl absorptions at mostly lower frequency values not exceeding 1715 cm^-1^. Their EI-mass spectra exhibit parent peaks at m/e 91, molecular ion peaks and M^.+^-CS(CO) \[A\] and M^.+^-PhCH~2~NCS(O) \[B\] fragments. The structures of **6a** and **6b,** which were believed to be the head to the tail dimerised products, was confirmed by comparison (m.p and IR) with authentic samples \[[@B11-molecules-06-00510]\].
{#molecules-06-00510-f002}
Configurational assignments of compounds **3** and **5** were based exclusively on ^1^H-NMR spectroscopy by comparing the observed chemical shift values of the olefinic protons with the incremented values \[[@B12-molecules-06-00510]\]; the olefinic protons of the Z-isomers are relatively deshielded by the 4-oxo- groups compared with the E-counterparts. The Z-configuration was assigned to **3b** and **3c** by comparing with the starting E-counterparts and to **5a** by comparing with a sample previously prepared \[[@B1-molecules-06-00510]\] upon treating a solution of **2a** in glacial acetic acid with bromine.
A role for pyridine alone in the isomerisation process can be ruled out as compounds **E,Z-3a-c** are recovered without detectable configurational change upon refluxing in this solvent. The larger proportions of Z-isomers observed in the compounds prepared as compared to the starting materials indicates that isomerisation has occured during the reactions. The great stability of the Z-isomers as compared with the E-counterparts is probably due to steric considerations Although isomerisation may be explained according to the hypothesis of formation of zwitterionic or biradical intermediates \[[@B13-molecules-06-00510]\], it is better explained in terms of successive addition and elimination of nitrile oxide at the β-terminus of the exocyclic α,β-unsatured carbonyl system of compounds **3** as outlined in **[Scheme 3](#molecules-06-00510-f003){ref-type="scheme"}**. The instability of the hypothetically formed adducts may be attributed to a sterically hindered transition state. The negative results reported for the reaction of tri- and tetra-alkyl ethylenes with nitrile oxides \[[@B14-molecules-06-00510]\] seem to be in accordance with our results.
{#molecules-06-00510-f003}
The formation of the 2,4-dioxo compounds **5** rather than the spiranes **7**; which could be produced by the attack of nitrile oxides at the thiono group of compounds **3** could be rationalised in terms of decomposition of the 1,4,2-oxathiazole ring of the spirane ring system by expelling 4-chlorophenyl- or 4- methoxyphenylisothiocyanates. The decomposition has occured most likely via a radical reaction as it has been reported by Husigen et al \[[@B15-molecules-06-00510]\] that all the 1,4,2-oxathiazoles which are formed via cycloaddition of nitrile oxides to thiocarbonyl compounds decompose exothermically at 90-150°C to form isothiocyanates and the oxygen analogues of the thiocarbonyl compounds. The molecular rearrangement leading to the isothiocyanate probably proceeds concurrently with the ring opening \[[@B15-molecules-06-00510]\] (**[Scheme 2](#molecules-06-00510-f002){ref-type="scheme"}**). The preference for the nitrile oxides to react with C=S rather than the C=O groups may be attributed to the polarizability of the sulfur function which is manifested by the great readiness with which several thiono containing compounds react with dipolar species \[[@B16-molecules-06-00510]\]. Formation of stable spiranes is reported upon reacting 2-thioxo-1,3,4-thiadiazole with nitrile imines \[[@B16-molecules-06-00510]\].
Experimental
============
General
-------
All melting points are uncorrected. IR spectra were measured on a Unicam SP1200 spectrometer as KBr discs. ^1^H-NMR spectra were recorded in d~6~-DMSO on a Varian Gemini 200 MHz instrument with chemical shifts (δ) expressed in ppm downfield from TMS. Mass spectra were recorded on Shimadzu GC-MS-Qp 1000 EX instrument operating at 70 ev. Column chromatography and TLC were run on Silica Gel Woelm, activity 113/30 mm according to Brockmann & Schodder and Silica Gel 60 F~254~ (Merck) aluminium backed TLC sheets.
Preparation of Starting Materials 3-Benzyl-5-\[2-(4-bromophenyl)-2-oxoethyl\]-4-oxo-2-thioxo-1,3-thiazolidine (**2b**).
-----------------------------------------------------------------------------------------------------------------------
Ammonium benzyl-dithiocarbamate (2.2 g, 11 mmol) in ethanol (10 mL) was added dropwise to a stirred solution of 3-(4-bromobenzoyl) propenoic acid \[[@B17-molecules-06-00510]\] (2.55 g, 10 mmol) in ethanol (10 mL) at room temperature. Concentrated hydrochloric acid (3 mL) was added portionwise to the stirred reaction mixture after 30 min. and the precipitated solid was filtered and recrystallised from benzene-light petroleum (b.p. 60-80°C) to give **2b** \[[@B1-molecules-06-00510],[@B18-molecules-06-00510]\].
E,Z-3-Benzyl-5-(4-bromobenzoylmethylene)-4-oxo-2-thioxo-1,3-**thiazolidine** (**3b**).
--------------------------------------------------------------------------------------
Bromine (20 mmol) was added to a solution of **2b** (3.4 g, 10 mmol) in glacial acetic acid (20 mL) and the resulting mixture was warmed on a water bath for 5 min. After cooling the reaction mixture was poured into cold water and filtered. The crude orange product (3.34 g, 80%) was recrystallised from glacial acetic acid to give **E,Z-3b.**
The analogous compounds *3-benzyl-5-\[2-(4-methylphenyl)-2-oxoethyl\]-4-oxo-2-thioxo-1,3-thiazolidine* (**2a**) and *3-benzyl-5-\[2-(4-chlorophenyl)-2-oxoethyl\]-4-oxo-2-thioxo-1,3-thiazolidine* (**2c**) \[[@B18-molecules-06-00510]\] *and E,Z- 3-benzyl-5-(4-methylphenylmethylene)-4-oxo-2-thioxo-1,3-thiazolidine* (**3a**) *and E,Z-3-benzyl-5-(4- chlorophenylmethylene)-4-oxo-2-thioxo-1,3-thiazolidine* (**3c**) \[[@B1-molecules-06-00510],[@B10-molecules-06-00510]\] were prepared by the same methods mentioned above for **2b** and **E,Z-3b**.
Reactions of Compounds **E,Z-3a-c** with Arylnitrile Oxides 4a and **b**: General Procedure
-------------------------------------------------------------------------------------------
Powdered α-hydroximino-4-chlorobenzyl chloride (**4a**) \[[@B19-molecules-06-00510]\] or the 4-methoxybenzyl counterpart (**4b**) \[[@B20-molecules-06-00510]\] (25 mmol) was added to a suspension of each of **E,Z-3a-c** (10 mmol) in anhydrous pyridine (10 mL) and the whole mixture was refluxed for 10 hrs. during which it acquired a violet colouration. The solid which precipitated after allowing the reaction mixture to stand at room temperature overnight was filtered off, washed with small portion of ether and chromatographed or recrystallised from an appropriate solvent to give **Z-3** and/or **Z-5** along with starting materials slightly enriched with the **Z**-configured isomers **E,Z-3**.
The crude product (2.82 g, 80%) obtained upon treating **E,Z-3a** with **4b** was crystallized from dioxane to give **E,Z-3a** as yellow crystals (0.71 g, 20%), m.p. 210-212°C containing ca. 20% of the Z- isomer. Dilution of the dioxane mother liquors gave **Z-5a** as brownish red crystals (1.77 g, 50%), m.p. 179-181°C (from dilute dioxane).
The reaction mixture of **E,Z-3b** with **4b** was concentrated (final volume ca. 5 mL), chromatographed over silica gel while monitoring by TLC. Elution with light petroleum (b.p. 40-60°C)-ether mixture (4:1 v/v) gave **Z-3b** (2.3 g, 55%) as red needles (from benzene). Elution with light petroleum (b.p. 40-60°C)- ether mixture (1:1 v/v) gave **Z-5b** (0.4 g, 10%) as brownish red crystals (from benzene-methanol). Elution with pure ether gave **E,Z-3b** (0.63 g, 15%) containing 30% of the Z- isomer.
The crude product (2.98 g, 80%) which was obtained upon reacting **E,Z-3c** with **4b** was crystallised from dioxane to give **E,Z-3c** (0.75 g, 20%) containing 90% of the Z-isomer as orange crystals m.p. 248- 250°C. On leaving the dioxane mother liquor to stand at room temperature for 24 hrs., it precipitated a yellow solid (1.96 g, 55%) which was recrystallised from benzene-methanol to give **Z-5c** as yellow crystals m.p. 226-228°C.
Recrystallisation of the crude product (1.49 g, 40%) obtained from the reaction of **E,Z-3c** with **4a** from dioxane precipitated successively **E,Z-3c** (0.6 g, 16%) containing 10% of the Z-isomer and **Z-3c** (0.67 g, 18%).
Upon allowing the water diluted original pyridine mother liquors to stand at room temperature for 48 hrs. therefrom precipitated colourless products which were recrystallised from dioxane to give 45% of 3,6-di-(4-methoxyphenyl)-1,4,2,5-dioxadiazine (**6a**), m.p. 178-180°C, undepressed on admixture with an authentic sample \[[@B11-molecules-06-00510]\] in all cases when **4a** was used and 5-10% of 3,6-di-(4-chlorophenyl)-1,4,2,5- dioxadiazine (**6b**), m.p. 222-224°C undepressed on admixture with an authentic sample \[[@B11-molecules-06-00510]\], when **4b** was the reactant.
Action of pyridine on E,Z-**3a-c**
----------------------------------
A suspension of each of **E,Z-3a-c** (3 mmol) in pyridine (10 mL) was refluxed for 15 hrs. while the solution acquired a violet colouration. The crude product which precipitated upon leaving the reaction mixture to stand at room temperature overnight (90-95%) was recrystallised from dioxane to give 90% of each **E,Z-3a-c** with similar E/Z ratios as the starting materials.
molecules-06-00510-t001_Table 1
######
**Reactions of E,Z-3a-c with Nitrile Oxides 4a and 4b**
------------- -------------------------- ---------------- ----------- ----------- --------
Reactants Products (% yield)^\*\*^
(E,Z)^\*^-3 Nitrile oxide (E,Z)^\*^-3\ (Z)^\*^-3 (Z)^\*^-5 6
\[Z%\]^\*\*\*^
3c 4a 3c(16)\[10\] 3c(18) \-\-- 6a(45)
3a 4b 3a(20)\[20\] \-\-- 5a(50) 6b(7)
3b 4b 3b(15)\[30\] 3b(55) 5b(10) 6b(5)
3c 4b 3c(20)\[90\] \-\-- 5c(55) 6b(10)
------------- -------------------------- ---------------- ----------- ----------- --------
^\*^ configurational assignment is based on ^1^H-NMR spectroscopy. ^\*\*^ yield of actually isolated compounds ^\*\*\*^ % of the Z-isomer in the E,Z-mixture.
molecules-06-00510-t002_Table 2
######
**Some Data of the Newly Synthesised Compounds**
------------------------------------------------------------------- ------------ ----------------------------------------- ------------------ ----------------- ----------------- ----------------- -----------
Compd. No. MP (°C) ^1^H-NMR Spectral data \[δ-values ppm\]
Olefinic AroylH^\*^ N-CH~2~[Ph]{.ul} N-[CH]{.ul}~2~Ph Me
H 2Ha 2Hb 5H 2H 3H
**2b ^a^ \[[@B1-molecules-06-00510],[@B18-molecules-06-00510]\]** 128-129 \-\-- 8.020(d^b^) 7.703(d^b^) 7.205(br.s) 5.15 (br s) \--
**E,Z-3a\[[@B1-molecules-06-00510]\]** 215-217 7.900(s^c^) 7.980(d^d^) 7.350-7.050 (m) 5.300 (br s^c^) 3.250 (s)
8.265(s^c^) 4.835 (br s^c^) 2.400 (s)
**Z-3a** 208-210 8.265(s^c^) 8.14(d^d^) 7.500-7.050 (m) 4.836 (br s) 2.400 (s)
**E,Z-3b** 250-253 8.158(s^e^) 8.168(d^f^) 7.713(d^f^) 7.352(br s) 5.274 (br s^e^) \-\--
8.275(s^e^) 4.870 (br s^e^)
**Z-3b** 188-190 8.274(d^e^) 8.144(d^e^) 7.831(br s) 7.351 (br s) 4.868 (br s) \-\--
**E,Z-3c\[[@B10-molecules-06-00510]\]** 251-252 8.135(s) 8.257(d^g^) 7.686 (d^g^) 7.353(br s) 5.288 (br s^c^) \-\--
8.161(s^c^) 5.050 (br s^c^)
**Z-3c** 239-240 8.161(s) 8.255(d^g^) 7.700(d^g^) 7.349(br s) 5.050 (br s) \--
**Z-5a** 179-181 8.271(s) 8.108(d^g^) 7.433(d^g^) 7.355(br s) 4.862 (br s) 2.420 (s)
**Z-5b** 226-228 8.280(s) 8.145(d^g^) 7.832(d^g^) 7.352(br s) 4.869(br s) \-\--
**Z-5c** 226-228 8.291(s) 8.234(d^f^) 7.689(d^f^) 7.352(br s) 4.850 (br s)
------------------------------------------------------------------- ------------ ----------------------------------------- ------------------ ----------------- ----------------- ----------------- -----------
\* H~a~ and H~b~ are the protons at the ortho- and meta positions of the aroyl group.^a^ The spectrum shows also the following signals characteristic for AMX system: δ: 5.505 (dd, H~A~), 4.815 (dd,H~M~), 3.920(dd,H~X~), JH~A~,H~M~ = 9.0 Hz, JH~A~,H~X~ = 3.5 Hz, JH~M~,H~X~ = 18.0 Hz.^b^ J = 7.5 Hz.^c^ Characteristic of E- and Z-isomers with integrated proton ratios of 15:1 respectively.^d^ J = 7.2 Hz.^e^ Characteristic of E- and Z-isomers with integrated proton ratios of 9:1, respectively;^f^ J = 8.6 Hz.^g^ J = 8.2 Hz.
molecules-06-00510-t003_Table 3
######
**Spectral and Analytical Data of Newly Synthesised compounds**
------------ ----------------- -------------- --------------- ----------------------- ----------------------- -------- ------- -------
Compd. No. IR (cm^-1^) C=O EI-MS m/e(%) Formula (M.W) Analysis: Calcd/Found
\[M^.+^\] \[A\] \[B\] C H N
**2b** 1710,1690 \-\-- \-\-- \-\-- C~18~H~14~BrNO~2~S~2~ 51.43/ 3.36/ 3.33/
(420.37) 51.60 3.40 3.39
**Z-3a** 1710,1680 353 309 204 C~19~H~15~NO~2~S~2~ 64.56/ 4.27/ 3.96/
(5.1) (39.3) (23.9) (353.47) 64.43 4.25 4.03
**E,Z-3b** 1710,1680 \-\-- \-\-- \-\-- C~18~H~12~BrNO~2~S~2~ 51.68/ 2.89/ 3.35/
(418.35) 51.65 2.96 3.42
**Z-3b** 1710,1680 417 373 268 C~18~H~12~BrNO~2~S~2~ 51.68/ 2.89/ 3.35/
(13.7) (15.8) (12.0) (418.35) 51.73 3.02 3.50
**Z-3c** 1715,1690 373 329 224 C~18~H~12~ClNO~2~S~2~ 57.82/ 3.23/ 3.75/
(3.0) (35.7) (22.2) (373.89) 57.91 3.15 5.62
**Z-5a** 1745,1690 337 309 204 C~19~H~15~NO~3~S 67.64/ 4.48/ 4.15/
(2.0) (39.3) (23.9) (337.40) 67.73 4.39 4.06
**Z-5b** 1750,1690 401 373 268 C~18~H~12~BrNO~3~S 53.74/ 3.01/ 3.48/
(3.7) (22.0) (12.2) (402.29) 53.711 3.10 3.52
**Z-5c** 1750,1690 357 329 224 C~18~H~12~ClNO~3~S 60.42/ 3.38/ 3.91/
(2.1) (35.7) (22.2) (357.83) 60.56 3.49 3.83
------------ ----------------- -------------- --------------- ----------------------- ----------------------- -------- ------- -------
*Samples Availability*: Samples are available from the authors.
| {
"pile_set_name": "PubMed Central"
} |
Waisberg DR, Abdala E, Nacif LS, et al. Liver transplant recipients infected with SARS-CoV-2 in the early postoperative period: Lessons from a single center in the epicenter of the pandemic. Transpl Infect Dis. 2020;00:e13418 10.1111/tid.13418
ABTO
: Brazilian Association of Organ Transplantation
ACR
: acute cellular rejection
COVID‐19
: coronavirus disease‐19
CT
: computed tomography
DDLT
: deceased‐donor liver transplantation
HCC
: hepatocellular carcinoma
ICU
: intensive care unit
LT
: liver transplantation
NASH
: non‐alcoholic steatohepatitis
NO
: naso‐oropharyngeal
OTI
: orotracheal intubation
PMT
: pulse methylprednisolone therapy
POD
: postoperative day
RT‐PCR
: real‐time polymerase chain reaction
SARS‐CoV‐2
: severe acute respiratory syndrome coronavirus‐2
TACE
: transarterial chemoembolization
1. INTRODUCTION {#tid13418-sec-0001}
===============
Brazil is currently one of the most affected countries in the world by the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and has become the epicenter of the coronavirus disease‐19 (COVID‐19) pandemic in Latin America.[^1^](#tid13418-bib-0001){ref-type="ref"} The impact of COVID‐19 in liver recipients remains largely unknown, as most data derive from case reports and small retrospective series.[^2^](#tid13418-bib-0002){ref-type="ref"}, [^3^](#tid13418-bib-0003){ref-type="ref"}, [^4^](#tid13418-bib-0004){ref-type="ref"}, [^5^](#tid13418-bib-0005){ref-type="ref"}, [^6^](#tid13418-bib-0006){ref-type="ref"}, [^7^](#tid13418-bib-0007){ref-type="ref"}, [^8^](#tid13418-bib-0008){ref-type="ref"}, [^9^](#tid13418-bib-0009){ref-type="ref"}, [^10^](#tid13418-bib-0010){ref-type="ref"}, [^11^](#tid13418-bib-0011){ref-type="ref"}, [^12^](#tid13418-bib-0012){ref-type="ref"}, [^13^](#tid13418-bib-0013){ref-type="ref"}, [^14^](#tid13418-bib-0014){ref-type="ref"}, [^15^](#tid13418-bib-0015){ref-type="ref"}, [^16^](#tid13418-bib-0016){ref-type="ref"} Moreover, most of them address patients who were transplanted years ago.[^2^](#tid13418-bib-0002){ref-type="ref"}, [^3^](#tid13418-bib-0003){ref-type="ref"}, [^4^](#tid13418-bib-0004){ref-type="ref"}, [^5^](#tid13418-bib-0005){ref-type="ref"}, [^6^](#tid13418-bib-0006){ref-type="ref"}, [^8^](#tid13418-bib-0008){ref-type="ref"}, [^9^](#tid13418-bib-0009){ref-type="ref"}, [^10^](#tid13418-bib-0010){ref-type="ref"}, [^12^](#tid13418-bib-0012){ref-type="ref"} Information regarding patients in the early postoperative period is very scarce.
Many authors recommend weaning of immunosuppression and some even advocate its complete withdrawn.[^2^](#tid13418-bib-0002){ref-type="ref"}, [^4^](#tid13418-bib-0004){ref-type="ref"}, [^9^](#tid13418-bib-0009){ref-type="ref"}, [^12^](#tid13418-bib-0012){ref-type="ref"}, [^16^](#tid13418-bib-0016){ref-type="ref"} Nevertheless, these suggestions are mostly based on patients in late postoperative period, for whom immunosuppression is already diminished and acute cellular rejection (ACR) is less likely. Another distinct feature is the impact of nosocomial infection, as these patients are generally infected in‐hospital. When the pandemic starts its declining phase, community viral spread is expected to decrease; however, nosocomial infection may persist longer and affect more routinely hospitalized patients who just underwent transplantation.
São Paulo is Brazil\'s largest city and the current epicenter of the disease in the country. Our institution is one of largest transplantation centers in Latin America, having performed more than 3000 liver transplants (LT), with an average of 160 LT per year. The aim of this study was to report a single‐center case series of consecutive patients in the early postoperative period of deceased‐donor liver transplantation (DDLT) who developed COVID‐19.
2. CASE REPORTS {#tid13418-sec-0002}
===============
During the first months of the city quarantine (March 24th to May 31st, 2020), we performed 19 DDLT in 18 patients and diagnosed 5 cases of COVID‐19 in the early postoperative period. In all cases, SARS‐CoV‐2 infection was confirmed via real‐time polymerase chain reaction (RT‐PCR) in naso‐oropharyngeal (NO) swab or tracheal secretion. Nucleic acid (RNA) was extracted with an automated method based on magnetic beads (mSample Preparation System RNA, Abbott). Reverse transcription, amplification, and detection were performed following an in‐house protocol validated in the Laboratory Division (College of American Pathologists accredited) comprising an E gene assay as the first‐line screening tool, followed by confirmatory testing with a N gene assay, as previously described.[^17^](#tid13418-bib-0017){ref-type="ref"} Endogenous gene RNAse P was used as internal control of extraction and amplification, as well as positive and negative external controls. Analytical sensitivity was 40 copies/mL, and specificity in samples containing other respiratory viruses RNA was 100%. Table [1](#tid13418-tbl-0001){ref-type="table"} depicts a summary of all cases, and Table [2](#tid13418-tbl-0002){ref-type="table"} shows laboratory assessment at time of COVID‐19 diagnosis.
######
Summary of cases with early postoperative COVID‐19
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
| Case | Age, sex, and BMI | Liver Disease | Comorbidities | Donor data | Surgical data | Immunosuppression protocol | COVID‐19 symptoms | COVID‐19 treatment and outcome |
+======+====================+================================+===============================+================================================+===============+=====================================================================================================================================================================================+=========================+================================================================+
| 1 | 69 y, female | HCV | SAH | Female, 45 y | GW: 1600 g | Basiliximab, tacrolimus, mycophenolate and intraoperative corticoid bolus and tampering | 9th POD | OTI on 12th POD |
| | | | | | | | | |
| | BMI: 34.71 kg/m^2^ | Downstaged HCC | Coronariopathy | BD: hemorrhagic stroke | TST: 285 min | | Fever | Azithromycin |
| | | | | | | | | |
| | | MELD: 15 | Pulmonary hypertension | | TIT: 585 min | | Mild dyspnea | Death on 13th POD due to due to refractory shock and acidosis. |
| | | | | | | | | |
| | | Child‐Pugh: A6 | | | WIT: 35 min | | Diarrhea | |
| | | | | | | | | |
| | | | | | BBP: None | | | |
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
| 2 | 67 y, male | NASH | SHA | Female, 22 y | GW: 1475 g | Basiliximab, tacrolimus, mycophenolate and intraoperative corticoid bolus and tampering | 36th POD | OTI on 37th POD |
| | | | | | | | | |
| | BMI: 32.81 kg/m^2^ | α1‐antitripsin deficiency | Obesity | BD: subarachnoid hemorrhage | TST: 435 min | | Fever | Azithromycin |
| | | | | | | | | |
| | | MELD: 13 | | 21 d ICU | TIT: 445 min | | Hypoactive Delirium | Hydroxychloroquine |
| | | | | | | | | |
| | | Child‐Pugh: B7 | | AST 156 UI/mL | WIT: 40 min | | Progressive dyspnea | Death on 56th POD due to secondary bacterial infection |
| | | | | | | | | |
| | | Hepatic encephalopathy | | ALT 296 UI/mL | BBP: None | | | |
| | | | | | | | | |
| | | | | GGT 584 mg/dL | | | | |
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
| 3 | 69 y, male | Alcoholic cirrhosis | SHA | Male, 54 y | GW: 1370 g | Tacrolimus and intraoperative corticoids bolus with tampering | 10th POD | Venturi mask |
| | | | | | | | | |
| | BMI: 27.58 kg/m^2^ | HCC | DM | BD: cranioencephalic trauma | TST: 425 min | | Fever | Supportive care |
| | | | | | | | | |
| | | MELD: 13 | | | TIT: 405 min | | watery diarrhea | Dyspnea worsened on 10th hospitalization day |
| | | | | | | | | |
| | | Child‐Pugh: A6 | | | WIT: 35 min | | dry cough | Discharged home on 17th hospitalization day |
| | | | | | | | | |
| | | | | | BBP: None | | Mild exertional dyspnea | |
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
| 4 | 59 y, male | Cryptogenic cirrhosis | Hepatosplenic schistosomiasis | Female, 52 y | GW: 1450 g | Tacrolimus, mycophenolate and intraoperative corticoid bolus and tampering. Received PMT for acute cellular rejection treatment | 11st POD | Supportive care |
| | | | | | | | | |
| | BMI: 24.38 kg/m^2^ | Ascites | | BD: hemorrhagic stroke | TST: 360 min | | Subfebrile temperature | Discharged on 27th POD |
| | | | | | | | | |
| | | Hepatic encephalopathy | | Presented cardiac arrest before organ recovery | TIT: 460 min | | dry cough | |
| | | | | | | | | |
| | | MELD: 10 | | | WIT: 35 min | | | |
| | | | | | | | | |
| | | Child‐Pugh: B7 | | | BBP: None | | | |
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
| 5 | 34 y, male | Sclerosing primary cholangitis | None | Female, 42 y | GW: 1425 g | Tacrolimus and intraoperative corticoids bolus with tampering. Mycophenolate, PMT, anti‐thymocyte globulin were later used due to steroid‐resistant severe acute cellular rejection | 18th POD: asymptomatic | Supportive care |
| | | | | | | | | |
| | BMI: 22.38 kg/m^2^ | MELD: 35 | | BD: ischemic stroke | TST: 395 min | | 24th POD: | Needle thoracocentesis drainage of pleural effusion |
| | | | | | | | | |
| | | Child B7 | | | TIT: 360 min | | Fever | Discharged home on 41st POD |
| | | | | | | | | |
| | | | | | WIT: 35 min | | Mild dyspnea | |
| | | | | | | | | |
| | | | | | BBP: None | | | |
+------+--------------------+--------------------------------+-------------------------------+------------------------------------------------+---------------+-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------+-------------------------+----------------------------------------------------------------+
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BBP, blood‐borne products; BD, brain death; BMI, body mass index; DM, diabetes *mellitus*; GGT, gamma‐glutamyl transferase; GW, graft weight; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; ICU, intensive care unit; MELD, model of end‐stage liver disease; NASH, non‐alcoholic steatohepatitis; PMT, pulse methylprednisolone therapy POD, postoperative day; SHA, systemic artery hypertension; TIT, total ischemic time; TST, total surgery time; WIT, warm ischemic time.
John Wiley & Sons, Ltd
This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.
######
Laboratory assessment on COVID‐19 diagnosis
Laboratory test Case 1 Case 2 Case 3 Case 4 Case 5
------------------------------------------------------------- ----------- ------------- -------------- ----------- ----------
Alkaline phosphatase (U/L) 571 398 103 646 906
gamma‐glutamyl transferase (U/L) 1288 1405 283 3211 2167
Total Bilirubin (mg/dL) 0.74 1.39 0.68 4.51 2.73
Direct Bilirubin (md/dL) 0.50 1.2 0.46 4.03 2.45
Aspartate aminotransferase (U/L) 251 121 53 116 43
Alanine aminotransferase (U/L) 150 137 106 209 65
Albumin (g/dL) 1.8 2.2 3.3 2.5 2.6
INR --- 1.1 1.07 1.06 1.10
Total leukocytes (/mm^3^) 29 340 12 910 3130 24 850 14 740
Lymphocyte count (/mm^3^) and percentage 1174 (4%) 387 (2.99%) 330 (10.54%) 1740 (7%) 737 (5%)
Creatinine (mg/dL)[^a^](#tid13418-note-0002){ref-type="fn"} 5.84 6.36 1.0 3.04 1.07
Urea (mg/dL)[^a^](#tid13418-note-0002){ref-type="fn"} 178 141 36 50 18
C‐reative protein (mg/L) 146.12 226.01 96.3 50.50 82.22
D‐dimer (ug/mL) 8477 19 354 4626 2253 6489
Lactate dehydrogenase (U/L) 895 691 280 --- ---
Ferritin (ng/mL) --- 2896 --- --- 1558
Tacrolimus serum level (ng/mL) 10.8 13.1 13.3 13.4 13.7
Patients 1, 2, and 4 required renal replacement therapy.
John Wiley & Sons, Ltd
This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.
2.1. Case 1 {#tid13418-sec-0003}
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A 69 years old female patient was presented with hepatitis C virus infection DDLT due to cirrhosis and hepatocellular carcinoma (HCC), which were downstaged to within Milan criteria after 4 transarterial chemoembolization (TACE). Her past medical history was remarkable for systemic arterial hypertension, coronary disease, and pulmonary hypertension. Myocardial perfusion scintigraphy did not show any inactive areas and ejection fraction was 69% on echocardiogram, even though coronary angiography identified important stenosis (\>80%) in the right posterior descending artery, descending anterior artery and circumflex artery.
The procedure went on uneventfully, and the patient was extubated on the 1st postoperative day (POD). Renal function was worsened, and dialysis was required from the 2nd POD. She presented with mild dyspnea, fever, and diarrhea on 9th POD. A thoracic computed tomography (CT) scan showed multiple bilateral ground‐glass pulmonary opacities, occasionally associated with interlobular septa thickening and fine reticulate, affecting approximately 50% of the lung. A RT‐PCR in NO swab confirmed SARS‐CoV‐2 infection, and she was started on azithromycin. The patient clinically worsened on 12th POD, presenting hypotension and massive dyspnea, and had to undergo orotracheal intubation (OTI). Despite immunosuppression discontinuation, she developed important hemodynamic instability and died on 13th POD due to refractory shock and acidosis.
2.2. Case 2 {#tid13418-sec-0004}
-----------
A 67 years old male patient underwent DDLT 10 days before the COVID‐19 quarantine was declared. He was a cirrhotic patient due to non‐alcoholic steatohepatitis (NASH) and α1‐antitripsin deficiency, who had additional points in waitlist because of hepatic encephalopathy. The postoperative course was remarkable for persistent need of hemodialysis. On 26th PO, liver enzymes were mildly elevated and he underwent a percutaneous liver biopsy, which did not show significant alterations, but cytomegalovirus infection was diagnosed (serum polymerase chain reaction: 213 458 IU/mL) and he was started on intravenous ganciclovir. On 36th POD, he was presented with fever, hypoactive delirium, and progressive dyspnea. Thoracic CT scan showed bilateral several ground‐glass pulmonary opacities affecting approximately 50% of the lungs (Figure [1A,B](#tid13418-fig-0001){ref-type="fig"}). He underwent OTI due to persistent hypoxemia and respiratory discomfort. A RT‐PCR in tracheal secretion was positive for SARS‐CoV‐2. He was then started on large‐spectrum antibiotics (meropenem and vancomycin) for 14 days as well as oseltamivir, azithromycin, and hydroxychloroquine for 5 days. Mycophenolate was withdrawn, and tacrolimus serum level was maintained at around 5 mg/dL.
{#tid13418-fig-0001}
He remained on mechanical ventilation for 20 days. Ventilator‐associated pneumonia was diagnosed on 53th POD with multiresistant *Acinetobacter baumannii* being identified on tracheal secretion, and he was started on colistin. His condition continued to deteriorate and he died of persistent hypoxemia and hemodynamic instability on 56th POD.
2.3. Case 3 {#tid13418-sec-0005}
-----------
A 69 years old male patient underwent DDLT due to alcoholic cirrhosis and HCC. Postoperative course was uneventful, and he was discharged home on 8th POD. He returned to the emergency department on 10th POD complaining of fever, watery diarrhea, dry cough, and mild exertional dyspnea. A thoracic CT scan showed bilateral multiple ground‐glass pulmonary opacities (Figure [1C](#tid13418-fig-0001){ref-type="fig"}), affecting less than 50% of the lungs. He was started on oseltamivir for 5 days, piperacillin/ tazobactam for 7 days, and oral metronidazole for 10 days. An RT‐PCR in NO swab was positive for SARS‐CoV‐2. Shortness of breath worsened on the 10th day of hospitalization. Another thoracic CT scan showed increase in number and dimensions of ground‐glass opacities, now affecting more than 50% of the lungs (Figure [1D](#tid13418-fig-0001){ref-type="fig"}). He could nonetheless sustain adequate levels of O~2~ saturation on Venture mask 50% and did not require further interventions, being discharged home on 17th hospitalization day.
2.4. Case 4 {#tid13418-sec-0006}
-----------
A 59 years old male patient with cryptogenic cirrhosis underwent an uneventful DDLT. The patient was extubated on 1st POD, but required hemodialysis from the 4th POD. Pulse methylprednisolone therapy (PMT) was used on 6th POD due to ACR. On 11st POD, he developed subfebrile temperature and dry cough, without need of oxygen therapy. A thoracic CT scan showed bilateral ground‐glass pulmonary opacities, sometimes associated with thickening of interlobular septa and fine reticulate. An RT‐PCR in NO swab confirmed SARS‐CoV‐2. Meropenen and Vancomycin were used for 3 days, and mycophenolate was withdrawn. The patient presented satisfactory evolution and did not require further interventions, being discharged on 27th POD.
2.5. Case 5 {#tid13418-sec-0007}
-----------
A 34 years old male patient with sclerosing primary cholangitis and cirrhosis underwent DDLT with biliodigestive anastomosis. Postoperative evolution was complicated by severe ACR, which did not respond to PMT. He underwent percutaneous liver biopsy, which complicated with hemorrhage and required exploratory laparotomy to evacuate blood clots on 11st POD. Owing to the diagnosis of steroid‐resistant severe ACR, mycophenolate and anti‐thymocyte globulin were introduced. On 18th POD, an abdominal CT scan performed to evaluate the presence of retained clots showed some ground‐glass opacities in the base of the left lung. He started with fever and mild dyspnea on 24th POD and RT‐PCR in NO swab confirmed SARS‐CoV‐2. A thoracic CT scan showed numerous bilateral peribronchovascular ground‐glass opacities, mainly in the upper lobes. Almost total atelectasis of the right lower lobe due to adjacent pleural effusion was also noted, which was eventually drained via needle thoracocentesis (Figure [1E,F](#tid13418-fig-0001){ref-type="fig"}). Despite the more aggressive immunosuppression, the patient remained well with adequate O~2~ saturation on oxygen catheter and was discharged home on 41st POD.
3. DISCUSSION {#tid13418-sec-0008}
=============
This report represents one of the largest series of liver transplant recipients in early postoperative with nosocomial COVID‐19. Fever and respiratory symptoms were present in all cases, and gastrointestinal manifestations were observed in two. We could identify two patterns of clinical evolution: some patients presented mild disease while others required mechanical ventilation and eventually died. As appointed for post‐transplant patients with long‐term metabolic complications,[^3^](#tid13418-bib-0003){ref-type="ref"} comorbidities seem to outweigh immunosuppression in determining prognosis even in the early postoperative period. In fact, one patient with unfavorable outcome had severe coronary disease and the other one experienced prolonged postoperative course complicated with CMV infection and kidney failure. Both were also obese. Whereas the first patient developed SARS‐CoV‐2 infection and rapidly progressed to death, the second exhibited a more insidious course and ultimately died of secondary bacterial infection. Regarding laboratory assessment, a wide variation in liver enzymes was observed, probably related to the particular evolution of each case before COVID‐19 diagnosis. However, inflammatory markers and D‐dimer were elevated in all cases, even though they are not specific for COVID‐19.
Regarding the specify treatment of COVID‐19, we followed our institution\'s protocol, in which most novel drugs, such as umifenovir, lopinavir/ritonavir, and tocilizumab,[^2^](#tid13418-bib-0002){ref-type="ref"}, [^4^](#tid13418-bib-0004){ref-type="ref"}, [^9^](#tid13418-bib-0009){ref-type="ref"}, [^12^](#tid13418-bib-0012){ref-type="ref"}, [^16^](#tid13418-bib-0016){ref-type="ref"} are used under clinical trials. Patients 1 and 2 developed severe COVID‐19 and required OTI. They were started on azithromycin, but hydroxychloroquine was used only in the second case, because the first one rapidly progressed to death. Hydroxychloroquine was initially recommended, but is no longer used given the latest evidence. Large‐spectrum antibiotics were used in patients 3 and 4 aiming to cover bacterial infection as well; however, as they presented satisfactory clinical evolution, no additional treatment was performed. Patient 5 experienced mild symptoms and successfully responded to supportive care.
Immunosuppression was completely withdrawn in case 1, due to the patient\'s dramatic worsening. In case 2, mycophenolate was discontinued and tacrolimus serum levels were lowered. In case 4, mycophenolate was also discontinued, but no changes were made in tacrolimus serum levels. In cases 3 and 5, immunosuppression was not altered. The risk of ACR is higher in early transplanted patients and, in fact, it occurred in 2 early cases previously described in literature.[^7^](#tid13418-bib-0007){ref-type="ref"}, [^13^](#tid13418-bib-0013){ref-type="ref"} It seems that immunosuppression must be tailored on a case‐by‐case basis according to the clinical evolution. For instance, patient 4 underwent PMT before COVID‐19 diagnosis due to ACR and patient 5 underwent PMT and anti‐thymocyte globulin infusion owing to steroid‐resistant ACR. Despite being severely immunosuppressed, they presented only mild symptoms.
As a public quaternary center, our institution was prepared to be the main reference center of COVID‐19 cases in São Paulo State at the beginning of April, 2020. As the number of transplants in the context of COVID‐19 pandemic has decreased substantially,[^16^](#tid13418-bib-0016){ref-type="ref"}, [^18^](#tid13418-bib-0018){ref-type="ref"}, [^19^](#tid13418-bib-0019){ref-type="ref"} strategies have been developed for ensuring the availability of transplantation activity, such as establishment of hospital areas especially dedicated to non‐COVID‐19 patients.[^20^](#tid13418-bib-0020){ref-type="ref"}, [^21^](#tid13418-bib-0021){ref-type="ref"} The main building of our institution was then designated exclusively for treating patients with COVID‐19 when the quarantine was declared, while the nearby institutes were considered areas with low exposure to SARS‐CoV‐2. Organ transplantation activities were supposed to continue in wards and ICUs without SARS‐CoV‐2‐infected patients. Furthermore, all deceased donors were screened to SARS‐CoV‐2 via RT‐PCR in NO swab and all recipients performed a thoracic CT scan before hospital admission, in accordance with Brazilian Association of Organ Transplantation (ABTO) recommendations, except patient 2 who was transplanted immediately before the implementation of these measures. All donors tested negative for SARS‐CoV‐2, and no alterations were noted on recipients CT scans.
The incubation period for SARS‐CoV‐2 is usually 5‐6 days; however, it may reach 14 days.[^21^](#tid13418-bib-0021){ref-type="ref"} Therefore, it is possible that 3 patients (cases 1, 3, and 4) might have been admitted to the hospital already infected. We consider this hypothesis unlikely, since all of them were clinically asymptomatic and with no radiological sign, and there was an ongoing nosocomial outbreak of COVID‐19 in our institution at that time, with many non‐transplant patients from others non‐COVID‐19 wards developing the disease. Therefore, it is reasonably safe to assume that we experienced a nosocomial spread of the virus, probably carried out by asymptomatic patients, or even by patient\'s visitors and healthcare personnel. Due to these events, infection control protocols were tightened, including a temporary ban on family visits, strengthening of barriers precautions, and mass SARS‐CoV‐2 screening for healthcare professionals. We also included the RT‐PCR for SARS‐CoV‐2 in NO swab in the recipients screening. Although those infection control protocols with strict barrier precautions may reduce SARS‐CoV‐2 nosocomial spread even in immunosuppressed patients,[^22^](#tid13418-bib-0022){ref-type="ref"} the challenge of maintaining large liver transplant programs in areas with high community transmission of SARS‐CoV‐2 will remain until a vaccine is developed.
In conclusion, we reported a case series of liver recipients who developed SARS‐CoV‐2 infection in the early postoperative period, probably related to a nosocomial outbreak. Our experience highlights that COVID‐19 may impact negatively the postoperative course, especially in elder and obese patients with comorbidities, and draws attention to COVID‐19 nosocomial spread.
CONFLICT OF INTEREST {#tid13418-sec-0009}
====================
The authors declare that they have no conflict of interest with regards to the content of this manuscript.
AUTHOR CONTRIBUTIONS {#tid13418-sec-0010}
====================
DRW, EA, and LSN involved in conceptualization and wrote the original draft. VR, LNG, RBM, RSP, and RMA investigated the study. CSL and LMM involved in resources. LBH, LD, and DRT involved in data curation. FHG and WA wrote, reviewed, and edited the study. LAC involved in supervision.
| {
"pile_set_name": "PubMed Central"
} |
The efficacy and safety of statin therapy are well established. However, despite their favourable profile, muscle symptoms reported with statin therapy limit their use negatively impacting adherence and cardiovascular benefits of this treatment. It is important that clinicians are cautious before attributing muscle symptoms to statin therapy, without further investigation of their cause. Moreover, this is complicated by the lack of internationally agreed clinical definitions of statin-induced myopathy and diagnostic tools.
Recently the European Atherosclerosis Society (EAS) Consensus Panel has circumvented the lack of consensus regarding the causality of statins with muscle symptoms by the use of the term statin-associated muscle symptoms, SAMS (*Table [1](#suz007-T1){ref-type="table"}*), thus providing an accessible resource for the diagnosis, assessment, and management of SAMS, as well as an update on current thinking about the aetiology of statin myopathy. Table 1**Definition of statin-associated muscle symptoms from reference**[@suz007-B1]
Understanding and clearly defining is critical for optimal treatment. Therefore, the management of SAMS is a key in the effective treatment of patients with cardiovascular disease (CVD), through achievement of maximum-tolerated statin dosing and other practical aspects. Combination therapy with ezetimibe and the addition of Proprotein convertase subtilisin/kexin type 9 inhibitors in high-risk patients with elevated LDL-C is supported by a pharmacological and clinical evidence and is the logical approach to maintain patients on statin therapy with the final aim to optimize the cardiovascular benefits of this treatment.
**Conflict of interest**: none declared.
| {
"pile_set_name": "PubMed Central"
} |
###### Strengths and limitations of this study
This protocol complies with the PRISMA-P guidelines.We will employ the Grading of Recommendations Assessment, Development and Evaluation approach to establish the certainty of the evidence on the effectiveness of reminders in improving childhood immunisation coverage.Non-randomised controlled trials are eligible for inclusion in this review. This study design tends to have a high risk of selection bias, but we plan to alleviate this risk by conducting sensitivity analyses to evaluate the robustness of the evidence to risk of bias by excluding studies with a high risk of selection bias.We anticipate a wide clinical variation across studies relating to participants, study settings and country income levels. We will mitigate the impact of such heterogeneity on the validity of our findings by pooling data only from clinically homogeneous studies.
Introduction {#s1}
============
Immunisation is a healthcare process in which an individual gains immunity or resistance to a disease through administration of a vaccine.[@R1] Immunisation is important in all stages of an individual\'s life as it helps children and adolescents fight infectious diseases and restores waning immunity in adults.[@R2] Besides benefits to the individual who receives the vaccine, immunisation helps protect his or her family members, friends and the community at large.[@R3] The WHO estimates that immunisation currently averts 2--3 million deaths every year in all age groups. These are deaths that would have been caused by diphtheria, tetanus, whooping cough (pertussis), measles and other vaccine-preventable diseases.[@R4]
The percentage of people who receive one or more vaccines of interest in relation to the target population is referred to as vaccination coverage. The vaccination coverage by 1 year of age for the third dose of a vaccine containing diphtheria, tetanus and pertussis (DTP3) is regarded as a proxy for childhood immunisation coverage worldwide.[@R5] Low childhood immunisation coverage is a significant public health issue, with great implication on children\'s health.[@R6] The WHO estimates that DTP3 coverage increased from 76% in 1990 to 86% in 2014 worldwide.[@R4] However, the increase in immunisation coverage is uneven; in 2014, DTP3 coverage was 96% in Europe and the Western Pacific and only 77% in Africa. The estimated number of children under 1 year of age who did not receive DTP3 was 18.7 million worldwide in 2014, with more than three-fifths of them residing in 10 low and middle-income countries: the Democratic Republic of Congo, Ethiopia, India, Indonesia, Iraq, Nigeria, Pakistan, the Philippines, Uganda and South Africa.[@R4] As a consequence of low immunisation coverage, 1.5 million children continue to die from vaccine-preventable diseases worldwide yearly.[@R7]
Immunisation coverage is affected by factors related to the health system, healthcare workers and caregivers (ie, parents or other persons assuming the parental role).[@R8] Caregiver factors that influence childhood immunisation coverage include (but are not limited to) low socioeconomic status, low parental education, younger maternal age, lack of knowledge about the importance of immunisation, negative attitudes towards immunisation, fear of side effects and forgetting vaccination schedules and appointments.[@R9] [@R10]
In line with the optimisation of childhood immunisation coverage, policymakers and healthcare workers need to put in place purposeful, structured, repeatable and adaptable approaches to sustain high immunisation coverage during childhood, irrespective of who the children are or where they live.[@R11] A potentially important strategy for achieving and sustaining high childhood immunisation coverage involves communicating the dates of scheduled vaccination visits and details of the vaccines on schedule to caregivers as well as re-engaging those caregivers who have missed vaccination appointments to bring their children to receive the vaccines due.[@R12]
Various systematic reviews have assessed the effectiveness of such reminder methods in improving childhood immunisation coverage, and we summarise the most relevant ones below.[@R13] Williams *et al*[@R13] conducted a systematic search and found 46 eligible studies published between 1980 and 2009 on strategies to optimise immunisation coverage among children under five in developed countries. Twenty-two studies focused on reminders to advise parents of upcoming vaccinations that are due and remind parents of those children who are overdue. The reminders ranged from automated telephone calls and generic postcards to personalised letters and home visits. There was heterogeneity of effects between types of reminders, with an overall effect being a median point change in coverage of +11% (range −11% to +24%). However, these data were collected six or more years ago and may not be applicable to low and middle-income countries where most unimmunised children live.
In another review, Oyo-Ita and coauthors assessed the effectiveness of intervention strategies to boost and sustain high-childhood immunisation coverage in low and middle-income countries.[@R14] The authors included six studies published between 1996 and 2009, but excluded studies focusing on reminder and recall methods because they were included in an existing review.[@R15] The latter assessed the effectiveness of patient reminder and recall systems in improving immunisation coverage, and compared the effects of various types of reminders in different patient populations. The authors included 47 studies available by May 2007 involving participants of all ages, and found that caregiver reminders improved childhood immunisation coverage: OR 1.47, 95% CIs 1.28 to 1.68). The review focused on high-income countries where (potential) recipients of care have primary healthcare providers whom they visit regularly.[@R15] Thus, the findings of the review would not be directly applicable to low and middle-income countries.
More recently, Harvey *et al*[@R16] conducted a systematic review and meta-analysis of parental interventions to improve early childhood (0--5 years) vaccine uptake. The authors conducted six pooled analyses involving 28 studies, and report that all interventions were somehow effective, although the effects were inconsistent, with a combination of postal and telephone reminders being the most effective reminder method: risk difference 0.1132, 95% CI 0.033 to 0.193. However, the authors excluded studies that did not provide outcome data in terms of the number of children completely immunised or up-to-date for their age from meta-analyses. In addition, interventions for which only one study was found were excluded from pooled analyses. Overall, included studies of reminder interventions in the analyses were published in 2011 or earlier.[@R16] The review therefore provides very low-certainty evidence on the effects of newer methods of reminders on childhood immunisation coverage. Recognising this limitation, the authors indicate that strategies used in most included studies may not be relevant to caregivers of today.
It would therefore be prudent to conduct a new systematic review that includes the newer methods of reminders such as text messages, twitter and other forms of social media. In addition, Harvey *et al* did not use Grading of Recommendations Assessment, Development and Evaluation (GRADE)[@R17] or a similar approach to assess the certainty of the evidence on the effectiveness of reminder methods in improving childhood immunisation coverage. We propose to fill this evidence gap by conducting a systematic review that will include all methods of sending reminders, and use the GRADE system to establish the certainty of the evidence and present the data in 'Summary of Findings' tables.[@R18] In addition, we will use a validated format to summarise the review findings for non-research end users, including (but not limited to) healthcare workers, programme managers and policymakers
Objectives {#s2}
==========
Our objective is to assess the effects of caregiver reminders on the uptake of the WHO-recommended vaccines in children less than 5 years of age.
Methods and analysis {#s3}
====================
Criteria for considering studies for this review {#s3a}
------------------------------------------------
### Types of studies {#s3a1}
We will include randomised controlled trials (RCTs, defined as studies in which participants were allocated to interventions at random) and non-randomised controlled trials (Non-RCTs, defined as trials that allocated participants to treatment arms by a non-random method such as alternation between groups and the use of birthdates or weekdays). We will include RCT and non-RCT trials with allocation at both individual and cluster levels. For trials with allocation at the cluster level, we will only include those with at least two intervention and two control clusters.
### Types of participants {#s3a2}
The participants of interest for this review will be caregivers (ie, parents or other persons fulfilling the parental role) of children in need of the WHO-recommended vaccines.
### Types of interventions {#s3a3}
Eligible interventions will be electronic mail (emails, text messages, twitter, other forms of social media, telephone calls) or postal mail (letters or postcards) delivered to caregivers to remind them of scheduled vaccination visits for their children or recall those who have missed vaccination visits. We will include multifaceted interventions involving any of the eligible interventions and conduct a subgroup analysis by nature of interventions (single or multifaceted). Eligible comparisons include no intervention, standard immunisation practices in the given setting, other interventions, or the same interventions delivered at a different level of intensity. Interventions meant only to inform or educate caregivers about the importance of vaccination are outside the scope of this review and will be excluded. In addition, we will exclude studies focusing on reminders for providers (rather than recipients) of care.
### Types of outcome measures {#s3a4}
The primary outcome for this review is vaccination coverage in children under five (as defined by the authors). Childhood vaccination coverage can be reported in a variety of ways, including coverage with individual vaccines, uptake of a combination of vaccines, DTP3 coverage, the proportion of fully immunised children and the percentage of children up-to-date with recommended vaccines.[@R6] [@R14] We will include studies with all these outcomes. The secondary outcomes will be the cost of the intervention, the incidence of vaccine-preventable diseases, the acceptability of the intervention, adverse events following immunisation and undesirable effects of the interventions.
Search methods for identification of studies {#s3b}
--------------------------------------------
We have developed a comprehensive search strategy for peer-reviewed literature (see online supplementary appendix). Searches will be conducted in PubMed, Scopus, Cochrane Central Register of Controlled Trials (CENTRAL), ISI Web of Science (Science Citation Index), Cumulative Index of Nursing and Allied Health (CINAHL) and PDQ Evidence. We will also check reference lists of relevant reviews and full-text articles assessed for eligibility in this review. In addition, we will search the WHO International Clinical Trials Registry Platform and Clinicaltrials.gov for ongoing trials.
Data collection and analysis {#s3c}
----------------------------
### Selection of studies {#s3c1}
Two review authors (MC and VM) will independently screen the search outputs for potentially eligible studies. We will obtain the full text of studies deemed potentially eligible by at least one of the two authors, and the two authors will independently assess them for eligibility against the study inclusion criteria (ie, types of studies, participants, interventions and outcomes). We will endeavour to obtain English translations for eligible articles published in languages other than English.
### Data extraction and management {#s3c2}
We will use the Cochrane Collaboration\'s Review Manager (RevMan: <http://ims.cochrane.org/RevMan>) for data management and statistical analyses. The two authors will use a predesigned pilot-tested data collection form to independently extract data on study design and methods, country setting (including income level as defined by the World Bank),[@R19] participant characteristics, intervention characteristics, study outcomes and study funding sources.
### Assessment of risk of bias in included studies {#s3c3}
Risk of bias in included studies will be assessed independently by MC and VM, using the Cochrane Risk of Bias tool.[@R20] The following domains will be assessed: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessors, completeness of outcome data, completeness of outcome reporting and other potential sources of bias (eg, baseline imbalance and conflicts of interest). Judgements will be made for each domain as low risk of bias, unclear risk of bias or high risk of bias according to the criteria in the Cochrane handbook. For cluster-randomised controlled trials, we will include additional domains for assessment such as 'loss of clusters'. We will categorise each included study into one of three levels of bias: low, moderate and high risk of bias. Studies classified as low risk of bias for all domains will be considered to be at low risk of bias. Studies with a high risk of selection, detection or attrition bias will be categorised as having high risk of bias. All other studies will be considered to have moderate risk of bias.
### Resolving discrepancies following duplicate assessments {#s3c4}
The two authors (MC and VM) conducting independent screening of search outputs, assessment of study eligibility, extraction of data from included studies and assessment of risk of bias in eligible studies will compare their results after each stage and resolve any differences by discussion and consensus. Should there be no consensus between the two, a third author (FM or CSW) will arbitrate.
### Measures of effect {#s3c5}
We will express study results for binary data as risk ratios and 95% CIs. For cost of interventions, we will report the costs in the currencies provided by the trial authors, with the current US\$ equivalents. Where the data are reported as a small number of ordinal categories, these data will be converted to binary data. For example, in the event that the acceptability of interventions is categorised as acceptable, moderately acceptable and not acceptable, the groups will be converted into two groups: acceptable (acceptable or moderately acceptable) versus not acceptable. For continuous data, such as the incidence of vaccine-preventable diseases, we will calculate unadjusted mean differences (MD) with their SDs.
### Data synthesis {#s3c6}
We will use both fixed-effect and random-effects methods to pool data from clinically homogeneous studies, and compare the results to assess the impact of statistical heterogeneity. We will then present the results from the random-effects method, unless it is contraindicated (eg, if there is funnel plot asymmetry). In the presence of funnel plot asymmetry, we will present the estimates of effects from both methods of meta-analyses, with the assumption that funnel plot asymmetry indicates that neither method is more appropriate. If both show a presence (or absence) of effect, we will be reassured; if they do not agree, we will report this. We will include data from eligible cluster RCTs in relevant meta-analyses after controlling for the design effect, using the intracluster correlation coefficient (ICC).
We will calculate overall intervention effects using generic inverse variance[@R20] and use the GRADE approach to categorise the certainty of the evidence for the effect of each intervention on each outcome as high, moderate, low or very low.[@R17]
### Unit of analysis issues {#s3c7}
We expect that controlled trials which allocated participants at cluster levels will appropriately control for clustering. However, if such data are instead reported as if allocation to interventions was performed at the level of the individual, we will request individual participant data to use in estimating the ICC. If individual participant data are not available, we will obtain external ICC estimates from external sources and use them to re-analyse the data.[@R20]
### Dealing with missing data {#s3c8}
We will contact the authors of included studies to supply relevant unreported data on our primary outcomes. For studies reporting outcomes only for participants who completed the trial or followed the protocol, we will request the additional information that would permit us to conduct intention-to-treat analyses. Should we not receive any requested missing data, we will describe the missing data and discuss the extent to which the missing data could alter our results. We will conduct sensitivity analyses to assess the impacts of missing data on estimates of effect.
### Assessment of heterogeneity {#s3c9}
We will assess clinical variation across studies by comparing the distribution of important characteristics relating to participants (eg, age), study settings (eg, single or sample of medical practice or clinic settings) and country income level according to the World (high, middle or low). We will pool data from clinically homogeneous studies, examine statistical heterogeneity between study results using the χ^2^ test of homogeneity (with significance defined at the α level of 10%), and describe any observed statistical heterogeneity between study results using the I^2^ statistic.[@R20]
### Assessment of reporting biases {#s3c10}
Should we include 10 or more studies in a meta-analysis, we will use funnel plots to assess the possibility of publication bias because when there are fewer studies, funnel plot asymmetry tests are not a reliable means of differentiating true asymmetry from the play of chance.[@R21] Funnel plot asymmetry can emanate from real inconsistency of effects of interventions; publication and other biases (eg, selective outcome reporting), poor methodological quality (poor methodological design, inadequate analysis, fraud) or chance.[@R22]
### Subgroup analysis {#s3c11}
We will carry out subgroup analyses for the primary outcome (vaccination coverage), with subgroups defined by intervention subtype (emails, telephone calls, text messaging, twitter, letters and postcards), nature of intervention (single or multifaceted intervention), country income level as defined by the World Bank (high-income, middle-income and low-income countries)[@R19] and study design (RCTs and non-RCTs). Where appropriate, the intention-to-treat principle and the available case principle will be used to analyse the data.
### Sensitivity analysis {#s3c12}
We will conduct sensitivity analyses to investigate the robustness of the results to risk of bias (low vs high risk of bias). For sensitivity analyses, we will define 'high risk of bias' based on each of three criteria, namely inadequate concealment of intervention allocation, inadequate blinding of outcome assessment and substantial loss to follow-up and/or withdrawals from allocated interventions. If we include cluster-randomised trials and insufficient information is available to adjust for clustering, we will use individuals as the unit of analysis and perform sensitivity analyses to assess the potential bias that may have occurred as a result of inadequately controlling for the effects of clustering. We will also perform sensitivity analyses if ICCs were obtained from external sources.
### Reporting of the review findings {#s3c13}
We have written this protocol and will report the review findings as recommended by relevant PRISMA guidelines.[@R23] In addition, we will present the outputs of our data analysis in forest plots and GRADE summary of findings tables.[@R18]
Ethics and dissemination {#s3d}
------------------------
The review protocol does not need approval by an ethics committee because we will use publicly available data without directly involving human participants. We published an outline of the protocol[@R26] in the PROSPERO International Prospective Register of Systematic Reviews in 2014, registration number: CRD42014012888. The results will provide updated evidence on the effects of electronic and postal reminders on immunisation coverage, and we will discuss the applicability of the findings to low and middle-income countries. We plan to disseminate review findings through publication in a peer-reviewed journal and present at relevant scientific conferences. In addition, we will prepare a policymaker-friendly summary using a validated format (eg, SUPPORT Summary)[@R27] and disseminate this through social media and email discussion groups.
The authors did not receive any external funding for this manuscript. They wrote this protocol during their routine work in their respective institutions, but the views expressed therein are those of the authors and not those of their institutions. Neither the authors' institutions nor any funder or sponsor played a role in developing the protocol. The authors acknowledge the Editor and the referees for critical and constructive comments on an earlier version of this manuscript.
**Twitter:** Follow Charles Wiysonge at [\@CharlesShey](http://twitter.com/CharlesShey)
**Contributors:** MC led the development of the protocol, wrote the first draft, coordinated and integrated comments from coauthors, and approved the final version for publication. FM and VM critically revised successive drafts of the manuscript and approved the final version for publication. CSW conceived the study, provided supervision and mentorship to MC, critically revised successive drafts of the manuscript, approved the final version for publication and is the guarantor of the manuscript.
**Competing interests:** None declared.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
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Introduction
============
In the last decade, the zebrafish (*Danio rerio*) was identified as a new genetic system in which to analyze hematopoietic development. Hematopoiesis in zebrafish is similar to that in mammals and other higher vertebrates whose representative blood cell types include the erythroid, thrombocytic, myeloid and lymphoid lineages. In mammals, primitive hematopoiesis occurs outside the embryo, in the blood islands of the yolk sac. Later in development, it moves to the aorta-gonadmesonephros (AGM) region, fetal liver and ultimately the bone marrow. By contrast, primitive hematopoietic stem cells (HSCs) in the zebrafish embryo are born intra-embryonically in ventral mesoderm-derived tissue called the intermediate cell mass (ICM). During this wave, the anterior part of the embryo generates myeloid cells, while the posterior part generates mostly erythrocytes and a number of myeloid cells. From 24 h post-fertilization (hpf), these primitive blood cells start to circulate throughout the embryo. Subsequently, the definitive HSCs emerge from the ventral wall of the dorsal aorta and migrate to the posterior region in the tail called the caudal hematopoietic tissue (CHT). From 3 days post-fertilization (dpf), lymphopoiesis initiates in the thymi. By 4 dpf, HSCs seed the kidney marrow, which is equivalent to bone marrow in mammals ([@b1-etm-05-04-1043]).
Myelopoiesis is the process of producing all types of myeloid cells including granulocytes and monocytes/macrophages. A number of zebrafish myelopoietic genes are expressed in patterns consistent with their mammalian orthologs, including myeloperoxidase (*mpo*), an enzyme that is a major component of human neutrophil and eosinophil granules. It is also a marker for zebrafish granulocytes ([@b2-etm-05-04-1043]). Myeloid cells have a wide spectrum of activities, including immune surveillance and tissue remodeling. Irregularities in myeloid cell development and their function are associated with the onset and the progression of a variety of human disorders, including leukemia ([@b3-etm-05-04-1043]). An in-depth study of *mpo* expression and function in zebrafish is likely to improve our ability to identify, isolate and culture hematopoietic cells to enhance our ability to use this simple organism to address disease biology.
Whole-mount *in situ* hybridization (WISH) is the method of choice to characterize the spatial distribution of gene transcripts during embryonic development. Initial protocols used non-radioactive digoxigenin (DIG)-labeled probes that permit for the first time visualization of global gene expression patterns in *Drosophila* embryos ([@b4-etm-05-04-1043]). We present a modified protocol, using a DIG-labeled probe to detect the spatio-temporal spectrum of *mpo* expression in zebrafish, which reduces the number of steps and obtains signal enhancement.
Materials and methods
=====================
Animals
-------
The AB zebrafish strain was maintained at 28.5°C as described by Westerfield ([@b5-etm-05-04-1043]). Embryos were staged as described by Kimmel *et al* ([@b6-etm-05-04-1043]). Developmental stages refer to hpf or dpf. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Research Center for Model Organisms (Shanghai, China) with approval ID 2012-0008.
Experimental materials
----------------------
Various restriction enzymes and the TOP10 *Escherichia coli* strain were purchased from Takara Bio Inc. (Japan). T4-DNA ligase was purchased from Promega Corporation (Madison, WI, USA) and DNA high fidelity polymerase KOD-Plus was purchased from Toyobo Co., Ltd. (Japan). The DIG-deoxyribonucleotide triphosphate (dNTP) labeling kit, blocking reagents, anti-DIG-AP and BM purple were purchased from Roche Diagnostics (Indianapolis, IN, USA). RNase inhibitor was purchased from Ambion (USA), levamisole, heparin and yeast RNA were purchased from Sigma (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA) and the plasmid Maxiprep kit was purchased from Qiagen (Hilden, Germany). The first-strand cDNA Quantscript RT kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). A fluorescence microscope (SMZ-1500; Nikon, Japan), ultra-violet spectrophotometer (ASP-3700; ACT Gene, Piscataway, NJ, USA), Biometra T personal polymerase chain reaction (PCR) amplification instrument (Goettingen, Germany), gel imaging analysis system (Tanon 3500, Shanghai, China) and SPX biochemical incubator (GNP-9160; Shanghai Jinghong Laboratory Instrument Co., Ltd., China) were also used.
Cloning and mpo/pBK-CML plasmid construction
--------------------------------------------
Total RNA was extracted from 40 embryos at 48 hpf, using TRIzol reagent according to the manufacturer's instructions. The cDNA was synthesized from 1 *μ*g total RNA using the first-strand cDNA Quantscript RT kit. The specific primers were designed according to the zebrafish *mpo* (*zmpo*) genomic sequence on the National Center for Biotechnology Information (NCBI) web site (gene ID, 337514); forward primer: 5'-TTCAAGTCCAGAACCAGTGAGCCT-3' and reverse primer: 5'-TTTAGCAGTGGCAGGAAGGATGGA-3'. The length of the amplified PCR product was 2442 bp, including two restriction enzyme cutting sites (*Xho* I at 216 bp and *Eco*RI at 2384 bp) and the probe sequence (at 737 bp). The probe sequence was designed to span exon borders of the gene. PCR was performed as follows: 95°C for 10 min; then 35 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 60 sec. The integrity of the PCR product was examined by 1% agarose gel electrophoresis. The purity was analyzed based on the absorbance ratio at 260 and 280 nm (A260/280). Then the *zmpo* fragment and the pBK-CML vector were digested with *Xho*I and *Eco*RI enzymes and connected at the same sticky end with the T4 ligase, which resulted in the construct of an *mpo*/pBK-CML plasmid.
Plasmid linearization and probe incubation
------------------------------------------
The *mpo*/pBK-CML plasmid was linearized with the *Sal*I restriction enzyme. DIG-antisense RNA probes were generated by T7 *in vitro* transcription (1 *μ*g linearization template DNA, 1 *μ*l DIG-dNTP mix, 2 *μ*l 100 mM dithiothreitol (DTT), 4 *μ*l 5X transcription buffer, 1 *μ*l RNase inhibitor, 1 *μ*l T7 RNA polymerase and diethylpyrocarbonate (DEPC) H~2~O to make 20 *μ*l, incubated for 2 h at 37°C). Then, 1 *μ*l DNaseI was added and incubated at 37°C for 20 min to purify the product. The final precipitation was stored at −20°C.
Embryo preparation
------------------
The required developmental stage of the embryos was selected according to the somite. Embryos younger than 48 hpf were dechorionated. The embryos were digested in 1 mg/ml pronase for ∼2--10 min. The digestion was stopped when \>10% embryos were free from their chorions. The chorions were broken with air from a pipe. If the chorions were difficult to remove, they were manually broken with a pair of tweezers. Embryos older than 24 hpf were decolored in 5% hydrogen peroxide and 5% potassium peroxide fish water for 15 min. Embryos (up to 40 embryos per 1.5 ml eppendorf tube) were fixed in fresh 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and agitated overnight at 4°C. Then, the embryos were washed twice in PBS with Tween-20 (PBST) for 5 min each and dehydrated with gradient (25, 50, 75 and 100%) methanal/PBST for 10 min each at room temperature (RT), then stored at −20°C.
First day of hybridization
--------------------------
The embryos were removed from the −20°C storage and rehydrated with gradient (75, 50, 25 and 0%) methanal/PBST. The embryos were fixed with 4% PFA for 20 min and rinsed twice with PBST for 5 min at RT. The embryos were digested with an appropriate concentration of proteinase K (10 *μ*g/ml in PBST) at 37°C for 3 min and then fixed and rinsed as above. They were then incubated at 68°C in negative hybrid liquid (HYB^−^, 50% formamide in 5X SSC buffer) for 15 min and prehybridized at 68°C in 300 *μ*l positive hybrid liquid (HYB^+^, 0.5 mg/ml yeast RNA and 50 *μ*g/ml heparin in HYB^−^) for 4 h. Following this, the HYB^+^ was replaced with fresh HYB^+^ containing the DIG-labeled probe (concentration, ∼1 ng/*μ*l) and incubated overnight at 68°C.
Second day of hybridization
---------------------------
The probe was removed and the embryos were washed twice for 30 min each with 50% formamide in 2X SSC buffer, then for 15 min in 2X SSC buffer and twice for 30 min each in 0.2X SSC buffer at 68°C. Then, the embryos were rinsed three times for 5 min each at RT in MABT (100 mM maleic acid, 150 mM NaCl and 0.1% Tween-20; pH 7.5) and blocked for 1 h at RT with blocking solution (10% blocking reagent and 50% lamb serum in MAB). Anti-DIG-AP (1:4000 dilution in blocking solution) was added and agitated overnight at 4°C.
Third day of hybridization
--------------------------
The embryos were washed with blocking solution for 30 min and MABT for 1 h at RT. Then, the embryos were soaked three times for 5 min each in staining buffer (100 mM Tris, 50 mM MgCl~2~, 100 mM NaCl, 0.1% Tween-20 and 1 mM levamisole; pH 9.5). The embryos were transferred to a 12-well plate, incubated in 1 ml/well BM purple stain for 30 min, monitored with a dissecting microscope every 30 min. The reaction was terminated by being washed twice in PBST and fixed with 4% PFA at 4°C to store. Images were taken in 3% methyl cellulose.
Results
=======
mpo/pBK-CML plasmid construction and probe synthesis
----------------------------------------------------
The cDNA of 48 hpf embryos was used as a template to amplify the *mpo* gemonic fragment by PCR. The PCR product was verified by 1% agarose gel electrophoresis and the single band at 2400 bp was observed as expected. The PCR product was cloned into the pBK-CML carrier and the *mpo*/pBK-CML recombinant plasmid was constructed. The plasmid was linearized by *Sal*I and the single band at 7300 bp was observed. The plasmid was digested with two enzymes (*Xho*I and *Eco*RI), producing two bands at 5200 and 2100 bp, respectively, which were the pBK-CML vector and *mpo* gene segment. This demonstrated that plasmid construction was successful ([Fig. 1A](#f1-etm-05-04-1043){ref-type="fig"}). The DIG-labeled antisense *mpo* mRNA probe was generated by T7 *in vitro* transcription and confirmed in the electrophoresis tank soaked with DEPC H~2~O overnight. There was a single band near 2100 bp ([Fig. 1B](#f1-etm-05-04-1043){ref-type="fig"}).
Temporal and spatial expression patterns of mpo
-----------------------------------------------
The expression patterns of *mpo* were investigated in zebrafish embryos from 12 to 72 hpf by *in situ* hybridization using the DIG-labeled antisense RNA probe. As shown in [Fig. 2](#f2-etm-05-04-1043){ref-type="fig"}, the earliest expression of zebrafish *mpo* was detected in cells of the ICM at 18 hpf and 1 to 2 h later, it was detected in cells in the rostral blood island (RBI). Strong signals were observed in the anterior ICM, then it spread over the yolk sac. By 72 hpf the *mpo*-expressing cells were in the circulation and distributed throughout the embryo. In our previous study, we established the transgenic enhanced green fluorescent protein \[Tg(*zlyz*:EGFP)\] zebrafish line ([@b7-etm-05-04-1043]), which expresses EGFP in primitive neutrophils (*mpo*^+^). The EGFP distribution coincided with the result of *in situ* hybridization of *mpo* ([Fig. 2](#f2-etm-05-04-1043){ref-type="fig"}).
In situ hybridization and cytological analyses
----------------------------------------------
To test whether *mpo* expression detected by *in situ* hybridization at an early stage could predict the myelopoiesis in zebrafish development, we compared the peripheral blood smear between wild type (WT) and Tg zebrafish. In our previous study, we established the Tg(*MYCN*:HSE:EGFP) zebrafish line, which suggested that *MYCN* converted erythropoiesis to myelopoiesis (Shen *et al*, the influence of MYCN gene on the transcriptional regulation of hematopoiesis. abs. 751, 9--12 June, 2011). As demonstrated by *in situ* hybridization, the *MYCN* gene increases the expression of *mpo* in embryos at 22 hpf ([Fig. 3A and C](#f3-etm-05-04-1043){ref-type="fig"}). For cytological analyses, blood cells collected from the zebrafish at 60 dpf were transferred onto glass slides using Cytospin, stained with Wright-Giemsa stain and examined under oil immersion by light microscopy ([@b8-etm-05-04-1043]). This revealed that the blood cells from WT fish were predominantly erythrocytes, with myeloid cells occasionally observed. By contrast, erythrocytes were significantly inhibited in Tg fish, which were enriched with granulocytes ([Fig. 3B and D](#f3-etm-05-04-1043){ref-type="fig"}).
Modified WISH
-------------
In order to avoid background staining, unincorporated nucleotides were removed from the probe preparation. We routinely used Ambion NucAway Spin Columns to purify the RNA probe according to the manufacturer's instructions (Cat. AM10070). Following the final precipitation, the hydrolyzed probe was placed in HYB^+^ (final concentration, ∼1 ng/*μ*l) and stored at −20°C. Each time the probe was used, it was degenerated in advance by placing it at 95°C for 5 min then on ice for 5 min to eliminate single RNA spontaneous formation of hairpin structures. Probes were reused for 4--5 times. When 24 hpf embryos were collected, the conventional decolorization method was used and they were placed in 0.03% phenylthiourea (PTU) in fish water at 12--36 hpf. We used the improved decolorizing liquid of 5% hydrogen peroxide and 5% potassium peroxide in fish water. As a result, the embryos only required soaking at 24 hpf for 10--15 min. The effect of decolorization was good without damaging the integrity of the embryo. Prior to hybridization, the embryos were permeabilized by digesting with proteinase K (10 *μ*g/ml in PBST) at RT for 5--30 min, depending on the developmental stage. The permeability of the probe was appropriate and the staining background was low. In addition, the traditional permeabilization method included digesting the embryos at RT for 5--30 min, depending on the developmental stage. This is difficult to control. We improved the digestion temperature to 37°C and the process was shorted to 5 min.
Discussion
==========
The zebrafish system has a number of unique advantages compared to other vertebrate model organisms. The embryos are externally fertilized and transparent, enabling *in vivo* visualization of early embryonic processes ranging from birth of HSCs in the mesoderm to migration of blood cells. In addition, large production of embryos makes phenotype-based forward genetics feasible ([@b9-etm-05-04-1043]). A steadily increasing number of hematopoietic-specific genes have been cloned in zebrafish, providing molecular reagents and markers for specific stages of hematopoietic differentiation and specific cell types. We constructed the DIG-labeled *mpo* RNA probe to investigate *mpo* gene expression in zebrafish embryos. The earliest *mpo* expression was detected in cells of the ICM at 18 hpf and 1--2 h later, it was detected in cells in the RBI. Strong signals were observed in the anterior ICM, then it spread over the yolk sac. By 72 hpf the *mpo*-expressing cells were in the circulation and distributed throughout the embryo, with a tendency for a subpopulation of *mpo*-expressing cells to be aggregated in the ventral vein region. Later-stage expression was difficult to distinguish by WISH.
We identified that the level of *mpo* expression detected by WISH at an early stage was consistent with the result of cytological analyses of adult fish ([Fig. 3](#f3-etm-05-04-1043){ref-type="fig"}). This method enabled us to track the gene changes that took place before morphological phenotypes were detected, as well as investigate the hematopoietic cell fate in mutational or transgenic models *in vivo*. Given the considerable morphological and functional parallels between zebrafish and mammalian myeloid cells, it is not surprising that zebrafish show conservation of the molecular regulation of myelopoiesis and the molecular tools for myeloid cell function ([@b3-etm-05-04-1043]).
In this study, we modified several steps of WISH. We carried out several pretreatment steps to ensure the purity and concentration of the probe, as well as shorten the digestion time by proteinase K at higher temperatures. In the decolorization step, we avoided using PTU, which simplified the zebrafish embryo culture steps and enhanced the environmental protection. Finally, we selected BM purple dye to simplify the staining step. The improved hybridization results demonstrated high specificity, distinct coloration and low background figures.
Using WISH in zebrafish, we have the ability to identify and study the lesions of myelopoiesis. Therefore, the powerful genetic approaches applicable in this model, the genomic resources being collected by the international zebrafish and genomic communities and the ability to study myeloid development in this model organism provide new insights into the myeloid arm of developmental hematology.
The authors thank all members of the Shanghai Research Center for Biomodel Organisms and the Shanghai Institute of Hematology for excellent technical support. This study was supported in part by the National Natural Science Foundation of China Grant (30900636), the Science and Technology Commission of Shanghai Municipality Grant (08JCl414900), the Science and Technology Commission of Shanghai Fund Project of the TCM Guide Project (12401906700) and the Science and Technology Fund Project of Shanghai Jiaotong University School of Medicine (09XJ21066).
{#f1-etm-05-04-1043}
{#f2-etm-05-04-1043}
{#f3-etm-05-04-1043}
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[^1]: Read at a Meeting of the Tuberculosis Society of Scotland on 21st April 1933.
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Background
==========
Cassava (*Manihot esculenta* Crantz) is an important tropical root crop that plays an important role not only in ensuring food security, but also in various bioindustrial applications such as animal feed, modified starch and biofuels \[[@B1]\],\[[@B2]\]. Because cassava is native to tropical regions, it is extremely cold-sensitive; its growth is drastically affected at temperatures lower than 18°C, and the plant cannot survive for long after exposure to freezing conditions \[[@B3]\]--\[[@B5]\]. Therefore, low temperatures and freezing conditions are the most important limiting factors for the cultivating location, as well as productivity. The damage of apical shoot seems be to more critical than other parts of the cassava plant \[[@B5]\]. Improving the tolerability of the cassava plant to multiple stresses has therefore, become a major objective of cassava breeders, especially in subtropical regions \[[@B4]\],\[[@B5]\]. Under cold conditions, up-regulation of reactive oxygen species (ROS) turnover and scavenging in cassava has been reported, and therefore, genetic manipulation of intracellular ROS level might be an effective approach in improving tolerance to abiotic stresses in this tropical crop \[[@B5]\]--\[[@B7]\].
In the ROS scavenging system that is responsible for homeostasis in plant cells, superoxide dismutases (SODs, EC 1.15.1.1), enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, provide the first line of defense against ROS in various subcellular compartments, i.e. chloroplast, mitochondria and cytosol \[[@B8]\]. Essentially, there are three types of SODs, each containing either manganese, iron, or copper plus zinc as a prosthetic group \[[@B9]\]. Along with other ROS scavenging mechanisms like catalase (CAT; EC 1.11.1.6), glutathione peroxidases (GPXs) and peroxiredoxin reductases (PrxRs), and the ascorbate--glutathione (ASC-GSH) cycle, the ROS levels are maintained in a homeostatic state. In the ASC-GSH cycle, using ascorbate as an electron donor, ascorbate peroxidase (APX, EC 1.11.1.1) scavenges potentially harmful hydrogen peroxide to water from the chloroplasts and mitochondria, as well as other organelles \[[@B10]\],\[[@B11]\]. Therefore, the formation of toxic hydroxyl radicals by superoxide and hydrogen peroxide can be controlled by the combined enzymatic actions of SOD and APX \[[@B12]\].
Transgenic plants that express SOD or APX have shown enhanced tolerance to multiple stresses \[[@B13]\]. For example, over-expression of different SODs (FeSOD, MnSOD or Cu/ZnSOD) in transgenic plants of tomato, rice, poplar, alfalfa, etc., showed increased tolerance to methyl viologen (MV), ozone, high salinity, chilling or other stresses \[[@B14]\]--\[[@B17]\]. Transgenic plants have also demonstrated an increased tolerance against various abiotic stresses by the expression of either cytosolic- or organelle-targeted cytosolic APX \[[@B18]\]--\[[@B22]\]. However, some reports suggest no change in response to oxidative or environmental stress with the expression of a single antioxidant enzyme \[[@B23]\],\[[@B24]\]. These contradictory findings may be due to the complex network of plant antioxidant defenses, which possibly confer a higher tolerance to oxidative stress by pyramiding or stacking of multiple genes in a single genotype \[[@B25]\]. The gene-stacking approach entails manipulation of two or more desirable enzymes mediating the ROS turnover and scavenging pathways, in improving the abiotic stress tolerance in plants.
Indeed, co-expression of two distinct ROS-scavenging enzymes, such as SOD and other ROS-scavenging enzymes, in the chloroplasts or cytosol in transgenic plants has a synergistic effect in increasing the levels of abiotic stress resistance. For example, coupled expression of Cu/ZnSOD and APX in transgenic plants of *Festuca arundinacea*, potato, tobacco, sweet potato and plum led to increased tolerance to multiple abiotic stresses, e.g., the herbicide methyl viologen (MV), chilling, high temperature and drought \[[@B12]\],\[[@B26]\]--\[[@B29]\]. Payton et al. \[[@B30]\] showed that co-expression of glutathione reductase (GR, EC 1.6.4.2) and APX in cotton improved antioxidant enzyme activity during moderate chilling at high light intensity, in chloroplasts \[[@B30]\]. Co-expression of the *Suaeda salsa* glutathione S-transferase (GST) and CAT1 in rice also caused tolerance to stresses caused by salt and paraquat \[[@B31]\]. Taken together, these data indicated that the combination of transgenes encoding different ROS-scavenging enzymes in various subcellular compartments might have a synergistic effect in improving stress tolerance.
Lately, plant breeders and biotechnologists have appreciated the molecular insights and advances in cassava abiotic stress resistance, on a global scale. Apart from the various approaches from traditional breeding to field evaluation \[[@B32]\], studies of cassava response to drought or cold stress at the molecular level have reportedly used the "omics" technology, e.g., expressed sequence tags, cDNAs and oligonucleotide microarray \[[@B5]\],\[[@B33]\]--\[[@B38]\]. However, few studies on improved tolerance to environmental stresses using genetic engineering have been reported \[[@B7]\],\[[@B39]\]. Senescence-induced expression of the isopentenyl transferase gene in cassava showed increased drought resistance, as observed by the elevated content of cytokinin in mature leaves, and prolonged leaf life \[[@B39]\]. Enhanced ROS scavenging by simultaneous expression of cytosolic *Me*Cu/ZnSOD and peroxisomal *Me*CAT1 in transgenic cassava also confirmed the improved tolerance towards drought and cold temperatures \[[@B7]\].
In the present study, transgenic cassava plants co-expressing both cytosolic *Me*Cu/ZnSOD (Genbank accession No. AY642137) and cytosolic *Me*APX2 (GenBank accession No. AY973622) showed enhanced ROS scavenging capacity, thereby leading to enhanced tolerance to oxidative stresses that was induced by MV, H~2~O~2~ as well as chilling. Our results suggest that manipulation of ROS-scavenging enzyme systems by the overexpression of both cytosolic SOD and APX is a worthwhile approach to produce transgenic plants with enhanced tolerance to a wide range of abiotic stresses.
Results
=======
Subcellular localization of *Me*Cu/ZnSOD and *Me*APX2
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Green fluorescent protein (GFP) was the fusion protein used for subcellular localization of *Me*Cu/ZnSOD and *Me*APX2. The control, CaMV 35S-eGFP construct, exhibited GFP fluorescence in the cytoplasm and nucleus of agroinfiltrated cells of *N. benthamiana* leaves. The *Me*Cu/ZnSOD has been reported as a cytosolic SOD isoform in our previous report \[[@B7]\]. The *Me*APX2 was located in the cytosol of the leaf cells, as indicated by the fused GFP fluorescence (Additional file [1](#S1){ref-type="supplementary-material"}) that was consistent with the signal peptide sequences predicted by Reilly et al. \[[@B38]\]. Therefore, both enzymes used in the study were cytosolic proteins.
Molecular characterization of the transgenic plants
---------------------------------------------------
Four independent transgenic plant lines (named SA1, SA2, SA4 and SA6) harboring the *MeCu/ZnSOD* and *MeAPX2* gene-expressing cassettes (Figure [1](#F1){ref-type="fig"}a) were produced by the use of *Agrobacterium*-mediated transformation and subcultured *in vitro* regularly. Confirmation of the single integration event of the pC-P54::MeCu/ZnSOD-35S::MeAPX2 T-DNA in these transgenic lines were carried out by the Southern blotting technique using *Xba*I-digested cassava genomic DNA, which were extracted from leaves of *in vitro* plants and hybridized with DIG-labeled *HPT* probe (Figure [1](#F1){ref-type="fig"}b, left panel). No signals were detected in the wild-type (WT) plants. However, when these WT plants were hybridized with the *MeAPX2* probe (Figure [1](#F1){ref-type="fig"}b, right panel), two bands of \~13 kb and \~6.6 kb were detected, indicating the possibility of two *APX* homologs in the cassava genome. The transgenic SA lines showed an additional band in their hybridization pattern, confirming that the transgenic plants were integrated at a single site (Figure [1](#F1){ref-type="fig"}b, right panel). All transgenic plants except SA2 line thrived successfully in the greenhouse and field, with a relatively normal leaf phenotype, growth capacity and root tuberization, similar to WT plants (Figure [1](#F1){ref-type="fig"}c); the SA2 line had a slightly dark-green and curled leaves when cultivated in the field. No significant differences of yield were found between WT and transgenic plant lines (Additional file [2](#S2){ref-type="supplementary-material"}).
{#F1}
The basic transcriptional levels of *MeCu/ZnSOD* and *MeAPX2* in cassava leaves were confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) (Figure [2](#F2){ref-type="fig"}a). The expressions of *MeCu/ZnSOD* and *MeAPX2* in transgenic lines were higher by up to 20- and 5-fold, respectively, when compared to that of WT plants. SOD expression level was about 5-times higher than APX2 in the SA lines, indicating that the *p54* promoter is stronger than CaMV 35S promoter in cassava, which is in agreement with the previous report \[[@B40]\]. Further, the changes in SOD and APX isoenzyme activity from leaves on non-denaturing gel were also confirmed (Figure [2](#F2){ref-type="fig"}b). A characteristic SOD isoenzyme banding pattern was found in all cassava leaves with Cu/ZnSOD, MnSOD and FeSOD \[[@B6]\], but the intensity of SOD bands was higher in all transgenic lines than the WTs. Similarly, a stronger APX isoenzyme band was also observed in all transgenic lines than that of WT (Figure [2](#F2){ref-type="fig"}b). These results indicate that the heightened production of SODs and APXs were achieved because of the transgene expression in transgenic cassava.
{#F2}
Higher protoplast viability and mitochondrial integrity of mesophyll cells under H~2~O~2~ stress
------------------------------------------------------------------------------------------------
Cell death and loss of mitochondrial integrity are indicators of stress damage. The viability of extracted transgenic and WT mesophyll protoplasts was above 95%, when stained by fluorescein diacetate (FDA). After 1 M H~2~O~2~ treatment, the viability of transgenic protoplasts from SA1, SA2, and SA4 decreased to 77%, 74%, and 80%, respectively, but the WTs showed 52% (Figure [3](#F3){ref-type="fig"}a), indicating significant improvement to stress tolerance. The mitochondrial integrity of mesophyll cells were observed by rhodamine 123 (Rh 123) staining. Strong Rh 123 fluorescent signals were observed in all cassava cells prior to H~2~O~2~ treatment, indicating normal mitochondrial activity in these cells. After H~2~O~2~ treatment, the protoplasts of the WT plants displayed diffuse and much weaker fluorescent signals, but the protoplasts derived from the transgenic plants retained their intense fluorescence activity (Figure [3](#F3){ref-type="fig"}b). The difference between transgenic lines and WT was evident, which indicated that the cells in transgenic plants displayed higher H~2~O~2~ tolerance than that in WT plants.
{#F3}
Enhanced tolerance to H~2~O~2~-mediated oxidative stress in transgenic leaves
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To evaluate the response of transgenic plants to oxidative stress, leaves showing a basal level of H~2~O~2~ production, as indicated by 3,3′-diaminobenzidine (DAB) staining (data not shown), were selected and treated with 0.5 M H~2~O~2~. After 24 hours, H~2~O~2~ molecules significantly accumulated in WT leaves (Figure [4](#F4){ref-type="fig"}a) at a concentration of 20.8 mmol/g, which was 1.6-times higher than that in the untreated leaves (Figure [4](#F4){ref-type="fig"}b). Leaves of transgenic plants showed much less H~2~O~2~ accumulation (Figure [4](#F4){ref-type="fig"}a), as observed by the elimination of the reaction product, DAB-H~2~O~2~, from a majority of leaves from the SA4 plants (Figure [4](#F4){ref-type="fig"}b). For example, the H~2~O~2~ concentration in the leaves of SA4 was 14.6 mmol/g, which was 11.6% higher than that in the untreated leaves.
{#F4}
The activities of SOD and APX in cassava leaves were assessed under normal and stressful conditions (Figure [4](#F4){ref-type="fig"}c, d). Enzyme activities were not significantly different between WT and transgenic plants under normal conditions. However, after stress induction, the SOD activity of WT plants decreased to 20% of the baseline value in 24 hours (1.7 U mg^−1^ protein, Figure [4](#F4){ref-type="fig"}c). In contrast, the SOD activity of all transgenic plant lines increased in 12 hours; for example, the activity level in SA4 showed a 2-fold increase as compared to 0 hours with 23.4 U mg^−1^ protein. However, their activities decreased at 24 hours; the lowest value was observed in SA1 plants with 6.6 U mg^−1^ protein, which was about 4-times higher than that of WT plants (Figure [4](#F4){ref-type="fig"}c). A consistent increase in APX activity was detected in all leaves, especially in SA1, with the activity level showing a 3-fold increased at 24 h as compared to 0 h; protein levels increased from 0.06 μmol · min^−1^ · mg^−1^ protein to 0.33 μmol · min^−1^ · mg^−1^ protein. The WT only showed an increase from 0.05 μmol · min^−1^ · mg^−1^ protein to 0.17 μmol · min^−1^ · mg^−1^ protein. At 24 hours, all transgenic plant leaves showed a significant increase in activity compared to the WT plant leaves (Figure [4](#F4){ref-type="fig"}d).
Enhanced tolerance to methyl viologen (MV)-mediated oxidative stress in transgenic leaves
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The effect of methyl viologen (MV), a superoxide-generating herbicide, on cassava was determined by subjecting the leaves to 100 μM MV for 2 days. Compared to WT plants, the extent of chlorophyll loss due to MV was significantly less in transgenic leaves (Figure [5](#F5){ref-type="fig"}a). As shown in Figure [5](#F5){ref-type="fig"}b, the chlorophyll content was not significantly different between WT and transgenic plants prior to MV treatment. After MV treatment, the chlorophyll content of WT plant leaves decreased by 75%, whereas in SA1, SA2 and SA4 leaves, the chlorophyll loss was only 21%, 37% and 38%, respectively. MV treatment causes membrane-lipid peroxidation, leading to an increase in the malondialdehyde (MDA) content. After MV treatment, the MDA content in WT plants increased to a maximum of 45%, whereas in transgenic lines the MDA content increased by approximately 14% (Figure [5](#F5){ref-type="fig"}c). The average amount of MDA in WT was 11.5 nmol/g Fresh Weight (FW), which had 2.8 nmol/g FW more than SA1 line. These data suggests that transgenic cassava has less lipid peroxidation because of timely ROS scavenging. The impact of ROS scavenging enzymes in transgenic cassava was further confirmed when both SOD and APX activities were significantly enhanced after treatment (Figure [5](#F5){ref-type="fig"}d and e). The SOD and APX activity was approximately 1.5-times higher in transgenic plants as compared to WT plants. The highest activity of SOD reached 21 U/mg protein in SA4 line; and the highest value of APX was found in SA1 line with 0.18 μmol · min^−1^ · mg^−1^ protein. These data confirmed that improved performance of transgenic cassava leaves against oxidative stress is due to elevated SOD and APX activities of the ROS scavenging system.
{#F5}
Improved cold tolerance of transgenic plants
--------------------------------------------
Two-month-old plants were given a chilling treatment by transferring into a growth chamber at 4°C for 2 days. After the treatment, the WT plants wilted severely, whereas the transgenic lines were slightly affected, with fewer leaves wilting (Figure [6](#F6){ref-type="fig"}a). Among the three transgenic cassava lines, the SA1 line was the least affected by the chilling treatment. The level of MDA increased by 7%, 8%, and 20% in SA1, SA2, and SA4 lines after chilling stress, respectively. However, WT showed a 40% increase in MDA content, which was significantly higher than that of transgenic lines (Figure [6](#F6){ref-type="fig"}b).
{#F6}
Other ROS-scavenging enzymes were monitored in both WT and SA transgenic cassava for their response to cold stress. Before treatment, both the WT and all the transgenic lines showed similar level of enzymatic activity. Upon cold treatment, increased activity of SOD, CAT, APX, MDHAR, DHAR and GR were confirmed in the transgenic lines as compared to the WT plants (Figure [7](#F7){ref-type="fig"}). The APX activity in SA lines showed \>2-fold increase than WT (Figure [7](#F7){ref-type="fig"}c). The increase in SOD and CAT activities in transgenic lines was about 1.5-fold that of WT after treatment (Figure [7](#F7){ref-type="fig"}a, b). In the ascorbate cycle, MDHAR, DHAR and GR increased up to 43.6%, 30.6% and 28.6%, respectively (Figure [7](#F7){ref-type="fig"}d, e, f). However, no significant changes in enzyme activity were observed in WT before and after the treatment.
{#F7}
Discussion
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The cassava plant is generally considered to be cold-sensitive, and abiotic stresses, such as low temperature and salinity, dramatically affect their growth, thereby, leading to reduced productivity \[[@B41]\],\[[@B42]\]. Excessive ROS generation, which might result in hypersensitive response and cell death in cassava, has been identified as an important indicator of such conditions \[[@B5]\],\[[@B36]\]. Therefore, maintaining ROS homeostasis via ROS production and scavenging mechanisms is critical \[[@B36]\],\[[@B43]\]. Optimum regulation of ROS generation and scavenging by the mobilization of various pathways has been proposed as a vital mechanism for managing stress in cassava \[[@B5]\],\[[@B36]\], and our previous study had confirmed that increased resistance to abiotic stress could be achieved in transgenic cassava by over-expression of cytosolic Cu/ZnSOD and peroxisomal CAT1 \[[@B5]\],\[[@B7]\]. In this study, we showed that transgenic cassava with coupled expression of cytosolic *Me*Cu/ZnSOD and cytosolic *Me*APX2 leads to improved oxidative and chilling stress resistance through elevated ROS scavenging. Our studies confirmed that the response to abiotic stress can be improved in cassava by genetic engineering techniques that combine two different ROS-scavenging pathways, i.e., SOD/CAT and SOD/ASC-GSH cycle.
Most of ROS scavenging enzymes co-expressed targeted the chloroplast \[[@B26]\]--\[[@B28]\]. Recently, Faize et al. \[[@B29]\] and Diaz-Vivancos et al. \[[@B12]\] reported that over-expression of cytosolic SOD and APX in tobacco and plums improved drought and salt tolerance. An interesting question raised is the effectiveness of the stacked ROS-scavenging enzymes with various subcellular targeting strategies in plants, undoubtedly their individual important function in stress defense \[[@B43]\],\[[@B44]\]. Since the substrate for APX activity is H~2~O~2~ that is a by-product of SOD action, their individual functions should be combined to achieve a synergistic effect on stress tolerance. Indeed, upon chilling, the enzymatic activity of MDHAR, DHAR and GR in the ASC-GSH cycle in the transgenic cassava was significantly increased (Figure [7](#F7){ref-type="fig"}). We noticed that, comparing to the transgenic cassava overexpressed cytosolic SOD and peroxisomal CAT \[[@B6]\], the SA transgenic cassava showed less tolerance to post-harvest physiological deterioration of their storage roots (data not shown). We suppose that the PPD occurrence in cassava storage root is directly linked to the oxidization process in the subcellular organelles of parenchyma cells; the action of in the peroxisome enables the timely scavenging of excess ROS generated *in situ*.
Both H~2~O~2~ and MV treatment in the presence of light lead to the generation of superoxide radicals and H~2~O~2~ in chloroplasts and mitochondria of plants. *In vivo* imaging of ROS using the fluorescent probe rhodamine-123, a stain readily sequestered by active mitochondria and mesophyll protoplasts, or DAB staining of cassava leaves showed improved tolerance of transgenic cassava cells to oxidative stress caused by H~2~O~2~ and MV (Figures [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}). At the cellular level, the viability of the mesophyll protoplast and integrity of the mitochondrial were clearly indicated by their tolerance to H~2~O~2~ in transgenic cassava cells (Figure [3](#F3){ref-type="fig"}). These results indicate that transgenic plants expressing both cytosolic SOD and cytosolic APX are able to rapidly scavenge superoxide and hydrogen peroxide at the site of generation, as well as prevent the formation of hydroxyl radicals, the most toxic ROS, prior to their interaction with target molecules, as noted in our previous report \[[@B6]\]. The changes were observed not only in cytosolic antioxidant system but also in the chloroplasts and mitochondria, which implied that changes in the cytosolic antioxidant defense impacts the subcellular compartments, consistent with previous reports \[[@B7]\],\[[@B12]\],\[[@B29]\],\[[@B45]\]. Therefore, ROS scavenging system in plant cells is an integrative network for developing an antioxidant machinery through cytosol and subcellular organelle interactions.
Under cold conditions, non-transformed cassava plants showed signs of oxidative stress-induced cellular damage such as wilting, and increased lipid peroxidation of leaves (Figure [6](#F6){ref-type="fig"}). Cassava is very sensitive to low temperature; cold temperatures disrupt the metabolic balance of plant cells, resulting in enhanced production of ROS, e.g. H~2~O~2~\[[@B5]\]. The timely detoxification of ROS is necessary for the maintenance of the Calvin cycle and transpiration \[[@B18]\]. Transcriptome profiling of low temperature-exposed cassava showed an increase in transcripts and enzyme activities of ROS scavenging genes and the accumulation of total soluble sugars \[[@B5]\]. In the SA transgenic lines, we confirmed that the enzyme activity of SOD and APX were significantly higher than that of WT during cold treatment (Figure [7](#F7){ref-type="fig"}). Increased catalytic activity of APX eliminated the endogenous H~2~O~2~ via the ASC-GSH cycle, which involves GR, DHAR and MDHAR, to restore the cellular redox state, thus, suggesting that the performance of ascorbate-glutathione cycle is essential for the regulation of an efficient system for scavenging the accumulated ROS in leaves during chilling stress. This response was correlated with the up-regulation of APX activity and by maintenance of ASC-GSH redox pools in cold-acclimated plants \[[@B46]\].
A higher CAT activity was also observed in the SA lines under stress conditions. This increase seemed to be involved in the removal of excess H~2~O~2~. Dong et al. \[[@B47]\] reported that peroxisomal metabolism responded to cold regulation through ROS by increasing the H~2~O~2~ production in the peroxisome \[[@B47]\]. H~2~O~2~ could also diffuse through the peroxisomal membrane into the cytosol \[[@B48]\], thus increasing the risk of oxidative damage in this compartment. It suggests that ROS scavenging system in cassava is a complex network involving multiple components of ROS production, turnover and scavenging as well as their cross-interactions.
Conclusions
===========
In conclusion, our results show the important role of cytosolic *Me*Cu/ZnSOD and cytosolic *Me*APX2 in cassava in improving ROS scavenging, thereby leading to reduced H~2~O~2~ accumulation and improved abiotic stress resistance. The results also confirm that the transgenic approach is effective in improving the stress resistance in cassava via proper gene stacking of ROS scavenging enzymes.
Methods
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Plasmid constructions, cassava transformation and phenotype evaluation of transgenic cassava
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The cDNA amplification of cassava *Cu/ZnSOD* (GenBank accession no. AY642137) and cassava *APX2* (GenBank accession no. AY973622) was determined by PCR using primers covering the full length of transcripts. The PCR fragment was sequenced and cloned into the binary vector pCAMBIA1301 under the control of vascular-specific promoter p54/1.0 promoter (GenBank accession no. AY217353.1) \[[@B40]\] and the ubiquitous CaMV 35S promoter, respectively to generate pC-P54::MeCu/ZnSOD-35S::MeAPX2 (Figure [1](#F1){ref-type="fig"}a). The plasmid was mobilized into *Agrobacterium tumefaciens* strain LBA4404 for cassava transformation using friable embryogenic callus of cultivar TMS60444. Embryogenic callus induction of cassava TMS60444, *Agrobacterium*-mediated genetic transformation and plantlet regeneration were performed by the methods described by Zhang et al. \[[@B49]\].
The uniform stem cuttings of field-grown cassava plants were used for phenotype and yield evaluation. Ten stem cuttings per transgenic line and WT were planted in late spring of 2012 in Wushe Plantation for Transgenic Crops, Shanghai, China, and harvested in early November, 2012. The performance of field plants was recorded regularly till harvest.
Subcellular localization
------------------------
The *MeAPX2* genes tagged with green fluorescent protein (GFP) were cloned into pCAMBIA1301, and the resulting constructs were used to transform *Agrobacterium tumefaciens* LBA4404. To identify the subcellular localization of the gene, *N. benthamiana* leaves were agroinfiltrated with the *Agrobacterium* strains harboring pC-35::MeAPX2-eGFP binary vectors, respectively, by the protocol described previously \[[@B50]\]. At 36 hours post-infiltration, the transiently transformed leaves were observed under a confocal microscope (FluoView FV1000, Olympus, Japan).
Southern blot and Real-time RT-PCR analyses of transgenic cassava plants
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Southern blot analysis entailed digestion of the genomic DNA (20 μg) of cassava leaves of *in vitro* plants with *Xba*I, followed by 0.8% (w/v) agarose gel electrophoresis and subsequent transfer to a positively charged nylon membrane (Roche, Mannheim, Germany). The PCR fragment of *MeAPX2* (1 Kb) was labeled with digoxigenin (DIG) using the PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). Hybridization and detection were performed by using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany), according to the manufacturer's instructions.
Gene expression was analyzed by real-time RT-PCR (qRT-PCR) of transgenic plants. Briefly, total RNA was extracted from fresh cassava leaves of *in vitro* plants using the RNA Plant plus Reagent (Tiangen, Beijing, China) essentially as described previously \[[@B51]\]. The RNA samples were digested with DNase I and the first strand of cDNA was synthesized from 5 μg total RNA from each sample using M-MLV reverse transcriptase (Toyobo, Osaka, Japan). qRT-PCR was carried out using the Bio-Rad CFX96 thermocycler SYBR Green I Master Mix (Toyobo, Osaka, Japan) according to the manufacturer's protocol, under the following PCR conditions: 95°C for 1 minute, followed by 40 cycles at 95°C for 15 seconds, 60°C for 15 seconds and 72°C for 20 seconds. The qRT-PCR primers were designed using Primer 3 Plus Software (<http://www.primer3plus.com>). The primers were: *MeCu/ZnSOD* (forward 5′- ATGTTCATGCCCTTGGAGAC -3′ and reverse 5′- GATCACCAGCATGACGAATG -3′), *MeAPX2* (forward 5′- CATTGATAAGGCCAGGAGGA -3′ and reverse 5′- TTGTTAGCAGCATGACCCTG - 3′), and *β-actin* (forward 5′- TGATGAGTCTGGTCCATCCA -3′ and reverse 5′- CCTCCTACGACCCAATCTCA -3′). Fold changes of RNA transcripts were calculated by the 2^-ΔΔCt^ method \[[@B52]\] with *β-actin* as an internal control \[[@B6]\]. A detailed method for qRT-PCR analysis is available in Additional file [3](#S3){ref-type="supplementary-material"}.
Isolation and viability assay of cassava mesophyll protoplasts
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*In vitro*-cultured cassava seedlings were grown at 25°C with light at 200 μmol · m^−2^ · s^−2^ and a photoperiod 16 hour/8 hour (light/dark). Protoplasts were isolated from the mature leaves of 30-day-old sterile plants, as described by Anthony et al. \[[@B53]\]. Leaves were sliced into 2 mm to 3 mm thin strips and incubated in digestion mixture (pH 5.8) containing 9% (w/v) mannitol, 1% Cellulose R-10 (Yakult Honsha Co., Tokyo, Japan), 1.5% macerozyme R-10 (Yakult Honsha Co., Ltd., Tokyo, Japan) and 25 mM morpholine ethanesulfonic acid (MES) in cell protoplast washing (CPW) medium (27.2 mg of KH~2~PO~4~, 101 mg of KNO~3~, 1480 mg of CaCl~2~ · 2H~2~O, 246 mg of MgSO~4~ · 7H~2~O, 0.16 mg of KI, 0.025 mg of CuSO~4~ · 5H~2~O per liter). After purification, the protoplasts were resuspended in CPW solution supplemented with 9% mannitol (CPW9M, pH 7.0) to a final concentration of 2 × 10^5^ cells/mL.
Protoplast viability was determined by fluorescein diacetate (FDA) staining \[[@B54]\]. The purified mesophyll protoplasts were treated for 5 minutes with CPW 9 M solution (pH 7.0) supplemented with 1 M H~2~O~2~ for 5 min, and then stained with FDA at a final concentration of 0.01%. Stained protoplasts were observed for fluorescence under a fluorescent microscope (Nikon TE2000-S, Japan). The viable percentage = (the number of protoplasts with green fluorescence)/(the number of total mesophyll protoplasts) × 100%.
Analysis of mitochondrial integrity
-----------------------------------
Mitochondrial integrity was measured by rhodamine 123 (Rh 123, Molecular Probes-Invitrogen CA, USA) fluorescence \[[@B55]\] after cassava mesophyll protoplasts were treated with 1 M H~2~O~2~ for 5 min. Fluorescence was detected using a confocal laser scanning microscope (FluoView FV1000, Olympus, Japan) with Ex/Em of 488 nm/515 nm. In each sample, 20--25 cells were scanned and viewed.
Treatment with MV, H~2~O~2~ and cold
------------------------------------
Fully expanded, healthy leaves were excised from the one-month-old plants in greenhouse and put in 10 cm-diameter petri dishes with different chemical solutions. For the MV treatment, leaves were allowed to float on 50 mL of 100 μM MV, and for the H~2~O~2~ treatment, leaves were exposed to 50 mL of 0.5 M H~2~O~2~. Leaves were incubated at 25°C under light conditions.
Four-week-old seedlings of transgenic and the control plants were transplanted into pots (30 cm in diameter, 45 cm in height) and grown in a greenhouse (16 h days, 30°C days and 22°C nights). Two-month-old plants were subjected to stress conditions. For cold treatment, plants with a uniform growth status were transferred to a chamber and incubated at 4°C for 48 hours under weak light (cool-white fluorescent light at approximately 35 μmol · m^−2^ · s^−1^).
Determination of chlorophyll content and lipid peroxidation
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Chlorophyll was isolated from WT and transgenic leaf segments according to the procedure described by Arnon et al. \[[@B56]\]. One-gram leaf disks from the one-month-old plants were homogenized in 10 mL of absolute ethyl alcohol and the homogenate was centrifuged at 3500 × *g* for 5 minutes. The supernatant was retained and the absorbance was recorded at 663 nm and 646 nm using a Nano-Drop spectrophotometer (Thermo Scientific, Scientific, Wilmington, DE, USA). Lipid peroxidation in leaf tissues was measured in terms of MDA content in the samples, according to method described by Heath and Packer \[[@B57]\]. One-gram leaves were homogenized in 10 mL of 10% (w/v) trichloroacetate (TCA) and centrifuged at 10,000 × *g* for 10 minutes. Thereafter, 2 mL of 10% trichloroacetic acid containing 0.67% (w/v) thiobarbituric acid was added to 2 mL of the supernatant. The mixture was boiled for 15 min, quickly cooled on ice, and centrifuged at 10,000 × *g* for 5 minutes. The absorbance of the supernatant was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm using a Nano-Drop spectrophotometer (Thermo Scientific).
Determination of H~2~O~2~ content and 3,3′-diaminobenzidine staining
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After determining the lipid peroxidation as above, the quantitative measurement of H~2~O~2~ in the leaves was carried out according to the method reported by Velikova et al. \[[@B58]\]. The 3,3′-diaminobenzidine (DAB) staining method was used to qualitatively detect the H~2~O~2~ generation in leaves after treatments, using the method described by Thordal-Christensen et al. \[[@B59]\].
Enzyme assays
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SOD isoenzyme was analyzed by separating the protein extracts in 10% native polyacrylamide gel with a 4% stacking gel in standard tris-glycine buffer (pH 8.3). Samples were electrophoresed at 100 V through the stacking gel for 20 min and 120 V through the separating gel for 60 min. After electrophoresis, the gel was immersed in 0.1% (w/v) nitroblue tetrazolium (NBT) solution for 15 min, briefly washed in ddH~2~O, then immersed in 100 mM potassium phosphate buffer (pH 7.0) containing 0.028 mM riboflavin and 28 mM TEMED (N,N,N\#,N\#-tetramethyl-ethylenediamine) for another 15 min. Gels were briefly washed in ddH~2~O and illuminated on a light box, with a light intensity of 30 mE · m^−2^ · s^−1^ for 15 min to initiate the photochemical reaction \[[@B60]\].
SOD activity was detected according to the method of Beauchamp and Fridovich \[[@B61]\]. A 3-mL reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 mM nitroblue tetrozulium (NBT), 2 mM riboflavin, 0.1 mM EDTA, and 100 mL enzyme extract. The reaction was initiated by placing the tubes under light intensity of 5000 lx. The absorbance was measured at 560 nm in a spectrophotometer and one unit of SOD is defined as the amount required to inhibit the photo reduction of NBT by 50%.
To analyze the APX isozymes, the protein extracts were separated in 10% native polyacrylamide gels with a 5% stacking gel in standard tris-glycine buffer with 2 mM ascorbate (pH 8.3). Samples were electrophoresed under conditions similar to the SOD isozyme electrophoresis. After electrophoresis, the gels were immersed in 50 mM potassium phosphate buffer, pH 7.0, containing 2 mM ascorbate for 10 min; then in 50 mM potassium phosphate buffer, pH 7.0, containing 4 mM ascorbate and 1 mM H~2~O~2~ for 20 min. After rinsing in water, the gels were stained in 50 mM potassium phosphate buffer, pH 7.8, containing 14 mM TEMED and 2.45 mM NBT for 10--30 min \[[@B62]\].
APX activity was determined as described by Nakano and Asada \[[@B63]\]. The reaction mixture contained 50 mmol/L potassium phosphate, pH 7.0, 1 mmol/L ascorbic acid (AsA), 2.5 mmol/L H~2~O~2~ and enzyme source (ca 15 μg protein) in a final volume of 2 mL at 25°C. Ascorbate oxidation was measured spectrophotometrically by a decrease of A290, using the absorption coefficient of 2.8 mM^−1^ · cm^−1^.
The activities of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and GR were assayed as described previously \[[@B64]\]--\[[@B66]\]. Protein was estimated according to Bradford \[[@B67]\].
Statistical analyses
--------------------
All data were represented as mean ± SD from at least three independent experiments with three replicates. Statistical analysis was done using SPSS 15.0 for Windows (SPSS, Chicago, IL), with two-tailed Student's *t*-tests for measuring significance. Double asterisks indicate significant differences between transgenic lines and WT at 1% level, one asterisk stands for at 5% level.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
JX carried out the transgenic cassava production, molecular and physiological analysis, and wrote the manuscript. JY, XD and YJ partially participated in the experiment and provided helpful suggestions. PZ was responsible for the overall concept, experimental design, data analysis, and revising this manuscript. All authors read and approved the manuscript.
Additional files
================
Supplementary Material
======================
###### Additional file 1:
**Subcellular localization of MeAPX2::GFP fusion protein and GFP control in*N. benthamiana*epidermal cells.** Scale bar = 50 μm.
######
Click here for file
###### Additional file 2:
**Yield of fresh storage roots in field-grown (5 months) wild type (WT) and SA transgenic plant lines.** No significant difference was found by *t*-test (*p* \< 0.05).
######
Click here for file
###### Additional file 3:
Supplemental methods for qRT-PCR analysis.
######
Click here for file
Acknowledgements
================
This work was supported by grants from the National Basic Research Program (2010CB126605), the National Natural Science Foundation of China (31271775), the National High Technology Research and Development Program of China (2012AA101204), the SIPPE-SCBG Joint Fund, the Earmarked Fund for China Agriculture Research System (CARS-12) and Shanghai Municipal Afforestation & City Appearance and Environmental Sanitation Administration (G102410, F132427).
| {
"pile_set_name": "PubMed Central"
} |
Abbreviations {#s1-1}
=============
DENV, dengue virus; GFAP, glial fibrillary acidic protein; HE, hematoxylin and eosin; MEM, minimum essential medium; p.i., post-infection; ZIKV, zika virus.
Introduction {#s1-2}
============
Zika virus (ZIKV) belongs to the *Flavivirus* genus within the *Flaviviridae* family \[[@R1]\], which includes a number of human pathogens such as dengue virus (DENV), yellow fever virus, West Nile virus and Japanese encephalitis virus and threats to global human health. Since 2007, ZIKV has infected several million people in more than 70 countries and caused thousands of microcephaly in newborns and Guillain--Barré syndrome in adults \[[@R3]\]. Clinical and animal experimental data have shown that the nervous system is the main target for ZIKV infection \[[@R5]\]. In addition, ZIKV can be detected in urine for a long time, suggesting that kidneys may be susceptible to ZIKV infection \[[@R9]\]. Testis damage caused by ZIKV infection in mice has recently been reported \[[@R10]\], but its implication in humans requires a long-term investigation. In the last year, great progress was achieved to reveal the pathogenesis of ZIKV. However, the molecular mechanism underlying the entry process of ZIKV still needs to be elucidated.
Several studies have speculated that Axl is a potential entry receptor for ZIKV. Axl is a receptor tyrosine kinase and together with two other similar proteins, Tyro3 and Mer, composes the family of TAM receptors which have been shown to mediate entry of DENV \[[@R13]\]. Axl is mainly expressed in monocytes and cells from the immune-privileged regions such as the nervous system and reproductive system \[[@R13]\]. Congruency of the distribution of Axl and the tissue tropism of ZIKV made researchers highlight its role as a candidate receptor for ZIKV \[[@R15]\], and some experimental results from different groups also supported this idea. Hamel *et al*. tested some entry and/or adhesion factors, which are crucial for flavivirus entry, and found that Axl is likely to play a major role in ZIKV infection \[[@R16]\]. Savidis *et al.* performed a functional genomic screen and conceived Axl as an entry factor for ZIKV infection \[[@R17]\]. Liu *et al.* showed that Axl could mediate productive infection of ZIKV in human endothelial cells \[[@R18]\]. Ma *et al.* indicated that the ZIKV infection was correlated well with the expression of Axl in testis and epididymis in mice \[[@R11]\]. Very recently, Meertens *et al.* used an engineered Axl decoy receptor and the Axl kinase inhibitor R428 to demonstrate Axl as a receptor for ZIKV entry into human glial cells \[[@R19]\]. Moreover, their results showed that ZIKV infection activated Axl kinase activity, which down-regulated IFN signalling and facilitated infection \[[@R19]\]. All of the above results suggested that Axl acts as a crucial factor for mediating ZIKV entry *in vitro.*
In spite of these results, there are very limited investigations regarding the *in vivo* role of Axl in ZIKV infection and the influence of Axl deficiency on the outcome of Zika disease. The impact of Axl deficiency has merely been tested on eyes \[[@R20]\] and the male reproductive system \[[@R10]\], and the contradictory results were documented regarding Axl's involvement in testis damage caused by ZIKV \[[@R10]\]. However, the anti-IFN receptor antibody used in these studies impaired the effects of Axl on IFN signalling and may underestimate the contribution of Axl during ZIKV infection. A strategy for a ZIKV animal model rather than IFN receptor disruption is thus needed to investigate the *in vivo* role of Axl.
In the current study, we used Axl-deficient mice (C57BL/6) and SJL mice which are susceptible to ZIKV infection \[[@R21]\] to generate Axl^−/−^ mice and their littermates (Axl^+/−^) with a SJL background. When intracerebrally injected with ZIKV, these mice gradually displayed clinical manifestations such as sloth, bradykinesia, which is a sign of moving slowly or with difficultly, and stopped body weight gain. We found that both Axl-deficient suckling mice and their littermates supported the replication of ZIKV and presented similar pathological changes in major relevant organs and similar survival rates. No significant difference was observed between Axl-deficient mice and their littermates. Our results therefore indicated that Axl is not an indispensable factor for ZIKV infection in mice.
Results {#s1-3}
=======
To investigate whether Axl plays a crucial role in ZIKV infection in mice, newborn Axl^−/−^ and Axl^+/−^ mice generated from Axl^−/−^ x Axl^+/−^ were characterized by genotyping PCR (Fig. S1, available in the online Supplementary Material) and were injected intracerebrally with 100 pfu ZIKV (CAS-ZK01 strain) within 72 h after birth. The body weight change and survival were observed every day in the following 30 days. Our results showed that both Axl^−/−^ and their littermates (Axl^+/−^) stopped weight gain at the ninth day post infection (p.i.) and remained unchanged for about 10 days. Then the weight gained recovered at about 19 days p.i. ([Fig. 1a](#F1){ref-type="fig"}). At 14 days p.i., mouse death occurred in both Axl^−/−^ and their littermates ([Fig. 1b](#F1){ref-type="fig"}). The body sizes of surviving mice were significantly smaller than the mock control injected intracerebrally with PBS ([Fig. 1c](#F1){ref-type="fig"}). By the end of the observation time, 41.6 % Axl^−/−^ mice (5/12) and 44.4 % (4/9) of their littermates died ([Fig. 1b](#F1){ref-type="fig"}). No statistically significant difference was found between Axl^−/−^ and their littermates in terms of the body weight change and survival rate.
{#F1}
Viral loads in major organs including the brain, heart, kidney, liver and spleen as well as serum were measured by qRT-PCR at 10 days p.i. The highest viral load with 10^8^ copies µg^−1^ total RNA was detected in brain ([Fig. 1d](#F1){ref-type="fig"}), similar to previous reports \[[@R5]\]. In all organs and serum tested, similar viral loads ranging from 10^6^--10^8^ copies µg^−1^ total RNA or ml^-1^ sera were detected in Axl^−/−^ mice and their littermates ([Fig. 1d](#F1){ref-type="fig"}). These results indicate that ZIKV has a similar organ distribution pattern and replicates at a similar level in Axl^−/−^ mice and their littermates, which results in a congruent infection outcome.
To analyse the pathological changes caused by ZIKV infection, organs that might be damaged by ZIKV infection, including the brain, heart, kidney, liver and spleen, were dissected at 10 days p.i., and sections were subjected to hematoxylin and eosin (HE) staining. The testis was not fully developed at the time, thus it was not analysed in our study. In contrast to mice injected intracerebrally with PBS, mice injected with ZIKV showed prominent pathological changes in both Axl^−/−^ and their littermates. Among all organs observed, there were obvious pathological changes in the brain including hyperemia, neuron death, neural cell disorganization and infiltration of inflammatory cells in the hippocampus and the cerebral cortex ([Fig. 2](#F2){ref-type="fig"}). There was remarkable atrophy of the glomerulus, and the renal cortex became thin and demonstrated infiltration of inflammatory cells and congestion. Moreover, enlarged white pulps and the appearance of germinal centres were observed in the spleen ([Fig. 2](#F2){ref-type="fig"}). The heart and liver showed no other conspicuous changes except for infiltration of inflammatory cells (Fig. S2) in the liver. All these pathological changes were observed in both Axl^−/−^ mice and their littermates to the same extent, indicating that Axl expression did not have an impact on the pathological changes induced by ZIKV infection.
{#F2}
To determine the distribution of ZIKV in different organs, cyrosections of these organs were subjected to immunofluorescence staining. In the brains of Axl^−/−^ mice and their littermates, ZIKV antigens were mainly distributed in the cerebral cortex and hippocampus ([Fig. 3](#F3){ref-type="fig"}). Cryosections of the major organs were further subjected to immunohistochemical staining. ZIKV antigens were detected in the brain with the highest signal intensity (Fig. S3), consistent with the highest level of viral load ([Fig. 1d](#F1){ref-type="fig"}) and obvious histopathological changes. Additionally positive immunoreactivity of ZIKV antigens was also observed in the kidney and spleen (Fig. S3), also consistent with high viral loads and histopathological changes. The heart and liver also showed some positive signal of ZIKV antigens although there were no obvious histopathological changes in the heart. However, some unspecific positive immunoreactivity was observed in the spleen of mock-treated mice, which may be due to non-specific binding of an antibody with an Fc receptor on the surface of immune cells such as B lymphocytes and macrophages, and these cells richly distribute in this organ. Importantly, no difference was found between the Axl^−/−^ mice and their littermates.
{#F3}
To further study the susceptible cell types for ZIKV infection in the brain, ZIKV antigens were co-immunostained with the cellular markers of neurons or gliocytes on the brain cyrosections of Axl^−/−^ mice and their littermates. Physiologically, Axl^−/−^ mice and their littermates displayed a similar distribution pattern of NeuN, the marker of the neuron \[[@R22]\] as well as glial fibrillary acidic protein (GFAP), the marker of the astrocyte \[[@R23]\] and ependymal cell \[[@R24]\] (Fig. S4). In all mice, ZIKV antigens were co-localized well with NeuN ([Fig. 4a](#F4){ref-type="fig"}), rather than with GFAP ([Fig. 4b](#F4){ref-type="fig"}), indicating that with or without the presence of Axl, the neuron was consistently the primary target cell type for ZIKV infection. In further experiments, co-immunostaining of ZIKV and Axl was performed in brain cyrosections of Axl^+/−^ mice. Although most of the ZIKV-infected cells expressed Axl in the hippocampus ([Fig. 4c](#F4){ref-type="fig"}, lower panels), the cells infected by ZIKV were negative for Axl in the cerebral cortex ([Fig. 4c](#F4){ref-type="fig"}, upper panels), showing that Axl was not an indispensable factor for ZIKV infection.
{#F4}
Discussion {#s1-4}
==========
In this study, by using Axl-deficient suckling mice and their heterozygous littermate controls, we showed that all these mice were able to support the replication of ZIKV and displayed clinical manifestations via intracerebral injection. No difference was found between Axl-deficient mice and their littermates, either in terms of the viral load, clinical manifestations, viral distribution or survival rate. Our results, therefore, provided the first infection outcomes and validated *in vivo* evidence arguing that Axl was not an indispensable factor for ZIKV infection.
Although many studies proclaimed that Axl is a receptor for ZIKV entry *in vitro*, there are indeed a few works alleging the opposite results. Miner *et al.* found that the development of conjunctivitis, panuveitis and infection of the cornea were independent of Axl in ZIKV-infected mice \[[@R20]\]. Govero *et al.* demonstrated high-level ZIKV infection in testis and epididymis in Axl-deficient mice \[[@R10]\]. In a recent study, Wells *et al.* found that the genetic ablation of Axl did not protect human neural progenitor cells from ZIKV Infection \[[@R25]\]. All of these results suggest that Axl may not be required for ZIKV infection in mice. Here, by characterizing in detail the infection of ZIKV in the presence or absence of Axl, we provide *in vivo* data showing that Axl has no essential role in ZIKV infection in mice. Very recently, Li and Hastings also reported that ZIKV replicated to a similar level in the brains of Axl wild-type and Axl knockout mice \[[@R26]\]. Comprehensively, the results further support the above-mentioned view: Axl is not an indispensable factor for ZIKV infection in mice.
It is notable that our results do not necessarily mean that Axl is not a receptor for ZIKV entry; it just implies that Axl does not contribute crucially during ZIKV infection *in vivo*. As the expression, distribution and interaction of cellular surface proteins in immortal cells are often different from that in primary cells *in vivo*, it is not surprising that some viral receptors identified *in vitro* have little contribution to viral infection *in vivo*. Therefore, before the viral receptors identified *in vitro* are designed as the target for an antiviral screen, it is necessary to evaluate their *in vivo* role, at least in small animals. Moreover, the variety and complexity of receptors have been reported for many viruses. For example, DENV, one of the best-studied flavivirus, is well known for its ability to use more than one receptor for viral entry \[[@R28]\] including heparan sulfate \[[@R29]\], DC-SIGN \[[@R30]\], β~3~-integrin \[[@R31]\]as well as Axl \[[@R14]\]. Meanwhile, the secretion of DENV also requires a KDEL receptor for the traffic from endoplasmic reticulum to Golgi apparatus \[[@R32]\]. The involvement of more than one receptor is possible to occur for ZIKV, which would also compromise the impact of Axl on ZIKV infection in mice. Thus, in the future, considerably more work is required to elucidate the molecular mechanism for ZIKV infection.
Methods {#s1-5}
=======
Virus and cells {#s2-5-1}
---------------
ZIKV (Asian lineage, CAS-ZK01 strain) was isolated from a patient with Zika fever and kindly provided by Dr George F. Gao (Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China). C6/36 cells (Aedes albopictus cells) were maintained at 28 °C in RPMI 1640 (Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, PAN, Germany). Vero cells (African green monkey kidney cell) were maintained in minimum essential medium (MEM, Gibco, USA) supplemented with 5 % FBS at 37 °C.
ZIKV was propagated in C6/36 cells in RPMI 1640 supplemented with 2 % FBS. And the virus in cultural supernatant was collected at the fifth, sixth and seventh day p.i. The viral titres were determined by a plaque assay on Vero cell monolayers under a fresh MEM overlay containing 1.2 % methylcellulose and 2 % FBS (MEM overlay) (Fig. S5). Viral stocks were stored at −80 °C until use.
For the plaque assay, the virus stock was serially diluted and incubated with Vero cell monolayers for 2 h with moderate shakings every 30 min. Afterwards, the virus was removed and the MEM overlay was added onto the Vero monolayer, which followed by 7 days of continuous incubation at 37 °C. After the MEM overlay was removed a final crystal violet staining was then utilized to visualize plaques formed by ZIKV infection.
Mice {#s2-5-2}
----
Mice deficient in Axl (F0 Axl^−/−^) were kindly provided by Professor Dan-Shu Han (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, PR China). The mice were bred and maintained under a specific pathogen-free animal facility at Capital Medical University. Male F0 mice (Axl^−/−^) was mated with female SJL mice (Axl^+/+^) to produce F1 mice (Axl^+/−^). Male F0 mice (Axl^−/−^) were then mated with female F1 mice (Axl^+/−^) to produce F2 mice (Axl^−/−^ for experiments and Axl^+/−^ as littermate controls). The average age of F2 suckling mice used in this study was 1.5 days.
Genotyping PCR {#s2-5-3}
--------------
Genotyping PCR was performed using a Mouse Tissue Direct PCR Kit (KG205, Tiangen) and a set of three primers (Wt: 5′-GCCGAGGTATAGTCTGTCACAG-3′, Mut:5′-TT TGCCAAGTTCTAATTCCATC-3′, and WtMut: 5′- AGAAGGGGTTAGATGAGGAC-3′). The genotyping experiments were carried out according to the manufacturer\'s instruction with using a few murine tail tissues. The size of PCR products are 350 bp for Axl^+/+^ mice, 350 and 200 bp for Axl^+/−^ mice and 200 bp for Axl^−/−^ mice.
Mouse experiments {#s2-5-4}
-----------------
For ZIKV infection, newborn mice were challenged with 100 pfu ZIKV in 20 µl PBS through intracerebral injection. Mice administered with 20 µl PBS served as mock controls. Survival indexes, including body weight, disease manifestation and survival rates of mice were recorded each day in the following 30 days or till death. Organs and serum of the infected mice were collected at the tenth day p.i. for determination of virus load and histological examination.
ZIKV mRNA quantification {#s2-5-5}
------------------------
The infected and mock control mice were euthanized by cervical dislocation and the major organs were harvested and homogenized in Trizol (Transgen, China, ET101-01). RNA was isolated from tissue lysates according to the manufacturer's protocol. The extracted RNA was dissolved in RNase free water and its purity and concentration were determined by NanoDrop 2000C (Thermo Scientific, USA) three times and represented as a mean value. A pair of primers (forward: 5′-TTGGGTTGTGTACGGAACCTG-3′, reverse: 5′-GTGCTTTGTGTATTCTCTTGA-3′) were designed to detect ZIKV genomic RNA. Real-time qPCR analysis was performed with Quant One Step qRT-PCR (SYBR Green I) Kit (FP303-01, Tiangen) on 7500 Real Time PCR System (Applied Biosystems, USA) according to the manufacturer's instruction. ZIKV genome RNA (MR766) transcripted *in vitro*, kindly provided by Professor Ai-Hua Zheng from CAS, was quantified and used as a standard template to establish the standard curve. Quantification of the copies of ZIKV mRNA was determined by the standard curve method and expressed as the copy number per µg total RNA (for organs) or ml (for sera).
Immunofluorescent staining {#s2-5-6}
--------------------------
The whole brains were embedded in OCT (Jung, Leica) immediately after excision and processed into 5 µm thick frozen sections. The tissue slices were air-dried and then fixed in ice cold acetone. Tissue slices were permeabilized in 0.5 % Triton-X100 solution for 10 min at room temperature and blocked in 5 % BSA for 2 h at 4 °C. The tissue slices were incubated with anti-ZIKV mouse sera (1 : 200), anti-NeuN antibody (ab104225, abcam, 1 : 500), anti-GFAP antibody (ab7260, abcam, 1 : 500) and/or anti-Axl antibody (AF854, Novus Biologicals, 1 : 500) for 3 h at 37 °C or overnight at 4 °C. Goat anti-mouse IgG Alexa Fluor 488 (A-11029, Invitrogen, 1 : 500) and Goat anti-rabbit IgG Alexa Fluor 594 (R37117, Invitrogen, 1 : 500) were used as secondary antibodies for 1 h at 37 °C. DAPI was used to display cell nuclei. All images were captured with a laser scanning confocal microscopy (Leica TCS SP5).
Hematoxylin and eosin staining {#s2-5-7}
------------------------------
The ZIKV-challenged mice were euthanized by cervical dislocation at 10 days p.i. and the major organs were harvested. The organs were divided into two parts. One was immediately fixed in modified Davidson's fluid solution (30 ml of 40 % formaldehyde, 15 ml of ethanol, 5 ml of glacial acetic acid and 50 ml of distilled water) overnight. Then the organs were embedded in paraffin, sectioned at 5 µm in thickness and subjected to a standard hematoxylin and eosin staining. The others were embedded in OCT (Jung, Leica) immediately and then subjected to 5 µm thick frozen sections for detection of ZIKV antigens by immunehistochemical staining.
Statistical analysis {#s2-5-8}
--------------------
Statistical analysis was performed with SPSS 19.0. Two-way ANOVA and log rank test were used to compare the body weight changes and survival rates of the two groups, respectively. The quantitative data of the two groups were compared using Student\'s *t*-test. The survival rates of the two groups were compared with calibrated Chi square test. Differences among the groups were considered to be significant at *P*\<0.05.
This work was supported by grants from the National Natural Science Foundation of China (No. 81471957, 81271839, 81671971, 81301435, 81372935, 81401676 and U1602223).
We thank Professor George F. Gao at Institute of Microbiology, Chinese Academy of Sciences (CAS), Professor Ai-Hua Zheng at Institute of Zoology, CAS, Dr Ting-Ting Deng at China-Japan Friendship Hospital for providing reagents and materials.
The authors declare that there are no conflicts of interest.
All animal experimental procedures were reviewed and approved by the Experimental Animal Welfare and Animal Ethics Committee of Capital Medical University, Beijing, PR China.
1038
###### Supplementary File 1
######
Click here for additional data file.
[^1]: These authors contributed equally to this work.
[^2]: Five supplementary figures are available with the online Supplementary Material.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S1}
============
Dendritic cells (DC) constitute a heterogeneous population of leukocytes that interconnect the innate and the adaptive immune response, in particular through their capacity to activate naïve T lymphocytes ([@B1]). DC depend on several growth factors for their proliferation, survival, and differentiation, most importantly Flt3L, GM-CSF, and M-CSF ([@B2]). Flt3L drives the development of various DC populations, in particular plasmacytoid and conventional DC (cDC), in peripheral tissues and lymphoid organs in the steady-state ([@B3]), whereas GM-CSF is important in generating inflammatory, monocyte-derived TNF/iNOS-producing DC (TipDC) ([@B1], [@B4]). Inactivation of M-CSF or its receptor *in vivo* results in a significant decrease in DC numbers ([@B5], [@B6]) and shift in DC subset composition ([@B7]), including a complete absence of epidermal Langerhans cells ([@B8]) and monocyte-derived DC in the intestinal lamina propria ([@B9]). Interestingly, M-CSF has been shown to induce also plasmacytoid and cDC development, in addition to development of macrophages, from BM cells of normal and Flt3L-knock out mice ([@B10], [@B11]). These observations underline a critical role of M-CSF signaling in the development of several DC populations.
Dendritic cells can initiate various types of T-cell responses, depending in part on the developmental status of the DC interacting with the T cells. Immature DC (iDC) characterized in mice as CD11c^+^MHC class II^low^CD86^low^ cells, are specialized in taking up and processing antigens but are poor immune stimulators and may induce tolerance. In contrast, mature DC (mDC), characterized as CD11c^+^MHC class II^hi^CD86^hi^ cells, induce cell-mediated and/or humoral immune responses ([@B12], [@B13]). Thus, tight regulation of DC maturation is required to maintain a proper immune balance.
MicroRNAs are an important class of regulators involved in differentiation and cell fate decisions ([@B14], [@B15]). They represent an extensive family of short (∼22 nt) single-stranded non-coding RNAs that regulate gene expression at a post-transcriptional level by binding to the 3′untranslated region (3′UTR) of mRNAs, thereby causing translational inhibition of the target mRNA primarily as a result of mRNA degradation ([@B16]). In recent years, microRNAs have emerged as important regulators of immune function, which has been demonstrated in particular by *in vivo* gain- or loss-of-function microRNA studies ([@B17], [@B18]). Thus far, however, most studies linking microRNAs with the immune system have focused on T and B lymphocytes, while only a limited number of studies have focused on their role in DC development and function ([@B19], [@B20]). Studies using human cells have shown that the microRNA expression profiles alter during DC development ([@B20]--[@B24]). Manipulating microRNA expression affects DC function in both human and mouse ([@B21], [@B25]). Here, we approached the question whether microRNAs are involved in regulating mouse monocyte-derived DC maturation focusing on the final stages where CD11c^+^MHC class II^low^CD86^low^ iDC develop into CD11c^+^MHC class II^hi^CD86^hi^ mDC. We determined the microRNA expression profiles of different mouse GM-DC maturation stages during GM-CSF-stimulated development *in vitro*. A set of microRNAs is described, which expression is prominently up-regulated during both the spontaneous and lipopolysaccharide (LPS)-induced transition of iDC to mDC. *Csf1r*, the gene encoding the growth factor receptor M-CSFR (c-Fms, M-CSFR, CD115), is identified as a predominant common target regulated by the induced miR-22, miR-34a, and miR-155. Moreover, we show that down-regulation of M-CSFR expression is a prerequisite for final DC maturation.
Materials and Methods {#S2}
=====================
Animals {#S2-1}
-------
Female C57BL/6J mice were obtained from Harlan (Horst, Netherlands) and were kept under specific pathogen-free conditions at the animal facility of the Erasmus MC, Rotterdam, Netherlands. Housing, care, and experimental handling were performed in accordance with Dutch legal regulations. Ethical approval was obtained after protocol review by the independent animal experiment committee DEC Consult, and registered under permit numbers EUR1408 (128-08-05), EUR1738 (128-09-02), EMC2135 (128-10-10), and EMC2759 (128-12-07).
DC maturation *in vitro* {#S2-2}
------------------------
Monocyte-derived DC were generated by GM-CSF stimulation of bone marrow (BM) precursors as described previously ([@B26]). These cells are indicated as GM-DC. Briefly, BM cells isolated from 8 to 13-week-old C57BL/6 mice were cultured in RPMI-1640 medium (Lonza, Belgium) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, and 20 ng/ml rmGM-CSF (Biosource International, Camarillo, CA, USA). Cells were kept in a humidified incubator at 37°C with 5% CO~2~. At day 0, BM leukocytes were seeded at 3 × 10^5^ per ml in either 100 mm dishes (BD Biosciences), 12-wells plates (Nunc) or 96-wells round-bottom plates (Nunc). At day 3, fresh culture medium was added to the plates and at day 6, half of the medium was replaced. To induce enforced GM-DC maturation, 100 ng/ml LPS (*Escherichia coli* strain 055:B5, Sigma) was added on day 6. Alternatively, plasmacytoid DC (pDC) and cDC were generated by Flt3L stimulation of BM precursors essentially as described by Naik *et al*. with minor modifications ([@B27]). To this end, BM cells isolated from 8 to 13-week-old female C57BL/6 mice and erythrocytes were lysed by treatment for 2 min with 0.155 M NH~4~Cl. Then, BM cells were extensively washed and cultured in RPMI-1640 (Lonza, Belgium) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, and 200 ng/ml Flt3L (Peprotech, Rocky Hill, NJ, USA), and seeded at 4 × 10^6^ per 2 ml in six-wells plates (Nunc). At day 7, cells were washed to remove free Flt3L, and stimulated for 24 h with 10 μg/ml CpG (ODN 2395, InvivoGen, San Diego, CA, USA).
To assess the role of TNF-α converting enzyme (TACE) in M-CSFR down-regulation, TACE inhibitors TMI-1 and TMI-2 ([@B28], [@B29]) were used in a final concentration of 10 and 15 μM, respectively (kindly provided by Dr. B. Scholte, Erasmus, MC, Netherlands). The functional inhibition during overnight cultures was tested by determining the TACE-mediated decrease of M-CSFR expression on BM monocytes. To that end, freshly isolated BM cells (10^6^ per ml) were cultured at 37°C for 24 h in a 12-well plate in RPMI-1640 with 10% FCS and antibiotics as described before, but without additional growth factors. Inhibitors were added from the start of the culture. As TACE inducer *E. coli* LPS (O55 B5, Sigma) was used in a final concentration of 100 ng/ml. Cells were harvested after 24 h. Similarly, TACE activity was inhibited in GM-DC cultures by adding inhibitors during the last 24 h of a 7-day culture in combination, either or not in the presence of LPS. Expression of M-CSFR/CD115 was determined as described below.
Flow cytometry and cell sorting {#S2-3}
-------------------------------
For cell labeling, incubations were performed in staining buffer (PBS pH 7.8, 1% BSA, 0.01% sodium azide) on ice for 30 min. Reagents used were fluorescent conjugates of CD11b (M1/70), CD11c (HL3), CD86 (GL1), CD115 (anti-M-CSFR, clone AFS98), mMGL/CD301 (ER-MP23), MHC class II I-A/I-E (M5/114.15.2), SiglecH (eBio440c), and rat-anti-mouse IgG-Alexa488 and streptavidin-Alexa633. These antibodies were obtained from BD Biosciences, eBioscience, Molecular Probes or prepared as purified Ig from hybridomas created in our lab. Cells were analyzed by flow cytometry using a FACSCalibur or FACSCanto II (Becton Dickinson) and FlowJo Analysis Software (Tree Star, Ashland, OR, USA). Sorting of cells was performed using a FACSAria Cell Sorter (Becton Dickinson).
MicroRNA microarray hybridization and analysis {#S2-4}
----------------------------------------------
Total RNA was extracted using acid-phenol:chloroform (Ambion) extraction and enriched for microRNAs using a mirVana microRNA isolation kit (Ambion) according to the manufacturer's protocols. RNA was labeled using a ULS™ aRNA labeling kit (Kreatech Diagnostics, Amsterdam). 1.5 μg of total RNA was incubated with Cy3-ULS for 30 min at 85°C and purified to remove unbound Cy3-ULS. Labeled RNA was hybridized on miRCURY LNA microRNA arrays (probe set 8.0; Exiqon, Vedbaek, Denmark) at 60°C for 16 h using a Tecan 4800 hybridization station. Slides were washed and immediately scanned using a Tecan LS Reloaded microarray laser scanner. Microarray data extraction, normalization, and data analysis were carried out as described ([@B30]). Heatmaps were generated using the TM4 microarray software suite ([@B31]). Significance Analysis of Microarrays (SAM) analysis was carried out on sorted DC populations obtained from three biological replicates, implementing a false discovery rate (FDR) ≤ 10% and a minimum 1.5-fold change in expression. All data are MIAME compliant. Raw data have been deposited in ArrayExpress and are accessible under numbers A-MEXP-2085 and E-MEXP-3311.
*Csf1r-*3′UTR luciferase reporter assay {#S2-5}
---------------------------------------
The full-length *Csf1r-*3′UTR was cloned into the *Xho*I/*Not*I site downstream the coding sequence of *Renilla luciferase* in the psiCHECK-2 luciferase reporter vector (Promega). Cloning primers *Csf1r-*3′UTR: FW 5′-GGATTCCTCGAGTCCTGCCGCT-CTCTACGT-3′ and RE 5′-GGATTCGCGGCCGCCTGGCTGTGTTAATGCTGTT-AGTT-3′. Mutant *Csf1r-*3′UTR constructs were generated by introducing three basepair mismatches into each seed region of the corresponding miR-22, -34a, and -155 binding sites (*Csf1r*-3′UTR mut all) or the miR-22 site alone (*Csf1r*-3′UTR 22mut) \[outsourced to Genscript (Piscataway, USA)\]. HEK293T cells, described by Stewart et al. ([@B32]), were plated in a 48-well plate at a density of 6 × 10^4^ cells per well and then co-transfected the next day with 10 ng psiCHECK-2 vector containing the full 3′UTR of *Csf1r* mRNA, together with miR-22, -34a, -155, and control over-expression oligonucleotides (Ambion) at 50 nM final concentration using Lullaby transfection reagent (Boca Scientific). Luciferase activity was measured 48 h later using the Dual Glow luciferase kit (Promega) in a TopCount NXT microplate luminescence counter (Packard Instrument Company, Connecticut, USA). Transfections were performed in duplicate and repeated three times in independent experiments. Statistical analysis was by unpaired two-tailed Student's *t* test; *p*-values of less than 0.05 were considered significant.
Transient microRNA inhibition in GM-DC {#S2-6}
--------------------------------------
3 × 10^5^ BM cells were cultured in 12-well plates to generate GM-DC. These cells were transfected on day 4 of culture with 50 nM anti-miR microRNA inhibitors (Ambion), mixed with siGLO Cy3-labeled non-targeting anti-miR oligonucleotides (Dharmacon) at a 5:1 ratio. Control samples were treated with a control non-targeting inhibitor (Dharmacon). Transient transfection was accomplished using DharmaFECT1 reagent (Dharmacon) according to the manufacturer's protocol.
Transient *Csf1r* over-expression in GM-DC {#S2-7}
------------------------------------------
The complete ORF of mouse *Csf1r* was purchased as a full-length cDNA clone (Open Biosystems, IMAGE accession no. 30436119). GM-DC (8 × 10^6^ cells) were co-electroporated at day 6 of culture with 8 μg of the *Csf1r* cDNA clone and 2 μg pEGFP-C1 control vector (Clontech) using an Amaxa nucleofector apparatus (Lonza; program Y-01) and Amaxa mouse macrophage nucleofector kit (Lonza) according to the manufacturer's instructions. For control experiments, cells were co-electroporated with 8 μg pCMV-SPORT6 vector and 2 μg pEGFP-C1 vector.
Quantitative real-time PCR of microRNAs and *Csf1r* {#S2-8}
---------------------------------------------------
The quantification of mature miR-155, -34a, and -22 expression levels was carried out with 1 μg of total RNA using the miScript PCR System (Qiagen) according to the manufacturer's instructions. ΔCt values for each microRNA were normalized to tubulin reference gene. miR-155 FW primer: 5′-TTAATGCTAATTGTGATAGGGG-3′. miR-34a FW primer: 5′-TGGCAGTGTCTTAGCTGGTTGT-3′. miR-22 FW primer: 5′-AAGCTGCCAGTTGAAGAACTGT-3′. Tubulin primers: FW 5′-CAGACCAACCACT-GCTACAT-3′ and RE 5′-AGGGAATGAAGTTGGCCAGT-3′.
*Ex vivo* analysis of M-CSFR expression {#S2-9}
---------------------------------------
FITC painting of mice was performed as described previously ([@B33]). Briefly, mice were painted on the shaved back with 250 μl of 1% FITC (Sigma) in 1:1 acetone:dibutylphthalate (Sigma) and draining axillary and brachial lymph nodes (LN) were collected 24 h afterward. Mesenteric LN were taken as a control. Cells were isolated by mechanical disruption of the LN, without enzymatic treatment, and stained with CD301/mMGL (ER-MP23), CD11b, CD86, anti-MHC class II I-A/I-E, and CD115 antibodies and analyzed by flow cytometry. To assess expression of M-CSFR expression in myeloid cells in the skin, ears from 8 to 13-week-old C57BL/6 mice were collected and frozen in Tissue-Tek OCT embedding medium (Sakura Finetek, Zoeterwoude, Netherlands) and cut into 6 μm-thick sections. Cryosections were fixed and stained as described earlier ([@B33]). Optimally titrated goat-anti-rat IgG-Alexa546 and streptavidin-Alexa633 were used to detect unlabeled antibodies and biotinylated antibodies, respectively. Images were acquired using a Leica TCS SP5 confocal microscope.
Statistical analysis {#S2-10}
--------------------
Statistical analysis between experimental and control groups was carried out using unpaired two-tailed Student's *t* test (unless stated otherwise) with the Graphpad Prism 5 software package. *P*-values of less than 0.05 were considered significant. Error bars represent mean ± SEM from at least three experiments.
Results {#S3}
=======
MicroRNA expression profiles change during DC development {#S3-11}
---------------------------------------------------------
To investigate which microRNAs are differentially expressed during monocyte-derived DC development *in vitro*, we performed microRNA profiling of distinct DC maturation stages isolated from 7 days GM-CSF-stimulated BM cultures, which were either or not additionally stimulated for 16 h with LPS (i.e., forced vs. spontaneous maturation). Different populations of GM-DC were sorted based on differential expression of maturation markers CD11c, MHC class II, and CD86 (Figure [1](#F1){ref-type="fig"}A). We then performed profiling of 328 different microRNAs using locked nucleic acid-based microRNA arrays. In total, 14 microRNAs were found to be differentially expressed in iDC to mDC development (Significance Analysis of Microarrays criteria: FDR ≤ 10%; fold change ≥ 1.5 or ≤−1.5, Figure [1](#F1){ref-type="fig"}B). The changes during spontaneous or LPS-induced maturation appeared to be very similar. Of all microRNAs that were screened, miR-155 showed the most abundant increase, reaching 11- and 17-fold up-regulation in the transition of iDC to mDC or iDC to LPS-mDC, respectively. These findings support data from Ceppi *et al*. ([@B34]) who found high levels of miR-155 in human monocyte-derived DC upon LPS stimulation. Conversely, miR-200b and -215 levels were down-regulated approximately threefold in both mDC and LPS-mDC compared to iDC. These results demonstrate that microRNA levels change during DC development *in vitro* and that iDC and mDC are characterized by distinct microRNA expression profiles.
{#F1}
M-CSFR is a target of miR-22, -34a, and -155, which are up-regulated during final DC maturation {#S3-12}
-----------------------------------------------------------------------------------------------
To investigate which developmental genes are regulated by microRNAs in the final maturation step from iDC to mDC we compared the list of differentially expressed microRNAs with one compiled for genes known to be involved in the development of myeloid cells (DC/macrophage/neutrophil) as listed in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database ([@B35]) (KEGG entry: mmu04640; Hematopoietic cell lineage -- *Mus musculus*). Using the Targetscan algorithm ([@B36]), we identified that 10 out of these 30 genes have predicted microRNA target sites in their 3′UTR conserved across mammals. Subsequently, we compared these 10 genes to our microRNA profiling data (Figure [1](#F1){ref-type="fig"}B), and found that only *Csf1r*, the gene encoding M-CSFR, and *Kitl* (SCF, stem cell factor) were potentially regulated by microRNAs differentially expressed in GM-DC. From these, M-CSFR was the most likely target for microRNA regulation in iDC to mDC transition as three out of four conserved predicted binding sites in the 3′UTR of *Csf1r* mRNA were targeted by differentially expressed microRNAs, i.e., miR-22, -34a, and -155 (Figure [2](#F2){ref-type="fig"}A). Other microRNA target prediction algorithms (incl. PicTar, EIMMo) confirmed the miR-22, -34a, and -155 binding sites in both human and mouse *Csf1r-*3′UTR (not shown). Interestingly, expression of all three microRNAs was up-regulated upon iDC to (LPS-) mDC transition in our array, which we could confirm by quantitative RT-PCR (Figure [2](#F2){ref-type="fig"}B). In accordance, we found a strong down-regulation of *Csf1r* mRNA levels in mDC compared to iDC (Figure [2](#F2){ref-type="fig"}C).
{#F2}
To test whether miR-22, -34a, and -155 actually can regulate *Csf1r* expression through direct 3′UTR interactions, we cloned the complete 3′UTR of *Csf1r* into the psiCHECK-2 reporter vector downstream the coding sequence of *Renilla luciferase*. This vector, which also encoded firefly luciferase as an internal control, was transfected into HEK293T cells. When miR-155- or miR-34a precursor microRNAs were co-transfected we observed that *Renilla luciferase* expression was repressed approximately 50% compared to controls (Figure [2](#F2){ref-type="fig"}D). Co-transfection of miR-22 precursor inhibited *Renilla luciferase* expression even almost 75%. The combination of all three microRNAs did not further down-regulate luciferase expression. These findings confirm and extend the work of O'Connell et al. ([@B37]) and Lu *et al*. ([@B24]) who have shown that *Csf1r* is a validated target of miR-155. To verify whether the repressive effects were microRNA-specific, we repeated these experiments with *Csf1r-*3′UTR reporter constructs harboring point mutations in all three (miR-22, -34a, and -155) microRNA binding sites or in just one (miR-22) microRNA binding site. Indeed, no repressive effects of microRNAs could be observed when mutants were tested containing alterations in all binding sites, indicating the specificity of microRNA targeting (Figure [2](#F2){ref-type="fig"}E). Mutation of the miR-22 binding site alone (but not the miR-34a and -155 binding sites) completely impaired miR-22-mediated *Csf1r-*3′UTR repression, whereas the repressive effects of miR-34a and -155 remained unaffected, with similar levels of repression as in our initial experiments with the wild-type mouse *Csf1r-*3′UTR (Figure [2](#F2){ref-type="fig"}E).
Taken together, these data demonstrate that miR-22, -34a, and -155 are up-regulated in the final step of iDC to mDC maturation and that all three microRNAs can directly regulate *Csf1r* expression by targeting its mRNA via 3′UTR interactions.
M-CSFR protein expression is down-regulated in the final phase of DC maturation {#S3-13}
-------------------------------------------------------------------------------
Based on the differential expression of *Csf1r*-regulating microRNAs and their *Csf1r* target mRNA, we predicted that M-CSFR protein expression decreased during the transition of iDC to mDC. Therefore, we followed M-CSFR expression in GM-CSF-stimulated BM cultures over time by flow cytometry. To induce final maturation, part of the cultures was treated with LPS on day 6. Indeed, M-CSFR expression was rapidly down-regulated during both spontaneous and LPS-induced transition of iDC to mDC, accompanied by the acquisition of high level MHC class II and CD86 expression (Figure [3](#F3){ref-type="fig"}A).
{#F3}
Although our results suggest that microRNAs are responsible for the observed down-regulation of M-CSFR expression, we cannot exclude other mechanisms that might also be involved in this process. In particular, LPS is known to stimulate activation of the transmembrane protease TNF-α-converting enzyme (TACE), which cleaves M-CSFR from the surface ([@B38]). To study a putative contribution of TACE-mediated M-CSFR cleavage to decreasing expression during final maturation, GM-DC were incubated with and without TACE inhibitors TMI-1 and -2 ([@B28], [@B29]) before enforcing DC maturation with LPS during overnight culture. We found that M-CSFR levels decreased significantly in LPS-stimulated DC compared to unstimulated cells, despite inhibition of TACE (Figure [3](#F3){ref-type="fig"}B). TMI-1 and -2 treatment of GM-DC by itself already caused a decrease in M-CSFR expression, which was not caused by decreased cell viability (not shown). The efficacy of the TACE inhibitors under these conditions is indicated by the inhibition of spontaneous and LPS-induced decrease of membrane-bound M-CSFR on BM monocytes \[compare left panels in Figure [3](#F3){ref-type="fig"}C to approximately 80% M-CSFR-expressing BM monocytes upon isolation ([@B39])\] (Figure [3](#F3){ref-type="fig"}C). Collectively, these data suggest that TACE-mediated shedding does not play a major role in reducing M-CSFR expression on maturing GM-DC.
To investigate whether down-regulation of surface M-CSFR expression is restricted to maturing GM-DC, we cultured freshly isolated BM in the presence of Flt3L to generate cDC (FL-cDC) and pDC and assessed surface M-CSFR levels by flow cytometry. We observed that only a minor subset of CD11c^+^SiglecH^−^ FL-cDC and CD11c^+^SiglecH^+^ pDC expressed M-CSFR (Figure [3](#F3){ref-type="fig"}D). Although enforced maturation with CpG in these cultures led to a further reduction in M-CSFR levels, we conclude that M-CSFR expression by iDC and down-regulation during their maturation is not a universal characteristic of Flt3L-generated cDC and pDC *in vitro*.
Next, we asked whether M-CSFR expression is regulated in DC *in vivo* as well. To that end, we focused on DC present in the skin, and assessed their M-CSFR expression *in situ* and after stimulated migration to skin-draining LN. The skin harbors various cells of myeloid origin, such as Langerhans cells in the epidermis, and macrophages/iDC in the dermis ([@B1], [@B33], [@B40]). Both epidermal and dermal populations of cells are able to incorporate skin-applied antigens and migrate subsequently to the draining LN, where they appear as phenotypically mDC ([@B33], [@B41]). Although flow cytometric analysis is the preferred method to quantify protein expression in mixed cell populations, we observed that the sample preparation of mouse skin tissue into single-cell suspensions using enzymatic digestion did not allow the use of flow cytometry due to enzymatic cleavage of surface-bound M-CSFR (not shown). Therefore, we analyzed M-CSFR expression by skin mononuclear phagocytes *in situ* by less sensitive confocal microscopy of tissue sections. We labeled mouse ear skin cryosections with CD115 -- an antibody directed against M-CSFR -- together with mMGL (ER-MP23) and CD11b antibodies to identify all dermal macrophages and iDC of the partially M-CSFR-dependent CD103^neg^ subset ([@B7]) and analyzed expression of these markers (Figure [3](#F3){ref-type="fig"}E). Cells that stain positively for the C-type lectin mMGL (CD301) are dermal macrophages and iDC that migrate to draining LN upon antigen uptake ([@B33], [@B42]). The images demonstrate that CD115 co-localizes with CD11b in the skin epidermis as well as with CD11b and mMGL in the dermis of the mouse ear. Similar observations were made in sections taken from mouse back skin (not shown). This indicates that both epidermal Langerhans cells and dermal mononuclear phagocytes uniformly express M-CSFR *in situ*, in agreement with previous findings by Hume *et al*. at the mRNA level ([@B43]).
To assess whether these CD115-positive mononuclear phagocytes in the skin down-regulate expression upon maturation during migration to draining LN, we skin-painted FITC onto shaved back skin of mice and analyzed the phenotype of the FITC^+^ cells that had emigrated from the skin to skin-draining LN 24 h later (Figure [3](#F3){ref-type="fig"}F). The majority of LN-immigrating, FITC-transporting cells from the skin express mMGL (ER-MP23) at a high level, reminiscent of their dermal origin. These findings are in agreement with our previous results ([@B33]) and with those of Irimura and colleagues ([@B42]). The skin-derived cells, expressing CD115 *in situ*, have lost CD115 expression upon LN arrival. Furthermore, FITC-positive LN cells have retained CD11b at an intermediate or low level, related to a DC nature, rather than the high level CD11b^+^ myelomonocytic cells, which are FITC-negative and partially M-CSFR^+^ (not shown). In agreement with their DC identity, gated FITC-positive LN cells show high levels of MHC class II and CD86 expression.
Together, our data demonstrate that M-CSFR protein is expressed by DC at the precursor and iDC stage, but down-regulated in the final stage of DC maturation. We show this during *in vitro* maturation of DC derived from BM precursors stimulated with GM-CSF, and our *in vivo* findings on maturation of connective tissue DC that migrate to skin-draining LN support this notion. In agreement with this, Cheong *et al*. have shown that monocytes down-regulate M-CSFR expression upon maturation to inflammatory LN DC *in vivo* ([@B44]).
Inhibition of miR-22, -34a, and -155 dysregulates M-CSFR expression and blocks DC maturation {#S3-14}
--------------------------------------------------------------------------------------------
At this point, it is reasonable to hypothesize that up-regulation of miR-22, -34a, and -155 is causally involved with *Csf1r* down-regulation. To substantiate this, we investigated whether M-CSFR protein down-regulation in maturing DC *in vitro* could be prevented by inhibiting miR-22, -34a, and -155 using microRNA inhibitor oligonucleotides. First, we used siGLO Cy3-labeled non-targeting anti-miR oligonucleotides to evaluate transfection efficiency, generally varying between 30 and 40% (Figure [4](#F4){ref-type="fig"}A). Then we inhibited microRNA-22, -34a, or -155 on day 4 of GM-DC culture and observed that M-CSFR down-regulation upon LPS-induced DC maturation at day 7 was significantly prevented (Figure [4](#F4){ref-type="fig"}B), demonstrating that miR-22, -34a, and -155 regulate M-CSFR expression in DC in the final stage of maturation. Interestingly, in these experiments we observed that not only down-regulation of M-CSFR was reduced on cells treated miR-22, -34a, or -155 inhibitors, compared to the cells transfected with control inhibitor, but that also the frequency of cells with a mDC phenotype (CD11c^+^MHCII^hi^CD86^hi^) was significantly lower (*p* \< 0.0001) (Figure [4](#F4){ref-type="fig"}C). This suggests that these microRNAs are directly involved with the final step in DC maturation. To investigate whether microRNA-mediated M-CSFR down-regulation was functionally involved in this step, we enforced *Csf1r* expression in developing GM-DC using an expression vector containing the complete ORF of mouse *Csf1r*. Cells were co-transfected with an EGFP expression vector at a 1:4 ratio (EGFP: *Csfr1*) to enable selection for successfully transfected cells (Figure [4](#F4){ref-type="fig"}D). In these experiments we found that enforced *Csf1r* expression significantly (*p* = 0.0013) impaired up-regulation of DC maturation markers MHC class II and CD86 upon stimulation with LPS (Figures [4](#F4){ref-type="fig"}E,F).
{#F4}
In summary, these results show that *in vitro* inhibition of miR-22, -34a, and -155 reduces LPS-induced M-CSFR down-regulation in GM-DC. Moreover, our findings suggest that down-regulation of M-CSFR in DC is required for full DC maturation.
Discussion {#S4}
==========
In this study we provide evidence that microRNAs play an important role in mouse GM-DC development by down-regulating M-CSFR expression in the final maturation step in which DC acquire the mature MHC class II^hi^ CD86^hi^ phenotype (Figure [5](#F5){ref-type="fig"}). We show that the transition from CD11c^+^ MHC class II^med^ CD86^−/lo^ iDC to CD11c^+^ MHC class II^hi^ CD86^hi^ mDC coincides with an up-regulation of some microRNAs and down-regulation of others. Noteworthy in this respect is that spontaneously *in vitro* matured mDC and LPS-induced mDC show remarkably similar microRNA expression profiles. This suggests that spontaneous, GM-CSF-induced and LPS-induced maturation proceed along similar routes, at least on a microRNA level.
{#F5}
Based on our profiling results, we have identified *Csf1r* as a primary target of microRNA-mediated regulation. The role of the *Csf1r* gene in myeloid cell development has been studied at several levels, but its function in DC development has remained underexposed ([@B45]). MacDonald *et al*. have demonstrated in the MacGreen mouse model, in which the *Csf1r* promoter directs the expression of EGFP, that the *Csf1r* gene is generally expressed by DC *in vivo* ([@B6]). Moreover, the significant reduction of DC in mice lacking *Csf1r* indicates that this gene is required for optimal expansion of DC ([@B5]--[@B7]). Although the MacGreen mouse model shows transcriptional activity of the *Csf1r* promoter throughout DC differentiation in GM-CSF-driven BM cultures, it does not necessarily indicate presence of the M-CSFR protein product ([@B6]). Our data demonstrate that *Csf1r* expression is actively down-regulated in the final phase of DC maturation at both the mRNA and protein level via microRNAs.
MicroRNAs are involved in the regulation of M-CSFR expression both directly and indirectly during myeloid cell differentiation. MicroRNAs 17-5p, -20a, and -106 regulate M-CSFR indirectly through targeting of the AML1/Runx1 transcription factor ([@B46]). In early myeloid precursor cells these microRNAs suppress AML1/Runx1 protein expression, leading to limited *Csf1r* transcription and inhibition of monocytic differentiation and maturation. In our profiling experiments, we observed no major changes in miR-17-5p, -20a, or -106 expression during DC development, suggesting that this regulation does not play an important role in later stages. O'Connell and colleagues have previously validated a significant number of targets of miR-155, including *Csf1r* ([@B37]), a finding that was recently confirmed by Lu *et al*. ([@B24]). However, these studies did not address the question whether microRNA-mediated regulation of *Csf1r* influences myeloid cell development. Our data provide evidence that final DC differentiation requires microRNA-mediated regulation of *Csf1r*. Additionally, we show here that miR-22 and miR-34a also directly target *Csf1r* and are up-regulated upon final DC maturation.
Tight regulation of *Csf1r* expression is essential for proper myeloid cell development and occurs at several levels. At a transcriptional level, the *Csf1r* gene is transactivated by several myeloid transcription factors, including PU.1, Runx1, C/EBPα, and several Ets family members through binding to its TATA box-deficient promoter located ∼300 bp upstream of the transcriptional start site ([@B47]--[@B49]). A highly conserved intronic enhancer named FIRE is located in the first intron downstream of the *Csf1r* promoter and contains additional Sp1 and Egr-2 binding motifs ([@B50], [@B51]). At the protein level, membrane-bound M-CSFR dimerizes after binding of its ligand M-CSF to macrophages, resulting in a number of modifications to the cell surface receptor including tyrosine/serine phosphorylation, ubiquitination, and subsequent internalization of the M-CSF/M-CSFR complex before its intralysosomal degradation ([@B52]). Additionally, various pro-inflammatory stimuli cause enzymatic cleavage of the M-CSFR ectodomain by TNFα-converting enzyme (TACE)-mediated shedding *in vitro* ([@B38], [@B53], [@B54]). However, *in vivo* down-regulation of surface M-CSFR expression under inflammatory conditions can also be TACE-independent, since TACE was not responsible for absence of M-CSFR expression from monocytes generated during severe *L. monocytogenes* infection ([@B39]). Our current experiments indicated that TACE also did not play a major role in M-CSFR down-regulation in final GM-DC maturation *in vitro*. Other post-translational modifications to the M-CSFR protein, such as ubiquitination or sumoylation, probably also contribute to M-CSFR instability. For instance, binding of M-CSF to M-CSFR induces conformational changes to the receptor leading to M-CSFR ubiquitination and subsequently altered protein half-life ([@B55], [@B56]).
Here, we have shown that an additional level of *Csf1r* expression regulation occurs, on top of transcriptional and post-translational control, through the action of microRNAs miR-22, -34a, and -155. Interestingly, two studies have indicated that DC from *Mir155^−/−^* mice show impaired expression of maturation markers after LPS stimulation and have impaired T cell stimulatory capacity ([@B57], [@B58]). In contrast, Lu *et al*. reported no such differences in miR-155-deficient DC after LPS stimulation ([@B24]), but the levels of LPS used to induce DC maturation in the latter study were at least 10-fold higher than those used in our and other studies ([@B57], [@B58]). Therefore, it is possible that hyper-stimulation by LPS annuls the attenuated DC maturation phenotype caused by miR-155 deletion. Direct comparison of M-CSFR-regulating microRNA levels and M-CSFR mRNA and protein expression in LPS- vs. spontaneously matured DC (Figures [2](#F2){ref-type="fig"}B,C, and [3](#F3){ref-type="fig"}A) indicates that differences at microRNA level, although important, do not provide a full explanation for differences at mRNA and protein level, and therefore post-translational mechanisms described above probably play an important additional role. Together, this shows that post-transcriptional microRNA-mediated regulation of *Csf1r* mRNA expression complements its transcriptional regulation, in line with the notion that *Csf1r* is stringently regulated during myelopoiesis.
To address the role of M-CSFR during final GM-DC maturation, we inhibited its down-regulation via microRNA inhibitors or transiently over-expressed *Csf1r* in GM-DC. Both approaches resulted in a significant inhibition of LPS-induced final DC maturation. Interestingly, mice lacking the *Csf1r* gene show major alterations in different subsets of DC, where especially the CD11b^+^ CD103^−^ subset is affected ([@B6], [@B7]). Therefore, both absence of *Csf1r* as well as enforced expression lead to aberrant DC development. This underscores that regulation of the levels of this receptor throughout DC differentiation is crucial. Our analysis of M-CSFR expression by skin and LN DC suggests that this regulation also occurs during *in vivo* maturation of DC. In agreement with these findings, Cheong and colleagues have shown that mouse blood monocytes, which are recruited to peripheral LN after i.v. injection of Gram-negative bacteria or LPS, also lose their M-CSFR expression upon *in vivo* differentiation to DC in the draining LN ([@B44]). It is tempting to speculate that especially expression of miR-155, stimulated by the inflammatory conditions ([@B59]), is involved with M-CSFR down-regulation in these infection-induced monocytes and monocyte-derived DC. The importance of post-transcriptional regulation of *Csf1r* is further supported by Sasmono *et al*. who have found that mouse neutrophilic granulocytes contain significant amounts of *Csf1r* mRNA, but do not express the corresponding protein product ([@B60]). Involvement of microRNAs in this regulation has not been shown, however.
How might M-CSFR-mediated inhibition of DC differentiation operate at the molecular level? Although speculative, a possible scenario is that M-CSFR triggering leads to sustained activity of the PI-3K/Akt pathway ([@B52]). This then inhibits LPS-induced activation of p38 MAPK, JNK and NF-κB which are important for the expression of mDC characteristics such as MHC class II and CD86 ([@B61], [@B62]). This view is supported by a study showing that GM-DC from mice lacking SHIP, a negative regulator of the PI-3K pathway, have an immature phenotype and mature poorly in response to LPS ([@B63]). Notably, this apparent block in DC maturation could be inverted by treating these cells with PI-3K inhibitors LY294002 or Wortmannin, indicating that PI-3K is a negative regulator of DC maturation. Therefore, M-CSFR-mediated activation of the PI-3K pathway might block final LPS-induced DC maturation. Additionally, M-CSFR-signaling elevates levels of c-Fos ([@B64]), whereas miR-155-mediated down-regulation of c-Fos was recently shown to be involved with DC maturation and function ([@B58]). It should be stressed, however, that M-CSFR signaling involves a variety of pathways, including those mediated by Ras, Jak/STAT, or β-catenin. Collectively, these downstream mechanisms are responsible for the final outcome of M-CSFR signaling in macrophages ([@B52], [@B65]). Which of these pathways is responsible for inhibition of DC maturation by M-CSFR activity remains to be demonstrated.
In summary, we have elucidated a previously unknown molecular mechanism regulating the final step in monocyte-derived DC maturation, which acts upstream of M-CSFR signaling and where the receptor itself is subject to microRNA-mediated control. Our work demonstrates that decreasing M-CSFR signaling contributes to enable final GM-DC maturation.
Conflict of Interest Statement {#S5}
==============================
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.
We wish to acknowledge Edwin de Haas and Benjamin Bartol for operating the cell sorter and Drs. G. van Cappellen and A. Houtsmuller for help with confocal microscopy. TACE inhibitors TMI-1 and -2 were kindly provided by Dr. B. Scholte (Erasmus MC, Department of Cell Biology).
[^1]: Edited by: Burkhard Ludewig, Kantonal Hospital St. Gallen, Switzerland
[^2]: Reviewed by: Sam Basta, Queen's University, Canada; Theresa T. Lu, Weill Cornell Medical Center, USA
[^3]: ^†^Joris Pothof and Pieter J. M. Leenen have contributed equally to this work.
[^4]: This article was submitted to Antigen Presenting Cell Biology, a section of the journal Frontiers in Immunology.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Hepatitis B virus (HBV) is a hepatotropic DNA virus infecting humans for more than 1500 years \[[@ref1]\]. To date, HBV is a leading cause of liver diseases worldwide, with more than 350 million people being persistently infected and at high risk developing liver failure, cirrhosis or hepatocellular carcinoma (HCC) \[[@ref2],[@ref3]\]. An increased proportion of chronic disease is due to the emergence of viral variants that either abrogate or reduce HBeAg production \[[@ref4]\]. One of the most critical mutations is the double A1762T/G1764A substitution in the basal core promoter (BCP) region and its appearance is usually followed by the development of the triple mutant A1762T/G1764A/C1766T \[[@ref5]\]. The most common mutation that results in an HBeAg-negative phenotype is the G1896A variant of the precore region and is usually accompanied by a second G1899A mutation \[[@ref6]\]. Variants with these mutations associate with more severe forms of liver damage and the development of HCC \[[@ref7],[@ref8]\]. Studies on the effect of BCP mutations on viral replication remain controversial. Among other studies we have shown that they induce low viral transcription and replication (unpublished data) \[[@ref9]-[@ref11]\]. Precore mutated variants associate with high viral replication or have no effect on viral load (unpublished data) \[[@ref11]-[@ref13]\].
The outcome of HBV infection depends on the intensity and type of anti-viral immunity produced by the host. In acute infection the adaptive immune system mounts a strong, multi-specific response, but its effectiveness depends on the quality of the earlier innate immune response. NK cells are important effectors of the innate antiviral immunity. In addition to the direct killing of viral-infected cells without antigen-specific priming, they regulate the adaptive immune response by producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α and immunoregulating cytokines. NK cells are enriched markedly in the liver, the site of HBV replication, implying that HBV has to evade NK cell-mediated immune responses to establish a persistent infection. In addition the high NK activity early in infection and during the incubation time suggests that they are key plays in viral clearance \[[@ref14]-[@ref17]\]. NK cells, however, can also negatively regulate specific antiviral immunity in CHB infection by contributing in the liver inflammation through TNF-related apoptosis-inducing ligand (TRAIL)- and Fas-mediated death \[[@ref17],[@ref18]\] and by the direct killing of HBV-specific CD8+ T cells, which triggers the recruitment of inflammatory cells that amplify the hepatic damage \[[@ref19],[@ref20]\].
The effector functions of NK cells are tightly regulated by the dynamic and coordinated balance of activating and inhibitory signals. In the present study, the NK cell receptors were selected on the basis of being known to control key cell functions. The main NK cell activating receptors studied in this respect include C-type lectin-like NK group 2D (NKG2D) receptor and immunoglobulin-like receptor NKp30. NKG2D is a potent activating receptor expressed in all NK cells, NKT cells and activated CD8+ T lymphocytes \[[@ref21],[@ref22]\]. It binds to a family of ligands (NKG2DL) including the major histocompatibility complex class-I polypeptide-related chain (MIC) protein family that contains MICA and MICB; and the cytomegalovirus UL16-binding proteins (ULBP) family, which consists of five members, ULBP1-4 and RAET1G \[[@ref23]\]. Another activating receptor involved in the direct regulation of NK cell-mediated recognition of cellular stress is the DNAX accessory molecule-1 (DNAM-1, CD226), which is constitutively expressed on NK cells, T cells, macrophages and a small subset of B cells \[[@ref24]\]. Similar to NKG2D, DNAM-1 not only promotes adhesion and activation of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets \[[@ref24]\]. Furthermore, lectin-like transcript 1 (LLT1, gene *clec2d*) is a type II transmembrane receptor that belongs to the C-type lectin like (CTL) superfamily of NK cell receptors. Six alternatively spliced transcripts of *clec2d* exist, with the isoform 1 (designed as LLT1) being the only one presented on the cell surface and hence the only isoform able to participate in the cell-to-cell transmission \[[@ref25]\]. LLT1 is expressed mainly on activated lymphocytes, including NK, T cells and B cells, as well as on antigen presenting cells (APC) \[[@ref26]\]. However, a decade ago, LLT1 was also identified as a physiological ligand of the NKR-P1 (CD161), expressed on all NK cells and subsets of T cells and represents the only member of the human NKR-P1 subfamily \[[@ref27],[@ref28]\]. NK cell cytotoxicity and IFN-γ production is inhibited upon engagement of NKR-P1 on NK cell with LLT1 on target cell \[[@ref27],[@ref28]\]. Upon viral infection, LLT1 expression is upregulated on surface of epithelial cells, while IFN-γ has been shown to increase LLT1 expression on B cells and APC \[[@ref29]\]. Hence, LLT1-mediated signaling is thought to represent a system that regulates both innate and adaptive responses. For activated NK cells to achieve successful lysis, strong adhesion to target cells that is required is mediated by the β2 integrin LFA1 \[[@ref30]\].
The objective of this study was to investigate the modulation of ligands for cell surface NK receptors in chronic HBV (CHB) patients and HepG2 cells transfected with BCP and precore mutants. Furthermore, we aimed to correlate patient's viral replication and the differential viral capacity of hotspot mutants with the expression of NK receptors and their ligands at the RNA level.
Patients and methods {#sec1-2}
====================
Patients {#sec2-1}
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Nineteen patients (11 men and 8 women; mean age 44.5±12 years) with CHB were studied ([Table 1](#T1){ref-type="table"}). Tissue samples were obtained by percutaneous needle biopsy, snap-frozen and stored in liquid nitrogen. From all the individuals included in the study a serum sample was collected at the time of liver biopsy, stored at -80°C and HBV DNA levels were measured using real-time polymerase chain reaction (qPCR). Individuals with a history of excess alcohol, or positive for HCV, HDV or HIV were excluded. Informed consent was obtained from all the patients (The department of Hepatology St Mary's Liver Tissue Bank, 06/Q0509/39). 1mg of liver tissue was processed using the gentleMACS Dissociator (Miltenyi Biotec SAS, Paris, France) prior to RNA extraction.
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Clinical characteristics of the studied patients. Cirrhosis was measured with according to the Ishak scoring system and necro-inflammation with the Knodel system. Expression of liver HBV RNA was normalized with h-GAPDH and data represents the mean±SEM from 3 RT PCR experiments. Patients were divided into four groups according to their serum viremia and intrahepatic HBV RNA levels
Transient transfection experiments {#sec2-2}
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The G1896A, G1899A, G1896A/G1899A, A1762T/G1764A and A1762T/G1764A/C1766T mutations were generated by splice extension mutagenesis using as a template a plasmid (p3.8II) containing a 1.2 genome length of HBV subtype *adr* (genotype C) \[[@ref31]\] in a pBluescript II KS (+) background (Stratagene, CA, USA) ([Table 2](#T2){ref-type="table"}). Full-length HBV genomes were amplified from wild-type (WT) or mutant HBV genomes according to the method described by Günther, using the P1 sense (HBV positions: nt1821-1841) and P2 antisense primers (HBV positions: nt1823-1806) modified to contain the *HindIII/SapI* sites and the *SacI/SapI* sites, respectively ([Table 2](#T2){ref-type="table"}) \[[@ref32]\]. PCRs were performed using the FastStart High Fidelity PCR System (Roche Applied Science, Germany) according to the manufacturer's instructions. Each of the 3.2kb amplified HBV fragments were purified from agarose gels using the GeneJET Gel Extraction Kit (Thermo Scientific, Life Science Research, Lutterworth, UK) and were cloned into a pCRII TA-vector (Invitrogen, Paisley, UK). All constructs were sequenced to confirm the presence of the mutations and to ensure that no additional mutations were introduced in the process.
######
Primers used full-length HBV genome generation by splice extension mutagenesis
Linear HBV monomers were released by *SapI* restriction digestion (New England Biolabs, Beverly, MA), gel purified, and then used for transient transfection of HepG2 by lipofection. HepG2 cells were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 IU penicillin mL^-1^ and 100 μg streptomycin mL^-1^. Cells were seeded at a density of 4x10^5^ cells in 60 mm diameter dishes (Corning, Flintshire, UK) and the following day were transfected with 500 ng HBV linear monomers using Lipofectamine Plus (Invitrogen) according to manufacturer's instructions. The culture medium was changed 24 h after transfection, and cells were harvested at 48 and 72 h.
All transfections included 1 μg of reporter plasmid expressing a green fluorescent protein to monitor transfection efficiency by fluorescence-activated cell sorting analysis and ranged 43-50% while the empty pUC19 vector used as a negative control.
RNA quantification {#sec1-3}
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Northern blot analysis {#sec2-3}
----------------------
For Northern blot analysis, 10 μg of total RNA per sample was separated on a 1% formaldehyde-agarose gel and blotted onto a Zeta-Probe GT membrane (BioRad, Bio-Rad Laboratories, Hercules, CA). Radioactive probes were prepared by random priming, using either full-length HBV DNA or 18S cDNA template and ^32^P labeled αdCTP (Amersham). After hybridization the membrane was washed and exposed to X-Omat film (Kodak, Rochester, NY) at -80°C.
Relative quantification by real-time (RT) PCR {#sec2-4}
---------------------------------------------
Total RNA was extracted from HepG2 cells 48 h after transfection with *SapI* -digested HBV DNA monomers and liver biopsies using Trizol reagent (Invitrogen) as recommended by the manufacturer. The RNA samples were treated with DNase (Qiagen, Crawley, UK) for 10 min at 25°C and were then purified using the RNeasy mini kit (Qiagen). RNA integrity was confirmed by agarose gel electrophoresis under UV, RNA concentrations were monitored by using the nanodrop and samples were stored at -80°C until further use.
Two μg RNA from each transfection and 500 ng RNA from each liver biopsy were reversed transcribed in 20 μL reactions using the RT^2^ First Strand kit (Qiagen). There were three biological replicates in each group for each transfection experiment and duplicates for each liver biopsy samples. The resulting cDNA samples were stored at -80°C until further use.
The relative levels of total HBV RNA transcripts concentrations were analyzed by real-time PCR with a TaqMan (Life technologies Paisley, UK) in a Light-Cycle (Roche Diagnostic, West Sussex, UK). Each PCR reaction was performed in a 75 μL reaction volume containing 5 μL cDNA, 0.5 mmol/L forward and reverse primers, 0.2 mmol/L 3' FL-labeled probe, and 0.4mmol/L LC-labeled probe. The forward and reverse primers used were 5'-CTCGTGGTGGACTTCTCTC-3'and 5'-cagcaggatgaagaggaa-3', and the probes were 5'-LC640-TGTCCTGGTTATCGCTGGATGTGTCT-PH-3' and 5'-CAC TCACCAACCTCCTGTCCTCCAA-FL-3'. The h-GAPDH housekeeping gene Light Cycler Set (Roche DNA control kit, Roche Diagnostics) was used to normalize RNA expression.
For the detection of MICA, ULBP2, ULBP3, B7H6, CD226 (DNAM-1), CLEC2D (LLT1), ITGAL (LFA-1) and TNFSF10 (TRAIL) we performed RT PCR arrays with customized RT^2^ custom arrays containing pre-dispensed primer assays on a StepOne Plus RT PCR (AB Applied Biosystems, Thermo Scientific, Loughborough, UK) using RT2 SYBR Green/qPCR Master Mix (Qiagen). The custom-made array included two housekeeping genes (GAPDH and β-Actin) and three internal controls (human genomic DNA contamination control, reverse transcription control and positive PCR control). Each PCR reaction contained cDNA synthesized from 125 ng of total RNA. The thermocycler parameters were 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 min.
Relative changes in gene expression for both viral and cell RNA transcripts were calculated using ΔΔC~t~ (threshold cycle) method. Threshold cycle numbers (Ct)^2^ above 35 were considered below detection level. The β-ACTIN housekeeping gene was used to normalize the RNA amounts. Results are expressed relative to β-ACTIN with NK markers as 2^-ΔΔCT^, where ΔΔC~t~ = ΔC~T-sample~ - ΔΔC~T-control~ and where ΔC~T~ = C~T-target\ gene~ - ΔC~T-ACT~.
Statistical analysis {#sec2-5}
--------------------
Two group comparisons of continuous variables were performed using the non-parametric Mann-Whitney test with two tailed values (GraphPrism 6). P values below 0.05 were considered statistical significant.
Results {#sec1-4}
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Liver and serum viremia in CHB patients {#sec2-6}
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We determined the expression of total HBV RNA in the liver of infected patients by RT PCR and compared this to the levels of serum viremia. It is well known that intrahepatic viremia does not correlate with serum viremia since the liver sampled cannot be representative. Indeed the levels of intrahepatic HBV RNA did not correlate with serum HBV viremia (r=0.34, P\>0.1) ([Fig. 1](#F1){ref-type="fig"}). We therefore divided the patients into four groups: 1) Low Serum Viremia patients, included those with serum viremia 1x10^1^ -- 1x10^4^ viral copies/mL; 2) High Serum Viremia group, included patients with serum viremia 1x10^6^ -- 1x10^10^ viral copies/mL; 3) Low Liver HBV RNA patients, included those liver samples with 1x10^1^ -- 1x10^3^ HBV RNA levels relative to β-ACTIN expression; and 4) High Liver HBV RNA samples with 1x10^4^ -- 1x10^5^ HBV RNA levels ([Table 1](#T1){ref-type="table"}). Twelve of 19 patients were HBeAg-negative and HBeAb-positive; all patients had minimal inflammation levels; 1/19 patient was cirrhotic and 8/19 patients had elevated alanine aminotransferase (ALT) levels.
{#F1}
Expression of NK regulatory molecules {#sec2-7}
-------------------------------------
**CHB patients**. We examined the expression of the key NK cells ligands MICA, ULBP2, ULPB3, B7H6, the NK receptors DNAM-1, LFA-1, TRAIL and LLT1 in the livers of CHB patients ([Fig. 2](#F2){ref-type="fig"}). In general, individuals with higher liver viral loads had higher expression of NK cell ligands and receptors. The exception to this was MICA, which was significantly downregulated in patients with high intrahepatic HBV RNA levels (P=0.02) and decreased in high viremic patients (P=0.05). We also compared the gene expression in 7 HBeAg(+) patients and 12 HBeAg(-) patients ([Fig. 3](#F3){ref-type="fig"}). The expression of ULBP2, ULBP3, LLT1, LFA-1, and DNAM-1 was upregulated in HBeAg(+) patients, the majority of which had increased serum and liver HBV levels, while MICA was downregulated in this group of patients. Interestingly LLT1 is a ligand for the inhibitory receptor CD161, expressed on NK cells and intrahepatic T cells. Thus, this together with the down-regulation of MICA, may represent potential escape mechanisms for HBV, counteracting the trends observed for the other NKG2D ligands and also B7H6 the ligand for NKp30. Consistent with the lack of correlation between serum and liver HBV viremia, we observed no consistent trends of NK cell ligand expression and serum viremia.
{#F2}
{#F3}
**HBV transfection experiments.** We had previously constructed using site directed mutagenesis, the replication-competent plasmids bearing the BCP mutations 1762/64 and 1762/64/66 and the precore 1896, 1899 and 1896/1899 (unpublished data) ([Table 2](#T2){ref-type="table"}). Using the same HBV constructs and plasmid-free transfection system, we have previously demonstrated that compared to the WT and precore variants, BCP mutations result in lower viral replication capacity while the precore variants show similar replication fitness to the WT. BCP mutants consistently produced the decreased accumulation of all HBV RNA transcripts (Northern blotting) ([Fig. 4](#F4){ref-type="fig"}); the viral replicative intermediates, including cccDNA (Southern blotting); and the culture HB surface antigen (HBsAg) levels (ELISA) (unpublished data).
{#F4}
To investigate whether the differential replication of HBV induces changes in the expression of ligands for NK cell receptors on transfected HepG2 we performed RT qPCR ([Fig. 5](#F5){ref-type="fig"}). Transfection of HepG2 cells induced the up-regulation of all NK cell ligands tested. MICA, ULBP3 and B7H6 transcript levels were not significantly altered after exposure to the WT and HBeAg mutant variants examined ([Fig. 5](#F5){ref-type="fig"}). However we observed a weak trend towards an association of ULBP2 with viremia in that there were reduced levels of ULBP2 mRNA with the BCP mutants compared to WT and increased ULBP2 levels expression with the pre-core variants. However, the expression of LLT-1 in HepG2 transfected with the WT genome was significantly decreased compared to 1762/74 (P=0.01) and 1762/64/66 (P=0.04). Thus, LLT-1 correlated with the BCP mutations and their low replication efficiency. This is consistent with our findings on intrahepatic LLT1 RNA levels.
{#F5}
Discussion {#sec1-5}
==========
Accumulating evidence suggest that activation of the NKG2D-ligand pathway contributes to the outcome of HBV infection. The unpredictable natural history of CHB infection makes it difficult to sample the immune correlates of viral replication activity longitudinally. The level of HBV DNA in serum is commonly used as a surrogate marker of HBV replication in the clinical setting but it does not reflect the levels of HBV in the liver \[[@ref33]\]. To monitor HBV levels, in addition to serum viremia we assessed HBV RNA expression in the same liver sample that we determined NK ligand RNA expression. We found that MICA transcripts were decreased in highly viremic liver samples of CHB patients whereas there was a trend towards increased expression of ULBP2 and ULBP3 with HBV liver viremia. Thus, a general trend towards upregulating expression of NK cell ligands, could be counteracted by decreasing expression of MICA and hence weakening NK surveillance of the infected cells. These patients had minimal inflammation on their liver biopsies and therefore downregulation of MICA could be both harmful by inhibiting NK-induced viral clearance but also protective by preventing the infected hepatocytes from becoming targets for NK cell-mediated killing and hence preventing the development of liver injury. The NK markers B7H6, DNAM-1, LFA-1 and TRAIL were also slightly elevated in high viremic liver samples. Our results indicate that high HBV replication induces the upregulation of gene expression of these NK ligands. A recent study by Lunemann showed lower levels of NKG2A, DNAM-1 and TRAIL on the NK cells of CHB patients as compared to acute patients \[[@ref34]\]. It has been previously shown that higher expression of NKG2D ligands on cancer cells is associated with down-regulation of NKG2D on NK cells related to soluble NKG2D ligand secretion \[[@ref35],[@ref36]\]. We therefore propose that a similar process occurs in CHB.
In the course of CHB infection, HBeAg-negativity concurs with the emergence of the precore mutation 1896 and 1899 while the BCP mutation 1762/64 and 1762/64/66 reduces HBeAg production. We have previously showed that the BCP mutations result in lower viral replication capacity, whereas the major precore mutations have no effect on viral replication. The expression of all NKG2DL was increased when HepG2 cells were transfected by HBV suggesting that HBV induces NKG2DL upregulation. This finding is consistent with our observation that NK cell ligands, with the exception of MICA, are increased in HBV infection. However, consistent trends for the ligands for activating receptors were not observed between *in vivo* and *in vitro* assays nor between HBeAg(+) and HBeAg(-) patients, with the possible exception of ULBP2. In general, NKG2D is a system thought to be important for clearing viral infection, and especially in cytomegalovirus infection, in which the virus has developed multiple mechanisms to down-regulate different NKG2D ligands \[[@ref37]\]. Consistent with previous studies, we found that MICA expression was reduced in the liver of CHB patients \[[@ref38],[@ref39]\]. Transfection of HepG2 cells with all HBV constructs induced its expression suggesting that early in infection MICA is upregulated but in the persistent infection HBV weakens the immune surveillance of NK cells by inhibiting MICA expression.
Conversely, expression of LLT1 was increased in patients with high serum and intrahepatic viremia. Likewise, in HepG2 transfections we found low but detectable levels of LLT1 that were positively correlated with the differential viral replication of the HBeAg mutated variants. Upregulation of LLT1 and its subsequent engagement to their CD161 receptor expressed on NK cells have been shown to inhibit NK-mediated cytotoxity and IFN-γ production by T cells \[[@ref27]\]. LLT1 expression has been reported to be absent in normal liver tissue but upregulated in HCC. Here we show for the first time that LLT1 is expressed in HBV infected liver and its levels associate with viral replication in HBV infection. Thus, HBV may circumvent the immunity of the host by inhibiting NK cytotoxicity and IFN-γ production via the upregulation of LLT1.
A detailed characterization of the molecular players that link the HBV stimuli to the regulation of NK cells will be critical to advance our knowledge on how HBV circumvents the host's immunity. The manipulation of ligand expression can provide new insight on CHB immunopathogenesis and can lead to possibilities of developing effective treatment strategies.
###### Summary Box
**What is already known:**
Natural killer (NK) cells play a key role in the outcome of hepatitis B virus (HBV) infectionHBV can circumvent host's immunity by modulating the expression of NK receptors and their ligandsMICA is ligand of the activating NKG2D receptor and is important in clearing viral infectionLLT1 is expressed on NK cells and is a ligand of the NKR-P1 receptor of NK cells
**What the new findings are:**
The gene expression of ULBP2, ULPB3, B7H6, DNAM-1, LFA-1 and TRAIL was elevated in high HBV replication *in vivo*HBV infection repressed MICA expression in the liver of chronic HBV patients whereas HBV transfection induced its expression, suggesting that early in infection MICA is upregulatedLLT1 expression was increased in patients with high serum and intrahepatic viremiaIn HepG2 transfections LLT1 levels were positively correlated with the differential replication of HBeAg mutated variantsA general trend towards upregulating expression of NK ligands can be counteracted by decreasing expression of MICA and hence weakening NK surveillance of the infected cells
This study was supported by grants from the European Association of the study of Liver (EASL).
Conflict of Interest: None
Imperial College, St. Mary's Campus, London, UK; University Hospital of Messina, Messina, Italy; University of Nicosia Medical School, Nicosia, Cyprus; Henry Welcome Laboratories, Southampton General Hospital, Southampton, UK
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The dataset supporting this article is available on the ORDA (Online Research Data) database (provided by figshare), DOI: [10.15131/shef.data.12732824](https://doi.org/10.15131/shef.data.12732824).
Introduction {#sec001}
============
During the last decade, the human-robot or robot environment interactions have been one of the main foci of research in robotics. Advances in tactile sensing \[[@pone.0237379.ref001], [@pone.0237379.ref002]\], compliant robotics \[[@pone.0237379.ref003], [@pone.0237379.ref004]\], soft robotics \[[@pone.0237379.ref005], [@pone.0237379.ref006]\] and new control methods \[[@pone.0237379.ref007]\], have been improving robot capability to interact with the environment. This trend is one of the promising advances that can bring new opportunities for robotic applications in the healthcare field. The possible outcomes of human-robot interaction in medical application range from robot-assisted medical imagery \[[@pone.0237379.ref008], [@pone.0237379.ref009]\], teleoperated surgery \[[@pone.0237379.ref010]\], medical training \[[@pone.0237379.ref011], [@pone.0237379.ref012]\] to robotic-assisted examination. This paper focuses on medical palpation of soft tissue to localize hard nodules.
Robot-assisted palpation aims to use a robot to perform haptic examinations of a patient in place of a medical practitioner. Often used as an early-stage examination, the goal of this haptic investigation is to estimate the mechanical properties such as the texture, the stiffness, or the consistency of an organ. It can also help to track the size and the position of a nodule or a tumor in soft tissues \[[@pone.0237379.ref013]\].
In such physical examinations, the compliance of the robotic probe plays an important role due to several reasons such as safety and stability under tissue uncertainty. Since the compliance is defined as the inverse of the stiffness, we mean by compliant system a physical system with low stiffness. By opposition, a stiff system is a system with low compliance. We will deliberately use the two words compliance and stiffness inconsistently in this paper since some ideas are more intuitive when expressed using the stiffness, while some others are more intuitive with the compliance. However, a stiff robotic probe to explore patient tissues relies only on compliance of the tissues which increases the risks of hurting the patient. Our previous study has shown that the compliance of the probe can be used to maximize haptic information gain during hard nodule depth estimation in soft tissue exploration \[[@pone.0237379.ref014]\]. There have been several approaches to give compliance to robots. These approaches can be based on mechanisms such as Variable Stiffness Actuators (VSA) \[[@pone.0237379.ref015], [@pone.0237379.ref016]\], passive elements integration \[[@pone.0237379.ref017]\], or based on control algorithms such as impedance controllers \[[@pone.0237379.ref018], [@pone.0237379.ref019]\]. The role of the compliance and the impedance have been widely studied for stability analysis, disturbance rejection or energy consumption, but the impact of such compliance on the perception remains an open question. Thus, the mechanical impedance of a system can, for instance, be used to filter the information and signals measured by a robot while interacting with the environment \[[@pone.0237379.ref020]\].
In the past, most approaches to robotics treated perception (sensing) and action (actuation) as decoupled phenomena. It seems clear that in the context of haptic exploration or robotic interaction with an uncertain environment, actions taken by the robot directly affect haptic perception. Our approach presented in this paper is inspired by recent studies that provide a better insight into the interaction between perception and action \[[@pone.0237379.ref014], [@pone.0237379.ref021]\]. In particular, we propose a novel control algorithm tested with the Variable Lever Mechanism (VLM) probe \[[@pone.0237379.ref022]\] to detect and localize an embedded nodule in soft tissues. Thanks to its controllable stiffness and its two sensing modalities (kinesthetic and haptic) we used the VLM probe to demonstrate that the compliance of the joints plays a significant role in the haptic detection, the localization, and the depth estimation of a nodule. The proposed algorithm uses the compliance of the VLM probe to perform either a local (longitudinal sweep) investigation using only the tip-end of the probe or a wide (lateral sweep) investigation using the palmar region of the probe's tip and its tactile sensor. The stiffness is tuned to maximize the information gain (entropy reduction in a random variable) during the explorations by using Bayes inference and likelihood functions. These likelihood functions are built from a prior knowledge obtained during previous palpation or thanks to Finite Element (FE) simulation.
The remainder of the paper is organized as follows. In the next section, the previous research done on robot-assisted palpation are presented. We will give an insight into the research we accomplished on variable stiffness palpation along with other approaches addressed in the literature. Then, we describe the methods used to study the effect of the stiffness of the VLM probe on the perception and detection of a hard nodule embedded in a soft tissue phantom. In particular, we detail the VLM probe's hardware, the phantom characteristics, the lateral and longitudinal sweep experiments, and the Finite Element (FE) simulation. The next section discusses the experimental and simulation results we obtained. The following section presents the likelihood functions obtained experimentally and from the FE simulations. The new algorithm to condition haptic perception and localize stiff inclusion in soft tissues is then detailed and tested in another section. Finally, we discuss and conclude about the performance of the algorithm and the impact of the stiffness variation on perception tasks.
Related work {#sec002}
------------
During the past two decades, we have witnessed an increasing trend to use Minimally Invasive Surgery (MIS) procedures over open surgeries. Even if we can find some exceptions such as robotic probes to measure the blood flow \[[@pone.0237379.ref023]\], most of the studies on robotically assisted palpation have been motivated to give local information during MIS that the surgeon would have obtained by manual palpation in open surgeries.
The majority of robotic palpation probes aims to detect hard inclusions in soft tissues. In particular, these probes are mainly designed to detect tumors or anomalies which are generally stiffer than the surrounding healthy tissue. The main types of sensors used to do so are the kinesthetic sensors and tactile sensors. In this paper, we refer to kinesthetic sensors, the sensors that aim to give a signal related to the force or the torque applied at a probe joint level. On the other hand, the tactile sensing is referring to fingertip contact sensing using taxel images. Tactile sensing is usually representing the behavior of the mechanoreceptor at the skin level. Finally, It should be noticed that in this section we omit work on medical imagery such as x-ray, CT scans, or ultrasound which are also solutions to detect hard inclusions but not based on haptic palpation.
Kinesthetic palpation probes measure the force and/or torque during the tissue indentation by the probe. For instance, Ahn *et al*. \[[@pone.0237379.ref024]\] developed a force sensing probe for prostate cancer detection and tested it on ex-vivo prostate tissue samples. They concluded on the interest of studying the tissue elasticity to estimate the presence of a nodule. Thus, they gave the likelihood of different elasticity levels in healthy and cancerous tissues respectively with the average Young's modulus they measured as well as the standard deviation. Liu *et al*. \[[@pone.0237379.ref025]\] and Sangpradit *et al*. \[[@pone.0237379.ref026]\] have developed a rolling indentation probe to detect different abnormalities embedded in porcine kidney samples. By computing a reaction force map after probing, they studied the role of indentation and nodule depth and size on the force measurement. They have shown in particular that the deeper and the smaller the nodule is, the hardest it is to detect it.
Tactile sensing is based on the measure of the local contact pressure between the probe and the tissue. Generally, the information given by these sensors is an array of values taken at spatially distributed measurement points also called taxels. Several technologies have been used to integrate tactile sensing in robotic palpation probes. For instance, Kwon *et al*. \[[@pone.0237379.ref027]\] designed a tactile sensor for robotic palpation based on an array of pressure-sensitive resistors. Xie *et al*. \[[@pone.0237379.ref028]\] used an array of optical sensors to measure the contact pressure during MIS palpation.
Trejos *et al*. \[[@pone.0237379.ref029]\] used a combination of tactile sensors and a kinesthetic sensor. The aim of the tactile sensor is to give a pressure map of the palpated region where the kinesthetic sensor is used only to control the robotic arm where the probe is attached to. They also mentioned using a hybrid impedance controller optimized for a stiff arm with kinematic redundancy. However, they did not investigate the impact of compliance during palpation. In our work, we use tactile and kinesthetic sensing in succession and use probe stiffness control to improve haptic perception efficacy during palpation.
Most of the previous work is based on a stiffness evaluation of the tissues and performed with a rigid probe. The interest of using a compliant probe is not only to avoid relying on the tissue compliance for safe robot-tissue interactions, but also to increase the robustness against misalignment of the probe with the tissue. For example, the probes designed by Jia *et al*. \[[@pone.0237379.ref030]\] or Faragasso *et al*. \[[@pone.0237379.ref031]\] are based on passive serial elastic components. A soft robotic approach has been followed by Pacchierotti *et al*. \[[@pone.0237379.ref032]\] using the BioTac sensor that is based on the measurement of the deformation and internal fluid pressure of a compliant fingertip.
Palpation behavior also affects the quality of haptic perception \[[@pone.0237379.ref014], [@pone.0237379.ref033]\]. Several palpation strategies have been proposed in the literature, but most of them have been developed for stiff palpation probes. Therefore, these strategies do not include stiffness control. A common strategy involves probing point by point to identify the local stiffness and refresh a stiffness map each time a new point is available. It is the strategy used by Ayvali *et al*. \[[@pone.0237379.ref034]\] who used a Bayesian optimization to determine where the next palpation point should be to reduce uncertainties. Garg *et al*. \[[@pone.0237379.ref035]\] also used a point by point method with a Gaussian process adaptive sampling in order to estimate the shape of the stiff inclusion efficiently. Using point by point strategy, Hoshi *et al*. \[[@pone.0237379.ref036]\] proposed an algorithm to optimize the stiffness estimation of the palpated tissues by coupling the force measurement with a predictive model based on the Finite Element Method. Nichols *et al*. \[[@pone.0237379.ref037], [@pone.0237379.ref038]\] have developed a point by point palpation strategy which is based on machine learning trained with ultrasonic elastography image. This algorithm is able to segment hard inclusion in soft tissues by giving the shape of the hard inclusion and an estimation of the local stiffness. Park *et al*. \[[@pone.0237379.ref039]\] presented the results of a similar machine learning control strategy to segment real cancerous breast samples with a micrometer scale probe. The point by point palpation strategy is really efficient for segmenting hard inclusions or detecting the shape of these abnormalities, but the uncertainties decrease with the number of points and it is often needed to use many points to obtain an accurate estimation. Multiplying the number of points also means increasing the examination time of the tissue, which can be inconvenient for the patient during an in-vivo examination.
Another approach to detect and diagnose hard inclusions in soft tissue is sweeping the probe on soft tissues. This method gives spatially continuous information of the palpated tissue, which reduces the time of examination, but the measured data often suffer from dynamic disturbance and non-linear effects due to the viscoelasticity of the tissues. Chalasani *et al*. \[[@pone.0237379.ref040]\] proposed a palpation strategy based on sweeping with a sinusoidal normal force profile. Since only the point where the force is maximum are used for the estimation of the hard inclusion, this strategy can be considered as a hybrid method between the sweeping method and point by point method. Salman *et al*. proposed an algorithm for a stiff probe that computes an optimal trajectory of sweeping palpation after based on prior knowledge obtained with point by point palpation \[[@pone.0237379.ref041]\]. They, in particular, have shown that switching from point by point to sweeping can save examination time. Ahn *et al*. \[[@pone.0237379.ref042], [@pone.0237379.ref043]\] have developed a probe for prostate cancer diagnostics. This probe performs a rotational sweep and measures the force during the sweep. The force is then compared to experimental and FE simulations to diagnose if the tissue contains a tumor or not. Based on the study of how humans perform palpation exploration, Konstantinova *et al*. \[[@pone.0237379.ref044]\] proposed an autonomous probing strategy following an auto-regressive force regulation to estimate the depth of a nodule in soft tissues. Nevertheless, all these sweeping strategies have been developed for stiff probes.
Our previous studies \[[@pone.0237379.ref014], [@pone.0237379.ref045]--[@pone.0237379.ref047]\] were also inspired by how humans regulate stiffness of fingers during soft tissue palpation. In the previous study \[[@pone.0237379.ref047]\], we have shown that the stiffness of the arm and hand joints is modified during the longitudinal sweeping exploration of soft tissues by varying the level of co-contraction of antagonistic muscles. The previous strategies to control the probe's stiffness was based on Markov chains and shown a reduction of the number of sweeps (5 Bayesian iterations to reach 80% of confidence) needed to estimate the depth of a nodule compared to a strategy with a random stiffness. The new algorithm proposed in this paper allows not only to estimate the depth of the nodule but also to perform the full 3D localization. Contrary to the previous algorithm, the proposed strategy conditioned the likelihood of the force peak prominence to minimize its variance and maximize the haptic information gain. The compliance of the probe is used to switch from lateral sweep palpation, which provides information for a wide area, to longitudinal sweep palpation, which investigates tissue along a straight line passing over the suspected location of the nodule, without reorienting the probe. Finally, with this new strategy which combines tactile and kinesthetic measurements with likelihood conditioning, we increased the size of the probed area, we added the localization of the nodule, we improved the resolution of the depth estimation from 3mm (in \[[@pone.0237379.ref047]\]) to 0.2mm without increasing the number of Bayesian iterations (on average).
Materials and methods {#sec003}
=====================
To study the role of the joints' stiffness on haptic perception during 3D localization of nodules in soft tissue palpation, we first describe the experimental setups and protocols. In the following subsections, the VLM probe hardware and phantom fabrication are presented. We then describe the sweeping directions studied in this paper. Finally, the FE simulation used to model the behavior of the probe during longitudinal sweep is detailed.
Variable stiffness palpation: The VLM probe {#sec004}
-------------------------------------------
As described in \[[@pone.0237379.ref022]\], the VLM probe (see [Fig 1](#pone.0237379.g001){ref-type="fig"}) is composed of a variable stiffness joint based on a variable lever mechanism and two sensors: a *Cyskin* tactile sensor \[[@pone.0237379.ref002]\] placed on the VLM probe fingertip and an *ATI NANO 25* placed at the equivalent wrist level. The stiffness variation of the VLM probe is based on position control of a flexible carbon rod. Changing the position of this carbon rod modifies the active length (the part which can bend) of the rod and, by cantilever effect, it changes the stiffness of the joint. An analytical model to correlate the carbon rod position to the equivalent angular stiffness of the joint has been given in our previous study.
