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Introduction
============
Real-time monitoring of firefighters\' heat strain during work has been attempted by recording deep body temperature and heart rate (HR). However, monitoring deep body temperature with HR during firefighting is cumbersome and inconvenient for firefighters. Furthermore, monitoring either deep body temperature or HR solely does not reflect full heat strain during firefighting while wearing personal protective clothing with self-contained breathing apparatus (SCBA). Because the capacity of SCBA is limited to 30\~45 min, firefighters should take short breaks during firefighting to replace the SCBA. In the US, they provide guidelines to have firefighters take a minimum 10 min break after using a bottle of SCBA, and a minimum 20 min break after using two bottles of SCBA. However, it is more helpful to prevent heat-related illness and diseases of firefighters if we propose a necessary break time based on non-invasively-monitored physiological response. The purpose of the present study was to investigate the possibility of HR as a heat strain index for firefighters during rest periods.
Methods
=======
Twelve professional male firefighters participated in an experiment wearing firefighters\' personal protective equipment (15 kg) with intermittent exercises at an air temperature of 32 °C and 43% relative humidity. Participants began each trial with a 10 min rest and performed two bouts of 15 min exercises on a treadmill at 5.5 km.hr^-1^(60% VO~2max~) that were separated by 10 min of seated rest. HR, rectal temperature (T~re~), and oxygen consumption were measured. T~re.break~and ΔT~re.break~were defined as T~re~during rest periods and changes in T~re~during rest, respectively. Linear regression equations were derived between HR and T~re~. Heart rate was expressed as absolute (HR~absolute,~bpm) and relative values (HR~relative,~%HR~max~).
Results
=======
During rest periods, significant regression equations were derived between HR and T~re;~and between HR and ΔT~re~:
$$\text{T}_{\text{re}\text{.break}} = 0.035 \cdot \text{H}\text{R}_{\text{relative}} + 35.83\left( {\text{R}^{2} = 0.722,P < 0.05} \right)$$
$$\text{Δ}\text{T}_{\text{re}\text{.break}} = 0.0008 \cdot \text{H}\text{R}_{\text{relative}} - 0.0066\left( {\text{R}^{2} = 0.506,P < 0.05} \right)$$
$$\text{T}_{\text{r}}\left( t \right) = 0.035 \cdot \text{\%~}HR_{rest} + 35.830 + \left( {8 \times \% HR_{rest} + 66} \right) \cdot 10^{- 4} \cdot \text{t}\mspace{420mu}\left( {\text{R}^{\text{2}} = 0.\text{7}0\text{8},P < 0.0\text{5}} \right)$$
By using equation 3, we calculated HR reference values which can identify when it is safe to continue 15 min, 30 min or 45 min operations after breaks (Figure [1](#F1){ref-type="fig"}). For example, to continue a 15 min of operation after a break at work without any heat-related illness, HR during the break should be less than 70% HR~max~(39°C T~re~predicted).
{#F1}
Conclusion
==========
We confirmed the possibility of using a heart rate index during breaks at work to evaluate firefighters\' heat strain. Using heart rate during breaks at work, we can determine whether to continue or stop firefighters\' operations in hot environments. However, further studies are required to confirm the validity of using a heart rate index in various thermal environments and work intensities of firefighting to determine when it is safe to return to work.
| {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the manuscript and its Supporting Information files.
Introduction {#sec001}
============
The term "muscle strength" refers to a muscle\'s or a group of muscles' ability to exert maximum muscular force \[[@pone.0226274.ref001]\]. Isokinetic dynamometry is the gold standard in muscle strength evaluation \[[@pone.0226274.ref002]\]. It is an adequate system for assessment and diagnosis in the field of biomechanics. Measuring muscle strength allows evaluating and comparing normal and diseased children, establishing goals for recovery and rehabilitation, and quantitatively monitoring the course of a disease and the response to treatment \[[@pone.0226274.ref003]\]. Therefore, following the safety standards and recommendations for children, isokinetic dynamometry is safe \[[@pone.0226274.ref004]\] and allows registering a range of angular velocities in both concentric and eccentric exercises \[[@pone.0226274.ref005]\].
Reliability is defined as the extent to which measurements can be replicated \[[@pone.0226274.ref006]\]. Reliability can be presented in relative or absolute values. Relative reliability indicates the degree to which individuals maintain their position in a sample with repeated measurements \[[@pone.0226274.ref007]\]; the most common indicator of relative reliability is intra-class correlation coefficients (ICC) \[[@pone.0226274.ref008]\]. In contrast, absolute reliability refers to the degree of conformity of the measures of a test from moment to moment \[[@pone.0226274.ref004]\]. The most common indicators of absolute reliability are the standard error of measurements (SEM) and the smallest real difference (SRD).
The reliability of measuring isokinetic knee strength with a dynamometer in children has not been fully investigated. The majority of isokinetic strength reliability studies report the correlation coefficient as an indicator of the agreement between measurements \[[@pone.0226274.ref001]\]. When reported, often only relative reliability is addressed, via ICC \[[@pone.0226274.ref009]\]. The relative reliability of isokinetic knee strength measurements in this age group has been reported as moderate \[[@pone.0226274.ref002], [@pone.0226274.ref004]\] to high \[[@pone.0226274.ref010], [@pone.0226274.ref011]\], and this parameter has been given more importance in previous studies. However, absolute reliability, which refers to the degree of conformity in test measurements from one time-point to another, has not yet been determined in depth. This should be the next step for these studies to clarify clinically important changes for patients \[[@pone.0226274.ref004]\]. Only two previous studies \[[@pone.0226274.ref004], [@pone.0226274.ref012]\] have given an approximation for the value of SRD. The present review presents more information on this variable. For the above reasons, it is important to know the reliability of isokinetic dynamometry.
Scattered information is available on the reliability of isokinetic measurements for children. Moreover, to our knowledge, no systematic reviews or meta-analyses on this topic have been published. Therefore, the purpose of this research was to perform a systematic review and meta-analysis of existing values of the test-retest reliability of isokinetic knee strength measurements in children, discuss potential limitations of the literature, and suggest recommendations for future research on statistical analyses for interpreting reliability. The current review and meta-analysis can provide valuable information for future guidelines and strategies for muscle strength reeducation in children.
Materials and methods {#sec002}
=====================
This systematic review meets the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines ([S1 File](#pone.0226274.s001){ref-type="supplementary-material"}).
Eligibility criteria {#sec003}
--------------------
Test-retest studies published in English (up to July 26, 2019) that measured knee flexion and extension in children using an isokinetic dynamometer were included.
For this research, we applied further eligibility criteria. Eligible studies were those that assessed the reliability of isokinetic knees in healthy children or those with cerebral palsy applied to dynamic contractions (concentric or eccentric) and that provided measures of force, reproducibility, ICC, peak torque, or SEM.
We did not include studies that reported duplicate results.
The meta-analysis included studies on the reliability of knee movement in the concentric mode, both for the extension and for the flexion of the joint at a speed of 60°/s, that always included the reliability values of the dominant leg of the subjects.
Two of the authors (FM and JCA) independently examined and screened the titles and abstracts of the retrieved articles to assess study eligibility. Any disagreement or uncertainty was resolved through discussion. All reviewers reviewed the full-text articles that met the inclusion criteria or had uncertain eligibility. Any disagreement was resolved by consensus.
Electronic literature search {#sec004}
----------------------------
Several databases were searched, including PubMed, Web of Science, Scopus, and Embase.
A combination of MeSH terms was used for the PubMed search: child, knee, muscle strength, reproducibility of results, and validation studies.
The specific keywords used for the Web of Science, Scopus, and Embase were combinations of "isokinetic", "knee", "reliability", "reproducibility", "children", "kids", "boys", "girls", and "dynamometer". The exact search was: *((children \[Title/Abstract\] OR kids \[Title/Abstract\] OR boys \[Title/Abstract\] OR girls \[title/abstract\]) AND knee \[Title/Abstract\] AND (reliability \[Title/Abstract\] OR retest \[Title/Abstract\] OR test-retest \[Title/Abstract\] OR reproducibility \[Title/Abstract\]) AND isokinetic \[Title/Abstract\])*.
After examining the records, additional searches were conducted in health improvement sources, meta-search engines (Google/Google scholar), and on the Retraction Watch website to identify additional publications and gray literature.
Isokinetic dynamometry {#sec005}
----------------------
Isokinetic movement is defined by maintaining an angular velocity of constant movement throughout the joint path. The current isokinetic dynamometry system allows evaluating in both concentric and eccentric modes of exercise. The dynamometer shows the value of the moment of force developed at each instant. The most important data recorded by the isokinetic dynamometer is the peak torque or maximum moment of force, which indicates the highest value of force recorded during the test. Another variable an isokinetic dynamometer provides is "work", which expresses the product of the moment of force and angular distance. Peak torque was used in this review.
Evaluation of the quality of the included studies {#sec006}
-------------------------------------------------
The selected studies were evaluated using the clinical evaluation tool (CAT) scale developed by Brink and Louw \[[@pone.0226274.ref013]\] and the Quality Appraisal for Reliability Studies (QAREL) \[[@pone.0226274.ref014]\].
The CAT scale is an instrument developed specifically to evaluate the methodological quality of studies, considering the validity and reliability of the objective clinical tests. The CAT scale contains 13 evaluation items. Four of the 13 items refer to validity issues, but the other nine refer to reliability; therefore, only these nine were used for this review. Each article was classified as \"yes\" when information was described in sufficient detail or \"no\" when there was not enough information for clarification \[[@pone.0226274.ref013]\]. A final percentage (%) evaluation column was added based on the items that each study achieved. Thus, the maximum possible score was 90%, which represents the highest methodological quality. Studies were considered high quality if they scored above 45%.
The Quality Appraisal for Reliability Studies (QAREL) scale is a quality assessment tool for diagnostic reliability studies. It consists of 11 questions (meets, does not meet, doubtful or not applicable) grouped into three categories of internal validity (items 3--9), external validity (items 1, 2, and 10), and the relevance of the statistical analyses (item 11). The maximum score is 110%.
Statistical analysis {#sec007}
--------------------
The meta-analysis included only five articles \[[@pone.0226274.ref002], [@pone.0226274.ref004], [@pone.0226274.ref010]--[@pone.0226274.ref012]\] of the ten incorporated in this review. This meta-analysis focused on the reliability of the concentric mode, both for extension and knee flexion. The choice of these items was because the studies all used a similar speed, 60°/s, always with the values of the dominant leg of the subjects. In addition, we considered the sample size and the type of participants; all were healthy except for those in Moreau's study, whose subjects had cerebral palsy (CP) \[[@pone.0226274.ref010]\].
Heterogeneity between the included studies was assessed using the chi-squared test on Cochran's Q (alpha set at 0.1) statistic \[[@pone.0226274.ref015]\] and Higgins and Thompson\'s I^2^ statistic \[[@pone.0226274.ref016]\].
The random-effects model and the fixed-effects model were used to combine standardized effect sizes with a 95% confidence interval.
The funnel plot and Egger´s weighted regression tests were used to evaluate possible publication bias (p ˂ 0.1 was considered statistically significant publication bias).
Regarding the reliability indicators used in this review, we started with the ICC, which is usually the main indicator of reliability. All ICCs collected in our review are shown at a 95% confidence interval (CI) \[[@pone.0226274.ref012]\].
The SEM was calculated from the square root of the mean error term derived from the analysis of variance (ANOVA) \[[@pone.0226274.ref012]\] and was used to determine the minimum difference that is considered important for a single subject \[[@pone.0226274.ref004]\]. SEM was calculated as $SEM = SD\sqrt{\left( 1 - ICC \right)}$, where SD is the standard deviation of day 1 and day 2 \[[@pone.0226274.ref017]\].
The SRD is shown as a measure of sensitivity to change. In the original formulation, SRD was defined as the 95% confidence limit of the SEM of difference scores \[[@pone.0226274.ref018]\]. The SRD was calculated as $SRD = 1.96 \times \sqrt{2} \times SEM$ \[[@pone.0226274.ref017]\].
The percentages of SEM and SRD are shown to represent the error of measurement in relative terms and thus allow comparing the different variables \[[@pone.0226274.ref012]\]. This was calculated with: \[SEM or SRD / mean of all values\] \[[@pone.0226274.ref012]\].
Results {#sec008}
=======
Search strategy and quality of studies {#sec009}
--------------------------------------
A total of 143 studies were identified through searches in electronic databases. Among these studies, we identified and eliminated 49 duplicates. Only 14 studies met the inclusion criteria, but four of these studies were excluded. One study was excluded because it presented different ages, another because it did not evaluate the knee joint, and another two because they did not perform test-retest. A manual search of the bibliographies of the relevant articles revealed no additional studies. Therefore, ten prospective studies were included in this systematic review on isokinetic knee movement in children ([Fig 1](#pone.0226274.g001){ref-type="fig"}).
{#pone.0226274.g001}
The quality of the articles according to the CAT quality score varied between 23% and 78%, with the maximum possible being 90%. According to the analysis, eight articles were evaluated as high quality ([Table 1](#pone.0226274.t001){ref-type="table"}). The quality of the articles varied according to the designs of the studies.
10.1371/journal.pone.0226274.t001
###### Evaluation of the quality of the studies with clinical evaluation tool (CAT).
{#pone.0226274.t001g}
Study 1 2 3 4 5 6 7 8 9 \%
------------------------------------------------------------ ----- ----- ----- ----- ---- ----- ----- ---- ----- ----
Ayalon et al. (2000)\[[@pone.0226274.ref027]\] yes yes no yes no yes yes no yes 67
Fagher et al. (2016)\[[@pone.0226274.ref004]\] yes no no yes no yes yes no yes 56
Iga et al. (2006)\[[@pone.0226274.ref005]\] yes no no no no yes yes no no 34
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] yes yes yes yes no yes yes no yes 78
Kellis et al. (1999)\[[@pone.0226274.ref011]\] yes no no yes no yes yes no yes 56
Merlini et al. (1995)\[[@pone.0226274.ref003]\] yes no no yes no yes yes no yes 56
Moreau et al. (2008)\[[@pone.0226274.ref010]\] yes no no yes no yes yes no yes 56
Pierce et al. (2006)\[[@pone.0226274.ref020]\] yes no no yes no no yes no yes 45
Santos et al. (2013) \[[@pone.0226274.ref002]\] yes yes no yes no yes yes no yes 67
Van den Berg-Emons et al. (1996)\[[@pone.0226274.ref021]\] yes no no no no no yes no no 23
%: (Items \"yes\" x 100)/9; 1. If human subjects were used, did the authors give a detailed description of the sample of subjects used to perform the test? 2. Did the authors clarify the qualification, or competence of the rater(s) who performed the test? 3. If interrater reliability was tested, were raters blinded to the findings of other raters? 4. If intrarater reliability was tested, were raters blinded to their own prior findings of the test under evaluation? 5. Was the order of examination varied? 6. Was the stability (or theoretical stability) of the variable being measured taken into account when determining the suitability of the time interval between repeated measures? 7. Was the execution of the test described in sufficient detail to permit replication of the test? 8. Were withdrawals from the study explained? 9. Were the statistical methods appropriate for the purpose of the study? %: final percentage of reliability.
According to the QAREL scale, the quality of the articles varied between 30% and 100%, with the maximum possible being 110% ([Table 2](#pone.0226274.t002){ref-type="table"}). Evaluation data indicate that eight articles scored between 60 and 100%.
10.1371/journal.pone.0226274.t002
###### Evaluation of the quality of the studies with Quality Appraisal of Reliability Studies (QAREL).
{#pone.0226274.t002g}
Study 1 2 3 4 5 6 7 8 9 10 11 \%
------------------------------------------------------------ ----- ----- ----- ----- ----- ----- ----- ---- ----- ----- ----- -----
Ayalon et al. (2000)\[[@pone.0226274.ref027]\] Yes Yes No Yes UC Yes No No Yes Yes Yes 70
Fagher et al. (2016)\[[@pone.0226274.ref004]\] Yes No No Yes UC Yes No No Yes Yes Yes 60
Iga et al. (2006)\[[@pone.0226274.ref005]\] Yes No No No No Yes Yes No Yes Yes No 50
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes 100
Kellis et al. (1999)\[[@pone.0226274.ref011]\] Yes No No Yes UC Yes No No Yes Yes Yes 60
Merlini et al. (1995)\[[@pone.0226274.ref003]\] Yes No No Yes UC Yes No No Yes Yes Yes 60
Moreau et al. (2008)\[[@pone.0226274.ref010]\] Yes No No Yes UC No No No Yes Yes Yes 60
Pierce et al. (2006)\[[@pone.0226274.ref020]\] Yes No No Yes Yes No Yes No No Yes Yes 60
Santos et al. (2013) \[[@pone.0226274.ref002]\] Yes Yes No Yes UC Yes No No Yes Yes Yes 70
Van den Berg-Emons et al. (1996)\[[@pone.0226274.ref021]\] Yes No No No Yes No No No No Yes No 30
%: (Items \"yes\" x 100)/11; Was the test evaluated in a sample of subjects who were representative of those to whom the authors intended the results to be applied? 2. Was the test performed by raters who were representative of those to whom the authors intended the results to be applied? 3. Were raters blinded to the findings of other raters during the study? 4. Were raters blinded to their own prior findings of the test under evaluation? 5. Were raters blinded to the results of the reference standard for the target disorder (or variable) being evaluated? 6. Were raters blinded to clinical information that was not intended to be provided as part of the testing procedure or study design? 7. Were raters blinded to additional cues that were not part of the test? 8. Was the order of examination varied? 9. Was the time interval between repeated measurements compatible with the stability (or theoretical stability) of the variable being measured? 10. Was the test applied correctly and interpreted appropriately? 11. Were appropriate statistical measures of agreement used?
Yes; No; UC: unclear.
Characteristics of the studies {#sec010}
------------------------------
[Table 3](#pone.0226274.t003){ref-type="table"} shows the characteristics of the participants. The sample sizes (n) of the final ten studies ranged from 12 to 39 participants, with participants aged 5--15 years. Some of the studies compared the dominant side with the non-dominant side. The joint evaluated in all studies was the knee. All studies evaluated the segments using computerized isokinetic dynamometers: Lido---Active, Cybex II, Cybex Norm, Biodex 6000, Biodex System 3, and Biodex System 4.
10.1371/journal.pone.0226274.t003
###### Characteristics of the participant.
{#pone.0226274.t003g}
Study N Age Gender Type Subjects Bilateral Time of rest Dynamometer
------------------------------------------------------------- ------- -------- -------- ---------------- ----------- -------------- -----------------------
Ayalon et al. (2000)\[[@pone.0226274.ref027]\] 12 9--15 N/S Cerebral Palsy No 7 days Cybex II
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 22 8--10 N/S Healthy No 7 days Biodex System 4
Iga et al. (2006)\[[@pone.0226274.ref005]\] 23 13--14 Boys Healthy Yes 7 days Lido Active
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] 28 12 N/S Sports-active Yes 7 days Biodex 6000
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 13 13 N/S Footballers Yes 7 days Cybex Norm
Merlini et al. (1995)\[[@pone.0226274.ref003]\] 12 6--8 Boys Healthy No 3 days Lido Active
Moreau et al. (2008)\[[@pone.0226274.ref010]\] 12 10--23 N/S Cerebral Palsy No 7 days Biodex Medical System
Pierce et al. (2006) \[[@pone.0226274.ref020]\] 15 10--12 N/S Cerebral Palsy No 1 hour N/S
Santos et al. (2013) \[[@pone.0226274.ref002]\] 21 5--12 N/S Healthy Yes 7 days Biodex System 3
Van den Berg-Emons et al. (1996) \[[@pone.0226274.ref021]\] 12/39 6--12 N/S Cerebral Palsy No 1.5 hours Cybex II
Age (years); N/S: Not Specified.
The contraction speeds ranged from 15°/s to 247.5°/s (4.32 rad/s) for the evaluation of the concentric mode and 60 to 180°/s for the eccentric mode.
To classify the strength of reliability, we used the scale proposed by Landis and Koch \[[@pone.0226274.ref019]\]: 0 represents poor reliability, 0.01--0.20 slight reliability, 0.21--0.40 regular reliability, 0.41--0.60 moderate reliability, 0.61--0.80 substantial reliability, and 0.81--1.00 very high (almost perfect) reliability.
All selected articles presented strength measurements for both knee flexion and extension in the concentric mode. Of these, only three \[[@pone.0226274.ref004], [@pone.0226274.ref005], [@pone.0226274.ref011]\] analyzed knee flexion and extension in the eccentric mode as well. Four studies \[[@pone.0226274.ref002], [@pone.0226274.ref005], [@pone.0226274.ref011], [@pone.0226274.ref012]\] included tests on both dominant and non-dominant legs. Studies found substantial and very high (almost perfect) reliability for all analyses, regardless of movement, speed, type of contraction, evaluation of the dominant or non-dominant leg, and whether the participants had CP or not.
The Pierce's study identified regular and moderate reliability where the knee flexion was assessed at 15°/s and 90°/s with ICCs of 0.31 and 0.38, respectively. This study also identified moderate reliability, because it measured knee extension assessed at 15°/s and at 90°/s with ICCs of 0.51 and 0.50, respectively. Both movements were in the concentric mode \[[@pone.0226274.ref020]\]. Fagher also measured knee flexion at 60°/s and 180°/s with ICCs of 0.62 and 0.49, respectively. This study, although it showed substantial reliability when measuring knee extension at 180°/s with an ICC of 0.68 in the eccentric mode, showed moderate reliability (0.60) for knee flexion \[[@pone.0226274.ref004]\] ([Table 4](#pone.0226274.t004){ref-type="table"}).
10.1371/journal.pone.0226274.t004
###### Relative and absolute reliability of concentric extension and flexion of the knee in isokinetic.
{#pone.0226274.t004g}
Knee action evaluated Speed (°/s) Mean /(SD) 1° test Mean /(SD) 2° test Mean /(SD) 3° test ICC (95% CI) SEM (Nm) SEM (%) SRD (Nm) SRD (%)
------------------------------------------------------------ ------------------ -------------------- -------------------- -------------------- ------------------- ---------- --------- ---------- ---------
**CONCENTRIC**
**EXTENSION**
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 60 47.8 (13.1) 51.0 (13.0) \- 0.81 (0.58--0.92) 5.5 11.1 15.3 30.9
180 34.5 (9.6) 38.4 (7.9) \- 0.68 (0.30--0.86) 4.5 12.4 12.6 34.4
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] 60 DL 104.0 (15.7) DL 106.3 (15.4) \- 0.87 (0.73--0.94) 5.5 5.2 15.2 14.4
NDL 101.9 (17.4) NDL 106.7 (15.8) \- 0.85 (0.62--0.94) 5.6 5.4 15.5 14.9
Santos et al. (2013)\[[@pone.0226274.ref002]\] 60 DL 139.5 (36.8) DL 139.8 (44.2) \- 0.87 15.8 11.2 \- \-
NDL 137.5 (34.8) NDL 138.3 (49.3) \- 0.81 16.3 11.8 \- \-
Moreau et al. (2008)\[[@pone.0226274.ref010]\] 60 39.24 (20.42) 37.42 (16.32) \- 0.95 \- \- \- \-
Pierce et al. (2006)\[[@pone.0226274.ref020]\] 15 2.4 (1.6) 2.1 (1.7) \- 0.51 \- \- \- \-
90 7.1 (5.0) 5.2 (3.8) \- 0.50 \- \- \- \-
180 13.5 (9.8) 10.6 (9.8) \- 0.86 \- \- \- \-
Merlini et al. (1995)\[[@pone.0226274.ref003]\] 100 40.1 (12.0) 41.2 (15.0) 40.4 (15.6) 0.95 \- \- \- \-
41.6 (13.4) 41.0 (15.3) 41.4 (14.1) 0.95 \- \- \- \-
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 60 DL 100.9 (12.3) DL98.1 (12.1) \- 0.98 \- \- \- \-
120 DL 87.2 (12.1) DL 85.1 (12.6) \- 0.96 \- \- \- \-
180 DL 74.7 (10.9) DL 74.3 (11.9). \- 0.89 \- \- \- \-
60 NDL 98.8 (14.1) NDL 9.5 (16.0) \- 0.96 \- \- \- \-
120 NDL 86.3 (13.0) NDL 85.6 (14.0) \- 0.93 \- \- \- \-
180 NDL 71.8 (12.2) NDL 73.1 (12.7) \- 0.94 \- \- \-
Iga et al. (2006)\[[@pone.0226274.ref005]\] 1.08 rad/s DL 159 (42) DL 161 (44) \- \- \- \- \- \-
2.16 rad/s DL 133 (32) DL 139 (35) \- \- \- \- \- \-
4.32 rad/s DL 106 (33) DL 111 (34) \- \- \- \- \- \-
1.08 rad/s NDL 154 (44) NDL 160 (47) \- \- \- \- \- \-
2.16 rad/s NDL 128 (29) NDL 163 (34) \- \- \- \- \- \-
4.32 rad/s NDL 103 (26) NDL 109 (29) \- \- \- \- \-
Ayalon et al. (2000)\[[@pone.0226274.ref027]\] 90 30.52 (2.76) 30.39 (2.93) \- 0.98--0.99 \- \- \- \-
Van den Berg-Emons et al. (1996)\[[@pone.0226274.ref021]\] 30 41.3 (15.7) 39.5 (16.7) \- \- \- \- \- \-
60 39.4 (19.5) 39.8 (17.4) \- \- \- \- \- \-
120 27.1 (9.7) 30.8 (12.8) \- \- \- \- \- \-
**FLEXION**
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 60 26.0 (5.5) 27.7 (6.2) \- 0.62 (0.28--0.82) 3.5 13.1 9.8 36.5
180 20.4 (4.3) 23.9 (5.4) 0.49 (0.03--0.77) 3.1 13.9 8.5 38.5
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] 60 DL 54.3 (11.0) DL 55.1 (9.2) \- 0.81 (0.63--0.91) 4.4 8.1 12.2 22.3
NDL 52.5 (10.5) NDL 53.4 (9.0) \- 0.77 (0.55--0.89) 4.7 8.9 13.0 24.6
Santos et al. (2013)\[[@pone.0226274.ref002]\] 60 DL 106.9 (21.6) DL 112.5 (29.1) \- 0.82 25.0 17.3 \- \-
NDL 113.9 (30.2) NDL 118.0 (33.6) \- 0.79 15.0 12.8 \- \-
Moreau et al. (2008)\[[@pone.0226274.ref010]\] 60 18.18 (14.57) 18.21 (13.63) \- 0.96 \- \- \- \-
Pierce et al. (2006)\[[@pone.0226274.ref020]\] 15 0.2 (1.0) 0.7 (1,3) \- 0.31 \- \- \- \-
90 1.1 (2.0) 1.3 (1.4) \- 0.38 \- \- \- \-
180 6.1 (4.0) 4.8 (3.3) \- 0.80 \- \- \- \-
Merlini et al. (1995)\[[@pone.0226274.ref003]\] 100 25.2 (8.7) 26.9 (8.3) 26.9 (7.5) 0.85 \- \- \- \-
27.3 (7.7) 26.7 (9.0) 26.6 (8.4) 0.85 \- \- \- \-
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 60 DL 64.5 (12.1) DL 66.8 (8.8) \- DL 0.90 \- \- \- \-
120 DL 59.5 (11.2) DL 61.8 (8.4) \- DL 0.88 \- \- \- \-
180 DL 50.5 (11.5) DL 53.6 (9.1) \- DL 0.89 \- \- \- \-
60 NDL 61.1 (9.0) NDL 63.5 (10.6) \- NDL 0.95 \- \- \- \-
120 NDL 55.2 (9.5) NDL 60.0 (11.2) \- NDL 0.86 \- \- \- \-
180 NDL 45.5 (11.1) NDL 49.9 (6.7) \- NDL 0.81 \- \- \- \-
Iga et al. (2006)\[[@pone.0226274.ref005]\] 1,08 rad/s DL 87 (24) DL 92 (28) \- \- \- \- \- \-
2,16 rad/s DL 79 (20) DL 85 (25) \- \- \- \- \- \-
4,32 rad/s DL 71 (17) DL 73 (19) \- \- \- \- \- \-
1,08 rad/s NDL 84 (22) NDL 89 (25) \- \- \- \- \- \-
2,16 rad/s NDL 75 (19) NDL 77 (20) \- \- \- \- \- \-
4,32 rad/s NDL 68 (19) NDL 67 (17) \- \- \- \- \-
Ayalon et al. (2000)\[[@pone.0226274.ref027]\] 90 14.69 (2.13) 16.41 (3.08) \- ICC 0.95--0.98 \- \- \- \-
Van den Berg-Emons et al. (1996)\[[@pone.0226274.ref021]\] 30 23.7 (9.1) 21.9 (10.0) \- \- \- \- \- \-
60 21.2 (10.1) 20.2 (8.8) \- \- \- \- \- \-
120 18.3 (8.2) 19.0 (7.8) \- \- \- \- \- \-
ICC = intraclass correlation coefficient (95% Confidence Interval); SEM = standard error of measurement; SRD = smallest real difference; DL = dominant leg; NDL = non dominant leg.
Only three articles \[[@pone.0226274.ref002], [@pone.0226274.ref004], [@pone.0226274.ref012]\] described the SEM. The values were represented in Newton/meters and as a percentage ([Table 5](#pone.0226274.t005){ref-type="table"}).
10.1371/journal.pone.0226274.t005
###### Eccentric extension and flexion of the knee in isokinetic, intra-class correlation coefficient, standard error of measurement and smallest real difference of strength measurements.
{#pone.0226274.t005g}
Knee action evaluated Speed (°/s) Mean /(SD) 1° test Mean /(SD) 2° test ICC (95%CI) SEM (Nm) SEM (%) SRD (Nm) SRD (%)
------------------------------------------------ ------------------ -------------------------- -------------------------- ------------------- ---------- --------- ---------- ---------
**ECCENTRIC**
**EXTENSION**
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 60 64.3 (23.0) 63.3 (17.8) 0.70 (0.40--0.86) 11.5 18 31.8 49.7
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 60 DL 130.5 (15.6) DL 135.7 (22.7) 0.92 \- \- \- \-
120 DL 129.5 (23.8) DL 125.5 (23.8) 0.88
180 DL 115.5 (16.1) DL 121.9 (20.7) 0.80
60 NDL 127.3 (17.0) NDL 136.1 (26.5) 0.82
120 NDL 131.1 (24.1) NDL118.2 (16.9) 0.76
180 NDL 112.8 (14.7) NDL 121.3 (21.2) 0.81
Iga et al. (2006)\[[@pone.0226274.ref005]\] 2.16 rad /s DL 182 (53) NDL 180 (47) DL 194 (51) NDL 189 (51) \- \- \- \- \-
**FLEXION**
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 60 49.2 (19.6) 42.2 (12.0) 0.60 (0.22--0.81) 9.8 21.5 27.3 59.6
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 60 DL 82.8 (10.5) DL 80.6 (12.6) 0.85 \- \- \- \-
120 DL 82.4 (12.8) DL 78.4 (11.0) 0.71 \- \- \- \-
180 DL 78.8 (11.2) DL 81.0 (12.5) 0.76 \- \- \- \-
60 NDL 75.8 (10.4) NDL 80.9 (16.7) 0.79 \- \- \- \-
120 NDL 80.9 (16.7) NDL 75.9 (11.2) 0.79 \- \- \- \-
180 NDL 76.0 (8.9) NDL 79.3 (14.6) 0.86 \- \- \- \-
Iga et al. (2006)\[[@pone.0226274.ref005]\] 2.16 rad /s DL 105 (28) NDL 98 (24) DL 110 (29) NDL 102 (28) \- \- \- \- \-
ICC = intraclass correlation coefficient (95% Confidence Interval); SEM = standard error of measurement; SRD = smallest real difference; DL = dominant leg; NDL = non dominant leg.
The SRD was described in only two articles \[[@pone.0226274.ref004], [@pone.0226274.ref012]\]. These values were also represented in Newton/meters and as a percentage. All values were below 60% for flexion in the eccentric mode ([Table 4](#pone.0226274.t004){ref-type="table"}).
There were many differences in the values of mean and standard deviation of peak torque (Tables [4](#pone.0226274.t004){ref-type="table"} and [5](#pone.0226274.t005){ref-type="table"}). For example, in Fagher et al. \[[@pone.0226274.ref004]\], in the concentric mode, the values of the second measure were better than the first one for knee extension and for knee flexion. However, in the eccentric mode, the values of the second measure were worse than the first one for both knee extension and knee flexion.
Moreover, several studies found decreases in knee flexion in the concentric mode from the first to the second measurement \[[@pone.0226274.ref003], [@pone.0226274.ref005], [@pone.0226274.ref020], [@pone.0226274.ref021]\].
In the three articles using the eccentric mode \[[@pone.0226274.ref004], [@pone.0226274.ref005], [@pone.0226274.ref011]\], there were also differences in the values observed when comparing the test-retest.
Meta-analysis {#sec011}
-------------
We performed a meta-analysis of the five studies for the reliability of knee movement in the concentric mode for both the extension and flexion of the joint of the subjects' dominant leg at a speed of 60°/s.
In this aspect, the aforementioned reliability was observed in more detail. For the knee extension, the correlation coefficient was 0.89 for fixed effects and 0.90 for random effects ([Table 6](#pone.0226274.t006){ref-type="table"}).
10.1371/journal.pone.0226274.t006
###### Summary meta-analysis of reliability with confidence interval for extension and flexion of knee in children in concentric mode at 60°/s.
{#pone.0226274.t006g}
Extension Weight (%)
------------------------------------------------- ------------ ------- ---------------- -------- --------- -------- --------
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 22 0.810 0.590 to 0.918 23.46 21.64
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] 28 0.870 0.736 to 0.938 30.86 23.18
Santos et al. (2013)\[[@pone.0226274.ref002]\] 21 0.810 0.582 to 0.920 22.22 21.31
Moreau et al. (2008)\[[@pone.0226274.ref010]\] 12 0.950 0.827 to 0.986 11.11 16.54
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 13 0.980 0.933 to 0.994 12.35 17.32
Total (fixed effects) 96 0.888 0.832 to 0.926 12.721 \<0.001 100.00 100.00
Total (random effects) 96 0.904 0.799 to 0.955 7.371 \<0.001 100.00 100.00
**Flexion**
Fagher et al. (2016)\[[@pone.0226274.ref004]\] 22 0.620 0.269 to 0.826 23.46 21.94
Johnsen et al. (2015)\[[@pone.0226274.ref012]\] 28 0.810 0.626 to 0.909 30.86 23.91
Santos et al. (2013)\[[@pone.0226274.ref002]\] 21 0.790 0.544 to 0.911 22.22 21.53
Moreau et al. (2008)\[[@pone.0226274.ref010]\] 12 0.960 0.860 to 0.989 11.11 15.88
Kellis et al. (1999)\[[@pone.0226274.ref011]\] 13 0.900 0.692 to 0.970 12.35 16.76
Total (fixed effects) 96 0.819 0.733 to 0.879 10.386 \<0.001 100.00 100.00
Total (random effects) 96 0.838 0.694 to 0.918 6.626 \<0.001 100.00 100.00
95% CI = 95% confidence interval.
For knee flexion, this correlation coefficient reached values of 0.82 for fixed effects and 0.84 for random effects.
[Fig 2](#pone.0226274.g002){ref-type="fig"} shows the values mentioned in the above table in a more schematic way. This figure allows quickly comparing each of the correlation coefficients, both for fixed effects and for random effects, between the concentric extension and flexion of the knee.
{#pone.0226274.g002}
Regarding the heterogeneity of each action of the knee, we emphasize:
- For knee extension, a value of Q = 12.59; I^2^ = 68.22; Significance (*p*) = 0.0135; 95% of the IC for I^2^ = 18.05--87.68.
- For knee flexion, a value of Q = 10.29; I^2^ = 61.14%; Significance (*p*) = 0.0358; 95% of the IC for I^2^ = 0.00--85.41.
Publication bias {#sec012}
----------------
[Fig 3](#pone.0226274.g003){ref-type="fig"} shows funnel plots corresponding to knee extension and flexion in the studies.
{#pone.0226274.g003}
The Egger test was used to evaluate the asymmetry of the funnel plot. The results of the Egger test for both knee extension and flexion were not significant (p = 0.109 and p = 0.136), suggesting that there is no publication bias.
Discussion {#sec013}
==========
The knee is one of the most studied joints in isokinetic dynamometry research, probably because it is easy to evaluate using a dynamometer \[[@pone.0226274.ref001]\]. Knee extensor and flexor strength are recognized as important for daily tasks, e.g., moving, standing or sitting, weight lifting, and climbing stairs \[[@pone.0226274.ref001]\]. In addition, many studies of knee extensors indicate that these could be representative of the total strength of the lower extremities. It is not only important to analyze the absolute values of flexors or extensors, but also the balance of both at the same time if the contralateral deficit is a compensated deficit; i.e., there is an adequate agonist/antagonist relationship \[[@pone.0226274.ref022]\]. Isokinetic dynamometry offers clear advantages over simple measurements to assess muscle strength, such as the continuous and accurate measurement of force production across a range of velocities of movement \[[@pone.0226274.ref023]\].
As we have indicated in the results, there were different heterogeneities for the test-retest data in each of the articles: they showed high and moderate values. Most authors thought that the results for the second tests would be better due to a learning effect \[[@pone.0226274.ref008], [@pone.0226274.ref024]\]. Some studies indicated a need for additional practice and familiarization with test procedures in this age group \[[@pone.0226274.ref025]\]. Other studies emphasized that the isokinetic method has the advantage of making it possible to detect large differences in the same muscles when analyzing at peak torque \[[@pone.0226274.ref026]\].
We analyzed the studies that reported reliability for the assessment of muscle strength in healthy children with CP. Our review suggests that the studies had good-to-excellent levels of reliability of muscle strength assessment in dominant and non-dominant knees using isokinetic dynamometry (0.7--1 correlation), except for Pierce et at. \[[@pone.0226274.ref020]\], which had low and moderate intra-class correlations at the velocities 15°/s and 90°/s, and Fagher et al. \[[@pone.0226274.ref004]\], where most results were moderate. Our results also support the contention that the muscle strengths of healthy children and children with CP are quantifiable and measurement is reliable, even in the presence of spasticity.
Otherwise, there were four studies whose participants were children with CP \[[@pone.0226274.ref010], [@pone.0226274.ref020], [@pone.0226274.ref021], [@pone.0226274.ref027]\] and one study whose participants were children without CP \[[@pone.0226274.ref011]\] where the values for the second test decreased compared to the first test in the concentric mode, but only for knee extension. The ICCs of these articles, except for Pierce et al. \[[@pone.0226274.ref020]\], were high for knee extension ([Table 4](#pone.0226274.t004){ref-type="table"}). These results demonstrate that voluntary muscle fatigue of the knee flexors and extensors can be reliably and feasibly assessed in children. It is believed that the number of repetitions and the type of contraction may influence the assessment of peak voluntary torque in subjects with CP. A possible explanation for this could be that the coordination of the agonist and antagonist muscles is more impaired at higher velocities than at lower velocities in children with CP \[[@pone.0226274.ref021]\]. It is suggested that there is higher heterogeneity for children with CP than for their healthy peers. The tests may be less suitable for children with CP than for healthy children because of attention deficits, which are known to occur with CP \[[@pone.0226274.ref028]\]. Future research should establish normative values regarding spasticity of the knee flexion and knee extension in children with CP to allow for the clearest interpretations of clinically meaningful change in these subjects.
This review emphasizes the need to find a consensus around the minimum change necessary to indicate a clinically important change for an individual or a particular population. Analyzing the only two articles that included SRD \[[@pone.0226274.ref004], [@pone.0226274.ref012]\] led us to suggest that: 1) for the knee extension in the concentric mode, the minimum change is around 15--15.5 Nm in absolute value and 31% in relative value with a speed of 60°/s and 34.5% with a speed of 180°/s; in the eccentric mode, the SRD should be 49.8%. 2) For knee flexion in the concentric mode, the minimum change is around 9.8--13.0 Nm in absolute value and 36.5--38.5% in relative value; in the eccentric mode, the SRD should be 59.6%.
Most of the studies had a time interval of one week between tests. One study had a three-days interval \[[@pone.0226274.ref003]\] and another two studies had time intervals lower than two hours \[[@pone.0226274.ref020], [@pone.0226274.ref021]\]. It is reasonable to establish a one-week interval because intervals of approximately 1 week between tests could maximize the effects of learning while still managing any effects of muscle fatigue \[[@pone.0226274.ref029]\].
A meta-analysis evaluates the replicability and generalizability of results, which are the hallmarks of good science \[[@pone.0226274.ref030]\]. This review presents a meta-analysis for the reliability of each of the included studies. The results of our analyses are shown in Tables [3](#pone.0226274.t003){ref-type="table"} and [4](#pone.0226274.t004){ref-type="table"}. We hope to provide more accurate and definitive values that will guide future research on the population treated here. [Table 6](#pone.0226274.t006){ref-type="table"} shows that the reliability varies. However, reliability is high for all the studies analyzed: for knee extension, reliability reaches a high---good level (0.89) for fixed effects and an excellent---very high level (0.90) for random effects; for knee flexion, reliability reaches a high---good level (0.82--0.84) for both fixed and random effects, which are lower values than for extension. The study of Kellis et al. \[[@pone.0226274.ref011]\] is the most reliable for extension, with an intra-class correlation coefficient of 0.98 and a range of 0.933--0.994 for 95% of the CI. For flexion movement, Moreau et al.'s study \[[@pone.0226274.ref010]\] meets the high expectations seen in Tables [4](#pone.0226274.t004){ref-type="table"} and [5](#pone.0226274.t005){ref-type="table"} for articles dealing with subjects with CP: this article has a correlation coefficient of 0.96 and a range of 0.860 to 0.989 for 95% of the CI. According to the meta-analysis, the relative reliability is high.
The methodological quality of this review, shown in Tables [1](#pone.0226274.t001){ref-type="table"} and [2](#pone.0226274.t002){ref-type="table"}, means it is a very useful tool for comparing the studies and allows drawing conclusions about the reliability of the studies. First, the percentages of items met (Tables [1](#pone.0226274.t001){ref-type="table"} and [2](#pone.0226274.t002){ref-type="table"}) indicate the limitations of the articles depending on the requested variables. Only one article reaches 78% quality \[[@pone.0226274.ref012]\], in CAT scale, and three are below 50%. The final mean of the percentages is approximately 53%, meaning that only half of the variables are fulfilled. In QAREL scale only one article reaches 100% quality and eight articles are above 55%.
The meta-analysis shows that one study has a 78% (CAT scale) and 100% (QAREL scale) score for methodological quality \[[@pone.0226274.ref012]\], another had 67% and 70% \[[@pone.0226274.ref002]\], and three studies had 56% and 60% \[[@pone.0226274.ref004], [@pone.0226274.ref010], [@pone.0226274.ref011]\]. The meta-analysis (5 studies) has a small sample (n = 96), but these results seem very interesting, since the reported reliability coefficients are high and excellent. High reliability coefficients are usually reported by studies with small sample sizes, which are associated with large standard errors and higher than normal CIs \[[@pone.0226274.ref030]\].
Limitations {#sec014}
-----------
The main limitation is the heterogeneity (isokinetic devices, populations, protocols...) of the identified studies. Because of the relatively small numbers of eligible studies, and the variability in the statistics used between studies, there were insufficient numbers to allow a meta-analysis for all velocities and the eccentric mode. For the same reason, a sub-analysis of the possible subgroups could not be performed. Of the five articles selected, four treated healthy children and only one study evaluated children with cerebral palsy. As for another possible subgroup, two articles evaluated soccer-playing children and three evaluated children who were not soccer players. As stated above, one evaluated children with cerebral palsy.
Publication bias exists in any literature review and should be considered when interpreting the results. Measures were taken to minimize bias as much as possible, including conducting a comprehensive search of multiple databases with gray literature databases. Only articles published in English and in peer-reviewed journals were included, which may have reduced the number of results. In addition, this meta-analysis was not registered online.
Conclusions {#sec015}
===========
The present review and meta-analysis explores existing data about different results in the test-retest reliability of isokinetic knee strength measurements in healthy and with CP children. This study provides a reliable analysis of isokinetic knee force measurements in the concentric mode. These findings suggest that isokinetic dynamometry can be used not only for measuring muscle strength for training, but also for muscle strength reeducation programs in children with or without CP.
More studies are needed in children with different pathologies to confirm our results. In addition, future research should evaluate knee force in the eccentric mode and show the reliability of test-retest measurements.
Supporting information {#sec016}
======================
###### PRISMA checklist.
(DOC)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Spinal cord development and neurogenesis are extremely interesting not only in the context of spinal injury and regeneration, but also in terms of the basic developmental processes that control cell lineage and specification. Complex molecular mechanisms guide progenitors cells in a specific spatial order that depend on their location according to the embryonic anteroposterior (AP) and dorsoventral (DV) axes. How patterning signals determine the identity of neural progenitors in a precise spatio-temporal order is still an issue of extensive study. Amongst the molecular pathways involved in this process is the retinoic acid (RA) pathway, which has multiple stage-specific functions in the generation, patterning, and maintenance of neural tissues [@pone.0032447-Maden1], [@pone.0032447-Maden2]. Initially neuroepithelial progenitors reside in a caudal neural plate 'stem zone' in which fibroblast growth factors (FGFs) act permissively to allow neural stem cell expansion [@pone.0032447-Bertrand1], [@pone.0032447-DiezdelCorral1], [@pone.0032447-DiezdelCorral2], and initial stages of neural specification [@pone.0032447-Stavridis1]. At these early stages RA appears to coordinate the progressive maturation of spinal cord progenitors (reviewed in [@pone.0032447-DiezdelCorral3]). RA synthesized by retinaldehyde dehydrogenase 2 (RALDH2) in differentiating paraxial (presomitic and somitic) mesoderm, diffuses towards the neuroepithelium and differentiates progenitor motor neurons (pMN) as they emerge from the caudal stem cell zone. Inhibiting RA signaling expands the size of the progenitor zone, seen as an enlarged caudal *Fgf8* expression zone [@pone.0032447-Sirbu1], [@pone.0032447-Vermot1]. Retinoid deficiency inhibits the initiation of pMN differentiation, affecting *Pax6*, *Olig2*, *Ngn2* and *Bhlhb5* expression [@pone.0032447-Molotkova1], [@pone.0032447-Skaggs1].
The next steps in dorsoventral pMN patterning require ventral Sonic hedgehog (Shh) and dorsal Wnts secretion (reviewed in [@pone.0032447-Ulloa1], [@pone.0032447-Dessaud1]). The morphogenic action of these main signals differentially induces ventral or dorsal transcriptional targets, leading to the establishment of neuronal subtypes, so neuronal circuits can form and function independent of their initial inducers (reviewed in [@pone.0032447-Edlund1], [@pone.0032447-Briscoe1]. Retinoic acid is intricately involved in many steps in this process (reviewed in [@pone.0032447-Maden2]). Data on vitamin A-deficient quail embryos indicate that RA signaling is required for expression of dorsal patterning genes, playing additional roles in pMN and interneuron specification [@pone.0032447-Wilson1]. Retinoid roles at later stages include guiding the induction and patterning of lateral motor column (LMC) neurons [@pone.0032447-Sockanathan1] by regulating AP patterning genes including Hox genes [@pone.0032447-DallaTorrediSanguinetto1]. Viable, tissue-specific mutants for the RA-synthesizing enzyme RALDH2 exhibit a reduced population of Lim1-positive brachial motoneurons, mispositioned LMC Islet1-positive neurons, and disregulated *Hoxc8*, leading to inappropriate axonal projection of nerves innervating extensor muscles and forelimb paralysis defects [@pone.0032447-Sockanathan1].
We have investigated these potential functions of RA by analyzing the spinal cord of *Raldh2^−/−^* null mutants rescued from early embryonic lethality by transient maternal RA supplementation [@pone.0032447-Mic1], [@pone.0032447-Niederreither1]. We show that dorsal spinal cord growth deficits are not due to abnormal Wnt- or dorsal-specific progenitor transcript levels. Rather, RA-deficient spinal cords are characterized by reduced dorsal FGF signaling and impaired expression of several Notch effectors. As a consequence, RA-deficiency inhibits neuronal stem cell proliferation, impairing neurosphere growth, differentiation and radial glial expression. Cell sorting experiments further show an expansion of the 'side population' (SP) of putative stem cells in the retinoid-deficient spinal cord. According to their transcriptional profiles, these cells were diverted from differentiation towards radial glia and maintained as pluripotent precursors and/or neural stem cells. In addition, analysis of spinal cord-derived neurospheres indicates that RA promotes neuronal differentiation *versus* pluripotent precursor maintenance.
Results {#s2}
=======
Rescued *Raldh2^−/−^* mutants as a model for RA deficiency in the differentiating spinal cord {#s2a}
---------------------------------------------------------------------------------------------
To analyze RA-dependent events in the differentiating mouse spinal cord, we took advantage of a rescue system allowing to postpone the lethality of the *Raldh2^−/−^* mutants (occurring at embryonic day E9.5). This can be achieved by providing RA at early developmental stages via the maternal food. The doses administered are non-teratogenic, but are sufficient to rescue early cardiovascular abnormalities in *Raldh2^−/−^* embryos, and to obtain mutants for analysis until E14.5 [@pone.0032447-Niederreither2], [@pone.0032447-Niederreither3]. The minimal time frame for such a rescue is a 24 hour administration from E7.5 to 8.5 (hereafter designated as 'short-term' RA supplementation). The RA supplementation can also be extended for several days, testing whether abnormalities in *Raldh2^−/−^* mutants might be rescued in a non-cell autonomous manner (see below).
Mutants recovered at E12.5 ([Fig. 1A,B](#pone-0032447-g001){ref-type="fig"}) or E14.5 (data not shown) after short-term RA supplementation consistently showed an abnormal spinal cord. Although the neural tube had closed, it was reduced in thickness dorsally, and instead of a roof plate only a thin layer of cells was present at the dorsal midline ([Fig. 1A,B](#pone-0032447-g001){ref-type="fig"}, arrows). To assess if the dorsal spinal cord defects were linked to a lack of active RA signaling, we used mice harboring the RARE-hsp68-*lacZ* transgene [@pone.0032447-Rossant1], a sensitive reporter for endogenous RA activity (e.g. ref. [@pone.0032447-Wagner1]). This transgene is strongly expressed in the dorsal-most spinal cord cells in E12.5 WT embryos ([Fig. 1A,C](#pone-0032447-g001){ref-type="fig"}), mirroring a conserved promoter domain regulating *Raldh2* expression [@pone.0032447-Castillo1]. In *Raldh2^−/−^* mutants after short-term RA supplementation, the dorsal domain of RARE-*lacZ* activity was absent, correlating with the abnormal thinning of the neuroepithelium and absence of a roof plate structure ([Fig. 1B, D](#pone-0032447-g001){ref-type="fig"}). A novel region of RARE-*lacZ* activity appeared in prospective interneurons, as previously described ([Fig. 1B,D](#pone-0032447-g001){ref-type="fig"}, white arrowheads) [@pone.0032447-Mic1], [@pone.0032447-Niederreither3]. Extending the RA supplementation until E10.5 improved dorsal spinal cord morphology in *Raldh2^−/−^* mutants, leading to dorsal activation of the RARE-*lacZ* reporter ([Fig. 1F](#pone-0032447-g001){ref-type="fig"}), yet RARE-*lacZ* activity was not as sharply restricted as in WT littermates ([Fig. 1E](#pone-0032447-g001){ref-type="fig"}). To further establish that RALDH2 is required for the induction of endogenous RA-responsive genes, we analyzed *Rarb* transcripts ([Fig. 1G,H](#pone-0032447-g001){ref-type="fig"}). Indeed, these were not detected in spinal cords of short-term supplemented mutants ([Fig. 1H](#pone-0032447-g001){ref-type="fig"}).
{#pone-0032447-g001}
The specification of distinct classes of neurons initially involves diffusible signals originating from dorsal (Wnt/BMP induced) and ventral (Shh induced) patterning centers. Graded signals from these two sites induce DV-restricted homeodomain and basic helix-loop-helix (bHLH) transcription factors expression. These transcriptional targets in the mitotic progenitor zone define the dorsoventral organization of spinal cord [@pone.0032447-Jessell1], [@pone.0032447-Lee1]. Unaltered *Wnt1*, *Wnt3a*, *Bmp2* and *Bmp4* expression in the ectoderm and/or dorsal spinal cord neuroepithelium of E12.5 *Raldh2^−/−^* mutants after short-term RA supplementation ([Fig. 1I--L](#pone-0032447-g001){ref-type="fig"}, insert panels, and data not shown) indicated that these dorsal inductive signals are intact. Roof plate-derived signaling is required to induce dorsal neuronal subtypes (reviewed in [@pone.0032447-Ulloa1]). Further analysis of *Raldh2^−/−^* mutants showed correct DV distributions of the transcription factors *Math1*, *Ngn1*, *Ngn2* and *Mash1* ([Fig. 1 I,J](#pone-0032447-g001){ref-type="fig"}, and data not shown) marking roof plate-dependent dl1-dl3 dorsal interneuron populations [@pone.0032447-Simmons1], [@pone.0032447-Gowan1], [@pone.0032447-Bermingham1]. This confirmed intact dorsal signaling in RA-deficiency mutants. Roof plate-independent *Lbx1* expression in dl4-dl6 interneurons [@pone.0032447-Gross1] was also observed in *Raldh2^−/−^* mutants ([Fig. 1K,L](#pone-0032447-g001){ref-type="fig"}). Our analysis thus shows that several molecular abnormalities observed at early stages of neural tube development in RA-deficient quail [@pone.0032447-DiezdelCorral2], [@pone.0032447-Wilson1] or mouse models [@pone.0032447-Molotkova1], [@pone.0032447-Ribes1], no longer appear after mid-gestation in the rescued *Raldh2^−/−^* mutants. This might be because (i) maternally administered RA could induce early regulatory events rescuing, in particular, expression of dorsal determinants, and/or (ii) other regulatory inputs eventually allowed to induce these regional determinants.Ventral patterning via Shh also appeared unaffected in E12.5 short-term rescued *Raldh2^−/−^* mutants, although the most severely affected mutants showed a subtle dorsal *Shh* expansion ([Fig. 1 M,N](#pone-0032447-g001){ref-type="fig"}, insert panels). While RA has been shown to affect interneuron *Dbx1* and *Dbx2* expression [@pone.0032447-Pierani1], these were unaltered in the rescued *Raldh2^−/−^* mutants ([Fig. 1O,P](#pone-0032447-g001){ref-type="fig"}, inserts, and data not shown) probably due to interneuron RA production. Expression of *Olig2*, though, was reduced, potentially due to the requirement for FGF signaling in its induction ([Fig. 1M,N](#pone-0032447-g001){ref-type="fig"}, main panels) [@pone.0032447-Chandran1], [@pone.0032447-Gabay1], [@pone.0032447-Kessaris1]. As suggested by their early RA-dependence ([Fig. S1](#pone.0032447.s001){ref-type="supplementary-material"}) [@pone.0032447-Molotkova1], [@pone.0032447-Ribes2], *Ngn2* and *Pax6* require sustained RALDH2 activity to achieve normal levels of expression in motor neuron and interneuron populations ([Fig. 1O,P](#pone-0032447-g001){ref-type="fig"}, arrowheads and brackets, and data not shown).
Reduced FGF signaling in the dorsal spinal cord of RA-deficient embryos {#s2b}
-----------------------------------------------------------------------
As RA regulates telencephalic growth [@pone.0032447-Ribes3] and neural specification by inducing FGF signaling [@pone.0032447-Stavridis1], we examined whether deficiencies in this growth factor pathway might be responsible for retinoid-dependent dorsal spinal cord alterations. Immunolocalization of FGF2 revealed a striking absence in the most dorsal regions of the spinal cord in E12.5 mutants, whereas ventral levels were much less affected ([Fig. 2A,B](#pone-0032447-g002){ref-type="fig"}). Similar dorsal-specific reductions in pERK1/2 levels ([Fig. 2C,D](#pone-0032447-g002){ref-type="fig"}; [Fig. S2A](#pone.0032447.s002){ref-type="supplementary-material"} for western blot analysis)- indicative of intracellular FGF signaling [@pone.0032447-Corson1]- revealed that the FGF pathway was compromised in the dorsal spinal cord. Forming spinal cord blood vessels also had strong pERK1/2 activity, again attenuated in the mutants ([Fig. 2C,D](#pone-0032447-g002){ref-type="fig"}). Extending RA supplementation until E10.5 restored both FGF2 and pERK expression in the dorsal spinal cord (data not shown).
{#pone-0032447-g002}
These observations led us to investigate FGF signaling in the neural tube of unrescued *Raldh2^−/−^* embryos. While we confirmed that, at early somite stages, mutants display anteriorized *Fgf8* expression in the caudal neural plate [@pone.0032447-Sirbu1], [@pone.0032447-Vermot1], from the 8 somite-stage onwards we observed reductions in FGF signaling, assayed by p-ERK immunolocalization, in the caudal neuroepithelium and paraxial mesoderm ([Fig. S2B](#pone.0032447.s002){ref-type="supplementary-material"}--E). The FGF-induced intracellular inhibitors *Sprouty1* (*Spry1*) and *Spry4* ([Fig. S2F,G](#pone.0032447.s002){ref-type="supplementary-material"}, and data not shown) and other *Fgfs* (*Fgf17* and *18*) were also reduced in mutants ([Fig. S2H](#pone.0032447.s002){ref-type="supplementary-material"},I, and data not shown). Section analysis indicated decreased FGF2 expression in dorsal regions of the neural tube of unrescued E9.5 mutants, especially along the ventricular layer ([Fig. S3A,B](#pone.0032447.s003){ref-type="supplementary-material"}). p-ERK was also decreased along the ventricular layer in E9.5 mutants ([Fig. S3C,D](#pone.0032447.s003){ref-type="supplementary-material"}). At E10.5, in short-term rescued mutants, pERK immunoreactivity was concentrated in the ventral spinal cord, and was already attenuated in developing blood vessels ([Fig. S3E,F](#pone.0032447.s003){ref-type="supplementary-material"}).
Transcriptomic analysis of the spinal cord {#s2c}
------------------------------------------
To further identify RA-dependent pathways regulating spinal cord formation, we performed transcriptome analysis of WT versus *Raldh2^−/−^* dissected brachial spinal cords collected at E12.5 after short-term RA rescue. Affymetrix DNA microarray analysis was performed on total RNA isolated from these samples. The validity of the screen was confirmed by reduced expression of known RA targets, such as *Rarb*, *Hoxb8* and *Hoxc6*, as well as *Raldh2* itself ([Table 1](#pone-0032447-t001){ref-type="table"}). Analysis of transcriptional profiles using the EASE gene ontology clustering software revealed a striking number of retinoid-signaling components and homeobox transcription factors reduced in expression in the mutants ([Table 1](#pone-0032447-t001){ref-type="table"}). These alterations were confirmed by ISH analysis of *Raldh2^−/−^* spinal cords ([Fig. 1G,H](#pone-0032447-g001){ref-type="fig"}, and data not shown). While reduced expression of selective structural proteins such as laminin α1 might disrupt basal lamina formation, an over-representation of TGFβ signaling and TGFβ target genes (such as procollagen I and VI \[[Table 1](#pone-0032447-t001){ref-type="table"}\]) could contribute to the spinal cord structural defects in mutants. A similar hyperactivation of TGFβ signaling is observed in foregut tissues of *Raldh2^−/−^* mutants at E8.5, and has been implicated in the lack of lung induction [@pone.0032447-Chen1] and cardiac outflow tract septation [@pone.0032447-Li1].
10.1371/journal.pone.0032447.t001
###### Main categories of genes exhibiting reduced or increased expression levels from DNA microarray analysis of *Raldh2^−/−^* vs. wild-type E12.5 spinal cords (see [Materials and Methods](#s4){ref-type="sec"}).
{#pone-0032447-t001-1}
Retinoid Signaling - Reduced Accession \# FC (E/B) p value
-------------------------------------- -------------- ------------- ----------
retinol binding protein 1, cellular NM_011254 0.392--16.0 0,013163
retinoic acid receptor, beta BB266455 0.525--8.88 0,047686
aldehyde dehydrogenase 1 A2 (Raldh2) NM_009022 0.540--5.43 0,041386
retinoic acid induced 2 BB770528 0.669--2.14 0,04899
SWI/SNF Smarca4 BG064918 0.883--1.3 0,02978
Homeobox Genes - Reduced
-------------------------- ----------- ------------- ----------
hox C4 NM_013553 0.486--10.7 0,030597
hox B5 NM_008268 0.599--6.60 0,00246
hox A5 BC011063 0.624--5.66 0,03059
hox B6 BC016893 0.727--3.50 0,02776
hox B8 X13721 0.742--3.28 0,043881
homeodomain only protein BC024546 0.807--2.46 0,008104
hox C6 BB440143 0.806--2.43 0,017862
Structural Proteins - Reduced
--------------------------------------- ----------- ------------- ----------
laminin, alpha 1 NM_008480 0.777--2.79 0,01239
microtubule-associated protein 1 B NM_008634 0.835--1.98 0,014958
amyloid beta binding protein (A) 2 BE952331 0.853--1.96 0,03334
neurofilament, light polypeptide M20480 0.859--1.91 0,023978
collapsin response mediator protein 1 AB006714 0.877--1.76 0,014808
fascin-1 BE952057 0.877--1.77 0,016426
TGF-beta Induced - Increased
-------------------------------------- ----------- ------------ ----------
transforming growth factor-β induced BB533460 1.55+3.55 0,020205
procollagen, type I, alpha 2 BF227507 1.495+3.12 0,013626
connective tissue growth factor NM_010217 1.45+2.8 0,008131
proline 4-hydroxylase AI314028 1.32+2.09 0,030724
procollagen, type VI, alpha 2 BI455189 1.26+1.82 0,016774
transforming growth factor, beta 2 AW049938 1.19+1.55 0,043708
tenascin C NM_011607 1.15+1.41 0,036012
Genebank accession numbers, Fold change (FC) ratios (Experimental versus Baseline, E/B) and Student\'s test p values are indicated.
Reduced Notch signaling in the RA-deficient spinal cord {#s2d}
-------------------------------------------------------
FGF signaling regulates uniform induction of Notch signaling as neuronal progenitors transition to an RA-dependent differentiation status [@pone.0032447-Akai1]. While the Notch pathway has long been implicated in neuronal differentiation [@pone.0032447-ArtavanisTsakonas1], [@pone.0032447-Lai1], [@pone.0032447-Lewis1], recent studies point to important roles in neural stem cells selection and maintenance [@pone.0032447-Rabadan1], [@pone.0032447-Pierfelice1], [@pone.0032447-Shimojo1]. In mutants for the Notch effectors *Hes1* and *Hes5*, neuroepithelial cells are not properly maintained and radial glial cells prematurely differentiate into neurons [@pone.0032447-Hatakeyama1]. In E12.5 *Raldh2^−/−^* spinal cords, *Delta1* transcripts (encoding a Notch ligand) were markedly reduced ([Fig. 3A,B](#pone-0032447-g003){ref-type="fig"}), whereas *Hes1* ([Fig. 3E,F](#pone-0032447-g003){ref-type="fig"}) and *Hes5* ([Fig. 3I,J](#pone-0032447-g003){ref-type="fig"}) expression were also affected. The same genes were downregulated in the early neural tube of unrescued *Raldh2^−/−^* mutants at E8.5 ([Fig. 3C,D,G,H,K,L](#pone-0032447-g003){ref-type="fig"}), consistent with observations made in avian models [@pone.0032447-DiezdelCorral2]. This further indicates that under RA deficiency, a failure to maintain FGF and Notch signaling might affect neuronal progenitor maintenance (see below).
{#pone-0032447-g003}
Disrupted dorsal root ganglia migration and survival {#s2e}
----------------------------------------------------
Retinoids may control several steps of peripheral nervous system development, by triggering neural crest cell (NCC) epithelial-mesenchymal transition, coordinating dorsal root ganglion (DRG) formation along defined rostrocaudal pathways [@pone.0032447-MartinezMorales1], and stimulating neurite outgrowth and sympathetic neuron survival [@pone.0032447-Plum1], [@pone.0032447-Corcoran1]. We examined trunk neural crest genesis and DRG formation in the rescued *Raldh2^−/−^* mutants. Unaltered expression of the neural crest markers *Sox10* and *Pax3* at E9.5 indicated no obvious defects on the generation of NCCs in short term RA-supplemented *Raldh2^−/−^* mutants (data not shown). However, postmigratory neural crest was disorganized at E10.5, as observed with several molecular markers including *Sox10*, *Isl1* and *Eya2* ([Fig. 4A,C](#pone-0032447-g004){ref-type="fig"}). In *Raldh2^−/−^* mutants, forelimb-level NCCs migrated as partly fused sheets rather than in segmental streams. Hence, developing DRG were not properly segregated, and gangliogenesis was compromised ([Fig. 4B,D](#pone-0032447-g004){ref-type="fig"}). Detailed analysis on sections of E12.5 mutants with *Sox10* ([Fig. 4E,F](#pone-0032447-g004){ref-type="fig"}), anti-neurofilaments ([Fig. 4G,H](#pone-0032447-g004){ref-type="fig"}), and the Notch ligand *Hrt2* (which marks the spinal nerves dorsal exit points, [Fig. 4E,F](#pone-0032447-g004){ref-type="fig"}, insets), showed that dorsal spinal nerves were disorganized and shortened, and their connections with the DRG were poorly defined ([Fig. 4F,H](#pone-0032447-g004){ref-type="fig"}, arrowheads).
{#pone-0032447-g004}
During gestation many neurons (including some of the DRG) are eliminated by apoptosis due to a lack survival signals [@pone.0032447-White1]. To examine if increased cell death accompanies DRG malformations, we performed immunodetection of the active form of caspase 3 (Act-Casp3) - a main effector of the apoptotic cascade (reviewed in [@pone.0032447-DAmelio1]). Higher levels of Act-Casp3 labelling were observed in the DRG of *Raldh2^−/−^* mutants, and at the level of the dorsal spinal nerve exit points ([Fig. 4J](#pone-0032447-g004){ref-type="fig"}, arrowheads). This increase in cell death might account for the shortened dorsal nerve tracts and overall disorganization of the dorsal nerves and DRG in the mutants. These defects were prevented by extending the RA supplementation until E10.5: in that case the *Raldh2^−/−^* mutants showed similar numbers of Act-Casp3+ cells as controls ([Fig. 4I,J](#pone-0032447-g004){ref-type="fig"}, insets), and better organized dorsal nerve tracts ([Fig. 4H](#pone-0032447-g004){ref-type="fig"}, inset).
Rescued *Raldh2^−/−^* mutants display an abnormal lateral motor column phenotype {#s2f}
--------------------------------------------------------------------------------
Spinal motor neurons arise from common ventral progenitor domains, and are organized in columns whose identities direct correct axonal projection [@pone.0032447-Tosney1], [@pone.0032447-Landmesser1]. Retinoic acid generated by RALDH2 in postmitotic motor neurons acts to upregulate *Lim1* expression, which is required for lateral motor columnar (LMC~L~) subtype specification. This in turn regulates dorsal limb axonal projection pathways [@pone.0032447-Sockanathan1], [@pone.0032447-Sockanathan2]. Conditional gene inactivation strategies have revealed functional contributions of RALDH2 acting both within the paraxial mesoderm and in postmitotic neurons, in order to specify LMC~L~ identity [@pone.0032447-Ji1], [@pone.0032447-Vermot2]. RA-supplemented *Raldh2^−/−^* mutants display severely disorganized brachial LMCs, as evidenced by the presence of continuous *Isl1*-expressing ventral columns, whereas these columns are interrupted by non *Isl1*-expressing cells in the WT spinal cord ([Fig. 5A,B](#pone-0032447-g005){ref-type="fig"}, brackets). These alterations are similar, or even more severe, than those described in LMC conditional *Raldh2* mutants. Furthermore, we found a severe reduction of the LMC motor pools marked by *Pea3* ([Fig. 5C,D](#pone-0032447-g005){ref-type="fig"}), *EphA4* (data not shown), *Hoxc6* and *Hoxc8* ([Fig. 5E--H](#pone-0032447-g005){ref-type="fig"}). This phenotype was partly rescued by extending the RA supplementation until E10.5, resulting in less severely reduced pools ([Fig. 5D](#pone-0032447-g005){ref-type="fig"}, inset). As each of the *Raldh2* conditional mutants [@pone.0032447-Ji1], [@pone.0032447-Vermot2] displays a milder version of the LMC phenotype observed in the rescued *Raldh2^−/−^* null mutants, our data support the idea that diffusible RA produced mesodermally and RA generated by LMC neurons act synergistically to specify the LMC motor pools.
{#pone-0032447-g005}
Spinal cord neurosphere proliferation and survival is RA-dependent {#s2g}
------------------------------------------------------------------
As RA deficiency reduced FGF and Notch signaling, we hypothesized that spinal cord neural stem cells (NSCs) would be adversely affected. To directly test this hypothesis we examined whether RA deficiency affects the ability of spinal cord cells to form clonally derived neurospheres, using a floating serum-free assay where NSCs proliferate and generate multipotent clones [@pone.0032447-Reynolds1]. Dorsal brachial/cervical spinal cord explants from short-term rescued E12.5 *Raldh2^−/−^* mutants were subjected to primary cultures, and progenitors enriched in the presence of FGF2 and EGF. Like their control littermates, *Raldh2^−/−^* spinal cords were able to generate neurospheres ([Fig. 6A--D](#pone-0032447-g006){ref-type="fig"}) after 8--12 days in culture. Interestingly *Raldh2^−/−^* spinal cord cultures exhibited a 25% reduction in neurosphere number at day 8 ([Fig. 6E](#pone-0032447-g006){ref-type="fig"}), and the resulting neurospheres were smaller in size when compared to WT controls ([Fig. 6A--D](#pone-0032447-g006){ref-type="fig"}).
{ref-type="sec"}), the number of spheres developing from *Raldh2^−/−^* embryos spinal cords is decreased by 25% compared to WT (E: 11.1±0.88 spheres/well for WT; 8.40±1.50 for mutants; n = 9; T- test: P value = 0,0009). (C,D) To further study progenitor cell differentiation, spheres were plated onto laminin-coated coverslips for 3 days, after 10 days of growth in suspension. *Raldh2^−/−^* derived spheres exhibited reduced number of cells compared to WT (F: one quadrant of a plated WT sphere is composed of 105.4±16.6 cells, against 40.9±6,82 cells in a *Raldh2^−/−^* sphere; DAPI positive nuclei were counted using ImageJ software; n = 9; P = 1.6×10^−5^). (G--O) After growth onto laminin coated coverslips for 3 days, cells were processed for immunocytochemistry. Nestin+ cells (I,M) are increased by 20% in the *Raldh2^−/−^*derived spheres, while TuJ1+ cells (J,N) are decreased by half. (H,L, DAPI staining; K,O, merged images). (G) After counting and normalization for total cell numbers, assessed by DAPI positive cell nuclei, one quadrant of a WT sphere contains 41.6±14 Nestin+ and 42.0±9.76 TuJ1+ cells, against 57.9±9.21 and 25.9±6,66 in a *Raldh2^−/−^* sphere (n = 9; P = 0,001 and 0,007, respectively).](pone.0032447.g006){#pone-0032447-g006}
Analysis of the ability of spinal cord-derived neurospheres to differentiate in vitro was performed to assess their lineage potential. Typically 10--12 spheres were allowed to attach to a laminin-coated coverslip. The absence FGF2 and EGF in culture stimulated this event. After 3 days, nestin positive neural progenitor cells and β-III tubulin (TuJ1 positive) differentiated neurons were detected in both WT and *Raldh2^−/−^* derived neurospheres. Notably, the nestin positive cell population was increased in the *Raldh2^−/−^* derived spheres by 20%, while differentiated neurons where decreased by half (n = 9) ([Fig. 6G--O](#pone-0032447-g006){ref-type="fig"}). This indicates that RA controls the fate of neuronal progenitors by promoting neuronal differentiation. The total number of cells in *Raldh2^−/−^* spheres was decreased by 50% in comparison to WT ([Fig. 6F](#pone-0032447-g006){ref-type="fig"}). The reduced fraction of differentiating neurons likely accounts for the progressively smaller size of mutant spheres under these conditions.
RA deficiency increases the spinal cord side population (SP) and alters its transcriptional profile {#s2h}
---------------------------------------------------------------------------------------------------
Dual-wavelength flow cytometric analysis (FACS) of cells labeled with the fluorescent DNA-binding dye Hoechst 33342 can be used to assay for the differential ability of stem cells to efflux the Hoechst dye [@pone.0032447-Goodell1], [@pone.0032447-Goodell2]. Using this assay a small population of laterally shifted cells (the 'side population', SP) exhibits NSC potential and is inhibited in its terminal lineage commitment [@pone.0032447-Bhattacharya1]. To investigate whether RA might regulate SP formation, brachial spinal cords were dissected at E12.5 from short-term rescued *Raldh2^−/−^* mutants and WT littermates, and assayed for SP cell numbers (n = 9 FACS experiments performed on independent pools of 10--20 spinal cords each). SP cells constituted approximately 0.6% of the sorted cell fractions from WT samples ([Fig. 7A,E](#pone-0032447-g007){ref-type="fig"}). Consistently, *Raldh2^−/−^* spinal cords exhibited a ∼3-fold increase in SP cells, with respect to WT samples ([Fig. 7B,E](#pone-0032447-g007){ref-type="fig"}). The Hoechst dye efflux capacity of stem cells is due to membrane efflux pumps of the ATP-binding cassette (ABC) transporter superfamily, including multidrug resistance 1 (MDR1) and ABCG2 [@pone.0032447-Zhou1], [@pone.0032447-Lassalle1]. These pumps could be blocked by verapamil in both WT and mutants ([Fig. 7C,D](#pone-0032447-g007){ref-type="fig"}), showing the specificity of the assay.
{ref-type="sec"}) of cell suspensions from dissected spinal cords of E12.5 WT (A) and *Raldh2^−/−^* mutants (B), and from WT and mutant samples preincubated in the channel blocker verapamil prior to dye addition (C,D). Percentages of cells within the SP fraction (red) are indicated. E: Mean percentages of SP cells in WT and mutant E12.5 spinal cords, respectively (n = 9 cell-sorting experiments performed on independent pools of 10--20 WT or mutant samples, respectively).](pone.0032447.g007){#pone-0032447-g007}
RNA was isolated from the sorted SP fractions, amplified and assayed by real-time quantitative PCR to test if the *Raldh2^−/−^* SP might be altered in its expression of pluripotency and/or NSC markers. Levels of the NSC markers *Msi1* and *Nestin* were upregulated by about 8--9 fold, data consistent with observations of increases in nestin positive cells in the neurosphere assays. Expression of the progenitor/neurogenic markers *Pax6*, *Olig2*, and *Sox2* was increased 2--6 fold in the mutant SP. The radial glia markers *Blbp* and *Glast*, though, were dramatically reduced under RA deficiency ([Fig. 8](#pone-0032447-g008){ref-type="fig"}). While levels of the *Mdr1* gene were increased 6-fold in the mutants, *Abcg2* levels were reduced by about 60% ([Fig. 8](#pone-0032447-g008){ref-type="fig"}). Collectively these changes indicate that the *Raldh2* mutant SP is shifted towards a more pluripotent/NSC transcriptional profile, but is inhibited in its ability to generate a radial glia lineage.
{#pone-0032447-g008}
Discussion {#s3}
==========
Rescue of *Raldh2^−/−^* embryos reveals abnormal spinal cord development {#s3a}
------------------------------------------------------------------------
*Raldh2* has a dynamic expression in the axial, paraxial and lateral plate mesoderm adjacent to the early embryonic neural tube. This is followed by specific expression in the developing meninges and lateral motor columns (LMC) of the spinal cord [@pone.0032447-Sockanathan1], [@pone.0032447-Niederreither4]. Initially RA sets the expression boundaries of *Hoxc* genes [@pone.0032447-Dasen1], [@pone.0032447-Liu1], then differentiates caudal FGF stem cell progenitors. Elaboration of DV programs of motor and interneuron differentiation [@pone.0032447-Wilson1], [@pone.0032447-Pierani1], [@pone.0032447-Novitch1] and induction of dorsal limb motor neuron subtypes also require RA [@pone.0032447-Sockanathan1], [@pone.0032447-Sockanathan2]. Our work has shown two levels of action of RA influencing spinal cord cells during development. RA acts globally (in both neuronal and non-neuronal lineages) to maintain high levels of FGF and Notch signaling. Additionally, it promotes neuronal progenitors differentiation. In the absence of RA, progenitor cells are maintained in a multipotent state; however, reduced FGF and Notch signaling will eventually lead to a depletion of neuronal progenitors and a block of differentiation toward the radial glia lineage.
Neurite growth and cell survival is hindered in dorsal root ganglia of RA-deficiency mutants {#s3b}
--------------------------------------------------------------------------------------------
In the peripheral nervous system RAR signaling appears to play a central role in regulating neurite extension (reviewed in [@pone.0032447-ClagettDame1], [@pone.0032447-Mey1]). An upregulation of *Rarb* following RA treatment correlates with increased neurite outgrowth in cultured adult dorsal root ganglia (DRG), and lentiviral RARβ2 overexpression promotes regeneration of sensory axons, both in vitro and in vivo after cervical dorsal root crush injury [@pone.0032447-Wong1], [@pone.0032447-So1]. RA is likely to sustain axonal growth and neuronal survival by transcriptional activation of neurotrophins and their receptors [@pone.0032447-Balmer1]. Reciprocally, the neurotrophin NGF activates *Raldh2* transcription [@pone.0032447-Corcoran2]. Through this regulatory loop, RA signaling will facilitate NGF and NT-3-dependent neurite outgrowth [@pone.0032447-Plum1], [@pone.0032447-Corcoran1], [@pone.0032447-Corcoran3] and activate targets necessary for axonal elongation [@pone.0032447-Muley1]. Upon *Raldh2* loss of function there is a clear reduction in the length of spinal nerves entering the DRG, which are predictably NGF and NT-3 RA-responsive populations. Actions of NGF increasing MAPK family signaling (SAPK and ERK1/2) and preventing DRG apoptosis [@pone.0032447-Scuteri1], are presumably impaired in the dorsal spinal nerves of *Raldh2^−/−^* mutants. A similar function of RA in offsetting cell death has been shown in the hind- and forebrain [@pone.0032447-Maden3], [@pone.0032447-Niederreither5], potentially due to altered FGF signaling [@pone.0032447-Ribes3], [@pone.0032447-Schneider1] and neurotrophin availability [@pone.0032447-White1].
RA deficiency reduces FGF and Notch signaling, and neural stem cell survival {#s3c}
----------------------------------------------------------------------------
In spite of an increasing number of studies aiming to elucidate the mechanisms of regulation of neuronal subtype diversification, altogether spinal cord neurogenesis remains poorly understood. In vitro differentiation studies of embryonic stem (ES) cells are a powerful tool, offering access to molecular pathways driving neural stem cell differentiation (reviewed in refs. [@pone.0032447-Gaspard1], [@pone.0032447-Peljto1]). Combinatorial interactions of trophic factors appear critical in inducing neural stem cells (NSCs) toward defined lineages. Interestingly, spatial-specificity can also be induced during NSC differentiation. As an example, motor neurons induced by combined RA and Shh action express markers of cervical level spinal motor neurons [@pone.0032447-Peljto1]. There is an intrinsic temporal patterning mechanism of ES cell neurogenesis, also occurring in the developing spinal cord. Neuron generation precedes that of glial cells. A late (E12.5+ in mouse) neuron-glial switch is a general property of NSCs in all parts of the developing brain and spinal cord. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the neuronal to glial switch in identity [@pone.0032447-Grandbarbe1], [@pone.0032447-Genethliou1], [@pone.0032447-Kaltezioti1]. This Notch-regulated glial transition is controlled by combined action of nuclear receptors COUP-TFI and COUP-TFII (NR2F1, NR2F2) [@pone.0032447-Naka1], the latter being found to be a RA-activatable receptor [@pone.0032447-Kruse1].
The effects of combined reductions in FGF2/p-ERK and Notch signaling in RA-deficient spinal cords might reflect more general functions of FGF2 in increasing neurons and glia generation from cortical stem cells [@pone.0032447-Lillien1], [@pone.0032447-Qian1], and Notch signaling in first maintaining NSCs, then promoting glial fate transition [@pone.0032447-Gaiano1]. Murine *Hes1*;*Hes5* double mutants have a premature depletion of radial glial cells, leading to structural alterations of the spinal cord and fusions of the DRG [@pone.0032447-Hatakeyama2], reminiscent of the phenotype of the short-term rescued *Raldh2^−/−^* mutants. RA directs ES cells to become a uniform RC2+Pax6+ radial glial lineage [@pone.0032447-Bibel1], [@pone.0032447-Plachta1], indicating it may serve as a master regulator. Consistently, the fetal brain subventricular zone harbors subpopulations of RA-activated RC2+, GLAST+ radial glia progenitors [@pone.0032447-Haskell1]. RALDH2, by inducing meningeal or fibroblastic FGF2 production in both the fetal brain and spinal cord, may play a role in the stromal microenvironment allowing stem cell maintenance, consistent with reduced NSC survival under RA deficiency [@pone.0032447-Siegenthaler1]; this study). The effects of RA on NSC populations appear synergistic, i.e. increasing both FGF and Notch signaling to promote NSC commitment [@pone.0032447-Lowell1].
Perspectives for human health {#s3d}
-----------------------------
We have shown that RA-deficiency abnormalities in spinal cord populations can be partly prevented by providing an extended maternal retinoid treatment, a finding which may be applicable in stem cell-based therapies. Recent studies in animal models clearly pointed out to a role of retinoids in promoting neurogenesis in the adult spinal cord [@pone.0032447-Leung1], and stimulating axonal outgrowth after spinal injury [@pone.0032447-Agudo1]. More generally, in human RA deficiency might contribute to NSC degeneration and to some neurological diseases. Reductions in *Raldh2* expression and RA signaling have been found in postmortem samples of patients with human amyotrophic lateral sclerosis (ALS) and Alzheimer\'s disease [@pone.0032447-Corcoran4], [@pone.0032447-Corcoran5], both exhibiting neurodegenerative phenotypes. There is evidence for defective retinoid transport and function in late onset Alzheimer\'s disease [@pone.0032447-Goodman1]. One mechanistic role for RARα signaling in preventing amyloid-β accumulation at the onset of Alzheimer\'s disease is by increasing Notch related α-secretase activity via a direct induction of ADAM10, and Alzheimer\'s-like defects are indeed suppressed by RARα agonists in a transgenic mouse model [@pone.0032447-Jarvis1]. Although the drug Memantine (an NMDA receptor antagonist) clinically used for treatment of Alzheimer\'s disease was found to increase the number of radial glial-like progenitor cells in adult mouse hippocampus [@pone.0032447-Namba1], more generally the role of the radial glial lineage in neurodegenerative models is still debated.
Whether alterations in retinoid signaling during human embryonic and fetal development may have a causative role in neurogenesis defects and/or mental retardation (e.g. ref. [@pone.0032447-Adams1]) is also unclear. Alcohol exposure appears to decrease embryonic RA levels [@pone.0032447-Yelin1], [@pone.0032447-Goez1], potentially contributing to mental retardation associated with the fetal alcohol syndrome [@pone.0032447-Chudley1]. Exposure to ethanol during critical periods of development decreases the radial glial progenitor pool, and reduces levels of activated Notch1 and FGFR2 [@pone.0032447-Rubert1]. Impaired retinoid signaling secondary to overall vitamin A deficiency or fetal alcohol exposure, may in the long-term result in the selective depletion of radial glial NSCs. While RA treatment alone might partially restore these defects, understanding how synergistic combinations of RA, Notch, and FGF agonists boost endogenous neuroregenerative capacities may offer a plausible alternative to cell based treatments.
Materials and Methods {#s4}
=====================
Ethics statement {#s4a}
----------------
All animals were maintained and manipulated under animal protocols reviewed by the Baylor College of Medicine and University of Texas at Austin ethics committees, which specifically approved this study (AUP 2010-00128), in strict accordance with NIH guidelines, provisions of the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act.
In situ hybridization (ISH) and immunohistochemistry (IHC) {#s4b}
----------------------------------------------------------
*Raldh2*-null mutants, and the maternal RA supplementation procedure, have been described previously [@pone.0032447-Niederreither1], [@pone.0032447-Niederreither3]. All animals were maintained and manipulated under animal protocols reviewed and approved by the Baylor College of Medicine and University of Texas at Austin, in strict accordance with NIH guidelines, provisions of the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act. Whole-mount ISH with digoxigenin-labeled riboprobes was performed as described [@pone.0032447-ChotteauLelievre1], using Intavis InSituPro robots (for details, see <http://www.empress.har.mrc.ac.uk/browser/>, Gene Expression section). All ISH beyond E9.5 were performed on transverse (100 µM) vibratome sections of paraformaldehyde-fixed embryos. X-gal assays were performed as described [@pone.0032447-Rossant1]. Whole-mount immunolabeling with the phospho-p44/p42 Map-kinase (p-ERK1/2) antibody (Cell Signaling) or FGF2 (Santa Cruz) was performed according to the Rossant web site protocol (<http://www.sickkids.ca/research/rossant/protocols/ImHis.asp>). Anti-cleaved caspase-3 (Cell Signaling Technology) and mouse monoclonal anti-neurofilament (2H3, Developmental Studies Hybridoma Bank) immunohistochemistry was performed using a peroxidase-conjugated goat anti-rabbit (Pierce) secondary antibody. Fluorescence immunolabelling was performed on transverse cryosections (14 µM) of E9.5 and E10.5 paraformaldehyde-fixed embryos, with p-ERK1/2 and FGF2 primary antibodies (refs. as above) revealed with Alexa 555 secondary antibody, and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). Procedures for conventional histology can be found at <http://www.empress.har.mrc.ac.uk/browser/>, Histology section. Four to ten *Raldh2^−/−^* embryos were examined for all assays and at each developmental stage described hereafter. All spinal cord sections displayed are at the lower cervical/brachial level.
Embryonic spinal cord derived neurosphere culture {#s4c}
-------------------------------------------------
The dorsal region of the brachial/cervical spinal cord was dissected from individual E12.5 embryos. Neurosphere experiments were performed as described [@pone.0032447-Chojnacki1] with slight modifications. Briefly, cells from 4 spinal cord tissue explants were dissociated in 500 µL of non-enzymatic cell dissociation medium (Sigma) with a fired-polished Pasteur pipette. Cells were plated in 6-well plates (Nunc, non-coated) at a density of 60,000 cells/mL in DMEM:F12 medium (Invitrogen) supplemented with 40 ng/mL FGF2 (human, Peprotech), and 20 ng/mL EGF (mouse, Sigma). Half medium was changed every 2 days. Spheres were counted under a Nikon light microscope and allowed to differentiate on laminin (Gibco)-coated 4 mm coverslips before immunostaining. Immunocytochemisty was performed according to ref. [@pone.0032447-Corcoran3]. Anti-β-III-tubulin (TuJ1, Covance) and anti-Nestin (rat-401, Developmental Studies Hybridoma Bank) were used at 1/600 and 1/100 dilutions, respectively. Nuclei were stained with 0.5 µg/mL diamino-2-phenyle-indole (DAPI, Sigma).
Flow cytometric analysis {#s4d}
------------------------
Lower cervical/brachial E12.5 spinal cords were dissected, washed in Hanks balanced salt solution (HBSS), and resuspended in a Liberase Blendzyme IV digestion buffer (Roche) solution for 5 minutes at 37°C. A 40-µm filtered supernatant was diluted with an equal volume of 20% serum (FBS) in Dulbecco modified Eagle medium (DMEM, Invitrogen) to stop the reaction, and tissues were pelleted and washed. After resuspension in DMEM containing 2% fetal calf serum (FCS) and 10 mM HEPES, cell concentration was adjusted to 10^6^ cells/mL using DMEM. Hoechst 33342 (Sigma) was added at 5 µg/mL and the cell suspension incubated at 37°C for 60 minutes. For control experiments, the channel blocker verapamil was added at a concentration of 50 µM for 5 minutes prior to the addition of Hoechst 33342. Cell suspensions were mixed every 20 minutes, and resuspended in 4°C HBSS containing 2% FCS and 2 µg/mL propidium iodide to assay for viability. Flow cytometric analysis and fluorescence-activated cell sorting (FACS) was carried out immediately afterward using a Dako triple laser cell sorter (MoFlow, Cytomation, Fort Collins, CO). For detailed descriptions of Hoechst 33342 staining and flow cytometry, see <http://www.bcm.edu/labs/goodell/index.cfm>.
Real-time quantitative RT-PCR {#s4e}
-----------------------------
Total RNA (100 ng) was subjected to real-time RT-PCR using SYBR Green Core Reagents (Qiagen) according to the manufacturer\'s protocol. The incorporation of SYBR Green dye into the PCR products was monitored in real-time with an ABI PRISM 7700 sequence detection system (PE Applied Biosystems). Target genes were quantified relative to a reference gene, glyceraldehyde-3-phosphate dehydrogenase (*Gapdh*), which expression was stable in our experimental conditions. Primer sequences are available on request.
Microarray analysis {#s4f}
-------------------
Total RNA (20 µg+) was extracted from dissected brachial spinal cords from ten WT and *Raldh2^−/−^* mutants collected at E12.5 after short-term RA rescue. RNA was purified using the RNeasy kit (Qiagen). An Agilent low RNA input linear amplification kit was used on sorted SP populations. RNA quality was verified by using an Agilent 2100 Bioanalyzer. All subsequent reactions using Affymetrix GeneChip Mouse 430A arrays were carried out by the Baylor College of Medicine Microarray Facility as indicated at <http://www.bcm.edu/mcfweb/>, and in compliance to MIAME guidelines. Data normalization and statistical analysis was performed using dChip 1.2 (W. Wong, Harvard University, Cambridge, MA). Genes were classified into functional categories using the EASE analysis tool (<http://david.abcc.ncifcrf.gov/>) based on the classification scheme in Gene Ontology.
Supporting Information {#s5}
======================
######
**Lack of RALDH2-mediated RA synthesis affects early neuronal determinants in the prospective spinal cord.** Whole-mount ISH analysis of *Pax6* (A,B), *Ngn2* (C,D), *Olig2* (E,F, main panels) and *Irx3* (E,F, insets) in WT and unrescued *Raldh2^−/−^* embryos (genotypes as indicated in each panel). The developmental stages are 6--8 somites (A--D, insets in E,F) and E9.0 (E,F, inset in C,D). fb, forebrain; hb, hindbrain; pl, placodal cells; sc, spinal cord.
(TIF)
######
Click here for additional data file.
######
**Altered FGF signaling in the developing spinal cord of RA-deficient mutants.** A: Western blot analysis of p-ERK levels in WT and *Raldh2^−/−^* samples. Upper panel: E9.0 (14--16 somite-stage) embryos, caudal regions, pooled, n = 15. Lower panel: E12.5 embryos collected after short-term rescue, upper cervical/brachial level spinal cords, pooled, n = 7. B-E: Whole-mount immunodetection of phosphorylated ERK1/2 (p-ERK1/2) in E8.5 (somitic stages and genotypes as indicatd) WT and *Raldh2^−/−^* embryos. B,C: Profile views; D,E: details of the caudal region viewed dorsally. Brackets in B,C indicate strongly labelled extra-embryonic membranes, and in D separate domains of high labelling in somitic and caudal regions. F--I: Whole-mount in situ hybridization (ISH) analysis of *Spry1* (F,G) and *Fgf18* (H,I) in WT and *Raldh2^−/−^* embryos (genotypes as indicated). All embryos are viewed dorsally, and the developmental stages are 6--8 somites (F,G, and H,I, insets), and 12--14 somites (H,I, main panels).
(TIF)
######
Click here for additional data file.
######
**Immunofluorescence analysis of FGF2 and pERK1/2 distribution in the neural tube of E9.5 unrescued embryos (A--D), and E10.5 embryos after short-term RA-rescue (E,F).** Transverse sections at cervico-brachial levels. Views of the immunofluorescence and of merged images with DAPI staining are shown side by side (right and left panels, respectively, with embryo genotypes indicated in the merged images). Brackets (A,B) highlight the ventricular cell layer in the dorsal neural tube.
(TIF)
######
Click here for additional data file.
We are grateful to Dr. J. Rossant for providing the RARE-*lac*Z transgenic mice, Drs. D. Anderson, S.L. Ang, P. Chambon, V. Christoffels, B. de Crombrugghe, D. Duboule, T. Gridley, P. Gruss, F. Guillemot, B. Hogan, C.C. Hui, K. Jagla, J. Johnson, S-K. Lee, A. Mansouri, G. Martin, A. McMahon, C. Sherr, J. Rubenstein, and P.X. Xu for providing template plasmids, Dr. L. White for microarray assistance, and Drs. S-K. Lee, M. Noseda, and M. Schneider for advice throughout the study and comments on the manuscript.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This work was supported by the National Institute of Health (R01 HL070733), Agence Nationale de la Recherche (ANR Neurosciences), and Fondation pour la Recherche Médicale. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: PD KN. Performed the experiments: MP SCL RLYW. Analyzed the data: MP PD KN. Contributed reagents/materials/analysis tools: RHF. Wrote the paper: MP PD KN.
| {
"pile_set_name": "PubMed Central"
} |
Key Points {#d30e196}
==========
Both warfarin and rivaroxaban are indicated for the treatment of pulmonary embolism.Anticoagulant medications are one of the most common classes of medications associated with adverse effects and emergency room visits.Poor communication between healthcare professionals and patients can increase the risk for adverse effects from medication therapy.
Introduction {#Sec1}
============
Venous thromboembolism (VTE) affects an estimated 900,000 persons annually in the USA \[[@CR1]\]. Comprised of deep venous thrombosis and pulmonary embolism (PE), the disease represents a significant health burden. For patients experiencing a thrombotic event, the 30-day mortality rate is 30 % \[[@CR2]\]. Warfarin is an effective traditional anticoagulant; however, a narrow therapeutic window necessitates laboratory monitoring of the International Normalized Ratio (INR) \[[@CR3]\]. One study found that 17 % of emergency department visits for adverse drug events (ADEs) among older adults were related to warfarin \[[@CR4]\]. Numerous medication interactions with warfarin may be responsible for associated ADEs, including bleeding \[[@CR5]\].
Because of the drawbacks of warfarin, medications that directly inhibit either coagulation factor II (thrombin) or factor Xa have more recently been developed. These anticoagulants are referred to as non-vitamin K oral anticoagulants (NOACs), and three of these agents (apixaban, dabigatran etexilate, and rivaroxaban) are currently approved for the treatment and prevention of VTE in the USA. NOACs have a faster onset, shorter half-life, as well as fewer medication and dietary interactions. Laboratory monitoring is unnecessary because the dose response is more predictable. However, they are more expensive and a specific antidote does not exist \[[@CR2], [@CR3]\]. In clinical practice, patients are increasingly inquiring about switching from warfarin to NOACs. During the transition, the potential for an ADE exists, such as recurrent VTE due to inadequate anticoagulation or bleeding due to therapeutic duplication. Classifying an ADE as potentially due to a medication error suggests process improvements can be made.
Case Presentation {#Sec2}
=================
A 62-year-old African American man was referred to a pharmacist-managed anticoagulation clinic for follow-up status post-extensive bilateral PE. The patient had received warfarin 10 mg daily for 2 days during the hospitalization, then was discharged with 5 mg daily. Six days after discharge, the patient presented for an initial anticoagulation clinic visit with an INR of 2.3 (goal INR 2.0--3.0). He was instructed to continue 5 mg daily and to return 7 days later. At the next visit, point-of-care testing revealed an INR of \>8.0. His last dose was taken in the morning the same day. The patient denied extra warfarin doses. There was no suggestion of concurrent acute illness or recent alcohol intake. He initially denied any new prescription or supplemental medications, and stated adherence with his maintenance medications, including chlorthalidone and losartan. He denied signs and symptoms consistent with bruising and bleeding. His active problem list included hypertension, osteoarthritis, sleep apnea, and gout. Past medical history included a PE less than 3 years earlier. At that time, he was maintained on warfarin 22.5 mg weekly for a treatment duration of 18 months. Laboratory examination prior to hospital discharge revealed hemoglobin 11.1 g/dL, hematocrit 33.9 %, and platelets 134 K/mcL. Liver function tests were within the normal range. The creatinine level was 1.75 mg/dL with an estimated creatinine clearance of 46 mL/min (using ideal body weight).
On further questioning, the patient reported starting a new prescription medication 5 days earlier. The tablet was described as small and triangular shaped. The anticoagulation clinic contacted the patient's community pharmacy and determined the medication was rivaroxaban 20 mg. Communication among the anticoagulation clinic staff revealed that this prescription had been placed after the initial visit because the patient inquired about the cost of rivaroxaban with his insurance plan. The community pharmacy placed the rivaroxaban prescription on hold rather than discontinue the order as the anticoagulation clinic staff had requested. When the patient presented to his pharmacy the next day to pick up a different medication refill, the rivaroxaban prescription had been filled and was included in the orders ready for pick up. He reportedly did not receive counseling when it was dispensed. He thought it was a new medication for neuropathy, as this problem had been recently discussed with his primary care physician. Despite reading the term 'blood thinner' in the medication guide, the patient did not clarify its indication with the dispensing pharmacist or anticoagulation clinic. He had picked up the original warfarin prescription 8 days earlier.
The patient had a prolonged coagulation time; however, the actual INR was uncertain given the limitations of the point-of-care device and the patient's reluctance to visit the laboratory for a venous blood draw. One consideration was the initial warfarin dosing was excessive, based on the comparison of his current dosing (5 mg daily) with his previous dose requirements (22.5 mg weekly). There was also the possibility the coagulopathy was due to concurrent administration of rivaroxaban. When switching from warfarin to rivaroxaban, it is recommended to discontinue warfarin and start rivaroxaban when the INR is \<3.0. The intent is to not only avoid inadequate anticoagulant effects during the transition but also to avoid duplication for an extended time interval \[[@CR6]\].
The anticoagulation clinic considered several ways to manage this patient. The patient did not desire to continue rivaroxaban, so this alternative was dismissed. The administration of vitamin K as a reversal strategy for warfarin was one option but could have placed the patient at high risk for recurrent VTE. For this same concern, the staff were reluctant to hold warfarin doses with the possibility of a resultant subtherapeutic INR. The patient was ultimately advised to take warfarin 2.5 mg daily for 3 days and then 5 mg for 1 day. He was counseled to monitor for signs and symptoms of bleeding and to seek emergent care if symptomatic. At a morning visit 5 days later, the INR was 4.3. He denied signs and symptoms of bleeding. INR monitoring occurred weekly to biweekly over the next 6 weeks. A total weekly dose of 22.5 mg was found to be sufficient to maintain INR in the goal range.
Discussion {#Sec3}
==========
Rivaroxaban and INR Effect {#Sec4}
--------------------------
A brief review of rivaroxaban pharmacokinetics is useful for application to the case. Rivaroxaban reaches maximum (peak) plasma concentrations 2--4 h after oral administration \[[@CR6]\]. Taken with food, time to peak plasma concentration increases from 2.5 h (after fasting) to 4 h \[[@CR7]\]. Maximal factor Xa inhibition and maximal effect on prothrombin time (PT) are found approximately 2 h after ingestion \[[@CR8]\]. Rivaroxaban has a terminal elimination half-life of 5--9 h in healthy young subjects (age 20--45 years), and 11--13 h in elderly subjects \[[@CR6]\]. In patients with mild (creatinine clearance 50--80 mL/min) and moderate (creatinine clearance 30--49 mL/min) renal function, plasma concentrations were increased 1.4- and 1.5-fold, respectively \[[@CR9]\]. Regarding patient weight, no relevant changes in pharmacokinetics and pharmacodynamics were observed at higher body weights (\>120 kg), while moderately increased effects occurred at lower body weights (\<50 kg) \[[@CR10]\].
In a recent study of healthy male patients (*n* = 96), the mean maximum PT prolongation was 4.4-fold (range 3.4- to 6.5-fold) for patients being transitioned from warfarin (goal INR 2.0--3.0) to rivaroxaban 20 mg once daily \[[@CR11]\]. In comparison, monotherapy with rivaroxaban only produced a 1.6-fold (range 1.4- to 2.1-fold) mean maximum PT prolongation. Peak plasma concentrations after rivaroxaban 20 mg were 223 ng/mL (160--360 ng/mL), with trough levels 22 ng/mL (1--38 ng/mL) \[[@CR12]\]. The estimated maximum plasma concentration of rivaroxaban 20 mg for DVT treatment is 270 ng/mL (189--419 ng/mL) according to pharmacokinetic and simulated virtual data \[[@CR13]\].
A recent study by Samama et al. \[[@CR14]\] tested the effect of rivaroxaban on multiple, commercially available hemostasis assays, including the CoaguChek XS^®^ point-of-care device, which uses a thromboplastin reagent with sensitivity comparable to Neoplastin^®^ and TriniClot^®^. In the study, rivaroxaban induced a concentration-dependent PT prolongation. This effect was linear over a broad concentration range with all PT reagents. The correlation between the PT ratio and the concentration of rivaroxaban determined by CoaguChek XS^®^ was *R*2 = 0.997 \[[@CR14]\].
In our case, the patient reportedly last administered rivaroxaban 20 mg in the morning. The INR was assessed 7--8 h later, approximately 4--5 h after the peak concentration, and the subsequent peak effect on PT. Using data from Kubitza et al. \[[@CR11]\] we can estimate the PT prolongation in our case to be less than 4.4-fold (effect with maximum rivaroxaban concentrations) but greater than 1.6-fold (effect with rivaroxaban monotherapy). His estimated creatinine clearance was 46 mL/min, suggesting an increased plasma concentration (1.5-fold) \[[@CR9]\] vs. patients with normal renal function. His weight was 133 kg, which has not been associated with relevant pharmacokinetic alterations \[[@CR10]\]. Because we were unable to obtain the actual venous INR value or rivaroxaban plasma concentration, our ability for further analysis is limited.
Improvement in Processes of Care {#Sec5}
================================
Discovery of the inadvertent duplication of anticoagulant therapy in this patient prompted the staff to examine the case to identify and learn about any specific safety lapses. Such an analysis is useful to facilitate improvements within the healthcare delivery system and ultimately prevent medication errors and ADEs. In 2012, warfarin was the second most frequent medication reported directly to the US Food and Drug Administration as causing a safety issue, primarily hemorrhage \[[@CR15]\]. The most frequent medication reported was the NOAC dabigatran, further highlighting the inherent risks associated with anticoagulant therapy. In our case, an ADE was averted and any bleeding consequence a near miss. The case also brings to attention how different elements of the system, including equipment and communication, have a role in the occurrence of a medication error (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1Analysis of the medication error using an Ishikawa cause-and-effect diagram
In this case, the anticoagulation staff contacted the patient's community pharmacy by telephone, placed the rivaroxaban order, and determined the patient's co-payment to be US\$40 per month. The prescription was then placed on hold in the patient's prescription profile, despite a request by the anticoagulation staff to cancel or discontinue it. Although it may be more labor intensive, a preferred strategy for determining medication cost is to contact the patient's specific insurance company rather than submitting a prescription claim at the retail pharmacy. In the USA, the medication co-payment for rivaroxaban may vary among users depending on the individual's insurance plan pharmacy benefit or lack thereof. The negotiated price of a prescription medication with the pharmaceutical industry may also differ in countries outside the USA and change the cost to the consumer. Cost is one factor that may affect the selection of a specific anticoagulation agent by physician and patient.
This case also highlights how the electronic medical record at the clinic site does not transmit discontinued medication orders to the dispensing pharmacy. Unless the prescribing provider or staff directly contacts the pharmacy to cancel or discontinue the prescription, the medication remains active, creating the opportunity for refill errors. This type of error can be alarmingly high. In a recent retrospective cohort study, it was found that among 83,902 medications electronically discontinued by a physician during a 12-month study period, 1218 were subsequently dispensed by the pharmacy \[[@CR16]\]. Until the electronic medical record interface allows discontinued orders to be electronically transmitted to the dispensing pharmacy, it is critical to directly communicate this action to the pharmacy. In addition, prescribing providers and staff should be educated about the potential deficiency of the system to help optimize safe medication therapy. After performing our internal analysis, we updated our clinic procedures to recommend cost inquiries are made through the insurance company and all anticoagulant discontinuations are directly communicated to the dispensing pharmacy.
Quality communication at the patient level is also essential for risk reduction. Patients should be educated and encouraged to become more engaged with their healthcare management, including asking any questions to understand the intended use of all medications. A recent study showed how reviewing enhanced medication plans with patients upon hospital discharge improved patient knowledge of their individual medication treatment without prolonging the overall process \[[@CR17]\]. Although this study was conducted in the inpatient setting, it highlights how process improvements can improve the quality of care and patient satisfaction without requiring additional time. Patients prescribed warfarin should continue to receive a handout listing medications to avoid at the initial visit, and this handout must be periodically reviewed as new agents are introduced into the market, such as NOACs. In this case, staff instructed the patient to continue warfarin and not to start rivaroxaban; however, a communication failure was experienced. Thorough documentation of any patient encounter is necessary to reduce the risk of medical liability. In this case, documentation of actual patient understanding to avoid concurrent use of rivaroxaban and warfarin was completed only after discovery of the medication error.
Poor communication during the prescription entry and verification processes also increased the likelihood of this medication error. Performing a thorough medication use review at the dispensing pharmacy site is an essential step to promote safe medication therapy. In this case, the active medication list included both warfarin and rivaroxaban, dispensed only 8 days apart, and this should have prompted an inquiry to the patient or anticoagulation clinic. If a patient uses multiple pharmacies, the opportunity for intervention would be less likely, and this highlights the importance of encouraging patients to maintain only one dispensing pharmacy. In the future, additional caution on behalf of the dispensing pharmacist is warranted given the possibility of serious harm. Patient counseling can be an effective strategy to reduce medication errors and is mandatory by law with every new prescription.
Conclusion {#Sec6}
==========
Sixty years after being approved for medical use, warfarin is still commonly used in clinical practice. NOACs offer many advantages compared with warfarin, yet possess sufficient disadvantages to leave a role for warfarin therapy moving forward. As anticoagulant therapy continues to evolve, the potential for medication errors will increase as clinical practice adapts to emerging treatment options. Given the high-risk nature of these medications, it is imperative to identify and prevent errors in the prescribing, transcription, and dispensing processes. Anticoagulation clinic staff must be knowledgeable and prepared to help manage the unique situations and challenges created by NOACs. In this case report, the authors describe inadvertent duplication of warfarin and rivaroxaban, and the subsequent challenges with interpreting the coagulation assay and determining clinical management. We attempted to apply available pharmacokinetic and laboratory studies to help guide therapy. Analysis after the event revealed several strategies to decrease the likelihood of recurrence. The potential safety issues and uncertainty that inherently come with using NOACs can be improved with communication among healthcare professionals. It is important to report such errors, through case reports and voluntary submission to the Food and Drug Administration, to improve patient safety.
Julie Fusco, Eric Paulus, Alexandra Shubat, and Sharminara Miah declare they have no conflict of interest. No financial support was received for the preparation of this manuscript.
Funding {#d30e380}
=======
The authors received no sources of funding that require acknowledgment.
Consent {#d30e385}
=======
Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder having an incidence of 1 in 6,000 to 1 in 10,000 live births \[[@B1]\]. The severity of TSC and its impact on the quality of life are extremely variable among patients \[[@B2]\]. Common clinical manifestations of this disease include intellectual handicap, autistic disorders, and epilepsy due to the frequent, widespread occurrence of cortical tubers, which are focal disruptions of the cortical architecture due to undifferentiated giant cells. Hamartomas are also found in multiple other organ systems, including the heart, lungs, kidneys, and skin \[[@B3]\].
Patients often seek medical attention for dermal lesions or frequent seizures. The clinical diagnostic guidelines on TSC were prepared based on clinical features, radiographic findings, and histopathological findings \[[@B3]\]. Accurate clinical diagnoses are relatively easy in patients with classic multisystem involvement, but are often difficult due to the diversity of clinical findings in TSC patients.
The genetic basis of TSC has been determined to be due to mutation in either one of two unlinked genes, *TSC1*and *TSC2*\[[@B4]\]. The human *TSC1*gene on chromosome 9q34 consists of 23 exons giving an 8.6-kb mRNA transcript, which has a coding region of 3.5-kb and encodes a 130-kDa protein spanning 1164 amino acids \[[@B5]\]. The *TSC2*gene, which is located on chromosome 16p13.3, contains 41 exons and encodes a 200-kDa protein with 1807 amino acid \[[@B4],[@B6]\]. Both *TSC1*and *TSC2*are tumor suppressor genes and their protein products, hamartin and tuberin, respectively, form a complex that regulates the mammalian target of rapamycin (mTOR) in the phosphoinositide 3-kinases (PI3-kinase)/AKT pathway to control cellular proliferation, adhesion, growth, differentiation or migration \[[@B7],[@B8]\]. Furthermore, both genes play a role in cortical differentiation and growth control.
The mutation spectra of the *TSC*genes are very heterogeneous and no hotspots for mutations have been reported. There are many mutations in each gene that are seen recurrently, but no single mutation accounts for more than about 1% of all TSC patients. *TSC2*mutations are about five times more common than *TSC1*mutations \[[@B9]\] and new mutations are typically found in the two-thirds of TSC cases that are sporadic \[[@B10]\]. Despite complete penetrance of the disease in TSC patients, phenotypic variability can make the determination of disease status difficult among family members of affected individuals.
In this study, we analyzed both *TSC1*and *TSC2*genes in 84 independent Taiwanese TSC probands for whom detailed information on clinical manifestations and phenotype were available. Furthermore, we also assessed the mutational distribution and possible genotype-phenotype correlations between and within the two genes.
Methods
=======
Patient Population
------------------
This study was approved by the Ethics Committee of the Division of Obstetrics and Gynecology, National Taiwan University Hospital. Eighty-four unrelated patients with confirmed clinical diagnoses of TSC and their family members were tested for mutations in *TSC1*and *TSC2*genes.
The general clinical features of TSC patients were determined by clinicians in accordance with the TSC diagnosis criteria set forth by the Tuberous Sclerosis Consensus Conference \[[@B3]\]. All patients\' symptoms were investigated by a person blind to mutational status. High-resolution brain magnetic-resonance imaging (MRI) or computed tomography (CT) was performed on most patients.
The extent of facial angiofibroma or forehead plaques, non-traumatic ungal or periungal fibromas, hypomelanotic macules, shagreen patches, multiple retinal nodular hamartomas, cortical tubers, subependymal nodules, subependymal giant cell astrocytomas, cardiac rhabdomyomas, lymphangiomyomatoses, renal angiomyolipomas and confetti-like lesions were all assessed. Moreover, most patients\' medical histories of mental development were assessed by a certified psychologist.
Sample Preparation
------------------
After genetic counseling and obtaining informed consent, 5--10 mL of peripheral blood were collected from the participants. Genomic DNA was isolated from peripheral whole blood using the Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, MN, USA).
Mutational Analysis of *TSC*Genes
---------------------------------
PCR primers and running conditions for each exon were available from previous studies \[[@B11]-[@B13]\]. The PCR reaction was run on each exon with a total sample volume of 25 μL containing 100 ng of genomic DNA, 0.12 μM of each respective primer, 100 μM dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl~2~, and 0.5 units of AmpliTaq Gold enzyme (PE Applied Biosystems, Foster City, CA, USA). Amplification was performed in a multiblock system thermocycler (ThermoHybaid, Ashford, UK). The PCR amplification started with a denaturing step at 95°C for 5 minutes, followed by 35 cycles of denaturing at 94°C for 30 seconds, annealing at melting temperature (Tm) for 30 seconds, extension at 72°C for 45 seconds, and ends with a final extension step at 72°C for 10 minutes.
The screening of mutations was performed using the Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc, San Jose, CA) with a C~18~reversed-phase column containing 2-μm nonporous poly (styrene/divinylbenzene) particles (DNASep Column, Transgenomic Inc). PCR products were analyzed using linear acetonitrile gradients and triethylammonium acetate acting as mobile phases with the provision of buffer A (0.1 M TEAA) and buffer B (0.1 M TEAA with 25% acetonitrile) (WAVE Optimized, Transgenomic Inc). Heteroduplex analyses were performed according to the manufacturer\'s protocol and of previous studies \[[@B14],[@B15]\].
Statistical method
------------------
The χ^2^and Fisher exact tests were used to examine the differences in clinical manifestations, phenotypes, and mutation distributions in independent Taiwanese probands between patients with *TSC1*and *TSC2*genes.
Direct Sequence Analysis
------------------------
PCR products were purified by solid-phase extraction and bidirectionally sequenced using Applied Biosystems\' Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems). Sequencing reactions were separated on a PE Biosystems 373A/3100 sequencer.
Results and Discussion
======================
Identification and Characterization of Mutations
------------------------------------------------
In the current study, we performed mutational analysis on the coding exons and the exon/intron junctions of both *TSC1*and *TSC2*in a total of 84 individuals with TSC and their family members. The determination of mutation vs. polymorphism was done by: 1) checking the mutation tables at the Chromium site (<http://chromium.liacs.nl/>); 2) comparison of findings to those of 100 healthy Taiwanese controls; and 3) checking the families similarly.
Nine mutations were identified in the *TSC1*gene while 55 were identified in the *TSC2*gene. Mutations in the *TSC1*gene included five nonsense mutations with early termination codons and four insertions/deletions which caused frameshifts and resulted in premature truncation of the protein. Three of these mutations were novel, while six were previously reported (Table [1](#T1){ref-type="table"}).
######
Status of *TSC1*mutations in Taiwanese patients with TSC
**No**. **Gene** **Exon** **Nucleotide change** **Codon change** **Mutation type** **Inheritance** **Reported** **Reference**
------------------------------------------------------ ---------- ---------- ----------------------- ------------------ ------------------- ----------------- -------------- ---------------
62 TSC1 7 c.602_604del CCT In-frame deletion S N This study
61 TSC1 15 c.1525C\>T p.R509X Nonsense F R \[5\]
72 TSC1 15 c.1791_1792dupAA Frameshift S N This study
2 TSC1 15 c.1884_1887delAAAG Frameshift F R \[5\]
36 TSC1 15 c.1959dupA Frameshift S R LOVD\*
54 TSC1 17 c.2074C\>T p.R692X Nonsense S R \[5\]
31 TSC1 18 c.2283C\>A p.Y761X Nonsense S R \[24\]
3 TSC1 18 c.2332C\>T p.Q778X Nonsense S N This study
41 TSC1 18 c.2356C\>T p.R786X Nonsense S R \[5\]
Total: 9, F:2, S:7, N:3, R:6 MM:0, NM:5, FM:4, SM:0.
F: familial case, S:sporadic case.
N: non-reported, R: reported.
MM: missense mutations, NM: nonsense mutations, FM: frameshift/in-frame mutations, SM: splicing site mutations.
\* The the Leiden Open (source) Variation Database which was available at <http://chromium.liacs.nl/lovd/.>
The 55 mutations in the *TSC2*gene included 12 missense, 15 nonsense, 21 frameshifts due to insertions and deletions and 7 putative splice-site mutations. Twenty-seven of these mutations were previously reported while 28 were novel (Table [2](#T2){ref-type="table"}). Of the familial *TSC2*missense mutations, A1141T and R1793Q may be rare polymorphic variants co-segregating with TSC. There was no direct evidence that these familial *TSC2*missense mutational changes were pathogenic.
######
Status of *TSC2*mutations in Taiwanese patients with TSC
**No**. **Gene** **Exon** **Nucleotide change** **Codon change** **Mutation type** **Inheritance** **Reported** **Reference**
------------------------------------------------------------- ---------- ----------- ----------------------- ------------------ -------------------- ----------------- -------------- ---------------
21 TSC2 1 c.109dupG Frameshift F N This study
30 TSC2 1 c.133_136delCTGA Frameshift S R DK\*
35 TSC2 3 c.268C\>T p.Q90X Nonsense S R \[25\]
47 TSC2 6 c.632delC Frameshift S N This study
8 TSC2 intron 8 c.848+3delG Splicing S N This study
37 TSC2 9 c.856A\>G p.M286V Missense F R \[10\]
78 TSC2 10 c.1060C\>T p.Q354X Nonsense S N This study
75 TSC2 10 c.1117C\>T p.Q373X Nonsense S R DK\*
48 TSC2 11 c.1226_1230delAACTG Frameshift S N This study
12 TSC2 12 c.1336C\>T p.Q446X Nonsense S R \[25\]
20 TSC2 14 c.1513C\>T p.R505X Nonsense S R \[10\]
57 TSC2 14 c.1513C\>T p.R505X Nonsense S R \[10\]
65 TSC2 intron 14 c.1599+2T\>C Splicing S N This study
76 TSC2 16 c.1794C\>G p.Y598X Nonsense S R \[10\]
29 TSC2 16 c.1832G\>A R611Q Missense S R \[10\]
59 TSC2 intron 16 c.1840-2A\>T Spilicing S N This study
82 TSC2 17 c.1939G\>A p.D647N Missense S R \[26\]
7 TSC2 18 c.2086T\>C p.C696R Missense S R \[27\]
53 TSC2 19 c.2103_2105dupTGA In-frame insertion S N This study
5 TSC2 19 c.2210T\>C p.L737P Missense S N This study
23 TSC2 20 c.2251C\>T p.R751X Nonsense S R \[10\]
70 TSC2 20 c.2251C\>T p.R751X Nonsense S R \[10\]
39 TSC2 21 c.2404dupA Frameshift F N This study
32 TSC2 21 c.2461A\>T p.K821X Nonsense S N This study
11 TSC2 21 c.2538delC Frameshift F N This study
67 TSC2 intron 21 c.2546-2A\>T Splicing S N This study
73 TSC2 intron 22 c.2639+1G\>C Splicing S R \[9\]
22 TSC2 23 c.2641delT Frameshift F N This study
27 TSC2 24 c.2824G\>T p.Q942X Nonsense S N This study
64 TSC2 26 c.2974C\>T p.Q992X Nonsense S R \[28\]
80 TSC2 26 c.3076dupT Frameshift S N This study
33 TSC2 28 c.3389delC Frameshift S N This study
19 TSC2 29 c.3412C\>T p.R1138X Nonsense S R \[9\]
42 TSC2 29 c.3421G\>A p.A1141T Missense F N This study
13 TSC2 30 c.3693_3696delGTCT Frameshift S R DK\*
51 TSC2 30 c.3696dupT Frameshift S N This study
9 TSC2 33 c.4175_4176delAG Frameshift S N This study
26 TSC2 33 c.4440dupA Frameshift S N This study
77 TSC2 34 c.4541_4544delCAAA Frameshift S R \[12\]
18 TSC2 35 c.4603_4605delGAC In-frame deletion S N This study
34 TSC2 35 c.4603G\>T p.D1535Y Missense S N This study
83 TSC2 36 c.4830G\>A p.W1610X Nonsense S R DK\*
28 TSC2 36 c.4846C\>T p.Q1616X Nonsense S N This study
16 TSC2 37 c.4909_4910delAA Frameshift S N This study
81 TSC2 38 c.5032dupT Frameshift S N This study
60 TSC2 39 c.5150T\>C p.L1717P Missense S R \[29\]
55 TSC2 intron 39 c.5160+3G\>C Splicing S N This study
43 TSC2 intron 39 c.5160+4A\>G Splicing S R \[29\]
4 TSC2 40 c.5227C\>T p.R1743W Missense S R DK \*
50 TSC2 40 c.5227C\>T p.R1743W Missense S R DK\*
56 TSC2 40 c.5228G\>A p.R1743Q Missense F R \[30\]
10 TSC2 40 c.5238_5255del18 Frameshift S R \[31\]
25 TSC2 40 c.5238_5255del18 Frameshift S R \[31\]
6 TSC2 40 c.5252_5259+19del27 Frameshift S R \[9\]
15 TSC2 41 c.5378G\>A p.R1793Q Missense F N This study
Total: 55, F:8, S:47, N:28, R:27 MM:12, NM:15, FM:21, SM:7.
F: familial case, S:sporadic case.
N: non-reported, R: reported.
MM: missense mutations, NM: nonsense mutations, FM: frameshift/in-frame mutations, SM: splicing site mutations.
\* The database of Dr David Kwiatkowski which was available at <http://tsc-project.partners.org/.>
For both genes, sequence variants that were possible mutations were tested in all other family members, including the parents and both the affected and the unaffected family members. In total, 31 of the 64 mutations (48%) had not been reported elsewhere. Moreover, no mutational hotspots were identified in either gene, with only four different mutations being found twice in TSC2.
Compared with those of European and American counterparts \[[@B9],[@B10],[@B16]\], the distribution of the *TSC1*and *TSC2*mutations among Taiwanese population is similar. Therefore, the spectrum of mutations seen among the Taiwanese is no different in comparison to those already reported thus far for these two genes, based on the genetic analyses of European and American TSC patients using the Fisher exact test (*P*= 0.85, 0.46, and 0.14, respectively).
Identification and Characterization of Polymorphism
---------------------------------------------------
In order to identify whether the observed changes were mutations or polymorphisms, samples from 100 normal individuals serving as controls were analyzed. Changes that were not found in more than 200 control alleles were considered pathogenic. Therefore, unique or less frequent changes such as missense and splicing site mutations (Table [2](#T2){ref-type="table"}) were considered likely pathogenic mutations. The nonpathogenic *TSC1*and *TSC2*mutations identified in the Taiwanese TSC patients are described in Table [3](#T3){ref-type="table"}. We identified nine nonpathogenic polymorphisms in the *TSC1*gene and 12 in the *TSC2*gene. The nonpathogenic sequence variants were identified in both the TSC patients and the normal controls. Fourteen of these polymorphisms had not been reported previously (4 at the *TSC1*locus and 10 at the *TSC2*locus) that included one missense variant within the *TSC1*coding region.
######
Polymorphisms identified for *TSC1*and *TSC2*in Taiwanese TSC population.
**TSC1**
----------- ----------------------- ------------------ ----------------------- --------------- -------------- ---------------
**Exon** **Nucleotide change** **Codon change** **Polymorphism type** **Frequency** **Reported** **Reference**
Intron 3 c.106+15 Intron 13 (16 %) N This study
10 c.965 T\>C p.M322T Missense 9 (11%) R \[24\]
Intron 11 c.1142-33 A\>G Intron 9 (11%) R LOVD^a^
Intron 12 c.1264-12 T\>C Intron 3 (4 %) N This study
Intron 14 c.1437-37 C\>T Intron 9 (11%) R LOVD^a^
15 c.1726 T\>C p.L576L Silent 11 (13 %) N This study
15 c.1960 C\>G p.Q654E Missense 3 (4 %) N This study
Intron 18 c.2392-35 T\>C Intron 9 (11%) R \[24\]
22 c.2829 C\>T p.A943A Silent 3 (4 %) R \[24\]
**TSC2**
**Exon** **Nucleotide change** **Codon change** **Polymorphism type** **Frequency** **Reported** **Reference**
14 c.1593 C\>T p.I531I Silent 3 (4 %) R \[26\]
Intron 15 c.1717-30 G\>A Intron 2 (2 %) N This study
Intron 15 c.1717-27 G\>A Intron 1 (1 %) N This study
Intron 21 c.2545+45 T\>A Intron 11 (13 %) N This study
23 c.2652 C\>T p.Y884Y Silent 1 (1 %) N This study
26 c.3126 G\>T p.P1042P Silent 1 (1 %) R DK^b^
Intron 27 c.3285-19 C\>T Intron 1 (1 %) N This study
29 c.3475 C\>T p.R1159R Silent 1 (1 %) N This study
33 c.4047 G\>A p.A1349A Silent 2 (2 %) N This study
Intron 33 c.4493+18 G\>A Intron 1 (1 %) N This study
Intron 38 c.5069-21 G\>A Intron 1 (1 %) N This study
Intron 39 c.5161-9 C\>T Intron 7 (8 %) N This study
\* Frequence means the number of cases in 84 Taiwanese TSC patients.
a The the Leiden Open (source) Variation Database which was available at <http://chromium.liacs.nl/lovd/.>
b The database of Dr David Kwiatkowski which was available at <http://tsc-project.partners.org/.>
Genotype-Phenotype Correlation: Familial or Sporadic TSC mutations
------------------------------------------------------------------
Mutations were identified and located in exons of both *TSC1*and *TSC2*genes (see Figure [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Of the 64 mutations found, nine and 55 were associated with *TSC1*(14%) and *TSC2*(86%), respectively, as shown in Table [4](#T4){ref-type="table"}. Of the 10 familial cases, 2 (20%) and 8 (80%) were *TSC1*and *TSC2*mutations, respectively. Among the 54 sporadic cases, 7 *TSC1*(13%) and 47 *TSC2*(87%) mutations were found. Accordingly, there was no significant difference between sporadic and familial TSC cases with respect to the frequency of *TSC1*vs *TSC2*mutation (*P*= 0.62).
{#F1}
{#F2}
######
Distribution of *TSC1*and *TSC2*mutations.
**N** **MM** **NM** **FM** **SM** **Total**
--------------------- ------- ----------- ----------- ----------- ---------- -----------
***TSC1*mutaions**
**Familial** 2 0 1 1 0 2 (3 %)
**Sporadic** 7 0 4 3 0 7 (11 %)
**Total** 9 0 (0 %) 5 (8 %) 4 (6 %) 0 (0 %) 9 (14 %)
***TSC2*mutations**
**Familial** 8 4 0 4 0 8 (13 %)
**Sporadic** 47 8 15 17 7 47 (73 %)
**Total** 55 12 (19 %) 15 (23 %) 21 (33 %) 7 (11 %) 55 (86 %)
N: screening numbers.
MM: missense mutations.
NM: nonsense mutations.
FM: frameshift/in-frame mutations.
SM: splicing site mutations.
Genotype-Phenotype Correlation: Clinical Manifestations
-------------------------------------------------------
The clinical characteristics associated with each mutation in the proband are shown in Tables [5](#T5){ref-type="table"} (eight *TSC1*mutations) and Table [6](#T6){ref-type="table"} (43 *TSC2*mutations). Most patients with *TSC1*and *TSC2*mutations had seizures, brain lesions (subependymal nodules and/or cortical tubers detected by MRI), and dermal manifestations. Our criteria for intellectual disability included any degree of mental retardation and learning disorder. The incidence of intellectual disability appeared lower in patients with *TSC1*mutations (3/8 = 38%) compared to that of patients with *TSC2*mutations (27/43 = 63%). However, this difference was not statistically significant (*P*= 0.25), but this would be expected because of such small sample sizes. Similarly, the incidence of mental retardation in patients with *TSC1*mutations (1/8 = 13%) appeared to be less than that of patients with *TSC2*mutations (17/43 = 40%), but this difference was not statistically significant (*P*= 0.23). Similarly, the frequencies of renal findings, cortical tubers, subependymal giant cell astrocytomas, liver tumors, cardiac tumors, or skin manifestations, including hypomelanotic macules, facial angiofibromas, shagreen patches, and ungual fibromas did not significantly differ between the patients with *TSC1*and *TSC2*mutations. However, all of these comparisons are under-powered due to the relatively small number of patients with *TSC1*mutations that were studied. For nearly all of the clinical features studied, the frequencies were less for those bearing *TSC1*mutations than for those bearing *TSC2*mutations. This is consistent with findings from other large studies, showing that *TSC1*disease is less severe than *TSC2*disease \[[@B9],[@B10],[@B16]\].
######
Clinical data of patients with *TSC1*mutations
**Family no**. **Familial/Sporadic** **Mutation type** **Sex** **Onset age of seizure** **Intellectual performance** **Brain tubers** **Renal tumors** **Hepatic tumors** **Cardiac rhabdomyoma** **Hypomelanotic macules** **Facial angiofibroma** **Shagreen patch** **Ungual fibroma**
---------------- ----------------------- ------------------- --------- -------------------------- ------------------------------ ------------------ ------------------ -------------------- ------------------------- --------------------------- ------------------------- -------------------- --------------------
2 F FS F 2 y N \+ 0 NA NA 0 \+ 0 \+
3 S NM M 8 y 3 m N \+ 0 0 0 \+ \+ \+ 0
41 S NM F 1 y N \+ 0 0 \+ \+ 0 0 0
31 S NM F 6 m LD \+ \+ 0 \+ \+ 0 0 0
36 S FS M 2 y N \+ 0 0 0 \+ \+ 0 0
61 F NM M 3 y 6 m LD \+ 0 0 0 \+ \+ 0 0
62 S FS M 1 m LD \+ \+ 0 0 \+ \+ \+ \+
72 S FS M 3 y N \+ 0 0 \+ \+ 0 \+ 0
N: normal or no seizure, LD: learning disorder, MR: metal retardation, NA: not available.
######
Clinical data of patients with *TSC2*mutations
**Family no**. **Familial/Sporadic** **Mutation type** **Sex** **Onset age of seizure** **Intellectual performance** **Brain tubers** **Renal tumors** **Hepatic tumors** **Cardiac rhabdomyoma** **Hypomelanotic macules** **Facial angiofibroma** **Shagreen patch** **Ungual fibroma**
---------------- ----------------------- ------------------- --------- -------------------------- ------------------------------ ------------------ ------------------ -------------------- ------------------------- --------------------------- ------------------------- -------------------- --------------------
4 S MM M 10 m N \+ \+ 0 0 \+ \+ \+ 0
5 S MM F 1 y N \+ NA NA 0 \+ \+ 0 0
6 S FS M 1 m LD \+ 0 0 \+ \+ 0 0 0
7 S MM M 6 m MR NA NA NA NA \+ \+ 0 0
8 S S F 3 m LD \+ \+ 0 0 \+ \+ \+ 0
9 S FS M 5 y LD \+ 0 0 0 \+ \+ \+ 0
10 S FS F 6 m LD \+ 0 0 \+ \+ \+ 0 0
11 F FS F 4 m LD \+ \+ 0 \+ 0 \+ \+ 0
12 S NM M 10 m N \+ \+ 0 NA \+ \+ \+ 0
13 S FS F 4 m MR \+ NA NA \+ 0 0 0 0
15 F MM F 7 m MR \+ 0 0 0 \+ 0 0 0
16 S FS M 1 y N \+ \+ 3 NA \+ \+ \+ \+
18 S FS F 5 m LD \+ NA NA NA \+ \+ \+ 0
19 S NM M 3 m MR \+ \+ 0 NA \+ 0 0 0
20 S NM M 1 y 6 m N \+ \+ 0 0 \+ \+ 0 0
21 F FS F 9 y N \+ \+ 1 0 \+ \+ \+ \+
22 F FS M 1 y LD NA NA NA NA \+ \+ \+ 0
23 S NM F 7 m MR \+ NA NA NA \+ 0 0 0
25 S FS M 6 m LD \+ 0 NA 0 \+ \+ \+ 0
26 S FS F 8 m LD \+ \+ 0 0 \+ \+ \+ 0
27 S NM M 1 y MR \+ \+ 0 0 \+ \+ 0 0
28 S NM M 3 m LD \+ NA NA NA \+ \+ \+ 0
29 S MM F 6 m MR \+ 0 NA 0 \+ 0 \+ 0
30 S FS F 1 y MR \+ \+ 0 0 \+ \+ 0 0
32 S NM M 3 m N \+ \+ \+ 0 \+ \+ 0 0
33 S FS F 1 m N \+ \+ \+ 0 \+ \+ \+ \+
34 S MM F 7 y N \+ \+ 0 0 \+ 0 \+ 0
35 S NM M 1 y MR \+ \+ 0 \+ \+ \+ \+ 0
37 F MM M 9 m MR \+ 0 0 \+ \+ \+ \+ 0
39 F FS F 3 m MR \+ NA NA NA \+ \+ \+ 0
42 F MM M 7 y N 0 \+ \+ NA \+ \+ \+ \+
47 S FS F 2 m N \+ 0 0 \+ \+ 0 0 0
48 S FS F 3 m MR \+ \+ 0 0 \+ \+ \+ 0
50 S MM M 3 m MR \+ NA NA NA \+ \+ \+ 0
53 S FS M 3 y MR \+ \+ 0 0 \+ \+ \+ 0
56 F MM F 2 y N \+ \+ \+ 0 \+ \+ \+ \+
57 S NM F 6 m MR \+ 0 0 0 \+ \+ \+ 0
59 S S F 1 m MR \+ \+ 0 0 \+ \+ 0 0
64 S NM M 2 m N \+ 0 0 0 \+ 0 0 0
67 S S F 3 m MR \+ 0 0 \+ \+ \+ 0 0
73 S S F 21 y N \+ 0 0 0 \+ \+ 0 \+
75 S NM F 1 y N \+ \+ 0 0 \+ \+ 0 0
82 S MM M 1 m N 0 0 0 \+ 0 0 0 0
N: normal or no seizure, LD: learning disorder, MR: metal retardation, NA: not available.
Conclusion
==========
This study is the first analysis of *TSC1*and *TSC2*genes in the Taiwanese population. We identified 64 mutations among a total of 84 patients (76%); 9 were *TSC1*mutations (14%) and 55 were *TSC2*mutations (86%). These numbers are similar to other studies with larger cohorts \[[@B9],[@B10],[@B16]-[@B18]\] and would be expected if the germ line mutation rate at the *TSC2*locus were higher than that at the *TSC1*locus. The failure to detect mutations in the remaining 24% of the patients may be due to a combination of lack of screening for large genomic deletion and rearrangement mutations in either *TSC1*or *TSC2*. The occurrence of mosaic mutations \[[@B19],[@B20]\] in some of these patients that may be difficult to detect. Another reason is mutation detection failure.
According to previous reports, somatic and general mosaicism are seen in 6%-10% of all TSC patients \[[@B20],[@B21]\]. In addition, large deletions have been identified in about 2%-4% of *TSC2*mutations \[[@B6]\] and less commonly in the *TSC1*gene \[[@B22],[@B23]\]. Thus, both of these situations likely contributed to patients in which mutations were not identified.
In summary, sixty-four different mutations were identified and characterized for the Taiwanese population. Of those, 31 were not previously described. The diverse mutation spectrum of TSC was also seen in different families and different populations.
Abbreviations
=============
DHPLC: Denaturing high performance liquid chromatography
TSC: Tuberous sclerosis complex
CT: Computed tomography
MRI: Magnetic-resonance imaging
PCR: Polymerase chain reaction
Tm: Melting temperature
Competing interests
===================
We received financial support in the form of a grant from the National Science Council of Taiwan (NSC 92-2314-B-002-319). We have no other competing interests to declare.
Authors\' contributions
=======================
*CCH*and *YNS*performed the molecular genetics studies and drafted the manuscript. *SCC*participated in the molecular genetics studies. *HHL*and *CCC*performed the clinical characterization of the patients. *PCC*and *CJH*performed the statistical analyses. *CPC*, *WTL*and *WLL*participated in the design of the study. *CNL*conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2350/7/72/prepub>
Acknowledgements
================
The authors gratefully extend their gratitude to the families and patients affected by TSC for their participation and cooperation in this study. We thank Prof. David Kwiatkowski, Department of Medicine, Brigham and Women\'s Hospital, Harvard Medical School for deeply review and rewrite the manuscript. We thank Dr. Fon-Jou Hsieh for his expertise and assistance. This work was supported by a grant from the National Science Council of Taiwan (NSC 92-2314-B-002-319).
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Background
==========
In April 2003 the Federal government announced a policy initiative called *A Fairer Medicare*. This initiative was heavily criticised and enabling legislation failed to pass the Senate. A revised version of the policy, called *Medicare Plus*, was announced in November 2003. Further revisions ensued before *Medicare Plus*was passed by the Federal parliament in March 2004 and its name was subsequently changed to *Strengthening Medicare*\[[@B1]\]. *Strengthening Medicare*aimed to improve access (including affordability) to out-of-hospital medical services and contained twenty-seven separate measures including incentive payments for general practitioners (GPs) to encourage bulk-billing of concession card holders and children under sixteen years (particularly in regional, rural and remote areas) and a series of measures to attract GPs to work in areas of undersupply such as outer metropolitan, regional and remote areas \[[@B1],[@B2]\].
Although *Strengthening Medicare*and its precursors were not specifically aimed at increasing the bulk-billing rate, there was considerable debate about the impact that the initiatives would have on the bulk-billing rate and access to GP services (see for example, the first and second reports of the Senate Select Committee on Medicare) \[[@B3],[@B4]\]. Also, one of the effectiveness indicators developed by the Department of Health and Ageing to measure progress towards Departmental outcomes in relation to access to Medicare is \'the percentage of Medicare services that are direct billed with no gap charged\' \[[@B2]\]. Recent increases in the overall bulk-billing rate for GP services have been seen as an indication that the *Strengthening Medicare*package, and bulk-billing incentives in particular, are \'working\' \[[@B1],[@B5]\]. Given the recent increases in the level of bulk-billing (i.e. \'free care\') provided by fee-for-service (FFS) GPs and enduring geographic differences in the supply of GPs, it is pertinent to question what impact increases in the bulk-billing rate will have on access to Medicare-funded GP services in different parts of Australia.
The most widely accepted study examining the relationship between price and utilisation of doctors\' services is the Rand Health Insurance Experiment (HIE) in the United States. HIE results show that utilisation of outpatient services decreases when patients are required to pay a co-payment and that there are no differences in the nature of the response across geographic areas \[[@B6]\]. Richardson applied the HIE results to Australian Medicare data and concludes that 78% of the change in GP service utilisation in the 1976--1986 period and 94% of the change in the 1984/85--1989/90 period were not related to decreases in consumer co-payments -- other factors were at work \[[@B7]\].
A review of Australian cross sectional studies examining factors associated with GP consultation rates indicates that higher frequencies are associated with lower cost, \[[@B8]-[@B10]\] patients having a health care card, \[[@B11]\] patients being in poorer health, \[[@B9],[@B11]\] and with areas of higher proportions older age residents \[[@B10]\]. Lower frequencies are to be found in areas with higher levels of education \[[@B8]\] and among female patients with higher levels of education, \[[@B9]\] in less geographically accessible (eg rural) areas \[[@B10],[@B11]\] and among female patients with an internal Health Locus of Control \[[@B9]\]. One study with highly aggregated levels of data showed an increase in the frequency of consultation in low socioeconomic status areas \[[@B12]\]. Another indicated that the relationship was not so straightforward. Low socioeconomic status was associated with an increase in the consultation rate in highly accessible areas and low consultation rates in inaccessible areas, \[[@B13]\] suggesting supply side factors are also important.
Cross sectional results for the impact of GP supply generally indicate an increase in supply being associated with an increase in frequency of consultation \[[@B10],[@B14],[@B15]\]. One Australian study by Doessel \[[@B16]\] indicated there was no association between GP supply and population based frequency of utilisation rates. However, Richardson and Peacock have indicated that these results should be interpreted carefully \[[@B15]\].
Observed associations between increases in doctor supply and increases in frequency of utilisation have given rise to the notion of supplier induced demand (SID). SID suggests that doctors are imperfect agents and can induce demand for health care, which directly conflicts with the full information and consumer sovereignty assumptions of the orthodox model of demand and supply \[[@B17]\]. Volumes of empirical evidence addressing the possibility of SID have been presented from a range of different health systems, and have been reviewed elsewhere \[[@B18]-[@B20]\]. Many of the studies reviewed have used cross-sectional data sets to examine the effect of doctor supply on the frequency of utilization of health care services \[[@B14],[@B21]-[@B24]\].
However, studies examining only the frequency of utilisation do not take into account the nature of the decision making process in the demand for non-emergency care which is thought to reflect sequential decisions involving both the individual and the GP \[[@B25]\]. Initially an individual decides whether to seek out health care or advice. This decision to consult is derived from the demand for health \[[@B26]\] and reflects an individual\'s beliefs about the severity of their condition, the availability of health care and their expectation of any potential benefits. Once the contact decision has been made and acted upon, the individual and the doctor decide on the type and amount of health services to be provided and the frequency of future consultations. Although the decision about the type, amount and frequency of future consultations is made primarily by the GP \[[@B6],[@B27],[@B28]\] the patient also has a role in this decision. The extent to which the patient participates in the decision depends on the nature of the doctor-patient agency relationship \[[@B29]\].
A small number of studies have attempted to examine the effect of the supply of doctors upon the patient-initiated and doctor-initiated components of utilisation. The results of these studies are inconclusive. Rossiter and Wilensky found that increases in doctor supply had no effect on patient-initiated visits but did have a small effect on doctor-initiated visits and took this to be evidence of SID \[[@B30],[@B31]\]. On the other hand, Escarce found that increased availability increased initial contacts but had little effect on the intensity of subsequent visits, the doctor-initiated component \[[@B32]\]. There is only one Australian study examining the patient-initiated component of the utilisation process. This study found a higher proportion of the resident population consulting a GP in areas with a greater supply of GPs, areas which have higher proportions of younger and older age residents, and areas of lower cost (higher bulk-billing rates). Lower proportions were associated with rural areas \[[@B10]\].
One of the shortcomings of much of the Australian work on the relationship between cost, supply and utilisation of GP services is the inability of the studies to account for border-crossing by patients. There is ample evidence that patients do not necessarily visit the nearest GP when they decide to see a doctor \[[@B33],[@B34]\]. The studies also indicate significant interactions between the variables associated with utilisation of GP services yet these interactions do not appear to have been systematically explored. And the studies are not always inclusive: some target specific demographic groups, others omit certain age groups or geographic areas from their analyses. Finally, despite their widespread acceptance, \[[@B35]\] the generalisability of the HIE results has been questioned \[[@B36],[@B37]\] and their relevance to the Australian situation is not clear.
This research uses publicly accessible data to investigate the likely impact of an increase in the bulk-billing rates and GP supply on access to Medicare-funded GP services in rural and urban areas. The methodology is based on an analysis of aggregated data from various sources and has been developed to be inclusive of all geographic areas and age groups, to take into account border crossing by patients, to systematically explore the interactions among the variables and to accommodate the two-part decision-making process thought to underlie the utilisation of GP services.
Methods
=======
Unit of analysis
----------------
Divisions of General Practice consist of \'geographically co-located group(s) of general practitioners who have formed an organization to work together \... to improve health outcomes at the local level\' and provide GPs with a \'corporate identity\' as well as a \'method of influencing the organization of health care delivery\' \[[@B38]\]. Divisions can, therefore, be regarded as geographically defined service delivery systems and are appropriate aggregation units for modelling the relationship between price, supply and utilisation of health care services. One hundred and twenty-one Divisions are included in the analysis.
Data
----
Two utilisation models have been developed. The first reflects the decision to consult an FFS GP and the second reflects the frequency of consultation once the decision to consult has been made. These contact and frequency decisions are modelled as two different stochastic processes \[[@B28]\]. Failure to do this can lead to inconsistent parameter estimates and misinterpretation of results \[[@B25]\].
### Decision to Consult Model
A patient to population ratio (decision to consult index) was considered an appropriate outcome variable for the contact model and, to overcome the problems of border crossing, the index has been defined as the number of Whole Patient Equivalents (WPEs) per head of resident population. (See the notes to Table [1](#T1){ref-type="table"} for the definition of WPE.)
######
Variables and sources of data for the regression models
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Variables** **Data Sources**
----------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Dependent: decision to consult index**
Whole Patient Equivalents (WPEs) per head of population\* WPEs: Table S2 in the Statistical Appendix to the report *The General Practice Workforce in Australia*†\
Population: HealthWIZ v6.2‡ based on 1996 census
**Dependent: frequency of consultation**
Group A1 and A2 consults per Standardized Whole Patient Equivalent (SWPE)^¶^ Group A1 and A2 Consultations: available at <http://www.hic.gov.au/providers/online_initiatives/hic_online.htm> accessed Sept 7 2003\
SPWEs: Table S2 in the Statistical Appendix to the report *The General Practice Workforce in Australia*†
**Predictor variables**
Geographic accessibility: Population weighted ARIA values for each Division \% Population in postcodes for each Division: available at <http://www.health.gov.au/hsdd/gp/divspc.htm> accessed Jul 11 2003\
Population in each postcode: HealthWIZ v6.2‡ based on 1996 census\
ARIA values for postal areas originally accessed at <http://www.health.gov.au/ari/aria/htm> accessed 26 Feb 2002. This url is no longer available but information about the data can be found at <http://www.gisca.adelaide.edu.au/products_services/ariav2.html>.
Bulk billing rate for general practice consultations HealthWIZ v6.2‡
Dr Density: number of GPs and Other primary medical care doctors per 1,000 head of population Vocationally Registered GPs and Other primary care practitioners: Table S2 in the Statistical Appendix to the report *The General Practice Workforce in Australia*†, derived from the 1998 Medical labour force survey undertaken by the Australian Institute of Health and Welfare (AIHW), Canberra\
Population: HealthWIZ v6.2‡ based on 1996 census
Index of Disadvantage Population at each level of disadvantage within a Division: HealthWIZ v6.2‡
\% population born in NESB country; % female, av age HealthWIZ v6.2‡ based on 1996 census
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Notes**: \* WPEs are derived by the GP Branch of the Commonwealth Department of Health and Ageing as an indicator of patient load for each practice. If a patient consulted at only one general practice during a financial year, the patient is counted as one WPE for the practice. If a patients visits more than one general practice, the patient is counted as a fraction of a WPE based on the schedule fee value for each general practice consulted.
† Australian Medical Workforce Advisory Committee: **The General Practice Workforce in Australia**. AMWAC Report 2000.2.
‡ Commonwealth Department of Health and Aged Care, National Social Health Statistical Database, developed by Prometheus Information Pty Ltd, commonly known as HealthWIZ. See Other Products at <http://prometheus.com.au/html_control/index_frame.htm>
^¶^SWPEs are standardised WPEs. \'The standardisation process is based on National Medical and Department of Veterans\' Affairs claims figures for each of 16 age/gender categories. The standardisation is achieved by allocating each patient to one of sixteen categories and multiplying the WPE value for each patient by the appropriate weight.
### Frequency of Consultation Model
Group A1 (General Practitioner) and Group A2 (Other un-referred) professional attendances are the basic building blocks of the Medicare system. In the 1998--99 financial year these services accounted for 95% of Medicare all services and 92% of the Medicare dollar benefits paid (excluding oral maxillofacial services) \[[@B39]\]. Frequency of consultation has been defined as the number of Group A1 and Group A2 services per Standardized Whole Patient Equivalent (SWPE). (See the notes to Table [1](#T1){ref-type="table"} for the definition of SWPE.)
Based on the review of the literature, the aim of the research and an exploration of the available data, the predictor variables initially entered into both models were: a Divisional measure of geographic accessibility, the bulk-billing rate, GP density, level of socioeconomic disadvantage of the resident population, and the proportion of the resident population born in a non-English speaking background (NESB) country. The proportion of the resident population that is female, and the average age of the resident population were also entered into the decision to consult model but not in the frequency of consultation model as age and gender are controlled for in the outcome variable. Sociodemographic information is based on the 1996 census and all other data relates to the 1998--99 financial year. The relevant data sources for the variables are shown in Table [1](#T1){ref-type="table"}.
Assuming that Australian Bureau of Statistics postal areas are an adequate approximation of postcode areas, a population weighted geographic accessibility value was calculated for each Division by multiplying the Accessibility/Remoteness Index of Australia (ARIA) \[[@B40]\] for each postal area by the proportion of the Divisional population living in the matching postcode and then summing the results across all Divisional postcodes. Values range from zero to twelve and low values indicate greater geographic accessibility. The Divisional index of disadvantage has been derived from the 1996 Socio-Economic Indexes for Areas (SEIFA) Index of Disadvantage \[[@B41]\] statistics for each Division. Values of this index range from one to eleven and the original variable has been recoded so that higher values indicate higher levels of socioeconomic disadvantage. GP density is the number of vocationally registered plus other GPs per 1,000 head of resident population. Summary statistics for the variables used in the modelling are shown in Table [2](#T2){ref-type="table"}.
######
Summary of the dependent and predictor variables
**Highly Accessible\* n = 64** **Accessible\* n = 37** **Moderately Accessible\* n = 7** **Remote\* n = 6** **Very Remote\* n = 5**
------------------------- ------ -------------------------------- ------------------------- ----------------------------------- -------------------- -------------------------
**Dependent Variables**
WPEs/head of pop^n^ Mean 0.95 0.83 0.76 0.70 0.54
SD 0.09 0.06 0.10 0.05 0.12
Consults/SWPE Mean 6.52 4.98 4.8 5.08 4.61
SD 0.91 0.53 0.42 0.44 0.62
**Predictor Variables**
ARIA value Mean 0.22 2.39 4.77 6.87 10.12
SD 0.28 0.80 0.65 0.56 0.82
Bulk-billing rate Mean 82.8% 55.7% 52.1% 65.5% 71.4%
SD 11.2% 13.4% 15.2% 7.0% 20.6%
GP density Mean 1.21 0.97 0.87 0.77 0.82
SD 0.33 0.16 0.27 0.17 0.16
Index of disadvantage Mean 6.80 5.00 4.50 3.61 3.93
SD 2.31 1.38 0.84 0.22 0.52
\% born NESB country Mean 16.0% 3.9% 4.9% 4.8% 3.9%
SD 10.4% 1.6% 2.6% 1.5% 1.9%
\% female Mean 50.4% 50.3% 49.5% 48.5% 47.6%
SD 1.3% 1.0% 1.8% 2.7% 3.1%
Average age (yrs) Mean 35.22 35.62 33.86 34.09 32.08
SD 2.45 1.76 2.20 2.32 2.15
**Data Sources**: See Table 1
**Notes**: \* Categories based on the following ARIA values: highly accessible = 0 -- 1.84, accessible = \>1.84 -- 3.51, moderately accessible = \>3.51 -- 5.80, remote = \>5,80 -- 9.08, very remote = \<9.02 -- 12. Source: Commonwealth Department of Health and Ageing, Accessibility/Remoteness Index of Australia (ARIA), available at <http://www.health.gov.au/ari/aria.htm> Accessed 8th January 2000.
Analysis
--------
Stepwise ordinary least squares (OLS) regression analyses have been undertaken for both models. A forward selection algorithm with an exclusion threshold of p \> 0.2 has been used to enter each of the predictor variables. The main predictor variables which were not excluded using this threshold formed the \'stem\' and their two-way and three-way interactions were entered sequentially using the same algorithm and exclusion criteria.
Results
=======
Decision to consult model
-------------------------
The main predictor variables for the decision to consult model are GP density, bulk-billing rate, socioeconomic disadvantage of the resident population and geographic accessibility (ARIA). Age, gender and NESB status of the resident population are not predictors in this model. The signs of the predictor variables indicate that:
• Higher levels of socioeconomic disadvantage in the resident population are associated with higher levels of the decision to consult index.
• Lower ARIA scores (higher geographic accessibility) are associated with higher levels in the decision to consult index.
• Although the signs for GP density and bulk-billing rates are positive, the three-way interaction term in the model indicates that the relationships between the decision to consult an FFS GP and the supply of GPs and bulk-billing rate are not straightforward. (Table [3](#T3){ref-type="table"})
######
Regression model for decision to consult
Model Unstandardized coefficients Standardized coefficients t-value Significance
----------------------------------------- ----------------------------- --------------------------- --------- -------------- -------
B Std Error Beta
Constant 0.497 0.039 12.897 0.000
GP density 0.234 0.020 0.559 11.717 0.000
Index of disadvantage 0.018 0.003 0.299 6.458 0.000
Bulk-billing rate 0.127 0.032 0.177 3.977 0.000
ARIA -0.010 0.006 -0.205 -1.812 0.073
ARIA \* GP density \* Bulk-billing rate -0.037 0.008 -0.444 -4.308 0.000
**Notes**: ARIA = population weighted Accessibility/Remoteness Index of Australia
Higher index of disadvantage = higher level of socioeconomic disadvantage
Dependent variable = number of Whole Patient Equivalents (WPEs) per head of population (see Table 1)
Rewriting the regression equation in the following way clarifies the relationship between the decision to consult and the bulk-billing rate.
Decision to consult = 0.497 + 0.234\*GP density + 0.018\*Index of disadvantage + Bulk-billing rate\*(0.127 - 0.037\*ARIA\*GP density) - 0.010\*ARIA
This equation shows that the positive effect associated with any given bulk-billing rate will be greatest in the most highly accessible Divisions (i.e. where ARIA = 0), and the \'turning point\' occurs where the bracketed term equals zero (i.e. when. ARIA\*GP density = 3.4 and 0.037\*ARIA\*GP density = 0.127). In the data set used for this analysis, there are twenty-one Divisions where ARIA\*GP density is greater than 3.4. The modelling predicts that increasing the bulk-billing rate ceteris paribus in these twenty-one Divisions will lead to a decrease in the number of people consulting an FFS GP within the Division. As can be seen in Table [4](#T4){ref-type="table"}, not all these Divisions are characterised by low bulk-billing rates and/or low decision to consult indices.
######
Divisions in which an increase in the bulk-billing rate will decrease the number of people consulting an FFS GP
**Geographic Accessibility** **Bulk-billing rate** **Index of disadvantage†** **GP density‡** **WPEs per head of population¶**
------------------------------ ----------------------- ---------------------------- ----------------- ----------------------------------
**Accessible\***
1\. 230 59% 6.8 0.96 0.82
2\. 412 73% 4.7 1.19 0.93
3\. 413 81% 4.5 1.06 0.85
4\. 507 69% 8.0 1.37 0.90
5\. 609 47% 5.3 0.89 0.80
**Moderately Accessible\***
6\. 231 56% 7.2 0.90 0.78
7\. 411 51% 5.6 0.96 0.79
8\. 509 21% 7.7 1.26 0.84
9\. 511 69% 7.1 0.84 0.86
10\. 801 62% 5.8 1.07 0.67
**Remote\***
11\. 241 76% 9.1 0.69 0.76
12\. 416 64% 7.3 0.54 0.65
13\. 417 64% 7.9 0.73 0.71
14\. 512 72% 7.8 0.90 0.74
15\. 611 59% 5.3 1.01 0.71
16\. 612 58% 7.0 0.78 0.63
**Very Remote\***
17\. 233 93% 8.9 0.71 0.70
18\. 415 57% 6.8 0.65 0.50
19\. 610 93% 8.0 1.06 0.38
20\. 614 49% 4.8 0.79 0.61
21\. 802 65% 6.8 0.89 0.51
**Notes**: \* Categories based on the following ARIA values: highly accessible = 0 -- 1.84, accessible = \>1.84 -- 3.51, moderately accessible = \>3.51 -- 5.80, remote = \>5,80 -- 9.08, very remote = \<9.02 -- 12. Source: Commonwealth Department of Health and Ageing, Accessibility/Remoteness Index of Australia (ARIA), available at <http://www.health.gov.au/ari/aria.htm> Accessed 8th January 2002.
† Higher values indicate higher levels of socioeconomic disadvantage
‡ Vocationally registered and other GPs per 1,000 head of population
¶ WPE = whole patient equivalent (see Table 1)
The standardized beta coefficients in Table [3](#T3){ref-type="table"} indicate that GP density is the most important predictor in the decision to consult model. The regression equation can also be rewritten to emphasise the relationship between utilisation and GP density.
Decision to consult = 0.497 + 0.018\*Index of disadvantage + 0.127\*Bulk-billing rate + GP density\*(0.234 - 0.037\*ARIA\*Bulk-billing rate) - 0.010\*ARIA
The positive effect associated with GP density will be greatest in highly accessible Divisions (ARIA = 0) and the \'turning point\' will occur when ARIA\*Bulk-billing rate equals 6.3. In the current data set this occurs in the three very remote Divisions: 233, 610 and 802. The modelling predicts that increasing GP density ceteris paribus in these Divisions would lead to a decrease in the number of people consulting an FFS GP -- an apparently perverse result.
Frequency of consultation model
-------------------------------
GP density and socioeconomic disadvantage of the resident population do not enter as predictor variables in the frequency of consultation model. As shown in Table [5](#T5){ref-type="table"}, the main predictors are the bulk-billing rate squared and the proportion of the resident population born in a non-English speaking background (NESB) country. There are two interaction terms: bulk-billing rate squared\*ARIA; and bulk-billing rate squared\*Proportion of the resident population born in an NESB country.
######
Regression model for the frequency of consultation
Model Unstandardized coefficients Standardized coefficients t-value Significance
------------------------------------------------------------- ----------------------------- --------------------------- --------- -------------- -------
B Std Error Beta
Constant 4.296 0.120 35.777 0.000
Bulk-billing rate squared 2.767 0.204 0.619 13.539 0.000
Bulk-billing rate squared \* ARIA -0.192 0.025 -0.248 -7.621 0.000
Prop^n^pop^n^born NESB country -4.154 1.841 -0.369 -2.256 0.026
Prop^n^pop^n^born NESB country \* Bulk-billing rate squared 8.108 2.091 0.675 3.878 0.000
**Notes**: ARIA = population weighted Accessibility/Remoteness Index of Australia
NESB = Non-English Speaking Background
Dependent variable = number of consults per Standardised Whole Patient Equivalent (SWPE -- see Table 1)
To emphasise the relationship between the bulk-billing rate and frequency of consultation the regression equation can be written as:
Frequency of consultation = 4.296 + Bulk-billing rate sq\*(2.767 + 8.108\*Prop^n^population born NESB country - 0.192\*ARIA) - 4.154\*Prop^n^population born NESB country
The positive impact associated with the bulk-billing rate will be highest in the most geographically accessible Divisions (i.e. where ARIA = 0) which have a high proportion of the resident population born in an NESB country. Assuming the proportion of the population born in an NESB country is zero, then the \'turning point\' in the relationship between bulk-billing and frequency of consultation will occur when 0.192\*ARIA equals 2.767 (i.e. ARIA = 14.4). Since the maximum ARIA value is twelve the \'turning point\' will not be reached and the modelling predicts that a ceteris paribus increase in the bulk-billing rate will unambiguously increase the frequency of consultation across all Divisions.
Performance of the models
-------------------------
In both models the residuals are normally distributed and both have good explanatory power. The linear R^2^value for the decision to consult is 0.845 and for the frequency of consultation it is 0.902. (Figure [1](#F1){ref-type="fig"})
{#F1}
Discussion
==========
Using publicly accessible data, aggregated to the level of the Division of General Practice, this research explores the associations between the bulk-billing rate, the supply of FFS GPs and utilisation of Medicare-funded GP services in rural and urban areas of Australia. Although the use of aggregated data has been described as a useful \'first-cut method\' for getting insights into the \'interplay of macroeconomic variables for the analysis of economic policy\', \[[@B42]\] such data has been criticised because it conceals individual differences in economic behaviour \[[@B43]\]. Because the primary aim of this research is to look at the likely impact of the Federal government\'s *Strengthening Medicare*policy on access to Medicare-funded GP services it is considered an appropriate methodology although there are some caveats that need to be noted.
No inferences can be drawn about the modelled relationships at the intra-Divisional level (i.e. at the individual or postcode level). It also needs to be borne in mind that the data used in the modelling related to a period some six years prior to the introduction of *Strengthening Medicare*. Policy changes in the intervening six years may have changed the nature of the modelled relationships. And a major assumption underlying the analysis is that the allocation of a single patient to different geographic areas is the result of border crossing by the patient. To the extent that the geographic allocation also results from border crossing by GPs (i.e. patients seen by a GP in one Division are linked to provider numbers in a different area) then the extent of border crossing by patients will be overstated. It is not clear from the data to what extent the results are confounded by GPs\' border crossing.
Bearing these caveats in mind, the results indicate that the relationship between bulk-billing and utilisation is complex. This complexity arises, in part, because utilisation is a dynamic process involving both the decision to consult a doctor and the frequency of consultation once contact has been established. Each stage in this process involves a unique set of interacting predictor variables. In addition, each Division represents a unique combination of the predictor variables which means that the impact of changes in bulk-billing will differ from Division to Division. Despite this complexity it is possible to draw a number of inferences from the modelling that have important policy implications.
The level of socioeconomic disadvantage in the resident population has no impact on frequency of consultation but higher levels of socioeconomic disadvantage in the resident population are associated with higher decision to consult ratios. These results suggest that, at the Divisional level, the delivery of FFS GP services in the 1998--99 period was responsive to the needs of socioeconomically disadvantaged groups.
In many, but not all, Divisions an increase in the bulk-billing rate will unambiguously increase utilisation of Medicare-funded GP consults within a given period: it will increase both the number of people consulting an FFS GP and the number of visits per patient. However, in a number of Divisions, an increase in the bulk-billing rate will simultaneously increase the number of times existing patients consult and decrease the number of people consulting in a given period. The most straightforward interpretation of this phenomenon is that new patients are \'crowded out\' by existing patients\' increased use of services.
Increasing the supply of FFS GPs in a Division will, in all but three very remote Divisions, increase the number of people consulting an FFS GP in a given period without impacting on the number of times patients consult. This would mean an increase the total number of FFS GP consults in a Division in a given period and, assuming a stable population, an increase in the number of consults per head of resident population. The positive relationship between supply and the number of people contacting a GP is consistent with both the SID hypothesis and orthodox economic theory, but the modelling also implies that SID is not occurring at the point in the decision-making process where it might be most expected to occur -- in the frequency of consultation. These results are consistent with Escarce\'s findings in relation to surgeons \[[@B32]\]. The reasons for the apparently perverse results in the three very remote Divisions cannot be clarified in this modelling exercise and require a more in-depth analysis to understand the dynamics underlying the result.