{#pone.0237379.g001}
[Fig 1](#pone.0237379.g001){ref-type="fig"} shows the design of the VLM probe. The VLM probe is based on a revolute variable stiffness joint composed of 2 rigid links (the base link and the tip link) connected with a revolute joint in parallel with a deformable carbon rod. This carbon rod acts as a variable spring that allows the stiffness of the joint to be controlled thanks to an *Actuonix L12-30-50-6-I* linear actuator. This actuator slides the carbon rod through the base link and the tip link changing the length of the carbon rod that can be bent (active length). As one can see, the hole in the base link has been designed such as that the carbon rod can slide axially but is constrained radially to prevent bending of the rod in the base link. On the other hand, the hole in the tip link is large enough to allow the carbon rod to bend in. A PTFE cylinder is used to transmit the radial forces between the tip link to the carbon rod. This PTFE cylinder has been designed to slide easily axially when the actuator is translating the carbon rod. Adjusting the active length of the carbon rod changes, by cantilever effect, the amount of force required to bend the rod and by consequence the angular stiffness of the probe.
In order to describe the movement of the probe, we need to define a frame. First, we define the axis **z** as the direction of the normal to the phantom surface. We then define the **x** axis as the intersection between the tangent surface of the phantom and the mid-sagittal plane of the probe. Finally, the **y** is defined in order to obtain a direct orthonormal frame (**x**,**y**,**z**). In the rest of the paper, this reference frame will be used to describe the directions of forces or displacement.
To move the VLM probe on the phantoms, the probe is attached to a 3 axis Cartesian robot. This robot is composed of an *Aerotech ANT130-XY* stage and an *Actuonix L16-50-150-12-P* linear actuator which allows the probe to be moved in the horizontal plane (**x**, **y**) and vertically (**z**) respectively. Two *National Instruments* cards are used to acquire the sensors' signals and control the vertical position and the stiffness of the probe. Especially, a *NI PCIe-6320* is used in order to acquire the force sensor signals whereas a *NI USB-6341* controls the two linear actuators positions. The programs to run the experiment and the algorithm have been implemented using C++.
In this paper, the indentation refers to vertical displacement of the actuator of the VLM probe along negative direction of **z** (positive when going down) instead of the depth of the probe's tip in the phantom. Additionally, the indentation 0mm refers to the point where the contact between the probe and the phantom starts. To deal with the issue of alignment between the phantom and the XY stage, the surface roughness of the phantom and possible deformation of the latter, the tangent surface of the phantom (0mm indentation position) is autonomously redefined each time the region of exploration is changed.
To autonomously detect the 0mm indentation position, the method is based on an indentation (without sweeping) and the detection of variation in the kinesthetic sensor measurement. This detection strategy aims to improve the robustness of the nodule detection by improving the accuracy of the indentation measurement. This is particularly important since related studies have shown that a variation of indentation can impact the nodule depth estimation \[[@pone.0237379.ref025], [@pone.0237379.ref047]\].
For all the experiments, the Cartesian coordinates (*x*, *y*, and indentation) of the probe are measured at 50Hz for *x*, *y*, and 10kHz for the indentation respectively. The forces and the position of the actuator which control the stiffness are also acquired at 10kHz. Finally, the tactile pressure is acquired at 20Hz. The acquisition rates are different, but all the signals are time-synchronized.
The angle between the surface of the phantom and the probe is set at 32°. This angle has been chosen to optimize the number of taxel in contact after indentation during the sweeps.
Soft tissue phantom with nodules {#sec005}
--------------------------------
The phantom is made of platinum-catalyzed silicone Ecoflex 00-10 (Smooth-On, Inc, USA), with 6 nodules embedded at a depth of 2 × 2mm, 2 × 4mm, 1 × 6mm and 1 × 8mm (the two additional nodules 2 and 4mm deep have not been used as prior knowledge in the likelihood functions but are used to test the proposed detection algorithm.). It has to be noticed that we define as nodule depth the distance between the surface of the phantom and the upper tangent plane to the nodule. All nodules are made of acrylic with a diameter of 16mm. The phantom is cast at room temperature with a width of 150mm and a thickness of 25mm while the distance between each nodule is 40mm. The ratio between the tissue thickness and the nodule diameter has been chosen accordingly to the one used in related studies in the literature \[[@pone.0237379.ref025], [@pone.0237379.ref029], [@pone.0237379.ref047]\]. The mechanical properties of the materials and components of the phantom are described in the Finite Element Simulation subsection.
In the presented study, we limited the nodule depth to 8mm to reduce the average palpation force level and minimize damage to the probe. Indeed, as shown in related works, detecting deeper nodules requires deeper indentation and by consequence, higher forces. Higher forces also lead to faster degradation of the tissue in repeated trials, making it difficult to compare results. Moreover, the lateral sweeps used an array of capacitive tactile sensors, that saturates if an excessive force is applied to locate the nodule. To avoid saturating the tactile sensor, we used a nodule depth that meets all hardware requirements to demonstrate the role of stiffness variation in conditioning the haptic perception during 3D localization of nodules in soft tissues.
These materials have been widely used to simulate human soft tissues mechanical properties. In particular, Ecoflex 0010 has been used in biomedical simulators to practice abdominal palpation \[[@pone.0237379.ref011]\] or needle insertion \[[@pone.0237379.ref048]\]. The size of the nodule represents the size of a tumor of type T1 (\<2cm) for the breast or liver cancers. This is, according to the TNM classification, the earliest stage where the nodules can be detected by palpation \[[@pone.0237379.ref049]\].
Ecoflex 00-10 is a rubber silicone that has a high coefficient of friction with the probe which affects the motion of the probe. For this reason and to protect the phantom, we wrapped the phantom in an additional layer of plastic film. It has to be noticed that, except for the friction coefficient, this layer of plastic film is not taken into account in the simulation.
Sweeping directions {#sec006}
-------------------
In this study, we aim to understand the role of the stiffness on the detection and 3D localization of a hard nodule embedded in soft tissues. Two types of sweeping with different aims are presented in this paper. These two palpation directions show distinct results that have been exploited in the Bayesian algorithm for nodule detection presented in a further section of this paper.
The aim of the two sweeping directions is to reproduce some human participants' palpation strategies that we observed during our previous study \[[@pone.0237379.ref044]\]. We have shown in this study that the palpation behavior of the participants is adapted to localize the nodule or to estimate the depth. From these observations and results, we found interesting to compare two types of sweeping strategies, one local with a light force applied to the phantom using the tip of the probe and one more global using the whole palmar region of the probe with the tactile sensor.
The first sweeping direction is the lateral sweep (along **y**). The aim of this sweeping strategy to perform the nodule detection on a wide surface. Therefore, during lateral sweeps, the probe is first significantly indented (along **z**) in the phantom to have contact between the whole palmar surface of the VLM probe and the phantom (see [Fig 2](#pone.0237379.g002){ref-type="fig"}).
{#pone.0237379.g002}
The second sweeping strategy, denoted longitudinal sweep, aims to improve the depth estimation of a hard nodule by performing a localized exploration. Indeed, during this longitudinal sweeps, the probe only slightly touches the surface of the phantom with the edge of its tip and is swept along the **x** direction.
The trajectories of the VLM probe during the lateral sweep and longitudinal analysis are shown in [Fig 2](#pone.0237379.g002){ref-type="fig"}.
During the study of the lateral sweeps, the probe is initially positioned to align the nodule with the end of the probe's tip and to have the nodule roughly centered on the trajectory. The probe is first indented at 25mm, then it is swept along **y** axis by 120mm at 30mm/s. The probe is then outdented and moved back at 100mm/s to the original position. This cycle is repeated 5 times, and after the fifth time, the VLM probe is shifted by 5mm along **x** axis to a new initial position. As the lateral sweeps are performed to localize nodule on a wide area, the aim of this shift is to observe the behavior of the probe when the latter is sweeping over a nodule at different distances. The next cycle is also repeated 5 times before applying a new shift. In total, 4 shifts are applied, the distance between the initial and last trajectories is then 20*mm*.
During the study of the longitudinal sweeps, the probe is first indented by 4mm. With a 4mm indentation, only the tip's edge of the probe is touching the phantom. The probe is then swept along the **x** axis by 120mm. The speed of the sweep is also set at 30mm/s. The probe has been initially positioned to sweep over the center of the nodules placed in the silicone. Finally, the probe is lifted up to avoid contact between the phantom and the probe and moved back to the initial position. The longitudinal sweeping is repeated 25 times.
The two sweeping directions have been tested for 15 different stiffnesses covering the range of stiffness that the probe can achieved with a carbon rod of 1.5mm.: 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.73, 0.74, 0.76, 0.77, 0.8, 0.83, 0.87, and 0.94Nm/rad and for 4 different nodule depths (2, 4, 6, and 8mm) and without nodule for a total of 1875 trials per sweeping direction. The supplemental [S1](#pone.0237379.s003){ref-type="supplementary-material"} and [S2](#pone.0237379.s004){ref-type="supplementary-material"} Videos show respectively some lateral sweeps and some longitudinal sweeps.
One can notice that the steps of the stiffness tested in this paper is not linear. This comes from the fact that for simplicity, we have chosen linear steps of 2mm in the active length of the carbon rod. This choice allows us to take advantage of the probe characterization performed in our previous study \[[@pone.0237379.ref022]\] and makes the stiffness control easier by relying on the closed-loop position control of the linear actuator. It also simplifies the implementation of carbon rod displacement in the FE simulation. However, since the relation between the stiffness and the active length of the carbon rod is nonlinear, it results in nonlinear steps of stiffness.
Finite Element simulation {#sec007}
-------------------------
The aim of our Finite Element (FE) model is to provide a further study on the impact of the joint stiffness variation during palpation exploration. The experimental results show that the variation of stiffness is more significant for the longitudinal sweeps than for the lateral sweep. As a consequence, we focused our FE simulation on longitudinal sweeps.
Using *COMSOL multiphysics*, a 2D Finite Element (FE) model has been developed to simulate the VLM probe and phantom behavior during the longitudinal sweeps. To reduce the complexity of this simulation performed the geometry of the probe has been simplified. [Fig 3](#pone.0237379.g003){ref-type="fig"} presents the mesh and the different material domains of the FE simulation.
{#pone.0237379.g003}
All the materials except the phantom silicone have been modeled as linear elastic materials. The *Ecoflex 0010* material, as several rubber silicones, follows a nonlinear behavior when it is significantly deformed. This is the reason why the material of the phantom has been modeled with a hyperelastic model in addition to a viscoelastic model. The chosen hyperelastic model is the Ogden model with the parameters obtained by Spark *et al*. \[[@pone.0237379.ref050]\]. The viscoelastic model is based on a Standard Linear Solid (SLS) model. The parameters have been obtained from experimental characterization during a previous study \[[@pone.0237379.ref011]\].
The bottom part of the phantom is constrained in position whereas a prescribed displacement is applied to the probe. To save some computing time, the sweeping distance has been reduced to 60mm (120mm in the experiments). The simulation has been performed for 15 active lengths of carbon rod (from 24mm to 52mm in steps of 2mm) which are equivalent to the 15 levels of stiffness studied in the experiment.
The contact between the probe and the phantom is modeled with two surface contact pairs covering the palmar region of the probe and the upper layer of the phantom, respectively. The pressure contact calculation is based on an Augmented Lagrangian Method, and the friction between the 2 contacts is modeled as Coulomb friction (*μ* in [Table 1](#pone.0237379.t001){ref-type="table"}). Finally, no rolling resistance is modeled for the contact between the probe and the phantom assuming pure sliding at the elements level.
10.1371/journal.pone.0237379.t001
###### Simulation parameters.
{#pone.0237379.t001g}
Domain Parameter Description Value Unit
------------------ ----------------------------------------------------------------------- ----------------------------- -------------- ------
General *v*~*x*~ Sweeping velocity 30 mm/s
*t*~*indent*~ Time to reach the maximum indentation 0.6 s
Δ~*t*~ Time step of the simulation 10^−2^ s
*z*~*max*~ Indentation 4 mm
*μ* Static Coulomb friction coefficient between the phantom and the probe 0.1 SI
3D printed parts *E*~1~ Young modulus 2.7 × 10^9^ Pa
*ρ*~1~ Density 1250 kg/m^3^
*ν*~1~ Poisson's ratio 0.36 SI
Nodule *E*~2~ Young modulus 3.2 × 10^9^ Pa
*ρ*~2~ Density 1180 kg/m^3^
*ν*~2~ Poisson's ratio 0.37 SI
PTFE cylinder *E*~3~ Young modulus 4 × 10^8^ Pa
*ρ*~3~ Density 2200 kg/m^3^
*ν*~3~ Poisson's ratio 0.48 SI
Carbon rod *E*~4~ Young modulus 10.2 × 10^9^ Pa
*ρ*~4~ Density 1000 kg/m^3^
*ν*~4~ Poisson's ratio 0.49 SI
Ecoflex 0010 *G*~5~ Shear modulus (Ogden model) 12605 Pa
*α*~5~ Strain Hardening Exponent (Ogden model) 4.32 SI
*τ*~5~ Relaxation time (SLS viscoelastic model) 2.30 s
*β*~5~ Energy factor (SLS viscoelastic model) 0.6 SI
The mesh of the FE model is composed of triangular elements (3933 elements for simulations with nodules and 3566 for the simulation without nodule). To increase the accuracy, a mesh refinement has been done around the nodule and around the probe joints. About 27 hours of computations on a 2.6GHz *Intel Xeon* 16 cores machine with 128GB of RAM were needed to simulate all the configurations proposed in the paper.
The main simulation parameters are given in [Table 1](#pone.0237379.t001){ref-type="table"}.
Results {#sec008}
=======
In this section, the results obtained during the experiments and from the FE simulation are presented. These results illustrate the differences between the two sweeping directions as well as the role of the VLM probe's stiffness variation on haptic perception. In the last subsection, we also describe how the likelihood functions of the force knowing the depth are generated. Thus, these likelihood functions can be seen as a knowledge obtain from past palpation or from the simulation and are used in the algorithm proposed in this paper.
Lateral sweep {#sec009}
-------------
The aim of this experiment is to analyze the suitability of the VLM probe for detecting a nodule concealed in soft tissues during a lateral sweep. The objective is also to understand if the stiffness is playing a role in nodule detection. Finally, it is explained how the bi-modal sensing (kinesthetic and tactile) helps in the localization of the nodule.
[Fig 4](#pone.0237379.g004){ref-type="fig"} shows an example of the signals acquired during lateral sweep experiments on a phantom without any nodule and with 16mm diameter nodules placed respectively at 2mm, 4mm, 6mm and 8mm from the surface. The signals shown on this figure are the force measured by the force/torque sensor along the **z** axis filtered with a Savitzky-Golay filter, the indentation and the *y* position of the probe. In this figure, four main periods can be distinguished:
1. **P1**: From *t* = 0s to *t* ≈ 0.6s. During this period, the VLM probe moves along the **z** axis but the probe is not in contact with the phantom, the indentation is then considered negative.
2. **P2**: From *t* ≈ 0.6s to *t* ≈ 5.2s. This is the indentation period. The probe continues moving along the **z** axis to reach an indentation of 25 mm. One can notice that the speed of the indentation reduces after 20mm of indentation, this phenomenon is due to the reaction force of the phantom and the frictions of the linear actuator and mechanism which counteract the proportional component of the regulator. It is then the integral action of the integrated controller which helps to converge to the desired position, this integral gain is set at a high value to ensure the convergence to the desired position.
3. **P3**: From *t* ≈ 5.2s and *t* ≈ 9.2s. This is the sweeping period. The probe moves along the **y** axis by 120mm with a speed of 30mm/s. It is important to take into account that since the acceleration and deceleration period are small, they are neglected in the rest of the paper. It can be noticed that at the beginning of the sweeping period, the force along **z** axis drops suddenly. This force drop can be explained by the action of several phenomena, in particular, the dynamic forces due to the acceleration but also the compliance of the joints and links. Indeed, all the joints except the variable stiffness joint are assumed to be rigid, but due to the play between some parts, the probe rotates slightly around the **y** axis which releases a bit the normal force applied to the phantom.
4. **P4**: The last period is for *t* ≥ 9.2s. The probe is static still indented. One can notice a small relaxation period after the probe stops.
{#pone.0237379.g004}
[Fig 4](#pone.0237379.g004){ref-type="fig"} shows that a force peak on **z** axis is measured when the probe sweeps over a nodule. This peak can easily be detected since it is also the maximum value measured during the period P3. It can be noticed that the force peak value is not necessarily suitable to estimate the depth of the nodule. Thus, in [Fig 4](#pone.0237379.g004){ref-type="fig"}, the force peak value for the nodule 8mm deep is higher than the force peak value for the nodule 4mm deep. This can be explained by the fact that a small error on the indentation or a small variation on the thickness of the phantom implies a big variation of the value. After studying the results of several trials, it seems more reliable to study the prominence of the peak. In this paper, we define the prominence of the peak as the difference between the force peak value and the force baseline value at the corresponding time of the peak. The baseline computation method used in this study is based on the Asymmetric Least Squares (ALS) method which is suitable for detecting a baseline of a signal with peaks \[[@pone.0237379.ref051]\]. The ALS baseline parameters have been tuned for the VLM probe signals but are kept constant for all the experiments presented in this paper.
[Fig 5](#pone.0237379.g005){ref-type="fig"} illustrates the force measured, the baseline computed, and the prominence of the peak for several combinations of stiffnesses and shifts when the probe is sweeping laterally over the nodule concealed at 4mm and the part of the phantom without nodule respectively. It has to be noticed that even if there is no peak due to a nodule, a peak prominence is computed from the sweep over the phantom without nodule. As for the other phantom sample, the prominence is computed from the distance between the baseline and the maximum force value detected. The peak prominences measured for the nodule are clearly higher than the prominences for the lateral sweep where no nodule is concealed.
{#pone.0237379.g005}
By comparing the different rows of [Fig 5](#pone.0237379.g005){ref-type="fig"}, one can see that the maximum force and the prominence increase with the shift. This can be explained by the fact that in the range covered in this paper, the higher the shift is, the closer the nodule is to the VLM probe joint. Then for the same tangent displacement (along **z**), if the point of application is closer to the center of rotation, the equivalent angular displacement is higher. Finally, if the angular displacement is higher, due to the stiffness, the reaction torque and forces applied are greater.
[Fig 6](#pone.0237379.g006){ref-type="fig"} shows the distributions of the force peak prominence per stiffness for each depth of nodule. It has to be noticed that the graph does not separate the shift so each box is obtained from 25 trials. These distributions presented with a boxplot standard confirm that there is a clear difference for the sweep with and without nodule. This figure also shows that the boxes for the different nodule depths are overlapping, this implies that from a single measure, it would be difficult to determine the nodule depth. To further support the interpretation of [Fig 6](#pone.0237379.g006){ref-type="fig"}, we detail, in the supplemental [S1 Appendix](#pone.0237379.s001){ref-type="supplementary-material"}, a comparison of the distributions obtained for each stiffness using statistical analysis.
{#pone.0237379.g006}
However, [Fig 7A](#pone.0237379.g007){ref-type="fig"} shows the contact pressure measured by several taxels of the Cyskin sensor during lateral sweeps over a 2mm deep nodule for different shifts. It can be seen on this figure that depending on the shift, a peak of contact pressure is measured when the probe sweeps over the nodule. [Fig 7B](#pone.0237379.g007){ref-type="fig"} exhibits the pressure contact prominence map measured by the tactile sensor at the force peak instant for different shifts. The latter shows once again that the region where the max contact pressure prominence is measured depends on the shift. By knowing the probe position and geometry, the localization of the nodule on **x** axis can then be estimated by interpolation. The position of the nodule on **y** axis is directly obtained from the position of the probe at the force peak instant.
{#pone.0237379.g007}
Longitudinal sweep {#sec010}
------------------
The previous section shows that the lateral sweep explores a large area and helps to find the location of the nodule but it only gives a rough estimate of the nodule depth. In this section, the simulation and experimental results for the longitudinal sweep that allows exploring the phantom more locally are presented. These results show that the longitudinal sweep is more suitable for estimating the depth of the nodule and highlights the role of the stiffness on haptic perception.
### Simulation results {#sec011}
Due to the nonlinear behavior of the soft material, coming from its hyperelasticity and its viscoelasticity, it is not simple to obtain an analytical model for the phantom. FE simulation is a way to have a better insight into what happens at the tissue level.
The supplementary [S1 Fig](#pone.0237379.s002){ref-type="supplementary-material"} and [S3 Video](#pone.0237379.s005){ref-type="supplementary-material"} shows some simulation frames for different nodule depth and stiffnesses. The stress in the soft tissues increases when the probe sweeps over the nodule. As can be seen, the deeper the nodule is, the smaller the stress is. Moreover, with the small forces applied during the longitudinal sweeps, the stress in the material under the nodule is not impacted as much as the stress in the material above the nodule. This phenomenon comes from the fact that during the palpation sweeps, the displacement of the nodule is small compared to the displacement of tissue above the nodule. This shows that the probe is more significantly affected by the amount of material above the nodule (the nodule depth) than the amount of material under the nodule.
From the FE model, the kinesthetic force measured by the F/T sensor can also be simulated. [Fig 8](#pone.0237379.g008){ref-type="fig"} shows an example of the evolution of the force during the sweeps for the same stiffness. It can be seen that due to the dynamics of the contact and the nonlinear behavior of the tissue, some oscillations appear in the simulated force signals. These oscillations are due to a stick-and-slip behavior coming from the friction between the probe and the phantom. These oscillations are more or less filtered by the mechanical impedance of the probe in which the stiffness plays an essential role. These oscillations can be observed on force signals measured during the experiments (see [Fig 10](#pone.0237379.g010){ref-type="fig"}). Furthermore, one can notice that the amplitude of these oscillations varies with the nodule depth, which means that these oscillations are not only dependent on the probe's internal dynamics but also on the ones from the phantom.
{#pone.0237379.g008}
[Fig 9](#pone.0237379.g009){ref-type="fig"} shows the distributions of the force peak prominence per stiffness for the different nodule depth obtained from the FE simulations. One can notice that this time the distributions for the different nodule depth can be distinguished from one to another. The simulation results are compared to the experimental results in the next subsection.
{#pone.0237379.g009}
### Experimental results {#sec012}
The force data presented in [Fig 10](#pone.0237379.g010){ref-type="fig"} shows once again that a peak can be detected when the probe sweeps over the nodule. This figure also shows that the force peak prominence seems more reliable than simply the peak height. For the longitudinal sweep, the peak detection is also simple since it corresponds to a maximum force measured during each trial.
{#pone.0237379.g010}
[Fig 11](#pone.0237379.g011){ref-type="fig"} presents with boxplot format the distribution of the force peak prominence for the 15 different stiffnesses and each nodule depth. It can be seen that compared to the results obtained for the lateral sweep ([Fig 6](#pone.0237379.g006){ref-type="fig"}) the distributions can be distinguished the majority of the cases. This observation confirms the results obtained from the FE simulations. Similarly to the lateral sweeps, the significance of the probe's stiffness variation on the force peak prominence distribution is further studied, using statistical analysis, in the supplemental [S1 Appendix](#pone.0237379.s001){ref-type="supplementary-material"}.
{#pone.0237379.g011}
The stiffness plays a role in the width of the distributions but also on the distance between the distributions. To compare the distance between the distributions for a given stiffness, the Standardized Euclidean Distance (SED) is used. The SED can be defined as follows: $$\begin{array}{r}
{d_{\text{SED}}\left( x,y \right) = \sqrt{\frac{\left( x - y \right)^{\intercal}\left( x - y \right)}{\sigma_{x}\sigma_{y}}},} \\
\end{array}$$ where *x* and *y* denotes two random vectors describing the distributions to be compared. *σ*~*x*~ and *σ*~*y*~ are the standard deviation of the random vectors *x* and *y* respectively. This metric is suitable to compare two distributions taking into account the standard deviation \[[@pone.0237379.ref052]\]. One can notice that the two vectors *x* and *y* must have the same dimension.
[Fig 12A](#pone.0237379.g012){ref-type="fig"} gives the minimum SED between the force peak prominence distribution without nodule and the force peak prominence distribution with nodule across the different stiffnesses of the probe. The results show that the distance varies depending on the stiffness. The higher this SED is the easiest it is to distinguish if there is a nodule. The figure shows that both sweeping directions give good results to distinguish if there is a nodule or not. In particular, the bests of the tested stiffnesses to detect the presence of a nodule in the stiffness range of the VLM probe are respectively *K*~*θ*~ = 0.68Nm/rad and *K*~*θ*~ = 0.76Nm/rad for the lateral sweep and for the longitudinal sweep. It can be noticed that since the longitudinal sweep is inspecting the phantom more locally, the distance between the force peak prominence distribution with nodule and without nodule is slightly higher, in particular for medium stiffness. Finally, the SED distance obtained from the FE simulation is overestimated. This can be explained by the fact that in the experimental approach, the phantom is not perfectly flat in contrast to the FE simulation where the phantom is virtually perfectly flat. This difference can also be observed by comparing the force peak prominence distributions, where it can be seen that the mean force peak prominence obtained during the simulation is closer to zero than the one obtained experimentally.
{#pone.0237379.g012}
[Fig 12B](#pone.0237379.g012){ref-type="fig"} presents the minimum SED distance between two distributions of the Force peak prominence when a nodule is present. This time, the minimum distance measures the ability to distinguish the depth of a nodule for the different stiffnesses. Thus, a clear difference between the two sweeping directions is observed. Indeed, Lateral sweep minimum is smaller than the longitudinal one. This highlights that a local investigation simplifies the nodule depth distinction. Finally, the SED distance obtained from the FE simulations gives a good insight into the role of the stiffnesses in the nodule depth distinction.
Likelihood functions {#sec013}
--------------------
From the previous experimental and simulation results, we can compute for each stiffness the probability density functions of the force peak prominence knowing the depth of the nodule: $p_{K_{\theta}}^{lat}\left( F \middle| d \right)$ and $p_{K_{\theta}}^{long}\left( F \middle| d \right)$ for the lateral sweep and the longitudinal sweep respectively. Where *F* refers to the force peak prominence, *d* refers to the depth of the nodule and *K*~*θ*~ refers to the stiffness of the probe. These functions are the results of a knowledge obtained by past or simulated explorations of the phantom. They are used in the nodule detection algorithm to update the depth estimation with Bayes inference.
To obtain the likelihood functions, we used a non-parametric Kernel distribution fitting using *Matlab 2018*. [Fig 13A and 13C](#pone.0237379.g013){ref-type="fig"} illustrate the obtained likelihood functions for each stiffness. The role of the stiffness on the sharpness of the distribution, as mentioned earlier, can also be seen in these figures.
{#pone.0237379.g013}
These likelihood functions are computed for the known nodule depth *d* ∈ {2, 4, 6, 8} and without nodule. Using these functions would be sufficient to estimate the depth of the nodule with a 2mm accuracy. However, to increase the resolution of the estimation, the probability density functions have been interpolated by linear interpolation of uncertain data \[[@pone.0237379.ref053]\]. The interpolated functions are shown in [Fig 13B and 13D](#pone.0237379.g013){ref-type="fig"} for the experimental results and on [Fig 13E](#pone.0237379.g013){ref-type="fig"} for the one generated from the simulation results.
Nodule detection algorithm {#sec014}
==========================
In this section, we propose the algorithm 1 for nodule detection that uses the property of the stiffness to condition the likelihood of the force peak prominence. This algorithm, based on Bayes inference and information gain theory, controls the VLM probe and its stiffness to autonomously palpate soft tissues and determine if there is or not a nodule. Moreover, the algorithm returns the position of the nodule in the (**x**,**y**) plane and the estimated depth. All the notations used in this algorithm and section are described in [Table 2](#pone.0237379.t002){ref-type="table"}.
10.1371/journal.pone.0237379.t002
###### Algorithm parameters.
{#pone.0237379.t002g}
Notations Description
----------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
$p_{K_{\theta}}^{lat}\left( F \middle| d \right)$ Probability density function of the force peak knowing the depth of the nodule *d* for a lateral sweep at stiffness *K*~*θ*~
$p_{K_{\theta}}^{long}\left( F \middle| d \right)$ Probability density function of the force peak knowing the depth of the nodule *d* for a lateral sweep at stiffness *K*~*θ*~
*P*(*N*) Probability of the presence of a nodule in the explored area.
*P*(*d*) Probability mass function of the depth of the nodule in the explored region.
*P*(*N*\|*F*) Posterior estimation of the probability of the presence of the nodule knowing the force peak prominence of the last sweep
*P*(*d*\|*F*) Posterior estimation of the probability mass function of the depth of the nodule knowing the force peak prominence of the last sweep
*dir* ∈ {*lat*, *long*} Direction of sweeping; lateral or longitudinal
$p_{K_{\theta}}^{dir}\left( F_{K_{\theta}} \right)$ Estimated probability density function of the force for the next sweep with the stiffness *K*~*θ*~.
$P_{N}^{th}$ Threshold on the probability of the presence of a nodule
$P_{d}^{th}$ Threshold on the probability of depth of a nodule
*x*~*n*~ Position of the nodule on the **x** axis.
*y*~*n*~ Position of the nodule on the **y** axis.
*IG* Information gain of the sweep, computed from the Kullback-Leibler divergence
*IG*^*th*^ Threshold on the information gain.
*K*~*θ*~ Stiffness of the probe for the sweep
$K_{\theta}^{detect}$ Best stiffness of the probe for distinguishing if there is a nodule
$\mathcal{K}$ Set of stiffness values *K*~*θ*~ that the probe can take. For the VLM probe $\mathcal{K} = \left\{ 0.65,0.66,0.67,0.68,0.69,0.7,0.71,0.73,0.74,0.76,0.77,0.8,0.83,0.87,and0.94 \right\}$
$\mathcal{D}$ Set of depth considered for the depth of the nodule. In this study $\mathcal{D} = \left\lbrack 2:0.2:8 \right\rbrack$
From the results described in the previous section, we obtained the probability density functions of the force knowing the depth for both sweeping directions and for each stiffness denoted $p_{K_{\theta}}^{lat}\left( F \middle| d \right)$ and $p_{K_{\theta}}^{long}\left( F \middle| d \right)$ respectively.
Description of the algorithm {#sec015}
----------------------------
**Algorithm 1**: **Proposed algorithm for nodule detection**
**Data**: Likelihood functions $P_{K_{\theta}}^{lat}\left( F \middle| d \right)$ and $P_{K_{\theta}}^{long}\left( F \middle| d \right)$
**Result**: Probability of the presence of a nodule *P*(*N*), Probability mass function of the depth of the nodule *P*(*d*), and the coordinates of the position of the nodule (*x*~*n*~,*y*~*n*~)
1 Initialization
2 **do**
3 **if** $P\left( N \right) < P_{N}^{th}$ **then**
4 the probe sweeps laterally over the region to explore with the stiffness $K_{\theta} = K_{\theta}^{detect}$
5 **else**
6 **foreach** $K_{\theta} \in \mathcal{K}$ **do**
7 Compute $p_{K_{\theta}}\left( F_{K_{\theta}} \right)$
8 **end**
9 the probe sweeps longitudinally over the region of the nodule with the stiffness $K_{\theta} = \underset{K_{\theta} \in \mathcal{K}}{\text{arg}\mspace{360mu}\text{min}}\left( \operatorname{Var}\left( F_{K_{\theta}} \right) \right)$
10 **end**
11 Filter force and tactile data
12 Find maximum force during the sweep;
13 Compute prominence;
14 **if** $P\left( N \right) < P_{N}^{th}$ **then**
15 Compute posterior estimations *P*(*d*\|*F*) and *P*(*N*\|*F*) with $p_{K\theta}^{lat}\left( F \middle| d \right)$
16 Compute information gain IG = *D*~*KL*~(*P*(*N*\|*F*) ∥ *P*(*N*))
17 **else**
18 Compute posterior estimations *P*(*d*\|*F*) and *P*(*N*\|*F*) with $p_{K\theta}^{long}\left( F \middle| d \right)$. Compute information gain IG = *D*~*KL*~(*P*(*d*\|*F*) ∥ *P*(*d*))
19 **end**
20 **if** $P\left( N \middle| F \right) < P_{N}^{th}$ **then**
21 Update nodule position (*x*~*n*~,*y*~*n*~) from probe position and tactile measurement at maximum force
22 **end**
23 Update *P*(*N*) and *P*(*d*) with the posteriori estimations
24 **while** $P\left( N \right) > 1 - P_{N}^{th}\&\underset{d \in \mathcal{D}}{\text{max}}\left( P\left( d \right) \right) < P_{d}^{th}\& IG > IG^{th}$;
25 return *P*(*N*), *P*(*d*), *x*~*n*~ and *y*~*n*~
Line 1 of the algorithm 1 refers to the initialization. During this step, the probe is manually positioned over the region of soft tissue that we want to explore. The probability of the presence of the nodule *P*(*N*) is set at 0.5 (same probability between the presence of a nodule and the non-presence of a nodule). The probability mass function of the nodule depth is set as flat distribution.
The condition in line 3 tests the likelihood of the presence of the nodule. If the probability *P*(*N*) is high enough, it means that a nodule has been detected during the precedent sweeps and that its position has been estimated. A longitudinal sweep is then performed over the region of the nodule to give a better estimation of the depth. On the other hand, if *P*(*N*) is still lower than the threshold, a lateral sweep is performed over the region to explore.
The stiffness chosen for the sweep is dependent on the direction and the depth estimation *P*(*d*). For lateral sweeps, the stiffness chosen is $K_{\theta}^{detect} = 0.68$ Nm/rad which is the stiffness that maximizes the SED between the distributions with nodule and the distribution without nodule during lateral sweep as shown on [Fig 12A](#pone.0237379.g012){ref-type="fig"}. For longitudinal sweeps the stiffness chosen is the one which minimizes the variance of the estimated peak force prominences $F_{K_{\theta}}$ with the probability distributions computed for all *K*~*θ*~ in $\mathcal{K}$ as follows: $$\begin{array}{r}
{p_{K_{\theta}}^{dir}\left( F_{K_{\theta}} \right) = \sum\limits_{d \in \mathcal{D}}P\left( d \right)p_{K_{\theta}}^{dir}\left( F \middle| d \right)} \\
\end{array}$$
The filters used at the line 11 are the same Savitzky-Golay filters used for post-processing the data in the results section. Similarly, the prominence is computed by subtracting the ALS baseline to the maximum force measured during the sweep.
Depending on the direction of the sweep the posterior estimation of the presence of a nodule and the probability mass function of the depth are computed with the Bayes inferences as follows: $$\begin{array}{r}
\begin{aligned}
{P\left( N \middle| F \right)} & {= \frac{P\left( N \right)p_{K_{\theta}}^{dir}\left( F \middle| N \right)}{P\left( N \right)p_{K_{\theta}}^{dir}\left( F \middle| N \right) + \left( 1 - P\left( N \right) \right)p_{K_{\theta}}^{dir}\left( F_{K_{\theta}} \right)}} \\
{P\left( d \middle| F \right)} & {= \frac{P\left( d \right)p_{K_{\theta}}^{dir}\left( F \middle| d \right)}{p_{K_{\theta}}^{dir}\left( F_{K_{\theta}} \right)}} \\
\end{aligned} \\
\end{array}$$ where the probability density functions are evaluated for the force peak prominence measured during the sweep and *dir* ∈ {*long*, *lat*} refers to the direction.
It can be noticed that $p_{K_{\theta}}^{dir}\left( F_{K_{\theta}} \right)$ appears in the denominator of the Bayes inference in ([3](#pone.0237379.e032){ref-type="disp-formula"}). Minimizing the variance of the expected force peak prominence at the line 9 increases the probability of having a sharp posterior estimation of the depth.
The information gain denoted *IG* is computed depending on the direction of the sweep using the Kullback-Leibler (KL) divergence which is a metric to evaluate how much information is gained when the probabilities *P*(*N*) and *P*(*d*) are updated from the prior to the posteriors. The KL divergence is often used in machine learning or with Bayes inference to evaluate the gain of information obtained with the update of the probability distributions. The KL divergences used in line 16 and 18 are given by the following equation: $$\begin{array}{r}
\begin{aligned}
{D_{KL}\left( P\left( N \middle| F \right) \right\| P\left( N \right))} & {= P\left( N \middle| F \right)\text{log}\mspace{720mu}\left( \frac{P\left( N \middle| F \right)}{P\left( N \right)} \right)} \\
{+ \left( 1 \right.} & {- {\left. P\left( N \middle| F \right) \right)\text{log}\mspace{720mu}}\left( \frac{1 - P\left( N \middle| F \right)}{1 - P\left( N \right)} \right)} \\
{D_{KL}\left( P\left( d \middle| F \right) \right\| P\left( d \right))} & {= \sum\limits_{d \in \mathcal{D}}P\left( d \middle| F \right)\text{log}\mspace{720mu}\left( \frac{P\left( d \middle| F \right)}{P\left( d \right)} \right)} \\
\end{aligned} \\
\end{array}$$
If the posterior estimation of the presence of the nodule is higher than the threshold, the position of the nodule in the (**x**, **y**) plane is updated. If the sweep is a lateral sweep, the position *y*~*n*~ is updated with the position where the maximum kinesthetic force has been detected, whereas *x*~*n*~ is estimated from the tactile sensor measures. Thus, the position is estimated from the normalized tactile prominence map at the time of the peak (as shown in [Fig 7B](#pone.0237379.g007){ref-type="fig"}). If the sweep is a longitudinal sweep, only the position *x*~*n*~ is updated from the position where the maximum force has been detected.
Line 24 gives the conditions which determine if the algorithm should continue sweeping. In other words, another sweep is needed if: 1) it is likely to have a nodule 2) the maximum probability of the depth *P*(*d*) is too low 3) the information gain of the last sweep is high enough. The respective thresholds $P_{N}^{th}$, $P_{d}^{th}$, and *IG*^*th*^ can be tuned to change the performances of the algorithm. Indeed, augmenting $P_{N}^{th}$, for instance, increases the confidence on the nodule detection but is likely to increase the number of lateral sweeps needed. $P_{d}^{th}$ and *IG*^*th*^ allows tuning the confidence on the depth estimation of the nodule and the minimal information gain respectively.
Several values of thresholds have been tried experimentally. $P_{N}^{th} = 0.8$, $P_{d}^{th} = 0.7$, and *IG*^*th*^ = 0.02 have been found to provide a good trade-off between the confidence level and number of sweeps. These parameters' values are the ones that have been used for the evaluation of the algorithm.
Evaluation of the algorithm {#sec016}
---------------------------
The evaluation of the proposed algorithm was carried out using the VLM probe. The evaluation was first performed using the likelihood function obtained experimentally, and then with the likelihood function generated from the FE simulations. It has to be noticed that for the 2mm and 4mm depth, the tests were done using the nodules which have not been used for computing the likelihood functions. Finally, the proposed palpation strategy aims to localize the nodule independently from the phantom orientation, so the algorithm has been tested for several orientations of the phantom.
[Fig 14A](#pone.0237379.g014){ref-type="fig"} shows representative results from the several trials performed with the algorithm. This figure shows the evolution of the probability mass function of the depth *P*(*d*) after each sweep. On average on all trials, the final expected value of the depth is in a range of 0.5mm around the real depth. The median of the number of sweeps is 5, with a minimum of 3 and a maximum of 10. The supplemental [S4 Video](#pone.0237379.s006){ref-type="supplementary-material"} also shows the behavior of the algorithm for a trial over a 4mm deep nodule.
{#pone.0237379.g014}
The two thresholds $P_{d}^{th}$ and *IG*^*th*^ can seem redundant at first but they are complementary to avoid too many sweeps. For instance, the majority of the trials are stopped by the threshold $P_{d}^{th}$, but for the 6mm deep nodule in [Fig 14A](#pone.0237379.g014){ref-type="fig"}, the evolution of *P*(*d*) over the sweeps is slow, and may not reach the threshold $P_{d}^{th}$. In this case, *IG*^*th*^ allows the algorithm to stop and give a result with lower confidence.
With the proposed threshold values, the algorithm was 100% accurate on the estimation of the presence of a nodule with a single lateral sweep. When a nodule was present, *P*(*N*) was over 0.98 after the first sweep, independently from the initial distance between the probe and the nodule. When there was no nodule, P(N) was in a range between 0 and 0.18 after the first lateral sweep. A trade-off needs to be found while tuning $P_{N}^{th}$ in order to maximize the detection rate but not increasing the number of false-positive detection, which would lead to unnecessary additional lateral explorations.
[Fig 14B](#pone.0237379.g014){ref-type="fig"} shows the evolution of the KL divergence of the nodule depth estimation and the variation of stiffness for the different sweeps for the same trials as shown in [Fig 14A](#pone.0237379.g014){ref-type="fig"}. It can be seen that with the set of thresholds, the KL divergence does not need to converge to stop the exploration. Concerning the stiffness, all the trials start with the same stiffness $K_{\theta}^{detect}$, while the first longitudinal sweep is usually performed with the same stiffness *K*~*θ*~ = 0.87Nm/rad. It is only after the second sweep when *P*(*d*) changes significantly that the stiffness selection for the sweeps differs from one nodule depth to another.
On the other hand, [Fig 15](#pone.0237379.g015){ref-type="fig"} shows the results of the algorithm for 3D localization of nodules using the likelihood functions obtained thanks to the FEM simulation instead of the one obtained experimentally. In particular, [Fig 15A](#pone.0237379.g015){ref-type="fig"} presents examples of nodule depth estimation for the same four nodules tested previously. One can see that the algorithm is able to detect the depth of the nodule but generally need more sweeps. Thus, the results obtained with the FEM based likelihood functions are particularly good for the 2 and 8mm deep nodules, but for the 4 and 6mm deep nodules the expected accuracy (mean of *P*(*d*)) and the confidence level (sharpness of *P*(*d*)) are lower than for the sweeps performed with the likelihood obtained experimentally. This is because the force peak prominence measured during the sweeps is further from the simulated expected value, so the information gained per sweep is generally smaller. As shown in [Fig 15C](#pone.0237379.g015){ref-type="fig"}, the threshold on the information gain is reached for the 4 and 6mm deep nodules. Moreover, this figure shows, that the probe can gain information within the first two sweeps with the exception that the 4mm deep nodule requires four sweeps to achieve significant information gain. It can be noticed that this important information gain corresponds to a significant change on the posterior distributions in [Fig 15A](#pone.0237379.g015){ref-type="fig"} from a relatively flat distribution to a sharper one and also to a variation of the probe stiffness. This shows once again the importance of stiffness variation for haptic 3D localization of nodules in soft tissues.
{#pone.0237379.g015}
The final depth estimate *d*~*est*~ can be computed as follows: $$\begin{array}{r}
{d_{est} = \sum\limits_{d \in \mathcal{D}}P\left( d \right)d} \\
\end{array}$$ The Root Mean Square Error (RMSE) between the estimated depths and the actual nodule depths for all the detections presented in Figs [14](#pone.0237379.g014){ref-type="fig"} and [15A](#pone.0237379.g015){ref-type="fig"} is 0.27mm. The highest absolute error is 0.53mm for the 6mm deep nodule with the likelihood function obtained from the FE simulation.
In addition, to complement the investigation on the effect of the probe's stiffness variation in the Bayesian nodule depth estimation, 2 trials have been run without the stiffness modulation strategy. During these trials performed for 4 and 6mm nodule depths, the stiffness is therefore maintained constant at the stiffness *K*~*θ*~ = 0.68Nm/rad (the same as the one used for lateral sweeps). The results for these trials are shown in [Fig 15B](#pone.0237379.g015){ref-type="fig"}. One can see that the accuracy stays similar to the case where the stiffness is updated from previous knowledge but the confidence level is significantly lower. It can also be observed in [Fig 15C](#pone.0237379.g015){ref-type="fig"} that the KL divergence quickly converges to 0, which means that the repetition of the sweeps with the same fixed stiffness did not bring much information on the nodule depth. These results confirm that the proposed strategy using stiffness variation helps in conditioning the force peak prominence likelihood and improves the nodule depth estimation.
Finally, since for all the trials presented in [Fig 15](#pone.0237379.g015){ref-type="fig"}, the likelihood functions for lateral sweep used are still the ones obtained from the experiments, there is no significant change in the detection of the nodule's presence.
Discussion {#sec017}
==========
In this study, we highlighted the importance of tuning the stiffness of a compliant palpation probe to shape the force peak prominence likelihood during a palpation task, and we proposed a controller that utilizes this principle to estimate the 3D localization of a nodule. This study highlights the role of compliance of a soft robot not only as a design parameter for safety but also as a control parameter for improved haptic perception \[[@pone.0237379.ref054]\].
There is still some remaining work to be done to implement this probing method on real scenarios like automated examination of biopsy samples or even remote patients. The main difficulty is that the likelihood functions obtained in this paper are valid only for the same thickness of soft tissue, the same size of the nodule, and the same sweeping speed since all these factors are impacting the force peak prominence dispersion. To solve this issue, new models able to predict the kinesthetic force uncertainties during a compliant palpation exploration would be needed. We have shown in this paper that FE simulation is one solution to predict the behavior and to generate likelihood functions. Indeed they can be used to generate the likelihood functions when no palpation has been performed on this type of tissue. However, they still require the knowledge of some tissue meta parameters such as the soft tissue elasticity. Also, increasing the accuracy of the tissue model or simulating the lateral sweeps would require developing a 3D FE model and repeating the simulation for several shifts (position along **x**). These modifications would increase the computational cost of the simulation significantly.
Another approach to generate the likelihood functions for different conditions is using mixture models \[[@pone.0237379.ref055], [@pone.0237379.ref056]\]. A mixture model linearly combines a set of nonlinear kernels to fit a given distribution of observations. In this particular scenario, likelihood functions for new materials can be constructed by learning a set of parameters of the mixture model already identified for a known tissue.
In this paper, the flat phantom has been designed to simplify the study and minimize assumptions. In real scenarios, such as breast or abdominal palpations, the surface of the tissue to be palpated is not likely to be flat and the breathing of the patient would add some disturbances. However, these issues have been widely addressed in the literature. Some control strategies can compensate in real time the motion due to the patient breathing \[[@pone.0237379.ref057]\] or to follow complex and moving surfaces \[[@pone.0237379.ref058]--[@pone.0237379.ref060]\]. In the future, we will implement these solutions and the Bayesian controller to test the probe on a soft robotic patient phantom with controllable and sensing organs.
More generally, this paper concludes that the probe stiffness matters in conditioning the shape of the likelihood function (sensor model) in a Bayesian framework to estimate a given feature in the tissue (in this case the nodule depth after having identified the location using lateral sweeps). However, when the tissue is inhomogeneous, multiple force prominence values will be present in the force profile. Then a suitable technique should be adopted to filter a target force prominence shape. One realtime solution is to convolve a target force shape on the measured force data. A data-driven approach can be used to build the target shape from a variety of tissue samples with a given feature in them.
In the proposed algorithm, the tactile sensor is currently used to help to find the location of the nodule. Removing the sensor would be possible, but it would require to increase the longitudinal sweeping distance by two times the length of the probe's tip link. This would then increase the exploration time and also the tissue region area that is probed. These two factors are not really suitable in the case of patient palpation.
Finally, one may notice that the stiffness variations (for the same nodule depth) during the trials with likelihoods functions computed from experimental do not necessary follow the same gradient as the variations during the trials with likelihoods function computed from the FE simulations. More generally, even across trials repeated for the same scenario, we observed different stiffness variations. This comes from the fact that the stiffness is adapted according to the current knowledge during the palpation exploration. The proposed method then presents a strategy that tunes the stiffness in order to increase the probability of getting new information based on prior knowledge (likelihood functions). Due to the stochastic behavior of the force peak prominence, even for the same stiffness and same nodule, the information obtained during a sweep is different from one sweep to another. As a consequence, for two trials on the same nodule, if the information collected so far is different, the stiffness selected to maximize the information gain of the next sweep will be different. Finally, this can also be supported by another study \[[@pone.0237379.ref014]\] where the variation of the human's joints stiffness during palpation tasks has been studied and clearly showed that this joint stiffness follows a random walk. In other words, during palpation exploration tasks, even humans do not follow a predictable gradient of joint's stiffness variation.
Conclusion {#sec018}
==========
In this paper, we have shown that the embodied stiffness of the robotic probe is conditioning the force peak prominence likelihood. In particular, we have shown that by controlling the stiffness of the probe it is possible to sharpen the probability distribution of the force peak prominence. This change of shape of the likelihood is seen as a haptic information gain and can be observed for both sweeping directions. For the lateral sweep, the impact of the joint's stiffness variation on the force peak prominence distribution is not as significant as for the longitudinal sweeps. This results in the fact that the haptic information gain is not sufficient to distinguish the depth of a nodule. However, the stiffness can be chosen to facilitate the detection of a nodule. In contrast, for the longitudinal sweeps, the haptic information gain from one depth to another is significant and helps to determine which stiffness is suitable for the depth estimation. To illustrate the importance of conditioning the likelihood of the force peak prominence, we proposed an algorithm that autonomously explores soft tissues using tactile for the localization of the nodule and kinesthetic sensing to estimate the nodule location and depth. This Bayesian algorithm selects the suitable stiffness by minimizing the variance of the expected force prominence based on prior knowledge that can be constructed from past palpations or generated from FE simulations. The algorithm has been tested on a soft tissue phantom with the VLM probe and shows a 100% reliability on the nodules detection and sub-millimeter accuracy in the 3D nodule localization.
Supporting information {#sec019}
======================
###### Statistical analysis.
This appendix provides the details of the statistical analysis performed to compare the distribution of the force peak prominence for different stiffnesses.
(PDF)
######
Click here for additional data file.
###### FE simulation results.
This supporting figure shows some results obtained during the 2D FE simulation used to model the VLM probe performing a longitudinal sweep on phantoms with a 2mm, with an 8mm nodules, and without nodule respectively. This figure shows the variation in the vertical force and the stress of the phantom (denoted *F*~*z*~ and *σ* respectively) at two different instants of the sweep, depending on the depth of the nodule and the stiffness.
(EPS)
######
Click here for additional data file.
###### Lateral sweeps on a 2mm deep nodule.
This video shows how the lateral sweeps have been performed over a 2mm deep nodule for 3 different shifts and 3 different stiffnesses.
(MP4)
######
Click here for additional data file.
###### Longitudinal sweeps.
This video shows how the longitudinal sweeps have been performed for 3 different stiffnesses. The video illustrates the sweeps over a 2mm deep nodule and a region of the phantom without nodule.
(MP4)
######
Click here for additional data file.
###### Simulation results.
This video shows some results obtained with the FE simulation. 3 different phantom conditions are compared for 2 different stiffnesses.
(MP4)
######
Click here for additional data file.
###### Algorithm test with a 4mm deep.
This video shows how the proposed Bayesian algorithm performs over a 4mm deep nodule.
(MP4)
######
Click here for additional data file.
10.1371/journal.pone.0237379.r001
Decision Letter 0
Ranzani
Tommaso
Academic Editor
© 2020 Tommaso Ranzani
2020
Tommaso Ranzani
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
23 Mar 2020
PONE-D-20-03370
Conditioned haptic perception for 3D localization of nodules in soft tissue palpation with a variable stiffness probe
PLOS ONE
Dear Dr. Herzig,
Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.
The paper is of interest and addresses the important issue of robotic detection and localization of hard nodules in soft tissues. However, all reviewers agreed that the paper need improvents in terms of rigor of experiments and clarity of presentation. In addition, Reviewer 3 raises important points related to the use of a variable stiffness mechanism for this specific application and some discrepancies between conclusions derived from the experiments versus the ones from FEM.
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Reviewer \#1: Line 5: Authors reported \"This trend is opening up new opportunities for robotic applications in the healthcare field.\" So, it is implied that the healthcare field with robotics is all about tactile feedback, or all the applications include tactile feedback, which is not true at all. It is true that with the tactile feedback, the robotic implications in healthcare field can be improved significantly - but authors should change their original sentence.
Line 37: what do the authors mean by \" force and tactile\" modalities? Kinesthetic and haptic? Please elaborate or clear these modalities.
Line 147: The previous sentence cites 4 different studies as the previous studies. Then, the authors start talking about presumably one of them saying \"In the previous study ......\" without specifying which study they are talking about. It should be clarified.
The VLM probe explains the design of the probe, which can be supported also visually. The directions and the definitions should be depicted clearly for the reader.
The pdf version I received had the actual Figure and the figure captions separated. I am not sure if this was a draft problem, or the final manuscript will be like this. If it\'s the later, numbering the sections make no sense, because the reader cannot follow. The authors should find a way to label the system parts on the images directly. Also, using the arrows with color code based on the motion direction might be impossible for the reader to capture, if they are reading from a black/white copy. Such an identification should be handled differently.
Why exactly Figure 1 (a) and (b) have different coordinate systems? I understand in both conditions, the sweeping action takes place in the y direction but what does that mean? What is the advantage of such rotation for the designer?
The motivation of having two conditions in Figure 1(a) and (b) are not clear. So, the direction of sweeping are different, but they are still tangential to the surface. What is the hypothesis or the expected outcome here? To have different \"depth estimation\", changing the direction of tangential sweep might be not enough. Also, given Figures have different orientations of component (6), resulting different contact areas with the surface. Is it intentional? If so, how is it related to the sweep direction? If not, why is it different?
Figure 2 is impossible to be understood - possibly because the coordinate system for both conditions are different. Still, this Figure must me improved!
The probe position seems to be changing between trials in the lateral sweep, but not in the longitudinal sweep. Why?
15 different stiffness values have been chosen for the experiment and these values seem random. It seems like these values are changing incrementally, but not linearly (there are some missing values) but it is curious how they are chosen! Is there a reason why all the values between 0.65 and 0.71 was tried, but 0.72 is ignored? Why is the differences between the last4 values are much bigger than the first 4?
FE model is only used for the longitudinal sweep but not for the lateral. Why?
Line 588 : Authors say \"This study highlights the role of compliance of a soft robot not only as a design parameter for safety, but also as a control parameter for improved haptic perception \" but in the paper, what we see is the comparison between different sweep directions. If these two things are connected, it means authors didn\'t do a very good job explaining how the sweep direction is related to the probe compliance. It would be also nice to mention this relationship in the discussion and/or conclusion section.
Citation numbers cannot be used as a subject of a sentence (\[35\] proposed ......)
Reviewer \#2: This paper presents a control algorithm for variable lever mechanism probe to detect and localize embedded nodules in soft tissues. Using this algorithm, the 3D position of the nodule can be estimated. In general, this paper is well-written. There are some questions and possible improves below.
1\. What is the material used to make the simulated nodules? What types of soft tissues and nodules do you simulate?
2\. How do you detect when the contact between the probe and the phantom starts? How does the accuracy of detection of the moments of the contact between the probe and the phantom affect the stiffness estimation?
Reviewer \#3: This paper addresses the problem of detecting hard nodules in soft tissue via robotic palpation, as well as assessing their depth. The central ideas are one, that the stiffness of the probe should matter; and two, that the proper stiffness can be found via a Bayesian search technique. The paper is clearly written and technically sound; however, I see very little evidence that supports the central ideas.
First, a few more details: the variable stiffness probe is mounted on a force sensor and equipped with a tactile sensor. Although Fig 7 shows that the latter provides some useful information, as far as I can tell, it is not used to support any of the paper\'s main points. Therefore, I see the tactile sensor as a bit of a distraction. I would recommend removing it altogether. In any event, the force sensor appears to give a clear indication of nodule location as well as depth. There is no doubt that the robotic probe succeeds!
My concern, however, relates to the importance of probe stiffness.
Figures 6, 9 and 11 illustrate the dependence of the \"force peak prominence\" on probe stiffness under lateral and longitudinal swiping, in simulation and experiment. With the exception of Fig 11, we see very little dependence on probe stiffness. Even with Fig 11, it would appear to suffice to pick a good stiffness (lower values appear better) and to fix it. The added value of varying the stiffness is by no means apparent.
This brings us to the Bayesian search, in which stiffness was updated trial-over-trial in a Bayesian fashion using likelihood functions at different stiffness values with prominence and nodule depth as variables. Two sets of likelihood functions, one obtained experimentally and one obtained from FEM. In both cases, the Bayesian technique clearly shows the best stiffness varying trial-over-trial. That variation, however, is not good evidence that an optimal stiffness is being obtained. To the contrary, it is notable that the best stiffness versus module depth behaves completely differently in Fig 14 (experimental likelihood) versus Fig 15 (FEM). The former tends toward a softer probe for deeper nodules and the latter tends toward a stiffer probe for deeper nodules. It is deeply concerning that the answers are just the opposite of one another. However, when looking at Fig 13, it appears that the likelihood functions simply don\'t vary much with stiffness. I suspect that the results are just noise.
Minor point: could the oscillations seen in Figs 8 and 10 be, in part, due to probe dynamics? Perhaps stick-slip excites those dynamics.
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10.1371/journal.pone.0237379.r002
Author response to Decision Letter 0
14 May 2020
Response to the Reviewer 1
1\. Line 5: Authors reported "This trend is opening up new opportunities for robotic applications in the
healthcare field." So, it is implied that the healthcare field with robotics is all about tactile feedback,
or all the applications include tactile feedback, which is not true at all. It is true that with the tactile
feedback, the robotic implications in healthcare field can be improved significantly - but authors should
change their original sentence.
The authors would like to thank the reviewer for this comment. The authors did not mean that it is
the only approach that contributes to healthcare robotics. To avoid confusion, the authors rephrased the
sentence as follows:
In section Introduction, paragraph 1 "This trend is one of the promising advances that can bring new
opportunities for robotic applications in the healthcare field."
2\. Line 37: what do the authors mean by " force and tactile" modalities? Kinesthetic and haptic? Please
elaborate or clear these modalities.
The authors effectively were meaning kinesthetic instead of force. This has been replaced in the highlighted
sentence but also in the rest of the paper. In order to clarify this point, the authors have also added
these definitions:
In section Related work, paragraph 2 "The main types of sensors used to do so are the kinesthetic sensors
and tactile sensors. In this paper, we refer to kinesthetic sensors, the sensors that aim to give a signal
related to the force or the torque applied at a probe joint level. On the other hand, the tactile sensing
is referring to fingertip contact sensing using taxel images. Tactile sensing is usually representing the
behavior of the mechanoreceptor at the skin level."
3\. Line 147: The previous sentence cites 4 different studies as the previous studies. Then, the authors start
talking about presumably one of them saying "In the previous study . . . . . . " without specifying which
study they are talking about. It should be clarified.
As suggested by the reviewer, the authors are now specifying the study that they were talking about:
In section Related work, paragraph 9 "In the previous study \[47\], we have shown that the stiffness of the
arm and hand joints is modified during the longitudinal sweeping exploration of soft tissues by varying
the level of co-contraction of antagonistic muscles."
4\. The VLM probe explains the design of the probe, which can be supported also visually. The directions
and the definitions should be depicted clearly for the reader.
If the authors understood well the comment, the reviewer is asking for a figure describing clearly the
design of the probe and the 2 sweeping directions. The authors have modified Figures 1 and 2. The
modified Fig 1 describes the VLM probe design while the modified Fig 2 clearly depicts the 2 sweeping
directions. We also revised the definitions in the Materials and methods section to clarify the description
of the sweeping directions. The modifications regarding the sweeping directions are detailed in the
comment 7. The modifications regarding the design are the following:
In section Variable stiffness palpation: the VLM probe, paragraph 2 "Fig 1 shows the design of the VLM
probe. The VLM probe is based on a revolute variable stiffness joint composed of 2 rigid links (the base
link and the tip link) connected with a revolute joint in parallel with a deformable carbon rod. This
carbon rod acts as a variable spring that allows the stiffness of the joint to be controlled thanks to an
Actuonix L12-30-50-6-I linear actuator. This actuator slides the carbon rod through the base link and
the tip link changing the length of the carbon rod that can be bent (active length). As one can see, the hole
in the base link has been designed such as that the carbon rod can slide axially but is constrained radially
to prevent bending of the rod in the base link. On the other hand, the hole in the tip link is large enough
to allow the carbon rod to bend in. A PTFE cylinder is used to transmit the radial forces between the tip
link to the carbon rod. This PTFE cylinder has been designed to slide easily axially when the actuator
is translating the carbon rod. Adjusting the active length of the carbon rod changes, by cantilever effect,
the amount of force required to bend the rod and by consequence the angular stiffness of the probe."
In section Variable stiffness palpation: the VLM probe, paragraph 3 "In order to describe the movement
of the probe, we need to define a frame. First, we define the axis z as the direction of the normal to the
phantom surface. We then define the x axis as the intersection between the tangent surface of the phantom
and the mid-sagittal plane of the probe. Finally, the y is defined in order to obtain a direct orthonormal
frame (x,y,z). In the rest of the paper, this reference frame will be used to describe the directions of forces
or displacement."
5\. The pdf version I received had the actual Figure and the figure captions separated. I am not sure if this
was a draft problem, or the final manuscript will be like this. If it's the later, numbering the sections
make no sense, because the reader cannot follow. The authors should find a way to label the system parts
on the images directly. Also, using the arrows with color code based on the motion direction might be
impossible for the reader to capture, if they are reading from a black/white copy. Such an identification
should be handled differently.
The figures were separated from the captions as explicitly defined in the PLOS submission guideline, the
final manuscript once edited should integrate the figures in the text. However, in order to simplify the
review process, the figures with section numbers have been edited. Also, the caption referring to colors
have been changed to help the reading from grayscale copies.
6\. Why exactly Figure 1 (a) and (b) have different coordinate systems? I understand in both conditions, the
sweeping action takes place in the y direction but what does that mean? What is the advantage of such
rotation for the designer?
The difference in the coordinate system was a mistake. Figures 1 and 2 have been modified to avoid
confusion with the reference frame. More details about the motivation of having the two sweeping
directions are given in the next answer.
7\. The motivation of having two conditions in Figure 1(a) and (b) are not clear. So, the direction of sweeping
are different, but they are still tangential to the surface. What is the hypothesis or the expected outcome
here? To have different "depth estimation", changing the direction of tangential sweep might be not
enough.
The authors thank the reviewer for this comment. The aim of having two different sweeping strategies
is to reproduce the exploration behavior of human participants observed during the previous study \[44\].
Indeed we observed that different strategies could be applied to localize the nodule and to estimate the
depth. In particular, this study showed that the force applied during the palpation varies according to
the aim of the exploration. Based on these observations and results we found interesting to compare the
two type of sweeping strategies, one local with a light force applied to the phantom and one more global
(with the whole palmar region of the probe's finger). The hypothesis behind the interest of the sweeping
direction is that one direction is more suitable for nodule localization in the tangential plane where the
other one gives better results to estimate the nodule depth. This hypothesis has been verified through the
study. The authors agree with the fact that changing the direction of the tangential sweep is not the only
impacting factor for depth estimation. The presented paper discusses the interest of stiffness variation as
well. To clarify the information regarding the two sweeping directions, the following modification has
been added to the paper:
In section Sweeping directions, paragraph 2 "The aim of the two sweeping directions is to reproduce
some human participants' palpation strategies that we observed during our previous study \[44\]. We
have shown in this study that the palpation behavior of the participants is adapted to localize the nodule
or to estimate the depth. From these observations and results, we found interesting to compare two types
of sweeping strategies, one local with a light force applied to the phantom using the tip of the probe and
one more global using the whole palmar region of the probe with the tactile sensor."
8\. Also, given Figures have different orientations of component (6), resulting different contact areas with
the surface. Is it intentional? If so, how is it related to the sweep direction? If not, why is it different?
The authors thank the reviewer for this comment. On figure 1, the orientation of the phantom and nodule
(previously labelled (6)) was, effectively, different between the subfigure (a) and (b). The proposed
probing strategy aims to detect the 3D location of the nodules independently from the orientation of the
phantom. This is also the reason why the phantom was also examined in different orientations during
the evaluation of the algorithm. In this regard, the two sweeping directions have been tested for different
orientations of the phantom. The modification of Figures 1 and 2 (presented in our answer to comment 4
of the reviewer 1) should suppress the confusion. To clarify that the strategy aims to be independent of
the initial phantom orientation, the following sentence has been added in the paper:
In section Evaluation of the algorithm, paragraph 1 "Finally, the proposed palpation strategy aims to
localize the nodule independently from the phantom orientation, so the algorithm has been tested for
several orientations of the phantom."
9\. Figure 2 is impossible to be understood - possibly because the coordinate system for both conditions are
different. Still, this Figure must me improved!
Fig 2 has been modified to improve clarity. The latter is presented in our answer for your comment 4.
10\. The probe position seems to be changing between trials in the lateral sweep, but not in the longitudinal
sweep. Why?
As explained in previous answers, the two sweeping directions have different objectives. The interest of
the lateral sweep is to detect the position of the nodule in the tangential plane. To localize the nodule in
the (x,y) plane, the probe utilizes both the kinesthetic sensor and the tactile sensor. Then, once a nodule
is localized, the longitudinal sweeping is used to improve the depth estimation thanks to the kinesthetic
feedback. In this regard, it is interesting to change the position for the lateral sweep (nodule position
detection) but not for the longitudinal sweep (nodule depth estimation). The following paragraph has
been rephrased to improve the clarity of the paper.
In section Sweeping directions, paragraph 6 "This cycle is repeated 5 times, and after the fifth time, the
VLM probe is shifted by 5mm along x axis to a new initial position. As the lateral sweeps are performed
to localize nodule on a wide area, the aim of this shift is to observe the behavior of the probe when the
latter is sweeping over a nodule at different distances. The next cycle is also repeated 5 times before
applying a new shift. In total, 4 shifts are applied, the distance between the initial and last trajectories is
then 20mm."
11\. 15 different stiffness values have been chosen for the experiment and these values seem random. It
seems like these values are changing incrementally, but not linearly (there are some missing values) but
it is curious how they are chosen! Is there a reason why all the values between 0.65 and 0.71 was tried,
but 0.72 is ignored? Why is the differences between the last 4 values are much bigger than the first 4?
The nonlinearity in the stiffness is coming from the VLM probe behavior. Indeed in a previous study
\[22\], the VLM probe stiffness has been modeled and characterized for several active lengths of the
carbon rod. Instead of using constant steps in stiffness, the authors have chosen linear steps of active
length of the carbon rod for three reasons: 1) we are using the same active carbon rod length as the one
we characterized in our previous study, 2) it is practically easier to control accurately the position of the
actuator thanks to its sensor (closed-loop control) 3) It is simpler to implement carbon rod displacement
in the FEM simulation. To clarify this information, the following paragraph has been added.
In section Sweeping directions, paragraph 9 "One can notice that the steps of the stiffness tested in this
paper is not linear. This comes from the fact that for simplicity, we have chosen linear steps of 2mm in
the active length of the carbon rod. This choice allows us to take advantage of the probe characterization
performed in our previous study \[22\] and makes the stiffness control easier by relying on the closed-loop
position control of the linear actuator. It also simplifies the implementation of carbon rod displacement
in the FE simulation. However, since the relation between the stiffness and the active length of the carbon
rod is nonlinear, it results in nonlinear steps of stiffness."
12\. FE model is only used for the longitudinal sweep but not for the lateral. Why?
The reasons why the FE model for the lateral sweeps is not proposed in the paper are multiple. First,
the aim of our FE model is to provide a further study on the impact of the joint stiffness variation during
palpation exploration. However, the experimental results show that the stiffness variation for the lateral
sweeps is less significant than for the longitudinal sweeps. As a consequence, there was less interest
to further carry the FE simulation for the lateral sweep. Second, to model the lateral sweeps, the FE
simulation needs to be modified from a 2D model to a 3D model. This implies an exponential increase
in computation cost (27 hours were already required to compute the simulation in 2D). Also, to follow
the experimental protocol, the simulation should be performed for the five different shifts, which also
increase the computational cost of the study. Finally, in the proposed algorithm, the lateral sweep is
generally used only once. According to the author, the complexity is not worth the information that the
lateral sweep FEM simulation would bring to this paper. To clarify, the following modifications have
been added to the paper:
In section Finite Element Simulation, paragraph 1 "The aim of our Finite Element (FE) model is to
provide a further study on the impact of the joint stiffness variation during palpation exploration. The
experimental results show that the variation of stiffness is more significant for the longitudinal sweeps
than for the lateral sweep. As a consequence, we focused our FE simulation on longitudinal sweeps."
In section Discussion, paragraph 2 "Also, simulating the lateral sweeps would require developing a 3D
FE model and repeating the simulation for several shifts (position along x). These modifications would
increase the computational cost of the simulation significantly."
13\. Line 588 : Authors say "This study highlights the role of compliance of a soft robot not only as a design
parameter for safety, but also as a control parameter for improved haptic perception " but in the paper,
what we see is the comparison between different sweep directions. If these two things are connected,
it means authors didn't do a very good job explaining how the sweep direction is related to the probe
compliance. It would be also nice to mention this relationship in the discussion and/or conclusion section.
The paper presents the analysis of the impact of the probe stiffness (or compliance) variation on the haptic
(kinesthetic and tactile) detection of nodules in soft tissues for two sweeping directions. The authors
would like to apologize if the confusion comes from the use of the word compliance. Indeed, compliance
is defined as the inverse of the stiffness. The authors then used from time to time compliance instead of
stiffness to avoid repetition. To avoid confusion, the authors have added the following modification in
the paper:
In section Introduction, paragraph 3 "Since the compliance is defined as the inverse of the stiffness, we
mean by compliant system a physical system with low stiffness. By opposition, a stiff system is a system
with low compliance. We will deliberately use the two words compliance and stiffness inconsistently in
this paper since some ideas are more intuitive when expressed using the stiffness, while some others are
more intuitive with the compliance."
14\. Citation numbers cannot be used as a subject of a sentence (\[35\] proposed . . . . . . )
As suggested, this has been corrected in the revised manuscript.
In section Related work, paragraph 7 "Using point by point strategy, Hoshi et al. \[36\] proposed an
algorithm to optimize the stiffness estimation of the palpated tissues by coupling the force measurement
with a predictive model based on the Finite Element Method"
Response to the Reviewer 2
This paper presents a control algorithm for a variable lever mechanism probe to detect and localize embedded
nodules in soft tissues. Using this algorithm, the 3D position of the nodule can be estimated. In general, this
paper is well-written. There are some questions and possible improves below.
1\. What is the material used to make the simulated nodules? What types of soft tissues and nodules do you
simulate?
The components used to simulate the nodule are acrylic spheres of 16mm diameter. The size of the
nodule represents the size of a tumor type T1 (\< 2cm) for breast or liver cancer. This is, according to
the TNM classification, the earliest stage where the nodules can be detected by palpation \[49\]. With the
platinum-catalyzed silicone (Ecoflex 00-10), the authors aim to simulate general human soft tissues, for
instance, abdominal organs such as liver or human breast. Indeed, this type of silicone is widely used to
mimic the mechanical properties of human tissues during palpation \[22\] or needle insertion \[48\]. This
information has been added in the paper
In section Soft tissue phantom with nodules, paragraph 2 "These materials have been widely used to simulate
human soft tissues mechanical properties. In particular, Ecoflex 0010 has been used in biomedical
simulators to practice abdominal palpation \[11\] or needle insertion \[48\]. The size of the nodule represents
the size of a tumor of type T1 (\<2cm) for the breast or liver cancers. This is, according to the TNM
classification, the earliest stage where the nodules can be detected by palpation \[49\]."
2\. How do you detect when the contact between the probe and the phantom starts? How does the accuracy
of detection of the moments of the contact between the probe and the phantom affect the stiffness
estimation?
The authors thank the reviewer for this question. The contact between the probe and the phantom is
detected every time the probe changes the palpation area thanks to its kinesthetic sensor. As described in
the paper, the method is based on an indentation (without sweeping) and the detection of force variation.
As shown in Fig 4, from the force sensor readings, it is simple to detect when the probe touches the
phantom. Since each actuator has its own position sensors, the position of the probe during the contact
can be found. The accuracy of the contact point detection is important; related studies have shown that a
variation of indentation can impact the stiffness estimation \[47, 25\]. The described detection routine and
the use of the force peak prominence (which takes into account the force measured around the peak) aims
to improve the robustness against an indentation error. More discussion on this point has been added in
the paper.
In section Variable stiffness palpation: the VLM probe, paragraph 6 "To autonomously detect the 0mm
indentation position, the method is based on an indentation (without sweeping) and the detection of
variation in the kinesthetic sensor measurement. This detection strategy aims to improve the robustness
of the nodule detection by improving the accuracy of the indentation measurement. This is particularly
important since related studies have shown that a variation of indentation can impact the nodule depth
estimation\[47, 25\]."
Response to the Reviewer 3
This paper addresses the problem of detecting hard nodules in soft tissue via robotic palpation, as well as
assessing their depth. The central ideas are one, that the stiffness of the probe should matter; and two, that
the proper stiffness can be found via a Bayesian search technique. The paper is clearly written and technically
sound; however, I see very little evidence that supports the central ideas.
1\. First, a few more details: the variable stiffness probe is mounted on a force sensor and equipped with a
tactile sensor. Although Fig 7 shows that the latter provides some useful information, as far as I can tell,
it is not used to support any of the paper's main points. Therefore, I see the tactile sensor as a bit of a
distraction. I would recommend removing it altogether. In any event, the force sensor appears to give a
clear indication of nodule location as well as depth. There is no doubt that the robotic probe succeeds!
The tactile sensor plays an important role in the nodule localization in the (x,y) plane. Indeed as explained
in the algorithm, the tactile sensor is used to locate the xn position of the nodule. The detection of xn after
a lateral sweep is not possible from the force sensor only. Without the x position computed after the
lateral sweep, the longitudinal sweeping distance would have to be increased by two times the length of
the tip of the probe. Increasing this distance would degrade the performances of the proposed method,
in terms of time (longer region to probe several times implies a longer time to find the nodule). In this
regard, the authors decided to keep the tactile sensor in the paper but added more discussion about it.
In section Discussion, paragraph 5 "In the proposed algorithm, the tactile sensor is currently used to
help to find the location of the nodule. Removing the sensor would be possible, but it would require to
increase the longitudinal sweeping distance by two times the length of the probe's tip link. This would
then increase the exploration time and also the tissue region area that is probed. These two factors are
not really suitable in the case of patient palpation."
2\. My concern, however, relates to the importance of probe stiffness. Figures 6, 9 and 11 illustrate the
dependence of the "force peak prominence" on probe stiffness under lateral and longitudinal swiping,
in simulation and experiment. With the exception of Fig 11, we see very little dependence on probe
stiffness. Even with Fig 11, it would appear to suffice to pick a good stiffness (lower values appear
better) and to fix it. The added value of varying the stiffness is by no means apparent.
The authors thank the reviewer for this comment. To provide a generic method that a user could reproduce
for any controllable stiffness probe, we present a detailed statistical analysis of the significance of
joint stiffness variation on the force peak prominence distribution. This statistical analysis supports the
authors' claim that the stiffness plays a significant role in the force peak prominence distribution during
longitudinal sweeps and by consequence on the nodule depth estimation. Also, the case where a low
fix stiffness (Kq = 0:68Nm/rad) is maintained across trials have been tested and added to the Fig 15 to
highlight the limitation of such a strategy in term of estimation confidence.
The presented statistical analysis tests the null hypothesis that the data from 2 different stiffnesses are
coming from the same distribution. Since the distribution for each stiffness is not normally distributed,
we use the Kruskal-Wallis test, which is particularly suitable for non-parametric distributions. The results
are summarized in the supplemental Appendix S1. These results of cross Kruskal-Wallis tests
show that, for the longitudinal sweeps with nodules, the force peak prominence is statistically different
(p-value\< 0:05). In addition, the more different the two stiffnesses are, the higher the significance of
the difference between their force peak prominence distribution is. Furthermore, the results from the
Kruskal-Wallis tests for data from the lateral sweeps show that the difference between the force peak
prominence's distributions is not statistically significant. These results support our claim that the stiffness
variation has a lower significance for the lateral sweeps. Finally, the results of the Kruskal-Wallis
tests for the longitudinal sweeps data when no nodule is embedded exhibits a lower number of statistically
different distributions than the ones from the data with a nodule. This can be interpreted as the fact
that the interaction between the nodule and stiffness impacts the force peak prominence significantly. In
order to make clearer that the stiffness variation is more significant for the longitudinal sweeps than for
the lateral sweeps, the authors have added the following paragraphs:
In section Conclusion, paragraph 1 "For the lateral sweep, the impact of the joint's stiffness variation on
the force peak prominence distribution is not as significant as for the longitudinal sweeps. This results in
the fact that the haptic information gain is not sufficient to distinguish the depth of a nodule. However, the
stiffness can be chosen to facilitate the detection of a nodule. In contrast, for the longitudinal sweeps, the
haptic information gain from one depth to another is significant and helps to determine which stiffness is
suitable for the depth estimation."
In section Lateral sweep, paragraph 10 "To further support the interpretation of Fig 6, we detail, in the
supplemental Appendix S1, a comparison of the distributions obtained for each stiffness using statistical
analysis."
In section Experimental results, paragraph 2 "Similarly to the lateral sweeps, the significance of the
probe's stiffness variation on the force peak prominence distribution is further studied, using statistical
analysis, in the supplemental Appendix S1."
To highlight the interest of the proposed algorithm compared to a palpation strategy with constant stiffness.
Two examples of Bayesian nodule depth estimation with a constant stiffness (chosen low as recommended
by the reviewer) have been added to the figure 15. These 2 examples clearly show that the
information gain (KL divergence) drops quickly, and the final confidence level of the depth estimation
stays low but with good accuracy. The discussion added to compare the scenario with a constant stiffness
with the proposed algorithm follows:
In section Evaluation of the algorithm, paragraph 8 "In addition, to complement the investigation on the
effect of the probe's stiffness variation in the Bayesian nodule depth estimation, 2 trials have been run
without the stiffness modulation strategy. During these trials performed for 4 and 6mm nodule depths,
the stiffness is therefore maintained constant at the stiffness Kq = 0:68Nm/rad (the same as the one used
for lateral sweeps). The results for these trials are shown in Fig 15B. One can see that the accuracy
stays similar to the case where the stiffness is updated from previous knowledge but the confidence level
is significantly lower. It can also be observed in Fig 15C that the KL divergence quickly converges to 0,
which means that the repetition of the sweeps with the same fixed stiffness did not bring much information
on the nodule depth. These results confirm that the proposed strategy using stiffness variation helps in
conditioning the force peak prominence likelihood and improves the nodule depth estimation."
3\. This brings us to the Bayesian search, in which stiffness was updated trial-over-trial in a Bayesian fashion
using likelihood functions at different stiffness values with prominence and nodule depth as variables.
Two sets of likelihood functions, one obtained experimentally and one obtained from FEM. In both cases,
the Bayesian technique clearly shows the best stiffness varying trial-over-trial. That variation, however,
is not good evidence that an optimal stiffness is being obtained. To the contrary, it is notable that the best
stiffness versus module depth behaves completely differently in Fig 14 (experimental likelihood) versus
Fig 15 (FEM). The former tends toward a softer probe for deeper nodules and the latter tends toward a
stiffer probe for deeper nodules. It is deeply concerning that the answers are just the opposite of one
another. However, when looking at Fig 13, it appears that the likelihood functions simply don't vary
much with stiffness. I suspect that the results are just noise.
The authors thank the reviewer for this comment. This paper shows how a set of favorable likelihood
functions can be used for fast convergence of the posterior to a higher information gain. The paper shows
that this likelihood function conditioning can be done just by changing the stiffness.
The authors did not claim that a global optimal stiffness exists. Indeed, the claim of the author is even
the opposite saying that the stiffness needs to be adapted according to the current knowledge during the
palpation exploration. The proposed method then presents a strategy that tunes the stiffness in order
to increase the probability of getting new information based on prior knowledge (likelihood functions).
Comparing the gradient of the selected stiffness between 2 different trials does not really make sense
since it is dependent on the past measurement of a random variable (the force peak prominence). Due
to the stochastic behavior of this variable, even for the same stiffness and same nodule, the information
obtained is different (which is the purpose of the paper). By consequence, it is not surprising that the
stiffness gradient followed during the trials with the likelihood functions obtained from the FEM (Fig 15)
and the likelihood functions obtained from experimental data (Fig 14) are different. This comes from the
fact that the prior knowledge is different and so is the information remained to be obtained. This can also
be supported by another study \[14\] where the variation of the human's joints stiffness during palpation
tasks has been studied and clearly showed that this joint stiffness follows a random walk. This confirms
that during palpation exploration tasks even humans do not follow a particular gradient of joint stiffness
variation.
Even if in Fig 13, the variation of the likelihood function for different stiffness is not visually obvious, the
added statistical analysis clearly shows that the variation of the force peak prominence distribution due
to a stiffness variation is statistically significant for the longitudinal sweeps. The discussions obtained
by addressing the reviewer's comments have been used to strengthen the discussions in the paper. The
modifications done are the following:
In section Discussion, paragraph 6 "Finally, one may notice that the stiffness variations (for the same
nodule depth) during the trials with likelihoods functions computed from experimental do not necessary
follow the same gradient as the variations during the trials with likelihoods function computed from
the FE simulations. More generally, even across trials repeated for the same scenario, we observed
different stiffness variations. This comes from the fact that the stiffness is adapted according to the current
knowledge during the palpation exploration. The proposed method then presents a strategy that tunes
the stiffness in order to increase the probability of getting new information based on prior knowledge
(likelihood functions). Due to the stochastic behavior of the force peak prominence, even for the same
stiffness and same nodule, the information obtained during a sweep is different from one sweep to another.
As a consequence, for two trials on the same nodule, if the information collected so far is different, the
stiffness selected to maximize the information gain of the next sweep will be different. Finally, this
can also be supported by another study \[14\] where the variation of the human's joints stiffness during
palpation tasks has been studied and clearly showed that this joint stiffness follows a random walk. In
other words, during palpation exploration tasks, even humans do not follow a predictable gradient of
joint's stiffness variation."
In section Experimental results, paragraph 5 "In particular, the bests of the tested stiffnesses to detect
the presence of a nodule in the stiffness range of the VLM probe are respectively Kq = 0:68Nm/rad and
Kq = 0:76Nm/rad for the lateral sweep and for the longitudinal sweep."to avoid the use of "optimal".
4\. Minor point: could the oscillations seen in Figs 8 and 10 be, in part, due to probe dynamics? Perhaps
stick-slip excites those dynamics.
These oscillations come from both the dynamics of the probe and the dynamics of the phantom (both
connected in series). Indeed, if they were coming exclusively from the probe dynamics, the oscillations
would vary with the stiffness variation but not with the nodule depth. Also, one can see that for the same
stiffness, the amplitude of the oscillations is varying with the nodule depth (also visible on simulation
from Fig 8); so, both dynamics are important. The authors strongly agree on the fact that the stick-slip of
the probe excites the probe and phantom dynamics. For improving clarity, more discussion about these
oscillations has been added in the paper.
In section Simulation results, paragraph 3 "Furthermore, one can notice that the amplitude of these
oscillations varies with the nodule depth, which means that these oscillations are not only dependent on
the probe's internal dynamics but also on the ones from the phantom."
######
Submitted filename: Response to Reviewers.pdf
######
Click here for additional data file.
10.1371/journal.pone.0237379.r003
Decision Letter 1
Ranzani
Tommaso
Academic Editor
© 2020 Tommaso Ranzani
2020
Tommaso Ranzani
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
8 Jun 2020
PONE-D-20-03370R1
Conditioned haptic perception for 3D localization of nodules in soft tissue palpation with a variable stiffness probe.
PLOS ONE
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Academic Editor
PLOS ONE
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Reviewers\' comments:
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Reviewer \#3: Thank you for the thorough response to my critique and for adding the constant stiffness case as a comparison. I also stand corrected on the behavior of the stiffness during Bayesian search. I now find the paper quite convincing.
Reviewer \#4: The authors present a new method for 3D localization of nodules in sot tissues based on a variable stiffness probe equipped with F/T sensors for kinesthetic perception and a tactile array for tactile perception. The authors proposed an exploration strategy based on a Bayesian approach to the detection and localization of the nodule that allows the authors to set the stiffness of the probe and the direction of the sweep to detect and localize the nodule.
The paper is well written and clear. However, I have some doubts and concerns that I would like the authors to clarify.
Major concerns
1\) The proposed study is based on the experimental results over a simplified setup in which the soft tissue is isotropic and homogenous, and its surface is flat. Since the method relies on fine-tuning of a few thresholds, how does it generalize to the real case, where the conditions on the soft tissue are not so clean, and the presence of other organs and tissues (even as stiff as bones) affects the sensed force?
2\) I don\'t see a great added value in the FEM simulations that the authors propose. First, I would like to have more details about how contacts were modeled (are there bearings, bushes, \... ?), and about the size of the soft tissue and its constraints to a fixed frame Second, if it can have a role in setting the initial value of the stiffness for a real case experiment, the soft tissue should be modeled more accurately.
3\) My third concern is about parameters. In particular the soft tissue thickness, the nodule Young modulus, and its depth. Since nodule diameter is 16mm, I would expect to have at least 32mm of soft tissue between the nodule and the supporting rigid plane. Even if the applied forces are small, it is a bit surprising that the nodule, especially at 2mm depth, does not influence the stress tensor of the surrounding soft tissue. For what regarding the depth, why the maximum selected depth is 8mm?
4\) The proposed algorithm seems quite sensitive to the thresholds. In all cases one lateral sweep allows the system to make the right decision whether the nodule is present or not. Did the authors try starting from a position far from the nodule? Is it so unlikely to have P(N)\<0.2 after the first sweep if it takes place far from the nodule?
5\) The authors claim submillimeter accuracy, but this is not supported by the results. Please note that it is even difficult to place the nodule in the soft body with such accuracy. Moreover, I can\'t see in the results where such accuracy has been achieved.
Minor concerns
6\) Algorithm1: I suggest the inclusion of an escape from the do-while (e.g. a timeout). Line 15: shouldn\'t dir be \"lat\" instead of \"long\"? Line 18: shouldn\'t dir be \"long\" instead of \"lat\"?
7\) line 221: what does it mean \"implemented in real-time\"?
8\) Typos: line 334: remove \"performed\", line 462: \"sweeps\", line 488: \"cases\", line 512: remove \"distance\"
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10.1371/journal.pone.0237379.r004
Author response to Decision Letter 1
29 Jun 2020
Response to the Reviewer 3
Thank you for the thorough response to my critique and for adding the constant stiffness case as a comparison.
I also stand corrected on the behavior of the stiffness during Bayesian search. I now find the paper quite
convincing.
The authors would like to thank again the reviewer for the valuable comments and feedback that helped us to
improve the clarity of the paper. The authors are happy to read that the previous answers reached the expectation
of the reviewer.
Response to the Reviewer 4
The authors present a new method for 3D localization of nodules in sot tissues based on a variable stiffness
probe equipped with F/T sensors for kinesthetic perception and a tactile array for tactile perception. The authors
proposed an exploration strategy based on a Bayesian approach to the detection and localization of the nodule
that allows the authors to set the stiffness of the probe and the direction of the sweep to detect and localize the
nodule. The paper is well written and clear. However, I have some doubts and concerns that I would like the
authors to clarify.
1\. The proposed study is based on the experimental results over a simplified setup in which the soft tissue
is isotropic and homogenous, and its surface is flat. Since the method relies on fine-tuning of a few
thresholds, how does it generalize to the real case, where the conditions on the soft tissue are not so
clean, and the presence of other organs and tissues (even as stiff as bones) affects the sensed force?
The aim of this paper is to quantify the role of compliance in the probe to accurately estimate the depth
of a stiff formation. Therefore, we idealized the scenario as much as possible to remove the effect of
any other artefacts introduced by the tissue. We even use a spherical hard nodule to remove the effect
of uneven geometry of the nodule. We do not claim that there is a specific probe stiffness that gives the
best result irrespective of the probe and the tissue condition. Therefore, what can be generalized is that
probe stiffness matters in conditioning the shape of the likelihood function (sensor model) in a Bayesian
framework to estimate a given feature in the tissue (in this case the nodule depth after having identified the
location using lateral sweeps). Therefore, when the tissue is inhomogeneous, multiple force prominence
values will be present in the force profile. Then a shape filter such as a convolution of a target shape over
the force profile should be developed to extract the target feature. This is beyond the scope of this paper.
We added the following text in the discussion to address this concern:
In section Discussion, paragraph 5 "More generally, this paper concludes that the probe stiffness matters
in conditioning the shape of the likelihood function (sensor model) in a Bayesian framework to estimate a
given feature in the tissue (in this case the nodule depth after having identified the location using lateral
sweeps). However, when the tissue is inhomogeneous, multiple force prominence values will be present in
the force profile. Then a suitable technique should be adopted to filter a target force prominence shape.
One realtime solution is to convolve a target force shape on the measured force data. A data-driven
approach can be used to build the target shape from a variety of tissue samples with a given feature in
them."
2\. I don't see a great added value in the FEM simulations that the authors propose. First, I would like to
have more details about how contacts were modeled (are there bearings, bushes, \... ?), and about the size
of the soft tissue and its constraints to a fixed frame Second, if it can have a role in setting the initial value
of the stiffness for a real case experiment, the soft tissue should be modeled more accurately.
The FEM simulations were done to understand how the probe stiffness variation leads to changes in
tissue stress dynamics that cannot be measured otherwise. Such insights are useful to understand why
probe stiffness variation and resulting differences in the shape of likelihood functions are underpinned
by the coupled dynamics between the probe and the tissue. Also, we have shown that the FE Analysis
can be used to generate the likelihood function. Using this likelihood functions, generated from simulations,
comes at a cost on the confidence level of the prediction, but can be useful in the case where
no experimental palpation data is available. Regarding the contact models; unfortunately, COMSOL
documentation does not specify if bearings or bushes models are used. However, the model is defined
with 2 contact pairs: the fingertip and the phantom. Then the pressure contact calculation between the
2 contact pairs is based on an Augmented Lagrangian Method, and the friction between the 2 pairs is
modeled as Coulomb friction. Finally, no rolling resistance is modeled for the contact between the probe
and the phantom, which is equivalent to assuming pure sliding. The authors agree that there is still room
for improvement in the soft tissue model. Yet, for the proposed application, the FE model is accurate
enough to perform the 3D localization of the nodules based on the likelihood functions obtained from the
simulation results. To improve the clarity of the model approach and support the interest of the proposed
FE simulation, the following modifications have been added to the paper:
In section Finite Element Simulation, paragraph 5 "The contact between the probe and the phantom is
modeled with two surface contact pairs covering the palmar region of the probe and the upper layer of the
phantom, respectively. The pressure contact calculation is based on an Augmented Lagrangian Method,
and the friction between the 2 contacts is modeled as Coulomb friction (m in Table 1). Finally, no rolling
resistance is modeled for the contact between the probe and the phantom assuming pure sliding at the
elements level."
In section Discussion, paragraph 2 "Also, increasing the accuracy of the tissue model or simulating
the lateral sweeps would require developing a 3D FE model and repeating the simulation for several
shifts (position along x). These modifications would increase the computational cost of the simulation
significantly."
3\. My third concern is about parameters. In particular the soft tissue thickness, the nodule Young modulus,
and its depth. Since nodule diameter is 16mm, I would expect to have at least 32mm of soft tissue between
the nodule and the supporting rigid plane. Even if the applied forces are small, it is a bit surprising that
the nodule, especially at 2mm depth, does not influence the stress tensor of the surrounding soft tissue.
For what regarding the depth, why the maximum selected depth is 8mm?
The phantom has been designed with a similar ratio tissue thickness to nodule diameter than the ones
used in published related works such as \[25, 47, 29\]. Also, it can be noticed that in the case of T1 tumors
in the kidney or in the left lobe of the liver, for instance, the ratio between the organ thickness and tumor
thickness can be inferior to 2. As the reviewer noticed, the FE simulation shows that with the small
forces applied during the longitudinal sweeps, the stress tensor under the nodule is not influenced even
for the 2mm deep nodule. This phenomenon comes from the fact that during the palpation sweeps, the
displacement of the nodule is negligible compared to the displacement of tissue above the nodule. In
other words, the amount of tissue compressed between the probe and the nodule (referred in this article
as the nodule depth) is more significant than the amount of tissue under the nodule. Finally, we decided
to limit the nodule depth to 8mm to reduce the average palpation force level to minimise damage to the
probe. Indeed, as shown in related works, detecting deeper nodules requires deeper indentation and by
consequence, higher forces. Higher forces also lead to faster degradation of the tissue in repeated trials,
making it difficult to compare results. Moreover, the lateral sweeps used an array of capacitive tactile
sensors, that saturate if an excessive force is applied during lateral sweeps to locate the nodule. Since
the development of a tactile sensor array for deep tissue exploration is not the focus of this paper, we
used a nodule depth that meets all hardware requirements to demonstrate the key scientific phenomenon
mentioned above. The discussion generated by the reviewer's comment have been added in the paper as
follows:
In section Soft tissue phantom with nodules, paragraph 1 "The ratio between the tissue thickness and
the nodule diameter has been chosen accordingly to the one used in related studies in the literature
\[25, 47, 29\]"
In section Soft tissue phantom with nodules, paragraph 2 "In the presented study, we limited the nodule
depth to 8mm to reduce the average palpation force level and minimize damage to the probe. Indeed,
as shown in related works, detecting deeper nodules requires deeper indentation and by consequence,
higher forces. Higher forces also lead to faster degradation of the tissue in repeated trials, making it
difficult to compare results. Moreover, the lateral sweeps used an array of capacitive tactile sensors, that
saturates if an excessive force is applied to locate the nodule. To avoid saturating the tactile sensor, we
used a nodule depth that meets all hardware requirements to demonstrate the role of stiffness variation
in conditioning the haptic perception during 3D localization of nodules in soft tissues. "
In section Simulation results, paragraph 2 "Moreover, with the small forces applied during the longitudinal
sweeps, the stress in the material under the nodule is not impacted as much as the stress in the
material above the nodule. This phenomenon comes from the fact that during the palpation sweeps, the
displacement of the nodule is small compared to the displacement of tissue above the nodule. This shows
that the probe is more significantly affected by the amount of material above the nodule (the nodule
depth) than the amount of material under the nodule."
4\. The proposed algorithm seems quite sensitive to the thresholds. In all cases one lateral sweep allows the
system to make the right decision whether the nodule is present or not. Did the authors try starting from
a position far from the nodule? Is it so unlikely to have P(N) \< 0:2 after the first sweep if it takes place
far from the nodule?
As any control strategy, the proposed algorithm relies on the tuning of some parameters (the threshold
here). In the sake of clarity, the authors described and discuss the role of each chosen threshold. One of
the advantages of the proposed threshold is that it is simple for the user to tune them according to their
desired level of confidence. The authors proposed this threshold because it is the one that gave the best
detection rate during the validation of the algorithm. The authors tested the algorithm at different starting
distance from the nodule, and the detection was successful independently from the starting point. The
limitation of this threshold comes more from the risk of false-positive than from missing a nodule due to
the initial position of the probe. Indeed, the algorithm continues to investigate even if the probability of
having a nodule is higher or equal 20%, which may result in some case in additional lateral exploration
when it was not required. The discussion from this question has been added to the paper:
In section Evaluation of the algorithm, paragraph 4 "With the proposed threshold values, the algorithm
was 100% accurate on the estimation of the presence of a nodule with a single lateral sweep. When
a nodule was present, P(N) was over 0.98 after the first sweep, independently from the initial distance
between the probe and the nodule. When there was no nodule, P(N) was in a range between 0 and 0.18
after the first lateral sweep. A trade-off needs to be found while tuning Pth
N in order to maximize the detection rate but not increasing the number of false-positive detection, which would lead to unnecessary additional lateral explorations."
5\. The authors claim submillimeter accuracy, but this is not supported by the results. Please note that it is
even difficult to place the nodule in the soft body with such accuracy. Moreover, I can't see in the results
where such accuracy has been achieved.
The authors disagree on this point with the reviewer. Indeed, the Root Mean Square Error (RMSE) for
the eight nodule detections using the presented algorithm (the two trials with fixed stiffness are removed
since they do not represent the algorithm) is 0:27mm. The highest absolute error is 0:53mm for the
6mm deep nodule with the likelihood function obtained from the FE simulation. Both the RMSE and the
maximum absolute error are under 1mm, which support the authors claim on submillimeter accuracy. The
accuracy of the nodule detection is computed from the estimated depth computed from the probability
distribution obtained after the last sweep (when the algorithm ends) as follow:
dest = sum(P(d)d)
Of course, the computed errors relies on the fact that the position of the nodule tested to generate the
likelihood functions are the ground truth for 2, 4, 6 and 8 mm deep. To better support the author claim,
the following modifications have been added to the paper:
In section Evaluation of the algorithm, paragraph 7 "The final depth estimate dest can be computed as
follows:
dest = dest = sum(P(d)d)
The Root Mean Square Error (RMSE) between the estimated depths and the actual nodule depths for all
the detections presented in Fig 14 and Fig 15A is 0.27mm. The highest absolute error is 0.53mm for the
6mm deep nodule with the likelihood function obtained from the FE simulation."
6\. Algorithm1: I suggest the inclusion of an escape from the do-while (e.g. a timeout). Line 15: shouldn't
dir be "lat" instead of "long"? Line 18: shouldn't dir be "long" instead of "lat"?
The authors would like to thanks the authors for his suggestion. Adding a timeout to the algorithm would
probably increase the robustness of the algorithm for real-world scenarios. However, for the purpose of
this paper, the author does not feel the need to add one since the threshold on the information gain already
act like a timeout. Indeed when the information gain during a new sweep is too low, the algorithm stops
the sweeps and returns the estimated position and depth probability distribution. Thank you very much
for pointing out the typos for line 15 and 18, the directions have corrected.
7\. line 221: what does it mean "implemented in real-time"?
The authors were using inappropriately "real-time", thank you for pointing it out. The sentence has now
been rephrased as follows:
In section Variable stiffness palpation: the VLM probe, paragraph 4 "The programs to run the experiment
and the algorithm have been implemented using C++."
8\. Typos: line 334: remove "performed", line 462: "sweeps", line 488: "cases", line 512: remove "distance"
The authors would like to thank the reviewer once again for pointing these typos out. They have been
corrected.
######
Submitted filename: Response to reviewers.pdf
######
Click here for additional data file.
10.1371/journal.pone.0237379.r005
Decision Letter 2
Ranzani
Tommaso
Academic Editor
© 2020 Tommaso Ranzani
2020
Tommaso Ranzani
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
27 Jul 2020
Conditioned haptic perception for 3D localization of nodules in soft tissue palpation with a variable stiffness probe.
PONE-D-20-03370R2
Dear Dr. Herzig,
We're pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.
Within one week, you'll receive an e-mail detailing the required amendments. When these have been addressed, you'll receive a formal acceptance letter and your manuscript will be scheduled for publication.
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Kind regards,
Tommaso Ranzani, PhD
Academic Editor
PLOS ONE
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10.1371/journal.pone.0237379.r006
Acceptance letter
Ranzani
Tommaso
Academic Editor
© 2020 Tommaso Ranzani
2020
Tommaso Ranzani
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
30 Jul 2020
PONE-D-20-03370R2
Conditioned haptic perception for 3D localization of nodules in soft tissue palpation with a variable stiffness probe.
Dear Dr. Herzig:
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| {
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} |
Introduction {#s1}
============
The new pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, disrupting health systems and critical care, even within the most developed heath systems and economies ([@B1]). As of April 22nd, 2020, 2.5 million confirmed cases of COVID-19 have been reported globally ([@B2]). Sustained community transmission is expected in low- and middle- income countries (LMICs) where COVID-19 containment strategies continue to be a challenge ([@B3]). Extreme mitigation strategies have been put in place in many countries to control COVID-19, to reduce disease transmission and to avoid overburdening healthcare systems including mass lockdowns, curfews, and social distancing measures ([@B4]). SARS-CoV-2 and interventions to reduce transmission are negatively impacting already impoverished communities in LMICs and will test heath systems that have little capacity for the management of high dependency patients, or sufficient PPE to protect health workers ([@B5]). Interventions will have long-lasting detrimental impacts on LMIC economies, and, in the absence of reliable and efficient tools for early detection of infected and exposed individuals, are likely to extend beyond 2020/21 including in Africa ([@B6]).
Africa is vulnerable to being overwhelmed by COVID-19. The World Health Organization (WHO) Director General Dr. Tedros Ghebreyesus, stated that the greatest concern was COVID-19 transmission in countries with weaker health systems than in developed nations ([@B7]). On Apr 17, 2020, the WHO estimated 10 million cases of COVID-19 spreading rapidly across Africa and up to 3 million deaths within 6 months ([@B8]). Cases are expected to rise quickly due to a chronic lack of testing, lack of personal protective equipment (PPE), and poor patient care facilities of basic equipment to contain the pandemic, such as PPE ([@B9], [@B10]). The ability to contain COVID-19 will depend on the success of social distancing and the ability to diagnose, isolate, and treat cases ([@B11]).
Case finding and reporting for COVID-19 in Africa is making less than ideal progress. Data from the African Centers for Disease Control (CDC) shows that while risk of importation of COVID-19 to Africa was lower than that to Europe (1 vs. 11%), response and reaction capacity are also lower; the latter being intrinsically linked to individual country wealth and resources for detection, prevention, and control ([@B12]). In late March, Africa had reported 41 local transmissions and only 9 imported cases, by 7 April 2020, 9,888 of 9,971 cases (99.2%) were community acquired with only 83/9,971 cases being imported. As of 18 April 2020, Africa had reported 1,000 deaths with COVID-19 and more than 19,800 cases in 52 out of 54 countries on the African continent ([@B13]). With travel restrictions in place, all cases of COVID-19 are considered community acquired ([@B14]). While many African nations have employed lessons learned from Ebola ([@B15]). COVID-19 is far more challenging to manage. Quantifying the pandemic growth across the African sub-continent and assessing the impact of interventions put in place will be compromised by the lack of diagnostic capacity ([@B16]).
Across East Africa, in April 2020, countries lack a coordinated response against COVID-19. While the WHO/AFRO are making strong recommendations, many governments are taking their own approach. The president of Tanzania encouraged people to "pray for 3 days" against COVID-19 and has not imposed any movement restrictions---places of worship remain open ([@B17]). In Kenya, only a partial lockdown is in place in major cities and many are not prepared for a total lockdown of the country ([@B18], [@B19]). In Uganda and Rwanda more extreme actions have been taken with total national lockdowns that have involved closure of all non-essential businesses, public transport, and the closure of schools and universities. Only local food stores, supermarkets, medical, and veterinary supplies are exempt and for these services a mandatory curfew has been imposed. No motor vehicles/motorists are permitted to use public roads unless they are listed among essential service providers authorized by the Office of the President ([@B20]--[@B22]). Countries not abiding with the social distancing guidelines as recommended by the WHO are putting neighboring countries at risk and compromising health security within the East African Community (EAC). The EAC continues to show a disorganized response against COVID-19 and this high level of disorganization has created confusion in the general public as to what is "true" and "false" COVID-19 information. Rumors and misinformation have spread widely within the community and the media; for example, a religious leader in Uganda claimed that there was no COVID-19 in Uganda and stated that it was just "simple flu" and an individual at Kampala City Council Football Club falsely claimed that Uganda had lost a patient to COVID-19; these culprits are currently in jail awaiting trial for spreading false information ([@B23], [@B24]). Risks and assumptions are causing disharmony in communities, fearful of risk of infection and of the economic consequences of government interventions.
To date, COVID-19 cases and deaths have been greatest in Europe, the Americas, the Western Pacific, the Eastern Mediterranean, South East Asia, and Africa, but the situation is fluid and will change as COVID-19 impacts new regions ([@B25]). While most COVID-19 patients have been of European, Asian, and African descent, data from the USA indicates that while African-Americans are at equal risk of infection with SARS-CoV-2, they are at higher risk of severe COVID-19 complications and death. In Illinois, USA, African Americans accounted for 29% of confirmed cases and 41% of deaths, yet comprise 15% of the state\'s population ([@B26]). Similar trends have been observed in Michigan and Wisconsin, USA ([@B27]). This is unlikely to have a basis in racial susceptibility but is more likely due to a vulnerability to infection and lack of access to quality medical care---it\'s not about race but about racism and poverty ([@B28]). These observations are of concern for developing countries in Africa and Latin America ([@B29]).
The prognosis for COVID-19 infection in Asia and Europe appears to be influenced by sex (being male), pre-existing health conditions (diabetes, cancers, and cardiovascular disease), and age (average age 81 years) ([@B30]); other risk factors may include air pollution and smoking ([@B31]). Reports released by the Chinese Center for Disease Control and Prevention show that men are more at risk than women ([@B32]). Sex differences in males and females in China are supported by the relatively higher antibodies titer generated in females against COVID-19 ([@B33]). Reports from Italy showed no significant differences between males and females infected with COVID-19 ([@B34]). Older males continue to be disproportionally affected by COVID-19 ([@B35]).
In young people (\<18 years), reported COVID-19 infections, hospitalization, and death are low ([@B36]). COVID-19 generally presents with milder symptoms in children than in adults ([@B3], [@B7]), but the evidence-base is unclear. This does not mean children are immune, and children are considered important asymptomatic carriers able to facilitate SARS-CoV-2 transmission within households ([@B37]).
While most COVID-19 infections are mild (with \<20% of cases being severe to critical) ([@B38]), communities of heavy smokers or those with lung function impairments are believed to be particularly susceptible to complications ([@B39]). The WHO has stated that women in Africa are most likely to die from COVID-19 due to sex inequity, chronic poverty among women, weak economic capacity, sexual, and gender-based violence ([@B40]). The elderly are more vulnerable to coronavirus ([@B41]) and underlying non-communicable diseases that are pervasive in Africa will predispose individuals to complications from SARS-CoV-2 infection.
This study aimed to identify perceptions of COVID-19 risk among Ugandans in order to identify novel strategies to guide the national COVID-19 Task Force (nCTF) to improve, control, and prevent COVID-19 infections.
Methods {#s2}
=======
This was a cross sectional study conducted with 161 Ugandan respondents in the second week of April 2020. During this period, COVID-19 infections started to increase in East Africa and Uganda was placed in total lockdown (March--May 2020). A pre-tested online questionnaire using Q-survey® (<https://www.qsurvey.qa/home/en>) was administered with study participants through online resources i.e., email, Facebook, Twitter, WhatsApp, and Viber. Only Ugandans were included in the study while international residents were excluded from the study, using phone IP addresses, which were automatically generated by the Q survey®. Study participants were encouraged to share the link to the questionnaire with family members and friends to enhance data collection using the same social media platforms. Financial challenges in this period implied that the response rate was low since a majority of Ugandans use prepaid mobile internet connection. The questionnaire was designed using major trending informal statements on COVID-19 to assess the knowledge and perceptions of COVID-19 ([Supplementary File 1](#SM1){ref-type="supplementary-material"}). Completing the questionnaire in full was not a mandatory requirement and some questions could be skipped. Metadata was collected and only study participants whose current location was in Uganda was preserved for statistical analysis.
Statistical Analysis
--------------------
Data was exported from Q survey in MS Excel and univariate statistics were conducted using WinPEPI® and significance was reported at a 95% confidence interval using questions which were asked to both males and females in Uganda.
Results {#s3}
=======
COVID-19 Perceptions Amongst Male and Female Ugandans
-----------------------------------------------------
Of 161 Ugandan respondents, 64% (*n* = 103, 95% CI: 56.3--71.1) were male and 36% were female (*n* = 58, 95% CI: 28.9--43.7). Most were between the ages of 18--30 years (70.5%, 110/161) ([Table 1](#T1){ref-type="table"}). A total of 52.7% of male respondents (54/102) considered males to be more vulnerable to infection with COVID-19 while 70.4% of female respondents (38/54) did not consider males to be more vulnerable; a significant difference between the groups (RR = 1.79, 95% CI: 1.14--2.8). Most female respondents considered infection risk to be equally distributed between genders and there was no significant difference in this perception between male and female respondents.
######
Description of the study participants in Uganda.
**Variables** **Statistic** **Percentage** **95% CI**
---------------------------- ----------------- -------------------------------------- ---------------- ------------
Sex Male 103[^a^](#TN1){ref-type="table-fn"} 64.0 56.3--71.1
Female 58[^a^](#TN1){ref-type="table-fn"} 36.0 28.9--43.7
Total 161[^a^](#TN1){ref-type="table-fn"} 100 98.2--100
Age 18--30 110[^a^](#TN1){ref-type="table-fn"} 70.5 60.8--75.2
31--55 46[^a^](#TN1){ref-type="table-fn"} 28.5 22.0--35.9
Undeclared 05[^a^](#TN1){ref-type="table-fn"} 3.1 1.2--6.8
Total 161[^a^](#TN1){ref-type="table-fn"} 100 98.2--100
Descriptive of age (years)
Mean 28.2[^b^](#TN2){ref-type="table-fn"} 27.1--29.2
Median 27.0[^b^](#TN2){ref-type="table-fn"} 25.0--29.0
Minimum 18.0[^b^](#TN2){ref-type="table-fn"}
Maximum 55.0[^b^](#TN2){ref-type="table-fn"}
25th Percentile 23.0[^b^](#TN2){ref-type="table-fn"}
75th Percentile 31.8[^b^](#TN2){ref-type="table-fn"}
SEM 0.6[^b^](#TN2){ref-type="table-fn"}
*Frequency*;
*counts. 95% CI, 95% confidence intervals*.
More male (52.5%, 52/99, 95% CI: 42.7--62.2) respondents believed they were more likely to die from COVID-19 than females (29.6%, 16/54, 95% CI: 18.6--42.8); however 70.2% (40/57) of females disagreed with them on who is more likely to die. Females were half as convinced that men were more likely to die than the male respondents (RR = 1.76, 95% CI: 1.1--2.7). There was no significant difference between the groups of male and female respondents on whether both sexes were equally at risk of death (RR = 0.8, 95% CI: 0.7--1.0). The majority of males (41.4%, 41/99) believed that males present with more severe signs; however, females (71.4%, 40/56) were over two times more likely to disagree with this statement. Female respondents (79%, 45/57) disagreed with the statement that symptoms presented more in males (OR = 1.8, 95% CI: 0.8--4.2). Most female respondents agreed that both sexes showed equal signs for COVID-19 (46/56) however no significant differences were found in males and females (RR = 0.9, 95% CI: 0.8--1.1). The majority of female respondents (*n* = 40/56, 71.4%, 95% CI: 58.6--82.1) did not think males present with more severe signs of COVID-19 than women, although no significant differences were found (RR = 1.5, 95% CI: 0.9--2.3). Most study participants indicated that both sexes shared the same symptoms ([Table 2](#T2){ref-type="table"}).
######
Frequency and percentage of respondents and their risk estimates on signs and symptoms of COVID-19 amongst male and female Ugandans.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Parameter compared to females** **Variables** **Frequency (%) of study participation response** **Risk estimates (95% CI)**
----------------------------------- --------------- --------------------------------------------------- ----------------------------- ------------ --------------- ------------ ------------
Age (years) 18--30 65 (65.0) 45 (80.4) 110 (70.5) −15.4\ 0.9\ 0.5\
(−30.7,0.0) (0.7--1.0) (0.2--1.0)
31--55 35 (35.0) 11 (19.6) 46 (29.5)
Total 100 (100) 56 (100) 156 (100)
Males more vulnerable Yes 54 (52.9) 16 (29.6) 70 (44.9) 23.3\ 1.8\ 2.7\
(6.3--40.3) (1.1--2.8) (1.3--5.5)
No 48 (47.1) 38 (70.4) 86 (55.1)
Total 102 (100) 54 (100) 156 (100)
Equal risk to infection Yes 68 (67.3) 44 (77.2) 112 (70.9) −9.9\ 0.9\ 0.6\
(−25.5--5.7) (0.7--1.1) (0.3--1.3)
No 33 (32.7) 13 (22.8) 46 (29.1)
Total 101 (100) 57 (100) 158 (100)
Males more likely to die Yes 52 (52.5) 17 (29.8) 87 (55.8) 22.7\ 1.8\ 2.6\
(5.9--39.5) (1.1--2.7) (1.3--5.3)
No 47 (47.5) 40 (70.2) 69 (44.2)
Total 99 (100) 57 (100) 156 (100)
Both sexes equally die Yes 61 (59.8) 42 (73.7) 103 (64.8) −13.9\ 0.8\ 0.5\
(−30.1--2.4) (0.7--1.0) (0.3--1.1)
No 41 (40.2) 15 (26.3) 56 (35.2)
Total 102 (100) 57 (100) 159 (100)
Males have more signs Yes 32 (32.3) 12 (21.1) 44 (28.2) 11.3\ 1.5\ 1.8\
(−4.1--26.7) (0.9--2.7) (0.8--4.2)
No 67 (67.7) 45 (78.9) 112 (71.8)
Total 99 (100) 57 (100) 156 (100)
Males show equal signs Yes 78 (76.5) 46 (82.1) 124 (78.5) −5.7\ 0.9\ 0.7\
(−20.0--8.7) (0.8--1.1) (0.3--1.6)
No 24 (23.5) 10 (17.9) 34 (21.5)
Total 102 (100) 56 (100) 158 (100)
Males with more severe signs Yes 41 (41.4) 16 (28.6) 57 (36.8) 12.8\ 1.5\ 1.8\
(−3.9--29.5) (0.9--2.3) (0.9--3.6)
No 58 (58.6) 40 (71.4) 98 (63.2)
Total 99 (100) 56 (100) 155 (100)
Both sexes equal to suffer signs Yes 69 (68.3) 39 (69.6) 108 (68.8) −1.3\ 1.0\ 0.9\
(−17.8, 15.1) (0.8--1.2) (0.5--1.9)
No 32 (31.7) 17 (30.4) 49 (31.2)
Total 101 (100) 56 (100) 157 (100)
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*Risk estimates conducted with males being the reference category; aR, attributable risk; RR, relative risk; OR, odds ratio; 95% CI, 95% confidence interval*.
Perceptions on COVID-19 Toward Children and Young Adults Amongst Ugandans
-------------------------------------------------------------------------
Overall, most respondents, 53.5%, 95% CI: 45.7--61.2 (84/157) considered children to be less vulnerable than adults; no significant differences were found between males and females (RR = 1.1, 95% CI: 0.8--1.5). Similar observations were found on participant responses for children being as equally vulnerable as adults. Most respondents considered mortalities to be less in children (male = 63.7%, 65/102, females = 55.4%, 31/56); however, no significant differences were found among those who disagreed with this perception (RR = 1.15, 95% CI: 0.9, 1.5). In addition, most respondents considered children to present with fewer signs of COVID-19 (82/158), and that the signs of infection are not the same in children (77/156), being less severe than in adults (80/155); there were no significant differences between males and females in these responses ([Table 3](#T3){ref-type="table"}).
######
Descriptive statistics and risk estimates on perceptions of COVID-19 in children and adults amongst male and female Ugandans.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Parameter compared to adults** **Variables** **Frequency (%) of study participation response** **Risk estimates (95% CI)**
---------------------------------- --------------- --------------------------------------------------- ----------------------------- ----------- --------------- ------------ ----------------
Children more vulnerable Yes 48 (47.5) 25 (44.6) 73 (46.5) 2.9\ 1.1\ 1.1\
(−14.8--20.5) (0.8--1.5) (0.6--2.2)
No 53 (52.5) 31 (55.4) 84 (53.5)
Total 101 (100) 56 (100) 157 (100)
Children equally vulnerable Yes 41 (40.2) 20 (36.4) 61 (38.9) 3.8\ 1.1\ 1.2\
(−10.9--18.6) (0.7--1.7) (0.6--2.5)
No 61 (59.8) 35 (63.6) 96 (61.1)
Total 102 (100) 55 (100) 157 (100)
Children die less Yes 65 (63.7) 31 (55.4) 96 (60.8) 8.4\ 1.2\ 1.4\
(−9.0--25.8) (0.9--1.5) (0.7--2.9)
No 37 (36.3) 25 (44.6) 62 (39.2)
Total 102 (100) 56 (100) 158 (100)
Children die equally Yes 44 (44.4) 26 (46.4) 70 (45.2) −2.0\ 1.0\ 0.9\
(−19.7--15.7) (0.−1.4) (0.4--1.9)
No 55 (55.6) 30 (30.0) 85 (54.8)
Total 99 (100) 56 (100) 155 (100)
Children less signs Yes 57 (55.9) 25 (44.6) 82 (51.9) 11.2\ 1.3\ 1.6\
(−6.3--28.8) (0.9--1.8) (0.8--3.2)
No 45 (44.1) 31 (55.4) 76 (48.1)
Total 102 (100) 56 (100) 158 (100)
Children equal signs Yes 52 (52) 27 (48.2) 79 (50.6) 3.8\ 1.1\ 1.2\
(−14.0--21.5) (0.8--1.5) (0.6--2.4)
No 48 (48) 29 (51.8) 77 (49.4)
Total 100 (100) 56 (100) 156 (100)
Children less severe signs Yes 55 (55) 25 (45.5) 80 (51.6) 9.5\ 1.2\ 1.5 (0.7--3.0)
(−8.2--27.3) (0.9--1.7)
No 45 (45) 30 (54.5) 75 (48.4)
Total 100 (100) 55 (100) 155 (100)
Children equally severe signs Yes 48 (48) 28 (50) 76 (76) −2.0\ 1.0\ 0.9\
(−19.7--15.7) (0.7--1.3) (0.5--1.9)
No 52 (52) 28 (50) 80 (80)
Total 100 (100) 56 (100) 156 (100)
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*Risk estimates conducted with males being the reference category; aR, attributable risk; RR, relative risk; OR, odds ratio; 95% CI, 95% confidence interval*.
Perceptions on COVID-19 Risks Among the Elderly
-----------------------------------------------
A significantly large proportion of female respondents (76.4%, 42/55) (compared to male respondents 59.4%, 60/101) did not believe mortality rates to be equal in the young and elderly (RR = 1.7, 95% CI: 1.0--2.9). Amongst those who believed that young adults show fewer signs than the elderly, a majority (92.7%, 51/55) of female respondents believed this to be true compared to 84.2% (85/101) of males. Furthermore, a majority of female respondents (71.4%, 40/56)---compared to only 53.1% (52/98) of male respondents agreed that elderly COVID-19 patients would show more severe signs than the young (OR = 2.2, 95% CI: 1.4, 4.8) as shown in [Table 4](#T4){ref-type="table"}. Among those who believed that young adults are more likely to die of COVID-19 than the elderly (19/156), 16% were males while 5.4% were females and there was no significant difference between them.
######
Perceptions on COVID-19 presentation in young adults and the elderly in Ugandans.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Parameter as compared to the elderly** **Variable** **Frequency (%) of study participation response** **Risk estimates (95% CI)**
-------------------------------------------------- -------------- --------------------------------------------------- ----------------------------- ------------ --------------- ------------ -------------
Young adults are more likely to die Yes 16 (16.0) 3 (5.4) 19 (12.2) 10.6\ 3.0\ 3.4\
(0.0--21.3) (0.9--9.8) (0.9--18.8)
No 84 (84.0) 53 (94.6) 137 (87.8)
Total 100 (100) 56 (100) 156 (100)
Young adults are equally vulnerable to dying Yes 41 (40.6) 13 (23.6) 54 (34.6) 17.0\ 1.7\ 2.2\
(0.8--33.1) (1.0--2.9) (1.0--5.0)
No 60 (59.4) 42 (76.4) 102 (65.4)
Total 101 (100) 55 (100) 156 (100)
Young adults show more signs Yes 16 (15.8) 4 (7.3) 20 (12.8) 08.6\ 2.2\ 2.4\
(−2.7--19.9) (0.8--6.2) (0.7--10.4)
No 85 (84.2) 51 (92.7) 136 (87.2)
Total 101 (100) 55 (100) 156 (100)
Young adults equally show signs Yes 45 (45.5) 17 (31.5) 62 (40.5) 14.0\ 1.4\ 1.8\
(−03.3--31.2) (0.9--2.3) (0.93.9)
No 54 (54.5) 37 (68.5) 91 (59.5)
Total 99 (100) 54 (100) 153 (100)
Young adults are more vulnerable to severe signs Yes 19 (19.4) 5 (9.3) 24 (15.8) 10.1\ 2.1\ 2.4\
(−02.3--22.6) (0.8--5.3) (0.8--8.6)
No 79 (80.6) 49 (90.7) 128 (84.2)
Total 98 (100) 54 (100) 152 (100)
Young adults equally show severe signs Yes 46 (46.9) 16 (28.6) 62 (40.3) 18.4\ 1.6\ 2.2\
(1.5--35.2) (1.0--2.6) (1.0--4.8)
No 52 (53.1) 40 (71.4) 92 (59.7)
Total 98 (100) 56 (100) 154 (100)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*Risk estimates conducted with males being the reference category; aR, attributable risk; RR, relative risk; OR, odds ratio; 95% CI, 95% confidence interval*.
Perceptions of Risk and Race Among Ugandans
-------------------------------------------
A majority of participants agreed that all races were at risk of COVID-19, however some participants thought that other races were more at risk than others. Amongst those who thought that COVID was a disease of "the whites," a majority of these were males (32.4%, 33/102, 95% CI: 23.82--41.88) compared to 26.3% females (*n* = 15/57, 95% CI: 16.1--38.9). Furthermore, no significant differences were found between males (5.9%, 6/102) and females (5.3%, 3/57) toward agreement that the disease also affects Blacks. Furthermore, a majority of respondents stated that all races show severe signs of COVID-19 and of these, a majority were females (57.9, 33/57%,) compared to 49.5% (50/101) who were males ([Table 5](#T5){ref-type="table"}).
######
Description of participant responses on race amongst male and female Ugandans.
**Parameter compared to race** **Variable** **Frequency (%) of respondents** **Risk estimates**
-------------------------------- -------------- ---------------------------------- -------------------- ----------- ------ ----- -----
Vulnerable White 33 (32.4) 15 (26.3) 48 (30.2) 6.5 1.3 1
All races 53 (52.0) 33 (57.9) 86 (54.1) −4.9 0.9 0.7
Blacks 6 (5.9) 3 (5.3) 9 (5.7) −1.0 0.9 0.7
Not Sure 10 (9.8) 6 (10.5) 16 (10.1) −0.5 0.9 0.8
Total 102 (100) 57 (100) 159 (100)
Susceptibility to death White 37 (36.6) 13 (23.2) 50 (31.8) 13.4 1.6 1
All races 50 (49.5) 32 (57.1) 82 (52.2) −7.6 0.9 0.5
Blacks 5 (5.0) 2 (3.6) 7 (4.5) 1.4 1.4 0.9
Not Sure 9 (8.9) 9 (16.1) 18 (11.5) −7.2 0.6 0.4
Total 101 (100) 56 (100) 157 (100)
More signs and symptoms White 34 (33.7) 15 (26.8) 49 (31.2) 6.7 1.3 1
All races 54 (53.5) 31 (55.4) 85 (54.1) −1.9 1.0 0.8
Blacks 6 (5.9) 2 (3.6) 8 (5.1) 2.4 1.7 1.3
Not Sure 7 (6.9) 8 (14.3) 15 (9.6) −7.4 0.5 0.4
Total 101 (100) 56 (100) 157 (100)
Severe signs White 38 (37.6) 16 (28.1) 54 (34.2) 1.0 1.3 1
All races 50 (49.5) 33 (57.9) 83 (52.5) −8.4 0.9 0.6
Blacks 6 (5.9) 3 (5.3) 9 (5.7) 0.7 1.1 0.8
Not Sure 7 (6.9) 5 (8.8) 12 (7.6) −1.8 0.8 0.6
Total 101 (100) 57 (100) 158 (100)
*Risk estimates conducted using a 2 x k analysis. aR, attributable risk; RR, relative risk; OR, odds ratio; 95% CI, 95% confidence interval*.
Discussion {#s4}
==========
In this study, a majority of study participants were young adults (64%, 103/161) and this was in agreement with statistical reports which have stated that a majority (51.3%) of Ugandans are in the age range of 15--64 years ([@B42]). The Uganda National Bureau of Statistics has classified age groups of Ugandans of adults using the 18--30 and 31--64 age groupings for adults ([@B43]), demonstrating the importance of young adults in epidemiological surveys. The study showed that a large proportion of males felt they were most vulnerable to COVID-19; however, these sentiments were not shared by women. Findings in the study demonstrate some disparities in COVID-19 risk perceptions at a time when COVID-19 cases are progressively increasing on the African continent ([@B13]). The differences in perception of vulnerability between men and women in our sample is concerning. The WHO has stated that women are at greater vulnerability in Africa ([@B25]), a risk perception not shared by our sample of Ugandan respondents (RR = 1.8, 95% CI: 1.14--2.8).
While both males and females recognize the importance of COVID-19 in their households, nearly half (52.9%, 54/102) of male respondents in our study perceived that they were more likely to die from COVID-19 than their female counterparts, while most women disagreed with this perception (RR = 1.8, 95% CI: 1.1--2.7). These findings in Uganda are worrying since a majority of males affected in Europe are elderly ([@B34]), and in this exploratory study, this was not the case. Furthermore, a majority of females disagreed with their male counterparts, demonstrating a level of superior knowledge; however, reasons for these disparities were not investigated by the current study. Perceptions of heightened male risk are likely to be influenced by online reports of more males dying than females, in Europe and China ([@B32]). It also appears to show an under-appreciation of the structural reasons why women are vulnerable to COVID-19, since they provide the most informal care in families, have limited economic opportunities, and less power in decision making ([@B44], [@B45]).
There is a need to develop novel strategies for communication of COVID-19 risk in Africa ([@B5]). The prognosis for COVID-19 patients in Asia and Europe appears to be affected mainly by sex, pre-existing health conditions, such as diabetes, cancers, and cardiovascular disease and being elderly ([@B30]), conditions which are also progressively rising in the African continent, despite poor prioritization of health service systems ([@B46]). However, women seek more health services than men ([@B47]). This may explain why women are able to mount stronger immunity than men ([@B48]); however, a young population in Africa is bound to have its own infection dynamics. Recent findings from Italy show no significant differences between genders ([@B34]). Symptomology in COVID-19 depends heavily on the immune status of a patient due to risky lifestyles like smoking and air pollution, and not necessarily on gender ([@B33]). In East African communities, household air pollution remains a public health threat especially in slums and rural communities where the use of firewood and charcoal continues to be routine ([@B49]). In Malaysia, knowledge has been shown to affect practice to minimize exposure to air pollutants ([@B50]), while a recent study in Uganda on COVID-19 has shown that knowledge affects practices promoted by the WHO against COVID-19 amongst market vendors ([@B51]).
Most male and female Ugandans in our sample believed that children below 18 years were less vulnerable to COVID-19 infections and that if they contracted the illness, they would be faced with a milder infection and that less would die from the disease. This is a common global perception that has been communicated by the media and many health agencies around the world in 2020 and is in agreement with studies that have shown a low infection rate in children ([@B36]). However, a significant number of women in this survey considered children to be equally vulnerable than adults and that children die more from the disease than adults (RR = 1.2, 95% CI: 0.9, 1.5). This may be due to maternal sentiments, communicated by women and not shared by men in Uganda, where men are often believed to show less empathy toward children ([@B52]).
The general opinion that elderly persons are more vulnerable to COVID-19 than young people is in agreement with recent epidemiological findings ([@B42]). Elderly individuals are known to be at high risk for COVID-19 ([@B30]). In general, women appear more knowledgeable than men on SARS-CoV-2; they may have a greater interest in health-related topics and show more health seeking behavior than men ([@B51]). Such misconceptions may have significant and far-reaching influence on health-seeking behavior ([@B53]).
It has been proposed that conception is functional and if people can solve problems within their existing conceptual environment, then the drive to change one\'s opinion becomes weak, although this does not help in solving a current problem ([@B54]). Thus, this theory of conceptual change is embedded in a set of epistemological assumptions that are far more generalizable than our application to misconceptions has exploited. These epistemological assumptions suggest that the basic problem of understanding cognitive development is to understand how the components of an individual\'s conceptual ecology interact and develop and how the conceptual ecology interacts with experience ([@B55]).
Of concern in this study is the perception among 30.2% (48/159) of respondents, particularly men, that COVID-19 is a "white-man\'s disease;" these feelings were strongest amongst males. These sentiments reflect the present disunified response against COVID-19 in East African states. In Tanzania and Burundi prayers are being promoted for people to seek divine intervention ([@B17]). Many believe that Africa will "be spared" COVID-19, since the disease originated in Asia before spreading to Europe ([@B1]), and following reports that malaria endemic regions would be protected, from generational exposure to chloroquine and hydroxychloroquine amongst Africans, since these drugs were reported to have some success in COVID-19 treatment ([@B56], [@B57]). That Ugandans perceive COVID-19 and infection of "whites" or the "other" is in direct contradiction of data coming from the USA and UK where Black, Asian, and minority ethnic (BAME) communities have been hit hard with infection.
Projections, from the WHO and others, indicate over 10 million cases of COVID-19 in Africa ([@B8]), and 3 million COVID-19 related deaths in the coming months of 2020 ([@B11]). Quantifying pandemic growth and assessment of interventions put in place ([@B16]) will not be straightforward in low- and middle- income settings faced with challenges of access to testing and reporting of cases and deaths. While many African nations have indeed learned from experiences of Ebola ([@B15]), SARS-CoV-2 is a far greater challenge. The virus itself and interventions that have been put in place are both new and difficult to manage long-term in LMICs. Disunity of policies across Sub-Saharan Africa ([@B17], [@B18], [@B20], [@B21]), will not enable disease containment necessary for economic and social recovery and will fuel sustained community transmission ([@B3]).
Study Limitations
-----------------
The study was conducted through an Online application, meaning that Ugandans without smart phones connected to the internet were not able to participate. Recruitment was achieved through sharing of the online link *via* social media and email platforms and data in this study was generally from the same age group i.e., 18--31 years. The sample size was small and further large-scale studies are needed to extend this exploratory study. Furthermore, results should be approached with caution until more large-scale studies are conducted which would include asymptomatic variables not investigated in the current study.
Data Availability Statement {#s5}
===========================
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/[Supplementary Material](#s9){ref-type="sec"}.
Ethics Statement {#s6}
================
Expediated ethical approval was acquired from the Institutional Review Board of Kampala International University under ID: Nr.UG-REC-023/201914. Consent to participate was acquired through online acceptance to participate in the study.
Author Contributions {#s7}
====================
KK and SW conceptualized the study. KK, SW, and FS designed the study. KK, FS, KM, GM, RS, IE, EA, RM, GN, HO, GZ, and JE conducted data acquisition. KK, EM, KB, MM, and SW conducted statistical analysis. KK, FW-S, EM, KB, MM, and SW conducted interpretation. KK, SW, and KB drafted the initial manuscript. KK, EM, KB, FS, MM, KM, GM, RS, FW-S, IE, EA, RM, GN, HO, GZ, JE, and SW revised the manuscript for intellectual content and approved the final version for publication. All authors agree to be accountable for all aspects of the work.
Conflict of Interest {#s8}
====================
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.
**Funding.** This work was supported by Zhejiang University Education Foundation Emergency Research Fund (SW and KB); Global Challenges Research Fund and the University of Edinburgh.
Supplementary Material {#s9}
======================
The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpubh.2020.00416/full#supplementary-material>
######
Click here for additional data file.
nCTF
: National COVID-19 Task Force
WHO
: World Health Organization
LMIC
: Low Middle income Countries
BAME
: Black, Asian, Minority Ethnic
CDC
: Center for Disease Control.
[^1]: Edited by: Zisis Kozlakidis, International Agency for Research on Cancer (IARC), France
[^2]: Reviewed by: Joseph Ntayi, Makerere University, Uganda; David Kitara Lagoro, Gulu University, Uganda
[^3]: This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Public Health
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Introduction {#sec1-1}
============
Posterior fossa extra dural hematoma (PFEDH) is rare, although it is the commonest traumatic lesion of the posterior fossa.\[[@ref1]\] The Posterior fossa is an unfavorable location of a hematoma. The relative infrequency combined with the paucity of early signs makes PFEDH dangerous because it may have an especially rapid downhill course if not promptly treated.\[[@ref2]\] Further, bilateral extradural haematomas (EDH) are particularly rare, and most of them are frontal.\[[@ref3]\] We report a case of bilateral PFEDH with bilateral occipito-temporal EDH and the technique used to minimize blood loss.
Case Report {#sec1-2}
===========
A twenty four year old unconscious male patient presented to our ER with GCS E2V2M5 following a road traffic accident. He fell down on his back while riding a bike without helmet. After resuscitation, an urgent CT head performed that revealed bilateral occipito-temporal EDH abutting transverse sinus with bilateral posterior fossa EDH \[Figure [1a](#F1){ref-type="fig"} and [b](#F1){ref-type="fig"}\]. There were fractures seen at the anterior cranial fossa base but no brain parenchymal injuries.
{#F1}
Patient was taken up for immediate surgery. In prone position, trifoliate incision was placed \[[Figure 2a](#F2){ref-type="fig"}\]. After raising the flaps, diastasis of lambdiod suture was noted on either side \[[Figure 2b](#F2){ref-type="fig"}\]. Bilateral craniotomies were done in the supratentorial region leaving strip of bone in the midline over the Superior Sagittal Sinus and the transverse sinuses. EDH was evacuated and a rent was seen in the bilateral transverse sinuses. Bilateral craniectomies were done in the posterior fossa and EDH was evacuated leaving a strip of bone over the bilateral transverse sinuses thus covering the torcular sinus and its confluence. \[[Figure 2c](#F2){ref-type="fig"}\]. Dural hitches were taken across the sinus over the bone strips in horizontal mattress pattern \[[Figure 2d](#F2){ref-type="fig"}\]. Absolute hemostasis was attained with this technique and bone flaps were replaced and wound closed in layers. The total blood loss in surgery for both sides was approximately 300 ml. The time taken for entire surgery was 70 minutes. Postoperatively patient became conscious with no deficits. Post operative CT scan head showed adequate decompression \[Figure [3a](#F3){ref-type="fig"} and [b](#F3){ref-type="fig"}\].
{#F2}
{#F3}
He developed CSF rhinorrhoea on 2^nd^ postoperative day that was managed conservatively. He was irritable for 3 days after which his GCS improved to 15 and was discharged. He is doing well and independent with excellent reovery at 12 month follow up.
Discussion {#sec1-3}
==========
Bilateral PFEDH is uncommon and its supratentorial extension is even rare.\[[@ref1]\] According to the speed of the clinical picture development, PFEDH can be divided into acute (\<24 hours), subacute (2-7 days) or chronic (\>7 days), depending on the onset of symptoms. The acute ones are caused by arterial injuries and chronic or subacute are secondary to venous injuries. However, in the index case there was venous sinus tear seen and the presentation was acute. Occipital bone fractures or diastasis of lambdoid sutures is seen in 40-86%.\[[@ref4]\] Swelling of soft tissue in occipital and retromastoid region is seen in almost all the patients.\[[@ref4]\] Distatic fracture of the lambdoid suture is usually associated with more complicated venous sinus injury making surgery more difficult.\[[@ref5]\]
Patients with occipital trauma should be evaluated immediately using cranial CT scans, and those having mass effect should be immediately treated surgically. PFEDH has a higher mortality than supratentorial localization. However, early diagnosis of PFEDH by urgent CT scan and prompt surgical evacuation provide excellent recovery.\[[@ref2]\] Patients with PFEDH with no mass effect, serial CT scanning is recommended especially in first 24 hours as repeat CT picks up delayed PFEDH even when the initial CT may be negative.\[[@ref1][@ref2][@ref6]\]
Classically, first one burr hole in the occipital region is made, immediately evacuating the hematoma from this burr hole, then performing the craniectomy. Bilateral or unilateral suboccipital craniectomy has been the standard approach.\[[@ref1]\] Strong dural attachment over the midline has been cited as the reason for bilateral PFEDH without continuity between the haematomas.\[[@ref3]\]
It is important to define mortality and morbidity risk factors in patients with PFEDH. In the previous studies, the risk factors were mentioned as pediatric age group, supratentorial extension of the hematoma, major sinus tear, cardiorespiratory instability, low admission coma score, and additional intracranial pathologies.\[[@ref1]\] However, in another series bilateral PFEDH, associated injuries, and hydrocephalus with effaced cisterns indicate poor prognosis and according to the authors, paediatric age group has better prognosis.\[[@ref2]\] In general, poor GCS at admission, older age and associated subdural and intracranial hematoma are associated with a poorer outcome.\[[@ref7]\]
In past, the mortality in PFEDH ranged between 0 and 50% in unilateral haematomas and 57% in bilateral cases.\[[@ref3]\] However, the recent studies have shown better outcome with early CT scan and aggressive management.\[[@ref1][@ref2][@ref5][@ref6][@ref7][@ref8]\]
Evacuating epidural hematoma has always been the most gratifying neurosurgery. However, PFEDH forms the bane of trauma neurosurgery. Its rapid progression, proximity to brain stem, involvement of sinuses requires expertise of an experienced neurosurgeon. Early CT scan is recommended for any trauma victim with soft tissue injury in occipital region, somnolence and occipital fracture/lambdoid diastasis.
In cases with both supratentorial and infratentorial EDH, the source of bleed is a tear in the transverse sinus with fracture of the overlying occipital bone. In such cases the bony strip over the transverse sinus provides a natural tamponade on the torn sinus and removing this bony strip disturb the tamponade causing moderate to massive blood loss during surgery.
The technique which gives speed and ease of decompression along with minimum blood loss will definitely portend the final outcome. Different methods have been described for removing both supratentorial and infratentorial EDH. However the literature stating the exact amount of blood loss in evacuating such lesions is sparse. These include making a supratentorial craniotomy, evacuating the EDH and then evacuating the infratentorial component by exploiting the space created by hematoma between the bone and sinus. This technique has been described by Chinese authors.\[[@ref9]\] However, we feel that the control of sinus bleed is little more difficult and also the infratentorial EDH evacuation becomes blind. Another technique is to make a single craniotomy involving both supratentorial and infratentorial regions, thus exposing the sinus. We noticed that in such a technique the blood loss was about 800-1000 ml depending upon the extent of sinus tear. Furthermore the lack of bony strip made the job of taking hitch sutures difficult. The likely criticism for our technique may be the time consumed in making four small craniotomies rather than a single large craniotomy plus the theoretical inadequate decompression. However we felt that major time is lost in controlling the bleed than making a craniotomy, also the plaguing issues such as air embolism, hypotension and hypoxia that come with sinus tear can be circumvented, literally. The target of any EDH evacuation is not only decompression but also the prevention of re-accumulation. By this method we maintain a natural bony framework across the area of hematoma to which dura can be adhered and recollection of blood can prevented.
Thus leaving the strip of bone provides a logical solution which not only gives a rigid frame against which sinus can be hitched but also saves the time lost in controlling the source of bleed.
Conclusion {#sec1-4}
==========
Early diagnosis of Posterior fossa EDH and prompt surgical evacuation especially in patients with mass effect provide excellent recovery. Leaving a strip of bone over the venous sinus avoids opening up the tears in it. Besides, this bone acts as a bridge over which dura on either sides of the sinus can to tied to each other to hitch the sinus up to the bone thereby, providing a good tamponade.
Financial support and sponsorship {#sec2-1}
---------------------------------
Nil.
Conflicts of interest {#sec2-2}
---------------------
There are no conflicts of interest.
| {
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Introduction {#Sec1}
============
Tropical cyclones (TCs) are one of the most costly natural hazards impacting on coastal regions across the world^[@CR1]--[@CR3]^ and there is an increasing trend of TC related billion-dollar disasters in the US^[@CR4]^. Over 70% of total tropical cyclone damage in the North Atlantic is caused by major TCs, category 3, 4 or 5 on the Saffir--Simpson scale, which make landfall^[@CR5]^.
In 2017, the North Atlantic hurricane season involved 6 landfalls and is the most costly season to date, with estimates of damage at over \$360bn^[@CR6],[@CR7]^. The season has broken several records. September had the largest Atlantic Accumulated Cyclone Energy (ACE) on record at 175, whilst the season ACE of 226 is the 7th highest. ACE (units, 10^4 ^Kn^2^) is defined as the sum of the maximum sustained surface wind speed squared at six‐hourly intervals for all periods when the TC is at least of tropical storm strength (\>=34 knots)^[@CR8]^. The duration of hurricanes in 2017 was also record-breaking, setting a new September record of 41 hurricane days, principally due to Irma, Jose and Maria, each of which lasted for over 9 days.
The active season which unfolded was not, however, well predicted in the early season forecasts^[@CR9]^. This suggests factors developed later in the season, leading to a higher level of activity than initially predicted. Here we compare the 2017 Atlantic hurricane season to the active seasons of 2005 and 2010 (as indicated in Fig. [1a](#Fig1){ref-type="fig"}). We explore the similarities and differences in the precursors to those seasons, predominantly from an ocean perspective, using a range of observational datasets. The seasons of 2005 and 2010 were chosen for their similarity in activity and intensity to 2017. The 2005 season has the 2nd highest Atlantic ACE on record at 250 with 15 hurricanes (\>=64 knots) and 7 major hurricanes (\>=96 knots). The 2010 season had an ACE of 165, 12 hurricanes (H) and 5 major hurricanes (MH) and was characterised by hurricanes with a significant duration, similar to 2017 (10 H, 6 MH), which formed further east. In addition, these seasons had the highest SSTAs in the MDR, between July and September for the period 1980--2017 (Fig. [1b](#Fig1){ref-type="fig"}).Fig. 1Atlantic tropical cyclone analysis. **a** Observed Atlantic tropical cyclone activity and ACE index from 1980--2017. **b** SSTA and vertical wind shear anomalies in the MDR between July and September for the period 1980--2017 and associated ACE Index (point size and colourbar). **c** Six hourly data from August to September Atlantic tropical cyclone tracks for the years 2005, 2010 and 2017, detailing the SSTA (°C), vertical shear anomaly, and associated wind speed (point size and colourbar)
As hurricanes intensify by extracting energy from the warm ocean surface, the underlying SSTs and ocean thermal structure are critical for their development^[@CR10]^ with local SSTs greater than 26.5 °C usually considered to be a necessary condition for tropical cyclone development, although it can vary slightly by ocean basin. Over 95% of TC in the Atlantic form in waters warmer than 25.7 °C^[@CR11]--[@CR13]^. Favourable (warm) ocean thermal structure in the MDR, together with a low vertical wind shear, and atmospheric low-pressure disturbances such as African Easterly Waves (AEW), are together conducive to intense and sustained hurricane development^[@CR5],[@CR9],[@CR11],[@CR14],[@CR15]^. The size of the Atlantic Warm Pool (AWP), where water is warmer than 28.5 °C, has also been shown to influence the TC track, with more TC genesis further east for a large AWP. Its size also affects the position and strength of the Azores high^[@CR16]^. When a large AWP is present, the Azores high weakens and shifts north eastwards, enabling TCs to track poleward and recurve towards the east.
Observed Atlantic hurricane frequency has been found to correlate with long-term variability of sea surface temperatures particularly as measured by the Atlantic Multi-decadal Variability (AMV) index^[@CR17]--[@CR23]^, which is the North Atlantic area-averaged (0--60°N, 0--80°W) Sea Surface Temperature Anomaly (SSTA). In addition, positive Ocean Heat Content Anomalies (OHCA) have been found to increase the hurricane intensity and track length^[@CR24]--[@CR26]^. Variations in SST are influenced by the heat balance in the mixed layer of the ocean which is governed by air-sea fluxes, together with horizontal advection and vertical advection/mixing processes, and may be written as^[@CR13],[@CR27]^:$$\documentclass[12pt]{minimal}
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\begin{document}$$\frac{{\partial T}}{{\partial t}} = \frac{{Q_{{\mathrm{net}}}}}{{(\rho C_{\mathrm{p}}H)}} + \left( {{\mathbf{U}}_{\mathrm{g}} + {\mathbf{U}}_{{\mathrm{ek}}}} \right) \cdot \nabla {\mathbf{T}} + \frac{{(w_{\mathrm{e}} + w_{{\mathrm{ek}}})(T - T_{\mathrm{b}})}}{H}$$\end{document}$$where *T* is the mixed layer temperature (equivalent to the SST), *Q*~net~ is the net surface heat flux i.e., the sum of the turbulent (sensible and latent) and radiative (solar and longwave) heat fluxes, *ρ* is the density of seawater, *C*~p~ is the specific heat of seawater, *H* is the mixed layer depth, **U**~g~ is the geostrophic current velocity, **U**~ek~ is the Ekman current velocity, *w*~e~ is the vertical entrainment rate, *w*~ek~ is the Ekman pumping velocity and *T*~b~ is the temperature of the water just below the mixed layer. The first term on the right hand side is determined by net surface heat flux, the second by horizontal advection and the third relates to vertical heat exchanges. Our analysis focuses on the respective contributions of surface heat fluxes and ocean circulation to the SST anomalies that can develop prior to hurricane seasons.
As an index of ocean heat transport, and convergence thereof, the observed Atlantic meridional overturning circulation (AMOC) strength at 26°N is used, as measured by the RAPID Array^[@CR28],[@CR29]^. Previous studies^[@CR30]--[@CR33]^ have identified links between ocean advection/AMOC strength and hurricane frequency, and also that the AMOC leads a SSTA dipole in the North Atlantic (with poles at 10--15°N and 45--60°N) that has maximum correlation strength at a 5-month lag^[@CR34]^. Anomalies of the surface net heat flux (SFX) and wind stress curl (WSC) have also been analysed for their impact on SST although they have not previously been identified as precursors to hurricane season strength. A reduction in the WSC in the tropical Atlantic can lead to maximum positive SSTAs around 2 months later^[@CR35]^, while SFX anomalies directly warm or cool the surface layer^[@CR34],[@CR36]--[@CR38]^.
Here we analyse SSTA alongside OHCA (using GODAS ocean re-analysis data^[@CR39]^), and compare these to related indices and data. Our results reveal that the positive SSTAs prior to the 2017 active hurricane season were generated by processes not previously recognised to be important indicators of hurricane season strength. Furthermore, in contrast to other recent strong seasons, in which the SSTAs were evident in March, the anomalously warm ocean surface in 2017 developed later between April and July, making prediction of the Atlantic hurricane season severity even more difficult.
Results {#Sec2}
=======
Atlantic hurricane season activity {#Sec3}
----------------------------------
The time series of Atlantic tropical cyclones from 1980 shows an increase in activity over the period (Fig. [1a](#Fig1){ref-type="fig"}). The number of landfalls has fluctuated with 6 or more recorded in 1985, 1996, 2005, 2008, 2010 and 2017. To better understand hurricane season activity, Fig. [1b](#Fig1){ref-type="fig"} highlights the ACE index for the years 1980--2017 and the associated SSTA and vertical wind shear anomaly in the MDR. Active seasons occur when there is a positive SSTA and a negative shear anomaly (i.e., weaker than average vertical wind shear) in the MDR (40% of years considered). Less active seasons occur when there is a negative SSTA and positive shear anomaly (43%) and when there is a positive SSTA and positive shear anomaly (14%). The seasonally averaged negative shear anomaly is greater in 2005 and 2010 compared to 2017. In both 2005 and 2010, negative anomalies persisted throughout the season, whereas in 2017 negative shear anomalies developed later in August and September. Previous work has linked ENSO variability / La Niña conditions with negative shear anomalies^[@CR40]--[@CR43]^, SSTA^[@CR44]--[@CR46]^ and a potential relationship between shear and SSTA^[@CR47]^. We note that in 2005, 2010 and 2017 La Niña persisted during August and September. There is also a significant correlation between SSTA and shear anomaly of --0.58 (*p* \< 0.01) indicating that positive (negative) SSTA are often associated with negative (positive) shear anomalies.
To composite the disparate influences on individual TC tracks (as opposed to season activity), Fig. [1c](#Fig1){ref-type="fig"} highlights conditions at 6 hourly intervals along the TC tracks for August and September of 2005, 2010 and 2017, incorporating SSTA, vertical shear anomaly and wind speed along the track. Again, the most favourable conditions for TCs are positive SSTAs and a negative vertical shear anomaly as the majority of the 6 hourly values fall within the corresponding quadrant of Fig. [1c](#Fig1){ref-type="fig"}. A substantial number of values coincide with positive SSTAs and low positive vertical shear anomalies. Far less favourable for TC tracks are negative SSTAs and even when the wind shear anomalies are clearly negative we only find few hurricane track points in the corresponding quadrant. The relationships between SSTA and vertical shear, and number of tropical cyclones are significant, with correlations of 0.75 (*p* \< 0.01) with SSTA and −0.67 (*p* \< 0.01) with vertical shear between July and September, for the period 1980--2017. Figure [1c](#Fig1){ref-type="fig"} also highlights that wind speeds over 100 knots are only observed when there are positive SSTAs present and usually a negative vertical shear anomaly, indicating that both positive SSTA and weak vertical shear are critical to the full intensification of hurricanes. Previous studies have shown that 70% of major storms in the North Atlantic undergo rapid intensification^[@CR48]^ and the interplay between SSTA and vertical shear in such intensification remains a matter for further research.
Of interest is the comparison between Fig. [1b and c](#Fig1){ref-type="fig"}, where the latter indicates TC tracks can exist where there are low positive values of vertical wind shear anomaly. The average vertical shear in the MDR in August and September is 6.14 ms^−1^ (for the period 1948--2017). Wind shear magnitudes of less than 10 ms^−1^ are generally considered favourable for TC genesis^[@CR9],[@CR14],[@CR49]^; accordingly, positive shear anomaly values up to 4 ms^−1^ are still likely to be conducive to TC development, consistent with a significant number of tracks observed in the top right quadrant (Fig. [1c](#Fig1){ref-type="fig"}).
Figure [2](#Fig2){ref-type="fig"} shows SSTA overlaid with sea level pressure (SLP) and tropical cyclone tracks for 2005, 2010 and 2017. Positive SSTA in the MDR is evident in each year, although strongest in 2010. The 2005 season featured hurricane activity in all months, whereas in 2010 and 2017, September was the most active month. The 2010 and 2017 seasons were characterized by large SST anomalies, persisting across the MDR from 10--20°N and 15--90°W, from July to September. Accordingly, some tropical cyclone (TC) genesis was located further east than is usual in both years, and as far east as 20°W, in contrast to 2005. In 2005, the largest SSTAs were further west at 40°W. In 2005 and 2010, positive temperature anomalies persisted to a depth of over 250 m during the season, whereas in 2017 the anomalies were generally only to a depth of 100 m between 10 and 60°W, although a positive anomaly to a depth of 250 m did exist during September 2017 between 60 and 80°W (Supplementary Fig. [1](#MOESM1){ref-type="media"}).Fig. 2Atlantic tropical cyclone tracks during July, August, September and October for 2005, 2010 and 2017. Tropical cyclone tracks (black) and observed SSTA (colour, °C) overlaid with SLP (contours, mb)
TC tracks are influenced by both SLP and SSTA. The positive SSTA across the MDR enabled an eastward shift in the genesis location in 2010 and 2017. In 2010, the Azores high was weaker and located further northeast in the Atlantic (especially in August), related to the large AWP^[@CR16]^, resulting in a lower pressure anomaly off the east coast of America (Supplementary Fig. [2](#MOESM1){ref-type="media"}) and enabling TC tracks to curve northwards. Accordingly, there were no US landfalls despite a very active season, and the 6 tracks which did make landfall in central America predominantly originated in the Gulf of Mexico or Caribbean Sea. In 2005 and September 2017, the lower pressure anomaly off the east coast of America during July to September also existed, but not to the same extent as 2010. TC tracks did curve northwards but there were 9 and 6 landfalls in the respective years.
Ocean and heat flux time series {#Sec4}
-------------------------------
Turning to the ocean variables and to highlight the connection between SSTAs, air-sea fluxes and the ocean circulation we focus on the period from 2000 to 2017 (Fig. [3b--d](#Fig3){ref-type="fig"}). Ocean temperature variability is characterised by SSTAs and OHCA105 (temperature anomalies averaged over the top 105 m). The impact of air-sea fluxes on SSTA and OHCA105 is dominated by latent heat fluxes (LHFX). To understand the timing of the development of SSTAs in different parts of the MDR, LHFX anomalies (LHFXA) are either averaged over the entire MDR (Fig. [3b](#Fig3){ref-type="fig"}), the southern part of the MDR (10--15°N, 40--60°W) or the north-eastern (NE) MDR (15--21°N, 24--36°W) (Fig. [3c](#Fig3){ref-type="fig"}). The location of each region is shown in Fig. [3a](#Fig3){ref-type="fig"}. The north-eastern and southern regions of the MDR were chosen because the air-sea fluxes reached a maximum in these areas in 2017 during April and May--August, respectively. The Meridional Overturning Circulation (MOC) is the main contributor to ocean heat transport in the North Atlantic and here we use observations of the MOC and of its Ekman component at 26°N for the period from 2004 to 2017 (Fig. [3d](#Fig3){ref-type="fig"}).Fig. 3Atlantic time series and map outlining the study region. **a** SST September 2017: green box: MDR region 10--20°N and 20--80°W, black box: southern MDR region 10--15°N and 40--60°W, blue box: north-eastern (NE) MDR 15--21°N, 24--36°W. **b**--**d** Observed Atlantic time series: **b** monthly SSTA, OHCA105m and LHFXA in the MDR; **c** monthly latent heat flux anomalies (LHFXA) in the southern MDR and NE MDR smoothed over 3 months; and **d** MOC and Ekman Transports 12-hourly data smoothed over 61 days by applying a low pass filter
SSTA and OHCA105m are closely aligned (Fig. [3b](#Fig3){ref-type="fig"}), although the strength of the anomaly is greater at the surface. Clear peaks in SSTA and OHCA105m are seen in 2005 and 2010, and also positive anomalies have been seen since 2015. LHFXA in the MDR (green line) is significantly correlated with SSTA and OHCA105m; correlation coefficients are respectively 0.52 and 0.38, both significant at the 0.01 level (*p* \< 0.01). The LHFXA variability is analysed further in Fig. [3c](#Fig3){ref-type="fig"} for the two areas (southern MDR and NE MDR) that experienced the strongest anomalies in 2017. The most extreme latent heat flux anomaly values occurred in 2017, with latent heat loss weaker by 33 Wm^--2^ sustained from May to August in the southern MDR region and weakening of over 60 Wm^−2^ in the NE MDR during April (unsmoothed data). In 2005 and 2010, the LHFXA values in the southern MDR region were noticeably smaller in magnitude (May--Aug mean: 7 Wm^−2^ in 2005; 13 Wm^−2^ in 2010).
A more detailed analysis of the April and May--Aug net surface heat flux anomalies (SFXA) for the MDRs from 1980 to 2017 is shown in Supplementary Fig. [3](#MOESM1){ref-type="media"}. In the NE MDR, the 2017 April SFXA of 93 Wm^−2^ was 3.4 standard deviations (SD) from the mean. In, the southern MDR, the 2017 May--August SFXA was 45 Wm^−2^, 2.5 SD from the mean. For each MDR, the 2017 SFXA was the most extreme in the period considered. Furthermore, for the NE MDR the April 2017 SFXA was nearly twice that for the next largest anomaly (55 Wm^−2^ in 2014) further emphasising the particularly unusual conditions prior to the 2017 season.
MOC values (Fig. [3d](#Fig3){ref-type="fig"}) are significantly below the seasonal mean values in February/March 2005 and 2010 at 11/13 Sv (1 Sv = 10^6 ^m^3^s^--1^) and 9/10 Sv, respectively. Average values for February and March are 15 Sv. Above average values are observed in February 2017 at 17 Sv (the latest available MOC data). Ekman transports are also below average in Feb/Mar 2005 and 2010 but are close to average in Feb/March 2017 and for the remainder of 2017. The February/ March values are important as Fig. [3b, d](#Fig3){ref-type="fig"} indicate that the observed MOC transport co-varies with the observed SSTA and OHCA105m in the MDR over the period from 2004 to 2017. The correlation between them is −0.35 for MOC-SSTA and −0.27 for MOC-OHCA105, when the MOC leads by 5 months, which is statistically significant at the 0.01 level (*p* \< 0.01). This is in line with the results of Duchez et al.^[@CR34]^ who found the strongest correlation between the SSTA and MOC occurred when the MOC leads by 5 months. In addition, Supplementary Fig. [4](#MOESM1){ref-type="media"} shows how the MOC transport anomaly in February and March co-varies statistically with SSTA and OHCA105m in July, August and September. Correlations over 0.5 are statistically significant at the 0.05 level (*p* \< 0.05). Anti-correlations over 0.6 are seen over large areas of the MDR between 10--20°N and 30--70°W during July--September. Importantly, the correlations are higher for OHCA105m than SSTA, highlighting that the variability of the MOC transport at 26°N influences the upper ocean layer in the MDR, which is of consequence for hurricane intensification as it potentially provides a significant heat source extending over the top 100 m of the ocean to power hurricane development.
Hurricane season precursors in 2005, 2010 and 2017 {#Sec5}
--------------------------------------------------
Figure [4a](#Fig4){ref-type="fig"} shows the 2017 March--August monthly surface heat flux anomalies (SFXA) overlaid with the surface wind anomaly. In April, a strong positive SFXA of 100--150 Wm^−2^ developed (i.e., 300--350 Wm^−2^ heat gain compared to the climatological mean of typically about 200 Wm^−2^) with a maximum at over 150 Wm^−2^ between 24--36°W and 16--18°N. This is associated with a reduction in the strength of the north easterly (NE) trade winds (30°W, 18--27°N) revealed by the wind vector anomalies. The weaker NE winds meant there was less cold, dry air over the region. As a consequence, the humidity gradient (between the sea surface and the overlying air) was lower than normal, and so the associated latent heat loss was reduced. In addition, the more humid air over the region enabled an increase in the downwards LongWave Radiative (LWR) flux. Anomalies in these two flux components largely account for the positive SFXA seen. Additionally, April 2017 is also characterised by a negative wind stress curl anomaly (WSCA) in the region associated with the anomalous winds (Fig. [4b](#Fig4){ref-type="fig"}). This results in a downward anomaly in Ekman pumping effectively reducing the upwelling of cold water at the eastern boundary and further assisting development of the positive SSTAs. Similarly, weaker than average NE/E winds persisted in June, July and August between 40--60°W, 9--15°N, along the southern boundary of the MDR. Again, the latent heat loss was lower and the LWR flux into the ocean was higher, largely explaining the positive SFXA and associated positive SSTA and OHCA in the southern MDR at this time.Fig. 4Atlantic Surface Heat Flux Anomalies (the sum of the net shortwave, net longwave, latent and sensible heat fluxes) during the build-up and early stages of the 2017 hurricane season. **a** Observed monthly surface heat flux anomaly from March to August 2017 (Wm^−2^) overlaid with the 1000 mb wind anomaly, red colours indicate stronger heat gain than normal. Blue box indicates the eastern Atlantic region. Black box indicates the southern MDR region. **b** Observed Atlantic surface heat flux anomaly in April 2017 (Wm^--2^) overlaid with the wind stress curl anomaly (WSCA). White contours indicate a negative WSCA (anomalous downwelling), grey contours indicate a positive or zero WSCA (anomalous upwelling). Contour units are 10^−8^ Nm^−3^
The extent to which the 2017 SFXA generated the observed SSTA, and how this compares to 2005 and 2010, is also explored (Fig. [5](#Fig5){ref-type="fig"}). In 2005 and 2010, positive SSTAs already existed in the MDR in March (Fig. [5a, b](#Fig5){ref-type="fig"}). The patterns of these SSTAs are consistent with those related to the below-average MOC and Ekman transport in February and March generating the dipole SSTA pattern observed, with negative anomalies in the northern part of the Atlantic and positive anomalies in the south^[@CR32],[@CR34],[@CR50]^. In 2017, in contrast, the MOC and Ekman transport was close to the 2004--16 mean, and SSTs were close to average (Fig. [5c](#Fig5){ref-type="fig"}). The SFXA forcing over April--July generates the SSTAs shown in Fig. [5d--f](#Fig5){ref-type="fig"}. In the MDR vicinity, SFXA-generated SSTAs are most substantial in 2017 (Fig. [5f](#Fig5){ref-type="fig"}), concentrated in the southern area of the MDR between 9--15°N and 30--60°W with a maximum around 1.4 °C.Fig. 5Surface Flux generated Temperature Anomaly April--July for 2005, 2010 and 2017. Initial condition---SSTA March (**a**--**c**). Estimated temperature anomaly April--July based on anomalous surface fluxes (**d**--**f**). Estimated SSTA in August formed by summing the initial condition and April--July surface flux generated temperature anomaly (**g**--**i**). Observed SSTA in August (**j**--**l**). Estimated minus observed August SSTA (**m**--**o**). Black box indicates MDR region. Colourbar units °C
Adding SFXA-generated temperature anomalies to the initial March SSTA patterns, we obtain estimates of the August SSTAs (Fig. [5g--i](#Fig5){ref-type="fig"}). When comparing the estimated to the observed SSTAs in August (Fig. [5j--l](#Fig5){ref-type="fig"}), the spatial SSTA patterns and sign in the MDR look similar, however the amplitude in 2005 and 2010 is higher than observed. Discrepancies (Fig. [5m−o](#Fig5){ref-type="fig"}) between surface flux based estimates and observations, can be attributed to the ocean circulation (advection) and mixing. The MOC recovered from its below-average strength in February and March during both 2005 and 2010 (Fig. [3d](#Fig3){ref-type="fig"}). This stronger MOC means that in spring and early summer more heat was transported northwards by the ocean, partly compensating for the MDR temperature changes linked to SFXA and assisting the development of average SST conditions in the north, whilst reducing the amplitude of the positive SSTAs in the south, seen during August in these years. Hence, the observed August SSTA in 2005 and 2010 are weaker than those estimated from surface heat flux alone. In 2017, the weaker NE/E trade winds observed from June to August in the MDR will have reduced the northward Ekman transport into the region. As less heat was transported north during those months it is likely to have led to the lower observed SSTA in August than predicted from surface fluxes alone. Additionally, along the eastern boundary between 9--18°N the weaker trade winds will have led to a reduced upwelling and the warmer observed SSTA in the area. From these results, we conclude that in 2005 and 2010, the initial ocean condition in March (MOC-related) was the main influence on SSTAs in August (Fig. [5j--l](#Fig5){ref-type="fig"}), whereas in 2017, the SFXAs that developed from April to July were the dominant factor. These results are not specific to the GODAS ocean temperature reanalysis dataset used here and we found similar results with NCEP SST data (Supplementary Fig. 5).
Our analysis of a range of observations reveals the important role that surface heat fluxes played in positive SSTA development in the MDR in 2017, which was critical for subsequent hurricane activity in September. Figure [6](#Fig6){ref-type="fig"} summarizes the mechanisms which were important during the season.Fig. 6Schematic of the mechanisms which contributed to the positive SSTA in the MDR, and an active hurricane season, in 2017. Orange arrows are the heat exchange anomalies. White arrows show the wind driven anomalies forcing the ocean. The green arrows show the wind driven Ekman transport anomaly response. The blue line indicates the notional surface temperature with suppressed upwelling (solid line) and without suppressed upwelling (dashed line)
The reduction in the NE trade winds between April and July enabled positive latent heat flux and LWR anomalies to develop. The negative wind stress curl generated downward Ekman pumping anomalies, suppressing the upwelling at the eastern boundary. These factors generated positive SSTAs. The reduction in the NE trade winds also reduced the Ekman transport of warm water into the MDR region, explaining why SFXA-predicted temperature anomalies are higher than observed, by August. Reduced vertical wind shear was also evident in late August and September, which together with the positive SSTAs, played a key role in the active hurricane season of 2017. In contrast, in 2005 and 2010, it was the reduced AMOC in February-March of those years which was the predominant cause of the positive SSTA in the MDR, which again combined with a weak vertical shear to favour very active hurricane seasons. These findings reveal for the first time that different precursors can generate positive SSTA in the MDR region, conducive to an active hurricane season. In 2017, surface fluxes were the dominant factor, whereas in 2005 and 2010 the AMOC played a key role.
In terms of hurricane prediction, the 2017 season was more difficult to forecast, as the surface flux anomalies developed between April and July shortly before the main season (August--September). For the other strong hurricane years considered, the reduction in the MOC / Ekman transport occurred earlier in February--March potentially enabling a longer lead time forecast given a sufficient observing system in place and noting also the importance of atmospheric conditions (vertical wind shear). In conclusion, our results have revealed that drivers of recent active hurricane seasons involving the ocean can take two forms: late winter changes in the ocean circulation and late spring/early summer changes in the air-sea heat flux. Developing forecast systems that adequately represent these processes will potentially aid preparedness and mitigation for the financial and societal consequences of hurricanes.
Methods {#Sec6}
=======
Hurricanes and ocean properties {#Sec7}
-------------------------------
The observed Atlantic tropical cyclone and hurricane track data for the years 1980--2016 were obtained from HURDAT2, the revised Atlantic hurricane database^[@CR51]^. Hurricane track data for 2017 was obtained from Unisys Weather (<http://weather.unisys.com/hurricanes/>). The NCEP Global Ocean Data Assimilation System^[@CR39]^ (GODAS) was used for the ocean temperatures at the surface and depth. All anomalies were based on the reference period 1980 to 2017 unless stated otherwise. The ocean heat content anomalies are based on the average temperature anomaly over the depth indicated. The observed AMOC strength and Ekman transport at 26°N, for the period 2004--2017, were obtained from the RAPID MOC monitoring project^[@CR52],[@CR53]^.
Surface meteorology and air-sea exchanges {#Sec8}
-----------------------------------------
The NCEP/NCAR reanalysis^[@CR54]^ was employed for sea level pressure (SLP), wind speeds and the air-sea heat flux. Wind speeds and associated anomalies were determined from the 1000 mb zonal and meridional components. The absolute vertical wind shear was calculated as the absolute difference between the 250-mb and 850-mb zonal wind using the reference period 1948 to 2017. The net surface heat flux (SFX) was determined as the sum of the net shortwave (*Q*~sw~), net longwave (*Q*~lw~), latent (*Q*~lat~) and sensible (*Q*~sen~) heat fluxes.$$\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{SFX}} = Q_{{\mathrm{sw}}} + Q_{{\mathrm{lw}}} + Q_{{\mathrm{lat}}} + Q_{{\mathrm{sen}}}$$\end{document}$$The estimated ocean temperature anomalies (Δ*T*) used in Fig. [5b](#Fig5){ref-type="fig"} and c are based on the surface flux anomalies (SFXA) and calculated as follows:$$\documentclass[12pt]{minimal}
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\begin{document}$$\Delta T\left( {x,y,t} \right) = \Delta T\left( {x,y,t_0} \right) + \frac{1}{{\rho DC_{\mathrm{p}}}}\mathop {\int }\nolimits_{\hskip -5pt t_0}^t {\mathrm{SFXA}}\left( {x,y,t} \right){\mathrm{d}}t$$\end{document}$$where *D* = mixed layer depth (100 m), *ρ* = density (1025 kg m^−3^), *C*~p~ = specific heat capacity 4182 JK^−1^ kg^−1^, *t*~0~: April, *t*: July. A mixed layer depth of 100 m was chosen for consistency with the findings of Cayan^[@CR38]^, who showed the MLD in the North Atlantic to be between 150 m and 75 m between April and July. The spatial temperature pattern does not vary with depth chosen. A more detailed calculation would require a spatially and temporally varying MLD but the aim here is to show the magnitude of the heat flux related signal in line with the approach adopted by Duchez et al.^[@CR55]^ The wind stress curl was calculated from NCEP/NCAR reanalysis^[@CR54]^ monthly surface wind stress.
Supplementary information
=========================
{#Sec9}
Supplementary Information Peer Review File
**Journal peer review information:** *Nature Communications* thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
=================================
**Supplementary Information** accompanies this paper at 10.1038/s41467-019-08496-4.
This work was supported by the Natural Environmental Research Council (NERC) \[grant number NE/L002531/1\], NERC projects DYNAMOC \[NE/M005097/1\] and ODYSEA (NE/M006107/1) and by the NERC programmes North Atlantic Climate System: Integrated Study (ACSIS) \[NE/N018044/1\], the RAPID-AMOC Climate Change (RAPID) programme, the European Union Horizon 2020 research and innovation programme BLUE-ACTION (Grant No. 727852). P.H. was supported by the Joint UK BEIS/Defra Met Office Hadley Centre Climate Programme (GA01101).
S.H. led the development of the study, carried out the analysis and was lead writer of the paper. All authors contributed to the evolution of the analysis and to the writing of the paper.
Hurdat2: <https://www.nhc.noaa.gov/data/hurdat/>; Hurricane track data 2017: <http://weather.unisys.com/hurricanes/>; GODAS: <http://www.cpc.ncep.noaa.gov/products/GODAS/>; AMOC and Ekman Transport data: <http://www.rapid.ac.uk/rapidmoc>; Sea Level Pressure and wind component data: <https://www.esrl.noaa.gov/psd/data/gridded/data.ncep.reanalysis.html>; Surface Flux data: <https://www.esrl.noaa.gov/psd/data/gridded/data.ncep.reanalysis.surfaceflux.html>.
Competing interests {#FPar1}
===================
The authors declare no competing interests.
| {
"pile_set_name": "PubMed Central"
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The planetary albedo and energy budget of the Earth is strongly regulated by clouds. An increase in anthropogenic aerosol typically increases cloud condensation nuclei and cloud droplet number concentration ($N_{d}$). As $N_{d}$ increases, the Twomey effect predicts an increase in cloud albedo and decrease in cloud droplet size for the specific condition that the amount of condensed water remains constant ([@r1]). Smaller droplets in polluted clouds can result in reduced collision coalescence and delay the onset of precipitation and accumulation of liquid water path. Over time, these responses can result in the expansion of cloud horizontal and vertical extents ([@r2]). By contrast, liquid water path and cloud albedo can also decrease under some conditions for clouds with larger concentrations of small droplets \[for example, in ship, volcano, and industrial pollution tracks ([@r3])\] owing to an enhanced entrainment--evaporation aerosol feedback in dry atmospheric conditions ([@r4]). The net effect of these processes has been hypothesized to influence the lifetime of clouds ([@r5]).
The existence of an aerosol effect on cloud lifetime is widely disputed ([@r6], [@r7]). The dispute partly arises from the lack of a clear definition and measurements that are resolved over the dimension of time. The forcing can be decomposed into liquid water path and cloud fraction adjustments, but these do not necessarily act in unison ([@r8]) to produce larger radiative effects and hence, longer cloud lifetimes. A common approach to quantifying the aerosol effect on cloud lifetime is to use statistical relationships in cloud micro- or macrophysical properties (e.g., precipitation rate, cloud liquid water content, or cloud fraction changes) to changes in aerosol concentration. However, typically these satellite-derived relationships are computed using a static Eulerian framework, which does not permit the analysis of cloud fields evolving through time. The A-Train constellation has been paramount for advancing process-level understanding of clouds and aerosols from its unique multisensor array (radiometers, a lidar, and a cloud-profiling radar), but short-term temporal changes cannot be quantified from a sun-synchronous orbit. The A-Train can provide only single snapshot images of cloud systems once per day at 13:30 local time in the afternoon (and at 01:30 but without optical property retrievals), thereby constraining the observations to an Eulerian framework. The connection of these variables to the dimension of time in a Lagrangian framework offers the capability to examine the aerosol influence on cloud development and lifetime. In this work, we use time-resolved observations from satellites in geostationary orbit and define cloud lifetime as the amount of time the cloud fraction computed over large spatial domains ($1^{○} \times 1^{○}$) exceeds a certain threshold (0.75).
The Lagrangian framework has traditionally been used to study the time-dependent response of the marine stratus-to-cumulus transition zone in observational ([@r9][@r10]--[@r11]) and modeling ([@r12][@r13]--[@r14]) studies. This transition typically follows a three-day equatorward redundant trajectory where the boundary layer proceeds through several diurnal cycles ([@r13]), increasingly warmer sea surface temperatures and unstable atmospheric conditions, stronger surface moisture and sensible heat fluxes, and more frequent precipitation. Sea surface temperature gradients and free-tropospheric subsidence rates are considered the primary drivers for the stratus-to-cumulus transition ([@r13]). However, changes in aerosol loading may also play a significant role in the development of clouds through their ability to modify $N_{d}$, precipitation rates, and cloud-top entrainment rates ([@r4]). Large eddy simulation experiments suggest that larger $N_{d}$ can extend the stratus-to-cumulus transition zone several hours by delaying the onset of precipitation and breakup of the stratocumulus layer ([@r14]). Furthermore, the transition can be hastened or slowed by the presence of solar-absorbing smoke layers. The response depends on a variety of factors: the distance between the top of the stratocumulus cloud and location of the overlying smoke layer, whether the smoke mixes with the planetary boundary layer (PBL), and whether the smoke is accompanied by an increase in moisture ([@r15]). A primary goal of the analysis undertaken here is to quantify the extent to which aerosols influence cloud properties along the stratus-to-cumulus transition zone using temporally resolved satellite observations.
Lagrangian Trajectory Framework {#s1}
===============================
Several operational satellite products are collocated to Lagrangian trajectories calculated using the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model ([*Materials and Methods*](#s7){ref-type="sec"}). Geostationary satellite retrievals of the radiative fluxes, cloud properties, and aerosol properties are taken from the Clouds and the Earths Radiant Energy System (CERES) Edition 4a (Ed4a) Synoptic (SYN) 1 degree 1 hour (1deg1hr) product ([@r16]). Precipitation estimates are temporally resolved every half hour in the Integrated Multi-satellitE Retrievals for Global Precipitation Measurement (IMERG) V06B product through an algorithm that integrates, interpolates, and intercalibrates infrared brightness temperature data from numerous satellites in geostationary orbit and from microwave retrievals from several satellites in nongeostationary satellite orbit. Sun-synchronous cloud and aerosol retrievals are obtained from the MODerate Resolution Imaging Spectroradiometer (MODIS) collection 6.1 product. Satellite retrievals are spatially aggregated (or linearly interpolated) to a $1^{○} \times 1^{○}$ region that moves along the centerline of the calculated positions in the HYSPLIT trajectory. This domain is small enough to avoid averaging over spatial gradients in aerosol properties ([@r17]) yet large enough to encompass a wide range of spatial resolutions from each satellite product. All products are averaged over hourly intervals along Lagrangian trajectories.
[Fig. 1](#fig01){ref-type="fig"} and [*SI Appendix*, Fig. S1](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental) demonstrate how the collocation method is implemented. The satellite image shows two Lagrangian trajectories covering the breadth of the stratus-to-cumulus transition zone. This case coincides with the emergence and dissipation of a pocket of open cells (POC) that formed on 5 September 2017 off the west coast of Namibia. POCs occasionally form along the stratus-to-cumulus transition and exhibit an open cellular hexagonal-like structure that results in relatively low planetary albedo compared with the bright surrounding deck of closed cell clouds. They typically form during the night after precipitation has become heavy enough to efficiently scavenge accumulation mode aerosol ([@r18]). Very low concentrations of accumulation mode aerosols, low $N_{d}$, and heavy precipitation rates are commonly observed in POCs. Cloud evolution is examined using a HYSPLIT back trajectory initialized from inside the location of the POC (red line) and in another trajectory located just to the east of the POC (blue) in the surrounding closed cell clouds.
{#fig01}
The Spinning Enhanced Visible and Infrared Imager (SEVIRI)-retrieved liquid cloud fraction rapidly increases to complete saturation over the trajectories as they are advected northwesterly over a four-day period. The initial aerosol optical depth ($AOD$) is similar in both trajectories with a value of $0.19 \pm 0.05$. The cloud fraction remains nearly at 100% until the POC begins to form on 5 September at 4 AM local time. Afterward, cloud fraction declines for roughly 24 h. The geostationary satellite observations show the unique ability to accurately determine the exact timing of the POC formation (within 1 h), while the sparse data from MODIS (Terra and Aqua satellites) only provide a rough estimate (within 12 h). Cloud droplet effective radius markedly increased over the development period of this POC ([Fig. 1*C*](#fig01){ref-type="fig"}). Similarly, the $N_{d}$ is markedly decreased inside the POC (24 $\pm$ 4 ${cm}^{- 3}$) compared with the surrounding cloud field (174 $\pm$ 29 ${cm}^{- 3}$) using [Eq. **2**](#eq2){ref-type="disp-formula"} described below. Satellite retrievals are generally more uncertain in broken cloudy conditions due to the general inability to account for complex three-dimensional (3D) radiative transfer and absorption ([@r19]). Higher effective radius and rain rates were observed inside this POC during the CLouds-Aerosol-Radiation Interactions and Forcing flight campaign (20 to 40 $\mu$m) compared with the surrounding cloud deck (10 to 20 $\mu$m) as described in Abel et al. ([@r20]). Similar behavior was observed in open cellular broken cloud conditions during the Variability of the American Monsoon Systems Ocean-Cloud-Atmosphere-Land Study campaign ([@r21]) and from a climatology of POCs in satellite observations ([@r20]). We conclude that there is confidence in the ability of the satellite data to capture microphysical responses along the trajectories used in this study.
Results {#s2}
=======
Time-resolved satellite retrievals of aerosol, cloud, and radiation are examined from several thousand 80-h forward trajectories for 10 y of observations (2008 to 2017). A key challenge to studying aerosol--cloud interactions is isolating the impact of confounding variables (e.g., meteorological variables such as sea surface temperature) on cloud properties so that the causal relationship can be quantified. Trajectories are initialized in clear-sky areas and sorted by clean and polluted conditions based on $AOD$ ([*Materials and Methods*](#s7){ref-type="sec"}). We use two strategies to account for confounders: 1) a regional-scale analysis of three subtropical oceanic basins in which all trajectories are constrained by wind direction to transect similar gradients in sea surface temperature and 2) a global-scale analysis where trajectories are stratified by gradients in the trajectory time series of lower troposphere stability. Finally, we determine whether aerosols can extend the average lifetime of a domain-wide population of clouds from timescale metrics applied to cloud development.
Regional-Scale Analysis. {#s3}
------------------------
Baja Californian, Chilean, and Namibian regions are used to characterize aerosol responses along the classic stratus-to-cumulus transition zone. Over 2,000 trajectories are initialized near the coast of Baja California ($125^{○}$W to $115^{○}$W; $20^{○}$N to $30^{○}$N) ([Fig. 2*A*](#fig02){ref-type="fig"}). The majority flow in a southwesterly direction ([Fig. 2*B*](#fig02){ref-type="fig"}) covering more than 1,000 km ([Fig. 2*C*](#fig02){ref-type="fig"}). Analysis was limited to those trajectories that flow along the median wind direction (210 $\pm$ 10°) and span a distance of at least 1,000 km from their point of origin. This method ensures that all trajectories transit through similar gradients in meteorological conditions as shown by the distribution of sea surface temperature in [Fig. 2*D*](#fig02){ref-type="fig"}. The ratio of polluted to clean trajectories varies from month to month ([Fig. 2*E*](#fig02){ref-type="fig"}). However, the aerosol effect on $N_{d}$ is robust in every season, and therefore, to preserve samples we use the full distribution of months shown here. The final distribution of the filtered trajectories, which is based on consistent wind direction and distance, is shown in [Fig. 2*F*](#fig02){ref-type="fig"}. Overall, the aim of this approach is to ensure the consistency in meteorological sampling between polluted and clean trajectories, and while the procedure results in the removal of roughly one-third of the tracks that deviate from the median wind, we retain several thousand trajectories to analyze aerosol--cloud responses.
{#fig02}
In [Fig. 3](#fig03){ref-type="fig"}, we observe the stratus-to-cumulus transition ([@r12]) from a gradual deepening of the boundary layer as indicated by an increase in cloud top height ($CTH$) and atmospheric destabilization. Changes in sea surface temperature, lower troposphere stability, boundary-layer moisture, and 500-hPa subsidence rates (subsidence rates for each region shown in [*SI Appendix*, Fig. S9](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)) as a function of time are nearly identical in both clean and polluted trajectories. Two robust cloud responses are observed: 1) the diurnal cycle and 2) the influence of $AOD$. Cloud fraction typically increases at night through more efficient mixing of moisture throughout the boundary layer by stronger cloud top radiative cooling ([@r12]). This is displayed by the oscillatory pattern of cloud fraction as shown in [Fig. 3*A*](#fig03){ref-type="fig"}. Aerosol is also correlated to an increase in cloud fraction. Cloud fraction increases at a faster rate in polluted trajectories as indicated by the steeper slope of cloud fraction over the first 20 h of the trajectory period. While we use a strict aerosol screening criteria ([*Trajectory Setup*](#s10){ref-type="sec"}), some influence of cloud contamination cannot be ruled out and could partially be responsible for the earlier formation of cloud at higher $AOD$. After this initial period of rapid cloud formation, the cloud fraction remains larger in the polluted trajectories throughout multiple diurnal cycles, albeit with smaller differences over time.
{#fig03}
Higher cloud fraction and $N_{d}$ in the trajectories forming in polluted conditions result in a significant increase in top of atmosphere (TOA) outgoing shortwave radiative flux ([Fig. 3*C*](#fig03){ref-type="fig"}). Higher cloud coverage in the polluted trajectories decreases the TOA outgoing long-wave radiative flux (by 5.2 W/$m^{2}$), but these decreases are much smaller compared with the outgoing shortwave flux (by 63.6 W/$m^{2}$). Therefore, radiative effects are based on shortwave flux changes in this paper. For deeper insight into cloud microphysical processes, we remove the nighttime portion of the diurnal cycle and focus on daily averages during daylight hours (9 to 16 local time) when the cloud optical property retrievals are most accurate from the satellite observations. $N_{d}$ and cloud fraction are significantly larger in the clouds along polluted trajectories in [Fig. 4](#fig04){ref-type="fig"}. Larger $N_{d}$ values in polluted trajectories are due to smaller cloud droplet effective radii (10% decrease) and larger cloud optical thicknesses (27.5% increase) on average. In addition, we observe some evidence for drizzle suppression in polluted clouds from the IMERG ([Fig. 4*F*](#fig04){ref-type="fig"}) and CloudSat ([*SI Appendix*, Fig. S16](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)) observations. The relatively heavier precipitation rates toward the end of day 3 may also explain the reduction in $N_{d}$ through wet deposition relative to day 2.
{#fig04}
This analysis has been extended to two additional stratocumulus cloud decks: those off the coasts of South America and southern Africa (e.g., [*SI Appendix*, Figs. S3--S8](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). In general, similar behavior is observed. $N_{d}$ and cloud fraction are significantly higher at larger $AOD$. However, *CTH* and liquid water path responses are either insignificant and/or reversed in these locations. Lower *CTH*s with larger liquid water path under absorbing aerosol layers off the coast of Africa have been observed ([@r22]), simulated ([@r15]), and generally hypothesized to be related to semidirect effects (increased stabilization and lowering of the PBL caused by heating of the atmosphere by absorbing aerosol above the boundary layer). When the lower tropospheric stability ($LTS$) is high, the cloud fraction response to increased $AOD$ is greatest across the three stratocumulus cloud decks on average ([Fig. 5](#fig05){ref-type="fig"}). We explore links to stability in greater detail in the following section.
{#fig05}
Global-Scale Analysis. {#s4}
----------------------
This section quantifies the role of lower troposphere stability in aerosol--cloud interactions using an ensemble of several hundred thousand trajectories across the global oceans ([*SI Appendix*, Fig. S10](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). Trajectories are not constrained by direction but rather, by $LTS$. Since $LTS$ primarily drives cloud fraction for marine stratus and cumulus cloud regimes ([@r13]), meteorological sorting is based on both its average value over the trajectory and its slope over time. Here, we select two initial starting values (16.5 $\pm$ 1.5 and 19.5 $\pm$ 1.5 K) for a range of slopes on the rate of change in $LTS$ over the trajectories (−2, −1, 1, 2 $\pm$ 1 K/d), thereby providing eight meteorological regimes. For reference, the median initial value and slope of the $LTS$ for the Baja Californian stratus region is 19.7 K and $- 1.5$ K/d, respectively.
Again, robust increases in cloud fraction ([*SI Appendix*, Fig. S11](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)) and $N_{d}$ ([*SI Appendix*, Fig. S12](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)) manifest in polluted trajectories, however, only under stable meteorological conditions. The dependence of $LTS$ on the cloud fraction response to increased $AOD$ has been observed in other studies ([@r23], [@r24]) with the caveat that some uncertainties involving aerosol humidification and increased cloud detection at high $AOD$ may drive an artificially strong $AOD$ cloud fraction response. The liquid water path response to increased $AOD$ has been shown to depend on the cloud base height ([@r25]), precipitation state (nonraining or raining), and meteorology ([@r23], [@r26]). As the atmosphere becomes more unstable, the PBL often becomes deeper ([@r27]) due to stronger updrafts. These updrafts produce more cumuliform-type clouds that may be more susceptible to an evaporation--entrainment feedback ([@r25]) if they are nonprecipitating, causing them to burn off and lead to decreases in liquid water path and cloud fraction ([@r6]) as aerosol concentrations increase. However, if the clouds are precipitating, an increase in aerosol concentration can lead to increases in liquid water path due to the suppression of drizzle ([@r23], [@r26]). As the atmospheric stability and capping inversion strength increase, the importance of collision--coalescence and of the evaporation--entrainment feedback weakens, and aerosol perturbations on decreasing water path become smaller ([@r26]). A drier free troposphere can also result in greater entrainment drying. Here, the mean relative humidity values at 850 hPa are about 20% lower in unstable trajectories, which may partially explain the lack of a strong decrease in cloud fraction and liquid water path in the unstable regime. Our results suggest that moistening by aerosol-induced drizzle suppression may be larger than the evaporation--entrainment feedback, thereby allowing the clouds to redistribute cloud water horizontally and increase cloud cover fraction under stable atmospheric conditions.
Total radiative forcing ([Eq. **3**](#eq3){ref-type="disp-formula"}) is calculated for each regime separately with mean values displayed in [Table 1](#t01){ref-type="table"} (and plotted in [*SI Appendix*, Fig. S13](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). Aerosols significantly increase cloud albedo and fraction particularly under stable atmospheric conditions during the early development of cloud formation along the trajectories. The Twomey effect comprises the bulk of the radiative forcing estimate consecutively for each day along the trajectories (over 50%) due to the large increase in $N_{d}$ ([*SI Appendix*, Fig. S12](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). The average radiative forcing attributed to aerosol--cloud interactions is $0.61 \pm 0.34$ W/$m^{2}$, which is in quantitative agreement with the estimates from other satellite-based studies ([@r26], [@r28]). Note that the total radiative effect is more than two times larger in the high-stability regimes partly owing to increases in cloud fraction compared with the low-stability regimes. Interestingly, the liquid water path response is weak and can be either negative or positive, which is in general agreement with several recent studies ([@r26], [@r28][@r29]--[@r30]). These observations support our assumption that strong temperature inversions limit dry air entrainment into the PBL, thereby enhancing the longevity of polluted clouds in stable meteorological regimes.
######
Total aerosol radiative forcing decomposed into Twomey, liquid water path, and cloud fraction components using [Eq. 3](#eq3){ref-type="disp-formula"} from thousands of trajectories initialized over global oceanic areas from $60^{○}$S to $60^{○}$N averaged over eight meteorological $LTS$ regimes
Forcing (W/$m^{2}$) Day 1 Day 2 Day 3
--------------------- ------------------- ------------------- -------------------
Twomey $- 0.34 \pm 0.28$ $- 0.42 \pm 0.31$ $- 0.61 \pm 0.41$
Liquid water path $- 0.06 \pm 0.03$ $+ 0.09 \pm 0.05$ $- 0.04 \pm 0.06$
Cloud fraction $- 0.25 \pm 0.08$ $- 0.08 \pm 0.03$ $- 0.08 \pm 0.03$
Total $- 0.66 \pm 0.30$ $- 0.42 \pm 0.31$ $- 0.76 \pm 0.42$
Cloud Lifetime Effect. {#s5}
----------------------
These results demonstrate that aerosols have a nonnegligible effect on cloud fraction in the stratus-to-cumulus transition zone. Do aerosols enhance cloud lifetime? We introduce two timescales to seek an answer to this question: 1) cloud-formation timescale as defined by the amount of time it takes to increase cloud fraction over a $1^{○} \times 1^{○}$ region from 0 to over 0.75 ($\tau_{f}$) and 2) cloud-persistence timescale defined as the amount of time the cloud fraction remains above 0.75 for a maximum period of 24 h ($\tau_{p}$). Several threshold values were examined; however, we selected these values as a means to optimize the total number of samples in this study. These conditions are met in approximately 80% of trajectories (i.e., cloud fraction remains below this threshold in the remaining cases). An example of a three-day trajectory off the coast of Africa in which the formation timescale is approximately 15 h (first occurrence where cloud fraction increases above 0.75) with 24-h persistence is displayed in [*SI Appendix*, Fig. S14](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental).
From the ensemble of trajectories off the coasts of Baja California, Chile, and Namibia, it takes approximately 20 h on average for cloud fraction to exceed 0.75 along trajectories ([Fig. 6*A*](#fig06){ref-type="fig"}). Interestingly, the cloud fraction threshold of 0.75 is reached roughly 5 h earlier when the atmosphere is polluted ($AOD > 0.10$) despite having similar meteorological conditions. An explanation for the shorter cloud-formation timescale may be due to faster destabilization and moistening of the dry boundary layer from enhanced long-wave radiative cooling rates of polluted clouds with larger droplet concentrations ([@r4]). However, larger values of $AOD$ may also contain more moisture commonly found in biomass burning plumes ([@r31]) that is missed in the European Center for Medium Range Weather Forecasts (ECMWF) Reanalysis (ERA) Interim (ERA-Interim) product or from missing cloud contamination in the satellite $AOD$ product despite the use of state-of-the-art methods ([@r32]) to reduce it ([*Trajectory Setup*](#s10){ref-type="sec"}).
{#fig06}
Regardless of the formation timescale, clouds also tend to live longer at higher $AOD$. [Fig. 6*B*](#fig06){ref-type="fig"} shows the histogram of the cloud-persistence timescale. Our results are broadly consistent with the Lagrangian decorrelation timescales described in Eastman et al. ([@r33]). On average, the cloud-persistence timescale is 2 h longer under polluted conditions. The differences were found to be significant at the 95th percentile using a two-tailed *t* test. We conclude that the larger cloud fraction associated with the polluted trajectories may manifest from the longer cloud lifetimes compared with unpolluted clouds.
This analysis has been extended to all regions and meteorological composites from the trajectories spanning the global oceans ([*SI Appendix*, Fig. S15](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). Slight differences in the timescales are observed between stratocumulus basins (e.g., polluted clouds live longer by about 1 h in Namibia compared with Baja California). The differences may be attributed to stronger shortwave heating by absorbing aerosols and slightly larger $LTS$ and amounts of planetary boundary-layer moisture for the polluted conditions in the Namibia region. In general, aerosols increase cloud lifetime, with the largest increases under stable atmospheric conditions in the main stratus-to-cumulus transition zones.
Conclusions {#s6}
===========
This study quantifies aerosol radiative forcing using a combination of geostationary satellite observations and Lagrangian trajectories. Polluted clouds are found to have larger cloud fraction, lifetime, and droplet concentrations under stable atmospheric conditions. These responses are robust across three separate oceanic basins and in several thousand trajectories stratified by gradients in lower troposphere stability, a meteorological variable that is a controlling factor on cloud fraction ([@r11], [@r13]). Evidently, clouds forming in high $AOD$ conditions have markedly larger $N_{d}$ under stable atmospheric conditions. These perturbations persist multiple days and lead to a significant radiative forcing-based estimate that has recently been suggested for warm boundary-layer clouds ([@r34]). However, the radiative forcing estimate in unstable atmospheric conditions is markedly smaller by comparison, presumably due to positive feedbacks between dry free-tropospheric air and entrainment ([@r4], [@r6]).
Large eddy simulation experiments following Lagrangian trajectories could be a useful tool to examine why cloud fraction exceeds a threshold of 0.75 earlier under high $AOD$ conditions along Lagrangian trajectories. We propose two hypotheses based on physical mechanisms: 1) the enhanced radiative cooling rates from polluted shallow clouds destabilize and moisten the PBL more rapidly than unpolluted clouds if the air above the boundary layer is sufficiently moist ([@r4]); 2) biomass burning plumes may increase boundary-layer humidity in addition to $AOD$ due to their moisture content ([@r31]), and this increase in humidity may be missing from the ECMWF ERA-Interim data or from the evaluation of cloud contamination despite efforts to prevent this ([@r32]) ([*Trajectory Setup*](#s10){ref-type="sec"}). Another possibility is the increase in subpixel cloud albedo may trigger earlier overcast cloudy pixel detection in the MODIS retrieval algorithm ([@r19]) than optically thinner clouds forming in unpolluted conditions.
Solar-absorbing aerosol layers above the boundary layer may result in semidirect effects along some of the trajectories in this study. Observations from NASA's Cloud Aerosol Transport System 1,064-nm lidar indicate that about 60% of the above-cloud aerosols off the coast of Africa are found within 360 m to the top of the underlying cloud ([@r35]). The sensitivity of low cloud cover change to elevated aerosols layers increases as the cloud--aerosol gap decreases ([@r31]). Furthermore, aerosol layers observed above the cloud top have less of an influence on decreasing cloud droplet size compared with when the aerosol--cloud layers touch ([@r36]). However, despite these potential semidirect effects off the coast of Africa, the general cloud responses to increased $AOD$ are remarkably consistent in all three regions examined in this study.
The stratus-to-cumulus transition is not, to first order, driven by changes in precipitation and aerosol concentration but by slowly varying gradients in sea surface temperature and large-scale subsidence ([@r13]). On average we find that aerosols increase cloud fraction along Lagrangian trajectories in stable atmospheric conditions. The extent to whether aerosols influence cloud morphology along the classic stratus-to-cumulus transition zone remains an open question. This transition zone contains a rich variety of cloud regimes (for example, open and closed cellular clouds displayed in [Fig. 1](#fig01){ref-type="fig"}) that manifest through rapidly changing processes like precipitation. Thermodynamic variables such as mixing ratio, cloud base height, and precipitation adjust on fast timescales (typically about 12 h) compared with the timescale of the inversion height that is set primarily by large-scale divergence (2 to 5 d) ([@r37]), which can take place gradually over the stratus-to-cumulus transition zone. It has been hypothesized that suppressing precipitation can transform broken open cellular cloud decks along this transition zone into closed overcast clouds. Such cloud regime transitions lead to significant local cloud radiative effects ([@r38]) and hence, may be critical for the development and maintenance of the stratus-to-cumulus transition. Future work linking Lagrangian trajectories to higher-resolution satellite imagery and cloud classification \[e.g., based on machine learning techniques ([@r39])\] may provide deeper clues to the role of aerosol on the stratus-to-cumulus transition and climate change.
Materials and Methods {#s7}
=====================
Data. {#s8}
-----
The CERES SYN Ed 4 product provides global temporally and spatially resolved aerosol, cloud, and radiation products. This product condenses petabytes of geostationary satellite data into a user-friendly gridded format ($1^{○}$ latitude by $1^{○}$ longitude resolved hourly) over the 2003 to 2018 period. The product is well suited to study the diurnal cycle of cloud properties and radiative fluxes in diverse locations over land and ocean ([@r16], [@r40]). This product uses 16 geostationary satellites. To ensure consistency across the various satellite sensors, cross-calibration of the instrument channels is tuned to match the cloud optical property retrievals from the MODIS collection 5 product ([@r41]). Top and bottom of atmosphere broadband radiative fluxes are calculated using the Langley Fu-Liou radiative transfer model using MODIS-assimilated aerosol retrievals in the Model for Atmospheric Transport and Chemistry and meteorological data from the Goddard Earth Observing System. Broadband radiative fluxes are normalized against the single-scanning footprint observations from CERES data acquired from Terra and Aqua satellites. The Langley Fu-Liou Ed. 4 model provides temporally resolved fluxes, which are used in the CERES Energy Balanced and Filled products ([@r42]). The monthly mean uncertainties are based on 37 land and 48 ocean buoy locations; the surface net shortwave and long-wave fluxes are 5.7 and 2.9%, respectively ([@r16]).
High-spatial resolution geostationary imagery data (3 km at the subsatellite point with a repeat cycle of 15 min) from SEVIRI on the Meteosat Second Generation satellite with 12 spectral channels (0.64, 0.84, 1.6, 3.9, 6.2, 7.3, 8.7, 9.7, 10.8, 12.0, 13, and high resolution visible broadband 0.4--1.1 $\mu$m) are used to retrieve cloud properties from the NASA Langley cloud retrieval for the ObseRvations of Aerosols above Clouds and their intEractionS (ORACLES) campaign. This algorithm uses the same architecture of the CERES SYN products. Due to limitations in data volume, we could only process the period during the ORACLES campaign to study the evolution of a POC (described by [Fig. 1](#fig01){ref-type="fig"}).
MODIS onboard Terra and Aqua satellites contain 36 spectral channels. The Terra satellite is in a descending node of the orbit crossing the equator at 10:30 and 22:30 local time. Similarly, Aqua is in an ascending node crossing the equator at 13:30 and 01:30 local time. Cloud and aerosol optical properties (cloud optical thickness, cloud effective radius) are provided twice per day in the sunlit part of the orbit, while *CTH*, pressure, and fraction are provided at nighttime too in the collection 6.1 products.
Precipitation is obtained from the IMERG V06B product, which combines passive microwave measurements from the Global Precipitation Measurement mission, observations from polar orbiting Advanced Microwave Scanning Radiometers, brightness temperature measurements from multiple satellites in geostationary orbit, and rain gauge data. The gridded product (0.$25^{○}$) provides precipitation retrievals every half hour on a 0.$1^{○}$ grid from 2015 to 2018, making it ideal to quantify precipitation changes along trajectories.
The narrow swath (effective footprint of $1.4 \times 1.8$ km) and limited temporal sampling of the w-band CloudSat cloud-profiling radar do not permit a detailed examination of precipitation in a Lagrangian framework. However, we use it here to quantify precipitation responses at 1:30 PM (only daytime observations are available in this study due to a battery failure in 2011) from the 2C-Column-Precip product ([@r43]). While CloudSat is more sensitive to detecting light precipitation (minimum detectable signal is −28 dBZ) compared with passive radars that make up the IMERG product (minimum detectable signal is approximately +15 dBZ), [*SI Appendix*, Fig. S16](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental) shows supporting evidence that less frequent occurrence of precipitation is identified under polluted conditions ($AOD > 0.1$) in both datasets.
Meteorological variables from the ECMWF ERA-Interim products are collocated to each trajectory; these include sea surface temperature, planetary boundary-layer specific humidity, and lower troposphere static stability ($LTS = \Theta_{\mathit{P700}} - \Theta_{Ps}$, where $\Theta_{\mathit{P700}}$ and $\Theta_{Ps}$ are the potential temperatures at 700 hPa and surface pressure levels, respectively).
Trajectory Model. {#s9}
-----------------
Trajectories are calculated using HYSPLIT ([@r44]). The trajectory model uses Air Resources Laboratory-generated Global Data Assimilation System (GDAS) meteorological data. GDAS outputs analysis time steps six times daily at 0.$5^{○}$ horizontal spatial resolution with 55 hybrid sigma-pressure levels. Each forward trajectory begins at 13:30 local time to coincide with the multisensor observations from the A-Train. Forward trajectories are run for a duration of 80 h to represent the average amount of time required to transition from stratus to cumulus ([@r13]).
To ensure that trajectories follow the mean motions of the PBL, they are initialized in the middle of the PBL (determined by the thermodynamic sounding) and constrained to flow along an isobaric surface to avoid escaping into the free troposphere. The HYSPLIT model as shown in [Fig. 1](#fig01){ref-type="fig"} ([Movie S1](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)) shows its seamless ability to track boundary-layer clouds over multiple days. The depth of the PBL is determined within HYSPLIT using the profiles of temperature, humidity, and wind velocity ([@r44]). Uncertainties associated with the particle trajectory positions are typically larger near converging winds and/or frontal systems. The uncertainty scales approximately 20% with distance from origin. Hence, for an 80-h period covering roughly 1,500 km along relatively stable subtropical atmospheric conditions, we estimate that the maximum displacements due to particle trajectory uncertainty are about 300 km in this study.
Trajectory Setup. {#s10}
-----------------
All forward trajectories are initialized over the ocean in clear-sky conditions as determined by the high-confidence cloud mask flag from the Aqua MODIS collection 6.1 (MYD08) product. Initializing in clear-sky conditions provides 1) an accurate baseline *AOD*, 2) known history of the aerosol-containing air mass prior to cloud formation, 3) a baseline cloud radiative effect for quantifying cloud system evolution, 4) a starting point to track the initial development and timescale of cloud formation and dissipation, and 5) ability to sort between clean and polluted trajectories. The clear-sky retrievals are defined by cloud fraction (over a $1^{○} \times 1^{○}$ degree region) being less than 5% and intercloud spacing being greater than at least 15 km. This criterion is established with the aim to reduce retrieval errors in high-cloud fraction scenes related to aerosol humidification from swelling near cloud edges, aerosol brightening due to 3D scattering from the sides of nearby clouds, and retrieval artifacts due to the presence of cloud contamination ([@r32]). The $AOD$ typically decreases away from cloud until leveling off at a length scale of approximately 15 km. The usage of potentially contaminated $AOD$ retrievals located within 15 km from nearby cloud significantly enhances the aerosol indirect effect ([@r32]). However, the gridded daily level 3 MODIS *AOD* product can be corrected following the above criteria to yield the statistically similar radiative forcing-based estimates compared with using high-resolution level 2 cloud and aerosol paired pixels. Therefore, our analysis restricts the use of gridded $AOD$ estimates in regions largely devoid of clouds and over dark ocean surfaces to increase the accuracy of the $AOD$ through limiting the possibility of cloud contamination and humidification. It is noteworthy that sometimes a significant portion of the $AOD$ can reside above the boundary layer, particularly for the Namibia region, and thereby contribute to greater uncertainty in the classification of clean and polluted conditions. Overall, these initial conditions provide the unique ability to link the history of the aerosol to the developing cloud system. Only the warm-cloud process is considered here; therefore, forward trajectories are rejected if they contain any multilayer cloud layers, ice cloud, or high-level cloud (cloud top pressure less than 500 hPa).