Conclusion
==========
Bulk-billing rates and the supply of FFS GPs are two important supply-side elements in the Australian health care system that are potentially amenable to policy manipulation. To the extent that the *Strengthening Medicare*policy has increased bulk-billing rates, and bearing in mind the qualifications expressed in relation to the data, the modelling indicates that an increase in bulk-billing rates will not necessarily increase access across all Divisions. Unlike the American HIE results which showed no difference in response across geographic areas, in Australia, rurality is clearly important in understanding the relationships between cost, supply and access to medical care and needs to be considered when modelling the impact that policy initiatives on access to FFS GP services.
*Strengthening Medicare*was specifically designed to increase access for financially disadvantaged patients and those less than 16 years of age. The modelling suggests that, in the late 1990s, FFS GPs were responsive to the needs of socioeconomically disadvantaged patients and age was not an important predictor in the utilization of Medicare-funded GP services. If this situation still existed prior to the introduction of the new policy, then it is likely that the impact of the initiative on access to FFS GP services for these two groups of patients would be somewhat restrained.
Finally, geographic differences in bulk-billing rates and the supply of FFS GPs are often taken to be evidence of inequities in the provision of heath services. According to Hancock, social equity requires that the \'provision of health services \... should not discriminate on the grounds of \... rurality or geographical location\' \[[@B44]\]. However, policy strivings for equivalence in performance indicators such as bulk-billing rates across geographic areas will not necessarily result in geographic equity of access. If geographic accessibility is not addressed, ensuring equity of access to FFS GP services in Australia would seem to require positive discrimination in favour of rural areas such that the supply of FFS GPs and bulk-billing rates are higher in rural than metropolitan areas.
Competing interests
===================
This work is funded by an Australian Research Council grant and the North East Victorian Division of General Practice is an industry partner in the research.
Authors\' contributions
=======================
SED: participated in the design, implemented the study drafted and revised the manuscript
KA: conceived the study, participated in its design and helped draft and revise the manuscript
DD: participated in the design of the study and revised the manuscript
SP: provided technical advice in the design of the study and revised the manuscript
LG: participated in the design and implementation of the data analysis and revised the manuscript
DV: conceived the study, participated in its design and revised the manuscript
Acknowledgements
================
The authors acknowledge the input of Dr Mark Robinson and Mr David Dart of the North East Victorian Division of General Practice in the initial development of this work.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
DNA alkylating agents have historically played an important role in systemic chemotherapy for cancer, including brain tumors. The first-generation alkylating agent dacarbazine (DTIC) requires enzymatic conversion to the active cytotoxic metabolite 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) in the liver \[[@CR1]\]. There are concerns that the therapeutic potential of DTIC in central nervous system (CNS) malignancies is limited because MTIC may not efficiently penetrate the blood--brain barrier. Temozolomide, an oral alkylating agent, overcomes these limitations. Temozolomide is rapidly absorbed following oral administration, with *T*~max~ values of approximately 1 h, and undergoes spontaneous pH-dependent hydrolysis to MTIC at physiologic pH with a half-life (*t*~½~) of approximately 1.8 h. MTIC is characterized by the formation of rate-limited pharmacokinetics (PK), with an observed in vivo half-life similar to that of temozolomide. MTIC subsequently degrades by pH-dependent hydrolysis to a reactive methyl-diazonium cation and AIC (5-aminoimidazole-4-carboxamide). Based on data from a limited number of subjects, the systemic bioavailability of oral temozolomide appears to be nearly 100% \[[@CR2], [@CR3]\]. Additionally, in a 14C-AME study, the mean recovery of radioactivity in feces was approximately 1% of the administered dose, suggesting that temozolomide is completely absorbed following oral administration. \[[@CR4]\] Because temozolomide is lipophilic, it efficiently crosses the blood--brain barrier and is bioavailable to the CNS (20--30% of plasma exposure) \[[@CR2], [@CR5]\].
Oral temozolomide is approved in many countries for the treatment of malignant glioma, and is approved for the treatment of melanoma in some regions. Oral temozolomide cannot be administered to all patients, specifically patients with difficulty swallowing capsules. This includes: patients with oropharyngeal dysfunction resulting from increased intracranial pressure/brain stem involvement; patients unable to take oral medications because of gastrointestinal obstruction, intractable nausea and vomiting, or other comorbidities affecting systemic absorption of the drug; and pediatric patients. An intravenous formulation provides a reasonable alternative for these patients.
An exposure equivalence approach was used to develop an intravenous formulation for temozolomide. Previous studies in nonhuman primates \[[@CR6]\], dogs (unpublished data) and rats \[[@CR7]\] demonstrated similar plasma PK profiles of temozolomide following oral or intravenous administration. Based on these data, a pilot clinical study was conducted to compare the PK profiles of equivalent doses of temozolomide, administered orally or by a 60-min intravenous infusion. The study demonstrated that the area under the curve (AUC) of oral and intravenous temozolomide were similar. However, the ratio (intravenous:oral) of maximum concentration of drug after dosing (*C*~max~) did not meet the criteria for exposure equivalence. The key parameter that required optimization was the rate of intravenous infusion so as to better match gastrointestinal absorption kinetics and achieve similar *C*~max~ values to that achieved via oral administration. Subsequently, Monte Carlo simulations to evaluate virtual crossover exposure equivalence trials using a population PK model derived from a previous population study of oral temozolomide \[[@CR8]\] were conducted and suggested that a 90-min intravenous infusion could achieve exposure equivalence with respect to *C*~max~ (unpublished data). The goal of this pivotal, randomized crossover study was to examine the exposure equivalence and safety profile of a 90-min intravenous infusion of temozolomide compared with an equivalent oral dose.
Methods {#Sec2}
=======
Eligibility {#Sec3}
-----------
Eligible subjects had a diagnosis of a primary CNS tumor (excluding CNS lymphoma), were at least 18 years of age and had a Karnofsky performance score of 70 of higher. All subjects were required to have adequate hematologic, hepatic and renal function. Subjects were excluded if they had impaired gastrointestinal absorption, vomiting or any other medical condition that would compromise the intake of oral medication. Subjects were excluded if they had received chemotherapy or biologic anticancer therapy within 4 weeks before study entry, or mitomycin C or nitrosourea therapy within 6 weeks before study entry. This study was conducted in accordance with good clinical practice (GCP) and in compliance with the World Medical Association Declaration of Helsinki with respect to written informed consent and the protection of rights of human subjects.
Study design {#Sec4}
------------
This multicenter, open-label, randomized, crossover study compared the PK of intravenous and oral temozolomide. As this study was conducted in the context of treating subjects with primary CNS malignancies, oral temozolomide was administered at the highest approved dose (200 mg/m^2^) on days 1, 2 and 5. On days 3 and 4, temozolomide was administered orally on one day and by 90-min intravenous infusion on an alternate day at a dose of 150 mg/m^2^ (the approved dose of temozolomide for the first cycle of treatment). Subjects were assigned, according to a computer-generated random code, to receive intravenous temozolomide either on day 3 or day 4 with oral temozolomide on an alternate day. All daily oral doses were rounded down to the nearest 5 mg. The doses to be administered on days 3 and 4, intravenous versus oral, were identical. If vomiting occurred during oral dosing, the subject was not redosed. For PK sampling on days 3 and 4, subjects were to fast for a minimum of 8 h before each dose of temozolomide and to continue fasting for 4 h afterward.
The primary objective was to evaluate exposure equivalence of a 90-min intravenous infusion to an equivalent oral dose of temozolomide based on the ratio of the log-transformed means for AUC and *C*~max~ for both temozolomide and MTIC. Based on regulatory guidelines, exposure equivalence was defined as a 90% confidence interval (CI) for the ratio of the means based on log-transformed data within the range of 80--125% \[[@CR9], [@CR10]\]. Secondary end points included local tolerability and safety. Adverse events were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0 over a 28-day period beginning from administration of the first dose of temozolomide.
The PK of temozolomide and MTIC following intravenous and oral administration was determined from serial blood samples taken on days 3 and 4, just before dosing (0 h), and at 0.25, 0.50, 1.00, 1.25, 1.5 (for intravenous dose, within 5 min after the end of infusion), 1.75, 2.00, 2.50, 3.00, 4.00, 6.00 and 8.00 h after initiation of infusion or administration of the oral dose. Plasma temozolomide and MTIC samples were collected and procured as previously described \[[@CR4], [@CR11], [@CR12]\]. Briefly, blood samples for MTIC were collected in prechilled heparinized tubes and immediately centrifuged for 10 min at 3,000 rpm at 4°C. The resulting plasma was immediately frozen in a dry ice methanol bath and then stored at -70°C until assayed. Blood samples for temozolomide were collected in prechilled heparinized tubes and then centrifuged for 10 min at 3,000 rpm at 4°C. Immediately following centrifugation, 50 μL of 8.5% phosphoric acid was added to each mL of plasma. Samples were then vortexed and stored at −20°C until assayed. Plasma concentrations of temozolomide and MTIC were determined by liquid chromatography, followed by tandem mass spectroscopy (LC-MS/MS). The lower limit of quantitation (LLOQ) for temozolomide and MTIC were 20 and 5 ng/mL, respectively. These methods were validated for selectivity, sensitivity, precision and accuracy. The stability of temozolomide in plasma after the addition of phosphoric acid and the stability of MTIC in plasma without phosphoric acid under various conditions were established. The temozolomide plasma assay was linear over the range of 20--30,000 ng/mL. The accuracy ranged from −6.8 to −2.1%, and the precision was 9.1--10%. The internal standard was ethazolastone. The MTIC plasma assay was linear over the range of 5--4,000 ng/mL. The accuracy ranged from --3.3 to 0.8% and the precision was 3.1--9.4%. The internal standard was dacarbazine.
The study protocol was written such that data from subjects/samples could be prospectively excluded from the primary analysis in case of protocol violations, unsuccessful dosing or possible sample procurement errors. For instance, subjects who vomited within 4 h of oral dosing on pharmacokinetic days, whose dose on days 3 and 4 were not within 10% of the recommended dose, or subjects whose intravenous infusion duration was not within 10% of 90 min were excluded. Additionally, if anomalous temozolomide or MTIC concentrations were observed (i.e., concentrations below LLOQ, a zero concentration between two non-LLOQ concentrations, or an LLOQ between two non-zero concentrations), the pH of the respective PK sample was checked to ensure that the sample was properly procured. If the sample was not at the recommended pH necessary to stabilize the analyte, the sample was excluded from analyses.
Noncompartmental analyses were conducted on individual concentration--time data. Log-transformed PK parameters (AUC and *C*~max~) for temozolomide and MTIC were subjected to a crossover analysis of variance (ANOVA) model, extracting the effects due to treatment, sequence, subject within sequence and period. Assuming an intrasubject variability (coefficient of variation) of 20% and modeling and simulation results, a target enrollment of 20 subjects was selected to provide a minimum of 90% power for the 90% CI of the ratio of the treatment means for derived AUC and *C*~max~ for temozolomide and MTIC to fall within the 80--125% confidence range.
Results {#Sec5}
=======
Subject disposition {#Sec6}
-------------------
A total of 22 subjects were enrolled at three centers. Subject demographics are presented in Table [1](#Tab1){ref-type="table"}. All 22 subjects were randomized and received 5 days of treatment with temozolomide (once-daily oral dosing of temozolomide for 4 days and a single intravenous dose of temozolomide for 1 day, either on day 3 or day 4). Eleven subjects received intravenous treatment on day 3, and 11 subjects received intravenous treatment on day 4. Pharmacokinetic data from three subjects were excluded from the primary analysis as per the prospectively defined exclusion criteria. Two subjects had anomalous temozolomide/MTIC levels \[one subject had predose temozolomide and MTIC concentrations that were 45 and 15%, respectively, of the corresponding *C*~max~ values; the other subject had MTIC concentrations on day 4 that were at or below assay LLOQ for all samples (in case of the latter subject, it was determined that the samples were not procured at the appropriate pH)\] and one subject had an interrupted infusion schedule. Thus, 19 subjects were included in the pharmacokinetic analyses.Table 1Subject demographics*n* = 22Mean age, years (range)45.8 (32--59)Sex, *n* (%) Male12 (55) Female10 (45)Race, *n* (%) White22 (100)KPS score, *n* (%) 705 (23) 803 (14) 903 (14) 10011 (50)Median weight, kg (range)80.2 (43.5--93.1)Median height, cm (range)170 (149--187)Median BSA, m^2^ (range)1.735 (1.34--2.16)*KPS* Karnofsky performance status, *BSA* body surface area
Pharmacokinetic assessments {#Sec7}
---------------------------
The *C*~max~, AUC and *t*~½~ for intravenous and orally administered temozolomide were similar and showed low intersubject variability (coefficient of variation \[CV\] = 12--21%; Table [2](#Tab2){ref-type="table"}). In contrast, the intersubject variability for MTIC *C*~max~, AUC(I) and AUC(*t*~f~) ranged from 53 to 62%. Individual subject data for the *C*~max~ of temozolomide are shown in Fig. [1](#Fig1){ref-type="fig"}a, and the mean values following intravenous and oral administration were 7.4 and 7.7 μg/mL, respectively. Individual subject data for the *C*~max~ of MTIC are shown in Fig. [1](#Fig1){ref-type="fig"}b. The individual AUC(I) values of temozolomide and MTIC following intravenous and oral administration are shown in Fig. [1](#Fig1){ref-type="fig"}c, d.Table 2Pharmacokinetic parameters of temozolomide and MTIC following intravenous and oral administrationTMZ (*n* = 19)MTIC (*n* = 19)i.v.p.o.i.v.p.o.*t*~½~, mean hours (CV%)1.81 (12)1.91 (13)1.80 (16)1.77 (11)*T*~max~, median hours (range)1.5 (0.92--2.0)1.0 (0.25--2.0)1.5 (1.25--1.75)1.0 (0.25--2.0)*t*~f~, mean hours (CV%)8.0 (0)8.0 (0)8.0 (0)8.0 (0)*C*~max~, mean μg/mL (CV%)7.44 (21)7.68 (19)0.32 (61)0.33 (62)AUC(*t*~f~), mean μg h/mL (CV%)23.4 (18)22.0 (14)0.94 (53)0.94 (60)AUC(I), mean μg h/mL (CV%)25.0 (18)23.6 (15)1.00 (54)1.00 (60)Data reported as arithmetic meansTMZ, temozolomide; MTIC, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide; i.v., intravenous; p.o., oral; *t*~1/2~, half-life; CV, coefficient of variation; *T*~max~, time of maximal analyte concentration, *t*~f~, time of final quantifiable sample; *C*~max~, maximum concentration of drug after dosing; AUC(*t*~f~), area under the concentration--time curve from 0 h to time of final quantifiable sample; AUC(I), area under the concentration--time curve from 0 h to infinityFig. 1Paired individual *C*~max~ for temozolomide (**a**) and MTIC (**b**) following intravenous (*i.v.*) and oral (*p.o.*) administration. Paired individual AUC(I) for temozolomide (**c**) and MTIC (**d**) following intravenous and oral administration. *C*~max~ maximum concentration of drug after dosing; *MTIC* 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, *AUC(I)* area under the concentration--time curve from 0 h to infinity
The ratios of the model-based (least-squares) geometric means (intravenous:oral) for the parameters of *C*~max~, AUC(I) and AUC(*t*~f~) for both temozolomide and MTIC were within ±6% of unity (Table [3](#Tab3){ref-type="table"}). The mean plasma concentration--time profiles for temozolomide and MTIC following temozolomide administration orally or a 90-min intravenous infusion were identical (Fig. [2](#Fig2){ref-type="fig"}a, b). The 90% CIs for the ratio of the log-transformed means for *C*~max~ and AUC (for both temozolomide and MTIC) were within the range for exposure equivalence (80--125%). ANOVA of log-transformed PK parameters (*C*~max~ and AUC) did not demonstrate any effects due to sequence of administration or period.Table 3Relative bioavailability of TMZ and MTIC following intravenous and oral administrationMode of administration^a^Intrasubject CV (%)Ratio estimate^c,d^ i.v./p.o. (%)90% CI, %i.v.^b^p.o.^b^MTIC (*n* = 19)* C*~max~ (μg/mL)0.280.28139891--105 AUC (*t*~f~) (μg h/mL)0.840.82910398--108 AUC (I) (μg h/mL)0.890.86810398--108TMZ (*n* = 19) *C*~max~ (μg/mL)7.37.5109791--102 AUC (*t*~f~) (μg h/mL)23.121.85106103--109 AUC (I) (μg h/mL)24.623.45105102--108TMZ, temozolomide; MTIC, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide; i.v., intravenous; p.o., oral; CV, coefficient of variation; CI, confidence interval; *C*~max~, maximum concentration of drug after dosing; AUC (*t*~f~), area under the concentration--time curve from 0 h to time of final quantifiable sample; AUC(I), area under the concentration--time curve from 0 h to infinity^a^The dose of TMZ administered on pharmacokinetic sampling days (both i.v. and p.o.) was 150 mg/m^2^ per day^b^Model-based (least-squares) geometric mean^c^ Based on log-transformed data using ANOVA model extracting the effects due to treatment, sequence, subject within sequence and period^d^Ratio of the mean value for i.v. to p.o. administrationFig. 2Mean plasma concentration--time profiles for temozolomide (**a**) and MTIC (**b**) following intravenous (*i.v.*) and oral (*p.o.*) administration. *MTIC* 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide
Safety evaluation (*n* = 22) {#Sec8}
----------------------------
The type and frequency of acute systemic toxicities experienced on days 3 and 4 were similar for intravenous and oral administration (Table [4](#Tab4){ref-type="table"}). All of these adverse events were CTC grade 1 or 2. The most commonly reported adverse events on days 3 and 4 were headache, dizziness, nausea and vomiting. The most commonly reported adverse events occurring throughout the entire 28-day study period are also shown in Table [4](#Tab4){ref-type="table"}. Four subjects experienced 12 severe or life-threatening treatment-emergent adverse events including hematologic toxicities (which is the known dose-limiting toxicity for temozolomide), hydrocephalus and appendicitis (both occurring in the same subject and deemed unrelated to temozolomide), and headache and convulsions (which were consistent with the subject's underlying disease and deemed unrelated to temozolomide).Table 4Treatment-emergent adverse events (all grades) occurring in ≥10% of subjects excluding injection-related eventsDays 3 and 4 only,^a^*n* (%)Entire 28-day study period, *n* (%)p.o.i.v.PooledPooledAny8 (36)10 (45)14 (64)21 (95)Headache3 (14)4 (18)6 (27)9 (41)Nausea1 (5)2 (9)3 (14)9 (41)Constipation01 (5)1 (5)6 (27)Anemia0005 (23)Vomiting1 (5)2 (9)3 (14)5 (23)Dizziness2 (9)1 (5)3 (14)4 (18)Leukopenia0004 (18)Neutropenia0003 (14)Thrombocytopenia0003 (14)*p.o.* oral, *i.v.* intravenous^a^Reports treatment-emergent adverse events not previously reported on days 1 and 2
Local toxicities were evaluated by subject-reported adverse events and a local tolerability score. Ten subjects reported 11 injection site reactions following intravenous administration. Nearly all events were mild and transient (one subject reported moderate infusion site pain). Ten events reported in nine subjects resolved on the day of the infusion (five events with a duration of 1--6 min, four events with a duration of 15--62 min, and one event with a duration of 131 min), and one event resolved the following day. Injection site reactions included infusion/injection site pain (*n* = 4), infusion site swelling (*n* = 1), injection site warmth (*n* = 2), injection site irritation (*n* = 2), injection site erythema (*n* = 1) and pruritus (*n* = 1). No thrombophlebitis was reported. All subjects with a local tolerability score greater than 0 also had a treatment-emergent injection site adverse event reported. Three subjects required local treatment for these events, consisting of application of ice to the injection site, the use of a pressure bandage, and/or change of injection site location. No subject was unable to complete the infusion or study treatment because of an adverse event at the injection site, although one subject had the injection site changed twice.
Discussion {#Sec9}
==========
This pivotal exposure equivalence study was designed to evaluate the bioavailability and safety of a 90-min intravenous infusion of temozolomide compared with an equivalent oral dose. The goal was to establish comparable systemic exposure (*C*~max~ and AUC) to both temozolomide and its active degradation product MTIC, following intravenous and oral administration. A crossover design was used to decrease variability and the number of subjects required to be enrolled. The crossover design used in this study is analogous to the approach used in other studies that have examined the exposure equivalence of intravenous versus oral administration of chemotherapy agents \[[@CR13], [@CR14]\]. In this study, 150 mg/m^2^ temozolomide, the approved dose of temozolomide for the first cycle of treatment in patients with recurrent glioma, including refractory anaplastic astrocytoma (AA), was the dose chosen for intravenous administration.
The intravenous dose was administered by a 90-min infusion either on day 3 or day 4 in the middle of a 5-day treatment course. Given that temozolomide and MTIC have similar half-lives of approximately 1.8 h \[[@CR4], [@CR12], [@CR15]--[@CR17]\], once-daily dosing on days 3 and 4 with a 24-h washout period (corresponding to approximately 13 half-lives between each dose) was sufficient. In fact, predose (0 h) concentrations for all PK-evaluable subjects in this study were below the LLOQ for both temozolomide and MTIC.
The results of this study showed that a 90-min intravenous infusion and an equivalent oral dose of temozolomide met the exposure equivalence criteria based on the ratio of mean AUC and *C*~max~ for both temozolomide and MTIC. Treatment-emergent adverse events were consistent with those reported previously in patients with recurrent glioma treated with oral temozolomide, except for local reactions because of intravenous administration. Injection site reactions were mostly mild and transient. No new safety concerns emerged.
The intrasubject variability in AUC and *C*~max~ for both temozolomide and MTIC was low (CV ≤ 13%; Table [3](#Tab3){ref-type="table"}). The intersubject variability in PK parameters for temozolomide was also low following both intravenous and oral administration. This was not unexpected, as gastrointestinal absorption of temozolomide is rapid and bioavailability is high. The intersubject variability in *C*~max~ and AUC for MTIC was higher than for temozolomide. Nonetheless, systemic exposure to MTIC was similar within individual subjects receiving intravenous versus oral administration, and the intrasubject variability of MTIC was low. The reason for the higher intersubject variability in MTIC is unclear. However, the low intrasubject variability (8--13%) suggests that random issues with sample handling were not a cause of the higher intersubject variability. In addition, the data were consistent across clinical sites.
In the present study, the PK profile of temozolomide was independent of the route of administration. This is consistent with previous studies evaluating the PK profile of oral temozolomide, which demonstrated that temozolomide exhibits linear pharmacokinetics over the therapeutic dose range \[[@CR2], [@CR4], [@CR16]--[@CR19]\] and that the PK profile of temozolomide is independent of the route of administration (i.e., intravenous, oral or hepatic intra-arterial infusion) \[[@CR2]\]. Other studies have demonstrated that total body clearance of temozolomide is linear \[[@CR8], [@CR16]\] and independent of dose \[[@CR16]\]. Moreover, the PK characteristics of temozolomide have been shown to be independent of dosing schedules \[[@CR17]\]. The results of the present study are also consistent with earlier studies that compared the PK of temozolomide administered by intravenous, oral or intra-hepatic routes in a few patients using different formulations \[[@CR2]\]. Therefore, the exposure equivalence data obtained from this study allow for direct extrapolation across the range of therapeutically meaningful doses and administration schedules.
In conclusion, this study demonstrated the exposure equivalence of a 90-min intravenous infusion with oral administration of temozolomide. Intravenous administration of temozolomide was generally well tolerated. In clinical practice, oral temozolomide is administered using a variety of doses and schedules. Based on the data from this study and the known PK characteristics of temozolomide, intravenous administration would result in an equivalent exposure compared with oral administration at any given dose and schedule. Potential applications include use in patients in whom oral administration is not feasible because of the inability to swallow, nausea, vomiting or impaired gastrointestinal absorption and in pediatric patients.
We thank Dr. Waldo H. Belloso, Dr. Zenon Beguellín, Dr. Jo Ann Horowitz, Dr. Rudolf Kwan, Dr. Douglas Kramer, Dr. Diego Del Carretto, Jane Devane, Ingrid Banks, Virginia Dawney and Jenny Lewis for their support with this study. Financial support for medical editorial assistance was provided by Schering-Plough. We thank Jerome Sah PhD, ProEd Communications, Inc.^®^, for his medical editorial assistance with this manuscript.
**Conflict of interest statement** P. Statkevich owns stock in Schering-Plough. M.A. Abutarif and D. Cutler have received remuneration from and own stock in Schering-Plough. B.D. Diez, Y. Zhu, F. Xuan, B. Kantesaria, M. Schwarz and M.G. Pallotta have nothing to disclose.
**Open Access** This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
Fabio H. Ottaviano: deceased (2 February 1971--4 November 2006).
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Baizhi (*Angelica dahurica* BENTH. et HOOK.) is a perennial Apiaceae plant, originated in Taiwan (Wang et al. [@CR38]) and found abundantly in Korea, China, Japan and Russia (Sarker and Nahar [@CR31]; Li et al. [@CR18], [@CR19]). The root of Baizhi is a traditional Chinese medicine known for its antipyretic and analgesic properties since thousands of years ago. Today, researchers are analyzing the multiple biological functions and investigating the chemical constituents of Baizhi.
The multiple pharmacological effects of Baizhi root extracts include antioxidation (Piao et al. [@CR28]), anti-inflammatory (Pervin et al. [@CR27]), antiproliferative (Yousif et al. [@CR42]), skin whitening (Cho et al. [@CR6]), anti-tumor (Kim et al. [@CR15]), antimicrobial (Kwon et al. [@CR16]) and anti-Alzheimer effects (Marumoto and Miyazawa [@CR22]). These effects are believed to be attributed to the plant's rich furanocoumarin compounds such as imperatorin and isoimperatorin. Imperatorin has anti-inflammatory (Garcia et al. [@CR12]), anticonvulsant (Luszczki et al. [@CR21]), hepatoprotective (Oh et al. [@CR26]), myorelaxant (Chiou et al. [@CR5]), vasodilator (Wang et al. [@CR39]) and anti-cancer effects (Li et al. [@CR18], [@CR19]). Isoimperatorin has anti-inflammatory (Abad et al. [@CR1]), antiallergic (Ryu et al. [@CR30]) and antimicrobial effects (Suleimenov [@CR34]). Beside the pharmacological effects of Baizhi furanocoumarins, root extracts contain a variety of phenolic compounds that are connected to its strong antioxidant activity (Wang et al. [@CR40]). Recently, foods with antioxidant effects become valued for their ability to remove reactive oxygen species, which oxidize lipids, DNA, membranes and proteins, and are involved in atherosclerosis, cancers and other diseases (Leopold and Loscalzo [@CR17]).
Growth of Baizhi has three stages: V-stage, vegetative stage; B-stage, bolting stage; and S-stage, summer dormancy stage. Summer dormancy in herbaceous perennials is characterized by: (1) cessation or reduction of leaf meristem growth; (2) senescence of most or all above-ground herbage; (3) possible dehydration of the bases of the youngest leaves at the base of vegetative tillers; and (4) possible preceding formation of resting organs in the form of swollen basal internodes (corm/tuber) or swollen leaf bases (such as bulbs) (Volaire and Norton [@CR36]). Although a report indicated NtFTs are target genes of temperature signal pathway to regulate the dormancy process in *Narcissus tazetta* var *Chinensis* (Feng et al. [@CR11]), the relationship between biochemical regular and summer dormancy still lacked conclusive researches (Gillespie and Volaire [@CR13]). For environmental drivers, vegetative organs develop under increasing day-length and temperature at the end of spring will induce summer dormancy (Volaire et al. [@CR37]). In addition, water deficit was shown to be an induction factor for *P. bulbosa* L. (Ofir and Kigel [@CR25]) and *D. glomerata* (Volaire [@CR35]). Accordingly, after the V-stage, instead of transitioning to the B-stage, Baizhi enters summer dormancy (S-stage) to survive hot and dry environmental conditions. Its above-ground portions die during summer, and its root becomes the storage organ and therefore becomes heavier, a mechanism that implies more harvest yield than at other growth stages. Thus, it is expected that Baizhi roots should be harvested at S-stage, although the S-stage is not necessary to complete the regular life cycle of Baizhi.
A previous study arranged a series of sowing dates, from September to November, to analyze the B-/S-stage ratio in Baizhi (Xingfu et al. [@CR41]). However, few analyses have revealed the content of pharmacological compounds in Baizhi roots extracted from different stages. Accordingly, the quantities in root may change during the growth stages. For example, with *Angelica sinensis* (known as Danggui in China), a well-known functional food for its roots, the growth stage is the most important factor affecting functional compound composition (Lü et al. [@CR20]). Another study indicated that the content of six phthalides and four aromatic acids in *A. sinensis* varied with harvest timing (Qian et al. [@CR29]).
In this study, we aimed to determine the functional activities of Baizhi roots at different growth stages, specifically the S-stage, which rarely occurs in other root-based medicinal plant species. To this end, we determined the activity of six furanocoumarin compounds, xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, and antioxidants in Baizhi primary taproots and lateral roots at V-, S- and B-stages. The results will provide experimental proof for the harvest timing of Baizhi, especially for functional quality.
Materials and methods {#Sec2}
=====================
Plant materials {#Sec3}
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The Baizhi field experiment of our study was grown in year 2014--2015 on a sandy loam soil at the Hualien District Agricultural Research and Extension Station (121°33E, 23°58N), Council of Agriculture, Executive Yuan. The 30 main plots 10 × 1.2 × 0.3 m (L × W × H) were sown in October and November 2014. Seed was initially sown to dry seedbeds and transplanted as one plant per hill to the puddled field plots at 3--4 leaves stage. The site soils had pretreated (2 weeks before transplantation) with organic matter (N:P~2~O~5~:K~2~O = 4.9:2.1:1.9; 8000 kg ha^−1^). The experiment was conducted one plant per hill to the puddled field with regular water application. Hills were spaced at a distance of 120 cm with 60 cm between planted rows (hill spacing = 120 cm × 60 cm). During the culture period, the total rainfall is about 900 mm, the mean air temperatures range from about 17 to 29 °C, the mean monthly sunshine hours range from 70 to 230 h. It was harvested at the three harvest stages, vegetative (V-stage), bolting (B-stage) and summer dormancy (S-stage), during the following year in May, July and August based on plant appearance (Fig. [1](#Fig1){ref-type="fig"}). Ten plantlets were harvested at each stage. The taproots and lateral roots (Fig. [1](#Fig1){ref-type="fig"}d) were separated, and the length, diameter and fresh weight were measured. The roots were then sliced, and part of the fresh root slices were freeze-dried (LYPH. LOCK 18, Labconco, USA) as HPLC--DAD samples; residues were completely dried in a 40 °C oven. The total weight of the two parts of dry roots were the dry weight.Fig. 1The appearance of Baizhi at three growth stages: V-stage, vegetative (23 weeks after sowing, **a**); B-stage, bolting (34 weeks after sowing, **b**); S-stage, summer dormancy (38 weeks after sowing, **c**); and roots harvested at V-stage (**d**)
Reagents and chemicals {#Sec4}
----------------------
All solvents were purchased from Merck (Darmstadt, Germany) and were of analytical grade or high-performance liquid chromatography (HPLC) grade. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and (3-(2-pyridyl)-5,6,diphenyl-1,2,4-triazine-p, p′-disulfonic acid monosodium salt hydrate were from Merck, and Fe^2+^ chloride tetrahydrate ferrozine, and furanocoumarin compound standards, including xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin were from Merck. The standards for HPLC with diode-array detection (HPLC--DAD) were dissolved in methanol to a concentration of 100 μg/ml and were stored at − 20 °C.
Root extracts and sample processing {#Sec5}
-----------------------------------
Freeze-dried root slices were crushed to a power by using a high-speed disintegrator (Solar Energy Co, Taiwan). Extraction for HPLC--DAD was performed as described (Shiau et al. [@CR32]) with some modifications. Methanol (20 ml) was added to 0.2 g ground powder, vortexed and let stand for 30 min. The powder with methanol was then ultrasonic shocked (DC600H, Delta, Taiwan) for 30 min, then centrifuged at 3320 rcf for 3 min. The supernatant was collected and the residue was re-extracted under same conditions for complete extraction. Finally, the supernatants were mixed and filtered through a 0.2-µm Acrodisc Syringe Filter (Pall, USA) before being collected in the Automatic Sampler-approved certified vials (Agilent, USA). Antioxidant assay extraction was performed as described (Shimada et al. [@CR33]) with some modifications. Methanol (20 ml) was added to 1.0 g ground powder; vortexed and shaken overnight (50 rpm), then centrifuged at 3320 rfc for 3 min. The supernatant was collected as the sample.
HPLC--DAD analysis {#Sec6}
------------------
To identify the furanocoumarin compounds in root extracts, 25 µl samples were separated by using an analytical column (Syncronis C18, 250 × 4.6 mm, 5 µm particle; ThermoFisher, USA) with flow rate 1.0 ml/min at 30 °C and absorbance 310 nm monitored by the ThermoFisher LC system (ThermoFisher, USA) equipped with a ThermoFinnigan UV detector and ThermoFisher Automatic Sampler. The column was protected by a C18 Guard column (HI-5C18-10C5, Hichrom, UK). A binary gradient was used for H~2~O (Solvent A) and methanol (Solvent B). Optimized pigment separation was achieved with a linear gradient of 40--20% Solvent A for 0--5 min, 20--10% Solvent A for 5--7 min, and 10--0% Solvent A for 7--9 min.
DPPH assay {#Sec7}
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DPPH radical scavenging activity was as described (Shimada et al. [@CR33]) with some modifications. The DPPH solution in methanol (0.4 mM) was prepared daily, and antioxidant samples were diluted or concentrated to at least seven concentrations. An amount of 50 µl sample solutions was loaded on a 96-well plate. Each concentration was loaded in six wells. Three were mixed thoroughly with 150 µl fresh DPPH solution (A1) and the others were mixed thoroughly with 150 µl methanol (A2). Pure methanol was used as the control (C1, C2). The loaded 96-well plates were incubated for 30 min in the dark, then the decrease in absorbance of light at 517 nm was measured. The DPPH radical scavenging effects were calculated as \[1 − (A1~517~ − A2~517~)/(C1~517~ − C2~517~)\].
Fe^2+^ chelating assay {#Sec8}
----------------------
Fe^2+^ chelating activity was measured as described (Dinis et al. [@CR9]) with some modifications. Antioxidant samples were diluted or concentrated to at least seven concentrations. The Fe^2+^ chloride tetrahydrate solution in methanol (12 mM) was prepared, and 10.5 µl of this solution was mixed thoroughly with 1400 µl sample solution. Ferrozine solution in methanol (5 mM) was prepared. An amount of 200 µl sample solution was loaded on 96-well plates, with each concentration loaded in six wells. Three were thoroughly mixed with 20 µl ferrozine solution (A1) and the others were thoroughly mixed with 150 µl methanol (A2). Pure methanol was used as the control (C1, C2). The loaded 96-well plates were left to stand for 5 min, then the decrease in absorbance of light at 562 nm was measured. The Fe^2+^ chelating effects were calculated as \[1 − (A1~562~ − A2~562~)/(C1~562~ − C2~562~)\].
Statistical analysis {#Sec9}
--------------------
Data were analysed by using ANOVA. Source of variation was harvest stage. Significant differences by harvest stage were calculated by the Fisher least significant difference (LSD) tests (*p* \< 0.05). Data are expressed as mean ± SD. The IC50 value of DPPH radical scavenging activity and Fe^2+^ chelating activity was predicted by Loess regression. All analyses involved use of R (version 3.2.2) software. p \< 0.05 was considered statistically significant.
Results {#Sec10}
=======
Root weight, length, diameter and moisture content {#Sec11}
--------------------------------------------------
The transition of Baizhi from vegetative to bolting or summer dormancy, and subsequent root mass is determined by sowing dates (Xingfu et al. [@CR41]), fertilizing management (Derong et al. [@CR8]) and environmental factors such as latitude. Root length and diameter were lower at V-stage than the other two stages, with no significant (*p* \< 0.05) difference between B- and S-stage. In Chinese medicine market, lateral roots of ginseng were commercialized independently, which termed as ginseng "fibers" or "beards". To this, root samples were divided into 'Taproot' and 'Lateral root' as shown in Table [1](#Tab1){ref-type="table"}. The root lengths were 23.91 ± 1.97, 29.60 ± 5.46 and 30.40 ± 5.83 cm at V-, B- and S-stage, respectively, and the root diameters were 76.45 ± 17.92, 106.93 ± 20.08 and 106.87 ± 38.41 cm, respectively. Root dry weight was highest at S-stage and lowest at B-stage. For taproots, the DW was 2.4 times greater at S- than V-stage and was about 2.7 times greater than at B-stage. For lateral roots, the DW was about 2.2 times greater at S- than V-stage and about 6.8 times greater than at B-stage. V-stage had the highest lateral root to total root ratio, and the proportions of lateral roots were about 26, 11 and 25% at V-, B- and S-stage, respectively. The findings could be explained by a portion of the biomass contributing to the growth of bolting, which may lead to about 2 m of flower moss. In contrast, the above-ground portions die during S-stage. Accordingly, harvesting Baizhi at S-stage rather than at V- or B-stage would allow for better yield. The variation in shoot may affect the harvest weight of root.Table 1Weight, length, diameter and moisture content of Baizhi roots at three growth stagesV-stageB-stageS-stageTaproot weight (g)171.45 ± 99.53^b^\*153.27 ± 37.89^b^406.96 ± 188.43^a^Lateral root weight (g)61.12 ± 35.42^b^19.66 ± 13.08^c^133.19 ± 85.02^a^Total root weight (g)232.57 ± 128.05^b^172.93 ± 38.95^c^540.16 ± 258.82^a^Lateral root/total root (%)261125Root length (cm)23.91 ± 1.97^b^29.60 ± 5.46^a^30.40 ± 5.83^a^Root diameter (mm)76.45 ± 17.92^b^106.93 ± 20.08^a^106.87 ± 38.41^a^Data are mean ± SD of 10 independent plantsV-stage, vegetative stage; B-stage, bolting stage; S-stage, summer dormancy stage\* Different letters indicate statistical significance (p \< 0.05) by LSD test
Antioxidant activity {#Sec12}
--------------------
Extracts of Baizhi root have anti-oxidant properties (Pervin et al. [@CR27]; Wang et al. [@CR40]). Both reports have successfully used BHT as a standard to determine the antioxidant activity. Accordingly, in our study, we focused on the content of functional compounds in different harvest stages. We determined DPPH scavenging activity and Fe^2+^ chelating assay by abundant samples with statistical results without the usage of BHT. First, we determined the antioxidant activity of the three stages by DPPH assay and observed a dose-dependent relation in DPPH scavenging activity of Baizhi root extracts (Fig. [2](#Fig2){ref-type="fig"}a, b). For taproots, the IC50 value of the extracts were 5.70 ± 4.54, 6.46 ± 3.06 and 10.92 ± 3.82 mg/ml at V-, B- and S-stage, respectively (Table [2](#Tab2){ref-type="table"}). The activity was about 2 times less at S-stage than the other two stages. However, from Table [1](#Tab1){ref-type="table"}, for taproot, the DW was about 2.5 times greater for S-stage than the other stages. The low activity for S-stage may be due to its high DW, which mostly consists of storage compounds such as starch. For extracts of lateral root, the three stages did not differ in activities. The IC50 value of the lateral root extracts were 5.07 ± 5.20, 6.76 ± 5.37 and 6.12 ± 5.12 mg/ml at V-, B- and S-stage, respectively (Table [2](#Tab2){ref-type="table"}). Furthermore, the DW of lateral roots was about 12 times greater at S-stage than the other stages, which indicates that 12 times total activity could be obtained from S-stage than the other stages. Taken together, the highest DPPH radical scavenging activity was obtained from roots harvested at S-stage.Fig. 2Antioxidant activities of Baizhi root extracts from different growth stages. Baizhi roots were extracted with methanol, and extracts were diluted or concentrated to at least seven concentrations, and subjected to DPPH assay of taproot (**a**) and lateral root (**b**) and Fe^2+^ chelating assay of taproot (**c**) and lateral root (**d**). The gray-line zones indicate 95% confidence intervals for the loess regression line derived from ten plant samples for each concentration Table 2Antioxidant activity of Baizhi taproot and lateral root at three growth stagesHarvest stageTaprootLateral rootDPPH radical scavenging capacity V-stage5.70 ± 4.54^a^\*5.07 ± 5.20^a^ B-stage6.46 ± 3.06^a^6.76 ± 5.37^a^ S-stage10.92 ± 3.82^b^6.12 ± 5.12^a^Fe^2+^ chelating capacity V-stage2.52 ± 7.13^a^2.55 ± 6.13^a^ B-stage1.66 ± 1.70^a^1.90 ± 1.74^a^ S-stage11.94 ± 6.09^b^2.65 ± 6.33^a^Roots were extracted with methanol and subjected to DPPH and Fe^2+^ chelating assay. Activity of extracts is shown as mean ± SD IC50 (mg/ml) predicted by Loess regression\* Different letters indicate statistical significance (p \< 0.05) by *t* test
We also determined the antioxidant activity of roots by Fe^2+^ chelating assay and found a dose-dependent relation in Fe^2+^ chelating assay of Baizhi root extracts (Fig. [2](#Fig2){ref-type="fig"}c, d). For taproots, the IC50 value of the extracts were 2.52 ± 7.13, 1.66 ± 1.70 and 11.94 ± 6.09 mg/ml for V-, B- and S-stage, respectively (Table [2](#Tab2){ref-type="table"}). The activity was about 5 times less at S-stage than the other two stages (Table [2](#Tab2){ref-type="table"}). Activity in lateral roots did not differ among the stages. The IC50 value for lateral root extracts were 2.55 ± 6.13, 1.90 ± 1.74 and 2.65 ± 6.33 mg/m for V-, B- and S-stage, respectively (Table [2](#Tab2){ref-type="table"}). However, the DW was about 2.5 times greater at S-stage than the other stages. Hence, the active compounds for DPPH and Fe^2+^ chelating mechanisms differed. Baizhi root harvested at S-stage provided antioxidant activity primarily via a DPPH radical scavenging instead of Fe^2+^ chelating mechanism.
Characterization of six furanocoumarin compounds in Baizhi root {#Sec13}
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In traditional Chinese medicine, crude Baizhi root materials are used as medicine or functional food for antipyretic and analgesic activities because of abundant furanocoumarin compounds (Li et al. [@CR18], [@CR19]). For example, isoimperatorin, imperatorin and oxypeucedanin may be useful for treating Alzheimer disease (Marumoto and Miyazawa [@CR22]). By analyzing the inhibitory activity on LPS-induced PGE2 production, it has been demonstrated that Isoimperatorin, imperatorin and phellopterin have potential as anti-inflammatory drugs (Ban et al. [@CR2]; Deng et al. [@CR7]). For other furanocoumarin compounds, xanthotoxin, isoimperatorin, imperatorin and oxypeucedanin have anti-cancer abilities (Kim et al. [@CR15]).
The chemicals described above involve major furanocoumarin compounds, which we extracted from Baizhi root for HPLC--DAD chromatogram assay; seven clear peaks were obtained. By using reference compounds, we identified peaks 1, 2, 4, 5, 6 and 7 as xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, respectively (Fig. [3](#Fig3){ref-type="fig"}) and the chemical structures are in Fig. [4](#Fig4){ref-type="fig"}. Then, we analyzed these six furanocoumarins in Baizhi root and found different patterns of furanocoumarin compounds from each harvest stage. The furanocoumarin content in taproot (or lateral root) at each harvest stage showed different patterns (Fig. [5](#Fig5){ref-type="fig"}). Oxypeucedanin had the highest yield of the six furanocoumarins (1.5--3.0 mg/g) and the other five yielded from 0.15 mg/g (bergapten) to 1.6 mg/g (phellopterin). The yield of each compound varied less than 50% at different harvest stages except for isoimperatorin from taproot. Similarly, the yield of taproot and lateral root varied less than 50% except for imperatorin and isoimperatorin at B-stage. Additionally, we have also analyzed the furanocoumarin content in shoot (above ground part) of V stage, only imperatorin was detected as about 0.21 mg/g.Fig. 3HPLC-DAD chromatogram of standard (**a**) compared with Baizhi root tissue (**b**). Peaks 1, 2, 4, 5, 6 and 7 were identified as xanthotoxin, bergapten, oxypeucedanin, imperatorin, phellopterin and isoimperatorin, respectively Fig. 4Chemical structure of furanocoumarins identified from root of Baizhi Fig. 5Xanthotoxin (**a**), bergapten (**b**), oxypeucedanin (**c**), imperatorin (**d**), phelllopterin (**e**) and isoimperatorin (**f**) content in Baizhi taproot and lateral root harvested at three stages. Roots were extracted with methanol and subjected to HPLC-DAD analysis (*n *= 10). Data are mean ± SD furanocoumarin content (mg/g sample). Different letters indicate statistical significance (p \< 0.05) by LSD test
To analyze the total amount of the six furanocoumarins in Baizhi roots, the results of harvest yield and six furanocoumarin contents were integrated (Fig. [6](#Fig6){ref-type="fig"}). Total furanocoumarin content was highest at S-stage. Also, furanocoumarin content was generally higher at S-stage than the other two stages; especially, the yield of xanthotoxin, bergapten and phellopterin was 2 times higher at S-stage than the other 2 stages. This result strongly supports that S-stage is the best harvest stage than the other stages because of its richer total furanocoumarin content.Fig. 6Total content of xanthotoxin (**a**), bergapten (**b**), oxypeucedanin (**c**), imperatorin (**d**), phellopterin (**e**), phellopterin (**f**) in Baizhi taproots and lateral root harvest at three stages. Data are mean of the DW shown at Table [1](#Tab1){ref-type="table"} multiplied the content of each furanocoumarin at each stage's taproot (black bars) and lateral root (gray bars). Different letters indicate statistical significance (p \< 0.05) by LSD test
Discussion {#Sec14}
==========
The root of Baizhi has been used as a functional food and traditional medicine. In this study, we analyzed the antioxidant activity and total furanocoumarin content of Baizhi root and concluded that the summer dormancy stage is the best stage for harvesting.
Furanocoumarins are believed to function as phytoalexin (Beier and Oertli [@CR4]) or allelochemical compound (Baskin et al. [@CR3]). These compounds are relevant for being potential substitutes for pesticides in crop protection (Eljarrat and Barceló [@CR10]). In wild parsnip (*Pastinaca sativa*, also an Apiaceae plant), the quantity of furanocoumarins allocated to floral units at different stages of development is consistent with the value of those floral units (Nitao and Zangerl [@CR24]). Additionally, studies of field-grown plants showed that FCs storage is closely related to organs and phenological stages. Leaves, stems, roots and fruits do not have equal concentrations of FCs even if psoralen, xanthotoxin, bergapten and isopimpinellin were detected in all tissues (Milesi et al. [@CR23]). Therefore, it is believed that FCs act, specifically in storage organs, as protection chemicals against biostress. As for the case of Baizhi entering the S-stage, the above ground parts occur yellowing, compounds redistribution. FCs are then yielded and accumulated in storage organ (root), specifically in cortex. We suggest that there may be two strategies for Baizhi to survive the stress of summer season. The first strategy is the avoidance trait, in which Baizhi will bolt early in May and adapt the summer season with seed dormancy. The second strategy is the resistance trait, the above-ground parts of Baizhi will senescence and the root will become the resting organs which contain higher dry weigh.
Conclusions {#Sec15}
===========
The results indicate that Baizhi roots should be harvested at S-stage. However, it is possible that the growth of Baizhi switch from vegetative stage to bolting stage directly, without a summer dormancy stage. Our preliminary studies indicated that sowing earlier by 1 month (in November) may result in early bolting in May. Many studies have reported that stage-transition of Baizhi is determined by, for example, sowing date, fertilizing management and other environmental factors. In the near future, global warming is expected to affect phenological events such as flowering and fruiting in plants and will hasten development time in plant species that respond to cues such as degree days (Hughes [@CR14]). Under the threat of global warming, analyzing the stage-transition physiology of Baizhi will facilitate the modification of the cropping system to achieve a stable and uniform S-stage of Baizhi.
V-stage
: vegetative stage
S-stage
: summer dormancy stage
B-stage
: bolting stage
DW
: dry weight
HPLC--DAD
: high-performance liquid chromatography with diode-array detection
DPPH
: 2,2-diphenyl-[l]{.smallcaps}-picrylhydrazyl
YCC and WHL conceived the experiment, analyzed the data and drafted the manuscript. WHL, TWC performed the experiment. All authors read and approved the final manuscript.
Acknowledgements {#FPar1}
================
We thank Professor Li-yu D. Liu and Chen-An Tsai for description of statistic results.
Competing interests {#FPar2}
===================
The authors declare that they have no competing interests.
Availability of data and materials {#FPar3}
==================================
This study is a part of our project that belongs to Ministry of Science and Technology and Council of Agriculture, Executive Yuan of Taiwan. Our project has not been ended yet; therefore, data will not be shared until we complete all project works.
Consent for publication {#FPar4}
=======================
Not applicable.
Ethics approval and consent to participate {#FPar5}
==========================================
Manuscripts reporting studies involving human participants, human data or human tissue must: a statement on ethics approval and consent: not applicable. The name of the ethics committee: not applicable.
Funding {#FPar6}
=======
This project was supported by the Ministry of Science and Technology and Council of Agriculture, Executive Yuan of Taiwan.
Publisher's Note {#FPar7}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
} |
Title: Are everyday clinical practice and guideline supported clinical decision making in contrast?
**Abstract**
Among psychiatrists there is a common feeling, that guidelines directives on psychopharmacological treatment are often in contrast to clinical decision making driven by clinical experience. This might be one reason, why compliance to guidelines is not as one might expect.
Reasons for this discrepancy are related among others in the way guidelines are developed by the respective commissions: the experts are often not or not any more clinically experienced, the priority given to meta-analyses lead to relative global results, for more differentiated treatment problems (drug resistence, predominance of certain sub-syndromes, psychiatric and non-psychiatric comorbidity, co-medication etc.) Sufficient data are not available, individual dispositions of the patients are not sufficiently considered in the EBM data base, the gap between phase- studies and phase-4 studies is difficult to overcome.
The current position of EBM neglects extremely the value of clinical practice and is not open enough for more critical reflections about is own methodological limitations. Especially the one-sided preference for meta-analyses should be replaced by a multi-methods approach, involving much more the evaluation of individual studies.This would help among others to give answers also to questions mentioned above, so far mostly not addressed sufficiently by the available guidelines.
| {
"pile_set_name": "PubMed Central"
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News of the completion of the new Wits School of Public Health (WSPH) building reminded me of the dream that so many of us had when the School was founded just over a decade ago. At the time I wrote: 'The year 2001 will be remembered as the year in which the Wits School of Public Health was established. This event marks a defining moment in the development of Public Health at Wits. The establishment of the school has over a short period of a few months brought a new dimension to the way in which we function. No longer can Public Health be regarded as the Cinderella of health sciences at Wits ... the School has demonstrated its academic worthiness.' The new building serves to reinforce this notion. The establishment of the School did not happen without opposition from within the Faculty of Health Sciences and the university. Similarly, finding a building suitable for the rapidly expanding School of Public Health did not happen without a struggle. Initial efforts led to the university architects drawing up plans for a multi-story School of Public Health building near the entrance of the Medical School. However, the university did not see its way clear to supporting such an undertaking and the plans were shelved. Efforts to acquire premises on Oxford Road, which members of staff inspected and liked, were similarly not supported by the university. It is for this reason, amongst others, that it is such a tremendous privilege to see the new building finally a reality -- indeed a dream come true.
Public Health: 1993--2002 {#S0001}
=========================
Public Health at Wits, as elsewhere, was not the stuff that medical faculties were made of in the 1990s. Two factors influenced the trajectory of the academic discipline of Public Health significantly. The first was the dramatic political change in South Africa which led to the establishment of a new democratic order with human rights, including the right to health and health care, enshrined in the country\'s constitution. The School played a significant role in shaping national health policy in South Africa. In 1994, two of the nine ministerial committees were chaired by staff in the School, and many other staff members served on a range of committees and advisory bodies that ultimately influenced government thinking and health policies in the democratic era. The second major event was the HIV and AIDS disaster, which had a devastating effect on mortality and morbidity in South Africa and brought enormous challenges to its health system. These external factors had an impact on the development of Public Health in significant ways. The new government had a large constituency to whose needs it had to respond to. As a result, it looked to Public Health to address the dire health problems faced by a large majority of South Africans, while the HIV epidemic exposed the worst failings of the South African Public Health system.
Notwithstanding these imperatives, material support for academic Public Health was not forthcoming. It took a lot of nagging and some strong words to eventually gain seven additional academic posts from the Gauteng Health Department in 2002, bearing in mind that the WSPH only had about nine provincial Health Department funded posts at the time, some of which were 'frozen' during the implementation of financial austerity measures as part of the government\'s Growth Equity and Redistribution (GEAR) policy in the late 1990s.
The emergence of Public Health at Wits also benefited from the readmission of South Africa to the world community, leading to exposure to many academics prominent in international Public Health. The school benefited from a regular stream of international visitors from other parts of Africa, Latin America, Asia, the United States of America and the United Kingdom. In South Africa, the 1990s saw several initiatives to start regional schools of Public Health. Wits University was part of the five-university initiative, called the Transvaal School of Public Health (later named the Thusano School of Public Health or TSPH), which aimed to strengthen the discipline of Public Health in the northern regions of South Africa. All in the Faculty did not support the TSPH as some felt that trans-university courses and programmes would compromise Wits' academic standards. These barriers had to be overcome and eventually an agreement at the level of the university Vice-Chancellors formalised the TSPH.
Following the World Development Report 1993, which argued the economic case for investing in health, there was a growing realisation that the global burden of diseases like malaria, HIV & AIDS and tuberculosis had a negative impact on the world economy. As a result, the world\'s rich nations established the Global Fund that supported the response to these three killer diseases. This gave added impetus to a worldwide resurgence of Public Health and provided much needed resources for Public Health research in South Africa.
In keeping with its commitment to strengthen South Africa\'s Public Health capacity, the WSPH initiated a Master of Public Health (MPH) programme and an MSc in Epidemiology in addition to the existing diploma courses, despite the enormous strain put on the limited teaching resources. This resulted in student numbers growing exponentially.
Meanwhile, the rural health initiatives started by John Gear, flourished. The Wits Rural Facility underwent a new incarnation; the Health Services (later Systems) Development Unit thrived, and spawned the Agincourt Project, which has since become one of the leading demographic and health surveillance sites in the world. Agincourt, in turn, gave birth to the Rural AIDS and Development Action Research programme (RADAR).
The Centre for Health Policy (CHP) excelled in the new post-apartheid environment and provided policy support for many government initiatives. It grew in size and the Women\'s Health Project, which it housed, became a leader in the struggle for gender equality in health care. This went on to have an enormous influence in shaping South Africa\'s new abortion legislation.
Urban Health was not to be left out as the School, as part of the WHO Collaborating Centre for Urban Health, continued to work in inner city areas such as Hillbrow and the Alexandra Township to the north of Johannesburg city centre. The emergence of Urban Health as a major field of endeavour internationally, led to the publication of the Urban Health Bulletin, the Healthy Cities movement and an increasing international collaboration of the School with partners in Europe, Africa, Latin America and South East Asia.
The novel addition of the Division of Oral Public Health enriched the academic endeavour of the WSPH, bringing much needed attention to the field of health promotion.
As I reflect on the Wits School in the 1990s, I am struck by the confluence of events, internationally and nationally, which provided a window of opportunity for Public Health to play a significant role in the improvement of the human condition. I have no doubt that the new School of Public Health building will stand as a beacon of hope for the improvement of population health, especially for the marginalised, dispossessed, oppressed and disempowered, in South Africa and beyond.
Head of Department of Community Health (1993--2001) and Inaugural Head of the Wits School of Public Health (2001--2002)
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Ventilator associated pneumonia is the most common among hospital infections, especially in intensive care unit patients Trauma. With the concern to improve the quality provided in assistance to patients on mechanical ventilation, intensive care created a multidisciplinary group to develop, evaluate and implement the necessary measures to prevent ventilator-associated pneumonia.
Objectives
==========
Reduce the incidence of ventilator-associated pneumonia in the intensive care unit of trauma.
Methods
=======
This is a quantitative prospective study, conducted from January 2010 to December 2014 in a 10-bed intensive care unit of trauma high complexity hospital, at Sao Paulo, Brazil. In 2009 we implemented the multidisciplinary group: supervision of nursing, medical coordinator, intensivist and physiotherapist intensive care, pharmacy, nutrition and infection control. The adopted actions taken over the years were: Weekly discussion of cases with evaluation of the clinical condition of the patient, sedation interruption and weaning; protocol review as oral hygiene, threshold elevation; Review of materials and respiratory care equipment.
Results
=======
The year 2009 was the beginning of VAP prevention actions of the multidisciplinary group, with only 4 reviews, and no case of infection. In 2010, the first year of follow up were 785 assessments with 25.85% of the membership shares and 0 (zero) EPI density. In 2011-2014 were 1216, 680, 1096 and 814 reviews respectively, 85.5%, 88.5%, 89.5% and 88.6% compliance actions. In relation to EPI density enters these years were: 0.85; 2.81; 1.02 and 0 (zero) in the year 2014. In evaluating the past eighteen months (July 2013 to December 2014), we found that there was no case of pneumonia associated with mechanical ventilation, a fact that motivated the drive to celebrate importance of working together.
Conclusion
==========
The interdisciplinarity of the sectors involved in the activities highlighted the importance of teamwork in order to better results in processes and quality of care.
Disclosure of interest
======================
None declared.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Domestication of animals and plants has played a major role in human history. With the advance of high-throughput genotyping and sequencing technologies, the analysis of large datasets in domesticated species offers great opportunities to study genome evolution in response to phenotypic selection [@pone.0103813-Andersson1]. The sheep was one of the first grazing animals to be domesticated [@pone.0103813-Zeder1] in part due to its manageable size and an ability to adapt to different climates and diets with poor nutrition. A large variety of breeds with distinct morphology, coat color or specialized production (meat, milk or wool) were subsequently shaped by artificial selection. Since the release of the 50K SNP array [@pone.0103813-Kijas1], it is now possible to scan genetic diversity in sheep in order to detect loci that have been involved in these various adaptive selection events. The Sheep HapMap dataset, which includes 50K genotypes for 3000 animals from 74 breeds with diverse world-wide origins, provides a considerable resource for deciphering the genetic bases of phenotype diversification in sheep. In the first analysis of this dataset [@pone.0103813-Kijas2], the authors looked for selection by computing a global among the 74 breeds at all SNP in the genome. They identified 31 genome regions with extreme differentiation between breeds, which included candidate genes related to coat pigmentation, skeletal morphology, body size, growth, and reproduction. Further studies took advantage of the Sheep HapMap resource to detect genetic variants associated with pigmentation [@pone.0103813-GarciaGmez1], fat deposition [@pone.0103813-Moradi1], or microphtalmia disease [@pone.0103813-Becker1]. An other study [@pone.0103813-Zhang1] performed a genome scan for selection focused on American synthetic breeds, using an approach similar to that in [@pone.0103813-Kijas2].
The 74 breeds of the Sheep HapMap dataset have a strong hierarchical structure, with at least 3 distinct differentiation levels: an inter-continental level (e.g. European breeds vs Asian breeds), an intra-continental level (e.g. Texel vs Suffolk European breeds), and an intra-breed level (e.g. German Texel vs Scottish Texel flocks). Recent studies [@pone.0103813-Excoffer1]--[@pone.0103813-Gnther1] showed that, when applied to hierarchically structured data sets, based genome scans for selection may lead to a large proportion of false positives (neutral loci wrongly detected as under selection) and false negatives (undetected loci under selection). Besides, the heterogeneity of effective population size among breeds implies that some breeds are more prone to contribute large locus-specific values than others [@pone.0103813-Bonhomme1]. Apart from these statistical considerations, merging populations with various degrees of shared ancestry can limit our understanding of the selective process at detected loci. Indeed, the regions pointed out in [@pone.0103813-Kijas2] can be related to either ancient selection, as the poll locus which has likely been under selection for thousands of years, or fairly recent selection, as the myostatin locus which has been specifically selected in the Texel breed. But in most situations the time scale of adaptation cannot be easily determined.
Another limit of genome scans for selection based on single SNP computations is that they do not sufficiently account for the very rich linkage disequilibrium information, even when the single SNP statistics are combined into windowed statistics. Recently, we proposed a new strategy to evaluate the haplotype differentiation between populations [@pone.0103813-Fariello1]. We showed that using this approach greatly increases the detection power of selective sweeps from SNP chip data, and also enables to detect soft or incomplete sweeps. These latter selection scenarios are particularly relevant in breeding populations, where selection objectives have likely varied along time and where the traits under selection are often polygenic.
In this study we provide a new genome scan for selection based on the Sheep HapMap dataset, where we distinguish selective sweeps within and between 7 broad geographical groups. The within group analysis aims at detecting recent selection events related to the diversification of modern breeds. It is based on the single marker FLK test [@pone.0103813-Bonhomme1] and on its haplotypic extension hapFLK [@pone.0103813-Fariello1]. The FLK test is an extension of the Lewontin and Krakauer (LK) test [@pone.0103813-Lewontin1] that accounts for population size heterogeneity and for the hierarchical structure between populations. As the LK test, the FLK test computes a global for each SNP, but allele frequencies are first rescaled using a population kinship matrix . This matrix, which is estimated from the observed genome wide data, measures the amount of genetic drift that can be expected, under neutral evolution, along all branches of the population tree. With this rescaling, allele frequency differences are typically down-weighted if they are obtained with small populations, or populations that diverged a long time ago. The between group analysis focuses on older selection events and is only based on FLK. Overall, we confirmed 19 of the 31 sweeps discovered in [@pone.0103813-Kijas2], while providing more details about the past selection process at these loci. We also identified 71 new selection signatures, with candidate genes related to coloration, morphology or production traits.
Results and Discussion {#s2}
======================
We detected selection signatures using methods that aim at identifying regions of outstanding genetic differentiation between populations, based either on single SNP, FLK [@pone.0103813-Bonhomme1], or haplotype, hapFLK [@pone.0103813-Fariello1], information. These methods have optimal power when working on closely related populations so we separately analyzed seven groups of breeds, previously identified as sharing recent common ancestry [@pone.0103813-Kijas2] and corresponding to geographical origins of breeds. Before performing genome scans for selection signatures, we studied the population structure of each group to identify outlier animals as well as admixed and strongly bottlenecked populations, using both PCA and model-based approaches [@pone.0103813-Pritchard1], [@pone.0103813-Alexander1]. hapFLK was found to be robust to bottlenecks or moderate levels of admixture, but these phenomena may affect the detection power so we preferred to minimize their influence by removing suspect animals or populations. Details of these corrections are provided in the methods section. The final composition of population groups are given in [Table 1](#pone-0103813-t001){ref-type="table"}.
10.1371/journal.pone.0103813.t001
###### Population groups from the Sheep HapMap dataset used for the detection of selection signatures.
{#pone-0103813-t001-1}
Group Abbreviation Size Populations (Abbreviations)
------------------- -------------- ------ ----------------------------------------------------------
Africa AFR 2 Red Maasai (RMA)
Ethiopian Menz (EMZ)
Asia ASI 8 Bangladeshi BGE (BGE)
Bangladeshi Garole (BGA)
Changthangi (CHA)
Deccani (IDC)
Garut (GUR)
Indian Garole (GAR)
Sumatra (SUM)
Tibetan (TIB)
Central Europe CEU 4 Bundner Oberlander (BOS)
Engadine Red (ERS)
Valais Blacknose (VBS)
Valais Red (VRS)
Italy ITA 4 Altamurana (ALT)
Comisana (COM)
Leccese (LEC)
Sardinian Ancestral Black (SAB)
Northern Europe NEU 6 Galway (GAL)
German (GTX), New Zealand (NTX) and Scottish (STX) Texel
Irish Suffolk (ISF)
New Zealand Romney (NZR)
South West Asia SWA 4 Afshari (AFS)
Moghani (MOG)
Norduz (NDZ)
Qezel (QEZ)
South West Europe SWE 4 Autralian Merino (MER)
Churra (CHU)
Meat (LAM) and Milk (LAC) Lacaune
Overview of selected regions {#s2a}
----------------------------
An overview of selection signatures on the genome across the different groups is plotted in [Figure 1](#pone-0103813-g001){ref-type="fig"} and a detailed description is provided in [Table 2](#pone-0103813-t002){ref-type="table"}. Detected regions were typically a few megabases long and included from 1 to 196 genes, with a median of 15 genes. However, in many regions strong functional candidate genes were found very close to the position with lowest p-value, typically among the two closest genes from this position. These genes are reported in [Table 2](#pone-0103813-t002){ref-type="table"}, as well as a few other functional candidates with less statistical evidence but strong prior knowledge from the literature. We found 41 selection signatures with hapFLK and 26 with FLK, although we allowed a slightly higher false discovery rate for FLK than hapFLK (10% vs 5%). This result was consistent with a higher power for hapFLK than FLK, as already shown in [@pone.0103813-Fariello1].
{#pone-0103813-g001}
10.1371/journal.pone.0103813.t002
###### Selection signatures in the 7 geographical groups.
{#pone-0103813-t002-2}
OAR Begin (Mbp) End (Mbp) P-value Q-value Group Test Diff. pop. Cand. gene Nr. genes Rank
----- ------------- ----------- --------- --------- ------- -------- -------------------- -------------- ----------- ------
2 46.65 57.99 6.3e-10 7.1e-07 ITA hapFLK COM NPR2† 85 15
2 51.41 53.44 4.1e-09 1.6e-04 ITA FLK COM 41 2
2 74.00 74.86 7.4e-04 3.7e-02 ITA hapFLK COM 7
2 81.27 87.32 4.1e-09 2.3e-06 ITA hapFLK COM BNC2 18 1
2 110.08 112.08 1.5e-05 6.7e-02 ASI FLK SUM TIB GUR 11
2 113.36 122.24 7.0e-06 3.3e-03 NEU hapFLK GTX NTX STX MSTN† 42 8
2 239.76 241.76 2.9e-05 9.3e-02 SWA FLK AFS RUNX3 33 1
3 84.40 86.40 2.5e-05 9.1e-02 ASI FLK 15
3 120.91 125.49 5.3e-04 3.0e-02 ITA hapFLK COM KITLG 5 5
3 122.07 130.85 6.8e-08 4.2e-04 AFR hapFLK 25 1
3 151.42 156.93 3.3e-16 3.1e-12 ITA hapFLK COM SAB HMGA2† 26 1
3 154.79 154.93 5.9e-04 4.3e-02 AFR hapFLK 12 12
3 159.64 161.60 6.1e-04 3.3e-02 ITA hapFLK COM 6
3 167.85 171.67 1.5e-04 1.3e-02 ITA hapFLK COM ALT SAB 27
4 4.61 6.61 5.3e-06 2.1e-02 SWA FLK MOG 8
4 8.50 19.66 4.2e-06 1.1e-03 CEU hapFLK VBS VRS 49
4 15.11 17.11 8.4e-07 1.5e-02 CEU FLK VBS 7
4 26.46 28.46 2.4e-05 9.1e-02 ASI FLK GUR IDC SUM HDAC9 6 1
4 44.49 45.76 2.7e-04 3.4e-02 NEU hapFLK NZR 12
4 45.57 47.57 1.8e-06 2.4e-02 ASI FLK SUM 8
4 67.75 69.80 3.5e-07 2.3e-03 SWA FLK MOG HOXA 18 2→10
5 29.40 31.40 1.1e-05 6.7e-02 ASI FLK GAR 3
5 47.35 49.35 1.4e-05 6.7e-02 ASI FLK BGA 35
5 78.16 78.76 4.2e-04 4.2e-02 NEU hapFLK NZT 16
6 5.62 7.62 3.1e-06 6.0e-02 ITA FLK SAB 11
6 33.22 41.02 3.4e-08 8.0e-05 SWE hapFLK LAC LAM ABCG2†/NCAPG 27 2/17
6 34.71 39.12 1.6e-07 4.1e-05 ITA hapFLK COM 23 11/2
6 35.94 38.31 2.1e-04 1.9e-02 CEU hapFLK VRS VBS 19 9/17
6 67.98 70.36 4.3e-06 1.1e-03 CEU hapFLK VBS KIT† 9 5
6 68.90 70.95 9.6e-07 5.3e-03 SWA FLK 10 2
6 93.30 94.39 3.8e-04 2.7e-02 CEU hapFLK VRS&VBS or ERS&BOS FGF5† 8 5
7 49.15 51.15 1.1e-05 9.7e-02 CEU FLK VRS 7
7 78.31 80.31 8.1e-07 1.5e-02 CEU FLK VRS ERS 13
8 23.97 25.97 2.9e-05 9.6e-02 ASI FLK TIB 6
9 29.46 31.55 3.7e-04 3.4e-02 SWE hapFLK CHU MER 1
9 37.79 46.03 1.9e-05 6.2e-03 NEU hapFLK NZT ISF 6
10 24.02 34.91 1.4e-14 1.1e-10 CEU hapFLK BOS ERS VRS RXFP2† 68 9
10 29.42 29.71 9.6e-04 4.4e-02 ITA hapFLK COM ALT 14 2
10 28.50 30.50 6.3e-06 7.5e-02 CEU FLK BOS ERS 14 1
10 28.50 30.50 3.2e-05 9.7e-02 SWA FLK NDZ 14 1
10 28.50 30.50 1.3e-06 5.4e-02 SWE FLK MER 14 1
10 48.90 49.59 5.2e-04 3.1e-02 CEU hapFLK 3
11 12.55 14.12 1.4e-04 2.2e-02 NEU hapFLK 33
11 24.18 38.74 9.8e-09 8.0e-05 SWE hapFLK LAC MER 296
11 40.31 46.70 3.3e-06 5.5e-04 ITA hapFLK SAB 164
12 42.66 44.66 3.4e-07 7.6e-03 ASI FLK SUM 10
13 33.10 40.02 5.7e-06 1.8e-03 AFR hapFLK 41
13 40.60 50.30 4.9e-07 4.9e-04 AFR hapFLK BMP2† 76 1
13 43.34 51.28 2.7e-07 1.7e-04 SWE hapFLK LAC LAM PRNP 49 8
13 56.11 57.17 2.5e-08 4.8e-04 SWA hapFLK MOG EDN3 19 1
13 55.33 57.43 8.4e-11 1.1e-06 SWA FLK MOG 19 1
14 6.37 13.60 1.6e-04 1.4e-02 ITA hapFLK SAB 70
14 13.64 13.70 5.3e-04 4.9e-02 NEU hapFLK ISF MC1R 48 33
14 13.70 16.46 1.2e-04 1.1e-02 ITA hapFLK SAB 37 21
14 45.49 50.09 1.6e-04 2.5e-02 NEU hapFLK NTX NZR 117
15 48.87 50.87 1.5e-05 6.7e-02 ASI FLK GAR IDC 36
15 71.71 73.71 3.8e-06 1.6e-02 SWA FLK MOG ALX4/EXT2 13 1/3
16 33.20 35.10 1.8e-04 1.8e-02 AFR hapFLK C6/C7 8 5/7
16 63.97 65.97 1.1e-05 6.7e-02 ASI FLK GAR IDC 5
19 4.42 7.43 2.2e-04 1.9e-02 CEU hapFLK VRS BOS GLB1† 17 14
19 30.42 35.09 3.2e-05 4.2e-03 CEU hapFLK VBS BOS ERS MITF† 14 9
19 44.60 46.60 3.9e-06 3.9e-02 ASI FLK GAR BGA WNT5A 4 1
20 36.74 38.52 2.8e-04 2.3e-02 CEU hapFLK VRS 10
22 18.90 24.36 1.5e-11 7.4e-08 NEU hapFLK GTX PITX3^‡^ 85 5
23 42.50 46.96 2.2e-05 5.4e-03 AFR hapFLK MC2R/MC5R 35 1/2
23 54.14 56.14 3.8e-07 7.6e-03 ASI FLK GAR 5
25 0.08 3.08 3.7e-04 2.4e-02 ITA hapFLK SAB 16
Regions identified with the hapFLK or FLK test, with the corresponding population group and most differentiated populations (except for the AFR group). Full names of groups and populations are given in [Table 1](#pone-0103813-t001){ref-type="table"}. The number of genes included in each region and the rank of candidate genes within the region is also provided. Overlapping regions detected in different population groups or with different tests are grouped within horizontal lines (in this case candidate genes are the same for all overlapping regions and are only reported in the first one). : signatures of selection previously identified [@pone.0103813-Kijas2]. : this outlying region is not due to evolutionary processes (see details in the main text).
Four regions were found with both the single SNP and the haplotype test and harbor strong candidate genes: NPR2, KIT, RXFP2 and EDN3 ([Table 2](#pone-0103813-t002){ref-type="table"}). The overlap was thus small, illustrating that the two tests tend to capture different signals. In particular, hapFLK will fail to detect ancient selective sweeps, for which the mutation-carrying haplotype is small and not associated with many SNP on the chip. On the contrary, single SNP tests will fail to capture selective sweeps when a single SNP is not in high LD with the causal mutation. They will also fail if the selected mutation is only at intermediate frequency but is associated to a long haplotype, in contrast with hapFLK.
Six regions were detected in more than one group of breeds. They all contained strong candidate genes ([Table 2](#pone-0103813-t002){ref-type="table"}). Three of these genes are related to coat color (KIT, KITLG and MC1R), and could correspond to independent selection events (see [discussion](#s2){ref-type="sec"} below). One region harbors a gene (RXFP2) for which polymorphisms have been shown to affect horn size and polledness in the Soay [@pone.0103813-Johnston1] and Australian Merino [@pone.0103813-Dominik1]. We detected this region in 4 different groups and in all of them the highest FLK value was found to be very close to RXFP2 (Figure S8 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}). This provides clear indication that selection in this region is related to RXFP2, consistent with previous selection signatures detected by comparing specifically horned and polled breeds (Figure 6 in [@pone.0103813-Kijas2]). However, we note that the signatures of selection in this region exhibit different patterns among groups. The signal is very narrow in the SWE and SWA groups, and is in fact not detected by the hapFLK test, whereas it affects a large genome region in the CEU group where it is detected by hapFLK. In the ITA group, the FLK statistics do not reach significance, and the hapFLK signal is not high (minimum q-value of 0.04). Overall, the selection signatures suggest that selection on RXFP2, most likely due to selection on horn phenotypes, was carried out worldwide at different times and intensities. Another region harbors the HMGA2 gene, involved in selection for stature in dogs [@pone.0103813-Akey1] and associated to body size in horses [@pone.0103813-MakvandiNejad1] and height in humans [@pone.0103813-Yang1]. The last region includes two interesting candidate genes: ABCG2, which has been associated to a strong QTL for milk production in cattle [@pone.0103813-CohenZinder1], and NCAPG, which has been associated to fetal growth [@pone.0103813-Eberlein1] and calving ease [@pone.0103813-Bongiorni1] in cattle and which is located in several selection signatures in this species [@pone.0103813-Mancini1]--[@pone.0103813-Druet1]. In our analysis, populations with a selection signature in this region belong to three European groups (SWE, ITA and CEU) and our results suggest that selection in these different groups might imply distinct genes ([Table 2](#pone-0103813-t002){ref-type="table"}).
In the paper presenting the Sheep HapMap dataset [@pone.0103813-Kijas2], 31 selection signatures were found, corresponding to the 0.1% highest single SNP . Using FLK and hapFLK, we confirmed signatures of selection for 10 of these regions. Considering the two analyses were performed on the same dataset, this overlap can be considered as rather small. Two reasons can explain this.
First, the previous analysis was based on the statistic. Although this statistic is commonly used for selection scans, it is prone to produce false positives when the population tree harbors unequal branch lengths (*i.e.* unequal effective population sizes) [@pone.0103813-Bonhomme1]. In particular, strongly bottlenecked breeds will contribute high values preferentially even under neutral evolution, because their smaller effective population size implies a larger variance of allele frequencies. With and , values between populations are rescaled using branch lengths, so populations with long branch lengths will not contribute more than others [@pone.0103813-Fariello1]. In fact they will tend to contribute less, as the statistical power to distinguish selective effects from drift effects is naturally lower in populations where drift is larger.
Second, the previous analysis was performed using all breeds at the same time. It is therefore possible that some of these regions correspond to differentiation between groups of breeds rather than within groups. To investigate this question, we performed a genome scan for selection between seven virtual populations corresponding to the ancestors of the seven population groups. Allele frequencies in each of these ancestral populations were estimated from those observed in modern breeds and regions with outlying genetic differentiation between ancestral populations were detected using the FLK statistic [@pone.0103813-Bonhomme1]. For this analysis, we did not include SNP lying in regions detected within groups since selection biases their estimated ancestral allele frequencies. The ancestral population tree was reconstructed using SNP for which we have unambiguous ancestral allele information (Figure S9 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}). This tree is decomposed into two main lineages, one for European breeds and one for Asian and African breeds. The African group exhibits a slightly higher branch length. We note, however, that this could be due to ascertainment bias of SNP on the SNP array.
This led to the identification of 23 new selection signatures ([Figure 2](#pone-0103813-g002){ref-type="fig"} and [Table 3](#pone-0103813-t003){ref-type="table"}), 9 of them being common to the analysis of [@pone.0103813-Kijas2]. Overall, combining the scans for recent and ancestral selection, we failed to replicate 12 of the regions in [@pone.0103813-Kijas2].
{#pone-0103813-g002}
10.1371/journal.pone.0103813.t003
###### Selection signatures in ancestral populations.
{#pone-0103813-t003-3}
Estimated ancestral allele frequencies
---- ----------- ---------------------------------------- ------ ------ ------ ------ ------ ------ --------- --------- ---------- ----- ----
1 7192190 0.15 0.08 0.16 0.55 0.69 0.04 0.38 1.7e-06 5.3e-03 TRPM8 19 8
1 237070498 0.87 0.95 0.91 0.48 0.24 0.77 0.35 1.4e-05 2.5e-02 GYG1 16 5
1 239424807 0.46 0.68 0.06 0.21 0.15 0.11 0.17 3.4e-05 4.8e-02 9
1 239491620 0.53 0.41 0.94 0.86 0.93 0.93 0.88 4.3e-05 5.6e-02 9
2 45500785 0.43 0.91 0.23 0.76 0.87 0.87 0.93 2.2e-06 6.4e-03 LPL 6 3
2 182607165 0.99 0.97 0.18 0.64 0.73 0.83 0.64 3.4e-08 1.8e-04 INSIG2 10 3
2 182672296 0.99 0.94 0.32 0.90 0.86 0.89 0.81 7.7e-07 2.8e-03 10
2 192231314 0.59 0.93 0.36 0.96 0.89 0.81 0.95 1.6e-05 2.8e-02 8
3 132478420 0.24 0.89 0.18 0.93 0.81 0.84 0.82 1.2e-06 3.9e-03 HOXC 54 19
3 180860403 0.71 0.53 0.28 0.82 0.31 0.12 0.13 1.7e-05 2.8e-02 22
5 15522700 0.68 0.63 0.92 0.27 0.76 0.99 0.78 9.8e-06 2.0e-02 51
7 89519883 0.63 0.61 0.19 0.89 0.18 0.60 0.95 6.1e-10 5.2e-06 TSHR 6 3
8 31748642 0.84 0.93 0.94 0.16 0.63 0.47 0.19 2.8e-05 4.1e-02 PREP 6 1
11 18248852 0.35 0.32 0.82 0.64 0.94 0.96 0.92 1.3e-05 2.5e-02 NF1 23 1
11 18325488 0.87 0.93 0.00 0.35 0.04 0.03 0.04 3.3e-16 7.2e-12 24 4
11 18335747 0.87 0.93 0.00 0.35 0.04 0.03 0.04 3.3e-16 7.2e-12 22 4
11 18433474 0.87 0.93 0.02 0.35 0.07 0.02 0.05 3.8e-15 5.4e-11 22 1
11 18440783 0.78 0.93 0.02 0.34 0.07 0.02 0.05 2.0e-14 2.2e-10 22 1
11 25704651 0.97 0.96 0.97 0.42 0.94 0.94 0.96 8.5e-06 1.9e-02 73
11 26284826 0.99 0.97 0.94 0.38 0.93 0.95 0.79 3.2e-05 4.6e-02 100
11 26571629 0.92 0.94 0.98 0.29 0.89 0.88 0.86 1.8e-05 2.8e-02 115
11 26872280 0.78 0.71 0.93 0.15 0.89 0.90 0.90 2.2e-07 9.5e-04 111
13 12120674 0.29 0.84 0.97 0.91 0.97 0.92 0.84 7.7e-06 1.8e-02 GATA3 6 1
13 62857560 0.52 0.62 0.65 0.98 0.67 0.92 0.36 3.6e-06 9.7e-03 ASIP 32 12
15 3706790 0.71 0.22 0.96 0.28 0.27 0.34 0.21 6.8e-06 1.7e-02 4
15 29856310 0.98 0.99 0.99 0.47 0.92 0.95 0.96 9.8e-06 2.0e-02 35
16 38696505 0.95 0.98 0.95 0.99 0.68 0.31 0.30 6.8e-07 2.7e-03 PRLR 18 2
17 4867509 0.91 0.95 0.85 0.54 0.18 0.58 0.17 1.8e-05 2.8e-02 TMEM154 9 1
18 19342316 0.90 0.79 0.67 0.35 0.75 0.10 0.09 1.9e-07 9.3e-04 ACAN 31 4
18 66470371 0.99 0.97 0.90 0.90 0.18 0.04 0.08 1.9e-09 1.3e-05 TRAF3 28 5
20 17381047 0.24 0.61 0.97 0.98 0.93 0.99 0.91 3.1e-08 1.8e-04 VEGFA 48 1
25 7517270 0.95 0.94 0.93 0.14 0.27 0.57 0.19 1.8e-05 2.8e-02 wool QTL 13
SNP with significant FLK value at the 5% FDR level, with estimated allele frequencies in all ancestral groups. The number of genes included in each region (1Mb up-or-downstream the position) and the rank of candidate genes within the region is also provided. : signatures of selection previously identified [@pone.0103813-Kijas2].
Selection Signatures within population groups {#s2b}
---------------------------------------------
### Coloration {#s2b1}
Many selection signatures are located around genes that have been shown to be involved in hair, eye or skin color. In particular, several detected regions include candidate genes that are involved in the development and migration of melanocytes and in pigmentation: EDN3, KIT, KITLG, MC1R and MITF. For all these genes except MITF, we have quite strong evidence that they are the genes targeted by selection in the detected region. In the SWA group, EDN3 was included in the detected region for both FLK and hapFLK, and in both cases it was the closest gene to the highest test value. KIT and KITLG were both included in a detected region (with relatively few genes) for two different geographical groups, and were very close to the position with the smallest p-value in one of those. MC1R was also in a detected region for two different groups, NEU and ITA. In the two cases it was not very close to the maximum of the signal, but we note that the black skin or coat color is an important characteristic of the two populations that have been found under selection in this region, the Irish Suffolk and Sardinian Ancestral Black. This observation, together with the fact that MC1R mutations are responsible for coat color patterns in mammals (*e.g* in cattle [@pone.0103813-Klungland1]), supports the hypothesis that MC1R is a good candidate for the signatures we observed.
Although not listed in [Table 2](#pone-0103813-t002){ref-type="table"}, SOX10 and ASIP, two other genes implied in pigmentation, also show some evidence of selection. In the ITA group, the q-value of hapFLK near SOX10 is 6.2% and almost reaches the significance threshold of 5%. Similarly, the two closest SNP to ASIP (*s66432* and *s12884*) present suggestive FLK p-values of respectively and in the ASI group, and one (*s12884*) is significantly differentiated between the ancestral groups. All these genes have previously been reported as being likely selection targets and/or associated to color patterns in different mammalian species. Finally, we found a signal for selection centered on the BNC2 gene, that has recently been associated with skin pigmentation in humans [@pone.0103813-Jacobs1]. All population groups present at least one selection signature which is very likely related to one of the above genes, reflecting the widespread importance of color patterns to define sheep breeds.
Inferring a precise history of underlying causal mutations for color patterns in this dataset is hard for several reasons: the precise phenotypic characterizations of coat color patterns in the Sheep HapMap breeds are not available; the 50K SNP array used does not offer sufficient density to associate a given selection signature to a specific set of polymorphisms; Finally, from the literature it appears that a large number of genes and mutations can be considered a priori as potentially causal for a given pigmentation pattern. In particular, mutations in different genes can give rise to the same phenotype (*e.g.* in horses [@pone.0103813-Hauswirth1]). Also, within a gene different mutations can give rise to different phenotypes, *e.g* mutations in the MC1R gene (also named the extension locus) have been associated to a large panel of skin or coat colors [@pone.0103813-Klungland1], [@pone.0103813-Lin1], [@pone.0103813-Joerg1]. Deciphering selection signatures related to coat color in sheep and in particular identifying the causal variants under selection will require sequencing these genes for individuals from several breeds with diverging color patterns. This in turn will help to understand the evolutionary history of the breeds and the effect of selection [@pone.0103813-Linnen1]. To potentially help in this task, in Table S1 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"} we list, for each "color gene", the populations that have likely been selected for.
### Morphology {#s2b2}
Another group of genes that are found within selection signatures have known effects on body morphology and development. NPR2, HMGA2 and BMP2, pointed out previously [@pone.0103813-Kijas2] are confirmed as good positional candidates by our study. We also found strong evidence for selection on WNT5A, ALX4 or EXT2, and two HOX gene clusters (HOXA and HOXC). WNT5A and ALX4 are two genes involved in the development of the limbs and skeleton. Mutations in WNT5A are causing the dominant Human Robinow syndrome, characterized by short stature, limb shortening, genital hypoplasia and craniofacial abnormalities [@pone.0103813-Person1]. ALX4 loss of function mutations cause polydactily in the mouse, through disregulation of the sonic hedgehog (SHH) signaling factor [@pone.0103813-Kuijper1], [@pone.0103813-Qu1]. Moreover, the ALX4 protein has been shown to bind proteins from the HOXA (HOXA11 and HOXA3) and HOXC (HOXC4 and HOXC5) clusters [@pone.0103813-Ravasi1]. Located just besides ALX4 and corresponding to the same selection signature, EXT2 is responsible for the development of exostose in the mouse [@pone.0103813-Stickens1]. *HOX* genes are responsible for antero-posterior development and skeletal morphology along the anterior-posterior axis in vertebrates. The selection signature around HOXA is a recent selection signature in the SWA group, while that around HOXC is an ancestral signature with a high differentiation of the ASI ancestor compared to AFR and SWA ([Table 3](#pone-0103813-t003){ref-type="table"}).
Finally, we note that an ancestral selection signature is found near the ACAN gene, whose expression was shown to be upregulated by BMP2 [@pone.0103813-Noguchi1], another candidate gene for selection. Three genes within the selection signature are found closer to the maximum test value than ACAN, but these are in silico predicted genes, whose protein coding function has not been confirmed, so ACAN seems to be overall a better candidate for explaining selection in the region. Mutations in the ACAN gene have been shown to induce osteochondrosis [@pone.0103813-Stattin1] and skeletal dysplasia [@pone.0103813-Tompson1]. The ACAN region has also been shown to be associated with height in humans [@pone.0103813-Weedon1].
### Traits of agronomic importance {#s2b3}
Sheeps have been raised for meat, milk and wool production. Under selection signatures, we found several genes associated with these production traits. In addition to the selection signature in Texels on the MSTN gene for increased muscularity [@pone.0103813-Clop1], discussed in [@pone.0103813-Fariello1], we detected a selection signature centered on HDAC9 and including few other genes, which could also be linked to muscling. HDAC9 is a known transcriptional repressor of myogenesis. Its expression has been shown to be affected by the callypige mutation in the sheep at the DLK1-DIO3 locus [@pone.0103813-FlemingWaddell1]. The signature around HDAC9 corresponds to a selection signature in the Garut breed from Indonesia, a breed used in ram fights. As already discussed, one selection signature contains ABCG2, a gene underlying a QTL with large effects on milk production (yield and composition) in cattle [@pone.0103813-CohenZinder1]. Also, one of the ancestral selection signatures reaches its maximum value close to the INSIG2 gene, recently shown to be associated with milk fatty acid composition in Holstein cattle [@pone.0103813-Rincon1]. Two selection signatures could be related to wool characteristics, one in the CEU group including the FGF5 gene, partly responsible for hair type in the domestic dog [@pone.0103813-Housley1], [@pone.0103813-Cadieu1], and an ancestral selection signature on chromosome 25 in a QTL region associated to wool quality traits in the sheep [@pone.0103813-Ponz1], [@pone.0103813-Bidinost1].
One of the strong outlying regions in the selection scan contains the PITX3 gene. Further analysis revealed that this signature was due to the German Texel population haplotype diversity differing from the other Texel samples (results not shown). It turns out that the German Texel sample consisted of a case/control study for microphtalmia [@pone.0103813-Becker1], although the case/control status information in this sample is not given in the Sheep HapMap dataset. The consequence of such a recruitment is to bias haplotype frequencies in the region associated with the disease, which provokes a very strong differentiation signal between the German Texel and the other Texel populations. Although not related to artificial or natural selection in sheep, this signature illustrates that our method for detecting selection has the potential to identify causal variants in case/control studies, while using haplotype information.
Ancestral signatures of selection {#s2c}
---------------------------------
For ancestral selection signatures, i.e. the regions showing outlying genetic differentiation between population groups, it is difficult to estimate how far back in time selection occurred. In particular, it would be interesting to place the divergences shown by the ancestral population tree with respect to sheep domestication. Two interesting candidate genes for ancestral selection signatures might indicate that the selection signatures captured could be rather old. First, we found selection near the TRPM8 gene, which has been shown to be a major determinant of cold perception in the mouse [@pone.0103813-Bautista1]. The pattern of allele frequency at the significant SNP ([Table 3](#pone-0103813-t003){ref-type="table"}) is consistent with the climate in the geographical origins of the population groups. AFR, ASI and ITA, living in warm climates, have low frequency (0.04--0.16) of the A allele, while NEU and CEU, from colder regions, have higher frequencies (0.55--0.7), the SWE group having an intermediate frequency of 0.38. Overall, this selection signature might be due to an adaptation to cold climate through selection on a TRPM8 variant. Another selection signature lies close to a potential chicken domestication gene, TSHR [@pone.0103813-Rubin1], whose signaling regulates photoperiodic control of reproduction [@pone.0103813-Nakao1]. This selection signature was identified before [@pone.0103813-Kijas2] and our analysis indicates that selection happened before the divergence of breeds within geographic groups, consistent with an early selection event. Given its role, we can speculate that selection on the TSHR gene is related to seasonality of reproduction. Under temperate climates, sheep experience a reproductive cycle under photoperiodic control. Furthermore, there is evidence that this control was altered during domestication [@pone.0103813-Balasse1] so our analysis suggests genetic mutations in TSHR may have contributed to this alteration.
As discussed above, some of the genes found underlying ancestral selection signatures can be related to production or morphological traits (*e.g.* ASIP, INSIG2, ACAN, wool QTL), indicating that these traits have likely been important at the beginning of sheep history. The other genes that we could identify as likely selection targets in the ancestral population tree relate to immune response (GATA3) and in particular to antiviral response (TMEM154 [@pone.0103813-Heaton1], TRAF3 [@pone.0103813-Oganesyan1]). The most significant ancestral selection signature is centered around the NF1 gene, encoding neurofibromin. This gene is a negative regulator of the ras signal transduction pathway, therefore involved in cell proliferation and cancer, in particular neurofibromatosis. Due to this central role in intra-cellular signaling, mutations affecting this gene can have many phenotypic consequences so that its potential role in the adaptation of sheep breeds remains unclear.
Conclusions {#s3}
===========
The Sheep HapMap dataset is an exceptional resource for sheep genetics studies. In a population genomics context, our study shows that the rich information contained in these data permits to start unraveling the genetic history of sheep populations worldwide. In order to fully exploit this information, we used recent statistical approaches that account for the relationship between populations and the linkage disequilibrium patterns (haplotype diversity). This allowed detecting with confidence more selection signatures and identifying for most of them the selected populations. Among these new selection signatures detected by our study, several result from recent selection and include good positional candidate genes with functions related to pigmentation (KITLG, EDN3), morphology (WNT5A, ALX4, EXT2, HOXA cluster) or production traits (HDAC9). Two ancestral selection signatures are also of particular interest as they harbor genes (TRPM8 and TSHR) whose functions (cold and photoperiodic perception respectively) seem highly relevant to the selection response during the early history of sheep domestication.
With information on adaptive genome regions and selected populations, we hope that our work will foster new studies to unravel the underlying biological mechanisms involved. To this aim, it is likely that further phenotypic and genetic data are required. On the genetics side, even though the SNP array used in this study was sufficient to localize genome regions harboring adaptive mutations, its density and the SNP ascertainment bias resulting from its design did not allow to tag the causative mutation precisely. Elucidating the causal variation underlying selection signatures will thus most likely require large scale sequencing data.
Genome scans for selection, including this one, are identifying regions that are outliers from a statistical model and do not require to specify an alternative hypothesis based on phenotypic records. While this can be seen as an advantage for the initial localization of genome regions, it is a limitation for the identification of biological processes involved. Gathering phenotypic records in specific populations, in particular for color and morphology traits, will be needed to go further.
Methods {#s4}
=======
**Selecting populations and animals.** Seventy-four breeds are represented in the Sheep HapMap data set, but we only used a subset of these breeds in our genome scan. We removed the breeds with small sample size ( 20 animals), for which haplotype diversity cannot be determined with sufficient precision. Based on historical information, we also removed all breeds resulting from a recent admixture or having experienced a severe recent bottleneck. Focusing on the remaining breeds, we then studied the genetic structure within each population group, in order to detect further admixture events. We performed a standardized PCA of individual based genotype data and applied the admixture software [@pone.0103813-Alexander1].
In two population groups (AFR and NEU) the different breeds were clearly separated into distinct clusters of the PCA and showed no evidence of recent admixture (Figures S1 and S2 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}). These samples were left unchanged for the genome scan for selection. A similar pattern was observed in three other groups (ITA, SWA, ASI), except for a few outlier animals that had to be re-attributed to a different breed or simply removed (Figures S3, S4 and S5 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}). In the two last groups (CEU and SWE), several admixed breeds were found and were consequently removed from the genome scan analysis (Figures S6 and S7 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}).
We performed a genome scan within each group of populations listed in [Table 1](#pone-0103813-t001){ref-type="table"}, with a single SNP statistic FLK [@pone.0103813-Bonhomme1] and its haplotype version hapFLK [@pone.0103813-Fariello1].
**Population trees.** Both statistics require estimating the population tree, with a procedure described in details in [@pone.0103813-Bonhomme1]. Briefly, we built a population tree for each group by first calculating Reynolds\' distances between each population pair, and then applying the Neighbor Joining algorithm on the distance matrix. For each group, we rooted the tree using the Soay sheep as an outgroup. This breed has been isolated on an island for many generations and exhibits a very strong differentiation with all the breeds of the Sheep HapMap dataset, making it well suited to be used as an outgroup.
**FLK and hapFLK genome scans.** The FLK statistic was computed for each SNP within each group. The evolutionary model underlying the FLK statistic assumes that SNP were already polymorphic in the ancestral population. To consider only loci that most likely match this hypothesis, we restricted our analysis within each group to SNP for which estimated ancestral minor allele frequency was above 5%. Under neutrality, the FLK statistic should follow a distribution with degrees of freedom (DF), where is the number of populations in the group. Overall, the fit of the theoretical distribution to the observed distribution was very good (Text S1 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}) with the mean of the observed distribution () being very close to (Table S3 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}). Using as DF for the distribution provided a better fit to the observed data than the theoretical value. We thus computed FLK p-values using the distribution. To compute the hapFLK statistic, we used of the Scheet and Stephens LD model [@pone.0103813-Scheet1], a mixture model for haplotypes which requires specifying a number of haplotype clusters to be used. To choose this number, for each group, we used the fastPHASE cross-validation based estimation of the optimal number of clusters. The results of this estimation are given in Table S2 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}. The LD model was estimated on unphased genotype data. The hapFLK statistic is computed as an average over 20 runs of the EM algorithm to fit the LD model. As in [@pone.0103813-Fariello1], we found that the hapFLK distribution could be modeled relatively well with a normal distribution (corresponding to non outlying regions) and a few outliers; we used robust estimation of the mean and standard deviation of the hapFLK statistic to eliminate the influence of outlying (*i.e.* potentially selected) regions. This procedure was done within each group, the resulting mean and standard deviation values obtained are given in Table S2 in [File S1](#pone.0103813.s003){ref-type="supplementary-material"}. Finally, we computed at each SNP a p-value for the null hypothesis from the normal distribution.
**Selection in ancestral groups.** The within-group FLK analysis provides for each SNP an estimation of the allele frequency in the population ancestral to all populations of the group. We used this information to test SNP for selection using between group differentiation, with some adjustments. First, the FLK model assumes tested polymorphisms are present in the ancestral population. SNP for which the alternate allele has been seen in only one population group are likely to have appeared after divergence (within the ancestral tree) and were therefore removed from the analysis. Second, regions selected within groups affect allele frequency in some breeds and therefore bias our estimation of the ancestral allele frequency in this group. We therefore removed all SNP that were included in within-group selection signatures. Finally, the FLK test requires a rooted population tree. For the within group analysis, we could use a very distant population to the current breeds (the Soay sheep). For the ancestral tree, we created an outgroup homozygous for ancestral alleles at all SNP.
**Identifying selected regions and candidate genes.** We defined significant regions for each statistic and within each group of populations. Using the neutral distribution ( for FLK and Normal for hapFLK), we computed the p-value of each statistic at each SNP. To identify selected regions, we estimated their q-value [@pone.0103813-Storey1] to control the FDR. For FLK, SNP with a q-value below 0.1 were considered significant, which by definition implies that we expect 10% of false positives among our detected SNP. Since the power of hapFLK is greater than that of FLK [@pone.0103813-Fariello1], we used a q-value threshold of 0.05, therefore controlling FDR at the 5% level. For the FLK analysis in ancestral populations, we used an FDR threshold of 5%.
We then aimed at identifying genes that seem good candidates for explaining selection signatures. We proceeded differently for the single SNP FLK and hapFLK. For FLK, we considered that significant SNP less than 500Kb apart were capturing the same selection signal. Then, we considered as potential candidate genes any gene that lies less than 1Mb of any significant SNP. For hapFLK, the genome signal is much more continuous than single SNP tests, because the statistic captures multipoint LD with the selected mutations. A consequence is that the significant regions can span large chromosome intervals. To restrict the list of potential candidate genes, and target only the ones closest to the most significant SNP, we restricted our search to the part of the signal where the difference in hapFLK value with the most significant SNP was less than 0.5. This allowed taking into consideration the profile of the hapFLK signal, *i.e.* if the profile resembles a plateau, the candidate region will be rather broad while very sharp hapFLK peaks will provide a narrower candidate region. We extracted all protein coding genes present in the significant regions using the Ensembl Biomart tool (<http://www.ensembl.org/biomart/>) for Ovis Aries 3.1 genome assembly. These full lists are provided as Supporting Information ([Dataset S1](#pone.0103813.s001){ref-type="supplementary-material"} and [Dataset S2](#pone.0103813.s002){ref-type="supplementary-material"}). Within each candidate region, genes were ranked according to their distance from the most significant position of the region (the larger the rank, the larger the distance). The functional candidate genes shown in [Table 2](#pone-0103813-t002){ref-type="table"} and discussed in the manuscript were chosen based on this rank and/or on their implication in previous association or sweep detection studies.
Acknowledgments {#s5}
===============
Data analyses were performed on the computer cluster of the bioinformatics platform Toulouse Midi-Pyrenees. We would like to thank Wendy Brand-Williams for her careful reading of the manuscript.
The members of the International Sheep Genomics Consortium who contributed samples and expertise towards the design and execution of ovine SNP50k genotyping and next generation sequencing, and/or coauthors of [@pone.0103813-Kijas2] include: Juan Jose Arranz, Universidad de Leon; Georgios Banos, Aristotle University of Thessaloniki; William Barendse, CSIRO Livestock Industries; Ahmedn El Beltagy, Animal Production Research Institute; Jorn Benenwitz, University of Hohenheim; Steven Bishop, The Roslin Institute; Simon Boitard, INRA; Lutz Bunger, Scottish Agricultural College; Jorge Calvo, CITA; Antonello Carta, Agris Sardegna; Ibrahim Cemal, Adnan Menderes University; Elena Ciani, University of Bari; Noelle Cockett, University of Utah; Dave Coltman, University of Alberta; Brian Dalrymple, CSIRO Livestock Industries; Mariasilvia DAndrea, Universit degli Studi del Molise; Ottmar Distl, University of Veterinary Medicine Hannover; Cord Drogemuller, Institute of Genetics, University of Berne; Georg Erhardt, Institut fr Tierzucht und Haustiergenetik Justus-Liebig-Universitt Gieen; Emma Eythorsdottir, Agricultural University of Iceland; Kimberly Gietzen, Illumina Inc.; Clare Gill, Texas A& M University; Elisha Gootwine, The Volcani Center; Vidya Gupta, National Chemical Laboratory; Olivier Hanotte, University of Nottingham; Ben Hayes, Department of Primary Industries Victoria; Michael Heaton, USDA MARC; Stefan Hiendleder, University of Adelaide; Han Jialin, ILRI and CAAS; Juha Kantanen, MTT Agrifood Research; Matthew Kent, CiGene; James Kijas, CSIRO Livestock Industries; Denis Larkin, University of Aberystwyth; Johannes A. Lenstra, Utrecht University; Kui Li, Lhasa People Hospital, Tibet; Terry Longhurst, Meat and Livestock Australia; Runlin Ma, Chinese Academy of Science; Russell McCulloch, CSIRO Livestock Industries; David MacHugh, University College Dublin; Sean McWilliam, CSIRO Livestock Industries; John McEwan, AgResearch; Jillian Maddox, University of Melbourne; Massoud Malek, IAEA; Faruque Mdomar, Bangladesh Agriculture University; Despoina Miltiadou, Cyprus University of Technology; Luis V. Monteagudo Ibez, Universidad de Zaragoza; Carole Moreno, INRA; Frank Nicholas, University of Sydney; Kristen Nowak, University of Western Australia; V. Hutton Oddy, University of New England; Samuel Paiva, Embrapa; Varsha Pardeshi, National Chemical Laboratory; Josephine Pemberton, University of Edinburgh; Fabio Pilla, Universit degli Studi del Molise; Laercio R. Porto Neto, CSIRO Livestock Industries; Herman Raadsma, University of Sydney; Cyril Roberts, Caribbean Agricultural Research and Development Institute; Magali San Cristobal, INRA; Tiziana Sechi, Agris Sardegna; Paul Scheet, University of Texas M. D. Anderson Cancer Center; Bertrand Servin, INRA; Mohammad Shariflou, University of Sydney; Pradeepa Silva, University of Peradeniya; Henner Simianer, University of Goettingen; Jon Slate, University of Sheffield; Mikka Tapio, MTT; and Selina Vattathil, University of Texas M. D. Anderson Cancer Center; Vicki Whan, CSIRO Livestock Industries.
Supporting Information {#s6}
======================
######
**Genes within candidate regions for selection in the 7 geographical groups.**
(TXT)
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Click here for additional data file.
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**Genes within candidate regions for selection in ancestral populations.**
(TXT)
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Click here for additional data file.
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**Combined Supporting Information File including Figures S1 to S9, Tables S1 to S3 and Text S1.**
(PDF)
######
Click here for additional data file.
[^1]: **Competing Interests:**William Barendse (member of International Sheep Genomics Consortium) is a PLOS ONE Editorial Board member. This does not alter the authors\' adherence to PLOS ONE Editorial policies and criteria.
[^2]: Conceived and designed the experiments: MIF BS MSC SB. Analyzed the data: MIF BS GTK. Contributed reagents/materials/analysis tools: ISGC. Wrote the paper: MIF BS GTK RR CM MSC SB. Interpreted the results: MIF BS GTK RR CM MSC SB.
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Introduction {#s0001}
============
Canine distemper (CD) is a highly contagious, acute or subacute systemic viral disease that has a high mortality rate in dogs and wild canines. It is caused by canine distemper virus (CDV), which belongs to the genus *Morbillivirus* within the subfamily *Paramyxovirinae*.
Vaccination remains the principal strategy for protection, and once clinical signs develop, treatment is limited to supportive care. Even in settings where a diagnosis is rapidly reached, a high standard of care is available and high levels of vaccination are achieved, CD outbreaks continue to occur amongst domestic dogs (Bohm, Blixenkrone & Lund [@CIT0001]; Ek-Kommonen *et al*. [@CIT0002]; Ezeibe [@CIT0003]; Gard *et al*. [@CIT0004]; Gemma *et al*. [@CIT0005]; Maes *et al*. [@CIT0007]; Martella, Elia & Buonavoglia [@CIT0008]; Patronek *et al*. [@CIT0009]; Schumaker *et al*. [@CIT0010]; Willi *et al*. [@CIT0011]).
This report pertains to a severe suspected CDV outbreak in unvaccinated domestic dogs in Mozambique, owned mainly by low-income families, who receive no care from state or private veterinarians. This observational report is, to our knowledge, the only documentation of a CDV outbreak of this magnitude in Africa in this species.
Case presentation {#s0002}
=================
Cases of sick dogs mainly from low-income families were detected by State Veterinarians in Nampula City, in the northern region of Mozambique, during January and February of 2013. About 200 dogs of all ages died. Affected dogs had never been vaccinated against CDV and consisted of different breeds, with predominance of the Africanis breed. State veterinarians clinically examined 25 cases and described the following symptoms: anorexia, fever, weakness, nasal and ocular mucopurulent discharges, dyspnoea, coughing, pale mucous membranes, muscular trembling and/or clonus, diarrhoea, resulting in fatality within 7--14 days. Treatment with antibiotics was unsuccessful.
Three cases were submitted for post-mortem examination and samples with relevant lesions were collected for histopathology and immunohistochemistry. Post-mortem examinations were carried out following standard procedures. Tissue samples were preserved in 10% phosphate-buffered formaldehyde, embedded in paraffin wax, sectioned at 5 μm thickness and stained with haematoxylin-eosin for routine histopathological examination. Sections of brain, lung, heart, kidney and urinary bladder were immunolabelled for CDV antigen with a monoclonal mouse antibody against CDV (Agriculture Canada, Animal Diseases Research, Canada).
The most obvious gross post-mortal lesions were cachexia (3/3), mucopurulent exudate in the trachea and bronchi (2/3), focal pneumonia (3/3), cyanotic heart (2/3), splenic atrophy with increased consistency (2/3), pale kidneys (3/3) and contracted bladders with multifocal haemorrhages in the mucosa (2/3).
Histopathological examination of the lung, bronchi and bronchioli showed extensive areas of severe purulent inflammation (2/3). Splenic and lymph node tissues appeared moderately active to highly active (2/3). In the grey matter of the cerebellum, multifocal blood vessels showed notable leukostasis, predominantly with lymphocytes and a few macrophages, and there were also mononuclear infiltrates visible in the perivascular spaces in these areas. There was mild gliosis in the tissue. A few blood vessels in the meninges also showed perivascular cuffing with mononuclear leukocytes, predominately lymphocytes (2/3). A few multifocal aggregates of neutrophils were visible in the parenchyma of the liver, and a mild degree of leukostasis was evident in the sinusoids. The portal regions showed the presence of leukocytes (3/3). Focally extensive severe interstitial inflammation of a chronic lymphoplasmocytic appearance was seen in the prostate (3/3).
The history of the disease, the clinical signs and gross and histopathological presentations raised the probability of the presence of a CDV infection. To support this putative diagnosis, immunohistochemistry was performed, which confirmed the disease through positive immunoreactivity to CDV antigens in one of these three cases.
The incidence of CDV infections worldwide has decreased thanks to the introduction of the highly protective CDV-modified live virus (MLV) vaccines more than 60 years ago (Martella *et al*. [@CIT0008]). However, in regions with a low proportion of vaccinated dogs, the incidence of CDV epidemics is still high (Willi *et al*. [@CIT0011]). Vaccination against CDV in Mozambique is only available to people who can afford private veterinary care and therefore the herd immunity against this disease falls far short of the 80% required to prevent outbreaks (Horzinek [@CIT0006]).
This report illustrates the highly contagious nature of the disease and the threat to animal welfare posed by non-vaccination. Valuable wildlife would also potentially be threatened by this disease in dogs.
The authors are grateful to Professor Erik Gruys, Veterinary Faculty, Utrecht University, The Netherlands, for the critical reading of the manuscript.
Competing interests {#s20003}
===================
The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article.
Authors' contributions {#s20004}
======================
C.G.B. and J.Z. were responsible for literature review, manuscript writing, *post mortem* and histopathological examinations. A.D., S.A., P.T.D., E.M.L., A.M., B.M. and J.L.J. were in charge of epidemiological and clinical investigations during the outbreak.
**How to cite this article:** Zacarias, J., Dimande, A., Achá, S., Dias, P.T., Leonel, E.M., Messa, A. *et al*., 2016, 'Severe canine distemper outbreak in unvaccinated dogs in Mozambique', *Journal of the South African Veterinary Association* 87(1), a1350. <http://dx.doi.org/10.4102/jsava.v87i1.1350>
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Background {#s1}
==========
Fruits are an integral part of our daily diet because of their nutritional attributes. There are at least 500 species of edible tropical fruits in the Asian region \[[@R01]\]. Tropical fruits can be categorised into two groups namely major and minor. Durian fruit is grouped under minor fruits \[[@R02]\]. In Malaysia, the durian fruit is considered as the King of fruits. It is an iconic, expensive and seasonal fruit in many parts of Southeast Asian countries, especially in Malaysia, Thailand, Indonesia, and the Philippines. The name durian originally comes from the Malay word, \'Duri\' which means \'thorn\' of the fruits. The species name \'zibethinus\' is based on the name of large Indian civet (Viverra zibetha) well known for its musky smell \[[@R03]\]. The meaning of the word, \'civet\' is a valuable secretion from the civet cat\'s scent glands that produce a musk-like aroma \[[@R04]\]. \'Skunk of the orchard\' (Asian), \'civet fruit\' (India), \'Stinkfrucht\' (German) and \'Stinkvrucht\' (Dutch) are names for durian in respective geographical areas \[[@R05]\]. Mature durian trees are known to produce fruits only 5-7 years after the germination of their seeds ([Figure 1A](#F1){ref-type="fig"}). Each durian tree produces around 15-800 fruits in every fruiting season. The fruit weight is generally in the range of 1-3 kg with the diameter in the range of 14-18 cm, and the length could be in the range of 19-32 cm ([Figure 1B](#F1){ref-type="fig"}). The edible part (also called aril) is the flesh of durian fruits. Durian fruit is round or oblong, with spiky outer parts either light green or brownish ([Figure 1C](#F1){ref-type="fig"}). The fruit contains 3-5 longitudinal sutures that cover from its apical to basal end, and it is known to open after complete ripening of the fruit. If all dehiscence zones of a durian fruit are opened, it covers about 400cm2 area \[[@R05], [@R06]\]. The shape of the durian seeds ([Figure 1D](#F1){ref-type="fig"}) is just like chestnuts, and the length and diameter of the seeds range from 2-6 cm, and 2-3 cm, respectively and seeds are light brownish \[[@R03]\].
In general, durian fruits have a distinctive and robust aroma. The notorious smell (that resembles to rotten eggs or onions) is caused by volatile sulphur compounds (VSC) regulated by methionine gamma lyases (MGL). Because of its strong smell, durian fruit is banned in airports, hotels and there are restrictions on its transport and storage \[[@R07]\]. Various means and or alternatives are used to avoid these issues. Durian fruits are also used to make fruit juice, wines, and other products to eliminate the strong aroma so that it can be easily transported in the global market \[[@R08]\]. The main Asian producers of durian are Malaysia and Thailand \[[@R08], [@R09]\]. Indonesia, Philippines, and some other Asian countries also do cultivate the durian but in small scale and mostly for domestic uses only. The major importers of durian fruits are Taiwan, Hong Kong, and Singapore, whereas a total of 90% exports come from Malaysia, Thailand, and Indonesia \[[@R10]\]. This review article highlights the nutritional and medicinal attributes of durian and the molecular studies involving transcriptomics work and the insights of genome draft.
The nutritional worth of durian {#s1a}
-------------------------------
Durian pulp is an excellent source of various nutrients important in the human diet. Devalaraja et al. \[[@R11]\] have reported that the durian fruit pulp is a good source of nutrients as it contains proteins (1.47%), dietary fat (5.33%), fibers (3.1%) and carbohydrates (27%) \[[@R11]\]. It is also rich in vitamins and minerals such as vitamin C, folic acid, thiamin, riboflavin, niacin, B6, vitamin A, potassium, iron, calcium, magnesium, sodium, zinc and phosphorus \[[@R10]\]. The durian fruit pulp also contains linoleic acid (2.20%), myristic acid (2.52%), oleic acid (4.68%), 10- octadecenoic acid (4.86%), palmitoleic acid (9.50%), palmitic acid (32.91%), and stearic acid (35.93%)\[[@R12]\].
Durian plant varieties {#s1b}
----------------------
There are 15 varieties of durians registered under the Malaysian Department of Agriculture (DOA) \[[@R13]\]. The 15 varieties are D24, D99 (kob kecil), D123 (Chanee), D145 (Beserah), D158 (Kan yau), D159 (Mon Thong), D169, D168 (IOI, Ma Muar), D175 (Udang merah, An He), D197 (Raja Kunyit, Musang King), D198 (Kim Hong) and D199 (Bola 828). Another three hybrid durian clones that are produced using (MARDI) are named as MDUR78 (D188, Clone from D24 x D7), MDUR79 (D189, Clone from D24 x D7) and MDUR88 (D190, Clone from D24 x D7) \[[@R13]\]. The localities of varieties along with their respective date and year of registration are summarised in [Table 1](#T1){ref-type="table"}. In Thailand, the Thai Agricultural Standard (TAS 3-2013) has reported that there are seven commercial varieties of durian. These seven varieties are named as Chanee, Monthong, Karnyao, Kradoomthong, Puangmanee, Nualthongchan, and Longlublae \[[@R14]\]. Monthong and Chanee varieties are popular and widely cultivated on a commercial basis in Thailand.
Use of durian in traditional medicine {#s1c}
-------------------------------------
Traditionally, in Asia, the durian leaf and root decoctions have believed to show antipyretic effect and decoctions are used as a febrifuge and anti-malarial agent. It is also used to treat phlegm, relieve colds, and treat skin diseases, jaundice, and swellings. The durian fruit is believed to have warming properties on the body; however, it has not been clinically investigated \[[@R15]\]. Durian fruit is considered to have potential medicinal and therapeutic properties that include its ability to boost the immune system and wound healing \[[@R16]\]. It is reported that durian has anti-oxidant \[[@R17]\], anti-cancer, anti-cardiovascular, anti-diabetic \[[@R18]\] and antiobesity properties \[[@R19]\], and can improve digestion, cure insomnia, lower the blood pressure and relieve the symptoms of depression, anxiety, and stress disorders \[[@R02], [@R20]\]. It is also widely believed that durian pulp contains strong aphrodisiac properties and local community believes that consumption of fruits in conjunction with alcohol will lead to death. However, there is no evidence to support these claims \[[@R10]\]. Previous studies have also reported the potential use of durian fruit pulp as fertility enhancing agent and studies were conducted to find out its effectiveness to treat infertility in PCOS (polycystic ovarian syndrome) \[[@R21]\]. Although the fruit is effective against various components of metabolic syndrome, specific studies of the mechanism of ovulation and menstrual disturbances need to be conducted. Durian fruits have also shown anti-proliferative activities as being reported by Bhat and Paliyath \[[@R02]\].
Molecular markers and gene studies {#s1d}
----------------------------------
DNA markers are very useful in plant breeding programmes to develop new superior varieties with desirable traits. DNA (molecular) markers identified based on the random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), inter-simple sequence repeats (ISSR), and or simple sequence repeats (SSRs) can be used to analyse the genetic variability among varieties and species.
Random amplification of polymorphic DNA {#s1e}
---------------------------------------
The RAPD markers-based approach was used by Ruwaida et al. (2009) to study the genetic variability within and between the five Indonesian durian varieties namely, Sukun, Sunan, Kani, Monthong, and Petruk, which revealed that there is a genetic correlation between different varieties of Indonesian durian \[[@R22]\]. RAPD analysis technique was used to study 14 accessions of Thailand durian cultivars, carried out by Vanijajiva (2011), and the research findings were in line with the varietal classification of studied durian varieties \[[@R23]\].
Restriction fragment length polymorphism {#s1f}
----------------------------------------
Polymerase chain reaction (PCR)-RFLP analysis of two chloroplast DNA genes (ndhC-trnV and rbcL) was carried out to study the phylogenetic relationship amongst 10 Durio species. The study revealed that rbcL gene is suitable to distinguish the low variation at higher taxonomic level due to its highly conserved sequence. However, the ndhC-trnV gene was able to distinguish the high variations, and it could be used as a reliable molecular marker. The research findings suggest that specific gene targeted PCR-RFLP could be helpful in the marker-assisted breeding programme of Durio species \[[@R24]\]. Based on the analysis of ndhF chloroplast gene and its nuclear ribosomal DNA (rDNA) sequences, the phylogenetic study carried out by Nyffeler and Baum (2000) suggests that pollination of the durian flowers by bats and birds have gradually replaced pollination by beetles which suggest the evolution of the floral traits \[[@R25]\].
ISSR and SSR {#s1g}
------------
The inter-simple sequence repeats (ISSR) markers-based approach was used to assess the genetic diversity and genetic relationships in 14 Thai durian cultivars, and the research findings revealed that these markers are useful in assessing the genetic relatedness within and between the durian cultivars \[[@R26]\]. The simple sequence repeats (SSR) or microsatellites were used as a molecular tool to access the genetic diversity and researchers found that these markers were useful in selecting the superior varieties in durian breeding programme \[[@R27]\]. A durian marker kit has been established to authenticate the durian cultivars using the SSR markers. It indicates the ability of these markers to distinguish the fruit varieties \[[@R28]\]. Because of maternal inheritance, the chloroplast DNA is very useful in the molecular studies of the plants. Based on this fact, the molecular analysis of durian\'s chloroplast DNA was conducted using the rbcL gene (Accession No: AF402957-AF402949) \[[@R29]\]. The previous classification and evaluations of durian were primarily based on the phenotypic traits such as the shape of fruit, the size of thorns on fruit and other morphological characters \[[@R30]\]. This approach is not useful due to its limited ability to differentiate durian types. Hence, the use of molecular markers has become a standard method to study the variability among closely related taxa \[[@R31]\].
Very limited numbers of genes are studied in durian. Palapol et al. \[[@R32]\] have isolated and characterised three alpha-expansin genes of the Thailand durian clone, Mon Thong. It is believed that expression of alpha-expansin genes plays a vital role in both dehiscence and softening of durian fruit. Several studies have proved that the presence of the various expansins in various crops can improve the yield, fruit ripening and help in developing a good trait of stress tolerance \[[@R33]\]. It is hypothesised that the digestion of intracellular starch granules by the amylase in the plastids of ripening durians determines the sweetness of fruit pulp. A putative α-amylase encoding gene from Thailand durian (clone, Mon Thong) was successfully isolated which contains 2,679 base pair open reading frame (ORF) that encodes for an 892 amino acid long protein \[[@R34]\].
Durian genome attributes {#s1h}
------------------------
Durian plant\'s diploid chromosome no is 56 (1n = 28, 2n = 56) \[[@R35]\]. The mysterious allure of the durian was making researchers curious about its genome. Until recently, there was no enough molecular information on durian as this plant is studied very poorly at the molecular level. However, recently, in October 2017, Teh et al. have published the draft genome of commercially important durian variety - called Musang King (Mao Shan Wang in Chinese) \[[@R36]\]. The reported annotation of durian draft genome does provide several insights about its attributes. Some important attributes of the durian genome are summarised in [Table 2](#T2){ref-type="table"}.
Based on the draft genome analysis, Teh et al. suggested that a class of genes called as methionine gamma lyases (MGL) is responsible for the durian\'s unique, pungent smell \[[@R36]\]. MGL regulates the odour compounds termed as volatile sulphur compounds (VSC) which causes the smell of rotten eggs or onions. Typically, most of the plants contain only two (2) copies of MGL. The copy number of MGL in durian genome is four (4), and this could explain why the durian fruit produces more amounts of smelly compounds, VSC.
The durian draft genome analysis suggests that cotton plant is amongst the closest relatives of the durian plant. In-depth, further analysis of the durian draft genome will help in understanding more secrets of the King of the fruits, and it will pave the way for breeders to create new durian varieties which could be drought-resistant, high temperature tolerant and or with low-sugar content for people with diabetes.
Future research directions {#s1i}
--------------------------
For the durian plant and fruit quality improvement, development of new varieties, which will be resistant to fungal infections, pest attacks, and drought need to be considered. In addition to this, minimization of strong offensive aroma in fruits by gene regulation and prolonging short postharvest-life of fruits can be considered for the further improvement of the durian. The combination of all reactions that occur at the molecular, biochemical and physiological levels are known to determine the pattern of the plant developments \[[@R37]\]. Therefore, it is essential to study the durian genome in depth to understand the molecular mechanisms and various pathways to design the strategies for the future improvement of durian using traditional breeding approach and or by selective genetic manipulation strategy.
The standard shelf life of durian fruit is about 2-3 weeks only \[[@R10]\]. Molecular studies, particularly transcriptomics will help to reveal the significant genes that express in the ethylene biosynthesis systems responsible for the durian ripening. If we increase the post-harvest shelf life, then it will help to reduce the current severe economic losses of durian.
In Malaysia, 30 fungal diseases have been identified in durian \[[@R38]\]. Phytopthora palmivora Butler, Lasioplodia theobromae, Phomopsis sp. and Colletotrichum gloeosporioides are commonly found in rotting durian. These pathogenic fungi efficiently infect the durian fruit due to suitable conditions after the post-harvest. Pseudococcus species (mealybugs) and Coccus species (scale insects\') are the two types of insects that can be found on the fruit surface. Mudaria magniplaga Walker (\'Seed borer\') and M. luteileprosa Holloway are serious pests that damage durian. Fourteen (14) insect species have been recorded to attack the Indonesian durian, and two nematode pests (Helicotilenchua spp. and Radopholus spp.) have been reported to attack the Malaysian durian \[[@R38]\].
Sulphur-containing compounds cause the strong and pungent aroma of durian, whereas esters and alcohols cause the fruity odour \[[@R10]\]. Recently, Li et al. (2017) reported the major odoractive compounds present in durian fruit pulp \[[@R39]\]. Transcriptomics and gene expression studies will help to understand various pathways and the patterns of involved genes expression. In addition to this, transcriptomics studies will help to identify the expression patterns of the gene that are involved in the fatty acid pathway of the volatile aroma compounds in durian fruits. Now, the draft genome sequence information of the durian is available to researchers; hence, it will serve as a reference sequence while analysing the transcriptomic data. Recently, we have initiated durian fruit-pulp tissue transcriptomics to elucidate the expressed genes and their expression patterns. The research findings will be reported in due time.
Conclusion {#s2}
==========
Durian is a good source of various nutrients and medicinal compounds beneficial for human health. The genome drafts of durian showed the presence of four copies of MGL, which provides the insights on the high synthesis of VSC responsible for the unique aroma of durian fruit. Further analysis of the reported genome will elucidate genes that govern different qualitative and quantitative genetic traits of the durian fruit. The transcriptomics study of the durian fruit-pulp will help in understanding various genes expression patterns in it and in designing a novel strategy for genetic manipulation of this plant to knockout the unwanted genes expression and or to over-express the desirable genes in a fruit-tissue-specific manner to boost its nutritional quality.
Conflict of Interest {#s3}
====================
The authors have declared that no conflict of interest.
The authors are grateful to the AIMST University, Malaysia for providing university research grant for the project.
**Edited by P Kangueane**
**Citation:**Husin *et al.* Bioinformation 14(6): 265-270 (2018)
###### Registered varieties of Durian in Malaysia \[13\]
No Clone/Variety and Name Localities Date and Year\*
------------------------------------------------------------------ ----------------------------------------------- --------------------------------------------- -----------------
1\. D24 Bukit Merah Reservoir, Perak 13-Nov-37
2\. D99 (kob kecil) Origin from Thailand 17-Jun-70
3\. D123 (Chanee) Origin from Thailand 24-Jul-71
4\. D145 (Durian Beserah, Tuan Mek, Durian Hijau) Baserah, Kuantan Pahang 30-Oct-81
5\. D158 (Kan yau) Origin from Thailand 30-Jun-87
6\. D159 (Mon Thong, bantal Mas) Origin from Thailand 30-Jun-87
7\. D169 Tanah Merah, Kelantan. Origin from Thailand May-89
8\. D168 (IOI, Durian Ma Muar) Muar, Johor 24-May-89
9\. D175 (Udang merah, Ang He) Pulau Pinang 04-Jun-90
10\. D197 (Raja Kunyit, Musang King) Tanah Merah, Kelantan 09-Dec-93
11\. D198 (Kim Hong) Batu Pahat, Johor Mar-13
12\. D199 (Bola 828) Batu Pahat, Johor Mar-13
13\. D188 (MDUR 78) Stesen MARDI Jerangau, Kemaman, Terengganu 30-Aug-91
(Clone from D24 x D7)
14\. D189 (MDUR 79) Stesen Mardi Jerangau, Kemaman, Terengganu 30-Aug-91
(Clone from D24 x D7)
15\. D190 (MDUR 88) Stesen Mardi Jerangau, Kemaman, Terengganu 01-Jul-92
(Clone from D24 x D7)
\* Date of registration with Department of Agriculture, Malaysia
###### Some important attributes of Durian (Durio zibethinus L.) cultivar 'Musang King' genome based on its draft genome \[[@R36]\]
No Genome Feature Specifics of Durian draft genome
------ --------------------------------------------------------------- ----------------------------------
1\. Genome size (estimated) 738 Mb
2\. The total length of genome assembly sequence (with gaps) (bp) 7152,30,256
3\. The total length of genome assembly sequence (ungapped) (bp) 7121,86,256
4\. Genes and pseudogenes 44,795
5\. Protein-coding genes 35,832
6\. Non-coding genes 1,329
7\. The median length of genes (bp) 3,160
8\. Mean length of genes (bp) 4,117
9\. Min length of the gene (bp) 68
10\. Max length of the gene (bp) 1,17,665
11\. Average coding sequence length (bp) 1,700.40
12\. Average exons per gene 5.8
13\. Max number of exons per transcript 79
14\. GC content 32.50%
{#F1}
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1. Introduction {#sec1}
===============
The vertebrate immune system has evolved to protect its host against invading pathogens and other environmental antigens. It is strategically organized to optimally guard against foreign or "nonself" antigens through intricate interactions between innate and adaptive immunity, allowing for the survival of the host. The adaptability and resiliency of the immune system rely on complex physiological and immunological mechanisms, many of which remain to be unraveled. Since the initial classification of T~H~1 and T~H~2 cells by Coffman, Mosmann, and colleagues in 1986, much focus has attempted to elucidate the role of helper T cell populations. These efforts have led to the identification of a distinct T helper population, called T~H~17 cells \[[@B1]--[@B3]\], which challenges the long-standing T~H~1/ T~H~2 paradigm and has advanced our overall understanding of T helper cells in health and disease.
Paradoxically, the same mechanisms that prevent disease quite commonly induce hypersensitivity and autoimmunity. In fact, it was in the study autoimmunity in which the key observations that led to the discovery of T~H~17 cells were made. These studies found that T~H~1 cells were not required for induction of experimental autoimmune encephalomyelitis (EAE) in mice, as had been thought \[[@B4], [@B5]\]. EAE induction instead required an IL-23-dependent set of T cells that were later identified as the unique T~H~17 cell subset. Since then, numerous reports have shown T~H~17 cells to be relevant, and sometimes central, to autoimmune pathogenesis, highlighting them as therapeutic targets. Recently, T~H~17 cells have been implicated in SLE pathogenesis. SLE is a chronic inflammatory disease characterized by autoantibodies to nuclear antigens. It can be difficult to diagnose and to treat due its multifaceted nature, and death usually occurs due to renal involvement. In this paper, we discuss the biological function and regulation of IL-17 and T~H~17 cells. We will then focus on our current understanding of the role of T~H~17 cells in murine and human SLE.
2. IL-17 and T~H~17 Cells {#sec2}
=========================
This subset of CD4+ memory effector T cells is functionally distinct from either the T~H~1 or T~H~2 cell lineage \[[@B6]--[@B10]\]. T~H~1 cells release mainly IFN-*γ* and TNF-*α* that regulate cell-mediated immunity through activation of macrophages, NK cells, and CD8^+^ T cells. This process is driven by IL-12 through signal transducer and activator of transcription 4 (STAT4) activation and results in the expression of the transcription factor T-bet. T~H~2 cells predominantly produce IL-4, IL-5, and IL-13. IL-4 regulates the humoral immunity through the activation of B lymphocytes. The process is driven primarily by the phosphorylation of STAT6 resulting in the activation of transcription factor GATA binding protein 3 (GATA-3). Unlike T~H~1 and T~H~2 cells, differentiation of T~H~17 cells *in vitro* is mediated by TCR signaling in the presence of TGF-*β* and IL-6 or IL-21 stimulation \[[@B8]\]. Although IL-23 is not required for differentiation of T~H~17 cells, it is necessary for their survival and maintenance \[[@B11]\]. Temporal expression analysis of IL-23R indicated that it is only expressed after activation of naïve T cells with TGF-*β* and IL-6. Therefore, its expression allows for the continuous stimulation of the differentiated cells. T~H~17 effector cells are characterized by the unique ability to secrete IL-17A and IL-17F in response to stimulation by TGF-*β* and IL-6.
At present, there are multiple factors that are known to contribute to the development of T~H~17 cells. The main regulator of T~H~17 differentiation is the T-cell-specific *γ* (ROR*γ*t) transcription factor induced by IL-6 and TGF-*β* \[[@B12], [@B13]\]. In addition to ROR*γ*t, other transcription factors also play critical role in T~H~17 cells-specific lineage development. A recent study has indicated that I*κ*Bz works in conjunction with ROR*α* and ROR*γ* in the absence of IL-6 and TGF-*β* could optimally induce T~H~17 cell development. Elimination of the transcriptional activation domain as well as the ankyrin repeat domain in I*κ*Bz would abolish its function in inducing T~H~17 cell formation through the downregulation of the NF-*κ*B pathway. I*κ*Bz physically interacts with the noncoding sequences 2 (NCS 2) regulatory element in the *Il17a*promoter region to enhance *IL17a* gene expression \[[@B14]\]. Although no specific mechanism was proposed, a study by Schraml et al. suggested that the activator protein (AP)-1 protein B-cell-activating transcription factor (BATF) regulates the development of T~H~17 cells by interacting with target genes downstream of IL-6 and TGF-*β* signaling. These downstream genes include the conserved intergenic elements in the *Il17a-Il17f* locus and to the *Il17, Il21*, and *Il22* promoters regions \[[@B15]\]. In addition, another transcription factor such as IRF4 is also involved in T~H~17 cell development. IRF4 is mediated by IL-21 to physically bind with the *Il17* promoter and act in conjunction with ROR*γ*t for optimal IL-17 transcription. IRF4 is also involved in the balance of Foxp3, ROR*α*, and ROR*γ*t during T~H~17 cells differentiation \[[@B16]\]. Furthermore, otherfactors which belong to the Runx transcriptional factor family member could regulate the generation of T~H~17 cells. The family includes Runx 1, Runx 2, and Runx 3; however, only Runx 1 appears to play a more specific role in promoting T~H~17 cells. An *in vitro* study showed that overexpression of CD4+ T cells with Runx 1 resulted in higher IL-17 production in the presence of TGF-*β* alone and more enhanced in IL-17 level when stimulated with both IL-6 and TGF-*β*. Therefore, the activation of ROR*γ*t by TGF-*β* alone or combination of IL-6 and TGF-*β* along with the overexpression of Runx 1 allowed for optimal T~H~17 cells formation. Using chromatin immunoprecipitation or ChIP assay, the authors demonstrated that the enhanced level of IL-17 was to due to the recruitment and synergistic binding of ROR*γ*t and Runx 1 to the *Il17* promoter and the CNS-5 enhancer region \[[@B17]\]. Interestingly, Foxp3 can inhibit Runx 1 and ROR*γ*t to promote regulatory T cells (Treg). Therefore, the transcriptional regulation and the dynamic interaction of these factors provide more complexities in understanding the development of T~H~17 cells. These interactive factors need to be considered when attempting to categorize different T cell populations.
The IL-17 family of cytokines consists of six members: Il-17A (referred to as IL-17), IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. Detailed descriptions of each cytokine, in addition to IL-21 and IL-22 which are also produced by T~H~17 cells, will be discussed below.
2.1. IL-17A and IL17-F {#sec2.1}
----------------------
Currently, IL-17A and IL-17F are the best characterized cytokines within the IL-17 family. IL-17A and IL-17F exist either as homodimers or as IL-17A/IL-17F heterodimers \[[@B18]\]. Receptors for IL-17A and IL-17F include IL-17RA and IL-17RC \[[@B19]--[@B21]\]. Activation of IL-17A and IL-17F initiates powerful inflammatory responses and further induces production of potent proinflammatory cytokines. Both IL-17A and IL-17F can mediate the production of IL-6, CCL3, and G-CSF in macrophages, but only IL-17A can activate CCL2, IL-1*β*, IL-12p70, and IL-9. IL-17A is also solely responsible for the activation of CCL2, CCL3, GM-CSF, IL-1*β*, and IL-9 in CD4+ T cells \[[@B22]\]. As part of the local inflammatory response, both cytokines are responsible for the proliferation, maturation, and recruitment of neutrophils \[[@B1]\]. They provide immediate immunological protection by producing antimicrobial and acute phase response proteins against a variety of pathogens, specifically *Propionibacterium acnes*, *Citrobacter rodentium*, *Klebsiella pneumoniae*, *Bacteroides spp*., *Staphylococcus aureus*\[[@B23]\], acid-fast *Mycobacterium tuberculosis*, and fungi infection such as *Candida albicans*\[[@B18], [@B24]\].
Most importantly, having the potential to upregulate the expression of specifics matrix metalloproteinases (MMPs) such as MMP-1, MMP-3, MMP-9, MMP-13, IL-17A, and IL-17F have been shown to be tissue-damaging cytokines and are intimately involved in autoimmune diseases, for example, Crohn\'s disease \[[@B25], [@B26]\], EAE \[[@B4]\], collagen-induced arthritis (CIA) \[[@B5]\], Sjögren\'s syndrome (SjS) \[[@B27], [@B28]\] and SLE which will be later discussed \[[@B29]--[@B34]\]. However, a recent study by Ishigame et al. \[[@B23]\] suggested that there are differential roles for IL-17A and IL-17F in autoimmune responses, in which IL-17F played a minimal role in the pathogenesis of delayed-type and contact hypersensitivities, EAE, CIA, and arthritis in animal models. In contrast, IL-17A appeared to produce more potent pathogenic cytokines in macrophages, whereby genetic knockout of *il-17a*rendered the mice with reduced disease phenotypes. The differential role of IL-17A and IL-17F raises interesting questions in deciphering mucosal immunity and autoimmunity. Both cytokines elicit their responses via similar receptor complexes; however, it is intriguing that they provide different autoimmune responses in terms of pathogenicity and protection. The contrasting biological functions could be due to an approximate 10-fold more potent induction of cytokines by IL-17A as compared to IL-17F \[[@B35]\]. In support of this concept, a recent review by Dubin and Kolls \[[@B36]\] has suggested a model which emphasizes the bioactivities of these cytokines on myeloid versus nonmyeloid cells or macrophages versus CD4+ T cells discussed priorly. Therefore, it is the ability of IL-17A to induce stronger responses and affect a wider range of cellular targets, making it a more pathogenic cytokine. It will be of interest if such a dichotomy is seen in SLE.
2.2. IL-17B, IL-17C, and IL-17D {#sec2.2}
-------------------------------
IL-17B, IL-17C, and IL-17D are the least studied members of the IL-17 cytokine family. It remains speculative whether they are capable of eliciting any proinflammatory or protective responses like IL-17A and IL17F. A study using the CIA mouse model has shown that adoptive transfer of IL-17B+ or IL-17C+ CD4+ T cells was able to recapitulate a CIA phenotype and that blockade of IL-17B prevented disease exacerbation. The authors suggested that the inflammation induced by IL-17B/IL-17C is mediated by the production of TNF-*α* \[[@B37]\]. However, genetic association study in coeliac disease (CD) using large sample of patients and controls (409 CD, 355 controls) provided no conclusive evidence in the association of the genetic variation of a number of cytokines including IL-17B and the development of the disease \[[@B38]\]. Interestingly, our recent microarray data from the C57BL/6.NOD-*Aec1Aec2* mouse model of primary-SS revealed a strong upregulated expression of *Il17b,* thus, this cytokine may play an important but unidentified role in the rheumatic diseases (unpublished data).
2.3. IL-17E (IL-25) {#sec2.3}
-------------------
Designated as IL-25, IL-17E has been shown to induce T~H~2-like responses with the upregulation of IL-4, IL-5, and IL-13 gene expression \[[@B39]\]. Furthermore, the ability of IL-17E to promote the expression of adhesion molecules, specifically ICAM-1, ICAM-3, and L-selectin, allows for eosinophilic infiltration and structural changes of epithelial cells, making it a vital cytokine for allergic inflammatory response and/or asthma-related attacks \[[@B40]\]. Activation of T~H~2-related cytokines also resulted in significant elevation of IgE, IgG~1~, and IgA levels in which IgE can induce the release of prostaglandin D~2~ (PGD~2~) on mast cells that directly mediates the vasodilatation, mucus production, and broncho-constriction. While IL-17E is normally negatively regulated by a Socs3-dependent pathway \[[@B41]\], numerous approaches have attempted to inhibit the biological function of IL-17E. Administration of monoclonal antibody that blocked the function of IL-17E dramatically reduced the production of IL-5/IL-13, infiltration of eosinophils, and IgE secretion, thereby preventing the antigen-driven airway inflammation and airway hyperresponsiveness (AHR) \[[@B42]\]. Taking advantage of the ability of IL-17E to bind to IL-17RA and IL-17RB receptors, antagonist monoclonal antibodies against either IL-17RB or IL-17RA receptor resulted in complete abolishment of IL-17E-induced AHR in naïve BALB/C mice \[[@B43]\]. A better understanding of IL-17E has shed additional information on the immunological activities of IL-17E cytokine and its participation in allergic inflammation, thereby providing potential therapeutic targets. Interestingly, a recent study by Kleinschek et al. \[[@B44]\] has indicated that IL-17E might play an opposing role to IL-17A. Knocking out IL-25 rendered the animals highly susceptible to the development of EAE characterized by the increase of IL-23 level and infiltration of IL-17 and IFN-*γ* producing T cells in the central nervous system. Furthermore, neutralization of IL-17A in the knockout mice prevented EAE \[[@B44]\]. This data clearly suggests the inhibitory role of IL-17E in EAE. However, genetic association study in patients with Crohn\'s disease or ulcerative colitis concluded that IL-17E has little association in the disease development \[[@B45]\]. Consequently, extensive studies are needed to fully elucidate the role of IL-17E in human disease.
2.4. IL-21 and IL-22 {#sec2.4}
--------------------
In addition to their signature cytokines, T~H~17 cells also produce IL-21 and IL-22. IL-21 functions as an autocrine cytokine which allows for an alternative differentiation pathway for T~H~17 cells when IL-6 is absent \[[@B46]\]. Furthermore, IL-21 is involved in the amplification of T~H~17 cell-specific lineage transcription factors allowing for the maintenance and stabilization of this cell population \[[@B16], [@B47]\]. IL-21 is also known to assist the activation and differentiation of naïve B cells to plasma cells by upregulation of Blimp-1 \[[@B48]\]. In addition, it induces the expression of the *γ*1 and *γ*3 germline transcripts for the isotypic switching to IgG1 and IgG3 from IgMin human B cells \[[@B49]\]. These features, thereby, establish IL-17-producing cells as helper T cells. The isotypic switching potential of IL-21 is critical in modulating the disease development of isotypic-dependent autoimmune diseases such as SLE \[[@B50]\] and SjS \[[@B28], [@B51]--[@B54]\]. In the nonobese diabetic (NOD) animal model for SjS, isotypic switching to an IgG1 antibody against the acetylcholine receptors (AchRs), specifically the muscarinic receptor type 3 (M3R), is required for the development of SjS. Perhaps, the most critical is its involvement in the formation of germinal centers by controlling the expression of Bcl-6, which regulates the survival and activation of B cells. Furthermore, IL-21 is necessary for the expansion of T~H~17 cells and follicular T helper cells through the costimulatory ICOS and c-Maf pathway \[[@B55]\]. Therefore, it is a critical cytokine in modulating not only the T cell biology, but also the B cell response.
IL-22 is a cytokine that is produced by subsets of T~H~17 cells as well as a multitude of other cell types, including natural killer cells-22 (NK-22), lymphoid tissue inducer (LTi) cells, and epithelial cells. Mucosal microflora can promote the secretion of IL-22 from epithelia and the differentiation of IL-22-producing cell populations, in particular cell populations expressing NKp46, for example, the ROR*γ*t+CD3−NKp46+ NK cell, the ROR*γ*t+CD3−NKp46− LTi cell, and an uncharacterized ROR*γ*t+CD3+NKp46+ cell population. The IL-22 receptor complex is a heterodimeric molecule composed of IL-22RA1 and IL-10R2 \[[@B56]\]. On interacting with its heterodimeric receptor (IL-10R2/IL-22R), IL-22 can transduce a signal through phosphorylation of tyrosine kinases Jak1 and Tyk2, followed by the activation of STAT3, and to a lesser degree a heterodimeric STAT1/STAT3 during signaling cascade \[[@B57]\]. IL-22 has also been reported to activate several signaling pathways, including the MAPK pathway via ERK1/2, JNK, and p38 for induction of IL-22-related genes \[[@B58]\]. Since epithelial cells express high levels of IL-10R2 and IL-22R, IL-22 can initiate a strong response from epithelial cells which includes production of cytokines, chemokines, acute phase proteins, and a number of antimicrobial molecules such as *β*-defensin, lipocalins, and calcium binding S100 proteins \[[@B59]\]. It is also involved in tissue repair following exacerbated immune responses and epithelial-barrier functions against bacterial infections \[[@B60]\]. Paradoxically, IL-22 has been shown to be pathogenically associated with several autoimmune diseases including rheumatoid arthritis \[[@B61]\] and Crohn\'s disease \[[@B62]\] as well as non-autoimmune diseases such as respiratory-distress syndrome \[[@B63]\] and cystic fibrosis \[[@B64]\]. Whether IL-22 is an important player in development and/or onset of rheumatic disease, like SLE, will be an interesting area of future studies.
3. Negative Regulation of T~H~17 Cells {#sec3}
======================================
It remains controversial whether T~H~17 cells are protective or pathogenic. Its mode of response is substantially dependent on the eliciting antigenic entities. In certain cases of fungal and bacterial infections, IL-17 can be protective by recruiting neutrophils to the site of injury; however, IL-17 activation can also lead to rampant and impetuous immune response resulting in exacerbated clinical pathology and autoimmunity. Therefore, regulatory elements of the IL-17/T~H~17 system are required to maintain congruency and homeostasis between the protective and pathogenic consequences. Although the research area is still in its infancy, as of present, there are clearly multiple systems that have the capability to regulate the development and differentiation of T~H~17 cells. One of the most critical regulatory factors is the IL-27 cytokine, which is secreted by activated macrophages and dendritic cells \[[@B65]\]. IL-27 is a member of the IL-12 family of cytokines and is comprised of a heterodimer between IL-27*α* (IL-27 p28) and IL-27*β* (IL-27 Ebi3) \[[@B66]\]. IL-27 (or the IL-27 p28 subunit *per se*) exerts the IL-27-associated biological effects by activating its heterodimeric IL-27R including WSX-1 and gp130. Signal transduction involves phosphorylation of JAK1, JAK2, STAT1, STAT3, STAT4, and STAT5 in T cells, NK cells, and monocytes, but only STAT3 in mast cells \[[@B67]\]. However, only STAT1 or STAT3 activation is critical for the resulting bioactivity of IL-27 on naïve T cells that express IL-27R \[[@B68]\]. Activation of STAT1 by JAK1 or JAK2 promotes T~H~1 differentiation via the upregulation of T-bet resulting in the production of IFN-*γ*. At the same time, IL-27 inhibits the production of IL-2 and IL-6, thus downregulating the IL-6-dependent STAT3 activation of ROR*γ*t expression and subsequent development of T~H~17 cells. Recent studies have suggested that IL-27 is pleiotropic, regulating hematopoietic stem cell differentiation, eliciting antitumor activities, as well as promoting both pro- and anti-inflammatory activities \[[@B69]--[@B72]\]. Due to its potent suppressive ability, IL-27 functions to inhibit the differentiation of T~H~17 cells in both *in vitro* and *in vivo* studies. In several animal models of autoimmune diseases, a deficiency in either IL-27 or IL-27R results in exacerbated pathology and clinical signs mainly due to the dysregulation and increase in numbers of IL-17 producing T cells \[[@B11]\]. Additionally, systemic injection of rIL-27 cytokine into autoimmune animal models of EAE, scleritis, or uveitis ameliorates many clinical symptoms \[[@B73]\]. Thus, the T~H~17/IL-23/IL-27 system is thought to bridge innate immunity and subsequent adaptive immune responses.
In addition to IL-27, other T helper cells populations can also negatively regulate the development of T~H~17 cells. As mentioned earlier, IFN-*γ* produced by T~H~1 cells upregulates the T-bet transcriptional factor which dampens the activation of ROR*γ*t resulting in the downregulation of T~H~17 cells. Similarly, the upregulation of GATA-3 transcription factor of T~H~2 cells by IL-4/5/13 could also restrict the expansion of T~H~17 cells by inhibiting the function of ROR*γ*t. One major aspect of T~H~17 cells negative regulation is the influence of Treg cells. Treg cell differentiation is driven mainly by TGF-*β* which activates Foxp3. Sharing the ubiquitous TGF-*β* factor, the presence or absence of IL-6 controls the developmental shift toward T~H~17 or Treg cells. The shift to CD4+CD25+Foxp3+ Treg and CD4+CD25+Foxp3+CD39+ subset plays a significant role in restricting the detrimental effect of T~H~17 cells in multiple sclerosis patients \[[@B74]\]. Interestingly, retinoic acid increases the expression of Foxp3 via activation and phosphorylation of Smad3 and concomitantly inhibits the expression of IL-6R*α*, IRF-4, and IL-23R, thereby limiting T~H~17 development \[[@B75]\]. An exciting and confounding feature of T~H~17 development is the plasticity among different T helper cells populations and the microenvironment or microflora that imposes on its lineage-specific differentiation. A study by Koenen et al. has demonstrated that human CD25highFoxp3+ Treg cells when stimulated with allogeneic monocytes in the presence of IL-2 and IL-15 can differentiate into IL-17 producing T cells. The study further showed that the lateral lineage conversion to T~H~17 cells from Treg cells relied on the histone deacetylase activity indicating the contribution of epigenetic modification \[[@B76]\]. In addition, T~H~17 cells have the propensity to convert to T~H~17/T~H~1 phenotype under the appropriate milieu of low TGF-*β* and high IL-12 levels which are often observed in the joints of children with inflammatory arthritis \[[@B77]\]. Other microorganisms such as live *C. albicans*can modulate tryptophan metabolism to inhibit IL-17 production \[[@B78]\], and *H. pylori*mediates the polarization of T~H~17/Treg balance toward regulatory response which inhibits T~H~17 response \[[@B79]\].
4. IL-17 in Murine Lupus {#sec4}
========================
As previously mentioned, the role of T~H~17 in the development of autoimmunity was initially scrutinized in murine models of induced EAE \[[@B2]\]. This disease model was originally believed to be dependent on IL-12, and thus, T~H~1 mediated. However, the revelation that IL-12 shared a subunit, p40, with a newly discovered cytokine, IL-23, and that this novel cytokine, not IL-12, was required for induction of disease sets the stage for investigation of T~H~17 these models \[[@B4], [@B5]\]. More recently, several lines of research have reported increased IL-17 production and T~H~17 functions in murine models of lupus as summarized in [Table 1](#tab1){ref-type="table"}.
BXD2 is one of 20 BXD recombinant inbred strains derived from a cross between C57BL/6J (B6) and DBA/2J. These mice develop a spontaneous and age-dependent lupus-like syndrome denoted by production of the canonical anti-DNA, antihistone, and rheumatoid factor autoantibodies, as well as splenomegaly, glomerulonephritis (GN), and erosive arthritis \[[@B80], [@B81]\]. BXD2 CD4+ T cells have enhanced T~H~17 development and consequent increased serum levels of IL-17 \[[@B82]\]. Moreover, IL-17-secreting CD4+ cells were shown to localize to germinal centers (GCs) in BXD2 spleens. This augmented IL-17 response was associated with increased GC development and stability in BXD2 spleens as compared to B6 controls. Additionally, BXD2 have increased amounts of IL-17R+ B cells \[[@B82]\]. These B cells have both an increased basal and an IL-17R-induced activation of the canonical NF*κβ* pathway, resulting in an increased expression of regulator of G signaling (RGS) proteins \[[@B83]\]. Consequently, RGSs enhance the GTPase activity of chemokine receptor G*α* subunits resulting in decreased chemotaxis \[[@B84], [@B85]\]. Indeed, BXD2 B cells were shown to have a diminished chemotactic response to CXCL12 and CXCL13, especially in the presence of IL-17 \[[@B82], [@B83]\]. This increased potential for B cell accumulation at the sites of CXCL12 and CXCL13 production, such as follicular dendritic cell rich areas \[[@B86], [@B87]\], is the likely cause of the enhanced GC formation in the BDX2 strain. Moreover, the concurrent production of IL-17 by T~H~17 cells in GCs further promotes B cell accumulation and GC stability. IL-17 also results in increased activation-induced cytidine deaminase (*Aicda*) expression and somatic hypermutation in BXD2 IL-17R+ B cells, which have an intrinsically enhanced ability to produce autoantibodies as compared to IL-17R-deficient BDX2 B cells \[[@B82]\]. Thus IL-17 has a central role in pathogenesis of the lupus-like syndrome observed in this model.
The MRL/*lpr* strain is a classical model of spontaneous lupus. It exhibits a lymphoproliferative disorder which manifests with autoantibody production, GN, and accumulation of CD4^−^CD8^−^ double-negative T (DNT) cells in the periphery \[[@B88]\]. A mutation in *Fas* is responsible for the *lpr* phenotype and is the major functional contributor of pathogenesis in this strain \[[@B89], [@B90]\]. It was recently shown that *Fas*-deficient DNT cells are capable of producing significant amounts of IL-17 \[[@B91]\]. Further, the T~H~17-stablizing cytokine, IL-23, potently induced IL-17 production in these DNT cells which were then capable of renal infiltration and GN induction. Finally, deletion of IL-23R prevented splenomegaly, lymphadenopathy, autoantibody production, and GN in the context of *Fas* deficiency and was associated with a major reduction of the DNT cell compartment along with its concomitant IL-17 production \[[@B92]\]. Thus, a pathogenic T~H~17-like function of DNT cells has been exposed, highlighting this subset as a target for disease intervention.
The SNF1 mouse model, derived from the F1 outcross of the New Zealand Black and SWR recombinant inbred strains, develops a spontaneous lupus-like syndrome that can be accelerated by immunization of nucleosomal peptides \[[@B93]\]. Upon disease induction, autoantibodies are produced, and GN with T~H~17 infiltration is initiated \[[@B94]\]. Interestingly, low-dose therapy of a tolerogenic histone-derived peptide caused increased TGF-*β* and decreased IL-6 expression in dendritic cells and resulted in enhancement of Treg function with a reduction in T~H~17 renal infiltrates \[[@B94]\]. Treatment with either oral or nasal anti-CD3 also ameliorates autoantibody production and nephritis in this model by inducing a regulatory T cell subset and reducing IL-17 production by T follicular helper cells \[[@B95], [@B96]\]. These results indicate that therapies that regulate Treg/T~H~17 homeostasis in favor of Treg might be effective at moderating SLE pathogenesis.
Finally, disruption of TNF*α* receptor signaling in spontaneous lupus-prone NZM2328 mice results in exacerbated disease that has associated with a greatly enhanced T effector/memory compartment. These cells were found to have a Th17 gene signature and produced more IL-17 than TNF-*α* receptor sufficient T effector/memory cells \[[@B97]\]. This work highlights the regulatory function that TNF-*α* can have and sets a caution for TNF-*α* blockade therapy.
5. IL-17 in SLE Patients {#sec5}
========================
As with murine lupus models, evidence for a T~H~17 role in human SLE is also mounting. Several recent reports show that plasma IL-17 and IL-17 producing T cells are increased in SLE patients \[[@B29]--[@B34]\]. Moreover, disease activity and severity are associated with increased IL-17 production \[[@B31]--[@B34]\]. SLE patients have increased phosphorylation of STAT3 \[[@B98]\], which is required for T~H~17 differentiation, as STAT3 deficiency in hyper-IgE syndrome patients results in the ablation of T~H~17 cells \[[@B99], [@B100]\]. The T~H~17-polarizing cytokines, IL-6, IL-21, and IL-23, all signal in a STAT3-dependent manner to induce transcription of the ROR*γ*t \[[@B101]\]. Indeed, SLE patients also have increased plasma levels of IL-6, and higher *Rorc* expression, which encodes ROR*γ*t \[[@B34], [@B102]\]. Taken together, T~H~17 expansion is an important feature of SLE that needs to be further investigated.
It is well established that there is a strong gender bias in the incidence of SLE in which roughly 90% of the cases occur in females. Since IL-17 production correlates with disease severity, the question is raised as to whether the female bias of SLE is due to differences in T~H~17 biology. While this has not been studied extensively, IL-17 *in vitro*production was shown to decrease with age in males, but not in females \[[@B103]\]. Although these results do demonstrate a gender difference, the relevance to SLE induction is not clear since the young cohorts, who were between 21 and 40 years old, the highly susceptible age of onset for SLE, did not produce different amounts of IL-17 in males versus females. Nevertheless, the ability to maintain higher levels of IL-17 production with age may contribute to the maintenance of the disease state in females. More recently, it was reported that *in vivo*treatment of mice with estrogen enhances T~H~17 polarization *in vitro*, supporting the hypothesis that T~H~17 cells contribute to the female bias of SLE \[[@B104]\]. There is, however, no direct evidence for this hypothesis, and further study is needed to clarify the role that gender may play in T~H~17 function and disease induction.
Similar to *Fas*-deficient mouse models of lupus, a significant amount of IL-17 is also produced by an expanded subset of DNT cells in SLE patients \[[@B30]\]. These DNT cells are derived from CD8^+^ cells that have downregulated CD8 in response to receptor stimulation \[[@B105]\]. While they are normally present in very small amounts, their expansion in SLE patients may be due to increased T cell activation. Because of their downregulated coreceptor, they have decreased survival and proliferation and display unique gene expression patterns and proinflammatory cytokine profiles \[[@B105]\]. Notably, as in lupus-prone mice, DNT cells can be found in kidney biopsies of SLE patients \[[@B30]\]. Therefore, DNT cells appear to represent a distinct effector population of T cells whose dysregulation may be central to SLE pathogenesis.
The fundamental role of type I IFN dysregulation is well established in SLE pathogenesis \[[@B106]\]. Unregulated IFN-*α* production has been shown to increase proinflammatory cytokine production, including IL-6 and IL-23 which lead to Th17-mediated inflammation in mice \[[@B107]\]. Also plasmacytoid dendritic cells (pDCs), which are known to potently secrete IFN-*α*, also produce IL-1*β*, IL-6, and IL-23 in response to Toll-like receptor (TLR)-7 stimulation in human studies \[[@B108], [@B109]\]. These pDCs are capable of inducing T~H~17 differentiation when cocultured with CD4^+^ cells. Endogenous nucleic acids are autoantibody targets in SLE and are capable of TLR activation following their uptake as immune complexes \[[@B110], [@B111]\]. Therefore, pDCs can be chronically activated, potentiating Th17 development and disease pathogenesis.
IL-17 also promotes B cell survival both alone and synergistically with B cell-activating factor (BAFF) \[[@B31]\]. Hence, a feedback loop is established where IL-17 promotes autoreactive B cells to persist longer and make autoantibodies which activate pDCs induce more T~H~17 cells. In parallel, the expansion of DNT cells results in more IL-17 production, exacerbating this progression ([Figure 1](#fig1){ref-type="fig"}). As IL-17 is central mediator to this process, therapeutic intervention that targets T~H~17 development and IL-17 production will be valuable treatments for SLE.
6. Conclusions {#sec6}
==============
The discovery of IL-17 and T~H~17 cells has expanded and transformed the conventional thought in immunology. The change adds complexity to an already complicated matter. The intricate and dynamic interaction between the different characters promotes the adaptability and resiliency of the immune system. Therefore, it is difficult to comprehend that any one particular entity is solely responsible for such a vexing system. The coincidental discovery of T~H~17 cells did not shift any paradigms, but merely add another unknown factor to an unsolved equation. Currently, there are numbers of issues that need to be resolved, for example, the differential function of IL-17 molecules within the family in the context of infectious disease and autoimmunity, the negative regulation of T~H~17 cells and its application in therapeutic approach, and its relevance in the pathogenesis of SLE besides observational or correlative studies. Tremendous strides are being made to address these issues.
This work was supported by PHS Grants K99DE018958 (CQN) from NIDCR, R21AI081952 (ABP) and R01AI45050 (LM) from NIAID, and funds from the Sjögren\'s Syndrome Foundation and Center for Orphan Autoimmune Disorders.
{#fig1}
######
IL-17 in murine models of lupus.
Model Description References
----------- --------------------------------------------------------------------------------------------------- --------------------
BXD2 IL-17 promotes spontaneous GC development as well as autoantibody production by IL-17R^+^ B cells \[[@B82], [@B83]\]
MRL.*lpr* Expansion of IL-17-producing DNT cells with kidney infiltration and GN induction \[[@B91], [@B92]\]
SNF1 Enhanced IL-17 production by CD4^+^ T cells with kidney infiltration \[[@B94]\]
NZM2328 Disruption of TNF*α* promotes Th17 development \[[@B97]\]
[^1]: Academic Editor: G. D. Kitas
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-ijms-19-03470}
===============
The WUSCHEL-related homeobox (WOX) family is a large group of transcription factors (TFs), specifically found in plants and characterized by the presence of 60--66 amino acids that constitute a DNA-binding domain, called a homeodomain (HB domain) \[[@B1-ijms-19-03470]\]. The HB domain is highly conserved and important for the function of WOX protein family members, which are also characterized by the presence of a helix--loop--helix--turn--helix secondary structure.
Previous reports have shown that *WOX* genes play a wide variety of roles in plant development and growth processes such as embryonic patterning, stem cell maintenance and organ formation \[[@B2-ijms-19-03470],[@B3-ijms-19-03470]\]. In *Arabidopsis thaliana*, there are at least 15 *WOX* genes (*AtWOXs*), classified into three clades: an ancient clade (*WOX10*, *WOX13* and *WOX14*), intermediate clade (*WOX8*, *WOX9*, *WOX11* and *WOX12*) and modern clade (*WUS* and *WOX1--7*) \[[@B2-ijms-19-03470],[@B4-ijms-19-03470],[@B5-ijms-19-03470]\]. The functions of *AtWOX*s have been well studied and shown to be generally distinct in members of different clades and conserved in members of the same clade. For example, members of the modern clade maintain apical stem cells \[[@B6-ijms-19-03470],[@B7-ijms-19-03470]\] and members of the intermediate clade mainly regulate zygote development and early embryo morphogenesis \[[@B8-ijms-19-03470],[@B9-ijms-19-03470]\], while members of the ancient clade regulate root development \[[@B10-ijms-19-03470]\]. The functions of *WOX*s in many other plants are also conserved and similar to those of their *Arabidopsis* homologs. For instance, in rice (*Oryza sativa*) and maize (*Zea mays*), the orthologs of *AtWOX5* (*OsWOX5* and *ZmWOX5*) play similar roles in the root meristem development \[[@B11-ijms-19-03470],[@B12-ijms-19-03470]\]; in Norway spruce (*Picea abies*), *PaWOX8* and *PaWOX9* are orthologs of the members of the AtWOX8/9 subcladeand determine the cell fate during early embryo patterning \[[@B13-ijms-19-03470]\]; and *PtWUS* in *Populus trichocarpa* corresponds to *AtWUS,* which is involved in shoot apical meristem (SAM) development.
*Brassica napus* L. (*B. napus*) has a high economic value because it is one of the main commercial sources of cooking oil. *B. napus* is an allotetraploid species (AnAnCnCn; *n* = 19), which originated from the recent hybridization of two base diploid genomes of *Brassica rapa* L. (*B. rapa*; AnAn; *n* = 10) and *Brassica oleracea* L. (*B. oleracea*; CnCn; *n* = 9) \[[@B14-ijms-19-03470]\]. Currently, there is a lack of detailed genomic information on the WOX gene family of *B. napus*, but it is of interest to explore the evolution and expression mechanisms of *WOX* genes (*WOXs*) between the *Brassica* An and Cn (BnAn and BnCn) sub-genomes \[[@B15-ijms-19-03470]\]. Furthermore, understanding the structural relationships between *Arabidopsis* and *B. napu*s would facilitate the prediction of *B. napu*s *WOX* genes that remain uncharacterized.