Hydroscopic growth results in greater attenuation of the incoming solar radiation than for the same number concentration of dry aerosol particles. To ensure that the clean and polluted trajectories are not influenced by the planetary boundary-layer humidity, we have examined the $AOD$ based on dry conditions (relative humidity =30%) using the Copernicus Atmosphere Monitoring Service (CAMS) reanalysis product. Mixing ratios of each aerosol constituent (CAMS aerosol composition variables: sea salt, dust, organic matter, black carbon, and sulfate) is provided for 25 vertical levels. Column integrated dry-mode *AOD* is calculated following $\tau = \sum_{n = 1}^{N}\int_{p_{s}}^{0}\beta_{e}r\left( p \right)dp/g$, where $N$ is the total number of aerosol species, $\beta_{e}$ (meters^2^ per gram) is the dry-mass extinction coefficient for each aerosol type using the look tables described in Benedetti et al. ([@r45]), $r\left( p \right)$ (grams per kilogram) is the mass mixing ratio of the aerosol species at each vertical level, $g$ (meters per second^2^) is the constant of gravity, $dp$ is the pressure of the model level, and $p_{s}$ is the surface pressure. [*SI Appendix*, Fig. S17](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental) shows a comparison between CAMS $AOD$ and CAMS dry-mass $AOD$ for the composite of trajectories initialized off the coast of California. On average, dry-mass $AOD$ is approximately 30% smaller than the assimilated $AOD$ due to smaller mass extinction coefficients and less hydroscopic growth when the air is dry. However, the strong correlation coefficient ($r^{2} = 0.91$) between dry and wet *AOD* implies that humidification effects are essentially linear and roughly the same between clean and polluted conditions as determined by MODIS satellite observations. We have taken this analysis a step further by using dry *AOD* as the proxy for our aerosol-cloud interaction metrics (instead of MODIS) and find similar results.
### Radiative Forcing. {#s11}
Aerosol radiative forcing is calculated based on the method following Quaas et al. ([@r46]) in which the forcing is decomposed into a Twomey effect and adjustments to Twomey are based on changes in liquid water path and cloud fraction associated with an increase in *AOD*. The CERES SYN product is used to calculate terms in the following equation:$$\frac{\Delta\alpha}{\Delta\ln AOD} = C\Delta\alpha_{c}\left( {1 - \alpha_{c}} \right)\frac{\Delta\ln N_{d}}{\Delta\ln AOD}\left( {\frac{1}{3} + \frac{5}{6}\frac{\Delta\ln L}{\Delta\ln N_{d}} + \frac{\Delta\ln C}{\Delta\ln N_{d}}} \right),$$where $C$ is the cloud fraction over the spatial domain, $\alpha_{c}$ is the average cloud albedo, $N_{d}$ is the cloud droplet concentration, $L$ is the liquid cloud water path, and $AOD$ is the aerosol optical thickness. Liquid water path is calculated using $L = 2/3\rho_{l}r_{e}\tau_{c}$ ([@r47]), where $r_{e}$ is the satellite-retrieved droplet effective radius and $\tau_{c}$ is the cloud optical thickness. The Twomey, liquid water path, and cloud fraction radiative effect contributions are shown as the first three terms in parentheses of [Eq. **1**](#eq1){ref-type="disp-formula"}. The $N_{d}$ is calculated as$$N_{d} = \gamma\sqrt{\tau_{c}}r_{e}^{- 2.5},$$where $\gamma = 1.37e - 5$ $m^{- 0.5}$ is related to the adiabatic condensation growth rate and adiabaticity factor typical for stratocumulus clouds. Grosvenor et al. ([@r47]) can be consulted for a comprehensive assessment of the uncertainties. Cloud property differences are calculated from the population of polluted and clean trajectories. Composites are based on the $AOD$ at the start of each trajectory within the clear-sky footprint of the satellite sensor. A value of 0.10 was chosen as it is the median value of the global $AOD$ distribution ([*SI Appendix*, Fig. S2](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental)). Our estimate of aerosol radiative forcing is calculated by multiplying [Eq. **1**](#eq1){ref-type="disp-formula"} with the incoming solar radiation and anthropogenic *AOD* following the method described in ref. [@r48] as$$\Delta F_{\mathit{TOA}} = - F^{\downarrow}\frac{\Delta\alpha}{\Delta\ln AOD}\ln AOD_{\mathit{anth}},$$where $F^{\downarrow}$ is the incoming solar radiative flux at the TOA is 340.2 W/$m^{2}$ ([@r49]) and $AOD_{\mathit{anth}}$ is the *AOD* attributed to anthropogenic activities. $AOD_{\mathit{anth}}$ is obtained from the ECMWF Monitoring Atmospheric Composition and Climate (MACC) II product. Natural anthropogenic aerosol contributions are provided by the integrated forecast system model constrained by satellite-retrieved $AOD$ at a wavelength of 0.55 $\mu$m from MODIS. On average, this leads to an anthropogenic aerosol fraction of approximately 20% globally (for example, see ref. [@r48]) ([Fig. 2](#fig02){ref-type="fig"}). Since MACC-II was only produced from 2003 to 2012, we use an annual climatology for this period to represent the spatially averaged anthropogenic aerosol concentration.
Data and Code Availability. {#s12}
---------------------------
CERES SYN Ed4a 4 product is available at <https://ceres.larc.nasa.gov>. MODIS collection 6 MYD08_D3 product is available at <https://earthdata.nasa.gov>. IMERG data are available from the NASA Goddard Space Flight Center (<https://pmm.nasa.gov>). CloudSat data are available from the Cooperative Institute for Research in the Atmosphere (<http://www.cloudsat.cira.colostate.edu>). ECMWF data were obtained from <https://www.ecmwf.int>. HYSPLIT trajectory code is available at <https://www.ready.noaa.gov/HYSPLIT.php>. All data and code availability websites were last accessed on 3 October 2020.
Supplementary Material
======================
This research was supported by European Research Council Project constRaining the EffeCts of Aerosols on Precipitation under the European Union's Horizon 2020 Research and Innovation Program Grant 724602. P.S. additionally acknowledges support from UK Natural Environment Research Council CLouds-Aerosol-Radiation Interactions and Forcing Project NE/L013479/1 and from the Alexander von Humboldt Foundation. We thank Paquita Zuidema for providing NASA ORACLES data and David Painemal for advice on using the CERES SYN product. The Centre for Environmental Data Analysis provided the computational infrastructure needed to process these data.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at <https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921231117/-/DCSupplemental>.
[^1]: Edited by John H. Seinfeld, California Institute of Technology, Pasadena, CA, and approved June 5, 2020 (received for review December 4, 2019)
[^2]: Author contributions: M.W.C., W.K.J., and P.S. designed research; M.W.C. performed research; M.W.C. and W.K.J. contributed new analytic tools; M.W.C. analyzed data; and M.W.C. wrote the paper with contributions from W.K.J. and P.S.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Rheumatoid arthritis (RA) is an immunologically driven, progressive, long-term condition. It is characterised by persistent synovitis, systemic inflammation and detectable autoantibodies, including rheumatoid factor and anti-cyclic citrullinated peptide antibody \[[@CR1]\]. Ongoing joint inflammation damages cartilage, bone and tendons; systemic inflammation causes extra-articular complications like vasculitis and lung disease. Uncontrolled active RA leads to disability, decreased quality of life (QoL) and increased co-morbidity. The end result is loss of work, major medical and social costs and high morbidity and mortality \[[@CR2], [@CR3]\].
RA management involves a multidisciplinary team including rheumatologists, specialist nurses, therapists and others. The team provide education, particularly on self-management, medication, psychological support, exercise and joint protection \[[@CR4]\]. RA outcomes are optimised by treating patients to pre-defined targets \[[@CR5]--[@CR7]\]; the most appropriate target is remission.
Drug treatment focusses on controlling joint inflammation with disease-modifying anti-rheumatic drugs (DMARDs). They reduce synovitis, systemic inflammation and disability. The dominant DMARD is methotrexate; others include sulfasalazine and leflunomide \[[@CR8]\]. The impact of DMARDs can be maximised by using them in combination. However, side effects limit DMARD use by both clinicians and patients \[[@CR4]\]. Steroids (glucocorticoids) also reduce joint inflammation \[[@CR9]\]. In the short-term steroids can be combined with DMARDs to reduce erosions and to treat systemic disease. Long-term steroid use has unacceptable toxicity \[[@CR10]\].
Biological agents, given when DMARDs cannot control RA, have revolutionised its management. Biologics such as tumour necrosis factor inhibitors (TNFi), rituximab, abatacept and tocilizumab are highly effective \[[@CR11]\]. They are mainly given with methotrexate to increase efficacy and reduce blocking antibodies \[[@CR12]\]. Their main risk is infection \[[@CR13]\] and use is limited by their high costs \[[@CR14]\]. Although they substantially improve RA outcome, they do not cure the disease.
RA patients are distinguished into categories according to their disease activity levels. This is currently undertaken on the basis of the Disease Activity Score for 28 joints (DAS28), which is a composite measure including assessment of tender and swollen joint (based on 28 joints), the erythrocyte sedimentation rate (ESR) and patient global assessments on a 100-mm Visual Analogue Scale (VAS) \[[@CR15]\].
DAS28 scores divide patients with established RA into four categories. These are as follows:High disease activity (DAS28 over 5.1)Intermediate (or moderate) disease activity (DAS28 over 3.2 to 5.1)Low disease activity (DAS28 2.6 to 3.2)Remission (DAS28 under 2.6)
Randomised clinical trials (RCTs) enrol patients with RA who have high disease activity. There is strong evidence from RCTs that patients with RA with high disease activity benefit from treatment with DMARDs and biologics. Remission or low disease activity are the goals of treatment. This approach, termed 'treat to target' \[[@CR16]\], is supported by a strong evidence base \[[@CR17], [@CR18]\]. When patients achieve sustained low disease activity or remission, most clinicians either maintain treatment or reduce treatment levels. However, many RA patients currently attending rheumatology clinics have intermediate (or moderate) disease activity levels \[[@CR19]\]. There is only limited evidence that intensive treatment strategies benefit such patients and there is uncertainty about how best to treat them. One key reason for these doubts is that such patients are not usually included within RCTs \[[@CR19]--[@CR22]\].
There are a number of clinical guidelines that summarise how best to manage patients with RA \[[@CR23]--[@CR25]\]. Those by the National Institute for Health and Clinical Excellence (NICE) are the most relevant for clinical practice in England \[[@CR25]\]. They make some general recommendations on the current management of patients with intermediate disease, which can be summarised as follows:Maintain suppressive treatment with DMARDs and steroidsMaintain symptomatic therapy (analgesics/non-steroidal anti-inflammatory drugs)Carry out annual specialist reviews, with urgent specialist reviews and treatment modification for flares (DAS28 over 5.1) or clinically significant adverse events
There has been discussion about the relative merits of giving treatments, such as biologics, to patients with intermediate disease activity, but no nationally agreed protocols exist in England. There is substantial international variation on the use of biological treatments and in some European countries and in North America, patients with intermediate disease activity are often prescribed biological treatment. The absence of any agreed treatment protocols for patients with intermediate disease activity is a major challenge in defining the most appropriate way to treat many RA patients attending specialist clinics in England \[[@CR25]\].
A number of strategy trials have shown the benefits of combining treatments -- DMARDs, steroids and, in some trials, biologics -- to optimise outcomes, and have confirmed the benefit of treating to target, where patients are treated until they reach the therapeutic target of remission or low disease activity \[[@CR16]\]. 'Intensive management' (IM) approaches used in these strategy trials give the best outcomes for RA so far reported \[[@CR17], [@CR18]\].
A characteristic feature of RA trials is their focus on increasing treatment in patients with high disease activity. One reason is that such patients have pressing needs to justify treatment change. A second is the relative ease of showing treatment benefit in patients with high disease activity. As a consequence, trials rarely enrol patients with intermediate disease activity \[[@CR19]--[@CR22]\]. A common feature of patients with intermediate disease is the duration of their RA and multiple historical treatment changes that have often been made to achieve acceptable levels of disease activity for the patient. Recruiting intermediate patients to a medication optimisation trial would require additional support components that would result in patients taking medications that they may have perceived to have been previously unsuccessful. Therefore, optimising treatment in intermediate disease requires components that would address knowledge, motivation and shared decision-making \[[@CR26]\].
Medication can alleviate symptoms and halt disease progression in rheumatoid arthritis (RA). Nevertheless, medication non-adherence is common in RA and poses a significant barrier to improving clinical outcomes in RA with only 58--82% of RA patients adhering to DMARDs \[[@CR27]\]. Factors that can influence adherence include patients' negative beliefs about medicines and their condition, as well as their degree of satisfaction with information about DMARDs \[[@CR28], [@CR29]\]. Similarly, adherence to biologic treatments, such as adalimumab, is driven by psychological factors, particularly medication beliefs \[[@CR30]\].
In addition to the impact that RA has on joints and physical disability, it also significantly affects QoL \[[@CR31]\]. Fatigue is reported in over 80% of RA patients \[[@CR32]--[@CR34]\] and 57% of RA patients identify fatigue as the most problematic symptom of their condition \[[@CR35]\]. Disease activity might not be the sole factor exerting a significant impact on fatigue; it may also result from a constellation of factors that include disease activity or pain, inactivity, depression, obesity and poor sleep \[[@CR36]\]. Due to the pervasive effect of RA on patient outcomes and its impact on health status and QoL, it was deemed crucial to incorporate a component of 'psychosocial support' in the intervention. 'Psychosocial support' aimed to address the challenging domains of coping with RA including medication adherence by influencing behaviour change.
Against this background, the key reasons for undertaking the TITRATE trial are:Remission is the most appropriate target in RA \[[@CR37]\]Intensive management regimens using DMARD combinations, steroids and sometimes biologics, together with a 'treatment support' programme of effective non-drug interventions and psychosocial support for coping with the various domains of RA are most likely to achieve remission \[[@CR38], [@CR39]\]The most important group of patients in which to investigate whether intensive management achieves remission are those with 'intermediate disease activity'; these patients currently continue to have persisting disease activity and as a consequence they develop progressive disability \[[@CR40]\]
The TITRATE trial is designed to show whether patients with intermediate disease activity benefit from such intensive management.
As a consequence of these considerations the trial will enrol patients with intermediately active RA defined by their DAS28 scores. The intervention will be intensive management with DMARDs and biologics given in a supportive manner agreed with individual patients. The control group will receive standard care following existing national guidance. The
primary outcome will be remission at 12 months assessed using DAS28. The hypothesis is that intensive management will increase the number of patients in DAS28 remission at 12 months compared with SC.
Methods {#Sec2}
=======
Aim and hypothesis {#Sec3}
------------------
TITRATE is a robust pragmatic clinical trial to improve outcomes for RA patients with intermediate disease activity by using an Intensive Management Programme. The trial will test the hypothesis that patients with established RA, who currently have intermediate disease activity (defined as DAS28-ESR of 3.2--5.1 with at least three active joints) and are currently receiving at least one DMARD, are more likely to achieve remission at 12 months if they receive intensive management than if they continue to have SC.
The primary objective is to improve outcomes defined through achieving remission at 12 months for RA patients with intermediate disease activity using intensive management.
The secondary objectives are as follows:
### Clinical outcomes {#Sec4}
To assess disability with the Health Assessment Questionnaire (HAQ)To determine the relative effect on quality of life (QoL)To assess acceptability of intensive management to RA patients with intermediate disease activityTo assess the risks of adverse events from intensive management
### Economic outcome {#Sec5}
To determine the cost-effectiveness of intensive management
Design {#Sec6}
------
The TITRATE trial is a 12-month, pragmatic, randomised, open-label, two-arm, parallel-group, multicentre trial undertaken at 35--40 specialist rheumatology clinics across England. The trial was designed by key stakeholders including rheumatologists with experience of treating RA, specialist nurses, GPs with a special interest in musculoskeletal disease, methodologists and two service users, who were patients with personal experiences of living with RA. The study design outlining the intensive management approach and standard treatment is shown in Fig. [1](#Fig1){ref-type="fig"}. A Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist is provided as Additional file [1](#MOESM1){ref-type="media"}, and a flow diagram is included as Fig. [2](#Fig2){ref-type="fig"}.Fig. 1Study design: outline of intensive management approach and standard treatment arms Fig. 2Standard Protocol Items Recommendations for Interventional Trials (SPIRIT) Schedule of enrolment, interventions and assessments
The trial assesses a treatment strategy rather than any particular drug therapy and all treatments will be used within their current marketing authorisation. Consequently, the Medicines and Healthcare Products Regulatory Agency decided that the trial did not meet their criteria for a Clinical Trial of an Investigational Medicinal Product (CTIMP) and that TITRATE was a non-CTIMP trial.
Ethical approval for this trial was obtained from the London -- West London and GTAC National Research Ethics Service (NRES) Committee.
Setting {#Sec7}
-------
The trial will be undertaken within routine rheumatology outpatient clinics in approximately 40 hospitals in England. A full list of participating centres is available at <https://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/rheumatology/research/clinical/current/titrate/introduction.aspx>.
Target population {#Sec8}
-----------------
The target population will be patients attending specialist rheumatology clinics who meet the most recent classification criteria for RA (American College of Rheumatology 2010 criteria); have established RA; currently have intermediate disease activity (DAS28-ESR of 3.2--5.1 and at least three active joints) and are receiving at least one conventional DMARD. Participants will need to meet the following eligibility criteria:
### Inclusion criteria {#Sec9}
Men and women aged over 18 yearsDiagnosis of RA (by American College of Rheumatology (ACR), 2010 criteria) \[[@CR41]\]Have received at least one DMARD for at least 6 months, and currently receiving at least one DMARDHave intermediate disease activity, defined by: (a) DAS28-ESR of 3.2--5.1; (b) at least three active joints (defined as swollen and/or tender) on 66/68 joint count, to include at least one swollen jointWilling and able to follow an Intensive Management ProgrammeAble and willing to give informed consent
### Exclusion criteria {#Sec10}
Major co-morbidities making intensive treatment inadvisable (e.g. heart failure)Previously failed multiple DMARDs (at least five treatments) or having received biologicsIrreversible disability from extensive joint damage (e.g. replacement of three or more major joints)Women who are pregnant, breast-feeding or at risk of conceivingCurrent or recent (within the 12 weeks prior to randomisation) participation in another interventional trialCurrently in an early RA pathway, which is a 12-month treatment programme for patients with early inflammatory arthritis, in which patients receive intensive treatment with DMARD combinations and steroids and are reviewed monthly by specialist nurses or equivalent members of the rheumatology team; this is one of the current NHS Best Practice Tariffs \[[@CR42]\]
Interventions {#Sec11}
-------------
### Standard care {#Sec12}
In the standard care arm, clinicians will follow their local pathways for managing RA patients with intermediate disease activity. These will be based on national guidance from NICE. The key components of the standard care algorithm comprise:Maintaining suppressive treatment with DMARDs and steroidsSeeing patients at least once each year in line with local pathways of careArranging urgent specialist reviews using routine approaches if there be a clinical need
### Intensive management {#Sec13}
In the intensive management arm, patients will be seen monthly by trained health practitioners, who will often, but not always, be a nurse identified by the principal investigator as being competent to provide the intervention, who will:Assess their RA and current general functioningEvaluate their drug treatmentModify the drug treatment according to a decision tool (or algorithm) in line with a 'shared treatment plan' formulated during the first visit. Shared treatment plans will involve agreements with patients about drugs, dosages and therapeutic sequences. Templates for potential shared treatment plans were developed with patients in advance of the trial as part of a preparatory study within the TITRATE programmeProvide supportive care according to the principles, knowledge and skills acquired in the training course which are underpinned by Motivational Interviewing techniques and are included in the Treatment Support Manual
The Intensive Management Programme will address the following:Provide information: there will be a handbook for patients \[[@CR43]\] which will describe in detail the various aspects of intensive treatment as well as information on psychoeducation such as details of medication side effects, ways of coping with the physical and emotional symptoms and the impact of RA on everyday life, contact details for support groups, and disability informationOptimise DMARDs/biologics: drug treatment will be modified following a treatment algorithm, which will recommend treatment options based on previous treatment, present treatment, contraindications, the patient's preferences and clinical assessments. The premise of the treatment algorithm is that, if the patient's disease is still active, whatever the current treatment strategy, the recommended strategy will always reflect potential intensification of treatment. All medication given to patients in the intensive management group will be in line with national guidance from NICE or the national specialist society (British Society for Rheumatology) \[[@CR44]\] the only differences being that patients will be reviewed more frequently than they would be under standard care and if their disease is not fully controlled may be given biological therapies in line with British Society for Rheumatology recommendations. Local safety screening for tuberculosis and other infections will be performed according to local guidelines for patients in the intensive management arm prior to starting biologics (if needed) in line with British Society for Rheumatology guidanceGive steroids: patients will receive intra-muscularly administered steroids (depomedrone or equivalent) if their arthritis is not fully controlled up to a maximum of 600 mg depomedrone (or equivalent) over the 12-month period. The dose of steroids given will range from 40 to 120 mg depending on specific clinical circumstancesProvide treatment support: along with treating patients more intensively with medication, the trained rheumatology practitioner will also provide patients in this group with supportive care. Patients will be educated and supported in a number of domains commonly affected by RA, with a particular focus on: pain and fatigue management; physical activity; medication adherence, sleep and low mood/anxiety. Patients will also have a handbook, co-developed with patients and national charities, which includes psychoeducation about their condition, treatment options and ways of coping with the physical and emotional symptoms
All specialist rheumatology practitioners involved in delivering the intensive management intervention will be trained according to a manualised training programme to ensure that the intervention provided is standardised across sites and participants. The training involves a 2-day course, with day 1 focussing solely on motivational interviewing techniques; specifically, open-ended questions, affirmations, reflections/reflective listening and summaries and the treatment algorithm for intensive management. The second day focusses on psychoeducation regarding various aspects of RA including preparation for behaviour change, goal-setting, diary keeping, self-monitoring, development of a shared treatment plan, standardisation of the DAS28 scoring process, explanation of the process of recording the sessions and the supervision that will be available to practitioners \[[@CR26]\]. To measure adherence to the intensive management intervention across sites, monthly sessions will be audiotaped and a randomly selected subset of these rated by a team of three independent assessors.
Assessments {#Sec14}
-----------
Assessments will be carried out by a member of the research team at each participating centre. Following consent and confirmation of eligibility, all patients will complete an initial baseline assessment. This will be followed by a midpoint and final assessment at 6 and 12 months from baseline, respectively. See Table [1](#Tab1){ref-type="table"} for a summary of the milestone research assessments.Table 1Summary of milestone assessmentsAssessmentBaselineMonth 6 (midpoint)Month 12 (final)Withdrawal^a^Extension study^b^DAS28 (ESR) and Extended Joint CountXXXXXCRPXXXXXAssessor global rating (VAS)XXXXXPain rating (VAS)XXXXXFatigue rating (VAS)XXXXXRA medicationXXXXXMedical historyXAlcohol consumptionXSmoking history/statusXXXXX-rays (plain, of hands and feet)XXXHAQXXXXXCSRIXXXXEQ-5D-5 LXXXXXPHQ-9XGAD-7XMARSXXXXBIPQXBMQXViews of RA treatmentXXXAdverse eventsXXXXDAS28-ESR will be calculated from joint count, patient global rating (VAS) and ESR during every visit; SDAI will be calculated from joint count, patient global rating (VAS), assessor global rating (VAS) and CRP at baseline, midpoint and final assessments^a^To be completed at any time during the trial if the patient withdraws. ^b^Extension study only*BIPQ* Brief Illness Perceptions Questionnaire, *BMQ* Beliefs about Medicines Questionnaire, *CRP* C-reactive protein*, CSRI* Modified Client Service Receipt Inventory, *EQ-5D-5 L* EuroQol 5-dimensional 5-level score, *ESR* erythrocyte sedimentation rate, *GAD-7* Generalized Anxiety disorder-7, *HAQ* Health Assessment Questionnaire, *MARS* Medication Adherence Rating Scale, *PHQ-9* Patient Health Questionnaire-9, *RA* rheumatoid arthritis, *SDAI* Simplified Disease Activity Index, *VAS* Visual Analogue Scale
### Primary outcome measure {#Sec15}
The primary outcome will be the number of patients in each treatment arm fulfilling the definition of remission as measured by DAS28-ESR (remission defined as DAS28-ESR \< 2.6) \[[@CR45], [@CR46]\] at 12 months.
### Secondary outcome measures {#Sec16}
These will assess the following outcomes at 6 and/or 12 months:*Alternative assessments of remission*: remission measured by the Disease Activity Score for 28 joints-C-reactive protein score (DAS28-CRP) \[[@CR46], [@CR47]\] the Simplified Disease Activity Index (SDAI) (remission defined as SDAI ≤ 3.3) \[[@CR48]\] at 12 months; remission assessed by all measures at 6 months*Assessment of individual components of remission*: tender joint counts (68 joints), swollen joint counts (66 joints), patient global assessments on 100-mm a Visual Analogue Scale (VAS), assessor global assessments on 100-mm VAS, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)*Disability*: Health Assessment Questionnaire (HAQ) \[[@CR49]\]*Joint imaging (predictor of future disability)*: plain X-rays of the hands and feet read by a modified Larsen's score \[[@CR50]\]*Quality of life*: EuroQoL 5-dimensional 5-level score (EQ-5D-5 L) \[[@CR51]\], fatigue rating (VAS)*Patient acceptability*: Modified Measuring Actual Patient-led Expectations in Rheumatoid Arthritis (MAPLe-RA) \[[@CR52]\], Medication Adherence Rating Scale (MARS) \[[@CR53]\]*Adverse eventsEconomic assessments*: Modified Client Service Receipt Inventory (CSRI) \[[@CR54]\]
### Potential predictor variables (psychosocial measures) {#Sec17}
Baseline assessments will include evaluation of a number of potential outcome predictors, which will be used in exploratory analyses. These will include:Lifestyle factors: alcohol consumption and tobacco smokingMood and anxiety: Patient Health Questionnaire-9 (PHQ-9) \[[@CR55]\] and Generalised Anxiety Disorder-7 (GAD-7) \[[@CR56]\]Health beliefs/illness perceptions: Beliefs about Medicines Questionnaire (BMQ) \[[@CR57]\] and Brief Illness Perceptions Questionnaire (BIPQ) \[[@CR58]\]
Withdrawal of participants {#Sec18}
--------------------------
Participants have the right to withdraw from the study at any time for any reason. Patients wishing to withdraw from the study will be asked to complete a withdrawal assessment, which is the equivalent of a final (month-12) assessment.
Patients in the intensive management arm who wish to withdraw will be given the following options:*Withdrawal from intervention*: revert to treatment as usual and complete 6- and/or 12-month follow-up assessments*Medical note review only*: revert to treatment as usual but consent to collection of data from routine medical notes (no follow-up assessments completed)*Withdrawal from research*: refuse any further collection of outcome data, either through follow-up assessments or medical note review
Patients in the standard care arm who wish to withdraw will be given the following options:*Medical note review onl*y: consent to collection of data from routine medical notes (no follow-up assessments completed)*Withdrawal from research*: refuse any further collection of outcome data, either through follow-up assessments or medical note review
Extension study for patients in the intensive management arm {#Sec19}
------------------------------------------------------------
To investigate whether disease activity and the impact of RA on general health remain stable after a period of 12 months of intensive management, we will undertake an exploratory study of the future outcome of patients in the intensive management arm of the trial. After completing 12 months in the intensive management arm, patients will return to receiving contemporary standard care treatment following local pathways for managing RA patients with intermediate disease activity. These patients will be invited to complete an additional assessment 6 months after completing the trial. This assessment will include measures of remission and a subset of patient-completed questionnaires used in the main trial. See Table [1](#Tab1){ref-type="table"} for further details.
Sample size {#Sec20}
-----------
One of the most relevant UK trials (TICORA) compared tight control versus standard treatment in patients with RA for less than 5 years; it reported that 16% of patients receiving standard care achieved DAS remission at the end of the trial \[[@CR59]\]. We therefore assume that with standard care 16% of patients will have achieved DAS remission at 1-year follow-up.
We will reject the null hypothesis (RA patients with intermediate disease activity (DAS28 3.2--5.1), despite DMARDs, will not have more remissions following 12 months of intensive management) if the difference in remission rates at 12 months between the intensive management arm and the standard care arm is 15% or greater. Demonstrating such a difference with 5% significance and 90% power requires randomising 358 patients in total, under 1:1 allocation (i.e. 179 patients per group). However, if we assume that 10% of patients will not provide follow-up information at the end of the study the required total sample size increases to 398 patients (199 per arm).
Randomisation {#Sec21}
-------------
Only when all baseline measures are complete and data is entered will patients be randomised. Randomisation will be at the level of the individual using block randomisation with randomly varying block sizes, to ensure pre-randomisation allocation concealment, stratified by site. Patients will be randomised to intensive management (IM) or standard care (SC) in a ratio of 1:1. All staff involved in the conduct of the trial will be unaware of the allocation sequence. The trial will not be blinded. Patients are an integral part of the intensive treatment algorithm and there is no possible way to blind such a trial.
Data analysis {#Sec22}
-------------
Analysis will be on an intention-to-treat (ITT) basis to reflect the randomisation process. We will also carry out two additional analyses populations: a complete case population: these will be observations that subjects complete the trial without missing data or violation of the protocol and, therefore, referred to as 'complete case analysis'. A per-protocol population: these will be observations that will be excluded from these analyses if patients are found to deviate from the protocol and referred to as 'per-protocol analysis'.
Baseline characteristics will be summarised by randomised group. The Multiple Imputations (MI) method will be used to impute missing primary or secondary outcomes. The robustness of the analyses performed to the missing at random assumption under MI model will be assessed by Linear Increment method of Diggle et al. \[[@CR60]\] to handle the missingness.
A logistic regression analysis will be used to analyse the primary outcome -- remission at 12 months. For secondary analyses that involve longitudinal measurements, generalised estimating equations (GEE) and/or mixed models will be used to estimate the effect of treatment, including baseline value as a covariate. Working correlation matrices will be unstructured, which is not unduly restrictive given that measurements will be taken at three time points. Valid/robust estimates of the precision of effects will be obtained through use of the information sandwich estimator for GEE analyses.
Treatment, and the demographic factors (age, ethnicity, gender, disease duration) as well as the design factor (region) will be included as explanatory variables in the multivariate analysis. The design factor will also be accounted for in the univariate model. The estimates for primary outcome will be presented as odds ratios (OR) with 95% confidence intervals for the effect of intensive management. Statistical significance will be determined at the 5% level using a two-sided test throughout.
Serious adverse event and adverse rates in the two arms will be compared using comparisons of two independent proportions.
Analysis of the exploratory extension study will use the final assessment in the TITRATE trial and the new 6-month follow-up data to compare the proportion of patients in remission at 12 and 18 months. Simple descriptive analysis will be used to determine the numbers of patients in remission and changes in the key clinical and functional outcomes.
A full statistical analysis plan was developed prior to the start of the trial.
Cost utility analysis {#Sec23}
---------------------
A cost-utility analysis will be undertaken to estimate the incremental cost per quality-adjusted life year (QALY) of intensive management compared to standard care in RA patients with intermediate disease activity, alongside the clinical trial.
The cost-utility analysis will be conducted in line with the NICE Guide to the Methods of Technology Appraisal (2013) \[[@CR61]\]. In particular, an NHS and Personal Social Services (PSS) perspective will be taken for costs, and health benefits will be quantified using QALYs.
The primary analysis will be an economic evaluation alongside the clinical trial, and will use the 1-year follow-up period of the trial to estimate expected 1-year costs and QALYs for the intervention and control groups. QALYs will be estimated using the EuroQol (5-level) questionnaire reported at baseline, 6 months and 12 months. The EuroQol will be valued using population tariff values to estimate EQ-5D-5 L scores. Although these are not yet published, they are expected to be available during 2013. QALYs will be estimated using the trapezium rule to calculate the area under the curve.
NHS resource use will be measured for each participant between baseline and final follow-up. This will include all medication costs, visits to health services and any social care and community support. Medical costs will be taken from the trial medication records, and other NHS and resources used will be self-reported using the widely used and validated Client Service Receipt Inventory (CSRI) questionnaire \[[@CR54]\]. Unnecessary questions in the CSRI will be removed to reduce the burden for patients; however, questions relating to personal costs incurred and time off work will be retained for a sensitivity analysis.
The estimate of cost-effectiveness will be reported as the incremental cost-effectiveness ratio (ICER). Patient variation in resource use and effectiveness will be captured by confidence intervals of the cost and outcome estimates separately. Due to the ratio property of the ICER, confidence intervals are less reliable and, therefore, bootstrapped estimates of the ICERs will be sampled to allow the probability of the intervention being cost-effective to be determined. This estimate of uncertainty will be reported using cost-effectiveness acceptability curves (CEACs), which report the probability that the intervention is cost-effective for any given level of willingness to pay.
A secondary analysis will be undertaken with a wider societal perspective. Personal costs and time off work will be included, as reported by patients using the CSRI questionnaire. Time off work will be valued as productivity losses using the Human Capital Method.
A potential tertiary analysis will include an extrapolation of the costs and benefits of the intervention, to allow a life-time estimate of expected costs and QALYs. Time to loss of efficacy of the intervention will be determined by a survival analysis of the within-trial data. This will be included in an established decision analytic model (The Sheffield RA Model) \[[@CR62]\]. The model will determine the future treatment pathway for the patient populations once a switch from intensive DMARD therapy is estimated, including biologics if patients progress to severe RA.
Discussion {#Sec24}
==========
TITRATE is intended to establish the clinical and cost-effectiveness of intensive treatment strategies in established RA patients with intermediate disease activity.
Three complex problems have come to light during our ongoing research into RA patients with intermediate disease linked to the TITRATE trial. The first of these is the potential heterogeneity of these patients. In some there is evidence of persisting inflammatory arthritis, with several swollen joints and an elevated ESR. In others the DAS28 mainly reflects high tender joint counts and high patient global assessments; these latter patients may have a pattern of fibromyalgic RA and they may not respond greatly to DMARDs \[[@CR63]\]. There is insufficient information about whether or not fibromyalgic RA represents a distinct group of patients or whether it reflects higher pain scores in a minority of patients who otherwise have a similar clinical phenotype. More information is needed to resolve this question.
The second problem is whether remission is the optimal target, or if low disease activity is adequate. Although disability is minimised and QoL is maximised when patients achieve remission, more patients will achieve low disease activity and remission than remission alone. For this reason, it might be preferable to have a broader target which is achieved more often than the narrower target of remission. Secondary analyses will examine both remission and low disease activity states. However, further research is needed to identify the optimal treatment target, particularly in patients with established RA in whom some joint damage and disability may already have become irreversible.
The third potential problem is the marked variability in the training and experience of rheumatology nurses in England. Some rheumatology nurses are very experienced with high skill levels and knowledge. However, many have relatively little experience in the specialty. The extent of this variation reflects the absence of any agreed accredited national training programme for nurses. One complexity in assessing the impact of training is the relative propensity of healthcare professionals to learn and implement a new approach, such as the intensive management intervention we have described. Whilst experienced nurses may have the greatest knowledge and skills they may also be the least likely to adopt new approaches. It is likely that several nurse/practitioner-related factors are implicated when applying innovative management approaches to long-term disorders like RA.
Many RA patients have intermediate disease activity, so we anticipate that the results of the TITRATE trial will inform the NHS on how best to manage these patients and the benefits of delivering a more intensive management approach provided such an approach is found to be both effective and cost-effective.
Several other factors may influence interpretation of the trial results. One issue is that intensive management might have positive impacts on some secondary outcomes alone; for example, improving QoL without increasing remissions. As a number of secondary outcomes are being measured caution will be used in interpretation such findings. Another issue is that patients or clinicians may be reluctant to sufficiently increase therapy in the intensive management group or may give more intensive treatment to the usual care group. Finally, there is the potential impact of evaluating different response thresholds; defining remission using DAS28 involves dichotomising patients based on where they fall on a continuous line and it is possible that views will change on the optimal threshold for this division. Caution will be needed in interpreting the assessment of different thresholds in the final analysis.
Trial status {#Sec25}
------------
Participants are currently being recruited. The first patient was enrolled in June 2014.
Additional file {#Sec26}
===============
Additional file 1:SPIRIT Checklist figure. (PPTX 333 kb)
ACR
: American College of Rheumatology
BIPQ
: Brief Illness Perceptions Questionnaire
BMQ
: Beliefs about Medicine Questionnaire
CRP
: C-reactive protein
CSRI
: Client Service Receipt Inventory
CTIMP
: Clinical Trial of an Investigational Medicinal Product
DAS
: Disease Activity Score
DMARD
: Disease-modifying anti-rheumatic drug
EQ-5D-5L
: EuroQoL 5-dimensional 5-level score
ESR
: Erythrocyte sedimentation rate
GAD-7
: Generalised Anxiety Disorder
GEE
: Generalised estimating equations
HAQ
: Health Assessment Questionnaire
IM
: Intensive management
ITT
: Intention-to-treat
MARS
: Medication Adherence Rating Scale
MI
: Multiple Imputations
NICE
: National Institute of Clinical Excellence
non-CTIMP
: non-Clinical Trial of an Investigational Medicinal Product
NRES
: National Research Ethics Service
OR
: Odds ratio
PHQ-9
: Patient Health Questionnaire-9
QALY
: Quality-adjusted life year
QoL
: Quality of life
RA
: Rheumatoid arthritis
RCT
: Randomised controlled trial
SC
: Standard care
SDAI
: Simplified Disease Activity Index
TNFi
: Tumour necrosis factor inhibitor
VAS
: Visual Analogue Scale
**Electronic supplementary material**
The online version of this article (doi:10.1186/s13063-017-2330-8) contains supplementary material, which is available to authorized users.
On behalf of TITRATE Programme Investigators
Work stream A: Heidi Lempp, Jackie Sturt, Sofia Georgopoulou and Louise Prothero; Work stream B: Naomi Martin, Richard Jenner, Isabel Neatrour, Rhiannon Baggott, Fowzia Ibrahim, Brian Tom, Allan Wailoo, Jonathan Tosh, James Galloway, Gabrielle Kingsley and David Scott; Work stream C: Brian Tom, Fowzia Ibrahim, Yujie Zhong, Aneela Mian, James Galloway and David L Scott.
Funding {#FPar1}
=======
This research has been funded by the National Institute for Health Research (NIHR) as one of its Programme Grants for Applied Research (Grant Reference Number: RP-PG-0610-10066; Programme title: Treatment Intensities and Targets in Rheumatoid Arthritis Therapy: Integrating Patients' And Clinicians' Views -- The TITRATE Programme). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
The TITRATE study is supported by the UKCRC-registered King's Clinical Trials Unit at King's Health Partners which is part-funded by the NIHR Biomedical Research Centre for Mental Health at South London and Maudsley, NHS Foundation Trust and King's College London and the NIHR Evaluation Trials and Studies Coordinating Centre (NETSCC).
Dr. Brian Tom is supported by the UK Medical Research Council (Unit Programme number MC_UP_1302/3).
Open access for this article was funded by King's College London.
Availability of data and materials {#FPar2}
==================================
The datasets generated during and/or analysed during the current study are not publicly available because data sharing would require institutional approval but are available from the corresponding author on reasonable request.
Trial sponsors {#FPar3}
==============
King's College London, Strand, London WC2R 2LS.
King's College Hospital NHS Foundation Trust, 161 Denmark Hill, London SE 8EF.
NHM led the writing of the protocol and manuscript, and coordinated the submission. FI provided statistical input to the protocol. BT is the lead statistician on the trial. JG contributed to the writing of the protocol. AW will lead on the health economic analysis of the trial and contributed to the writing of the protocol. JT contributed to the writing of the protocol. HL coordinated service users' input to the Intensive Management Programme. LP supported the intensive management training programme. SG devised and delivered the intensive management training programme to nurses/practitioners. JS devised the intensive management training programme. DLS, as chief investigator of the trial, designed the trial and was a major contributor to the writing of the protocol and co-led the writing of the manuscript. All authors contributed to the writing of the manuscript, and read and approved the final manuscript.
Ethics approval and consent to participate {#FPar4}
==========================================
Ethical approval for this trial was obtained from the London -- West London and GTAC National Research Ethics Service (NRES) Committee (13/LO/1308). This Ethics Committee approved the study for all participating centres. All participants provide written informed consent before participating in the trial or extension study.
Consent for publication {#FPar5}
=======================
Not applicable.
Competing interests {#FPar6}
===================
A list of investigators with competing interests and the details they have declared is as follows: Dr. James Galloway received consultancy fees for educational talks from UCB, Pfizer, Napp and MSD. Dr. Heidi Lempp received funding over the last 5 years from the National Institute of Health Research; European Union; Guy's and St Thomas' Charity, London; The Health Foundation, London; Arthritis Research UK; and the South London Membership Council. Professor Jackie Sturt received funding from Eli Lilly for consultancy activities in 2016 and will continue to do so in 2017.
Pfizer made Enbrel freely available for use outside NICE guidance in this study.
The following authors declare that they have no competing interests: Dr. Naomi Martin, Ms. Fowzia Ibrahim, Dr. Brian Tom, Professor Allan Wailoo, Mr. Jon Tosh, Ms. Louise Prothero, Dr. Sofia Georgopoulou, Professor David Scott.
Publisher's Note {#FPar7}
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Introduction {#Sec1}
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Synaptonemal complex protein 1 (SYCP1) belongs to a group of proteins that form the synaptonemal complex, which is crucial to the process of meiotic recombination (Page and Hawley [@CR55]; Zickler and Kleckner [@CR84]). Specifically, SYCP1 forms the transverse filaments that link the lateral elements of the complex and is made up of coiled-coil domains (Meuwissen et al. [@CR49]). Such is the propensity of this protein to form these ordered transverse filaments that SYCP1 has been observed to form synaptonemal-like structures ex vivo, forming 'polycomplexes' made up of stacks of transverse filaments (Liu et al. [@CR45]), highlighting this important structural role. Indeed, in *SYCP1−/−* mice, meiotic synapses are unable to form and meiosis does not progress. Whilst previously only identified within the vertebrates, work carried out by Fraune et al. (Fraune et al. [@CR22]) has shown *SYCP1* to be more ancient and widely conserved across the Metazoa, with *SYCP1* present in the cnidarian *Hydra vulgaris*, the poriferan *Amphimedon queenslandica* and the ctenophore *Pleurobrachia pileus*.
Due to its crucial role in meiosis, *SYCP1* expression is observed within the germ cells of vertebrates (Iwai et al. [@CR35]; Zheng et al. [@CR83]) and also within the basal-most cells of the *Hydra* testis, highlighting this deeply conserved role of SYCP1 in meiosis (Fraune et al. [@CR22]). This localisation of SYCP1 to the germ cells is such that a promoter fragment of *SYCP1* is sufficient to drive germline expression in zebrafish without requiring additional regulatory elements (Gautier et al. [@CR28]). It is this expression of *SYCP1* within the germ cells that may have led to the multiple instances of *SYCP1* retrogene formation (Ferrier et al. [@CR21]; Sage et al. [@CR62]), with the mouse alone having at least one *SYCP1* retrocopy. The first of these, *Sycp1-ps1*, is present across several related *Mus* sub-species but has accumulated multiple point mutations and deletions and is no longer transcribed. The second, *Sycp1-ps2,* is transcribed and represents a much younger pseudogene. Interestingly, this second retrocopy is found only within lab strains of *Mus musculus* and is absent even from wild *Mus musculus* populations, highlighting the much more recent nature of this second *Sycp1* retrotransposition event (Sage et al. [@CR62]).
The role of SYCP1 in meiosis makes it a candidate for the 'out-of-testis' route of retrogene production, in which functional retrogenes often emerge from genes expressed within the testis, whether there is function within the testis or not (Kleene et al. [@CR40]). Indeed, most genes that give rise to retrogenes are often found to be originally expressed within the testis (Marques et al. [@CR48]; Vinckenbosch et al. [@CR76]), and it seems that many retrogenes may even be initially transcribed within the testis before gaining additional functions, due to the promiscuous transcription in this tissue. Many examples exist of retrogenes becoming bona fide functional genes that play important roles. The FGF4 retrogene present in short-legged dog breeds is responsible for the common occurrence of chondrodysplasia in these breeds (Parker et al. [@CR56]), whilst the vertebrate RHOB gene plays a role as a tumour suppressor gene (Prendergast [@CR57]) and originates from a retrotransposition early in vertebrate evolution (Sakai et al. [@CR65]). Indeed, severe disease phenotypes can arise from mutations in retrogenes, such as the gelatinous drop-like corneal dystrophy arising from mutations that disrupt *TACTSTD2*, which leads to blindness (Tsujikawa et al. [@CR74]), or the deletion of the retrogene UTP14B which leads to a severe recessive defect in spermatogenesis (Bradley et al. [@CR8]). In some cases, the retrogene has not only become functional or retained functionality, but has also replaced the parental gene copy in function and resulted in the loss of the parental copy from the genome. This phenomenon, known as retrogene replacement (Krasnov et al. [@CR41]), or alternatively as 'orphaned retrogenes' (Ciomborowska et al. [@CR12]), has been documented largely by single-exon gene copies lying in a different locus from that of the ancestral multi-exonic parent copy. Though few in number, studies of retrogene replacement can give unique insight into the regulatory environment of genes and genomic loci. This is especially true in the case of the *Iroquois-Sowah* locus of Bilateria (Maeso et al. [@CR47]), in which the ancestrally linked *Iroquois* and *Sowah* genes have been decoupled in the tetrapods. Despite this syntenic block being maintained for over 600 million years of evolution, the parental *Sowah* gene has been pseudogenised and lost from the *Iroquois* locus in tetrapods, whilst a retrogene copy now exists elsewhere in the genome. Interestingly, *Iroquois* cis-regulatory modules remain within the pseudogenised remnants of *Sowah* genes next to the *Iroquois* locus (Maeso et al. [@CR47]).
*SYCP1* is a gene that has undergone retrogene formation in multiple chordate lineages but has also undergone retrogene replacement within the cephalochordate amphioxus (Ferrier et al. [@CR21]), making this a particularly intriguing case. Here, a single-exon retrogene copy of *SYCP1* has inserted just upstream of the ParaHox gene *Gsx*, a feature unique to the amphioxus ParaHox cluster. Though previously named *AmphiSCP1* by Ferrier et al. ([@CR21]), this amphioxus *SYCP1* gene will be amended to *AmphiSYCP1* here, in order to maintain consistency across species in light of the detailed analysis by Fraune et al. ([@CR22]). With no multi-exonic copy present elsewhere in the amphioxus genome, it appears that the single-exon *AmphiSYCP1* retrogene is the only *SYCP1* gene present in amphioxus (Ferrier et al. [@CR21]). The ParaHox cluster of chordates has previously been shown to be open to invasion by retrotransposons (Osborne and Ferrier [@CR52]; Osborne et al. [@CR53]), perhaps due to *Cdx* transcription within the germline opening up the cluster to transposable elements (Kurimoto et al. [@CR43]). With complex and perhaps long-range or even pan-cluster regulatory mechanisms directing ParaHox gene expression across the cluster (Garstang and Ferrier [@CR27]; Osborne et al. [@CR51]), *AmphiSYCP1* presents an excellent case with which to study how the regulation and expression of retrogenes are affected when they enter a new locus. The likely dense regulatory landscape of the amphioxus ParaHox cluster provides an opportunity to examine both the regulation of *AmphiSYCP1* and the surrounding genes.
Here, we show that *SYCP1* underwent retrogene replacement prior to the divergence of the *Branchiostoma* lineage of amphioxus, with the *AmphiSYCP1* retrogene inserting adjacently to the amphioxus ParaHox cluster. Despite its proximity to the ParaHox gene *Gsx*, *AmphiSYCP1* does not display ParaHox-like expression but does display embryonic expression in addition to the expected gonadal expression, which is altogether atypical for a gene family whose only known role is in meiosis. Identification of a transcribed multi-exonic *AmphiSYCP1* 5′ untranslated region (UTR) with multiple isoforms suggests the de novo evolution of a regulatory 5′ UTR after retrogene insertion. Finally, the proximity of the *AmphiSYCP1* 5′ UTR to the 5′ of the adjacent *AmphiCHIC* gene, similar expression of *AmphiCHIC* to *AmphiSYCP1* and the high support for a bidirectional promoter region overlapping the transcriptional start site of these two genes suggest the co-option of regulatory information from *AmphiCHIC* by *AmphiSYCP1*.
Results {#Sec2}
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*AmphiSYCP1* is a retrogene adjacent to the ParaHox cluster that has led to retrogene replacement of the ancestral parental copy prior to the divergence of the *Branchiostoma* {#Sec3}
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The previously identified *SYCP1* coding sequence (Ferrier et al. [@CR21]) was used to confirm that *Branchiostoma floridae SYCP1* was indeed upstream of *Gsx* and present as a single coding exon within the *B. floridae* genome (Fig. [1](#Fig1){ref-type="fig"}a). Furthermore, *SYCP1* is also present in the same location and as a single coding exon, in both the *Branchiostoma lanceolatum* and *Branchiostoma belcheri* genomes (see the "Materials and methods" section) (Fig. [1](#Fig1){ref-type="fig"}b, c), revealing that the *SYCP1* retrotransposition event must have occurred prior to the divergence of the *Branchiostoma* genus. Comprehensive searches against genomic and transcriptomic databases from all three *Branchiostoma* species reveal no other *SYCP1* gene copies or transcripts bar the *AmphiSYCP1* retrogene. The single-coding-exon organisation of amphioxus *SYCP1* genes is in stark contrast to the multi-exonic arrangement of *SYCP1* genes in most other species (Table S1), consistent with the amphioxus gene originating via retrogene replacement.Fig. 1Comparison of *SYCP1* position across amphioxus species. **a** A schematic of the *B. floridae* SYCP1 gene with relative positions of coding sequence and identified 5′ and 3′ UTRs with respect to the surrounding genes. **b** A schematic of the *B. lanceolatum* SYCP1 gene with relative positions of coding sequence with respect to the surrounding genes. **c** A schematic of the *B. belcheri* SYCP1 gene with relative positions of coding sequence with respect to the surrounding genes. *B. belcheri* SYCP1 is missing both the 3′ and 5′ ends of the coding sequence. Coding exons are represented in black, whilst UTR is represented in white. Chevron lines linking exons show the intron-exon structure of genes based on mRNA transcripts. Grey denotes artefacts resulting from genome scaffold assembly errors. Right-angle arrows indicate known transcriptional start sites and orientation of transcription
Whilst the whole coding sequence for *SYCP1* is present in both *B. floridae* and *B. lanceolatum*, *B. belcheri* Sc0000020 contains only the central region of *SYCP1* coding sequence as the 5′ adjacent sequence does not match *SYCP1* and seems to be an unrelated non-coding sequence, and the 3′ adjacent sequence is represented by a string of N's. This is likely due to the low-quality sequence in this region or problems with the assembly within v15h11.r2 rather than *B. belcheri SYCP1* being incomplete. The position of amphioxus *SYCP1* genes is given relative to the flanking *CHIC* and *Gsx* genes in Fig. [1](#Fig1){ref-type="fig"} for *B. floridae* (Fig. [1](#Fig1){ref-type="fig"}a), *B. lanceolatum* (Fig. [1](#Fig1){ref-type="fig"}b) and *B.belcheri* (Fig. [1](#Fig1){ref-type="fig"}c).
*AmphiSYCP1* is expressed during embryogenesis in addition to the expected expression within the adult gonads {#Sec4}
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In order to examine if *AmphiSYCP1* has come under the influence of any nearby ParaHox regulatory elements, in situ hybridisation of *AmphiSYCP1* was carried out on a time course of *B. lanceolatum* embryos, ranging from mid-gastrula to pre-mouth larvae, as these are the stages that display collinear ParaHox expression. This revealed extensive expression of *AmphiSYCP1* throughout the stages examined (Fig. [2](#Fig2){ref-type="fig"}a--i), within both the mesoderm and endoderm, though this expression does not show ParaHox-like collinearity (Osborne et al. [@CR51]) and is much more extensive throughout the embryo than might be expected if *AmphiSYCP1* were being controlled by ParaHox regulatory elements. This expression is observed within the mid-gastrula (Fig. [2](#Fig2){ref-type="fig"}a) throughout the mesendoderm (black and white arrowheads) and continues into the late gastrula (Fig. [2](#Fig2){ref-type="fig"}b) where the mesendoderm is beginning to differentiate into the ventral endoderm (black arrowhead) and dorsal mesoderm (white arrowhead) but is absent from the ectoderm and neurectoderm in both stages. As embryogenesis progresses to the early neurula (Fig. [2](#Fig2){ref-type="fig"}c, d), expression is restricted more towards the central region of the embryo along the anterior-posterior axis, again present in the endoderm and mesoderm, but with expression notably absent from the extreme posterior of the embryo, ectoderm and developing neural plate. By the mid-late neurula stages (Fig. [2](#Fig2){ref-type="fig"}e, h), expression is undetectable in the posterior tailbud but still present throughout the central mesoderm (white arrowheads) and endoderm (black arrowhead). Expression remains absent from the ectoderm and neural tube. During the pre-mouth stage (Fig. [2](#Fig2){ref-type="fig"}i), *AmphiSYCP1* expression is present throughout the mesoderm and endoderm but absent from the posterior tailbud region, the ectoderm and the extreme anterior tip of the embryo. Finally, in addition to embryonic expression, *AmphiSYCP1* transcripts were cloned via RT-PCR from adult *B. lanceolatum* gonadal cDNA, confirming the expression of *AmphiSYCP1* within adult gonads as expected for a meiotic gene (Fig. [2](#Fig2){ref-type="fig"}j).Fig. 2Expression of *B. lanceolatum SYCP1* transcripts within embryos and gonadal tissue. **a--i** Embryonic expression of *B. lanceolatum SYCP1* is shown from the mid-gastrula (**a**) to the pre-mouth (**i**) stages of development. Expression begins in the endoderm (black arrowheads) and dorsal mesoderm (white arrowheads) at the mid-gastrula stage to the late gastrula (**a**, **b**) before becoming more restricted to the centre of the animal and excluded from the extreme posterior in the early neurula (**c**, **d**). This expression pattern continues into the mid-late neurula (**e**, **f**). Expression reaches anteriorly to a region below the forming cerebral vesicle throughout the late neurula--pre-mouth (**g**--**i**), whilst expression elsewhere becomes much more diffuse throughout the somites and endoderm. **a**, **b**, **c**, **e**, **g**, **i** represent lateral views, whilst **d**, **f**, **h** represent dorsal views. **j** shows the 3121-bp *SYCP1* mRNA transcript cloned from *B. lanceolatum* gonadal total mRNA. mg mid-gastrula, lg late gastrula, en early neurula, mn mid-neurula, ln late neurula, pm pre-mouth. Scale bar represents 100 μm
*AmphiSYCP1* has evolved a de novo 5′ UTR with distinct isoforms {#Sec5}
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*B. floridae SYCP1* has previously been described as a retrogene, as it contains a single open reading frame with no introns within the amphioxus ParaHox PAC clones 33B4 and 36D2 (Ferrier et al., [@CR21]). Searches for *B. floridae SYCP1* in the *B. floridae* expressed sequence tagged (EST) database (<http://amphioxus.icob.sinica.edu.tw>/) (Yu et al. [@CR81]) revealed a *B. floridae* cDNA clone, bfad022l10, containing 5′ and 3′ ESTs that align to *B. floridae SYCP1* coding sequence and immediately flanking non-coding sequence (Fig. [3](#Fig3){ref-type="fig"}a). This EST clone was obtained from whole adult animal, which would be consistent with *SYCP1* expression within meiotic cells within the gonads.Fig. 3Amphioxus *SYCP1* has a multi-exonic 5′ UTR. A schematic depicting the relative positions of exons within the *CHIC-SYCP1* region of *B. floridae* and *B. lanceolatum* genomes. Black boxes represent coding sequence, white boxes represent UTR and grey boxes represent sequenced transcripts. Right-angle arrows indicate transcriptional start sites and orientation of transcription. **a** The *B. floridae* EST transcript bfad022\|10 identifies both a multi-exonic 5′ UTR, as well as a 3′ UTR, that is adjacent to the single-exon *SYCP1* coding sequence. **b** Whilst *B. lanceolatum* also has a multi-exonic 5′ UTR, two different isoforms are present, one with four UTR exons whilst the second isoform lacks 5′ UTR exon 2. **c** Agarose gel depicting the two cloned *B. lanceolatum SYCP1* 5′ UTR fragments seen in schematic **b**. The longer is 307 bp in length, whilst the shorter is 246 bp in length. Reamplification of individual bands is shown to enable better visualisation of these independent clones. These fragments were obtained via PCR from *B. lanceolatum* gonadal cDNA
The 3′ EST, bfad022l10 3′ (accession number BW716295.1), encompasses a 685-bp 3′ UTR downstream of the coding sequence of *SYCP1*. This represents a single exon containing the *SYCP1* coding sequence and 3′ UTR. As expected, the 5′ EST, bfad022l10 5′ (accession number BW697675.1), aligned to the most 5′ coding sequence of *B. floridae SYCP1*, with a 334-bp alignment covering this region. Additionally, a short 53-bp region immediately 5′ and adjacent to the coding sequence also matched the EST, designating 5′ UTR sequence present in the same exon as the coding sequence.
In addition, the 5′ EST, bfad022l10 5′, also aligned to further regions upstream of the *SYCP1* coding exon, with the mRNA sequence indicating three exons spread throughout the 3259 bp between the coding regions of *SYCP1* and *CHIC* (Fig. [3](#Fig3){ref-type="fig"}a). The three additional 5′ UTR exons were identified with discontiguous MegaBLAST, in order to accommodate sequence polymorphisms within these short exons relative to the genomic sequence. In total, only 16 nucleotides across the entire 599 bp of bfad022l10 5′ did not show a match to the *B. floridae* ParaHox genomic sequence.
In order to identify if this novel 5′ UTR was also present in *B. lanceolatum*, the EST data collected from *B. floridae* was then aligned to the *B. lanceolatum* ParaHox scaffold and primers designed against the beginning of 5′ UTR exon 1 and the 5′ of the coding region. These were used to clone the 5′ UTR region from adult *B. lanceolatum* gonadal cDNA. This not only isolated a transcribed, spliced *AmphiSYCP1* 5′ UTR transcript, but also identified two distinct isoforms of this transcript (Fig. [3](#Fig3){ref-type="fig"}b, c). The first of these is a long isoform (307-bp polymerase chain reaction (PCR) fragment) that contains all four *AmphiSYCP1* 5′ UTR exons, with exon 4 contiguous with the coding exon sequence. The second is a shorter isoform (246-bp PCR fragment) that lacks *AmphiSYCP1* 5′ UTR exon 2 but is otherwise identical to the longer isoform.
*AmphiSYCP1* shares a bidirectional promoter with the adjacent *AmphiCHIC* gene {#Sec6}
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As the 5′ UTR of *AmphiSYCP1* must have evolved post-insertion of the ancestral amphioxus *SYCP1* single-exon retrogene, a promoter region driving the transcription of this 5′ UTR sequence must have either been co-opted from an existing nearby promoter sequence or evolved de novo. In order to establish which of these was the case, a total of 7000 bp, starting from within *AmphiCHIC* intron 1 to the end of the *AmphiSYCP1* coding exon, were analysed for promoter sequences. This ensured that the transcriptional start sites of *AmphiSYCP1* and its neighbour *AmphiCHIC* were both included as well as any possible overlap of promoter sequences. Three independent promoter prediction algorithms, Neural Network Promoter Prediction (NNPP), TSSW and ProScan1.7, were used across both *B. floridae* and *B. lanceolatum* to look for consistency across algorithms, which should increase confidence in the validity of any predicted promoters.
Within *B. floridae*, a total of five 50-bp predicted promoter sequences were identified by NNPP (Fig. [4](#Fig4){ref-type="fig"}a) (Table S2), with the prediction with the highest support located surrounding the start of *AmphiSYCP1* 5′ UTR exon 1. This region, annotated as NNPP3 in Fig. [4](#Fig4){ref-type="fig"}a, was the only sequence predicted in all three Promoter prediction programs and had the highest support value in both NNPP and ProScan 1.7 (Table S2). This was also the only region predicted by TSSW and is identified as 50 bp in length using NNPP and 250 bp in ProScan. It also lies on the negative strand and spans the start of *AmphiSYCP1* 5′ UTR exon 1, in the same orientation as the *CHIC* gene, and is located 56 bp upstream of *AmphiCHIC* (Fig. [4](#Fig4){ref-type="fig"}a).Fig. 4Promoter analysis of the *B. lanceolatum* and *B. floridae CHIC/SYCP1* loci. For the *SYCP1/CHIC* locus of both *B. lanceolatum* and *B. floridae*, promoter sequences predicted by either NNPP, TSSW or ProScan 1.7 are visualised relative to the surrounding *CHIC* and *SYCP1* exon-intron structures. The size and position of each predicted promoter identified are indicated by a grey box/black vertical line. In addition, black arrowheads indicate the direction of the DNA strand the promoter was identified upon. **a** For *B. floridae*, five promoters were predicted by NNPP (NNPP1--5), one by TSSW (TSSW promoter TSS) and two by ProScan 1.7 (ProScan1, ProScan 2). Only one promoter region, including NNPP3, TSSW promoter TSS, and ProScan 1 and 2, agrees across all three prediction models. **b** For *B. lanceolatum*, nine promoters were predicted by NNPP (NNPP1--9), two by TSSW (TSSW promoter 1 TSS, TSSW promoter 2 TSS), whilst only one was predicted by ProScan 1.7 (ProScan1). Only one promoter region, including NNPP5, TSSW promoter 1 TSS, TSSW promoter 2 TSS and ProScan 1, agrees across all three prediction models. This region also agrees across both *B. floridae* and *B. lanceolatum* (**a**, **b**), further supporting the confidence of this region as a bona fide promoter. In addition, this promoter is predicted in both directions within both species, suggesting a bidirectional promoter. Back boxes represent coding exons, whilst white boxes represent UTR exons. Right-angle arrows indicate known translational start sites and the orientation of transcription
Within *B. lanceolatum*, a total of nine 50-bp predicted promoter sequences were identified by NNPP (Fig. [4](#Fig4){ref-type="fig"}b) (Table S2), with the prediction with the highest support again located surrounding the start of *AmphiSYCP1* 5′ UTR exon 1, annotated as NNPP5 in Fig. [4](#Fig4){ref-type="fig"}b. This region was also identified in TSSW (TSSW2) and overlaps a second TSSW hit facing in the opposite orientation towards *AmphiCHIC* (TSSW1). Finally, this region was identified in both NNPP and TSSW and is also identified as a 250-bp region oriented in the direction of *AmphiSYCP1* 5′ UTR in ProScan 1.7. This ProScan-predicted promoter also overlaps the first exons of both *AmphiCHIC* and *AmphiSYCP1* 5′ UTRs.
All three prediction programs thus agree on a strong candidate promoter region that overlaps the first exons of both *AmphiCHIC* and *AmphiSYCP1* 5′ UTRs. Also, transcription is predicted from this region in both directions in both species. This implies that *AmphiSYCP1* has co-opted a promoter with bidirectional capability from the neighbouring *AmphiCHIC* gene.
*AmphiCHIC* is expressed in the same tissues as *AmphiSYCP1* {#Sec7}
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As both *AmphiCHIC* and *AmphiSYCP1* appear to share the same bidirectional promoter, it is possible that the two would share some similarities in their expression. In order to examine this, the expression of *AmphiCHIC* was assayed by both in situ hybridisation and RT-PCR in the same stages and tissues examined for *AmphiSYCP1* (Fig. [5](#Fig5){ref-type="fig"}a--i).Fig. 5Expression of *B. lanceolatum CHIC* transcripts within embryos and gonadal tissue. **a--i** Embryonic expression of *B. lanceolatum CHIC* is shown from the mid-gastrula (**a**) to the pre-mouth (**i**) stages of development. Expression begins in the ventral endoderm (black arrowheads) and dorsal mesoderm (white arrowheads) at the mid-gastrula stage to the late gastrula (**a**--**c**) but is clearly absent from the ectoderm. This expression continues through the early-late neurula (**d**--**g**), with expression absent from both the ectoderm and neural tube, as well as becoming restricted from the far anterior of the embryo. Throughout the late neurula--pre-mouth stages (**g--i**), expression reaches as far anteriorly as the first somite dorsally and ventrally up to the pharynx but is still notably absent from the neural tube, cerebral vesicle and ectoderm. **a**, **b**, **d**, **f**, **h** represent lateral views, whilst **c**, **e**, **g**, **i** represent dorsal views. **j** shows the 455-bp *AmphiCHIC* mRNA transcript cloned from *B. lanceolatum* gonadal total mRNA that was used to create an antisense RNA hybridisation probe. mg mid-gastrula, lg late gastrula, en early neurula, mn mid-neurula, ln late neurula, pm pre-mouth. Scale bar represents 100 μm
In the mid-gastrula, *AmphiCHIC* expression can be seen throughout the mesendoderm but is absent from the ectoderm (Fig. [5](#Fig5){ref-type="fig"}a). This continues into the late gastrula/early neurula (Fig. [5](#Fig5){ref-type="fig"}b, c). In the mid-neurula, *AmphiCHIC* expression appears to be excluded from the anterior and posterior extremes of the embryo (Fig. [5](#Fig5){ref-type="fig"}d, e). Whilst the mesoderm expression (white arrowhead) extends almost all the way to the anterior, the ventral endoderm expression is much more restricted to the posterior of the embryo (black arrowhead) (Fig. [5](#Fig5){ref-type="fig"}d). In the late neurula, expression is similar to that of the mid-neurula, though expression now extends into the posterior tailbud but is still absent from the ectoderm (Fig. [5](#Fig5){ref-type="fig"}f, g). In this stage, the lack of anterior expression is now more noticable, and though the dorsal mesoderm expression (white arrowhead) extends further anteriorly than the ventral endoderm expression (black arrowhead), the far anterior portion of the embryo is clearly lacking any *AmphiCHIC* expression (Fig. [5](#Fig5){ref-type="fig"}f, g). This expression pattern continues to the pre-mouth stage, with expression only reaching as far anteriorly as the presumptive pharynx along the ventral side and into the first somite dorsally (Fig. [5](#Fig5){ref-type="fig"}h, i). In concurrence with *AmphiSYCP1* gonadal expression, a 455-bp spliced *AmphiCHIC* transcript, spanning *AmphiCHIC* exon 1 to exon 6, was amplified and cloned from adult *B. lanceolatum* gonadal cDNA (Fig. [5](#Fig5){ref-type="fig"}j).
*SYCP1* is widely conserved across the Metazoa {#Sec8}
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Fraune et al. ([@CR22]) showed that *SYCP1* was much more highly conserved across the metazoans than previously thought, greatly extending the evolutionary history of this gene. With the ever-growing list of genome sequences available, greater taxon sampling can now be achieved to further address *SYCP1* evolutionary history. With this aim, a multiple alignment of SYCP1 proteins was produced, highlighting the conserved CM1 domain identified by Fraune et al. ([@CR22]) and aiming to better sample underrepresented phyla. The chimaera (*Callorhinchus milii*) was added to the Vertebrata as a basal fish lineage, as well as additional echinoderm species, including a second echinoid (*Lytechinus variegatus*) and two members of the asteroids (starfish) (*Asterias amurensis* and *Pateria pectinifera*). Additionally, a single hemichordate SYCP1 sequence from *Saccoglossus kowalevskii* was identified, giving examples of SYCP1 from all three main deuterostome phyla. In the Protostomia, lophotrochozoan sequences were expanded greatly within the Mollusca with the addition of a gastropod (*Pomacea canaliculata*), two bivalves (*Mytilus galloprovincialis* and *Ruditapes philippinarum*) and three cephalopods (*Octopus bimaculoides*, *Hapalochlaena maculosa* and *Sepiella maindroni*). Two additional annelid sequences were also obtained (*Lamellibrachia satsuma* and *Olavius algarvensis*). No additional ecdysozoan members were obtained (beyond the highly divergent and short *Petrolisthes cinctipes* sequence fragment found by Fraune et al. [@CR22]). Several additional members of the Cnidaria were obtained beyond *Hydra vulgaris* (*Orbicella faveolata*, *Hydractina symbiolongicarpus* and *Turritopsis sp*.), and a full length *Nematostella vectensis SYCP1* sequence was obtained to replace the short EST read previously used by Fraune et al. ([@CR22]). The Ctenophora was expanded to include the sea walnut *Mnemiopsis leidyi* as well as *Pleurobrachia pileus.* Finally, the poriferan *Amphimedon queenslandica* represents the sole example of SYCP1 so far identified in this phylum. Full species names, groups and accession numbers are given in Supplementary file 4. Whilst a full SYCP1 protein alignment can be found in Fig. S1, the CM1 conserved motif (see the "Materials and methods" section) provides much better resolution for distinguishing SYCP1 sequences from other coiled-coil proteins. Figure [6](#Fig6){ref-type="fig"} illustrates the high level of conservation of the CM1 motif across the Metazoa.Fig. 6The CM1 motif of SYCP1 is highly conserved across the Metazoa. A CLUSTALW protein multiple alignment of the CM1 domains of SYCP1 shows a high level of conservation across an 83-aa motif across the metazoan species examined. Conservation is visualised with false colour using the Zappo colour table for amino acids. Effort made to identify transcripts from phyla underrepresented within (Fraune et al. [@CR22]). A consensus sequence made up of the most abundant amino acid for each position is given in black. The names of species used are given to the left of the alignment, and species are organised roughly according to the current known phylogeny with amphioxus species as the focus. The numbers in parentheses indicate the position of the CM1 motif amino acids within the obtained native peptide sequence
To deduce possible evolutionary relationships between these SYCP1 sequences, phylogenetic trees were produced for the SYCP1 CM1 domain (see the "Materials and methods" section). Bootstrap support values are low on many branches, for both NJ (Fig. [7](#Fig7){ref-type="fig"}a) and ML (Fig. [7](#Fig7){ref-type="fig"}b) analyses. Nevertheless, the vertebrates group together with significant support, as do the different vertebrate groups such as mammals and fish. The remainder of the phylogeny grouped roughly as expected according to known species relationships, albeit with very low support. The paucity of significant node support values is likely due to the short length of the CM1 motif. Several smaller clades do, however, show consistently high support, often where better representation of more closely related species can be found. These include the Branchiostomidae, Asteroidea, Echinoidia, Cephalopoda, Tunicata, Hydrozoa and Ctenophora. The Cephalopoda and Tunicata are often grouped together, likely due to long-branch attraction. *Alvinella* also often groups with the Asteroidea rather than the Lophotrochozoa, in this case due to several key amino acid similarities at positions 65--70 seen in Fig. [6](#Fig6){ref-type="fig"}. Whether this convergent similarity has any functional significance remains to be seen.Fig. 7Phylogeny of metazoan SYCP1 CM1 motifs. **a** Neighbour-joining tree built using the 83-aa CM1 domain of SYCP1 proteins, using the JTT + G matrix with 1000 bootstraps, a gamma shape parameter of 2.157 and a 95% partial-deletion cutoff. **b** Maximum-likelihood tree built using the 83-aa CM1 domain of SYCP1 proteins, using the LG + G model with four discrete gamma categories, using all sites, and branch support calculated using the aLRT SH-like statistic (Anisimova and Gascuel [@CR2]), with bootstrap support values provided as a function of the aLRT statistic. CCDC39 proteins were used as an outgroup to SYCP1. Bootstrap values over 70% are given. Longer branch lengths equate to a further evolutionary distance between nodes. NJ trees were built using MEGA7, whilst ML trees were built using PHYML (see the "Materials and methods" section). The analysis involved a total of 45 amino acid sequences
Discussion {#Sec9}
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Amphioxus *SYCP1* is a transcribed retrogene that replaced its parental multi-exonic copy before the divergence of the *Branchiostoma* genus {#Sec10}
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Comparisons between the three amphioxus genomes show that amphioxus *SYCP1* is present as a single coding exon within *B. floridae*, *B. lanceolatum* and *B. belcheri* (Fig. [1](#Fig1){ref-type="fig"}). Thus, we can conclude that an *SYCP1* retrogene must have been present upstream of the ParaHox cluster, between *CHIC* and *Gsx*, before the divergence of these three species. It will be necessary to examine the ParaHox cluster of both *Asymmetron* (Yue et al. [@CR82]) and *Epigonichthys* (Nohara et al. [@CR50]), as the only two other amphioxus groups known besides *Branchiostoma* species, to determine if this instance of retrogene replacement is typical for all amphioxus.
The presence of multi-exonic *SYCP1* genes throughout the rest of the Bilateria, within the vertebrates, echinoderms and Lophotrochozoa (Table S1), makes it highly likely that both amphioxus and *Ciona intestinalis SYCP1* genes evolved via retrotransposition and replaced a multi-exonic ancestral parent gene. Indeed, in much the same manner as *AmphiSYCP1*, there is only one single-exon copy of *SYCP1* within the *Ciona*, though it has inserted into a different locus and does not lie next to any of the ParaHox genes (Table S1**)**. Retrogene replacement appears to be a relatively common mechanism in *Ciona* (Kim et al. [@CR37]), and with the genome compaction, dispersal and gene loss in tunicates (Berna and Alvarez-Valin [@CR5]; Dehal et al. [@CR18]; Hughes and Friedman [@CR34]), this mechanism may contribute to their fast genome evolution. In addition, the existence of multiple instances of *SYCP1* retrogene copies within the mouse (Sage et al. [@CR62]) also suggests that *SYCP1* is perhaps prone to retrotransposition, at least within the chordates. The expression of *SYCP1* within the germ line may very well make *SYCP1* a target for the 'out-of-the-testis' route of retrogene production (Kleene et al. [@CR40]; Vinckenbosch et al. [@CR76]) and eventual replacement of the parent gene by the retrocopy (Ciomborowska et al. [@CR12]).
Amphioxus *SYCP1* has evolved a de novo multi-exonic 5′ UTR that may originate from a co-opted bidirectional *CHIC* promoter {#Sec11}
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Our RT-PCR data, combined with transcriptome and genome sequence data, indicates the presence of a multi-exonic 5′ UTR stretching upstream from the *SYCP1* coding sequence between *SYCP1* and *CHIC* (Fig. [4](#Fig4){ref-type="fig"}). Promoter analysis revealed no promoter present immediately upstream of the *SYCP1* coding region; however, it did reveal a putative promoter lying upstream of *CHIC* exon 1 (Fig. [4](#Fig4){ref-type="fig"}). This putative promoter was identified with high support values in all three of the programs used for prediction (NNPP, TSSW and ProScan 1.7). These three programs were used in order to provide multiple alternative methods of both identification and support for putative promoter sequences (Prestridge [@CR58]; Reese [@CR60]; Solovyev et al. [@CR70]; Solovyev et al. [@CR71]) and mitigate against any shortcomings of each program. This approach leads to the prediction of one promoter region that is common to all three approaches, making it much more likely that this site is indeed a bona fide promoter sequence. To corroborate this, the same analysis was carried out upon both *B. floridae* and *B. lanceolatum*, with both species producing very similar results despite the high levels of polymorphism that exist in non-coding sequence in amphioxus species (Huang et al. [@CR32]).
Intriguingly, promoter predictions across both species indicate a promoter on both positive and negative strands at this site, raising the possibility that this may be a bidirectional promoter. The presence of this promoter overlapping the first exons of both *CHIC* and *SYCP1* 5′ UTR is certainly consistent with this (Fig. [4](#Fig4){ref-type="fig"}). This raises the possibility of an interesting evolutionary scenario, in which *AmphiSYCP1* has co-opted a *CHIC* promoter, whilst retaining its essential germline expression. *SYCP1* would then have either evolved its own de novo 5′ UTR in order to take advantage of this bidirectional promoter or co-opted UTR sequence from the adjacent *CHIC* gene. It is likely that the orientation of the two genes, as well as the position of the predicted promoter sequence, precludes the co-option of 5′ UTR elements from *CHIC*. Also, we find no evidence for, but cannot conclusively exclude, a third possibility that amphioxus *SYCP1* inserted into an intervening gene between *CHIC* and *Gsx*, possibly replacing all of this gene except for these few 5′ non-coding exons such that *AmphiSYCP1* inherited these non-coding exons from this additional, but now absent, gene. Whilst it may seem a large evolutionary leap for a retrogene to evolve a 5′ UTR or co-opt an existing nearby regulatory element, this has been seen to occur with other bilaterian retrogenes. For example, a genome-wide screen of retrogenes within *Drosophila* revealed that several regulatory motifs were overrepresented in the cis-regulatory elements of testis-expressed retrogenes and that specific regulatory motifs had been selectively recruited by retrogenes from their new genomic loci (Bai et al. [@CR4]). Indeed, it seems that retrogenes rarely bring along any active regulatory elements of their own when inserting into their new locus (Bai et al. [@CR3]). Another key study selectively looked at the evolution of introns within retrogenes of mammals and found that most introns found associated with retrogenes occurred in the 5′ flanking sequence to the retrogene insertion site, which is linked to the recruitment of distal promoters (Fablet et al. [@CR20]). There may even be selective pressure for the evolution of multi-exonic 5′ UTRs within retrogenes, as those with introns display higher transcription levels and broader expression patterns than those without. Fablet et al. ([@CR20]) propose a scenario where 5′ exon-intron structures evolve de novo or through fusion to the 5′ UTR of a neighbouring gene as a direct link to the recruitment of a distant promoter by a retrogene. It is also noteworthy that of those recruited by distant promoters and that gained 5′ exon-intron UTR structures, most were recruited by bidirectional CpG promoters (Fablet et al., [@CR20]). It is becoming clear that the phenomena of retrogenes recruiting regulatory elements from regions flanking their insert site, as well as retrogenes gaining introns, may not be as rare as they once seemed (Kang et al., [@CR36]; Sorourian et al., [@CR72]). There is an abundance of general transcription occurring within cells to which no functional role can be attributed, and lots of non-coding, non-functional RNA is produced (Struhl [@CR73]). It is entirely possible that retrogenes could be co-opting the sequences involved in this pervasive transcription to facilitate their own transcription as part of retrogene evolution.
The combination of 5′ UTR transcript, precise placement of a predicted promoter (perhaps bidirectional) adjacent to both *CHIC* exon 1 and *SYCP1* 5′ UTR exon 1 and broad *CHIC-*like somatic expression of *AmphiSYCP1* in embryos are all consistent with recruitment of a bidirectional *CHIC* promoter by the *AmphiSYCP1* retrogene. *SYCP1* would then have evolved a de novo 5′ intron-exon structure to make use of the distant promoter. A preliminary check for CpG islands within the *CHIC-SYCP1* 5' UTR region yielded no results, but the identified promoter region could nonetheless still display bidirectionality. Indeed, it is now thought that bidirectionality is an inherent feature of promoters (Wei et al. [@CR78]). Further work could examine this promoter region in a reporter background to test both its bidirectionality and its similarity to *AmphiSYCP1* expression.
Expression of *AmphiSYCP1* is much broader than expected for a meiosis gene {#Sec12}
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It is clear from the in situ hybridisation of *AmphiSYCP1* that expression is by no means limited to the germ cells, and typical germ cell markers such as *nanos* and *vasa* show markedly different embryonic expression patterns to *SYCP1* (Dailey et al. [@CR16]; Wu et al. [@CR79]). As *SYCP1* expression is limited to meiotic cells in both vertebrates (Casey et al. [@CR10]; de Vries et al. [@CR17]; Iwai et al. [@CR35]), including primordial germ cells (Zheng et al. [@CR83]), and *Hydra* (Fraune et al. [@CR22]), it was expected that no embryonic expression would be observed, as the testis and ovaries have not yet formed in amphioxus, or that *SYCP1* would display *nanos*-/*vasa*-like germ cell expression (Wu et al. [@CR79]). Furthermore, if *AmphiSYCP1* had transposed along with its own regulatory elements, such as a promoter region, it might even be expected that germ cell expression is the most likely outcome, as previous work has shown the zebrafish *SYCP1* promoter region to be sufficient to drive GFP transgenes within germ cells (Gautier et al. [@CR28]).
*AmphiSYCP1* is expressed in the endoderm and mesoderm in a broad pattern throughout these tissues and also seems to exhibit spatio-temporal changes in expression. *AmphiSYCP1* is notably absent not only from the ectoderm and the posterior tailbud, but also from the extreme anterior in all stages (Fig. [2](#Fig2){ref-type="fig"}). This expression pattern, which is much broader than expected for *SYCP1*, suggests that *AmphiSYCP1* has co-opted regulatory elements from its new genomic locus. It does not appear to have come under the influence of ParaHox regulatory elements, however, as the broad expression pattern observed is not reminiscent of ParaHox expression, and there is no CNS expression, a hallmark of ParaHox genes (Brooke et al. [@CR9]; Osborne et al. [@CR51]). In addition, *AmphiSYCP1* does not exhibit any of the patterns of collinear expression, either spatial or temporal, expected if it had co-opted pan-cluster regulatory elements from the adjacent amphioxus ParaHox cluster. It may, however, have gained some of this somatic expression from its co-option of regulatory elements from the neighbouring *AmphiCHIC* gene.
Our study provides the first description of *AmphiCHIC* expression, and though it is not identical to that of *AmphiSYCP1*, certain similarities can be observed. These are particularly evident in the early stages of development (Figs. [2](#Fig2){ref-type="fig"}a--f and [5](#Fig5){ref-type="fig"}a--d), where expression is limited to the mesoderm and endoderm and excluded from the ectoderm and neural tube. As embryogenesis progresses, differences in expression become more apparent after closure of the blastopore, though many similarities remain. Expression becomes broader throughout the mesoderm and the endoderm for both *AmphiSYCP1* and *AmphiCHIC* whilst still remaining absent from the ectoderm and neural tube in each case (Figs. [2](#Fig2){ref-type="fig"}f--i and [5](#Fig5){ref-type="fig"}f--i).
The expressions of *AmphiSYCP1* and *AmphiCHIC* are difficult to compare within other chordate phyla, as neither have been examined in an embryonic spatio-temporal context, with *SYCP1* somatic expression not yet observed at all within the vertebrates. Very little expression data exists even for the vertebrate *CHIC* genes. However, *CHIC1* and *CHIC2* were both originally identified as *Brain x-linked protein* (*Brx*) and *BrX-like translocated in leukaemia* (*BTL*), respectively, and their roles in the regulation of nuclear hormone receptors (Kino et al. [@CR39]) and exocytosis (Cools et al. [@CR15]) have been described. Though the expression of vertebrate CHIC genes was first identified in the brain, both *CHIC1* and *CHIC2* also exhibit expression in the testis, ovary, uterus, endomesoderm, intestine, ectoderm, many secretary organs of the digestive tract, thyroid, prostate and pineal gland (data from <http://www.proteinatlas.org>/ (Uhlén et al. [@CR75])). CHIC genes seem to show expression in a range of tissues, many of which have secretory functions. This may be linked to the described role in plasma membranes and vesicles and exocytosis (Cools et al. [@CR15]). This expression also holds true for the protostome CHIC homologues *TAG-266* (*Caenorhabditis elegans*) (Consortium CeS [@CR14]) and *CG5938* (*Drosophila melanogaster*) (Hoskins et al. [@CR31]). Since bilaterian CHIC genes are expressed in the testis and ovaries, co-option of *CHIC* regulatory elements would still allow *AmphiSYCP1* to carry out its meiotic function and also give the potential to evolve new expression domains within somatic tissues.
One other example of bilaterian *SYCP1* expression is particularly noteworthy with respect to the expression of *AmphiSYCP1* within the embryonic somatic tissue. In the sea urchin *Strongylocentrotus purpuratus*, *SYCP1* is found to be expressed in the larvae throughout the adult rudiment (Yajima et al. [@CR80]). This structure goes on to form most of the adult animal, and the larvae is largely cast off or reabsorbed. Determining the function of sea urchin *SYCP1*, along with other meiotic genes that are expressed throughout the adult rudiment, awaits further research. It remains to be seen whether the embryonic expression of meiotic genes is a more widespread phenomenon, or indeed whether *SYCP1* carries out a yet unknown function within embryogenesis or somatic cells. It is possible, however, that transcription of *SYCP1* is not indicative of any function in somatic cells. Mammalian studies have indicated that meiotic genes can be activated in initially broad domains and only later become restricted to germ cells (Saitou et al. [@CR63]; Saitou et al. [@CR64]), with transcription often beginning prior to the initiation of meiotic events (Kimble and Page [@CR38]). As such, it is entirely possible that the somatic expression of *AmphiSYCP1* transcripts merely represents non-functional transcription. It is also possible that *SYCP1* transcription is allowed to proceed in somatic tissues as it has no negative effect or that the improvement to transcription in target tissues granted by co-opted regulatory elements outweighs any transcriptional costs in somatic tissues.
*SYCP1* is widely conserved across the Metazoa, except for its absence from the Ecdysozoa {#Sec13}
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As Fraune et al. ([@CR22]) showed, SYCP1 proteins are much more deeply conserved across the Metazoa than previously believed, along with several other components of the synaptonemal complexes, suggesting deep conservation of meiotic machinery (Fraune et al. [@CR22]; Fraune et al. [@CR23]; Fraune et al. [@CR24]; Fraune et al. [@CR25]). This work on both *Hydra* (Fraune et al. [@CR22]; Fraune et al. [@CR23]; Fraune et al. [@CR24]; Fraune et al. [@CR25]) and sea urchin (Yajima et al., [@CR80]) synaptonemal complex proteins has not only identified these genes, but also confirmed their expression. The phylogenetic study carried out here has sought to extend the work of Fraune et al. ([@CR22]), identifying *SYCP1* genes and proteins throughout the Metazoa, by utilising the wealth of new genome sequences that have become available. This has allowed a broader sampling of *SYCP1* from within the non-chordate deuterostomes, specifically, with the addition of an echinoid, two asteroids and one hemichordate sequence from the Ambulacraria, providing at least one example of *SYCP1* from each deuterostome phylum, as well as much greater representation within both the Lophotrochozoa and Cnidaria.
The Ecdysozoa are notably absent from the list of *SYCP1*-possessing taxa. Fraune et al. ([@CR22]) included a *Petrolisthes cinctipes* sequence as the sole ecdysozoan representative. This sequence was included in initial phylogenetic analysis, but consistently groups basal to all lineages other than *Pleurobrachia* and *Amphimedon*, including the Cnidaria. Further examination of this sequence fragment shows it to be both short and highly divergent even in comparison to cnidarian, poriferan and ctenophore sequences. Indeed, when included in phylogenies, this sequence proved to be unstable, and iterations of the alignment carried out with CLUSTALW (Larkin et al. [@CR44]) and MUSCLE (Edgar [@CR19]) did not align the *Petrolisthes* ESTs to the conserved CM1 domain at all. Instead, this crustacean sequence aligned further towards the coiled-coil containing-C terminus of other SYCP1 proteins.
To attempt to validate this sequence as a bona fide SYCP1, we searched for *SYCP1* from other ecdysozoan groups, including more basal arthropod lineages such as the myriapod *Strigamia maritima* (Chipman et al. [@CR11]) and spiders (Sanggaard et al. [@CR66]). This search provided no *SYCP1* candidates. Multiple peptide sequences, including mouse, amphioxus, *Hydra* and *Amphimedon* SYCP1 sequences, were all used as queries when looking for ecdysozoan sequences, as well as BLAST searches using only the conserved CM1 domains. This is even more relevant in light of the lineage-specific components of synaptonemal complexes of well-studied ecdysozoans such as *Drosophila melanogaster* and *Caenorhabditis elegans*, both species having independently evolved functionally similar, but novel, synaptonemal complex proteins that fulfil the same functional role as SYCP1 in other metazoans, (Bogdanov I et al. [@CR6]; Bogdanov et al. [@CR7]; Colaiácovo et al. [@CR13]; MacQueen et al. [@CR46]; Page and Hawley [@CR54]; Schild-Prufert et al. [@CR67]; Smolikov et al. [@CR69]). The complete lack of SYCP1 proteins in any other ecdysozoan and evolution of lineage-specific synaptonemal proteins in both *D. melanogaster* and *C. elegans* suggest that the *Petrolisthes* sequence could be a case of misidentification or contamination. It is also possible that the 'SYCP1' hits are not, in fact, SYCP1 and that a longer sequence would reveal a lack of homology. This sequence could also simply be an instance of another coiled-coil protein, of which there are many, with the small sequence preventing proper identification. The precise point in animal evolution at which the transition was made from the typical metazoan SYCP1 system to the ecdysozoan alternatives remains to be resolved.
Conclusion {#Sec14}
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In this study, the amphioxus retrogene *AmphiSYCP1* has been characterised, highlighting its expression and regulation in relation to the surrounding genomic locus into which it has inserted. In situ hybridisation of *AmphiSYCP1* revealed widespread embryonic and somatic expression unexpected for a meiotic gene, whilst promoter and transcriptional analyses reveal that *AmphiSYCP1* seems to have not only co-opted a bidirectional promoter from the adjacent gene *AmphiCHIC*, but also evolved a de novo multi-exonic 5′ UTR in order to make use of this promoter. The conservation of this regulatory structure between *B. lanceolatum* and *B. floridae*, as well as the presence of two different *AmphiSYCP1* isoforms with differing 5′ UTR exon structures, implies an important role for this 5′ UTR structure in the regulation and expression of *AmphiSYCP1*. We also describe the expression of the adjacent gene *AmphiCHIC*. This supports the hypothesis that *AmphiSYCP1* has co-opted a bidirectional *AmphiCHIC* promoter, with *AmphiCHIC* displaying a similar expression pattern to that of *AmphiSYCP1* during embryonic development. *AmphiSYCP1* does not appear to have co-opted regulatory patterns from the adjacent ParaHox cluster, however, despite its proximity to *AmphiGsx*. Finally, phylogenetic analysis of *SYCP1* proteins from across the Metazoa supports the ancient origin of *SYCP1* even though resolution is poor outside of the Vertebrata, but in contrast to Fraune et al. ([@CR22]), we conclude that *SYCP1* has been lost within the Ecdysozoa.
Materials and methods {#Sec15}
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Origin and culture of *B. lanceolatum* individuals {#Sec16}
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Live adult *B. lanceolatum* were collected by the Plymouth Marine Laboratory, UK, and were transferred in 2011 to the aquarium system of the Gatty Marine Laboratory at the University of St. Andrews, UK, where they were kept in culture with continual aeration and circulating ambient-temperature seawater under a 16:8-h (light/dark) photoperiod until harvested. Animals were fed once or twice a day with a mixed diet of unicellular red algae *Rhinomonas reticulata* supplemented with MarineSnow (Two Little Fishies, Inc), a planktonic solution for filter-feeding marine invertebrates. Gravid animals used for gonadal RNA extraction were fixed directly in RNAlater for 24 h, and the gonads were then dissected. Embryos were collected by spawning of ripe amphioxus at the facilities of Laboratoire Aragó in the summer of 2010. These were induced by heat stimulation as described in Fuentes et al. ([@CR26]), and embryonic stages (gastrula, early neurula, mid-neurula, late neurula and early larval stages) were collected at regular intervals and fixed in 4% (*m*/*v*) paraformaldehyde in MOPS buffer for 1 h at room temperature or overnight at 4 °C and then transferred into 70% ethanol and stored at − 20 °C until use (Holland et al., [@CR30]). Embryos of mid-late neurula stages were kindly gifted by Dr. Ildiko Somorjai (University of St. Andrews).
Isolation of adult *B. lanceolatum* gonadal cDNA {#Sec17}
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Ripe gonads were dissected from a single gravid adult *B. lanceolatum* individual stored in RNAlater (Sigma). This was then rinsed in RNase-free water (Fisher Scientific) several times before being transferred to 1 ml TriReagent (Sigma) on ice. The tissue was homogenised in a D-Matrix tube (MP Biomedicals) in a Fastprep FP120 cell homogeniser (Thermo Savant) at 6 m/s for 40 s. Phenol/chloroform extractions were carried out until no denatured protein material could be observed at the aqueous/chloroform interface. The aqueous phase was then taken and precipitated with an equal volume of isopropanol, followed by a 70% ethanol wash. The dry RNA pellet was then resuspended in RNase-free water and stored at − 80 °C for long-term storage. An aliquot was stored at − 20 °C for immediate use. Due to the lack of introns within the *AmphiSYCP1* coding region, an additional DNase I treatment was carried out upon the RNA to ensure the removal of any possible genomic DNA contamination. One microlitre of DNase I (Fermentas) was added to an aliquot of the RNA solution and incubated at 37 °C for 30 min. One microlitre of 50 mM EDTA was then added to this, and the sample was heat-deactivated at 65 °C for 10 min. Pure, uncontaminated RNA was then repurified using the Isolate RNA mini kit (Bioline) according to the manufacturer's instructions. cDNA was produced from this purified adult *B. lanceolatum* gonadal RNA sample using the Tetro cDNA synthesis kit (Bioline) following the manufacturer's instructions, using oligo(dT)s to prime the reaction.
Cloning of *SYCP1* and *CHIC* transcripts {#Sec18}
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*B. lanceolatum SYCP1* coding sequence, *SYCP1* 5′ UTR and *CHIC* transcripts were obtained by PCR using BIOTAQ polymerase (Bioline) from adult *B. lanceolatum* gonadal cDNA preparations. PCRs were set up with a total volume of 50 μl in a 0.2-ml PCR tube. All reactions used 5 μl 10 × NH~4~ buffer, 2 μl 50 mM MgCl~2~ solution, 2 μl (5 μl for *AmphiSYCP1*) 10 mM dNTPs, 1 μl 20 μM forward primer, 1 μl 20 μM reverse primer, 1 μl of a one-tenth dilution of adult *B. lanceolatum* gonadal cDNA, 0.5‐1 μl 5 U/μl BIOTAQ and ddH~2~O up to a total volume of 50 μl. Primer sequences, annealing temperatures and elongation times used were as follows: AmphiSYCP1 F (**GCAGGTGTRTYATCAGCAAGAG**) and AmphiSYCP1 R (**ACTCRAAGAAGCCAAAAACAGT**) at 56 °C annealing temperature with 3-min extension time, B.la_SYCP15'UTR_v2 F (**AGAGAGGAGGAACAGAGGGATTTT**) and B.la_SYCP15′UTR_v2 R (**CCTCAACATTAGCAGCATGATCTTT**) at 58 °C annealing temperature with 45-s extension time, B.la_CHIC_ex1 F (**GAGCGGCTTATGGAGGAACA**) and B.la_CHIC_ex6 R (**AGTCTGGTCTGTGGATGGGA**) at 60 °C with 45-s extension time. PCR products for *B. lanceolatum SYCP1* coding (3121 bp), *SYCP1* 5′ UTR (307 and 246 bp) and *CHIC* (455 bp) were then gel-purified and cloned into pGEM-T Easy according to the manufacturer's instructions. These clones were then sequenced in both forward and reverse orientations using 3.2 μM T7 and SP6 primers, with the additional 3.2 μM B.la SYCP1-centre F (**AGTCTCTTCAAGATCAGCTGCAA**) and B.la SYCP1-centre R (**CTTTATCTTCGATGGTTTTCTTCA**) primers used to sequence the centre of the large 3121-bp SYCP1 coding product. Accession numbers for cloned sequences are provided in the methods below.
In situ hybridisation {#Sec19}
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PCR templates were synthesised from the *B. lanceolatum SYCP1* coding region (3121 bp), *SYCP1* 5′ UTR (307 bp) and *CHIC* (455 bp) pGEM-T Easy clones using M13 primers, and DIG labelled antisense RNA probes then synthesised from these templates using T7 polymerase. The large *SYCP1* coding antisense probe underwent an additional partial alkaline hydrolysis step by adding 30 μl 200 mM Na~2~CO~3~, 20 μl 200 mM NaHCO~3~ and RNAse-free water up to a total volume of 100 μl followed by incubation for 15 min at 60 °C. Antisense RNA probes were purified using mini quick-spin columns (Roche) according to the manufacturer's protocol. In situ hybridisation was carried out upon gastrula to pre-mouth stage *B. lanceolatum* embryos according to Holland et al. ([@CR30]) with the following modifications. Amphioxus embryos were rehydrated through an ethanol series into PBT and then digested for 5 min at room temperature in 2 μg/ml proteinase K, except for pre-mouth embryos and 2-day larvae which were proteinase K-treated for 10 min. After triethanolamine/acetic anhydride washes, embryos were washed once in PBT for 1-min with rotation, then again in PBT for 5-min with rotation. This was then changed for 100 μl of hybridisation buffer pre-warmed to 60 °C and rotated for 1 min. This was changed for fresh hybridisation buffer and rocked for 2 h. Antisense RNA probe was mixed in 1/50 dilutions in fresh warm hybridisation buffer and denatured at 70 °C for 10 min, before being added to the embryos. These were then rocked overnight at either 60 or 62 °C. RNase steps were carried out with 2 μl 10 mg/ml RNaseA and 1 μl RNaseT1 (10,000 U/ml) in 1 ml of wash solution 3, and 250 μl was added per well. After wash solution 5, 200 μl of blocking solution was added to the embryos and rotated for 3 h at room temperature. Blocking solution was replaced with 1:2000 antidigoxigenin-alkaline phosphatase (Alkaline Phosphatase) Fab fragments in blocking solution and incubated overnight at 4 °C. Embryos were washed four times NaPBT for 20 min each at room temperature, before three washes in AP− followed by three washes in AP+. AP+ was exchanged for staining buffer, and embryos were left in the dark at room temperature for the colour to develop. The final post-staining procedure consisted of three washes in AP− for 10 min each, rotating in the dark, followed by three washes in NaPBT for 10 min each, rotating in the dark. Embryos were finally fixed in 4% PFA in NaPBS for 1 h at room temperature, washed twice in NaPBT for 10 min each and transferred to 80% glycerol to clear.
Bioinformatic prediction of candidate *SYCP1* promoters {#Sec20}
-------------------------------------------------------
In order to utilise a more robust approach to promoter prediction, three independent promoter prediction programs utilising different prediction algorithms were employed: NNPP (Reese et al. [@CR59]; Reese and Eeckman [@CR61]), TSSW (Solovyev et al. [@CR71]) and WWW Promoter Scan (Prestridge [@CR58]), which uses ProScan 1.7. Default settings were used for all three prediction software programs.
Analysis of SYCP1 conservation {#Sec21}
------------------------------
The position of the retrogene *AmphiSYCP1* adjacent to the *B. floridae* ParaHox cluster was confirmed by TBLASTN search against the *B. floridae* genome, using the *M. musculus SYCP1* peptide sequence as a query sequence, and also through a BLASTN search using the previously identified *AmphiSYCP1* nucleotide sequence from the *B. floridae* ParaHox PACs 33B4 and 36D2 (Ferrier et al. [@CR21]). The resulting *B. floridae SYCP1* nucleotide and peptide sequences were then used as a query to perform both BLASTN and TBLASTN searches against the *B. lanceolatum* (*B. lanceolatum* genome consortium, unpublished) and *B. belcheri* (Huang et al. [@CR33]) genomes to confirm the presence of the *AmphiSYCP1* retrogene adjacent to the *B. lanceolatum* and *B. belcheri* ParaHox clusters. *B. floridae SYCP1* 5′ and 3′ EST reads were obtained through BLASTN searches against the NCBI EST database using the *B. floridae SYCP1* nucleotide sequence.
SYCP1 protein sequences were acquired by either TBLASTN or BLASTP searches using the *B. floridae SYCP1*, *M. musculus SYCP1* or *Hydra SYCP1* peptide sequences as a query against protein, transcriptomic shotgun assembly, whole-genome shotgun assembly and EST databases using NCBI, UNIPROT and JGI databases. Sequences were then aligned using CLUSTAL Omega (Sievers et al. [@CR68]) within Jalview (Waterhouse et al. [@CR77]), using the default settings. An 83-amino acid (aa) 'CM1' conserved domain, identified within (Fraune et al. [@CR22]), was extracted and used to determine evolutionary relationships. The analysis involved 45 amino acid sequences in total. ProtTest3.2 (Abascal et al. [@CR1]) and PHYML (Guindon et al. [@CR29]) were used to infer the best-fit model for building phylogenetic trees. Neighbour-joining and maximum-likelihood trees were determined using MEGA7 (Kumar et al. [@CR42]) and PHYML (Guindon et al. [@CR29]), respectively. A neighbour-joining tree was built using the JTT + G model with 1000 bootstraps, a gamma shape parameter of 2.157 and a 95% partial-deletion cutoff. A maximum-likelihood tree was built using the LG + G model with four discrete gamma categories, using all sites, and branch support calculated using the aLRT SH-like statistic (Anisimova and Gascuel [@CR2]), with bootstrap support values provided as a function of the aLRT statistic. CDCC39 sequences from human, sea urchin and fruit fly were obtained and used as an outgroup to help root the phylogenetic trees. This outgroup was chosen as a related coiled-coil domain protein and to maintain comparison with the results of Fraune et al. ([@CR22]).
Accession numbers {#Sec22}
-----------------
GenBank accession numbers for sequences cloned within this study are as follows:
*B. lanceolatum SYCP1* 5′UTR + coding region isoforms: \[SYCP1_3Exon_5primeUTR_Isoform_mRNA: **MF076789**, SYCP1_2Exon_5primeUTR_Isoform_mRNA: **MF076790**\].
*B. lanceolatum CHIC* mRNA fragment: \[Blan_CHIC_gonadal_mRNA: **MF399210**\].
Electronic supplementary material
=================================
{#Sec23}
Figure S1: Full-length protein alignment of metazoan SYCP1 proteins. A CLUSTAL W protein multiple alignment of SYCP1 proteins from across the Metazoa. Conservation is visualised with false colour using the Zappo colour table for amino acids. Effort was made to identify transcripts from phyla underrepresented within (Fraune et al. [@CR22]). A consensus sequence made up of the most abundant amino acid for each position is given in black. Names of species used are given to the left of the alignment and species are organised roughly according to the current known phylogeny with amphioxus species as the focus. Table S1: Metazoan SYCP1 intron length comparison. Intron, exon, and transcribed gene lengths for the *SYCP1* gene of several metazoan species are shown in base pair length (bp). The genome version and scaffold number are given for each species, or accession numbers where sequences were obtained from a clone. Table S2: Amphioxus SYCP1 predicted promoter sequences. Sequences for the predicted promoter sequences seen in figure [4](#Fig4){ref-type="fig"} are given, with bold italics denoting the position of predicted transcriptional start site (TSS) where possible. Table S3: Metazoan SYCP1 sequences. Sequences collected for protein alignment and phylogeny are listed. The species is given with the common name in brackets, along with the phylogenetic grouping of the species. Finally accession numbers for all sequences used are given. (PDF 16315 kb)
Communicated by Caroline Brennan
**Electronic supplementary material**
The online version of this article (10.1007/s00427-017-0600-9) contains supplementary material, which is available to authorized users.
MGG was supported by the University of St Andrews, School of Biology, Biotechnology and Biological Sciences Research Council DTG, and the Wellcome Trust ISSF. Work in the authors' laboratory is also supported by the Leverhulme Trust. The authors thank the members of the Ferrier and Somorjai labs (University of St Andrews) for fruitful discussions. Additionally, the authors would like to thank Dr. Hector Escriva (Observatoire Oceaneologique de Banyuls) and the *B. lanceolatum* genome consortium for allowing us to use unpublished genomic data in this study.
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"pile_set_name": "PubMed Central"
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Background {#Sec1}
==========
*Rehmannia glutinosa* Libosch, one of the most common and important medicinal plants in China, is recorded in Chinese medical classic 'Shennong's Herba'. *R. glutinosa* based on the different processing methods is classified into three categories, namely fresh roots, dried roots (Dihuang), and steamed roots (Shu Dihuang), and they are applied to treat different diseases clinically. Shu Dihuang is usually applied to in the traditional Chinese medicine formulas that can nourish the Yin deficiency of liver, kidney and heart \[[@CR1]--[@CR3]\]. Many clinical and experimental studies have reported that the root of *R. glutinosa* possesses hypoglycaemic \[[@CR4]\], anti-oxidant \[[@CR5], [@CR6]\] anti-cancer \[[@CR7]\], anti-inflammatory \[[@CR8]\], and immune-enhancement effects \[[@CR9]\].
Iridoid glycosides are considered the main active ingredients in Dihuang. At present, a number of chemical constituents from Dihuang, such as iridoids, ionone glucosides, sesquiterpenes and phenylethanoid glycosides \[[@CR10]--[@CR12]\], have been isolated and identified. In the Chinese Pharmacopoeia (Ch. P), catalpol and verbascoside were quantified by HPLC methods to control the quality of Dihuang and Shu Dihuang, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging method was applied to distinguish verbascoside through thin layer chromatography (TLC) \[[@CR13]\]. However, these methods might not be able to reveal efficiently the bioactivity of the compounds in Dihuang and Shu Dihuang.
Free radicals may be involved in cancer, ageing and cardiovascular diseases \[[@CR14]\]. There has been a large research focus on antioxidants, especially plant-derived antioxidants. In order to identify efficiently antioxidants in complex extracts, the combinational techniques such as HPLC-DAD-DPPH, HPLC-DAD-ABTS, DPPH-CE-DAD and ABTS-CE-DAD for the analysis of DPPH^•^ and ABTS^+•^ scavenging activities, have been developed to screen radical scavengers in herbal medicines \[[@CR15]--[@CR19]\]. In these on-line methods, antioxidants separated by HPLC and CE will produce their negative peaks in real time detected at 517 nm for DPPH^•^ or at 414/734 nm for ABTS^+•^. This avoids the long and tedious process of bioassay-guided fractionation and isolation for structure elucidation in searching antioxidants from complex matrixes. Although the on-line methods have been used to evaluate the antioxidant activity of some herbal medicines, their effectiveness and extensive application need to be further verified.
In this study, an on-line HPLC-UV-ABTS method was developed to screen antioxidants in Dihuang and Shu Dihuang. The chromatographic and active fingerprints could be simultaneously obtained, and antioxidants from the two herbs were compared and identified by HPLC-FTMS. The angle cosine method was used to analyze the similarities of the chromatographic and active fingerprints. Thus, the quality of the herbs could be assessed comprehensively through the proposed research methods.
Methods {#Sec2}
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Materials and reagents {#Sec3}
----------------------
The information of the Dihuang and Shu Dihuang samples was listed in Additional file [1](#MOESM1){ref-type="media"}. Thirteen batches of Dihuang and 13 batches of Shu Dihuang samples were purchased from eleven manufacturers in China. Seven batches of Dihuang samples from the five manufacturers in Hubei province were recorded as S01, S02, S03, S04, S05, S06 and S12. Four batches of Dihuang samples from the four manufacturers in Anhui province were labeled as S07, S08, S09 and S13, while S10 and S11 were collected from the manufacturers of Guangzhou and Hangzhou, respectively. The thirteen of batches of Shu Dihuang samples from the same manufacturers were labeled as P01--13. Verbascoside was purchased from Chromadex (Irvine, America). ABTS was purchased from TCI (Shanghai, China).
HPLC and MS grade acetonitrile were obtained from Merck Drugs & Biotechnology (Darmstadt, Germany). Formic acid (FA) was purchased from Aladdin industrial corporation (Shanghai, China). Sodium chloride (NaCl), sodium dihydrogen phosphate (NaH~2~PO~4~), potassium chloride (KCl), potassium persulfate (K~2~S~2~O~8~), disodium hydrogen phosphate (Na~2~HPO~4~), sodium dihydrogen phosphate (NaH~2~PO~4~), sodium phosphate (Na~3~PO~3~) and acetic acid were analytical reagent grade. The water used was purified from a Millipore water purification system (Millipore, Bedford, MA, USA).
Preparation of sample and standard solutions {#Sec4}
--------------------------------------------
All of the Dihuang and Shu Dihuang samples were dried at 60 °C under reduced pressure and pulverized to coarse powder. Sample powder (3 g) was extracted twice with 70 mL methanol for 40 min by ultra-sonication at room temperature. The extract solutions were filtered and mixed, then evaporated to dryness under vacuum and diluted with 15% ethanol to 10 mL. After being centrifuged at 10000 rpm for 10 min, an aliquot of 20 μL solution was analyzed by HPLC.
Verbascoside (7.78 mg and 3.81 mg) was weighed accurately and dissolved in 50 mL by methanol as the stock solutions. The different concentrations solutions, such as 3.05、6.10、18.7、50.3、99.6 and 155.6 μg/mL, were obtained through diluting the stock solutions with methanol. The standard solutions were used to set up the correlative graphs and equations between inhibiting peak areas and the concentrations of verbascoside.
Preparation of ABTS solution {#Sec5}
----------------------------
A stock solution was prepared with ABTS (0.110 g) dissolved in 100 mL of PBS solution (4.1 g NaCl, 0.135 g NaH~2~PO~4~, 0.7 g Na~2~HPO~4~ and 0.075 g KCl in 500 mL) containing 0.3 mM K~2~S~2~O~8~. A 2.0 mM stock solution was diluted using PBS solution containing 10% methanol to 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mM, respectively. In on-line analysis, ABTS solution was freshly prepared and protected from light and cooled in an ice bath.
HPLC-UV-ABTS assay {#Sec6}
------------------
The HPLC-UV-ABTS system was reported in our previous publication \[[@CR20]\]. The on-line instrumentation consisted of a Waters HPLC for chromatographic fingerprint analysis at 334 nm or 250 nm and an Ultimate 3000 UV detector for ABTS^+•^ scavenging analysis at 734 nm. ABTS solution was delivered with an injection pump of Pickering PCX Della (Pichering Laboratories Inc., USA) at the flow rate of 0.5 mL/min after PDA detector, and then the elution was mixed with ABTS solution after PDA detector through a reaction coil with 1.4 mL volume. The reaction products were determined at 734 nm by UV detector (Ultimate 3000). All other parts of the HPLC-UV-ABTS system were interconnected using polyether ether ketone (PEEK, 0.5 mm i.d.) tubes and T-shaped PEEK tubes.
Agilent Extend C~18~ columns (250 mm × 4.6 mm, 5 μm) were used for all chromatographic separations. The mobile phase comprised 0.1% (v/v) acetic acid (A) and acetonitrile (B). Gradient elution was performed as follows: 2--4% B in 0--10 min, 4--15% B in 10--40 min, 15--25% B in 40-55 min, 25--30% B in 55-60 min and 30--95% B in 60-65 min. The flow rate was 1.0 mL/min and column temperature was maintained at 30 °C. The detection wavelength was set at 334 and 250 nm for acquiring chromatograms. An aliquot of 20 μL sample solution was injected for HPLC-UV-ABTS analysis.
HPLC-FTMS analysis {#Sec7}
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Thermo Scientific Orbitrap Fusion Tribrid HPLC-MS system was controlled by Xcalibur software (Version 3.0). The chromatographic conditions were the same as those in the HPLC-UV-ABTS analysis. The mobile phase was split after the DAD detector by a T-tube connected using two PEEK tubes with the same inner i.d. and different length. The split solution with a flow rate of 0.2 mL/min finally arrived at the MS detector.
The MS experiments were performed to get an accurate MS and MS^2^ of the new analogue. The ionization source was operated in the negative ionization modes with the flow rates of the sheath gas and auxiliary gas at 40 and 10 arbitrary unit, respectively, capillary temperature at 320 °C, ion spray source capillary at 2.5 kV, source current at 100 μA. Six scan events were selected in the MS experiment. Scan event 1 was used for full scan with scan range from 100 to 1000 m/z and resolution 60,000. Scan events 2--6 were used to produce MS^2^ through dependent scans selecting the 1th to 5th most intense ions in scan event 1 and resolution 15,000. Collision energy was set at 35 V using High Energy Collision Dissociation (HCD).
Similarity of the two-dimensional fingerprint {#Sec8}
---------------------------------------------
It is well known that the samples with similar chromatographic fingerprint possess likely similar properties. Therefore, the consistency of herbal medicines can be tested through comparing the similarity between the chromatographic fingerprints of samples and the reference/standard fingerprints.
In this paper, a data analysis method was employed to evaluate the similarity of the two-dimensional fingerprint \[[@CR21]\]. The chromatographic and active fingerprints from HPLC-UV-ABTS method were represented mathematically by a vector of their chromatographic peak areas and inhibiting peak areas. Thus, taking the two-dimensional fingerprint as an example, assume that vector *X* (*x*~1~, *x*~2~, *x*~3~, ..., *x*~n~) (\| *X* \| = $\documentclass[12pt]{minimal}
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\begin{document}$$ \sqrt{{x_1}^2+{x_2}^2+{x_3}^2+\cdots \cdots {x_n}^2} $$\end{document}$) represents the chromatographic or active fingerprint and the other vector *Y* (*y*~1~, *y*~2~, *y*~3~,..., *y*~n~) (\| *Y* \| = $\documentclass[12pt]{minimal}
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\begin{document}$$ \sqrt{{y_1}^2+{y_2}^2+{y_3}^2+\cdots \cdots {y_n}^2} $$\end{document}$) represents the reference fingerprints, while the vector angle of *X* and *Y* is calculated by the formula. The two vectors are more similar when the cosine values are near 1. $$\documentclass[12pt]{minimal}
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\begin{document}$$ \cos \theta =\frac{X\cdot Y}{\mid X\mid \times \mid Y\mid } $$\end{document}$$
Where *x*~i~ denotes absolute peak area of chromatographic or active fingerprints, and *y*~i~ denotes mean area of the peak.
Results {#Sec9}
=======
Optimization of HPLC-UV-ABTS analysis conditions {#Sec10}
------------------------------------------------
HPLC-UV-ABTS was developed for the determination of free radical scavengers in the complex matrixes due to a relatively simple and stable instrument system. Methanol/acetonitrile-acid aqueous solution used as mobile phases were compatible with the on-line detection of ABTS^+•^ inhibition after column or UV detector. Baseline was relatively stable due to the buffer effect of ABTS dissolved in buffer solutions to mobile phases.
The concentrations of the ABTS solution affected directly the sensitivity of the HPLC-UV-ABTS method. In view of this, the different concentrations of ABTS including 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mM were investigated. The results showed that the stronger inhibition peaks were produced when the lower concentrations of ABTS were selected, while the higher concentrations induced strong baseline draft. Finally, 0.6 mM ABTS was used to analysis the antioxidants in the two herbs. Additionally, the flow rates at 0.25 and 0.5 mL/min for the ABTS solution were optimized. The flow rate of 0.5 mL/min was finally selected owing to a long time delay from the lower flow rate for ABTS^+•^ scavenging analysis.
Method validation for HPLC-UV-ABTS {#Sec11}
----------------------------------
The analytical methods of the chromatographic and active fingerprints were validated based on retention times, peak areas and ABTS^+•^ inhibition peak areas. The intra-day precisions of HPLC-PDA were 0.086--0.75% (*n* = 5) for retention times and 0.70--1.46% (*n* = 5) for peak areas, while the inter-day precisions were 0.10--0.83% (*n* = 5) for retention times and 1.18--3.37% (*n* = 5) for peak areas. The intra-day precisions of HPLC-UV-ABTS were within the range of 0.028--0.93% (*n* = 5) for retention times and 0.069--0.95% (*n* = 5) for negative peak areas, whereas the inter-day precisions were 1.23--4.73% (*n* = 5) for retention times and 1.66--5.71% (*n* = 5) for negative peak areas.
On-line HPLC-UV-ABTS analysis {#Sec12}
-----------------------------
The chromatographic and active fingerprints of Dihuang and Shu Dihuang were shown in Fig. [1](#Fig1){ref-type="fig"}. Although chromatographic fingerprints of Dihuang and Shu Dihuang were similar, their active fingerprints were different. Fourteen negative peaks were observed in Dihuang (Fig. [1](#Fig1){ref-type="fig"}a), while there were 17 inhibition peaks in Shu Dihuang (Fig. [1](#Fig1){ref-type="fig"}b). The negative peaks 1, 2, 3, 4, 8, 9, 10 and 16 in Shu Dihuang were stronger than those in Dihuang. In addition, several new negative peaks were found in Shu Dihuang such as 5, 6 and 7, and negative peak 12 in Dihuang disappeared. Fig. 1The chromatographic fingerprints and ABTS^+•^ inhibition profiles of Dihaung S07 (**a**) and Shu Dihuang P07 (**b**) at 334 nm and the partial chromatograms of the two samples at 250 nm. A partial chromatogram at 250 nm from 15.0 min to 21.0 min in chromatogram of Dihuang, a partial chromatogram at 250 nm from 12.0 min to 24.0 min in chromatogram of Shu Dihuang
In Fig. [1](#Fig1){ref-type="fig"}a and b, few peaks were observed from 10 min to 40 min in the chromatographic fingerprints of the two herbs detected at 334 nm, while some weak peaks could be found at 250 nm and these peaks in Shu Dihuang showed obvious ABTS^+•^ inhibition activity, such as peaks 3--9. In view of stronger chromatographic signal at 334 nm for iridoid glycosides as main components in the two herbs, the chromatographic fingerprints were detected at this wavelength. Furthermore, ABTS^+•^ scavenging activity was detected usually at 414 nm and 734 nm, but the wavelength at 734 nm was selected in order to avoid the interference from the herb extracts at 414 nm.
The activity evaluation of antioxidants in samples {#Sec13}
--------------------------------------------------
As shown in Fig. [1](#Fig1){ref-type="fig"}, there were 18 antioxidants in the Dihuang and Shu Dihuang samples and their capacities for scavenging ABTS^+•^ were obviously different. However, it was difficult to evaluate and compare the activity of the antioxidants due to the impossibility of all the standards obtained. Some publications reported that the relative potencies of the antioxidants in complex extracts could be evaluated by the "quantity-effect" equation of a positive control \[[@CR22], [@CR23]\]. Thus the potencies of the antioxidants against free radical could be compared and antioxidant activity of a complex extract could be obtained through calculating the total potency of all the antioxidant components.
In Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}, verbascoside showed strong ABTS^+•^ scavenging activity in all the Dihuang and Shu Dihuang samples, so it was applied as a positive control. The functional equation of verbascoside y = 2E-07 × ^4^ - 8E-05 × ^3^ + 0.0079 × ^2^ + 0.5755x + 1.4754 (*R*^2^ = 1) was generated using negative peak areas (x) and C (y) (Fig. [3](#Fig3){ref-type="fig"}) (Additional file [2](#MOESM2){ref-type="media"}). If verbascoside (1 μg/ml) was presumed as a potency unit, the potencies of 18 antioxidants in the samples could be calculated by the functional equation (Additional file [3](#MOESM3){ref-type="media"}). Fig. 2The chromatographic fingerprints and ABTS^+•^ inhibition profiles of 13 batches of Dihuang and 13 batches of Shu Dihuang samples. **a** Dihuang, **b** Shu Dihuang. The chromatographic fingerprints detected at 334 nm. ABTS+• inhibition profiles detected at 734 nm Fig. 3The correlative graphs and equations between the negative peak areas and concentrations for verbascoside against ABTS^+•^
The potencies of 18 antioxidants in all of the Dihuang samples and Shu Dihuang samples were shown in Fig. [4](#Fig4){ref-type="fig"}a and b. The potencies of the antioxidants in Dihuang and Shu Dihuang were markedly different. In Fig. [4](#Fig4){ref-type="fig"}a, peak 2, 11, 12, 13 and 14 in the Dihuang samples showed strong ABTS^+•^ inhibition activity, while the antioxidant potencies of peak 1, 2, 3, 4, 11, 13, 14 and 16 in the Shu Dihuang samples were twice to ten times than those in the Dihuang samples (Fig. [4](#Fig4){ref-type="fig"}b). In addition, the activity of peak 1, 2, 3, 4, 8 and 16 in Shu Dihuang increased and this might attributed to the processing action of Dihuang. In our previous study, the HPLC-UV-DPPH method was developed to compare DPPH^•^ scavengers in Dihuang and Shu Dihuang \[[@CR24]\], and the results were consistent with the above the results of HPLC-UV-ABTS analysis. Fig. 4The potencies of the chromatographic peaks in the Dihuang and Shu Dihuang samples. **a** Dihuang, **b** Shu Dihuang
Main antioxidants in Dihuang and Shu Dihuang {#Sec14}
--------------------------------------------
The eighteen negative peaks in Fig. [1](#Fig1){ref-type="fig"} were identified by HPLC-FTMS and comparison with reference standards and the data in related literatures \[[@CR12]\], and the MS data were listed in Table [1](#Tab1){ref-type="table"}. As shown in Table [1](#Tab1){ref-type="table"}, the most common inhibition peaks were iridoid glycosides, such as peaks 6--18 except for peak 8. In the Dihuang samples, the stronger negative peaks 11, 12, 13, 14 and 16 were identified as echinacoside, isomer of echinacoside, jionoside A1/A2, verbascoside and isoverbascoside, respectively. While peak 1, 4, 8 and 9 showing stronger activity in the Shu Dihuang samples were identified as glutinoside, geniposidic acid, mussarnosidic acid, and syringic acid-4-O-α-*L*-rhamnoside, respectively. Peaks 2 and 3 needed to be further identified. Table 1Identification of active components in Dihuang and Shu Dihuang sample by HPLC-FTMSPeakRT\[M-H\]-Chemical formulaMS^2^Identification16.22397.0238C~15~H~23~O~10~Cl379.1123,276.9031,252.2112glutinoside28.55495.1254C~26~H~23~O~10~477.1181,379.1190, 217.0684, 199.0581unknown314.37731.2100C~29~H~33~O~14~N~9~505.1492, 323.0935, 263.0734,221.0633unknown417.25373.1073C~16~H~22~O~10~283.0792,211.0580,167.0687,123.0431geniposidic acid518.61375.1246C~16~H~24~O~10~315.1030,255.0831,213.0735,169.0843, 125.05868-epiloganic acid619.60389.1168C~17~H~26~O~10~183.0634,165.0531,139.0377loganin720.55461.1592C~20~H~30~O~12~315.1039,297.0937,161.0429,135.0429decaffeoylacteoside821.94375.1246C~16~H~24~O~10~315.1021,213.0738,169.0843,151.0740mussarnosidic acid929.57343.0262C~15~H~20~O~9~299.1118,284.0858,197.0424,182.0192syringic acid-4-*O*-α-*L-*rhamnoside1032.87475.1747C~21~H~32~O~12~329.1194,311.1093,143.0583,161.0429darendoside B1141.02785.2394C~35~H~45~O~20~623.2108,461.1603,161.0218echinacoside1243.60785.2394C~35~H~45~O~20~623.2108,461.1603,161.0218isomer of echinacoside1345.74799.2570C~36~H~48~O~20~623.2102,605.1965,461.1597,315.1040jionoside A1/A21450.14623.1865C~29~H~36~O~15~477.1354,461.1599,443.1508,315.1039,179.0321,161.0218verbascoside1550.98813.2729C~37~H~50~O~20~637.2254,491.1701,193.0477,175.0373jionoside B1/B21652.17623.1865C~29~H~36~O~15~461.1600,315.1040,251.0525,179.0322,161.0218isoverbascoside1754.61637.2048C~30~H~38~O~15~491.1492,475.1773,461.4599,443.1497jionoside D/leucosceptoside A1859.89651.2176C~31~H~40~O~15~505.1632 475.1754 193.0477martynoside/isomer
Iridoid glycosides are main ingredients in Dihuang and Shu Dihuang, and verbascoside exhibiting stronger antioxidant activity is used to control quality of the two herbs in Ch. P. However, the activity of the other iridoid glycosides can't be ignored. The contributions of 18 antioxidants in the Dihuang and Shu Dihuang samples to the total activity were shown in Fig. [5](#Fig5){ref-type="fig"} (Additional file [4](#MOESM4){ref-type="media"}). In Fig. [5](#Fig5){ref-type="fig"}a, peaks 2, 11 and 14 in the Dihuang samples exhibited their high contributions, and the total contribution percentage of the three main active components in the Dihuang was 39.2--58.1%. Peaks 1, 2, 3, 11, 14 and 16 in the Shu Dihuang samples showed their high contributions (Fig. [5](#Fig5){ref-type="fig"}b), and the total contribution percentage of peak 2, 11 and 14 was 55.9--69.4%. In addition, the contributions of peak 1, 2, 3, 4, 8 and 16 in the Shu Dihuang samples were twice to twenty times than those in the Di Huang samples, while the contributions of peak 11, 13 and 14 in the Shu Dihuang samples decreased. The results indicated that the antioxidant activity of Dihuang might be changed after being processed. Fig. 5The contributions of the chromatographic peaks in the Dihuang and Shu Dihuang samples to the total activity. **a** Dihuang, **b** Shu Dihuang
Similarity for Dihuang and Shu Dihuang samples {#Sec15}
----------------------------------------------
The chromatographic and active fingerprints of the thirteen batches of the Dihuang and Shu Dihuang samples from the different manufacturers were displayed in Fig. [2](#Fig2){ref-type="fig"}. As shown in Fig. [2](#Fig2){ref-type="fig"}a and b, the chromatographic and active fingerprints among the different samples were similar. In order to evaluate objectively the quality of the samples, the angle cosine method was employed to analyze the similarities of the two fingerprints. The angle cosine values of each chromatographic and active fingerprint to their respective reference chromatogram were calculated and listed in Table [2](#Tab2){ref-type="table"}. The raw data were shown in Additional file [5](#MOESM5){ref-type="media"} and Additional file [6](#MOESM6){ref-type="media"}. In Table [2](#Tab2){ref-type="table"}, the different common peaks were used to evaluate the similarities of the two fingerprints. The peak 12 was not detected in the Shu Dihuang samples, so 8 common peaks were selected in the similarity evaluation of the chromatographic fingerprints. Fifteen and seventeen common peaks were used to analyze the similarity of the active fingerprints of the Dihuang and Shu Dihuang samples, respectively. Table 2Similarity of Dihuang and Shu Dihuang samplesSamplesDihuangSamplesShu DihuangHPLC (9 common peaks)ABTS (15 common peaks)HPLC (8 common peaks)ABTS (17 common peaks)S010.9940.982P010.9910.969S020.9540.957P020.9640.959S030.9030.898P030.9020.854S040.9520.958P040.9520.980S050.9880.978P050.9680.961S060.9750.969P060.9760.955S070.9940.974P070.9840.984S080.9890.974P080.9320.944S090.9920.988P090.9870.991S100.9910.934P100.9830.966S110.9410.946P110.9460.975S120.8400.716P120.9740.984S130.9940.993P130.9930.989
As shown in Table [2](#Tab2){ref-type="table"}, the chromatographic and active fingerprints of the samples represented their differences to some extent. The similarity values of S03 and S12 in the two fingerprints were lower than those of the other samples, and their similarity values in the active fingerprints were less than 0.9. Moreover, the similarity value of P03 in the chromatographic fingerprints was lower than that of the other samples and less than 0.9 in the active fingerprints. Although the similarity values of the two fingerprints were differences, the quality of the Dihuang and the Shu Dihuang samples were basically consistent except for the several samples.
Discussion {#Sec16}
==========
In this study, the HPLC-UV-ABTS method was developed to evaluate the main antioxidants and quality of the two herbs. Sensitivity of the on-line method correlated mainly concentration and flow rate of ABTS solution. Lower concentrations of ABTS solution were used, and stronger inhibition peaks in the samples could be obtained. Considering strong baseline drift induced by the higher concentrations of ABTS solution, 0.6 mM within the range of 0.3--0.8 mM was selected for the on-line analysis. Moreover, flow rate of ABTS solution affected directly the reaction times of the compounds and ABTS^+•^ when volume of reaction coil (1.4 mL) and flow rate of HPLC elution were stable. Flow rate of 0.5 mL/min for ABTS solution was applied due to a long time delay and lower resolution of the active fingerprints from the lower flow rates. Under the above analysis conditions, the method validation of HPLC-UV-ABTS was investigated through the intra-day and inter-day precisions of the chromatographic peaks, the negative peaks and retention times. The results indicated that the on-line method was stable and reproducible.
The chromatographic fingerprints of all the samples were detected at 334 nm, while some minor peaks were found in the chromatographic fingerprints of Shu Dihuang at 250 nm and showed their ABTS^+•^ scavenging activity. These minor peaks are hardly focused on owing to their weak signals in the chromatographic fingerprints. Chromatographic fingerprint mainly showing characteristics of strong peaks gave too much weight to major peaks than to minor peaks. In view of this, the similarities of the chromatographic and active fingerprints were evaluated and compared by the vector angle cosine method. Similarity values of several samples were less than 0.9. This might imply their different quality with the other samples. Thus, the combination method of the chromatographic and active fingerprints could evaluate objectively the quality of herbal medicines.
In order to assess and compare the activity of the components in the Dihuang and Shu Dihuang samples, verbascoside was used as a positive control to obtain the relative activity of the ABTS^+•^ inhibition peaks. The results found that the active components in the two herbs were markedly different. Contents of some components in Dihuang were changed after being processed so as to induce their different activity in the two herbs. This might imply the different therapeutic effects of the two herbs in clinical to some extent. Thus, the "quantity-effect" research method displayed its important action in the quality evaluation and identification of the active compounds of herbal medicines.
Conclusion {#Sec17}
==========
In this study, the on-line HPLC-UV-ABTS method and a "quantity-effect" research idea were developed to evaluate the quality and antioxidant activity of the Dihuang and Shu Dihuang samples from the different manufacturers. The results revealed that the antioxidants in the Shu Dihuang samples were obviously different from those in the Dihuang samples owing to the contents of some components having changed after Dihuang was processed. Iridoid glycosides were the main antioxidants and their contributions to the total activity were higher, while the new produced ingredients in Shu Dihuang displaying significant ABTS^+•^ inhibition activity could not be ignored. In short, the HPLC-UV-ABTS method was simple, rapid and reliable and could be applied widely to screen the antioxidants and evaluate the antioxidant activity of the complex extracts. The combination method of the chromatographic and active fingerprints could evaluate integratedly the active components of the complex matrixes and might be valuable and meaningful for improving the quality control of herbal medicines.
Supplementary information
=========================
{#Sec18}
**Additional file 1.** A table for manufacturer source of the Dihuang and Shu Dihuang samples. **Additional file 2.** The raw data for the functional equation of verbascoside as a positive control, negative peak area (x), concentration (y). **Additional file 3.** The peak areas and potencies of the negative peaks in the thirteen batches of Dihuang and Shu Dihuang samples. Potency of each negative peak calculated by the functional equation of verbascoside. **Additional file 4.** The raw data of Figs. [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}. The potencies of 18 negative peaks in 13 batches of Dihuang and Shu Dihuang samples. **Additional file 5.** The potencies of the negative peaks in the active fingerprints and their similarity evaluation. The similarity values of the active fingerprints of 13 batches of Dihuang and Shu Dihuang samples calculated by the angle cosine formula. **Additional file 6.** The peak areas of the chromatoghraphic fingerprints and their similarity evaluation. The similarity values of the chromatographic fingerprints of 13 batches of Dihuang and Shu Dihuang samples calculated by the angle cosine formula.
ABTS
: 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
BCD
: Biological chemistry detection
Ch. P.
: Chinese Pharmacopoeia
DPPH
: 2,2-diphenyl-1-picrylhydrazyl hydrate
HPLC
: high performance liquid chromatographic
HPLC-FTMS
: HPLC-fourier-transform mass spectrometry
HPLC-PDA
: HPLC-photo-diode array
HPLC-UV-ABTS
: high performance liquid chromatographic-ultraviolet detection-ABTS
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Hong-Ying Li and Jiang-Ji Fang are contributed equally to this work and should be considered co-first authors.
Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1186/s12906-019-2798-8.
We are pleased to thank Thermo Fisher Scientific for technical support.
HL and JF contributed equally to this work. HL and JF conceived and designed the study. HS performed the HPLC-FTMS analysis experiment and collected the data. XZ analyzed the data and supervised the work. XD collected samples from different manufactories and wrote the manuscript. JL involved in the initial experimental design, and provided a great help and careful guide for the twice modification of the manuscript including the data processing analysis and grammar part. All authors read and approved the final manuscript.
This work was financially supported by Natural Science Foundation of China (Grant no. 81403093), Science Foundation of Hubei Food and Drug Administration (Grant no. 2016010 + 02) and Natural Science Foundation of Hubei Province (No. 2015CFB493). The on-line HPLC-UV-ABTS method derived from the study of Natural Science Foundation of China. This work provided research data to finish Science Foundation of Hubei Food and Drug Administration. The design and idea of this study were based on Natural Science Foundation of Hubei Province.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
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Introduction {#s1}
============
Innate-like T cells (CD1-restricted T cells or MHC-related molecule-1 (MR1)-restricted T cells) are classified as innate lymphoid cells that have features similar to those of the cells involved in acquired immunity, such as T cell receptor (TCR) expression ([@B1]). However, their TCR repertoire is very limited, and they recognize self or exogenous non-peptide antigens presented by a family of non-polymorphic and MHC class I-related molecules ([@B1]).
NKT cells are characterized by the expression of TCRs with a limited repertoire, consisting of Vα14 and Jα18 (in mice) or Vα24 and Jα18 (in humans) ([@B2]). In addition, their sets of Vβs are also skewed toward mainly Vβ8.2 (in mice) and Vβ11 (in humans). Since NKT cells have limited TCRs, they are called invariant natural killer T (iNKT) cells. α-galactosylceramide (α-GalCer) presented by CD1d is the most potent and well-analyzed ligand that activates iNKT cells ([@B2]). Activated iNKT cells regulate various immune responses to protect us from tumors or infectious diseases ([@B3], [@B4]). However, these cells can also contribute to chronic inflammatory disease, such as allergic inflammation and autoimmune responses ([@B5], [@B6]).
Like iNKT cells, mucosal-associated invariant T (MAIT) cells express a semi-invariant TCR with a unique TCRα chain (Vα19-Jα33 in mice, Vα7.2-Jα33 in humans) and a restricted set of TCRβ chains ([@B7]). MAIT cells are activated by a bacterial riboflavin derivative presented by MR1 ([@B8]). Although MAIT cells have been suggested to play a role in antibacterial immunity through sensing MR-1-bound microbial products, it has been speculated that these cells may also be involved in regulating beneficial host commensal interactions in the intestine and potentially in the lung ([@B9], [@B10]). As well as participating in antimicrobial immunity, MAIT cells may be involved in the control of chronic inflammation ([@B11]).
Another interesting feature of these MAIT and iNKT cell populations is their memory-like phenotype ([@B12]). They are able to produce effector cytokines, cytolytic molecules, and growth factors at early time points of immune responses. Therefore, they are considered to play an essential role in the host defense immune responses. Both populations have distinct and characteristic tissue localization, as NKT cells reside in the thymus, spleen, lung and liver, while MAIT cells are preferentially found in the gut lamina propria, lung and liver ([@B13], [@B14]). NKT cells are relatively abundant in mice and show a lower frequency in humans, whereas the opposite situation is true for MAIT cells ([@B15]).
Upon activation, iNKT cells produce a large amount of both Th1 and Th2 cytokines in addition to inflammatory cytokines, such as interleukin (IL)-17 and tumor necrosis factor α (TNFα). iNKT cells show heterogeneity in their transcriptional factors. Three different subsets of iNKT cells have been shown to produce distinct cytokines, defined as NKT1, NKT2 and NKT17 ([@B16], [@B17]). iNKT cells are also distinguished by their surface molecules, such as CD4 and IL-17RB. CD4^+^IL-17RB^+^ iNKT cells in particular produce IL-13, IL-9, IL-10, IL-17A, and IL-22 ([@B18]). Since these cytokines exert different immunoregulatory functions, certain populations of iNKT cells might contribute to the development of chronic disorders, such as allergic diseases ([@B18]). MAIT cells produce IFN-γ, IL-4, IL-17, and TNFα ([@B12], [@B19]). Lepore et al. suggested that IL-5 and IL-13 can be produced by MAIT clones ([@B20]). It would be possible to identify distinct subsets of MAIT cells, that can produce specific cytokines such as is observed in the case of iNKT cells.
Asthma is a chronic inflammatory disease in the lung that causes recurring periods of wheezing, chest tightness, shortness of breath and coughing. It is well established that allergic asthma is induced by Th2 cell-mediated immune responses ([@B21]). Studies by our group and other authors have revealed that chronic airway inflammation in asthma patients is caused by pathogenic memory Th2 cells, which express high levels of IL-33 receptor ST2 and have a CD161^high^CRTH2^high^ phenotype in human ([@B22]--[@B25]).
Memory T cells are considered to play a beneficial role by responding immediately and strongly to the secondary invasion by the same antigen of a microorganism ([@B26]). However, memory T cells can induce adverse effects in cases of chronic inflammatory disease if they respond to allergens or self-antigens repeatedly for a long duration ([@B27]). Thus, allergen-specific memory Th2 cells, particularly the pathogenic subpopulation of ST2^+^ Th2 cells paly important roles in the pathogenesis of IL-5-induced eosinophilic inflammation and fibrotic responses ([@B24], [@B28]). However, it is also recognized that asthmatic patients show heterogeneous phenotypes, including so-called type-1 and type-2 mixed inflammation with neutrophilic infiltration. Thus, other cell types, such as NKT cells likely contribute to the development or exacerbation of asthma ([@B29]).
This review describes and discusses the immunoregulatory roles of innate-like T cells in asthma in animal models and human patients.
Beneficial and detrimental effects of iNKT cells for allergic asthma {#s2}
====================================================================
Many investigators have tried to determine the roles of iNKT cells in asthma over the past 20 years. To this end, Akbari et al. assessed OVA-induced airway hyperactivity (AHR) and allergic airway inflammation in iNKT cell-deficient Jα281 knockout (KO) and CD1d KO mice ([@B30]). They noted a significant defect in the development of AHR and inflammation in these NKT cell deficient mice. The defects were corrected by the adoptive transfer of iNKT cells in an IL-4- and IL-13-dependent manner. Therefore, iNKT cells were considered to contribute to the development of AHR and airway inflammation independent of Th2 cells. In addition, the same group showed that non-classical NKT cells, which are restricted to a β2-microgloblin-independent form of CD1d, also contribute to the development of AHR ([@B31]). Woo et al. further suggested that iNKT cells are also required for the generation of Th2 cells by recruiting CD103^+^ dendritic cells (DCs) to the lung via the XCL1-XCR1 axis ([@B32]). Furthermore, another group suggested that iNKT cells act as an adjuvant to enhance allergic asthma, as systemic iNKT cell activation by α-GalCer administration or adoptive transfer of iNKT cells before OVA challenge significantly augmented the Th2 inflammatory responses ([@B33]). These results indicate that iNKT cells have detrimental effects in allergic asthma.
Simultaneously, other groups reported experimental results indicating that iNKT cells are not involved in the development of allergic asthma. OVA-induced allergic inflammation was not reduced in CD1d-deficient mice or β2-microgloblin KO mice lacking iNKT cells ([@B34], [@B35]). Moreover, a protective role of iNKT cells in allergic asthma was suggested. Subsequent AHR in these models can be suppressed by the systemic activation of iNKT cells by α-GalCer treatment or the transfer of α-GalCer-loaded bone marrow-derived DCs before OVA challenge in an IFN-γ-dependent manner ([@B36], [@B37]). In addition, Grela et al. reported that IFN-γ-producing iNKT cells stimulated with toll like receptor (TLR) 7 agonist (R848) attenuated allergic asthma, which is consistent with the finding that TLR7 stimulation not only enhances viral responses but also alleviates experimental asthma ([@B38]).
Thus, iNKT cells display either beneficial or detrimental effects in allergic asthma. These conflicting effects may be due to the various cytokine production patterns of iNKT cells under different conditions. IL-4 or IL-13 production from iNKT cells is required for the development of allergic asthma in mouse models, while iNKT cells can produce IFN-γ, which can suppress the Th2 response and thereby prevent allergic asthma. However, even when employing similar protocols, different institutes obtained completely different findings ([@B33], [@B36], [@B37]). Since iNKT cells can detect bacterial components through their invariant TCRs or Toll-like receptors, the difference in the lung microbiota may affect the function of distinct iNKT cell subsets, such as NKT1 and NKT2.
Although inducing Th1 bias by iNKT cell activation may result in the inhibition of AHR and eosinophilic infiltration, our recent study shed light on how NKT cell activation can suppress Th2 type inflammation. While immunological memory plays a central role in providing protection against infection or cancer, antigen-specific memory CD4 T cells contribute to the pathogenesis of allergic and autoimmune disorders by recognizing allergens or self-antigens ([@B24], [@B39]). Our data showed that the activation of iNKT cells with α-GalCer during the memory phase resulted in the downregulation of IL-4, IL-5, and IL-13 and up-regulation of IFN-γ in memory Th2 cells ([@B40]). These functionally altered memory Th2 cells display a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. We therefore concluded that activated iNKT cells directly regulate memory Th2 cell function *in vivo*. Chang et al. showed another inhibitory mechanism for allergic disorder by iNKT cells. They found that influenza infection in neonates helped prevent allergic asthma by inducing CD4^neg^CD8^neg^ iNKT cell activation, which is associated with the expansion of regulatory T cells ([@B41]). The inhibitory effect required T-bet and TLR7 expression in iNKT cells. Furthermore, the administration of α-GalCer or glycolipid derived from *Helicobacter pylori* to neonates recapitulated the result ([@B41]), suggesting that infection with certain microorganisms can prevent the subsequent development of allergic asthma by expanding a specific subset of iNKT cells. Therefore, the authors proposed that treatment of children or allergic patients with compounds such as α-GalCer or other glycolipids derived from microorganisms might be effective in preventing or improving the development or symptoms of allergic asthma.
Lung iNKT cell-dependent allergic or non-allergic asthma {#s3}
========================================================
Lung iNKT cells are relatively abundant compared to iNKT cells in the peripheral blood ([@B14]). The activation of pulmonary iNKT cells by the intranasal α-GalCer administration rapidly induced AHR and eosinophilic inflammation in naïve mice, and this effect was independent of conventional CD4 T cells ([@B42]). Michel et al. showed that NK1.1^neg^ iNKT cells produced high levels of IL-17 and induced neutrophilic infiltration following the intranasal administration of α-GalCer in a murine model ([@B43]). In addition, the development of AHR was observed in non-human primates by the direct activation of pulmonary iNKT cells with α-GalCer, indicating that pulmonary iNKT cells are critical effector cells in these animal models ([@B44]). Our previous study showed that α-GalCer induced AHR and neutrophilic infiltration, and the neutrophilic infiltration was significantly attenuated in CD69-deficient mice, indicating that activated iNKT cells-mediated asthmatic responses were dependent on CD69 expression ([@B5]). We recently identified myosin light chain (Myl) 9 and Myl12 as functional ligands for CD69 ([@B45]). We also showed that the interaction between CD69 on Th2 cells and Myl9 expressed on the luminal side of endothelial cells in the blood vessels recruits activated Th2 cells to the inflammatory site, resulting in airway inflammation ([@B45], [@B46]). CD69 on iNKT cells might therefore induce the migration of iNKT cells to the lung by binding to Myl9 or Myl12 and also play a critical role in the development of AHR and airway inflammation (Figure [1](#F1){ref-type="fig"}).
{#F1}
Even if iNKT cell activation in the lung does contribute to asthma, we are unlikely to be exposed to α-GalCer, a component of marine sponge, in our daily lives. Several studies have indicated that substances naturally existing in our environment, such as allergens, pathogens and air pollution, might activate iNKT cells and cause or exacerbate airway inflammation. Glycolipids from bacteria, such as *Sphingomonas, Borrelia*, and *Leishmania* species, are recognized by invariant TCR of iNKT cells ([@B47]). In particular, glycolipids purified from *Sphigomona* cell walls were shown to induce rapid AHR after respiratory administration in wild-type mice but not iNKT-deficient mice ([@B42]). Although a glycolipid that can induce iNKT cell activation has not been identified in viruses, Kim et al. suggested that viruses may facilitate CD1d antigen presentation and induce iNKT cell activation in an indirect manner ([@B48]). The authors also showed that IL-13 production from macrophages stimulated by iNKT cells during respiratory virus infection induces the development of AHR and mucus production independent of the adaptive immune response. *Aspergillus fumigatus* is a saprophytic fungus that is ubiquitous in the environment and is commonly associated with allergic asthma ([@B49]). Albacker et al. reported that the *Aspergillus funmigatus*-derived glycosphingolipid asperamide B directly activates iNKT cells in a CD1d-restricted, Myd88-independent, and dectin-1-independent manner ([@B50]). The intranasal administration of asperamide B rapidly induced AHR and neutrophil infiltration into the lung, suggesting that fungi can contribute to the induction of asthmatic symptoms by iNKT cells. Therefore, iNKT cells activated by glycolipids from microorganisms may contribute to the development and exacerbation of asthma symptoms in humans.
It was recently revealed that non-glycolipid stimulation could also activate iNKT cells, resulting in the induction of AHR. House dust extract (HDE) contains antigens and is capable of inducing airway inflammation by activating mouse Vα14 or human Vα24 NKT cells ([@B51]). The stimulation of mouse Vα14 iNKT cells was shown to be CD1d-dependent and not dependent on TLR agonist present in HDE. Although the antigen in HDE remains incompletely characterized, the authors suggested that the immunostimulatory material in HDE was of neither bacteria nor glycolipid origin ([@B51]). Ozone is an air pollutant that has also been reported to be associated with asthma ([@B52], [@B53]). The development of AHR was found to be inducible even in healthy individuals following exposure to ozone, which causes airway epithelial damage, and increased numbers of neutrophils ([@B54], [@B55]). Furthermore, asthmatic patients are more susceptible to the detrimental effects of this pollutant. A murine model of ozone induced-asthma revealed the indispensable role of IL-17-producing iNKT cells for the induction of AHR ([@B56]). Although how ozone activates iNKT cells is unclear at present, NKT cells activated by ozone can induce a form of asthma that is characterized by cellular infiltration and AHR.
In addition to the naturally existing molecules in the environment, extracellular factors are also known to activate iNKT cells. T cell immunoglobulin and mucin domain-1 (TIM-1) is an important asthma susceptibility gene and also a receptor for phosphatidylserine (PtdSer) ([@B57]), an important marker of cells undergoing programed cell death or apoptosis ([@B58]). NKT cells can activate, proliferate, and produce cytokines through recognition of PtdSer by TIM-1 ([@B59]). Furthermore, the apoptosis of airway epithelial cells activates pulmonary NKT cells, resulting in AHR and suggesting that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern ([@B60]). Previous studies have shown that apoptosis induced by virus infection or ozone exposure can trigger NKT activation ([@B48], [@B56], [@B60]), as infection with some viruses triggers apoptosis and externalization of PtdSer. In addition, it has been reported that TLR signaling enhances the activation of iNKT cells. Vultaggio et al. showed that systemic dsRNA (poly (I:C)) selectively upregulates the IL-17 production from iNKT cells activated by α-GalCer. The authors therefore expected that the exacerbation of airway inflammation might be induced by certain virus infections ([@B61]). Furthermore, several cytokines involved in the initiation and amplification of Th2 responses have been reported ([@B62]). IL-25 is capable of enhancing AHR and is produced by activated Th2 cells, epithelial cells, basophils, and mast cells ([@B63]). The administration of recombinant IL-25 induced Th2-type responses, including increased serum IgE levels, eosinophilia, pathological changes in the lung, and AHR. These symptoms induced by IL-25 were not observed in iNKT cell-deficient mice ([@B64], [@B65]). Moreover, iNKT cells expressing IL-17 receptor B were shown to be essential for IL-25-induced AHR using an adoptive transfer model ([@B65]). Thymic stromal lymphoprotein (TSLP) is also considered to play an important role in the iNKT cell-dependent asthma model ([@B66]). While the targets of TSLP are T cells, mast cells, basophils, and DCs, Nagata et al. demonstrated that TSLP also acts on iNKT cells to enhance AHR by up-regulating their production of IL-13 ([@B67]). IL-33 enhanced the production of Th1 and Th2 cytokines in activated NKT cells ([@B68], [@B69]). These results indicate that natural ligands in the environments act as antigens for iNKT cells to induce allergic asthma, and TCR-independent stimuli to iNKT cells may exacerbate the asthmatic symptoms such as AHR (Figure [1](#F1){ref-type="fig"}).
Although it is obvious that the direct activation of lung iNKT cells causes lung inflammation, which types of inflammation are induced is still controversial. Two groups claimed that the intranasal administration of α-GalCer induced allergic airway inflammation because eosinophil infiltration into the lung, a feature of type 2-mediated responses, was observed in IL-4- and IL-13-dependent manners ([@B42], [@B70], [@B71]). However, neutrophil infiltration, which represents non-allergic airway inflammation, is frequently observed in severe or Th17-mediated asthma ([@B72], [@B73]). The activation of iNKT cells by the intranasal administration of α-GalCer, asperamide B or PtdSer induces pulmonary neutrophil infiltration, suggesting that iNKT cell may contribute to non-allergic airway inflammation ([@B5], [@B43], [@B50], [@B59]). In contrast, equivalent numbers of eosinophils and neutrophils have been noted with ozone or poly (I:C) stimulation ([@B56], [@B61]). This discrepancy in outcomes may be due to the activation of distinct subsets of iNKT cells: one produces IL-13 and IL-5, which activate and recruit eosinophils; the other produces IL-17, thereby inducing the recruitment of neutrophils. Additional flow cytometry single cell analyses addressing the precise production profiles of cytokines in iNKT cells are needed in order to discriminate the infiltrated subsets.
With many clinical and experimental examinations, it has been revealed that asthma is more heterogeneous and complex than previously thought. While allergic asthma is induced by allergens and mediated by Th2 cells, a non-allergic form of asthma is caused independent of Th2 responses ([@B29]). Non-allergic asthma is induced by multiple environmental factors, such as air pollution (smoke, ozone, and diesel particles) and virus infection. Although the immunological pathways of non-allergy asthma are still unclear, the activation of iNKT cells with their specific ligands or cytokines may contribute to the development of non-allergy asthma.
Taken together, these findings suggest that different types of iNKT cell ligands may activate distinct subsets of iNKT cells, thereby resulting in distinct patterns of airway inflammation. Therefore, lung iNKT cell activation may contribute to the development of various types of asthmatic inflammation (Figure [1](#F1){ref-type="fig"}).
Therapeutic intervention for iNKT cell-dependent allergic asthma {#s4}
================================================================
As we pointed out above, iNKT cells may play have a critical role in the development or exacerbation of asthma. Although further investigations are needed, Dimaprit (H2 histamine receptor agonist) or intravenous immunoglobulin treatment does appear to suppress iNKT cell-dependent allergic asthma ([@B74], [@B75]). The administration of anti-mouse CD1d monoclonal antibodies (20H2) or CD1d-dependent antagonist has also been shown to suppress OVA-induced AHR and inflammation in murine models ([@B76], [@B77]). Indeed, McKnight et al. reported that anti-mouse CD1d monoclonal antibody (20H2) treatment before the intranasal administration of α-GalCer impaired iNKT cell-induced AHR in an experimental mouse model of asthma, while this antibody did not suppressed OVA-induced allergic asthma. These results suggest that this antibody may attenuate non-allergic asthma ([@B35]). Anti-human CD1d antibody (NIB.2) possesses a high affinity for human and cynomolgus macaque CD1d and inhibits NKT cell activation by inhibiting the interactions of the TCRβ chain of iNKT cells with CD1d ([@B78]). NIB.2 treatment significantly reduced the cytokine levels and numbers of lymphocytes and macrophages in the bronchoalveolar lavage fluid (BALF) in a primate model of asthma ([@B78]). However, this antibody may affect other CD1d-restricted T cells that are not involved in airway inflammation ([@B79]). Therefore, the development of a more specific method will pave the way for therapeutic interventions to alleviate symptoms.
Mouse invariant monoclonal antibody, NKT14 was found to specifically bind to invariant TCR of mouse iNKT cells and deplete iNKT cells in mice via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity for 3 weeks ([@B80]). The elimination of iNKT cells was sufficient to prevent murine AHR and pulmonary eosinophilic inflammation elicited by the oropharyngeal inhalation with α-GalCer ([@B71]). In addition, NKT14 administration prior to sensitization abrogated either antigen-mediated AHR alone or both AHR and pulmonary inflammation ([@B71], [@B80]).
Role of iNKT cells in asthma patients {#s5}
=====================================
In order to determine the role of iNKT cells in human asthma, many investigators have examined iNKT cells in asthma patients with regard to their numbers and the production of cytokines (Table [1](#T1){ref-type="table"}). An initial report published in 2006 by Akbari et al. stated that more than 60% of CD4 T cells in the BALF from severe asthmatic patients were iNKT cells, while the infiltration of iNKT cells was not observed in patients with other pulmonary diseases, such as sarcoidosis, or in healthy controls ([@B81]). Three other supportive reports showed that asthmatic patients display higher frequency of iNKT cells in BALF as compared to healthy control donors do ([@B82], [@B83], [@B91]). However, the very high numbers of iNKT cells (\~60% among CD4 T cells) reported by Akbari et al. have not been replicated by other investigators.
######
iNKT cells in patients with asthma.
**Frequency of iNKT cells are**
------ --------------------------------- --------------------------- -------- ------------------------------------------------------- ------------------------------------------------------------------- ---------------------------- --------------------------- -------- -------------------------------------------------------------------- -----------------------------------------------
2006 Akbari et al. ([@B81]) CD1d-restricted NKT cells BALF Healthy controls 0.9% of CD3^+^ cells (mean)
Patients with asthma treated with corticosteroids 73% of CD3^+^ cells (mean)
Patients with asthma not treated with corticosteroids 72% of CD3^+^ cells (mean)
Hamzaoui et al. ([@B82]) CD56^+^ Vα24^+^ NKT cells Sputum Healthy controls 0.09 ± 0.02% in sputum
Severe asthma 0.82 ± 0.07% in sputum
Mild asthma 0.07 ± 0.05% in sputum
Pham-Thi et al. ([@B83]) CD1d-restricted NKT cells BALF Non-atopic controls 0.116 ± 0.03% in T cells
Asthma 0.435 ± 0.09% in T cells
2007 Vijayanand et al. ([@B84]) Vα24^+^ Vβ11^+^ NKT cells Sputum Control subjects 0--0.6% of CD3^+^ cells
Subject with moderately severe asthma treated with corticosteroids 0--1.3% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0--0.6% of CD3^+^ cells
Vα24^+^ Vβ12^+^ NKT cells BALF Control subjects Data not shown
Subject with moderately severe asthma treated with corticosteroids 0--0.2% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0% of CD3^+^ cells
CD1d-restricted NKT cells Sputum Control subjects 0--0.1% of CD3^+^ cells
Subject with moderately severe asthma treated with corticosteroids 0% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0 of CD3^+^ cells
CD1d-restricted NKT cells BALF Control subjects Data not shown
Subject with moderately severe asthma treated with corticosteroids 0--0.8% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0 or 0.3% of CD3^+^ cells
6B11^+^ NKT cells Sputum Control subjects 0--0.6% of CD3^+^ cells
Subject with moderately severe asthma treated with corticosteroids 0 or 0.5% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0% of CD3^+^ cells
6B11^+^ NKT cells BALF Control subjects Data not shown
Subject with moderately severe asthma treated with corticosteroids 0--0.6% of CD3^+^ cells
Subject with mild asthma not treated with corticosteroids 0% or 2.7% of CD3^+^ cells
Mutalithas et al. ([@B85]) 6B11^+^ NKT cells BALF Controls 0.12 ± 0.02% of CD3^+^ cells
Asthma 0.37 ± 0.1% of CD3^+^ cells
2009 Matangkas-ombut et al. ([@B86]) CD1d-restricted NKT cells BALF No asthma 0.07--0.68% of CD3^+^ cells
Well controlled asthma 0.05--9.57% of CD3^+^ cells
Severe asthma 2.43--7.18% of CD3^+^ cells One patient shows 64.5% of iNKT cells
2010 Brooks et al. ([@B87]) 6B11^+^ NKT cells Sputum Controls Median of 0.06% (IQR, 0-0.2%) in all T cells
Asthma Median of 0.07% (IQR, 0-0.17%) in all T cells
Koh et al. ([@B88]) CD56^+^ NKT cells Sputum Controls 0.005 ± 0.005% in CD3^+^ cells Koh et al. ([@B88]) CD56^+^ NKT cells PBMCs Controls 1.81 ± 0.33% in CD3^+^ cells
Asthma 0.27 ± 0.08% in CD3^+^ cells Asthma 1.98 ± 0.37% in CD3^+^ cells
6B11^+^ NKT cells Sputum Controls 0.32 ± 0.15% in CD3^+^ cells 6B11^+^ NKT cells PBMCs Controls 0.22 ± 0.05% in CD3^+^ cells
Asthma 0.76 ± 0.3% in CD3^+^ cells Asthma 0.24 ± 0.04% in CD3^+^ cells
Vα24^+^ NKT cells Sputum Controls 0.03 ± 0.02% in CD3^+^ cells Vα24^+^ NKT cells PBMCs Controls 0.42 ± 0.05% in CD3^+^ cells
Asthma 0.14 ± 0.03% in CD3^+^ cells Asthma 0.48 ± 0.04% in CD3^+^ cells
2012 Yan-Ming et al. ([@B89]) Vα24^+^ Vβ11^+^ NKT cells PBMCs Controls 0.135 ± 0.061% in blood
Asthma 0.051 ± 0.041% in blood
2014 Shim et al. ([@B90]) Vα24^+^ Vβ11^+^ NKT cells PBMCs Controls 0.2 ± 0.1% in blood
Asthma 0.2 ± 0.2% in blood
6B11^+^ Vβ12^+^ NKT cells PBMCs Controls 0.07 ± 0.01% in blood
Asthma 0.2 ± 0.1% in blood
*BALF, bronchoalveolar lavage fluid; PBMCs, peripheral blood mononuclear cells; HCs, healthy controls*.
In contrast, a similar study by other group found that the number of iNKT cells was not increased in patients with asthma ([@B91]). Another group reported that iNKT cells were found in low numbers in the sputum or BALF of patients with asthma, chronic obstructive pulmonary disease and healthy controls, with no significant differences among the three groups ([@B84]). Mutalithas et al. also reported similar results in the BALF ([@B85]). Furthermore, the influx of iNKT cells in the airways was not observed after segmental allergen challenge ([@B92], [@B93]). To explain this discrepancy, Thomas et al. ([@B93]) and Vijayanand ([@B84]) pointed out that 6B11 antibody was able to stain alveolar macrophages nonspecifically. They suggested that the higher frequency of iNKT cells was due to the non-specific binding to the cells, and that the lymphocyte population should be gated for the analysis of iNKT cells ([@B83], [@B91]). However, Akbari et al. argued that they had already gated the lymphocyte population and used a CD1d-tetramer instead of 6B11 antibody to stain iNKT cells. In addition, those authors readdressed the issue regarding the number of iNKT cells in BALF from patients with severe asthma the next year ([@B86]). They confirmed that patients with severe asthma had a significantly increased number of iNKT cells compared to healthy controls. In this report, however, CD1d-restricted iNKT cells accounted for 2--7% of total CD3^+^ cells in the BALF of asthmatic patients, and only 1 patient with severe asthma had an iNKT cell proportion of 64.5% ([@B93]). The findings of Reynolds et al. supported the increase in the number of iNKT cells in the lung using biopsies with allergen challenge ([@B94]). Nevertheless Brooks et al. subsequently suggested that the high frequency of iNKT cells detected in BALF was due to the non-specific staining of dead cells ([@B87]). In addition, they also indicated that there was no marked difference in the frequency of 6B11^+^ iNKT cells in sputum even when including dead cells in the samples.
After 2010, it was suggested that a reduced iNKT cell frequency in the PBMCs of asthmatic patients did not imply that iNKT cells were irrelevant to the development of asthma. Koh et al. showed that the numbers of NKT cells in peripheral blood did not differ markedly between patients and control groups ([@B88]). However, in sputum, the numbers of iNKT cells were significantly increased in patients with asthma. Their subsequent study demonstrated the negative correlation between blood iNKT cell number and eosinophils, cytokines, or chemokines in sputum ([@B95]). These results suggested that iNKT cell might be mobilized to the lung during the exacerbation. Two other groups also demonstrated the profound reduction or no increase in iNKT cells in the blood of asthma patients compared to the normal control group ([@B89], [@B90]). However, they also showed an increased IL-4 production in iNKT cells of asthma patients compared to controls. Pedroza showed that pediatric asthmatic patients undergoing exacerbations of asthma displayed increased numbers of iNKT cells in the blood that also produced less IFN-γ and more IL-4 than children with stable asthma or in healthy control children ([@B96]). These results suggest that Th2-like iNKT cells might be involved in the development of asthmatic exacerbations.
At present, studies on iNKT cells in asthma patients have provided conflicting results. The frequency of iNKT cells in the lungs is particularly hotly debated. As such, we conclude that the frequency of iNKT cell does not always reflect the severity of the diseases. Although there are some recent reports that suggest no correlation between the blood iNKT cell number and clinical asthma severity ([@B97]), it is becoming more widely recognized that iNKT cells likely play a role in the development and possibly exacerbation of allergic asthma. In addition, the studies of iNKT cells in other asthma etiologies, such as chronic, occupational, steroid-resistant, exercise-induced, and aspirin-induced asthma, where Th2 cells may not paly a major role, may provide new insights into these type of diseases. We therefore suggest a few experimental design approaches to adopt when studying the role of iNKT cells in particular diseases. First, in the flow cytometry analysis of iNKT cells in patients, lymphocytes, particularly live cells, should be gated for the analysis, and control staining, including with isotype controls, should be performed, with the results compared. This will prevent the contamination of cells with non-specific staining patterns. Second, more than two staining protocol should be employed. At least three different approaches have been established for identifying iNKT cells, such as CD1d-tetramer, anti-Vα24 antibody and 6B11 antibody recognizing the CDR3 region of Vα24-JαQ TCR. Although these approaches should theoretically provide similar results, using multiple staining protocols may help clear up any confusion if controversial results are obtained. Third, in addition to assessing the frequency of iNKT cells, their cytokine production (IL-2, IL-4, IFN-γ, or IL-17) should also be examined by flow cytometry. As we discussed above, it would be difficult to demonstrate the relevance of iNKT cells to diseases by analyzing only the frequency of such a small population. Examining changes in their function may therefore be useful for elucidating their contribution to the pathology of diseases.
MR1-restricted cells {#s6}
====================
MAIT cells are a subset of innate-like T lymphocytes first described in 1999 ([@B98]). These MR-1-restricted cells are abundant in humans and can rapidly express a variety of pro-inflammatory cytokines ([@B12]). While iNKT cells are suggested to play critical roles in murine models of allergic airway diseases, they are rare in human airways. MAIT cells, by contrast, are 5- to 10-fold more abundant in humans than in mice ([@B15]). Since MAIT cells exist in the lung and may be able to produce Th2 cytokines ([@B19], [@B20]), these cells may contribute to the development of asthma. However, several reports have indicated a different role for these cells. Hinks et al. observed a striking deficiency of Vα7.2^+^ CD161^+^ T cells in blood, sputum, and bronchial biopsy samples, suggesting that the deficiency correlated with the severity of asthma ([@B11], [@B99]). A similar deficiency in humans was observed in autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, Crohn\'s disease, ulcerative colitis, or chronic inflammatory disease, such as type 2 diabetes) ([@B100]--[@B103]). In addition, it was reported that an increased MAIT cell frequency at 1 year of age was associated with a decreased risk of asthma by 7 years of age ([@B104]). These results suggest that MAIT cells may play a protective role against chronic inflammation.
Given that MAIT cells respond to bacterial metabolites, it is possible that MAIT cell activation by gut or lung microbiota is required to prevent asthma. If MAIT cells can exert a suppressive function against chronic inflammation, this hypothesis would be inconsistent with their ability to produce various inflammatory cytokines. In addition, it was also reported that the numbers of MAIT cells producing IL-17 are increased in asthmatic patients ([@B105]). Since MAIT-deficient mice have been generated ([@B106]), investigations into the function of MAIT cells infiltrating the inflammatory site in mouse models may help provide answers.
Conclusion {#s7}
==========
Studies investigating the roles of iNKT cells in allergic responses have helped to explain the Th2-dependent mechanisms underlying the development of allergic asthma. However, iNKT cells also have been suggested to be associated with the development of non-allergic airway inflammation that is induced and/or exacerbated by non-Th2 factors, such as viruses, air pollution and inflammatory cytokines (IL-17 or TNFα). Furthermore, recent studies have suggested that NKT cells or MAIT cells may play a critical role in the inhibition of asthmatic symptoms. Although a clear conclusion has not been reached due to inconsistent results, innate-like T cells apparently have critical and varied roles in regulating immune responses. As such, more intensive studies will be required in order to elucidate the mechanisms underlying the induction of various types of asthma by innate-like T cells and establish innovative therapeutic strategies.
Author contributions {#s8}
====================
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
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.
**Funding.** Grants-in Aid for Scientific Research (S) (TN\#26221305), the Practical Research Project for Allergic Diseases and Immunology (Research on Allergic Diseases and Immunology) from Japan Agency for Medical Research and development, AMED (JP18ek0410030) (TN).
MR1
: MHC-related molecule-1
TCR
: T cell receptor
iNKT
: invariant natural killer T
α-GalCer
: α-galactosylceramide
MAIT
: mucosal-associated invariant T
IL
: interleukin
TNFα
: tumor necrosis factor α
AHR
: airway hyperactivity
KO
: knockout
TLR
: toll like receptor
Myl
: myosin light chain
HDE
: house dust extract
TIM-1
: T cell immunogloblin and mucin domain-1
PtdSer
: phosphatidylserine
TSLP
: thymic stromal lymphoprotein
DCs
: dendritic cells
BALF
: bronchoalveolar lavage fluid.
[^1]: Edited by: Luc Van Kaer, Vanderbilt University, United States
[^2]: Reviewed by: Maria Leite-de-Moraes, INSERM U1151 Institut Necker Enfants Malades Centre de Médecine Moléculaire (INEM), France; Seddon Y. Thomas, National Institute of Environmental Health Sciences (NIEHS), United States; Shin-ichiro Fujii, RIKEN Center for Integrative Medical Sciences (IMS), Japan; Rosemarie DeKruyff, Stanford University, United States
[^3]: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology
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{#sp1 .277}
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Background
==========
Minocycline is a second-generation tetracycline antibiotic derivative that effectively crosses the blood-brain barrier \[[@B1]\], and which has a proven safety record in humans \[[@B2]\]. Minocycline has anti-inflammatory and neuroprotective effects in animal models of cerebral ischaemia \[[@B3],[@B4]\], traumatic injury \[[@B5]\], glutamate-induced neurotoxicity \[[@B6]\], experimental autoimmune encephalonmyelitis \[[@B7]\], Huntington\'s disease \[[@B8]\] and Parkinson\'s disease \[[@B2],[@B9],[@B10]\]. Minocycline\'s efficacy has been bolstered by studies showing decreased secondary neuronal damage via the inhibition of microglial activation. Recently, it has been demonstrated that this minocycline-mediated microglial inhibition attenuates the development of pain hypersensitivity by inhibiting proinflammatory cytokine expression in rat models of both neuropathic pain and spinal immune activation by intrathecal human immunodeficiency virus-1 (HIV-1) gp120 \[[@B11],[@B12]\]. Also, minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium chloride co-transporter 2 (KCC2) in the spinal cord \[[@B13]\]. In addition, we confirmed that minocycline attenuates tactile hypersensitivity following the trigeminal sensory nerve injury by inhibiting microglial p38 mitogen activated protein kinase (MAPK) activation \[[@B14]\], and that minocycline markedly reduced formalin-induced inflammatory pain by inhibition of excitatory postsynaptic currents (EPSCs) in the substantia gelatinosa \[[@B15]\]. These findings raise the possibility of the potential of minocycline as an analgesic for other types of pain models.
Visceral pain is the most common form of pain produced by disease, for which medical care is sought. Despite the conventional belief that visceral pain is a variant of somatic pain, it differs in neurological mechanisms and transmission pathways. Visceral pain is characterized by referral hyperalgesia and also it is not always linked to tissue injury \[[@B16],[@B17]\]. Also, visceral pain that reflects the enhanced perception of physiological signals from the gut and/or the enhanced perception of experimental visceral stimuli along with hypervigilance to these aspects, is commonly considered to play a major role in the pathophysiology of irritable bowel syndrome (IBS) \[[@B17],[@B18]\]. Various studies have evaluated the underlying mechanisms of visceral hypersensitivity and the influence of various stresses on the visceral pain pathways \[[@B17],[@B18]\]. Recent studies have revealed the activation of ERK in spinal cord after noxious visceral stimulation \[[@B19],[@B20]\].
ERK, a MAPK, could play a role in regulating nociceptive activities in primary sensory pathways after pathologic irritation of the peripheral system, such as peripheral nerve injury or inflammation \[[@B21]-[@B23]\]. Phosphorylation of ERK is observed in spinal dorsal horn neurons in response to noxious stimulation of the peripheral tissue, such as the injection of complete Freund\'s adjuvant (CFA) into a hindpaw \[[@B22]\], an intense noxious peripheral or C-fiber electrical stimulus \[[@B21]\], L5 spinal nerve ligation \[[@B23]\], and the injection of cyclophosphamide into the urinary bladder \[[@B24]\]. ERK was also shown to be phosphorylated in the spinal cord of a murine model of visceral pain and hyperalgesia, intracolonic instillation of either capsaicin or mustard oil \[[@B25]\], and in a model of acute inflammation and distention of the colon \[[@B26]\]. Intrathecal injection of specific inhibitor (U-0126 or PD-98059), which specifically attenuates ERK activity, can reduce nociceptive response behavior in the inflammatory pain, CFA-induced joint inflammation \[[@B27]\], and visceral pain by intracolonic capsacin \[[@B19]\]. These studies suggest an essential role of ERK in the development and maintenance of inflammatory or neuropathatic hyperalgesia. However, very little is known about the molecular signaling mechanisms evoked by acute visceral pain and there is no information on the involvement of ERK in the spinal processing in this type of pain.
The present study focused on the role of minocycline on spinal ERK in modulating acute visceral pain. The study hypothesis was that minocycline attenuates the acetic acid-induced visceral nociception by inhibiting the phosphorylation of neuronal ERK in the spinal cord.
Results
=======
Minocycline inhibits acetic acid-induced abdominal contraction
--------------------------------------------------------------
Acetic acid injection into the abdomen produces an acute visceral pain response \[[@B28]\]. In this study, for the 60 minutes following acetic acid administration (1.0%, 250 μl), the number of abdominal constrictions or writhes, such as lengthwise stretches of the torso with a concomitant concave arching of the back \[[@B28]\], were counted and totaled every 5 minutes. Saline-treated mice showed the typical abdominal stretching and constriction behaviors, which peaked at 10-15 minutes (15.1 ± 1.2), and then gradually declined. However, the peak pain responses by peritoneal irritation were significantly inhibited by pretreatment of minocycline in a dose dependent manner (4 mg/kg, 11.0 ± 1.5; 10 mg/kg, 6.1 ± 1.1; 40 mg/kg, 3.5 ± 0.8) (Figure [1A](#F1){ref-type="fig"}). The total number of writhing responses during the 60 minutes after acetic acid injection in the saline pretreatment group was 84.5 ± 8.7. However, the total number of writhes was significantly decreased by pretreatment of minocycline in a dose dependent pattern (4 mg/kg, 52.7 ± 4.6; 10 mg/kg, 32.0 ± 3.3; 40 mg/kg, 18.9 ± 3.5) (Figure [1B](#F1){ref-type="fig"}). Intraperitoneal (i.p.) injection of either saline or minocycline by themselves did not alter the behavior of the animals (data not shown). The results suggest that minocycline has an anti-nociceptive effect on acetic acid-induced acute visceral pain.
{#F1}
Minocycline reduces acetic acid-induced c-Fos expression
--------------------------------------------------------
Because c-Fos, an immediate early gene protein product, is a neuroactive marker that can be used to analyze nociceptive pathways \[[@B25],[@B29],[@B30]\], we compared c-Fos expression between the saline- or acetic acid-treated mice (n = 8, in each group) after the time at which acetic acid maximally affected visceral pain (30 minutes after acetic acid injection) (Figure [1](#F1){ref-type="fig"}). c-Fosimmunoreactivity (IR) was evaluated in the T5-L2 spinal cord where primary afferents from splanchnic nerves innervate the entire gastrointestinal tract \[[@B17],[@B31]\]. c-Fos-IR in the spinal cord was very scarce in normal mice (saline-administration alone) (I-X, 41.7 ± 2.2; I-II, 5.9 ± 0.5; III-IV, 24.0 ± 1.3; V-VI, 5.5 ± 0.6; VII-IX, 5.3 ± 0.6; X, 1.0 ± 0.1). The number of c-Fos positive cells in T5-L2 spinal cord was extensively increased by i.p. injection of acetic acid (I-X, 187.2 ± 5.0; I-II, 36.3 ± 1.9; III-IV, 69.2 ± 2.3; V-VI, 34.8 ± 1.3; VII-IX, 37.8 ± 1.7; X, 9.3 ± 0.5), but acetic acid-induced c-Fos-IR enhancement was significantly decreased by minocycline-pretreatment 1 hour prior to acetic acid administration (I-X, 114.2 ± 3.1; I-II, 21.6 ± 1.3; III-IV, 40.3 ± 1.3; V-VI, 22.8 ± 1.0; VII-IX, 24.1 ± 1.2; X, 5.4 ± 0.4). Minocycline, itself did not exert any effect on c-Fos expression in the spinal cord (Figure [2C](#F2){ref-type="fig"}). Taken together, the results suggest that minocycline has an inhibitory action for the excitation of spinal neurons in acetic acid-induced acute visceral pain. In addition, to investigate whether c-FOS was specifically activated in the T5-L2 spinal cord, we compared the expression of c-Fos in the samples from C1-C7, T5-L2 and L4-S1 spinal cord 30 minutes after acetic acid injection. c-Fos expression was upregulated in the T5-L2 levels, but not in the C1-C7, L4-S1, and normal T5-L2 segments (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1).
{#F2}
Minocycline attenuates acetic acid-induced neuronal p-ERK expression
--------------------------------------------------------------------
It has been demonstrated that spinal ERK is activated in experimental visceral pain models \[[@B19],[@B26],[@B27]\]. Therefore, we investigated whether minocycline could produce its effects through the spinal ERK pathway in the acetic acid-induced acute visceral pain. As shown in Figure [3A](#F3){ref-type="fig"}, 30 minutes after acetic acid administration, phosphorylation of ERK was clearly evident in the T5-L2 spinal cord. However, the elevated level of phospho (p)-ERK was decreased by minocycline-administration (Figure [3A](#F3){ref-type="fig"}). In addition, we examined the spinal distribution of p-ERK expression (Figures [3B-E](#F3){ref-type="fig"}). Immunohistochemical analysis indicated that p-ERK positive cells in the T5-L2 spinal cord were very scarce in saline-administrated mice (I-X, 13.2 ± 3.4) (Figures [3B, E](#F3){ref-type="fig"}). The number of p-ERK positive cells in the lamina I to X of the spinal cord was significantly increased by acetic acid-administration (I-X, 46.7 ± 3.4), but these acetic acid-stimulated p-ERK enhancement was significantly decreased by minocycline-pretreatment (I-X, 26.9 ± 1.3) (Figures [3C-E](#F3){ref-type="fig"}). To investigate the nature of the p-ERK positive cells, we examined whether ERK was activated in neuron, microglia, or astrocytes using multiple immunofluorescence method. Interestingly, the p-ERK immunofluorescence in the spinal cord from saline-treated mice was found exclusively in neuron (83.4%; 243 p-ERK IR and NeuN IR neurons/290 p-ERK IR neurons) (Figures [4AC](#F4){ref-type="fig"}), but was not clear in microglia or astrocytes (Figures [4D-I](#F4){ref-type="fig"}). Microglia and astrocytes were not sufficiently activated 30 minutes after acetic acid treatment (Figures [4E, H, J](#F4){ref-type="fig"} and [4K](#F4){ref-type="fig"}). These results suggest that minocycline attenuates neuronal ERK activation in the acetic acid-induced acute visceral pain. In addition, to investigate whether p-ERK is specifically increased in T5-L2 spinal segments, we compared the expression of p-ERK in the samples from C1-C7, T5-L2 and L4-S1 spinal segments 30 minutes after acetic acid injection. As expected, the phosphorylation of ERK was specifically increased in the T5-L2 levels, but not in the C1-C7, L4-S1, and normal T5-L2 spinal segments (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1).
{#F3}
{#F4}
Microglia and astrocytes are not morphologically activated by acetic acid treatment
-----------------------------------------------------------------------------------
Because spinal glia (microglia and astrocytes) are activated in inflammatory pain \[[@B15],[@B32],[@B33]\] and neuropathic pain \[[@B12],[@B34]-[@B36]\], and since minocycline attenuates behavioral hypersensitivity through the inhibition of microglial activation in these pain models \[[@B12],[@B15],[@B32]\], we examined whether spinal microglia and astrocytes were activated by acetic acid and, if so, whether the activated spinal microglia could be inhibited by minocycline pretreatment. Activated microglia usually display CD11/b or Iba-1 (a marker for microglia/macrophage lineage cells)-IR with enlarged cell body and much shorter and thicker processes \[[@B15],[@B37]\]. However, when we analyzed CD11/b positive cells 30 minutes following acetic acid injection, microglial activation by acetic acid was not clearly found compared to normal mice (Figures [4E, J](#F4){ref-type="fig"}). In addition, we also confirmed that astrocytes similar to microglia were not enough activated 30 minutes following acetic acid injection, using glial fibrillary acidic protein (GFAP) antiserum (Figures [4H, K](#F4){ref-type="fig"}). The results indicate that the morphology of spinal microglia and astrocytes are not distinctly influenced by either acetic acid in acute visceral pain, and that spinal microglia and astrocytes do not directly contribute to acute visceral pain.
Intrathecal administration of PD-98059 reduces acetic acid-induced abdominal constriction
-----------------------------------------------------------------------------------------
After acetic acid administration, abdominal pain response was increased and p-ERK expression was up-regulated mainly in spinal neurons, but not in glial cells, and the elevated pain response and p-ERK expression was reduced by pretreatment of minocyclin (Figures [1](#F1){ref-type="fig"} and [4](#F4){ref-type="fig"}). This observation suggests that neuronal p-ERK expression contributes to acetic acid-induced abdominal pain. To address this issue, we directly introduced PD-98059, an ERK upstream kinase (MEK) inhibitor, to normal mice intrathecally 20 minutes before the injection of acetic acid. In the vehicle-treated mice, the number of acetic acid-induced writhes peaked at 10-15 minutes (16.7 ± 3.1) then gradually declined. The total number of writhes was 78.0 ± 10.5 for 60 minutes, similar to the result of Figure [1](#F1){ref-type="fig"} (Figure [5A, B](#F5){ref-type="fig"}). However, these abdominal pain responses and total number of writhes were almost completely blocked by pretreatment of PD-98059 in a dose dependent manner at the peak time (0.1 μg, 5.6 ± 2.2;
0.5 μg, 2.6 ± 1.1) and total number of writhes (0.1 μg, 42.4 ± 15.6; 0.5 μg, 14.6 ± 5.2) (Figures [5A, B](#F5){ref-type="fig"}). These results indicate that intrathecal introduction of PD-98059 inhibits acetic acid-induced abdominal pain.
{#F5}
Discussion
==========
In this study, we investigated the potential of minocycline as an analgesic for acute visceral pain. Using the writhing test as an experimental model, we examined the effect of minocycline on acetic acid-induced pain response and spinal mechanism. The results indicate that (1) ERK activation is involved in nociceptive behavior and the excitation of spinal neurons in acetic acid-induced acute abdominal pain, (2) almost all cells with ERK activation are spinal neurons (T5-L2), (3) minocycline administrated 1 hour before acetic acid injection into the peritoneum markedly decreases c-Fos and p-ERK expression in the spinal cord (Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}), and (4) the intrathecal introduction of the MEK inhibitor, PD-98059, reduces acetic acid-induced abdominal pain (Figure [5](#F5){ref-type="fig"}). Collectively the data indicate that the ERK activation is involved in the pain behavior and neuronal response of acute abdominal pain, and that minocycline remarkably attenuates acute abdominal pain behavior and spinal c-Fos-IR, and that these effects might be a consequence of reduced spinal ERK activations.
As in other areas of pain research, the use of animal models is a necessary step to elucidate the underlying neurophysiological and neuropharmacological mechanisms of visceral pain. Over the past few years, a number of animal models have been developed that, to a large extent, mimic the nociception originating in the viscera \[[@B18],[@B31]\]. These models have helped advance our understanding of the acute physiological responses associated with mechanical or inflammatory visceral nociception. It has become apparent that visceral pain and somatic pain are different, although some similarities exist \[[@B31]\]. In the present study, used the acetic acid-induced nociceptive model was developed for the screening of analgesic drugs and described as the writhing test \[[@B38]\] to exam the anti-nociceptive effect of minocycline in acute visceral pain. I.p. injection of acetic acid is a well known noxious chemical visceral stimulus in animals \[[@B38]-[@B44]\]. The nociceptive stimulation of the peritoneum by i.p. acetic acid produces abdominal contractions or writhes, and gastrointestinal ileus characterized by inhibition of gastric emptying and small intestine transit. Both procedures are associated with visceral pain \[[@B45]-[@B47]\]. This model is also used as a model of somatic-visceral pain \[[@B48],[@B49]\].
In a recent study, systemically administered minocycline (15, 30, 45, or 100 mg/kg) reduced carrageenan- or formalin-induced paw edema, formalin-induced inflammatory nociceptive responses, and tactile hypersensitivity by following the sensory nerve injury \[[@B15],[@B50]\]. In the present study, minocycline (4, 10, or 40 mg/kg), when intraperitoneally administrated 1 hour before acetic acid injection reduced writhing behavior in a dose dependent pattern (Figure [1](#F1){ref-type="fig"}). However, interestingly, a previous publication reported the contradictory results; a single dose of minocycline (30 or 100 mg/kg, i.p.) 30 minutes before acetic acid or zymosan injection did not attenuate the nociceptive behavior in mice. It had no effect on the early events of peritoneal inflammation (vascular permeability, inflammatory cell infiltration, and release of pro-inflammatory cytokines) in acetic acid or zymosan-injected mice. In addition, minocycline did not alter basal nociceptive responses in the tail immersion test \[[@B51]\]. They used male Swiss mice (20-26 g), and we used male ICR mice (20-25 g), they administrated minocycline 30 minutes prior to nociceptive stimuli, and we injected 1 hour before acetic acid stimulation, and they prepared minocycline by suspension them in one or two drops of Tween 80 in normal saline and we used normal saline. Furthermore, it has been reported that the concentration of minocycline in brain peaks at 1-2 hours after i.p. injection in phosphate buffered saline \[[@B52]\]. We suggest that differences in animal strain, preparation of drug, and duration for drug delivery could influence the nociceptive response.
c-Fos, an immediate-early gene protein product, is a neuroactive marker that can be used to indicate the activity of the spinal and supraspinal structures by many kind of peripheral pain including the visceral pain \[[@B49],[@B53],[@B54]\]. Previous studies showed that acetic acid enhanced central sensitivity with number of c-Fos-immunoreactive nuclei in the spinal cord \[[@B49],[@B55]\]. Recent studies showed that ERK phosphorylation by noxious stimuli contributes to the neuronal c-Fos expression in the spinal cord. ERK phosphorylation is involved in the establishment as well as maintenance of long-term neuronal alterations associated with chronic pain following noxious stimulation, such as c-fiber/capsaicin stimulation \[[@B56]\], chronic constrictive injury-induced neuropathic pain \[[@B57]\], noxious bladder stimulation \[[@B58]\], and colorectal distension stimulation \[[@B26]\]. In this study, p-ERK expression was consistent with the high expression of c-fos in the laminae I-X of the spinal segments (T5-L2) which innervate gastrointestinal tract. These results suggest that ERK activation is correlated with the expression of spinal c-Fos in acetic acid-induced mice.
Phosphorylation of spinal ERK has a substantial role in nociception \[[@B22],[@B59],[@B60]\]. Recent studies have shown that ERK phosphorylation in the spinal dorsal horn neurons might also participate in some visceral pain processing by in a model of visceral pain and hyperalgesia induced by cororectal distension \[[@B61]\], bladder hyperalgesia by cyclophosphamide \[[@B20]\] and intracolonic instillation of irritants (capsaicin, mustard oil) in adult mice \[[@B19]\]. However, the role of spinal ERK in response to acetic acid-induced acute visceral pain has not been previously described. Our study clearly demonstrates that acetic acid can rapidly induce phosphorylation of ERK in the spinal cord (Figure [3](#F3){ref-type="fig"}). The p-ERK-IR neurons were specifically localized to T5-L2 segments, which is consistent with gastrointestinal innervations \[[@B17]\]. Therefore, we suggest that phosphorylation of spinal ERK is responsible for nociceptive transmission from the gastrointestinal tract stimulated by acetic acid.
Spinal microglia are activated in inflammatory and neuropathic pain \[[@B15],[@B24]\]. Activated microglia secrete proinflammatory mediators such as prostaglandins, proteases, excitatory amino acids and cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1β, and IL-6, and the released substances participate in pain processing \[[@B52]\]. Recently, it has been demonstrated that minocycline attenuates the development of pain hypersensitivity by the inhibition of microglial activation and proinflammatory cytokine expression in inflammatory and neuropathic pain \[[@B11],[@B24]\]. Our previous studies confirmed that minocycline attenuates tactile hypersensitivity following trigeminal sensory nerve injury through the inhibition of microglial p38 MAPK activation \[[@B60]\], and that minocycline markedly reduces formalininduced inflammatory pain by inhibition of EPSCs in the substantia gelatinosa \[[@B15]\]. Recently, it was reported that minocycline completely reverses mechanical hyperalgesia in diabetic rats through microglia-induced changes in the expression of the potassium chloride co-transporter 2 (KCC2) at the spinal cord \[[@B13]\]. These findings raise the possibility that minocycline may behave as a potential analgesic for other types of pain model. Therefore, in the present study, we examined the anti-nociceptive effects of the minocycline in an acetic acid-induced acute visceral pain models. The results show that minocycline attenuates behavioral hypersensitivity through the inhibition of neuronal ERK activation in the spinal cord of acute visceral pain.
After peripheral nerve inflammation or injury, spinal microglia is initially activated and subsequently the sustained activation of astrocytes is precipitated. Activated astrocytes, as well as microglia, produce proinflammatory cytokines and chemokines, which are implicated in the induction and maintenance of pathological pain \[[@B62]\]. In our immunohistochemical study, the morphological activation of microglia and astrocytes were not clear 30 min after acetic acid injection, compared to normal brain (Figure [4](#F4){ref-type="fig"}). These findings agree with previous reports that the activation of microglia and astrocytes were not discernable as early as 1 h following formalin injection (acute pain) \[[@B15],[@B33],[@B63]\] and 2 h following the transection of the inferior alveolar nerve (chronic pain) \[[@B14]\], respectively. But in these reports, activated microglia and astrocytes were only increased after 1 day and peaked 3-7 days following formalin injection or peripheral nerve transection \[[@B14],[@B15],[@B33],[@B63]\]. Therefore, our findings suggest that microglia and astrocytes might not be a causal factor in the central hypersensitivity of acetic acid-induced acute visceral pain.
MAPKs signalling pathways are likely important mechanism for development and maintenance of central sensitization \[[@B64],[@B65]\]. The anti-nociceptive effect of an ERK upstream kinase (MEK) inhibitor, PD-98059, has been reported in various pain models including chronic constriction injury \[[@B66]\], complete Freund\'s adjuvant-induced monoartritis \[[@B67]\], and formalin-, capsaicin-, or mustard-induced inflammatory pain \[[@B19],[@B21]\]. More recently, it was reported that increased spinal ERK1/2 phosphorylation in 3-week monosodium iodoacetate (MIA)-osteoarthritis (OA) rats was blocked by the PD-98059, when examined 30 minutes following acute intrathecal administration \[[@B68]\]. Moreover, the observations in MIA-OA rats that PD-98059 treatment partially blocks pain behaviour and reduces grip force strength \[[@B68]\] supports the potential involvement of ERK1/2 phosphorylation in the dorsal horn spinal cord in mediating nociceptive-induced central sensitization associated with this model of osteoarthritis. In the present study, because the acetic acid-stimulated acute abdominal pain was reduced by inhibiting neuronal p-ERK expression in spinal cord by pretreatment of minocycline (Figure [3](#F3){ref-type="fig"}), we studied whether the directly introduction of PD-98059 to subarachnoid space could reduce the acetic acid-induced acute abdominal pain. Consistent with previous findings, we confirmed that the intrathecal pre-administration of PD-98059 markedly blocks the excitation of spinal neuron in the mouse model of acetic acid-induced acute abdominal pain.
It is interesting to note that minocycline inhibits the early activation of neuronal ERK MAPK in spinal cord although the effect of minocycline is generally considered to be on glia. Based on the early/acute response of the visceral pain, the acute inhibitory effect of minocycline on ERK activation might not be due to its inhibitory actions on spinal glial activation. Minocycline might have a direct inhibitory effect on neuronal ERK rather than glial ERK. Thus, these results suggest that the anti-nociceptive effect of minocycline for acute visceral pain is associated with neuronal ERK activation in the spinal cord.
Conclusions
===========
Increased spinal ERK phosphorylation is important for pain behaviors based on the MEK inhibitor studies. However, the direct link between minocycline\'s inhibitory effects in visceral nociceptive responses and its modulating effects on p-ERK expression has been unclear. In this study, minocycline attenuated the abdominal nociception, and the activation of spinal c-Fos and ERK 1/2 in the acetic acid-induced acute visceral pain. The intrathecal introduction of PD-98059, a MEK inhibitor, reduced the nociceptive behavior by acetic acid. These results strongly suggest that minocycline has an anti-nociceptive effect on acetic acid-induced acute visceral pain by inhibiting neuronal ERK activation in the spinal cord.
Methods
=======
Animals
-------
The male ICR mice (weight, 20-25 g) were kept at a constant temperature of 23 ± 2°C with a 12-h light-dark cycle (light on 08:00 to 20:00), and fed food and water *ad libitum*. The animals were allowed to habituate to the housing facilities for 1 week before the experiments. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) in College of Oriental Medicine, Kyung Hee University and animal treatments were performed according to the guidelines of the International Association for the Study of Pain \[[@B69]\].
Acetic acid-induced writhing test
---------------------------------
The acetic acid-induced visceral pain model is widely used in experimental research to produce abdominal contractions \[[@B38]-[@B44]\]. Mice were randomly assigned to two groups, saline-treated group (control group, n = 12) and minocycline-treated group (n = 31). Minocyclinetreated mice received minocycline (Sigma-Aldrich, U.S.A.) either 4 mg/kg (n = 10), 10 mg/kg (n = 10) or 40 mg/kg (n = 11) intraperitonially (i.p.) 1 hour before acetic acid i.p. injection (1.0%, 250 μl), respectively. Control mice received an equal volume of saline vehicle. The dosage of minocycline was determined based on the previous reports of therapeutic effects of minocycline \[[@B3],[@B4],[@B10],[@B12],[@B15]\]. The time point of acetic acid injection was determined based on a previous report of optimal delivery of minocycline in rats \[[@B70]\]. Following the i.p. injection of acetic acid, mice were placed in a clear plastic cage (20 × 26 × 12 cm), and the number of writhes per mice was counted in 5 minutes interval for 60 minutes. The behavioral tests were performed blinded under the constant condition (temperature, 23 ± 2°C; humidity, 55 ± 5%) between 9:00 am and 12:00 am in a quiet room.
Immunocytochemical evaluation
-----------------------------
At 30 minutes after the i.p. injection of acetic acid, the mice used for immunohistochemistry (n = 8/group) were anesthetized with 40 mg/kg sodium pentobarbital (i.p.), and perfused with fresh 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The T5-L2 spinal segment was removed and postfixed at 4°C overnight and then cryoprotected in 0.1 M PBS (pH 7.4) containing 30% sucrose for 48 hours at 4°C. Immunostaining was carried out according to previously established procedures \[[@B15],[@B37]\]. Briefly, cryosections (10 μm thickness) were mounted onto gelatin-coated slide glass. Six transverse sections in 700 μm intervals were selected from each animal \[saline, n = 8; minocycline (40 mg/kg), n = 8\] and incubated for 30 minutes with 3% H~2~O~2~, and then blocked with a solution containing 5% normal goat/or horse serum, 2% BSA, 2% FBS and 0.1% triton X-100 for 2 hours at room temperature (RT). The sections were incubated overnight at 4°C with either rabbit anti-c-Fos (1:10,000; Oncogene, U.S.A.), or rabbit anti-phospho ERK (1:500; Cell Signaling, U.S.A.). Sections were then incubated with biotinylated rabbit IgG antibody (1:200; Vector Laboratories, U.S.A) for 1 hour at RT. After rinsing, the sections were incubated with avidin-biotinylated HRP complex (1:200; Vector Laboratories, U.S.A) for 1 hour at RT and visualized with DAB. Sections were rinsed, and dehydrated and cover-slipped. Immunostained images were captured using image system (DP 70 digital camera, Olympus, Japan) under light microscope. Photo-images from the right side of T5-L2 were drawn using manual technique, and the numbers of c-Fos positive cells were counted. Each section was divided into (1) superficial dorsal horn (laminae I-II), (2) deep dorsal horn (laminae III-IV), (3) neck region (laminae V-VI), (4) ventral horn (laminae VII-IX), and (5) central canal region (lamina X) \[[@B71]\]. Evaluation of the immunostained sections were performed by an experimenter unaware of the experimental condition.
Immunofluorescence evaluation
-----------------------------
For double immunofluorescent staining, sections were incubated overnight at 4°C with a mixture of rabbit anti-p-ERK antibody (1:500; Cell Signaling, U.S.A.) and mouse anti-NeuN (1:500; Chemicon, U.S.A)/or rat/mouse anti-CD11/b (1:200; Serotec, U.S.A.)/GFAP (1:1,000; Chemicon, U.S.A) antibody. The sections were then incubated for 1 hour at RT with mixture of Cy3- and FITC-conjugated rabbit/rat/mouse IgG antibody (1:200; Jackson ImmunoResearch, U.S.A.), and then examined with confocal imaging system (LSM 5 PASCAL; Carl Zeiss, Germany).
Western blot analysis
---------------------
To investigate the level of p-ERK expression, minocycline (40 mg/kg) was injected intraperitoneally 1 hour before the i.p. injection of acetic acid. And at 30 minutes after the i.p. injection of acetic acid, the mice used for Western blot analysis (n = 4/group) were anesthetized, and the C1-C7, T5-L2, and L4-S1 spinal segments were removed with lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, and protease inhibitor mixture). A total of 50 μg of tissue lysate from each sample was resolved by electrophoresis on a 10% SDS-PAGE. The proteins were then transferred to PVDF membranes and blocked with 5% nonfat dry milk in Tween 20-containing Tris-buffered saline (TBST, 20 mM Tris, pH 7.4, 0.1% Tween 20, and 150 mM NaCl). The membranes were probed overnight with rabbit anti-c-Fos (1:1,000; Merck KGaA, Germany) or rabbit anti-p-ERK (1:2,000; Cell Signaling, U.S.A.) antibodies at 4°C, which was followed by incubation with HRP-conjugated secondary antibody at RT for 1 hour prior to enhanced chemiluminescence ECL (Amersham Pharmacia Biotech, U.S.A.) treatment and exposure to x-ray film. For normalization of antibody signal, the membranes were stripped and reprobed with antibodies for actin (1:2,000; Santa Cruz, U.S.A.) or ERK 1/2 (1:2,000; Cell Signaling, U.S.A.). After Western blot was performed several times, the density of each band was converted to numerical values using an Adobe Photoshop CS2 program (Adobe, San Jose, CA, U.S.A.), subtracting background values from an area of film immediately adjacent to the stained band. Data are expressed as the ratio of c-Fos or p-ERK against actin or total ERK 1/2 for each sample.
Intrathecal (i.t.) administration of PD-98059
---------------------------------------------
The i.t. injections were performed under light isofluran anesthesia (1-2%). The dorsal fur of each mouse (saline + acetic acid, n = 10; 0.1 μg PD-98059 + acetic acid, n = 8; 0.5 μg PD98059 + acetic acid, n = 10) was shaved, the spinal column was arched, and a 30-gauge needle was directly inserted into the subarachnoid space, between the L5 and L6 vertebrae \[[@B72]\]. Correct i.t. positioning of the needle tip was confirmed by manifestation of a characteristic tail flick response. The 0.1 and 0.5 μg of PD-98059 (2-amino-3\'-methoxyflavone; Calbiochem, USA), an ERK upstream kinase (MEK) inhibitor, or vehicle (saline alone or 10% DMSO) were slowly injected into the mice with a 50 μl Hamilton micro syringe in a total volume of 5 μl. The entire injection procedure, from the induction of anesthesia until recovery of consciousness, took 4-5 minutes. Preliminary injections were performed with a similar volume of 10% India ink solution, and then the reliability and accuracy of this method was confirmed by subsequent dissection of the lumbar spinal cord. The success rate for the prior injections with this technique was over 97.5%. The same investigator performed all injections. The i.p. injections of acetic acid and behavioral test were performed 20 minutes after i.t. injection of PD-98059, as described above.
Statistical analysis
--------------------
The statistical significance of differences between the values was determined using an ANOVA with a Fisher\'s post hoc test. All data are presented as the mean ± S.E. and a statistical difference was accepted at the 5% level unless indicated otherwise.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
IHC conceived all experiments, performed immunohistochemistry, analyzed the results and prepared figures, and wrote the manuscript. MJL performed behavioral experiments and Western blot. MJ, NGG and KYL assisted with behavioral experiments. HSJ participated in the design of the study and drafted the manuscript with IHC. All authors have read and approved the final manuscript.
Supplementary Material
======================
###### Additional file 1
**Figure S1**. Expression of c-Fos and p-ERK in the spinal cord of acetic acid-induced visceral pain models. c-Fos (A, B) and p-ERK (C, D) are specifically upregulated in the T5-L2 spinal segments of acetic acid-treated mice. Values, expressed as relative intensities, represent the mean ± SEM. *\*P*\< 0.01 *versus*five control groups except acetic acid-treated T5-L2 segments (ANOVA test with a Fisher\'s post hoc test). S, saline-treated normal mice; AA, acetic acid-treated mice.
######
Click here for file
Acknowledgements
================
This work was supported by a grant from the Kyung Hee University in 2010 (KHU20100701).
| {
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Introduction
============
Obstructive sleep apnea (OSA), the most common type of sleep apnea, is characterized by repeated episodes of complete or partial obstruction of the upper airways during sleep, despite efforts to breathe, and is associated with a reduction in blood oxygen saturation. OSA associated with excessive daytime sleepiness is commonly called OSA syndrome or OSA-hypopnea syndrome \[[@REF1]\]. The recurrent episodes of upper airway collapse during sleep in subjects with OSA are associated with recurrent oxyhemoglobin desaturation and arousals from sleep \[[@REF1]\].
Signs and symptoms of OSA include unexplained daytime sleepiness, restless sleep, and loud snoring (with periods of silence followed by gasps). Less common symptoms are morning headaches; insomnia; trouble concentrating; mood changes such as irritability, anxiety, and depression; forgetfulness; increased heart rate and/or blood pressure; decreased sex drive; unexplained weight gain; and increased urination and/or nocturia, and heavy night sweats. Awakenings in subjects with OSA are usually too brief for subjects to remember. Awakenings are frequently accompanied by shortness of breath, which corrects itself quickly within one or two deep breaths. Subjects may also make snorting, choking, or gasping sounds. These patterns can repeat five to more than 30 times per hour throughout the entire night \[[@REF2]\]. These disruptions may impair subjects' abilities to reach the desired deep, restful phases of sleep, with subjects likely feeling sleepy during waking hours. Subjects with OSA may be unaware that their sleep was interrupted, with many of these subjects thinking that they slept well throughout the night \[[@REF3]\].
Diagnosis and treatment of OSA depend on subjects' knowledge, awareness, and attitudes toward this condition. The present study, therefore, evaluated the knowledge, awareness, and attitudes toward OSA in persons living in the Asir region of Saudi Arabia. This study also assessed risk factors associated with OSA in this population, including obesity, hypertension, smoking, and family history; the main sources of information from which the public obtains information about sleep and how to improve it; and attitudes toward OSA, including its complications and awareness of the seriousness of these complications.
Materials and methods
=====================
This self-funded descriptive online cross-sectional study involved a nonrandomized sample of the general population of the Asir region of Saudi Arabia. Questionnaires were administered online over 30 days to subjects living in the Asir region who were aged \>18 years. The validated questionnaire survey was sent to social media sites used by the Saudi population, including Twitter, Facebook, WhatsApp, Telegram, and Snapchat, and conducted online via Google survey. The questionnaire included demographic aspects such as age, gender, and level of education, as well as several health-related characteristics, such as health status, body mass index, physical activity, and smoking.
The raw data were recorded on appropriate designed excel spreadsheets and processed in accordance with the best practices for raw data management to identify any inaccuracies prior to statistical analysis. Outlying data were flagged. Categorical variables were treated similarly to identify any potential anomalies. All identified anomalies were discussed with the biostatistics team and were corrected prior to initial statistical analysis. Descriptive statistics were recorded as numbers, percentages, means, and standard deviations.
The study protocol was approved by the ethics review board of our institution, and all participants provided written informed consent. Subjects were informed of their right to withdraw from the study at any time without any obligation toward the study team. The data were collected and used for research purposes only, with only the principal investigator having access to the data. Confidentiality was maintained throughout the study by keeping the participants' identity anonymous, with the names and personal information of participants not obtained for ethical reasons.
Results
=======
The study sample consisted of 626 participants. Of these, 37% were aged ≥45 years, 27% were aged 36-45 years, 20% were aged 26-35 years, and 16% were aged 18-25 years (Table [1](#TAB1){ref-type="table"}). Slightly less than two-thirds of respondents were males (61%), while 39% of participants were females. Analysis of respondent education level showed that most subjects were highly educated, with 64% having a bachelor\`s degree, 6% having a master's degree, and 6% having a Ph.D. In addition, 20% of participants had completed secondary school, whereas only 2% completed intermediate schools, and 1% completed primary schools (Table [2](#TAB2){ref-type="table"}). Moreover, 28% of respondents worked in the healthcare field, whereas 72% did not.