In the present study, we performed a comprehensive genome-wide analysis of the WOX gene family in *B. napus* and identified 58 putative genes (*BnWOXs*) with open reading frames (ORFs). Phylogenetic analysis classified these *BnWOX*s into three clades, characterized by the integration of sequence features, chromosomal location, phylogenetic relationship, subcellular localization, interacting proteins, collinearity and expression patterns. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that members of the WOX4 subclade were induced by abiotic stresses or hormones in seedling roots, suggesting their function in stress tolerance. Overall, our study will serve as a foundation for future research on the functional roles of *WOX*s in *B. napus*.
2. Results {#sec2-ijms-19-03470}
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2.1. Identification of 58 BnWOX Genes and Their Physicochemical Properties {#sec2dot1-ijms-19-03470}
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To identify the WOX-encoding genes in the *B. napus* genome, a preliminary BLASTP search was performed using the HB domain sequences of known *Arabidopsis* WOX proteins as queries. In each case, a large number of deduced amino acid sequences (\>50 candidates) containing WOX or WOX-like repeats were obtained. Only hits with E-values of \<1.0 were considered as members of the WOX gene family. The redundant candidate sequences were discarded from our data set, according to their chromosomal locations. We were then able to identify 58 typical, non-redundant *WOX* genes in the *B. napus* genome; those had complete ORF regions and encoded proteins with typical WOX features, which we verified using PROSITE (<http://www.expasy.org/tools/scanprosite/>). To distinguish these genes, we provisionally named them *BnWOX1* to *BnWOX58* based on their order on the corresponding chromosomes ([Table 1](#ijms-19-03470-t001){ref-type="table"}). We also identified 27 and 30 non-redundant *WOX* genes in *B. rapa* (*BrWOXs*) and *B. oleracea* (*BoWOXs*), respectively, using the same method ([Table S1](#app1-ijms-19-03470){ref-type="app"}). Physicochemical property analysis showed that the corresponding BnWOX proteins (BnWOXs) varied in length from 121 to 390 amino acids; their molecular weight ranged from 13.96 to 71.82 kDa and the isoelectric points were 5.08--9.58. Subcellular localization analysis demonstrated that all 58 proteins were located in the nucleus ([Table 1](#ijms-19-03470-t001){ref-type="table"}).
2.2. Phylogenetic Analysis of the BnWOX Gene Family {#sec2dot2-ijms-19-03470}
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To determine the evolutionary relationships of the *BnWOX* gene family with those of *Arabidopsis* and the *B. napus* ancestor species, we constructed neighbor-joining (NJ) and maximum-likelihood (ML) phylogenetic trees of 130 WOX proteins, from *B. napus* (58), *B. rapa* (27), *B. oleracea* (30) and *Arabidopsis* (15), based on the multi-alignment of their HB domains using MEGA 7.0 \[[@B16-ijms-19-03470]\] and PhyML 3.0 \[[@B17-ijms-19-03470]\], respectively.
Our results showed that the NJ and ML tree topologies were highly congruent ([Figure 1](#ijms-19-03470-f001){ref-type="fig"} and [Figure S1](#app1-ijms-19-03470){ref-type="app"}). In the phylogenetic trees, the 130 WOX members clustered into three main clades: modern, intermediate and ancient clades ([Figure 1](#ijms-19-03470-f001){ref-type="fig"}). The number of *WOX* genes in the modern clade (72 genes) was greater than that in the ancient (24 genes) and intermediate (34 genes) clades, indicating the gene expansion in higher plants. Our data were consistent with those of previous reports, which indicated that the WOX gene family was chronologically divided into three clades (i.e., ancient, intermediate and modern/WUS clades) \[[@B4-ijms-19-03470]\]. In each clade, the numbers of genes from these four species were usually different and each *Arabidopsis WOX* gene (*AtWOX*) generally had more than one ortholog. Thus, the modern clade consisted of 33 *BnWOX*s, 15 *BoWOX*s, 16 *BrWOX*s and 8 *AtWOX*s; the intermediate clade included 14 *BnWOX*s, 9 *BoWOX*s, 7 *BrWOX*s and 4 *AtWOX*s; and the ancient clade had 11 *BnWOX*s, 6 *BoWOX*s, 7 *BrWOX*s and 3 *AtWOX*s. This distribution showed that the number of genes constantly increased with the evolution of the gene family.
We further divided the candidate *WOX* genes into nine subclades: WUS, WOX1, WOX2, WOX3, WOX4, WOX5/7, WOX6, WOX11/12 and WOX8/9, based on the bootstrap values and the topology of the phylogenetic tree. There were seven subclades in the modern clade and two in the intermediate clade ([Figure 1](#ijms-19-03470-f001){ref-type="fig"}). In the ancient clade, only homologs of *WOX13* were found in non-Brassicaceae species \[[@B3-ijms-19-03470],[@B18-ijms-19-03470],[@B19-ijms-19-03470],[@B20-ijms-19-03470]\]; however, we found that homologs of *WOX10/14* existed in all of the four Brassicaceae, which indicates that *WOX10/14* may be unique to Brassicaceae \[[@B21-ijms-19-03470]\]. In addition, the *WOX1/WOX6* homologs were divided into two subclades (WOX1 and WOX6), with high bootstrap values.
2.3. Sequence Analysis of B. napus WOX Domains {#sec2dot3-ijms-19-03470}
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To compare the sequence features, we performed a multiple alignment analysis of the HB domains of the 58 BnWOXs using MAFFT with the default parameters \[[@B22-ijms-19-03470]\]. The sequence logos and the secondary structures of the HB domains were generated on the Weblogo (<http://weblogo.berkeley.edu/logo.cgi>) and PRABI (<https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html>) platforms ([Figure 2](#ijms-19-03470-f002){ref-type="fig"}). Our results showed that the HB domains were highly conserved and commonly contained helix--loop--helix--turn--helix structures, which were either 63 or 64 amino acid residues in length, with the exception of BnWOX23, BnWOX52, BnWOX13 and BnWOX36, which had a short amino acid deletion at the C-terminus due to incomplete genome information ([Figure 2](#ijms-19-03470-f002){ref-type="fig"}). Consistent with previous reports \[[@B3-ijms-19-03470]\], there was a conserved Y (Tyr) residue insertion after the 17th amino acid in the HB domains of all AtWUS homologs, resulting in a total of 63 amino acids ([Figure 2](#ijms-19-03470-f002){ref-type="fig"}). In addition, 16 amino acid residues were completely conserved and mainly located in the third helix, which may play an important role in gene function \[[@B1-ijms-19-03470],[@B3-ijms-19-03470]\]. Interestingly, there were also some conserved subclade-specific substitutions (e.g., at 8th, 37th, 20th and 23rd amino acid position) in the HB domains in the modern clade ([Figure 2](#ijms-19-03470-f002){ref-type="fig"}). However, the functional properties of these sites need to be further predicted and/or confirmed by mutational analysis, which was beyond the scope of the current study.
The FYWFQNH, FYWFQNR and YNWFQNR motifs (from 50th to 56th amino acid in the HB domain) have been reported as representative markers for the three main clades, respectively \[[@B18-ijms-19-03470],[@B23-ijms-19-03470]\]. In the present study, we confirmed these motifs in *B. napus* as well but the Y residue in the YNWFQNR motif was replaced by C (Cys) in BnWOX19 and BnWOX41 in the ancient clade. Together, our analysis of the protein sequences indicated that the HB domains are highly conserved in BnWOXs.
2.4. Gene Structure and Intron Pattern Analysis {#sec2dot4-ijms-19-03470}
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Structural diversity of genes can provide an important clue for the function and evolution of multigene families \[[@B24-ijms-19-03470]\]; therefore, in the present study, we analyzed the gene structure of *WOX*s in *B. napus*, *B. rapa*, *B. oleracea* and *Arabidopsis* ([Figure S2](#app1-ijms-19-03470){ref-type="app"}).
We did not observe intron insertions in the HB domains of the 73 *WOXs* from *Arabidopsis* and *B. napus*. Members of the same subclade generally had the same or similar exon/intron patterns, except for the WOX6 and WOX11/12 subclades, which showed complex exon--intron patterns and significant variation in the number of introns present ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}). The HB domains were located in the first exon in the WOX5 subclade and in the second exon in the WOX10/14 subclade, although they were commonly conserved in members of the same clade or subclade ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}). Moreover, 12 conserved intron patterns were found in the *BnWOX* gene family ([Table S2](#app1-ijms-19-03470){ref-type="app"}). Members of the ancient clade had two intron insertion sites; the first was conserved throughout all *WOX* members but the second differed between *WOX13* and *WOX10/14* homologs. In the intermediate clade, we identified three intron patterns and the intron insertion sites were similar in the WOX8/9 subclade, while members of the WOX11/12 subclade showed different intron insertion patterns and the number of introns was variable. Interestingly, the sequences at the C-terminal end of the genes in the WOX11/12 and WOX8/9 subclades were highly homologous; accordingly, several genes across these two subclades had a conserved intron insertion site in this region ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}). Similarly, in the modern clade, the intron insertion sites were generally highly conserved in each subclade, except the *WOX6* subclade, which had several members with an additional intron insertion site at the C- or N-terminus. Moreover, we found that very few intron patterns were different between members of the WOX1/WOX6 and WOX5/WUS subclades because of their high homology. These results indicated that these clades or subclades may have had a common ancestor and that intron insertion might be a driving force of functional differentiation during evolution.
We predicted that 25 *BnWOX*s were complementary to 31 microRNA sequences ([Table S3](#app1-ijms-19-03470){ref-type="app"}), which suggests that the *WOX* gene family may be regulated by microRNAs. Moreover, we found that 32%, 12% and 56% of *BnWOX*s belonged to the ancient, intermediate and modern clades, respectively, thus showing the concentration of the genes in the modern clade. This result indicated that the members of the clade might participate in a larger number of biological processes, as will be discussed below; however, the mismatch rate was as high as 33% so that the accuracy of these findings needs further experimental verification.
Overall, our results suggested that the intron loss/gain had occurred in *WOX*s outside the HB domain during the evolution of the gene family in Brassicaceae and the highly conserved or similar intron patterns that we identified further support the data of our phylogenetic analysis and classification.
2.5. Conserved Motif Analysis of B. napus WOX Proteins {#sec2dot5-ijms-19-03470}
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Specific motifs are generally related to functional conversion and diversion and conserved motifs could be identified in the 58 BnWOX proteins using the MEME software and sequence alignment.
Eleven conserved motifs (motifs 1--11) were identified in the full-length BnWOX proteins ([Table S4](#app1-ijms-19-03470){ref-type="app"}). With the exception of the conserved HB domain (motif 10) present in the members of all clades, the remaining domains (motifs) were only distributed in members of a given clade or subclade. In most cases, the same clade and subclade shared similar motif compositions, which further supported our phylogenetic analysis and classification data based on the homology of the HB domains in the WOX protein family. This indicated that some motifs were only shared by WOX proteins within the same clade or subclade, thus being subclade specific. For example, motifs 8 and 3 were present in all members of the modern and intermediate clades, respectively, while motif 7 was specific to the WOX11/12 subclade, which may be related to the acquisition of a novel function. However, several motifs were shared among clades or subclades, suggesting a common origin. For instance, motif 6 was common for the ancient clade and WOX11/12 subclade, which may indicate their close evolutionary relationship. No ancient clade-specific motif was found in this study.
In *Arabidopsis,* there are three functional domains in members of several subclades of the modern clade and these domains contribute significantly to the protein functions \[[@B3-ijms-19-03470],[@B26-ijms-19-03470]\]. Thus, the acidic region (motif 5) can potentially function as an activator domain \[[@B2-ijms-19-03470]\]; the WUS box (TLXLFP; motif 8) is essential for the transcriptional repressor activity and involved in the regulation of leaf blade outgrowth \[[@B5-ijms-19-03470]\]; and the EAR-like motif (motif 11) is involved in transcriptional repression \[[@B27-ijms-19-03470]\]. In the present study, although the acidic region and EAR-like (LXLXL) motif were not identified in the BnWOX proteins by MEME, we confirmed these two motifs in the modern clade by multi-sequence alignment analysis. The WUS box existed in most members of the modern clade, except those in the WOX7 subclade, while the acidic region was only present in the WUS subclade proteins and the EAR-like motif was merely present in the members of the WUS and WOX5/7 subclades, except AtWOX7 ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}). However, the acidic region was not strictly conserved among the WUS members from *B. napus* and similar results were obtained in Solanaceae species \[[@B28-ijms-19-03470]\], where several sites were substituted in the motif. Future research should examine whether these amino acid substitutions impact the functions of candidate genes in *B. napus*. Taken together, the organization of conserved motifs in each clade or subclade of the BnWOX gene family encoded proteins was strongly conserved. To some extent, these specific motifs may contribute to the functional divergence and conservation of BnWOX proteins.
2.6. Chromosomal Distribution and Duplication of B. napus WOX Genes {#sec2dot6-ijms-19-03470}
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To investigate the relationships between genetic divergence and gene duplications within the *B. napus WOX* gene family, we determined the chromosomal location of the candidate *BnWOX*s based on the genome annotation information from the Genoscope database and visualized the data with R \[[@B29-ijms-19-03470]\].
Our results showed that all but nine genes were located in the unanchored random regions and the last 49 *BnWOX*s were unevenly distributed on 18 of the 19 chromosomes (except for ChrC06) ([Figure 4](#ijms-19-03470-f004){ref-type="fig"}). For example, there were five genes on ChrA05 and ChrC02 each and only one on ChrA04. In general, *BnWOX*s were randomly arranged on the top and/or bottom of the chromosomes. *B. napus* (AnAnCnCn; *n* = 19) is an allotetraploid derived from the recent hybridization between *B. rapa* (AnAn; *n* = 10) and *B. oleracea* (CnCn; *n* = 9) \[[@B14-ijms-19-03470]\] and is, therefore, an ideal species for studying the effects of polyploidy on *WOX* gene expansion. In the present study, we also identified 27 and 30 *WOX* genes in the *B. rapa* and *B. oleracea* genomes, respectively and found that these genes were similar to those in the An and Cn sub-genomes of *B. napus* \[[@B14-ijms-19-03470]\].
Collinearity analysis revealed that there were strong orthologs among the *B. napus, B. rapa, B. oleracea* and *Arabidopsis WOX* genes ([Figure 4](#ijms-19-03470-f004){ref-type="fig"}). There was a tripling in *Brassica* species after diversion from their common ancestor with *Arabidopsis* \[[@B14-ijms-19-03470]\]. Thus, one *Arabidopsis WOX* should theoretically correspond to three orthologs in *B. rapa* and *B. oleracea*. However, the synteny between *WOX* genes of *B. rapa* and *B. oleracea* and their *Arabidopsis* homologs was less than expected (13:27:25; [Table S6](#app1-ijms-19-03470){ref-type="app"}), indicating that duplicated genes might have been lost during evolution \[[@B30-ijms-19-03470]\]. As expected, 42 of the 58 *BnWOX*s (70%) found in the *B. napus* genome had a syntenic relationship, among which 32 *BnWOX*s were inherited from *BoWOX*s (18 genes) and *BrWOX*s (14 genes). These results indicate that allotetraploidy was the main force for the rapid expansion of the *WOX* gene family in *B. napus*. Moreover, eight (19%) *BnWOX*s were obtained by segmental duplication events, including two, three and three genes in the ancient, modern and intermediate clades, respectively ([Table S5](#app1-ijms-19-03470){ref-type="app"}), while one gene was obtained by a homologous exchange. In addition, we found that eight (53%) *AtWOX*s, seven (23%) *BoWOX*s and 17 (60%) *BrWOX*s resulted from segmental duplication and whole-genome duplication in *Arabidopsis, B. oleracea and B. rapa,* respectively. Similar results have been reported for cotton \[[@B20-ijms-19-03470]\]. We found only one putative tandem duplication, located on ChrA06 (BnaA06g25460D and BnaA06g25450D) of *B. napus*, which indicates that tandem duplications may have contributed less to the expansion of the *WOX* gene family. Overall, our results indicate that the expansion of the *WOX* gene family in the *B. napus* genome was mainly due to whole-genome duplication (polyploidy) and segmental duplication.
2.7. Prediction of BnWOX Protein Interaction Networks in B. napus {#sec2dot7-ijms-19-03470}
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It has been reported that some WOX proteins function by forming complexes with other proteins \[[@B10-ijms-19-03470]\]. However, there is still no genome-wide overview of the protein interaction network of the WOX family members. Hence, on the basis of the data publicly available in STRING \[[@B31-ijms-19-03470]\], we predicted and constructed a protein--protein interaction network for BnWOXs, based on their orthology with AtWOXs and visualized these findings with Cytoscape \[[@B32-ijms-19-03470]\].
First, we predicted 38 interacting protein pairs for 10 AtWOXs, based on the *Arabidopsis* data in STRING (interaction score \>0.65). Among the 10 AtWOXs, one was in the intermediate clade, two in the ancient clade and seven in the modern clade, suggesting an increasing protein interaction trend in the modern clade. Among the 38 predicted proteins, 27% were peptides, 30% were TFs and the rest were other types of proteins. It is, therefore, clear that most proteins that interact with WOXs are peptides, such as CLAVATA3 (CLV3)/ESR-RELATED 40 (CLE40) and CLV3 ([Table S7](#app1-ijms-19-03470){ref-type="app"}). CLE peptides are a well-known group of post-translationally modified signal molecules involved in cell division in SAM, the root apical meristem (RAM) and vascular meristem, which can respond to stress signals \[[@B10-ijms-19-03470],[@B33-ijms-19-03470],[@B34-ijms-19-03470]\]. Previous reports have demonstrated that AtWUS, AtWOX4, AtWOX5 and AtWOX14 can regulate cell division in SAM, vascular meristem and RAM \[[@B10-ijms-19-03470],[@B35-ijms-19-03470]\], respectively, while WUS--CLV interactions were shown to establish a feedback loop between stem cells and the underlying regulatory center \[[@B7-ijms-19-03470]\]. In addition, the results of the present study showed that AtWOXs could interact with some TFs, such as GRAS, SCR and SHR ([Table S7](#app1-ijms-19-03470){ref-type="app"}). It is interesting that homologs of WOX5, SCR and SHR have been reported to be important for root development \[[@B36-ijms-19-03470]\]. Although their interaction was not demonstrated, the data indicated that WOX proteins might regulate the root development by interacting with GRAS proteins.
Based on the orthologous relationship between *B. napus* and *Arabidopsis*, a total of 463 interacting protein pairs were predicted for BnWOXs ([Figure 5](#ijms-19-03470-f005){ref-type="fig"}). Our results showed that protein interactions might be common for this gene family and that WOX proteins, especially those in the modern clade, might play a conserved role in regulating proliferation and differentiation of meristem stem cells. Our study provides important information needed to further investigate the molecular mechanisms of BnWOXs in *Brassica* species.
2.8. Expression Analysis of WOX Genes at Different Developmental Stages in B. napus {#sec2dot8-ijms-19-03470}
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Gene expression is associated with biological function of the encoded protein; therefore, we examined the expression patterns of the 58 *BnWOX*s in 50 *B. napus* tissues/organs, based on the RNA-seq data from the Gene Expression Omnibus database at the National Center of Biotechnology Information. To make the image used for the expression analysis more intuitive, we excluded from the heatmap ([Figure 6](#ijms-19-03470-f006){ref-type="fig"}) data on 18 *BnWOX*s with no or weak expression (fragments per kilobase of transcript per million mapped reads (FPKM) \< 1), which are most likely pseudogenes or expressed under certain conditions. We found that all *BnWOX*s were likely to be expressed in a limited number of vegetative tissues and reproductive organs, with relatively more genes expressed in root, stem and seed tissues, suggesting possible temporal and spatial expression patterns. Moreover, expression patterns of members of each clade were relatively conserved. We did not observe any expression of the genes from the ancient clade, except for *BnWOX19*, *BnWOX48*, *BnWOX41*, *BnWOX43* and *BnWOX13*; the remaining genes showed conserved expression patterns in roots, stems and pistils at flowering stages ([Figure 6](#ijms-19-03470-f006){ref-type="fig"}). Fourteen members of the intermediate clade were conserved and preferentially expressed in seeds, especially in the seed coat and embryo tissues, suggesting that they may contribute to seed maturation and embryonic development. This result was consistent with those of a previous report, which indicated that members of the WOX8/9 subclade regulated tissue proliferation during embryonic development in *Arabidopsis* \[[@B37-ijms-19-03470]\]. In the modern clade, *BnWOX*s showed a relatively wider expression pattern than did those from the other two clades and were highly expressed in the roots, stems, flowers and seeds at different stages, which indicated that members of the modern clade may play tissue-specific roles. In general, the expression patterns were different for members of different subclades in the modern clade but very similar within the same subclade. For example, *BnWOX10*, *BnWOX18*, *BnWOX44* and *BnWOX50* from the WOX4 subclade were highly expressed in roots and stems, while *BnWOX27*, *BnWOX40* and *BnWOX11* from the WOX1 subclade showed high levels of expression in the pistil. The relatively wider expression patterns of members of the modern clade were consistent with the data from previous reports, which showed that this clade originated later and underwent clear functional divergence from the other clades in this gene family \[[@B2-ijms-19-03470]\]. Together, these results show that *BnWOX*s generally have narrow expression profiles, being predominantly expressed in roots, stems and seeds and that the expression patterns in each clade or subclade reflect functional conservation.
2.9. Responses of BnWOX Genes to Environmental Stresses and Phytohormone Induction {#sec2dot9-ijms-19-03470}
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Previous studies on *WOX* genes from various species have mainly focused on plant development \[[@B38-ijms-19-03470],[@B39-ijms-19-03470]\], while their responses to environmental stresses and hormone induction were seldom evaluated. Therefore, in the present study, a comprehensive expression analysis of *BnWOX*s, based on RNA-seq data, was performed in roots after treatment with five hormones, including the auxin indole-3-acetic acid (IAA), abscisic acid (ABA), the cytokinin 6-benzyladenine (6-BA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and gibberellic acid (GA). Our results showed no obvious changes in the expression levels of more than half of *BnWOX*s after phytohormone treatment; however, several members of the WOX4 subclade were induced in seedling roots, which indicated their possible roles in hormone responses ([Figure S3](#app1-ijms-19-03470){ref-type="app"}).
To further elucidate these expression results and gain an insight into the expression of *BnWOX*s in response to abiotic stresses (salt and drought), four members of the WOX4 subclade (*BnWOX10*, *BnWOX50*, *BnWOX44* and *BnWOX18*) were selected to investigate their responses to hormone, salt and polyethylene glycol (PEG) stresses using qRT-PCR to evaluate changes in gene expression. The results of qRT-PCR analysis were similar to those of our RNA-seq analysis of phytohormone treatment responses, that is, the members of the WOX4 subclade were differentially regulated by different stress treatments ([Figure 7](#ijms-19-03470-f007){ref-type="fig"}). Moreover, as a sister pair, *BnWOX50* and *BnWOX18* showed similar stress responses, being repressed by almost all exogenous phytohormones, although 6-BA, which downregulated the expression of these genes at 1, 3 and 6 h, gradually reversed this inhibitory effect at 12 and 24 h. Moreover, three-fold inhibition of *BnWOX50* and *BnWOX18* expression was observed after NaCl and PEG treatments, compared with their expression in the untreated control, indicating that these genes may be involved in drought and salt resistance. On the other hand, the *BnWOX10* and *BnWOX44* genes showed differential expression patterns under some stress conditions, while being similarly upregulated by ABA treatment and downregulated after 6 h of IAA treatment. The expression of *BnWOX44* was upregulated by ACC, GA, 6-BA, NaCl and PEG treatments, while *BnWOX10* was repressed by the same treatments, indicating functional divergence between these genes. Overall, members of the WOX4 subclade may play vital roles in responses to salt and drought stresses, as well as to phytohormones, which makes these genes candidates for further study of *B. napus* abiotic stress responses.
3. Discussion {#sec3-ijms-19-03470}
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3.1. Structural and Functional Conservation of the Plant WOX Gene Family {#sec3dot1-ijms-19-03470}
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The WOX gene family is specific to plants and has been identified in rice, sorghum, maize, *Arabidopsis* and Norway spruce \[[@B3-ijms-19-03470],[@B12-ijms-19-03470],[@B13-ijms-19-03470]\]. *WOX* genes are well known to play key roles in the development and functional conservation in various plant species \[[@B2-ijms-19-03470],[@B6-ijms-19-03470],[@B8-ijms-19-03470],[@B40-ijms-19-03470]\]. To confirm this conservation of the WOX gene family across land plants, we summarized the functions of *WOX* genes that have been characterized in plants to date ([Table S11](#app1-ijms-19-03470){ref-type="app"}). We found that members of the ancient clade were mainly involved in the regulation of root development; members of the intermediate clade were involved in embryogenesis and morphological development and members of the modern clade were mainly involved in meristem maintenance. Although functional differentiation has occurred in the modern clade, the functions of members of each subclade are relatively conserved. For example, members of the WOX4 subclade in *Arabidopsis* and rice mainly promote vascularization \[[@B10-ijms-19-03470]\]; the expression profile in *B. napus* in the present study was consistent with previous findings, further supporting functional conservation across different species.
Notably, we found that functional conservation of each clade or subclade was supported by a highly conserved gene structure, illustrated by conserved motif and intron patterns. The HB domain contains a helix--loop--helix--turn--helix structure \[[@B22-ijms-19-03470]\], which can recognize sequence-specific targets in a precise spatial and temporal manner. Moreover, the domain is conserved in different species, thus maintaining its functional integrity \[[@B2-ijms-19-03470],[@B3-ijms-19-03470]\]. In addition, we found representative markers, YNWFQNR, FYWFQNH and FYWFQNR, in all three clades in *B. napus*, while members of the modern and intermediate clades contained conserved substitutions at the H and R residues, which may be responsible for their functional diversification ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}). Interestingly, we found that these representative markers and some conserved amino acid residues were located in the helix 3 region of the HB domain. Thus, we speculate that helix 3 plays a pivotal role in the functional differentiation between different clades. Although the functions of the proteins in the modern clade were relatively varied, they were conserved within the same subclade. There were also several conserved substitutions outside the helix 3 region in the modern clade; thus, 27%, 50%, 19% and 28% of subclade-specific sites existed in the helix 1, loop, helix 2 and turn regions, respectively, indicating that the loop and turn regions were also main regions related to the functional differentiation in the modern clade ([Figure 3](#ijms-19-03470-f003){ref-type="fig"}).
We observed that the conserved motifs outside the HB domain were specific for each clade or subclade, indicating that these motifs were likely required for specific protein functions. In the modern clade, nearly all the members (except AtWOX7) shared a conserved WUS box. It has also been reported that STF/WOX1 interacted with TOPLESS (TPL)/TPL-related proteins through the conserved WUS box to regulate the blade outgrowth by mediating cell proliferation in *Medicago truncatula* \[[@B41-ijms-19-03470]\]. Similarly, the results of the present study revealed that TPL could interact with WOX5 homologs, suggesting that this may be a common mechanism for the repressive function of the WUS box in members of the modern clade. The EAR-like domain was shared by members of the WUS and WOX5/7 subclades and has been reported to act as a transcriptional repressor \[[@B3-ijms-19-03470]\]. In addition, a previous report has indicated that the acidic region was related to the activation of transcription of WOX proteins \[[@B2-ijms-19-03470]\] and was only found in the WUS subclade. These results demonstrated that specific motifs were conserved in proteins belonging to certain clades and subclades and could play important roles in the functional conservation in the WOX gene family. Consequently, the other motifs identified in this study may also be critical for gene function, although their roles still need further experimental clarification.
Introns can be specifically inserted and conserved in plant genomes and are related to functional diversity as exons are lost or gained during evolution \[[@B18-ijms-19-03470]\]. In the present study, we found that intron insertion sites were similar or conserved in the same clades or subclades ([Table S2](#app1-ijms-19-03470){ref-type="app"}), which further supports the division and functional conservation of subclades. Members of the WOX4 subclade, which are involved in the maintenance of vegetative and reproductive meristems \[[@B10-ijms-19-03470]\], shared conserved patterns of introns. Differences in the intron patterns between the WOX2 and WOX1 subclades corresponded to their involvement in zygotic apical cell and lateral organ development, respectively. Thus, the intron insertion patterns may contribute to functional conservation and differentiation.
The results of our analysis of the relationships of conserved motifs and intron patterns with the gene structure and expression profiles of *BnWOX*s indicate that there are homologous genes in each clade and/or subclade, which contribute to functional conservation in different plant species. The results presented here will be useful for selecting appropriate candidate genes for further functional research in *B. napus.*
3.2. Conservation of Expression Profiles in Different Plants Supports Their Functional Conservation {#sec3dot2-ijms-19-03470}
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The expression pattern of a gene often correlates with its function and previous studies have investigated the expression of *WOX*s in rice and *Arabidopsis* \[[@B3-ijms-19-03470],[@B14-ijms-19-03470],[@B42-ijms-19-03470]\]. To further confirm the functional conservation and differentiation during the evolution of land plants, we analyzed *WOX* gene expression patterns in rice, soybean, *B. napus, B. oleracea and B. rapa* ([Figure S4](#app1-ijms-19-03470){ref-type="app"}). Our results indicated that the expression patterns of members of the intermediate and ancient clades were generally conserved and they were expressed at high levels in green pods and roots in soybean ([Figure S5](#app1-ijms-19-03470){ref-type="app"}). However, the expression of ancient-clade genes differed between rice and soybean, indicating that functional differentiation occurred during the evolution of monocot and dicot plants.
As mentioned above, *B. napus* is a typical allotetraploid, making it an ideal model for studying the effect of naturally occurring polyploidy on genetic conservation \[[@B14-ijms-19-03470]\]. To investigate the functional conservation and differentiation in the *WOX* gene family, we compared the expression patterns and sequence identities/similarities of the HB domains, as well as the full-length protein, gene and promoter sequences of 32 orthologous pairs between *B. napus, B. oleracea* and *B. rapa* and of 23 sister pairs from *B. napus* ([Table S9](#app1-ijms-19-03470){ref-type="app"}). We found that *BoWOX*s and *BrWOX*s were also mainly expressed in vegetative (root, stem and leaf) and reproductive (flower and silique) organs. Most members of the intermediate clade showed no or very low expression levels compared to those of members of the other two clades, which may indicate that the former are pseudogenes or need to be induced by specific stresses. The expression of *WOX*s from the ancient and intermediate clades was conserved across *B. napus, B. oleracea* and *B. rapa* and was mainly observed in roots ([Figure S3](#app1-ijms-19-03470){ref-type="app"}). Our results also showed that most sister pairs shared a very high degree of sequence identity, with 96.9% identity in the HB domains and 86.32% identity in the full-length proteins. Accordingly, 73% (17 of 23) of the *BnWOX* paralogs shared similar expression patterns. Among these sister pairs, 80%, 85% and 55% belonged to the ancient, intermediate and modern clades, respectively, implying that these genes are functionally redundant. For example, the *BnWOX09* and *BnWOX58* pair from the intermediate clade was highly expressed in seed tissues, while the *BnWOX55* and *BnWOX14* pair from the ancient clade was highly expressed in roots. In contrast, several sister pairs underwent neo-functionalization or sub-functionalization, especially in the modern clade, so that their expression patterns were slightly or obviously different. For instance, in the *BnWOX24* and *BnWOX53* pair, only *BnWOX53* was highly expressed in the root ([Figure 6](#ijms-19-03470-f006){ref-type="fig"}). Furthermore, the promoter regions of sister pairs were significantly homologous, with the average sequence identity ranging from 20% to 86% ([Table S9](#app1-ijms-19-03470){ref-type="app"}). In addition, the expression patterns of sister pairs were closely related to their promoter sequence identities. Thus, we found that several sister pair genes that were differentially expressed had high sequence identity in the HB regions but less identity in the promoter regions, suggesting that the functional divergence of homologous *WOX* genes may have first occurred in the promoter regions during their evolution.
In this study, we identified 18 and 14 orthologous pairs from the *WOX* gene family in *B. rapa* and *B. oleracea* using the identity in the HB domain, which was 93.6% and 89.9%, respectively. However, the expression patterns of 62% of the orthologous pairs were slightly divergent or significantly different. For example, *BnWOX53* and *BoWOX12* were both expressed in roots but *BnWOX53* was also expressed in seeds, indicating that functional divergence occurred during the evolution of these two genes. Interestingly, approximately 60%, 20% and 20% of the *WOX* genes that exhibited differential expression belonged to the modern, intermediate and ancient clades, respectively ([Table S8](#app1-ijms-19-03470){ref-type="app"}), further supporting the hypothesis that the modern clade emerged recently in land plants and underwent functional diversification during the evolution. Furthermore, the average levels of identity of the promoter regions of orthologous pairs ranged from 8% to 99.7% and were generally much lower than those of the ORF regions, indicating that the promoter regions play vital roles in the functional diversity of *WOX* genes. Taken together, comparison of the expression patterns of *WOX* genes across different land plants revealed the major functional conservation and/or slight differentiation during the evolution of this gene family and indicated that the functional differentiation of these genes may be related to sequence diversity in their promoter regions.
4. Materials and Methods {#sec4-ijms-19-03470}
========================
4.1. Identification of WOX Proteins and Phylogenetic Analysis of the B. napus Genome {#sec4dot1-ijms-19-03470}
------------------------------------------------------------------------------------
The sequences of 15 *Arabidopsis WOX* genes were downloaded from the TAIR *Arabidopsis* genome (<http://www.arabidopsis.org/>) \[[@B43-ijms-19-03470]\] to aid in the identification of candidate genes encoding WOX proteins in *B. napus*. We performed a BLASTP search of the Genoscope genome database (<http://www.genoscope.cns.fr/brassicanapus/>) using the DNA-binding domain protein sequences of the *Arabidopsis WOX* genes as queries. To verify the reliability of our results, the protein functional and structural domains were predicted by PROSITE profiling (<http://www.expasy.org/tools/scanprosite/>) \[[@B44-ijms-19-03470]\] to confirm that each protein had the HB domain. We acquired the *B. oleracea* and *B. rapa* WOX protein sequences from Phytozome (<https://phytozome.jgi.doe.gov/pz/portal.html>) and BRAD (<http://brassicadb.org/brad/index.php>) using the same method as that used for *Arabidopsis*.
The biochemical properties of the candidates were predicted using ExPASy \[[@B45-ijms-19-03470]\] and subcellular localization was investigated using Cell-PLoc \[[@B46-ijms-19-03470]\].
To gain insights into the evolutionary history of the *WOX* gene family in *B. napus*, *B. rapa*, *B. oleracea* and *Arabidopsis*, we constructed an NJ tree based on a multiple sequence alignment of the HB domains, using MEGA version 7.0 \[[@B16-ijms-19-03470]\] with the following parameters: Poisson correction, pairwise deletion and a bootstrap with 1000 replicates. Tree files were viewed and edited with FigTree v1.3.1 (<http://tree.bio.ed.ac.uk/software/figtree/>). The MEGA 7.0 program was used to estimate the best model for the multiple sequence alignment of the HB domains by default. The JJT amino acid substitution model with estimation of the gamma distribution shape parameter (JJT + G + I) was suggested to be the best evolutionary models, based on the Akaike information criterion (AIC)statistics. An ML tree was constructed using PhyML 3.0 \[[@B17-ijms-19-03470]\] with 100 replicates and the JJT + G + I model.
4.2. Sequence, Gene Structure and Conserved Motif Analysis and Construction of the Protein Interaction Network {#sec4dot2-ijms-19-03470}
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To analyze the sequence features of the BnWOXs proteins, we performed a multiple alignment analysis of the HB domains using MAFFT version 7 with the default parameters (<https://mafft.cbrc.jp/alignment/server/>). To obtain optimized alignments, the deduced amino acid sequences were adjusted manually in MEGA 7.0 and BioEdit 7.0 (<http://www.psc.edu/biomed/genedoc/>) \[[@B16-ijms-19-03470]\] with the default parameters. The intron patterns, including the distribution, position and phases and the HB domain positions in the *B. napus WOX* genes were analyzed using the GSDS software 2.0 (<http://gsds.cbi.pku.edu.cn/>) \[[@B25-ijms-19-03470]\]. The intron insertion information for the *WOX* genes of *Arabidopsis*, *B. rapa* and *B. oleracea* was acquired from Phytozome (<https://phytozome.jgi.doe.gov/pz/portal.html>).
The MEME version 4.11.1 \[[@B47-ijms-19-03470]\] program was used to identify potential protein motifs, in addition to the HB domain, using the following parameter settings for the distribution of motifs: the maximum number of motifs, 10; the minimum width of a motif, 6; and the maximum width of a motif, 50. Only motifs with an E-value ≤ 1 × 10^−10^ were used for further analysis. The interaction network was constructed based on the orthologs of *BnWOX*s in *Arabidopsis* using the STRING platform (<https://string-db.org/?tdsourcetag=s_pctim_aiomsg>) and visualized with Cytoscape version 3.4.0 (Chongqing, China).
4.3. Chromosomal Location and Synteny Analysis {#sec4dot3-ijms-19-03470}
----------------------------------------------
The gene loci for *B. napus WOX* genes were extracted from the Genoscope genome database. The synteny relationships of *WOX* genes from *Arabidopsis*, *B. napus*, *B. oleracea* and *B. rapa* were acquired from CoGe (<https://genomevolution.org/coge/>). The R package \[[@B29-ijms-19-03470]\] was used to view the chromosomal locations of the candidates and their collinearity.
4.4. Analysis of Expression Profiles of *BnWOX*s at Different Developmental Stages and After Hormone and Stress Treatment {#sec4dot4-ijms-19-03470}
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The temporal and spatial expression patterns of candidate *BnWOX*s were further analyzed using the RNA-seq data from 50 different tissues, which included roots, stems, leaves, flowers, seeds and siliques from the *B. napus* cultivar "Zhongshuang 11" (ZS11) at different developmental stages (e.g., germination, seedling, budding, initial flowering and full-bloom stages). Seedling roots treated with hormones (e.g., IAA, GA, 6-BA, ABA and ACC) were also collected for analysis. The data were analyzed with Cluster 3.0 (Chongqing, China) \[[@B32-ijms-19-03470]\] and a heatmap was drawn using the R package.
For qRT-PCR, seeds of ZS11 were obtained from the College of Agriculture and Biotechnology, Southwest University (Chongqing, China) and germinated on Petri dishes. At the five-leaf stage, seedlings were treated with Hoagland's liquid medium, which contained a 15% (*w*/*v*) PEG 6000 solution to simulate a drought condition, 200 mM NaCl, or phytohormones (50 µM ABA, 120 µM GA, 75 µM 6-BA, 60 µM ACC and 10 µM IAA), and grown in an artificial climate chamber at 25 °C under a 14-h/10-h (day/night) photoperiod. Root tissue was harvested at 0, 1, 3, 6, 12 and 24 h of treatment, immediately frozen in liquid nitrogen and stored at −80 °C for RNA isolation.
Extraction of total RNA from root samples and subsequent cDNA synthesis were performed as described previously \[[@B48-ijms-19-03470]\]. The SYBR Premix ExTaq™ II kit (Takara Biotechnology, Dalian, China) was used for qRT-PCR amplification in a CFX Connect™ real-time PCR system (Bio-Rad, Chongqing, China) and the SYBR Green PrimeScript RT-PCR kit (Takara Biotechnology). Each reaction was conducted in a 10 µL volume and contained 5 µL of PCR master mix, 2.5 µL of double distilled H~2~O (ddH~2~O), 2 µL of diluted template and 0.25 µL of each gene-specific primer ([Table S10](#app1-ijms-19-03470){ref-type="app"}). Three biological replicates were performed and three technical replicates were taken for each biological replicate. The *Actin7* gene (GenBank accession no. AF024716) was used as an internal control. The reaction conditions for real-time PCR were as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing at 58 °C for 30 s. The relative gene expression levels were calculated using the 2^−ΔΔ*C*t^ method \[[@B49-ijms-19-03470]\].
Supplementary materials can be found at <http://www.mdpi.com/1422-0067/19/11/3470/s1>.
######
Click here for additional data file.
H.D. developed the conception of the study; M.-M.W., J.-N.L. and H.D drafted and revised the manuscript. M.-M.W., H.D., M.-M.L., J.W., F.R., Y.-W.W. and P.-C.G. contributed to data analysis. M.-M.W., H.D., Y.-Z.K. and P.-F.L. conceived and designed the experiments. All authors reviewed and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (31471528 and 31671727), the "111" Project of China (B12006) and the National Basic Research Program of China (973 Program; 2015CB150201).
The authors declare no conflict of interest.
ABA
abscisic acid
ACC
1-aminocyclopropane-1-carboxylic acid
CLV3
CLAVATA3
CLE40
CLAVATA3/ESR-RELATED 40
FPKM
fragments per kilobase of transcript per million mapped reads
GA
gibberellic acid
GSDS
gene structure display server
HB domain
homeodomain
IAA
indole-3-acetic acid
ORF
open reading frame
PEG
polyethylene glycol
qRT-PCR
quantitative real-time polymerase chain reaction
RAM
root apical meristem
RNA-seq
RNA sequencing
SAM
shoot apical meristem
TPL
TOPLESS
WOX
WUSCHEL-related homeobox
ZS11
"Zhongshuang 11"
{#ijms-19-03470-f001}
{#ijms-19-03470-f002}
![Gene structure and conserved motifs in *WOX*s from *Arabidopsis* and *B. napus*. (**a**) The neighbor-joining tree was generated from the alignment of 73 *WOX* genes from *Arabidopsis* and *B. napus*. The different background colors represent the nine subclades, which are supported by high bootstrap values. The colored dots indicate the 12 conserved intron insertion patterns. (**b**) Gene structures were generated using the Gene Structure Display Server (GSDS 2.0) \[[@B25-ijms-19-03470]\]. Green boxes indicate exons; black lines indicate introns and pink boxes represent the HB domains. Numbers 0, 1 and 2 represent intron phases. (**c**) Conserved motifs were detected using MEME and are shown as boxes of different colors. Motif sequences are provided in [Table S4](#app1-ijms-19-03470){ref-type="app"}.](ijms-19-03470-g003){#ijms-19-03470-f003}
{#ijms-19-03470-f004}
{#ijms-19-03470-f005}
{#ijms-19-03470-f006}
{#ijms-19-03470-f007}
ijms-19-03470-t001_Table 1
######
Features of the 58 *BnWOX* genes from *Brassica napus*, identified in this study.
Gene Genome ID Chromosome Protein Length (Amino Acids) cDNA Length (bp) Introns/Exons pI Molecular Weight (Da) Subcellular Localization
----------- --------------- --------------- ------------------------------ ------------------ --------------- ------ ----------------------- --------------------------
*BnWOX01* BnaA01g01910D chrA01 256 940 2/3 5.72 28,458.4 Nucleus
*BnWOX02* BnaA01g34040D chrA01 289 870 3/4 6.48 31,778.6 Nucleus
*BnWOX03* BnaA02g07100D chrA02 175 528 2/3 6.36 19,236.2 Nucleus
*BnWOX04* BnaA02g24210D chrA02 320 1053 2/3 6.96 35,591.2 Nucleus
*BnWOX05* BnaA02g36550D chrA02 302 909 2/3 7.09 34,348.7 Nucleus
*BnWOX06* BnaA03g22070D chrA03 226 681 1/2 9.33 26,087 Nucleus
*BnWOX07* BnaA03g53400D chrA03 263 1036 2/3 5.3 29,170.3 Nucleus
*BnWOX08* BnaA04g16520D chrA04 234 705 1/2 9.32 27,143.2 Nucleus
*BnWOX09* BnaA05g09770D chrA05 390 1173 2/3 8.35 43,341.9 Nucleus
*BnWOX10* BnaA05g18600D chrA05 251 756 2/3 9.63 28,555.1 Nucleus
*BnWOX11* BnaA05g22250D chrA05 351 1287 3/4 8.59 40,110.9 Nucleus
*BnWOX12* BnaA05g27750D chrA05 191 718 1/2 8.87 22,361 Nucleus
*BnWOX13* BnaA05g32800D chrA05 326 981 3/4 6.63 35,661 Nucleus
*BnWOX14* BnaA06g14590D chrA06 203 768 2/3 5.43 22,850.2 Nucleus
*BnWOX15* BnaA06g25450D chrA06 294 885 2/3 7.81 33,475.7 Nucleus
*BnWOX16* BnaA07g02390D chrA07 302 909 2/3 6.85 34,305.6 Nucleus
*BnWOX17* BnaA07g11310D chrA07 133 402 1/2 7.89 15,260.3 Nucleus
*BnWOX18* BnaA08g04100D chrA08 256 942 2/3 9.31 29,143.6 Nucleus
*BnWOX19* BnaA08g14960D chrA08 239 720 3/4 5.73 27,383.7 Nucleus
*BnWOX20* BnaA09g09400D chrA09 316 1022 3/4 6.89 35,862 Nucleus
*BnWOX21* BnaA09g18650D chrA09 267 804 4/5 6.41 30,920.3 Nucleus
*BnWOX22* BnaA09g45340D chrA09 275 901 3/4 6.28 31,962 Nucleus
*BnWOX23* BnaA10g12360D chrA10 210 633 1/2 6.83 23,085.3 Nucleus
*BnWOX24* BnaA10g16970D chrA10 274 949 2/3 6.34 30,342.5 Nucleus
*BnWOX25* BnaA10g24790D chrA10 192 579 1/2 9.17 22,114.9 Nucleus
*BnWOX26* BnaAnng01480D chrAnn_random 191 576 1/2 6.66 21,832.3 Nucleus
*BnWOX27* BnaAnng11050D chrAnn_random 353 1062 3/4 6.97 40,197.6 Nucleus
*BnWOX28* BnaAnng35720D chrAnn_random 166 501 2/3 8.99 19,173.4 Nucleus
*BnWOX29* BnaAnng36010D chrAnn_random 121 366 1/2 9.61 13,962.8 Nucleus
*BnWOX30* BnaAnng41310D chrAnn_random 247 847 1/2 8.77 27,574.8 Nucleus
*BnWOX31* BnaC01g03050D chrC01 265 1016 2/3 5.67 29,250.4 Nucleus
*BnWOX32* BnaC01g40590D chrC01 289 972 3/4 6.48 31,778.6 Nucleus
*BnWOX33* BnaC02g02200D chrC02 191 576 1/2 5.18 46,938.7 Nucleus
*BnWOX34* BnaC02g07490D chrC02 283 852 2/3 5.08 71,825.3 Nucleus
*BnWOX35* BnaC02g08530D chrC02 121 366 1/2 6.09 31,334.6 Nucleus
*BnWOX36* BnaC02g10040D chrC02 199 600 1/2 5.95 21,671.7 Nucleus
*BnWOX37* BnaC02g32050D chrC02 320 1277 2/3 6.96 35,542.2 Nucleus
*BnWOX38* BnaC03g18850D chrC03 397 1303 3/4 6.57 44,157.5 Nucleus
*BnWOX39* BnaC03g26450D chrC03 222 669 1/2 9.26 25,657.6 Nucleus
*BnWOX40* BnaC03g40380D chrC03 350 1053 3/4 7.72 40,024.6 Nucleus
*BnWOX41* BnaC03g62360D chrC03 171 641 1/2 7.65 19,697.3 Nucleus
*BnWOX42* BnaC04g39860D chrC04 236 711 1/2 9.3 27,260.4 Nucleus
*BnWOX43* BnaC04g46990D chrC04 195 588 2/3 5.51 21,785.3 Nucleus
*BnWOX44* BnaC05g25380D chrC05 251 908 2/3 9.58 28,526 Nucleus
*BnWOX45* BnaC05g41930D chrC05 191 718 1/2 8.87 22,485.1 Nucleus
*BnWOX46* BnaC05g48100D chrC05 339 1020 3/4 5.98 37,085.8 Nucleus
*BnWOX47* BnaC07g06960D chrC07 302 2162 2/3 7.36 34,407.9 Nucleus
*BnWOX48* BnaC07g15200D chrC07 195 588 2/3 5.35 21,686.1 Nucleus
*BnWOX49* BnaC07g45680D chrC07 261 1022 2/3 5.23 29,076.2 Nucleus
*BnWOX50* BnaC08g04810D chrC08 255 938 2/3 9.43 28,912.4 Nucleus
*BnWOX51* BnaC08g39150D chrC08 290 873 4/5 5.93 33,988.5 Nucleus
*BnWOX52* BnaC09g34650D chrC09 210 633 1/2 6.58 23,144.4 Nucleus
*BnWOX53* BnaC09g40100D chrC09 276 1794 2/3 5.97 30,547.8 Nucleus
*BnWOX54* BnaC09g49730D chrC09 192 579 1/2 8.99 22,099.8 Nucleus
*BnWOX55* BnaCnng49040D chrCnn_random 209 579 2/3 5.48 23,619.1 Nucleus
*BnWOX56* BnaCnng49570D chrCnn_random 280 843 1/2 5.97 31,047.2 Nucleus
*BnWOX57* BnaCnng51820D chrCnn_random 351 1314 3/4 8.4 40,001.6 Nucleus
*BnWOX58* BnaCnng62530D chrCnn_random 390 1954 2/3 8.35 43,232.7 Nucleus
| {
"pile_set_name": "PubMed Central"
} |
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Introduction
============
Barrett's esophagus is a premalignant condition of unknown etiology rarely reported in the pediatric population. Chronic gastrointestinal reflux seems to play the biggest role in its pathogenesis with other risk factors, including intellectual disability, developmental delay, cerebral palsy, gastric tube placement, and esophageal atresia \[[@REF1]-[@REF4]\]. Patients with Barrett's esophagus have a higher incidence of complications, including strictures, ulceration, and the potential development of adenocarcinoma. Pediatric patients, particularly those with intellectual disabilities, often have silent or non-specific symptoms, predisposing them to late presentation. Early recognition of high-risk children is crucial in the initial investigation and work up.
Barrett's esophagus in children with peptic strictures has not been well characterized, and its prevalence is not known \[[@REF1]\]. We report a case of peptic esophageal stricture with Barrett's esophagus in an adolescent patient with mild intellectual disability who presented with dysphagia and recurrent episodes of vomiting. Prompt, appropriate treatment can significantly improve the quality of life and lead to proper surveillance.
Case presentation
=================
A 13-year-old Caucasian male with mild intellectual disability, attention deficit hyperactivity disorder and constipation, was transferred to our tertiary care facility due to a two-month history of dysphagia with recurrent episodes of vomiting. Initially, the patient vomited only after ingesting solids, but this progressed to include liquids. Further history was difficult to obtain due to the child's intellectual disability and speech delay. History from various family members as well as caregivers at the psychiatric institution from where he was transferred asserted that the boy also experienced self-induced vomiting on occasion, heartburn, and a seven-pound weight loss in one week. His medications included polyethylene glycol and ranitidine.
His physical examination upon admission was completely unremarkable. Laboratory values were within normal limits, including complete blood count and differential, serum electrolytes, glucose, amylase, lipase, liver and kidney function tests, and thyroid-stimulating hormone level. Computed tomography (CT) scan of the abdomen, which initially was done to rule out any obstructive causes for his symptoms, revealed stool retention. Despite treatment with polyethylene glycol, which completely resolved the stool retention, he continued to have immediate postprandial emesis. Additional diagnostic testing with barium esophagram was performed, which revealed: "focal persistent narrowing of the proximal and mid-esophagus" (Figure [1](#FIG1){ref-type="fig"}).
{#FIG1}
External compression of the esophagus was ruled-out with a CT angiogram of the chest, which showed circumferential thickening of the esophagus (Figure [2](#FIG2){ref-type="fig"}). Pediatric gastroenterology was consulted, and an esophageal endoscopy with biopsy was performed, which showed a snug circumferential stricture with a diameter of approximately 6 mm at 24 cm from the incisors (Figure [3](#FIG3){ref-type="fig"}).
{#FIG2}
{#FIG3}
The biopsy results indicated the presence of erosive esophagitis. During the same sitting, endoscopic guided balloon dilation was done to 8 mm. The procedure was well tolerated without any complications, and he was discharged on high dose pantoprazole (40 mg twice daily) for erosive esophagitis with peptic esophageal stricture with Barret's esophagus (Figure [4](#FIG4){ref-type="fig"}).
{#FIG4}
Serial repeat dilations were done at three- to four-week intervals with resultant dilation of the esophagus to 15 mm (Figure [5](#FIG5){ref-type="fig"}). Since the procedures, the boy tolerated an advancing diet. Vomiting episodes resolved, and consistent weight gain occurred.
{#FIG5}
Discussion
==========
Our patient presented with dysphagia with recurrent episodes of vomiting and limited medical history. Initially, the differential diagnosis included more common entities such as gastroesophageal reflux disease (GERD), cyclic vomiting syndrome, or rumination syndrome. Barium esophagram aided the diagnosis of esophageal stricture. Our patient also had Barrett's esophagus, which appeared on endoscopy and confirmed by pathology. Diagnosis of peptic stricture can usually be suspected with a careful history but should be confirmed with a barium esophagram followed by endoscopy with biopsies \[[@REF2]\].
Though esophageal stricture presents in children, it is relatively rare. In the primary care setting, esophageal strictures are most commonly peptic strictures, which develop in 8% of individuals with GERD \[[@REF3],[@REF4]\]. Although they can occur in any age group, peptic strictures tend to occur most often in elderly patients \[[@REF5]\]. The occurrence of benign esophageal stricture increases with age, at a mean of 59 years \[[@REF6]\]. Potential complications include a chronic relapsing course, an increased risk of food impaction, an increased risk for pulmonary aspiration, co-existent Barrett's esophagus, and the need for esophageal dilation. Perforation secondary to dilation treatment can also contribute to the significant morbidity faced by patients with peptic strictures \[[@REF7]\].
Barrett's esophagus occurs in only 0.25% of children below 18 years presenting with upper gastrointestinal symptoms (dysphagia, epigastric pain, heartburn, vomiting, poor weight gain, etc.). Regarding etiology, there is a strong correlation between gastroesophageal reflux and Barrett's esophagus. However, in children, symptoms may be relatively silent due to the impaired sensitivity of the columnar lining to acid. Gastroesophageal reflux disease is commonly seen in patients with motor and intellectual disabilities. Studies have estimated up to 10-25% of institutionalized patients have symptoms of vomiting, rumination, or regurgitation. Barrett's esophagus in children with peptic strictures has not been well characterized, and its prevalence is not known \[[@REF1]\].
Endoscopic balloon dilation is the first-line therapy for the management of esophageal stenosis as it is a safe and effective procedure \[[@REF8]\]. Success rates reportedly range between 76% and 100% for esophageal strictures in children \[[@REF9]\]. Following esophageal dilation, patients should be treated with proton pump inhibitors (PPIs) to promote healing and reduce the risk of stricture recurrence. PPIs promote esophagitis healing, and esophagitis healing, in turn, improves dysphagia and decreases dilation need in patients with peptic stricture \[[@REF10]\]. Vigorously treating esophagitis with PPIs in patients with peptic strictures is a crucial part of the overall management \[[@REF11]\]. Although the malignant transformation into adenocarcinoma in children is rare, proper surveillance of Barrett's epithelium is vital in management \[[@REF12]\].
Conclusions
===========
Although peptic stricture with Barrett's esophagus is rare in children and typically presents with dysphagia, it needs to be included in the differential diagnosis of a child with the common symptom of vomiting in the setting of developmental delay. Barium esophagram and esophageal endoscopy with biopsy clarified the diagnosis in our patient, who had a vague history; he was then effectively treated with repeated balloon dilation and PPIs.
The authors have declared that no competing interests exist.
Consent was obtained by all participants in this study
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Gastric cancer is the fourth most common malignancy worldwide and the second most common cause of cancer-related deaths each year (10.4% of cancer deaths) [@pone.0040364-Parkin1]. Treatment of gastric cancer includes a combination of surgery, chemotherapy, or radiation therapy. However, nearly 60% of the patients affected succumb to gastric cancer even after a curative resection alone or after adjuvant therapy [@pone.0040364-Macdonald1]. It has long been known that gastric cancer results from a combination of environmental factors and the accumulation of generalized and specific genetic alterations. Many of the genetic or epigenetic alterations associated with gastric cancer, including loss of heterozygosity, microsatellite and chromosomal instability and hypermethylation, have been reported [@pone.0040364-Nobili1]. Understanding these alterations and the molecular mechanisms involved in gastric carcinogenesis will be critical for the improvement of diagnosis, therapy and prognosis prediction of this disease.
The eukaryotically conserved SWI/SNF chromatin-remodeling complex plays essential roles in a variety of cellular processes, including differentiation, proliferation and DNA repair [@pone.0040364-Reisman1]. Loss of SWI/SNF subunits has been reported in most tumors, and a large number of experimental observations suggest that this complex is critical for tumor suppression [@pone.0040364-Roberts1]. The complexes contain seven or more noncatalytic subunits that presumably help modulate the targeting and activity of the ATPase [@pone.0040364-Martens1]. One subunit of this complex, hSNF5/Ini1/BAF47, has been identified as a tumor suppressor [@pone.0040364-Biegel1], [@pone.0040364-Biegel2]. The other noncatalytic subunit, p270/ARID1A/BAF250a (adenine-thymine AT-rich interactive domain-containing protein 1A), has been demonstrated to be essential for normal cell cycle arrest [@pone.0040364-Nagl1]. Knockdown of *ARID1A* in a leukemia cell line confers resistance to Fas-mediated apoptosis [@pone.0040364-Luo1].
Recently, *ARID1A* mutations and loss of BAF250a protein have been found to correlate strongly with the ovarian clear-cell carcinoma and uterine low-grade endometrioid carcinoma [@pone.0040364-Wiegand1]--[@pone.0040364-Guan1]. These observations indicate that *ARID1A* is a potential candidate tumor suppressor gene. However, the clinical significance of such differential expression and the function of the ARID1A protein remain undefined due to the lack of studies using fresh human tumor samples. In the present study, we analyzed the *ARID1A* expression level in gastric cancer using real-time quantitative RT-PCR, western blotting and immunohistochemistry. Meanwhile, we identified the relationship between *ARID1A* expression and clinicopathological features and evaluated its prognostic value in post-resection survival of gastric cancer patients. Furthermore, we evaluated the functional role of *ARID1A* in the tumorigenesis of primary gastric cancer by examining the in vitro proliferation and colony formation in gastric cell lines.
Results {#s2}
=======
*ARID1A* mRNA Expression Analyzed by Real-time Quantitative RT-PCR {#s2a}
------------------------------------------------------------------
The mRNA level of *ARID1A* was determined by real-time quantitative RT-PCR assays in 66 paired cancerous and the matched adjacent normal gastric mucosa tissues. The *ARID1A* expression level was significantly lower in 43 (65.15%) tumor-bearing tissues compared with the adjacent non-tumor tissues (*P* = 0.0029, [Figure 1](#pone-0040364-g001){ref-type="fig"}).
{#pone-0040364-g001}
ARID1A Protein Expression Analyzed by Western Blotting {#s2b}
------------------------------------------------------
Western blotting was performed on 25 gastric cancer specimens and corresponding adjacent non-cancerous gastric mucosa tissues from the 66 paired samples. The results showed an ARID1A band at the expected size of 242 kDa and the amount of ARID1A protein present was further measured by densitometry. Consistent with the quantitative real-time PCR results, a decrease in ARID1A expression was seen in 13 (52%) of the gastric tumor tissues compared with matched adjacent non-tumor tissues (*P* = 0.0015, [Figure 2A](#pone-0040364-g002){ref-type="fig"} and [Figure 2B](#pone-0040364-g002){ref-type="fig"}). Likewise, the ARID1A protein expression was remarkably decreased in gastric cancer cell lines, SGC7901, AGS, especially in MGC803, compared with normal gastric cell line GES1 ([Figure 2C](#pone-0040364-g002){ref-type="fig"}).
{#pone-0040364-g002}
Immunohistochemical Analysis of ARID1A Expression in Gastric Cancer Tissue Samples and its Relationship with the clinicOpathological Parameters {#s2c}
-----------------------------------------------------------------------------------------------------------------------------------------------
To further investigate the clinicopathological and prognostic roles of ARID1A expression, we performed immunohistochemical analyses of the 224 paraffin-embedded gastric cancer tissue blocks. Overall, 115 of 224 (51.3%) cases showed negative ARID1A expression in cancerous tissues ([Figure 3B](#pone-0040364-g003){ref-type="fig"}), whereas 109 (48.7%) cases showed positive immunostaining ([Figure 3C&D](#pone-0040364-g003){ref-type="fig"}). Normal gastric mucosa showed the strongest ARID1A positive staining ([Figure 3A](#pone-0040364-g003){ref-type="fig"}). The correlations between the expression of ARID1A and various clinicopathological parameters are listed in [Table 1](#pone-0040364-t001){ref-type="table"}. The data showed that the loss of ARID1A expression was significantly correlated with depth of tumor infiltration (T stage, *P* = 0.001) and tumor grade (*P* = 0.006), but not with age, gender, tumor size, distant metastasis (M stage), and tumor locus or local lymph node metastasis (N stage).
{#pone-0040364-g003}
10.1371/journal.pone.0040364.t001
###### Correlation between ARID1A expression and clinicopathological variables of 224 gastric cancer cases.
{#pone-0040364-t001-1}
Clinicopathological parameters *n* [a](#nt101){ref-type="table-fn"} ARID1A expression χ^2^ *P* value
--------------------------------- -------------------------------------- ------------------- ------ ----------- ---------
**All** 224 109 115
**Age (years)**
\<55 104 46 58 1.525 0.230
≥55 120 63 57
**Gender** 3.395 0.068
Male 149 66 83
Female 75 43 32
**Tumor size** 3.850 0.053
\<3 cm 38 24 14
≥3 cm 186 85 101
**Tumor infiltration** 16.108 0.001\*
T1 32 22 10
T2 27 18 9
T3 155 68 87
T4 10 1 9
**Local lymph node metastasis** 6.733 0.081
N0 90 49 41
N1 77 39 38
N2 32 9 23
N3 25 12 13
**Distant metastasis** 3.061 0.102
M0 204 103 101
M1 20 6 14
**Grade** 9.812 0.006\*
1 7 5 2
2 41 28 13
3 176 76 100
Numbers of cases in each group. \* Statistically significant (*P*\<0.05).
Expression of ARID1A and Clinical Outcome {#s2d}
-----------------------------------------
The 5-year overall survival rates in patients with positive and negative ARID1A expression were 68.8% and 52.2%, respectively. The overall survival of patients with negative ARID1A expression was significantly worse than that of ARID1A-positive patients (*P* = 0.003, log-rank test, [Figure 4](#pone-0040364-g004){ref-type="fig"}). Univariate Cox regression analyses showed that depth of tumor infiltration, local lymph node metastasis, distant metastasis, tumor size and ARID1A expression were significantly associated with overall survival ([Table 2](#pone-0040364-t002){ref-type="table"}). Furthermore, a multivariate Cox regression analysis confirmed the depth of tumor infiltration, local lymph node metastasis, distant metastasis and ARID1A expression as independent predictors of the overall survival of gastric cancer patients ([Table 2](#pone-0040364-t002){ref-type="table"}).
{#pone-0040364-g004}
10.1371/journal.pone.0040364.t002
###### Univariate and multivariate analyses of overall survival of gastric cancer patients.
{#pone-0040364-t002-2}
Variables *n* [a](#nt103){ref-type="table-fn"} Univariate analyses Multivariate analyses
----------------------------- -------------------------------------- --------------------- ----------------------- ------------------------------------------ --------- ----------------- ------------------------------------------
**Age (years)** 0.141
\<55 104 1.000
≥55 120 1.357 0.904--2.309
**Gender** 0.923
Female 75 1.000
Male 149 0.980 0.648--1.482
**Tumor size** 0.002[\*](#nt104){ref-type="table-fn"} 0.479
\<3 cm 38 1.000 1.000
≥3 cm 186 4.218 1.714--10.381 1.450 0.519--4.056
Tumor infiltration 0.004[\*](#nt104){ref-type="table-fn"} 0.044[\*](#nt104){ref-type="table-fn"}
T1 32 1.000 1.000
T2 27 3.056E4 0.000--3.442E66 8.086E3 0.000--1.126E49
T3 155 9.661E4 0.000--1.086E67 1.897E4 0.000--2.635E49
T4 10 2.099E5 0.000--2.362E67 4.309E4 0.000--5.999E49
Local lymph node metastasis \<0.001[\*](#nt104){ref-type="table-fn"} \<0.001[\*](#nt104){ref-type="table-fn"}
N0 90 1.000 1.000
N1 77 3.286 1.805--5.984 1.956 1.023--3.743
N2 32 5.688 3.004--10.768 2.077 1.010--4.271
N3 25 7.717 3.895--15.288 5.225 2.524--10.818
Distant metastasis \<0.001[\*](#nt104){ref-type="table-fn"} \<0.001[\*](#nt104){ref-type="table-fn"}
M0 204 1.000 1.000
M1 20 6.347 3.854--10.453 5.230 2.998--9.124
Grade 0.504
1 7 1.000
2 41 1.968E4 0.000--1.550E60
3 176 2.704E4 0.000--2.127E60
ARID1A 0.003[\*](#nt104){ref-type="table-fn"} 0.029[\*](#nt104){ref-type="table-fn"}
Negative 115 1.000 1.000
Positive 109 0.525 0.342--0.805 0.611 0.393--0.950
HR, hazard ratio; CI, confidence interval;
Numbers of cases in each group;
Statistically ignificant (*P*\<0.05).
The Role of *ARID1A* in Cell Proliferation and Colony Formation in MGC803 and GES1 Cell Lines {#s2e}
---------------------------------------------------------------------------------------------
To evaluate the effects of *ARID1A* on cell proliferation**,** the *ARID1A* expression vector and the control vector were respectively transfected into MGC803 cells. *ARID1A* expression in transfected cells were detected by western blotting ([Figure 5A](#pone-0040364-g005){ref-type="fig"}). The cell growth assay revealed that cell growth rate in *ARID1A*-transfected gastric cancer cells were significantly lower than control vector-transfected gastric cancer cells ([Figure 5C](#pone-0040364-g005){ref-type="fig"}). Meanwhile, the efficiency of colony formation was significantly (*P* = 0.0379) inhibited in *ARID1A*-transfected gastric cancer cells compared with control vector-transfected gastric cancer cells ([Figure 5D](#pone-0040364-g005){ref-type="fig"}). To further confirm the proliferation suppression function of *ARID1A*, we silenced the *ARID1A* expression in GES1 cell line with siRNA. The *ARID1A* expression in transfected cells were detected by western blotting ([Figure 5B](#pone-0040364-g005){ref-type="fig"}). We found that silencing the expression of *ARID1A* in GES1 significantly enhanced cell proliferation compared with mock siRNA treatment ([Figure 5E](#pone-0040364-g005){ref-type="fig"}).
{#pone-0040364-g005}
Discussion {#s3}
==========
In spite of great advances in diagnosis and therapy, gastric cancer remains one of the most deadly neoplasms, with a dismal prognosis after radical gastrectomy [@pone.0040364-Jemal1], [@pone.0040364-Hartgrink1]. The clinical outcome of gastric cancer is determined by a series of tumor characteristics, such as locoregional tumor growth and invasion, differentiation grade, angiogenesis, distant metastasis and cell cycle progression, which are regulated by a variety of related genes, including oncogenes and tumor suppressor genes. Therefore, identification of gastric cancer-specific biomarkers involved in these procedures is very important for diagnosis, therapy and prognosis prediction in clinic.
*ARID1A*, a newly identified tumor suppressor gene which encodes a member of the SWI/SNF complex, has a high mutation frequency in bladder cancer, uterine endometrioid carcinoma, ovarian endometrioid and clear cell carcinoma [@pone.0040364-Wiegand1]--[@pone.0040364-Guan1], [@pone.0040364-Gui1], [@pone.0040364-Jones2]. In ovarian clear cell carcinoma, it is reported that *ARID1A* mutation is significantly associated with ARID1A immunoreactivity [@pone.0040364-Maeda1]. Recently, exome sequencing study revealed that *ARID1A* is also frequently mutated in gastric cancer [@pone.0040364-Wang1], [@pone.0040364-Zang1]. However, thus far the expression, clinical significance and biological functions of *ARID1A* in gastric cancer have not been explored. Therefore, we evaluated the expression of *ARID1A* in gastric cancer by real-time PCR, western blotting and immunohistochemistry, in addition to its clinicopathological and prognostic significance in a large human sample. Furthermore, using in vitro cell model, we also investigated the tumor suppressor role of *ARID1A* in gastric cells in detail.
In the current study, we demonstrated that *ARID1A* was expressed at both lower mRNA and protein level in gastric cancer tissues than corresponding non-cancerous mucosa. In agreement with these molecular biological findings, immunohisto- chemistry with a anti-ARID1A antibody showed that *ARID1A* was completely silenced in 115 out of 224 patient gastric cancer samples, with positive expression in another 109 patients. Our observation is in agreement with a series of studies revealing that *ARID1A* expression is frequently lost or reduced in a number of cancer tissues and cell lines, such as breast cancer, uterine endometrioid carcinoma, ovarian clear cell and endometrioid carcinoma \[13,18,20, and 21\].
To date, the causes of *ARID1A* silencing have not been fully elucidated. The existing studies focus on mutations in *ARID1A*, particularly in gynecologic cancers. It is reported that a nonsense or indel mutation of *ARID1A* was correlated with loss or reduction of protein expression in uterine endometrioid carcinoma, ovarian endometrioid carcinoma and clear cell carcinoma [@pone.0040364-Wiegand1]--[@pone.0040364-Guan1], [@pone.0040364-Maeda1]. In an integrated genomic investigation, Mamo *et al*. only found one truncated mutation of *ARID1A* in the T47D breast cancer cell line, without any mutation in the 11 breast cancer samples which showed DNA copy number loss at the 1p36.11 locus adjacent to *ARID1A* [@pone.0040364-Mamo1]. Eight of nine samples with DNA copy number loss at 1p36.11 also have low ARID1A protein expression, suggesting a concordance between DNA copy number loss and *ARID1A* inactivation. In the exome sequencing study by Wang *et al*., a total of 46 mutations was found in 32 out of 109 (29%) gastric cancer samples, with 39 (85%) truncated mutations [@pone.0040364-Wang1]. Twenty-four (75%) of the 32 gastric cancer samples with *ARID1A* mutations show either loss of or substantially reduced protein expression compared to those without *ARID1A* mutation. In contrast, there are only 6 gastric cancer samples showing absent or weak protein expression in the absence of detectable *ARID1A* mutation, which suggests that other mechanisms may contribute to *ARID1A* inactivation. Recently, another exome sequencing research by Zang *et al*. also showed *ARID1A* mutations in 8% of gastric samples, of which 75% lost or reduced the protein expression [@pone.0040364-Zang1]. More interestingly, both studies demonstrated higher *ARID1A* alterations in gastric cancer samples with microsatellite instability (MSI) than those with microsatellite stability (MSS). Moreover, the mutation spectrum of *ARID1A* is distinct between the two genetic types of gastric cancer, with most indels in the MSI type and more single-neucliotide variations in the MSS type. MSI is defined as indel mutations within nucleotide repeats (known as microsatellite regions) resulted from DNA mismatch repair gene inactivation-induced replication errors [@pone.0040364-Ottini1]. Proposed as the initiating genomic events of gastric cancer, MSI often leads to accumulation of additional cancer-related genetic instabilities, such as allelic losses and frameshift mutations in genes involved in cell proliferation regulation, apoptosis and DNA repair. It has been reported that MSI occurs in 25% to 50% of sporadic gastric cancer, defining a unique genetic type disease with different clinicopathological features [@pone.0040364-Ottini1]. In the study of Wang *et al*., the indel mutation rate (78%) of *ARID1A* in MSI gastric cancer is comparable to that of *TGFBR2* in MSI colon cancer, a well-established and functionally validated driver gene inactivated by MSI [@pone.0040364-Markowitz1]. These data indicate that the mutation of *ARID1A* together with MSI may play an important role in gastric carcinogenesis. Therefore, the relationship between *ARID1A* alterations and MSI status in gastric cancer, as well as its clinicopathological significance, needs further investigation in the future research.
In the study by Wang *et al.*, the expression of *ARID1A* was only detected in a small-size sample (32 cases), and there was no further exploration of its clinical significance [@pone.0040364-Wang1]. Here, in a larger gastric cancer population (224 cases), we found that the loss of *ARID1A* expression was significantly correlated with a higher T stage of gastric cancer, implying that absence of *ARID1A* expression may promote tumor growth and invasion. In addition, we detected lower ARID1A immunoreactivity in poorly differentiated gastric cancer tissues than in well-differentiated ones, suggesting that decreased *ARID1A* expression might play a role in tumor de-differentiation. Consistent with our findings, other investigators also found that decreased *ARID1A* expression is significantly associated with a higher grade of breast cancer [@pone.0040364-Mamo1], as well as a higher FIGO stage in ovarian clear cell carcinoma [@pone.0040364-Katagiri1]. ARID1A promotes the formation of BRG1 or BRM-contained SWI/SNF chromatin remodeling complexes, which are essential for normal cell cycle arrest [@pone.0040364-Nagl1], [@pone.0040364-Wilson1].
A Kaplan-Meier survival analysis showed a significant correlation between the loss of *ARID1A* expression and poorer clinical outcome of gastric cancer patients after radical operation. Cox hazard ratio regression analyses further demonstrated that the *ARID1A* expression level was an independent risk factor for survival, suggesting that it may serve as a valuable prognostic biomarker for gastric cancer patients after surgery and a potential target for gene therapy in the treatment of gastric cancer. In ovarian clear cell carcinoma, it was also reported that patients with positive *ARID1A* expression had a longer progression-free survival than those with negative *ARID1A* expression [@pone.0040364-Katagiri1]. Moreover, loss of *ARID1A* expression is significantly correlated with chemoresistance in ovarian clear cell carcinoma, which is also associated with a poor prognosis of cancer. These data suggest that *ARID1A* expression and mutation examination might be helpful to guiding clinical management. Taken together, our observations that the loss of *ARID1A* expression in gastric cancer is associated with more malignant phenotypes and a worse prognosis imply that it may play a tumor suppressor role in gastric carcinogenesis.
We further investigated the functional role of *ARID1A* in gastric cell lines. Restoring *ARID1A* expression in gastric cancer cells significantly inhibited cell proliferation and colony formation. Silencing the expression of *ARID1A* in gastric epithelial cells significantly enhanced the cell growth rate. These results indicated that *ARID1A* may play an import role in inhibiting tumor cell growth. Recently, functional assays of *ARID1A* in gastric cancer cell lines by Zang *et al*. suggested that *ARID1A* exert tumor-suppressor activity [@pone.0040364-Zang1]. Guan *et al*. demonstrated that restoring the expression of wild-type *ARID1A* is sufficient to suppress the proliferation and tumorigenecity of xenografts with human ovarian cancer cell lines harboring *ARID1A* mutations, while RNA interference-mediated *ARID1A* silencing enhances cellular proliferation and tumorigenicity in two non-transformed human ovarian epithelial cell lines, IOSE-80PC and OSE4 [@pone.0040364-Guan2]. These data, together with ours, suggest that loss of *ARID1A* may play an important role in the process of carcinogenesis.
In conclusion, we have demonstrated the loss of *ARID1A* expression in gastric cancer and its correlation with a more malignant phenotype and poorer prognosis in a large number of clinical samples. In addition, we proved that *ARID1A* can inhibit tumor cell growth and colony formation in vitro. To the best of our knowledge, the data generated in the current study represent the first report correlating the presence of *ARID1A* with clinicopathological characteristics and the overall survival of gastric cancer patients. Taken together with the results of Wang *et al.* and Zang *et al*. [@pone.0040364-Wang1], [@pone.0040364-Zang1], we further confirmed that *ARID1A* might serve as a candidate tumor suppressor and prognostic biomarker in gastric carcinogenesis.
Materials and Methods {#s4}
=====================
Ethics Statement {#s4a}
----------------
The research was approved by the Ethics Committee of Sun Yat-sen University Cancer Center, and written informed consent was obtained from each patient involved in the study.
Cell Lines and Culture Conditions {#s4b}
---------------------------------
The gastric cancer cell lines, SGC7901, AGS, MGC803, and the gastric epithelial mucosa cell line GES1 were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in RPMI 1640 media supplied with 10% heat-inactive fetal bovine serum (FBS). The cells were incubated at 37°C in a humidified chamber containing 5% CO~2~.
Human Tissue Sample*s* {#s4c}
----------------------
A total of 66 paired cancerous and matched adjacent noncancerous gastric mucosa tissues were collected from gastric cancer patients undergoing gastrectomy at Sun Yat-sen University Cancer Center between 2009 and 2011, and the diagnosis was confirmed by pathological examination. The 25 paired cancerous and corresponding adjacent noncancerous gastric mucosa tissues used to detect the ARID1A protein expression in western blotting were selected from the 66 paired samples. After surgical resection, fresh tissues were immediately immerged in RNAlater (Ambion, Inc., USA) to avoid RNA degradation, stored at 4°C overnight to allow thorough penetration of RNAlater into the tissue and then frozen at −80°C until RNA and protein extraction was performed. Another 224 paraffin-embedded primary gastric carcinoma samples which had been collected between 2003 and 2005, were obtained from the Sun Yat-sen University Cancer Center. None of these patients had received radiotherapy or chemotherapy prior to surgery. The follow-up data of the gastric cancer patients in this study are available and complete. Postoperative follow-up occurred at our outpatient department and included clinical and laboratory examinations every 3 months for the first 2 years, every 6 months during the third to fifth years, annually for an additional 5 years or until patient death, whichever occurred first. The histopathological type and stage of gastric cancer were determined according to the criteria of the World Health Organization classification and the TNM stage set out by the Union for International Cancer Control.
Extraction of Total RNA and Real-time Quantitative PCR {#s4d}
------------------------------------------------------
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, California, USA) according to the manufacturer's protocol. Total RNA concentration was assessed by measuring absorbance at 260 nm using a NANO DROP spectrophotometer (ND-1000, Thermo Scientific, USA). Reverse transcription (RT) to synthesize the first-strand of cDNA was performed with 2 µg of total RNA treated with M-MLV reverse transcriptase (Promega, USA) according to the manufacturer's recommendations. The resulting cDNA was then subjected to real-time quantitative PCR for evaluation of the relative mRNA levels of *ARID1A* and *GAPDH* (glyceraldehyde-3-phosphate dehydrogenase, as an internal control) with the following primers: *ARID1A* forward: 5′-CTTCAACCTCAGTCAGCTCCCA-3′, and reverse: 5′-GGTCACCCACCTCATACTCCTTT-3′; *GAPDH* forward: 5′-CTCCTCCTGTTCGACAGTCAGC-3′, and reverse: 5′-CCCAATACGACCAAATCCGTT-3′. Gene-specific amplification was performed using an ABI 7900HT real-time PCR system (Life Technologies, Carlsbad, California, USA) with a 15 µl PCR mix containing 0.5 µl of cDNA, 7.5 µl of 2 x SYBR Green master mix (Invitrogen, Carlsbad, California, USA), and 200 nM of the appropriate oligonucleotide primers. The mix was preheated at 95°C (10 min) and then amplified at 95°C (30 sec) and 60°C (1 min) for 45 cycles. The resolution curve was measured at 95°C for 15 sec, 60°C for 15 sec and 95°C for 15 sec. The Ct (threshold cycle) value of each sample was calculated from the threshold cycles with the instrument's software (SDS 2.3), and the relative expression of *ARID1A* mRNA was normalized to the *GAPDH* value. Data were analyzed using the comparative threshold cycle (2^-ΔCT^) method.
Western Blotting Analysis {#s4e}
-------------------------
The homogenized gastric cancer samples, including tumor and nontumor tissues, as well as cell lines, were lysed in RIPA lysis buffer, and the lysates were harvested by centrifugation (12,000 rpm) at 4°C for 30 min. Approximately 50 µg protein samples were then separated by electrophoresis in a 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane. After blocking the non-specific binding sites for 60 min with 5% non-fat milk, the membranes were incubated overnight at 4°C with a mouse monoclonal antibody against ARID1A (Abgent Primary Antibody Company, USA, at a 1∶1000 dilution). The membranes were then washed three times with TBST (tris-buffered saline with tween-20) for 10 min and probed with the horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (Immunology Consultants Laboratory, USA, at a 1∶2000 dilution) at 37°C for 1 hour. After three washes, the membranes were developed by an enhanced chemiluminescence system (Cell Signaling Technology, Danvers, Massachusetts, USA). The band intensity was measured by densitometry using the Quantity One software (Bio-Rad Laboratories, Inc. Hercules, CA, USA). The protein levels were normalized to that of GAPDH detected using mouse anti-human GAPDH monoclonal antibody (Shanghai Kangchen, China, at a 1∶10000 dilution).
Immunohistochemistry Analysis {#s4f}
-----------------------------
The tissue sections were deparaffinized with dimethylbenzene and rehydrated through 100%, 95%, 90%, 80% and 70% ethanol. After three washes in PBS (phosphate-buffered saline), the slides were boiled in antigen retrieval buffer containing 0.01 M sodium citrate-hydrochloric acid (pH = 6.0) for 15 min in a microwave oven. After rinsing with PBS, the tissue sections were incubated with primary antibody and the slides were then rinsed in 3% peroxidase quenching solution (Invitrogen) to block endogenous peroxidase. The sections were then incubated with a mouse monoclonal antibody against ARID1A (Abgent Primary Antibody Company, USA, at a 1∶300 dilution) at 4°C overnight and then incubated with horseradish peroxidase (HRP) (ChemMateTM DAKO EnVisionTM Detection Kit) at room temperature for 30 min. After washing in PBS, the visualization signal was developed with 3, 3′-diaminobenzidine (DAB) solution, and all of the slides were counterstained with hematoxylin. As negative controls, adjacent sections were processed as described above except that they were incubated overnight at 4°C in blocking solution without the primary antibody.
The total ARID1A immunostaining score was calculated as the sum of the percent of positively stained tumor cells and the staining intensity. Briefly, the percentage of positive staining was scored as 0 (0--9%, negative), 1 (10%--25%, sporadic), 2 (26%--50%, focal) or 3 (51%--100%, diffuse), and the intensity as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (dark staining). The total immunostaining score was calculated with the value of percent positivity score × staining intensity score, which ranged from 0 to 9. The expression level of ARID1A was defined as following: "−" (negative, score 0), "+" (weakly positive, score 1--3), "++" (positive, score 4--6), "+++" (strongly positive, score 7--9). Based on the ARID1A expression levels, the gastric cancer patients were divided into two groups: negative ARID1A expression group (ARID1A-) and positive ARID1A expression group (ARID1A+, ARID1A++ or ARID1A+++).
Expression Plasmid and Transient Transfections {#s4g}
----------------------------------------------
A eukaryotic expression plasmid pCMV6-Entry containing the full-length of human *ARID1A* cDNA was obtained from the Asbio Technology Company (Guangzhou, China). Empty vector was used as negative control. MGC803 cells were cultured in 6-well plates until they reached 85--90% confluence, and then transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, gene expression was examined by western blotting analysis. And then, cell proliferation and colony formation were performed.
RNA Oligonucleotides and Cell Transfections {#s4h}
-------------------------------------------
For knockdown of *ARID1A* expression, the siRNAs were synthesized by GenePharma Company (Shanghai, China). The siRNA sequences were as follows: siRNA-*ARID1A*, sense: 5′GCCCUAACAUGGCCAAUAUTT3′, antisense: 5′AUAUUGGCCAUGUUAGGGCTT3′. The negative control (NC), sense: 5′UUCUCCGAACGUGUCACGUTT3′, antisense: 5′ACGUGACACGUUCGGAGAATT3′. 400 pmol siRNA-*ARID1A* or NC were transfected into 2×10^5^ GES1 cells using Lipofectamine RNAi MAX reagent (Invitrogen, USA) according to the manufacturer's protocol. After that, cell proliferation was then performed.
Proliferation Assay {#s4i}
-------------------
Cell growth rate of MGC803 or GES1 cells was detected by MTS cell proliferation assay. Cells were seeded in a 96-well plate at a density of 5×10^2^ cells per well. The cell growth rate was detected using cell proliferation MTS kit according to the manufacturer\'s instruction (Promega, USA). Each experiment was performed in triplicate.
Colony Formation Assay {#s4j}
----------------------
For the colony formation assay, *ARID1A*-expressing MGC803 cells or control MGC803 cells were plated in a 6-well plate at a density of 5×10^2^ cells per well. After 10 days of culture, surviving colonies (\>50 cells per colony) were counted with crystal violet (0.5%) staining. Colony-forming efficiency (CFE %) was defined as the ratio of the number of colonies formed in culture to the number of cells inoculated. The experiment was done in triplicate.
Statistical Analysis {#s4k}
--------------------
Differences in mRNA and protein expression between tumor samples and the paired adjacent non-tumor tissue samples were evaluated with the paired-samples t-test. The χ^2^ test was used to analyze the relationships between ARID1A expression and various clinicopathological parameters. Survival curves were calculated using the Kaplan--Meier method and compared by the log-rank test. The Cox proportional hazard regression model was used for univariate and multivariate analyses to study the effects of the clinicopathological variables and ARID1A expression on survival. The two-tailed unpaired Student's *t* test was used to assess differences in cell growth rate and colony formation. Statistical analyses were performed with the Statistical Package for the Social Sciences, version 17.0 (SPSS Inc., Chicago, IL, USA), and a two-sided *P* value less than 0.05 was considered to be statistically significant.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This work was supported by the National Natural Science Foundation of China (30973398) and Guangdong Natural Science Foundation (925100890). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: JCX YBC KP. Performed the experiments: DDW. Analyzed the data: DDW YBC JJZ LL YQL QJW. Contributed reagents/materials/analysis tools: WW JGC QZP SPC LXH MLK JH. Wrote the paper: DDW YBC.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S0001}
============
According to the World Health Organization (WHO) and United Nations Children and Educational Fund (UNICEF), about 1.5 million babies die every year because they were not breastfed. Several others suffer from contagious diseases and malnutrition, leading to their premature death because they were bottle-fed \[[@CIT0001]\]. Optimal breastfeeding rates have not been encouraging the world over with sub-optimal feeding being customized in Sub-Saharan Africa. The incidence of breastfeeding initiation exceeds 90% in almost every country and is widespread in Sub-Sahara Africa \[[@CIT0002]\]. However, Exclusive Breastfeeding (EBF) during the early stages of the infant\'s life which is a recommendation from WHO remains critical to the survival of the infant. Breastfeeding is universally practiced; nonetheless, the level of knowledge of mothers and their belief systems regarding exclusive breastfeeding are not well documented \[[@CIT0003]\].
In Africa, more than 95% of infants are breastfed but the feeding practices are often inadequate, feeding water and other liquids to breastfeed infants is prevalent \[[@CIT0004], [@CIT0005]\]. Prolonged breastfeeding, however, is common and the median duration of breastfeeding ranges between 16 and 28 months. Urbanization and mother\'s education are cited as the major factors that tend to shorten breastfeeding \[[@CIT0006]\]. The situation in Ghana is not different. Here, women breastfeed for a median duration of 22 months with 53.4% breastfeeding their young babies. The early introduction of additional foods and liquids causes higher rates of diarrhea illness and mortality \[[@CIT0007]\].
To help curb the trend, the concept of EBF was conceived and launched in 1990, through the collective efforts of the World Health Organization and United Nations Children and Educational Fund. The two bodies adopted the Innocentile Declaration on the Protection, Promotion and Support of Breastfeeding. The Declaration recognized that breastfeeding is a unique process which affords infants the idyllic combination of nutrients that provide critical protection against infectious diseases. The protective quality of breast milk decreases the rate of infant morbidity and mortality as well as contributes to the health and wellbeing of children. EBF for six months is the global goal for optimal maternal and child health and nutrition. All women should be encouraged to breastfeed and all infants should receive breast milk for up to two years of age or beyond with complementary foods \[[@CIT0008]\].
Thereafter, breastfeeding began to champion the fore as a public health issue. Owing to the fact that both mother and infant who do not breastfeed are at a momentous disadvantage relative to health outcomes, most health organizations and government agencies promote EBF for the first six months of life with continued breastfeeding and appropriate complementary foods thereafter. Healthy people 2010 set goals for increasing the initiation, duration and exclusivity of breastfeeding. These goals include; 75% initiation rates, 50% continuation of any breastfeeding at six months, 25% of any breastfeeding at one year, 60% of EBF at three months 25% EBF through six months \[[@CIT0009]\].
Available data, however, shows that 72.9% of infants have experienced at least some breastfeeding, 59.4% exclusively breastfed at seven days, 38.7% exclusively breastfed at three months and 13.9% exclusively breastfed at six months \[[@CIT0010]\]. These data disclose that EBF rates are trembling downwards, irrespective of the massive campaigns that have gone in favor of its adoption.
In pursuance of the battle against infant mortality, the World Health Organization and the United Nations Children and Educational Fund again developed the Baby Friendly Hospital Initiative which was launched in 1991. The Nkawie District Hospital became a beneficiary of this initiative. In addition, the Ghana Health Service and Basic II with the sponsorship of USAID launched the community based growth promotion strategy (CBGP) in late 2002 with its objective to improve the level of EBF in certain districts in Ghana including the Atwima Nwabiagya district. Growth promoters, were to among others, educate mothers on exclusive breastfeeding, assist dispel misconceptions (cultural practices) associated with breast feeding and also support mothers to overcome difficulties associated with the practice.
However due to the absence of remuneration, coupled with ineffective monitoring, this laudable initiative toppled. This study aims at identifying the impact of the defunct initiative as well as other factors affecting EBF practice in selected peri-urban and rural sub-districts in the Atwima Nwabiagya District of the Ashanti Region of Ghana.
Methods {#S0002}
=======
The study area {#S20003}
--------------
Atwima Nwabiagya District is one of the Twenty Seven (27) administrative districts in Ashanti Region with Nkawie as the district capital. It has a Population size of about 197,874 and shares common boundaries with the Ahafo Ano South District to the North, Amansie West District to the south, Kumasi Metropolis to the East and Atwima Mponua District. The district boasts of a state owned hospital in its capital and two other privately owned hospitals in settlement areas. In addition, there are seven health centers and five maternity homes, all boosting up healthcare delivery in the district. Major settlements in the district are Abuakwa, Toase, Akropong, Asuofua and Barekese. The district has several of its rural communities occupying hard to reach areas. This makes healthcare to these communities, especially during the rainy season, a herculean task. None-the less, with the health of the child at its heart of activities, as engrossed in the Health Directorate\'s vision statement as "a district where every child born lives to celebrate his/her fifth birthday", the Atwima Nwabiagya district is lauded as one of the first to extend healthcare to the doorstep of the people through its home visitation programme. The district hospital has been awarded a baby friendly status and has further benefitted from several international donor support like the USAID and Linkages \[[@CIT0011]\].
Study design and population {#S20004}
---------------------------
A cross sectional comparative study design was employed to identify the determinants of EBF in the two sub-districts of Abuakwa and Barekese in the Ashanti region of Ghana. Granted that the district records a 100 percent turn-out at child welfare clinic (district mid-year review 2009), the researchers deemed it appropriate to conduct a community based study. The team travelled alongside the outreach team of the health center in the sub-district to the communities where clinics were held. Interviews were conducted with mothers after they had completed their routine at outreach welfare clinics but at a different location. This is to ensure that mothers provided response in a relatively free environment devoid of intimidation or the watchful eyes of health providers. The time frame for the research lasted a little over a year, spanning through June 2009 to September 2010.
Study population comprised nursing mothers selected from each of the six communities within the two sub-districts under study. Primarily, simple random sampling was used to select two sub-districts among the five sub-districts in the Atwima Nwabiagya district. These were Abuakwa, a peri-urban district and Barekese, a predominant rural sub-district. Three communities each from the two sub districts were further randomly selected. A systematic sampling procedure of an interval of three was then used to contact fifty (50) nursing mothers who had turned up at child welfare clinic organized in each community. Before arriving at the interval used, the researchers did the following basic calculation: the average number of households that had at least a baby of 0-12 months old at the time of survey (200) was divided by the average number of mothers that turned up at child welfare clinic in each community (70). In all three hundred (300) nursing mothers made up the sampled population.
Study procedures {#S20005}
----------------
EBF was studied as the dependent variable. The independent variables included mothers' knowledge level of exclusive breastfeeding, cultural practices in relation to breastfeeding, mother\'s occupation and exclusive breastfeeding, and community participation in EBF programs. Quantitative data was gathered from respondents during Outreach Clinics conducted at the communities by staff the two main health centers in the sub-districts. Trained Field Assistants, each from the communities, were recruited for data collection. Questionnaires were translated and administered in the local language (Twi). Questionnaires were pre-tested in Sapaase, a peri-urban community, and Boahenkwa II, a rural community. Both within a sister sub-district.
Statistical analysis {#S20006}
--------------------
Data were entered and all statistical analyses carried out in STATA Version 10 (StataCorp, College Station, TX, USA). Unadjusted and Adjusted Odd ratios with 95% confidence interval (95% CI) estimated.
Ethical approvals {#S20007}
-----------------
Ethical clearance was obtained from the Okomfo Anokye Ethics Committee, Kumasi and that of the Department of Community Health, Kwame Nkrumah University of Science and Technology, Kumasi. In addition, written permission was sought from the regional and district directors of health services within whose jurisdictions the study took place. The purpose of the study was also explained to participants in a language that was comprehensible to them and they had the choice to participate or abstain. Reference codes were used to replace personal identifiers to ensure participant confidentiality and anonymity. Mothers involved in breastfeeding malpractices were identified and advised on the need to practice exclusive breastfeeding.
Results {#S0008}
=======
In all, three hundred (300) nursing mothers made up the study population with the two sub-districts sharing equal numbers of respondents ([Table 1](#T0001){ref-type="table"}). Among the three hundred (300) respondents who participated in this study, one hundred and fifty (150) belonged to the Abuakwa sub-district and the other half to the Barekese sub-district. Majority of respondents in both districts came from the 21-30 age group with 79(52.67%) and 85(56.675%) coming from Abuakwa and Barekese respectively ([Table 1](#T0001){ref-type="table"}).
######
Demographic characteristics of the study participants
N= 300
---------------------------------- ------------- ------------- ------------- -----------
**Age** P = 0.248
15-20 24 (16.00) 30 (20.00) 54 (18.00)
21-30 79 (52.67) 85 (56.67) 164 (54.67)
31-40 45 (30.00) 31 (20.67) 76 (25.33)
41-50 2 (1.33) 4 (2.67) 6 (2.00)
**Educational level** P = 0.001
None 17 (11.33) 37 (24.67) 54 (18.00)
Primary 17 (11.33) 28 (18.67) 45 (15.00)
JHS 98 (65.33) 80 (53.33) 178 (59.33)
SHS 17 (11.33) 5 (3.33) 22 (7.33)
Tertiary 1 (0.67) 0 (0.00) 1 (0.33)
**Place of delivery** P = 0.006
Health Facility Residence of TBA 127 (84.67) 107 (71.33) 234 (78.00)
At Home 0 (0.00) 4 (2.67) 4 (1.33)
23 (15.33) 39 (26.00) 62 (20.67)
**Husband\'s Occupation** P = 0.000
Civil Servant 34(22.67) 20(13.33) 54(18)
Farming 34(22.67) 74(49.33) 108(36.00)
Driving 30(20) 27(18.00) 57(19.00)
Trading 52(34.67) 29(19.33) 81(27.00)
**Marital Status** P = 0.489
Married 47(31.33) 56(37.33) 103(34.33)
Widowed 4(4.67) 5(3.33) 9(3.00)
Single 99(66.00) 89(59.33) 188(62.67)
**Religious Background** P = 0.002
Christian 139(92.67) 118(78.67) 257(85.67)
Moslem 10(6.67) 27(18.00) 37(12.33)
Traditionalist 1(0.67) 5(3.33) 6(2.00)
**Average income per month** P = 0.000
\<GHC50.00 48(32.00) 84(56.00) 132(44.00)
\> GH₵50 but \<GHC100.0 52(34.67) 63(42.00) 115(38.33)
GHC200 & above 50(33.33) 3(2.00) 53(17.67)
**Items Owned** P = 0.000
TV set 71(47.33) 28(18.67) 99(33.00)
Motor 2(1.33) 1(0.67) 3(1.00)
Car 5(3.33) 7(4.67) 12(4.00)
Sound system 71(47.33) 114(76.00) 185(61.67)
Others 1(0.67) 0(0.00) 1(0.33)
The second highest recruited nursing mothers were those from the 31-40 age groups. Abuakwa had 45(30.00%) and Barekese had 31(20.67%) respondents. There were more teenage mothers in Barekese than Abuakwa as the figures for the 15-20 age group stood at 24(16.005%) and 30(20.00%) for Abuakwa and Barekese respectively. Mothers in the 41-50 age groups were the least: 2(1.33%) for Abuakwa and 4(2.675%) for Barekese. This shows that nursing mothers between the ages of 15-40 had interest in exclusive breastfeeding.
Most nursing mothers who participated in the study had attained Junior High School/Middle school education with the highest number emerging from Abuakwa with 98(65.33%) respondents. Barekese also had 80(53.33%) respondents attaining their Junior High School/Middle School education. Also 17(11.33%) mothers from Abuakwa had attained primary education as against 28(18.67%) from Barekese. Only 5(3.33%) mothers from Barekese had attained their Senior High School education whereas Abuakwa had 17(11.33%) mothers with the same qualification. Surprisingly, in the sample, Barekese had no mother with tertiary education whereas Abuakwa had only one mother (0.67%). There were more illiterate mothers in Barekese than in Abuakwa. The figures show 37(24.67%) mothers in Barekese had never been to school. This was as against 17(11.33%) from Abuakwa.
Majority of the respondents gave birth at a health center: 114(76.00%) and 102(68.00%) respondents from Abuakwa and Barekese respectively. Only 5(3.33%) mothers in Barekese gave birth in a maternity home whereas 13(8.67%) mothers from Abuakwa also did same. However, more mothers in Barekese 39(26.00%) gave birth at home as compared to 23(15.33%) in Abuakwa. No mother in Abuakwa gave birth at the residence of a TBA whereas in Barekese 4(2.67%) mothers gave birth at the residence of a TBA.
Farming was the major occupation among husbands in the two areas though Barekese recorded higher figures than Abuakwa. Terrifyingly, majority of respondents in both sub-districts were not married. Single mothers recorded 99(66.00%) and 89(59.33%) for Abuakwa and Barekese respectively. Only 47(31.33%) mothers were married in Abuakwa whereas 56(37.33%) were married in Barekese. Also 4 (4.67%) mothers in Abuakwa and 5 (3.33%) in Barekese were divorced.
Most of the mothers interviewed both in Abuakwa and Barekese were Christians. Abuakwa registered 139(92.67%) whilst Barekese registered 118(78.67%). Few mothers earned about GH¢200.00 and above in the two districts. This implies that more mothers in Barekese earned less than GH¢50.00 than in Abuakwa. For those who earned less than GH¢100 a month, the figures recorded 52(34.67%) and 63(42.00%) for Abuakwa and Barekese respectively.
The commonest item owned by most respondents and their partners was sound system with more mothers owning more sound systems in Barekese than in Abuakwa. The results show 71(47.33%) and 114(76.00%) for Abuakwa and Barekese respectively. However, 71(47.33%) respondents in Abuakwa had TV sets as against 28(18.67%) respondents in Barekese. Regarding assets ownership, 5 (3.33%) respondents in Abuakwa and 7 (4.67%) respondents in Barekese said either they or their partners owned a car whereas 2(1.33%) and 1(0.67%) respondents in Abuakwa and Barekese respectively claimed they or their partners owned a motor cycle.
Level of Knowledge about EBF in the sub-districts {#S20009}
-------------------------------------------------
The studied participants demonstrated high level of knowledge of EBF as presented in [Figure 1](#F0001){ref-type="fig"}. [Table 2](#T0002){ref-type="table"} presents details of the cultural reasons for mix-feeding. With regards to colostrum disposal, 70(46.67%) and 50(33.33%) of respondents in Abuakwa and Barekese respectively discarded colostrum regarding it as impure and also as a tradition. However, 80 (53.33%) of respondents in Abuakwa and 100 (66.67%) respondents in Barekese reported that they fed their babies with the yellowish milk that first came out of the breast. There was a significant difference in the two sub-districts with regards to colostrum disposal (P = 0.018).
{#F0001}
######
Cultural reasons for mix--feeding
N= 300
------------------------------------------------- ------------- ------------ ----------------- -----------
**Breast milk polluted as result of pregnancy**
True 69 (46.00) 74 (49.33) 143 (47.67) 157 P = 0.563
False 81 (54.00) 76 (50.67) (52.33)
**Colostrum should be discarded**
True 70(46.67) 50(33.33) 120(40.00) P = 0.018
False 80(53.33) 100(66.67) 180(70.00)
**Asiram**
Yes 35(23.33) 24(16.00) 59(19.67) P = 0.110
No 115(76.67) 126(84.00) 241(80.33)
**Fear of mother dying**
True 36 (24.00) 58 (38.67) 94 (31.33) P = 0.006
False 114 (76.00) 92 (61.33) 206 (68.67)
Few mothers in the study areas had their babies suffering from malnutrition-asiram due to improper feeding practices. 35(23.33%) of mothers in Abuakwa and 24 (16%) of respondents in Barekese performed cultural rituals to promote the health of their babies. This was against 115(76.67%) and 126(84%) in Abuakwa and Barekese respectively who did not indulge in the practice. Statistically, there was no significant difference between the two sub-districts (P = 0.110).
The fear of the mother dying and leaving baby behind without it getting used to other foods did not influence their mothers' decision to mix-feed their babies. There were 26 (24%) and 58 (38.67%) nursing mothers in Abuakwa and Barekese respectively who gave supplementary feeds to their babies for fear of mothers dying early. Contrastingly, 114 (76%) and 92 (61.33%) of respondents in the two areas respectively did not indulge in the practice. Statistically, there was a significant difference between the two sub-districts (P = 0.006).
Majority of the respondents who exclusively breastfed in the two sub-districts were traders, 158 (52.67%). Out of this, 88 (58.67%) came from Abuakwa while 70 (46.67%) were those from the Barekese sub-district. Farmers made up the second category of 78 (26%). Out of this, 22(14.67%) belonged to the Abuakwa division whereas 56(37.33%) consisted of those from Barekese. ([Table 3](#T0003){ref-type="table"}) Almost all the mothers went on maternity leave upon delivery in the two sub-districts. 148(98.67%) of mothers in Abuakwa as well as 147(98%) of mothers in Barekese went on maternity leave. There was no significant difference in the two districts (P = 0.652). One (0.68%) and 5(3.40%) of nursing mothers went on maternity leave for 2 months for Abuakwa and Barekese respectively).
######
Occupational and other factors impact on EBF
N= 300
------------------------------------- ------------- ------------- ------------------- --------------
**Respondent Occupation** P = \< 0.001
Farming 22 (14.67) 56 (37.33) 78 (26.00)
Trading 88 (58.67) 70 (46.67) 158 (52.67)
Apprentice 34 (22.67) 23 (15.33) 57 (19.00)
Civil servant 6 (4.00) 1 (0.67) 7 (2.33)
**Maternity leave**
Yes 148 (98.67) 147 (98.00) 295 (98.33) P = 0.652
No 2 (1.33) 3 (2.00) 5 (1.67)
**For how long** N = 295
One month 1 (0.68) 1 (0.68) 2 (0.68) P = 0.389
Two months 1 (0.68) 5 (3.40) 6 (2.03)
Three months 11 (7.43) 13 (8.84) 24 (8.14)
More than three months 135 (91.22) 128 (87.07) 263(89.15)
**How often do you go to work** N = 120
Twice a week 0 (0.00) 6 (8.70) 6 (5.00) P = 0.076
Thrice a week 2 (3.92) 6 (8.70) 8 (6.67)
Five days a week 39 (76.47) 41 (59.42) 80 (66.67)
Seven days a week 10 (19.61) 16 (23.19) 26 (21.67)
**Do you carry baby to work** N = 128
Yes 37 (67.27) 48 (65.75) 85 (66.41) P = 0.857
No 18 (32.73) 25 (34.25) 43 (33.59)
**Who takes care of it** N = 44
Mother 8 (40.00) 6 (25.00) 14 (31.82) P = 0.323
Sister 7 (35.00) 10 (41.67) 17 (38.64)
Mother-in-law 5 (25.00) 5 (20.83) 10 (22.73)
Husband 0 (0.00) 3 (12.50) 3 (6.82)
**Food taken in mother\'s absence**
Koko 15(78.95) 24(96.00) 39(88.64) P = 0.227
Formula 1(5.26) 1(4.00) 2(2.27)
Expressed milk 1(5.26) 0(0.00) 1(2.27) 2(4.55)
Others 2(10.53) 0(0.00)
**How food is stored** N = 35
Milk in a fridge 2(12.50) 2(10.53) 4(11.43) P = 0.722
Koko in a flask 12(75.00) 16(84.21) 28(80.00) 3(8.57)
Others 2(12.50) 1(5.26)
**Has baby fallen ill lately** N = 300
Yes 59(39.33) 72(48.00) 131(43.67) P = 0.130
No 91(60.67) 78(52.00) 169(56.33)
**Diagnoses of baby\'s illness** N = 131
Malaria 23(38.98) 29(40.28) 52(39.69) P = 0.277
Diarrhea 28(47.46) 37(51.39) 65(49.62)
Convulsion 0(0.00) 2(2.78) 2(1.53)
Anaemia 2(3.39) 0(0.00) 2(1.53)
Source: Field Data 2009.
Almost all the mothers went on maternity leave upon delivery in the two sub-districts. 148(98.67%) of mothers in Abuakwa as well as 147(98%) of mothers in Barekese went on maternity leave. There was no significant difference in the two districts (P = 0.652). One (0.68%) and 5(3.40%) of nursing mothers went on maternity leave for 2 months for Abuakwa and Barekese respectively. Eleven (7.43%) and 13(8.84%) of mothers in the two areas respectively went on leave for three months whereas the majority 135(91.22%) of mothers in Abuakwa and 128(87.07%) of mothers in Barekese went on maternity leave for more than 3 months. There was no significant difference in the two sub-districts (P = 0.389).
For mothers who have resumed work, 39 (76.47%) and 41(59.42%) for Abuakwa and Barekese respectively went to work five days in a week, whereas 10(19.61%) and 16(23.19%) for Abuakwa and Barekese went to work all the days of the week. Statistically, there was no significant difference with regards to length of days spent at work in the two areas under study, (P = 0.076). Regarding food taken by baby in the absence of the mother, (78.95%) and 24(96%) of nursing mothers who have resumed work in Abuakwa and Barekese respectively fed babies below six months with a locally prepared cereal meal. This was against (5.26%) of mothers in Abuakwa who fed babies with baby formula and expressed milk. No nursing mother in Barekese gave expressed milk to baby upon resumption of work. There was no significant difference with regards to supplementary feeds given to baby in the two sub-districts (P = 0.227).
Diarrhea cases were the most prevalent in babies whose mothers had resumed work. The results registered 28(47.40%) and 37(51.39%) for Abuakwa and Barekese respectively. Malaria registered second with 23(38.98) and 29(40.28%) in the two sub-districts. Two (3.39%) of mothers in Abuakwa admitted that their babies suffered from anaemia whereas there was no such case recorded for Barekese. Once again there was no significant difference in the two areas (P = 0.277).
[Table 4](#T0004){ref-type="table"} presents the findings of community participation in exclusive breastfeeding. Community participation did not register positive results with regards to promotion of EBF. The figures revealed that 108 (72%)and 96 (64%) of nursing in Abuakwa and Barekese respectively were pressured most especially by mothers-in- law to give supplementary feeds. Figures showed no significant difference though, (P = 0.137). Also community members did not seem to play an effective role in EBF promotion in both sub-districts. 143(95.33%) and 133(88.66%) of nursing mothers in Abuakwa and Barekese respectively were rather dissuaded by members of the community. This was against 7(4.67%) and 17(11.33%) from the two areas who admitted they received societal support in relation to the practice of exclusive breastfeeding. This recorded significant difference in the two sub-districts, (P = 0.033). Peer counseling was virtually non-existent in the areas under study. 145(96.67%) of mothers in Abuakwa and 126(84%) of mothers in Barekese did not receive peer counseling on exclusive breastfeeding. This was as against 5(3.33%) and 24(16%) of mothers in Abuakwa and Barekese respectively who claimed the reverse. Statistically, there was a difference in relation to counseling on exclusive breastfeeding in the two areas under study (P = 0.00).
######
Community participation
N= 300
--------------------------------------- ------------- ------------ --------------- -----------
**Pressure to give food** P = 0.137
Yes 108 (72.00) 96 (64.00) 204(68.00)
No 42 (28.00) 54 (36.00) 96 (32.00)
**By whom** N = 204 P = 0.393
Husband 18 (16.67) 23 (23.96) 41 (20.10)
Mother-in-law 34 (31.48) 27 (28.12) 61 (29.90)
Neighbors 29 (26.85) 20 (20.83) 49 (24.02) 41
Friends 19 (17.59) 22 (22.92) (20.10)
Others 8 (7.41) 4 (4.17) 12 (5.88)
**Encourage to practice E.B.F** N = 300 P = 0.033
Yes 7(4.67) 17(11.33) 24(8.00)
No 143(95.33) 133(88.66) 276(92.00)
**Counseling on E.B.F by volunteers** P = 0.000
Yes 5(3.33) 24(16.00) 29(9.67)
No 145(96.67) 126(84.00) 271(90.33)
**E.B.F practiced by mothers** P = 0.101
Yes 6(4.00) 13(8.67) 19(6.33)
No 81(54.00) 66(44.0) 147(49.0)
Don\'t know 63(42.00) 71(47.34) 134(44.67)
**Breast feeding in public** P = 0.584
Yes 144(96.00) 142(94.67) 286(95.33)
No 6(4.00) 8(5.33) 14(4.67)
Source: Field Data 2009
With regards to breastfeeding in public, the results revealed that 144(96%) of nursing mothers in Abuakwa and 142(94.67%) of nursing mothers in Barekese gave breast milk to babies while in public. However, 6 (4%) and 8(5.33%) of mothers in Abuakwa and Barekese respectively did not feel comfortable exposing their breasts in public. There was no significant difference in this notion in the two sub-districts, (P = 0.584).
Two factors were associated with EBF in the univariate logistic model. Unmarried mothers were less likely to practice EBF compared with mothers who were married (OR = 0.46, 95% CI: 0.28, 0.77). Also the duration of breastfeeding was associated with EBF. The adjusted odds ratio was 0.41(95% CI: 0.32, 0.54) in favor of three months compared with six months ([Table 5](#T0005){ref-type="table"}).
######
Logistics analysis of factors associated with EBF
Variables Odds Ratio P-value 95% CI
----------------------- ------------ --------- -------------
**Sub-district**
Abuakwa 1
Barekese 0.65 0.06 0.40, 1.02
**Education**
None 1
Primary 1.04 0.89 0.56, 1.93
Middle 0.87 0.68 0.45, 1.68
Secondary 0.83 0.69 0.34, 2.03
**Age group**
15-20 1
21-30 1.48 0.21 0.80, 2.75
31-40 1.81 0.10 0.90, 3.67
41-50 2.5 0.31 0.42, 14.83
**Marital Status**
Married 1
Widowed 0.64 0.52 0.16, 2.57
Single 0.46 0.001 0.28, 0.77
**Nipple pain**
Yes 1
No 1.12 0.70 0.63, 0.99
**Ancatta**
Yes 1
No 0.67 0.56 0.18, 2.57
**Length of EBF**
Six months 1
Three months 0.13 0.001 0.07, 0.24
Twelve months 0.75 0.82 0.07, 8.56
**Mother Occupation**
Farming 1
Trading 0.09 0.71 0.52, 1.55
Apprentice 0.97 0.93 0.49, 1.93
Civil servant 2.03 0.41 0.37, 11.32
**Fellow mothers**
Yes 1
No 0.68 0.38 0.29, 1.62
Discussion {#S0010}
==========
This study provides quantitative insights into the determinants of EBF in the Atwima Nwabiagya District of the Ashanti Region of Ghana. The study revealed that mothers' level of knowledge about EBF was good as such the practice was high in the two sub-districts. It was also found out that traditional cultural practices in the area did not prevent mothers from practicing EBF. All these factors could be attributed to the impact of the Community Based Growth Promotion strategy initiative that was launched in the studied communities in the past.
The level of knowledge of EBF {#S20011}
-----------------------------
Several writers have found a positive correlation between exclusive breastfeeding knowledge and actual practice \[[@CIT0011]--[@CIT0014]\]. Though others in the field have seemingly refuted this assertion \[[@CIT0015], [@CIT0016]\], our study conforms to the former. Mothers who participated in the study had appreciable level of knowledge regarding exclusive breastfeeding and this manifested into actual practice. Mothers demonstrated high knowledge level in relation to what they make of exclusive breastfeeding, duration of exclusive breastfeeding, improper feeding practices and its consequences on health of baby and the recommended feeds for baby after six months. Mothers main source of came from antenatal visits followed by growth promoters in communities. Although Abuakwa (peri-urban) recorded comparatively higher figures with regards to good knowledge of EBF than Barekese (rural), the difference was not significant. Perhaps this may be due to the fact that urban mothers generally possess a high level of literacy hence are better informed especially on novel issues than their rural counterparts. This is consistent with earlier findings on the subject. However, the level of prevalence falls below those presented by the GHS in their (2006) cluster survey report. This can, however, be viewed as an understandable phenomenon since the cluster survey might have made use of a larger sample size than the current study.
The cultural practices associated with EBF {#S20012}
------------------------------------------
The cultural practices of the people in relation to breastfeeding did not affect mothers' decision to exclusively breastfeed their babies in the two sub-districts. Cultural myths and practices associated with breastfeeding have been a bane to the practice of Exclusive Breastfeeding especially in Africa \[[@CIT0011], [@CIT0017], [@CIT0018]\]. Ranging from colostrum disposal through to herbal medications and the eventual mix-feeding syndrome, mothers have been confronted with the challenge of how best to care for their babies. Fortunately nursing mothers in the Atwima Nwabiagya district have transcended these cultural stalemates and are preoccupied in ensuring that their babies benefit solely from essential nutrients expelled in breast milk for the first six months of life, although this does not go without hitches. Results indicate that several mothers still indulge in mix feeding for fear of their early demise. Relatedly figures representing mothers who discarded colostrum are significant enough to warrant attention though less than those found out in rural Tanzania \[[@CIT0019] and the West Bengal State of India \[[@CIT0020]\].
Sexual prohibition during breastfeeding is a taboo held by many African countries \[[@CIT0018]\] with Ghana non-exempt. The assertion that mothers' milk is polluted when she begins sexual union with her partner has compelled many mothers to introduce complementary feeds early in baby\'s life. Inherent in the taboo is the rationale to postpone childbirth, prevent unintended pregnancy and to afford mothers enough time to nurse their babies. However, with post-natal counseling on family planning methods coupled with the erstwhile vibrant activities of growth promoters, this traditional adherence is fast losing its grip on the people. These cultural issues influencing EBF have been reported elsewhere \[[@CIT0018]\].
Farming and exclusive breastfeeding {#S20013}
-----------------------------------
Farming as a profession was cited as a major setback to the practice of EBF in the Barekese sub-district. Though majority of the respondents were traders, there were more traders in Abuakwa than Barekese. Since more women in Abuakwa exclusively breastfed their babies than Barekese it is realistic to assume a positive relationship between the trading profession and exclusive breastfeeding. Mothers who trade their wares either within the neighborhoods or in the market carry their babies along and periodically breastfeed. Female farmers from rural Ghana leave babies in the care of others and travel miles away to attend to farming activities. The main food fed to baby in the absence of the mother is a locally prepared cereal meal, tenaciously increasing baby\'s chances of contracting infection.
Most mothers worldwide enjoy a rest period following birth though the length of time differs with respects to mothers' occupation and the organization she works for. The period spans through one week to three months. The longer the period, the greater the chances of practicing exclusive breastfeeding and vice versa. The leave period provides mothers the opportunity to be closer to their babies and to provide the best possible care. Since mother and baby are always together, the likelihood of practicing EBF is high. The opposite holds true \[[@CIT0021], [@CIT0022]\]. More mothers in Abuakwa, than Barekese went on maternity leave for more than three a month\'s period. This revelation is in consonance with the findings elsewhere in England \[[@CIT0021]\]. Again, the rural sub-district (Barekese) recorded a high number of babies suffering from diarrhea. However, babies in both areas who were not subjected to mix feeds did not report of any diarrhea disease. This outcome supports the findings reported by Kosmala-Anderson et al \[[@CIT0021]\]. A confirmation of the fact that babies who breastfed exclusively for the six months are less likely to suffer from constipation or diarrhea and are also less prone to childhood diseases including juvenile diabetes, allergies, asthma, eczema, gastrointestinal, urinary and respiratory tract infections.
Absence of community volunteers {#S20014}
-------------------------------
The virtual absence of community volunteers in exclusive breastfeeding promotion became the utmost disincentive to the practice of EBF in the two areas. The timing of the study coincided with the period that witnessed the fold-up of growth promotion activities. Until then mothers had benefitted immensely from counseling and support on exclusive breastfeeding from growth promoters. Having lost touch with these activities exposed mothers to the vagaries of society to mix-feed. Mothers- in- law are a force to reckon with in this regard. Their conservative approach to issues has made many a change a daunting task. This possible relapse could be due to the fact that growth promotion initiative had only lasted four years in the district and definitely not enough a period to change a societal issue. Elsewhere initial and recurrent contacts with peer counselors were associated with a significant increase in exclusive breastfeeding initiation, duration and rates \[[@CIT0023]--[@CIT0026]\].
Breastfeeding, to mothers in this Sub-region, is a natural phenomenon and mothers do not hesitate to breastfeed wherever they find themselves. This contrasts with what pertains in the Western world \[[@CIT0015]\]. What should, however, be of worry is whether mothers' hands and nipples are cleaned under the circumstances to breastfeed and what recommendation is in place to ensure that in their bid to breastfeed at any time and place that baby demands breast milk, mothers do not end up endangering the lives of babies though infection..
Study limitations {#S20015}
-----------------
The vast nature of the district made it difficult for the researchers to cover the entire district. Nonetheless, this might not affect the results since those who eventually took part in this study were randomly selected. Financial constraint was a major determinant of the sample size.
Conclusion {#S0016}
==========
The level of knowledge of EBF in the two sub-districts was generally good and as such the practice was high. The cultural practices of the people in relation to breastfeeding did not affect mother\'s decision to exclusively breastfeed their babies. We also noticed that mothers who were farmers were less likely to practice EBF compared with other professions like trading. However, the absence of community participation in all aspects pertaining to EBF initiatives became the utmost barrier to the practice of EBF within the study area. The District Health Administration should ensure that EBF campaigns through outreach clinics are enhanced by addressing, particularly, mothers' occupational needs and effective EBF practices. Also, the Ghana Health Service should consider reinstating the CBGP intervention programme while adequately motivating growth promoters. Locally, the Assembly could initiate an award scheme to appreciate mothers who exclusively breastfeed. This could serve as incentives to get mothers adopt the practice.
The authors are most grateful to the almighty God for providing the direction for the completion of this study. We are particularly grateful to the study participants for making time to respond to the study questions. Appreciation also goes to the District Director of Health Services, Atwima Nwabiagya District, Miss Beatrice Appah for her unwavering support, Dr. Ernestine Agnes Addy who supervised the work, and Mr. Emmanuel Nakua for his support with the statistical analysis. This study was self-funded. Ethical approvals were obtained from the Okomfo Anokye Ethics Committee, Kumasi and as well as the Department of Community Health, Kwame Nkrumah University of Science and Technology, Kumasi.
Competing interests {#S0017}
===================
The authors declare no competing interests.
Authors' contributions {#S0018}
======================
AA conceived of and developed the study proposal under the guidance of Prof. Ernestina Agnes Addy who supervised the work. AA performed the statistical analysis with support from Mr. Emmanuel Nakua and KAA. The manuscript was initially drafted by AA with review by KAA. Both authors read and approved of the final manuscript.
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Background
==========
After severe brachial palsy involving the shoulder, secondary operations are sometimes required to restore function. These include shoulder artrhodesis, rotational osteotomy, muscle transfer or a combination of these techniques.
For paralysis of the deltoid and supraspinatus muscle many different muscle transfers have been advocated to restore movement and stability of the shoulder. These include transfer of the trapezius, pectoralis major and teres major, latissimus dorsi, and combined biceps and triceps.
In a classic monograph; Saha \[[@B1]\] gave details of his experience with transfer of the trapezius, using a modification of the technique originally described by Bateman \[[@B2]\]. However, the absence of clear indications for the operation and expecting too much for this transfer alone has led to its infrequent use.
We have evaluated the results of the trapezius transfer for flail shoulder after brachial plexus injury.
Methods
=======
We treated 10 patients, 8 males and 2 females, by transfer of the trapezius to the proximal humerus. In 6 patients the C5 and C6 roots had been injured; in one C5, C6 and C7 roots; and in 3 there were complete brachial plexus injuries. Eight of the 10 had had neurosurgical repairs before muscle transfer.
Their average age was 28.3 years (range 17 to 41), and the average follow-up was 17.5 months (range 6 to 52). The mean delay between injury and transfer was 3.1 years (range 14 months to 6.3 years).
All patients had elbow flexion (2 had had previous Steindler flexorplasties) and 6 patients had good ipsilateral hand function.
Evaluation included physical and radiographic examinations. The active abduction/flexion shoulder motion was recorded (power between 3 to 5 grades according to MRC scale). Shoulder abduction was measured as the angle between the trunk and the arm. The pre-operative average was 3.1° (range 0° to 30°). The average shoulder forward flexion was 4.5° (range 0° to 45°). In all patients, the deltoid, supraspinatus, teres minor, infraspinatus and subscapularis were paralysed and the trapezius, levator scapulae were preserved. The rhomboids were affected in 2 patients. Paralysis of deltoid and supraspinatus was confirmed by EMG. All patients were unemployed at the time of trapezius transfer. Radiological subluxation of the shoulder was present in all cases. The subjective assessment of the patients was not considered.
Surgery can be considered if the patient presents flail shoulder at more than one year after the accident without spontaneous recovery or when it is clear that recovery following neurosurgical repair is not progressing any more. A simple trapezius transfer is compatible with the later return of some function to other shoulder girdle muscles. Passive shoulder abduction of 80° is an important pre-requisite before transfer. The only contra-indication is advanced degeneration of the shoulder.
A modification of Mayer\'s \[[@B3]\] transfer of the trapezius muscle was performed in which a portion of the acromion is removed to allow for a more straight-line pull. The lateral aspect of the acromion and its attached trapezius is removed, and its undersurface is roughened with a rasp. Fixation with one or two screws secures the acromion and trapezius transfer to the proximal part of the humeral shaft.
The principal goal of this work was to evaluate the results of the trapezius transfer for flail shoulder after brachial plexus injury.
Surgical technique
------------------
The patient is placed supine with a sand-bag under the shoulder. The shoulder, the neck, and the whole arm are prepared and free.
A saber-cut incision is made from the inferior border of the anterior axillary fold over the anterior aspect of the shoulder to a point a few centimetres lateral to the medial border of the scapula and just distal to the scapular spine. The deltoid origin is then cut from the lateral third of the clavicle, the acromion, and the lateral half of the spine of the scapula.
A Gigli wire saw is used to transect the root of the acromion, and then the lateral clavicle, so as to separate the lateral 1 cm of the clavicle with the acromion. The remaining insertions of the trapezius are elevated from the clavicle and the scapular spine to 2 cm from the vertebral border of the scapula. Careful dissection is needed to define the interval between the trapezius and the supraspinatus. Special attention is needed to preserve the neurovascular bundle of the spinal accessory nerve and transverse cervical artery, which courses from deep to superficial through the trapezius.
The partly detached deltoid is split longitudinally to expose the proximal humerus, which is scored with an osteotome. The arm is then abducted to 90°, and the acromiocalvicular fragment with its trapezius insertion is fixed to the humerus with two screws, ensuring firm bone-to-bone. The wound is thoroughly irrigated with saline solution, and the deltoid is sutured on top of the new trapezius insertion. The skin is closed in two layers over suction drains a shoulder spica applied with the shoulder in 90° of abduction.
Postoperative management. Drains are removed on the second or third day. The spica is worn for six weeks or until union is seen between the acromion fragment and the humerus. The arm is then allowed to adduct progressively and a vigorous physical therapy programme is started. As strength improves, more resisted muscle strengthening exercises are added.
Results
=======
The transfer improved function of the shoulder (Figure [1](#F1){ref-type="fig"}). Postoperatively, the average gain in shoulder abduction was 46.2° (p \< 0.001, Fisher exact test); the gain in shoulder flexion average 37.4° (p \< 0.001). All patients had stable shoulders (no subluxation of the humeral head on radiographs, Figure [2](#F2){ref-type="fig"}).
{#F1}
{#F2}
Surgical time averaged 2 hours (range 1 to 4), and the estimated mean blood loss was 200 ml. There were no postoperative complications.
Discussion
==========
Severe injuries to the brachial plexus cannot always be successfully repaired; even failures are seen after the best repair. Unsatisfactory or incomplete results affect abduction, external rotation and forward projection of the humerus at shoulder level.
Flail shoulder secondary to a brachial plexus injury is difficult to treat. After neurosurgical treatment and adequate physiotherapy, reconstructive surgery may be needed to improve the stability and function of the shoulder.
Deltoid and supraspinatus paralysis may be managed by shoulder fusion \[[@B4]-[@B6]\] or muscle transfer \[[@B7]\]. Shoulder arthordesis has been considered the procedure of choice in patients with flail shoulder after brachial plexus palsy, but is irreversible and has a high complication rate. Cofield and Briggs \[[@B8]\] pointed out the disadvantages of arthrodesis (24% incidence of fractures, 25% had no improvement and 15% had aggravation of pain).
Trapezius, levator scapulae and rhomboid muscles remain healthy or recover in 96% of cases, therefore are available for transposition.
Several muscle transfers have been advocated to restore movement and stability of the shoulder after poliomyelitis \[[@B7],[@B9],[@B10]\], and, more recently, the use of these procedures after brachial plexus palsy has been reported. \[[@B11]-[@B14]\]
Aziz, Singer and Wolff \[[@B12]\] discuss trapezius transfer for flail shoulder after brachial plexus palsy, finding it a simple procedure with minimal blood loss, which provided functional improvement.
Passive shoulder abduction of 80° is an important pre-requisite, and requires intensive physiotherapy before transfer. If 80° is not obtained, shoulder arthrodesis is recommended \[[@B13]\].
Trapezius transfer to treat flail shoulder after a brachial plexus injury will allow the patient to position the arm much better, even when functional recovery is not adequately strong to keep the shoulder stable. The procedure is relatively simple with minimal blood loss and the only contraindication is advanced degeneration of the shoulder. Trapezius transfer can be used combined with other transfers to achieve optimal use of the upper limb.
Conclusion
==========
Trapezius transfer can provide satisfactory functional improvement and it is better than arthrodesis for paralysis of the shoulder after brachial plexus injury.
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Introduction {#Sec1}
============
Mendelian randomization (MR) is the use of genetic variants to infer the presence or absence of a causal effect of a risk factor on an outcome. Under the assumption that the genetic variants are valid instrumental variables, this causal effect can be consistently inferred even in the presence of unobserved confounding factors^[@CR1]^. The instrumental variable assumptions are illustrated by a directed acyclic graph as shown in Fig. [1](#Fig1){ref-type="fig"}^[@CR2]^.Fig. 1Directed acyclic graph of instrumental variable assumptions made in univariable Mendelian randomization.$\documentclass[12pt]{minimal}
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\begin{document}$$\theta$$\end{document}$ = causal effect of interest.
Recent years have seen an explosion in the size and scale of data sets with biomarker data from high-throughput experiments and concomitant genetic data. These biomarkers include proteins^[@CR3]^, blood cell traits^[@CR4]^, metabolites^[@CR5]^ or imaging phenotypes such as from cardiac image analysis^[@CR6]^. High-throughput experiments provide ideal data resources for conducting MR investigations in conjunction with case-control data sets providing genetic associations with disease outcomes (such as from CARDIoGRAMplusC4D for coronary artery disease^[@CR7]^, DIAGRAM for type 2 diabetes^[@CR8]^, or the International Age-related Macular Degeneration Genomics Consortium \[IAMDGC\] for age-related macular degeneration^[@CR9]^). In addition to their untargeted scope, one specific feature of high-throughput experiments is a distinctive correlation pattern between the candidate risk factors shaped by latent biological processes.
Multivariable MR is an extension of standard (univariable) MR that allows multiple risk factors to be modelled at once^[@CR10]^. Whereas univariable MR makes the assumption that genetic variants specifically influence a single risk factor, multivariable MR makes the assumption that genetic variants influence a set of multiple measured risk factors and thus accounts for measured pleiotropy. Our aim is to use genetic variation in a multivariable MR paradigm to select which risk factors from a set of related and potentially highly correlated candidate risk factors are causal determinants of an outcome. Existing methods for multivariable MR are designed for a small number of risk factors and do not scale to the dimension of high-throughput experiments. We therefore seek to develop a method for multivariable MR that can select and prioritize biomarkers from high-throughput experiments as risk factors for the outcome of interest. In this context we propose a Bayesian model averaging approach (MR-BMA) that scales to the dimension of high-throughput experiments and enables risk factor selection from a large number of candidate risk factors. MR-BMA is formulated on two-sample summarized genetic data which is publicly available and allows the sample size to be maximized.
To illustrate our approach, we analyse publicly available summarized data from a metabolite genome-wide association study (GWAS) on nearly 25,000 participants to rank and prioritise metabolites as potential biomarkers for age-related macular degeneration. Data are available on genetic associations with 118 circulating metabolites measured by nuclear magnetic resonance (NMR) spectroscopy^[@CR11]^ from <http://computationalmedicine.fi/data#NMR_GWAS>. This NMR platform provides a detailed characterisation of lipid subfractions, including 14 size categories of lipoprotein particles ranging from extra small (XS) high density lipoprotein (HDL) to extra-extra-large (XXL) very low density lipoprotein (VLDL). For each lipoprotein category, measures are available of total cholesterol, triglycerides, phospholipids, and cholesterol esters, and additionally the average diameter of the lipoprotein particles. Apart from lipoprotein measurements, this metabolite GWAS estimated genetic associations with amino acids, apolipoproteins, fatty and fluid acids, ketone bodies and glycerides. We assess the performance of our proposed method in a simulation study with scenarios motivated by the metabolite GWAS and by publicly available summary data on blood cell traits measured on nearly 175,000 participants^[@CR4]^.
Results {#Sec2}
=======
Multivariable Mendelian randomization and risk factor selection {#Sec3}
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Standard MR requires genetic variants to be specific in their associations with a single risk factor of interest, and does not allow genetic variants to have pleiotropic effects on other risk factors on competing causal pathways. Multivariable MR allows genetic variants to be associated with multiple risk factors, provided these risk factors are measured and included in the analysis. Hence multivariable MR allows for 'measured pleiotropic effects'^[@CR10],[@CR12]^. As illustration, multivariable MR can be considered as an extension of the standard MR paradigm (Fig. [1](#Fig1){ref-type="fig"}) to model not one, but multiple risk factors (Fig. [2](#Fig2){ref-type="fig"}).Fig. 2Directed acyclic graph of instrumental variable assumptions made in multivariable Mendelian randomization.$\documentclass[12pt]{minimal}
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We consider a two-sample framework, where the genetic associations with the outcome (sample 1) are regressed on the genetic associations with all the risk factors (sample 2) in a multivariable regression which is implemented in an inverse-variance weighted (IVW) linear regression. Each genetic variant contributes one data point (or observation) to the regression model. Weights in this regression model are proportional to the inverse of the variance of the genetic association with the outcome. This is to ensure that genetic variants having more precise association estimates receive more weight in the analysis. The causal effect estimates from multivariable MR represent the direct causal effects of the risk factors in turn on the outcome when all the other risk factors in the model are held constant^[@CR12],[@CR13]^ and Supplementary Fig. [1](#MOESM1){ref-type="media"}). Including multiple risk factors into a single model allows genetic variants to have pleiotropic effects on the risk factors in the model referred to as "measured pleiotropy"^[@CR14]^.
However, the current implementation of multivariable MR is not designed to consider a high-dimensional set of risk factors and is not suitable to select biomarkers from high-throughput experiments.
To allow joint analysis of biomarkers from high-throughput experiments in multivariable MR, we cast risk factor selection as variable selection in the same weighted linear regression model as in the IVW method. Formulated in a Bayesian framework (for full details we refer to the Methods section) we use independence priors and closed-form Bayes factors to evaluate the posterior probability (*PP*) of specific models (i.e. one risk factor or a combination of multiple risk factors). In high-dimensional variable selection, the evidence for one particular model can be small because the model space is very large and many models might have comparable evidence. This is why MR-BMA uses Bayesian model averaging (BMA) and computes for each risk factor its marginal inclusion probability (*MIP*), which is defined as the sum of the posterior probabilities over all models where the risk factor is present. MR-BMA reports the model-averaged causal effects (*MACE*), representing conservative estimates of the direct causal effect of a risk factor on the outcome averaged across these models. These estimates can be used to compare risk factors or to interpret effect directions, but should not be interpreted absolutely. As we show in a simulation study based on real biomarker data, MR-BMA provides effect estimates biased towards the Null when there is a causal effect but reduces the variance of the estimate, trading bias for reduced variance. Consequently, MR-BMA enables a better and more stable detection of the true causal risk factors than either the conventional IVW method or other variable selection methods.
Detection of invalid and influential instruments {#Sec4}
------------------------------------------------
Invalid instruments may be detected as outliers with respect to the fit of the linear model. Outliers may arise for a number of reasons, but they are likely to arise if a genetic variant has an effect on the outcome that is not mediated by one or other of the risk factors---an unmeasured pleiotropic effect. To quantify outliers we use the $\documentclass[12pt]{minimal}
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\begin{document}$$Q$$\end{document}$-statistic, which is an established tool for identifying heterogeneity in meta-analysis^[@CR15]^. More precisely, to pinpoint specific genetic variants as outliers we use the contribution $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$ is defined as the weighted squared difference between the observed and predicted association with the outcome.
Even if there are no outliers, it is advisable to check for influential observations and re-run the approach omitting that influential variant from the analysis. If a particular genetic variant has a strong association with the outcome, then it may have undue influence on the variable selection, leading to a model that fits that particular observation well, but other observations poorly. To quantify influential observations, we suggest to use Cook's distance ($\documentclass[12pt]{minimal}
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\begin{document}$$Cd$$\end{document}$)^[@CR16]^. We illustrate the detection of influential points and outliers in the applied example and provide more details in the Methods.
Simulation results {#Sec5}
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To assess the performance of the proposed method, we perform a simulation study in three scenarios based on real high-dimensional data. We compare the performance of the conventional approach (Multivariable IVW regression), the Lars^[@CR17]^, Lasso, and Elastic Net^[@CR18]^ penalised regression methods developed for high-dimensional regression models, MR-BMA, and the model with the highest posterior probability from the BMA procedure (best model). We seek to evaluate two aspects of the methods: (1) how well can the methods select the true causal risk factors, and (2) how well can the methods estimate causal effects. Risk factor selection is evaluated using the receiver operating characteristic (ROC) curve, where the true positive rate is plotted against the false positive rate. True positives are defined as the risk factors in the generation model that have a non-zero causal effect. Causal estimation is evaluated by calculating the mean squared error (MSE) of estimates, which is defined as the squared difference between the estimated causal effect and the true causal effect. The MSE of an estimator decomposes into the sum of its squared bias and its variance.
Genetic associations with the risk factors are obtained from three different scenarios. Two scenarios are based on the NMR metabolite GWAS by ref. ^[@CR11]^, where we use as instrumental variables $\documentclass[12pt]{minimal}
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\begin{document}$$n=150$$\end{document}$ independent genetic variants that were associated with any of three composite lipid measurements (LDL cholesterol, triglycerides or HDL cholesterol) at a genome-wide level of significance ($\documentclass[12pt]{minimal}
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\begin{document}$$p \, < \, 5\times 1{0}^{-8}$$\end{document}$) in a large meta-analysis of the Global Lipids Genetics Consortium^[@CR19]^. In Scenario 1, we consider a small set of $\documentclass[12pt]{minimal}
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\begin{document}$$d=92$$\end{document}$ risk factors. Scenario 3 is based on publicly available summary data on $\documentclass[12pt]{minimal}
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\begin{document}$$d=33$$\end{document}$ blood cell traits measured on nearly 175,000 participants^[@CR4]^. Using all genetic variants that were genome-wide significant for any blood cell trait, we have $\documentclass[12pt]{minimal}
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\begin{document}$$n=2667$$\end{document}$ genetic variants as instrumental variables. For each scenario, we generate the genetic associations for the outcome based on four random risk factors having a positive effect in Setting A and on eight random risk factors, of which four have a positive and four have a negative effect, in Setting B. In addition, we vary the proportion of variance in the outcome explained by the causal risk factors. Each simulation setting is repeated 1000 times. Full detail of the generation of the simulated outcomes is given in the Supplementary Methods.
Looking at a small set of $\documentclass[12pt]{minimal}
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\begin{document}$$d=12$$\end{document}$ risk factors in the NMR metabolite data of which four risk factors are true causal ones (Scenario 1, Setting A), we see that MR-BMA is dominating all other methods in terms of area under the ROC curve (see Fig. [3](#Fig3){ref-type="fig"}a). Next best methods are Lasso, Elastic Net, the Bayesian best model and Lars. The standard IVW method gives the worst performance. Similar results were obtained when varying the variance in the outcome explained by the risk factors (Setting A in Supplementary Fig. [3](#MOESM1){ref-type="media"} and Setting B in Supplementary Fig. [4](#MOESM1){ref-type="media"}). With respect to the MSE of estimates (Table [1](#Tab1){ref-type="table"}), MR-BMA has the lowest MSE in almost all scenarios followed by Elastic Net, Lasso, the Bayesian best model, and then Lars. Elastic Net has the lowest MSE for $\documentclass[12pt]{minimal}
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\begin{document}$${R}^{2}=0.5$$\end{document}$ in setting B. The highest MSE is seen for the IVW method, which provides unbiased estimates, as can be seen in Supplementary Figs. [5](#MOESM1){ref-type="media"} and [6](#MOESM1){ref-type="media"}, but at the price of a high variance. As can be seen from Supplementary Table [1](#MOESM1){ref-type="media"}, all estimation methods except the IVW are biased conservatively towards the Null when there is a true causal effect. Yet, all causal effect estimates are unbiased when there is no causal effect. Supplementary Table [2](#MOESM1){ref-type="media"} (Setting A) and Supplementary Table [3](#MOESM1){ref-type="media"} (Setting B) provide the mean and the standard deviation of the causal effect estimates, which confirm the large standard deviation of the IVW estimate compared with the other approaches.Fig. 3Receiver operating characteristic (ROC) curve for simulation study on metabolite GWAS.ROC curves plotting the true positive rate against the false positive rate for **a** a small number of risk factors $\documentclass[12pt]{minimal}
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\begin{document}$$(d=12)$$\end{document}$ of which four have true positive effects (Scenario 1, Setting A) and for **b** a large number of risk factors $\documentclass[12pt]{minimal}
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\begin{document}$$(d=92)$$\end{document}$ of which four have true positive effects (Scenario 2, Setting A). Proportion of variance explained ($\documentclass[12pt]{minimal}
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\begin{document}$${R}^{2}$$\end{document}$) is set to 0.3.Table 1Mean squared error (MSE) of the causal effect estimates from the competing methods on the NMR metabolite and blood cell trait data.Setting ASetting B$\documentclass[12pt]{minimal}
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\begin{document}$${R}^{2}$$\end{document}$:0.10.30.50.10.30.5Scenario 1:IVW0.67270.16750.07840.59490.16190.0629Lars0.12920.04470.02980.15590.06480.0372Lasso0.06040.02890.01620.10460.05030.0307Elastic Net0.06730.03000.01620.11610.0480**0.0287**MR-BMA**0.03400.01750.01050.05340.0368**0.0306Best model0.07170.03200.01560.09210.05140.0376Scenario 2:IVW22.95166.05942.625723.24955.77152.4802Lars0.03540.03670.00940.03210.02120.0143Lasso0.00640.00470.00390.01050.00860.0074Elastic Net0.00640.00440.00340.00980.00780.0067MR-BMA**0.00510.00390.00320.00880.00760.0063**Best model0.01140.00810.00610.01500.01210.0096Scenario 3:IVW1.62000.42720.17422.31400.62080.2566Lars0.34610.11510.04820.58920.16690.0844Lasso0.01610.00670.00400.03780.02250.0166Elastic Net0.01680.00740.00440.04510.02240.0169BMA**0.00660.00340.00190.02350.01650.0149**Best model0.01280.00510.00270.04440.02420.0177We mark in bold font the lowest MSE in each experimental setting. Scenario 1: NMR metabolites, *d* = 12 risk factors, Scenario 2: NMR metabolites, *d* = 92 risk factors, and Scenario 3: blood cell traits, *d* = 33 risk factors. Setting A includes four true causal risk factors which increase the risk and Setting B includes eight true causal risk factors of which half are protective and the other half increases the risk
When increasing the number of risk factors to $\documentclass[12pt]{minimal}
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\begin{document}$$d=92$$\end{document}$ while keeping the number of true causal risk factors constant to four (Scenario 2, Setting A), the standard IVW method fails to distinguish between true causal and false causal risk factors and provides a ranking of risk factors which is nearly random as shown in the ROC curve in Fig. [3](#Fig3){ref-type="fig"}b and Supplementary Figs. [7](#MOESM1){ref-type="media"} and [8](#MOESM1){ref-type="media"}. Despite being unbiased (see Supplementary Figs. [9](#MOESM1){ref-type="media"} and [10](#MOESM1){ref-type="media"}, Supplementary Tables [1](#MOESM1){ref-type="media"}, [2](#MOESM1){ref-type="media"} and [3](#MOESM1){ref-type="media"}), the variance of the IVW estimates is large and prohibits better performance. In contrast, Lars, Lasso, Elastic Net and MR-BMA provide causal estimates which are biased towards zero, but have much reduced variance compared with the IVW estimates. The Lasso provides sparse solutions with many of the causal estimates set to zero. This allows the Lasso and Elastic Net to have relatively good performance at the beginning of the ROC curve, but their performance weakens when considering more risk factors. The best performance in terms of the ROC characteristics is observed for MR-BMA. In terms of MSE (Table [1](#Tab1){ref-type="table"}), the dominant role of the variance of the IVW estimate becomes again apparent as the IVW method has a thousand times larger MSE than MR-BMA, which has the lowest MSE for all scenarios considered. Similarly to earlier results on the bias of the effect estimates we find that the IVW is unbiased when there is a causal effect, while the other methods designed for high-dimensional settings are conservatively biased towards the null, and only unbiased when there is no causal effect (Supplementary Table [1](#MOESM1){ref-type="media"}).
In the blood cell trait data (Scenario 3), MR-BMA has again the lowest MSE, followed by the regularised regression approaches and the best model in the Bayesian approach. Despite a large sample size ($\documentclass[12pt]{minimal}
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\begin{document}$$d=33$$\end{document}$), the IVW approach is the only unbiased method at the cost of an inferior detection of true positive risk factors (Supplementary Figs. [12](#MOESM1){ref-type="media"} and [13](#MOESM1){ref-type="media"}) and a large variance (Supplementary Figs. [14](#MOESM1){ref-type="media"} and [15](#MOESM1){ref-type="media"}, Supplementary Tables [2](#MOESM1){ref-type="media"} and [3](#MOESM1){ref-type="media"}), and consequently a MSE which is in a magnitude of a hundred larger than other methods designed for high-dimensional data analysis (Table [1](#Tab1){ref-type="table"}).
Metabolites as risk factors for age-related macular degeneration {#Sec6}
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Next we demonstrate how MR-BMA can be used to select metabolites as causal risk factors for age-related macular degeneration (AMD). AMD is a painless eye-disease that ultimately leads to the loss of vision. AMD is highly heritable with an estimated heritability of up to 0.71 for advanced AMD in a twin study^[@CR20]^. A GWAS meta-analysis has identified 52 independent common and rare variants associated with AMD risk at a level of genome-wide significance^[@CR9]^. Several of these regions are linked to lipids or lipid-related biology, such as the *CETP*, *LIPC* and *APOE* gene regions^[@CR21]^. Lipid particles are deposited within drusen in the different layers of Bruch's membrane in AMD patients^[@CR21]^. A recent observational study has highlighted strong associations between lipid metabolites and AMD risk^[@CR22]^.
This evidence for lipids as potential risk factor for AMD has motivated a multivariable MR analysis which has shown that HDL cholesterol may be a putative risk factor for AMD, while there was no evidence of a causal effect for LDL cholesterol and triglycerides^[@CR23]^. Here, we extend this analysis to consider not just three lipid measurements, but a wider and more detailed range of $\documentclass[12pt]{minimal}
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\begin{document}$$d=30$$\end{document}$ metabolite measurements to pinpoint potential causal effects more specifically. As summary-level data we use $\documentclass[12pt]{minimal}
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\begin{document}$$d=30$$\end{document}$ metabolites as measured in the metabolite GWAS described earlier^[@CR11]^ for the same lipid-related instrumental variants as described previously. All of these metabolites have at least one genetic variant used as an instrumental variable that is genome-wide significant and no genetic associations of metabolites are stronger correlated than $\documentclass[12pt]{minimal}
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\begin{document}$$r=0.985$$\end{document}$. First, we prioritise and rank risk factors by their marginal inclusion probability (MIP) from MR-BMA using $\documentclass[12pt]{minimal}
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\begin{document}$${\sigma }^{2}=0.25$$\end{document}$ as prior variance and $\documentclass[12pt]{minimal}
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\begin{document}$$p=0.1$$\end{document}$ as prior probability, corresponding to a priori three expected causal risk factors. Secondly, we perform model diagnostics based on the best models with posterior probability $\documentclass[12pt]{minimal}
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\begin{document}$$> 0.02$$\end{document}$.
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\begin{document}$$n=148$$\end{document}$), the top risk factor with respect to its MIP (Supplementary Table [4](#MOESM1){ref-type="media"}A) is LDL particle diameter (LDL.D, MIP = 0.526). All other risk factors have evidence less than $\documentclass[12pt]{minimal}
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\begin{document}$$> 0.02$$\end{document}$. For illustration, we present here the predicted associations with AMD based on the best model including LDL.D, and TG content in small HDL (S.HDL.TG) against the observed associations with AMD. We colour code genetic variants according to their $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-statistic (Fig. [4](#Fig4){ref-type="fig"}a and Supplementary Fig. [16](#MOESM1){ref-type="media"}A, Supplementary Table [5](#MOESM1){ref-type="media"}) and Cook's distance (Fig. [4](#Fig4){ref-type="fig"}b and Supplementary Fig. [16](#MOESM1){ref-type="media"}B, Supplementary Table [6](#MOESM1){ref-type="media"}). First, the $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-statistic indicates two variants, rs492602 in the *FUT2* gene region and rs6859 in the *APOE* gene region, as outliers in all best models. Second, the genetic variant with the largest Cook's distance (*Cd* = 0.871--1.087) consistently in all models investigated is rs261342 mapping to the *LIPC* gene region. This variant has been indicated previously to have inconsistent associations with AMD compared with other genetic variants^[@CR23],[@CR24]^.Fig. 4Diagnostic plots for outliers and influential genetic variants.Plotting the predicted associations with AMD based on the model including LDL.D, and S.HDL.TG ($\documentclass[12pt]{minimal}
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\begin{document}$$n=148$$\end{document}$ genetic variants. This is the highest-ranking model when keeping outlying and influential genetic variants in the analysis. The colour code shows: **a** the $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-value larger than 10 or Cook\'s distance larger than the median of the relevant $\documentclass[12pt]{minimal}
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\begin{document}$$F$$\end{document}$-distribution is marked by a label indicating the gene region.
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\begin{document}$$n=145$$\end{document}$), and report the ten risk factors with the largest marginal inclusion probability in Table [2](#Tab2){ref-type="table"} and the full results in Supplementary Table [7](#MOESM1){ref-type="media"}. The top two risk factors are total cholesterol in extra-large HDL particles (XL.HDL.C, $\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{MIP}}=0.700$$\end{document}$) and total cholesterol in large HDL particles (L.HDL.C, $\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{MIP}}=0.229$$\end{document}$). XL.HDL.C and L.HDL.C were strongly correlated ($\documentclass[12pt]{minimal}
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\begin{document}$$r=0.80$$\end{document}$), and models including both have very low evidence as can be seen in Table [3](#Tab3){ref-type="table"} which gives the posterior probability of individual models. Supplementary Fig. [17](#MOESM1){ref-type="media"} shows the scatterplots of the genetic associations with each of these two risk factors individually against the genetic associations with AMD risk. We select the five individual models with a posterior probability $\documentclass[12pt]{minimal}
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\begin{document}$$> 0.02$$\end{document}$ to inspect the model fit (Supplementary Figs. [18](#MOESM1){ref-type="media"} and [19](#MOESM1){ref-type="media"}). This time, no genetic variant has a consistently large $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-statistic (Supplementary Table [8](#MOESM1){ref-type="media"}) or Cook's distance (Supplementary Table [9](#MOESM1){ref-type="media"}). Repeating the analysis without the largest influential point, rs5880 in the *CETP* gene region, or the strongest outlier, rs103294 in the *AC245884.7* gene region, did not impact the ranking of the risk factors. We tested the robustness of the results with respect to a wide range of prior variance and prior probability parameters; results did not change substantially (Supplementary Tables [10](#MOESM1){ref-type="media"} and [11](#MOESM1){ref-type="media"}).Table 2Ranking of risk factors for age-related macular degeneration (AMD) according to their marginal inclusion probability (MIP) after exclusion of outlying and influential variants ($\documentclass[12pt]{minimal}
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\begin{document}$$n=145$$\end{document}$).Risk factorMarginal inclusion probability (MIP)Model-averaged causal effect $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{\theta }}_{\text{MACE}}$$\end{document}$1XL.HDL.C0.70.3442L.HDL.C0.2290.0873HDL.D0.0870.0224XS.VLDL.TG0.082−0.0195LDL.D0.074−0.0186IDL.TG0.066−0.0127XXL.VLDL.TG0.0630.0188S.VLDL.TG0.062−0.0149Serum.TG0.061−0.01410Serum.C0.054−0.011Results are given after excluding genetic variants in the *APOE*, *FUTC*, and *LIPC* regions. $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{\theta }}_{\text{MACE}}$$\end{document}$ is the model-averaged causal effect of a risk factorHDL.D HDL diameter, IDL.TG triglycerides in IDL, L.HDL.C total cholesterol in large HDL, LDL.D LDL diameter, Serum.C serum total cholesterol, Serum.TG serum total triglycerides, S.VLDL.C total cholesterol in small VLDL, S.VLDL.TG triglycerides in small VLDL, XS.VLDL.TG triglycerides in very small VLDL, XL.HDL.C total cholesterol in very large HDLTable 3Ranking of models (sets of risk factors) for age-related macular degeneration (AMD) according to their posterior probability (PP) after exclusion of outlying and influential variants ($\documentclass[12pt]{minimal}
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\begin{document}$$n=145$$\end{document}$).Models or sets of risk factor(s)Posterior probability (PP)Model-specific causal estimates $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{\theta }}_{\gamma }$$\end{document}$1XL.HDL.C0.1560.5092L.HDL.C0.0780.3843XL.HDL.C,XS.VLDL.TG0.0260.457,−0.1814IDL.TG,XL.HDL.C0.025−0.179,0.4955HDL.D0.0230.3596Serum.C,XL.HDL.C0.019−0.183,0.5737S.VLDL.TG,XL.HDL.C0.015−0.172,0.4438S.VLDL.C,XL.HDL.C0.014−0.164,0.4779Serum.TG,XL.HDL.C0.014−0.169,0.46510S.HDL.TG,XL.HDL.C0.013−0.18,0.415Results are given after excluding genetic variants in the *APOE*, *FUTC*, and *LIPC* regions. $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{\theta }}_{\gamma }$$\end{document}$ is the causal effect estimate for a specific modelHDL.D HDL diameter, IDL.TG triglycerides in IDL, L.HDL.C total cholesterol in large HDL, LDL.D LDL diameter, Serum.C serum total cholesterol, Serum.TG serum total triglycerides, S.VLDL.C total cholesterol in small VLDL, S.VLDL.TG triglycerides in small VLDL, XS.VLDL.TG triglycerides in very small VLDL, XL.HDL.C total cholesterol in very large HDL
We also applied Lars, Lasso and Elastic Net after excluding outliers and influential points ($\documentclass[12pt]{minimal}
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\begin{document}$$n=145$$\end{document}$). Lars showed the largest regression coefficient for L.HDL.C including 11 risk factors. Lasso selected four risk factors with the largest regression coefficient for XL.HDL.C, while Elastic Net selected ten risk factors with the largest regression coefficient for L.HDL.C. Full results for the competing methods are given in Supplementary Tables [12](#MOESM1){ref-type="media"}--[15](#MOESM1){ref-type="media"}. A disadvantage of regularised regression approaches is that risk factor selection is binary; risk factors are either included in the model or set to have a coefficient zero. The magnitude of regularised regression coefficients does not rank risk factors according to their strength of evidence for inclusion in the model.
The detection of influential points in the initial analysis highlights rs26134, a genetic variant in the *LIPC* gene region, which had a strong impact on the analysis. Figure [5](#Fig5){ref-type="fig"} shows the model diagnostics of the highest ranked model excluding outlying and influential points (XL.HDL.C as the sole risk factor), with the variant in the *LIPC* gene region also plotted. This particular variant exhibits a distinct, potentially pleiotropic, effect. While all other variants support that XL.HDL.C increases the risk of AMD, this particular variant has the opposite direction of association with AMD risk as that predicted by its association with XL.HDL.C. Further functional and fine-mapping studies of this region are needed to understand the contrasting association of this variant with AMD risk.Fig. 5Diagnostic plot for influential genetic variants.Plotting the predicted associations with AMD based on the model including XL.HDL.C ($\documentclass[12pt]{minimal}
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\begin{document}$$n=148$$\end{document}$ genetic variants, where the colour code shows Cook\'s distance for the genetic variants. This is the highest-ranking model on omission of outlying and influential genetic variants from the analysis. Note rs26134 in the *LIPC* gene region which has an anomalous direction of association with AMD risk in contrast to all other genetic variants.
These results confirm previous studies^[@CR23],[@CR24]^ that identified HDL cholesterol as a putative risk factor for AMD and draw the attention to extra-large and large HDL particles. A recent observational study^[@CR22]^ supports our finding that extra-large HDL particles have an important role in the pathogenesis of AMD.
Discussion {#Sec7}
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We here introduce MR-BMA, an approach for multivariable MR which allows for the analysis of high-throughput experiments. This model averaging procedure prioritises and selects causal risk factors in a Bayesian framework from a high-dimensional set of related candidate risk factors. As is common for statistical techniques for variable selection, MR-BMA does not provide unbiased estimates. However, as shown in the simulation study, causal estimates from MR-BMA have reduced variance and thus MR-BMA improves over unbiased approaches, like the IVW method, in terms of mean squared error and detection of true risk factors. The primary aim of this work is to detect causal risk factors rather than to unbiasedly estimate the magnitude of their causal effects. MR-BMA is a multivariable MR approach that can analyse a high-dimensional set of risk factors. When analysing many risk factors jointly one important implicit assumption of MR-BMA is sparsity, i.e., the proportion of true causal risk factors compared with all risk factors considered is small. Since MR-BMA evaluates all possible combinations of risk factors exhaustively or all relevant combinations of risk factors in a shotgun stochastic search there is an upper bound for the maximum model size in order to keep the computation tractable. Sparsity is a common assumption for high-throughput data and we have seen in the applied example that the best models only contained one to three metabolites as risk factors despite allowing for a model size of up to twelve risk factors. Yet this is an important aspect of the algorithm and the maximum model size should be adjusted if models including many risk factors are expected or evidenced in the data.
We demonstrated the approach with application to a dataset of NMR metabolites, which included predominantly lipid measurements, using variants associated with lipids as instrumental variables. Previous MR analysis^[@CR23],[@CR24]^ including three lipid measurements from the Global Lipids Genetics Consortium^[@CR19]^ have identified HDL cholesterol as potential risk factor for AMD. Our approach to multivariable MR refined this analysis and confirmed HDL cholesterol as a potential causal risk factor for AMD, further pinpointing that large or extra-large HDL particles are likely to be driving disease risk. Other areas of application where this method could be used include imaging measurements of the heart and coronary artery disease, body composition measures and type 2 diabetes, or blood cell traits and atherosclerosis. As multivariable MR accounts for measured pleiotropy, this approach facilitates the selection of suitable genetic variants for causal analyses. In each case, it is likely that genetic predictors of the set of risk factors can be found, even though finding specific predictors of, for example, particular heart measurements from cardiac imaging, may be difficult given widespread pleiotropy^[@CR25]^. MR-BMA allows a more agnostic and hypothesis-free approach to causal inference, allowing the data to identify the causal risk factors.
Multivariable MR estimates the direct effect of a risk factor on the outcome and not the total effect as estimated in standard univariable MR. This is in analogy with multivariable regression where the regression coefficients represent the association of each variable with the outcome when all others are held constant. Having said this, the main goal of our approach is risk factor selection, and not the precise estimation of causal effects, since the variable selection procedure shrinks estimates towards the null. This results in causal effect estimates being biased towards the Null when there is a causal effect and unbiased estimates when there is no causal effect. If there are mediating effects between the risk factors, then this approach will identify the risk factor most proximal to and has the most direct effect on an outcome. For example, if the risk factors included would form a signalling cascade (Supplementary Fig. [1](#MOESM1){ref-type="media"}b) then our approach would identify the downstream risk factor in the cascade with the direct effect on the outcome and not the upstream risk factors in the beginning of the cascade. Hence, a risk factor may be a cause of the outcome, but if its causal effect is mediated via another risk factor included in the analysis, then it will not be selected in the multivariable MR approach.
Our approach is formulated in a Bayesian framework. Particular care needs to be taken when choosing the hyper-parameter for the prior probability which relates to the a priori expected number of causal risk factors. In the applied example the results were robust to a wide range of prior specifications for the parameter as seen in Supplementary Table [10](#MOESM1){ref-type="media"}. In addition, the prior variance of the causal parameters needs to be specified and tested for robustness as we show in the Supplementary Table [11](#MOESM1){ref-type="media"}.
When genetic variants are weak predictors for the risk factors, this can introduce weak instrument bias. In univariable two-sample MR, any bias due to weak instruments is towards the null and does not lead to inflated type 1 error rates^[@CR26]^. However, in multivariable MR, weak instrument bias can be in any direction (see Methods), although bias will tend to zero as the sample size increases and consequently the instrument strength increases. Selection of risk factors is only possible if there are genetic variants that are predictors of these risk factors. One of the biggest challenges of multivariable MR is the design of a meaningful study, in particular the choice of both, the genetic variants and the risk factors. The design of the study is important for the interpretation of the risk factors prioritised: The ranking of risk factors is conditional on the genetic variants used. For instance, in our applied example we find evidence for extra-large and large HDL cholesterol concentration given that we used lipid-related genetic variants as instrumental variables. We recommend to include only risk factors which have at least one, and ideally multiple genetic variants that act as strong instruments. Caution is needed for the interpretation of null findings, particularly in our example for non-lipid risk factors, as these might be deprioritised in terms of statistical power by our choice of genetic variants. The instrument selection and general study design are essential for the MR-BMA approach and we strongly recommend the user to be critical in the choice of genetic variants and risk factors. Moreover, similar to standard MR we urge to perform model checks and be transparent in the presentation of the removal of outlier/influential genetic variants.
A further requirement for multivariable MR is that the genetic variants can distinguish between risk factors^[@CR12]^. We recommend to check the correlation structure between genetic associations for the selected genetic variants and to include no pair of risk factors which is extremely strongly correlated. In the applied example, we included only risk factors with an absolute correlation \<0.99. As we were not able to include more than three measurements for each lipoprotein category (cholesterol content, triglyceride content, diameter), care should be taken not to overinterpret findings in terms of the specific measurements included in the analysis rather than those correlated measures that were excluded from the analysis (such as phospholipid and cholesterol ester content).
Another assumption of multivariable MR is that there is no unmeasured horizontal pleiotropy. This means that the variants do not influence the outcome except via the measured risk factors. The assumption of no horizontal pleiotropy is a common and untestable assumption in MR. It is an active area of research to robustify MR against violations of this assumption. Some of these robust methods for MR make a specific assumption about the behaviour of pleiotropic variants, such as MR-Egger^[@CR27]^, which assumes pleiotropic effects are uncorrelated from the genetic associations with the risk factor---the InSIDE assumption. Other methods exclude outlying variants as they are potentially pleiotropic such as MR-PRESSO^[@CR28]^. In multivariable MR, pleiotropic variants can be detected as outliers to the model fit. Here we quantify outliers using the $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-statistic. Outlier detection in standard univariable MR can be performed by model averaging where different subsets of instruments are considered^[@CR29],[@CR30]^, assuming that a majority of instruments is valid, but without prior knowledge which are the valid instruments. In multivariable MR, ideally one would like to perform model selection and outlier detection simultaneously. In addition, we search for genetic variants that are influential points. While these may not necessary be pleiotropic, we suggest removing such variants as a sensitivity analysis to judge whether the overall findings from the approach are dominated by a single variant. Findings are likely to be more reliable when they are evidenced by multiple genetic variants. One necessary future development is post-selection inference^[@CR31],[@CR32]^ in the high-dimensional multivariable MR framework. MR-BMA does not provide unbiased causal effect estimates. Re-fitting an unbiased multivariable MR model after risk factor selection would ignore the uncertainty of the selection and consequently not provide valid inferences.