###### Age distribution of the participants in the study
------- -------- ---------
Number Percent
18-25 102 16.3
26-35 124 19.8
36-45 171 27.3
45+ 229 36.6
Total 626 100.0%
------- -------- ---------
###### Educational level of respondents
----------------------- -------- ---------
Number Percent
Primary 6 1.0
Intermediate 15 2.4
Secondary 124 19.8
University (Bachelor) 401 64.1
Master 40 6.4
PhD 40 6.4
Total 626 100.0%
----------------------- -------- ---------
The most frequent chronic diseases in our study respondents were diabetes mellitus (12%), hypertension (10%), vision problems (9%), and arthritis (7%), with 6% having other chronic diseases (Table [3](#TAB3){ref-type="table"}). Most respondents (62%) were in good health, reporting no chronic diseases.
###### Percentages of participants with chronic diseases
---------------------- -------- ---------
Number Percent
Diabetes mellitus 73 11.7
Hypertension 60 9.6
High cholesterol 2 0.3
Depression 14 2.2
Heart disease 15 2.4
Arthritis 45 7.2
Vision problems 57 9.1
Respiratory diseases 30 4.8
Cancer 1 0.2
Others 39 6.2
None 389 62.1
Total 626
---------------------- -------- ---------
Most subjects (54%) reported sleeping for four to six hours per day, and 41% reported sleeping for seven to nine hours per day. Only 3% reported sleeping for more than nine hours per day, whereas 2% reported sleeping for three or fewer hours per day. Assessments of participants' knowledge about the number of hours of sleep needed per day showed that the majority (82%) regarded that sleep for seven to nine hours was needed, with 14% reporting that four to six hours per day were needed (Table [4](#TAB4){ref-type="table"}). Only 4% reported that sleep for more than nine hours per day was needed, and only 0.2% reported that with three or fewer hours were required. About 70% of the respondents reported that the time they slept was sufficient for performing daily activities without difficulties, whereas 30% did not. Assessment of participants' knowledge of the most important benefits of adequate sleep found that 51% regarded physical benefits, 41% enhanced thinking, 5% reported emotional benefits, and 3% regarded social benefits as most important.
###### Participants' knowledge of sleep and need to sleep
------------------------------------------------------------------------------- -------- ---------
Hours of sleep needed per day
Time (hours) Number Percent
≤3 1 0.2
4-6 87 13.9
7-9 514 82.1
9+ 24 3.8
Sleep time is sufficient for performing daily activities without difficulties
Yes 433 69.2
No 193 30.8
Most important benefits of adequate sleep
Physical benefits 317 50.6
Enhanced thinking 259 41.4
Social benefits 16 2.6
Emotional benefits 34 5.4
Lack of sleep affects work
Yes 611 97.6
No 15 2.4
Drowsiness during the day
Yes 375 59.9
No 251 40.1
Nap during the day
Yes 468 74.8
No 158 25.2
Nap during the day affects sleep at night
Yes 319 51.0
No 307 49.0
Relationship between sleep and aging
Yes 444 70.9
No 45 7.2
Do not know 137 21.9
Total 626 100.0
------------------------------------------------------------------------------- -------- ---------
Almost all respondents (98%) believed that lack of sleep affected their work, whereas 60% reported drowsiness during the day. Although 75% reported taking a nap during the day, 51% were aware that naps during the day affect sleep at night. In addition, 71% of respondents reposted knowledge of a relationship between sleep and aging, whereas 22% were unaware of this relationship.
Of the study participants, 64% had heard of OSA, whereas 36% had not. About 75% regarded OSA as dangerous, whereas 24% did not know whether it is dangerous or not. Moreover, 81% of respondents reported never hearing about methods of diagnosing OSA, with 84% being unaware of methods used to treat OSA. The majority of respondents (96%) had never been diagnosed with OSA, with 80% regarding the diagnosis and treatment of sleep apnea as important.
Discussion
==========
This cross-sectional study was undertaken to determine the knowledge, awareness, and attitudes toward OSA among the general population of the Asir region of Saudi Arabia. Investigations of the presence or absence of risk factors for OSA (e.g., obesity, hypertension, smoking, family history), the main sources of information to the public about sleep and how to improve it, general attitudes toward OSA and its complications, and public awareness of the seriousness of these complications may help in the diagnosis and treatment of OSA.
Many studies worldwide have assessed awareness and knowledge of OSA, with one study in China reporting that 21.5% of the respondents were aware of OSA. A total of 77 (5.9%), 158 (12.1%), 150 (11.5%), and 110 (8.4%) respondents were able to correctly list at least one risk factor, symptom, health consequence, and treatment options for OSA, respectively \[[@REF4]\]. Among the study participants of the Asir region of Saudi Arabia, 64% had heard of OSA; 93%, 89%, 73%, and 16% answers were correct regarding risk factors, symptoms, health consequence, and treatment options for OSA; the population education level of the first research showed 9.3% with no former education, 36.4% with secondary and below education, and 54.2% with post-secondary education. However, in this research, the education level showed secondary and post-secondary more than 95%.
The reason for the population to seek healthcare services is their understanding and knowledge regarding the symptoms and the complications; a study involving the general population in the Lorraine region of France showed despite encouraging results regarding OSA symptoms, the general population showed limited awareness of its complications. Innovative educational campaigns must be organized to inform practitioners and the general public about the disease and raise awareness of its complications \[[@REF5]\]. A study in the USA found that OSA is a serious illness affecting about 12% of adults, with most of these patients being undiagnosed, posing serious healthcare and economic burdens. The STOP-BANG score (Snoring, Tiredness, Observed apnea, blood Pressure, Body mass index, Age, Neck circumference, and Gender) was reported to be a reliable screening tool to identify patients with probable OSA \[[@REF6]\]. Early diagnosis by in-lab polysomnography or home sleep apnea test should be followed by an appropriate therapeutic modality. Currently, continuous positive airway pressure therapy remains the treatment of choice for patients with moderate and severe OSA.
The importance of lifestyle modifications such as smoking cessation, weight reduction, and physical activity found to be a way of treatment. Controlled intervention trials strongly suggest that weight reduction together with a healthy diet and increased physical activity may correct or at least improve the symptoms of OSA \[[@REF7]\].
Knowledge sources about OSA are very important. In this study, we found (26%) of respondents seeking the doctors for information, while (23%) they seek it from social media. Research done about the quality of YouTube as a source of information showed that YouTube is a promising source of information for OSA patients. Educational and news videos are of the highest quality. General quality measures like search position, views, and likes are not correlated with formally scored value. Sleep surgery and otolaryngologists are minimally mentioned, representing an opportunity for improvement \[[@REF8]\].
Conclusions
===========
The present study showed a poor level of awareness regarding all aspects of OSA among the general population of the Asir region of Saudi Arabia. Most subjects lacked sources of knowledge about this condition, indicating the need for medical personnel to improve awareness. Health education campaigns about OSA and its importance are needed, as is the training of primary healthcare physicians to raise awareness in their communities. Additional studies are also needed to assess the level of community awareness about OSA and its complications.
The authors have declared that no competing interests exist.
Consent was obtained by all participants in this study
**Animal subjects:** All authors have confirmed that this study did not involve animal subjects or tissue.
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Background
==========
The circadian system supplies organisms with a means to adapt their internal physiology to the continuously changing environmental stimuli that exist on a rotating planet \[[@B1]\]. The central pacemaker is located in the suprachiasmatic nucleus (SCN) of the hypothalamus and coordinates, via neural and humoral signals, multiple peripheral clocks situated in tissues throughout the animal \[[@B2]\]. These peripheral clocks consist of a group of highly conserved 'clock' genes and their protein products functioning within tightly controlled autoregulatory transcription-translation feedback loops \[[@B3]\] that ultimately give rise to 24-h alterations in gene expression and behavioural outputs.
Circadian clock gene expression is detectable not only in the SCN but in almost all peripheral tissues. The molecular clockwork mechanism comprises a series of feedback loops that encompass a set of core clock genes: *ARNTL* (aryl hydrocarbon receptor nuclear translocator-like), *CLOCK* (circadian locomotor output control kaput), *PER1* (period homolog 1), *PER2* (period homolog 2), *PER3* (period homolog 3), *CRY1* \[cryptochrome 1 (photolyase-like)\], and *CRY2* \[cryptochrome 2 (photolyase-like)\] \[[@B4]\].
The positive axis of the loop is created by transcription factors *CLOCK* and *ARNTL* as they undergo transcription and translation \[[@B3]\]. *CLOCK* and *ARNTL* proteins heterodimerize and bind to E-box enhancers upstream of *PER* and *CRY* genes in order to trigger transcription \[[@B5]\]. Following this, a complex is formed by *PER* and *CRY* proteins that relocates to the nucleus to inhibit *CLOCK/ARNTL* activity. This leads to the repression of their own transcription completing the negative axis of the feedback loop \[[@B2]\]. *RORA*, (RAR-related orphan receptor A) *NR1D1* (nuclear receptor subfamily 1, group D, member 1), and *NR1D2* (nuclear receptor subfamily 1, group D, member 2) are orphan nuclear receptors that make up a secondary feedback loop. *RORA* instigates *ARNTL* transcription whilst *NR1D1* and *NR1D2* repress its expression \[[@B6]\]. Each cycle of the molecular clock within a tissue gives rise to the simultaneous upregulation of a subset of clock-controlled genes \[[@B7]\] activated by the transcriptional activity of the ARNTL/CLOCK heterodimer.
Markers of circadian phase in humans include melatonin \[[@B8]\] and body temperature \[[@B9]\]; however melatonin has been established as not circadian in the horse \[[@B10]\]. Previous studies in humans have used white blood cells or oral mucosa as a method of detecting human clock gene expression \[[@B11],[@B12]\]. These methods have several reported drawbacks \[[@B11],[@B13]\]. Physical stimuli and time delays due to the processing of cell separation may affect levels of expression of clock genes and the overall quality of the isolated mRNA. For instance with white blood cells, the issue relates to time delays due to the processing of cells prior to transcriptional inactivation which in turn may affect the levels of mRNA of clock genes. A similar concern can be seen with the collection of oral mucosa cells. RNA samples were shown to be severely fragmented and thus the results were discarded \[[@B12]\]. There are additional impracticalities in collecting oral mucosal cells from horses. These could be avoided through the collection of hair follicles as an alternative sampling method \[[@B13]\]. The main advantages of using hair follicle cells are that they can be obtained noninvasively and cells can be collected and the RNA stabilised simply by plucking hairs and adding them directly to an RNA stabilisation buffer.
A non-invasive and effective method for detecting peripheral equine clock gene expression has yet to be established. This hinders progress in areas of equine circadian research. Currently, research investigating equine peripheral clocks has relied on collecting muscle biopsies or blood samples. These methods can often be time consuming, costly and can occasionally lead to animal welfare concerns as this approach to collecting samples can be invasive.
In this report we examine a convenient and non invasive method for detecting equine clock gene expression through the use of hair follicle cells collected from the mane of the horse.
Materials and methods
=====================
Sample collection
-----------------
Five healthy, non-pregnant mares (*Equus caballus*) of various lightweight breeds were individually housed in standard 12 ft x 12 ft stalls for 24-h under a light--dark (LD) cycle that mimicked the environmental photoperiod for that time of year. Mares were chosen for their availability on UCD's Lyons Research Farm. Stallions were not available for the study and castrated males (geldings) are considered unsuitable as their neuroendocrine system is compromised. The experiment was conducted in March where the times of dawn and dusk were 06:00 and 18:00 respectively, corresponding to a 12 h Light : 12 h Dark LD cycle at longitude 138 W6.8, latitude N53.2 (County Kildare, Ireland). While stabled, horses had access to hay and water *ad libitum.* We collected mane hair samples at 4-h intervals from 16:00 \[Zeitgeber Time (ZT) 9, where time of lights on defines ZT 0\] for a period of 24 h.
Each hair sample consisted of 10--20 hair follicles that were trimmed to remove excess hair and carefully placed in a 2 mL screw-cap tube containing 400 ul of binding buffer from the High Pure RNA Isolation Kit (Roche, Indianapolis, Indiana).
Quantitative polymerase chain reaction (qPCR)
---------------------------------------------
Total RNA was isolated using the High Pure RNA Isolation Kit (Roche, Indianapolis, INdiana) according to the manufacturer\'s instructions with minor modification: The hair follicles were placed in a 2.0 mL screw cap tube, containing 400 uL of binding buffer from the High Pure RNA Isolation Kit (Roche) and 200 uL of PBS. A single 5 mm stainless steel bead (Qiagen) was added to each tube and the samples were homogenised at maximum speed (30 Hertz) for 2 min using the Qiagen TissueLyser system. Following homogenisation the samples were spun for 1 min at maximum speed to reduce foaming, the homogenate was then applied to the filter column and RNA was extracted as per instructions for the High Pure Isolation Kit. RNA was eluted in 50 uL and stored at -80 deg C.
RNA quantity was measured using the NanoDrop ND1000 spectrophotometer V 3.5.2 (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed using the Agilent Bioanalyser RNA Chip (Santa Clara, California). All samples were shown to have a RIN value in excess of 7.5. The RNA was converted to complementary (c) DNA and a cDNA pool containing 3.5 ul from each sample was prepared and used to generate a 7 point, 1 in 4 serial dilution. This serial dilution was used to test the efficiency of each primer pair used in the study. The remaining cDNA was diluted to 2.0 ng/ul of RNA equivalents and stored at −20°C. A number of minus reverse transcription (RT) controls were included during the cDNA preparation.
Quantitative PCR assays were performed using Biosystems 7500 Sequence Detection System and the Sensi Mix SYBR Kit (Bioline, Taunton, Massachusetts). A panel of eight putative reference genes was assessed for stability using the GeNorm algorithm with the qBase Pair Softwear package. Results showed that *ARTB* and *HPRT* were suitable reference genes and the optimal normalization factor was calculated as the geometric mean of these two reference targets. Each PCR reaction was prepared in duplicate and in a volume of 20ul \[10 ul master mix, 5.0 ul cDNA, 1.2 ul forward (300 uM), 1.2 ul reverse (300 uM) and 2.6 ul water\]. A panel of five core clock genes was selected; *PER1* (period homolog 1), *PER2* (period homolog 2), *ARNTL* (aryl hydrocarbon receptor nuclear translocator-like), *CRY1* \[cryptochrome 1 (photolyase-like)\], *NR1D2* (nuclear receptor subfamily 1, group D, member 2) and the clock controlled gene (CCG) *DBP* (D-site of albumin promoter binding protein). Candidate genes were selected based on prior evidence of their cyclic expression in human hair follicles \[[@B13]\] and where equine primer sequences were previously published \[[@B14]\]. Primers sequences were commercially synthesised by Eurofins MWG Operon (Ebersberg, Germany).
Thermal cycling consisted of one cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Melt curves were examined to confirm specificity of each PCR product. Primer efficiencies were shown to be between 90% and 110%. Transcript abundance was determined relative to *ACTB* and *HPRT1* using the Q Base Plus Software package (Biogazelle, Belgium). CNQR results were analysed using SPSS.
Data analysis
-------------
One-way repeated measures ANOVA (GraphPad Prism Version 5.0 for Mac, GraphPad software, San Diego, California, USA, <http://www.graphpad.com>) was used to determine whether the temporal pattern of expression for each transcript varied significantly over the 24-h period. The presence of diurnal (24-h) temporal variation for transcript means was evaluated using a Cosinor programme \[[@B15]\] based on the least squares cosine fit method \[[@B16]\]. In all cases significance was assessed as *p \< .05*. Data are presented as means ± SEM.
Results
=======
The expression patterns of five core clock genes *PER1, PER2, ARNTL, CRY1* and *NR1D2* and the clock controlled gene, *DBP* (D-site of albumin promoter binding protein) were investigated in this study. We detected mRNA expression of all six genes in equine hair follicles. ANOVA revealed significant 24-h variation for three of the genes *PER1, PER2* and *DBP (p = .024, p = .02* and *p = .036,* respectively*;* Figure [1](#F1){ref-type="fig"}) but not for the remaining three genes *ARNTL, CRY1* and *NR1D2 (p = .58, p = .32, p = .37,* respectively; Figure [1](#F1){ref-type="fig"}).
{#F1}
Cosinor analysis confirmed a significant 24-h cyclic component for *PER1 (p = .002), DBP (p = .0033)* and *NR1D2 (p = .0331)* while the 24-h cosine fit for *PER2* (p = .0643) was just shy of significance. As the inverse oscillatory relationship between *PER2* and *ARNTL* expression is considered a characteristic component of the molecular clockwork mechanism, we further examined whether any individual expression profiles of *ARNTL* exhibited a circadian waveform. No individual 24-h expression profiles for *ARNTL* exhibited significance via cosinor analysis (*p* \> .05) Results of cosinor analysis including estimated acrophases are presented in Table [1](#T1){ref-type="table"}.
######
Results of cosinor analyses of mRNA expression profiles
**Gene transcript** **Robustness (%)** **Acrophase (24 h)** **Zeitgeber Time (ZT)** ***p*value**
--------------------- -------------------- ---------------------- ------------------------- --------------
*PER1* 94 10:33 4.5 *0.002*
*DBP* 92 08:37 2.5 *0.0033*
*NR1D2* 65 08:57 3.0 *0.0331*
*PER2* 51 09:33 3.5 *0.0643*
*CRY1* 0 n/a n/a *0.8944*
*ARNTL* 0 n/a n/a *0.3066*
For each gene transcript the robustness values, acrophase time, corresponding Zeitgeber Time and and significance values (p) are presented.
Discussion
==========
In this study, we demonstrate that RNA extraction from equine hair follicle cells yields high quality RNA and is suitable for detection of 24 h oscillations of core components of the equine molecular clock. To demonstrate that oscillating clock gene expression in hair follicle cells can be used as markers to assess an equine peripheral clock, we examined gene expression in hair follicle cells collected from the manes of five mares over a 24 h period. This is the first study to investigate a non-invasive method of assessing an equine peripheral tissue clock.
We show that mRNA levels of *PER1, PER2* and *DBP* vary significantly over time as determined by ANOVA. However, of these three, only *PER1* and *DBP* are shown to exhibit a 24-h sinusoidal profile as determined by cosinor analysis. Furthermore, although non-significant by ANOVA, the expression profile of *NR1D2* is positive for 24-h rhythmicity by cosinor analysis. These apparent discrepencies can be explained by understanding the nature of each analysis. Cosinor analysis is more sensitive than ANOVA at picking up sinusoidal patterns in the data associated with a specified period as that is precisely what it is designed to do. The ANOVA can take into account the repeated measures design in partitioning out the variance but has no means of accounting for the temporal relationship (angular proximity) of the time points and no model for temporal variation against which the observed variation is compared. In the case of *PER2,* the highly variable means at specific time points were detected by ANOVA but the angular proximity of the time points were only weakly sinusoidal. Conversely, while the mean expression of *NR1D2* at specific time points varied to a lesser extent, the angular proximity of the data points more closely reflected a sinusoidal pattern. Of the six gene transcripts examined, we find that *PER1* and *DBP* may serve as the most reliable markers for this peripheral clock in the horse with limited sampling frequency.
Furthermore, we report that as few as 10 equine hairs are sufficient for high quality RNA yield. A significant advantage of this technique is that it can be carried out by an untrained person and eliminates the need for tissue biopsies.
A comparison of our results with a previous study evaluating clock gene expression from human hair plucked from the scalp and chin \[[@B13]\] revealed that a similar temporal pattern of expression of *PER2, NR1D2* and *DBP* exists between species when maintained under LD12:12. Although the LD cycle under which the human subjects were maintained is not reported, the three oscillating genes in common to the two studies, *PER1, DBP* and *NR1D2*, exhibit peaks in the morning between 07:00 and 09:30, suggesting a similar phase relationship between these genes in the two species.
Importantly, in a previous investigation of clock gene expression profiles in equine gluteal muscle conducted by our lab \[[@B14]\], peak values of *PER2, NR1D2* and *DBP* were observed at 07:00. Although further studies are required to accurately identify the phase relationship between the peripheral clocks in muscle tissue and hair follicles, these findings suggest that hair follicle expression profiles may provide a valuable marker with a similar phase relationship to the envitronmental LD cycle as a more performance relevant equine tissue, gluteal muscle.
It was surprising to fail to find rhythmic expression of the core clock genes, *ARNTL* and *CRY1*, in hair follicle cells. However, it has become clear from studies in peripheral tissues that the specific contributions and interactions between specific clock components vary in a tissue-specific manner \[[@B4]\]. Moreover, in the recent human study of hair follicle clock gene expression, only "slight oscillations" of *ARNTL* were detected while *CRY1* was not examined \[[@B13]\]. It cannot be excluded that the cycling amplitude of *ARNTL* and *CRY1* in follicle cells may be inherently small and undetectable in the current assays, or that post-translational processing plays a more pivotal role in this tissue clock.
Circadian rhythms regulate hundreds of functions relevant to the athletic horse including body temperature and hormone production \[[@B10],[@B17]\], immune function \[[@B18]\] and muscle metabolism \[[@B14]\]. Disruption of the circadian system could have a profound influence on equine health. In humans, the disruption of circadian rhythms has been linked to jetlag \[[@B19]\], insomnia \[[@B20]\], stomach ailments \[[@B21]\] and depression \[[@B22]\]. With more research into the characterisation of the circadian clock, it may be possible to pin point the exact consequences of disruption to the circadian cycle on the equine system.
The need for a non-invasive marker of circadian phase is particularly relevant for furthering equine research in relation to transmeridian transportation of horses for international competition. It is well accepted that the circadian clock regulates activity and also muscle metabolism in mammals \[[@B23],[@B24]\]. This is supported by evidence that nocturnally active rats experience peak expression of *PER2* in skeletal muscle at the onset of dark \[[@B23],[@B24]\] whereas diurnally active horses have an opposing peak of *PER2* expression at dawn \[[@B14]\]. The capacity of the molecular clock within the SCN to reset following an abrupt 6-h LD shift, as occurs during transmerdian travel across six time zones, was shown to take up to eigth days in mice \[[@B25]\]. The gradual re-entrainment of clock genes within specific areas of the SCN was correlated with, and mirrored, a disruption in circadian behavioral output, as measured by locomotor activity rhythms in a further study \[[@B26]\].
In addition to desynchronized clock gene expression within the master pacemaker in the SCN, rhythmic gene expression in peripheral tissues, which rely on SCN signals for synchrony, are also significantly disrupted. This was clearly demonstrated in rats when it was reported that clock gene cyclicity in skeletal muscle, liver and lung shifted more slowly than the SCN following both LD cycle advances and delays \[[@B27]\]. The authors concluded that this likely further explained the physical malaise in humans associated with rapid transmeridian travel.
The capacity to evaluate markers of circadian phase, as determined by clock gene expression patterns, to determine the extent and duration of circadian misalignment in response to abrupt changes in the 24-h LD cycle, as occurs during transmeridian travel, represents a valuable experimental tool. Our results suggest that equine hair follicles may be used in future studies as a reliable and non-invasive method to detect the time duration required for a peripheral equine tissue to adjust to a new time zone. If it is found that the phase of the equine hair follicle clock closely mirrors that in skeletal muscle, which our results suggest, and exhibit similar re-entrainment rates following simulated jet lag, then there clearly exisits the opportunity to quantify the duration of potential performance deficits and to develop a molecular test for time zone resynchronization in these valuable global athletes. This in turn could help horse trainers determine how much time in advance to transport their racehorse to a temporary location before an important race.
Conclusions
===========
We demonstrate that RNA extraction from equine hair follicle cells is a suitable method of evaluating certain core clock genes and a clock-controlled gene to assess an equine peripheral clock. In particular, our findings support the evaluation of the gene transcripts *PER1* and *DBP* in hair follicle cells as suitable markers for evaluating the phase of a peripheral clock in the horse.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
LMW collected the data, performed the gene expression assays, analysed data and drafted the manuscript. BAM conceived of the project, assisted with data collection and analysis and drafted the manuscript. JAB supervised gene expression assays and assisted with drafting manuscript. All authors approved the final manuscript.
Acknowledgments
===============
The authors are grateful to Lisa Dunne for assistance with data collection. We would like to thank the staff at UCD Lyons Research Farm.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Diabetes is a chronic disease requiring substantial patient engagement and self-management for successful control. Despite well-established clinical guidelines, most persons with diabetes struggle with managing their diet, physical activity, and glucose self-monitoring [@pone.0048549-Glasgow1], [@pone.0048549-Grzywacz1]. Diabetes risk factors are modifiable with health behavior change, yet meaningful health promotion counseling is uncommon in primary care [@pone.0048549-Jansink1]--[@pone.0048549-Vickers1] and few physicians are extensively trained in the counseling techniques known to promote self-efficacy and support self-management [@pone.0048549-Burke1], [@pone.0048549-Finocchio1]. In conditions such as diabetes, where psychosocial barriers are common and challenging, and fostering self-efficacy and self-care is critical, identification of clinical strategies that successfully promote behavior change would be particularly valuable.
One type of health care provider, naturopathic physicians (NDs), who practice a broad spectrum of complementary and alternative medicine (CAM), routinely include clinical counseling on wellness and prevention and prioritize health promotion [@pone.0048549-Fleming1]--[@pone.0048549-Hawk1]. NDs treat a wide variety of health concerns similar in scope to those seen by conventional primary care [@pone.0048549-Cherkin1]. NDs are currently licensed in 16 states, the District of Columbia, and the United States territories of Puerto Rico and the United States Virgin Islands. Although NDs can use a variety of conventional diagnostic techniques and pharmaceutical agents, including oral anti-diabetic medications and insulin, their therapies focus on natural products, mind-body techniques, detailed nutrition counseling, physical activity prescription, and stress management recommendations delivered through individualized health promotion counseling [@pone.0048549-Hawk1]--[@pone.0048549-Bradley1]. The clinical effectiveness of ND care for diabetes has been recently explored [@pone.0048549-Hellquist1]--[@pone.0048549-Oberg1] but patients' experiences with ND care have never been investigated qualitatively.
To be effective, treatment approaches for diabetes and other complex chronic conditions need to effectively engage patients in their own care [@pone.0048549-Bodenheimer1], [@pone.0048549-Glasgow2]. In order to better characterize patients' experiences using CAM for diabetes, and to better understand the patient experience with naturopathy, this study explored the experiences and perceptions of persons who had previously used only conventional medical care for their diabetes following a year of adjunctive naturopathic care (ANC).
Methods {#s2}
=======
This qualitative analysis was conducted as part of an evaluation of an observational study of the effects of a course of adjunctive naturopathic care (i.e., in addition to continued medical care) on persons with sub-optimally controlled (HbA1c \>7.5%) type 2 diabetes. Details of the study and the characteristics of ANC are available elsewhere [@pone.0048549-Bradley2]. In summary, in that study, participants could receive up to 8 visits to one of four naturopathic physicians (3 female and 1 male) over a one-year period, as an adjunct to continuing their usual medical care. The mean number of ANC visits actually made was 3.9+/−2.1, most of which were made within the first 6 months. None of the participants had previously received care from a naturopathic physician. All had access to naturopathic medicine as part of their current insurance benefits, although many may not have been aware of this. Following initiation of ANC, this observational study found improvements in patient-reported outcomes (e.g., glucose monitoring, diet, self-efficacy, motivation, and mood) and reductions in blood glucose that exceeded those for similar patients who do not receive ANC [@pone.0048549-Bradley2]. Approval for this study was obtained from the IRB at Group Health Cooperative.
Selection of Participants and Recruitment {#s2a}
-----------------------------------------
The 37 adults with diabetes who participated in the parent study and made at least one visit to an ND were eligible for this study. After completing their final outcomes assessment 12 months after entering the study, participants were invited to participate in either a focus group or an in-depth telephone interview focusing on their experiences and opinions about the naturopathic care they had received. Twenty-two (59%) agreed to participate; 17 attended one of three focus groups and 5 were interviewed by telephone. Characteristics of study participants are presented in [Table 1](#pone-0048549-t001){ref-type="table"}.
10.1371/journal.pone.0048549.t001
###### Demographics of participants and non-participants.
{#pone-0048549-t001-1}
Characteristics Participants(n = 22) Non-participants(n = 15) P value
--------------------------------------------------------------------------------- ----------------------------------------- ------------------------------------------------ -------------------------------------- ------
Gender (Male, % ) 50 40 0.16
Age (Years, mean/SD) 57.1 (7.6) 57.9 (7.5) 0.48
Highest Education (%) High school, GED or less 9.1 12.5 0.69
Some college, incl. technical 40.9 42.5
College graduate 50.0 45.0
Ethnicity (White/Non-Hispanic %) 59.1 65.0 0.39
Annual Family Income (%) \<\$60,000 45.4 38.5 0.41
\$60,000--85,000 13.6 20.5
\>\$85,000 40.9 41.0
Years of Diabetes (mean/SD) 6.55 (3.95) 7.22 (4.19) 0.27
Baseline Hemoglobin A1c (mean/SD) 7.91 (0.58) 8.01 (0.56) 0.23
Rating of Conventional Healthcare for diabetes[\*](#nt101){ref-type="table-fn"} Perceived Effectiveness + (mean/SD) 1.82 (0.85)[+](#nt102){ref-type="table-fn"} 1.81 (0.91)^+?^ 0.98
Moderately/very satisfied with care (%) 90.9 81.2[?](#nt103){ref-type="table-fn"} 0.38
Rating of Naturopathic Healthcare for diabetes[\*](#nt101){ref-type="table-fn"} Perceived Effectiveness + (mean/SD) 2.24 (SD 0.95)[+](#nt102){ref-type="table-fn"} 2.86 (0.95)^+?^ 0.07
Moderately/very satisfied with care (%) 95.2 57.4[?](#nt103){ref-type="table-fn"} 0.01
at 6-month follow-up,
0 to 5 scale: 5 = most effective,
n = 14 for perceived effectiveness/satisfaction items.
Development of the Interview Guide {#s2b}
----------------------------------
Three popular health behavior models describing the processes of decision-making, self-efficacy, and behavioral outcomes (Social Cognitive Theory [@pone.0048549-Bandura1], the Health Beliefs Model [@pone.0048549-Rosenstock1], and Self-Determination Theory [@pone.0048549-Ryan1]) informed development of our interview guide \[the complete interview guide is available in Focus Group Guide S1\]. The interviews explored: 1) motivations and reasons for participating; 2) general descriptions of their experience, including anything unexpected; 3) positive and negative experiences with ANC; 4) new information or insights gained from the ND experience; 5) whether and how the ND addressed health behaviors; 6) behavior changes made or contemplated as a result of the experience; and 4) comparisons of naturopathic care and conventional care for diabetes.
Focus Groups and Interviews {#s2c}
---------------------------
Three trained qualitative researchers conducted the interviews and focus groups. Focus groups were approximately 90 minutes and interviews ranged from 25 to 60 minutes. All were tape-recorded and transcribed by a professional transcriptionist. Transcripts were imported into Atlas.ti version 6.2 (Berlin, Germany), a qualitative software that assists with coding and data management. Participants received a small incentive payment.
Analysis {#s2d}
--------
We used an inductive/deductive content analysis approach [@pone.0048549-Elo1]. We first abstracted information pertaining to the key elements identified in the interview guide and developed an a priori code list based on the topics of inquiry. A second researcher reviewed the code list and additional codes were added based on her initial read of the transcripts; this yielded 127 unique codes. All 3 researchers then coded transcripts separately. Atlas.ti mapping features were used to aggregate codes into themes, eliminating responses that were off-topic or were isolated opinions not expressed by at least 3 participants. Redundant responses and shared opinions were aggregated into super-codes and sorted by code density as well as by participant ID (to account for repetitive statements by individual participants). The analysis team used an iterative process to discuss the themes, clarify and expand upon interpretations of findings, and contextualize the coded responses back into the transcripts of the full conversation. When disagreements were identified, the researchers reviewed the original transcripts to achieve consensus on the intent of the participants' comments. Concepts and themes were also organized by participant ID to quantify the relative frequency of different characteristics of the care experience.
The characteristics of study participants and non-participants were compared using unpaired T-tests ([Table 1](#pone-0048549-t001){ref-type="table"}). The only statistically significant difference was that participants were more likely to have been satisfied with their naturopathic care.
Results {#s3}
=======
Our findings first describe why patients wished to try ND care and then highlight the nine major themes that emerged from the analyses. Three of these themes characterize the ND-patient *interaction* (patient-centered, holistic, and collaborative) and five characterize the *content* of the encounters (individualized and detailed health promotion, counseling that promoted self-efficacy, pragmatic and practical self-care recommendations, novel treatment options that fostered hopefulness, and patient education that addressed both diabetes self-care and general health). The final theme - contrast between ND care and conventional medical care - was often mentioned in the context the other themes but warranted description as a separate theme.
Reasons for Participation in a Study of Naturopathic Care for Diabetes {#s3a}
----------------------------------------------------------------------
Three major reasons were reported for participating in a study of ND care for diabetes ([Table 2](#pone-0048549-t002){ref-type="table"}). Most respondents wanted to try a different approach to diabetes. About 40% expressed frustration with their current diabetes care and were concerned about progression of the disease, especially the need to increase medication or initiate insulin injections. About a third (32%) were curious about naturopathic medicine and were attracted to a more natural approach to health and disease.
10.1371/journal.pone.0048549.t002
###### Reasons people with diabetes chose to participate in a study of naturopathic care (n = 22).
{#pone-0048549-t002-2}
Reason \%[\*](#nt104){ref-type="table-fn"} Example quotes
------------------------------------------------------------------------------------------ ------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Desire for a different approach** **(not specifically CAM)** 59 "I was interested in something else. I agree with medicine up to a point -- I had it coming out of my ears. I wanted something different."
"If it was another MD, I don't know -- you know, I have an MD. I have gone through the typical medical array of people that you go to when you have diabetes. I thought it would be a different perspective, maybe helpful."
**Frustration with perceived ineffectiveness/** **limitations of conventional medicine** 41 "It seemed like all my doctor was pushing was medicine, get that A1c down, more medicine, more medicine, insulin. Even though I knew there were these other things, like exercise and eating better and all that, we don't discuss that"
"I just didn\'t see to be doing any good with standard medicine"
**Specific interest in trying naturopathic** **medicine or CAM** 32 "I was just curious and I always wanted to see a naturopathic doctor"
"I prefer just to go natural with the body"
"I wanted to reassess my lifestyle, the things I do."
Sums to \>100% because some participants offered multiple responses.
Themes Related to the Nature of Naturopathic Care ([Table 3](#pone-0048549-t003){ref-type="table"}) {#s3b}
---------------------------------------------------------------------------------------------------
10.1371/journal.pone.0048549.t003
###### Themes related to the nature of naturopathic care (n = 22).
{#pone-0048549-t003-3}
Theme \%[\*](#nt105){ref-type="table-fn"} Example codes and patients' terminology
---------------------- ------------------------------------- ---------------------------------------------------------------------------------------
**Patient-centered** 95 Not feeling rushed through the appointment
Felt ND was present and focused on them during encounter.
Feeling listened to and understood
Empathic
**Holistic** 73 In-depth intake procedure contributed to feeling understood as a whole person
Other health problems were addressed as influences on the management of diabetes
Psychosocial influences on health were addressed
**Collaborative** 68 Patient preferences and input were solicited
Feeling supported, "cheered on"
Treatment recommendations were individualized
Accountability was established with individualized follow-up and monitoring schedules
Sums to \>100% because some participants offered multiple responses.
### Theme 1: Patient Centered {#s3b1}
Various aspects of the patient-centeredness of the interaction, a major focus of CAM and integrative medicine [@pone.0048549-Barrett1], [@pone.0048549-Maizes1], were noted by 95% of participants. Participants commented that the relatively long visits with NDs (30--60 minutes) created an opportunity for a different type of interaction.
> *"It's like they \[MDs\] have a set amount of time allocated for each patient -- maybe 15 minutes. So I have a choice between my broken finger and my diabetes. That's what it feels like. \[The ND\] was present in the moment. I couldn't tell if she was thinking of the next patient and \[thinking\] 'I have to finish here so I can do the next thing'. She was very thorough too. She would look me in the eye; she was comfortable with her position. And I guess I liked the power and control she gave me over my disease."*
>
> *"I think the medical doctor is maybe too accustomed to seeing so many people with diseases and the progressions \-- I didn\'t feel like the experiences or the symptoms that I was having were, I don\'t know, weren\'t maybe as worthy of treatment. He had seen people dealing with worse things than what I was dealing with. With the naturopath it was \-- if it affects you, then it\'s real and let\'s take care of that."*
Patients reflected on characteristics of the therapeutic relationship they developed with the naturopathic physicians. They used words like "casual," "relaxed," "personal" to describe NDs' communication styles in contrast to using words like "formal," "limited" -- both in time and focus, and "structured" to characterize their experiences with conventional medicine.
> *"I\'m more at ease when I talk to my naturopath. I\'m very honest with her. We have more time, and she would really listen and give me more detail about what will happen. With my primary doctor, she is wonderful too, it\'s just the time limit that she can talk with me. The next step would be insulin is what my primary doctor said; that\'s what pushed me to go into the study -- I didn't want to go see my primary doctor because I know she just goes with whatever one is saying about statistics or numbers that come out from the lab test results. It\'s a different atmosphere, a different way of expressing myself to the two, the primary and the naturopath."*
>
> *"She was very easy to talk to. I felt that she really listened and she was present with me in the moment. And that she was not holding back any information. She was genuinely forthcoming with the information that she had. There was, well, if it gets worse, we'll try this. It's like, let's take these steps; and it was really kind of laid out."*
### Theme 2: Holistic {#s3b2}
Patients often said they were surprised by the "whole" or "holistic" nature of ND care. Several commented that the ND attended not only to their diabetes but also to other aspects of their health and life that might impact their diabetes and their ability to engage in self-care. Several participants noted the beneficial effects of the ND's attention to their psychoemotional state.
> *"I would say that \[the naturopathic\] approach is dealing with your whole person, not just the specific ailment like diabetes...if you fix all these other things, everything else is going to be better. Your numbers are going to be better and so on. So she really helped resolving those by recommending other things to do."*
>
> *"I think one difference is, my medical doctor seems to want to manage symptoms. And the naturopath wants to get kind of to the bottom of things, take all the layers off to get to and address the root issue."*
### Theme 3: Collaborative {#s3b3}
Patients reported that the NDs employed a collaborative approach that respected patient autonomy and engaged them in making decisions about following different treatment approaches to improve glycemic control. A subtheme reflected the educational component of the ND-patient interaction; patients are not only engaged in selecting the course of treatment but they are engaged as active participants in evaluating the effects of the treatment, consistent with the emerging societal prioritization of "patient centered outcomes" [@pone.0048549-Stange1], [@pone.0048549-Ferrante1].
> *"It was a two-way street, not a one way street. My PCP, I love him, he is still my physician, but I felt that on a professional level, he was so squeezed...So with the naturopath, she always said what did you do? Let\'s look at the results. I always had the results. 'Okay, this is okay, this needs work', and she would always tell me something else. At the end she would always ask 'what would you like to do?'"*
>
> *"\[The MDs\] look at numbers and then prescribe a pill. I thought that was perfectly normal until \[I worked with\] the naturopath who said I think we can fix this with a change of diet. That got my attention. The changes were sort of like why don\'t you try this and so rather than what my pill doctor said, which is you will take this, this, and this. I didn\'t know what the ramifications were of missing it or taking too much or anything like that. It was a much different experience with the naturopath."*
Themes Related to the Content of Naturopathic Care ([Table 4](#pone-0048549-t004){ref-type="table"}) {#s3c}
----------------------------------------------------------------------------------------------------
10.1371/journal.pone.0048549.t004
###### Themes related to the content of naturopathic care (n = 22).
{#pone-0048549-t004-4}
Theme \% [\*](#nt106){ref-type="table-fn"} Example codes and patients' terminology
----------------------------------------------------------------------------- -------------------------------------- -------------------------------------------------------------------------------------------------------------------
**Health promotion counseling: detailed** **& individualized** 100 Exercise prescriptions common and goals established with patient's input
Naturopathic diet recommendations given [@pone.0048549-Oberg1]
Stress management routinely discussed
Other lifestyle change addressed such as promoting positive mental/emotional health
**Counseling promoted patient empowerment** 95 Learning to engage in self-reflection/self-awareness
Increased behavioral capabilities/competence
Strategies for prioritizing self-care
Understanding and executing self-management activities
**Recommendations were pragmatic** 82 "Tips" about diet, exercise and other health behaviors emphasized real-world implementation
Problem-solving (barriers to behavior change, reasons for unexpected blood sugar changes) occurred
**Novel & complementary treatment options** **were offered** 82 Alternatives to pharmaceutical management (i.e. lifestyle change, dietary supplements) created hopefulness
Feeling open to trying new things like mind-body approaches
Trying dietary supplements & natural products for both glycemic control and other health concerns
**Information about diabetes and self-management improved health literacy** 77 Felt better educated about diabetes as a disease and goals for self-management
Felt better educated about the role of psychosocial, behavioral, and emotional factors in relation to blood sugar
Received educational materials from ND
Sums to \>100% because some participants offered multiple responses.
### Theme 4: Health promotion counseling that is detailed and individualized {#s3c1}
When describing the topics discussed with the NDs, all participants noted the emphasis on health promotion. Many participants emphasized that the diet, exercise, and stress management strategies they learned differed in quantity and specificity from those they had previously received in diabetes education classes or from their primary care provider:
> *One of the things that naturopath did was explain things about lifestyle. \[Other providers have\] tried to talk to me about that. I did go to conferences \[at the hospital\] for diabetes and stuff like that. Stress does figure into it. One of the practical impacts of the stress is you don\'t get enough sleep. What I found is it was diet, stress, sleep. It\'s kind of a package; everything went together. The naturopath, even though he didn\'t quite explain it that way, that\'s what he was trying to get at. All these pieces fit together.*
>
> *I thought it was more personal in nature and more personal directions for me to go, and here is a book on nutrition, here\'s a book on diabetes. And so on. It was: here\'s something you can do, what you need to change as far as eating goes, and exercise, and all that. More specific to me.*
Regarding the results of working with an ND on her diabetes, another participant reported:
> *Well, probably some dietary changes, and paying more attention\-- for some reason when you go to the dietitian, things just don\'t seem to come together. So trying to eat every 4 hours, that type of thing. Not that it always works. I probably eat too much. But I\'m paying more attention to the diet. The times \[I eat\]. What\'s in the diet. Like proteins for sure, and trying to eat more vegetables and fruits and stuff. I joined a gym, which I thought I would never do.*
### Theme 5: Counseling promoted patient empowerment {#s3c2}
Participant reports suggested that the naturopathic counseling style helped them increase their locus on control, with numerous patients reporting experiencing new "confidence," feeling "less overwhelmed," and feeling "in control."
> *"I was surprised at how much it depended on me. You know, I guess this process has shown me how much my health is in my hands. That I'm not having to give it over to an MD to watch me deteriorate, and that certain markers mean certain things because this is the way the disease progresses. \[The ND\] was more like, take these steps and these things can change. Keep you as healthy as we can, as long as we can."*
>
> *"My naturopath was very encouraging. She would encourage me to take the power and control she was trying to give me. And I would say that was for the best. I felt very encouraged to take control."*
>
> *"For me it \[diabetes\] is less of an obstacle. It\'s more something I can deal with and work with. Yes, I will fall off the wagon periodically, but it won\'t be the end of the world if I do. He \[the naturopath\] helped me stop making it something that wasn\'t ... a huge obstacle, something to get stressed out about."*
### Theme 6: Recommendations were pragmatic {#s3c3}
The third most common topic was the practical nature of the suggestions about self-management and implementing the health promoting behavioral changes. Participants described the advice they received as "specific tips," "practical for real-life," and focused on "problem-solving" experiences like elevated blood sugar readings and barriers to self-care. Patients felt these "tips" influenced their confidence in self-care by making changes seem less overwhelming and more feasible.
> *"It felt like she had made a point of showing me all the little tricks that you generally overlook, because no one ever taught them to us. Just taking a small piece of protein at bedtime will bring your morning numbers down by 10 or 25 points; I never heard that. Or like a teaspoon of cinnamon will help your numbers. And I tried that and it worked. She had all these things."*
>
> *"I got specifics from the naturopath. You know, it's hard for me to fit exercise into my daily activities. I would fall down on that and beat myself up on it. And then I wouldn't want to tell \[the ND\] exactly what I have done. She helped me decide on walking as a regular program and when I couldn't get there, she did follow up with me. She suggested I do the minimum I can do and that will be fine to start. Don't beat myself up about it. So that was different from that I would have gotten from my GP."*
### Theme 7: Novel and complementary treatment options were offered {#s3c4}
Another topic of the clinical interaction reported by participants was exposure to new therapeutic options. These new options appeared to give patients a new sense of hopefulness or optimism about their diabetes, both in terms of self-management and in an expanded view of their medical team.
> *He prescribed supplements. And I already have a regimen of medication that I take \-- and supplements \-- you know, just the general multivitamin minerals. But they weren\'t specific to diabetes. \[The ND recommended ones that\] were more specific. And they made such a difference that \-- it was more a realization that when I ran out and I had to wait a week for payday and going back to the way it was, knowing that that supplement had created such difference was kind of surprising to me.*
>
> *I was complaining about not having time, and she said but you have a Wii, the game machine. She said play the Wii, and if you are doing the boxing thing or you are doing any of those things, it\'s exercise. She showed me that there are alternatives to getting on a treadmill or going to a gym. It\'s accessible whenever you want it. The weather doesn\'t have to be nice. That for me was really helpful, alternative ways of looking at things and doing things.*
### Theme 8: Information about diabetes and self-management improved health literacy {#s3c5}
Patients reported that they learned useful new information from the NDs. Additional time spent on fundamental health education appeared to help participants develop a better understanding of their chronic disease, evaluate a variety of treatment options, and make informed decisions regarding their self-care and treatment options.
> *I'm not a big supplement guy and so she was talking about fiber. Fiber does this and that -- \[the ND\] had a fairly convincing argument of why that was important and what it could do for me. And so I was willing to try it. The \[medical\] doctor said: yeah, fiber is important but never really got the full explanation. So now I get my 100% daily requirement of fiber everyday and then some. I believe it's had some effect on my health.*
>
> *She gave me 4 different things to try. \[The ND said\] well then, let\'s play with fruit. So we had berries with sugar or coffee sweetener. And the cream. You could take berries, cream with no sugar, and Splenda, that reduces my sugars 20 to 50 points in the morning. And we played with that. And we tried apples. And we had a different experiment going for 3 weeks to find out what works with my body.*
### Theme 9: Contrast between naturopathic care and conventional medicine {#s3c6}
As illustrated by many of the quotes included for the themes described above, nearly all participants contrasted their NM experience with conventional medicine. The attributes that stood out as most different included the focus on psychosocial factors, especially stress, as contributors to glycemic control; the use of dietary supplements and natural products; and the emphasis on education and developing an understanding of self and health. Participants also commented about the greater emphasis by NDs on individualized goals (e.g. starting with a walking program or minimal exercise and building upon it).
Additionally, participants reported the benefits of having both naturopathic and conventional providers on their care team. Many reported both approaches made useful contributions to their overall diabetes management, with some reporting their experience with the ND helped them re-engage with their conventional primary care providers and/or improve their adherence to prescription medication:
> *"You've got to know about yourself and know what your numbers are and ask questions and force them to pay attention...I'm going to go back to my GP and ask him "Are you in the driver's seat? Are you going to help me?"*
>
> *"I was concerned that I could become dependent on insulin. \[The ND\] was encouraging: that in most cases, if you lose weight, then your dependency on insulin also goes down. So not to be afraid of taking insulin. She was actually the one that got me taking the insulin regularly in the first place."*
The only complaint about their naturopathic care experience was the high costs of recommended dietary supplements, which were not covered by insurance.
Discussion {#s4}
==========
This study is unique in that it captures the opinions of naturopathic-naïve patients who were exposed to ND care in a pragmatic trial conducted in a community-based setting. The percentage of patients with diabetes across the U.S. using naturopathic medicine as a specific form of CAM is unknown, but among naturopathic medicine users, 16.8% have diabetes which is almost twice the national prevalence of diabetes [@pone.0048549-Hawk1], [@pone.0048549-Centers1]. The quotations reported here provide insight into what patients' might experience when working with naturopathic physicians to address diabetes and improve glycemic control.
Aspects of both the nature and the content of ND care emerged as important themes in patients' perceptions of their first experiences with naturopathic care. Many of these perceptions contrasted ND care with MD care, highlighting key distinctions that may have important implications for understanding how care for diabetes might be improved.
The characteristics of the nature and content of naturopathic medicine reflect features of care that are well-supported by the literature as necessary for effective behavior change counseling, including being intensive [@pone.0048549-Lindstrom1]--[@pone.0048549-Ornish1], multi-factorial [@pone.0048549-Daubenmier1]--[@pone.0048549-Toobert1], collaborative [@pone.0048549-Anderson1], [@pone.0048549-Berger1], and targeted to the patient and their individual needs [@pone.0048549-Daubenmier1], [@pone.0048549-Jones1]. There were many examples of how patients increased self-efficacy during their experience working with an ND; 95% reported experiencing a sense of empowerment. Examples of self-efficacy changes reflected in the narrative include "it's up to me," "I feel more in control now," "I can succeed in managing it."
The characteristics of care delivered by naturopathic physicians appear congruent with domains of health promotion theory known to be important to effect behavior change. For example, according to Social Cognitive Theory [@pone.0048549-Bandura1], to change behaviors, people must have the confidence, or *self-efficacy*, to persist even when faced with obstacles. If they do not feel that they can exercise control over their health behavior, they will not be motivated to act and behavior does not change.
Other domains of the Social Cognitive Theory (SCT) of health behavior change are also reflected in patients' experiences with NM. For example, the "tips" and strategies patients learned from NDs contributed to their *Behavioral Capability*--their knowledge and skill in mastering healthy behaviors. Patients described how they had been engaged in *Observational Learning* by experimenting with different dietary strategies to see how their blood sugar control would be affected. *Reciprocal Determinism,* which refers to the dynamic interaction between the person, their environment and the behavior, was reflected in participant's comments about attention to psychosocial barriers and the holistic approach that addressed more than blood sugar control. *Outcome Expectations,* the final domain of the SCT, may have been clarified by the ND emphasis on health education and individualized goal-setting. Future research should further examine if the congruence between characteristics of ANC with conceptual models of health behavior change can be quantified. If so, the findings may be useful logic models for designing health promotion interventions to improve diabetes self-care.
Patients reported they had a unique experience working with the naturopathic physicians, even though they had diabetes for an average of seven years and were receiving care in a managed healthcare delivery system known for its high quality care, including access to diabetes education programs and nutritionists. Many reported that they loved their MDs, but may not have realized the limitations of medical care until they experienced something different. Previous reports have emphasized the similarities in ND and MD care delivery, but these studies have been largely quantitative (i.e. types of diagnoses, categorical similarities in visit content such as history taking, lab ordering, prescribing) [@pone.0048549-Cherkin1], [@pone.0048549-Boon1]. An obvious difference between ND and MD care is the average visit duration (about 40 minutes for NDs [@pone.0048549-Cherkin1] and 21 minutes for primary care MDs [@pone.0048549-Chen1], although within the healthcare system studied, primary care visits are allocated 20--30 minutes per patient. While longer visits do appear to reduce laboratory orders and prescribing, studies have not found improved care in diabetes or other chronic conditions simply by allowing for more time [@pone.0048549-Greenfield1], [@pone.0048549-Dugdale1] suggesting the approach and content of the ANC visits set this experience apart, rather than time *per se*.
The findings of this small exploratory study are limited by the small number of NDs (3 female and 1 male) involved and restriction to patients in an integrated health care delivery system in Western Washington. Enrollees in this health system are known to have more years of education than the national average [@pone.0048549-Spangler1]. In addition, although the 22 participants in this qualitative study were not significantly different than the 15 who declined to participate on a variety of sociodemographic and diabetes-related variables, they had been more satisfied with their experience of naturopathic care. As a result, the views of the participants described in this study are almost certainly more positive than would have been the case had we had a higher participation rate. However, given that 48% of patients with sub-optimally controlled diabetes in our target population said they would be very likely to try ND care for their diabetes if covered by their health plan, there appears to be a large pool of individuals interested in and open to complementary approaches to care of their diabetes [@pone.0048549-Bradley3].
As our current healthcare system struggles to meet the needs of patients with chronic diseases such as diabetes, effective strategies are needed to help patients gain self-efficacy to engage in health promoting behaviors. Based on the results reported here, the utilization of naturopathic physicians as an adjunct (in addition) to conventional primary care has benefit beyond primary care alone, however, future research is needed to confirm these benefits and if confirmed, elucidate whether the benefits observed are additive, substitutive, or synergistic to usual care. Care delivered by naturopathic physicians appears to be a useful model that has some preliminary evidence from observational studies of beneficial effects on patient behaviors and clinical outcomes [@pone.0048549-Bradley2]. Additional research using a randomized clinical trial design should test the utility of this unique approach in larger, most diverse populations. If more rigorous studies show that adjunctive naturopathic care provides substantial benefits beyond those of medical care alone for people with diabetes, new models of integrative care will warrant serious exploration.
Supporting Information {#s5}
======================
######
The full text of the focus group interview guide.
(DOC)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: EO RB DC CH KS CC SC. Performed the experiments: EO DC CH. Analyzed the data: EO DC CH KS RB. Wrote the paper: EO RB DC. Reviewed and edited the manuscript: EO RB CH KS SC CC DC.
| {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Indigo is one of the most popular dyestuffs widely used in dyeing by the textile, food and pharmaceutical industries. Although it used to be produced by extraction of plants such as *Indigofera* and *Polygonum tinctorium* \[[@pone.0138455.ref001],[@pone.0138455.ref002]\], indigo is mainly produced by chemical synthesis. Because the toxic wastewater generated by chemical processes contains aniline, cyanide and high levels of chemical and biological oxygen demands (COD and BOD), researchers have been trying to develop greener methods for indigo production \[[@pone.0138455.ref003],[@pone.0138455.ref004]\]. Microbial production of indigo could be a competitive alternative owing to the environmentally-friendly nature \[[@pone.0138455.ref003]\].
Since 1983, a lot of indigo-producing bacterial strains, belonging to the genera *Pseudomonas* \[[@pone.0138455.ref001],[@pone.0138455.ref005]--[@pone.0138455.ref008]\], *Rhodococcus* \[[@pone.0138455.ref009]\], *Sphingomonas* \[[@pone.0138455.ref010]\], *Acinetobacter* \[[@pone.0138455.ref011],[@pone.0138455.ref012]\] and *Comamonas* \[[@pone.0138455.ref013]\], have been isolated from soils, polluted sediments, intertidal sediments and activated sludge. The enzymes responsible for indigo bio-production (mono- or di-oxygenases) have been used for the construction of genetically engineered microorganisms (GEMs) \[[@pone.0138455.ref014]--[@pone.0138455.ref017]\], and the mechanisms of indigo production from indole by both wild strains and GEMs have been studied with the help of advanced chemical analytical methods \[[@pone.0138455.ref005],[@pone.0138455.ref016],[@pone.0138455.ref018]\]. In recent study we found that microbial communities stimulated by different aromatics could also produce indigo from indole, but the indigo production was dissimilar among different groups \[[@pone.0138455.ref019]\]. However, compared with the pure-culture studies, the role of microbial communities in indigo production still remains poorly understood.
The use of microbial communities in an industrial process has many advantages over the use of pure cultures. For example, microbial communities can perform complicated functions that a single population cannot, and they are more stable and resilient to complex environmental conditions \[[@pone.0138455.ref020]--[@pone.0138455.ref022]\]. In addition, bioaugmentation with specific strains may further improve the performance of indigenous microbial communities, which has been successfully applied in a variety of bioremediation processes \[[@pone.0138455.ref023],[@pone.0138455.ref024]\]. However, the role and fate of the introduced inocula are still debated \[[@pone.0138455.ref025]\]. With the rapid development of high-throughput sequencing technologies, it is possible to obtain a detailed picture on the composition, structure and dynamics of microbial communities \[[@pone.0138455.ref026]--[@pone.0138455.ref029]\]. Such information may have important implications on understanding microbial communities for indigo bio-production from indole.
In previous studies we showed that the wild strain *Comamonas* sp. MQ (a genus within *Betaproteobacteria*), the recombinant *Escherichia coli* ~*nagAc*~ carrying the naphthalene dioxygenase gene (*nag*) from strain MQ, and the activated sludge systems were capable of producing indigo from indole \[[@pone.0138455.ref013],[@pone.0138455.ref017],[@pone.0138455.ref019]\]. In this study, we tried to address the following questions: (i) Does inoculation of strain MQ/*E*. *coli* ~*nagAc*~ affect the composition of the indigenous microbial community and alter the efficiency of indigo production? (ii) What microorganisms play key roles in indigo production in these AS systems? To answer these questions, three AS systems, i.e. non-augmented AS (G1), AS plus *Comamonas* sp. MQ (G2) and AS plus *E*. *coli* ~*nagAc*~ (G3), were prepared to examine indigo biosynthesis from indole, and the microbial communities were analyzed using Illumina MiSeq sequencing technology. This study should provide a new insight to understand the microbial production of indigo.
Materials and Methods {#sec002}
=====================
Bacterial strains {#sec003}
-----------------
*Comamonas* sp. MQ (CGMCC No. 6865) was isolated from activated sludge of a local sewage farm (Dalian, China) \[[@pone.0138455.ref013]\]. The activated sludge used in this study was collected from Chunliu River Wastewater Treatment Plant (Dalian, China) under the permission of Dalian Drainage Department, and the field studies did not involve endangered or protected species. The recombinant *E*. *coli* ~*nagAc*~ carrying the naphthalene dioxygenase gene (GeneBank ID JN655512) from strain MQ was constructed previously \[[@pone.0138455.ref017]\]. Strain MQ was cultivated in Luria-Bertani (LB) medium with 300 mg/l naphthalene and incubated at 30°C with continuous shaking until the bacteria reached the late logarithmic phase of growth. The recombinant *E*. *coli* ~*nagAc*~ was cultured according to the methods described in the previous study \[[@pone.0138455.ref017]\]. Both strains were harvested by centrifugation at 8,000 × *g* for 5 min. The cells were washed twice with sterile sodium phosphate buffer (PBS, 0.2 M, pH 7.0), and the cell pellets were used for inoculation.
Experimental design and operation conditions {#sec004}
--------------------------------------------
Three sequencing batch reactors (SBRs) were simulated with 250-ml flasks containing 100 ml synthetic wastewater, which consisted of 6 g/l Na~2~HPO~4~, 3 g/l KH~2~PO~4~, 0.5 g/l NaCl, 1 g/l NH~4~Cl, 0.011 g/l CaCl~2~, 0.24 g/l MgSO~4~ and 0.2 g/l naphthalene. The SBRs were seeded with the activated sludge (0.54 g, dry weight at 105°C), and domesticated with the wastewater for 15 days. Then, the SBRs were inoculated with different bacteria: (1) group 1 (G1), non-augmented SBR; (2) group 2 (G2), augmented SBR with *Comamonas* sp. MQ (0.22 g, dry weight); (3) group 3 (G3), augmented SBR with recombinant *E*. *coli* ~*nagAc*~ (0.24 g, dry weight). The whole operation process was divided into three stages: T1 stage (0--30 d), indole was 73--85 mg/l; T2 stage (30--81 d), indole was 168--185 mg/l; and T3 stage (81--132 d), indole was 277--290 mg/l. Each operation cycle of SBR was performed for 72 h, including 2 h filling, 66 h reacting, 2 h settling and 2 h decanting. At the end of each SBR operation cycle, samples were taken to monitor the yields of indigo and the residual concentrations of indole. The pure culture controls were performed by inoculating the same amount of strain MQ and recombinant *E*. *coli* ~*nagAc*~ into the synthetic wastewater, respectively, and the operation processes were carried out under the identical conditions as the AS systems.
Chemical analysis {#sec005}
-----------------
The concentrations of indole and indigo were measured using high performance liquid chromatography (HPLC) (Shimadzu LC20A; Thermo Hypersil ODS-2 column, 5 μm, 250×4.6 mm). The pigments were also analyzed by HPLC-mass spectroscopy (MS) to identify the products. HPLC and MS were conducted as described previously \[[@pone.0138455.ref013],[@pone.0138455.ref017]\].
DNA extraction, PCR amplification, and sequencing {#sec006}
-------------------------------------------------
During the operation, activated sludge samples were taken concurrently from the three reactors for high-throughput sequencing, and 11 samples were collected from each group (Table A in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). The genomic DNA was extracted using the protocol previously described \[[@pone.0138455.ref030]\]. DNA concentration was determined by Pico Green assay using a FLUOstar OPTIMA fluorescence plate reader (BMG Labtech, Germany), and each DNA sample was diluted to 10 ng/μl for PCR amplification. The V4 region of the 16S rRNA gene was amplified using the methods previously described \[[@pone.0138455.ref019]\]. High-throughput sequencing of the 16S rRNA gene was conducted on Illumina MiSeq platform for 300 cycles at the Institute for Environmental Genomics, University of Oklahoma.
Data analysis {#sec007}
-------------
After sequencing, the bar-codes and primers were removed, and the paired-end (PE) reads were overlapped using the Flash program to assemble the final V4 tag sequences \[[@pone.0138455.ref031]\]. The low-quality fragments were all eliminated, including the sequences without exactly matching the forward primer, the sequences containing ambiguous reads (N), and the variable tags shorter than 240 bp. The clean sequences were then subjected to Chimera detection by UCHIME \[[@pone.0138455.ref032]\]. Each sample was randomly re-sampled and normalized at 27,530 sequences. Operational taxonomic units (OTU) were classified at 97% similarity level using CD-HIT \[[@pone.0138455.ref033]\], and the reads from singleton OTUs were removed. In addition, the taxonomic assignment of OTUs was performed by RDP classifier with a confidence threshold of 50% \[[@pone.0138455.ref034]\]. The Shannon index (H), Pielou's evenness index (J), species richness estimator of Chao1 and rarefaction curves were analyzed by Mothur for each sample \[[@pone.0138455.ref035]\]. The detrended correspondence analysis (DCA) was performed by Canoco 4.5 \[[@pone.0138455.ref036]\]. The dissimilarity tests based on Bray-Curtis similarity distance matrices were performed by the Vegan package in R 3.1.2 (<http://www.r-project.org/>), including permutational multivariate analysis of variance (Adonis), analysis of similarity (ANOSIM) and multiresponse permutation procedure (MRPP) \[[@pone.0138455.ref037]\]. A phylogenetic tree was constructed by MEGA 5.1 using neighbor-joining algorithm with 1,000 bootstrap replicates. The 10 most abundant genera in each group were depicted in a heat map conducted by R 3.1.2. Pearson correlation was calculated to determine the relationship between the relative abundances of microbial taxa and indigo yields. The sequencing data have been deposited into the NCBI Sequence Read Archive database under the accession number SRX897059.
Quantitative real-Time PCR assays {#sec008}
---------------------------------
The naphthalene dioxygenase gene (*nagAc*) of *Comamonas* sp. MQ was selected for qPCR assays, which were conducted in triplicate using TaKaRa PCR Thermal Cycler Dice Real Time System (TaKaRa, China) with the primer set nagF (5'-CAG CGC ACT TTC GGA ACC-3') and nagR (5'-CTG GTA GGC GCG GTA AAA G-3'). The qPCR mixture (25 μl) contained 12.5 μl of SYBR Premix Ex *Taq* (TaKaRa, China), 1 μl of each primer (10 μM), and 2 μl of template DNA. The thermal profile included 30 s of initial denaturation at 95°C, and 40 cycles of 5 s at 95°C and 30 s at 60°C. Amplicons were visualized and checked by electrophoresis on agarose gel (1.5%, w/v).
Results {#sec009}
=======
Indigo production from indole in the AS systems {#sec010}
-----------------------------------------------
Three AS systems for indigo production were constructed using naphthalene and indole as the inducible substrates (Table A in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). A blue product was produced by three reactors in the presence of indole. Metabolite analysis by HPLC-MS showed that the blue product had a prominent molecular ion (MH^+^) peak at m/z 263, which was confirmed to be indigo (Fig A in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). The results demonstrated that all three AS systems were able to produce indigo from indole. [Fig 1](#pone.0138455.g001){ref-type="fig"} depicts the performances of indigo production and indole consumption over the 132-day operation, which have been divided into three stages based on the changes in indole concentration, i.e. T1 (0--30 d), T2 (30--81 d) and T3 (81--132 d). In T1 stage, G2 produced the highest yields of indigo (nearly 15.7 ± 0.8 mg/l) compared to negligible production in G1 and G3. Subsequently, the indigo yields in G1 (17.3 ± 0.9 mg/l) and G3 (45.9 ± 2.3 mg/l) displayed modest increases in the T2 stage. All three treatments exhibited better capabilities for indigo production in T3 stage when the influent indole concentration was raised to 280 mg/l. After Day 90, indigo yields dramatically increased to approximately 99.5 ± 3.0 mg/l in G3, whereas the yields of G1 and G2 were only 19.2 ± 1.2 and 27.3 ± 1.3 mg/l, respectively. Indole was completely transformed at the end of each SBR cycle over the entire period of operation ([Fig 1B](#pone.0138455.g001){ref-type="fig"}). In the pure culture controls, strain MQ could produce 33.2 ± 2.5 mg/l indigo from indole at the first operation cycle due to the good activities of naphthalene dioxygenase induced in the cells (Fig B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). Similarly, the recombinant *E*. *coli* ~*nagAc*~ could produce 68.4 ± 3.1 mg/l indigo from indole, while the non-recombinant *E*. *coli* without *nag* gene was unable to produce indigo (Fig B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). Then, strain MQ was able to grow in the synthetic wastewater medium when indole concentration was below 100 mg/l, but low yields of indigo (\<10 mg/l) were produced due to the toxicity of indole to bacterial strains (Fig B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). When the concentrations of indole increased to above 100 mg/l, strain MQ could hardly grow. Whereas, the recombinant *E*. *coli* ~*nagAc*~ was unable to grow due to the lack of nutrients and almost no indigo was produced after the first operation cycle. These results indicated that both inoculated strains might contribute to indigo production in the AS systems at early days of operation process. The differences in indigo production among the three treatments suggested the harbored microbial communities were the driving force in indigo production.
{#pone.0138455.g001}
Overview of sequencing analysis of 16S rRNA gene amplicons {#sec011}
----------------------------------------------------------
A total of 1,721,342 effective reads with an average length of 253 bp were obtained from 36 samples, resulting in 1,422 OTUs at 97% sequence identity cutoff. Even at high depth (\~27,500 sequences per sample) of sequencing, the rarefaction curves did not approach saturation, indicating that some microbial taxa remained undetected (Fig C in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). Microbial richness (the observed OTU number and Chao1 estimator) was similar among the samples from G1, G2 and G3 (ANOVA, *P* \> 0.05) (Table B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). However, both Shannon (H) and evenness (J) indices were significantly different among the three treatments in T1 stage (*P* \< 0.05), probably owing to the inoculation of strain MQ and *E*. *coli* ~*nagAc*~ at the beginning of operation. The H and J indices showed little difference in T2 stage (*P* \> 0.05), but then became distinctly different in T3 stage (*P* \< 0.05) (Table B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). Compared to the original sludges, the microbial richness and evenness were significantly lower in all three treatments (*P* \< 0.001), leading to lower Shannon indices (Table B in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}), which indicated that the addition of indole reduced microbial diversity in the AS systems.
Survival of the inoculated strains in the AS systems {#sec012}
----------------------------------------------------
The relative abundances of *Comamonas* sp. and *E*. *coli* were investigated to determine the survival rates of the inoculated strains. In G1 and G3, *Comamonas* sp. maintained about 40% of the population throughout operation process (Fig D in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). However, in G2, before Day 10, 87% of the sequences belonged to *Comamonas* sp. (Fig D in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}), most likely due to the inoculation of *Comamonas* sp. MQ. From Day 10 to 30, the proportion of *Comamonas* sp. declined to around 30% and remained at that level (Fig D in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). In contrast, very few *E*. *coli* sequences were detected in any of the groups based on taxonomic classification by the RDP classifier.
The *nagAc* gene from both inoculated strains was detected by qPCR at each sampling time point. In the non-augmented G1, there were less than 10 copies/ng *nagAc* at all the time points (Fig E in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). In G2, the gene abundance peaked at Day 10, reaching 4.5×10^4^ copies/ng, which corresponded well with the high proportion of *Comamonas* sp. as shown above, yet dropped to 45 copies/ng at Day 30, and below 1 copy/ng at Day 130 (Fig E in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). This gene was also detected in G3 with relatively high abundance within 10 days (200--500 copies/ng) (Fig E in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). As indicated above, the relative abundances of *Comamonas* sp. in G1 and G3 were almost the same (Fig E in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}), but the *nagAc* abundances in G2 and G3 was much higher than those in G1. Thus, the *nagAc* gene in G3 should be from *E*. *coli* ~*nagAc*~ added at the beginning, while the gene in G2 was probably from *Comamonas* sp. MQ. The indigo yields significantly correlated with the abundance of the *nagAc* gene (in log10 unit) in the T1 stage (R^2^ = 0.62, *P* \< 0.001), but showing no significant relationship at the T2 and T3 stages (*P* \> 0.10) ([Fig 2](#pone.0138455.g002){ref-type="fig"}). Based on the combination analyses of high-throughput sequencing and qPCR, it suggested that the inoculated strain MQ could remain in the AS systems for at least 30 days.
{#pone.0138455.g002}
Community composition and dynamics of three AS systems {#sec013}
------------------------------------------------------
DCA was performed to visualize the succession of microbial communities over time. The microbial communities from three treatments diverged from the original AS after receiving indole ([Fig 3](#pone.0138455.g003){ref-type="fig"}). The three treatments clustered separately in early days of T1 stage (Day 6 to 12), but converged by late days of T1 stage (Day 24 to 30) ([Fig 3](#pone.0138455.g003){ref-type="fig"}), during which the inoculated strains had almost disappeared. Then in T2 stage, the groups separated, forming three clusters ([Fig 3](#pone.0138455.g003){ref-type="fig"}). In T3 stage, G2 and G3 were clustered, but distinctly separated from G1 ([Fig 3](#pone.0138455.g003){ref-type="fig"}). Results of three dissimilarity tests, Adonis, ANOSIM and MRPP, suggested that the community structures of the original AS and the three treatments were significantly different (*P* \< 0.05) (Table C in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). Furthermore, the communities of the three treatments were also distinctly different between T1 and T2 stages (*P* \< 0.05), but not between T2 and T3 stages (*P* \> 0.05) (Table C in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}).
{ref-type="supplementary-material"}.](pone.0138455.g003){#pone.0138455.g003}
The microbial community composition of the three treatments are illustrated in [Fig 4](#pone.0138455.g004){ref-type="fig"}. Among the total 1,422 OTUs detected, 351, 197 and 143 OTUs were shared by the three treatments in T1, T2 and T3 stages, respectively (Fig F in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). These shared OTUs accounted for 99% of the classified sequences in each stage. As a result, the downstream analysis focused primarily on the shared OTUs. Although 16 phyla were detected in T1 stage, *Proteobacteria* and *Bacteroidetes* covered over 97% of the shared sequences ([Fig 4A](#pone.0138455.g004){ref-type="fig"}). In T2 and T3 stages, more than 96% of the shared sequences belonged to *Proteobacteria* ([Fig 4A](#pone.0138455.g004){ref-type="fig"}), among which *Betaproteobacteria* was the most dominant class (43.8--77.5% in T2 stage, 49.7--84.0% in T3 stage). Compared with the three treatments, the original sludge communities had higher percentages of *Gammaproteobacteria* (54.8%, compared to 22.4% on average) and *Bacteroidetes* (14.0%, compared to 1.6% on average).
{#pone.0138455.g004}
A total of 144 families were classified based on the RDP database and 87 families were shared by three treatments. Ten of these families represented over 90% of sequences. In the original AS, *Pseudomondaceae* was the most abundant and accounted for 48.3% of the sequences, followed by *Comamonadaceae* (11.9%), *Xanthomonadaceae* (6.0%), *Chitinophagaceae* (4.5%), *Flavobacteriaceae* (3.5%), *Phyllobacteriaceae* (2.9%), *Alcaligenaceae* (1.5%) and *Rhodobacteraceae* (1.4%) ([Fig 4B](#pone.0138455.g004){ref-type="fig"}). The community compositions of G1, G2 and G3 were distinct from the original AS ([Fig 4B](#pone.0138455.g004){ref-type="fig"}). For instance, *Comamonadaceae* was detected in all three treatments with considerably higher relative abundance (more than 40% on average). In G2, *Comamonadaceae* was extremely enriched in early days of T1 stage (83--90%) due to the addition of *Comamonas* sp. MQ, yet the abundance decreased in T2 and T3 stages and was even lower than those in G1 and G3 ([Fig 4B](#pone.0138455.g004){ref-type="fig"}). The abundance of *Pseudomonadaceae* decreased dramatically in T1 stage (3.7--6.5%) and further decreased in T2 (below 0.6%) and T3 (below 0.2%) stages. In contrast, the abundance of *Alcaligenaceae* was below 5% in T1 stage of each treatment, while increased to above 30% in T2 and T3 stages of G2 and G3, becoming the predominant population ([Fig 4B](#pone.0138455.g004){ref-type="fig"}). The relative abundance of *Phyllobacteriaceae* also increased in all three treatments (4.3--18.0%), especially G1 (10.5--31.0%). The most abundant genera from the original AS and three treatments are shown in [Fig 4C](#pone.0138455.g004){ref-type="fig"}. Four genera were abundant (\> 1% on average) in all three treatments, i.e. *Comamonas* (family *Comamonadaceae*), *Pseudomonas* (family *Pseudomonadaceae*), *Alcaligenes* (family *Alcaligenaceae*) and *Aquamicrobium* (family *Phyllobacteriaceae*), which represented major fractions of their corresponding families (Table D in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}) and therefore shared similar dynamic patterns.
Linking community composition to indigo production {#sec014}
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To discern the relationship between community composition and indigo production, Pearson correlation test was conducted at both genus and family levels ([Fig 5](#pone.0138455.g005){ref-type="fig"}). Among the four major genera (*Comamonas*, *Pseudomonas*, *Alcaligenes and Aquamicrobium*), the most abundant genus *Comamonas* had no significant correlation with indigo yields in all groups (*P* \> 0.10) ([Fig 5](#pone.0138455.g005){ref-type="fig"}). The abundance of *Pseudomonas* did not have correlations with indigo yields in G1 or G2 (*P* \> 0.05) either, but was negatively correlated with indigo yields in G3 (r = -0.66, *P* \< 0.05) ([Fig 5](#pone.0138455.g005){ref-type="fig"}). In contrast, the relative abundance of *Alcaligenes* exhibited a strong positive relationship with indigo yields in G3 (r = 0.97, *P* \< 0.001), and the relative abundance of *Aquamicrobium* was also positively correlated with indigo yields in G1 (r = 0.92, *P* = 0.001) ([Fig 5](#pone.0138455.g005){ref-type="fig"}). The relative abundances of family *Comamonadaceae*, *Pseudomonadaceae*, *Alcaligenaceae* and *Phyllobacteriaceae* showed similar correlations with indigo yields as the corresponding genus in general (*P* \< 0.05) (Fig G in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}).
{#pone.0138455.g005}
The correlation analysis was also performed with other dominant families with more diverse genera. The relative abundances of *Rhodobacteraceae* and *Xanthobacteraceae* were positively correlated with indigo yields in G1 (*P* \< 0.05), whereas the abundances of *Beijerinckiaceae* showed positive effects on indigo production in G2 and G3 (*P* \< 0.05) (Fig G in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}). In contrast, the abundances of *Xanthomonadaceae* decreased with indigo yields in G1 and G3 (*P* \< 0.05) (Fig G in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}).
Discussion {#sec015}
==========
While the microbial production of indigo has been comprehensively studied using wild strains or GEMs, less has been done to characterize the indigo-producing capacity of microbial communities. In this study, we successfully achieved indigo production from indole by AS and bioaugmented AS, and the associated microbial communities were revealed by Illumina MiSeq sequencing technology. The two bioaugmented groups produced higher yields of indigo (27.3 ± 1.3 and 99.5 ± 3.0 mg/l) compared with the non-augmented AS (19.2 ± 1.2 mg/l). Sequencing analysis revealed that the inoculated *Comamonas* sp. MQ was dominant early in the time course, while the recombinant *E*. *coli* ~*nagAc*~ was not detected in the bioaugmented system. Similar results were observed in bioaugmentation studies on 3-chloroaniline and polyurethane biodegradation, in which the inoculated strains accelerated the removal of undesired compounds, but did not remain in activated sludge or soil communities \[[@pone.0138455.ref023],[@pone.0138455.ref038],[@pone.0138455.ref039]\]. qPCR analysis showed that the indigo yields were closely associated with *nagAc* gene abundance in T1 stage ([Fig 2](#pone.0138455.g002){ref-type="fig"}), and thus naphthalene dioxygenase might be responsible for the production of indigo. However, the abundance of *nagAc* gene was greatly reduced by Day 80 (Fig E in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}), and it had no significant relationship with indigo yields in T2 and T3 stages ([Fig 2](#pone.0138455.g002){ref-type="fig"}). The indigo yields, by contrast, increased significantly after Day 70, especially in G3 ([Fig 1](#pone.0138455.g001){ref-type="fig"}). Therefore, we would expect other oxygenases in the AS systems that might catalyze the biotransformation of indole to indigo, and the indigenous bacteria were primarily responsible for the high yields of indigo in the late period of operation. These results were similar to those reported by Bai et al. \[[@pone.0138455.ref025]\], who found that indigenous bacteria played the most significant roles in degrading pyridine and quinoline in their bioaugmented systems.
Based on DCA ordination and dissimilarity tests, the communities from the three treatment groups changed when indole was added. Both deterministic and stochastic processes could be involved in shaping the assembly and succession of the communities \[[@pone.0138455.ref040]--[@pone.0138455.ref043]\]. The separation of the three treatments in early days of T1 stage could be due to the addition of exogenous strains *Comamonas* sp. MQ and *E*. *coli* ~*nagAc*~. But in late days of T1 stage, both inoculated strains were almost gone (Fig D in [S1 File](#pone.0138455.s001){ref-type="supplementary-material"}), and each treatment started to recover their own communities, during which the stochastic processes of colonization and extinction might be involved \[[@pone.0138455.ref041],[@pone.0138455.ref043]\]. Thereafter, distinct communities had been established in T2 and T3 stages, and each community was assembled under the high indole pressure. A recent study by Zhou et al. \[[@pone.0138455.ref042]\] found that, even under identical conditions, the same source community could evolve into different communities with different structures and distinct functions due to stochastic processes. Chase \[[@pone.0138455.ref040]\] also found that stochastic processes were likely to play a strong role in the assembly of high-productivity communities. Therefore, we speculated that the differences in indigo production among the three treatments in T2 and T3 stages might be a consequence of stochastic processes in community assembly.
Previous studies have shown that *Comamonadaceae* (genus *Comamonas*) \[[@pone.0138455.ref013]\], *Moraxellaceae* (genus *Acinetobacter*) \[[@pone.0138455.ref011],[@pone.0138455.ref012]\], *Nocardiaceae* (genus *Rhodococcus*) \[[@pone.0138455.ref009]\], *Pseudomonadaceae* (genus *Pseudomonas*) \[[@pone.0138455.ref001],[@pone.0138455.ref005]--[@pone.0138455.ref008]\] and *Sphingomonadaceae* (genus *Sphingomonas*) \[[@pone.0138455.ref009]\] were able to produce indigo from indole. However, only a few sequences of *Moraxellaceae*, *Nocardiaceae* and *Sphingomonadaceae* were detected in all three treatments. The dominant families *Alcaligenaceae*, *Phyllobacteriaceae*, *Beijerinckiaceae*, *Rhodobacteraceae* and *Xanthobacteraceae* have not been reported to participate in indigo bio-production, and owing to the fact that the abundances of those families were positively correlated with indigo yields, there may be some new indigo-producing strains in these treatments.
Among the bacterial strains reported to produce indigo, *Pseudomonas* sp. is most commonly studied \[[@pone.0138455.ref001],[@pone.0138455.ref005]--[@pone.0138455.ref008]\]. Although *Pseudomonas* has been widely used as a model microorganism in the study of naphthalene degradation, a previous study showed that *Pseudomonas* was not the dominant degrader in naphthalene-amended soil microcosms \[[@pone.0138455.ref044]\]. Likewise, in this study, *Pseudomonas* abundance decreased during the operation in all three treatments, thus displaying no positive relationship with indigo production. In our previous study, *Comamonas* sp. MQ exhibited the ability to produce indigo from indole \[[@pone.0138455.ref013]\]. As indigo yields were significantly positively correlated with the abundance of *nag* gene from *Comamonas* sp. MQ in T1 stage, *Comamonas* could play an important role in indigo production in the early stage. However, *Comamonas* displayed no significant correlations with indigo yields over the whole period of operation (*P* \> 0.10). Therefore, there were likely other populations that could also produce indigo from indole. *Alcaligenes* and *Aquamicrobium*, with the ability to degrade aromatic compounds \[[@pone.0138455.ref045],[@pone.0138455.ref046]\], might serve as good candidates for indigo production in this study. During the past several decades, only one study found trace amounts of indigo in the culture broth of *Alcaligenes* sp. In3 when growing with indole \[[@pone.0138455.ref047]\]. While *Aquamicrobium* sp. was reported to be capable of degrading thiophene-2-carboxylate and biphenyls \[[@pone.0138455.ref046],[@pone.0138455.ref048]\], indigo production has been rarely described. In the present study, the relative abundance of *Alcaligenes* increased dramatically in T2 and T3 stages of G3, during which the system reached the maximal indigo production. A significantly positive correlation was obtained between the abundance of *Alcaligenes* and indigo yields in G3 ([Fig 5](#pone.0138455.g005){ref-type="fig"}). Similarly, a strong positive correlation of relative abundance with indigo yields was also found for *Aquamicrobium* in G1 ([Fig 5](#pone.0138455.g005){ref-type="fig"}). In previous report \[[@pone.0138455.ref019]\], we have found that *Aquamicrobium* could serve as a new biocatalyst for indigo production in AS systems. Therefore, it was presumed that *Alcaligenes* and *Aquamicrobium* were likely to be the functional bacteria in the AS communities responsible for indigo production. The present study also suggests that there are more bacteria capable of producing indigo from indole as revealed by high-throughput sequencing technologies. Nevertheless, the duplicate measurements in a single reactor run does not make up for the lack of a second or third replicate reactor with the exact same conditions, and further investigation needs to be carried out in triplicate in order to provide a more effective analysis of microbial communities.
In summary, the possibility of bioaugmented microbial communities producing indigo was demonstrated, and our studies also supplied the Illumina sequencing data for the microbial communities associated with indigo production during 132-day operation. The structure and diversity of the communities changed greatly over time. Our results suggested several populations might participate in indigo production, such as *Alcaligenes* and *Aquamicrobium*, which had not been widely observed previously. This study should provide important information on microbial communities for the production of indigo from indole.
Supporting Information {#sec016}
======================
###### This includes Tables A-D and Figures A-G.
Table A. Experimental setup and sampling in this study. Table B. Diversity indices of the original AS and three treatments. Table C. Dissimilarity tests of microbial communities from three treatments at different stages. Table D. Relative abundances of the major genera (\>1% on average) in the original AS and three treatments. Fig A. Mass spectra of the products produced by activated sludge systems. Fig B. Indigo production performance of the pure culture controls. Fig C. Rarefaction curves based on 16S rRNA gene amplicon sequencing of microbial communities. Fig D. Relative abundance of *Comamonas* sp. in the original AS and three treatments. Fig E. Relative abundance of *nagAc* gene in the original AS and three treatments. Fig F. Venn diagrams based on all detected OTUs at a cutoff of 3% sequence similarity. Fig G. Correlations between the relative abundances of major phylotypes and indigo yields in each treatment.
(PDF)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: YYQ XZ QM Jizhong Zhou. Performed the experiments: XZ QM. Analyzed the data: XZ QM JD YD LW YJQ. Contributed reagents/materials/analysis tools: YYQ Jiti Zhou Jizhong Zhou. Wrote the paper: YYQ XZ JD ZH JVN.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Cisplatin is a widely used anti-cancer agent that has been used in non-small cell lung, esophagus, bladder, endometrium, ovary and testicular cancers, as well as many other types of cancers^[@CR1],[@CR2]^. However, its side effects, such as ototoxicity, renal toxicity and neurotoxicity, limit its clinical use^[@CR3]--[@CR5]^; among these effects, nephrotoxicity is the most concerning. Nephrotoxicity induced by cisplatin mainly occurs in renal tubular proximal tubular epithelial cells and is characterized by tubular cell necrosis, oxidative damage, inflammatory injury and acute renal failure^[@CR6]--[@CR8]^, which is related to high morbidity and mortality^[@CR9]^. As the mechanisms implicated in cisplatin-induced AKI are still unclear, an effective clinical agent for preventing cisplatin-induced AKI is needed. Recently, dexamethasone (Dex) has been used as a potent therapeutic agent for acute kidney injury induced by cardiac surgery^[@CR10],[@CR11]^, multiple myeloma^[@CR12],[@CR13]^ and drug-induced nephrotoxicity^[@CR14],[@CR15]^. However, the shortened survival of solid tumor patients due to corticosteroids restricts its application^[@CR16]^.
Huaiqihuang (HQH) extractum is a Chinese herbal complex that consists of *Trametes robiniophila* Murr., *Lycium barbarum* and *Polygonatum sibiricum*, all of which have been used intensively in China for more than 1, 000 years. Among them, *Trametes robiniophila* Murr. is one of the important anti-tumor herbs and is widely used in the adjuvant therapy of primary liver cancer, breast cancer, stomach cancer, colon cancer and other cancers^[@CR17]--[@CR20]^. HQH extractum has been used in the treatment of nephrotic syndrome, bronchial asthma and recurrent upper respiratory tract infection in children. An *in vitro* study showed that HQH extractum protects against podocyte injuries by regulating the p-ERK/CHOP signaling pathway to promote podocyte proliferation and suppress podocyte apoptosis^[@CR21]^. Liu H *et al*. reported that HQH extractum could increase podocyte nephrin expression, inhibit the NF-κB signaling pathway and suppress glomerular and tubular apoptosis in rats with adriamycin-induced nephropathy^[@CR22]^. All these studies suggest that HQH extractum is a potent nephroprotective agent; however, little is known about the protective effect of HQH in AKI.
In this study, the renoprotective effects of HQH against cisplatin-induced AKI was surveyed in mice and we found that pretreatment with HQH demonstrated renoprotective effects similar to those of Dex via similar mechanisms to relieve cisplatin-induced renal injury. Furthermore, it was shown that HQH did not impair cisplatin-mediated killing of HeLa tumor cells *in vitro*. Taken together, these results suggest that HQH is a potent renoprotective agent for preventing the renal toxicity caused by cisplatin without affecting the anti-tumor effects of cisplatin.
Results {#Sec2}
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Effects of Huaiqihuang (HQH) Extractum on Renal Damage in Cisplatin-Treated Mice {#Sec3}
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The experimental design of the renoprotective effect of HQH on cisplatin-induced renal injury in C57BL 6 male mice is summarized in Fig. [1A](#Fig1){ref-type="fig"}. Urea levels were significantly increased in the cisplatin-treated group, but this effect was significantly reversed by HQH and Dex pretreatment (Fig. [1B](#Fig1){ref-type="fig"}). An obvious medullary congestion zone was seen in cisplatin-treated kidneys when the mice were sacrificed on day 3, which was significantly attenuated after HQH pretreatment (Fig. [1C](#Fig1){ref-type="fig"}).Figure 1HuaiQihuang extractum (HQH) or Dexamethasone (Dex) pretreatment attenuated acute renal injury induced by cisplatin (Cis). (**A**) Scheme: The group of HQH + Cis received 6 g/kg HQH for two consecutive days before intraperitoneal injection with 20 mg/kg cisplatin and the group of Dex + Cis intraperitoneally injected with 4 mg/kg Dex 30 minutes before intraperitoneal injection with cisplatin. (**B**) Serum urea was measured at day 3 after cisplatin injection (\*P \< 0.05, \*\*P \< 0.01, n = 5/group). (**C**) Representative medullary congestion after Cisplatin injury with or without HQH and Dex pretreatment. Graphs indicate congestion area. (**D**) Representative photomicrographs and graphs of Cisplatin induced kidney injury, with or without HQH and Dex pretreatment. Injured tubules was showed by PAS staining, Bar = 50 μm. LTL (lotus tetragonolobus lectin) identifying proximal tubules (green), Bar = 100 μm. Kim-1 expression (red) identifying tubular injury, Bar = 50 μm. Statistics analyzed using the Mann-Whitney U test. \*\*P \< 0.01, \*\*\*\*P \< 0.0001, n = 5/group. Values are means ± SEM.
The morphological changes in the kidneys are shown in Fig. [1D](#Fig1){ref-type="fig"}. Cisplatin-induced cast formation, tubular dilation and brush border loss were significantly alleviated by HQH pretreatment (Fig. [1D](#Fig1){ref-type="fig"}). The HQH group showed less tubular cast than the Dex group.
To assess the injury of proximal renal tubule cells (TECs), we detected the brush border with immunofluorescence staining of lotus tetragonolobus lectin (LTL) and kidney injury molecule 1 (Kim-1). As shown in Fig. [1D](#Fig1){ref-type="fig"}, the loss of LTL-positive tubules in the cisplatin-treated kidneys was restored by HQH pretreatment. Compared to the Dex group, the HQH group had more LTL-positive tubules. Compared to its expression in cisplatin-treated control mice, Kim-1 was less expressed in the HQH group, similar to the Dex group, (Fig. [1D](#Fig1){ref-type="fig"}). These data suggested that HQH pretreatment alleviated cisplatin-induced AKI better than the dexamethasone.
Effects of Huaiqihuang (HQH) Extractum on Proliferation, Apoptosis and Necroptosis of TECs in Cisplatin-Treated Mice {#Sec4}
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Previous studies have shown that cisplatin-induced AKI is accompanied by proliferation of renal TECs^[@CR23]--[@CR25]^. We found that cisplatin induced a significant increase in Ki67-positive nuclei in TECs, which was not further increased in the HQH- and Dex-pretreated group (Fig. [2A](#Fig2){ref-type="fig"}), suggesting that HQH pretreatment-induced renoprotection does not occur through the increased renal regeneration capacity related to TEC proliferation. Apoptosis in TECs was assessed using the terminal deoxynucleotidyl transferase--mediated dUTP nick-end labeling (TUNEL) assay (Fig. [2B](#Fig2){ref-type="fig"}). TUNEL-positive cells were almost undetectable in the control kidneys, while the number of the TUNEL-positive cells was significantly increased after cisplatin administration. Pretreatment of both HQH and Dex reduced the cisplatin-induced apoptosis; however, there was no difference between the HQH-pretreated group and the Dex-pretreated group. We further examined the expression of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, which showed that HQH pretreatment attenuated cisplatin-induced Bax expression (Fig. [2C](#Fig2){ref-type="fig"}) and enhanced the expression of anti-apoptotic Bcl-2. This result suggests that HQH pretreatment protected the kidneys against cisplatin-induced apoptosis. Tubular necroptosis is another characteristic of kidney tissues in cisplatin-treated mice and is characterized by high levels of receptor interacting-protein 3(RIP3) and mixed-lineage kinase domain-like protein (MLKL) in TECs^[@CR26]^. To assess this necroptosis, we observed the expression of RIP3 and MLKL by western blotting (Fig. [2C](#Fig2){ref-type="fig"}). We found that both HQH and Dex pretreatment attenuated kidney necroptosis induced by cisplatin, suggesting that HQH treatment protected the kidneys against cisplatin-induced necroptosis.Figure 2HQH pretreatment protected mice from cisplatin induced tubular apoptosis and necroptosis, but not improved renal tubular epithelial cell proliferation. (**A**) Representative photomicrographs and graphs of immunofluorescence staining for Ki-67. Bar = 50 μm. (\*\*P \< 0.01 versus control, n = 8/group). Ki-67 was stained in red and nucleus was counterstained in blue by DAPI. (**B**) Representative photomicrographs and graphs of TUNEL (green) assay for analysis of tubular apoptosis (\*\*P \< 0.01, n = 5/group). Bar = 50 μm. (**C**) Representative Western blot and graphs for pro-apoptotic proteins Bax (**C** and **D**, \*P \< 0.05, n = 3/group), anti-apoptotic protein Bcl-2 (**E** and **F**, \*P \< 0.05, n = 3/group) and the key element of necroptosis RIP3 (\*P \< 0.05, n = 3/group) and MLKL (\*P \< 0.05, n = 3/group). Each group of blots is the same exposure of a gel, each group of results repeated more than three times.
Effects of Huaiqihuang (HQH) Extractum on inflammation of kidneys in Cisplatin-Treated Mice {#Sec5}
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Previous study confirmed that cisplatin-induced acute renal injury is related to the release of pro-inflammatory cytokines, including high mobility group box-1 protein (HMGB1) and tumor necrosis factor-α (TNF-α)^[@CR27]^. As shown in Fig. [3A](#Fig3){ref-type="fig"}, cisplatin injection resulted in obvious release of HMGB1 into the cytoplasm in TECs, which was restored by HQH and Dex pretreatment (Fig. [3A](#Fig3){ref-type="fig"}). We further used RT-PCR to detect the expression of TNF-h (Fig. [3B](#Fig3){ref-type="fig"}). We found that both HQH and Dex pretreatment reduced the expression of TNF-N induced by cisplatin. These results were further confirmed by Western blotting, as shown in Fig. [3C](#Fig3){ref-type="fig"}. NFκB and TNF-N are the downstream signaling pathways of HMGB1 and TLR4^[@CR28]--[@CR30]^. We found that HMGB1 in the cytoplasm, NF-κB P56 in the nucleus and TLR4 in total protein, were highly induced after cisplatin administration and these effects were reversed by HQH and Dex pretreatment (Fig. [3C](#Fig3){ref-type="fig"}). We also assessed the MAPK signaling pathway; however, the expression of p-Erk/Erk was increased in all three groups and there was no significant difference between them (Fig. [3C](#Fig3){ref-type="fig"}). This result suggests that HQH pretreatment protected the kidneys against cisplatin-induced inflammation through the HMGB1/TLR4/NFκB pathway.Figure 3HQH protected the kidney through inhibition of HMGB1/TLR4/NF-κB/TNF-α pathway. (**A**) Representative photomicrographs and graphs for the expression of HMGB1 by immunohistochemical staining (\*\*P \< 0.01, n = 5/group). Bar = 50 μm. (**B**) Real-time PCR analyses of TNF-α (\*\*P \< 0.01, n = 3 \~ 7/group). (**C**) Representative Western blot and graphs for HMGB-1 in cytoplasmprotein (\*\*P \< 0.01, \*P \< 0.05, n = 3 \~ 4/group) and p65 in nucleoprotein (\*P \< 0.05, n = 3 \~ 4/group), and TLR4 (\*P \< 0.05, n = 4/group), p-Er and Erk (\*P \< 0.05, n = 3/group) in total protein. Each group of blots is the same exposure of a gel, each group of results repeated more than three times.
HQH Extractum does not Bind to Cisplatin {#Sec6}
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To observe the change in HQH after the addition of cisplatin, we applied nuclear magnetic resonance to detect the change in the one-dimensional proton spectrum. If cisplatin binds to HQH, it will cause chemical shifts, which will cause a change in the proton spectrum. However, we found that these two lines substantially overlapped (the red line indicates the hydrogen spectra of HQH and the blue line indicates the hydrogen spectra of Cis added to HQH) (Fig. [4](#Fig4){ref-type="fig"}), suggesting that HQH and cisplatin do not undergo chemical or physical reactions and that neither agent produces chelates or degradation. In other words, HQH does not react with cisplatin to prevent cisplatin from entering the cell.Figure 4HQH does not bind with cisplatin. (**A**) The nuclear magnetic resonance showed that HQH and Cis does not interact with each other. The red line indicates the hydrogen spectra of HQH. The blue line indicates the hydrogen spectra of which Cis into HQH. These two lines substantially overlapped, suggesting that HQH does not interact with Cis. (nuclear magnetic resonance spectrometer, agilent 600 DD2).
HQH Extractum does not Impair Cisplatin-Mediated Killing of HeLa Tumor Cells *In Vitro* {#Sec7}
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To verify the protective effect of HQH on the kidney at the cellular level, we performed a CCK 8 experiment in cisplatin-treated renal tubular epithelial cells after HQH pretreatment, which showed that HQH pretreatment increased the viability of TECs in a dose-dependent manner from 0 mg/mL to 12 mg/mL (Fig. [5a](#Fig5){ref-type="fig"}). Therefore, we pretreated TECs with HQH at a concentration of 12 mg/mL and showed that, at such a dose, HQH pretreatment significantly inhibited cisplatin-induced necroptosis *in vitro* (Fig. [5b](#Fig5){ref-type="fig"}). Thus, we used 12 mg/mL HQH for the following *in vitro* experiments in tumor cells (HeLa cells). We found that HQH does not inhibit the anti-tumor activity of cisplatin in HeLa cells (Fig. [5c](#Fig5){ref-type="fig"}), which suggests that HQH pretreatment protects against cisplatin-induced renal toxicity without affecting the anti-tumor effect of cisplatin.Figure 5HQH reduced the damage of renal tubular epithelial cells induced by cisplatin, but did not affect the antitumor activity of cisplatin in Hela cells. (**a**) CCK 8 experiment in renal proximal tubules after HQH (0/6/12/18 mg/ml) pretreatment 12 h before cisplatin (75 μM) treatment 12 h (\*P \< 0.05, n = 5/group). (**b**) RIP3 expression are decreased in renal proximal tubules after HQH (12 mg/ml) pretreatment 12 h before cisplatin (75 μM) treatment 12 h (\*P \< 0.05, n = 5/group). (**c**) CCK8 cell proliferation assay for Hela cell. Con: Without stimulus; Cis: 30 mM; HQH: 10 mg/ml; Dex: 50 μM; (\*\*P \< 0.01, n = 5/group).
HQH Extractum Pretreatment Attenuates AKI In Two Other Models {#Sec8}
-------------------------------------------------------------
To verify that Huaiqihuang also provides protection in other nephrotoxic models, we established a folic acid (FA)-induced AKI model and an ischemia-reperfusion (IRI)-induced AKI model. The experimental design of the renoprotective effect of HQH on FA- or IRI-induced renal injury is summarized in Fig. [6A and C](#Fig6){ref-type="fig"}, respectively. FA-induced (Fig. [6C](#Fig6){ref-type="fig"}) and IRI-induced (Fig. [6D](#Fig6){ref-type="fig"}) cast formation, tubular dilation and brush border loss were significantly alleviated with HQH and Dex pretreatment. Urea levels were significantly increased by FA and IRI treatment, which were restored by HQH and Dex pretreatment (Fig. [6C and D](#Fig6){ref-type="fig"}). There was no difference between the HQH- and Dex-pretreated groups, suggesting that HQH pretreatment attenuated acute renal tubular injury and improved kidney function in various models of AKI in mice.Figure 6HQH and Dex pretreatment attenuate renal injuries induced by folic acid (FA) or unilateral kidney ischemia reperfusion (IRI). (**A**) Scheme: Male C57BL/6 mice with body weight about 25 g underwent intraperitoneal injection with NaHCO3(150 mM) or 150 mg/kg folic acid (n = 4/group). Mice received gavage of 6 g/kg HQH extractum every day or one dose of Dex with 4 mg/kg before intraperitoneal injection with 150 mg/kg folic acid. (**B**) Representative photomicrographs and graphs of kidney injury outcomes induced by folic acid (\*P \< 0.05, n = 4/group). Bar = 100 μm. Serum urea was measured at day 3 (\*P \< 0.05, n = 4/group). (**C**) Scheme: Male C57BL/6 mice underwent left kidney Ischemia Reperfusion Injury (IRI) (ischemia 20 min, n = 5/group). The group of HQH + IRI mice received 10 g/kg HQH extractum gavage two consecutive days before IRI surgery on left kidney and sacrificed three days later (ischemia 20 min, n = 5/group). The group of Dex + IRI mice intraperitoneal injected with 4 mg/kg Dex one hour before left kidney IRI surgery (ischemia 20 min, n = 5/group). (**D**) Representative photomicrographs and graphs of the kidney outcomes of the IRI models (\*P \< 0.05,\*\*P \< 0.01, n = 4 \~ 5/group). Bar = 50 μm. Serum urea was measured at day 3 after IRI (\*P \< 0.05, n = 4 \~ 5/group).
Discussion {#Sec9}
==========
This study identified the renoprotective properties of HQH extractum on cisplatin-induced acute kidney injury. HQH does not bind to cisplatin directly *in vivo*. It ameliorated cisplatin-induced TECs apoptosis and necrosis and preserved renal function by inhibition of the HMGB1/TLR4/NFκB/TNF-α pathway. We found that HQH pretreatment did not inhibit the anti-tumor activity of cisplatin and it demonstrated similar renoprotective effects in various models of AKI, similar to Dex. In considering the reports that corticosteroids retard the treatment effectiveness of solid tumors and shorten survival to a certain extent^[@CR16],[@CR31],[@CR32]^, the present findings opened a new market for the prevention of kidney injury in patients with solid tumors.
Cisplatin-induced nephrotoxicity involves a complex multifactorial process. First, cisplatin has a direct toxic effect on the renal tubular cells and renal blood vessels, resulting in a decline in renal blood flow and glomerular filtration rate. Second, cisplatin induces renal interstitial inflammation and chronic interstitial fibrosis, leading to irreversible renal damage. Additionally, cisplatin activates a variety of cellular pathways, cytokines and signaling molecules in TECs, which causes renal inflammation, apoptosis and necrosis^[@CR9],[@CR33]--[@CR35]^.
Huaiqihuang (HQH) extractum is a Chinese herbal complex composed of *Trametes robiniophila* Murr. (Huaier), *Lycium barbarum* (Gouqi) and *Polygonatum sibiricum* (Huangjing). It has been identified that Huaiqihuang may protect against proteinuria by preventing MPC5 podocyte damage via targeting the p-ERK/CHOP pathway^[@CR21]^ and enhancing Nephrin expression and the necrosis factor κg signaling pathway^[@CR22]^. HQH not only activates macrophages, neutrophils, natural killer cells but also promotes T lymphocyte division, proliferation, maturation and differentiation^[@CR36]^. Based on the immunoregulatory function of HQH extractum and the possible direct protective effect on injured kidneys, mice were pretreated with HQH before the injection of cisplatin. As a result, the number of renal tubular epithelial cell injuries, swelling, apoptosis and necrosis was decreased and after pretreatment with HQH, the renal tubular lesion was alleviated, which was significantly lower than that of the cisplatin-treated control group (P \< 0.05). Such protective effects were further identified in FA- and IRI-induced AKI models (Fig. [6](#Fig6){ref-type="fig"}), suggesting that HQH is a broad-spectrum nephroprotective agent for AKI.
Glucocorticoids have been largely used in the clinical treatment of acute renal injury with a significant protective effect^[@CR37]--[@CR39]^, but the side effects of glucocorticoids for cancer patients, such as hair loss, endocrine disorders, weight gain and osteoporosis, make them unsuitable for long-term use. The more serious concern is that corticosteroids even inhibit the effectiveness of cancer treatment and shorten survival of solid tumor patients^[@CR16],[@CR31],[@CR32]^. However, the key component of HQH, *Trametes robiniophila* Murr. (Huaier), has been identified as an effective chemotherapeutic agent for certain tumors, 'with safe and broad anti-cancer spectrum'^[@CR40]--[@CR44]^. In the present study, we found that pretreatment with HQH and dexamethasone showed a similar renoprotective effect for cisplatin-induced acute kidney injury treatment. Thus, HQH can eventually be used in clinical prevention or treatment of cisplatin chemotherapy-induced kidney injury.
In summary, the current study indicated that HQH extractum protects the kidneys from cisplatin-induced injuries by inhibiting the HMGB1/TLR4/NFκB/TNF-α pathway in renal tubular cells. It shows potent therapeutic activity superior to that of dexamethasone for AKI in cancer patients. HQH extractum might be a potential adjuvant for clinical cisplatin therapy.
Materials and Methods {#Sec10}
=====================
Cisplatin-Induced Acute Kidney Injury Model {#Sec11}
-------------------------------------------
Male C57BL/6 mice (7 weeks old, 20--23 g) were purchased from the Hubei Experimental Animal Research Center. All mice were housed in the experimental animal center at the Tongji Medical College, Huazhong University of Science and Technology with a 12/12-h light/dark cycle. After a minimum of 1 week of acclimatization, the mice (n = 5/group) were randomly divided into four groups. Two groups of mice were gavage pretreated daily with normal saline (NS) for 3 days. The third group was gavage pretreated daily with HQH (6 g/kg; Qidong GaiTianLi Pharmaceutical Co.; dissolved by NS, 0.4 g/ml) for 3 days. A single nephrotoxic dose of cisplatin (20 mg/kg; Nanjing Pharmaceutical Co.) was administered via intraperitoneal injection (ip) to the HQH-pretreated group and one NS-pretreated group on the third day 30 minutes before the pretreatment. A single dose of NS (0.015 ml/g) was administered ip to one NS-pretreated group as a control. The last group was administered ip a single dose of dexamethasone (Dex) (4 mg/kg; MaAnShan Fengyuan Pharmaceutical Co.) 30 minutes before cisplatin (20 mg/kg) ip. The mice were sacrificed on Day 3. All studies were approved by the Animal Care and Use Committee (ACUC) in Tongji Hospital and conducted in accordance with NIH guidelines.
FA-Induced Acute Kidney Injury Model {#Sec12}
------------------------------------
Male C57BL/6 mice (7 weeks old, 20--23 g) were purchased from the Hubei Experimental Animal Research Center. All mice were housed in the experimental animal center at the Tongji Medical College, Huazhong University of Science and Technology with a 12/12-h light/dark cycle. After a minimum of 1 week of acclimatization, the mice (n = 4/group) were randomly divided into four groups. Two groups of mice were gavage pretreated daily with normal saline (NS) for 3 days. The third group was gavage pretreated with HQH (6 g/kg; Qidong GaiTianLi Pharmaceutical Co.; dissolved by NS, 0.4 g/ml) daily for 3 days. A single nephrotoxic dose of FA (150 mg/kg; SIGMA; dissolved by 150 mM NaHCO~3~) was administered ip to the HQH-pretreated group and one NS-pretreated group per day 30 minutes after the gavage. A single dose of NaHCO~3~ (150 mM, 0.2 ml) was administered ip to one NS-pretreated group as a control. The last group was administered ip a single dose of Dex (4 mg/kg; MaAnShan Fengyuan Pharmaceutical Co.) 30 minutes before FA (150 mg/kg) ip. The mice were sacrificed on Day 3. All studies were approved by the Animal Care and Use Committee (ACUC) in Tongji Hospital and conducted in accordance with NIH guidelines.
IRI-Induced Acute Kidney Injury Model {#Sec13}
-------------------------------------
Male C57BL/6 mice (7 weeks old, 20--23 g) were purchased from the Hubei Experimental Animal Research Center. All mice were housed in the experimental animal center at the Tongji Medical College, Huazhong University of Science and Technology with a 12/12-h light/dark cycle. After a minimum of 1 week of acclimatization, the mice (n = 5/group) were randomly divided into four groups. Two groups of mice were gavage treated daily with normal saline (NS) for 5 days. The third group was gavage treated with HQH (10 g/kg; Qidong GaiTianLi Pharmaceutical Co.; dissolved by NS, 0.4 g/ml) daily for 5 days. Left renal ischemia-reperfusion surgery was performed on mice in the HQH-pretreated group and one NS-pretreated group one hour after the gavage on Day 0. No surgery was performed on the mice in one NS-treated group, used as controls. The last group was administered ip of a single dose of Dex (4 mg/kg; MaAnShan Fengyuan Pharmaceutical Co.) one hour before the left renal ischemia-reperfusion surgery. The mice were sacrificed on Day 3. All studies were approved by the Animal Care and Use Committee (ACUC) in Tongji Hospital and conducted in accordance with NIH guidelines.
Serum Metabolites {#Sec14}
-----------------
Commercial kits were used to measure urea (Bioassay Systems, Hayward, CA).
Histologic, immunohistochemical and immunofluorescence staining {#Sec15}
---------------------------------------------------------------
Paraffin-embedded renal sections (3 μm) were subjected to periodic acid-Schiff (PAS) staining to evaluate tubular injury. The tubule injuries include cast formation, tubular dilation and brush border loss, which was analyzed by the quantities of injury tubules in \>10 random high-power fields (HPF). LTL (Lotus Tetragonolobus Lectins) carrying green fluorescence could be seen on the tubular brush border with specific coloring at the proximal end. However, when external factors or internal immune factors lead to renal tubular injury the brush edge is removed and there is no fluorescence expression. Therefore, LTL low coloring can be used as an indicator of renal tubular injury. For immunohistochemical (IHC) analysis, renal sections (3 μm) were incubated with 3% H~2~O~2~ for 20 min and nonspecific proteins were blocked with 10% goat serum for 30 min at room temperature. The slides were incubated with primary antibodies against either Kim-1 (1:1000, R & D), Ki-57 (1:200, Abcam), or HMGB-1 (1:200, Abcam) at 4 °C overnight. The slides were then incubated with the appropriate secondary antibody (HRP-conjugated secondary antibody for IHC and fluorescent-labeled secondary antibody for IF).
Western blot analysis {#Sec16}
---------------------
Renal tissues were homogenized in RIPA lysis buffer containing two kinds of protease inhibitors: cocktail (50×) and phenylmethanesulfonyl fluoride (PMSF, 100×). The extraction of nucleoprotein and cytoplasm protein was performed according to the manufacturer's instructions (KeyGEN BioTECH). Total protein concentrations were determined using a BCA assay kit according to the manufacturer's instructions. The kidney tissue total protein was separated by SDS-PAGE and the separated proteins were transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature and then probed with antibodies against either Bax (1:1000, Abclonal), Bcl-2 (1:1000, Abclonal), RIP3 (1:200, BioVision), MLKL (1:1000, Abgent), TLR-4 (1:500, Abgent), β-actin (1:2000, Google Bio.), p-Er/Erk or GAPDH (1:2000, Google Bio.) at 4 °C overnight and with p65 (1:1000, Rui Ying Bio.) for nucleoprotein and HMGB-1 (1:1000, Abcam) for cytoplasmic proteins. After being washed with TBST, the blots were incubated with an HRP-conjugated anti-IgG and the target bands were visualized with ECL plus reagents according to the manufacturer's instructions. The intensities of the target bands were analyzed by densitometry and were normalized to GAPDH or β-actin using Quantity One software (BioRad, CA, USA); prior to analysis, the intensities of the target bands were normalized to the corresponding values of the control group.
Real-time quantitative RT-PCR {#Sec17}
-----------------------------
Total RNA extraction and reverse transcription were conducted using a GoScript reverse transcription system (Promega, USA). PCR enzymes and master mixes (Thermo Scientific, USA) were used for real-time PCR with primers specific to mouse GAPDH and TNF-α. The sequences for all primers are shown in Table [1](#Tab1){ref-type="table"}. Relative expression levels were normalized to GAPDH and calculated using the 2^−ΔΔCt^ method.Table 1Primer sequences.ForwardReverseMouse GAPDH5′-TCAACGATTTGGTCGTATT-3′5′-CTGTGGTCATGAGTCCTTCC-3′Mouse TNF-α5′-CCCTCACACTCAGATCATCTTCT-3′5′-GCTACGACGTGGGCTACAG-3′Mouse RIP35′-CAGTGGGACTTCGTGTCCG-3′5′-CAAGCTGTGTAGGTAGCACATC-3′
Nuclear Magnetic Resonance (NMR) {#Sec18}
--------------------------------
First, 450 μl cisplatin (1 mg/ml) was taken and then 50 μl D~2~O was added; the samples were thoroughly mixed and placed in NMR tubes. One-dimensional ^1^H NMR spectra were obtained. Then, 500 μl H~2~O (containing 10% D~2~O) was added to 152.9 mg HQH and thoroughly mixed and after centrifugation, the supernatant was placed in NMR tubes. One-dimensional ^1^H NMR spectra were obtained. Next, 450 μl cisplatin with 50 μl D~2~O was added to 156.5 mg HQH and thoroughly mixed and after centrifugation, the supernatant was placed in NMR tubes. One-dimensional ^1^H NMR spectra were obtained.
*In vitro* cell culture in TECs {#Sec19}
-------------------------------
Male C57BL/6 mice (4 weeks old, 12--15 g) were sacrificed and kidneys were removed; then, tubular epithelial cells were isolated and cultured in DMEM/F12 (Gibco) with 10% FBS medium at 37 °C in 5% CO~2~. Tubular epithelial cells were split into 96-well plates with HQH (0/6/12/18 mg/ml) when the cells were confluent. Then, a CCK8 experiment in renal proximal tubules was performed after HQH (0/6/12/18 mg/ml) pretreatment 12 h before cisplatin (75 μM) treatment for 12 h. The CCK8 experiment was performed according to the manufacturer's instructions.
*In vitro* cell culture in HeLa cells {#Sec20}
-------------------------------------
HeLa cells were cultured in DMEM-H (Gibco) with 10% FBS medium at 37 °C in 5% CO~2~. The HeLa cells were split into 96-well plates with DMEM-H medium (with 1% FBS), 30 mM cisplatin, 10 mg/ml HQH + 30 mM cisplatin, or 50 μM dexamethasone + 30 mM cisplatin when the cells were confluent; then, a CCK8 cell proliferation assay for HeLa cells was performed. The CCK8 experiment was performed according to the manufacturer's instructions.
Statistical analysis {#Sec21}
--------------------
All statistics were analyzed using the Mann-Whitney U test; \*P \< 0.05, \*\*P \< 0.01, \*\*\*P \< 0.001, \*\*\*\*P \< 0.0001. Values are the means ± SEM. \*P \< 0.05 was considered to indicate a significant difference.
Electronic supplementary material
=================================
{#Sec22}
Supplementary Information
**Electronic supplementary material**
**Supplementary information** accompanies this paper at 10.1038/s41598-018-25610-6.
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This work was supported by Qidong Gaitianli Pharmaceutical Co., Ltd. Thanks to Prof. Gang Chen, Professor of Gynecologic Oncology Laboratory, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, who donated HELA cells.
The experiments were designed by Ying Yao, Rui Zeng, Gang Xu and Yujiao Guo and performed by Yujiao Guo, Meng Wang, Jingyi Mou and Zhi Zhao. Data analysis was carried out by Yujiao Guo, Guangchang Pei, Juan Yang, Fengming Zhu, Han Zhu, Meng Wang, Jingyi Mou and Zhi Zhao. Reagents were contributed by Guangchang Pei, Juan Zhan, Meng Wang, Zhi Zhao, Jingyi Mou, Yuxi Wang and Qian Yang. The manuscript was written by Yujiao Guo and Rui Zeng. The manuscript was read and approved by all of the authors.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
The intrauterine hyperglycaemia in women with gestational diabetes mellitus (GDM) is supposed to be an important factor that predisposes offspring to obesity and type 2 diabetes mellitus (T2D) \[[@B1], [@B2]\]. However, the mechanisms connecting intrauterine exposure to hyperglycaemia with subsequent development of metabolic diseases are not clear enough.
There is some suggestion that the exposure to diabetes in utero increases the risk of offspring obesity via alterations in the "adipoinsular axis," the endocrine loop, linking the brain and endocrine pancreas with insulin- and leptin-sensitive tissues in the control of eating behaviour and energy balance \[[@B1], [@B3]\].
Adipokines play an important role in the energy metabolism regulation \[[@B4]\]. Leptin (LEP) and adiponectin (ADIPOQ) are well-recognised obesity- and diabetes-related candidate genes through which the adipose tissue influences the regulation of several important physiological functions, including appetite, satiety, energy expenditure, insulin sensitivity, fat distribution, and endothelial function \[[@B4]\]. Adiponectin and leptin are also factors associated with fetal growth \[[@B5]\] and shown as predictors of early-life weight gain \[[@B6], [@B7]\].
Another promising adipokine is angiopoietin-like protein 4 (ANGPTL4), a multifunctional signal protein expressed in many tissues. ANGPTL4 is involved in the regulation of multiple physiological processes, including energy metabolism \[[@B8]\], plasma glucose level and tolerance regulation \[[@B9]\], fat storage, and lipid metabolism \[[@B10]\]. The association of ANGPTL4 expression with obesity was confirmed in a study of monozygotic twins \[[@B11]\]. Robciuc et al. revealed that ANGPTL4 expression in the adipose tissue and circulation was inversely correlated to body weight, suggesting a role for ANGPTL4 in acquired obesity \[[@B11]\].
The change in the expression of the abovementioned genes in the fetal tissues may serve as a marker of subsequent metabolic diseases of the offspring. The association between the presence of hyperglycaemia in the mother and altered cord blood levels of leptin, adiponectin, and ANGPTL4 has been identified in previous studies \[[@B5], [@B12], [@B13]\]. An increased placental LEP expression level has been also described in women with GDM \[[@B14]\].
However, it is not obvious that maternal hyperglycaemia causes such alterations. Perhaps, on the contrary, the altered gene expression functions in GDM pathogenesis (e.g., due to activation of hormone-encoding genes evoking insulin resistance or reduction of insulin secretion). It is also possible that both phenomena (maternal hyperglycaemia and changes in the expression of adipokines in the fetus and/or the mother) result from other pathological processes.
Randomised controlled trials (RCT) comparing changes of newborn gene expression level in groups of women with different target glucose levels during the treatment of GDM are supposed to help clarifying the cause-and-effect relations.
The human umbilical vein endothelial cells (HUVECs) represent a good cellular model for studying the effect of maternal hyperglycaemia on the fetal cardiovascular system and can serve as a marker of the predisposition of the fetus to metabolic diseases \[[@B15]\].
In this study, we investigated the alterations in ANGPT4, ADIPOQ, LEP, and leptin receptor gene (LEPR) expression levels in HUVECs and concentrations of these adipokines in the cord blood from newborns of women with GDM with different glycaemic targets compared to healthy women.
2. Materials and Methods {#sec2}
========================
This study was carried out at the Almazov National Medical Research Centre (ANMRC) as part of the ongoing RCT "Genetic and epigenetic mechanisms of developing gestational diabetes mellitus and its effects on the fetus" (GEM GDM) which started in July 2015. This study was approved by the local ethical committee (Protocol 119); informed written consent was obtained from all subjects.
2.1. Design and Study Population {#sec2.1}
--------------------------------
Forty-one women with GDM and 25 controls were randomly selected to assess the levels of expression of genes in HUVECs. The women with GDM were randomised to 2 groups according to target glycaemic levels: group 1 (target fasting blood glucose \< 5.1 mmol/L and \<7.0 mmol/L 1-hour postprandial) (GDM1, *N* = 21) and group 2 (target fasting blood glucose \< 5.3 mmol/L and \<7.8 mmol/L 1-hour postprandial) (GDM2, *N* = 20).
GDM was diagnosed according to the Russian National Consensus \[[@B16]\] and the recommendations of the International Association of Diabetes and Pregnancy Study Groups (IADPSG) based on the results of 2-hour oral glucose tolerance test (OGTT) performed at 24th--28th week of gestation \[[@B17]\]. Pregnant women without diabetes were included as controls.
None of the patients had previous history of diabetes mellitus or any known medical condition affecting glucose metabolism.
They were all followed until delivery at ANMRC. Anthropometric variables (height and blood pressure) were measured using standardised procedures. Prepregnancy body mass index (BMI) was calculated based on the prepregnancy weight recalled by participants. Women with GDM were consulted by the endocrinologist and provided the results of their self-measurements of blood glucose every 2-3 weeks. In case of exceeding the target blood glucose levels (in 2 or more measurements per week in group 1 and in more than 1/3 of measurements per week in group 2), insulin therapy was started. The participants were asked to keep electronic nutrition and glycaemic control diaries with the help of a specially developed mobile application and send data to the doctor. The mobile application is described elsewhere \[[@B18]\]. According to the personal diaries, automatic calculations of the integral indicators characterising the self-control of glycaemia (fasting, postprandial, and average glycaemia) were accomplished. Electronic diary data were available for almost all women with GDM (*N* GDM1 = 19, *N* GDM2 = 20) and 8 women from the control group.
2.2. Blood Sample Processing and Analysis {#sec2.2}
-----------------------------------------
Cord blood samples were collected immediately after delivery. Blood glucose measurements were made on fresh plasma samples. The cord blood serum samples were stored at −80°C for further analysis of C-peptide, leptin, adiponectin, and ANGPTL4. Plasma glucose (PG) concentration was determined by the glucose oxidase method. Serum C-peptide level was measured by the chemiluminescent microparticle immunoassay (Architect C-peptide assay, Abbott Laboratories, IL, USA). Serum adiponectin (BioVendor Laboratory Medicine Inc., Modrice, Czech Republic) and leptin (Diagnostics Biochem Canada Inc., Canada) levels were measured using an enzyme-linked immunosorbent assay (ELISA) as recommended by the manufacturer. Serum level of ANGPTL4 was determined by DuoSet ELISA Development kits (DY3485) from R&D Systems (USA). The limit of detection for ANGPTL4 is 1.25 ng/mL. The detection range is 1.25 ng/mL--80 ng/mL. The following factors prepared at 800 ng/mL were assayed and exhibited no cross-reactivity or interference: recombinant human angiopoietin-1, angiopoietin-2, angiopoietin-4, and angiopoietin-like 3 and recombinant mouse angiopoietin-3 and angiopoietin-like 3.
The limit of detection for Leptin is 0.5 ng/mL. The detection range is 0.5--100 ng/mL.
The following substances were tested at 1000 ng/mL and exhibited no cross-reactivity: mouse leptin, TNF-α, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-16, GM-CSF, CSF, and EGF.
The limit of detection for adiponectin is 26 ng/mL. The detection range is 26 ng/mL--100 ug/mL. No cross-reactivity has been observed for human leptin and leptin receptor.
Intra-assay coefficients of variation (CVs) for leptin assay were between 3.7% and 5.5%, and interassay CVs were 5.8--6.8%. For adiponectin assay, the intra- and interassay CVs were 3.9--5.9% and 6.3--7.0%, respectively.
2.3. Isolation and Identification of the HUVECs {#sec2.3}
-----------------------------------------------
The HUVECs were isolated using a standard collagenase digestion method \[[@B19]\] as we do routinely in our laboratory \[[@B20]\]. Immediately after isolation, the cells were cultured and expanded in endothelial cell medium (ECM cat number 1001, ScienCell, San Diego, CA) containing 5% fetal bovine serum, 1% penicillin/streptomycin, and endothelial cell growth supplement in a humidified atmosphere of 95% air and 5% CO~2~ at 37°C. For this study, 80% of confluent HUVEC monolayers (passages 2-3) were used.
The purity of primary HUVEC cultures was evaluated by flow cytometry analysis performed on Guava EasyCyte8. Briefly, detached cells were resuspended in 200 *μ*L of PBS containing 1% bovine serum albumin (Sigma-Aldrich, Saint Luis, MO, USA) and incubated for 15 min at 20°C with the following antibodies (Ab): FITC-conjugated anti-CD31, PE-A-conjugated anti-CD144, PE-Cy7-A-conjugated anti-CD146 (BioLegend, San Diego, CA, USA), PE-A-conjugated anti-CD105 (Bioscience Pharmingen, San Jose, CA, USA), and APC-A-conjugated anti-CD45 (DAKO, Santa Clara, CA, USA). Data files were collected and analysed using the FACSDiva software program (version 6.1.3; BD Bioscience, San Jose, CA, USA).
2.4. Evaluation of Apoptosis and Immunocytochemical Assay {#sec2.4}
---------------------------------------------------------
The viability of HUVEC was assessed by flow cytometry with the determination of the number of viable cells, as well as those in early and late apoptosis and necrosis evaluated by Annexin-V/PI (BioLegend, San Diego, CA, USA) double staining.
The expression of von Willebrand factor and CD146 (BioLegend, San Diego, CA, USA) in HUVECs was detected by immunocytochemical staining. Cell nucleuses were stained with 4′,6-diamidino-2-phenylindole (DAPI).
2.5. RT-qPCR {#sec2.5}
------------
Total RNA was extracted from HUVEC using ExtractRNA reagent (BC032, Evrogen, Moscow, Russia) according to the manufacturer\'s protocol. One microgram of total RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) kit (SK021, Evrogen, Moscow, Russia). After cDNA synthesis, quantitative real-time PCR was performed in 25 *μ*L reaction mixture containing: 5x qPCRmix-HS LowROX (PK154L, Evrogen, Moscow, Russia) diluted to a final concentration of 1x, 20x primers diluted to 1x, 50 ng cDNA, and deionised distilled water. Reaction mixtures were incubated for an initial denaturation at 95°C for 10 min, which was followed by 40 PCR cycles, each consisting of exposure to 95°C for 15 sec and 60°C for 1 min. Gene expression was evaluated by real-time PCR using Applied Biosystems TaqMan Gene Expression Assays (ADIPOQ: Hs00605917_m1; LEP: Hs00174877_m1; LEPR: Hs00174497_m1; and ANGPTL4: Hs01101127_m1). All data are expressed as ratio to the reference gene GAPDH (forward AATGAAGGGGTCATTGATGG, reverse AAGGTGAAGGTCGGAGTCAA) (AlkorBio, Saint-Petersburg, Russia).
Relative expression was evaluated according to the 2^−ΔΔCt^ method \[[@B21]\]. In order to confirm the correctness of the method of detection of LEP and ADIPOQ expression, we used RNA samples derived from our previous adipose differentiation experiments \[[@B22]\]. RNA samples from adipose tissue multipotent mesenchymal stromal cells (MSC) and from differentiated in vitro adipose tissue were used as negative and positive controls, respectively.
2.6. Data Analysis {#sec2.6}
------------------
Statistical analysis was performed using SPSS 22.0 (SPSS Inc., USA). Mean and standard deviation are reported for continuous variables, and numbers and percentages are reported for categorical variables. Differences among the groups were analysed by Mann--Whitney test (for comparison between two groups), Kruskal-Wallis test (for comparison of more than two groups) or chi-square test. A *p* value \< 0.05 was considered statistically significant.
3. Results {#sec3}
==========
3.1. Characteristics of the Participants {#sec3.1}
----------------------------------------
Baseline characteristics of the participants are described in [Table 1](#tab1){ref-type="table"}. The women from all three groups did not differ in terms of age and prepregnancy BMI. The GDM1 group had higher diastolic BP compared to controls (*p* = 0.003). The GDM1 and GDM2 groups had higher levels of fasting PG (*p* = 0.004 and *p* = 0.003, resp.) and higher levels of PG 1 h and 2 h in OGTT (*p* \< 0.001 in comparison with controls).
Mean levels of fasting, 1-hour postprandial and average blood glucose measured by the participants during the study are described in [Table 2](#tab2){ref-type="table"}. The GDM1 group achieved significantly lower average and 1-hour postprandial glucose levels compared to GDM2. Mean postprandial BG was lower in GDM1 even compared to the control group, though the difference did not reach statistical significance (*p* = 0.088). Gestational weight gain did not differ between GDM1 and GDM2 groups and was lower in both groups compared to controls ([Table 2](#tab2){ref-type="table"}). The percentage of women treated with insulin was 40% and 29% in the GDM1 and GDM2 groups, respectively, and did not significantly differ (*p* = 0.495).
3.2. Pregnancy Outcomes {#sec3.2}
-----------------------
Pregnancy outcomes and biochemical markers in cord blood are shown in [Table 3](#tab3){ref-type="table"}.
There was no statistically significant difference among the groups in terms of pregnancy outcomes (percent of large for gestational age (LGA) and small for gestational age (SGA) newborns, delivery by caesarean section) and the level of C-peptide, adiponectin, and ANGPTL4 in cord blood serum and glucose in cord blood plasma. The level of leptin in cord blood serum was higher in the GDM2 group than in the GDM1 (*p* = 0.036) and the control group, but the difference from the control group did not reach statistical significance (*p* = 0.066). After adjustment by insulin therapy, age, and prepregnancy BMI, the difference in the level of leptin between GDM1 and GDM2 remained significant (*p* = 0.01). The leptin/adiponectin ratio (LAR) in cord blood serum was higher in the GDM2 group compared to controls (*p* = 0.011) with no difference between the GDM1 and control group (*p* = 0.404).
3.3. HUVEC Characterisation {#sec3.3}
---------------------------
HUVECs were obtained from the umbilical vein, expanded in vitro, and characterised for expression of endothelial markers by flow cytometry and immunohistochemistry. All samples demonstrated characteristic endothelial morphology and immunophenotype CD45−/CD144+/CD31+/CD146+/CD105+ and stained positively for endothelial markers, von Willebrand factor, and CD146 (as demonstrated in our previous work \[[@B20]\]). There was no difference in the parameters of viability and replicative aging of HUVEC cultures from different patient groups (data not presented).
3.4. Gene Expression in HUVECs {#sec3.4}
------------------------------
ANGPTL4 expression was downregulated in the HUVECs derived from GDM patients compared to control group (23.11 ± 5.71, 21.47 ± 5.64, and 98.33 ± 0.92, respectively, for GDM1, GDM2, and control groups; *p* \< 0.001 for comparison among the 3 groups) while no difference between GDM1 and GDM2 groups was observed ([Figure 1(a)](#fig1){ref-type="fig"}).
We did not detect the expression of LEP in HUVEC but found out that they expressed LEPR, and the expression of LEPR demonstrated a decline in the GDM1/GDM2 groups compared to the control group, though the differences were not statistically significant ([Figure 1(c)](#fig1){ref-type="fig"}). The expression of LEPR did not correlate with the level of LEP in cord plasma ([Figure 1(d)](#fig1){ref-type="fig"}) which indicates that there is no reciprocal regulation between LEP and its receptor in HUVECs. The expression of ADIPOQ was detected in HUVEC samples, but the level of its expression was as low as in negative control samples and lower by four orders of magnitude in HUVEC compared to adipocytes (positive control) ([Figure 1(b)](#fig1){ref-type="fig"}).
4. Discussion {#sec4}
=============
Our RCT has shown that GDM treated according to the most widely accepted current guidelines was associated with the increased LAR in cord blood and that LAR did not differ from the control group if GDM was treated aiming at tighter glycaemic targets. The GDM1 group (with tighter glycaemic targets) had lower levels of leptin compared to GDM2. We also found a decrease in the level of expression of ANGPTL4 in HUVEC of newborns from women with GDM in comparison with the control group. However, there was no difference in the level of expression of ANGPTL4 between the two groups of GDM with different glycaemic targets.
The most appropriate target levels of glycaemia for the management of GDM are not universally defined \[[@B23]\]. Most organisations \[[@B24]--[@B26]\] suggest the targets for glycaemic control for women with GDM based on recommendations from the Fifth International Workshop-Conference on Gestational Diabetes Mellitus \[[@B27]\].
These targets were used by the Maternal-Fetal Medicine Units Network (MFMU) trial showing benefit for the treatment of GDM \[[@B28]\]. We used these targets for group 2.
However, there are no reliable data from controlled trials of lower versus higher target levels of glycaemia to identify ideal glycaemic targets for prevention of fetal risks \[[@B29]\].
The glycaemic targets used in our study for group 1 were tighter in accordance with current Russian guidelines \[[@B16]\]. Our data suggest that achieving tighter glycaemic targets during GDM treatment reduces LAR in the cord blood. However, this data should be interpreted with caution considering the small sample size. The full information about pregnancy outcomes is needed to guide clinical practice regarding target glycaemic levels during pregnancy. Our current study with a small sample size was not designed for this purpose. However, our findings may serve as a confirmation of the cause-and-effect relationship between maternal hyperglycaemia and alteration of LAR in newborns. Our results are in line with the data by Pirc et al. that reported treatment of mild GDM reduces cord blood leptin \[[@B12]\]. The authors hypothesise that hyperleptinaemia of the babies born to women with untreated mild GDM may persist for some time. It could reduce appetite and food intake and might contribute to the phenomenon of catch-down growth seen in macrosomic infants following birth \[[@B12], [@B30]\].
Follow-up studies are needed to understand the impact of tight glycaemic targets during pregnancy on obesity development in the offspring of women diagnosed with GDM according to IADPSG criteria. It is especially important taking into consideration the evidence that low early-life leptin concentrations may promote faster weight gain in infancy \[[@B31]--[@B33]\].
There is controversy about the association of adiponectin levels in cord blood with GDM. Pirc et al. reported decreased levels of adiponectin in the cord blood of newborns from mothers with GDM \[[@B12]\], whereas several other studies, like ours, find no effect of maternal DM on cord blood adiponectin concentration \[[@B5], [@B34], [@B35]\]. The reasons for these differences are unclear but may reflect different assay methodologies, different study populations, and different criteria used to diagnose GDM.
The level of expression of LEP in HUVECs turned out to be below the detection threshold. The expression of ADIPOQ was detected in HUVEC samples, but the level of its expression was as low as in negative control samples, which confirms that umbilical vein endothelium is not a place of adiponectin production. We are not aware of any other study addressing the expression of these genes in HUVEC. However, our results are in line with some of the previous studies which have shown that ADIPOQ is not expressed in the placenta \[[@B14], [@B36]\]. Thus, the levels of cord adiponectin may be attributed to other fetal tissues, while LEP has been shown to be expressed in the placenta \[[@B14]\]. It should be noted that there are also conflicting results of the studies which indicated the expression of ADIPOQ in human placenta \[[@B36], [@B37]\].
In contrast to LAR changes associated with tight glycaemic targets of treatment of GDM, the level of expression of ANGPTL4 was lower in both the GDM groups regardless of glycaemic targets compared to the control group. Possibly, it is due to the fact that the difference in target glycaemic levels is not significant enough to affect the expression level of ANGPTL4, at least on such a small sample. Another plausible explanation is that the reduced level of activity of ANGPTL4 is transmitted at the genetic level to newborns from their mothers. Maybe, the reduced level of activity of ANGPTL4 contributes to the development of GDM in the mothers, that is, it is the cause, not the consequence of hyperglycaemia.
This hypothesis is supported by the data of Xu et al. on the lower level of ANGPTL4 in patients with type 2 diabetes whose pathogenesis is close to GDM \[[@B9]\]. However, this hypothesis is contradicted by the data of Ortega-Senovilla et al., indicating that maternal serum ANGPTL4 concentrations showed no difference between the control and GDM women \[[@B13]\].
Moreover, opposite to our data, Ortega-Senovilla et al. showed that serum ANGPTL4 concentrations in cord serum were higher in those from GDM than those from control pregnancies \[[@B13]\]. We found no difference in the level of ANGPTL4 in the cord serum. There seems to be no correlation between the levels of ANGPTL4 protein in the cord serum and ANGPTL4 gene expression in one of the fetal tissues (HUVEC). It is plausible that the main source of cord serum ANGPTL4 is some other fetal tissue (e.g., the liver). The functional consequences of the downregulation of ANGPTL4 mRNA levels in HUVECs in GDM remain to be identified.
In addition, other factors besides intrauterine hyperglycaemia may affect the activity of a number of genes, including ANGPTL4, in the fetus. Known factors that affect the weight of the newborn are the body mass index (BMI) of the mother and maternal gestational weight gain. Obviously, these parameters are influenced by the mother\'s lifestyle (the quantitative and qualitative composition of the diet and the level of physical activity).
It is known that ANGPTL4 can be regulated by diet \[[@B38], [@B39]\]. The diet interventions leading to NEFA increase in the blood (high-fat diet, a very low-energy diet, and fasting) were shown to upregulate the plasma level of ANGPTL4 \[[@B38]\].
This upregulation has been confirmed likewise in vitro as the expression of ANGPTL4 is upregulated in response to exposure to fatty acids in cell studies \[[@B40]\].
Our study established a significantly lower pregnancy weight gain in GDM patients compared to controls which is obviously due to diet adherence by patients.
Further studies are needed to clarify the cause-and-effect relationship between GDM and the level of expression of ANGPTL4 gene in HUVEC.
The level of C-peptide in the cord blood is commonly used as a marker of fetal hyperinsulinemia \[[@B41]\]. The data presented by HAPO study has shown associations between increasing levels of fasting, 1-hour, and 2-hour plasma glucose obtained on oral glucose-tolerance testing and cord blood serum C-peptide level above the 90th percentile \[[@B41]\]. We did not reveal any difference in the level of C-peptide among the groups. It could be a result of the treatment that was efficient to reduce fetal hyperinsulinemia in both GDM groups or it may be due to a small sample size.
The strength of our study is the design of the RCT of different glycaemic targets for women with GDM which allows at testing cause-and-effect relationships. The weakness of the study, besides its relatively small sample size, is the lack of information about maternal levels of the studied gene expression.
5. Conclusion {#sec5}
=============
Our study established positive association of cord leptin levels and LAR with target levels of glycaemia during pregnancy in women with GDM. Further investigation into long-term consequences of cord leptin concentrations is required.
We also found a decrease in the expression of ANGPTL4 in HUVECs of neonates from mothers with GDM. However, we could not prove the causal relationship between intrauterine hyperglycaemia and the expression of the ANGPTL4 gene, given the absence of differences between the level of expression of ANGPTL4 in groups with different glycaemic targets. This relationship remains to be clarified.
The study was funded by the Russian Science Foundation (Project no. 17-75-30052).
Data Availability
=================
The data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval
================
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008, and have been approved by the institutional committee of ANMRC (Protocol 119).
Conflicts of Interest
=====================
The authors declare no conflict of interest.
{#fig1}
######
Characteristics of the participants at study entry.
--------------------------------------------------------------------------------------------------------------------------------------
GDM 1\ GDM2\ Control\ *p* *p* control-GDM1 *p* control-GDM2 *p* GDM1-GDM2
(*N* = 20) (*N* = 21) (*N* = 25)
---------------------------- ------------- ------------- ------------- --------- ------------------ ------------------ ---------------
Maternal age, years 30.9 ± 5.4 32.3 ± 5.0 30.8 ± 4.2 0.566
Prepregnancy BMI, kg/m^2^ 25.4 ± 7.2 26.1 ± 6.5 23.4 ± 4.2 0.287
BP syst, mmHg 120 ± 13 118 ± 12 112 ± 14 0.114
BP diast, mmHg 76 ± 8 73 ± 10 69 ± 8 0.016 0.003 0.155 0.195
Fasting PG, mmol/L 5.1 ± 0.8 5.0 ± 0.6 4.5 ± 0.4 0.007 0.004 0.003 0.396
OGTT 1 h PG, mmol/L 10.2 ± 1.4 9.9 ± 1.6 6.9 ± 1.9 \<0.001 \<0.001 \<0.001 0.454
OGTT 2 h PG, mmol/L 8.0 ± 1.6 8.8 ± 1.6 5.9 ± 1.5 \<0.001 \<0.001 \<0.001 0.231
Fasting leptin, ng/mL 22.2 ± 20.7 29.5 ± 26.2 26.6 ± 17.0 0.561
Fasting adiponectin, ng/mL 7.2 ± 3.3 9.1 ± 3.3 8.9 ± 2.5 0.077
--------------------------------------------------------------------------------------------------------------------------------------
Note: BMI: body mass index; BP: blood pressure; PG: plasma glucose; OGTT: oral glucose tolerance test.
######
Blood glucose data from electronic diaries and gestational weight gain.
-------------------------------------------------------------------------------------------------------------------------------------
GDM 1\ GDM2\ Control\ *p* *p* control-GDM1 *p* control-GDM2 *p* GDM1-GDM2
(*N* = 20) (*N* = 21) (*N* = 25)
-------------------------------- ------------ ------------ ------------ ------- ------------------ ------------------ ---------------
Gestational weight gain, kg 9.9 ± 4.9 9.5 ± 5.9 15.2 ± 7.8 0.006 0.023 0.023 0.970
BG average, mmol/L^∗^ 5.6 ± 0.3 5.9 ± 0.4 6.0 ± 0.5 0.004 0.110 0.893 0.005
Fasting BG, mmol/L^∗^ 4.7 ± 0.4 4.8 ± 0.3 4.7 ± 0.3 0.499 0.835 0.421 0.735
1 h postprandial BG, mmol/L^∗^ 5.9 ± 0.3 6.4 ± 0.5 6.5 ± 0.7 0.002 0.088 0.818 0.002
Number of BG measurements 140 ± 78 147 ± 60 42 ± 21 0.001 \<0.001 \<0.001 0.946
\% (*N*) treated with insulin 40% (8) 29% (6) N/A 0.495
-------------------------------------------------------------------------------------------------------------------------------------
Note: ^∗^derived from electronic diaries filled in by participants (*N* GDM1 = 19, *N* GDM2 = 20, *N* control = 8) during the study period. BG: blood glucose; N/A: nonapplicable.
######
Pregnancy outcomes, biochemical markers in the cord blood, and ANGPTL4 gene expression in HUVECs.
------------------------------------------------------------------------------------------------
GDM 1\ GDM2\ Control\ *p*
(*N* = 20) (*N* = 21) (*N* = 25)
--------------------------------------- ---------------- ---------------- -------------- -------
Gestational age at delivery, weeks 39.2 ± 1.5 39.3 ± 1.0 39.7 ± 1.0 0.261
Caesarean section, % (*N*) 30% (6) 19% (4) 20% (5) 0.723
Birth weight, g 3572 ± 488 3584 ± 577 3513 ± 555 0.856
Height, cm 52.1 ± 2.5 52.4 ± 2.3 52.1 ± 2.5 0.990
LGA, % (*N*) 20% (4) 23% (5) 12% (3) 0.235
SGA, % (*N*) 5% (1) 9.5% (2) 4% (1) 0.819
Apgar score 1 min 7.5 ± 0.7 7.7 ± 1.1 7.7 ± 0.6 0.204
Apgar score 5 min 8.6 ± 0.5 8.7 ± 0.9 8.8 ± 0.4 0.208
Glucose, mmol/L 4.7 ± 1.2 5.3 ± 1.3 4.5 ± 1.2 0.203
C-peptide, ng/mL 0.8 ± 0.5 1.0 ± 0.6 0.9 ± 0.4 0.379
Leptin, ng/mL 8.8 ± 6.6^a^ 18.3 ± 16.1 10.6 ± 10.4 0.042
Adiponectin, ng/mL 15.9 ± 11.5 16.3 ± 14.4 18.3 ± 14.3 0.843
LAR 0.97 ± 1.31 1.70 ± 1.66^b^ 0.72 ± 0.46 0.038
ANGPTL4 in cord serum, ng/mL 19.9 ± 15.0 14.1 ± 4.5 13.9 ± 5.2 0.248
ANGPTL4 relative expression in HUVECs 23.1 ± 25.6^c^ 21.5 ± 25.8^c^ 98.3 ± 104.6 0.001
------------------------------------------------------------------------------------------------
Notes: LAR: leptin/adiponectin ratio; LGA: large for gestational age; SGA: small for gestational age. LGA was defined by a birth weight exceeding the 90th percentile on standard charts. SGA was defined by a birth weight below the 10th percentile on standard charts. ^a^*p* \< 0.05 versus GDM2; ^b^*p* \< 0.05 versus the control group; ^c^*p* \< 0.01 versus the control group.
[^1]: Academic Editor: Agnieszka Rak
| {
"pile_set_name": "PubMed Central"
} |
Obesity is a major long-term concern in human immunodeficiency virus (HIV)-positive (HIV+) persons given their increased cardiovascular risk \[[@OFU040C1]\], and the pathogenic link of obesity to diabetes mellitus, cardiovascular disease \[[@OFU040C2], [@OFU040C3]\], and all-cause mortality \[[@OFU040C4]\]. Soon after the introduction of antiretroviral treatment (ART), regional body fat distribution abnormalities became important metabolic concerns \[[@OFU040C5], [@OFU040C6]\]. With the use of the thymidine-analogs zidovudine and stavudine essentially disappearing in Western countries, lipoatrophy \[[@OFU040C7]\] has been receiving less attention and concern has shifted towards lipohypertrophy and obesity. Obesity is now increasingly recognized as a problem in HIV+ persons, and it has been attributed to improved health due to effective ART, normal aging, and obesity trends similar to those seen in the general population \[[@OFU040C8]--[@OFU040C17]\]. However, large-scale, longitudinal analyses are not available.
In this study, we describe obesity trends and concurrent demographic changes from 1990 to 2012 in the nationally representative Swiss HIV Cohort Study (SHCS), which currently follows more than 8000 active patients \[[@OFU040C18]\]. We also investigate the contribution of demographics, HIV-related factors, and the most commonly used ART combinations to body mass index (BMI) change after 1 and 4 years of ART in 1601 participants who started a first ART regimen after January 1, 1998. This study represents the most comprehensive longitudinal BMI analysis undertaken in HIV+ persons.
METHODS {#s2}
=======
Study Participants {#s2a}
------------------
Participants included HIV+ persons, aged ≥16 years who were followed in the SHCS ([www.shcs.ch](www.shcs.ch)) \[[@OFU040C18]\]. The study was approved by local ethics committees. All participants provided written informed consent.
We present 2 main types of analyses: first, a population-based descriptive analysis of all SHCS patients with at least 1 available BMI from 1990 to 2012 (Figure [1](#OFU040F1){ref-type="fig"}; [Supplementary Figure 1](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1); [Supplementary](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1) Table [1](#OFU040TB1){ref-type="table"}); second, detailed analyses of BMI changes from 0--1 and 1--4 years after starting ART in participants initiating a first ART regimen after January 1, 1998 ([Tables 1](#OFU040TB1){ref-type="table"} and [2](#OFU040TB2){ref-type="table"}; [Supplementary Tables 2 and 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1); [Supplementary Figure 2](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)). To limit confounding, pregnant females and patients with incident cancer or acquired immune deficiency syndrome (AIDS)-defining events after ART start were excluded. We also excluded participants with missing CD4 (+120/−30 days at ART start) or BMI information (+120/−120 days at ART start and at 1 and 4 years thereafter), those with ≥62 days of ART interruption, and injection drug users (IDUs), due to low numbers, increased rates of dropout, and missing data. A common reason for missing BMI information was ART initiation before SHCS enrollment. Table 1.Baseline Characteristics of 1601 SHCS Participants Who Started a First ART Regimen After January 1, 1998AllMSM\*Heterosexual maleHeterosexual female*P*-value\*Participants, *n* (%)1601 (100)892 (56)390 (24)319 (10)Age, median years (IQR)40 (34--47)40 (34--45)44 (36--53)37 (32--45)\<0.001Ethnicity White, *n* (%)1280 (80)808 (91)315 (81)157 (49)\<0.001 Black, *n* (%)181 (11)10 (1)65 (17)106 (33) Asian, *n* (%)83 (5)38 (4)4 (1)41 (13) Other, *n* (%)57 (4)36 (4)6 (2)15 (5)Smoking ever, *n* (%)613 (38)377 (42)149 (38)87 (27)\<0.001HCV coinfection, *n* (%)21 (1)7 (1)9 (2)5 (2)0.08CD4 nadir, cells/µL, median (IQR)217 (131--289)235 (161--305)199 (103--271)194 (93--265)\<0.001HIV-RNA at start of ART, log~10~ copies/mL4.8 (4.2--5.4)4.9 (4.3--5.3)4.9 (4.2--5.5)4.7 (4.0--5.3)0.04[^2][^3] Table 2.Bi- and Multivariable Linear Regression of BMI Changes During Year 0--1 and Years 1--4 of ART Among 1601 SHCS Participants Who Started a First ART Regimen After January 1, 1998CovariablesYear 0-1 of ARTYears 1--4 of ARTCoefficient Bivariable models (95% CI)^a^*P* ValueCoefficient Multivariable Models (95% CI)^b^*P* ValueCoefficient Bivariable Models (95% CI)^c^*P* ValueCoefficient Multivariable Models (95% CI)^b^*P* ValueBaseline BMI^d^−0.09 (−.11 to .07)\<.001−0.09 (−.11 to −.06)\<.001−0.02 (−.03 to −0.01)\<.001−0.01 (−.02 to −.00).002Delta BMI change, first year of ART--------−0.05 (−.07 to −.03)\<.001−0.06 (−.07 to −.04)\<.001HIV transmission group MSM1--1--1--1-- Het male0.46 (.24 to .68)\<.0010.20 (−.02 to .42).070.06 (−.02 to 0.13).140.02 (−.05 to .10).53 Het female0.18 (−.06 to .41).140.07 (−.18 to .32).580.05 (−.03 to .13).24−0.03 (−.11 to .06).52Age per 10 years^e^0.25 (.16 to .34)\<.0010.15 (.06 to .24).0020.01 (−.02 to .04).350.02 (−.01 to .05).14Ethnicity White1--1--1--1-- Black0.04 (−.25 to .33).80−0.05 (−.36 to .26).740.26 (.16 to .35)\<.0010.28 (.16 to .37)\<.001 Asian−0.60 (−1.0 to −.18).005−0.79 (−1.2 to −.39)\<.001−0.01 (−.15 to .13).870.01 (−.13 to .15).88 Other−0.44 (−.93 to .05).08−0.25 (−.71 to .21).29−0.01 (−.17 to .15).90−0.03 (−.19 to .13).71CD4 nadir 0--99 cells/µL1.73 (1.45 to 2.02)\<.0011.64 (1.4 to 1.9)\<.0010.40 (.14 to .66).0020.44 (.19 to .70).001 100--199 cells/µL0.50 (.24 to .76)\<.0010.46 (.19 to .72).001−0.04 (−.16 to .07).440.05 (−.07 to .17).40 200--349 cells/µL−0.00 (−.21 to .21).99−0.02 (−.23 to .19).820.04 (−.03 to .11).280.07 (−.00 to .14).05 ≥350 cells/µL1--1--1--1--Smoking, ever−0.46 (−.65 to −.27)\<.001−0.29 (−.47 to −.11).002−0.04 (−.10 to .02).20----Smoking cessation−0.11 (−.78 to .56).75----0.15 (.03 to .26).010.16 (.04 to .27).006HCV coinfection−0.73 (−1.5 to .06).07----−0.26 (−.46 to −0.06).01----Peginf0.56 (−3.1 to 4.2).76----−0.72 (−1.1 to −.37)\<.001--0.71 (−1.0 to −.37)\<.001Most frequent ART regimens TDF, xTC, EFV1−1−1−1− ABC, xTC, EFV0.27 (−.20 to .75).260.21 (−.23 to .64).35−0.01 (−.16 to .15).95−0.01 (−.16 to .14).89 ABC, xTC, LPV0.51 (−0.05 to 1.1).080.22 (−.30 to .74).410.14 (−.07 to .35).210.15 (−.06 to .36).16 ABC, xTC, ATV, RTV0.15 (−0.50 to .80).660.19 (−.41 to .79).530.16 (−.04 to .36).110.14 (−.05 to .33).16 AZT, xTC, EFV0.04 (−.30 to .39).81−0.13 (−.45 to .20).44−0.16 (−.28 to −.04).01−0.17 (−.29 to −.05).006 AZT, xTC, LPV0.54 (.19 to .89).0030.29 (−.04 to .62).08−0.16 (−.30 to −.02).03−0.12 (−.25 to .02).09 AZT, xTC, NFV0.27 (−.27 to .80).33−0.09 (−.59 to .41).73−0.25 (−.46 to −.03).02−0.22 (−.43 to −.01).04 TDF, xTC, LPV0.45 (.10 to .80).010.30 (−.03 to .62).080.08 (−.07 to .23).300.06 (−.08 to .21).38 TDF, xTC, NVP−0.09 (−.60 to .42).730.06 (−.42 to .54).810.00 (−.14 to .14).970.03 (−.10 to .16).65 TDF, xTC, ATV, RTV0.04 (−.30 to .39).800.12 (−.20 to .44).450.01 (−.11 to .13).890.06 (−.05 to .17).30 Other^f^0.27 (−.01 to .55).060.14 (−.12 to .40).29−0.06 (−.14 to .03).17−0.04 (−.12 to .04).36[^4][^5][^6][^7][^8][^9][^10] Fig. 1.Longitudinal trends of population body mass index (BMI) categories and median BMI trajectory, entire Swiss HIV Cohort Study population, 1990--2012. Note: Confidence intervals are omitted for visual clarity. Abbreviations: AIDS, acquired immune deficiency syndrome; IDU, injection drug user; ART, antiretroviral therapy.
Definitions and Measures {#s2b}
------------------------
Body mass index (kg/m^2^) was stratified into \<18.5 (underweight), 18.5--24.9 (normal), 25--29.9 (overweight), and 30 (obese), without modification for Asians \[[@OFU040C19]\]. Hepatitis C coinfection was defined as detectable hepatitis C virus (HCV) RNA. Smoking was stratified into ever, never, and smoking cessation during follow-up. xTC refers to ART containing either 3TC (lamivudine) or FTC (emtricitabine).
Statistical Analysis {#s2c}
--------------------
Categorical variables were compared with χ^2^ tests, and continuous variables were assessed with non-parametric methods (Kruskal-Wallis equality-of-populations rank test or tests for trend over calendar year, whichever was applicable). We performed bi- and multivariable linear regression analyses to investigate the effects of HIV transmission groups, age, CD4 nadir, smoking, HCV coinfection, and ART agents on BMI after ART initiation, presented as mean BMI changes (95% confidence intervals \[CIs\]). Antiretroviral agents are recorded with start and stop dates in the SHCS, and ART exposure was assessed at every follow-up visit.
Assumptions underlying these analyses were verified in scatter diagrams. In exploratory analyses, BMI trajectories after ART start were typically biphasic, with an initial rapid BMI gain corresponding approximately to the first year, and a smaller BMI gain during years 1--4 of ART. Therefore, subsequent detailed analyses were performed separately for years 0--1 and 1--4 of ART. Multivariable linear regression models for BMI change during year 1 were adjusted for BMI at ART start, HIV transmission group, age per 10 years, ethnicity (white, black, Asian, other), CD4 nadir (0--99, 10--199, 200--349, \>350 cells/µL), smoking (never, ever, smoking cessation), and ART regimen. During year 1--4 of ART, additional adjustments were for BMI after 1 year of ART and pegylated interferon treatment. Antiretroviral treatment exposure was considered in 3 ways: most frequent ART regimens (Table [2](#OFU040TB2){ref-type="table"}; [Supplementary](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1) Table [2](#OFU040TB2){ref-type="table"}), nucleoside reverse-transcriptase inhibitor (NRTI) backbones ([Supplementary Table 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)), and third agents ([Supplementary Table 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)), each defined as being administered during ≥50% of the respective time periods (years 0--1 and 1--4). Antiretroviral treatment regimens with \<50 person-years of exposure were summarized in an "other" category.
Based on multivariable linear regression analyses, we made standardized predictions of mean BMI changes \[kg/m^2^ per year\] for different HIV transmission categories (men who have sex with men \[MSM\], male heterosexual, female heterosexual), for different ethnicities (white, black, Asian), and different age groups (30--40, 41--50, 50+ years). For predictions, we fixed the remaining variables CD4 at 200--350 cells/µL, smoking status at never smoking, and baseline BMI at 22 kg/m^2^. Interactions between BMI changes and ART, transmission groups, and ethnicity were analyzed with a likelihood ratio test. We performed sensitivity analyses adjusting for calendar periods (2003--2005, 2006--2008, and 2009 and after). All analyses were done using Stata (version 13.1; StataCorp, College Station, TX).
RESULTS {#s3}
=======
Population BMI Trends, 1990--2012 {#s3a}
---------------------------------
The BMI distribution in the entire SHCS population stratified by calendar year is shown in Figure [1](#OFU040F1){ref-type="fig"} and [Supplementary Figure 1](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1), respectively. From 1990 to 2012, the percentage of normal weight and underweight participants decreased. Median BMI and the percentage of obese and overweight participants increased (Figure [1](#OFU040F1){ref-type="fig"}; [Supplementary](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1) Table [1](#OFU040TB1){ref-type="table"}). In 2012, the median BMI was 24 kg/m^2^ (interquartile range, 22--27), and 57% (95% CI, 56%--58%) of the population were normal weight, 4.3% (3.9--4.7) were underweight, 29% (28--30) were overweight, and 9.3% (8.6--9.9) were obese. From 1990 to 2012, the percentage of white participants (*P* \< .001), IDUs (*P* \< .001), and participants with AIDS-defining illnesses decreased (*P* \< .001). The percentage of women (*P* = .03), participants on ART (*P* \< .001), the median CD4 cell count at ART initiation (*P* \< .001), and the median age of participants increased (*P* \< .001).
BMI Change After ART Initiation {#s3b}
-------------------------------
From January 1, 1998 to December 31, 2012, 5415 SHCS participants with MSM or heterosexual HIV transmission started a first ART regimen. Stringent inclusion criteria resulted in a substantial reduction of participants ([Supplementary Figure 2](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)). Subsequent analyses are based on 1601 participants (Table [1](#OFU040TB1){ref-type="table"}; [Supplementary](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1) Table [2](#OFU040TB2){ref-type="table"}), 6250 person-years of follow-up, and 4803 BMI measurements. Median age at ART start was 40 years, 80% were white, and 19% were female. Body mass index change after ART initiation was biphasic; the mean BMI change during year 0--1 of ART was 0.92 kg/m^2^ per year (95% CI, .83--1.0), and during years 1--4 was 0.31 (0.29--0.34) kg/m^2^ per year. The mean interval between BMI determinations was 0.99 years (range, 0.43--1.5 years) between baseline and year 1 after ART start, and 2.9 years (1.7--4.2) between years 1 and 4.
Body Mass Index Change, Year 0--1 After ART Start {#s3c}
-------------------------------------------------
In the final, multivariable adjusted model, there was a trend towards larger BMI gain in heterosexual males compared with MSM. Compared with whites, annualized BMI change was not different in black and other ethnicities, but smaller in Asians (−0.79 \[95% CI, −1.2 to −.39\] kg/m^2^; *P* = .001). Body mass index change was associated with older age (0.15 \[0.06--0.24\] kg/m^2^ per 10 years older; *P* = .002). Compared to CD4 nadir \>350 cells/µL, a CD4 nadir of 0--99 and 100--199 cells/µL had the largest effect on BMI change (1.64 \[1.4--1.9\] kg/m^2^ per year; *P* \< .001 and 0.46 \[0.19--0.71\] kg/m^2^ per year; *P* = .001, respectively). Smoking was associated with a BMI decrease (−0.29 \[−0.47 to 0.11\] kg/m^2^ per year; *P* = .002). Body mass index change was similar with the ten most commonly prescribed ART regimens. Body mass index change was also similar for different NRTI backbones and third drugs, except for raltegravir, which was associated with a BMI change of 1.0 (\[0.17--1.8\] kg/m^2^ per year; *P* = .018), compared with efavirenz ([Supplementary Table 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)). These multivariable associations remained unchanged after adjustment for calendar period. No significant interactions were detected between BMI changes and ART, transmission groups, and ethnicity.
Body Mass Index Changes, Years 1--4 After ART Start {#s3d}
---------------------------------------------------
In the final, multivariable adjusted model, BMI change was similar in heterosexual males (0.02 \[95% CI, −.05 to 0.10\] kg/m^2^ per year; *P* = .53) and females (−0.03 \[−0.11 to 0.06\] kg/m^2^ per year; *P* = .52) compared with MSM. No age effect on BMI change was identified during years 1--4 (0.02 \[−0.01 to 0.05\] kg/m^2^ per year; *P* = .14). Body mass index gain was larger in black compared with white participants (0.28 \[0.16--0.37\] kg/m^2^ per year; *P* \< .001). Body mass index change was also associated with CD4 nadir 0--99 cells/µL (0.44 \[0.19--0.70\] kg/m^2^ per year; *P* = .001) compared to CD4 nadir \>350 cells/µL, with smoking cessation (0.16 \[0.04--0.27\] kg/m^2^ per year; *P* = .006) and with pegylated interferon (−0.71 \[−1.0 to −0.37\] kg/m^2^ per year; *P* \< .001). Compared to calendar period 1998--2002, BMI change was similar in 2003--2005 (*P* = .57), but it increased in 2006--2008 (0.12 \[0.03--0.21\] kg/m^2^; *P* = .008) and in 2009 and after (0.19 \[0.09--0.28\] kg/m^2^ per year; *P* \< .001). No significant interactions were detected between BMI changes and ART, transmission groups, and ethnicity. Body mass index change was similar for the 10 most commonly prescribed ART regimens (Table [2](#OFU040TB2){ref-type="table"}). As regards NRTIs and third agents ([Supplementary Table 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)), compared with tenofovir/xTC and efavirenz, zidovudine was associated with a smaller BMI increase, in combination with either efavirenz/xTC (−0.17 \[−0.29 to −0.05\] kg/m^2^ per year; *P* = .006) or with nelfinavir/xTC (−0.22 \[−0.43 to −0.01\] kg/m^2^ per year; *P* = .04). The NRTI backbones zidovudine/abacavir/xTC, zidovudine/xTC, and d4T/xTC were associated with a BMI decrease when comparing them with tenofovir/xTC (−0.20 \[−0.32 to −0.08\], 0−.20 \[−0.28 to −0.13\], and −0.35 \[−0.67 to −0.04\] kg/m^2^ per year, respectively). There was a larger BMI increase with atazanavir/ritonavir compared with efavirenz (*P* = .03) ([Supplementary Table 3](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1)).
The effects of transmission category, ethnicity, and age groups are further illustrated in Figure [2](#OFU040F2){ref-type="fig"}, which shows predicted BMI changes during years 0--1 and 1--4 of ART. The BMI increase was biphasic in white patients and black women, and the age effect was restricted to year 0--1 of ART. Fig. 2.Adjusted predicted annualized body mass index (BMI) changes during years 0--1 and years 1--4 of ART start. Separate predictions of BMI changes/year are presented for human immunodeficiency virus transmission category, gender, ethnicity, and age groups. Results are based on multivariable regression models adjusted for transmission category, ethnicity, age group, CD4 categories, and smoking status. For predictions, we fixed the remaining variables CD4 at 200--350, never smoking, and baseline BMI of 22 kg/m^2^. Abbreviations: hetero, heterosexual; MSM, men who have sex with men.
DISCUSSION {#s4}
==========
This study has 3 major findings; first, obesity rates in the SHCS have been increasing since 1995 in the setting of demographic changes, earlier ART start, and more widespread population ART coverage; second, BMI increase after ART initiation typically was biphasic, and the BMI increase in year 0--1 was as large as the increase in years 1--4 combined and was related most notably to the CD4 nadir; and third, we found little evidence that the individual ART regimen selected is a major driver of the extent of BMI change after ART start.
We describe a number of changes in demographics and in HIV management that may be partially responsible for the increasing obesity prevalence in the entire SHCS population over time, including an aging population, fewer IDU, initiation of ART at higher CD4 counts, and increasingly complete ART coverage. Injection drug use has been associated with lower BMI, more weight loss, and less weight gain after ART start, compared with other HIV+ persons \[[@OFU040C20]\], but not in all studies \[[@OFU040C11]\]. Before ART start, BMI typically declines with advanced HIV infection \[[@OFU040C21]\] and may predict survival and progression to AIDS \[[@OFU040C22]\]. In contrast, earlier ART start in recent years may prevent opportunistic complications, wasting, and the associated weight loss. Successful ART has produced an aging HIV+ population that is in much better health \[[@OFU040C23]\]. Advancing age is an important contributor to BMI in the general population \[[@OFU040C14], [@OFU040C15], [@OFU040C17], [@OFU040C24]--[@OFU040C26]\]. Increasing obesity rates have previously been recorded in HIV+ populations \[[@OFU040C11], [@OFU040C13], [@OFU040C23], [@OFU040C27]\], and the percentages of normal weight, underweight and overweight persons in the SHCS in 2012 was very similar to the percentages reported in the Swiss general population in 2012 \[[@OFU040C28]\].
Our study represents the most comprehensive assessment of longitudinal BMI change after ART start to date. In detailed analyses, we found BMI change after ART initiation to be a biphasic process that included a mean increase during the first year of ART that was as large as the mean increase during the 3 subsequent years combined. A biphasic BMI behavior has not previously been described; it likely represents a rapid re-gain of weight lost during untreated HIV infection \[[@OFU040C6], [@OFU040C10]\], perhaps similar to the return of plasma lipid levels to preinfection levels after ART start \[[@OFU040C29]\]. The biphasic BMI increase resembles the biphasic CD4+ lymphocyte increase after starting ART, which is initially rapid and followed by a second phase of slower CD4 increase \[[@OFU040C30]\]. We observed a biphasic BMI increase after ART start in white participants and in black women; there were limited numbers of patients of other ethnicities in our study and CIs were wide.
The largest contributor to BMI increase was the CD4 nadir. A larger degree of weight loss before ART that may occur with profound immunosuppression presumably correlates with a larger degree of weight re-gain after ART start. The BMI effect of a CD4 nadir \< 100 cells/µL persisted into years 1--4 of ART. An effect of the CD4 nadir on weight gain during the first \[[@OFU040C31]\] and the first 2 years \[[@OFU040C20]\] of ART has been previously reported. Contrary to our expectations, smoking and advancing age had no effect on BMI behavior during years 1--4 of ART, whereas smoking cessation was associated with larger BMI gain, consistent with data from the general population \[[@OFU040C32]\].
We found a limited contribution of the selected ART regimen to BMI change. Potential differences between individual drugs were not apparent when the BMI effect of commonly used ART combinations was examined during prolonged exposure (years 1--4). Our findings extend previous reports including a randomized study (422 participants; median follow-up, 5 years) in which median BMI increase did not differ with protease inhibitor (PI) vs non-NRTI (NNRTI) treatment, even though individual PIs and NNRTIs were not compared \[[@OFU040C33]\]. In AIDS Clinical Trials Group (ACTG) Study A5142 (753 participants), the median BMI increase did not differ between treatment arms at 96 weeks \[[@OFU040C34]\]. A5142 compared lopinavir/ritonavir vs efavirenz vs both, and stavudine vs tenofovir vs zidovudine. In ACTG A5224s, a larger mean BMI gain was noted at 96 weeks in patients randomized to atazanavir/ritonavir (*n* = 109) compared with efavirenz (*n* = 94) \[[@OFU040C35]\]. In an observational study of 681 patients, PI treatment was associated with greater weight gain than NNRTI treatment after 2 years of ART, but individual PIs and NNRTIs were not assessed \[[@OFU040C20]\].
Zidovudine-containing regimens were associated with smaller BMI increases in the SHCS compared with other regimens. This result is consistent with the well recorded, sustained effect of thymidine analogs on peripheral fat loss \[[@OFU040C7], [@OFU040C36], [@OFU040C37]\] and suggests that lipoatrophy may result in an attenuated BMI gain, as previously reported \[[@OFU040C10]\]. The increased BMI gain in our study in more recent calendar periods may partially be related to little use of thymidine analogs \[[@OFU040C9]\]. We note that 2 studies conducted during 1999--2002 and 1999--2003, respectively, reported no longitudinal BMI increase in HIV+ persons \[[@OFU040C38], [@OFU040C39]\]. Possible explanations for this include much less widespread ART use during the early ART era and, if ART was given, it typically included thymidine analogs.
Strengths of this study include longitudinal data collection in an established, large observational study. Due to the prospective design of the SHCS, we were able to control for important cofactors. Limitations include that information on diet and certain psychiatric comedications potentially associated with weight gain are not yet systematically captured; alcohol use and physical activity has been recorded since 2009 and was not considered in the present study. Moreover, the SHCS does not collect information on poverty, an important social determinant of BMI change.
In conclusion, increasing obesity rates in the SHCS population have occurred at the same time as trends towards restoration of health and normal aging in the setting of increasingly widespread population ART coverage. Ethnicity and immune suppression (CD4 nadir) had important effects on BMI behavior after ART start, but there was no clear evidence that frequently used ART regimens differ in their influence on BMI change during 4 years of follow-up. Similar to the general population, HIV+ patients should be motivated to consider a healthy lifestyle including diet, exercise, and behavioral modifications.
Author Contributions {#s5}
====================
P. E. T. and M. I. designed the study. B. L. analyzed the data. B. H. and M. I. wrote the first draft, and B. H., M. I., B. L., and P. E. T. wrote the final version of the manuscript. All investigators contributed to data collection and interpretation of the data, reviewed drafts of the manuscript, and approved the final manuscript.
Supplementary Data {#s6}
==================
[Supplementary material](http://ofid.oxfordjournals.org/lookup/suppl/doi:10.1093/ofid/ofu040/-/DC1) is available online at *Open Forum Infectious Diseases* (<http://OpenForumInfectiousDiseases.oxfordjournals.org/>).
###### Supplementary Data
We thank all involved physicians, study nurses, and, most importantly, participants of the SHCS. The members of the Swiss HIV Cohort Study are as follows: Aubert V, Barth J, Battegay M, Bernasconi E, Böni J, Bucher HC, Burton-Jeangros C, Calmy A, Cavassini M, Egger M, Elzi L, Fehr J, Fellay J, Furrer H (Chairman of the Clinical and Laboratory Committee), Fux CA, Gorgievski M, Günthard H (President of the SHCS), Haerry D (deputy of \"Positive Council\"), Hasse B, Hirsch HH, Hösli I, Kahlert C, Kaiser L, Keiser O, Klimkait T, Kouyos R, Kovari H, Ledergerber B, Martinetti G, Martinez de Tejada B, Metzner K, Müller N, Nadal D, Pantaleo G, Rauch A (Chairman of the Scientific Board), Regenass S, Rickenbach M (Head of Data Center), Rudin C (Chairman of the Mother & Child Substudy), Schöni-Affolter F, Schmid P, Schultze D, Schüpbach J, Speck R, Staehelin C, Tarr P, Telenti A, Trkola A, Vernazza P, Weber R, Yerly S.
***Financial support.*** This study was financed within the framework of the Swiss HIV Cohort Study (Study no. 627), supported by the Swiss National Science Foundation (grant no. 134277).
***Potential conflicts of interest.*** B. H. has received travel grants from Essex Chemicals, Wyeth, Astra Zeneca, and Janssen. B. L. has received travel grants, grants, or honoraria from Abbott, Aventis, Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Merck Sharp & Dohme, Roche, and Tibotec. E. B. has received travel grants, grants, and honoraria from Abbott, BMS, Boehringer Ingelheim, Gilead Sciences, Janssen, MSD, and ViiV. A. C. received travel grants from Boehringer Ingelheim and received unrestricted educational grants from Janssen Cilag, Gilead, MSD, ViiV, BMS, and AbbVie. P. E. T.′s institution has received advisory fees from MSD and honoraria from ViiV. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
[^1]: B. H. and M. I. contributed equally to this manuscript.
[^2]: Abbreviations: ART, antiretroviral therapy; HIV, human immunodeficiency virus; IQR, interquartile range; MSM, men who have sex with men; SD; standard deviation; SHCS, Swiss HIV Cohort Study.
[^3]: \* MSM are compared with heterosexual males and females.
[^4]: Abbreviations: ABC, abacavir; ART, antiretroviral therapy; ATV, atazanavir; AZT, zidovudine; BMI, body mass index; CI, confidence interval; EFV, efavirenz; HCV, hepatitis C virus; Het, heterosexual; LPV, lopinavir; NFV, nelfinavir; NRTI, nucleoside reverse-transcriptase inhibitor; NVP, nevirapine; Other, patients with no backbone or no third drug for 50% of the time or patients with 3 class regimens; Peginf, pegylated interferon; RAL, raltegravir; RTV, ritonavir; SHCS, Swiss HIV Cohort Study; TDF, tenofovir; xTC, lamivudine or emtricitabine.
[^5]: ^a^ All covariables of the bivariable model were adjusted for baseline BMI.
[^6]: ^b^ Multivariable models were adjusted for all variables listed.
[^7]: ^c^ All covariables of the bivariable model were adjusted for BMI after 1 year of ART.
[^8]: ^d^ Baseline BMI at start of ART and BMI after 1 year of ART, respectively.
[^9]: ^e^ Age at baseline (January 1, 1998 or at registration in the SHCS, whichever is the later) and after 1 year of ART.
[^10]: ^f^ Contains all ART regimens with \<50 person-years of exposure.
| {
"pile_set_name": "PubMed Central"
} |
Data-independent acquisition (DIA)[^1^](#G1){ref-type="fn"} is gaining more and more interest as a method for reliable and comprehensive label-free quantification (LFQ) of proteomics data and was already successfully applied in several clinical and biomarker discovery studies ([@B1][@B2][@B3]--[@B4]). So far, most liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) approaches are relying on data-dependent acquisition (DDA) in which typically the most abundant precursor ions of an MS1 survey scan are selected for fragmentation and acquisition of respective MS2 spectra ([@B5][@B6]--[@B7]). Thus, only selected precursors can be identified leading to a loss of information, especially for low abundant precursors ([@B8]). In addition, the stochastic nature of data-dependent precursor selection results in only partially reproducible LC-MS/MS data ([@B9], [@B10]). At the time of writing this manuscript, quantification of DDA data is usually achieved on the basis of MS1 chromatographic peak area, which is prone to interferences, especially in complex samples ([@B11], [@B12]). To reliably determine relative protein abundances in a label-free experiment, targeted approaches such as selected reaction monitoring (SRM, ([@B13])) or parallel reaction monitoring (PRM, ([@B14])) are being used. In SRM a predefined set of up to 100 precursors is chosen for fragmentation before acquisition, resulting in highly accurate quantification even at low abundance at the cost of the depth of the analyzed proteome ([@B5], [@B13]). Although DDA is commonly used for discovery proteomics, SRM/PRM can be applied for *e.g.* the verification of a predetermined set of proteins.
In contrast to DDA and SRM/PRM, in which only a subset of all precursor ions present in a complex sample is fragmented and analyzed in MS2, DIA methods allow the fragmentation of all possibly generated precursor ions by cycling through predefined *m*/*z* windows along the whole survey scan range ([@B15], [@B16]). Although this allows acquisition of nearly complete MS2 data, the direct correlation between precursor and its fragment ions is lost resulting in the need for more complex data analysis algorithms. Typically, spectral libraries, which are generated from previous DDA measurements, are used to infer the precursor peptide - fragment connection and thus allow peptide and protein identification ([@B17], [@B18]). In addition, spectral library-free approaches are being developed (*e.g.* DIA-Umpire and DirectDIA), but currently these cannot identify as many precursors as the spectral library-based approaches ([@B19], [@B20]). DIA allows quantification on MS2 level through extraction of fragment ion chromatograms, which are less prone to interference than MS1 peak areas. This makes the quantification more accurate and reliable than in DDA ([@B15], [@B21], [@B22]).
Although DIA was first described in 2004 ([@B23]) proteomics scientists show increasing interest in it for the past few years mainly because of vast improvements in instrumentation and software. Several studies revealed the advantages of DIA against DDA methods. Most of them demonstrated an improved peptide and protein identification rate using spectral library-based DIA (*e.g.* ([@B24], [@B25])) even in single shot analyses ([@B16]). In addition, a few studies showed an improved quantification reproducibility based on the coefficient of variation (CV) between technical replicates ([@B1], [@B26]). But more detailed analyses especially of quantification performance regarding specificity and accuracy of differential abundance detection are rarely performed because they rely on the use of suitably tailor-made samples.
Because DIA performance relies on the use of suitable spectral libraries different methods for the library generation were proposed and evaluated: Although Rosenberger *et al.* developed a generic large-scale human spectral library database ([@B27]), it was shown later on that the use of large external library repositories is inferior in terms of number of identifications ([@B28]). The in-house generation of project and sample-specific libraries benefits from the use of the same LC-MS setup as used in DIA measurements ([@B1], [@B28]). It is common to combine repeated DDA measurements of the same sample to maximize peptide coverage, but libraries can also be extended by sample pre-fractionation. For instance, Govaert *et al.* evaluated fractionation on protein, peptide and acquisition level and showed that all methods increased the library size, whereby protein fractionation using SDS-PAGE proved to be the most effective method ([@B29]). The quality of a spectral library often is assessed on the basis of library size (number of included precursors, peptides and proteins) and the number of yielded extractions from DIA data instead of the accuracy of the contained spectra. Just recently, methods to create libraries *in-silico* were developed ([@B30][@B31]--[@B32]). These take a suitable protein sequence database as input and computationally predict peptide fragments, which can be compiled into spectral libraries.
In this study, we exhaustively evaluated the quality of different spectral libraries regarding their identification and especially their quantification potential in DIA data analysis. Therefor we created a gold standard spike-in sample set consisting of C2C12 (immortalized mouse myoblast) cell lysate as constant background spiked with 13 proteins in five different concentrations. These standardized samples, although mimicking complex clinical samples with the proteins of interest present over a broad concentration range, allowed a detailed analysis of quantification reproducibility as well as specificity of and accuracy in differential abundance detection. The five samples were measured by DDA and DIA in triplicate, whereby the following approaches for spectral library usage were realized for DIA data analysis: On the one hand we used repeated DDA runs of in-solution digests and on the other hand prefractionation of samples on protein and peptide level to generate a range of different spectral libraries in Spectronaut 11 (SN, Biognosys AG, Schlieren, Switzerland) using the identification results of Proteome Discoverer 2.2 (PD, Thermo Fisher Scientific, Dreieich, Germany). In addition, we used Spectronaut Pulsar\'s protein identification algorithm for spectral library generation of two representative DDA data sets. These spectral library-using approaches were compared with the spectral library-free DirectDIA analysis by SN Pulsar, which has not been thoroughly described in literature so far, and an analysis of DDA data using OpenMS ([@B33], [@B34]). The generated data were thoroughly evaluated regarding peptide and protein identification and especially regarding quantification performance. In addition to CVs, we determined the significance of the induced spike-in abundancy changes and their correlation with the theoretical values. Furthermore, we analyzed the effectiveness to identify proteins that were differentially abundant (true positives) and the analysis specificity in terms of false positive rate. The focus of the presented analyses was the evaluation of the impact of differently generated spectral libraries on one DIA data set, not to benchmark different MS technologies or tools on the same sample respectively data set, as was for example done in ([@B35]). In the latter Navarro *et al.* benchmarked the quantitative performance of different mass spectrometers and analysis tools but used the same libraries for DIA extraction. The main part of this work was performed using data generated by Spectronaut. Nevertheless, we additionally analyzed the data set with OpenSWATH ([@B36]) and will highlight some of the results (for detailed results, see supplemental document).
Experimental Procedures
=======================
Sample Preparation
------------------
Frozen C2C12 cells were warmed up within a water bath (37 °C) and taken into culture in a 58 cm^2^ Petri dish (Sarstedt, Nümbrecht, Germany) within 10 ml DMEM (Gibco®, Thermo Fisher) standard medium containing 15% (v/v) FCS (Gibco®), 2% (v/v) sodium pyruvate (Biochrom, Berlin, Germany), 1% (v/v) non-essential amino acids (Biochrom) and 1% (v/v) penicillin/streptomycin (Pan Biotech, Aidenbach, Germany). The cells were cultivated in an incubator (37 °C, 5% CO~2~), the medium was changed every 2 days and the cells were split at a confluency of approx. 70%. For this, the cells were washed with 5 ml PBS (Gibco®), then detached with 1.5 ml 0.05% Trypsin/1 M EDTA (Gibco®) for 3 min inside the incubator and further the activity of trypsin was stopped by the addition of standard medium.
Before lysis, cells were pelleted by centrifugation at 16,000 × *g* for 10 min and then lysed in 30 m[m]{.smallcaps} TrisHCl, pH 8.5, 7 [m]{.smallcaps} urea and 2 [m]{.smallcaps} thiourea using glass beads and sonication (4 × 1 min on ice). After lysate transfer into a fresh tube, glass beads were washed with distilled water, the resulting solution was combined with the lysate (resulting in 5.3 [m]{.smallcaps} urea and 1.5 [m]{.smallcaps} thiourea concentrations) and cleared by centrifugation at 16,000 × *g* for 10 min.
As spike-in proteins we chose 13 non-mouse proteins with varying physico-chemical properties (see [supplemental Table S1](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for detailed information and UniProt accessions): human α-synuclein, β-lactoglobulin from bovine milk, fibrinogen α, β and γ from human plasma (Merck Millipore, Darmstadt, Germany), glucose oxidase from *Aspergillus niger*, human hemoglobin α and β, lipases 1, 2 and 3 from *Candida rugosa*, lysozyme from chicken egg white and myoglobin from equine skeletal muscle (Sigma-Aldrich, part of Merck KGaA, Darmstadt, Germany, unless otherwise stated). The fibrinogen α, β and γ were contained in the same solution, as were the hemoglobin α and β and the lipases 1, 2 and 3 respectively. Therefore, the relative amounts of these proteins are equal in all spike-in levels. The spike-ins were combined in a manner to yield in a comparable overall sample amount and physiologically plausible protein concentrations.
The gold standard spike-in sample set (GS) consisting of five samples was prepared as follows: a constant amount of C2C12 lysate as background matrix was spiked with varying amounts of the 13 spike-in proteins in 50 m[m]{.smallcaps} ammonium bicarbonate (AmBic) as specified in [Table I](#TI){ref-type="table"}.
###### Composition of the gold standard spike-in sample set consisting of a constant C2C12 cell lysate background and varying amounts of 13 non-mouse spike-in proteins. Shown are the pmol amounts of the spike-in proteins for each of the five sample states
UniProt accession Amount of spike-in proteins (pmol)
------------------------ ------------------------ ------------------------------------ ----- ----- ----- -----
α-synuclein (pmol) P37840 1 10 0.5 0.1 5
β-lactoglobulin (pmol) P02754 0.5 0.1 5 10 1
Fibrinogen (pmol) P02671, P02675, P02679 10 5 1 0.5 0.1
α, β, γ each
Glucose oxidase (pmol) P13006 0.1 1 10 5 0.5
Hemoglobin (pmol) P69905, P68871 0.5 5 10 1 0.1
α, β each
Lipase (pmol) P20261, P32946, P32947 0.1 0.5 1 5 10
1, 2, 3 each
Lysozyme (pmol) P00698 5 10 0.1 0.5 1
Myoglobin (pmol) P68082 1 0.1 5 10 0.5
C2C12 lysate (μg) 20 20 20 20 20
For spectral library generation (in-solution digest, protein fractionation and peptide fractionation) C2C12 lysate was mixed with equal amounts of the spike-in proteins (called master mix, MM, in the following) as specified in [supplemental Table S2](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1).
###
#### In-solution Tryptic Digestion
GS samples, MM for the in-solution and peptide fractionation library (sample composition as specified in [Table I](#TI){ref-type="table"} and [supplemental Table S2](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)) were prepared as follows.
After reduction with dithiothreitol (DTT, final concentration of 5 m[m]{.smallcaps}) for 20 min at 56 °C proteins were alkylated with iodoacetamide (13.75 m[m]{.smallcaps} final concentration) at ambient temperature for 30 min in the dark. Samples were diluted with 50 m[m]{.smallcaps} AmBic to an urea concentration \<1.5 M and digestion was carried out using trypsin (Serva, Heidelberg, Germany) at an enzyme to substrate ratio of approx. 1:27 at 37 °C overnight. The digestion was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 0.5%. After centrifugation the supernatant was collected, and the peptide concentration was determined by in-house amino acid analysis (AAA) ([@B37]). Before LC-MS/MS analysis was performed, the iRT kit provided by Biognosys, which is required for DIA analysis using Biognosys\' SN, was added according to the manufacturer\'s instructions. In brief, solubilized iRT peptides were diluted 1:10 in 0.1% TFA and 1 μl was added to each sample.
#### Protein Fractionation by SDS-PAGE and In-gel Tryptic Digestion
For protein fractionation by SDS-PAGE the MM sample (composition as specified in [supplemental Table S2](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)) was reduced and alkylated as described in In-solution Tryptic Digestion. Per lane, 40 μg were loaded onto an Invitrogen™ Bolt™ 4--12% Bis-Tris Plus Gel (Thermo Fisher Scientific) and proteins were separated at 200 V in MOPS buffer. After staining with SimplyBlue SafeStain (Thermo Fisher Scientific) each lane was cut into 10 slices, which were destained before subjection to overnight trypsin digestion using 0,12 μg trypsin per gel slice. Peptides were eluted twice using 0.1% TFA: acetonitrile (ACN) 1:1. The supernatants of each fraction were combined, vacuum dried and resuspended in 0.1% TFA 10 different fractions. Before LC-MS/MS analysis, 0.5 μl of solubilized iRT peptides were added to each sample.
#### Peptide Fractionation by High pH Reversed Phase Chromatography
Peptide fractionation was done using the "Pierce High pH Reversed-Phase Peptide Fractionation Kit" (Thermo Fisher Scientific) according to the manufacturer\'s manual. In brief, the peptide sample prepared as described in In-solution Tryptic Digestion was vacuum-dried, resuspended in 300 μl 0.1% TFA and loaded onto the equilibrated column. Peptides were eluted stepwise with increasing ACN concentration in 0.1% triethylamine into 8 fractions, that were vacuum-dried and resuspended in 0.1% TFA. Finally, peptide concentrations were determined by in-house AAA. Before LC-MS/MS analysis, 0.5 μl of solubilized iRT peptides were added to each sample.
Mass Spectrometric Acquisition
------------------------------
All samples (library samples, DDA and DIA analysis samples) were analyzed using the following LC-MS/MS setup: For LC separation the nanoHPLC system Ultimate 3000 (Thermo Fisher Scientific) was used with a PepMap 100 C18 (100 μm ID × 2 cm, particle size 5 μm, pore size 100 Å; Thermo Fisher Scientific) as precolumn and a PepMap C18 (75 μm × 50 cm, particle size 2 μm, pore size 100 Å; Thermo Fisher Scientific) as analytical column. Peptides were separated by a 120 min gradient using 0.1% formic acid (FA) as buffer A and 84% ACN in 0.1% FA as buffer B. The gradient was run from 5% to 40% buffer B. Subsequently, peptides were ionized by electrospray ionization and transferred into a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The capillary temperature was set to 250 °C and the spray voltage to 1600 V. The lock mass polydimethylcyclosiloxane (445.120 m/z) was used for internal recalibration.
For DDA MS runs (library generation and DDA analyses), the mass range of MS1 full scans was set to 350--1400 *m*/*z* with a resolution of 60,000 at 200 *m*/*z* (AGC 3 × 10^6^, 80 ms maximum injection time). HCD fragmentation of the Top10 abundant precursor ions was performed at 27% NCE. The fragment analysis (MS2) was performed with a resolution of 30,000 at 200 *m*/*z* (AGC 1 × 10^6^, 120 ms maximum injection time, 2.2 *m*/*z* isolation window).
For DIA MS runs, the MS1 full scans were performed at a mass range of 350--1400 *m*/*z* with a resolution of 120,000 at 200 *m*/*z* (AGC 3 × 10^6^, 20 ms maximum injection time). Fragment analysis (MS2) was subdivided into 22 DIA isolation windows of equal width (49 *m*/*z* wide) using a resolution of 30,000 at 200 *m*/*z* (AGC 3 × 10^6^, auto maximum injection time). Stepped collision energy was used (25.5%, 27 and 30% NCE).
Sample analyses, containing the spike-in proteins, in DDA and DIA mode were performed with 200 ng sample peptide amount (corresponding to ∼1 to 100 fmol spike-ins), whereas for library generation up to 800 ng of peptides per sample were injected.
The raw spectrometric data and the spectral libraries have been deposited to the ProteomeXchange Consortium (<http://proteomecentral.proteomexchange.org>) via the PRIDE partner repository ([@B38]) with the data set identifiers PXD012986, PXD012987, and PXD012988.
###
#### Spectral Library Generation
DDA data were searched with PD using Mascot 2.5 (Matrix Science Ltd, London, UK, ([@B39])) as peptide search engine and Percolator ([@B40]) for the refinement of target-decoy estimation.
For all searches, DIA and DDA, the following common settings were used- as fixed modification, only aarbamidomethylation at C was set, whereas as variable modifications oxidation (M), Gln-\>pyro-Glu (N-terminal Q), deamidated (NQ), ammonium (DE) and ammonia-loss (N, N-terminal C) were allowed because of sample preparation A maximum of two missed cleavages was allowed.The precursor tolerance was set to 5 ppm and the fragment tolerance to 20 mmu.The cleavage enzyme Trypsin (cleavage at each K and R, unless followed by P) was used.
For DDA identifications, a database consisting of the UniProt reference mouse proteome (release 2017_12, 52548 protein entries), the cRAP contaminants (unchanged since January 2015, 115 entries), the iRT protein for DIA retention time calibration and a database of the spike-in proteins was used. Additionally, to the 13 spiked-in proteins several proteins that were identified together with them in prior experiments (data not shown) were added to the database, containing altogether 160 protein accessions in the spike-in database (compare Statistical Analysis). Thus, the complete protein database used in this study contained 52,824 entries. The searches to create the spectral library were refined using Percolator, allowing only high confidence peptides in PD. Spectral libraries were generated using the spectral library generation function of SN using mostly the default settings. In brief, PD confidence level "high" was used for identification with SN protein inference enabled. Fragment ions between 300--1800 *m*/*z*, with minimum 3 amino acid length and minimum relative intensity of 5 were considered. Precursors with less than 3 fragment ions were removed. For the generation of SN Pulsar spectral libraries, DDA raw data were directly loaded into SN Pulsar and spectral library generation was done using the same settings as described for SN 11. An overview on all spectral libraries used in this work regarding library creation can be found in [Fig. 2](#F2){ref-type="fig"} and [Table II](#TII){ref-type="table"} ([supplemental Table S3](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) shows some additional information like preparation time), and regarding library content (their constituting precursors, peptides and protein groups) in [supplemental Table S5](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1). In total, eight different libraries are described here; whereby the most complex spectral library "IS and F all 78" was created from DDA data of the other 5 non-Pulsar libraries and 6 additional measurements of the "MM IS 6" sample.
###### Overview of analyzed DIA libraries. The names of each library consists of the spike-in types (MM: master mix, i.e. mixture of background and spike-ins in fixed amount, GS: gold standard, i.e. the same samples as measured by DIA), the digest type (IS: in-solution, F: fragmented and therefore in-gel), whether fractionation was performed and on which level (Prot: protein fraction, Pep: peptide fractionation), finally, the number represents the number of MS runs for the library. The libraries generated by Pulsar instead of ProteomeDiscoverer are indicated by the respective prefix
Name Spike-in type Digest type Fractionation No of runs Software
--------------------- --------------- ------------- --------------- ------------ -----------
MM IS 6 Constant In-solution No 6 PD+SN11
GS IS 15 Varying In-solution No 15 PD+SN11
GS IS 30 Varying In-solution No 30 PD+SN11
MM F Prot 20 Constant In-gel Protein 20 PD+SN11
MM F Pep 16 Constant In-solution Peptide 16 PD+SN11
IS and F all 78 Combined Combined Combined 78 PD+SN11
Pulsar GS IS 15 Varying In-solution No 15 SN Pulsar
Pulsar MM F Prot 20 Constant In-gel Protein 20 SN Pulsar
#### DDA Data Analysis Using OpenMS
DDA data were analyzed with a KNIME ([@B41]) workflow using OpenMS and PIA ([@B42], [@B43]) nodes (workflows are deposited inside PRIDE with the data). In brief, MS/MS spectra were first converted into centroided mzML using the vendor algorithm of msConvert (ProteoWizard version 3.0.10112 ([@B44])). Afterward, they were identified using the target-decoy approach with Mascot 2.5, MS-GF+ ([@B45]) and X!Tandem ([@B46]) and searches were combined using PIA, maintaining a peptide spectrum match and protein level false discovery rate of 1%. Peptide-features were detected and quantified using the FeatureFinderMultiplex. This algorithm uses the areas under the curves of the peptide isotope ion trails in the MS1 scans for the respective quantifications. The identifications were mapped to the features, aligned and normalized using the respective OpenMS nodes. Before the protein quantification using Top3 peptide abundancies, protein inference was conducted using PIA. The quantities for purely sequence based peptides was inferred from the quantities of peptides distinguishing different modifications and charge states by summing up the respective raw quantities, which is the default approach in OpenMS. The resulting peptide and protein quantifications were statistically analyzed as described below (see Statistical Analysis).
#### DIA Data Analysis Using Spectronaut 11 and Pulsar
DIA data were analyzed with SN 11 using the following settings. Calibration was set to non-linear iRT calibration with precision iRT enabled. Identification was performed using 1% q-value cutoff on precursor and protein level whereas the maximum number of decoys was set to a fraction of 0.5 of library size. The mass tolerance for matching precursor and fragment ions was set to dynamic (default), which lets SN determine the optimal value. For quantification interference correction was enabled with at least three fragment ions used per peptide, major and minor group quantities were set to mean peptide and mean precursor quantity, respectively with Top3 group selection each. Quantity was determined on MS2 level using area of XIC peaks with enabled cross run normalization. A complete description of all parameters can be found in [supplemental Table S4](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1).
In addition, DirectDIA was performed on the DIA data set with SN Pulsar using the same settings as described above.
#### Statistical Analysis
The quantitative data were exported from SN using an export schema, which allowed a statistical analysis for peptide and protein group relative abundancies. In the following we used all quantities, which had a valid value for at least one run, which is analogous to using the "sparse" setting in SN, unless stated otherwise. All data from the DIA and DDA measurements were analyzed by the same workflow using KNIME and R. The respective workflow can be found in the ProteomeXchange upload. On both levels, peptide and protein, the following analyses were conducted: first, missing values were imputed to a value of 0. The data were transformed using the inverse hyperbolic sine function (arcsinh), which has similar characteristics as the logarithm in the given numeric range but is defined for 0. Afterward, an analysis of variance (ANOVA) model was fitted to the transformed data. As a post-hoc test Tukey\'s honest significance test was conducted, to determine, which spike-in states were significantly differential. Finally, the ANOVA *p* values were corrected for multiple testing using the Benjamini-Hochberg procedure. To calculate the fold changes and log2 ratios between the spike-in states, the average quantities between the replicates for each state were calculated, leaving out the missing values instead of trying to impute them.
To account for impurities introduced by the spike-in protein solutions additionally to the 13 spike-in proteins 147 protein sequences were added to the protein database used for identifications. These proteins were identified by prior MS analyses of the spike-in protein solutions (data not shown), which were not highly purified. These "spike-in contaminants" are not expected to be found in all spike-in samples because of their low amount, but are nevertheless added to the protein database to allow an identification of the respective spectra. Thus, they are neither considered true positives (TP) nor false positives (FP) in the remaining analyses.
Besides these spike-in contaminants, there might be more peptides in the spike-in solutions, which could be detected and mapped to mouse proteins. To account for these FP, the abundancies of proteins, which are detected to be differential, were correlated to the known spike-in abundancies, using Pearson\'s correlation coefficient. The data were filtered if the correlation was higher than 0.9. Additionally, a fold change (FC) filter was applied, filtering out all candidates with relatively small FCs (FC \< 1.3).
In all analyses, unless stated otherwise, the TP are the spike-in proteins, which were correctly identified to be differentially abundant. FP are detected as differential but are none of the spike-in proteins. To calculate the FP rate of the differential proteins, the number of differential FPs was divided by all differential proteins, *i.e.* the number of TP, the spike-in contaminants (which are regarded as TP in this calculation, as they obviously are regulated) and the FP.
The accuracy of differential abundance detection was assessed based on the mean absolute percentage error (MAPE), which is a percental measure for the deviation of all spike-in ratios from the theoretical ones. The formula for the calculation is: $$MAPE = \frac{1}{n}{\sum\limits_{t = 1}^{n}\left| \frac{A_{t} - F_{t}}{A_{t}} \right|}$$ where *n* is the number of possible abundance ratios (ten ratios for the five different states), *A~t~* is the theoretical log2 value of the abundance ratio and *F~t~* the median of the measured log2 ratios for the respective states. This allows to compare the accuracy of the quantifications using one objective value.
To compare the SN results with results generated by another tool, we performed an analysis using OpenSWATH. The analysis followed the basic tutorial settings of OpenSWATH using Mascot search results, iProphet ([@B47]) for the combination of results, Mayu ([@B48]) for the FDR estimation and spectraST ([@B49]) for the actual library generation. The DIA data was analyzed by OpenSWATH and PyProphet ([@B50]) and the quantified features were aligned using TRIC ([@B51]). For more information on the commands and parameters, see the supplementary file. The same KNIME workflow as for the SN and OpenMS data was used for the final analyses.
Experimental Design and Statistical Rationale
---------------------------------------------
As described in the prior paragraphs a total of 78 LC-MS/MS runs were analyzed for the spectral library generation (compare [supplemental Table S3](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1), the number in the library names reflects the number of MS runs used for the creation of the library, the "MM IS 6" were additionally replicated with 800 ng and analyzed with the "IS and F all 78" library). For the DDA and DIA analysis, the described five spike-in states were measured in triplicates to reflect a common replicate number, resulting in 15 LC-MS/MS runs per method. For the retention time alignment of the DIA analyses, the iRT kit provided by Biognosis was applied, as described in Sample Preparation. MS1 and MS2 spectra were acquired for the DDA and DIA analyses as described in Mass Spectrometric Acquisition.
Results
=======
To thoroughly analyze the performance of different label-free quantification approaches, we generated a gold standard spike-in sample set (GS) consisting of C2C12 cell lysate as constant background and 13 non-mouse proteins each in five different concentrations (study design see [Fig. 1](#F1){ref-type="fig"}). By using spike-in proteins with varying physico-chemical properties, we were able to mimic a complex biological sample containing the proteins of interest---the proteins to be relatively quantified---over a broad concentration range. The individual spike-ins were combined in a way to keep the overall protein concentration constant between the five samples to facilitate good comparability---a prerequisite for successful normalization for relative quantification. We used this sample set to compare identification and quantification performance, on the one hand between DDA, DirectDIA, and spectral library-based DIA and on the other hand we evaluated the influence of different spectral libraries. Because the actual differentially abundant proteins (true positives, TPs)---the spiked-in non-mouse proteins---can clearly be distinguished from false positive (FP) background proteins, the quantification specificity can be evaluated in detail. Regarding relative quantification, the GS enabled us to compare the protein differential abundance ratios obtained by the various analyses with the theoretic values to estimate quantification accuracy.
{#F1}
Generation of the Gold Standard Spike-in Sample Set
---------------------------------------------------
For the creation of a sample set suited for the verification of quantification reproducibility, specificity and accuracy, we used whole mouse cell lysate as constant background, representing a complex biological sample, complemented with 13 purified non-mouse proteins in varying concentrations. In summary, six human, three animal and four fungal proteins with varying characteristics in terms of length, hydrophobicity and isoelectric point (pI) as well as with minimal tryptic peptide overlap with the C2C12 cell lysate were selected. Proteins consisting of more than one subunit (hemoglobin and fibrinogen) and the fungal lipases were present in sample mixtures, whereas all other proteins were present in separate solutions (see [Table I](#TI){ref-type="table"} and [supplemental Table S1](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for sample composition and spike-in protein details, respectively). Before preparation of the GS, each protein sample was characterized individually by LC-MS/MS verifying the presence of the 13 spike-in proteins. Beside these, 147 additional proteins were identified (in the following termed "spike-in contaminants," see supplemental FASTA file containing the spike-in protein sequences and the spike-in contaminants). The thirteen proteins result theoretically in 948 possible tryptic peptides (considering amino acid sequence lengths ranging from 6 to 50 and allowing up to two missed cleavages) ensuring enough statistical power for data analysis.
Altogether, the GS consisted of five different samples that contained the same amount of C2C12 lysate but varying concentrations of each spike-in protein. The individual spike-in amounts were shuffled to keep the overall protein concentration constant (see [Table I](#TI){ref-type="table"}). We used five physiologically plausible spike-in amounts of 1.25 to 125 n[m]{.smallcaps} per spike-in protein (as deduced from plasma protein concentration ranges ([@B52])) in a 0.27 μg/μl overall protein concentration, generating a broad range of protein ratios between 2 and 100. For LC-MS/MS analysis of the GS, each sample was measured in triplicate in DDA and DIA mode using the same instrumental setup (nano-LC coupled to Q-Exactive HF).
Spectral Libraries
------------------
For DIA data analysis of the GS, eight different spectral libraries were generated using (1) two different sample types, (2) varying sample preparation methods, and (3) two different search engines (see [Fig. 2](#F2){ref-type="fig"} for an overview). In addition to the samples of the GS, we used a protein master mix (MM) containing the spike-in proteins in equal amounts within the C2C12 matrix (1), thus ensuring all proteins of interest to be present for successful identification. Although the GS was used for in-solution (IS) digest followed by mass spectrometric acquisition only, the MM sample was also subjected to fractionation (F) on protein (Prot) or on peptide (Pep) level to create more complex libraries (2). For spectral library generation the individual IS and F samples were analyzed by LC-MS/MS in DDA mode. As the most comprehensive library, a combined library containing all DDA runs was generated (1+2). All the above described libraries were created using Proteome Discoverer (PD) identification with Mascot as search engine and Spectronaut (SN) for spectral library creation. To examine the influence of the search engine, two representative libraries were also generated using SN Pulsar (3).
{#F2}
The simplest spectral library generated with six DDA runs of IS digested MM sample ("MM IS 6") contained the least number of peptides and protein groups (23,020 peptides and 4046 protein groups, see [Fig. 3](#F3){ref-type="fig"}, for the complete numbers see [supplemental Table S5](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). This MM sample mimics a sample pool, which can be generated if the amount of each individual sample is too low for multiple MS analyses. We additionally created the "MM IS 6" library with 4-fold increased peptide concentration, but this resulted only in a marginal increase in library size (data not shown). In contrast, increasing the number of MS runs from 15 in the "GS IS 15" library (24,457 peptides and 4,126 protein groups) to 30 ("GS IS 30") resulted in a 25 and 13% larger library on peptide and protein level, respectively. Sample fractionation is often performed as a strategy to increase the spectral library depth ([@B29]). We performed fractionation on protein and peptide level ("MM F Prot 20" and "MM F Pep 16"), which further increased the spectral library size. Here protein fractionation yielded in higher number of peptides than fractionation on peptide level (41,322 peptides and 5859 protein groups in "MM F Prot 20" and 30,838 peptides and 5919 protein groups in "MM F Pep 16"). We observed an improvement of library size of up to 39% on peptide level by measuring each fraction twice instead of just once (data for single measurements not shown). As expected, combining all sample runs into one spectral library ("IS and F all 78") resulted in the largest library with 7718 protein groups and 71,008 peptides. We also observed an impact of the used spectrum search engine on library size. SN Pulsar, although using the same raw data, was able to extract considerably more peptides and proteins than the respective combined PD and SN approach (see [Fig. 3](#F3){ref-type="fig"} and [supplemental Table S5](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for actual numbers).
{#F3}
Peptide and Protein Identification
----------------------------------
To evaluate the performance of DIA for the analysis of the GS data set, we used different DIA approaches including spectral library-free "DirectDIA" and spectral library-based analyses using the different spectral libraries described above. These were compared with a standard DDA approach using OpenMS examining ten analyses in total. Initially, we assessed the number of identified peptides and protein groups. Overall, spectral library-based analyses resulted in higher numbers of identified protein groups compared with the DDA- and DirectDIA-based analyses. The latter led to the lowest number of identified protein groups with 2979 over all 15 analyzed samples (see [Fig. 3](#F3){ref-type="fig"}*B*, for the actual numbers see [supplemental Table S6](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). Among the spectral library-based analyses a library size dependent increase in the number of identified proteins and peptides was observed for the DIA analysis using libraries generated with the combined PD and SN approach from IS samples. In brief, the simplest IS library "MM IS 6" library extracted 3757 proteins and 22,397 peptides, whereas the "GS IS 15" library extracted 3938 proteins and 23,911 peptides. Including additional DDA runs within spectral library generation, as realized in the "GS IS 30" library, resulted in 4522 proteins and 30,086 peptides. As the results using the "MM IS 6" and the "GS IS 15" libraries are comparable, using a sample pool for library generation seems to be a suitable approach for DIA data analysis if *e.g.* only small amounts of sample are available. Surprisingly, although the fractionation libraries ("MM F Prot20" and "MM F Pep 16") were larger than the IS libraries ("MM IS 6," "GS IS 15," and "GS IS 30"), lower peptide and protein group identifications were observed (compare numbers in [supplemental Table S5 and S6](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). The highest number of peptides and protein groups was identified with the most complex library "IS and F all 78" (47,646 peptides and 5044 protein groups). In terms of identifications the use of Pulsar instead of PD+SN for spectral library generation proved to be beneficial, *e.g.* 63% more peptides and 15% more protein groups were extracted with the library "Pulsar MM F Prot 20" than with "MM F Prot 20." This must be cautioned though: even as it is possible to set the FDR filters in Pulsar, the analysis is rather a black box. The actual decoys and FDR calculations cannot be inspected, at least when using the Spectronaut implementation of Pulsar.
###
#### Library Recovery
Investigating the library recovery in more detail - which is defined as the number of peptides or protein groups actually identified from the GS data set compared with the respective library content - we observed a very high library recovery when using the IS libraries (over 95% peptide and 89% protein recovery, see [supplemental Fig. S1*A* +S1*B*](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). In contrast, significantly fewer peptides and proteins present in the fractionation libraries were identified in the GS data set (about 58--70%). This indicates that the IS libraries might not cover all measurable peptides from the GS sample. The fractionation libraries, especially "IS and F all 78" and "Pulsar MM F Prot 20," on the other hand included many unrecoverable peptide spectra.
#### Completeness
For DDA it is known that when measuring comprehensive sample sets, e.g. from a clinical study, many peptides are not recorded for all samples (so called missing values ([@B9])). Therefore, we evaluated the portion of consistently identified peptides and proteins, termed "completeness," within each analysis and compared those among all approaches (see [supplemental Fig. S1*C*](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). With DDA only 52% of the peptides and 65% of the protein groups were identified in all samples, whereas the DirectDIA approach provided 91% peptide and almost 99% protein completeness. In spectral library-based DIA analyses 82% to 91% of identified proteins were present in all 15 samples regardless which spectral library was used. However, on peptide level the completeness was considerably affected by the library choice. Among the PD+SN libraries the IS libraries yielded 80% to 83% completeness whereas in case of the fractionation libraries about 63% completeness was achieved. Although the peptide completeness in percent was quite low when using the most complex "IS and F all 78" library (56%), the actual number of consistently identified peptides was higher than with any other analysis. A similar effect was observed with the Pulsar spectral libraries. When comparing the Pulsar with the respective PD+SN libraries the peptide completeness dropped by about 10% whereas the Pulsar generated libraries yielded about 6700 more consistently identified peptides. As expected, peptides found in only one sample out of the 15 were mostly low abundant ones (data not shown).
In many analysis strategies, proteins or peptides must be present in at least a certain percentage of the analyzed runs to not be rejected because of too many missing values. Therefore, in [supplemental Table S8](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) we show the percentage of protein groups and peptides, which were found in all and at least 80%, 66%, and 50% of the runs. The data shows, that the percentages on the peptide level increase with decreasing required completeness but maintains the same trend. On protein level though, requiring an identification in only 80% of the runs already results in a completeness above 90% in all analyses, except for the DDA.
Besides the examination of peptide and protein completeness within each individual analysis, we further inspected the overlap of identified peptides and proteins between the ten different analyses (see [Fig. 4](#F4){ref-type="fig"}). In total 6787 peptides and 2258 proteins were identified by all methods, whereas 3976 peptides and 205 proteins were identified in one analysis only - in the following termed unique identifications (see [supplemental Table S6](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). The highest number of unique peptide identifications were achieved using the "IS and F all 78" (3,431 peptides) and the two Pulsar libraries ("Pulsar MM F Prot 20" 3976 and "Pulsar GS IS 15" 2652 peptides). These spectral libraries also identified the most unique proteins together with "GS IS 30". The abundance of the uniquely identified peptides and proteins was significantly lower than the abundance of all identified peptides and proteins (see [supplemental Fig. S2](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for abundance box plots of those analyses with the most unique identifications). This indicates that with DIA analyses using comprehensive spectral libraries ("GS IS 30," "IS and F all 78," "Pulsar GS IS 15," and "Pulsar MM F Prot 20") detection of low abundant proteins was improved.
{#F4}
To evaluate the identification of the 13 spike-in proteins we examined the number of runs in which the respective protein was identified ([supplemental Table S7](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). In total, all spike-in proteins were identified in each analysis. Individual proteins (three lipases, alpha-synuclein and myoglobin) could not be identified consistently in each run, except for the DirectDIA analysis, in which all proteins were identified throughout. This is because the DirectDIA algorithm matches and inspects all runs simultaneously for the identification of peptides and thus generally has a higher completeness than the other analyses. As expected, an abundancy-dependent identification pattern was observed. Alpha-synuclein was congruently not identified in the lowest spike-in concentration (0.1 pmol), except with DirectDIA, "GS IS 30" and DDA. The most heterogeneous identification pattern was observed for Lipase 2, which was identified in all 15 runs when using DirectDIA, but not consistently in the other analyses.
In summary, for peptide and protein identification we conclude that with spectral-library based analyses more identifications were achieved than with DDA or DirectDIA, even with the simplest library The larger the spectral library, the more peptide and protein identifications.Using SN Pulsar identifies yields in higher peptide and protein identification numbers.Pooling of samples for spectral library generation is a good alternative to the measurement of each individual sample.
Peptide and Protein Quantification
----------------------------------
The focus of this work was to compare quantification performance among the different analyses described in 3.2 and 3.3 in terms of reproducibility, specificity and accuracy.
###
#### Reproducibility
First, we evaluated the reproducibility between replicates based on global coefficient of variation (CV) on peptide and protein group level (see [Fig. 5](#F5){ref-type="fig"} and [supplemental Table S9](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). For the calculation of the CVs, the missing values were not imputed but simply left out. Imputing the values to 0 yields in much higher CVs and imputing to mean would lead to lower CVs (data not shown), whereas both alternatives would not reflect the actual data. In agreement with prior publications ([@B1], [@B26]) we found DDA showing much higher median CVs (29% on peptide and 24% on protein level) compared with DIA which ranged from 6% for DirectDIA to 10% for the "Pulsar GS IS 15" on peptide level, whereas the CVs on protein level were even lower (between 5 and 9%). In general, we observed a trend toward increasing CVs with increasing library size, which might be a result of detecting more low abundant species that naturally entail a higher variation. This was also true in case of the Pulsar libraries. Both contained more peptides and proteins than the respective libraries generated using PD+SN. An exception was the analysis using the simplest library "MM IS 6" that, although exhibiting the lowest protein CV (7%) among the spectral library-based analyses, resulted in mediocre peptide CV (8%). The difference between the PD+SN and Pulsar identification was particularly evident in case of the "GS IS 15" library, which resulted in quite low CVs on peptide and protein level (8 and 7% respectively), whereas the "Pulsar GS IS 15" analysis showed the highest peptide CV (10%) among the DIA analyses.
{#F5}
#### Specificity
Next, we evaluated the quantification data obtained by the different analyses regarding differential abundance detection specificity - *i.e.* the ability to differentiate between true positives (TPs, the spike-in proteins) and false positives (FPs, the mouse matrix). For this, a differential abundance test consisting of ANOVA with post hoc Tukey test was applied to the MS1 precursor peak areas for DDA and the MS2 extracted ion chromatogram peak areas for DIA data (details see Statistical Analysis). To maintain very high statistical significance, we filtered for a relatively strict corrected *p* value of 0.01. In all analyses spike-in proteins (TP) and C2C12 background proteins (FP) were found to be differentially abundant.
Of the 13 spike-in proteins, 9 to 12 were detected as differentially abundant in DDA or DIA analyses (see [Fig. 6](#F6){ref-type="fig"} and [supplemental Table S10](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). The maximum of 12 TP detections was achieved by "DirectDIA," "MM IS 6," "GS IS 15," "MM F Pep 16, and "Pulsar MM F Prot 20." It must be considered, that though we quantified all proteins with each method (see Peptide and Protein Identification) not all passed the *p* value threshold. The differential abundance of the TP proteins was detected with all analyses, except for the one of the three lipases, myoglobin and alpha-synuclein (see also [supplemental Table S11](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). In addition to the 13 TPs, we found spike-in contaminants to be differentially abundant (see [supplemental Table S10](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). Because these were present in the samples, they were not counted as FPs. The lowest number of TP proteins was found with the "IS and F all 78" analysis that identified only 9 spike-in proteins as differentially abundant. At the same time the highest number of TP peptides (163) was detected, which cover all 13 spike-in proteins. The same was observed with the other DIA analyses. This indicates that the applied approach of taking the mean of the top three peptide intensities to infer protein quantities is insufficient.
{#F6}
Moreover, we analyzed the misdetection (FP) of C2C12 mouse proteins as differentially abundant. Surprisingly, with DDA the lowest number of FP (15 protein groups and 172 peptides) was achieved whereas DirectDIA resulted in the highest number of FP, both on protein (151) and peptide level (730, see [Fig. 6](#F6){ref-type="fig"} and [supplemental Table S10](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1)). The library-based DIA analyses detected between 56 ("MM F Pep 16") and 121 ("Pulsar GS IS 15") FP protein groups, whereas the analyses using the fractionation libraries ("MM F Pep 16" and "MM F Prot 20") exhibited the lowest number of FP, both on protein and peptide level.
To investigate why we found much more FP with DIA than with DDA we had a closer look at the quantification data. We found that most FP could be classified into two categories: group I showed differential abundance trends very similar to the spike-in proteins (see [supplemental Fig. S3](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for an example). The peptides of these "correlating" proteins most likely originated from one of the spike-in solutions but the corresponding non-mouse proteins were not included in our database and have shared peptides with (probably homolog) mouse proteins. Even though we performed the initial check for spike-in contaminants (see Statistical Analysis), we used an MS with lower resolution for this than the one used for the DIA experiment. Therefore, we might have missed some of the contaminants in the preliminary checks. The second and larger group (II) encompassed proteins exhibiting a very small fold-change over the five samples with usually only one sample exhibiting a slightly higher or lower abundance than the other four. These "low fold change" protein hits are probably found to be significantly differentially abundant because of a lower CV of DIA compared with DDA and statistical effects emerging from the limited number of replicates. In DDA these were not found to have a significant differential abundance at *p* \< 0.01 (data not shown). Both groups were filtered out by A) a correlation analysis and B) requiring a minimal fold change of 1.3 (details see Statistical Analysis). After applying both filters to all analyses (DDA and DIA), the number of FP was reduced significantly in all DIA analyses, so that they now exhibited comparable values to DDA (see [Table III](#TIII){ref-type="table"}). On peptide and protein level the lowest FP rates were achieved using the "GS IS 15" and "GS IS 30" libraries for DIA analyses, whereas the highest FP rates were observed with "Pulsar MM F Prot 20" and "MM F Pep 16." The OpenSWATH analysis yielded in about the same numbers of TP and FP, both with the unfiltered and filtered approach. Therefore, a tool induced bias can be excluded.
###### False positive rate. Number of FP and resulting FP rate after correlation and low-fold change filtering on protein and peptide level
Protein level Peptide level
--------------------- --------------- --------------- ----- --------
DDA 5 20.00% 124 42.61%
DirectDIA 13 28.89% 118 35.22%
MM IS 6 8 25.00% 119 40.61%
GS IS 15 5 14.29% 123 34.36%
GS IS 30 6 16.22% 125 32.81%
MM F Prot 20 14 29.79% 167 43.04%
MM F Pep 16 12 30.77% 129 43.58%
IS and F all 78 14 28.57% 226 42.56%
Pulsar GS IS 15 12 26.67% 188 40.26%
Pulsar MM F Prot 20 14 30.43% 214 43.50%
To further analyze the specificity of differential abundance detection, ROC (receiver operator characteristics) curves, displaying the number of TP against the number of FP of a q-value sorted list, together with the respective AUC (area under the curve) were calculated (see [Table IV](#TIV){ref-type="table"}). The higher the AUC the better the specificity of the analysis, which means that the TP are detected with higher significance than the FP. We found specificities with AUCs ranging from 0.61 to 0.92 on peptide level and from 0.8. to 0.97 on protein level (where an AUC of 1.0 is the theoretically best specificity). The best specificities on protein level were achieved in DIA analyses using libraries with a high similarity to the actual samples: the "MM IS 6" (AUC: 0.97) and the "GS IS 15" (AUC: 0.95, see [supplemental Fig. S4](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1) for exemplary ROC curves) whereas "Pulsar MM F Prot 20" and "IS and F all 78" achieved almost equally good results (AUCs 0.94 and 0.93 respectively). DDA showed a medium specificity compared with all other analyses on protein level, whereas DIA with "MM F Pep 16" had the overall lowest specificity. "Pulsar MM F Prot 20" showed a very high specificity on protein level (AUC: 0.94), but not on peptide level (AUC: 0.63).
###### AUCs and mean absolute percentage errors. Table showing the AUC of the ROCs and the mean absolute percentage error (MAPE) of the respective method on the peptide and protein level. The MAPE was calculated between the median of achieved and expected protein/peptide fold changes, on log2 transformed ratios. The values can be interpreted as deviation between the achieved and expected ratio in percent
Peptide Protein
--------------------- --------- --------- ------ --------
DDA 0.61 65.20% 0.88 57.77%
DirectDIA 0.92 16.16% 0.87 21.67%
MM IS 6 0.85 18.45% 0.97 23.15%
GS IS 15 0.92 13.99% 0.95 22.84%
GS IS 30 0.88 17.08% 0.87 25.92%
MM F Prot 20 0.73 16.10% 0.82 21.41%
MM F Pep 16 0.68 15.39% 0.80 27.21%
IS and F all 78 0.63 15.12% 0.93 27.52%
Pulsar GS IS 15 0.76 13.78% 0.88 17.69%
Pulsar MM F Prot 20 0.63 12.91% 0.94 19.53%
#### Accuracy
Because the amount of spiked-in protein was known for every sample and each of the 13 spike-in proteins, the theoretical FCs can be compared with the relative quantification results of the DDA and DIA data analyses (see box plots in [Fig. 7](#F7){ref-type="fig"} and supplementary accuracy plots). The accuracy of differential abundance detection was assessed based on the mean absolute percentage error (MAPE, see 2.2.4). The MAPE varied greatly among the different analyses (see [Table IV](#TIV){ref-type="table"}). DDA, although able to detect 74% of all possible differential states showed the highest deviation from the actual abundance ratios of spike-in proteins based on the MAPE (see [Fig. 7](#F7){ref-type="fig"}*A* + [7](#F7){ref-type="fig"}*C*). The median FCs were more accurate with higher spike-in amounts, but higher deviations were observed with low spike-in amounts, especially for 0.1 pmol. For example, the fold-change of 10 was hit more accurate, when the spike-in amounts 10 pmol and 1 pmol were compared, whereas the ratios of 1 pmol to 0.1 pmol and 5 pmol to 0.5 pmol were less accurate. This trend was not observed in the DIA approach, indicating a higher accuracy and linear range - at least in the concentration range discussed here. The MAPE of all DIA analyses was much lower for all ratios and amounts, both on peptide and protein level, compared with DDA. The lowest deviation from the actual abundance ratios on peptide and protein level was observed with the Pulsar libraries (see [Fig. 7](#F7){ref-type="fig"}*B* + [7](#F7){ref-type="fig"}*D* and [Table IV](#TIV){ref-type="table"}) whereas "MM F Pep 16" and "IS and F all 78" exhibited the highest MAPE on protein level (supplementary accuracy plots). Besides the Pulsar libraries, DirectDIA and "MM F Prot 20" exhibited the best accuracies on protein level. In general, the accuracy on peptide level was better than on protein level.
{#F7}
In summary these quantification data show The larger the spectral library, the higher the CV on peptide and protein level---In DIA small FCs might entail the risk of more FP---Quantification performance of DIA is superior to DDA, especially in terms of reproducibility and accuracy---Quantification accuracy is decreased when considering low protein/peptide amounts in DDA, but not in DIA---Quantification on peptide level is preferable because of better differential abundance detection and higher accuracy---
#### Discussion
In this study, we compared the identification and especially the quantification characteristics of DDA and different DIA approaches including a spectral library-free DirectDIA analysis and various spectral library-based analyses. Among these, we used sample-specific libraries of differing complexity. As we performed the analysis with two independent software packages---Spectronaut and OpenSWATH---we could confirm, that the results were not specific for one tool. Even though both approaches yielded different numbers, the general trends were in the same directions. As our focus was the impact of different libraries, not the comparison of tools, we only give the numbers and values generated by the SN analysis in the following.
For a thorough comparison between the different analysis approaches we created the gold standard spike-in sample set (GS) mimicking characteristics of complex biological samples. The constant background of C2C12 cell lysate was spiked with physiological levels of 13 non-mouse proteins in varying ratios. The total spike-in amount was kept constant for all samples. The combination of the libraries and the GS sample set allowed a comprehensive in-depth evaluation of quantification performance of DIA in terms of reproducibility, specificity and accuracy.
Several studies already showed that spectral library-based DIA surpasses shotgun DDA in terms of number of identified peptides and proteins ([@B1], [@B53]) and has a significantly greater run-to-run identification overlap ([@B54], [@B55]). Here, we confirmed both points independent of the library complexity although especially peptide identification and consistency varied between the used spectral libraries. Using larger spectral libraries, either by combining many DDA runs ("GS IS 30") or different sample preparation strategies ("IS and F all 78") or by using a particular search engine (Pulsar), resulted in high numbers of extractions from DIA data (on average 4700). Smaller libraries of up to 15 repetitive DDA measurement of non-fractionated samples ("MM IS 6" or "GS IS 15" libraries) identified about 3800 protein groups. Sample fractionation for library creation on peptide or protein level, although enlarging library size, did not prove to be very beneficial in extracting DIA data. Fractionation on protein level (using the "MM F Prot 20" library) achieved more identifications than fractionation on peptide level ("MM F Pep 16"). Govaert *et al.* also observed that an increase in identifications from DIA data is not proportional to the increase in library size when sample prefractionation is used ([@B29]). The authors also showed that high-pH reverse phase peptide fractionation is less effective than SDS-PAGE protein fractionation in terms of number of identified peptides and proteins. Nonetheless, this technique is often applied in clinical studies to increase library depth (*e.g.* ([@B56], [@B57])). When looking at the identification of specific proteins - here the spike-in proteins - a higher identification consistency over all 15 DIA runs was achieved with libraries generated from DDA data of non-fractionated samples (especially "Pulsar GS IS 15," "GS IS 30," and "GS IS 15"). In summary, we recommend using libraries from repetitive DDA measurements of individual samples or a sample pool for protein identification. The use of a sample pool is of advantage especially if only small sample amounts are available. As DirectDIA shows similar identification numbers as DDA, this might also be an option in case of very limited sample amounts as it is often the case in clinical studies. To maximize identifications, complex libraries deriving from different sample preparation strategies should be used.
In quantitative proteome studies a high quantification reproducibility is of utmost importance. In terms of peptide and protein quantification, DIA outperforms DDA reproducibility in this study. A similar superiority of DIA compared with DDA was already described in other studies ([@B1], [@B16]). Because of the stochastic nature of data-dependent Top10 acquisition and a higher interference when using MS1 peak areas for quantification, the reproducibility of DDA-based analyses is limited in general.
In addition, quantification specificity---the ability to clearly differentiate between regulated and unregulated proteins---greatly influences the outcome of differential proteome studies. Our GS data set is especially suited to assess the specificity of each analysis method based on the number of true (TP) and false positives (FP) as well as good discrimination of these as deduced from ROCs. We found, that although OpenMS analysis of DDA data led to an acceptable high number of TP peptides and proteins and low number of FP, the discrimination of both on peptide level was difficult. This means that when quantification data are sorted by *p* value the real candidates would not necessarily be the first on the list; making manual inspection and subsequent verification indispensable. Even when applying a relatively strict *p* value cutoff of 0.01, which was corrected for multiple testing, DIA exhibits many FP. The application of a FC filter (here we applied 1.3) drastically reduced the number of FP to 4--19% of the unfiltered value. Obviously, the applied correlation filter cannot be used in real life analyses, as the differentially expressed proteins and their ratios are not known.
Interestingly, in DIA data analysis we found that the used spectral library also had a great effect on the quantification specificity although raw data were the same. Altogether, using small libraries ("MM IS" and "GS IS") resulted in the best discrimination between TP and FP both on peptide and protein level whereas the most complex libraries ("Pulsar MM F Prot 20" and "IS and F all 78") led to lower specificity especially on peptide level. Using sample fractionation or Pulsar for library creation does not seem beneficial but also not very disadvantageous. It was also found by Wu *et al.* ([@B28]) that libraries should not be excessively large to achieve good quantification specificity.
Quantification accuracy, describing how well the detected FC matches the theoretical FC, was much higher in all DIA analyses compared with DDA. Especially when considering low protein, respectively peptide amounts the DDA accuracy was affected, independent from the FC itself. Only small differences in accuracy were observed between the various DIA analyses ranging from 13--18% on peptide and 18--28% on protein level (DDA: 65 and 58%, respectively). Here, the use of Pulsar seemed to be slightly beneficial resulting in the highest accuracies both with "Pulsar MM F Prot 20" and "Pulsar GS IS 15." The most complex library "IS and F all 78" resulted in the lowest accuracy on protein level, whereas the smallest library "MM IS 6" exhibited the lowest accuracy on peptide level. These discrepancies between peptide and protein level quantifications point toward an insufficient protein inference. This becomes even more evident when inspecting the detection of TPs: whereas on protein level not all 13 spike-in proteins could be detected as differentially abundant, on peptide level all spike-in proteins could be successfully quantified. This highlights the importance of developing more accurate methods to infer protein quantities from peptide intensities.
Altogether, our analyses showed that DIA is superior to DDA. Using spectral libraries for DIA analysis always increases identification and quantification depth compared with DDA. When the aim of a study is only identification the use of large sample-specific libraries is beneficial, whereas for quantification smaller libraries perform better. The use of Pulsar seems to be a good compromise for both, identification and quantification. Nevertheless, its application in Spectronaut is debatable: the number of identifications and quantifications are raised substantially compared with other methods, but Pulsar has no publication of the underlying algorithms yet. Also, the FDR estimation in Pulsar is a black box in the used implementation, which makes deeper inspection and analysis of the correctness impossible. DirectDIA could be a good option when no libraries can be created, *e.g.* because of limited sample availability. In general, we recommend quantification and statistical analyses on peptide level for differential proteome studies.
DATA AVAILABILITY
=================
Data has been deposited to the ProteomeXchange repository (<https://www.ebi.ac.uk/pride/archive>) with identifiers PXD012986, PXD012987, PXD012988, and PXD014956.
Reviewer access data is provided in the respective document.
Supplementary Material
======================
###### Supplemental file 01
###### Supplemental file 02 - spike-in FASTA
###### Supplemental file 03 - additional plots
\* This work was funded by FoRUM Program of Ruhr University Bochum (F871--2016), by the de.NBI project (FKZ 031 A 534A) of the Bundesministerium für Bildung und Forschung (BMBF), PURE (Protein Unit for Research in Europe), a project of North Rhine-Westphalia, Germany, by the European Union H2020 project NISCI, GA no. 681094 and by the Internal Security Fund (ISF) co-financed by the European Union.
^^ This article contains [supplemental Figures and Tables](http://www.mcponline.org/cgi/content/full/RA119.001714/DC1). The authors declare that they have no conflicts of interest with the contents of this article.
^1^ The abbreviations used are:
DIAdata-independent acquisitionAAAamino acid analysisACNacetonitrileAGCautomated gain controlAmBicammonium bicarbonateANOVAanalysis of varianceAUCarea under the curveCVcoefficient of variationDDAdata-dependent acquisitionDTTdithiothreitolFAformic acidFCfold changeFPfalse positiveFfractionationGSgold standardHCDhigher-energy collision dissociationin-solin-solutionISin-solutionLC-MS/MSliquid chromatography coupled tandem mass spectrometryLFQlabel-free quantificationMAPEmean absolute percentage errorMMmaster mixNCEnormalized collision energyPDproteome discovererPeppeptideProtproteinROCreceiver operator characteristicSNspectronautSRMselected reaction monitoringTFAtrifluoroacetic acidTPtrue positive.
We thank Britta Eggers and Timo Sticher (Ruhr University Bochum, Medizinisches Proteom-Center, Germany) for the cultivation and provision of the analyzed C2C12 cells. Further, we thank Caroline May for the many discussions regarding DIA analyses and library generation.
[^1]: ¶ these authors contributed equally to the work.
[^2]: Author contributions: K.B., S.P., K.P., S.S., M.E., K.M., and J.U. performed research; K.B., S.P., K.P., K.M., and J.U. analyzed data; K.B., S.P., M.E., K.M., and J.U. wrote the paper; S.P., S.S., M.E., K.M., and J.U. designed research.
| {
"pile_set_name": "PubMed Central"
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INTRODUCTION {#SEC1}
============
Many bioinformatics workflows, especially those that process genomic and epigenomic next generation sequencing (NGS) data, produce expansive data sets in the form of genomic ranges defined by chromosome, start position and end position and can represent phenomena such as somatic mutations, copy number variations, DNA-- or RNA--protein binding sites and epigenetic state changes. The objective of Goldmine is to provide biologically-relevant annotation to genomic range sets and is motivated by two characteristics of such data. First, the range sets can be very large in size and require automated processing. For example, the number of peaks from a ChIP-seq experiment can range from the 100s to the 100 000s ([@B1]) and differentially methylated regions (DMRs) among human tissues can number in the 700 000s, even after stringent filtering criteria ([@B2]). Second, genomic ranges are not limited to gene bodies and can overlap with non-gene regulatory elements distal to genes, such as those established by large scale reference sequencing efforts ([@B1],[@B3],[@B4]). Analyzing how query genomic ranges from new studies relate to both known gene models and genomic features present in reference data can greatly facilitate hypothesis generation (Supplementary Figure S1A). Goldmine addresses the need to add interpretability, summarization and filtering to large sets of genomic ranges by annotating user-supplied genomic ranges with respect to known gene models and putative functional elements (Supplementary Figure S1B).
Existing tools for the analysis of genomic ranges fall into three categories. Goldmine belongs to a class of tools that provide detailed annotation of a query set of ranges to reference sets of ranges and gene models. Two existing tools with a similar concept are ChIPpeakAnno ([@B5]) and HOMER\'s annotatePeaks.pl ([@B6]). While these tools are ChIP-seq centric and provide nearest gene annotations, Goldmine is designed to accept genomic ranges from any source and also provides detailed feature annotation (Supplementary Table S1). A second category links genomic ranges to genes for the purposes of performing gene ontology enrichment and includes tools such as GREAT ([@B7]) and ChIP-ENRICH ([@B8]). Goldmine complements these tools by providing additional annotation of non-gene elements from reference data and can be used as a pre-filter to create query range sets to be provided to these other tools. For example, Goldmine could be used to stratify all query ranges that fall into known exons, and only these ranges are provided to GREAT for gene ontology analysis. The third category performs statistical enrichment calculations globally between a query set of ranges and a reference set of ranges to determine if range overlaps occur more than expected by chance. These tools include LOLA ([@B9]), GenometriCorr ([@B10]) and regioneR ([@B11]). Goldmine can work together with these existing tools for global enrichment calculations by providing a companion annotation that details the exact overlap for each individual range with combinations of reference set ranges, enabling the next level of candidate filtering and prioritization after an enrichment has been observed. Additionally, the range set enrichment tools can establish statistical significance if a frequent overlap is observed from manual inspection of a Goldmine annotation table. In summary, Goldmine provides detailed annotations and accountings of overlaps with both gene models and features, and automates complex tasks that would otherwise require manual download of data tables and custom programming.
MATERIALS AND METHODS {#SEC2}
=====================
Obtaining and caching reference genomic and epigenomic data {#SEC2-1}
-----------------------------------------------------------
In addition to user-supplied genomic ranges, Goldmine supports the direct loading into R of data from UCSC Genome Browser tables ([@B12],[@B13]). These tables (viewable at <https://genome.ucsc.edu/cgi-bin/hgTables>) contain the bulk of extant annotation available for most species with assembled genomes. In the case of the human genome, the *hg19* assembly contains data for multiple gene databases (including RefSeq, UCSC knownGene and ENSEMBL), non-coding RNA databases, the GWAS catalog, dbSNP, all data from the ENCODE project and various other feature sets including repeat elements, CpG islands and conserved elements. All tables can be loaded directly into R using the Goldmine function getUCSCTable(). The fread() function from the data.table package ([http://CRAN.R-project.org/package = data.table](http://CRAN.R-project.org/package%20=%20data.table)) is employed for memory-efficient storage of large tables. To conserve bandwidth, tables can be cached and stored in a local repository, and only re-downloaded if an updated version is available. Each version downloaded by Goldmine is named with a date stamp corresponding to the last modified date on UCSC\'s FTP server, and specific tables can be loaded by date stamp or synchronization can be disabled to ensure reproducibility of results. Otherwise, the latest available versions of tables are always obtained. Goldmine is not limited to using feature sets from UCSC, and any desired range set from other sources can be utilized. The input features list to Goldmine can be a user-generated list of GenomicRanges objects from any source. GenomicRanges objects can be created from BED files using the included makeGRanges() function.
Annotating sets of genomic ranges {#SEC2-2}
---------------------------------
Automated annotation of a set of genomic ranges is performed using the goldmine() function (Supplementary Figure S1B). Internally, functions and data structures from the GenomicRanges package are employed for fast overlap operations ([@B14]). Goldmine reports quantitative overlap results, enabling the user to filter the extent of overlap as desired for downstream analysis. For the analyses presented here, we have defined overlap as any overlap between a query range and a range from a reference set (1 bp or more). The functions getGenes() and getFeatures() can be used to customize the gene and feature sets used by goldmine() and allow simplified loading of commonly used tables. The goldmine() function reports two types of annotation tables. The 'wide' format has the same number of rows as the query set. The much more detailed 'long' format reports each pair of overlapping query range and gene/feature range as a row (analogous to an inner join in a relational database, keyed by range overlap). The 'wide' format provides an easy to view summary of contexts, where each query range is annotated with the percent overlap with each gene model component or feature set. These percentages can be used to divide query ranges into categories based on genomic and feature context. A simple category call is made based on the gene models automatically, and multiple overlaps are resolved using the priority order promoter \> gene 3′ end \> exon \> intron \> intergenic. By default, promoters are defined as −1000 bp to +500 bp of a transcription start site and gene 3′ ends are defined as 1000 bp flanks both upstream and downstream of a transcription end site. Both definitions are user-adjustable. The distance to nearest gene, genes directly overlapped by the range and the genes that generated the context call are also reported. The 'long' format is useful for viewing individual overlaps with certain features or gene isoforms in full detail, as it captures all the complexity of the overlaps that produced the percentages reported in the 'wide' format.
Annotation of ENCODE ChIP-seq peaks using goldmine {#SEC2-3}
--------------------------------------------------
The Goldmine function getFeatures() was used to obtain the 'wgEncodeRegTfbsClusteredV3' supertrack table. A copy of this data was generated and split into a list with one range set per factor using split(). The list of all sites was given as the query to goldmine(), and the split list was given as the features list. The context calls were aggregated to fractions of binding sites called by Goldmine in each context using data.table and plotted using ggplot2. Because this run annotated each binding site with the fraction of overlap with binding sites from each other factor, this output was also used to analyze co-occurrence biases on a per-context level. The feature annotation fraction columns were extracted and made into a matrix. The matrix was made Boolean by considering any overlap (fraction \> 0) as TRUE, and FALSE otherwise. The fractions of these overlaps were then aggregated within each context for pairwise combinations of each factor with each other factor. Fractions were computed as the number of sites where factor A sites overlap with factor B sites divided by the total number of factor A sites ([@B1]). Experiment-specific peak lists used to ascertain context changes for the same factor across individual cells and conditions were created by parsing the supertrack based on the index available in the 'wgEncodeRegTfbsClusteredInputsV3' table at the UCSC genome browser. Goldmine was then applied separately for the list of peaks from each cell line. Results were aggregated by cell line, factor and context using data.table and plotted using ggplot2.
DNA methylation sequencing data processing {#SEC2-4}
------------------------------------------
Methylated DNA immunoprecipitation sequencing (MeDIP-seq) read alignments in BED format were obtained from the Roadmap Epigenomics Projects under GEO accessions GSM543025, GSM613913, GSM669607, GSM543027, GSM613917 and GSM669609. The samples used are pairs of 'CD4, Naïve Primary Cells' and 'CD8, Naïve Primary Cells' from three human donors. The BED format alignments were converted to BAM format using the 'bamtobed' function of bedtools ([@B15]) and were sorted and indexed using samtools ([@B16]). The read depths for both cell types from one donor (TC009) were nearly twice as large as the other two donors, and these samples were downsampled to match the mean of the depth (57 349 008 reads) from the other samples using samtools.
Detection of differentially methylated regions (DMRs) {#SEC2-5}
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DMRs were detected using MethylAction ([@B17]). A window size of 50 bp and a fragment size of 266 bp were selected based on the protocol referenced in the GEO records. A subject-level effect was added to the testing model to account for the paired nature of the samples. Chromosome X and Y were excluded from the analysis. The methylaction() function was run with all other options as default, and the resulting DMR list was filtered to retain only those with ANODEV.padj \< 0.01 and \|log~2~(fold change)\| \> log~2~(1.5).
Annotation of DMRs {#SEC2-6}
------------------
The getGenes() function was used to obtain the ENSEMBL genes for *hg19* for annotation. The getFeatures() function was used to obtain the feature sets for annotation from the tables: 'wgEncodeRegDnaseClusteredV3', 'wgEncodeRegTfbsClusteredV3' and 'gwasCatalog'. These features were concatenated with the output from getCpgFeatures() resulting in one feature list with the genomic ranges each for ENCODE DNaseI hypersensitive sites, ENCODE ChIP-seq peaks, GWAS catalog SNPs and CpG islands/shores/shelves. The drawGenomePool() option was used to draw a length-matched genomic null set of regions to the DMRs, sampling 100 times more regions than the query. These ranges were concatenated with the filtered MethylAction DMR set. The goldmine() function was run using these gene and feature lists and the GenomicRanges object containing both the DMRs and null regions as the query ranges. Resulting annotated data was saved for viewing using the gmWrite() function. The frequencies of each DMR pattern and the null set for overlapping with each gene model or feature context were aggregated and plotted using the R packages data.table and ggplot2.
Curation of DMRs with known and potential functions {#SEC2-7}
---------------------------------------------------
The 'call_genes' column in the 'context.csv' file saved by Goldmine\'s gmWrite() function was used to search for DMRs in the promoters of known lineage factors for the CD4+ versus CD8+ fate decision. The 'genes.csv' file was filtered for rows with promoter fraction \> 0, and a list of all unique ENSEMBL gene IDs (ENSG numbers) was saved. This list was provided to GeneMANIA ([@B18]) with the setting of zero related genes and attributes. The gene ontology (GO) term enrichment table was saved and plotted using ggplot2 for all terms with FDR \< 15%. Plots of DMR regions were generated using ggbio ([@B19]) and the UCSC genome browser ([@B13]). ChromHMM and H3K27ac data for CD4+ and CD8+ T cells were obtained from the 'Roadmap Epigenomics Data Complete Collection at Wash U VizHub' track hub available from the UCSC browser.
Enrichment of consensus ChIP-seq peaks in DMRs {#SEC2-8}
----------------------------------------------
Enrichment was computed as the odds ratio between observed and expected frequencies of per-bp overlap rates between DMRs and all-methylated regions using a standard equation ([@B20]). The all-methylated regions were defined as the set of 50 bp windows with 4 or more reads in all 6 samples. These windows were overlapped with the genome-wide context GenomicRanges produced by the getGeneModels() function from Goldmine and categorized using the same priority order as Goldmine (promoter \> gene 3′ end \> exon \> intron \> intergenic). The 'wgEncodeRegTfbsClusteredV3' was obtained using getFeatures() and was converted into a list with one range set per factor using the split() function. For each DMR pattern and for each ChIP-seq factor, the fraction of bp in the DMR set that overlap with the given factor was compared to the same fraction in the all-methylated region set, and the odds ratio was calculated. Therefore, the background set used for each enrichment calculation is the set of all-methylated regions that fall in the same genomic context. This comparison is justified because promoters are compared to promoters, introns to introns and so forth. In other words, each enrichment is above that expected for any non-differentially methylated region in the same genomic context. Enrichments were considered significant if the lower bound of the 95% confidence interval (CI) of the odds ratio was \>1 and more than 5% of base pairs in the DMR set were covered by the factor\'s ChIP-seq binding site ranges. The lower bound of the 95% CI was plotted on the heatmap. Non-significant enrichments were excluded from the heatmap (white squares).
RESULTS {#SEC3}
=======
Using a reference gene database, Goldmine classifies genomic ranges as one of promoter, exon, intron, gene 3′ end, or intergenic (Figure [1A](#F1){ref-type="fig"}). To illustrate how this classification can capture biological information, we annotated a supertrack of cross-cell line ChIP-seq peaks from the ENCODE project. This analysis was enabled by Goldmine\'s capability to annotate any set of ranges with the percent overlap with any set of query ranges. The annotation revealed a spectrum of context-biased binding profiles (Figure [1B](#F1){ref-type="fig"} and Supplementary Table S2). We also found numerous examples where genomic contexts shift across cell lines and cell treatments for a given transcription factor (Supplementary Figure S2), and these suggest that the DNA-binding properties of such factors can be dynamic across biological conditions. For example, the binding sites of RE1-silencing transcription factor (REST) show a range of context biases from 15.5% promoter in H1-ESC cells to 50.5% promoter in HEPG2 cells, without a substantial change in total binding site number (Figure [1C](#F1){ref-type="fig"}). Because REST has known developmental roles ([@B21]), these shifts in context may reveal a balance between distal regulatory versus promoter regulatory functions throughout development ([@B22]).
{#F1}
DNA binding factors often function in complexes that can be revealed by ChIP-seq peak co-occurrence modules ([@B1]), and we identified multiple examples of co-binding preferences that are specific to certain genomic contexts using Goldmine (Supplementary Figure S3). A single command produced a co-occurrence matrix among all peaks by requiring a simple filter of \>1 bp overlap. Because Goldmine provides detailed raw annotation, users can select custom thresholds for filtering and analyzing overlaps. One example of a factor with context-specific co-binding preferences is CCCTC-Binding Factor (CTCF) (Figure [1D](#F1){ref-type="fig"}). The genomic context and binding partners of given CTCF sites can delineate among the multiple transcriptional and structural functions of the protein ([@B23]). Using Goldmine, we identified that co-binding occurs with transcription factors MAX, MYC and YY1 highly at promoters but at lower levels in all other genomic contexts, suggesting promoters may be co-regulated by these factors and CTCF acting as a transcription factor ([@B24]). CTCF can also function as an insulator element at the boundaries of topological domains ([@B25]), and in contrast to the aforementioned transcription factors, Goldmine detected a co-occurrence module with chromatin interaction regulators ZNF143 ([@B26]), RAD21 and SMC1 ([@B27]) relatively evenly across all genomic contexts. This demonstrates how Goldmine can help stratify a set of genomic ranges based on co-occurrence with factors associated with distinct functions. By providing a unified and simple tool for annotating any set of genomic ranges with respect to gene model contexts, Goldmine enables global insights into the dynamics of phenomena mappable using NGS.
To further demonstrate how Goldmine\'s annotation can facilitate biological and functional interpretation of a genomic range set, we derived DMRs between CD4+ and CD8+ T cells from Roadmap Epigenomic Project MeDIP-seq data using MethylAction ([@B17]) and annotated the results using Goldmine (Supplementary Table S3). As the CD4+ versus CD8+ lineage decision is a model for bivalent differentiation patterns ([@B28]), an analysis of this methylome-wide data can reveal how epigenetics interacts with both known and novel drivers of this developmental process. MethylAction provided as output the genomic ranges for 910 CD4+ hypermethylation DMRs and 1005 CD8+ hypermethylation DMRs (Figure [2A](#F2){ref-type="fig"}), and Goldmine annotation showed that the DMRs distributed in all genomic contexts (Figure [2B](#F2){ref-type="fig"}). While DNA methylation is commonly studied in a promoter-centric manner, the context analysis reveals widespread DNA methylation changes outside of the promoter. Such revelation can aid in context-specific hypothesis generation. For example, gene body DMRs may be associated with gene activation ([@B29]) or alternative splicing ([@B30]). Intergenic DMRs may target distal regulatory elements, such as enhancers and repressors, that could regulate many genes and be dynamic throughout developmental processes ([@B31]). Goldmine can also annotate input genomic ranges with reference feature ranges. Using this functionality on the T cell DMR data, a bias against CpG islands is evident (Figure [2C](#F2){ref-type="fig"}), and the DMRs are also enriched for overlap with ENCODE ChIP-seq and DNaseI-seq data (Figure [2D](#F2){ref-type="fig"}). Such feature annotation can be particularly useful for generating functional hypotheses for intergenic genomic ranges, as feature sets that capture regulatory elements and variation can be employed.
{#F2}
By sectioning gene models into components (Figure [2B](#F2){ref-type="fig"}), Goldmine reveals detailed information about the overlap of the DMRs with transcription units that could be missed by simply overlapping with gene bodies as single units. The annotation immediately revealed the presence of promoter hypermethylation at key lineage genes *CD4* ([@B32]) (Figure [2E](#F2){ref-type="fig"}) and *CD8A*, which corresponds to the expected expression patterns of these genes in the two T cell lineages. The promoter-overlapping gene list saved directly from Goldmine is enriched for gene ontology terms related to T cell receptor and immunity (Figure [2F](#F2){ref-type="fig"}). Additionally, Goldmine\'s 'long format' annotation provides a detailed accounting of the complex relationships between query regions and gene models by describing all isoforms, introns, exons, nested and overlapping genes. This demonstrates how Goldmine\'s gene model annotation streamlines and automates the process of deriving biologically relevant loci from a large set of anonymous genomic ranges.
In addition to summary annotations, Goldmine provides detailed descriptions for each pair of overlaps between query ranges and genomic feature sets. This is important because intergenic regulatory elements often involve multiple DNA binding factors with side-by-side binding sites. By filtering the DMR list to those annotated as intergenic and overlapping with both ChIP-seq and DNaseI-seq sites, we identified an intergenic DMR that correlates with a putative enhancer (Figure [2G](#F2){ref-type="fig"}). Goldmine\'s gene model and feature annotations can also function in tandem to provide information about regions that might otherwise be overlooked. We computed enrichment of CD4+ hypermethylation DMRs for consensus ChIP-seq peaks from all of ENCODE and found variable enrichment levels across gene model contexts (Figure [2H](#F2){ref-type="fig"}). When performing the analysis on all DMRs together, only 3 factors achieve statistical significance. However, when the analysis is stratified by context annotations, enrichments unique to each gene context are discovered. Of note, binding sites for RUNX3, a known master regulator in the CD4+ versus CD8+ fate decision ([@B33],[@B34]) were found to be enriched in promoters and introns. Additionally, *RUNX3* expression is known to be repressed in CD4+ cells, and Goldmine annotation identified *RUNX3* promoter to be hypermethylated in CD4+ cells (Supplementary Table S3). Taken together, these observations, made possible by Goldmine, suggest that *RUNX3* expression may be directly regulated by promoter methylation and that its transcriptional function may also be modulated by DNA methylation in CD8+ cells. The application of Goldmine to the DMR\'s between CD4+ and CD8+ T cells illustrates the usefulness of annotation to divide relevant subsets of genomic ranges into those of biological interest and to work in tandem with existing tools for motif and gene set enrichment analysis.
DISCUSSION {#SEC4}
==========
Compared with other tools for genomic range annotation, a key distinction of Goldmine is that it enables real-time synchronization with the latest annotation tables, so gene models can be used from the latest builds of reference databases. While this automation applies to any tables available from the UCSC Genome Browser, the user has complete flexibility to use any set of genomic ranges as a reference. Any ranges that can be input to R and stored as GenomicRanges ([@B14]) can be used, enabling Goldmine to annotate with respect to any reference range sets of interest that can be derived from existing Bioconductor ([@B35]) annotation packages or generated from external file formats such as BED files. In Figure [2G](#F2){ref-type="fig"}, we used the ChromHMM ([@B3]) calls derived from a BED file available from the Roadmap Epigenomics Project ([@B4]). Another unique feature is that Goldmine provides annotation on the level of transcripts in the format of a detailed table with a list of the specific introns and exons overlapped by a genomic range. To our knowledge, no existing tool provides this level of detail. Transcript-level annotation can be valuable, such as in the case of linking epigenetic phenomena to co-transcriptional RNA processing ([@B30],[@B36]). Additionally, RNA-centric techniques such as HITS-CLIP can map RNA--protein binding, and Goldmine can be a valuable tool to examine the diversity of transcripts produced by each peak event. While we note the limitation of this analysis is that the results are correlative, candidate selection is a requisite step before embarking on detailed experimental studies of novel mechanisms at specific loci. Goldmine should be considered as a tool to establish and prioritize these candidate sets. Moreover, Goldmine\'s detailed annotation complements existing genomic range analysis tools focused on global gene set and region set enrichment.
As demonstrated in Figure [2G](#F2){ref-type="fig"}, Goldmine is also distinguished by the fact that it is not limited to gene-centric annotation. In this example, Goldmine was used to identify an epigenetic change that directly overlaps with an annotated enhancer and correlates with the activity of this enhancer as derived from reference histone modification data in the relevant cell types. Not only can Goldmine detect individual loci with such specific overlaps of interest to the biologist, it can comprehensively catalog sites with desired overlap characteristics, whether they involve gene models or not. Thus, Goldmine is an information integration tool that can narrow large sets of genomic ranges into those that match specific configurations in relationship to reference data, and can be used to derive comprehensive lists of candidate loci for further experimental testing.
In summary, because Goldmine is designed to work for any set of genomic ranges, regardless of source or type, it is widely applicable to genomic ranges produced from genetic mutation data, RNA-focused assays such as Ribo-seq and HITS-CLIP, and many epigenome-wide sequencing data including DMRs, ChIP-seq peaks, differential histone modification/positioning, and DNaseI hypersensitive sites. Goldmine also simultaneously provides feature-level annotations that can be used to leverage recent epigenome-wide data sets, which are of particular utility in describing intergenic genomic ranges that may co-occur with functional elements. As a result, Goldmine reduces the complexity of the extant genomic and epigenomic annotation to aid in the prioritization of candidate loci for experimental testing.
Supplementary Material
======================
###### SUPPLEMENTARY DATA
The authors acknowledge the Roadmap Epigenomics Consortium (<http://nihroadmap.nih.gov/epigenomics/>) and the ENCODE Consortium (<https://www.encodeproject.org/>) for generating data used in this report.
SUPPLEMENTARY DATA {#SEC5}
==================
[Supplementary Data](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkw477/-/DC1) are available at NAR Online.
FUNDING {#SEC6}
=======
National Cancer Institute of the National Institutes of Health \[R01CA154356 to A.T., F31CA195887 to J.M.B\]; Scott Hamilton CARES Initiative; J.M.B. is a predoctoral student in the Molecular Medicine PhD Program of the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, funded in part by the 'Med into Grad' initiative of the Howard Hughes Medical Institute (HHMI). Funding for open access charge: National Cancer Institute \[R01CA154356\].
*Conflict of interest statement*. None declared.
| {
"pile_set_name": "PubMed Central"
} |
[^1]: **Session:** 235. Diagnostics - Diarrheal Disease
*Saturday, October 7, 2017: 12:30 PM*
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Methadone was developed in Germany in 1937 and introduced to the United States (US) in 1947 \[[@CR1]\]. It is a prescription opioid and is currently one of the primary options for the medication-assisted therapy of opioid use disorder \[[@CR2]\] and for alleviating chronic pain. However, it has a high abuse potential \[[@CR3]\]. Methadone has the highest rate of mortality in overdose of prescribed opioid analgesics in the US \[[@CR4]\] and is an important cause of opioid-related deaths in many other countries \[[@CR5]\]. In 2011, in the US alone, approximately 180,000 patients were reported to be on methadone maintenance therapy \[[@CR6]\]. Another survey in the US reported that the number of individuals receiving methadone increased from about 227,000 in 2003 to over 350,000 in 2015 \[[@CR7]\].
Along with the increased use of methadone comes an increased risk of side effects or adverse reactions related to overdose, such as rhabdomyolysis, sedation, sweating, respiratory depression, dizziness, nausea, dysrhythmias, vomiting, itching, constipation, orthostatic hypotension, prolongation of the QT interval, and death \[[@CR1]\]. Despite the large numbers of potentially serious sequelae of methadone use, few studies have evaluated trends in methadone toxicity in the US. Therefore, we aimed to evaluate the trends and patient-related factors associated with cases of methadone exposure seen in US hospitals by utilizing a well-established prospective clinical database.
Methods {#Sec2}
=======
We queried the Toxicology Investigators Consortium (ToxIC) Case Registry for all cases of methadone poisoning recorded between Jan 1, 2010, and December 31, 2017. The ToxIC Registry prospectively records cases cared for by participating medical toxicologists. It was designed to collect data by medical toxicologists and thus is felt to represent toxicologically accurate information. Members of the Consortium consist of all medical toxicologists from participating sites. Although this has varied from year to year, there are currently 49 sites participating in ToxIC, comprising the majority of US medical toxicology training programs and practices.
Patient data is entered into the ToxIC Registry via an online interface on which information is recorded on the substances involved, patient demographics, presenting signs and symptoms, toxidromes, treatments administered, and outcomes. The ToxIC Registry has been described in detail previously \[[@CR8]--[@CR10]\].
For the current study, we queried the ToxIC Registry for the following patient variables: gender, age, race, data pertaining to the cause of the exposure, agents involved, route of exposure (e.g. oral, parenteral), clinical manifestations, including toxidromes, abnormalities of vital signs, renal, cardiovascular, nervous system, gastrointestinal, metabolic, pulmonary, hematologic, muscle, and dermatologic effects; therapeutic interventions, including antidotes, medication treatment, decontamination, elimination techniques, and pharmacologic and non-pharmacologic support. Intentional methadone exposure was defined as any ingestion taken for therapeutic purposes, self-harm, or misuse/abuse.
Our inclusion criterion was any inpatient case in the ToxIC Registry where methadone was an implicated causal agent. Patients were excluded if the data related to age, sex, or cause of toxicological consultation was missing, if they were outpatients, or if they were seen for methadone withdrawal. Accidental ingestions, and those due to pediatric exploratory behavior, were classified as unintentional. All other cases were classified as intentional exposures. The analyses included all patients of any age meeting inclusion criteria who were registered during the study period.
The ToxIC project took place after review by the Western IRB and individual IRBs of ToxIC sites. All data in the ToxIC Registry is patient-deidentified and collected during routine clinical care. It does not involve any patient interventions.
Statistical and analytical methods {#Sec3}
----------------------------------
Descriptive statistics and relative frequencies plus graphical techniques were applied for investigating the patients' features. Data analysis was performed using SPSS version 19 and Prism software. Descriptive data, including frequency, percentage, mean, and standard deviation, were extracted and analyzed. Variables are reported as mean or median ± standard deviations.
Using the Chi-square test, we investigated the frequency distribution of clinical manifestations (coma, respiratory depression, seizure, etc.) in single- and co-exposure cases. Also, using this test, we examined and compared the frequency distribution of administered treatment patients with single- and co-exposures. For comparing the mean methadone dose consumed, after investigating the normality using the Kolmogorov-Smirnov test through the Mann-Whitney nonparametric test, we compared the methadone doses, QTc, and the effect rates in patients with single- or co-exposure to methadone. Also, the frequency distribution of clinical effects was reported based chronicity of use in all methadone patients.
Distribution of the total number of intentional and unintentional methadone poisoning cases reported to the ToxIC Registry 2010--2017 was assessed by the Chi-square test. *P* values of smaller than 0.05 were regarded as significant, although data were presented without regard to formal statistical significance.
Results {#Sec4}
=======
Nine hundred and seventy-three patients who met our inclusion criteria, with a mean age of 41.9 ± 16.6 years (range: 11 months-78 years), and a median age of 45.0 years were analyzed. Seven cases were excluded based on our exclusion criteria (6 were outpatients, and one patient had missing data). Eight hundred and thirty-one (86.0%) patients were 19--65 years old, and 28 (2.9%) were under the age of 2 years. Five hundred eighty-two (60.2%) were male.
Three hundred and fifty-four cases (36.4%) had methadone-only exposures, and 619 (63.6%) had had co-ingestants (Table [2](#Tab2){ref-type="table"}). The mean dose of methadone in all cases was 111 ± 122 mg (mg) (range: 3--800), with a median dose of 90 mg. In the methadone-only group, the mean methadone dose was 114 ± 129, with a median of 95 mg. For the group with co-ingestants, the mean methadone dose was104 ± 124, with a median of 80 mg. The mean methadone dose in patients who received naloxone was 112 ± 108, with a median of 91 mg. In patients who did not receive naloxone, the mean methadone dose was 110 ± 137, with a median of 90 mg. The Mann-Whitney test did not show any significant difference in the methadone dose between groups that did or did not receive naloxone (*p* = 0.18).
The route of exposure was known in 437 (44.9%) patients. Of these, 420 (44.8%) patients consumed methadone orally, and 17 (1.8%) used a parenteral route. The chronicity of exposure, known in 607 patients, was acute in 411 (67.7%) patients, acute on chronic in 136 (14.5%), and chronic in 60 (6.4%) patients.
Six hundred and fifty-two (67.6%) patients had been referred to the medical toxicology service by the emergency department, 120 (12.5%) by the admitting service, 86 (8.9%) by another hospital service, and 66 (6.8%) were transferred from outside hospitals (Table [1](#Tab1){ref-type="table"}). The in-hospital mortality rate of methadone poisoning was 1.4% (14 patients). Table 1Frequency of sources of referral to medical toxicology services and demographic informationVariableFrequency /meanPercent /SD**Age (**year)41.916.6**Dose** (milligram)111.34121.78**Source of referral** Emergency department (ED)65267.5 Admitting Service12012.4 Outside Hospital Transfer666.8 Poison center121.2 Request from another hospital service868.9 PCP or other Outpatient Treating MD141.4 Self-Referral10.1 Unknown151.5**Gender** Male58460.0 Female38940.0**Role of medical toxicologist and location of toxicology consultation** Attending (Inpatient)17919.9 Consult (ED/Inpatient)75780.1**Chronicity of exposure** Acute41167.7 Acute on chronic13614.5 Chronic606.4**Reason for methadone use** Withdrawal management515.2 Abuse20921.4 Attempt at self-harm50852.2
Seven hundred and sixty-eight (79.5%) patients had intentional methadone exposures. Of these, 51 (6.6%), 209 (27.3%), and 508 (66.1%) were due to avoidance of withdrawal, drug abuse, and attempts at self-harm, respectively. As shown in Fig. [1](#Fig1){ref-type="fig"}, the highest rate of methadone poisoning was observed in 2013. There was an increasing rate of methadone exposures in 2010--2013, followed by a decline in 2014--2017. The highest number of intentional methadone poisoning cases was clearly increasing in 2010 and peaked in 2013--2014 with 137 and 136 cases, respectively. After 2014, there was a decline. The highest frequency of unintentional methadone poisoning was reported in 2011 (44 patients). Fig. 1Distribution of the total number of methadone poisoning cases reported to the ToxIC Registry 2010--2017
Chi-square testing comparing the different years during our study period indicated that the causes of methadone toxicity significantly varied with time (X2 = 295.81, *p* \< 0.001). The highest percent of methadone cases reported to the ToxIC Registry relative to the overall number of cases reported to the Registry for each year was obtained in 2013 (n: 169 \[17.5% of all methadone cases reported\]), and the lowest rate was in 2015 (n: 77 \[7.9%\] \[Fig. [2](#Fig2){ref-type="fig"}\]). As is evident in Fig. [2](#Fig2){ref-type="fig"}, the frequency distribution of methadone poisoning cases in 2015 had a significant difference from the years 2011, 2012, 2013, 2014 (*p* \< 0.001). The frequency distribution of methadone poisoning cases in 2017 had a significant difference from the years of 2011, 2012, 2013, 2014 (*p* \< 0.001). Frequency distribution of methadone poisoning cases in 2010, as well as 2016, had a significant difference from the years of 2011, 2012, 2013, 2014 (*p* \< 0.001). Tables [2](#Tab2){ref-type="table"} and [3](#Tab3){ref-type="table"} show the distribution of clinical effects and mean doses associated with various clinical manifestations for single-agent and co-ingestant exposure to methadone. In patients with co-ingestants, 88 (14.3%) co-ingested sedative-hypnotics, 35 (3.6%) oxycodone, 27 (2.8%) heroin, and 353 (34.0%) other substances. Fig. 2The percent of all methadone cases/total cases to the ToxIC Registry by year. Frequency distribution of methadone poisoning cases in 2015 had a significant difference with years of 2011, 2012, 2013, 2014 (*p* \< 0.001). Frequency distribution of methadone poisoning cases in 2017 had a significant difference with years of 2011, 2012, 2013, 2014 (*p* \< 0.001). Frequency distribution of methadone poisoning cases in the 2010 as well as 2016 had a significant difference with years of 2011, 2012, 2013, 2014 (*p* \< 0.001).Table 2The percent of clinical effects in methadone alone poisoning and co-ingestionVariableTotalSingle exposure of methadone (*n* = 354)Co exposure of Methadone with other drugs (*n* = 619)Coma/CNS472 (48.6%)354 (100%)118 (19.1%)Agitation69 (7.1%)39 (11.1%)30 (4.84%)Seizures21 (2.2%)13 (3.67%)8 (1.29%)Weakness6 (0.6%)2 (0.56%)4 (0.64%)pH \< 757 (5.8%)43 (12/0%)14 (2.26%)Bradycardia41 (4.2%)26 (7.28%)15 (2.42%)Hypertension34 (3.5%)24 (6.72%)10 (16.1%)Hypotension38 (3.9%)28 (7.91%)10 (16.1%)Tachycardia22 (2.3%)13 (3.67%)9 (1.45%)QTc \> 500 milliseconds24 (2.9%)20 (5.64%)4 (0.64%)Acute Kidney injury92 (9.5%)70 (19.7%)22 (3.55%)Rhabdomyolysis43 (4.4%)35 (9.88%)8 (1.29%)Aspiration pneumonitis53 (5.4%)44 (12.4%)9 (1.45%)Respiratory depression327 (33.6%)251 (70.9%)76 (12.3%)Hepatotoxicity (AST \> 1000)36 (3.7%)28 (7.91%)8 (1.29%)Values are frequency and percentageThe percentages in the methadone only and methadone plus coingestants are calculated in each group*Abbreviations*: *AST* aspartate aminotransferase, *CNS* central nervous systemTable 3Comparison of methadone doses and effect rates in patients with single or co-ingestant exposure of methadoneComplicationsDose of methadone in methadone alone (mg)Dose of methadone in methadone plus other drugs (mg)Coma/Central nervous system (CNS) depressionYes96.2 \[56.8--125.0\]97.2 \[72.3--136.0\]No65.9 \[38.6--84.1\]124.3 \[100.1--168.7\]AgitationYes132.5 \[98.3--140.7\]119.3 \[108.9--127.1\]No125.0 \[96.2--156.4\]37.5 \[29.8--65.3\]No66.6 \[54.8--89.2\]121.3 \[95.6--163.8\]SeizuresYes138.2 \[96.5--165.1\]136.9 \[103.6--172.3\]No145.2 \[100.2--189.0\]120.1 \[89.6--160.3\]WeaknessYes43.7 \[35.6--57.6\]43.6 \[32.1--68.9\]No14.2 \[9.6--25.6\]119.6 \[100.0--156.3\]pH \< 7Yes40.2 \[24.3--50.8\]45 ± 24.2No69.8 \[57.3--99.2\]124.3 \[100.6--154.9\]BradycardiaYes42.3 \[36.8--60.2\]64.5 \[49.6--87.0\]No38.6 \[30.1--45.7\]119.9 \[96.3--157.6\]HypertensionYes40.2 \[32.3--50.9\]126.3 \[100.5--160.0\]No39.6 \[28.9--61.2\]120.3 \[95.6--156.8\]HypotensionYes32.0 \[25.3--41.9\]26.3 \[14.6--38.9\]No40.9 \[32.9--53.6\]126.5 \[105.3--153.6\]TachycardiaYes74.9 \[62.3--90.4\]147.1 \[125.3--175.1\]No36.0 \[28.9--51.4\]123.6 \[98.0--168.6\]QTc \> 500 millisecondsYes74.2 \[52.8--88.6\]32.1 \[29.6--36.5\]No34.5 \[28.6--58.9\]128.7 \[106.5--150.6\]Acute Kidney injuryYes42.5 \[14.8--65.3\]16.8 \[9.8--26.1\]No117.3 \[84.0--140.4\]102.3 \[86.7--123.9\]RhabdomyolysisYes37.5 \[29.7--50.0\]42.3 \[19.0--57.8\]No43.8 \[34.6--59.9\]124.3 \[97.8--150.1\]Aspiration pneumonitisYes24.6 \[16.7--36.8\]37.8 \[26.3--49.0\]No28.6 \[14.6--34.5\]124.8 \[100.1--148.6\]Respiratory depressionYes80.3 \[60.3--100.2\]46.6 \[34.8--67.2\]No128.7 \[100.2--168.7\]108.9 \[85.3--125.6\]Hepatotoxicity (AST \> 1000)Yes45.2 \[25.6--59.9\]43.2 \[29.7--60.0\]No62.4 \[48.7--81.0\]122.3 \[99.3--145.6\]Dose values are mean ± Standard deviation\*: Mann-Whitney testz = test statistics Mann-Whitney
The median (25th%-75th%) QTc in patients with methadone poisoning was 446.0 \[430.0--480.0\] milliseconds. The median (25th%-75th%) QTc in single-exposure and co-exposure methadone patients were 449.9 \[438.0--462.9\] and 435.2 \[389.9--480.0\] milliseconds, respectively (*p* = 0.28).
The most common clinical manifestations in methadone poisoned patients were coma (48.6%) and respiratory depression (33.6%) (Table [2](#Tab2){ref-type="table"}), occurring at mean doses of 96.2 \[56.8--125.0\] and 37.5 \[29.7--50.0\] mg, respectively. Two percent of patients experienced seizures. The median dose of methadone in patients with seizures who ingested methadone alone was 138.2 \[96.5--165.1\] mg, similar to the dose of methadone in patients with seizures in the co-ingestion group, which was 136.9 \[103.6--172.3\] mg (Table [3](#Tab3){ref-type="table"}). The median (25th%-75th %) QTc in patients with and without seizure was 453.0 \[438.0--481.0\] and 441.0 \[401.0--495.0\] milliseconds respectively (*p* = 0.49).
Table [4](#Tab4){ref-type="table"} shows the frequency of treatments, stratified by single and polydrug methadone poisoning cases. Among all patients, naloxone was the most commonly used antidote. Four hundred and forty-two patients (45.4%) received naloxone. Benzodiazepines, the second most frequently administered class of agents, were given to 119 (12.3%) patients. Of those receiving benzodiazepines, 20 patients had seizures, 32 patients had agitation, 46 had withdrawal, and the reason for receiving benzodiazepine in 21 patients was unknown. Table 4Comparison of administered treatments in patients with single and co-ingestant exposures to methadoneVariableTotalSingle exposure of methadone (*n* = 354)Co exposure of Methadone with other drugs (*n* = 619)NaloxoneYes442 (45.4%)125 (35.3%)317 (51.2%)No531 (54.6%)232 (65.5%)299 (48.3%)N-acetylcysteineYes40 (4.2%)27 (7.6%)13 (2.1%)No933 (95.8%)330 (93.2%)603 (97.4%)FlumazenilYes22 (2.3%)9 (2.5%)13 (2.1%)No951 (97.7%)348 (98.3%)603 (97.4%)Sodium bicarbonateYes21 (2.2%)13 (2.77%)8 (1.29%)No952 (97.8%)344 (97.2%)608 (98.2%)VasopressorsYes34 (3.5%)26 (7.34%)8 (1.29%)No939 (96.5%)331 (93.5%)608 (98.2%)AnticonvulsantsYes13 (1.4%)8 (2.3%)5 (0.81%)No960 (98.6%)349 (98.6%)611 (98.7%)AntipsychoticsYes25 (2.6%)10 (2.8%)15 (2.42%)No948 (97.4%)347 (98.2%)601 (97.1%)BenzodiazepineYes119 (12.3%)66 (18.6%)53 (8.56%)No854 (87.7%)291 (81.5%)563 (90.9%)OpioidsYes38 (4.0%)27 (8.22%)11 (1.77%)No935 (96.0%)330 (93.2%)605 (97.7%)
Sixty-six percent of patients who experienced coma had acute poisoning, while 47.8% had acute on chronic poisoning. Of patients who experienced respiratory depression, 49.4% had acute poisoning, while 66.9% had acute on chronic poisoning, and 5% had chronic poisoning. (Table [1](#MOESM1){ref-type="media"} in supplemental data). Three-hundred and eight (69.8%) patients with coma and 226 (51.3%) patients with respiratory depression received naloxone (Table [2](#MOESM1){ref-type="media"} in supplemental data).
Discussion {#Sec5}
==========
Methadone has been used therapeutically to alleviate pain in patients with chronic disease and to reduce and control withdrawal syndrome in patients who suffer opioid dependency syndrome in methadone maintenance treatment (MMT) clinics \[[@CR11]\]. It has a high potential for abuse and may be used illicitly by opioid-dependent patients \[[@CR11]\].
Methadone is well known for its long duration of action and potential for fatality in overdose. This places significant health care and economic burdens on society, especially where death occurs. Mortality costs attributed to methadone accounted for approximately 6.5 million dollars in 2009 in the US \[[@CR12]\]. Other studies showed that patients who receive methadone to alleviate chronic pain in pain clinics are at higher risk of mortality \[[@CR11]\]. Patients who have chronic pain tend to be older individuals in poorer health who may be receiving multiple medications and experiencing high levels of depression and anxiety. Methadone may be abused by individuals with opioid misuse disorder, which increases its risks \[[@CR11]\]. Our study demonstrated that rates of methadone toxicity in the US, as reflected in the ToxIC database, appeared to increase until 2013--2014, after which there was a decline. Since the ToxIC database is a reflection of cases for which medical toxicology consultation was required, it is likely that more trivial cases are not included. Thus, these data should be interpreted as reflecting significant poisonings.
Another study with different study period has shown that hospital discharge frequency for methadone poisoning rose dramatically through 1997--2007, and then significantly declined through 2007--2014 \[[@CR13]\]. This discrepancy may be due to the different study periods and study populations. In that study, the authors analyzed national trends in inpatient and emergency department discharges for opioid abuse, dependence, and poisoning, but in this study, we analyzed just inpatient methadone poisoning cases. In 2005, the Researched Abuse, Diversion and Addiction-Related Surveillance (RADARS) System reported that there was a correlation between the increasing trend in methadone prescriptions and the degree of diversion and abuse, with no meaningful difference in the number of people on methadone maintenance therapy \[[@CR14]\]. More recently, the number prescriptions for all opioids has decreased, associated with awareness by practitioners of the dangers of opioids, and national, state, and local measures for reducing the prescribing of opioids \[[@CR13], [@CR15]--[@CR17]\].
The US Centers for Disease Control and Prevention reported that prescriptions for opioids peaked in 2012, with greater than 255 million filled (81.3 prescriptions for every 100 persons). The total national opioid prescription frequency then decreased between 2012 and 2017, with the lowest rate in the last ten years in 2017, at 58.7 prescriptions for every 100 persons. However, this still represents more than 191 million opioid prescriptions filled \[[@CR18]\]. This is consistent with our data. We showed a peak in methadone poisoning in 2013 and 2014, and after that, there was a decline in the number of methadone poisoning cases.
In our study, the mortality rate due to methadone poisoning was 1.4%. However, our patients were admitted to hospitals and thus were alive at presentation. It is possible, however, that the rate of death from methadone poisoning in cases that do not reach a hospital is much higher \[[@CR19]\].
Methadone abuse is an important ongoing epidemic, and the 2017 data from the National Poisoning Data System illustratively contains 1054 single methadone poisonings, 456 intentional methadone overdoses, and 56 deaths associated with methadone \[[@CR20]\]. Similarly, in a study by Dart et al., methadone was the leading opioid as a cause of death in the NPDS, with 178 cases in 2012 \[[@CR4]\].
Coma and respiratory depression were the most common signs of severe methadone toxicity in our study. In a cohort of prescription opioid overdose patients, methadone was the second most commonly prescribed opioid after oxycodone. They showed that the risk factors for severe respiratory depression in patients with prescription opioid overdose include drug misuse (pertinent for methadone), increased age, and the specific opioid medication involved. In that study, methadone had a much higher risk of severe respiratory depression \[[@CR21]\]. Other studies revealed that a history of a substance use disorder was closely associated with the development of opioid-induced respiratory depression, with an odds ratio of 12.7 \[[@CR22]\].
Patients experiencing these complications ingested a mean of 104 mg, which is a lower average dose than our entire cohort. This is likely because non-opioid-tolerant patients are the most vulnerable to adverse effects of opioids, even at lower doses, and there was an over-representation of acute ingestions in the group with coma and respiratory depression. Almost 3% of patients had QTc prolongation, a known, yet uncommon, adverse effect of methadone \[[@CR1]\]. It should be noted that the ToxIC Registry does not record minor prolongations of the QT interval. The criterion for QTc prolongation in our database is for it to be over 500 milliseconds. Thus, the actual number of cases that had less consequential QT prolongation was undoubtedly higher. However, complications such as torsade de pointes are unlikely at these lower QTc intervals. Nine and a half percent of our patients had acute kidney injury (AKI). Methadone-induced AKI, which may be a consequence of rhabdomyolysis, has been previously reported \[[@CR23]\].
Interestingly, we found that 2% of patients experienced seizures. Most of these patients ingested a high dose of methadone. Methadone-induced seizure has been previously reported \[[@CR24]\]. Few studies have evaluated the convulsive effects of methadone and the mechanism behind it. Animal studies showed that acute administration of methadone could substantially reduce the seizure threshold. NMDA and μ-opioid receptors may be involved in methadone's convulsive activity in the acute methadone overdose \[[@CR25]\].
In our study, it was found that just half of the patients with respiratory depression received naloxone. Similarly, Aghabiklooei et al. evaluated 322 serious pure methadone-poisoned patients. In their study, naloxone was administered for the treatment of respiratory depression to 40% of cases in the emergency department or during hospitalization \[[@CR26]\]. As with any opioid poisoning, patients with respiratory depression or hypoxia require either naloxone administration or mechanical respiratory support \[[@CR27]\].
Limitations {#Sec6}
-----------
The number of centers in the ToxIC Registry has changed over time. This is because the quality control procedures in ToxIC have caused poorly performing centers to be dropped, while new centers have joined the Consortium. The total number of cases reported each year has not varied widely, suggesting that the time trends we observed were not due to changes in the total number of cases reported to the ToxIC Registry. Further, as reviewed above, our time trends of serious methadone poisoning cases comport with those seen for all methadone poisonings in other national studies.
Secondly, we have reported the rates of consultations to medical toxicology services and not actual poisoning rates. Thus, our report likely represents the frequency of more serious cases of methadone toxicity. Because the treatment of methadone poisoning has not changed substantially over the study period, it is unlikely that the rate of consultation for serious cases could explain the decline starting in 2015. This suggests that professional and national efforts to curtail opioid toxicity have resulted in a trend of decreasing numbers of cases of serious methadone intoxication.
Understanding the pattern of opioid use in the US is necessary before effective measures to reduce morbidity and mortality from opioid use can be instituted. The opioid epidemic continued to increase after 2017; however, we could not present that data after 2018. Despite this, the time trends represent a component of the overall dynamic of "waves of the opioid epidemic." By 2018, the "third wave" created by fentanyl and its analogs was underway, with methadone playing a lesser role.
Conclusion {#Sec7}
==========
Our data demonstrate that rates of methadone poisoning increased in 2010--2014, followed by a decline in 2015--17.
Supplementary information
=========================
{#Sec8}
######
**Additional file 1: Table 1.** Distribution of clinical effects based on the chronicity of use in methadone poisoning patients **Table 2.** Frequency of Receiving naloxone based on clinical effects
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1186/s40360-020-00435-0.
Not applicable.
OM, CH, AA, JB contributed to conception, design, and preparation of the manuscript. OM and JB conducted the data collection and contributed to acquisition, and interpretation. OM, CH, AA, JB made substantial contributions in drafting the manuscript, and revising it critically for important intellectual content. All authors have read and approved the final version of manuscript.
No funding was obtained for this study.
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All data used for this study was obtained from the Toxicology Investigators Consortium ([https://www.ToxICRegistry.org](https://www.toxicregistry.org)) with permission by ToxIC after an application by the first author, who is currently in possession of the data set used in this analysis. Because the Western Institutional Review Board (WIRB) has concluded that the data collection in ToxIC does not meet the definition of Human Subjects Research and was approved by ToxIC no further ethics committee permission was deemed necessary.
This study complies with national guidelines. It was done on a subset of data from the Toxicology Investigators Consortium's (ToxIC's) database. The data collection for ToxIC was reviewed by the Western Institutional Review Board (WIRB) and because no personal health information was collected and there was patient intervention it was judged not to be human subjects research. All institutions contributing data did so after review and acceptance by their Institutional Review Boards.
Not Applicable.
The corresponding author of this article is one of the associate editors of the journal of BMC pharmacology and toxicology. Other co-authors have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
I. Introduction
===============
Angiogenesis is observed in inflammatory reactions, wound healing, and immune reactions. Within tumors, new blood vessel growth is essential for progression and metastasis[@B1]. The term angiogenesis was first used in 1971 by Folkman, and researchers have advised that tumors can grow by forming new blood vessels from the existing blood vascular system, and that angiogenesis is closely related to not only tumors, but also various other diseases such as proliferative retinopathies, age-related macular degeneration, and rheumatoid arthritis[@B2],[@B3]. In the case of mammals, oxygen and nutrient supply are indispensable for survival; since diffusion-controlled oxygen supply to cells cannot function when cells are located farther than 100 to 200 µm from blood vessels, tumor cells cannot grow more than several cm or metastasize to other organs without angiogenesis[@B4],[@B5]. This angiogenetic process is activated by factors produced by tumors to form new blood vessels through basal membrane decomposition by the protease secreted by tumor cells and to support the movement and proliferation of vascular endothelial cells[@B6].
Approximately 20 different factors that induce the angiogenesis process are known, such as basic fibroblastic growth factor, placenta growth factor (PIGF-1), epidermal growth factor, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF)[@B7],[@B8]. The most important angiogenic factor is VEGF, which was first identified by Ferrara and Henzel[@B9] in a bovine pituitary gland follicle cell culture medium.
VEDF is a 46 kDa heparin-binding homodimeric glycoprotein. To date, in addition to VEGF-A, PIGF-1, VEGF-B, VEGF-C, and VEGF-D are known[@B10],[@B11],[@B12]. These VEGFs respectively bind to VEGF receptor-1 (VEGF-1 or Flt-1) and VEGF receptor-2 (VEGF-2 or KDR/Flk-1) to promote endothelial cell differentiation and proliferation. Many studies have demonstrated increases in the expression of VEGFs in the processes of carcinoma progression and proliferation[@B13],[@B14],[@B15],[@B16].
Oral cancers show lower than 50% long-term survival rates because of their high metastasis and recurrence rates, and their prognoses have not greatly improved, despite the development of various treatment methods, due to their high probability of local recurrence and metastasis[@B17]. In addition, although the treatment plans and prognoses of patients clinically diagnosed with oral squamous cell carcinoma mainly follow the TNM classification, treatment results do not coincide with these criteria in many cases. Accordingly, studies are needed to identify markers that will enable a more accurate patient prognosis based on the molecular biological characteristics of carcinomas. They are also elements that affect the occurrence, progression, and metastasis of carcinomas.
Therefore, the present study was conducted to examine the expression profiles of VEGFs involved in angiogenesis, which is important for carcinoma progression according to the histological characteristics of oral squamous cell carcinoma, and to find a correlation between patient clinical information and differences in VEGF expression according to TNM classification. In addition, for an indirect comparison of the relationship between carcinoma progression and VEGF expression, differences in the expression of oral squamous cell carcinoma and intraepithelial carcinoma were examined.
II. Materials and Methods
=========================
1. Study materials
------------------
For study materials, we used 20 tissue slices excised after surgery from 20 patients diagnosed with oral squamous cell carcinoma in the Department of Oral and Maxillofacial Surgery, College of Dentistry, Dankook University, and 20 tissue slices excised from 20 patients diagnosed with intraepithelial carcinoma. The specimens were divided into three groups of well, moderate, and poor histological differentiation. Tumor size classification based on TNM staging was performed, and the specimens were divided into four groups, T1 to T4.([Table 1](#T1){ref-type="table"})
The excised tissues were fixed for eight to 12 hours using 10% neutral-buffered formalin and then made into paraffin blocks in the usual method.
2. Study methods
----------------
### 1) Immunohistochemical staining of VEGF
The excised tissues were fixed and made into 4 µm paraffin slices on poly-L-lysine-treated slides. After removing the paraffin using the usual method, the tissues were treated with 0.01 M citrate buffer (pH 6.0) for 15 minutes in a pressure cooker to retrieve the antigens, treated with hydrogen peroxide/methanol for 15 minutes, and then treated with normal goat serum for 20 minutes to prevent nonspecific binding with endogenous peroxidase. A polyclonal antibody (Abcam, Cambridge, MA, USA) against VEGF was diluted 1 : 25 and applied to the tissues, which were then incubated for at least eight hours at 4℃. Thereafter, the tissues were washed three times with phosphate-buffered saline (PBS, pH 7.0), incubated for 20 minutes in a primary antibody enhancer in a Lab Vision kit (Thermo Scientific, Waltham, MA, USA), washed three times with PBS, and incubated with polymers for 40 minutes at room temperature. Then, the tissues were again washed three times using PBS, color-developed using diaminobenzidine, control-stained using hematoxylin, and observed using an optical microscope. When the staining was determined by a pathologist to be weak or negative, it was classified as low-level staining, and when the staining was even and strong, it was classified as high-level.
### 2) VEGF quantitative real-time polymerase chain reaction (qRT-PCR)
#### (1) Total RNA extraction
Three 15-µm slices from a paraffin block were placed into a xylene solution three times for five minutes each to remove paraffin and were then washed three times in 100% ethanol for five minutes each to remove xylene. Thereafter, the tissues were immersed in a graded ethanol solution prepared using diethyl pyrocarbonate (DEPC)-treated distilled water. Then, the tissues were briefly stained for 10 seconds using hematoxylin and washed with DEPC-treated distilled water. Thereafter, tumor tissues were placed into an Eppendorf tube for extraction of total RNA using a High Pure RNA Paraffin kit (Roche, Penzburg, Germany). The quantity and quality of the extracted RNA were measured using a NanoDrop spectrophotometer (Thermo Scientific).
#### (2) cDNA synthesis
Using 1 to 2 µg of total RNA as a template, cDNA was synthesized using a Maxime RT PreMix kit and random primers (iNtRON Biotechnology, Seongnam, Korea). That is, 20 µL of reactant was reacted for 60 minutes at 45°, and the reverse transcriptase was inactivated for five minutes at 95°.
#### (3) PCR
A PCR primer and probe were designed using Primer Express (PE Applied Biosystems, Foster City, CA, USA) software. The primer and probe sequences are shown below. An AccuPower DualStar PCR PreMix kit (Bioneer, Daejeon, Korea) was used for 20 µL reactions. The 20 µL of synthesized cDNA reactant was diluted in 80 µL of DEPC-treated distilled water, and 3 µL of the solution was used as a PCR template. The reaction medium composition was as follows: PCR forward primer, 10 pmol, 1 µL; PCR reward primer, 10 pmol, 1 µL; Taqman probe, 10 pmol, 1 µL; Template, 3 µL; DEPC-treated distilled water, 14 µL.
PCR was performed in an ExiCycler (Bioneer), and the individual reaction conditions were as follows: treatment for five minutes at 95°, followed by denaturation for 20 seconds at 95° and 50 cycles of annealing/extension for 30 seconds at 60°. Thereafter, each specimen was prepared in triplicate and analyzed.
The primers and probes of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and VEGF gene were as follows:
VEGF forward primer: 5\'-GCACCCATGGCAGAAGG-3\'VEGF reverse primer: 5\'-CTCGATTGGATGGCAGTAGCT-3\'VEGF probe: 5\'-FAM-ACGAAGTGGTGAAGTTCATGGATGTCTATCAC-TMARA-3\'GAPDH forward primer: 5\'-GAAGGTGAAGGTCGGAGTC-3\'GAPDH reverse primer: 5\'-GAAGATGGTGATGGGATTTC-3\'GAPDH probe: 5\'-FAM-CAAGCTTCCCGTTCTCAGCCTAMRA-3\'
The results of ΔC~T~=C~T~(VEGF)-C~T~(GAPDH), the relative calculation of the housekeeping gene GAPDH and VEGF expressions through analyses using a 96-channel optical unit, was converted into 2^-ΔC~T~^ and is indicated as such.
### 3) Statistical analysis
Pearson\'s correlation analysis was used to examine the relationship between VEGF expression based on the results of immunohistochemical staining and the clinical and histological profiles of the carcinoma, and the significance level was set to a matching coefficient r\>0.5 at *P*\<0.05.
In addition, Student\'s t-test was used to examine the relationship between relative value of VEGF mRNA (VEGF/GAPDH) and difference in expression between carcinoma clinical profile and intraepithelial carcinoma tissues, and the significance level was set to *P*\<0.05.
III. Results
============
1. Findings from immunohistochemical staining
---------------------------------------------
In normal oral squamous epithelial tissues or intraepithelial carcinoma, little VEGF expression was observed.([Fig. 1](#F1){ref-type="fig"})
On the other hand, increased VEGF expression was observed in the cytoplasm of invasive tumor cells in moderately differentiated oral squamous cell carcinoma, remarkably increased VEGF expression was observed in marginally differentiated oral squamous cell carcinoma compared to normal tissues and intraepithelial carcinoma tumor cells.([Fig. 2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"})
2. Clinical and histological relationships between VEGF expression and carcinoma according to immunohistochemical staining
--------------------------------------------------------------------------------------------------------------------------
Nine of 20 cases (45%) of VEGF staining were low-level, and the remaining 11 cases (55%) were high-level. The correlation between the profile of histological differentiation of carcinoma and VEGF expression was significant, as was the correlation between item classification according to tumor size of TNM classification and differences in VEGF expression. No correlation between any other factor and differences in VEGF expression was statistically significant.([Table 1](#T1){ref-type="table"})
3. qRT-PCR of VEGF
------------------
Although the relative VEGF mRNA expression (average 0.79) was weak in the intraepithelial carcinoma tissues used in the experiment, stronger relative VEGF mRNA expression (average 2.26) was observed in all 20 oral squamous cell carcinoma tissues.([Fig. 4](#F4){ref-type="fig"})
4. Relationship between relative VEGF mRNA level (VEGF/GAPDH) and clinical and pathological profiles of carcinomas
------------------------------------------------------------------------------------------------------------------
VEGF mRNA expression in the carcinoma was higher than that in intraepithelial carcinoma tissue, and the difference was statistically significant (Student\'s t-test, *P*\<0.05). In addition, among tumor types classified according to size, T2 and larger tumors showed a significant increase in VEGF mRNA expression compared to that in T1. On the other hand, none of the correlations between clinical factors such as gender, age, nodal or remote metastasis, or TNM stage and VEGF expression were significant.([Table 2](#T2){ref-type="table"})
IV. Discussion
==============
Angiogenesis is an indispensable requisite for tumor growth, infiltration, and metastasis[@B1]. Although early-stage tumors are avascular, the cells in tumors 1 to 2 mm or larger or infiltrated fibroblasts around tumor cells secrete substances that stimulate angiogenesis to proliferate new micro-vessels. The proliferated micro-vessels supply nutrients to tumor cells, and vascular endothelial cells secrete growth factors such as basic fibroblast growth factor (bFGF), insulin-like growth factor-2, and PDGF to help tumor growth. In addition, these factors produce breakdown enzymes such as urokinase, collagenase, and plasminogen activator that contribute to infiltration into surrounding tissues[@B4],[@B18],[@B19],[@B20],[@B21].
Many factors are involved in angiogenesis. VEGF is secreted by diverse cells and has specificity to vascular endothelial cells. VEGF receptors such as VEGF-1 and VEGF-2 are known to play a role in this specificity. These factors are located in the cell membranes of endothelial cells and are activated after binding to other factors in the extracellular matrix. They are known to promote cell nucleus division and contribute to angiogenesis through extracellular matrix dissolution and endothelial cell movement[@B5],[@B22].
The genes of human VEGFs are composed of eight exons separated into seven introns and are located on chromosome 6p21.3. Four different isoforms, VEGF~121~, VEGF~165~, VEGF~189~, and VEGF~206~, exist due to diverse exon splicing; of these, VEGF~165~ has been reported as the most functionally important isoform[@B23],[@B24]. Ferrara and Davis-Smyth[@B25] reported that factors that control the expression of VEGF genes include tissue oxygen tension, growth factors, hormones, and oncogenes, and that the expression increases when tissue pO~2~ concentration is low due to the effects of growth control factors such as TGF-α, TGF-β, and FGF or adrenal cortical hormones. That is, the low oxygen states in the environment around a tumor characterized by fat growth produce reversible increases in VEGF mRNA transcription, leading to increases in expression within the tumor. As tumor sizes increases, the distances between the nearest blood vessels increase, causing cells in expanding tumors to experience oxygen deficiency and producing low-oxygen areas within the tumor. In response to this, tumor cells produce endothelial growth factors. Through this mechanism, VEGF expression increases within tumors, in particular, in low-oxygen areas in necrotic regions. Therefore, VEGF overexpression due to low oxygen can be thought of as a compensatory mechanism that enables tumor tissues to increase oxygen through vascular proliferation[@B26].
Many studies have reported that VEGF expression increases with micro-blood vessel density in diverse tumors such as colon cancer, ovarian cancer, hepatocellular carcinoma, gastrointestinal malignancy, renal cancer, breast cancer, and head and neck cancer, and that tumor cells express VEGF mRNA and secrete VEGF-like proteins[@B27],[@B28],[@B29],[@B30]. In addition, study results indicate that tumors with rich VEGFs recur in much shorter periods of time after operation than do those with insufficient VEGFs, supporting the fact that VEGFs contribute to tumor occurrence and angiogenesis and affect prognosis. For the same reason, studies have reported that, in many tumors, increases in expression of VEGF receptor appear in proportion to increases in expression of vascular endothelial cell growth factor[@B31],[@B32]. In a study of the correlation between diverse clinicopathological profiles and VEGF expression in breast cancer, Maeda et al.[@B33] stated that prognosis worsened as the expression increased. Smith et al.[@B34] reported that VEGF overexpression was the most influential factor on poor prognosis of oral squamous cell carcinoma. As such, studies conducted to examine the relationship between VEGF expression and carcinoma prognosis have noted that the relationship may be an ancillary measure for determining carcinoma prognosis with limitations that had been following existing histological classification or clinical TNM classification.
According to the present results of VEGF immunohistochemical tests, very little expression of VEGF was observed in normal oral squamous cell tissues. This is consistent with the results of other studies indicating that, when VEGF is normal, its expression will be limited in endothelial cells[@B25],[@B35]. In addition, based on histopathological findings, little VEGF expression was observed in well-differentiated and less-invasive intraepithelial carcinoma tissues or highly differentiated oral squamous cell carcinoma than in normal cells. On the other hand, strong VEGF expression was observed in less-differentiated invasive oral squamous cell carcinoma. These results suggest that the degree of VEGF expression is correlated with the degree of differentiation or invasiveness of carcinoma; this was supported by the statistical analysis conducted with VEGF expression levels based on the results of immunohistochemical staining and clinical and histological profiles of carcinomas. The correlation coefficient r of the Pearson correlation analysis, which was the statistical method used to that end, can be interpreted as indicating a very high correlation when its value is 0.5 or higher, moderate correlation when its value is between 0.4 and 0.5, and very low correlation when its value is lower than 0.4. Consequently, in the present study, the correlation between the degree of histological differentiation and VEGF expression was significant, as was the correlation between classification according to tumor size and VEGF expression.
In addition, VEGF qRT-PCR was conducted to quantify relative VEGF gene expression; based on the analysis of the relationship between relative level of VEGF mRNA (VEGF/GAPDH) and the clinical and pathological profiles of the carcinoma, VEGF expression increased with tumor size, leading to increased oxygen demand compared to cases with small tumors (T1). In addition, compared to intraepithelial carcinoma tissues in the early stage, the amount of VEGF expression increased in oral squamous cell carcinoma tissues with progression of tumor cell invasion into connective tissues, and the difference was shown to be statistically significant. This result is similar to the results of immunohistochemical tests, suggesting that VEGF expression is activated with tumor growth, and it can be assumed that increases in VEGF expression are involved in the angiogenesis of tumors.
In conclusion, VEGF gene expression was more highly increased in progressed oral squamous cell carcinoma than in normal tissue cells or intraepithelial carcinoma, and the correlation between VEGF expression and the degree of histological differentiation of oral squamous cell carcinoma according to tumor size was significant. These results lead to the inference that VEGF expression in carcinomas is involved in angiogenesis and progression and affects the prognosis. However, since the number of samples tested was small, a study with a larger number of samples is needed to support the correlation between VEGF gene expression and clinicopathological factors in oral squamous cell carcinoma.
V. Conclusion
=============
VEGF binds to VEGFR-1 and VEGFR-2, which are receptors in vascular endothelial cells, and is involved in endothelial cell differentiation and migration as well as vascular proliferation, and plays important roles in the angiogenesis of tumors. In addition, VEGF produces plasma fibers outside of blood vessels, causing changes in the extracellular matrix through cellulose deposition. The matrix then promotes the growth of macrophages, fibroblasts, and endothelial cells in tumors, in particular, promoting vascular endothelial cell proliferation, and are thereby involved in the angiogenesis of tumors.
In this study, we used 20 tissue slices excised after surgery from 20 patients diagnosed with oral squamous cell carcinoma. To identify VEGF expression in each tissue slice, we performed immunohistochemical tests, conducted VEGF gene qRT-PCR analysis, and statistically analyzed the results.
1\. Immunohistochemical test: In normal oral squamous cells, VEGF expression was observed only in the vascular endothelial cells in the mesenchymal tissues. On the other hand, greatly increased VEGF expression was observed in weakly differentiated oral squamous cell carcinoma.
2\. VEGF gene expression: VEGF gene expression was observed in all 20 tumor tissue slices, with only weak VEGF gene expression observed in intraepithelial carcinoma tissues.
3\. Differences in VEGF expression among different tumor sizes were significant, and VEGFs were overexpressed in highly invasive carcinomas compared to intraepithelial carcinoma tissues.
In conclusion, VEGF expression was increased in insufficiently differentiated invasive carcinomas and was overexpressed in invasive oral squamous cell carcinoma but not in intraepithelial carcinoma tissues. These findings suggest that VEGF likely plays a role in angiogenesis of oral squamous cell carcinoma.
No potential conflict of interest relevant to this article was reported.
{#F1}
{#F2}
{#F3}
{#F4}
###### Correlation between immunohistochemical VEGF expression and clinical and pathological factors
(VEGF: vascular endothelial growth factor)
^1^Pearson correlation analysis, significance r\>0.6, *P*\<0.05.
Values are presented as number.
###### Relationship between relative level of VEGF mRNA (VEGF/GAPDH) and clinical and pathological factors
(VEGF: vascular endothelial growth factor, GAPDH: glyceraldehyde-3-phosphate dehydrogenase)
^1^VEGF mRNA expression derived from quantitative real-time polymerase chain reaction.
^2^Student\'s t-test, *P*\<0.05.
Values are presented as number or mean±standard deviation.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
The blood-feeding female of phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae) include natural vectors of protozoa of the genus *Leishmania* (Kinetoplastida: Trypanosomatidae), which are the parasitic causative agents of mammalian leishmaniasis ([@b6-ijad-3-22]). There are approximately 700 species of phlebotominae sand flies divided among 6 genera, of which only two, i.e. *Phlebotomus* in the old world (OW) and *Lutzomyia* in the new world (NW) are medically importance ([@b9-ijad-3-22], [@b12-ijad-3-22], [@b10-ijad-3-22], [@b28-ijad-3-22]).
Only 10% of these Phlebotominae sand flies act as disease vector. Further, only 30 species of these are important from public health point ([@b28-ijad-3-22]). A total of about 21 *Leishmania* spp. have been identified to be pathogenic to human ([@b29-ijad-3-22]). However, recent studies based on molecular data have provided evidence that the genus *Phlebotomus* is not monophyletic ([@b2-ijad-3-22]). Furthermore, [@b25-ijad-3-22] have recently revised the definition of morphological characters states. In the Mediterranean Region, species of the subgenus *Larroussius* are the main vectors of *Leishmania infantum*, which cause Visceral Leishmaniasis (VL) in humans and dogs ([@b6-ijad-3-22], [@b5-ijad-3-22]). The subgenus is readily identified by the development long extension of the spermathecal neck in females ([@b23-ijad-3-22]).
Description of the subgenus *Larroussius* created by Nitzulescu (1931) with *P. major* Annandale, 1910 as the type species ([@b15-ijad-3-22], [@b12-ijad-3-22]).
Among 27 species of Phlebotominae sand flies in the OW ([@b1-ijad-3-22], [@b25-ijad-3-22], [@b26-ijad-3-22], [@b27-ijad-3-22]), at least 12 species of the subgenus *Larroussius* are proven or probable vectors of leishmaniasis ([@b6-ijad-3-22]). The subgenus *Larroussius* is closely related to the subgenera *Transphlebotomus* and *Adlerius*, and the proliferation of species within *Larroussius* and *Adlerius* is probably recent ([@b26-ijad-3-22], [@b24-ijad-3-22]).
Although, identification of male specimens of *Larroussius* are not very difficult, but determination of some females of this subgenus has been considered impossible based on morphological characters.
[@b11-ijad-3-22] showed that three sympatric species of *Larroussius* in Greece could readily be separated by the morphology of base of the spermathecal ducts.
[@b7-ijad-3-22] separated 13 species of *Larroussius* by the morphology of the base of the spermathecal ducts. Recent studies showed that molecular tools could help resolve phylogenetic relationships between species of subgenus *Larroussius* ([@b3-ijad-3-22], [@b4-ijad-3-22], [@b13-ijad-3-22], [@b16-ijad-3-22], [@b14-ijad-3-22]).
There are four species of subgenus *Larroussius* including *P. kandelakii*, *P. perfiliewi*, *P. major* and *P. tobbi* in Meshkinshahr district, north western Iran. The first species is a proven vector and the second is the probable vector of viscerotropic *Leishmania* spp. in this area ([@b17-ijad-3-22], [@b18-ijad-3-22], [@b19-ijad-3-22], [@b20-ijad-3-22], [@b21-ijad-3-22]).
The objective of the present study was to identify and distinguish the females of *P. perfiliewi*, *P. major* and *P. tobbi*. They are similar in morphological characters and indistinguishable. We analyzed the sequences of cytochrome b (mtDNA) and elongation factor-1α (nDNA) genes of these three species and compared with morphological characteristics. Complete phylogenetic information is available for these genes and they are useful for the study of molecular systematic and speciation.
Material and methods
====================
Study area
----------
Meshkinshahr District (48° 17′ N, 38° 15′ E) is located at 1890 masl in Ardebil Province, Iran. The district occupies the northern foothills of the Sabalan Mountains, which rise to an altitude 4881 masl. Temperature varies from −27 °C in winter to 41 °C in summer. The human population was 156141 in 2006 and the principal economic activity is sheep farming.
Sand fly sampling
-----------------
Sand fly sampling was carried out from Jun--October 2006 (during the period of peak activity), in 30 villages distributed throughout the Meshkinshahr District. Sand flies were collected once every 15 d from indoor habitats (bedroom, stable, toilet, bath room, hen nest, hay loft and store room) and outdoor habitats (yard, rodent burrow, stone and wall crevices, fox burrows and dog kennel, rubble and riverbanks) using sticky papers, CDC light trap and Aspirator. Sand fly specimens were stored in 96% ethanol.
Dissection, Mounting and Morphological identification
-----------------------------------------------------
After recording the sampling data and locations, sand fly specimens were washed in 1% detergent then twice in sterile distilled water. Each specimen was then dissected in fresh drop of sterile normal saline by cutting off the head and abdominal terminalia with sterilized forceps and single used mounted needles. The remainder of the body was stored in the sterile eppendorf microtubes.
Specimens were mounted in Puri's or Berlese's medium and identified using the identification keys of [@b31-ijad-3-22], [@b15-ijad-3-22], and [@b12-ijad-3-22]. Morphological characters, which used in this study, included pharyngeal armature, spermathecal segments number, length of spermathecal neck, palpal and ascoids formula. First and second are more important characters.
DNA extraction
--------------
DNA was extracted from the dissected thorax and attached anterior abdomen of individual sand flies using the method of Ish-Horowiz ([@b22-ijad-3-22]). In the 1.5 ml microtubes, the thorax plus anterior abdomen of each sand fly was frozen and defrosted twice to break up tissue using a sampler tips or pestel, with grinding mix. Then SDS mix was used to denature proteins associated with the DNA, then ice cold 8M KOAc was added to remove effectively the SDS proteins from solution. Cell debris and proteins were separated from the DNA by centrifugation then the DNA in the supernatant was precipitated over night at −20 °C in 96% ethanol. Following ethanol precipitation, the DNA was dissolved in 15μl 1X TE (10mM Tris-HCl, 1mM EDTA pH= 8.0) and stored at −20 °C.
PCR amplification of Cyt b and EF-1α
------------------------------------
For Cyt b one pair primers were used. CB3-FC (forward) (5′-CA(C/T) ATTCAA-CC (A/T)GAATGATA-3′) with CB-R06 (reverse) (5′-TATCTAATGGTTTCAAAACA ATTGC-3′) to amplify an overlapping 3′ fragment of 499 bp without primers (CB3 fragment). Also for EF-1α one pair primers were used, EF-F05 (forward) (5′-CCTGG ACAT-CGTGATTTCAT-3′) with EF-F08 (reverse) (5′-CCACCAATCTTGTAGACATCCTG-3′) to amplify of 454 bp without primers.
The PCR reaction conditions were identical for both Cyt b and EF-1α. 2μl 10X PCR buffer, 1.2 μl MgCl~2~, 0.15μl primers (F and R), 2μl dNTPs, 1.5μl DNA with the reaction volume completed to 20 μl by distilled water, followed by initial denaturation of Cyt b at 94 °C for 3 min. PCR consisted of 35 cycles of denaturation at 94 °C for 30 sec, annealing1 at 40 °C for 30 sec, annealing 2 at 44 °C for 30 sec, extension at 72 °C for 90 sec and then final extension at 72 °C for 10 min. For EF-1α, initial denaturation at 94 °C for 3 min. PCR consisted of 35 cycles of denaturation at 94 °C for 30 sec, annealing at 48°C for 30 sec, extension at 72 °C for 30 sec and then final extension at 72 °C for 10 min.
Direct sequencing of PCR products
---------------------------------
One hundred nanograms of each purified DNA sample was cycle-sequenced using an ABI Parsim® Big Dye™ Terminator cycle sequencing Ready Reaction Kit (version 2.0) and ABI 373/377 sequencing systems (ABI, PE Applied Biosystems), with 3.2 Pmol of the same primers that were used for PCR.
Aligning and phylogenetic analysis of DNA sequences
---------------------------------------------------
DNA sequences from both strands were aligned and edited using Sequencher Demo 4.7 and BioEdit softwares. Multiple or pairwise sequence alignments of DNA were used with CLUSTAL W PPC: Clustalw version 1.7. Phylogenetic analyses were done using Parsimony PAUP. Relationships were inferred based on genetic distances using the Neighbor Joining (NJ) option with default settings.
Results
=======
Species composition of subgenus *Larroussius*
---------------------------------------------
Out of the 1743 sand fly specimens collected, 660 specimens (37.9%) belonged to the subgenus *Larroussius*, including: *Phlebotomus* (*La.*) *kandelakii* (31%), *P.* (*La.*) *major* (1.5%), *P.* (*La.*) *tobbi* (1.5%), *P.* (*La.*) *perfiliewi* (1.7%) and *P.* (*La.*) *major* group (2.1%).
Aligning and phylogenetic analysis of *Larroussius* DNA sequences
-----------------------------------------------------------------
PCR amplification of Cyt b and EF-1α was successfully achieved for all 3 species of the subgenus *Larroussius*.
Seven sequences for Cyt b and four for EF-1α were compared for *P. major*. Comparison of pairwise genetic similarity or score of sequences showed 87%--100% similarity for Cyt b and 96%--100% similarity for EF-1α. Also 2.7% genetic diversity for Cyt b and 2.8% for EF-1α was observed.
There were two haplotypes for Cyt b from seven sequences ([Table 1](#t1-ijad-3-22){ref-type="table"}) and for EF-1α, there were two haplotypes within four sequences ([Table 2](#t2-ijad-3-22){ref-type="table"}). The Neighbor-joining (NJ) phylogram for Cyt b showed two lineages, and each of these had subgroups with short branches. One of the lineages had one haplotype from sand flies from the same habitat (Stone crevices) but different locations (Ur kandi, Mueel, Agh daragh). The second lineage had one haplotype from sand flies from different habitats (Rodent burrow and Fox burrows) and locations (Ghurt tappeh, Alni, Niaz suee) ([Fig. 1](#f1-ijad-3-22){ref-type="fig"}). In addition, phylogenetic tree for EF-1α showed two lineages and only one haplotype had subgroups. One of the lineages had one haplotype from sand flies from the same location (Ur kandi) but different habitats (Stone crevices, bedroom, and hen nest). The second lineage had one haplotype from one specimen in rodent burrow ([Fig. 2](#f2-ijad-3-22){ref-type="fig"}).
Seven sequences for Cyt b and three for EF-1α were compared for *P. tobbi*. Comparison of pairwise genetic similarity of sequences indicated the 100% similarity for both Cyt b and EF-1α sequences. Genetic diversity and unique haplotype for Cyt b and EF-1α were not observed. For Cyt b and EF-1α one haplotype was obtained ([Table 1](#t1-ijad-3-22){ref-type="table"} and [2](#t2-ijad-3-22){ref-type="table"}). The Neighbor-joining (NJ) phylogram for both Cyt b and EF-1α showed one lineage for all specimens in the same location and habitat ([Fig. 1](#f1-ijad-3-22){ref-type="fig"} and [2](#f2-ijad-3-22){ref-type="fig"}).
Six sequences for Cyt b and three for EF-1α were compared for *P. perfiliewi*. Comparison of pair wise genetic similarity of sequences indicated 100% similarity for both Cyt b and EF-1α sequences. In this species genetic diversity for Cyt b and EF-1α were not observed ([Table 1](#t1-ijad-3-22){ref-type="table"} and [2](#t2-ijad-3-22){ref-type="table"}). Phylogenetic tree for both Cyt b and EF-1α showed one lineage for all specimens in the different location and habitats ([Fig. 1](#f1-ijad-3-22){ref-type="fig"} and [2](#f2-ijad-3-22){ref-type="fig"}). The Neighbor-joining (NJ) phylogram in combination of Cyt b and EF-1α for seven specimens showed two lineages for *P. perfiliewi* and *P. tobbi* and one lineage for *P. major* ([Fig. 3](#f3-ijad-3-22){ref-type="fig"}).
Identification of the female *Larroussius* species using morphological characters
---------------------------------------------------------------------------------
In the present study, the morphological characteristics of the three species female of *Larroussius*1647 were described as follows:
### Phlebotomus major and Phlebotomus neglectus
Palpal formula: 1, 4, 2, 3, 5 and the formula of ascoids: 2/3--8, 1/9--5. Pharyngeal armature has occupied ⅓ of pharynx space and punctiform, arranged in several rows and anterior elements have serrated margin ([Fig .4](#f4-ijad-3-22){ref-type="fig"}). Spermatheca with 14--16 segments and length of spermatheca neck ⅔ of spermatheca capsule ([Fig. 5](#f5-ijad-3-22){ref-type="fig"}).
### Phlebotomus tobbi
Palpal formula: 1, 4, 2, 3, 5 or 1, (2, 4), 3, 5 and the formula of ascoids: 2/3--8, 1/9--15. Pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several concave irregular rows of large dots and anterior part of pharynx have a fine dots ([Fig. 6](#f6-ijad-3-22){ref-type="fig"}). Spermatheca with 11--13 segments. Length of spermatheca neck as long as spermatheca capsule ([Fig. 7](#f7-ijad-3-22){ref-type="fig"}).
### Phlebotomus perfiliewi
Palpal formula: 1, 4, 2, 3, 5 and the formula of ascoids: 2/3--9, 1/10--15. Pharyngeal armature has occupied over ½ of pharynx space and punctiform, arranged in several concave regular rows of large dots ([Fig. 8](#f8-ijad-3-22){ref-type="fig"}). Spermatheca with 17--20 segments. Length of spermatheca neck ½ of spermatheca capsule ([Fig. 9](#f9-ijad-3-22){ref-type="fig"}).
Identification of the male *P. major* and *P. neglectus* using morphological characters
---------------------------------------------------------------------------------------
Among 26 male specimens in this species 17 specimens of *P. major* (65.4%) with 20--30 ventrally directed and long hairs stand densely on coxite and Palpal formula 1, 4, (2, 3), 5 ([Fig. 10](#f10-ijad-3-22){ref-type="fig"} and [11](#f11-ijad-3-22){ref-type="fig"}). In 9 specimens of *P.neglectus* (34.6%) with less than 20 ventrally directed hairs, widely spaced and sparser and Paplal formula 1, 4, 2, 3, 5 ([Fig. 12](#f12-ijad-3-22){ref-type="fig"} and [13](#f13-ijad-3-22){ref-type="fig"}).
Discussion
==========
According to the results of previous studies, the vectors of viscerotropic *Leishmania* spp. in OW mainly belong to the subgenera *Larroussius*, *Adlerius*, *Euphlebotomus*, *Synphlebotomus* and *Paraphlebotomus*. In the subgenus *Larroussius*, *P.perniciosus* in Algeria, France, Italy, Malta, Spain, *P.ariasi* in France, Spain, Italy, *P. perfiliewi* in Italy, East Mediterranean, North of Africa, Greece, Azerbaydzhan, Tunisia, *P. tobbi* in Cyprus, East Mediterranean, Sicil, *P. kandelakii* in Afghanistan, Russia, and *P. neglectus* in Greece, Albania, Portugal ([@b6-ijad-3-22]). In North West of Iran *P. kandelakii* and *P. perfiliewi transcaucasicus* are proven and probable vector of viscerotropic *Leishmania* spp. ([@b21-ijad-3-22]).
According to the finding of the present investigations, the phylogeny of Cyt b and EF-1α haplotypes confirms the morphological relationships among the three species *P. perfiliewi*, *P. major* and *P. tobbi* in the subgenus *Larroussius*. The males of these three species have a several diagnostic morphological characters, whereas the females of these species show very similarities in morphology of spermathecal segment number, pharyngeal armature and other characteristics.
Although morphological characteristics are the most practical methods for species distinguishing, new molecular techniques are very useful to resolve problems of identification in the cases with morphologically similarities.
Access on the genetic diversity and molecular systematic of the *Larroussius* sand flies species not only useful to find the taxonomic status of them, but also indicates the ecological and geographical differences.
In our analysis, all of the Cyt b and EF-1α sequences, the monophyly of the subgenus *Larroussius* was confirmed, in concordance with the morphologically and molecularly based phylogenies of [@b24-ijad-3-22], [@b26-ijad-3-22] and [@b3-ijad-3-22]. All of the branches of the parsimony and distances trees had strong support and showed identical relationships and indicated the validity of many of characters in inferring evolutionary relationships. The trees were also topologically similar to the parsimony tree of [@b3-ijad-3-22].
On morphological characteristics, the present study confirmed the observations of [@b15-ijad-3-22], [@b12-ijad-3-22], [@b11-ijad-3-22], and [@b7-ijad-3-22].
Phylogenetic analyses of *P. major* populations showed 2 lineages in different locations such as rodents burrow and stone crevices ([Table 3](#t3-ijad-3-22){ref-type="table"}).
According to our molecular and morphological finding, it seems that there are both *P. major* and *P. neglectus* in Meshkinshahr district. This is the first report of *P. neglectus* in this area as well as in Iran ([Table 3](#t3-ijad-3-22){ref-type="table"})
Further studies needs to resolve the taxonomic status of this species. *P. major* has many geographical variants the females of which have conventionally been distinguished by differences in pharyngeal armature. Adler (1933) stressed the tendency of *P. major* to form geographical races. He showed (1946) that they differ mainly in the form of the pharyngeal armature of the females. He also mentioned ecological differences between the races and stresses that some races may have a different importance in the epidemiology of VL.
Our study on phylogenetic analyses of *P. tobbi* populations showed one lineage with single haplotype for either cytB or EF-1α gene. *P. tobbi* was first found in Iran and Israel, and was described as a variety of *P. perniciosus*. Parrot (1934) reported that it is a single species with the name of *P. tobbi*. This was accepted also by [@b30-ijad-3-22].
*Phlebotomus tobbi* is mainly found in burrows and is rather common in Transcaucasia and it seems to be identical with the sand flies from Iran and southwest Asia. Therefore, *P. tobbi* is considered a separate species.
*Phlebotomus perfiliewi* is the probable vector of VL in the north west of Iran. In the present study, this species had one lineage with a single haplotype. Phylogenetic tree retrieved from cytB or combination of cytB and EF-1α showed close relationship of *P. perfiliewi* with *P. tobb*i.
Perfiliev (1966) recognized three subspecies of *P. perfiliewi* (*perfiliewi*, *galilaeus* and *transcaucasicus*) which were distinguished by minor morphological differences in the aedeagus of the male. [@b12-ijad-3-22] listed the same three subspecies but commented that the distinction of the male *galilaeus* from *transcaucasicus* uncertain. [@b1-ijad-3-22] raised *galilaeus* and *transcaucasicus* to level species. The spermatheca of *galilaeus* and *perfiliewi* are indistinguishable, suggesting that these allopatric sand flies are taxonomically very close and are perhaps two subspecies ([@b12-ijad-3-22]).
To find an exact distinction and identification of the female species of *Larroussius*, needs more investigations in different parts of the world as well as Iran.
The author would like to appreciate very much for kind collaboration of Health of Center staffs of Meshkin-shahr district. This study was M.Sc thesis and financially was supported by the School of Public Health. Tehran University of Medical Science: Project No.: 240. 6129.
{#f1-ijad-3-22}
{#f2-ijad-3-22}
{#f3-ijad-3-22}
{#f4-ijad-3-22}
{#f5-ijad-3-22}
{#f6-ijad-3-22}
{#f7-ijad-3-22}
{#f8-ijad-3-22}
{#f9-ijad-3-22}
{#f10-ijad-3-22}
{#f11-ijad-3-22}
{#f12-ijad-3-22}
{#f13-ijad-3-22}
######
Sequences comparison of Cyt b-mtDNA gene in the subgenus *Larroussius*
**Species** **Total Sequences** **Pairwise genetic similarity (%)** **Total aplotype** **Unique haplotypes** **Genetic diversity (%)**
--------------------- --------------------- ------------------------------------- -------------------- ----------------------- ---------------------------
***P. major*** 7 87 -- 100 2 0 2.7
***P. tobbi*** 7 100 1 0 0.3
***P. perfiliewi*** 6 100 1 0 0
######
Sequences comparison of EF-1α-nDNA gene in the subgenus *Larroussius*
**Species** **Total Sequences** **Pairwise genetic similarity (%)** **Total aplotype** **Unique haplotypes** **Genetic diversity (%)**
--------------------- --------------------- ------------------------------------- -------------------- ----------------------- ---------------------------
***P. major*** 4 96--100 2 0 2.8
***P. tobbi*** 3 100 1 0 0
***P. perfiliewi*** 3 100 1 0 0
######
Collected sand flies of the subgenus *Larrossius* in different location and habitats in Meshkinshahr District, Iran
**Species** **Code No.** **Sex** **Location of capture** **Altitude of capture (masl)** **Habitat** **Date of capture** **Method of catching** **Type of Geue** **Geubank accession number**
------------------------------------ -------------- --------- ------------------------- -------------------------------- ---------------- --------------------- ------------------------ ------------------ ------------------------------
*P.major* MSH-1201 Male Agh daragh 1390 Stone crevices 05.08.06 SP Cyt b GQ169331
*P.major* MSH-297 Male Ur kandi 1551 Stone crevices 09.07.06 SP Cyt b GQ169332
*P.major* MSH-619 Male Mueel 2780 Stone crevices 10.07.06 SP Cyt b GQ169333
*P.major* MSH-861 Male Niaz suee 1080 Fox burrows 22.07.06 SP Cyt b GQ169334
*P.major* MSH-007 Female Alni 1267 Fox burrows 16.06.06 SP Cyt b GQ169335
*P.major* MSH-052 Male Ghurt tappeh 1547 Rodent burrow 17.06.06 SP Cyt b GQ169336
*P.major* MSH-1397 Male Mueel 2780 Stone crevices 07.08.06 SP Cyt b GQ169337
*P. neglectus* (GenBank) Cyt b AF161191
*P. neglectus* (GenBank) Cyt b AF161193
*P.perfiliem* MSH-721 Male Dushan lu 1209 Rodent burrow 22.07.06 SP Cyt b GQ169338
*P.perfiliewi* MSH-738 Male Dushan lu 1209 Rodent burrow 22.07.06 SP Cyt b GQ169339
*P.perfiliewi* MSH-756 Female Dushan lu 1209 Yard 22.07.06 CDC Cyt b GQ169340
*P.perfiliewi* MSH-795 Female Niaz suee 1080 Yard 22.07.06 CDC Cyt b GQ169341
*P.perfiliewi* MSH-812 Female Niaz suee 1080 Yard 22.07.06 CDC Cyt b GQ169342
*P.perfiliewi* MSH-836 Male Niaz suee 1080 Stone crevices 22.07.06 SP Cyt b GQ169343
*P.perfiliewi* (GenBauk) Cyt b AF161201
*P.tobbi* MSH-480 Male Ghurt tappeh 1547 Rodent burrow 09.07.06 SP Cyt b GQ169344
*P.tobbi* MSH-551 Male Ghurt tappeh 1547 Stone crevices 09.07.06 SP Cyt b GQ169345
*P.tobbi* MSH-903 Male Ghurt tappeh 1547 Stone crevices 23.07.06 SP Cyt b GQ169346
*PJobbi* MSH-1292 Male Ghurt tappeh 1547 Stone crevices 06.08.06 SP Cyt b GQ169347
*P.tobbi* MSH-1481 Male Ghurt tappeh 1547 Stone crevices 20.08.06 SP Cyt b GQ169348
*P.tobbi* MSH-1657 Male Ghurt tappeh 1547 Stone crevices 03.09.06 SP Cyt b GQ169349
*P.tobbi* MSH-899 Female Ghurt tappeh 1547 Stone crevices 23.07.06 SP Cyt b GQ169350
*P,tobbi* (GenBank) Cyt b AF161212
*P.tobbi* (GenBank) Cyt b AF161210
*P.caucasicus* (GenBank/Out group) Cyt b FJ217390
*P.sergenti* (GenBank/Out group) Cyt b EU980367
*P.major* MSH-297 Male Ur kandi 1551 Stone crevices 09.07.06 SP EF-1α GQ169351
*P.major* MSH-312 Male Ur kandi 1551 Bed room 09.07.06 ASP EF-1α GQ169352
*P.major* MSH-317 Male Ur kandi 1551 Hen nest 09.07.06 SP EF-1α GQ169353
*P. major* MSH-522 Male Ghurt tappeh 1547 Rodent burrow 09 07 06 SP EF-1α GQ169354
*P. major* (GenBauk) EF-1α EF416834
*P*.neglectus (GenBank) EF-1α AF160802
*P.neglectus* (GenBank) EF-1α AF160801
*P.perfiliewi* MSH-721 Male Dushan lu 1209 Rodent burrow 22.07.06 SP EF-1α GQ169355
*P.perfiliewi* MSH-738 Male Dushan lu 1209 Rodent burrow 22.07.06 SP EF-1α GQ169356
*P.perfiliewi* MSH-836 Male Niaz suee 1080 Stone crevices 22.07.06 SP EF-1α GQ169357
*P.perfiliewi* (GenBauk) EF-1α AF160805
*P.tobbi* MSH-903 Male Ghurt tappeh 1547 Stone crevices 23.07.06 SP EF-1α GQ169358
*P.tobbi* MSH-1292 Male Ghurt tappeh 1547 Stone crevices 06.08.06 SP EF-1α GQ169359
*P.tobbi* MSH-1481 Male Ghurt tappeh 1547 Stone crevices 20.08.06 SP EF-1α GQ169360
*P.tobbi* (GenBank) EF-1α AF160810
*P.caucasicus* (GenBank/Out group) EF-1α EF416836
SP = Sticky paper ; CDC = CDC Light trap ; Asp = Aspirator
Cyt b = Cytochrome b ; EF-lα = Elongation Factor 1-α
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Plant mating systems are highly diverse and have profound effects on reproductive success under variable ecological conditions. Self-fertilization (selfing) may be adaptive when mates or pollinators are limited, as originally proposed by Darwin, because it confers reproductive assurance [@pone.0057902-Darwin1]. This advantage may be offset by inbreeding depression, the reduced fitness of offspring in comparison to outcrossed progeny [@pone.0057902-Darwin1]--[@pone.0057902-Charlesworth1]. There are several obvious advantages of outcrossing, including avoidance of inbreeding depression by reducing the expression of deleterious recessive mutations, increased potential for rapid adaptation to changing ecological conditions, and maintenance of long-term adaptive capacity during evolution [@pone.0057902-Darwin1]--[@pone.0057902-Goldberg1]. As a consequence, most plants have evolved a wide variety of physiological or morphological mechanisms to ensure or facilitate outcrossing [@pone.0057902-Darwin1], [@pone.0057902-Stebbins1], [@pone.0057902-Barrett1].
Herkogamy, the spatial separation of male and female reproductive organs within flowers, is almost ubiquitous in animal-pollinated hermaphroditic plants. In self-incompatible species, herkogamy may function primarily to reduce sexual interference, whereas in self-compatible plants, it is usually considered an adaptive character that decreases the likelihood of self-pollination and increases the opportunity for outcrossing [@pone.0057902-Barrett1], [@pone.0057902-Webb1]. Indeed, a number of studies have reported evidence for a monotonically increasing relationship between the degree of herkogamy and outcrossing rate across diverse plant species, including *Nicotiana* [@pone.0057902-Breese1], *Clarkia* [@pone.0057902-Holtsford1], *Turnera* [@pone.0057902-Belaoussoff1], *Mimulus* [@pone.0057902-Karron1], *Aquilegia* [@pone.0057902-Brunet1], and *Datura* [@pone.0057902-Motten1], although there are exceptions (e.g., *Narcissus*, [@pone.0057902-Medrano1], [@pone.0057902-Medrano2]). Notably, these self-compatible plant species are commonly with relatively large floral displays and, in these cases, the role of herkogamy in promoting outcrossing may be complex. On the one hand, large floral displays can increase outcrossing rates, probably through enhanced pollinator attraction [@pone.0057902-Klinkhamer1]--[@pone.0057902-Button1]. On the other hand, large floral displays could promote geitonogamy, self-pollination as a consequence of pollen dispersal among flowers on the same plant [@pone.0057902-Brunet1], [@pone.0057902-Harder1], [@pone.0057902-Karron2]. To clarify the role of herkogamy in promoting outcrossing, ideally it requires a highly selfing species with phenotypic variation in herkogamy and with a small floral display.
Herkogamy demonstrates moderate to high heritability across different plant species and may be able to respond to selection rapidly [@pone.0057902-Motten1], [@pone.0057902-Shore1]--[@pone.0057902-Herlihy1]. The molecular basis of herkogamy, however, remains largely unexplored across diverse species (but see [@pone.0057902-Chen1]). This may be primarily attributable to the limited genomic resources that are available for those plant species, which are generally non-model organisms. Hence, studying ecologically important and complex characters (e.g., herkogamy) in model organisms such as *Arabidopsis thaliana* may be particularly beneficial.
*A. thaliana* is a model plant used for a wealth of studies, ranging from molecular genetics and genomics to evolution and ecology [@pone.0057902-MitchellOlds1], [@pone.0057902-Weigel1]. It is a predominantly selfing species with a strong selfing syndrome -- a characteristic set of floral morphology and function (e.g., small, unscented, and inconspicuous flowers without effective separation of sexual organs) that facilitates self-pollination [@pone.0057902-Darwin1], [@pone.0057902-Ornduff1], [@pone.0057902-Sicard1]. Typically estimated outcrossing rates ranged from 0.3% to 2.5% [@pone.0057902-Abbott1]--[@pone.0057902-Platt1], but could exceed 10% in some populations [@pone.0057902-Bomblies1], [@pone.0057902-Platt1]. The size of a mature *A. thaliana* flower is as small as 2--3 mm and anthers are typically positioned closely above the stigma ([Figure 1A](#pone-0057902-g001){ref-type="fig"}). This arrangement is known as reverse herkogamy [@pone.0057902-Barrett1]. Approach herkogamy, in which the stigma is positioned above the anthers, has not been reported to date in natural *A. thaliana* accessions.
{#pone-0057902-g001}
In this study we demontrate the existence of approach herkogamy in natural *A. thaliana* accessions collected from alpine regions in Europe and Africa and elucidate the function of herkogamy. Specifically, we address the following scientific questions: (1) What is the phenotypic variation and plasticity in herkogamy in *A. thaliana*? (2) What is the reproductive consequence of the degree of herkogamy in this highly selfing plant species under different environmental conditions? (3) What is the genetic architecture underlying this complex trait?
Results {#s2}
=======
Characterization of approach herkogamy under greenhouse conditions {#s2a}
------------------------------------------------------------------
In an analysis of natural *A. thaliana* accessions collected from alpine areas in Europe and Africa ([Table S1](#pone.0057902.s004){ref-type="supplementary-material"}), we observed substantial variation in self-fertilization rates (ranging from ∼0 to ∼100%) under pollinator-free greenhouse conditions. In particular, two of the investigated accessions, called BRA and SIM, exhibited strongly reduced seed set in comparison to the lab standard line Columbia (Col-0; [Figure 2A](#pone-0057902-g002){ref-type="fig"}). BRA showed a gradient of increasing spontaneous self-fertilization throughout the inflorescence and SIM largely failed to set seeds autonomously, while Col-0 displayed incomplete self-fertilization only in the first flowers along the main stem ([Figure 2B](#pone-0057902-g002){ref-type="fig"}). Analyses of floral morphology revealed that, in contrast to Col-0, BRA and SIM displayed approach herkogamy in the greenhouse ([Figures 1A--D](#pone-0057902-g001){ref-type="fig"}). Moreover, we observed phenotypic variation in the degree of approach herkogamy in BRA individuals ([Figures 1B & 1C](#pone-0057902-g001){ref-type="fig"}), while the phenotype was highly stable in SIM ([Figure 1D](#pone-0057902-g001){ref-type="fig"}).
{#pone-0057902-g002}
To test whether reduced spontaneous self-fertilization is due to deficiencies in sexual functions such as male or female sterility, we manually pollinated flowers with their self pollen (assisted selfing) and used the pollen to pollinate flowers of a male-sterile line (pollen siring). The resulting fertilization rates were high (\>80%) for all examined accessions ([Figure 2A](#pone-0057902-g002){ref-type="fig"}), indicating that neither male nor female sterility can account for the strongly reduced spontaneous self-fertilization. Moreover, the success of assisted selfing excluded the possibility of dichogamy, the temporal separation of the sexes. These results suggest that the observed approach herkogamy is likely the major factor underlying the strongly reduced spontaneous self-fertilization rates in BRA and SIM under greenhouse conditions.
We measured anther height and stigma height in Col-0, BRA, and SIM in the greenhouse ([Figure S1](#pone.0057902.s001){ref-type="supplementary-material"}A). In BRA, the anther height did not differ from Col-0 (*P* = 0.996), whereas the stigma height was significantly increased in comparison to Col-0 (*P*\<0.001; [Figure 2C](#pone-0057902-g002){ref-type="fig"}). In contrast, SIM had significantly reduced anther height (*P*\<0.001) but increased stigma height than Col-0 (*P*\<0.05; [Figure 2C](#pone-0057902-g002){ref-type="fig"}). We then quantified the degree of separation between anthers and stigmas (SAS) as the difference between stigma height and anther height (i.e., SAS = stigma height -- anther height), and found that both BRA and SIM had positive SAS values (i.e., approach herkogamy), whereas Col-0 had a negative SAS value (i.e., reverse herkogamy; [Figure 2D](#pone-0057902-g002){ref-type="fig"}). Hybrids between Col-0 and either BRA or SIM exhibited similar anther or stigma height, and thus had SAS values close to zero (i.e., no herkogamy; [Figures 1E, 1F](#pone-0057902-g001){ref-type="fig"}, [2C & 2D](#pone-0057902-g002){ref-type="fig"}). Hybrids between BRA and SIM resembled the strong phenotype of approach herkogamy in BRA ([Figures 1G](#pone-0057902-g001){ref-type="fig"}, [2C & 2D](#pone-0057902-g002){ref-type="fig"}), suggesting that BRA alleles are dominant. Developmental differences in floral organs between BRA, SIM, and hybrids suggest that different genetic networks may control approach herkogamy in these two accessions.
Herkogamy and its effects on mating patterns under natural conditions {#s2b}
---------------------------------------------------------------------
To assess the effects of approach herkogamy on mating patterns in BRA and SIM under natural conditions, we conducted field experiments in which we grew BRA and SIM together with 13 control accessions that exhibit reverse herkogamy ([Table S1](#pone.0057902.s004){ref-type="supplementary-material"}). Because both BRA and SIM were originally collected at high altitudes, we considered whether the expression of approach herkogamy in these two accessions was related to climatic conditions at different altitudes, and therefore grew one experimental population at high altitude and one at low altitude. Since each of the 13 control accessions displayed reverse herkagomy and was different from both BRA and SIM, we grouped them as control for comparison. Overall, substantial variation in SAS (a proxy for herkogamy) and outcrossing rates were observed within and among accessions as well as field sites ([Figures 3A & 3B](#pone-0057902-g003){ref-type="fig"}). Firstly, the outcrossing rates detected in our experimental populations were high and variable. We randomly chose 3--4 test plants from each accession out of each field site for estimating their outcrossing rates. In total, we identified 717 outcrossed offspring from the 3502 progeny that were genotyped using molecular markers. The average outcrossing rates for the populations were 22.39% (low altitude) and 14.57% (high altitude), and the accession means ranged from 4.17% to 74.87% (low altitude) and from 2.81% to 36.81% (high altitude). Secondly, at low altitude, SIM exhibited approach herkogamy (i.e., positive SAS) and had the highest outcrossing rate. At high altitude, however, the degree of approach herkogamy in SIM was highly reduced and most flowers expressed no herkogamy (i.e., SAS values close to zero; but see [Figure 1H](#pone-0057902-g001){ref-type="fig"}). Accession BRA, in contrast, did not express approach herkogamy at either high or low altitude, but instead had no herkogamy. Nevertheless, both BRA and SIM had higher outcrossing rates than the control accessions. This indicates that an absence of reverse herkogamy may reduce the efficiency of self-pollination and thus enhances the opportunity for cross-pollination in nature. Thirdly, at both high and low altitude, the control accessions stably expressed reverse herkogamy (i.e., negative SAS) and had significantly lower outcrossing rates in comparison to BRA and SIM. This suggests that efficient self-pollination may be a benefit of reverse herkogamy in this small plant with a pronounced selfing syndrome. Importantly, the degree of herkogamy expressed under field conditions was highly correlated with outcrossing rates (*R* ^2^ = 0.82; [Figure 3C](#pone-0057902-g003){ref-type="fig"}), suggesting a causal relationship.
{#pone-0057902-g003}
A further common-garden experiment that was carried out in a rural field site with only three accessions (i.e., Col-0, BRA, and SIM) consistently revealed a positive and linear relationship between the degree of herkogamy and outcrossing rates ([Figure 3D](#pone-0057902-g003){ref-type="fig"}). These results together indicate that the degree of herkogamy has a strong effect on mating patterns in *A. thaliana*: approach herkogamy can effectively promote outcrossing, no herkogamy may facilitate outcrossing, and reverse herkogamy likely ensures efficient autonomous self-pollination.
Expression of herkogamy is temperature sensitive {#s2c}
------------------------------------------------
We observed both phenotypic variation and plasticity in herkogamy in our field experiments ([Figure 3A](#pone-0057902-g003){ref-type="fig"}). Because temperature changes substantially with increasing altitude, we experimentally tested the influence of ambient temperature on the expression of herokogamy. For this purpose, we grew Col-0, BRA, and SIM plants in climate chambers that differ only in ambient temperature and estimated SAS. Highly significant genotype by temperature interactions were found (two-way ANOVA, *F~4,71~* = 12, *P*\<0.001). At 22°C or 30°C, SIM displayed approach herkogamy (i.e., positive SAS) and set few seeds autonomously, whereas BRA had negative SAS and set seeds, similar to Col-0. At 16°C, accessions SIM, BRA, and Col-0 displayed reverse herkogamy and produced full seed set through autonomous self-pollination ([Figure 4](#pone-0057902-g004){ref-type="fig"}). These findings indicate that the expression of herkogamy in BRA and SIM is highly temperature dependent, and the fact that no approach herkogamy in BRA was found in this experiment indicates the influence of other environmental factors in this accession.
{#pone-0057902-g004}
Genetic architecture underlying approach herkogamy {#s2d}
--------------------------------------------------
To investigate the genetic architecture underlying approach herkogamy we performed segregation analysis and QTL mapping in an F~2~ population derived from a cross between SIM and Col-0 in the greenhouse. We analyzed four traits -- anther height, stigma height, self-fertilization rate, and SAS -- in 227 F~2~ individuals and found that self-fertilization rates were positively correlated with SAS (*R* ^2^ = 0.66; [Figure 5A](#pone-0057902-g005){ref-type="fig"}). In total, we identified 9 QTLs underlying SAS that were distributed across all 5 chromosomes ([Figure 5B](#pone-0057902-g005){ref-type="fig"}). Each QTL explained between 2% and 18% of the variance, for a total of 55% of the variance in SAS. The QTLs controlling self-fertilization rate were largely the same as those underlying SAS, suggesting a common genetic basis and thus a causal relationship ([Figure 5B](#pone-0057902-g005){ref-type="fig"}). QTLs affecting anther height and stigma height represented a subset of those underlying SAS, in an additive pattern, indicating that anther height and stigma height are independently controlled and collectively confer approach herkogamy in SIM ([Figure 5B](#pone-0057902-g005){ref-type="fig"}).
{#pone-0057902-g005}
Discussion {#s3}
==========
Natural genetic variation in outcrossing in *A. thaliana* {#s3a}
---------------------------------------------------------
Given that *A. thaliana* is generally considered to be a highly selfing plant, the outcrossing rates detected in our experimental populations were surprisingly high and variable. As we cannot recognize outcrossing events when the pollen donor has the same genotypes as the pollen recipient at the examined loci, the detected outcrossing rates may be conservative. In nature, many factors can influence outcrossing rates in a population, such as population density, pollinator availability, and floral morphology. It remains unknown whether some *A. thaliana* populations exhibit such high outcrossing rates in nature as observed in our experimental populations. Therefore, it would be interesting to examine the outcrossing rates of these *A. thaliana* populations (i.e., BRA and SIM) in their native habitats, ideally over several continuous growth seasons. However, outcrossing events might go undetected if one examines only a limited number of genetic loci, because of insufficient genetic variation within natural populations of *A. thaliana*; a colonizing species usually characterized by small local population sizes and strong population structure [@pone.0057902-Bomblies1], [@pone.0057902-Franois1]. Accordingly, in this study we designed experimental arrays consisting of multiple *A. thaliana* genotypes in random arrangements that facilitated the detection of outcrossed progeny through marker-assisted analyses.
Nevertheless, the remarkable variation in outcrossing rates among accessions within each experimental population cannot simply be attributed to external factors, and thus indicates that natural genetic variation among the studied accessions has a major effect on outcrossing. Even in the high-altitude area (alpine climatic conditions), the outcrossing rates in two alpine accessions (BRA and SIM) were high ([Figure 3B](#pone-0057902-g003){ref-type="fig"}). Therefore, it may be possible that substantial outcrossing events occur in their native populations. The existence of "outcrossing hotspots" in *A. thaliana* observed in a previous study reinforces our speculation [@pone.0057902-Platt1]. Hence, we consider that mixed mating in natural *A. thaliana* populations may be more common than has been acknowledged to date.
Herkogamy and its reproductive consequences {#s3b}
-------------------------------------------
The main components of herkogamy in most flowering plants can be divided into vertical and horizontal separation of stigmas and anthers within flowers ([Figure 6A](#pone-0057902-g006){ref-type="fig"}). Two types of herkogamy that focus on the vertical separation, approach and reverse herkogamy, as well as their reproductive consequence have been well investigated in diverse plant species (see the review in [@pone.0057902-Barrett1]). The functional relevance of the horizontal separation, in contrast, has received less attention. In *A. thaliana*, horizontal stigma-anther separation has been lost, most likely during the evolution of the selfing syndrome, in contrast to its outcrossing relatives (e.g., *A. lyrata*; [Figure 6B](#pone-0057902-g006){ref-type="fig"}). As a consequence, ripe pollen in a normal *A. thaliana* flower can be efficiently deposited on the receptive stigma by gravity for self-pollination. This could be quite different to those self-compatible plants with relatively large floral displays, in which there is horizontal separation to some degree, and thus is an important point when considering the function of herkogamy.
{#pone-0057902-g006}
In this study, we included a relatively large number of control accessions with different origins representing 10 natural accessions collected in the same areas as BRA, together with another high altitude accession (Sha), the lab standard line (Col-0), and another widely used accession (Bay-0). We found that all control accessions displayed reverse herkogamy and had relatively low outcrossing rates in our experiments, compared to the high outcrossing rates in accessions BRA and SIM ([Figures 3A & 3B](#pone-0057902-g003){ref-type="fig"}). These results indicate that reverse herkogamy facilitates efficient self-pollination in the absence of horizontal stigma-anther separation, whereas approach herkogamy (or no herkogamy) can reduce the efficiency of self-pollination and thus promote outcrossing in *A. thaliana*. Importantly, the observed change in the degree of herkogamy in *A. thaliana* was minute in the investigated accessions and SAS values typically ranged from −0.5 mm to 0.5 mm ([Figures 2D](#pone-0057902-g002){ref-type="fig"} & [3A](#pone-0057902-g003){ref-type="fig"}). Nevertheless, these subtle floral modifications caused a dramatic change in the mating patterns of these *A. thaliana* accessions.
Possible significance of environment-dependent herkogamy {#s3c}
--------------------------------------------------------
The expression of approach herkogamy in accessions BRA and SIM was highly environment-dependent. What is the ecological significance of such a trait? We hypothesize that the environment-dependency of approach herkogamy in *A. thaliana* may be adaptive and thus maintained by natural selection if approach herkogamy is expressed under suitable ecological conditions and confers the advantages of outcrossing, while it is repressed under less suitable conditions, such as during cold and rainy weather periods, when pollinators are scarce. A previous study on *Collinsia verna* showed that populations had a dynamic mating system, adapting to different pollination environments, which resulted in mixed mating and reproductive assurance [@pone.0057902-Kalisz1]. Together, these findings suggest that environment-dependent flexible mating systems may be advantageous.
Molecular basis of approach herkogamy {#s3d}
-------------------------------------
Approach herkogamy has previously been reported in laboratory mutants of *A. thaliana* such as *opr3*, *hth* and *rdr6*, where it is usually associated with reduced fertility [@pone.0057902-Stintzi1]--[@pone.0057902-Tantikanjana1]. The natural examples of approach herkogamy analyzed in the present study are different from these reported single-gene mutants. Our natural accessions displaying environment-dependent approach herkogamy have normal fecundity and approach herkogamy in SIM is controlled by multiple QTLs, similar to the situation reported in other species [@pone.0057902-Chen1], [@pone.0057902-Fishman1], [@pone.0057902-Chen2]. Moreover, BRA and SIM were collected from disjunct geographical regions in the Swiss and Ethiopian mountains and approach herkogamy is achieved differently in the two accessions ([Figure 2C](#pone-0057902-g002){ref-type="fig"}). Thus, approach herkogamy in natural *A. thaliana* accessions has a complex genetic basis and may have evolved independently multiple times. As a first step towards harnessing the power of *A. thaliana* for a molecular dissection of approach herkogamy, we here analyzed the developmental characteristics, environmental control, genetic architecture, and the effects of this trait on the mating patterns. Detailed analysis of molecular evolutionary mechanisms of approach herkogamy seems promising and must await further study.
Materials and Methods {#s4}
=====================
Plant material and growth conditions {#s4a}
------------------------------------
Detailed information about the *A. thaliana* accessions used in this study is listed in [Table S1](#pone.0057902.s004){ref-type="supplementary-material"}. Seeds of Col-0 (N1092), Bay-0 (N954), Sha (N929), and a male-sterile accession (N75) were obtained from the European Arabidopsis Stock Centre (<http://arabidopsis.info/>). Seeds were stratified at 4°C for 4--5 days to break seed dormancy, and were germinated and grown at 22/20°C (day/night) under long-day light conditions \[16 h/8 h (day/night)\] for 7 days. All plants were then vernalized at 4°C under short-day light conditions \[8 h/16 h (day/night)\] for 4--5 weeks and subsequently transplanted into individual pots and used for the experiments.
The expression of herkogamy is environment-dependent, and details of the growth conditions are therefore provided here. The pollinator-free greenhouse compartments used received natural light, supplemented by artificial light under low illumination, and had temperature/humidity control systems. We used long-day light conditions and 22/20°C (day/night) for plant growth in the greenhouse. Only artificial light was available in climate chambers with temperature/humidity control systems.
Measurement of herkogamy {#s4b}
------------------------
We measured anther height and stigma height in a freshly opened flower under a microscope ([Figure S1](#pone.0057902.s001){ref-type="supplementary-material"}A), and the degree of separation between anther and stigma (SAS, as the proxy of herkogamy) was estimated as the difference between stigma height and anther height (i.e., SAS = stigma height -- anther height). For each individual, three to five freshly opened flowers were measured and for each genotype, at least three individuals were typically measured.
To assess changes in SAS during floral development and to determine the optimal stage for measurement, we first estimated SAS in flowers of Col-0, BRA, and SIM grown in a climate chamber at various developmental stages. For each inflorescence, we measured 6 flowers simultaneously and assigned the most recently opened flower to stage 0 (approximately equal to stage 13 described in [@pone.0057902-Smyth1]); three older flowers were consecutively assigned to stages 1, 2, and 3, respectively; whereas 2 younger flowers were assigned to stages -1 and -2. For each accession, mean SAS values were determined by measuring flowers from 6--7 inflorescences in 3 individuals. A polynomial regression was fitted to the SAS values against different developmental stages. Our results indicate that SAS values peaked at stage 0 in each investigated accession ([Figure S1](#pone.0057902.s001){ref-type="supplementary-material"}B), and this stage was subsequently used for all measurements.
Estimation of fertilization rates of specific siliques {#s4c}
------------------------------------------------------
To assess the fertilization rate of a specific silique, we dissected the silique under a microscope and manually counted the numbers of fertilized and non-fertilized ovules ([Figure S1C](#pone.0057902.s001){ref-type="supplementary-material"}). The fertilization rate of each silique was calculated by dividing the number of fertilized ovules by the total number of ovules. The first 30 siliques along the main stems from three individuals of each accession (i.e., Col-0, BRA or SIM) were measured, and used for regression modeling and calculating overall self-fertilization rates ([Figures 2A & 2B](#pone-0057902-g002){ref-type="fig"}). In the assisted selfing and pollen siring groups, 15--40 siliques (mean = 28) and 11--30 siliques (mean = 24), respectively, were scored.
Influence of ambient temperature on the expression of herkogamy {#s4d}
---------------------------------------------------------------
The effects of different ambient temperatures on herkogamy were tested in three climate chambers set to identical light conditions (i.e., long-day) at 16°C, 22°C, or 30°C. Eight replicates per accession (i.e., Col-0, BRA or SIM) were grown in a randomized design within each chamber. SAS was determined from three flowers per individual.
QTL analysis {#s4e}
------------
An F~2~ population, derived from a cross between SIM (mother) and Col-0 (father), was grown in the greenhouse. SAS was estimated in three flowers of each F~2~ individual, and the self-fertilization rate of each individual was estimated as the proportion of fertilized siliques (judged by the morphology of siliques) among the first 20 siliques along the main stem. In total, 227 F~2~ individuals were phenotyped and further genotyped using 47 fluorescent-labeled microsatellite markers (markers were selected from the following website based on their genomic positions: <http://www.inra.fr/internet/Produits/vast/msat.php>) distributed across the *A. thaliana* genome. Linkage maps were constructed using JoinMap 4 [@pone.0057902-VanOoijen1] and QTL analysis was performed with MapQTL 5 [@pone.0057902-VanOoijen2]. To improve the normality of phenotypic data, anther height and stigma height were box-cox transformed, while SAS was arcsin transformed. QTL mapping was initially performed on transformed data with interval mapping (IM) and followed by composite interval mapping, referred to as MQM mapping in MapQTL 5, where markers outside the test interval in the genome were used as cofactors to increase the power and precision of QTL identification. The significant cofactors for each MQM model were determined through an iterative automatic cofactor selection. The genome-wide logarithm of odds (LOD) significance threshold was obtained from permutation tests with 1000 replicates as implemented in MapQTL 5. The percentage of variance explained, and the additive and dominance effects of each QTL were estimated at the LOD peak using MapQTL 5.
Field experiments at high and low altitudes {#s4f}
-------------------------------------------
The field experiments were performed in the Swiss Alps (in the area of Flims, Switzerland) at one low altitude site (Boefel; 640m a.s.l.; 46°52′23.45′′N/9°31′07.01′′E) and one high altitude site (Nagens; 2170 m a.s.l.; 46°51′52.11′′N/9°14′11.83′′E). All necessary permits were obtained for these field studies: Boefel, permitted by Christian Gerber (contact address: Canalweg 21, 7023 Haldenstein, Switzerland. Tel: 0041 81 353 1493); Nagens, permitted by Laax-Gemeinde (contact person: Claudio Coray, Via Principala 91, 7031 Laax, Switzerland. Tel: 0041 81 921 4616). At each site, 130 plants from 15 accessions ([Table S1](#pone.0057902.s004){ref-type="supplementary-material"}; 8--9 replicates per accession) were randomized and transplanted into a rectangular block (filled with identical soil) with a distance of 10 cm between direct neighbors ([Figure S2](#pone.0057902.s002){ref-type="supplementary-material"}). Plants were watered 0--2 times in the first week after transplantation when necessary. Flowers on the main stems from healthy plants (from the 15^th^--25^th^ flowers) were chosen for measuring anther height and stigma height. Three flowers from three to five individuals of each accession were measured. Towards the end of the flowering season, all aboveground material of each plant was harvested, stored in a big envelope and dried in a climate chamber at 25°C for one week. Subsequently all seeds of each plant were collected.
Common-garden experiment in a rural field site {#s4g}
----------------------------------------------
The common-garden experiment was performed in an experimental station close to Zurich (47°27′00.41′′N, 8°40′57.22′′E; 550 m a.s.l.; No specific permissions were required because it is the experimental station of ETH Zurich and our plant materials were natural germplasms). The plants (i.e., Col-0, BRA, and SIM) were vernalized at 4°C for different time periods so as to synchronize flowering among the different accessions. Plants were then transplanted into large pots (35 cm in diameter) prior to flowering. In each pot, one test plant (Col-0, BRA, or SIM) was planted in the center and 5 plants with different genotypes were planted as potential pollen donors in a circle around the test plant at a distance of 10 cm ([Figure S3](#pone.0057902.s003){ref-type="supplementary-material"}). The pots were placed at random positions in grassland with distances of approximately 1.2 m among pots. After three weeks, all pollen-donor plants and unopened flower buds or inflorescences on the test plants were removed. Mature siliques were collected from each test plant.
Estimation of outcrossing rates {#s4h}
-------------------------------
We randomly chose 3--4 test plants from each accession out of each field site for estimating their outcrossing rates. For each test plant, 32--48 progenies were grown and genotyped at four microsatellite loci. For each multiplex set, four highly polymorphic makers labeled with different fluorescent dyes (i.e., FAM or HEX) were selected from 50 loci examined in all parental accessions ([Table S2](#pone.0057902.s005){ref-type="supplementary-material"}). Labeled PCR products were subjected to fragment analysis with a 3730xl DNA Analyzer (Applied Biosystems), and the results were analyzed using GeneMapper 4.0 (Applied Biosystems) software as follows: (**i**) for each individual, we first checked if there was a maternal allele for each of the four loci. If not, we regarded it as seed contamination and excluded it. Overall, we found very low seed contamination. (**ii**) We then examined if there were additional alleles to the maternal alleles. An individual offspring was regarded as outcrossed progeny (hybrid between two inbred accessions) if more than two additional alleles were present among the four investigated loci. Individuals with one additional allele, which was rarely observed in our samples, were considered possible technical errors, and were excluded from outcrossed progeny. (**iii**) To determine if we could effectively identify real outcrossed progeny for each outcrossed individual, we examined if the potential father could be found in one of the included accessions by comparing alleles. We analyzed 169 outcrossed individuals from 414 samples in details, and successfully identified potential fathers for 98.2% of the examined hybrids. Additional alleles in three exceptional individuals could be partially found in the studied accessions. This confirmed that real hybrid offspring were actually identified in our study. (**iv**) The outcrossing rate for each test plant was then estimated as the proportion of outcrossed progeny.
Statistical analysis {#s4i}
--------------------
Statistical analyses were performed using R [@pone.0057902-R1]. Tukey\'s post-hoc significance tests were used for multiple comparisons, whereas Pearson\'s correlation tests were used to analyze trait associations. Two-way ANOVA was performed to assess the effects of genotype, ambient temperature and their interaction on SAS.
Non-linear least-square regression models for the self-fertilization rates of siliques along the main stems of the three studied accessions (i.e., Col-0, BRA, and SIM) were fitted in R. The function is: y = exp(a+b\*x)/(1+exp(a+b\*x)). *x* is a vector of predictors, indicating the position of a specific silique in the main stems, and *y* is the observation, i.e., the autonomous self-fertilization rate. *a* and *b* are regression parameters.
Supporting Information {#s5}
======================
######
**Illustrations of the phenotypic measurements.**
(TIF)
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Click here for additional data file.
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**Illustration of the box design for the field experiments.**
(TIF)
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Click here for additional data file.
######
**Illustration of the pot design for the common-garden experiment.**
(TIF)
######
Click here for additional data file.
######
Information on *A. thaliana* accessions used in this study.
(XLSX)
######
Click here for additional data file.
######
Information on the markers used in this study and their polymorphisms in the studied accessions.
(XLSX)
######
Click here for additional data file.
We thank M. Leuzinger, B. Blattmann, M. Frei, F. Berger, Dr. S. Guesewell and Dr. J. Jansa and the Genetic Diversity Center (GDC) at ETH Zurich for technical support. We thank Dr. M. Koch for providing various *Arabidopsis* species for a close observation of herkogamy and Dr. M. Nordborg, Dr. D. Luo, and Dr. J. Levine for comments on earlier versions of the manuscript.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: YL AW. Performed the experiments: YL. Analyzed the data: YL. Contributed reagents/materials/analysis tools: YL AW. Wrote the paper: YL AW.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Development of a safe and effective prophylactic HIV vaccine remains enormously challenging, due to the virus\'s high diversity and our limited understanding of immune correlates of protection. While most effective vaccines are designed to mimic natural infection and protective immune responses to it, such a template for HIV vaccine design remains elusive, since sterilizing immune responses to natural infection have not been observed. A priority of HIV vaccine development is, therefore, to identify settings where natural infection elicits some immune functions desired in a vaccine. For example, HIV-infected individuals who spontaneously control viral replication have provided insights into immune mechanisms of HIV control [@ppat.1003593-Baker1]. However, models where the response, rather than delaying disease, prevents infection -- the ultimate goal of a prophylactic vaccine -- remain less well characterized. Studies of superinfection (reinfection from a different partner) provide a unique model in which to investigate the impact of pre-existing responses on susceptibility to infection by diverse circulating viral variants, which include multiple subtypes with up to 30% sequence variation.
HIV superinfection has been reported in a number of settings [@ppat.1003593-Chohan1]--[@ppat.1003593-Redd2], implying that HIV acquisition can occur despite the immune response to initial infection. However, it remains an open question whether pre-existing infection affords some protection from superinfection, and individuals who do become superinfected are a select subset deficient in a particular aspect of immunity. Published estimates of superinfection incidence vary from no identified cases [@ppat.1003593-Baker1], [@ppat.1003593-Tsui1]--[@ppat.1003593-Gonzales1] to rates roughly similar to initial infection [@ppat.1003593-Chohan1]--[@ppat.1003593-Redd2], [@ppat.1003593-Piantadosi2], [@ppat.1003593-Kraft1]. These discrepancies are largely explained by differences in participant inclusion criteria and study design. The studies that have directly compared initial and superinfection incidence have had limited statistical power due to cohort size [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2], [@ppat.1003593-Kraft1] or number of cases of superinfection identified [@ppat.1003593-Smith1], [@ppat.1003593-Redd1]. Additionally, methods used to identify superinfection have evolved. Superinfection is most reliably detected in longitudinal samples by the presence of a single viral clade initially followed by introduction of a second phylogenetically distinct clade [@ppat.1003593-vanderKuyl2]. Detection sensitivity is dependent on the number of genomic regions analyzed [@ppat.1003593-Piantadosi1], as well as sequencing depth [@ppat.1003593-Pacold1]. Until recently, sequences were obtained by limiting dilution amplification and Sanger sequencing [@ppat.1003593-Chohan2], [@ppat.1003593-Altfeld1], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2], which limits detection to cases where the second virus is relatively abundant. The development of next generation sequencing (NGS) has enabled higher-throughput, deeper sequencing of large cohorts [@ppat.1003593-Pacold1], [@ppat.1003593-Redd3].
To date, the largest study to examine the rate of superinfection in a prospective seroincident cohort was a NGS screen by Redd *et al.* of 149 individuals in which 7 cases were identified [@ppat.1003593-Redd1]. No statistically significant difference was found between the incidences of initial infection and superinfection, though the relatively small number of cases may have resulted in limited statistical power. A greater number of cases was found in a high-risk cohort in Mombasa, Kenya, with 12 cases of 56 women screened [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2]. However, this study used Sanger sequencing to sample ∼7 clones per sample, which could miss lower frequency variants, and was not powered to compare incidences. In the present study, we developed a NGS method for identification of superinfection, and used it to screen 129 women in the same Mombasa cohort, including those classified as singly infected in the prior study. We identified 9 additional cases of superinfection, for a total of 21 cases in this cohort. These combined data enabled comparison of the incidence rates of initial infection and superinfection.
Results {#s2}
=======
Identification of superinfection cases in a NGS screen {#s2a}
------------------------------------------------------
In order to conduct a sensitive, high-throughput screen for superinfection in the Mombasa cohort, we developed a pipeline for amplification, next-generation sequencing (NGS), data cleaning, and phylogenetic and sequence diversity analysis of longitudinal plasma RNA ([Fig. 1](#ppat-1003593-g001){ref-type="fig"}). One-hundred thirty-two women met our selection criteria for the NGS superinfection screen, with a median follow-up time of 2046 days (IQR 1265--2848). We successfully amplified *gag*, *pol* and *env* at three timepoints in 115 women and at least two genomic regions in at least the first and last timepoints in 129. The remaining 3 women were dropped from analysis. In total, ∼1.7 million raw sequencing reads were obtained, with ∼1.25 million passing quality filtering: a median of 901 per amplicon per sample.
{#ppat-1003593-g001}
Women were considered putative superinfection cases if the posterior probability of monophyly supported single infection at the earliest studied timepoint followed by introduction of a distinct viral clade and increased viral diversity consistent with that seen in simulated dual infection ([Fig. 1e&f](#ppat-1003593-g001){ref-type="fig"}). Putative cases of superinfection were confirmed and their timing specified by analyzing intervening timepoints. Nine cases of superinfection were detected and their timing specified. One case of suspected dual infection was detected, in which two clades were detected at the earliest sample analyzed (60 days post-infection (dpi)) and throughout infection (data not shown).
Characteristics of superinfection cases {#s2b}
---------------------------------------
Example data from two cases of superinfection are summarized in [Figures 2](#ppat-1003593-g002){ref-type="fig"} and [3](#ppat-1003593-g003){ref-type="fig"}. Initial screening of subject QD151 ([Fig. 2](#ppat-1003593-g002){ref-type="fig"}) showed monophyletic subtype A infection at 39 dpi and two subtype A clades in all three genes at 938 and 1701 dpi. In subsequent analysis of intervening timepoints the second clade was first detectable at 801 dpi ([Fig. 2a](#ppat-1003593-g002){ref-type="fig"}). At this time, pairwise distance increased sharply, for example in *gag* from 0.27% at 241 dpi, to 12.75% at 801 dpi ([Fig. 2b](#ppat-1003593-g002){ref-type="fig"}), into the range observed in simulated dual infections. These observations supported introduction of a second subtype A variant between 241 and 801 dpi. The initial clade was no longer detectable in *pol* at 1701 dpi, suggestive of a genomic recombination event ([Fig. 2c](#ppat-1003593-g002){ref-type="fig"}). Similarly, subject QB210 ([Fig. 3](#ppat-1003593-g003){ref-type="fig"}) showed initially monophyletic infection with a subtype A/D virus, followed by introduction of a subtype C/D virus at 163 dpi, evidenced by polyphyly and a shift in pairwise distance (\>10%) in all 3 genes ([Fig. 3a and 3b](#ppat-1003593-g003){ref-type="fig"}). In intervening timepoints, the second variant could be detected in all genes at 163--170 dpi, but was undetectable in *gag* and *pol* after 170 dpi, indicating recombination ([Fig. 3c](#ppat-1003593-g003){ref-type="fig"}).
{#ppat-1003593-g002}
{#ppat-1003593-g003}
Characteristics of the 9 new cases of superinfection are summarized in [Table 1](#ppat-1003593-t001){ref-type="table"} and [Figure S2](#ppat.1003593.s002){ref-type="supplementary-material"}. In all but two cases the superinfecting variant was detected in all 3 amplicons in at least one timepoint. In all cases, the superinfecting variant was detected at multiple timepoints in at least one amplicon. In one case (QC369), the initial variant became undetectable in any amplicon following superinfection, suggesting it was replaced, to our detection limit, by the superinfecting variant. Both variants were detected at two timepoints each, the initial variant at 17 dpi and 28 dpi, and the superinfecting variant at 143 dpi and 451 dpi ([Fig. S2](#ppat.1003593.s002){ref-type="supplementary-material"}), indicating this result was not due to contamination. Further, the possibility of sample mix-up was excluded by HLA-typing (data not shown). As illustrated in [Figures 2](#ppat-1003593-g002){ref-type="fig"}, [3](#ppat-1003593-g003){ref-type="fig"} and [S2](#ppat.1003593.s002){ref-type="supplementary-material"}, in the other 8 cases, variants were intermittently detected in different amplicons at different times, suggestive of genomic recombination and dynamic turnover of the circulating viral population.
10.1371/journal.ppat.1003593.t001
###### Summary of 9 new superinfection cases in the Mombasa cohort.
{#ppat-1003593-t001-1}
ID Window SI \[midpoint\] (dpi) Initial subtype SI subtype Virus outcome
--------------------------------------------- ------------------------------ ----------------- ------------ --------------- ----------------------------------- ----------------------------------- --- ---------------
QB210 17--163 \[90\] A D D C[†](#nt102){ref-type="table-fn"} C[†](#nt102){ref-type="table-fn"} D Recombination
QC369 29--143 \[86\] A A A A A A Replacement
QD149 996--1086 \[1041\] A A A \- \- A Recombination
QD151 241--801 \[521\] A A A A A A Recombination
QD696 49--174 \[112\] A A A \- A A Recombination
QF441 255--444 \[350\] A A A D[†](#nt102){ref-type="table-fn"} D[†](#nt102){ref-type="table-fn"} A Recombination
QF564 17--1270 \[644\] A A A D[†](#nt102){ref-type="table-fn"} D[†](#nt102){ref-type="table-fn"} A Recombination
QG262 59--144 \[102\] A A A A A A Recombination
QG284 155--260 \[208\] A A A A A A Recombination
Proportion subtype A (%) 100.0 88.9 88.9 57.1 62.5 88.9
Proportion subtype D (%) 0.0 11.1 11.1 28.6 25.0 11.1
Proportion subtype C (%) 0.0 0.0 0.0 14.3 12.5 0.0
Proportion intersubtype superinfections (%) 42.8 37.5 0.0
-superinfecting variant not detected.
intersubtype superinfection.
Combining the data here with those from previous studies in the Mombasa cohort [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2], a total of 146 women were examined for superinfection: 90 were tested using NGS, 39 using both NGS and Sanger sequencing, and 17 using only Sanger sequencing. Among the 39 women previously identified as singly infected by Sanger sequencing and tested by NGS here, no new cases of superinfection were identified, suggesting older methods were sensitive enough to detect superinfection. Twenty-one cases of superinfection were confirmed based on detection of the superinfecting virus in two or more samples. The timing windows of all 21 superinfection events are summarized in [Figure 4](#ppat-1003593-g004){ref-type="fig"} and [Table S2](#ppat.1003593.s005){ref-type="supplementary-material"}. The midpoint of the timing window of the 9 new cases ranged from 81 to 1041 dpi, with 6 occurring within the first year of infection. The window of superinfection events was defined to a median of within 127 days, with window sizes of 90 to 1253 days. Timing of all 21 cases ranged from 63 to 1895 dpi, defined to a median of within 146 days.
![Summary of timing of superinfection events relative to initial infection events.\
Subject identifiers for the 21 cases of superinfection are listed with the 9 cases identified here listed first, followed by the 12 cases from prior studies [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2]. Time since initial infection (years) is represented as blue bars. The red rectangles represent the interval between the last timepoint at which only the initial variant was detected and the first timepoint at which the superinfecting variant was detected. The blue line marks the interval midpoint. \*QB045 was HIV RNA-positive at enrollment and was therefore excluded from the incidence analysis.](ppat.1003593.g004){#ppat-1003593-g004}
Sequence similarity between initial and superinfecting viruses {#s2c}
--------------------------------------------------------------
We detected both inter-subtype and intra-subtype superinfections. In 6 of 9 cases identified by NGS, the superinfecting variant was the same subtype as the initial variant in every gene where both were detected. In all 9 cases, the variants were the same subtype in the *env* amplicon ([Table 1](#ppat-1003593-t001){ref-type="table"}). Among all 21 cases of superinfection ([Table S2](#ppat.1003593.s005){ref-type="supplementary-material"}), the majority of superinfection events we detected were intrasubtype, regardless of genomic region: 53.8% were intrasubtype based on *gag* sequence, 62.5% based on *pol*, and 70.6% based on *env*.
We further investigated the possibility of a bias in sequence similarity of superinfecting variants to initial variants by analyzing amino acid diversity. We compared the pairwise amino acid distance between initial and superinfecting variants within each superinfection case to the distance that would be expected by chance. The latter was modeled by simulated mixtures of sequences from all possible pairs of singly infected individuals in the Mombasa cohort ([Fig. 5](#ppat-1003593-g005){ref-type="fig"}). Using NGS data from the 9 superinfection cases and 120 singly infected women screened here, we found no significant differences between the sequence similarity within superinfected individuals and that expected by chance ([Fig. 5a](#ppat-1003593-g005){ref-type="fig"}). Including Sanger sequencing data from the additional 12 superinfected women previously screened yielded a similar result ([Fig. 5b](#ppat-1003593-g005){ref-type="fig"})
![Sequence similarity between initial and superinfecting variants.\
Boxplots of pairwise amino acid distances in each genomic region are displayed, comparing the distance between the initial and superinfecting sequences within each superinfection case (red) to the distance between sequences in simulated mixtures of randomly selected pairs of singly-infected individuals (green). **A.** Analysis of 454 sequences from NGS screen. **B.** Analysis of 454 sequences from NGS screen and Sanger sequences from 12 previously identified cases of superinfection [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2].](ppat.1003593.g005){#ppat-1003593-g005}
Incidence and timing of initial infection and superinfection {#s2d}
------------------------------------------------------------
The incidence of superinfection among women who were screened was compared to the incidence of initial infection in the entire cohort at risk. Only incident HIV infections (occurring after enrollment in the cohort) were included. Fourteen women who were seronegative but HIV RNA positive at enrollment were excluded for this reason. Seven of these had been screened for superinfection, and one was found to be superinfected, which mirrors the frequency of superinfection observed in the entire group. The individual with evidence of dual infection at the earliest timepoint was also excluded, since we were unable to distinguish coinfection from superinfection. After exclusions, 1910 women were at risk of initial infection, contributing 5124 person-years, and 138 women were screened for superinfection, contributing 764py following first infection. There were 295 initial infections, giving a crude incidence rate of 5.7 per 100pys, and 20 superinfections, giving a crude incidence rate of 2.61 per 100 pys.
The incidence of superinfection and initial infection over time is summarized in [Figure 6](#ppat-1003593-g006){ref-type="fig"}. We used Andersen-Gill proportional hazards analysis to generate a hazard ratio (HR) relating the incidence of superinfection to that of initial infection. The unadjusted HR for this comparison was 0.49 (CI 0.31--0.76, p = 0.0018). Variables previously shown to influence HIV exposure risk in this cohort [@ppat.1003593-Martin1], [@ppat.1003593-Baeten1] were included as adjustments in the model (summarized in [Table 2](#ppat-1003593-t002){ref-type="table"}). These included self-reported sexual risk behavior, place of work, hormonal contraceptive use, genital tract infections, years in sexwork, age at first sex, total follow-up time in the cohort and calendar year. The HR for superinfection compared to initial infection, adjusted for these variables, was 0.47 (CI 0.29--0.75, p = 0.0019). Since proportional hazards analysis is based on time to infection and the precision with which superinfection timing was determined varied between cases, we performed sensitivity analyses setting infection timing for all cases to the start or midpoint of the timing windows rather than the end, as done for the above analysis. In both of these analyses, significant differences in incidence were also observed: setting infection timing to the start of the windows, the adjusted HR was 0.33 (CI 0.18--0.58, p = 0.00012); using the window midpoints, the adjusted HR was 0.39 (CI 0.23--0.63, p = 0.00016).
{#ppat-1003593-g006}
10.1371/journal.ppat.1003593.t002
###### Sociodemographic and clinical characteristics of women at risk of initial infection and screened for superinfection.
{#ppat-1003593-t002-2}
Initial infection risk set n = 1910 Superinfection risk set (screened) n = 138
------------------------------------------------------- ------------------------------------- --------------------------------------------
Age 26.0 (23.0--21.0) 28 (24.5--33.4)
Years education 8 (7--10) 7 (7--10)[†](#nt105){ref-type="table-fn"}
Parity 1 (1--2) 2 (1--2)[†](#nt105){ref-type="table-fn"}
Bar worker, no. (%) 1410 (74) 117 (85)[†](#nt105){ref-type="table-fn"}
Alcohol, no. (%) 1459 (76) 116 (84)[†](#nt105){ref-type="table-fn"}
Years in sexwork 1.0 (0.1--3.0) 3.2 (1.5--5.7)
Age at first sex 17 (15--18) 17 (15--18)
Sexual risk behavior in past week
Sex frequency 2 (1--3) 1 (0--2)
Unprotected sex frequency 0 (0--1) 0 (0--1)
Number sexual partners 2 (1--3) 1 (0--1)
Hormonal contraception in past 70 days, no. (%)
OCP 234 (12) 20 (14)
Depot 400 (21) 53 (38)
Norplant 39 (2) 2 (1)
IUD 41 (2) 3 (2)
STI in past 70 days, no. (%)
Bacterial vaginosis 665 (35) 71 (51)
Cervicitis 235 (12) 30 (22)
Genital ulcer disease 35 (2) 10 (7)
Gonorrhea 85 (4) 16 (12)
Trichomoniasis 105 (5) 22 (16)
Any 900 (47) 95 (69)
Total years follow-up[∧](#nt106){ref-type="table-fn"} 1.6 (0.3--5.3) 6.7 (4.3--9.8)
Calendar year, median (IQR) 1997 (1995--2002) 1997 (1995--1999)
Values shown are median (IQR), unless otherwise specified.
Data shown were collected at time of entry into each risk set (seroincident cohort enrollment for initial infection risk and initial HIV infection for superinfection risk), except where marked.
at seroincident cohort enrollment.
Time from seroincident cohort enrollment to censoring, regardless of infection events.
We assessed whether the risk of superinfection varied with time since initial infection by dividing our data into infection events occurring early or late in follow-up and estimating the HR, as above, in each subset. We found that within the first 6 months at risk, the incidence rates of initial and superinfection did not differ significantly (adjusted HR 0.73, p = 0.51), whereas after 6 months the rate of superinfection was lower than that of initial infection (adjusted HR 0.40, p = 0.0017). A similar result was observed when considering events within or beyond one year at risk: within the first year, the incidence rates of initial and superinfection did not differ significantly (adjusted HR 0.54, p = 0.14), but after one year the rate of superinfection was significantly lower (adjusted HR 0.43, p = 0.0059). Sensitivity analyses setting infection time to the start and midpoint of the timing windows as above reproduced the same results (data not shown).
We noted that the previous screens in the cohort appeared to detect a higher frequency of superinfection than the NGS screen (12 cases of 56 women screened, compared with 9 cases of 90), with a greater fraction of the events occurring later after initial infection ([Fig. 4](#ppat-1003593-g004){ref-type="fig"}). Since the NGS screen spanned later years in the cohort than the previous studies, such a difference could be due to the known decline in infection risk in the cohort over calendar time [@ppat.1003593-Martin1], [@ppat.1003593-Baeten1]. However, the numbers of events are small when the datasets are considered separately and the difference both in superinfection incidence rate and post-infection timing between the two studies was not statistically significant (data not shown).
Discussion {#s3}
==========
In this study we used NGS to screen for superinfection in 129 high-risk women and identified 9 cases of superinfection. Combined with previous studies[@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2], a total of 21 cases of superinfection were detected among 146 women screened in this cohort. There was a statistically significant difference between the incidence of superinfection (2.61 per 100pys) and initial infection (5.75 per 100 pys), with a hazard ratio of 0.47 after adjusting for potential confounding factors. This suggests that HIV infection provides partial protection from subsequent infection.
The relatively large size of this cohort and high number of superinfection cases enabled us to detect for the first time a statistically significant difference between the incidence of initial infection and superinfection. This possibility has been proposed previously, though the studies were not designed and/or powered to detect a difference [@ppat.1003593-Piantadosi2], [@ppat.1003593-Kraft1]. In the largest incidence study prior to the present study, Redd *et al.* screened a comparable number of individuals (149) in a lower-risk cohort and identified 7 cases of superinfection. The incidence of superinfection was not found to differ significantly from initial infection, but there was a trend for lower incidence of superinfection when controlling for baseline sociodemographic differences between the groups at risk of initial and superinfection. Analysis of our data using the same methods as Redd *et al.* -- Poisson regression with propensity score matching [@ppat.1003593-Redd1] -- was consistent with the results of our Andersen-Gill analysis, showing a significant difference in incidence, with an estimated incidence ratio of 0.48 (p = 0.011) comparing superinfection to initial infection.
In addition to sample size, two strengths of our incidence analysis were our specification of infection timing to within a few months on average and our comparison of initial and superinfection risk within the same cohort. These enabled us to adjust for the same potential confounding factors in both the initial infection and the superinfection risk sets, using frequently collected time-varying covariate data. Particularly important, given the sequential nature of superinfection, was adjustment for calendar year to control for decline in infection risk in the cohort over time. The distributions of initial and superinfection events over calendar time were similar ([Fig. S3](#ppat.1003593.s003){ref-type="supplementary-material"}), suggesting community-level changes over time did not severely bias our analysis.
The ∼two-fold reduction we found in the incidence of superinfection has a number of possible interpretations. First, it may indicate that the adaptive immune response elicited by initial infection provides partial protection from second infection. If this were the case, superinfection might preferentially occur early in infection, before the response has matured [@ppat.1003593-Chohan1], [@ppat.1003593-Redd2], [@ppat.1003593-Smith2]. In support of this idea, we found that, although superinfection occurred throughout the course of first infection, the incidence of superinfection was significantly lower than initial infection after the first 6 months of infection, but not earlier. This suggests that susceptibility to superinfection decreased over time, coincident with broadening and strengthening of HIV-specific immunity. Indeed, this has been suggested by two earlier studies, each documenting three cases of superinfection that occurred within the first year after initial infection [@ppat.1003593-Smith1], [@ppat.1003593-Kraft1].
If the difference in incidence we observed is due to a partially protective adaptive immune response, we would anticipate superinfection would preferentially occur with more distantly related viruses, more likely to escape the response. Using viral subtype and pairwise amino acid distance as surrogate measures of antigenic distance, our data provided no evidence of this effect. The majority of the 21 superinfection events we detected were intrasubtype, and the proportion of subtype A, C and D viral sequences was similar for the initial and superinfecting viruses, consistent with the subtype distribution in this cohort [@ppat.1003593-Rainwater1]. The pairwise distance between initial and superinfecting variants was no higher than the distribution of distances between random pairs of singly-infected individuals from the Mombasa cohort. This may potentially be explained by limited sample size or insufficient simultaneously circulating subtypes. It also may be that sequence relatedness is a poor indicator of susceptibility to the immune response or the genome regions we analyzed are not critical antigenic determinants of protection.
Alternatively, it is possible that protective immune responses are not driving the protective effect we observed. Another potential explanation for the lower risk of superinfection is that HIV infection itself may reduce infection risk by depleting permissive target cells. On the other hand, chronic immune activation and immunodeficiency following HIV infection could increase susceptibility, potentially blunting protective effects [@ppat.1003593-Klasse1]. Thus, there may be a complex interplay of biological factors impacting HIV risk in an HIV-positive individual.
So far, studies of immune correlates of superinfection have yielded variable results -- some suggesting neutralizing antibody deficits in superinfection [@ppat.1003593-Smith3], [@ppat.1003593-Basu1], while others, including studies in the Mombasa cohort, detected no differences in antibody [@ppat.1003593-Blish1], [@ppat.1003593-Forthal1] or cellular [@ppat.1003593-Blish2] responses. A major challenge in these studies has been the identification and analysis of large enough numbers of superinfection cases: the small sample sizes in studies to date (three to twelve superinfected individuals) would restrict detection to only very large effects. Small sample size is just one factor that has made detecting immune deficits associated with superinfection challenging and contributed to variable results among studies. There has also been variation among published studies in the control groups used for comparison, including the time at which the response was analyzed relative to the time of superinfection and initial infection. Given the dynamic nature of the immune response, sample timing could impact measures in both controls and cases. Furthermore, precision in the estimated timing of superinfection varies between studies, and between cases, providing an additional variable. Divergent findings between studies may also reflect differences in the assays used and subtleties in the immune parameters they capture.
Our finding of lower risk of superinfection than initial infection provides greater impetus for larger-scale comprehensive analysis of multiple immune mechanisms, including both those analyzed in the smaller studies to date and, perhaps of more interest, those not characterized in prior studies. If the discrepancies in earlier studies reflect the fact that multiple immune parameters are at play, then examining a variety of immune responses in the same individuals in a larger cohort may be needed to define responses that contribute to HIV susceptibility following initial infection.
Like all studies, the study presented here has a number of limitations. Firstly, while our screening methods are among the most sensitive developed, it remains possible that some cases of superinfection were missed. In particular, reinfection by the same source partner is not captured by any existing methods. Additionally, our specification of the timing of superinfection was limited by the samples available to us. While follow-up was generally frequent in this study population, there were six superinfection cases where sample availability limited our ability to define the time of superinfection to within a one-year period. This uncertainty in superinfection timing did not affect our findings, as we found that whether we assumed in the incidence analysis that the true timing of superinfection was at the start, midpoint or end of the timing window, the results indicated that the incidence of superinfection was significantly lower than that of initial infection. Finally, as in all observational studies, residual confounding of our incidence estimate by behavioral changes and sexual network-level factors not measured or accounted for in our analyses remains a possibility. However, the fact that we compared initial and superinfection risk within the same cohort and collected covariate data at frequent intervals enabled us to minimize this issue to an extent not possible in previous studies.
This study provides the first robust evidence that HIV infection reduces the risk of subsequent infection. The underlying mechanism remains unclear, but this finding prompts exploration of correlates of protection from HIV in high-risk individuals who continue to be exposed after first infection. Furthermore, this study reinforces that superinfection occurs at a considerable rate, calling for studies of its impact on the clinical progression, transmission, and epidemiology of HIV.
Materials and Methods {#s4}
=====================
Ethics statement {#s4a}
----------------
The study was approved by the ethical review committees of the University of Nairobi, the University of Washington and the Fred Hutchinson Cancer Research Center. Written informed consent was obtained from all participants.
Study population {#s4b}
----------------
Seronegative women in Mombasa, Kenya, attended monthly visits, at which clinical examinations, interviews and sample collection took place, as previously described [@ppat.1003593-Martin1]. Following seroconversion, sample collection took place quarterly. Individuals were selected for superinfection screening based on sample availability \<6 months and \>2 years post-initial HIV infection, and an approximately equally spaced intervening sample. Within these limitations, samples with maximal plasma viral load, \>1000 copies/ml, and prior to initiation of antiretroviral therapy were selected. Thirty-nine of 44 women previously screened for superinfection by Sanger sequencing and identified as singly infected [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2] were rescreened; the remaining 5 women did not have adequate samples available.
Viral amplification and sequencing {#s4c}
----------------------------------
HIV virions were isolated from heparinized plasma using the μMACS VitalVirus HIV Isolation kit (Miltenyi Biotec) and viral RNA extracted from 140--420 µl, depending on viral load, using the Qiamp viral RNA Mini kit (Qiagen). Nested RT-PCR of ∼500 bp in *gag*, *pol* and *env* was conducted in duplicate (see [Table S1](#ppat.1003593.s004){ref-type="supplementary-material"}). RNA input into each reaction was normalized to 3000 viral genomes according to plasma viral load, or the maximum possible where viral load was too low. RT-PCRs for the three genes were multiplexed. Nested PCR reactions were carried out separately for each region with primers containing adaptors for Roche 454 sequencing and a unique 8 bp barcode sequence to identify each sample. PCR products were purified using AMPure XP PCR purification beads (Agencourt) and quantified using the Qubit dsDNA HS assay (Invitrogen). PCR products were sequenced on the Roche 454 GS-Junior or GS-FLX titanium platform. Where initial sequencing suggested superinfection (see below), timing was inferred by sequencing intervening timepoints. Sequences are available upon request from the authors.
Bioinformatic pipeline {#s4d}
----------------------
454 sequences were error-corrected using AmpliconNoise [@ppat.1003593-Quince1]. Chimeric sequences were identified and removed using UCHIME [@ppat.1003593-Edgar1]. Cross-contamination between samples sequenced together and contamination by other lab samples was identified by all-against-all BLAST against a local database of published HIV sequences and sequences from the same sequencing run. Sequences with high identity hits to known laboratory stains or other samples from the same sequencing run were removed. Sequences with abundance \<5 reads or 0.5% of the sample, whichever was higher, were excluded from further analyses as lower abundance variants were not reproducibly detected in repeated deeper sequencing of two selected samples where rare variants formed a distinct phylogenetic clade. An amplicon-specific profile HMM was created from an alignment of representative sequences from multiple subtypes. For each subject and amplicon, 20 reference sequences were selected by placing 454 reads on a tree of candidate reference sequences [@ppat.1003593-Matsen1] and minimizing the average distance to the closest leaf [@ppat.1003593-Matsen2]. These reference sequences, representatives from subtypes common to the region, and 454 reads were aligned to the HMM using hmmalign [@ppat.1003593-Eddy1] and non-consensus columns removed. Any sequences \<200 bp long after alignment and trimming were removed. We used BEAST [@ppat.1003593-Drummond1] to calculate a posterior probability of monophyly for the sequences. A posterior sample of trees was obtained using a strict molecular clock, Bayesian Skyline Plot population model and the HKY substitution model. Each MCMC chain ran 20 million iterations, sampling every 2000, discarding the initial 25% of samples as burn-in. Chains were assessed for convergence by examining effective sample size (ESS) and by visual inspection of traces of key parameters. A strict clock was used as poor mixing was frequently observed under relaxed clock models. BEAST runs with intermediate posterior probabilities (0.2--0.8) were manually examined for recombinant sequences and run again with putative recombinants removed. Pairwise distances were calculated for all sequence pairs under the TN93 model using APE [@ppat.1003593-Paradis1], reporting the maximum within-subject distance. For comparison, 95% confidence limits of pairwise distances were calculated for sequences from known single infections (previously screened in [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2]) and simulated dual infections. Dual infections were simulated by combining all pairs of sequences from previously screened singly infected samples. Pairwise distances calculated from 454 sequences obtained in this study were compared to the upper bound of the 95% quantile of single infection distances, and the lower bound of the 95% quantile of simulated dual infection distances. This pipeline was validated and refined by processing monophyletic viral isolates, known mixtures of isolates, and known cases of superinfection detected by Sanger sequencing [@ppat.1003593-Piantadosi2]. These methods were found to be sensitive enough to distinguish two subtype A isolates mixed at abundances of 5%∶95% genome copies in all three genomic regions, and at 1%∶99% in two of three genomic regions ([Fig. S1](#ppat.1003593.s001){ref-type="supplementary-material"}).
Analysis of amino acid distance in superinfection {#s4e}
-------------------------------------------------
Sequences were aligned as for the phylogenetic analysis. Insertions relative to the reference alignment were removed, and sequences with \<60% coverage or identified as recombinants between initial and superinfecting variants upon visual inspection were excluded. For each case of superinfection, viral sequences were annotated as the initial strain or the superinfecting strain. We calculated the mean Hamming distance between amino acid sequences of the superinfecting strain from the time of superinfection detection and sequences of the initial strain up to and including this time. In calculating the mean distance, each pairwise comparison was weighted using the product of the multiplicities of the two reads. To investigate whether these distances deviated from what would be expected by chance, an artificial set of mock superinfections was generated by combining sequences from singly infected individuals. All pairs of singly infected individuals screened by 454 sequencing were enumerated. In each pair, one individual was randomly chosen to be the source of the 'initial' virus in the simulated superinfection. A time of 'superinfection' was chosen randomly from the available sampled timepoints and sequences from all timepoints up to and including this time were used for analysis. The other individual in the pair acted as the source of the 'superinfecting' virus. A time of 'transmission' was chosen randomly from the available sampled timepoints and sequences from this timepoint were used. Mean distances within pairs were calculated as above. The analysis was repeated including *gag* and *env* Sanger sequences from previously published cases [@ppat.1003593-Chohan2], [@ppat.1003593-Piantadosi1], [@ppat.1003593-Piantadosi2], trimmed to the genome region amplified for NGS, and given unit weight. A two-sample Wilcoxon test was used to test for a difference between the distances observed in true superinfections and those simulated in mock superinfections.
Incidence analysis {#s4f}
------------------
Statistical analysis was performed using R ([www.r-project.org](http://www.r-project.org)). The incidences of initial and superinfection were compared by Andersen-Gill proportional hazards analysis. The predictor was inclusion in the screen for superinfection, modeled as a time-dependent variable, and the outcome was time to HIV infection (initial and super). Timing of infection events for the incidence analysis was set to the study visit of their detection (for initial infection events the visit after inferred infection timing; for superinfection events, the time at which the superinfecting virus was first detected). Individuals who were HIV infected but not screened for superinfection were censored after acquisition of initial infection. Individuals who became superinfected were censored after acquisition of superinfection. Individuals who were screened and not found to be superinfected were censored at the last timepoint screened. Since samples after initiation of antiretroviral treatment were excluded from superinfection screening, no follow-up after treatment initiation was included. The model was adjusted for time-varying variables at each visit: calendar year, age, years in sexwork, number of weekly sexual partners, number of weekly unprotected sex acts, hormonal contraceptive use in the prior 70 days and any genital tract infection in the prior 70 days (bacterial vaginosis, cervicitis, genital ulcer disease, gonorrhea, trichomoniasis); place of work and age at first sex recorded at enrollment; and total follow-up time in the study. Incidences of initial and superinfection were also estimated as described in [@ppat.1003593-Redd1], using Poisson regression and propensity score matching to select a subset of women at risk of initial infection whose baseline risk profiles most closely matched those of women screened for superinfection.
Supporting Information {#s5}
======================
######
**Detection of control viral mixtures at 95∶5 & 99∶1.** Phylogenetic trees representing viral sequences in *gag*, *pol* and *env* from known mixtures of plasma from two individuals (QA966 and QF927) at ratios of 95∶5 and 99∶1.
(PDF)
######
Click here for additional data file.
######
**Schematics of viral variants detected in 9 cases of superinfection.** Detection of initial and superinfecting variants is indicated in each of the three genomic regions at each timepoint.
(PDF)
######
Click here for additional data file.
######
**Calendar year at the estimated time of initial infection and superinfection in superinfection cases.**
(PDF)
######
Click here for additional data file.
######
**PCR primers and conditions.**
(XLSX)
######
Click here for additional data file.
######
**Characteristics of all 21 cases of superinfection.**
(XLSX)
######
Click here for additional data file.
We are indebted to the participants of the Mombasa cohort and staff at the study clinic. We thank Frederic Bushman, Stephanie Grunberg, Christian Hoffmann and Erik Toorens at the University of Pennsylvania for use of the 454 sequencing facility and technical assistance and advice. We also thank Jan Albert and his group for helpful discussions on NGS methods during the initial phases of this study.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JO KR. Performed the experiments: KR DFB SE. Analyzed the data: COM FAM KR KOD BAR RSM JO. Contributed reagents/materials/analysis tools: COM FAM KM WJ. Wrote the paper: KR JO.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Alcohol consumption is one of the major risk factors for death and disease worldwide.^[@r01],[@r02]^ Korea is among the countries with the highest alcohol consumption in the world, and alcohol is the second leading cause of disability-adjusted life years (DALY).^[@r02]^ Since Koreans consume not only internationally popular alcoholic beverages, such as beer, whiskey, and wine, but also traditional alcoholic beverages, how various alcoholic beverages influence total alcohol consumption should be evaluated.
The impact of alcohol consumption on health varies depending on the amount of alcohol and types of alcoholic beverages consumed. The World Health Organization (WHO) has suggested that alcohol drinking has an impact on the chronic harm it causes according to the amount of alcohol consumed.^[@r03]^ Several studies have confirmed that heavy drinking is known to cause various illnesses or trauma.^[@r01],[@r04]--[@r07]^ The types of alcoholic beverages consumed are also known to be associated with mortality or risk of diseases. For example, it has been suggested that the consumption of beer or strong spirits in Western countries has more harmful effects on mortality, CVD, or cancer than wine.^[@r08]--[@r10]^
Previous studies evaluated the prevalence or correlates of alcohol consumption depending on distinctive alcohol-related cultures.^[@r11]--[@r18]^ Younger age, higher socioeconomic status (SES), and smoking were consistently found to have positive associations with alcohol consumption in various countries.^[@r15]--[@r18]^ It was reported that beer drinkers are more likely to be high-risk drinkers compared to wine drinkers,^[@r19]^ while consumers of strong spirits are more likely to be high-risk drinkers compared to beer drinkers.^[@r20]^ In Korea, it was reported that the trends in high-risk drinking fluctuated between 2008 and 2014 in men but not in women,^[@r21]^ and associations were observed between a younger age and a lower educational level and the amount of alcohol consumption.^[@r22],[@r23]^ However, the prevalence and correlates by types of alcoholic beverages have not been assessed.
Therefore, we evaluated the trends in age-standardized prevalence and correlates of high-risk consumption of alcohol according to WHO guidelines^[@r03]^ and the types of alcoholic beverages.
MATERIALS AND METHODS
=====================
Study population
----------------
The baseline information of participants in the Health Examinees-Gem (HEXA-G) study, who were derived from the Health Examinees study, a component of the Korean Genome and Epidemiology Study (KoGES_HEXA), which recruited participants aged 40--69 years at 38 general hospitals and health examination centers in eight regions from 2004 through 2013, was used for analysis. The design of the KoGES_HEXA cohort study has been described elsewhere.^[@r24],[@r25]^ Well-trained interviewers used a structured questionnaire to collect information on socio-demographic characteristics, medical history, medication usage, family history, and lifestyle factors, including smoking habits, alcohol consumption habits, weight control, and regular exercise participation. Information on reproductive history was also collected from the women, and skilled medical staff performed a physical examination on all the participants. During recruitment, all the participants voluntarily signed a consent form before entering the study, and the Institutional Review Board (IRB) of Seoul National University Hospital, Seoul, Korea approved the study (IRB No. 0608-018-179). In the HEXA-G study, 139,348 participants, comprising 46,978 men (33.7%) and 92,370 (66.3%) women, were included at baseline, after excluding participants recruited at 21 centers (*n* = 30,374). Exclusion criteria for the recruiting center were described in a previous study.^[@r26]^ Briefly, sites that only operated in the pilot study years, that have different processes for quality control and biospecimen collection, and that have been participating for fewer than 2 years were excluded. Among the HEXA-G participants, those who did not provide information on alcohol consumption status or duration of alcohol cessation were excluded (*n* = 9,524). Those missing data on any of the other variables were not excluded. Finally, 129,824 participants, including 43,927 men (33.8%) and 85,897 women (66.2%), were included in the analyses.
Alcohol consumption
-------------------
Alcohol consumption was assessed by asking whether the participants drank alcohol. Never drinkers were defined as those who had never drank alcohol in their lives for any reasons; former drinkers were those who had not drank alcohol in the prior 12 months, but had consumed it in the past; and current drinkers were those who had drunk alcohol in the prior 12 months. Because the former drinkers were more likely to have a diagnosis of disease ([eTable 1](#sm01){ref-type="supplementary-material"} and [eTable 2](#sm01){ref-type="supplementary-material"}), only the results for the current drinkers were presented in the main analysis. Among the current drinkers, information on the frequency of alcohol consumption per month, week, and day and the number of drinks each time were collected according to the type of alcoholic beverage, such as soju (the most widely consumed distilled liquor in Korea), beer, makgeolli (a traditional fermented rice wine), strong spirits, wine, and cheongju (a distilled rice liquor).^[@r20]^ Those who reported drinking each type of alcoholic beverage at least once in the prior year were regarded as drinkers of the alcoholic beverage, whereas those who drank alcohol but did not drink a certain type of alcoholic beverage were categorized as drinkers of other types of beverages. If there was no information on the frequency of alcohol consumption, the status of drinking each type of alcoholic beverage was categorized as missing. The participants were not exclusively categorized as drinkers of each type of beverage because some drank more than one type of alcohol. For example, a participant who drank beer and wine was counted twice, once as a beer drinker and once as a wine drinker. The total amount of alcohol consumed per day was assessed by summing the alcohol consumed per day from all types of alcoholic beverage after multiplying the number of drinks each time, the average frequency per day, and the standard ethanol content of one drink (soju: 19%; beer: 5%; makgeolli: 6%; strong spirits: 43%; wine: 13%; and cheongju: 13%).^[@r27]^ The amount of alcohol consumption was categorized as low-, medium-, and high-risk according to the criteria based on WHO guidelines for monitoring alcohol consumption (40 and 60 g per day for men and 20 and 40 g per day for women for medium- and high-risk, respectively).^[@r03]^
Potential correlates
--------------------
Demographic, behavioral, and perceived health-related factors, social relationships, and the diagnoses of diseases known to be associated with alcohol consumption a priori were selected as potential correlates and grouped into categories.^[@r15]--[@r18],[@r22],[@r23],[@r28],[@r29]^ The demographic factors included the following: age (40--44, 45--49, 50--54, 55--59, 60--64, and 65--69 years), education (≤middle school, high school, and ≥college), household income in Korean currency (\<2 million won, 2--3.9 million won, and ≥4 million won), marital status (living with spouse or living alone), and current occupation (office, manual, unemployed, or housewife). The behavioral factors included the following: smoking status (never, former, and current), body mass index (BMI; \<18.5, 18.5--24.9, 25.0--29.9, and ≥30 kg/m^2^) as an indicator of weight control, and duration of regular exercise (none, \<150, and ≥150 min/week). The perceived health-related factors included the following: self-rated health (good, normal, and poor) and perceived stress in the prior month (not at all, often, and frequent). Other information was also collected: social relationships including contact frequency with family (none, \<8, and ≥8 times per month) and close friends (none, \<4, and ≥4 times per month); and diagnoses of the following diseases (no and yes): diabetes, myocardial infarction, stroke, cancer, acute liver disease, fatty liver, and cirrhosis.
Statistical analysis
--------------------
All of the analyses were processed separately by sex. An age distribution of the Korean mid-year population in 2005 (in 5-year groups) was used as a standard population to estimate the age-standardized prevalence. Joinpoint regression was used to estimate the annual percentage change (APC) of the age-standardized prevalence of never, former, and current drinkers and risk level of alcohol consumption in the total population.^[@r30]^ In current drinkers, the trends in the age-standardized mean of the total amount of alcohol consumption (g/day) and the percentage of alcohol brought by each type of alcoholic beverage were evaluated.
Odds ratios (ORs) and 95% confidence intervals (CIs) for association of each potential correlate with alcohol consumption or low-, medium-, and high-risk drinking were evaluated using a multinomial logistic regression model compared to never drinkers. To avoid multicollinearity, we checked the variance inflation factors (VIF) for potential correlates that were associated with alcohol consumption ([eTable 1](#sm01){ref-type="supplementary-material"} and [eTable 2](#sm01){ref-type="supplementary-material"}) in men or women and excluded the variables with VIF greater than 10 from the model. Finally, all potential variables were included in the model. Differences between the groups according to low- and high-risk were estimated by testing linear hypotheses about the regression coefficients.^[@r31]^ Because the association of potential correlates with medium- and high-risk consumption were not different ([eTable 3](#sm01){ref-type="supplementary-material"} and [eTable 4](#sm01){ref-type="supplementary-material"}), those two categories were combined as high-risk. In the analysis by types of alcoholic beverage, the association of each potential correlate with consumption of soju, beer, makgeolli, strong spirits, and wine were compared to the consumption of other types of beverages using a logistic regression model because the drinkers of each type of alcoholic beverage were not exclusively categorized. The same model previously described was used. Because the prevalence of consuming cheongju was less than 5% in both men and women, the results for cheongju are not shown.
The JoinPoint Regression Program, version 4.4.0 (National Cancer Institute, Rockville, MD, USA), was used to conduct the joinpoint regression. SAS, version 9.4 (SAS Inc., Cary, NC, USA), was used to conduct the multinomial and multivariate logistic regression. All the tests were two-sided. Because the JoinPoint Regression Program only provided whether the *P* value was less than 0.05, the *P* value was considered an indicator of statistical significance in the joinpoint regression.
RESULTS
=======
Prevalence and trends in alcohol consumption
--------------------------------------------
The mean and standard deviation (SD) of age in the study population was 53.7 (SD, 8.4) for the men and 52.4 (SD, 7.8) for the women. The age-standardized prevalence of current alcohol consumption was 75.7% in the men and 31.9% in the women. The trends in the age-standardized prevalence of alcohol consumption and low- or high-risk alcohol consumption appear in Figure [1](#fig01){ref-type="fig"}. The prevalence of alcohol consumption did not change in the men. In women, the prevalence of current drinkers (27.1% in 2005 and 31.5% in 2013, APC = 3.06, *P* \< 0.05) and both low- (23.0% in 2005 and 29.5% in 2013, APC = 4.28, *P* \< 0.05) and high-risk drinkers (0.9% in 2005 and 1.9% in 2013, APC = 8.35, *P* \< 0.05) increased. In the current drinkers, the mean age-standardized amount of alcohol consumption was 21.4 g/day in the men and 5.5 g/day in the women during the study period. There was a trend in the increase in the amount of alcohol consumption only in the women (4.6 g in 2005 and 5.8 g in 2013, APC = 2.35, *P* \< 0.05), in whom the percentage of alcohol from beer (27.4% in 2005 and 35.4% in 2013, APC = 3.53, *P* \< 0.05) and makgeolli increased (5.0% in 2005 and 7.8% in 2013, APC = 20.55, *P* \< 0.05), while strong spirits (1.3% in 2005 and 0.4% in 2013, APC = −12.69, *P* \< 0.05) and wine decreased (7.2% in 2005 and 2.2% in 2013, APC = −14.22, *P* \< 0.05) (Figure [2](#fig02){ref-type="fig"}). Among the five types of alcoholic beverages, soju was the most frequently consumed (Table [1](#tbl01){ref-type="table"}). Those who drank strong spirits were most likely to be high-risk drinkers among both the men and women (25.6% in the men and 18.6% in the women).
{#fig01}
{#fig02}
###### Frequency of consuming each type of alcoholic beverage and total alcohol consumption according to the types of alcoholic beverages consumed by 58,977 current drinkers (32,475 men and 26,502 women) aged 40--69 in the HEXA-G study
Soju Beer Makgeolli Strong spirits Wine
-------------------------------------------------------- -------- -------- ----------- ---------------- -------
Men
Total,^a^ *N* 29,817 14,023 6,256 2,518 1,745
Frequency of alcohol consumption, %
\<1 time/week 27.1 43.5 52.2 79.9 73.4
1 time/week 22.8 26.0 20.3 10.5 12.3
2--3 times/week 35.3 23.9 17.5 7.1 9.6
≥4 times/week 14.8 6.6 10.0 2.4 4.8
Amount of alcohol consumption^b^, g/day, lsmean^c^ 19.4 22.4 28.5 33.2 22.2
Risky amount of alcohol according to WHO guideline, %
\<40 g/day 83.4 80.6 72.6 67.8 80.1
≥40 g/day 14.1 16.2 22.9 25.6 13.2
Women
Total,^a^ *N* 18,824 14,711 3,925 748 2,906
Frequency of alcohol consumption, (%)
\<1 time/week 65.2 66.8 73.3 79.1 77.5
1 time/week 19.1 18.4 15.9 9.6 11.4
2--3 times/week 13.1 11.9 8.3 7.8 8.2
≥4 times/week 2.6 3.0 2.4 3.5 2.9
Amount of alcohol consumption^b^, g/day, lsmean^c^ 7.8 8.7 10.7 16.4 7.7
Risky amount of alcohol according to WHO guideline, %
\<20 g/day 91.5 90.8 87.3 73.4 89.6
≥20 g/day 5.9 6.5 8.5 18.6 3.9
WHO, World Health Organization.
Results of cheongju were not shown because of low prevalence (\<5% in men and women). The total percentage of each category does not equal 100% because there were missing data.
^a^Drinkers of each alcoholic beverage were not exclusively categorized.
^b^Total amount of alcohol consumption among those who drink each type of alcoholic beverage.
^c^Least squares means (lsmean) were adjusted for age, education, household income, current occupation, marital status, smoking, body mass index, duration of regular exercise, self-rated health, stress, and contact frequency with family and close friends, and diagnosis history of diabetes, myocardial infarction, stroke, acute liver disease, fatty liver, and cirrhosis.
Correlates of high-risk consumption of alcohol and the types of alcoholic beverages
-----------------------------------------------------------------------------------
Figure [3](#fig03){ref-type="fig"} presents the summary of the associations of potential correlates with low- or high-risk alcohol consumption and each type of alcoholic beverage in the men, women, and both (detailed ORs and 95% CIs are presented in [eTable 5](#sm01){ref-type="supplementary-material"}, [eTable 6](#sm01){ref-type="supplementary-material"}, [eTable 7](#sm01){ref-type="supplementary-material"}, [eTable 8](#sm01){ref-type="supplementary-material"}, [eTable 9](#sm01){ref-type="supplementary-material"}, and [eTable 10](#sm01){ref-type="supplementary-material"}). Among the demographic factors, the older participants were less likely to be high-risk drinkers in both the men and women. They preferred to drink makgeolli, whereas preference for soju or beer was lower than that of the younger participants. A higher educational level was negatively associated with high-risk drinking and soju drinking, although it was positively associated with other beverages. Office job employees and the unemployed were also negatively associated with high-risk drinking and positively associated with wine consumption. In the men, a higher level of household income was positively associated with high-risk drinking and the consumption of beer, strong spirits, and wine. In the women, living alone was positively associated with high-risk drinking and the consumption of beer, strong spirits, and wine.
{#fig03}
Among the behavioral factors, smoking and regular exercise were positively associated with high-risk drinking in both the men and women. Smoking, the most strongly associated factor, was associated with the consumption of soju and strong spirits. Regularly exercising more than 150 minutes per week and having frequent stress were associated with all types of beverages except soju. A BMI higher than 25 was associated with high-risk drinking and the consumption of soju and strong spirits in the men.
Among the perceived health-related factors, frequent stress was positively associated with high-risk drinking and the consumption of makgeolli, strong spirits, and wine in both the men and women. The women who rated their health as "good" were more likely to be high-risk drinkers and consumers of soju.
Among the social relationship factors, frequent contact with family was negatively associated with high-risk drinking and the consumption of strong spirits; however, frequent contact with friends was positively associated with high-risk drinking and the consumption of soju.
DISCUSSION
==========
Sex-specific trends in alcohol consumption were influenced by demographic, behavioral, and perceived health-related factors. Soju was the most frequently consumed alcoholic beverage. Those who drank strong spirits were more likely to be high-risk drinkers. There were different associations between correlates and alcohol consumption according to the types of alcohol beverages. The present study further identified how each correlates relate to the consumption of various alcoholic beverages in Korea and the alcoholic beverages that may influence the trends in alcohol consumption in the Korean population.
The findings for the trends in the prevalence of alcohol consumption in our study were concordant with those of the Korean National Health and Nutrition Examination Survey (KNHANES), a nationally representative survey of participants aged 15 years or older. In the KNHANES, the age-standardized prevalence of alcohol consumption remained at a high level, from 72.6% to 75.2% in men, and increased from 36.9% to 46.5% in women between 2005 and 2015.^[@r32]^ In terms of the amount of alcohol, Korea is one of the countries with the highest alcohol consumption by men aged 15 years or older; they consume 45.6 g/day of pure alcohol, whereas women drink 13.0 g/day of pure alcohol according to a report by the WHO.^[@r01]^ However, those amounts were higher than those in our study population aged 40--69 years (21.4 g/day in the men and 5.5 g/day in the women). This difference could result from the presence of young participants aged 15 to 39 years, who had a higher prevalence of high-risk drinking, and from unrecorded alcohol consumption from sources such as homemade or informally produced alcohol, which added 20% to the WHO's report.^[@r01]^ In our study, 91.8% of the male drinkers consumed soju and 71.0% of the women mainly consumed beer or spirits, unlike in many Western countries.^[@r19],[@r33]^ The WHO reported that 70.5% of the alcohol consumption in the 2010 Korean population came from the "other beverage" category, which is comparable to our results showing the consumption of soju and makgeolli at 77.5% by men and 57.6% by women.^[@r01]^
Similar demographic factors, that those who were younger were more likely to be drinkers or high-risk drinkers, were consistently reported in previous studies.^[@r17],[@r22],[@r23],[@r34]^ We found that beer and makgeolli were inversely associated with age, which was also reported in China, where beer consumption was higher in the younger generation, and consumption of a Chinese traditional alcoholic beverage, baijiu, was higher in older people.^[@r17]^ Higher education was negatively associated with high-risk drinking in both men and women, and higher levels of household income were positively associated with high-risk drinking in men. This finding reflects that educational inequalities have stronger effects on health behaviors than income levels in developed countries.^[@r23],[@r34]^ The greater consumption of strong spirits and wine in those with higher education or household incomes may reflect the higher price of strong spirits and wine compared to other beverages in Korea.