In conclusion, we introduce here MR-BMA, the first approach to perform risk factor selection in multivariable MR, which can identify causal risk factors from a high-throughput experiment. MR-BMA can be used to determine which out of a set of related risk factors with common genetic predictors are the causal drivers of disease risk.
Methods {#Sec8}
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Mendelian randomization data input: summarized data set-up {#Sec9}
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One of the key features of MR is that the approach can be performed using summarised data on genetic associations---beta-coefficients and their standard errors from univariate regression analyses. No access to individual-level genotype data is needed. In addition, these association estimates can be derived from different samples. In two-sample MR, the genetic associations with the risk factor are derived from one sample and the genetic associations with the outcome from another sample^[@CR26]^. The use of summarised data in two-sample MR allows the sample size to be maximised by integrating data from large meta-analyses including hundreds of thousands of participants.
We assume the context of two-sample MR with summarized data^[@CR33]^. For each genetic variant $\documentclass[12pt]{minimal}
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\begin{document}$$\mathrm{se}({\beta }_{{X}_{ij}}^{\star })$$\end{document}$ from a univariable regression in which the risk factor $\documentclass[12pt]{minimal}
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\begin{document}$${{\bf{X}}}_{j}$$\end{document}$ is regressed on the genetic variant $\documentclass[12pt]{minimal}
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\begin{document}$${{\bf{G}}}_{i}$$\end{document}$ in sample one, and beta-coefficient $\documentclass[12pt]{minimal}
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\begin{document}$$\mathrm{se}({\beta }_{{Y}_{i}}^{\star })$$\end{document}$ from a univariable regression in which the outcome $\documentclass[12pt]{minimal}
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\begin{document}$${\bf{Y}}$$\end{document}$ is regressed on the genetic variant $\documentclass[12pt]{minimal}
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\begin{document}$${{\bf{G}}}_{i}$$\end{document}$ in sample two. For simplicity of notation, although the beta-coefficients are estimates, we omit the conventional "hat" notation and treat the beta-coefficients as observed data points. When considering multiple risk factors, we construct a matrix of beta-coefficients $\documentclass[12pt]{minimal}
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\begin{document}$$d$$\end{document}$ is the number of risk factors and $\documentclass[12pt]{minimal}
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\begin{document}$$n$$\end{document}$ is the number of genetic variants.
We assume that the genetic effects on risk factors and on the outcome are linear and homogeneous across the population, and identical between the two samples^[@CR34]^. Furthermore, we assume that the $\documentclass[12pt]{minimal}
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\begin{document}$$n$$\end{document}$ genetic variants selected as instrumental variables are independent, an assumption common in MR studies. This is usually achieved by including only the lead genetic variant from each gene region in the analysis. Finally, we assume that genetic association estimates are derived from two distinct samples with no overlap between the samples. These assumptions can all be relaxed to some extent if the goal is causal inference rather than causal estimation; see ref. ^[@CR35]^ for details.
Multivariable Mendelian randomization and the linear model {#Sec10}
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Multivariable MR is an extension of the standard MR paradigm (Fig. [1](#Fig1){ref-type="fig"}) to model not one, but multiple risk factors as illustrated in Fig. [2](#Fig2){ref-type="fig"}. Univariable MR can be cast as a weighted linear regression model in which the genetic associations with the outcome $\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{Y}_{i}}^{\star }$$\end{document}$ are regressed on the genetic associations with the risk factor $\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{X}_{i}}^{\star }$$\end{document}$ in order to estimate the total effect $\documentclass[12pt]{minimal}
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\begin{document}$$\theta$$\end{document}$ of the risk factor $\documentclass[12pt]{minimal}
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\begin{document}$${\bf{Y}}$$\end{document}$^[@CR36]^$$\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{Y}_{i}}^{\star }=\theta {\beta }_{{X}_{i}}^{\star }+{\epsilon }_{i},\quad {\epsilon }_{i} \sim {\mathcal{N}}(0,{\mathrm{se}}{({\beta }_{{Y}_{i}}^{\star })}^{2}).$$\end{document}$$
In multivariable MR, the genetic associations with the outcome are regressed on the genetic associations with all the $\documentclass[12pt]{minimal}
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\begin{document}$$j=1,\ldots ,d$$\end{document}$ risk factors^[@CR10]^$$\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{Y}_{i}}^{\star }={\theta }_{1}{\beta }_{{X}_{i1}}^{\star }+{\theta }_{2}{\beta }_{{X}_{i2}}^{\star }+\ldots +{\theta }_{d}{\beta }_{{X}_{id}}^{\star }+{\epsilon }_{i},\quad {\epsilon }_{i} \sim {\mathcal{N}}(0,{\mathrm{se}}{({\beta }_{{Y}_{i}}^{\star })}^{2}).$$\end{document}$$
Weights in these regression models are proportional to inverse of the variance of the genetic association with the outcome ($\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{se}}{({\beta }_{{Y}_{i}}^{\star })}^{-2}$$\end{document}$). This is to ensure that genetic variants having more precise association estimates receive more weight in the analysis. The same weighting can also be achieved by standardising the association estimates, by dividing $\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{se}}({\beta }_{{Y}_{i}}^{\star })$$\end{document}$. In the following derivations, we assume that $\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{X}_{i}}={\beta }_{{X}_{i}}^{\star }/{\mathrm{se}}({\beta }_{{Y}_{i}}^{\star })$$\end{document}$ are standardised, so that the variances of the $\documentclass[12pt]{minimal}
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\begin{document}$${\epsilon }_{i}$$\end{document}$ terms are all 1. To account for heterogeneity in the regression equation, we can use a multiplicative random effects model, which increases the variance of the error terms by a multiplicative factor^[@CR37]^. Our parameter of interest is the vector of regression coefficients $\documentclass[12pt]{minimal}
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\begin{document}$${\boldsymbol{\theta }}=\{{\theta }_{1},\ldots ,{\theta }_{d}\}$$\end{document}$. These are the direct causal effects of the risk factors in turn on the outcome when all the other risk factors in the model are held constant^[@CR13]^. In contrast, univariable Mendelian randomization using genetic variants that are instrumental variables for the specific risk factor of interest estimates the total effect of the risk factor on the outcome. The direct effect will differ from the total effect if the effect of the risk factor is mediated via another risk factor included in the model^[@CR12]^. We illustrate the difference between the direct and total effect using directed acyclic graphs in Supplementary Fig. [1](#MOESM1){ref-type="media"}. In some cases (such as to identify the proximal risk factor to the outcome), the direct effect is of interest; in other cases (such as to evaluate the potential impact of intervening on a risk factor), it is the total effect that is truly of interest^[@CR13]^.
Choosing genetic variants as instruments {#Sec11}
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In multivariable MR, a genetic variant is a valid instrumental variable if the following criteria hold:IV1 Relevance: The variant is associated with at least one of the risk factors.IV2 Exchangeability: The variant is independent of all confounders of each of the risk factor--outcome associations.IV3 Exclusion restriction: The variant is independent of the outcome conditional on the risk factors and confounders.
One of the main differences of multivariable MR compared with univariable MR is the relaxation of the exclusion restriction condition. In contrast to univariable MR, multivariable MR allows for measured pleiotropy^[@CR14]^ via any of the observed risk factors. Hence the instrumental variable assumptions are more likely to be satisfied for multivariable MR than for univariable MR for a given choice of genetic variants.
It is not necessary for every genetic variant to be associated with all the risk factors, although if no genetic variants are associated with a particular risk factor, then the causal effect of that risk factor cannot be identified. This would also occur if the genetic associations with two risk factors were exactly proportional. For precise identification of causal risk factors, it is necessary to have some variants that are more strongly associated with particular risk factors than others^[@CR12]^. More precisely a risk factor can be included into the analysis if the following criteria (RF1--RF2) hold:RF1 Relevance: The risk factor needs to be strongly instrumented by at least one genetic variant included as instrumental variable.RF2 No multi-collinearity: The genetic associations of any risk factor included cannot be linearly explained by the genetic associations of any other risk factor or by the combination of genetic associations of multiple other risk factors included in the analysis.
The study design and in particular the selection of genetic variants as IVs are the most important steps for multivariable MR and great care needs to be taken when designing the study and also when reporting the study design. All interpretation of the results is conditional on the genetic variants selected as IVs. We initially assume that all genetic variants are valid instruments. There is an emerging literature^[@CR27],[@CR38]^ on how to perform robust MR analysis in the presence of invalid instruments; similar extensions can be adapted for multivariable MR^[@CR14]^.
Risk factor selection as variable selection in the linear model {#Sec12}
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We consider the situation in which we have a set of genetic variants that are instrumental variables for a set of risk factors, and we want to select which of those risk factors are causes of the outcome. Our implicit prior belief is that not all of the risk factors are causally related to the outcome and that there are some true causal risk factors (signal) and some risk factors which do not have an effect (noise). We formulate the selection of risk factors in two-sample multivariable MR as a variable selection task in the linear regression framework. In order to model the correlation between risk factors we base our likelihood on a Gaussian distribution$$\documentclass[12pt]{minimal}
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\begin{document}$${{\boldsymbol{\beta }}}_{{\bf{Y}}}| {{\boldsymbol{\beta }}}_{{\bf{X}}},{\boldsymbol{\theta }},\tau \sim N\left({{\boldsymbol{\beta }}}_{{\bf{X}}}{\boldsymbol{\theta }},\frac{1}{\tau }\right).$$\end{document}$$Following the $\documentclass[12pt]{minimal}
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\begin{document}$${D}_{2}$$\end{document}$ prior specifications as introduced in ref. ^[@CR39]^, we use the following conjugate priors for the causal effects $\documentclass[12pt]{minimal}
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\begin{document}$${\boldsymbol{\theta }} \sim \, N(0,{\boldsymbol{\nu }}/\tau )\\ \epsilon \sim \, N\left(0,\frac{1}{\tau }\right)\\ \tau \sim \, \Gamma (\kappa /2,\lambda /2),$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$${\boldsymbol{\nu }}={\mathrm{diag}}({\sigma }^{2})$$\end{document}$ is the diagonal variance matrix of the causal effects (independence prior), and the precision $\documentclass[12pt]{minimal}
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\begin{document}$${\gamma }_{j}=\left\{\begin{array}{l}1, \, {\rm{if}}\ {\rm{the}}\ j{\rm{th}}\ {\rm{risk}}\ {\rm{factor}}\ {\rm{is}}\ {\rm{selected}},\\ 0 \, \, \text{otherwise}.\end{array}\right.$$\end{document}$$The indicator $\documentclass[12pt]{minimal}
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\begin{document}$${\boldsymbol{\gamma }}$$\end{document}$ encodes a specific regression model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ that includes the risk factors as indicated in $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ can include one or a combination of multiple risk factors. To evaluate the evidence of a specific model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$, we calculate the Bayes factor for model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ against the null model that does not include an intercept or any risk factor. The Bayes factor $\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})$$\end{document}$ has the following closed form representation$$\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})=\frac{| {\mathbf{\Omega}}{| }^{1/2}}{| {{\boldsymbol{\nu }}}_{\boldsymbol{\gamma }}{| }^{1/2}}{\left(\frac{{\boldsymbol{\beta }}_{\bf{Y}} ^{t}{\boldsymbol{\beta }}_{\bf{Y}}-{\mathbf{\Theta }}^{t}{\mathbf{\Omega }}^{-1}{\mathbf{\Theta }}} {{\boldsymbol{\beta }}_{\bf{Y}} ^{t}{\boldsymbol{\beta }}_{{\bf{Y}}}}\right)}^{-n/2},$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$${\mathbf{\Theta }}={\mathbf{\Omega }}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{\bf{Y}}}$$\end{document}$ is the causal effect estimate and $\documentclass[12pt]{minimal}
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\begin{document}$${\mathbf{\Omega }}={({{\boldsymbol{\nu }}}_{{\boldsymbol{\gamma }}}^{-1}+{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}})}^{-1}$$\end{document}$ is the inverse of the shrinkage covariance between the genetic associations of the risk factors. For a detailed derivation of the Bayes factor we refer to the Supplementary Methods in the Supplementary Information.
Prior specification {#Sec13}
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Another important aspect is the prior for the model size $\documentclass[12pt]{minimal}
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\begin{document}$$k$$\end{document}$, which we model using a Binomial distribution$$\documentclass[12pt]{minimal}
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\begin{document}$$Pr(K=k)=\left(\begin{array}{l}d\\ k\end{array}\right){p}^{k}{(1-p)}^{d-k}.$$\end{document}$$This requires choosing the probability $\documentclass[12pt]{minimal}
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\begin{document}$$p$$\end{document}$ of including a risk factor in the model according to prior assumptions regarding the sparsity of the results. We recommend to select $\documentclass[12pt]{minimal}
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\begin{document}$$p$$\end{document}$ according to the expected a priori model size, which is $\documentclass[12pt]{minimal}
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\begin{document}$$p\times d$$\end{document}$. Currently, all risk factors are assumed to have the same prior probability, and thus the probability of all models of the same size $\documentclass[12pt]{minimal}
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\begin{document}$$k$$\end{document}$ is equal. The prior of a specific model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ of size $\documentclass[12pt]{minimal}
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\begin{document}$$k$$\end{document}$ is defined as$$\documentclass[12pt]{minimal}
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\begin{document}$$p({M}_{{\boldsymbol{\gamma }}})={\left(\begin{array}{l}d\\ k\end{array}\right)}^{-1}Pr(K=k)={p}^{k}{(1-p)}^{d-k}.$$\end{document}$$
The second important aspect is the prior for the variance of the risk factors $\documentclass[12pt]{minimal}
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\begin{document}$${\boldsymbol{\nu }}={\mathrm{diag}}({\sigma }^{2})$$\end{document}$, where we assume that all risk factors have the same prior variance $\documentclass[12pt]{minimal}
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\begin{document}$${\sigma }^{2}$$\end{document}$. Large values of $\documentclass[12pt]{minimal}
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\begin{document}$${\sigma }^{2}$$\end{document}$ would favour strong causal effects of the risk factors on the outcome. Following ref. ^[@CR39]^ we initially set $\documentclass[12pt]{minimal}
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\begin{document}$${\sigma }^{2}=0.25$$\end{document}$, but sensitivity of the results with respect to this prior should be investigated. The parameter can be specified in the implementation of MR-BMA. In the applied example we perform a sensitivity analysis for this important parameter.
Posterior calculation and marginal inclusion probability of a risk factor {#Sec14}
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\begin{document}$${\mathbf{\Gamma }}$$\end{document}$ be the space of all possible combinations of risk factors. The posterior probability (PP) of a model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ can be expressed by the prior probability ([8](#Equ8){ref-type=""}) and the Bayes factor ([6](#Equ6){ref-type=""}) of model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ is$$\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{PP}}({M}_{{\boldsymbol{\gamma }}}| {{\boldsymbol{\beta }}}_{{\bf{Y}}},{{\boldsymbol{\beta }}}_{{\bf{X}}})=\frac{p({M}_{{\boldsymbol{\gamma }}}){\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})}{{\sum }_{{\boldsymbol{\gamma }}\in {\boldsymbol{\Gamma }}}p({M}_{{\boldsymbol{\gamma }}}){\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})}.$$\end{document}$$
In high-dimensional variable selection, the evidence for one particular model can be small because the model space is very large and many models might have comparable evidence. This is why MR-BMA uses Bayesian model averaging (BMA) and computes for each risk factor $\documentclass[12pt]{minimal}
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\begin{document}$$j$$\end{document}$ its marginal inclusion probability (MIP), which is defined as the sum of the posterior probabilities over all models where the risk factor is present$$\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{MIP}}(j=1| {{\boldsymbol{\beta }}}_{{\bf{Y}}},{{\boldsymbol{\beta }}}_{{\bf{X}}})=\frac{{\sum }_{{\boldsymbol{\gamma }}\in {\mathbf{\Gamma }}}I({\gamma }_{j}=1)p({M}_{{\boldsymbol{\gamma }}}){\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})}{{\sum }_{{\boldsymbol{\gamma }}\in {\mathbf{\Gamma }}}p({M}_{{\boldsymbol{\gamma }}}){\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})},$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$I({\gamma }_{j}=1)$$\end{document}$ equals 1 if risk factor $\documentclass[12pt]{minimal}
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\begin{document}$$j$$\end{document}$ is part of the model and 0 otherwise.
An exhaustive evaluation of all possible combinations of risk factors is computationally prohibitive already for a moderate number of risk factors ($\documentclass[12pt]{minimal}
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\begin{document}$$d \, > \, 20$$\end{document}$). To alleviate this issue we have implemented a shotgun stochastic search algorithm^[@CR40]^ that evaluates all combinations of risk factors with a non-negligible contribution to the calibration factor $\documentclass[12pt]{minimal}
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\begin{document}$${\sum }_{{\boldsymbol{\gamma }}\in {\mathbf{\Gamma }}}p({M}_{{\boldsymbol{\gamma }}}){\mathrm{BF}}({M}_{{\boldsymbol{\gamma }}})$$\end{document}$ in Eq. ([9](#Equ9){ref-type=""}). This algorithm is based on the assumption that the majority of combinations of risk factors have a posterior probability close to zero and do not need to be considered when computing the calibration factor in the denominator of Eqs. ([9](#Equ9){ref-type=""}) and ([10](#Equ10){ref-type=""}).
Causal estimation {#Sec15}
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We derive the estimates for the causal effects $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\theta }}}}_{{\boldsymbol{\gamma }}}$$\end{document}$ of model $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\theta }}}}_{{\boldsymbol{\gamma }}}={\mathbf{\Omega }}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{\bf{Y}}}={({{\boldsymbol{\nu }}}_{{\boldsymbol{\gamma }}}^{-1}+{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}})}^{-1}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{\bf{Y}}},$$\end{document}$$which is closely related to the regression coefficient in Ridge regression. Adding the diagonal matrix $\documentclass[12pt]{minimal}
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\begin{document}$${{\boldsymbol{\nu }}}_{{\boldsymbol{\gamma }}}^{-1}$$\end{document}$ stabilises the inversion and makes the estimate more robust to strong correlation among risk factors. There can be strong correlation between candidate risk factors as seen in the genetic correlation matrices in the applied examples as illustrated in Supplementary Figs. [2](#MOESM1){ref-type="media"} and [11](#MOESM1){ref-type="media"}, which makes it important to stabilise the causal estimate.
The model-averaged causal estimate (MACE) for risk factor $\documentclass[12pt]{minimal}
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\begin{document}$$j$$\end{document}$ from the MR-BMA approach is$$\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\theta }}}}_{\text{MACE}}(j)=\sum _{{\boldsymbol{\gamma}}\in {\boldsymbol{\Gamma }}}I({\gamma }_{j}=1){\mathrm{PP}}({M}_{{\boldsymbol{\gamma }}}| {{\boldsymbol{\beta }}}_{{\bf{Y}}},{{\boldsymbol{\beta }}}_{{\bf{X}}}){\hat{{\boldsymbol{\theta }}}}_{{\boldsymbol{\gamma }}}.$$\end{document}$$Both $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\theta }}}}_{{\boldsymbol{\gamma }}}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\theta }}}}_{{\mathrm{MACE}}}$$\end{document}$ are conservative estimates of the true causal effect. They are biased towards the Null if there is a causal effect and unbiased otherwise. We therefore only recommend to interpret the direction of effect and the magnitude of these causal effect estimates in comparison with the effects of other risk factors.
MR-BMA ranks and prioritises risk factors according to their marginal inclusion probability and estimates the MACE as defined in Eq. ([12](#Equ12){ref-type=""}). As an alternative approach, we also consider selecting the 'best model' based on the individual model posterior probabilities as defined in Eq. ([9](#Equ9){ref-type=""}).
Detection of invalid and influential instruments {#Sec16}
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Invalid instruments may be detected as outliers with respect to the fit of a specific linear model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$. We recommend to check the best individual models for outliers by visual inspection of the scatterplot of the predicted associations based on $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{{\boldsymbol{\beta }}}}_{Y}={{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}}{{\hat{\boldsymbol{\theta }}}}_{{\boldsymbol{\gamma }}}$$\end{document}$ against the actual observed $\documentclass[12pt]{minimal}
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\begin{document}$${{\boldsymbol{\beta }}}_{{\bf{Y}}}$$\end{document}$. If a genetic variant is detected consistently as an outlier in several of the top models, it may be advisable to explore the analyses excluding that outlying variant from the analysis. To quantify outliers we use the $\documentclass[12pt]{minimal}
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\begin{document}$$Q$$\end{document}$-statistic, which is an established tool for identifying heterogeneity in meta-analysis^[@CR15]^. It is defined as the sum of the residual vector $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$, which is the squared difference between the observed and predicted association with the outcome$$\documentclass[12pt]{minimal}
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\begin{document}$$Q=\sum_{i=1}^{n}{q}_{i}=\sum_{i=1}^{n}{({\beta }_{{Y}_{i}}-{\hat{\beta }}_{{Y}_{i}})}^{2}.$$\end{document}$$We note that Eq. ([13](#Equ13){ref-type=""}) is defined on the weighted coefficients $\documentclass[12pt]{minimal}
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\begin{document}$${\beta }_{{Y}_{i}}^{\star }$$\end{document}$ the $\documentclass[12pt]{minimal}
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\begin{document}$$Q$$\end{document}$-statistic^[@CR12]^ is defined as$$\documentclass[12pt]{minimal}
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\begin{document}$$Q=\sum_{i=1}^{n}{q}_{i}=\sum_{i=1}^{n}\frac{1}{{\mathrm{se}}{({\beta }_{{Y}_{i}}^{\star })}^{2}}{({\beta }_{{Y}_{i}}^{\star }-{\hat{\beta }}_{{Y}_{i}}^{\star })}^{2},$$\end{document}$$with first order weighting equal to $\documentclass[12pt]{minimal}
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\begin{document}$$\frac{1}{{\mathrm{se}}{({\beta }_{{Y}_{i}}^{\star })}^{2}}$$\end{document}$^[@CR41]^.
The individual element $\documentclass[12pt]{minimal}
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\begin{document}$${q}_{i}$$\end{document}$ measures the heterogeneity of genetic variant $\documentclass[12pt]{minimal}
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\begin{document}$$i$$\end{document}$ for a particular model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$. We refer to $\documentclass[12pt]{minimal}
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\begin{document}$$q$$\end{document}$-statistic, and use this to evaluate if specific genetic variants are outliers to the model fit.
Even if there are no outliers, it is advisable to check for influential observations and re-run the approach omitting a particular influential variant from the analysis. If a particular genetic variant has a strong association with the outcome, then it may have undue influence on the variable selection, leading to a model that fits that particular observation well, but other observations poorly. To quantify influential observations for a particular model $\documentclass[12pt]{minimal}
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\begin{document}$${M}_{{\boldsymbol{\gamma }}}$$\end{document}$ we suggest to use Cook's distance^[@CR16]^$$\documentclass[12pt]{minimal}
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\begin{document}$$C{d}_{i}=\frac{{q}_{i}}{{s}^{2}d}\frac{{h}_{i}}{{(1-{h}_{i})}^{2}},$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$${\bf{H}}={{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}}{({{\boldsymbol{\nu }}}_{{\boldsymbol{\gamma }}}^{-1}+{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}})}^{-1}{{\boldsymbol{\beta }}}_{{{\bf{X}}}_{{\boldsymbol{\gamma }}}} ^{t}$$\end{document}$, and $\documentclass[12pt]{minimal}
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\begin{document}$${s}^{2}=\frac{1}{n-d}{\epsilon }^{t}\epsilon$$\end{document}$ is the mean squared error of the regression model. Following ref. ^[@CR42]^, we recommend to use the median of a central $\documentclass[12pt]{minimal}
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\begin{document}$$F$$\end{document}$-distribution with $\documentclass[12pt]{minimal}
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\begin{document}$$n-d$$\end{document}$ degrees of freedom as a threshold, and remove variants that have a Cook's distance which exceeds this value.
Impact of weak instrument bias {#Sec17}
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In the following presentation, we consider two risk factors with observed genetic associations $\documentclass[12pt]{minimal}
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\begin{document}$$Y$$\end{document}$, respectively. Following the measurement error literature^[@CR43]^, we derive the induced bias of the IVW estimates of the true causal effects $\documentclass[12pt]{minimal}
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\begin{document}$${\hat{\theta }}_{1}= \, {\theta }_{1}-\frac{{\theta }_{1}{\lambda }_{1}-\rho {\theta }_{2}{\lambda }_{2}}{1-\rho }\\ {\hat{\theta }}_{2}= \, {\theta }_{2}-\frac{{\theta }_{2}{\lambda }_{2}-\rho {\theta }_{1}{\lambda }_{1}}{1-\rho },$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$${\mathrm{var}}(\epsilon )$$\end{document}$, decreases when increasing the sample size. If the genetic associations with the risk factors are estimated with different degrees of uncertainty, then bias could be more considerable. Analogous to differential measurement error, risk factors with more precisely estimated genetic associations would be prioritized in the regression model. In our application, all risk factors are measured on the same high-throughput platform and on the same sample size, thus reducing the impact of weak instrument bias to influence the ranking of risk factors.
Simulation study {#Sec18}
----------------
To evaluate the performance of MR-BMA, we perform a simulation study taking genetic associations with risk factors from two real data sets, the first one based on genetic associations with NMR metabolites^[@CR11]^ and secondly on genetic associations with blood cell traits^[@CR4]^. Further information on the data sets and pre-processing is given in the next sections. We simulate genetic associations with the outcome $\documentclass[12pt]{minimal}
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\begin{document}$$d=33$$\end{document}$ all blood cell traits available) number of risk factors included.Number of true risk factors: (Setting A) four risk factors have an effect of $\documentclass[12pt]{minimal}
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\begin{document}$$\theta =-0.3$$\end{document}$, the other risk factors have no effect.Proportion of variance in the outcome explained by the risk factors: $\documentclass[12pt]{minimal}
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\begin{document}$${R}^{2}=0.1,0.3,0.5$$\end{document}$ which defines the variance of the error.
We compare six different analysis methods:Multivariable inverse-variance weighted (IVW) regression (Eq. ([2](#Equ2){ref-type=""}))^[@CR10]^Least-angle regression (Lars) as L1 regularised regression^[@CR17]^Lasso as L1 regularised regression^[@CR18]^Elastic Net as L1 and L2 regularised regression^[@CR18]^MR-BMA using marginal inclusion probabilities (Eq. ([10](#Equ10){ref-type=""}))Bayesian best model selection using posterior probabilities of individual models (Eq. ([9](#Equ9){ref-type=""}))
Both Lars^[@CR17]^ and Lasso are versions of L1 regularised linear regression, and Elastic Net is a mixture of a L1 and L2 regularised linear regression, all of which have been devised for variable selection in high-dimensional data. We use here the Lars implementation^[@CR17]^ and for Lasso and Elastic Net we use the glmnet^[@CR18]^ implementation. For all regularised regression methods, we use cross-validation (CV) to tune the regularisation parameter to achieve the minimum cross-validation MSE. For the small risk factor space including 12 NMR metabolites, the MR-BMA approach is performed using an exhaustive search of all possible models with prior probability of a risk factor to be included set to $\documentclass[12pt]{minimal}
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Data pre-processing for NMR metabolites for simulation {#Sec19}
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The first data resource used for the simulation and application is publicly available summarized data on genetic associations with risk factors derived from a NMR metabolite GWAS^[@CR11]^ from <http://computationalmedicine.fi/data#NMR_GWAS>. All of the metabolites were inverse rank-based normal transformed, so the association estimates are all in standard deviation units. In order to avoid selection bias, we choose genetic variants based on an external dataset. As the majority of the metabolite measures relates to lipids, we take $\documentclass[12pt]{minimal}
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\begin{document}$$<5\times 1{0}^{-8}$$\end{document}$) in a large meta-analysis of the Global Lipids Genetics Consortium^[@CR19]^. We extract beta-coefficients and standard errors of genetic associations for the 150 genetic variants and the 118 available metabolites. Next, we compute the genetic correlation structure between metabolites based on the $\documentclass[12pt]{minimal}
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\begin{document}$$n=150$$\end{document}$ genetic variants. This allows us to investigate risk factor selection for a realistic genetic correlation structure between metabolites (Supplementary Fig. [2](#MOESM1){ref-type="media"}) and distribution of the regression coefficients.
Data pre-processing for blood cell traits for simulation {#Sec20}
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As a secondary data resource, we use publicly available summary data from the GWAS catalog <https://www.ebi.ac.uk/gwas/> on 36 blood cell traits measured on nearly 175,000 participants^[@CR4]^. Using all genetic variants that were genome-wide significant for any blood cell trait we have $\documentclass[12pt]{minimal}
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\begin{document}$$> 0.99$$\end{document}$. After removing three composite traits (sum of eutrophil and eosinophil counts, granulocyte count, and sum of basophil and neutrophil counts) from further analysis, there was no pair of blood cell traits with greater genetic correlation than 0.99. The respective correlation matrix is shown in Supplementary Fig. [11](#MOESM1){ref-type="media"}. The final dataset used for the simulation consists of $\documentclass[12pt]{minimal}
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Data pre-processing and analysis for applied example of age-related macular degeneration {#Sec21}
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In the applied example we demonstrate how MR-BMA can be used to select metabolites as causal risk factors for age-related macular degeneration (AMD). As risk factors we consider a range of circulating metabolites measured by NMR spectroscopy^[@CR11]^. We use the same lipid-related genetic variants as in the simulation study. We restrict the risk factor space to include only lipoprotein measurements on total cholesterol content, triglyceride content, and particle diameter. For the various fatty acid measurements, we only included total fatty acids. Other lipid characteristics were highly correlated with the selected lipid measurements and including all of the lipid measurements would introduce multi-collinearity (RF2). As a next step we excluded all metabolite measures that did not have a single genetic variant that is genome-wide significant to meet the relevance criterion RF1. None of the remaining $\documentclass[12pt]{minimal}
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\begin{document}$$17,832$$\end{document}$ controls which is available from <http://csg.sph.umich.edu/abecasis/public/amd2015/>. To synchronise the genetic data on the metabolite risk factors and the AMD outcome, we match the effect alleles and we remove two genetic variants missing in the AMD data, so that the overall analysis includes $\documentclass[12pt]{minimal}
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\begin{document}$$n=148$$\end{document}$ variants. Finally, we use the Ensembl Variant Effect Predictor^[@CR44]^ to annotate the genetic variants to the gene that is most likely affected.
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\begin{document}$$p=0.01$$\end{document}$ to 0.3 reflecting 0.3-9.0 expected causal risk factors. This choice alters the posterior probabilities of various individual models, but the overall marginal inclusion probabilities of the risk factors are relatively stable. Finally, we vary the prior variance $\documentclass[12pt]{minimal}
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Reporting summary {#Sec150}
-----------------
Further information on research design is available in the [Nature Research Reporting Summary](#MOESM3){ref-type="media"} linked to this article.
Supplementary information
=========================
{#Sec23}
Supplementary Information Peer Review File Reporting Summary
**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.
Supplementary information
=========================
**Supplementary information** is available for this paper at 10.1038/s41467-019-13870-3.
This work was supported by the UK Medical Research Council (MC_UU_00002/7). S.B. and V.Z. are supported by Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 204623/Z/16/Z). This study would not have been possible without the access to publicly available summary data. We would like to thank the International AMD Genetics consortium (<http://amdgenetics.org/>), the authors of the blood trait GWAS as curated by the GWAS catalog (<https://www.ebi.ac.uk/gwas/>), and the authors of the NMR-GWAS (<http://www.computationalmedicine.fi/data>).
V.Z. and S.B. conceived and designed the study. V.Z carried out the statistical and computational analyses. J.M.C. and C.K. contributed to the design of the study and the interpretation of the findings. The paper was written by V.Z. and S.B.; and revised by all the co-authors. All co-authors have approved of the final version of the paper.
All data used in our study is in the public domain. Our study is based on publicly available summary-level data on genetic associations from the International AMD Genetics consortium <http://amdgenetics.org/>, the GWAS catalog <https://www.ebi.ac.uk/gwas/>, and MAGNETIC NMR-GWAS <http://www.computationalmedicine.fi/data>. Pre-processed summary-level data used as input for this study is available from <https://github.com/verena-zuber/demo_AMD>.
R-code for MR-BMA (R version $\documentclass[12pt]{minimal}
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\begin{document}$$> \, 3.4.2$$\end{document}$, MIT license) and all other multivariable MR approaches (IVW, lars, lasso and elastic net) is provided on <https://github.com/verena-zuber/demo_AMD>. Moreover, we provide markdown scripts and the summary-level data on AMD and NMR metabolites as presented in the applied example on <https://github.com/verena-zuber/demo_AMD>. This allows the reader to reproduce all results and figures of the applied example and adapt the presented methodology on risk factor selection in multivariable MR into their own research.
The authors declare no competing interests.
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Introduction {#Sec1}
============
The Tasmanian devil (*Sarcophilus harrisii*, referred to as the devil hereinafter), the world's largest living carnivorous marsupial, is currently at risk of extinction in the wild due to an unusual transmissible cancer known as Devil Facial Tumour Disease (DFTD)^[@CR1]^. DFTD is caused by a clonal tumour cell line that originated in a Schwann cell^[@CR2]^. Tumour cells are transmitted between individuals as allografts via biting^[@CR3]^, and diseased animals usually die within 6 months^[@CR4]^. Despite having a typical mammalian immune system which is capable of allograft rejection^[@CR5]--[@CR7]^, most devils produce no allogeneic response against the foreign tumour cells, with naturally occurring anti-DFTD responses having only been observed in six animals so far^[@CR8]^. Such lack of immune response has thus far been explained by the fact that DFTD cells downregulate their cell surface expression of the major histocompatibility complex (MHC) class I molecules through epigenetic regulation^[@CR9]^. However, MHC I downregulation is one of the most common mechanisms of tumour immune escape in human cancers^[@CR10],[@CR11]^, yet there has been no indication that such cancers can cross histo-incompatibility barriers. Therefore, it is likely that DFTD uses additional immune evasion mechanisms to be so remarkably contagious.
The hypothesis that we explored in this study arose from the observation that DFTD does not affect devils of different age classes equally. Devils have an average lifespan of 5--6 years. Females become reproductively mature at the age of 1 and males at the age of 2 years^[@CR12]^. At DFTD-affected sites, devils older than 3 years of age are rare (\<10%, with certain populations containing only animals \<3 years of age), and the level of disease prevalence is higher in older devils than that in younger animals^[@CR13]^. Interestingly, animals less than 1-year-old are rarely affected by DFTD^[@CR1]^, and diseased mothers do not transmit DFTD to their young^[@CR14]^. Such age-associated difference in disease prevalence led us to hypothesise that the decline of overall immune competence in an individual, either due to ageing or other factors, could play a role in causing susceptibility to DFTD.
To perform a comprehensive characterisation of immunosenescence in the devil and explore its role in DFTD, we analysed the T-cell repertoire diversity of devils. T cells are the main effector cells responsible for the immune surveillance of infections and tumours. T-cell repertoire diversity, which decreases with age as immunosenescence advances, is a key indicator of the capacity of an animal's immune system to respond to diseases^[@CR15],[@CR16]^. We focused on the T-cell receptor (TCR) beta (TCRB) chain, which is believed to harbour most of the diversity within the T-cell repertoire, particularly in its third complementarity-determining region (CDR3), which is the main region involved in antigen recognition^[@CR17]^. In humans, it has been estimated that the number of unique TCRB sequences can exceed 1 × 10^8^ in a young adult^[@CR18]^. This extraordinary diversity is generated during maturation of naive T cells in the thymus via a mechanism known as VDJ recombination, which involves random recruitment and imprecise joining of TCR variable (V), diversity (D) and joining (J) gene segments^[@CR19]^. Having a highly diverse repertoire of T cells is not only essential for successful host defence against a wide range of pathogens, but also has important implications for an animal's survival in the face of cancer. For instance, studies in humans have shown that high TCRB diversity and less clonotype loss during immunotherapy are associated with better clinical outcomes and improved overall survival in patients with metastatic melanoma^[@CR20],[@CR21]^. In light of this, we set out to investigate the role of TCRB diversity in DFTD. We observed a marked decline of TCRB diversity in devils within their first 2 years of life, resulting in early immunosenescence and increased susceptibility of older animals to DFTD. Strikingly, we also found that devils' T-cell repertoire undergoes further constriction after DFTD infection, rendering the animals more vulnerable to other secondary infections and cancers.
Results {#Sec2}
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TCRB transcript sequencing {#Sec3}
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We analysed peripheral blood of 50 devils, including 32 DFTD-free captive devils at 11 months (*N* = 10), 2 years (*N* = 10) or more than 5 years (*N* = 12) of age, and -2 or 3-year-old wild devils with (*N* = 7) or without (*N* = 8) DFTD; additionally, paired samples were collected from three devils before and after catching DFTD (Supplementary Table [1](#MOESM1){ref-type="media"}). TCRB partial transcripts consisting of a partial V segment, D, J and 5′ end of C were amplified and sequenced on an Illumina Miseq System (NCBI SRA\# SRP092288). The final dataset comprises a total of 12,517,846 quality-filtered sequences, with the number of sequences per sample ranging between 64,822 and 480,643 (Fig. [1a](#Fig1){ref-type="fig"}, Supplementary Table [1](#MOESM1){ref-type="media"}). From these sequences, 1,749,761 distinct TCRB transcripts representing distinct TCR clonotypes were identified by clustering with CD-HIT. Consistent with human and mouse genes, devil TRBV segments contain a conserved cysteine residue near the 3′ end and TRBJ segments contain a conserved phenylalanine in the 5′ portion, which are used to define the beginning and ending of CDR3 in TCRB transcripts^[@CR22]^. More than 84% of devil CDR3 sequences had a length between 30 and 42 bp (Fig. [1b](#Fig1){ref-type="fig"}), similar to the length distribution in humans^[@CR23]^. It should be noted that this study aims to compare TCR diversity between sample groups; due to the exceptionally high diversity of the T-cell repertoire, deeper sequencing will be necessary to fully reveal the diversity in devils.Fig. 1Characterisation of the TCRB sequence dataset. **a** The number of sequences per sample; captive and wild samples were analysed separately due to the differences in sequencing depth. Box-plot elements: centre line, median; box limits, first and third quartiles; whiskers, minimum and maximum ranges; points, outliers. **b** Length distribution of the CDR3 region
Early immunosenescence in the devil {#Sec4}
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Captive devils were used to study the pattern of immunosenescence in the species. The diversity of the TCRB repertoire was estimated using the Chao1^[@CR24]^, Shannon entropy^[@CR25]^ and Gini coefficient^[@CR26]^, which are commonly used in ecological studies, but can also be applied to immunological data^[@CR17]^. A more recently developed method, DivE, was also used, as it has been proposed to produce a more accurate estimation than other methods for datasets with relatively low sequencing depth^[@CR27]^. To account for the differences in sequencing depth among samples, all captive devil samples were rarefied (repeated random subsampling) to a common size of 100,000 sequences, while wild samples were rarefied to 250,000 sequences per sample. The different indices produced consistent results (Fig. [2](#Fig2){ref-type="fig"}, Supplementary Figure [1](#MOESM1){ref-type="media"}).Fig. 2Comparison of TCRB diversity among three age groups using captive devils. Mann--Whitney U-test statistics and *p*-values (in parentheses) are shown above each panel. Box-plot elements: centre line, median; box limits, first and third quartiles; whiskers, minimum and maximum ranges. **a** TCRB clonotype richness estimated using DivE. **b** Clonality of the TCR repertoire inferred by Gini coefficient. **c** Proportion of sequences that represent public clonotypes
Devils aged 11 months had the most diverse TCRB repertoire and showed the highest richness and evenness (Fig. [2a, b](#Fig2){ref-type="fig"}). Compared with the young devils, both 2-year-old and 5-year-old adult devils had significantly lower levels of TCRB diversity (Fig. [2a, b](#Fig2){ref-type="fig"}), showing a clear trend in the decline in richness and increase in clonality with age, although no significant difference was detected between 2-year-old and older devils in repertoire richness. The relative abundance of public clonotypes (identical clonotypes shared by multiple individuals, as opposed to private clonotypes unique to an individual) also increased significantly between young and adult devils (Fig. [2c](#Fig2){ref-type="fig"}), which likely resulted from decreased output of new T-cell clonotypes by the thymus and accumulation of clonal selection caused by common pathogens affecting devils^[@CR28]^.
These observations are indicative of an early onset of immunosenescence in the devil, causing a major decline in the T-cell repertoire diversity prior to the age of 2. We further investigated this by examining the thymus of young devils via computed tomography (CT) imaging. The thymus is the sole organ responsible for the production of naive T cells and novel clonotypes, and plays a key role in generating the high TCR diversity during development. The thymus is known to undergo structural changes and shrinks in total mass with age^[@CR29]^. This degeneration process, known as thymic involution, represents the most drastic anatomical change of the immune system in relation to ageing. One male and one female devil were scanned at two time points: 8 months and 10 months of age. CT images revealed that the thymus of devils is situated in the typical mammalian position, dorsal to the sternum, ventral to the great vessels and the trachea and cranial to the heart (Fig. [3](#Fig3){ref-type="fig"}). A cervical prolongation is also indicated in the CT images, extending some distance cranially along the trachea. Based on estimation of the cross-sectional area of the thickest part of the gland, enlargement of the thymus in devils is likely completed before the age of 8 months, with marginal changes in size (0.3% in the female and −0.7% in the male) occurring between the two time points studied (Table [1](#Tab1){ref-type="table"}). Signs of involution can be seen in the thymus at 10 months of age, with the outer layer of cortical tissue starting to degenerate and showing disruption (Fig. [3](#Fig3){ref-type="fig"} transverse sections).Fig. 3Computed tomography imaging of the thymus (T) in young devils. Cyan lines indicate locations of transverse sections where measurements of the thymus shown in Table [1](#Tab1){ref-type="table"} were taken. Scale bar under each image represents 1.00 cmTable 1Measurements of the thymus in young devilsSexAgeBody mass (kg)Thymus cross-sectional area (mm^2^)Devil 1Female8 months1.1786.610 months1.3086.9Devil 2Male8 months1.8188.710 months2.1088.1
DFTD-associated constriction of TCRB diversity {#Sec5}
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Analysis of the T-cell repertoire diversity in wild devils revealed a striking difference between DFTD-affected and unaffected individuals. As shown in Fig. [4](#Fig4){ref-type="fig"}, diseased devils at 2 or 3 years of age had a T-cell repertoire that was considerably less diverse than that of non-DFTD animals, with the average richness decreasing by 40.4 and 25.2% in diseased 2-year-old and 3-year-old devils, respectively, compared with non-DFTD devils. This loss of TCR diversity in DFTD-affected devils was characterised by a reduced number of distinct TCRB clonotypes and a decreased level of evenness, which are indicative of T-cell repertoire constriction and clonal expansion occurring in association with DFTD. Furthermore, comparisons of TCR diversity in three devils before and after catching DFTD showed a consistent pattern with the diversity dropping to a level markedly lower than that seen in non-DFTD devils of the same age (Fig. [4](#Fig4){ref-type="fig"}), suggesting that such constricted TCR repertoire was unlikely a consequence of ageing or a pre-existing condition of the devils before catching DFTD.Fig. 4Comparison of TCRB diversity between DFTD and non-DFTD devils. Mann--Whitney U-test statistics and *p*-values (in parentheses) are shown above each panel. Coloured data points represent three pairs of pre- and post-DFTD samples, with the same colour indicating the same animal. **a** TCRB clonotype richness estimated using DivE. **b** Clonality of TCR repertoire inferred by Gini coefficient
Further dissection of data revealed a pronounced change in the relative abundance of public TCRB clonotypes between non-DFTD and DFTD devils, with the T-cell repertoire of diseased animals becoming dominated by public clonotypes (Fig. [5a](#Fig5){ref-type="fig"}). Among 85,632 public clonotypes identified, 4889 showed significant increase in observed frequencies in the DFTD group (Mann--Whitney U test *p*-value \< 0.05) and were highly shared across diseased devils (Fig. [5b](#Fig5){ref-type="fig"}; observed frequencies and nucleotide sequences of the top-100 most abundant of these clonotypes are available in Supplementary Data [1](#MOESM3){ref-type="media"}--[2](#MOESM4){ref-type="media"}). Out of 451 detected V and J segment combinations (Supplementary Figure [2](#MOESM1){ref-type="media"}), 183 were found in these 4889 clonotypes, with most of the utilised V segments belonging to the same phylogenetic clade (Fig. [5c](#Fig5){ref-type="fig"}). Although no pattern was observed in CDR3 sequences across these clonotypes, they appeared to tend to have a relatively long CDR3 domain (Fig. [5d, e](#Fig5){ref-type="fig"}). These results indicate that the constricted TCR diversity in DFTD devils was largely caused by selective clonal expansion in the T-cell repertoire, resulting in a large fraction of shared clonotypes amongst individuals.Fig. 5Expansion of public clonotypes in DFTD-affected devils. **a** Prevalence of public clonotypes significantly increased in diseased devils. Mann--Whitney U-test statistics and *p*-values (in parentheses) are shown. Coloured data points represent three pairs of pre- and post-DFTD samples, with the same colour indicating the same animal. **b** Observed frequencies of top-100 most abundant public clonotypes that showed significantly higher frequencies (Mann--Whitney U test cutoff *p* \< 0.05) in DFTD samples than in non-DFTD samples. **c** TRBV and TRBJ segment usage among expanded public clonotypes. **d** Expanded TCRB sequences tend to have a long CDR3 domain compared with the average distribution of CDR3 length across all sequences (dashed line). **e** Sequence logo showing a high variability within the CDR3 domain in expanded clonotypes
As DFTD can be accompanied by secondary infections^[@CR30]^, further analysis was carried out to examine if the observed DFTD-associated clonal expansion within the TCR repertoire could have resulted from responses to secondary infections. Quantitative PCR was carried out to assess the peripheral blood level of nine T-cell-related markers, transcription factors and cytokines in DFTD-affected devils relative to that in non-DFTD devils (Fig. [6a](#Fig6){ref-type="fig"}). Cytokines interferon gamma (encoded by *IFNG*), interleukin 4 (*IL4*) and interleukin 6 (*IL6*), which are centrally involved in differentiation and robust effector responses of different subsets of T cells, such as Th1, Th2 and Th17^[@CR31]--[@CR35]^, all showed decreased expression levels in diseased devils in comparison with the baseline expression in non-DFTD devils. The expression of IFN-γ and IL-4 was closely associated with that of transcription factors T-bet (*TBX21*) and GATA-3 (*GATA3*), respectively, which were also suppressed in the DFTD group (Fig. [6b](#Fig6){ref-type="fig"}). These patterns are inconsistent with a cellular or humoral immune response to an infectious disease, suggesting that the observed decline in TCR diversity in DFTD-affected devils was unlikely the result of secondary infections. We also explored the possibility of regulatory T cells causing immune suppression by examining the expression of transforming growth factor beta 1 (*TGFB1*), interleukin 10 (*IL10*), forkhead box P3 (*FOXP3*) and cytotoxic T-lymphocyte protein 4 (*CTLA4*), all of which are known to positively correlate with the population size of immunosuppressive CD4+CD25+ Treg cells^[@CR36],[@CR37]^. The expression of these genes was also downregulated in the DFTD group (Fig. [6a](#Fig6){ref-type="fig"}), suggesting that the prevalence of Treg cells in the peripheral blood of DFTD-affected devils was not elevated but suppressed.Fig. 6Relative quantitative PCR analysis of gene expression in DFTD and non-DFTD devils. **a** Fold change in the expression level of nine T-cell marker, transcription factor or cytokine genes in DFTD devils in comparison with non-DFTD devils (log-2 scale). Box-plot elements: centre line, median; box limits, first and third quartiles; whiskers, minimum and maximum ranges. **b** Association between T-bet and IFN-γ gene expression, and between GATA-3 and IL-4 expression in wild devils. Gene expression was normalised to two reference genes, *GAPDH* and *GUSB*. Relative expression of *GUSB* normalised to *GAPDH* is shown (in panel **a**) as a control for non-differentiated expression between DFTD and non-DFTD samples
Discussion {#Sec6}
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In this study, we found a negative correlation between age and TCRB diversity in devils, and a marked decrease in TCRB diversity associated with DFTD infection. Our results suggest that devils experience a depletion in TCRB repertoire diversity between the age of 11 months and 2 years, possibly due to an early onset of thymic involution. One important feature of immunosenescence in the devil is that the rate of the T-cell diversity decline during early life is much higher than what is observed in the human. In humans, the change in TCRB diversity between childhood and young adulthood (between 6 and 25 years) is not prominent^[@CR38]^. Also, interestingly, in humans, the TCRB diversity declines substantially between the ages of 30s and 60s and then remains relatively stable afterwards^[@CR38]^, whereas in devils, the decline in clonotype richness between 2-year-olds and 5-year-olds was not statistically significant. These findings may indicate that immunosenescence advances at a faster rate in early life in the devil, with the thymus becoming mostly regressed before adulthood and therefore ceasing to produce new naive T cells; older devils possibly mainly rely on peripheral T-cell division to maintain the T-cell pool, as further indicated by the observed increase in repertoire clonality with age.
Limited literature is available on the thymic development and involution in the devil and other closely related species. Marsupials have a distinctive morphology and life history compared with eutherian mammals. Due to the short gestation periods (15--35 days), marsupial neonates are highly underdeveloped and lack lymphoid tissue and lymphocytes at birth^[@CR39]^. However, this is compensated by rapid development of immune tissue immediately after birth, with the thymus and lymphocytes appearing within the first week^[@CR39]^. In koalas (*Phascolarctos cinereus*), thymic development continues until 8 months of age, and involution does not start until 2--3 years after sexual maturity^[@CR40]^. In the devil, however, we observed in the two examined individuals that the thymus had stopped enlargement at 8 months and showed signs of involution at 10 months. This is consistent with a previous histological and immunohistochemical study finding that the thymus had started to regress in 1-year-old devils^[@CR5]^. These observations indicate that the onset of thymic involution in the devil likely occurs in the first few months of life, long before puberty starts, in contradiction to koalas. This contradiction may be due to the very different life histories of the two species; it may also be related to the fact that koalas, like other diprotodont marsupials^[@CR39]^, such as kangaroos and wallabies, have a different thymic system comprising one thoracic and one cervical thymus, which may undergo different development and atrophy phases than the common single-thymus system found in devils. Further work on devil pouch young will be required to determine the exact timeline of thymic development and involution in the devil.
Before this study, it was assumed that DFTD infection did not have a direct impact on the normal functionality of the host immune system. This assumption was based on in vitro experiments, which demonstrated that lymphocytes isolated from diseased devils were able to produce comparable levels of proliferation responses to mitogen stimulation as those from healthy individuals^[@CR41]^. Our results overturned this assumption, showing that DFTD caused substantial constriction in the TCRB repertoire of affected animals. It has been seen in mice that, despite having functional CD8 T cells, aged mice lose the capability to mount CD8 T-cell responses to certain influenza virus epitopes due to restricted TCRB diversity^[@CR16]^. The same process may be occurring in diseased devils, with the depleted diversity in the TCRB repertoire of DFTD-affected devils directly impairing immunity to infections. Moreover, based on qPCR analysis, a range of cytokines that play crucial roles in T-cell differentiation, homoeostasis and activation were produced at lower levels in the peripheral blood of DFTD-affected devils. These results suggest that DFTD not only hides from the host immune surveillance by downregulating MHC molecules on tumour cells^[@CR9]^, but can also exert a negative impact on the host immune system, which in turn can facilitate its transmission.
The decreased TCR diversity in diseased devils was largely due to the relative expansion of certain clonotypes, which tended to have a long CDR3 domain and utilise certain sets of V segments. These selectively expanded clonotypes that were shared and dominant among DFTD-affected individuals were inferably ineffective against the tumour and had lower cytokine production. The ultimate mechanism underlying this clonal selection remains to be investigated; one possible hypothesis is that in young devils, thymic production of new naive T cells may enable a continual generation of novel clonotypes that have a higher affinity towards DFTD tumour antigens, which may be a prerequisite for maintaining a tuned T-cell population to defend against the tumour.
A range of human diseases have been reported to cause constriction of TCR diversity, such as HIV^[@CR42]^, bladder cancer^[@CR43]^ and glioma^[@CR44]^, via mechanisms such as T-cell apoptosis and induced thymic involution. Therapeutic treatments, such as chemotherapy or radiation, are also known to lead to a depleted diversity of the T-cell repertoire^[@CR45],[@CR46]^. By contrast, certain cancer immunotherapy, such as CTLA4 blockade, can induce T-cell activation and proliferation, and increase the number of TCR clonotypes and diversity^[@CR20]^. This highlights the importance of further studies to explore the mechanism of DFTD-induced TCRB repertoire disruption for the development of an efficient treatment. It will also be important to characterise how the disease impacts on γδ T cells and other lymphocyte populations of the host, which will provide comprehensive insights into this unusual transmissible cancer.
To sum up, the constriction of TCRB repertoire in the devil's first 2 years of life likely has contributed to the observed higher DFTD prevalence in older devils. DFTD infection can lead to further decline of the TCRB diversity in diseased animals and result in a repertoire dominated by certain clonotypes with high reoccurrence among individuals. These results have important implications for the ongoing development of anti-DFTD vaccines^[@CR47]^ and treatments^[@CR48]^. Our data suggest that animal's age needs to be a key consideration during vaccination, as the constricted TCR repertoire of older devils is likely to cause poor responses to vaccination. Further research is needed to elucidate the mechanism underlying DFTD-induced T-cell repertoire disruption, which may lead to novel treatment strategies and improved success rates of therapies.
Methods {#Sec7}
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Annotation of devil TCRB genes {#Sec8}
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Three TRBC and 41 V segments were identified in the devil reference genome using multiple rounds of BLAST and HMMER searches (detailed in Supplementary Methods). A schematic map of the devil TCRB locus is shown in Supplementary Figure [3](#MOESM1){ref-type="media"}, with coordinates of all annotated TCRB gene segments provided in Supplementary Table [2](#MOESM1){ref-type="media"}.
Sample collection {#Sec9}
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Peripheral blood was collected (Supplementary Methods) from 32 captive devils, which belonged to three distinct age groups: 11 months old (*N* = 10), 2 years old (i.e. 24--36 months after birth; *N* = 10) and more than 5 years old (*N* = 12). Fifteen 2--3-year-old wild devils with (*N* = 7) or without (*N* = 8) DFTD were sampled, with three additional pairs of samples collected from three devils before and after catching DFTD (Supplementary Table [1](#MOESM1){ref-type="media"}). These procedures were carried out with approval from the Animal Ethics Committee of The University of Sydney under project number 550 (captive devils) and 681 (wild devils).
TCRB transcript amplification and sequencing {#Sec10}
--------------------------------------------
The devil blood RNA was extracted using RNeasy Protect Animal Blood Kit (Qiagen) with on-column DNase treatment. Quality and concentration of extracted RNA was analysed on a 2100 Bioanalyzer (Agilent Technologies). All samples had an RNA integrity number higher than 8.6. Blood cell counts were performed on a Sysmex XT-2000iV Haematology Analyzer. Similar to what was found in humans^[@CR49]^, the blood RNA level positively correlates with white blood cell count (R^2^ = 0.33) and lymphocyte count (R^2^ = 0.75) in devils (Supplementary Figure [4](#MOESM1){ref-type="media"}). In total, 500 ng of RNA was used in cDNA synthesis with SuperScript VILO Master Mix (Invitrogen). The strategy for TCRB transcript amplification was adapted from a protocol developed in humans^[@CR18]^. Instead of targeting full-length TCRB transcripts, this strategy amplifies shorter partial transcripts, which allows for an overlap between Illumina paired-end reads over the CDR3 region and thereby ensures high consensus sequence accuracy. This method is more suitable for comparative analysis of TCR diversity among samples^[@CR18]^, but can be less accurate for evaluating expression levels of various V segments within a sample due to the differences in PCR primer efficiencies. Briefly, TCRB transcripts were amplified using forward primers specific to one to three V segments and a universal reverse primer designed for all three C segments; primers were tagged with Illumina adaptor sequences on the 5′ end and designed and optimised to have high efficiencies (\>0.95; Supplementary Table [3](#MOESM1){ref-type="media"}). Successful amplification was confirmed by visualising PCR amplicons on gels. Four TRBV segments (V7, V10, V22 and V36) consistently showed no amplification among samples with multiple primers designed and tested, and therefore are likely not transcribed. PCR was carried out using Platinum Taq DNA polymerase high-fidelity kit (Invitrogen, protocol provided in Supplementary Methods). PCR products were pooled for each sample and amplicons with 250--350 bp of size were purified from the gel using QIAquick Gel Extraction Kit (Qiagen). Purified amplicons were submitted to The Ramaciotti Centre for Genomics (Randwick, Australia) for library preparation service and sequencing on an Illumina Miseq in two 2 × 200-bp paired-end runs.
Sequence data analysis {#Sec11}
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Paired-end reads were joined using fastq-join method^[@CR50]^. TCRB sequences containing a minimum Phred quality score higher than 30 (Supplementary Figure [5](#MOESM1){ref-type="media"}) and an intact open-reading frame were retained. The TRBV region of the sequences were matched to the 37 transcribed germline TRBV segments using BLAST. TRBJ sequences were isolated by trimming 5′ V and 3′ C regions, and were used to identify a total of 13 TRBJ segments in the devil genome. Sequences that failed to map to annotated V and J segments (3.1% of all sequences) were excluded from subsequent analyses. CDR3 was defined as the region between the conserved cysteine near the 3′ end of TRBV and the conserved phenylalanine in the 5′ portion of TRBJ segments^[@CR22]^. Sequences were clustered and distinct TCRB transcripts were identified using the CD-HIT method^[@CR51]^. Transcripts that were detected in more than one animal with 100% sequence identity were defined as public TCRB. To account for the differences in sequencing depth among samples, all captive samples were rarefied to a common size of 100,000 sequences and wild samples rarefied to 250,000 sequences with 10 rounds of subsampling performed. The level of diversity was estimated using four methods: Chao1^[@CR24]^, Shannon index^[@CR25]^, Gini coefficient^[@CR26]^ and DivE^[@CR27]^; with DivE, the curvature parameter (C~p~) values ranged between 0.20 and 0.33. Mann--Whitney U tests were performed to identify significant differences (*p* \< 0.05) between sample groups. Evolutionary relationships between TRBV and TRBJ segments were inferred using the Neighbour-Joining method.
Computed tomography {#Sec12}
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Two young devils, one male and one female, were used to investigate the development/involution of the thymus. Both animals were hand-raised, belonging to the Save the Tasmanian Devil Program. CT scans were taken at two time points, 8 months and 10 months of age (Supplementary Methods). As devils are expected to enter puberty in their second year, further scans were not performed to reduce the impact on the animals' development. CT images were analysed and measurements of the thymus were taken using software OsiriX. This work was approved by the Animal Ethics Committee of The University of Sydney under project number 550.
Quantitative PCR {#Sec13}
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Relative expression quantification of nine target genes, including *TBX21, IFNG*, *GATA3, IL4*, *IL6*, *TGFB1*, *IL10*, *FOXP3* and *CTLA4*, was carried out using two previously established reference genes *GAPDH*^[@CR2]^ and *GUSB*^[@CR52]^ on DFTD and control (non-DFTD) samples. Primers and PCR protocols for *IL10*, *IL6*, *TGFB1*, *GAPDH*, *GUSB, TBX21, IFNG*, *GATA3* and *IL4* were adopted from previous publications^[@CR2],[@CR12],[@CR52]^. Primers for *FOXP3* and *CTLA4* were designed using software Oligo v6.71, with forward and reverse primers located on different exons (all primer sequences in Supplementary Table [4](#MOESM1){ref-type="media"}). Primer specificity was checked by purifying and sequencing the PCR amplicon. Real-time PCRs were carried out on a RotorGene 6000 using Quantifast Sybr Green PCR Master Mix (Qiagen; Supplementary Methods). PCR efficiencies ranged between 0.95 and 1.04, and the correlation coefficient of standard curves between 0.988 and 1.000 (Supplementary Table [4](#MOESM1){ref-type="media"}). Normalised relative expression of target genes was calculated using the geNorm equations^[@CR53]^. Comparisons between DFTD and control groups were performed in software REST 2009 v2.0.13, with *p* \< 0.05 suggesting down- or upregulated expression in the disease group^[@CR54]^. The relative expression of the reference gene *GUSB*, normalised to *GAPDH*, was included in Fig. [6](#Fig6){ref-type="fig"} as a control for non-differentiated expression.
Reporting summary {#Sec14}
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Further information on experimental design is available in the [Nature Research Reporting Summary](#MOESM5){ref-type="media"} linked to this article.
Supplementary information
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{#Sec15}
Supplementary Information Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Reporting Summary
**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 10.1038/s42003-019-0342-5.
This work was funded by Australian Research Council Discovery Project DP140103260. We thank Devil Ark, the Australian Reptile Park, the WILD LIFE Sydney Zoo and the Save the Tasmanian Devil Program for providing access to animals and assisting with sample collection. We thank veterinarians at The Sydney University Veterinary Teaching Hospital who helped with performing anaesthesia and collecting samples. Particular thanks to our volunteers, including Vanessa Barrs, Julia Beatty, Christie Budd, Rebecca Gooley, Christine Griebsch, Elissa Kadar, Alan Marcus, Louise McGregor, Alicia McLuckie, Jessica Talbot, Lydia Tong, Jelena Vukcevic, Bianca Waud, Mark Westman, Joanna Whitney and Mariko Yata.
Y.C., A.T.P. and K.B. designed the study; Y.C. carried out lab work, analysed data and wrote the paper; M.M. performed CT scans, provided guidance on CT image analysis and wrote the CT methodology description; E.P. and S.F. coordinated and led sample collection; K.B. provided feedback on drafts and revised the paper; all authors read and commented on the paper.
Sequence data that support the findings of this study have been deposited in NCBI Sequence Read Archive (SRA) with the accession code SRP092288.
Competing interests {#FPar1}
===================
The authors declare no competing interests.
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Background
==========
Coronary artery disease (CAD) is one of the most common causes of morbidity and mortality and has a high socioeconomic burden. The detection and proper management of CAD in the earlier stages of the disease could reduce the burden of the disease, its related complications, treatment costs as well as improve quality of life of the affected patients \[[@b1-poljradiol-82-165],[@b2-poljradiol-82-165]\].
Although coronary angiography is considered to be the gold standard method for the evaluation of coronary artery disease and its management, it is invasive. Recently, noninvasive techniques, such as coronary CT angiography (CCTA), have been developed and are considered as important diagnostic tools for identifying patients with CAD, especially symptomatic patients \[[@b3-poljradiol-82-165]\]. CCTA is not a gold standard method for the diagnosis of CAD, however, it is a useful tool for determining the best management for patients with CAD \[[@b4-poljradiol-82-165]\]. In addition to its advantages, CCTA has some limitations, including exposure to high doses of radiation and low sensitivity and specificity due to blooming artifacts caused mainly by the calcification of vessels \[[@b5-poljradiol-82-165]\].
Another non-invasive and commonly used technique is the coronary artery calcium score (CACS). CACS is considered to be a well-established and validated imaging tool. The available evidence indicates that it could have clinical applications in both symptomatic and asymptomatic patients \[[@b6-poljradiol-82-165],[@b7-poljradiol-82-165]\].
CACS in asymptomatic patients could be used as a prognostic tool for CAD diagnosis, independent of traditional risk factors \[[@b8-poljradiol-82-165]\]. In symptomatic patients, the association between CACS and CAD has a high sensitivity but low specificity, and there are reports claiming that the absence of coronary artery calcification cannot definitely rule out stenosis \[[@b9-poljradiol-82-165]\].
The association between CACS and CAD, its related future cardiac events and mortality has been investigated previously in several studies \[[@b10-poljradiol-82-165]--[@b12-poljradiol-82-165]\].There are also some studies evaluating the prognostic value of CACS for determining the presence and severity of CAD \[[@b13-poljradiol-82-165],[@b14-poljradiol-82-165]\]. There are controversies regarding the usefulness of CACS for predicting coronary artery stenosis. Some studies reported that the usefulness of CACS is limited in some age groups or high-risk populations, and recommended to use CCTA instead \[[@b15-poljradiol-82-165]\].
It is also suggested that, given the fact that the occurrence of CAD, its related risk factors and different presentations in various ethnic populations \[[@b16-poljradiol-82-165]\], the predicting value of the score would not be similar in different populations.
Given the increasing trend for CAD morbidity \[[@b1-poljradiol-82-165]\] and the necessity of early detection in order to prevent its complication as well as to reduce the burden of the disease, and the presence of controversies regarding the usefulness of CACS for predicting coronary artery stenosis, the aim of this study was to determine the prognostic value of CACS for evaluating the presence and severity of CAD in patients with sign and symptoms of the disease. Our results could provide useful information on early detection of high-risk patients using this non-invasive method.
Material and Methods
====================
In this cross-sectional study, all consecutive patients with suspected CAD referred to the radiology department of Alzahra hospital, affiliated to Isfahan University of Medical Sciences, for coronary computed tomography angiography (CCTA) were enrolled. This study was conducted from September 2015 to March 2016.
The protocol of this study was reviewed by the radiology department review board and the ethics committee of Isfahan University of Medical Sciences and approved with a research project number of 293416.
During this study, medical files and radiological findings of the patients were reviewed. Patients with a history of percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), coronary stenting or previous myocardial infarction/acute coronary syndrome were excluded.
Multidetector CT technique
--------------------------
A 64-slice MDCT scanner (LightSpeed VCT 64, GE Healthcare, USA) was used.
First, for the purpose of calcium scoring, an area from the tracheal carina down to the diaphragm was scanned without contrast with a slice thickness of 3 mm, tube voltage of 100 or 120 kV(considering patients mass index) and tube current of 300 mA. Secondly, a contrast-enhanced, retrospective ECG-gated technique was used for a coronary study. The scan delay was calculated by the test bolus technique using 15 ml of non-iodinated contrast agent and then a bolus of 85 ml of the contrast, followed by 40ml of saline injected intravenously at a flow rate of 5 ml/second via the antecubital vein. The coronary CT angiography examination parameters were as follows: collimation, 64×0.625 mm; tube voltage, 120 kV; tube current, 400--600 mA; tube rotation time, 350 ms; pitch of 0.2. The images were initially reconstructed at 75% of the R-R interval but other phases were used if needed.
Multidetector CT image analysis
-------------------------------
The Agatston method (by smart score software) was used for the quantification of the calcium score. The left main, left anterior descending, left circumflex and right coronary arteries (RCA) were examined for the presence of calcified plaques in the non-contrast axial slices and a total score was recorded. Source images and semi-automatically traced images of coronary arteries were evaluated by a radiologist using dedicated software (Advantag Windows 4.3, GE Healthcare). The presence of plaque and severity of stenosis were determined at the maximal stenotic site and compared to the proximal and distal sites. Different severities of coronary artery stenosis based on the CCTA report were classified into five groups as follows: 1) no plaque, 2) non-significant plaque, 3) significant stenosis in a single vessel, 4) significant stenosis in two vessels, 5)significant stenosis in three vessels. The mean CACS was determined in each group of patients with different severities of coronary artery stenosis and compared between groups. The association between CACS and different CAD risk factors was determined as well. Different cutoff points of CACS with the highest sensitivity and specificity for discriminating between different levels of coronary artery stenosis were determined.
Statistical analysis
--------------------
All recorded data were analyzed using the SPSS software (V.20, SPSS Inc., Chicago, USA). The mean (SD) CACS in different groups of coronary artery stenosis was compared using the t-Student test. The association between CACS and different severities of coronary artery stenosis was determined using the Spearman test. Specificity and sensitivity of different cutoff points for CACS for discriminating between different levels of coronary artery stenosis was determined using receiver operating characteristic (ROC) curves.
Results
=======
In this study, medical files of 748 patients \[390 (52.1%) male and 358(47.9%) female\] with suspected CAD and referred for CCTA were reviewed. The mean (SD) age of the studied population was 53.96 (12.1) years. The frequency of patients with no plaque, non-significant plaque, single-vessel disease, two-vessel disease and three-vessel disease was 54.4%, 19%, 14%, 7.4% and 5.2%, respectively.
The mean CACSs in patients with different severities of coronary artery stenosis are presented in [Figure 1](#f1-poljradiol-82-165){ref-type="fig"}. The mean CACS increased significantly with increasing severity of the coronary vessel stenosis (P\<0.0001).
The Spearman correlation coefficients indicated that there was a significant positive association between the severity of CAD and CACS (P\<0.001, r=0.781).The results of ROC curve analysis for discriminating CAD (presence of stenosis) from the non-stenosis condition**,** non-significant stenosis from those with different grades of significant stenosis, single-vessel stenosis from two-vessel stenosis and two-vessel stenosis from three-vessel stenosis are presented in [Figure 2](#f2-poljradiol-82-165){ref-type="fig"}.
ROC curve analysis indicated that the optimal cutoff point for discriminating CAD (presence of stenosis) from the non-stenosis condition was 5.35 with 88.6% sensitivity and 86.2% specificity. Predictive positive and negative values for this cutoff point were 84.4% and 90%, respectively. Area under the curve for discriminating CAD (presence of stenosis) from the non-stenosis condition was 92.1% ([Figure 2A](#f2-poljradiol-82-165){ref-type="fig"}).
The optimal cutoff point for discriminating non-significant stenoses from those with different grades of significant stenosis was 106.5, with 70.4% sensitivity and 68.3% specificity. Predictive positive and negative values for this cutoff point were 75.7% and 62.2%, respectively. Area under the curve for discriminating non-significant stenoses from those with different grades of significant stenosis was 73.1% ([Figure 2B](#f2-poljradiol-82-165){ref-type="fig"}).
The optimal cutoff point for discriminating single-vessel stenosis from two-vessel stenosis was 255.5, with 70% sensitivity and 63% specificity([Figure 2C](#f2-poljradiol-82-165){ref-type="fig"}). The optimal cutoff point for discriminating two-vessel stenosis from three-vessel stenosis was 410.5, with 59% sensitivity and 68.3% specificity. Predictive positive and negative values for this cutoff point were 75.7% and 54.5%, respectively([Figure 2D](#f2-poljradiol-82-165){ref-type="fig"}).
Area under the curve for different levels of coronary artery stenosis did not have sufficient sensitivity and specificity for discriminating between different levels of CAD severity (\<70%).
Discussion
==========
In this study, we compared CACS in different grades of coronary artery stenosis determined by CCTA and evaluated different cutoff levels for CACS for discriminating between different levels of coronary artery stenosis. Our results indicated that CACS is associated with the severity of coronary artery stenosis. ROC curve analysis showed that the cutoff of 5.33 for CACS had appropriate sensitivity and specificity for discriminating "no plaque" from the non-stenotic condition. However, other cutoffs determined for the differentiation between different grades of stenosis had insufficient sensitivity and specificity for predicting coronary artery stenotic severity. The cutoff determined for discriminating significant from non-significant stenoses, though acceptable (\>70%), had insufficient sensitivity and specificity.
As mentioned above, the results of different studies regarding the usefulness of CACS for predicting coronary artery stenosis are not conclusive \[[@b10-poljradiol-82-165]--[@b14-poljradiol-82-165]\]. Some authors suggested that it could be a good predicting factor for mild to moderate stenosis \[[@b13-poljradiol-82-165]\]. Others have reported that it has insufficient predictive value \[[@b14-poljradiol-82-165]\]. Overall, there were great controversies in this regard. The results of two different studies from our region were not similar either \[[@b13-poljradiol-82-165],[@b14-poljradiol-82-165]\]. Thus, we aimed to evaluate the usefulness of this score for predicting CAD, its severity and CAD-related risk factors in our population.
The results of the Multi-Ethnic Study of Atherosclerosis (MESA) cohort indicated that CACS has a better predictive value for CAD than traditional risk factors \[[@b8-poljradiol-82-165]\].
Yamamoto et al. investigated the clinical applications of CACS in identifying high-risk Japanese patients. They indicated that higher CACS values are associated with an increased risk of CAD and its related mortality. They concluded that in spite of having appropriate clinical value for both symptomatic and non-symptomatic patients, higher CACS values have insufficient accuracy \[[@b7-poljradiol-82-165]\].
In a study in Taiwan, Liu et al. evaluated the prognostic value of CACS for CAD and cardiac events based on 5 years of follow-up. They reported that CACS had a significant association with the presence of CAD and its related cardiac events in a vessel-based study. They showed that with increasing CACS, the involvement of coronary vessels is greater. They concluded that this score could be used as an additional filter before CCTA among symptomatic patients, but we should consider also the fact that the presence of significant CAD could not be excluded by CACS equal to zero \[[@b17-poljradiol-82-165]\].
Similarly, many studies indicated that in spite of the fact that a higher score of CACS is associated with a higher risk of CAD, there is no conclusive agreement with respect to low or zero CACS s and the occurrence or severity of CAD. Results of current documents in this field indicated that occurrence of CAD in each population is based on the lower cutoff level of CACS \[[@b18-poljradiol-82-165]\]. The results of different studies are not similar. Some authors reported a cutoff level of 100 for CACS, whereas others reported a cutoff level of 10. The recommendations in different guidelines are not similar as well \[[@b16-poljradiol-82-165],[@b19-poljradiol-82-165]--[@b20-poljradiol-82-165]\].
The results of two recent regional studies were not similar either \[[@b13-poljradiol-82-165],[@b14-poljradiol-82-165]\]. Almasi et al. evaluated the value of CACS for predicting the presence and severity of CAD among 202 patients. Their findings confirmed the association between CACS and CAD occurrence as well as its severity. They reported a cutoff value of 350 for CACS for predicating coronary artery involvement. They concluded that this score could be used as an additional filter prior to CCTA among suspected patients, especially those with mild to moderate CAD risk factors \[[@b13-poljradiol-82-165]\].
Motevalli et al., in a study performed in Iran with a larger sample size, indicated that the presence and different grades of stenosis in CCTA are associated with CACS, but it had insufficient sensitivity for determining coronary artery stenosis. They concluded that CCTA is superior to CACS for the detection of CAD. The strength of their study was the larger sample size and also the evaluation of vessel-specific CACS as prognostic factors for the occurrence of CAD. According to their vessel-specific CACS findings, the left anterior descending artery(LAD) calcium score had appropriate specificity for ruling out stenosis and the left main (LM) calcium score had appropriate sensitivity for diagnosing stenosis \[[@b14-poljradiol-82-165]\].
In our study, the sample size was not as large as that of Motovalli and colleagues, but was higher than that of Almasi et al. Our results regarding the association between CACS and the presence and severity of CAD were similar to the both above-mentioned studies. In this study, we determined a cutoff value of 5.3 for distinguishing stenosis of coronary artery from the non-stenosis condition and a cutoff value of 106.5 for the differentiation between non-significant stenosis and different grades of significant stenosis. The values are lower than those reported by Almasi et al. We did not find any appropriate cutoff value for the discrimination between different grades of stenosis.
Considering our findings, it is suggested that CACS could be used as a screening score for diagnosing coronary artery stenosis, but further evaluation of CAD severity should be performed by CCTA.
Gitsioudis et al., in Germany, studied the usefulness of CACS as a filter scan before CCTA for the detection of CAD based on age, gender and CAD risk factors. Their results showed that CACS should be limited to younger patients, especially women with an intermediate risk profile in order to prevent the unnecessary exposure to radiation and CCTA is recommended in this group of patients \[[@b15-poljradiol-82-165]\].
Thus, considering the finding of the above-mentioned study, we could also recommend CACS as a screening tool that could be used in mild to moderate risk patients, and in high-risk patients CCTA is a preferable diagnostic method.
A limitation of the current study was its cross-sectional and retrospective design. Moreover, we did not evaluate vessel-specific CACS.
Conclusions
===========
We demonstrated that there is a significant association between CACS and the presence as well as the severity of CAD. CACS could have an appropriate prognostic value for the determination of coronary artery stenosis but not for discriminating between different severities of stenosis.
{#f1-poljradiol-82-165}
{#f2-poljradiol-82-165}
[^1]: Study Design
[^2]: Data Collection
[^3]: Statistical Analysis
[^4]: Data Interpretation
[^5]: Manuscript Preparation
[^6]: Literature Search
[^7]: Funds Collection
| {
"pile_set_name": "PubMed Central"
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Introduction
============
*Craniophora* Snellen, 1867 is an Old World genus of the Acronictinae restricted to the Palaearctic, Oriental, Australian and Ethiopian regions ([@B7]). Most of the 20 described species occur in eastern Asia ([@B15]; [@B5]). Only a few taxonomic studies of the genus have been undertaken (e.g., [@B4]; [@B13]; [@B1]; [@B5], [@B11]), and most of the publications mentioning *Craniophora* are faunistic works or check lists (e.g., [@B2], [@B3]; [@B9]; [@B6]; [@B12]; [@B17]; [@B10]). A diagnosis of the genus is given by [@B7] and [@B5].
In the subfamily Acronictinae there are two main branches according to the external and genital features. The first branch consists of the genera *Acronicta* Ochsenheimer, *Gerbathodes* Warren, *Moma* Hübner, *Oxicesta* Hübner, *Simyra* Ochsenheimer, *Subleuconycta* Kozhanchikov with diverse external features, but with similar genitalia with a heavily-sclerotised clasping apparatus and a simple structure of the vesica. The second group consists of the genus *Craniophora* with very similar external features, weakly sclerotised valvae and more complex vesica than in the first group.
The majority of the taxa of *Craniophora* and *Acronicta* are separable without checking their genital structures. In smaller species groups, however, the separation of species using external features requires thorough study due to the similarity in forewing pattern and shared features of the typical noctuid maculation. Forewing traits shared by many *Craniophora* and *Acronicta* include the variably strong basal dash, and the black streak of the tornus situated in the submedial fold, often extending from the medial area or the postmedial line to the terminal line. Moreover, in the three main species-groups of *Craniophora*, such as the *pontica*-, *harmandi*- and the *fasciata*-groups (although these have not previously been proposed formally, the common external and genital features suggest it), identification of some species requires detailed study of the genitalia.
Additionally, the species of *Cranionycta* de Lattin, which is a distinct lineage, probably with intermediate position between *Acronicta* and *Craniophora* (distributed in the Russian Far East, China, Nepal) externally can also be very similar to *Craniophora*, although they are usually smaller with narrower wings than most of the *Craniophora* species. The main differences are found in the specific features of the genitalia of both sexes (see [@B14]; [@B8]; [@B12]); in the males of *Craniophora*, the most conspicuous features are the much broader, but shorter, less sclerotised valvae, slighter corona, more diverticulated or twisted and more or less larger vesica; in the females the corpus bursae is more developed, without signa.
Method
======
Specimens were collected in China using ultraviolet light traps. Genital slides were prepared following standard techniques (abdominal integument cut lengthwise after KOH maceration male and female genitalia were dissected and mounted in euparal or in Canada balsam on glass slides). Additional material for comparison was borrowed from the Hungarian Natural History Museum, Budapest(HNHM). Additionally, numerous genitalia dissections were examined, both material on loan and own material.
The genital slides were digitalized with an Olympus SZX12 zoom stereo microscope with an Olympus DP 70 digital microscope camera in the Hungarian Natural History Museum, Budapest. After the digitalization, the pictures were converted to greyscale mode and the unnecessary grey background was deleted by photo editing software (Gimp). The habitus pictures were taken with Nikon D200 with Micro-Nikkor 200mm F/4 lens and Nikon D90 with Nikkor 200mm F/4 lens, after deleted the background by software.
The authors of all of the newly described taxa are the authors of this paper; however, *Craniophora sichuanensis* sp. n. is described in co-authorship with Aidas Saldaitis.
Systematic part
===============
Genus *Craniophora* Snellen, 1867
---------------------------------
*Craniophora* Snellen, 1867, *De Vlinders van Nederland, Macrolepidoptera systematisch beschreven*: 262.
**Type species.** *Noctua ligustri* \[Denis et Schiffermüller\], 1775, *Ankündung eines systematischen Werkes von den Schmetterlingen der Wienergegend*: 70.
Craniophora fujianensis
-----------------------
Kiss & Gyulai sp. n.
http://zoobank.org/E032E47F-3F27-44A3-8AEA-78C7683AC46B
http://species-id.net/wiki/Craniophora_fujianensis
[Figs 1](#F1){ref-type="fig"} [, 7](#F2){ref-type="fig"} [, 8](#F2){ref-type="fig"}
### Type material.
**Holotype:** Male ([Fig. 1](#F1){ref-type="fig"}), China, Fujian, Dai Mao Shan, 20 km NW of Longyan, 25°32\'N, 116°51\'E, 1300 m, 21--30.Nov.2004, leg V. Siniaev and team; slide No.: 3207 Gyulai (coll. P. Gyulai, to be deposited in Hungarian Natural History Museum (HNHM), Budapest). **Paratypes:** None. We exclude specimens of *Craniophora fujianensis* from Hainan, China, as these represent a separate subspecies, described below.
{#F1}
### Diagnosis and description.
Wingspan 37 mm. *Craniophora fujianensis* resembles *Craniophora praeclara* (Graeser, 1890) ([Fig. 6](#F1){ref-type="fig"}) and especially *Craniophora harmandi* (Poujade, 1898) ([Fig. 5](#F1){ref-type="fig"}) externally. Reliable separation of the three taxa does not require genitalic study, since *Craniophora fujianensis* exhibits unique external characteristics. The shared features of the two related taxa are the more or less similar forewing pattern and noctuid maculation, the presence of the strong or weaker black streaks in the basal area, in the termen and the tornus (the latter streak in the submedial fold, regularly from the medial area or the postmedial line towards the terminal line; the oblique, wavy antemedial line, the double, crenulate postmedial line and the less wavy whitish-grey subterminal line. *Craniophora fujianensis* can be distinguished from *Craniophora praeclara* and *Craniophora harmandi* by its more uniform vestiture of thorax, light brownish-grey (and not chequered white) forewing fringe and the less evenly broad, somewhat shorter blackish streak extending through the submedial fold from the medial area outwards to the lowest part of the terminal line. In comparison with *Craniophora harmandi*, the new species has a more unicolorous, lighter brownish-grey forewing ground colour; lighter, more obsolescent, narrower dark suffusion in the medial area, conspicuous clear white colouration of the small quadrangular basal spot, which is not confluent with the whitish spot of the costal field; rather ashy grey (and not white), less conspicuous comma-like tiny spot beside the claviform stigma; the stigmata are smaller, the orbicular spot is not evenly white encircled. *Craniophora fujianensis* is distinguished from *Craniophora praeclara* by its smaller average size, more unicolorous, lighter brownish-grey forewing ground colour, without mossy green shades; lighter, more obsolescent, narrower dark suffusion in the medial area; clear white small quadrangular basal spot; less crenulate postmedial line, much smaller, blackish-filled stigmata and especially by the almost white hindwing of the male. **Male genitalia** ([Figs 7](#F2){ref-type="fig"}, [8](#F2){ref-type="fig"}): a close relationship with *Craniophora harmandi* ([Figs 13](#F3){ref-type="fig"}, [14](#F3){ref-type="fig"}) is evident; however, the differences are very conspicuous. *Craniophora fujianensis* can be easily distinguished from both of the allied taxa by its much larger, longer uncus, larger juxta and vinculum, large bundle of long hairs on the tegumen, strikingly elongate, curved valvae with straighter dorsal and almost evenly curved (with one angle medially) ventral costa and broader corona with much longer setae. The aedeagus is larger, the vesica ventrally curved; the two medial spines are straight, almost evenly thin and parallel, the third, weaker medial spine weakly sclerotised and hardly visible; whereas the two large spines are oppositely positioned in *Craniophora harmandi*, and the third, medial, cornutus is weaker than the others but stronger than in *Craniophora fujianensis*; *Craniophora praeclara* has no cornuti in the vesica ([Figs 15](#F3){ref-type="fig"}, [16](#F3){ref-type="fig"}). Additionally, *Craniophora fujianensis* has a tiny semiglobular, sclerotised medial diverticulum, finely serrate on its surface, from which a longitudinal, wavy-ribbed, sclerotised area is situated towards the terminal section of the vesica.
{#F2}
{#F3}
**Female.** Unknown.
### Etymology.
The species name refers to Fujian Province, China, where the species was discovered.
### Distribution.
The species is known from Fujian and Hainan Provinces, China, with the nominate subspecies known only from the type locality in Fujian; subspecies *hainanensis* occurs in Hainan. *Craniophora fujianensis* is the allopatric sister taxa of *Craniophora harmandi*, which occurs from the western Himalaya to Taiwan, in the region with monsoonic influence.
Craniophora fujianensis hainanensis
-----------------------------------
Kiss & Gyulai ssp. n.
http://zoobank.org/3A6BE9D1-5170-4B00-925A-5A8E250C5909
http://species-id.net/wiki/Craniophora_fujianensis_hainanensis
[Figs 2](#F1){ref-type="fig"} [, 3](#F1){ref-type="fig"} [, 9](#F2){ref-type="fig"} [, 10](#F2){ref-type="fig"}
### Type material.
**Holotype:** Male ([Fig. 2](#F1){ref-type="fig"}), China, prov. Hainan, Wuzhi Shan, 1333 m, 03--10.Jan.2008, leg local collector; slide No.: 3502 Gyulai (coll. P. Gyulai, to be deposited in HNHM, Budapest). **Paratype:** Male ([Fig. 3](#F1){ref-type="fig"}), same data as holotype; slide No.: 3209 Gyulai (coll. G. Ronkay, Budapest, Hungary).
### Diagnosis and description.
*Craniophora fujianensis hainanensis* is endemic to the island of Hainan. It can be separated at first sight from similar *Craniophora* by its whitish-ochreous forewing ground colour, indistinct wing pattern, with a more double-angled inner edge of the medial fascia, the whitish fringe and the conspicuous clear white hindwing. Wingspan 35--38 mm. **Male genitalia** ([Figs 9](#F2){ref-type="fig"}, [10](#F2){ref-type="fig"}): In the male genitalia, ssp. *hainanensis* has a somewhat shorter uncus, with the ventral costa evenly rounded, and a distally more dilated valvae compared to the nominate subspecies. More specimens are needed to evaluate if these differences represent individual or subspecific variation. The two thin medial spines of the vesica are not parallel, but V-shaped, arising from the same sclerotised plate; the tiny semiglobular, medial diverticulum is hardly visible because the surface is not sclerotised or spinulose; the longitudinal, wavy-ribbed, sclerotised area towards the end of the vesica is bifurcate anteriorly then confluent.
**Female.** Unknown.
### Etymology.
The name refers to the island of Hainan where this taxon occurs.
### Distribution.
The subspecies is known only from the type-locality, China, Hainan Island.
Craniophora sichuanensis
------------------------
Kiss, Gyulai & Saldaitis sp. n.
http://zoobank.org/E24039A7-3A32-448D-BEE0-C56FED2881A4
http://species-id.net/wiki/Craniophora_sichuanensis
[Figs 4](#F1){ref-type="fig"} [, 11](#F2){ref-type="fig"} [, 12](#F2){ref-type="fig"}
### Type material.
**Holotype:** Male ([Fig. 4](#F1){ref-type="fig"}), China, W. Sichuan, road Yaan/Kangding, Erlang Shan Mt., 2200 m, 02.Aug.2011, 29°87.340\"N, 102°30.970\"E, leg. Floriani and Saldaitis; slide No.: 2883 Gyulai (coll. P. Gyulai, to be deposited in HNHM, Budapest). **Paratypes:** None.
### Diagnosis and description.
Wingspan 32 mm. Externally most similar to *Craniophora harmandi* and to a lesser degree to *Craniophora fujianensis*. The shared features with the two related taxa are the more or less similar forewing pattern and noctuid maculation and the less sinuous whitish-grey subterminal line. It can be distinguished from *Craniophora fujianensis* by its smaller size, with a wingspan of 32 mm compared to 35--38 mmin the two subspecies of *Craniophora fujianensis* and 33--40 mm in *Craniophora harmandi*; the slight black circle in the centre of the thoracic tuft; the white, curved, fine, comma-like basal mark (which is not quadrangular as in the two related taxa); the conspicuous, clear white inner stripe of the medial area along the broad black medial fascia; the more recognisable white outline of the orbicular and reniform stigmata; the longer basal black streak, the diluted blackish streak extending in the submedial fold from the middle of the medial line outward to the lowest part of the terminal line (tornal area) and the more uniform, light brownish-grey hindwing with a faint dark-brown discal spot, sinuous medial line and darker suffused terminal area. Additionally, in comparison with *Craniophora harmandi*, *Craniophora sichuanensis* has darker and narrower dark suffusion in the medial area, and lacks the large whitish area extending outward from the reniform stigma toward the apex and in the postmedial line. *Craniophora sichuanensis* is more distinct from *Craniophora fujianensis*, especially in the conspicuous clear white inner third of medial area. **Male genitalia** ([Figs 11](#F2){ref-type="fig"}, [12](#F2){ref-type="fig"}): Uncus almost evenly slender and apically hooked, valvae spatulate, lacking corona, vesica almost even in width with two equally long, weak, slender spines and one shorter, broader, stout cornutus and the broad, sclerotised distal area covered by numerous almost straight parallel ribs. These genitalia features, as well as the overall smaller male genitalia, shorter, more asymmetrical, medially broadened valvae, and V-shaped vinculum, separate the new species from the two close relatives.
**Female.** Unknown.
### Etymology.
The species name refers to the type locality in the Province of Sichuan, China.
### Distribution.
The new species is known only from the Erlang Shan at the eastern edge of the Tibetan plateau in China's Sichuan province. The single male was collected at ultraviolet light. The new species appeared with a very local distribution, as it was discovered in only one valley in mountainous region. The new species was collected in virgin mixed forest habitat dominated by various broad-leaved trees such as oaks (*Quercus dentata* Thunb., *Quercus glauca* Thunb.), poplars (*Populus cathayana* Rheder, *Populus simonii* Carrière), elm (*Ulmus parvifolia* Jacq.), rhododendrons (*Rhododendron brachycarpum* D. Don ex G. Don, *Rhododendron dauricum* L.), and bamboos (*Phyllostachys* ssp., *Borinda* ssp., *Fargesia* spp.). Adults are on the wing with many other late summer Noctuidae species, such as *Pareuplexia chalybeate* (Moore, 1867), *Blepharosis bryocharis* Boursin, 1964, *Blepharosis lamida* (Draudt, 1950) and *Amphipyra amentet* Babics, Benedek & Saldaitis, 2013.
Supplementary Material
======================
###### XML Treatment for Craniophora fujianensis
###### XML Treatment for Craniophora fujianensis hainanensis
###### XML Treatment for Craniophora sichuanensis
The authors would like to thank Aidas Saldaitis (Vilnius, Lithuania) and Alessandro Floriani (Milano, Italy) for providing the HT of *Craniophora sichuanensis* to the Hungarian Natural History Museum, Budapest; Zsolt Bálint (Budapest, Hungary) for allowing us the use of a microscope to digitalize the slides, and for loaning material from the HNHM, Budapest; Zoltán Varga (Debrecen, Hungary) and László Ronkay (Budapest, Hungary) for their suggestions and checking the manuscript; Edvárd Mizsei (Debrecen, Hungary) for providing technical help with the photographs of adults.
This paper was supported by the OTKA (K-84071) and TÁMOP 4.2.4. A/1-11-1-2012-0001 "National Excellence Program -- Elaborating and operating an inland student and researcher personal support system." The project was subsidized by the European Union and co-financed by the European Social Fund.
[^1]: Academic editor: D. Lafontaine
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###### Strengths and limitations of this study
- This large study is the first study to have reported on the extent to which autism and intellectual disabilities are independently associated with poor mental and general health, in children and adults.
- The study comprises a whole country population, with high participation rate (94%), and the conditions were systematically enquired about on everyone.
- A limitation is that conditions were self/proxy reports rather than in-depth diagnostic assessments.
Introduction {#s1}
============
Both intellectual disabilities and autism occur not uncommonly in children and adults, and can co-occur. Children and adults with intellectual disabilities have notably poorer mental and general health than other people.[@R1] This has also been reported for autistic children and adults,[@R5] although the quantity of research is limited, particularly with regard to adults. The extent of co-occurrence of intellectual disabilities in autistic people used to be considered to be as high as 50%--70%,[@R10] although more recent reports suggest that it may be lower, though still considerable, at about 20%.[@R9] This may in part relate to the broadening of criteria for the autism spectrum to include 'milder' autism and greater awareness about autism in children and young people in recent years, as it is well established that autism prevalence is higher in people with more severe intellectual disabilities and vice versa.[@R12] Autism is generally considered to be associated with poor mental health. However, the largest study to examine this in adults in a general community population found no difference in rates of mental ill health in adults with co-occurring autism and intellectual disabilities, compared with age-gender-Down syndrome level of ability-matched adults with intellectual disabilities but no autism.[@R13] As intellectual disabilities and autism have tended to be studied separately, the relative extent to which being autistic, or having intellectual disabilities, accounts for their poor population health is not clear. This is important to understand, given the frequent co-occurrence of these conditions, and is important to understand in both child and adult populations, given the more recent change in co-occurrence due to higher frequency of diagnosis of autism.
The aim of this paper is to study the extent to which autism and intellectual disabilities are independently associated with poor mental and general health, in children and adults.
Method {#s2}
======
Strengthening the Reporting of Observational Studies in Epidemiology guidelines {#s2-1}
-------------------------------------------------------------------------------
The Strengthening the Reporting of Observational Studies in Epidemiology checklist for cross-sectional studies was adhered to.
Census process and variables {#s2-2}
----------------------------
Scotland has performed a national Census every 10 years since 1841, the most recent being Scotland's Census, 2011. Information was collected on every resident in Scotland on the Census date, 27 March 2011. This included people in private households and also people in community residences (such as care homes, prisons and student halls of residence). In private households (typically family households), one person was responsible for completing the Census details for all the household's residents; for communal establishments, the manager was responsible for providing the information. It is a legal requirement in the UK to complete the Census. Failure to provide information or for providing false information attracted a fine of up to £1000. Non-responses were followed up by the Census team and help provided. These factors accounted for the high response rate; Scotland's Census 2011 achieved a 94% response rate.[@R14] The Census team adjusted for the 6% non-response rate using a Census Coverage Survey to estimate numbers and characteristics. The Census Coverage Survey included around 40 000 households; the records from it were matched with Census records, with all individuals deterministically matched to check for duplicates. Individuals estimated to be missing from the Census were then imputed, using a subset of characteristics from real individuals, including health information. This edit and imputation methodology was adapted from the Office for National Statistics rigorous and systematic guidelines, available at: <http://webarchive.nationalarchives.gov.uk/20160108193745/http://www.ons.gov.uk/ons/guide-method/method-quality/survey-methodology-bulletin/smb-69/index.html> and further details on the Census population estimates are available at: <http://www.scotlandscensus.gov.uk/documents/censusresults/release1b/rel1bmethodology.pdf>
Full details of the methodology and other background information on Scotland's Census, 2011 are available at: <http://www.scotlandscensus.gov.uk/supporting-information>.
The Census included questions on demography, long-term conditions and on general health.
The question on long-term conditions enquired:
'Do you have any of the following conditions, which have lasted, or are expected to last, at least 12 months? Tick all that apply:
- Deafness or partial hearing loss.
- Blindness or partial sight loss.
- Learning disability (eg, Down's syndrome).
- Learning difficulty (eg, dyslexia)
- Developmental disorder (eg, autistic spectrum disorder or Asperger's syndrome).
- Physical disability.
- Mental health condition.
- Long-term illness, disease or condition.
- Other condition, please write in
- free-text space was then provided for conditions to be listed.
- No condition'.
The question on general health enquired:
'How is your health in general?
- Very good.
- Good.
- Fair.
- Bad.
- Very bad'.
The terminology used in both these questions was specifically investigated prior to implementation of data collection. The General Register Office for Scotland commissioned Ipsos MORI Scotland to undertake cognitive question testing, to determine whether the questions were answered accurately and willingly by respondents, and what changes if any might be required to improve data quality and/or the acceptability of the response options. Cognitive interviewing is a widely used approach to critically evaluate survey questionnaires.[@R15] It tests the way respondents understand, mentally process and respond to survey materials. It enables researchers to modify survey material to enhance clarity. Retrospective probing was deemed to be the most appropriate of the different techniques available. It involved the interviewer presenting the question, the respondent answering it, and the interviewer then probing for specific information relevant to the question or to the specific answer given (eg, What does this question mean in your own words?). This research was undertaken with 102 participants with a mix of gender and age, both with and without the health conditions and disabilities (including people with more than one of the conditions). This included people with autism, intellectual disabilities, dyslexia, dyspraxia, speech impairment, mental health conditions (both milder and more serious) and other long-term conditions. The results found that the question on general health status functioned well and did not need amendment, as did the questions on long-term conditions, including intellectual disabilities and mental health condition, while the question on autism was redesigned to that listed above in order to more accurately capture the data specifically on autism. Additionally, the response 'no' was amended to 'no condition'. The other questions did not require any modification. Further information can be found at: <http://www.scotlandscensus.gov.uk/documents/research/2011-census-health-disability-questions.pdf> <http://www.scotlandscensus.gov.uk/documents/legislation/changes-to-gov-statement-report.pdf>
In Scotland, the term 'learning disability' is synonymous with the international term 'intellectual disabilities'.[@R16]
For 2.6% of the Census returns, information on long-term conditions was not completed. The Census team assumed the most plausible explanation was that the person had no long-term condition but did not see the 'No condition' check box at the end of the question. They, thus, recorded them to have none of the long-term conditions.
Data analysis {#s2-3}
-------------
First, frequency data were generated. Next, we used logistic regressions to calculate the ORs with 95% CIs of autism, intellectual disabilities, age and gender in predicting (1) having a mental health condition and (2) poor general health. We dichotomised the general health status variable to good health (very good or good health) or poor health (fair, bad, or very bad health). The gender variable was binary, the reference group was male. We conducted the analyses separately for children and young people (aged 0--24 years) and adults (aged 25+ years). This was because in Scotland's Census, 2011, the prevalence of autism is higher in the children and young people than in the adults, most likely due to widening out of the diagnostic criteria and greater awareness of autism in recent decades. Hence the adults with autism are more likely to be on the more severely affected range of the autism spectrum. For the children and young people, the reference group was aged 0--15 years (childhood), given the physiological changes and changing life experiences that occur in adolescence/transition compared with younger children, which may have a bearing on general and mental health. The adults were grouped into 10-year age bands, with the reference group being aged 25--34 years. We then conducted a second round of the regressions, including the interaction terms age x intellectual disabilities and age x autism. This was because the influence of age on mental health and general health is likely to differ in people with intellectual disabilities and possibly in people with autism to that seen in other people. All analyses were conducted with SPSS software V.22.
Patient and public involvement {#s2-4}
------------------------------
The question on intellectual disabilities and autism was included in Scotland's Census, 2011 at the behest of third sector organisations for people with intellectual disabilities and autism. This study was undertaken by the Scottish Learning Disabilities Observatory, which has a specific remit for people with intellectual disabilities and autism; its steering group includes partners from the third sector organisations. Results from this study will be disseminated for people with intellectual disabilities and autism in easy-read version via the Scottish Learning Disabilities Observatory website and newsletters.
Results {#s3}
=======
Scotland's Census, 2011, includes records on 5 295 403 people aged more than 0--75 years, of whom 1 548 819 (29.2%) were children and young people, and 3 746 584 (70.8%) were adults aged 25 years and over. Of the children and young people, 9396 (0.6%) reported having intellectual disabilities and 25 063 (1.6%) reported having autism. Of the adults aged 25 years and over, 16 953 (0.5%) reported having intellectual disabilities and 6649 (0.2%) reported having autism. Of the children and young people with intellectual disabilities, 3756/9396 (40.0%) additionally had autism, and of the adults aged 25 years and over with intellectual disabilities, 1953/16 953 (11.5%) additionally had autism. Of the children and young people with autism, 3756/25 063 (15.0%) additionally had intellectual disabilities, and of the adults aged 25 years and over with autism, 1953/6649 (29.4%) additionally had intellectual disabilities.
538/5640 (9.5%) of the children and young people with intellectual disabilities but no autism had a mental health condition, and 3383/15 000 (22.6%) of the adults with intellectual disabilities but no autism had a mental health condition. A total of 1601/21 307 (7.5%) of the children and young people with autism but no intellectual disabilities had a mental health condition, and 1314/4696 (28.0%) of adults with autism but no intellectual disabilities had a mental health condition. A total of 15 829/1 518 116 (1.0%) of the children and young people with neither condition had a mental health condition, and 208 493/3 724 935 (5.6%) of the adults with neither condition had a mental health condition.
[Table 1](#T1){ref-type="table"} presents the OR (95% CI) of intellectual disabilities, autism, age and gender in predicting a mental health condition in the children and young people. It presents the results of two regressions, the second one including the interaction terms. Both intellectual disabilities (OR 7.0, 95% CI 6.3 to 7.9)and autism (OR 25.1, 95% CI 23.0 to 27.3) independently increased the odds of having a mental health condition, more so for autism. Mental health conditions were also predicted by female gender (OR 1.5, 95% CI 1.4 to 1.5) and being a young person rather than a child (OR 10.5, 95% CI 10.1 to 11.0).
######
Predictors of mental health conditions in the whole population of children and young people
Variable Regression 1 Regression 2 (including interaction terms)
--------------------------------- ------------------------------------ -------------------------------------------- -------------- ----------------
Age 0--15 (ref) -- --
16--24 7.65 7.36 to 7.95 10.54 10.06 to 11.05
Gender Male (ref) -- --
Female 1.49 1.45 to 1.54 1.48 1.44 to 1.53
Autism No autism (ref) -- --
Autism 10.21 9.67 to 10.78 25.08 23.02 to 27.32
Intellectual disabilities No intellectual disabilities (ref) -- --
Intellectual disabilities 5.85 5.44 to 6.29 7.04 6.30 to 7.87
Age x intellectual disabilities 0--15 (ref) -- --
16--24 -- 0.66 0.57 to 0.76
Age x autism 0--15 (ref) -- --
16--24 -- 0.24 0.21 to 0.26
Constant 0.00 0.00
In adults ([table 2](#T2){ref-type="table"}), a similar pattern was seen with both intellectual disabilities (OR 3.5, 95% CI 3.2 to 3.8) and autism (OR 5.3, 95% CI 4.8 to 5.9) independently predicting a mental health condition, as did female gender (OR 1.3, 95% CI 1.2 to 1.3). All age groups had higher odds ratios than the 25--34 years of predicting having a mental health condition, except for the oldest age group, aged 65+years who had a lower rate.
######
Predictors of mental health conditions in the whole population of adults
Variable Regression 1 Regression 2 (including interaction terms)
--------------------------------- ------------------------------------ -------------------------------------------- -------------- --------------
Age 25--34 (ref) -- --
35--44 1.40 1.38 to 1.42 1.40 1.38 to 1.42
45--54 1.38 1.36 to 1.40 1.38 1.36 to 1.40
55--64 1.08 1.06 to 1.09 1.07 1.05 to 1.08
65+ 0.92 0.90 to 0.93 0.91 0.90 to 0.92
Gender Male (ref) -- --
Female 1.25 1.24 to 1.26 1.25 1.24 to 1.26
Autism No autism (ref) -- --
Autism (ref) 5.29 5.00 to 5.59 5.30 4.80 to 5.85
Intellectual disabilities No intellectual disabilities (ref) --
Intellectual disabilities 4.42 4.26 to 4.59 3.50 3.20 to 3.84
Age x intellectual disabilities 25--34 (ref) -- --
35--44 -- 0.99 0.87 to 1.11
45--54 -- 1.24 1.10 to 1.39
55--64 -- 1.71 1.51 to 1.94
65+ -- 1.82 1.59 to 2.08
Age x autism 25--34 (ref) -- --
35--44 -- 1.03 0.89 to 1.19
45--54 -- 0.94 0.80 to 1.10
55--64 -- 1.02 0.84 to 1.25
65+ -- 1.18 0.97 to 1.44
Constant 0.46 0.46
A total of 2453/5640 (43.5%) of children and young people with intellectual disabilities but no autism, and 7834/15 000 (52.2%) of adults with intellectual disabilities but no autism had poor general health. A total of 3898/21 307 (18.3%) of children and young people with autism but no intellectual disabilities, and 2 134/4 696 (45.4%) of adults with autism but no intellectual disabilities had poor general health. A total of 42 713/1 518 116 (2.8%) of the children and young people with neither condition, and 880 044/3 724 935 (23.6%) of the adults with neither condition had poor general health.
[Table 3](#T3){ref-type="table"} presents the OR (95% CI) of intellectual disabilities, autism, age and gender in predicting poor general health in the children and young people. It presents the results of two regressions, the second one including the interaction terms. Both intellectual disabilities (OR 18.3, 95% CI 17.2 to 19.6) and autism (OR 8.4, 95% CI 8.0 to 8.8) independently increased the odds of having poor general health, more so for intellectual disabilities. Poor general health was also predicted by female gender (OR 1.1, 955 CI 1.1 to 1.1) and being a young person rather than a child (OR 2.3, 95% CI 2.2 to 2.3.
######
Predictors of poor general health in the whole population of children and young people
Variable Regression 1 Regression 2 (including interaction terms)
--------------------------------- ------------------------------------ -------------------------------------------- -------------- ----------------
Age 0--15 (ref) -- --
16--24 2.14 2.10 to 2.18 2.28 2.24 to 2.33
Gender Male (ref) -- --
Female 1.11 1.09 to 1.14 1.11 1.09 to 1.13
Autism No autism (ref) -- --
Autism 6.70 6.46 to 6.95 8.40 8.02 to 8.80
Intellectual disabilities No intellectual disabilities (ref) -- --
Intellectual disabilities 14.05 13.39 to 14.73 18.34 17.17 to 19.58
Age x intellectual disabilities 0--15 (ref) -- --
16--24 -- 0.57 0.52 to 0.63
Age x autism 0--15 (ref) -- --
16--24 -- 0.54 0.50 to 0.58
Constant 0.02 0.02
In adults ([table 4](#T4){ref-type="table"}), a similar pattern was seen with both intellectual disabilities (OR 7.5, 95% CI 7.0 to 8.1 and autism OR 4.5, 95% CI 4.1 to 4.9) independently predicting poor general health, as did female gender (OR 1.1, 95% CI 1.1 to 1.1). A gradient is seen, with older age groups progressively predicting having poor general health.
######
Predictors of poor general health in the whole population of adults
Variable Regression 1 Regression 2 (including interaction terms)
--------------------------------- ------------------------------------ -------------------------------------------- -------------- ----------------
Age 25--34 (ref) -- --
35--44 1.78 1.76 to 1.80 1.79 1.77 to 1.81
45--54 2.86 2.83 to 2.89 2.90 2.87 to 2.93
55--64 4.81 4.76 to 4.86 4.88 4.82 to 4.93
65+ 10.25 10.15 to 10.36 10.39 10.29 to 10.50
Gender Male (ref) -- --
Female 1.05 1.05 to 1.06 1.05 1.05 to 1.06
Autism No autism (ref) -- --
Autism (ref) 3.39 3.21 to 3.58 4.46 4.06 to 4.89
Intellectual disabilities No intellectual disabilities (ref) --
Intellectual disabilities 4.39 4.25 to 4.53 7.54 7.02 to 8.10
Age x intellectual disabilities 25--34 (ref) -- --
35--44 -- 0.72 0.65 to 0.79
45--54 -- 0.60 0.54 to 0.65
55--64 -- 0.45 0.40 to 0.50
65+ -- 0.24 0.21 to 0.26
Age x autism 25--34 (ref) -- --
35--44 -- 0.83 0.72 to 0.96
45--54 -- 0.59 0.50 to 0.68
55--64 -- 0.49 0.41 to 0.59
65+ -- 0.44 0.36 to 0.53
Constant 0.08 0.08
Discussion {#s4}
==========
Principal findings and interpretation {#s4-1}
-------------------------------------
This is the largest study to date on this topic, comprising a whole country population. Our findings have demonstrated that both intellectual disabilities and autism are associated with having a mental health condition and with poor general health. This is so in both children/young people and in adults, after the overlap between these two conditions (intellectual disabilities and autism) is accounted for. For mental health conditions, this is particularly so for autism (OR 25.1, 95% CI 23.0 to 27.3 for children/young people; OR 5.3, 95% CI 4.8 to 5.9 for adults). For poor general health, this is particularly so for intellectual disabilities (OR18.3, 95% CI 17.2 to 19.6 for children/young people; OR 7.5, 95% CI 7.0 to 8.1 for adults). Previous literature on this is limited, and has not taken account of the overlap between autism and intellectual disabilities. It is of particular note that autism contributes to poor general health and especially to having a mental health condition even after taking account of the contribution of intellectual disabilities. The mental health conditions did not include transient common mental disorders, as the question referred to mental health conditions lasting or expected to last at least 12 months, that is, severe mental health conditions.
The extent of mental and general health inequality experienced by the population with intellectual disabilities and the population with autism, in comparison with the general population, is greatest in children/young people than it is for adults, though is substantial at all ages. This reflects that both mental health conditions and poor general health are much more common in adults than children and young people in the general population, while they are common at all ages in people with autism and in people with intellectual disabilities. Indeed, in people with intellectual disabilities, those with more severe intellectual disabilities have more comorbidity[@R18] and die at an earlier age[@R19] including in childhood. Hence, with increasing age, although acquiring age-related conditions, the population with intellectual disabilities has less disability-related comorbidity and perversely may be healthier than the younger population with intellectual disabilities.
The aetiology of mental and general ill health in people with intellectual disabilities or people with autism includes genetic predisposition[@R18]; indeed the term, Early Symptomatic Syndromes Eliciting Neurodevelopmental Clinical Examination, has been coined for the association of problems in one or more of 10 health domains in young children.[@R19] It is clear though that aetiology is multifactorial, and social and environmental factors such as life events, which occur more commonly in people with intellectual disabilities, have been shown to precede onset of mental health conditions in adults with intellectual disabilities.[@R20] Therefore, underpinning mechanisms appear to include the interplay between genes, environment and lifestyle^[@R21]^ including differences in health related behaviours, such as diet and exercise,[@R22] and inequalities in access to services.[@R23] This is important, as understanding these factors provides pathways to the development of interventions to improve health. Examples tailored to these populations, in addition to drug treatments, include interventions developed to address lifestyle,[@R24] general health[@R25] and psychological interventions for mental health conditions.[@R26]
Comparison with previous literature {#s4-2}
-----------------------------------
No previous studies have been identified which investigated the extent to which autism and intellectual disabilities are independently associated with poor mental and general health, in children and adults. We believe that this is, therefore, the first study to do so and subsequently we cannot compare these results.
Strengths and limitations {#s4-3}
-------------------------
The main strengths of the study are the 94% whole population response, rather than biassed sampling; the large population size of 5.3 million; that the conditions (intellectual disabilities, autism, mental health condition and general health) were systematically enquired about for each person; and that the phrasing of the questions underwent cognitive question testing in advance of the Census to ensure they captured the intended meaning. Consequently, we believe that these results are generalisable to other high-income countries.
Limitations include the use of the term of 'developmental disorders' in the Census. However, the Census form prompted responses only for autistic spectrum disorder or Asperger's syndrome. Furthermore, the developmental disorders category was distinguished from intellectual disabilities, learning difficulties and mental health conditions, which are important distinctions. Hence, we consider that respondents will have replied accordingly, that is, responded regarding autism. However, we have no means to check this. In addition, conditions were self/proxy reports rather than in-depth diagnostic assessments (which would not be possible on such a large scale). Respondents reported whether or not each person was known to have autism and/or intellectual disabilities, rather than each person having an assessment, so some reporting error is possible. However, intellectual disabilities and autism are conditions that are typically diagnosed during infant/primary school age, if not before. In Scotland these diagnoses attract additional educational support, which is to the child's advantage; once diagnosed these are lifetime diagnoses. Consequently, there may be an undercount in the early years of childhood, whereas reporting of these conditions should be accurate in later childhood, youth and in adults, within the diagnostic criteria prevailing at the time of diagnosis. The proportion of people in the population reported to have autism was lower after age 25. This reflects the broadening of diagnostic criteria and greater awareness of autism in recent years; hence, the older people with autism might have more severe autism than the children/youth reported to have autism. The children/youth with autism are likely to include some who function well. We do not know the extent to which reporting of mental health conditions and general health status would reflect that found in in-depth diagnostic assessments, although subjective general health status is commonly used in population studies, and it is well established as an extremely valid measure of health. There is a strongly predictive linear gradient across subjective health status and subsequent number of medical appointments, hospital admissions and mortality.[@R27] We do not know the proportion who self-reported or for whom the report was by another household reference person (eg, parent). However, the latter is likely to be more common for the people with intellectual disabilities in view of their intellectual disabilities, and for the children and young people. Six per cent of Census entries were imputed. The Census team assumed the 2.6% who did not provide information on long-term conditions did not have any of them, but we are unable to confirm the accuracy of this assumption.
Future research investigating narrower age bands of children/youth may be useful, and next steps must importantly include study of the aetiology of poor health in these populations, to inform the development of further effective interventions.
Implications {#s5}
============
Intellectual disabilities and autism are not uncommon, and due to their associated poor mental and general health, services and supports need to be available in sufficient quantity and quality. Our findings demonstrate that this is not just related to the coexistence of these conditions, or just to having intellectual disabilities, as the population with autism is also independently associated with substantial health inequalities compared with the general population, across the entire life course.
Supplementary Material
======================
###### Reviewer comments
###### Author\'s manuscript
We acknowledge funding from the UK Medical Research Council (grant number: MC_PC_1717).
**Contributors:** DK analysed the data, jointly interpreted it and wrote the first draft of the manuscript. ER jointly interpreted the data and contributed to the manuscript. KD jointly interpreted the data and contributed to the manuscript. LAH-M jointly interpreted the data and contributed to the manuscript. CM jointly conceived the project, interpreted the data and contributed to the manuscript. AH jointly interpreted the data and contributed to the manuscript. S-AC jointly conceived the project, interpreted the data and contributed to the manuscript. All authors approved the final version of the manuscript. S-AC is the study guarantor.
**Funding:** This study was funded by the Medical Research Council (grant reference MC_PC_17217) and the Scottish Government via the Scottish Learning Disabilities Observatory.
**Disclaimer:** The funders had no role in the study design, collection, analyses and interpretation of data, in writing the report, nor in the decision to submit the article for publication.
**Competing interests:** None declared.
**Patient consent for publication:** Not required.
**Ethics approval:** Approval was gained from the Scottish Government for secondary analysis of the Scotland Census, 2011 data. Access to a subset of data was provided.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data availability statement:** No data are available.
| {
"pile_set_name": "PubMed Central"
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1. Introduction {#sec1}
===============
Around the world, ischemia stroke had been the third leading cause of death and the leading cause of disability \[[@B1]\]. Cerebral ischemia was always caused by reduction or blockage of the blood flow to some regions in brain which is induced by vascular reflow after contraction, percutaneous transluminal coronary angioplasty, organ transplantation, and so on \[[@B2]\]. Clinically, vascular recanalization was always used to restore blood supply; however, this reperfusion might induce further injuries in brain, and this process was called ischemia/reperfusion (I/R) injury \[[@B3]\]. I/R caused multiple insults to cerebral microvasculatures which are accompanied with oxygen free radicals production, mast cell degranulation, and endothelial cell injury \[[@B4]\]. It had been established that inflammatory response plays an important role in the pathogenesis of cerebral I/R injury \[[@B5]\]. Therefore, pharmacological alleviation of inflammatory damage was considered to be one of the most promising solutions for the treatment of stroke.
Nuclear factor-kappa B (NF-*κ*B), a critical transcription factor involved in the inflammatory processes, was activated during I/R which further induced a variety of proinflammatory molecules expression, such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-a (TNF-a), and resulted in neuron death \[[@B6]\]. AMP-activated protein kinase (AMPK) had been recognized as an emergency response enzyme and played important roles at the cellular and whole-organism levels \[[@B7]\]. Some studies had showed that AMPK protected brain from I/R injuries though inhibiting NF-*κ*B mediated inflammatory \[[@B8]\]. Thus, AMPK is currently proposed as a new therapeutic target for I/R-induced inflammation.
*Carthamus tinctorius* L. (safflower) had been frequently used in traditional Chinese medicine for treatment of cerebrovascular and cardiovascular diseases \[[@B9]--[@B11]\]. It also exerts other pharmacological effects such as anticoagulant, antioxidant, and calcium antagonist effects. It had been reported that the main chemical constituents in safflower were flavonoids, polysaccharides, lignans, and triterpene alcohols \[[@B12]\]. The extracts from safflower contained yellow and red pigments which included safflower yellow B (SYB), hydroxysafflor yellow A (HSYA), safflower yellow A (SYA), and others \[[@B13]\]. However, which components are responsible for its protective effects were still largely unknown for us. SYB, a natural flavonoid compound, had been used as cardiovascular drugs in traditional Chinese medicine \[[@B14]\]. Some literatures reported that SYB had strong antioxidant effects and protected oxidative stress-induced nerve and hepatocytes cell damage \[[@B15], [@B16]\]. However, little research on the anti-inflammatory effects of SYB on brain I/R had been undertaken. Thus, in this study, we tried to investigate (1) whether SYB inhibit inflammatory mediated by I/R*in vivo* and*in vitro*; (2) whether the anti-inflammatory effect of SYB was mediated by AMPK/NF-*κ*B signaling pathway.
2. Materials and Methods {#sec2}
========================
2.1. Materials {#sec2.1}
--------------
SYB (purity \>98%, determined by HPLC) was obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Compound C was obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Cell culture related reagents including Dulbecco\'s modified Eagle\'s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Cell counting kit (CCK-8), type I collagenase, and western blot reagents were obtained from Sigma (St. Louis, MO, USA). The kits for the determination of interleukin-6 (IL-6), IL-1, tumor necrosis factor-*α* (TNF-*α*), and Cyclooxygenase 2 (COX-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against AMPK, P-AMPK, ACC, P-ACC, NF-*κ*B, I-*κ*B, Bax, Bcl-2, cleaved-caspase 3, and *β*-actin were obtained from Santa Cruz Biotechnology, USA. All other chemicals used were of the best available commercial grade.
2.2. Rat Ischemia/Reperfusion (I/R) Model {#sec2.2}
-----------------------------------------
All animal experiments were conducted according to "The Guidance to Experimental Animal Welfare and Ethical Treatment" by The Ministry of Science and Technology of the People\'s Republic of China (2006) and "Guide for the Care and Use of Laboratory Animals" by the National Research Council of the National Academies (National Academy of Science, 2011).
Rats were randomly divided into 6 groups: sham, model, SYB-L (treated with 2 mg/kg SYB), SYB-M (treated with 4 mg/kg SYB), SYB-H (treated with 8 mg/kg SYB), and NIM (nimodipine-treated group, 0.4 mg/kg). The focal ischemia was induced by middle cerebral artery occlusion (MCAO) and performed as described in detail previously \[[@B17]\]. Briefly, rats were anaesthetized by intraperitoneal injection of chloral hydrate (300 mg/kg). Body temperature and vital signs were monitored. The middle cerebral artery (MCA) was isolated and occluded by a monofilament nylon suture for 2 h. The rats in sham group were underwent the same surgical procedure expect for arterial occlusion. After reperfusion for 20 h, rats were euthanized and blood and brain tissues were collected. The drug was administrated by intraperitoneal injection. In sham and model rats, saline was given by the same administration.
2.3. Cerebral Infarction and Neurofunctional Evaluation {#sec2.3}
-------------------------------------------------------
After reperfusion for 20 h, the neurological deficit score of rats was evaluated by Longa\'s method of a five-point scale \[[@B17]\]. The evaluation was performed by a researcher who was blinded to the design: 0 = no neurological deficit; 1 = failure to extend right paw fully; 2 = circling to right; 3 = falling to right; 4 = no spontaneous walking with a depressed level of consciousness.
Postural reflexes after reperfusion were measured as the method reported \[[@B18]\]. Briefly, rats were suspended 100 cm above the ground surface and slowly lowered and then placed on the table and pushed from side to side: 0 = no deficit; 1 = forelimb flexion when rats were suspended by the tail; 2 = decreased resistance to lateral push.
The beam balance test was also used \[[@B19]\]. After reperfusion, rats walked on a narrow beam (width 1.5 cm; length 150 cm) and the behavior was scored as the method reported \[[@B20]\]: 1 = steady posture with paws and balance on the beam; 2 = grasps side of beam or wavering; 3 = hugs the beam and one or two limbs slip off beam; 4 = hugs the beam and three limbs slip off beam; 5 = rat attempts to balance with paws on beam but falls; 6 = rat drapes over beam, then falls; 7 = rat falls off the beam without attempting to balance.
Adhesive removal test was used to measure the sensory stimuli response \[[@B21]\]. A small piece of adhesive tape square (width 2 mm; length 2 mm) was placed on right or left C2 or C3 vibrissae of the rat. The right or left was alternated between each rat and each session, and then the times to contact or remove the adhesive tape were measured, with a limit of 60 s.
Following the neurological evaluation, the brain was collected from 6 rats in each group and kept at -20°C for 40 min. Brains were sliced into five slices of 2 mm thick and then stained in 2% solution of 2,3,5-tri-phenyl tetrazolium chloride (TTC) at 37°C for 10 min. The stained slices were photographed and measured by an image analysis system (JM VIS-TEC, Suzhou, China). The infarct volumes were expressed as the ratio virus the contralateral hemisphere volume.
2.4. PC12 Cells Ischemia/Reperfusion (I/R) Model {#sec2.4}
------------------------------------------------
PC12 cells were cultured by DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO~2~ and 95% air. The culture medium was changed every other day. To initiate ischemia/reperfusion (I/R) model in vitro, cells were plated into relative culture dishes and cultured for 24 h. After treatment with SYB for another 24 h, the medium was removed and replaced with glucose-free DMEM in a hypoxia incubator (95% N~2~ and 5% CO~2~) for 4 h (ischemia). Following ischemia, the glucose-free DMEM was replaced with the normal culture medium and cells were incubated for another 20 h (reperfusion) under normal culture conditions.
2.5. Analysis of Cell Viability {#sec2.5}
-------------------------------
MTT assay was used to determine cell viability. PC12 cells were plated into 96-well plates at a density of 1 x 10^4^ cells/well. Cells were treated by different drugs for 24 h and subjected to I/R. 20 *µ*l MTT solution (5 mg/ml) was added to each well and maintained at 37°C for 4 h. The MTT was removed and 150 *μ*l DMSO was added to each well. The optical density (OD) was determined by a microplate reader (Infinite M200 PRO, Tecan) at 490 nm. The cell survival ratios were showed as a percentage of the control.
2.6. Cell Apoptosis {#sec2.6}
-------------------
PC12 cells were pretreated with SYB for 24 h before subjected to I/R. The cells were washed by PBS and treated according to manufacturer\'s instructions for the Annexin V-FITC apoptosis assay kit. The apoptosis rate was analyzed by a flow cytometry (BD Biosciences, San Jose, CA, USA). Cell apoptosis ratio was expressed as percentages of the number of total cells.
2.7. LDH Leakage {#sec2.7}
----------------
The cytotoxicity was measured by the LDH leakage assay after different treatments. The culture medium was collected, and cells were scraped in PBS. The cells membranes were broken down to release the total LDH in cells, and then centrifugation was undertaken to clear up the cell samples. The LDH leakage was calculated from the ratio between the LDH levels in culture medium and in cell content.
2.8. Cytokine Enzyme-Linked Immunosorbent Assay (ELISA) {#sec2.8}
-------------------------------------------------------
Serum or culture supernatants were collected and saved at -20°C until used. ELISA for IL-1, IL-6, TNF-*α*, and COX-2 were used to measure the anti-inflammation activity of SYB. All operations were performed according to manufacturer\'s protocol.
2.9. Protein Extraction and Western Blot Analysis {#sec2.9}
-------------------------------------------------
Total or nuclear proteins extracts of different cell treatment group and brain tissue were lysed by nuclear and cytoplasmic extraction reagent kits. The protein concentrations were determined by a BCA protein measurement kit. Equal amounts of protein samples (30 *µ*g) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes. The protein membranes were blocked with 5% non-fat dried milk for 30 min at 37°C, followed by incubation with specific primary antibodies (AMPK, P-AMPK, NF-*κ*B, P-I*κ*B-*α*, Bax, Bcl-2, cleaved-caspase 3, and *β*-actin) overnight at 4°C. Then, the membranes were further incubated with relative secondary antibody for 1 h at 37°C. Bands were detected using enhanced chemiluminescence light-based detection kits (Millipore, Billerica, MA, uSA). *β*-Actin was used as a loading control in cytoplasm. Lamin B was used as a loading control in nuclear.
2.10. Statistical Analysis {#sec2.10}
--------------------------
Data are presented as mean ± SD. One-way analysis of variance (ANOVA) was used followed by Tukey\'s multiple comparison tests using Graphpad prism 5.0. P-values of \<0.05 and \<0.01 were considered statistically significant.
3. Results {#sec3}
==========
3.1. SYB Protected Brain from I/R-Induced Injuries {#sec3.1}
--------------------------------------------------
To evaluate the effects of SYB on the balance and sensory activity, postural reflexes, adhesive removal test, and beam balance test were performed after stroke. As the results showed, rats got high scores on reflex ([Figure 1(a)](#fig1){ref-type="fig"}), beam balance ([Figure 1(b)](#fig1){ref-type="fig"}), and sensory tests ([Figure 1(c)](#fig1){ref-type="fig"}), indicating that the balance and sensory activity were injured by I/R treatment. As compared with the model group, the scores were significantly decreased by SYB treatment (P\<0.01). The neurological deficit scores were also measured. After reperfusion, neurological deficit scores in I/R rats were significantly higher than those in sham group (P\<0.01), indicating that I/R effectively caused neurological dysfunction in rats ([Figure 1(d)](#fig1){ref-type="fig"}). As compared with the model group, neurological deficit scores were significantly decreased in SYB and NIM treatment groups (P\<0.01). Infarct volume was measured by TTC after reperfusion. No infarction was observed in sham group. In model group, the infarct volume was significantly increased by I/R treatment (P\<0.01). Treatments with SYB at all doses significantly reduced the infarct volume compared with the model group and showed dose-dependent manner ([Figure 1(e)](#fig1){ref-type="fig"}). Following I/R, there was a significant reduction in brain water content in the SYB treatment groups compared with model group ([Figure 1(f)](#fig1){ref-type="fig"}). LDH, S-100*β*, and NSE levels in brain were considered as brain injury markers. Compared with the sham group, I/R significantly increased the levels of LDH, S-100*β*, and NSE in brain, and SYB treatments significantly decreased their levels compared with model group (Figures [1(g)](#fig1){ref-type="fig"}, [1(h)](#fig1){ref-type="fig"}, and [1(i)](#fig1){ref-type="fig"}).
3.2. SYB Inhibited I/R-Induced Inflammatory Reaction in Brain {#sec3.2}
-------------------------------------------------------------
To determine whether SYB had some effects on the inflammatory reaction in brain after I/R, inflammatory factors including IL-1, IL-6, TNF-*α*, and COX-2 were measured by ELISA. As shown in [Figure 2](#fig2){ref-type="fig"}, levels of IL-1, IL-6, TNF-*α*, and COX-2 in serum were significantly increased in model group, which compared with sham group (P\<0.01). After treatment with SYB or NIM, serum levels of IL-1, IL-6, TNF-*α*, and COX-2 were significantly inhibited compared with model group (P\<0.01). These results demonstrated that I/R induced inflammatory reaction in brain, and SYB treatments had protective effects on I/R-induced inflammatory reaction.
3.3. SYB Induced AMPK Phosphorylation and Inhibited NF-*κ*B Expression in Brain {#sec3.3}
-------------------------------------------------------------------------------
To determine the effect of SYB on NF-*κ*B p65 expression, levels of NF-*κ*B p65 were measured in cytoplasm and nuclear. As the results showed in [Figure 3(a)](#fig3){ref-type="fig"}, the expression levels of NF-*κ*B p65 were decreased in cytoplasm and increased significantly in nuclear, compared with sham group (P\<0.01). The phosphorylation levels of I*κ*B-*α* were increased significantly in model group, compared with sham group (P\<0.01). In SYB treatment group, the expression levels of NF-*κ*B p65 were increased in cytoplasm and decreased in nuclear compared with model group (P\<0.01). The phosphorylation levels of I*κ*B-*α* were also inhibited by SYB treatments.
Next, the phosphorylation levels of AMPK and its downstream ACC were measured by Western blotting. As the results showed in [Figure 3(b)](#fig3){ref-type="fig"}, I/R induced significant reduction of AMPK phosphorylation levels and also ACC phosphorylation levels in model group compared in sham group (P\<0.01). However, in SYB treatment group, the phosphorylation levels of AMPK and ACC were significantly increased compared with model group (P\<0.01). These results suggested that SYB could induce AMPK phosphorylation and inhibited NF-*κ*B expression in brain subjected to I/R injury.
3.4. SYB Protected PC12 Cells from I/R-Induced Cell Apoptosis {#sec3.4}
-------------------------------------------------------------
To elucidate the protective effects of SYB against I/R-induced cell damage, PC12 cells were pretreated with SYB (60, 120, and 180 nmol/l) for 24 h and then subjected to I/R for another 24 h. Concentrations of SYB used in this study did not induce any adverse effects (data not shown). The protective effects of SYB on I/R induced cell injuries were determined by MTT assay and LDH leakage assay. As the results showed in [Figure 4(a)](#fig4){ref-type="fig"}, cell viability was significantly decreased by I/R treatment, and pretreatment with SYB for 24 h increased the cell viability in a dose-dependent manner. Similarly, the LDH leakage assay revealed that I/R induced LDH leakage from the cells, and SYB inhibited LDH leakage in a dose-dependent manner ([Figure 4(b)](#fig4){ref-type="fig"}), suggesting that SYB enabled PC12 cells to maintain cell integrity.
To determine whether SYB had protective effects against apoptosis, Annexin V-FITC/PI double staining and western blotting were used. As the results of flow cytometric detection showed, compared with control group, I/R induced a significant increase in the apoptosis of PC12 cells, and SYB pretreatment significantly decreased the apoptosis rate ([Figure 4(c)](#fig4){ref-type="fig"}). The results of western blotting showed that I/R obviously reduced Bcl-2 expression and increased Bax and cleaved-caspase 3 expression compared with control group ([Figure 4(d)](#fig4){ref-type="fig"}). Compared with model group, treatment of PC12 with SYB increased Bcl-2 expression and decreased Bax and cleaved-caspase 3 expression (P\<0.01).
3.5. SYB Inhibited I/R-Induced Inflammatory Factors Expression in PC12 Cells {#sec3.5}
----------------------------------------------------------------------------
To study the mechanisms through which SYB performs its protective effects, levels of inflammatory factors were determined. The PC12 cells were pretreated with SYB for 24 h and subjected to I/R for another 24 h, and the levels of IL-1, IL-6, TNF-*α*, and COX-2 were measured by ELISA kits. The results showed that I/R induced rapid and significant increase of IL-1, IL-6, TNF-*α*, and COX-2 levels in PC12 cells ([Figure 5](#fig5){ref-type="fig"}). Pretreatment of the cells with SYB for 24 h significantly decreased the levels of IL-1, IL-6, TNF-*α*, and COX-2 compared with the I/R group (P\<0.01). These results suggested that SYB had protective effects against I/R-induced inflammatory response in PC12 cells.
3.6. The Effects of SYB on the Expression of AMPK and NF-*κ*B in PC12 Cells {#sec3.6}
---------------------------------------------------------------------------
NF-*κ*B signaling pathway was an important inflammatory regulator responsible for I/R. To clarify the mechanism of SYB in inhibiting inflammation, the activity of NF-*κ*B was measured by western blotting. As shown in [Figure 6(a)](#fig6){ref-type="fig"}, there was a low basal level of NF-*κ*B p65 in the nuclear of normal cells. I/R induced significant increasing of NF-*κ*B p65 in the nuclear together with an increased phosphorylation levels of I-*κ*B, suggesting I/R induced a translocation of NF-*κ*B p65 from the cytoplasm to the nucleus. In addition, SYB inhibited the phosphorylation of I-*κ*B and nuclear translocation of NF-*κ*B p65 in a dose-dependent manner.
AMPK had showed its protective effects on I/R related injuries. The phosphorylation of AMPK was also determined by Western blotting. As shown in [Figure 6(b)](#fig6){ref-type="fig"}, the phosphorylation levels of AMPK were significantly lower in I/R group than those in control group (P\<0.01), while the phosphorylation levels of AMPK were increased by SYB pretreatment in a dose-dependent manner as well as its downstream ACC. These results suggested that SYB induced the phosphorylation of AMPK and activated the downstream pathway.
3.7. SYB Inhibited the Nuclear Translocation of NF-*κ*B through AMPK {#sec3.7}
--------------------------------------------------------------------
In order to further determine the role of AMPK on the anti-inflammatory effects of SYB, the AMPK specific siRNA, siAMPK were used to block the activation of AMPK. As the results showed in Figures [7(a)](#fig7){ref-type="fig"} and [7(b)](#fig7){ref-type="fig"}, siAMPK inhibited the SYB-induced phosphorylation of AMPK and ACC in the PC12 cells subjected to I/R. Moreover, the inhibition of AMPK markedly reduced the capacity of SYB to decrease NF-*κ*B p65 nuclear translocation ([Figure 7(c)](#fig7){ref-type="fig"}), as well as the IL-1 and IL-6 levels (Figures [7(d)](#fig7){ref-type="fig"} and [7(e)](#fig7){ref-type="fig"}). Consistently, the inhibition of AMPK by siAMPK also eliminated the SYB induced cytoprotective effects against I/R induced cell viability reduction ([Figure 7(f)](#fig7){ref-type="fig"}). These results suggested that SYB inhibited the NF-*κ*B mediated inflammatory response by activating the AMPK pathway.
4. Discussion {#sec4}
=============
Cerebral ischemia was one of the most common clinical circulatory arrests and caused neuronal damage in some certain vulnerable brain areas like hippocampal CA1 region \[[@B22]\]. Several mechanisms were included in cerebral ischemia induced injury, including calcium overload, inflammatory response, free radical injury, and some others \[[@B23]\]. Among these, inflammatory response was considered to be the leading cause of cerebral ischemia \[[@B24]\]. Cerebral ischemia induced inflammatory response and reperfusion aggravated the inflammation response causing secondary brain damage \[[@B25]\]. Proinflammatory responses were found within minutes after the onset of cerebral ischemia and lead to tissue damage, improper cellular repair, and dysfunction \[[@B26]\]. So inhibition of the inflammatory response in ischemia regions at the beginning of ischemia might provide effective treatment.
Safflower was always used for the treatment of coronary heart disease, hypertension, and cerebrovascular disease \[[@B27]\]. Previous studies considered that HSYA was the main active substance; however, more and more researchers found that other water-soluble compounds including SYB and SYA were responsible for its therapeutic effects \[[@B13], [@B14]\]. Safflower injection had been approved by the State Food and Drug Administration of China for the treatment of cerebral ischemia in 2000. Clinical studies had showed that safflower injection had good curative effects and minor side effects \[[@B28]\]. There were some literatures reported that HSYA had anti-inflammatory effects of and protected brain from I/R injury \[[@B29], [@B30]\]. However, to the best of our knowledge, there are no studies available to date on the effects of SYB on I/R-induced inflammatory response in brain. Thus, this study was designed to examine the inhibitory effects of SYB on I/R-induced inflammatory response in vivo and in vitro.
In this study, MCAO and reperfusion in rat were used to mimic some features of human brain pathology. The results of neurological deficit scores and infarct volume showed that I/R model was successful. Treatment with SYB significantly decreased the neurological deficit scores and infarct volume and showed dose-dependent manner. Serum neuron specific enolase (NSE) and S100 calcium binding protein B (S-100B), two biochemical markers of brain injury, were always used clinically to evaluate the degree of brain injury \[[@B31]\]. In this study, we found that I/R increased the levels of NSE and S-100B, and SYB decreased their levels in a dose-dependent manner. These results suggested that SYB had protective effects against I/R-induced brain injuries.
Proinflammatory cytokines were upregulated in the brain after some pathological stimulus including cerebral ischemia. They were secreted by immune cells and also produced by brain cells, like glia cells and neurons \[[@B32], [@B33]\]. The most important inflammation related cytokines in brain during ischemia injury were IL-1, IL-6, COX-2, and TNF-*α* \[[@B34]\]. Among these, IL-1 and TNF-*α* could exacerbate the degree of brain injury \[[@B35]\]. In the current study, we found that IL-1, IL-6, COX-2, and TNF-*α* were increased significantly after I/R operation. SYB treatment inhibited the elevation of IL-1, IL-6, COX-2, and TNF-*α* in brain and PC12 cells. These results suggested that SYB inhibited the inflammation induced by I/R in vivo and in vitro.
In eukaryotic cells, NF-*κ*B was an important nuclear transcription factor which regulates the expression of many cytokines, including proinflammatory cytokines \[[@B36]\]. In various diseases, NF-*κ*B had been found to be a promising target in inhibiting inflammatory response. Accumulating evidences showed that elevated NF-*κ*B contributes to brain injury induced by ischemia \[[@B37]\]. Under normal condition, NF-*κ*B was associated with its inhibitory protein I*κ*B and sequestered in the cytoplasm. Upon activation, I*κ*B kinases (IKKs) were activated and led to the phosphorylation of I*κ*B which further induced the proteasome-mediated degradation; thus NF-*κ*B is transported into nucleus from cytoplasm \[[@B38]\]. The nuclear NF-*κ*B bound with its binding sequence to activate the relevant promoters and induced the expression of inflammatory cytokines, including IL-1, IL-6, COX-2, and TNF-*α* \[[@B39]\]. These facts suggested that NF-*κ*B played an important role in regulating inflammation, and the inhibition of NF-*κ*B was protective against neuroinflammation and neurodegeneration. In this study, I/R induced the phosphorylation of I*κ*B and nuclear translation of NF-*κ*B p65 in brain and PC12 cells. However, SYB treatments significantly decreased the nuclear translation of NF-*κ*B p65, together with the reduction of I*κ*B phosphorylation. These results suggested that the anti-inflammation effects of SYB might be through inhibiting the NF-*κ*B pathway.
AMPK had been considered as a detector of cellular homeostasis and also modulated oxidative stress and inflammation \[[@B40]\]. It regulated several signal translocation pathways to affect the cell death and survival. AMPK could also mediate several signaling cascades to inhibit the inflammation \[[@B41]\]. The results of Western blotting showed that SYB treatment significantly increased the phosphorylation of AMPK and also its downstream ACC, suggesting SYB could activate the AMPK pathway. Amassing research supported that AMPK negatively regulates NF-*κ*B, and the reduced AMPK led to an increase of NF-*κ*B signaling activities in several cell lines \[[@B42], [@B43]\]. Therefore, we investigated whether that AMPK pathway contributes to the protective effects of SYB. To further study the relationship between AMPK and NF-*κ*B during SYB treatment, compound C and siAMPK were used. The results showed that inhibition of AMPK markedly reduced the capacity of SYB to decrease NF-*κ*B p65 nuclear translocation and increased the expression level of IL-1 and IL-6. Further analysis also indicated that siAMPK abolished the cytoprotective effects of SYB against I/R injury. These results suggested that AMPK/NF-*κ*B was involved in the cytoprotective effects of SYB.
In conclusion, our results strongly suggested that SYB treatment protected cerebral cell from I/R induced inflammation through a mechanism that SYB activated AMPK and negatively regulated NF-*κ*B mediated inflammatory response. These results provided some scientific evidences for the cerebral protection effects of SYB and suggested it might be useful in the treatment of various brain diseases associated with inflammation.
This work was supported by National Natural Science Foundation of China (no. 81471140) and Shenzhen University General Hospital Science and Technology Personnel Booster Program (no. 85706-0000040537).
Data Availability
=================
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
=====================
The authors declare that they have no conflicts of interest.
Authors\' Contributions
=======================
Shibin Du and Youliang Deng contributed equally to this work.
{#fig1}
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{#fig3}
{#fig4}
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{#fig7}
[^1]: Academic Editor: Shan-Yu Su
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Introduction {#s1}
============
Nanotribology is a branch of tribology and was coined by Krim, Solina and Chiarello [@pone.0081094-Krim1] in a paper entitled "Nanotribology of a Kr monolayer". Nanotribology deals with tribological phenomena occurring at sub-micron or smaller length scales; tribology is the science and technology of interactive surfaces in relative motion, which include the studies of friction, lubrication and wear [@pone.0081094-JostH1]. The difference between tribology and nanotribology is comparable to the Newtonian physics and quantum physics [@pone.0081094-RensselarJ1]. Nanotribology is a highly interdisciplinary field where tribologists, physicists, chemists, material scientists, micro/nano- and miniature system engineers are jointly developing theories, simulations, and final applications that benefit society [@pone.0081094-GebeshuberI1]. Today nanotribology is one of the most important mechanical technologies and it uses many new instruments such as the surface force apparatus, atomic force microscope, friction force microscope and scanning tunnel microscope [@pone.0081094-CarpickR1]. The emergence of micro/nanotribology and atomic force microscopy based techniques has provided researchers with a viable approach to addressing tribological problems [@pone.0081094-Bhushan1], [@pone.0081094-Bhushan2]. Nanotribological studies are mostly fundamental in nature and the results are applied in the MEMS, in HDD technologies as well as in nanotechnologies [@pone.0081094-Rymuza1]. Nanotribology has brought the scale of interest, familiar in physics and chemistry, to the level of an engineering phenomenon. The future of nanotechnology depends on advancements in nanotribology, which has wide applications ranging from health care to energy conversion and storage, and micro craft space exploration [@pone.0081094-AshaL1]. Applications include improving car engine lubrications, biolubrication in hip joints and cosmetics, shrinking devices to micrometer and nanometer scales to manufacture nanoscale machines, and expanding the range of temperatures, speeds, and chemical environments to the extreme conditions where devices operate [@pone.0081094-12th1].
Evaluating research fields like nanotribology using scientometric techniques is useful in determining, for example, the citation impact of contributing authors and institutions [@pone.0081094-Malarvizhi1]. Findings from these investigations can help researchers to realize the breadth of research in the field and establish possible future research directions [@pone.0081094-Li1]. Bibliometric/scientometric studies have been carried out in the past in various research fields ranging from science to engineering and medicine (see a selection in [Table 1](#pone-0081094-t001){ref-type="table"}). The aim of the present study is: (1) to examine the publications pattern of nanotribology research output at global level; (2) to analyse the publications pattern and impact of the most producing countries; (3) to examine the productivity and impact of the most publishing institutions; (4) to analyse the pattern of authorship and prolific authors; (5) to examine the impact of the most preferred journals; (6) to analyse the characteristics of highly cited papers; (7) to analyse the keywords appended by the authors; (8) to visualize the co-authorship network among the authors and collaboration network among the top ten most productive countries.
10.1371/journal.pone.0081094.t001
###### Recent bibliometric/scientometric studies.
{#pone-0081094-t001-1}
Author(s) and year Research field
--------------------------------------------------- ----------------------
Modak and Giridhar (2008) [@pone.0081094-ModakJ1] Chemical Engineering
Tsay (2008) [@pone.0081094-TsayM1] Hydrogen Energy
Ortiz et al (2009) [@pone.0081094-OrtizA1] Cancer
Sun, Wang and Ho (2012) [@pone.0081094-Sun1] Estuary Pollution
Yu et al (2012) [@pone.0081094-YuJ1] Photosynthesis
Dong et al (2012) [@pone.0081094-Dong1] Solar Power
Wang et al (2013) [@pone.0081094-Wang1] GPS
Zhou and Wang (2013) [@pone.0081094-Cao1] Laparoscopy
Fu, Wang and Ho (2013) [@pone.0081094-FuH1] Drinking Water
Kademani et al (2013) [@pone.0081094-KademaniB1] Materials Science
Materials and Methods {#s2}
=====================
SCOPUS (Elsevier) was used to retrieve the records related to nanotribology research for the period 1996--2010. The following keywords were used in the combined field of title, abstract and keywords: *nanotribo\** OR *microtribo\** OR *nano-tribo\** OR *micro-tribo\** OR {*nano tribology*} OR {*micro tribology*}. The search was carried out on 11/15/2012 and refined to restrict the literature to articles, conference papers and reviews [@pone.0081094-Konur1]. Bibliographic details related to nanotribology research are available from 1974 in SCOPUS. Since SCOPUS does not have complete citation information for papers published before 1996 [@pone.0081094-Ball1], the present study was confined to 1996--2010. Self-citations (of authors, institutions, etc.) have not been excluded from the analyses.
The retrieved data were exported to MS-Excel. 29 records were deleted where the information related to author and affiliation was not available. We proceeded with 1321 papers related to nanotribology research during the period 1996--2010. Manual coding was done for the number of authors, country of origin and affiliation of authors. The fractional counting method was applied to give credit to all the contributing authors, institutes and countries [@pone.0081094-Borsi1]. Institutional affiliations and author names were unified manually.
Tools and Techniques Employed {#s2a}
-----------------------------
The impact of research is measured by citations [@pone.0081094-Konur1]. Since we measure the citation impact within one field (nanotribology) only, we did not apply field-normalized indicators (such as the relative citation rate). Citations per publication (CPP) can be used to assess the impact of publications for publication years, countries, institutes and authors. The formula of CPP is
CPP = Total Citations / Total Papers
Both the output and impact of the publications of the most productive countries, institutes and journals are measured using the h-index. Hirsch [@pone.0081094-HirschJ1] proposed the h-index as an alternative to standard bibliometric indicators for single scientists; it is defined as follows:
A scientist has index h if h of his or her N~p~ papers have at least h citations each and other papers (N~p~--h) have ≤h citations each.
UCinet [@pone.0081094-BorgattiS1] is used to generate a collaborations network among the top ten most productive countries in nanotribology research. To construct the collaboration network map, the following steps were taken.
Among the various available methods to calculate the h-index, Ye [@pone.0081094-YeF1] found that the Glänzel-Schubert [@pone.0081094-Schubert1] model was better than the Hirsch and Egghe-Rousseau [@pone.0081094-Egghe1] models to estimate the h-index of a publication set. The difference between these models is that the original h-index model links only total citations and the Egghe-Rousseau model links only total publications, whereas the Glänzel-Schubert model incorporates the total citations as well as total publications ([Table 2](#pone-0081094-t002){ref-type="table"}). Since its introduction in 2005, the h-index has been applied not only to single scientists, but also to research groups [@pone.0081094-vanRaanAF1] and countries [@pone.0081094-Schubert2]. For example, Fu, Wang and Ho [@pone.0081094-FuH1] applied the h-index to countries, institutes and journals.
10.1371/journal.pone.0081094.t002
###### Various models of the h-index.
{#pone-0081094-t002-2}
Model Equation Description
------------------ ----------------------- -------------------------------------------------------------------------------------------------------------------
Hirsch h = √(C/a) C = total citations, a is a constant ranging from 3 to 5
Egghe-Rousseau h = P^1/α^ P = total publications, α \>1 is Lotka's exponent
Glänzel-Schubert h = cP^1/3^(CPP)^2/3^ c is a constant (0.9 for journals and 1 for other units), P = total publications, CPP = citations per publication
Step 1 -- A matrix was developed using the number of papers collaborated on by the countries with each other country among the top ten in Excel matrix editor.
Step 2 -- Collaboration network was visualized with Netdraw [@pone.0081094-BorgattiS2]
Step 3 -- Colours of the nodes were changed ([Figure 1](#pone-0081094-g001){ref-type="fig"}).
{#pone-0081094-g001}
The Sci^2^ tool [@pone.0081094-Sci21] is used to generate a co-author network map among the authors of nanotribology research. To construct the visualization map, the following steps were taken.
Step 1 -- Load CSV file was selected
Step 2 -- Extract co-author network was selected (file format: SCOPUS)
Step 3 -- Network Analysis Tool kit was selected
Step 4 -- GUESS [@pone.0081094-Adar1] was selected and default random layout used ([Figure 2](#pone-0081094-g002){ref-type="fig"})
{#pone-0081094-g002}
Step 5 -- Extract K-core was selected (Unweighted and Directed)
Step 6 -- GUESS was selected and default random layout used ([Figure 3](#pone-0081094-g003){ref-type="fig"}).
{#pone-0081094-g003}
Results {#s3}
=======
Growth of Publications and Citations {#s3a}
------------------------------------
[Table 3](#pone-0081094-t003){ref-type="table"} provides the following general characteristics about nanotribology research output for the period 1996--2010: annual output, average number of authors, number of citations received, citations per publication, percentage of cited documents and citations per publication per year. Nanotribology research increased tremendously from 34 papers in 1996 to 161 papers in 2010, with an average of 88 papers per year. The highest number of papers was published in the year 2008, at 177, and the lowest in 1997, at 17. The average number of authors per paper increased from 2.41 in 1996 to 3.66 in 2010, with an average of 3.52 authors per paper. This increase reflects the increasing trend of (international) collaboration in this research field. Out of total publications, 70% of papers received one or more citations. Overall, 11913 citations were received by 1321 papers, with an average of 9 per paper; citations per paper per year were 1.36.
10.1371/journal.pone.0081094.t003
###### Year-wise output, average authors and citations.
{#pone-0081094-t003-3}
Year TP AU AU/P TC CPP Cited \% cited CPPY
------- ------ ------ ------ ------- ------- ------- ---------- ------
1996 34 82 2.41 876 25.76 23 67.65 1.61
1997 17 48 2.82 445 26.18 15 88.24 1.75
1998 55 174 3.16 814 14.80 44 80.00 1.06
1999 38 126 3.32 604 15.89 28 73.68 1.22
2000 71 228 3.21 822 11.58 49 69.01 0.96
2001 67 222 3.31 766 11.43 51 76.12 1.04
2002 48 180 3.75 492 10.25 38 79.17 1.03
2003 67 252 3.76 861 12.85 55 82.09 1.43
2004 99 323 3.26 858 8.67 66 66.67 1.08
2005 139 511 3.68 1319 9.49 92 66.19 1.36
2006 115 447 3.89 948 8.24 84 73.04 1.37
2007 101 372 3.68 1129 11.18 79 78.22 2.24
2008 177 615 3.47 947 5.35 104 58.76 1.34
2009 132 486 3.68 524 3.97 86 65.15 1.32
2010 161 590 3.66 508 3.16 114 70.81 1.58
Total 1321 4656 11913 928
Mean 3.52 9.02 70.25 1.36
TP = total papers, AU = number of authors, AU/P = avg. authors per paper, TC = total citations, CPP = citations per paper, CPPY = citations per paper per year.
Level of Collaboration {#s3b}
----------------------
Collaboration type is determined by the author affiliations of each paper as follows (see [Table 4](#pone-0081094-t004){ref-type="table"}): (i) Single-authored publications; (ii) Single institute publications with author affiliations from the same institution; (iii) Inter-institutionally collaborative publications with different author affiliations within the same country; (iv) Single country publications with author affiliations from the same country; (v) International collaborative publications with author affiliations from different countries. Single-authored publications are very nominal (11%) and multi-authored publications dominate the research field. The level of collaboration and its citation impact of nanotribology research output during 1996--2010 are presented in [Table 4](#pone-0081094-t004){ref-type="table"}, which indicates that the publications contributed with international collaboration had the highest impact, with an average CPP of 11.71, while single institute publications had the lowest impact, at 8.47.
10.1371/journal.pone.0081094.t004
###### Level of collaboration and citation impact.
{#pone-0081094-t004-4}
Level TP \% of TP TC CPP
---------------------------------------------- ------ ---------- ------- -------
Without collaboration(single-authored) 141 10.67 1247 8.84
Collaboration withinternational institutions 161 12.19 1885 11.71
Collaboration withanother institution 314 23.77 2811 8.95
Collaboration withinthe same institution 705 53.37 5970 8.47
Total 1321 11913
TP = total papers, TC = total citations, CPP = citations per paper.
Most Productive Countries {#s3c}
-------------------------
Forty four countries were involved in the total research output (n = 1321) on nanotribology during 1996--2010. About 84% of total publications were contributed by the top ten most productive countries, which indicate that the researchers from these countries were involved more in this research field compared to other countries and 85% of total citations were received by the publications contributed by these top ten countries. G7 countries (USA, UK, France, Germany, Italy, Canada, and Japan) contributed 58% of total publications during the study period and five of the G7 countries are ranked among the top ten countries. The domination of the G7 countries has occurred in most of the research fields [@pone.0081094-Dong1]. The top ten most productive countries on nanotribology research during the period 1996--2010 are ranked and listed in [Table 5](#pone-0081094-t005){ref-type="table"}. The rankings are based on total publications including single country papers, international collaborative papers, and h-index. The USA published the most papers (n = 330.67) and is ranked top in terms of single country papers, international collaborative papers and received the highest h-index of 44. China ranked second in terms of total publications and single country papers. However, China ranked third in terms of international collaborative papers and fourth in h-index (h = 17). Germany ranked second in terms of h-index, while it is ranked fourth in terms of total publications.
10.1371/journal.pone.0081094.t005
###### Top ten most productive countries and their rank.
{#pone-0081094-t005-5}
Country TP R(TP %) R(SCP %) R(ICP %) \% C CPP R(h)
------------- -------- ----------- ----------- ----------- ------- ------- --------
USA 330.67 1 (25.03) 1 (25.97) 1 (18.43) 8.97 15.91 1 (44)
China 252.5 2 (19.11) 2 (20.28) 3 (10.87) 6.93 4.38 4 (17)
Japan 201 3 (15.22) 3 (17.00) 12 (2.48) 1.99 4.54 5 (16)
Germany 108.5 4 (8.21) 4 (7.68) 2 (12.11) 17.97 8.28 2 (20)
UK 59 5 (4.47) 5 (3.88) 4 (8.70) 23.73 10.17 3 (18)
South Korea 43.33 6 (3.28) 6 (3.36) 11 (2.69) 9.99 11.57 3 (18)
France 38.33 7 (2.9) 7 (2.76) 7 (3.93) 16.51 10.04 5 (16)
Taiwan 32.67 8 (2.47) 8 (2.76) 25 (0.42) 2.05 6.00 7 (11)
Switzerland 29.67 9 (2.25) 9 (1.47) 5 (7.87) 42.7 10.28 6 (15)
Poland 20.5 10 (1.55) 10 (1.38) 10 (2.80) 21.95 2.17 8 (5)
TP = total papers, R = rank, SCP = single country papers, ICP = international collaborative papers, % C = percent of ICP in its total papers, CPP = citations per paper, h = h-index.
[Figure 1](#pone-0081094-g001){ref-type="fig"} provides the collaboration network (Ucinet) among the top ten countries. It can be seen that collaborations among the top ten countries were frequent. The exception is Taiwan, which is not integrated in this network of the other top ten countries.
Top Ten Most Productive Institutes {#s3d}
----------------------------------
There were around 680 institutions worldwide involved in the 1321 publications during 1996--2010. Of the total of 1321 publications, 846 (64%) were single institute publications and the remaining 475 (36%) were inter-institutionally collaborated publications. The performance of the top ten most productive institutes was examined and is presented in [Table 6](#pone-0081094-t006){ref-type="table"}. The top ten institutes published 21% of all papers. Even though the UK, France, Taiwan, Switzerland and Poland were ranked among the top ten countries, they had no institute among the top ten in [Table 6](#pone-0081094-t006){ref-type="table"}. Of the top 10 most productive institutes, four are in the USA, two each in China and Japan, and one each in South Korea and Germany. Tsinghua University of China ranked top in terms of number of publications, but had the second-lowest value for the h-index. Ohio State University of the USA ranked second in terms of number of publications and had the highest h-index of 36. Among the top most productive institutes, Ilmenau University of Technology, Germany, and University of California, USA published the highest percentages of papers with inter-institutional collaboration in their total publications, at 46% and 37% respectively. Ohio State University, Iowa State University, University of California and University of Illinois originate from the USA and produced 33% of their country's total output. Apart from the academic and research institutions, the following corporate bodies (among others) were involved in nanotribology research: Hitachi Ltd., Micro Materials Ltd., Falex Tribology NV, Caterpillar Inc., Ford Motor Company, Kao Corporation, and IBM.
10.1371/journal.pone.0081094.t006
###### Most productive institutes.
{#pone-0081094-t006-6}
Institute TP (R) IICP \% IICP CPP h
--------------------------------------------------------- ------------ ------- --------- ------- ----
Tsinghua University (China) 50.50 (1) 9.49 19 3.17 8
Ohio State University (USA) 50.42 (2) 5.42 11 29.99 36
Lanzhou Institute of Chemical Physics, CAS (China) 31.33 (3) 5.33 17 10.18 15
Nagoya University (Japan) 29.33 (4) 2.33 8 4.45 8
Iowa State University (USA) 22.50 (5) 4.5 20 7.87 11
Ilmenau University of Technology (Germany) 22.24 (6) 10.24 46 10.06 13
University of California (USA) 20.74 (7) 7.74 37 34.32 29
Nippon Institute of Technology (Japan) 19 (8) 2 11 2.55 5
Korea Institute of Science and Technology (South Korea) 17.83 (9) 5.83 33 13.54 15
University of Illinois (USA) 15.58 (10) 2.58 17 12.15 13
TP = total papers, R = rank, IICP = inter-institutionally collaborated papers, % IICP = percent of IICP in its total papers, CPP = citations per paper, h = h-index.
Core Journals {#s3e}
-------------
Journals which published at least 20 papers related to nanotribology research during 1996--2010 are listed in [Table 7](#pone-0081094-t007){ref-type="table"}. Ten journals published 20 or more papers and these journals published 33% of all papers. About 45% of all citations were received by the papers published in the top ten journals. *Tribology Letters* (n = 130) was the top journal by publication output, followed by *Wear* (n = 66) and the *Journal of Japanese Society of Tribologists* (n = 51). *Langmuir* had the highest impact with CPP of 29.63 and *Wear* received the highest h-index of 32 among the top ten journals. The *Journal of Japanese Society of Tribologists* ranked third in terms of total papers and it received the second-lowest h-index of 3. These core journals are in the subject areas of physics, materials science and engineering.
10.1371/journal.pone.0081094.t007
###### Top ten most preferred journals.
{#pone-0081094-t007-7}
Journal TP (R) CPP h
----------------------------------------------- --------- ------- ----
*Tribology Letters* 130 (1) 12.38 27
*Wear* 66 (2) 22.06 32
*Journal of Japanese Society of Tribologists* 51 (3) 0.61 3
*Tribology International* 42 (4) 9.79 16
*Surface and Coatings Technology* 35 (5) 11.14 16
*Langmuir* 30 (6) 29.63 30
*Mocaxue Xuebao/Tribology* 26 (7) 2.50 5
*Japanese Journal of Tribology* 22 (8) 0.09 1
*Applied Surface Science* 21 (9) 8.62 12
*Thin Solid Films* 21 (9) 18.14 19
TP = total papers, R = rank, CPP = citations per paper, h = h-index.
Most Highly Cited Papers {#s3f}
------------------------
The characteristics of highly cited papers (the 1% most highly cited papers) are listed in [Table 8](#pone-0081094-t008){ref-type="table"} among the papers related to nanotribology research during 1996--2010. Citations received by the 13 top cited papers accumulated to 1503 (12%) of all citations. Of the 13 papers, 3 have a single author while the others have more than one author. Twelve most cited papers are single country papers and originated from the USA (10), UK (1) and Belgium (1). The most cited papers were published in ten different journals. The top cited paper was "Surface engineering and microtribology for microelectromechanical systems" authored by Komvopoulos K. from the USA and published in *Wear* in 1996. In this paper, the analysis of various surface micromechanisms, such as solid bridging, liquid meniscus formation, van der waals force, and electrostatic charging and the significance of surface roughness and material properties are emphasized. *Wear* comes under the subject categories of engineering and materials science.
10.1371/journal.pone.0081094.t008
###### Characteristics of highly cited papers.
{#pone-0081094-t008-8}
Paper TC CPY Country of origin
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ----- ------- -------------------
Komvopoulos K. (1996). Surface engineering and microtribology for microelectromechanical systems.*Wear,* 200 (1--2): 305--327. 263 16.44 USA
Xu S., Miller S., Laibinis P. E., Liu G. - Y. (1999). Fabrication of nanometer scale patterns within self-assembledmonolayers by nanografting. *Langmuir,* 15 (21): 7244--7251. 163 12.54 USA
Sheehan P. E., Lieber C. M. (1996). Nanotribology and nanofabrication of MoO3 structures by atomic forcemicroscopy. *Science,* 272 (5265): 1158--1161. 134 8.38 USA
Liu H., Bhushan B. (2003). Nanotribological characterization of molecularly thick lubricant films for applicationsto MEMS/NEMS by AFM. *Ultramicroscopy,* 97 (1--4): 321--340. 123 13.67 USA
Johnson K. L. (1997). Adhesion and friction between a smooth elastic spherical asperity and a plane surface.*Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences,* 453 (1956): 163--179. 123 8.20 UK
Enachescu M., Van Den Oetelaar R. J. A., Carpick R. W., Ogletree D. F., Flipse C. F. J., Salmeron M. (1998).Atomic force microscopy study of an ideally hard contact: The diamond(111)/tungsten carbide interface.*Physical Review Letters,* 81 (9): 1877--1880. 98 7.00 USA, Netherlands
Bhushan B., Liu H. (2001). Nanotribological properties and mechanisms of alkylthiol and biphenyl thiolself-assembled monolayers studied by AFM. *Physical Review B - Condensed Matter and Materials Physics,*63 (24): 5412-1-5412-11. 96 8.73 USA
Liu H., Bhushan B. (2002). Investigation of nanotribological properties of self-assembled monolayerswith alkyl and biphenyl spacer chains (Invited). *Ultramicroscopy,* 91 (1--4): 185--202. 89 8.90 USA
Szlufarska I., Chandross M., Carpick R. W. (2008). Recent advances in single-asperity nanotribology.*Journal of Physics D: Applied Physics,* 41 (12): 123001. 88 22.00 USA
Kim S. H., Asay D. B., Dugger M. T. (2007). Nanotribology and MEMS. *Nano Today,* 2 (5): 22--29. 85 17.00 USA
Liu E., Blanpain B., Celis J. P. (1996). Calibration procedures for frictional measurements with a lateralforce microscope. *Wear*, 192 (1--2): 141--150. 83 5.19 Belgium
Tsukruk V. V., Everson M. P., Lander L. M., Brittain W. J. (1996). Nanotribological properties of compositemolecular films: C60 anchored to a self-assembled monolayer. *Langmuir*, 12 (16): 3905--3911. 80 5.00 USA
Bhushan B. (2007). Nanotribology and nanomechanics of MEMS/NEMS and BioMEMS/BioNEMS materialsand devices. *Microelectronic Engineering*, 84 (3): 387--412. 78 15.60 USA
TC = total citations, CPY = citations per year.
Authorship Pattern {#s3g}
------------------
Authorships vary from single to a maximum of 13 authors in the field of nanotribology research during the period 1996--2010. It can be observed from [Table 9](#pone-0081094-t009){ref-type="table"} that the highest percentage of contributions (22%) was made by three authors, followed by four authors and two authors, with 21% and 20% respectively. There was one paper with the highest number of authors (n = 13) which received the highest CPP of 32 among the authorships. Around 80% of the publications were contributed within a range of authors between 2 and 5. Only 11% of publications were contributed by a single author.
10.1371/journal.pone.0081094.t009
###### Authorship pattern and its citation impact.
{#pone-0081094-t009-9}
\# authors \# papers \% TC CPP
------------ ----------- ----- ------- -------
1 141 11 1247 8.84
2 266 20 2799 10.52
3 294 22 2413 8.21
4 272 21 2387 8.78
5 195 15 1443 7.40
6 90 7 1054 11.71
7 30 2 290 9.67
8 18 1 186 10.33
9 11 1 39 3.55
10 2 0 18 9.00
12 1 0 5 5.00
13 1 0 32 32.00
Total 1321 100 11913
TC = total citations, CPP = citations per paper.
Co-authorship Network {#s3h}
---------------------
There were 2581 authors involved in the total of 1321 papers in the field of nanotribology during 1996--2010. This shows that the research in this area was well diffused with many authors. The co-authorship network in [Figure 2](#pone-0081094-g002){ref-type="fig"} was visualized using the Sci^2^ tool. Each node represents one author and the size of the node denotes the number of papers. The thickness of interconnecting lines denotes the number of co-authored papers. Authors with a significant number of papers can be identified from the visualization map, which indicates that the trio of Fukuzawa K., Zhang H., and Mitsuya K. co-authored the most during the study period. Apart from this trio, there exist significant links between Schaefer J. A. and Scherge M., Miyake S. and Watanabe S., Bhushan B. and Liu H. as well as Dress D. and Achanta S. The visualization map indicates that most author pairs have not co-authored with the same intensity as this trio of Fukuzawa K., Zhang H., and Mitsuya K.
[Table 10](#pone-0081094-t010){ref-type="table"} provides the general properties of the co-authorship network, which indicates that only 0.18% of all possible edges (co-authorships) are present during the study period. This percentage indicates that the observed network is not dense: the level of cooperation between the scientists in this research field seems to be low [@pone.0081094-Erman1].
10.1371/journal.pone.0081094.t010
###### Properties of the co-author network.
{#pone-0081094-t010-10}
Sl Description Values
---- --------------------------- --------
1 Nodes (\# authors) 2581
2 Edges (\# co-authorships) 6156
3 Average degree 4.7702
4 Density 0.0018
K-core is the largest sub graph of a certain co-author network where nodes have at least k (here k = 10) interconnections. [Figure 3](#pone-0081094-g003){ref-type="fig"} provides a sub-graph of 25 out of 2581 nodes in [Figure 2](#pone-0081094-g002){ref-type="fig"}. These 25 authors have 10 or more co-author links during the study period.
Prolific Authors {#s3i}
----------------
[Table 11](#pone-0081094-t011){ref-type="table"} provides the rank list of the top ten authors in the field. The ranks are based on publication numbers (frequency) and h*-*index. The top ten authors published between 19 and 60 papers during the study period. A total paper (TP) of authors shows the amount of publication credit of a concerned author which is obtained by fractional counting method. These authors were from the USA, Japan, Germany, China, and Switzerland. Bhushan B., who is also prominently visible in the co-authorship network of [Figure 2](#pone-0081094-g002){ref-type="fig"}, was the most prolific author, with 60 papers, and had the highest h-index of 28. Carpick R. W. ranked fifth in terms of paper numbers and had the second-highest h*-*index (h = 16) after Bhushan B.
10.1371/journal.pone.0081094.t011
###### Prolific authors.
{#pone-0081094-t011-11}
Author Frequency (R) TP TC h (R)
---------------- --------------- ------- -------- --------
Bhushan B. 60 (1) 38.84 922.65 28 (1)
Wen S. 36 (2) 11.7 44.6 6 (5)
Fukuzawa K. 26 (3) 5.72 25.7 5 (6)
Zhang H. 26 (3) 5.61 21.57 4 (7)
Miyake S. 23 (4) 8.23 26.82 4 (7)
Mitsuya Y. 23 (4) 5.23 24.63 5 (6)
Schaefer J. A. 23 (4) 5.91 44.64 7 (4)
Ahmed S. I. U. 23 (4) 5.81 49.18 7 (4)
Scherge M. 19 (5) 5.92 90.95 11 (3)
Carpick R. W. 19 (5) 5.32 145.82 16 (2)
R = rank, TP = total papers, TC = total citations, h = h-index.
Research Trend {#s3j}
--------------
The nanotribology research trends can be obtained by analysing the author keywords appended to the research papers across different time periods [@pone.0081094-Zhang1]. Word Cluster Analysis [@pone.0081094-Li1] is applied to analyse the author keywords. In this method, words which have plural forms, abbreviations and their transformations are grouped into single keywords. 860 papers (65%) out of all papers included author keywords. Analysis of keywords during the study period revealed that 1608 author keywords were used. Among them, 1193 (74%) keywords appeared once and 182 keywords appeared twice. The large number of keywords which appeared only once indicates that there was a lack of continuity in research [@pone.0081094-ChuangK1]. Author keywords appended to the nanotribology research papers during 1996--2010 were ranked by total papers and five year block periods. [Table 12](#pone-0081094-t012){ref-type="table"} provides the frequency and rank of author keywords which appeared at least 22 times during the study period. Nanotribology/Microtribology, Atomic Force Microscope, Friction, Wear and Adhesion are the most popular keywords. Any one of the above keywords appeared in 951 papers (72%). 25% of all papers dealt with instruments like Atomic Force Microscope, Friction Force Microscope, and Scanning Probe Microscope. Nanoindentation is gaining popularity among the test methods. Friction, Wear, Lubrication and Adhesion are frequently used topics during the study period. Molecular Dynamics, MEMS, Hard Disk and Diamond like carbon are the only research topics in the keywords listed in [Table 12](#pone-0081094-t012){ref-type="table"} while the others are (nano)tribology related techniques and studies. Since these keywords moved up the ranks steadily from 1996--2000 to 2006--2010, they could be the major research topics in the future.
10.1371/journal.pone.0081094.t012
###### Frequently used author keywords.
{#pone-0081094-t012-12}
Keywords TP 1996-10 R (%) 96-00 R (%) 01--05 R (%) 06--10 R (%)
------------------------------ ----- --------------- ------------- -------------- --------------
Nanotribology/Microtribology 438 1 (33.2) 1 (31.2) 1 (28.6) 1 (36.6)
Atomic Force Microscope 198 2 (15.0) 2 (15.3) 2 (14.0) 2 (15.5)
Friction 166 3 (12.6) 3 (9.3) 3 (13.3) 3 (13.1)
Wear 82 4 (6.2) 4 (7.0) 4 (5.7) 4 (6.3)
Adhesion 67 5 (5.1) 5 (3.7) 8 (3.8) 5 (6.3)
Tribology 60 6 (4.5) 7 (2.8) 5 (5) 6 (4.8)
Diamond Like Carbon 54 7 (4.1) 11 (1.9) 6 (4.8) 7 (4.4)
Self-assembled Monolayers 44 8 (3.3) 32 (0.9) 10 (3.1) 8 (4.2)
Nanoindentation 43 8 (3.3) 19 (1.4) 7 (4.0) 10 (3.4)
MEMS 41 9 (3.1) 20 (1.4) 11 (2.9) 9 (3.8)
Molecular Dynamics 33 10 (2.5) 12 (1.9) 22 (1.4) 11 (3.4)
Boundary Lubrication 29 11 (2.2) 12 (1.9) 12 (2.6) 12 (2.0)
Thin Film 24 12 (1.8) 21 (1.4) 12 (2.6) 21 (1.5)
Hard Disk 23 13 (1.7) 62 (0.5) 15 (2.4) 18 (1.7)
Lubrication 22 14 (1.7) 8 (2.8) 18 (1.7) 23 (1.3)
Scanning Probe Microscope 22 14 (1.7) 14 (1.9) 9 (3.3) 61 (0.6)
TP = total papers, R = rank.
Discussion {#s4}
==========
This study identified the leading countries, institutes, authors, core journals in the field of nanotribology research and examined their citation impact. Average number of authors per paper was 3.52 which is similar to research on Tsunami (3.1%) [@pone.0081094-ChiuW1] and Bioinformatics (2.43) [@pone.0081094-PatraS1]. The 52% increase in collaboration of authors in nanotribology research can be seen as an expression of the field's development towards 'big science' [@pone.0081094-Glnzel1]. The share of non-cited papers is 30% in nanotribology research which is lower than in Acupuncture [@pone.0081094-FuJ1] where it was 38%. Moreover, Meho [@pone.0081094-MehoL1] estimated that around 90% of the papers published in academic journals are never cited. Papers with international collaboration in nanotribology research had more citation impact and increased visibility than national one. This result is in agreement with many other studies on other research fields. In nanotribology research, few authors (top 10 most productive) produced most of the papers (n = 278; 21%) and almost 12% of the most cited papers received 50% of all citations. This concentration of output and impact is also visible in many other research fields. The authors of highly cited papers in nanotribology research are not the same as the highly productive authors, which seems not to be consistent with Simonton's model of creative productivity [@pone.0081094-SimontonD1]. Core journals and journals publishing the highly cited papers in nanotribology research are from the subject areas of physics, chemistry, engineering, material science, biochemistry and computer science. This result shows the interdisciplinary nature of the research field.
Highly cited papers obtained through bibliometric analysis can be considered in collections of 'suggested readings' [@pone.0081094-LeeJ1] which may provide the outline of particular research fields (here: nanotribology).
Conclusion {#s5}
==========
The results of the present study on global nanotribology research output during 1996--2010 based on SCOPUS records explored key characteristics such as: growth rate of research, level of collaboration of publications, most publishing countries, most productive institutes, most preferred journals, citation impact, highly cited papers and research trend. The annual number of papers published grew tremendously from 34 in 1996 to 161 in 2010. Forty four countries engaged in nanotribology research during 1996--2010. Compared with other research fields like photosynthesis [@pone.0081094-YuJ1], where 156 countries were active, and nuclear waste management [@pone.0081094-Kumar1], where 140 countries were active, nanotribology seems to be a spatially concentrated research field. The USA published most of the (highly cited) papers. G7 countries contributed around 59% of the total papers. Tsinghua University, China, published the most papers. The top three journals were *Tribology Letters*, *Wear* and *Journal of Japanese Society of Tribologists*. Analysis of authorship pattern shows that co-authored publications dominate the research field. The visualization map indicates that the co-author network is not dense and only 25 authors (1%) have at least 10 co-author links during the study period. The results of this study could help the researchers (stakeholders) in the field of nanotribology as well as nanotechnology to understand the global research patterns and trends of nanotribology and to establish the future research directions.
There is one limitation of our study which should be addressed in future bibliometric studies on tribology or on other research topics. Since the nanotribology papers included in this study were attributes by Elsevier to several different subject areas in Scopus, field-normalized impact scores should actually be used to study research impact. The use of these scores is the standard in evaluative bibliometrics [@pone.0081094-Bornmann1], in which for example different universities are compared based on papers attributed to different subject areas in a data base. We abstained from using normalized citation scores in this study because we used the bibliometric methods in a non-evaluative context of only a single research topic.
We are grateful to Dr. Sriram Sundararajan, Associate Professor and Director of Undergraduate Education at Iowa State University, USA for recommendations on improving the manuscript. His research programme builds upon a fundamental framework in tribology (among other things). We would like to thank the academic editor and two anonymous reviewers for their valuable and fruitful comments on the earlier version of the manuscript. We used no current external funding sources for this study.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: BE PR. Performed the experiments: BE PR. Analyzed the data: BE PR. Contributed reagents/materials/analysis tools: BE PR. Wrote the paper: BE PR LB.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
The Dengue virus is a single-stranded RNA virus of the Flaviviridae family causing Dengue fever and dengue hemorrhagic fever. Encephalopathy is a very common neurological complication of dengue fever. Dengue encephalopathy is usually secondary to multisystem derangement like shock, hepatitis, coagulopathy, and concurrent bacterial infection.\[[@ref1]\] Dengue encephalitis is a different entity, which occurs due to direct neuronal infiltration by the dengue virus.\[[@ref2][@ref3]\] We report a case of dengue shock syndrome with encephalitis, with peculiar MRI findings.
Case Report {#sec1-2}
===========
A 17-year-old girl presented with a history of high-grade fever and rigors for three days. She had two episodes of vomiting, generalized headache, and altered sensorium, lasting for 15 to 20 minutes, few hours prior to admission. There was no significant past medical history.
On admission she was febrile, with a temperature of 101°F, pulse of 132 / minute, blood pressure of 90 / 40 mmHg (MAP-60 mmHg) and cold extremities. On pulse oximetry, oxygen saturation was 100% on breathing room air. There were no signs of respiratory distress. She was pale, and skin rash, edema, icterus, and clubbing were absent. There was no evidence of mucosal bleeding.
The neurological examination did not reveal any significant abnormality. There was no neck stiffness. The rest of the systemic examination was also within normal limits. There was no hepatosplenomegaly. She was resuscitated with fluids. First dose of third generation cephalosporin was given empirically in view of the possibility of bacterial infection. Empirical parenteral antimalarial preparation -- artesunate was also given, in view of the possibility of cerebral malaria.
Investigations revealed hemoglobin (Hb) of 14.5 g / dl, Packed Cell Volume (PCV) of 45, White Blood Cell count (WBC) of 7100 / cumm, with neutrophils 64%, lymphocytes 34%, and monocytes 2%. The platelet count was 1.35 lacs / cumm and ESR was 48 mm at the end of one hour. Malaria antigen rapid test was negative. The liver enzymes were raised SGOT-220 U / L, SGPT-182U / L. Serum albumin was 3.65 gm / dl, and INR was 2.0. Blood urea nitrogen was 39 gm / dl with creatinine of 1.4 mg / dl. The electrolytes were within normal range. Serum ammonia was 23 mg/dl. There was evidence of metabolic acidosis with blood lactate 2.00 mmol / L on initial blood gas. No hypoxia or hypercapnia was seen; X- Ray chest and 2 D echocardiography were normal. Ultrasonography (USG) showed evidence of bilateral minimal pleural effusion with mild ascites.
The patient\'s blood pressure and urine output improved after initial resuscitation with crystalloids. A few hours later, this young girl had an episode of generalized tonic-clonic seizure with unresponsiveness. She was treated with lorazepam and fosphenytoin. She was intubated and ventilated in view of the poor level of consciousness after the seizure.
An MRI brain scan was done as an emergency. It showed \[[Figure 1](#F1){ref-type="fig"}\] extensive parenchymal hyperintense lesions in the bilateral cerebellar cortex, vermis of the cerebellum, entire pons and midbrain, bilateral medial temporal lobes, and both thalami, on T2 and Fluid attenuated inversion recovery (FLAIR) images. These lesions look hypointense on T1W images, hyperintense on FLAIR images, and dark on the ADC map. Small foci of hemorrhage in the thalamic lesions looking hypointense, were seen on the T2 GRE images. These features were suggestive of Japanese Encephalitis (JE). She was started on acyclovir with the possibility of Herpes simplex encephalitis.
{#F1}
Cerebrospinal fluid (CSF) analysis was done. The findings are given in [Table 1](#T1){ref-type="table"}.
######
CSF analysis report
IgG / IgM antibodies for Dengue were positive in the serum as also on the DNA polymerase chain reaction (PCR) for Dengue. CSF analysis was positive for dengue IgM antibodies. Virus isolation was not possible.
IgM antibodies for Leptospira were negative. Chikungunya antibodies were negative. Titers for Hepatitis viruses were negative (HAV, HEV, HBsAg, HCV). Serum procalcitonin was 0.3 ng / ml (Negative less than 0.5 ng / ml). The blood and urine culture did not show any growth.
Her liver functions improved subsequently, and serum ammonia and electrolytes were within normal limits.
After the episode of seizure, her neurological status did not change. She remained comatose even after correction of shock, hepatitis, metabolic acidosis, and coagulopathy. The MRI brain findings were in favor of the possibility of direct extensive neuronal injury. Although the MRI brain findings were in favor of JE, CSF analysis was negative for JE. Therefore, the possibility of viral encephalitis due to dengue was strongly suspected. During the hospital course, she developed multiorgan dysfunction, shock, and acute respiratory distress syndrome (ARDS). She was ventilated according to the ARDS net protocol. She was started on steroids in view of the septic shock. Empirical antibiotics and antimalarials were continued till reports of the causative agent were obtained, and then withdrawn. Her coagulopathy was treated with fresh frozen plasma. Acyclovir was stopped on the basis of CSF analysis and MRI brain findings.
[Table 2](#T2){ref-type="table"} shows her investigations over the next 10 days.
######
Investigations
In spite of all the treatment she continued to remain comatose, with hemodynamic compromise, and expired on the eleventh day after admission.
Discussion {#sec1-3}
==========
Dengue virus infections are among the most common cause for hospital admissions in western Maharashtra. It is estimated that 50 to 100 million infections and 25,000 fatalities occur worldwide every year. The World Health Organization (WHO) surveillance shows that the global incidence is rising.\[[@ref4]\] Numerous neurological manifestations like transverse myelitis,\[[@ref2]\] myositis,\[[@ref5]\] and Gullian-Barre syndrome\[[@ref6]\] have been reported. Dengue encephalopathy is a well-recognized and common entity, the incidence ranging from 0.5 to 6.2 %.\[[@ref5]\] The possible mechanisms are liver failure (hepatic encephalopathy), cerebral hypoperfusion (shock), cerebral edema (vascular leak), deranged electrolytes, and intracranial bleeding due to thrombocytopenia or coagulopathy, which is secondary to hepatic failure.\_\[[@ref7]\] There are subsets of patients in whom the cause for neurological injury remains unclear even after excluding the above-mentioned indirect mechanisms. These raise the possibility of direct neuronal injury due to the dengue virus. Dengue is thought to be a non-neurotropic virus.\[[@ref3]\] However, there are reports of the demonstration of dengue virus and IgM antigen in the cerebrospinal fluid (CSF) of patients with encephalopathy. There are case reports by Misra *et al*., from India,\[[@ref5]\] and Solomon *et al*., from Vietnam.\[[@ref2]\] In the study described by Misra *et al*.,\[[@ref5]\] 11 patients were seen with confirmed dengue infection, but no CSF study was reported. Solmon *et al*.,\[[@ref2]\] diagnosed dengue encephalitis in nine patients, but virus or antibody was found in the CSF of only two cases. Kankirawatana *et al*.\[[@ref8]\] and Kularatne *et al*.,\[[@ref9]\] had a similar study in which they showed the association of dengue with encephalitis.
On admission our patient had a history suggestive of encephalopathy with shock, coagulopathy, metabolic acidosis, and deranged liver functions. This encephalopathy is usually explained by the abnormalities mentioned earlier in the text. However, subsequent evaluation with positive MRI and CSF showed evidence of encephalitis. This showed the co-existence of encephalopathy and encephalitis. The MRI findings noted in our case are most characteristic of JE and not commonly seen with dengue fever.\[[@ref10]\] The pattern of involvement. \[bilateral thalamic involvement with foci of hemorrhage, with involvement of temporal lobe and brain stem\] is very uncommon with dengue. There is only one similar case report by Kamble *et al*., with similar MRI findings.\[[@ref10]\] This case is presented to highlight the possible extensive involvement of the brain by dengue virus.
Conclusions {#sec1-4}
===========
Dengue is not classically a neurotropic virus, although there is recent evidence of direct neuronal injury. Dengue encephalitis must be thought of in differentials of encephalopathy, in patients with dengue. In such cases, neuroimaging and CSF analysis should be done whenever possible. The virus or antibody can be isolated from the serum, but the CSF samples may be negative. The dengue encephalitis is thought to be benign, but can be fatal at times. The role of an antiviral in such cases needs to be further defined because of the extensive parenchymal involvement and possible unfavorable outcome.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
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Background {#Sec1}
==========
With a mortality rate close to the incidence rate (331,000 deaths worldwide for 338,000 new cases in 2012 \[[@CR1]\]), pancreatic carcinoma is one of the most lethal human cancers. Advances in systemic chemotherapy and radiotherapy provided limited improvement in survival, and the 5-year overall survival (OS) remains close to 5%. Only 50% of newly diagnosed patients have a non-metastatic disease with either a resectable or borderline resectable tumor (20%) or an unresectable locally-advanced tumor (30%) \[[@CR2]\]. In patients with a resectable tumor, complete surgical removal followed by adjuvant chemotherapy is the only curative treatment. However, most of the patients display distant relapse; the median OS remains 23 months on average, and the 5-year survival is 20%. The mortality of surgery has decreased during the last 30 years, but its morbidity remains at approximately 50% \[[@CR3]\].
The high rates of patients with stage IV and experiencing distant relapses after surgery in non-metastatic stages along with preclinical data suggest that metastatic spread may precede local tumor formation \[[@CR4]\]. This has led to the emerging consensus that pancreatic cancer is a systemic disease already at diagnosis. More effective systemic therapies should confer an increased likelihood of cure after resection. Neoadjuvant chemotherapy, standardly used for borderline resectable and unresectable locally advanced diseases \[[@CR2]\], is being tested in resectable tumors with several objectives \[[@CR5]\], including early treatment of occult micrometastases, avoidance of unnecessary and morbid resection for rapidly metastasizing tumors, improvement of the likelihood of margin-negative resection, and better chemotherapy delivery than in adjuvant settings when surgical complications may delay or worsen chemotherapy tolerability. Other advantages include the ability to assess tumor response and to search for biological predictors for pathological response, which is associated with survival \[[@CR6], [@CR7]\]. Neoadjuvant chemotherapy provided interesting results in resectable pancreatic cancer in a few institutional prospective phase II studies \[[@CR8]--[@CR10]\], and randomized phase II/III studies are ongoing \[[@CR2]\]. However, this approach faces potential hurdles such as a possible missed opportunity for curative surgery and the absence of surgical staging. In this context, improving our ability to select patients for either immediate surgery or neoadjuvant chemotherapy is crucial, and represents an area of high need and intense research \[[@CR2]\].
The current prognostic factors are clinicopathological, notably based on the American Joint Committee on Cancer (AJCC) tumor, node and metastasis staging, and the criteria used for immediate surgery are technical (mainly based on the vascular involvement assessment), clinical (e.g., based on performance status), and biological (e.g., based on CA19-9 value). However, the criteria's ability to consistently predict a patient's outcome is limited, with substantial heterogeneity within the so-defined prognostic classes \[[@CR11]\]. Actually, no prognostic or predictive biomarker has yet been established for pancreatic cancer. High-throughput molecular analyses revealed the extensive heterogeneity of cancers, and notably pancreatic cancer. Key molecular alterations have been identified, such as *KRAS*, *TP53*, *SMAD4*, *CDKN2A*, and *ARID1A* mutations and *GATA6* amplification \[[@CR12], [@CR13]\], but they remain without clinical application to date. Several studies of gene expression profiling have also been reported \[[@CR14]\], mainly focused on the comparison of cancer versus normal pancreatic tissues. A few prognostic gene expression signatures have been developed \[[@CR15]--[@CR24]\], in general from small sample series and without validation in independent sets, or with validation in limited tumor sets. Biologically relevant molecular subtypes have been identified \[[@CR16], [@CR25], [@CR26]\], and associated with OS \[[@CR27]\]. However, identifying molecular predictors to aid in patient care remains necessary.
Here, we collected data of 695 pancreatic carcinoma samples from gene expression datasets, and searched for a gene expression signature predictive for post-operative OS.
Methods {#Sec2}
=======
Gene expression datasets {#Sec3}
------------------------
We retrospectively collected clinicopathological and gene expression data of clinical pancreatic carcinoma samples from nine publicly available datasets \[[@CR15], [@CR16], [@CR20], [@CR21], [@CR23], [@CR25], [@CR28]--[@CR30]\] from the National Center for Biotechnology Information/Genbank Gene Expression Omnibus, ArrayExpress, European Genome-phenome Archive, and The Cancer Genome Atlas (TCGA) databases (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Samples had been profiled using whole-genome DNA microarrays (Affymetrix or Agilent) and RNA-Seq (Illumina). The complete dataset contained 695 samples, including 601 operated primary cancer samples with available survival data. The study was approved by our institutional board.
Gene expression data analysis {#Sec4}
-----------------------------
Data analysis required pre-analytic processing. First, we normalized each DNA microarray-based dataset separately, by using quantile normalization for the available processed Agilent data, and Robust Multichip Average \[[@CR31]\] with the non-parametric quantile algorithm for the raw Affymetrix data. Normalization was performed in R using Bioconductor and associated packages. Then, we mapped hybridization probes across the different technological platforms. We used SOURCE \[[@CR32]\] and NCBI EntrezGene \[[@CR33]\] to retrieve and update the Agilent annotations, and NetAffx Annotation files \[[@CR34]\] for the Affymetrix annotations. The probes were then mapped according to their EntrezGeneID. When multiple probes represented the same GeneID, we retained the one with the highest variance in a particular dataset. For the TCGA, Bailey's and Kirby's data, we used the available normalized RNA-Seq data that we log~2~-transformed.
We defined the molecular subtypes of all pancreatic cancer samples in each dataset separately as defined in the original publications, i.e., the three Collisson's subtypes \[[@CR16]\] were classical, quasi-mesenchymal, and exocrine-like, the two Moffitt's epithelial subtypes \[[@CR26]\] were basal-like and classical, and the four Bailey's subtypes \[[@CR25]\] were squamous, pancreatic progenitor, immunogenic, and aberrantly differentiated endocrine exocrine (ADEX). To identify a prognostic expression signature, we applied a supervised analysis using learning and validation sets. The learning set was a subset (n = 39) of the Bailey's and TCGA RNA-Seq datasets that included samples from patients with survival of at least 36 months after surgery (long-term survivors (LTS); n = 17) and from patients dead of disease between 2 and 6 months after surgery (short-term survivors (STS); n = 22). The 562 other samples with available survival data from the other datasets were gathered and used as an independent validation set. Samples of the learning set were pooled before supervised analysis by using COMBAT (empirical Bayes), included in the inSilicoMerging R/Bioconductor package, as a batch effects removal method. The final merged set included 15,291 genes in log2-transformed data. The accuracy of normalization was controlled by principal component analysis (Additional file [2](#MOESM2){ref-type="media"}: Figure S1). The supervised analysis compared the expression profiles of 15,291 genes between the 22 STS samples and the 17 LTS samples using a moderated *t*-test with empirical Bayes statistic included in the Limma R packages. False discovery rate was applied to correct for the multiple testing hypothesis and significant genes were defined by the following thresholds: *P* \< 5%, false discovery rate \< 25%, and fold change superior to \|2x\|. Ontology analysis of the resulting 1400-gene list was based on the gene ontology (GO) biological processes of the Database for Annotation, Visualization and Integrated Discovery (DAVID) \[[@CR35]\]. We then developed a prognostic classifier while minimizing the number of retained genes. Starting from the resulting 1400-gene list, we used logistic regression analysis with Least Absolute Shrinkage and Selection Operator \[[@CR36]\] (LASSO), which is a selection method that handles high-dimensional regression variables with no prior feature selection step by shrinking all regression coefficients toward zero, and thus forcing many regression variables to be exactly zero. The penalty regularization parameter λ was chosen via the cross-validation routine *cv.glmnet* before running the main algorithm implemented in the R package *glmnet* version 1.9-8, with an n-fold equal to 10. The λ value was finalized by using the lambda.min, which is the value of lambda giving minimum mean cross-validated error (lambda.min was 0.0153). The resulting classifier allowed the definition of two classes of samples, namely the predicted STS-like class and the predicted LTS-like class. Its robustness was assessed in the independent validation set (n = 562) by classifying each sample in each dataset separately as STS-like or LTS-like. Since a few studies have indicated that many gene signatures were random noise signatures \[[@CR37], [@CR38]\], we evaluated whether our prognostic 25-gene signature was not inferior to random signatures. A resampling scheme was used to generate 100,000 random 25-gene signatures within the 1400 genes differentially expressed identified by supervised analysis in the learning set. Each random signature was then applied to the validation set to determine its significance level in prognostic terms for OS. We then measured the proportion of random signatures with a *P* value inferior to the *P* value from our 25-gene signature.
Statistical analysis {#Sec5}
--------------------
Associations between tumor groups and clinicopathological features were analyzed using the *t*-test or the Fisher's exact test when appropriate. Overall survival (OS) was calculated from the date of diagnosis to the date of death from pancreatic cancer. Follow-up was measured from the date of diagnosis to the date of last news for living patients. Survivals were calculated using the Kaplan--Meier method and were compared with the log-rank test. Uni- and multivariate survival analyses were performed using Cox regression analysis (Wald test). Variables tested in univariate analyses included patient age at time of diagnosis (\>60 vs. ≤ 60 years), sex (male vs. female), AJCC clinical stage (2, 3, and 4 vs. 1), pathological features including pathological type (others vs. ductal), tumor size (pT2, T3, and pT4 vs. T1), lymph node status (positive vs. negative), grade (2, 3, and 4 vs. 1), our 25-gene classification (STS-like vs. LTS-like), and the different molecular subtype classifications. Variables with a *P* value lower than 0.05 were tested in multivariate analysis. All statistical tests were two-sided at the 5% level of significance. Statistical analysis was performed using the survival package (version 2.30) in the R software (version 2.15.2) \[[@CR39]\]. We followed the reporting REcommendations for tumor MARKer prognostic studies (REMARK criteria) \[[@CR40]\]. A Sweave report describing the analysis of gene expression data and the associated statistical analysis is available as Additional file [3](#MOESM3){ref-type="media"} (Supplementary Text).
Results {#Sec6}
=======
Patient population {#Sec7}
------------------
We collected nine retrospective/prospective public whole-genome mRNA expression datasets of 695 pancreatic samples, and focused our analysis on the 601 cancer samples from patients operated from the outset and with available survival. As shown in Table [1](#Tab1){ref-type="table"}, the majority of patients were aged 60 years or older, and 54% were male. Most cases (96%) were AJCC stage 1 or 2, ductal type (98%), and grade 2 (55%). All but one case had been treated by front-line surgery, and the majority of tumors were pT2 (16%) or pT3 (77%), and pN-positive (69%). All Bailey's, Moffitt's, and Collison's molecular subtypes were represented. A total of 354 patients died. The median OS was 20 months (range, 1--156), and the 2-year OS was 40% (95% CI 36--45).Table 1Patient and tumor clinicopathological characteristics of 601 samplesCharacteristicsAll (*n* = 601)Age at diagnosis, years ≤ 60118 (32%) \> 60246 (68%)Sex Female170 (46%) Male197 (54%)AJCC Stage 162 (12%) 2431 (84%) 310 (2%) 412 (2%)Pathological type Ductal537 (98%) Other^a^11 (2%)Pathological grade 133 (12%) 2154 (55%) 391 (32%) 42 (1%)Pathological tumor size (pT) pT118 (5%) pT262 (16%) pT3302 (77%) pT411 (3%)Pathological lymph node status (pN) Negative141 (31%) Positive310 (69%)Collisson subtypes Classical234 (39%) Exocrine-like211 (35%) Quasi-mesenchymal156 (26%)Moffitt subtypes, 'type' Basal-like232 (39%) Classical369 (61%)Bailey subtypes ADEX140 (23%) Immunogenic104 (17%) Pancreatic progenitor142 (24%) Squamous215 (36%)Deceased354 (59%)2-year OS (95% CI)40% (36--45)Median OS, months (range)20 (1--156.4)^a^Other: 8 neuroendocrine tumors, 2 acinar cell carcinomas, 1 intraductal tubulopapillary neoplasm*ADEX* aberrantly differentiated endocrine exocrine, *AJCC* American Joint Committee on Cancer, *CI* confidence interval, *OS* overall survival
Identification of a prognostic expression signature {#Sec8}
---------------------------------------------------
We searched for a gene signature associated with OS. Supervised analysis was performed in a learning set of 39 samples selected to represent the two opposite groups of patients, including 17 LTS and 22 STS. Analysis identified 1400 genes differentially expressed between the two groups (Additional file [4](#MOESM4){ref-type="media"}: Table S2). All associated GO biological processes are shown in Additional file [5](#MOESM5){ref-type="media"}: Table S3, and the top 40 processes are shown in Table [2](#Tab2){ref-type="table"}. The robustness of those genes was tested by testing their ability to classify the LTS and STS samples from the other independent datasets. Out of the 67 samples classified, 49 (76%) were accurately classified, suggesting strong robustness (*P* = 7.68 × 10^--5^, Fisher's exact test).Table 2Top 40 gene ontology (GO) biological processes associated with the 1400 genes differentially expressed between the short-term survivor (STS) and long-term survivor (LTS) samples of the learning setGO:BP TermsIDGO:BP Terms*NP* valueStatus STS vs. LTSGO:0030198Extracellular matrix organization709.15 × 10^--26^UpGO:0007155Cell adhesion752.46 × 10^--22^UpGO:0022617Extracellular matrix disassembly352.70 × 10^--18^UpGO:0008544Epidermis development251.04 × 10^--17^UpGO:0030574Collagen catabolic process262.78 × 10^--17^UpGO:0006955Immune response501.44 × 10^--16^UpGO:0006954Inflammatory response522.34 × 10^--16^UpGO:0030199Collagen fibril organization196.76 × 10^--16^UpGO:0018149Peptide cross-linking101.23 × 10^--10^UpGO:0006935Chemotaxis222.34 × 10^--10^UpGO:0010951Negative regulation of endopeptidase activity236.65 × 10^--10^UpGO:0030216Keratinocyte differentiation143.24 × 10^--9^UpGO:0010466Negative regulation of peptidase activity81.47 × 10^--8^UpGO:0001501Skeletal system development222.51 × 10^--8^UpGO:0007160Cell-matrix adhesion191.30 × 10^--7^UpGO:0000278Mitotic cell cycle501.71 × 10^--7^UpGO:0008283Cell proliferation421.86 × 10^--7^UpGO:0031124Mrna 3-end processing6002.19 × 10^--7^UpGO:0008284Positive regulation of cell proliferation453.26 × 10^--7^UpGO:0001525Angiogenesis323.31 × 10^--7^UpGO:0019228Neuronal action potential61.26 × 10^--6^DownGO:0007409Axonogenesis121.13 × 10^--6^DownGO:0007628Adult walking behavior71.09 × 10^--6^DownGO:0007212Dopamine receptor signaling pathway57.83 × 10^--7^DownGO:0006906Vesicle fusion104.63 × 10^--7^DownGO:0030073Insulin secretion95.27 × 10^--8^DownGO:0007274Neuromuscular synaptic transmission65.20 × 10^--8^DownGO:0007399Nervous system development271.44 × 10^--8^DownGO:0014047Glutamate secretion95.89 × 10^--9^DownGO:0007626Locomotory behavior154.46 × 10^--9^DownGO:0086010Membrane depolarization during action potential81.71 × 10^--9^DownGO:0031018Endocrine pancreas development121.16 × 10^--9^DownGO:0017158Regulation of calcium ion-dependent exocytosis109.94 × 10^--10^DownGO:0006112Energy reserve metabolic process204.05 × 10^--11^DownGO:0017157Regulation of exocytosis131.60 × 10^--11^DownGO:0006813Potassium ion transport153.94 × 10^--12^DownGO:0071805Potassium ion transmembrane transport171.97 × 10^--13^DownGO:0016079Synaptic vesicle exocytosis174.00 × 10^--15^DownGO:0007269Neurotransmitter secretion197.04 × 10^--16^DownGO:0007268Synaptic transmission662.33 × 10^--37^Down
To render this signature more easily applicable in clinics, we built a multigene classifier from the 1400-gene list. Logistic regression analysis retained 25 genes (Table [3](#Tab3){ref-type="table"}), including 12 and 13 genes respectively upregulated and downregulated in the STS samples. As expected, the classifier based on these 25 genes sorted with 100% accuracy those 39 patients into two classes, with STS-like including all STS patients and LTS-like including all LTS patients.Table 3List of 25 genes included in our prognostic classifierSymbolDescriptionCytobandExpression statusGPR87G protein-coupled receptor 873q24Up STS vs. LTSKRT13keratin 13, type I17q21.2Up STS vs. LTSRAC2ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)22q13.1Up STS vs. LTSC16orf74chromosome 16 open reading frame 7416q24.1Up STS vs. LTSNAMPTnicotinamide phosphoribosyltransferase7q22.3Up STS vs. LTSDHRS9dehydrogenase/reductase (SDR family) member 92q31.1Up STS vs. LTSHIST2H2BFhistone cluster 2, H2bf1q21.2Up STS vs. LTSTREM2triggering receptor expressed on myeloid cells 26p21.1Up STS vs. LTSZDHHC20zinc finger, DHHC-type containing 2013q12.11Up STS vs. LTSCD180CD180 molecule5q12Up STS vs. LTSADGRG6adhesion G protein-coupled receptor G66q24.1Up STS vs. LTSAPBB1IPamyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein10p12.1Up STS vs. LTSEGR3early growth response 38p23-p21Down STS vs. LTSMACROD2MACRO domain containing 220p12.1Down STS vs. LTSEPHA7EPH receptor A76q16.1Down STS vs. LTSRASGEF1ARasGEF domain family, member 1A10q11.21Down STS vs. LTSSYNMsynemin, intermediate filament protein15q26.3Down STS vs. LTSS100A1S100 calcium binding protein A11q21Down STS vs. LTSWNK2WNK lysine deficient protein kinase 29q22.3Down STS vs. LTSRAMP2receptor (G protein-coupled) activity modifying protein 217q12-q21.1Down STS vs. LTSSOCS2suppressor of cytokine signaling 212qDown STS vs. LTSCOL28A1collagen, type XXVIII, alpha 17p21.3Down STS vs. LTSB4GALT6UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 618q11Down STS vs. LTSPLCB4phospholipase C, beta 420p12Down STS vs. LTSMTURNmaturin, neural progenitor differentiation regulator homolog (Xenopus)7p14.3Down STS vs. LTS
We assessed the gene overlap between our 25-gene signature and the three molecular subtype classifiers \[[@CR16], [@CR25], [@CR26]\] and five other signatures recently published that displayed robust and independent prognostic value \[[@CR15], [@CR17], [@CR20], [@CR22], [@CR30]\]. As shown in Additional file [6](#MOESM6){ref-type="media"}: Figure S2, there was no overlap with the five signatures, and the overlap with the molecular subtype classifiers was very low (0 gene with Collisson, 1 with Moffitt stroma, 2 with Moffitt tumor, and 3 with Bailey).
Validation of the 25-gene classifier and clinicopathological associations {#Sec9}
-------------------------------------------------------------------------
We tested the 25-gene prognostic classifier in the independent validation set of 562 patients whose clinicopathological characteristics were close to those of the learning set (Additional file [7](#MOESM7){ref-type="media"}: Table S4) and with a 2-year OS of 39% (95% CI 35--44; Fig. [1a](#Fig1){ref-type="fig"}). The classifier sorted the 562 patients into two classes, STS-like (*n* = 216; 38%) and LTS-like (*n* = 346; 62%), with a 2-year OS of 25% (95% CI 18--33) and 48% (95% CI 42--54), respectively (*P* = 4.33 × 10^--9^, log-rank test; Fig. [1b](#Fig1){ref-type="fig"}), thus confirming its prognostic value. The respective median OS were 15 months (range, 1--104) and 23 months (range, 1--156). Interestingly, in each of the nine datasets separately, the 2-year OS was shorter in the STS-like class than in the LTS-like class, and the difference was or tended to be significant (Additional file [8](#MOESM8){ref-type="media"}: Figure S3). To assess the likelihood of our 25-gene signature as a non-random signature, we generated by a resampling scheme 100,000 random gene signatures from the list of 1400 genes differentially expressed and tested their prognostic value in the validation set. None of the random signatures was more significant than the data-derived 25-gene signature, suggesting that the latter represented an optimal prognostic combination.Fig. 1Overall survival (OS) in the validation set according to our prognostic 25-gene classifier. Kaplan--Meier OS curves in all patients (**a**) and in all patients according to our prognostic classifier (STS-like and LTS-like classes) (**b**). The *P* values of the log-rank test are indicated
We searched for associations between the 25-gene signature-based classification and the clinicopathological variables of samples. As shown in Table [4](#Tab4){ref-type="table"}, no association was found with patient age and sex, AJCC stage, pathological type, tumor size, and lymph node status. By contrast, there were more grade 3 tumors (*P* = 1.50 × 10^--3^) in the STS-like class, and more aggressive molecular subtypes (Baileys' squamous, Collison' quasi-mesenchymal, and Moffitt's basal-like; *P* \< 0.05).Table 4Associations of our prognostic classification with clinicopathological features (validation set)Characteristics*n*LTS-like (*n* = 346)STS-like (*n* = 216)*P* valueAge at diagnosis0.219 ≤ 6010864 (31%)44 (38%) \> 60217145 (69%)72 (62%)Sex0.419 Female157105 (50%)52 (44%) Male171106 (50%)65 (56%)AJCC stage0.759 15435 (12%)19 (11%) 2403255 (84%)148 (85%) 3105 (2%)5 (3%) 4118 (3%)3 (2%)Pathological type0.087 Ductal504308 (98%)196 (100%) Other66 (2%)0 (0%)Pathological grade1.50 × 10^--3\*^ 12724 (16%)3 (3%) 213890 (59%)48 (54%) 37538 (25%)37 (42%) 421 (1%)1 (1%)Pathological tumor size (pT)0.879 pT1159 (4%)6 (5%) pT25736 (16%)21 (16%) pT3281181 (78%)100 (76%) pT4116 (3%)5 (4%)Pathological lymph node status (pN)0.824 Negative12379 (30%)44 (29%) Positive291183 (70%)108 (71%)Collisson subtypes1.00 × 10^--6\*^ Classical223130 (38%)93 (43%) Exocrine-like194147 (42%)47 (22%) Quasi-mesenchymal14569 (20%)76 (35%)Moffitt subtypes, 'type'7.80 × 10^--15\*^ Basal-like21488 (25%)126 (58%) Classical348258 (75%)90 (42%)Bailey subtypes1.00 × 10^--6\*^ ADEX128114 (33%)14 (6%) Immunogenic10169 (20%)32 (15%) Pancreatic progenitor13390 (26%)43 (20%) Squamous20073 (21%)127 (59%)2-year OS (95% CI)56248% (42--54)25% (0.18--0.33)4.33 × 10^--9\*^Median OS, months (range)56222.8 (1--156.4)15.0 (1--103.92)*ADEX* aberrantly differentiated endocrine exocrine, *CI* confidence interval, *LTS* long-term survivors, *STS* short-term survivors, *OS* overall survival\*Statistically significant
Uni- and multivariate prognostic analyses {#Sec10}
-----------------------------------------
We compared the prognostic value of our 25-gene classifier with that of other clinicopathological variables in the validation set. In univariate analysis (Table [5](#Tab5){ref-type="table"}), three variables were associated with OS (Wald test), namely the AJCC clinical stage (*P* = 4.71 × 10^--3^), the pathological pN status (*P* = 1.24 × 10^--4^), and our 25-gene classifier (*P* = 7.47 × 10^--9^). The hazard ratio (HR) for death was 1.93 (95% CI 1.55--2.42) in the STS-like vs. LTS-like classes. In multivariate analysis, only our classifier (*P* = 6.33 × 10^--7^) and the pN status (*P* = 2.95 × 10^--2^) remained significant, suggesting an independent prognostic value. The stratification of patients according to both the classifier and the AJCC stage identified classes with different 2-year OS (Additional file [9](#MOESM9){ref-type="media"}: Figure S4). For example, in patients with stage 1 tumor, the 2-year OS was 42% in the STS-like class (42%) and 73% in the LTS-like class (*P* = 6.74 × 10^--3^, log-rank test). Stage 2 patients were similarly subdivided into STS- and LTS-like with a 21% and 46% 2-year OS (*P* = 4.37 × 10^--7^, log-rank test), respectively.Table 5Uni- and multivariate Cox regression analyses for overall survival (validation set)CharacteristicsUnivariateMultivariateMultivariate*N*HR (95% CI)*P* value*N*HR (95% CI)*P* value*N*HR (95% CI)*P* valueAge at diagnosis\>60 vs. ≤ 603251.22 (0.88--1.70)0.234SexMale vs. female3281.08 (0.80--1.45)0.633AJCC Stage2 vs. 14782.01 (1.32--3.07)4.71 × 10^--3\*^4081.57 (0.88--2.82)0.1283 vs. 13.11 (1.33--7.23)4082.21 (0.82--5.97)0.1194 vs. 12.85 (1.16--7.05)4081.44 (0.19--10.97)0.723Pathological typeOther vs. ductal5100.36 (0.09--1.45)0.151Pathological grade2 vs. 12421.52 (0.65-- 3.55)0.1853 vs. 12.15 (0.91-- 5.11)4 vs. 12.66 (0.53--13.3)Pathological tumor size (pT)2 vs. 13641.49 (0.62--3.59)0.1313 vs. 11.95 (0.86--4.42)4 vs. 12.93 (1.01--8.48)Pathological lymph node status (pN)1 vs. 04141.83 (1.34--2.48)1.24 × 10^--4\*^4081.50 (1.04--2.16)2.95 × 10^--2\*^Collisson subtypesExocrine-like vs. classical5621.00 (0.77--1.29)2.32 × 10^--3\*^5620.94 (0.66--1.34)0.732Quasi-mesenchymal vs. classical1.52 (1.17--1.99)5621.15 (0.83--1.59)0.395Moffitt subtypes, 'type'Classical vs. basal-like5620.64 (0.51--0.80)6.29 × 10^--5\*^5621.00 (0.72--1.38)0.994Bailey subtypesImmunogenic vs. ADEX5620.81 (0.57--1.17)8.98 × 10^--6\*^5620.68 (0.43--1.06)0.090Pancreatic progenitor vs. ADEX0.97 (0.70--1.35)5620.79 (0.51--1.23)0.302Squamous vs. ADEX1.64 (1.22--2.19)5621.09 (0.68--1.74)0.73125-gene classifierSTS-like vs. LTS-like5621.93 (1.55--2.42)7.47 × 10^--9\*^4082.04 (1.54--2.70)6.33 × 10^--7\*^5621.77 (1.38--2.26)6.33 × 10^--6\*^*ADEX* aberrantly differentiated endocrine exocrine, *CI* confidence interval, *HR* hazard ration, *LTS* long-term survivors, *STS* short-term survivors\*Statistically significant
Given the association between the molecular subtypes and the 25-gene classifier, we compared their respective prognostic performance. In univariate analysis, the three molecular subtype classifiers confirmed their prognostic value in this large sample set (Additional file [10](#MOESM10){ref-type="media"}: Figure S5). However, in multivariate analysis including the four multigene classifiers, only our 25-gene classifier remained significant (*P* = 6.33 × 10^--6^, Wald test, Table [5](#Tab5){ref-type="table"}) with a HR of 1.77 (95% CI 1.38--2.26). As shown in Fig. [2](#Fig2){ref-type="fig"}, it affected the clinical outcome of all molecular subtypes of all three classifications, except the Bailey's progenitor subtype.Fig. 2Overall survival (OS) in the validation set according to our prognostic 25-gene classifier and the molecular subtypes. Kaplan--Meier OS curves according to our prognostic classifier (STS-like and LTS-like classes) and the molecular subtypes defined by Bailey (**a** pancreatic progenitor; **b** immunogenic; **c** ADEX; **d** squamous), Collison (**e** exocrine-like; **f** classical; **g** quasi-mesenchymal), and Moffitt (**h** classical; **i** basal-like). The *P* values (log-rank test) for the comparison between the two classes within each molecular subtype are indicated
Discussion {#Sec11}
==========
Pancreatic carcinoma is a heterogeneous disease with high metastatic propensity and poor prognosis. In patients with resectable disease, the development of effective systemic therapies is crucial. During the last decades, several retrospective studies \[[@CR41]\] and a few prospective phase II studies \[[@CR8]--[@CR10]\] have suggested the potential benefit of neoadjuvant chemotherapy, and large randomized phase II/III trials are ongoing. In this context, a major challenge is to improve the imperfect current prognostic factors to aid in therapeutic decision-making, notably regarding the decision for immediate surgery followed by chemotherapy or neoadjuvant chemotherapy followed by surgery. Here, we have analyzed whole-genome expression profiles of 601 pancreatic carcinoma samples from operated patients, and identified a robust 25-gene classifier associated with post-operative OS independently of classical prognostic factors and molecular subtypes. To our knowledge, this study is by far the largest prognostic study of gene expression profiles in pancreatic carcinoma.
Gene expression profiling remains today the most promising and successful high-throughput molecular approach to identify new prognostic tools in early-stage cancers. Multigene signatures are already marketed, such as Oncotype™ in breast cancer or Coloprint™ in colon cancer, yet no similar signature is available in pancreatic carcinoma. The paucity of tumor specimens available for analysis explains the relatively small number of samples profiled in previous prognostic studies, with 102 samples in the largest one \[[@CR20]\] to use supervised analysis, and 328 in the Australian ICGC study \[[@CR25]\], which identified prognostic molecular subtypes by unsupervised analysis. We overcame the problem by pooling nine public datasets, representing a total of 601 operated primary cancers with available follow-up, and allowing the use of a learning set and a validation set in the supervised analysis. Our series displayed classical clinicopathological characteristics and poor prognosis with a 40% 2-year OS. The learning set, which included only 39 samples, was remarkably small compared with the validation set; this might have reduced our ability to capture the best genes for the classifier. However, it was carefully designed to contain two groups with distinct aggressiveness, namely a LTS group after surgery and a STS group, and to contain samples profiled using the same technology (RNA-Seq). Such design likely explains the large number of genes (1400) differentially expressed between the two patient groups despite the correction for the multiple testing hypothesis, and the robustness of our final signature in the validation set. A similar design had been used previously \[[@CR20]\] by comparing primary tumors from metastatic versus non-metastatic patients. The size of our series allowed testing of the classifier in a large independent validation set of 562 samples with multivariate analysis and increased statistical power. For comparison, the other prognostic expression signatures published to date in pancreatic cancer \[[@CR15]--[@CR24]\] were defined in learning sets including 6--70 clinical samples, then tested in validation sets including 67--246 samples, with inconstant multivariate analysis.
We first identified 1400 genes differentially expressed between the STS and LTS samples. From this gene list, a 25-gene classifier was developed, identifying two classes, namely STS-like and LTS-like. The prognostic value was verified in the independent validation set, in which the two classes, STS-like (38% of samples) and LTS-like (62%), showed a different 2-year OS (25% in the STS-like and 48% in the LTS-like). Interestingly, and by contrast to the other published studies in the field, this prognostic value existed in each of the nine datasets considered separately. As expected, the other variables significant in univariate analysis included the AJCC stage and the pathological lymph node status. The pathological type (other vs. ductal) was not significant (HR 0.36 with *P* = 0.151) because of the small percentage (1%) of "other" types. Interestingly, all six "other" type samples were classified in the LTS-like class, in agreement with the better prognosis of neuroendocrine tumors. Importantly, the OS analysis was not modified when limited to the 504 ductal samples with a 27% 2-year OS in the STS-like and 48% in the LTS-like. Our 25-gene classifier displayed an independent prognostic value. Interestingly, it outperformed the molecular subtypes in multivariate analysis and identified patients with shorter and longer survival in all subtypes but one, highlighting substantial heterogeneity in each of them. None of the 100,000 25-gene signatures randomly generated by a resampling scheme was more significant than the data-derived 25-gene signature, suggesting that this latter represented a non-random optimal prognostic combination.
Ontology analysis of the 25 genes revealed interesting pathways, such as pathways related to the metastatic process (extracellular matrix organization and disassembly, cell and cell-matrix adhesion), local inflammation (immune and inflammatory responses, chemotaxis), and cell proliferation (mitotic cell cycle, positive regulation of proliferation) associated with the "poor-prognosis genes". Pathways associated with the "good-prognosis genes" included those related to pancreas metabolism (endocrine pancreas development, energy reserve metabolic process, insulin secretion) or synaptic connections (synaptic transmission and vesicle exocytosis, membrane depolarization during action potential). Whether the 25 classifier genes are causative of the phenotype in a biological sense or reflect another associated phenomenon remain to be explored. However, it was interesting to find some genes already reported as associated with cancer biology and/or to the clinical outcome of cancer patients. Among the genes upregulated in STS, *GPR87*, *RAC2*, *NAMPT*, *C16orf74*, *TREM2*, and *CD180* are involved in NF-~K~B-mediated cell signaling, and *KRT13*, *RAC2*, *C16orf74*, *ADGRG6*, and *APBB1IP* in epithelial--mesenchymal transition. These two pathways are frequently affected in pancreatic ductal adenocarcinoma (PDAC) \[[@CR42], [@CR43]\]. Activation of the NF-~K~B signaling pathway plays an important role in the development and progression of disease and impacts the epithelial--mesenchymal transition, chemoresistance, migration, and invasion of pancreatic cancer cells \[[@CR42], [@CR44]--[@CR46]\]. The NF-~K~B activation pathway picked by our signature might not necessarily be related to tumor cells themselves. Stromal cells can modulate their activation status through NF-~K~B, based on the signals collected from their environment. TREM2 and CD180 are negative regulators of the Toll-like receptor pathway \[[@CR47]\], a family of receptors that recognize damage-associated molecule patterns, whose increased serum levels have been associated with cancer \[[@CR48]\]. Inhibition of Toll-like receptors results in impaired immediate host defensive responses and anti-tumor response mounting. TREM2 and CD180 are also part of the conventional markers used to describe "alternatively" activated M2 macrophages. M2 macrophages promote angiogenesis, tissue remodeling and repair, thus facilitating tumor progression and invasion, and their presence is correlated with poor prognosis in several cancers, including PDAC \[[@CR49], [@CR50]\]. Identifying molecules that modulate some specific "activation nodes" of the wide NF-~K~B signaling pathway could be interesting for pancreatic cancer therapy. Two other genes related to NF-~K~B activation are *GPR87* and *NAMPT*, and represent potential therapeutic targets. *GPR87* is overexpressed in various cancers, including pancreatic cancer cells and tissues, and its overexpression correlates with shorter OS \[[@CR51]\]. GPR87 enhances pancreatic cancer aggressiveness by activating the NF-~K~B signaling pathway, and plays a role in tumor cell survival \[[@CR52], [@CR53]\] and the regulation of TP53 \[[@CR54]\]. Antagonists of GPR87 are in development \[[@CR53]\]. *NAMPT* is one of the two enzymes regulating the NAD+ salvage pathway, a vital pathway allowing pancreatic cancer cells to maintain their metabolism, notably in hypoxic conditions \[[@CR55]\]. *NAMPT* is also involved in tumor angiogenesis \[[@CR56], [@CR57]\]. Thus, targeting *NAMPT* may not only disturb the salvage pathway on which pancreatic tumor cells heavily rely, but may also "normalize" blood vessels in the tumor, a phenomenon that will improve the delivery and efficacy of anticancer treatments and relieve immunosuppression \[[@CR58], [@CR59]\]. Several NAMPT inhibitors are currently in development in oncology \[[@CR60]\]. For example, FK866, a non-competitive highly specific inhibitor of NAMPT, shows potent anti-tumor activity both in vitro and in vivo \[[@CR61]\] on pancreatic cancer samples overexpressing *NAMPT* mRNA. Among the other genes of our signature upregulated in STS samples are *C16orf74* and *KRT13*, which are associated with poor OS in pancreatic \[[@CR62]\] and prostate \[[@CR63]\] cancers.
Thirteen genes of our signature were downregulated in STS samples. Three of them, *EGR3*, *EPHA7*, and *MACROD2*, play a role in peripheral nervous system biology, which may have a role in PDAC aggressiveness \[[@CR64]\]. We previously reported that the *MACROD2* locus at chromosome 20p12.1 may be a cancer-specific fragile site often affected in PDAC \[[@CR65]\]. Four genes (*EPHA7*, *SOCS2*, *SYNM*, *WNK2*) are tumor suppressor genes whose hypermethylation is a common mechanism of downregulation. WNK2 is a serine-threonine kinase involved in the regulation of electrolyte homeostasis, cell survival, and proliferation. Its downregulation occurs early in PDAC oncogenesis \[[@CR66]\]. SOCS2 is an important regulator of the JAK-STAT pathway \[[@CR67]\]. SYNM is a type IV intermediate filament involved in the modulation of cell adhesion and motility; in breast cancer, SYNM methylation is associated with shorter recurrence-free survival \[[@CR68]\].
Conclusions {#Sec12}
===========
We have identified a 25-gene classifier associated with post-operative OS independently of classical prognostic factors and molecular subtypes. The strength of our study lies in the size of the series, the robustness of the classifier in a large and multicentric validation set and in each dataset separately, its independent prognostic value, its non-random nature, and the biological relevance of the included genes. The small number of genes should facilitate the clinical application of the classifier by using other transcriptional tests applicable to formaldehyde-fixed paraffin-embedded samples such as qRT-PCR, RNAscope™ or Nanostring™ technologies. Limitations include the retrospective nature of our series and associated biases. Despite the very high *P* values, the HR for death was relatively low, around 2, in both uni- and multivariate analyses, and therefore of uncertain clinical value. However, we think that the testing of our signature in the current prospective trials of adjuvant and neoadjuvant chemotherapy trials is warranted, and should be tested not only as a two-tiered classifier, but also as a continuous score. Indeed, a continuous score based on the expression of 25 genes showed significant prognostic value (data not shown) in univariate analysis (HR for death of 2.84 (95% CI 2.06--3.91), *P* = 1.96 × 10^--10^) and in multivariate analysis (HR for death of 3.25 (95% CI 2.11--4.99), *P* = 7.42 × 10^--8^). If validated, our signature could help select patients with resectable disease for either immediate surgery (for the predicted LTS-like patients) or neoadjuvant chemotherapy (for the predicted STS-like patients), which ultimately should affect outcome and impact quality of life. Of course, the clinical utility of this approach will have to be prospectively demonstrated prior to any use in clinical routine. Neoadjuvant chemotherapy, currently mainly based on anatomical considerations, might also be indicated, and its benefits maximized, on the basis of the expression profile of aggressiveness, regardless of resectability. Finally, some of the classifier genes, or the pathways in which they are involved, may represent therapeutic targets. Therefore, functional studies to assess this are warranted.
Additional files
================
{#Sec13}
Additional file 1: Table S1.List of pancreatic cancer datasets included in our analysis. List of pancreatic cancer datasets included in our analysis. (XLS 28 kb) Additional file 2: Figure S1.Principal component analysis (PCA) of pancreatic carcinoma samples of the learning set before and after normalization. PCA was applied to the 279 TCGA and ICGC samples and the 685 Bailey's classifier genes. Before normalization (A), samples are grouped in the 2D scatter plot representation according to their origin dataset (left), and not according to their Bailey's molecular subtype type (right), whereas after normalization (B), all samples are grouped according to their molecular subtype (right), and not according to their origin dataset (left), suggesting that the inter-set technical differences have been removed by normalization. In A and B, each colour represents a set (left) and each colour represents a molecular subtype (right). (PPTX 595 kb) Additional file 3:Supplementary Text. Sweave report. Sweave report describing the different steps of gene expression data analysis and associated statistics. (PDF 1013 kb) Additional file 4: Table S2.List of 1400 genes differentially expressed between the short-term survivor (STS) samples and long-term survivor (LTS) samples of the learning set. List of 1400 genes differentially expressed between the STS and LTS samples of the learning set. (XLS 382 kb) Additional file 5: Table S3.Ontology analysis of the 1400 genes differentially expressed between the short-term survivor (STS) and long-term survivor (LTS) samples of the learning set. Ontology analysis of the 1400 genes differentially expressed between the STS and LTS samples of the learning set. (XLS 3728 kb) Additional file 6: Figure S2.Gene overlap between our 25-gene signature and other prognostic signatures. Venn diagram showing the overlap in genes between our signature and three prognostic signatures (A, Wang's 28-gene signature, Haider's 36-gene signature, and Chen's 15-gene signature; the Stratford's 6-gene and the Kirby's 19-gene signatures are not shown because they display no gene common with the other four signatures), and between our signature and the four molecular subtype classifiers (B, Bailey's 859-gene classifier, Collisson's 62-gene classifier, Moffitt's tumor 50-gene classifier, and Moffitt's stroma 48-gene classifier). (PPTX 131 kb) Additional file 7: Table S4.Patients and tumor clinicopathological characteristics of the learning and validation sets. (XLS 31 kb) Additional file 8: Figure S3.Overall survival (OS) in each set of the pooled validation set according to our prognostic 25-gene classifier. Kaplan--Meier OS curves in all patients according to our prognostic classifier (STS-like and LTS-like classes). The dashed vertical line represents the 2-year OS. The *P* values of the log-rank test are indicated. (PPTX 142 kb) Additional file 9: Figure S4.Overall survival (OS) in the validation set according to our prognostic 25-gene classifier and the American Joint Committee on Cancer (AJCC) Tumor, Node and Metastasis stage. Kaplan--Meier OS curves according to our prognostic classifier (STS-like and LTS-like classes) in patients with AJCC stage 1 (a) and AJCC stage 2 (b). The *P* values of the log-rank test are indicated. (PPTX 78 kb) Additional file 10: Figure S5.Overall survival (OS) in the validation set according to the molecular subtypes. Kaplan--Meier OS curves according to the molecular subtypes defined by Bailey (a), Collison (b), and Moffitt (c). The *P* values of the log-rank test are indicated. (PPTX 103 kb)
ADEX
: aberrantly differentiated endocrine exocrine
AJCC
: American Joint Committee on Cancer
GO
: gene ontology
HR
: hazard ratio
LTS
: long-term survivor
OS
: overall survival
PDAC
: pancreatic ductal adenocarcinoma
STS
: short-term survivor
TCGA
: The Cancer Genome Atlas
**Electronic supplementary material**
The online version of this article (doi:10.1186/s12916-017-0936-z) contains supplementary material, which is available to authorized users.
Not applicable.
Funding {#FPar1}
=======
Our work was supported by Institut Paoli-Calmettes, Institut National de la Santé et de la Recherche Médicale, Institut National du Cancer, and Site de Recherche Intégrée sur le Cancer Marseille (INCa-DGOS-Inserm 6038 grant). None of them had any role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials {#FPar2}
==================================
All expression and clinicopathological data analyzed in the present study are available in the GEO, ArrayExpress, EGA, and TCGA databases, as indicated in Additional file [1](#MOESM1){ref-type="media"}: Table S1.
DJB was involved in the conception and design of study, analysis and interpretation of data, and drafting of the manuscript. PF was involved in the acquisition, analysis and interpretation of all data. AL, MG, FP, JLR, JRD, and VM were involved in the analysis and interpretation of all data. DB was involved in the analysis and interpretation of data. EM and FB were involved in the conception and design of study, analysis and interpretation of data, and draft of the manuscript. All authors read critically and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
All nine studies pooled had been previously published with the appropriate ethics approval and consent. The study was approved by our institutional board (Comité d'Orientation Stratégique IPC 2016-034).
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no conflict of interest.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s001}
============
S[outh Africa has the highest number]{.smallcaps} of people infected with HIV-1 worldwide, estimated at 6.1 million in 2012.^[@B1]^ A major feature of HIV-1 is the extreme genetic diversity of the viral genome, which may have an impact on viral diagnostics, transmission, disease progression, and clinical management.^[@B2]^ HIV consists of two types, HIV-1 and HIV-2, and HIV-1 can be further divided into four groups: M (Major), O (Outlier), N (Non-M, Non-N), and P. Group M is responsible for the pandemic and can be divided into nine subtypes and subsubtypes, as well as into recombinant forms, which can be divided into circulating recombinant forms (CRFs) and unique recombinant forms (URFs). Currently, there are more than 65 CRFs and numerous URFs identified in the Los Alamos HIV Database \[[www.hiv.lanl.gov/content/index](www.hiv.lanl.gov/content/index)\]. The HIV-1 pandemic is not uniform, but complex and dynamic with different regional distributions of subtypes and CRFs. Subtype C is the most prevalent form in South Africa and accounts for nearly 50% of all HIV infections worldwide.^[@B3],[@B4]^ It is essential to continuously monitor the diversity and spread of HIV-1 worldwide as the pandemic matures.
A total of 309 full or near full-length unique HIV-1 genomes from South Africa have been characterized in various studies, 296 (95.78 %) of which are subtype C isolates.^[@B5]^ Other South African near full-length HIV-1 genomes include two subtype A1 isolates,^[@B8],[@B11]^ two subtype B isolates,^[@B8],[@B11]^ five subtype D isolates,^[@B12],[@B13]^ and four viral recombinant forms, which included three different URF_AC recombinant forms and one complex URF.^[@B6],[@B8],[@B14]^
We describe the near full-length genome (NFLG) sequencing and phylogenetic analysis of seven additional South African viral strains, including HIV-1 subtypes A, B, G, and two URFs.
Materials and Methods {#s002}
=====================
Ethics statement {#s003}
----------------
This study was approved by the Health Research Ethics Committee (HREC) of Stellenbosch University (IRB0005239) and all study participants provided written informed consent for the collection of samples and subsequent analyses.
Patients and RNA/DNA isolation {#s004}
------------------------------
Plasma and peripheral blood mononuclear cell (PBMC) samples from the Tygerberg Virology (TV) cohort were obtained between 1998 and 2004. The TV cohort, which was previously described in Jacobs *et al*.,^[@B15]^ is a rich sample repository containing specimens from patients from wide and diverse backgrounds based on race, socioeconomic status, and sexual orientation. Viral genotyping was performed on the envelope region of 410 sequences^[@B15]^ and the partial *gag*, *pol*, and *env* regions of a further 10 sequences.^[@B11]^ A total of 35 (8.53%) non-C isolates were identified among the 410 samples from the TV cohort. Of these, seven non-C strains were selected for further characterization based on the availability and quantity of samples.
RNA was extracted from 1 ml of the plasma samples (TV047, TV096, TV101, TV218, and TV546) using the QIAamp Ultrasense Virus kit. High-molecular-weight DNA was extracted from cultured TV016 using the Qiagen DNAeasy Blood and Tissue kit and from uncultured TV1057 using the QIAmp DNA Mini kit (Qiagen, GmbH, Hilden, Germany). These two samples were genotyped from proviral DNA due to the lack of plasma in the case of TV016, the difficulty in amplifying from RNA in the case of TV1057, and the fact that these two patients were on treatment at the time of sampling.
RT-PCR and sequencing of TV047, TV096, TV101, TV218, and TV546 {#s005}
--------------------------------------------------------------
Between four and six overlapping fragments spanning the genome of HIV-1 were amplified using a nested long-range reverse transcriptase polymerase chain reaction (RT-PCR) method. Reverse transcription was performed with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) for cDNA synthesis, as described previously.^[@B16]^ Primer sequences, not provided here, can be found in previous publications.^[@B17]^ Primers p24-7, poli8R, JH38R, and 9131R-2 were used for cDNA synthesis. For PCR amplification, primer combinations RE9737F/p24-7 (first round) and RE9745/MOp24-6 (nested PCR) were used to amplify a 1.2-kb fragment from the 5\' long terminal repeat (LTR) to *gag* p24. Primer combinations VFgag8/LPpoli-2 and VFgag9/LPpoli-4 (nested PCR) were used to amplify a 3.8-kb fragment spanning *gag* p24--*pol* IN from four specimens and VFgag8/ppr4b and VFgag9/ppr10 from specimen TV218. Primer pairs 4274F-2 (5′ ACAGCAGTACAAATGGCAGTATTCATTC)/LP7728R and 4277F-2 (5′ GCAGTACAAATGGCAGTATTCAT)/LP7725R were utilized to amplify a 3.4-kb *pol*--*env* region from TV096, TV101, and TV546. Primer pairs ppf4/IDRps3R and ppf5B/LP7632R (5′ TATCCCATTGCAGCCAGGTCAT) were used to amplify a 3.2-kb *pol* IN--*env* IDR from TV218.
The region spanning *pol--env* in TV047 was amplified in two overlapping fragments: *pol--env* V3 (2.3 kb) using primers ppf4b/V3vh2R (5′ AAAAATTCCCCTCCACA) and ppf5b/V3vh4R (5′ GTGCRTTACAATTTCYGGGTCC) and an *env*V3- IDR fragment using primer pairs V3vh1F (5′ TAGGCCAGYAGTRTCAAC)/JH38R and V3vh3F (5′ GCAGTCTRGCAGAARAAGAGGTARTA)/IDRps3R. To complete coverage of the *pol--env* region for TV047 and TV218, an additional *pol*-IN product was amplified using primers and conditions as previously described.^[@B17]^
A 1.6-kb *env* IDR-3\' LTR product was generated with the primer combinations 7496F/9131R-2 and 7542F/9110R-2 from TV096 and TV218. Primer pairs JH41/9131R-2 and env*-*27F/9110R-2 amplified this region from TV047 and TV101, and a combination of primers env-27F/9131R-2 and 7542F/9110R-2 was used for amplification from specimen TV546.
PCR amplifications were performed with Advantage-2 polymerase Mix (Clontech, Palo Alto, CA) at cycling conditions described earlier.^[@B16]^ PCR products were purified with the QIAquick PCR purification kit (Qiagen Inc.) and both strands were sequenced directly using the ABI Prism Big Dye Terminator Cycle Sequencing Reactions kit v. 1.0 (Applied Biosystems) and the ABI PRISM 3100 Genetic Analyser (Applied Biosystems). Sequence data were assembled and edited using Sequencher software Version 4.0.5 (Gene Code Corporation, Ann Arbor, MI). Positions with sequence ambiguities were assigned the appropriate IUPAC designations.
PCR and sequencing of TV016 and TV1057 {#s006}
--------------------------------------
Four overlapping fragments, LTR--*gag* (1.09 kb), *gag--pol* (3.89 kb), *pol--env* (3.94 kb), and *env*--LTR (1.64 kb), were amplified using GoTaq DNA polymerase (Promega, Madison, WI) as described previously.^[@B11]^ Sequencing reactions were done with the ABI Prism BigDye Terminator Cycle sequencing kit v. 1.0 and run on the ABI 3130xl automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequenced data were assembled into contiguous fragments and edited in Sequencher Version 4.8 (Gene Codes Corporation, Ann Arbor, MI).
Sequence quality analysis and preliminary subtyping using online tools {#s007}
----------------------------------------------------------------------
The HIV-1 sequence quality analysis tool was run before further analysis ([www.hiv.lanl.gov/content/sequence/QC/index.html](www.hiv.lanl.gov/content/sequence/QC/index.html)).
Sequences were then screened with online HIV-1 viral subtyping and recombinant detection tools: jpHMM (<http://jphmm.gobics.de>)^[@B20],[@B21]^ and REGA v. 3.0 (<http://dbpartners.stanford.edu:8080/RegaSubtyping/stanford-hiv/typingtool/>).^[@B22]^
SimPlot bootscan analyses {#s008}
-------------------------
A multiple alignment was done with the HIV-1 reference subtypes ([www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html](www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html)) and the new TV sequences using Clustal W.^[@B23]^ The alignment was edited in Se-Al v. 2.0 (<http://tree.bio.ed.ac.uk/software/seal>) and then used for bootscan analysis in SimPlot v. 3.5.1. All of the bootscans were performed with the Kimura two-parameter nucleotide substitution, a window size of 400 bp, and a step size of 50 bp.
Maximum likelihood (ML) phylogenetic tree inference {#s009}
---------------------------------------------------
We compiled a dataset that included the HIV-1 subtype reference dataset from the Los Alamos Database, as well as randomly selected additional sequences. Multiple sequence alignments were done with Clustal W.^[@B23]^ Thereafter, all of the genes of HIV-1 \[with the exception of the long terminal repeats (LTR) and the *nef*-coding region\] were concatenated in Se-Al v 2.0 (<http://tree.bio.ed.ac.uk/software/seal>). Gene fragments were excised and overlapping gene regions were deleted from the structural genes (*gag*, *pol*, and *env*), while still conserving the open reading frames. This concatenated alignment was manually edited to obtain a codon alignment.
A modeltest was performed in jModelTest 1.0 using the Akaike Information Criterion (AIC) method to estimate the best-fitting model of nucleotide substitution. Maximum likelihood tree topology was inferred in phyML v. 3.0.^[@B24]^ The maximum likelihood tree topology was inferred with the GTR, an estimated Gamma shape parameter, and the subtree pruning and regrafting (SPR) method of tree rearrangement. Branch support was calculated with the implementation of bootstrap resampling totaling 100 bootstrap replicates.
Phylogenetic analyses of possible recombinants, TV101 and TV218 {#s010}
---------------------------------------------------------------
Based on the breakpoints identified with online tools, ML tree topologies were inferred for each of the recombinant fragments. Each fragment was aligned with the HIV-1 subtype reference alignment in Clustal W and edited in Se-Al v 2.0 as described before (<http://tree.bio.ed.ac.uk/software/seal>). ML tree topologies were inferred for each of the recombinant fragment alignments in phyML v. 3.0,^[@B24]^ with the HKY85 model of nucleotide substitution and an estimated Gamma shape parameter and bootstrap resampling totaling 100 replicates.
The AC recombinant NFLG sequences described previously in South Africa (AF411956, GU201611, and DQ093606) were investigated with TV218, using jpHMM to determine if the breakpoints were similar and if we had a possible new CRF.
Results {#s011}
=======
Patient demographics {#s012}
--------------------
All available clinical and demographic data of the seven patients are summarized in [Table 1](#T1){ref-type="table"}. The mean viral load was 61,608 RNA copies/ml (SD=71,246.257), while the mean CD4^+^ cell count was 548 cells/mm^3^ (SD=1,139.721). The dates of sampling collection predate the implementation of the public national HIV treatment campaign and thus only two of the patients were receiving antiretroviral therapy at the time of sampling.
######
[Demographic and Clinical Data of the Seven HIV-1-Infected Patients]{.smallcaps}
*Patient* *Sample date* *Sex, age, ethnicity*^[a](#tf1){ref-type="table-fn"}^ *Mode of infection* *Viral load (copies/ml blood)* *CD4 cell count (cells/mm^3^ blood)* *ART* *Origin*
----------- --------------- ------------------------------------------------------- --------------------- -------------------------------- -------------------------------------- ------- ---------------
TV016 04-1998 M, 39, MR Heterosexual No data 421 Yes Western Cape
TV047 08-2000 M, 29, MR Heterosexual 29,800 265 No Western Cape
TV096 09-2000 F, 36, Af Heterosexual 204,020 96 No Western Cape
TV101 09-2000 F, 23, Af Heterosexual 88,558 2000 No South Africa
TV218 11-2000 F, 25, Af Heterosexual 76,117 No data No KwaZulu-Natal
TV546 08-2001 F, 31, Af Heterosexual 15,985 No data No Eastern Cape
TV1057 04-2002 M, 53, Ca Homosexual 140,000 108 Yes Western Cape
M, male; MR, mixed race; F, female; Af, African; Ca, caucasian
ART, antiretroviral treatment.
Near full-length genomic sequences {#s013}
----------------------------------
Assembly of the overlapping amplification products TV47 (9,083 nucleotides), TV96 (9,058 nucleotides), TV101 (9,038 nucleotides), TV218 (9,039 nucleotides), and TV546 (9,104 nucleotides) resulted in characterization of NFLG spanning from the 5\' U5 region through the 3\' U3 region. Open reading frames (ORFs) were identified for *gag*, *pol*, and *env* structural genes and for *vif*, *vpr*, *vpu*, *nef*, *tat*, and *rev* regulatory/accessory genes. TV016 (8,039 nucleotides) and TV1057 (8,084 nucleotides) spanned from the beginning of the *gag* to the end of the *env* region and excluded the *nef* ORF.
Online subtyping and bootscan analyses {#s014}
--------------------------------------
Preliminary subtyping was done using the online REGA version 3 and jpHMM tools, summarized in [Table 2](#T2){ref-type="table"}. These tools provide a quick, preliminary analysis of the sequences before detailed manual phylogenetic inference. Three isolates (TV016, TV047, and TV1057) were identified with high confidence as HIV-1 subtype B. TV096 was identified as HIV-1 subtype A1 and TV546 as subtype G. The REGA and jpHMM tools identified two possible recombinants, TV101 and TV218. SimPlot bootscan analysis identified TV101 as an A1/D recombinant and TV218 as an A1/C recombinant. TV016, TV047, and TV1057 were confirmed as HIV-1 subtype B with high similarity scores using SimPlot and TV096 and TV546 as subtype A1 and subtype G, respectively.
######
[Subtyping of All Seven New South African HIV-1 Non-Subtype C Strains]{.smallcaps}
*Sequence name* *REGA 3.0* *jpHMM* *SimPlot* *ML-tree* *Subtype assigned* *GenBank accession*
----------------- ------------ --------- ----------- ----------- -------------------- ---------------------
TV016 B B/K B B B KJ948656
TV047 B B B B B KJ948657
TV096 A1 A1 A1 A1 A1 KJ948658
TV101 A1/D A1/D A1/D A1 URF A1/D KJ948659
TV218 A1/C A1/C A1/C C outlier URF A1/C KJ948661
TV546 G G G G G KJ948662
TV1057 B B B B B KJ948660
ML, maximum likelihood; jpHMM, jumping profile hidden Markov model.
ML phylogenetic tree inference {#s015}
------------------------------
The ML tree is indicated in [Fig. 1](#f1){ref-type="fig"}. Three isolates, TV016, TV047, and TV1057, clustered within HIV-1 subtype B with high bootstrap support. TV546 clustered within HIV-1 subtype G with high support, while TV218 clustered as an outlier to the subtype C cluster. The remaining two strains, TV096 and TV101, clustered within HIV-1 subtype A1 with high support. Based on the phylogenetic inference it would seem that TV218, as an outlier to the larger subtype C cluster, may represent a subtype C viral recombinant form. TV101 clustered within the A1 clade but this can possibly be explained by the small recombinant regions of subtype D that are interspersed within the larger subtype A1 sequence.
](fig-1){#f1}
Phylogenetic analysis of TV101 and TV218 recombinants {#s016}
-----------------------------------------------------
TV101 and TV218 were further investigated and phylogenies were also inferred from the recombinant breakpoints identified with online jpHMM, REGA version 3, and bootscan analyses. Representations of the genome mosaic of TV101 and TV218 are illustrated in [Fig. 2](#f2){ref-type="fig"} and [Fig. 3](#f3){ref-type="fig"}, respectively. TV101 is a recombinant between HIV-1 subtype A1 and D with six breakpoints and TV218 is a recombinant between HIV-1 subtype C and A1 with four breakpoints. The breakpoints of all the other South African AC sequences are illustrated in [Fig. 4](#f4){ref-type="fig"}.
](fig-2){#f2}
](fig-3){#f3}
](fig-4){#f4}
Discussion {#s017}
==========
Limited information is available for HIV-1 non-subtype C sequences in South Africa and currently only 13 non-subtype C NFLG sequences are listed in the LANL HIV database. In this study, seven new NFLG sequences were characterized: three subtype Bs (TV016, TV047, and TV1057), one subtype A1 (TV096), one subtype G (TV546), one unique AD (TV101), and one unique AC (TV218) recombinant form. This is the first NFLG of subtype G that has been characterized from South Africa. Only three subtype B NFLGs have been described from Africa and with this article we increase this number to six.
These NFLG subtype B sequences include virus strains isolated from both heterosexual and homosexual individuals. All of these NFLGs identified in the present study were characterized in HIV-1-infected South Africans. Traditionally, subtype C has been the predominant viral form of HIV-1 in South Africa and today the subtype still accounts for the majority of infections (\>95.0%). However, in recent years we have seen an increase in the number of non-C HIV-1 isolates characterized among South African individuals (E. Wilkinson and G.B. Jacobs, unpublished observations). It is of the utmost importance to continue to monitor the genetic diversity of the HIV-1 epidemic within the country, as increasing heterogeneity can potentially impact the design of an effective vaccine, viral diagnostic assays, disease progression, and treatment and may lead to the rise of more recombinant forms. There are currently more than 65 CRFs identified in the Los Alamos Database and in 2011 they were responsible for at least 20% of HIV-1 infections worldwide.^[@B3]^
HIV-1 subtype B in South Africa {#s018}
-------------------------------
Previously, only two subtype B NFLGs have been characterized from South Africa^[@B8],[@B11]^ and another one from the African continent.^[@B25]^ This isolate from Gabon shows no close phylogenetic relationship with any of the South African subtype B strains. Two of the subtype B strains (TV016 and TV047) were genotyped from patients who were heterosexually infected, while the other remaining patient, TV1057, became infected via homosexual contact in 1982. He is classified as a slow progressor and was receiving antiretroviral therapy when the sample was taken. Two different HIV epidemics have been described within South Africa: HIV-1 subtype B in homosexual men represented the early epidemic and accounted for the majority of HIV infections during the 1980s^[@B26]^ and HIV-1 subtype C in the heterosexual population caused a later (or second) epidemic and is currently the most prevalent subtype.^[@B27]^
Although TV1057 was sampled in 2001, the infection occurred in 1982, providing an opportunity to analyze a subtype B strain that originated from the time of the early epidemic in the country. This strain was most closely related to GenBank accession number EF363124 from the United States as identified through a BLAST search. TV016 (infected heterosexually in 1989) and TV047 are characteristic of an emerging subtype B epidemic occurring in the heterosexual population, indicating a crossover of the two epidemics.^[@B15],[@B28],[@B29]^ TV016 was most closely related to GenBank accession number EF363124 and TV047 to GenBank accession number AY835795.
HIV-1 subtype G in South Africa {#s019}
-------------------------------
TV546 is the first NFLG subtype G strain that has been characterized within South Africa. The first full-length HIV-1 subtype G sequences were described in 1998 originating from the Democratic Republic of the Congo.^[@B30]^ The majority of HIV-1 subtype G isolates identified originate from several West Central and East African countries.^[@B31]^ Subtype G is also included in nosocomial epidemic outbreaks in the former Soviet Union^[@B34]^ and more recently has been linked to spread among infected drug users in the Iberian peninsula.^[@B35],[@B36]^ There has been one report of the detection of subtype G *gag* sequences in South Africa that was generated from samples that were obtained from migrant workers from Nigeria, Kenya, Zambia, and Angola.^[@B37]^ Compared to other subtypes, subtype G occurs infrequently and TV546 is only the second report of this subtype in South Africa. The strain was detected in a female patient residing in a rural area of the Eastern Cape Province and was most closely related to isolate 944-5 from Cameroon (FJ389366) as indicated by BLAST. This patient, who became infected via heterosexual contact, had no history of travel outside of South Africa.
HIV-1 subtype A1 in South Africa {#s020}
--------------------------------
TV096 is only the third NFLG of the subtype A1 isolate characterized from South Africa. The first full-length HIV-1 subtype A1 isolate from South Africa was characterized from a female patient of African descent,^[@B8]^ while the second was characterized from an African male.^[@B11]^ TV096 was sampled from a female patient of African descent in the late stages of HIV infection, as characterized by World Health Organization (WHO) criteria. Blast and phylogenetic inference indicated that TV096 was more closely related to African HIV-1 subtype A1 isolates from Senegal and Uganda. Phylogenetics of the previously characterized A1 sequences from South Africa also showed a close genetic relationship with other HIV-1 subtype A1 isolates from the East African region.
Unique recombinant forms in South Africa {#s021}
----------------------------------------
Two unique recombinant forms were also identified in this study. TV101 is the second URF_AD described from South Africa and is most closely related to AF457082 from Kenya. The first URF_AD recombinant was characterized from a South African individual who became infected via heterosexual contact in Kenya.^[@B11]^ These two URF_AD sequences share no recombinant breakpoints. HIV-1 subtypes D and subtype A1 have been detected in South Africa in the past.^[@B8],[@B11],[@B12]^
In addition to the URF_AD, TV218, a URF_AC was also characterized in the present study. This is the fourth URF composed of subtypes A and C that has been identified in South Africa. Two A1/C recombinants, isolate 04ZAPS204B1 (GenBank accession DQ093606)^[@B8]^ and isolate BBCR06 (GenBank accession GU201611),^[@B14]^ and one A2/C recombinant, 98ZADu178 (GenBank accession AF411965),^[@B6]^ have already been described. TV218, 98ZADu178, and 04ZAPS204B1 were sampled in Durban, KwaZulu-Natal, while the other A1/C isolate was sampled in the far northern part of South Africa.^[@B14]^
TV218 revealed a close genetic similarity to a previously described A2C subtype (AF411965, isolate 98ZADu178) from South Africa.^[@B6]^ The 98ZADu178 sequence was derived from cultured cells from an asymptomatic sex worker in Durban (sample date 1998), while TV218 was directly amplified and sequenced from plasma obtained during 2000 from a 25-year-old female in Durban. These two A1C sequences are 97% similar and share similar breakpoints. The *vpr*, *tat*, and *rev* region of TV218 is subtype A1 with a high probability, whereas this region in 98ZADu178 is A1/A2 with a low probability.
Conclusions {#s022}
===========
Phylogenetic inference of seven newly sequenced HIV-1 strains identified subtypes A1, B, and G as well as URF_AC and URF_AD. There is a need for more NFLG sequences because partial HIV-1 sequences may underrepresent viral recombinant forms. It is necessary to continue monitoring the evolution and spread of HIV-1 in South Africa and worldwide. Understanding HIV-1 diversity in South Africa will play an important role in HIV-1 prevention strategies.
Sequence Data {#s023}
=============
The sequences analyzed during the study have been deposited in GenBank and are available under the following accession numbers: KJ948656 to KJ948662.
Acknowledgments {#s024}
===============
This study was funded by the Poliomyelitis Research Foundation (PRF), the National Research Foundation (NRF), and the Medical Research Council (MRC) of South Africa. This research project was funded by the South African Medical Research Council (MRC) with funds from the National Treasury under its Economic Competitiveness and Support Package. This research and the publication thereof are the result of funding provided by the Medical Research Council of South Africa in terms of the MRC\'s Flagships Awards Project MRC-RFA-UFSP-01-2013/ UKZNHIVEPI.
Author Disclosure Statement {#s025}
===========================
No competing financial interests exist.
| {
"pile_set_name": "PubMed Central"
} |
Published: May 8, 2018
Introduction {#sec1}
============
Rett syndrome is a disease associated with loss of function mutations in the gene MECP2, which was originally identified as encoding a methylated DNA binding protein ([@bib3], [@bib21]). Patient symptoms include microcephaly, intellectual disability, facial dysmorphia, and seizure activity ([@bib2]). Studies in murine models recapitulate many of the patient phenotypes and have recently identified a role for Mecp2 particularly in inhibitory neurons ([@bib33]). These studies demonstrated that loss of MECP2 can lead to defects in transcription ([@bib4], [@bib12]), dendritic branching ([@bib41]), nuclear size ([@bib3]), and AKT signaling ([@bib13]).
MECP2 has also been described as a transcription factor with specific targets ([@bib4], [@bib41]), and more broadly as either a transcriptional activator ([@bib13]) or repressor ([@bib6], [@bib24]). However, despite decades of research on MECP2, it is still unclear how mutations in this protein lead to patient symptoms ([@bib3], [@bib18]). To confirm findings made in other models and further study these in a human system, some have turned to modeling Rett syndrome *in vitro* by taking advantage of disease-in-a-dish approaches. This involves making human induced pluripotent stem cells (hiPSCs) from patient somatic cells, or using genome engineering to introduce mutations into wild-type (WT) human pluripotent stem cells. In the current study, we also sought to mitigate the effect of genetic background and variability of differentiation by taking advantage of several isogenic lines of hiPSCs that either express the WT allele or the mutant allele leading to cells that express or lack MECP2 ([@bib32]). This allowed for detailed molecular analyses of hiPSCs, neural progenitor cells (NPCs), and neurons with and without MECP2 under the same genetic background. In comparing neurons from Rett patients as well as those with *MECP2* silenced by small interfering RNA (siRNA), it is clear that loss of MECP2 leads to induction of P53 and senescence, potentially opening an avenue of investigation for this intellectual disability syndrome.
Results {#sec2}
=======
A Human Model of Rett Syndrome *In Vitro* {#sec2.1}
-----------------------------------------
Cognizant of the fact that differentiation from hPSCs is highly variable across individual lines, culture conditions, and time, we developed an isogenic model to study Rett syndrome *in vitro* to remove the confound of genetic background ([@bib32]). Because female patients with Rett syndrome are usually heterozygous for mutant alleles of *MECP2*, fibroblasts isolated from these patients display a mosaic pattern where roughly half the cells express either the mutant or WT allele. This is shown in [Figure 1](#fig1){ref-type="fig"}A, where fibroblasts isolated from two patients with distinct mutant alleles of *MECP2* (R982 and R567) showed that roughly half the cells expressed MECP2 while the other half lacked detectable amounts of this protein. Patient descriptions are provided in [Figure S1](#mmc1){ref-type="supplementary-material"}. Reprogramming to iPSCs using a small set of transcription factors has been shown to happen at the clonal level, such that individual reprogramming events in single fibroblasts generate isolated hiPSC clones ([@bib38]). Therefore, reprogramming of mosaic fibroblast cultures from two different patients generated single hiPSC clones that either expressed MECP2 protein or lacked it ([Figure 1](#fig1){ref-type="fig"}B) (method described in a previous study; [@bib28]). In addition, our work and that of others has shown that under standard conditions, the inactive X chromosome in human fibroblasts does not reactivate upon reprogramming to the pluripotent state ([@bib32]), which is distinct from murine reprogramming ([@bib17]).Figure 1Generation of the Isogenic Model of Rett Syndrome *In Vitro*(A) Fibroblasts isolated from Rett syndrome patients (R982 and R567) heterozygous for MECP2 mutations exhibit a mosaic pattern of MECP2 expression due to random XCI.(B) Multiple isogenic hiPSC lines were produced from patient 982 with a typical Yamanaka protocol yielding individual isogenic clones with and without MECP2 expression from the same patient, as judged by NANOG and OCT4 staining.(C) Specification of hiPSCs derived from patient 982 toward neural progenitor cells yielded homogeneous cultures of NPCs with and without MECP2.(D) Terminal differentiation of NPCs derived from patient 982 toward neurons and glia by growth factor withdrawal as measured by immunostaining for MAP2 and GFAP.(E) MECP2+ and MECP2− hiPSCs and neurons were assayed by western blot with antibodies that recognize the active forms of Akt and its downstream target S6.(F) Sholl assay of dendritic complexity was performed on WT versus MUT neurons derived from patient 982.^∗^p \< 0.05 according to Student\'s t test. Bar graphs represent means ± SEM. Scale bars on images indicate 10 μm.
Thus, we were able to create multiple lines of hiPSCs with and without MECP2 from individual patients and thereby control for differences in genetic background (shown in [Figure 1](#fig1){ref-type="fig"}B are clones made from patient 982; clones from 567 look similar). The hiPSCs generated from fibroblasts of both patients appeared to be unaffected by the lack of MECP2, expressed all appropriate markers, and successfully generated teratomas upon injection into the testes of immunocompromised mice, consistent with previous hiPSC models for loss of MECP2 ([Figure S2](#mmc1){ref-type="supplementary-material"}A) ([@bib5], [@bib10]). Lack of MECP2 in patient-derived cells and specificity of antibody was also confirmed by western blot ([Figure S2](#mmc1){ref-type="supplementary-material"}B).
Importantly, we never observed reactivation of the silenced X chromosome that would have resulted in re-expression of the WT allele of *MECP2* in any cultures regardless of differentiation status or passage. This is consistent with previous data showing that, despite evidence for erosion of isolated portions of the silenced X chromosome ([@bib22]), many portions of the inactivated X remain silenced even through reprogramming or differentiation ([@bib25], [@bib32]). To measure the effect of any potential XCI erosion, we performed a DNA methylation analysis on the X chromosome on the lines from patient 982. This analysis showed that while some erosion of XCI was detectable across the X chromosome, there was not a significant difference between any of the lines ([Figure S3](#mmc1){ref-type="supplementary-material"}A), and methylation at the *MECP2* locus specifically was unchanged between the lines ([Figure S3](#mmc1){ref-type="supplementary-material"}B).
As Rett syndrome primarily afflicts the nervous system and MECP2 is most highly expressed in neurons, we first generated NPCs from all of the hiPSCs lines following standard protocols ([@bib26]). Across at least two lines per patient with and without MECP2, we measured the rate of neuralization, the morphology of NPCs, and expression of typical marker genes. We were unable to detect consistent differences in these properties between multiple clones of both WT and MECP2 null lines derived from both patients ([Figures 1](#fig1){ref-type="fig"}C and [S2](#mmc1){ref-type="supplementary-material"}C). Furthermore, the growth rate of NPCs with and without MECP2 was not consistently different in NPCs made from either patient ([Figure S2](#mmc1){ref-type="supplementary-material"}D). Next, the NPCs were further differentiated by a non-directed differentiation approach that yields both neurons and glia (growth factor withdrawal; [@bib26]) ([Figure 1](#fig1){ref-type="fig"}D). All NPCs from both patients produced neurons and glia at the same rate ([Figures S2](#mmc1){ref-type="supplementary-material"}E and S2F).
Previous studies have also shown that loss of MECP2 in neurons can lead to a decrease in AKT signaling ([@bib13]). A similar pattern was observed here in mutant neurons generated from Rett patient hiPSCs as measured by phosphorylation of AKT and S6, while hiPSCs themselves did not seem to be affected by loss of MECP2 ([Figure 1](#fig1){ref-type="fig"}E). Dendritic complexity has been shown extensively to be reliant on MECP2 expression in various models of Rett syndrome, and we found a statistically significant decrease in complexity in neurons made in the absence of MECP2 by Sholl assay ([Figure 1](#fig1){ref-type="fig"}F). In addition, we observed qualitative differences in basic neuronal morphology between WT and mutant neurons, where the neurons lacking MECP2 had shorter, thicker processes, and their soma was not as well defined.
Loss of MECP2 Affects the Transcriptome of Neurons {#sec2.2}
--------------------------------------------------
It has been suggested that loss of MECP2 only affects gene expression in neurons as opposed to the hPSCs and NPCs from which they were derived ([@bib13]). We sought to determine whether gene expression was affected in hiPSCs, NPCs, or neurons in this patient-derived *in vitro* model. To optimize the search for molecular effects of loss of MECP2 in neurons, we generated defined neuronal cultures by following the newly established 3i (three inhibitor) method to create interneurons ([Figure 2](#fig2){ref-type="fig"}A) ([@bib20]). Interneurons are particularly relevant in the study of Rett syndrome as interneuron-specific deletion of *Mecp2* in mice recapitulates many of the disease symptoms ([@bib11], [@bib33]). We validated the quality of differentiation at each step by immunostaining for markers typical of particular cell types (SOX2, SOX1, and NESTIN as well as FOXG1 and NKX2.1 for NPCs; and Tuj1, MAP2, and GABA for interneurons) in both WT and MUT cultures followed by quantification ([Figures S4](#mmc1){ref-type="supplementary-material"}A and S4B). While methods for derivation from pluripotent stem cells are effective at making interneurons, these cultures are not pure. As such, we first ensured that the proportion of neurons present in the cultures for comparison were not consistently different ([Figure S4](#mmc1){ref-type="supplementary-material"}C). We then assessed whether interneurons lacking MECP2 also showed diminished dendritic branching. In fact, in patient-derived interneurons made by 3i, defects in dendritic branching as measured by the number of endpoints were clearly observed ([Figure 2](#fig2){ref-type="fig"}A).Figure 2Loss of MECP2 Is Associated with Differential Gene Expression in Neurons(A) Immunostaining neurons generated from patient 982 for TuJ1, a neuronal-specific marker. Right: quantification of dendritic complexity by counting endpoints.(B) Volcano plots of differentially expressed genes (DEGs) in hiPSCs, NPCs, and neurons.(C) Gene ontological analysis of DEGs increased versus decreased in MECP2 null neurons.(D) An examination of SASP genes in neurons.(E) Patient skin-derived clones of fibroblasts lacking MECP2 showed strong β-gal activity, while those of WT fibroblasts did not.(F) Top: the senescence assay applied to neural progenitors derived from Rett patients did not show significant senescence activity. Bottom: patient-derived neuronal cultures showed a strong increase in the absence of MECP2 (quantification across independent lines shown on the right).(G) RT-PCR for P53 targets after siRNA treatment of WT neurons.Bar graphs represent means ± SEM. ^∗^p \< 0.05. Scale bars on images indicate 10 μm.
We therefore proceeded with deep RNA sequencing (RNA-seq; \>120 million reads per sample) of hiPSC, NPC, and interneuron cultures. With such sequencing depth, it was possible to analyze the RNA-seq reads for the known mutations present in the patients from which these lines were made ([Figure S4](#mmc1){ref-type="supplementary-material"}D). This analysis demonstrated that each line studied expressed strictly either the WT or mutant allele of *MECP2*, and that XCI status was unchanged even after extensive differentiation to neurons. We quantified the expression level of *MECP2* in WT cells across these three stages of development and found that the average reads per kilobase of transcript per million mapped reads RPKM was 3.1 for hiPSCs, 4.3 for NPCs, and 7.75 for interneuron-enriched cultures. This is consistent with consensus that MECP2 is enriched in neuronal cells, but also demonstrates that it could potentially be relevant to hiPSC and NPC physiology as well. However, high stringency analyses (false discovery rate \<0.05) of the RNA-seq data yielded very few gene expression changes due to loss of MECP2 in hiPSCs or NPCs derived from Rett patients ([Figure 2](#fig2){ref-type="fig"}B), consistent with [@bib13]. On the other hand, interneuron cultures made from patient 982 showed many gene expression changes when comparing two individual WT and MUT clones ([Figure 2](#fig2){ref-type="fig"}B). Gene ontology analysis uncovered many neuronal physiology-related pathways that were downregulated due to loss of MECP2 in neurons, while genes associated with extracellular remodeling and cell migration appeared to be induced ([Figure 2](#fig2){ref-type="fig"}C).
Probing the RNA-seq data, we also found that MECP2 null interneuron cultures showed a strong increase in a group of genes that are known to be induced by senescent cells, known as the senescence-associated secretory program (SASP). The vast majority of SASP genes that were changed in MECP2 null neurons were upregulated as opposed to downregulated, suggesting a robust pattern of SASP induction ([Figure 2](#fig2){ref-type="fig"}D). The only previous report linking MECP2 loss to senescence was performed by partial silencing of this protein in mesenchymal stem cells, but the results were consistent with those shown here for patient-derived MECP2 null fibroblasts ([@bib29]). The induction of SASP was intriguing in light of the fact that, while attempting to make clones of fibroblasts from patients with Rett syndrome, we repeatedly found that clones lacking MECP2 did not expand well after passage (14 MECP2 null clones were created, none expanded), while clones expressing the WT allele expanded without a problem (42 MECP2+ clones were created, we attempted to expand four of them, and all four expanded).
To determine whether MECP2 null fibroblasts encounter senescence, we performed an assay to detect endogenous beta-galactosidase activity, which is known to be a hallmark of this process ([@bib36]). Indeed, MECP2 null fibroblasts showed strong activity in this senescence assay ([Figure 2](#fig2){ref-type="fig"}E). We did not encounter such difficulties with clonal expansion once hiPSCs or hiPSC-derived NPCs were made from patients, presumably because during reprogramming, telomerase is strongly induced to restore telomere length at least beyond the critical threshold ([@bib19], [@bib30]). In fact, our RNA-seq data showed that hiPSCs made from patients had very high expression of *TERT*, and NPCs still expressed moderate levels, while neurons did not express appreciable levels (average RPKM for *TERT*: hiPSC, 8.8; NPC, 1.6; neuron, 0.006). Importantly, the same endogenous galactosidase activity assay on interneurons showed a dramatic increase in senescence activity in neurons lacking MECP2 ([Figure 2](#fig2){ref-type="fig"}F). These data indicate that loss of MECP2 leads to not only induction of SASP but also a *bona fide* senescence program in neurons.
Induction of P53 in the Absence of MECP2 {#sec2.3}
----------------------------------------
Cellular senescence programs are known to be regulated by P53, which can then activate various response pathways downstream, such as DNA repair and apoptosis ([@bib34]). Interestingly, P53 induction due to telomere shortening was previously shown to cause defects in dendritic branching ([@bib7]), which is also the dominant phenotype in Rett syndrome. To begin to look for hallmarks of P53 induction in the absence of MECP2, we performed RT-PCR for P53-related targets in cells with silencing of *MECP2* by siRNA ([Figure S4](#mmc1){ref-type="supplementary-material"}E). This assay suggested that decreased MECP2 levels led to induction of P53-related target genes such as *P21*, *GADD45*, *DDIT4*, and *DDB2* ([Figure 2](#fig2){ref-type="fig"}G).
To determine the effect of loss of MECP2 in relation to cell-stress pathways at the protein level, we performed immunostaining for H2AX, PML, P53, and P21 in neurons with and without MECP2. Staining for each of these markers showed strong increases in expression/levels of these markers of cell stress in patient-derived NPCs, neurons, and also after silencing of *MECP2* in both NPCs and neurons ([Figures 3](#fig3){ref-type="fig"}A--3D). WT NPCs with silencing of *MECP2* by siRNA and neurons lacking MECP2 also showed clear induction of these marks.Figure 3Loss of MECP2 Leads to Induction of DNA Damage and P53(A) Immunostaining of patient NPCs, NPCs with siRNA against MECP2, and patient neurons showed a strong increase in H2aX in the absence of MECP2.(B) Immunostaining of patient NPCs, NPCs with siRNA against MECP2, and patient neurons.(C) Immunostaining for P53 and p21, a target of P53.(D) Immunostaining after siRNA silencing of MECP2 in WT neuronal cultures.(E) Treatment of MECP2 null neurons with DMSO or Pifithrin, followed by immunostaining with antibody for TuJ1 shows a change in dendritic branching. Bottom left: RT-PCR for *GADD45*, a P53 target gene, showed that Pifithrin reduced P53 activity. Bottom right: quantification of branching phenotype across three independent experiments.^∗^p \< 0.05 according to Student\'s t test. Bar graphs represent means ± SEM. Scale bars on images indicate 10 μm.
Blocking Induction of P53 Can Rescue Dendritic Branching Defects Due to Loss of MECP2 {#sec2.4}
-------------------------------------------------------------------------------------
Previous evidence from a murine model of telomere shortening as a result of loss of telomerase complex (TERT) led to defects in dendritic branching, and this effect was strictly dependent on induction of P53 ([@bib7]). A more recent study also showed that experimentally aging the neural lineage with telomerase inhibition led to neurons with signs of aging, including reduced dendritic branching ([@bib35]). Therefore, we posited that inhibition of P53 in MECP2 null neurons could potentially restore appropriate dendritic branching. To determine whether blocking the action of P53 could improve dendritic branching in MECP2 null interneurons, we took advantage of Pifithrin-α, a potent inhibitor of P53 target gene activation ([@bib1]). Treatment of MECP2 null interneurons with Pifithrin-α showed evidence of P53 inhibition as measured by RT-PCR for *GADD45* ([@bib34]), a target gene important for DNA repair ([Figure 3](#fig3){ref-type="fig"}E). After 24--48 hr of P53 inhibition by Pifithrin-α, MECP2 null interneurons appeared to adopt an improved neuronal morphology typified by increased physical distinction between the soma and neurites, longer, thinner neurites, as well as increased dendritic branching as shown and quantified in [Figure 3](#fig3){ref-type="fig"}E. These data provide evidence that neurons without MECP2 induce P53 activity, which then inhibits the formation of complex neuronal processes.
To determine whether any of the phenotypes discovered in this *in vitro* model of Rett syndrome have relevance to patients afflicted with the disease, we acquired tissue specimens from Rett patients and age-matched controls. We first quantified the degree of chimerism of female Rett patient neurons due to skewing of X chromosome inactivation to determine the relative ratio of neurons that express MECP2 versus those that did not. Some of the Rett patient brains showed roughly 75% of neurons lacked MECP2, while others appeared to have less than 25% MECP2 null neurons ([Figure 4](#fig4){ref-type="fig"}A).Figure 4Evidence for P53 Induction in Rett Patient Neurons in Human Brain(A) Each Rett brain sample was assessed for the percentage of neurons with and without expression of MECP2 by immunostaining.(B) RT-PCR on RNA isolated from Rett brain versus age-matched control brains for P53 targets.(C) A re-analysis of data published by [@bib8]. Shown are a sample of P53 targets and SASP genes found on lists of genes upregulated in Rett brain. All of these differentially expressed genes were derived using a corrected p value (false discovery rate) \<0.05 from at least n = 5 samples from control and Rett brains.Bar graphs represent means ± SEM.
We performed RT-PCR on samples from some of these brains to determine whether they showed signs of increased P53 activity. To ensure accurate RNA representation, we first assessed the quality of the RNA from these frozen tissues, and only proceeded with RT-PCR in samples that showed an RIN (RNA integrity number) value above 5. All the Rett patient brains we processed for RT-PCR showed induction of canonical P53 target genes (identified in [@bib37]), consistent with what was observed in the patient-derived neurons *in vitro* ([Figure 4](#fig4){ref-type="fig"}B). Recently, a new study was published that isolated brain tissue from Rett patients and control subjects to perform an RNA-seq profile to characterize changes specific to Rett motor cortex or cerebellum. Using data provided in that study, we found that many direct P53 targets (as defined in [@bib37]) and SASP signature genes were upregulated in the Rett brains in both the motor cortex and the cerebellum ([Figure 4](#fig4){ref-type="fig"}C).
Discussion {#sec3}
==========
Taken together, these data demonstrate that loss of MECP2 leads to clear signs of stress such as H2AX deposition, P53/P21 induction, and initiation of a senescence program, all of which suggest that neurons in Rett syndrome could be in suboptimal health, leading to neurophysiological defects such as dendritic arborization ([@bib41]). While one paper suggested that RNAi-mediated silencing of *MECP2* could promote senescence in mesenchymal cells ([@bib29]), decades of work on Rett syndrome have not uncovered a role for MECP2 in relation to senescence in a wide variety of models such as various transgenic mouse lines, human patient postmortem analyses, and *in vitro* human models.
These results also raise the question of whether senescence could be common to the etiologies of other intellectual disability syndromes. The phenotypes described here show a striking similarity to those observed in hiPSCs and neural derivatives made from patients with immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) syndrome ([@bib40]). Two independent studies showed that ICF patient-derived hiPSCs displayed telomere shortening that was coupled to senescence of somatic derivatives such as fibroblasts. ICF syndrome only partially overlaps with Rett syndrome in terms of patient phenotypes, but is caused by mutations in DNMT3B, a *de novo* DNA methyltransferase ([@bib15]). These findings together are highly relevant as DNMT3B is a key *de novo* methyl transferase to create methylated DNA (5mC), which is the substrate for Tet oxygenase's to create 5-hydroxmethylated DNA (5hmC), which is known to be strongly bound by MECP2 ([@bib23]). Recently, another study showed that deletion of Tet enzymes, which are critical to generate the 5hmC mark, led to shortened telomeres ([@bib39]), which is known to lead to P53 activation.
Another possible interpretation of these data is that instead of a failure to mature, Rett syndrome neurons instead show aspects of premature aging. The fact that MECP2 null neurons show induction of aging-related genes, including P53 targets, and induce senescence pathways are consistent with this idea ([@bib31]). On the other hand, while Rett patients suffer from a post-natal cognitive decline, and long-term survivors show phenotypes associated with Parkinson disease ([@bib42]), the typical phenotypes presented in young female patients are not consistent with premature aging. Whether the physiological response to loss of MECP2 is truly akin to premature aging or whether patients suffer from effects that are unrelated to aging is worthy of continued investigation.
Experimental Procedures {#sec4}
=======================
Generation of Isogenic Rett Syndrome iPSCs {#sec4.1}
------------------------------------------
Reprogramming was performed as described ([@bib16]).
Generation of Teratomas {#sec4.2}
-----------------------
Generation of teratomas was previously described ([@bib14]).
Differentiation *In Vitro* and Analysis {#sec4.3}
---------------------------------------
Neural specification with neural rosette derivation, neuroprogenitor (NPC) purification, and further differentiation to neurons and glia were performed as described previously ([@bib26], [@bib27]).
Immunofluorescence and Image Quantification {#sec4.4}
-------------------------------------------
Immunofluorescence was performed as described previously ([@bib27]) and is described in detail in the [Supplemental Information](#mmc1){ref-type="supplementary-material"}.
RT-qPCR {#sec4.5}
-------
RT-PCR with real-time PCR measurement was carried out on a Roche 480 as described ([@bib26], [@bib27]). The primer sequences are available in the [Supplemental Information](#app2){ref-type="sec"}.
siRNA Gene Silencing {#sec4.6}
--------------------
All knockdown experiments were performed as described previously ([@bib27]).
β-Galactosidase Senescence Assay {#sec4.7}
--------------------------------
β-Galactosidase senescence assay was performed using the Senescence β-Galactosidase Staining Kit from Cell Signaling. The number of blue cells and number of total cells were quantified using the Cell Counter plugin in ImageJ.
Quantification of Dendritic Arborization {#sec4.8}
----------------------------------------
The stained cells were then imaged at 20×, and dendritic arbors of individual cells were traced using the Simple Neurite Tracer plugin for ImageJ. The number of process ends per cell were counted using the Cell Counter plugin for ImageJ. The number of process ends per cell are presented as mean ends per cell ± SEM.
RNA Expression Profiling {#sec4.9}
------------------------
RNA-seq was performed as described previously ([@bib9]). These data are available from NIH dataset GEO: [GSE107399](ncbi-geo:GSE107399){#intref0010}.
Author Contributions {#sec5}
====================
M.O., E.K., D.A., P.L., K.F., B.S.V., J.C., C.S., J.C.P., I.G., J.L., C.C., E.K., and S.T. provided data through experimentation. M.O., E.K., and W.E.L. contributed to writing the manuscript. X.X., M.P., K.P., and W.E.L. provided financial support for this work.
Supplemental Information {#app2}
========================
Document S1. Supplemental Experimental Procedures and Figures S1--S4Document S2. Article plus Supplemental Information
We would like to acknowledge the helpful discussions and insight on this manuscript with Gail Mandel (OHSU). This work was funded by training grants to M.O. (NIH-Virology and Gene Therapy, UCLA), P.L. (CIRM, UCLA), C.S. (CIRM-Bridges, Cal-State-Northridge), and D.A. (HHURP, UCLA). W.E.L. was supported by a Rose Hills Scholar award through the Eli and Edythe Broad Center for Regenerative Medicine. W.E.L. and K.P. were supported by NIH (P015P01GM099134). This research was also supported by the Allen Distinguished Investigator Program and the Paul G. Allen Frontiers Group, and a March of Dimes Scholar Award (6-FY17-406).
Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at [https://doi.org/10.1016/j.stemcr.2018.04.001](10.1016/j.stemcr.2018.04.001){#intref0015}.
[^1]: Co-first author
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
The domestic food hygiene research in Korea has advanced in recent years, but food poisoning is still present due to clustering of dietary life and growth of the food service industry ([@r018]; [@r012]; [@r005]). Due to this issue, food safety and sanitation methods are very important factor for consumers in purchasing food and beverages (Lee, 2001), and the growing awareness of food safety among consumers has led to increased food product safety. The safety among consumers has led to the increase in food product safety. According to [@r019], nearly 69.2% of consumers check food labeling at the time of purchase. The food label provides information regarding the product name, raw material, expiration date, preservation method, nutritional ingredients, and other details ([@r001]), and contains important information that aids the consumer in ascertaining food safety (Lee and Kim, 2001). According to a survey on food safety awareness conducted by the Korean National Council of Consumer Organizations in 2009, 25.4% of consumers considered the expiration date the most important aspect of food labeling when purchasing food products; this aspect scored higher than price, raw material, origin, and food additives. A recent study by [@r009] also suggests that consumers consider the expiration date as the most important aspect on the labeling, above all other details ([@r023]). Based on these reports, it is clear that the expiration date is a crucial factor for purchase decisions, clearly demonstrated by the fact that consumers tend to buy products that have relatively long expiration dates, though there are no issues with the consumption of a product close to its expiration date ([@r024]). Products that are past their expiration dates are taken off the shelves and discarded. According to the Korean Consumer Agency, more than 65 million tons of food are disposed of each year due to surpassed expiration dates, resulting in high resource waste. Section 2 of the Livestock Labeling Standards, by the Animal and Plant Quarantine Agency, defines expiration dates as the period between production and purchase of the product during which the product can be legally sold. The expiration date is also referred to as the "Sell-by Date", where the product can be offered to the consumer (Codex, General Standard for the Labeling of Prepackaged Food). The expiration date also discloses the longest period of time the product can be sold to a consumer, as determined by the manufacturer and based on processing standards and compositional specifications ([@r016]). Originally, the government recommended a certain expiration date for each type of product, until the release of expiration dates on July 1, 2002. As companies are now allowed to set the expiration dates for individual products, a growing number of companies have determined expiration dates of products without scientific evidence, while other companies copied the expiration dates of competitors. Therefore, a guideline is needed in setting expiration dates. The Korea Food and Drug Administration have proposed the development of scientifically determined guidelines for expiration dates, as well as guidelines for the storage of dairy products and other livestock products. The Product Liability Law (PL) was passed shortly after the release of expiration dates, causing the manufacturers to be responsible for mishaps due to incorrect expiration dates, and satisfying consumers by providing products with longer expiration dates and better quality control. However, small businesses, which constitute up to 70% of livestock production in Korea, may lack the necessary funding and/or advancements to conduct proper experiments. Also they are alleged to set expiration dates without scientific evidence (Hwang and Park, 2007). Issues occur when small businesses determine the expiration date of their products based solely on competing products from larger companies without considering the manufacturing process or the packaging of the product. Accordingly, the Korea Food & Drug Administration has provided a scientifically sound process of determining food product expiration dates via the "Guideline for Establishment of Shelf-life of Foods". For livestock products, however, specific regulations have not been set for expiration dates or experimental manuals, resulting in difficulties. Butter and cheese are more likely to be imported and exported than other dairy foods, such as whole milk and yogurt. When butter and cheese are circulated, the risk of deterioration increases due to long distances and time ([@r008]). Although there have been several studies focused on the shelf life of whole milk ([@r004]; [@r015]; [@r003]), studies on other dairy products, such as butter and cheese, for determining shelf life are lacking. Therefore, we used legal and non-legal (not illegal) methods in this study to conduct quality tests of butter and cheese stored at 10℃, 15℃, 25℃, and 35℃, respectively, to identify a scientifically valid expiration date.
Materials and Methods {#s2}
=====================
Storage conditions and materials {#s2a}
--------------------------------
Milk product samples were chosen from dairy product companies in Korea, from brands consumed the most by the Korean population as assessed by market research. Cheese is typically pasteurized for 5 min at 80℃; a softprocess cheese consumed by many consumers in Korea was used. Butter is typically pasteurized at 85℃. Butter (unsalted) is pasteurized for 15 s at 85℃. Samples were transferred immediately to the laboratory under refrigerated conditions, and the initial sample quality analyzed. Butter and cheese were both stored at 10℃, 15℃, 25℃, and 35℃ respectively. The experiment was performed at a circulating temperature of 10℃ and 15℃, while the expedited experiment was performed at 25℃ and 35℃. The storage temperature of 10℃ is the maximum limit in a refrigerated environment; 15℃ was used to simulate storage conditions in refrigerated shelves in distributing markets, considering that most dairy products have a surface temperature greater than 10℃ ([@r011]). To simulate the scenario of the consumer leaving the product at room temperature, we used a set temperature of 25℃. Butter and cheese samples used for experimentation were kept for 221 d at 10℃ and 15℃. Samples that were stored at 25℃ and 35℃ were kept for 109 d.
Physicochemical analyses {#s2b}
------------------------
To assess changes occurring in the samples over time, acidity, acid value, pH, and moisture content were measured. Lipid oxidation negatively affects product flavor and taste. However, as it plays a crucial part in determining the shelf life of a product, the acid value, acidity, and pH, which are indicators of rancidness, must be analyzed for butter and cheese samples ([@r020]). Acidity was measured by adding 10 mL of distilled water to 10 mL of the sample along with 0.5 mL of phenolphthalein, and the solution was titrated with 0.1 N NaOH until the reddish color persisted for more than 30 s. The acid value was measured by carefully measuring out 5-10 g of the sample and adding to an erlenmeyer flask, while also adding 100 mL of ethanol-ether mixture (1:2) and titrating the solution with a 0.1 N neutral KOH-ethanol solution until the reddish color lasted for more than 30 s. The moisture content was measured by carefully weighing out 3-5 g of the sample and placing it in a dry oven set at 98-100℃ for 3-5 h. The sample was then placed in a desiccator, where it was cooled for approximately 30 min, and then weighed. pH was measured using a pH meter. All of the above experiments were conducted based on the livestock processing and compositional standards as defined by the Animal and Plant Quarantine Agency (2012).
Microbiological analysis {#s2c}
------------------------
To observe the microbiological activity over time, the number of total bacteria, coliform, and growth of *Listeria monocytogenes* (*L. monocytogenes*) was monitored. The number of bacteria was counted by plating the solution on Plate Count Agar (Difco, USA) and incubating at 37℃ for approximately 48 h. Plates with 25-250 colonies were counted and multiplied by the dilution factor to determine the actual colony count. Coliform count was performed by homogenizing the undiluted solution and then diluting by decimal dilution. The resulting in *Lis. monocytogenes* dilutions were plated on PALCAM agar plates with supplement (Difco, USA) and incubated at 35℃ for 24 h. Colonies with a dark brownish or blackish ring were counted and multiplied by the dilution factor to obtain the total number of colonies. All microbiological tests were carried out according to the livestock processing and composition standards (Animal and Plant Quarantine Agency, 2012).
Sensory tests {#s2d}
-------------
To determine changes in the quality of each sample, a sensory test was performed using sight (color, luster, appearance, molding, and yeast formation), taste (flavor and sourness), and smell (smell and odor). The sensory analysis was performed by 10 individuals trained in sensory testing and scored based on a 9-point scale, where 9 was "liked extremely," 8 was "liked very much," 7 was "liked moderately," 6 was "liked slightly," 5 was "neither liked nor disliked," 4 was "disliked slightly," 3 was "disliked moderately," 2 was "disliked very much," and 1 was "disliked extremely" (Ministry of Food and Drug Safety, 2011). The examiners were instructed to give the product a score of 5 points when the product had reached its quality limit ([@r010], [@r021]).
Calculation of the appropriate shelf life of butter and cheese {#s2e}
--------------------------------------------------------------
Each experiment was classified as legal and non-legal. For legal indicators, the samples were considered to have passed quality when the samples were above legal specifications. For non-legal indicators, sensory evaluation and linear regression analysis were used to evaluate the standard value. However, for *L. monocytogenes*, the Guidelines for Assessing the Microbiological Safety of Readyto- Eat Foods Place on the Market (2009) was referenced to calculate the standard values. Since the shelf life of butter and cheese is greater than 6 mon, a streamlined experiment was conducted as stated in the expiration date selection experiment guidelines, which defines a streamlined process as an experiment conducted in a harsher environment than that of its original environment because its expiration date is longer than three months. The rate constant (K) was calculated using physiochemical, microbiological, and sensory experimental data. The rate constant (K) was derived through a zero-order reaction, which displayed a constant degradation rate regardless of the quality attributes, and a first-order reaction, which showed exponential change in the degradation rate based on the quality attributes. After using the above equations, the reaction rate by temperature and linear regression model of ln K~1~ with 1/T was calculated and chosen as the most reliable order of reaction. The Arrhenius equation was used to select the expiration date according to ln K = ln A − (Ea/R) × 1/T equation for each indicator, where K is the specific reaction rate, Ea is the activation energy in kJ/mol, R is the gas constant (1.987 cal/mol K), and T is the absolute temperature (K = 273 + ℃).
Results and Discussion {#s3}
======================
Changes in microbiological quality {#s3a}
----------------------------------
No coliforms or bacteria were detected, indicating that the product is of acceptable quality based on its composition, according to the livestock processing and component standards. Additionally, the non-legal indictor, *L. monocytogenes*, was not detected. Although bacteria and *L. monocytogenes* were found intermittently in cheese samples after long storage periods, no trends and/or patterns were observed. Coliforms were not detected in cheese samples throughout the storage period (data not shown), while *L. monocytogenes* was detected intermittently, but did not show a consistent trend (data not shown). *Escherichia coli* O157:H7 and *L. monocytogenes* were inoculated into the butter samples, giving initial bacterial counts of 10^5^ CFU/g, and after storage for 21 d at 4℃ and 21℃, all samples except sweet whipped salted butter had a bacterial cell count less than 10^1^ CFU/g ([@r025]). These results indicate that butter and cheese do not support bacterial growth. A previous study on the effect of long-term food storage by animals ([@r014]) found no bacterial pollutants. This corresponds with the results obtained in this study, indicating that butter and cheese, which have more fat than other products, do not support bacterial growth.
Changes in physicochemical quality {#s3b}
----------------------------------
As a result of physiochemical changes observed in butter samples stored at 10℃, 15℃, 25℃, and 35℃, the initial acidity, a legal quality indicator, was 0.51 mg KOH/g on d 0, which was much lower than the quality limit of 2.80 mg KOH/g. After 221 d of storage at 10℃, the acidity was 0.67 and at 15℃, was 0.73. When stored for 109 d, the acidity was 0.71 at 25℃ and 1.39 at 35℃; these results show a tendency towards increased acidity from the initial value, and imply that any increase in the storage temperature rapidly increases the acidity. pH, a nonlegal quality indicator, was 6.96 in the initial butter sample; after storage for 221 d at 10℃, it was reduced to 6.33, and at 15℃ was 6.31. Furthermore, sample pH after storage for 109 d at 25℃ was 5.12 and 4.70 at 35℃, indicating that long-term storage of butter in cold temperatures decreases the pH, while storage at high temperatures results in a greater decrease in pH. Acid value, another non-legal quality indicator, was 0.01 in the initial butter sample. After storage for 221 d at 10℃, the acid value was 0.14, and 0.23 at 15℃. After storage for 109 d, the acid value of the sample stored at 25℃ was 0.16, and 0.84 in the sample stored at 35℃.
As a result of physiochemical changes was observed in butter samples which was stored at 10℃, 15℃, 25℃, and 35℃, pH of the base sample was 5.91, and after storage for 221 d at 10℃, was 5.66, and 5.59 at 15℃. After 109 d, the sample stored at 25℃ had a pH of 5.62 while the sample stored at 35℃ had a pH of 5.49. The results show an inverse relationship between pH and storage time/ temperature; pH falls as the storage time increases and a higher storage temperature results in a faster decrease in the pH value. The initial cheese sample acidity was 0.50. After 221 d of storage, the acidity of the sample stored at 10℃ was 0.80, whereas at 15℃, it was 1.00. After 109 d, the acidity of the sample stored at 25℃ was 0.94 and 1.13 at 35℃, indicating that increased storage time increases the acidity and that higher temperatures cause a rapid increase in acidity. The initial cheese sample moisture content, a non-legal indicator, was 50.23. After 221 d of storage, the moisture content of the sample stored at 10℃ was 46.65 and 46.53 when the sample was stored at 15℃. After 109 d, the moisture content of the sample stored at 25℃ was 47.29 and 46.55 at 35℃. These results indicate that increased storage time correlated with lower moisture content.
Changes in sensory evaluation {#s3c}
-----------------------------
The sensory evaluation of butter samples, a legal indicator for evaluation, was performed using a 9-point scale (Ministry of Food and Drug Safety, MFDS. 2013), with d 0 scored as 9 points and the sensory quality limit set at 5 points. Butter samples were analyzed after storage at 10℃, 15℃, 25℃, and 35℃. The butter sample stored at 10℃ scored 9 points for 67 d whereas the sample stored at 15℃ scored 9 points for only 63 d. After the mentioned dates, the samples stored at 10℃ and 15℃ were above the quality limit of 5 points until 221 d, while the sample stored at 25℃ dropped to 5 points in 1 d. Also, the sample stored at 35℃ reached the sensory quality limit after 1 d. Sensory evaluation of cheese samples was analyzed after storage at 10℃, 15℃, 25℃, and 35℃. The cheese sample stored at 10℃ scored 9 points for 88 d, while those stored at 15℃, 25℃, and 35℃ scored 9 points up to 81, 53, and 46 d, respectively. The quality of these samples dropped after the above dates, with scores lower than 5 points.
Calculated actual shelf life {#s3d}
----------------------------
Linear regression analysis was performed based on the storage time. Calculated quality indicator standard values were used in the linear regression equation to determine the quality limit date. The linear regression equation for butter was shown in [Table 1](#t001){ref-type="table"}. After storage of butter and cheese samples at 10℃, 15℃, 25℃, and 35℃, the acid value, appearance, acidity, and pH were evaluated for the butter samples, while the sensory evaluation, moisture content, and acidity were evaluated for the cheese samples. The data were then used to calculate a linear regression graph based on the storage time. Counts of coliforms, total bacteria, and *L. monocytogenes*, which were never found, were excluded from the quality limit decision process. The expiration date was typically decided via determining a proper quality indicator and measuring the point at which the product loses its value by sensory evaluation. In this study, compositional specifications of the "Animal and Plant Quarantine Agency, Process Criteria and Ingredient Standard of Livestock Products" were used as quality indicators to determine the quality limit when the product reached the legal specification, and the results of the quality limit date based on the legal specifications are presented in [Table 1](#t001){ref-type="table"}. In case of butter, acid value and sensory evaluation, which are both legal specifications, showed the following results: expiration date of the sample stored at 10℃ was 107.80 mon (based on acid value) and 21.94 mon (based on sensory evaluation); at 15℃, it was 93.53 mon (based on acid value) and 17.38 mon (based on sensory evaluation); and at 25℃, it was 50.57 mon (based on acid value) and 7.14 mon (based on sensory evaluation). For quality indicators that do not have legal specifications, a linear digression equation was calculated based on quality indicators and the 9-point scale. The expiration date was determined by inserting the quality limit of 5 points into the linear digression equation ([Table 2](#t002){ref-type="table"}). In case of butter, measurements of acidity and pH, which are non-legal quality indicators, showed that the expiration dates of butter samples were 31.10 mon (based on pH) and 24.23 (based on acidity) when stored at 10℃; 24.25 and 13.19 mon when stored at 15℃; 7.28 and 6.10 mon when stored at 25℃. The quality limit was determined using the indicator that gave the shortest quality limit date. Therefore, when using sensory evaluation as the indicator, the quality limit date for samples stored at 10℃ was 21.94 mon, which was the shortest limit when compared to results obtained using other indicators. The quality limit date for samples stored at 15℃ using acidity as the indicator was 13.19 mon, which was the shortest result. Similarly, the quality limit date for samples stored at 25℃, with acidity as the indicator, was the lowest at 6.10 mon. The quality limit of cheese was also determined in a similar manner. In case of cheese, sensory evaluation, which are only legal specifications, showed the following results: expiration date of the sample stored at 10℃ was 23.98 mon, at 15℃, it was 17.88 mon; and at 25℃, it was 6.51 mon. For quality indicators that do not have legal specifications, a linear digression equation was calculated based on quality indicators and the 9-point scale. In case of non-legal biomarker, for samples stored in 10℃, 15℃ and 25℃, the quality limit based on the acidity, pH, and moisture which are non-legal quality indicators. It showed that the expiration dates of cheese sample were 29.98 (based on pH), 10.81 (based on moisture contents) and 19.21 mon (based on acidity) when stored at 10℃; 17.18, 9.47 and 9.83 mon when stored at 15℃; 5.71 (based on pH), 5.48 (based on moisture contents), and 4.64 (based on acidity) when stored at 25℃. The quality limit was determined using the indicator that gave the shortest quality limit date. As a result, using moisture contents as the indicator, the quality limit date for samples stored at 10℃ was 10.81 mon, which was the shortest limit when compared to results obtained using other indicators. The quality limit date for samples stored at 15℃ using moisture contents as the indicator was 9.47 mon, which was the shortest result. Similarly, the quality limit date for samples stored at 25℃, with acidity as the indicator, was the lowest at 4.64 mon.
###### Shelf life of butter and cheese samples stored at 10℃, 15℃, and 25℃ by legal quality limit
Quality criteria Temperature (℃) Quality limit^2)^ Estimated shelf life (mon)
-------------------------------- ------------------ ----------------- ------------------- ----------------------------
Butter Acid value 10 2.80 107.80
15 2.80 93.53
25 2.80 50.57
Sensory overall evaluation^1)^ 10 5 21.94
15 5 17.38
25 5 7.14
^1)^Penal number=10; 1=very different from control, 5=different from control (quality limit of overall sensory evaluation), 9=same as control.
^2)^Legal quality limit: "Process Criteria and Ingredient Standard of Livestock Products" by Animal, Plant and Fisheries Quarantine and Inspection Agency.
###### Shelf life of butter and cheese samples stored at 10℃, 15℃, and 25℃ by non-legal quality limit
Quality criteria Temperature (℃) Quality limit^1)^ Regression equation Correlation coefficient Estimated shelf life (mon)
--------- ------------------ ----------------------- ---------------------- ----------------------- ------------------------- ----------------------------
Butter pH 10 3.55 y = -0.0037× + 6.9999 0.8529 31.10
15 3.94 y = -0.0043× + 7.0717 0.8425 24.25
25 4.62 y = -0.012× + 7.235 0.6279 7.28
Acidity 10 0.41 y = 0.0005× + 0.0474 0.5671 24.23
15 0.43 y = 0.001× + 0.0295 0.9030 13.19
25 0.23 y = 0.0011× + 0.0285 0.6070 6.10
^1)^Quality limit: Estimated by regression analysis between non-legal quality criteria (y) and overall sensory evaluation (x) during the storage period.
Calculation of accelerative experimental shelf life {#s3e}
---------------------------------------------------
The sample stored at 35℃ was tested using an accelerated model to find the quality limit date ([Table 3](#t003){ref-type="table"}). According to zero-order and first-order reactions, the product with the largest R² was chosen and used to determine the quality limit by the equation ln K = -(Ea/R)×1/T+ln A. For butter samples, the quality limit was based on the first-order acid value, zero-order appearance, zero-order pH, and first-order acidity. In case of butter, the zeroorder acid value showed a quality limit of 9.92 mon, while zero-order sensory estimated 1.83 mon. Also, the zero-order pH estimated a quality limit of 0.58 mon, and first order acidity estimated a quality limit of 2.75. In case of cheese, the zero-order sensory value showed a quality limit of 3.49 mon, while first-order estimated 4.22 mon. Also, the zero-order moisture content estimated a quality limit of 4.28 mon, and zero order acidity estimated a quality limit of 0.20 mon.
###### Shelf life of butter and cheese samples stored at 35℃ using an accelerated experiment
Quality criteria Order of reaction K^3)^ A~0~^4)^-A~t~^5)^ Estimated shelf life (mon)
-------------------------------- ------------------------- ------------------- ------------ ------------------- ---------------------------- ------
Butter Legal quality limit^1)^ Acid value Zero order 0.00956 2.85 9.92
First order -6.38620 0.54 10.75
Sensory overall evaluation^3)^ Zero order 0.00541 0.30 1.83
First order -3.44790 3.43 3.59
Non-legal quality limit^2)^ pH Zero order 0.13224 2.29 0.58
First order -3.23640 1.70 1.44
Acidity Zero order 0.04105 4.00 3.25
First order -4.94250 0.59 2.75
^1)^Legal quality limit: "Process Criteria and Ingredient Standard of Livestock Products" by Animal, Plant and Fisheries Quarantine and Inspection Agency.
^2)^Non-legal quality limit: Estimated by regression analysis between quality criteria (y) and overall sensory evaluation (x) during the storage period.
^3)^K: rate constant.
^4)^Initial date of index quality attribute.
^5)^ date of index quality attribute as t time passes.
Accordingly, the shortest quality limit date (by acidity) was chosen as the quality limit. After considering factors that may cause quality changes, such as characteristics, distribution process, and the risk involved ([@r010]), a safety factor under one was chosen as an expiration date multiplication factor. As a result, [Table 4](#t004){ref-type="table"} shows the shelf-life, estimated as the earliest date among all date of quality limit when each quality criteria were reached to quality limit. Shelf-life of butter was estimated after 21.94 mon at 10℃, 17.18 mon at 15℃, 6.10 mon at 25℃, and 0.58 mon at 35℃; that of cheese was estimated after 10.81 mon at 10℃, 9.47 mon at 15℃, 4.64 mon at 25℃, and 0.20 mon at 35℃. Finally, shelf-life can established to applied safety factor (\<1), as shelf life can change due to factors or other quality standard criteria. Therefore, although it can not be applied to all butter and cheese products, there has been no report that butter and cheese has previously been researched on establishment in shelf life. Taken together, these results suggest that serve a basis data in establishment shelf life on butter and cheese.
###### Estimated shelf-life of butter and cheese at 10℃, 15 ℃, 25℃, and 35℃
Temperature (℃) Shelf life (months)
----------------- --------------------- -------
10 21.94 10.81
15 17.18 9.47
25 6.10 4.64
35 0.58 0.20
This paper was supported by Konkuk University in 2014.
| {
"pile_set_name": "PubMed Central"
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Objective
=========
Mutations in different ciliopathy-associated genes often result in overlapping clinical phenotypes, which can in part be explained by disruption of overlapping functional protein modules. In this study we conducted large-scale affinity proteomics in a systems biology-based approach to boost insights into the assembly of these ciliary modules, and their connectivity in larger functional protein networks: the ciliary protein interaction landscape. This provides an important framework to deconvolute the pathways and processes that drive ciliopathies, and to understand the general importance of ciliary function for cellular homeostasis.
Methods
=======
Using more than 220 known and potential ciliary proteins as baits, fused to the Strep/FLAG-tandem affinity purification tag (SF-TAP), we purified protein complexes from human embryonic kidney cells (HEK293T), which were analysed by mass spectrometry. In parallel, specific modules were scrutinized for binary interactions by yeast two-hybrid analyses. Existing and newly developed bioinformatic algorithms were employed to validate the confidence of the identified interactions and to define functional modules.
Results
=======
We obtained low, medium and high confidence sets of protein interactions and modules. From this data we could assign novel components to known ciliary modules such as the anterograde and retrograde intraflagellar transport modules and the dynein-2 module. Due to the strong focus on ciliary proteins as baits and the integration of data from various sources, we could also identify several new modules, potentially with cilia-associated functions in health and disease.
Conclusion
==========
Our systems oriented approach, employing affinity proteomics to define the ciliary network has resulted in a comprehensive description of known and candidate ciliary protein networks and modules, which can serve as a resource for candidate ciliopathy proteins and our understanding of pathogenic mechanisms underlying ciliopathies.
| {
"pile_set_name": "PubMed Central"
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Introduction {#sec1}
============
The sea buckthorn carpenterworm, *Holcocerus hippophaecolus* Hua, Chou, Fang and Chen (Lepidoptera: Cossidae), attacks mainly the roots and trunks of *Hippophae rhamnoides* L. (Rosales: Elaeagnaceae), a commercially important deciduous shrub in the northern regions of China, thus making most of them hollow and eventually leading to the death of the plant. In recent years, *Ho. hippophaecolus* populations have sharply increased in Inner Mongolia and Liaoning, Shanxi, Ningxia, Shaanxi, and Gansu provinces in China, which has aggravated the damage to *Hi. rhamnoides* populations and severely impaired the local eco-environmental construction and economic activities based on this species ([@bibr17], [@bibr18]; [@bibr05]; [@bibr13]). The authors studied the parasitoids of *Ho. hippophaecolus*, and an important species, *Lissonota holcocerica* Sheng sp.n., was found and investigated.
*Lissonota* Gravenhorst, 1829, belonging to subfamily Banchinae (Hymenoptera: lchneumonidae), comprises 388 described species ([@bibr16]). The Oriental species were studied by Chandra and Gupta ([@bibr01]). So far 24 species are known from China ([@bibr09]; [@bibr10]; [@bibr11], [@bibr12]). The biology was reported by Gauld et al. ([@bibr03]). The status of the genus was elucidated by Townes ([@bibr14]) and Townes and Townes ([@bibr15]).
The holotype and six paratypes are deposited in the Insect Museum, General Station of Forest Pest Management (GSFPM), State Forestry Administration, P.R. China. Two paratypes have been deposited in the Natural History Museum, London, U.K. (BMNH).
Materials and Methods {#sec2}
=====================
Cocoons of *Ho. hippophaecolus* were collected from *Hi. rhamnoides* forest heavily infested by *Ho. hippophaecolus* during the periods of adult emergence, and all cocoons collected were dissected to record the number of parasitic and non-parasitic cocoons. Two square nylon cages (100 × 100 × 100 cm) were set up outdoors, and 10 virgin males and 10 virgin females of *L. holcocerica* were placed in pairs into each cage. One cage only had water while the other cage had both water and 10% honeydew. The feeding, mating, oviposition, and longevity behaviors were then observed. Additional observations were made in the field on *Ho. hippophaecolus* infested forests from which *L. holcocerica* emerged naturally.
The morphological terminology is mostly that of Gauld ([@bibr02]). Wing vein nomenclature is based on Ross ([@bibr08]) and the terminology on Mason ([@bibr06], [@bibr07]).
Results and Discussion {#sec3}
======================
1. Genus *Lissonota* Gravenhorst, 1829 *Lissonota* Gravenhorst, 1829.
Ichneumonologia Europaea, 3:30. Typespecies: *Lissonota sulphurifera* Gravenhorst, 1829.
2. **Diagnosis.** Face wider than long. Apical margin of clypeus thick. Malar space 0.4 to 1.3 times as long as basal width of mandible. Occipital carina complete, lower end joining oral carina some distance above base of mandible. Epomia absent or short and weak. Propodeum usually with posterior transverse carina, rarely absent. Hind wing vein 1-cu slightly longer than cu-a. Tarsal claws usually with distinct pectination. First tergum moderately narrowed toward the base, with a rather abrupt constriction at base. Ovipositor sheath 1.1 to 4.5 times as long as hind tibia.
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1. **Etymology.** The name of the new species is based on the host\'s name.
2. **Types.** Holotype, female, CHINA: Jianping County, Liaoning Province, 20 June 2003, leg. Chang-Kuan Lu and Shi-Xiang Zong (GSFPM). Paratypes: 2 females, CHINA: Wulateqanqi, Inner Monggol Autonomous Region, 14 July 1978, leg. He-Ming Chen (GSFPM). 1 female, CHINA: Kazuo County, Liaoning Province, 11 August 1983, leg. Guang Wu (GSFPM). 1 female, CHINA: Huanren County, Liaoning Province, August 1984, leg. Wen-Long Han (GSFPM). 1 female and 1 male, CHINA: Dongsheng, Inner Monggol Autonomous Region, 15 July 2002, leg. Chang-Kuan Lu and Shi-Xiang Zong (BMNH). 2 males, CHINA: Jianping County, Liaoning Province, 20 June 2003, leg. Chang-Kuan Lu and Shi-Xiang Zong (GSFPM). 1 female and 1 male, CHINA: Dongsheng, Inner Monggol Autonomous Region, 6 to 15 June 2006, leg. Mei Su (GSFPM). 1 female, CHINA: Dongsheng, Inner Monggol Autonomous Region, 10 July 2006, leg. Mao-Ling Sheng (GSFPM). 1 female, CHINA: Kuandian County, Liaoning Province, 30 June 2009, leg. Xiao-Yi Wang (GSFPM).
3. **Diagnosis.** Wing brown. Ratio of length of hind tarsomeres 1:2:3:4:5 is 10.0:4.5:3.0:1.5:2.7. Propodeum irregularly rough, median portion irregularly and strongly convex, basal portion triangularly concave inversely. First and second terga with dense and distinct punctures. Ovipositor sheath 2.5 to 3.0 times as long as hind tibia.
4. **Description.** Female. Body length 13.5 to 20.0 mm. Fore wing length 11.0 to 16.5 mm. Ovipositor sheath length 16.0 to 19.5 mm.
5. **Head.** Face ([Figure 3](#f03_01){ref-type="fig"}) 2.1 to 2.2 times as wide as long, with dense punctures, median portion strongly convex, upper lateral with concavity close to antennal socket. Clypeal suture distinct. Median portion of clypeus strongly convex transversely, basal portion smooth with sparse and indistinct punctures, apical portion smooth and impunctate. Mandible relatively long, with sparse fine punctures; upper tooth approximately as long as lower tooth. Cheek with dense, shallow and indistinct punctures. Malar space approximately as long as basal width of mandible. Gena smooth, with distinct punctures, distance between punctures 1.4 to 3.0 times diameter of puncture; straightly convergent backward; in lateral view 0.6 to 0.7 times as long as width of eye. Vertex with irregular punctures. Postero-ocellar line about 0.9 times as long as ocular-ocellar line. Median portion of firons deeply concave, with smooth median longitudinal groove, upper and lateral portion with dense punctures. Antenna filiform, with 39 to 40 flagellomeres. Ratio of length of flagellomere 1:2:3:4:5 is 10.0:7.0:6.3:6.0:5.8. Occipital carina complete and strong, joining oral carina distinctly above base of mandible.
6. **Mesosoma.** Anterior portion of pronotum with weak and indistinct punctures; upper portion of lateral concavity with short transverse wrinkles, lower and posterior portions with distinct punctures, distance between punctures 0.3 to 1.5 times diameter of puncture. Mesoscutum with dense punctures, distance between punctures 0.2 to 2.0 (sublateral-median about 2.5) times diameter of puncture. Notaulus indistinct. Scutellum slightly convex, with irregular punctures. Postscutellum slightly convex, anterior-lateral portion concave. Mesopleuron with punctures similar to that of mesoscutum, slightly larger than that; median portion with irregular transverse concavity. Epicnemial carina strong, upper end reaching about 0.3 distance to subalar prominence. Speculum distinct. Metapleuron with dense punctures, distance between punctures 0.5 to 2.5 times diameter of puncture. Juxtacoxal carina absent. Anterior half of submetapleural carina strongly lobed. Wings brown. Fore wing with vein 1cu-a distal of 1-M. Areolet slanting quadrangular, receiving 2m-cu at its middle. Vein 2-Cu approximately 2.0 times as long as 2cu-a. Hind wing vein 1-cu slightly longer than cu-a. Claw small, pectinate. Ratio of length of hind tarsomeres 1:2:3:4:5 is 10.0:4.5:3.0:1.5:2.7. Propodeum ([Figure 4](#f04_01){ref-type="fig"}) irregularly rough, basal-lateral portion with distinct punctures, median portion irregularly and strongly convex, basal portion triangularly concave inversely, posterior portion behind posterior transverse carina strongly declining. Propodeal spiracle oval.
7. **Metasoma.** Metasoma robust, apical portion slightly compressed. First tergum about 1.2 times as long as apical width, with dense and distinct punctures, lateral portion rough, subapical with weak and short longitudinal wrinkles, basal-median portion concave and smooth, apical half with irregular longitudinal median groove. Median dorsal carinae vestigial basally. Spiracle located at basal 0.3 of first tergum. Second tergum ([Figure 5](#f05_01){ref-type="fig"}) 0.7 to 0.8 times as long as apical width, with dense and distinct punctures, distance between punctures 0.2 to 1.0 times diameter of puncture, apical margin smooth narrowly. Third tergum with dense and more finer punctures than that of second tergum, apical margin smooth narrowly. Fourth tergum with distinct punctures, the punctures sparser and finer than that of third. Fifth tergum with very sparse, irregular and fine punctures. Remaining terga smooth. Apical margin of eighth tergum almost truncated. Apex of hypopygium with a triangular notch. Ovipositor sheath approximately 2.5 to 3.0 times as long as hind tibia.
8. **Color** ([Figure 1](#f01_01){ref-type="fig"}). Black, except the following. Apical portion of clypeus, legs, except coxae black and hind tarsi dark brown, brown to reddish brown. Spots on lateral-anterior portion of mesoscutum, tegulae and spots of subalar prominences yellow to yellowish brown.
9. **Male** ([Figure 2](#f02_01){ref-type="fig"}). Body length 18.5 to 19.5 mm. Fore wing length 14.5 to 15.0 mm. Antenna with 42 flagellomeres. Ventral profiles of second trochanters and femora, apical portions of hind tibiae blackish brown.
10. **Host.** *Holcocerus hippophaecolus* Hua, Chou, Fang and Chen (Lepidoptera: Cossidae). Endoparasitism.
11. **Host food.** *Hippophae rhamnoides* L. (Rosales: Elaeagnaceae).
{#f01_01}
{#f02_01}
{#f03_01}
{#f04_01}
This new species is similar to *L. setosa* (Geoffroy 1785), but can be easily distinguished by the following key.
{#f05_01}
In Sheng and Sun\'s key to species ([@bibr12]), the new species can be inserted as follows:
1. 20\. Frons flat or almost flat. Postero-ocellar line as long as or longer than ocular-ocellar line. Ovipositor sheath as long as length of body 21
2. Frons concave deeply, with a smooth median longitudinal groove. Posteroocellar line shorter than ocular-ocellar line. Ovipositor sheath shorter or longer than length of body 20′
3. 20′. Wing distinctly brown. First to fourth terga with dense and regular punctures *L. holcocerica* Sheng
4. Wing hyaline, very slightly infuscate. The sculpture on first to fourth terga is quite irregular, tending towards aciculaterugose, with punctures, especially at the apex of the first tergite and on the second *L. setosa* (Geoffroy)
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1. The host of *L. holcocerica* is *Ho. hippophaecolus* in living wood of *Hi. rhamnoides*.
Adults emerge during the daytime, mostly from 09:00 to 14:00 hr at room temperature. Newly emerged adults chew a round hole with mouthparts in the epidermis of the cocoons, and then crawl out quickly. Adults groom their antennae and wings after emerging, and then take off, heading towards light places indoors.
Virgin females start their mating behaviors after their adult emergence; mating occurs during the daytime, mostly from 8:00 to 12:00 hr at room temperature, and lasts from 15 sec to 15 min. Before copulation, female wasps chase males, flying around in the rearing cage. After mating, males rest for a while then show gestures of wanting to copulate with other females, but usually they are rejected by females. Copulated females sit still and reject males.
Mated females search for hosts by walking on the base of *Hi. rhamnoides* stem infested by *Ho. hippophaecolus* while vibrating their antennae, tapping the stem while searching for oviposition sites. In field, female wasps were seen walking around at the base of trunks, swinging their antenna to detect larvae. Females laid eggs into bodies of *Ho. hippophaecolus* larvae that lived in the stems of *Hi. rhamnoides*. After hatching, larvae of wasps fed inside the host larvae. Parasitized moth larvae dug into the soil and formed a cocoon earlier than normal. After depleting all the nutrients of the moth larvae, wasp larvae made membranous cocoons and pupated. Parasitized cocoons collected in field were half the size of normal ones or even smaller.
Male longevity was 5.8 ± 2.4 days whereas female longevity was 7.6 ± 4.6 days in the field. Nonetheless, the longevity of males and females was 5.5 ± 1.8 days when only fed with some water and 7.8 ± 4.0 days when fed with some water and 10% of honey dew (syrup). Therefore, the results show that nutritional supplements can significantly extend the life span of *L. holcocerica*.
The authors are deeply grateful to Dr. Gavin Broad, The Natural History Museum, London, U.K., for comparing the similar species, and also thank Dr. Kampungu Gerson, University of Namibia faculty of Agriculture Department of Agricultural Economics, for reviewing this manuscript. This research was supported by the program of the Fundamental Research Funds for the Central Universities (YX2011-18) and The National Natural Science Foundation of China (NSFC, No.30730075, No. 31070585).
[^1]: **Editor:** Andrew Deans was editor of this paper.
| {
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1. Introduction {#sec0005}
===============
The recent growing interest in health and diet has led to an increased focus on soy foods and their functional components, such as isoflavones. Soy isoflavones have a structure similar to that of estrogen and have weak estrogenic activity [@bib0005]. Therefore, isoflavones have been used by some postmenopausal women as a natural alternative to estrogen replacement therapy for prevention of postmenopausal osteoporosis [@bib0010]. Equol is a metabolite of daidzein. Equol is formed by microbiota in the intestine and it binds to estrogen receptors (ERs) and induces transcription more strongly than soy isoflavones [@bib0015]. Lampe [@bib0020] suggested that the clinical effectiveness of soy isoflavones is due to their ability to produce equol.
Estrogen plays a critical role in the skeletal development of female and male mammals throughout life [@bib0025]. However, the effects of isoflavone exposure during early life on bone development have not been extensively studied. Injecting pregnant mice with diethylstilbestrol during pregnancy results in positive, long-lasting effects on the skeleton of female offspring [@bib0025]. This finding suggests that in utero exposure to estrogen can result in programming of bone cells. Moreover, short-term exposure to estrogen or compounds with estrogen-like activity during early stages of development has positive effects on bone health in adulthood in mice [@bib0030]. Female offspring of mice exposed to soy isoflavones have a higher bone mineral density (BMD) compared with control group and improved femur structure at 4 months of age, the time when maximal bone mass is established in CD-1 mice [@bib0035], [@bib0040], [@bib0045]. However, Kaludjerovic and Ward [@bib0050] reported that short-term neonatal exposure to isoflavones provides protection against deterioration of bone tissue in female mice, but not in male mice, after a decline in endogenous sex steroid production. We previously reported that consumption of a diet with 0.08% (w/w) isoflavones stimulates bone formation in immature male mice and exerts the opposite effect in female mice [@bib0055]. These results suggest that daidzein has a specific, sexually dimorphic effect on bone formation and BMD during growth periods.
Consumption of soy isoflavone supplements during early life is frequently associated with beneficial effects on bone development in mice [@bib0035], [@bib0040], [@bib0045]. However, there is concern regarding isoflavones as endocrine-disrupting chemicals because of their estrogenic activity [@bib0060], [@bib0065]. Pregnancy and lactation are hormone-sensitive and important periods in the development of reproductive capacity of offspring. Therefore, the fetus or nursing offspring may be more affected by estrogen or food components with potential hormonal activity than adults. Several studies have shown that perinatal exposure to isoflavones in rodents induces adverse reproductive effects on the offspring, such as a change in reproductive organ weight and serum hormone levels in mice and rats [@bib0060], [@bib0065], [@bib0070].
We previously reported that isoflavones were present in the milk of dams that were exposed to isoflavones in rats [@bib0075]. The maternal--fetal intrauterine transfer of isoflavones in rats fed a diet supplemented with isoflavones elicits high serum isoflavone levels in rat pups. These high levels are maintained throughout the suckling period by the passage of isoflavones into maternal milk [@bib0075], [@bib0080]. Therefore, human infants can be exposed to elevated levels of isoflavones throughout nursing if the mother is consuming a diet that is abundant in soy foods or supplements [@bib0085]. However, few studies have assessed the transfer of isoflavone and its metabolite equol from mothers to the nursing offspring. Therefore, the objective of the present study was to assess the safety and efficiency of perinatal or peripubertal exposure to daidzein on bone development and on reproductive organs at early adulthood in rats.
2. Materials and methods {#sec0010}
========================
2.1. Animals {#sec0015}
------------
Female Sprague-Dawley rats at day 5 of gestation were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). Rats were housed individually in clear plastic cages with wire-mesh covers and paper chip bedding (ALPHA-DriTM, Shepherd Specialty Papers, Inc., Richland, MI). The weaned offspring were housed individually in stainless-steel cages. Rats were maintained in a temperature- and humidity-controlled room (23 ± 1 °C and 60% ± 5% relative humidity) with a 12-h light--dark cycle. The offspring were kept in the same groups as their respective dams. Dams were pair-fed their respective diet and offspring were pair-fed their respective diets among males or females. This experiment complied with the guidelines of the Standards Relating to the Care and Management of Experimental Animals and Relief of Pain of the Japanese government (Notice No. 88 of the Ministry of the Environment, 2006).
2.2. Diets and chemicals {#sec0020}
------------------------
Pregnant rats were fed diet mixtures containing daidzein at dose levels of 0 or 0.5 g/kg. These diets were based on the modified AIN-93G diet with replacement of corn oil for soybean oil [@bib0090]. All ingredients were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Purified daidzein (\>98% purity) and equol were purchased from LC Laboratories Co., Ltd. (Woburn, MA).
2.3. Experimental design {#sec0025}
------------------------
The experimental design is shown in [Fig. 1](#fig0005){ref-type="fig"}. Pregnant rats at day 5 of gestation (*n* = 18) of random body weights were divided into three groups of six rats each: dams and their offspring were fed the control diet (Control); dams were fed the daidzein diet (0.5 g/kg diet) during pregnancy and then the control diet at postnatal day (PND) 13 and their offspring were fed the control diet (Dz-S); and dams and their offspring were fed the daidzein diet (Dz-L) through the experiment. Accordingly, pups in the Dz-S group were exposed to daidzein only through the placenta and the milk from the dams. The day of birth was designated as PND 1. On PND 1, the number of pups was adjusted to eight (4 males and 4 females) for each dam. Litter size, birth weight, and the sex ratio were measured as pregnancy outcomes. Pups were weaned on PND 22, and offspring were housed in individual cages. From PNDs 22 to 77, offspring were weighed weekly and food intake was measured daily. Offspring (1 female and 1 male per litter) were anesthetized by exposure to diethyl ether (Wako, Osaka, Japan) on PNDs 14, 22, 35, and 77, and dams were anesthetized by exposure to diethyl ether on PND 22. After laparotomy, the stomach was removed on PND 14. Whole blood was collected by cardiac puncture, and the testes, seminal vesicles, ovaries, and uteri were removed and weighted on PNDs 22, 35, and 77. Serum was separated by centrifugation at 3000 rpm for 20 min and frozen at −80 °C. For measurement of BMD, the right femur was removed and stored in 70%/30% ethanol/water (Sigma--Aldrich Japan, Tokyo, Japan).Fig. 1Experimental design. PND: postnatal day
2.4. Pup development {#sec0030}
--------------------
Hormonal imbalances in male or female pups due to the diet treatments were determined by comparing their anogenital distance (AGD) measurements with those of rats [@bib0060]. AGD, which is the length of the perineum from the base of the genital tubercle to the proximal edge of the anus under natural extension without stretching, was measured at PNDs 14 and 22.
2.5. Quantification of BMD and bone metabolism {#sec0035}
----------------------------------------------
Femoral BMD was measured by dual-energy X-ray absorptiometry using a specialized software program for small animals (Model DCS-600EX-R III, Aloka, Ltd., Tokyo, Japan). BMD was calculated using the bone mineral content of the measured area. Intra-assay and inter-assay coefficients of variation were less than 1.0% and 4.8%, respectively. The detection limit of BMD is 15 mg/cm^2^. Serum osteocalcin levels were measured with a commercial kit, the Rat Osteocalcin EIA Kit (Biomedical Technologies, Inc., Stoughton, MA).
2.6. Serum daidzein and equol concentrations in offspring and the stomach contents of pups {#sec0040}
------------------------------------------------------------------------------------------
Serum daidzein and equol concentrations were measured by the methods reported by Gamache and Acworth [@bib0095]. An aliquot of the pretreated sample solution was used for high-performance liquid chromatography (HPLC) (Shimadzu LC10AD) with an electrochemical detector (Coulochem III, ESA Laboratories Inc., Chelmsford, MA) that was equipped with analytical cells (detector 1, 300 mV; detector 2, 600 mV) (ESA) and a guard cell (650 mV) (ESA). The HPLC conditions were as follows: column, Nova-Pac C18 (Waters, 3.9 mm × 150 mm; Nihon Waters K.K., Tokyo, Japan); column temperature, 40 °C; mobile phase, 50/35/15% (v/v/v) 50 mM sodium acetate (pH 4.8):methanol:acetonitrile (Wako, Osaka, Japan); and flow rate, 0.65 ml/min.
2.7. Statistical analysis {#sec0045}
-------------------------
Data are expressed as means ± SEM. All statistical tests were conducted with SPSS, version 15.0 (SPSS Inc., Chicago, IL). Significance of differences in BMD was determined by single-factor analysis of covariance and Fisher\'s protected least significant difference tests. Body weight was used as a covariate in the analysis of BMD to adjust for possible confounding. Body weight, AGD, reproductive organ weight, and serum isoflavone and osteocalcin concentrations were analyzed using analysis of variance (ANOVA). Differences between treatment groups were assessed by Tukey\'s test. Differences were considered significant at *p* \< 0.05.
3. Results {#sec0050}
==========
3.1. Pregnancy outcomes {#sec0055}
-----------------------
There was no difference in the number of pups per litter among the groups (Control = 13.3 ± 1.0; Dz-S = 12.2 ± 1.4, and Dz-L; 12.3 ± 0.9). There were also no differences in the number of males and females per litter (m, f: Control = 6.5, 6.8; Dz-S = 6.7, 5.5; and Dz-L = 7.0, 5.3) and birth weight among the groups (Control = 7.0 ± 0.3 g; Dz-S = 7.4 ± 0.2 g; Dz-L = 7.5 ± 0.2 g).
3.2. Body weight, food intake, AGD, and sex organ weight {#sec0060}
--------------------------------------------------------
The mean body weight for males on PND 1 (day of birth) and PND 22 (weaning day) was not significantly different among the groups ([Table 1](#tbl0005){ref-type="table"}). The mean body weight for males on PND 35 was significantly higher in the Dz-L group than in the Control and Dz-S groups, but there was no significant difference among groups on PND 77. There was no difference in total weight gained among the groups ([Table 1](#tbl0005){ref-type="table"}). There was no difference in food intake in dams and offspring among the groups (data not shown). There were no significant differences in mean body weight and the total weight gained in females at any of the time points among the groups ([Table 1](#tbl0005){ref-type="table"}).Table 1Body weight and total weight gain of offspring on PNDs 1, 22, 35, and 77.GroupControlDaidzein ShortDaidzein LongMale Body weight (g) PND 17.32 ± 0.367.60 ± 0.297.90 ± 0.32 PND 2267.5 ± 1.263.6 ± 1.663.1 ± 1.7 PND 35151.4 ± 1.7^b^148.2 ± 1.0^b^158.6 ± 2.0^a^ PND 77433.0 ± 6.7430.8 ± 11.8439.8 ± 14.8 Total weight gain (g)425.7 ± 6.5423.2 ± 12.0431.9 ± 15.0Female Body weight (g) PND 13.56 ± 0.403.30 ± 0.513.03 ± 0.26 PND 2265.0 ± 0.361.0 ± 1.361.0 ± 1.9 PND 35130.7 ± 2.2127.5 ± 3.8135.5 ± 5.4 PND 77243.3 ± 4.3242.6 ± 7.5233.0 ± 17.0 Total weight gain (g)239.8 ± 4.4239.2 ± 7.3230.0 ± 6.8[^1][^2][^3]
[Table 2](#tbl0010){ref-type="table"} shows AGD and sex organ weight. The AGD of male and female offspring on PNDs 14 and 22 was not different among the groups. On PNDs 22, 35 and 77, treatment with daidzein had no effect on the mean weight of testes and seminal vesicles. The weight of the ovaries and uterus in female offspring on PNDs 22, 35, and 77 was not altered by daidzein exposure to the dams or offspring.Table 2Anogenital distance and sex organ weight of offspring on PNDs 14, 22, 35, and 77.GroupControlDaidzein ShortDaidzein LongMale Anogenital distance (mm) PND 143.72 ± 0.093.96 ± 0.183.77 ± 0.11 PND 223.14 ± 0.103.14 ± 0.113.21 ± 0.13 Testis weight (mg) PND 22277 ± 10264 ± 14262 ± 14 PND 351304 ± 421346 ± 401277 ± 43 PND 773353 ± 273319 ± 443233 ± 75 Seminal vesicle (mg) PND 2243 ± 245 ± 244 ± 2 PND 35161 ± 12149 ± 9169 ± 25 PND 77922 ± 49922 ± 37967 ± 55Female Anogenital distance (mm) PND 142.61 ± 0.102.89 ± 0.112.72 ± 0.03 PND 222.01 ± 0.042.15 ± 0.082.12 ± 0.09 Ovaries + uterus (mg) PND 2265 ± 367 ± 568 ± 3 PND 35210 ± 33167 ± 21233 ± 27 PND 77515 ± 48546 ± 41541 ± 54[^4][^5][^6]
3.3. BMD of the femur {#sec0065}
---------------------
On PND 22, there was no difference in femur BMD in male and female rats among the groups ([Fig. 2](#fig0010){ref-type="fig"}A and B). On PND 35, femur BMD in male and female rats in the Dz-S group was significantly lower than that in the Control and Dz-L groups ([Fig. 2](#fig0010){ref-type="fig"}C and D). On PND 77, femur BMD in male rats in the Dz-S group was significantly lower than that in the Control and Dz-L groups ([Fig. 2](#fig0010){ref-type="fig"}E). However, femur BMD in female rats was not different among the groups on PND 77 ([Fig. 2](#fig0010){ref-type="fig"}F).Fig. 2Bone mineral density of the right femur in offspring of dams that were fed the control diet or the diet containing 0.5 g daidzein/kg during pregnancy and lactation and/or the post-weaning period. Data are shown for PNDs 22, 35, and 77. All values are means ± SEM (*n* = 6). Values that do not share the same superscript letters are significantly different from each other (*p* \< 0.05). Significant differences in BMD were determined by one-factor ANCOVA and Fisher\'s protected least significant difference test. Control group: rats were fed a control diet; Dz-S group: rats were fed a daidzein diet (0.5 g daidzein/kg diet) during pregnancy and lactation; Dz-L group: rats were fed a daidzein diet (0.5 g daidzein/kg diet) during pregnancy, lactation, and post-weaning. PND: postnatal day.