Among the behavioral factors, consumption of beverages with higher alcohol content than other beverages, such as soju and strong spirits, was associated with smoking, which has been established as the most influential factor in high-risk drinking.^[@r14]--[@r18],[@r22],[@r34]^ The association of regular exercise with high-risk alcohol consumption was found in several previous cross-sectional studies,^[@r18],[@r22],[@r35],[@r36]^ whereas null associations were found in longitudinal studies.^[@r37]--[@r39]^
Among the perceived health-related factors, those who reported their health as "good" were more likely to be high-risk drinkers than those who reported their health as "poor" in a previous study of 230,715 Koreans.^[@r22]^ Because previous prospective studies have shown that alcohol consumption does not affect the change in self-rated health,^[@r40],[@r41]^ these results may reflect that those who think their own health is "good" tend to be inattentive to their health or overestimated their own health. Association of stress with alcohol consumption was consistently reported in a previous Korean study^[@r22],[@r34]^ and in a meta-analysis of 11 European studies.^[@r42]^ These studies suggested that people drink alcohol to relieve stress, although alcohol consumption is a poor strategy for coping with stress, because alcohol itself may increase stress levels.^[@r43]^ Programs to educate people on coping with stress should be promoted.
Among the social relationship factors, our results for contact with family or friends reflected the evidence reported in previous studies, which reported the protective effects of family on high-risk alcohol consumption and the increased possibility of alcohol consumption when friends around the person were drinkers.^[@r23],[@r44]^ Bosary et al suggested that the quality of relationships with family, friends, or significant others has a central role in substance use behaviors.^[@r45]^
There are many policies in Korea to reduce alcohol consumption, such as the regulation of advertising, mandatory warnings on alcoholic beverages, juvenile protection, and drunken driving control, but they have not been effective.^[@r46]^ The results of the present study suggest that it may be more efficient to promote policies or campaigns while considering correlates of high-risk alcohol consumption and specific alcoholic beverages.
This study had limitations that should be considered when interpreting its results. First, all of the variables were collected using a self-reported questionnaire. This can cause the underreporting of alcohol consumption or other unhealthy behaviors because of social desirability bias and recall bias. Second, only information on the six main types of alcoholic beverages was covered because information on other types of beverages was available only for a few recruiting years or there was no standard volume for those beverages. However, the results were expected to be unchanged because of the total frequency of those who reported drinking other types of alcoholic beverages was approximately less than 3% among the current drinkers. Third, the total frequency of those who drank two or more types of alcoholic beverages could not be assessed because the frequencies were collected by types of alcoholic beverages; we could not distinguish those who drank two or more types of beverages at once from those who drank them separately.
To conclude, this study found that there were sex-specific trends in alcohol consumption and increased percentages of alcohol from certain types of alcoholic beverages. Because there were inverse associations of age with beer and makgeolli, age-specific interventions should be performed to reduce these trends. Appropriate programs for at-risk populations, such as those with younger age, low education, smoking, exercising, stress, and frequent contact with friends could reduce alcohol-related problems in Korea. The findings of the present study provide information for establishing and promoting country-specific policies and campaigns.
This work was supported by the Research Program funded by the Korea Centers for Disease Control and Prevention (2004-E71004-00, 2005-E71011-00, 2005-E71009-00, 2006-E71001-00, 2006-E71004-00, 2006-E71010-00, 2006-E71003-00, 2007-E71004-00, 2007-E71006-00, 2008-E71006-00, 2008-E71008-00, 2009-E71009-00, 2010-E71006-00, 2011-E71006-00, 2012-E71001-00, and 2013-E71009-00) and the Education and Research Encouragement Fund of Seoul National University Hospital (2017).
Conflicts of interest: None declared.
APPENDIX A. SUPPLEMENTARY DATA {#sm01}
==============================
The following is the supplementary data related to this article:
eTable 1. Alcohol consumption according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 43,927 men aged 40--69 in the HEXA-G study
eTable 2. Alcohol consumption according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 85,897 women aged 40--69 in the HEXA-G study
eTable 3. Amount of alcohol consumption based on WHO guideline according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 43,927 men aged 40--69 in the HEXA-G study
eTable 4. Low-, medium-, and high-risk alcohol consumption according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 85,897 women aged 40--69 in the HEXA-G study
eTable 5. Alcohol consumption lower or more than 40 g/day (threshold for low-risk alcohol consumption) according to demographic and behavioral factors, perceived health, social relations, and diagnosis history of diseases among 43,927 men aged 40--69 in the HEXA-G study
eTable 6. Alcohol consumption lower or more than 20 g/day (threshold for low-risk alcohol consumption) according to demographic and behavioral factors, perceived health, social relations, and diagnosis history of diseases among 85,897 women aged 40--69 in the HEXA-G study
eTable 7. Frequency of type of alcoholic beverage consumed according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 32,475 current male drinkers aged 40--69 in the HEXA-G study
eTable 8. Associations of demographic and behavioral factors, perceived health, social relations, and diagnosis history of diseases with the consumption of each type of alcoholic beverage compared to consumption of other types among 32,475 male current drinkers aged 40--69 in the HEXA-G study
eTable 9. Frequency of type of alcoholic beverage consumed according to demographic and behavioral factors, perceived health-related factors, social relations, and past diagnosis of diseases among 26,502 current female drinkers aged 40--69 in the HEXA-G study
eTable 10. Associations of demographic and behavioral factors, perceived health, social relations, and diagnosis history of diseases with the consumption of each type of alcoholic beverage compared to with consumption of other types among 26,502 female current drinkers aged 40--69 in the HEXA-G study
| {
"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
============
Congenital diaphragmatic hernia (CDH) is a rare developmental defect of the diaphragm, causing considerable morbidity and mortality^[@CR1],[@CR2]^. Morphological changes are present from an early developmental stage, consisting of increased muscularization of the pulmonary vessels, rarefication of pulmonary arterioles and capillaries, and a decreased alveolar density^[@CR3]^. Consecutively, most CDH newborns present with pulmonary hypertension (PH) and lung hypoplasia which are associated with mortality^[@CR4]^. The severity of PH and lung hypoplasia may subsequently contribute through several mechanisms, such as mechanical ventilation-induced lung damage, to long-term oxygen dependency and the development of chronic lung disease (CLD), which can be defined as oxygen dependency at 28 days of life. The cellular and molecular mechanisms initiating and contributing to PH and CLD are only partially understood^[@CR5]^. However, morphological changes are likely being enhanced in response to hypoxia, inflammation, oxidative stress and injury of the bronchoalveolar system and the pulmonary vessels^[@CR6]^.
Therefore, the development of CLD can be considered to be, in a large proportion, induced by the morphological changes leading to PH and lung hypoplasia, and mechanical ventilation-induced lung damage^[@CR7],[@CR8]^. The introduction of lung protective ventilation strategies (e.g. permissive hypercapnia) has improved the outcome of CDH newborns and supportive techniques such as extracorporeal membrane oxygenation (ECMO) may limit lung trauma and improve cardiopulmonary dysfunction^[@CR9],[@CR10]^.
The understanding of the underlying mechanisms for the development of CLD in CDH might lead to the identification of novel biomarkers and drug targets to therapeutically promote lung and vascular growth, which may improve outcome of CDH^[@CR11]^. MicroRNAs (miRNAs), a class of non-coding RNAs that suppress gene translation (most commonly through promoting mRNA degradation or disrupting mRNA translation), might serve as biomarker for disease severity in CDH^[@CR5]^. Animal and cellular models of PH suggest that dysregulations of certain miRNAs contribute to the pathogenesis of PH, which might be a predecessor of the development of CLD in CDH^[@CR5]^. Moreover, miR-200b and miR-10a have been identified to play a crucial role in hypoplastic CDH lungs, and to be associated with survival after fetoscopic endoluminal tracheal occlusion (FETO)^[@CR12]^. In the present preliminary study, we sought to identify circulating miRNAs as a potential biomarker associated with severe outcomes in CDH newborns, and to uncover specific contributor genes and biological pathways that may aid our understanding of the condition.
Results {#Sec2}
=======
Cohort characterization {#Sec3}
-----------------------
About 72% of patients in our study survived to discharge. ECMO rate was 55.6% (10/18). In 16/18 patients (88.9%), the diagnosis was established prenatally, and 13/18 patients had a left-sided hernia. Eight patients (44.4%) were allocated to the Death/CLD group, while 10 (55.6%) to the No-CLD group. Patients were similar according to sex, gestational age, birth weight and umbilical artery pH. Although patients in the Death/CLD group had a lower O/E lung-to-head ratio (LHR), a higher proportion of intrathoracic liver herniation and presented more right-sided hernias, these differences did not reach statistical significance. However, outcome data were significantly worse in infants in the Death/CLD group, in terms of overall survival, use of ECMO, surgical/patch repair, and duration of mechanical ventilation and oxygen supplementation (Table [1](#Tab1){ref-type="table"}). The individual clinical condition, including the cause of death, of the 18 CDH newborns at the time of blood sample acquisition is demonstrated in Supplementary Table [1](#MOESM1){ref-type="media"}.Table 1Sample demographics and clinical characteristics.Death/CLDNo-CLDp-value(n = 8)(n = 10)***Demographics***Male, n (%)4 (50%)8 (80%)0.315Gestational age, weeks (range)37.6 (34.3--38.4)38.2 (35.1--39.9)0.274Birth weight, kg (range)3.0 (2.5--3.9)3.1 (2.2--4.2)0.897Umbilical artery pH, median (range)7.30 (7.25--7.41)7.35 (7.27--7.45)0.315O/E LHR, % (range)32 (29--46)42 (35--55)0.094Left-sided CDH, n (%)4 (50%)9 (90%)0.213Intrathoracic liver, n (%)7 (87.5%)4 (40%)0.101FETO, n (%)1 (13%)1 (10%)0.911***Time of Sampling***Age at blood sampling, hours (range)24.9 (23.3--29.9)24.1 (23.4--27.9)0.315PaO2 at time of sampling, Torr (IQR)75 (45--83)173 (72--193)0.021Mild PH, n (%)05 (50%)0.084Moderate PH, n (%)4 (50%)3 (30%)0.503Severe PH, n (%)4 (50%)2 (20%)0.308***Outcome/Therapies***Alive at 28 days, n (%)5 (63%)10 (100%)0.203Overall survival, n (%)3 (38%)10 (100%)0.027ECMO support, n (%)8 (100%)2 (20%)0.003Duration ECMO support, days (range)12.1 (5.2--34.6)4.7 (3.1--6.3)0.267Surgical Repair, DOL (range)11 (7--36)5 (2--11)0.001Patch Repair, n (%)7/7 (100%)4 (40%)0.043Mechanical ventilation, days (range)36.4 (11.1--53.6)7.8 (5.3--11.84)0.001Oxygen supplementation, days (range)49.2 (16--262.5)13.4 (5.8--25.9)0.005CLD: chronic lung disease, O/E: observed vs. expected, LHR: lung-to-head ratio, FETO: fetoscopic endoluminal tracheal occlusion, IQR: interquartile range, PH: pulmonary hypertension, ECMO: extracorporeal membrane oxygenation, DOL: day of life.
Circulating miRNAs associated with severe outcomes in CDH {#Sec4}
---------------------------------------------------------
Through array profiling of mature miRNAs in blood of CDH newborns, collected 24 h after birth, we identified 33 circulating miRNAs that were significantly changed (p \< 0.05) in those children who died or developed CLD within 28 days (Death/CLD group) (Fig. [1A](#Fig1){ref-type="fig"}, Supplementary Table [2](#MOESM1){ref-type="media"}), compared to those who survived with no signs of CLD until up to 28 days of life (No-CLD group). Considering the reduced size of our sample, we used a combined endpoint of death and CLD as a representation of severe disease outcomes. Seven of the 33 significant miRNAs survived correction for multiple comparisons (BH-adjusted p \< 0.05, Table [2](#Tab2){ref-type="table"}, Fig. [1B](#Fig1){ref-type="fig"}) with an expression change of at least 1, and were thus considered differentially expressed (DE). We observed down-regulation of most of the significant miRNAs in the Death/CLD group. Most miRNAs also appear to have, in average, a reported medium expression in the lung, with considerable numbers of target mRNAs and proteins in this organ, according to the IMOTA database.Figure 1(**A**) Heatmap of significantly changed circulating mature miRNAs in CDH-PH children who died or developed CLD during the first 28 days after birth (Death/CLD at 28 days, n = 8), compared to those children who survived without developing CLD during this period (No-CLD at 28 days, n = 10). Blood samples were drawn 24 h after birth. Statistical significance was obtained through a moderated t-test (unadjusted p \< 0.05). Hierarchical clustering was applied to samples and miRNAs using Euclidean distances with complete linkage. **(B)** Expression levels of those differentially expressed miRNAs (Benjamini-Hochberg-adjusted p \< 0.05 and expression change ≥1) in the Death/CLD and No-CLD groups of CDH children. P-values are represented by the yellow line.Table 2Differentially expressed miRNAs in blood of CDH children with poor outcome at 28 days after birth.Transcript ID/miRNAMeanMean Death/CLDChange*Diff*. *expressionLung-specific information (IMOTA)*No-CLDPvalAdj PvalExpressionmRNAsProteinshsa-let-7b-5p6.429.893.472.59E-050.009High1960463hsa-let-7c-5p6.078.662.608.83E-050.014High1381271hsa-miR-1307-3p6.044.82−1.221.14E-040.014Medium19142hsa-miR-185-3p4.303.15−1.158.90E-040.046Not expressed6211hsa-miR-80842.711.39−1.321.08E-030.046Not found in databasehsa-miR-331-3p6.095.09−1.001.09E-030.046Medium617154hsa-miR-210-3p5.123.68−1.441.14E-030.046Medium12927\*Only miRNAs with Adj Pval \<0.05 from a moderated t-test and expression change ≥1 were considered differentially expressed. Blood samples were drawn at a 24 h after birth time-point. CLD: chronic lung disease. Death refers to intra-hospital death between 24 h and 28 days after birth.
Interestingly, miR-6511b-3p (r = 0.621) and miR-25-5p (r = −0.77) showed a relatively high correlation with death, although the number of death cases in our study is too low to consider this a truly significant finding, warranting further exploration. Additional potentially interesting relationships include the negative correlation of miR-8084 expression with moderate to severe PH (r = −0.549), using an established classification of PH severity in this population, and the positive correlation of miR-744-5p levels with PaO~2~ measures at 24 h (r = 0.741) (Fig. [2A](#Fig2){ref-type="fig"}). As expected, most significant miRNAs correlated with the outcome, represented as either the binomial No-CLD or Death/CLD classification, or as a graded No-CLD → CLD → Death classification. Furthermore, some DE miRNAs mildly correlated as well with the use of ECMO support and PaO~2~, including let-7b-5p (r~ECMO~ = 0.727, r~PaO2~ = −0.601), let-7c-5p (r~ECMO~ = 0.755, r~PaO2~ = −0.64) and miR-8084 (r~ECMO~ = −0.693, r~PaO2~ = 0.537). However, it is difficult to tell whether these relationships could be the result of ECMO-induced changes already present by the time of sample collection, or could truly result from the abnormal biological processes that ultimately determined the need for ECMO support.Figure 2Correlation heatmaps of expression levels of significantly changed miRNAs with clinical classifications/measures **(A)** and the expression of other miRNAs **(B)**. Color grading corresponds to the positive (red) and negative (blue) Pearson's correlation coefficients. Rows and columns were hierarchically clustered by Euclidean distance with complete linkage. Clinical classifications/measures were mostly represented as absent (N = 0) or present (Y = 1), except for the graded outcome (where no-CLD = 0, CLD = 1 and death = 3), PH severity (where mild = 0, moderate = 1 and severe = 3), and the continuous values of PaO~2~ at 24 h.
Four major clusters of significant miRNAs can be appreciated from the correlation patterns amongst their expression levels, with the seven DE miRNAs distributed in three of these clusters (Fig. [2B](#Fig2){ref-type="fig"}), which might relate to differential functions between clusters and/or to different sources of the circulating miRNAs.
Functional implications of miRNA dysregulation in the development of CLD in CDH children {#Sec5}
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We desired to use the miRSystem online tool to investigate target genes of our DE miRNAs, because of the advantages provided by its integrative analysis. However, only four of the seven DE miRNAs (let-7b-5p, let-7c-5p, miR-210-3p and miR-331-3p) were present in this system, for which the target genes for the remaining DE miRNAs (miR-185-3p, miR-1307-3p and miR-8084) were obtained from independent queries in miRTargetLink. Our integrated list, consisting of 857 genes with validated status or a minimum of three hits from miRSystem, and genes from miRTargetLink with some degree of evidence, resulted in the identification of 411 genes predicted to be targeted by more than one of our seven DE miRNAs (Supplementary Table [3](#MOESM1){ref-type="media"}). No common target for all DE miRNAs was identified; the largest hits were observed for *SEMA4G*, which was predicted to be targeted by four DE miRNAs, while the highest observed/expected (O/E) ratios, based on the miRSystem integrative analysis, were observed for *NRTN* and *ESPL1*, targeted by the let-7 family of DE miRNAs (Table [3](#Tab3){ref-type="table"}).Table 3Top target genes for the seven differentially expressed miRNAs in the Death/CLD group of CDH children.GeneHitsmiRNAsPathways\*O/E\*SEMA4G4let-7b-5p, let-7c-5p, miR-210--3p, miR-331--3p12.34SOCS13let-7b-5p, let-7c-5p, miR-331--3p268.45UNC5A3let-7b-5p, let-7c-5p, miR-331--3p93.57TBKBP13let-7b-5p, let-7c-5p, miR-331--3p13.37HMGA13let-7b-5p, let-7c-5p, miR-185--3p133.11IGF2BP23let-7b-5p, let-7c-5p, miR-331--3p22.79KCNC43let-7b-5p, let-7c-5p, miR-331--3p32.77SLC6A13let-7b-5p, let-7c-5p, miR-210--3p92.43POU2F23let-7b-5p, let-7c-5p, miR-210--3p22.36EGR33let-7b-5p, let-7c-5p, miR-331--3p21.85VAV33let-7b-5p, let-7c-5p, miR-185--3p301.64ACSL63let-7b-5p, let-7c-5p, miR-1307--3p101.56THBS13let-7b-5p, let-7c-5p, miR-8084181.54TGFBR13let-7b-5p, let-7c-5p, miR-331--3p231.51MEF2D3let-7b-5p, let-7c-5p, miR-331--3p121.45ADCY93let-7b-5p, let-7c-5p, miR-185--3p591.44RAB5B3let-7b-5p, miR-331--3p, miR-185--3p41.40ABCC53let-7b-5p, let-7c-5p, miR-185--3p31.09NRTN2let-7b-5p, let-7c-5p423.67ESPL12let-7b-5p, let-7c-5p222.19GNG52let-7b-5p, let-7c-5p4816.90ERCC62let-7b-5p, let-7c-5p1116.90QARS2let-7b-5p, let-7c-5p616.90AKR1B102let-7b-5p, let-7c-5p416.90HTR1E2let-7b-5p, let-7c-5p913.65NGF2let-7b-5p, let-7c-5p4011.45IL82let-7b-5p, let-7c-5p453.29RANBP22let-7b-5p, let-7c-5p433.20TP532let-7b-5p, let-7c-5p642.31NRAS2let-7b-5p, let-7c-5p932.25CHUK2let-7b-5p, let-7c-5p1092.23PAK12let-7b-5p, let-7c-5p552.23POLR2D2let-7b-5p, let-7c-5p512.18CASP32let-7b-5p, let-7c-5p562.05NUP982let-7b-5p, let-7c-5p402.05RB12let-7b-5p, let-7c-5p511.95CDKN1A2let-7b-5p, let-7c-5p681.88CCND12let-7b-5p, let-7c-5p521.49STAT32let-7b-5p, let-7c-5p511.39MAP3K12let-7b-5p, let-7c-5p691.31ITGB32let-7b-5p, let-7c-5p401.28MAP3K7IP22let-7b-5p, let-7c-5p501.25\*Based only on miRSystem report for let-7b/c-5p, and miR-210-3p, -331--3p. O/E: ratio observed/expected.Selection: O/E \>= 1.0, Hits \> 1, Pathways \>= 1; if Hits = 2, then Pathways \>= 40 or O/E \> 10.
We further explored the functional interactions between common targets of DE miRNAs, as well as their functional implications, by constructing a PPI network and performing pathway analyses on it. This allowed us to identify not only functional types of interaction (i.e. activation/catalysis, inhibition, complex formation/binding, or predicted interaction), but also functionally related target clusters (network modules), as well as proteins likely linking different biological processes/pathways (network hubs, i.e. proteins showing the larger numbers of interactions within the network). The resulting full network consisted of 227 nodes and was enriched for pathways highly related to responses to growth factors, adhesion molecules and immunological stimulation, as well as morpho-/organogenesis, including the PI3K-Akt, MAPK, FoxO, Ras, IL4-mediated, TGF-β, integrin, Jak-STAT and p53 signaling pathways, focal adhesion and ECM-receptor interactions, among the most significantly enriched terms, and cell cycle regulation by BTG proteins, signaling by activin, Fas, SMAD2/3, EPHA, growth hormone and TNF receptors, regulation of hypoxia and oxygen homeostasis by HIF-1-α, and oxidative stress response, among the pathways with the higher proportions of proteins from the network in the pathway (Supplementary Table [4](#MOESM1){ref-type="media"}, Module: All).
The main network (Fig. [3](#Fig3){ref-type="fig"}, Supplementary Table [5](#MOESM1){ref-type="media"}) was conformed by 208 nodes, 480 edges (interactions), and 13 modules (clusters). As network hubs, we identified MAPK1 (38 interactions), STAT3 (31 interactions), ITGB3 and SMAD2 (22 interactions). In a lesser extent, NRAS and TP53 (18 interactions), as well as CHUK and PDGFB (16 interactions), were also highlighted. With the exception of MAPK1, which was a predicted target of miR-1307-3p and miR-185-3p, these network hub molecules were predicted targets for let-7b/c-5p miRNAs (Table [3](#Tab3){ref-type="table"}). Pathway enrichment analysis of the network modules revealed that the largest clusters of proteins are involved in the responses to immune stimulation and growth signals, cell cycle regulation and tissue/organ morphogenesis, while smaller clusters play roles in hypoxia, oxytocin signaling, platelet homeostasis and cardiac conduction (Table [4](#Tab4){ref-type="table"}, Supplementary Table [4](#MOESM1){ref-type="media"}). All these modules are closely linked through the aforementioned hub proteins. To illustrate this functional connectivity further, we mapped those miRNA targets observed within the Pathways in Cancer KEGG diagram as, due to the integrative nature of this pathway and its relation to cellular proliferation, growth and differentiation, we observed the highest proportion of proteins from the network in this term (Supplementary Fig. [1](#MOESM1){ref-type="media"}). In summary, the results from our analysis support the involvement of the TGF-β family of growth and differentiation factors, inflammation and semaphorin signaling, which may impair organ fetal organogenesis and postnatal lung and cardiac functions, in the severe pathophysiology of CDH with poor outcomes.Figure 3Protein-protein interaction network generated for shared miRNA target genes. After integration of miRSystem and miRTargetLink target gene lists, those genes predicted to be targeted by more than one of the differentially expressed miRNAs served as input for the network creation using the Reactome FI app for Cytoscape. Disconnected nodes and isolated clusters of less than 4 proteins were excluded from the network view and module enrichments. Information on paired protein interactions is available in the Supplementary Table [3](#MOESM1){ref-type="media"}. Top results from the pathway enrichment analyses for the network and network modules can be found in Table [3](#Tab3){ref-type="table"}, and Supplementary Table [4](#MOESM1){ref-type="media"}.Table 4Summary of main findings from the pathway analysis (by module) of the network created for shared target genes between top differentially expressed miRNAs in the Death/CLD group of CDH children.NodesTop PathwaysProteins227Signaling pathways activated in response to growth factors and adhesion molecules; tissue and organ morphogenesis.36TNF receptor and MAPK signaling pathways; signaling by NGF; innate immune response; response to hormones and growth factorsC2, CD86, CHUK, DAPK1, DUSP16, DUSP3, DUSP4, DUSP9, EGR3, FAS, FASLG, GAB2, HMGB1, IKBKAP, IKBKE, MAP3K1, MAP3K3, MAP4K3, MAP4K4, MAPK1, MAPK6, MASP1, NRAS, NRK, NTRK3, PCYT1B, PLA2G3, RICTOR, RPS6KA3, SLC20A1, SYNGAP1, TBKBP1, TNFAIP3, TNFRSF1B, TSC1, ULK228Jak-STAT, FoxO and PI3K-Akt signaling pathways; inflammationB3GAT3, CCL22, CCL3, CCL7, CCR7, CYP19A1, DICER1, DUSP1, FGF11, GHR, GIPC1, IGF1, IGF1R, IL10, IL13, IL22RA1, IL6, INSR, IRS2, NGF, OSMR, PAPPA, POU2F2, RDX, SOCS1, SOCS4, STAT3, TARBP225Cell cycle regulation and p53 signalingBCL2L1, BTG2, CBFA2T3, CCND1, CCND2, CDC25A, CDKN1A, CNOT6L, CREM, E2F2, E2F5, HMGA1, MYB, PAX3, PSME1, RALB, RB1, RNMT, RRM2, SH2B3, STEAP3, TNKS, TP53, ZBTB7A, ZNF24820ECM organizaion, focal adhesion, integrin signaling; plateletsCBL, COL15A1, COL1A1, COL1A2, COL24A1, COL3A1, COL4A1, COL4A2, COL4A5, COL4A6, COL5A2, DMP1, DTX1, DTX2, E2F6, ITGB3, ITGB8, PAG1, PDGFB, THBS119TGF-β, Wnt and Hippo signaling pathwaysACVR1B, ACVR1C, ACVR2A, ACVR2B, APC2, BRF2, CHD7, CHRD, DVL3, GDF6, MEF2D, NLK, POLR3D, SENP2, SMAD2, SMARCC1, TGFBR1, VANGL2, ZFYVE1616EPH-Ephrin signaling, axon guidance; cell migrationABL2, ACTA1, ARHGEF15, CAP1, CASP3, CLDN1, DMD, EPHA3, EPHA4, MLLT4, PAK1, PPP1R12B, SGCD, UNC5A, UTRN, VAV315Lipid and energy metabolismACSL6, ADIPOR2, CHD9, CRY2, EEF2K, MED6, MED8, NCOA1, NKD1, PPARA, PPARGC1A, PRKAB2, RORC, SCD, WNT112Intracellular signalingADCY9, ADRB1, ADRB2, ADRB3, EDN1, FZD4, GABBR2, GNAL, GNG5, HTR1E, HTR4, OPRM111MitosisAHCTF1, CLASP2, DIAPH2, ESPL1, GEMIN7, NUP98, NXT2, PIAS4, RANBP2, RNF8, SMC1A10Hypoxia; oxygen homeostasisCHD4, EGLN2, EGLN3, ERCC6, HIF3A, POLR2D, TAF5, TAF9B, ZNF354A, ZNF4317MAPK, oxytocin, calcium signalingANAPC13, CACNA1D, CACNA1E, CACNG4, CDC14B, PPP3CA, RASGRP16Protein transport and modificationCD59, COPZ1, CTSC, SCN5A, SEC. 24 C, SPTBN44Platelet homeostasis; cardiac conductionATP2A2, ATP2B1, ATP2B3, ATP2B4
Circulating DE miRNAs as potential prognostic biomarkers and/or therapeutic targets for CLD in CHD children {#Sec6}
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Finally, because of the high overlapping and redundant nature of miRNA target genes and pathways, we wished to investigate the relative importance of dysregulations at 24 h of the circulating DE miRNAs for the outcomes of Death/CLD at 28 days after birth in CDH children, in order to rank our DE miRNAs for future studies on their potential as prognostic biomarkers for CLD in CDH, and/or therapeutic targets. For this, we used C&RTs and found that most Death/CLD cases (7/8) could be differentiated from those No-CLD cases through the expression values of miR-1307-3p (importance rank: 79, split constant: 5.31), while miR-185-3p was important to separate the remaining set of Death/CLD cases initially classified as No-CLD within the first split (importance rank: 100, split constant: 3.305) to achieve a complete (100%) classification accuracy (Fig. [4A](#Fig4){ref-type="fig"}), using the lower cost (cross-validation cost: 0.167, resubstitution cost: 0), most suitable tree (Fig. [4B](#Fig4){ref-type="fig"}). From this algorithm, we obtained then a relative higher ranking of miR-185-3p and miR-1307-3p, closely followed by miR-210-3p (importance rank: 77), and the let-7b/c-5p miRNAs (importance rank: 76) (Fig. [4C](#Fig4){ref-type="fig"}), even though these latter were not included in the split criteria.Figure 4Classification of cases was used to obtain the relative importance of differentially expressed circulating miRNAs for the Death/CLD condition outcome. The corresponding chosen classification tree **(A)**, the costs of the cross-validation procedure **(B)** and the predictor importance ranking **(C)** for the chosen tree (number 1, \*) are shown.
Discussion {#Sec7}
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In this study, we identified seven miRNAs differentially expressed at 24 h of life in the blood of CDH newborns who developed CLD or died within 28 days after birth, and showed that these not only correlate with different measures of pulmonary dysfunction, but also interconnect to participate in processes crucial to establish proper morphology and function of the heart and lungs. Our results suggest that a subset of these miRNAs might hold the potential to serve as prognostic circulating biomarkers for the development of CLD in CDH newborns, which should be properly evaluated in an extended cohort, and implicate TGF-β and semaphorin signaling, as well as inflammatory responses, in the development of CLD in CDH.
The rate of mortality and disability in the long-term among CDH patients depends, in great extent, on the presence and severity of PH^[@CR19]^. Persistent problems in surviving children, such as long-term oxygen dependency, need for mechanical ventilation, persistent wheezing, and increased risk for pulmonary infections^[@CR20]--[@CR22]^, suggest that a combination of mechanical ventilation-induced damage and primary defects of the lung and pulmonary vascular structures are responsible for the cardiopulmonary abnormalities observed in CDH children^[@CR2]^. The contributions of miRNAs to lung morphogenesis during fetal development, and to inflammatory processes during pulmonary disease, have been attractive topics for research in past years. This led to the identification of a number of these molecules, including miR-210 and let-7 family members, as important regulators of normal and abnormal lung development and function^[@CR23]^, and of pulmonary arterial remodeling in response to vessel injury and cellular stressors, such as hypoxia^[@CR24]^. Consistently, members of the let-7 family of miRNAs and miR-1307 have been implicated in the severity of PH in systemic scleroderma^[@CR25]^, miR-185 has been shown to contribute to the oxidative stress-mediated, hyperoxia-induced death of lung epithelial cells during acute lung injury and acute respiratory distress syndrome^[@CR26]^ and, together with miR-210, contribute to the rapid progression of pulmonary fibrosis^[@CR27]^. Further, miR-210, together with miR-331, has been associated with cardiovascular disease and markers of systemic inflammation in human immunodeficiency virus (HIV)-1 infection^[@CR28]^.
The hypoxia-induced miR-210 has been previously implicated in PH, other lung diseases and ischemic heart disease^[@CR29]--[@CR32]^. We suggest that this miRNA also contributes to the development of CLD and the severity of PH as outcomes in CDH newborns. Interestingly, though, miR-210 presented a lower importance than miR-185 and miR-1307 to differentiate CDH cases within the Death/CLD group from those in the No-CLD group. We can speculate that these latter miRNAs regulate distinctive pathways that contribute to progression of CLD in a greater extent than the former, which might be implicated in pathways contributing to PH pathogenesis. Similarly, the previously reported PH-associated miR-21, miR-204, and clusters miR-143/145, miR-17-92 and miR-130/301, as well as the CDH-associated miR-200b and miR-10a might not have shown significance in our study because of a reduced relevance for severity and progression of CLD in CDH newborns, compared to the pathogenesis of each individual disease. Moreover, most observations on miRNA dysregulations in CDH and PH come from studies in animal or cellular models, which may not fully translate to the human pathobiology.
It is well-known that the TGF-β superfamily plays crucial roles in pre- and post-natal lung development, importantly shaping alveolarization and controlling the extracellular matrix composition and tissue homeostasis, among other functions. Not surprisingly, its involvement in pulmonary and cardiovascular diseases has been largely described^[@CR33]--[@CR36]^. Because the TGF-β canonical and non-canonical signaling pathways overlap with a number of pathway terms highly enriched in our PPI network^[@CR34],[@CR37]^, our results provide evidence for the involvement of the TGF-β superfamily in the development of CLD in CDH newborns, providing support to the hypothesis that a primary disruption in lung vasculature and airway development accompany the diaphragmatic defect in CDH, and that the extent of the resulting cardiopulmonary dysfunction contributes to the development of CLD in CDH newborns. Further support to this hypothesis is found through our observation that major genes participating in the semaphorin signaling, involved in development and regulation of immune responses through guidance cues^[@CR38],[@CR39]^, are predicted targets of the miRNAs we found dysregulated in blood of CDH children with severe outcomes.
The O/E ratio of fetal lung volume measured by ultrasound or magnetic resonance imaging (MRI) has been proposed to serve as prognostic biomarker for mortality, the need for ECMO and development of CLD^[@CR40],[@CR41]^. Our study is, to our knowledge, the first to assess the differential miRNA blood profiles related to death and the development of CLD in CDH, and provide an extensive analysis of functional implications and hierarchies of miRNA dysregulations which not only suggests potential for measurements of circulating miRNAs as prognostic biomarkers, but also uncovers crucial regulators with potential for therapeutic purposes. It has already been demonstrated that less severe lung hypoplasia in CDH is associated with compensatory upregulation of miR-200b, and that treatment with this miRNA at an early developmental stage improves lung growth in the nitrofen-induced CDH rodent model^[@CR42]^.
We acknowledge the small sample size of our study is an important limitation, providing small power for statistical testing. Our miRNA candidates would require validation in a larger, well-characterized cohort. Another limitation in our study is the lack of repeated measures in a time series, which would have allowed us to identify changes induced by ECMO and other external factors that influence the development of CLD in CDH newborns. Similarly, the unavailability of tissue-specific miRNA expression limits our ability to interpret the biological meaning of our pathway and network analyses. However, all of these pathways have been described in the context of lung development, PH, and inflammation, all being a part of the pathophysiology of CDH. Considering array measures of small non-coding RNAs may be biased only to those miRNAs and other small RNAs that are part of the array, it is possible that using RNA-seq would have led to discovery of additional, novel circulating miRNAs associated with severe outcomes in CDH. This is an important avenue of future research. Finally, we also acknowledge the potential overfitting of our C&RTs and, although we obtained similar results using two different algorithms (data not shown) for this same reason, the aim of this analysis was not to provide proof of the biomarker performance of our identified miRNAs, but to help us in ranking them for future follow-up studies.
In conclusion, we identified several miRNAs differentially expressed at 24 h of life in the blood of CDH newborns who developed CLD or died within 28 days after birth. Whether these miRNAs might serve as prognostic circulating biomarkers for the development of CLD in CDH newborns, and might be useful therapeutic targets, needs to be investigated in a larger cohort. Despite the aforementioned limitations, we believe our study provides encouraging evidence to ensure further validation of our present findings.
Methods {#Sec8}
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Sample cohort {#Sec9}
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Eighteen CDH patients with PH admitted to our neonatal intensive care unit (NICU) were prospectively enrolled in this study. Written informed consent was obtained from parents or legal representatives. The study was approved by the Institutional Review Board of the University Hospital Bonn and performed in accordance with the ethical standards described in the Declaration of Helsinki. Patients with severe concomitant malformations or insufficient echocardiographic data were excluded from the study.
Standard CDH treatment protocol {#Sec10}
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Infants were intubated after delivery. Mechanical ventilation was started using gentle ventilation and permissive hypercapnia (if tolerated by the patient). The initial inspired oxygen fraction (FiO~2~) was 1.0, and infants received inhaled nitric oxide (iNO) therapy during the initial period of stabilization (iNO 20 ppm). FiO~2~ was titrated to achieve an arterial postductal partial oxygen pressure (PaO~2~) of 80--150 mmHg, and ventilator settings were adjusted to reach a partial pressure of carbon dioxide (PCO~2~) of 45--60 mmHg. Dobutamine and milrinone were administered to treat cardiac dysfunction, and norepinephrine and vasopressin were added to achieve a mean arterial blood pressure ≥40 mmHg. Infants were sedated with fentanyl and midazolam during the first days of life. Criteria for ECMO followed those published in the guidelines of the CDH Euro Consortium^[@CR13]^: preductal oxygen saturation \<85% or postductal saturation \<70%, oxygenation index (OI) ≥40 and consistently present, increased PaCO~2~ \> 70 mmHg with a pH \< 7.15, a peak inspiratory pressure ≥28 cm H~2~O or mean airway pressure ≥17 cm H~2~O, or persistent systemic hypotension (mean arterial pressure \<40 mmHg).
Assessment of PH severity {#Sec11}
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Echocardiography was performed using a Philips CX50 CompactExtreme Ultrasound System with a S12-4 sector array transducer (Philips Healthcare, Best, The Netherlands). The pulmonary arterial pressure (PAP) was graded as \<2/3 systemic pressure (mild PH), 2/3 systemic pressure to systemic pressure (moderate PH) or suprasystemic pressure (severe PH), as described by Keller *et al*.^[@CR14]^. Assessment of PH included: (1) Ductus Arteriosus flow pattern; (2) intraventricular septum position; and (3) calculation of right ventricular systolic pressure from the tricuspid regurgitation (TR) jet, with an estimation of 5 mmHg for right atrial pressure. The angle of insonation was kept below 20°.
Outcome data {#Sec12}
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Study personnel prospectively documented information on vital signs, blood gases and treatment data. The OI was calculated using the formula: OI = (MAP × FiO~2~ × 100)/PaO~2~, where MAP represents the mean airway pressure in cm H~2~O. For group allocation according to disease severity, a combined primary clinical endpoint was used, consisting of death or development of CLD within the first 28 days after birth (Death/CLD), or survival without CLD at 28 days of life (No-CLD). CLD was defined as oxygen dependency at 28 days of life according to the definition of bronchopulmonary dysplasia of Jobe and Bancalari^[@CR15]^.
RNA extraction and miRNA array hybridization {#Sec13}
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Post-pulmonary arterial blood samples were collected from 18 CDH newborns 24 hours after birth, via an indwelling arterial catheter in the femoral artery and using the PAXgene Blood RNA System (QIAGEN, Germany). Total RNA was isolated using the chemagic RNA Blood Kit special (CMG-1083, PerkinElmer, Baesweiler, Germany). The quantity and quality of the isolated RNA were assessed using an ND-1000 spectrophotometer (Peqlab Biotechnologie, Erlangen, Germany) and a BioAnalyzer 2100 (Agilent Technologies, Waldbronn, Germany), respectively. All samples had a concentration ≥20 ng/µl and an RNA integrity number (RIN) ≥6.9. Profiling of miRNAs was carried out from 250 ng of total RNA hybridized to Affymetrix GeneChip miRNA 4.0 arrays (Affymetrix, Santa Clara, CA), following manufacturer's instructions. Poly(A) tailing and biotinylation were performed using the Affymetrix GeneChip Hybridization, Wash, and Stain Kit.
Data analysis {#Sec14}
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Affymetrix CEL files were pre-processed following the robust multi-array analysis (RMA) and detection above background (DABG) workflow in the Affymetrix Expression Console (Affymetrix Santa Clara, CA) software. miRNA expression data was used as background-substracted, quantile-normalized and log2-transformed values. Individual probesets were considered present in a given sample if detection p \< 0.05. After annotation, all probesets corresponding to RNAs other than mature miRNAs were removed. A miRNA was considered expressed in the dataset if the corresponding probeset was present in at least 12 of the 13 samples from newborns with 2/3 of systemic pressure. All expressed probesets were present in at least 16 of the 18 total samples.
Statistical analysis {#Sec15}
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Statistical comparisons of demographic/clinical data and circulating miRNA expression levels between children in the Death/CLD and No-CLD groups was performed by means of two tailed Mann-Whitney U-tests, and a moderated t-test adjusted for multiple comparisons through the Benjamini-Hochberg (BH) method, respectively. Statistical significance was set to raw p \< 0.05 values for demographic/clinical data and "significantly changed" miRNAs, whereas "differentially expressed" (DE) miRNAs were considered those with BH-adjusted p \< 0.05 and expression change ≥1.0. Correlation coefficients between clinical parameters and expression levels of DE miRNAs were obtained through the Pearson's method.
Target genes {#Sec16}
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DE miRNAs were submitted for "miRNAs to Target Genes" analysis on miRSystem^[@CR16]^, using default settings and the expression changes between Death/CLD and No-CLD groups as weights. DE miRNAs not found in the miRSystem database were queried individually on miRTargetLink^[@CR17]^ to obtain the evidence-based target genes. Overlaps between individual target lists were then identified. Furthermore, it was investigated whether the significantly changed miRNAs are expressed in lung tissue and/or there is evidence that they have targets in lung, by searching each miRNA in the interactive multi-omics-tissue atlas (IMOTA)^[@CR18]^.
Protein-protein interactions and biological pathways {#Sec17}
----------------------------------------------------
To investigate the functional implications of miRNA dysregulations in severe CDH outcomes, we created a protein-protein interaction (PPI) network of molecules targeted by at least two of our DE miRNAs with pathway annotations in miRSystem using the Reactome FI app for Cytoscape 3.5.1. We clustered the network and fetched the annotations to show the types of functional relationships. All unconnected nodes, as well as clusters conformed by less than four proteins, were removed from the network view. The full network and network modules (clusters) not hidden from view were analyzed for enriched biological pathways. Enrichment results were filtered to keep only those terms with false discovery rate (FDR) \<0.01 and a number of module genes in the term \> 2.
Relative importance of DE miRNAs {#Sec18}
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To investigate the relative importance of DE miRNAs for condition outcome, we employed a classification and regression tree (C&RT)-style algorithm to apply an exhaustive search for univariate splits for Death/CLD or No-CLD class prediction. The tree was generated using STATISTICA 7 (StatSoft), using all seven DE miRNAs as continuous predictor variables and the following settings: chi-square goodness of fit, estimated prior probabilities, pruning on misclassification error, and a seed for random number generator = 10, plus 3-fold cross-validation.
Electronic supplementary material
=================================
{#Sec19}
Supplementary Dataset 6 Supplementary Dataset 1 Supplementary Dataset 2 Supplementary Dataset 3 Supplementary Dataset 4 Supplementary Dataset 5
**Electronic supplementary material**
**Supplementary information** accompanies this paper at 10.1038/s41598-018-29153-8.
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This work was supported by the grants RE 1723/1-3 and RE 1723/4-1 from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) to HMR, and the Cluster of Excellence ImmunoSensation.
M.H.R.: Concept, analysis, interpretation and manuscript preparation. R.Z.: Profiling of circulating miRNAs, data collection and manuscript writing. S.H.H., A.M., S.B., T.D., L.S., S.H.: Study design, data collection and critical manuscript revision. H.M.R., F.K.: Study design, sampling of patients, data collection and manuscript writing.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
| {
"pile_set_name": "PubMed Central"
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Background {#Sec1}
==========
Osteoarthritis (OA) is characterized by degeneration of articular cartilage, sclerosis of the subchondral bone, and marginal osteophyte formation. OA is associated with chronic pain, stiffness, decreased range of motion and joint deformity, muscle wasting, and tendonitis. The diagnosis of OA relies on clinical symptoms, physical findings, and radiographic findings. Discordance is often found between radiological findings and symptomatic findings of OA \[[@CR1]\]. The etiology of OA appears to be multifactorial with hereditary, metabolic, hormonal, developmental, and mechanical components \[[@CR2]\]. Apart from mechanical and genetic factors that contribute to development of OA, age has been a primary risk factor \[[@CR3]\]. Age-associated changes in cartilage extracellular matrix (ECM), increase in inflammatory cytokine milieu locally, and increased free radical species have been implicated in the loss of ability of cartilage to adapt to mechanical stress or load \[[@CR4]\]. Interestingly, depletion of the mesenchymal stem cells (MSCs) in local stromal population has been indicated to be associated with OA \[[@CR5], [@CR6]\]. Whether it is a cause or an effect of OA remains to be elucidated. Current treatments include lifestyle modifications and diet together with pain relief using paracetamol or non-steroidal anti-inflammatory drugs (NSAIDs) in early OA. However, treatment of the advanced disease relies on total joint replacement which is found to be associated with complications. The lifespan of the prosthesis is limited; therefore, surgery is delayed until symptoms limit a patient's lifestyle and is avoided in younger patients. Since most of the abovementioned modalities only provide symptomatic relief, the regenerative potential of stem cells to repair injured and damaged tissue is a promising new strategy in the field of orthopedics. Of these options, patient-derived (autologous), minimally manipulated, MSCs, for the treatment of chronic diseases such as OA is being investigated to achieve clinical significance \[[@CR7]\].
Here we describe a case report on the healing effect of adipose-derived stromal vascular fraction (SVF) in a woman with peripheral spondyloarthritis, grade 2 sacroiliitis, enthesitis, and ankylosing spondylitis (AS) that were confirmed by X-ray, magnetic resonance imaging (MRI), computed tomography (CT) scan, and ultrasound scan. AS is a form of arthritis that primarily impacts the spine, causing inflammation, leading to chronic pain. AS symptoms often include disturbances of sleep, fatigue, depression, and anxiety \[[@CR8]\]; therefore, they have a profound impact on the patient's quality of life. AS is predominantly detected in patients between the ages of 20 and 40 years and can be triggered by multiple factors that are similar to those for OA. Conditions associated with inflammation such as OA and AS are controlled by NSAIDs and common analgesic medication \[[@CR9]\].
MSCs can be found throughout the body. Adipose tissue is an excellent source of stem cells having 10--100 times more MSCs than bone marrow \[[@CR10]\]. SVF can be easily obtained from loose connective tissue that is associated with adipose tissue by a process of liposuction under local anesthesia. SVF is a collection of a heterogeneous population of: MSCs; hematopoietic stem cells (HSCs); regulatory T cells (T~reg~); pericyte-endothelial cells (ECs); mast cells; a complex microvascular structure of fibroblasts, white blood cells (WBC), dendritic cells (DCs), and intra-adventitial smooth muscle-like cells; and ECM. The immunomodulatory, anti-inflammatory, and regenerative properties of SVF are not attributed to a single type of population residing within, although they are effects of all the types of cells constituting the SVF. The use of SVF as a medical treatment is increasing due to the abundance of its cellular properties, ease of collection, immunomodulatory properties, and safety \[[@CR11]\].
The aim of this treatment was to reduce pain associated with OA and improve our patient's quality of life without the need for NSAIDs or analgesics. Our patient was expected to show significant improvements in her OA, along with some minor improvements to general inflammation present within her body. Interestingly, short-term follow-up presented improvements in her OA as well as comorbidities of AS, depression, anxiety, and fatigue. A 3-year follow-up (in June 2017) including multiple injections of SVF indicated significant changes in her quality of life. All conditions maintained their improvements at the follow-up intervals.
Case presentation {#Sec2}
=================
Our patient was a 27-year-old Australian woman with grade IV OA confirmed by X-ray images of her pelvis; ultrasound scans showed right knee joint effusion, enthesitis, and synovitis; a CT scan of her spine indicated annulus bulges at L3/4 and L4/5, and bilateral grade 2 sacroiliitis changes; a background of AS (human leukocyte antigen-B27 negative) confirmed by MRI imaging; chronic pain syndrome with pain amplification; and post-traumatic stress disorder. Her body mass index (BMI) was 39.4 kg/m^2^. She did not have any: infection with hepatitis B, hepatitis C, or human immunodeficiency virus (HIV); malignancy; previous history of allergic reaction to any component of our therapeutic measure; active cardiac, respiratory, neurologic or endocrine disease necessitating receipt of medication. She was not pregnant or in lactating condition. A written and informed consent was obtained from our patient. Arthritic symptoms were measured using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Hip Disability and Osteoarthritis Outcome Score (HOOS) by scoring for pain intensity, walking ability (distance), joint stiffness, physical function, sports and recreation, and quality of life. Changes to her AS symptoms were measured using the Ankylosing Spondylitis Quality of Life (ASQoL) questionnaire. For liposuction and stem cell treatment, she was admitted to Macquarie Stem Cells. Under light sedation and using aseptic technique, 450 ml of fat was harvested from her abdomen. Cell isolation was performed in PC II safety cabinet. Cells were isolated using collagenase digestion using Liberase GMP grade (enzyme blend).
Our patient's preoperative HOOS score (baseline score) for both hips was 122 (range 0--168), WOMAC for her right knee was 70 (range 0--90), and the baseline ASQoL questionnaire was 18 (range 0--18). We obtained 2.058 billion nucleated cells with a viability of 89.10% using Muse® Cell Analyzer. A total of 738 million cells were injected on the day: 100 million cells injected into each hip and right knee intra-articular under ultrasound guidance, and 438 million cells were administered as an intravenous infusion. The remaining 1.320 billion cells were cryogenically frozen into four separate vials of 330 million cells following the protocols of Thirumala *et al.* \[[@CR12]\]. Follow-up intravenous infusions of 330 million cells were provided at 3 months, 12 months, and 36 months. Our patient's follow-up intervals were performed at 1 day, 3 months, 6 months, 12 months, 24 months, and 36 months respectively. Neither local nor systemic adverse events were observed during the follow-up and she was satisfied with the therapy after 3 months with an increasing trend over the period. At 3-month post-treatment, she exhibited increased mobility. Her HOOS and WOMAC scores decreased to 82 and 37, respectively from her baseline scores. She also noted that pain in her spine, hips, and right knee associated with OA and AS had decreased. Interestingly, in addition to her decreased pain and increased mobility, she was feeling more energetic. Within 6 months after the first SVF infusion, her HOOS and WOMAC questionnaire scores had decreased to 79 and 31, respectively. She showed dramatic improvements over 2 years after her first SVF infusion and presented with decreased dependency on a wheelchair or walking stick (HOOS and WOMAC scores not available). Her dependency on pain relief and anti-depressant medications was found to be decreased as is evident from Table [1](#Tab1){ref-type="table"}. At the 36-month follow-up, she presented significant improvements overall. She remained free from NSAIDs and her pain levels were minimal. Follow-up HOOS and WOMAC scores had decreased to 32 and 20, respectively. Her pre-treatment to post-treatment ASQoL score had decreased to 3 signifying increased quality of life. She still presents some symptoms of depression; however, her anxiety appears to have resolved almost completely. Her progressive improvement is observed over 3 years with WOMAC, HOOS, and ASQoL (Fig. [1a, b](#Fig1){ref-type="fig"}).Table 1Medical history timelineTimeline200620082011201220142014201520162017Age19 years21 years25 years27 years27 years28 years29 years30 yearsDiseaseAS (HLA-B27 negative)MVAOAOA grade IVOA grade IVSymptomsSpinal pain involving shoulders and interscapular region and lower lumbar spineSevere fracture of right kneeEffusion, reduced range of movement, low grade enthesitis, bilateral grade 2 sacroiliitis, peripheral arthritisEffusion, reduced range of movement, low grade enthesitis, bilateral grade 2 sacroiliitis, peripheral arthritisEffusion, reduced range of movement, low grade enthesitis, bilateral grade 2 sacroiliitis, peripheral arthritis, OA of hips and right kneeDiagnosisMRICT scan and ultrasound scanX-rayMedication as per medical historyData not availableData not availableAllegron (nortriptyline; 25 mg X 2)Xenacort A (Triamcinolone acetonide)40Allegron (nortriptyline; 25 mg X 2)Allegron (nortriptyline; 25 mg X 1)Allegron (nortriptyline)Mirtazon (mirtazapine; 30 mg)Ketoprofen (200 mg)Mirtazon (mirtazapine; 30 mg)Mirtazon (mirtazapine; 30 mg)Mirtazon (mirtazapine)Cymbalta (duloxetine; 60 mg X 5)Cymbalta (duloxetine; 60 mg X 3)Cymbalta (duloxetine; 60 mg X 3)Cymbalta (duloxetine; 60 mg X 3)Cymbalta (duloxetine)Valdoxan (agomelatine; 25 mg X 3)Endone (oxycodone; 5 mg X 3)Diazepam (5 mg X 3)Endone (oxycodone; 5 mg X 3)Endone (oxycodone; 5 mg X 3)Endone (oxycodone)Tramadol (50 mg X 2, 150 mg X 1)Lyrica (pregabalin; 150 mg X2)Endone (oxycodone; 5 mg X 4)Lyrica (pregabalin; 150 mg X 5)Lyrica (pregabalin; 150 mg X 3)Lyrica (pregabalin)Lyrica (pregabalin; 300 mg X 2)Nexium (esomeprazole; 40 mg)Lyrica (pregabalin; 150 mg X 2)Nexium (esomeprazole; 40 mg)Nexium (esomeprazole; 40 mg)Nexium (esomeprazole)Orudis SR (ketoprofen; 200 mg X 3)Nexium (esomeprazole; 40 mg)Orudis SR (ketoprofen; 200 mg X 3)Orudis SR (ketoprofen; 200 mg)Orudis (ketoprofen)Oxycontin (oxycodone; 30 mg)Oxycontin (oxycodone; 30 mg)Oxycontin (oxycodone; 30 mg)Oxycontin (oxycodone)Panadol Osteo (paracetamol; 665 mg PRN)Remeron (mirtazapine; 30 mg)Panadol Osteo (paracetamol; 665 mg PRN)Panadol Osteo (paracetamol; 665 mg PRN)Panadol Osteo (paracetamol)Somac (pantoprazole; 20 mg X 1)Seroquel (quetiapine; 25 mg X 3)Panadol Osteo (paracetamol; 665 mg × 3) PRN)Seroquel (quetiapine; 25 mg X 3)Seroquel (quetiapine; 25 mg X 3)Seroquel (quetiapine; 25 mg X 3)Seroquel (quetiapine; 25 mg X 3)Seroquel (quetiapine; 25 mg)Ostelin (ergocalciferol; 25 mcg)Ostelin (ergocalciferol; 25 mcg)Ostelin (ergocalciferol)Ostelin (ergocalciferol)PantoprazolePantoprazolePantoprazolePhysiotens(moxonidinePhysiotens (moxonidine)Physiotens (moxonidineValium (diazepam; 5 mg PRN)Valium (diazepam; 5 mg PRN)Valium (diazepam)Methadone (10 mg X 6)Methadone (10 mg X 6)MethadoneMethadone (10 mg X 5)Uremide (furosemide)Uremide (furosemide)Uremide (furosemide)Tresos B multivitaminTresos B multivitaminTresos B multivitaminTresos B multivitaminBioMagnesium high doseBioMagnesium high doseBioMagnesium high doseBioMagnesium high doseHumira (adalimumab) injectionHumira (adalimumab) injectionHumira (adalimumab) injectionOroxine (levothyroxine)Oroxine (levothyroxine)Oroxine (levothyroxine)Walking aidWheelchair usageWheelchair usageWheelchair usageStick/WalkerInterventionSVF infusion July 2014 \--\> Oct 2014 \--\> Aug 2015 \--\> June 2017*AS* ankylosing spondylitis, *CT* computed tomography, *HLA* human leukocyte antigen, *MRI* magnetic resonance imaging, *MVA* motor vehicle accident, *OA* osteoarthritis, *PRN* as necessary, SVF stromal vascular fractionFig. 1Western Ontario and McMaster Universities Osteoarthritis Index and Hip Disability and Osteoarthritis Outcome Score progressive improvement chart. **a** Percentage improvement of Western Ontario and McMaster Universities Osteoarthritis Index (*red*) and Hip Disability and Osteoarthritis Outcome Score (*blue*) on *Y*-axis over 36 months (on *X*-axis). The graph highlights the measurements taken at 0 (baseline or pre-treatment score), 3 months, 6 months and at 36 months after first stromal vascular fraction infusion. **b** Patient improvement based on subjective questionnaires after stromal vascular fraction treatment. Improvement of patient in terms of Western Ontario and McMaster Universities Osteoarthritis Index and Hip Disability and Osteoarthritis Outcome Score that measure osteoarthritis and Ankylosing Spondylitis Quality of Life questionnaire that measures quality of life of a patient with ankylosing spondylitis over baseline. The percentage improvement over baseline (on *Y*-axis) and questionnaires used in the study (on *X-*axis). *ASQoL* Ankylosing Spondylitis Quality of Life, *HOOS* Hip Disability and Osteoarthritis Outcome Score, *SVF* stromal vascular fraction, *WOMAC* Western Ontario and McMaster Universities Osteoarthritis Index
Discussion and conclusions {#Sec3}
==========================
Consistent with our findings, positive therapeutic effects of SVF have been shown in treatment of OA of hip and knee \[[@CR7]\]. In 2011, based on a case series of 339 patients treated with SVF, Centeno *et al.* reported that 69% of patients were candidates for knee replacement \[13\]. However, after treatment with MSCs only 6.9% took up the option for replacement. Out of all the patients, 60% reported \> 50% pain relief and 40% reported \> 75% pain relief at 11 months \[[@CR13]\]. We report here that SVF infusion given intravenously as well as intra-articularly was not only safe but effective in alleviating the pain associated with grade IV OA of hips and knee. HOOS and WOMAC scores showed 73% improvement over baseline (Fig. [1b](#Fig1){ref-type="fig"}). HOOS and WOMAC questionnaires are both subjective measurements used to observe function following joint arthroscopy and arthroplasty procedures and are proven to be useful for the evaluation of patient-relevant outcomes \[[@CR14]\]. Interestingly, her AS also showed improvements based on ASQoL: her ASQoL score decreased from 18 down to 3 after SVF treatment. We used the ASQoL to assess our patient's AS; the ASQoL questionnaire is a feasible method of determining a patient's quality of life \[[@CR15]\].
This is a first study reporting improvement of AS, a comorbidity, along with OA. In 2014, Wang *et al.* conducted an AS allogeneic stem cell treatment 20-week follow-up (ASAS20) study, which involved treating patients with AS via intravenous infusion of allogeneic cells with a 20-week follow-up \[16\]. The findings confirmed improvements via both objective MRI evaluations as well as subjective questionnaires. The responders showed 77.4% improvement based on the questionnaire assessment, and the MRI reports confirmed a decrease in inflammation \[[@CR16]\]. These results are similar to the findings of the study described here, except for the use of allogeneic bone marrow-derived stem cells. Allogeneic and autologous cells are similar in nature, however, previous publications have shown autologous cells can be better in performance and safety when compared to allogeneic cells \[[@CR17], [@CR18]\]. SVF contains a high number of adipose-derived stem cells (ASCs) that are reported to preferentially migrate toward injured, inflamed, or hypoxic tissues to promote regeneration \[[@CR19]\]. Chemokines and cytokines play an important role in cell activation, survival, and differentiation as well as cell migration. Inflammation is a key biomarker in driving depression \[[@CR20], [@CR21]\]. SVF plays a key role in suppressing inflammation; it thereby aids in repair and regeneration. Medications such as Lyrica (pregabalin), Nexium (esomeprazole), and Orudis (ketoprofen) have been associated with minor side effects of depression. We need to consider that possible reasons for improved depression could be related to a decrease in medication use, along with the immunomodulatory effects of SVF. The hypothesis in improving a patient's quality of life is not only due to improvements in arthritic changes, it can also be associated with suppressing the inflammatory biomarkers that are linked to depression. This original case report provides insight into the fact that SVF treatment has the potential to improve patients' quality of life by improving joint function and mobility, and decrease pain in patients with OA as well as AS. Further study utilizing multiple patients is required to arrive at conclusions on the effectiveness of this treatment for AS.
AS
: Ankylosing spondylitis
ASQoL
: Ankylosing Spondylitis Quality of Life
CT
: Computed tomography
ECM
: Extracellular matrix
HOOS
: Hip Disability and Osteoarthritis Outcome Score
MRI
: Magnetic resonance imaging
MSCs
: Mesenchymal stem cells
NSAIDs
: Non-steroidal anti-inflammatory drugs
OA
: Osteoarthritis
SVF
: Stromal vascular fraction
WOMAC
: Western Ontario and McMaster Universities Osteoarthritis Index
The authors acknowledge all the staff at Macquarie Stem Cells for contributing directly or indirectly toward the collection of data for this study.
Funding {#FPar1}
=======
This retrospective study did not require any funding. The patient included in this study was a private patient who chose to undertake the SVF procedure at her own cost. These data have not been presented at any meetings or conferences.
Availability of data and materials {#FPar2}
==================================
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article.
BB performed data collection and analysis, RB conducted surgical procedures and designed the study, PB performed cell isolation, and AL performed data analysis and manuscript writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
According to Therapeutic Goods (Excluded Goods) Order No. 1 of 2011 (the Order), autologous human cells and tissues that are used: for use in the patient from which they were taken; used under the supervision of a medical practitioner who is caring for that patient and for a single indication in a single course of treatment. They have been declared not to be therapeutic goods for the purpose of the Therapeutic Goods Act 1989 (the Act) by the Secretary of the Department of Health (the Secretary), therefore, they are not regulated by the TGA\[1\]. \[1\] A copy of the Order can be found at This regulation was valid until 31st May 2019 before the new regulation comes into effect. Hence, an ethics approval was not required for this study. This patient has consented to participate in the treatment of OA with autologous adipose-derived SVF. The patient has provided informed consent for this case report to be published. The written consent was signed on the date of initial treatment and again at the 3-year follow-up interval. It was not signed separately to the routine medical manuscript submissions. Patient privacy and confidentiality has been taken into consideration and the patient wishes for her personal details to remain private.
Consent for publication {#FPar4}
=======================
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-in-Chief of this journal.
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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"pile_set_name": "PubMed Central"
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Background
==========
Iron is a critical micronutrient for both plant and animal nutrition, serving as a required co-factor for a variety of cellular processes. Iron deficiency anemia is one of the leading human nutritional disorders worldwide, affecting 43% of the population of developing countries \[[@B1]\]For most of the world\'s population, legumes are a major source of dietary iron \[[@B1],[@B2]\]. Though iron composes 5% of the earth\'s crust \[[@B3]\] it is largely unavailable to plants, particularly in calcareous soils with a pH greater than 7.5. Calcareous soils are especially prevalent in the upper Midwest of the US \[[@B4],[@B5]\] and have been implicated in iron deficiency in soybeans. Iron deficiency chlorosis (IDC) in soybeans is characterized by interveinal chlorosis of the developing trifoliates \[[@B6]\]contributing to yield loss directly proportional to the severity of the chlorosis \[[@B6]\].
Plants have evolved two systems to uptake iron from the soil. These systems are termed strategy I and II \[[@B7],[@B8]\]. Soybeans and other dicots utilize strategy I, in which the rhizosphere is acidified by the release of protons to produce a favorable environment for the release of iron from chelating agents in the soil. A membrane bound reductase reduces iron to the usable Fe^+2^form. The iron is then transported across the plasma membrane by a specific transporter for distribution and use within the plant. The reduction of the iron from Fe^3+^to Fe^2+^has been shown to be the rate-limiting step in IDC \[[@B9]\]. Graminaceous monocots utilize strategy II, whereby the roots release chelators called phytosiderophores to bind Fe^+3^ions. Once bound, the entire complex is transported into the root where it is uncoupled. The Fe^+3^ion is reduced to Fe^+2^and the phytosiderophores are re-released into the soil.
The quantitative nature of IDC makes field studies problematic. Previous studies have identified multiple Quantitative Trait Locus (QTL) associated with IDC \[[@B4],[@B10]\]. Many of the same QTL have been identified in both field and greenhouse studies, where plants are grown in a hydroponics system designed specifically to induce IDC\[[@B10]\]. Growing plants in a controlled greenhouse environment with regulated nutritional availability allows for reproducible induction of iron deficiency stress. In addition, the advent of microarray technology now allows for the identification of individual transcripts whose expression levels are affected by iron availability\[[@B11],[@B12]\]. The availability of a whole-genome sequence assembly for the soybean genome has, for the first time, allowed us to genetically position differentially expressed genes induced by iron deficiency.
Genomic studies in many organisms have shown genes in close proximity to one another in the genome are often co-expressed. These co-expressed genes create clusters of expression neighborhoods \[[@B13]\] which are conserved by natural selection \[[@B14]\] A study in Arabidopsis showed clusters of up to 20 different genes were coordinately regulated, with a median cluster size of 100 kb \[[@B15]\]. In rice, approximately five percent of the genome has been associated with co-expressed gene clusters \[[@B16]\]. Initially co-expressed genes were thought to belong to similar biological pathways \[[@B15]\], but further studies have shown co-functionality to be a poor predictor of co-expression \[[@B17]\]. Instead, promoter analysis has found co-regulated genes are often regulated by common transcription factors \[[@B13],[@B17],[@B18]\] The co-expression of clustered genes may be partially regulated by the interaction of common promoter elements and transcription factors \[[@B18]\]. Co-regulated genes often have common transcription factors \[[@B17]\], so an increase in the number of transcription factor binding sites in promoter regions would increase the likelihood of the transcription factor binding and aiding in the expression of the gene cluster.
The objectives of our research are to identify a list of candidate genes with a potential involvement in soybean iron deficiency and to associate these genes with the genome sequence to determine any correlation with previously identified QTL. We also wanted to determine whether the changes in candidate gene expression were due to structural or sequence differences in the candidate genes. The results from these analyses confirmed the co-expressed genes were co-localized and possibly coordinately regulated.
Results
=======
Candidate Gene Identification and GO analysis
---------------------------------------------
RNA from the second trifoliate of both iron efficient Clark and iron inefficient IsoClark grown under iron limiting conditions (50 uM Fe(NO~3~)~3~, iron inefficient plants show severe chlorotic symptoms) and iron sufficient conditions (100 uM Fe(NO~3~)~3~, no chlorotic symptoms in either genotype) were hybridized to the Affymetrix^®^GeneChip^®^Soybean Genome Array. Eight hundred and thirty-five transcripts were differentially expressed between Clark plants grown under iron sufficient and iron limiting conditions (Additional file [1](#S1){ref-type="supplementary-material"}). By comparison 200 transcripts differentially expressed between IsoClark plants grown under the same conditions (Additional file [2](#S2){ref-type="supplementary-material"}). Only 18 transcripts were common between the two lists (data not shown). Under iron deficient growth conditions, there were 179 genes differentially expressed between the two NILs (Additional file [3](#S3){ref-type="supplementary-material"}). However, an analysis of the data revealed only 21 transcripts met or exceed the two fold difference required to be considered differentially expressed between Clark and IsoClark genotypes grown under iron sufficient conditions (Table [1](#T1){ref-type="table"}). This result confirms the NILs probably differ by only a limited number of genes.
######
Differentially Expressed Genes between Clark and IsoClark Genotypes Grown Under Iron Sufficient Conditions
Affymetrix Probe ID Fold Change UniProt ID Plant GOSlim AtHomolog PlantGOSlim
-------------------------- ------------- ------------ ------------------------ ----------------------------------------
GmaAffx.93650.1.S1_s\_at -12.383 Q6WE90 No Homolog
Gma.12096.1.A1_at -9.9 No UniProt No Homolog
Gma.18.1.S1_at -9.416 Q39819 AT4G10250 response to stress
Gma.17141.1.S1_at -8.776 Q9ZSA7 AT4G10490 other metabolic
Gma.14554.1.S1_at -7.959 Q1SJ63
GmaAffx.62046.1.S1_at -5.922 Q5CAZ5 AT1G34210 developmental
Gma.2185.3.S1_at -5.724 Q9FJL3 AT3G25230 response to stress
Gma.10282.1.A1_at -5.591 Q1T3Y4 AT4G27670 response to stress
GmaAffx.90956.1.S1_s\_at -5.297 No UniProt AT5G53740 biological process
Gma.11793.1.S1_at -3.469 No UniProt No Homolog
GmaAffx.89665.1.A1_s\_at -3.365 Q9AY32 No Homolog
Gma.12660.1.A1_at -3.173 Q8H2B1 AT1G56300 protein metabolism
Gma.10282.2.S1_at -2.899 Q1T3Y4 AT4G27670 response to stress
GmaAffx.93424.1.S1_x\_at -2.884 Q9S7H2 AT5G20620 protein metabolism
GmaAffx.56241.2.S1_at -2.739 Q9SCW4 AT5G62020 transcription
Gma.8636.1.S1_at -2.459 O80982 AT2G26150 transcription
GmaAffx.72322.1.S1_at -2.32 Q7F1F2 AT5G48570 protein metabolism
Gma.1727.1.S1_at -2.297 Q1SVQ0 AT2G39730 response to abiotic or biotic stimulus
GmaAffx.5924.1.S1_at -2.295 Q8L7T2 AT1G52560 response to stress
GmaAffx.74022.1.S1_at -2.267 Q1RY14 AT4G28480 protein metabolism
GmaAffx.93424.1.S1_s\_at -2.261 Q9S7H2 AT5G20620 protein metabolism
Fold Change between Iron Sufficient and Iron Deficient: Change in expression levels between Clark and IsoClark plants grown in iron sufficient conditions. A negative fold change is a down regulation of gene expression in the IsoClark genotype in comparison to the Clark genotype.
UniProt ID: Identifier for the UniProt sequence most similar to the consensus sequence used to create the Affymetrix probe set
Plant GO Slim At Homolog: The Arabidopsis homolog of the soybean consensus sequence represented by the Affymetrix probe set.
Plant GO Slim: The GO Slim annotation for the identified Arabidopsis homolog.
GO slim categories that were over represented in our lists of differentially expressed genes were identified for both the Clark and IsoClark comparisons. Transcripts with GO slim classifications that are over represented on our list of differentially expressed genes should be representative of the processes and pathways being affected in both the iron efficient and iron inefficient plants. The Clark genotype had 488 out of 835 unique transcripts with GO slim IDs. Of the corresponding GO slim IDs, 24 were over represented in our list of differentially expressed genes (Table [2](#T2){ref-type="table"}) in comparison with the entire chip. The over represented GO slim categories could be further divided into 14 biological process IDs, 9 molecular function IDs, and 1 cellular component processes (Table [2](#T2){ref-type="table"}). Of the 200 differentially expressed genes in the IsoClark genotype, 49 had corresponding Arabidopsis GO slim IDs. Of these, 21 genes had GO annotations that were over represented. These GO categories fell into two molecular function categories and three biological process categories (Table [3](#T3){ref-type="table"}).
######
GO Slim Terms Over Represented in Candidate Genes from Comparison Between Clark Plants Grown in Iron Sufficient and Iron Deficient Hydroponics Solutions
GO Slim ID GO Term Description Number of Genes with GO ID Bonferroni Corrected P-Value
--------------- ------------------------------------ ---------------------------- ------------------------------
GO:0000004 BP Unknown Function 128 0
GO:0006270 BP DNA Replication Initiation 10 0
GO:0009611 BP Response to Wounding 36 0
GO:0009695 BP Jasmonic Acid Biosynthesis 24 0
GO:0006826 BP Iron Ion Transport 9 1.2E-07
GO:0006879 BP Iron Ion Homeostasis 10 3.6E-07
GO:0010039 BP Response to Iron Ion 9 5.28E-06
GO:0009617 BP Response to Bacterium 8 0.000018
GO:0006275 BP Regulation of DNA Replication 4 0.00303
GO:0006972 BP Hyperosmotic Response 4 0.00303
GO:0030397 BP Membrane Disassembly 8 0.004381
GO:0008299 BP Isoprenoid Biosynthesis 10 0.006706
GO:0009408 BP Response to Heat 20 0.014334
GO:0019373 BP Epoxygenase P450 5 0.045066
GO:0008199 MF Ferric Iron Binding 9 0
GO:0008094 MF DNA-dependent ATPase Activity 10 1.29E-06
GO:0016165 MF Lipoxygenase Activity 12 0.000128
GO:0047763 MF Cafeate O-Methyltransferase 8 0.001014
GO:0030337 MF DNA Polymerase Processivity Factor 4 0.001426
GO:0005544 MF Calcium Dependent Lipid Binding 8 0.001965
GO:0009978 MF Allene Oxide Synthase 5 0.017949
GO:0046423 MF Allene Oxide Cyclase 4 0.020157
GO:0008815 MF Citrate (Pro-3S) Lyase 5 0.029946
GO:0009346 CC Citrate Lyase 5 0.002807
GO Slim ID\'s statistically over or under represented in the list of differentially expressed genes comparing Clark plants grown in iron sufficient and iron limiting hydroponics conditions in comparison to their presence on the Affymetrix^®^GeneChip^®^. The GO Slim Column provides the GO Slim ID number and corresponding category. GO Slim categories are Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). The GO Term Description column contains the best description for the GO Slim ID. The number of genes with GO ID column is the number of genes differentially expressed between Clark plants grown in iron sufficient and iron deficient hydroponics solutions with the corresponding GO Slim ID. The Bonferroni Corrected P-value is the statistical significance of the GO Slim ID. Only GO Slim IDs with P-values equal to or less than 0.5 were considered statistically over or under represented in the list of differentially expressed genes in comparison to the presence of the GO Slim ID on the GeneChip^®^.
######
GO Slim Terms Over or Under Represented in Candidate Genes from Comparison Between IsoClark Plants Grown in Iron Sufficient and Iron Deficient Hydroponics Solutions
GO Slim GO Term Description Number of Genes with GO ID Bonferroni Corrected P Value
--------------- --------------------------- ---------------------------- ------------------------------
GO:0000004 BP Unknown Function 49 0
GO:0006809 BP Nitric Oxide Biosynthesis 4 0.0006102
GO:0010025 BP Wax Biosynthesis 5 0.00736524
GO:0019953 BP Sexual Reproduction 5 0.01223184
GO:0008940 MF Nitrate Reductase 4 0.00006192
GO:0008382 MF Iron Superoxide Dismutase 3 0.01245882
GO Slim ID\'s statistically over or under represented in the list of differentially expressed genes comparing Clark plants grown in iron sufficient and iron limiting hydroponics conditions in comparison to their presence on the Affymetrix^®^GeneChip^®^. The GO Slim Column provides the GO Slim ID number and corresponding category. GO Slim categories are Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). The GO Term Description column contains the best description for the GO Slim ID. The number of genes with GO ID column is the number of genes differentially expressed between IsoClark plants grown in iron sufficient and iron deficient hydroponics solutions with the corresponding GO Slim ID. The Bonferroni Corrected P-value is the statistical significance of the GO Slim ID. Only GO Slim IDs with P-values equal to or less than 0.5 were considered statistically over or under represented in the list of differentially expressed genes in comparison to the presence of the GO Slim ID on the GeneChip^®^.
Examining the GO terms associated with the candidate genes provides further insight into the disparity of the number of differentially expressed genes between genotypes. The IsoClark (inefficient) genotype does not appear to induce the full complement of genes induced in Clark in response to the iron deprivation stress. The most prevalent GO term in all three classifications for both genotypes was \'unknown function\' (Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). However, the Clark (efficient) genotype also had a high proportion of GO terms (and thus, transcripts) specifically related to iron availability and usage, (ferric iron binding (GO:0008199), iron ion transport (GO:0006826), and iron ion homeostasis (GO:0006879)) that were over-represented on our lists of candidate genes responding to iron stress. In addition, Clark genes encoded a number of GO terms not specifically related to iron, but which are associated with a more general stress response (GO:0009611 -- response to wounding, GO:00099 jasmonic acid biosynthesis, and GO:0009408 -- response to heat).
Real Time PCR Confirmation
--------------------------
The differential expression observed through sqRT-PCR analysis mirrored, in direction, the expression differences observed in the microarray study (Table [4](#T4){ref-type="table"}). The difference in expression levels seen in between the sqRT-PCR and the microarray experiment is most likely due to cross hybridization. Multiple members of the same gene family may hybridize to the same spot on the microarray, while the sqRT-PCR experiment is designed to amplify only single members of the gene family. The sqRT-PCR experiments confirmed the iron deficient plants had lower expression levels of the transcripts than the iron sufficient plants, replicating the results seen in the microarray data.
######
Semi Quantitative Real Time PCR Results
Affymetrix Probe Set Annotation Conditions of Differential Expression Differential Expression in Microarray Diff Express in sqRT-PCR
----------------------- -------------------- --------------------------------------- --------------------------------------- --------------------------
Gma.13296.3.S1_at Lipid Transfer CSSvCSD 190.34 2.62
Gma.17724.3.S1_at GDSL motif CSSvCSD 6.26 8.46
Gma.17825.1.A1_at GDSL motif CSSvCSD 4.69 10.26
GmaAffx.89896.1.S1_at Heat Shock Protein CSSvCSD 23.45 2.04
GmaAffx.93268.1.S1_at Heat Shock Protein CSSvCSD 19.91 1.36
GmaAffx.51733.1.A1_at Ribonuclease T2 CSSvCSD -13.35 -2.98
GmaAffx.88242.1.S1_at SnRNP protein CSSvCSD 3.58 11.18
Gma.16500.1.S1_at Lipoxygenase CSSvCSD 11.36 16.9
Gma.3705.1.S1_at Nitrate Reductase CSDvICSD -3.53 -1.101
Gma.9609.1.S1_at Reductase CSDvICSD 2.26 2.23
GmaAffx.36066.1.S1_at Replication Factor CSDvICSD 2.63 3.58
Details of sqRT-PCR experiments. Affymetrix Probe set denotes the differentially expressed sequence for which primers were designed. Conditions of Differential Expression represent the genotype and growth conditions in the comparison where the sequence was differentially expressed (CSS -- Clark Shoots Iron Suficient, CSD -- Clark Shoots Iron Deficient, ICSD -- IsoClark Shoots Iron Deficient). Differential Expression in Microarray, the change in gene expression for the sequence from the microarray (in fold change). Differential Expression in sqRT-PCR, the change in gene expression for the sequence seen in the sqRT-PCR experiment (in fold change).
Positioning Candidate Genes on the 7X Genome Assembly
-----------------------------------------------------
Sequencing of the soybean genome by the Department of Energy, Joint Genome Institute currently has produced 7X sequence coverage of the genome <http://www.phytozome.net>, which has been assembled by USDA-ARS researchers into twenty draft pseudo chromosomes based on marker homology, allowing us to place our candidate genes on specific chromosomes (Figure [1](#F1){ref-type="fig"}).
{#F1}
The sequences of transcripts identified as differentially expressed by microarray analysis (see above) were obtained from the Affymetrix^®^website <http://www.affymetrix.com>. These sequences were then queried against the 7X soybean genome using BLASTN \[[@B19]\] and an e-value cutoff of 10E^-50^to ensure a high sequence similarity between the aligned sequences. The same parameters were used to compare the sequences of SFPs to the 7X genomic sequence assembly. Markers used in previous iron QTL studies were also identified on the pseudo chromosomes to delineate known iron QTL regions (Figure [1](#F1){ref-type="fig"}). The iron efficiency QTL were scaled to the 7X build and used to determine if any of the candidate genes from the microarray experiment were encoded within the iron QTL regions. Fifty-eight genes in the Clark genotype and twenty-one genes in the IsoClark genotype were located within previously identified iron QTL (Figure [1](#F1){ref-type="fig"}, Additional files [1](#S1){ref-type="supplementary-material"} and [2](#S2){ref-type="supplementary-material"}).
Sequences of the delineated iron QTL regions were analyzed using FGENESH <http://www.softberry.com> using Arabidopsis as the training model to identify gene structure predictions. The identified gene sequences were mined from the genome sequence and compared to known transcription factors in Arabidopsis <http://datf.cbi.pku.edu.cn/download.php>\[[@B20]\] rice, soybean, barley, and medicago <http://planttfdb.cbi.pku.edu.cn/index.php>\[[@B21]\]. This comparison identified 780 predicted genes within the previously identified QTL regions that show high sequence similarity (10E^-50^) to known transcription factors. One of these, within a QTL region on chromosome 12, showed a 100% identity to the Arabidopsis FIT gene (AT2G28160).
Cluster Analysis
----------------
The gene distribution simulation randomly placed genes across the assembled genome. A second analysis assumed 36% of the genome was heterochromatic as proposed by Singh and Hymowitz \[[@B22]\] and reflected in the distribution of predicted gene locations <http://www.phytozome.net>. This analysis further constrained the algorithm, reducing the probability of candidate genes exhibiting clustering within the genome. If the candidate genes were randomly located throughout the genome, we would expect the experimental results to closely mirror the simulated data study. However our results, for both clustering analyses, strongly indicate that the differentially expressed genes exhibit clustering of two or more genes within 1,000,000 bp, 100,000 bp, and 10,000 bp in the genome (Tables [5](#T5){ref-type="table"} and [6](#T6){ref-type="table"}, Additional files [4](#S4){ref-type="supplementary-material"} and [5](#S5){ref-type="supplementary-material"}).
######
Clusters of Candidate Genes on 7X build of Soybean Genome from Clark genotype assuming 36% of the genome is heterochromatic.
1,000,000 base bins
--------------------- ------------------ ---------------- ------------------- -------------------
0 523 7.94 569 5.79
1 221 11.92 188 -2.77
2 137 9.34 99 -4.07
3 56 6.37 44 -1.88
4 17 3.76 36 5.05
5 4 1.96 13 4.59
6 1 0.90 2 1.11
7 0 0.41 2 4.88
8 0 0.14 1 7.14
100,000 base bins
\# of Genes Stimulation Mean Stimulation SD Experimental Data Sds from Sim Mean
0 8885 6.47 8926 6.34
1 671 12.50 486 -14.8
2 42 6.05 98 9.26
3 2 1.29 22 15.5
4 0 0.22 2 9.09
5 0 0.03 1 33.33
100 base bins
\# of Genes Stimulation Mean Stimulation SD Experimental Data Sds from Sim Mean
0 95243 2.16 94678 -2.62
1 751 4.31 593 -36.66
2 5 2.16 71 30.55
3 0 0.10 7 70
4 0 0
5 0 1
Details of sqRT-PCR experiments. Affymetrix Probe set denotes the differentially expressed sequence for which primers were designed. Conditions of Differential Expression represent the genotype and growth conditions in the comparison where the sequence was differentially expressed (CSS -- Clark Shoots Iron Suficient, CSD -- Clark Shoots Iron Deficient, ICSD -- IsoClark Shoots Iron Deficient). Differential Expression in Microarray, the change in gene expression for the sequence from the microarray (in fold change). Differential Expression in sqRT-PCR, the change in gene expression for the sequence seen in the sqRT-PCR experiment (in fold change).
######
Clusters of Candidate Genes on 7X build of Soybean Genome from IsoClark Genotype assuming 36% of the genome is heterochromatic.
1,000,000 base bins
--------------------- ------------------ ---------------- ------------------- -------------------
0 757 4.98 783 5.22
1 165 8.81 118 -5.33
2 33 4.40 38 1.14
3 4 2.02 8 1.98
4 0 0.66 7 10.60
100,000 base bins
\# of Genes Stimulation Mean Stimulation SD Experimental Data Sds from Sim Mean
0 9359 2.13 9325 -15.96
1 236 4.21 178 -13.78
2 5 2.07 28 11.11
3 0 0.25 4 16
100 base bins
\# of Genes Stimulation Mean Stimulation SD Experimental Data Sds from Sim Mean
1 95754 0.72 95128 -869.44
1 245 1.44 199 -31.94
2 1 0.72 22 29.17
3 0 0.03 1 33.33
Details of sqRT-PCR experiments. Affymetrix Probe set denotes the differentially expressed sequence for which primers were designed. Conditions of Differential Expression represent the genotype and growth conditions in the comparison where the sequence was differentially expressed (CSS -- Clark Shoots Iron Suficient, CSD -- Clark Shoots Iron Deficient, ICSD -- IsoClark Shoots Iron Deficient). Differential Expression in Microarray, the change in gene expression for the sequence from the microarray (in fold change). Differential Expression in sqRT-PCR, the change in gene expression for the sequence seen in the sqRT-PCR experiment (in fold change).
The candidate genes do not show a high concordance with known iron QTL regions, but do serve to identify additional genomic regions of IDC transcriptional importance (Figure [1](#F1){ref-type="fig"}). The largest cluster contained eight candidate genes located within 1 MB on chromosome 6 (Figure [1](#F1){ref-type="fig"}). There was also a cluster of seven genes on chromosome 2 (Figure [1](#F1){ref-type="fig"}). Chromosome 2 contained six clusters of four or more genes within 1,000,000 bases, as did chromosome 13 (Figure [1](#F1){ref-type="fig"}). None of these clusters were located within known iron QTL. However, another cluster of seven genes falls on chromosome 12 (Figure [1](#F1){ref-type="fig"}), which has three known iron QTL, together spanning 24 cM of the linkage group. Chromosome 7 contained the most gene clusters, eight separate clusters, each with four candidate genes (Figure [1](#F1){ref-type="fig"}). Again, chromosome 7 has not been previously shown to contain regions of genetic importance to soybean IDC. A number of the gene clusters contain multiple copies of genes encoding proteins with similar functions. A cluster of six candidate genes on chromosome 9 all encode proline rich proteins while three of the genes in a cluster of 5 on chromosome 6 encode caffeic acid O-methyltransferases (Additional file [4](#S4){ref-type="supplementary-material"}). The close physical proximity of co-expressed genes of the same function provides further support for the clustering of co-expressed and co-functional gene clusters.
The identification of these gene clusters on chromosomes not previously known to be involved in soybean IDC opens new regions of genetic interest to investigate in future studies. The majority of the candidate genes identified were not within the iron QTL, nor were the largest clusters of differentially expressed genes. Genes affecting chlorosis and yield loss may not be confined to the previously identified QTL regions. However, the iron QTL must contain sequence of importance to IDC. It is likely a transcription factor(s) controlling the expression of genes required to induce an iron stress response is encoded within the QTL regions. We have identified 780 predicted transcription factors, including the soybean homolog to the Arabidopsis FIT gene, within the previously identified QTL. This, or another of these transcription factors may be responsible for inducing a cascade of gene expression changes due to limited iron conditions. The transcription factor/factors may also affect the expression of the canonical iron genes, such as IRT and FRO, none of which are encoded within the previously identified QTL, nor identified as differentially expressed in our microarray experiment.
Motif Analysis
--------------
Previous research has demonstrated that genes clustered in close proximity in a genome may be coordinately regulated. To determine if clusters of IDC genes were coordinately regulated, we examined 500 bases from the 5\' untranslated regions (putative promoters) of all differentially expressed IDC genes and used these as input into the MEME software program. As an internal control, sequences were not analyzed as members of clusters; rather, all sequences were analyzed as a single large group. If IDC genes were coordinately regulated, MEME could also be used to independently identify potential gene clusters. In total, the putative promoters of 835 iron deficiency induced genes from the Clark genotypic comparison and 200 genes from the IsoClark comparison were analyzed using MEME. There were no motifs found using MEME in the IsoClark (inefficient) promoter regions. All motifs identified by MEME were found in the promoter regions of genes differentially expressed due to iron deficiency in the Clark (efficient) genotype. Twenty-one motifs with E-values more significant than 10E^-6^were identified by MEME analysis. Following visual inspection, this number was reduced to 11. Motifs were eliminated if they contained repetitive sequence or had lower significance E-values. The 11 motifs were identified in 248 IDC genes, representing 129 of the clusters of two or more genes as mentioned above. One mechanism by which genes can be coordinately regulated is through the action of transcription factors that bind to the promoter to regulate gene expression. Therefore, the 11 motifs identified above were compared to known transcription factor binding sites in the TRANSFAC \[[@B23]\] database (Table [7](#T7){ref-type="table"}). Three motifs showed significant sequence similarity (99% identity) to known transcription factor binding sites (Table [7](#T7){ref-type="table"}). These three sites bind a helix-loop-helix transcription factor (bHLH), an elongation factor (EF2), and a Myb transcription factor. These binding sites were identified in the promoter regions of 42, 40, and 28 iron responsive genes respectively. Both helix loop helix and Myb transcription factors are known to be involved in regulating the iron stress response and general stress responses in other plant species.
######
Motifs in Promoter Regions of Differentially Expressed Genes Identified by MEME and Their Similarity to Transcription Factor Binding Sites in the TRANSFAC Database
Identified Motif Sequence \# Seqs withMotif Motif E-Value TRANS FAC Hit ID TRANS AC Binding Site Sequence TRANS FAC annotation
--------------------------- ------------------- --------------- ------------------ -------------------------------- ----------------------
CATCCAACGGC 29 1.2E-1 M00227 TCCAACGGC Myb
CCCGCCACGCGCCAC 48 5.1E-26 M00187 GCCACG[T]{.ul}GCC Helix Loop Helix
TGGCGGGA 50 5.8E-13 M00024 TGGCG[C]{.ul}GA Elongation Factor 2
CCAAACCC 50 2.7E-5 No Hit
CCACCACCACC 48 3.8E-16 No Hit
ACACAACACAC 45 2.2E-10 No Hit
AAAATAAAAATAAAA 9 2.27E-7 No Hit
AATAAAAAAATAAAA 8 1.51E-7 No Hit
AGCTAGCTAGC 6 1.47E-7 No Hit
AGCGAGCGAGC 4 6.23E-8 No Hit
AGCAAGCTAGC 3 2.47E-7 No Hit
Identified Motif Sequence: The motif sequence identified by MEME analysis in the promoter region of differentially expressed genes from the Clark genotype.
\# of Seqs with Motif: The number of differentially expressed genes whose promoter regions contain the identified motif.
Motif E-Value: E-value of the motif assigned by MEME.
TRANSFAC Hit ID: The TRANSFAC ID of the transcription factor binding site showing high homology to the identified motifs. Only three identified motifs showed homology to known transcription factor binding sites in the TRANSFAC database.
TRANSFAC Binding Site Sequence: The transcription factor binding site sequence. Mismatched bases are between the identified motif and the reported binding sites are underlined.
TRANSFAC Annotation: The type of transcription factor that binds to the identified TFBS according to TRANSFAC.
SFP Analysis
------------
The SFP analysis identified 170 single feature polymorphisms, seventy-two SFPs were unique to the Clark genotype and 98 unique to the IsoClark genotype. A number of the Affymetrix^®^sequences found to contain an SFP perfectly matched more than one genomic location, giving a potential of 208 predicted genes with an SFP (Additional file [6](#S6){ref-type="supplementary-material"}). Only one of the 170 SFPs identified in this study (GmaAffx.41460.1.S1_at) was encoded within a gene identified as differentially expressed between IsoClark plants grown under iron sufficient and iron deficient conditions. The remaining SFPs were not in differentially expressed genes in either Clark or IsoClark genotypes. This suggests most of the SFPs are encoded in regulatory elements, which would not necessarily be differentially expressed. GO slim ID analysis, as previously described, was performed with the gene sequences containing SFPs. Of the genes containing SFPs, 20% had an unknown biological process annotation. The most prevalent group with known annotations was related to transcriptional regulation. Genes involved with electron transport, ATP binding, ligases, and transferases were also identified as over-represented by their GO IDs (data not shown).
Discussion
==========
Microarray Analysis
-------------------
Through a combination of a suite of analyses we have extended the fundamental understanding of the genetics underlying iron uptake and homeostasis in plants, but specifically soybean. Affymetrix gene chip analysis allowed us to identify candidate genes that are induced by iron deficiency in the leaf tissue of two NILs, complementing previous studies done in roots \[[@B11],[@B12]\]. The Clark genotype analysis identified 835 differentially expressed genes when grown under iron sufficient and iron insufficient conditions while in IsoClark only 200 were identified (Additional files [1](#S1){ref-type="supplementary-material"} and [2](#S2){ref-type="supplementary-material"}). These genes have been aligned with the genomic sequence to determine their location. A sliding window analysis determined the co-expressed genes are clustered in the genome, suggesting co-regulation. The SFP analyses determined the differentially expressed genes are not a result of structural differences in the genes between the two NILs, providing further support that the differentially expressed genes are being co-regulated. Finally, motif analysis identified 11 short conserved motifs in the promoter regions of the candidate genes, which are most likely transcription factor binding sites. The cumulative results of all analyses leads us to propose the differential iron response in the NILs is a result of a mutation in the iron inefficient NIL of a transcription factor, or factors, probably encoded within one or more of the previously identified QTL, that prevents the induction of the iron deficiency gene expression responses seen in the iron efficient NIL.
The candidate genes identified with the microarray experiment suggest the Clark genotype is capable of recognizing the iron deficiency and eliciting a change in transcription patterns as a response to the stress. We hypothesize that the iron deficient Clark plant compensates for the lack of iron availability by adjusting its physiological processes to conserve available iron. Alternatively, the IsoClark genotype does not appear to initiate an effective response to the iron deficient conditions. The lack of differentially expressed genes in the IsoClark genotype, when comparing iron sufficient and iron deficient conditions, implies the iron deficient IsoClark plant continues to function as if still in iron sufficient conditions. However, the lack of iron as a cofactor in many of the basic biological processes results in a multitude of biological pathway failures, resulting in chlorotic plants.
In Arabidopsis, iron deficiency stress causes an increase in the transcription of electron transport chain components. Specifically, cytochromes are upregulated \[[@B24]\]. Our experiment identified seventeen genes associated with cytochrome P450 in iron stressed Clark plants. All seventeen genes were down-regulated in iron stressed tissue compared to non stressed tissue, the opposite response as seen in Arabidopsis plants \[[@B24]\]. Thimm et al. proposed a correlation between iron deficiency stress and in induction of phosphoenolpyruvate carboxylase activity \[[@B24]\]. Four genes associated with phosphoenolpyruvate activity were identified as differentially expressed in the Clark genotype by microarray analysis. All four of these genes were down regulated in plants grown under iron stress rather than in iron sufficient conditions. Iron deficiency has also been shown to induce glycolytic activity \[[@B25]\]. Three enzymes involved in glycolysis; glyceraldehydes 3 phosphate dehydrogenase (G3PD), pyruvate kinase (PK), and fructose 6 phosphate kinase (F6PK) have been shown to be up-regulated in Arabidopsis \[[@B24]\] and cucumber \[[@B25]\] under iron deficiency stress. Microarray analysis comparing Clark plants grown in iron sufficient and iron stressed conditions only identified a single G3PD and a single F6PK, both of which were down-regulated in the iron stressed tissue compared to iron sufficient tissue. Seven genes associated with PK were identified in our microarray. Again, all seven were down regulated. The down regulation of the three main components of glycolysis suggests soybean, unlike Arabidopsis, does not increase non photosynthetic carbon fixation or phosphoenolpyruvate carboxylase activity under iron stressed conditions. The contrasting results support the hypothesis proposed by Zocchi et al. that soybeans do not follow canonical iron deficiency responses \[[@B26]\].
Soybean does follow some of the established responses to iron deficient stress conditions. It has been proposed that under iron deficient conditions citrate provides a carbon skeleton for chlorotic leaves to allow for sustained growth and respiration \[[@B27]\]. Clark iron deficient stressed plants show a down regulation of citrate lyase (GO: 0008815) in comparison to non-stressed plants. The reduced breakdown of citrate in iron stressed plants lends credence to this hypothesis. Additionally, iron deficient conditions cause decreased activity of lipoxygenases \[[@B24]\]. All thirteen lipoxygenases identified by microarray analysis in the Clark genotype showed decreased expression in the iron stressed tissue compared to the iron sufficient tissue.
The discrepancies between previously reported literature and the soybean iron deficiency response highlight the complexity of the iron stress response. However, it is important to remember that transcriptional regulation is only one form of regulation. Post-transcriptional modification may be an important component to understanding soybean\'s iron deficiency response, but that is beyond the scope of this investigation.
GO Slim ID Analysis
-------------------
The Clark (iron efficient) genotype had an over-representation of genes in GO slim categories specific to iron availability/usage and categories associated with a more general stress response (Table [2](#T2){ref-type="table"}). This reinforces the hypothesis that Clark responds specifically to iron but also to a more general stress response. A similar pattern was observed in a cDNA microarray experiment \[[@B12]\]. Additionally, the Clark genotype showed a statistically significant number of GO slim IDs that were over-represented related to DNA replication and DNA binding activity. The increased expression levels of genes involved in these processes is probably a result of the DNA repair required to prevent lethal mutations from ROS \[[@B28]\], which are more prevalent under conditions of stress \[[@B28]\]. DNA binding activity suggests the activity of transcription factors, which lead to dramatic expression changes downstream. However, the down regulation of genes related to translation ie: GO0006412 (translation) and GO: 0006468 (protein amino acid phosphorylation) is indicative that the plant is not synthesizing proteins at a normal rate as it would under optimal growth conditions and is instead reducing the expression of genes involved in cellular processes not imperative to survival.
The IsoClark genotype had many fewer GO categories significantly over represented on our lists of candidate genes (Table [3](#T3){ref-type="table"}) in comparison to Clark. Only two of the GO classifications were related to iron GO:0008940 (nitrate reductase activity) and GO:0008382 (iron superoxide dismutase activity). The remaining GO categories show little association to either a general or an iron specific stress response. It appears the IsoClark genotype is unable to recognize or respond to the iron stress. The IsoClark genotype had fewer genes differentially expressed due to iron deficiency and most of the genes that were differentially expressed are not associated with stress related pathways.
Clusters of Co-Expressed Genes
------------------------------
Expression analysis has been used in some model organisms to identify differentially expressed genes that are clustered together within the genome \[[@B13]-[@B18],[@B29],[@B30]\]. These genomic neighborhoods are thought to be conserved by natural selection \[[@B14]\] but are not entirely explained by co-functionality \[[@B17]\]. The combined use of expression data with known QTL positions and expression clusters should further narrow the list of candidate genes to identify functionally important differences in the soybean genome affecting iron efficiency.
Co-expressed genes show a non random distribution throughout the genome \[[@B14],[@B17]\]; where similarly expressed genes are located in clusters. Localized co-expression of genes has been reported in many different species including (but not limited to Arabidopsis \[[@B15],[@B29],[@B30]\], rice \[[@B16]\], human \[[@B13],[@B31]\]and yeast \[[@B14]\]). Williams et al (2004) found genes located nearby in the genome and genes involved in the same pathways are more likely to be co-expressed. The incidence of co-expressed gene clusters has been widely studied \[[@B13]-[@B15],[@B17],[@B31]\]. One proposed explanation is that the co-expressed genes are regulated by a common transcription factor. Grouping these genes creates an increase in the abundance of binding sites specific to that transcription factor \[[@B31]\]. A related hypothesis suggests the co-expressed genes are regulated by similar promoter sequences, so a co-expression \'neighborhood\' would increase the availability of these promoter sequences \[[@B18]\]. However, genomic studies have, as of yet, been unable to confirm either of the two hypotheses.
Cluster analysis, as first reported by Grant et al.(2000), was performed to determine if candidate genes identified by the microarray experiment were randomly distributed across the genome. Iterative simulations modeling our data showed our candidate genes were not distributed evenly throughout the genome. Using a sliding window of 1,000,000 bases, we identified more genes in smaller regions of the genome than expected by a random distribution of the differentially expressed genes with 3 -- 8 candidate genes per 1,000,000 bases (Tables [5](#T5){ref-type="table"} and [6](#T6){ref-type="table"}). The same patterning held true when the sliding window was reduced to 100,000 and 100 bases. The statistical significance, from comparing the experimental data to the simulated data, is found in the number of simulated standard deviations (SDs) the experimental data is from the simulated data (Simulation SD column in Tables [5](#T5){ref-type="table"} and [6](#T6){ref-type="table"}). When comparing clusters of three or more genes in either Clark or IsoClark, there are only four instances (3 and 6 genes per cluster, Table [7](#T7){ref-type="table"}; 2 and 3 genes per cluster, Table 8) where the difference between the experimental data and the simulation study is not statistically different.
In the Clark genotype, with a window of 1,000,000 bases, there were thirty-six clusters of four genes and thirteen clusters of five genes per window identified in the experimental data. There were only seventeen clusters of four genes and only four occurrence of five genes clustering together in the simulation study. The difference in SDs is 5.05 and 4.59 respectively, indicating a highly statistically significant difference. The million base window allowed larger gene clusters to be identified in the experimental data (two clusters of seven genes and a single cluster containing eight genes). No clusters of these sizes were identified in the simulation study, further supporting the clustering hypothesis. When the window size is decreased to 100,000 or 100 bases, three genes in a cluster become significantly over represented in the experimental data compared to the simulation study. The microarray experiment identified 22 clusters of three genes per 100,000 bases. No clusters of three or more genes were identified in the simulation study at either window size.
The IsoClark genotype identified fewer candidate genes in the microarray experiment, which reduces the number of gene clusters identified. However, even with a reduced number of candidates, IsoClark still exhibited clustering of co-expressed genes. With a window of 1,000,000 bases, there were eight clusters of three genes identified in the experimental data, but only four clusters are identified in the simulation study. There were seven clusters of four genes per million bases identified in the IsoClark simulation, but none in the simulated data. The retention of clusters, even among so few candidate genes, lends further support that the soybean genome has conserved genomic regions with co-expressed genes.
Individual gene clusters are interesting because so many of them contain multiple copies of similar genes (Additional files [4](#S4){ref-type="supplementary-material"} and [5](#S5){ref-type="supplementary-material"}). For example, all six genes in the cluster on chromosome 9 encode proline rich proteins while three of the five genes in a cluster on chromosome 6 encode caffeic acid O-methyltransferase. The co-expression of these genes coupled with their close physical proximity lends further credence to the hypothesis that they are regulated by a common transcription factor.
Single Feature Polymorphisms (SFPs)
-----------------------------------
Identifying candidate genes for a trait of interest is the most widely used method of analyzing the data provided by microarray experiments. However, mining the hybridization data to identify single feature polymorphisms (SFPs) provides a high throughput platform for detecting polymorphisms \[[@B32]\]. Single Nucleotide Polymorphisms (SNPs) are the most commonly recognized polymorphism, but identification is labor intensive and SNP coverage across the genome is fairly sparse \[[@B33]\]. It has been suggested that there is a greater probability of identifying a causal polymorphism for the trait of study using SFPs than traditional SNPs \[[@B33]\], perhaps due to better genic coverage.
To date, only 3 molecular markers (Satt 481, Satt114, and Satt239) segregate with the iron efficiency trait in soybean across multiple populations \[[@B34],[@B35]\]. The 72 SFPs identified in the Clark genotype and the 98 SFPs identified in the IsoClark genotype relative to Williams 82 in this study have the potential to be developed into molecular markers specific to IDC. Initially, we hypothesized the SFPs would correlate with the differentially expressed genes. However, only one SFP (GmaAffx.41460.1.S1_at) was found in a gene differentially expressed in IsoClark leaf tissue. In Arabidopsis this gene is essential for NADH mediated reduction of the plastiquinone pool in respiratory electron transport and is up-regulated under mild heat stress \[[@B36]\]. It is logical that this gene might be differentially expressed in the iron inefficient plant as photosynthesis slows due to a lack of iron serving as electron transporters. The remaining169 SFPs were not differentially expressed due to iron stress. The majority of the sequences identified containing an SFP have an unknown function and the largest class of annotated SFPs is transcription factors (Additional file [6](#S6){ref-type="supplementary-material"}). These SFP polymorphisms may alter transcription and/or translation rates of key genes and proteins, or serving some other regulatory function in soybean iron homeostasis.
Promoter Motifs
---------------
Analysis of the 500 bp upstream of the start codon for the predicted genes in soybean <http://www.phytozome.net> that coordinate with the differentially expressed candidate genes identified 11 conserved motifs (Table [7](#T7){ref-type="table"}). These small motifs were notable for both their highly conserved sequences and conserved positions. A comparison of these motif sequences to the TRANSFAC database identified three of the 11 motifs as transcription factor binding sites (TFBS). It is likely that the remaining eight motifs represent previously uncharacterized transcription factor binding sites. One of the three motifs that showed high similarity to a TRANSFAC TFBS was a Myb TFBS. Myb transcription factors have been implicated in inducing the stress response in plants in response to various abiotic stresses including phosphate stress \[[@B37]\] and asian soybean rust \[[@B38]\]. The identification of the Myb TFBS in the promoter region of candidate genes from the Clark genotype supports the idea that Clark is able to induce both an iron specific stress response and a more generalized stress response.
The identification of a basic helix loop helix (bHLH) TFBS motif in the promoter region of candidate genes from the Clark genotype may be indicative of the iron specific stress response induced in Clark under iron deficient conditions. In Arabidopsis, bHLH proteins have been identified as essential components in mediating iron uptake under iron stress conditions. Specifically, AtbHLH38 and AtbHLH39 both form heterodimers with AtbHLH29 to regulate iron uptake gene expression under iron deficient conditions in Arabidopsis \[[@B39]\]. AtbHLH29 encodes a transcription factor known as FIT (FER like iron deficiency induced transcription factor \[[@B40]\]), which dimerizes with either AtbHLH38 or 39 to induce FRO2 and IRT1 gene expression \[[@B39]\]. Though the soybean FIT homolog was not identified as differentially expressed due to iron deficiency, it was one of the 780 transcription factors predicted to be encoded within previously identified QTLs. The importance of bHLH transcription factors in regulating iron uptake gene expression makes the identification of a bHLH TFBS in the promoter region of iron deficiency induced genes particularly exciting. This is the first evidence that iron uptake gene expression may be similarly regulated in Arabidopsis and soybean.
Iron QTL and the Soybean Genome
-------------------------------
QTL mapping and marker assisted selection have been utilized by plant breeders for decades in the pursuit of crop improvement. This approach has been especially important for quantitative traits such as iron deficiency chlorosis \[[@B4],[@B34],[@B35],[@B41]\]. Only in recent years have scientists been able to utilize microarray technology to examine gene expression on a global scale to identify candidate genes for their trait of interest. The development of the Affymetrix^®^GeneChip^®^Soybean genome array \[[@B42]\], representing approximately 75% of the predicted genes in soybean (data not shown), means repeatable precision, providing more confidence to the microarray experiments than cDNA arrays.
The availability of the whole-genome soybean sequence has provided the ability to visualize the placement of candidate gene sequences within the genome. This view will allow further insight into soybeans\' response to iron deficiency stress. Nineteen QTL regions have been previously identified for iron deficiency chlorosis, both in field and hydroponic studies \[[@B4],[@B10],[@B41]\]. These regions represent approximately 182 cM of genetic information. Our initial hypothesis was that the majority of the genes identified in the microarray experiment would map within known iron QTL regions. However, only 58 of the 835 (7%) candidates in the Clark genotype and 21 of the 200 (10%) in the IsoClark genotype mapped within known QTL regions (Figure [1](#F1){ref-type="fig"}, Additional files [1](#S1){ref-type="supplementary-material"} and [2](#S2){ref-type="supplementary-material"}). Thus, the majority of the candidate genes identified in this study lie outside of regions identified as iron QTL. However, given the evidence of coordinate gene expression, gene clustering and conserved promoter motifs in our data, we have revised our previous hypothesis. We now propose the previously identified QTL regions likely correspond to transcription factors that regulate gene expression during iron stress. While microarray experiments would identify IDC regulated genes whose expression changes in response to a transcription factor, they may not identify the transcription factor itself. In contrast, QTL mapping would identify a mutation in a transcription factor, which is at the top of the signaling pathway. The mutation would effect either the expression of the transcription factor or its ability to bind to target promoters. This hypothesis is supported by our data. The clustering of co-expressed genes suggests they are being coordinately regulated. This is supported by the conserved motifs identified in the promoter regions of candidate genes. Most often, motifs are conserved throughout a previously identified cluster of genes in Clark. It is unlikely these motifs are missing or are altered in the promoter regions of the IsoClark genome. More likely, IsoClark may have a mutation in the transcription factor that controls the expression of these genes. Only by combining QTL analyses, microarray analyses of NILs, and the genome sequence could this conclusion be reached.
Conclusion
==========
The use of near isogenic soybean lines, microarray analysis, SFP identification, and the sequence of the soybean genome has allowed us to identify individual genes lying within known iron efficiency QTL whose expression levels are affected by iron availability. We have also identified 11 conserved motifs in the promoter sequence of genes differentially expressed due to iron deficiency stress. The 58 differentially expressed genes identified in Clark and 21 in IsoClark, located within known QTL regions, are the first genes identified by microarray analysis within QTL regions specific to iron deficiency stress. The conserved motifs throughout the promoter regions of the differentially expressed genes in the Clark genotype provide compelling evidence that the differential iron response is likely due to the differential expression or binding of a transcription factor. Co-expressed genes clustered either by physical proximity (Tables [5](#T5){ref-type="table"} and [6](#T6){ref-type="table"}) or through shared promoter motifs (Table [7](#T7){ref-type="table"}) provide new regions of genetic interest in the study of iron deficiency chlorosis in soybean. Additionally, both types of clustering suggest the control of soybeans\' iron deficiency response is regulated by the differential expression of a transcription factor or a mutation within the transcription factor, which affects its ability to bind to target promoter regions. This implies the eight transcription factors differentially expressed in Clark under iron deficiency stress which are located within known iron QTL regions are likely candidate genes for the QTL. An analysis of the 780 transcription factors predicted within the IDC QTL regions, specifically the FIT homolog, additional 52 bHLH transcription factors, and the other 50 genes in Clark and 21 genes in IsoClark that map within the QTL regions may further elucidate the response induced in soybean due to iron deficiency stress. Additionally, the conserved motifs identified by MEME in the promoter regions of iron deficiency induced genes can be used to mine the soybean genome for additional genes potentially affected by IDC, but which are not represented on the soybean Affymetrix^®^GeneChip^®^.
Materials and methods
=====================
Plant Growth and RNA Extractions
--------------------------------
NILs developed for their characteristic response to limited iron conditions, were developed by the USDA-ARS \[[@B43]\]. The iron efficient PI548533 \[[@B18]\] was crossed with iron inefficient T203 (PI54619). Five repeated backcrosses to Clark yielded the iron inefficient line PI547430 (IsoClark). Both the iron efficient Clark and the iron inefficient IsoClark were germinated in sterile vermiculite and transferred to a DTPA buffered nutrient hydroponics system 7 days after planting. Each 10L hydroponic unit contained 2 mM MgSO~4~\*7H~2~O, 3 mM Mg(NO~3~)~2~\*6H~2~O, 2.5 mM KNO~3~, 1 mM CaCl~2~\*2H~2~O, 4.0 mM Ca(NO~3~)~2~\*4H~2~O, 0.020 mM KH~2~PO~4~, 542.5 μM KOH, 217 μM DTPA, 1.52 μM MnCl~2~\*4H~2~O, 4.6 μM ZnSO~4~\*7H~2~O, 2 μM CuSO~4~\*5H~2~O, 0.20 μM NaMoO~4~\*2H~2~0, 1 μM CoSO~4~\*7H~2~O, 1 μM NiSO~4~\*6H~2~O, 10 μM H~3~BO~3~, and 20 mM HCO~3~. A pH of 7.8 was maintained by the aeration of a 3% CO~2~: air mixture. A supplemental nutrient solution containing 16 mM potassium phosphate, 0.287 mM boric acid and 355 mM ammonium nitrate was added daily to maintain proper plant nutrition. Both iron efficient and iron inefficient plants were grown in iron sufficient (100 uM Fe(NO~3~)~3~) and iron limiting (50 uM Fe(NO~3~)~3~) hydroponic conditions. Leaf tissue from the 2^nd^trifoliate was collected 21 days after planting, or after 14 days in the hydroponics system. Tissue was flash frozen in liquid nitrogen and stored at -80°C until RNA could be extracted. Three independent biological replicates were used as the experimental tissue. RNA extractions were performed using the Qiagen RNeasy Plant Mini Kit (catalog \# 74904). RNA samples were submitted to the Iowa State University GeneChip^®^facility to be hybridized and scanned using the Soybean Affymetrix^®^GeneChip^®^. Chip data has been uploaded to Gene Expression Omnibus as accession number GSE10730. Hybridization data was visualized using Bioconductor to ensure all hybridizations had normal distributions. The data was then loaded into the Gene Traffic Microarray Analysis program where it was normalized using the invariant set command, using the Clark 100 uM Fe as the control group, and a model based expression index (MBEI) \[[@B44]\] analysis was performed on perfect match probes only. Hybridization statistics were used to determine a two-fold change in expression, consistent across all replicates, reflected a statistically significant (p values and standard errors generated by analysis not shown) difference in expression between genotype and iron concentrations. An analysis of Clark plants grown in iron sufficient and iron deficient conditions showed 835 transcripts differentially expressed at two-fold or greater (Supplemental Table 1). IsoClark plants grown in identical conditions showed 200 transcripts that met the criteria for differential expression (Supplemental Table 2).
Candidate Gene Annotation
-------------------------
The candidate genes were queried against the SoyBase Affymetrix^®^GeneChip^®^Soybean Genome Array Annotation page, publicly available at <http://www.soybase.org/AffyChip/>. Here, researchers with the USDA-ARS have used BLASTX and TBLASTX \[[@B19]\] to compare the sequences from which all Affymetrix probes were derived to the UniProt database and the Arabidopsis genome gene calls (TAIR7, <http://www.arabidopsis.org/>). The top three UniProt BLAST hits and the Arabidopsis best hit GO annotation is reported for each Affymetrix probe set. To assign a putative function and classification to the differentially expressed genes (Table [1](#T1){ref-type="table"}, Additional files [1](#S1){ref-type="supplementary-material"}, [2](#S2){ref-type="supplementary-material"}, and [3](#S3){ref-type="supplementary-material"}) the three UniProt annotations were compared. If all three were identical that annotation was assigned to the gene. If the top three BLAST hits were not in concordance, that sequence was re-examined to determine if one of the annotations was more likely correct than the others. If no annotation could be confidently identified by BLAST analysis with UniProt, the differentially expressed gene was annotated as an unknown. If the gene sequence for the Affymetrix^®^probe showed no sequence homology to any of the proteins in the UniProt database, the sequence was annotated as No UniProt Hit.
GO Slim Term Analysis
---------------------
For expressed genes with homology greater than 10e^-6^to an Arabidopsis gene, custom perl scripts were written to parse and tally each transcript GO slim ID for biological process, molecular function, and cellular process. The same scripts were used to tally GO slim IDs for the entire chip. Differences between the expressed genes and the entire chip were compared using a Fisher exact test \[[@B45]\]. This test was performed to identify the GO slim terms within each of the three GO slim classifications that were over-or under-represented in the lists of differentially expressed genes in relation to their presence on the soybean Affymetrix^®^chip. A Bonferroni correction \[[@B46]\], using the number of identifiers present on the Affymetrix^®^chip, was applied to the two-tailed probability value (p-value) of each GO slim identifier. GO slim identifications with a p-value of less than or equal to 0.05 after the Bonferroni correction were considered statistically over-or under-represented in our list of differentially expressed genes (Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). This correction is likely to underestimate the number of categories of genes either over-or under-represented on the lists of differentially expressed genes in comparison to their prevalence on the Affymetrix^®^chip.
Real Time PCR Confirmation
--------------------------
The differential expression observed in the microarray experiment to identify candidate genes was confirmed using semi quantitative Real Time Reverse Transcriptase PCR (sqRT-PCR). Eleven transcripts identified as differentially expressed in the microarray experiment were tested using sqRT-PCR (Table [4](#T4){ref-type="table"} and Additional file [7](#S7){ref-type="supplementary-material"}). Genes for sqRT-PCR confirmation were chosen based on differential expression levels in the microarray. We tested genes showing both extreme differential expression and those just exceeding the two-fold criteria. Primers were designed from the EST sequence used to construct the Affymetrix probe to produce 250 bp amplicons. The sqRT-PCR was conducted as described by the Stratagene protocol (Catalog \#600532) using the Stratagene Brilliant qRT-PCR kit with 25 uL reactions. For each experimental reaction, 200 ng of total RNA was added as initial template along with 125 mM MgCl~2~and 100 nM forward and reverse primers. Cycling parameters were as follows: 45 min at 42°C for reverse transcription, 10 min at 95°C to denature reverse transcriptase StrataScript, 40 cycles of 30 sec at 95°C, 1 min at proper annealing temperature, 30 sec at 72°C. All sqRT-PCR reactions were performed in the Stratagene Mx3000P followed by a dissociation curve, taking a fluorescence reading at every degree between 55°C and 95°C to ensure only one PCR product was amplified. As controls, a passive reference dye was added to each reaction to ensure the increase in fluorescence was due to an increase in amplicon and not an artifact of the PCR. Additionally, each sample was run in triplicate and normalized against tubulin amplification to ensure differential expression was not due to differing amounts of initial template RNA added to each sample.
To be considered differentially expressed, samples had to differ in cycle thresholds (Ct) by more than 1 cycle, which corresponds to the two-fold difference in gene transcripts between the NILs identified by the microarray experiment. The resulting fold change of the sqRT-PCR was calculated from the differences in Ct using the 2^Δ^Ct method \[[@B47]\].
SFP Identification and Association with known IDC QTL on Soybean Genome
-----------------------------------------------------------------------
Single Feature Polymorphisms (SFPs) were identified following the protocol outlined by West et al. 2006 \[[@B48]\]. In brief, the microarray data from plants grown under iron sufficient conditions was transformed by robust multichip analysis (RMA) \[[@B49]\]. Custom perl scripts were used to examine each of the ten individual probes comprising a single perfect match probe. These perl scripts assigned each perfect mach probe set an SFPdev score by subtracting the average hybridization signal from the other ten probes from the hybridization signal of the probe in question and dividing that by the hybridization signal of the probe being examined ((hyb signal probe 1 - (hyb signal probe 1+ hyb signal probe 2 + hyb signal probe 3 + hyb signal probe 4 \...hyb signal probe 10)/10)/hyb signal probe 1). SFPdev scores with an absolute value greater than or equal to two on all replicates indicated an SFP (Additional file [6](#S6){ref-type="supplementary-material"}).
Statistical Modeling and Cluster Analysis
-----------------------------------------
To determine if gene distribution along the assembled genome could be explained by random chance, a simulation program originally reported by Grant \[[@B50]\]was applied to a theoretical genome. A genome of 996,903,313 bp (the combined size of the 7x genome assembly which has been assigned to soybean molecular linkage groups) was partitioned into 1,000,000 bp, 100,000 bp, and 10,000 bp windows resulting in 953 bins, 9,530 bins and 95,300 bins respectively. The program positioned 760 or 200 genes depending on the genotype (see below) being simulated on the genome and determined the number of genes within the window. The simulation was repeated 1,000 times. The mean number of bins with 0 -- 8 genes was calculated for the 1,000 repetitions. A standard deviation for each gene bin size was also calculated. To determine how this compared with our experimental data, the sequences assigned to chromosomes were concatenated together and the sliding window analysis was performed to identify clusters. Chromosomes are designated as shown in <http://www.soybase.org>. The difference between the microarray data and the simulated data is calculated in terms of the number of simulated data standard deviations \[[@B43]\]. A difference greater than two SD is considered statistically significant. The sign of the difference is indicative of whether there are more or fewer genes than expected.
Motif Identification and Analysis
---------------------------------
The consensus sequence used by Affymetrix^®^to generate the probes on the Soybean GeneChip^®^identified as differentially expressed between Clark plants grown under iron sufficient and iron deficient conditions were queried against the 7X genome gene calls. The top hit for each differentially gene was used as the gene call for the differentially expressed sequence on the Affymetrix^®^GeneChip^®^. The 835 differentially expressed sequences in the Clark genotype correlated with 760 of the predicted genes in the 7X genome release <http://www.phytozome.net> while the 200 predicted genes from the IsoClark genotype correlated with 200 predicted genes from the 7X genome. Custom perl scripts identified the 500 bases upstream of the start codon for each gene from the 7X genome assembly. The reverse complement of each of the 500 bp promoter regions was also identified. The program MEME (Multiple Em for Motif Elicitation \[[@B51]\]) was run against the 500 base promoter regions of all IDC genes to identify short conserved sequences in the promoter regions of the differentially expressed genes using the -dna -mod anr -evt 1 commands. Identified motifs with E-values \< 1E^-6^were then compared against a modified TRANSFAC database using BLASTN \[[@B19]\] to determine if identified motifs contained any known transcription factor binding sites (Table [7](#T7){ref-type="table"}).
Authors\' contributions
=======================
JAO carried out the sample collection and preparation, RT PCR validation, data analysis, and drafted the manuscript. RTN wrote custom Perl scripts and performed various bioinformatic analyses. SC, JS, and JG performed the genome sequence alignments and assembly. DG performed the genome cluster analysis. CPV. provided comments, suggestions, and revisions to the manuscript. MAG performed various bioinformatic analyses, did the SFP identification, GO category analysis, wrote custom Perl scripts for various analyses, and provided editorial assistance. RCS conceived the study, coordinated the design of the project, and drafted the manuscript. This research was funded in part by the North Central Soybean Research Program.
Supplementary Material
======================
###### Additional file 1
**Differentially expressed genes in the Clark genotype comparing plants grown in iron sufficient and iron deficient conditions**. A table of differentially expressed genes in the Clark genotype comparing plants grown in iron sufficient and iron deficient conditions including the identified fold changes and gene annotations.
######
Click here for file
###### Additional file 2
**Differentially expressed transcripts in the IsoClark genotype between plants grown in iron sufficient and iron deficient conditions**. A table of differentially expressed genes in the IsoClark genotype comparing plants grown in iron sufficient and iron deficient conditions including the identified fold changes and gene annotations.
######
Click here for file
###### Additional file 3
**Differentially Expressed Genes between Clark and IsoClark genotypes grown under Iron Deficient Conditions**. A table of differentially expressed genes between Clark and IsoClark genotypes grown under iron deficient conditions including the identified fold changes and gene annotations.
######
Click here for file
###### Additional file 4
**Differentially Expressed Genes in Clusters identified in the Clark genotype with a sliding window of 1,000,000 bases**. A table of differentially expressed genes in the Clark genotype illustrating the identified gene clusters using a sliding window of 1,000,000 bases, their chromosomal location, and gene annotation.
######
Click here for file
###### Additional file 5
**Differentially Expressed Genes in Clusters identified in the IsoClark genotype with a sliding window of 1,000,000 bases**. A table of differentially expressed genes in the IsoClark genotype illustrating the identified gene clusters using a sliding window of 1,000,000 bases, their chromosomal location, and gene annotation.
######
Click here for file
###### Additional file 6
**Identified and annotated SFPs between two NILs Soybean Genome Chip consensus sequences**. A table identifying the Affymetrix probes containing a SFP between the Clark and IsoClark genotypes. The data also identifies the chromosome containing the identified SFP and the annotation of the gene containing the SFP.
######
Click here for file
###### Additional file 7
**Primer sequences used for semi quantitative real time RT-PCR**. A table listing the Affymetrix probe IDs and the associated forward and reverse sequences of the primers used in the semi quantitative real time RT-PCR.
######
Click here for file
| {
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Correction to: *Nature Communications*; 10.1038/s41467-018-05461-5, published online 06 August 2018
The original version of this Article contained errors in the second sentence in the legend of Fig. 1, which incorrectly read 'These two elastic insulators are identical in lattice constant *a* (3*a*~0~), plate thickness (0.4*a*~0~), and radius of perforated holes *r* (0.18*a*~0~) but different hole-center distance characterized by *b*.' The correct version states 'plate thickness ($\documentclass[12pt]{minimal}
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\begin{document}$$\sqrt 3$$\end{document}$ × 0.4*a*~0~)' in place of 'plate thickness (0.4*a*~0~)' and 'radius of perforated holes *r* ($\documentclass[12pt]{minimal}
\usepackage{amsmath}
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\begin{document}$$\sqrt 3$$\end{document}$ × 0.18*a*~0~)' rather than 'radius of perforated holes *r* (0.18*a*~0~)'.
The first sentence of the 'Sample preparation' section of the Methods originally incorrectly read 'Our samples are prepared exclusively on polished stainless-steel plates (Type 201, mass density 7803 kg m^−3^) with a fixed plate thickness 7.82 mm.' In the corrected version, 'mass density 7903 kg m^−3^' replaces 'mass density 7803 kg m^−3^'.
The second sentence in the legend of Supplementary Fig. 3, originally incorrectly read 'The symmetry of the phononic crystal remains unchanged as C~6*ν*~, and thickness of the substrates *H* (equals to 0.4*a*~0~), lattice constant a (equals to 3*a*~0~) and radius of perforated holes *r* (equals to 0.18 *a*~0~) maintain constant.' The correct version states '√3 × 0.4*a*~0~' in place of '0.4*a*~0~' and '√3 × 0.18*a*~0~' rather than '0.18*a*~0~'.
This has been corrected in both the PDF and HTML versions of the Article.
| {
"pile_set_name": "PubMed Central"
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INTRODUCTION
============
The Xizang (Tibet) autonomous region, located in the southwestern area of China, is the main part of the Tibetan Plateau known as "the roof of the world", averaging 4,000 m in elevation. The region is of key importance to the ecological security of China and southeastern Asia, and contains some of the most sensitive and fragile ecosystems. Grasslands are the principal ecosystems of the region and the utilization of the forage they produce for animal production is the leading industry. The area of natural grassland in Tibet is 820×l0^6^ ha, accounting for 20.5% of the total grassland area of China, the largest area of grassland in China. In recent decades, excessive utilization and destruction by overgrazing has led to serious degeneration of the grasslands in Tibet. It has been reported that 43 million ha \[[@b1-ajas-18-0966]\], about 26% to 46% of the total grassland area in Tibet, has already been degraded significantly. This situation has detrimentally affected the region's environment and ecology and its sustainable use for pastoral livestock production.
Sheep are an important livestock species, providing daily necessities, financial income and livelihood for nearly 9.8 million farmers over the entire area of the Xizang-Qinghai Plateau \[[@b1-ajas-18-0966]\]. Livestock productivity is low \[[@b2-ajas-18-0966]\], owing to the serious degeneration of natural grasslands and long-established traditional rearing systems in which farmers focus on animal survival for much of the year with short periods of livestock growth over summer. To improve the ecological environment and livestock production, the "Retire Livestock and Restore Grassland Policy", introduced by the government, is being implemented, and traditional grazing management is being transitioned to a semi-grazing and semi-housing system, using animal shelters for much of the year. The major problem for pastoral livestock production on the Tibetan grasslands is managing yearlong production systems in a grassland with a short (3- to 4-month) growing season \[[@b3-ajas-18-0966]\] over late spring and summer, when more than 70% of the annual precipitation falls, followed by dry and cold periods from autumn through to early spring. Animals are traditionally grazed all year, but grasslands can only maintain or increase their live weights in summer. In winter, farmers in these regions traditionally take livestock out to graze daily on poor quality pasture. The animals, especially pregnant ewes, are exposed to cold and wind stress that causes body heat to be quickly lost. Feeding indoors in winter is an effective way to alleviate grazing pressure on native grassland and to protect the ecosystem for sustainable utilization of grassland resources. In addition, housing and indoor feeding allows the animals to maintain their body temperature during daily grazing and reduces the extra energy requirement of these animals \[[@b4-ajas-18-0966]\]. This, consequently, reduces weight loss and increases financial incomes during winter and spring \[[@b5-ajas-18-0966],[@b6-ajas-18-0966]\]. Previous studies have focused on animal liveweight changes; however, little is known about physiological status and blood metabolites when animal management is changed from traditional grazing systems to semi-grazing and semi-housing systems.
The aim of the present study was to assess the effects of traditional daily grazing (G) and dry lot feeding (D, no grazing) on the growth performance, blood physiological and biochemical profiles, as well as antioxidant capacity and metabolites in the serum of Pengbo semi-wool sheep ewes during winter--spring in a Tibetan alpine pastoral area.
MATERIALS AND METHODS
=====================
Study site
----------
The experiment was conducted at a sheep raising cooperative in Linzhou County (91°16 E, 29°53 N; altitude 3,926 m above sea level), Tibet, China, from December 2016 to April 2017. Linzhou County is typical of the "One River and Two Streams region" in Tibet, belonging to a plateau monsoon and semi-arid climate regime, with mean annual temperature and precipitation of approximately 2.4°C to 5.8°C and 440 mm. The main rainfall occurs in summer (between June and August), and daily air temperatures are often below 0°C in winter; in the coldest period (December to January) it can be −15°C at night. The daily range of the surface ground temperature is large, and alternating negative and positive temperatures are frequent, occurring on about 150 to 230 days every year \[[@b7-ajas-18-0966]\]. It is one of the places with the highest number of strong-wind days in China. Generally, the annual average wind speed is 3.0 to 4.0 m/s. The duration for which the wind speed is greater than 17.2 m/s is 30 to 129 days, and 40% to 50% of this occurs in spring \[[@b7-ajas-18-0966]\], which accelerates the lowering of the temperature. The frost-free period is 120 days, from May through September. Grassland below 4,200 m above sea level is dominated by *Pennisetum centrasiaticum* Tzvel. The aboveground biomass of the desirable species is 225 to 300 kg dry matter (DM)/ha in summer and it is here that the yield is very low in winter--spring. The Pengbo semi-wool sheep is the major source of income for local farmers. These sheep typically produce 2.5 to 3.3 kg medium fine wool, and have a mature liveweight of 23 to 45 kg.
Animals and management
----------------------
All procedures performed in studies involving animals were in accordance with the ethical standards of the Welfare and Ethics Committee of the Chinese Association for Laboratory Animal Sciences (SAC/TC 281). Sixty Pengbo semi-wool ewes, approximately 1.5 years of age with an average body weight of 31.33±0.43 kg, were randomly assigned to two treatment groups (n = 30 sheep in each), either the grazing (G) or the dry lot feeding (D) group. In summer and early autumn, all ewes were grazed on available grassland during the day and returned to separate individual sheds in the evening. During winter and spring, the ewes in the G group were grazed continuously each day and housed in one shed each evening over the experimental period. The ewes in the D group were housed in another shed each day and allowed access to a walled yard through the middle of the day. The structure and condition of the individual sheds in each group was the same. The sheds were adjacent and their floor areas were 40 m^2^ with a 60-m^2^ walled yard outside each shed, potential wind speed differences between the walled yard and the sheep grazing areas. Three troughs made of iron were placed in each walled yard allowed the ewes free access to supplements. The sheds had the inclined front sunlight-roof and gaps were filled with blankets to minimize airflow. A pair of windows on the front wall could be opened when appropriate to improve airflow. The average night temperatures inside the sheds of groups G and D were 2.03°C and 2.42°C, respectively.
All animals in each group were fed the same amount of commercial concentrate and *Secale cereale* grass hay. The commercial concentrate ration of 400 g/d per ewe was fed twice daily, at 9:00 and 19:00. Grass hay, salt licks, and water were available *ad libitum* to all ewes throughout the experimental period. The nutritive value of the commercial concentrate and grass hay is given in [Table 1](#t1-ajas-18-0966){ref-type="table"}. All ewes were weighed once a month, prior to feeding or grazing in the morning.
Air temperature recording
-------------------------
Temperatures in the shed were recorded at 30-min intervals with a recorder (JQA-1100DB, Qingdao Jiaqi Electron Equipment Co., Ltd., Qingdao, China) which was located in the middle of each roof beam. Temperatures in the field in which animals in the G group were grazed daily was recorded hourly using a HOBO weather station logger (H21, Onset Computer Corp., Bourne, MA, USA).
Blood sample preparation
------------------------
Blood samples were collected on the last day of the experiment, prior to the morning feed. The samples were taken from the jugular vein of six ewes in each group. For each ewe, 5 mL of blood was collected for evaluation of physiological and biochemical parameters. In addition, the serum was separated from 10 mL blood centrifuged at 3,000 r/min for 15 min at room temperature. The samples were collected and aliquoted into 2-mL plastic vials, which were stored at −80°C for subsequent determination of antioxidant capacity and metabolites.
Chemical analyses
-----------------
The physiological profile of the blood samples, including white blood cells (WBC), red blood cells, hemoglobin, and platelets, was determined with an automated hematology analyzer (Celltac E MEK-7222K, Nihon Kohden, Tokyo, Japan); the biochemical profiles, including total protein, albumin, globulin, glucose, urea, cholesterol, triglyceride, high density lipoprotein-cholesterol (HDL-C), and low density lipoprotein cholesterol were determined using an automatic biochemistry analyzer (Hitachi 7020, Hitachi High Technologies, Inc., Ibaraki, Japan). Serum antioxidant capacity, including total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) activity, was tested using a commercial biochemical reagent kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.
Metabolomics analysis
---------------------
### Non-targeted metabolomics analysis
All serum samples were acquired by the liquid chromatography mass spectrometry (LC-MS) system following the machine instructions. First, all chromatographic separations were performed using an ultra performance liquid chromatography (UPLC) system (Waters, Elstree, UK). An ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm, Waters, UK) was used for the reversed phase separation. The column oven was maintained at 50°C. The flow rate was 0.4 mL/min and the mobile phase consisted of solvent A (water+0.1% formic acid) and solvent B (acetonitrile+0.1% formic acid). Gradient elution conditions were set as follows: 0 to 2 min, 100% phase A; 2 to 11 min, 0 to 100% B; 11 to 13 min, 100% B; 13 to 15 min, 0 to 100% A. The injection volume for each sample was 10 μL.
A high-resolution tandem mass spectrometer, Xevo G2 XS QTOF (Waters, UK), was used to detect metabolites eluted from the column. The QTOF was operated in both positive and negative ion modes. For positive ion mode, the capillary and sampling cone voltages were set at 3 kV and 40 V, respectively. For negative ion mode, the capillary and sampling cone voltages were set at 1 kV and 40 V, respectively. The mass spectrometry data were acquired in Centroid MSE mode. The time-of-flight mass range was from 50 to 1,200 Da and the scan time was 0.2 s. For the MS/MS detection, all precursors were fragmented using 20 to 40 eV, and the scan time was 0.2 s. During the acquisition, the LE signal was acquired every 3 s to calibrate the mass accuracy. Furthermore, in order to evaluate the stability of the LC-MS during the whole acquisition, a quality control sample (pool of all samples) was acquired after every 10 samples.
### Metabolic profiling and metabolite analysis
The raw data acquired were analyzed using Progenesis QI v2.2 software (Nonlinear Dynamics, Newcastle, UK) and metaX as an R package for peak detection and alignment. The intensity of each ion was normalized with regard to the total ions, using the count to generate a data matrix that consisted of the retention time, m/z value, and the normalized peak area. The retention time and m/z data for each peak were determined by the aforementioned software. Further analyses of the data matrix were performed using SIMCAP+software 12.0 (Umetrix AB, Umea, Sweden) for the principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA).
Identified metabolites were further functionally and metabolically characterized using the Kyoto encyclopedia of genes and genomes (KEGG) and the human metabolome database (HMDB) database. Furthermore, the construction, interaction and pathway analysis of potential biomarkers was performed with software based on database sources, including the KEGG and HMDB, to identify the metabolic pathways.
Statistical analysis
--------------------
The data on the monthly changes in the temperature in the field and each treatment during December to April were analyzed using repeated measures in the MIXED procedure of SAS, with a model that included fixed and interactive effects of treatment and month. When the factors were significant (p\<0.05), means were compared using Tukey's test. The body weight, as well as physiological and biochemical parameters and antioxidant capacity in the blood of the ewes, were examined using a two-sample t-test for means with a model that included treatment effects and experimental error, and the results were expressed as mean±standard error. Differences were considered significant when p\<0.05.
For serum metabolites, PCA, an unsupervised method, was used to find the direction of maximum variance in a complex collection of data. PLS-DA, a supervised method, was used to perform classification and feature selection. Furthermore, a variable important for the projection score was used to rank the metabolites on the basis of their importance in discriminating treatment groups. To assess the statistical significance of class discrimination in the PLS-DA model, a permutation test was performed. In addition, a one-way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) test was performed on the metabolomics data, in order to assess which metabolites were mainly involved in each of the various groups. The threshold of significance was set at p\<0.05.
RESULTS
=======
Differences in ambient temperatures in the field and sheepfold
--------------------------------------------------------------
There were significant differences in air temperature between the field and sheep sheds ([Figure 1](#f1-ajas-18-0966){ref-type="fig"}). The average temperature value from December to April was higher (p\<0.001) inside the two sheds (4.92°C and 6.0°C) than that in the outside field (1.89°C), but no significant difference was observed between the two sheds.
Differences in the weight gain of ewes
--------------------------------------
The live weight and weight gain of ewes were significantly affected by treatment ([Table 2](#t2-ajas-18-0966){ref-type="table"}). The final weight was 28.8 kg in the G group, compared with 32.4 kg in the D group (p = 0.020). Consequently, the weight gain of the G group (−22.8 g/d) was significantly lower (p = 0.011) than that of the D group (5.59 g/d).
Differences in the blood physiological and biochemical parameters of ewes
-------------------------------------------------------------------------
Most of the physiological parameters did not significantly differ according to the treatment, but the number of WBCs and platelets were higher in the D group than in the G group (p = 0.037, p = 0.023; [Table 3](#t3-ajas-18-0966){ref-type="table"}), whereas the ewes in the G group had a higher platelet-large cell ratio than the D group ewes (p = 0.028). The values of cholesterol and HDL-C were lower in the D group than in the G group (p = 0.039; p = 0.008), and there were no significant differences in other biochemical parameters between the groups.
Differences in the serum antioxidant capacity of the ewes
---------------------------------------------------------
The antioxidant capacity was measured in serum samples from the ewes given different treatments. The ewes in the G group had higher GSH-Px activity compared with the ewes in the D group (p = 0.041; [Table 4](#t4-ajas-18-0966){ref-type="table"}), but there was no significant difference in T-AOC, SOD, and MDA values between the groups.
Differences in serum metabolites of the ewes
--------------------------------------------
The PCA score scatter plot is shown in [Figure 2](#f2-ajas-18-0966){ref-type="fig"}. The PCA 3D score plot showed that the metabolic profiles in the G and D groups did not have a clear separation ([Figure 2A, 2B](#f2-ajas-18-0966){ref-type="fig"}). The supervised analysis, PLS-DA, was subsequently performed to maximize the separation and identify those metabolites contributing to the observed separation. In the PLS-DA score plot ([Figure 2C, 2D](#f2-ajas-18-0966){ref-type="fig"}), the separation between the G and D groups was more prominent. In order to confirm the specificity and the significance of important metabolites identified from PCA and PLS-DA, we performed univariate analysis using one-way ANOVA and Tukey's HSD test on each metabolite. Thirteen metabolites were significantly different between the groups (p≤0.05). In addition, a total of three potential metabolites, abscisic acid (ABA), xanthoxin and 3,4-dihydroxy-5-polyprenylbenzoate (DHHB), were selected for further investigation. Interestingly, all these compounds were upregulated (p\<0.05) in the G group and acted to disturb metabolic pathways ([Figure 3](#f3-ajas-18-0966){ref-type="fig"}, [Table 5](#t5-ajas-18-0966){ref-type="table"}).
DISCUSSION
==========
Effects of indoor housing on ewe liveweight
-------------------------------------------
Low winter temperatures and chilling winds cause cold stress by increasing the energy requirements of livestock, leading to large decreases in productivity. For adult sheep, the lower critical temperature is −3°C \[[@b8-ajas-18-0966]\], below which daily metabolisable energy requirements increase by 0.14 to 0.64 MJ for each 1°C decrease in ambient temperature from January to December (60-kg sheep) \[[@b9-ajas-18-0966]\]. Our previous study has found that the appropriate ambient temperature for adult ewes in northwest pastoral regions of China should be above 2°C \[[@b10-ajas-18-0966]\]. In Linzhou County in Tibet, where high altitude, low winter temperatures and wind chill factors result in high energy demands, the animals will lose weight. During the entire experimental period, the average temperature inside the two sheds did not differ significantly, but average temperature outdoors in winter (December to February) was −0.61°C, well below the indoor temperature (3.65°C) and the range reported by Zhang et al \[[@b10-ajas-18-0966]\]. Such low winter temperatures led to a large weight loss (nearly 23 g/d) in the ewes that were grazed daily, which means that considerable amounts of energy from high-quality supplements would be needed to prevent any weight loss and maintain body temperature. As estimated by Young \[[@b11-ajas-18-0966]\], feed requirements for overwintering beef cows are elevated by 30% to 70% under adverse winter conditions. However, in the present trial, the ewes in the G and D groups were fed the same quantity of concentrate (400 g/d), and the ewes grazed outdoors still lost weight at 22.8 g/d although medium quality grass hay (with 71.9 g/kg DM crude protein and 706 g/kg DM neutral detergent fiber, respectively) was supplied freely during the experimental period. This is mainly attributed to the harsh wind and low temperature in winter--sping in the local area. During the coldest month, the wind speed is about 15 km/h (= 4.2 m/s) and the outdoor temperature averages −2.29°C, which resulted in remarkable weight loss (22.8 g/d) by the ewes grazed outside daily. However, housing in a mild shed without grazing can protect sheep from acute cold during winter \[[@b12-ajas-18-0966]\] and reduce their weight loss \[[@b4-ajas-18-0966]\]. As the report by Tuo et al \[[@b13-ajas-18-0966]\] found, yaks (6 to 8 years of age) fed in a warm shed had a higher live weight than yaks grazing (+283 g/d), and the daily weight gain of the warm-shed yak calves was 189 g/d greater than that of calves from grazing yaks. Similar findings were observed for the marketing rate of Tibetan sheep and yaks by Zhen et al \[[@b6-ajas-18-0966]\], who reported that the marketing rate for Tibetan sheep and yaks, respectively, increased by 3% to 9% and 2% to 3% following implementation of a crop-forage regime plus warm sheds. Additionally, the warm sheds also resulted in more animals to rear and a greater capability to avoid mortality, especially for young animals and adult females, which led to higher incomes.
In the present study, the ewes allocated to the indoor feeding maintained significantly higher live weight and increased weight gain by 125%, as compared with the ewes kept grazing outdoors (5.59 vs −22.8 g/d). This is because the indoor feeding reduced the long-term cold stress during daily grazing as well as reducing exposure to the harsh wind chill. The benefits of indoor feeding may also be related to the reduced energy requirements due to walking activity and the low quality forage available to the sheep. Therefore, local herders should focus more on housing sheep indoors, preferably in a warm shed, and feeding animals rather than grazing them outdoors during winter--spring, which would decrease the weight loss of ewes, and allow better management of degraded grasslands.
Effects of indoor housing on blood physiological and biochemical profiles in the ewes
-------------------------------------------------------------------------------------
Hematological examination reflects an animal's responses to its external and internal environments. The normal values of physiological and biochemical indexes are used for evaluating stress and the well-being of an animal. In the present study, winter grazing of ewes resulted in a decreased WBC count, in line with the report by Zakari et al \[[@b14-ajas-18-0966]\], who found that the WBCs of donkeys decreased during the cold dry season. The physiological responses of animals to environmental stress and their energy balance have profound effects on serum biochemical parameters. Soon after the beginning of stress or during mild short-term stress, stress hormones, such as glucocorticoids, induce the release of leukocytes from organs, and these enter the blood vessels and lymphatics \[[@b15-ajas-18-0966]\]. This is a consequence of increased blood leukocyte numbers. As the stress response continues, stress hormones induce leukocytes to exit the blood and redistribute in the lungs, liver and lymph nodes in preparation for immune challenges \[[@b16-ajas-18-0966]\], which results in a decrease in blood leukocyte numbers \[[@b17-ajas-18-0966]\]. Low temperature was one of the most serious threats for the winter grazing sheep used in this trial. The long-term (5 months) cold grazing induced a cold stress response, resulting in a decrease in WBCs in the grazing sheep (G group). It is important to note, however, that the WBC values were above the reference range for animals in other regions in China \[[@b18-ajas-18-0966]\]. This effect is highly likely to be attributable to the adaptation of these animals to the high altitude conditions in Tibet.
Compelling evidence suggests that platelets may act as one of the regulators of the immune response by activating the immune system during autoimmune processes \[[@b19-ajas-18-0966]\]. In this study, all platelet parameters, in both the G and the D sheep, were within the reference range \[[@b20-ajas-18-0966]\]. However, lower numbers of platelets were observed in ewes grazed outdoors, which is in line with the finding of Zhao et al \[[@b21-ajas-18-0966]\], who found that the number of platelets in Holstein cattle decreased from 279 ×10^9^ to 60.8×10^9^/L when the temperature decreased from −5°C to −20°C.
Moreover, the serum metabolomic data showed that the grazing ewes had higher ABA and xanthoxin concentrations than indoor housed ewes. Xanthoxin is a natural precursor of ABA \[[@b22-ajas-18-0966]\], which is a 15-C weak acid involved in the response to environmental stress in both plants and animals \[[@b23-ajas-18-0966]\]. ABA can be produced and released from many animal and human cells (immune cells, cardiovascular cells, and pancreatic cells) under physiological or pathological conditions, and regulates cell growth, development and immune responses to various stimuli through a signaling pathway \[[@b24-ajas-18-0966]\].
From the above findings regarding the physiological profile, it can be deduced that the grazing sheep exposed to long-term cold stress showed induction of immunoprotective responses through changing levels of WBCs and platelets, as well as serum metabolites. This long-term stressor may reduce the health of ewes.
As demonstrated by Zhang et al \[[@b4-ajas-18-0966]\], cold grazing in winter leads to high levels of heat loss from ewes and serious energy imbalance. The ewes in G group suffered severe cold stress during daily grazing, and the higher serum cholesterol and HDL-C concentrations were probably due to the thermogenesis used by the sheep to adapt to the cold environment. Cholesterol is synthesized as a response to energy metabolism in the liver. There is some evidence that cholesterol and total lipid concentrations were higher when fat-tailed sheep are reared in cold stress conditions \[[@b25-ajas-18-0966]\]. Calves exposed to cold environments had an increase in blood lipid concentrations when compared with calves exposed to warm environments \[[@b26-ajas-18-0966]\]. These findings further suggested the involvement of blood lipids in cold thermogenesis. HDL-C transports cholesterol from peripheral cells to the liver for recycling and disposal \[[@b27-ajas-18-0966]\], and evidence suggests that exercise will be effective in increasing HDL-C \[[@b28-ajas-18-0966]\]. The ewes used in this trial that grazed outdoors daily experienced long durations and long distances of walking exercise every day and this may have increased the concentration of HDL-C.
Effects of indoor housing on the serum antioxidant capacity of ewes
-------------------------------------------------------------------
Animals have evolved highly complex antioxidant systems (enzymic and nonenzymic), which work synergistically to protect the cells and organ systems of the body against free radical damage. The most efficient enzymatic antioxidant systems involve SOD and GSH-Px. As expected, indoor housing resulted in decreased GSH-Px activity in the ewes in the current study. GSH-Px contains Se, which is tightly bound, and its activity in various tissues from several species was found to be dependent on dietary Se intake \[[@b29-ajas-18-0966]\]. In this study, the grazing ewes had higher GSH-Px activity, which may be ascribed to their intake of diverse herbage compounds, compared with the single simple forage source provided to the housed ewes. The lack of differences in serum T-AOC and MDA concentrations between the groups implied that the winter housing feeding system in this trial may not have been sufficient to influence the total antioxidant ability and lipid peroxidation.
The results from the metabolomics analysis showed that the sheep fed indoors had decreased DHHB, as compared with those grazed outdoors. DHHB is an intermediate that participates in the biosynthesis pathway of ubiquinone and plays a pivotal role in inner mitochondrial membrane electron transport; it also serves as an important endogenous antioxidant, preventing lipid peroxidation in cell membranes \[[@b30-ajas-18-0966]\]. The higher serum DHHB concentration in the G group reflects the increased energy production and better cell antioxidant activity, which is consistent with the above results. It suggests that grazing sheep can supply the extra energy consumption required by long distance walking, accompanied by the chill and wind in the cold season, by thermogenesis through increasing lipid metabolism. At the same time, along with the enhancement of mitochondrial energy metabolism, free radical production also increased. Therefore, the grazing ewes showed increased ubiquinone content and GSH-Px activity as a compensatory antioxidant mechanism.
Nevertheless, the temperatures both inside and outside the sheds were below the optimum temperature range for sheep in the cold season, leading to low efficiency in the growth performance of local sheep, even when given the same supplementation. In this study, low numbers of metabolites in serum were tested, and the nutrient metabolites did not show differences. Therefore, the differences in serum metabolites in two groups were focused on the stress reaction.
CONCLUSION
==========
In Tibet, keeping livestock in sheds in winter--spring, in combination with feeding a moderate (or high) quantity of fodder, is a more efficient management strategy than daily grazing through winter that will enhance animal performance, leading to increased farmer incomes. Most importantly, winter--spring housing will reduce the cold stress on grazing animals, enhancing the blood physiological and biochemical profiles, and thereby possibly contribute to reduced management costs.
**CONFLICT OF INTEREST**
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
The work was funded by the National Natural Science Foundation of China (No. 31402119) and the Achievements Transferring Projects of Xizang Autonomous Region, China (no grant number).
{#f1-ajas-18-0966}
{#f2-ajas-18-0966}
{#f3-ajas-18-0966}
######
Chemical composition values of individual feeds on dry matter basis
Feed Chemical composition
---------------------------------------------------------- ---------------------- ------- ------ ------- -------
Concentrate[1)](#tfn2-ajas-18-0966){ref-type="table-fn"} 921.7 161.2 11.2 96.8 60.1
*Secale cereale* hay 935.4 71.9 7.25 696.1 392.0
DM, dry matter; CP, crude protein; ME, metabolisable energy; NDF, neutral detergent fiber; ADF, acid detergent fiber.
Concentrate consisted of chopped maize 65%, soybean meal 10%, cottonseed meal 8%, rapeseed meal 6%, wheat bran 10% and premix 1%.
######
Changes in liveweight of ewes grazed outside (G) and fed indoors (D)
Item Treatment p-value
---------------------- -------------------------------------------------------- -------------------------------------------------------- -------
Starting weight (kg) 31.2±0.5 31.5±0.3 0.858
Final weight (kg) 28.8±1.2[\*](#tfn3-ajas-18-0966){ref-type="table-fn"} 32.4±0.9[\*](#tfn3-ajas-18-0966){ref-type="table-fn"} 0.020
Weight gain (g/d) −22.8±7.8[\*](#tfn3-ajas-18-0966){ref-type="table-fn"} 5.59±7.36[\*](#tfn3-ajas-18-0966){ref-type="table-fn"} 0.011
Means within each row differ significantly (p\<0.05).
######
Physiological and biochemical parameter in blood of ewes grazed outside (G) and fed indoors (D)
Item Treatment p-value
---------------------------------------------- --------------------------------------------------------- --------------------------------------------------------- -------
Physiological parameters
White blood cell (×10^9^/L) 138.5±7.2[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 159.3±5.4[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 0.037
Hemoglobin (g/L) 128.2±10.9 135.5±4.3 0.441
Red blood cell (×10^12^/L) 8.35±0.72 8.40±0.33 0.882
Platelet (×10^9^/L) 192.8±72.4[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 379.0±53.2[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 0.023
Mean platelet volume (fL) 6.07±0.49 5.70±0.50 0.229
Platelet distribution width (%) 14.7±0.2 14.6±0.1 0.893
Plateletcrit (%) 0.13±0.03 0.17±0.02 0.211
Platelet-large cell ratio (%) 13.1±3.5[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 7.8±1.7[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 0.028
Biochemical parameters
Total protein (g/L) 72.6±5.7 70.4±4.4 0.472
Albumin (g/L) 25.3±1.2 24.4±1.4 0.187
Globulin (g/L) 49.2±4.1 48.0±5.5 0.693
Glucose (mmol/L) 3.25±0.37 3.02±0.52 0.418
Urea (mmol/L) 8.74±1.13 7.52±1.23 0.105
Cholesterol (mmol/L) 1.66±0.26[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 1.36±0.18[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 0.039
Triglyceride (mmol/L) 0.31±0.13 0.29±0.16 0.731
High-density lipoprotein cholesterol (g/mL) 1.40±0.05[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 1.29±0.06[\*](#tfn4-ajas-18-0966){ref-type="table-fn"} 0.008
Low density lipoprotein cholesterol (g/mL) 0.40±0.10 0.34±0.08 0.381
Means within each row differ significantly (p\<0.05).
######
Serum antioxidant capacity of ewes grazed outside (G) and fed indoors (D)
Item Treatment p-value
------------------------------------------- -------------------------------------------------------- ------------------------------------------------------- -------
Total antioxidant capacity (nmol/mg prot) 3.72±0.20 3.54±0.15 0.485
Superoxide dismutase (U/mg prot) 117.9±4.2 110.2±3.7 0.202
Glutathione peroxidase (nmol/mg prot) 123.4±8.9[\*](#tfn5-ajas-18-0966){ref-type="table-fn"} 97.8±5.2[\*](#tfn5-ajas-18-0966){ref-type="table-fn"} 0.041
Malondialdehyde (nmol/mg prot) 4.26±0.30 3.85±0.20 0.138
Means within each row differ significantly (p\<0.05).
######
Identification and trends of change for differential metabolites
Metabolite Formula p-value VIP Retained time m/z Trend Metabolic pathway
------------------------------------ ------------------------------ ---------------------------------------------------- ------ --------------- ----- ------- -----------------------------------------------------
Abscisic acid C~15~H~20~O~4~ 0.004[\*](#tfn7-ajas-18-0966){ref-type="table-fn"} 6.99 4.55 247 ↑ Metabolic pathways
Xanthoxin C~15~H~22~O~3~ 0.008[\*](#tfn7-ajas-18-0966){ref-type="table-fn"} 5.96 5.37 233 ↑ Metabolic pathways
3-Polyprenyl-4,5-dihydroxybenzoate C~12~H~14~O~4~·\[C~5~H~8~\]n 0.044[\*](#tfn7-ajas-18-0966){ref-type="table-fn"} 5.11 5.37 291 ↑ Ubiquinone and other terpenoid-quinone biosynthesis
VIP, variable important for the projection.
Means within each row differ significantly (p\<0.05).
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Solar driven photoelectrochemical (PEC) water splitting is a promising route to directly store solar energy in the chemical bonds of hydrogen. Due to the limitation of available materials capable of overall PEC water splitting, a tandem cell is likely required to efficiently convert solar energy into hydrogen.^[@cit1],[@cit2]^ One promising tandem cell configuration is comprised of an n-type semiconductor as a photoanode to drive the oxygen evolution reaction that is electrically connected to a p-type photocathode to drive the hydrogen evolution reaction ([Scheme 1](#sch1){ref-type="fig"}). This type of PEC cell is advantageous as it allows researchers to independently investigate and optimize each half-cell. Although many semiconductor metal oxides have been proposed as a photoanode for solar water oxidation, the majority of their band gaps lie in the UV region which covers a negligible portion of solar spectrum.^[@cit3]--[@cit6]^ Metal oxide materials with narrower optical band gap and absorption edges that extend to the visible region, *e.g.* Fe~2~O~3~,^[@cit7],[@cit8]^ WO~3~,^[@cit9],[@cit10]^ and BiVO~4~,^[@cit11],[@cit12]^ have therefore attracted a lot of attention. The state-of-the-art electrodes using these materials have produced promising water oxidation photocurrent densities, with the best examples producing approximately 5 mA cm^--2^ at 1.23 V *vs.* RHE. For example, Wang and co-workers recently employed a solution processed hematite photoanode in combination with an amorphous Si electrode to achieve overall water splitting at an efficiency of 0.91%. No metal oxide photoanode, however, has produced a photocurrent density that would enable achieving ∼10% water splitting efficiency.
{#sch1}
Domen *et al*. have recently introduced a new class of nitride semiconductors, specifically tantalum nitride (Ta~3~N~5~), as promising alternative candidates to oxides for PEC water oxidation.^[@cit13]--[@cit19]^ In one impressive example, they demonstrated 1.5% efficient solar water splitting with Ba-doped Ta~3~N~5~ nanorods nitridized at 1000 °C for 2 h.^[@cit20]^ Tantalum nitride is intrinsically an n-type semiconductor with an optical band gap of 2.1 eV that theoretically corresponds to a maximum photocurrent density of 12.5 mA cm^--2^.^[@cit21],[@cit22]^ If it is coupled with an appropriate photocathode in a PEC tandem cell, it could perform unassisted water splitting at a solar-to-hydrogen efficiency of ∼15%.^[@cit23]^ Strikingly, Li and coworkers recently reported a Ta~3~N~5~ photoanode on Ta foil prepared by ammonolysis at 950 °C for 6 h that produced a photocurrent density of ∼12.1 mA cm^--2^ at 1.23 V *vs.* RHE with a photocurrent onset potential of ∼0.7 V *vs.* RHE.^[@cit24]^
Implementing a Ta~3~N~5~ photoanode in a tandem configuration to achieve efficient overall water splitting is hindered by the lack of a synthetic procedure to prepare Ta~3~N~5~ electrodes under conditions compatible with a transparent conductive oxide (TCO) substrate. Most of the studies on tantalum nitride (Ta~3~N~5~) share a similar synthetic route, beginning with the oxidation of Ta(0) to Ta([v]{.smallcaps}), followed by ammonolysis at elevated temperatures (\>800 °C) for long periods of time (\>6 h), as noted in the best literature examples provided above. Despite the simplicity and great performance that comes with this method, there are multiple negative consequences. The initial oxidation of tantalum metal is very energy intensive. Importantly, the subsequent ammonolysis prevents the use of a TCO substrate as noted by others.^[@cit18]^ In our lab, we found that when conventional TCOs such as FTO (F-doped SnO~2~), ITO (Sn-doped In~2~O~3~), and AZO (Al-doped ZnO) are exposed to ammonia at 600 °C or higher, they are reduced to metallic phases and become flaky with weak adhesion to the substrate. Consequently, their important properties of conductivity and transparency are lost. Therefore, Ta~3~N~5~ is commonly prepared on Ta foil which excludes the applicability of the Ta~3~N~5~ as a photoanode in a tandem configuration ([Scheme 1](#sch1){ref-type="fig"}), since the substrate is not transparent to subbandgap light. Furthermore, high temperature ammonolysis makes it difficult to control the morphology, interfaces and the inherent properties of this semiconductor.
In order to overcome these issues, we synthesized Ta-doped TiO~2~ (TTO) films *via* atomic layer deposition (ALD) which we found to be a stable TCO in reducing atmospheres. In addition, to circumvent the high temperature ammonolysis, ALD was also used to directly deposit thin films of Ta~3~N~5~ on the TTO substrates. While initial as-deposited films are primarily amorphous TaO~*x*~N~*y*~, these films can be nitridized to Ta~3~N~5~ at far more moderate nitridation conditions, *i.e.* 750 °C for 30 minutes, compared to previous reports where hours (\>6 h) of nitridation at temperatures higher than 800 °C were necessary. The photoelectrochemical properties of the Ta~3~N~5~ films deposited on TTO were investigated and the PEC water oxidation performance was analyzed. The excellent material control reported here allowed for a detailed material structure--function relationship to be determined and a path to improved performance elucidated.
Experimental
============
Film preparation
----------------
Thin films of Ta-doped TiO~2~ TCO films were prepared on quartz substrates (Advalue Technology) by alternating the deposition of TiO~2~ and TaO~*x*~ with four different ratios of TaO~*x*~ : TiO~*x*~ ALD cycles, 1 : 200, 1 : 150, 1 : 100, and 1 : 50, to modify the dopant concentration. TiO~2~ was deposited using a modified literature procedure;^[@cit25]^ briefly, titanium isopropoxide (99.9%, Aldrich) was heated to 80 °C and pulsed for 2 s. After purging for 10 s, water was pulsed for 15 ms followed by purging for another 10 s. The growth rate of TiO~2~ at 250 °C was found to be 0.2 Å per cycle. The deposition of the TaO~*x*~ sub-cycles is described below. The as-deposited TTO films were subsequently annealed under an ammonia atmosphere at 750 °C for 30 minutes with a heating rate of 35 °C min^--1^ and cooled down to the room temperature by opening up the top cover of the tube furnace.
TaO~*x*~N~*y*~ and TaO~*x*~ films were deposited on quartz, silicon (University Wafer, with ∼16 Å native SiO~2~) or the TTO coated quartz substrates described above using ALD (Savannah 200, Cambridge Nanotech Inc). All substrates were sequentially sonicated for 15 minutes in soap, DI water and isopropyl alcohol, then blown dry under a nitrogen flow and loaded into the ALD chamber. High purity nitrogen was used as a carrier gas, which was further dried and deoxygenated by in-line molecular sieves 3 Å (Sigma Aldrich) and an O~2~ scrubber (Restek), respectively. Throughout the deposition, the N~2~ flow rate was adjusted at 5 SCCM, providing a constant pressure of ∼350 mTorr. Pentakis(dimethylamine)tantalum([v]{.smallcaps}), Ta(N(CH~3~)~2~)~5~ (PDMAT), (99.9%, Aldrich) was used as the tantalum precursor. Monomethyl hydrazine, CH~3~NHNH~2~ (MMH), (99.9%, Aldrich) or DI water (Millipore, 18 MΩ m) were used as the co-reactants. The tantalum precursor, PDMAT, was kept at 90 °C and consecutively pulsed 5 times for 2 s duration with 10 s purging in between pulses. The MMH and DI water co-reactants were kept at ambient temperature. Nitridation or oxidation was performed by a 15 ms pulse of MMH or water followed by purging for 15 s to complete one ALD cycle. Films were annealed in an ammonia atmosphere at 750 °C for 30 min to complete the nitridation and crystallize the films.
Film characterization
---------------------
Film thicknesses were determined *via* spectroscopic ellipsometry (SE) using a Horiba Jobin Yvon, Smart-SE instrument. X-ray photoelectron spectroscopy (XPS) was performed with a Perkin Elmer Phi 5600 ESCA system using a monochromatic Mg Kα source to illuminate the sample at a takeoff angle of 45°. Survey scans of 0--1100 eV binding energy and detailed scans for C 1s, O 1s, N 1s and Ta 4f, Ti 2p regions were measured for all samples. The binding energies were corrected in reference to C 1s peak (284.8 eV) and Shirley background subtraction was performed for fitting for each sample. Absorbance spectra were collected on a Perkin Elmer Lambda35 UV-vis spectrometer equipped with a Labsphere integrating sphere. Raman spectra were recorded using a LabRam Armis, Horiba Jobin Yvon instrument equipped with a 532 nm laser and a ×50 microscope to focus the laser on the film surface. X-ray diffraction (XRD) patterns were obtained on a Bruker D8 Advanced diffractometer using Cu radiation with a Kα1 wavelength of 1.5418 Å. 4-Probe electrical measurements were performed using a computer controlled Pro4-440N system equipped with Keithley 2400, and Pro4 software. The film thickness was also measured by cross-section SEM (Carl Zeiss Auriga, Dual Column FIBSEM) and was taken at a tilt angle of 90°.
All electrodes were coated with the Co-Pi co-catalyst *via* photoelectrodeposition prior to carrying out further PEC measurements. The Co-Pi co-catalyst was deposited in a solution with 0.5 mM Co(NO~3~)~2~ in a 0.1 M potassium phosphate buffer at pH 7 at a constant potential of 1.06 *vs.* RHE for 180 s under AM 1.5 G simulated sunlight. A Ag/AgCl and high surface area platinum mesh were used as the reference and counter electrodes, respectively.
Photoelectrochemical measurements were made with an Eco Chemie Autolab potentiostat coupled with Nova electrochemical software. The light source was a 450 W Xe arc lamp (Horiba Jobin Yvon). An AM 1.5 solar filter was used to simulate sun light at 100 mW cm^--2^ (1 sun). All the photoelectrochemical measurements were performed by shining light on the electrodes through electrolyte. Current--voltage curves were measured using a scan rate of 10 mV s^--1^. The incident light was chopped using a computer controlled Thor Labs solenoid shutter. Electrodes were masked with a 60 μm Surlyn film (solaronix) with a 0.28 cm^2^ hole which was adhered to the electrode by heating to 120 °C. The protected electrodes were clamped to a custom made glass electrochemical cell with a quartz window. A homemade saturated Ag/AgCl electrode was used as the reference electrode and was frequently calibrated to a commercial saturated calomel electrode (Koslow Scientific). Potentials *vs.* Ag/AgCl were converted to reversible hydrogen electrode (RHE) by the equation *E* ~RHE~ = *E* ~Ag/AgCl~ + 0.197 V + (0.059 V)pH. An aqueous solution of 0.5 M K~2~HPO~4~ was used as the electrolyte. The pH of the electrolyte was adjusted to 13 by adding KOH. A high surface area platinum mesh was used as the counter electrode.
Results
=======
ALD of TTO
----------
ALD was used to deposit Ta-doped TiO~2~ (TTO) on quartz substrates. Different Ta concentrations were introduced by varying the relative number of ALD cycles of TiO~2~ and TaO~*x*~. Samples with TaO~*x*~ : TiO~2~ ALD sub-cycle ratios of 1 : 50, 1 : 100, 1 : 150 and 1 : 200 were prepared to produce a series of decreasing Ta dopant concentrations in TiO~2~. In addition, pure TiO~2~ films were prepared as control substrates. The total number of cycles were controlled to keep the final film thickness constant at 100 nm. Energy dispersive spectroscopy (EDS) was used to determine the resultant concentration of Ta in TiO~2~. Since the Si (Kα: 1.739 eV) signal from the quartz coincides with Ta (M: 1.809 keV), TTO films were also deposited on Al (Kα: 1.486 eV) substrates which have a well-separated EDS peak (Kα(Al): 1.486 eV).^[@cit26]^ The EDS spectra of these films with different concentration of Ta are shown in Fig. S1.[†](#fn1){ref-type="fn"} The Ta concentration in the 1 : 200 film was below the detection limit of the instrument, so it was not included in this plot, however, all the observed signals for the other three films are readily assigned to Ta, Ti, O, and Al (substrate). As shown in Fig. S1,[†](#fn1){ref-type="fn"} the atomic percentage of Ta was found to increase linearly with the ALD sub-cycle ratio of TaO~*x*~ : TiO~2~. The atomic percentages of Ta in the films, then were calculated from a linear fit of these data and used to assign the following percentages of Ta contained in the films: 5.0 (±0.32), 2.5 (±0.16), 1.67 (±0.11) and 1.25 (±0.08). We note that the actual concentration of Ta contained in the TTO films does not correspond simply to the pulse ratios of Ta and Ti precursors. The difference can largely be accounted for by the different growth rates: ∼0.25 Å per cycle for TiO~2~ compared to ∼0.79 Å per cycles for TaO~*x*~, *vide infra*.
The resistivity of the as-deposited Ta-doped TiO~2~ films on quartz were on the order of MΩ cm. In addition, consistent with a previous study, we observed that when the Ta-doped TiO~2~ films were annealed in air or oxygen, they became more insulating.^[@cit27]^ Prior examples of Ta-doped TiO~2~ were prepared at low oxygen pressure, *e.g.* 10^--5^ Torr, or the films were annealed in vacuum.^[@cit27]--[@cit29]^ Since our ultimate goal is to realize TCO films coated with Ta~3~N~5~, which may have to be annealed under ammonia, *vide infra*, all TCO films were annealed under a reducing ammonia atmosphere at 750 °C for 30 minutes.
XPS measurements were performed on samples deposited on quartz both before and after annealing in ammonia. The surface concentration of Ta for the as-deposited films is higher compared to the results from EDS measurements (see Fig. S2[†](#fn1){ref-type="fn"}). Since XPS is a surface sensitive technique, this higher apparent concentration of Ta may be attributed to the fact that the deposition of TaO~*x*~ was the last ALD cycle of these films. After annealing in ammonia, however, the atomic ratio of Ta/Ti determined by XPS was within error of the ratio determined by EDS on the as-deposited samples. Thus, annealing allows Ta to diffuse and be homogeneously distributed throughout the film. We therefore take the surface compositional analysis done by XPS after annealing as a good approximation of bulk composition. Details of the XPS analysis of as-deposited and annealed TTO films with different concentrations of Ta are discussed following Fig. S3 in the ESI.[†](#fn1){ref-type="fn"} The atomic percentages of oxygen and nitrogen as a function of Ta concentration after annealing in ammonia are shown in [Fig. 1a](#fig1){ref-type="fig"}. After annealing in ammonia the atomic percentage of O decreased and a new N signal emerged which indicates oxygen is substituted by nitrogen in the films. Thus, the annealing step results in TiO~2~ co-doped with Ta and N. Interestingly, at high concentration of Ta, *i.e.* ∼5%, another N signal is detectable which can be assigned to a Ta--N bond. Further, the Ta signal from the same film shows two types of Ta present in the films. Therefore, we attribute this to the formation of TaN~*x*~ as a separate phase at high Ta concentrations. This observation is supported by the XRD results of the films and the resistivity of the films discussed below.
{#fig1}
The XRD diffraction patterns of all annealed samples were unambiguously assigned to anatase TiO~2~. A detailed analysis of the XRD patterns of the N- and Ta- co-doped TiO~2~ films with different Ta concentrations is discussed in the ESI following Fig. S4.[†](#fn1){ref-type="fn"} Depending on the dopant concentration, however, the peak positions of anatase are shifted to the lower angles which indicates an increase in cell volume as expected from doping Ta into TiO~2~.^[@cit30]^
The resistivity of the TTO films as a function of the concentration of Ta is shown in [Fig. 1a](#fig1){ref-type="fig"}. The resistivity decreases sharply with introduction of Ta, reaching a minimum for the film with 1.6% Ta. This is ∼3 times smaller than the optimum Ta concentration reported in the literature.^[@cit28],[@cit31]^ The main difference between the Ta-doped TiO~2~ synthesized in this study to those reported in literature is the annealing atmosphere. As noted above, the use of ammonia as the reducing atmosphere results in TiO~2~ films co-doped with Ta and N. As depicted in [Fig. 1a](#fig1){ref-type="fig"}, the resistivity of the films has a strong correlation to the atomic concentration of oxygen and nitrogen. The film without Ta exhibits a surprisingly low resistivity which results from the formation of nitrogen-doped TiO~2~ or segregation of metallic TiN phases. The lowest resistivity occurs for the film with 1.6% Ta, which has the highest concentration of nitrogen and the lowest concentration of oxygen, *i.e.* the highest concentration of oxygen vacancies. Based on the formal charge of oxygen and nitrogen, it can be inferred that substitution of oxygen with nitrogen induces an increase in the concentration of oxygen vacancies. On the other hand, substitution of Ti^4+^ with Ta^5+^ may reduce the number of oxygen vacancies. Therefore, co-doping of N and Ta into TiO~2~ may have an opposing influence on carrier concentration and conductivity, which explains the difference between the optimal doping concentration found here compared to prior reports.^[@cit27]^
The optical transmittance of un-doped and Ta-doped TiO~2~ thin films after annealing in ammonia is shown in [Fig. 1b](#fig1){ref-type="fig"}. Note that these transmittance values were not corrected for reflectance, which accounts for ∼25% loss of incident photons (Fig. S5[†](#fn1){ref-type="fn"}). The transmittance of the TiO~2~ without Ta was below 50% in the visible region, which is in line with numerous reports of N-doped TiO~2~.^[@cit32],[@cit33]^ The substitution of oxygen with nitrogen introduces new states in the band gap which results in absorption edge tailing to the visible region. Upon Ta-doping, however, the average transmittance values in the visible region are increased with a maximum transmittance value of ∼70% for 1.6% Ta doped in TiO~2~.
ALD of Ta~3~N~5~
----------------
TaO~*x*~ films were deposited from 50--500 ALD cycles at 250 °C. The resultant film thickness increases linearly with the number of ALD cycles (Fig. S6[†](#fn1){ref-type="fn"}). The growth rate was found to be 0.79 Å per cycle, which is in good agreement with the previous report of the ALD deposition of TaO~*x*~ (0.85 Å per cycle).^[@cit34]^ The ALD of tantalum nitride using PDMAT and MMH has previously been studied, where an ALD deposition temperature window between 200 and 300 °C was found with a growth rate of ∼0.3 Å per cycle.^[@cit35]^ It has been previously reported that PDMAT suffers from thermal decomposition at temperatures above 300 °C, therefore, to avoid the decomposition of the precursor and ensure an ALD process, 280 °C was used as the maximum deposition temperature.^[@cit34],[@cit35]^ Interestingly, while we confirmed an ALD temperature window over 175 to 280 °C, we found a growth rate approximately three times larger with a small temperature dependence. Plots of thickness *vs.* number of ALD cycles are provided in Fig. S7.[†](#fn1){ref-type="fn"} From the slope of these plots, the growth rates were found to reproducibly vary from 0.86 Å per cycle at 175 °C to 1.04 Å per cycle at 280 °C, as shown in [Fig. 2](#fig2){ref-type="fig"}.^[@cit35],[@cit36]^ These growth rates were confirmed by cross section SEM measurements of the films grown with 1000 ALD cycles at 175 and 280 °C, which are shown in Fig. S8.[†](#fn1){ref-type="fn"} The images indicate a ∼28 nm difference in film thicknesses which is consistent with the different growth rates displayed in [Fig. 2](#fig2){ref-type="fig"}.
{#fig2}
In addition to the growth rate, the temperature affected the composition of the deposited films. The bulk composition of the as-deposited films was analyzed by EDS (Fig. S9[†](#fn1){ref-type="fn"}). Silicon with ∼16 Å SiO~2~ was used as the substrate. The Ta and Si signals overlap which prevents accurate determinations of these individual elements. The atomic percentages of nitrogen and oxygen were calculated based on the signal of these two elements and are shown in [Fig. 2](#fig2){ref-type="fig"}. Oxygen was detected in all films. We note that only a minimal amount of O can be attributed to the ∼16 Å SiO~2~ substrate since the film thicknesses are ∼100 nm. Thus, despite the lack of oxygen in either ALD precursors, and the use of high purity nitrogen as a carrier gas, all deposited films are actually amorphous, TaO~*x*~N~*y*~. Thus, there must be some source of oxygen which we were not able to fully eliminate despite significant efforts to control the ALD atmosphere. Further, as the deposition temperature increases from 200 to 250 °C there is a change in the relative percentage of oxygen and nitrogen; the relative amount of O compared to N decreases from ∼65% to ∼25%.
The surface composition of the as-deposited films was also analyzed by XPS. Fitted spectra are shown in [Fig. 3](#fig3){ref-type="fig"}. As the deposition temperature increases, the N 1s signal grows and it can only be fitted to a single Ta--N peak. The oxygen signal was fitted to three peaks. Two peaks with binding energies \>531 eV were assigned to carbon species, *i.e.* C--OH and C0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000O groups. The peak at 529--530.5 eV was assigned to the Ta--O group which was correlated to the Ta 4f peak. The peak positions of Ta 4f~7/2~ and Ta 4f~5/2~ strongly depend on the immediate surrounding atoms, *e.g.* ∼26.6 and 28.5 eV for Ta--O and 25.0 eV and 26.9 eV for Ta--N, respectively.^[@cit37]^ Therefore to avoid complexity arising from carbon species, the surface atomic percentages of Ta--O and Ta--N were estimated from the Ta 4f peaks ([Fig. 3b](#fig3){ref-type="fig"}). As it can be seen, at lower temperatures the film is mostly composed of Ta--O groups. On the other hand, at higher deposition temperatures, the Ta--N becomes the dominant composition. This result is in line with the EDS analysis discussed earlier. These combined results are also in agreement with the previous study by Ritala *et al.* who studied the deposition of thin films of Ta~3~N~5~ at temperatures from 200 to 500 °C *via* ALD using TaCl~5~ and NH~3~ as the reactants.^[@cit38]^ Their results showed that the composition of the films was strongly correlated to the deposition temperature and the concentration of oxygen was decreased from 25 to ∼5% as the deposition temperature increased from 200 to 500 °C.
{#fig3}
The composition and growth rates of the films deposited at 280 and 250 °C are similar; since we found 280 °C to be the edge of the ALD temperature window, all the subsequent depositions of TaO~*x*~N~*y*~ were performed at 250 °C unless otherwise mentioned. A lack of diffraction peaks in XRD and phonon modes in the Raman spectrum of as deposited films indicate that these films are in fact amorphous TaO~*x*~N~*y*~ (Fig. S10[†](#fn1){ref-type="fn"}). Therefore, to improve the crystallinity and to modify the composition, they were annealed ammonia. There are three parameters which control the results of annealing; temperature, time and flow rate of ammonia. It was found that the optimum conditions (details discussed surrounding Fig. S11--S13[†](#fn1){ref-type="fn"}) to form pure crystalline Ta~3~N~5~ films from the as-deposited films is ammonolysis at 750 °C for 30 min with an ammonia flow rate of ≥200 mL min^--1^. It is worth noting that both ALD deposited thin films of TaO~*x*~ and TaO~*x*~N~*y*~ were nitridized to Ta~3~N~5~ (Fig. S14[†](#fn1){ref-type="fn"}) at far more moderate conditions compared to previous reports.^[@cit20],[@cit39],[@cit40]^
Four TaO~*x*~N~*y*~ films of different thicknesses were deposited on quartz followed by ammonolysis at 750 °C for 2 hours. Based on the XRD patterns of the films (Fig. S15[†](#fn1){ref-type="fn"}), they can all be unambiguously matched to Ta~3~N~5~. The thicknesses of Ta~3~N~5~ films were evaluated *via* both cross section SEM and SE (Fig. S16[†](#fn1){ref-type="fn"}). As shown in Fig. S16d,[†](#fn1){ref-type="fn"} the growth rate found by both methods are in good agreement. However, the growth rate of pure Ta~3~N~5~, *i.e.* ALD deposition followed by ammonolysis, was ∼0.77 Å per cycle while the growth rate of the as-deposited films is ∼1.0 Å per cycle. This discrepancy in the growth rates is due to the fact that the as-deposited films are amorphous TaO~*x*~N~*y*~, whereas ammonolysis transforms the films to crystalline Ta~3~N~5~ which has 22% smaller molar volume per Ta atom than Ta~2~O~5~.^[@cit41],[@cit42]^
The absorbance of Ta~3~N~5~ as a function of the thickness is plotted in [Fig. 4](#fig4){ref-type="fig"} (absorptance, transmittance and reflectance are shown in Fig. S17[†](#fn1){ref-type="fn"}). The absorbance was corrected for the substrate using a previously developed model.^[@cit43]^ The absorbance scales linearly with the film thickness confirming a linear growth of tantalum nitride by ALD/ammonolysis. The absorption coefficient, *α*(*λ*) (cm^--1^), was calculated from absorbance using the average film thicknesses from SEM and SE (Fig. S18a[†](#fn1){ref-type="fn"}). Ta~3~N~5~ has two optical transitions, located at ∼2.10 eV and ∼2.50 eV. A recent study on optoelectronic properties of Ta~3~N~5~ suggests that both electronic transitions of Ta~3~N~5~ are direct.^[@cit44]^ The corresponding Tauc plot for direct transitions is shown in Fig. S18b.[†](#fn1){ref-type="fn"}
{#fig4}
To study the PEC performance, ∼75 nm (1000 cycles) of TaO~*x*~N~*y*~ was deposited on 100 nm TTO films with different Ta concentrations, followed by ammonolysis at 750 °C for 30 minutes. Attempts to increase the ammonolysis time to 2 hours resulted in transformation of the anatase-TiO~2~ to rutile-TiO~2~ in the TTO, based on the XRD patterns of the films, which resulted in an electrode with negligible photocurrent. Our initial results produce a photocurrent density of ∼0.77 mA cm^--2^ at 1.23 V *vs.* RHE with an onset photocurrent potential of ∼0.8 V *vs.* RHE. The PEC performance of these electrodes is strongly correlated to the conductivity of the TTO substrates. The photocurrent response of the electrodes at 1.23 V *vs.* RHE as a function of Ta concentration is shown in Fig. S21.[†](#fn1){ref-type="fn"} Remarkably, the observed photocurrents are in total agreement with the conductivity of TTO shown in [Fig. 1a](#fig1){ref-type="fig"}. This performance falls short of the recent report by Li and coworkers,^[@cit24]^ who reported a photocurrent density of ∼12.1 mA cm^--2^ at 1.23 V *vs.* RHE with a photocurrent onset potential of ∼0.7 V *vs.* RHE for the electrode prepared on a Ta foil which was nitridized under ammonia at 950 °C for 6 h. Van de Krol *et al.* also recently studied the formation of Ta~3~N~5~ as a function of ammonolysis conditions on Pt foil.^[@cit14]^ The maximum photocurrent density of ∼1.1 mA cm^--2^ at 1.23 V *vs.* RHE with an onset photocurrent potential of ∼0.9 V *vs.* RHE was found for the Ta~3~N~5~ film prepared at 800 °C for 10 h with the addition of IrO~2~ cocatalysts. To the best of our knowledge, however, this is the first report of PEC water oxidation of Ta~3~N~5~ on any TCO ([Fig. 5](#fig5){ref-type="fig"}).
{#fig5}
Finally, since we have not yet eliminated the ammonolysis step in the synthesis, we compared the behavior of the TaO~*x*~N~*y*~ deposited films to TaO~*x*~. 40 nm of TaO~*x*~ was deposited on the best TTO (1.6% Ta) followed by ammonolysis at 750 °C for 30 minutes. The transmittance/reflectance spectra of the corresponding films are compared to that of the TaO~*x*~N~*y*~-derived film in Fig. S22.[†](#fn1){ref-type="fn"} The TaO~*x*~-derived film is colorless with a take-off transmittance at ∼450 nm. On the other hand, the TaO~*x*~N~*y*~-derived film is orange with a take-off transmittance at ∼590 nm which corresponds to the known band gap of Ta~3~N~5~, *i.e.* 2.1 eV, discussed above. The PEC performance of these electrodes are compared in Fig. S23.[†](#fn1){ref-type="fn"} The TaO~*x*~-derived film shows negligible photocurrent superimposed on a large dark current. Therefore, it can be concluded that the TaO~*x*~-derived films require harsher nitridization conditions (higher temperature and longer durations) where the TCO is not chemically stable.
Conclusions
===========
The realization of photoactive Ta~3~N~5~ films on a TCO electrode was demonstrated for the first time. This required two breakthroughs. First, we established the ALD of TTO and found that it is structurally and chemically stable (unlike conventional TCO materials including FTO, ITO and AZO) under the reducing atmosphere employed (ammonolysis at 750 °C for 30 min), and can therefore be used as a conductive transparent layer for tantalum nitride electrodes. The TTO films are not able to withstand harsher nitridation conditions required for the conversion of TaO~*x*~ to Ta~3~N~5~, however, which is the synthetic route of all prior examples of Ta~3~N~5~ photoelectrodes. Therefore, the second necessary breakthrough consisted of the direct deposition of TaO~*x*~N~*y*~ films *via* ALD which can be crystallized and completely converted to Ta~3~N~5~ under sufficiently milder ammonolysis conditions to maintain the TCO properties. The resultant Ta~3~N~5~ films on TTO showed promising solar water oxidation performance, especially considering that the films are quite thin and not yet optimized. We found that the performance of the photoelectrodes correlated to the conductivity of the TCO. Thus, it would be beneficial to utilize state-of-the-art TCOs such as FTO. Since these are not stable under even the mildest ammonolysis procedures utilized here, it would clearly be advantageous to directly deposit crystalline Ta~3~N~5~ films under sufficiently mild conditions on a TCO, which do not require a subsequent annealing (thus ammonolysis) step. While we are actively working on such an ideal synthetic method, the results reported herein represent a significant step towards realizing a high-efficiency solar water oxidation electrode which can be employed in a tandem configuration.
Author contributions
====================
The experiments were designed by H. H., O. Z. and T. W. H. The experiments were carried out by H. H. and O. Z. The manuscript was prepared by H. H. and T. W. H. All authors have approved the final version of the manuscript.
T. W. H. thanks the National Science Foundation (CHE-1150378) for support of this research.
[^1]: †Electronic supplementary information (ESI) available. See DOI: [10.1039/c6sc02116f](10.1039/c6sc02116f) Click here for additional data file.
| {
"pile_set_name": "PubMed Central"
} |
Introduction to *Paenibacillus* {#Sec1}
===============================
Bacteria belonging to the genus *Paenibacillus* have been isolated from a variety of environments, with many of the species being relevant to humans, animals, plants, and the environment. The majority of them are found in soil, often associated with plants roots: these rhizobacteria promote plant growth and can be exploited for use in agriculture. Many species of *Paenibacillus* produce antimicrobial compounds that are useful in medicine or as pesticides, and many yield enzymes that could be utilized for bioremediation or to produce valuable chemicals. Some species are pathogens to honeybees or other invertebrates; while others are occasional opportunistic infectors of humans. In fact, many of these pertinent characteristics overlap within the same species. In accordance with their diverse characteristics, members of *Paenibacillus* have been discovered in disparate habitats, from polar regions to the tropics, and from aquatic environments to the driest of deserts (Additional file [1](#MOESM1){ref-type="media"}).
Species of *Paenibacillus* were originally included in the genus *Bacillus*, which historically was defined based on morphological characteristics in common with the type species *Bacillus subtilis*, isolated in 1872. Any bacterium was classified as *Bacillus* if it was rod-shaped, aerobic or facultatively anaerobic, and could form endospores allowing it to remain dormant in inhospitable conditions. However, these characteristics are actually very ancient and not suitable for grouping species into a single genus \[[@CR1]\]. A study in 1988 using numerical taxonomy based on 188 unit characters suggested a framework for splitting *Bacillus* into several genera \[[@CR2]\]. A more accurate representation of phylogenetic relationships among these bacteria was attained in 1991, when 16S rRNA gene sequences were determined for standard strains of 51 species then defined as *Bacillus* \[[@CR2], [@CR3]\]. Phylogenetic analyses showed that these sequences segregated into at least five distinct clusters, one of which was reassigned to the novel genus *Paenibacillus* in 1993 \[[@CR4]\] and includes the type species *Paenibacillus polymyxa* \[[@CR5]\]. The name *Paenibacillus* is derived from the Latin adverb *paene*, meaning almost; almost a *Bacillus* \[[@CR4]\].
Not long after the creation of the genus, organisms previously thought of as separate *Paenibacillus* species were re-classified as equaivalent: for example, *Paenibacillus gordonae* was determined to be a synonym of *Paenibacillus validus*, and *Paenibacillus pulvifaciens* was determined to be a subspecies and later a synonym for *Paenibacillus larvae* \[[@CR6], [@CR7]\]. Several species were also reclassified into the genus *Paenibacillus*, including *Clostridium durum*, *Bacillus alginolyticus*, *Bacillus chondroitinus*, *Bacillus curdlanolyticus*, *Bacillus glucanolyticus*, *Bacillus kobensis*, and *Bacillus thiaminolyticus* \[[@CR8], [@CR9]\]. In 1997, a proposed emendation described *Paenibacillus* as having 16S rDNA sequences with more than 89.6% similarity, being motile by means of peritrichous flagella (projecting in all directions), and being non-pigmented on nutrient agar, among other characteristics. Species of this genus can be gram positive, gram negative, or gram variable, in addition to sharing the basal characteristics ascribed to *Bacillus* \[[@CR8]\].
The number of useful or otherwise relevant *Paenibacillus* species has spawned genome sequencing of 212 strains representing 82 species, as well as 79 uncharacterized strains (Additional file [2](#MOESM2){ref-type="media"}), and a variety of patents (Table [1](#Tab1){ref-type="table"}). Genome size ranges from 3.02 Mbp (for *P. darwinianus* Br, isolated from Antarctic soil \[[@CR10]\]) to 8.82 Mbp (for *P. mucilaginosus* K02, implicated in silicate mineral weathering \[[@CR11]\]) and genes number from 3064 (*P. darwinianus* Br) to 8478 (*P. sophorae* S27, a rhizobacterium). Like *P. darwinianus*, the insect pathogens *P. larvae* and *P. popilliae* have genomes on the smaller side (4.51 and 3.83 Mbp, respectively), perhaps reflecting their niche specialization. The DNA G + C content of *Paenibacillus* ranges from 39 to 59 mol% \[[@CR12]\].Table 1Selection of active patentsPatent numberPatenting countryPatenting agencyPatent titleIssue dateUS9017692B2United StatesOhio State Innovation FoundationAntimicrobial agent, bacterial strain, biosynthesis, and methods of use28-Apr-15US8822179B2United StatesUniversity of Florida Research Foundation, Inc.Nucleic acid compositions and the encoding proteins21-Feb-12US8652819B2United StatesUniversity of Georgia Research Foundation, Inc.*Paenibacillus* spp. and methods for fermentation of lignocellulosic materials18-Feb-14US8329430B2United StatesKorea Research Institute of Bioscience and BiotechnologyPolymyxin synthetase and gene cluster thereof11-Dec-12US8084418B2United StatesDow AgroSciences LLCMethods of inhibiting insects by treatment with a complex comprising a *Photorhabdus* insecticidal protein and one or two Xenorhabdus enhancer proteins27-Dec-11US7935335B2United StatesKaken Pharmaceutical Co., Ltd.Strains belonging to the genus *Paenibacillus* and method of controlling plant disease by using these strains or culture thereof03-May-11CN104255816AChinaQingdao Jinxiu Shuiyuan Commerce And Trade Co LtdNon-polluted antibacterial peptide biopesticide07-Jan-15KR1020140115022KoreaMOS Co., LtdNovel *Paenibacillus* peoriae gsoil 1119 having an excellent antifungal effect against plant pathological fungi, and microorganism preparation for controlling containing same30-Sep-14WO2014146881A1InternationalWacker Chemie AGMicroorganism strain and method for the fermentative production Of C4 compounds from C5 sugars27-Feb-14CN201410255003ChinaStanley Fertilizer Stock Co., Ltd.Special functional biological slow-release fertilizer for chilies13-Aug-14CN201410181469ChinaShandong UniversityMixed flora microbial preparation and application thereof in sewage treatment16-Jul-14KR1020130055820KoreaEcodream Farm Service Corporation*Paenibacillus* Kribbesis T9 having clubroot control effect for cabbage04-Jul-14WO2014099525A1InternationalDanisco US Inc*Paenibacillus* curdlanolyticus amylase, and methods of use, thereof26-Jun-14CN201210538699ChinaHenan Academy Of Agricultural SciencesBio-control bacterium for preventing and treating sesame wilt disease, separation method, inoculant, and application of the inoculant18-Jun-14CN201410006084ChinaNortheastern UniversityMethod for removing heavy metal ions in water by using fermentation broth of bacterium producing flocculant04-Jun-14CN201410018001ChinaYancheng Institute Of TechnologyGene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as construction method and application thereof30-Apr-14CN201310579323ChinaTianjin Wuqing District Plant Protection StationBiological straw decomposition agent and preparation method thereof05-Mar-14KR1020120076529KoreaKorea Research Institute Of Bioscience And BiotechnologyMethod for increasing potato production using novel *Paenibacillus* Sp.22-Jan-14KR1020120064809KoreaGeon-Nong Co., Ltd.Deodorant for decreasing bad smell of septic tank, garbage dump, sewerage, and compost producing apparatus using complex microbial agent27-Dec-13KR1020120022400KoreaMoon, Byung Woo Yeongam Fig Cluster AgencyNutrient solution composition for box culturing ficus carica capable of easily adjusting electrical conductivity (ec) and ph according to the amount of the nutrient composition and using method thereof13-Sep-13CN201210212246ChinaTaicang Zhoushi Chemical Product Co., Ltd.Method for producing ethanol by adopting mixed culture organism by means of glycerol fermentation03-Oct-12KR1020100133442KoreaKonkuk University Industrial Cooperation Corp.Novel strain *Paenibacillus* xylanexedens Sk2925 With excellent activity of decomposing xylan03-Jul-12JP2009004013JapanYan; Sun ChanWastewater treatment method22-Jul-10JP2004059241JapanKubota CorpMethod for cleaning contamination caused by polychlorinated biphenyls/polycyclic aromatic hydrocarbons15-Sep-05EP1490468GermanyAladin Gesellscaft für Innovative mikrobiologische Systeme GmbHBiological pipe cleaner29-Dec-04DE19706598A1GermanyKnoell Hans Forschung EV Univ Schiller JenaMacrolide antibiotic apidiothricin27-Aug-98WO2016020371A1GermanyBASF SEAntifungal *Paenibacillus* strains, fusaricidin-type compounds, and their use11-Feb-16WO2016019480A1ChileUniversidad de Santiago de Chile*Paenibacillus* polymyxa SCHC 33 bacterial strain, and use thereof to combat phytopathogenic fungi in fruits, vegetables, or plants11-Feb-16WO2016019480A1United StatesDanisco US Inc.*Paenibacillus* and *bacillus* spp. mannanases17-Mar-16EP2871073A1GermanyRoche Diagnostics GmbhRecombinantly produced neutral protease originating from *Paenibacillus polymyxa*16-Jan-14WO2015153956A1United StatesThe Board of Regents of the Nevada System of Higher Education on Behalf of the University of Nevada, Las Vegas*Paenibacillus larvae* treatment with phage lysin for American Foulbrood Disease08-Aug-15WO2015092548A3DenmarkDupont Nutrition Biosciences Aps*Paenibacillus* strains and compositions thereof that inhibit microorganisms17-Sep-15WO2015101116A1ChinaBright Dairy & Food Co., LTDNew *Paenibacillus* sp. strain, as well as culture method and use thereof07-Jul-15US9085789B2United StatesUniversity of Georgia Research Foundation Inc.*Paenibacillus* spp. and methods for fermentation of lignocellulosic materials18-Feb-2014EP2999801A1United StatesBiowish Technologies, Inc.Microbial-based waste water treatment compositions and methods of use thereof30-Mar-2016EP2999340A1JapanHisataka GodaDrug-resistant microbe and variant microbe disinfectant containing chlorous acid aqueous solution30-Mar-2016WO2016054176A1United StatesDanisco US Inc.Compositions comprising beta-mannanase and methods of use07-Apr-2016US8956842B2KoreaKorea Research Institute of Chemical Technology*Paenibacillus* sp. HPL-3 strain producing xylanase having heat-resistance, a wide range of optimum pH and high activity, a novel xylanase separated from the strain, and a method for mass-production of the same using the transformant originated from the strain17-Feb-2015EP2929022A1United StatesDanisco US Inc.Compositions and methods of use14-Oct-2015EP2759600A4JapanHayashibara CoProduction method for powder containing crystalline, -trehalose dehydrate08-Jul-2015EP2556835A1JapanJapan Eco-Science Co. Ltd.Mixture, dissolving solution and pharmaceutical agent each comprising thermophilic microorganism13-Feb-2013WO2015114552A1South AfricaUniversity of PretoriaPlant growth promoting rhizobacterial strains and their uses06-Aug-2015WO2015123752A1CanadaGreen On Industries, Inc.Composition and method for hydrocarbon and lipid degradation and dispersal27-Aug-2015WO2015109766A1ChinaBright Dairy & Food Co., Ltd.Method for preparing fermentation broth extracts having chymosin activity and products thereof30-Jul-2015WO2015113175A1ChileUniversidad Del DesarrolloBiocide composition for controlling pests affecting European honey bees, comprising a water-soluble *Olea europaea* extract06-Aug-2015WO2015023662A1United StatesBio-Cat Microbials LlcCompositions comprising bacillus strains and methods of use to suppress the activities and growth of fungal plant pathogens19-Feb-2015US9187381B1United StatesBio AG Corp.Composition and method for formulating a biofertilizer and biopesticide17-Nov-2015US9113605B2United StatesCore Intellectual Properties Holdings, LlcMethods and compositions to aggregate algae25-Aug-2015Patents were searched at the World Intellectual Property Organization (WIPO), European Patent Office (EPO), United States Patent and Trademark Office (USPTO), and Japan Patent Office (JPO)
Currently, *Paenibacillus* is one of eight genera included in the family Paenibacillaceae. However, a phylogram of the family suggests that *Paenibacillus* is paraphyletic, with the other genera (*Aneurinibacillus*, *Brevibacillus*, *Cohnella*, *Fontibacillus*, *Oxalophagus*, *Saccharibacillus*, and *Thermobacillus*) forming subsidiary clades. The genus *Paenibacillus* is therefore expected to undergo significant taxonomic subdivision in the future \[[@CR1]\]. Conversely, the number of novel species being identified as *Paenibacillus*, and established species being reclassified as such, continues to grow, and the genus currently comprises around 200 species (Additional file [1](#MOESM1){ref-type="media"}). Despite this complexity, the present review attempts to summarize all members currently classified as *Paenibacillus* with respect to the characteristics---both positive and negative---that are most relevant to humankind.
Plant growth promotion {#Sec2}
======================
The genus *Paenibacillus* contains many species which are known to promote the growth of plants including maize \[[@CR13]\], *Populus* \[[@CR14]\], pumpkin \[[@CR15]\], rice \[[@CR16]\], switchgrass \[[@CR17]\], and many others. Like other plant growth-promoting bacteria, they accomplish this through various facets. Plant-associated species of *Paenibacillus* can directly influence plant growth by producing indole-3-acetic acid (IAA) and other auxin phytohormones, solubilizing inaccessible phosphorous into form that can be taken up by plant roots, and some species can also fix atmospheric nitrogen \[[@CR18]\]. In addition, *Paenibacillus* helps to control phytopathogens by triggering induced systemic resistance (ISR) and/or producing a variety of biocidal substances (see "[Biocontrol](#Sec7){ref-type="sec"}" and "[Antimicrobial peptides](#Sec11){ref-type="sec"}" sections).
While some plant growth promoting bacteria, including *P. macerans*, are used in commercial biofertilizers, their use is currently limited. The establishment and performance of these microorganisms in the field can be affected by numerous environmental variables, such as soil pH, salinity, moisture content, and temperature \[[@CR19]\]. Despite these limitations, continuing research may enable more widespread use of these biofertilizers. One benefit of inoculating fields with endospore-forming bacteria such as *Paenibacillus* is their capacity to survive for long periods in the soil under adverse environmental conditions \[[@CR20]\].
Nitrogen fixation {#Sec3}
-----------------
Atmospheric nitrogen (N~2~) is relatively inert, and must be fixed to a usable chemical form before being incorporated into amino acids, nucleotides, and other metabolites. As eukaryotes do not have the ability to fix their own nitrogen, its bioavailability in the soil is a major limiting factor for plant growth, and farmers routinely apply nitrogen fertilizers to ensure crop productivity. Commercial nitrogen fertilizers are produced by the Haber-Bosch process through reducing nitrogen gas, which uses fossil fuels for the energy needed, and the resulting carbon dioxide emissions and pollution contributing to global warming and adverse effects on human health. Additionally, over 50% of synthetic nitrogen fertilizer is not taken up by crops, and is instead lost to the environment where it contributes to eutrophication, greenhouse gas production, and acid rain \[[@CR21]\]. Such nitrogen pollution is considered to be the second most important driver of anthropogenically induced global change, next to the perturbation of the carbon cycle \[[@CR22]\]. However, its effects can be lessened by inoculating fields or crops with microorganisms, including some strains of *Paenibacillus*, that fix nitrogen in or around plant roots, where it is actually needed.
Nitrogen-fixing (diazotrophic) bacteria and archaea primarily use a molybdenum (Mo)-dependent nitrogenase (Nif) to catalyze the reduction of N~2~ to bioavailable NH~3~. Synthesis of this enzyme requires a minimum of three structural genes and three genes for FeMo-cofactor biosynthesis \[[@CR23]\]. However, optimum activity requires the presence of additional genes, as the specific activity of a nitrogenase expressed in *Escherichia coli* was only about 10% of that observed in the *Paenibacillus* species from which it was derived; and improvements were made with additional transgenes \[[@CR24]\]. Alternatives to Nif have active site cofactors that lack Mo, instead containing both vanadium and iron (Vnf), or iron only (Anf) \[[@CR23]\].
More than 20 *Paenibacillus* species can fix nitrogen \[[@CR25]\], with single species comprising both diazotrophic and non-diazotrophic strains \[[@CR26], [@CR27]\]. The *nif* gene cluster is highly conserved among nitrogen-fixing *Paenibacillus*, with most clusters containing 9 genes within 10.5--12 kb, and exhibiting over 80% identity \[[@CR25]\]. Most of the Nifs appear monophyletic, having likely been derived from a single horizontal gene transfer, followed by duplications and gene cluster loss in some lineages \[[@CR27], [@CR28]\]. At least two strains, *P. riograndensis* SBR5^T^ and *P. durus* DSMZ1735, have additional *nif*s from a potentially independent, more recent horizontal transfer, although these genes may not be functional \[[@CR28]\]. The alternative nitogenase Vnf is encoded in the genomes of *P. zanthoxyli* JH29 and *P. azotofixans* ATCC 35681, while Anf is encoded by *P. sophorae* S27, *P. forsythia* T98 \[[@CR25]\], and *P. riograndensis* SBR5^T^; all of which also contain the *nif* cluster. Having Vnf or Anf, in addition to Nif, may provide an selective advantage in certain circumstances, as *anf* shows higher expression under molybdenum-depleted conditions \[[@CR28]\].
The ability of organisms to fix nitrogen can be identified by growth on nitrogen-free medium, while reliable estimates of nitrogenase activity can be obtained using ^15^N~2~ fixing assays. However, acetylene reduction assays are less costly than ^15^N~2~ and are sufficient to estimate relative nitrogenase activities. These assays are based on the ability of nitrogenases to also reduce acetylene (C~2~H~2~) to ethylene (C~2~H~4~). Such assays have found a wide range of relative activities among nitrogen-fixing *Paenibacillus* species, for example, with *P. zanthoxyli* DSM 18202 producing 140 times more activity than *P. peoriae* DSM 8320 \[[@CR29]\].
Phosphate solubilization {#Sec4}
------------------------
Next to nitrogen, phosphorus is the second most important element limiting plant growth and productivity. Although it is abundant in soils, only 0.1% exists in a soluble form that can be taken up by plant roots; the remainder forms insoluble mineral complexes, or is immobilized in organic matter. Chemical phosphorus fertilizers are therefore used to supplement soluble phosphorus on most agricultural soils, but these are costly and adversely impact the environment \[[@CR19]\].
Unlike nitrogen, phosphorus fertilizer is a finite resource obtained from rock phosphate, and once high-quality deposits are used, the shift to lower grade rock will result in even higher costs. The manufacturing process emits poisonous hydrogen fluoride gas. Once applied to the field, less than 30% of the chemical fertilizer is typically used by the plant, with much of the rest incorporating into inorganic mineral complexes within the soil. The rise in insoluble phosphorus can disturb microbial diversity, eventually leading to reduced soil fertility; and trace amounts of heavy metal impurities in the fertilizer can accumulate over time. Eutrophication can also result as phosphorus-rich soil erodes into aquatic systems \[[@CR19]\].