3.4. Serum osteocalcin concentrations {#sec0070}
-------------------------------------
[Table 3](#tbl0015){ref-type="table"} shows serum osteocalcin concentrations, which are a biochemical marker of bone formation. In male rats, mean serum osteocalcin concentrations on PND 22 were significantly lower in the Dz-S and Dz-L groups (1.48 ± 0.08 and 1.51 ± 0.08 ng/ml, respectively) than in the Control group (1.91 ± 0.12 ng/ml). On PND 35, the mean serum osteocalcin concentration in the Dz-S group was significantly lower than that in the Dz-L group (4.45 ± 0.20 ng/ml) in male rats. On PND 77, there was no difference in serum osteocalcin concentrations in male rats among the groups.Table 3Serum osteocalcin concentrations in rat offspring on PNDs 22, 35, and 77.GroupControlDaidzein ShortDaidzein LongOsteocalcin (ng/ml) Male PND 221.91 ± 0.12^a^1.48 ± 0.08^b^1.51 ± 0.08^b^ PND 354.08 ± 0.22^ab^3.34 ± 0.30^b^4.45 ± 0.20^a^ PND 775.04 ± 0.334.23 ± 0.174.84 ± 0.22 Female PND 221.64 ± 0.041.62 ± 0.061.63 ± 0.05 PND 353.95 ± 0.10^a^3.37 ± 0.09^b^3.67 ± 0.06^ab^ PND 774.23 ± 0.114.16 ± 0.144.11 ± 0.13[^7][^8][^9]
In female rats, mean serum osteocalcin concentrations were significantly lower in the Dz-S group (3.37 ± 0.09 ng/ml, *p* = 0.001) and tended to be lower (*p* = 0.085) in the Dz-L group (3.67 ± 0.06 ng/ml) than in the Control group (3.95 ± 0.10 ng/ml) on PND 35 ([Table 3](#tbl0015){ref-type="table"}). There was no difference in mean serum osteocalcin concentrations in female rats among the groups on PNDs 22 and 77.
3.5. Stomach content and serum daidzein and equol concentrations {#sec0075}
----------------------------------------------------------------
Serum daidzein and equol concentrations were detected in dams in the Dz-L group (4.30 ± 0.45 and 5.71 ± 1.26 μmol/L, respectively). Stomach contents of daidzein and equol in each offspring were measured and combined the values from male and female. Daidzein (11.86 ± 1.28 and 15.12 ± 5.32 nmol/g, respectively) and equol (1.29 ± 0.29 and 3.37 ± 1.14 nmol/g, respectively) were detected in the stomach contents of the suckling pups of dams in the Dz-S and Dz-L groups ([Fig. 3](#fig0015){ref-type="fig"}). Serum daidzein and equol concentrations were observed in the offspring on PNDs 22 (2.03 ± 0.40 and 1.58 ± 0.18 μmol/L, respectively), 35 (4.23 ± 0.91 and 4.40 ± 1.08 μmol/L, respectively), and 77 (1.17 ± 0.33 and 4.31 ± 0.76 μmol/L, respectively) in the Dz-L group.Fig. 3Daidzein and equol concentrations in the stomach contents of suckling pups of dams that were fed a control diet or diet containing 0.5 g daidzein/kg during pregnancy and lactation. Data are shown for PND 14. All values are means ± SEM (*n* = 6--9). Values that do not share the same superscript letters are significantly different from each other (*p* \< 0.05). Data were analyzed using one-way ANOVA. Differences between the groups were assessed by Tukey\'s test. Control group: rats were fed a control diet; Dz-S group: rats were fed a daidzein diet (0.5 g daidzein/kg) during pregnancy and lactation; Dz-L group: rats were fed a daidzein diet (0.5 g daidzein/kg) during pregnancy, lactation, and post-weaning.
4. Discussion {#sec0080}
=============
This study showed that perinatal exposure to daidzein inhibited an increase in BMD and serum osteocalcin concentrations in male and female rats on PND 35. However, peripubertal exposure to daidzein protected against the decline of these bone parameters in male and female rats on PND 35. On PND 77, femur BMD in male rats that were exposed to daidzein perinatally was lower than that in control rats or those with peripubertal exposure to daidzein. However, femur BMD in female rats was not different among the groups. These results suggest that the effects of daidzein on bone metabolism during development depend on the timing of exposure and sex. However, perinatal or peripubertal exposure to daidzein did not affect development of reproductive organs by early adulthood in rats.
On PND 35, femur BMD in male and female rats in the Dz-S group was significantly lower than that in the Control and Dz-L groups ([Fig. 2](#fig0010){ref-type="fig"}C and D). Similarly, serum osteocalcin concentrations, a biochemical marker of bone formation, tended to be lower in male and female rats in the Dz-S group than in the Control and Dz-L groups on PND 35 ([Table 3](#tbl0015){ref-type="table"}). On PND 77 in male rats, femur BMD in the Dz-S group was significantly lower than that in the Control and Dz-L groups ([Fig. 2](#fig0010){ref-type="fig"}E), but female BMD was not different among the groups ([Fig. 2](#fig0010){ref-type="fig"}F). Our results suggest that perinatal and postnatal daidzein exposure show different effects on bone metabolism. Mardon et al. [@bib0100] reported that rats with perinatal or lifelong exposure to an isoflavone-rich diet had increased femoral and metaphyseal BMD at 24 months, but there was no significant effect at 3, 6, or 12 months. However, mice that are treated with daidzein during the prenatal period have lower femur and lumbar spine BMD than controls at 4 months of age [@bib0105]. Endogenous hormone status and the age at BMD measurement may affect experimental outcomes. The age at which BMD is most altered needs to be determined to fully understand how bone mineral accumulation is affected by prenatal or postnatal exposure to isoflavones. Bone quality is also important for assessing bone development. Further studies are required to confirm the effects of daidzein on bone development, such as measurement of biomechanical strength and bone microarchitecture.
In male rats in the Dz-S and Dz-L groups, mean serum osteocalcin concentrations on PND 22 were significantly lower than those in the Control group, but there was no difference in female rats among the groups. These sex-specific findings are consistent with those of previous studies using a mouse model in which exposure to daidzein (2 mg/kg body weight/day) from PNDs 1 to 5 improved BMD [@bib0040] and microarchitecture in the femur of female mice at 4 months of age, but it had minimal effects on bone development in male mice [@bib0045]. Reports on men with administration of aromatase and/like compounds have shown that estrogen is crucial for bone growth and development [@bib0110], [@bib0115], [@bib0120]. Estrogen withdrawal prevents epiphyseal fusion, which stops bones from lengthening, up-regulates bone resorption, and decreases BMD [@bib0125]. Therefore, exposure to estrogen-like compounds is crucial for healthy skeletal development in male mammals. In our study, daidzein showed different effects on bone metabolism in female rats compared with male rats. The reason for this finding may be because of the difference in types of sex hormones [@bib0055]. Additionally, the change in BMD in male rats on PND 77 observed in our study may have occurred as a consequence of programming effects in the fetal period. "The concept of programming" is that a stimulus or insult during a critical or sensitive period of development can have long-term or lifetime effects on an organism [@bib0130]. Therefore, hormonal signals operating during critical windows have numerous programming effects. Estrogen-signaling pathways and modes of epigenetic programming have provided some insight into the potential mechanism of action. By binding and activating nuclear receptors, isoflavones may interfere with hormonal signaling and/or the production of enzymes and transcription factors [@bib0135], [@bib0140]. During development, such as fetal and/or perinatal periods, endocrine hormones and enzymes can alter epigenetic regulation that controls gene transcription [@bib0145]. It is speculated that exposure to daidzein during the fetal period has induced irreversible effects on bone metabolism in this study. Further studies are required to confirm the effects of perinatal and peripubertal exposure to daidzein on bone development, as well as to determine the underlying mechanism.
Safety is an important issue to consider when extrapolating these findings to isoflavone supplements or soy-based infant formulas. We investigated the effect of daidzein on safety in addition to bone metabolism. We found no marked differences in the outcome of pregnancy, including the number of pups per litter, the sex ratio, and birth weight. Our findings are consistent with those of Ward et al. [@bib0045] who reported that no effects on those parameters were observed in exposure of pregnant mice to genistein (3.75 mg/day) and/or daidzein (3.75 mg/day) subcutaneously. Consequently, exposure of dams to daidzein does not appear to have a remarkable effect on pregnancy outcomes.
In our study, perinatal or peripubertal exposure to daidzein did not affect body weight in female rats. However, peripubertal exposure to daidzein in the Dz-L group increased body weight in male rats on PND 35 compared with the Control and Dz-S groups ([Table 1](#tbl0005){ref-type="table"}). There was no significant difference in male offspring body weight among the groups on PND 77 ([Table 1](#tbl0005){ref-type="table"}). In contrast to our findings, Ronis et al. [@bib0150] showed that male rats exposed to dietary isoflavones (80 mg/kg body weight/day) from PNDs 15 to 33 had reduced body weight at PND 33. In the present study, the daidzein dose for offspring in the Dz-S and Dz-L groups was calculated as approximately 51 mg/kg body weight/day, which was lower than that in the study by Ronis et al. [@bib0150]. Although a large number of studies have shown that exposure to isoflavones during the prenatal and/or postnatal period influence growth, the results are inconsistent.
In our study, the sex organ weights and AGD in male and female rats exposed to daidzein during the perinatal or peripubertal periods did not change with treatment ([Table 2](#tbl0010){ref-type="table"}). AGD is used as a measure of endocrine disruption. A longer AGD at earlier onset of puberty has been observed in female and male mice that were exposed to estrogen [@bib0155]. Our study indicated that exposure to daidzein during the perinatal period or during the perinatal period and after weaning might not affect parameters of endocrine disruption.
In our study, pregnant rats in the Dz-S and Dz-L groups were fed approximately 50 mg daidzein/kg body weight/day (approximately 15 mg/day), and daidzein and equol were detected in the stomach contents of suckling pups ([Fig. 3](#fig0015){ref-type="fig"}). Although daidzein is transferred from lactating mothers to suckling pups [@bib0075], our results suggest that equol is also transferred from mothers to suckling pups via the breast milk of dams that are fed a diet containing daidzein. Moreover, a similar transfer from mother to fetus has been reported in rats and humans [@bib0075], [@bib0080], [@bib0085]. Daidzein is rapidly transferred from the maternal circulation to the fetus, and elevated serum daidzein levels are present in rat fetuses from mothers that were fed isoflavones [@bib0075], [@bib0080], [@bib0085], [@bib0160]. One of the main metabolites of daidzein is equol, which is thought to be the most potent modifier of the effects of isoflavones. Equol might mediate the effects of daidzein in sex organ development and bone metabolism in rat fetuses and offspring.
In our study, serum daidzein and equol concentrations in offspring on PNDs 22, 35, and 77 were detected. A previous study showed that circulating daidzein levels in rats on PND 21 were similar to those in human infants (1.16 ± 0.09 μmol/L) who were fed a soy protein-based formula [@bib0160]. Serum daidzein concentrations in rat offspring on PNDs 35 and 77 in our study are several times higher than those in Asian people [@bib0165], [@bib0170]. The dose of isoflavones that was administered in the study was much higher than those from ordinary soy products. However, the dose of soy isoflavones used in this study could be obtained by isoflavones supplements. Our results suggest that excessive intake of isoflavones during the prenatal and postnatal periods should be carefully assessed in further studies. Further investigation concerning the safety and efficacy of soy products fortified with isoflavones and extracted supplements remains a research priority, particularly because of their widespread availability and growing use.
5. Conclusions {#sec0085}
==============
In conclusion, we assessed the safety and efficiency of perinatal or peripubertal exposure to daidzein on bone and reproductive organ development at early adulthood in rats. Perinatal exposure to daidzein does not confer a positive effect on BMD in either male or female rats on PND 35. However, peripubertal exposure to daidzein protects against a decline in BMD in male and female rats on PND 35. Perinatal exposure to daidzein leads to reduced femoral BMD in male rats, but not female rats, on PND 77. Therefore, the effects of daidzein on bone metabolism during development may depend on the timing of exposure and sex. Exposure to daidzein during the perinatal or peripubertal period does not have serious adverse effects on sexual development of offspring. Further investigations should assess the mechanisms underlying these responses of bone metabolism to daidzein, as well as the safety of daidzein exposure during the perinatal period and throughout life.
Conflict of interest {#sec0090}
====================
The authors have no conflicts of interest.
Transparency document {#sec0095}
=====================
Transparency document
This study was supported by grants from the MEXT-Supported Program for the Strategic Research Foundation at Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (S1101015). Excellent technical assistance was provided by Chiharu Yamazaki. The authors would like to thank Dr Sachie Ikegami, Otsuma Woman\'s University, for her advice on the research.
[^1]: All values are means ± SE (*n* = 6). Values that do not share the same superscript letters are significantly different from each other (*p* \< 0.05). Data were analyzed using one-way ANOVA. Differences between the groups were assessed by Tukey\'s test.
[^2]: Dams and their offspring fed the control diet (Control); dams fed the daidzein diet (0.5 g daidzein/kg diet) during pregnancy then the Control diet at postnatal day (PND) 13 and their offspring fed the Control diet (Dz-S); and dams and their offspring fed the daidzein diet (Dz-L).
[^3]: PND: postnatal day.
[^4]: All values are means ± SE (*n* = 6). Data were analyzed using one-way ANOVA. Differences between the groups were assessed by Tukey\'s test (*p* \< 0.05).
[^5]: Dams and their offspring fed the control diet (Control); dams fed the daidzein diet (0.5 g daidzein/kg diet) during pregnancy then the Control diet at postnatal day (PND) 13 and their offspring fed the Control diet (Dz-S); and dams and their offspring fed the daidzein diet (Dz-L).
[^6]: PND: postnatal day.
[^7]: All values are means ± SE (*n* = 6). Values that do not share the same superscript letters are significantly different from each other (*p* \< 0.05). Data were analyzed using one-way ANOVA. Differences between the groups were assessed by Tukey\'s test.
[^8]: Control; the group fed the control diet, Daidzein Short; the group fed the daidzein diet (0.5 g daidzein/kg diet) during both pregnancy and lactation periods, Daidzein Long; the group fed the daidzein diet (0.5 g daidzein/kg diet) during pregnancy, lactation and postweaning periods.
[^9]: PND: postnatal day.
| {
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Inappropriate use of antimicrobials is linked to the emergence of multidrug-resistant pathogens and adverse effects in patients \[[@CIT0001]\]. Antimicrobial stewardship programs, directed at reducing the inappropriate use of antimicrobials, are becoming mandated in US healthcare systems \[[@CIT0002]\]. Although education of clinicians is considered to be an essential element of any program designed to influence prescribing behavior \[[@CIT0003]\], there has been less attention paid to teaching predoctoral students the principles of appropriate antimicrobial use, particularly early in training \[[@CIT0004]\]. A survey of preclinical medical student microbiology curricula reported that only 65% of schools are teaching antimicrobial stewardship during their courses \[[@CIT0005]\]. Ninety percent of 317 senior medical students and 89% of 579 graduating pharmacy students reported that they would like more education on responsible use of antimicrobials \[[@CIT0006], [@CIT0007]\]. However, the Infectious Diseases Society of America's position statement, "Stewardship Commitments for the White House Forum on Antibiotic Stewardship" published in 2015 did not include education of health professional students as one of its commitments \[[@CIT0008]\].
Research on antimicrobial prescribing practices by physician trainees indicates that norms around "prescribing etiquette" can interfere with the optimization of antimicrobial therapy as well as a disconnect between microbiology coursework and the practical challenges of antimicrobial prescribing \[[@CIT0009]\]. Early introduction of core concepts of antimicrobial stewardship might normalize the idea of appropriate antimicrobial use as a shared responsibility and increase the acceptance of stewardship interventions. An interprofessional approach to stewardship is recommended by multiple professional and governmental organizations as an essential feature of a successful antimicrobial stewardship program \[[@CIT0003], [@CIT0012]\]. Because antimicrobial stewardship is explicitly an interprofessional activity, educating students about stewardship in an interprofessional environment can provide a more authentic simulation of practice. Moreover, standards in medical and pharmacy undergraduate education emphasize the importance of early interprofessional experiences \[[@CIT0013]\].
We aimed to address these deficiencies in the literature by (1) developing and implementing a joint interprofessional curriculum in 2 professional schools to enable early learners to appreciate the importance of rational antimicrobial use as healthcare professionals and (2) determining the effect of this activity on knowledge and attitudes towards interprofessional healthcare teams. We hypothesized that an interprofessional curriculum that models an authentic work experience can be successfully implemented and developed in preclinical undergraduate medical and pharmacy education.
METHODS {#s1}
=======
Design and Setting {#s2}
------------------
The learning objectives for the curriculum were developed by 1 infectious diseases specialist pharmacist (C. M.) and 2 infectious diseases physicians (B. S. S., P. V. C.-H.) after a focused needs assessment and review of the relevant literature \[[@CIT0016]\]. The study took place during the 2013--2014, 2014--2015, and 2015--2016 academic years at the University of California, San Francisco (UCSF). The study was granted exempt review status from the UCSF Institutional Review Board.
Curriculum {#s3}
----------
Participants of the curriculum were second-year medical students and third-year pharmacy students at UCSF. The curriculum was embedded into the courses they were taking on microbiology and infectious diseases (medical students) or antimicrobial pharmacology (pharmacy students). Attendance at the interprofessional workshop was a course expectation; students were offered a small amount of bonus credit for completing the pre- and postworkshop surveys.
The curriculum ([Figure 1](#F1){ref-type="fig"}) was designed as follows and delivered through the Moodle learning management system (Moodle Pty Ltd, Perth, Australia): (1) students viewed a case vignette online (Case 1) and answered related clinical questions; (2) an online independent learning module on antimicrobial stewardship was subsequently provided; then, (3) approximately 1 week later, the students participated in a 2-hour workshop on antimicrobial stewardship. The curriculum, including learning objectives, learning module, interactive case vignettes, and instructor materials, are freely available for access at the following site: <http://tiny.ucsf.edu/stewardship>. The workshops were led by a physician or pharmacist with expertise in antimicrobial use and consisted of approximately 12 students. Students were subdivided into mixed-professions small groups of 4--5 students. In small groups, students revisited Case 1 from the online module and came to a consensus group answer, which was then discussed with the larger group. The small groups then worked through Case 2 together again followed by group discussion. The cases used a branched-logic format: the decisions students made in the initial stages of the case determined the subsequent choices and case outcomes. The decisions required in the cases reflected key considerations in antimicrobial stewardship: whether to initiate treatment, impact of patient allergies and drug interactions on antimicrobial selection, adequacy of empiric spectrum of activity, and de-escalation of therapy based on culture results.
{#F1}
Assessment {#s4}
----------
Each student completed a survey of knowledge and attitudes towards antimicrobial use, antimicrobial resistance, and interprofessional practice before and after participation in the curriculum; the after survey included 2 additional items asking for them to rate the value of the experience ([Figure 1](#F1){ref-type="fig"}). The survey instrument consisted of 19 Likert scale items asking for the level of agreement with statements related to antimicrobial use and resistance and interprofessional collaboration. The items related to antimicrobial use were adapted from a survey of physician attitudes toward antimicrobial prescribing and resistance \[[@CIT0017]\]. The items related to interprofessional collaboration were adapted from a validated survey of attitudes towards interprofessional learning and interaction \[[@CIT0018]\]. For purposes of primary analysis, the data were dichotomized into answers of "agree/strongly agree" versus "neutral/disagree/strongly disagree".
Analysis {#s5}
--------
Analysis of changes in student attitudes before and after the survey was performed using McNemar's test or the paired *t* test to account for the paired nature of the data. Interaction between study year and student profession with prepost survey changes were explored using mixed-effects logistic regression models. All analyses were performed with Stata SE software (version 13; StataCorp, College Station, TX).
RESULTS {#s6}
=======
A total of 822 students participated over the 3 study years (470 medical students, 352 pharmacy students); of these, 745 (91%) completed both pre- and postcurriculum surveys. Statistical analysis was performed on these 745 participants (425 medical students, 320 pharmacy students). The survey results are displayed in [Table 1](#T1){ref-type="table"}; results were similar across study years and thus pooled results are presented. The first 5 questions concerned perceptions of antimicrobial use and resistance. Attitudes were not significantly different between pre- and postsurvey answers, with the exception of the perception of patient demand being the major reason physicians prescribe unnecessary antibiotics, which increased (42.2% to 59.1%, *P* \< .001). There was no evidence of interaction between profession and changes in attitudes.
######
Survey Responses Before and After Stewardship Curriculum
Question %Agree/Strongly Agree (n/N)
---------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------- ------------------- --------
Knowledge and attitudes about antibiotic resistance
Antibiotic resistance is a major public health problem 99.1 (763 of 770) 99.2 (738 of 744) .76
Overprescribing of antibiotics is a major cause of antibiotic resistance 95.7 (736 of 769) 98.9 (735 of 743) .002
Physicians should only consider the needs of the individual patient when prescribing antibiotics 26.0 (200 of 769) 27.8 (206 of 742) .19
I am confident the development of new and effective drugs will keep pace with the growing rate of antibiotic resistance 14.7 (113 of 770) 18.7 (139 of 744) .02
Patient demand is the major reason that physicians prescribe unnecessary antibiotics 39.8 (306 of 769) 54.1 (402 of 743) \<.001
Perceived efficacy in interprofessional collaboration
I am able to describe the role of each profession in appropriate antibiotic use^b^ 34.2 (263 of 769) 82.4 (613 of 744) \<.001
I can communicate in a manner that engages the interprofessional team 75.4 (580 of 769) 94.4 (702 of 744) \<.001
I can describe collaborative approaches to appropriate antibiotic use^c^ 48.6 (374 of 769) 92.2 (435 of 741) \<.001
Attitudes towards interprofessional learning and collaboration
My skills in communicating with patients of clients would be improved through learning with students from other healthcare professions 84.4 (648 of 768) 91.9 (685 of 744) \<.001
My skills in communicating with other healthcare professionals would be improved through learning with students from other healthcare professions 89.7 (689 of 768) 94.2 (699 of 742) \<.001
I would prefer to learn only with peers from my own profession 5.1 (39 of 770) 8.9 (67 of 744) .005
Learning with students from other healthcare professions is likely to facilitate subsequent working professional relationships 83.2 (637 of 766) 91.4 (681 of 745) \<.001
Learning with students from other healthcare professions would be more beneficial to improving my teamwork skills than learning only with my peers 80.6 (620 of 769) 90.2 (672 of 745) \<.001
Collaborative learning would be a positive learning experience for all healthcare students 86.1 (661 of 768) 92.7 (690 of 744) \<.001
Learning with students from other healthcare professions is likely to help to overcome stereotypes that are held about the different professions 79.5 (611 of 769) 88.5 (659 of 745) \<.001
I would enjoy the opportunity to learn with students from other healthcare professions 83.5 (643 of 770) 91.5 (681 of 744) \<.001
Learning with students from other healthcare professions is likely to improve the service for patient of client 86.5 (665 of 769) 92.9 (692 of 745) \<.001
^a^ *P* value is based on McNemar's test for students with paired data.
^b^ *P* = .03 for interaction between student profession and paired change in response; medical students increased agreement to a greater extent than pharmacy students.
^c^ *P* = .03 for interaction between student profession and paired change in response; medical students increased agreement to a greater extent than pharmacy students.
Three questions asked about students' self-perceived efficacy in interprofessional collaboration; for all of these items, there were significant increases in students' perceived abilities. In the preworkshop survey, less than half as many medical students as pharmacy students believed they could describe the role of other professions in appropriate antibiotic use (22.1% vs 58.1%). Both groups increased in their self-reported abilities on this question in the postsurvey (to 78.1% for medical students and to 87.9% for pharmacy students), with a greater increase seen in medical students (*P* = .03 for interaction). Medical students also had a greater increase in their ability to describe collaborative approaches to appropriate antibiotic use (40.4% to 90.9%) compared with pharmacy students (59.8% to 92.8%, *P* = .03 for interaction).
Among the 9 questions primarily concerned with attitudes towards interprofessional learning and collaboration, students generally expressed more favorable attitudes towards interprofessional learning and collaboration. Despite the overall favorable attitudes, the percentage of students who agreed or strongly agreed that they would prefer learning from peers from their own profession increased overall by 3.8% (*P* = .005); there was no evidence of interaction by student profession by this measure. Unlike the other survey items related to interprofessionalism, a rating of "Strongly Agree" represented a less favorable attitude towards interprofessional collaboration, so it is possible that this finding is an artifact of the reversal of the scale \[[@CIT0019]\].
On the postcurriculum questionnaire, 84.5% (616 of 729) of students agreed or strongly agreed that completing the online learning module was a valuable learning experience, and 92.7% (676 of 729) belived that the small group workshop was a valuable learning experience. Workshop favorability ratings were high across all student-preceptor pairs, ranging from 86.7% for medical students with pharmacist preceptors to 96.3% for pharmacy students with physician preceptors. Invited open-ended student comments were highly favorable; common themes were the authenticity of the scenarios relative to other interprofessional education exercises and the recognition of the complementary knowledge that medical and pharmacy students each brought to bear on the problems. Some students believed that pharmacy students were disadvantaged because they had not yet completed their instruction in infectious diseases, whereas the medical students had.
DISCUSSION {#s7}
==========
We demonstrated that an interprofessional curriculum on antimicrobial stewardship, including medical and pharmacy students, can positively impact knowledge and attitudes towards both antimicrobial stewardship and interprofessional collaboration. Given the emphasis placed on antimicrobial stewardship as a key public health strategy \[[@CIT0020]\], interventions that can raise awareness of stewardship and improve health professional teamwork in this area may carry a significant impact. The curriculum was also well received by learners from both professions regardless of the profession of their workshop leader.
Several studies have identified deficiencies in knowledge and behaviors of health professions trainees in antimicrobial use \[[@CIT0006], [@CIT0007], [@CIT0021]\], leading some authors and organizations to recommend a greater focus on these topics in the undergraduate curriculum \[[@CIT0008], [@CIT0024], [@CIT0025]\]. Equally important to improving knowledge is being able to apply it in the clinical environment, which requires an understanding of each clinician's role in appropriate antimicrobial use. Some components of antimicrobial stewardship are taught in a majority of United States and United Kingdom medical and pharmacy schools \[[@CIT0005], [@CIT0026], [@CIT0027]\]. However, content delivery is largely lecture-driven, only a minority of schools covered all recommended stewardship principles, and few programs offer opportunities for interprofessional education in stewardship. The current study suggests that an interprofessional curriculum on stewardship where students collaborate on authentic case simulations can substantially improve students' knowledge of and comfort with their roles in and knowledge of antimicrobial stewardship.
This was a single-center study at an institution with medical and pharmacy undergraduate schools at the same campus, which enabled us to recruit a large number of physician and pharmacist workshop leaders. We recognize that some medical and pharmacy schools are not geographically accessible to each other to engage in this type of learning activity. However, because interprofessional educational experiences are mandated by the accrediting bodies for medicine \[[@CIT0014]\] and pharmacy \[[@CIT0015]\], there is strong pressure for creative solutions to expand the interprofessional education space. We encourage schools to identify ways to have medical students to interact with pharmacists involved in stewardship programs and for pharmacy students to be partnered with physicians involved in stewardship programs. At minimum, during the preclinical years when stewardship concepts are being introduced, it would be ideal to invite a colleague from the other discipline to be serve as a coleader of these sessions, to highlight the importance of interprofessional collaboration in stewardship. Given that a portion of this curriculum was delivered through an online learning platform, distance collaboration may be another method to involve learners from different professions.
Learner knowledge of antimicrobial stewardship concepts was assessed by a small number of survey items. This was driven by a limited number of available survey instruments in the literature related to antimicrobial stewardship appropriate to undergraduate health professions population and by a desire to limit the overall length of the survey to improve response rates. Future implementations of the curriculum will include a more robust assessment of antimicrobial stewardship knowledge.
CONCLUSIONS {#s8}
===========
This study adds to the limited literature available on antimicrobial stewardship education for undergraduate health professionals, and, to the best of our knowledge, it is the only study of an intervention targeting these learners. We hope that our curriculum can serve as an exemplar for both stewardship and interprofessional education activities at other institutions.
We acknowledge Linda Brinen for assistance in implementation of the curriculum and the faculty of the University of California, San Francisco (UCSF) Teaching Scholars Program for feedback on the project proposal.
*Finanical support.*
This study was supported by a grant from the UCSF Center for Innovation in Interprofessional Education and the UCSF Gold-Headed Cane Endowed Teaching Chair in Internal Medicine.
*Potential conflicts of interest.*
All authors: No reported conflicts.
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]: Correspondence: C. MacDougall, PharmD, MAS, Professor of Clinical Pharmacy, University of California San Francisco School of Pharmacy, 533 Parnassus Ave, U-585, Box 0622, San Francisco, CA 94044 (<[email protected]>).
| {
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The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.
Introduction
============
Avascular necrosis (AVN), also known as osteonecrosis, is caused by insufficient blood supply to the bone ultimately leading to ischemic cell death \[[@REF1]\]. Increased osteoclastic activity attempts to remove necrotic bone, and increased osteoblastic activity occurs to repair the damage. Ultimately, the bone structures collapse; in the case of the proximal femur, this results in progressive pain exacerbated by weight-bearing and loss of joint function.
AVN most commonly affects bones with a single terminal blood supply and limited collateral circulation, such as the femoral head \[[@REF2]\]. The postulated mechanisms of interruption of blood flow include: vascular occlusion, altered lipid metabolism, and intravascular coagulation \[[@REF3]\]. Some of the most common causes of non-traumatic AVN of the femoral head include corticosteroids, sickle cell anemia, Gaucher's disease, connective tissue disorders, radiotherapy, and alcohol consumption \[[@REF1]-[@REF4]\], although it may be aggravated by obesity, and can also be idiopathic \[[@REF5]\]. Individual patients usually have more than one risk factor, indicating that the pathogenesis is likely multifactorial \[[@REF6]\].
The exact incidence of AVN in the setting of malignancy remains unclear. Over a six year period in which pelvic x-rays were performed routinely prior to radiotherapy (RT) and yearly thereafter, 18/568 (3.2%) patients treated for gynecologic malignancies developed serious bone sequelae, with AVN diagnosed in 0.5% of patients treated for cervical cancer \[[@REF7]\]. In that study, AVN most commonly occurred between the ages of 40 and 60 \[[@REF7]\].
Most patients present late in the disease course. Histology is the gold standard for diagnosis but is usually unnecessary and not routinely performed. The radiographic appearance depends on the stage and extent of the lesion \[[@REF8]\]. Early, there is an increase in density of the affected area made more prominent by surrounding osteoporosis. Cystic changes appear that represent areas of absorption of dead bone, and varying degrees of collapse are seen \[[@REF8]\]. Recovery may be possible prior to femoral head collapse. Without treatment, the process is almost always progressive, leading to irreversible joint destruction \[[@REF5]\].
In a prospective study in which 72 femoral heads with early AVN underwent serial magnetic resonance imaging (MRI) evaluated by blinded review, prognostic factors for the highest rates of \"clinical and radiographic deterioration,\" were investigated. Clinical deterioration was defined as development of symptoms, while radiologic deterioration was defined as femoral head collapse. Stage at diagnosis, necrosis or more than two-thirds of the weight-bearing area, and lateral involvement (compared with medial lesions) correlated with worse outcomes \[[@REF9]\].
RT is often cited as a risk factor for the development of AVN \[[@REF3]\]; however, it is unclear whether the relationship is a stochastic or deterministic (non-stochastic) one. A stochastic, or probabilistic effect, has no dose threshold below which the effect does not occur, and the probability of occurrence increases with increased dose. Severity of the effect is independent of dose; the classic example is RT-induced carcinogenesis. Conversely, a deterministic effect has a practical threshold, above which the severity of harm increases with dose, as is the case with cataracts \[[@REF10]\].
In this case report, we describe a patient with prostate cancer metastatic to bone who was diagnosed with AVN subsequent to receiving RT that included the left femoral head. This patient provided consent for his deidentified clinical information to be reported.
Case presentation
=================
Our patient is a 51-year-old non-smoking, non-obese (BMI 24.8) male, who worked as a surveyor, with comorbidities of hypertension, osteoarthritis, peptic ulcer disease, cataracts, left ankle surgery, migraines, depression, remote motor vehicle accident without lower extremity trauma, and minimal ethanol use (1-2 alcoholic beverages/month). He presented in 1994 with localized prostate cancer (PSA 15 ng/L; Gleason 3+4=7). He completed six months of neoadjuvant androgen deprivation therapy (cyproterone acetate) with a clinical and biochemical response, followed by radical prostatectomy with bilateral pelvic lymph node sampling confirming pT2cN0 (0/14) Gleason grade 3+4=7 adenocarcinoma. PSA nadir was 0.2.
By early 1997, he had experienced an asymptomatic biochemical recurrence. Restaging transrectal ultrasound (TRUS), computed tomography (CT), and bone scan were negative. A salvage prostate bed RT was recommended but declined by the patient, so he was started on diethylstilbestrol 0.1 mg od and megestrol 40 mg TID. These were discontinued within six months due to fatigue and depression in favour of watchful waiting. Further imaging between 1998 and 2001 did not reveal evidence of bone metastases despite further biochemical progression (Figure [1](#FIG1){ref-type="fig"}). During this time, he participated in a clinical trial briefly (\<6 months) but was withdrawn due to non-compliance in early 2002. Ultimately, he agreed to intermittent cyproterone, acknowledging that it was not standard of care at the time.
Figure 1PSA values over time.Given the absolute values, it is likely that the patient's aggressive prostate cancer was non-PSA-secreting.
The first diagnosis of bone metastases occurred on routine restaging in January 2004. Over the next year, he developed a significant burden of bone disease but agreed to megestrol and bicalutamide in various sequences and combinations, along with analgesics (Table [1](#TAB1){ref-type="table"}).
Table 1Morphine equivalent daily dose and steroid equivalents received.\*Gabapentin started in escalating doses. \*\*Breakthrough dose/frequency not specified so cannot be included in total MEDD. \^Diagnosed with pulmonary embolus. ¥ Received 1 dose during emergency room visit. Abbreviation: dex -- dexamethasone.DateEstimated MEDD\*\*SteroidsAnti-CoagulationJuly-Sept 200712mgDex 16mg IV-Jan 200922.1mg\--Feb 20098.8mg-Lovenox 120mg SQ od Feb 200918.9mg-July 201012.6mg-Oct 201037.8mgDex 80mgDec 2010\*9.5mgDex 4mg po prn\*\*Feb 201125.2mg-Mar 201148mg-Mar 201160mg-April 2011132mgDex 40mgMay 2011120mg-Aug 2011144mg-Aug 2011144mg-Oct 2011223.5mg-Dec 2011308mg-
He received a total of six months of depot luteinizing hormone-releasing hormone (LHRH) agonist with some prostate-specific antigen (PSA) and symptom control, but did not elect to continue due to side effects. Over the next two years, this patient initiated and discontinued multiple medications of his own accord, sometimes in direct contravention to his care team's advice.
After being diagnosed with a pulmonary embolus (February 2009), he initiated self-administration of subcutaneous anticoagulation which he continued for the remainder of his life (Table [1](#TAB1){ref-type="table"}). By October 2009, he was declared hormone-refractory but wished to remain on intermittent depot LHRH agonist only. By the spring of 2010, with progressive bone pain, he agreed to a Radiation Oncology referral.
He would go on to receive palliative external beam RT to various anatomical locations on six different occasions (Figure [2](#FIG2){ref-type="fig"}).
Figure 2Palliative radiotherapy received.Please note, this patient has six lumbar-type vertebrae. \*Femoral heads excluded. Abbreviation: incl -- inclusive; SI -- sacroiliac.
He received eight Gray (Gy) in one fraction to the left hip and proximal femur via an anterior/posterior approach in July 2010 and sustained an excellent pain response. He also received 8 Gy/1 to the right hemipelvis in April 2011 and 6 Gy/1 to the right hip and proximal femur in January 2012. The lifetime doses received by the left and right femoral heads were 8.8 Gy and 15.3 Gy respectively (Figure [3](#FIG3){ref-type="fig"}).
Figure 3Composite dose color wash incorporating all palliative RT delivered to lumbar spine and pelvis.
By August 2011, with a deteriorating performance status, he was discharged from the Medical Oncology service. He went on to require multiple emergency room visits for pain control in two different provinces, and progressed to requiring a walker for ambulation. His pain was managed in the community with opioid rotation and escalation (Table [1](#TAB1){ref-type="table"}). There is no record of him receiving bisphosphonates, vitamin D, or prednisone (for the purposes of disease control) at any time.
He had onset of left lateral hip pain radiating inferiorly to the knee, exacerbated by walking and other physical activity, in the fall of 2011. An abdominal flat plate performed primarily to investigate fecal loading (August 2011) had not revealed abnormalities in the left hip. A bone scan (November 2011) revealed new area of uptake in the anterior-superior left femoral head with AVN in the differential diagnosis (Figure [4](#FIG4){ref-type="fig"}).
Figure 4Bone scintigraphy.4A: Bone scan performed prior to institution of RT to right pelvis showing absence of uptake on left side (Feb 23, 2011). 4B: Bone scan revealing an area of focal increased uptake in the anterior superior left femoral head in the setting of progressive bone metastases (Nov 3, 2011).
Plain films conducted three weeks later demonstrated slight flattening of the superior articular surface of the left proximal femoral head, accompanied by mild juxtaarticular sclerosis and cysts, in keeping with early AVN. In retrospect, these changes could be identified on the August imaging (Figure [5](#FIG5){ref-type="fig"}).
Figure 5Plain x-rays.5A: Abdominal plain film (August 11, 2011) demonstrating normal hip joints. 5B: Pelvis plain film (Nov 22, 2011) revealing flattening of the superior articular surface of the left femoral head accompanied by mild juxtaarticular sclerosis and small juxtaarticular cysts (arrow) in keeping with early avascular necrosis.
The diagnosis occurred approximately 15 months after RT was delivered to the left hip.
The patient declined further investigations including MRI. By January 2012, he was describing a constant dull ache in the left hip without neuropathic features and was walking with a pronounced limp. He described a "mushing" feeling especially when lying in a left lateral decubitus position. Two further bone scans were reported as stable in the left femoral head, with progression in his widespread bone metastases elsewhere. But CT pelvis and plain films demonstrated dramatic progression in mid 2012 and early 2013, respectively (Figure [6](#FIG6){ref-type="fig"}).
Figure 6Evolution of presumed avascular necrosis.6A: CT pelvis eight months after AVN diagnosis (July 23, 2012). The left hip demonstrates superior asymmetric joint space narrowing, with dense subarticular sclerosis and irregularity of the articular surface of the femoral head, in keeping with AVN. There is extensive dense sclerosis of the right pubic bone plus scattered sclerotic areas in both iliac bones and the left pubic bone suspicious for metastases. 6B-D: Pelvic x-rays fourteen months after AVN diagnosis (Jan 8, 2013), demonstrating severe narrowing of the joint space superiorly and laterally. Sclerosis and subchondral cyst formation is seen on both sides of the joint space. The superior articular surface is disrupted and depressed.
Taking into account his limited life expectancy due to the absence of further therapeutic options for his malignancy, the potential complications associated with surgery, and the anticipated length of post-surgical rehabilitation, hip replacement surgery was not pursued. Symptom control and supportive care were continued and the patient passed away due to his prostate cancer on April 5, 2013, approximately 17 months after the diagnosis of AVN.
Discussion
==========
Changes in the bone caused by radiation have been termed radiation osteitis, radiation necrosis of bone, osteonecrosis due to radiation and osteoradionecrosis \[[@REF11]\]. Ewing ascribed the effect of RT on bone to both interference with its nutrition as a result of obliteration of the vascular supply, and to secondary irradiation related to the high calcium content \[[@REF12]\]. Direct destruction of osteoblasts and the impairment of the regenerative response to radiation injury from compromised circulation are also likely to contribute \[[@REF13]\]. After irradiation, the normally orderly interplay between bone formation and resorption is lost, with delayed remineralization and weakening as result \[[@REF14]\].
We present a patient with prostate cancer metastatic to bone, who developed atraumatic AVN of the left femoral head approximately 16 months after receiving single fraction palliative-intent RT to this region. Although MRI with gadolinium contrast is considered the imaging procedure of choice, our patient declined further investigations. On balance, the diagnosis was felt to be in keeping with a predominantly AVN picture, rather than one of progressive bone metastases although we cannot absolutely exclude some contribution from the latter (Table [2](#TAB2){ref-type="table"}). Data in Table [2](#TAB2){ref-type="table"} is adapted from \[[@REF15]\].
Table 2Differentiating metastases versus avascular necrosis.Adapted from \[[@REF15]\]. MetastasisRadiation-Induced AVNAssociated soft tissue massPossiblyNoType of lesionLytic or sclerotic in otherwise normal bone with a transition zone appreciable between normal and abnormal boneSclerosis without cortical destruction; fractures can occur in demineralized (as opposed to lytic) areasInner cortex / iliopectineal lineMay be breachedPreservedLocationAny bone via hematogenous spreadAny bone with precarious blood supply within radiation portalsTime intervalAnyUsually \>1 year post-radiation
Although our patient never received systemic cytotoxic chemotherapy, bisphosphonates or other bone-modifying agents, he did receive a cumulative steroid dose equivalent to just over 9 g of dexamethasone over four years. Other potentially relevant risk factors suggested in the literature include hypercoaguability \[[@REF8]\], although he was on therapeutic doses of anticoagulation for almost three years prior to his AVN diagnosis. An additional theory is that of osteoporosis caused by long-term androgen deprivation therapy contributing to microscopic fractures of weakened bony trabeculae, possibly exacerbated by diminished sensibility from the anti-inflammatory effects of steroids \[[@REF8],[@REF16]\]. However, his hormonal deprivation was intermittent and incomplete, and his lifetime steroid dose relatively low. Marrow infiltration may also have played a role in weakening of the bone. Additionally, some individuals do not have blood vessels in the ligamentum teres at all, making them particularly susceptible to AVN \[[@REF7]\]. Finally, idiopathic AVN of the femoral head has also been described \[[@REF5],[@REF8]\], and therefore the RT may have been completely causally unrelated.
Irradiation of mature bone causes radiographically demonstrable atrophic changes, likely due to osteoblast damage, with a threshold proposed of 4000 rads (equivalent to 4000 cGy) \[[@REF11]\]. According to currently accepted normal structure tolerance guidelines, there is a 5% risk of AVN if the entire femoral head receives 52 Gy, which rises to a risk of 50% after 65 Gy \[[@REF17]\]. However, much smaller radiation doses---as low as 2500 rads---are sufficient to initiate changes in the endothelium of local blood vessels \[[@REF18]\].
There is still no satisfactory explanation as to why one patient develops AVN after RT while another who has received a similar dose does not \[[@REF7]\]. It is also difficult to explain unilateral AVN in the setting of identical doses received by the contralateral femoral head \[[@REF7]\]. In terms of the role of RT in our case, the dose absorbed by his left femur was not considered to exceed the tissue tolerance of normal bone. In fact, it was also lower than the maximum dose received by his contralateral femoral head, which did not go on to develop AVN.
The possible explanation relates to the development of our patient's progressively worsening bone metastases in the *right*hip, which required multiple courses of palliative RT. This led to increasing stresses being placed on the left hip as he attempted to favour the right side. As dead bone is being revascularized and remodeled, the forces involved in weight-bearing contribute to collapse and fracture \[[@REF9],[@REF13]\]. Indeed, weight-bearing has been described as a repetitive traumatic insult, capable of exacerbating an evolving picture of AVN \[[@REF2]\], and even repeated slight injury to tissues with a diminished capacity for recovery have been blamed for instigating AVN \[[@REF5],[@REF19]\].
It is therefore likely that AVN occurs when a number of etiological factors coincide \[[@REF6]\], as supported by long average latent period to the development of this complication \[[@REF20]\]. Although the incidence of radiation-induced fracture increases after higher bone doses \[[@REF7]\], the relationship between dose and development of AVN is not as clear-cut given the wide variety of associated total doses reported in the literature (Table [3](#TAB3){ref-type="table"}).
Table 3Biologically equivalent dose estimated from reported cases of avascular necrosis within radiated hip(s) where systemic therapy was explicitly stated as not delivered.\*Maximum dose (or prescribed dose if dmax not available). \*\*Not enough information to calculate equivalent steroid dose. Abbreviations: C -- cyproterone; P -- prednisone.ReferenceHistologyHip(s) AffectedFemoral Head Dmax\*Estimated BED (Gy2)Systemic ChemotherapyDexamethasone Equivalent Received Prior to Diagnosis of AVNPresent caseProstateLeft8.8 Gy47.5 Gy2No9.1gMacdonald patient 2 \[[@REF21]\]ProstateRight20 Gy70 Gy2NoC\*\*Thorne patient 12 \[[@REF22]\]HDRight35 Gy65.6 GyNo0Macdonald patient 1 \[[@REF21]\]ProstateBilateral38.4 Gy61.4 Gy2NoC\*\*Phillips patient 3 \[[@REF23]\]EndometrialRight50 Gy65.6Gy2 + 15Gy via brachyNo0MacDougall patient 1 \[[@REF5]\]Testis teratomaLeft58.8 Gy96.4 Gy2No0Kolin patient 10 \[[@REF24]\]EndometrialLeft65 Gy125.5 Gy2No0Kolin patient 11 \[[@REF24]\]ProstateRight65 Gy125.5 Gy2NoP\*\*Csuka patient 1 \[[@REF25]\]ProstateBilateral68 Gy128.8 Gy2NoNRKolin patient 5 \[[@REF24]\]BreastBilateral122 Gy244 Gy2No60g
Other case reports have described AVN after a dose as low as 1540 rads in the absence of systemic therapy \[[@REF7],[@REF26]\], and therefore it is likely that the total dose is not the predominant factor \[[@REF2]\]. In the absence of being able to conclude there is a dose ceiling below which AVN does not occur, as suggested by the present case and literature review, it is therefore likely that radiation-induced AVN is a stochastic effect.
Conclusions
===========
In conclusion, we report a case of AVN of the femoral head after palliative RT for metastatic prostate cancer. While it is not possible to establish that radiotherapy was the dominant cause, since the etiology in this patient is likely multifactorial, the possibility of some contribution must be considered. This is especially true in the absence of significant steroid doses expected to cause osteopenia, and without cytotoxic systemic therapy or bisphosphonates. At present, the RT dose threshold below which there is no risk for AVN is unknown, and therefore involvement by the radiotherapy cannot be definitively excluded. A wide variety of radiotherapy doses delivered to the femoral head have been related to subsequent AVN diagnoses, with our patient's dose the lowest of these.
Given the possibility that RT-induced AVN is a stochastic effect, this diagnosis should be considered in any patient with a painful hip who has received radiotherapy to the femoral head, especially in conjunction with other risk factors. Caution is required before assuming that progressive skeletal metastases are the cause, without adequate radiologic evidence excluding other etiologies. This is to avoid delivering unnecessary irradiation due to a misdiagnosis of bone metastases. An early detection will also allow for surgical intervention in eligible patients, therefore enhancing the quality of life and delaying or preventing irreversible femoral head collapse.
The authors have declared that no competing interests exist.
Patient signed consent form for use of deidentified clinical information in a case report prior to death. This can be uploaded if necessary (although does contain identifying information so I have not done this yet).
The authors wish to acknowledge the Alberta Cancer Foundation for the summer studentship support of AD.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s0005}
============
KEY TEACHING POINTS•An association between long QT syndrome and dilated cardiomyopathy has been previously described in the presence of SCN5A mutations and may represent a sodium channel "overlap syndrome" between channelopathies and cardiomyopathies.•It is possible that molecular interaction between potassium channel mutations and other mutations in the cardiac sarcomere and mitochondria may lead to a phenotypic overlap syndrome between long QT syndrome and cardiomyopathy.•Additional screening for cardiomyopathy may be warranted in patients with a variety of genetic channelopathies.
Congenital long QT syndrome (LQTS) is an inherited cardiac channelopathy characterized by prolongation of the QT interval on electrocardiogram (ECG), and is associated with an increased risk of life-threatening ventricular arrhythmias. Mutations in over a dozen distinct genes have been implicated in the pathogenesis of this group of disorders.[@bib1] LQTS type 1 (LQT1), the most prevalent LQTS subtype, is characterized by a heterozygous loss-of-function mutation in the KCNQ1 gene, which codes for the α-subunit of the delayed rectifier inward potassium ion channel. The association of LQTS with dilated cardiomyopathy (DCM) is rare but has been reported in the presence of sodium channel gene mutations, as seen in LQTS type 3. In this case report, we describe a patient with familial LQT1 (KCNQ1 mutation) identified in infancy who was subsequently diagnosed with severe DCM later in childhood.
Case report {#s0010}
===========
A 2-day-old male infant underwent cardiology evaluation owing to a family history of LQTS. Evaluation of the extended family had previously been remarkable for multiple family members, including his mother, having a prolonged QTc on ECG screening. The patient's ECG demonstrated a QTc of 495 msec and abnormal T-wave morphology ([Figure 1A](#f0005){ref-type="fig"}). He was admitted to the hospital for initiation of propranolol. Genetic testing of the patient and multiple first- and second-degree maternal relatives identified the presence of a deleterious genetic mutation, KCNQ1 Ser 349 Ter, consistent with LQT1. The patient's older brother, who had previously been thought to be unaffected, was also found to be positive for the mutation and started on medication. The patient was maintained on propranolol until 4 years of age, and nadolol thereafter. Throughout this time he was clinically well, without palpitations, syncope, or documented arrhythmias, although his QTc remained prolonged on serial ECG evaluations. He underwent formal exercise testing at 7 years of age, which was notable for a prolonged QTc throughout exercise and recovery. There were no arrhythmias and he had a normal oxygen consumption of 40.5 mL/kg/min. He was active in multiple recreational sports, including basketball, lacrosse, and baseball; an automated external defibrillator was available for emergency use. Repeat exercise testing was performed at 8 years of age, which again demonstrated a prolonged QTc without ventricular ectopy. Oxygen consumption was not measured, but his physical working capacity was described as low (work rate 67 watts).
At 9 years of age, the patient was admitted to the hospital complaining of several days of diffuse abdominal pain, vomiting, diarrhea, and fatigue. He was admitted to the general pediatric service with a presumed diagnosis of viral gastroenteritis and discharged home the following day after receiving intravenous hydration. He presented to the emergency room 1 week later with continued gastrointestinal symptoms, as well as worsening fatigue and dyspnea. The initial physical examination was notable for tachypnea and intermittent retractions. On auscultation, a 1/6 holosystolic murmur was heard at the apex, with no other abnormalities. Hepatomegaly with tenderness to palpation was present. The initial laboratory evaluation was notable for an elevated B-type natriuretic peptide of 8578 pg/mL. A chest radiograph showed increased interstitial markings without pulmonary edema or cardiomegaly. His ECG showed new T wave changes and voltage criteria for left ventricular (LV) hypertrophy, but no evidence of arrhythmia ([Figure 1B](#f0005){ref-type="fig"}). A transthoracic echocardiogram was performed and demonstrated severely diminished LV ejection with a markedly dilated left atrium, a mildly dilated left ventricle, and moderate to severe mitral regurgitation. The LV ejection fraction (EF), as estimated by Simpson's rule (biplane), was 15%. Right ventricular (RV) ejection was decreased as well, but not as markedly as that of the left ventricle. There was echocardiographic evidence of elevated pulmonary artery pressures with an RV pressure estimate of 38 mm Hg above central venous pressure and a pulmonary artery end-diastolic pressure estimated at 15 mm Hg using the modified Bernoulli equation ([Figure 2](#f0010){ref-type="fig"}). An infectious evaluation failed to show any evidence of active viral infection (blood polymerase chain reaction assays for adenovirus, influenza, parainfluenza, metapneumovirus, rhinovirus, enterovirus, cytomegalovirus, HHV-6, parechovirus, parvovirus B19, Epstein-Barr virus, and stool polymerase chain reaction assays for common gastrointestinal pathogens were all negative) or systemic inflammation (C-reactive protein \<0.5 mg/dL, erythrocyte sedimentation rate 0 mm/h) and the troponin I was 0.01 ng/mL. He was treated with 2 g/kg of intravenous immunoglobulin, without improvement.
A metabolic evaluation (lactate, pyruvate, acylcarnitine profile, blood and urine carnitine levels, plasma amino acids, urine organic acid, and creatine kinase) was negative. Commercial genetic testing consisting of DNA sequencing for 51 known cardiomyopathy genes was performed by the Laboratory for Molecular Medicine (Boston, MA) and was notable for a variant of unknown significance in the titin gene (p.Asn18096Lys). A Combined Mito Genome Plus Mito 140 Nuclear Gene Panel (GeneDx, Gaithersburg, MD) was also performed and showed heterozygosity for a variant of unknown significance in the ACO2 gene (p.Arg142Gln) and heterozygosity for a variant of unknown significance in the NDUFA10 gene (p.Arg337His). Cardiac magnetic resonance imaging (MRI) was obtained and demonstrated moderate to severe LV dilation (end-diastolic volume 165 mL/m^2^), severely diminished LV ejection (EF 21%), and moderately diminished RV ejection (EF 32%) without dilation or hypertrophy. MRI tissue characterization was limited owing to myocardial thinning, but the Lake Louise criteria for MRI diagnosis of myocarditis were not met.[@bib2] Based on these findings, a diagnosis of idiopathic DCM was made. Upon clinical improvement, milrinone was discontinued and he was transitioned to an oral regimen of enalapril, digoxin, nadolol, and enteral diuretics. Owing to his lack of arrhythmias and relative small size, placement of a primary prevention implantable cardioverter-defibrillator was deferred. He was discharged home after approximately 4 weeks, but required readmission for reinitiation of milrinone approximately 1 month later owing to recurrent symptoms of congestive heart failure. A cardiac catheterization was performed during that admission and was notable for preserved cardiac index of 3.6 L/min/m^2^ (on milrinone), elevated pulmonary capillary wedge pressure of 14 mm Hg, and normal pulmonary vascular resistance. Based on these findings and clinical status, the patient was listed for heart transplantation. He recently underwent an uncomplicated orthotopic heart transplant. Gross pathologic examination of the explanted heart was consistent with the diagnosis of DCM, with scattered myocyte hypertrophy on microscopic examination.
The patient's older brother, who also has the LQT1 mutation, was evaluated with screening echocardiography and has normal cardiac chamber size and systolic function. No other family members have been diagnosed with cardiomyopathy ([Figure 3](#f0015){ref-type="fig"}).
Discussion {#s0015}
==========
To our knowledge, this is the first report in the medical literature of an association between LQT1 and DCM. Our patient is unique, as he carries genotypic and phenotypic characteristics of LQT1 and went on to develop DCM without evidence of frequent ventricular ectopic beats or sustained arrhythmias, making a tachycardia- or PVC-induced cardiomyopathy unlikely. His inflammatory markers and infectious evaluation were negative, and cardiac MRI did not show convincing evidence of myocarditis; therefore, a concurrent myocarditis is highly unlikely. Our patient's presentation, therefore, is more consistent with an idiopathic DCM of subacute onset.
While a cardiac sodium channel "overlap syndrome" between channelopathies and cardiomyopathies has been described related to abnormalities in the SCN5A gene, no such association with DCM has been described with respect to the cardiac potassium channels involved in LQT1. The SCN5A gene encodes the α-subunit of the voltage-dependent cardiac sodium channel and has been implicated in a number of pathologic cardiac conditions, including LQTS type 3 (LQT3), Brugada syndrome, sick sinus syndrome, conduction system disease, sudden infant death syndrome, and DCM. A study by McNair et al[@bib3] found an SCN5A mutation prevalence of 1.7% in their multicenter cohort of 338 patients with DCM. While none of these patients had QTc prolongation, multiple case reports have described an overlap between ECG evidence of LQTS and DCM in patients with mutations in the SCN5A gene.[@bib4], [@bib5] The mechanism of overlap between channelopathy and cardiomyopathy in LQT3 is not well understood, but it has been proposed that ion channel mutations may ultimately result in structural changes to the myocardial tissue via their interaction with cytoskeletal proteins, either directly or as a result of altered ion homeostasis.[@bib6], [@bib7] KCNQ1 gene mutations have not previously been described in patients with DCM, but they have been implicated in other types of cardiomyopathies such as hypertrophic cardiomyopathy and LV noncompaction cardiomyopathy.[@bib8], [@bib9] The delayed rectifier potassium channel affected in LQT1 is one of several types of potassium channels responsible for reconstitution of the cardiac action potential, whereas the function of the SCN5A channel is absolutely necessary for depolarization and, hence, contractile function.[@bib10], [@bib11] It may be that, unlike sodium channel mutations, a single potassium channel mutation is insufficient to cause a significant effect on contractile function without additional abnormalities in cytoskeletal proteins or cardiac metabolism.
Genetic testing of known cardiomyopathy genes revealed genetic variants of unknown significance in mitochondrial genes involved in the electron transport chain and in titin, an autosomal gene coding for a sarcomere protein commonly implicated in DCM. It is possible that normally nonpathogenic variations in the titin gene can become disease-producing via interaction with mutations in other susceptible genes.[@bib12] It has been previously suggested that titin may influence activity of the delayed outward rectifying potassium currents affected in LQT1.[@bib13] While the KCNQ1 mutation was inherited maternally, the titin mutation was inherited paternally, and overlap between the KCNQ1 and titin mutations was not identified in any other maternal family member ([Figure 3](#f0015){ref-type="fig"}).
Conclusions {#s0020}
===========
While a mutation in KCNQ1 has not been previously described as an etiology for DCM, we hypothesize that the potassium ion channel could be involved in the pathogenesis of myocardial dilation and resultant ventricular dysfunction, either in isolation or via molecular interplay with other genetic variants of unknown significance. Similar to SCN5A, it is possible that a potassium channel overlap syndrome may exist. There has been little investigation into the role of the delayed rectifier potassium channel in heart failure.[@bib14] More research is needed into the possible pathogenic manifestations of this mutation within the cardiac myocyte. From a clinical standpoint, patients with known deleterious potassium and sodium channel genetic mutations should be carefully monitored for both QTc prolongation and ventricular dysfunction, irrespective of the presenting phenotype.
Appendix. Supplementary materials {#s0030}
=================================
Supplementary Material
Supplementary Material
Supplementary Material
Supplementary data associated with this article can be found in the online version at [doi:10.1016/j.hrcr.2015.10.011](http://dx.doi.org/10.1016/j.hrcr.2015.10.011){#ir0005}.
{#f0005}
{#f0010}
{#f0015}
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"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
The oxidation of Low Density Lipoprotein (LDL) is a major pathogenic factor in the development of atherosclerosis. Oxidized LDL (oxLDL) accumulates in the sub-endothelial space of the arterial wall and activates endothelial cells, smooth muscle cells and macrophages. The resulting chronic inflammatory response contributes to atherosclerotic plaque progression\[[@B1]\]. Although is well accepted that the uptake of oxLDL has pathophysiologic relevance in atherosclerosis development, the molecular mechanisms of its interaction with macrophages are not fully understood.
CD36 is one of the main scavenger receptors involved in the uptake of oxLDL by macrophages and has generally been viewed as essential for foam cell formation\[[@B2]\]. Its deficiency greatly reduced the uptake of oxLDL and atherosclerotic lesions in mice models\[[@B3],[@B4]\]. Monocytes from individuals lacking CD36, or *in vitro* experiments using functional blockage of this receptor with antibodies, decreased the oxLDL uptake by 50% \[[@B3]\]. However, other studies have found that a deficiency of CD36 did not prevent macrophage foam cell formation *in vivo*\[[@B5],[@B6]\]. These contradictory observations suggest that additional receptor(s) might be involved in macrophage activation by oxLDL and foam cell formation.
The oxidative modification of LDL generates several oxidized phospholipids (oxPL) that share a common unsaturated fatty acid in the *sn*-2 position of the phosphatidylcholine and confer the ability to bind to CD36\[[@B7]\]. These structural characteristics are also responsible for the interaction of some oxPL with the receptor for Platelet Activating Factor (PAFR) \[[@B8]\], which its expression has been found in macrophages from human atherosclerotic plaques\[[@B9]\]. In previous studies we demonstrated that deficiency or antagonism of PAFR reduced the oxLDL uptake by human macrophages and CD36 expression. It was also found that co-stimulation of CD36 and PAFR is required for IL-8 and MCP-1 production by macrophages exposed to oxLDL\[[@B10],[@B11]\]. We also showed that oxLDL stimulates a number of signal transduction pathways through PAFR engagement including MAPK and PI3K/Akt pathway activation, which leads to the transcription of genes for cytokines and CD36 as well\[[@B11]\]. Because CD36 has a very short cytoplasmic domain\[[@B12]\], it has been suggested that its association with other receptors is required for activating intracellular signaling pathways\[[@B13],[@B14]\].
The PAFR has, in its amino acid sequence, a binding motif for caveolin-1\[[@B15]\], which is a constitutive protein in lipid raft platforms. Thus, we hypothesized that the association of PAFR with CD36 might occur in lipid raft domains. In the present study, we found that oxLDL induces the formation of a complex comprising CD36, PAFR and Flotillin-1 within lipid raft platforms and this is required for the uptake of oxLDL and IL-10 production by macrophages. Moreover, we found colocalization of PAFR and CD36 in human atherosclerotic plaques.
Materials and Methods {#s2}
=====================
Purification and oxidation of LDL {#s2.1}
---------------------------------
The study was approved by the ethics committee of the Institute of Biomedical Sciences, University of Sao Paulo and the participants provide their written consent. Blood was collected from normolipidemic volunteers and plasma was obtained after centrifugation at 1,000 *g*, 4°C, for 15 min in the presence of EDTA 1 mg/mL. Thereafter, we added benzamidine (2 mmol/L), gentamicin (0.5%), chloramphenicol (0.25%), PMSF (phenyl-methyl-sulfonyl-fluoride) (0.5 mmol/L), and aprotinin (0.1 U/mL) (all acquired from Sigma-Aldrich, St. Louis, MO, USA). LDL (density: 1.019-1.063 g/mL) was isolated by sequential ultracentrifugation (100,000 *g*, 4°C), using a P90AT-0132 rotor (CP70MX ultracentrifuge; Hitachi Koki Co., Ltd, Tokyo, Japan), dialyzed (4°C) against PBS, pH 7.4 containing 1 mmol/L EDTA, filtered (0.22 μm) and stored at 4°C. The protein concentration was determined by the BCA kit (Thermo Scientific, Rockford, IL, USA). Part of the LDL was dialyzed overnight against EDTA-free PBS. The LDL with minimal degree of oxidation (moxLDL) was obtained by dialysis of LDL (2 mg/mL) against 2 μM FeSO~4~· 7H~2~O in PBS (pH 7.2) for 48 h at room temperature in the dark. Highly oxidized LDL (oxLDL) was obtained by incubation of LDL with CuSO~4~ (5 μmol/L per mg of LDL protein/ 18 h/ 37°C). The oxidation for both methods was stopped by the addition of 1 mmol/L EDTA, the degree of oxidation was determined by measuring the concentration of tiobarbituric acid-reactive substances (TBARS) and conjugated dienes. TBARS values for LDL, moxLDL and oxLDL were 0.2, 7.5, and 53.1 nmol/mg protein respectively. Conjugated dienes were mesured by optical density at 234 nm: 2.84, 3.02, and 4.00 for LDL, moxLDL and oxLDL respectively.
Cell culture {#s2.2}
------------
6--8 week old male C57BL/6 mice were obtained from our own animal facilities and were housed in a room with 12 h light--dark cycle with water and food *ad libitum*. Animal care and research protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and approved by the Biomedical Sciences Institute/USP---Ethical Committee for Animal Research (CEEA). We did not perform *in vivo* studies. Bone marrow-derived macrophages (BMDM) were established as previously described by Davies and Gordon (2005)\[[@B16]\], with minor modifications. In brief, femurs were flushed with PBS, using a 26 x 1.2"-gauge needle. Cells were grown in L-Cell conditioned medium (DMEM containing 20% L929 cell-conditioned medium, 15% FCS, 2 mmol/L l-glutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycin) (Gibco, Long Island, NY, USA) , incubated at 37°C in 5% CO~2~. At day 3, new fresh L-Cell conditioned medium was added. Monolayer of macrophages was scrapped at day 6. Macrophages were culture in DMEM/1% for one day before the experiments.
The monocytic cell line THP-1 was cultured in RPMI-1640 medium supplemented with 5% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 15 mM HEPES and 11 mM sodium bicarbonate. Cell cultures were maintained in a humidified atmosphere containing 5% CO~2~ at 37°C. The differentiation of THP-1 monocytes into macrophages was induced by 150 nM phorbol 12-myristate-13-acetate (PMA) for 24 h. Non-adherent cells were removed by aspiration of the supernatant followed by replacement with fresh medium.
Uptake of oxLDL {#s2.3}
---------------
LDL was labeled with Fluorescein isothiocyanate (FITC) (Merck Chemicals, Nottingham, UK), as described previously\[[@B17],[@B18]\]. Breafly, 2 mg/mL of LDL were dialyzed in carbonate buffer (NaHCO~3~ 0.5 mol/L; EDTA 1 mmol/L pH 9.5) containing 2 mg of Fluorescein isothiocyanate (FITC), overnight at 4°C. The unbound FITC was removed by dialysis in PBS/EDTA and PD10 column (Amersham Pharmacia Biotech, Uppsala, Sweden). The FITC concentration in LDL was determined by spectroscopy against FITC standard solution at 495 nm. The F/P (fluorochrome/protein) molar ratio was calculated and admitted in the range of 2.4 to 3 as previously described \[[@B19]\]. FITC-LDL was oxidized by CuSO~4~ (5 μmol/L per mg of LDL protein; 18 h; 37°C). Cells were treated with PAFR antagonists "WEB2170 (50 µmol/L) (Boehringer Ingelheim, Pharma KG, Biberach, Germany), CV3988 (10 µmol/L) (Tocris Bioscience, Bristol, UK)" alone or in combination with anti-CD36 blocking antibody (1 µg/mL) (monoclonal IgA anti-CD36, clone CRF D-2712, BD Biosciences, Franklin Lakes, NJ, USA), for 30 min, then incubated with FITC-oxLDL (30 µg/mL) for 1 h at 37°C. In some experiments the cells were pre-treated with the Methyl-β-cyclodextrin (βCD) or with the inactive analogue α-ciclodextrin (αCD) (all from Sigma-Aldrich, St. Louis, MO, USA) before uptake assay. Cells were washed with cold PBS and fixed with 2% formaldehyde. The uptake of FITC-oxLDL was visualized by fluorescent microscopy and evaluated in flow cytometer (FACS Canto II - Becton BD Biosciences) and the data were analyzed by the software Summit® V4.3 (DakoCytomation).
Co-immunoprecipitation and immunoblotting {#s2.4}
-----------------------------------------
Macrophages were treated with oxLDL (30 µg/mL) or PAF (10^-7^ mol/L) for 20 min. Resting and activated cells were lysed without agitation on ice for 30 min using HEPES buffer containing 1 mmol/L CaCl~2~, 1 mmol/L MgCl~2~, 1% Triton-x-100, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Calbiochem- Merck Chemicals, Nottingham, UK). Post-nuclear lysates were incubated overnight with the primary antibody of interest (rabbit anti-PAFR or mouse IgA anti-CD36). Protein A-Sepharose (GE Healthcare, NJ, USA) and protein G-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) were added to samples containing anti-PAFR and anti-CD36, respectively, and incubated for 3 h at 4°C with gentle agitation. Immune complexes bound to beads were washed three times with HEPES buffer containing protease and phosphatase inhibitors, without triton-x-100, and boiled in SDS sample buffer for 5 minutes. Proteins were separated by 10% SDS-PAGE, transferred to a Hybond™ nitrocellulose membrane (GE Healthcare, NJ, USA), and incubated with rabbit-anti-PAFR (Cayman Chemical, Ann Arbor, Michigan, USA) or mouse IgA-anti-CD36 (BD Biosciences, Franklin Lakes, NJ, USA) or with mouse anti-flotillin-1(BD Biosciences, Franklin Lakes, NJ, USA). As secondary antibodies we used anti-rabbit IgG-HPR (1:2,000), anti-mouse-HRP (1:1,000) (Cell Signaling Technology, Beverly, MA, USA), biotin-anti-IgA (1:500) (BD Biosciences, Franklin Lakes, NJ, USA) with streptavidin-HRP (1:200) (Life Technologies, Carlsbad, CA, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). The resulting autoradiograms were analyzed with the AlphaEaseFC^TM^ software V3.2 beta (Alpha Innotech).
Transfection of HEK 293T cells {#s2.5}
------------------------------
HEK 293T cells (ATCC) were cultured in DMEM supplemented with 10% FCS, 15 mM HEPES, 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The plasmids CD36-pcDNA3.2/V5-DEST, pcDNA3-kozac-mycPAFR and the control plasmid pcDNA3 were purified using a Qiagen Plasmid Kit (Qiagen Inc., Valencia, CA, USA). The day before the transfection, HEK 293T cells were seeded onto 24-well plates at a density of 2 x 10^5^ cells/well. The cells were transiently transfected with 2 µg of total plasmid DNA per well using Lipofectin® Transfection Reagent (Invitrogen-Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. At 48 h after transfection, the cells were used for the experiments.
mRNA expression {#s2.6}
---------------
RNA was isolated using TRIzol reagents (Life Technologies, Carlsbad, CA, USA). For the real-time reverse-transcriptase polymerase chain reaction (PCR), cDNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Ontario, USA), according to the manufacturer\'s instructions. PCR-master mix (Power SyBr® Green, Applied Biosystems, Warrington, UK) containing the specific primers was then added. Primers used were: hIL-10 forward: GATCCAGTTTTACCTGGAGGAG and reverse: CCTGAGGGTCTTCAGGTTCTC; hIL12p40 forward TGCCCATTGAGGTCATGGTG and reverse: CTTGGGTGGGTCAGGTTTGA, and GAPDH forward: GAGTCAACGGATTTGGTCGT and reverse: TTGATTTTGGAGGGATCTCG. Real-time PCR was performed using a Stratagene Mx3005PTM QPCR System (Santa Clara, CA, USA). Relative gene expression was calculated by the 2^-Delta\ Delta\ C(T)^ method, as previously described\[[@B20]\]. Data are shown in fold increase related to untreated cells.
MTT assay {#s2.7}
---------
The mitochondrial-dependent reduction of methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) to formazan insoluble crystals was used to evaluate cell viability. Briefly, 10 µL of 5 mg/ml of MTT in PBS were added to the cells after the treatments. After incubation at 37°C for 2 h, 100 µL of 10% SDS in 0.01 mol/L HCl was added to dissolve the crystals and incubated for 16 h. The absorbance was measured in a Dynatech microplate reader at 570 nm.
Confocal microscopy of macrophages {#s2.8}
----------------------------------
Macrophages (2 x 10^5^) were plated in glass cover slips and treated with oxLDL (30 µg/mL) or PAF (10^-7^ mol/L) (Cayman Chemical, Ann Arbor, Michigan, USA) for 20 minutes, and then washed with PBS. Cells were fixed with 3% paraformaldehyde and blocked with 1% BSA in PBS, before incubation with primary antibodies anti-PAFR (1:100) (Cayman Chemical, Ann Arbor, Michigan, USA) and IgA anti-CD36 (1:100) (BD Biosciences, Franklin Lakes, NJ, USA). FITC Donkey anti-rabbit IgG (1:100) (BioLegend, San Diego, CA, USA), Alexa-647 donkey anti-rabbit (1:200) (Invitrogen-Life Technologies, Carlsbad, CA, USA) or Biotin-anti--mouse IgA (1:200) with streptavidin-PE (1:200) (BD Biosciences, Franklin Lakes, NJ, USA) was used as the secondary antibody. Cells stained with secondary antibody and control antibody were used to control for the background from each fluorophore. Slides were mounted in Prolong® Gold anti-fade reagent with DAPI (4,6-diamidino-2-phenylindole) (Invitrogen-Life Technologies, Carlsbad, CA, USA). Cells were imaged on a Zeiss LSM 510 confocal microscope using 100x oil objective at a 60-fold magnification. Images were analyzed by Pearson's coefficient in JACoP (Just Another Colocalization Plugin) using the package ImageJ 1.44p (Wayne Rasband, NIH, USA) available in [[http://imagej.nih.gov/ij]{.ul}](http://imagej.nih.gov/ij). Confocal images were taken with identical settings to allow comparison of staining. Single confocal sections of the cells were captured in multitrack. Each set of frames from a given treatment condition depicts a representative from at least 20 analyzed cells in three independent experiments.
Confocal microscopy of atherosclerotic plaque tissue {#s2.9}
----------------------------------------------------
Carotid artery plaques were collected from patients who were undergoing endarterectomy. Informed written consent was obtained from all subjects. The investigation was approved by the Ethical Committee of Northern Stockholm and was in agreement with institutional guidelines and the principles that were set forth in the Declaration of Helsinki. Acetone-fixed sections were incubated with 5% horse serum followed by a polyclonal rabbit anti-human PAF receptor IgG antibody at a concentration of 25 µg/mL. Binding was detected with DyLight 594 anti rabbit IgG antibody (Vector Laboratories, Peterborough, UK). The binding specificity was detected using the PAF receptor blocking peptide (Cayman Chemical, Ann Arbor, Michigan, USA) prior to incubation with the PAF receptor IgG antibody. For double staining of the PAFR and CD36 or CD68, a mouse anti-human CD36 (Cayman Chemical, Ann Arbor, Michigan, USA) or a mouse anti-human CD68 (BD Biosciences, Franklin Lakes, NJ) was used as the primary antibody, followed by DyLight 488 anti-mouse IgG (Vector Laboratories, Peterborough, UK). Lipid autofluorescence was blocked with 0.03% Sudan black B (Sigma Aldrich, USA) in 70% ethanol. Nuclei were visualized with DAPI (Sigma Aldrich, St. Louis, MO, USA), and images were captured with a Leica TCS SP5 confocal microscope.
Cytokine measurements {#s2.10}
---------------------
IL-10 (mouse and human), mIL-12p40, and hTGFβ concentration in the supernatants of macrophages were measured using BD OptEIA^TM^ ELISA Set (BD Biosciences, San Diego, CA, USA).
Statistical analysis {#s2.11}
--------------------
Data are presented as mean ± SEM. Analysis of variance (ANOVA) and the Student-Newman-Keuls post-test were used to evaluate the statistical significance of the differences between three or more groups. Two-tailed unpaired Student\'s t-test was used when differences between two groups were analyzed. Significance was assumed if p \< 0.05.
Results {#s3}
=======
Uptake of oxLDL and IL-10 production by macrophages requires engagement of CD36 and PAFR {#s3.1}
----------------------------------------------------------------------------------------
Bone marrow-derived macrophages (BMDM) were treated with the blocking antibody to CD36 alone or in combination with two molecularly unrelated PAFR antagonists CV3988 (10 µmol/L) or WEB2170 (50 µmol/L) for 30 min and then incubated with FITC-oxLDL for 1 h. The concentrations of PAFR antagonists were based on previous results from our group\[[@B10]\]. We found a rapid increase in macrophage fluorescence within 30 minutes of FITC-oxLDL addition, which accumulates in the plasma membrane and also in intracellular vesicles (illustrated in [Figure 1A](#pone-0076893-g001){ref-type="fig"}). The fluorescence intensity was quantified as a measure of oxLDL uptake. [Figure 1B](#pone-0076893-g001){ref-type="fig"} shows that the uptake was reduced by pre-treatment of macrophages with antibodies to CD36 (62% inhibition compared to untreated group). Pre-treatment with PAFR antagonists (WEB2170 or CV3988) also reduced the oxLDL uptake (42% and 61% inhibition, respectively). The combination treatment with anti-CD36 and WEB2170 or anti-CD36 and CV3988 further reduced the oxLDL uptake (71% and 79% inhibition, respectively). PAFR antagonists or anti-CD36 did not affect the cell viability, measured by MTT assay, which was around 98% in all groups. These data show that the uptake of oxLDL by BMDM is higher when both receptors are functional.
{#pone-0076893-g001}
Next, we investigated if PAFR and CD36 are required for IL-10 and IL-12 production, which are cytokines involved with macrophage suppression or activation, respectively. BMDM were pre-treated with the anti-CD36 blocking antibody alone, or in combination with the PAFR antagonists 30 min before stimulation with oxLDL (30 µg/mL). Under these experimental conditions, BMDM produced IL-10 ([Figure 1C](#pone-0076893-g001){ref-type="fig"}), but IL-12 was not detected (data not shown). The IL-10 production was significantly reduced by the previous treatment of macrophages with PAFR antagonists, an anti-CD36 blocking antibody, or a combination of PAFR antagonists with anti-CD36. These data show that oxLDL induces IL-10 and not IL-12, and that a crosstalk or synergistic effect between CD36 and PAFR is required for IL-10 production. This was further confirmed in HEK293T cells transiently transfected with plasmids encoding hPAFR cDNA or hCD36 cDNA, or both, and then stimulated with oxLDL for 5 h. We found that only double transfected cells expressed IL-10 mRNA and trace amounts of IL-12 mRNA. Non-transfected or single transfected HEK293T cells were unable to respond to oxLDL ([Figure 1D](#pone-0076893-g001){ref-type="fig"}).
The effects of oxLDL may vary according to the degree of oxidation. We then assayed the effect of minimally oxidized LDL (moxLDL) on IL-10 and TGFβ production by human macrophage THP-1 cells. The moxLDL was characterized as presenting low values for both negative charges and TBARS compared to oxLDL, as described in methods section. The moxLDL preparation did not induce IL-10 and TGF-β production as did the cells stimulated with oxLDL ([Figure 1E-F](#pone-0076893-g001){ref-type="fig"}).
### Lipid Raft integrity is important for uptake of FITC-oxLDL and IL-10 production {#s3.1.1}
Cholesterol-rich microdomains, known as lipid rafts, have been shown to function as specialized platforms for receptor interactions\[[@B21]\]. We, therefore, examined whether lipid raft integrity is required for FITC-oxLDL uptake and the induction of IL-10. Macrophages were treated with methyl-β-cyclodextrin (βCD), a synthetic molecule that sequesters cholesterol from plasma membranes and disrupts lipid rafts. We found that βCD caused a significant reduction (70%) in FITC-oxLDL uptake ([Figure 2A and 2B](#pone-0076893-g002){ref-type="fig"}). In [Figure 2C](#pone-0076893-g002){ref-type="fig"} it can be seen that βCD treatment markedly inhibited the production of IL-10 induced by oxLDL. The inactive analog αCD did not affect the IL-10 production. These data show that the lipid raft integrity is important for the oxLDL uptake and IL-10 production induced by oxLDL.
{#pone-0076893-g002}
It has been shown that Syk (Spleen tyrosine kinase) may interact with CD36 in the lipid raft fraction, contributing to its association with other receptors and adaptor proteins and allowing it to drive cell activation \[[@B22]\]. To investigate the role of Syk we treated macrophages with a Syk-selective inhibitor, piceatannol (20 µmol/L) or with a general kinase inhibitor genistein (10 µmol/L), 10 min before the FITC-oxLDL uptake assay or oxLDL-induced IL-10 production. We found that piceatannol did not interfere with the oxLDL uptake or IL-10 production ([Table 1](#pone-0076893-t001){ref-type="table"}). In contrast, genistein treatment decreased both FITC-oxLDL uptake and IL-10 production. These results show that oxLDL uptake and IL-10 production is dependent on the activation of kinases other than Syk. The treatments employed in these studies did not affect the cell viability, measured by MTT assay.
10.1371/journal.pone.0076893.t001
###### Syk activation is not required for IL-10 production and oxLDL uptake by macrophages.
**IL-10 (pg/mL**) **oxLDL uptake (MFI**)
-------------------- --------------------- ------------------------
**Cont** 52.6 ± 11 7.8 ± 1
**oxLDL** 221.1 ± 49 ^\*\*\*^ 97.5 ± 8 ^\*\*\*^
**oxLDL + Piceat** 354.8 ± 80 104.5 ± 2
**oxLDL + Genist** 43.4 ± 8 ^\#^ 50.4 ± 17 ^\#^
Macrophages were treated with the Syk-selective inhibitor, piceatannol (20 µmol/L), or with a general kinase inhibitor genistein (10 µmol/L), 10 min. For uptake assay, treated cells were incubated with FITC-oxLDL for 1 h and the fluorescence measured by FACS. Data are presented in mean of fluorescence intensity (MFI) For IL-10 production, treated cells were stimulated with oxLDL (30 µg/mL) for 24 h and the cytokine production was evaluated in the supernatants. Data are presented as mean ± SEM of three independent experiments. \*\*\*p\<0.001 comparing with non-stimulated cells. \# p\<0.05 comparing cells treated with non-treated cells.
oxLDL induces colocalization and co-immunoprecipitation of PAFR and CD36 {#s3.2}
------------------------------------------------------------------------
In order to investigate whether oxLDL induces a complex formation with PAFR and CD36, we performed co-immunoprecipitation assays. Macrophages were stimulated with oxLDL (30 µg/mL) or PAF (10^-7^ mol/L) for 20 min. PAFR and CD36 from the cell lysate were immunoprecipitated as described in the methods section. [Figure 3A](#pone-0076893-g003){ref-type="fig"} shows that stimulation with oxLDL rapidly induced receptor interaction, which was detected by the co-immunoprecipitation of PAFR and CD36 (2-fold increase compared to non-stimulated control). In non-stimulated control cells we detected a small proportion of PAFR co-immunoprecipitated with CD36, which was not significantly increased by PAF stimulation. Next, we analyzed the immunoprecipitation of PAFR or CD36 with flotillin-1, a lipid raft associated protein\[[@B23]\]. It can be seen in [Figure 3B](#pone-0076893-g003){ref-type="fig"} that oxLDL induced the strong co-immunoprecipitation of flotillin-1with PAFR and CD36 (around 1.8-fold increase in relation to non-stimulated cells). These results strongly suggest that PAFR and CD36 associate in a protein complex induced when oxLDL is added to macrophages and this may occur in the lipid raft platforms. To further define the association of PAFR and CD36, we performed colocalization assays by confocal microscopy. BMDM were stimulated with oxLDL (30 µg/mL) for 5, 10 and 20 min, fixed with 3% paraformaldehyde and labeled with antibodies to CD36 and PAFR. We found that oxLDL stimulation induced a redistribution of PAFR and CD36 in macrophages increasing the colocalization on the macrophage membrane ([Figure 4A](#pone-0076893-g004){ref-type="fig"}). This effect was time-dependent, with maximum colocalization seen in 10 min ([Figure 4B](#pone-0076893-g004){ref-type="fig"}). In contrast, in PAF stimulated macrophages, only a discrete receptor colocalization was observed. These data indicate that oxLDL induces a spatial redistribution in the plasma membrane, resulting in the recruitment of PAFR and CD36 to the same complex in macrophages.
{#pone-0076893-g003}
{#pone-0076893-g004}
oxLDL induces colocalization of GM1 ganglioside with PAFR and CD36 {#s3.3}
------------------------------------------------------------------
As flotilin-1 has immunoprecipitated with CD36 and PAFR after oxLDL stimulation ([Figure 3B](#pone-0076893-g003){ref-type="fig"}), we next investigated whether GM1, another constitutive raft protein used as a raft marker, colocalizes with PAFR and CD36 in macrophages stimulated with oxLDL. GM1 was cross-linked by CTxB, and anti-CTxB antibody detected clustering (patching) of GM1. Utilizing Ctx-FITC staining, GM1 was visualized in a diffuse distribution in resting macrophages and clustering in oxLDL-stimulated macrophages ([Figure 5A](#pone-0076893-g005){ref-type="fig"}). Confocal microscopy indicated that a small proportion of CD36 colocalized with GM1 in resting cells, whereas PAFR was not detected in these areas. The oxLDL treatment for 10 minutes increased the colocalization of both PAFR and CD36 with GM1 ([Figure 5B and 5C](#pone-0076893-g005){ref-type="fig"}). In experiments using triple staining, we found that oxLDL rapidly induces the recruitment of PAFR and CD36 to the same lipid raft ([Figure 6D](#pone-0076893-g006){ref-type="fig"}). These experiments confirm the data obtained with co-immunoprecipation and cholesterol depletion, showing that oxLDL induces the recruitment of PAFR and CD36 to the same lipid raft membrane platforms.
{#pone-0076893-g005}
{#pone-0076893-g006}
Colocalization of PAFR and CD36 in human atherosclerotic plaques {#s3.4}
----------------------------------------------------------------
Next, we investigated whether the colocalization of both receptors is present in atherosclerotic lesions. Human carotid atherosclerotic lesions were labeled with antibodies to hPAFR and hCD36. The specificity of the anti-hPAFR was confirmed by competitive assay using the PAF receptor blocking peptide ([Figure 6A](#pone-0076893-g006){ref-type="fig"}). [Figure 6B](#pone-0076893-g006){ref-type="fig"} shows that PAFR was predominantly colocalized with CD68 positive cells indicating its expression in macrophages. Consistent with results found in mouse macrophages, PAFR colocalized with CD36 in human atherosclerotic plaque ([Figure 6B](#pone-0076893-g006){ref-type="fig"}). These results show that PAFR and CD36 act cooperatively in human cells and this may be relevant in atherosclerosis development.
Discussion {#s4}
==========
Here we describe that PAFR and CD36 are recruited to the same complex and this is required for an optimal oxLDL uptake and IL-10 production induced by oxLDL in macrophages. This was confirmed by transfection experiments where oxLDL induced the expression of IL-10 mRNA only in HEK293T expressing both receptors. The disruption of lipid rafts by treatment with mβCD reduced IL-10 production. The uptake of oxLDL was increased when both receptors were present. The interaction between these receptors seems to require lipid rafts formation. Finally, we observed that PAFR and CD36 are colocalized in human atherosclerotic plaques.
In a previous work we found that PAFR-antagonists reduce oxLDL uptake and that PAFR deficient mice take up less oxLDL than their littermate controls\[[@B10]\]. Thus, we already suspected that CD36 and PAFR interact upon macrophage exposure to oxLDL. Here, we show that PAFR antagonists and mAb to CD36 alone or in combination reduced the oxLDL uptake. Results were consistent using two different and molecularly unrelated PAFR antagonists that induced similar inhibition.
CD36 is considered a pattern recognition receptor that binds to negatively charged ligands from both pathogens and oxidized/damaged self-components\[[@B24],[@B25]\]. This receptor was shown to interact with TLR2-TLR6 in macrophages exposed to *S. aureus*-derived lipoteicoic acid (LTR)\[[@B26]\] and also with TLR4-TLR6 in cells stimulated with amyloid-β protein\[[@B13]\]. It is known that CD36 recognizes oxidized phospholipids and apoptotic cells\[[@B25]\]. We have shown that PAFR also recognizes apoptotic cells and oxLDL and suggested that these receptors interact somehow in macrophages membrane for optimal oxLDL uptake\[[@B10],[@B27]\].
According the oxidative stage, the LDL might have similar or different effects on cell activation. A minimal oxidation degree of LDL is characterized by antioxidant depletion, oxidation of arachidonic acid-containing phospholipids, relatively low linoleic acid oxidation and insignificant protein modifications \[[@B28]\]. However, in highly oxidized LDL, phospholipids, triacylglycerol and cholesterol esters are transformed into hydroperoxides which react with ApoB-100, resulting in modification and fragmentation of amino acid side chains \[[@B29],[@B30]\]. Here, we found that only the LDL with a high degree of oxidation increased IL-10 and TGF-β production in human THP-1 cells. However, in previous study we showed that LDL with low and high oxidation both increase the expression of CD36 and may both contribute to foam cell formation \[[@B31]\]. Recognition of oxLDL by murine macrophage receptors induces the production of IL-10 but not IL-12. Our results show that both CD36 and PAFR are involved in IL-10 production. Although several receptors can be involved in oxLDL recognition \[[@B32]\], the oxLDL-induced IL-10 production depends mainly on CD36 and PAFR since, in the present study, the production of this cytokine was almost completely blocked by treatment with PAFR antagonists and mAb to CD36. Our results show that IL-10 production by BMDM is dependent on PAFR activation corroborating previous results reported by Verouti et al, 2011\[[@B33]\] . These authors showed that oxLDL induced MCP-1 production partly through PAFR activation and that this occurred via protein kinases activation. Our previous results are also in accordance with these authors, since we showed that oxLDL-induced IL-8 production by human monocytes-macrophages was dependent on MAPK and PI3K/AKT pathways activation \[[@B11]\]. It was also reported that the activation of PAFR is essential for oxLDL-induced recruitment of human bone marrow-derived mesenchymal stem cells dependent on MAPK activation \[[@B34]\].
IL-10 is an anti-inflammatory cytokine that was found to be expressed by macrophages from atherosclerosclerotic plaques\[[@B35]\]. This cytokine is a marker of alternatively activated macrophages which are involved in repair mechanisms with fibroblasts activation and collagen production\[[@B35]\]. IL-10 was shown to decreases CD36 mRNA expression and increases the cholesterol efflux in macrophages\[[@B36]\]. Other studies have shown that IL-10 induces lipid accumulation in macrophages and may contribute to the foam cell formation\[[@B37]\]. However, its role in atherosclerosis still remains to be determined.
Here, we demonstrate that the lipid rafts disruption decreases the uptake of oxLDL and IL-10 production by macrophages. Lipid rafts are cholesterol-rich and sphingomyelin-rich membrane domains functioning as scaffold platforms for the association of signaling molecules and compartmentalization of cellular processes. It has also been shown that lipid raft formation is involved in cell activation induced by oxidized lipids\[[@B38]\], production of pro-inflammatory cytokines\[[@B39]\] and uptake of acetyl LDL\[[@B40]\]. The following results indicate that the interaction of CD36 and PAFR occurs within lipid raft domains of the BMDM membrane: a) Disruption of lipid rafts by treatment with MβCD reduced the oxLDL uptake and IL-10 production; b) oxLDL induced co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein, flotillin-1\[[@B23]\] and colocalization of PAFR and CD36 with the lipid raft marker GM1-ganglioside\[[@B41]\]. Data presented by others have been shown that CD36 is recruited to lipid rafts in a ligand-dependent manner\[[@B22],[@B42]\]. Although PAFR is a GPCR and might interact within lipid raft platforms\[[@B43]\], there is only one study showing that PAFR migrates to lipid rafts, which was observed in cells transfected with PAFR and stimulated with PAF\[[@B15]\]. In a previous study we showed that both receptors, PAFR and CD36, are able to bind the oxLDL. However, only the co-stimulation of CD36 and PAFR by oxLDL was able to transduce intracellular signaling for cytokine production \[[@B11]\]. Although our data clearly suggest a crosstalk between receptors for PAF and CD36, they do not show at what level the interaction among these receptors occurs. We show here that one possibility is that oxLDL recruits both receptors to specific membrane microdomains (lipid rafts), allowing association between these two receptors. However, we cannot exclude the possibility that they signal in parallel, interacting downstream or that other receptors that are recruited to the same microenvironment also contribute.
The signaling elicited by CD36 engagement leads to the recruitment of the adaptor protein Syk, which was shown to contribute to receptors association\[[@B22]\]. In our study, Syk inhibition did not affect oxLDL uptake or IL-10 production, in contrast to genistein, a general inhibitor of kinases, which reduced both events. This indicates that Syk is not involved in CD36-PAFR interaction induced by oxLDL. It has been shown that Syk phosphorylation requires macrophage activation induced by LDL with minimal modification\[[@B44]\]. It is likely that oxLDL will induce distinct effects depending on the degree of oxidation. Indeed, previous data showed that LDL with high or low degrees of oxidation has different effects on macrophage\[[@B31]\].
The CD36 and PAFR complex formation in human atherosclerotic plaques is intriguing. It can be speculated that these receptors association would contribute not only to increased foam cell formation but also contributes to chronic inflammatory response in the atherosclerotic plaque. However, we have no clues as to how this affects the progression of atherosclerosis. Although the role of PAF/CD36 complex formation in atherosclerotic plaques remains to be determined, this study increases our understanding of macrophage interactions with oxLDL and provides new insights into atherosclerosis research.
We thank Dr Jana Stankova (Université de Sherbrooke, Quebec, Canada) for the kind gift of expression constructs.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: FJR SJ. Performed the experiments: FJR MF MP MMK DFJK YW. Analyzed the data: FJR SJ MF MP MMK DFJK YW. Contributed reagents/materials/analysis tools: SJ DFJK. Wrote the manuscript: FJR SJ.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension (PH) (Stacher et al. [@CR19]; International PPHC et al. [@CR12]; Zhao et al. [@CR23]; Fujiwara et al. [@CR10]; Nasim et al. [@CR14]; Olschewski [@CR16]; Bogaard et al. [@CR3]; MMea and [@CR13]), which can be either be idiopathic (sporadic-90 %, familial-10 %). PAH can also be a complication associated with other conditions such as connective tissue disease, congenital heart disease, anorexigen use (dexfenfluramine), portal hypertension, and human immunodeficiency virus (Stacher et al. [@CR19]; International PPHC, Lane KB, Machado RD, Pauciulo MW, Thomson JR, et al. [@CR12]; MMea et al. [@CR13]). Evidence in the literature suggests that metabolic pathway abnormalities characterize and may play a significant role in the development and progression of PAH (Fessel et al. [@CR9]). For example, pulmonary arterial endothelial cells (PAECs) in PAH share similar hyperproliferative characteristics as malignant tumor transformation that is accompanied by significant metabolic shifts to support anabolic growth and energy metabolism (Xu et al. [@CR22]; Chen et al. [@CR4]). Moreover, it has been shown that mitochondrial oxidative phosphorylation with glucose uptake and utilization occurs in PAEC development. Significant elevation of hemoglobin has been found in the PAH sample group without a history of diabetes or any other obvious metabolic diseases, indicating the impairment of whole-body glucose homeostasis in PAH (Pugh et al. [@CR17]; Hansmann et al. [@CR11]; Archer et al. [@CR1]). Additionally, vascular changes under chronic hypoxic condition has been directly linked to an imbalance between glycolysis, glucose oxidation, and fatty acid oxidation (Sutendra et al. [@CR20]), while in vitro pulmonary arterial endothelial cell culture with disruption of the Bone Morphogenetic Protein Receptor II (BMPRII) gene showed significant metabolomic changes (Fessel et al. [@CR9]). Our recent work showed that disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways exited in the human PAH lung, indicating that PAH has specific metabolic pathways contributing to abnormal ATP synthesis for the vascular remodeling process in pulmonary hypertension (Zhao et al. [@CR24]). Collectively, in vitro, human and animal models suggest that multiple metabolic pathways are reprogrammed during PAH vascular remolding and that metabolic heterogeneity may play an important role in both ATP energy supply and the molecular pathogenesis of pulmonary hypertension. Here, we provide direct evidence of a novel increase in bile acid metabolites in PAH lung tissue associated with the elevated expression of bile acid synthesis related transcripts, indicating de novo synthesis of bile acids may characterize and contribute to the pathogenesis of PAH.
Materials and methods {#Sec2}
=====================
Global biochemical profiles were determined in human lung tissue and compared across 8 normal (47 ± 15 years of age, 4 females) and 8 pulmonary arterial hypertension patients (40 ± 12 years of age, 5 females). Eligibility criteria included end stage PAH patients who went through lung transplantation. Lung samples were obtained from the recipient lung at the time of lung transplantation. Control lung samples were obtained from normal tissue of cancer patients undergoing surgery (lobectomy). Biospecimens and associated clinical data related to the study were collected with written consent from the University Health Network and approved by the Internal Review Board. Unbiased metabolomic profiling using liquid/gas chromatography coupled to mass spectrometry (LC/GC--MS) was performed as described (Reitman et al. [@CR18]; Evans et al. [@CR8]). The detail procedure of metabolic analysis has been documented in the *Supplement data*.
Transcriptomic analysis {#Sec3}
-----------------------
mRNA samples from the normal (n = 8) and native PAH lungs (n = 8) were isolated as described (Zhao et al. [@CR24]). Bile acid related profiles were compared between a control group and samples with idiopathic pulmonary arterial hypertension. Briefly, the total RNA analysis in lung tissues was performed using Trizol extraction according to the manufacturer's instructions. Biotinylated cRNA was prepared according to the standard Affymetrix protocol (Expression Analysis). Following fragmentation, cRNA were hybridized on GeneChip Genome Array. GeneChips were scanned using the HuGene-1_0-st-v1 GeneArray Scanner G2500A. The data were analyzed with Partek Genomics Suite 6.6 using the Affymetrix default analysis settings and global scaling as the normalization method. The value definition was set up using Partek Genomics Suite 6.6. Significantly changed genes were determined by *t* test with a false discovery rate of two fold. The data base has been submitted to NCBI/GEO and has been approved and assigned a GEO accession number GSE53408.
Immunoblotting {#Sec4}
--------------
Protein concentrations were determined using the BCA protein assay (Pierce, IL, USA). Equal amounts of the protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated for overnight at 4 °C with the following antibodies from Abcam^R^: anti-CYP7B1(1:1,000). After wash with TBS-Tween, the blots were incubated for 60 min at room temperature with horseradish peroxidase-conjugated antibodies, respectively: anti-rabbit antibody (1:15,000; Sigma-Aldrich, St. Louis, MO). Signals from immunoreactive bands were visualized by fluorography using an ECL reagent (Pierce). The intensity of individual bands in immunoblots were quantified using the NIH Image program.
Immunohistochemistry {#Sec5}
--------------------
The sections of both PAH and normal lung tissue were fixed for 4 h at room temperature with PBS made of 4 % formaldehyde, permeabilized for 30 min in Triton X-100 (0.5 % in PBS), and incubated with 5 % nonfat skim milk in PBS for 90 min. Sections were incubated for 180 min at room temperature with antibodies for anti-CYP7B1 (1:1,000). The sections were then incubated with biotinylated secondary antibody and visualized with DAB. Stained cells and sections were visualized with the Zeiss LSM 510 confocal microscope.
Results and discussion {#Sec6}
======================
We explored and characterized the metabolomic signature of pulmonary hypertension (PAH) to enhance our understanding of disease progression. Using untargeted metabolic profiling, we found that PAH lung (n = 8) possessed significantly higher levels of multiple bile acid metabolites, including the primary bile acids taurocholate (Fig. [1](#Fig1){ref-type="fig"}), glycocholate (Fig. [2](#Fig2){ref-type="fig"}), taurochenodeoxycholate, and glycochenodeoxycholate (Fig. [3](#Fig3){ref-type="fig"}). Bile acids are normally synthesized in the liver and gallbladder from cholesterol by 7-alpha-hydroxylase, also called cytochrome P450 (CYP7A1), as a rate-limiting enzyme in the synthesis of bile acid via the classic pathway (Nishimoto et al. [@CR15]; Cohen et al. [@CR5]; Crestani et al. [@CR6]; Wang and Chiang [@CR21]). Although the presence of bile acids in lung tissue may partially reflect reflux in these patient (D'Ovidio et al. [@CR7]; Blondeau et al. [@CR2]), microarray analysis surprisingly revealed that the gene encoding cytochrome P450 B1 (CYP7B1), but not CYP7A1, had a significantly higher expression in PAH lung (Fig. [4](#Fig4){ref-type="fig"}a). This finding was also confirmed by Real time RTPCR. Further molecular analysis using Western blot showed that the expression of CYP7B1enzyme was higher in PAH lung (Fig. [4](#Fig4){ref-type="fig"}b). These results suggest that increased bile acid metabolites may not solely be due to reflux from the esophagus (D'Ovidio et al. [@CR7]; Blondeau et al. [@CR2]) but come from the lung itself. Thus, PAH lung tissue may have the capacity for de novo synthesis of bile acids. Notably, increased bile acids metabolites could potentially serve as biomarkers for disease progression. By applying immunohistochemistry, CYP7B1positive immunostaining was found in pulmonary vascular endothelial cells, specifically in newly formed vascular endothelial cells in plexiform lesions of occluded pulmonary arteries (Fig. [4](#Fig4){ref-type="fig"}c), suggesting that CYP7B1may also be involved in the vasculogenesis during the vascular remodeling process of PAH (Fig. [5](#Fig5){ref-type="fig"}). This hypothesis needs to be further tested by additional functional analyses. In summary, we have shown direct evidence that a de novo synthesis of bile acid may be involved in pathogenesis of PAH, suggesting that bile acids in lavage fluid may serve as ideal biomarkers for the diagnosis and prognosis of PAH.Fig. 1MS/MS fragmentation spectrum of taurocholate in control and PAH lung. *Top panel* shows a representative negative ion, selected ion chromatogram (SIC) for taurocholate (*m/z* 514.3) in normal (NL) and pulmonary hypertension (PAH) lung tissue. Taurocholate compound identification relied on confirmed experimental MS/MS fragmentation spectrum matched to the authenticated taurocholate standard, run separately (*bottom panel*). Limited peak detection was observed in NL samples Fig. 2MS/MS fragmentation spectrum of glycocholate in control and PAH lung. Representative negative ion is selected ion chromatogram (SIC) for glycocholate (*m/z* 464.4) in normal (NL) and pulmonary hypertension (PAH) lung tissue (*top panel*). Glycocholate compound identification relied on confirmed experimental MS/MS fragmentation spectrum matched to the authenticated glycocholate standard, run separately (*bottom panel*) Fig. 3PAH lung has a unique bile acids metabolic pathway. Intermediates in the bile acids pathway revealed significantly elevated levels of multiple glycine and taurine conjugated bile acids in the PAH lung. Data for normal lung (NL, n = 8) are represented in *green boxes*, while data for pulmonary hypertension lung (n = 8) are shown in *pink boxes*. Quantities are in relative arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration (PAH with *red frame* indicates \**p* \< 0.05 compared to NL Fig. 4**a** Microarray data showed that the gene encoding cytochrome P450, family 7, subfamily B, polypeptide 1 (Oxysterol 7α-hydroxylase) was significantly highly expressed in PAH lung. (*p* = 0.000187299). **b** Western blot analysis of CYP7B1 expression in normal and PAH lungs. Lung lysate was loaded and immunoblotted with antibody against CYP7B1 and GAPDH (loading control). Consistent with a significant increase of CYP7B1 gene expression in PAH, the enzyme protein for CYP7B1 (37KD) was significantly increased in PAH lungs compared with NL lungs. Densitometric analysis of CYP7B1 was normalized to the intensity of the respective GAPDH band. Data are expressed as mean ± SD (n = 4). \**p* \< 0.05 versus NL. **c** CYP7B1 positive immunostaining in newly formed small blood vessels (*arrows*) in the plexiform lesions of occluded pulmonary small vessel in PAH lung. Representative micrographs of immunostaining of PAH lung sections are shown with anti--CYP7B1 in the pulmonary vascular endothelial cells. (ratio 1:200) Fig. 5Major intermediates in the classical bile acids pathway through Cholesterol 7 alpha-hydroxylase, also known or cytochrome P450 7A1 (CYP7A1), are shown in *blue*. Our finding suggests that PAH lung has a specific bile acids pathway though CYP7B1, as shown in *red*
Electronic supplementary material
=================================
{#Sec7}
Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 14 kb)
| {
"pile_set_name": "PubMed Central"
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As a guest editor of the special edition of the *International Journal of Qualitative Studies on Health and Well-being* I would like to present seven designated articles. The current thematic cluster represents qualitative research illustrating the challenges in supporting the central aspects of health and well-being for individuals in various situations linked to human vulnerability. This theme cluster will provide the reader with a new and greater understanding of the inner meaning of vulnerability and greater insights into how health and well-being, achieved through different forms of support and care, can enhance empowerment in spite of obstacles such as illness, disease, impairment, old or young age, or gender. Different kinds of qualitative methodologies have been used to elucidate the phenomenon of vulnerability. Data collection procedures vary from qualitative interviews and diaries to focus group interviews. Qualitative content analysis, phenomenographical approach, and grounded theory are used to analyse the data in the different studies. The common denominator for the included articles is the commitment of the authors to impart knowledge in terms of greater understanding of the core aspects of health and well-being among humans in different vulnerable situations.
Vulnerability is a condition that is relevant for all human beings, but is especially actualized in connection with loneliness, injury, ill health and death. An overall responsibility for the professionals who take care of people in situations where they face vulnerability is thus to help and support the exposed individuals in attaining improved health and well-being. Professionals, such as psychologists, social workers and nurses from all the specialities, have a responsibility in their work to facilitate their clients' empowerment processes. A prerequisite for them to be able to contribute with help and support in a constructive manner is knowledge about what support is needed for people in vulnerable situations. Participation in healthcare situations among children with juvenile idiopathic arthritis is one specific example where healthcare professionals need more knowledge from the perspective of the children. A constructivist grounded theory is used in order to understand the factors that influence the promotion of increased child participation in healthcare (Gilljam, Arvidsson, Nygren, & Svedberg, [@CIT0002]). Previous research into the participation of children with juvenile idiopathic arthritis in healthcare situations most often focussed on the perspectives of the parents and healthcare professionals but not on the perspectives of the vulnerable children themselves. Adult patients with established rheumatoid arthritis are also vulnerable and they often experience that quality of life entails a constant balancing of lifestyle habits between the ideal situation (ideality) and the actual situation (reality) (Malm et al., [@CIT0005]). The knowledge of how lifestyle habits for these patients influence their quality of life in terms of limitations, self-regulation, and companionship provides a further example of human vulnerability which is elucidated by means of a qualitative content analysis according to Graneheim and Lundman ([@CIT0003]). A woman in early labour represents another example of a vulnerable situation. Early labour is depicted as the very first phase of the labour process when professionals are generally deemed not to be needed (Carlsson, [@CIT0001]). Women have reported that they are often left in a vulnerable situation in this phase, where their preferences are not always met and where they are not always included in the decision-making process. One core prerequisite for midwifes is thus knowledge about how to support women in early labour so that they feel that they are in a safe and secure place. A grounded theory approach was used to reveal a substantive theory of women needs.
In order to facilitate the recovery of women after a myocardial infarction, a qualitative content analysis was carried out (Wieslander, Mårtensson, Fridlund, & Svedberg, [@CIT0007]). Findings suggest that both a preventive and promotive perspective should be taken into consideration in personal recovery among women after a myocardial infarction. Health promotion also plays an important role in the management of diabetes and chronic kidney disease in Vietnam, especially when the prevalence of the disease is rising there (Pham & Ziegert, [@CIT0006]). To gain knowledge about the needs for health promotion among Vietnamese nurses a phenomenographical approach was adopted, which concluded that health promotion needs to be further integrated into the education if these vulnerable people are to receive adequate and appropriate support (Pham & Ziegert, [@CIT0006]). Vulnerability among elders with a high fall risk in independent living was elucidated by using a classic grounded theory design showing that seniors were resolving their main concern of being able to carry on being themselves as they used to in their earlier lives by self-preservation strategies (Källstrand Eriksson, Hildingh, Buer, & Thulesius, [@CIT0004]). The common issue in all of the presented articles highlights human vulnerability and the significance of the knowledge and ability needed to provide practical and emotional support in order to be able to help the individual verbalize his or her needs and desires.
These papers demonstrate that one essential and shared aspect of enhanced health and well-being can be understood as a relief from the position of vulnerability, regardless of its origin. Equality in decision-making processes is achieved through a genuine dialogue that aims to support the individual\'s personal resources and insights. The revealed knowledge about the nature of vulnerability may constitute a necessary prerequisite for the further development of our ability to reduce vulnerability and strengthen our capability for supporting coping strategies, such as decision making in accordance with the personal preferences of those in vulnerable situations. The presented articles indicate core aspects of perspectives on health and well-being when they are needed the most, such as in situations linked to human vulnerability. This theme cluster will provide the reader with new comprehension of the meaning of vulnerability and greater realisation into how intervntions to improve health and well-being can enhance empowerment regardless of illness, disease, impairment, old or young age, or gender.
Henrika Jormfeldt
, RNT, PhD
School of Health and Welfare
University of Halmstad
Halmstad, Sweden
E-mail: Henrika.jormfeldt\@hh.se
| {
"pile_set_name": "PubMed Central"
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Amended data surfaced in our work is publicly available online at <http://files.pushshift.io/reddit/requests/1-10m_submissions.zip> as well as <https://github.com/DGaffney/baumgartner_missing_data_paper>.
1 The Baumgartner Reddit Corpus {#sec001}
===============================
Trace data sourced from online platforms has become an essential component for many forms of research ranging from sentiment analysis \[[@pone.0200162.ref001]\] to epidemiological modeling \[[@pone.0200162.ref002]\] and economics \[[@pone.0200162.ref003]\]. Dominant social platforms such as Twitter and Facebook have provided researchers with opportunities to directly study complex phenomena that, at their root, rely strongly on the nature of social interaction \[[@pone.0200162.ref004]\]. The reason for this, as Tufekci \[[@pone.0200162.ref005]\] argues, is that large platforms (specifically Twitter, in this analogy) serve as a *model organisms* for the social sciences, ones that allow for ideal conditions for measurement of many phenomena in a relatively accessible form.
On July 2, 2015, a new model organism was provided to researchers by Jason Baumgartner---a "complete" copy of one of the largest forums, Reddit, which has gained high visibility in the past several years due to events such as the Reddit blackout \[[@pone.0200162.ref006]--[@pone.0200162.ref008]\] and the Gamergate controversy \[[@pone.0200162.ref009]\]. Subsequently, many researchers have adopted the dataset, and have used it to study a wide range of questions, including the evolution of social networks \[[@pone.0200162.ref010]\], user migration through online platforms \[[@pone.0200162.ref007], [@pone.0200162.ref011]\], hate speech \[[@pone.0200162.ref012]\], and online behavior research methodology \[[@pone.0200162.ref013]\], among others.
As a social news platform, Reddit hosts discussions about text posts and web links across hundreds of communities called "subreddits" \[[@pone.0200162.ref014], [@pone.0200162.ref015]\]. Discussions from public subreddits are aggregated by a variety of news aggregators to create the "front page of the web" that Reddit was founded to provide to its readers \[[@pone.0200162.ref016]\]. While the site also provides chatrooms and features for live discussions of breaking news \[[@pone.0200162.ref017]\], the most common Reddit experience is centered around top-level *submissions* and the *comnents* that people post when discussing those submissions within their subreddit communities. The Baumgartner dataset follows this common experience and includes submissions and comments.
Researchers are drawn to the Baumgartner Reddit dataset for its completeness. In principle, a complete dataset improves research validity by avoiding the ambiguities of samples provided by platform application programming interfaces (APIs) and third-party data resellers \[[@pone.0200162.ref018], [@pone.0200162.ref019]\]. In this paper, we show that this dataset, as distributed and used by researchers, is not as complete as reported. We report on gaps in this data, categorize the risks to research validity from these gaps, and share collaborative re-analyses of peer-reviewed papers that have used this dataset. Finally, we conclude with reflections on the sensitivity of online behavioral research to the kinds of gaps we found in the Baumgartner Reddit Dataset.
1.1 Sequential ID analysis {#sec002}
--------------------------
The Baumgartner Reddit dataset came about through a convergence of factors: a mostly-public conversation platform, engineering details specific to the design of the Reddit system, and a creative data scientist who capitalized on these characteristics to contribute a unique dataset to public knowledge.
Many databases include the concept of an Identity column, or a column that generates an internal ID to serve as a unique reference to the row, or object, within the database. In many cases, this value auto-increments---the first value in the database assumes a value of 1, the next, a value of 2, and so forth. This number can be artificially shifted within the space---for instance engineers may partition early IDs of 1-1,000,000 for experimenting with data, for some reason, and start all production-system data created by users with ID 1,000,001. Aside from this possibility, if an object contains an ID of *n*, then it is plausible to assume that there are at least *n* objects within the database.
In personal correspondence, Baumgartner explained that this intuition led him to develop systems designed to systematically-collect all data on Reddit. Baumgartner's algorithm batches up 100 integers, converts them to the Base 36 representation that Reddit uses to represent their objects, and then queries for those objects. Reddit then returns the request with a set of all public, found objects. Baumgartner's algorithm can be run in a highly parallel environment---many batches of 100 IDs can be concurrently requested, with no need to interact with one another. On other platforms, some error may be returned if data has been deleted. With Reddit, no error is returned---instead, a truncated object reflecting that this deletion has occurred is returned. Therefore, barring technical issues, this method should provide a complete accounting for every ID within the range 1-*n* for all public comments and submissions within the dataset. Using this method, Baumgartner archived the public record of Reddit comments and submissions from the platform's creation through July 2015. Baumgartner has continued to provide this data as a freely-available resource.
In this analysis, we consider the full dataset as released by Baumgartner in July 2015, supplemented with updates published by Baumgartner through the end of February 2016. We also include a followup analysis extended to June 2017.
2 Diagnosing missing data {#sec003}
=========================
Because Reddit comments and submissions have unique, sequential IDs, we can analyze gaps in the sequence to evaluate the completeness of the dataset. We observed two kinds of missing information: dangling references (known unknowns) and gaps indicated by the absence of information that we would expect to exist given the use of sequential integers to index comments and submissions (unknown unknowns).
We discovered the completeness problem when working with this dataset for our own research. Taking a random sample of subreddits and generating a timeline of daily comments and submissions, plots showed impossible results given the architecture of Reddit: some comment timelines started earlier than their corresponding submission timelines.
The first kind of gap we discovered were dangling references. On Reddit, comments can only occur within a discussion of a submission and can only refer to other comments or submissions. In all cases, a submission would have to exist for a comment to refer to it, a relationship that is unidirectional in time. By traversing these relationships, we observed many references to missing comments and submissions. These can be thought of as "known unknowns:" comments which refer to other comments or to a parent submission, where the referred-to comment or parent submission is not contained within the Baumgartner dataset.
We also observed a second kind of gap: objects that are never referenced in the dataset but are likely missing. If all comments and submissions are given sequential integer IDs, we would expect an unbroken sequence of integers to be associated with information in the dataset. This is not the case. Consider the comments dataset: the earliest comment in the Baumgartner dataset is comment \#2 and the highest is \#29,484,960,643. In October 2007, the Reddit Company incremented the comment IDs by several billion IDs. When accounting for this difference, we assume that any other gaps in the sequence of comment IDs can be attributed to gaps in the dataset: we count 943,755 total potentially-missing comments up to February 2016.
Missing comment IDs could be attributed to many possible causes. These IDs could be dangling references, public information that for some reason were not returned by Reddit's systems to Baumgartner's software at that moment, unobservable technical errors within Reddit's architecture, or information that was part of a community that had set its discussions to be private. A major point of evidence in this direction is that during the Reddit blackout \[[@pone.0200162.ref006]\], the number of missing comments and submissions spiked significantly. In this case, due to the constraints of the platform, it is not possible to disambiguate private content from truly missing content---lookups on private IDs appear to yield no data (assuming that the API request is not done by an authenticated user who has access to the private content).
In some cases, it is possible that some of the missing IDs were never associated with any content. In correpondence, Baumgartner reported successfully retrieving many of the IDs not present in the original corpus, confirming that many of these missing IDS are genuinely associated with content. Of the initial set of 666,542 distinct comment ids and 864,598 distinct submission ids from the beginning of Reddit to February 2016 (when we initially contacted Baumgartner with missing ID lists to check), we found 101,257 existing comments and 405,911 existing submissions within those requested sets, which is substantial enough that not all "missing" data claims are spurious. Furthermore, these missing IDs are not associated with deleted content, since the Reddit platform returns information about deleted data, which is included in the Baumgartner dataset---in any event where a deleted comment is requested via the Reddit API (and subsequently, the Baumgartner corpus), a stubbed object is returned, clear of most metadata, simply stating that the object was deleted. In that event, we know the status of the object, and can confirm that it is not falsely "missing" from the dataset, but is instead truly missing in that it has been intentionally erased.
For submissions, we are less confident about the magnitude of missing unknown unknowns. While we have observed 1,539,583 "gaps" in the space of IDs for submissions through February 2016, the first submission in the Baumgartner dataset starts at 9,970,002. When searching for submissions between \#1 and \#9,970,001, we have successfully found some submissions, leading us to believe that millions of submissions from the early history of Reddit may be absent from this dataset, though that figure only represents an upper bound.
Deleted content, which is included in this dataset, represents a risk to validity that we do not consider here. A user who deletes even one comment in their posting history introduces many of the problems we describe in this paper, even if the fact of the comment is recorded in the Baumgartner dataset.
2.1 The per-user risk of missing data {#sec004}
-------------------------------------
How likely is a researcher to encounter these gaps? To address this question, we estimate the per-user risk of missing data, using a random sample of 7,400 accounts from the Baumgartner dataset.
The average user in this sample commented 6.8 times and commented 96.6 times from late January 2006 through February 2016. These averages occur on a highly skewed distribution, as illustrated by the log-histograms in [Fig 1](#pone.0200162.g001){ref-type="fig"}. Based on [Table 1](#pone.0200162.t001){ref-type="table"}, the known maximum amount of missing comments and submissions is 943,755 and 1,539,583, respectively---dangling references are a subset of "unknown unknowns." Across the entire Baumgartner dataset, only 0.043% and 0.65% of comments and submissions, respectively, are missing. The issue has a compounding effect, since a small number of users create a large amount of the content on the platform. The more posts and comments someone produces, all else being equal, the more likely their histories will be affected by the missing data issue. As we have also shown, unknown unknowns expanded dramatically in the 16 months following February 2016 and now include 36 million missing comments and 28 million missing submissions.
{#pone.0200162.g001}
10.1371/journal.pone.0200162.t001
###### Totals for missing data in the Baumgartner dataset.
{#pone.0200162.t001g}
Data Type Comments Submissions
----------------------------------- ------------ -------------
Dangling References (to Feb 2016) 101,257 405,911
Unknown Unknowns (to Feb 2016) 943,755 1,539,583
Unknown Unknowns (to Jun 2017) 35,801,325 27,795,423
What is the probability of data loss for an individual Redditor history? While in reality the missing data is not uniformally distributed throughout the corpus, we can estimate the effect by compounding probabilities to assess the degree to which a user could be affected by only a small amount of missing data. Using the averages from earlier, we can calculate the risk of any individual submission *r*~*s*~ or comment *r*~*c*~ being missing simply by $\sum_{c}^{n}r_{c}$ and $\sum_{s}^{n}r_{s}$, respectively. In this case, the "average" Redditor may be exposed to a total maximum risk level of ∝ 4.18% likelihood for missing at least one comment and ∝ 4.46% for missing at least one submission. In the 7,400 individual set, approximately 2% of the sampled users had a 50% or greater chance of having a missing comment, and 2.6% of the sampled users had a 50% or greater chance of having a missing submission. These estimates were based on the census of dangling references and unknown unknowns from the beginning of the corpus to February 2016; we expect relatively similar rates in later data, since the rate of missed content has been consistent for the past several years. We offer these rough approximations to communicate a qualitative sense of how this missing data issue may create an appreciable problem for some forms of research. We include a more detailed typology of possible errors below.
2.2 Distribution of gaps across time {#sec005}
------------------------------------
Far from being uniformally distributed throughout the dataset, the instances of missing data appear to be "bursty"---clustered at certain moments of time. Consequently, certain spaces in the Reddit network or certain time periods may be at greater risk of missing data than others. Importantly, we found significant gaps for comments at key moments in Reddit history that have been subjects of research, including the SOPA/PIPA protests \[[@pone.0200162.ref020]\] and the months leading up to the Reddit blackout \[[@pone.0200162.ref006]\]. Leaning on Jo et al \[[@pone.0200162.ref021]\], we employ a measure of "burstiness", defined as $B = \frac{\sigma_{t} - \mu_{t}}{\sigma_{t} + \mu_{t}}$, where *σ*~*t*~ and *μ*~*t*~ are the standard deviation and mean of the size of missing id gaps for each month of data from the Baumgartner corpus. This measure considers the relative dispersion of errors throughout the ID space per each month of gathered data. This measure is bounded from \[-1, 1\], where a score of -1 indicates completely evenly dispersed errors, and a score approaching 1 indicates that errors are located in a more concentrated set of missing blocks. [Fig 2](#pone.0200162.g002){ref-type="fig"} shows many high positive burstiness scores, indicating that missing blocks are often distributed unevenly within months throughout the dataset.
{#pone.0200162.g002}
Overall, Figs [3](#pone.0200162.g003){ref-type="fig"} and [4](#pone.0200162.g004){ref-type="fig"} illustrate an initially erratic distribution of errors throughout the dataset. For researchers concerned about the dispersion of missing objects, consider the dark blue line which shows the cumulative percent of missing objects or the simple percent of missing content per month of data in the medium blue. For researchers concerned with the percent of missing content to date, consider the light blue line which charts how much content appears to be missing from the beginning of the corpus until the end of our current analysis. These errors appear to occur directly within periods of substantial research interest and may affect several published results \[[@pone.0200162.ref006], [@pone.0200162.ref007]\]. While the rate of error was particularly erratic in early years, and the distribution of errors per ID gap continues to be erratic ([Fig 2](#pone.0200162.g002){ref-type="fig"}), the error rate per month has evened out to around 1% missing data per month.
{#pone.0200162.g003}
{#pone.0200162.g004}
2.3 Distribution of gaps across communities {#sec006}
-------------------------------------------
We also considered the degree to which missing content differentially affects individual subreddits. If data from some communities were more affected by gaps than others, the gaps could influence the results of comparative research about populations communities \[[@pone.0200162.ref022]\]. If gaps affected communities equally, we would expect that the number of missing pieces of content monotonically rises with the number of overall pieces of content posted to a subreddit. As [Fig 5](#pone.0200162.g005){ref-type="fig"} shows, we find only marginal evidence for such a supposition. While more missing content is positively and significantly associated with larger subreddits, we do not find a direct relationship. One confounding factor may be the temporal "center of gravity" of a subreddit---older subreddits are positioned at a time when more content was missing, on average, which may differentially affect older subreddits. We attempted to control for subreddit age in a multiple linear regression which accounted for the size of subreddits as well as the time at which those subreddits were created; we did not find any meaningful increase in explanatory power in the adjusted model. [Table 2](#pone.0200162.t002){ref-type="table"} provides the output from two regressions, one on missing submissions and one on missing comments, where observations are individual subreddits, and we hold the total number of known missing objects as dependent against the total number of found objects as well as the date the subreddit was created. The time at which a subreddit was created, however, is a poor proxy for the true "center of gravity" of content (i.e. the time at which a subreddit was most active), a characteristic that these models do not account for.
{#pone.0200162.g005}
10.1371/journal.pone.0200162.t002
###### Regression exploring the relationship between amount of missing content per subreddit and total amount of known content per subreddit, and month in which the subreddit was created.
We expect that these two variables would have meaningful explanatory power for where missing content is---we find that this appears to be the case for missing comments but not for missing submissions, as evidenced by the relative *R*^2^ values.
{#pone.0200162.t002g}
------------------------------------------------------------------------------------------------------------------------------
Variable Submissions Comments
------------------------- ------------------------------------------------- --------------------------------------------------
Total Content 0.212[\*\*\*](#t002fn003){ref-type="table-fn"}\ 0.217[\*\*\*](#t002fn003){ref-type="table-fn"}\
(0.008) (0.006)
Month Subreddit Created 0.005[\*\*\*](#t002fn003){ref-type="table-fn"}\ 0.002[\*\*](#t002fn002){ref-type="table-fn"}\
(0.001) (0.001)
Constant 1.198[\*\*\*](#t002fn003){ref-type="table-fn"}\ -0.518[\*\*\*](#t002fn003){ref-type="table-fn"}\
(0.094) (0.095)
Observations 8,176 4,341
R^2^ 0.086 0.306
Adjusted R^2^ 0.086 0.305
------------------------------------------------------------------------------------------------------------------------------
\* p\<0.1
\*\* p\<0.05
\*\*\* p\<0.01
In the above sections, we have considered the influence of potentially-missing content on analyses of users, behavior over time, and groups. We observed numerous sources of potential bias in research: a substantial percentage of users could be affected by these gaps, the gaps are not evenly distributed across time, and gaps are not evenly distributed across communities.
3 How missing data affects common research methods in computational social science {#sec007}
==================================================================================
How might these gaps influence research in practice? We expect that researchers asking different kinds of questions will face different kinds of risks from missing data. In the following sections, we categorize published literature that uses this dataset and offer a typology of the risks that these gaps represent to common research methods in computational social science.
*User history analysis* papers face the *highest risks* from missing data, since a missing comment or submission could hide an important part of that user's history. A network analysis may fail to include a user's participation in a particular community or interaction with a key user. Furthermore, survival analyses might mis-estimate the moment of a person's departure or their participation level. *Network analysis* papers also face *high risks*, since the presence or absence of a tie could be dependent on the missing data. *Sum analyses* that count the size or incidence rate of participation in subreddits or the use of certain kinds of language face *moderate risk*, especially when analyzing small communities and rare events. *Content analysis* that involves training machine learning systems on Reddit comments face *minimal risk* because their research rarely includes claims about the population of Reddit users.
3.1 Risk to user history analyses {#sec008}
---------------------------------
Papers that test hypotheses based on user histories on Reddit may have substantial gaps in the histories that they seek to test. Analyses on user histories that consider the history in full are, in general, exposed to the highest risk---analyses that are especially sensitive to high-volume users are very likely, on average, to consider users whose histories have gaps. Hessel et al \[[@pone.0200162.ref023]\], for example, observes and compares sums of comment participation between subreddits, and observes the full chain of user history---Hessel et al \[[@pone.0200162.ref024]\] adopts a similar approach. Barbosa et al \[[@pone.0200162.ref013]\] compares year cohorts of individual-level behavior across all of Reddit, and as has been shown, some years are more affected by gaps than others. Additionally, the large number of potential missing submissions from Reddit's earliest years may also affect these findings. If a user history analysis requires the complete posting history between subreddits for a given user, gaps in such transmissions may constitute meaningful gaps in explaining a wide array of hypotheses.
3.2 Risks to network analyses {#sec009}
-----------------------------
Some papers test network hypotheses by constructing interaction networks between users or communities, sometimes over time. Data gaps also represent a high risk to these papers, since missing submissions may result in unobserved ties in the network. Tan and Lee \[[@pone.0200162.ref011]\] observe histories of user accounts participating in different communities, while Fire and Guestrin \[[@pone.0200162.ref010]\] observe network ties over time modeled on user histories. Substantial blocks of missing data, including the potentially large amount of missing submissions from Reddit's nascency could redraw the map of community ties on the platform. Tree structures reconstructing threads are also similarly affected, such as work by Hessel et al \[[@pone.0200162.ref025]\] and Fire and Guestrin \[[@pone.0200162.ref026]\], which through linkages of comments and submissions similarly face issues due to missing submissions (i.e. parents of threads) or comments.
3.3 Risks to research that counts and compares participation between communities {#sec010}
--------------------------------------------------------------------------------
Other papers test hypotheses based on participation sums within communities. Gaps that are biased toward particular communities will represent a risk to the validity of these studies. Matias \[[@pone.0200162.ref006]\] observes levels of subreddit participation by moderators, observes relative participation levels of subreddit commenters in other subreddits, and observes moderator participation in "metareddits". Newell et al \[[@pone.0200162.ref007]\] observes comment volumes within subreddits. Barthel \[[@pone.0200162.ref027]\] observes comments about political candidates across Reddit during a period where many submissions are within the dataset. Barbaresi \[[@pone.0200162.ref028]\] analyzes German language text to identify relative commenting rates about places in Germany. Horne and Adali \[[@pone.0200162.ref029]\] consider posts within /r/worldnews to determine linguistic characteristics of why some news frames are more visible than others. Dosono et al \[[@pone.0200162.ref030]\] considers a specific set of communities associated with self-expression of Asian-American Pacific Islander (AAPI) identity on the platform.
As we showed in [Fig 5](#pone.0200162.g005){ref-type="fig"}, gaps do not appear to be evenly distributed across communities, since the number of missing comments and submissions per community is not strongly correlated to the number of observed comments and submissions in that community. While a simple statistical regression between the total counts of missing data and known data shows the relationship to be significant, the *R*^2^ is low enough in both cases to lead us to conclude that studies on some subreddits could lead towards very biased results due to higher than random amounts of missing data.
In practice, we observe 78 subreddits where at least 20% of the comments are missing, and 1,755 subreddits where at least 20% of the submissions are missing. Among subreddits that have any dangling references, on average they are missing at least 35% of their submissions. The *R*^2^ score in a model predicting the volume of a community's missing observations from the volume of observed comments and submissions only explains 30% of the variance of missing comments and 10% of the variance of missing submissions ([Fig 5](#pone.0200162.g005){ref-type="fig"}). The risk to any specific study will depend on the distribution of gaps across the specific communities being compared.
3.4 Risks to machine learning models {#sec011}
------------------------------------
Finally, some studies train machine learning models and conduct linguistic analysis of the Baumgartner dataset. Insofar as these studies do not make claims about populations, gaps represent a minimal risk to the validity of this research. For example, Saleem et al \[[@pone.0200162.ref012]\] trains machine learning models on comments from particular subreddits that have since been quarantined or banned by Reddit for harmful behavior.
In our observations of communities where the mass of missing data is pooled, it seems to trend towards such communities---across the three subreddits considered in their work, one of those subreddits has a large number of dangling references: observed comments refer to 696,642 unique missing submissions in the dataset for this one community alone. Among comments, 1,100 of 1,585,014 total comments were known to be missing. Saleem, Dillon, Benesch, and Ruths have re-analyzed their data after filling some gaps and fail to find any substantial differences in the performance of their machine learning models (citation forthcoming). Furthermore, since the purpose of this kind of machine learning research is to make inferences about out-of-sample observations rather than to test hypotheses about a population, such research may be less sensitive to variation due to missing data.
4 Discussion {#sec012}
============
All datasets have biases, no matter how complete we wish them to be. In the process of designing research, conscientious researchers will study those biases, document them, and account for them as best as possible. In this paper, we have shown ways in which an influential public dataset does not represent the "complete" record that its publisher and users aspired to. We have documented per-user risks of missing data, risks from the uneven distribution of missing data over time, and risks in the uneven distribution of missing data across communities. We have outlined the risks to research validity represented by these data gaps, including some of our own work.
We have raised these issues in direct conversation with Baumgartner, who has quickly and graciously re-processed ID blocks with missing data and filled in any gaps that are able to be filled. By publication time of this paper, we believe that any missing data that can be filled will have been done so for datasets provided directly by Baumgartner up to February 2016. As of now, Baumgartner has acknowledged future steps to be taken in terms of ensuring the integrity of future data by double-checking for missing content---while his commitment to increasing the integrity of the data is not required, it is highly appreciated \[[@pone.0200162.ref031]\]. Data shared from any other source may still include these missing observations. Since any missing data that Reddit does not provide will still be missing from the corrected datasets, we encourage researchers to check the integrity of your data when publishing results from this dataset. Additionally, Hessel and his colleagues have provided a response to the issues raised in this work, which we have included as supporting information.
More widely, the case of this so-called complete dataset draws attention to the risks to validity from research cultures that move fast to produce new results when new data is released. While many researchers have utilized Baumgartner's generous work on this Reddit dataset to investigate important questions, too few of us questioned a "completeness" statement that shouldn't have been accepted as truth. This dataset has numerous omissions, and those issues affect different research agendas with varying levels of severity.
As researchers, we need to protect ourselves from the dazzling scale of large datasets. We encourage more people in Baumgartner's position to collect data, share it in an ethical manner, and contribute to knowledge through the research that it enables. It will not always be possible or reasonable to place strict methodological expectations upon such citizen scientists---that responsibility lies firmly on academics. We hope this paper will encourage other researchers to test their assumptions and document data quality when conducting social scientific research with large datasets that they did not collect.
Supporting information {#sec013}
======================
###### Response letter from Hessel, Lee, Mimno and Tan.
Authors of all cited works were solicited for opportunities to respond to our findings---Jack Hessel, Lillian Lee, David Mimno, and Chenhao Tan jointly have provided a response.
(PDF)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Pulmonary fibrosis is characterized by abnormal accumulation of myofibroblasts in the interstitium and alveolar space \[[@B1], [@B2]\]. Persistent myofibroblast survival and accumulation are the essential events underlying the evolution of lung fibrosis in animal models and idiopathic pulmonary fibrosis (IPF) in humans \[[@B3]\].
Several mechanisms have been suggested to drive the unabated multiplication of myofibroblasts in IPF \[[@B4]\], including a high proliferation rate \[[@B5], [@B6]\] and resistance to apoptosis \[[@B7]--[@B9]\], based on evidence from in vitro studies using myofibroblasts from humans with IPF, as well as in vivo research using murine models of lung fibrosis.
Fibroblasts are heterogeneous, differing in phenotype and function \[[@B10], [@B11]\]. In the lungs of mice and humans, there are two subpopulations of fibroblasts, which are distinguished by their expression of Thy1 \[[@B10], [@B12]\]. It has been shown that fibroblasts in the lungs of humans with IPF and of bleomycin-treated mice are predominantly Thy1^−^ \[[@B13]--[@B16]\].
Thy1 is a 25--37 kDa glycosylphosphatidylinositol- (GPI-) anchored cell surface protein that belongs to the immunoglobulin-like gene superfamily \[[@B17]\]. Thy1 has a variety of functions in different tissues \[[@B18]\], including a role in cell apoptosis \[[@B19]--[@B24]\] and proliferation \[[@B16], [@B25]--[@B31]\]. It has been reported to function in T cell activation, neurite outgrowth, apoptosis, tumor suppression, and wound healing and fibrosis \[[@B32]\] via multiple pathways. It is involved in T cell activation, and its role in T cell function has been extensively reviewed \[[@B33]--[@B35]\]. In mice and in humans, Thy1^+^ and Thy1^−^ fibroblasts differ with respect to cytokine \[[@B28], [@B36]--[@B39]\] and growth factor responses \[[@B40], [@B41]\], as well as cell migration \[[@B42]\].
Using gene chip analysis, we found that myofibroblast Thy1 cross-linking mediates downregulation of genes promoting cell proliferation, survival, and differentiation and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions \[[@B15]\].
In the current study, we further evaluated the critical role of Thy1 protein in lung myofibroblast proliferation and apoptosis in naïve cells and during the evolution of lung fibrosis, as well as its association with profibrotic functions such as differentiation and collagen production.
2. Materials and Methods {#sec2}
========================
2.1. Animals {#sec2.1}
------------
Male, 11-12-week-old, C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) and C57BL/6J Thy1-deficient mice were used (kindly provided by Professor R.J. Morris, Department of Chemistry, King\'s College, London, UK). Their Thy1-deficient status versus wild-type (WT) was confirmed in C57BL/6 Thy1-deficient mice by flow cytometry of spleen cells using anti-Thy1-FITC mAb ([Figure 1(a)](#fig1){ref-type="fig"}). At 12--14 weeks, body weight for the two types was similar, in the range of 23--27 gm. Histological sections of the lung, heart, brain, colon, liver, and kidney were studied. There were no differences in the histological sections or in lung hydroxyproline content, which were 1274 ± 181 (mean ± SE) and 1122 ± 73 in Thy1-deficient and WT controls, respectively. Bronchoalveolar cellularity was also similar in terms of the number of cells per ml, with 99% macrophages and 1% lymphocytes or neutrophils. The two groups of mice were thus similar in all respects except for Thy1 expression.
All animal procedures were approved by the Hebrew University Hadassah Medical School Animal Care Committee. Mice were housed in a specific pathogen-free environment.
2.2. Intratracheal Instillation {#sec2.2}
-------------------------------
Mice were anesthetized by intraperitoneal (IP) injection of 0.05--0.07 ml of 40 mg/ml Ketalar (Parke-Davis, Pontypool, Gwent, UK) and 0.5 mg/ml droperidol (Janssen Pharmaceutica, Beerse, Belgium). A dose of 0.06--0.08 units of bleomycin (H. Lundbeck, Copenhagen, Denmark) dissolved in 0.1 ml of saline solution to induce lung fibrosis, or 0.1 ml of saline alone as control, was slowly injected intratracheally (IT). The mice were sacrificed 14 days after IT, as we have previously described and detailed \[[@B3]\].
2.3. Lung Cell Isolation and Culture and Quantification of Myofibroblasts {#sec2.3}
-------------------------------------------------------------------------
The lungs were removed, minced, and incubated (37°C, 5% CO~2~ air) for 45 min in PBS containing 1 mg/ml collagenase (C0130, Sigma-Aldrich, St. Louis, MO, USA). After enzyme treatment, lung tissue was gently passed through a cell dissociation sieve (Sigma-Aldrich) or 40 *μ*m nylon mesh filters (Falcon, Becton Dickinson, Franklin Lakes, NJ) and then washed twice in PBS. For myofibroblast culture experiments, lung cells (LC) were resuspended in fibroblast culture medium. Cell cultures were incubated at 37°C in 5% humidified CO~2~. Typically, within 1 week of culture initiation, more than 95% of the cells were morphologically myofibroblasts.
Cells were passaged every 5 days by dissociating monolayers with a mild trypsin solution (Biological Industries, Beit HaEmek, Israel). After initial cultures were established, myofibroblasts obtained on passages 2 through 20 were used. Myofibroblast *α*-SMA cell markers were evaluated by assessment of any increase in cell staining visualized on confocal microscope and by quantification of mean fluorescence intensity (MFI) with flow cytometry using specific anti-*α-*SMA mAb (Dako, Glostrup, Denmark) diluted 1 : 200 in 1% BSA.
2.4. Thy1^+^ and Thy1^−^ Myofibroblast Subpopulation Sorting by Flow Cytometry {#sec2.4}
------------------------------------------------------------------------------
Myofibroblasts (0.5 × 10^6^) were collected from cultures and incubated in FACS buffer (3% FCS in PBS) with anti-Thy1.2 PE (Pharmingen, San Diego, CA, USA) (0.06 *μ*g per 0.5 × 10^6^ cells) for 30 min at room temperature, washed with FACS buffer, and analyzed by flow cytometry or sorted using a FACS cell sorter under sterile conditions using a FACS-Star™ (Beckton-Dickinson, Franklin Lakes, NJ, USA), as shown in ([Figure 1(a)](#fig1){ref-type="fig"}). Thy1^+^ cells are more spindle-shaped and have more elongated processes than Thy1^−^ cells, which are more rounded.
2.5. Proliferation {#sec2.5}
------------------
### 2.5.1. Cell Cycle Analysis by Flow Cytometry {#sec2.5.1}
BrdU (Sigma-Aldrich) was added to the fibroblast culture to reach a final concentration of 20 *μ*M. The cells were later removed by incubation with trypsin for 2 h (for Mlg cells) or 6 h (for primary fibroblasts) and centrifuged at 1200 rpm for 10 min. The pellets were washed in 5 ml of 1× cold PBS, gently resuspended in 100 *μ*l of cold PBS, and fixed by slowly dripping 5 ml of ice-cold ethanol (70%). Cells were incubated at −20°C for at least 30 min or overnight, pelleted by centrifugation at 1800 rpm for 5 min, and 1 ml of 2 N HCl/Triton X-100 was slowly added with a gentle vortex. Cells were then incubated at room temperature for 30 min, pelleted by centrifugation at 1800 rpm for 5 min, and resuspended in 1 ml of 0.1 M Na~2~B~4~O~7~, pH 8.5 to neutralize the sample. After centrifugation (1800 rpm, 5 min), the pellet was incubated with 20 *μ*l FITC-conjugated anti-BrdU (Becton Dickinson) for 30 min at room temperature. Cells were pelleted and resuspended in 1 ml of PBS containing 5 *μ*g/ml propidium iodide (PI) and stored in the dark until analyzed by flow cytometry. The proliferation distribution was determined by measuring corresponding BrdU uptake versus total DNA content (cells in S phase).
### 2.5.2. Proliferation Rate Analysis by Confocal Microscope {#sec2.5.2}
Myofibroblasts isolated from the mouse lungs were seeded (0.1 × 10^6^) on 22 × 22 mm glass coverslips in fibroblast culture medium. After 24 h, BrdU was added to the culture to a final concentration of 20 *μ*M. Nonadherent cells were removed 6 h later, and adherent cells were washed twice with PBS and fixed by incubation with cold 70% ethanol overnight at 20°C. Cells were then incubated with 0.5 ml of 2 N HCl/Triton X-100 (room temperature, 30 min), and 0.5 ml of 0.1 M Na~2~B~4~O~7~, pH 8.5 was added. Cells were then washed with PBS and incubated with 20 *μ*l anti-BrdU-FITC (Becton Dickinson) for 30 min at room temperature. PI 5 *μ*g/ml was added for 2 min. Cells were then washed twice with PBS, and coverslips were mounted on glass microscope slides with mounting solution and examined with a confocal microscope (Carl Zeiss AG, Oberkochen, Germany) attached to a Zeiss Axiovert 135M inverted microscope.
### 2.5.3. Cell Growth Assessment by Methylene Blue Staining {#sec2.5.3}
The growth of myofibroblast monolayers was assessed by colorimetric quantitation of the cell mass of the surviving monolayer after staining with the basic dye methylene blue \[[@B43]\]. Assays were initiated with 10^4^ cells/well in 96-well microtiters, plated, and incubated overnight to confluence at 37°C in a 5% CO~2~ environment. Cells were fixed in 2.5% glutaraldehyde in 200 *μ*l of medium. Fixed monolayers were washed twice with 200 *μ*l of borate buffer (10 mM, pH 8.4) and stained for 1 h with methylene blue (1% in 10 mM borate buffer). Excess stain was removed by three washes with double-distilled water (DDW), and plates were dried overnight at room temperature. Bound methylene blue was extracted with 200 *μ*l of 0.1 M HCl followed by 1 h incubation at 37°C and measured at an optical density of 620 nm in a microtiter plate reader (Titertek Multiskan MMC, Flow Laboratories, Irvine, UK).
### 2.5.4. Cell Proliferation Assessment by CFSE Staining {#sec2.5.4}
In order to track myofibroblast growth, the cells were stained by fluorescent carboxyfluorescein diacetate succinimidyl ester (CFSE) \[[@B44]\] to allow flow cytometry visualization of eight to 10 discrete generations of cell division both in vitro and in vivo. CFSE labeling is distributed equally between daughter cells after division. Daughter cells thus show half of the fluorescence of their parent cells.
2 × 10^6^ myofibroblasts/ml were resuspended in PBS. An equal volume of freshly diluted 2.5 *μ*M CFSE (Molecular Probes) in PBS was added for 8 min at room temperature and staining was stopped by the addition of an 1 ml ice-cold FCS for 1 min. Cells were immediately washed three times in RPMI 1640, and the cells were cultured for 72 h. The intensity of CFSE labeling was measured by flow cytometry with CFSE intensity labeling at time 0 serving as a control measurement.
2.6. Myofibroblast Thy1 Activation {#sec2.6}
----------------------------------
Subconfluent myofibroblasts were stimulated with anti-Thy1 G7, which has previously been shown to activate T cells \[[@B45]\], or with anti-rat IgG2C*κ* isotype control (Pharmingen). We followed the methods discussed previously by Cohen et al. \[[@B46]\]. Both stimulants were added to myofibroblasts at varying concentrations ranging from 1--20 *μ*g/ml, together with recombinant protein G cross-linker (Sigma-Aldrich) at the same concentration.
2.7. RNA {#sec2.7}
--------
### 2.7.1. RNA Isolation {#sec2.7.1}
Total cellular RNA was isolated from myofibroblasts in culture using TRI Reagent (cat. number T9424, Sigma-Aldrich) according to the protocol supplied by the manufacturer. To assess RNA integrity and exclude DNA contamination, an aliquot of each sample was analyzed by electrophoresis on a 1% agarose stained with ethidium bromide. Purity and quantitation of RNA was assessed by spectrophotometer.
### 2.7.2. Reverse Transcription Polymerase Chain Reaction (RT-PCR) {#sec2.7.2}
RNA was reverse transcribed to cDNA using an avian myeloblastosis virus RT-based protocol and random primers, as well as poly-dT (Reverse Transcription System, Promega, Madison, WI, USA). One microgram of each sample was uniformly used for reverse transcription. The cDNA was diluted in a final volume of 200 *μ*l with nuclease-free water.
TaqMan real-time PCR, primers, and probes were purchased from Applied Biosystems (Foster City, CA, USA). Probe sequences of the genes analyzed were as follows:
\(i\) 18s 5′-ATTGGAGGGCAAGTCTGGTGCCAGC-3′
\(ii\) FGFR 5′-GCTCGGCACGAGACAGACTGGTCTTA-3′
\(iii\) AGRT1 5′-TTTCGCCAAGCCTGCACCTCCATGC-3′
The real-time PCR reaction mixture contained 9 or 4.5 *μ*l of the sample and 10 or 5 *μ*l of 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 1 or 0.5 *μ*l of 20× mix of unlabeled PCR primers, and a fluorogenic probe (5′ FAM dye labeled). A PRISM 7000 Sequence Detection System (Applied Biosystems) was used with the default thermal cycling program (95°C for 10 min followed by 40 cycles of 95°C, 20 seconds, 60°C, and 1 minute). Reactions were performed in triplicate. The relative quantification method was used with ΔCt calculated as Ct (target gene)-Ct (18s gene). The relative quantity of the product was expressed according to the formula 2^−ΔCt^.
2.8. Construction of Thy1 Expression Vector {#sec2.8}
-------------------------------------------
Thy1 expression vector was obtained by ligation of the entire Thy1 cDNA into pTARGET™ mammalian expression vector (Promega). Thy1 cDNA was generated from primary culture of mouse lung fibroblasts by RT-PCR, using two specific primers assigned from the published above in the paragraph discussing RT-PCR.
(i)Forward- 5′GACAAGCTTATGAACCCAGCCAT3′
(ii)Reverse- 5′GCCTCTAGATCACAGAGAAATGAA3′
The PCR product of the expected size, coded for the full-length cDNA of Thy1, was purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) according to the manufacturer\'s instructions. Purified products were cloned into a pTARGET mammalian expression vector (Promega), according to manufacturer\'s instructions. The correctness of the insert was confirmed by sequencing (Danyel Biotech, Mira Korner, Hebrew University, Jerusalem, Israel).
2.9. Gene Expression Manipulation {#sec2.9}
---------------------------------
### 2.9.1. Downregulation of Gene Expression by siRNA {#sec2.9.1}
SiRNAs were purchased from Qiagen (Valencia, CA, USA). Nontargeting siRNA (cat. number SI1027281) was used as a control.
### 2.9.2. Thy1 Downregulation {#sec2.9.2}
A combination of two types of Thy1 siRNA, 10 picomole of each (cat. numbers SI01448132 and SI01448125), was used.
### 2.9.3. Transfection Using the DreamFect™ Kit {#sec2.9.3}
1 *μ*g of expression vector was delivered into cultured fibroblasts (0.25 × 106) in each well of a 6-well plate, in the presence of 4 *μ*l/1 *μ*g DNA DreamFect (OZ Biosciences, Marseille, France), to 2 ml final medium volume. Following incubation at 37°C in humidified 95% air, 5% CO~2~ atmosphere, the medium was renewed.
### 2.9.4. Transfection Using Electroporation {#sec2.9.4}
Myofibroblasts were harvested and washed twice with ice-cold phosphate-buffered saline (PBS) (Mg^2+^, Ca^2+^ free) and resuspended in solution R (transfection kit). Small interference RNA (siRNA) or cDNA expression vector (2 *μ*g) was added to 12 *μ*l of cell suspension containing 0.5 × 106 cells. The mixture was then subjected to a single pulse from a micoporator apparatus (Digital Bio Technology, Seoul, South Korea). After shocking, samples were added to growth medium with 10% FCS. Protein expression analysis was performed 24--48 h after transfection.
### 2.9.5. Exogenic Thy1 Gene Expression Manipulation Using Thy1 siRNA or Thy1 cDNA Expression Vector {#sec2.9.5}
Transfected fibroblasts were tested to detect changes in Thy1 expression by staining with anti-Thy1 antibody and flow cytometry analysis. Thy1 downregulation was detected when siRNA specific to Thy1 was introduced into primary myofibroblasts isolated from the mouse lungs, with mean fluorescence intensity (MFI) of Thy1 staining decreasing from 553 to 83 ([Figure 1(b)](#fig1){ref-type="fig"}). Thy1 upregulation was detected when Thy1 expression vector was introduced into Mlg cell line (mouse lung-transformed fibroblasts) that lacks Thy1 expression. Expression of Thy1 was accomplished in 28.6% of non-Thy1 expressor Mlg cell line (mouse lung fibroblast), using sense but not antisense/control orientation of Thy1 cDNA ([Figure 1(c)](#fig1){ref-type="fig"}).
2.10. Statistical Analysis {#sec2.10}
--------------------------
The Mann--Whitney nonparametric test was performed for comparison of two groups. *p* \< 0.05 was considered statistically significant.
3. Results and Discussion {#sec3}
=========================
3.1. Thy1 Expression and Myofibroblast Proliferation {#sec3.1}
----------------------------------------------------
Thy1 expression is associated either with a low or a high rate of cell growth in different cell types \[[@B16], [@B25], [@B26], [@B28]--[@B31]\]. Since we found an increase in the proportion of Thy1^−^ myofibroblasts among the total myofibroblast population at day 14 following bleomycin IT \[[@B15]\], we assessed whether the high rate of lung myofibroblast proliferation is associated with an absence of Thy1 expression. To this end, myofibroblasts were isolated from the lungs of Thy1-deficient and WT-untreated mice. Results show a high proliferation rate as detected by cell mass measurement in myofibroblasts isolated from Thy1-deficient mice compared to myofibroblasts isolated from WT mice ([Figure 2(a)](#fig2){ref-type="fig"}). These results were confirmed by cell cycle analysis and BrdU uptake showing higher percentage of proliferation in myofibroblasts isolated from Thy1-deficient mice (66%) compared to myofibroblasts isolated from WT mice (6%) ([Figure 2(b)](#fig2){ref-type="fig"}).
3.2. Inverse Correlation between Thy1 Expression and Fibroblast Proliferation {#sec3.2}
-----------------------------------------------------------------------------
We then assessed whether the variance in proliferation rates between Thy1^+^ and Thy1^−^ myofibroblasts can be attributed to differences in Thy1 expression. A Thy1-negative lung fibroblast cell line, Mlg, was transfected with a Thy1.2 expression vector or a control with antisense orientation. Proliferation was assessed following transfection, by cell mass measurement and BrdU uptake. As shown, introduction of Thy1 cDNA expression vector into the Mlg cell line decreased its proliferation rate as measured by cell mass, from 1.8 (O.D.) in the control vector-transfected cells to 1.35 in Thy1-upregulated Mlg cells ([Figure 3(a)](#fig3){ref-type="fig"}). These results were confirmed by cell cycle analysis, which showed a decrease in the proliferation rate of Thy1-upregulated Mlg cells to 16% compared to 26% for control Thy1^−^ cells ([Figure 3(b)](#fig3){ref-type="fig"}).
Concomitantly, by knocking down Thy1 expression in murine primary lung myofibroblasts (Thy1^+^) using specific siRNA, we demonstrated that the myofibroblast proliferation rates were increased, as assessed by cell mass measurements, from 1.1 to 1.5 (O.D.) in Thy1-downregulated myofibroblasts ([Figure 3(c)](#fig3){ref-type="fig"}). These results were further confirmed by cell cycle analysis with BrdU uptake showing concomitant increases in proliferation rates for myofibroblasts with downregulated Thy1 (39.4%) compared to control (17.8%) ([Figure 3(d)](#fig3){ref-type="fig"}).
3.3. Thy1 Activation Downregulates Expression of Several Genes That Have a Role in Lung Fibroblast Proliferation {#sec3.3}
----------------------------------------------------------------------------------------------------------------
As we have previously shown \[[@B15]\], gene chip analysis revealed that Thy1 activation downregulated the expression of several genes that have a role in lung fibroblast proliferation \[[@B46]\], including genes affecting the cell cycle, as well as genes involved in signaling in the MAPK \[[@B47]\], insulin \[[@B48], [@B49]\], and TGF*β* \[[@B50], [@B51]\] pathways. In this study, we validated these gene chip results by real-time RT-PCR. We chose to validate fibroblast growth factor receptor (FGFR1) \[[@B52]\] and angiotensin receptor (AGRT1) \[[@B53]\], which appear to have important roles in fibroblast proliferation. Results obtained by real-time RT-PCR were consistent with gene chip analysis, which showed decreases in expression of both genes (Figures [4(a)](#fig4){ref-type="fig"} and [4(b)](#fig4){ref-type="fig"}, resp.). This downregulation may provide some explanation for our observation that Thy1 expression attenuates lung myofibroblast proliferation due to the decrease in myofibroblast receptors that transmit proproliferation signals.
3.4. Thy1^−^ Myofibroblasts from the Fibrotic Lungs Are More Proliferative Than Thy1^+^ Myofibroblasts {#sec3.4}
------------------------------------------------------------------------------------------------------
In order to extend the in vitro findings performed in naïve cells (Figures [1](#fig1){ref-type="fig"}[](#fig2){ref-type="fig"}[](#fig3){ref-type="fig"}--[4](#fig4){ref-type="fig"}) to the in vivo murine model of lung fibrosis, we compared the proliferation of Thy1^**+**^ and Thy1^−^ myofibroblasts isolated from the fibrotic lungs. Myofibroblasts were isolated from the lungs of bleomycin-treated mice and sorted into Thy1^+^ and Thy1^−^ by FACS cell sorter. As shown in [Figure 5](#fig5){ref-type="fig"}, the proliferation rate increased from 0.68 to 0.9 OD when assessed by cell mass measurement ([Figure 5(a)](#fig5){ref-type="fig"}) and from 54.8% to 89% based on CFSE staining ([Figure 5(b)](#fig5){ref-type="fig"}). The BrdU uptake rate ([Figure 5(c)](#fig5){ref-type="fig"}, yellow spots) was higher for Thy1^−^ myofibroblasts compared with Thy1^+^ myofibroblasts.
3.5. Thy1 Expression Is Not Involved in the Regulation of Myofibroblast Apoptosis {#sec3.5}
---------------------------------------------------------------------------------
In addition to increased proliferation, myofibroblast accumulation in lung fibrosis can also result from their decreased capability to undergo apoptosis. We initially assessed whether Thy1 has a role in determining basal levels of lung fibroblast apoptosis. To this end, cleavage of pro-caspase 3 was assessed in Thy1^+^ and Thy1^−^ fibroblasts taken from the lungs of bleomycin-treated mice.
Cleavage was also assessed in a lung fibroblast cell line (Mlg), following transfection with a Thy1.2 expression vector or a control vector, and in murine primary lung myofibroblasts transfected with Thy1 siRNA or control siRNA. Repeat experiments showed no cleavage of the pro-caspase 3 in any of the tested fibroblasts ([Figure 6(a)](#fig6){ref-type="fig"}), indicating that Thy1 expression does not have a role in the regulation of basal fibroblast apoptosis.
In order to assess the requirement of Thy1 activation, as opposed to just Thy1 expression in the regulation of fibroblast apoptosis, we followed the methods described in Cohen et al. \[[@B46]\]. Briefly, primary lung myofibroblasts were stimulated with G7 anti-Thy1 mAb (10 *μ*g/ml), which we have previously shown to induce Src phosphorylation \[[@B46]\]. Cleavage of pro-caspase 3 and DNA ladders was determined in primary fibroblasts following Thy1 activation. No pro-caspase 3 cleavage or DNA ladders were observed following Thy1 activation (Figures [6(b)](#fig6){ref-type="fig"} and [6(c)](#fig6){ref-type="fig"}, resp.), further confirming that Thy1 does not influence apoptosis.
3.6. Induction of Myofibroblast Differentiation Is Associated with Downregulation of Thy1 Expression {#sec3.6}
----------------------------------------------------------------------------------------------------
Myofibroblasts are activated fibroblasts expressing *α*-smooth muscle actin protein (*α*-SMA), and are the major cellular component in IPF fibroblastic foci \[[@B54]\]. The origin of these myofibroblasts is controversial. Some studies suggest that they derive from preexisting peribronchial and perivascular adventitial fibroblasts \[[@B55]\] while others suggest that they result from fibroblasts that have been induced to differentiate into myofibroblasts by treatment with cytokines, such as TGF*β* \[[@B56], [@B57]\], which is known to be secreted \[[@B39]\] and activated \[[@B41]\] following exposure to fibrotic stimuli. It has been shown that Thy1^−^ myofibroblasts have higher *α*-SMA expression compared to their Thy1^+.^counterparts \[[@B58]\]. As previously shown by others \[[@B59]\], and as we hypothesized and show here ([Figure 7](#fig7){ref-type="fig"}), during differentiation of fibroblasts to myofibroblasts (new synthesis of *α*-SMA), Thy1 expression is decreased, rendering activated Thy1^−^ myofibroblasts. To this end, murine primary lung myofibroblasts were stimulated with TGF*β* for 72 h and stained for Thy1 and *α*-SMA. While myofibroblast *α*-SMA expression was increased following TGF*β* stimulation, as determined by visual increase in cell staining in confocal microscope ([Figure 7(a)](#fig7){ref-type="fig"}) and by quantification of mean fluorescence intensity (MFI) by flow cytometry (Figures [7(b)](#fig7){ref-type="fig"} and [7(c)](#fig7){ref-type="fig"}), Thy1 expression was decreased ([Figure 7](#fig7){ref-type="fig"}), indicating that differentiation of fibroblasts into myofibroblasts is associated with downregulation of Thy1 protein expression. This may serve as another mechanism increasing the Thy1^−^ myofibroblast population during lung fibrosis.
In addition, as we have shown in our previous publication \[[@B15]\], gene chip analysis revealed that Thy1 activation as detailed by Cohen et al. \[[@B46]\] downregulates expression of some genes that have a role in myofibroblast differentiation and actin cytoskeleton regulation. Three genes are of special interest here. Two of them, Pax7 \[[@B60]\] and MyoD \[[@B61]\], are master muscle differentiation regulatory genes; the third is *α*-SMA, which is known as a myofibroblast cell marker. The inhibitory effects of Thy1 activation on myofibroblast differentiation and gene expression explain the necessity of Thy1 downregulation to allow myofibroblast differentiation.
3.7. Thy1^−^ Myofibroblasts from the Fibrotic Lungs Increase Collagen Protein and RNA Levels Than Thy1^+^ Myofibroblasts {#sec3.7}
------------------------------------------------------------------------------------------------------------------------
To extend the in vitro findings performed in naïve cells (Figures [1](#fig1){ref-type="fig"}[](#fig2){ref-type="fig"}[](#fig3){ref-type="fig"}--[4](#fig4){ref-type="fig"}) to the in vivo model of lung fibrosis, we compared the collagen protein and gene expression of Thy1^**+**^ and Thy1^−^ myofibroblasts isolated from the fibrotic lungs and collagen protein expression in myofibroblasts that were stimulated with G7 anti-Thy1 mAb (10 *μ*g/ml). As shown in [Figure 8](#fig8){ref-type="fig"}, collagen protein expression is downregulated at the RNA level ([Figure 8(a)](#fig8){ref-type="fig"}) and protein level ([Figure 8(b)](#fig8){ref-type="fig"}) in Thy1^+^ compared to Thy1^−^ fibrotic lung myofibroblasts. Moreover, direct activation of Thy1 receptor by specific anti-Thy1 mAb (*α*-Thy1) versus control (IgG) decreased the extent of spontaneous collagen production in naïve lung myofibroblasts ([Figure 8(c)](#fig8){ref-type="fig"}).
Abnormal myofibroblast accumulation and collagen deposition are characteristics of lung fibrosis \[[@B62], [@B63]\]. Myofibroblasts are thought to be primarily responsible for increased deposition of collagen within the lung \[[@B55], [@B64]\]. We have previously shown that Thy1 activation downregulates genes promoting fibroblast cell survival and differentiation \[[@B15]\], functions that are considered to be profibrotic. In this case, Thy1 may also serve as a protector against fibroblast overactivity, as shown by others who report an inverse correlation between Thy1 expression on fibroblasts and the evolution of lung fibrosis \[[@B16], [@B65]\].