As is the case for nitrogen, the application of chemical phosphorus fertilizer could be reduced by inoculating fields with phosphorus-solubilizing microorganisms, such as *Paenibacillus*. While bacteria may have drawbacks when compared to phosphorus solubilizing fungi, such as lower activity and a tendency to lose activity after repeated sub-culturing \[[@CR19]\], some may nonetheless become preferred bio fertilizers due to additional qualities that simultaneously benefit plant growth.
Phosphorus solubilizing microorganisms use a variety of mechanisms that make phosphorus available to plants, with the principal method being organic acid (especially gluconic acid) production. Such acids can directly dissolve mineral phosphorus through anion exchange, chelate metal ions, or lower soil pH to release phosphorus from the mineral complex. Microorganisms also release phosphorus from their substrates during enzymatic degradation, and from their own cells upon death \[[@CR19]\].
Genomic analyses suggest that most *Paenibacillus* strains can solubilize phosphorus through gluconic acid production: a study of 35 strains comprising at least 18 species found that all but two strains have genes involved in gluconic acid production, encoding glucose-1-dehydrogenase and gluconic acid dehydrogenase. The strains apparently lacking these genes were *P. beijingensis* 1--18 and *P. terrae* HPL-003; while those having the genes included strains from *P. azotofixans*, *P. curdlanolyticus*, *P. dendritiformis*, *P. elgii*, *P. forsythia*, *P. graminis*, *P. lactis*, *P. massiliensis*, *P. mucilaginosus*, *P. peoriae*, *P. polymyxa*, *P. sabinae*, *P. sonchi*, *P. sophorae*, *P. vortex*, and *P. zanthoxyli* \[[@CR27]\]. Genes for the uptake and degradation of phosphonates, which containing the highly stable C-P bond, and for a phosphate-specific transport system, were found in all analysed genomes \[[@CR27]\]. Phosphorus solubilisation has been confirmed by *P. elgii* \[[@CR30]\], *P. kribbensis* \[[@CR31]\], *P. macerans* \[[@CR32]\], *P. mucilaginosus* \[[@CR33]\], *P. polymyxa* \[[@CR32]\], *P. xylanilyticus* \[[@CR34]\], and several unclassified strains.
Iron acquisition {#Sec5}
----------------
Like phosphorus, iron is abundant in soil in mainly in non-bioavailable form. Particularly in alkaline or chalky soils, it forms largely insoluble Fe^3+^ oxy-hydroxides, which are not readily used by either microorganisms or plants. Most microorganisms therefore reduce Fe^3+^ to Fe^2+^ using ferrireductases or solubilize it with extracellular, low molecular weight Fe^3+^ chelators called siderophores \[[@CR35]\], which are released under iron limited conditions. The soluble Fe^3+^-siderophore complexes are available to plants as well as microorganisms \[[@CR36]\], being recognized by specific membrane receptors and transported into cells \[[@CR37]\].
Siderophores are synthesized mainly by nonribosomal peptide synthetases (NRPSs), which are encoded by gene clusters. These multienzyme complexes consist of various modules that each incorporate one or more specific amino acids into a peptide backbone (see also "[Nonribosomal lipopeptides](#Sec13){ref-type="sec"}" section). The three types of siderophores are classified based on their functional groups, being catecholates, hydroxamates, and α-hydroxy carbolates \[[@CR38]\]. However, few siderophores have thus far been characterized from *Paenibacillus*.
*Paenibacillus larvae* produces a catecholate -type siderophore called bacillibactin, which is also made by *Bacillus subtilis* and members of the *Bacillus cereus* sensu lato group \[[@CR38]\]. Bacillibactin is a cyclic trimeric lactone of 2,3-dihydroxybenzoate (DHB)-glycine-threonine. Similar to bacillibactin, paenibactin is a cyclic trimeric lactone of 2,3-DHB-alanine-threonine produced by *P. elgii* B69, the difference being the amino acid inserted between the DHB and threonine units \[[@CR37]\]. Catecholates have not been detected in cultures of *P. polymyxa* SQR-21, which instead produces hydroxamate-type siderophores at low concentrations in the late log phase \[[@CR35]\]. Siderophore synthesis gene clusters are present only in some strains of some *Paenibacillus* species, and are thought to have been obtained from fairly recent events of horizontal gene transfer \[[@CR26], [@CR37]\].
Aside from siderophore production, *Paenibacillus* may promote iron uptake by plants via other mechanisms. *P. polymyxa* BFKC01 transcriptionally activates plant genes involved in iron deficiency responses, including the membrane bound ferric chelate reductase *fro2* and the divalent metal transporter *irt1*, as well as genes involved in the synthesis of iron-mobilizing phenolic compounds \[[@CR39]\]. *Paenibacillus*-produced organic acids, such as oxalic acid, could also conceivably contribute to iron uptake, but release of such acids does not appear to be iron regulated \[[@CR35]\].
Phytohormone production {#Sec6}
-----------------------
Auxins are hormones that are crucial regulators of gene expression and development throughout a plant's life, participating in cell division, elongation, fruit development and senescence. There are multiple classes of auxins, but the first identified and most abundant in nature is indole-3-acetic acid (IAA) \[[@CR40]\]. Although plants are able to produce their own phytohormones, they can also utilize foreign sources produced by other organisms.
In addition to plants, IAA is synthesized by fungi and bacteria, including *Paenibacillus* and most other plant-associated bacteria \[[@CR20]\]. The production of IAA by *Paenibacillus* is thought to contribute to plant growth promotion \[[@CR41]\]. However, this hormone has a complex relationship with plants, being produced by both plant-growth-promoting and phytopathogenic bacteria. Lower levels of exogenous IAA typically increase plant growth and productivity, while high levels lead to disease susceptibility \[[@CR20]\]. Perhaps auspiciously, the tryptophan precursor of IAA is energetically costly, and considerable amounts are only made with an excess of tryptophan, which may be exuded from the plant, typically during the stationary phase of bacterial growth \[[@CR41]\].
Three different enzymatic pathways have been identified that convert tryptophan to IAA; in bacteria, the most common is the indole-3-pyruvic acid pathway, which is also present in plants. In this pathway, an aminotransferase deaminates tryptophan to yield indole-3-pyruvic acid, which is then converted to indole-3-acetaldehyde by a decarboxylase in the rate-limiting step. Finally, indole-3-acetaldehyde is oxidized to IAA via an unknown enzyme \[[@CR20], [@CR41]\].
Genes encoding putative indolepyruvate decarboxylase (IpdC), a key enzyme in the indole-3-pyruvic acid pathway, are present in all analyzed *Paenibacillus* genomes, with over 96% amino acid identity between strains across 98% of the sequence \[[@CR27]\]. IpdC belongs to a family of enzymes that exhibit some level of substrate promiscuity, and substitution of only a few amino acids in the active site can shift the primary affinity to another substrate. The active site must therefore be analysed in detail to confirm a gene's identity as an *ipdC* homologue whose primary function is IAA synthesis \[[@CR41]\]. However, the *Paenibacillus* genomic analysis did not identify genes involved in the other IAA pathways, that is, tryptophan monooxygenase or indole-3-acetamide hydrolase, suggesting that this genus most likely relies on the indole-3-pyruvic acid pathway \[[@CR27]\].
Biocontrol {#Sec7}
==========
Perhaps the most notable plant-growth promoting feature of *Paenibacillus* species comes from their numerous biocontrol capabilities. By inducing the plant's own resistance mechanisms or by producing biocidal substances, *Paenibacillus* can neutralize a diverse variety of phytopathogens and insect herbivores. *P. polymyxa* alone has been shown to provide protection to cauliflower \[[@CR42]\], pea \[[@CR43]\], ginseng \[[@CR44]\], cucumber \[[@CR45]\], chickpea \[[@CR46]\], peanut \[[@CR47]\], soybean \[[@CR48]\], pepper \[[@CR49]\], and more. Other species of *Paenibacillus* that have biocontrol properties include *P. alvei* \[[@CR50]\], *P. brasilensis* \[[@CR51]\], *P. dendritiformis* \[[@CR52]\], *P. ehimensis* \[[@CR53]\], *P. elgii* \[[@CR54]\], *P. kobensis* \[[@CR55]\], *P. lentimorbus* \[[@CR56]\], *P. macerans* \[[@CR57]\], *P. peoriae* \[[@CR58]\], and *P. thiaminolyticus* \[[@CR59]\].
Tolerance of some *Paenibacillus* species to commercial fungicides and insecticides \[[@CR60]\] indicates the possibility of using these microorganisms in combination with existing control solutions. However, when considering *Paenibacillus* or its compounds for biocontrol, it is important to note that competition can occur both ways. For example, while low levels of fusaric acid, a toxin produced by the fungus *Fusarium oxysporum*, increase production of the antifungal enzyme β-1,3-glucanase by *P. polymyxa* strains WR-2 and SQR-21, higher levels of fusaric acid actually decrease their production and result in reduced *P. polymyxa* growth \[[@CR61]\]. Furthermore, *Paenibacillus* may compete with other beneficial organisms \[[@CR62], [@CR63]\].
Induced systemic resistance {#Sec8}
---------------------------
Many beneficial rhizobacteria and root-associated mutualists, including members of *Paenibacillus*, can trigger induced systemic resistance (ISR) when present in high enough population densities. ISR is a latent defense mechanism occurring in plant tissues that are spatially separated from the inducer, providing enhanced protection against a range of pathogens or pests. Rather than immediately activating a defensive state, ISR primes for faster and stronger defenses by hypersensitizing the plant to potential threats \[[@CR64]\]. A variety of *Paenibacillus* species seem to elicit ISR against pathogenic bacteria \[[@CR49], [@CR65]\], fungi \[[@CR50], [@CR66]--[@CR68]\], nematodes \[[@CR69]\], and viruses \[[@CR70]\].
The ISR pathway begins when the plant recognizes elicitors from the beneficial microorganism, such as structural proteins, enzymes, reactive oxygen species, or volatile organic compounds \[[@CR64], [@CR71]\]. ISR can lead to increased systemic levels of the plant hormone salicyclic acid (SA-dependent response), or to an SA-independent response. The latter can include increased transcription of genes that are regulated by the plant hormones jasmonic acid, or enhanced expression of genes that are responsive to jasmonic acid or ethylene, which are then induced upon attack. In addition, SA-independent ISR can prime for physical responses such as enhanced callose deposition at sites of pathogen entry, which is regulated by abscisic acid and creates a structural barrier against further attack. While there are exceptions, SA-dependent ISR typically induces mechanisms against biotrophic pathogens (those requiring that the host cells remain alive), while the jasmonic acid/ethylene pathway protects against cell death-provoking necrotrophs and against insect herbiviores \[[@CR64]\].
Among *Paenibacillus* species, ISR has been demonstrated for *P. polymyxa*, *P. alvei*, *P. elgii*, and *P. lentimorbus*. For example, *P. polymyxa* strain KNUC265 was shown to protect against the bacterial pathogens *Xanthomonas axonopodis* and *Erwinia carotovora* in pepper and tobacco, respectively, using bacterial volatiles and diffusible metabolites as elicitors \[[@CR49]\]. *P. polymyxa* E681 was shown to use volatile organic compound elicitors to protect *Arabidopsis thaliana* against the bacterium *Pseudomonas syringae* via primed transcription of salicylic acid, jasmonic acid, and ethylene signaling genes \[[@CR65]\]. Consistent with the typical roles of SA-dependent and jasmonic acid/ethylene pathways, *Pseudomonas syringae* is described as a hemibiotrophic pathogen, with both biotrophic and necrotrophic stages \[[@CR72]\]. Similarly, *Arabidopsis thaliana* is primed by *P. alvei* K165 against a hemibiotrophic fungus \[[@CR54]\], *Verticillium dahlia*, by both salicylate and jasmonate-dependent pathways \[[@CR68]\]; while cucumber is primed by *P. elgii* MM-B22 against the hemibiotrohic fungus \[[@CR54]\] *Colletotrichum orbiculare*, responding to attack with defense-related enzymes and H~2~O~2~-induced cell death \[[@CR67]\]. Priming of tobacco by *P. lentimorbus* B-30488 also results in accumulation of defense-related enzymes in response to cucumber mosaic virus infection \[[@CR70]\].
Insecticides {#Sec9}
------------
*Paenibacillus* species have been shown to kill larvae of pest insects including beetles \[[@CR73]\] and lepidopterans \[[@CR74]\]. *Paenibacillus popilliae*, which infects larvae of the Japanese beetle, *Popillia japonica*, was the first microbial control agent registered in the US for use against an insect. However, its use was never widespread due in part to the inability of this particular species to be cultured in synthetic media \[[@CR75]\]. Nonetheless, research continues into the potential of *Paenibacillus* species for insect pest control, with the effective proteins including chitinase and crystal protein (Cry).
Chitinase enzymes produced by *Paenibacillus* hydrolyse chitin, which is a structural polysaccharide of insect exoskeletons and gut linings, leading to low feeding rates and death of infected insects. Both *Paenibacillus* sp. D1, from a seafood industry effluent treatment plant, and its isolated chitinase have been shown to cause concentration-dependent mortality of cotton bollworm (*Helicoverpa armigera*) when coated onto leaves fed to the larvae \[[@CR76]\]. *Paenibacillus* sp. D1 and its chitinase are tolerant to common insecticidal chemicals, and the chitinase itself is also highly stable in the field at 40 °C \[[@CR60]\], indicating the potential of the organism or its enzyme to be used as insecticide in the field.
Cry proteins are best known for conferring insecticidal activity to the bacterium *Bacillus thuringiensis* and to genetically modified crops. Following ingestion, these proteins form pores in the insect midgut epithelial cells, resulting in cell lysis and death \[[@CR77]\]. Cry homologs from *Paenibacillus lentimorbus* strain Semadara, which was isolated from larvae of the beetle *Blitopertha orientalis*, have been shown to cause mortality of beetle larvae \[[@CR78]\]. Genes encoding Cry have also been found in *P. popilliae* \[[@CR79]\] and *Paenibacillus* spp. Kh3 \[[@CR80]\]. In addition, *P. polymyxa* NMO10 has been genetically engineered with Cry1C from *B. thuringiensis* in order to combine the insecticidal activity of Cry and the growth promotion properties of *P. polymyxa*. The modified strain demonstrated greater toxicity than *B. thuringiensis* against lepidopteran insects \[[@CR81], [@CR82]\].
While insecticidal activities of *Paenibacillus* species may contribute to insect pest control, it is important to note that interactions between species in the field can be complex and non-target effects of biocontrol agents needs to be considered. For example, large populations of *Paenibacillus* species, or *P. polymyxa* alone, near the root system can actually increase the susceptibility of plants to aphids, possibly via increased levels of the plant growth promoting hormone IAA \[[@CR83]\]. Furthermore, while various *Paenibacillus* species suppress parasitic nematodes, most notably the root-knot nematode *Meloidogyne incognita*, *P. nematophilus* has been found to impede dispersal of the beneficial nematode *Heterorhabditis megidis* and reduce its infectivity of moth larvae \[[@CR63]\].
Antimicrobials {#Sec10}
--------------
Many *Paenibacillus* species compete with other microorganisms through the production of a wide range of antimicrobial compounds. In one study, 25 of 55 isolates from water and soil exhibited a broad inhibition spectrum against tested bacteria and pathogenic fungi Lorentz RH, Ártico S, Da Silveira AB, Einsfeld A and Corção G \[[@CR84]\], suggesting that a good proportion of *Paenibacillus* species are likely to produce antimicrobials. Yet diversity exists even within the same species: of 25 strains of *P. polymyxa*, 15 were strongly inhibitory to the oomycete pathogen *Phytophthora capsici*, while 10 showed weak or no antimicrobial effect \[[@CR85]\]; and genome sequencing confirms the diversity of antimicrobial-encoding gene clusters among *P. polymyxa* strains \[[@CR26], [@CR27]\].
Various *Paenibacillus* strains, or their isolated antimicrobial compounds, could therefore be useful in controlling phytopathogenic microorganisms, leading to lower usage of chemical biocides which can have negative environmental effects. Soilborne fungal pathogens, in particular, require high doses of chemical fungicides for control due to their wide host spectra and persistence in soil \[[@CR53]\]. The antimicrobial activities of *Paenibacillus* may also be useful for post-harvest control of food-borne bacteria, such as *Salmonella*, that are pathogenic to humans \[[@CR86]\].
The antimicrobials produced by *Paenibacillus* include peptides, enzymes, and volatile organic compounds (VOCs). While antimicrobial peptides are extremely significant for biocontrol in agriculture, the purified or synthesized peptides also have realised and potential uses in medicine and food processing, and are therefore discussed further in a separate section (see "[Antimicrobial peptides](#Sec11){ref-type="sec"}" section).
Hydrolytic enzymes of *Paenibacillus* can attack the cell walls of fungal and oomycete competitors. Cell walls of filamentous fungi contain a large fraction of β-1,3-glucan and chitin, while those of oomycetes consist primarily of β-1,3-glucan, β-1,6-glucan and cellulose; and both contain up to 11% protein. Various species of soil-dwelling *Paenibacillus* have been found to produce glucanses, chitinases, cellulases, and proteases that are implicated in the destruction of eukaryote cell walls. For example, crude enzyme extract from *P. ehimensis* KWN38 was shown to deform hyphal morphology and prevent growth of the basidiomycete fungus *Rhizoctonia solani*, the ascomycete fungus *Fusarium oxysporum*, and the oomycete *Phytophthora capsici*, all of which are phytopathogens \[[@CR53]\]. Glucanases and chitinases from strains including *P. ehimensis* IB-X, *P. ehimensis* MA2012, and *P. polymyxa* A21 have been shown to damage cell wall structures and/or inhibit *R. solani*, the oomycete *Pythium aphanidermatum*, and the ascomycetes *Alternaria alternata*, *Botrytis cinerea*, *Colletotrichum gloeosporioides*, and *Drechlera sorokiniana* \[[@CR87]--[@CR90]\]. A chitinase from *Paenibacillus* sp. D1 also exhibited high stability in presence of commonly used fungicides, suggesting the potential of some hydrolytic enzymes as additives to chemical fungicides \[[@CR91]\].
VOCs can enhance interactions between soil-dwelling microorganisms, as these compounds diffuse through air-filled pores in the soil to reach physically separated organisms \[[@CR92]\]. A large number of VOCs are produced by *Paenibacillus* species \[[@CR93], [@CR94]\] as well as other microorganisms \[[@CR92]\]. For example, *P. polymyxa* WR-2 was found to produce 42 VOCs, over 30 of which had some degree of antifungal activity against *F. oxysporum*, including 13 that completely inhibited its growth. The compounds included benzenes, aldehydes, keytones, and alcohols, although some were produced in low quantities; with benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol being the main antifungal compounds \[[@CR94]\]. Antimicrobial VOCs have application for biocontrol of agricultural pathogens, post-harvest diseases (particularly of fruit), and building molds \[[@CR92]\].
In addition to the antimicrobials described above, a non-volatile organic compound, methyl 2,3-dihydroxybenzoate, from *P. elgii* HOA73 was found inhibited growth of *B. cinerea*, *F. oxysporum*. *P. capsici*, and *R. solani* \[[@CR95]\].
Antimicrobial peptides {#Sec11}
======================
Further to "[Antimicrobials](#Sec10){ref-type="sec"}" section, *Paenibacillus* produces antimicrobial peptides with realised or potential applications in agriculture, medicine, and food processing. These peptides are of two types: ribosomally-synthesized bacteriocins, and non-ribosomally synthesized peptides, where amino acids are incorporated independently of messenger RNA.
Bacteriocins {#Sec12}
------------
Bacteriocins are ribosomally synthesized, proteinaceous toxins that inhibit the growth of bacteria that are related to the producer. *Paenibacillus* species are known to produce at least two of the three classes of bacteriocins, being lantibiotics and pediocins.
Lantibiotics, also known as Class I bacteriocins, contain the non-coded amino acid lanthionine \[[@CR96]\]. They are typically active against Gram-positive bacteria, as the outer membrane of Gram-negative bacteria presents a natural barrier. However, some gram negatives can be affected at high concentrations \[[@CR97]\].
Lantibiotics are usually expressed at the late exponential phase or early stationary phase of bacterial growth, and are encoded in a cluster along with genes required for their extensive post-translational modifications \[[@CR96]\]. In *P. polymyxa* OSY-DF, for example, the paenibacillin prepropeptide is encoded in a cluster that also contains putative genes for lantibiotic dehydratase, lantibiotic cyclase, acetylase, peptidase, and an ATP-binding cassette (ABC) transporter that may function for export to the extracellular space \[[@CR96]\]. Other lantibiotics include paenicidin A produced by *P. polymyxa* NRRL B-30509 \[[@CR98]\] and penisin produced by *Paenibacillus* sp. strain A3 \[[@CR99]\].
Lantibiotics in *Paenibacillus* are fairly recent discoveries, with Paenibacillin first reported in 2007 \[[@CR100]\]. However, the lantibiotic Nisin, produced by *Lactococcus lactis*, has been in commercial use since the 1950s, both as a food preservative and in veterinary medicine. Lantibiotics have low toxicity toward mammals and are poorly immunogenic, meaning there is little risk of adverse effects \[[@CR97]\]. Paenibacillin has the advantages of pH and heat stability, as well as activity against a broad range of foodborne pathogens and spoilage bacteria, making it an attractive candidate for food preservation \[[@CR101]\].
Compared to these lantibiotics, less research has been done on *Paenibacillus*-produced pediocins, also known as Class II bacteriocins. Pediocins are nonmodified, linear peptides, and include SRCAM 37 and SRCAM602 produced by *P. polymyxa* \[[@CR101]\].
Nonribosomal lipopeptides {#Sec13}
-------------------------
In contrast to bacteriocins, many antimicrobial peptides produced by *Paenibacillus* are synthesized nonribosomally, independently of RNA. Here, the amino acid residues are pieced together by nonribosomal peptide synthetases (NRPSs), which are multienzyme complexes that can incorporate a mixture of [d]{.smallcaps} and [l]{.smallcaps} amino acids. Each module of an NRSP incorporates one or more specific amino acids into the peptide chain. Resulting peptides show great diversity in sequence and structure, and an enhanced resistance to proteolytic enzymes. The nonribosomal lipopeptides act primarily by disrupting membranes of the target cells, and because it is difficult for target organism to reorganize their membranes, development of resistance is usually slow \[[@CR102]\].
Nonribosomal lipopeptides can be categorized as linear cationic, cyclic cationic, or cyclic noncationic. Although they are the most amenable to chemical synthesis, limited research has been conducted on linear cationic nonribosomal lipopeptides. In *Paenibacillus*, these include saltavalin, jolipeptin, and tridecaptins \[[@CR102]\].
### Cyclic cationic lipopeptides {#Sec14}
The most thoroughly researched cyclic cationic lipopeptides from *Paenibacillus* are the polymyxins, first isolated from *P. polymyxa* in 1947, although they are also produced by strains of *P. alvei* \[[@CR103]\], *P. kobensis* \[[@CR55]\], and possibly other species. These are a family of peptides each consisting of a polycationic diaminobutyryl-containing heptapeptide ring and tripeptide side chain with a fatty acid derivative at the N-terminus \[[@CR104]\]. Members of the family include polymyxin A, B, C, D, E (also called colistin), M (also called mattacin), P, S, and T \[[@CR103]--[@CR106]\]. These polymyxins differ from each other in amino acid composition at residues 3, 6, and 7, including D vs L stereochemistry of amino acids. Subgroups (e.g. polymyxin E~1~ and E~2~) differ in the lipid moiety and/or the amino acid at residue 7 \[[@CR103]\]. Amino acid diversity is thought to arise from combinatorial chemistry, due to mixing and matching of alleles of the NRPS modular domains \[[@CR104]\]. Polymyxin gene clusters found to date each encode three multi-modular NRPSs and two ABC transporters \[[@CR103], [@CR107]\].
Polymyxins bind to the lipid A component of lipopolysaccharide on the outer membrane of gram-negative bacteria to disrupt the outer membrane, then permeabilize and disrupt the inner membrane. Most cases of resistance occur in strains that have modified lipid A to reduce its net negative charge, thereby reducing affinity for polymyxin \[[@CR102]\].
Polymyxins B and E are produced industrially from *P. polymyxa* strains \[[@CR103]\]. They are used in ointments, such as the antibiotic creams Neosporin and Polysporin (both contain polymyxin B), for the treatment and prevention of topical skin infections; and as last-resort treatments for multidrug resistant internal infections \[[@CR108]\]. While polymyxins were used extensively from the 1940s until the 1970s to treat gram-negative bacterial infections, their clinical uses are currently limited primarily because of toxicity to the human central nervous system and kidneys. While this toxicity may be less severe than previously reported \[[@CR102]\], synthetic production with modifications can create new polymyxins with improved pharmacokinetic properties as well as activity against resistant bacteria \[[@CR109]\].
Other cyclic cationic lipopeptides produced by *Paenibacillus* include octapeptins (e.g. battacin), paenibacterin, polypeptins (e.g. pelgipeptin), and gavaserin. Octapeptins have the structure of truncated polymyxins, but are active against both Gram-negative and Gram-positive bacteria, and are less toxic \[[@CR102]\]. Paenibacterin is a cyclic 13-residue amino acid produced by *P. thiaminolyticus* OSY-SE, with activity against Gram negative and positive bacteria \[[@CR59]\].
### Cyclic noncationic lipopeptides {#Sec15}
The cyclic noncationic lipopeptides found so far in *Paenibacillus* are fusaricidins, first reported in *P. polymyxa* KT-8 in 1996 \[[@CR110]\]. These are hexapeptide rings that contain one or more ester bonds in addition to the amide bonds (depsipeptides), with an attached guanidinylated ß-hydroxy fatty acid \[[@CR104], [@CR111]\]. A single operon produces a variety of fusaricidins, differing in their incorporation of amino acids at three of the six positions in the peptide ring. The diversity here is due to relaxed substrate specificity of the NRPS \[[@CR112]\], in contrast to the modular mixing of polymyxin synthases. Fusaricidins are active against fungi, including many important phytopathogens, and a variety of gram-positive bacteria. Both naturally occurring structures and synthetic modifications can be chemically synthesized, creating improved stability and decreased nonspecific cytotoxicity toward human cells \[[@CR111]\].
Other medical applications {#Sec16}
==========================
In addition to a diverse array of antimicrobials, *Paenibacillus* produce other compounds that may be useful in medicine and dentistry. Their exo-polysaccharides (EPS) have antioxidant and anti-tumour properties, while mutanase enzymes may help to reduce tooth decay.
Microbial EPSs are water-soluble polymers that attach to the cell surfaces or are released into the medium. Strains of *Paenibacillus* produce EPSs with varying characteristics that may be medically useful. For example, those from *P. polymyxa* SQR-21 and *P. polymyxa* EJS-3 have superoxide scavenging activity and inhibit lipid peroxide \[[@CR113], [@CR114]\]. Some of these EPSs have been found to reduce oxidative stress in the livers of mice and inhibit in vitro growth of gastric cancer cells \[[@CR114]\].
Mutanases, also called (1→3)-α-glucanases, from *Paenibacillus* may be useful to help prevent tooth decay. These enzymes break down branched (1→3),(1→6)-α-[D]{.smallcaps}-glucans (mutans) which are produced by commensal streptococci and which form a major component of dental biofilm (plaque) that can harbor cariogenic bacteria. In contrast to the other polysaccharides and proteins in the biofilm, mutans are resistant to enzymes produced by oral microorganisms, and are substantially rigid and water-insoluble, thus are not dissolved and washed away by oral fluid \[[@CR115]\].
Mutanases are produced by fungi and bacteria, including *P. curdlanolyticus*, *P. glycanilyticus*, and *P. humicus*. However, these enzymes are not widespread in nature and are not produced by oral microorganisms, with producing bacteria usually isolated from soil. The bacterial enzymes are typically endo-(1→3)-α-glucanases, which cleave internal (1→3)-α-linkages at random sites along the glucan chain. These mutanases tend to be more stable than their fungal counterparts, and are active at a higher pH that is more consistent with the oral environment \[[@CR115]\].
A marketed oral rinse, Biotene PBF (Laclede Professional Products), contained mutanase among other components, but the product's effectiveness was not proven, which may be due to a variety of reasons. In many cases, mutanase production requires inducing (1→3)-α-glucans, which have been difficult to make in quantities large enough to support products with high enzyme concentrations. However, (1→3)-α-glucan components of fungal cell walls have more recently been shown to induce mutanase expression in *Paenibacillus* and other organisms \[[@CR115]\].
Process manufacturing {#Sec17}
=====================
*Paenibacillus* strains produce a variety of enzymes with potential applications in industrial process manufacturing for detergents, food, textiles, paper, and biofuel; including amylases, cellulases, hemicellulases, lipases, pectinases, and lignin-modifying enzymes (Additional file [3](#MOESM3){ref-type="media"}). Although enzymes sourced from *Paenibacillus* are not presently used in these processes, the search is ongoing for enzymes that are highly active under industrially-relevant conditions, have improved stability, or can be produced at a lower cost than currently available alternatives.
The laundry and dish detergents industry is the primary consumer of industrial enzymes. Proteases, lipases, amylases, and sometimes hemicellulases, are used to break down food and other organic residues, such as blood and grass stains. Cellulases are also used in laundry detergents to restore the smooth look and feel cotton-based fabric, by removing small balls of fibers that form on the cloth during wearing and washing. The other major industry to use enzymes is food, feed, and beverages. Here, amylases convert starches into sugar sweeteners such as high-fructose corn syrup, and create precursors for brewing alcoholic beverages. Cellulases and pectinases are used to extract and clarify juices, and, along with hemicellulases, to improve the nutritional quality of animal feeds. In the textile industry, pectinases, proteases, and lipases remove impurities from cotton and enhance wettability for dyeing and finishing; amylases remove coating agents from yard after it is woven (desizing); and cellulases can produced a "stonewashed" denim finish. For paper products and cellulosic biofuel, microbial enzymes can help remove lignin that causes paper to yellow and reduces the availability of fibers for saccharification and fermentation to biofuels. After lignin removal, cellulases and hemicellulases can alter fiber properties for paper manufacturing, or hydrolyze fibers for bioethanol or biobutanol production. For an alternative biofuel, lipases can serve as transesterification catalysts for biodiesel production \[[@CR116]\].
Cold-active enzymes are desirable for laundry detergents, as washing with cold water reduces energy consumption and fabric wear. Fittingly, cold-active protease, amylase, xylanase, and cellulase are efficiently produced by *P. terrae* \[[@CR117]\]. For most other applications, thermostability and activity under harsh conditions are desired. Accordingly, a cellulase from *P. chitinolyticus* CKS1 has optimal activity at 80 °C and pH 4.8 \[[@CR118]\]; while another from *P. tarimensis* retains high activity from pH 3.0 to 10.5, from 9 mM to 5 M NaCl, at 80 °C in high salt, and in the presence of organic solvents, EDTA, and heavy metals \[[@CR119]\].
Bioremediation {#Sec18}
==============
A variety of industries including petroleum, textiles, pulp and paper, and other chemical industries can unintentionally or intentionally release large amounts of organic pollutant compounds and heavy metals. *Paenibacillus* species may be utilized in the removal or degradation of these environmental pollutants, through bioflocculation or enzymatic activities.
Often used for wastewater treatment, flocculation is a process that removes suspended particles from liquids, frequently through the addition of chemicals (flocculants) that promote aggregation. Many microorganism including bacteria, fungi, and algae can produce biological flocculants consisting of polysaccharides, proteins, or other macromolecules. Strains of *P. jamilae*, *P. macerans*, *P. polymyxa*, and *P. validus* have been shown to promote bioflocculation of heavy metal ions or acid dyes; and *P. elgii* B69 produces an exopolysaccharide (EPS) bioflocculant that can remove multiple pollutants including heavy metal ions, dyes, and kaolin clay over a wide pH range \[[@CR120]\]. *Paenibacillus* could therefore be used to help remove contaminants from a variety of wastewaters.
To degrade contaminants, either in wastewaters or at sites of environmental spills, *Paenibacillus* can produce various enzymes that metabolize aliphatic and aromatic organic pollutants, including oxygenases, dehydrogenases, and ligninolytic enzymes \[[@CR121], [@CR122]\]. The textiles industry produces multiple chemicals that can pollute natural waters and soils, most notably dyes that are released due to inadequate wastewater treatment \[[@CR123]\]. Strains of *Paenibacillus*, either on their own or in concert with other bacteria, are able to degrade many textile dyes \[[@CR124]--[@CR126]\] and polyvinyl alcohol (PVA), which is used as a coating for textile and paper fibers \[[@CR127]\]. Other hazardous effluents produced by pulp and paper mills can be decontaminated by *Paenibacillus* \[[@CR128]\].
*Paenibacillus* strains can also degrade pollutants derived from extracting, refining, and transporting petroleum and coal tar, including crude oil \[[@CR129]\], diesel fuel \[[@CR130]\], bitumen \[[@CR131]\], disulfide oils \[[@CR132]\]; and the polycyclic aromatic hydrocarbons (PAHs) naphthalene \[[@CR133]\], phenanthrene \[[@CR134]\], and pyrene \[[@CR135]\]. Strains can also degrade the chemical gasoline additives ethyl tert-butyl ether \[[@CR136]\] and benzene \[[@CR134]\], the latter of which is also used in the production of chemicals and plastics, including nylon.
Pathogenicity {#Sec19}
=============
Some *Paenibacillus* species are known to infect various organisms, including honeybees and the parasite vector *Biomphalaria glabrata*, and occasionally present as opportunistic infections in humans.
Honeybee disease {#Sec20}
----------------
The most studied disease associated with *Paenibacillus* is American Foulbrood (AFB), caused by *P. larvae*. AFB afflicts honeybee (*Apis* species) colonies globally, and is the most destructive brood disease \[[@CR137]\].
Although tylosin, lincomycin, and oxytetracycline \[[@CR138]\] are effective antibiotics against *P. larvae*, antibiotics are poorly metabolized by honeybees, and their residues or those of their metabolites can be stable in honey for over a year. Elimination of the residues can only occur when the honeybees consume all of it or when the beekeeper removes the contaminated food \[[@CR139]\]. Residual antibiotics or their metabolites cause issues when the honey is meant for human consumption, as they can trigger allergic reactions, harm healthy microbiota, and confer resistance to pathogenic bacterial strains. Additionally, *P. larvae* resistance to antibiotics such as oxytetracycline has become increasingly more common in recent years \[[@CR140]\]. The current most typical solution to deal with an AFB affliction is to burn the entire hive.
AFB is caused by four strains of *P. larvae*, named ERIC I-IV based on the identities of their enterobacterial repetitive intergenic consensus (ERIC) sequences. ERIC I and II are the types typically isolated from afflicted hives, as ERIC III and IV are less virulent \[[@CR38], [@CR140]--[@CR145]\]. While ERIC II is the most virulent strain, ERIC I is more prevalent globally, likely because it can infect all honeybee subspecies, whereas ERIC II is restricted to certain subspecies \[[@CR137]\].
As the most virulent strain, *P. larvae* ERIC II has a larval LT~100~ (time it takes the entire colony's larvae to die) of just 7 days \[[@CR145]\]. This strain produces a unique functional S-layer protein which facilitates attachment of the bacteria to the peritrophic matrix that lines the honeybee's midgut epithelium \[[@CR146]\]. By contrast, *P. larvae* ERIC I has a larval LT~100~ of 12 days \[[@CR145]\] and produces a toxin known as the C3larvin toxin which may contribute to its pathogenicity. Although its target substrate has not been determined, this toxin acts as an ADP-ribosyltransferase and is lethal when expressed in yeast in vitro \[[@CR147]\].
Both ERIC I and ERIC II have an invasive spore stage and a non-invasive vegetative stage. Initial ingestion of spore-contaminated food by the larvae marks the beginning of the non-invasive phase. The spores travel to the midgut lumen and germinate, after which the bacteria proliferate rapidly. Honeybee larvae are most susceptible to infection within the first 36 h after hatching, and only a few spores are needed to initiate infection. After this window, the peritrophic matrix is too thick for the bacteria to penetrate and colonization is never achieved. Both strains use chitinases to degrade the peritrophic matrix for both nourishment and access to the midgut epithelium \[[@CR146]\].
The S-layer protein expressed by ERIC II facilitates initial association of the bacteria to the peritrophic matrix, and ERIC I may have a similar protein. If the bacteria are successful in degrading the peritrophic matrix, they eventually penetrate into the midgut epithelium and the haemocoel using chitinases and proteases, marking the invasive phase. The host larvae dies soon after from bacteremia \[[@CR145]\]. Adult honeybees are not susceptible to infection, and so in the process of cleaning contaminated cells they tend to transfer spores all over the hive. In this way the infection spreads horizontally. The infection can also spread vertically when a contaminated mother colony infects its daughter swarm upon establishment of a new colony. The horizontal mode of transmission is much more virulent and can occur both within and between hives \[[@CR148]\]. Further attributing to the pathogenicity of *P. larvae* are the paenilimicins and paenilarvins that it produces. Paenilimicins are antimicrobials that fight ecological niche competitors and are not directly involved in killing the bee larvae, while paenilarvins are antifungal compounds which also negatively affect bee larvae \[[@CR142], [@CR143]\]. As such, paenilicmins and paenilarvins promote the survival and colonization of *P. larvae* within honeybee larvae, while paenilarvins have the added disadvantage of directly impacting the health of the bee. ERIC II produces four paenilimicins: A1, A2, B1, and B2. In addition, paenilarvins A and B are produced as a secondary metabolite during the infection of *P. larvae* \[[@CR143]\]. Other nonribosomal peptides and peptide-polyketide hybrids are *P. larvae* secondary metabolites which are expected to have additional roles in the bacteria's pathogenicity \[[@CR149]\].
Several naturally isolated substances and compounds have shown potential in *P. larvae* inhibition. Both monofloral and polyfloral honey demonstrate in vitro activity against *P. larvae* strains, and this is likely due in part to the high concentration of sugar causing extreme osmotic stress for the bacteria. In addition, nectar contains several antimicrobial secondary plant metabolites whose presence can be detected in processed honey. Because these secondary metabolites are species-specific in their antagonism, polyfloral honey tends to confer more resistance than monofloral honey, as it contains the metabolites from several plant nectars \[[@CR140]\]. Propolis, a substance derived from plant resins, is used by bees in the construction of their hives and also shows inhibitory activity against *P. larvae* \[[@CR150], [@CR151]\]. It was also discovered that multiple Hypericum extracts have the potential to inhibit *P. larvae* in vitro, including hyperforin, uliginosin A and B, 7-epiclusianone, albasidin AA, and drummondin E \[[@CR144]\]. Other compounds demonstrating potential to control *P. larvae* infections include *Azadirachta indica, Vitex trifolia*, *Calendula officinalis*, and *Nasturtium officinale* extracts \[[@CR152], [@CR153]\], a collection of essential oils \[[@CR154]\], and frozen as well as freeze-dried sunflower bee pollen \[[@CR155]\]. Certain other bacterial species demonstrate antagonistic activity to *P. larvae*, including *Bacillus polymachus* which shows potential as a biocontrol agent \[[@CR156]\]. Multiple *Enterococcus* species have demonstrated antagonistic activity to *P. larvae*, and this is believed to be due to the bacteriocin genes found in their genomes \[[@CR157]\]. A novel *P. larvae* phage phiIBB-Pl23 produces an endolysin which effectively kills *P. larvae* while at the same time remains nontoxic to bees \[[@CR158]\].
Different species of honeybees glycosylate their royal jelly proteins in distinct patterns, which seems to have an influence on their vulnerability to *P. larvae* infections. *Apis mellifera* lingustica's royal jelly proteins display higher antihypertension activity than those produced by *Apis cerana* cerana and is active at lower levels. Interestingly, this difference in activity grants *Apis mellifera* lingustica optimized molecular functioning and enhanced immune activity, and so the strain is more resistant to *P. larvae* than is *Apis cerana* cerana in that larval exposure to the spores results in infection less frequently \[[@CR159]\]. Although the strain tested was ERIC I, this may help to explain the specificity observed in *P. larvae* ERIC II infections. These findings have implications when exploring potential techniques to control AFB, as well as in discovering novel techniques for controlling human hypertensive disease.
Another species of bacteria believed to put the honeybee at risk is *P. apiarius* \[[@CR160]\]. Although not the primary cause of disease, *P. alvei* acts as a secondary infector in cases of European Foulbrood, along with other species of pathogenic bacteria including *Enterococcus faecalis*, *Brevibacillus laterosporus*, *Bacillus pumilus*, *Achromobacter euridice* \[[@CR140]\], and *P. dendritiformis* \[[@CR161]\].
Snail disease {#Sec21}
-------------
*P. glabratella* was originally isolated from visible white nodules on snails in a population with high mortality. This species causes significant mortality in infected *Biomphalaria glabrata* snail populations, and is highly contagious among members of the population. Moreover, infected snails produce infected eggs causing decreased levels of hatching. These findings hold promise for *P. glabratella* as a potential biocontrol agent against the tropical parasitic disease Schistosomiasis which commonly uses these snails as vectors \[[@CR162]\]. This has implications in developing countries where parasites are a significant cause of death. Schistosomiasis ranks second globally among parasitic diseases of public health and socio-economic importance and is endemic in Africa \[[@CR163]\].
Opportunistic infections of humans {#Sec22}
----------------------------------
Several *Paenibacillus* species have been isolated from humans globally (Additional file [1](#MOESM1){ref-type="media"}). Although the majority of these colonisations are not harmful to their host, some have demonstrated pathogenicity to humans. In almost every case, *Paenibacillus* infections are opportunistic and tend to infect immunocompromised people. Diseases or syndromes associated with *Paenibacillus* infection include chronic kidney disease \[[@CR164]\], sickle cell disease \[[@CR165]\], premature birth \[[@CR166]\], Whipple's disease \[[@CR167]\], hydrocephalus \[[@CR168]\], skin cancer, chronic interstitial nephropathy, and acute lymphoblastic leukemia \[[@CR169]\]. In many of these cases it is unclear whether the relationship between the infection and the disease was correlated or causal, but it is likely that *Paenibacillus* was simply occupying suitable niches opportunistically. Many human-recovered *Paenibacillus* isolates come from elderly patients whose immune systems are generally weak.
A significant risk factor to *Paenibacillus* infection is the use of intravenous drugs. Intravenous drug use grants bacteria and other contaminants entry into the blood stream which would normally be unexposed to these pathogens. In one case, *P. amylolyticus* coinfection with *Lysinibacillus fusiformis* caused bacteremia leading to sepsis in an immunocompromised patient known to use intravenous heroin \[[@CR170]\]. Additionally, several cases of *P. larvae* bacteremia have been caused by the use of intravenous methadone prepared with honey from hives infected with *P. larvae.* Some pharmacies are known to prepare methadone using a viscous substance such as honey to prevent patients from misusing it through injection. If residual *P. larvae* spores in the honey happen to be injected through intentional misuse, they can germinate under proper conditions and cause infection \[[@CR171]\].
*Paenibacillus* isolated from humans have demonstrated a variety of strain-dependent drug resistances including resistances to norfloxacin, clindamycin, ampicillin, and ticarcillinclavulanic acid. Successful drug treatments have included cefotaxime, ceftrixone, and a treatment involving amikacin and zosyn followed by po Levofloxacin \[[@CR164], [@CR165], [@CR172]\].
Dairy spoilage {#Sec23}
==============
Another well-known negative aspect of *Paenibacillus* is its role in the spoilage of milk and other dairy products. *Paenibacillus* is among the most important bacterial genera that produce spoilage enzymes in the dairy industry, along with *Bacillus* and *Viridibacillus*. Endospores of these genera are able to survive extreme conditions including high heat, pressure, biocides, and UV irradiation, allowing them to withstand pasteurization and persist in industrial equipment. Small numbers of *Paenibacillus* spores can therefore be found in both raw and pasteurized milk \[[@CR173]\].
Many *Paenibacillus* strains also grow well at refrigeration temperature. *Paenibacillus* represents more than 95% of bacteria in raw milk after 10 days of refrigerated shelf life \[[@CR173]\], while spoilage of pasteurized milk due to *Paenibacillus* is delayed by the germination process of spores and usually occurs after 17--21 days \[[@CR174]\]. Strains that are able to grow at low temperature (6 °C) share numerous genetic features including genes that encode peptidases with cold-adapted features and cold-adaptation related proteins \[[@CR175]\].
While *Paenibacillus* enzymes can be beneficial to other industries (see "[Process manufacturing](#Sec17){ref-type="sec"}" section), their proteases, lipases, and phospholipases negatively impact the texture of dairy products, such as curdling caused by proteases, as well as the flavour \[[@CR173]\]. However, not all dairy-isolated strains produce these activities \[[@CR174]\]. *Paenibacillus* species isolated from dairy products include *P. amylolyticus*, *P. lactis*, *P. lentimorbus*, *P. lucanolyticus*, *P. odorifer*, *P. peoriae*, and *P. stellifer* \[[@CR173], [@CR174], [@CR176]\].
Conclusions {#Sec24}
===========
*Paenibacillus* was separated from *Bacillus* in 1993, but is a paraphyletic genus that is likely to undergo further subdivision in the future. This diverse genus has relevance in many areas, spurring numerous genome sequencing projects and patents. Many species are well-known plant-growth promoters, with various strains capable of promoting plant nutrient uptake, controlling phytopathogens, and producing phytohormones. The usefulness of inoculating *Paenibacillus* in the field can be restricted by various environmental conditions, but further research into the establishment and performance of these species within complex soil ecosystems may allow for their widespread use as biofertilizer. In addition to agricultural applications, *Paenibacillus* produces a diversity of antimicrobials, enzymes, and exopolysaccharides with relevance in medicine, process manufacturing, and bioremediation, some of which have already been commercialized. However, the genus is not purely beneficial, with some species causing food spoilage, honeybee disease, or opportunistic infections in humans. Still, the future can expect more discoveries and optimizations that will allow *Paenibacillus* to contribute positively to health and sustainable processes.
Additional files {#Sec25}
================
**Additional file 1.** All discovered *Paenibacillus* species along with their countries, environments, and years of isolation. **Additional file 2.** List of *Paenibacillus* genome sequencing projects and their progress. **Additional file 3.** Secreted compounds and significant enzymes produced by members of the genus *Paenibacillus.*
ABC
: ATP-binding cassette
AFB
: American Foulbrood
Anf
: iron-dependent nitrogenase
Cry
: crystal protein
DHB
: dihydroxybenzoate
EPS
: exo-polysaccharide
ERIC
: enterobacterial repetitive intergenic consensus
IAA
: indole-3-acetic acid
IpdC
: indolepyruvate decarboxylase
ISR
: induced systemic resistance
LT~100~
: time required to kill 100% of subjects
Nif
: molybdenum-dependent nitrogenase
NRSP
: nonribosomal peptide synthetase
PAH
: polycyclic aromatic hydrocarbon
PVA
: polyvinyl alcohol
Vnf
: vanadium- and iron-dependent nitrogenase
VOC
: volatile organic compound
Elliot Nicholas Grady and Jacqueline MacDonald contributed equally to this work
EG performed preliminary literature searches, created Additional files [1](#MOESM1){ref-type="media"} and [2](#MOESM2){ref-type="media"}, wrote the section on pathogenicity, much of the introduction, and contributed content to most other sections. JM researched and wrote most of the final content. LL helped collect and organize research for Additional files [1](#MOESM1){ref-type="media"} and [2](#MOESM2){ref-type="media"}. AR performed patent searches and created Table [1](#Tab1){ref-type="table"}. ZCY conceived of and edited the report. All authors read and approved the final manuscript.
Acknowledgements {#FPar1}
================
This research was funded by the Agriculture and Agri-Food Canada Growing Forward-AgriFlex (RBPI number 2555); Agriculture and Agri-Food Canada Growing Forward II project 1670; as well as a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN-2015-06052) awarded to Z-C Yuan. The funding body was not involved in the design of the study; in the collection, analysis, and interpretation of data; or in writing the manuscript. Due to space limitation, we are not able to include all important *Paenibacillus* works.
Competing interests {#FPar2}
===================
The authors declare that they have no competing interests.
Funding {#FPar3}
=======
This research was funded by the Agriculture and Agri-Food Canada Growing Forward-AgriFlex (RBPI number 2555); Agriculture and Agri-Food Canada Growing Forward II project 1670; as well as a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN-2015-06052) awarded to Z-C Yuan. The funding body was not involved in the design of the study; in the collection, analysis, and interpretation of data; or in writing the manuscript.
| {
"pile_set_name": "PubMed Central"
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###### Key questions
What is already known?
======================
- Rapid transfer to a medical facility is a key mechanism through which ambulance-based emergency medical systems (EMS) improve trauma outcomes.
What are the new findings?
==========================
- Trauma victims are rapidly transported in Delhi, India, to a hospital by bystanders and police.
- India's medico-legal system imposes substantial burdens on good Samaritans by forcing them to interact with police.
- For-profit hospitals often decline to provide first aid and stabilise patients delaying access to care.
What do the new findings imply?
===============================
- Formal layperson-EMS should get official sanction from police and carry visible symbols of their authority to assist with emergency transport, and the system should work with empanelled hospitals that have agreed to participate.
- Delhi already has several key EMS components that could be integrated and expanded.
Introduction {#s1}
============
In India and many low- and middle-income countries (LMICs), ambulance-based emergency medical systems (EMS) are uncommon. Over half of the global population lives in areas without formal EMS.[@R1] Victims of traffic crashes in LMICs are typically transported from the crash scene to a hospital by bystanders and passing vehicles.[@R4] In India, surveillance at medical facilities suggests that most victims are brought to hospital by taxis or police, with ambulances accounting for less than 5% of transport --- a pattern that has shifted little in 30 years ([figure 1](#F1){ref-type="fig"}). Despite strong advocacy,[@R13] the high equipment and operational costs of ambulance-based EMS suggests they will not be adopted or expanded to scale in many LMICs.[@R1]
![Mode of transport to hospital for victims of traffic crashes in India. ('Others' includes transport by private vehicles, bicycles, cycle rickshaws and other modes. Data sources: St. Stephens Hospital, unpublished data; TITCO: combines data from five major trauma centres in Delhi, Mumbai (two trauma centres), Kolkata and Chennai.[@R42])](bmjgh-2019-001963f01){#F1}
In principle, EMS aim to improve trauma outcomes by providing: (1) medical interventions in the field, and (2) rapid transport to a definitive care facility. However, evidence suggests that many prehospital interventions are ineffective and that the main benefits of EMS likely accrue from rapid transfer. Increasingly, studies have questioned the benefits of emergency endotracheal intubation,[@R15] intravenous drug therapy,[@R16] fluid resuscitation[@R17] and spine immobilisation.[@R19] In fact, studies show that trauma victims transported by advanced life support ambulances have similar or worse outcomes than basic life support, likely due to interventions that are poorly performed and which delay transfer to definitive care.[@R22] Recognising the weak evidence for advanced training of first responders, the WHO recommends that LMICs should train lay responders, such as commercial drivers, to provide basic first aid and rapid transfer to hospitals.[@R13] Several studies have reported training of commercial drivers, police, community leaders and others in LMICs (Ghana,[@R29] Madagascar,[@R30] Iraq[@R31] and Uganda,[@R33] among many others[@R12]).
The second key aspect of EMS --- coordination of lay responders to reduce transfer time --- has received very little attention in advocacy efforts. Although advanced communication networks have been previously viewed as expensive components of the most advanced systems,[@R3] recent developments in smartphone technology have made coordinated taxi fleets common in LMICs.[@R34] In fact, there have been several unsuccessful attempts to develop a 'layperson-EMS' by using peer-to-peer or dispatcher-coordinated networks of lay responders. For instance, in recent years, ridesharing companies in India (Uber in Hyderabad[@R35] and Wagon Cab in Delhi[@R36]) have launched emergency ride options. Similarly, some services have attempted to build dispatcher-coordinated networks of existing private ambulances (AMBER Health[@R37] and LifeHover[@R38] in Delhi, Dial242 in Mumbai,[@R38] StanPlus, eSahai and Call Ambulance in Hyderabad[@R38]). A review of the websites for these services suggest that few have sustained after the initial launch. This indicates that layperson-EMS may face legal, medical and social barriers that need to be systematically addressed.
In 2017, researchers at the University of Chicago and the Indian Institute of Technology (IIT) Delhi initiated a project funded by the US National Institutes of Health to assess the feasibility of developing a coordinated system of lay first-responders that could improve population-level outcomes of traffic crashes in Delhi, India. One aspect of the project aimed to understand how trauma victims currently access medical care, and the barriers to such a system being formalised into a coordinated layperson-EMS. In particular, we sought to understand the following aspects from the perspective of frontline stakeholders, policymakers and experts: (i) who helps victims, (ii) how are they transported to hospitals, (iii) what deters help from good Samaritans, (iv) how can these barriers be addressed and (v) could a layperson-EMS improve outcomes. In this paper, we report our findings and share recommendations for EMS policy in Delhi and similar settings.
Methods {#s2}
=======
Study setting and context {#s2-1}
-------------------------
Delhi is the capital of India and has an estimated population of 20 million. It has a complex administrative structure, with federal, state and local government agencies overseeing various aspects of governance.[@R39] For example, while Delhi Police is overseen by the federal government, health services are primarily administered by the state government.[@R39] Delhi's formal EMS consists of a public ambulance service with a fleet of 265 ambulances (31 Advanced Life Support, 110 Basic Life Support and 124 Patient Transport)[@R40] but it is common for emergency transport to be provided by police, taxis (especially auto rickshaws) and private vehicles.[@R41] Healthcare in Delhi is provided by a mix of for-profit hospitals, and relatively low-cost public hospitals.[@R43] Patients often leave for-profit hospitals against medical advice for financial reasons.[@R44] It is standard medico-legal practice across India to treat all injuries as potentially criminal cases that are registered as medico-legal cases and reported to the police. Surveys suggest that the fear of getting entangled in long-drawn legal processes deters bystanders from helping trauma victims.[@R46] In 2014, following public interest litigation, the Supreme Court of India instructed the federal government to develop legislation to protect good Samaritans and compel for-profit hospitals to stabilise victims and provide first aid.[@R48] Subsequently the government issued an executive order to this effect ('Good Samaritan Notification').[@R49]
Study design and methods {#s2-2}
------------------------
We used qualitative methods to elicit viewpoints of five groups of stakeholders --- (1) drivers of taxis (three-wheeled auto rickshaws and four-wheeled taxis), (2) medical personnel (doctors, nurses and hospital administrators), (3) legal experts, (4) police personnel and (5) other stakeholders (including policymakers, ambulance providers, non-governmental organisations (NGOs), hospital stretcher bearers and security guards and public health researchers). We acquired research data through:
### Key informant interviews {#s2-2-1}
#### Sampling {#s2-2-1-1}
We used a combination of purposeful sampling techniques.[@R50] All respondents had experience with transport, medical or legal aspects of trauma either as professional practitioners or through policy engagement (criterion sampling). We relied on recommendations from interviewees to identify additional respondents (snowball sampling), and used convenience sampling to identify certain taxi drivers and medical personnel.
#### Interview guides {#s2-2-1-2}
We designed interview guides to understand respondents' experiences and viewpoints towards emergency transport, barriers to bystander assistance and a coordinated layperson-EMS ([figure 2](#F2){ref-type="fig"}). The guides were informed by preliminary discussions with a sample of stakeholders prior to data collection. We refined and added questions as data collection progressed. Interview guides are provided as [online supplementary appendix A1](#SP1){ref-type="supplementary-material"}.
10.1136/bmjgh-2019-001963.supp1
{#F2}
In order to anchor reactions about a coordinated layperson-EMS, we presented respondents with a hypothetical taxi-EMS ([figure 2](#F2){ref-type="fig"}) consisting of a fleet of taxis with drivers trained in first aid and dispatcher-coordinated using a smartphone application (app). When an emergency call is received at the control room and no ambulance is available, the taxi closest to the crash site is recruited through the app. The app provides navigation to the crash site and the closest participating hospital, where the driver is paid for their effort using mobile-to-mobile cash transfer.
#### Data collection {#s2-2-1-3}
Four researchers (RA, RAr, KB, VS) with backgrounds in transportation, engineering and/or public health collected data during December 2017 to March 2019. Two researchers had training in qualitative methods (VS, RAr), while one underwent a short training in qualitative methods before the project began (RA). We conducted 50 interviews (26 recorded), typically lasting 45 to 60 min, in Hindi, English or a combination ([table 1](#T1){ref-type="table"}). These included five short interviews (\<15 mins) with individuals with knowledge about a specific topic but who were not suitable for full length interviews. We took handwritten notes, which were typed and stored electronically.
######
Data sources: interviews, stakeholder consultations and documents
Type of interviewees Interviews Details
----------------------- ------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Legal experts 5 Incl. four that participate in policy dialogue\*
Medical practitioners 16† Incl. 11 doctors, three nurses, one hospital administrator and two EMTs; nine from government hospitals, two from a charitable private hospital and five from for-profit private hospitals; five participate in policy dialogue
Police personnel 8† Incl. three that participate in collecting evidence used in accident claims tribunals; two senior police officials; one police control room van driver, one from a public hospital police post; two participate in policy dialogue
Taxi drivers 10 Incl. eight auto rickshaw drivers, two that drive both auto rickshaws and four-wheeled taxis and one Uber driver
Other stakeholders 11 Incl. two academic researchers, two policymakers, two NGO representatives, two ER stretcher bearers, two ER security guards and one public ambulance administrator
EMTs, emergency medical technicians; ER, emergency room; Incl., including; NGO, non-governmental organisation.
Stakeholder meeting Participants Details
--------------------- -------------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Meeting 23 Including five legal experts, seven academic researchers, three government bureaucrats, one ambulance administrator, four trauma physicians and one senior police personnel
Document review Documents Details
----------------- ----------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Documents 36 Including three legal judgements, five laws and regulations, eight online articles and news stories, six monographs and academic articles, nine policy briefs and agency reports.
\*Individuals who participate in policy dialogue related with emergency medical response, trauma care and good Samaritan protections.
†Including short interviews with one medical practitioner and four police personnel.
**Stakeholder consultation**: Halfway through data collection (June 2018), we held a stakeholder workshop to share emerging ideas and get feedback. The workshop had 16 external participants ([table 1](#T1){ref-type="table"}) in addition to seven from our team, and included legal experts, academic researchers, government bureaucrats, ambulance administrators, physicians and senior police personnel. We took notes which informed our data collection and analysis.
**Document review**: Thirty-six documents were reviewed to gather information on legal judgements, national and state policies and operational procedures. Documents were identified through internet searches and discussions with participants.
Analysis {#s2-3}
--------
Drawing on principles from general thematic analysis,[@R51] recorded interviews were transcribed by a contracted transcriber, and cleaned and checked by a team member (RA). We combined deductive and inductive approaches in the process of developing the codebook, using some predefined codes and allowing others to emerge during coding. We first applied the draft codebook to three transcripts, and further refined the codes. Next, two of the four coders (RA, RAr, VS, KB) coded all transcripts and notes. We consolidated our coding in NVivo and partitioned the data. Team members reviewed the coded data and developed early themes. Two members (KB, VS) further refined the themes, and finalised them after discussion with the entire research team.
Data quality {#s2-4}
------------
We strengthened data quality by: (1) Triangulation --- we discussed concepts across stakeholder categories and cross-checked respondent statements with key documents; (2) Prolonged engagement --- we spent 14 months collecting data at regular intervals, allowing us to develop a deeper understanding of the topics; (3) Peer debriefing --- we had regular group meetings throughout the research process and (4) Respondent validation through a stakeholder consultation.
Reflexivity {#s2-5}
-----------
Our study was underpinned by a constructivist epistemology, where our goal was to capture the perspective of respondents, many of whom held different viewpoints. However, we also attempted to gain a positivist understanding of relevant processes. The research team was multidisciplinary, and we had regular discussions on epistemological approaches and the impact of these different approaches on data collection and interpretation.
Patient and public involvement {#s2-6}
------------------------------
We did not involve patients in the development of the research question. A stakeholder consultation midway through the study was used to share emerging findings, get feedback and refine interview questions, and inform analysis. Findings will be disseminated at opportune district and national meetings.
Results {#s3}
=======
We start by presenting themes related with what happens at the scene, and at the hospital, where we focus particularly on medico-legal practices and inter-hospital transfers, which emerged as the primary deterrents to help from good Samaritans and a source of delays in access to medical care. Finally, we present themes related with strengthening the system of prehospital transport and care in Delhi. [Boxes 1--4](#B1 B2 B3 B4){ref-type="boxed-text"} present selected quotes organised by thematic headings.
###### Selected quotes related with what happens at the crash scene
[CROWD BEHAVIOUR AT SCENE]{.ul}
- 'it\'s mostly police and to some extent there are some you know bystanders who will help. It is also seen that the auto rickshaw drivers or the taxi drivers they help more than the normal general public.' MP3
- 'sometimes the situation becomes worst ... public start beating the driver. Sometimes their family members arrive. Patient is serious, bleeding too much, their relative does not bear after seeing this ... The Gujjar, Jatt, of Delhi do not talk, they start beating ... These people are well educated so start beating us.' TD9
[MODES OF TRANSPORT]{.ul}
- '40% to 50% (of trauma cases) are brought still by the police. Right? Rest are being brought by bystanders or relatives ... other common modes of private transport or auto rickshaws which are very common, three wheeler auto rickshaws, taxis and then private four wheelers or private two wheelers also in fact, motorised two wheelers. Only about 3% to 5% ... are being brought in by the CATS ambulance services.' MP16
- 'I will say in Delhi, in the most cases PCR van, this is the reality, that lots of time passersby ignores, ambulance also do not reach, hospitals do not send but in 90% cases police's PCR van transport him to the hospital.' PP1
[CHOICE OF HOSPITAL]{.ul}
- 'we do not feel courage to take him to the private hospital as we are unaware of his situation and who will pay for it. In private hospitals money has to be deposited before the treatment. And for the unknown I will transport to the public hospital because how will I pay, I can't afford it.' TD5
[MEDICAL CONCERNS WITH LAYPERSON TRANSPORT]{.ul}
- '...ambulances are used as a transport vehicle. ... even in USA when there are highly trained paramedics, they are not supposed to do lot of interventions on the scene because that can harm the patient. So ultimately the primary objective is that patient reaches the definitive care facility as quickly as possible.' MP11
- 'Autos are not the ideal mode of transport because you can't make the patient lie down flat on the back ... but you know in our condition I think that is even better because rather than no transport the patient lies on the roadside bleeding to death which happens so many times over here. I think even if they just pick up the guy and bring I think it is great job done.' MP13
- '... the liability of the auto rickshaw driver for causing further damage is not addressed by the law. ... And if it has been raised somewhere, they might have beaten up the auto rickshaw driver, and that is all. But it has not been raised as a legal issue in a court of law.' LE1
CATS, Centralised Accident and Trauma Services; LE, legal expert; MP, medical practitioner; PCR, Police Control Room; PP, police personnel; TD, taxi driver.
###### Selected quotes related with what happens at the hospital
[MEDICO-LEGAL PROCESSES]{.ul}
- 'Policemen say you sit inside let him become conscious, you go after that. That's why the auto drivers hesitate even more.' TD1
- 'The police might want to settle the matter --- take a certain amount from each party and close the case.' LE4
- 'The image of the police, and its practices, are such that people do not want to get caught in a situation where the police might extort money from them and harass them just because they helped the victim.' Stakeholder Consultation
- '... they feel there would be problems related to police case, it is a medico-legal case, ... Sometimes they want to help but they are more concerned about their own safety and not get entangled to the unnecessary problems.' MP11
- '(Medico-legal process) doesn't happen over here because it is a very cumbersome process and we can't afford to waste our time getting into those legalities. So we always prefer if patients, they are referred back to us, ... So we don't get involved in those formalities directly.' MP13, doctor at private hospital
- 'It is my personal view that if we are helping someone our job is only to drop him to hospital and handover him to the hospital staff and after that we are free to leave. If this happens then all will help.' OS7, stretcher bearer
- 'as medical professional we find it distressing to have the police in the ER.' Stakeholder Consultation
- 'if they are put down as a witness, then they would be required ... to appear and provide evidence. ... That is what keeps them away from taking a patient to the hospital. ... I wouldn't, for instance, if I was a lawyer or a businessman, take somebody in my car, if I fear that I might have to go to court half-a-dozen times.' LE1
[INTER-HOSPITAL TRANSFERS]{.ul}
- 'I think it is fairly common from private hospitals to be referred to government hospitals and one major reason is not the expertise, because expertise may be available in these private hospitals ... lot of trauma patients are transferred because they cannot afford the treatment in private facilities.' MP11
- 'We usually don't refer. Most people leave against medical advice after stabilisation sometimes for financial reasons etc after stabilisation. There is zero charge for stabilisation.' MP12; doctor; private hospital
- 'Private hospitals are very clever. They know the provision. They will give you first aid and then ask you to deposit 50 000 rupees for further treatment or take him away. And you can't do anything. ... There have been two to three times cases where they (private hospitals) did refuse to give the body of the deceased to the family until and unless they deposit the expenses. ... after giving the first aid and some treatment they will ask the patient to deposit the money otherwise they will ask him to take the patient away. ... They (private hospitals) will just admit him and that's all, rest depends on the patient. He has to bring the money for depositing, if he doesn't on some pretext or the other the hospital refuse to admit him.' LE5
[LOGISTICAL AND OTHER PROBLEMS]{.ul}
- 'I am transporting him but who will go with him? ... Because we face this major problem in hospital that either we ... should carry the injured person on stretcher or should park my three-wheeler in parking. If I will go to parking then 10 to 15 min would be waste for sure. Parking is not nearby.' TD5
- 'These days in (hospitals) five to six bouncers are posted for the safety of doctors that no one attacks them. When (the doctors/nurses) are sitting and gossiping, not helping anyone, then what will one do? ... will you not feel angry? Yes or no? ... If you want to do the real study then go to Safdarjung and see, the female is pregnant and in trouble but no doctor is helping her, four to six doctors, ladies are sitting and gossiping there ... Many times the doctors and female doctors are beaten up by the public, why did they beat them? Because what they do is just sit and gossip.' TD7
LE, legal expert; MP, medical practitioner; OS, other stakeholder; TD, taxidriver.
###### Selected quotes related with the effects of new legal protections for good samaritans
Effects of new legal protections for good samaritans
----------------------------------------------------
- 'It is the duty of the hospitals to actually proclaim in big bold letters that have no fear, bring the injured here, so it's their duty in law. They are not doing it.' LE2
- 'how much impact that has made after about 2 years? ... I don't think it has got big impact ... it has not drastically changed the pattern of people who are bringing in patients to be very frank.' MP16
- 'earlier, it was such that if there was an accident, 'What if we only get caught?' People used to get scared. But now, it is not like that ... Now, the laws have ... changed. Because of that, they help.' MP1
- 'the police chowki (post) guy will ask for name/number. While the Supreme Court says that you do not need to give your information, these people will still ask.' LE4
- 'As far as awareness (of legal protections for good Samaritans) is concerned is minimal in the last 1 year we have trained about 3500 police officers across 10 states. Not a single one of them knew about this. So it is a huge uphill task to ... let the police and other authorities know that they now have some obligations and duties towards the good Samaritan ... the awareness level is still quite low and for us that reveals a big challenge in terms of implementation of the law.' OS5, NGO representative
- 'Yes they do have to appear before the court and they will even after the good Samaritan law comes in. They will be cited as witnesses.' LE2
LE, legal expert; MP, medical practitioner; NGO, non-governmental organisation; OS, other stakeholder.
###### Selected quotes related with recommendations for improving the system
Recommendations for improving the system
----------------------------------------
- 'we are worried about duplication‚ we\'re talking about integration. We do not want any new numbers. ... One number that works for all emergencies.' Policymaker, Stakeholder Consultation
- 'if they (taxis) become part of the umbrella then obviously you can have any amount of ferrying vehicles ... control room knows that ok if there is not an ambulance but there is a taxi ... they will definitely improve the response time.' MP16
- 'if I phone and say my son has swallowed a marble, can you refuse? It's unethical to have a system only for road traffic injuries (RTIs).' Academic, Stakeholder Consultation
- '(ambulances) are preferred, but if they take time to reach, it is better that the patient be shifted to the hospital as quickly as possible in any available vehicle.' MP1
- 'If we should encourage a structured system of transport (by autos/taxis) then it should be done properly. Whether it is a neck or back injury, an auto will be impossible to fit a person in. Maybe in a taxi...especially one like a PCR -- with a length-wise seat. Spine stability is important.' MP12; doctor; private hospital
- 'Taxi is much better than auto. It has more space, jerking movements are also less.' MP7
- 'They should be trained, so they do not harm the patient during the transport and they know about the hospitals and the facility.' MP10
- 'If he (auto rickshaw driver) has an identity card, then he won't be stopped at all. Without an ID card the cops will stop and ask whether he (auto rickshaw driver) hit the injured.' PP5
- 'People don't mind giving their time for a cause ... They should be incentivised. ... But not too much.' MP5
MP, medical practitioner; PP, police personnel; RTIs, road traffic injuries
At the crash scene {#s3-1}
------------------
**Crowd behaviour**: Respondents commonly noted that large crowds usually gather at crash scenes. While most people are primarily onlookers, many respondents noted that usually someone steps forth to manage the situation and/or help the victim. Such help includes calling the police, persuading drivers of passing vehicles drivers to transport the victim to a hospital, and contacting family members using information obtained from the victim or their belongings.
**Modes of transport**: Many respondents asserted that police vans and auto rickshaws are the vehicles most commonly used for transport, with police vans being used more for victims with extensive visible trauma.
> Police will be called first if the condition is very serious but sometimes what happens is a person is transported in auto and he dies on the way, then it will be asked that why haven't you called at 100 ... therefore first of all police should be called. TD8
Many respondents described the police as providing a de facto EMS for trauma. Senior police officers explained that Delhi Police started using Police Control Room vans equipped with stretchers and first aid supplies, and training patrol staff in first aid, starting in the 1980s. This infrastructure was eventually expanded and integrated into routine police operations. Many respondents strongly perceived that the Delhi Police operate a robust system with a widely known number (100), large fleet of vehicles and rapid response. Respondents familiar with other Indian cities observed that such coordinated use of police for emergency transport is unique to Delhi.
Some respondents noted that public and private ambulances are common in Delhi but are used primarily for inter-hospital transfers. Some added that public awareness of public ambulances is growing and bystanders increasingly also call their number (102), but many noted that police vans arrive on the scene much sooner and transport victims without waiting for the ambulance.
> ...one must be aware of the ambulance number but I don't know ... (we know that) if you call 100 number police vehicle will come transport. TD4
Taxi drivers (TD) cited humanitarian and spiritual reasons, as motivating factors for helping (*'as we are sowing so shall we reap later.'* TD4) but some noted that taxi drivers may be persuaded by bystanders or police to help. Police personnel noted, and two taxi drivers confirmed, that police sometimes pay taxi drivers to transport crash victims. Compared with four-wheeled taxis, respondents noted that auto rickshaw drivers helped more often because they are ubiquitously available, easier to accost and easier to clean if soiled by blood and bodily fluids.
**Choice of hospital**: Most respondents agreed that the choice of hospital is determined by victims or their companions if they are alert, and the decision includes considerations of injury severity, ability to pay, types of facilities in the vicinity and where they know a doctor. If they are unable to decide, bystanders make the choice based on similar considerations. Taxi drivers predominantly preferred public hospitals and noted that despite the law and government assurances, for-profit hospitals don't initiate care without a guarantee of payment.
> In private hospitals money has to be deposited before the treatment. And for the unknown I will transport to the public hospital because how will I pay (at a private hospital). TD5
Police typically transport victims to one of Delhi's nine designated public trauma centres unless the victim or their companions request otherwise, or in exceptional circumstances, such as a particularly severe crash that occurs near a major for-profit hospital.
**Medical concerns with layperson transport**: Many respondents raised concerns with the use of auto rickshaws for emergency transport. Medical respondents often stated that laypersons can exacerbate neck and spine injuries. Some noted that auto rickshaws have a jerky motion and are not wide enough to accommodate supine patients, arguing that emergency transport should be provided by equipped ambulances and trained providers.
> ... in auto space is very less. Patient's leg will get folded, hand will get fractured, neck will also get tilted. MP6; nurse
However, when further probed, they usually added that despite such concerns they had never received a patient whose injuries were worsened because of transport by auto rickshaw.
> ... the patient gets an injury, and by the time they get here, it is worse because of the movement ... But in my shift, none like that have come. MP1; nurse
Such objections were more often raised by non-medical professionals or junior medical professionals. Senior medical respondents and/or those who participate in policy dialogue on EMS raised such concerns less frequently, and when they did, usually noted that the benefits of rapid response by laypersons outweighed the potential of exacerbating injuries. Two senior medical respondents also noted that in practice even ambulance crew do not take adequate spinal precautions.
At the hospital {#s3-2}
---------------
On arrival at the casualty or emergency department (ED) entrance, patients are transferred to stretchers and wheeled in, where they are triaged and directed to diagnostic services or treatment. Two processes of registration, medical and medico-legal, start nearly simultaneously with medical care.
### Medico-legal processes {#s3-2-1}
Most major public hospitals have a police post, where a message is sent from the ED requesting the medico-legal register. Smaller public hospitals and private hospitals that do not have a police post maintain a medico-legal register, and hospital security contacts police when a medico-legal case is received. The doctor who first examined the patient fills the medico-legal form, documenting the event time, date, location (which determines police jurisdiction) and a medical description of injuries. Medico-legal registration is a starting point for police involvement, which almost all respondents noted is a deterrent to bystander assistance and medical care.
#### Police presence in hospitals {#s3-2-1-1}
Medico-legal processes result in a high-level of police presence in the hospital. We observed that police posts at major public hospitals were usually a busy site. Typical activities included police officers questioning companions of victims to determine crash location, making calls to police stations with jurisdiction to initiate investigations, obtaining contact information of witnesses and recording statements and overseeing the medico-legal register. We also observed substantial police presence at the ED, especially in the ambulance bay, the triage area and at the bedside. Here, in addition to obtaining testimony, police were helping negotiate settlements.
> the police come between the person who has hit and who has been hit and then they negotiate. They will tell this ... 'ok I will leave you if you give me this much. MP16
If the parties don't settle, the victim, or their representative, registers a 'First Information Report' (FIR), which officially initiates a police investigation, and legal proceedings that can last years.
> The major concern is that (it) would require them to make several journeys as witnesses to the trial courts, (which) are notorious for being ... slow ... courts and hospitals are two places where Indians don't want to go. LE2
Police respondents claimed that such negotiations are for cases involving mild injuries and that serious cases are 'cognisable' (ie, police officer has the authority to independently initiate an investigation) and are always investigated. However, other respondents noted that even serious injuries are settled with police mediation but carry the risk that the offending party may renege. Therefore, victims often wait for 10 to 15 days to confirm payment before asking the police to file an FIR.
Several medical respondents noted that police presence is distressing to patients and medical staff and interferes with medical care. Further, they stated that medico-legal registration and testifying in court adds a substantial burden on doctors. (*'If an FIR is filed then the court will call the doctor ... It is definitely burdensome but we have to oblige.'* MP2) They cited this as a reason why some doctors and private facilities avoid engaging with medico-legal cases.
#### Harassment of good samaritans {#s3-2-1-2}
Forced interactions with police and subsequent visits to court were the most commonly cited reason that deterred people from transporting crash victims to hospital.
> Police will call you, ask you, who, what, why ... you would be called in police station again and again. TD5
In addition to describing police interactions as unpleasant, respondents often pointed to more serious concerns. Taxi drivers, legal experts (LEs) and some other respondents commonly talked about good Samaritans being wrongly accused, and sometimes convicted, of causing the crash. Anecdotes from taxi drivers commonly included being unwillingly held at the hospital until the victim regained consciousness and absolved them of responsibility (*'Policemen say you sit inside, let him become conscious, you go after that.'* TD8). Many talked about police intentionally accusing innocent bystanders in an attempt to extort money, or because they had received bribes from the guilty party. Several respondents noted that police try to coerce dishonest testimony or rely on professional witnesses and close cases quickly. For example, one lawyer with experience in handling such cases indicated that victims are sometimes coerced to implicate bystanders, and that 'professional eyewitnesses' who provide false testimony are common.
The police were not the only source of such harassment. Taxi drivers talked about doctors and nurses persuading them to share their contact information, which two nurses corroborated.
> ... giving a contact number is not a big deal. We try to convince him. MP7, nurse
### Inter-hospital transfers {#s3-2-2}
Medical and non-medical respondents talked extensively about patients being shunted between hospitals, leading to delays in medical care. Among the common reasons for transfers from public hospitals and small private hospitals was the absence of medical specialists, unavailability of beds and/or lack of medical services. For major for-profit hospitals that typically have the facilities and expertise to provide care, inability to pay was the most common reason. Respondents mentioned that often patients remain at private hospitals until they have exhausted their resources before moving the patient to a public facility.
> referrals come from hospitals which are corporate or private hospitals, and patients when they exhaust their money, when their financial situation becomes very bad, they come to us. MP9; public hospital
Some respondents noted that some hospitals violate the law by refusing to release the dead body, or retaining property (such as vehicles) until payments are made.
Several respondents, including one from a private hospital, mentioned that private hospitals prefer not to accept trauma cases brought directly from the field to avoid medico-legal processes. Instead, some private hospitals encourage patients to go to a public hospital for medico-legal registration before returning for medical care.
Most respondents mentioned that private hospitals are required by law to stabilise patients and provide first aid. However, many argued that for-profit hospitals would not be financially viable unless they charged for such services.
> ultimately they are private hospitals. They run for profit. MP16
One respondent noted that for-profit hospitals have security guards at the gate that informally advise taking the patient to other public facilities. Others mentioned that in cases where the victim is seen by a medical professional, they often receive inadequate care because stabilisation, resuscitation and first aid are poorly defined concepts. A few respondents from public hospitals described instances where the condition of the patient worsened because they were transferred without being stabilised. Many noted that inter-hospital transfers occur without any communication leaving the receiving hospital ill-prepared to respond.
### Logistical and other problems {#s3-2-3}
Several taxi drivers mentioned loss of work hours as a problem if they help take a victim to hospital. Many also raised other issues, especially parking. Unlike ambulances, which can be left unattended at the ED entrance, private vehicles have to park in the visitor's parking lot, which may be some distance and require parking fees. Importantly, taxi drivers mentioned (and we observed) that patient companions play an important role in unloading the patient from transporting vehicles, pushing the stretcher in the hospital and, more generally, advocating for resources (eg, medical attention, hospital beds, access to imaging services). Taxi drivers are unable to play this role while they are looking for parking. Many taxi drivers noted that they are much more willing to transport crash victims if there is an accompanying person.
Beyond forced interactions with police, taxi drivers and other respondents also pointed to other forms of harassment at the hospital. Some mentioned hostile questioning by doctors and medical staff (*'Who are you? From where have you come? They ask like this...'* TD10), and, in some instances, being assaulted by agitated family members when they arrive at the hospital. (*'Sometime their family members arrive, and the patient is serious, bleeding too much, their relative does not bear after seeing this ... they start beating us.'* TD9). More generally, EDs were described by some as unpleasant environments. Respondents described incidents of violence at the ED, often directed at hospital staff who are perceived as not doing enough for patients, but sometimes also directed at good Samaritans who bring patients to hospitals. Our observations at the EDs showed numerous means for managing such violence, such as limited entry points, lots of security guards, signs that prohibited some items that can be used as weapons and heavily fortified doors and windows.
Effects of new legal protections for good samaritans {#s3-3}
----------------------------------------------------
The government's recent Good Samaritan Notification instructed police and hospitals to protect good Samaritans, and ensure that private hospitals provide critical care.[@R49] However, our respondents stated conflicting opinions about whether the Notification had affected practices. Although several mentioned that good Samaritans were no longer detained at hospitals, or forced to provide their contact information, many respondents said that practices perceived as harassment remained unchanged. This was usually attributed to a lack of awareness of the new legal protections. However, legal respondents who participate in court proceedings noted that the Notification was unlikely to help because police practices that are perceived as harassment have always been unlawful. In particular, they noted that the law has always been interested only in the testimony of witnesses, and the Notification doesn't change this. Detaining non-witnesses and coercing testimony has always been unlawful. Nevertheless, most respondents agreed that the Notification has positive features, such as limiting how often witnesses and medical professionals are required to testify in court. The Notification also requires hospitals to post signs highlighting legal protections for good Samaritans but, consistent with our observations, respondents said that it was very rare for hospitals to post such signage.
> We had put up a noticeboard saying that nobody had to give their name but we have removed. ... maybe because of some renovation. MP2; doctor; private charitable hospital
The Notification also emphasised a previous Supreme Court ruling that compels all hospitals to stabilise crash victims and provide first aid but many respondents noted that hospital practices were unchanged. Respondents noted that the problem is unlikely to be fixed unless financial mechanisms are created to reimburse private hospitals for the costs of providing care. They described several attempts by state governments to create cashless insurance systems and current attempts by the federal government to establish universal health coverage that may help.
Recommendations for improving prehospital care and transport in Delhi {#s3-4}
---------------------------------------------------------------------
Almost all respondents agreed that lay responders had an important role in transporting trauma victims to hospital especially since it is already common practice. However, a few cautioned against inadvertently doing harm to the current system of police-based transport in Delhi. Most respondents emphasised that attempts at strengthening prehospital care should be part of a coordinated system of emergency response. The following issues consistently emerged in our interviews.
- Single call number: Many respondents commented on the proliferation of emergency numbers (fire, police, ambulance, women safety, etc) and the need for a single nationwide number.
- Responding to all emergencies: Some respondents noted that an EMS should respond to all emergencies because restricting to specific causes (eg, accidents) is not ethically justified.
- Include private ambulances: Some respondents recommended integrating private ambulances into the coordinated EMS. Private ambulances for inter-hospital transfers are common in Delhi.
- Use of appropriate transport modes based on injury severity: Most respondents felt that taxis were appropriate for minor injuries but diverged in their views about severely injured victims, which many felt should be handled by paramedics and ambulances.
- Importance of rapid transfer to hospital: Many senior respondents commented on the trade-off between providing medical care in a prehospital setting and in rapid transport. Most agreed that since it is difficult to provide advanced interventions in the field, victims should be transferred to hospital by whatever means available.
- First aid training: All respondents noted the importance of training of first responders (lay or professional).
- Appropriate financial compensation: On the issue of monetary incentives for lay responders (such as taxi drivers), all taxi drivers felt that they should be compensated for their time. Other respondents took more nuanced positions. Many felt that public recognition is the most important incentive, and that monetary rewards should be carefully calibrated to not create perverse incentives. Some felt that monetary rewards shouldn't be offered at all. Respondents also noted that the new policy of the Delhi government to give rupees 2000 (\~\$30) to anybody who transports a crash victim to hospital is unlikely to work unless there are real protections for good Samaritans. Medical respondents noted that so far, the reward is rarely claimed.
- Managing EMS costs: Respondents who participate in policy dialogue pointed to the high cost of ambulances and the savings expected from using lay responders. Cost issues weren't raised by other respondents.
- Mechanisms to give legitimacy to lay responders: Some respondents alluded to class perceptions, asking if victims, families, bystanders and police would trust taxi drivers to behave responsibly. (*'the first thing is trust development ... between the taxi drivers and the victim because ... pregnant victims and ... ladies ... don't enjoy much of a trust with taxi drivers'* MP9). They suggested that participating drivers should carry visible symbols (eg, badges, medallions, certificates) of their association with the EMS. Several respondents noted that such legitimacy should be sanctioned by police and the medical system, and provide participating taxi drivers with several privileges, including leaving their vehicles unattended in the ambulance bay, and not being coerced into sharing contact information, providing statements or being identified as witnesses. Although such issues related with establishing trust and legitimacy were not raised by taxi drivers, their responses suggested that they assumed that their participation in a taxi-EMS would confer protections from harassment. We asked all interviewed taxi drivers if they would be willing to participate in such a system. All agreed, often with enthusiasm.
- Legal feasibility of a taxi-EMS: We asked legal experts and policymakers if they expected the drivers of a purpose-built taxi-EMS to have legal protections intended for good Samaritans. Respondents agreed they would because participation was voluntary. They agreed that the tort liability of those who help has never been raised in the courts in India but one respondent added that if the taxi-EMS is legally challenged, it is possible that some forms of transport, such as auto rickshaws, may not be viewed as appropriate by the judiciary.
Discussion {#s4}
==========
India is in the midst of making substantial changes to national health and transportation policies,[@R52] and improving trauma outcomes is the focus of significant policy dialogue. In the last few years, there has been extensive advocacy from NGOs,[@R47] Supreme Court judgements,[@R48] executive orders[@R49] and substantial national and international news coverage focused on improving access to care for trauma victims.[@R55] Our study provides important insights relevant to this dialogue by providing a rich description of what happens to trauma victims in the field, at hospitals and in the medico-legal system. In the discussion that follows we highlight key legal, medical and social issues, focusing specially on implications for a layperson-EMS.
Addressing legal barriers {#s4-1}
-------------------------
In India, deeply entrenched medico-legal practices force good Samaritans into extended and often onerous interactions with police. Surveys suggest that over half of law-abiding Indian citizens fear the police,[@R57] and our results illustrate why. Our respondents shared many anecdotes where good Samaritans were coerced into being witnesses, held against their will or much worse, wrongfully implicated in causing the crash. Furthermore, medical professionals dislike adhering with medico-legal practices because they interfere with patient care.
India's recent Good Samaritan Notification[@R49] tries to circumscribe allowable police practices but our findings suggest that the Notification appears not to have affected medico-legal processes substantively. This is likely because the executive order doesn't stop police from questioning witnesses, and the most irksome police practices, such as coercing testimony, were already illegal. The Notification also instructs local governments to give rewards to bystanders, which pale in comparison to the medico-legal burdens. This may explain why only 4% of good Samaritans who helped trauma victims have claimed the financial incentive of rupees 2000 (\~\$30) being offered by the Delhi government.[@R15]
While WHO recommends that countries establish legislations that protect good Samaritans,[@R13] our study highlights that the issues and legal remedies will likely vary based on country-specific conditions. Globally, good Samaritan laws are typically framed as either duty-to-assist or protection-against-liability,[@R58] which may not make sense in many LMICs. Consider, for instance, that good Samaritan laws in the USA successfully helped alleviate physician concerns that they would be sued for malpractice if they helped at accident sites.[@R59] However, China's new law protecting good Samaritans from the liability of exacerbating injuries fails to address their main concern of being implicated of causing the accident.[@R60] Thus, it is important that LMICs assess their particular situations and evolve appropriate countermeasures for protecting good Samaritans. For our specific example, protecting taxi drivers against police harassment would likely require the taxi-EMS to have official police sanction, and the taxis to carry visible symbols (eg, medallions or certificates) of their authority to provide emergency transport.
Addressing healthcare system barriers {#s4-2}
-------------------------------------
Although a layperson-EMS aims to address transportation barriers, our findings highlight other important sources of delays in India's healthcare system. Notably, care at for-profit hospitals is often delayed until it is established that somebody will pay for victims who may be poor, unaccompanied and/or disoriented. Although an amendment to the Motor Vehicles Act has required all hospitals to stabilise patients since 1994,[@R62] our respondents described many anecdotes of how for-profit hospitals sidestep requirements. Such practices will likely continue until there is a mechanism for hospitals to recoup costs, such as through no-fault universal health coverage, as recommended by the Sustainable Development Goals.[@R63] India is in the midst of major legislative and policy reforms to its healthcare[@R43] and motor vehicle insurance sectors,[@R53] whose implementation could create such a financial mechanism. However, until it is established that for-profit hospitals reliably provide care, good Samaritans should take victims with uncertain financial means to public hospitals. For a layperson-EMS, this likely means transporting to hospitals that have agreed to participate and have the requisite capacity.
Addressing social barriers {#s4-3}
--------------------------
Our findings suggest that a layperson-EMS could face opposition from various stakeholders. The underlying rationale for a layperson-EMS is that rapid transfer to hospital is of overriding importance for improving outcomes. Although our senior medical respondents understood this well, junior medical respondents commonly expressed concern about exacerbating spinal injuries. Similar apprehensions were also raised by some of our non-medical respondents. Although such concerns are likely overstated,[@R19] the social acceptability of a layperson-EMS will likely require its medical legitimacy to be accepted by the public.
Social class issues were the subtext of many reactions to our proposed taxi-EMS. Often these took the form of a strong bias against the vehicle (especially auto rickshaws) and the entrenched class hierarchies of cities such as Delhi, but also concern of whether taxi drivers were responsible actors who would be trusted by, for instance, pregnant women. Regardless of whether such apprehensions have merit, ensuring social acceptability of a taxi-EMS may require that the system is viewed as a semi-professional operation with visible symbols of authority to act in an emergency, and with trained drivers who appear to be following a prescribed protocol.
Building a complete system of emergency care {#s4-4}
--------------------------------------------
Our study finds that Delhi already has key EMS components that could potentially be integrated and expanded into a complete system of emergency care. Notably, our study highlighted that the police in Delhi already transports trauma victims to hospital as a routine part of operations. In effect, they respond to accidents like a coordinated non-medical EMS, with a control room and dispatch protocols, fleet of vehicles equipped with first aid and victim transport capabilities, providers with rudimentary training and empanelled hospitals.[@R64] In fact, contrary to a popular belief that trauma victims face large delays in getting to hospital, many of our frontline stakeholders noted that most severely injured victims in Delhi are already transported rapidly by police. Partly for this reason, they tended to view the taxi-EMS as an additional component to police operations intended for less severely injured victims. Although the use of equipped police vehicles to transport trauma victims is unique to Delhi, dispatcher-coordinated police operations are common in many major urban centres in India,[@R65] and likely, globally. If these capacities can be extended to emergency transport, they may provide LMICs a cost-effective pathway to coordinated EMS.
Delhi's coordinated police response, as well as our hypothetical taxi-EMS, focus primarily on accidents. However, traumatic injuries only accounts for about one-fifth of all emergency health conditions.[@R66] Our senior respondents noted that it would be unethical for an EMS to not respond to all health emergencies. Although it is unclear how much the mandate of a police-based operation can be expanded to incorporate other medical services, it may be possible to efficiently link it with other services. Delhi's police already contact the Centralised Accident and Trauma Services public ambulance system, which is often unable to meet demand. However, the supply of ambulances in the EMS could be dramatically increased by including private ambulances, which are commonly available in Delhi but used primarily for inter-hospital transfers. Other governance challenges associated with developing a coordinated system in Delhi include its unique political system (Delhi's governance is shared between the federal government, Delhi's state government and four municipal governments, and the broader National Capital Region includes two other states with their own ambulance systems). Thus, other LMIC settings interested in developing an EMS should assess their current situation of prehospital transport and the availability of existing components of an EMS system that may be integrated and expanded.
Finally, in addition to strengthening prehospital transport, it is important to improve the quality of care provided at hospitals. Consider that recent efforts by the Indian government (including a conditional cash transfer) succeeded in increasing births at facilities but had no effect on maternal and neonatal mortality.[@R67] Similarly, Gupta *et al* [@R68] describe substantial delays in care for traumatic brain injuries at major trauma facilities in India reducing gains from improving the prehospital system. In summary, improving trauma outcomes in India and LMICs requires understanding how victims access care at present, and developing systems that help victims get quickly to a medical facility where they receive high-quality care.
We are grateful to the many researchers and practitioners who helped us identify relevant documents and connect with stakeholders.
**Handling editor:** Stephanie M Topp
**Contributors:** KB, VS, MV, GT and DM contributed to study conception. KB and VS developed the research protocol. RA, RAr, VS, KB designed interview guides, conducted interviews, coded data and developed preliminary themes. KB and VS refined and finalised themes. KB and VS wrote the first draft of the manuscript. All authors generated hypotheses, interpreted findings and critically reviewed the manuscript.
**Funding:** This work was supported by the US National Institutes of Health (NIH)/Fogarty (grant number 5R21TW010168).
**Competing interests:** None declared.
**Patient consent for publication:** Not required.
**Ethics approval:** This project was approved by the Institutional Ethics Committees/Boards of the University of Chicago, St. Stephen's Hospital and Indian Institute of Technology, Delhi.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data availability statement:** No data are available.
| {
"pile_set_name": "PubMed Central"
} |
Adolescent alcohol problems are an important public health issue. Research has indicated an increasing prevalence of adolescent alcohol use disorders (AUDs) (i.e., alcohol abuse and alcohol dependence) over recent decades. Approximately 40 percent of people with an AUD developed their first symptoms between the ages of 15 and 19 ([@b10-arh-22-2-95]). People with an earlier age of onset of AUDs tend to experience more severe alcohol problems and are more likely to have other psychiatric disorders (e.g., [@b3-arh-22-2-95]). At the same time, longitudinal research has shown that drinking status and the presence of alcohol-related problems can change considerably across adolescence and into young adulthood. Much remains to be learned about the nature and development of alcohol problems during the teenage years.
The diagnostic criteria for AUDs have largely been developed based on research and clinical experience with adults. This article summarizes the role of diagnostic classification in the treatment and research of AUDs and describes the current diagnostic criteria for AUDs as defined in the *Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition* (DSM--IV) ([@b2-arh-22-2-95]). Next, the article reviews epi-demiological and clinical research on DSM--IV AUD criteria among adolescents and potential limitations of these criteria when applied to this age group. Finally, the article describes some of the diagnostic interviews and screening tools that can be used to assess AUDs among adolescents.
The Diagnosis of AUDs Among Adolescents
=======================================
For any type of medical or psychiatric disorder, a valid diagnostic system is necessary to advance both treatment and research. Psychiatric disorders, including AUDs, are best viewed as evolving constructs that organize and describe a constellation of symptoms and behaviors. An accurate diagnostic system informs the clinician about course, prognosis, and the most effective treatment approaches. For researchers, diagnostic classification allows identification of subgroups and developmental pathways to the disorder. The standardized definitions provided by specific diagnostic criteria facilitate communication among and between researchers and clinicians. Although alcohol problems occur along a continuum of severity, specific diagnostic boundaries must be defined to guide both research and the allocation of limited health care resources.
DSM--IV Diagnostic Criteria for AUDs
------------------------------------
The DSM--IV describes two primary AUDs: alcohol abuse and alcohol dependence. A person receives a diagnosis of alcohol abuse if he or she experiences at least one of four abuse symptoms (i.e., role impairment, hazardous use, legal problems, and social problems) (see [table](#t1-arh-22-2-95){ref-type="table"} below) that lead to "clinically significant impairment or distress." These symptoms reflect either pathological patterns of alcohol use, psychosocial consequences, or both.
The framework for the diagnosis of alcohol dependence in the DSM--IV was influenced by the concept of the Alcohol Dependence Syndrome (ADS) developed by [@b7-arh-22-2-95]. In the ADS, alcohol dependence is defined rather broadly---that is, as a constellation of symptoms related to physical dependence as well as compulsive and pathological patterns of alcohol use. To qualify for a DSM--IV diagnosis of alcohol dependence, a person must exhibit within a 12-month period at least three of the following seven dependence symptoms: (1) tolerance, (2) withdrawal or drinking to avoid or relieve withdrawal, (3) drinking larger amounts or for a longer period than intended, (4) unsuccessful attempts or a repeated desire to quit or to cut down on drinking, (5) much time spent using alcohol, (6) reduced social or recreational activities in favor of alcohol use, and (7) continued alcohol use despite psychological or physical problems (see [table](#t1-arh-22-2-95){ref-type="table"}, p. 96). No single criterion is necessary or sufficient for an alcohol dependence diagnosis. Alcohol dependence is subtyped in DSM--IV as with or without physiological features, defined by tolerance or withdrawal symptoms.
######
Symptoms of Alcohol Abuse and Alcohol Dependence as Defined in the *Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition* (DSM--IV)
Alcohol Use Disorder Brief Identifier of Symptom Abstracted DSM--IV Definition
---------------------- ------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------
Alcohol Abuse Role impairment Frequent intoxication leading to failure to fulfill major role obligations (e.g., at school, work, or home)
Hazardous use Recurrent use when it is physically hazardous (e.g., driving while intoxicated)
Legal problems Recurrent alcohol-related legal problems
Social problems Continued drinking despite knowledge of persistent or recurrent social or interpersonal problems caused or exacerbated by alcohol use
Alcohol Dependence Tolerance Need to increase consumption by 50 percent or more to achieve the same effects; markedly reduced effects when drinking the same amount
Withdrawal Signs of alcohol withdrawal; drinking to avoid or relieve withdrawal
Using more or longer than intended Recurrent drinking of larger amounts or for a longer period of time than intended
Quit/cut down Unsuccessful attempts or a persistent desire to quit or cut down on drinking
Much time spent using alcohol Much time spent using, obtaining, or recovering from the effects of alcohol
Reduced activities Important social or recreational activities given up or reduced in favor of alcohol use
Psychological/physical problems Continued drinking despite knowledge of a recurrent or persistent psychological or physical problem caused or exacerbated by alcohol use
In contrast to previous versions of the DSM, the symptoms of alcohol abuse and alcohol dependence are mutually exclusive in DSM--IV. Moreover, the diagnoses of alcohol abuse and alcohol dependence are arranged hierarchically, such that a dependence diagnosis precludes an abuse diagnosis. Although not stated explicitly in DSM--IV, this hierarchical design implies that compared with alcohol dependence, alcohol abuse should be relatively mild and should onset at an earlier age.
The DSM--IV diagnostic criteria for AUDs are similar to the DSM--IV criteria for other drug use disorders (although some important differences do exist). Although this article focuses on adolescent AUDs, many of the diagnostic and assessment issues that are discussed apply to other drug use disorders as well. Because adolescent drinking and AUDs are strongly associated with other drug use and drug use disorders (e.g., [@b15-arh-22-2-95]), both alcohol and other drug use behaviors should be assessed in research and clinical settings.
Studies of DSM--IV AUDs Among Adolescents
=========================================
Several recent epidemiological and clinical studies have assessed DSM--IV AUD symptoms and diagnoses among adolescents. Epidemiological studies are important, because they provide estimates of the rates of symptoms and diagnoses in the general adolescent population. Clinical studies are equally important and complement epidemiological research by characterizing symptom patterns among adolescents who present for addiction treatment. Furthermore, clinical studies often provide detailed assessment of a large number of subjects with symptoms and diagnoses.
Findings of Epidemiological Studies
-----------------------------------
Not surprisingly, the prevalence rates of adolescent AUDs vary according to age and gender. For example, [@b5-arh-22-2-95] examined age-and gender-specific prevalences of AUDs in a representative household sample of 776 youth ages 10 to 20 in New York State. That study, which used DSM--III--R criteria, found that AUD prevalences jumped from 3.5 percent at ages 14 to 16 (3.1 percent of girls and 4.1 percent of boys) to 14.6 percent at ages 17 to 20 (8.9 percent of girls and 20.3 percent of boys). The observation that the prevalence of AUDs is higher in boys than in girls, particularly during late adolescence, has been confirmed in several studies.
Several recent studies have assessed DSM--IV alcohol symptoms and diagnoses in general population samples of adolescents. [@b12-arh-22-2-95] assessed DSM--IV AUD symptoms and diagnoses by interviewing a representative sample of 1,507 students ages 14 to 18 from urban and rural high schools in Oregon. Approximately 23 percent of the respondents had experienced at least one DSM--IV alcohol abuse or dependence symptom during their lifetime. The most common symptoms were dependence symptoms (i.e., tolerance, drinking larger amounts or for a longer period of time than intended, and reduced activities in favor of alcohol use) rather than abuse symptoms. The dependence symptoms of withdrawal and alcohol-related medical problems, and the abuse symptom of alcohol-related legal problems, were relatively rare. AUD diagnoses occurred in 6.2 percent of the sample at some time in their lives (1.9 percent of the sample had alcohol abuse and 4.3 percent had alcohol dependence). Another 16.7 percent of the sample had experienced some alcohol-related problems. This included 13.5 percent of participants who met the criteria for one or two DSM--IV dependence symptoms and no abuse symptoms, who therefore did not fulfill the DSM--IV criteria for an AUD.
Another large epidemiological study of alcohol and other drug use disorders assessed 74,008 9th and 12th grade high school students in Minnesota ([@b9-arh-22-2-95]). The lifetime presence of DSM--IV symptoms (all except withdrawal) was assessed by questionnaire; subjects were asked to respond affirmatively to questions about symptoms if they applied to either alcohol or other drugs. Among the 9th graders who had ever used alcohol or other drugs (approximately one-half of all 9th graders), 13.8 percent met the criteria for drug abuse and 8.2 percent met the criteria for drug dependence. Another 13 percent had one or two dependence symptoms and no abuse symptoms. The most common symptoms among the 9th graders were dependence symptoms: tolerance, using alcohol or drugs in greater amounts or for a longer time than intended, and much time spent using.
Approximately two-thirds of the 12th graders in the study had ever used alcohol or other drugs. Of those, 22.7 percent met the criteria for alcohol or other drug abuse, 10.5 percent met the criteria for alcohol or other drug dependence, and an additional 9.9 percent had one or two dependence symptoms and no abuse symptoms. The most common symptom in 12th graders was the abuse symptom of hazardous use (e.g., driving while intoxicated), followed closely by the dependence symptoms of tolerance, using alcohol or drugs in greater amounts or for a longer time than intended, and much time spent using, as well as the abuse symptom of social problems. Conversely, symptoms of alcohol-related medical and legal problems were rare. However, because questions about symptoms were not asked separately for alcohol versus other drugs in this study, the rate of alcohol and drug use disorders probably was overestimated, because in some cases positive answers about different symptoms may have applied to different substances. Therefore, these results should be interpreted cautiously. In addition, this study reported the prevalence of alcohol and drug use disorders only for adolescents who had ever used alcohol or other drugs. Accordingly, the results are not directly comparable to prevalence estimates for the entire adolescent population.
Findings of Clinical Studies
----------------------------
Several recent studies have evaluated DSM--IV criteria for AUDs among clinical samples of adolescents. [@b14-arh-22-2-95] used an adapted version of the Structured Clinical Interview for the DSM ([@b24-arh-22-2-95]) to examine DSM--IV symptoms of AUDs among adolescents ages 13 to 21 who were recruited from both clinical and community sources. The most common symptoms were the dependence symptoms of tolerance, drinking in greater amounts or for a longer period of time than intended, and much time spent using alcohol, as well as the abuse symptom of continued use despite social problems. Conversely, the dependence symptoms of withdrawal and alcohol-related medical problems and the abuse symptoms of hazardous use and alcohol-related legal problems were uncommon. For example, only 23 percent of the adolescents diagnosed with alcohol dependence (and none of the subjects without alcohol dependence) had experienced alcohol withdrawal. The high rates of tolerance and the low rates of withdrawal in this study are consistent with the results of a clinical study of adolescents by [@b25-arh-22-2-95] that used DSM--III--R criteria. The study by [@b14-arh-22-2-95] also identified five domains of recurrent alcohol-related problems not contained in the DSM--IV that were highly prevalent among adolescents with AUDs. Those problems were blackouts, passing out, risky sexual behavior, craving, and an alcohol-related drop in school grades.
Another investigation focused on DSM--IV criteria for AUDs in a clinical sample of 772 adolescents ages 12 to 19 ([@b30-arh-22-2-95]). AUD symptoms and diagnoses were assessed using the Adolescent Diagnostic Interview (ADI). The most common AUD symptoms in this study were the dependence symptoms of drinking in greater amounts or over longer periods than intended, unsuccessful attempts or a repeated desire to quit or cut down on drinking, and much time spent using alcohol. In contrast, the prevalence of withdrawal and alcohol-related legal problems was relatively low. Unlike the studies by [@b25-arh-22-2-95] and [@b14-arh-22-2-95], this investigation detected relatively low rates of tolerance, possibly because the ADI criteria for tolerance may be more conservative than those of other instruments.
Limitations of the DSM--IV Criteria for AUDs in Adolescents
-----------------------------------------------------------
In general, the DSM--IV criteria for AUDs have shown some validity in adolescents, in that groups classified as having alcohol dependence, alcohol abuse, and no diagnosis tend to differ on measures of alcohol use, other drug use, and independent measures of alcohol problem severity ([@b12-arh-22-2-95]; [@b14-arh-22-2-95]; [@b30-arh-22-2-95]). However, the available data also suggest potential limitations of the DSM--IV criteria for AUDs when applied to adolescents. Some of these limitations may apply to adults as well.
One potential limitation is that the DSM--IV criteria appear to include several symptoms that are not typically experienced by adolescent problem drinkers. Some symptoms have a very low prevalence, even in clinical samples, and thus may have only limited utility. Those symptoms include withdrawal and alcohol-related medical problems, which generally emerge only after years of heavy drinking. Other symptoms may have limited utility because they tend to occur only in particular subgroups of adolescents. For example, the alcohol abuse symptom of hazardous use, which is usually assigned due to driving while intoxicated, is rare in early adolescence and then increases after age 16, although presumably only in youths with access to automobiles. [@b11-arh-22-2-95] reported that among adolescents, the symptoms of hazardous use and alcohol-related legal problems were highly related to male gender, increased age, and symptoms of conduct disorder.
Another limitation is that some DSM--IV symptoms may have low specificity for adolescents---that is, their presence does not clearly distinguish among adolescents with different levels of drinking problems. For example, the development of some tolerance to alcohol's effects is likely a normal developmental phenomenon that occurs in most adolescent drinkers. The DSM--IV criteria define tolerance, in part, as the need to increase consumption by 50 percent or more to achieve the same effects. Thus, a need to consume three drinks to produce the same effect previously produced by two drinks would qualify as "tolerance" according to DSM--IV. Such a change in consumption at these relatively moderate drinking levels, however, likely occurs in most adolescent drinkers. [@b14-arh-22-2-95] found that tolerance was highly prevalent in adolescent drinkers with and without AUDs, even though this symptom was assigned only in subjects who consumed an average of five or more standard drinks per drinking occasion. Although marked tolerance to alcohol is an important aspect of alcohol dependence, difficulty in specifying and measuring this phenomenon makes it a problematic symptom for adolescents.
Other limitations of the DSM--IV criteria are related to the alcohol abuse category. The one-symptom threshold for the DSM--IV diagnosis of alcohol abuse, combined with the broad range of problems covered by the abuse symptoms, produces a great deal of heterogeneity among persons in this diagnostic category. A related issue is the lack of an accepted conceptual definition of alcohol abuse ([@b11-arh-22-2-95]). Furthermore, the mutually exclusive DSM--IV categories of alcohol abuse and alcohol dependence symptoms are not clearly distinguished either conceptually or empirically. Some of the abuse and some of the dependence symptoms measure impaired control over drinking in the face of negative consequences. [@b9-arh-22-2-95] found that measures of sensitivity, specificity and predictive power did not support the diagnostic distinction between abuse and dependence symptoms. Other investigators, however, have found results more supportive of the DSM--IV's categorization of alcohol abuse and alcohol dependence symptoms ([@b12-arh-22-2-95]).
A similar limitation of the DSM--IV AUD criteria among adolescents involves sequencing in the age of onset of alcohol abuse and alcohol dependence symptoms. Because alcohol abuse is usually considered as a relatively mild category relative to alcohol dependence, the onset of abuse symptoms would be expected to precede the onset of dependence symptoms. A study of sequencing in the age of symptom onset among adolescents, however, did not support the DSM--IV system ([@b16-arh-22-2-95]). The results suggested that DSM--IV alcohol symptoms developed in three distinct stages among adolescents, with some dependence symptoms occurring before some abuse symptoms, as follows:
- The first stage was characterized by three dependence symptoms (i.e., tolerance, drinking larger amounts or for a longer period of time than intended, and much time spent using alcohol) and two abuse symptoms (i.e., role impairment and social problems).
- The second stage was characterized by three dependence symptoms (i.e., unsuccessful attempts or a persistent desire to quit or cut down on drinking, reduced activities because of alcohol use, and continued use despite physical or psychological problems) as well as two abuse symptoms (i.e., hazardous use and alcohol-related legal problems).
- The third stage, which had the longest time to symptom onset, was characterized by the dependence symptom of withdrawal.
Finally, another apparent limitation of the DSM--IV criteria for AUDs is the existence of "diagnostic orphans"---that is, persons who exhibit one or two alcohol dependence symptoms and no alcohol abuse symptoms, who therefore do not qualify for a DSM--IV AUD. (For more information on diagnostic orphans, see [sidebar](#bx1-arh-22-2-95){ref-type="boxed-text"}, p. 100.)
###### Diagnostic Orphans: Adolescents With Alcohol Symptoms but Without a DSM--IV Alcohol Use Disorder
The *Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition* (DSM--IV) ([@b31-arh-22-2-95]) describes two alcohol use disorders (AUDs), alcohol abuse and alcohol dependence, whose symptoms do not overlap. DSM--IV defines alcohol abuse by the presence of at least one of four symptoms and alcohol dependence by the co-occurrence of at least three of seven symptoms within a 1-year period (see [table](#t1-arh-22-2-95){ref-type="table"}, p. 96). [@b34-arh-22-2-95] coined the term "diagnostic orphans" to describe adolescents with one or two of the alcohol dependence symptoms and none of the alcohol abuse symptoms, who therefore do not qualify for either a DSM--IV alcohol abuse or alcohol dependence diagnosis.
Epidemiological studies suggest that a substantial portion of adolescents are diagnostic orphans. [@b12-arh-22-2-95] found that 13.5 percent of high school students were diagnostic orphans. [@b32-arh-22-2-95] found that, among those students who had ever used alcohol or other drugs, 13 percent of 9th graders and 9.9 percent of 12th graders were diagnostic orphans. Diagnostic orphans also have been described in a representative household sample of adults ([@b33-arh-22-2-95]).
In a study of adolescents drawn from clinical and community sources, [@b36-arh-22-2-95] found that diagnostic orphans represented about 31 percent of regular drinkers (i.e., adolescents who drank at least once a month for at least 6 months) who did not qualify for a DSM--IV alcohol abuse or dependence diagnosis. Diagnostic orphans were equally common among male and female regular drinkers. The most common symptoms exhibited by this group were tolerance, drinking larger amounts or for a longer period of time than intended, much time spent using alcohol, and unsuccessful attempts or a persistent desire to quit or cut down on drinking. Diagnostic orphans reported levels of drinking and other drug use and rates of drug use disorders that were similar to those of adolescents with an alcohol abuse diagnosis and significantly higher than those of adolescent regular drinkers without any DSM--IV alcohol symptoms. These results do not support the distinction between those with alcohol abuse (who do have a DSM--IV AUD) and diagnostic orphans (who do not have a DSM--IV AUD).
It is possible to conclude that adolescent diagnostic orphans have "fallen through the cracks" of the DSM--IV system for AUDs. Alternatively, the results could be interpreted as indicating that the one-symptom threshold for the DSM--IV diagnosis of alcohol abuse is too liberal, and that some adolescents with alcohol abuse diagnoses should not be classified as having an AUD. More research is needed to address these issues. Adolescent diagnostic orphans likely are an important group for treatment and prevention efforts.
***---Christopher S. Martin and Ken C. Winters***
The Assessment of AUDs Among Adolescents
========================================
Clinicians and researchers use various approaches to assess alcohol problems in adolescents. The comprehensiveness of the assessment depends upon the purposes of the evaluation. One approach is the use of brief screening instruments---most commonly self-report questionnaires---to determine the possible presence of alcohol problems. (For more information on screening instruments, see [sidebar](#bx2-arh-22-2-95){ref-type="boxed-text"}, pp. 102--103.) If an initial screening indicates the need for further assessment, clinicians and researchers can employ diagnostic interviews to assign AUDs and to measure the nature and severity of alcohol problems. While this article emphasizes alcohol, most of these screening instruments and diagnostic interviews assess consumption patterns, problems and/or diagnoses for both alcohol and other drugs.
###### Screening Instruments for Adolescent Alcohol Use Disorders
In contrast to diagnostic interviews, which serve to establish a diagnosis of an alcohol use disorder (AUD), the aim of screening tools is to identify the *possible* presence of an alcohol problem or AUD. Thus, screening tools are used to determine whether a more complete assessment of a person's condition and treatment needs is appropriate. Screening tools are typically self-report questionnaires that employ scoring cutoffs. The use of screening tools requires caution. A score above the cutoff point does not necessarily indicate the presence of an AUD but merely suggests that a more detailed assessment should be performed. Similarly, a score below the cutoff point does not necessarily indicate the absence of an AUD, but merely suggests that this is likely.
The following sections summarize some of the available screening tools that have been used widely with adolescents. Some of these instruments assess both alcohol and other drug use and problems, whereas others are specific to alcohol.
***Screening Tools for Alcohol and Other Drug Use Disorders***
***Client Substance Index---Short (CSI--S).*** The CSI--S ([@b47-arh-22-2-95]) was developed and evaluated as part of a larger drug abuse screening protocol through the National Center for Juvenile Justice. The instrument is a 15-item yes/no questionnaire that is designed to identify juveniles within the court system who need additional assessment for alcohol and other drug problems. The CSI--S has shown good reliability. Scores on the CSI--S are consistent with other measures of adolescent alcohol and other drug problems, and the instrument discriminates among adolescent groups defined according to the severity of their criminal offenses.
***Drug and Alcohol Problem (DAP) Quick Screen.*** This 30-item questionnaire has been tested in a pediatric practice setting ([@b45-arh-22-2-95]). Studies have indicated that these items measure overall alcohol and other drug problem severity. The reliability and validity of the DAP Quick Screen, however, have not been evaluated.
***Drug Use Screening Inventory---Revised (DUSI--R).*** The adolescent version of the DUSI--R ([@b46-arh-22-2-95]) assesses alcohol and other drug use patterns as well as psychosocial functioning in different life areas using 159 true/false questions. This tool, which was developed from the same initial pool of items as was the Problem Oriented Screening Instrument for Teenagers (described below), yields scores on 10 functional adolescent problem areas: alcohol and other drug use, physical health, mental health, family relations, peer relationships, educational status, vocational status, social skills, leisure and recreation, and aggressive behavior/delinquency. The DUSI--R also includes a lie scale and has lifetime, past-year, and past-month versions. The adolescent version of the DUSI--R has shown good reliability and validity ([@b39-arh-22-2-95]). For example, the scores on certain DUSI--R subscales are related to alcohol and other drug use disorder diagnoses among adolescents.
***Perceived Benefit of Drinking and Drug Use.*** This 10-item questionnaire, which asks questions about the perceived benefits of alcohol and other drug use, was developed as a nonthreatening problem severity screen. It is based on the approach that beliefs about drug use, particularly the expected personal benefits of using alcohol and other drugs, tend to be associated with actual alcohol and other drug use. The validity of this instrument is supported by findings that in both school samples and adolescent psychiatric inpatient samples, test scores are related to other measures of alcohol and other drug use and associated problems ([@b42-arh-22-2-95]).
***Personal Experience Screening Questionnaire (PESQ).*** The PESQ is a 40-item questionnaire that provides measures of overall problem severity, alcohol and other drug use history, certain psychosocial problems, and response-distortion tendencies (i.e., the tendency to exaggerate or minimize responses about alcohol and other drug use behaviors) ([@b49-arh-22-2-95]). Cutoff scores indicating the need for further assessment have been established and validated for normal adolescents, juvenile offenders, and adolescents in addiction treatment.
***Problem Oriented Screening Instrument for Teenagers (POSIT).*** This 139-item yes/no questionnaire is part of the Adolescent Assessment and Referral System developed by the National Institute on Drug Abuse ([@b43-arh-22-2-95]). The POSIT was developed from the same pool of initial items as the DUSI--R (described previously). It addresses 10 areas of adolescent functioning (e.g., alcohol and other drug use, mental health, family relations, educational status, and aggressive behavior/delinquency). Cutoff scores indicating the need for further assessment have been established. Several investigators have reported evidence supporting the validity of the POSIT (e.g., [@b37-arh-22-2-95]).
***Substance Abuse Subtle Screening Inventory (SASSI).*** [@b41-arh-22-2-95] adolescent version of the SASSI consists of 81 questions pertaining to the severity of alcohol and other drug problems. The SASSI yields scores for alcohol problems, other drug problems, and defensiveness (i.e., the tendency to minimize or deny problems). Validity data indicate that the SASSI cutoff score suggesting "chemical dependency" corresponds highly with diagnoses of alcohol and other drug use disorders obtained upon treatment entry ([@b44-arh-22-2-95]).
***AUD-Specific Screening Tools***
***Adolescent Alcohol Involvement Scale (AAIS).*** The AAIS is a 14-item questionnaire that examines current and past alcohol consumption, drinking context, short-and long-term effects of drinking, and perceptions about drinking ([@b40-arh-22-2-95]). An overall score describes the degree of alcohol involvement. The AAIS scores are significantly related to AUD diagnoses, independent clinical assessments of severity, and parental reports. Cutoff scores have been established for 13- to 19-year-olds from both clinical and nonclinical samples.
***Adolescent Drinking Index (ADI).*** The ADI measures adolescent problem drinking using 24 items addressing alcohol problems related to psychological, physical and social functioning, as well as impaired control over drinking behavior. The instrument yields an overall severity score as well as two subscale scores reflecting self-medicating drinking and rebellious drinking. Studies have confirmed the reliability and validity of this tool. Scores on the ADI are associated with alcohol consumption levels and differ significantly among adolescents with different levels of alcohol problem severity ([@b38-arh-22-2-95]).
***Rutgers Alcohol Problem Index (RAPI).*** The RAPI is a 23-item questionnaire that focuses on consequences of alcohol use pertaining to family life, social relations, psychological functioning, delinquency, physical problems, and neuropsychological functioning ([@b48-arh-22-2-95]). Positive responses to the RAPI questions were found to correlate with AUD diagnoses.
***---Christopher S. Martin and Ken C. Winters***
Self-Reports and Their Validity
-------------------------------
Self-reports provide the most direct information about a person's alcohol and other drug use and associated problems, which is often not available from any other source. As such, self-reports are critical for diagnostic assessment. The validity of self-reported alcohol and other drug use behaviors, however, has been the subject of considerable debate. In addition to purposely distorting the truth, clients may provide inaccurate responses because of lack of insight, inattentiveness, or misunderstanding of a question. Furthermore, adolescents with alcohol and other drug problems are sometimes developmentally delayed in terms of their cognitive, social, and emotional functioning, which may affect their perception of problems and their willingness to report them. However, the literature does provide some support for the validity of adolescent self-reports of alcohol and other drug use and related problems ([@b13-arh-22-2-95]), as follows:
- A large proportion of youth in addiction treatment settings admit to the use of alcohol and other drugs and associated problems.
- Few adolescents in treatment endorse questions that indicate the faking of responses (e.g., high scores on "lie" scales of questionnaires or admitting to the use of a fictitious drug).
- The information provided by adolescents is usually in general agreement with information obtained from other sources (e.g., parents, peers, and archival records).
- The information provided in adolescent self-reports generally remains consistent over time.
Nevertheless, inconsistent self-reports have been noted in the literature. When adolescents were asked about infrequent past alcohol and drug use and when queried over a 1-year interval about the age of initial alcohol and other drug use, significant inconsistencies have been observed (e.g., [@b23-arh-22-2-95]). Furthermore, clinical experience suggests that many adolescents entering treatment tend to minimize the extent of their alcohol and other drug use and the severity of associated problems. In fact, some investigators have observed that adolescents sometimes report greater past alcohol and other drug use and related problems at treatment completion than at treatment entry (e.g., [@b26-arh-22-2-95]).
The complex issues regarding the validity of self-reports warrant further research. Researchers should consider the effects of how the information is gathered on the degree of self-disclosure. Several studies have indicated that questionnaires administered by computer and pencil-and-paper methods tend to yield equivalent responses concerning alcohol and other drug use behaviors. Some research with adults has shown slightly higher reports of alcohol and other drug use when the information is obtained through questionnaires as opposed to interviews. Similar studies have not yet been conducted with adolescents.
Parent Reports and Their Validity
---------------------------------
Another commonly used information source regarding adolescent alcohol and other drug use and associated problems are the youths' parents. Clinical experience has long suggested, however, that many parents cannot provide meaningful details about their child's alcohol and other drug use behaviors. Studies on this topic have yielded inconsistent results. In studies comparing diagnoses of alcohol and other drug use disorders based on parent reports with those based on self-reports, diagnostic agreement has ranged from 17 percent ([@b27-arh-22-2-95]) to 63 percent ([@b6-arh-22-2-95]). Another recent study of adolescents in addiction treatment compared self-reports and mother reports regarding a wide range of alcohol and other drug use behaviors ([@b29-arh-22-2-95]). In that study, the concurrence of self-reports and mother reports of alcohol and drug use and related consequences was modest. The findings showed that most often the mother under-reported alcohol and drug use behaviors compared to the adolescent's report.
Diagnostic Interviews
---------------------
Diagnostic interviews, in which clients are asked a set of predetermined questions, are considered by many researchers and clinicians to be the most comprehensive measures of alcohol and other drug use disorders. With the advent of definable diagnostic criteria for these disorders, such as those delineated in DSM--IV, diagnostic interviews can more precisely and reliably elicit the information needed to make a diagnosis. Furthermore, diagnostic interviews use standardized symptom definitions and question formats, which help minimize variability in responses. The use of followup questions provides important information that cannot always be obtained through the more rigid format of a questionnaire.
Diagnostic interviews are often described as either structured or semi-structured, based on the way in which they are administered and the degree of clinical judgment that the interviewer must employ when asking questions and when assigning symptoms and diagnoses. Highly structured interviews direct the interviewer to read verbatim a series of questions in a decision-tree format. In the decision-tree format, different responses lead to specific followup questions that assess the nature, persistence, duration, and clinical impact of alcohol- and drug-related problems. The interviewer rates each symptom as either absent or present, according to detailed written symptom definitions. Most structured interviews can be administered with acceptable reliability by a well-trained lay person. Semi-structured interviews require the interviewer to elicit an initial response and then determine, through additional unstructured probing, whether a symptom is present or absent. Such interviews allow considerable latitude in adapting questions to suit the respondent, and therefore usually require more advanced training in assessment. In terms of consistency of results across different interviewers, semi-structured interviews are often at a disadvantage compared with structured interviews, because they involve greater clinical judgment in scoring the responses. Many professionals believe, however, that this format can produce more comprehensive information than can fully structured interviews.
Selecting Diagnostic Interviews for Adolescent AUDs
---------------------------------------------------
Given the considerable amount of expertise, time, and resources needed to develop sound diagnostic instruments, it is generally most cost-effective to use interviews whose properties have already been assessed. Many recent instruments have been extensively studied and have proven to be reliable and valid. Several handbooks and review articles are available that can guide the selection of an appropriate interview (e.g., [@b1-arh-22-2-95]; [@b17-arh-22-2-95]).
A number of criteria should be considered when selecting a diagnostic interview for adolescent AUDs and other drug use disorders, including the following:
- The diagnostic interview should have demonstrated adequate measurement properties of reliability and validity. Reliability refers to the consistency of results across different interviewers or assessments. Validity refers to whether an interview measures what it is supposed to measure. Validity is often assessed by comparing interview results with other measures that are known to accurately assess diagnoses.
- Assessment should preferably involve both lifetime and recent (e.g., within the past year) time frames, because a lifetime perspective can provide important information about the course and chronicity of a disorder, while the profile of recent symptoms has obvious clinical, research, and diagnostic value.
- For positive responses, the instrument should contain questions related to the ages of both onset and offset of symptoms.
- The interview should provide questions to assess whether a problem was sufficiently persistent, recurrent, or clinically significant to warrant a positive symptom rating.
- Unless some of the information is provided by other assessment tools, the diagnostic interview should assess a wide variety of alcohol-and drug-related behaviors, including alcohol and other drug use, problems not contained in the diagnostic criteria, prior treatment experiences, family and peer alcohol and drug use, school functioning, and mental health status.
- The interview's ease of administration, length, and training requirements must be compatible with the assessment goals. For example, researchers generally require interviews that efficiently yield reliable and detailed data. In contrast, some clinicians may be more interested in measures that are not excessively time consuming and require only modest training to administer.
Commonly Used Diagnostic Interviews for Adolescents
===================================================
A number of diagnostic interviews can be used to assess adolescent alcohol and other drug use disorders. Some of those instruments focus primarily on alcohol and other drug use disorders, whereas others are general psychiatric interviews that contain specific sections for assessing those disorders. The following sections summarize some of those diagnostic interviews. The list emphasizes interviews that have been adapted for DSM--IV criteria and are widely used in the United States.
General Psychiatric Interviews
------------------------------
### Diagnostic Interview for Children and Adolescents (DICA)
The DICA is a long-standing structured psychiatric interview. A revised version incorporating the DSM--IV criteria now exists ([@b20-arh-22-2-95]). Although no studies have specifically evaluated the DICA's measurement properties regarding AUDs, general findings indicate that this instrument is reasonably reliable and valid.
### Diagnostic Interview Schedule for Children (DISC--C)
The structured DISC--C has undergone several adaptations, the most recent of which is based on the DSM--IV ([@b22-arh-22-2-95]). A separate version of the interview exists for parents. Both the child and the parent versions of the DISC have shown good sensitivity in identifying youth who have received an independent medical diagnosis of an alcohol or drug use disorder ([@b8-arh-22-2-95]). However, the DISC--C has shown only modest reliability for DSM--III--R alcohol and other drug use disorders ([@b21-arh-22-2-95]).
### Kiddie SADS (K--SADS)
This popular semi-structured interview, which is organized around the Research Diagnostic Criteria, is a child and adolescent version of the Schedule for Affective Disorders and Schizophrenia. Symptoms of alcohol and other drug use disorders are contained in the version of the interview that addresses lifetime symptoms (K--SADS--E) ([@b18-arh-22-2-95]). A DSM--IV version now exists (K--SADS--E--5) ([@b19-arh-22-2-95]). Reliability and validity studies of the K--SADS--E provide no data regarding alcohol and other drug use disorders, so the use of this interview among youth with alcohol and drug problems should proceed cautiously.
### Structured Clinical Interview for the DSM (SCID)
The SCID is a structured interview developed to assesses psychiatric disorders according to DSM criteria in adults. The SCID provides specific operational definitions for each symptom and verbatim questions in a decision-tree format. The interviewer rates each symptom as absent, subclinical, or clinically present. The SCID is used widely, and the DSM--III--R section on alcohol and other drug use disorders has shown good reliability with adults. [@b14-arh-22-2-95] modified the DSM--III--R version of the drug use disorders section of the SCID ([@b24-arh-22-2-95]) to assess DSM--IV alcohol and other drug use disorders among adolescents. Symptoms and diagnoses established with this version of the SCID have shown good concurrent validity (i.e., are associated with measures of drinking and problem severity assessed at the same time). In addition, preliminary analyses have suggested moderate to high agreement among interviewers (i.e., inter-rater reliability).
Interviews Focusing on Alcohol and Other Drug Use Disorders
-----------------------------------------------------------
### Adolescent Diagnostic Interview (ADI)
The ADI assesses the symptoms of alcohol and other drug use disorders as defined in both the DSM--III--R and DSM--IV. The ADI also measures sociodemographic information; alcohol and other drug use history; and psychosocial functioning, including mental health. The ADI's reliability and validity are moderate to high ([@b28-arh-22-2-95]).
### Customary Drinking and Drug Use Record (CDDR)
The CDDR is a structured interview that measures alcohol and other drug use for both recent (i.e., past 3 months) and lifetime periods, the presence of DSM--III--R and DSM--IV dependence symptoms for alcohol and other drug use disorders, and several negative consequences that are similar to DSM--III--R and DSM--IV alcohol and other drug abuse symptoms. The CDDR has high reliability across all major content domains and good concurrent validity. The CDDR has been found to discriminate between youth in the general population and those in treatment and produces results consistent with those of other diagnostic instruments ([@b4-arh-22-2-95]).
Summary
=======
Any diagnostic system applied to adolescent alcohol problems should reflect current knowledge of the nature and development of those problems. The diagnostic criteria for AUDs in the DSM--IV, however, were developed largely from research and clinical experience with adults. Although the number of studies is small, the available data suggest important limitations of the DSM--IV AUD criteria when applied to adolescents. More research is needed to evaluate potential changes in diagnostic criteria that may better represent the nature and development of adolescent alcohol problems. It is an open question whether future changes in diagnostic criteria for AUDs can provide a unified system that is equally valid for both adults and adolescents, or whether adolescent-specific clinical and research criteria for AUDs should be developed.
Research has generally supported the validity of self-reports of alcohol and other drug problems obtained from teenagers in clinical settings. Future research should identify characteristics of the individual adolescent and of the setting in which the information is obtained that influence the validity of self-reports. Clinicians and researchers have numerous options when selecting diagnostic instruments and screening measures for adolescent AUDs. Many of these instruments have favorable reliability and validity, and several reviews provide an overview to help guide the choice of an appropriate assessment instrument (e.g., [@b1-arh-22-2-95]; [@b17-arh-22-2-95]).
Work on this article was supported by National Institute on Alcohol Abuse and Alcoholism (NIAAA) grants P50--AA--08746 to the University of Pittsburgh, AA--00249 to Christopher S. Martin, and by National Institute on Drug Abuse grants DA--04434 and DA--05104 to Ken C. Winters.
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The plateau zokor, Myospalax baileyi, is small subterranean rodent that inhabits the Qinghai-Tibet Plateau at elevations ranging from 2000 to 4200 m[@b1]. Zokors spend 85--90% of their lives in underground nests, which are usually greater than 2 m deep for females and approximately 1.5 m deep for males[@b2]. The depths of the burrows means that the zokor experiences even lower levels of oxygen than those found on the surface of the Qinghai-Tibet Plateau and higher levels of carbon dioxide[@b3].
The plateau zokor exemplifies a successful adaptation to an extreme environmental condition. Compared to pikas (*Ochotona curzniae*) and Sprague-Dawley rats (*Rattus norvegicus*), the plateau zokor exhibit remarkably increased red blood corpuscle counts and contents of hemoglobin and myoglobin[@b3][@b4][@b5][@b6]. In contrast, they have markedly lower pulse rates[@b6], decreased lactate dehydrogenase activity, and lower 2,3-bisphosphoglycerate-content in their blood compared with pikas, mice and rats that live in the same area[@b3]. The oxygen pressure in the arterial blood of the plateau zokor is about 1.5 times higher than that of pikas and rats, and about 0.36 and 0.26 times, respectively, greater in venous blood[@b3]. Partial pressure for carbon dioxide in arterial and venous blood of the plateau zokor is 1.5-fold and 2.0-fold higher than in rats and pikas, respectively[@b3]. The difference in oxygen saturation of arterial to venous blood was about 2 and 4.5-fold higher in plateau zokor than in pikas and rats, respectively[@b3]. However, oxygen saturation of the plateau zokor is nearby 5.7 and 9.3 times lower in venous blood than that of pikas and rats, respectively[@b3]. These observations suggest that the plateau zokor has a strong capability to acquire oxygen from their hypoxic-hypercapnic environment.
To elucidate adaptations, and their genetic bases, of animals to extreme environments is a primary mission of modern evolutionary biology[@b7]. Recently developed bioinformatics help us comparatively analyze massive amounts of data generated by next-generation sequencing technologies at differential scales (e.g. genomics, transcriptomics and proteomics) across diverse species[@b8][@b9][@b10]. High altitude environments, especially low temperature, low oxygen density and intense UV radiation, result in harsh physiological challenges for animals[@b11][@b12]. A series of studies suggested that genes involved in the hypoxia-inducing factor (HIF) signaling pathway play key roles in the plateau adaptation of mammals[@b13][@b14][@b15][@b16]. Here, we examined the plateau zokor, a small endothermic mammal that experiences dual stress from high altitude hypoxia and sealed burrow hypoxia, and thus may become an excellent model for studying adaptations to low oxygen density and hypercapnic concentrations. Revealing the genetic bases of these adaptations in the plateau zokor therefore should be helpful to develop a comprehensive understanding of plateau adaptations of whole endotherms.
The naked mole rat, *Heterocephalus glaber*, is another strictly subterranean rodent[@b17]. Like the plateau zokor, they also live in full darkness, at low oxygen and high carbon dioxide concentrations and possess extremely similar habitats and physiological characteristics[@b18]. Thus, the plateau zokor and the naked mole rat represent a pair of functionally convergent homoplasies (evolutionary analogues) in adapting to the hypoxic environment of sealed burrows[@b19]. Utilization of the draft genome of the naked mole-rat[@b18] together with the transcriptomes of multiple tissues from the plateau zokor should help us deeply explore genetic adaptations allowing the survival of these rodent species to harsh subterranean environments.
Results
=======
*De novo* assembly and annotation of transcriptomes
---------------------------------------------------
We attained about 272.45 million (25.01 Gb sequence data) raw reads for five tissues (brain, liver, kidney, skeletal muscle and retina) of a plateau zokor. About 256.10 million (19.67 Gb) high-quality reads remained after quality control. The transcriptomes of the five tissues (brain, liver, kidney, skeletal muscle and retina) were pooled to generate an improved *de novo* assembly. The contig N50 parameters of the *de novo* assembly of the pooled data were superior to the single tissue transcriptome *de novo* assemblies, resulting in the identification of 208,451 non-redundant transcripts with a contig N50 length of 2433 base pairs ([Table 1](#t1){ref-type="table"}). The transcripts were aligned to the NCBI Non-Redundant Protein Database (NR database) using Blastx program (E-value 1e^−5^), allowing the annotation of 73,116 transcripts, based on 40,528 NCBI non-redundant proteins, including 34,523 (\~85.2 %) that mapped to 19,736 NCBI genes ([Supplementary Dataset 1](#S1){ref-type="supplementary-material"}). NCBI gene2go database (<ftp://ftp.ncbi.nih.gov/gene/DATA>) was then utilized to retrieve the GO ids for each of the annotated genes. In summary, 56 GO terms, mainly covering biological GO categories at three ontologies levels (23 GO terms in Biological Process, 15 GO terms in Molecular Function and 18 GO terms in Cellular Component), were identified using WEGO[@b20], a web tool for plotting GO annotations. Our analyses of GO terms generally represented the main biological GO classification and ensured the integrity of the downstream functional analyses of the candidate genes.
Predicted orthologous genes
---------------------------
We identified 8,217 genes that were one-to-one orthologous gene pairs between the plateau zokor (M. baileyi), and each of rat (*R. norvegicus*), kangaroo rat (*Dipodomys ordii*), guinea pig (*Cavia porcellus*), naked mole-rat (*H. glaber*) and human (*Homo sapiens*). After removing low quality and short sequences, 8,020 high-confidence one2one genes were retained for the downstream analyses. Our final dataset, therefore, included six species and 8020 genes.
Phylogenetic tree and divergence time
-------------------------------------
By integrating the 8020 single-copy orthologs among the six species (M. baileyi, *R. norvegicus*, *D. ordii*, *C. porcellus*, *H. glaber* and *H. sapiens*) and one-to-one orthologous gene pairs from *H. sapiens* and *Oryctolagus cuniculus*, downloaded from ENSEMBL 75 BIOMART database, a total of 7011 high-confidence single-copy orthologous gene pairs were obtained from the seven species (M. baileyi, *R. norvegicus*, *D. ordii*, *C. porcellus*, *H. glaber*, *O. cuniculus* and *H. sapiens*). After multiple sequence alignment (MSA) and quality trimming, 7011 one-to-one orthologous genes were concatenated into a single supergene to extract four-fold degenerate sites for construction of a phylogenetic tree using the Mrbayes_v3.0 software[@b21]. High Bayesian posterior probabilities in this constructed phylogenetic tree have well resolved the phylogenetic relationships between the plateau zokor and other rodent species ([Fig. 1a](#f1){ref-type="fig"}). Based on this reliable phylogenetic tree, we estimated the divergence time of each node using BEAST v1.7.5[@b22]. As expected, the plateau zokor groups with the rat with an ancestor approximately \~52 million years ago, whereas the ancestor of the plateau zokor, rat and kangaroo rat diverged from the ancestor of the guinea pig and naked mole-rat approximately \~55 million years ago ([Fig. 1b](#f1){ref-type="fig"}). Our estimated divergence time are similar to those of a previous report[@b23].
Evolutionary rate in plateau zokor
----------------------------------
We utilized the free ratio model (M1) in PAML4[@b24] to calculate the selective constraints acting on each of the orthologous genes using our accepted tree topology ([Fig. 2a](#f2){ref-type="fig"}). Because outlier-genes with larger *d*~*N*~/*d*~*S*~ may generate deviations in evaluating the overall selective constraint in species, our dataset was filtered to remove genes with *d*~*N*~/*d*~*S*~ \> 4, yielding a set of 7117 othologs shared by the plateau zokor and rat lineages. As saturation of substitutions influences the estimates of *d*~*N*~*/d*~*S*~, we used previously described methods[@b25] to test for saturation and plotted the ratio of transitions and transversions to *d*~*S*~; these relationships were linear up to the *d*~*S*~ of 1, thus orthologues with *d*~*S*~ \> 1 were excluded from further analyses ([Fig. 3](#f3){ref-type="fig"}). After the saturation test, 7077 orthologous genes were remained, with these highly reliable genes used to evaluate the overall selective constraints in these two related rodent species. The low overall *d*~*N*~/*d*~*S*~ mean value in the plateau zokor (0.0904) and the rat (0.1360) demonstrates that the majority of genes experienced purifying selection on both lineages. Compared to the rat, the plateau zokor has a significantly lower mean *d*~*N*~/*d*~*S*~ value at the genomic level (Wilcoxon rank sum test, *P* \< 2.2E--16) ([Fig. 2b](#f2){ref-type="fig"}), suggesting that genes evolved a lower rate in the plateau zokor compared to rat since their split from a common ancestor. The mean d~*N*~/*d*~*S*~ value (0.1360) for the rat calculated in this study is very similar to that of a previous study (0.137)[@b26], suggesting that our analysis is reliable.
Accelerated evolution of genes in specific GO categories
--------------------------------------------------------
Average *d*~*N*~*/d*~*S*~ values for genes in each GO category were calculated based on our reconstructed tree topology ([Fig. 2a](#f2){ref-type="fig"}) using the free ratio model (M1) implemented in PAML4[@b24]. Among the Go categories, 95 harbor significantly higher average *d*~*N*~*/d*~*S*~ values in the plateau zokor lineage compared to the rat (*P* \< 0.05, binomial test), with these categories including those involved in energy metabolism including glucose transport (GO:0015758, P = 6.58E--15), regulation of glucose transport (GO:0010827, P = 3.09E--11), mitochondrion (GO:0005739, P = 1.34E--06), fatty acid beta-oxidation (GO:0006635, P = 0.0004) ([Fig. 2c](#f2){ref-type="fig"} and [Supplementary Dataset 2](#S1){ref-type="supplementary-material"}). In contrast, the rat possessed only 65 categories exhibiting significantly higher *d*~*N*~*/d*~*S*~ values compared to the plateau zokor, which included those for collagen (GO:0005581, P = 3.22E--58) and protein polyubiquitination (GO:0000209, P = 1.50E--06).
Positive selection
------------------
The improved branch-site model in PAML4[@b24] was used to detect positively selected genes (PSGs) among the 8,020 orthologous genes along the plateau zokor branch using our reconstructed tree topology ([Fig. 2a](#f2){ref-type="fig"}). A total of 346 PSGs (4.3%) were identified with signals of positive selection ([Supplementary Dataset 3](#S1){ref-type="supplementary-material"}). G:Profiler[@b27] enrichment analyses showed that the candidate PSGs were significantly enriched into categories that included response to stress (P = 4.28E--16), respiratory gaseous exchange (P = 9.31E--05), blood vessel development (P = 1.54E--03) and ATPase activity (P = 1.32E--04) ([Supplementary Dataset 4](#S1){ref-type="supplementary-material"}). These categories appear to be biologically relevant to adaptation to high altitudes. According to previous studies, angiogenesis is largely initiated by the HIF pathway and is a crucial response to hypoxia[@b28][@b29]. Here, 12 PSGs (*ANGPTL3*, *PRCP*, *SEC24B*, *EPHA1*, *MCAM*, *KAT6A*, *MYH9*, *CYSLTR2*, *MYLK*, *SPINT1*, *PPAP2B* and *STRA6*) involved in blood vessel development were identified in the plateau zokor and they may represent adaptive responses to hypoxia. Since ATPase genes have a role in providing energy in conditions of low PO~2~, and show evidence of adaptation in the Tibetan antelope (*Pantholops hodgsonii*)[@b30], another sympatric plateau species, PSGs associated with ATPase activity also may be adaptive to supply energy in the extremely hypoxic sealed burrow environment at high altitudes for the plateau zokor.
In particular, 5 PSGs (*SFTPA2*, *CSF2RB*, *MAPK8IP3*, *PASK* and *MTG2*) involved in respiratory gaseous exchange were over-represented. This category was not found to over-enriched in PSGs in studies of other plateau species, such as Tibetan antelope (*P. hodgsonii*)[@b30], Tibetan boar (*Sus scrofa*)[@b31] and yak (*Bos grunniens*)[@b16], thus these genes may be specific to the plateau zokor and involved in the adaptive evolution of the respiratory system response to hypoxic-hypercapnic environment. In addition, tissue-specific analyses of the expression of PSGs showed the tendency that the brain hosted a higher proportion of PSGs with highest expression than any other tissue (kidney, retina, muscle and liver) ([Fig. 2d](#f2){ref-type="fig"}).
Convergent/parallel evolution
-----------------------------
Adaptive evolution at the molecular level also can be studied by detecting convergent/parallel evolution at the amino acid sequence level[@b32]. Since both the plateau zokor and naked mole-rat live in sealed burrows and possess extremely similar phenotypic physical characteristics, we detected convergent/parallel evolved genes to reveal possible adaptive mechanisms on these two lineages. In summary, no convergently evolved genes were identified between the plateau zokor and naked mole-rat, however 787 candidate parallel evolved genes, including 700 having a single parallel amino acid change, 72 with two parallel amino acid substitutions and 15 having multiple (\>2) parallel changes were identified on the two branches ([Supplementary Dataset 5](#S1){ref-type="supplementary-material"}).
Our GO enrichment analyses of these candidate PEGs showed that they were significantly overrepresented in GO categories such as response to hypoxia (P = 3.27E--04), oxygen homeostasis (P = 5.85E--03), erythrocyte homeostasis (P = 3.25E--02), angiogenesis (P = 5.63E--08), blood vessel development (P = 1.69E--10), respiratory tube development (P = 8.75E--03) and ATPase activity (P = 6.79E--07) ([Supplementary Dataset 6](#S1){ref-type="supplementary-material"}). Some of these GO categories are similar to the GO categories identified in the PSG analysis, suggesting that positively selected and parallel evolved genes exist in the same pathways (e.g. blood vessel development, respiratory tube development and ATPase activity) hinting that these genes related to energy metabolism and the development of the respiratory tube may contribute vital roles to adapting to the extremely high-elevation hypoxic burrow environment. However, in contrast, some significant GO categories (e.g. erythrocyte homeostasis, response to hypoxia and oxygen homeostasis) only were specifically enriched in the PEGs list and these specific GO categories possibly directly contribute to the burrow adaptation at high altitude because of their functional importance. Among these categories, 12 genes (*EPAS1*, *SLC29A1*, *MYO5B*, *AJUBA*, *TGFBR3*, *VASN*, *ACAA2*, *PINK1*, *SIRT1*, *SIRT2*, *SOD2* and *NARFL*) are involved in response to hypoxia; 5 genes (*EPAS1*, *DNASE2*, *ADAR*, *TGFBR3* and *SMAP1*) are involved in erythrocyte homeostasis and two genes (*SOD2* and *NARFL*) are involved in oxygen homeostasis. Since the response to hypoxia GO category is generally associated with high-elevation adaptation[@b15][@b33], this suggests that their parallel evolved sites may not have appeared by random, but instead possess important functions, although the functions of these sites remain unclear. The gene *AJUBA* is an example, where an examination of homologous coding sequences in diverse mammals showed that the parallel evolved site (Glu76Asp) in the plateau zokor (is homologous to 86Glu site in human) occurs at an otherwise extremely conserved site in other species ([Fig. 4](#f4){ref-type="fig"}).
Protein-Protein Interaction (PPI) Network
-----------------------------------------
As mapping of candidate genes to protein-protein interaction (PPI) networks is essential to understand their biological functions, we combined the PSGs and PEGs, resulting in a total of 1,133 candidate genes, and mapped them to the PPI network database (InnateDB)[@b34] to further explore their functional roles. Sub-networks of more than 5 nodes were retained and our analyses resulted in the identification of 935 seed proteins (queries) (82.5%) mapped to sub-networks consisting of 5,954 nodes (proteins) and 15,871 edges (protein-protein interactions) ([Fig. 5](#f5){ref-type="fig"}). Of the 23 genes with the highest degree of interaction (\> 100), 14 (*FN1*, *ITGA4*, *SIRT1*, *SHC1*, *ACTB*, *IQCB1*, *XRCC5*, *HTT*, *CASP8*, *XIAP*, *EPS15*, *PSMD1*, *EPAS1* and *SIRT2*) underwent parallel evolution, with 3 of these (*RAF1*, *EEF2 and MYH9*) also experiencing positive selection. Our results suggest that PSGs and PEGs may play central roles in maintaining these sub-networks.
Discussion
==========
The plateau zokor is a specialized species endemic to the Qinghai-Tibet Plateau[@b35], which may have evolved a series of physiological strategies to counter the effects of hypoxia[@b3] and can be regarded as an alternative evolved pattern to study adaptation to high elevation. Here, we report the transcriptomic data for a plateau zokor with a total of 20,8451 non-redundant transcripts from pooled multiple-tissue data with a Contig N50 attained length of 2,433 base pairs. The length of our sequences allowed the high-confident identification of orthologous genes, which could then be used to perform evolutionary analyses for plateau adaptation. Our analyses meaningfully contribute to the study of the genetic bases of adaptive evolution to high elevation in plateau mammalian species.
After diverged from their ancestor, compared to the rat lineage, overall the plateau zokor displayed a slower mean evolutionary rate in genomic level, although it possesses genes with more rapid evolution in GO categories related to energy metabolism. This result supports the hypothesis that highland adaptation of endothermic species mainly involves several features, including expanded gene families, elevated evolutionary rates and positive selection on genes involved in hypoxia and energy metabolism[@b16][@b30][@b36]. Significantly accelerated evolution of genes in energy metabolism also support the conclusion that the plateau zokor obtains most of its energy by aerobic oxidation instead of anaerobic glycolysis[@b37]. Adaptive evolution at the molecular level was well reflected in our screens for positive selection that identified genes involved adaptation[@b38]. In the plateau zokor lineage, genes involved in energy metabolism, such as ATPase activity experienced positive selection and thus we suggest that this category of genes may be relevant to adaptation to high altitudes. PSGs associated with blood vessel development were also over-represented and may contribute their function to hypoxic responses as angiogenesis is usually considered to be a target of the HIF pathway[@b28][@b29]. Several genes are of particular interest due to their functional implications. For example, *ANGPTL3* stimulates endothelial cell adhesion and migration via integrin and induces blood vessel formation *in vivo*[@b39]. This gene experienced positive selection suggesting an important function in angiogenesis. Another gene, *PRCP* (serine protease prolylcarboxypeptidase), might influence systemic blood pressure and vascular anticoagulation and contributes to cell proliferation and angiogenesis[@b40], promoting the health or impair the repairs of blood vessels.
Our study also identified a potentially new GO category for plateau adaptation, respiratory gaseous exchange, as it was significantly enriched in our PSGs list. This category was not found in previous analyses for positive selection in other plateau species (yak (*B. grunniens*), Tibetan boar (*Sus scrofa*), Tibetan antelope (*P. hodgsonii*) and ground tit (*Parus humilis*))[@b16][@b30][@b31][@b36], suggesting that it might be specific to the plateau zokor. For example, the candidate genes *SFTPA2* and *CSF2RB* are involved in surfactant structure and the regulation of surfactant homeostasis in the lung[@b41][@b42]. These genes may explain at molecular level why the plateau zokor possess a large capacity for lung gas diffusion. The number of PSGs with tissue-specific highest expression in brain is higher than in other tissues (e.g. liver, kidney, muscle and retina), suggesting that PSGs prefer to be highly expressed in the brain, since the brain might contribute more than other tissues to the response to hypoxia. We know for humans that the brain has high energy requirements, and consumes nearly 20% of the oxygen and 25% of the glucose of the entire body[@b43]. Thus, in the plateau zokor, the brain may similaily consume a high proportion of the energy and oxygen.
Adaptive evolution at the molecular level can also be studied by detecting convergent/parallel evolution at the amino acid sequence level[@b32]. Species living in similar ecological environments could be shaped by convergent evolution to form physiological or morphological similarities[@b44]. The naked mole-rat also lives in a strictly subterranean habitat that involves full darkness and low oxygen and high carbon dioxide concentrations[@b18][@b45][@b46]. Our GO functional analyses showed parallel evolved genes were significantly enriched in the following GO categories: response to hypoxia, ATPase activity and angiogenesis. These GO categories illustrate that in order to survive their shared harsh environment, the plateau zokor and naked mole-rat may experience convergent functional improvements in the supply of optimum oxygen levels by influencing the function of genes related to the hypoxia response, energy mechanism and angiogenesis. For example, the gene *EPAS1* encodes HIF-2α, which primarily regulates the production of erythropoietin (EPO). In turn, EPO is a key hormone that stimulates and regulates the production of erythrocytes[@b47]*. EPAS1* is widely reported as a candidate adapted gene for living at high elevations[@b13][@b15][@b48]. Additionally, analysis of Protein-Protein interaction (PPI) networks also demonstrates that *EPAS1* possesses strong interactions with other proteins. Taken together, *EPAS1* in the plateau zokor and naked mole-rat lineages exhibited parallel changes of amino acid sequences that are likely not due to random chance, but are an adaptive mechanism in response to decreased oxygen in their sealed environments. Another gene, *AJUBA*, is also involved in response to hypoxia and is a key regulator of the hypoxic response regulating the cellular and physiological changes to oxygen levels by controlling the degradation, and transcriptional activity of hypoxia-inducible transcriptional activity, of hypoxia-inducible transcription factors (HIFs)[@b49]. In particular, this gene hosts a highly conserved parallel evolved site (E76D) (identified in comparison of homologous sequences among multiple species) that suggests that it is also associated with highland adaptation, although the function of this particular amino acid substitution is unknown. Future functional experiments will be necessary to validate its importance. Thus, the plateau zokor exhibits multiple strategies to adapt to harsh highland environments. Although we did not directly examine the role of differentially expressed genes, due to the limitations of our sampling strategy, changes in gene expression likely also play important roles in the phenotypic evolution of this species[@b50].
Materials and Methods
=====================
Methods were carried out in accordance with approved guidelines.
Sample collection and library preparation
-----------------------------------------
The care and treatment of the plateau zokor comply with the guidelines for the National Care and Use of Animals approved by the National Animal Research Authority (P. R. China). Experimental protocols involving live animals were approved by the Ethics and Experimental Animal Committee of Kunming Institute of Zoology, Chinese Academy of Science, China (Approval ID: SYDW-2015012).
A plateau zokor individual was collected in Qinghai Province and after euthanasia, the brain, liver, kidney, skeletal muscle and retina tissue were quickly biopsied, placed in liquid nitrogen, and stored at −80 °C upon return to the laboratory. Total RNA from these tissues was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). RNA purifications were performed using an RNeasy Mini Kit (Qiagen, Chatsworth, CA). Library constructions from the brain, liver and kidney of a plateau zokor (no replicates) followed the Illumina Genome Analyzer II RNA sample preparation kit (GA IIx, Illumina, Inc.) and Library preparation for skeletal muscle and retina (no replicates) were according to the Illumina Hiseq2000 RNA sample preparation kit (Illumina, San Diego, CA). All original data were deposited in the NCBI Sequence Read Archive database (Accession Number: SRP057676; Alias: PRJNA282349).
*De novo* assembly and annotation of transcriptomes in plateau zokor
--------------------------------------------------------------------
Sequencing adaptors used for cDNA library construction were trimmed using Cutadapt[@b51] (version_1.2.1), which removed adapter sequences from the high-throughput sequencing reads. We employed Btrim64[@b52] (version_0.1.0) to delete regions with average quality scores of less than 20 and impose a minimal length equal or great than 20 bp. High quality pooled paired end reads from multiple tissues were *de novo* assembled using Trinity[@b53] (version_2013_08_14) by default parameters. Transcripts of pooled transcriptome were aligned to the NCBI Non-redundant protein database (NR) using BLASTX program (E-value 1E^−5^) to produce annotation results. NCBI gene2accession and gene2go databases (<ftp://ftp.ncbi.nih.gov/gene/DATA>) were then used, respectively, to retrieve gene ID and GO ID for each annotated gene. WEGO software[@b20] (<http://wego.genomics.org.cn/cgi-bin/wego/index.pl>) was used to perform functional annotation analyses at three gene ontology levels (Biological Process, Molecular Function and Cellular Component).
Prediction of open reading frames
---------------------------------
Coding sequences of the plateau zokor were predicted based on the assumption that the longest open reading frame in the longest transcript per gene had a greatest chance of being a protein-coding region. GETORF in EMBOSS[@b54] (version_6.4.0) was applied to obtain the nucleic sequences between the start and stop codons and limit the minimum size of a fragment to 120 bp. The transeq program in EMBOSS[@b54] (version_6.4.0) was then used to obtain translated protein sequences.
Identification of one-to-one orthologous genes
----------------------------------------------
We used the longest protein sequences per gene to perform a best reciprocal hit (BRH) (E-value 1E^−5^) methods to identify one-to-one orthologous genes among six species: plateau zokor (M. baileyi), rat (*R. norvegicus*), kangaroo rat (*D. ordii*), guinea pig (*C. porcellus*), naked mole-rat (*H. glaber*), and human (*H. sapiens*). Protein sequences of the naked mole rat were downloaded from the naked mole rat database (<http://mr.genomics.org.cn/page/species/index.jsp>). Genomic protein sequences of the rat, kangaroo rat, guinea pig and human were downloaded from ENSEMBL 75 (http://www.ensembl.org/). Predicted orthologous genes were submitted to multiple alignments using PRANK[@b55][@b56][@b57] (Parameters: −f = fasta -F -codon -noxml -notree -nopost). We aligned the longest ORFs for the longest transcript-pairs across the six species. Gblocks[@b58][@b59] (version_0.91b; Parameters: −t = c −b3 = 1 −b4 = 6 −b5 = n) was employed to reduce the rate of false positive predictions by filtering out sequencing errors, incorrect alignments and no-orthologous regions based on codons. After trimming, we removed alignment lengths shorter than 10 bp to obtain one2one orthologous genes.
Phylogenetic tree and divergence time
-------------------------------------
To establish phylogenetic relationship of the plateau zokor to other rodent mammals, one-to-one orthologous gene pairs between the human (*H. sapiens*) and rabbit (*O. cuniculus*) were downloaded from ENSEMBL 75 BIOMART database and added to dataset of one-to-one orthologous described above, generating one-to-one orthologous gene pairs for seven species. After multiple sequence alignments and trimming by the programs PRANK[@b55][@b56][@b57] and Gblocks[@b58][@b59], respectively, these single-copy orthologous genes were concatenated into one supergene and 4-fold degenerate sites were extracted and used to construct a phylogenetic tree. Modeltest_0.1[@b60] was used to select the best substitute model and Mrbayes_v3.0[@b21] was utilized to reconstruct the phylogenetic tree. Chain length was set to 10,000,000, with the first 1,000 samples treated as burned in, and the other parameters were set as defaults. Nodal ages within rodent mammals were evaluated, along with their 95% confidence intervals (CIs), from our 4-fold degenerate site data from the supergene sequence using the Bayesian relaxed molecular clock method implemented in BEAST v1.7.5[@b22]. Chain length was set to 10,000,000, with the first 1,000 samples treated as burned in. We utilized an indirect estimate[@b18] of the divergence time as a calibration point with the split of the ancestor of rabbit from the ancestor of mouse, rat and naked mole-rat to estimate the internal nodal ages.
Evolutionary rate and accelerated evolution of GO categories
------------------------------------------------------------
Free ratio model (M1) (Parameters: model = 1, NSsites = 0, fix_omega = 0, omega = 1) in PAML4[@b24] was used to calculate selective constraint of each orthologous gene using our constructed tree topology. Genes with *d*~*N*~/*d*~*S*~ \>4 were first filtered and then a saturation test was performed to produce a final dataset to evaluate the overall selective constraints in the tested species. GO term information was downloaded from ENSEMBL (<http://www.ensembl.org>), and only those GO categories with more than 20 orthologs were included in our analyses. Lineage-specific mean *d*~*N*~/*d*~*S*~ values were estimated by concatenating alignments from all orthologs for each GO category. Relatively accelerated evolutionary GO terms were identified using a binomial test according to a previous study[@b26].
Positive selection analyses
---------------------------
Branch-site model (Parameters: Null hypothesis: model = 2, NSsites = 2, fix_omega = 1, omega = 1; Alternative hypothesis: model = 2, NSsites = 2, fix_omega = 0, omega = 1) in PAML4[@b24] was used to detect positive selection in the one-to-one orthologous genes using our tree topology as the guide tree. Compared to other estimation models, the Branch-site model has the advantage of detecting positive selection that affects only a few sites on a pre-specified (foreground) branch of the species tree. The likelihood rate test (LRT) was used to detect positive selection on the foreground branch. PSGs were inferred only if their *P* values were less than 0.05. After identifying positively selected genes, the Bayes empirical Bayes (BEB) method was implemented to calculate posterior probabilities and to record positively selected sites. P-values of all PSGs also were normalized by FDR using Benjamini & Hochberg approach[@b61]. G:Profiler software[@b27] was utilized to perform functional enrichment analyses for the PSGs by using 'all known genes' of human as the statistical background domain size. Additionally, we deduced that the variable trend of tissue-specific highest expressed PSGs in plateau lineage. Here, our analyses controlled the strict gradient thresholds with folds in expression level (\>1.5fold, \>3fold and \>4.5fold) to ensure this trend more accurate. In brief, if log~2~ (fpkm + 1) of the PSGs in one tissue respectively was greater 1.5, 3 and 4 fold of the expressions of all other four tissues, then the PSG was regarded to be the tissue-specific highest expressed gene.
Convergent/parallel evolution
-----------------------------
Convergent/Parallel evolved changes were identified using the PAML4[@b24] package to reconstruct the most likely ancestral states, and then a PERL program was used to calculate the number of parallel amino acid replacements for a specified pair of branches. If the posterior probability of the reconstructed ancestral amino acid site was more than 90%, then they were retained and their states were deemed as reliable. In addition, G:Profiler software[@b27] was used to perform functional enrichment analyses for these PEGs utilizing 'all known genes' of human as the statistical background domain size.
Protein-Protein Interaction (PPI) Network
-----------------------------------------
We mapped the combined PSGs and PEGs candidate gene lists to the protein-protein interaction (PPI) network database (InnateDB)[@b34]. Sub-network topologies were obtained by NETWORKANALYST software[@b62] (<http://www.networkanalyst.ca/NetworkAnalyst>). Sub-networks with node counts ≤5 were filtered and the statistics of total nodes, edges and seed proteins were obtained from the mapping overview.
Additional Information
======================
**How to cite this article**: Shao, Y. *et al.* Genetic adaptations of the plateau zokor in high-elevation burrows. *Sci. Rep.* **5**, 17262; doi: 10.1038/srep17262 (2015).
Supplementary Material {#S1}
======================
###### Supplementary Dataset 1
###### Supplementary Dataset 2
###### Supplementary Dataset 3
###### Supplementary Dataset 4
###### Supplementary Dataset 5
###### Supplementary Dataset 6
We thank Pro. Yong-Yi Shen for his help in data analysis. This work was supported by grants from the National Natural Science Foundation of China, and the Animal Branch of the Germplasm Bank of Wild Species of Chinese Academy of Sciences (the Large Research Infrastructure Funding).
**Author Contributions** Y.P.Z. designed the experiments. Y.S. analyzed the data and wrote the paper. J.X.L. and L.Z. performed the separation, purification and sequencing of RNA. R.L.G. provided the data samples. D.M.I. and R.W.M. revised this paper. All authors reviewed the manuscript.
![(**a**) Phylogenetic placement of the plateau zokor derived from an analysis of 4-fold degenerate sites from concatenated orthologous sequence by Mrbayes algorithm[@b21]. Substitutions/site and Bayesian posterior probabilities are shown. (**b**) The evaluation of the divergence times across seven mammalian species. The Bayesian relaxed molecular clock method was implemented using the BEAST v1.7.5 software[@b22]. We utilized an indirect estimate[@b18] of the divergence time as the calibration point for the split of the ancestor of rabbit from the ancestor of mouse, rat and naked mole-rat. All nodal ages were indicated by medians and 95% HPD intervals (blue bars).](srep17262-f1){#f1}
{#f2}
![Saturation test of the one-to-one orthologous gene pairs.\
Each ratio (transition/transversion) of gene was calculated by the free-ratio model of PAML4[@b24]. (**a**) Plot of the ratio (transition/transversion) vs. *d*~*S*~in the plateau zokor lineage. (**b**) Plot of the ratio (transition/transversion) vs. *d*~*S*~ in rat lineage.](srep17262-f3){#f3}
![(**a**) Topology of the species tree. In this species tree, the two parallel evolving branches (plateau zokor and naked mole rat) were highlighted in red. (**b**) Multiple sequence alignments (MSAs) of the predicted coding sequences of *AJUBA* plateau zokor and other species. The MSAs were generated by PRANK[@b55][@b56][@b57] with the aligned coding sequence translated into protein sequence using MEGA5[@b63]. In the protein sequences, the parallel evolved site (E76D) in the plateau zokor is indicated by the red arrow.](srep17262-f4){#f4}
{#f5}
###### The summary of the transcriptome data for a plateau zokor.
Tissue Raw Reads Number Clean Reads Number Contigs Number Contigs N50 ([\*](#t1-fn1){ref-type="fn"}bp)
--------- ------------------ -------------------- ---------------- ----------------------------------------------
Brain 60,294,120 57,366,679 66674 2371
Kidney 44,738,308 43,607,559 51306 2275
Liver 55,713,348 53,250,755 43874 1617
Muscle 67,915,990 62,924,369 99059 884
Retina 43,793,426 38,956,940 90362 812
Mixture 272,455,192 256,106,302 208451 2433
^\*^bp = base pair.
| {
"pile_set_name": "PubMed Central"
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Introduction {#s01}
============
Activation-induced deaminase (AID) is a crucial enzyme for the immune response because it generates high-affinity and switched antibodies in germinal center (GC) B cells by somatic hypermutation (SHM) and class switch recombination (CSR; [@bib15]; [@bib32]). AID initiates SHM and CSR through the deamination of deoxycytidine residues into deoxyuridines on the DNA of Ig genes ([@bib15]; [@bib23]; [@bib3]; [@bib40]). The resulting U:G mismatch can be alternatively recognized and processed by base excision repair (BER) or mismatch repair (MMR) pathways, leading either to point mutations, in the case of SHM, or to double-strand breaks (DSBs) followed by a recombination reaction, in the case of CSR ([@bib3]; [@bib40]; [@bib33]; [@bib13]). Although AID activity has a strong preference for Ig genes, it can also target other genes, giving rise to point mutations ([@bib38]; [@bib20]; [@bib9]) or oncogenic chromosome translocations (TCs; [@bib29], [@bib30]; [@bib34]). Understanding AID specificity, or targeting, has been hindered by the technical challenge of detecting AID-induced mutations, which occur at very low frequencies. Here, we have used next generation sequencing to directly measure raw AID mutational activity on a broad representation of the genome and thus gather conclusions on AID specificity, DNA repair, and lymphomagenesis.
Results and discussion {#s02}
======================
Capture-based deep sequencing allows high-throughput identification of AID targets {#s03}
----------------------------------------------------------------------------------
To explore the scope of AID-induced mutations at a high-throughput scale, we designed a capture library against 1,588 regions corresponding to 1,379 different genes as a representation of the B cell genome (Table S1; see Design of DNA capture library in the Materials and methods). Genomic DNA from GC B cells was isolated, captured, and deep sequenced (Fig. S1, A and B). We made use of a mouse model deficient for both BER and MMR pathways (*Ung^−/−^Msh2^−/−^* mice). In the absence of BER and MMR, AID-induced U:G mismatches remained unprocessed and were replicated over, thus leaving behind almost solely C→T and G→A transitions, the footprint of AID deamination events on DNA ([@bib28]; [@bib13]). This approach allowed an extremely efficient enrichment and sequencing depth (Fig. S1, A and B). We found a set of 291 genomic regions (corresponding to 275 different genes) that were reproducibly mutated in *Ung^−/−^Msh2^−/−^* GC B cells when compared with *Aicda*^−/−^ GC B cells (q ≤ 0.05; [Fig. 1 A](#fig1){ref-type="fig"}; Fig. S1, C--E; and Table S2; representative targets were validated by Sanger sequencing; [Fig. 1 B](#fig1){ref-type="fig"} and Table S3). Importantly, the 275-gene target collection included 30 of the 35 previously known AID targets, such as *Bcl6*, *Pim1*, *RhoH*, *Pax5*, and *Cd83* ([Fig. 1 C](#fig1){ref-type="fig"} and Table S2; [@bib20]; [@bib9]; [@bib13]). Mutations detected in the 291-target regions strongly accumulated in AID mutational hotspots (WR[C]{.ul}(Y)/(R)[G]{.ul}YW; underlined letters specify deaminated nucleotides; W = A/T; R = A/G; Y = C/T; [Fig. 1 D](#fig1){ref-type="fig"}; [@bib36]). Finally, we found that our 275-target set included a big proportion of genes subject to DSBs or chromosome TCs (Fig. S1 F; [@bib1]; [@bib8]; [@bib39]; [@bib25]; [@bib4]). Thus, our deep sequencing approach has allowed the discovery of an unprecedented, massive collection of AID targets.
![**High-throughput analysis of AID-induced mutations.** DNA from Peyer\'s patch GC B cells was captured with a probe library for 1,588 genomic regions (Table S1) and deep sequenced. AID targets were identified as those regions accumulating significantly more C→T transition mutations in *Ung^−/−^Msh2^−/−^* than in *Aicda^−/−^* mice (Table S2; FDR ≤0.05, one-tail Fisher test and Benjamini-Hochberg correction; two independent experiments; see Materials and methods). **(A)** Circos plot representation of the AID targets identified in this study and their associated molecular features. The outer ring shows chromosome location and is followed by C→T transition mutation frequency in *Ung^−/−^Msh2^−/−^* (red) and *Aicda^−/−^* (gray) mice. **(B)** Validation of representative AID targets by Sanger sequencing (one-tail Fisher test; Table S3). **(C)** Overlap between the targets discovered in this study and previously reported AID targets. **(D)** Mean transition frequency in total C/G nucleotides and in C/G within WR[C]{.ul}(Y)/(R)[G]{.ul}YW hotspots (W = A/T; R = G/A; Y = C/T) of the 291 AID targets (two-tailed Student's *t* test; two independent experiments). **(E)** Logo representation of the sequence context of mutated cytosines (mutation frequency ≥4 × 10^−3^). Statistically significant enrichment of nucleotides surrounding the mutated C is indicated (\*, FDR ≤10^−3^, one-tail Fisher test and Bonferroni correction; see Materials and methods), and numbers indicate percentages. **(F)** Mean mutation frequency of cytosines within the indicated motifs (dark blue bar, newly identified hotspot; gray bar, control motif for newly identified hotspot; light blue bars, WRCY hotspots; red bars, random four-nucleotide motifs; two-tailed Mann-Whitney test). \*, P ≤ 0.05; \*\*\*, P \< 10^−3^; \*\*\*\*, P \< 10^−4^. Error bars depict SEM.](JEM_20171738_Fig1){#fig1}
Identification of AG[C]{.ul}TNT as a novel AID hotspot {#s04}
------------------------------------------------------
To gain insights into the local sequence preference of AID, we first analyzed the mean mutation frequency at individual WR[C]{.ul}Y/R[G]{.ul}YW hotspots across all 291 AID targets and found a wide range of mutability, with AA[C]{.ul}T and AG[C]{.ul}T as the top mutated hotspots in both strands of DNA, which may reflect an intrinsic preference for AID deaminase activity. Next, we performed an unbiased analysis of the sequence context of mutated cytosines. We found that A, G, and T nucleotides were the preferred nucleotides at −2, −1, and +1 positions ([@bib22]; [@bib43]; [@bib45]), respectively, but we further uncovered a significant preference for T at +3 ([Fig. 1 E](#fig1){ref-type="fig"} and Fig. S2). Indeed, cytosines lying at the AG[C]{.ul}TNT motif were significantly more mutated than those in AG[C]{.ul}TNV (where V is A, C, or G) or than other WR[C]{.ul}Y/R[G]{.ul}YW hotspots ([Fig. 1 F](#fig1){ref-type="fig"} and Fig. S2, A and B). Thus, our study has revealed AG[C]{.ul}TNT as a novel and the most highly mutated AID hotspot identified so far.
Prediction of AID targets {#s05}
-------------------------
Using the uniquely large set of AID-mutated genes identified in this study, we performed a comprehensive analysis of molecular features that associate with SHM, including transcription, epigenetic marks, and regulatory sequences ([Fig. 1 A](#fig1){ref-type="fig"}; [@bib41]; [@bib13]). We first observed that transcription levels and transcription rates are significantly higher in AID targets than in nontargets and that this difference is even higher for highly mutated targets ([Fig. 2 A](#fig2){ref-type="fig"}). We also found that RNAPolII and the stalling factor Spt5, previously described to associate with AID ([@bib17]; [@bib21]), show higher binding density within AID mutational targets ([Fig. 2 B](#fig2){ref-type="fig"}). Likewise, AID targets were enriched in marks of active enhancers and transcriptional elongation, such as Med12, H3K36me3, and H3K79me2 ([Fig. 2 C](#fig2){ref-type="fig"}). Finally, we found that primary AID targeting, as measured by AID mutations in the absence of repair, also focuses preferentially in the vicinity of superenhancers ([Fig. 2 D](#fig2){ref-type="fig"}) and in regions subject to convergent transcription ([Fig. 2 E](#fig2){ref-type="fig"}; [@bib12]; [@bib25]). Together, our mutagenesis study shows that several mechanisms linked to transcription are critical for AID activity, as suggested in previous studies ([@bib17]; [@bib21]; [@bib12]; [@bib25]; [@bib42]). Our data also indicate that AID targeting cannot be defined by any of these features alone. To approach whether a combination of these molecular features could be used to predict AID targeting, we developed a prediction model using a machine-learning algorithm, fed with the collection of genes analyzed here together with the set of molecular features described in [Fig. 2 (A--E)](#fig2){ref-type="fig"} (Fig. S3, A and B; see Machine learning to predict AID targets in the Materials and methods for details). We found that a combination of high-density RNAPolII and Spt5 binding, found in 2.3% of genes in the whole genome (Fig. S3 B), predicts AID specificity with 77% probability (P \< 0.001; [Fig. 2 F](#fig2){ref-type="fig"} and Fig. S3 A). Conversely, low RNAPolII binding combined with low gene expression predicted the absence of mutations for 95% of genes ([Fig. 2 F](#fig2){ref-type="fig"}). To test the accuracy of our prediction model, we analyzed the mutation frequency of a new collection of genes (not included in our capture library) with high-density RNAPolII and Spt5 binding (Fig. S3 C and Table S4). We found that 11/12 of the analyzed genes were significantly mutated (Table S4 and [Fig. 2 G](#fig2){ref-type="fig"}). Indeed, two genes (*Hist1h1c* and *Clec2d*) were mutated at the range of the top 20% mutated genes at frequencies similar to those found in *Pax5* or *Rhoh* (Table S2 and Table S4). Thus, we have built a powerful predictive tool for AID activity.
{#fig2}
BER and MMR back up each other to faithfully repair AID-induced lesions {#s06}
-----------------------------------------------------------------------
BER and MMR act downstream of AID-induced U:G mismatches so that UNG is critical for the generation of transversions at C:G pairs while MSH2 facilitates the introduction of mutations at A:T pairs ([@bib6]; [@bib24]; [@bib26], [@bib27], [@bib28]; [@bib13]). UNG and MSH2 can also promote conventional, faithful repair of AID-induced U:G mismatches ([@bib9]; [@bib22]). To explore the contribution of BER and MMR to AID mutagenic activity, we analyzed GC B cells from single-deficient *Ung^+/−^Msh2^−/−^* and *Ung^−/−^Msh2^+/−^* mice and from control *Ung^+/−^Msh2^+/−^* mice and compared the mutation frequency of the 291 AID target regions identified in this study (Table S2). We found similar mean mutation frequencies in B cells deficient for UNG alone, MSH2 alone, or proficient for both, whereas AID targets harbored significantly more mutations in the combined absence of UNG and MSH2 ([Fig. 3, A and B](#fig3){ref-type="fig"}). Indeed, only a small proportion (∼6%) of the genes mutated in *Ung^−/−^Msh2^−/−^* cells was also mutated in single-knockout and double-heterozygous cells ([Fig. 3 C](#fig3){ref-type="fig"} and Table S2). Moreover, we found that classical AID off targets, such as *Bcl6* or *Pim1*, although mutated in all genotypes analyzed, harbored a significantly bigger load of mutations in *Ung^−/−^Msh2^−/−^* cells than in *Ung^+/−^Msh2^−/−^*, *Ung^−/−^Msh2^+/−^*, or *Ung^+/−^Msh2^+/−^* cells ([Fig. 3 D](#fig3){ref-type="fig"}). Together, these data indicate that BER and MMR back up each other to faithfully repair most of the AID-induced lesions in GC B cells.
{#fig3}
AID targets are recurrently mutated in human lymphomas {#s07}
------------------------------------------------------
We next assessed the contribution of AID off-target mutations to B cell--derived malignancies by making use of available sequencing data on human lymphomas. We found that AID targets are significantly enriched in genes mutated in human B cell lymphomas (see Annotation of AID targets in the Materials and methods for details; [Fig. 4 A](#fig4){ref-type="fig"}). Indeed, 21/275 (7.6%) of our set of AID target genes are mutated in diffuse large B cell lymphomas (DLBCLs; [Fig. 4 B](#fig4){ref-type="fig"}), a highly prevalent, aggressive form of lymphoma ([@bib37]). Lymphoma genes mutated by AID included *Bcl6*, *RhoH*, *Pim1*, *Ebf1*, *Eif4a2*, and *Pax5*, which is in agreement with previous studies ([@bib38]; [@bib20]; [@bib9]). In addition, we identified nine novel genes mutated in human DLBCLs that accumulate AID-induced mutations ([Fig. 4 B](#fig4){ref-type="fig"}), including *Mef2b*, *Lyn*, *Tnfaip3*, *Gna13*, and *Irf8*. Remarkably, we found many instances where the exact same mutations described in human lymphoma genes were also found in the AID targets identified in this study in nontransformed mouse B cells ([Fig. 4 C](#fig4){ref-type="fig"} and Table S5). Together, these results suggest that off-target AID mutagenic activity can contribute to GC-associated lymphomagenesis.
![**AID targets are recurrently mutated in human lymphomas. (A)** AID targets are enriched in genes involved in lymphoma development. Percentage of lymphoma genes within AID target and nontarget genes. Annotation was done from public data on human lymphoma sequencing (see Materials and methods; two-tailed Fisher test; \*\*\*\*, P \< 10^−4^). **(B)** Mutation frequency in total C/G nucleotides and C/G nucleotides within WR[C]{.ul}(Y)/(R)[G]{.ul}YW hotspots (W = A/T; R = G/A; Y = C/T) of the 21 AID target genes involved in human DLBCL development analyzed in *Ung^−/−^Msh2^−/−^* mice (mean of two independent experiments; see Materials and methods). **(C)** Mutation profiles of representative DLBCL genes analyzed in *Ung^−/−^ Msh2^−/−^* mice. Blue bars indicate mutations identical to those found in human lymphoma tumor samples (Table S5); asterisks indicate mutations occurring in a WR[C]{.ul}(Y) hotspot. The diagrams below the graphs represent the complete gene (not to scale), and blue boxes indicate the region depicted above. Mutation frequency found in each nucleotide in *Aicda^−/−^* mice was subtracted before plotting.](JEM_20171738_Fig4){#fig4}
Until now, the study of AID specificity has been hindered by the technical challenge of detecting AID-induced mutations; indeed, only a limited number of genes has been directly interrogated for AID-mediated mutagenesis ([@bib20]; [@bib9]; [@bib13]). However, genome-wide AID specificity has been inferred from high-throughput analysis of AID binding, which does not warrant AID activity, AID-induced DSBs, or chromosomal TCs, which involve complex processing of the initial lesion induced by AID ([@bib1]; [@bib8]; [@bib39]; [@bib44]; [@bib12]; [@bib25]). The strategy developed in this study has provided an unprecedented scope to the analysis of AID targeting: we describe here the broadest collection of AID mutational targets (275 genes) to date, 10-fold larger than the previously known targets. The strength of this analysis is well supported by the confirmation of the vast majority of previously identified AID targets and the validation of targets by conventional Sanger sequencing.
Here, we have integrated our mutation data with a collection of molecular features of GC B cells to feed a machine-learning algorithm. According to the machine-learning tree generated here, the combined binding of Spt5 and RNAPolII at high density is the best predictor for AID mutability, although additional combinations of transcriptional traits bear some predictive power as well. Furthermore, we have performed independent experimental validation showing that randomly picked Spt5^high^RNAPolII^high^ genes indeed are very frequently mutated by AID. This is, to our knowledge, the first instance of a tool that successfully predicts the potential of a gene to be targeted by AID. Regarding the fate of AID-induced lesions, BER and MMR have long been known to broaden the diversity of SHM with an apparent perverted recruitment of error-prone polymerases and to do so in a cooperative manner ([@bib28]; [@bib3]; [@bib13]). The mechanisms responsible for the error-free versus error-prone activity of UNG and MSH2 are far from understood, and both gene-specific and local sequence contexts may play a role in defining the fate of the U:G resolution ([@bib9]; [@bib22]; [@bib43]). Strikingly, here we show that the fate of the majority of off-target lesions induced by AID is to undergo faithful repair by BER and MMR and that, again, both pathways can back up each other in this task with only a minor fraction of the mutations escaping them. Whether this reflects gene-specific qualities or is the consequence of excessive mutation load will deserve further investigation. We would speculate that a minor fraction of unrepaired mutations in prolymphomagenic genes could provide cell growth advantage and account for the predominance of AID-mediated mutations in lymphomas. Regardless of oncogenic relevance, it is remarkable that even though our mutation analysis was performed in nontransformed cells, we could detect individual AID-induced mutations that are recurrently mutated in lymphoma. Thus, our results yield a novel perspective on the contribution of AID activity to B cell transformation through the introduction of mutations.
We expect our mutational study will be valuable for other research questions, including validation of novel molecular mechanisms involved in AID targeting, prediction of novel targets, or assessment of cancer-associated mutations. Furthermore, similar approaches would be of immediate interest to broaden our knowledge on the role of AID or other mutagenic activities not only in B cell lymphomas, but also in malignancies from any origin.
Materials and methods {#s08}
=====================
Mice {#s09}
----
*Ung* and *Msh2* mutant mice used in this study were generated by crossing *Ung^−^*^/^*^−^* mice ([@bib18]) and *Msh2*^−/−^ mice ([@bib31]). *Aicda*^−/−^ mice have been previously described ([@bib15]). Mice were housed in specific pathogen-free conditions. Male and female mice between 20 and 28 wk were used for the experiments. The number of animals per group to detect biologically significant effect sizes was calculated using an appropriate statistical sample size formula. All experiments were done in concordance with EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under RD 53/2013.
Design of DNA capture library {#s10}
-----------------------------
A set of 1,379 mouse genes was selected as a representation of the genome (Table S1). 85% of all genes were randomly picked, ensuring even representation of chromosomal location by bioinformatic analysis and unbiased biological function. ∼15% of the library corresponded to previously known AID targets ([@bib16]; [@bib20]; [@bib7]; [@bib9]; [@bib35]; [@bib21]), IgH probes, and other controls. Probes were designed in eArray (Agilent) to capture the first 500 bp downstream of each transcriptional start site (TSS) of each of the 1,379 genes. Because various genes contained more than one predicted TSS, the library includes a total of 1,588 different genomic regions. Library design included 50 extra nucleotides at both ends of each region to optimize the capture yield. A custom target enrichment capture library was then synthesized by the manufacturer (SureSelectXT; Agilent).
DNA capture and sequencing {#s11}
--------------------------
GC (*Cd19^+^Fas^+^GL7^+^*) B cells were isolated from Peyer's patches of *Ung^+/−^Msh2^+/−^* (*n*~1~ = 10; *n*~2~ = 11), *Ung^−/−^Msh2^+/−^* (*n*~1~ = 46; *n*~2~ = 8), *Ung^+/−^Msh2^−/−^* (*n*~1~ = 46; *n*~2~ = 2), and *Ung^−/−^Msh2^−/−^* (*n*~1~ = 37; *n*~2~ = 8) littermates and *Aicda^−/−^* (*n* = 31 mice) mice by sorting in a FACSAria cell sorter (BD Biosciences) after staining with anti--mouse antibodies to *Cd19*, *Fas*, and *GL7* (BD Biosciences). Genomic DNA was isolated by standard procedures and quantified in a fluorometer (Qubit; Invitrogen). DNA capture, library preparation, and DNA sequencing were performed by the Genomics Unit at Centro Nacional de Investigaciones Cardiovasculares (CNIC). In brief, DNA was fragmented in a sonicator (Covaris) to ∼200 nucleotide-long (mean size) fragments and purified using AMPure XP beads (Agencourt). Quality was assessed with the 2100 Bioanalyzer (Agilent). Then, fragment ends were repaired, adapters were ligated, and the resulting library was amplified and hybridized with our custom SureSelectXT library of RNA probes. DNA--RNA hybrids were then captured by magnetic bead selection. After indexing, libraries were single-end sequenced in a HiSeq 2500 platform (Illumina).
Target enrichment assessment by quantitative RT-PCR {#s12}
---------------------------------------------------
*Noxa1*, *Ostn*, and *Pcna* amplifications were quantified with green assay (SYBR; Applied Biosystems) in a real-time PCR system (AB7900 Standard; AbiPrism). *Gapdh* amplifications were used as normalization controls. The following primers were used: *Gapdh* (forward), 5′-TGAAGCAGGCATCTGAGGG-3′; *Gapdh* (reverse), 5′-CGAAGGTGGAAAGTGGGAG-3′; *Ostn* (forward), 5′-CATAGTGTTGCTGTGGTT-3′; *Ostn* (reverse), 5′-CATTATATTGGTCTGCTGTT-3′; *Noxa1* (forward), 5′-CGCGGGACAGCAATGAGAAG-3′; *Noxa1* (reverse), 5′-CCATCTACTCAGTTTCAAGGA-3′; *Pcna* (forward), 5′-CTCCAGCACCTTCTTCAG-3′; and *Pcna* (reverse), 5′-TCTCATCTAGTCGCCACA-3′.
SDS software (Applied Biosystems) was used for analysis of the data.
Sanger sequencing {#s13}
-----------------
Regions to be sequenced were amplified from 160--200-ng genomic DNA in four independent reactions to minimize possible PCR biases. The following primers were used: *Hist1h1b* (forward), 5′-ATGCCTTAGACTTCACCGCC-3′; *Hist1h1b* (reverse), 5′-TTGTAACCTTGAGTCGCCGC-3′; *miR142* (forward), 5′-CGGTCCCTGGGAAGTTACAC-3′; *miR142* (reverse), 5′-AACGAGAGGCAAACAGTCTTCA-3′; *Cd19* (forward), 5′-GCCCCTCTTCCCTCCTCATA-3′; *Cd19* (reverse), 5′-CCTGCACCCACTCATCTGAA-3′; *Cdk4* (forward), 5′-TCTGGCAGCTGGTCACATGG-3′; and *Cdk4* (reverse), 5′-GATCACCAGCTAGTCGTCCC-3′. Amplification reactions were carried in a final volume of 25 µl using 2.5 U Pfu Ultra HF DNA polymerase (Agilent) and the following PCR setup: 95°C for 2 min, 25 (*Cd19* and *Cdk4*) or 26 cycles (*miR142* and *Hist1h1b*) of denaturation at 94°C for 30 s, annealing at 57°C (*miR142* and *Hist1h1b*) or 58°C (*Cd19* and *Cdk4*) for 30 s, extension at 72°C for 1 min, and a final stage of 72°C for 10 min. PCR products were purified from a 1% agarose gel (Illustra Gel Band Purification kit; GE Healthcare) and cloned into pGEMT vector (Promega). Competent DH5α *Escherichia coli* bacteria were transformed with the constructs, and individual colonies (192--288 per gene) were grown in 96-well plates. Plasmidic DNA was then isolated (Plasmid MiniPrep kit; Millipore) and sequenced by Sanger sequencing using SP6 universal primer. Sequence analysis was performed using SeqMan software (Lasergene).
PCR-Seq to validate the machine-learning approach {#s14}
-------------------------------------------------
40--50 ng of genomic DNA was amplified using the following primers: *Apobec3* (forward), 5′-GTCTTCCATAGCCTGCTCACA-3′; *Apobec3* (reverse), 5′-TAGCTGACTGGTGTGGTTCC-3′; *Aurkaip1* (forward), 5′-ACTTGTCACTTCCGCAGTCC-3′; *Aurkaip1* (reverse), 5′-CCATCCCCAAGTCAGGTGTG-3′; *Ccdc17* (forward), 5′-TCTTTTCTGTCCAGTCCGCC-3′; *Ccdc17* (reverse), 5′-ACAAATGGGCAGAGTCAGGG-3′; *Cd52* (forward), 5′-TACTGCCGCACACATGACTC-3′; *Cd52* (reverse), 5′-TGAGGTGGGAAGCCAAACAT-3′; *Cd68* (forward), 5′-AGGGGCTGGTAGGTTGATTG-3′; *Cd68* (reverse), 5′-GGAGTCAGGACTGGATTTGAC-3′; *Cd69* (forward), 5′-TCTAAAGGTTTTGAGACCCCC-3′; *Cd69* (reverse), 5′-TGAAGCCTCATCAACGCACT-3′; *Clec2d* (forward), 5′-GGCTCCTGACCTTGAAATGC-3′; *Clec2d* (reverse), 5′-AGGCAACTTCTGCCACTATGC-3′; *Coro1a* (forward), 5′-AGGGCTCTGGGGTTCTACTT-3′; *Coro1a* (reverse), 5′-GGAAATGACCACGGGGGTTT-3′; *Hist1h1c* (forward), 5′-CTCTATCGGCGTACTGCCAC-3′; *Hist1h1c* (reverse), 5′-ATCGAGTCCCTTGCAACCTT-3′; *Il4i* (forward), 5′-ATTCCCGAGGGAGGTGAGTG-3′; *Il4i* (reverse), 5′-GGTAGCTTCTCTCCGTCACAC-3′; *Maz* (forward), 5′-GTCAACAAAGAACCCCTCCCT-3′; *Maz* (reverse), 5′-CACCTGTCCCCTGAGTTGTG-3′; *Trex1* (forward), 5′-GCCTAACAGGTTTGATTGTCC T-3′; and *Trex1* (reverse), 5′-TAGGCTGAGCACTCCCAGTC-3′. Amplification reactions were carried in a final volume of 25 µl using 2.5 U Pfu Ultra HF DNA polymerase (Agilent; 95°C for 2 min, 26 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and a final stage of 72°C for 10 min). PCR products were purified and fragmented using a sonicator (Covaris), and libraries were prepared by the CNIC Genomics Unit according to the manufacturer's instructions (NEBNext Ultra DNA Library Prep; New England Biolabs). Sequencing was performed in a HiSeq 2500 platform (Illumina). Analysis was performed as previously described ([@bib22]).
Gene expression profiling by RNA-Seq {#s15}
------------------------------------
GC (*CD19^+^Fas^+^GL7^+^*) and resting (*CD19^+^Fas^−^GL7^−^*) B cells were sorted from Peyer's patches of littermate 12-wk-old WT C57BL/6 mice. Three biological replicates were analyzed, each composed of a pool of five female mice. RNA was purified from pellets of 2--2.5 × 10^4^ cells, and DNaseI treatment was applied to avoid DNA contamination (RNAeasy MiniKit; Qiagen). RNA quality was assessed with the 2100 Bioanalyzer, showing high RNA purity and integrity. Sequencing libraries were prepared by the CNIC Genomic Unit according to the manufacturer's protocol (NEB NEXT Ultra RNaseq Library Prep kit; New England Biolabs) from 100 ng RNA per replicate and sequenced in a HiSeq 2500 platform.
Computational analysis {#s16}
----------------------
### Pipeline to identify and annotate AID-induced mutations {#s17}
Raw reads were demultiplexed by Casava v1.8 to generate a fastq file that was aligned to the mouse genome (NCBIm37 v61 Feb 2011) with Novoalign 2.08.01 (command line options: -o SAM -F ILM1.8 -H -r None -q 2). Sam files were processed with Samtools 0.1.19 to generate a sorted bam file that was piped to a custom Perl script for the analysis of AID mutations. In brief, the software analyzes the regions of interest in the bam file, annotates hotspots, localizes and suppresses annotated single nucleotide polymorphism positions (Sanger Mouse Genomes Project SNP and Indel Release v2), and reports relevant information about AID activity. AID targets were identified as those genes accumulating significantly more C→T transition mutations in *Ung^−/−^Msh2^−/−^* than in *Aicda^−/−^* mice (false discovery rate \[FDR\] ≤0.05, one-tail Fisher test and Benjamini-Hochberg correction).
Mutation frequencies were calculated as follows:$${Total~mutation~freq} = \frac{Total~number~of~mutations}{Total~sequenced~length},$$$${Mutation~freq~}_{C/G} = \frac{\left( {{Mutated~cytosines} + {Mutated~guanines}} \right)}{\left( {{Seq~length~cytosines} + {Seq~length~guanines}} \right)},$$and
Mutation freq
WRC
(
Y
)
/
(
R
)
GYW
=
(
Mutated cytosines
WRC
(
Y
)
\+
Mutated guanines
(
R
)
GYW
)
(
Seq length cytosines
WRC
(
Y
)
\+
Seq length guanines
(
R
)
GYW
)
.
(Only cytosines in WR[C]{.ul}(Y) and guanines in (R)[G]{.ul}YW were considered to calculate mutation frequency at hotspots.)
### Integration of AID targets with public data on TC and DSB occurrence {#s18}
The bar graph included in Fig. S1 F represents overlaps in the 1,375 genes analyzed in this study (divided into mutated and nonmutated genes) and genes where TCs or DSBs occur in B cells: [@bib12] refer to TC sites identified by HTGTS in αCD40+IL4-activated B cells as published in Table S2 from their study; [@bib8] refer to TC sites identified by TC-Seq in IgH^I-Sce^ LPS+IL4-activated B cells as published in Table S4 from their study; [@bib1] refer to TC sites identified by HTGTS in c-myc^25xI-SceI^ αCD40+IL4-activated B cells as published in Table S3 (significant hits at P ≤ 0.05) from their study; [@bib25] refer to DSBs identified by replication protein A (RPA) differential recruitment (RPA--chromatin immunoprecipitation \[ChIP\]) in Ig*k*AID 53BP1^−/−^ in vitro activated B cells as published in Table S1 A from their study; and [@bib39] refer to DSBs identified by Nbs1 binding (ChIP-on-ChIP) in LPS+αIgD-dextran+BLySS-activated B cells as published in Table S1 (P ≤ 0.05) from their study.
### Sequence context of mutated cytosines {#s19}
The sequence context of mutated cytosines (C→T transition frequency ≥4 × 10^−3^) was analyzed in a window of 10 nucleotides. Logo representation was done using WebLogo3, and the percentage of each nucleotide in each position surrounding the mutated cytosine was calculated by a custom Perl script. Enrichment for adenosine, guanine, cytosine, or thymine was tested against the sequence context of all cytosines present in the 1,588 regions analyzed in this study (one-tailed Student's *t* test + Bonferroni correction).
### Gene expression profiling by RNA-Seq {#s20}
After demultiplexing by Casava v1.8, read quality was assessed by FASTQC, and sequencing adapters were removed from sequence reads by cutadapt v1.9. The resulting reads were aligned to and quantified on the mouse transcriptome (NCBIm38 v75, Feb 2014) using RSEM v1.2.25 with the following parameters: -p 3--time--output-genome-bam--sampling-for-bam--bowtie-e 60--bowtie-m 30--bowtie-chunkmbs 512--fragment-length-mean 180--fragment-length-sd 50.
### Transcription rate analysis (GRO-Seq) {#s21}
Reads were mapped to the mouse genome (mm9/NCBI37) using bowtie2, and uniquely mapped, nonredundant reads were kept. Reads mapping in ±1 kb from TSSs were quantified and summarized at the gene level using HTSeq.
### PolII and Spt5 recruitment {#s22}
Quantification of PolII and Spt5 recruitment was extracted from Table S3 A in [@bib21].
### Superenhancer analysis {#s23}
Data were extracted from the catalog of superenhancers that overlap with gene bodies identified in GC B cells as published in Table S3 in [@bib12] (GEO accession no. [GSE62296](GSE62296)).
### Epigenetic mark analysis {#s24}
Sequencing data (fastq files) for each epigenetic mark were aligned to the mouse genome (NCBIm37 v61, Feb 2011) using bowtie 1.1.1 (command line options:--best -m1 -n2 -p2). Alignment files were processed by Samtools 0.1.19 to generate a sorted bam file. Peak calling was done using MACS2 (v2.1.0.20140616) according to the optimal parameters for a histone modification status profiling as reported by the creators of the tool ([@bib5]). Mapping of annotated peaks to genes was done using GREAT (version 3.0.0).
### Convergent transcription analysis (GRO-Seq) {#s25}
Convergent transcription data analysis was performed as described in [@bib12]. In brief, reads were mapped to the mouse genome (mm9/NCBI37) using bowtie2, and uniquely mapped, nonredundant reads were kept. HOMER (v4.6) was used with default parameters to identify transcribed regions from both strands and bedtools (v2.24) to find and annotate ConvT regions (regions where \>100 bp of sense and antisense transcription overlap occurs).
### Machine learning to predict AID targets {#s26}
The conditional inference tree for classification was built using the *ctree* function from the party R package with default parameters. Genes with a background mutation frequency \>5 × 10^−4^ were excluded to avoid artifacts. The following variables were fed into the model for each of the 1,339 genes analyzed: expression, transcription rate, PolII recruitment, and Spt5 recruitment (quantitative, continuous); Med12 recruitment, H3K4me1 recruitment, H3K36me3 recruitment, H3K79me2 recruitment, regulation by superenhancers, and occurrence of convergent transcription (qualitative, discrete). All variables were assigned equal weights to fit the model.
### Annotation of AID targets {#s27}
Annotation of AID targets was performed based on public data on sequencing of human DLBCLs, Burkitt lymphomas, and follicular lymphoma tumors ([@bib10]; [@bib11]; [@bib14]; [@bib46]; [@bib2]; [@bib19]).
Data availability {#s28}
-----------------
Sequencing data generated for this study are available through the GEO database: targeted DNA deep sequencing (accession no. [GSE102944](GSE102944){#dblnk77b326cb-e178-28cc-2064-510c786d96c2}) and RNA-Seq (accession no. [GSE98086](GSE98086)).
The rest of the datasets analyzed in the current study are publicly available through the GEO and/or Sequence Read Archive: GRO-Seq (accession no. [GSE62296](GSE62296)), GC B cells (accession nos. [SRR1611832](SRR1611832), [SRR1611833](SRR1611833), and [SRR1611834](SRR1611834)), naive B cells (accession nos. [SRR1611829](SRR1611829), [SRR1611830](SRR1611830), and [SRR1611831](SRR1611831)), ChIP-Seq of PolII and Spt5 (accession no. [GSE24178](GSE24178)), and ChIP-Seq data of epigenetic marks Med12 (accession no. [SRX347810](SRX347810)), H3K4me1 (accession no. [SRX347815](SRX347815)), H3K36me3 (accession no. [SRX185869](SRX185869)), and H3K79me2 (accession no. [SRX185843](SRX185843)).
Statistical analysis {#s29}
--------------------
Statistical analyses were performed with stats R package v3.1.1. Error bars in figures represent SEM. Student's *t* test was applied to continuous data, and a Fisher test was used to assess differences between categorical variables. P-values were corrected for multiple hypothesis testing by Benjamini-Hochberg or Bonferroni method where appropriate. Differences were considered statistically significant at P ≤ 0.05 or q ≤ 0.05.
Online supplemental material {#s30}
----------------------------
Fig. S1 shows the experimental workflow used to identify AID targets and technical controls. Fig. S2 shows mutation analysis of WR[C]{.ul}Y/R[G]{.ul}YW hotspots. Fig. S3 shows details on the machine-learning classification tree used for the prediction of AID targets. Table S1 contains a list of the genes included in the capture library. Table S2 A contains a detailed mutation analysis of AID targets in *Ung^+/−^Msh2^+/−^*, *Ung^−/−^Msh2^+/−^*, *Ung^+/−^Msh2^−/−^*, and *Ung^−/−^Msh2^−/−^*. Table S2 B contains a list of the 18 AID targets mutated in repair-proficient GC B cells. Table S3 shows mutation analysis of genes validated by Sanger sequencing. Table S4 shows mutation analysis of the genes selected for machine-learning validation. Table S5 contains a list of the mutations found in *Ung^−/−^Msh2^−/−^* GC B cells that have been identified in cohorts of human lymphoma patients.
Supplementary Material
======================
###### Supplemental Material
###### Tables S1-S5 (zipped Excel files)
We thank all members of the B Cell Biology Laboratory for useful discussions, V. Barreto for critical reading of the manuscript, F. Sánchez-Cabo for advice on statistics analysis, J.M. Ligos for help with flow cytometry, and A. Dopazo for advice on DNA capture and sequencing.
A. Pérez-García was a fellow of the research training program funded by the Ministerio de Educación, Cultura y Deporte (grant FPU-AP2009-1732); A.F. Álvarez-Prado and A.R. Ramiro are supported by Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). This work was funded with the following grants to A.R. Ramiro from Plan Estatal de Investigación Científica y Técnica y de Innovación 2013--2016, Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Retos Investigación: Proyectos I+D+i 2016, Ministerio de Economía, Industria y Competitividad (MEIC; grants SAF2013-42767-R and SAF2016-75511-R). This work is cofunded by Fondo Europeo de Desarrollo Regional and the European Research Council Starting Grant program (grant BCLYM-207844). The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505).
The authors declare no competing financial interests.
Author contributions: A.F. Álvarez-Prado, P. Pérez-Durán, and A.R. Ramiro designed experiments; A.F. Álvarez-Prado, P. Pérez-Durán, A. Pérez-García, and V.G. de Yébenes performed experiments; A. Benguria performed DNA sequencing; A.F. Álvarez-Prado and C. Torroja developed scripts for data analysis; A.F. Álvarez-Prado and A.R. Ramiro analyzed data and prepared figures; and A.F. Álvarez-Prado and A.R. Ramiro wrote the manuscript.
[^1]: P. Pérez-Durán's present address is Institute for Cancer Genetics, Columbia University, New York, NY.
[^2]: A. Pérez-García's present address is Beatson Institute for Cancer Research, Glasgow, Scotland, UK.
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Background {#Sec1}
==========
Infections of the respiratory tract are a major risk to patients with chronic respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). The majority of acute exacerbations of COPD (AECOPD) is associated with an acute respiratory infection, with viral and/or bacterial pathogens being detected in more than half of all exacerbations \[[@CR1], [@CR2]\]. Experimental infections of COPD and asthma patients with human rhinoviruses (HRV) have moreover provided evidence for a causal relationship between an acute infection and the onset of symptoms characteristic for acute exacerbations in both diseases \[[@CR3], [@CR4]\]. Other viruses that are detected during AECOPD include respiratory syncytial virus (RSV), influenza viruses and adenovirus strains \[[@CR5], [@CR6]\].
Next to viral infection, also bacterial infections can be involved in the development of acute exacerbations. Among the bacterial species that are frequently detected during such episodes are strains of non-typeable (NT) *Haemophilus influenzae*, *Pseudomonas aeruginosa* and *Streptococcus pneumoniae* \[[@CR1]\]. Importantly, colonization with these bacteria is also frequently observed in the stable state of the disease. Potential pathogenic microorganisms (PPMs) have been detected in approximately 25 % of COPD patients during stable disease, even when rather insensitive culture-dependent techniques were employed \[[@CR7]--[@CR10]\]. Likewise, increased load of PPMs has also been described for other chronic lung diseases, such as asthma and cystic fibrosis \[[@CR11]--[@CR13]\].
Not only is bacterial colonization associated with an increased risk to develop an acute exacerbation, it is also associated with increased levels of inflammatory markers in the stable state \[[@CR14]--[@CR16]\]. Furthermore, pro-inflammatory cytokines, such as IL-6 and IL-8, have been shown to be elevated in the sputum of frequent exacerbators and during exacerbation \[[@CR17]\]. Changes in IL-6 between stable state and exacerbation were found to be particularly pronounced, if the exacerbations were associated with a viral infection \[[@CR17]--[@CR19]\].
AECOPD associated with the detection of a combination of bacterial and viral pathogens have been reported to be particularly severe in terms of inflammation and decline in lung function \[[@CR20]\]. Moreover, these events on average required longer hospitalizations \[[@CR2]\]. Presence of both, potential pathogenic bacteria and viruses, during the same period of exacerbations have been observed in as much as 12 to 25 % of AECOPD \[[@CR21], [@CR22]\]. When specifically looking at AECOPD associated with a positive culture of NT *H. influenzae*, almost half of the patients were also tested positive for at least one respiratory virus \[[@CR23]\]. Considering these studies made use of rather insensitive conventional culturing techniques to detect bacteria, these numbers are most likely even underestimating the prevalence of simultaneous presence of bacteria and viruses.
Numerous studies have investigated how primary viral infections increase susceptibility to secondary bacterial infections, for example by disrupting epithelial barriers \[[@CR24]\], increasing bacterial attachment or colonization \[[@CR25]--[@CR28]\], or modulating innate and adaptive immune responses \[[@CR29]--[@CR33]\]. On the other hand, consequences of a primary exposure to bacteria on the outcome of secondary viral infections have been less extensively studied \[[@CR34]\]. Considering the substantial proportion of patients chronically colonized with bacteria, this concept might - though less well understood - be equally important.
We therefore hypothesized that the response of airway epithelial cells to respiratory viral infections is influenced by pre-exposure to bacterial pathogens. In the present study we investigated how exposure to different respiratory bacteria influences the inflammatory response and susceptibility of respiratory epithelial cells to a secondary infection with respiratory syncytial virus (RSV), influenza B virus and adenovirus.
Methods {#Sec2}
=======
Cell culture {#Sec3}
------------
BEAS-2B cells (ATCC CRL-9609) were cultured in RPMI-1640 (life technologies, Carlsbad, USA) supplemented with 10 % fetal bovine serum (FBS; Lonza, Basel, Switzerland). For experiments, BEAS-2B cells were used for up to 15 passages after cryopreservation (passage number 20 to 35). Culture flasks and multiwell plates were pre-coated with a mixture of bovine collagen (30 μg/ml), human fibronectin (10 μg/ml; both BD Biosciences, San Jose, USA) and bovine serum albumin (BSA, 10 μg/ml, Sigma Aldrich, St Louis, USA). For experiments, BEAS-2B cells were seeded at 10^5^ cells per well in collagen/fibronectin-coated 48-well plates the day before the experiment. Cell layers were approximately 80--90 % confluent at the start of the experiment.
Primary bronchial epithelial cells (pBECs) were kindly provided by the Primary Lung Cell (PLUC) facility Maastricht University Medical Center + (MUMC+, Maastricht, The Netherlands). Lung tissue used for the isolation of pBECs was obtained from the Maastricht Pathology Tissue Collection (MPTC) and originated from tissue resected during lobectomies or pneumonectomies of patients who underwent surgery for lung cancer. Collection, storage and use of tissue and patient data were performed in agreement with the "Code for Proper Secondary Use of Human Tissue in the Netherlands" ([http://www.fmwv.nl](http://www.fmwv.nl/)). The scientific board of the MPTC approved the use of materials for this study under code MPTC2010-019. PBECs were isolated from bronchus rings that were macroscopically free of cancer. Isolation, culture and characterization of cells was performed as previously described \[[@CR35]\]. Epithelial character of the cells was confirmed by immunohistochemical staining for cytokeratins 5, 6, 8 and 17. Experiments were performed on cells isolated from seven donors. All donors (median age 67 years, range 55--78 years) were never-smokers or former smokers, and had not been diagnosed with COPD, cystic fibrosis or asthma.
For the seeding of pBECs, the methodology was adapted to account for different generation times of cells derived from different donors and to meet growth requirements of the primary cells. PBECs were seeded at 1.5 × 10^4^ cells per well in pre-coated 24-well plates in B/D medium consisting of 50 % DMEM (life technologies) and 50 % Bronchial Epithelial Basal Medium (BEBM; Lonza), supplemented with bronchial epithelial growth medium (BEGM) singlequots (Lonza), and BSA (1.5 mg/ml, Sigma Aldrich). Experiments were started once cell layers reached approximately 80 % confluence.
Virus culture {#Sec4}
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Virus strains were provided by the Dutch National Institute for Public Health and the Environment (RIVM, Bilthoven, the Netherlands).
RSV (strain A2) was propagated in Vero cells and concentrated by precipitation in polyethyleneglycol 6000 as described previously \[[@CR36]\]. Influenza B (Yamagata lineage) were propagated in MDCKII cells. Human adenovirus 2 was propagated in HeLa cells.
The infectivity of viruses was quantified by determining the 50 % Tissue Culture Infective Dose (TCID~50~) using the Spearman Karber Method \[[@CR37]\].
Bacterial culture {#Sec5}
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All bacterial strains used in this study were reference strains obtained from ATCC and were cultured at 37 °C and 5 % CO~2~. NT *H. influenzae* (ATCC 49247) was cultured on Vitox-supplemented chocolate agar plates (Oxoid, Wesel, Germany). *P. aeruginosa* (ATCC 27853) and *S. pneumoniae* (ATCC 49619) were cultured on B/D Columbia blood agar plates (Becton Dickinson, Franklin Lakes, USA).
Infection protocols {#Sec6}
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### Preparation of inactivated bacterial suspensions {#Sec7}
Bacterial suspensions were prepared by adding several colonies of an overnight culture to RPMI-1640 medium. These suspensions were heat-inactivated at 65 °C for 1 h. Inactivation was confirmed by plating out aliquots of the suspension on agar plates. Bacteria were then pelleted by centrifugation at 4500 x g for 10 min, washed once with PBS and re-suspended in infection medium. The composition of the infection medium was dependent on the cell type and virus used. Stimulation and infection of BEAS-2B cells with bacteria in combination with RSV and adenovirus was performed in RPMI-1640 supplemented with 2 % FBS (Lonza). For subsequent infection with Influenza B, bacterial suspensions were prepared in serum-free medium consisting of Minimal Essential Medium (life technologies) supplemented with 1 mg/ml proteose peptone, 0.1 mg/ml BSA, 0.2 mg/ml D-glucose monohydrate (all Sigma Aldrich, St Louis, USA) and 0.05 ‰ trypsin/EDTA (life technologies). For experiments on primary cells, infection medium consisted of B/D medium supplemented with BEGM singlequots (both Lonza) except human epidermal growth factor and bovine pituitary extract. The turbidity of the bacterial suspensions was adjusted to 0.5 McFarland (equivalent to approximately 1.5 × 10^8^ cfu/ml).
### Continuous stimulation {#Sec8}
Bacterial suspensions were further diluted 1:10 in infection medium. Cells were first stimulated with bacteria for 4 h, and subsequently infected with the respective virus. For virus infection, culture supernatants were aseptically collected from each well and preserved while cells were exposed for one hour to diluted virus to yield a multiplicity of infection (MOI) of one. After this initial attachment period, cells were washed once with PBS and the original culture medium (including bacteria) was added to back the wells.
### Pre-exposure protocol {#Sec9}
Bacterial suspensions of 0.5 McFarland were prepared as described above. For stimulation of cells, 0.5 ml of the bacterial suspension or control medium were added to each well. After 24 h of exposure, cell layers were washed with PBS and infected with RSV at an MOI of one. Virus particles were allowed to attach for 1 h, after which unbound virus was aspirated, cells layers were washed with PBS and fresh medium was added to the wells.
Cell-free supernatants and cell lysates were collected 24, 48 and 72 h after viral infection and stored at −80 °C until use.
ELISA {#Sec10}
-----
Levels of IL-6 and IL-8 were determined using an enzyme linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Ready-SET-Go, eBioscience, San Diego, USA). Lower limits of detection were 2.4 pg/ml for IL-6 and 3.2 pg/ml for IL-8.
Determination of RSV RNA copies {#Sec11}
-------------------------------
Total RNA from cell lysates was isolated using the FavorPrep Tissue Total RNA purification kit (Favorgen, Pingtung, Taiwan). Remaining genomic DNA was degraded by DNase treatment (Ambion Turbo DNAfree, life technologies). For each sample, 500 ng of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, USA). Real-time PCR was then performed on a MyIQ real-time PCR system (Bio-Rad) using the following primers: Forward 5′- TTTCCACAATATYTAAGTGTCAA-3′; Reverse 5′- TCATCWCCATACTTTTCTGTTA-3′. Viral RNA copy numbers were calculated by using a standard curve derived from in vitro transcribed RSV RNA.
Statistical analyses {#Sec12}
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Statistical analysis was performed using Prism 5 (GraphPad, La Jolla, USA) and SPSS 22.0 (IBM, Armonk, USA). Data are presented as mean ± standard error of the mean (SEM) of five to seven independent experiments. For better visualization of the synergistic effects, the additive effect of two pathogens is indicated as a dashed line in some graphs. This theoretical additive effect for a given combination of pathogens was calculated as the sum of cytokine levels of the individual infections. Statistical significance of bacterial and viral challenges, as well as interactions between the two, were determined using a two way analysis of variance (ANOVA) and a Bonferroni post-hoc analysis. To determine statistical significance of bacterial-viral interactions in primary epithelial cells, relative changes over the additive effect were calculated and tested for statistical significance using a one sample *t*-test. Differences in viral RNA copies and infectious particles were tested for statistical significance using a one-way ANOVA. In all cases, effects were considered statistically significant if *p* \< 0.05.
Results {#Sec13}
=======
*H. influenzae* and *P. aeruginosa* synergistically enhance the release of the pro-inflammatory cytokines IL-6 and IL-8 during RSV infection {#Sec14}
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We first investigated if exposure to respiratory bacterial pathogens can alter the epithelial response to subsequent RSV infection. To this end, we made use of the immortalized bronchial epithelial cell line BEAS-2B, which is frequently used to study airway epithelial responses to pathogens and displays similar expression of pattern recognition receptors (PRRs) as primary bronchial epithelial cells \[[@CR38]\]. BEAS-2B cells were stimulated with heat-inactivated preparations of NT *H. influenzae*, *P. aeruginosa* or *S. pneumoniae*. Four hours later, cells were additionally infected with RSV and secretion of IL-6 and IL-8 was monitored over a total period of 72 h.
Exposure of cells to heat-inactivated *H. influenzae* or *P. aeruginosa* caused significant release of the pro-inflammatory cytokines IL-6 and IL-8, as did infection with RSV alone. When cells were first exposed to heat-inactivated *H. influenzae* (Fig. [1a](#Fig1){ref-type="fig"}) or *P. aeruginosa* (Fig. [1b](#Fig1){ref-type="fig"}) and subsequently infected with RSV, IL-6 release was synergistically increased: Seventy-two hours after infection, experimentally determined values of IL-6 in the culture supernatants were on average increased by 56 ± 3 % (*H. influenzae*) or 46 ± 9 % (*P. aeruginosa*) compared to the sum of cytokines released by either bacteria or virus alone (indicated as bacteria + virus (theoretical), dashed line). For *S. pneumoniae* on the other hand, there was no significant interaction of bacteria and virus on the induction of IL-6 release at any of the assessed time points (Fig. [1c](#Fig1){ref-type="fig"}). Of note, the cytokine response of BEAS-2B cells to heat-inactivated pneumococci was clearly lower than the cells' response to the other two tested bacterial species and there was no significant effect of *S. pneumoniae* exposure on cytokine release (Fig. [1c, f](#Fig1){ref-type="fig"}).Fig. 1Co-stimulation of epithelial cells with bacteria and virus potentiates inflammatory cytokine release. BEAS-2B cells were first exposed to heat-inactivated preparations of non-typeable (NT) *H. influenzae* (**a**, **d**), *P. aeruginosa* (**b**, **e**) or *S. pneumoniae* (**c**, **f**) for 4 h and subsequently infected with RSV and incubated for up to 72 h in the presence of bacteria. Concentrations of IL-6 (**a--c**) and IL-8 (**d--f**) in culture supernatants were determined by ELISA. The theoretical additive effect (*dashed line*) was calculated as the sum of bacteria and virus-induced cytokine secretion. *Symbols* indicate a statistically significant effect of bacteria alone (\*), virus alone (\#) and a significant interaction between bacteria and virus (&) as determined by Two Way Repeated Measures ANOVA (*p* \< 0.05). hpi: hours post viral infection
Similar to IL-6, also IL-8 release was synergistically enhanced in cells infected with RSV in the presence of either *H. influenzae* (Fig. [1d](#Fig1){ref-type="fig"}) or *P. aeruginosa* (Fig. [1e](#Fig1){ref-type="fig"}), whereas we did not detect a significant interaction of *S. pneumoniae* with this virus (Fig. [1f](#Fig1){ref-type="fig"}).
RSV and *H. influenzae* also synergistically increased IL-6 release when applied in the reverse order, i.e., when cells were first infected with RSV and subsequently co-stimulated with heat-inactivated bacteria, suggesting interactions between the two pathogens are not exclusive to primary bacterial stimulation (data not shown). However, since we were primarily interested in the effect of a primary bacterial exposure (as a model for bacterial colonization) on secondary viral infections, all results presented here are derived from experiments in which cells were first exposed to bacteria and subsequently infected with virus.
Stimulation with heat-inactivated bacteria and/or RSV did not significantly affect metabolic activity of the cells; differences in cytokine secretion were thus unlikely to be caused by pathogen-specific effects of the treatments on cell viability (Additional file [1](#MOESM1){ref-type="media"}: Figure S1A).
Due to the limited possibilities for bacterial control or clearance in our in vitro setting, inactivating the bacterial suspension was inevitable to avoid bacterial overgrowth of the epithelial cultures during the 3 day experimental period. To exclude any effect of the mode of inactivation on our readouts, we repeated the experiments using a low dose of gentamicin (4 μg/ml) to inhibit growth of *H. influenzae,* instead of heat-inactivating the bacteria. Although the results failed to meet statistical significance, we still observed a trend towards a synergistic interaction between *H. influenzae* and RSV on IL-6 release (mean increase 65 % ± 39 %, *p* = 0.065; Additional file [1](#MOESM1){ref-type="media"}: Figure S1B).
Pre-exposure of cells to heat-inactivated *H. influenzae* is sufficient to synergistically increase RSV-induced cytokine release {#Sec15}
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Several respiratory viruses, including RSV, have previously been shown to enhance attachment of a variety of bacterial species to cells of the respiratory tract \[[@CR27], [@CR28], [@CR39]--[@CR41]\]. Due to the prolonged incubation during which the cells are exposed to both pathogens (bacteria and viruses), it cannot be excluded that viral infection enhances bacterial binding also in our experimental setting. Such augmented bacterial attachment and concomitant enhanced recognition might also explain an increased inflammatory response. In a second set of experiments, we therefore investigated if the altered inflammatory response of co-stimulated cells required the presence of bacteria during viral infection.
Cells were pre-exposed to bacteria for 24 h, after which cell layers were washed thoroughly with PBS and infected with RSV. This pre-exposure of cells to *H. influenzae* was sufficient to synergistically increase RSV-induced IL-6 release at all assessed time points. Pre-exposure also increased IL-8 release after subsequent infection with RSV, with the interaction reaching statistical significance at 72 h after infection and a trend towards statistical significance at the earlier time points (24 h: *p* = 0.077; 48 h: *p* = 0.089; Fig. [2d](#Fig2){ref-type="fig"}).Fig. 2Pre-exposure of cells to NT *H. influenzae* increases production of inflammatory cytokines upon RSV infection. BEAS-2B cells were first exposed to heat-inactivated preparations of non-typeable (NT) *H. influenzae* (**a**, **d**), *P. aeruginosa* (**b**, **e**) or *S. pneumoniae* (**c**, **f**) for 24 h, after which bacteria were washed away and cells were infected with RSV for up to 72 h. Concentrations of IL-6 (**a--c**) and IL-8 (**d--f**) in culture supernatants were determined by ELISA. The theoretical additive effect (*dashed line*) was calculated as the sum of bacteria and virus-induced cytokine secretion. *Symbols* indicate a statistically significant effect of bacteria alone (\*), virus alone (\#) and a significant interaction between bacteria and virus (&) as determined by Two Way Repeated Measures ANOVA (*p* \< 0.05). hpi: hours post viral infection
In cells pre-exposed to *P. aeruginosa* before RSV-infection, we only detected a small, yet statistically significant synergistic effect on IL-6 production 24 h after infection (mean increase over additive effect 8 % ± 2 %, *p* = 0.002), but not at the later time points (Fig. [2b](#Fig2){ref-type="fig"}). IL-8 secretion in response to RSV, however, was still synergistically enhanced after pre-exposure of cells to *P. aeruginosa* at 72 h after infection (Fig. [2e](#Fig2){ref-type="fig"}). In line with the results obtained from a continuous exposure to bacteria, pre-exposure of cells to heat-inactivated preparations of *S. pneumoniae* did not significantly affect cytokine release after RSV infection (Fig. [2](#Fig2){ref-type="fig"} c and f). As for the continuous exposure protocol, also during the pre-exposure protocol there was no evidence for a significant effect of the treatments on cell viability (Additional file [1](#MOESM1){ref-type="media"}: Figure S1C).
Taken together, these results indicate that pre-exposure of cells with NT *H. influenzae,* is sufficient to potentiate RSV-induced cytokine release and that continuous presence of bacteria is not required. The increased inflammatory response of cells pre-exposed to NT *H. influenzae* was thus unlikely to be triggered by enhanced bacterial attachment to the cells.
Influence of previous exposure to bacteria on RSV-induced IL-6 production by primary bronchial epithelial cells {#Sec16}
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We next sought to confirm our findings in primary bronchial epithelial cells (pBECs). Therefore, bronchial epithelial cells were isolated from seven non-smoking individuals without chronic respiratory diseases such as COPD, asthma or cystic fibrosis (median age 67 years; range 55--78 years). Presence of chronic respiratory diseases was excluded based on medical history, pulmonary function tests and imaging. The cells were treated with suspensions of heat-inactivated bacteria and subsequently infected with RSV according to the pre-exposure protocol described in the [Methods](#Sec2){ref-type="sec"} section of this article.
Cytokine release, particularly in response to RSV infection, varied considerably between cells from different donors (Table [1](#Tab1){ref-type="table"}).Table 1IL-6 release by pBECs pre-exposed to heat-inactivated bacteria and subsequently infected with RSV24 hpi48 hpi72 hpi- RSV+ RSV- RSV+ RSV-RSV+ RSVctrl6.1 ± 1.1179.1 ± 77.28.4 ± 2.6208.5 ± 83.36.6 ± 1.8234.3 ± 91.6+hiNTHI8.4 ± 1.6206.8 ± 88.011.4 ± 2.6258.7 ± 116.310.3 ± 2.7324.2 ± 147.3+hiPA19.5 ± 14.9225.0 ± 123.021.4 ± 16.3288.7 ± 120.618.6 ± 13.9335.3 ± 139.7+hiSP4.5 ± 0.9177.8 ± 74.86.0 ± 1.7210.0 ± 88.06.2 ± 1.6238.7 ± 100.1Values are given in pg/ml and represent the mean ± SEM (*n* = 7). hpi: hours post (viral) infection, *hi* heat-inactivated, *NTHI* non-typeable *H. influenzae*, *PA P. aeruginosa*, *SP S. pneumoniae, RSV* respiratory syncytial virus
In order to account for inter-individual variability, results in Fig. [3](#Fig3){ref-type="fig"} are expressed as percent changes relative to a calculated additive effect (i.e., relative to the sum of cytokine concentrations released in response to each pathogen alone) for each donor. Pre-exposure to *H. influenzae* increased RSV-induced IL-6 release by cells from four out of seven donors above this additive effect (Fig. [3a](#Fig3){ref-type="fig"}). Likewise, pre-exposure to *P. aeruginosa* synergistically enhanced the IL-6 release following RSV infection in four out of seven donors, with a trend towards statistical significance 72 h after infection (*p* = 0.09, Fig. [3b](#Fig3){ref-type="fig"}). Moreover, and also in line with the results obtained in BEAS-2B cells, *S. pneumoniae* did not further increase IL-6 release after RSV-infection in pBECs (Fig. [3c](#Fig3){ref-type="fig"}). In contrast, however, pre-exposure of pBECs to either bacterial species did not significantly affect virus-induced IL-8 secretion (data not shown).Fig. 3Exposure of pBECs to NT *H. influenzae* or *P. aeruginosa* enhances IL-6 release. PBECS of seven donors were pre-exposed to heat-inactivated preparations of non-typeable (NT) *H. influenzae* (**a**)*, P. aeruginosa* (**b**)*,* or *S. pneumoniae* (**c**) for 24 h, after which bacteria were washed away and cells were infected with RSV for up to 72 h. IL-6 levels in culture supernatants 24, 48 and 72 h after viral infection were determined by ELISA. Due to considerable inter-individual variation in absolute cytokine levels, data are shown as relative changes of experimentally determined cytokine concentrations over the theoretical additive effect for each donor. Results obtained using cells from each donor are denoted with the same symbol in all datasets. hpi: hours post viral infection
Up to 48 h after viral infection, there was no effect of the treatments on metabolic activity of pBECs; Seventy-two hours after infection, cell viability was modestly decreased in virus-infected cells, yet there were no differences between cells pre-treated with the different bacteria that would explain altered cytokine secretion (Additional file [1](#MOESM1){ref-type="media"}: Figure S1D).
Enhanced release of inflammatory mediators is not linked to enhanced viral replication {#Sec17}
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Exposure to bacterial pathogens has previously been suggested to enhance viral attachment and replication in lung epithelial cells \[[@CR36], [@CR42]\]. We therefore tested if the enhanced inflammatory response of BEAS-2B cells towards RSV after pre-exposure with bacteria was due to enhanced viral replication. Neither intracellular viral RNA levels (Fig. [4a](#Fig4){ref-type="fig"}) nor the release of infectious progeny (Fig. [4b](#Fig4){ref-type="fig"}) was significantly altered when cells were pre-exposed to either of the three bacteria used in this study. Nevertheless, viral replication seemed at least indirectly to be required to potentiate cytokine release, since *H. influenzae* in combination with UV-inactivated virus did not cause increased secretion of IL-6 (Additional file [1](#MOESM1){ref-type="media"}: Figure S1E). In agreement with this, synergy between the pathogens was most pronounced after cells mounted a cytokine response against the virus itself, i.e., at 48 h after viral infection and later.Fig. 4Pre-exposure of cells to bacteria has no significant effect on RSV replication. BEAS-2B cells were exposed to heat-inactivated bacteria for 24 h, after which cell layers were washed and infected with RSV for another 72 h. **a** Intracellular RSV RNA copies were determined by real-time PCR. **b** Release of infectious progeny in culture supernatants was measured by determining the TCID~50~. NTHI: non-typeable *H. influenzae*, PA: *P. aeruginosa*, SP: *S. pneumoniae*, n.s.: not significant
To test if exaggerated inflammation was linked to increased expression of key PRRs involved in the recognition of bacterial and/or viral structures, we next measured mRNA levels of TLR2, TLR3, TLR4 and RIG-I. However, there was no significant interaction between bacteria and viruses on the expression levels of any of these genes (Additional file [1](#MOESM1){ref-type="media"}: Figure S2).
Changes in the virus-induced inflammatory response after bacterial pre-exposure are species-specific {#Sec18}
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Because of the pathogen-specificity we observed when investigating different bacteria in combination with RSV, we next evaluated the inflammatory response of bacterially challenged epithelial cells during infection with different viruses. Next to RSV, rhinoviruses, influenza viruses and specific adenoviruses are among the most frequently detected respiratory viral pathogens \[[@CR5], [@CR43]\]. Since the influence of bacterial exposure on infection with human rhinovirus has already been the subject of previous studies \[[@CR36], [@CR42]\], we now assessed combinations of different bacteria with influenza B virus and a type 2 human adenovirus.
In our hands, infection with influenza B virus only induced modest IL-6 release (Fig. [5](#Fig5){ref-type="fig"} a--c). Although we found a statistically significant interaction between *H. influenzae* and influenza B virus (measured cytokine levels lower than the calculated additive effect 24 and 72 h after infection), as well as between *P. aeruginosa* and influenza B (measured cytokine levels lower than the calculated additive effect at all time points), co-stimulated cells still released cytokines at levels comparable to those of cells stimulated with bacteria only.Fig. 5Co-stimulation of cells with bacteria and viruses influences cytokine release in a pathogen-specific manner. BEAS-2B cells were first pre-exposed to either NT *H. influenzae* (**a**, **d**), *P. aeruginosa* (**b**, **e**) or *S. pneumoniae* (**c**, **f**) for 4 h and subsequently infected with Influenza B (**a--c**) or Adenovirus (**d--f**) for up to 72 h in the presence of bacteria. Concentrations of IL-6 in culture supernatants were determined by ELISA. The theoretical additive effect (*dashed line*) was calculated as the sum of bacteria and virus-induced cytokine secretion. *Symbols* indicate a statistically significant effect of bacteria alone (\*), virus alone (\#) and a significant interaction between bacteria and virus (&) as determined by Two Way Repeated Measures ANOVA (*p* \< 0.05). hpi: hours post viral infection
Human adenovirus on the other hand did not induce IL-6 production by itself, but significantly reduced cytokine release induced by the different bacteria. When compared to the additive effect of the pathogens, stimulation of cells with adenovirus after bacterial exposure reduced IL-6 release at 72 h by 57 % ± 6 % in combination with NT *H. influenzae* (Fig. [5d](#Fig5){ref-type="fig"}) and 68 % ± 4 % in combination with *P. aeruginosa* (Fig. [5e](#Fig5){ref-type="fig"}). For IL-8, there was a significant interaction between all three bacteria in combination with human adenovirus 72 h after viral infection. Measured IL-8 levels were on average 39 % ± 9 % (for NT *H. influenzae*), 42 % ± 4 % (for *P. aeruginosa*) and 83 % ± 2 % (for *S. pneumoniae*) below the additive effect of these pathogens.
In summary, the effect of subsequent challenges with bacteria and viruses on release of inflammatory mediators was not only dependent on the bacterial species used, but also virus-dependent.
Discussion {#Sec19}
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*H. influenzae* is the most commonly found PPM in the lower respiratory tract of COPD patients. We here demonstrate that exposure of the bronchial epithelial cell line BEAS-2B to heat-inactivated *H. influenzae* significantly augments RSV-induced release of the pro-inflammatory cytokines IL-6 and IL-8. *H. influenzae* has previously been described to enhance the secretion of IL-8 in response to certain strains of another major respiratory virus, human rhinovirus (HRV), by up-regulating expression of TLR3 \[[@CR42]\]. In our experimental model of bacterial exposure and RSV-infection, the synergy between heat-inactivated *H. influenzae* and RSV on the release of pro-inflammatory cytokines was not reflected in the expression levels of TLR3, or other pattern recognition receptors (PRRs) relevant for the detection of RSV and/or *H. influenzae* \[[@CR44]--[@CR47]\].
Increased bacterial or viral binding is considered a major feature in the pathogenesis of polymicrobial infections. RSV infection has previously been described to promote fimbriae-mediated attachment of *H. influenzae* to epithelial cells \[[@CR41], [@CR48]\]. However, considering that the pro-inflammatory effects of pre-exposure to heat-inactivated *H. influenzae* were sustained even when unbound bacteria were washed away prior to RSV infection, the increased release of IL-6 and IL-8 is most likely not a consequence of greater bacterial binding. Conversely, exposure of lung epithelial cells to *H. influenzae* has been shown to increase the cells' susceptibility to secondary infection with major group HRV by upregulating their cellular entry receptor, ICAM-1 \[[@CR36], [@CR42]\]. Although neutralizing antibodies for ICAM-1 can reduce RSV infection in vitro, it is not the main functional receptor for this virus \[[@CR49], [@CR50]\]. Correspondingly, the exaggerated inflammation we observed in our model was not linked to increased viral endpoint titers nor increased levels of intracellular viral RNA, suggesting alterations in intra- or intercellular signaling rather than a generalized increase in viral replication as a cause. Nevertheless, viral replication was at least indirectly required, since UV-inactivated virus did not further increase the secretion of inflammatory cytokines in our experimental model. Interestingly, treatment of epithelial cells with heat-killed *H. influenzae* can reduce bacterial and fungal burden after secondary infection with several pathogens, and also limit influenza virus replication in a mouse model of influenza virus pneumonia \[[@CR51], [@CR52]\]. Effectiveness against other viruses has yet to be evaluated, but was not evident for RSV in our model.
Similar to *H. influenzae*, also *P. aeruginosa* elicited a strong increase in the secretion of pro-inflammatory cytokines IL-6 and IL-8, and like *H. influenzae*, potentiated RSV-induced cytokine release. However, in BEAS-2B cells, continuous presence of *P. aeruginosa* during viral infection was required to observe a sustained synergistic effect on IL-6 release. *P. aeruginosa* and RSV have been described to interact directly and RSV has hence been suggested as a coupling agent between bacteria and cells, thus enhancing bacterial attachment \[[@CR53]\]. Such an interaction might explain the diverging results we obtained using different exposure protocols.
The third bacterial pathogen we investigated in this study was *S. pneumoniae*, a Gram-positive opportunistic pathogen of the respiratory tract. In our experimental setting, heat-inactivated preparations of *S. pneumoniae* did not increase secretion of IL-6 or IL-8. This lack of an epithelial reaction towards *S. pneumoniae* might be attributable to the fact that we used heat-inactivated preparations of bacteria, preventing the expression of important virulence factors. Also, we did not observe a consistent significant interaction between *S. pneumoniae* and RSV in our model in terms of eliciting an inflammatory response. Importantly, RSV has recently been found to directly interact with *S. pneumoniae* \[[@CR54]\]*.* Bacterial binding to the viral G protein triggered profound alterations in the bacterial transcriptome and lead to increased expression of a set of virulence factors. Unfortunately, in our experimental setting inactivation of bacteria was inevitable to prevent bacterial overgrowth and synergistic effects of *S. pneumoniae* and RSV might therefore be obscured. Altered bacterial virulence due to direct interactions between pathogens adds another layer of complexity to the study of polymicrobial infections.
Our experiments in pBECs revealed considerable inter-individual differences between cells from different donors, not only in terms of absolute cytokine concentrations, but also with respect to the effect of bacterial exposure on subsequent infection. Although we aimed to include pBEC donors of a relatively homogenous group in terms of age, smoking-status and the absence of COPD, asthma and cystic fibrosis as underlying pulmonary diseases, differences that arise due to their clinical background or the isolation procedure cannot fully be excluded as a source of variability. Notably, cytokine release was evidently lower in primary cells than in BEAS-2B cells, particularly in response to bacteria. This less responsive phenotype might contribute to the differences we observed between primary cells and the immortalized cell line. Nonetheless, at 72hpi, results obtained with cells from four out of seven donors were in line with the data obtained in BEAS-2B cells, showing a more than additive effect of pre-exposure with *H. influenzae* or *P. aeruginosa* and RSV infection.
The effect of an exposure to bacteria prior to viral infection on cellular inflammatory responses was not only dependent on the bacterial species, but was also virus-specific. Cells exposed to bacteria and subsequently infected with influenza B still released cytokine levels comparable to those of cells exposed to bacteria alone. Even though secondary bacterial infection is a major contributor to influenza virus pathology the reverse order of sequential infections does not seem to have such detrimental effects \[[@CR34]\]. This is in line with previous data by Lee et al., who demonstrated a lack of effect on the course of the disease when mice were exposed to *H. influenzae* type b before an influenza challenge, despite a strong lethal synergism when mice were infected in the reverse order \[[@CR55]\]. Application of *H. influenzae* lysates can even confer a temporary protection against subsequent influenza infection by boosting local innate responses \[[@CR52]\].
In contrast, cytokine levels produced by cells infected with adenovirus after an initial bacterial exposure were significantly reduced. Decreased levels of IL-6 and IL-8 after exposure to bacterial stimuli in combination with adenovirus might be attributed to the adenoviral proteins E1A and E1B, which have previously been reported to reduce the induction of IL-6 in response to bacterial and inflammatory stimuli in human epithelial cells \[[@CR56], [@CR57]\].
Using an in vitro model of bronchial epithelial cells allowed us to investigate pathogen specific interactions in a controlled environment. Being the first tissue to be exposed to infectious particles, the airway epithelium is crucial for initiating a first immune response. However, the possibilities to mimic a chronic exposure to bacteria, as would be the case during a bacterial colonization of the lungs, are limited in vitro. Therefore, more studies are needed to examine, for example, the impact of immune cells and the development of tolerance in this context. Extrapolating biological significance from the magnitude of an effect observed in vitro may be difficult. In a model of bacterial co-colonization, Ratner et al. showed that interactions of pathogens on the release of cytokines in vitro which were in a similar order of magnitude as the ones we observed in our study, could indeed translate to a biologically relevant effect in vivo \[[@CR58]\].
Even though culture-dependent techniques usually only revealed a limited number of bacterial genera in the lower respiratory tract of colonized individuals, molecular techniques suggest that the lung microbiota is in fact far more complex. Furthermore, changes in the lung microbiome in asthma and COPD have been reported in several studies \[[@CR59]--[@CR61]\]; particularly pathogenic proteobacteria, such as *H. influenzae*, were more frequently found in the lungs of patients suffering from these diseases than in control subjects. It can be assumed that not only the presence of certain bacterial species, but also the composition of the local microbiota in the respiratory tract affects the response towards viral pathogens. In view of the pathogen-specific effects we observed in our study, it would therefore be interesting to expand this concept further towards a more complex representation of the lung microbiome in health and disease.
Conclusions {#Sec20}
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We show that pre-exposure of airway epithelial cells to heat-inactivated NT *H. influenzae* and *P. aeruginosa* aggravates the production of pro-inflammatory cytokines in response to subsequent infection with RSV, but not influenza B virus or adenovirus. Taken together, our data provide experimental evidence for the pleiotropic effects of microbial interactions on pulmonary inflammation. If these findings can be translated into the clinical setting, it might enable clinicians to identify patients at high risk for developing more severe viral infections on the basis of their bacteriological status.
Additional file {#Sec21}
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Additional file 1:Supplemental information. (ZIP 83 kb)
The authors would like to thank Danny Goudkade, MD (Department of Pathology, Maastricht University Medical Center) for his help in collecting the lung tissue used for isolation of pBECs, as well as Dinette Agterhuis, MD (Department of Respiratory Medicine, Maastricht University Medical Center) for gathering the data on donor characteristics.
Part of the work described in this manuscript has been presented as an abstract at the Annual Meeting of the European Respiratory Society 2015 (European Respiratory Journal Sep 2015, 46 (suppl 59) DOI: 10.1183/13993003.congress-2015.PA2604).
Authors' contributions {#FPar1}
======================
CB designed and performed experiments, analyzed data and drafted the manuscript. FG designed experiments, interpreted data and revised the manuscript. AH performed experiments and analyzed data. MD, PS and EW interpreted data and revised the manuscript. GR and FS conceived and designed the study, interpreted data and revised the manuscript. All authors read and approved the final version of the manuscript.
Competing interests {#FPar2}
===================
The authors declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
To the Editor: We report a successful treatment of a case diagnosed with double outlet right ventricle (DORV) with absent pulmonary valve (APV) and bronchiarctia.
The patient is a female of 14-year-old with body weight 44 kg, who was referred to our hospital for cardiac murmur and history of exercise intolerance and recurrent respiratory tract infections. She presented cyanotic. Electrocardiogram showed right ventricle hypertrophy and right bundle branch block. The chest X-ray showed right pulmonary artery dilation, pulmonary blood vessels decreased, heart shape enlargement and cardio/thoracic ratio at 0.52. Echocardiography and cardiac computerized tomography (CT) scan supported the diagnosis. The CT scan also showed severe left bronchial stenosis \[[Figure 1a](#F1){ref-type="fig"}\]. The diagnosis was confirmed in the operation. The operation of total correction was performed under cardiopulmonary bypass with hypothermia. Operative findings included right ventricle enlargement, the aorta overriding the ventricle septal defect (VSD) more than 90%, aorta/main pulmonary artery ratio at 1:1 in diameter, right ventricular outflow tract, and the pulmonary valve annulus stenosis. The absence of pulmonary valve is presented in [Figure 1b](#F1){ref-type="fig"}. After clamping the aorta, the cardioplegia solution was given through the aortic root. Oblique incision was made in the right ventricle outlet tract, abnormal muscles were resected, and the VSD was repaired with autologous pericardial. The right ventricular outflow tract and the main pulmonary artery were widened transannulusly using autologous pericardial with three leaflets. Inotropic drugs were used to maintain cardiac function postoperatively. The patient recovered well during 1-year follow-up.
{#F1}
To our knowledge, DORV with VSD, right ventricle outlet and pulmonary artery annulus stenosis, APV, and bronchiarctia has not been reported in literature. DORV with VSD and pulmonary artery or valve stenosis is a kind of congenital heart disease. The surgical treatment result is not satisfactory.\[[@ref1]\] However, the girl needed to undergo the surgical treatment due to complex pathological changes. During the operation, we reconstructed the main pulmonary valve, repaired the VSD, and reconstructed the left and right ventricle outflow tract. We repaired the VSD with autologous pericardial to make the left ventricle outflow tract elastic, considering patient\'s condition, growth, and development. The autologous pericardium with three leaflets was used to widen the right ventricle outflow tract to avoid the stenosis and reduce the pulmonary valve regurgitation.\[[@ref2][@ref3]\] Residual shunt should also be avoided. It is quite difficult for patients with bronchiarctia to recover, but most of them can recover uneventfully by strengthening the airway care, taking physical therapy, and preventing and managing infection postoperatively.\[[@ref4]\] The girl\'s recovery showed that the bronchiarctia is not the surgical contraindication but will affect the period of the recovery. The patient needs to be followed up for cardiac function, and the recurrence of left and right ventricle outflow tract stenosis as well as pulmonary valve regurgitation increases.
Financial support and sponsorship {#sec2-1}
=================================
This study was supported by a grant from the National Natural Science Foundation of China (No. 81370328).
Conflicts of interest {#sec2-2}
=====================
There are no conflicts of interest.
**Edited by:** Yi Cui
| {
"pile_set_name": "PubMed Central"
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1. Introduction {#sec-1}
===============
The outcome of in vitro fertilization (IVF) is strongly dependent upon ovarian response to gonadotropin stimulation \[1\]. Ovarian stimulation can delivery 2 unpleasant results as either hyper or low response \[2\]. Due to these conditions, ovarian response prediction before stimulation is important for clinicians. Several methods can be used to predict ovarian stimulation results. Antral follicle count (AFC), day 3 (D3) follicle stimulating hormone (FSH) level, D3 estradiol (E2) level, anti-Müllerian hormone (AMH) level are the most used parameters \[3\]. High FSH levels or decreased AMH levels could predict the poor response for gonadotrophin stimulation but could not foretell for fecundity \[4\].
There is a negative correlation between female's chronological age and ovarian reserve. The first finding of ovarian aging is a prominent increment in FSH levels compared to luteinizing hormone (LH) levels. This increment makes the FSH/LH ratio increased \[5\]. Increased FSH/LH ratio is a new method to determine the cycle outcome before starting stimulation \[6\]. Researchers reported that increased FSH/LH ratio can predict decreased ovarian reserve and lower pregnancy outcomes \[7--9\].
In this study we aimed to compare the cycle outcomes of patients' experienced controlled ovarian hyperstimulation-embryo transfer (COH-ET) with either FSH/LH ratio higher than 2 or not.
2. Materials and methods {#sec-2}
========================
This retrospective cohort study was conducted with 648 COH-ET cycles performed between October 2012 and October 2014 after approval of local ethical committee (2014/13-04). Preparation of all patients before stimulation consisted of detailed anamnesis, physical examination, transvaginal ultrasonography, day 3 (D3) hormone profile \[follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol (E2), progesterone (P4), thyroid stimulating hormone (TSH), prolactin\], semen analysis, hysterosalpingography or hysteroscopy. Antral follicle count (AFC) were determined on day 2/3 before starting stimulation. COH was performed with long GnRH agonist, GnRH antagonist, and micro dose flare-up protocols as appropriate.
The FSH/LH ratio cut-off is changing among studies, but the most used cut-off is 2 \[1,3\]. Due to this condition, we determined cut-off ratio as 2 and cycles were divided into 2 groups according to D3 FSH/LH ratio. Group 1 (G1) was consisted of 473 cycles with FSH/LH ratio \< 2 and Group 2 (G2) was consisted of 175 cycles with FSH/LH ratio ≥ 2.
GnRH agonists were initiated on luteal phase and gonadotropin stimulation was started on day 2/3 of the proceeding cycle. Gonadotropin doses \[recombinant FSH (rFSH), human menopausal gonadotropin (HMG) or both\] were determined according to age, FSH level, and AFC of women. In antagonist cycles, gonadotropin stimulation was started on day 2/3 of fresh cycle and when the leading follicle reached 12 mm in diameter or blood E2 level reached 300 pg/mL, cetrorelix or ganirelix were started. In micro dose flare-up protocol, oral contraceptive pill was started on day 2 of previous cycle for 21 days and on day 1 of proceeding cycle GnRH agonist was initiated (subcutaneous leuprolide acetate 40 µg/daily). On day 2 of cycle, exogenous gonadotropins were started. When at least 3 follicles reached 17 mm in diameter, recombinant human chorionic gonadotropin (hcg) was administered for final maturation. Both of GnRH antagonists and agonists were continued until the day of hcg injection. Oocyte pick-up (OPU) was performed at 34--36 h after hcg injection.
In embryology, mature oocytes were inseminated by intracytoplasmic sperm injection (ICSI) after cumulus separation. Fertilization was defined as the observation of 2 pronuclei 24 h after ICSI. Embryo transfer (ET) was performed on day 3 with single cleavage embryo or on day 5 with single blastocyst. Luteal support was done with daily application of vaginal 8% progesterone gel and intramuscular 50 mg progesterone. Pregnancy was checked with blood beta hcg test 14 days after ET. Blood beta hcg test was repeated 2 days later for confirming the healthy increment. Transvaginal ultrasonography was performed for visualization of gestational sac 10 days later. Ongoing pregnancy was defined as the presence of more than 24 weeks of gestation with a live foetus. Implantation rate (ImR) was calculated as the ratio of gestational sac number/transferred embryo number. Fertilization rate (FR) was calculated as the ratio of fertilized oocyte number/mature oocyte number. Pregnancy rate (PR) was calculated as the ratio of beta hcg test positivity/transferred embryo number.
Age, body mass index (BMI), infertility aetiology, D3 FSH-LH-E2 levels, AFC, stimulation protocol type, amount of the used gonadotropins, E2 and P4 levels on the day of hcg injection, retrieved total oocyte count (TOC), mature oocyte count (MOC), fertilised oocyte count (FOC), transferred embryo number, and pregnancy results were recorded from patient files. Primary outcome of this study is the comparison of mature oocyte number and fertilisation rate between groups. Secondary outcome is the comparison of implantation rate and live birth rates between groups.
Statistical analysis was performed with SPSS 21.0 version (IBM Corp., Armonk, NY, USA). Comparison of continuous variables between groups were done with student t-test or Mann--Whitney U test according to distribution normality of data. Comparison of categorical variables were done with chi-square test or Fisher exact test where applicable. For investigation of possible relation and interaction, correlation and regression analysis were performed respectively. P-value smaller than 0.05 was accepted as statistically significant.
3. Results {#sec-3}
==========
The demographic characteristics of groups are presented on Table 1. The mean age, infertility duration, BMI and D3 FSH level of G1 were significantly lower than those of G2. AFC of G1 was significantly higher than G2. The infertility aetiologies of groups are presented on Table 1. Decreased ovarian reserve and unexplained infertility were significantly lower in G1 compared to those in G2, but anovulation rate was significantly higher in G1 than G2.
######
Demographic characteristics of groups.
---------------------------- ------------------------- ------------------------ ---------
G1 (FSH/LH \< 2)N = 473 G2 (FSH/LH ≥ 2)N = 175 P-value
Age (years) 30.7 ± 5.2 33.9 ± 5.5 \<0.01
Infertility period (years) 5.9 ± 4.2 6.9 ± 4.8 0.02
BMI (kg/m2) 25.3 ± 4.3 26.3 ± 4.6 0.02
Previous cycle number 1.5 ± 0.9 1.7 ± 1.2 0.6
D3FSH (mIU/mL) 6.4 ± 4.4 9.1 ± 6.4 \<0.01
AFC 10.1 ± 7.4 6.5 ± 5.8 \<0.01
TSH (mIU/mL) 1.7 ± 1.1 1.5 ± 0.9 0.1
Infertility aetiology (%)
-Male factor 38.1 32 0.1
-Tubal factor 10.1 5.5 0.06
-Anovulation 17.5 3.4 0.04
-Endometriosis 7.7 9.8 0.14
-Decreased ovarian reserve 9.7 22.9 0.03
\- Unexplained infertility 17 26.4 0.04
---------------------------- ------------------------- ------------------------ ---------
Note: Values are presented as mean ± SD and n (%).
The stimulation characteristics of groups are presented on Table 2. While the GnRH agonist protocol rate was significantly higher in G1 than G2, the remaining protocol rates did not show significant difference between groups. Total and start HMG doses of G1 were significantly lower than G2. Oestradiol level on hcg day was significantly higher in G1 compared to that in G2. TOC, MOC, and FOC were significantly higher in G1 compared to those in G2. There was no significant difference between groups for parameters of FR, PR, ImR, and transferred embryo number.
######
Stimulation and embryology characteristics of patients.
------------------------------------------------------------------------- ------------------------- ------------------------ --------------
G1 (FSH/LH \< 2)N = 473 G2 (FSH/LH ≥ 2)N = 175 P-value
Pituitary suppression type (%)-antagonist -agonist -micro dose flare-up 63.3370.7 71.6280.4 0.060.010.12
Oestradiol level on hcg day (pg/mL) 2305.1 ± 1250.1 1656.4 ± 1144.8 \<0.01
Endometrial thickness on hcg day (mm) 10.4 ± 2.3 10.1 ± 2.5 0.1
Progesterone level on hcg day (ng/mL) 0.9 ± 0.4 0.8 ± 0.4 0.23
Stimulation duration (day) 9.0 ± 1.8 9.3 ± 1.9 0.06
HMG start dose (IU) 76.9 ± 146.8 168.4 ± 199.4 \<0.01
FSH start dose (IU) 251.9 ± 124.7 245.7 ± 173.3 0.3
HMG total dose (IU) 582.0 ± 1045.4 1236.1 ± 1377.5 \<0.01
FSH total dose (IU) 1831.6 ± 1016.5 1749.6 ± 1342.3 0.6
Total oocyte number 13.1 ± 7.9 8.8 ± 6.4 \<0.01
Mature oocyte number 9.7 ± 6.7 6.5 ± 4.6 \<0.01
Fertilized oocyte number 7.1 ± 5.1 4.9 ± 3.7 \<0.01
Mature/total oocyte rate (%) 73 76 0.12
Fertilization rate (%) 77 79 0.4
Implantation rate (%) 66 58 0.1
Transferred embryo number 1.9 ± 0.7 1.8 ± 0.6 0.3
Pregnancy rate (%) 37.6 35.4 0.4
------------------------------------------------------------------------- ------------------------- ------------------------ --------------
Note: Values are presented as mean ± SD and percentage.
The pregnancy outcomes are presented on Table 3. There were no differences between groups for parameters of live birth rate and abortion rate.
######
Pregnancy outcomes.
------------------- --------------------------- --------------------------- ---------
G1 (FSH/LH \< 2)(N = 178) G2 : (FSH/LH ≥ 2)(N = 62) P-value
Chemical abortion 36 (21%) 9 (16%) 0.72
Missed abortion 29 (16%) 12 (20%) 0.24
Ectopic pregnancy 2 (0.5%) 2 (0.5%) 0.16
Preterm delivery 3 (0.5%) 0 0.55
Live birth 108 (62%) 39 (63.5%) 0.85
------------------- --------------------------- --------------------------- ---------
Note: Values are presented as n (percentage).
Correlation (R = 0.08, P = 0.02) and regression analysis (OR = 0.6, 95% CI = 0.188--0.222, P \< 0.01) revealed no relation between FSH/LH ratio and mature/total oocyte ratio. There was no relation between FSH/LH ratio and rates of fertilization and implantation.
4. Discussion {#sec-4}
=============
In this study we observed that increased FSH/LH ratio has no detrimental effect on pregnancy outcomes of infertile women. Increased FSH/LH ratio brings the costs of increased gonadotropin doses for stimulation with decreased total collected oocyte count. Fertilisation, implantation, and pregnancy rates of women with increased FSH/LH ratio did not show difference from women with normal FSH/LH ratio. Our results showed that decreased ovarian reserve did not have negative influence on pregnancy outcomes. In our study, we determined the live birth rate of our population and we observed that high FSH/LH ratio did not show negative impact on live birth rate.
Low ovarian response despite appropriate ovarian stimulation is an undesirable result of IVF programme \[10\]. The underlying causes of ovarian resistance to gonadotropin stimulation are still unclear. POSEIDON group (Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number) improved a new classification system in patients with decreased ovarian reserve or unexpected inappropriate ovarian response to exogenous gonadotropins \[11\]. The application of POSEIDON classification system with retrospective analysis would be more appropriate for prognosis evaluation \[12\]. AMH estimation, AFC/TOC ratio, FSH/LH ratio are the most recent methods that are used to predict stimulation outcome \[13,14\]. In our study, we compared the results of cycles between women with increased FSH/ LH ratio and normal. We observed decreased total and mature oocyte counts in women with increased FSH/LH ratio. But interestingly, this decrement did not show negative impact on pregnancy and live birth rates. Khan et al. compared infertile and fertile women for age related ovarian reserve decrement among the population under 40 years. They observed no significant difference for serum AMH levels and AFC between infertile and fertile women \[15\]. Similar to our results, Prasad et al. observed increased gonadotropin doses requirement with decreased MOC on women with FSH/LH ratio higher than 2. However contrary to our results, Prasad et al. reported lower pregnancy rates on women with increased FSH/LH ratio \[7\]. Zhen et al. investigated the outcomes of 472 IVF-ET cycles belong to 426 poor ovarian response patients. They detected significantly lower clinical pregnancy rate in women aged \>40 years than in those aged \< or = 40 years \[16\].
Ho et al. used the cut-off value of 3 for FSH/LH ratio and they observed that women with FSH/LH ratio bigger than 3 revealed less mature oocytes \[17\]. Johnson et al. reported that increased FSH/LH ratio despite normal basal FSH level pointed to decreased ovarian reserve and poor response \[18\]. Liang et al. compared basal FSH/LH ratio between cycles with cancellation or not. They observed significantly increased FSH/LH ratio in the cycles ended with cancellation \[19\]. Mukherjee et al. compared the cycle outcomes of 74 patients either with FSH/LH ratio bigger than 3.6 or not. They observed significantly lower total oocyte count and increased cycle cancellation rate in the group with FSH/LH ratio bigger than 3.6 \[20\]. Liu et al. investigated retrospectively 297 cycle outcomes of women either with FSH/LH ratio bigger than 2 or not. They reported increased cycle cancellation rate and lower pregnancy rate in women with FSH/LH ratio bigger than 2 \[21\]. Rehman et al. evaluated the cycle outcomes of 282 women retrospectively. They classified women into 2 groups according to median FSH/LH ratio of their population. They compared cycle outcomes between women with FSH/LH ratio bigger than 1.26 or not. They reported lesser oocyte and embryo quality in women with FSH/LH ratio bigger than 1.26 \[3\]. There was no consensus among the researchers about the FSH/LH ratio to predict the stimulation outcome. In our study, we excluded the cycles ended with cancellation. Due to this criterion, we did not make a comment for cycle cancellation rate on the basis of increased FSH/LH ratio.
Huang et al. researched the factors affecting live birth rate with a population consisted of 2277 IVF/ICSI-ET cycles. They observed significant differences between cycles ended either with live birth or not for the parameters of maternal and paternal age, body mass index, poor ovarian response, TOC, number of transferred embryos. They emphasized that maternal age and number of embryos transferred were independent factors affecting live birth rate \[22\]. Our results differed from those of Huang et al. This difference may arise from our small population. In a multicentre study, the researchers investigated the effect of TOC on cumulative live birth rate of patients experienced frozen-thawed ET after fresh cycle. They observed that the probability of live birth increased from 7 oocytes \[23\]. In another study, investigators analysed the impact of ICSI on cumulative live birth rate among couples without male factor infertility. The median time spent by couples from oocyte pick-up to live birth was similar between IVF and ICSI cycles \[24\]. Interestingly Rao et al. observed that physically active women experienced higher live birth rates compared to physically inactive women in IVF/ICSI cycles \[25\].
Orvieto et al. compared cycle outcomes between women stimulated with either HMG or rFSH on a population with FSH/LH ratio bigger than 2. In HMG group, they observed significantly higher number of top-quality embryos and higher implantation and clinical pregnancy rates compared to those in rFSH group \[26\]. In our study, the women with FSH/LH ratio bigger than 2 received more HMG compared to women with FSH/LH ratio smaller than 2. This treatment modification might improve pregnancy outcomes in our population. Barroso et al. investigated cycle outcomes of infertile women with either FSH/LH ratio bigger than 3 or not. After adjustment for transferred embryo number and age, women with FSH/LH ratio bigger than 3 showed significantly lower MOC, implantation, and pregnancy rates compared to women with FSH/LH ratio smaller than 3 \[1\]. In our study clinical pregnancy and live birth rates of women with FSH/LH ratio bigger than 2 did not show significant difference from those of women with FSH/LH ratio smaller than 2.
In conclusion, in our population, increased FSH/LH ratio did not affect the rates of pregnancy and live birth negatively. Expanded and prospectively organised studies are needed to discuss our results.
Acknowledgments {#sec-ack}
===============
We thank staff of Fırat University IVF Unit for their support during our study.
[^1]: **CONFLICT OF INTEREST:**
The authors declare that there is no conflict of interest to disclose.
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Protocol
========
1. Removal of the Abdominal Mammary Gland and Preparing a Whole Mount
---------------------------------------------------------------------
1. Euthanize the animal according to the IACUC guidelines. Pin the animal on its back from its legs with needles, or tape the legs onto the surface board , and wipe the skin wet with ethanol (EtOH). Lift the skin a little bit with forceps and with sharp scissors make a midline incision into the skin starting between the pair \#5 nipples and cut towards the neck . Make an inverted Y incision from the midline towards the hind legs . If thoracic mammary glands are also to be collected, make also an Y incision from the midline between the pair \#2 nipples towards the front legs.
2. Dissect the skin open with scissors on one side of the inverted Y incision to expose the abdominal \#4 mammary gland; the mammary glands are attached to the skin. Pin the skin with needles onto the surface board , to completely expose the gland. Work one side and gland at the time.
3. Dissect the mammary gland free from the skin either using sharp scissors and/or a scalpel, starting from the proximal area close to the nipple and working towards the distal end of the gland towards the spine of the animal . (This can take several minutes, especially in an older rat.)
4. Immediately spread the detached gland onto an appropriately labeled glass slide (use a proper permanent marker pen and/or \'a diamond pen\'), and spread the gland carefully corresponding to its original size and shape *in situ* . The glass slide must be bigger than the gland. After spreading the gland onto the slide, let it sit on the table for a while, so that the gland sticks onto the slide, but don\'t let it dry.
5. Put the slide into a jar containing Carnoy\'s fixative (75% glacial acetic acid, 25% absolute ethanol=EtOH) , let it get fixed at room temperature (RT) in the fume hood for 2 days or longer. Slides can also be left in the fixative for a longer period of time.
6. Wash the slides in 70% EtOH for 1 hour at RT.
7. Rinse in distilled water for 30 min at RT.
8. Stain in Carmine Alum stain\*) for 2 days or longer (until you see that the lymph nodes have stained through; look at the back side of the slide).
9. Wash in increasing series of EtOH, 1 hour in each: 70% -\> 95% -\> 100% . After the last wash in absolute EtOH put the glands in xylene . Let sit in the fume hood in RT at least for 2 days. This last step is clearing of the mammary gland meaning delipidation of the mammary fat pad, and subsequent increase in transparency. The fattier the gland is the longer clearing time is required.
10. Mount with cover-slips using a mounting media, such as Permount . Let the slides dry well (several days) before observing under a stereo microscope.\*Carmine Alum Stain:Place 1 g carmine (Sigma C1022) and 2.5 g aluminum potassium sulfate (Sigma A7167) in 500 mL distilled water and boil for 20 min. Adjust final volume to 500 mL with water. Filter and refrigerate. Solution can be used for several months. Discard when color becomes weak.
2. Analysis of Mammary Gland Whole Mount Morphology
---------------------------------------------------
Mammary gland whole mounts morphology is analyzed according to the following end-points and the results correlated with mammary cancer risk.
1. Mammary gland whole-mount at PND 21 (pre-pubertal age): Epithelial growth: Distance from the nipple to the end of epithelial tree (in millimeters), measured using a rulerPotential for malignant transformation: Number of TEBs (terminal end buds), counted under a light microscope . TEBs are the largest bulbous structures located only at the distal end of the mammary epithelial treeDifferentiation: AB1 (alveolar bud) score (0-5). Alveolar buds are spread across the epithelium.AB2 score (0-5) Wholes mounts are scored under a light microscope. The score values from AB1 and AB2 are added for a final differentiation score.
2. Mammary gland whole-mount collected at PND 50 (post-pubertal age) : Epithelial growth: Distance from the lymph node to the end of epithelial tree (in millimeters), measured using a ruler.Distance from the tip of the epithelial tree to the end of fat pad, measured using a ruler.Potential for malignant transformation: Number of TEBs, counted under a light microscopeDifferentiation: AB1 score (0-5)AB2 score (0-5)Lobules score (0-5) Wholes mounts are scored under a light microscope. Differentiation will be assesses in two ways. First, the score values from AB1, AB2 and lobules are added for a final differentiation score. In addition, the ratio between lobules score and AB1+AB2 score will be calculated. ABs differentiate to lobules, and the higher the ratio between lobules and ABs, the more differentiated the gland is.
3. Palpation and Mammary Tumor Measurement in Rats and Correlation to Mammary Gland Morphology
----------------------------------------------------------------------------------------------
1. To begin this procedure, hold the rat by grasping the whole body with the palm over the back, with forefinger behind the head and the thumb and second finger under the opposite axilla. Turn the rat so it is lying on its posterior, and palpate to detect any mammary tumors; palpable tumors should feel like a \"lump\".
2. Next use a caliper to measure the width, length, and height of the tumor. Calculate the tumor volume using the ellipsoid formula, Volume =1/6πabc, where \'a\'= width, \'b\' = length, and \'c\' = height.
3. Manually record the location and size of the tumor in a notebook on a weekly basis. This data will be transferred to a spreadsheet later.
4. Representative Results
-------------------------
Careful dissection of the mammary fat pad and its processing to wholemount will allow assessment of developmental state of the mammary gland. When each step is done correctly, the whole mammary epithelial tree is clearly visible within the fat pad, and this allows easy determination of the number of TEBs and calculation of the density of alveolar buds and lobules. Missteps in preparing wholemounts may include failure to dissect the whole fat pad, insufficient fixing, and inadequate clearing.
Assessment of mammary gland morphology will provide information about the number of structures present that can give rise to mammary tumors (TEBs), degree of differentiation of the epithelial structures (alveolar buds and lobules), and measure of growth (ductal elongation). It is important to separate TEBs to terminal ends; the latter are located at the distal end of the epithelium, similarly to TEBs, but they are smaller than TEBs and do not give rise to malignant tumors. Terminal ends are also seen within the epithelial tree.
**Figure 1: Representative result of well prepared mammary gland whole mount.** All components of the gland have been properly dissected and carmine staining is optimal.
**Figure 2: Representative result of a poorly prepared mammary gland whole mount.** The gland has been properly dissected (the distal portion of the gland is missing), has not been properly stretched and delipidation has not completely occurred.
Discussion
==========
Assessment of mammary gland morphological end-points and the growth of the epithelial tree can be used to predict whether early life dietary manipulations, or other manipulations which alter *in utero* or prepubertal hormonal environment, modify later mammary cancer risk. Since breast cancer in humans is initiated in mammary structures (terminal ductal lobular units, TDLUs) similar to TEBs in rats, this technique can be used to determine the potential of early life exposures to affect breast cancer risk. Further, in women high mammographic density increases the risk of breast cancer by 4-6 fold, and assessment of the growth of mammary epithelial tree can be used to identify factors which determine mammographic density and factors which reduce this density. To obtain this information, mammary gland should be fully dissected, properly stretched on a glass slide so that all its components can be visualized under a microscope. In addition, proper carmine staining and delipidation in xylene will provide whole mounts that are suitable for mammary cancer risk assessment.
Disclosures
===========
No conflicts of interest declared.
NCI (U54 CA00100970), NCI (1 R03 CA150040-01), ACS(116602-PF-09-018-01-CNE)
[^1]: Correspondence to: Sonia de Assis at <[email protected]>
| {
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Introduction
============
Signaling by the phosphatidylinositol-3-kinase (PI3K)-Akt pathway is a central regulator of cell growth, metabolism, and survival ([@B89]; [@B183]). PI3K-Akt signaling is involved in a wide range of physiological processes in many different cells and tissues, and at various stages of development including homeostasis of adult tissues. Disruptions of PI3K-Akt signaling also contribute to disease, such as the insulin resistance and type II diabetes that results from impaired PI3K-Akt signal transduction in insulin-responsive tissues ([@B30]; [@B183]). In contrast, inappropriate amplification of PI3K-Akt signaling is present in many different types of cancer cells thereby driving cell growth and tumor progression ([@B52]; [@B44]; [@B168]; [@B90]; [@B282]; [@B89]; [@B183]).
PI3K-Akt signaling occurs following a wide range of cues emanating from the plasma membrane, such as receptor tyrosine kinases, G-protein coupled receptors and immune receptors ([@B89]; [@B183]). Activation of these receptors, typically but not exclusively resulting from ligand binding, leads to signals that classically activate class I PI3K, leading to the production of phosphatidylinositol-3,4,5-trisphosphate (PIP~3~) from phosphatidylinositol-4,5-bisphosphate (PI45P~2~). In addition, receptor signaling can lead to the production of phosphatidylinositol-3,4-bisphosphate (PI34P~2~), either by dephosphorylation of PIP~3~ by 5-phosphatases or by activation of class II PI3Ks that phosphorylate phosphatidylinositol-4-phosphate (PI4P). PIP~3~ and PI34P~2~ trigger membrane recruitment and potentiate signals that lead to the activation of Akt. In turn, Akt exerts control of \>100 substrates distributed throughout the cell including the plasma membrane, various endomembrane compartments, the mitochondria, cytosol, and the nucleus ([@B300]; [@B240]; [@B75]), thus directing multiple facets of cell physiology.
A central question in the regulation of receptor signaling is the control of these signals by their spatiotemporal organization, such as afforded by the endomembrane system. Many receptor complexes initiate signaling at the plasma membrane and subsequently undergo endocytosis upon ligand binding leading to the transit of active receptor signaling complexes through various endomembranes, which depending on the membrane traffic itinerary of each receptor can include early, recycling and late endosomes ([@B257]; [@B99]; [@B16]). This endocytic movement has led to the central hypothesis that the plasma membrane and various internal membrane compartments represent distinct signaling environments, such that activated receptors may trigger unique signals from each membrane locale. Moreover, the plasma membrane, and by extension various endomembrane compartments are non-homogenous lipid bilayers, comprised of various nanodomains defined by the presence of unique proteins such as clathrin, caveolin, flotillin and tetraspanins, as well as actin-dependent nanodomains, each of which also represent unique signaling nano-environments for activated receptors ([@B66]; [@B173]).
Distinct signaling environments, ranging from nanoscale domains at the plasma membrane to microscale endomembrane compartments represent opportunities for distinct activation, regulation or functional outcome of PI3K-Akt signaling. In this review, we first examine the identity of the molecular players that trigger the initial activation of and/or regulate PI3K-Akt signaling by cues that initiate at the cell surface. Although Akt activation also occurs in response to other intracellular cues, such as in response to DNA damage ([@B169]), we focus here on signals triggered at the plasma membrane by receptor tyrosine kinases (RTKs). We direct the reader to several excellent recent reviews that examine the spatiotemporal organization of signaling by GPCRs and other receptors ([@B146]; [@B274]; [@B93]; [@B39]). We examine the evidence for specific localization of PI3K-Akt signals to various subcellular compartments, including nanoscale domains of the plasma membrane and various endomembrane compartments. Subsequently, we examine the evidence that the spatiotemporal organization of PI3K-Akt signaling within these various compartments may result in distinct outcomes of Akt signaling at each locale.
Molecular Mechanisms of PI3K-AKt Signal Activation
==================================================
Receptor tyrosine kinases are a family of 58 human proteins that are critical for a wide range of physiological processes, from development to maintenance of tissue homeostasis in adults ([@B159]; [@B160]). RTK activation can trigger signals that promote cell growth, proliferation, survival, migration or differentiation, and these outcomes depend on the specific ligand, receptor, and cellular context. RTKs for the most part bind extracellular ligands, which then leads to activation of intrinsic kinase domains that then relay the signal from ligand binding to intracellular signaling networks ([@B314]; [@B159]). RTK activation typically occurs either by ligand binding to a constitutive receptor dimer (as is the case for insulin receptor) ([@B188]; [@B261]), or by stabilization of a receptor dimer (as is the case for the epidermal growth factor receptor, EGFR) ([@B4]).
For many RTKs, activation of their intrinsic kinase domain results in phosphorylation of multiple tyrosine residues within cytosolic motifs of the receptor itself, which in turn serve as ligands to recruit signaling adaptors or enzymes harboring phospho-tyrosine binding (PTB) or Src-homology 2 (SH2) domains. This, in turn, leads to activation of a vast network of signals, which has been extensively reviewed elsewhere ([@B315]; [@B159]; [@B292]; [@B302]). Here, we focus on signals that lead to activation of PI3K, leading in turn to the production of specific phosphoinositides, such as PIP~3~ and PI34P~2~, and subsequently Akt activation. To this end, we discuss PI3K enzymes, how these are activated by direct binding to RTKs or to scaffolding proteins, and how this leads to the activation of Akt. We also consider negative regulation by relevant lipid and protein phosphatases.
PI3K Isoforms
-------------
Phosphatidylinositol-3-kinase enzymes are classified into three classes (I--III). Class I PI3K are comprised of one of several p110 catalytic subunits and one regulatory subunit of variable size ([@B139]; [@B282]). RTKs primarily activate a subset within this group, Class 1A PI3K, which are comprised of a heterodimer of one p110α, β, or δ catalytic subunit and one p85α (or splice variants p50α and p55α), p85β or other regulatory subunits. Class 1B PI3Ks are comprised of the p110γ catalytic subunit and the p101 and p87 regulatory subunits and are largely activated by GPCR signaling ([@B288]), and thus will not be examined here as we focus on RTK signaling. For Class IA PI3Ks, the interaction of regulatory and catalytic subunits is constitutive and in the absence of signals serves to suppress the p110 subunits ([@B195]; [@B287]). Binding of the regulatory subunit to specific motifs harboring phosphorylated tyrosines (e.g., on RTKs or scaffolding proteins, see below) via its SH2 domains relieves the inhibition on p110 subunits. Specific regulatory subunits have additional domains that expand the mechanisms of activation, such as the p110 subunits that can also be activated by binding to Ras via N-terminal Ras-activating domains ([@B291]). The activation of Class IA PI3Ks leads to production of PIP~3~ from PI45P~2~.
Class II PI3Ks are comprised of three isoforms in humans, PI3KC2α, β, and γ ([@B83]; [@B224], [@B225]; [@B184]). These isoforms are each comprised of C2 and PX domains that mediate binding to lipids, especially PI45P~2~ ([@B167]; [@B269]; [@B297]), and a kinase domain that catalyzes the formation of PI34P~2~ from PI4P, as well as phosphatidylinositol-3-phosphate (PI3P) from phosphatidylinositol ([@B83]; [@B224]). Additional protein interaction domains and activities are present in each isoform, such as binding to clathrin for PI3KC2α and β ([@B73]; [@B94]; [@B224]). The regulation of Class II PI3Ks is less well understood. Class II PI3Ks may constitutively associate with membranes and require additional activation signals such as the conformational change in PI3KC2α induced by binding to specific proteins and PI45P~2~ ([@B297]).
Class III PI3K has a sole member, Vps34, which functions at the early endosome to produce PI3P from PI ([@B254]; [@B147]; [@B12]). Vps34 and PI3P are essential for membrane traffic from the plasma membrane to early endosomes and also regulate a number of sorting phenomena, including assembly of the retromer cargo retrieval complex ([@B120]). As PI3P produced by Vps34 does not appear to contribute to activation of Akt signaling directly, we here focus on Class I and II PI3Ks and discuss Vps34 only in the context of its requirement for membrane traffic regulation of Akt signaling.
### PI3K Activation by RTKs
As a result of decades of intensive research, there is considerable insight into the mechanism of activation of Class IA PI3K by RTKs. Some receptor tyrosine kinases can directly bind and thus activate PI3Ks, while others require a scaffolding or binding protein. Here, we illustrate the latter with EGFR and the former mechanism with ErbB3.
In the case of EGFR, binding to ligands leads to activation of the kinase domain, which in turn, leads to the phosphorylation of a number of residues on the C-terminal tail of the receptor ([@B24]; [@B160]; [@B88]). The phosphorylation of Y1068 is essential for binding of Grb2 via its SH2 domain, which then recruits Grb2-associated binder1 (Gab1) via an SH3-proline rich domain interaction ([@B171]). The phosphorylation of Gab1 on Y447, Y472, and Y589 leads to recruitment of Class IA PI3Ks, and production of PIP~3~ leading to Akt activation ([@B124]; [@B187]; [@B148]). Gab1 possesses a PH domain that binds PIP~3~, and as Gab1 membrane binding contributes to PI3K activation, this mechanism of activation of PI3K is subject to positive feedback regulation ([@B234]). Other RTKs such as MET use a similar mechanism of activation, but MET can directly bind Gab1 via a Gab1-binding module found in the cytosolic portion of this receptor ([@B244]). Gab1 is related to other scaffolding or docking proteins that function to control PI3K activation by RTKs, including insulin-receptor substrate (IRS) and fibroblast growth factor receptor substrate 2 (FRS2), which either recruit class IA PI3K directly ([@B36]; [@B259]; [@B30]) or via Gab1 recruitment upon activation of specific RTKs ([@B150]; [@B191]; [@B248]; [@B64]; [@B106]).
In contrast to adaptor-mediated recruitment, other RTKs can directly bind PI3K. Upon binding its ligand, such as neuregulin ([@B45]), and phosphorylation, ErbB3 interacts directly with class IA PI3K leading to PI3K activation ([@B119]; [@B18]; [@B265]). Given the distinction of direct receptor binding *versus* adaptor-dependent PI3K-Akt signal activation by various RTKs, spatial-temporal differences and strength of the signal may occur; however, this remains to be explicitly examined.
Class II PI3Ks can also be activated downstream of RTK activation. PI3KC2α and β are recruited to EGF and platelet-derived growth factor receptor (PDGFR) signaling complexes ([@B9]), via recruitment to adaptor protein complexes that include Grb2 ([@B303]; [@B143]). The detailed mechanisms for this class of PI3K remain much less well understood than class I PI3K, and additional insight into the regulation of class II PI3K would be very informative.
Akt Activation by RTK Signaling
-------------------------------
The production of either PI34P~2~ or PIP~3~ is essential for the recruitment and activation of Akt by RTKs. There are three isoforms of Akt (1--3), each comprised of an N-terminal pleckstrin homology (PH) domain, an internal kinase domain, and a C-terminal regulatory domain. The isolated PH domain of each isoform of Akt is able to bind either PI34P~2~ or PIP~3~ with similar affinity, but in the context of the full-length protein, Akt1 and 3 preferentially bind PIP~3~ while Akt2 preferentially binds PI34P~2~ ([@B170]). In each case, binding to either PI34P~2~ or PIP~3~ elicits recruitment of Akt to the membrane, in parallel to similar membrane recruitment by these lipids of 3-phosphoinositide-dependent kinase 1 (PDK1) via its PH domain. PDK1 phosphorylates Akt on T308, which together with membrane binding leads to a substantial increase in Akt activity ([@B272]; [@B246]; [@B121]). Phosphorylation of Akt on S473 further enhances Akt activity, which in the context of RTK signaling is elicited by the mechanistic target of rapamycin complex 2 (mTORC2) ([@B241]; [@B212]; [@B98]). Alternatively, phosphorylation can also be mediated by DNA-PK in other contexts ([@B84]; [@B31]; [@B276]). Regardless, the dually phosphorylated Akt has a substantial increase in activity ([@B114]).
While membrane binding and phosphorylation on T308 and S473 represent the canonical activation of Akt by RTKs, there is an increasing appreciation of many different modifications and regulators of Akt ([@B231]). Notably, K63-ubiquitinylation of Akt on K8 and K14, mediated by TRAF6 upon IGF1 stimulation ([@B312]) or Skp2 SCF upon EGF stimulation ([@B49]) is required for Akt activation, membrane recruitment and substrate phosphorylation, a modification that is negatively regulated by the deubiquitinase CYLD ([@B311]). This K63-ubiquitinylation does not appear to control Akt degradation. Further adding to the complexity of regulation of Akt activation by post-translational modification, Akt methylation on K64 by SETDB1 promotes the binding of JMJD2A, which functions as an adaptor to recuit TRAF6 or the Skp2 SCF and K63-ubiquitinylation of Akt, required for Akt activation ([@B296]). While mechanisms such as K63-mediated ubiquitinylation control Akt activation, much remains to be determined about how these mechanisms are regulated, and how these impact Akt cellular localization.
All three isoforms of Akt follow this activation mechanism and share some overlapping substrates. However, there are substrates of Akt that are isoform-specific, and in many contexts, Akt isoforms are non-redundant with distinct roles in cellular and systemic physiology ([@B270]; [@B102]; [@B256]; [@B238]). For example, Akt1 but not Akt2 phosphorylates palladin, an actin-bundling protein ([@B54]). This may contribute to the distinct ability of Akt1 to promote breast tumor initiation and impair invasion and migration ([@B130]; [@B133]), while Akt2 enhances the invasive and metastatic capabilities of breast tumors ([@B8]; [@B133]).
Attenuation of PI3K-Akt Signaling Downstream of RTKs
----------------------------------------------------
Given the impact on various aspects of cell physiology, mitogenic signaling emanating from RTKs and propagated through PI3K-Akt signaling is subject to negative regulation at various levels. A large number of phosphatases negatively regulate phosphorylation of RTKs and their adaptors and scaffold/docking proteins ([@B159]; [@B313]; [@B206]). RTKs are also subject to negative regulation by degradation secondary to internalization. Examination of this aspect of RTK signaling is beyond the scope of this review, but we direct the reader to several excellent comprehensive reviews on this subject ([@B235]; [@B266]; [@B267]; [@B129]; [@B38]; [@B62]).
Several lipid and protein phosphatases regulate these signals directly at the level of PI3K-Akt. PTEN is a lipid phosphatase that negatively regulates PI3K-Akt signaling by dephosphorylation of PIP~3~ to produce PI45P~2~ ([@B158]), and also negatively regulates PI34P~2~ ([@B180]). Given its central role in negative regulation of PI3K-Akt signaling, PTEN is a potent tumor suppressor, and disruptions of PTEN actively promote tumor growth and progression ([@B158]). In addition, SH2-domain containing inositol phosphatase 2 (SHIP2, also known as INPP1L) dephosphorylates the 5-position of PIP~3~, leading to the production of PI34P~2~ ([@B108]; [@B170]). While regarded in some ways as a negative regulator of PI3K-Akt signaling by catalysis of turnover of PIP~3~, that SHIP2 leads to the production of PI34P~2~ may potentiate the activation of specific isoforms of Akt. Indeed SHIP2 is responsible for the production of PI34P~2~ that selectively activates Akt2 ([@B170]). An additional phosphatase, INPP4B, has recently emerged as a negative regulator of PIP~3~ and PI34P~2~ ([@B149]), yet other studies have noted that INPP4B promotes Akt signaling by relieving negative regulation of Class I PI3K ([@B227]). Hence, while PTEN and INPP4B are potent suppressors of PI3K-Akt signaling, SHIP2 has a more complex role in the regulation of this pathway.
Several phosphatases act to directly regulate phosphorylation of Akt. Protein phosphatase 2A (PP2A) is a well-established negative regulator of Akt that elicits Akt dephosphorylation, in particular on the T308 site ([@B233]; [@B258]). In addition, PH domain leucine-rich repeat protein phosphatase (PHLPP) 1 and 2 are two phosphatases that act selectively on the S473 site ([@B95]). Interestingly, these two PHLPP isoforms exhibit specificity for different Akt isoforms, such that PHLPP1 regulates signaling by Akt2 and PHLPP2 regulates signaling by Akt3 ([@B35]).
With this framework of activators and negative regulators of PI3K-Akt signaling, we next discuss the critical contributions of spatiotemporal activation of Akt signals at different scales: within nanodomains at the plasma membrane, and within endomembrane compartments. Subsequently, we examine how Akt functionally controls cell and systemic physiology, with a focus on compartment-specific activation and functions of Akt.
Localization of PI3K-AKt Signaling Within Plasma Membrane Nanodomains
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The initiation of signaling at the plasma membrane involves the spatiotemporal organization of receptors and cytoplasmic proteins that transduce extracellular signals to the appropriate intracellular destination. Recent technological advances, particularly in the field of live-cell fluorescence microscopy, have revealed that signaling receptors are heterogeneously distributed in the plasma membrane, as a result of enrichment in distinct plasma membrane nanodomains. These nanodomains vary in lifetime and composition and include cholesterol-rich structures (caveolae and flotillin), clathrin structures, tetraspanin-enriched nanodomains, dorsal actin ruffles, and Ras nanoclusters. From this vantage, these nanodomains serve to compartmentalize signaling complexes into transient signaling hotspots on the plasma membrane. In the case of caveola and clathrin, which are capable of forming *bona fide* endocytic vesicles, endocytosis of receptor/nanodomain complexes might also serve as a checkpoint for the redistribution of active signaling complexes to distinct subcellular locales or termination of the signal through degradative pathways. The following examines the evidence for localized PI3K-Akt signaling within specific plasma membrane nanodomains.
Clathrin
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Cells have adopted several unique mechanisms for the internalization of extracellular material, membrane proteins, lipids, and solutes. Perhaps the best-described mechanism involves the formation of clathrin-coated pits (CCPs) at the plasma membrane and subsequent clathrin-mediated endocytosis (CME). CCPs initiate by the recruitment of the clathrin adaptor protein complex 2 (AP2), to the plasma membrane by recognition of internalization motifs on cargo proteins destined for CME and by binding PI45P~2~ ([@B249]; [@B194], [@B193]; [@B189]; [@B279]; [@B57]; [@B142]). This is followed by the assembly of other components the clathrin coat, which in addition to clathrin includes accessory proteins, eventually leading to scission from the membrane by the GTPase dynamin. Following this internalization, nascent vesicles undergo uncoating, followed by membrane traffic and sorting. CME has been described in the context of cell signaling as an essential regulator of EGFR signaling dynamics, whereby EGF (ligand) stimulation of EGFR leads to internalization of the ligand/receptor complex ([@B266]; [@B99]; [@B251]; [@B62]). Internalized ligand/receptor complexes are delivered to the endosomal system, which can lead to ubiquitin-dependent lysosomal degradation or recycling back to the plasma membrane. Thus, depending on the cellular context, RTK internalization by CME can attenuate signaling through receptor internalization, lead to degradation, or prolong signaling by receptor recycling.
Beyond their role in endocytosis, recent evidence suggests that a subset of plasma membrane CCPs may also represent unique clathrin nanodomains that directly influence Akt signaling by orchestrating the assembly of transient receptor signaling complexes on the plasma membrane. This previously unrecognized role of clathrin nanodomains as signaling scaffolds suggests another critical level of control over receptor signaling. EGF treatment of ARPE-19 cells leads to the accumulation of EGF and phosphorylated Gab1 (pY627), the most receptor-proximal upstream activator of PI3K/Akt, in clathrin structures on the plasma membrane ([@B97]; [@B175], [@B174]). Perturbation of CCP formation, but not receptor endocytosis, attenuates Gab1 (pY307 and pY627) and Akt (pT308 and pS473) phosphorylation ([@B97]), supporting a role for some clathrin structures as signaling nanodomains required for PI3K-Akt signaling. Interestingly, the engineered expression of ErbB2 in ARPE-19 cells, which normally express little ErbB2, rescues the inhibitory effects of clathrin perturbation on Akt (pS473) phosphorylation ([@B97]).
In addition, phosphorylated Akt and PTEN preferentially localize to short-lived CCPs in MCF10A breast epithelial cells ([@B237]). Deletion of PTEN in these cells or addition of supplemental PIP~3~ enhanced the initiation of short-lived CCPs. These effects were mirrored in MDA-MB-231 and SUM149PT triple negative breast cancer cells, which lack ErbB2 and functional PTEN, respectively. Together, these studies suggest that a distinct subpopulation of clathrin structures at the plasma membrane form nanodomains required for PI3K-Akt activation in cells lacking ErbB2. In contrast, co-expression of ErbB2 leads to EGFR-dependent Akt activation that is clathrin-independent ([@B97]). Furthermore, PI3K/Akt signaling at clathrin nanodomains is directly influenced by the phosphatase activity of PTEN, through the control of PIP~3~ abundance on the plasma membrane ([@B237]). Collectively, these studies support the notion that a subset of clathrin structures function as signaling nanodomains at the cell surface, as has been proposed for certain aspects of GPCR signaling ([@B79], [@B78]; [@B80]).
Components of the PI3K/Akt signaling pathway impinge on different stages of CCP formation and play a reciprocal role in regulating CME. Akt activity positively regulates CME through a mechanism that leads to dephosphorylation and thus activation of dynamin-1 (pS774) ([@B228]). Collectively, these studies establish the existence of a reciprocal regulation network in which plasma membrane clathrin nanodomains directly facilitate Akt activation at the plasma membrane, followed by modulation of ligand/receptor complex traffic after CME. In turn, multiple levels of signaling converge to control CME.
Caveolin and Flotillin Membrane Nanodomains
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Caveolae are 50--100 nm bulb-shaped invaginations on the plasma membrane that are primarily composed of oligomers of the integral membrane protein caveolin-1, and the cavin proteins, which are essential for caveolae formation ([@B122]). Caveolae are typically thought of as a type of membrane rafts. This is due to the ability of caveolins to bind cholesterol, the sensitivity of caveolae to disruption of membrane cholesterol, and the low buoyant density of isolated caveolae ([@B264]). Insights into the role of caveolae in PI3K/Akt signaling has mostly been inferred through rather harsh disruption of the cell surface by cholesterol depletion or overexpression of caveolin-1 in cell lines without endogenous caveolin-1 expression ([@B219]; [@B85]) but given the limitations of these approaches, the interpretation of such results should be taken with caution ([@B324]). Furthermore, caveolin-1 knockout mice are viable suggesting that essential growth factor signaling remains intact ([@B74]; [@B220]). While silencing of caveolin-1 has been reported to enhance Akt activity in endothelial cells ([@B103]), the limitations of methods used to alter caveola have contributed to the inconsistencies in the literature concerning the effects of caveolin-1 on PI3K/Akt signaling, and thus results should be interpreted with caution.
Many studies revealed interaction of receptor tyrosine kinases with caveolin proteins or incorporation of receptors within caveolae ([@B66]). The impact of this nanoscale compartmentalization is complex and in some cases impacts regulation at the level of the receptors themselves, thus broadly impacting multiple signaling pathways ([@B309]; [@B208]; [@B19]; [@B289]; [@B58],[@B60], [@B59]; [@B86]; [@B298]; [@B34]; [@B66]; [@B308]; [@B173]). In addition, while there is little direct evidence that EGFR is detected within caveolae ([@B66]) interactions of EGFR within caveolin-1, perhaps in the context of non-caveolar assemblies of caveolin-1 proteins ([@B118]; [@B154]; [@B204]; [@B144]) negatively regulate EGFR ([@B61]; [@B81]; [@B218]; [@B305]; [@B154]; [@B155]). Indeed, caveolin proteins harbor a caveolin-scaffolding domain (CSD, amino acids 82-101) that allows interaction with many different proteins ([@B141]), suggesting a mechanisms for how protein-protein interactions involving caveolins may control RTK signaling to PI3K-Akt.
While these studies establish several different possible modalities of control of receptor tyrosine kinase signaling by caveolins and caveolae at the level of the receptors, there is also evidence of enrichment and control of PI3K/Akt signaling intermediates in these structures. Both PI45P~2~ and PIP~3~ are detected in membrane nanodomains ([@B299]), and insulin-like growth factor (IGF1) stimulation triggers PIP~3~ partitioning into membrane nanodomains that are sensitive to cholesterol perturbation ([@B156]). While these studies establish the non-homogenous partitioning of PIP~3~ in the plasma membrane, it is not clear how these lipid nanoclusters relate to caveolae. Caveola or caveolin proteins may also regulate Akt activity through interactions with Akt kinases and phosphatases. In prostate cancer cells, caveolin-1 sustains Akt signaling by inhibiting the Akt phosphatases PP1 and PP2A through direct interaction with the CSD ([@B161]). Growth factor stimulation by EGF or PDGF activates PDK1 in membrane raft nanodomains defined by Lyn localization that are spatially distinct from membrane regions of PTEN recruitment ([@B96]). This spatial segregation of activating kinase (PDK1) and negative regulation by phosphatases (PTEN) was proposed to be critical for Akt signaling, and disruption of this compartmentalization by ceramide impaired Akt activation ([@B104]; [@B110]; [@B96]).
Flotillin nanodomains are a subset of membrane rafts distinct from caveolae that may also control RTK signaling. These structures are composed of oligomers of the highly conserved flotillin-1 and flotillin-2 proteins ([@B153]; [@B15]). While these nanodomains may also control PI3K/Akt signaling, much less is known about this phenomenon. Several studies that perturbed flotillin function observed impaired Akt signaling ([@B5]; [@B136]; [@B166]), yet it is not clearly established if this represents specific effects on PI3K-Akt signaling or broad regulation of RTKs. Consistent with the latter possibility, flotillins may function to control receptor membrane traffic such as that of IGF1R ([@B136]), or expression of specific receptor tyrosine kinases ([@B226]; [@B11]). Thus, both caveolae and flotillin nanodomains can contribute to control of Akt signaling by receptor tyrosine kinases at many levels, including at the level of the receptor thus broadly impacting many aspects of signaling. Much remains to be learned about the mechanism by which flotillins, caveolins and/or nanodomains formed by these proteins compartmentalize signals leading to Akt activation by receptor tyrosine kinases.
Tetraspanin-Enriched Nanodomains
--------------------------------
Tetraspanins are a large family of 33 proteins in humans, each with four membrane-spanning domains, that form membrane nanodomains through the interaction with other tetraspanins, integral membrane proteins, and cytoplasmic signaling proteins. Given the number of tetraspanin family members and their ubiquitous or tissue-specific distribution, it is no surprise that tetraspanins have been implicated in a diverse array of (patho)physiological processes and signaling pathways involved in immunity, angiogenesis, cancer, and many others ([@B310]; [@B51]; [@B21]; [@B66]; [@B22]; [@B281]; [@B290]; [@B245]).
One of the primary functions of tetraspanins is the organization of plasma membrane receptors to facilitate signaling. Various tetraspanins, including CD9, CD63, CD81, CD82, and CD151 ([@B210], [@B211]; [@B277]; [@B198]; [@B69]; [@B286]; [@B22]), have been reported to interact with specific RTKs. Early studies on tetraspanin CD82 demonstrated that ectopic CD82 expression in cells alters the plasma membrane distribution of EGFR ([@B211]). More recent studies using single particle approaches have revealed that CD82 confines EGFR in distinct regions of the plasma membrane, and loss of CD82 results in enhanced clathrin-mediated endocytosis of the receptor and impaired receptor signaling ([@B63]).
Consistent with the ability of tetraspanins to control the dynamic nanoscale localization of RTKs, perturbations or alterations of tetraspanins impact certain aspects of RTK signaling, from the activity of receptors to specific signaling pathways. For instance, CD82 depletion or blocking antibody treatment attenuates Akt activation, resulting in induction of a pro-apoptotic phenotype ([@B207]). Furthermore, in endothelial cells lacking CD151, Akt activation is attenuated, resulting in impaired angiogenesis, which may be related to the requirement for tetraspanins for the activation of eNOS signaling ([@B278]; [@B321]). This regulation is complex, as perturbation of CD82 can also promote EGFR signaling to Akt activation in other contexts ([@B162]).
Mechanistically, there remains much to be learned about how tetraspanin nanodomains control PI3K/Akt signaling. Tetraspanin domains are indeed enriched in specific signaling regulators such as certain PKC isoforms ([@B319]), lipid enzymes such as phosphatidylinositol-4-kinase ([@B23]; [@B316]; [@B43]; [@B55]) and specific glycosphingolipids ([@B283]; [@B111]; [@B112]). The latter function in conjunction with CD82 to control signaling by EGFR and MET ([@B162]). As with caveolin and flotillin nanodomains, it remains to be determined how tetraspanins may control RTK signaling leading to Akt activation, which could result either from specific recruitment and regulation of PI3K-Akt signaling intermediates or by broad control of RTKs controlling many signaling pathways. Consistent with the latter and with a context-dependent regulation of RTK signaling by tetraspanins, CD151 deletion attenuates ERK but not Akt activation in breast cancer epithelial cells expressing ErbB2 ([@B68]). It is plausible that the differences in Akt activation in response to CD151 perturbation are the result of cell-type specific effects, such as the expression of ErbB2, which is known to modulate the dependence of Akt activation on clathrin ([@B97]).
Future studies aimed at understanding how each of these can impact activation of PI3K/Akt signaling, either directly or in the context of complex regulation of signaling intermediates by compartmentalization within other nanodomains, will be very informative.
Dorsal Ruffles (DRs) and Other Actin-Based Structures at the Plasma Membrane
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Receptor tyrosine kinase signaling triggers dynamic remodeling of the cytoskeleton; in particular, activation of EGFR, PDGFR, IR, MET, and others leads to the rapid formation of actin-rich dorsal ruffles(DRs) at the plasma membrane ([@B2]; [@B125]; [@B66]; [@B317]). While not observed in all cells and contexts, DRs can serve to compartmentalize RTKs into spatially distinct membrane nanodomains for signaling. In addition, DRs act in parallel to RTK internalization by CME as a clathrin-independent mechanism for RTK internalization by macropinocytosis ([@B214]; [@B2]; [@B109]; [@B317]). Internalization of RTKs by DRs through the macropinocytic pathway requires Arp2/3-dependent actin polymerization and PI3K; disruption of each results in attenuated DR formation and RTK macropinocytosis ([@B70]).
In cells in which they are observed, DRs also serve as a platform for PI3K/Akt signaling. In L6 myotubes, insulin stimulates recruitment of the Class I PI3K subunits p110α and p85 to DRs, leading to the production of PIP~3~ and subsequently, Akt1 recruitment to these structures ([@B145]; [@B221]). Similarly, EGF stimulation of A431 cells leads to enrichment of PI45P~2~ and PIP~3~ in DRs, suggesting increased activity of class I PI3K in this compartment ([@B7]). In NIH3T3 cells, PIP~3~ production in DRs leads to the recruitment of the adaptor protein SH3YL1 along with SHIP2, which generates PI34P~2~; knockdown of either SH3YL1 or SHIP2 attenuated DR formation suggesting that PI3K/Akt signaling is essential for DR maturation ([@B115]). Finally, it was recently shown that EGF and PDGF stimulate Akt activation in DRs, with distinct requirements for microtubules ([@B317]). Taken together, DRs comprise a distinct membrane-associated nanodomain that serves as a platform for Akt activation and signal termination via RTK macropinocytosis; this platform requires dynamic remodeling of the actin cytoskeleton and in certain contexts, microtubules.
Akt Activation at the Primary Cilia
-----------------------------------
The primary cilia is a microtubule-based protrusion at the plasma membrane that acts as a hub for the integration of a number of important mechanochemical signals which regulate cellular growth, development, and quiescence. The primary cilia, of which there is typically only one per cell, is anchored via its assembly of microtubules to a centriolar anchor known as a basal body ([@B216]; [@B181]). The assembly of the primary cilia, known as ciliogenesis, occurs following mitosis largely in quiescent cells ([@B105]). Illustrating the importance of this structure, at least 35 human diseases (ciliopathies) are caused by mutations in genes involved in the function of cilia, including the primary cilia ([@B229]). Many signaling pathways are either activated in a localized manner at the primary cilia, or impact ciliogenesis, or both, including those triggered by Hedgehog, Notch, Wnt, Hippo, and certain GPCR ligands ([@B14]; [@B304]). Here, we highlight studies that examined RTK-mediated Akt signaling in the primary cilia.
Platelet-derived growth factor (PDGF) receptor α is a RTK that localizes to the mature primary cilia, and following PDGF treatment, Akt is activated in the primary cilia of NIH 3T3 fibroblasts ([@B253]). Genetic disruption of IFT88 or IFT20, two different component of the primary cilium intraflagellar transport system, attenuates or enhances PDGFRα and Akt activation at the ciliary basal body in fibroblasts, respectively, thus altering wound healing ([@B252]; [@B56]; [@B250]). As with caveolin and flotillin nanodomains, it remains to be determined if cilia specifically control PI3K-Akt signaling by localized recruitment of these signals to cilia, or by a broader regulation that occurs at the level of RTKs themselves, thus impacting many signaling pathways. New insight in the former possibility may obtained from observations that PI3KC2α is localized at the primary cilia basal body ([@B87]) and that PDGFRα stimulation of fibroblasts also leads to recruitment of phosphorylated Akt to the basal body, where it can phosphorylate its substrate Inversin to control ciliogenesis ([@B275]).
Ras Nanoclusters
----------------
The Ras proteins (H-Ras, K-Ras, and N-Ras) are a family of small GTPases that are important downstream effectors of RTK signaling ([@B255]; [@B263]). Following RTK activation by external stimuli, Ras proteins are recruited by Son of sevenless (SOS)-Grb2 to the tyrosine phosphorylated cytoplasmic residue of the receptor. From here Ras is activated by SOS GEF activity, leading to canonical activation of the Raf-MEK-ERK pathway. Single molecule and EM imaging revealed that Ras proteins form short-lived signaling nanodomains at the plasma membrane ([@B113]; [@B1]; [@B138]; [@B322]). These short-lived Ras nanodomains might regulate PI3K/Akt signaling independently of RTK-Grb2-Gab1 axis, as Ras proteins interact with the p110 catalytic subunit of Class I PI3K isoforms leading to its activation ([@B201]), and genetic disruption of the Ras-PI3K interactions leads to impaired growth and development ([@B46]). The Ras-PI3K p110α interaction is mediated by phosphorylation of K-Ras S191, and this causes formation of phosphorylated K-Ras nanoclusters that are distinct from the membrane population of non-phosphorylated K-Ras ([@B17]). From here the Ras-PI3K interaction can stimulate the production of membrane PIP~3~ ([@B92]), which presumably triggers Akt activation. Consistent with control of PI3K-Akt signaling by Ras nanoclusters, a G12V mutant of K-Ras exhibits enhanced clustering of PI45P~2~ and an enhanced ability to elicit Akt activation compared to wild-type K-Ras ([@B323]), but the mechanism by which this control occurs remains to be fully elucidated. Importantly, while these mechanisms of protein-protein interaction predicts that PI3K-Akt signaling can be triggered by Ras and thus controlled within Ras nanoclusters, this remains largely speculative and is in need of further investigation.
As discussed in the preceding sections, there are numerous mechanisms by which PI3K-Akt activation can depend on or be facilitated by plasma membrane nanodomains. That multiple such distinct nanodomains operate for different RTKs is perhaps not surprising given the diversity of signals and activation mechanisms of these receptors. However, there are also numerous examples of specific RTKs for which different studies have identified a role of distinct nanodomains in the activation of PI3K-Akt signals. For example, EGFR-dependent Akt activation requires clathrin, flotillin nanodomains and is also regulated by caveolin-1 and dorsal ruffles. This suggests several possibilities: (i) that several distinct nanodomains operate simultaneously to coordinate different specific molecular events in sequence that collectively lead to Akt activation or (ii) that distinct molecular and cellular contexts underlie unique requirements for specific nanodomains in specific situations. To this end, studies that extend beyond the establishment of a functional requirement for specific nanodomains to also resolve the specific molecular mechanisms by which nanodomains facilitate RTK signaling are very informative. Indeed, we previously showed that clathrin nanodomains are required for activation of PI3K-Akt signaling by EGFR only in the absence of ErbB2 ([@B97]), providing a molecular explanation for the context-dependent requirement for clathrin vs. other nanodomains for EGFR-dependent activation of PI3K signaling. Also, studies that undertake a systematic analysis of RTK signaling within nanodomains at the cell surface will further illuminate how each type of receptor and/or intracellular signal may engage and require distinct nanodomains either simultaneously or selectively, in a context-dependent manner. Such studies will reveal the molecular details of the nanoscale organization of the plasma membrane that is essential for PI3K-Akt signal activation.
Localization of PI3K-AKt Signaling Among Endomembrane Compartments
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A fundamental, yet still incompletely answered question is how is Akt able to synthesize diverse inputs into a response that targets only the appropriate substrates at the proper location. The conventional mechanism of Akt activation involves recruitment of inactive Akt to membrane sites of PI34P~2~ or PIP~3~ through interaction with its PH domain. Indeed, structural studies elucidated that binding of the Akt PH-domain to membrane phosphoinositides results in a conformational change that relieves the autoinhibitory interaction between the PH and catalytic domains, resulting in enhanced substrate binding capacity and Akt activation ([@B75]; [@B177]). However, there are conflicting models regarding the ability of Akt to phosphorylate non-plasmalemmal substrates. The first model involves Akt activation on a particular membrane resulting in Akt becoming "locked" in an active conformation, which then allows redistribution of this active Akt to a multitude of other subcellular locales where it acts on its effectors ([@B190]; [@B41],[@B42]; [@B6]). A second more recent model suggests that Akt is activated in a compartment-specific fashion, whereby inactive Akt is recruited to distinct subcellular locales for activation and is mostly unable to redistribute to other compartments once activated ([@B75]). Here, we first examine the evidence for plasma membrane activation of PI3K-Akt signals, then the evidence for activation of Akt on various specific endosomes, then lastly discuss the evidence for Akt activation in and/or redistribution to other compartments.
Plasma Membrane Akt Activation
------------------------------
Consistent with the role of plasma membrane nanodomains in regulating Akt signaling, there is substantial evidence to implicate the plasma membrane as the primary site of Akt activation ([Figure 1](#F1){ref-type="fig"}). Given that the substrate for the class I PI3K, PI45P~2~ is highly enriched at the plasma membrane ([@B71]; [@B13]), it follows that PIP~3~ production occurs mainly within this compartment ([@B202]). The recent development of sophisticated probes for lipid imaging revealed that PIP~3~ is indeed produced exclusively at the plasma membrane ([@B170]). Moreover, several studies identified that the vast majority of PI34P~2~ is generated by SHIP2-mediated dephosphorylation of PIP~3~ ([@B108]; [@B170]), suggesting that PI34P~2~ is produced secondary to Class I PI3K, and as opposed to production by Class II PI3K ([Figure 1](#F1){ref-type="fig"}). Importantly, two distinct probes identified either exclusive plasma membrane ([@B108]) or plasma membrane and endosome ([@B170]) production of PI34P~2~. Notably, this localized production of PIP~3~ and PI34P~2~ was associated with isoform-specific localized activation of Akt, with Akt2 selectively activated at the plasma membrane or endosomal sites by PI34P~2~ while Akt1 and Akt3 display a preference for plasma membrane PIP~3~ ([@B170]). Thus, these findings support the model of activation of class I PI3Ks at the plasma membrane, leading to PIP~3~ production therein, which can then be coupled to direct activation of Akt at the plasma membrane (e.g., Akt1,3) or transport of PIP~3~ from the plasma membrane to endosomes, allowing for PI34P~2~ production and Akt2 activation in both compartments ([@B170]).
{#F1}
In addition to the roles of PI34P~2~ and PIP~3~ in Akt membrane recruitment and activation, other species of membrane lipids have also been implicated in driving the spatiotemporal organization of PI3K/Akt signaling at the plasma membrane. The PH domain of PDK1 binds PIP~3~ ([@B202]), but it also displays a high affinity for phosphatidylserine (PS), and PI3K inhibition (and thus attenuation of PIP~3~ synthesis) fails to attenuate PDK1-PH domain association with the plasma membrane, while PS depletion results in accumulation of PDK1-PH in the cytoplasm ([@B176]). Similarly, PS depletion renders the Akt-PH domain insensitive to stimulation by IGF1, resulting in its cytoplasmic accumulation ([@B127]). Akt activation is attenuated in fibroblasts expressing a mutant PDK1-PH domain that fails to associate with the membrane ([@B176]), while mutation of the Akt-PH domain to yield a PS-binding defective mutant fails to activate Akt in response to IGF1 ([@B127]). In the latter study, the authors suggest that PS binding to Akt promotes a conformational change that facilitates PIP~3~ binding for subsequent T308 and S437 phosphorylation by PDK1 and mTORC2, respectively ([@B127]). PS is detected in the plasma membrane and on a variety of organelles ([@B82]), thus suggesting that modulation of Akt activation by PS could occur at the plasma membrane as well as other compartments.
Insulin stimulation results in a PI3K-dependent accumulation of the Akt2 isoform at the plasma membrane in adipocytes, while Akt1 was localized to the cytoplasm ([@B242]; [@B101]). Akt1 was unable to elicit phosphorylation of the RabGAP AS160 to control membrane traffic of the facilitative glucose transporter GLUT4, while a mutant of Akt1 that promotes its plasma membrane accumulation rescued phosphorylation of AS160. The preferential recruitment of Akt2 to the plasma membrane following insulin stimulation may be partially mediated by ClipR-59, a CAP-gly domain-containing protein that directly interacts with phosphorylated Akt ([@B72]). Furthermore, insulin-stimulated plasma membrane Akt recruitment was not only influenced by PIP~3~ production therein, but also controlled by ubiquitin-like protein 4A (Ubl4A), which by association with Arp2/3, generates actin structures that assist in delivering Akt to the plasma membrane for subsequent ligand-stimulated activation ([@B320]). These studies support a model where Akt localization to the plasma membrane, either by PIP~3~ production or via delivery by other mechanisms, is critical for Akt activation.
The investigation of EGFR signaling supports further evidence for the plasma membrane-centric view of Akt activation. As previously mentioned in the section on clathrin nanodomains, interfering with clathrin (but not receptor endocytosis *per se*) attenuates Akt signaling, suggesting that the clathrin structures on the plasma membrane orchestrate Akt signaling ([@B97]). In cells depleted of all dynamin isoforms and thus with effectively completely arrested EGFR endocytosis, EGF stimulation leads to sustained, apparently normal Akt signaling ([@B268]). In support of these findings, interfering with EGFR endocytosis or ubiquitination, both of which cause EGFR membrane retention, results in an upregulation of an EGF-dependent gene expression program, some of which is Akt-dependent ([@B33]). Together, these studies suggest that plasma membrane EGFR signaling is sufficient to trigger normal Akt phosphorylation in the cell types examined, at least when assessing the overall activation of Akt and primary transcriptional outcomes.
Activated Akt in Multiple Distinct Endosomes
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Endocytosis of RTKs (EGFR, PDGFR, and others) by CME from the cell surface provides the cell with a means for regulating cell surface signaling while also directing signaling complexes to the various endomembrane compartments via fusion with the endosomal system ([Figure 1](#F1){ref-type="fig"}). Throughout this process, endosomes are characterized by interactions with proteins involved in trafficking and sorting that facilitate the maturation of the nascent endosome. These proteins include members of the Rab GTPase family (e.g., Rab5, Rab7, and Rab11), APPL1, EEA1, and WDFY2 ([@B271]; [@B215]; [@B100]). Beyond merely providing a molecular signature to the endosomes that they occupy; these proteins are also involved in the spatiotemporal regulation of signaling proteins (including Akt) and lipids (including PI3P and PI34P~2~) that may originate at the endosomal level. As such, numerous studies have investigated the mechanisms of Akt signaling throughout the endosomal compartments that are traversed by signaling receptors after ligand binding.
Differences in the subcellular localization of the Akt isoforms have been documented, suggesting that Akt may be activated in a compartment-specific fashion. In general, activated Akt1 and Akt3 occupy similar compartments within the cell, including the plasma and nuclear membranes ([@B170]). Akt2, on the other hand, is consistently found in the cytoplasm, intracellular membranes, where it interacts with early endosomes, as demonstrated by colocalization with the early endosomal markers Rab5, APPL1, EEA1, and WDFY2 ([@B196]; [@B295]; [@B163]; [@B170]). However, as noted above, some studies have also reported that Akt2 is selectively recruited to the plasma membrane upon insulin stimulation ([@B242]; [@B101]).
APPL1 demarks a subpopulation of early endosome through which some signaling receptors such as EGFR transit before eventually undergoing traffic to classical EEA1- and PI3P-positive early endosomes ([@B325]; [Figure 1](#F1){ref-type="fig"}). Importantly, depletion of APPL1 attenuates Akt activation at this site ([@B247]; [@B228]). APPL1 may contribute to sustained Akt activation in endosomes by facilitating interaction with the actin cytoskeleton, through the actin-binding protein MYO6 ([@B185]). Loss of MYO6 results in the shuttling of APPL1- and/or Rab5-positive endosomes from actin ruffles in the cell periphery to the perinuclear space, and impaired Akt activation in response to EGF stimulation. In addition, transition of cargo from PI3P-negative, APPL1-positive early endosomes to APPL1-negative, PI3P-positive early endosomes is regulated by Beclin-1, and as such signaling to Akt is controlled by Beclin-1, perhaps as a result of gating the duration of signaling from APPL1 early endosomes ([@B236]). Thus, APPL1 might contribute to early endosomal Akt signaling by bringing inactive Akt into contact with early endosomes and delaying the transit of endosomes harboring active Akt through the endocytic pathway.
Subsequent to transit through APPL1 endosomes, some RTKs such as EGFR traffic to so-called classical early endosomes, demarked by EEA1, which associates with these compartments through PI3P binding by its FYVE domain ([@B200]; [Figure 1](#F1){ref-type="fig"}). Both EEA1 and APPL1 are individually associated with Rab5-positive early endosomes, but they are typically found as distinct subpopulations ([@B185]), yet Akt can be detected in EEA1 endosomes in some circumstances ([@B205]). The Class II PI3K PI3KC2γ colocalizes with Rab5 positive endosomes ([@B32]) suggesting a mechanism for localized production of PI34P~2~ at Rab5-positive early endosomes, which could lead to Akt activation therein. Indeed, loss of PI3KC2γ results in impaired Akt activation on endosomes. The inositol-3-phosphatase INPP4B is enriched in Rab5 positive early endosomes and loss of INPP4B results in enhanced Akt2 activation in this subpopulation of endosomes ([@B163]). This observation supports the model of localized PI34P~2~ production at early endosomes that is under the compartment-specific tight control of lipid phosphatases. Consistent with this model, Rab5 potentiates the activity of inositol-4-phosphatases, and loss of this endosomal phosphatase leads to enhanced PI34P~2~ levels ([@B260]).
In addition to early endosome subpopulations defined by the presence of APPL1 or EEA1, an additional, early endosomal population that lacks these markers but contains WDFY2 are essential for endocytosis of some receptor cargo ([@B116]). WDFY2, through its FYVE domain, binds PI3P with high affinity and can interact with and co-localizes with Akt2, but not Akt1. Loss of WDFY2 results in loss of Akt2 protein levels and phosphorylation, which suggested a model in which initial activation of Akt2 at the plasma membrane leads to the protection of Akt2 protein from degradation by subsequently binding to WDFY2 endosomes ([@B295]).
Some RTKs transit from early endosomes to late endosomes *en route* to degradation in the lysosome ([@B266]; [@B99]; [@B62]; [Figure 1](#F1){ref-type="fig"}). The transition from early to late endosome to lysosome is characterized by the loss of Rab5 and recruitment of Rab7 ([@B230]); this switch in identity is also accompanied by changes in the phosphoinositide populations on the endosome. Early endosomes are abundant in PI3P, which is produced by the class III PI3K and Rab5 effector VPS34 ([@B134]). PI3P is involved in the homotypic fusion of early endosomes, the recruitment of Rab7 GEFs, and components of the ESCRT complex. Collectively, the actions of many proteins facilitate the sorting and shuttling of the endosome along the endocytic pathway ([@B294]), as well as degradation of material by incorporation of specific cargo such as RTKs into intralumenal vesicles destined for the lysosome ([@B223]; [@B29]).
The late endosome/lysosome has the potential to regulate Akt activation in two broad ways: (i) control of lysosomal sorting that impacts the rate of RTK degradation could modulate signals emanating from late endosomal receptors, thus affecting Akt signal duration, or (ii) the unique phosphoinositide profile of late endosomes/lysosomes may facilitate Akt activation separate from the plasma membrane or early endocytic compartments. Many instances of the former have been presented ([@B159]; [@B99]). For example, alterations in EGFR membrane traffic that lead to accelerated lysosomal degradation result in shorter duration of receptor signaling, including that leading to Akt activation ([@B262]). The regulation of RTK degradation is sophisticated, as in some circumstances perturbation of lysosomal degradation by depletion of Rab7 surprisingly enhanced EGFR/HER2 proteasomal degradation and attenuated EGF-stimulated Akt phosphorylation (S473) ([@B301]). In this view, RTKs signals can remain active and highly relevant to sustain Akt activation at the late endosomes, resulting in enhanced pro-survival Akt signaling in cancer cells.
As previously mentioned, mTORC2 localizes to a subset of early (Rab5+) and late (Rab7+) endosomes in a PI3K-dependent manner ([@B76]). The PI3P-binding protein, Phafin-2, was identified as a positive regulator of EGFR degradation in response to EGF, by promoting EGFR shuttling through the endocytic/lysosomal degradation pathway ([@B222]). Induction of autophagy in cells triggers the lysosomal interaction between Phafin-2 and Akt, which leads to sustained Akt activation to control autophagy ([@B186]). Lysosomal mTORC2 leads to Akt activation in this compartment, thus suppressing chaperone-mediated autophagy (CMA) ([@B10]). Although it is unclear why the function of Akt activation on macro- or chaperone-mediated pathways differs, these studies suggest that late endosomes/lysosomes are sites of Akt activation.
Localized Akt Activation in Distinct Compartments vs. Redistribution of Activated Akt
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Given the requirement for PIP~3~ and PI34P~2~ for activation of Akt (as described above), a model of Akt activation initially emerged, which stipulated that Akt activation occurs at the plasma membrane or in endosomes enriched with PI34P~2~. Once activated in these specific compartments, this model proposes that Akt may adopt a "locked-active" conformation that permits redistribution of the active kinase to the nucleus ([@B190]) or other cellular compartments. This model was largely inferred by the vast number of Akt substrates and their diverse subcellular distributions. Additionally, the high rate of PIP~3~ turnover followed by sustained whole-cell Akt activation observed in early studies supported a model where transient PIP~3~ synthesis at the cell surface activated Akt for redistribution to the rest of the cell ([@B6]). Supporting this model, a FRET-based biosensor of Akt activity demonstrated that Akt activation in the plasma membrane preceded that in the cytosol, suggesting a redistribution of Akt once activated ([@B152]). Using a different FRET-based approach, Akt was found to form a complex with PDK1 prior to activation, such that upon ligand activation, the conformational change in Akt that occurs upon membrane binding allows phosphorylation of Akt, followed by dissociation from the membrane and redistribution of active Akt to distal sites ([@B40]).
Several studies that revealed that Akt activation by RTKs occurs primarily at the cell surface and does not outright require receptor endocytosis *per se* ([@B33]; [@B268]; [@B97]) are broadly consistent with a model in which Akt is activated at the plasma membrane and then redistributes to other locales. However, directly studying active Akt redistribution is technically challenging, and only a small number of studies have directly examined this "locked-active" Akt redistribution model. In endothelial cells, internalization of the complement membrane attack complex (MAC) is mediated by CME and Rab5, leading to recruitment of activated Akt to MAC-containing endosomes. In these cells, inhibition of CME by clathrin siRNA had no effect on Akt phosphorylation, suggesting that in this case, Akt is activated at the membrane, independently of clathrin, from where it is then redistributed ([@B135]).
The redistribution of "locked-active" Akt may occur via microtubules. Active Akt can associate with microtubules through interaction with the microtubule binding protein dynactin p150 ([@B140]). Inhibiting microtubule polymerization with nocodazole did not inhibit initial Akt activation by IGF1 stimulation; however, nocodazole treatment attenuated Akt signaling after the initial IGF1 stimulus. This suggests that microtubule polymerization possibly acts as a mechanism for redistributing active Akt to other parts of the cell to sustain Akt signaling.
Compared to the well-established mechanisms for Akt activation at the plasma membrane and in a subset of endosomes, much remains to be understood about the mechanisms that gate Akt activation that results in localized Akt activity in other membrane compartments. An important recent study found that while PI34P~2~ or PIP~3~ binding to the Akt PH-domain allosterically activates this kinase, the activity of Akt rapidly returns to basal upon dissociation from PIP~3~ or PI34P~2~ ([@B75]). This study thus challenges the "locked-active" Akt model in which the kinase is capable of redistribution from the plasma membrane to other compartments. Instead, the findings of this study suggest that Akt activity is restricted to specific membrane compartments in which it is initially activated, as dissociation of Akt from lipid binding would render Akt inactive. This model suggests instead that Akt is only activated in compartments enriched with compatible lipid ligands, such as PI34P~2~ and PIP~3~.
While the evidence that Akt requires ongoing association with membranes to sustain activity is thus strong, it is less well understood how certain compartments in which Akt has been reported, such as the late endosome or lysosome, could support Akt activation, due to the paucity of information of enrichment or production of PIP~3~ or PI34P~2~ in these compartments. Further studies have implied that Akt can be activated at other distinct subcellular locales, including the nucleus ([@B300]; [@B240]), mitochondria ([@B240]), and the endoplasmic reticulum ([@B25]), again begging the question of how PIP~3~ or PI34P~2~ can be produced or enriched in these compartments to support Akt activation. Indeed the Class II PI3K PIK3C2β is recruited to the lysosome to produce PI34P~2~, but this did not appear to regulate Akt and instead suppressed mTORC1 signaling ([@B184]). It is possible that additional mechanisms such as chaperones could support redistribution of Akt among compartments once activated. Future studies that can glean further insight into the mechanisms of localized Akt activation in specific compartments, and/or in possible modes of redistribution of active Akt will be very informative.
Compartment-Specific AKt Functions and Outcomes
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Given the sheer number of Akt substrates that have been described in the literature, it is no surprise that Akt exhibits specific effects in different subcellular locales. A detailed analysis of the effects of Akt on each substrate, in each subcellular compartment is beyond the scope of this review, which can instead be found in several recent reviews ([@B182]; [@B183]). Instead, the following section highlights some key regulatory functions of Akt in distinct organelles and how dysregulated Akt activity in these locations can contribute to disease. A particular emphasis is placed on how localized Akt activity coordinates the activity of key signaling nodes in the regulation of cellular energy metabolism for regulation of cell growth and apoptosis.
Plasma Membrane Akt May Support Formation of Invadopodia for Cancer Cell Metastasis
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Invadopodia are actin-rich, plasma membrane-associated structures that play a crucial role in remodeling of the extracellular matrix (ECM) for cell migration and invasion. The formation of invadopodia is particularly relevant in cancer metastasis, whereby degradation of the ECM by invadopodia-associated matrix metalloproteases (MMPs) facilitates dissemination of cancer cells to other compartments ([@B126]; [@B77]; [Figure 2](#F2){ref-type="fig"}). Invadopodia formation occurs in stages and is often triggered by RTK signaling; stimulation with EGF, VEGF, PDGF, and other RTK ligands can promote the initiation of invadopodia ([@B126]). Following RTK activation, invadopodia initiation requires recruitment of the regulators of actin polymerization, N-WASP, Arp2/3, cofilin, to the actin-cortactin complex ([@B126]; [@B77]). This facilitates actin polymerization, which is then anchored to the plasma membrane by interaction with the PI34P~2~-binding scaffold protein Tks5 ([@B199]). From here, the invadopodia mature via additional Cdc42 (or other Rho-family GTPases)-mediated actin polymerization and recruitment of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP), which leads to rapid degradation of the underlying ECM ([@B126]; [@B47]; [@B77]).
{#F2}
Phosphoinositides play a critical role in the regulation of invadopodia formation and activity, at least in part due to the regulation of Akt activation ([Figure 2](#F2){ref-type="fig"}). The balance between formation of PIP~3~ and PI34P~2~ by Class I PI3Ks and phosphatases appears to modulate the metastatic potential of many cancer cell lines ([@B91]; [@B180]). In MDA-MB-231 breast cancer cells, knockdown of the Class IA PI3K p110α catalytic subunit or chemical inhibition of class I PI3K attenuates invadopodia formation and Akt activation, while activating mutations of PIK3CA (which encodes p110α) promote invadopodia formation and Akt activation ([@B307]). The control of invadopodia formation requires Akt downstream of Class I PI3K activity, as knockdown of either PDK1 or Akt recapitulated the effects of p110α deletion ([@B307]). Interestingly, expression of a chimeric Akt that is constitutively active but lacks targeting specific to invadopodia (myristoylated Akt) attenuated invadopodia formation, suggesting that localized Akt at the site of invadopodia initiation and not general Akt activation on cellular membranes is required for invadopodia formation ([@B307]). Also in MDA-MB-231 breast cancer cells, knockdown of SHIP2 and PTEN differentially impacted invadopodia formation ([@B91]). This yet again links PI3K/Akt signaling to invadopodia formation. Similarly, loss of PTEN or the 4-phosphatase INPP4B results in accumulation of PI34P~2~ at the plasma membrane in MCF10a breast cancer cells and loss of both PTEN and INPP4B substantially enhanced Akt activity and invadopodia formation ([@B180]). While a role for a plasma membrane-specific pool of Akt in the control of invadopodia formation and dynamics remain to be thoroughly investigated, given that invadopodia are protrusions of the plasma membrane, it is perhaps expected that these structures are controlled by plasma membrane pools of Akt.
Akt Control of GSK3 at Early Endosomes
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GSK3 is a ubiquitously expressed serine/threonine kinase that was first identified as a regulator of glycogen synthase activity. Similar to Akt, GSK3 acts on over 100 known substrates with unique tissue and subcellular distributions, and as such, it plays an important role in regulating a diverse array of cellular processes. One of the overarching themes of GSK3-mediated regulation is the concept that upon phosphorylation by GSK3, many substrates are inactivated or targeted for degradation ([@B53]; [@B26]; [@B183]; [@B20]). GSK3 is negatively regulated by phosphorylation at S21 and S9, found within a conserved motif in GSK3α and GSK3β, respectively.
Akt is one of a number of regulatory kinases that can phosphorylate GSK3 in response to stimuli, and there is evidence to suggest that this event occurs at the level of early endosomes, which provides regulatory feedback cues to components of the endocytic pathway ([Figure 3](#F3){ref-type="fig"}). GSK3 phosphorylation occurs in a unique subset of endosomes that are APPL1 positive and TSC2 negative, and this requires APPL1 and PI3K activity ([@B247]; [@B228]). The phosphorylation of GSK3 by Akt is mediated by the Akt2 isoform at early endosomes ([@B32]). Notably, inhibition of CME, which prevents PI34P~2~ delivery to early endosomes, attenuates GSK3β phosphorylation but not that of FoxO or S6K, suggesting that early endosomes are a critical site for GSK3β regulation but perhaps not that of other Akt substrates ([@B170]). Accordingly, disrupting CME-derived membrane traffic results in accumulation of phosphorylated Akt in APPL1 endosomes and enhanced GSK3 phosphorylation, which leads to reduced dynamin-1 phosphorylation that promotes clathrin assembly ([@B228]). As such, endosomal Akt-GSK3 signaling may function as part of a positive feedback loop with clathrin-dependent Akt activation ([@B97]; [@B251]) to potentiate Akt signaling.
![The reciprocal regulation of Akt activation, endocytosis, and APPL1 endosomes. Shown is an active RTK within a clathrin nanodomain (some of which lead to production of clathrin-coated vesicles for receptor internalization), with associated active class 1A PI3K (e.g., linked via phosphorylated Gab1 to EGFR, as in [@B97]). Also enriched within clathrin nanodomains are the phosphatases SHIP2 and PTEN, which control clathrin nanodomain dynamics and may thus control PI3K-Akt signaling, and Akt itself. Following internalization, some RTKs and associated signals are delivered to APPL1 endosomes, which also harbor active Akt that serves to phosphorylate (and inactivate) GSK3ß, a phenomenon which relieves the inhibition of GSK3ß on dynamin-1, which in turn regulates formation of clathrin structures and endocytosis at the cell surface. Hence, the activation of Akt at the plasma membrane and within APPL1 endosomes both requires clathrin structures at the cell surface and promotes formation of clathrin structures for internalization, establishing a positive feedback loop to amplify PI3K-Akt signal propagation downstream of certain RTKs.](fcell-07-00070-g003){#F3}
Akt Regulation of TSC2 and Other Processes at Lysosomes
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Several lines of evidence suggest that Akt has compartment-specific functions that involve localization to the lysosome. As the site of mTORC1 activation, the lysosome is at the confluence of pathways that integrate nutrient and growth factor signaling for the control of cell growth. Activation of mTORC1 at the lysosome is accomplished through the combined effects of two separate, yet equally important pathways that converge upon mTORC1 to facilitate its full activation. In the first pathway, Rag GTPases are stimulated in the presence of amino acids and form a heterodimer that facilitates recruitment of mTORC1 to the lysosomal surface through interaction with Raptor ([@B67]; [@B165]; [@B239]; [@B243]; [@B306]). From here, mTORC1 is activated by PI3K-Akt signaling which impacts the activation of Rheb, a second class of GTPase ([@B280]; [@B172]). In the absence of mitogenic signaling, Rheb activity is suppressed by the GAP activity of the TSC complex, which is comprised of TSC1, TSC2, and TBC1D17. Mitogenic signaling impairs TSC-mediated suppression of Rheb, thus leading to activation of mTORC1 ([@B132], [@B131]; [@B280]; [@B172]).
Specifically, the activation of Akt by many RTKs elicits phosphorylation of TSC2, resulting in its inactivation and dissociation from the lysosome ([@B192]), thus allowing Rheb-dependent mTORC1 activation ([Figure 4](#F4){ref-type="fig"}). The highly contextualized nature of mTORC1 activation at the lysosome by growth factor signaling through Akt and nutrient availability ensures that mTORC1 is only activated when the cellular microenvironment favors growth. Given the localization of TSC2 to the lysosome, Akt recruitment to or activation at the lysosome would seem to be required for phosphorylation of TSC2 and thus activation of mTORC1.
{#F4}
This compartment-specific role of Akt activation is consistent with the activation of Akt at the lysosome proposed to contribute to the regulation of CMA ([@B10]; [@B123]; [Figure 4](#F4){ref-type="fig"}). Indeed, lysosomal Akt promotes autophagy through interactions with lysosomal proteins such as Phafin2 and the serine/threonine kinase VRK2 ([@B186]; [@B123]). Lysosomal Akt and VRK2 promote lysosomal acidification and silencing VRK2 by siRNA attenuates lysosomal Akt activation ([@B123]). While many factors such as ubiquitinylation also control Akt turnover, such as by the proteasome or by caspases ([@B164]), these studies highlight that Akt also thus exerts control over global protein stability, likely as a result of lysosome-specific functions. Hence, multiple lines of evidence indicate lysosomal-specific functions of Akt.
Nuclear Akt Promotes Cancer Growth and Chemotherapeutic Resistance
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Upon stimulation of some RTKs, active Akt is detected in the nucleus ([@B300]; [@B240]), and this can be delayed ∼30 min subsequent to initial Akt activation detected at the plasma membrane ([@B190]). Active nuclear Akt in the nucleus is protected from nuclear export and degradation by interaction with B23/NPM in the nucleus, a phenomenon which has a net effect of promoting cell cycle progression ([@B157]). Akt activity in the nucleus has been best described in the context of regulating nuclear/cytoplasmic localization of the Forkhead Box O Family (FoxO) members, a set of highly conserved transcription factors that control apoptosis, cell division, and metabolism ([@B183]). Akt regulates FoxO localization by directly phosphorylating three conserved regions on the FoxO proteins, which results in their association with 14-3-3 and retention in the cytoplasm ([@B37]). As a result of FoxO cytoplasmic retention, nuclear Akt activity attenuates expression of the FoxO gene expression program, including genes involved in promoting apoptosis (FasL, Bim) and cyclin-dependent kinase inhibitors (p27) ([@B37]; [@B285]; [@B318]). Thus, the net effect of active Akt in the nucleus is suppression of FoxO-mediated expression of genes that promote apoptosis and inhibit growth, and thus enhanced cell cycle progression and cell survival.
This role of nuclear Akt has important implications for cancer treatment. Akt activating mutations (in genes such as PIK3CA, PIK3CB, PIK3R1, PTEN, AKT, TSC1/2, and mTOR) are common in many tumors and are viewed as attractive therapeutic targets ([@B213]; [@B137]). However, prolonged Akt inhibition in cancer cell lines leads to FoxO-mediated upregulation of the RTK oncogenes Her3, IGF-1R, and the insulin receptor, possibly through relief of the negative feedback associated with RTK signaling ([@B50]). In lung cancer xenografts, Akt inhibition alone fails to prevent tumor growth, and this is correlated with enhanced RTK activity. Interestingly, the sensitivity to Akt inhibitors is improved with adjuvant administration of EGFR/ErbB2 inhibitors, suggesting that targeting different components of the EGFR/PI3K/Akt signaling axis can overcome the compensatory RTK activity that accompanies inhibition of Akt alone ([@B50]).
Akt is also active in the nucleus in response to various forms of DNA damage ([@B276]). Doxorubicin and other chemotherapeutic agents as well as ionizing radiation exert their cytotoxic effects, in part, by inducing DNA damage and subsequent cell cycle arrest and apoptosis ([@B209]). These effects are countered by repair pathways that respond to DNA damage, including activation of Akt by DNA-dependent protein kinases (DNA-PKs), which collectively activate various DNA repair pathways ([@B84]; [@B217]; [@B169]; [@B284]; [@B276]). Further studies that contribute to the understanding of how the nuclear functions of Akt activated by DNA damage may interface with Akt activated by RTK signaling will be valuable, given that active nuclear Akt is a central driver of cancer signaling and chemotherapeutic resistance and represents an attractive target for cancer therapy ([@B3]; [@B128]).
Mitochondrial Akt Coordinates Energy Metabolism and Apoptosis
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Nutrients and growth factors trigger Akt mobilization to the mitochondria where it plays a central role in regulating cellular energy metabolism and apoptosis ([@B240]; [@B25]; [Figure 5](#F5){ref-type="fig"}). This role of Akt is particularly important in cancer cell signaling, where dysregulated mitochondrial energy metabolism and apoptosis have been implicated in promoting tumor growth ([@B293]). Mitochondrial signaling is required for the localized activation or redistribution of Akt to mitochondria, as disruption of mitochondrial membrane potential attenuates IGF1-stimulated Akt activation in the mitochondria ([@B28]). Furthermore, disruption of mTORC2 signaling by Rictor deletion attenuates Akt activation at mitochondrial-associated ER membranes ([@B25]).
{#F5}
Accumulation of active Akt in the mitochondria following growth factor stimulation leads to the phosphorylation of a number of substrates responsible for energy metabolism, including hexokinase-2 (HK2), ATP-synthase, and GSKβ3 ([@B107]; [@B28]; [@B27]; [@B197]; [@B232]; [@B48]; [Figure 5](#F5){ref-type="fig"}). Further, hypoxia induced activation of mitochondrial Akt, which in turn led to phosphorylation of a wide range of mitochondrial substrates including pyruvate dehydrogenase kinase I ([@B48]). It remains to be determined to what extent the hypoxia-induced Akt program is recapitulated by RTK activation of Akt. Moreover, while translocation of Akt to mitochondria upon insulin and IGF1 stimulation has been reported ([@B28]), how Akt activated outside of the mitochondria may translocate into mitochondria is unclear, as are possible mechanisms by which RTK-derived signals may trigger activation of Akt within the mitochondria. The net effect of these individual phosphorylation events elicited by Akt at the mitochondria is a shift in cellular metabolism toward glycolytic pathways ([@B232]; [@B48]), reduction in oxidative damage and suppression of apoptosis ([@B48]). The effects of mitochondrial Akt on HK2 are central to the coupling of energy metabolism and apoptosis; as the first committed step in glycolysis, HK2 phosphorylates glucose to glucose-6-phosphate (G6P). G6P negatively regulates HK2, resulting in its dissociation from the mitochondria. However, HK2 is stabilized on the mitochondria by Akt phosphorylation at T473 ([@B232]), thus potentiating glycolytic metabolism.
Akt-mediated phosphorylation of HK2 also attenuates apoptosis by preventing cytochrome c release from the mitochondria ([@B107]; [@B197]). The anti-apoptotic effects of Akt/HK2 on promoting mitochondrial integrity represents a separate arm of the intrinsic apoptosis pathway, as dissociation of Akt/HK2 from the mitochondrial membrane is sufficient to induce apoptosis in the absence of Bax/Bak ([@B179]). Mitochondrial Akt also inhibits apoptosis by blocking mitochondrial caspase-3 activation, an effect specific to mitochondrial Akt, as overexpression of a constitutively active, mitochondrial-targeted Akt blocked caspase activation in the presence of PI3K inhibitors ([@B273]). Thus, the concerted effects of active Akt at the mitochondria promotes glycolysis and inhibits the intrinsic apoptosis pathway, such that in cancer, these features promote cell growth and survival.
Conclusion and Perspectives
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The many and diverse substrates of Akt require tight control and regulation of this important kinase. This regulation is afforded in part by the intricate orchestration of Akt activation and function in space and time in many different endomembrane compartments, including the plasma membrane, endosomal compartments, mitochondria and the nucleus. This compartmentalization of Akt functions provides an important mechanism to allow context-specific outcomes of Akt activation. While this is an attractive model, several important aspects of this spatial compartmentalization of Akt activation and function are still poorly understood.
There is indirect evidence of Akt activation on some internal membranes, such as from perturbations of late endosome/lysosomal traffic that disrupt Akt activation, and the possible activation of Akt on or in mitochondria. However, there is little direct evidence of PIP~3~ and PI34P~2~ production on some of these internal membrane compartments that are proposed to support Akt activation. For the case of lysosomes, the localization of mTORC2 to this compartment is indeed consistent with localized activation of Akt, yet the strict requirement of Akt activation on PIP~3~ or PI34P~2~ indicate that our understanding of Akt activation at the lysosome and other internal organelles remains incomplete. Interestingly, while PI3KC2 is present at the lysosome, this lysosome-localized lipid kinase did not appear to contribute to Akt activation ([@B184]). Resolving how PIP~3~ and/or PI34P~2~ can be produced in any cellular compartment other than the plasma membrane and early endosomes as part of canonical Akt activation will be very informative. Indeed, the development and use of novel lipid biosensors as recently reported for PI34P~2~ ([@B108]; [@B170]) or resolving other aspects of the mechanism of activation of Akt specific to various compartments will be very informative.
Several lines of evidence suggest that endocytic portals, likely to be clathrin-coated structures and vesicles, are important points of spatial convergence of many aspects of PI3K-Akt signaling. Plasma membrane clathrin structures are sites of signaling that lead to PI3K activation ([@B66], [@B65]; [@B97]), and Akt itself is enriched in these structures ([@B237]). In addition to these signals that positively regulate PI3K-Akt activation, clathrin structures are also enriched in lipid phosphatases including PTEN ([@B237]) and SHIP2 ([@B203]), thus making clathrin nanodomains an important platform for spatial coordination of both positive and negative regulation of PI3K activation. Notably, the accumulation of PI34P~2~ on internal membranes leading to activation of Akt2 required both SHIP2 and internalization (perhaps of PIP~3~) from the plasma membrane ([@B170]). This model proposes that early endosome PI34P~2~ is thus derived from plasma membrane PIP~3~. While this is an interesting model, this is difficult to reconcile with the well-established recruitment of many lipid phosphatases within clathrin-coated structures and vesicles, and the very rapid turnover of plasma membrane phosphoinositides in vesicles upon scission from the plasma membrane ([@B151]; [@B325]; [@B117]). Hence, plasma membrane clathrin nanodomains and endocytic vesicles may indeed be spatial bottlenecks that both allow PI3K-Akt activation in coordination with negative regulation of this signaling axis to prevent aberrant signaling. However, much remains to be determined about how PI3K-Akt signals are coordinated in plasma membrane clathrin nanodomain platforms and endocytic vesicles derived therefrom.
In addition, many of the functions of Akt that are thought to be compartment-specific are inferred, largely from the localization of Akt to specific compartments that each harbor unique substrate pools. The development of methods to specifically perturb or alter Akt in a compartment specific manner ([@B178]) will be instrumental in resolving how compartment-specific activation or redistribution of Akt impacts cell physiology. With the important roles that Akt plays in the context of normal human health and development, as well as the mounting evidence for a critical role of disruption of Akt function in diseases such as cancer and type II diabetes, understanding the molecular mechanisms that underlie the compartment-specific activation and functions of Akt over the coming years will have substantial impact.
Author Contributions
====================
MS, GF, and CA wrote and edited this review manuscript.
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.
**Funding.** This work was supported by a project grant from the Canadian Institutes of Health Research (PJT-156355) to GF and CA.
We would like to acknowledge the work of many colleagues in the field whose excellent work we were not able to discuss due to space limitations.
[^1]: Edited by: Steve Caplan, University of Nebraska Medical Center, United States
[^2]: Reviewed by: Brian Paul Ceresa, University of Louisville, United States; Adriano Marchese, Medical College of Wisconsin, United States; Wei Guo, University of Pennsylvania, United States
[^3]: This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology
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Abbreviations {#nomen0010}
=============
CHP
: Combined Heat and Power
DC
: District Cooling
DH
: District Heating
EE
: Energy Efficiency
EED
: Energy Efficiency Directive
EEOS
: Energy efficiency obligation scheme
EMS
: Energy management systems
ESR
: Effort Sharing Regulation
EUR
: Euros
EUROSTAT
: Statistical office of the European Union
FNEE
: National Energy Efficiency Fund (*Fondo Nacional de Eficiencia Energética)*
GHG
: Greenhouse gas
IDAE
: Institute for the Diversification and Saving of Energy (*Instituto para la Diversificación y el Ahorro de Energía)*
Ktoe
: Thousand tonnes of oil equivalent
M
: Million
MITECO
: Ministry for Ecological Transition and Demographic Challenge (*Ministerio de Transición Ecológica y el Reto Demográfico)*
Mtoe
: Million tonnes of oil equivalent
NECP
: National energy and climate plan
NEEAP
: National energy efficiency action plan
RES
: Renewable Energy Sources
SME
: Small and medium sized enterprise
TJ
: Terajoule
TOE
: Tonnes of oil equivalent
1. Introduction {#sec1}
===============
The European Commission has a strategic long-term vision to lead the transition towards a climate-neutral economy by 2050 in line with the objectives of the Paris Agreement. Building on the EU's international commitments, in 2009 the EU adopted a wide-ranging package where it located legislation to ensure the EU meets its climate and energy targets for the year 2020. They are the so-called EU's '20-20-20' targets -- 20% increase in energy efficiency, 20% cut in greenhouse gas emissions (from 1990 levels), and 20% renewables by 2020. In 2016, in the Clean Energy for all Europeans package \[[@bib1]\] the European Commission set tasks to bring EU energy legislation into line with the new 2030 climate and energy targets stressing the 'Energy efficiency first' as one of the key principles of the Energy Union. This is because energy efficiency is the most cost effective way to reduce emissions, improve energy security, enhance competitiveness and make energy consumption more affordable for all consumers. It also presents one of the key factors in achieving the long-term energy and climate goals. With Article 194 of TFEU (Treaty on Functioning of the European Union) the EU promotes energy efficiency and energy saving and the development of new and renewable forms of energy. Specifically, the 2012 Energy Efficiency Directive (EED) \[[@bib2]\] is an overarching directive, which introduced a number of measures, policy requirements and tools (i.e. energy efficiency obligations schemes or alternative measures) to enable the EU to reach its 20% energy efficiency target by 2020. The EU Member States are required to use energy more efficiently at all stages of the energy chain, including energy generation, transmission, distribution and end-use consumption. Further revisions were set for long-term trajectories by the amended EED \[[@bib3]\] with the energy efficiency target for 2030 set of at least 32.5% (to be achieved collectively across the EU). Within the framework of the Paris Agreement, the European Commission also issued a strategy for a climate neutral economy by 2050 by updating its roadmap towards a systematic decarbonisation of the economy \[[@bib4]\] and by providing a cost-efficient trajectory towards the attainment of the target of net-zero emissions. The EU also operates the EU Emissions Trading Systems, which puts a price on greenhouse gas emissions to create financial incentives for industry and businesses to reduce emissions \[[@bib5]\].
While the EU is on track to meet its 2020 targets on GHG emission reductions and renewable energy \[[@bib6]\], it seems that this is not the case for energy efficiency, which will be discussed in this paper while exploring the EU recent trends in terms of primary and final energy consumption and noting the gaps in reaching the set targets. The specific emphasis will be placed on the industrial sector, which is one of the largest users of energy (after the transport sector) in the EU and in most Member States, including Slovenia and Spain. This sector requires further attention not only because of the incline of energy consumption in recent years but also because of its potential of energy efficiency. Notably, this article will review the most recent EU updates on energy efficiency in relation to the measures imposed by Articles 7, 8, and 14 revised EED, which are the most relevant to the industrial sector. It will then explore the extent to which the national regulatory frameworks (including the most recent national plans) in the selected jurisdictions, such as Slovenia and Spain, promote energy efficiency and what measures are employed to address that. These will be placed in a broader national context in terms of strategy developments and institutional settings. While embracing a "stick and carrot" approach where regulations are combined with incentivising instruments to ensure that energy-intensive industries implement more advanced technology to save energy, this article will also identify good practices to improve energy efficiency in these countries.
Specifically, the paper is structured as follows. After this introduction (section [1](#sec1){ref-type="sec"}) and the background and methodology (section [2](#sec2){ref-type="sec"}), section [3](#sec3){ref-type="sec"} outlines the recent EU regulatory framework and policies on energy efficiency (including the updated EED), the EU energy consumption as well as prospects to meet the energy efficiency targets for 2020 and 2030. This will follow by energy efficiency obligation schemes as well as alternative measures (Article 7 EED), mandatory audits and energy management systems (Article 8 EED) and industrial waste heat recovery heating and cooling (Article 14 EED). Given that the EU Member States are obliged to transpose the EU regulatory framework (i.e. the EED) and contribute towards the overall EU target, the paper will also discuss the national measures and policies employed at national level to enhance energy efficiency, namely in two Member States -- Slovenia (section [4](#sec4){ref-type="sec"}), and Spain (section [5](#sec5){ref-type="sec"}). Finally, section [6](#sec6){ref-type="sec"} summarises the discussions presented in the paper, whereas the conclusions and recommendations are singled out in section [7](#sec7){ref-type="sec"}.
2. Background and methodology {#sec2}
=============================
Energy efficiency is a highly debated topic mainly due to its significant importance to achieve the sustainability goals. Underpinned by interdisciplinary by its very nature, energy efficiency has been analysed from different perspectives. For instance, there is comprehensive scholarship literature on technological advancements to improve energy efficiency, most importantly, from various renewable energy sources \[[@bib7], [@bib8], [@bib9], [@bib10]\], including the technologies for industrial waste heat recovery \[[@bib11],[@bib12]\] with a specific emphasis being placed either on one \[[@bib13]\], or several industrial branches, such as the aluminium, ceramic, and steel industries \[[@bib14]\]. Some reports focused on the larger scale of EU and beyond in attempt to identify best practices and case studies for industrial energy efficiency improvements \[[@bib15]\]. There have also been scientific considerations and suggestions to move away from single-sector thinking to a coherent energy systems in terms of smart energy system demonstrating the benefits from the integration of all sectors and infrastructures \[[@bib16]\]. An economic perspective should not be undermined as well, for instance, the improvements of energy efficiency have been discussed within enterprise \[[@bib17],[@bib18]\], industrial sub-sectors in a selected jurisdiction \[[@bib19]\], or the standpoints of whole society \[[@bib20], [@bib21], [@bib22]\]. There has been a critical view expressed that in order to limit energy consumption, there is a need for energy sufficiency (or conservation) rather than energy efficiency to avoid any the 'rebound' effects \[[@bib23]\]. There have been also studies undertaken at the larger scale, such as the EU level, where Connolly, Lund et al. proposed a new heat strategy with the expansion of district heating, along with more heat savings, heat recycling, and the use of more renewable energy resources, which would result in heating and cooling costs being reduced by approximately 15% \[[@bib24]\].
Further studies have addressed the EU regulatory frameworks and policies on energy efficiency \[[@bib25]\] quite often with an economic connotation, as all European policies and initiatives must be economically justified. For instance, while using the new methodology built on an econometric model Bertoldi and Mosconi \[[@bib26]\] evaluated energy efficiency policies in the EU Member States in the period 1990--2013 and discovered the energy savings induced by these policies counted to a remarkable 12%. Some papers have focused on energy efficiency improvements by national measures in one selected Member State, such as Lithuania \[[@bib27]\], Slovenia \[[@bib28]\], and Spain \[[@bib29]\]. There have also been more specialised studies conducted on the EU energy efficiency policies, especially the EED and its separate aspects; for instance, the EU energy efficiency obligation schemes \[[@bib30]\], in particular, White Certificate \[[@bib31],[@bib32]\]; the EED requirements on mandatory energy audits \[[@bib33]\], industrial waste heat recovery \[[@bib34]\]. Even though the EED has a horizontal application and covers broader themes, the focus of this paper is on the selected measures of the EED which are the most relevant to the industrial sector, such as the measures imposed by Articles 7, 8, and 14 EED. Building on the previous studies, the EU policies on the selected measures on energy efficiency together with statistical data on EU energy consumption are discussed to provide the foundation of this paper, as the EU is a driving force for change in national Member States. Yet, its objectives and targets cannot be achieved without national contributions. Therefore, Slovenia and Spain have been chosen as case studies in this paper. Given that the 'rebound' effects are less likely in the industrial sector, the common terminology of 'energy efficiency' will be used.
As far as the methodology is concerned, the review process involved analysing the primary sources, such as the EU regulatory framework, policies, and assessment reports published by the European Commission, and the European Environment Agency as well as national government strategy and policy documents, the mandatory reports, such as the National Energy Efficiency Action Plans (NEEAPs), with a specific emphasis being placed on the most recent plan - the Integrated National Energy and Climate Plan (NECP). All Member States of the EU had to submit their draft NECP for the Commission's evaluation to ensure that the countries' national energy efficiency contributions and their proposed measures would collectively achieve the EU target. Therefore, the evaluation of these new plans (which are currently available in national languages) is essential, as they define national measures and policies that support energy efficiency in different sectors, including industry, as well as the quantification of achieved and forecast energy savings. This enables businesses operating in the industrial sector to better understand their government's priorities and future trajectories. A template with specific questions centred on four typologies, such as i) energy consumption (including energy consumption in industry), ii) obligatory energy savings schemes (and alternative measures), iii) audits and energy management systems, and finally, iv) efficiency in heating and cooling (including industrial waste heat recovery) was provided for each national rapporteur for data collection. Qualitative data analysis was then undertaken within each typology, which then facilitated consistency in comparative analysis between the two jurisdictions in attempt to identify any good practices. In addition, comparison took place not only at jurisdictional level, but also within a jurisdiction in terms of the energy efficiency measures and policies introduced by the previous NEEAPs (effective until the end of 2020) and the future orientated NECPs.
3. The EU regulatory framework and policies on energy efficiency and energy consumption {#sec3}
=======================================================================================
3.1. EU energy consumption {#sec3.1}
--------------------------
Energy consumption in the EU is assessed in the context of primary energy consumption (gross inland consumption, excluding non-energy uses) \[[@bib2]\] and/or final energy consumption (the total energy consumed by end users - all energy supplied to industry, transport, households, services and agriculture, yet, excluding deliveries to the energy transformation sector and the energy industries themselves) as defined in Articles 2 (2) and 2 (3) EED respectively \[[@bib2]\]. A 20% EU energy efficiency target by 2020 translates into a primary energy consumption of no more than 1483 Mtoe and a final energy consumption of no more than 1086 Mtoe. Whereas the 2030 energy efficiency target is 32.5%, meaning that a primary energy consumption should not be more than 1273 Mtoe and a final energy consumption -no more than 956 Mtoe. Given that the UK is leaving the EU at the end of 2020, the Union's energy consumption figures have been adjusted accordingly, to the situation of the remaining 27 Member States; with a primary energy consumption of no more than 1312 Mtoe in 2020 and 1128 Mtoe in 2030 and a final energy consumption of no more than 959 Mtoe in 2020 and 846 Mtoe in 2030 \[[@bib35]\].
Given that energy needs are influenced by a number of factors, including economic developments as well economic/financial/health crises (i.e. the current pandemic of coronavirus), the structural changes in industry, the implementation of energy efficiency measures and also the specific weather situation, the primary energy consumption has fluctuated over the years. While energy consumption in the EU has been gradually decreasing in the 2000s (with some exceptions), this trend has changed in 2014. Between 2014 and 2017 the primary energy consumption has increased by 4% each year, but it decreased by 0.7% in 2018.
[Fig. 1](#fig1){ref-type="fig"} indicates that the gap between the actual level of primary energy consumption and the target level in 2020 was 15.2% in 2006, 1.5% in 2014 and in 2018 it was 4.9% and the distance to the 2030 target was 22.0% in 2018 (in the revised EU-27 Member States). Specifically, primary energy consumption in the EU-28 in 2017 was 1675 Mtoe, which is 1.6% higher than in 2016 \[[@bib35]\].Fig. 1Distance to 2020 and 2030 targets for primary energy consumption in the EU-27 \[[@bib36]\].Fig. 1
As far as final energy consumption is concerned, final energy consumption in the EU was 5.7% lower in 2017 in comparison to 2005, accounting to an average annual decline of 0.5%. It was lower in all sectors (save the services sector) and the highest decrease was in the industry (14.7%) and households (7.1%) sectors \[[@bib35]\]. This overall decrease of final energy consumption since 2005 can be down to the various factors similar to primary energy consumption, including the changes in economic performance (the economic recession), structural changes in various end-use sectors, in particular less energy intensive industrial sectors, improvements in end-use efficiency, and favourable climatic conditions \[[@bib37]\]. However, alongside to primary energy consumption, final energy consumption has also been increasing in the EU since 2014. For instance, in 2018 it was 5.5% higher than in 2014.
While in 2014 the final energy consumption was 2.2% below the 2020 target level, in 2018 it was 3.2% above the 2020 target level and the distance to the 2030 target was 17.0% in 2018 (in the revised EU-27 Member States, see [Fig. 2](#fig2){ref-type="fig"} ) \[[@bib35]\]. In 2018, the energy consumption increased in 15 Member States, in comparison to 2017 with the biggest energy-consumption increase being recorded in Poland and Spain, with a rise of 13.7% and 7.5% respectively from 2013 \[[@bib5]\].Fig. 2Distance to 2020 and 2030 targets for final energy consumption in the EU-27 \[[@bib36]\].Fig. 2
3.2. EU policies on energy efficiency {#sec3.2}
-------------------------------------
### 3.2.1. Overview {#sec3.2.1}
The European Commissioner for Climate Action and Energy in 2016 expressed that "the cheapest energy, the cleanest energy, the most secure energy is the energy that is not used at all" \[[@bib38]\]. Therefore, energy efficiency needs to be considered as a source of energy in its own right. To ensure this is the case, the European Commission has set ambitious policies and targets on energy efficiency. The EED \[[@bib2]\], adopted in 2012, embraced a set of measures such as: legal obligations to establish energy saving schemes and/or alternative measures in the Member States, the provisions on the setting of energy-efficiency targets, general energy-efficiency policies, energy audits, combined heat and power (CHP), management systems for enterprises, consumer behaviour etc. 2019 marked the completion of the new energy rulebook -- so called the Clean Energy for all European package \[[@bib1]\]. As part of this package, the revised EED \[[@bib3]\] updated the policy framework for 2030 and beyond. It set a binding energy efficiency target of at least 32.5% by 2030 (which can be increased subject to a review in 2023). As stated above, the 2030 binding target is to be achieved collectively across the EU. The EU Member States must set their own national contributions for 2030. Specifically, the Governance Regulation \[[@bib39]\] requires the Member States to design integrated 10-year NECPs, which should, *inter alia*, define how they intend to meet their energy efficiency and other targets for 2030 and provide future directions to all, including industries.
### 3.2.2. Obligatory energy savings schemes and alternative measures {#sec3.2.2}
Pursuant to Article 7, the revised EED has extended the Member States' annual energy savings obligations; while from January 2014 to December 2020 they had to ensure savings each year from of 1.5% of annual energy sales to final customers by volume, the new savings each year from January 2021 to December 2030 is 0.8% of annual final energy consumption. The Member States have flexibility on how they achieve their energy savings, such as by introducing energy savings obligation schemes, alternative measures or a combination of the two. Alternative measures embrace CO~2~ taxes, financing schemes, fiscal incentives, regulations or voluntary agreements that lead to the application of energy-efficient technology, training and education, energy efficiency standards, norms and labelling to achieve or even go beyond the targets mandated by EU law \[[@bib2]\]. For instance, relying on the MURE database, Bertoldi and Mosconi (2020) reported that during the 1990--2013 period the average number of energy policy measures was 66 in the Member States, whereas Spain indicated 139 measures (in comparison to Denmark and its 27 measures). As far as the high impact measures are concerned, Spain reported 91 "high impact" measures while Denmark only 3 \[[@bib26]\]. Therefore, the evaluation of the effectiveness of these measures is complex.
Furthermore, the Member States may institute minimum efficiency standards to reduce industrial energy demand, as they address the main obstacles to the take-up of cost-effective energy-efficiency measures in industry (i.e. risk aversion and uncertainty). Yet, these should be designed with an awareness of possible interactions or overlaps with the EU emissions trading scheme \[[@bib40]\]. The updated EED clarified that the calculation of the amount of energy savings either as a result of energy efficiency obligations schemes or alternative policy measures are on equal footing. Therefore, due to the flexibility of energy efficiency obligation schemes the Member States can design them in a variety of ways to meet national needs and to fit within their different policy mixes in order to achieve sustained energy savings and find the system that is best suited to particular circumstances of a Member State \[[@bib41]\]. However, the Member States need to take into account energy poverty and other social aspects when they design their energy efficiency schemes and alternative measures. They also should assess and take appropriate measures (where applicable) to minimise the impact of the direct and indirect costs of energy efficiency obligation schemes on the competitiveness of energy-intensive industries exposed to international competition \[[@bib2]\]. Notably, countries with energy-intensive, trade-exposed industries (e.g. aluminium smelting), may decide to exclude (or 'carve out') such industries from an energy efficiency obligatory schemes on the grounds that their competitiveness in international markets may be adversely affected \[[@bib40]\]. While the EED has a horizontal application, the Member States have the flexibility to target one or more specific sector(s) in order to meet the energy savings obligation, by introducing policy measures, for instance, in the industrial sector \[[@bib40]\]. According to the estimations based on the Member States national plans, the biggest proportion of savings will be achieved by measures targeting buildings followed by the second and third biggest proportions coming from industry and transport respectively; and the energy efficiency obligatory scheme (a cross-sector policy) will generate the highest share of savings per policy measure \[[@bib42]\].
### 3.2.3. Audits and energy management systems {#sec3.2.3}
Article 8 EED imposes an obligation of independent, cost-effective, high-quality energy audits to all large enterprises, which defined as non-SMEs (small and medium sized enterprises). The EU definition of SMEs is fewer than 250 employees and a turnover of no more than EUR 50 M or a balance sheet of no more than EUR 43 M. These audits must be conducted at least every four years. They are designed to provide individual firms with valuable advice on their energy consumption. Indeed, energy audits and energy management are identified as important instruments to explore economic energy efficiency potentials; to gain knowledge and develop a strategy to improve energy efficiency in businesses \[[@bib5]\]. Large businesses are exempted from this audit obligation if they have implemented energy management systems (EMS) (Article 8 (6) EED), where an EMS whereby 'means a set of interrelated or interacting elements of a plan which sets an energy efficiency objective and a strategy to achieve that objective' \[[@bib2]\]. An EMS creates a structure to monitor energy consumption and improve energy efficiency in an industrial firm and the use of it in industry is currently increasing \[[@bib5]\]. The Member States should also encourage energy audits and EMS to SMEs.
### 3.2.4. Efficiency in heating and cooling (including industrial waste heat recovery) {#sec3.2.4}
There are also requirements addressing efficiency in heating and cooling (as defined in Article 14 EED), requiring the Member States to carry out a comprehensive assessment of the cost effective potential for energy efficiency in heating and cooling, principally through the use of cogeneration, efficient district heating and cooling and the recovery of industrial waste heat. Specifically, the Member States need to identify the technological solutions used to supply heating and cooling, while making distinctions between on-site (i.e. heat-only boilers; high-efficiency heat and power generation; heat pumps; and other on-site technologies) sources and off-site sources (i.e. high-efficiency heat and power generation; waste heat; and other off-site technologies) and between renewable and fossil energy sources. To analyse the economic potential for efficiency in heating and cooling, an assessment should include the identification of suitable technologies for supplying low-carbon and energy-efficient heat and cold on the national territory using a cost-benefit analysis and a baseline and alternative scenarios among other things \[[@bib43]\]. The European Commission has also published some soft legislation (non-binding) to help the Member States to fully transpose the different elements of the 2018 amending EED into national law, including the recommendation on transposing the energy savings obligations under the energy efficiency directive and annex \[[@bib40]\]; the recommendation on the implementation of the new metering and billing provisions of the energy efficiency directive and annex \[[@bib44]\]; and recommendation on the content of the comprehensive assessment of the potential for efficient heating and cooling under Article 14 of the energy efficiency directive and annex \[[@bib45]\].
4. Energy efficiency: a case study of Slovenia {#sec4}
==============================================
4.1. Overview {#sec4.1}
-------------
Slovenia is one of the smallest EU Member States (followed by Estonia, Cyprus, Luxembourg and Malta in descending order) with its population of just over 2 million. Given that it is not rich in fossil fuels, it imports all of its oil and natural gas \[[@bib46]\], referring to energy dependency at 48% as in 2018 \[[@bib47]\]. This dependency results in economic, political, and social vulnerability within the EU \[[@bib48]\]. In 2018, domestic energy production in Slovenia was 148 299 TJ (3.542 Mtoe) with nuclear energy accounting for the largest share of 42%, followed by renewable energy sources (including hydro energy) with 32%, coal with 25%, and with less than 0.5% of energy being produced from other sources \[[@bib49]\]. Slovenia has a great potential for the sustainable development of energy policy embracing energy efficiency.
As previously discussed, for the EU to meet their 20-20-20 targets, the Member States must deliver on their national targets. While the EU emission target is 20% less compared to 1990 levels, Slovenia's national target is 4% more compared to 2005 levels (for sectors falling outside the EU Emission Trading System) \[[@bib50]\]. Meanwhile, the national target for renewables is 25% by 2020 and primary energy consumption 7.125 Mtoe \[[@bib51]\], which was revised from the previously stated target of 7.3 Mtoe. In its final integrated NECP \[[@bib52]\] published in February 2020, Slovenia indicated that its primary energy consumption will not exceed 6.36 Mtoe by 2030 (see [Table 1](#tbl1){ref-type="table"} ). The NECP also anticipates 20% GHG emission reduction by 2030 in comparison to 2005 in sectors not included in the Emission Trading scheme. The target of energy efficiency improvement is defined at 35% by 2030 with final energy consumption not exceeding 4.72 ktoe (see [Table 1](#tbl1){ref-type="table"}).Table 1National targets of Slovenia.Table 1National targets and contributions20202030GHG compared to 2005 under the Effort Sharing Regulation (ESR)+4%−20%Renewable Energy25%27%Energy Efficiency (improvements from 2007):20%35%Primary energy consumption (Mtoe)7.1256.36Final energy consumption (Mtoe)5.1184.72
The total investment required for the implementation of all the NECP measures is estimated at EUR 22 829 M, of which EUR 6139 M are dedicated to energy efficiency \[[@bib52]\].
4.2. Energy consumption {#sec4.2}
-----------------------
As defined in the Slovenian NEEAP \[[@bib51]\], primary energy consumption for Slovenia should not exceed 7.125 Mtoe in 2020 ([Fig. 3](#fig3){ref-type="fig"} ). This means that it may not exceed the 2012 figure by more than 2%. By the end of 2015, final energy savings of 4949 GWh were achieved, which was 16% above the target for 2016 \[[@bib51]\]. More recently, in 2018, the primary energy consumption decreased by 1.8% compared to 2012.6.88 Mtoe of primary energy consumption in 2018 \[[@bib47]\] indicates that it is still within the indicative target ([Fig. 3](#fig3){ref-type="fig"}).Fig. 3Primary energy for the period 2000--2018 and the EED target goal for 2020 \[[@bib47]\].Fig. 3
With regard to final energy consumption, Slovenia's national target should not exceed 5.118 Mtoe in 2020 \[[@bib51]\]. With 4.972 Mtoe of final energy consumption in 2018, Slovenia is 3% below the indicative target for 2020, as presented in [Fig. 4](#fig4){ref-type="fig"} \[[@bib47]\].Fig. 4Final energy consumption for the period 2000--2018 and the indicative goal for 2020 \[[@bib47]\].Fig. 4
During the period from 2014 to 2018, there was an increase in the final energy consumption of all final energy sectors. The largest increase in 2018, in comparison to 2014, can be attributed to the industry sector (12.5%), followed by transport (10.0%), households (4.7%), and other (0.4%). The largest share in the total final energy consumption belongs to the transport sector, namely 40%, followed by industry with 28%, households with 21% and other users with 11%. In the period 2000--2018, the share in final energy consumption decreased the most in industry, namely, by 4% \[[@bib47]\]. Final energy consumption in Slovenia slightly increased in 2018 (by 0.5%) in comparison to 2017 \[[@bib49]\].
### 4.2.1. Energy consumption in industry {#sec4.2.1}
For the industrial sector, Slovenia has set an indicative target for final energy consumption in 2020, namely that the total energy consumption in this sector will not exceed 1.307 Mtoe \[[@bib51]\] by 2020 and 1.283 Mtoe \[[@bib52]\] by 2030. Final energy consumption in industry has been increasing since 2013. Notably, from 2013 to 2018 it has increased by 15.6%. Therefore, in 2018, it was about 2% above the target value for 2020, as presented in [Fig. 5](#fig5){ref-type="fig"} \[[@bib52]\]. Due to the increasing use of final energy in the industrial sector, the trajectory towards achieving the indicative target in the industry is currently unfavourable. Therefore, energy productivity in this sector has to be improved.Fig. 5Final energy consumption in industry for the period 2000--2018 and the indicative goal for 2020 \[[@bib47]\].Fig. 5
As far as specific industrial branches are concerned, the final energy consumption in basic metal production (C24) has been increasing since 2010. On the contrary, from 2008 to 2018 there have been the significant decrease in energy consumption in the production of non-metallic mineral products (C23 -- 40% reduction) and the paper industry (C17 -- 27% reduction) (see [Fig. 6](#fig6){ref-type="fig"} ).Fig. 6Final energy consumption in industry per industrial branch in the period 2008--2018 \[[@bib53]\].Fig. 6
It is important to note that energy-intensive branches in Slovenia account for about 25% of total added value in industry (data for 2018) \[[@bib53]\]. The key contribution, almost 12.3% of the total added value of Slovenian industry is generated by pharmaceutical companies (C21) with the remaining energy-intensive sectors contributing as follows: production of chemicals and chemical products (C20) 4.0%, production of metal production (C24) 3.7%, non-metallic mineral products (C23) 3.5% and paper and pulp production (C17) 1.6% \[[@bib53]\].
4.3. National policies on energy efficiency {#sec4.3}
-------------------------------------------
Building on the EU energy policy, the Slovenian energy policy goals also aim to ensure a reliable, safe and competitive energy supply in a sustainable manner by ensuring the transition to a low carbon society \[[@bib54]\]. The Ministry of Infrastructure and Spatial Planning, Directorate for Energy is the government body responsible for Slovenian energy policy, which is supported by the Energy Agency (*Agencija za energijo*), the regulatory authority supervising and regulating the energy field that, *inter alia*, is also responsible for promoting efficient use of energy \[[@bib55]\]. Additionally, within the ambit of the [Ministry of Environment](http://www.buildup.eu/en/explore/links/department-energy-efficiency-and-renewable-energy){#intref0015} and Spatial Planning, there is also the national public entity Eco Fund (*Eko sklad*), which is the only specialised institution in Slovenia that provides financial supports for environmental projects \[[@bib56]\]. The Eco Fund is also responsible for the preparation and implementation of energy efficiency measures at national level and provides financial aids to natural and legal persons (i.e. energy refurbishment of buildings, replacement of old inefficient heating systems, energy advising, energy poverty alleviation programmes, co-financing investments in renewable energy sources, subsidies for electric car etc.) \[[@bib57]\].
The main legislative framework for the energy sector in Slovenia is postulated in the Energy Act \[[@bib58]\], which transposed a number of EU directives concerning electricity and gas markets, energy efficiency and renewable energy sources. It also contains the principles of energy policy, measures ensuring energy security, and measures regulating energy infrastructure and heat distribution. There is also a new long-term development strategy document -- the Energy Concept of Slovenia (*Energetski koncept Slovenije*), containing the national energy programme \[[@bib58]\]. This document, on the basis of the projections of the country's economic, environmental and social development and the adopted international commitments, defines the objectives of the reliable, sustainable and competitive energy supply for the next 20 years (and tentatively for the next 40 years) \[[@bib59]\].
The current measures for achieving the 2020 targets in Slovenia are contained in the NEEAP 2020, which aims to meet the long term climate and energy targets with the highest possible efficiency and the lowest possible costs \[[@bib51]\]. Future trajectories are now incorporated in the integrated NECP which sets the overall target to improve energy efficiency by at least 35% in 2030 from the 2007 baseline scenario (in line with the revised EED) \[[@bib52]\].
4.4. Obligatory energy savings schemes and alternative measures {#sec4.4}
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Slovenia meets the binding requirements of Article 7 EED through a combination of both energy efficiency obligation scheme and alternative measures. The two main measures are the energy efficiency obligation scheme (EEOS) and the Eco Fund, where the obligation is divided as follows: half of the 1.5% energy savings goes to the Eco-Fund and the other half to EEOS. Article 318 of the Energy Act defines that obligated parties are suppliers of electricity and heat and suppliers of solid, liquid or gaseous fuels to final customers irrespective of their size. They should ensure the achievement of energy savings among final customers \[[@bib58]\]. The obliged entities may, however, meet their obligations by remitting funds to the Eco Fund in an amount equal to the total savings that should have been achieved by final customers and the additions to the price of heat and fuel prices to increase Eco-Fund's energy efficiency. Furthermore, these measures may be implemented in all sectors, including transport and industry that are included in the emission allowances trading scheme \[[@bib51]\]. The Energy Agency is responsible for reporting on and monitoring the achievement of energy savings.
According to the Energy Agency report for 2018 \[[@bib60]\], the obliged entities reached 24.4 ktoe of final energy savings, hence, exceeding the 2018 target of 21.5 ktoe. The industrial sector accounted for 37% of all achieved savings because of the main measures being employed, such as the introduction of CHP systems, energy efficient lightning, use of efficient electrical devices (i.e. electrical motors, ventilation systems), introduction of EMS and the use of excess heat. Evaluation of the effectiveness of the NEEAP2020 implementation noted that the overall sectoral comparison for the period 2016--2018 indicates the largest savings being made in the industry sector (i.e. 179 GWh in 2016 and 133.3 GWh in 2017) and the housing sector (163.5 GWh in 2016 and 171.8 GWh in 2017) \[[@bib61],[@bib62]\].
With regard to alternative measures, Slovenia chose the implementation of the Eco Fund's programme for promoting energy efficiency measures \[[@bib58]\]. The obliged parties and their contributions provide funds for Eco Fund, which is supplemented by funds from the Climate Change Fund. The Eco Fund's target in individual years within the 2014--2020 period amounts to 0.75% (22.53 ktoe) of the annual savings target. Therefore, the savings made by the Eco Fund with the help of the measures implemented across the entire 2014--2020 period should reach 157.7 ktoe in 2020, giving cumulative savings of 630.8 ktoe across the entire period \[[@bib51]\]. The energy savings provided by the Eco Fund as an alternative measure in 2018 amounted 19.9 ktoe \[[@bib63]\]. Even though savings are attained in any given year, Slovenia has not reached the set goals yet, since the surpluses achieved by the obliged entities are transferred to future years, and the objectives set in the framework of alternative measures (22.5 ktoe of final energy savings per year) were not achieved \[[@bib51]\]. For the forthcoming period of 2021--2030 Slovenia will continue energy efficiency improvements through a combination of the contributions from the obliged parties and alternative measures, such as the implementation of the energy efficiency measures from the NEEAP 2020, for instance, the Operational Program for reduction of GHG emissions until 2020, the Operational Program for the implementation of European Cohesion Policy Eco Fund programmes and tax mechanisms \[[@bib52]\]. A revision of the methods for calculating energy savings is also foreseen, thus, eliminating irregularities that could lead to unrealistically high savings and therefore, low market prices \[[@bib52]\].
To conclude, it has been noted that there is a need to improve monitoring of the implementation of the scheme and to ensure the conditions for its stable functioning. This relates primarily to checking the reporting by the obliged entities of the achieved savings (i.e. through the implemented projects), and on-going modification of methods for calculating energy savings. With regard to the implementation of alternative measures, the most important problem remains the effective allocation of funds.
4.5. Audits and energy management systems {#sec4.5}
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The obligations arising from Article 8 EED in Slovenia were also transposed in the Energy Act \[[@bib58]\] supported by the Rules on the methodology for the production and the content of energy audits adopted in June 2016, compelling large companies to conduct an energy audit every four years \[[@bib64]\]. Given that the target group in this article is large enterprises - defined as "enterprises that are not SMEs" (Article 8 (4) EED), gave the Member States flexibility either to follow the EED verbatim or provide their own definition. Nabitz and Hirzel discovered that different approaches to translating the target groups were taken across the Member States; in some cases, further criteria for a delimitation of large companies were applied which resulted in either an extended or reduced target group regarding the obligation \[[@bib65]\]. For instance, Slovenia adjusted and lowered the financial thresholds for the definition of SMEs, which led to cover businesses that are SMEs. Specifically, the Companies Act (ZGD-I) provides that large companies have to fulfil two conditions: i) have at least 250 employees; ii) an annual income of more than EUR 35 000 000; and the value of assets of more than EUR 17 500 000 \[[@bib65]\]. To guarantee compliance with the requirements Slovenia decided to impose the penalty (EUR 10 000) on the management level (i.e. director of the company) \[[@bib33]\].
The Energy Agency is responsible for the monitoring of the fulfilment of this obligation. During the 2013--2014 period, 138 energy audits were conducted \[[@bib51]\]. According to the Statistical Office of Slovenia, there were 120 large industrial companies in Slovenia in 2018, 30 companies (including non-industrial large companies) had ISO 50001 certificate in 2018 \[[@bib66]\]. The measures of management system were also implemented, as businesses with a certified EMS are excluded from the mandatory audit requirement. Slovenia has also introduced a successful education and training program for energy managers (EUREM), which drives future energy efficiency projects \[[@bib67]\]. This educational element is largely supported by the European Commission, especially in newer Member States.
In addition, in 2017, the Eco Fund announced a public call for granting of non-refundable financial incentives to carry out an energy audit for SMEs \[[@bib51]\]. In 2018, Eco fund has supported by financing (50% of the incurred cost) of energy audits for 10 SMEs \[[@bib63]\]. In the area of energy audits and EMS, the continuation of the implementation of existing measures and their upgrading has been planned. The incentive scheme is about to be upgraded with the establishment of a scheme to promote energy governance in SMEs. EMS are also supported with financial incentives to raise energy efficiency and consumption of renewable energy sources (RES) in the industry through Eco Fund \[[@bib51]\]. This is reflected in the NECP, which proposes the continuation of the ongoing measures and foresees the expansion of EMS \[[@bib52]\].
4.6. Efficiency in heating and cooling (including industrial waste heat recovery) {#sec4.6}
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In compliance to Article 14 EED, Slovenia has developed a comprehensive strategy for heating and cooling providing guidelines for the development of efficient DH (District Heating), DC (District Cooling) and the use of CHP \[[@bib51]\] According to the Energy Act \[[@bib58]\], the ministry responsible for energy, every five years, prepares a comprehensive assessment of the possibilities for the use of CHP and effective DH and DC with the overall assessment, which also includes a cost-benefit analysis. For instance, the first comprehensive assessment of the possibilities for the use of CHP and effective DH and DC has estimated the technical potential for CHP only in DH systems at up to 600 GWh of heat \[[@bib51]\]. The Centre for RES/CHP Support provides information, training and awareness-raising brochures to various target groups in relation to the benefits and practical aspects of the development and use of energy efficiency and renewable energy technologies \[[@bib58]\].
The support schemes in Slovenia were improved in 2009 which significantly expanded the promotion of CHP to all sectors (primarily in DHs), set more support for the production of electricity from RES (several RES resources and size classes of support) and defined clear methodological starting points for determining the amount of support. As a result, in the period up to 2014, around 140 MWe of new CHP generating plants were built mostly in DH systems, in recent years the number of smaller units in the service sector has also increased significantly \[[@bib51]\].
There are financial incentives intended for investments in new DH-RES systems and DH-RES microsystems, as well as the expansion of existing DH-RES systems and construction of new boiler rooms with wood biomass boilers, CHP on wood biomass or solar systems \[[@bib51]\], which is implemented under the Operational Programme for the Implementation of the European Cohesion Policy for the period 2014--2020 \[[@bib68]\]. Furthermore, the Eco Fund has prepared a comprehensive financial incentive programme for the period from 2018 to 2020 to promote the sustainable development of existing DH systems and increase their competitiveness by implementing various measures \[[@bib51]\], where the exploitation of excess heat from the industry is also highlighted. The NECP continues the implementation of all energy efficiency measures from the NEEAP 2020 and provides guidelines and the basis for the improvements of energy efficiency (e.g. 33 ktoe excess heat recovery in industry by 2030 and 61 ktoe by 2040). The NECP plans at least a 30% share of RES in industry, taking into account the use of excess heat, and also anticipates a 1.3% annual increase in the share of RES in heating and cooling in industry, including excess heat and cold \[[@bib52]\], which is in accordance with the requirements of Article 23 REDII (Recast Renewable Energy Directive) \[[@bib69]\].
In terms of technological developments, the intake of several technologies that are considered in European industrial strategy until 2020 as key technologies were extremely low in the Slovenian manufacturing companies \[[@bib70]\]. Slovenia was also named as a traditionalist when it comes to the readiness of the countries towards Industry 4.0 \[[@bib71]\]. Nevertheless, empirical evidence undertaken by Hojnik and Ruzzier \[[@bib72]\] highlighted that competitive pressure was the most influential driving force of eco-innovation process, followed by managerial environmental concern and customer demand in Slovenia. Slovenia has recently started introducing the new technological developments. Some examples include the ERDF funded project OPERH2, which is designed to deal with the optimisation of energy conversion in order to reduce the share of fossil fuel used with hydrogen in industrial glass melting. There have also been several successful projects in terms of heat waste recovery. For instance, company SIJ (Slovenian Steel Industry with high energy intensive production), implemented a project to capture waste heat from the cooling system of the electrical arc furnace in steel production (in Metal Ravne) for the purpose of district heating. A heat exchanger with heat power of 4.5 MW~th~ was installed for heat recovery. The potential of the heat utilisation for district heating presents about 25% of the heat waste and is estimated at 8000 GWh per year. This was the first project of cooperation between the steel industry, the district heating system operator and local community in Slovenia. As discussed in 4.2.1 section Slovenia is unlikely to meet its energy efficiency target set to the industrial sector. This is mainly due to the perpetually increasing final energy consumption, especially in the basic metal production branch. Therefore, the energy efficiency measures are essential. Most recently, as part of the Horizon 2020 funded project ETEKINA, a heat exchanger prototype will be installed in one of the Slovenian steel company in order to significantly improve the plant's heat management by recovering of at least 40% of the waste heat streams \[[@bib73]\]. This in turn will reduce environmental impact and the company's energy bills. Given that traditionally, the Slovenian steel undertakings are producers of special steel products and mainly oriented to export \[[@bib74]\], this new technology will further lead to the increase of the company's global competitiveness, will secure local jobs and most importantly, will also contribute in meeting the EU's climate and energy goals \[[@bib73]\].
5. Energy efficiency: a case study of Spain {#sec5}
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5.1. Overview {#sec5.1}
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Spain, which is divided into seventeen autonomous communities (comunidades autónomas) and two African autonomous cities with their own parliaments and regional governments, is now the fourth largest EU Member States (after Germany, France and Italy, as the UK is no longer counted as a Member State) with its population of 47.1 million (2019 data) \[[@bib75],[@bib76]\]. It is a member of 'Union for the Mediterranean', which also has a focus on renewable energies and energy efficiency \[[@bib77]\], providing a platform for the regional cooperation. As far as energy is concerned, Spain is one of the largest net importers of energy in absolute numbers alongside Germany, Italy and France, due to its prevalence of fossil fuels in the energy mix \[[@bib78]\]. For instance, in 2017 its primary energy consumption was 132 Mtoe, 99 Mtoe of which was fossil fuels (almost all imported) \[[@bib77]\]. It is estimated that renewables, efficiency and reduction of imports (mainly coal and oil) will lower the degree of energy dependency from 74% in 2017 to 61% in 2030 \[[@bib77]\].
General State Administration as well as Governments of Autonomous Communities have embarked on ambitious policies, have set the programmes, measures and actions falling under ambit of their competence \[[@bib79], [@bib80], [@bib81], [@bib82]\] in order to comply with the EED. While autonomous communities introduce specific measures for energy efficiency, the strategic energy and climate framework, which includes the NECP 2021--2030, the Law on Climate Change and Energy Transition, and the Just Transition Strategy \[[@bib83]\] is set for the whole of Spain. It seems that the mandatory requirement to introduce the NECP has facilitated the long overdue national Climate Change and Energy Transition Law, approved in 2020 alongside the development of long-term and Just Transition strategies \[[@bib84]\]. For instance, the new Climate Change and Energy Transition Law embraces some bidding and other market mechanisms as well as mandatory measures such as phase-outs, deadlines, bans and ambitious targets. However, while new fossil fuel subsidies are banned, they are allowed on social grounds, such as to protect Spain's economic interests or to offset for the lack of adequate technological alternatives \[[@bib85]\], therefore, leaving a loophole for new subsidies for fossil fuel in the future. The Just Transition strategy, which accompanies the NECP, is designed to anticipate and manage the consequences on the regions and people directly linked to technologies to be progressively displaced as a result of the transition \[[@bib77]\].
In terms of the binding national targets, in its NECP Spain has set the targets for renewables, aiming at installing 122.7 GW renewable energy capacity by 2030, primarily wind and solar, leading to renewables making up 42% of the final energy consumption, whereas a 74% share of renewables in the electricity generation and a 28% share of renewables in transport by 2030 \[[@bib77],[@bib85]\]. In line with the EU's climate-neutral strategy, Spain also aims to achieve a 100% renewables share in the electricity sector by 2050. The plan is also to obtain a 23% reduction in GHG emissions compared to the 1990 levels. Even though initially the target was set at 40% similar to the ambition of most EU countries (Germany (−55%), Ireland (−40%), France (−40%), and Spain's closest neighbour Portugal (−45%), yet, it was scaled down and settled at 23% arguing it was a fair, achievable and balanced goal (see [Table 2](#tbl2){ref-type="table"} ) \[[@bib84]\]. Energy efficiency plays a key role in energy planning policies in Spain due to its strategic importance in the transition towards a more competitive and sustainable economy. The target of primary energy consumption by 2030 in Spain is projected at a 39.5% reduction compared to the baseline, the reduction in primary energy consumption equivalent to an improvement of the primary energy intensity of 3.5% per year until 2030 \[[@bib77]\].Table 2National targets of Spain.Table 2National targets and contributions20202030GHG compared to 2005 under the Effort Sharing Regulation (ESR)[a](#tbl2fna){ref-type="table-fn"}−27%−49%GHG compared to 1990 under the ESR−11%−23%Renewable Energy over final energy consumption20%42%Energy Efficiency improvement:24,2%39,5%Primary energy consumption (Mtoe)123,498,5Final energy consumption (Mtoe)86,373,6[^1]
The objectives set in the NECP cannot be attained during the period of 2021--2030 without investments, which is estimated to be EUR 241 412 M, most of which being disbursed by the private sector (approximately 80%). The plan will promote accumulated savings in fossil fuel imports of EUR 67 381 M (or 28% of the total budget) \[[@bib77]\]. However, those values may alter due to the new economic circumstances generated by the COVID-19 crisis. During the writing of this paper there have not been any modifications confirmed.
5.2. Energy consumption {#sec5.2}
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As far as energy consumption in Spain is concerned, after a long upward trend in energy demand, primary energy consumption followed a downwards trend for consecutive years from 2007 until 2014. This downward trend was reversed in 2015 by a 4.1% increase in consumption, which has been further increasing annually \[[@bib86]\]. The years of 2017 \[[@bib77]\] and 2018 \[[@bib87]\] (see [Fig. 7](#fig7){ref-type="fig"} ) have also observed continuous energy consumption growth in Spain, mainly boosted by greater economic and industrial activity \[[@bib77]\]. In terms of primary energy and energy sources, there was an increase in renewable energy with a 3.4% increase in consumption, ahead of oil (+2.7%), nuclear power (+2.2%) and natural gas (+2%) in 2016 \[[@bib88]\].Fig. 7Evolution of Primary Energy Consumption by sources in Spain, 2007--2018 \[[@bib87]\].Fig. 7
The trend in final energy demand by energy source is also consistent with primary energy, both demonstrating common peculiarities. For instance, in 2015, final energy consumption (excluding non-energy uses) rose to 80 461 ktoe, an increase of 1.6% over the previous year, which marked a break in the downward trend during the 2007--2014 period (with the exception of the upturn in 2010) \[[@bib86]\]. With regard to energy sources, the increase in 2018 is mainly due to the increased consumption of oil products (+2.1%) and natural gas (5.8%), which jointly accounted for 67.4% of overall demand (see [Fig. 8](#fig8){ref-type="fig"} ) \[[@bib89]\].Fig. 8Trends of final energy consumption by sources, 2000--2018 \[[@bib77],[@bib89]\].Fig. 8
As previously discussed, Spain set the efficiency target of 39.5% by 2030. The National Energy Efficiency target in Spain has been defined in terms of primary energy consumption and final energy consumption. Specifically, the target of primary energy consumption is 98.5 Mtoe for the 2030 \[[@bib77]\].
With regard to the final energy consumption, it is planned to be reduced to 73.5 Mtoe in 2030. The target accumulated energy saving from January 2021 to December 2030 is 36 809 ktoe from which 10 256 ktoe belongs to the industry sector, whereas the target accumulated energy saving from January 2014 to December 2020 was set at 15 979 ktoe \[[@bib90]\].
The targets set for 2030 meant that Spain had to review and update its objective of improving energy efficiency by 2020 with respect to the objective in its previous plan - the NEEAP 2017--2020. That plan defined that primary energy consumption should not exceed 122.6 Mtoe by 2020, referring to a 24.7% improvement in energy efficiency. However, in line with the new NECP 2021--2030, the 2020 target had to be formulated as an improvement of 24.2%, which means that 123.4 Mtoe should not be exceeded in terms of primary energy consumption (see [Fig. 9](#fig9){ref-type="fig"} ) \[[@bib77]\].Fig. 9Reduction objective for the consumption of primary energy (Mtoe/year) \[[@bib77]\].Fig. 9
### 5.2.1. Energy consumption in industry {#sec5.2.1}
In the context of sector-specific demand, similar to Slovenia, [Fig. 10](#fig10){ref-type="fig"} indicates that transport continued to be the highest energy consumption of 40% of total final demand (i.e. 32 531 Ktoe), followed by the industrial sector - 25% (20 219 Ktoe) with the remaining 35% shared by the residential, tertiary and agriculture sectors in 2018. In 2016 approximately 75.6% of energy consumption in the industrial sector was concentrated in five branches - metallurgy, non-metallic minerals, chemistry, food, beverages and tobacco, and pulp and paper \[[@bib88]\]. For the industrial sector, Spain has set a target scenario for final energy consumption (excluding non-energetic uses) of 19 771 ktoe \[[@bib77]\] in 2020 and 19 570 ktoe \[[@bib77]\] in 2030.Fig. 10Final energy consumption by sectors in Spain in 2018 \[[@bib77]\].Fig. 10
Spanish policy of financial support for industrial investment has provided the framework to promote industrial competitiveness. Consequently, the measures implemented by the previous NEEAP 2017-2020 and its predecessor contributed to an improvement in energy efficiency. For instance, during the period 2004--2016 it was quantified an annual decrease of 2% in final energy intensity in the industry sector \[[@bib77]\]. Final energy consumption in the industry sector had slightly increased in 2017 but stabilised in 2018 (see [Fig. 11](#fig11){ref-type="fig"} ). This suggests that further investments in this sector are essential.Fig. 11Final energy consumption by sectors in Spain 2007--2018 \[[@bib77]\].Fig. 11
5.3. National policies on energy efficiency {#sec5.3}
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The EED was transposed in different national regulations in Spain. For instance, the Law 18/2014 of 15 October established the system of energy saving obligations, whereas Royal Decree 56/2016 transposed the EED regarding energy audits, accreditation of service providers and energy auditors and promotion of efficiency of energy supply \[[@bib91]\].
As per institutions, the Ministry for Ecological Transition and Demographic Challenge (*Ministerio de Transición Ecológica y el Reto Demográfico, MITECO*); Institute for the Diversification and Saving of Energy (*Instituto para la Diversificación y el Ahorro de Energía, IDAE*), which is responsible for the objectives and commitments regarding improvements in energy efficiency, renewable energy and other low carbon cost technologies; Autonomous Communities and local institutions work together in the implementation and monitoring of targets and measures defined in the NECP. The Climate Change Policy Coordination Commission was created in 2005 to coordinate the collaboration between the General State Administration and Autonomous Communities with regard to all climate change policies \[[@bib92]\].
The National Energy Efficiency Fund (*Fondo Nacional de Eficiencia Energética, FNEE*), established by Law 18/2014 of October 15, 2014, introduced urgent measures for growth, competitiveness and efficiency. This Fund falls under auspice of the MITECO and is an instrument of the implementation mechanisms for financial and economic support, technical assistance, training and information or other measures in order to increase energy efficiency across all energy-intensive sectors. The FNEE consists of the financial contributions from the European Structural Funds, contributions of the obligated parties required by the EEOS, other contributions from the State budget as well as any other resources intended to finance actions of energy savings and efficiency \[[@bib90]\]. Specifically, the financial contributions from the obligated parties for 2014 was EUR 103 M, which has increased significantly for the two consequent years of 2015 and 2016 at EUR 207 M, followed by the other consequent years of 2017 and 2018 set at EUR 205 M with the most recent years of 2019 and 2020 reaching EUR 203 and EUR 207 M respectively \[[@bib93], [@bib94], [@bib95], [@bib96], [@bib97], [@bib98], [@bib99]\]. The total amount of the aid program for 2020 is EUR 307 M \[[@bib100]\]. The FNEE is supervised and monitored by Supervision and Monitoring Committee and managed by the IDAE.
5.4. Obligatory energy savings schemes and alternative measures {#sec5.4}
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The cumulative savings target in Spain are reached by implementing the national EEOS and by applying a combination of alternative measures of a regulatory, fiscal or economic nature. EEOS in Spain are identified within the FNEE, which has now been extended until December 31, 2030 as defined by the updated EED \[[@bib3]\]. In terms of saving obligation, pursuant to the respective laws \[[@bib101], [@bib102], [@bib103], [@bib104]\], the obligated parties (i.e. the energy distributors and retail energy sales companies, namely, gas and electricity retailers, petroleum product wholesalers and liquified petroleum gas wholesalers) are obliged each year to contribute to the FNEE an amount which is calculated by taking into account the investments required to achieve their respective obligation (i.e. assigned annual energy savings quota). Additionally, Article 7 EED and the updated EED require the Member States to demonstrate the achievement of cumulative energy savings in the two main periods: January 1, 2014--December 31, 2020 and January 1, 2021--December 31, 2030 ensuring that new savings are attained each year. It is estimated that Spain for the first period would achieve 15 979 ktoe, whereas for the second period it would be equivalent of 36 809 ktoe. This in turn means that new and additional energy savings would be equivalent to 669 ktoe each year from January 2021 to December 2030 \[[@bib77]\], demonstrating the importance of the EEOS in terms of generating new savings in Spain. The distribution by sectors of the planned 36 809 ktoe accumulative savings is shown in [Fig. 12](#fig12){ref-type="fig"} .Fig. 12Distribution by sectors of the planed 36 809 ktoe savings \[[@bib77]\].Fig. 12
The NECP incorporates the ten principal energy efficiency measures designed to meet the obligation arising from the application of Article 7 EED by taking a sectoral approach. One of the measures (No. 2.5 funded by the FNEE) is to facilitate improvements in the technology and management systems of industrial processes for SMEs and large enterprises (particularly to those not included in the EU ETS) \[[@bib86]\]. Specifically, this measure promotes:i)the investments for replacing low energy efficient equipment or installations by high energy-efficient technologies or by best available technologies or for replacing the auxiliary energy consuming systems.ii)the investments in the implementation of energy management systems in industry \[[@bib77]\] which will be further discussed in the following section.
In the past, there were some successful alternative measures. For instance, the Energy Diversification and Saving Investment Fund which is part of JESSICA (Joint European Support for Sustainable Investment in City Areas) with a budget of EUR 123 million financed, *inter alia*, urban energy-efficiency and energy management projects, falling within one of the eligible sectors (construction, industry, transport and energy-related public service infrastructure). CLIMA projects financed by Spanish FES-CO2 (Carbon Fund for a Sustainable Economy) also applicable to industry aimed at reorienting the Spanish production system towards a low-carbon model. Order IET/274/2015 of February 13, 2015 introduced the Industrial competitiveness incentive program to stimulate investments that contribute significantly towards generating added value in the industrial sector. Specifically, it aimed to help companies shift towards newer production models which were more advanced, efficient and environmentally friendly, and to manufacture products with greater added value, intended to have a significant impact on businesses' competitiveness therefore, enabling them to gain access to and increase their presence in international markets \[[@bib86]\]. There are also initiatives at local level to promote energy efficiency. For instance, in Basque Autonomous Community jurisdiction, Basque Energy Cluster regularly organises events to promote the energy efficiency in industry where companies show their experiences of different energy efficiency measures applications (i.e. fumes heat recovery for air combustion pre-heating, water for cooling moulds, use of frequency variable devices in injection machines, using of advanced sensors and monitoring in forging processes, TIC technology integration for energy efficiency in Industry 4.0, etc.) \[[@bib105]\].
Building on the most recent measures as defined in the NECP, the industrial sector is expected to achieve 10 256 ktoe of cumulative final energy savings during the 2021--2030 period \[[@bib77]\]. These planned energy-saving targets should be attained through a) public support programmes (i.e. aid through grants or repayable low-interest loans in compliance with the EU State Aid regulations); or b) voluntary agreements \[[@bib77]\]. The authorities responsible of this measure is MITECO and IDAE (coordinated with other Ministry Departments having competencies in industry policy) and the Governments of Autonomous Communities under their respective jurisdiction.
5.5. Audits and energy management systems {#sec5.5}
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Article 8 EED was implemented in Spain by the adoption of Royal Decree 18/2014 and Royal Decree 56/2016 of February 12 on energy efficiency concerning energy audits, accreditation of providers of energy services and auditors, and promotion of the efficiency of energy supply. For instance, audits must be conducted by properly qualified energy auditors \[[@bib106]\]. The Energy Audit Register was created in the MINETAD based on the information provided by the regional authorities \[[@bib107]\].
As discussed above, only large companies have the obligation to perform an audit every four years under Article 8 EED. Spain adopted a similar approach to the EU by using the same employee and financial thresholds. Yet, it defines large companies with at least 250 employees and a turnover of more than EUR 50 million or a balance sheet of more than EUR 43 million \[[@bib106]\]. Furthermore, an audit is applicable to corporate groups which, taking into account the aggregate size of the corporations from that consolidated group, meet the large enterprise criteria. They must undergo an energy audit every four years, which covers a least 85% of the total final energy consumption of the group of facilities located within national territory \[[@bib106]\]. The penalties can be imposed on large companies in case of non-compliance. Spain has a two-fold test: i) up to EUR 60 000; or ii) up to 10% of the company's turnover, therefore, linking with a profitability criterion \[[@bib33]\]. To ensure compliance, the Member States must have a mechanism in place of continuous monitoring. Most EU Member States, including Spain, conduct random checks on spot and large companies must provide the relevant documents on request. In addition, these obligated companies also proactively submit the information about their energy audit \[[@bib33]\]. By December 2018 there have been around 35 000 energy audits listed \[[@bib108]\].
In compliance with the EED, the implementation of an energy or environmental management system is considered equivalent to the above obligation, provided that the management system includes an energy audit in accordance with the minimum energy audit criteria \[[@bib86]\]. As discussed above, there is also an aid programme available for SMEs and large industrial enterprises, financed by the FNEE, which can facilitate the implementation of the energy saving and efficiency measures proposed by energy audits to reduce energy consumption in industrial processes.
5.6. Efficiency in heating and cooling (including industrial waste heat recovery) {#sec5.6}
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Royal Decree 56/2016 of February 12, 2016 also transposed Article 14 EED to promote energy efficiency in the production and in the use of heating and cooling energy. Based on the statistics provided by IDAE the co-generated installed capacity in Spain at the end of 2016 was 5618 MW with cogeneration having a strong presence in the industrial sector, with an installed capacity of 5197,1 MW \[[@bib109]\] (i.e. approximately 92% of the installed capacity, followed by the remaining 8% in the service and residential sectors). The fuel mainly consumed by co-generation plants is natural gas, which represents 84% of electricity production and 86% of heat production \[[@bib108]\].
Given that approximately 2400 MW of co-generated capacity will have exceeded its regulatory life span by 2030, Spain is planning to transform ageing CHP units into high efficiency CHP units. Additionally, Royal Decree 15/2018 introduced measures for the energy transition and consumer protection, which should in turn drive the return to electricity self-consumption from renewable sources or from co-generation facilities connected to the network. A measure has been proposed aimed at the transition of co-generation to high efficiency in a total of 1200 MW of co-generation facilities using natural gas with an optimised design based on: useful heat, electrical self-consumption, flexibility of operation with regard to the electricity system and high efficiency. The mechanism is based on the competitive tendering procedure with a multi-annual schedule of tenders, to ensure a cost-effective remuneration scheme for the application of public funding, supported by the necessary administrative measures to take advantage of the existing infrastructure. It is estimated that savings associated with this measure will be 1471 ktoe of cumulative primary energy during the 2021--2030 period.
Spain also has some initiatives to promote technology for heating and cooling (such as novel thermal insulations and passive strategies \[[@bib110]\], geothermal closed-loop heat exchange \[[@bib111]\], district heating/cooling \[[@bib112]\], trigeneration systems \[[@bib113]\], retrofitting strategies in manufacturing processes and devices \[[@bib114]\] etc.). For instance, IDAE offers a Series of Technical Guidelines with the objective of increasing energy efficiency of the thermal installations in buildings \[[@bib109]\]. There are also guides prepared by the Autonomous Communities, such as Basic Guide to Heating and Cooling networks, prepared by Catalan Institute of Energy and COFELY AG \[[@bib115]\], which promote and disseminate the technology of district heating and/or cooling networks, as well as advise the potential promoters of this type of installations from a methodology that establishes the criteria that should be considered when designing a district network \[[@bib116]\]. The EU-funded projects, such as the Horizon 2020 also facilitates the promotion of new technologies. For instance, the I-Therm project has been identified by the European Commission as one of good practices in terms of industrial waste heat recovery \[[@bib38]\], which developed technologies and processes for the efficient and cost effective heat recovery in industrial facilities, including in a company in Spain active in steel production \[[@bib12],[@bib117]\]. In the most recent project ETEKINA, the heat exchanger technology using heat pipes for thermal recovery will be installed in one aluminium automotive parts producer in Spain enabling it to recover the lost heat (of approximately 40%), therefore, increasing the overall energy efficiency and reducing the energy costs, which especially for primary aluminium producers are significant -representing between 22% and 29% of the production costs \[[@bib73]\].
6. Summary of the discussions {#sec6}
=============================
*EU level*. 2019 marked the completion of the Clean Energy for all European package (also known as the Winter package) \[[@bib1]\] with the final regulations and directives being issued. As part of this package is one of the key principles of "Putting energy efficiency first" embedded in the revised EED, which has expanded the obligatory measures defined in Article 7 EED up to 2030, yet, with the reduced target of 0.8% (for the forthcoming 2021--2030 period) rather than 1.5% as in the previous period of 2014--2020. This way the European Commission forces obliged parties to achieve new energy savings each year. Given that the Member States have flexibility on how they achieve their energy savings, such as by introducing energy savings obligation schemes, alternative measures or a combination of the two, there is a vast variation of these measures across the Member States. In the context of the industrial sector, while the Member States may incorporate some alternative measures (i.e. through tax reduction) to incentive energy-intensive industries to implement energy efficiency measures, the EED has more coercive measures, such as audit obligations on all large enterprises (including businesses operating in the industrial sector). Large businesses are only exempted from this audit obligation if they have implemented an energy management systems, which create a structure to monitor energy consumption and improve energy efficiency in an industrial firm and the use of it in industry is currently increasing \[[@bib118]\]. Finally, there are also provisions to address efficiency in heating and cooling requiring the Member States to conduct a comprehensive assessment of the cost effective potential for energy efficiency in heating and cooling, principally through the use of cogeneration, efficient district heating and cooling and the recovery of industrial waste heat \[[@bib119]\].
There is an expectation that all these measures will contribute (or continue to contribute) in achieving national energy efficiency targets, which in turn will lead to the accomplishment of the EU overall energy efficiency target of 32.5% by 2030. To ensure this is the case, all the Member States had to present their integrated NECP, *inter alia*, outlining how they intend to meet the energy efficiency targets for 2030 and what measures will be in place. Statistically speaking, it seems the EU may fall short of meeting its targets, as the final energy consumption was 2.2% below the 2020 target level in 2014, in 2018 it was 3.2% above the 2020 target level and the distance to the 2030 target was 17.0% based on 2018 data.
*National level.* While Slovenia and Spain differ in size, they both have large energy dependency set at 48% and 79% respectively with a great potential for the sustainable energy development, including energy efficiency. Spain aims to reduce its dependency to 61% by 2030. In both countries industry is the second biggest energy consumer, with the transport sector taking the first place. Therefore, both Slovenia and Spain set their more energy efficiency targets to be achieved by 2030, 35% and 39.5% respectively (see [Table 3](#tbl3){ref-type="table"} ).Table 3Summary of the main provisions discussed in Slovenia and Spain.Table 3MeasureDescription and evaluation**Slovenia**EE targetsMore ambitious targets to be achieved by 2030: for its primary (6.36 Mtoe) and final (4.72 Mtoe) energy consumptions; and overall improvement by 35% in comparison to only 20% by 2020.EEOS/alternative measuresA combination of energy efficiency obligation schemes and alternative measures;\
Need of the improvement of the monitoring of the scheme;\
Utilisation of Eco Funds.Audits/Management systemsExpanded definition of large companies to ensure wider coverage of the Audit obligation;\
Support mechanism to conduct Audits to SMEs.Efficiency in Cooling & HeatingDeveloped a comprehensive strategy for heating and cooling providing guidelines for the development of efficient DH, DC, and the use of CHP;\
Various financial incentives intended for investments in new (or expansion) of DH-RES systems/microsystems, construction of CHP etc.**Spain**EE targetsMore ambitious targets to be achieved by 2030; for its primary (98.5 Mtoe) and final (73.6 Mtoe) energy consumptions; and overall improvement by 39.5% in comparison to its revised target of 24.2% by 2020.EEOS/alternative measuresA combination of energy efficiency obligation schemes and alternative measures;\
A sectorial approach;\
Utilisation of the FNEE, including the investments to replace low energy equipment/to implement energy management systems.Audits/Management systemsRelies the EU definition of SMEs, to define its large companies for the Audit obligation.Efficiency in Cooling & HeatingEssential focus on transforming ageing CHP units into high efficiency CHP units;\
Support mechanism based on the competitive tendering procedure which incorporates more market-based principles.
Both Slovenia and Spain meet their binding requirements of Article 7 EED through a combination of energy efficiency obligation schemes and alternative measures. Slovenia noted that it has problems in monitoring of the implementation of the scheme (i.e. checking the reporting by the obliged entities of the achievement of energy savings from their implemented projects), as well as the implementation of alternative measures, in the context of the effective allocation of funds to those which have the greatest potential to increase energy efficiency \[[@bib51]\]. Spain has a clear sectorial approach, as the measures designed to meet the obligations arising of Article 7 EED are set for each sector (i.e. No 2.5 is applicable for the industrial sector). Interestingly, neither of these countries has white energy certificates (employed in other EU Member States, such as Italy, France, etc) \[[@bib5]\], even though the Spanish Law 18/2014 provides a possibility for the establishment of an accreditation mechanism for achieving of energy saving -- Energy Saving Certificates.
With regard to the audit obligation, Slovenia has modified the EU definition of SMEs. While Slovenia has expanded the obligation scope by lowering the financial thresholds for the definition of SMEs therefore, covering firms that SMEs, Spain on the other hand, adopted a similar to the EU approach, by defining large companies instead. Most likely Slovenia made these adjustments to reflect its small size of its economy and that most businesses in Slovenia belongs to the SMEs category. Given that the Member States are allowed to lay down national rules on penalties to guarantee compliance with the requirements of Article 8 EED, both Slovenia and Spain can impose fines. While Spain places penalties on businesses themselves and links its fine up to EUR 60 000 with a profitability value (not exceeding 10% of the company's turnover), whereas Slovenia imposes fines (EUR 10 000) on the directors (or other senior person at managerial level).
As indicated by [Table 3](#tbl3){ref-type="table"} both countries contain measures to promote energy efficiency in the production and in the use of heating and cooling energy as required by the EED. It seems that the co-generated installed capacity in Spain is most visible in the industrial sector (i.e. approximately 92% of the installed capacity).
7. Conclusions {#sec7}
==============
The EU has a clear long-term strategy towards carbon-neutral economy. To achieve this, the Clean Energy for all Europeans package \[[@bib4]\] was issued and set tasks to bring EU energy legislation into line with the new more ambitious 2030 climate and energy targets stressing the 'Energy efficiency first' as one of the key principles of the Energy Union. This is because energy efficiency is the most cost effective way to reduce emissions, improve energy security, enhance competitiveness, therefore, enabling to achieve the long-term energy and climate goals. Given that the Member States come in different shape and size of the economies and their development level, they present different national contributions to the EU energy efficiency target of 32.5% by 2030. To ensure that the EU energy efficiency target will be achieved, all the Member States had to present their integrated NECPs, which, *inter alia*, must outline how they intend to meet the energy efficiency targets for 2030 and what measures will be employed to secure them. Specifically, this paper has focused on two EU Member States, namely, Slovenia and Spain. Both countries analysed in this paper have high domestic dependence on imported energy sources, therefore, showing a great potential for energy efficiency. To address this, Slovenia and Spain have set more ambitious national energy efficiency contributions of 35% and 39.5% respectively for 2030 in comparison to their 2020 targets. They also committed to continue their national measures and schemes (including obligatory energy efficiency schemes) imposed on the obliged parties from their NEEAP 2020 as well energy efficiency support mechanisms, therefore, embracing a 'carrot and stick' approach. The paper has also identified some good practices to improve energy efficiency in the industrial sector, especially through the EU-funded projects, therefore, outlining the importance of funding initiatives. Additionally, the EU requirement to submit the integrated NECP have had a broader impact, inspiring additional national developments in both countries. For instance, the Energy Concept of Slovenia was introduced in Slovenia, whereas in Spain it facilitated the launch of the national Climate Change and Energy Transition Law as well as the development of long-term and Just Transition strategies.
Finally, both Slovenia and Spain have energy efficiency incentive programmes, which may be affected by the COVID-19 crisis. Given that during the writing of this paper there have not been any adjustments confirmed by the governments in both countries, the near future will demonstrate the extent to which the developments of energy efficiency have been impact by this crisis.
Authorship statement {#sec8}
====================
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in Energy.
Authorship contributions {#sec9}
========================
Please indicate the specific contributions made by each author (list the authors' initials followed by their surnames, e.g., Y.L. Cheung). The name of each author must appear at least once in each of the three categories below.
Jurgita Malinauskaite: Conception and design of study, acquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content. Hussam Jouhara: Conception and design of study, acquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content. Bakartxo Egilegor: acquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content. Fouad Al-Mansour: acquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content. Lujean Ahmad: acquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content. Matevz Pusnikacquisition of data, Formal analysis and/or interpretation of data, Drafting the manuscript, revising the manuscript critically for important intellectual content, Approval of the version of the manuscript to be published
Acknowledgements {#sec10}
================
All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are named in the Acknowledgements and have given us their written permission to be named. If we have not included an Acknowledgements, then that indicates that we have not received substantial contributions from non-authors.
**This statement is signed by all the authors (**a photocopy of this form may be used if there are more than 10 authors**):** Author's nameAuthor's signatureDateHussam JouharaHussam JouharaJuly 02, 2020Jurgita MalinauskaiteJurgita MalinauskaiteJuly 02, 2020Bakartxo EgilegorBakartxo EgilegorJuly 02, 2020Fouad Al-MansourFouad Al-MansourJuly 02, 2020Matevz PusnikMatevz PusnikJuly 02, 2020Lujean AhmadLujean AhmadJuly 02, 2020
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.
This work has been funded by Innovation and Networks Executive Agency (10.13039/501100012290INEA, European Commission under project H2020 ETEKINA (heat pipE TECHnologies for INdustrial Applications) Contact number: 768772.
[^1]: The emissions in 2005 were higher than in 1990 (54%) and the projections of GHG emission in 2020 are also higher than in 1990 (11%). This explains the reason behind the high percentage figure of GHG reduction in 2030 referred to 2005. The GHG reduction plan by 2030 compared to the 1990 is set at a 23%.
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