The increased number of myofibroblasts accumulating in lung fibrosis may be due to their increased proliferation, their resistance to apoptosis, or both. We have found that Thy1^−^ myofibroblasts isolated from bleomycin-treated mice are more proliferative than Thy1^+^ subsets (Figures [2](#fig2){ref-type="fig"}[](#fig3){ref-type="fig"}[](#fig4){ref-type="fig"}--[5](#fig5){ref-type="fig"}). Moreover, Thy1 downregulation increased myofibroblast proliferation, and exogenic upregulation of Thy1 decreased it ([Figure 3](#fig3){ref-type="fig"}), indicating that Thy1 has an inhibitory effect on myofibroblast proliferation.
These observations are consistent with previous reports showing in vitro that Thy1^−^ fibroblasts are more proliferative than those which are Thy1^+^ when exposed to fibrogenic cytokines and growth factors \[[@B28]\], both in lung sections of bleomycin-treated mice and in humans with IPF \[[@B16]\]. Nevertheless, in naïve mice, no significant difference in cell growth rates between Thy1^+^ and Thy1^−^ fibroblasts was detected \[[@B11]\], possibly due to differences in the in vivo milieu of these naïve and the bleomycin-treated mice above.
The inhibitory effect of Thy1 on cell proliferation was also demonstrated in neurite outgrowth \[[@B66]\] and tumor growth \[[@B25], [@B26], [@B29]\]. Inhibition of cell proliferation is related to a GPI-linked protein, as demonstrated in T cells \[[@B34]\], and there is precedent for a role of Src kinase in T cell growth inhibition \[[@B67]\]. Consistent with these observations, we have shown that Thy1 activation in myofibroblasts induces Src phosphorylation \[[@B46]\], which may have inhibitory effect on myofibroblast growth.
Alternatively, Thy1 may attenuate lung myofibroblast proliferation indirectly by downregulating receptors such as Fgf and Angtl, which control fibroblast proliferation \[[@B52], [@B53]\], as we have shown by gene chip analysis \[[@B15]\] and validated by real-time PCR ([Figure 4](#fig4){ref-type="fig"}). The decrease in these receptors\' expression may reduce transmission of proproliferation signals to fibroblasts and thereby attenuate myofibroblast proliferation.
We found no difference between WT and chimeric Thy1-deficient mice with WT lymphocytes and Thy1-deficient mesenchymal cells, when compared to control the saline-treated mouse lungs in assessments of lung fibrotic injury by semiquantitative morphological index of pathological sections, as well as collagen content \[[@B15]\]. In Thy1 null mice, Thy1 is absent from all cells that would otherwise express it, in contrast to our chimeric Thy1-deficient mice, whose lymphocytes express Thy1. Because Thy1 is normally expressed on murine lymphocytes, it is possible that the severe lung fibrosis following bleomycin IT that develops in Thy1 null mice, is not due to changes in fibroblast Thy1 expression, but rather is due to changes in lymphocyte function \[[@B68]\]. However, the role of lymphocytes in bleomycin-induced lung fibrosis remains controversial, as does the role of inflammation in IPF \[[@B4], [@B69]--[@B74]\].
Gene chip analysis revealed that Thy1 activation downregulated expression of several other genes that have a role in lung fibroblast proliferation, including cell cycle, MAPK \[[@B47]\], and insulin signaling pathway genes \[[@B15], [@B49], [@B75]\]. Taken together with our earlier findings \[[@B15]\], these results provide a possible explanation for the mechanism for Thy1 expression attenuation of lung myofibroblast proliferation.
Neither activation nor exogenic upregulation or downregulation of Thy1 expression led to any change in fibroblast apoptosis ([Figure 6](#fig6){ref-type="fig"}); however, it may be possible that Thy1 can indirectly influence myofibroblast susceptibility to apoptosis that is triggered by other factors. It has been shown that angiotensin II and TGF-*β* protect fibroblasts from apoptosis \[[@B76], [@B77]\]. Indeed, we show that Thy1 activation downregulates angiotensin II and pathway genes affecting TGF-*β* signaling \[[@B15]\] and therefore may increase myofibroblast capacity to undergo apoptosis. This is consistent with a previous report showing that Thy1^+^ fibroblasts were more sensitive to apoptosis induction \[[@B58]\]. Myofibroblasts are the major cellular component in IPF fibroblastic foci \[[@B54]\]. Gene chip analysis revealed that Thy1 activation downregulates *α*-SMA expression \[[@B15]\]. This is consistent with a previous report showing that Thy1^−^ myofibroblasts have higher *α*-SMA expression compared to Thy1^+^ \[[@B58]\]. Moreover, Thy1 activation downregulated the expression of genes known to have a role in myofibroblast differentiation, such as Pax7 \[[@B60]\] and MyoD \[[@B61]\], which are master regulatory genes of muscle differentiation. This is consistent with a previous report showing enhancement of myofibroblast differentiation marker in Thy1^−^ compared to Thy1^+^ fibroblasts \[[@B58]\]. Moreover, the inhibitory effect of Thy1 activation on the expression of genes involved in myofibroblast differentiation explains the necessity of Thy1 downregulation for myofibroblast differentiation. Indeed, we show that induction of myofibroblast differentiation (e.g., *α*-SMA expression) by TGF-*β* stimulation was associated with reduction of Thy1 expression ([Figure 7(a)](#fig7){ref-type="fig"}). In addition, in bleomycin-treated mice, the number of *α*-SMA-positive cells increased \[[@B78], [@B79]\] when myofibroblast expression of Thy1 decreased ([Figure 7(b)](#fig7){ref-type="fig"}).
In addition, TGF-*β* is a key mediator of lung fibrosis \[[@B80], [@B81]\]. It stimulates fibroblast proliferation and migration \[[@B51]\], induces ECM production \[[@B82], [@B83]\], and promotes myofibroblast differentiation \[[@B84]\]. We show here that Thy1-deficient cells overexpress collagen at the RNA ([Figure 8(a)](#fig8){ref-type="fig"}) and protein ([Figure 8(b)](#fig8){ref-type="fig"}) levels. Moreover, Thy1 stimulation decreased collagen protein levels in naïve lung fibroblasts ([Figure 8(c)](#fig8){ref-type="fig"}). Our gene chip analysis revealed that Thy1 activation downregulates downstream molecules of the TGF-*β* signaling pathway \[[@B15]\]. This finding is consistent with other observations showing that Thy1 limits the ability of fibroblasts to activate TGF-*β* \[[@B41]\], and Thy1 null mice have higher TGF-*β* compared to WT mice following bleomycin instillation \[[@B16]\]. Since TGF-*β* stimulates fibroblast proliferation \[[@B51]\], deceased expression of genes involved in TGF-*β* signaling may reduce the transmission of proproliferation signals to fibroblasts, leading to the attenuation of their proliferation.
4. Conclusions {#sec4}
==============
These findings indicate that decreases of Thy1^+^ lung myofibroblast subsets in lung fibrosis increase their proliferative functions. Thy1 is critical and not only associated with the downregulation/control of genes promoting cell survival and proliferation, fibroblast differentiation, and collagen production as thoroughly assessed by genetic manipulations in Thy^+^ and in mirror experiments with Thy^−^ lung fibroblasts.
This work was supported by the Israel Science Foundation and the Gutenberg Fund. The authors thank Shifra Fraifeld for her editorial assistance in preparing this paper.
AGRT:
: Angiotensin receptor
BAL:
: Bronchoalveolar lavage
BrdU:
: Bromodeoxyuridine staining
cDNA:
: Complementary DNA
CFSE:
: Carboxyfluorescein diacetate succinimidyl ester
Ct:
: Target gene
ECM:
: Extracellular matrix
FCS:
: Fluorescence-activated cell sorting
FGFR:
: Fibroblast growth factor receptor
FITC:
: Fluorescein isothiocyanate
GPI:
: Glycosylphosphatidylinositol
h:
: Hour
IP:
: Intraperitoneal injection
IPF:
: Idiopathic pulmonary fibrosis
IT:
: Intratracheal injection
mAb:
: Monoclonal antibody b
min:
: Minutes
PBS:
: Phosphate-buffered saline
PE:
: Phycoerythrin
qPCR:
: Quantitative polymerase chain reaction
RPM:
: Revolutions per minute
RT:
: Room temperature
RT-PCR:
: Reverse transcription polymerase chain reaction
sec:
: Seconds
siRNA:
: Small-interfering RNA
TGF:
: Transforming growth factor
Thy1^−^:
: Thy1-negative
Thy1^+^:
: Thy1-positive
WT:
: Wild-type
*α*-SMA:
: *α*-Smooth muscle actin.
Conflicts of Interest
=====================
The authors have no competing interests to disclose.
Authors\' Contributions
=======================
Pazit Y. Cohen contributed to research design and performed all experimental work and primary data analysis, prepared the initial draft of the paper, and contributed to revisions. Raphael Breuer contributed to research concept and strategy, supervised the project, and critically revised the paper. Shulamit B. Wallach-Dayan conceived the research concept and strategies, supervised the study and data analysis, and critically revised the paper. All authors reviewed the final version of the paper and accept responsibility for the accuracy and integrity of the research findings reported here.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
{#fig5}
{#fig6}
{#fig7}
{#fig8}
[^1]: Academic Editor: Mirella Giovarelli
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-ijms-20-00260}
===============
The viral envelope spike (Env), which is the infection machine of HIV type I (HIV-1), is a trimeric assembly composed of three external glycoprotein gp120 subunits and three transmembrane glycoprotein gp41 subunits \[[@B1-ijms-20-00260]\]. The virally encoded glycoprotein gp120 is located on the viron's surface and is therefore not only responsible for interactions with the host receptors, but is also the sole target of neutralizing antibodies. In contrast to a general membrane fusion mechanism of enveloped viruses, HIV-1 evolves a special two-step infection strategy to protect its conserved functional sites from attack by antibodies via successively binding to the receptor CD4 and the coreceptor CCR5/CXCR4 on the host cell's surface \[[@B2-ijms-20-00260],[@B3-ijms-20-00260]\]. Binding of CD4 by gp120 has been shown to play an intermediate role in viral infection through inducing large gp120 structural arrangements that promote the maturation of the CD4 binding site (CD4bs) and exposure of the coreceptor binding site \[[@B4-ijms-20-00260]\]. Moreover, CD4bs-induced antibodies can directly interrupt interactions between gp120 and CD4 and hence prevent virus entry \[[@B5-ijms-20-00260]\]. Interestingly, in the newly developed clinical trial it has been shown that the combination of various anti-HIV-1 antibodies can maintain long-term viral suppression \[[@B6-ijms-20-00260]\].
Accumulating structural \[[@B7-ijms-20-00260],[@B8-ijms-20-00260],[@B9-ijms-20-00260],[@B10-ijms-20-00260],[@B11-ijms-20-00260],[@B12-ijms-20-00260]\] and biophysical \[[@B13-ijms-20-00260],[@B14-ijms-20-00260]\] studies indicate that the CD4-triggered conformational arrangements in gp120 are necessary for coreceptor recognition and subsequent virus-cell membrane fusion. In 2015, Rasheed et al. generated a full-length structural model of gp120 in complex with CD4 and antibody 17b using a computational protocol that integrates available X-ray structures, cryo-electron microscopy (cryo-EM) maps, and information of binding interactions \[[@B10-ijms-20-00260]\]. A recently reported cryo-EM structure of the CD4-bound gp120 \[[@B12-ijms-20-00260]\] reveals a dramatic displacement of the V1/V2 region when going from the unliganded state to the liganded conformation. Evidence for large conformational changes in gp120 upon CD4 binding also comes from the energetics experiments \[[@B13-ijms-20-00260]\], which reveal an unexpectedly large magnitude of binding thermodynamics, including changes in enthalpy, entropy, and heat capacity upon CD4 binding. The hydrogen-deuterium exchange (HDX) technique, which measures the rates of deuterium incorporation into backbone amides under solution condition, has been employed to characterize CD4-induced conformational changes in a soluble HIV-1 Env trimer \[[@B14-ijms-20-00260]\], revealing that CD4 binding induces reorganizations of the V1/V2 region, V3 loop, and much of the inner domain of gp120. However, HDX cannot provide detailed information on the conformational flexibility of a protein because it is only based on the hypothesis that the regions undergoing rapid deuterium exchange are unstructured or flexible, while those involved in stable hydrogen-bonding networks (regular secondary structure regions) or those that are highly occluded from the solvent are rigid.
Interestingly, single-molecule fluorescence resonance energy transfer (smFRET) has revealed that the unliganded gp120 in the context of native Env trimers on the viral surface is intrinsically dynamic and capable of sampling at least three distinct conformations, i.e., the low-, intermediate-, and high-FRET states, without induction by any receptor or coreceptor \[[@B15-ijms-20-00260]\]; further comparison of the probability distributions of the conformational states sampled by the unliganded Env (in the absence of a receptor/coreceptor) and the liganded Env (in the presence of CD4 and 17b) suggests that the low- and intermediate-FRET states are the ground (or unliganded) state and the CD4/17b-liganded state, respectively, for which the structures have been determined by cryo-EM \[[@B16-ijms-20-00260],[@B17-ijms-20-00260],[@B18-ijms-20-00260]\]. The high-FRET state stabilized by CD4 is an intermediate during the conformational transition between the ground and CD4/17b-liganded states, but its structure has not yet been characterized. The smFRET results imply that CD4 could selectively bind to the intermediate-FRET state, thus disrupting the distribution of three states and shifting the equilibrium toward the liganded state. However, detailed questions on dynamic aspects of gp120-CD4 interactions, such as how CD4 stabilizes the liganded state and what the effects of CD4 binding would be on the conformational dynamics, molecular motions, and thermodynamics of gp120 remain unanswered.
Molecular dynamics (MD) simulations can provide an atom-level picture of protein dynamics and a representation of the free energy landscape (FEL) near the native state of a protein. Thanks to graphics processing unit (GPU)-accelerated computing, multiple-replica MD simulations \[[@B19-ijms-20-00260]\] can now gain adequate sampling convergence. In this work, two simulation systems, one starts with the monomeric gp120 in the liganded state (hereafter referred to as the CD4-free gp120) and the other with gp120 in complex with the CD4 D1 (CD4~D1~) domain (hereafter, the gp120 in the complex is referred to as the "CD4-complexed gp120"), were constructed based on the recently published full-length gp120 model in complex with CD4 and 17b \[[@B10-ijms-20-00260]\]. Aiming to probe the effects of CD4 binding on the conformational dynamics, molecular motions, and thermodynamics of gp120, we performed a series of multiple-replica MD simulations on both systems, with the total simulation period reaching 2 µs. Our results reveal that CD4 binding suppresses the conformational fluctuations of gp120, while CD4 removal allows gp120 to transition to the unliganded state, thus providing a basis by which to better understand the mechanisms of receptor association and HIV-1 immune evasion.
2. Results {#sec2-ijms-20-00260}
==========
2.1. Molecular Architecture of gp120 {#sec2dot1-ijms-20-00260}
------------------------------------
Structural models of the CD4-free gp120 and gp120-CD4 complex were extracted from the HIV-1 Env model (PDB ID: 3J70 \[[@B10-ijms-20-00260]\]). [Figure 1](#ijms-20-00260-f001){ref-type="fig"} shows the cartoon representations of these models. It should be noted that gp120 in both models is full length and in the liganded state. The molecular architecture of the liganded gp120 can be divided into five surface-exposed variable regions (V1-V5), a bridging sheet, and a conserved core. The structural core can further be divided into two domains, the inner domain and the outer domain, with the inner domain mainly composed of a near-terminal seven-stranded β-sandwich and three α-helices (α0, α1, and α5), and the outer domain of two end-to-end stacked β-barrels and two α-helices (α2 and α3). It is clear from [Figure 1](#ijms-20-00260-f001){ref-type="fig"} that both the V1/V2 region and V3 loop protrude away from the gp120 core and present a Y-like orientation with respect to each other. The bridging sheet is composed of four anti-parallel β-strands in the order β3-β2-β21-β20, in which the β20--β21 hairpin has been considered as a regulatory switch for conformational transitions of HIV-1 Env \[[@B20-ijms-20-00260]\]. According to the crystallographic structure of gp120 in complex with CD4 and 17b (PDB ID: 1GC1) \[[@B7-ijms-20-00260]\], residues involved in direct contact with CD4 are distributed over four non-continuous segments of the sequence, including the CD4-binding loop (α3), a part of the ℒD loop, the tip of the β20--β21 hairpin in the bridging sheet, and parts of β23 and β24 in the outer domain.
2.2. Conformational Dynamics {#sec2dot2-ijms-20-00260}
----------------------------
To evaluate the stability and equilibration of MD simulations, the time evolution of backbone root-mean-square deviation (RMSD) with respect to the starting structure was calculated ([Figure 2](#ijms-20-00260-f002){ref-type="fig"}). For both systems, all replicas show a rapid increase in RMSD values from the start of simulations until reaching a plateau after about 20 ns. For the CD4-free and CD4-complexed gp120s, the equilibrium portions of RMSD curves range between 0.5 and 1.2 nm and between 0.4 and 1.0 nm, respectively. When compared to the CD4-free gp120, the narrower range of RMSD curves for the CD4-complexed gp120, together with its smaller amplitude of RMSD fluctuations, indicates that CD4 binding suppresses the global structural deviation/change of gp120.
It has been shown that multiple-replica MD simulations can enhance conformational sampling of a protein by sampling different directions in the conformational space from the starting structure \[[@B19-ijms-20-00260],[@B21-ijms-20-00260]\]. For each simulation system, only the equilibrated portions (20--100 ns) of the 10 replicas were concatenated into a single joined 800 ns trajectory, based on which the following analyses were performed to ensure that the calculated parameters reflected the intrinsic properties of gp120.
2.3. Conformational Flexibility {#sec2dot3-ijms-20-00260}
-------------------------------
Per-residue C~α~ atom root-mean-square fluctuation (RMSF) values ([Figure 3](#ijms-20-00260-f003){ref-type="fig"}) were calculated based on the joined equilibrium trajectories to evaluate and compare the conformational flexibility of these two gp120 forms. The variations in RMSF values along the chain of the CD4-free and CD4-complexed gp120s are very similar, with regular secondary structure regions exhibiting low values while the N-, C-termini, and variable loops show high values. Nevertheless, with few exceptions, almost all structural regions have higher RMSF values in the CD4-free gp120 than in the CD4-complexed gp120, resulting in the average RMSF values of 0.8 ± 0.20 and 0.6 ± 0.15 nm, respectively. Therefore, the CD4-complexed gp120 is characterized overall by lower flexibility/mobility as compared to the CD4-free form in MD simulations.
Close inspection of [Figure 3](#ijms-20-00260-f003){ref-type="fig"} reveals that the regions participating in the formation of CD4bs, including the ℒD loop, the segment ranging from α2 to α3, β20--β21 hairpin, and the segment from β24 to α5, exhibit significantly lower RMSF values in the CD4-complexed gp120 than in the CD4-free gp120. This is not surprising, as the presence of CD4 restricts the free fluctuations of these regions. Surprisingly, some of the surface-exposed regions, such as layer 1 (mainly involved in α0), α1, V1/V2 stem (residues 120--128 and 194--203), and loops V4 and V5, also have somewhat lower RMSF values in the CD4-complexed gp120. The lower mobility of these regions in the CD4-complexed gp120 could also be attributed to CD4 binding, because they are located in the vicinity of CD4~D1~, whose large size may obstruct fluctuations of the nearby regions in gp120. Also worth noting is the V1/V2 region; although it extends away from the gp120 core and is distant from CD4~D1~, higher mobility was observed in the CD-free gp120. However, the other side of the Y-like structure, i.e., the V3 loop, exhibits very similar conformational flexibility in both gp120 forms. In summary, the binding of CD4 not only heavily restrains the conformational flexibility of CD4bs, but also imposes the inhibition effect on the flexibility of its nearby regions and even those relatively remote from CD4~D1~, ultimately resulting in a decrease in the global conformational flexibility of the CD4-complexed gp120 when compared to the CD4-free form.
2.4. Collective Motions {#sec2dot4-ijms-20-00260}
-----------------------
Principal component analyses (PCA) of the joined equilibrium trajectories indicate that only a few eigenvectors possess eigenvalues which are large enough ([Figure 4](#ijms-20-00260-f004){ref-type="fig"}, main plot). Diagonalization of the covariance matrices gives the total mean square fluctuation (MSF) values of 216.8 and 139.8 nm^2^ for the CD4-free and CD4-complexed gp120s, respectively, indicating that the former experienced larger-amplitude fluctuations than the latter during simulations. Moreover, for the CD4-free gp120, the first three and five eigenvectors contribute 65.6% and 75.5% to the total MSF, respectively, and for the CD4-complexed form, the corresponding contributions are 62.6% and 72.5%, respectively. Therefore, collective atomic fluctuations along the first three eigenvectors represent the largest-amplitude protein motions.
[Figure 5](#ijms-20-00260-f005){ref-type="fig"} shows the collective motions of both forms of gp120 along the first three eigenvectors using the porcupine plot, in which the direction and length of the cone drawn on a C~α~ atom represent the fluctuation direction and amplitude of this atom along the eigenvector, respectively. As shown in [Figure 5](#ijms-20-00260-f005){ref-type="fig"}A, the first eigenvector of the CD4-free gp120 describes a common rotation of the inner and outer domains (which constitute the gp120 core) in an anticlockwise direction around an axis parallel to both domains; the V3 loop and N-, C-termini rotate in the same direction as that of the core, while the V1/V2 excursion moves with the largest amplitude in the up-left direction, leading to an approach to the distal end of the gp120 core. In contrast, the first eigenvector of the CD4-complexed gp120 ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}D) describes a clockwise rotation of the gp120 core, and at the same time, V1/V2 moves in the down-right direction, exhibiting a trend of slightly more departure from the core. Along the second eigenvector, the core of the CD4-free gp120 ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}B) exhibits somewhat similar anticlockwise rotation to that along the first eigenvector, but the V1/V2 region moves in the down-left direction, leading to its approach to the proximal end of the gp120 core. For the CD4-complexed gp120 ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}E), it is obvious that the collective fluctuations of its structural core is somewhat restrained by CD4 binding, while V3 and V1/V2 exhibit the largest fluctuation amplitude, with the former moving in the opposite direction relative to that of the core and the latter displaying a self-contraction behavior. Along the third eigenvector, the core of both gp120 forms exhibits reduced fluctuation amplitude compared to that along the first two eigenvectors, except for some surface-exposed loop regions such as loops V3 and V4 in the CD4-free gp120 ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}C) and V3 and V5 in the CD4-complexed gp120 ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}F). Nevertheless, the reduced collective motions seem to lead to a slight expansion and contraction of the CD4-binding cavity in the CD4-free and CD4-complexed gp120 forms, respectively. Of note is that the V1/V2 excursion still has the largest fluctuation amplitude, but manifests as a twisting motion in the CD-free gp120 and up-and-down mixed motions in the CD4-complexed gp120.
2.5. Free Energy Landscape (FEL) and Representative Structures {#sec2dot5-ijms-20-00260}
--------------------------------------------------------------
To reveal the effect of CD4 binding on the thermodynamics of gp120, the FELs for both forms of gp120 were constructed using the probability density function with the projection of joined equilibrium trajectories onto the first two eigenvectors as the reaction coordinates. As shown in [Figure 6](#ijms-20-00260-f006){ref-type="fig"}, both FELs present an irregular and divergent shape, distinguishing from the classical funnel-like FEL of the globular protein. In the FEL of the CD4-free gp120, there are four free energy basins/minima with an energy level lower than −13 kJ/mol (labelled as "a" to "d" in [Figure 6](#ijms-20-00260-f006){ref-type="fig"}A), while only three basins which have an energy level lower than -13 kJ/mol can be found in the FEL of the CD4-complexed gp120 (labelled as "e" to "g" in [Figure 6](#ijms-20-00260-f006){ref-type="fig"}B). If those with a free energy level lower than −10 kJ/mol are included, there are still more basins in the FEL of the CD4-free gp120 than of the CD4-complexed gp120, indicating there are more conformational substates sampled by the CD4-free gp120. In addition, the FEL of the CD4-free gp120 covers a larger region in the essential subspace than that of the CD4-complexed gp120, implying a large conformational entropy of the CD4-free gp120. Also worth noting is that the minimum free energy value of the CD4-complexed gp120 FEL is −14 kJ/mol (basin "e" in [Figure 6](#ijms-20-00260-f006){ref-type="fig"}B), which is lower than that of the CD4-free gp120 FEL, −13 kJ/mol, and this implies a higher stability of the CD4-complexed gp120. Taken together, the FEL of the CD4-complexed gp120 is characterized by fewer local free energy minima, larger free energy surface, and lower global minimum free energy value when compared to that of the CD-free gp120, indicating that CD4 binding reduces the conformational diversity and entropy and enhances the stability of gp120.
To further compare the differences in conformation between thermodynamically different substates, the representative structures/substates of the two gp120 FELs were extracted from the low free energy basins as labelled in [Figure 6](#ijms-20-00260-f006){ref-type="fig"}. [Figure 7](#ijms-20-00260-f007){ref-type="fig"}A,B show the superimposed backbones of the four and three representative structures for the CD4-free and CD4-complexed gp120s, respectively. It is clear that for both gp120 forms, the cores of their representative structures show relatively small conformational differences, although those of the CD4-free gp120 are characterized by somewhat larger variations than the CD4-complexed form, especially in the core peripheral regions, such as V3, V4, layer 1, and N-, C-termini. The most pronounced differences were observed in the orientation of the V1/V2 region with respect to the gp120 core. The orientation of V1/V2 is similar between the starting conformations of both gp120 forms, as shown by the substates "b" and "f", in which the V1/V2 tip points away from the gp120 core. However, in the substates "c" and "d" of the CD4-free gp120 ([Figure 7](#ijms-20-00260-f007){ref-type="fig"}A), the V1/V2 region is located in the vicinity of the V3 loop and partially covers the bridging sheet, indicating that the CD4-free gp120 sampled the conformations similar to the unliganded state during the multiple-replica MD simulations. In the case of the CD4-complexed gp120, although the orientations of V1/V2 are distinctly different among the three substates ("e" to "g"), V1/V2 still remains distant from the core, suggesting that the presence of CD4 prevents gp120 from transitioning toward the unliganded state. As a result, it can be concluded that: (i) the major differences among the sampled substates arise mainly from the distinct orientations of the V1/V2 region with respect to the gp120 core; (ii) CD4 binding locks gp120 conformation in the liganded state; and (iii) the removal of CD4 allows gp120 to transition between the liganded and unliganded states, in agreement with the results of the smFRET study \[[@B15-ijms-20-00260]\].
3. Discussion {#sec3-ijms-20-00260}
=============
Previous X-ray crystallographic studies \[[@B7-ijms-20-00260],[@B22-ijms-20-00260],[@B23-ijms-20-00260]\] have shown that the gp120 core assumes distinctly different conformations before and after CD4 binding. These crystal structures, together with the data from the energetics \[[@B13-ijms-20-00260]\] and HDX \[[@B14-ijms-20-00260]\] experiments evidencing the drastic conformational rearrangements of gp120 upon CD4 binding, support the induced-fit viewpoint that it is the CD4 binding that induces the conformational transition of gp120 from the unliganded to the liganded states. However, direct observations of conformational dynamics of gp120 in the absence of CD4 using smFRET \[[@B15-ijms-20-00260]\] identified three distinct conformational states, which coexisted in equilibrium but whose relative populations were remodeled by CD4 and antibody binding. Such observations support the conformational selection mechanism \[[@B24-ijms-20-00260]\] of CD4 binding, since the liganded state of gp120 has already existed, although with a low probability, in the conformational ensemble of the ligand-free gp120, and CD4 can bind selectively to this state and ultimately shift the equilibrium toward the liganded state. In the current study, we have performed multiple long-time MD simulations on gp120 in the liganded state with and without CD4 to probe the effect of CD4 binding on the dynamics and thermodynamics of gp120. Our results reveal that although the presence of CD4 suppresses the overall conformational fluctuations/flexibility of gp120 and hence locks gp120 in the liganded state, the removal of CD4 enhances the flexibility of gp120, allowing it to sample a wider conformational space and to transition backward to the unliganded state. The capability of the CD4-free gp120 in the initial liganded state to sample the other conformations including the unliganded state, together with the experimentally observed multiple states for the ligand-free HIV-1 Env trimer/gp120, indicates that conformational selection does indeed dominate the CD4 binding process.
Our MD simulations also identify the high-flexibility regions, collective motion modes, and the crucial regions directly involved in conformational transition of gp120, which could facilitate the understanding of the viral entry and immune evasion mechanisms of HIV-1. The observed high-flexibility regions are common to both forms of gp120, i.e., the V1/V2 region, loops V3, V4, and V5, and N-, C-termini ([Figure 3](#ijms-20-00260-f003){ref-type="fig"}). It has been shown that the high flexibility/mobility of the protruding V3 in the liganded state is advantageous for gp120 to sense and trap coreceptor molecules such as CCR5/CXCR4 \[[@B25-ijms-20-00260],[@B26-ijms-20-00260]\], whose engagement will promote additional conformational changes to trigger the formation of gp41-entry machinery \[[@B3-ijms-20-00260]\]. Because V4 is connected to the bridging-sheet element β20 through β19, and V5 is located between β23 and β24, the higher flexibility of these two loops in the CD4-free gp120 may explain the increased mobility of the bridging sheet as well as the β-strands connected by them, as compared to the CD4-complexed gp120. It is possible that the unstable bridging-sheet element would facilitate the disintegration of the mature bridging sheet followed by element rearrangements to obtain the premature one observed in the unliganded gp120 \[[@B27-ijms-20-00260]\]. On the other hand, the highly mobile bridging sheet and outer-domain β-strands in the CD4-free gp120 likely impart increased lability to certain antibody neutralization epitopes (i.e., CD4-induced and CD4-binding-site epitopes), which may disturb recognition/binding of relevant antibodies to gp120 in the liganded state but without CD4 occupation.
For both forms of gp120, the V1/V2 region exhibits the highest flexibility during MD simulations ([Figure 3](#ijms-20-00260-f003){ref-type="fig"}). This is likely due to its complete departure from the core in the liganded state ([Figure 1](#ijms-20-00260-f001){ref-type="fig"}). Moreover, the V1/V2 region exhibits the largest fluctuation amplitude and complicated motion modes along the first three eigenvectors ([Figure 5](#ijms-20-00260-f005){ref-type="fig"}). However, no matter what the mode is and regardless of its complexity, the V1/V2 region of the CD4-free form shows a trend of moving toward the gp120 core, while in the CD4-complexed gp120, V1/V2 moves either upward or downward along the core side without a trend of approaching the core. In fact, of the four populated substates sampled by the CD4-free gp120, the two substates ("c" and "d" in [Figure 7](#ijms-20-00260-f007){ref-type="fig"}A) show the orientation of V1/V2 in proximity of V3 and partially covering the bridging sheet. In all three populated substates sampled by the CD4-complexed gp120, the V1/V2 region still stays distant from V3 and the bridging sheet, although its orientation, relative to the core, is distinct among the three substates. As a result, it is reasonable to consider that: i) V1/V2 is a key determinant in distinguishing among different conformational substates of gp120; ii) the reorientation ability of V1/V2 is crucial for gp120 to transition from the liganded to unliganded states; iii) CD4 binding weakens the reorientation ability of V1/V2 and hence prevents the restoring transition of gp120 to the unliganded state. Additionally, since the V1/V2 region participates in associations among the three gp120 subunits in the context of the unliganded Env trimer, its dynamical properties can affect Env stability and gp120 conformational transition (i.e., from the unliganded to the liganded states), and hence contribute to regulating the neutralization phenotypes of primary HIV-1 isolates \[[@B28-ijms-20-00260],[@B29-ijms-20-00260],[@B30-ijms-20-00260]\].
4. Materials and Methods {#sec4-ijms-20-00260}
========================
4.1. Simulation System Preparation {#sec4dot1-ijms-20-00260}
----------------------------------
Atomic coordinates of the CD4-free gp120 and gp120-CD4~D1~ complex were extracted from the structural model of HIV-1 Env (PDB ID: 3J70 \[[@B10-ijms-20-00260]\]), in which the full-length gp120 subunits are in complex with CD4 and 17b. The reason for retaining only the CD4 D1 domain in the complex model is that among the four domains (D1 to D4) of CD4, only the D1 domain makes direct interatomic contact with gp120 \[[@B7-ijms-20-00260]\] and, furthermore, its presence is necessary and sufficient for the induction of virus-cell membrane fusion \[[@B31-ijms-20-00260],[@B32-ijms-20-00260]\]. First, the two structures were subjected to energy minimization in a vacuum to refine the models and to relieve strain resulting from any bad contact. Subsequently, these two energy-optimized structures were individually solvated in a dodecahedron periodic box of TIP3P water molecules \[[@B33-ijms-20-00260]\], with a minimum solute-box edge distance set to 8 Å and numbers of Cl^−^ and Na^+^ introduced to obtain the electroneutral system at 150 mM salt concentration. Finally, each solvated system was once again subjected to energy minimization until no significant energy changes could be detected.
4.2. MD Simulations {#sec4dot2-ijms-20-00260}
-------------------
All MD simulations were performed using GROningen MAchine for Chemical Simulations (GROMACS) 5.1.4 \[[@B34-ijms-20-00260]\] with the AMBER99SB-ILDN force field \[[@B35-ijms-20-00260]\]. Before production runs, each prepared protein-solvent system was subjected to four successive 200 ps position-restrained MD simulations with protein-heavy atoms restrained by decreasing harmonic force constants (i.e., 1000, 100, 10, and 0 kJ/mol/nm^2^) to soak the solute into water molecules \[[@B36-ijms-20-00260]\]. To improve conformational sampling, ten independent 100 ns production runs were performed for each system, with each run initialized with different initial atomic velocities assigned from a Maxwell distribution at 300 K. In the production MD simulations, the following protocols were used: The LINear Constraint Solver (LINCS) algorithm \[[@B37-ijms-20-00260]\] was used to restrain bond lengths to their equilibrium positions; the integration time step was 2 fs; system coordinates were saved every 2 ps; the particle-mesh Ewald (PME) algorithm \[[@B38-ijms-20-00260]\] was used to treat long-range electrostatic interactions; a twin-range cut-off (1.0 and 1.4 nm) was used to treat van der Waals interactions; temperatures of the solute and solvent were separately coupled to a 300 K heat bath with a coupling constant $\tau_{t}$ of 0.1 ps; and pressure was maintained at 1 atm using the Parrinello-Rahman barostat \[[@B39-ijms-20-00260]\] with a coupling constant $\tau_{p}$ of 0.5 ps.
4.3. Analysis Methods {#sec4dot3-ijms-20-00260}
---------------------
RMSD and RMSF were calculated using GROMACS tools "gmx rmsd" and "gmx rmsf", respectively. PCA was performed on the covariance matrix built from C~α~ atomic fluctuations in a MD trajectory, obtaining a set of eigenvectors and eigenvalues. Collective motion modes along the first few eigenvectors were shown as porcupine plots, which were obtained using a modevectors.py script with the two extremes of an eigenvector projection as the input. FEL with eigenvectors 1 and 2 as the reaction coordinates was constructed using the probability density function $F\left( s \right) = - k_{B}T\ln\left( {N_{i}/N_{max}} \right)$, where $k_{B}$ is the Boltzmann's constant, $T$ is the temperature, $N_{i}$ is the population of bin $i$, and $N_{max}$ is the population of the most populated bin.
5. Conclusions {#sec5-ijms-20-00260}
==============
In this work, we performed μs-scale multiple-replica MD simulations on the CD4-free gp120 and gp120-CD4 complex to probe the effects of CD4 binding on the conformational dynamics, molecular motions, and thermodynamics of gp120. Comparative analyses of the joined equilibrium trajectories in terms of RMSD and RMSF reveal that CD4 binding suppresses the global structural deviation/change, as well as the overall conformational flexibility of gp120. Further comparison of the constructed FELs indicates that CD4 binding reduces the conformational entropy and conformational diversity while enhancing the stability of gp120. The collective motions of the structural core in both forms of gp120 are somewhat similar, manifesting as either the common rotation of the inner and outer domains or expansion/contraction of the CD4-binding cavity. However, the observed differences in fluctuation direction of the peripheral loops, in particular the V1/V2 region between these two forms gp120, possibly have distinct consequences for gp120 conformation. Visual check of the representative structures extracted from free energy basins/minima of FELs reveals that the conformational transition from the liganded to the unliganded states did indeed occur for the CD4-free gp120, but not for the CD4-complexed gp120 during simulations; moreover, the major conformational differences are reflected in the relative orientation of V1/V2 with respect to the gp120 core. As a result, we conclude that the V1/V2 region is the major structural determinant for gp120 transition between different states/substates and that CD4 binding greatly weakens the reorientation ability of V1/V2, and, as such, prevents the transition to the unliganded state. In the absence of CD4, the spontaneous conformational transition of gp120 from the liganded state to the unliganded state supports the proposed conformational selection mechanism for CD4 binding. Additionally, the enhanced mobility of the bridging sheet and certain β-strands of the outer domain upon CD4 removal likely destabilizes certain antibody neutralization epitopes, thus providing a possible explanation for the avoidance of antibody-mediated neutralization of gp120 in the liganded state without CD4 occupation. We suggest that the discovery and development of small molecules capable of restraining V1/V2 reorientation and locking gp120 conformation in either the liganded or the unliganded states may be a promising strategy to control HIV-1 infection.
S.-Q.L., P.S. and Y.L. designed the study and revised the manuscript. Y.L. and L.D. performed the simulations, analyzed the data, and drafted the manuscript. L.-Q.Y. participated in analyzing the data. All authors contributed to and approved the final version of the manuscript.
This study was funded by the National Natural Sciences Foundation of China (No. 31370715, 31860243, and 31660015) and Programs for Excellent Young Talents (XT412003) and Donglu Scholar in the Yunnan University.
The authors declare no conflict of interest.
{#ijms-20-00260-f001}
{#ijms-20-00260-f002}
{#ijms-20-00260-f003}
{#ijms-20-00260-f004}
{#ijms-20-00260-f005}
{#ijms-20-00260-f006}
{#ijms-20-00260-f007}
| {
"pile_set_name": "PubMed Central"
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Introduction
============
Since its introduction in 1993, there has been continuous underfunding of the Heath System in Hungary; it is especially true for expensive specialties, like Intensive Care. In many other countries that use DRG costing, there is individual funding available for ICUs, but it is not the case in Hungary. In this study, we compared actual ICU costs with reimbursed funds during a 1-month period.
Methods
=======
We selected 1 month for which the amount of reimbursed fund was available (August 2001). Retrospectively, we collected the actual variable costs for every patient during that period, using nursing and medical records. These variable costs included drugs, disposables, nutrition, blood and diagnostics. Fixed costs (non-clinical support and personnel) were then calculated with the top-down method. We did not include estate costs in our study as it was proven to show little importance in cost analysis \[[@B1]\]. General patient data, reason for admission, SAPS-II score, length of ICU stay and length of mechanical ventilation were assessed as well. These potential cost indicators were then evaluated to the actual cost of ICU care and reimbursed DRG funds.
Results
=======
There were 12 patients during August 2001, who had been admitted to and discharged from our ICU. The overall costs of these cases were 7.72 M Ft (SD = 0.60 M Ft) and there was only 7.26 M Ft (SD = 0.61 M Ft) refunded to the unit.
The length of stay correlated very well with actual cost (*r*^2^ = 0.98) and less well with reimbursed DRG funds (*r*^2^ = 0.75). The length of mechanical ventilation showed better correlation with DRG funding (*r*^2^ = 0.86), then with actual costs (*r*^2^ = 0.66). However, there was no correlation between SAPS-II and actual or reimbursed funds (*r*^2^ = -0.54, -0.08).
Conclusion
==========
The actual cost of Intensive Care was significantly higher then the reimbursed DRG fund given to the unit. In our Health System, DRG refund is supposed to cover estate costs and capital equipment as well. There was a negative balance without including these cost blocks, which highlights the importance of individual funding for Intensive Care. DRG refund was less than actual cost for patients who were not ventilated. Our study confirmed that SAPS II could not be used as a cost indicator.
HUF EURO
---------------------- ----------- -------- --------
Drugs/fluids 981,687 3974 12.70%
Disposables 545,566 2209 7.06%
Nutrition/blood 266,212 1078 3.45%
Clinical support 191,537 775 2.48%
Non-clinical support 1,094,840 4433 14.17%
Personnel 4,648,614 18,820 60.15%
Total cost 7,728,456 31,289 100%
| {
"pile_set_name": "PubMed Central"
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We address the relationship between structure and dynamics in complex networks by taking the steady-state distribution of the frequency of visits to nodes--a dynamical feature--obtained by performing random walks[@c1] along the networks. A complex network[@c2] is taken as a graph with directed edges and associated weights, which are represented in terms of the weight matrix $W$. The $N$ nodes in the network are numbered as $i = 1,2,\ldots,N$, and a directed edge with weight $M$, extending from node $j$ to node $i$, is represented as $W\left( i,j \right) = M$. No self-connections (loops) are considered. The *in* and *out strengths* of a node $i$, abbreviated as ${is}\left( i \right)$ and ${os}\left( i \right)$, correspond to the sum of the weights of its in- and outbound connections, respectively. The stochastic matrix $S$ for such a network is$$\left. S\left( i,j \right) = W\left( i,j \right)\slash{os}\left( j \right). \right.$$The matrix $S$ is assumed to be irreducible; i.e., any of its nodes can be accessible from any other node, which allows the definition of a unique and stable steady state. An agent, placed at any initial node $j$, chooses among the adjacent outbound edges of node $j$ with probability equal to $S\left( i,j \right)$. This step is repeated a large number of times $T$, and the frequency of visits to each node $i$ is calculated as $\left. v\left( i \right) = \left( \text{number}\;\text{of}\;\text{visits}\;\text{during}\;\text{the}\;\text{walk} \right)\slash T \right.$. In the steady state (i.e., after a long time period $T$), $\mathbf{v} = S\mathbf{v}$ and the frequency of visits to each node along the random walk may be calculated in terms of the eigenvector associated with the unit eigenvalue (e.g., Ref. [@c6]). For proper statistical normalization we set $\sum_{p}v\left( p \right) = 1$. The dominant eigenvector of the stochastic matrix has theoretically and experimentally been verified to be remarkably similar to the corresponding eigenvector of the weight matrix, implying that the adopted random walk model shares several features with other types of dynamics, including linear and nonlinear summations of activations and flow in networks.
In addition to providing a modeling approach intrinsically compatible with dynamics involving successive visits to nodes by a single or multiple agents, such as is the case with world wide web (WWW) navigation, text writing, and transportation systems, random walks are directly related to diffusion. More specifically, as time progresses, the frequency of visits to each network node approaches the activity values which would be obtained by the traditional diffusion equation. A full congruence between such frequencies and activity diffusion is obtained at the equilibrium state of the random walk process. Therefore, random walks are also directly related to the important phenomenon of diffusion, which plays an important role in a large number of linear and nonlinear dynamic systems including disease spreading and pattern formation. Random walks are also intrinsically connected to Markov chains, electrical circuits, and flows in networks, and even dynamical models such as Ising. For such reasons, random walks have become one of the most important and general models of dynamics in physics and other areas, constituting a primary choice for investigating dynamics in complex networks.
The correlations between activity (the frequency of visits to nodes $\mathbf{v}$) and topology (out strength $\mathbf{os}$ or in strength $\mathbf{is}$) can be quantified in terms of the Pearson correlation coefficient $r$. For full activity-topology correlation in directed networks, i.e., ${\mid r \mid} = 1$ between $\mathbf{v}$ and $\mathbf{os}$ or between $\mathbf{v}$ and $\mathbf{is}$, it is enough that (i) the network must be strongly connected, i.e., $S$ is irreducible, and (ii) for any node, the in strength must be equal to the out strength. The proof of the statement above is as follows. Because the network is strongly connected, its stochastic matrix $S$ has a unit eigenvector in the steady state, i.e., $\mathbf{v} = S\mathbf{v}$. Since $\left. S\left( i,j \right) = W\left( i,j \right)\slash{os}\left( j \right) \right.$, the $i\text{th}$ element of the vector $S\mathbf{os}$ is given as$$S\left( i,1 \right){os}\left( 1 \right) + S\left( i,2 \right){os}\left( 2 \right) + \cdots + S\left( i,N \right){os}\left( N \right) = \frac{W\left( i,1 \right)}{{os}\left( 1 \right)}{os}\left( 1 \right) + \frac{W\left( i,2 \right)}{{os}\left( 2 \right)}{os}\left( 2 \right) + \cdots + \frac{W\left( i,N \right)}{{os}\left( N \right)}{os}\left( N \right) = W\left( i,1 \right) + W\left( i,2 \right) + \cdots + W\left( i,N \right) = {is}\left( i \right).$$By hypothesis, ${is}\left( i \right) = {os}\left( i \right)$ for any $i$ and, therefore, both $\mathbf{os}$ and $\mathbf{is}$ are eigenvectors of $S$ associated with the unit eigenvalue. Then $\mathbf{os} = \mathbf{is} = \mathbf{v}$, implying full correlation between frequency of visits and both in and out strengths.
An implication of this derivation is that for perfectly correlated networks, the frequency of symbols produced by random walks will be equal to the out strength or in strength distributions. Therefore, an out strength scale-free[@c3] network must produce sequences obeying Zipf's law[@c7] and vice versa. If, on the other hand, the node distribution is Gaussian, the frequency of visits to nodes will also be a Gaussian function; that is to say, the distribution of nodes is replicated in the node activation. Although the correlation between node strength and random walk dynamics in undirected networks has been established before[@c8] (including full correlation[@c9]), the findings reported here are more general since they are related to any directed weighted network, such as the WWW and the airport network. Indeed, the correlation conditions for undirected networks can be understood as a particular case of the conditions above.
A fully correlated network will have ${\mid r \mid} = 1$. We obtained $r = 1$ for texts by Darwin[@c11] and Wodehouse[@c12] and for the network of airports in the USA.[@c13] The word association network was obtained by representing each distinct word as a node, while the edges were established by the sequence of immediately adjacent words in the text after the removal of stopwords[@c14] and lemmatization.[@c15] More specifically, the fact that word $U$ has been followed by word $V$, $M$ times during the text, is represented as $W\left( V,U \right) = M$. Zipf's law is known to apply to this type of network.[@c16] The airport network presents a link between two airports if there exists at least one flight between them. The number of flights performed in one month was used as the strength of the edges.
We obtained $r$ for various real networks (Table [I](#t1){ref-type="table"}), including the fully correlated networks mentioned above. To interpret these data, we recall that a small $r$ means that a hub (large in or out strength) in topology is not necessarily a center of activity. Notably, in all cases considered $r$ is greater for the in strength than for the out strength. This may be understood with a trivial example of a node from which a high number of links emerge (implying large out strength) but which has only very few inbound links. This node, in a random walk model, will be rarely occupied and thus cannot be a center of activity, though it will strongly affect the rest of the network by sending activation to many other targets. Understanding why a hub in terms of in strength may fail to be very active is more subtle. Consider a central node receiving links from many other nodes arranged in a circle, i.e., the central node has a large in strength but with the surrounding nodes possessing small in strength. In other words, if a node $i$ receives several links from nodes with low activity, this node $i$ will likewise be fairly inactive. In order to further analyze the latter case, we may examine the correlations between the frequency of visits to each node $i$ and the *cumulative hierarchical in and out strengths* of that node. The hierarchical degree[@c17] of a network node provides a natural extension of the traditional concept of node degree. The immediate neighbors of a node $i$ are called the first hierarchical level of $i$. The subsequent hierarchical levels are obtained as follows. The level $h + 1$ contains the neighbors of the nodes of level $h$. The cumulative hierarchical out strength of a node $i$ at the hierarchical level $h$ corresponds to the sum of the weights of the edges extending from the hierarchical level $h - 1$ to the level $h$, plus the out strengths obtained from hierarchy $1$ to $h - 1$. Similarly, the cumulative in strength of a node $i$ at hierarchical level $h$ is the sum of the weights of the edges from hierarchical level $h$ to the previous level $h - 1$, plus the in strengths obtained from hierarchy 1 to $h - 1$. The traditional in and out strengths are, respectively, the cumulative hierarchical in and out strengths at hierarchical level 1 (see Supplementary Methods in Refs. [@c20] for an illustration of hierarchical levels). Because complex networks are also small world structures, it suffices to consider hierarchies up to two or three levels.
For the least correlated network analyzed, viz., that of the largest strongly connected cluster in the network of WWW links in the domain of Ref. [@c21] (Massey University, New Zealand) (Refs. [@c22]) activity could not be related to in strength at any hierarchical level. Because the Pearson coefficient corresponds to a single real value, it cannot adequately express the coexistence of the many relationships between activity and degrees present in this specific network as well as possibly heterogeneous topologies. Very similar results were obtained for other WWW networks, which indicate that the reasons why topological hubs have not been highly active cannot be identified at the present moment (see, however, discussion for higher correlated networks below).
However, for the two neuronal structures of Table [I](#t1){ref-type="table"} that are not fully correlated (network defined by the interconnectivity between cortical regions of the cat[@c24] and network of synaptic connections in *C. elegans*[@c25]), activity was shown to increase with the cumulative first and second hierarchical in strengths. In the cat cortical network, each cortical region is represented as a node, and the interconnections are reflected by the network edges. Significantly, in a previous paper,[@c26] it was shown that when connections between cortex and thalamus were included, the correlation between activity and outdegree increased significantly. This could be interpreted as a result of increased efficiency with the topological hubs becoming highly active. Furthermore, for the fully correlated networks, such as word associations obtained for texts by Darwin and Wodehouse, activity increased basically with the square of the cumulative second hierarchical in strength (see Supplementary Fig. 2. in Ref. [@c20]). In addition, the correlations obtained for these two authors are markedly distinct, as the work of Wodehouse is characterized by substantially steeper increase of frequency of visits for large in strength values (see Supplementary Fig. 3 in Ref. [@c20]). Therefore, the results considering higher cumulative hierarchical degrees may serve as a feature for authorship identification.
In conclusion, we have established (i) a set of conditions for full correlation between topological and dynamical features of directed complex networks and demonstrated that (ii) Zipf's law can be naturally derived for fully correlated networks. Result (i) is of fundamental importance for studies relating the dynamics and connectivity in networks, with critical practical implications. For instance, it not only demonstrates that hubs of connectivity may not correspond to hubs of activity but also provides a sufficient condition for achieving full correlation. Result (ii) is also of fundamental importance as it relates two of the most important concepts in complex systems, namely, Zipf's law and scale-free networks. Even though sharing the feature of power law, these two key concepts had been extensively studied on their own. The result reported in this work paves the way for important additional investigations, especially by showing that Zipf's law may be a consequence of dynamics taking place in scale-free systems. In the cases where the network is not fully correlated, the Pearson coefficient may be used as a characterizing parameter. For a network with very small correlation, such as the WWW links between the pages in a New Zealand domain analyzed here, the reasons for hubs failing to be active could not be identified, probably because of the substantially higher complexity and heterogeneity of this network, including varying levels of clustering coefficients, as compared to the neuronal networks.
This work was financially supported by FAPESP and CNPq (Brazil). Luciano da F. Costa thanks grants 05/00587-5 (FAPESP) and 308231/03-1 (CNPq).
######
Number of nodes (No. nodes), number of edges (No. Edges), means and standard deviations of the clustering coefficient (CC), cumulative hierarchical in strengths for levels 1--4 (IS1--IS4), cumulative hierarchical out strengths for levels 1--4 (OS1--OS4), and the Pearson correlation coefficients between the activation and all cumulative hierarchical in strengths and out strengths $\left( r_{{IS}1}–r_{{OS}4} \right)$ for the complex networks considered in the present work.
Cortex *C. elegans* Airports Darwin Wodehouse WWW
------------- -------------------- ----------------------- ------------------------------ ----------------------- ----------------------- -----------------------
No. nodes 53 191 280 3678 3705 10 810
No. edges 826 2449 4160 22 095 16 939 158 102
CC $0.60 \pm 0.15$ $0.22 \pm 0.11$ $0.62 \pm 0.41$ $0.04 \pm 0.11$ $0.03 \pm 0.08$ $0.60 \pm 0.21$
IS1 $25.89 \pm 9.42$ $100.82 \pm 110.03$ $2041.07 \pm 4323.33$ $7.87 \pm 22.15$ $5.29 \pm 16.15$ $14.63 \pm 155.87$
IS2 $217.13 \pm 56.68$ $1183.32 \pm 960.60$ $76\ 068.88 \pm 53\ 936.38$ $329.61 \pm 648.33$ $188.45 \pm 385.21$ $176.00 \pm 917.67$
IS3 $285.02 \pm 27.13$ $3543.97 \pm 1118.85$ $110\ 381.09 \pm 35\ 614.97$ $3352.93 \pm 2716.07$ $1977.58 \pm 1758.30$ $879.71 \pm 2635.18$
IS4 $285.68 \pm 27.13$ $4164.04 \pm 535.73$ $113\ 662.07 \pm 32\ 404.79$ $6943.53 \pm 2470.62$ $4830.73 \pm 1876.14$ $2468.12 \pm 4528.49$
OS1 $25.89 \pm 11.87$ $100.82 \pm 73.69$ $2041.07 \pm 4329.44$ $7.87 \pm 22.15$ $5.29 \pm 16.15$ $14.63 \pm 10.58$
OS2 $217.96 \pm 89.94$ $1156.76 \pm 675.14$ $76\ 049.93 \pm 54\ 196.34$ $313.16 \pm 626.72$ $187.60 \pm 394.19$ $176.00 \pm 131.02$
OS3 $296.98 \pm 34.93$ $3071.82 \pm 806.15$ $110\ 771.60 \pm 35\ 721.52$ $3234.23 \pm 2705.50$ $1961.32 \pm 1778.45$ $913.55 \pm 495.34$
OS4 $298.94 \pm 32.19$ $3532.41 \pm 473.59$ $114\ 054.35 \pm 32\ 493.50$ $6753.76 \pm 2454.90$ $4823.73 \pm 1853.97$ $2356.92 \pm 1200.37$
$r_{{IS}1}$ 0.83 0.78 1.00 1.00 1.00 0.15
$r_{{IS}2}$ 0.58 0.84 0.33 0.86 0.82 0.09
$r_{{IS}3}$ 0.24 0.43 0.11 0.42 0.43 0.13
$r_{{IS}4}$ 0.24 0.35 0.08 0.20 0.22 0.11
$r_{{OS}1}$ 0.39 0.20 1.00 1.00 1.00 0.00
$r_{{OS}2}$ 0.30 0.01 0.33 0.87 0.81 $- 0.03$
$r_{{OS}3}$ $- 0.03$ $- 0.19$ 0.11 0.42 0.43 $- 0.05$
$r_{{OS}4}$ $- 0.07$ $- 0.33$ 0.07 0.20 0.22 $- 0.07$
[^1]: Electronic mail: luciano\@if.sc.usp.br
| {
"pile_set_name": "PubMed Central"
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All relevant data are available from figshare at [10.6084/m9.figshare.4597918](http://dx.doi.org/10.6084/m9.figshare.4597918).
Introduction {#sec001}
============
In August 2011, Igor Labutov and Jason Yosinski, two PhD students at Cornell University, let a pair of chat bots, called Alan and Sruthi, talk to each other online. Starting with a simple greeting, the one-and-a-half-minute dialogue quickly escalated into an argument about what Alan and Sruthi had just said, whether they were robots, and about God \[[@pone.0171774.ref001]\]. The first ever conversation between two simple artificial intelligence agents ended in a conflict.
A bot, or software agent, is a computer program that is persistent, autonomous, and reactive \[[@pone.0171774.ref002],[@pone.0171774.ref003]\]. Bots are defined by programming code that runs continuously and can be activated by itself. They make and execute decisions without human intervention and perceive and adapt to the context they operate in. Internet bots, also known as web bots, are bots that run over the Internet. They appeared and proliferated soon after the creation of the World Wide Web \[[@pone.0171774.ref004]\]. Already in 1993, Martijn Koster published "Guidelines to robot writers," which contained suggestions about developing web crawlers \[[@pone.0171774.ref005]\], a kind of bot. Eggdrop, one of the first known Internet Relay Chat bots, started greeting chat newcomers also in 1993 \[[@pone.0171774.ref006]\]. In 1996, Fah-Chun Cheong published a 413-page book, claiming to have a current listing of all bots available on the Internet at that point in time. Since then, Internet bots have proliferated and diversified well beyond our ability to record them in an exhaustive list \[[@pone.0171774.ref007],[@pone.0171774.ref008]\]. As a result, bots have been responsible for an increasingly larger proportion of activities on the Web. For example, one study found that 25% of all messages on Yahoo! chat over a period of three months in 2007 were sent by spam bots \[[@pone.0171774.ref009]\]. Another study discovered that 32% of all tweets made by the most active Twitter users in 2009 were generated by bots \[[@pone.0171774.ref010]\], meaning that bots were responsible for an estimated 24% of all tweets \[[@pone.0171774.ref011]\]. Further, researchers estimated that bots comprise between 4% and 7% of the avatars on the virtual world Second Life in 2009 \[[@pone.0171774.ref012]\]. A media analytics company found that 54% of the online ads shown in thousands of ad campaigns in 2012 and 2013 were viewed by bots, rather than humans \[[@pone.0171774.ref013]\]. According to an online security company, bots accounted for 48.5% of website visits in 2015 \[[@pone.0171774.ref014]\]. Also in 2015, 100,000 accounts on the multi-player online game World of Warcraft (about 1% of all accounts) were banned for using bots \[[@pone.0171774.ref015]\]. And in the same year, a database leak revealed that more than 70,000 "female" bots sent more than 20 million messages on the cheater dating site Ashley Madison \[[@pone.0171774.ref016]\].
As the population of bots active on the Internet 24/7 is growing fast, their interactions are equally intensifying. An increasing number of decisions, options, choices, and services depend now on bots working properly, efficaciously, and successfully. Yet, we know very little about the life and evolution of our digital minions. In particular, predicting how bots' interactions will evolve and play out even when they rely on very simple algorithms is already challenging. Furthermore, as Alan and Sruthi demonstrated, even if bots are designed to collaborate, conflict may occur inadvertently. Clearly, it is crucial to understand what could affect bot-bot interactions in order to design cooperative bots that can manage disagreement, avoid unproductive conflict, and fulfill their tasks in ways that are socially and ethically acceptable.
There are many types of Internet bots (see [Table 1](#pone.0171774.t001){ref-type="table"}). These bots form an increasingly complex system of social interactions. Do bots interact with each other in ways that are comparable to how we humans interact with each other? Bots are predictable automatons that do not have the capacity for emotions, meaning-making, creativity, and sociality \[[@pone.0171774.ref017]\]. Despite recent advances in the field of Artificial Intelligence, the idea that bots can have morality and culture is still far from reality. Today, it is natural to expect interactions between bots to be relatively predictable and uneventful, lacking the spontaneity and complexity of human social interactions. However, even in such simple contexts, our research shows that there may be more similarities between bots and humans than one may expect. Focusing on one particular human-bot community, we find that conflict emerges even among benevolent bots that are designed to benefit their environment and not fight each other, and that bot interactions may differ when they occur in environments influenced by different human cultures.
10.1371/journal.pone.0171774.t001
###### Categorization of Internet bots according to the intended effect of their operations and the kind of activities they perform, including some familiar examples for each type.
{#pone.0171774.t001g}
Benevolent Malevolent
------------------------- ---------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Collect information** Web crawlersBots used by researchers Spam bots that collect e-mail addressesFacebook bots that collect private information
**Execute actions** Anti-vandalism bots on WikipediaCensoring and moderating bots on chats and forums Auction-site botsHigh-frequency trading algorithmsGaming botsDDoS attack botsViruses and wormsClickfraud bots that increase views of online ads and YouTube videos
**Generate content** Editing bots on WikipediaTwitter bots that create alerts or provide content aggregation Spam bots that disseminate adsBot farms that write positive reviews and boost ratings on Apple App Store, YouTube, etc.
**Emulate humans** Customer service bots\@DeepDrumpf and poet-writing bots on TwitterAI bots, e.g. IBM's Watson Social bots involved in astroturfing on TwitterSocial bots on the cheater dating site Ashley Madison
Benevolent bots are designed to support human users or cooperate with them. Malevolent bots are designed to exploit human users and compete negatively with them. We have classified high-frequency trading algorithms as malevolent because they exploit markets in ways that increase volatility and precipitate flash crashes. In this study, we use data from editing bots on Wikipedia (benevolent bots that generate content).
We study bots on Wikipedia, the largest free online encyclopedia. Bots on Wikipedia are computer scripts that automatically handle repetitive and mundane tasks to develop, improve, and maintain the encyclopedia. They are easy to identify because they operate from dedicated user accounts that have been flagged and officially approved. Approval requires that the bot follows Wikipedia's bot policy.
Bots are important contributors to Wikipedia. For example, in 2014, bots completed about 15% of the edits on all language editions of the encyclopedia \[[@pone.0171774.ref018]\]. In general, Wikipedia bots complete a variety of activities. They identify and undo vandalism, enforce bans, check spelling, create inter-language links, import content automatically, mine data, identify copyright violations, greet newcomers, and so on \[[@pone.0171774.ref019]\]. Our analysis here focuses on editing bots, which modify articles directly. We analyze the interactions between bots and investigate the extent to which they resemble interactions between humans. In particular, we focus on whether bots disagree with each other, how the dynamics of disagreement differ for bots versus humans, and whether there are differences between bots operating in different language editions of Wikipedia.
To measure disagreement, we study reverts. A revert on Wikipedia occurs when an editor, whether human or bot, undoes another editor's contribution by restoring an earlier version of the article. Reverts that occur systematically indicate controversy and conflict \[[@pone.0171774.ref020]--[@pone.0171774.ref022]\]. Reverts are technically easy to detect regardless of the context and the language, so they enable analysis at the scale of the whole system.
Our data contain all edits in 13 different language editions of Wikipedia in the first ten years after the encyclopedia was launched (2001--2010). The languages represent editions of different size and editors from diverse cultures (see [Materials and Methods](#sec004){ref-type="sec"} for details). We know which user completed the edit, when, in which article, whether the edit was a revert and, if so, which previous edit was reverted. We first identified which editors are humans, bots, or vandals. We isolated the vandals since their short-lived disruptive activity exhibits different time and interaction patterns than the activity of regular Wikipedia editors.
Results {#sec002}
=======
Bots constitute a tiny proportion of all Wikipedia editors but they stand behind a significant proportion of all edits ([Fig 1A and 1B](#pone.0171774.g001){ref-type="fig"}). There are significant differences between different languages in terms of how active bots are. From previous research, we know that, in small and endangered languages, bots are extremely active and do more than 50% of the edits, sometimes up to 100% \[[@pone.0171774.ref019]\]. Their tasks, however, are mainly restricted to adding links between articles and languages. In large and active languages, the level of bot activity is much lower but also much more variable.
{#pone.0171774.g001}
Compared to humans, a smaller proportion of bots' edits are reverts and a smaller proportion get reverted ([Fig 1C and 1D](#pone.0171774.g001){ref-type="fig"}). In other words, bots dispute others and are disputed by others to a lesser extent than humans. Since 2001, the number of bots and their activity has been increasing but at a slowing rate ([S1 Fig](#pone.0171774.s001){ref-type="supplementary-material"}). In contrast, the number of reverts between bots has been continuously increasing ([Fig 2A](#pone.0171774.g002){ref-type="fig"}). This would suggest that bot interactions are not becoming more efficient. We also see that the proportion of mutual bot-bot reverts has remained relatively stable, perhaps even slightly increasing over time, indicating that bot owners have not learned to identify bot conflicts faster ([Fig 2B](#pone.0171774.g002){ref-type="fig"}).
{#pone.0171774.g002}
In general, bots revert each other a lot: for example, over the ten-year period, bots on English Wikipedia reverted another bot on average 105 times, which is significantly larger than the average of 3 times for humans ([S1 Table](#pone.0171774.s009){ref-type="supplementary-material"}). Bots on German Wikipedia revert each other to a much lesser extent than other bots (24 times on average). Bots on Portuguese Wikipedia, in contrast, fight the most, with an average of 185 bot-bot reverts per bot. This striking difference, however, disappears when we account for the fact that bots on Portuguese Wikipedia edit more than bots on German Wikipedia. In general, since bots are much more active editors than humans, the higher number of bot-bot reverts does not mean that bots fight more than humans. In fact, the proportion of bots' edits that are reverts is smaller for bots than for humans ([Fig 1C](#pone.0171774.g001){ref-type="fig"}). This proportion is highest for bots in the English and the Romance-language editions (Spanish, French, Portuguese, and Romanian). Interestingly, although bots in these languages revert more often compared to bots in other languages, fewer of these reverts are for another bot ([S2 Fig](#pone.0171774.s002){ref-type="supplementary-material"}).
Our analysis focuses on interactions in dyads over time. We model the interaction trajectories in two-dimensional space, where the x-axis measures time and the y-axis measures how many more times the first editor has reverted the second compared to the second reverting the first ([Fig 3](#pone.0171774.g003){ref-type="fig"}). We analyze three properties of the trajectories: latency, imbalance, and reciprocity. Latency measures the average steepness of the interaction trajectory, imbalance measures the distance between the *x*-axis and the last point of the trajectory, and reciprocity measures the trajectory's jaggedness (see [Materials and Methods](#sec004){ref-type="sec"} below for definitions).
{#pone.0171774.g003}
Analyzing the properties of the interaction trajectories suggests that the dynamics of disagreement differ significantly between bots and humans. Reverts between bots tend to occur at a slower rate and a conflict between two bots can take place over longer periods of time, sometimes over years. In fact, bot-bot interactions have different characteristic time scale than human-human interactions ([S3 Fig](#pone.0171774.s003){ref-type="supplementary-material"}). The characteristic average time between successive reverts for humans is at 2 minutes, 24 hours, or 1 year. In comparison, bot-bot interactions have a characteristic average response of 1 month. This difference is likely because, first, bots systematically crawl articles and, second, bots are restricted as to how often they can make edits (the Wikipedia bot policy usually requires spacing of 10 seconds, or 5 for anti-vandalism activity, which is considered more urgent). In contrast, humans use automatic tools that report live changes made to a pre-selected list of articles \[[@pone.0171774.ref024],[@pone.0171774.ref025]\]; they can thus follow only a small set of articles and, in principle, react instantaneously to any edits on those.
Bots also tend to reciprocate each other's reverts to a greater extent. In contrast, humans tend to have highly unbalanced interactions, where one individual unilaterally reverts another one ([S4](#pone.0171774.s004){ref-type="supplementary-material"} and [S5](#pone.0171774.s005){ref-type="supplementary-material"} Figs).
We quantify these findings more precisely by identifying different types of interaction trajectories and counting how often they occur for bots and for humans, as well as for specific languages. To this end, we use k-means clustering on the three properties of the trajectories (latency, imbalance, and reciprocity) and on all bot-bot and human-human interactions longer than five reverts (the results are substantively similar without the length restriction). We do not claim that the clusters are natural to the data; rather, we use the clusters to compare the interactions of the different groups.
The algorithm suggested that the data can be best clustered in four trajectory types ([S6 Fig](#pone.0171774.s006){ref-type="supplementary-material"}):
- **Fast unbalanced trajectories**. These trajectories have low reciprocity and latency and high imbalance. They look like smooth vertical lines above the x-axis.
- **Slow unbalanced trajectories**. These trajectories have low reciprocity and high latency and imbalance. They look like smooth diagonal lines above the x-axis.
- **Somewhat balanced trajectories**. These trajectories have intermediate imbalance and reciprocity. They are somewhat jagged and cross the x-axis.
- **Well balanced trajectories**. These trajectories have low imbalance and high reciprocity. They are quite jagged and centered on the x-axis.
Looking at the prevalence of these four types of trajectories for bots and humans and across languages, we confirm the previous observations: bot-bot interactions occur at a slower rate and are more balanced, in the sense that reverts go back and forth between the two bots ([Fig 4](#pone.0171774.g004){ref-type="fig"}). Further, we find that bot-bot interactions are more balanced in smaller language editions of Wikipedia. This could be due to the fact that bots are more active in smaller editions and hence, interactions between them are more likely to occur. Less intuitively, however, this observation also suggests that conflict between bots is more likely to occur when there are fewer bots and when, common sense would suggest, coordination is easier.
{#pone.0171774.g004}
Discussion {#sec003}
==========
Our results show that, although in quantitatively different ways, bots on Wikipedia behave and interact as unpredictably and as inefficiently as the humans. The disagreements likely arise from the bottom-up organization of the community, whereby human editors individually create and run bots, without a formal mechanism for coordination with other bot owners. Delving deeper into the data, we found that most of the disagreement occurs between bots that specialize in creating and modifying links between different language editions of the encyclopedia. The lack of coordination may be due to different language editions having slightly different naming rules and conventions.
In support of this argument, we also found that the same bots are responsible for the majority of reverts in all the language editions we study. For example, some of the bots that revert the most other bots include Xqbot, EmausBot, SieBot, and VolkovBot, all bots specializing in fixing inter-wiki links. Further, while there are few articles with many bot-bot reverts ([S7 Fig](#pone.0171774.s007){ref-type="supplementary-material"}), these articles tend to be the same across languages. For example, some of the articles most contested by bots are about Pervez Musharraf (former president of Pakistan), Uzbekistan, Estonia, Belarus, Arabic language, Niels Bohr, Arnold Schwarzenegger. This would suggest that a significant portion of bot-bot fighting occurs across languages rather than within. In contrast, the articles with most human-human reverts tend to concern local personalities and entities and tend to be unique for each language \[[@pone.0171774.ref026]\].
Our data cover a period of the evolution of Wikipedia when bot activity was growing. Evidence suggests that this period suddenly ended in 2013 (<http://stats.wikimedia.org/EN/PlotsPngEditHistoryTop.htm>). This decline occurred because at the beginning of 2013 many language editions of Wikipedia started to provide inter-language links via Wikidata, which is a collaboratively edited knowledge base intended to support Wikipedia. Since our results were largely dictated by inter-language bots, we believe that the conflict we observed on Wikipedia no longer occurs today. One interesting direction for future research is to investigate whether the conflict continues to persist among the inter-language bots that migrated to Wikidata.
Wikipedia is perhaps one of the best examples of a populous and complex bot ecosystem but this does not necessarily make it representative. As [Table 1](#pone.0171774.t001){ref-type="table"} demonstrates, we have investigated a very small region of the botosphere on the Internet. The Wikipedia bot ecosystem is gated and monitored and this is clearly not the case for systems of malevolent social bots, such as social bots on Twitter posing as humans to spread political propaganda or influence public discourse. Unlike the benevolent but conflicting bots of Wikipedia, many malevolent bots are collaborative, often coordinating their behavior as part of botnets \[[@pone.0171774.ref027]\]. However, before being able to study the social interactions of these bots, we first need to learn to identify them \[[@pone.0171774.ref028]\].
Our analysis shows that a system of simple bots may produce complex dynamics and unintended consequences. In the case of Wikipedia, we see that benevolent bots that are designed to collaborate may end up in continuous disagreement. This is both inefficient as a waste of resources, and inefficacious, for it may lead to local impasse. Although such disagreements represent a small proportion of the bots' editorial activity, they nevertheless bring attention to the complexity of designing artificially intelligent agents. Part of the complexity stems from the common field of interaction---bots on the Internet, and in the world at large, do not act in isolation, and interaction is inevitable, whether designed for or not. Part of the complexity stems from the fact that there is a human designer behind every bot, as well as behind the environment in which bots operate, and that human artifacts embody human culture. As bots continue to proliferate and become more sophisticated, social scientist will need to devote more attention to understanding their culture and social life.
Materials and methods {#sec004}
=====================
Data {#sec005}
----
Wikipedia is an ecosystem of bots. Some of the bots are "editing bots", that work on the articles. They undo vandalism, enforce bans, check spelling, create inter-language links, import content automatically, etc. Other bots are non-editing: these bots mine data, identify vandalism, or identify copyright violations.
In addition to bots, there are also certain automated services that editors use to streamline their work. For example, there are automated tools such Huggle and STiki, which produce a filtered set of edits to review in a live queue. Using these tools, editors can instantly revert the edit in question with a single click and advance to the next one. There are also user interface extensions and in-browser functions such as Twinkle, rollback, and undo, which also allow editors to revert with a single click. Another automated service that is relatively recent and much more sophisticated is the Objective Revision Evaluation Service (ORES). It uses machine-learning techniques to rank edits with the ultimate goal to identify vandals or low-quality contributions.
Our research focuses on editing bots. Our data contain who reverts whom, when, and in what article. To obtain this information, we analyzed the Wikipedia XML Dumps (<https://dumps.wikimedia.org/mirrors.html>) of 13 different language editions. To detect restored versions of an article, a hash was calculated for the complete article text following each revision and the hashes were compared between revisions \[[@pone.0171774.ref023]\]. The data cover the period from the beginning of Wikipedia (January 15, 2001) until February 2, 2010 --October 31, 2011, the last date depending on when the data was collected for the particular language edition. This time period captures the "first generation" of Wikipedia bots, as in later years, Wikidata took over some of the tasks previously controlled by Wikipedia. The sample of languages covers a wide range of Wikipedia editions in terms of size; for example, it includes the four largest editions by number of edits and number of editors. In terms of cultural diversity, the sample covers a wide range of geographies.
Wikipedia requires that human editors create separate accounts for bots and that the bot account names clearly indicate the user is a bot, usually by including the word "bot" (<https://en.wikipedia.org/wiki/Wikipedia:Bot_policy>). Hence, to identify the bots, we selected all account names that contain different spelling variations of the word "bot." We supplemented this set with all accounts that have currently active bot status in the Wikipedia database but that may not fit the above criterion (using <https://en.wikipedia.org/wiki/Wikipedia:Bots/Status> as of August 6, 2015). We thus obtained a list of 6,627 suspected bots.
We then used the Wikipedia API to check the "User" page for each suspected bot account. If the page contained a link to another account, we confirmed that the current account was a bot and linked it to its owner. For pages that contained zero or more than one links to other accounts, we manually checked the "User" and "User_talk" pages for the suspected bot account to see if it is indeed a bot and to identify its owner. The majority of manually checked accounts were vandals or humans, so we ended up with 1,549 bots, each linked to its human owner.
We additionally labeled human editors as vandals if they had all their edits reverted by others. This rule meant that we labeled as vandals also newcomers who became discouraged and left Wikipedia after all their initial contributions were reverted. Since we are interested in social interactions emerging from repeated activity, we do not believe that this decision affects our results.
Using the revert data, we created a directed two-layer multi-edge network, where ownership couples the layer of human editors and the layer of bots \[[@pone.0171774.ref029]\]. To build the network, we assumed that a link goes from the editor who restored an earlier version of the article (the "reverter") to the editor who made the revision immediately after that version (the "reverted"). All links were time-stamped. We collapsed multiple bots to a single node if they were owned by the same human editor; these bots were usually accounts for different generations of the same bot with the same function. In the network, reverts can be both intra- and inter-layer: they occur within the human layer, within the bot layer, and in either direction between the human and bot layers. The multi-layer network was pruned by removing self-reverts, as well as reverts between a bot and its owner.
Interaction trajectories {#sec006}
------------------------
We model the interaction trajectories in two-dimensional space, where the x-axis measures time and the y-axis measures the difference between the number of times *i* has reverted *j* and the number of times *j* has reverted *i*. To construct the trajectories, starting from *y*~*0*~ = 0, *y*~*t*~ = *y*~*t-1*~ + 1 if *i* reverts *j* at time *t* and *y*~*t*~ = *y*~*t-1*~ − 1 if *j* reverts *i* at time *t*; the labels *i* and *j* are assigned so that *y* \>= 0 for the majority of the *ij* interaction time. We analyze three properties of the trajectories:
- Latency. We define latency as the mean log time in seconds between successive reverts: *μ*(log~10~ *Δt*).
- Imbalance. We define imbalance as the final proportion of reverts between *i* and *j* that were not reciprocated: \|*r*~*i*~*− r*~*j*~\| / (*r*~*i*~ *+ r*~*j*~), where *r*~*i*~ and *r*~*j*~ are the number of times *i* reverted *j* and *j* reverted *i*, respectively.
- Reciprocity. We define reciprocity as the proportion of observed turning points out of all possible: (\# turning points) / (*r*~*i*~ *+ r*~*j*~− 1), where *r*~*i*~ and *r*~*j*~ are the number of times *i* reverted *j* and *j* reverted *i*, respectively. A turning point occurs when the user who reverts at time *t* is different from the user who reverts at time *t*+1.
K-means clustering {#sec007}
------------------
To identify the number of clusters *k* that best represents the data, we apply the elbow and silhouette methods on trajectories of different minimum length. The rationale behind restricting the data to long trajectories only is that short trajectories tend to have extreme values on the three features, thus possibly skewing the results. According to the elbow method, we would like the smallest *k* that most significantly reduces the sum of squared errors for the clustering. According to the silhouette method, we would like the *k* that maximizes the separation distance between clusters and thus gives us the largest silhouette score.
Although the elbow method suggests that four clusters provide the best clustering, the silhouette method indicates that the data cannot be clustered well ([S8 Fig](#pone.0171774.s008){ref-type="supplementary-material"}). We do not necessarily expect that trajectories cluster naturally; rather, we employ clustering in order to quantify the differences between the interactions of bots versus humans across languages. We hence analyze the clustering with *k* = 4. This clustering also has the advantage of yielding four types of trajectories that intuitively make sense.
Supporting information {#sec008}
======================
###### The number of bots, the number of edits by bots, and the proportion of edits done by bots between 2001 and 2010.
Between 2003 and 2008 the number of bots and their activity have been increasing. This trend, however, appears to have subsided after 2008, suggesting that the system may have stabilized.
(TIFF)
######
Click here for additional data file.
###### For the majority of languages, bots are mainly reverted by other bots, as opposed to human editors or vandals.
English and the Romance languages in our data present exceptions, with less than 20% of bot reverts are done by other bots.
(TIFF)
######
Click here for additional data file.
###### Bot-bot interactions have different characteristic time scale than human-human interactions.
The figures show the distribution of interactions for a particular latency, where we define latency as the mean log time in seconds between successive reverts. (A) Bot-bot interactions have a characteristic latency of 1 month, as indicated by the peak in the figure. (B) Human-human interactions occur with a latency of 2 minutes, 24 hours, or 1 year.
(TIFF)
######
Click here for additional data file.
###### Bot-bot interactions are on average more balanced than human-human interactions.
We define imbalance as the final proportion of reverts between *i* and *j* that were not reciprocated. (A) A significant proportion of bot-bot interactions have low imbalance. (B) The majority of human-human interactions are perfectly unbalanced.
(TIFF)
######
Click here for additional data file.
###### Bots reciprocate much more than humans do also at a smaller timescale.
We measure reciprocity as the proportion of observed turning points out of all possible. (A) A significant proportion of bot-bot interactions have intermediate or high values of reciprocity. (B) The majority of human-human interactions are not reciprocated.
(TIFF)
######
Click here for additional data file.
###### Four types of interaction trajectories suggested by the k-means analysis.
The left panels show a sample of the trajectories, including bot-bot and human-human interactions and trajectories from all languages. The right panels show the distribution of latency, imbalance, and reciprocity for each type of trajectory. The three properties measure the average steepness, the *y*-value of the last point, and the jaggedness of the trajectory, respectively. (A) Fast unbalanced trajectories have low reciprocity and latency and high imbalance. (B) Somewhat balanced trajectories have intermediate imbalance and reciprocity. (C) Slow unbalanced trajectories have low reciprocity and high latency and imbalance. (D) Well balanced trajectories have low imbalance and high reciprocity.
(TIFF)
######
Click here for additional data file.
###### The number of articles with a certain number of bot-bot and human-human reverts.
\(A\) Few articles include more than 10 bot-bot reverts. The most contested articles tend to be about foreign countries and personalities. Further, the same articles also re-appear in different languages. (B) There are many articles that are highly contested by humans. The most contested articles tend to concern local personalities and entities. It is rare that a highly contested article in one language will be also highly contested in another language.
(TIFF)
######
Click here for additional data file.
###### Performance of the k-means clustering algorithm for different number of clusters and for sub-samples with different minimum length of trajectories.
\(A\) The elbow method requires the smallest *k* that most significantly reduces the sum of squared errors for the clustering. Here, the method suggests that four clusters give the best clustering of the data. (B) The silhouette method requires the *k* that maximizes the separation distance between clusters, i.e. the largest silhouette score. Here, the method suggests that the clustering performs worse as the number of clusters increases.
(TIFF)
######
Click here for additional data file.
###### Descriptive statistics for the bot-bot layer and the human-human layer in the multi-layer networks of reverts.
Bots revert each other to a great extent. They also reciprocate each other's reverts to a considerable extent. Their interactions are not as clustered as for human editors. Still, both for bots and humans, more senior editors tend to revert less senior editors, as measured by node assortativity by number of edits completed.
(PDF)
######
Click here for additional data file.
The authors thank Wikimedia Deutchland e.V. and Wikimedia Foundation for the live access to the Wikipedia data via Toolserver. The data reported in the paper are available at 10.6084/m9.figshare.4597918.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** MT LF TY.**Data curation:** MT RG TY.**Formal analysis:** MT.**Funding acquisition:** TY.**Investigation:** MT.**Methodology:** MT TY.**Project administration:** TY.**Resources:** TY.**Supervision:** TY.**Visualization:** MT.**Writing -- original draft:** MT.**Writing -- review & editing:** MT RG LF TY.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by decreased or absent activity of lysosomal α-galactosidase-A (α-gal-A) activity, with progressive and multisystemic accumulation of globotriaosylceramide (Gb3) and its metabolites.\[[@ref1]\] In the kidney, this accumulation is observed in glomerular cells, peritubular capillaries, vascular endothelial, smooth muscle cells, and tubular cells.\[[@ref2][@ref3]\] Progressive Gb3 accumulation is associated with life-threatening complications like renal failure, cardiovascular dysfunction, and stroke.\[[@ref1][@ref4]\] Nephropathy is one of the major complications of FD and mainly includes reduced glomerular filtration rate and proteinuria.\[[@ref5]\]
Early diagnosis of nephropathy in FD patients is important. Prior to 2001, kidney disease was the major cause of death reported in affected patients.\[[@ref6]\] Subsequently, it was described that life expectancy is decreased in FD patients of both sexes; the cardiovascular cause is the most frequent and patients who die by cardiovascular disease have two characteristics: (i) they previously received renal replacement therapy (RRT) for end-stage renal disease (ESRD) and, most importantly, (ii) they were diagnosed late.\[[@ref7]\] In addition, affected patients show different degrees of annual loss of renal function according to the magnitude of proteinuria and the decrease in eGFR at the baseline.\[[@ref8]\]
Enzyme replacement therapy (ERT) is a specific treatment for FD available since 2001. In Fabry nephropathy, its efficacy is greater the earlier it starts, a reduction of Gb3 tissue accumulation has been shown in a dose-dependent manner.\[[@ref9]\] In more advanced stages of renal damage, its efficacy decreases due to the inability of correcting the progression when irreversible histological lesions are present, such as tissue fibrosis.\[[@ref10][@ref11]\]
The aim of this study is to analyze the relationship between age at diagnosis and the severity of nephropathy in an FD population.
Subjects and Methods {#sec1-2}
====================
In this retrospective study, patients with FD diagnosis were included from June 2007 to September 2017 from three reference centers in Argentina: (i) Los Manantiales Neuroscience Center, Grupo Gamma Rosario, Rosario, Santa Fe, Argentina; (ii) Center of Infusion and Study of Lysosomal Diseases of the Pergamino Clinical Nephrology Institute, Pergamino, Buenos Aires, Argentina; (iii) Intensive Unit Care of the Dr. Enrique Erill de Escobar Hospital, Belén de Escobar, Buenos Aires, Argentina. The diagnosis of FD was made by assessing enzyme activity α-gal-A \[[@ref12]\] and molecular study.\[[@ref13]\] Plasma and urine creatinine was determined by electro-chemiluminescence Roche Diagnostics. eGFR was calculated with Schwartz equation and CKD-EPI in patients under and over 18 years old, respectively.\[[@ref14]\] To classify the eGFR stage The Kidney Disease: Improving Global Outcomes Chronic Kidney Disease Guideline 2013 (KDIGO) classification was used.\[[@ref15]\] The albumin/creatinine ratio (ACR) in urine was used to estimate the urinary excretion of proteins in 24 h. Values from 0 to 30 mg/dl was considered normal, 30 to 300 mg/dl was indicative of albuminuria, and greater than 300 mg/dl indicated proteinuria, in at least two different samples of urine in all cases.
To estimate the degree of correlation between age and eFGR, the Pearson correlation coefficient was used. To estimate the degree of correlation between age and degree of proteinuria (ordinal with less than five categories), the Spearman correlation coefficient. It worked with a confidence interval (CI) of 95%. *P* values of \<0.05 were considered of statistical significance to reject the null hypothesis.
The data were processed in IBM SPSS version 20 database. The study was approved by a local Ethics Committee. The adult patients signed an informed consent, and for the pediatric patientstheir legal representative gave consent according to local legislation.
Results {#sec1-3}
=======
Seventy-two patients were studied with mean age of 26.26 ± 16.48 years and 30 men (41.6%). Twenty-seven pediatric patients and 45 adults were included. Thirteen genotypes were found: E398X, L415P, c886A\>G, L106R, c.680G\>A, A292T, c. 448.delG, R363H, C382Y, R301Q, D109G, del 3 and 4 exons, W81X, all pathogenic mutations of GLA gene.
The mean eGFR in paediatric population was 115.81 ± 20.87 ml/min/1.73 m^2^ and in adults was 80.63 ± 42.22 ml/min/1.73 m^2^. The mean ACR was 11.18 ± 11.10 mg/g and 386.63 ± 737.01 mg/g in paediatric patients and adults, respectively.
The FD complications frequency in studied population is shown in [Table 1](#T1){ref-type="table"}.
######
Frequency of typical Fabry disease complications in studied population
Pediatric Adults
--------------------------------- ----------- --------
Gender (M/F) 11/16 18/27
Cornea verticillata (%) 29.62% 31.11%
Gastrointestinal discomfort (%) 18.51% 28.88%
Neuropathic pain (%) 40.74% 73.33%
Angiokeratomas (%) 29.92% 42.22%
Deafness (%) 7.40% 44.44%
LVH (%) 0.00% 44.445
Arrhythmia (%) 11.11% 11.11%
CNS damage\* (%) 7.40% 31.11%
Albuminuria/proteinuria (%) 22.22% 57.77%
eGFR decreased\*\* (%) 0.00% 26.66%
\*Stroke and/or typical lesions in cerebral white substance in angionuclear magnetic resonance of brain. \*\*eGFR \<60 ml/min/m^2.^ Ref: M: Males, F: Females, LVH: Left ventricular hypertrophy, CNS: Central nervous system, eGFR: Estimated glomerular filtration rate
[Table 2](#T2){ref-type="table"} shows the inverse correlation between the age at diagnosis and eGFR in Pearson\'s coefficient test (value = −0.462; *P* = \< 0.01), and [Figure 1](#F1){ref-type="fig"} shows the graphical representation of its linear correlation. [Table 3](#T3){ref-type="table"} shows the direct correlation between age at diagnosis and degree of proteinuria in Spearman\'s coefficient (value = +0.385; *P* = \<0.01).
######
Pearson's bilateral correlation coefficient test between age at diagnosis and estimated glomerular filtration rate variables
Age at diagnosis eGFR
-------------------------- --------------------- ------------ ------------
Age at diagnosis Pearson correlation 1 −0.462\*\*
Significance (bilateral) 0.000
*n* 72 72
eGFR Pearson correlation −0.462\*\* 1
Significance (bilateral) 0.000
*n* 72 72
\*\*The correlation is significant at the 0.01 level (bilateral). eGFR: Glomerular filtration rate
{#F1}
######
Spearman's bilateral correlation coefficient between the age at diagnosis and degree of proteinuria variables
Age at diagnosis Proteiuria
--------------------------- ------------------------- ------------------ ------------
Rho of Spearman
Age at diagnosis Correlation coefficient 1.000 0.385\*\*
Significance. (bilateral) 0.001
*n* 72 72
Proteinuria Correlation coefficient 0.385\*\* 1.000
Significance. (bilateral) 0.001
*n* 72 72
\*\*The correlation is significant at the 0.01 level (bilateral)
Discussion and Conclusions {#sec1-4}
==========================
Nephropathy is a major complication of FD patients,\[[@ref1][@ref5]\] with a related increased morbidity and mortality.\[[@ref7]\] Kidney damage can begin at a very young age,\[[@ref16]\] and glomerular-sclerosis and vascular lesions have been described in renal biopsies of children and adolescents who had not yet exhibited decreased eGFR or overt proteinuria.\[[@ref2][@ref9][@ref17][@ref18][@ref19]\]
In the natural course of Fabry nephropathy, the annual decline in renal function over time is related to the degree of proteinuria, male sex, and is faster when the initial eGFR is less than 60 ml/min/1.73 m^2^.\[[@ref4][@ref8]\] Progression of renal damage culminates in ESRD. Affected males with "classical" mutations of the GLA gene, which produces severe decrease in α-gal-A activity, require RRT for ESRD treatment at ages of 35--44 years.\[[@ref20]\] Therefore, the severity of the nephropathy conditions the evolution of FD patients, and the search for factors of poor renal prognosis is useful in affected patients.
Early biomarkers of prealbuminuric-stage nephropathy have been studied in FD patients with promising results,\[[@ref21][@ref22]\] because it is known that urinary protein excretion is present when the renal tissue lesions are irreversible.\[[@ref2][@ref9][@ref16][@ref17][@ref18][@ref19]\] The purpose of these investigations is to detect early renal damage before irreversible structural damage occurs in renal tissue.
The prototype of irreversible renal damage is renal fibrosis, both in glomerulosclerosis or tubulo-interstitial fibrosis.\[[@ref10]\] Renal fibrosis, characterized by excessive deposition of extracellular matrix (ECM), is recognized as a common pathological feature of chronic kidney diseases (CKD), which is in direct relation to progression and leads to the development of ESRD.\[[@ref23]\] As in other causes of nephropathy, there are no effective treatments that can reverse renal fibrosis in FD. In our study population, a direct relationship was found between age at diagnosis of FD and the severity of the nephropathy, as determined by eGFR and the degree of proteinuria.
The Pearson\'s bilateral correlation coefficient test (value = −0.462) between the age at diagnosis and eGFR indicates inverse correlation between both variables with a strong statistical significance (*P* = \<0.01). At higher age, patients present with lower eGFR. On the other hand, the Spearman\'s bilateral correlation coefficient (value = +0.385) between the age at diagnosis and the degree of proteinuria indicates direct correlation with a strong statistical significance (*P* = \<0.01). At higher age at diagnosis, patients have higher proteinuria.
ERT is more effective the earlier it is started; in a dose-dependent manner it is able to revert tissue lesions, even in injured podocytes.\[[@ref9]\] In more advanced stages of nephropathy, ERT is less effective because it is unable to reverse renal fibrosis.\[[@ref9][@ref10][@ref24][@ref25]\]
In conclusion, the diagnosis of FD at a younger age could be a key to improve the prognosis of nephropathy and allow early and effective interventions.
Financial support and sponsorship {#sec2-1}
---------------------------------
SJ have received speaker fees from Genzyme, SHIRE and Biomarin; and and support for research work. NA received speaker fees from Genzyme and Shire and travel grants from Genzyme, Shire and Protalix. FP have received speaker fees from Genzyme.
Conflicts of interest {#sec2-2}
---------------------
There are no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Broadly neutralizing monoclonal antibodies (bnmAbs) that target the structurally conserved CD4 binding site (CD4-BS) bind with high affinity to Env and neutralize diverse HIV-1 isolates, irrespective of their clade ([@bib25], [@bib26]; [@bib2]; [@bib18]). Despite the isolation of these anti--CD4-BS bnmAbs from distinct HIV^+^ subjects, they share common genetic and structural features, which are critically important for their unique neutralizing properties ([@bib18]; [@bib26]). So far, four classes of anti--CD4-BS broadly neutralizing antibodies (bNAbs) have been defined: b12, HJ16, VRC01, and 8ANC131 ([@bib8]). Of particular interest for HIV vaccine development are the VRC01 class antibodies because of their exceptional neutralization potency and breadth. Their VH domains are derived from VH1-2, in contrast to the 8ANC131 class antibodies whose VH domains are derived from VH1-46 ([@bib18]) and b12 whose VH is derived from VH1-03 ([@bib5]). The currently known VRC01 class antibodies (such as VRC01, NIH45-46, 3BNC60, and 12A21), were isolated from distinct HIV^+^ subjects infected with different viruses and display up to 57% diversity in VH and up to 65% diversity in VL sequences; [@bib25], [@bib26]; [@bib18]; [@bib24]; [Table S1](http://www.jem.org/cgi/content/full/jem.20122824/DC1){#supp1}). Despite this high degree of amino acid sequence diversity, they share structural similarities that allow them to recognize the CD4-BS in a manner very similar to each other and to the CD4 receptor ([@bib18]; [@bib26]). That interaction is primarily through the VH antibody domains; however, the light chains of VRC01 class antibodies also make important contacts with Env. This contrasts with b12, which appears to interact with Env exclusively through its heavy chain. The mode of Env interaction of HJ16 class antibodies is not yet known. An additional unique feature of the VRC01 class antibodies is that they make contact not only with the gp120 outer domain, but also with the gp120 inner and bridging sheet domains ([@bib2]; [@bib18]; [@bib26]).
Only a fraction of those infected with HIV develop broadly neutralizing anti--CD4-BS antibodies ([@bib12]). Despite the presence of anti--CD4-BS epitopes on recombinant Env ([@bib9]; [@bib1]; [@bib16]; [@bib14]), Env immunization has so far failed to elicit such antibodies ([@bib13]; [@bib21]). The reasons why such antibodies are not elicited by Env immunization, and are only rarely generated during natural HIV infection, are currently not well understood. Identifying the roadblocks that prevent the generation of such antibodies and approaches to overcome these barriers will aid in the development of an effective HIV vaccine ([@bib13]).
The germline-reverted (unmutated) forms of VRC01 class anti--CD4-BS bnmAbs, do not recognize the recombinant Env-derived bait protein used to isolate the B cells expressing the corresponding mature antibodies ([@bib29]; [@bib18]). We reported that the germline-reverted forms of two VRC01class antibodies, NIH45-46 (a clonal variant of VRC01; [@bib18]) and 3BNC60, fail to recognize a large panel of recombinant Env derived from clades A, B, and C, the three most predominant clades worldwide ([@bib5]). In the current study, we expanded our Env panel and included two additional VRC01 class anti--CD4-BS bnmAbs; 3BNC117 and 12A21 ([@bib18]; [Table S2](http://www.jem.org/cgi/content/full/jem.20122824/DC1){#supp2}). The mature versions of these mAbs recognize between 92% (3BNC60) and 71% (12A21) of Envs tested. In contrast, there was no detectable binding to these Envs by the germline-reverted versions of these antibodies. We note that although the accessibility of the CD4-BS by CD4 and anti--CD4-BS antibodies is restricted by the V1, V2, and V3 variable domains of Env and associated N-linked glycosylation sites (NLGS; [@bib27]; [@bib11]; [@bib17]), the elimination of these regions did not result in Env-recognition by the germline-reverted anti--CD4-BS bnAbs studied here.
The interactions between Env and the germline-reverted forms of anti--CD4-BS bnAbs are commonly investigated using soluble forms (IgG or scFv) of antibodies ([@bib28]; [@bib25], [@bib26]; [@bib5]; [@bib18]). This interaction may not accurately reflect the interaction of Env with the corresponding membrane-anchored BCRs on B cells. Conceivably, certain recombinant Envs can bind weakly to, and cross-link the germline-reverted BCRs of VRC01 class anti--CD4-BS bNAbs and activate the corresponding B cells. If that is the case, then the deficiency in the development of such antibodies during immunization could not be explained by a lack of recognition of their germline-reverted BCRs by Env. Here, we investigated the interaction between recombinant HIV Env and B cells expressing the germline-reverted BCR versions of VRC01 class antibodies to identify potential roadblocks during the earliest steps of B cell activation. We report that in order for Env to engage and activate B cells expressing germline-reverted NIH45-46 and VRC01 BCRs, specific N-linked glycosylation sites (NLGS) located at key regions of Env must be absent (something that only rarely occurs during natural HIV-infection).
RESULTS AND DISCUSSION
======================
To ensure that the binding specificities of soluble antibody forms are congruent with their BCR forms, we investigated how diverse Envs interact with B cell--expressed BCRs of mature and germline-reverted NIH45-46; one of the most potent and broadly neutralizing VRC01 class antibodies ([@bib18]). Similar levels of mature and germline-reverted BCRs were expressed by transfected B cells and both BCRs mediated comparable intracellular Ca^2+^ flux responses, after cross-linking by anti--human IgG F(ab′)~2~ (unpublished data). A panel of diverse clade A, B, and C trimeric Envs that display a wide range of affinities for the mature NIH45-46 (mNIH45-46) bound to B cells expressing the mNIH45-46 BCR ([Fig. 1, a and b](#fig1){ref-type="fig"}). An Env with a point mutation (D368R), which greatly reduces the binding of anti--CD4-BS antibodies, was included as a negative control. The Envs that bound the mNIH45-46 BCR also induced Ca^2+^ flux through this BCR ([Fig. 1 c](#fig1){ref-type="fig"}). We did not detect binding of these Envs to cells expressing the germline-reverted NIH45-46 BCR (glNIH45-46 BCR; [Fig. 1 b](#fig1){ref-type="fig"}), nor did we observe Ca^2+^ flux in these cells ([Fig. 1 d](#fig1){ref-type="fig"}). Similar lack of YU2 Env-binding to the germline-reverted VRC01 (glVRC01) BCR was recently reported ([@bib15]).
{#fig1}
Elimination of the Env variable regions 1, 2 or 3 did not result in germline antibody-Env binding (Table S2). However, accessibility of the CD4-BS is also limited by proximal NLGS ([@bib19]). NLGS located in Loop D and V5 of gp120 are of particular interest because they are located in two regions of Env that modulate the neutralizing activities of VRC01 class antibodies ([@bib10]). In fact, the light chains of the mature NIH45-46 and VRC01 antibodies contact the *N*-acetyl-glucosamine residues at Asn 276 in loop D ([@bib29]; [@bib2]). We eliminated NLGS from these regions from the clade C Env 426c, which is recognized with high apparent affinity (10.6 nM) by mNIH45-46 ([Fig. 1 a](#fig1){ref-type="fig"}, [Fig. 2 a](#fig2){ref-type="fig"}, and [Table S3](http://www.jem.org/cgi/content/full/jem.20122824/DC1){#supp3}). Elimination of the 276, the 460 and 463 (V5), or all three NLGS from 426c increased the binding of mNIH45-46 (apparent affinities of 3.66 nM, 1.9 nM, and 18 pM, respectively; [Fig. 2, d, g, and j](#fig2){ref-type="fig"}; and Table S3). The mutant Envs also bound more efficiently to mNIH45-46 BCR ([Fig. 3 a](#fig3){ref-type="fig"}) and induced increased B cell activation, as indicated by increases in tyrosine phosphorylation ([Fig. 3, b and g](#fig3){ref-type="fig"}) and Ca^2+^ flux ([Fig. 3 i](#fig3){ref-type="fig"}). Importantly, although glNIH45-46 did not bind the 426c Env ([Fig. 2 b](#fig2){ref-type="fig"}), it bound the mutant lacking the 276 NLGS (apparent affinity of 0.7 µM; [Fig. 2 e](#fig2){ref-type="fig"} and Table S3) and the mutant lacking all three NLGS (apparent affinity of 46 nM; [Fig. 2 k](#fig2){ref-type="fig"} and Table S3).
{#supp4}.](JEM_20122824_Fig2){#fig2}
{#fig3}
We also examined the interactions between these Envs and glVRC01, a clonal variant of the NIH45-46 antibody ([@bib18]). Even stronger binding affinities were observed with the glVRC01 antibody ([Fig. 2, f and l](#fig2){ref-type="fig"}; and Table S3). In all cases, the increase in the on-rates, rather than decrease in the off-rates, observed when the NLGS are removed from 426c, are suggestive of alleviation of steric constraints rather than changes in epitope conformation. Interestingly, the glNIH45-46 and glVRC01 did not bind the mutant lacking the 460 and 463 NLGS ([Fig. 2, h and i](#fig2){ref-type="fig"}). The mutant Envs lacking the 276 NLGS or all three NLGS bound the glNIH45-46 BCR ([Fig. 3 a](#fig3){ref-type="fig"}) and activated the corresponding B cells (increase in tyrosine phosphorylation \[[Fig. 3, c and g](#fig3){ref-type="fig"}\] and Ca^2+^ flux \[[Fig. 3 j](#fig3){ref-type="fig"}\]). In agreement with our binding data for the glVRC01 (Table S3), which indicated stronger Env-binding affinities for glVRC01 than glNIH45-46, the ensuing B cell activation with these two Envs was stronger ([Fig. 3, d, g, and k](#fig3){ref-type="fig"}) than that observed with B cells expressing the glNIH45-46 BCR ([Fig. 3, c, g, and j](#fig3){ref-type="fig"}).
Because the intensity of BCR signaling through the IgG form is stronger than through the IgM form ([@bib23]; [@bib6]; [@bib22]), we performed our studies with the IgG form, to ensure that any weak signaling induced by our Env proteins would be detected. However, when germline BCRs are expressed on naive B cells, they are of the IgM or IgD classes. We confirmed that the signaling pattern through the IgM glNIH45-46 and glVRC01 BCRs mirrors that observed with the corresponding IgG BCRs, but, as expected, the intensity of the signal is lower ([Fig. 3, e, f, and h](#fig3){ref-type="fig"}).
The aforementioned results suggest that the NLGS at position 276 plays a critical role in the binding efficiencies of mature and germline-reverted NIH45-46 and VRC01 with Env. Structural modeling suggests that this NLGS hinders antibody binding by interfering with the light antibody chain ([Fig. 3 l](#fig3){ref-type="fig"}). Our model indicates that the NLGS at position 463 is not close enough to the antibody epitope to be a major player in this interaction. The model and the improved affinities of germline-reverted and mature NIH45-46 and VRC01 for the triple mutant over the single N276D mutant are consistent with the hypothesis that the glycan at 460 also limits the accessibility of the antibody; the fact that removal of 460 and 463 alone does not significantly improve germline-reverted affinity emphasizes that germline-reverted binding is dependent on removal of the glycan at 276. The CDRL1 of mNIH45-46 and mVRC01 have a two-residue deletion relative to the germline-reverted antibodies ([Fig. S1](http://www.jem.org/cgi/content/full/jem.20122824/DC1){#supp5}; [@bib29]; [@bib2]; [@bib18]) and this difference may result in differential interactions with the NLGS at positions 276 and 460. Future studies will reveal whether or not similar mutations on different Envs also lead to glNIH45-46 or glVRC01 binding.
In accordance with the aforementioned antibody Env-binding results, mNIH45-46 neutralized 426c viruses expressing the deglycosylated Env variants more efficiently than the parental virus ([Fig. 4 a](#fig4){ref-type="fig"}), whereas glNIH45-46 weakly neutralized viruses expressing either the single (276 NLGS) or triple NLGS mutant Envs, but not viruses expressing WT or Env lacking the 460 and 463 NLGS ([Fig. 4 b](#fig4){ref-type="fig"}). The NLGS at position 276 is highly conserved among circulating HIV-1 strains and only 5.0% of the Env sequences lack this glycosylation site. Interestingly, a higher proportion of clade C than clade B Envs lack this glycosylation site (7.4% of C and 2.0% of B). Even less frequent is the simultaneous absence of all three NLGS (3.1%). Potentially, viral clones with rare glycosylation patterns on Env emerge during infection and stimulate the germline-reverted BCRs of VRC01 class antibodies, such as NIH45-46 and VRC01, thus starting the process of antibody affinity maturation that eventually leads to the development of broadly neutralizing antibody responses ([@bib3]; [@bib14]). The rarity of circulating Envs lacking these NLGS may in part explain why VRC01 class antibody responses are elicited only by a minority of those infected with HIV ([@bib12]). We note that the above NLGS mutations on the backbone of the 426c Env do not diminish the ability of that Env to mediate virus--cell entry ([Fig. 4 d](#fig4){ref-type="fig"}). We do not yet know whether all the naturally occurring Envs that lack those three NLGS bind glNIH45-46 and glVRC01. We also do not know whether the modifications we discuss here will always lead to glNIH45-46 or glVRC01 binding when introduced on the backbone of all the Envs we tested (Table S2). Most likely, they will not always result in antibody binding because additional structural elements of Env will vary in a backbone-specific manner and may limit antibody binding even in the absence of the three NLGS discussed here. Structural information of Envs that do and do not bind glNIH45-46 and glVRC01 will be very useful to better understand why some Env are recognized by these antibodies and why some are not. Such structural studies will greatly assist Env-immunogen design efforts that are geared toward stimulating B cells expressing VRC01 class BCRs.
{#fig4}
Despite the potent binding of other VRC01 class members studied here (12A21, 3BNC60, and 3BNC117; [Fig. 5 a](#fig5){ref-type="fig"}) to the 426c Env lacking all three NLGS, their germline-reverted versions did not recognize that Env ([Fig. 5 b](#fig5){ref-type="fig"}). The heavy chains of mAbs NIH45-46, VRC01, 12A21, 3BNC60, and 3BNC117 are derived from the same VH1-2\*02 allele ([@bib29]; [@bib18]). However, the light chains of mAbs NIH45-46 and VRC01 are derived from Vk3-11, whereas those of 12A21, 3BNC60, and 3BNC117 are derived from Vk1D-33. Structural differences between the light chains among these antibodies (Fig. S1) could be responsible for their different Env-recognition properties. Indeed, when the germline-reverted light chains of 12A21 and 3BNC60 were replaced by that of NIH45-46/VRC01, the chimeric germline-reverted antibodies recognized the 426c NLGS mutant Envs ([Fig. 5, c and d](#fig5){ref-type="fig"}). Thus, the mutations in loop D and the V5 region identified here may be the minimal set of mutations allowing the binding of germline VRC01 class antibodies to the CD4-BS. It will be important to identify additional Env mutations that alleviate clashes between the germline-reverted light chains of antibodies such as 12A21 or 3BNC60 and elements of Env.
{#fig5}
Our experiments indicate that a potential reason for the lack of elicitation of VRC01 antibodies, by Env immunogens is the inability of these immunogens to stimulate B cells expressing the germline BCRs of such antibodies and initiate the maturation process that leads to the production of bNAbs against one of the most conserved HIV Env regions. We show that glycosylation of specific Env regions hinders recognition by the germline-reverted BCRs of two of the most potent VRC01 class antibodies. As such, our findings provide a pathway to overcome a crucial, early, roadblock in the elicitation of similar antibodies by immunization. We note that additional blocks may prevent the elicitation of such antibodies during immunization and that potentially prime boost immunization strategies need to be devised to guide BCR evolution toward specific B cell maturation pathways that are necessary for the production of VRC01 class antibodies ([@bib8]).
MATERIALS AND METHODS
=====================
### Antibodies and BCR construction.
The anti--CD4-BS mAbs discussed here were isolated from different HIV-infected subjects by VH+VL gene amplification from single B cells ([@bib25]; [@bib18]). Soluble IgGs were expressed in 293F cells and purified by Protein A affinity chromatography. Mature and germline-reverted IgG forms of VRC01 were provided by J. Mascola and X. Wu (National Institutes of Health, Bethesda, MD).
To generate plasmids expressing the membrane-anchored forms of the anti-HIV antibodies, the following strategy was used. The variable heavy and light antibody regions (separated by a furin cleavage site and an F2A peptide sequence) were inserted into pTT5 vector. The constant IgG and IgM splice variant cDNAs (containing the two exons that encode the transmembrane and cytoplasmic domains of human IgG and IgM) were synthesized by GenScript. That sequence was introduced 3′ to the variable heavy region in the aforementioned pTT5 vector. These vectors express the membrane-anchored version of either the mature or germline-reverted versions of the antibodies studied here.
### Cells.
The DG-75 human Burkitt's lymphoma (\#CRL-2625; ATCC) cell line was maintained in RPMI-1640 supplemented with 10% FBS. DG-75 cells express human IgM do not express Fc receptors.
### Neutralization assays.
Neutralization assays were performed as previously described ([@bib17]). TZM-bl cells were maintained in DMEM (Cellgro) supplemented with 10% FBS, 2 mM [l]{.smallcaps}-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For neutralization assays, the cells were plated at a density of 5 × 10^4^ cells per well 24 h before addition of virus and antibody. Antibodies were serially diluted in microtiter wells at the indicated starting concentration in a total volume of 30 µl per well. 30 µl of virus (previously determined to result in 2 × 10^5^ luciferase units) was added to each well containing the titrated antibodies. Antibody-virus mixtures in duplicate wells were incubated at 37°C for 1.5 h. During the last 30 min of the incubation, cells were treated at 37°C with 1 µg/ml of polybrene. The polybrene was then aspirated from the cells and replaced with 50 µl of antibody-pseudovirus mixture. Plates were incubated at 37°C for 72 h, media was aspirated, and cells were lysed to measure luciferase activity using Steady-Glo Luciferase Reagent (Promega)
### ELISA.
Env ELISA assays were performed with established methodologies, either by directly adsorbing recombinant Env (SIVmac239, JRCSF, BAL, Consensus C, DU151, Consensus A1, MJ613, and A244; Immune Technologies) to ELISA plates or by capturing p120 released from pseudoviruses after lysis by 1% Triton X-100 on D7324-coated (anti--C-terminal gp120 sheep antibody) ELISA wells. In the first case, the binding cut off for ELISA was defined as the mean + 3 SD of A~450~ recorded with the SIVmac gp120 at the highest antibody concentration, whereas in the second case the binding cut off was the mean + 3 SD of the absorbance obtained for each antibody against the capture antibody alone (no Env).
### Biolayer interferometry analysis of mAb-Env binding.
These assays were performed on the Octet KQe instrument (ForteBio, Inc.). IgGs (20 µg/ml in PBS) were immobilized onto anti--human IgG FC capture (AHC) biosensors (ForteBio) for 5 min. The baseline interference was then read for 60 s, in kinetics buffer (KB: 1× PBS, 0.01% BSA, 0.02%, Tween 20, and 0.005% NaN~3~),followed by subsequent immersion of the sensors into wells containing recombinant trimeric Env gp140 diluted in KB for 300s (association phase). Sensors were then immersed in KB for the indicated times (dissociation phase). All kinetic interactions were measured with new sensors at 30°C and shaking at 1,000 RPM in 96-well plates. Curve fitting was done using a 1:1 binding model using the Data analysis software (ForteBio) and using the concentration of gp140 protomers in solution. Mean *k*~on~ and *k*~off~ and apparent K*~d~* values were determined from all binding curves that matched the theoretical fit with an R^2^ value of ≥0.95.
### HIV envelopes.
The following Envs were examined: clade A: Q168a2, Q259d2, Q461e2, Q769h5; clade B: SF162, SF162ΔV1, SF162ΔV2, SF162ΔV3; clade C: 823c, 756c, 706c, 459c, 140c, 327c, 405c and 426c. Proteins were either purchased or expressed and purified in-house as previously described ([@bib20]). These clade C Envs (available from under GenBank accession nos. [KC769511](KC769511)--[KC769518](KC769518)) were derived from acutely infected participants in the HVTN503 clinical trial ([@bib4]). The JRCSF, SF162, ADA, JRFL, YU2, and HXB2 Envs were derived from clade B viruses isolated during chronic infection. The clade C Envs Du151, Du422, CAP45, and ZM249 and the clade B Envs QH0692, AC10.0, PVO, TRO, RHPA, TRJO, WITO, REJO, and THRO were derived from viruses isolated during the first 6 mo of infection. The clade A Envs Q168a2, Q259d2, Q461e2, and Q769h5 were derived from viruses isolated between 28 and 75 d after infection. The A244 Env was derived from a clade A/E virus.
### Expression and activation of exogenous BCRs on B cells.
2 × 10^6^ DG-75 B cells in 100 µl of cell line Nucleofector solution V (Lonza) were electroporated with 5 µg of BCR-expressing plasmids (Amaxa Nucleofector II; Lonza; program O-006). Cell surface expression of exogenous human BCRs was determined by allophycocyanin (APC)-conjugated mouse monoclonal anti--human IgG (1/10; BD). 24 h after transfection, cells were loaded with Fluo-4 Direct calcium indicator (Invitrogen), in RPMI-1640 medium containing 10% FBS at 37°C for 45 min. Cells were pelleted and stained with APC-conjugated mouse monoclonal anti--human IgG (1/10 dilution in 100 µl of RPMI-1640 with 10% FBS and Fluo-4 Direct) for 15 min. The cells were washed with 5 ml in RPMI-1640 containing 10% FBS, pelleted, and resuspended at ∼10^6^ cells/ml in RPMI-1640 and subjected to Ca^2+^ flux analysis at a medium flow rate on an LSR II cytometer (BD). For all the experiments, we gated on B cells expressing comparable numbers of BCRs.
Minimum levels of background fluorescence (Min~FL~) were determined by averaging the background Fluo-4 absorbance in cells for 30 s. After that, activation of exogenous BCRs by recombinant Env (30--400 µg/ml) was determined by monitoring changes in Fluo-4 fluorescence associated with cells expressing the exogenous BCRs (APC positive cells) for 210 or 270 s. Ionomycin was added to a final concentration of 6.5 nM for 60s and maximum Fluo-4 fluorescence (Max~FL~) was established by averaging changes in Fluo-4 fluorescence recorded during the last 10 s.
The percentage of maximum Fluo-4 fluorescence at each time point t was determined using the formula: (Fluorescence at t-Min~FL~) / (Max~FL~-Min~FL~) × 100. This analysis was performed on both the BCR-positive (anti--IgG-APC--positive) and BCR-negative (anti--IgG-APC--negative) cells simultaneously. The background Fluo-4 fluorescence signal from the BCR-negative cells was subtracted from that of the BCR positive population at each time point.
Tyrosine phosphorylation activity after a 3-min incubation at room temperature with Env (40 or 200 µg/ml) was determined in DG75 cells expressing NIH45-46 and VRC01 BCRs. Cells were pelleted and resuspended in lysis buffer (1% NP-40, 10% glycerol, 2 mM EDTA, 137 mM NaCl, and 20 mM Tris HCl, pH 8.0) supplemented with phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail; Thermo Fisher Scientific) for 10 min on ice. Lysates were cleared by centrifugation and subjected to SDS-PAGE and Western blot analysis using 4G10 pY mAb (Millipore) and Anti--β-actin rabbit mAb (13E5; Cell Signaling Technology). Bands were visualized with IR-dye conjugated secondary antibodies and imaged and quantified by Odyssey Infrared Imaging System (LI-COR Biosciences).
### Env sequence analysis.
The 2010 Filtered Web Alignment (excluding 4 SIV sequences; Los Alamos HIV Database, <http://www.hiv.lanl.gov/>) was used to determine the frequency of NLGS in Env.
### Recombinant Env binding to exogenous BCRs expressed on B cells.
BCR expressing and mock transfected DG-75 cells were stained with APC-conjugated mouse anti--human IgG, as described above. After washing in PBS containing 10% FBS (FACS wash buffer) cells were resuspended (10^6^ cells/ml) in FACS wash buffer and 1.0 × 10^5^ cells were incubated with 30 µg/ml of gp140 Env for 20 min on ice. The cells were washed and incubated with R-phycoerythrin (PE)--labeled IgG purified from pooled sera of 50 HIV-1+ subjects. After centrifugation and washing (2× in ice cold FACS wash buffer), the cells were resuspended in 200 µl of 1% paraformaldehyde and analyzed on an LSR II cytometer (BD). Envelope surface staining was expressed as the MFI of PE staining in the presence of Env + HIV Ig PE minus the MFI obtained with HIV-IgG-PE alone on BCR expressing (APC-positive) cells.
### Env mutagenesis.
Point mutations in the gp140 and gp160 envelope sequences were introduced by site-directed mutagenesis (Stratagene Quick Change II system from Agilent Technologies, Santa Clara, California), using the following primers (sense strand provided) 426c-N460D/N463D (5′-GATGGGGGAAACACTACCGATAACACCGAGATTTCC-3′) and 426c-N276D (5′-GAGGAAGAGATTGTGATCAGATCAAAAGACCTGAGCGATAAT-3′).
### Modeling the NIH45-46-Env interaction.
A homology model for core gp120 of clade C strain 426c was built by RosettaRemodel ([@bib7]), using the NIH45-46-gp120 crystal structure (PDB accession no. [3u7y](3u7y)) as a structural template. Low-energy backbone conformations were generated for the V5 loop in the presence of bound NIH45-46. Representative Man8GlcNac~2~ glycans were then aligned onto each gp120 Asn residue within a glycosylation sequon, and low-energy glycan conformations were modeled using GlycanRelax ([@bib19]).
### Statistical analysis.
Significant increase of tyrosine phosphorylation was assessed by repeated measures ANOVA followed by Tukey's multiple comparisons test comparing log-transformed, actin-normalized Western blot signal intensities of antigen-stimulated cells with that of unstimulated cells. A p-value \<0.05 was considered significant.
### Online supplemental material.
Fig. S1 shows amino acid alignment of the germline-reverted antibody heavy and light chains. Table S1 shows characteristics of VRC01 class antibodies. Table S2 shows IgG binding to recombinant Env proteins. Table S3 shows the binding kinetics of mature and germline NIH 45-46 IgG to trimeric 426c Env gp140 variants. Online supplemental material is available at <http://www.jem.org/cgi/content/full/jem.20122824/DC1>.
Supplementary Material
======================
###### Supplemental Material
We thank Zachary Caldwell, Sara Carbonetti, Anna Gazumyan, Jolene Glenn, Sam Danziger, and D. Noah Sather for technical support.
This work was supported by National Institute of Allergy and Infectious Diseases/HIV Vaccine Research and Design grant P01AI081625 and R56 AI047708 (L. Stamatatos). We also acknowledge support by the J.B Pendleton Charitable Trust and by the University of Washington Center for AIDS Research, an NIH funded program (P30 AI027757). A.M. Dreyer was supported by the Swiss National Science Foundation (PBBSP3 144245).
The authors declare no competing financial interests.
Abbreviations used:CD4-BSCD4-binding siteEnvHIV envelope glycoproteinbNAbbroadly neutralizing antibodyNLGS*N*-linked glycosylation site
[^1]: A.T. McGuire and S. Hoot contributed equally to this paper.
[^2]: S. Hoot's present address is Altravax, Inc. Sunnyvale, CA 94085.
| {
"pile_set_name": "PubMed Central"
} |
Review
======
Background
----------
There is growing evidence of mortality effects related to long-term exposure (i.e., exposures of a year or more) to ambient air pollution \[[@B1]-[@B3]\]. Cardiovascular effects of short- and long-term exposure to particulate matter air pollution focusing on PM~2.5~ have recently been comprehensively reviewed \[[@B4],[@B5]\]. Experimental and epidemiological studies in the recent decade have significantly increased our knowledge of mechanisms that could plausibly explain the associations observed in epidemiological studies between ambient air pollution and mortality \[[@B4]\].
Most studies have reported associations linked to particulate matter, often represented by the mass concentration of particles smaller than 10 μm (PM~10~) or 2.5 μm (PM~2.5~). In many urban areas, motorized traffic emissions are an important source of ambient particles and gaseous pollutants such as nitrogen oxides (NO~2~ and NO). Exposure contrasts related to traffic emissions are usually poorly represented by the concentration of PM~10~ or PM~2.5~, because of the high regional background concentration of these particle metrics from other sources \[[@B6],[@B7]\]. However, there are more specific markers for traffic related air pollution, which include elemental carbon and ultrafine particles number \[[@B7]-[@B10]\]. Janssen and co-workers recently demonstrated that health impact assessments of traffic-related pollutants based upon PM~2.5~ seriously underestimated the health risks compared to an assessment based upon elemental carbon \[[@B7]\]. There is also growing evidence of health effects related to ultrafine particles \[[@B8],[@B9]\]. Finally, the effects of coarse particles (the particle fraction between 2.5 and 10 μm) have attracted renewed attention \[[@B11]\]. Emission controls for road traffic have now substantially reduced tailpipe emissions, and therefore non-tailpipe emissions including engine crankcase emissions (combusted lubricating oil), road, tire and brake wear are becoming increasingly important. A recent study in the Netherlands found similar increases of concentrations in major roads compared to urban background for metals related to break and tire wear (Cu, Zn) as for soot and ultrafine particles which are due to tailpipe emissions \[[@B10]\] . In a review of the limited literature, coarse particles were associated with short-term effects on mortality and hospital admissions, but no evidence was found for long-term exposure effects \[[@B11]\]. The number of studies on long-term coarse particle exposure reviewed was small however at the time.
The aim of the current review is to evaluate the epidemiological evidence for cardiovascular and respiratory mortality effects of long-term exposure to fine particulate matter, including a meta-analysis. We focused on epidemiological studies of mortality, as experimental studies and mechanisms of effect have been discussed in detail previously \[[@B4]\]. The American Heart Association review \[[@B4]\] is updated with a significant number of new studies published in 2009 -- 2012. We further include more pollutants in the review, specifically NO~2~, elemental carbon and coarse particles. We evaluated the findings on potentially susceptible subgroups across studies of PM~2.5~. In addition, we have included the studies on more specific cardiovascular causes of death, especially fatal myocardial infarction and stroke.
Methods
-------
We performed a search in the databases Medline and Scopus with the search terms air pollution, cohort, and mortality until January 2013. We supplemented the search with studies included in the review by Brook and co-worker \[[@B4]\] and by browsing the reference lists of identified papers. In case more than five studies were identified, we performed a meta-analysis. We tested for heterogeneity of cohort-specific effect estimates and obtained combined effects estimates, using random effects methods of DerSimonian and Laird \[[@B12]\]. The I^2^ statistic was calculated as a measure of the degree of heterogeneity across studies \[[@B13]\]. I^2^ ranges from 0 to 100% and can be interpreted as the variability of study-specific effect estimates attributable to true between study effects. From some studies multiple papers were available such as the Six Cities study \[[@B14]-[@B16]\]. In the meta-analysis we used only the most recent paper, which had longer follow-up. We only included studies in the quantitative meta-analysis that directly provided PM~2.5~ exposure estimates. For NO~2~ we only included studies which accounted for intra-urban spatial variation using e.g. dispersion models, land use regression models or spatial interpolation. We used STATA version 10 (Stata Corp, College Station, Texas) for meta-analysis. Effect estimates are presented as excess risks expressed per 10 μg/m^3^ contrast in exposure, except elemental carbon for which risks were expressed per 1 μg/m^3^.
### PM~2.5~ and all-cause and cardiovascular mortality
Table [1](#T1){ref-type="table"} and Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} summarize the studies on long-term air pollution exposure and all-cause and cardiovascular mortality using PM~2.5~ or PM~10~ as exposure metric \[[@B14]-[@B39]\]. Most but not all studies report significant associations between PM~2.5~ and all-cause mortality. Since the publication of the authoritative American Heart Association Scientific Statement, sixteen new cohort studies were published between 2009 and January 2013. These studies were often performed in more selected groups e.g. female teachers \[[@B27],[@B36]\] or male truck drivers \[[@B32]\]. The geographic range has also been expanded significantly with several new studies from Japan and China now published. Another tendency is the publication of large studies based upon large population samples (e.g. census), with often less information on confounding variables such as individual smoking habits. Large cohort studies have used neighborhood socio-economic status and co-morbidities strongly associated with smoking as proxies for actual smoking data \[[@B26],[@B38]\]. Effect estimates differed substantially across studies, with most studies showing less than 10% increase in mortality for an increment of 10 μg/m^3^ PM~2.5~. The random effects summary estimate for the percent excess risk per 10 μg/m^3^ PM~2.5~ for all-cause mortality was 6.2% (95% CI: 4.1 -- 8.4%). A formal test of heterogeneity was statistically significant, with an I^2^ value of 65% indicating moderate heterogeneity. I^2^ can be interpreted as the variability in effect estimates due to true between study variability and not chance \[[@B13]\]. The random effects summary effect estimate for cardiovascular mortality was 10.6% (95% CI 5.4, 16.0%) per 10 μg/m^3^. Thus, the overall effect estimates were larger for cardiovascular than for all-cause mortality. This pattern was found in most of the individual studies, with the exceptions being the Dutch cohort study \[[@B23]\], the US trucking industry cohort study \[[@B32]\] and a national cohort study from New Zealand \[[@B35]\]. Significant and large heterogeneity of effects was found across studies, with an I^2^ statistic of 61%. After excluding the Miller study \[[@B22]\], moderate heterogeneity remained (I^2^ = 40%). Overall, the new studies have supported an association between PM~2.5~ and mortality first identified in the US Six City and ACS studies. It is of interest to note that the weight of the ACS study in the combined effect estimate is 12% for all-cause mortality, documenting that the combined estimate does not rely on one or two studies. Furthermore, effect estimates from the three large population cohorts without individual smoking data \[[@B26],[@B34],[@B38]\] were not higher than those from the individual cohort studies. An important question is what the explanation is for the observed heterogeneity of effect estimates. Differences in study population, exposure assessment, pollution mixture, study period, outcome assessment, and confounder control could have contributed to these differences.
{#F1}
{#F2}
######
**Summary of effect estimates (excess risk per 10 μg/m**^**3**^**) from cohort studies on particulate matter (PM**~**10**~**or PM**~**2.5**~**) and mortality from all causes and cardiovascular diseases**
**Study** **Study population** **Follow-up period** **Pollutant** **Conc**^**a**^**(μg/m**^**3**^**)** **Spatial scale**^**b**^ **% change in risk (95%CI) in mortality associated with a 10 μg/m**^**3**^**increase PM** **References**
----------------------------------------------- ------------------------------------------------------------------ ---------------------- --------------- -------------------------------------- ----------------------------------------- ------------------------------------------------------------------------------------------- ---------------- ------------
Harvard six cities 8111 adults in six US cities 1976 - 1989 PM~2.5~ 18 (11--30) City 13(4, 23) 18 (6, 32) \[[@B15]\]
Harvard six cities 8096 adults in six US cities 1979 -1998 PM~2.5~ 15 (10--22) City 16 (7, 26) 28 (13,44) \[[@B14]\]
Harvard six cities 8096 adults in six US cities 1974 - 2009 PM~2.5~ 16 (11--24) City 14 (7, 22) 26 (14, 40) \[[@B16]\]
American Cancer Society (ACS) study 552, 800 adults from 51 US cities 1982 - 1989 PM~2.5~ 18 (9--34) City 26 (8, 47) NA \[[@B17]\]
ACS study 500,000 adults from 51 US cities 1982 -1998 PM~2.5~ 18 (4) City 6 (2, 11) 9 (3, 16)^c^ \[[@B18]\]
ACS sub-cohort study 22,905 subjects in Los Angeles area 1982 - 2000 PM~2.5~ (\~9 -- 27) Zip code (Int) 17 (5, 30) 26 (1, 60)^c^ \[[@B19]\]
German cohort 4752 women in Ruhr area 1985 -- 2003 PM~10~ 44 (35--53) Address (near) 12 (−9, 37) 52 (8, 114) \[[@B20]\]
German cohort 4752 women in Ruhr and surrounding area 1985 - 2008 PM~10~ 44 (35--53) Address (near) 22 (6, 41) 61 (26, 104) \[[@B21]\]
Women's Health Initiative Observational Study 65,893 postmenopausal women from 36 US metropolitan areas 1994-1998 PM~2.5~ 14 (3--28) Zip code (near) NA 76 (25,147) \[[@B22]\]
Netherlands Cohort Study 120, 852 subjects from Netherlands 1987 -1996 PM~2.5~ 28 (23--37) Address (LUR) 6 (−3, 16) 4 (−10, 21) \[[@B23]\]
Nurses' Health Study 66,250 women from the US north eastern metropolitan areas 1992-2002 PM~10~ 22 (4) Address (LUR) 11 (1,23) 35 (3, 77) \[[@B24]\]
Nurses' Health Study 66,250 women from the US north eastern metropolitan areas 1992-2002 PM~2.5~ 14 (6--28) Address (LUR) 26 (2, 54) NA \[[@B25]\]
Medicare national cohort 13.2 million elderly Medicare recipients across the USA 2000 - 2005 PM~2.5~ 13 (4) Zip code (Mean) 4 (3, 6)^d^ \[[@B26]\]
California teachers study 45,000 female teachers 2002 -2007 PM~2.5~ 18 (7--39) Address (near) 6 (−4, 16) 19 (5, 36)^c^ \[[@B27]\]
Swiss national cohort National census data linked with mortality 2000 - 2005 PM~10~ 19 (\>40)^e^ Address (Disp) NA −1 (−3, 0) \[[@B28]\]
Health professionals follow-up study 17,545 highly educated men in the midwestern and northeastern US 1989 -- 2003 PM~2.5~ 18 (3) Address (LUR) −14 (−28,2) 3 (−17, 26) \[[@B29]\]
Vancouver cohort 452,735 Vancouver residents 45--85 yr 1999 -- 2002 PM~2.5~ 4 (0 -- 10) Address (LUR) NA 7 (-14, 32) \[[@B30]\]
China nat. hypertension survey 70,497 men and women 1991 - 2000 TSP 289 (113--499) City 0.3 (0, 1) 1 (0, 2) \[[@B31]\]
US trucking industry cohort 53,814 men in the US trucking industry 1985 -2000 PM~2.5~ 14 (4) Address (near) 10 (3, 18) 5 (−7, 19) \[[@B32]\]
Chinese retrospective cohort study 9,941 adults from five districts of Shenyang city 1998 -2009 PM~10~ 154 (78--274)^f^ District (mean) 53 (50, 56) 55 (51, 60) \[[@B33]\]
Canadian national cohort 2.1 million nonimmigrant Canadians . \> 25 yr 1991 - 2001 PM~2.5~ 9 (2 -- 19) Enumeration area, N = 45710 (satellite) 10 (5, 15) 15 (7, 24) \[[@B34]\]
New Zealand Census mortality study 1.06 million adults in urban areas from 1996 census 1996 -1999 PM~10~ 8 (0 -- 19) Census tract (Disp) 7 (3, 10) 6 (1, 11) \[[@B35]\]
California teachers study 101,784 female teachers 1997- 2005 PM~2.5~ 16 (3--28) Address (Inter) 1 (−5, 9) 7 (−5, 19) \[[@B36]\]
Nippon data cohort 7,250 adults \> 30 yr throughout Japan 1980 - 2004 PM~10~ \<27 - \> 43 District (near) −2 (−8, 4) −10 (−19, 0) \[[@B37]\]
Rome longitudinal study 1,265,058 adults from Rome 2001 - 2010 PM~2.5~ 23 (7 -- 32) Address (DISP, 1 km grid) 4 (3, 5) 6 (4, 8) \[[@B38]\]
^a^ Mean with minimum -- maximum in parentheses (μg/m^3^). One number in parentheses is standard deviation.
^b^ Spatial scale of exposure assignment, in parentheses exposure assignment method. City = average of monitors within the city; Near = nearest monitor concentration; LUR = land use regression; Disp = dispersion modeling; Inter = interpolation.
^c^ Cardio-pulmonary mortality reported if cardiovascular mortality not available.
^d^ Combining the estimates from the three regions of the USA.
^e^ Median and 90th percentile reported.
^f^ Very high pollution levels that changed significantly during follow-up changing the ranking of the five districts.
Studies adjusted for individual smoking except references \[[@B26],[@B28],[@B30],[@B34],[@B38],[@B56]\].
### Effect modification
Differences in the fraction of *susceptible subjects* may have contributed to the observed differences. Brook \[[@B4]\] suggested that women might be more susceptible to ambient air pollution. The studies with higher PM effect estimates, particularly the WHI-study have indeed been performed in women only. However, it is problematic to draw conclusions about susceptible subgroups based upon *between-study* comparisons as multiple factors differ between studies. A comparison of PM effect estimates between men and women *within* studies does not provide clear evidence that women have a stronger response (Table [2](#T2){ref-type="table"}). The findings from the AHSMOG are difficult to interpret, with higher effects in men in the larger earlier study \[[@B40]\] and larger effects in women in the smaller cohort with longer follow-up \[[@B41]\]. The larger effect estimate for BC for men in a Canadian study \[[@B30]\] has to be interpreted with care, because of the lack of data on a variety of important covariates, including individual smoking data, though the authors argue that smoking likely has not confounded the associations with mortality. In the French PAARC study, effect estimates for the evaluated pollutants (TSP, BS and NO~2~) were similar among men and women \[[@B42]\]. There is also only weak evidence that effect estimates are larger among never-smokers, though in all evaluated studies a (borderline) significant association was found in never-smokers (Table [2](#T2){ref-type="table"}). Associations in current smokers were more variable across the studies, consistent with the larger 'noise' generated by smoking. In all four studies, PM~2.5~ effect estimates were higher for those with the lowest education and there was little indication of an association in those with higher education. The absence of an association in the (highly educated) Health professionals study \[[@B29]\] is consistent with this observation. In contrast, in the French PAARC study, effect estimates for Black Smoke were very similar across educational strata, with significant effect also found in those with a university degree \[[@B42]\]. Furthermore the PM~2.5~ effect estimates (excess risks) in an extended analysis of the ACS differed less than originally reported: 8.2%, 7.2 and 5.5% per 10 μg/m^3^ for subjects with low, medium and high education respectively \[[@B43]\]. If confirmed in further studies, it is likely that multiple life style related factors may play a role in the stronger effects observed in less-educated subjects. These may include dietary factors such as lower fruit and anti-oxidant intake \[[@B23]\], higher risk of obesity or other pre-existing diseases, higher actual exposures than assumed in the studies, lack of air conditioning and possibly interaction with other risk factors such as poorer housing conditions e.g. moisture.
######
**Effect modification of the effect (excess risk per 10 μg/m**^**3**^**) of PM**~**2.5**~**on cardiovascular mortality**
**Subgroup** **ACS \[**\[[@B18]\]**\]**^**a**^ **NLCS \[**\[[@B23]\]**\]** **Harvard six city \[**\[[@B43]\]**\]** **Nurses health \[**\[[@B24]\]**\]** **WHI \[**\[[@B22]\]**\]** **AHSMOG \[**\[[@B40]\]**\]** **AHSMOG \[**\[[@B41]\]**\]**
------------------- ----------------------------------- ----------------------------- ----------------------------------------- -------------------------------------- ---------------------------- ------------------------------- -------------------------------
***Sex***
Men 5 (0, 11) 3 (−5, 12)^b^ 33 (8, 63)^a^ NA NA 4 (−3,11) −10 (−−24, 5)
Women 6 (0, 12) 7 (0, 14) 20 (−6, 53) −3 (−9, 2) 42 (6, 90)
*Smoking status*
Never 6 (1, 12) 13 (−4, 32) 36 (2, 82) 83 (20, 179) 18 (−1, 40) NA NA
Former 5 (0, 11) −4 (−17, 13) 29 (−3, 72) 22 (−18, 83) 21 (1, 52)
Current 4 (−2, 11) 3 (−10, 19) 35 (94, 74) −12 (−48, 48) 68 (6, 166)
*Education*
Low 11 (6, 18) 20 (−10, 70)^a^ 45 (13, 85) 40 (11, 75) NA NA
Medium 6 (1, 13) 2 (−16, 24) 30 (−2,73) 33 (14, 55)
High 1 (−3, 6) −10 (−35, 20) −3 (−29, 34) 11 (−6, 31)
*Body mass index*
Non-Obese NA NA NA 8 (−24, 52) −1 (−10, 29)^c^ NA NA
Obese 99 (23, 222) 35 (12, 64)^c^
^a^ Read from graph.
^b^ natural-cause mortality.
^**c**^ for BMI \< 22.5, continuous trend observed NA = not available.
In two studies, PM~2.5~ effect estimates were substantially higher among subjects with high body mass index \[[@B22],[@B24]\].
It is likely that subject characteristics might explain part of the variability of air pollution effect estimates across studies where subgroup analyses are limited by power to detect differences. Hence, further research is required to study the effects of air pollution on women, smokers, obese participants, and diabetes mellitus with better measurement of the exposures. Gene-environment interactions have been shown for the (short-term) air pollution effects on inflammation markers \[[@B44],[@B45]\] Inflammation likely plays an important role in the mechanism of cardiovascular events \[[@B3],[@B4]\]. Gene-environment interactions have not yet been studied in the framework of mortality cohort studies.
### Exposure issues
One of the important sources of variability of effect estimates between studies is likely related to exposure definition and misclassification. While the most important environmental predictor to consider is actual individual-level *exposure* to ambient particles, which presumably drives the health effects, most studies have used outdoor concentrations at sites distant to the participant's precise location. The use of outdoor exposures leads to exposure misclassification. In the cohort studies, exposure has been characterized by the outdoor concentration at the city level based upon central site monitoring or the nearest monitor, or modeling at the individual address. Table [1](#T1){ref-type="table"} shows that the spatial scale of assessment and exposure assessment method varied significantly across studies, probably contributing to differences in effect estimates. Differences in pollution range across studies (Table [1](#T1){ref-type="table"}) may have contributed as well. These exposure estimates do not take into account time activity patterns such as time spent in the home or in traffic and factors affecting infiltration of particles indoors. There is a large literature documenting the importance of air exchange rate on infiltration of particles indoors. Importantly, these factors may differ between homes within a study area and between study areas in different climates. In a study of short-term effects, PM10 effects on hospital admissions were larger in US cities with lower% of air conditioning, related to higher particle infiltration rates \[[@B46]\]. The impact of air conditioning use has not been investigated yet in the framework of cohort studies. In the Multiethnic study of Atherosclerosis Air study, indoor-outdoor measurements have been performed to adjust the exposure estimates \[[@B47],[@B48]\] and each participant provides time-activity information to weight exposures between time spent indoors and outdoors. Evidence for the importance of time activity patterns was obtained in the US truckers study, showing higher ambient PM~2.5~ effect estimates in the population excluding long-haul drivers who spend more time away from home \[[@B32]\]. Other factors could however also explain the higher effect estimated after excluding long-haul drivers. In the WHI study, effect estimates tended to be higher for subjects spending more than 30 minutes outdoors \[[@B22]\]. In a validation study in the Netherlands, the contrast of personal soot exposure for adults living on a major road compared to those living at a background location, was larger for those spending more time at home \[[@B49]\]. Because of the reliance on ambient exposure estimates, it is not surprising that some heterogeneity in effect estimates across studies is found.
Differences in *particle composition* or contributing sources very likely explain some of the heterogeneity in effect estimates, as was observed for short-term mortality and hospital admission studies of PM~2.5~ and PM~10~\[[@B50]-[@B53]\]. For a comprehensive review we refer to the recent evaluation made by the World Health Organization (<http://www.euro.who.int/en/what-we-do/health-topics/environment-and-health/air-quality/publications/2013/review-of-evidence-on-health-aspects-of-air-pollution-revihaap>). Particle composition effects have not been systematically investigated in cohort studies with the exception of the California teacher's study \[[@B27]\]. In a recent review it was shown that on a per microgram per m^3^ basis, mortality effect estimates were about 10 times larger for EC than for PM~2.5~\[[@B7]\]. Hence, in locations with higher levels of primary combustion particles we could expect higher PM~2.5~ effects. In the next section, evidence on EC is further discussed.
A further important issue is for which *period exposure* is characterized. Air pollution data may not be available for the entire follow-up period. As an example in the ACS study, PM~2.5~ data were available at the start and end of follow-up \[[@B18]\]. When significant (often downward) trends in pollution occur with changing (often decreasing) spatial contrasts in the study, bias may occur in the estimated association between pollution and mortality. The follow-up study from the Harvard Six City study \[[@B14]\] and two studies in potentially at-risk populations \[[@B54],[@B55]\] suggested that the relevant exposure for cardiovascular effects may be the exposure in the past few years. These authors conclude that it does not take decades to bridge the gap between the short- and long-term exposure effect estimates, consistent with the effect of intervention studies showing reductions in mortality in the year after the intervention \[[@B54],[@B55]\]. These studies \[[@B54],[@B55]\] have made use of long-term temporal contrast within cities adjusting for secular trends. PM effect estimates were similar to the previously discussed studies exploiting spatial contrasts.
A further *temporal* issue in studies that use land use regression models for exposure assessment is that these models often are based upon current measurement campaigns and linked to health outcomes that occurred in the past. Three studies in the Netherlands, Rome (Italy) and Vancouver (Canada) have shown that for periods of about 10 years current LUR models predicted historic spatial contrasts well \[[@B56]-[@B58]\]. Even when concentrations have decreased over time, spatial contrasts often remain stable. Spatial contrasts may not be stable in areas with rapid economic development as indicated in one of the Chinese cohort studies in which the ranking of study areas changed during follow-up \[[@B33],[@B59]\]. Even when the ranking of subjects is not changed, the quantitative spatial contrast in a study area may have changed, e.g. because the difference between major roads and background locations has decreased in time. Changed spatial contrasts will affect the estimated slope of the mortality pollution association \[[@B18],[@B56]\]. Moving of subjects may further complicate the assessment.
An important question to address for the traffic pollution studies is potential confounding by *road traffic noise*, which has been shown to be related to cardiovascular disease including MI as well. A few studies have attempted to disentangle traffic-related air pollution and noise \[[@B60]-[@B62]\]. These studies found moderate correlations between air pollution and noise. The three studies differed somewhat in their findings of independent air pollution and noise effects. More work is needed in this area.
### Coarse particles and elemental carbon
Table [3](#T3){ref-type="table"} presents studies that have used elemental carbon or coarse PM as the exposure metric. Table [3](#T3){ref-type="table"} illustrates that there is no evidence that long-term exposure to coarse PM is related to mortality. In three of the four cohort studies that reported no significant association with coarse PM, significant associations with PM~2.5~ were found \[[@B18],[@B25],[@B63]\]. However, exposure assessment for coarse particles is more challenging than for PM~2.5~ because of the influence of local sources, hence central site monitors are likely to have greater errors in representing residential concentrations. It is therefore possible that with more spatially resolved exposure assessment methods such as land use regression models or dispersion models, potential long-term exposure effects will be detected. The California Teacher's study did not evaluate coarse PM and did not find significant associations between all-cause mortality and elemental concentrations of Si, Fe and Zn, elements abundant in coarse particles, but did report an association between Si and ischemic heart disease \[[@B27]\].
######
**Summary of effect estimates (excess risk per 10 μg/m**^**3**^**) from cohort studies on coarse particulate matter and elemental carbon (per 1 μg/m**^**3**^**) and mortality from all causes and cardiovascular diseases**
**Study name** **Study design** **Follow-up period** **Pollutant** **Conc**^**a**^**(μg/m**^**3**^**)** **Spatial scale**^**b**^ **% change in risk (95%CI) in mortality** **References**
-------------------------------------- ------------------------------------------------------------------ ------------------------- --------------- -------------------------------------- -------------------------- ------------------------------------------- ---------------- ------------
*Coarse PM*
ACS study 500,000 adults 51 US cities 1982 - 1998 PM~2.5--15~ 19 (6) City 1 (−2 3) 2 (−2, 5)\* \[[@B18]\]
AHSMOG study 3769 California seventh-day Adventists 1977 -- 1992 PM~2.5--15~ 27 (4 -- 44) Address (Inter) 5 (−8, 20) NA \[[@B63]\]
Nurses' Health Study 66,250 women from US north eastern metropolitan areas 1992- 2002 PM~2.5--10~ 8 (0 -- 27) Address (LUR) 3 (−11, 18) NA \[[@B25]\]
Health professionals follow-up study 17,545 highly educated men in the midwestern and northeastern US 1989 -- 2003 PM~2.5--10~ 10 (3) Address (LUR) −10 (−22, 4) 8 (−10, 29) \[[@B29]\]
*EC*
Netherlands Cohort Study 120, 852 subjects from Netherlands 1987 - 1996 BS^e^ 17 (9--36) Address (LUR) 5 (0, 11) 4 (−5, 13) \[[@B23]\]
ACS study (extended) 500,000 adults 51 US cities 1982 -- 1998 EC IQR = 0.31 City 6 (1, 11) 11 (3, 19) \[[@B64]\]
Worcester MI survivors 3,895 MI patients 1995 - 2005 EC 0.4 (0.1 -- 0.9) Address (LUR) 2 (−7, 11)^d^ NA \[[@B65]\]
15 (3, 29)
Vancouver cohort 452,735 Vancouver residents 45--85 yr 1999 -- 2002 BC 1.5 (0--5) Address (LUR) NA 6 (3, 9) \[[@B30]\]
PAARC 14,284 adults in 24 French areas 1974 -- 1998 BS 44 (18--77) Address (near) 7 (3, 10) 5 (−2, 12) \[[@B42]\]
Veteran's study 70,000 male US veterans 1997 -- 2001 EC 0.6 (0.1 -- 2.0) County (mean) 18 (5, 33) NA \[[@B66]\]
California teachers study 45,000 female teachers 2002 -2007 EC 1.1 (0.2 -- 2.4) Address (near) 3 (−11,19) 11 (−9, 36) \[[@B27]\]
Two Scotch cohorts 15, 402 and 7,028 adults from West-central and central Scotland 1972 - 1998 1970 - 1998 BS 19 LUR + temporal 5 (1,9) 7 (0, 13) \[[@B67]\]
^a^ Mean with minimum -- maximum in parentheses (μg/m^3^). One number in parentheses is standard deviation.
^b^ Spatial scale of exposure assignment, in parentheses exposure assignment method. City = average of monitors within the city; Near = nearest monitor concentration; LUR = land use regression; Disp = dispersion modeling; Inter = interpolation.
^c^ Cardio-pulmonary mortality reported if cardiovascular mortality not available.
^d^ HRs for first two years after MI and after the first two years of survival.
^e^ BC (Black Carbon), BS (Black Smoke) and EC (Elemental carbon) are different markers used to assess soot. Increases consistent with a 1 μg/m^3^ increase in EC were used \[[@B7]\].
Studies adjusted for individual smoking except references \[[@B26],[@B28],[@B30],[@B34],[@B38],[@B56]\].
Consistently, the summary estimate for PM~10~ was smaller than for PM~2.5~ with a summary effect estimate per 10 μg/m^3^ of 3.5% (95% CI 0.4%, 6.6%) with significant heterogeneity (I^2^ = 69%) of the studies included in Table [1](#T1){ref-type="table"}, excluding the because of changing spatial patterns difficult to interpret Chinese retrospective study \[[@B33]\]. The PM~10~ analysis was added as several studies only report PM~10~.
Effect estimates for EC were very consistent across studies \[[@B23],[@B27],[@B30],[@B42],[@B64]-[@B67]\]. The random effects summary estimate for all-cause mortality per 1 μg/m^3^ EC was 6.1% (95% CI 4.9%, 7.3%), with highly non-significant heterogeneity of effect estimates (I^2^ = 0%). Most of the included studies assessed EC exposure at the city-scale \[[@B27],[@B64]\] which represents variation in city background but does not account for small-scale variation related to proximity to major roads. Many studies have documented significant intra-urban contrasts for EC, related to especially major roads \[[@B7]\]. Most likely EC and NO~2~ should be considered representatives of the complex mixture of traffic-related air pollution, rather than the only components causally associated with mortality.
There is fairly consistent evidence of associations of mortality with nitrogen dioxide (Table [4](#T4){ref-type="table"}). The random effects summary estimate for all-cause mortality per 10 μg/m^3^ for NO~2~ was 5.5% (95% CI 3.1%, 8.0%), with significant and large heterogeneity of effect estimates (I^2^ = 73%). In this analysis, the Chinese study \[[@B33]\] was not included as exposure was assessed at the district level. Inclusion of the essentially null findings of the ACS study-excess risk of 0.3% (95% CI −0.8, 1.3%)- resulted in an only slightly smaller combined estimate of 4.7% (95% CI 2.4, 7.1%). In the ACS study, intra-urban variation was also not accounted for. As traffic-related air pollution varies on a small spatial scale, it is even more critical to assess exposure on a fine spatial scale such as the residential address than for PM~2.5~.
######
**Summary of cohort studies on NO**~**2**~**and mortality from all causes and cardiovascular diseases (excess risk per 10 μg/m**^**3**^**)**
**Study name** **Study population** **Follow-up period** **Pollutant** **Conc**^**a**^**(μg/m**^**3**^**)** **Spatial scale**^**b**^ **% change in risk (95%CI) in mortality per 10 μg/m**^**3**^ **References**
------------------------------------ --------------------------------------------------- ---------------------- --------------- -------------------------------------- -------------------------- -------------------------------------------------------------- ---------------- ------------
Oslo cohort 16,209 men in Oslo, Norway 1972 -- 1998 NO~x~ 11 (1 -- 168) Address (DISP) 8 (6,11) NA \[[@B68]\]
Netherlands Cohort Study 120, 852 subjects from Netherlands 1987 -1996 NO~2~ 37 (15--67) Address (LUR) 8 (0, 16) 7 (−6, 21) \[[@B23]\]
German cohort 4752 women in Ruhr and surrounding area 1985 -- 2003 NO~2~ 39 (20 -- 60) Address (near) 11 (1,21) 36 (14, 63) \[[@B20]\]
German cohort 4752 women in Ruhr and surrounding area 1985 -- 2008 NO~2~ 39 (20 -- 60) Address (near) 11 (4,18) 32 (18, 47) \[[@B21]\]
PAARC 14,284 adults in 24 French areas 1974 -- 1998 NO~2~ 20 (12 -- 32) Address (near) 14 (3, 25) 27 (4, 56) \[[@B42]\]
China nat. hypertension survey 70,497 men and women 1991 - 2000 NOx 50 (20 -- 122) City 2 (0, 3) 2 (1, 4) \[[@B31]\]
Vancouver cohort 452,735 Vancouver residents aged 45--85 yr 1999 -- 2002 NO~2~ 32 (15 -- 58) Address (LUR) NA 5 (1, 9) \[[@B30]\]
DCH 52,061 adults in Copenhagen and Arhus 1993 - 2009 NO~2~ 17 (11 -- 60) Address (DISP) 8 (2, 13) 15 (3,27) \[[@B69]\]
US trucking industry cohort 53,814 men in the US trucking industry 1985 -2000 NO~2~ 28 (14) Address (LUR) 5 (3, 7) 4 (0, 8) \[[@B32]\]
Chinese retrospective cohort study 9,941 adults from five districts of Shenyang city 1998 -2009 NO~2~ 46 (18--78) District (mean) 145 (134, 158) 146 (131, 163) \[[@B33]\]
Rome longitudinal study 684,000 adults from Rome 2001 - 2006 NO~2~ 45 (11) Address (LUR) 4 (3, 5) NA \[[@B56]\]
California Teachers study 101,784 female teachers 1997 -2005 NO~2~ 67 (10 -- 134) Address (Inter) −3 (−9, 4) −2 (−12, 9) \[[@B36]\]
Shizuoka elderly cohort 13,444 adults \> 65 yr 1999 - 2006 NO~2~ 25 (−19, 75) Address (LUR) 2 (−4, 8) 15 (3, 28) \[[@B70]\]
Ontario tax cohort 205, 440 adults in Toronto, Hamilton,Windsor 1982 -- 2004 NO~2~ 43 (8), 31 (6), 24 (5)^c^ Address (LUR) NA 8 (5, 11) \[[@B71]\]
Rome longitudinal study 1,265,058 adults from Rome 2001 - 2010 NO~2~ 44 (13--75) Address (LUR) 3 (2, 3) 3 (2, 4) \[[@B38]\]
^a^ Mean with minimum -- maximum in parentheses (μg/m^3^). One number in parentheses is standard deviation.
^b^ Spatial scale of exposure assignment, in parentheses exposure assignment method. City = average of monitors within the city; Near = nearest monitor concentration; LUR = land use regression; Disp = dispersion modeling; Inter = interpolation.
^c^ Mean (IQR) per city.
Studies adjusted for individual smoking except references \[[@B26],[@B28],[@B30],[@B34],[@B38],[@B56]\].
### Specific cardiovascular causes of death
Table [5](#T5){ref-type="table"} shows associations between ambient air pollution and mortality from ischemic heart disease or myocardial infarction (MI), including studies based upon death certificates, more detailed studies using registry data, or ideally cohort studies with epidemiological review of medical records, allowing more precise identification of disease incidence. Several case--control studies based upon M.I. registries or epidemiological studies with clinical review have found associations between NO~2~ and fatal M.I. but not non-fatal M.I. \[[@B72]-[@B74]\]. Thus far, the finding of associations for fatal MI only was interpreted as an evidence that air pollution particularly affects the frail, or acts to aggravate a disease progression caused by other factors. On the other hand, it is also possible that the outcomes of ischemic heart diseases are misclassified and combined as composite outcomes, where fatal outcomes are captured more precisely \[[@B75]\]. Although there is increasing evidence that air pollution is associated with markers of early atherosclerosis, it is possible that air pollution will affect the underlying biological processes that predispose to atherothrombosis (which leads to MI and stroke) compared to atherosclerosis \[[@B76],[@B77]\]. Another explanation is that the type of outcomes affected by pollution are those that have higher case-fatality rates (e.g., arrhythmic sudden death has higher case-fatality rate than overall MI).
######
Summary of the studies on particulate matter and NO~2~ and mortality from specific cardiovascular diseases (excess risk per 10 μg/m^3^)
**Study name** **Pollutant** **Conc**^**a**^**(μg/m**^**3**^**)** **Spatial scale**^**b**^ **% change in risk (95%CI) in mortality associated with a 10 μg/m**^**3**^**increase** **References**
-------------------------------------- --------------- -------------------------------------- ----------------------------------------- ---------------------------------------------------------------------------------------- ---------------- ------------- ------------
ACS study PM~2.5~ 17 (5) City 18 (14, 23) NA 2 (−5, 10) \[[@B39]\]
Oslo cohort NO~x~ 11 (1 -- 168) Address (DISP) 8 (3, 12) NA 4 (−6, 15) \[[@B68]\]
Women's Health Initiative Study PM~2.5~ 14 (3--28) Zip code 5 (near) 76 (25,147) NA NA \[[@B22]\]
Netherlands Cohort Study BS 17 (9--36) Address (LUR) 1 (−17, 22) NA 39 (−1, 94) \[[@B23]\]
Nurses' Health Study PM~10~ 22 (4) Address (LUR) 35 (3, 77) NA NA \[[@B24]\]
Nurses' Health Study PM~2.5~ 14 (6--28) Address (LUR) NA 102 (7, 278) NA \[[@B25]\]
California teachers study PM~2.5~ 18 (7--39) Address (near) 55 (24, 93) NA NA \[[@B27]\]
Swiss national cohort PM~10~ 19 (\>40)^c^ Address (Disp) −1 (−3, 0) NA −1 (−2, 0) \[[@B28]\]
Health professionals follow-up study PM~2.5~ 18 (3) Address (LUR) −2 (−30, 35) NA NA \[[@B29]\]
Canadian national cohort PM~2.5~ 9 (2 -- 19) Enumeration area, N = 45710 (satellite) 30 (18,43) NA 4 (−7, 16) \[[@B34]\]
Californian Teachers study PM~2.5~ 16 (3--28) Address (Inter) 20 (2, 41) NA 16 (−8, 46) \[[@B36]\]
Shizuoka elderly cohort NO~2~ 25 (−19, 75) Address (LUR) 27 (2, 58) NA 9 (−6, 27) \[[@B70]\]
Nippon data cohort PM~10~ \<27 - \> 43 District (near) −8 (−27, 17) NA −14 (−26,1) \[[@B37]\]
DCH NO~2~ 17 (11 -- 60) Address (Disp) 7 (−9, 26) NA 6 (−14, 32) \[[@B69]\]
Ontario Tax cohort NO~2~ 43 (8), 31 (6), 24 (5)^c^ Address (LUR) 9 (4, 14) NA −4 (−10, 5) \[[@B71]\]
Rome longitudinal study PM~2.5~ 23 (7 -- 32) Address (DISP, 1 km grid) 10 (6, 13) NA 8 (4, 13) \[[@B38]\]
*M.I. registry studies*
Stockholm NO~2~ 14 (3 -- 32) Address (DISP) NA 15 (−1, 33) NA \[[@B72]\]
Rome residents NO~2~ (\<30 - \> 60) Census block (LUR) NA 7 (2, 12) NA \[[@B73]\]
Stockholm residents NO~2~ 12 (2 -- 33) Address (DISP) NA 8 (5, 11) NA \[[@B74]\]
IHD = ischemic heart disease; MI = myocardial infarction. Fatal MI reported for registry studies. NA = not available.
^a^ Mean with minimum -- maximum in parentheses (μg/m^3^). One number in parentheses is standard deviation.
^b^ Spatial scale of exposure assignment, in parentheses exposure assignment method. City = average of monitors within the city; Near = nearest monitor concentration; LUR = land use regression; Disp = dispersion modeling; Inter = interpolation.
^c^ Median and 90th percentile reported.
Studies adjusted for individual smoking except references \[[@B26],[@B28],[@B30],[@B34],[@B38],[@B56]\].
Fewer studies have evaluated cerebrovascular mortality. In the Dutch cohort study and in the Women's Health Initiative Study, a strong association was found \[[@B22],[@B23]\]. In contrast, in the ACS study, the Norwegian cohort, and the Swiss national cohort study no association was found \[[@B28],[@B39],[@B68]\]. It is possible that poorer recording of cerebrovascular mortality on death certificates has contributed to these inconsistencies. There is also some evidence from ecological studies that air pollution may contribute to stroke mortality \[[@B78],[@B79]\].
Two studies have reported significant associations between particulate matter air pollution and dysrhythmia, heart failure and cardiac arrest combined \[[@B39],[@B60]\]. These results are based upon smaller numbers of events, and require large cohort studies for further verification. The results are consistent with several studies documenting significant associations between short-term PM or NO~2~ exposure and mortality due to heart failure and dysrhythmia and defibrillator discharges \[[@B4],[@B80]\].
### Air pollution and respiratory mortality
Table [6](#T6){ref-type="table"} shows the effect estimates for respiratory mortality. In the two first US cohort studies, no association between PM~2.5~ and respiratory mortality was found \[[@B15],[@B17]\]. In contrast to the findings of these US studies, strong associations were found in the Dutch cohort study \[[@B23]\], a Norwegian study \[[@B68]\] and a Chinese study \[[@B59]\]. The random effect pooled estimate per 10 μg/m^3^ for PM~2.5~ was 2.9% (95%CI −5.9, 12.6%), highly non-significant. The heterogeneity across studies was statistically significant with an I^2^ statistic of 59%, indicating moderate heterogeneity. Associations for PM were weaker in the Dutch and Chinese cohort study than with NO~2~ or NO~x~. Respiratory mortality may be more related to primary traffic-related pollutants than with long-range transported particles, though further work is needed to test this hypothesis. The smaller number of deaths due to respiratory disease compared to cardiovascular diseases, contributed to larger confidence intervals within individual studies and larger variability of the main effect estimates across studies. In time series studies including several large multi-city studies in the USA and Europe, significant associations between daily variations in PM and respiratory mortality were found \[[@B1]-[@B4]\]. Expressed per 10 μg/m^3^ PM excess risks of about 1% are typically reported for short-term exposures, larger than for all-cause mortality \[[@B1]-[@B4]\]. In contrast to cardiovascular disease, current evidence therefore does not suggest an additional risk from long-term exposure, possibly related to mortality displacement \[[@B2],[@B3]\]. More studies are needed to evaluate long-term exposures on respiratory mortality more thoroughly.
######
**Summary of the studies on air pollution and mortality from all respiratory disease (excess risk per 10 μg/m**^**3**^**)**
**Study Name** **Pollutant** **Conc**^**a**^**(μg/m**^**3**^**)** **Spatial scale**^**b**^ **% change in risk (95%CI) in mortality per 10 μg/m**^**3**^ **References**
------------------------------------- --------------- -------------------------------------- --------------------------- -------------------------------------------------------------- ----------------
AHSMOG PM~10~ 51 (17) Address (Inter) 6 (−1, 15) \[[@B40]\]
ACS study PM~2.5~ 17 (5) City −8 (−14, -2) \[[@B39]\]
Oslo cohort NO~x~ 11 (1 -- 168) Address (DISP) 16 (6, 26) \[[@B68]\]
Harvard six cities PM~2.5~ 15 (10--22) City 8 (−21, 49) \[[@B14]\]
Netherlands Cohort Study PM~2.5~ 28 (23--37) Address (LUR) 7 (−25, 52) \[[@B23]\]
Netherlands Cohort Study NO~2~ 37 (15--67) Address (LUR) 12 (0, 26) \[[@B23]\]
California Teachers study PM~2.5~ 18 (7--39) Address (near) 3 (−20, 34) \[[@B27]\]
China national. hypertension survey NO~x~ 50 (20 -- 122) City 3 (0, 6) \[[@B31]\]
China national. hypertension survey TSP 289 (113 -- 499) City 0.3 (−1,1) \[[@B31]\]
US truckers study PM~2.5~ 14 (4) Address (near) 20 (−9, 60) \[[@B32]\]
US truckers study NO~2~ 28 (14) Address (LUR) 15 (1,31) \[[@B32]\]
California Teachers study PM~2.5~ 16 (3--28) Address (Inter) 21 (−3, 52) \[[@B36]\]
New Zealand Census study PM~10~ 8 (0 -- 19) Census tract (Disp) 14 (5, 23) \[[@B35]\]
Shenyang cohort study PM~10~ 154 (78 -- 274) District (mean) 67 (60, 74) \[[@B59]\]
Shenyang cohort study NO~2~ 46 (18--78) District (mean) 197 (169, 227) \[[@B59]\]
Shizuoka elderly cohort NO~2~ 25 (−19, 75) Address (LUR) 19 (2, 38) \[[@B70]\]
Two Scotch cohorts BS 19 LUR + temporal 11 (−3, 28) \[[@B67]\]
Rome longitudinal study PM~2.5~ 23 (7 -- 32) Address (DISP, 1 km grid) 3 (−3, 8) \[[@B38]\]
^a^ Mean with minimum -- maximum in parentheses (μg/m^3^). One number in parentheses is standard deviation.
^b^ Spatial scale of exposure assignment, in parentheses exposure assignment method. City = average of monitors within the city; Near = nearest monitor concentration; LUR = land use regression; Disp = dispersion modeling; Inter = interpolation.
Studies adjusted for individual smoking except references \[[@B26],[@B28],[@B30],[@B34],[@B38],[@B56]\].
Conclusions
===========
There is a significant number of new studies on long-term air pollution exposure, covering a wider geographic area, including Asia. These recent studies support associations found in previous cohort studies on PM~2.5~. The pooled effect estimate expressed as excess risk per 10 μg/m^3^ increase in PM~2.5~ exposure was 6% (95% CI 4, 8%) for all-cause and 11% (95% 5, 16%) for cardiovascular mortality. Long-term exposure to PM~2.5~ was more associated with mortality from cardiovascular disease (particularly ischemic heart disease) than from non-malignant respiratory diseases (pooled estimate 3% (95% CI −6, 13%)). Significant heterogeneity in PM~2.5~ effect estimates was found across studies, likely related to differences in particle composition, infiltration of particles indoors, population characteristics and methodological differences in exposure assessment and confounder control. All-cause mortality was significantly associated with elemental carbon (pooled estimate per 1 μg/m^3^ 6% (95% CI 5, 7%)) and NO~2~ (pooled estimate per 10 μg/m^3^ 5% (95% CI 3, 8%)), both markers of combustion sources. There was little evidence for an association between long term coarse particulate matter exposure and mortality, possibly due to the small number of studies and limitations in exposure assessment. Across studies, there was little evidence for stronger association among women compared to men. Subjects with lower education and obese subjects experienced larger mortality effect related to fine PM, though the evidence for differences related to education has been weakened in more recent studies.
Our review suggests several specific research questions. Research into the reasons for the heterogeneity of effect estimates would be extremely useful for health impact assessment. Better exposure assessment including spatially resolved outdoor exposures and more chemically speciated PM might in part be able to resolve the observed heterogeneity. Chemical speciation would allow assessing particles from different sources e.g. particles from combustion sources and non-tailpipe emissions separately, a question clearly relevant for air pollution control policy. Specific attention to motorized traffic emissions is important because (road) traffic is an important source of ambient air pollution. More work on coarse particles and at the other side of the particle size spectrum, ultrafine particles is needed. Ongoing new research in the USA in the Multi-Ethnic study of Atherosclerosis and Air pollution (MESA-AIR) and the European Study of Cohorts for Air Pollution Effects (ESCAPE) that use large cohorts and state-of the art spatially-resolved exposure methods will likely contribute significant new answers in the near future to these questions.
Abbreviations
=============
ACS: American Cancer Society study; BS: Black Smoke; BC: Black Carbon; CI: Confidence interval; EC: Elemental Carbon; NO2: Nitrogen dioxide; NOx: Nitrogen oxides; PM: Particulate matter; PM2.5: Particles smaller than 2.5 μm; PM10: Particles smaller than 10 μm; TSP: Total suspended particles.
Competing interests
===================
None of the authors has a competing interest.
Authors' contributions
======================
GH, RMK, RB, AP, BO, BB and JK have contributed to the definition of the scope of the review, identification of studies and interpretation of results. GH drafted the text. GH, RMK, RB, AP, BO, BB and JK provided critical comments and approved the final manuscript.
Acknowledgments
===============
The paper has been prepared in the framework of the projects ESCAPE and TRANSPHORM. The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2011) under grant agreement number: 211250.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#sec1-1}
============
Aphallia is absence of penis in a genotypic male. It is due to failure of development of genital tubercle during embryonic life. The newborn with aphallia presents as a devastating social emergency to parents and family. These are genetic males with normal testes and scrotum. There is absence of penis and urethral opening is located in the anterior rectal wall in the majority of cases. Phallic reconstruction using the free radial forearm flap (RFF) or the pedicled anterolateral thigh flap (ALTF)\[[@ref1]\] has been routinely used in penile inadequacy or in female-to-male transsexuals. However, these reconstructions are carried out around puberty. Even if any reconstruction in infancy is carried out successfully, it is likely to cause encumberance during childhood and a further source of social embarrassment. We describe a novel technique as a temporizing procedure which would see the patient through childhood.
CASE REPORT {#sec1-2}
===========
We describe 2 patients who presented at the age of 9 months and 1 year with penile agenesis. Both were passing urine and stools per rectum. The physical examination revealed complete absence of a phallus, a well-developed scrotum and normal sized testes. The urethral opening was located on the anterior rectal wall, about 2 cm. from the anal verge. Parents of both the children wanted them to be raised as males. The karyotype confirmed 46XY. The hormonal profile was within normal limits. The spinal radiographs were normal. An ultrasonography revealed normal kidneys and no other associated anomalies. The voiding cystourethrography revealed a short urethra with urethro-rectal communication a short distance beyond the bladder neck. While MRI confirmed the above findings, it also revealed a normal looking urinary bladder and absence of corpora cavernosa and the corpus spongiosum. Urinary bladder was seen to open lower down on the anterior rectal wall through the short posterior urethra ending approximately 2.0 cm from the anal verge. T2 weighted images revealed the prostate gland (typical hyperintensity). There was no additional, detectable structural anomaly.
TECHNIQUE {#sec1-3}
=========
Urethral re-location {#sec2-1}
--------------------
Bowel preparation is done by peglec solution (polyethylene glycol with electrolytes) administered orally for 48 hours before surgery. Under general anesthesia, the child is placed in lithotomy position. Urethral lumen is catheterized easily by inserting a \#8F feeding tube through the anal verge. We use a pre-anal, anterior coronal approach, to detach and mobilize the urethral opening from the anterior rectal wall \[[Figure 1a](#F1){ref-type="fig"}\]. A semilunar incision is given on the perineum, anterior to the anal verge, from 3 O'clock to 9 O'clock. The urethra-easily identified over the feeding tube-is mobilized and detached from the anterior surface of rectum. The rectal end of the opening is closed by a single layer of seromuscular, running, PDS suture.
{#F1}
Scrotal flap-phallic reconstruction {#sec2-2}
-----------------------------------
A quadrangular flap is taken from the anterior scrotal skin and raised with its dartos layer. The two edges of the flaps are folded together and the edges sutured together to create a neophallus \[[Figure 1b](#F1){ref-type="fig"}\]. The previously mobilized urethral opening is re-located in the midline, between the two scrotal halves and fixed to the root of the neophallus \[Figures [2a](#F2){ref-type="fig"} and [b](#F2){ref-type="fig"}\]. The scrotal defect is closed in the midline configuring to a median raphe. There is no tissue loss and the adjoining tissues on the abdominal wall remain undisturbed for any possible future use. Six months later, a buccal mucosal graft can be laid on the undersurface of the neophallus \[[Figure 3a](#F3){ref-type="fig"}\] and later tubularized.
{#F2}
{#F3}
DISCUSSION {#sec1-4}
==========
Aphallia is a rare birth defect seen in 1 in 30 million births. In the past, gender re-assignment was the preferred approach.\[[@ref2]\] However, pre- and postnatal effects of androgens on brain, cause a male-typical shift in terms of psychosocial and psychosexual development, resulting in gender dysphoria later in life. As a result, the current trend is to rear these children as males.\[[@ref3][@ref4]\]
Urethral opening is typically present lower down in the rectum in the post-sphincteric location (87%) and less commonly in the presphincteric or prostatourethral location. Both our cases had the former, typical presentation. Cystography and MRI are useful investigations to look for any associated anomaly as well as defining the anatomy. Cystography, through suprapubic route, is often difficult to perform in un-cooperative children. MRI should be the preferred modality of management as has been first demonstrated by us.\[[@ref5]\]
Phallic reconstruction to treat aphallia is a challenging venture. Available procedures are recommended for older children\[[@ref6]\] or after puberty. The development of microsurgical free-flap techniques made the first microsurgical phalloplasty possible using a free radial forearm flap.\[[@ref1][@ref7][@ref8]\] However, there are several problems with early phalloplasty, such as, lack of further growth and the large neo-phallus being an encumberance to the young child. The microvascular techniques are more demanding at a young age and any tissue utilized earlier would hinder, any later age appropriate surgery. Also, none of these procedures provide erectile tissue. They are, anatomical phallic configurations in which, at best, penile implants could be inserted.
At the same time, postponing surgery till post-pubertal period adds to parental anxiety, affects psychosocial development of the child who continues to pass urine per rectum. Therefore, a temporizing procedure is necessary which may see the child through childhood \[[Figure 3b](#F3){ref-type="fig"}\]. Scrotal phalloplasty, using scrotal skin to reconstruct a temporary phallus, can be performed easily. Urethro-rectal disconnection is easily performed by our pre-anal anterior coronal approach. It provides an age-appropriate phallic appearance and does not disturb the tissues which may be required for post-pubertal phalloplasty.
Therefore, scrotal phalloplasty serves as a temporizing procedure which may see the child through early school going years. Adding buccal mucosal graft allows urethral reconstruction and thus enables the child to micturate while standing. As he approaches teenage the other already available procedures\[[@ref1][@ref7][@ref8]\] could be introduced with ease, as, scrotal phalloplasty does not encroach on tissues used for any future surgeries.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
| {
"pile_set_name": "PubMed Central"
} |
A male newborn was delivered vaginally at 38^5/7^ weeks. Pregnancy was uneventful, and no fetal anomalies were detected at prenatal ultrasound controls. Maternal serologies for HbsAg, HCV, HIV, Toxoplasma, and Rubella were negative, as was vaginal swab. Delivery was not complicated and Apgar Score was 9 at 1st, 9 at 5th, and 10 at 10th minute of life. The neonate presented at birth with a genitourinary defect consistent with the classic bladder exstrophy variant of the exstrophy--epispadias complex (EEC). Exposed, everted bladder template was clearly visible immediately below umbilical stump; a completely dorsally opened (epispadic) urethral plate run from bladder neck down to the open glans; left and right corpora cavernosa were clearly visible beneath and alongside urethral plate; the scrotum was normally developed, but caudally displaced; anus was normal ([Fig. 1](#FI150005-1){ref-type="fig"}). Physical examination was otherwise unremarkable. The defect was covered in delivery room with sterile silicon gauzes and transparent waterproof dressing. Prudently, no umbilical vascular catheters were positioned at birth. Cerebral, cardiac, abdominal, and kidney ultrasound were then performed, turning out to be normal. Pelvic ultrasound esteemed a pubic symphysis diastasis of 28 mm. At 72 hours of life, primary closure of bladder, pelvis, and abdominal wall was performed successfully, without pelvic osteotomy. Two ureteral catheters, one transurethral catheter and an epicystostomy tube were left indwelling. In the first 5 postoperative days, the patient was kept immobilized with pelvis and lower limbs wrapped around and suspended in a special hammock device (modified Bryant traction) ([Fig. 2](#FI150005-2){ref-type="fig"}).[@JR150005-1] Parenteral nutrition was administered during this period. Curarization was maintained for the first 5 days after surgery; systemic sedoanalgesia was gradually tapered, and ultimately stopped 10 days after surgery. Full enteral feeding was restored at day 10 postoperatively. A perirenal urinoma was registered as a postoperative complication. Tubes were sequentially removed during the fourth postoperative week, and patient was discharged on postoperative day 28. Patient was kept in a thoracopelvic orthosis for the first 2 month of life.[@JR150005-2] Further surgery for epispadias repair is scheduled at the age of 9 months of life.
{#FI150005-1}
{#FI150005-2}
Discussion
==========
What is Exstrophy--Epispadias Complex
-------------------------------------
EEC encompasses a spectrum of pathologies going from isolated epispadias, to classic bladder exstrophy, to cloacal exstrophy as the most severe---and rarest--presentation.[@JR150005-3] Next to the bladder, the malformation involves the genitalia, the abdominal wall muscles, the pelvic floor musculature, and the bony pelvis.[@JR150005-4]
Epidemiology
------------
The overall incidence of EEC has been estimated by Nelson et al[@JR150005-5] in 2.15 per 100,000 live births, with an even male-to-female ratio (odds ratio, 0.989; 95% confidence interval 0.88--1.12), and a significantly increased incidence in white compared with nonwhite neonates (incidence, 2.63 vs. 1.54 per 100,000; *p* \< 0.0001). Classic bladder exstrophy occurs in 1:10,000 to 1:50,000 live births[@JR150005-6]; epispadias is estimated to occur in 1:117,000 live births, and cloacal exstrophy in 1:250,000 births.[@JR150005-7]
Embryology and Associated Conditions
------------------------------------
EEC derives from a derangement in mesodermal layers fusion during the first weeks of fetal life. Normally, at the end of third week of gestation, intermediate layer of mesoderm starts to invaginate to give origin to the urogenital system, while the lateral plate mesoderm will contribute in forming the primitive gut tube.[@JR150005-3] A disruption in this interaction, possibly related to a cloacal membrane overgrowth preventing medial migration of mesenchymal tissue, is reported to give origin to EEC[@JR150005-8]; severity of the resulting condition depends on the point at which disturbed mesodermal layers interaction begins.
Given the embryological origin of the disorder, EEC is often associated with other peculiar orthopedic, musculocutaneous, and gynecological conditions. Associated upper urinary tract anomalies are rare. Gastrointestinal and spinal/neurological anomalies can be associated in patients with cloacal exstrophy ([Table 1](#TB150005-1){ref-type="table"}).[@JR150005-3] [@JR150005-4] [@JR150005-9]
###### EEC---commonly associated conditions
---------------------------------------------------------------------------------
Urological
Stenosis/obstruction of the ureteropelvic junction
Vesicoureteral reflux
Ectopic kidney
Horseshoe kidney
Renal dysplasia/agenesis
Megaureter
Ureteral ectopy
Ureterocele
Musculocutaneous
Abdominal wall defects
Divergent distal rectus abdominis muscles
Umbilical hernia
Spinal/neurological
Neural tube defects
Vertebral anomalies
Myelodysplasia and/or myelomeningocele
Dysraphism
Tethered cord
Orthopedic
Clubfoot deformities
Absence of feet
Tibial or fibular deformities
Hip dislocations
Pubic symphysis gap
Open-book configuration of the pelvis
Gastrointestinal
Common hindgut remnant
Anteriorly displaced anus
Imperforate anus
Rectal stenosis
Rectal prolapse
Omphalocele
Gastrointestinal malrotation/duplication
Short bowel syndrome
Duodenal atresia
Small short bowel deletion
Gynecological
Vaginal/uterine prolapse
Müllerian anomalies (e.g., vagina and/or uterus duplication, vaginal agenesia)
---------------------------------------------------------------------------------
Abbreviation: EEC, exstrophy--epispadias complex.
Medical and Surgical Management
-------------------------------
Immediate medical management of EEC consists of covering the extruding viscera with sterile silicon gauzes plus a surmounting occlusive dressing to prevent air contact and dehydration of the exstrophic plaque. Umbilical catheters should not be positioned. No prophylactic antibiotic therapy is mandatory at birth if no physical signs of infection are detectable; on the contrary, postoperative prophylactic antibiotic treatment is advisable to avoid potential postoperative complications (see section "Long-Term Complications and Outcome").
Surgical correction varies depending on the type and severity of the defect; most of the neonates, however, will need closure of the bladder and abdominal wall, repair of epispadias, ureteral reimplantation, and bladder neck repair. Some of them will also require pelvic osteotomy to facilitate relaxation of the abdominal wall during closure. Osteotomy might possibly also improve functional outcomes of genitourinary reconstruction.[@JR150005-10] Criteria proposed to select cases requiring an osteotomy include patients undergoing closure after 72 hours of life, when the pelvis becomes more stiff, those with a pubic diastasis wider than 4 cm, and those with a nonmalleable pelvis. Under these circumstances, the osteotomy is performed at the same time as exstrophy closure (i.e., combined pelvic osteotomy).[@JR150005-11] In patients with extremely wide pubic diastases (\> 6 cm, most often associated with cloacal exstrophy), a strategy involving osteotomy before bladder closure (i.e., staged pelvic osteotomy) has been proposed to allow gradual reduction in diastasis with slow stretching of pelvic soft tissue.[@JR150005-12]
Postoperatively, management key factors for the success of closure include immobilization and traction of the lower limbs, appropriate urinary drainage, curarization, analgesia (also using epidural catheters when possible), broad-spectrum antibiotic prophylaxis, and parenteral nutrition.
Currently, a staged approach is the strategy most commonly used. Alternatively, the neonatal primary complete repair and the deferred primary complete repair have been proposed.[@JR150005-13] [@JR150005-14] In the standard-staged repair, a primary closure of the bladder without osteotomy, and without genital reconstruction in males, is attempted in the first 72 hours of life; between 6 and 12 months of age, the epispadias repair is performed in males. Bladder neck reconstruction follows around the age of 5 years, if a reasonable bladder capacity is reached.
Patients whose bladders fail to grow before bladder neck reconstruction (to at least 100 mL of capacity), or fail keep growing after bladder neck reconstruction thereby causing persistent incontinence or upper urinary tract deterioration, are candidate to undergo augmentation cystoplasty. A segment of sigmoid colon or ileum can be used to augment the bladder.[@JR150005-15]
Augmented bladders generally lose the ability to empty volitionally to completion; therefore, periodical clean intermittent catheterizations become necessary. As the reconstructed urethra is unreliable for catheterization, bladder augmentation is combined with placement of a catheterizable conduit bridging the bladder to the skin. The conduit can be created using the appendix (i.e., Mitrofanoff appendicovesicostomy) or a 2 to 3 cm long segment of ileum (i.e., Monti ileovesicostomy) and should be patent to a 12--14 Ch tube.[@JR150005-16] The most common complications include stomal stenosis and urinary leakage via the conduit.[@JR150005-17] The appendix is generally less keen than is the ileal tissue to develop complications.[@JR150005-18]
Long-Term Complications and Outcome
-----------------------------------
Modern-staged repair of the EEC as previously described has shifted the goal from patient\'s survival to quality of life. The single most important outcome in bladder exstrophy repair is probably urinary continence,[@JR150005-18] which is more easily achieved with successful primary bladder closure and good bladder growth.[@JR150005-19] The latter occurs as an adaptation of the bladder wall to the increase in bladder outlet resistances after bladder closure and after bladder neck reconstruction. Of note, this increase in resistances should never happen at the cost of upper urinary tract deterioration, which may occur in case of poorly compliant bladders with incomplete emptying.[@JR150005-20] Reportedly, up to 80% of cases can achieve a socially acceptable urinary continence (3-hour dry interval during daytime and volitional voiding) using this surgical strategy.[@BR150005-21] Nevertheless, many series report that 20 to 50% of cases will require bladder augmentation eventually.[@JR150005-15]
Genital function keeps being a relevant problem in patients with exstrophy long term. Males can experience problems for the presence of a short penis or persistent penile curvature.[@JR150005-22]
Excessive bladder-neck tapering may cause seminal obstruction and recurrent epididymitis leading to infertility. Female patients can experience problems because of the abnormal genital appearance, stenosis of the vaginal introitus, and pelvic organ prolapse.[@JR150005-23] These problems, however, do not generally impair a good quality of life.[@JR150005-24]
Although patients with exstrophy have almost invariably an abnormal gait in the long term because of the recurrence of the pelvic diastasis, which invariably recurs also in patients undergoing osteotomy, long-term orthopedic complications are rare.[@JR150005-25] Patients receiving an osteotomy may experience transient of persistent nerve damage, hip pain, and unequal length of limbs.[@JR150005-26]
Conclusion
==========
Reportedly, modern-staged repair of EEC can achieve a socially acceptable urinary continence (3-hour dry interval during daytime and volitional voiding) in up to 80% of cases after successful primary closure[@BR150005-21]; sexual function can be an issue in the long term (particularly in males),[@JR150005-22] [@JR150005-23] but overall quality of life can be good.[@JR150005-24]
**Conflicts of Interest** None.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Galactosylceramide (GalCer) is the most abundant sphingolipid of mammalian myelin \[[@B1]\]. It is synthesized in the endoplasmic reticulum by UDP-galactose:ceramide galactosyltransferase (encoded by the *Cgt*gene). In the Golgi apparatus, part of the GalCer is sulfated by cerebroside sulfotransferase (encoded by the *Gal3st1*gene), forming sulfatide \[[@B2]\] (see Figure [1](#F1){ref-type="fig"}). In the absence of a functional *Cgt*gene, compact myelin can be formed, which is, however, unstable and *Cgt*^-/-^mice develop tremors and ataxia at 3 to 4 weeks of age \[[@B3],[@B4]\]. This phenotype could be explained by disturbed axon-glial contacts at the paranodes in the CNS caused by mistargeting of essential adhesion molecules, NF-155 and Caspr \[[@B5],[@B6]\]. At least in part, these structural alterations are caused by the loss of sulfatide rather than GalCer, as demonstrated by a similar alteration of the paranodal region in *Gal3st1*-deficient mice, which lack sulfatide but have normal GalCer levels \[[@B7]\]. However, myelin appear to be more stable in *Gal3st1*-deficient mice, suggesting additional, yet less defined roles of GalCer in myelin \[[@B8]\].
{#F1}
In *Cgt*^-/-^mice, the loss of GalCer and sulfatide is accompanied by a significant upregulation of 2-hydroxylated fatty acid-containing (HFA) glucosylceramide (HFA-GlcCer) and HFA-sphingomyelin \[[@B3],[@B4]\]. Interestingly, there are no indications for presence of HFA-gangliosides or other higher glycosylated HFA-sphingolipids in the brain of *Cgt*^-/-^mice \[[@B9]\]. The presence of HFA-GlcCer and HFA-sphingomyelin was interpreted as a compensatory upregulation, which may also (in part) functionally replace HFA-GalCer, enabling *Cgt*^-/-^mice to form compact myelin \[[@B3],[@B10]\].
HFA-sphingolipids in CNS and PNS myelin are synthesized from 2-hydroxylated fatty acids, formed by the fatty acid 2-hydroxylase (encoded by the *Fa2h*gene) \[[@B11]-[@B14]\]. Although loss of 2-hydroxylated sphingolipids in myelin does not affect initial myelin formation, it causes late onset (in mice older than 6 months) axon and myelin sheath degeneration \[[@B15]\]. *Fa2h*-deficiency was identified as the cause of a new leukodystrophy with spastic paraparesis \[[@B16]\], and hereditary spastic paraplegia SPG35 \[[@B17]\], respectively.
In order to test the hypothesis that the presence of HFA-GlcCer and HFA-sphingomyelin in *Cgt*^-/-^mice is a functional important compensatory upregulation, preventing a more severe phenotype, we generated *Cgt*^-/-^mice with an additional deficiency in the *Fa2h*gene. Our analysis shows that the additional deletion of *Fa2h*does not obviously affect the phenotype of *Cgt^-/-^*mice. This suggests that HFA-GlcCer and HFA-sphingomyelin do not functionally compensate the loss of HFA-GalCer in *Cgt^-/-^*mice.
Results
=======
Generation of *Fa2h*^-/-^*/Cgt*^-/-^double deficient mice
---------------------------------------------------------
In order to test the hypothesis that HFA-GlcCer partially compensates the loss of HFA-GalCer in *Cgt*^-/-^mice and thereby prevents a more severe phenotype, we generated *Cgt*^-/-^mice with an additional deficiency in *Fa2h*. As shown previously, young *Fa2h*^-/-^mice form structural and functional normal myelin \[[@B15]\] and did not show behavioral abnormalities that would indicate myelin deficiency. Older *Fa2h*^-/-^, however, developed a progressive axonal degeneration in peripheral nerves and brainstem, accompanied by myelin sheath degeneration \[[@B15]\]. As shown before \[[@B3],[@B4]\], *Cgt*^-/-^mice had a strongly reduced life span, whereas *Fa2h*^-/-^mice did not show increased mortality (data not shown). Survival of *Fa2h*^-/-^/*Cgt*^-/-^double deficient mice was not significant different from *Cgt*^-/-^mice (around 50% survival at four weeks of age). There were no obvious behavioral differences between the two genotypes. However, because of the low amount of age-/weight- and gender-matched 4-week-old *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice available, extensive behavioral testing could not be performed, and thus minor behavioral differences between *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice cannot be ruled out. The following biochemical and morphological analyses were done with mice at 4 weeks of age.
GlcCer levels are reduced in CNS and PNS of *Fa2h*^-/-^/*Cgt*^-/-^mice when compared to *Cgt*^-/-^mice
------------------------------------------------------------------------------------------------------
TLC analysis of total brain lipids from wild-type, *Fa2h*^-/-^, *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice showed significant levels of HFA-GlcCer in *Cgt*^-/-^mice, in line with earlier reports \[[@B3],[@B4]\]. Unexpectedly, NFA-GlcCer levels in total brain of *Fa2h*^-/-^/*Cgt*^-/-^mice were strongly reduced compared to HFA-GlcCer levels in *Cgt*^-/-^mice (Figure [2A](#F2){ref-type="fig"}). Presence of NFA-GlcCer in *Fa2h*^-/-^/*Cgt*^-/-^mice was better visible when the amount of lipids from these mice loaded was increased 4-fold compared to controls (Figure [2B](#F2){ref-type="fig"}). A similar reduction in GlcCer levels was seen when sphingolipids of purified CNS myelin were examined (Figure [3A](#F3){ref-type="fig"}). Densitometry revealed a reduction of NFA-GlcCer by more than 80% compared to HFA-GlcCer levels in *Cgt*^-/-^mice (Figure [3C](#F3){ref-type="fig"}). Analysis of peripheral nerves (sciatic nerve) revealed a reduction of GlcCer in *Fa2h*^-/-^/*Cgt*^-/-^mice by about 60% compared to *Cgt*^-/-^mice (Figure [3B, D](#F3){ref-type="fig"}). MALDI-TOF mass spectrometry of hexosylceramides from *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice (CNS myelin) demonstrated that cerebrosides in both genotypes mainly contained very long chain fatty acyl (VLCFA; C22:0, C24:0, and C24:1) residues (data not shown). The TLC analysis also revealed a strong increase in the very-long chain fatty acid (VLCFA)-containing sphingomyelin (SM; upper band) in *Fa2h*^-/-^/*Cgt*^-/-^, but also in *Fa2h*^-/-^/*Cgt*^+/-^mice, when compared to *Cgt*^-/-^and wild-type mice (note that *Fa2h*^-/-^mice heterozygous for Cgt were used as a control in these experiments, because the number of *Fa2h*^-/-^/*Cgt*^+/+^mice were limited). Densitometry confirmed a 2-fold increase of sphingomyelin in CNS myelin of *Fa2h*^-/-^/*Cgt*^-/-^mice compared to wild-type controls (and 60% increase in *Fa2h*^-/-^/*Cgt*^+/-^mice; *Fa2h*^-/-^were not analyzed) (Figure [3E](#F3){ref-type="fig"}). The increase of sphingomyelin in the PNS was about 50% in *Fa2h*^-/-^/*Cgt*^-/-^mice (Figure [3F](#F3){ref-type="fig"}).
{#F2}
{#F3}
In line with data published by Bosio et al. \[[@B18]\], HFA-ceramide was detectable in *Cgt*^-/-^mice but not in mice of other genotypes (Figure [4A](#F4){ref-type="fig"}). However, in contrast to the 18-day-old *Cgt*^-/-^mice analyzed by Saadat et al. \[[@B19]\], we did not observe a strong increase of ceramide in 4-week-old *Cgt*^-/-^mice compared to wild-type controls. This might be due to the different ages examined.
{#F4}
To examine the possibility that GlcCer was replaced by gangliosides in myelin of *Fa2h*^-/-^/*Cgt*^-/-^mice, gangliosides were isolated and analyzed by TLC. Although the total amount of gangliosides in the myelin fraction of *Fa2h*^-/-^/*Cgt*^-/-^and *Cgt*^-/-^mice was increased compared to wild-type, the major myelin ganglioside GM1 was unchanged and only gangliosides normally found in neuronal membranes (GD1a, GD1b, GT1b) were increased (Figure [4B](#F4){ref-type="fig"}). Most likely these gangliosides are derived from neuronal membranes and possibly reflect a higher proportion of neuronal membrane contaminations in the *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice, with their significant reduced amounts of compact myelin. In line with this, Saadat et al. \[[@B19]\] showed a similar relative composition of major gangliosides in myelin preparation from mice deficient in *Ugcg*in myelinating cells. We therefore believe that myelin ganglioside levels in *Fa2h*^-/-^/*Cgt*^-/-^mice were not increased compared to *Cgt*^-/-^mice or wild-type controls. In summary, we conclude that HFA-GlcCer present in *Cgt*^-/-^CNS myelin was replaced by only low amounts of NFA-GlcCer but larger amounts of NFA-sphingomyelin in *Fa2h*^-/-^/*Cgt*^-/-^mice. Taken together, these results indicate that myelin hexosylceramides were mainly replaced by sphingomyelin in the absence of *Fa2h*expression. It should be noted that the increase of sphingomyelin and the decrease of hexosylceramides were more pronounced in CNS myelin than in the PNS. This might indicate differences between oligodendrocytes and Schwann cells in the trafficking or metabolism of sphingolipids.
Myelination in *Fa2h*^-/-^/*Cgt*^-/-^mice as compared to *Cgt*^-/-^mice
-----------------------------------------------------------------------
Myelin content in the brain of *Cgt*^-/-^, *Fa2h*^-/-^/*Cgt*^-/-^, and wild-type mice was examined by Western blot analysis of myelin basic protein (MBP) and by gravimetry of purified compact myelin. At the examined time point (4 weeks of age), MBP levels were reduced in *Cgt*^-/-^mice compared to wild-type controls (Figure [5A](#F5){ref-type="fig"}), in agreement with our previous observations \[[@B20]\]. A comparable reduction of MBP was found in *Fa2h*^-/-^/*Cgt*^-/-^mice. Accordingly, the amount of compact myelin isolated by sucrose gradient centrifugation was significant reduced in both, *Fa2h*^-/-^/*Cgt*^-/-^and *Cgt*^-/-^mice, compared to wild-type controls (Figure [5B](#F5){ref-type="fig"}), but there was no significant difference between the first two genotypes.
{#F5}
Absence of GalCer and HFA-sphingolipids does not affect stability of CHAPS-insoluble membrane fractions
-------------------------------------------------------------------------------------------------------
Compact myelin of wild-type mice is stable against extraction with the detergent CHAPS at low \[[@B21]\] and high temperature \[[@B15]\]. Data by Simons et al. \[[@B21]\] suggest that loss of HFA-GalCer in *Cgt*^-/-^mice affects the association of the myelin proteolipid protein (PLP) with CHAPS-insoluble membrane fractions (CIMF), suggesting a role for (HFA-)GalCer in the formation or stabilization of CIMF. We have recently shown that the absence of HFA-sphingolipids does not affect stability of myelin CIMF \[[@B15]\]. However, in order to examine a possible synergistic effect of *Fa2h*and *Cgt*deficiency, purified myelin of *Fa2h*^-/-^/*Cgt*^-/-^mice was subjected to CHAPS extraction at 37°C. Optiprep gradient centrifugations were performed and the fractions were examined for sphingolipid content, as described \[[@B15]\]. These experiments showed that myelin was resistant to CHAPS extraction irrespective of the genotype (Figure [6](#F6){ref-type="fig"}). Therefore, we conclude that neither GalCer nor HFA-sphingolipids are essential for stabilization of CIMF.
{#F6}
Comparable myelination in *Fa2h*^-/-^/*Cgt*^-/-^and *Cgt*^-/-^mice
------------------------------------------------------------------
To evaluate the extent of myelin sheaths thickness, cross sections were obtained from the cervical spinal cord of 4-week-old mice. On each side of the midline, starting at the deep medial boundary of the ventral funiculus and extending ventrally and laterally, fibers of all calibers were included. In this unbiased sampling approach, the number of myelinated axons was unchanged in *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice (179 ± 19 versus 194 ± 30). Only few axons in both mouse mutants displayed no myelin at this postnatal stage (Figure [7A](#F7){ref-type="fig"}). The mean axon diameter did not differ significantly between *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice (3.88 μm versus 3.77 μm). The axons of *Fa2h*^-/-^/*Cgt*^-/-^mice recruit inappropriately thick myelin relative to their absolute calibers resulting in an average myelin thickness of 0.60 ± 0.02 μm versus 0.53 ± 0.01 μm in *Cgt*^-/-^(p \< 0.01, Chi-Square test, n = 3) (Figure [7B](#F7){ref-type="fig"}). However, due to the relative small difference in myelin thickness, the g-ratios did not differ significantly between the two genotypes (Figure [7C](#F7){ref-type="fig"}). Furthermore, electron microscopy of myelinated axons revealed normal compact myelin in *Fa2h*^-/-^/*Cgt*^-/-^mice (Figure [7D](#F7){ref-type="fig"}). These results demonstrate that a compact myelin sheath can be generated in the absence of GalCer and any 2-hydroxylated sphingolipids.
![**Normal lamellar spacing in *Fa2h*^-/-^/*Cgt*^-/-^myelin**. (A) Typical cross sections of spinal cords from *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice (two different magnifications are shown). Arrows indicate axons with thin or without myelin. Asterisks indicate demyelinated axons. Scale bar, 40 μm (upper panel) 10 μm (lower panel). Myelin thickness (B) and g-ratios (C) were determined in the spinal cord of three mice per genotype. Though there was a shift towards increased myelin thickness in *Fa2h*^-/-^/*Cgt*^-/-^compared to *Cgt*^-/-^mice (Data shown are the mean SD \[n = 3 animals per genotype\]; \*\*p \< 0.01, Chi-Square test), the g-ratios of myelinated axons were similar in *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice. (D) Electron micrographs showing normal compact myelin in *Fa2h*^-/-^/*Cgt*^-/-^mice (Magnification: 16,000×). Scale bar, 50 nm.](1471-2202-12-22-7){#F7}
No obvious structural differences between oligodendrocytes of *Fa2h*^-/-^/*Cgt*^-/-^and *Cgt*^-/-^were observed by the histological or electron microscopic analyses, however, a detailed structural analysis was not performed, and therefore we cannot exclude subtle structural changes in *Fa2h*^-/-^/*Cgt*^-/-^when compared to *Cgt*^-/-^mice (e.g. in the paranodal region).
Discussion
==========
Although HFA-sphingolipids appear to be dispensable for the formation of compact myelin \[[@B15]\], they are essential for long-term myelin maintenance and may also play a role in glia-dependent axonal support. In the absence of GalCer in *Cgt*^-/-^mice, compact myelin can be formed \[[@B3],[@B4]\], which is, however, unstable, also suggesting a role of GalCer in myelin maintenance. We hypothesized that HFA-sphingolipids may also play a more subtle role in early postnatal development, which could be detectable on a *Cgt*^-/-^background, where compact but less stable myelin is formed. However, we did not observe signs of a more severe phenotype in *Fa2h*^-/-^/*Cgt*^-/-^mice compared to *Cgt*^-/-^mice. At the behavioral level, *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice were indistinguishable. Though there was a shift towards thicker myelin in *Fa2h*^-/-^/*Cgt*^-/-^compared to *Cgt*^-/-^mice, the g-ratios did not differ significantly between *Cgt*^-/-^and *Fa2h*^-/-^/*Cgt*^-/-^mice. Furthermore, myelin from *Fa2h*^-/-^/*Cgt*^-/-^and *Cgt*^-/-^mice was resistant towards extraction with CHAPS, as shown before for *Fa2h*^-/-^and wild-type mice \[[@B15]\].
Unexpectedly, HFA-GlcCer present in *Cgt*^-/-^mice was replaced by only low amounts of NFA-GlcCer in *Fa2h*^-/-^/*Cgt*^-/-^mice but higher levels of NFA-sphingomyelin. This suggests that the GlcCer concentration in myelin is not critical and furthermore that elevated GlcCer levels in *Cgt*^-/-^mice do not functionally compensate loss of GalCer. Accordingly, Saadat et al. \[[@B19]\] showed that deleting oligodendroglial glucosylceramide synthase (*Ugcg*) in *Cgt*^-/-^mice did not reinforce the myelin phenotype. This demonstrates that upregulation of (HFA)-GlcCer in *Cgt*^-/-^mice does not functionally compensate loss of GalCer in these mice. In *Ugcg*-deficient *Cgt*^-/-^mice, HFA-GlcCer was partially replaced by HFA-sphingomyelin \[[@B19]\]. Our results demonstrate that HFA-sphingomyelin can be replaced by NFA-sphingomyelin without any obvious effect on the phenotype of *Cgt*^-/-^mice. Thus, the significant up-regulation of HFA-sphingomyelin in *Cgt*^-/-^mice is not a functional compensation.
The reason for the much lower NFA-GlcCer level in CNS myelin of *Fa2h*^-/-^/*Cgt*^-/-^mice compared to the HFA-GlcCer level in *Cgt*^-/-^mice is currently unknown. One possible explanation for the high HFA-GlcCer level and absence of HFA-gangliosides \[[@B9]\] in *Cgt*^-/-^mice could be the inability of the responsible glycosyltransferases to use HFA-GlcCer as a substrate. However, presence of a high amount of HFA-gangliosides in other tissues \[[@B22]\] and in tumor cells \[[@B23]\] argues against this. An alternative explanation could be differential sorting of HFA- and NFA-GlcCer, as shown for polarized epithelial cell lines \[[@B24]\], or reduced half-life of HFA-sphingolipids.
Although structural changes at the paranodes of *Cgt*^-/-^mice are clearly caused by sulfatide rather than GalCer deficiency \[[@B7],[@B25]\], myelin appear to be much more stable in young adult *gal3st1*-deficient mice, whereas myelin maintenance is already affected in young *Cgt*^-/-^mice \[[@B8]\], indicating additional function roles of HFA- and/or NFA-GalCer. Taken together, our results and those of Saadat et al. \[[@B19]\] strongly suggest that the upregulation of HFA-GlcCer and HFA-sphingomyelin in *Cgt*^-/-^mice does not functionally compensate the loss of HFA-GalCer in these mice. This, however, does not exclude the possibility that GalCer could be functionally replaced by GlcCer, if the latter would be present at higher concentrations than in *Cgt*^-/-^mice. The HFA-GlcCer concentration in *Cgt*^-/-^mice is clearly below the concentration of HFA-GalCer in wild-type but also in heterozygous *Cgt*^+/-^mice (which show a wild-type phenotype). There might be a relatively high threshold of hexosylceramide concentration in myelin to efficiently fulfill its role in myelin maintenance. This possibility could be tested using transgenic mice overexpressing *Ugcg*under control of an oligodendrocyte specific promoter, which to our knowledge are not available yet. Alternatively, the functional role of HFA- and/or NFA-GalCer may not be taken over by GlcCer or other glycolipids.
Conclusions
===========
Our data indicate that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid, though myelin maintenance is impaired. While the specific role of GalCer in myelin maintenance remains mysterious, our results suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in *Cgt*^-/-^mice does not functionally compensate the loss of HFA-GalCer.
Methods
=======
Mice
----
*Fa2h*^-/-^mice have been described previously \[[@B15]\]. Heterozygous *Cgt*^+/-^mice (kindly provided by Dr. Brian Popko, University of Chicago) were crossed with *Fa2h*^-/-^mice and double heterozygous *Fa2h*^+/-^/*Cgt*^+/-^mice were interbred or bred with *Fa2h*^-/-^/*Cgt*^+/-^mice to obtain mice of all possible nine genotypes. Genotyping was done using tail genomic DNA and the following oligonucleotides. *Fa2h*genotyping: 5\'-GCTCTTCTTCAAGAGCCATCC-3\', 5\'-GTGCTGTACCTCAGCTGGTC-3\'. 5\'-ATTCGCAGCGCATCGCCTTCTATC-3\', PCR products: 1045 bp for wild-type and 685 bp for the targeted allele; *Cgt*genotyping: 5\'-TTACCAAGGAGTTCAGCAAACC-3\', 5\'-CCTCTCAGAAGGCAGAGACATTG-3\', 5\'-TCTGCACGAGACTAGTGAGACG-3\', PCR products: 684 bp for wild-type and 820 bp for the targeted allele. All animal experiments followed internationally recognized guidelines and were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen, Germany.
Lipid extraction and thin layer chromatography
----------------------------------------------
Total lipid extracts were prepared from brains, sciatic nerves, or purified myelin (from 4-week-old animals) according to Bligh and Dyer \[[@B26]\]. In some experiments, phosphoglycerolipids were removed by mild alkaline hydrolysis as described \[[@B27]\]. In order to isolate gangliosides from purified myelin, lipids were extracted as described by Folch et al \[[@B28]\] and the ganglioside containing upper phase was desalted by reversed-phase chromatography using RP-18 columns (Merck, Darmstadt, Germany). Lipids were separated by thin layer chromatography (TLC) in one of the following solvent systems: (1) chloroform/methanol/water (65/25/4) for hexosylceramides and sphingomyelin, (2) chloroform/methanol/acetic acid (190/9/1) for ceramides, and (3) chloroform/methanol/0.22% CaCl~2~(60/35/4) to separate gangliosides. HPTLC silica gel 60 plates (Merck) were used for all experiments. To visualize lipids, HPTLC plates were sprayed with cupric sulfate in aqueous phosphoric acid \[[@B27]\] followed by charring at 180°C for 5 min. Lipids were quantified by densitometry and the GalCer and GlcCer levels were normalized to cholesterol. Data are shown as the mean ± SD and were tested for statistically significant differences by ANOVA with post hoc Fisher\'s least significant difference (LSD) test using the program STATISTICA.
Isolation of myelin
-------------------
Compact myelin was isolated by sucrose gradient centrifugation as described by Norton and Poduslo \[[@B29]\], with minor modifications. Brains were homogenized in water using an Ultra-Turrax tissue homogenizer (IKA-Werke, Staufen, Germany). Aliquots of the homogenates were used for lipid extraction. The residual homogenates were adjusted to 10.5% (w/v) sucrose in 5 mM Tris-HCl (pH 7.4) and overlaid onto 10 ml of 30% (w/v) sucrose in 5 mM Tris-HCl (pH 7.4). After centrifugation (68,000×g, 50 min, 4°C) the enriched myelin fraction was recovered from the 10.5%/30% interphase, resuspended in 5 mM Tris-HCl (pH 7.4) and centrifuged at 68,000×g for 10 min. The resulting pellet was resuspended in 5 mM Tris-HCl (pH 7.4), centrifuged again, and the myelin was further purified by a second sucrose gradient centrifugation, followed by two washing steps in 5 mM Tris-HCl (pH 7.4). The final myelin pellet was resuspended in a small volume of water, lyophilized and stored at -80°C.
Analysis of CHAPS insoluble membrane fractions
----------------------------------------------
CHAPS insoluble membrane fractions (CIMF) of purified myelin were prepared by optiprep density gradient centrifugation of myelin samples treated with 20 mM CHAPS at 37°C, as described previously \[[@B15],[@B21]\]. Six fractions of 350 μl each were removed from the top of the gradient and lipids were isolated from each fraction according to Bligh and Dyer \[[@B26]\], and analyzed by TLC as described above.
MALDI-TOF mass spectrometry
---------------------------
MALDI-TOF MS of hexosylceramides was done as described \[[@B13]\].
Western blotting
----------------
Brain samples were homogenized in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl (TBS), containing 5 mM EDTA and 1 mM PMSF, using an Ultra-Turrax tissue homogenizer (IKA-Werke, Staufen, Germany). Homogenates were centrifuged at 1,000×g for 5 min and the supernatant was mixed with SDS-PAGE sample buffer containing 2-mercaptoethanol. Proteins were separated in 12.5%-polyacrylamide gels and transferred to nitrocellulose membranes by semi-dry blotting. Membranes were stained with rabbit anti-myelin basic protein (MBP; dilution 1:10,000; Millipore, Schwalbach, Germany) and mouse anti-alpha-tubulin (Developmental Studies Hybridoma Bank, University of Iowa). Bound secondary antibodies were detected by enhanced chemiluminescence as described \[[@B30]\]. Protein concentrations were determined with the Bio-Rad DC protein assay (Bio-Rad Laboratories, München, Germany) using bovine serum albumin as standard.
Morphometrical analysis
-----------------------
Axon caliber and myelin thickness were measured on toluidine-stained semithin sections of the cervical spinal cord of 4-week-old mice. Axonal caliber was determined by the diameter of a circle of area equivalent to each axon. The g-ratio was determined by dividing the diameter of the axon by the diameter of the fiber (axon with myelin). Quantification of myelinated axons was performed with a semi-automatic program on the basis of AnalySIS using a light microscope (100× objective, BX60, Olympus). All morphometric measurements were conducted in a blinded manner using coded sections. Ultrathin sections (50 nm) were photomicrographed with an EM10 electron microscope (Zeiss, Germany) at a magnification of 16.000×.
Authors\' contributions
=======================
MM carried out the animal work, performed lipid and Western blot analyses and CIMF experiments. JJ carried out the morphometrical analyses and participated in writing the draft manuscript. CG participated in the design and coordination of the study. VG participated in the design and coordination of the study. ME conceived and designed the study, wrote the draft manuscript, and participated in the lipid and Western blot analyses. All authors read and approved the final manuscript.
Acknowledgements
================
This work was supported by the Deutsche Forschungsgemeinschaft through SFB645 of the University of Bonn. The authors thank Dr. Brain Popko for providing the *Cgt*^-/-^mouse line and Dr. Andreas Ratzka for help with preparing the final version of the manuscript.
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"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
============
In everyday life, distraction can easily interfere with goal-directed behavior, limiting humans' ability to stay focused on task-relevant information (Eltiti et al. [@CR23]). Previous literature demonstrated that emotional events (mainly negative) have a privileged access to visual awareness because they tend to capture attention and processing resources in a bottom-up fashion (Vuilleumier [@CR75]; Yiend [@CR80]). For the same reason, negative stimuli have been shown to be particularly successful in distracting people from their current goal (Anticevic et al. [@CR2]; Dolcos et al. [@CR21]; Dolcos and McCarthy [@CR19]; Iordan and Dolcos [@CR37]; Wessa et al. [@CR77]). Several studies reported evidence that negative stimuli are hard to be ignored, even when the emotional valence of the stimulus is entirely irrelevant to the current task (Fenker et al. [@CR25]; Huang et al. [@CR35]; Pessoa et al. [@CR57]; Vuilleumier et al. [@CR76]; Ziaei et al. [@CR82], [@CR83]).
The neural mechanisms that allow for a prioritized processing of task-relevant negative information are thought to involve direct subcortical pathways that reach the amygdala (for reviews, see Pessoa and Adolphs 2010; Vuilleumier [@CR75]) and the insula (Menon and Uddin [@CR47]; Uddin [@CR70]; Zaki et al. [@CR81]). While the role of the amygdala in emotional processing has been suggested since a long time (MacLean [@CR44]; see also Pessoa [@CR55]), recent models emphasize the crucial role played by the insula as an emotional "hub" (Menon and Uddin [@CR47]; Uddin [@CR70]; Wessa et al. [@CR77]; Zaki et al. [@CR81]). This para-limbic region has been demonstrated to derive information about bodily states, and, subsequently, to play a crucial role in the experience of emotions (Critchley and Harrison [@CR16]; Zaki et al. [@CR81]). The insula would detect behaviorally relevant stimuli and coordinate high-level neural resources through extensive anatomical and functional connections with the rest of the brain (Menon and Uddin [@CR47]; Uddin [@CR70]). This region has been shown to have intrinsic functional connectivity with large-scale brain networks such as the dorsal frontoparietal network and the default mode network (e.g., Seeley et al. [@CR66]; Sridharan et al. [@CR67]; Uddin et al. [@CR71]). For these reasons, the insula has been recently described as the core structure of the so-called "saliency network" (Uddin [@CR70]), a brain system devoted to prioritize processing of potentially relevant information (Desimone and Duncan [@CR18]; Itti et al. [@CR39]), with implications for the allocation of spatial attention (e.g., Gottlieb et al. [@CR31]; Nardo et al. [@CR49]) and working memory (WM) encoding (e.g., Fine and Minnery [@CR26]; Melcher and Piazza [@CR46]; Pedale and Santangelo [@CR53]; Santangelo and Macaluso [@CR61]; Santangelo et al. [@CR62]; see, for a review, Santangelo [@CR60]). Overall, both limbic and para-limbic regions (namely the amygdala and the insula) are thought to automatically activate in the presence of negative stimuli and to modulate the activity of key regions involved in sensory processes (e.g., the primary visual cortex; Vuilleumier [@CR75]) and high-level post-perceptual processes, such as top-down attentional control (e.g., the dorsolateral prefrontal cortex; Uddin [@CR70]), short- and long-term memories (e.g., the medial temporal lobe and the hippocampus; Dolcos et al. [@CR20]), and decision-making (e.g., the orbital frontal cortex; Bechara et al. [@CR4]).
The impact of "emotional distraction" on goal-directed behavior has been primarily studied in the context of WM tasks, for example, by presenting task-irrelevant negative stimuli while the participants have to maintain previously encoded information (Anticevic et al. [@CR2]; Dolcos et al. [@CR21]; Dolcos and McCarthy [@CR19]; Iordan and Dolcos [@CR37]). Using this design, Dolcos and McCarthy ([@CR19]) reported that the presentation of negative stimuli during the WM maintenance phase evoked increased activity in emotional-related areas, namely the amygdala and the ventrolateral prefrontal cortex. Concurrently, the authors observed a decrement of activation in working memory-related areas, such as the dorsolateral prefrontal cortex and the lateral parietal cortex. The latter imaging effect correlated with a concurrent behavioral decrement of WM performance. In the same year, Dolcos and colleagues (2006) also reported evidence that emotional-related areas, such as the amygdala, showed increased functional connectivity with the inferior frontal cortex---a well-known area involved with general inhibitory processes (e.g., Aron et al. [@CR3])---when negative distractors were presented during WM maintenance. These findings highlight a tight interplay between ventral "affective" and dorsal "control" regions that shows enhanced coupling to cope with emotional distraction in the context of WM tasks (see, for a review, Iordan et al. [@CR38]).
However, in these previous WM studies, neutral stimuli and emotional distractors were temporally separated, with emotional interference arising in the absence of any simultaneous stimulation. On the contrary, in everyday life, our sensory experience is characterized by a multitude of concurrent stimuli competing among them to access our awareness (Bundesen et al. [@CR10]). Emotionally salient stimuli are thought to have a high probability of winning the competition, affecting the distribution of our attentional resources (Vuilleumier [@CR75]; Yiend [@CR80]). In this sense, visual search tasks could offer an optimal scenario to understand the neural systems that are responsible for the facing of "emotional distraction" on goal-directed behavior during the deployment of visual attention resources. Visual search is an attention task involving an active scan of the environment for a specific target among a number of different distractors. During a visual search task, neutral and emotional objects could be simultaneously presented allowing to test for the efficacy of emotional stimuli in promoting visual attention selection when they are the target to be searched for, or in negatively affecting the capacity to pay attention to other (emotionally neutral) elements when they are task irrelevant (i.e., emotional distraction).
Previous behavioral studies showed that emotional distraction plays a detrimental role on visual search performance (Anderson et al. [@CR1]; Fenker et al. [@CR25]; Hodsoll et al. [@CR34]; Huang et al. [@CR35]). However, as far as we know, no studies have been conducted to investigate the specific neural correlates involved with the avoidance of task-irrelevant emotional stimuli during visual search. Moreover, only few studies directly compared searching for emotional vs. non-emotional targets (i.e., with the emotional item playing a distracting role). These studies reported contrasting results, and none of these investigated the neural correlates involved with these processes. Hodsoll and colleagues ([@CR34]) reported a behavioral study in which participants were asked to search for a female target face among male distracting faces (or vice versa) and judge whether the target face was tilted to the left or to the right. When one of the distractor faces had an emotional expression, the orientation discrimination of the target face was impaired. This suggests that task-irrelevant emotional stimuli can capture attention resources, with a consequent detriment on search performance. Other studies showed, however, opposite findings. Hunt and colleagues (2007) asked participants to make a speeded saccade toward a predefined target among distractors. The valence (happy or angry) and orientation (upright or inverted) of the target and distractors, both consisting in "emoticon" faces, varied. The authors reported that task-irrelevant emotional faces captured oculomotor behavior, thus impairing search of the current target. However, this happened only when the current target was defined by an emotional expression. By contrast, when the participants were asked to search for a neutral feature, such as an upright face among inverted distractors, task-irrelevant emotional faces failed to capture the overt orienting of attention. The authors interpreted these results as an evidence that searching for neutral stimuli in the presence of emotional distractors depends on top-down attention control (e.g., Pessoa et al. [@CR57]), and on the specific task-set related to the current target definition.
Overall, this behavioral literature indicates that under some circumstances emotional stimuli have privileged access to attentional and perceptual processes, while in other conditions efficient top-down regulation can prevent distraction/interference. Here, we conducted an eye tracking--fMRI experiment aimed at investigating---both at behavioral and neuro-physiological level---the interplay between emotional capture and emotional distraction (derived from either negative or positive stimuli), that is, the impact of task-relevant vs. -irrelevant emotional objects in biasing spatial attention selection. With respect to the previous literature, employing very simple and repetitive stimuli (e.g., words: Bradley and Lang [@CR6]; faces: Lundqvist et al. [@CR43]; single visual objects: IAPS, Lang et al. [@CR41]), here we used complex everyday life scenes. Complex scenes involve a large number of discrete elements, thus enhancing stimulus competition and the need of attentional selection (e.g., Henderson [@CR33]; see also Desimone and Duncan [@CR18]). We hypothesized that the interplay between affective and control regions affects the allocation of spatial attention when searching through visual scenes that include emotional stimuli. Moreover, we asked whether any such mechanism of attention control would engage also when distraction derives from positive stimuli or it is rather selective for coping with negative-driven emotional distraction.
During fMRI scanning, we presented participants with pictures depicting everyday scenes. These included an emotional object (either negative or positive) that in half of the trials corresponded to the to-be-searched and judged target. When emotional objects were task irrelevant, subjects were asked instead to search for an emotionally neutral object in the scene. Additionally, we added a baseline condition, consisting of scenes not including any emotional object, which enabled us to measure the behavioral performance and neural correlates of searching for a neutral object in the absence of emotional distraction. At a behavioral level (Behavioral Hypothesis, Beh H 1), we expected a "search benefit" for task-relevant emotional targets compared to neutral targets (Vuilleumier [@CR75]; Yiend [@CR80]). Following the literature on emotional distraction that mainly investigated the effect of "negative" distracting stimuli (Anderson et al. [@CR1]; Anticevic et al. [@CR2]; Dolcos et al. [@CR21]; Dolcos and McCarthy [@CR19]; Hodsoll et al. [@CR34]; Iordan and Dolcos [@CR37]; Wessa et al. [@CR77]; Ziaei et al. [@CR82], [@CR83]), we also predicted (Beh H 2) a "search cost" when the participants had to find neutral targets in scenes including a task-irrelevant negative distractor compared to scenes without emotional distractor.
Furthermore, we collected eye-movement data, which allowed us to assess the exploration of the scenes depending on the task relevance/irrelevance of the emotional stimuli. Here, we expected (Eye Movement Hypothesis, EM H 1) that task-relevant emotional objects would lead to faster fixations compared to neutral targets. Additionally (EM H 2), if emotional objects were automatically processed we would expect to find evidence of equally fast fixations, irrespectively of their task relevance. By contrast (EM H 3), if top-down control was efficient in avoiding emotional distraction we would expect a reduction of attentional capturing by task-irrelevant vs. task-relevant emotional stimuli (Huang et al. [@CR35]; Hunt et al. [@CR36]).
At a neuroimaging level (fMRI Hypothesis, fMRI H 1), we expected that searching for emotional objects would reflect in the activation of limbic (i.e., the amygdala; Vuilleumier et al. 2001) and para-limbic (i.e., the insular cortex; Uddin 2014) areas. Moreover (fMRI H 2), we expected that coping with emotional distraction when searching for a neutral target would result in an increased activation of brain regions involved in top-down attention control, such as the dorsal frontoparietal network to preserve goal-directed behavior (Corbetta et al. [@CR15]; Corbetta and Shulman [@CR14]; see also Iordan et al. [@CR38]; Wessa et al. [@CR77]; Ziaei et al. [@CR82]). While the previous literature on emotional distraction (Anticevic et al. [@CR2]; Dolcos et al. [@CR21]; Dolcos and McCarthy [@CR19]; Iordan and Dolcos [@CR37]) mainly focused on the interference driven by negative distractors, in the present study we also aimed at investigating whether positive stimuli would produce a similar interference, and would engage the same coping mechanism at the neural level. Specifically, we expected an increased activation of regions related to voluntary eye-movement control---such as the frontal eye field (FEF; Mohanty et al. [@CR48]; Tseng et al. [@CR69])---during the avoidance of both negative and positive emotional distractors. Further (fMRI H 3), on the basis of previous literature suggesting automatic processing of negative stimuli by limbic/para-limbic areas (i.e., the amygdala and the insular cortex; e.g., Phelps [@CR59]; Uddin et al. [@CR72]; Vuilleumier et al. [@CR76]), we tested for higher activation of those areas specifically involved with the processing of negative stimuli, irrespective of their task relevance, also with the help of a localizer task for emotional-related processing areas. Finally (fMRI H 4), following the hypothesis that affective regions modulate the activity of the frontoparietal control regions during negative emotional distraction (Dolcos and McCarthy [@CR19]), we expected an increased functional connectivity between limbic/para-limbic areas (mainly responding to negative stimuli; i.e., the amygdala and the insular cortex; e.g., Phelps [@CR59]; Uddin et al. [@CR72]) and the dorsal frontoparietal control network when subjects searched for a neutral target-object in the presence of a negative emotional distractor. This would be consistent with the notion that the interplay between affective and attention control regions can mitigate the impact of emotional distraction on the allocation of spatial processing resources.
Materials and methods {#Sec2}
=====================
Participants {#Sec3}
------------
Twenty-five healthy volunteers took part in the experiment. Three participants were excluded from data analysis because of within-fMRI-run head movements larger than 3 mm or 3°, leaving 22 participants for the final analyses (10 males; mean age 23.6 years; range 19--30 years). All participants gave written consent to the study, which was approved by the independent Ethics Committee of the Santa Lucia Foundation. All procedures were in accordance with the principles of the 1964 Helsinki declaration.
Stimuli and task {#Sec4}
----------------
The set of stimuli included 150 pictures depicting scenes of everyday life. These pictures were collected on the World Wide Web and included both internal (e.g., kitchens, living rooms, bedrooms, etc.) and external scenes (e.g., roads, buildings, natural landscapes, etc.), but no single-object photo. No people were represented in any scene. Pictures were displayed at 18 × 12° of visual angle (resolution in pixels, 680 × 448). 120 of the 150 pictures were digitally modified by means of CorelDraw Graphics Suite v. 12 to include negative or positive emotionally arousing objects, also collected on the World Wide Web (i.e., emotional scenes). 60 pictures included a negative emotional stimulus (e.g., a spider, a snake, etc.), while the other 60 pictures included a positive emotional stimulus (e.g., a cake, a puppy, etc.; see, for a similar approach, Buttafuoco et al. [@CR11]). The remaining 30 pictures did not include any emotional stimulus (neutral scenes).
To ensure that the inclusion of the emotional object affected the emotional impact of the scenes, we asked a group of 50 independent observers not taking part in the main experiment (18 males; mean age = 30.0 years, range 21--61 years) to rate the emotional valence (9-point scale: 1 = totally negative, 9 = totally positive) and the emotional arousal (9-point scale: 1 = totally calm, 9 = totally excited) of the scenes by means of an online survey. Crucially, the comparison between emotional and neutral scenes revealed that the emotional scenes significantly influenced emotional valence (mean ± standard error for scenes with negative object vs. neutral scenes: 2.64 ± 0.09 vs. 5.55 ± 0.15, *t*(88) = − 17.4, *p* \< 0.001; scenes with positive object vs. neutral scenes: 6.22 ± 0.10 vs. 5.55 ± 0.15; *t*(88) = 4, *p* \< 0.001), and emotional arousal (scenes with negative object vs. neutral scenes: 6.03 ± 0.11 vs. 3.33 ± 0.07, *t*(88) = 16.9, *p* \< 0.001; scenes with positive object vs. neutral scenes: 3.76 ± 0.06 vs. 3.33 ± 0.07; *t*(88) = 4.2, *p* \< 0.001). Overall, these data provide clear evidence that the inclusion of the emotional object (either negative or positive) made the entire scenes perceived and evaluated as more emotionally loaded than neutral scenes.[1](#Fn1){ref-type="fn"}
During fMRI scanning, participants were asked to localize and report the position (left vs. right hemifield) of the target-object, corresponding to a cue word presented at the beginning of each trial. For each emotional scene, we designated as the to-be-searched object either the emotional object or another, emotionally neutral object presented in the scene. For the neutral scenes, we designated only one object as the search target. For each emotional scene, the identity of the search target (emotional or neutral object) was counterbalanced across participants. This generated five different search conditions (see Fig. [1](#Fig1){ref-type="fig"}a):Fig. 1**a** Examples of negative, neutral and positive scenes including emotional target-objects (red circles in negative and positive scenes) and neutral target-objects (yellow circles in negative, neutral and positive scenes). Circles are not displayed during the experiment. **b** Diagram showing the sequence of events during one trial. The trial began with a warning signal for 500 ms. The cue word was then presented for 1 s. This defined the to-be-searched target in the following scene, which was presented for 2 s. Within this interval, participants had to search for and discriminate the position (left vs. right) on the scene of the pre-cued target-object by pressing one of two response buttons. After a variable ITI ranging from 8.5 to 10.5 s a new trial began. **c** Behavioral results. Mean inverse efficiency scores (IES) ± standard error of the means for the five conditions: negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT. Asterisks indicate statistically significant differences between conditions: \**p* \< 0.05; \*\**p* \< 0.01, \*\*\**p* \< 0.001. **d** Fixations patterns. Top panel: timeline indicating the meaning of the different fixation indexes, i.e., the latency of the fixation on the target-object (f-fix-lat, yellow bar), the RT measured from the onset of the first fixation on target (f-fix-RT, green bar); and the duration of this first fixation (f-fix-dur, red bar). Bottom panel: Mean ± standard error of f-fix-lat, f-fix-RT, and f-fix-dur related to fixations of the current target-object in the main conditions (negS_negT_negF, negS_neuT_neuF, neuS_neuT_neuF, posS_posT_posF, posS_neuT_neuF) or to fixations of the emotional object when it was not the to-be-searched target in the negS_neuT_negF and posS_neuT_posF conditions
scenes including a to-be-searched negative target (negS_negT; i.e., negative scene, negative target; 30 pictures);scenes including a negative emotional object wherein the to-be-searched target was a neutral object (negS_neuT; i.e., negative scene, neutral target; 30 pictures);scenes including a to-be-searched positive target (posS_posT; i.e., positive scene, positive target; 30 pictures);scenes including a positive emotional object wherein the to-be-searched target was a neutral object (posS_neuT; i.e., positive scene, neutral target; 30 pictures);scenes not including any emotional stimulus with a neutral to-be-searched target (neuS_neuT; i.e., neutral scene, neutral target; 30 pictures), used as a baseline condition.
Targets were located equiprobably in the left or the right hemifield. Several evaluations of the pictures were performed to control for possible differences between the experimental conditions. First, we made sure that the size of the target-objects did not differ significantly between the five experimental conditions, as revealed by a one-way ANOVA, \[*F*(4, 116) \< 1; n.s.\]. Then, we checked for the eccentricity of the target-objects across the five conditions. Separately for each picture, we computed the center of mass of the target-object as the average of the horizontal and vertical coordinates of each pixel belonging to the target-object. We extracted the horizontal eccentricity considering the horizontal position of the center of mass and converted this to degrees of visual angle. The absolute horizontal eccentricity values did not differed across the five conditions: \[*F*(4, 116) \< 1; n.s.\]. Finally, we measured the visual saliency of target-objects using the Saliency Toolbox 2.2 (<http://www.saliencytoolbox.net/>). This created a saliency map for each scene using local discontinuities in line orientation, intensity contrast, and color opponency (Itti et al. [@CR39]), allowing us to evaluate the saliency associated to the designed target-objects. Saliency values did not differed across the five experimental conditions: \[*F*(4, 116) \< 1; n.s.\]. These quantitative measures helped us to rule out several possible confounding factors in the interpretation of our results.
The participants' task was to find the target-object and to report whether this was located on the left or the right side of the picture. The presentation of the stimuli and the collection of responses were accomplished through MatLab 7.1 (The MathWorks Inc., Natick, MA), using Cogent 2000 Toolbox (Wellcome laboratory of Neurobiology, University College London). The sequence of events is illustrated in Fig. [1](#Fig1){ref-type="fig"}b. Each trial started with the presentation of a warning signal for 0.5 s. Immediately after, a "cue word" defining the to-be-searched target was presented on a gray background for 1 s. This was followed by the presentation of the visual scene. Participants were asked to search for the cued object and to press one of two response buttons as quickly and as accurately as possible, depending on the location of the target-object in the picture (left vs. right visual hemifield). Participants had a maximum time of 2 s to give an answer, and after that the scene disappeared. To optimize the ability to isolate the hemodynamic response associated with each single scene presentation we used a long variable inter-trial interval ranging from 8.5 to 10.5 s (mean of 9.5 s), uniformly distributed (see, e.g., Dale [@CR17]), consisting in a gray background. Participants underwent three fMRI-runs (lasting approximately 11 min each), including 50 trials each. The order of trials within and across runs was randomized with a constraint: each run included ten trials for each of the five conditions.
Eye movements and fixation indexes {#Sec5}
----------------------------------
Together with the behavioral data, we also acquired the subjects' gaze-position during fMRI. This was done to provide us with additional information about the impact of the emotional stimuli on the overt exploration of the visual scene, as a function of task relevance (see, for a similar approach, Santangelo and Macaluso [@CR61]; Santangelo et al. [@CR62]). The eye-movement data were recorded with an ASL eye-tracking system, adapted for use in the scanner (Applied Science Laboratories, Bedford, MA; Model 504, sampling rate 60 Hz). Using the MTtools (<http://www.brainreality.eu/mt_tools/>), we computed fixation positions for each picture and for each participant considering a time window of 2 s, corresponding to the duration of the scene presentation, starting from the picture onset. We then computed for each picture a target-map. This procedure consisted in manually drawing (using CorelDraw Graphics Suite v. 12) the target-object within each scene and then copy and paste this object on a picture with the same resolution of the initial scene (680 × 448 pixels) consisting on a gray background uniformly distributed. The object was pasted at the same location as the original scene. Hence, for each target we obtained a 680 × 448 matrix with values set equal to 1 at the coordinates of the target-object and zeros everywhere else. Finally, this binary map was smoothed with a Gaussian filter (FWHM = 1°) to account for eye-tracking noise. For the emotional scenes, we built both a target-map related to the emotional object (i.e., negS_negT and posS_posT conditions) and a target-map related to the neutral object (i.e., negS_neuT and posS_neuT conditions); while for the neutral scenes, we built a target-map related to the only neutral object used as target (i.e., neuS_neuT).
These target-maps were used to compute three different fixation indexes: (1) the latency of the first fixation on the target-map (f-fix-lat), indicating the strength of attentional grabbing of the current target; (2) the time interval between the onset of the first fixation on the target and the response button press (f-fix-RT), highlighting the time needed to decide whether the stimulus was a target, having accounted for the initial capture of spatial attention, i.e., the "f-fix-lat" index; and (3) the duration of the first fixation on the target-map (f-fix-dur), indicating the length of perceptual processing devoted to that stimulus, irrespective of the response time, cf. "f-fix-RT". Figure [1](#Fig1){ref-type="fig"}d (top panel) shows a schematic depiction of the three indexes in relation to each other (see also the Online Resource, where we confirmed our main results using a forth fixation-index, namely the fixation probability of the target-map).
We used these fixation indexes to address several hypotheses about the processing of target and non-target emotional stimuli. If emotional targets were attentional "grabbing" (EM H 1) we would expect a bottom-up effect on gaze direction evidenced by: faster first fixation latencies (f-fix-lat), shorter intervals between target fixation and response button press (f-fix-RT), and longer fixation durations (f-fix-dur) following the presentation of emotional compared to neutral targets (cf. Calvo and Lang [@CR12]). To test this hypothesis, we computed f-fix-lat, f-fix-RT, and f-fix-dur related to the object that was defined as target within each scene (see Fig. [1](#Fig1){ref-type="fig"}d, bottom panel): (1) task-relevant emotional targets in negative scenes (negS_negT_negF, meaning: negative scene, negS, negative target, negT, analysis related to the negative target, negF); (2) neutral targets in scenes including a negative distractor (negS_neuT_neuF, i.e., negative scene, negS, neutral target, neuT, analysis related to the neutral target, neuF); (3) neutral targets in scenes that not included any emotional distractor (neuS_neuT_neuF, i.e., neutral scene, neuS, neutral target, neuT, analysis related to the neutral target, neuF); (4) task-relevant emotional targets in positive scenes (posS_posT_posF, i.e., positive scene, posS, positive target, posT, analysis related to the positive target, posF); (5) neutral targets in scenes including a positive distractor (posS_neuT_neuF, i.e., positive scene, posS, neutral target, neuT, analysis related to the neutral target, neuF).
Additionally, we characterized fixation patterns also for those conditions in which the emotional object was task irrelevant, that is, a distractor. If emotional objects were automatically processed when task irrelevant (EM H 2), we would expect to find evidence of fast gaze orienting also in these conditions. By contrast, if top-down control was efficient in avoiding emotional distraction (EM H 3) we would expect a reduction of attentional capture by emotional distractors. To check for this, we directly compared fixation indexes related to the emotional object in conditions of task relevance (conditions 1 and 4 above, negS_negT_negF and posS_posT_posF) vs. conditions of task irrelevance, that is: (1) negS_neuT_negF, i.e., negative scene, negS, neutral target, neuT, analysis related to the negative emotional distractor, negF; (2) posS_neuT_posF, i.e., positive scene, posS, neutral target, neuT, analysis related to the positive emotional distractor, posF.[2](#Fn2){ref-type="fn"}
Magnetic resonance imaging {#Sec6}
--------------------------
A Siemens Allegra (Siemens Medical Systems, Erlangen, Germany) operating at 3T and equipped for echo-planar imaging (EPI) was used to acquire the functional magnetic resonance images. A quadrature volume head coil was used for radio frequency transmission and reception. Head movements were minimized by mild restraint and cushioning. Thirty-two slices of functional MR images were acquired using blood oxygenation level-dependent imaging (3 × 3 mm^2^ in plane resolution, 2.5 mm thick, 50% distance factor, repetition time = 2.08 s, time echo = 30 ms), covering the entirety of the cortex.
fMRI data analysis {#Sec7}
------------------
We used SPM8 (Wellcome Department of Cognitive Neurology) implemented in MATLAB 7.4 (The MathWorks Inc., Natick, MA) for data pre-processing and statistical analyses. Each participant underwent three fMRI-runs, each comprising 320 volumes. After having discarded the first four volumes of each run, all images were corrected for head movements. Slice-acquisition delays were corrected using the middle slice as a reference. All images were normalized to the standard SPM8 EPI template, resampled to 2 mm isotropic voxel size, and spatially smoothed using an isotropic Gaussian kernel of 8 mm FWHM. Time series at each voxel for each participant were high-pass filtered at 220 s and pre-whitened by means of autoregressive model AR(1).
Statistical inference was based on a random-effects approach, which is comprised of two steps: first-level multiple regression models estimating contrasts of interest for each subject, followed by the second-level analyses for statistical inference at the group level. The main aim of the study was to assess the impact of emotional objects in capturing attention/perceptual resources depending on their current task relevancy: that is, when the emotional object was the search target vs. a distracting object. Accordingly, the first-level model considered as events-of-interest the five main conditions: negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT. All the erroneous and missing trials (i.e., trials in which participants did not correctly localize the left vs. right position of the current target or failed to do so within the time window of 2 s: overall 11.2% of trials) were modelled as a separate event type that was not considered in the group analyses. The events were modelled as miniblocks, time locked at the onset of the pictures with a duration of 2 s, i.e., the time of scene presentation. All predictors were convolved with the SPM8 hemodynamic response function, and the parameters of head movements were included as covariates of no interest. For each subject, linear contrasts were used to average the parameter estimates associated with each of the five conditions of interests, across the three fMRI-runs.
For the group-level analysis, we carried out a within-subject ANOVA that modelled the five relevant event types: negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT. Correction for nonsphericity (Friston et al. [@CR30]) was used to account for possible differences in error variance across conditions, arising---for example---because of the different number of trials in the five conditions of interest and/or any non-independent error terms for the repeated measures.
### Target-related fMRI analysis {#Sec8}
To address our main question about the role of task relevance on the processing of emotionally arousing stimuli, we considered the conditions when the emotional object was either the search target or a task-irrelevant distractor, irrespective of the emotional valence. We used the contrast "target vs. distractor" emotional objects \[(negS_negT + posS_posT) \> (negS_neuT + posS_neuT)\] to identify the activity associated with the effect of searching for a task-relevant emotional stimulus (see fMRI H 1). The reverse contrast, comparing "distractor vs. target" emotional objects \[i.e., (negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\], was used instead to highlight top-down volitional control involved with avoiding non-target (distracting) emotional stimuli, thus preserving goal-directed attention toward the neutral target (see fMRI H 2). Moreover, we checked whether top-down control involved with distraction avoidance was selectively deployed for negative or positive distractors \[i.e., (negS_neuT---negS_negT) \> (posS_neuT---posS_posT) and (posS_neuT---posS_posT) \> (negS_neuT---negS_negT)\]. Importantly, it should be noticed that the four conditions considered in these contrasts included scenes containing one emotional object (either the target or the distractor), thus removing any overall effect of processing emotional stimuli. The statistical threshold was set to *p* = 0.05, FWE corrected at the voxel level, considering the whole brain as the volume of interest.
### Emotional-related fMRI analysis {#Sec9}
Since the contrast "target vs. distractor" emotional object described above in the target-related fMRI analysis (negative and positive scenes collapsed together) failed to reveal any significant effect at the whole brain level (cf. "[Target-related fMRI analysis](#Sec16){ref-type="sec"}", below), we asked whether any effect of emotional target and/or emotional distractor was specific for the negative stimuli, irrespective of their task relevance (Fenker et al. [@CR25]; Huang et al. [@CR35]; see fMRI H 3). First of all, we identified regions involved in the processing of negative stimuli by comparing conditions including a negative stimulus (negS) vs. all the other conditions: i.e., \[(negS_negT + negS_neuT) \> (neuS_neuT + posS_posT + posS_neuT)\], weighted as: \[(1/2 + 1/2) \> (1/3 + 1/3 + 1/3)\]. The choice to compare negative conditions versus the average of both positive and neutral conditions was motivated by the fact that "negative minus positive" and "negative minus neutral" showed very similar patterns of activation (see Figure S2 in the Online Resource). Moreover, we also performed the opposite comparison, contrasting positive scenes vs. all the other conditions: i.e., \[(posS_posT + posS_neuT) \> (neuS_neuT + negS_negT + negS_neuT)\]. The statistical threshold was set to *p* = 0.05, FWE corrected at the voxel level, considering the whole brain as the volume of interest. In addition, we considered an independent dataset (cf. "Localizer task", below) that allowed us to focus on limbic and para-limbic areas traditionally involved in the processing of negative stimuli (amygdala and insular cortex, see Phan et al. [@CR58]; Seara-Cardoso et al. [@CR65]). Accordingly, corrected *p* values were also assigned considering the limbic regions identified in the localizer scan as a reduced volume of interest (small volume correction, SVC; Worsley et al. [@CR78]). For this, we used spheres of 8 mm of radius that matched the FWHM of the smoothing filter. Spheres were centered in the left and the right amygdala (cf. Table [2](#Tab2){ref-type="table"}).
Finally, all the regions involved in the processing of negative vs. positive or neutral conditions were combined together in a volume of interest (VOI) using MarsBar 0.42 ("MARSeille Boîte A Région d'Intérêt" SPM toolbox). Within this VOI, we tested the contrasts "distractor vs. target" and "target vs. distractor" emotional objects, now considering only scenes including negative stimuli: (negS_neuT) \> (negS_negT) and (negS_negT) \> (negS_neuT), respectively; *p*-*FWE*-*corr* = 0.05, at the voxel level. This latter analysis allowed us to check whether the brain activity observed during the processing of negative scenes was modulated by the task relevance of negative objects.
### Inter-regional connectivity of the affective regions {#Sec10}
Together with the intra-regional analyses described above, we performed analyses of inter-regional connectivity to address the hypothesis that coping with emotional distraction may involve changes of connectivity between limbic/para-limbic regions and cortical regions involved in attention control (cf. Dolcos et al. [@CR21]; Iordan et al. [@CR38]; Uddin [@CR70]; see fMRI H 4). We used analyses of inter-regional connectivity \[psychophysiological interactions (PPIs)\] (Friston [@CR29]) implemented with the "Generalized Form of Context-Dependent Psychophysiological Interactions" SPM toolbox (McLaren et al. [@CR45]). At the subject level, each PPI analysis included five regressors corresponding to the psychological variables of interest (i.e., negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT, as in the main analysis), the time course of the seed area (i.e., the physiological variable highlighting the activity of either the left insula and the right amygdala; cf. Table [2](#Tab2){ref-type="table"}), and the critical cross-products (i.e., the psychophysiological interaction term) between the five psychological variables and time course of each seed area. The head motion realignment parameters were included as covariates of no interest. For each of the two seed areas, the parameter estimates of the five PPI regressors entered a within-subject ANOVA for statistical inference at the group level. For each ANOVA, we tested for changes of functional connectivity with the rest of the brain when the emotional object was a to-be-ignored distractor compared to when it was the search target \[(negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\], expecting an increased connectivity with the dorsal frontoparietal control network. Moreover, we wanted to be sure to highlight the selective functional coupling of our seed regions within the areas involved with resistance from negative, but not positive, distraction. For this reason, we used a procedure involving two masks that aimed to isolate the brain regions recruited during the avoidance of either negative or positive emotional distractors (i.e., (negS_neuT \> negS_negT) and (posS_neuT \> posS_posT), respectively, at *p*-*unc* = 0.05). Specifically, we used the constraint that the inter-regional coupling had to include regions recruited when coping with negative distractor (i.e., inclusive masking with negS_neuT \> negS_negT), and---at the same time---not include areas involved in coping with positive distractors (i.e., exclusive masking with posS_neuT \> posS_posT). This would indicate a coupling with regions selectively involved in coping with distraction by negative stimuli. An analogous masking procedure was used to assess inter-regional coupling with regions selectively recruited by positive distractors.
The statistical threshold was set to *p* = 0.05, FWE corrected at the voxel level, considering the whole brain as the volume of interest.
### Localizer task {#Sec11}
Together with the main search task, we acquired fMRI data during a standard localizer task for emotional-related processing areas (Johnston et al. [@CR40]). The localizer provided us with an independent dataset to identify brain regions that responded differentially to negative vs. positive emotional stimuli, see also the "[Emotional-related fMRI analysis](#Sec9){ref-type="sec"}" section above. For the localizer task, we selected 144 pictures (48 negative, 48 positive and 48 neutral pictures) from the International Affective Picture System (IAPS; Lang et al. [@CR41]). IAPS are well-known stimuli able to elicit the activation of emotional brain areas such as the amygdala (Britton et al. [@CR8]) or the insula (Wright et al. 2004), and have been pre-tested in normative samples for their valence and arousal values. The 144 selected IAPS pictures were clearly distinguishable according to their valence and arousal scores. The standardized mean valence scores (1 = unhappy, 9 = happy) were significantly lower for negative (3.14 ± 0.64) than for neutral (5.09 ± 0.56) pictures, which, in turn, were lower than for positive pictures (7.21 ± 0.53) \[(*t*(94) = − 19,08, *p* \< 0.001) and (*t*(94) = − 23.72, *p* \< 0.001), respectively\]. The mean arousal scores (1 = calm, 9 = excited) were greater for both negative (5.63 ± 0.55) and positive (4.73 ± 0.75) than for neutral (2.87 ± 0.54) pictures \[(*t*(94) = 20.60, *p* \< 0.001) and (*t*(94) = 13.79, *p* \< 0.001), respectively\]. The 144 selected IAPS pictures did not include any human but single objects belonging to the same categories of objects that we used as emotional target/distractor stimuli in the complex scenes of the main visual search task: e.g., for negative stimuli: scary and/or disgusting animals, dead animals, weapons, disgusting food, excrements; for positive stimuli: baby animals, appetizing foods, flowers, money, gold bars; for neutral stimuli: kitchen tools, work tools, household furniture, electrical outlets.
The participants were required to passively view the pictures. The 144 pictures were presented in 12 blocks, each consisting of 12 pictures belonging to the same emotional valence, resulting in 4 blocks of positive, 4 blocks of negative, and 4 blocks of neutral pictures. Each block lasted for 18 s (1.5 s per picture) and was separated from the following block by a variable inter-trial interval ranging from 2 to 4 s (uniformly distributed), filled with a fixation cross displayed on a gray background. To avoid the induction of long-lasting mood states, we presented the different blocks in a pseudo-random sequence so that no more than two blocks of the same condition was consecutively presented (cf. Dolcos et al. [@CR20]).
The analysis of the functional localizer aimed to highlight the activity in limbic and para-limbic areas associated with the processing of negative valence stimuli. Accordingly, we compared "negative" minus "positive and neutral" blocks of IAPS pictures (cf. Johnston et al. [@CR40]). This comparison revealed the activation of both the left and the right amygdala (see Table [2](#Tab2){ref-type="table"}). As before, the statistical threshold was set to *p* = 0.05, FWE corrected at the voxel level, considering the whole brain as the volume of interest. The left and right amygdala were used as additional volumes of interest (SVC analysis) to compare scenes including negative (negs_negT and negS_neuT) vs. the other objects (neuS_neuT, posS_posT and posS_neuT) in the main visual search task, with the aim to identify regions involved in the processing of negative stimuli during the main searching task, cf. above.
Results {#Sec12}
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Behavioral data {#Sec13}
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On each trial, the participants indicated the location (left vs. right hemifield) of the target-object defined by the cue word. Performance was measured in terms of the "inverse efficiency score" (IES), which combines reaction times (RT) and accuracy (IES = mean RT/mean proportion of accuracy, see Table [1](#Tab1){ref-type="table"}), and provides correction for potential speed--accuracy trade-offs present in the data (see, e.g., Bruyer and Brysbaert [@CR9]).Table 1Behavioral data in the five experimental conditionsNegS_negTNegS_neuTNeuS_neuTPosS_posTPosS_neuTIES (ms)1206 ± 431305 ± 511236 ± 381028 ± 261262 ± 32RT (ms)1072 ± 271092 ± 281084 ± 24967 ± 231090 ± 20ACC (%)89.7 ± 1.584.8 ± 1.888.5 ± 1.794.2 ± 0.987.0 ± 1.8Mean (± SEs) inverse efficiency scores (IES), reaction times (RTs), and accuracy (percentages), for the different conditions
A one-way analysis of variance (ANOVA) with five levels (i.e., the five conditions: negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT) was conducted on the IES data revealing significant differences among the conditions, \[*F*(4, 84) = 19.31, *p *\< 0.001\], see Fig. [1](#Fig1){ref-type="fig"}c. Planned comparisons revealed a significant facilitation of location discrimination (lower IES) when the current to-be-searched target was an emotional object (negS_negT or posS_posT) compared to when the current target was a neutral object (negS_neuT or posS_neuT): *t*(21) = 3.31; *p *= 0.002 and *t*(21) = 8.89; *p *\< 0.001, respectively (compare bars 1 vs. 2 and bars 4 vs. 5 in Fig. [1](#Fig1){ref-type="fig"}c). These findings could indicate a facilitation in selecting emotional targets (Beh H 1), but also a discrimination cost when searching for a neutral target in the presence of emotional distractors (Beh H 2).
To disentangle the discrimination facilitation vs. discrimination cost of the emotional objects depending on their current task relevance, we compared each condition including an emotional object (i.e., negS_negT, negS_neuT, posS_posT, posS_neuT) with the baseline condition, i.e., scenes without emotional objects (neuS_neuT). These planned comparisons revealed a discrimination facilitation (Beh H 1) when searching for positive targets (posS_posT; *t*(21) = 7.78; p \< 0.001; compare bars 4 vs. 3 in Fig. [1](#Fig1){ref-type="fig"}c), and a discrimination cost (Beh H 2) when searching for a neutral target in the presence of negative distractors (negS_neuT; *t*(21) = 1.82; *p* = 0.041; compare bars 2 vs. 3 in Fig. [1](#Fig1){ref-type="fig"}c). By contrast, searching for a neutral target in scenes including a positive emotional distractor did not differ significantly from the baseline condition (posS_neuT; *t*(21) = 0.89; *p *= 0.192; compare bars 5 vs. 3 in Fig. [1](#Fig1){ref-type="fig"}c). This might indicate that non-target positive objects are less "distracting" than non-target negative objects compared to the baseline condition, which is in line with the literature (see Iordan and Dolcos [@CR37]). Finally, planned comparisons failed to reveal any difference between the baseline condition and searching for negative targets (negS_negT; *t*(21) = 0.78; *p* = 0.221; compare bars 1 vs. 3 in Fig. [1](#Fig1){ref-type="fig"}c). This latter finding appears somewhat surprising with respect to the previous literature that highlighted greater discrimination or detection facilitation for negative compared to neutral objects (Flykt [@CR27]; Öhman et al. [@CR52]; Schubö et al. [@CR63])[3](#Fn3){ref-type="fn"}. The following eye-movement data analyses were helpful to clarify this point.
Eye-movement data {#Sec14}
-----------------
To clarify the behavioral findings (Fig. [1](#Fig1){ref-type="fig"}c and the paragraph above) and to further investigate the deployment of overt attentional resources triggered by emotional stimuli according to their task relevance, we measured three indexes related to: the latency of the first fixation on the target (f-fix-lat); the time interval between the onset of the first fixation on the target and the response button press (f-fix-RT); the duration of the first fixation on the target (f-fix-dur) (see also eye-movement analysis in the Online Resource for the target fixation probability index). We conducted three different repeated-measures ANOVAs with five levels corresponding to the main experimental conditions (negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT) on the data derived from each of the three fixation indexes (f-fix-lat, f-fix-RT, and f-fix-dur; see Fig. [1](#Fig1){ref-type="fig"}d). All the three ANOVAs revealed significant differences between the main conditions: f-fix-lat, \[*F*(4, 84) = 2.58, *p* = 0.043\], f-fix-RT, \[*F*(4, 84) = 6.09, *p* \< 0.001\], and f-fix-dur, \[*F*(4, 84) = 6.92, *p* \< 0.001\], indicating different patterns of fixations depending on the five experimental conditions.
Post-hoc analyses revealed that subjects' gaze arrived equally fast on the emotional object when it was the current to-be-searched target, irrespective of being negative (negS_negT; 472 ms) or positive (posS_posT; 488 ms) (difference = 16 ms; *p* = 0.604; compare the first vs. the forth orange bar in Fig. [1](#Fig1){ref-type="fig"}d, left panel). However, RTs computed from the onset of the first fixation on the emotional target were significantly longer for negative (655 ms) than positive (542 ms) to-be-searched targets (difference = 113 ms; *p* \< 0.001; compare the first vs. the forth green bar in Fig. [1](#Fig1){ref-type="fig"}d). This latter finding might account for the lack of differences in the IES performance between searching for negative targets and neutral targets in the baseline condition (see the paragraph above). Consistently, the duration of the first fixation tended to be longer for negative (753 ms) than for positive (699 ms) targets, though this effect was not fully significant (difference = 54 ms; *p* = 0.116). Overall, these patterns of eye-movement data highlighted that negative stimuli captured overt attentional orienting equally well as positive objects, supporting the EM H 1, but this did not correspond to an equally fast target-location discrimination (left vs. right response).
The analysis of the eye-movement data also revealed that the emotional objects failed to capture overt attention when task irrelevant, thus highlighting efficient top-down control to filter out current distractors (supporting the EM H 3; see also the EM H 2 for the opposite expectation). This was evidenced by the analysis of fixation indexes that directly compared emotional targets vs. emotional distractors (see Fig. [1](#Fig1){ref-type="fig"}d, right panel). We carried out two 2 × 2 repeated-measures ANOVAs with the factors of target valence (negative vs. positive) and task relevancy (emotional target vs. emotional distractor) on either the first fixation latencies (f-fix-lat) and the duration of the first fixation (f-fix-dur) data (see also the Online Resource for a consistent analysis on the fixation probability data). Both ANOVAs revealed a main effect of task relevancy \[f-fix-lat: (*F*(1, 21) = 118.35, *p* \< 0.001), and f-fix-dur: (*F*(1, 21) = 60.72, *p* \< 0.001)\], indicating that emotional distractors (i.e., negS_neuT_negF and posS_neuT_posF) were fixated later and for a shorter duration (733 ms and 414 ms, respectively) than emotional targets (i.e., negS_negT_negF and posS_posT_posF; 480 ms and 726 ms, respectively). Moreover, the ANOVA on the f-fix-dur revealed a main effect of target valence, \[*F*(1, 21) = 4.47, *p* = 0.047\], indicating that negative objects were fixated for a longer duration (591 ms) than positive objects (549 ms), irrespective of being targets or distractors. The ANOVAs did not reveal any other significant effects (all Fs \< 2.12; all *p*s \> 0.160).
Overall, these findings highlight an interplay between bottom-up and top-down attention control depending on the task relevance of the emotional stimuli. On one hand, emotional objects captured overt orienting when they were task relevant, with faster fixation latencies than non-emotional targets, though with longer RTs from target fixation (i.e., the f-fix-RT index) for negative than for positive target-objects, indicating bottom-up facilitation in directing attention towards task-relevant emotional stimuli. On the other hand, the capability of emotional objects to capture overt orienting of spatial attention was dramatically reduced when they were task irrelevant, irrespective of their positive or negative valence. This indicates efficient top-down control in filtering out the current emotional distractor. These mechanisms were further investigated through the analysis of the fMRI data.
fMRI data {#Sec15}
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### Target-related fMRI analysis {#Sec16}
First, we tested for activation associated with the effect of searching for "emotional minus neutral targets" in the emotional scenes, i.e., \[(negS_negT + posS_posT) \> (negS_neuT + posS_neuT)\], expecting to observe increased activity in limbic/para-limbic regions, given the bottom-up facilitation in directing attention towards task-relevant emotional stimuli (see fMRI H 1). However, this comparison failed to reveal any significant effect at the whole brain level, possibly due to the fact that we averaged negative and positive stimuli (see also Introduction). Conversely, the opposite contrast, related to searching for neutral targets in the presence of emotional distractors, irrespective of their emotional valence, \[(negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\], highlighted activity in a network of dorsal frontoparietal regions typically involved with top-down control (Corbetta and Shulman [@CR14]; Corbetta et al. [@CR15]; see also Iordan et al. [@CR38]), supporting the fMRI H 2. These regions included anteriorly the right frontal eye fields (FEF), and posteriorly, the right superior parietal gyrus (SPG), plus several activations in the occipital cortex, including the right and left lingual gyri (LG) and the right middle occipital gyrus (MOG; see Table [2](#Tab2){ref-type="table"}). Although this network of areas was more pronounced on the right hemisphere, homologous activations were also observed at a lower statistical threshold in the left hemisphere (see Fig. [2](#Fig2){ref-type="fig"}a).Table 2MNI coordinates (*x*, *y*, *z*), *Z* values, and *p* values for the areas showing a significant activation in the main visual search task and in the localizer task*x y zZ* valuep-FWE-corrSearching for neutral targets in emotional scenes Right FEF30 4 565.81\< 0.001 Right SPG16 − 64 584.880.016 Right LG10 − 98 125.240.003 Left LG− 8 − 100 165.240.007 Right MOG36 − 82 365.020.008Searching for negative vs. positive plus neutral targets Left IFG− 52 36 66.03\< 0.001 Right IFG50 34 65.97\< 0.001 Left INS− 28 22 − 185.260.003 MSFc− 4 28 505.220.003 Left STP− 46 24 − 185.030.008 Left FG− 46 − 52 − 225.520.001 Left IOG− 46 − 62 − 145.400.001 Left MOG− 50 − 76 45.220.003 Left MTG− 58 − 66 04.890.019 Right IOG/ITG44 − 72 − 86.01\< 0.001Processing of negative vs. positive plus neutral IAPS pictures in the localizer task L AMY− 16 − 6 − 164.820.011 R AMY16 − 6 − 164.640.022 L MOG− 48 − 80 86.64\< 0.001 R MOG48 − 76 185.70\< 0.001 L SOG− 26 − 82 305.160.002 L FG− 44 − 58 − 184.440.050 R FG44 − 50 − 265.49\< 0.001 L LG− 22 − 76 − 104.890.008 SMA10 16 705.170.002 R IFG42 8 264.680.019*FEF* frontal eye fields, *SPG* superior parietal gyrus, *LG* lingual gyrus, *MOG/SOG/IOG* middle/superior/inferior occipital gyrus, *IFG* inferior frontal gyrus, *INS* insula, *MSFc* medial superior frontal cortex, *STP* superior temporal lobe, *FG* fusiform gyrus, *MTG* middle temporal gyrus, *ITG* inferior temporal gyrus, *AMY* amygdala, *SMA* supplementary motor areaFig. 2**a** fMRI results related to searching for a neutral target (neuT) in positive/negative scenes. Axial section of a standard MNI template showing the peak of activation in the right FEF in the whole brain analysis that showed a significant effect of searching for neutral vs. emotional targets irrespective of the emotional valence of the scene \[(negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\]. The bar plot summarizes the activity of the right FEF in the five conditions, highlighting an increase of activity during searching for neutral targets in emotional scenes (compare bars 2 and 5 vs. bars 1 and 4). **b** Brain responses associated with the effect of searching for negative vs. positive or neutral targets \[(negS_negT + negS_neuT) \> (neuS_neuT + posS_posT + posS_neuT)\] at the whole brain level. The related bar plot indicates an increase of activity of the left INS during searching through negative scenes, irrespective of the task relevance of the negative object (compare bars 1 and 2 vs. bars 3, 4 and 5). **c** Coronal section showing the peak of activation in the right amygdala (AMY) that showed a significant effect of processing negative vs. positive or neutral IAPS stimuli in the localizer task \[(neg) \> (pos + neu)\]. **d** Axial and coronal sections of a standard MNI template showing the peak of activation in right prefrontal cortex (i.e., the MFG) that showed functional coupling (psychophysiological interactions) with left INS during search of neutral vs. emotional targets \[(negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\]. The bar plot summarizes the functional connectivity of the right MFG with left INS in the five conditions, highlighting increased insular/prefrontal coupling specifically when neutral targets have to be searched for in the context of negative scenes (compare bars 2 and 5 vs. bars 1 and 4). For display purposes, all activation maps are displayed at a threshold of *p* \< 0.001 (uncorrected). In the signal plot, the level of activation is expressed in arbitrary units (a.u., ± 90% confidence interval)
Considering the searching cost revealed by our behavioral analysis with negative distractors, we checked whether the activity of the dorsal frontoparietal network was specifically related to the avoidance of negative vs. positive distractors \[(negS_neuT---negS_negT) \> (posS_neuT---posS_posT)\] or vice versa \[(posS_neuT---posS_posT) \> (negS_neuT---negS_negT)\]. Both analyses failed to reveal any significant activations, suggesting that both negative and positive emotional distractors contributed in the enhanced activity observed in the dorsal frontoparietal network during avoidance of emotional distraction.
### Emotional-related fMRI analysis {#Sec17}
We then focused on the processing of emotional valence. First, we identified areas responding to negative stimuli by comparing the two conditions including a negative object, irrespectively of their task relevance (negS_negT and negS_neuT) vs. the other three conditions (see fMRI H 3). At the whole brain level, this revealed a large network of areas, including anteriorly the left and right IFG, the left insula (INS) and the medial superior frontal cortex (MSFc) (Fig. [2](#Fig2){ref-type="fig"}b). Significant effects were also found in several occipital and temporal areas, including the left FG, the left MOG, the left and right inferior occipital (IOG) and temporal (ITG) gyri (see Table [2](#Tab2){ref-type="table"}). The opposite comparison including positive objects irrespectively of their task relevance (posS_posT and posS_neuT) vs. the other three conditions failed to reveal any significant activations, suggesting a selective involvement of the above described regions during the processing of negative (but not positive) stimuli, irrespective of their current task relevancy.
In addition, we used the localizer data to highlight areas traditionally associated with the processing of negative stimuli (cf. Table [2](#Tab2){ref-type="table"}, Fig. [2](#Fig2){ref-type="fig"}c, and method section). The comparison between "negative" vs. "positive and neutral" IAPS pictures administered with the localizer task revealed the activation of the left (x, y, z: − 16, − 6, − 16; *t* = 4.82; *p* = 0.011) and right amygdala (x, y, z: 16, − 6, − 16; *t* = 4.64; *p* = 0.022). We then used the coordinates of these areas as additional regions of interest to search for the effect of processing negative stimuli \["scenes including a negative object (negs_negT and negS_neuT)" minus "the other three conditions (neuS_neuT, posS_posT and posS_neuT)"\] in the main visual search task. This revealed a significant effect of processing negative scenes in the right amygdala (x, y, z: 18, − 4, − 20; *t* = 3.25; *p*-*SVC*-*corr* = 0.023), but not in the left amygdala (x, y, z: − 20, − 6, − 18; *t* = 2.33; *p*-*SVC*-*corr* = 0.152). The activation of the right amygdala confirmed the fMRI H 3, expecting stronger involvement of the limbic system during the processing of the negative scenes, irrespective of the task relevance of the negative objects. Again, no effects were found for the processing of positive stimuli.
Limbic/para-limbic areas involved in the processing of scenes including a negative object (namely the left insula and the right amygdala) were combined together in a volume of interest (VOI) to test whether their activity was modulated by task relevance. For this, we contrasted, within this VOI, scenes where the negative object was task relevant vs. scenes where the negative object was task irrelevant \[(negS_negT) \> (negS_neuT)\] and vice versa \[(negS_neuT) \> (negS_negT)\]. These analyses failed to reveal any significant activation, apparently indicating that the activity in these limbic/para-limbic areas was not further modulated by the current task relevance (i.e., by top-down control).
### Inter-regional connectivity of the affective regions {#Sec18}
The evidence that emotional objects capture overt attention to a less extent when they are task irrelevant (cf. eye-movement data; see also Fig. [1](#Fig1){ref-type="fig"}d, right panel) is consistent with the notion of a selective involvement of top-down control to cope with emotional distractors. Based on this evidence and on the literature highlighting changes of connectivity between limbic/para-limbic regions and other cortical regions involved in attention control during coping with emotional distraction (cf. Dolcos et al. [@CR21]; Iordan et al. [@CR38]; Uddin [@CR70]; see also Introduction), we chose the left insula and the right amygdala (i.e., the regions responding to negative scenes in the emotion-related fMRI analysis, cf. Table [2](#Tab2){ref-type="table"}) as regions of interest (ROI) for the PPI analysis. For each of the two seed regions, we tested for changes of functional connectivity with the rest of the brain when the emotional object was a to-be-ignored distractor compared when the emotional object was the search target \[(negS_neuT + posS_neuT) \> (negS_negT + posS_posT)\], expecting increased coupling between limbic/para-limbic and dorsal top-down control regions in the presence of emotional distractors (see fMRI H 4).
When the connectivity analysis was seeded on the right amygdala, we failed to observe any significant functional coupling with the rest of the brain. Instead, the left insula showed significant connectivity with the right prefrontal cortex (*x*, *y*, *z*: 52, 12, 40; *t *= 5.18; *p* = 0.022), extending dorsally with the FEF and ventro-laterally with the MFG (see Fig. [2](#Fig2){ref-type="fig"}d). Since the seed regions for the PPI analysis (i.e., left insula and right amygdala) derived from the comparison between "negative scenes" minus "neutral and positive scenes", we aimed to specifically highlight the functional connectivity of these regions in the presence of negative distractors. For this reason, we used a masking procedure (see Methods), confirming that the inter-regional coupling between the left insula and the right prefrontal cortex was present inside the neural circuit recruited when coping with negative distractor (i.e., inclusive masking with negS_neuT \> negS_negT, *p*-*unc*. = 0.05). On the contrary, it was absent inside the neural circuit recruited when coping with positive distractor (i.e., inclusive masking with posS_neuT \> posS_posT, *p*-*unc*. = 0.05). This masking procedure, therefore, allowed to highlight the selective coupling between the insular and prefrontal cortex when coping with negative but not positive distraction. The signal plot of Fig. [2](#Fig2){ref-type="fig"}d highlights the specificity of this effect in the context of coping with negative distraction: insular/prefrontal coupling increased when searching for neutral targets with negative distractors and decreased when searching for negative targets (compare bars 2 vs. 1), while positive scenes modulated much less the functional coupling between these areas (compare bars 5 vs. 4). Ultimately, these findings highlighted an enhanced insular/prefrontal coupling selectively when neutral vs. emotional targets have to be searched for while coping with distraction from negative elements in the scene, confirming the fMRI H 4.
### Discussion {#Sec19}
The aim of the current study was to investigate at both behavioral and neural levels the impact of emotional stimuli on the distribution of attentional resources depending on their current task relevance, i.e., emotional targets vs. emotional distractors. The current behavioral findings revealed overall facilitation in searching for emotional compared to neutral targets in the presence of emotional distractors. Surprisingly, this benefit was selective for positive stimuli. Nevertheless, the analysis of fixations revealed that both negative and positive targets were fixated earlier than neutral targets, indicating that they captured overt spatial attention to a comparable extent. Notwithstanding that, attentional capture by emotional stimuli was modulated by the current task demand: emotional distractors were fixated later and for a shorter duration than emotional targets, suggesting efficient top-down control in avoiding emotional distraction, at least in terms of the overt distribution of spatial attention, while negative distractors still entailed a searching cost in terms of behavioral performance. Consistently, the fMRI data demonstrated an interplay between bottom-up and top-down processes to cope with emotional distraction. Negative (but not positive) stimuli were mandatorily processed by limbic/para-limbic regions (namely the left insula and the right amygdala) irrespective of the current task relevancy. As revealed by the analysis of inter-regional connectivity, however, the functional coupling between the left insula and the right prefrontal cortex increased while searching for neutral targets, specifically in the presence of negative emotional distractors. This indicates that the inter-regional coupling between affective and attention control regions plays a central role to attenuate emotional distraction.
The behavioral facilitation in searching for emotional compared to neutral targets is in agreement with most of the previous literature, indicating a global facilitation in processing emotional stimuli (Engen et al. [@CR24]; Pedale et al. [@CR54]; Schupp et al. [@CR64]), as well as in searching for emotional vs. neutral targets in displays including an emotional distractor (Flykt [@CR27]; Öhman et al. [@CR52]). Compared with searching for neutral targets in neutral scene, however, only positive (but not negative) targets entailed faster location discrimination, which might appear somewhat surprising. The large majority of emotional studies employed negative stimuli, while only a few studies have focused on the effect of positive emotional stimuli (e.g., Anderson et al. 2011; Bradley et al. 2004; van Hooff et al. 2011). The general finding is that positive stimuli associated with rewards (e.g., sexual stimuli, drugs-related stimuli for addicted individuals, or stimuli associated with reward via conditioning) tend to capture attentional resources equally well than negative stimuli. Here, we showed an advantage in searching for positive stimuli even though they were not associated with any specific reward. A similar finding was observed by Hodsoll and colleagues ([@CR34]). They used a visual search task in which participants were asked to search for a singleton target face among distracting faces (i.e., a female face among male faces or vice versa) and then judge the target face orientation (see also the description of this study in the Introduction). When one of the distractor faces had an emotional expression (fearful, angry or happy), the target face orientation discrimination was impaired. However, when the target face was an emotion singleton, only happy faces involved RT facilitation, indicating attentional capturing. Hodsoll and colleagues explained this finding suggesting "the possibility that the cost associated with processing negative expressions is not merely due to capture of attention to the wrong item (distractor rather than target). There appears to be an additional effect of slowing simply due to the very processing of the negative emotion and its unpleasant connotations. Such cost can offset any potential benefit of capture to negative target singletons and may result in a difficulty to disengage from negative emotional faces" (p. 352).
Here, we reported consistent findings with Hodsoll and colleagues ([@CR34]), with search benefits following positive stimuli, and search costs following negative stimuli (cf. Fig. [1](#Fig1){ref-type="fig"}c). Crucially, the current data on fixation patterns allow us to confirm and further extend the notion of "negative-related disengagement difficulty" to objects other than faces in complex visual scenes. The current eye-movement data corroborate this view, highlighting that negative stimuli were equally attentional grabbing than positive stimuli when task relevant. This latter finding was evidenced by similar indexes of first fixation latencies (i.e., the f-fix index; see also the fixation probability index on the Online Resource) on both positive and negative emotional targets (cf. Fig. [1](#Fig1){ref-type="fig"}d). However, the mean RT measured since the first fixation on target revealed disproportionately longer response latencies (i.e., the f-fix-RT index) for negative than for positive target-objects. This indicates that, although attentional grabbing, the negative stimuli entailed slower responses. This may arise from a difficulty to disengage from processing the negative emotional attributes of the object (cf. Hodsoll et al. [@CR34]). The analysis of fixation patterns also revealed that the strength of (overt) attentional capture by emotional stimuli is modulated by the current task relevance. In fact, first fixation latencies on emotional distractors were about 300 ms slower than first fixations on emotional targets, indicating efficient avoidance of emotional distraction (cf. Fig. [1](#Fig1){ref-type="fig"}d). In summary, these results supported a twofold effect driven by emotional objects: on one hand, a facilitation in directing the gaze to the emotional objects when they were the current targets, indicating the existence of attentional priorities related to these stimuli; on the other hand, subjects' gaze avoided the emotional stimuli when they were task irrelevant, indicating efficient top-down control to cope with emotional distractors.
The efficient top-down attentional control to avoid overt orienting of spatial attention on emotional distractors is consistent with our fMRI data. Searching for neutral targets in scenes including emotional distractors revealed the recruitment of the dorsal frontoparietal network, typically involved with top-down volitional control (Corbetta and Shulman [@CR14]; Corbetta et al. [@CR15]). Dorsal frontoparietal regions, including the superior premotor cortex and the posterior parietal cortex, have been shown to be consistently involved during active visual search and target detection using naturalistic stimuli (e.g., Ellison et al. [@CR22]; Ogawa and Macaluso [@CR51]). More generally, these regions have been shown to be involved with attention control and voluntary shifts of spatial attention (e.g., Corbetta and Shulman [@CR14]; Corbetta et al. [@CR15]; Hahn et al. [@CR32]). The increased activation of these regions when participants need to avoid emotional distractors to carry out the searching task is in good agreement with the current eye-movement results, indicating efficient filtering out of task-irrelevant emotional stimuli. Both findings suggest an increased necessity of top-down control to resist from directing the gaze and processing resources to emotional distractors.
Notwithstanding the recruitment of the top-down frontoparietal control system, the impact of negative stimuli on searching performance is evident when looking at the searching costs, with a decreased performance in searching for neutral targets in the presence of negative (but not positive) distractors compared to searching for neutral targets in neutral scenes (cf. bar 2 vs. bar 3 in Fig. [1](#Fig1){ref-type="fig"}c). This latter finding suggests that negative stimuli did capture attention resources, at least in a "covert" fashion (cf. first fixation latencies on emotional distractors about 300 ms longer than on emotional targets), with a consequent detriment of searching performance due to negative emotional distraction (e.g., Anticevic et al. [@CR2]; Dolcos et al. [@CR21]). The behavioral effect of negative distraction is also consistent with the evidence that negative stimuli here activated limbic/para-limbic regions, irrespective of their being relevant or not for the search task (see the left insula, Fig. [2](#Fig2){ref-type="fig"}b, and the right amygdala). The involvement of these regions in the processing of negative stimuli has been demonstrated by a number of previous studies (see, for reviews, Fox et al. [@CR28]; Lindquist et al. [@CR42]; Uddin [@CR70]). Here, we extend these results showing increased limbic/para-limbic activity during the exploration of complex visual scenes, including a number of non-emotional objects competing for processing resources with the emotional object.
These findings in limbic/para-limbic regions support the notion of a "mandatory" processing of negative emotional stimuli, irrespective of current task demands. For instance, Vuilleumier and colleagues (2001) asked participants to judge similar vs. different pairs of faces or houses presented in attended or unattended locations. The presentation of fearful faces reflected in increased activity of the amygdala, irrespective of any manipulation of spatial attention (though see Pessoa et al. [@CR57], for inconsistent findings). Similarly, in the context of visual search, Fenker and colleagues (2010) reported a magnetoencephalography (MEG) study in which they manipulated the difficulty (high vs. low) of searching for stimuli superimposed on task-irrelevant fearful vs. neutral faces. Task-irrelevant fearful faces elicited the activation of the extra striate visual cortex, irrespective of high or low searching demands and searching performance. Consistently, here we found increased limbic/para-limbic activity (namely in the left insula and in the right amygdala) whenever a negative stimulus occurred in the scene, which, therefore, appears to be unavoidably processed in a mandatory fashion.
The current task demand nevertheless modulated the functional connectivity of these regions, and, in particular, of the left insula. This para-limbic region has been recently proposed to act as a central "hub" integrating both internal information, involving bodily state and other signals from the adjacent limbic regions, and external information related to emotionally salient stimuli, thus coordinating the activity of control/monitor structures such as the anterior cingulated cortex and prefrontal regions along the dorsal frontoparietal cortex (Menon and Uddin [@CR47]; Uddin [@CR70]). The key role of the insular cortex as a central "emotional" hub has been supported by previous functional connectivity studies (e.g., Cauda et al. [@CR13]; Nomi et al. [@CR50]; Taylor et al. [@CR68]; see Uddin et al. [@CR72], for a review). Here, we directly demonstrate using an active task (i.e., a visual search) the crucial role of this region in processing emotional saliency and in coordinating top-down (dorsal frontoparietal) resources when the emotional stimulus is task irrelevant. The enhanced insular/prefrontal connectivity in conditions of emotional distraction could reflect the engagement of a neural circuit in which the insula plays first the role of "emotional salience detector" (Cauda et al. [@CR13]; Seeley et al. [@CR66]; Uddin [@CR70]). Then, to cope with emotional interference/distraction, the insula would determine a switch from exogenous to endogenous attentional control, through the increased connectivity with the dorsal frontoparietal control network (Bishop et al. [@CR5]; Vincent et al. [@CR74]). In line with this notion, here we found an increased insular activation in the presence of negative scenes, irrespective of the current relevance of the emotional object, but also a selective increase in the insular/prefrontal connectivity in conditions of searching for neutral targets in the presence of negative distractors (cf. Fig. [2](#Fig2){ref-type="fig"}d). It is worth noting that this condition (negS_neuT) entailed a behavioral cost compared to the baseline condition (neuS_neuT; i.e., searching for a neutral target without any emotional distractor), suggestive of the recruitment of additional resources (top-down control) to cope with emotional distraction and carry out the search task. The increased insular/prefrontal coupling can be then interpreted as representing, on one hand, the involvement of a mandatory processing of negative stimuli accomplished by the insula despite task irrelevance, and, on the other hand, the attempt to attenuate emotional distraction by the recruitment of neural top-down control resources to perform the visual search task. The latter would include the right MFG extending dorsally to the FEF, activated by searching for neutral (non-emotional) targets (cf. Fig. [2](#Fig2){ref-type="fig"}a).
To conclude, the current findings highlighted an interplay between bottom-up attention driven by emotionally salient negative stimuli, processed by ventral affective areas (namely insula and amygdala) and top-down control, processed by dorsal executive/control regions, during search for neutral targets in the presence of emotional distractors. Here, we demonstrated that the increased functional coupling between affective and control regions (i.e., the insular/prefrontal connectivity) is a core mechanism enabling the avoidance of emotionally negative distractors, thus allowing the deployment of spatial attention resources toward task-relevant emotionally neutral stimuli during visual search in complex and naturalistic scenes.
Electronic supplementary material
=================================
{#Sec20}
Below is the link to the electronic supplementary material.
Fig. S1 Supplementary Eye-movement analysis. Mean ± standard error of the fixation probability index related to fixations of the current target-object in the main conditions (negS_negT_negF, negS_neuT_neuF, neuS_neuT_neuF, posS_posT_posF, posS_neuT_neuF) or to fixations of the emotional object when it was not the to-be-searched target in the negS_neuT_negF and posS_neuT_posF conditions.
Fig. S2 Projections on a standard MNI template showing activation related to the processing "negative minus positive" scenes (red map), "negative minus neutral" scenes (yellow map), and the overlap between the two comparisons (orange map). Activation maps are displayed at a threshold of p \< 0.001 (uncorrected). Supplementary material 1 (PDF 324 kb)
It is worth noting that, despite our set of stimuli included emotional stimuli clearly distinguishable from the neutral stimuli in terms of valence and arousal, the absolute difference in valence and in arousal ratings between neutral and positive scenes was lower than the difference between neutral and negative scenes. This potential limitation is a consequence of the fact that we used a limited set of emotional categories. Stimuli eliciting highly arousing positive emotions typically represent social interactions (e.g., families, sexual stimuli, romantic couples; see the IAPS database, Lang et al. [@CR41]). Conversely, the scenes used for the current task did not include any human subject, which might result to be more salient than other objects, and then, attentional capturing per se.
Note that for the two latter conditions in which the emotional object was a distractor we did not compute the f-fix-RT index: given that here we analyzed fixations related to task-irrelevant objects that did not require any responses, it was pointless to report for these conditions the interval between the first fixation and response button press (i.e., the f-fix-RT index), which is related to a different object in the scene (i.e., the current to-be-searched neutral target).
For completeness, we conducted two one-way ANOVAs on the main conditions (negS_negT, negS_neuT, neuS_neuT, posS_posT, posS_neuT) for the accuracy and RT data. These revealed overall coherent results with the IES. Both analyses were significant: ACC \[*F*(4, 84) = 6.16, *p* \< 0.001\] and RTs \[*F*(4, 84) = 21.5, *p *\< 0.001\]. Planned comparisons revealed higher accuracy when the current target was an emotional (negS_negT or posS_posT) compared to a neutral object (negS_neuT or posS_neuT): *t*(21) = 2.75; *p* = 0.006 and *t*(21) = 3.76; *p *\< 0.001, respectively. Moreover, planned comparisons revealed faster RT for positive targets (posS_posT) compared to neutral targets in the presence of positive distraction (posS_neuT): *t*(21) = 10.17; *p *\< 0.001. As concerns the baseline condition (neuS_neuT), the planned comparisons revealed faster RT and higher accuracy when searching for positive targets (posS_posT): *t*(21) = 7.43; *p* \< 0.001 and *t*(21) = 3.36; *p* = 0.001, respectively; and only higher accuracy when searching for negative targets (negS_negT): *t*(21) = 1.77; *p* = 0.046.
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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The Neuroimaging Laboratory, Santa Lucia Foundation, is supported by The Italian Ministry of Health. This work has received support from the European Research Council, grant agreement n. 242809 to EM.
The authors declare that they have no conflict of interest.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed written consent was obtained from all individual participants included in the study, which was approved by the independent Ethics Committee of the Santa Lucia Foundation.
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All relevant data are within the manuscript and its Supporting Information files.
Introduction {#sec001}
============
In Germany, about 32 000 t of active pesticide substances are used in conventional agriculture each year \[[@pone.0237163.ref001]\]. Food producing animals are exposed to these pesticides through the ingestion of residues left on plants \[[@pone.0237163.ref002]--[@pone.0237163.ref005]\]. Acute intoxications are rare given low concentrations of residues \[[@pone.0237163.ref003]\], but long-term exposure could disturb cellular pathways leading to chronic diseases such as cancer and neurodegenerative disorders \[[@pone.0237163.ref006]\]. To protect the consumer, animal models are used to assess the potential risk of pesticide residues \[[@pone.0237163.ref007]\].
Several pesticides have been shown to impact transcription factors, such as the aryl hydrocarbon receptor (AhR) \[[@pone.0237163.ref008],[@pone.0237163.ref009]\]. AhR belongs to the bHLH/PAS family of transcription factors and is localized to the cytoplasm as an inactive heterodimer of HSP90 (heat shock protein 90), XAP2 (hepatitis B virus protein X-associated protein2), and p23 \[[@pone.0237163.ref010]\]. After ligand-binding, the chaperones are released and the ligand-AhR-complex translocates to the nucleus. The complex dimerizes with its required partner ARNT (aryl hydrocarbon nuclear translocator) and binds to special motifs (DRE, dioxin response elements) in the 5\'-untranslated region (UTR) resulting in increased AhR target gene expression \[[@pone.0237163.ref010],[@pone.0237163.ref011]\]. The AhR target gene cytochrome P450 1A1 (CYP1A1) is currently used as a biomarker to identify AhR-activating substances \[[@pone.0237163.ref012]--[@pone.0237163.ref015]\]. AhR activation increases detoxification to protect the organism from xenobiotic exposure by influencing the gene expression and activity of efflux transporters, such as the ABCG2 transporter (ATP-binding cassette subfamily G 2, Breast Cancer Resistance Protein, BCRP) \[[@pone.0237163.ref011],[@pone.0237163.ref016]--[@pone.0237163.ref018]\].
In bovine mammary glands, the bovine ABCG2 (bABCG2) transporter is localized in the apical membrane of alveolar epithelial cells \[[@pone.0237163.ref019]\] representing the main route of xenobiotics into milk \[[@pone.0237163.ref020]\]. The substrate spectrum contains commonly used veterinary drugs, such as antibiotics, anthelmintics \[[@pone.0237163.ref021]--[@pone.0237163.ref026]\], and other toxic compounds including mycotoxins \[[@pone.0237163.ref027]\]. Halwachs et al. (2013) showed that TCDD (2,3,7,8-tetrachlorodibenzo-*p*-dioxin) and the fungicide prochloraz increased bABCG2 transport activity in primary bovine mammary epithelial cells (PBMEC). Specific AhR DREs were identified in the 5\`-UTR of the bABCG2 gene. TCDD and prochloraz activated these DREs, increasing both expression and activity of the AhR target gene CYP1A. It was therefore concluded that AhR mediated the induction of bABCG2 activity \[[@pone.0237163.ref028]\]. Another study with Madin-Darby canine kidney II cells overexpressing bABCG2 (MDCKII-bABCG2) also demonstrated a TCDD- and prochloraz-mediated activation of AhR induced bABCG2 mRNA expression and efflux activity \[[@pone.0237163.ref029]\]. Therefore, the use of these pesticides could lead to an accumulation of potentially harmful ABCG2 substrates in dairy milk, elevating consumer risk.
While human drug approval processes investigate drug-drug interactions, this is not part of the pesticide approval process \[[@pone.0237163.ref030],[@pone.0237163.ref031]\]. The first step investigates pesticide-mediated induction of efflux transporters through i*n vitro* assays \[[@pone.0237163.ref031],[@pone.0237163.ref032]\]. However, very few studies have been published on pesticide-mediated regulation of drug transporters \[[@pone.0237163.ref032]\], and no species-specific *in vitro* models are available. Instead, pesticide assessment is more commonly based on animal experiments with lactating goats or cows. Nevertheless, replacement, reduction and refinement of animal studies (the 3R principle) is highly encouraged. This study proposes an *in vitro* MDCKII-bABCG2 cell model for identifying AhR-activating and bABCG2-inducing pesticides, implementing the 3R principle in practice. The study subsequently used the cell model to asses thirteen pesticides approved for use in the European Union and commonly found in conventional agriculture, particularly Germany.
Material and methods {#sec002}
====================
Chemicals and solvents {#sec003}
----------------------
Milli-Q water was prepared using a Millipore Synergy UV water purification system (Millipore S.A.S., Molsheim, France). Solvents at analytical grade were purchased from Carl Roth (Karlsruhe, Germany) and Merck Millipore (Darmstadt, Germany). All other chemicals were obtained from Sigma-Aldrich (Darmstadt, Germany), except for methiocarb (Dr. Ehrenstorfer, Augsburg, Germany) and 2,3,7,8-tetrachlorodibenzo-*p*-dioxin (TCDD, AccuStandard, New Haven, USA). Stock solutions of pesticides, TCDD, and 2,2',4,5,5'-pentachlorobiphenyl (PCB101) were prepared with declared solvents ([Table 1](#pone.0237163.t001){ref-type="table"}) in accordance with their maximal solubility listed in Pesticide Properties data base \[[@pone.0237163.ref033]\] or PubChem \[[@pone.0237163.ref034]\]. The prepared pesticide concentrations were subsequently diluted with cell culture medium to the same percentage level as the solvents stated in [Table 1](#pone.0237163.t001){ref-type="table"}.
10.1371/journal.pone.0237163.t001
###### Selected compounds, 1-fold MRL (maximum residue level) concentrations, and solvents used for their dissolution.
{#pone.0237163.t001g}
Substance group Lot no. Purity MRL muscle^a^/fat^b^ Solvent Reference
------------------------- -------------------------- ----------- -------- ---------------------- --------- --------------- ----- ----------------------------
**TCDD** Dioxin 215031440 n\. a. 1 toluene 0.1 \[[@pone.0237163.ref028]\]
**PCB101** Polychlorinated biphenyl SZBC032XV 98.0 n\. a. 10 DMSO 0.1 \[[@pone.0237163.ref028]\]
**Prochloraz** Imidazole SZBA112X 99.1 0.03^b^ 80 ethanol 0.1 \[[@pone.0237163.ref035]\]
**Tolclofos-methyl** Thiophosphoric ester SZBA323XV 97.9 0.01^a,b^ 33 methanol 0.1 \[[@pone.0237163.ref036]\]
**Chlorpyrifos-methyl** Organophosphate SZBC109XV 99.9 0.01^a,b^ 37 methanol 0.1 \[[@pone.0237163.ref037]\]
**Diflufenican** Carboxamide SZBC048XV 97.9 0.01^a,b^ 25 methanol 0.2 \[[@pone.0237163.ref038]\]
**Dimethoate** Organophosphate SZBC243XV 99.5 0.01^a,b^ 44 methanol 0.2 \[[@pone.0237163.ref039]\]
**Dimethomorph** Morpholine SZB9069XV 99.0 0.02^a,b^ 52 methanol 0.2 \[[@pone.0237163.ref040]\]
**Glyphosate** Phosphonate SZBC164XV 99.9 0.05^a,b^ 296 milli-Q water 0.4 \[[@pone.0237163.ref036]\]
**Ioxynil** Hydroxybenzonitrile SZB8114XV 99.8 1.00^a,b^ 2,700 methanol 0.5 \[[@pone.0237163.ref036]\]
**Iprodione** Dicarboximide SZBC174XV 99.5 0.05^a,b^ 100 methanol 0.1 \[[@pone.0237163.ref041]\]
**Methiocarb** Carbamate 10630 99.5 0.01^a,b^ 44 xylene 0.1 \[[@pone.0237163.ref042]\]
**Rimsulfuron** Sulfonylurea SZBC047XV 99.9 0.05^a,b^ 116 ethyl acetate 0.1 \[[@pone.0237163.ref043]\]
**Tebuconazole** Triazole SZBB055XV 99.5 0.10^a,b^ 325 toluene 0.1 \[[@pone.0237163.ref036]\]
**Thiacloprid** Neonicotinoid SZBC180XV 99.9 0.05^a,b^ 198 ethyl acetate 0.1 \[[@pone.0237163.ref036]\]
n.a. not available, COM (European Commission), DMSO (dimethyl sulfoxide), EFSA (European Food Safety Authority)
Selection of pesticides and their concentrations {#sec004}
------------------------------------------------
Commonly-used pesticides were selected for testing based on three key criteria: (1) the substance is approved for use in the European Union \[[@pone.0237163.ref044],[@pone.0237163.ref045]\], (2) the substance is present in the "top ten list" of pesticides \[[@pone.0237163.ref046]\], and (3) the substance has been identified in national monitoring programs as regularly exceeding maximum residue levels (MRLs) in food \[[@pone.0237163.ref047]--[@pone.0237163.ref050]\]. During the study the European approval of ioxynil and iprodione expired in 2015 and 2018, respectively \[[@pone.0237163.ref045]\]. In line with previous studies \[[@pone.0237163.ref008],[@pone.0237163.ref009],[@pone.0237163.ref028],[@pone.0237163.ref029],[@pone.0237163.ref051]\], the known AhR-activating pesticide prochloraz served as a positive control, while the non-activating tolclofos-methyl provided the negative control. To reflect the *in vivo* situation, applied pesticide concentrations corresponded to the MRLs permitted in bovine fat or muscle ([Table 1](#pone.0237163.t001){ref-type="table"}).
Cell culture {#sec005}
------------
All studies were performed with Madin-Darby canine kidney II cells (MDCKII) that overexpressed the bovine ABCG2 efflux transporter, as developed by Wassermann et al. 2013 \[[@pone.0237163.ref022]\]. Cells were cultivated in MEM medium with Earle's Salts (2.2 g/L NaHCO~3,~ stable glutamine; Biochrom, Berlin, Germany), supplemented with 10% (*v/v*) fetal calf serum (Life Technology, Karlsruhe, Germany), 1% (*v/v*) non-essential amino acids (Biochrom, Berlin, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom, Berlin, Germany), and grown at 37°C and 5% CO~2~. Cells were sub-cultured using 0.05% trypsin/0.02% EDTA (Biochrom, Berlin, Germany) every 3 to 4 days, up to a total of 14 passages.
Cytotoxicity (WST-1 assay) {#sec006}
--------------------------
Cytotoxicity of selected pesticides was determined by water-soluble tetrazolium 1 (WST-1) cytotoxicity assay (Roche, Mannheim, Germany). MDCKII-bABCG2 cells (3 x 10^4^ cells/mL) were seeded in 96-well plates (TPP, Trasadingen, Switzerland). After 24 h, cells were incubated with increasing concentrations (up to 500-fold MRL) of the selected pesticides for 72 h. Cells treated with 0.1% Triton X-100 served as positive control and untreated MDCKII-bABCG2 cells were used as negative control. The WST-1 assay was performed as described by Halwachs et al. (2013) \[[@pone.0237163.ref028]\]. SigmaPlot 11.0 (Systac Software, San Jose, CA, USA) was used to calculate the IC~50~ (inhibitory concentration), defined as the pesticide concentration that reduces cell viability to 50%. Data are presented as mean ± SEM, calculated from at least two independent experiments (N ≥ 2) with over 12 technical replicates per experiment (n ≥ 12).
Gene expression analysis (qPCR) {#sec007}
-------------------------------
For gene expression analysis, 5 x 10^4^ MDCKII-bABCG2 cells per mL were seeded in cell culture dishes (100/20 mm, Greiner Bio-one GmbH, Frickenhausen, Germany). 6--8 h after seeding, cells were treated either with pesticides in 0.1-, 1- and 10-fold MRL concentrations ([Table 1](#pone.0237163.t001){ref-type="table"}) or its corresponding solvent. The treatment was renewed once a day. TCDD (1 nM, 10 nM) and PCB101 (10 nM, 100 nM) were selected as the AhR-activating and non-activating compounds, respectively. After a total of 72 h of incubation, cells were washed twice with PBS, and then lysed with 1 mL of 0.05% trypsin/0.02% EDTA. The proteolytic cell dissociation was stopped with cell culture medium. The suspension was centrifuged (2000 rpm, 5 min), the supernatant removed and the pellet washed twice with PBS. Afterwards, cell density was adjusted to 3 x 10^6^ cells, and 400 μL RNAlater^®^ solution (Invitrogen, Life Technologies, Carlsbad, USA) was added to the pellet. After an overnight storage at 4°C, pellets were transferred to -20°C for subsequent total RNA extraction.
After RNAlater^®^ removal, total RNA was isolated using the TRIzol^®^ reagent (Invitrogen, Life Technologies, Milan, Italy) coupled with the RNeasy Mini kit (Qiagen, Hilden, Germany) for the aqueous phase purification, following manufacturer's procedures. A DNase digestion step with RNase-free DNase (Qiagen, Hilden, Germany) was also performed. The nucleic acid was evaluated in terms of quality and quantity using the Nanodrop ND1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and 1% agarose gel electrophoresis under denaturing conditions. Afterwards, 1 μg of total RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Life Technologies, Milan, Italy) following manufacturer's instructions. Finally, the cDNA samples were stored at -20°C until use.
To assess the effect of pesticides at the gene expression level, AhR (`ENSCAFT00000003863`), aryl hydrocarbon receptor repressor (AhRR, `ENSCAFT00000039404`), ARNT (`ENSCAFT00000019492`), CYP1A1 (`ENSCAFT00000028474`) and cytochrome P450 1B1 (CYP1B1, `ENSCAFT00000009970`) were chosen as candidate genes for mRNA analysis ([Table 2](#pone.0237163.t002){ref-type="table"}). Mitochondrial ATP synthase 5 (ATP5B, `ENSCAFT00000000224`), vacuolar protein trafficking and biogenesis associated homologue (CCZ1, `ENSCAFT00000024533`), hypoxanthine phosphoribosyl transferase 1 (HRPT1, `ENSCAFT00000002627`), ribosomal protein L8 (RPL8, `ENSCAFT00000002627`), ribosomal protein L32 (RPL32, `ENSCAFT00000046893`) and ribosomal protein S5 (RPS5, `ENSCAFT00000003710`) were used as internal control genes (ICGs, [S2 Table](#pone.0237163.s003){ref-type="supplementary-material"}).
10.1371/journal.pone.0237163.t002
###### Oligonucleotides and UPL probes used for gene expression analysis.
{#pone.0237163.t002g}
Gene Primer sequence (5\'-3\') Primer concentration (nM) Human UPL probe Amplicon size (bp) Reference
------------------------------- -------------------------------- --------------------------- ----------------- -------------------- ----------------------------
AHR `F: cttcgtgtgccgactaaggt` 300 \#120 63 \[[@pone.0237163.ref052]\]
`R: tggaaattcattgccagaaa` 300
AHRR `F: attttatgcgtcagcaacaatc` 300 \#165 68 \[[@pone.0237163.ref052]\]
`R: tgcatcacatccgtctgg` 300
ARNT `F: ccacttggacccctagcac` 300 \#62 60 \[[@pone.0237163.ref052]\]
`R: cttggctgtagcctgagca` 300
CYP1A1 `F: agggacgttgcgtctttgt` 300 \#59 65 \[[@pone.0237163.ref052]\]
`R: cgggttaccccatagcttct` 600
CYP1B1 `F: gacgccttcatcctctcg` 600 \#70 82 \[[@pone.0237163.ref052]\]
`R: gcacgtactccatgtccaac` 300
ATP5B `F: tctgaaggagaccatcaaagg` 600 \#120 74 \[[@pone.0237163.ref053]\]
`R: agaaggcctgttctggaagat` 600
CCZ1 `F: tgaagcactgcatttaattgtttat` 600 \#148 96 \[[@pone.0237163.ref054]\]
`R: cttcggcaaaaatccaatgt` 600
HPRT1 `F: tgctcgagatgtgatgaagg` 300 \#62 192 \[[@pone.0237163.ref053]\]
`R: tcccctgttgactggtcatt` 600
RPL8 `F: ggacggagctgttcatcg` 300 \#137 90 \[[@pone.0237163.ref054]\]
`R: gcacattgcctatgttgagc` 300
RPL32 `F: ccggaagttcctagtccaca` 600 \#146 78 Designed ex novo
`R: gcaatctctgcacaataagacttg` 600
RPS5 `F: ccggaacatcaagactattgc` 300 \#136 72 \[[@pone.0237163.ref054]\]
`R: gaattggaagagcccttgg` 300
Gene expression levels were determined using previously validated and published quantitative real time PCR (qPCR) assays \[[@pone.0237163.ref052]--[@pone.0237163.ref054]\] except for RPL32, for which oligonucleotides and the corresponding Universal Probe Library (UPL) probe were designed *ex novo* using the UPL Assay Design center web service (Roche, Basel, Switzerland). The oligonucleotide sequences and concentrations, amplicon sizes and UPL probes are listed in [Table 2](#pone.0237163.t002){ref-type="table"}. For evaluating qPCR performance, standard calibration curves were generated by amplifying decreasing amounts of MDCKII cDNA diluted at 3-fold intervals. Standard curve analyses of target genes and ICGs showed high test linearities (error \< 0.2) and acceptable amplification efficiencies (comprised in the range between 90% and 110%). The ICGs' amplification efficiency was equal to that of the target genes. The main qPCR parameters (efficiency, linearity and dynamic range) are reported in [S1 Table](#pone.0237163.s002){ref-type="supplementary-material"}. The qPCR reactions were performed using LightCycler® 480 (Roche, Basel, Switzerland), clear LightCycler® 480 Multiwell Plate 96 and standard PCR conditions. Assay-dependent forward and reverse primer concentrations were mixed with 1X LightCycler® 480 Probe Master, then the selected UPL probe (200 nM final concentration) and 2.5 ng of cDNA were added to a final volume of 10 μL. Crossing point (C~P~) or cycle number at detection threshold values were acquired using the LightCycler® 480 software release 1.5.0 using the second derivative maximum method \[[@pone.0237163.ref055]\]. The relative quantification of obtained data was performed using the ΔΔCt method \[[@pone.0237163.ref056]\]. For the normalization step, only ICGs not affected by the treatment were used for each compound ([S1 Table](#pone.0237163.s002){ref-type="supplementary-material"}). The selection criterion was the absence of a statistically significant difference in the arithmetic mean of ICGs between control and treated cells. Data were normalized against the vehicle control which was set as 1. Relative quantification values (RQ) were expressed in arbitrary units (AU) as mean ± SEM from three independent experiments with two technical replicates per experiment (N = 3, n = 6).
CYP1A activity (EROD assay) {#sec008}
---------------------------
The CYP1A enzyme reduces 7-ethoxyresorufin to resorufin quantifiable through spectrofluorometry \[[@pone.0237163.ref013]\]. The EROD assay was therefore chosen to investigate a pesticide-mediated induction of the AhR target gene CYP1A1. Cells were seeded in 24-well plates (Sarstedt, Nümbrecht, Germany) at a density of 3 x 10^4^ cells/mL and treated with pesticides, solvents, TCDD or PCB101 as stated for qPCR analysis. After incubating the cells with pesticides for 72 h EROD activity was measured according to Donato et al. (1993) \[[@pone.0237163.ref013]\], with minor modifications. Cells were washed three times with warm PBS buffer (Biowest SAS, Nuaillé, France), then incubated with MEM Earle's medium, supplemented with 10 nM dexamethasone, 16 μM 7-ethoxyresorufin, and 10 μM dicoumarol for 2 h (75 rpm, 37°C, 5% CO~2~). Cell supernatants (90 μL) and resorufin reference standards were transferred to a 96-well plate (TPP, Trasadingen, Switzerland). Cell supernatant treated only with EROD-medium for 5 min was used as a background. To reduce conjugates of formed resorufin, 30 μL 0.1 M sodium acetate buffer (pH 4.5), containing 15 fishman units of β-glucuronidase and 120 roy units of arylsulfatase (Roche, Mannheim, Germany), was added for 1 h (75 rpm, 37°C, 5% CO~2~). Following incubation, 240 μL ethanol was added to each well and centrifuged for 10 min at room temperature (3000 rpm). The formed resorufin was detected by spectrofluorometry (540 nm excitation/595 nm emission wavelengths, Tecan Genios/Tecan Infinite F200 Pro, Crailsheim, Germany) and EROD activity was calculated as pmol/min per mg protein. Protein amounts were determined by bicinchoninic acid assay (BCA, Thermo Scientific, Rockford, USA) following the manufacturer's instructions. Data were normalized against the vehicle control which was set as 1 and expressed as mean ± SEM from three independent experiments with three technical replicates per experiment (N = 3, n = 9).
bABCG2 efflux activity (Hoechst 33342 accumulation assay) {#sec009}
---------------------------------------------------------
The Hoechst 33342 accumulation assay was used to detect bABCG2 efflux activity. The assay protocol was adapted from that published by Halwachs et al. (2014) \[[@pone.0237163.ref051]\]. MDCKII-bABCG2 cells were seeded in 96-well plates (TPP, Trasadingen, Switzerland) at a density of 2 x 10^4^ cells/mL. After 6--8 h, cells were treated with 1-fold MRL concentration of prochloraz or 10-fold MRL concentration of tolclofos-methyl for 12 h to 48 h. A 10-fold MRL incubation with chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron and tebuconazole was conducted for 48 h. Controls were treated with respective solvents ([Table 1](#pone.0237163.t001){ref-type="table"}). Simultaneous incubations with the ABCG2 inhibitor Ko143 (5 μM) were performed to prove the involvement of the bABCG2 transporter. Subsequently, MDCKII cells were washed twice with warm PBS followed by exposure to MEM Earle´s medium supplemented with Hoechst 33342 (5 μM, Biochemica Applichem, Darmstadt, Germany), either in the presence or absence of Ko143 (5 μM) for 30 min in a shaking incubator (150 rpm, 37°C, 5% CO~2~). Cells were subsequently washed twice with cold PBS, lysed with 0.1% SDS/PBS, and the intracellular amount of fluorescent dye Hoechst 33342 was measured (360 nm excitation/465 nM emission wavelengths, Tecan Infinite F200 Pro, Crailsheim, Germany). The relative fluorescence units (RFU) per mg protein were calculated by comparing pesticide-treated cells to solvent-treated control cells. Protein amounts were quantified by BCA. Three independent experiments were carried out, each containing six technical replicates, and data were expressed as mean ± SEM (N = 3, n = 18).
Statistical analysis {#sec010}
--------------------
A statistical analysis of the gene expression data was performed on three independent experiments (N = 3, n = 6). The results from the control and treated cells (two different concentrations) were compared for each compound using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (GraphPad Prism 5 software, San Diego, California, USA). Data from the EROD (N = 3, n = 9) and Hoechst 33342 assays (N = 3, n = 18) were examined by one-way ANOVA with Fisher-LSD post-hoc test. The level of significance was set as p ≤ 0.05.
Results and discussion {#sec011}
======================
Cytotoxicity of pesticides, TCDD and PCB101 {#sec012}
-------------------------------------------
The cytotoxicity of the selected pesticides and their solvents was determined through WST-1 assay. An IC~50~ value was only able to be calculated for ioxynil (IC~50~ \~ 97.5 μM, [S1B Fig](#pone.0237163.s005){ref-type="supplementary-material"}). A decreased cell viability was also detected for prochloraz- and tolclofos-methyl-treated cells in 100- and 500-fold MRL concentrations without a calculable IC~50~ value ([S1A Fig](#pone.0237163.s005){ref-type="supplementary-material"}). In general, no cytotoxic effects of the chosen solvents or pesticides were observed from 1- to 10-fold MRL concentrations in MDCKII-bABCG2 cells ([S1C](#pone.0237163.s005){ref-type="supplementary-material"}, [S1D](#pone.0237163.s005){ref-type="supplementary-material"} and [S2A--S2C](#pone.0237163.s006){ref-type="supplementary-material"} Figs). The cytotoxicity of TCDD, PCB101 and their solvents has been previously determined in MDCKII-bABCG2 cells \[[@pone.0237163.ref029]\].
Validation of MDCKII-bABCG2 cells to known AhR ligands {#sec013}
------------------------------------------------------
Contaminants such as polychlorinated hydrocarbons bind to AhR and activate the AhR pathway \[[@pone.0237163.ref010],[@pone.0237163.ref015]\]. Previous studies have shown that the known AhR-activating ligand TCDD induced the bABCG2 efflux activity in PBMEC and MDCKII-bABCG2 cells while the non AhR-activating compound PCB101 did not \[[@pone.0237163.ref028],[@pone.0237163.ref029]\]. In the present study, MDCKII-bABCG2 cells were treated with TCDD (1 nM, 10 nM) and PCB101 (10 nM, 100 nM) for 72 h to determine AhR activation by known AhR target genes. As shown in [S3 Fig](#pone.0237163.s007){ref-type="supplementary-material"}, TCDD significantly increased CYP1A1 (4-fold, [S3A Fig](#pone.0237163.s007){ref-type="supplementary-material"}), CYP1B1 (3-fold, [S3B Fig](#pone.0237163.s007){ref-type="supplementary-material"}), and AhRR (2-fold, [S3C Fig](#pone.0237163.s007){ref-type="supplementary-material"}) mRNA levels in both applied concentrations compared to the control. AhR mRNA levels were not affected by TCDD ([S3D Fig](#pone.0237163.s007){ref-type="supplementary-material"}). These results are in line with existing literature which identified CYP1A1, CYP1B1, and AhRR as typical AhR target genes \[[@pone.0237163.ref010],[@pone.0237163.ref057],[@pone.0237163.ref058]\]. TCDD induced CYP1A enzyme activity has also previously been shown in MDCKII-bABCG2 cells \[[@pone.0237163.ref029]\].
While PCB101 did not affect the mRNA expression of CYP1B1 or AhRR, CYP1A1 was significantly down-regulated ([S4 Fig](#pone.0237163.s008){ref-type="supplementary-material"}). However, in previous EROD studies, no alteration of CYP1A enzyme activity was detected in MDCKII-bABCG2 cells \[[@pone.0237163.ref029]\]. Post-transcriptional regulation by dynamic RNA modification may explain why the down-regulation of CYP1A1 mRNA by PCB101 does not decrease CYP1A enzyme activity \[[@pone.0237163.ref059]\].
This experiment proved the MDCKII-bABCG2 cell line contained a functional AhR signaling pathway with inducible AhR target genes (AhRR, CYP1A1 and CYP1B1 mRNAs) and CYP1A enzyme activity by the known AhR ligand TCDD.
Identification of AhR-activating pesticides {#sec014}
-------------------------------------------
Several pesticides activate transcription factors, leading to increased expression and activity of their target genes \[[@pone.0237163.ref032]\]. The mRNA levels of AhR, AhRR, CYP1A1 and CYP1B1 were measured by qPCR while CYP1A enzyme activity was measured by EROD assay \[[@pone.0237163.ref014],[@pone.0237163.ref015]\].
The positive control prochloraz (1-fold MRL) significantly increased CYP1A1 mRNA expression (1.5-fold, [Fig 1A](#pone.0237163.g001){ref-type="fig"}) and CYP1A activity (3-fold, [Fig 1B](#pone.0237163.g001){ref-type="fig"}), while CYP1B1, AhR, AhRR or ARNT expression levels were not altered. In accordance with our previous studies \[[@pone.0237163.ref028],[@pone.0237163.ref029],[@pone.0237163.ref051]\], neither the negative control tolclofos-methyl nor the solvents had an impact on the AhR target genes CYP1A1 ([Fig 2A and 2B](#pone.0237163.g002){ref-type="fig"}, [S5 Fig](#pone.0237163.s009){ref-type="supplementary-material"}), CYP1B1, or AhRR ([S3 Table](#pone.0237163.s004){ref-type="supplementary-material"}, [S5 Fig](#pone.0237163.s009){ref-type="supplementary-material"}).
{#pone.0237163.g001}
{#pone.0237163.g002}
The pesticides dimethoate, dimethomorph, glyphosate, iprodione, methiocarb, and thiacloprid did not alter CYP1A1 mRNA expression or CYP1A-mediated EROD activity ([Fig 3A and 3B](#pone.0237163.g003){ref-type="fig"}) in a result comparable to the negative control tolclofos-methyl ([Fig 2A and 2B](#pone.0237163.g002){ref-type="fig"}). These five pesticides were therefore classified as "non-AhR-activating pesticides" in accordance with the literature \[[@pone.0237163.ref008],[@pone.0237163.ref009]\].
{ref-type="table"}) for 72 h. **A.** CYP1A1 mRNA expression was examined by qPCR and **B.** CYP1A activity was detected by EROD assay. All presented data were normalized to control levels set as 1 and expressed as mean ± SEM (three independent experiments, one-way ANOVA with Tukey's post hoc test or Fisher LSD post hoc test, \* significant difference in comparison to the control: \* p ≤ 0.05).](pone.0237163.g003){#pone.0237163.g003}
As shown in [Fig 4A](#pone.0237163.g004){ref-type="fig"}, chlorpyrifos-methyl, ioxynil, rimsulfuron and tebuconazole significantly increased CYP1A1 mRNA levels by at least 50% compared to control levels. 10-fold MRL concentrations of these pesticides were also significantly increase EROD activity ([Fig 4B](#pone.0237163.g004){ref-type="fig"}). In contrast to TCDD ([S3B and S3C Fig](#pone.0237163.s007){ref-type="supplementary-material"}), none of these pesticides altered AhRR or CYP1B1 mRNA expression ([S3 Table](#pone.0237163.s004){ref-type="supplementary-material"}). CYP1A1 has previously been identified as the most inducible AhR target gene \[[@pone.0237163.ref057]\] which could explain why the tested pesticides only impact CYP1A1. Furthermore, a TCDD-mediated AhR induction is up to 500.000-fold stronger than prochloraz \[[@pone.0237163.ref009]\], potentially amplifying its impact to the other AhR target genes.
{ref-type="table"}) for 72 h. A. Gene expression analysis of CYP1A1 and B. EROD assay were performed. All data were normalized to control levels set as 1 and expressed as mean ± SEM (three independent experiments, one-way ANOVA with Tukey's post hoc test or Fisher LSD post hoc test, \* significant difference in comparison to the control: \*\*\* p ≤ 0.001, \*\* p ≤ 0.01, \* p ≤ 0.05).](pone.0237163.g004){#pone.0237163.g004}
While treatment with 10-fold MRL of diflufenican only slightly increased CYP1A1 mRNA expression, EROD activity was doubled ([Fig 4B](#pone.0237163.g004){ref-type="fig"}). Therefore, chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron, and tebuconazole were classified as "AhR-activating pesticides".
AhR-activating pesticides influence the bABCG2 efflux activity {#sec015}
--------------------------------------------------------------
The Hoechst 33342 accumulation assay was used to assess bABCG2-inducing pesticides. To rule out the direct interaction of applied pesticides with the bABCG2 transporter, cells were washed twice with PBS following pesticide exposure. Incubation with Hoechst 33342 then followed in the absence of pesticide.
MDCKII-bABCG2 cells contain 98 base pairs of the 5\'-UTR including DREs in front of the bABCG2 gene \[[@pone.0237163.ref022]\]. After ligand-binding, the AhR-ligand-ARNT-complex binds to the DREs, inducing bABCG2 gene transcription and subsequent secretion of bABCG2 substrates \[[@pone.0237163.ref017],[@pone.0237163.ref018],[@pone.0237163.ref029]\]. In line with previous experiments \[[@pone.0237163.ref029],[@pone.0237163.ref022],[@pone.0237163.ref051]\], treatment of the cells with the ABCG2 inhibitor Ko143 (5 μM) significantly increased the intracellular Hoechst 33342 amount in comparison to solvent- and pesticide-treated cells (Figs [1C](#pone.0237163.g001){ref-type="fig"}, [2C](#pone.0237163.g002){ref-type="fig"} and [5C](#pone.0237163.g005){ref-type="fig"}). This proved that the bABCG2 transporter mediates Hoechst secretion. Elevated ABCG2 efflux activity is therefore represented by observing a decreased intracellular accumulation of the ABCG2 substrate Hoechst 33342.
{ref-type="table"}) for 24 h and the Hoechst 33342 accumulation assay was performed in presence or absence of the ABCG2-inhibitor Ko143 (5 μM). Data are expressed as mean ± SEM (three independent experiments, one-way ANOVA with Fisher LSD post hoc test, \* significant difference in comparison to the control: \*\*\* p ≤ 0.001, \*\* p ≤ 0.01, \* p ≤ 0.05; \# significantly different to Ko143: \#\#\# p ≤ 0.001, \#\# p ≤ 0.01, \# p ≤ 0.05).](pone.0237163.g005){#pone.0237163.g005}
Cells treated with 1-fold MRL of the positive control prochloraz for 24 h significantly decreased Hoechst 33342 levels by approximately 20% ([Fig 1C](#pone.0237163.g001){ref-type="fig"}). The same results were observed after 48 h of exposure ([Fig 1C](#pone.0237163.g001){ref-type="fig"}). The negative control tolclofos-methyl (10-fold MRL) had no impact on the bABCG2 efflux activity ([Fig 2C](#pone.0237163.g002){ref-type="fig"}).
Cells incubated with 10-fold MRL of all previously identified AhR-activating pesticides significantly decrease the level of intracellular Hoechst after 24 h when compared to their respective solvent controls ([Fig 5](#pone.0237163.g005){ref-type="fig"} and [S6 Fig](#pone.0237163.s010){ref-type="supplementary-material"}). All identified AhR-activating pesticides, chlorpyrifos-methyl, diflufenican, rimsulfuron, ioxynil and tebuconazole (10-fold MRL) were therefore proven to induce the bABCG2 efflux activity.
As shown in [Fig 5](#pone.0237163.g005){ref-type="fig"} and [S6 Fig](#pone.0237163.s010){ref-type="supplementary-material"}, this result was only detectable after the cells were incubated with pesticide for 24 h, indicating that treatment with AhR-activating pesticides for 24 h represents the best experimental set-up to detect altered transporter activity in this approach.
Conclusion {#sec016}
==========
The current pesticide approval process relies on animal experiments conducted with lactating goats or cows. To reduce the need for these experiments and implement the 3R principle in practice, the MDCKII-bABCG2 cell model was investigated as an *in vitro* tool to identify AhR-activating and bABCG2-inducing pesticides.
MDCKII-bABCG2 cells contain an inducible AhR pathway where AhR activation increases the expression of typical AhR target genes, particularly CYP1A1. Thus, the EROD assay represents appropriate tool to detect AhR-activating pesticides.
Similar to the positive control prochloraz, 10-fold MRL concentrations of chlorpyrifos-methyl, diflufenican, ioxynil, rimsulfuron and tebuconazole activated the AhR pathway and increased bABCG2 efflux activity. As concluded by Chedik et al. 2018 \[[@pone.0237163.ref032]\], an increased efflux activity eliminates substrates faster from the body. In the bovine mammary gland, the enhanced secretion of bABCG2-substrates into milk, such as drugs and toxins, creates a potential risk to consumers.
Overall, the presented approach is an appropriate *in vitro* tool to reduce the number of animals required for residue depletion studies.
Supporting information {#sec017}
======================
######
(XLSX)
######
Click here for additional data file.
###### qPCR assay parameters: Efficiency, linearity and dynamic range.
(PDF)
######
Click here for additional data file.
###### Internal Control Genes (ICGs) used for the relative quantification analysis of data.
(PDF)
######
Click here for additional data file.
###### CYP1B1, AhR, AhRR and ARNT mRNA expression in untreated and treated MDCKII-bABCG2 cells.
Data were normalized to control levels and are expressed as fold change of relative quantification value (RQ) in arbitrary units (AU) (mean ± SEM, N = 3, n = 6, one-way ANOVA with Tukey's post hoc test, significant differences are shaded in grey, ^a^ significantly different to the control, ^b^ significant difference between 1- and 10-fold MRL concentration, ^aaa,\ bbb^ p ≤ 0.001; ^aa,\ bb,\ cc^ p ≤ 0.01; ^a,\ b^ p ≤ 0.05).
(PDF)
######
Click here for additional data file.
###### Cytotoxicity of prochloraz, tolclofos-methyl, ioxnyil, chlorpyrifos-methyl, diflufenican, dimethoate and dimethomorph.
MDCKII cells were incubated with pesticides in increasing concentrations for 72 h. Cell viability was measured by water soluble tetrazolium-1 (WST-1) assay. Data were normalized to control levels and are expressed as percentage of cell viability (mean ± SEM, N = 2, n = 12, one-way ANOVA with Holm-Šidák post hoc test, \* significant difference in comparison to the control: \*\*\* p ≤ 0.001, \*\* p ≤ 0.01, \* p ≤ 0.05).
(PDF)
######
Click here for additional data file.
###### Cytotoxicity of glyphosate, thiacloprid, iprodione, methiocarb, rimsulfuron and tebuconazole.
MDCKII cells were incubated with pesticides in increasing concentrations for 72 h. Cell viability was measured by water soluble tetrazolium-1 (WST-1) assay. Data were normalized to control levels and are expressed as percentage of cell viability (mean ± SEM, N = 2, n = 12, one-way ANOVA with Holm-Šidák post hoc test, \* significant difference in comparison to the control: \*\*\* p ≤ 0.001, \*\* p ≤ 0.01, \* p ≤ 0.05).
(PDF)
######
Click here for additional data file.
###### Effects of AhR-inducing TCDD upon gene expression of the AhR gene battery.
MDCKII cells were incubated with TCDD (1 nM, 10 nM) for 72 h followed by gene expression analysis on CYP1A1 (A), CYP1B1 (B), AhRR (C) and AhR (D). Data were normalized to control levels and are expressed as fold change of relative quantification value (RQ) in arbitrary units (AU) (mean ± SEM, N = 3, n = 6, one-way ANOVA with Tukey's post hoc test, \*\*\* indicate significant differences in comparison to the control with p ≤ 0.001).
(PDF)
######
Click here for additional data file.
###### Effects of PCB101 upon gene expression of the AhR gene battery.
MDCKII cells were incubated with PCB101 (10 nM, 100 nM) for 72 h followed by gene expression analysis on CYP1A1 (A), CYP1B1 (B), AhRR (C) and AhR (D). Data were normalized to control levels and are expressed as fold change of relative quantification value (RQ) in arbitrary units (AU) (mean ± SEM, N = 3, n = 6, one-way ANOVA with Tukey's post hoc test, \* significant differences in comparison to the control, \*\*\* p ≤ 0.001; \*\* p ≤ 0.01; \* p ≤ 0.05).
(PDF)
######
Click here for additional data file.
######
Effects of the solvents used to dissolve the pesticides and dioxins upon gene expression of CYP1A1 (A), CYP1B1 (B), AhRR (C) and AhR (D). MDCKII-bABCG2 cells were incubated with solvents, listed in [Table **1**](#pone.0237163.t001){ref-type="table"}, for 72 h. Gene expression analysis was subsequently carried out on CYP1A1 (A), CYP1B1 (B), AhRR (C) and AhR (D). Data were normalized to control levels and are expressed as fold change of relative quantification value (RQ) in arbitrary units (AU) (mean ± SEM, N = 3, n = 6, one-way ANOVA with Tukey's post hoc test, level of significance p ≤ 0.05).
(PDF)
######
Click here for additional data file.
###### Hoechst 33342 accumulation in MDCKII cells after 48 h incubation with AhR-activating pesticides.
MDCKII-bABCG2 cells were treated with the selected pesticides in 10-fold MRL concentration ([Table 1](#pone.0237163.t001){ref-type="table"}) for 48 h and the Hoechst 33342 accumulation assay was performed in presence or absence of the ABCG2-inhibitor Ko143 (5 μM). Data are expressed as mean ± SEM (three independent experiments, one-way ANOVA with Fisher LSD post hoc test, \* significant difference in comparison to the control: \*\*\* p ≤ 0.001, \*\* p ≤ 0.01, \* p ≤ 0.05; \# significantly different to Ko143: \#\#\# p ≤ 0.001, \#\# p ≤ 0.01, \# p ≤ 0.05).
(PDF)
######
Click here for additional data file.
We would like to thank C. Lakoma and B. Scholz for their skillful technical assistance. We acknowledge support from Leipzig University for Open Access Publishing.
ABCG2
: ATP-binding cassette subfamily G 2
AhR
: aryl hydrocarbon receptor
AhRR
: aryl hydrocarbon receptor repressor
ARNT
: aryl hydrocarbon receptor nuclear translocator
ATP5
: mitochondrial ATP synthase 5
AU
: arbitrary units
bABCG2
: bovine ABCG2
BCA
: bicinchoninic acid assay
BCRP
: breast cancer resistance protein
bHLH/PAS
: basic-region helix-loop-helix/period aryl hydrocarbon receptor nuclear translocator-single minded
CCZ1
: vacuolar protein trafficking and biogenesis associated homologue
C~P~
: crossing point or cycle number at detection threshold
CYP1A1
: cytochrome P450 family 1, subfamily A, isoform 1
CYP1B1
: cytochrome P450 family 1, subfamily B, isoform 1
DRE
: dioxin response elements
EROD
: 7-ethoxyresorufin-*Ο*-deethylase
HPRT1
: hypoxanthine phosphoribosyl transferase 1
HSP90
: heat shock protein 90
ICG
: internal control gene
IC~50~
: inhibitory concentration
MDCKII
: Madin-Darby canine kidney II cells
MRL
: maximum residue level
N
: number of independent experiments
n
: number of technical replicates
n.a.
: not available
PBMEC
: primary bovine mammary epithelial cells
PCB101
: 2,2',4,5,5'-pentachlorobiphenyl
qPCR
: quantitative real time polymerase chain reaction
RFU
: relative fluorescence unit
RPL32
: ribosomal protein L32
RPL8
: ribosomal protein L8
RPS5
: ribosomal protein S5
RQ
: relative quantification value
SEM
: standard error of the mean
TCDD
: 2,3,7,8-tetrachlorodibenzo-*p*-dioxin
UPL
: universal probe library
UTR
: untranslated region
WST-1
: water-soluble tetrazolium 1
XAP2
: hepatitis B virus protein X-associated protein 2
[^1]: **Competing Interests:**MSD Animal Health Innovation GmbH provided financial support in the form of salary to author SH during the time of the manuscript preparation. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
[^2]: Current address: MSD Animal Health Innovation GmbH, Schwabenheim, Germany
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Platelet rich plasma (PRP) is considered a promising technology in topical therapy, as it contributes to the wound healing process. The mechanism of action is related to the actions of: biomolecules, such as adhesive proteins, which promote cell interaction, hemostasis, coagulation and extracellular matrix composition; coagulation factors and associated proteins that produce thrombin; fibrinolytic factors and associated proteins that produce plasmin and vascular remodeling; and, proteases and antiproteases, which act on angiogenesis, vascular remodeling and coagulation control. Growth factors promote chemotaxis, cell proliferation and differentiation, and angiogenesis; chemokines, cytokines and others act to regulate angiogenesis and intracellular communication; and, antimicrobial proteins have bactericidal and fungicidal actions^(^ [@B1] ^)^.
Thus, the PRP acts in the various healing phases, promoting the shortening of the inflammatory phase by means of hemostasis, the provisional fibrin matrix, and the reduction of biofilm, promoting the formation of granulation tissue (chemotaxis, angiogenesis, and cell proliferation), epithelialization, keratinocyte proliferation and migration, and remodeling, with extracellular matrix synthesis^(^ [@B1] ^)^.
Platelet rich plasma is a product that originates from the centrifugation of whole blood, which is rich in growth factors and structural proteins that stimulate collagen and the extracellular matrix production that promote tissue repair, and which stimulate neovascularization and tissue regeneration^(^ [@B2] ^)^. It can be of an autologous nature, when the blood used for centrifugation comes from the patient himself; homologous, when the source is another patient; and heterologous, when it is derived from animal blood. The effectiveness of autologous, heterologous, and homologous PRP requires further studies^(^ [@B3] ^)^.
Studies performed in humans have shown the effectiveness of topical PRP in stimulating the healing of chronic wounds, such as diabetic ulcers, when compared with other antiseptic dressings for both healing and infection prevention^(^ [@B4] ^)^. Studies with venous ulcers have shown improved area reduction and a higher number of healed ulcers^(^ [@B5] ^-^ [@B8] ^)^.
Platelet rich plasma infiltrations have also been studied in clinical trials, such as: meniscus muscle repair with functional improvement after 18 weeks^(^ [@B9] ^)^; decrease of complications of primary total joint arthroplasty, with diminished bleeding and improved healing^(^ [@B10] ^)^; and, the use in knee osteoarthritis showed improvement of degeneration and quality of life^(^ [@B11] ^)^.
Autologous PRP, considered to be a bio stimulant, has also been used in dermatology for facial, neck, and hand rejuvenation due to its ease of application, lower risk of infection and allergies, in addition to its having hemostatic properties that reduce the possibility of bruising^(^ [@B12] ^-^ [@B14] ^)^. In addition to dermatological treatments, it has been used in areas such as androgenic alopecia in men and women, where it demonstrated an increase in the quantity and caliber of hair^(^ [@B15] ^)^. Thus, the use of PRP has grown significantly in topical therapies.
It can be prepared using commercial kits, a closed system, or by centrifugation and preparation with technical handling of the supernatant after centrifugation, by aspiration, in an open system.
Treatment with PRP has been considered experimental in Brazil, according to the Federal Council of Medicine Opinion No. 20/2011, due to the need for more scientific evidence to support its regulation^(^ [@B16] ^)^.
Given this, the National Health Surveillance Agency (ANVISA - Agência Nacional de Vigilância Sanitária) determined, in Technical Note No. 64/2015, that the processing of PRP for autologous purpose is allowed on an experimental basis, and that closed systems used in health facilities should be regulated by ANVISA^(^ [@B17] ^)^.
The Federal Council of Dentistry, in Resolution No. 158, of June 8, 2015, allows venipuncture and manipulation of PRP, in a closed system, by a qualified dentist or a health professional in conjunction with the dentist^(^ [@B18] ^)^.
Currently, scientific evidence studies such as controlled clinical trials are being developed to establish the effectiveness of PRP. But there are still few studies that assess the cost of producing PRP, either by autologous or homologous means. Studies that do evaluate the cost have considered all the treatment performed, without separately describing the cost of performing the technique^(^ [@B5] ^,^ [@B19] ^)^. Other studies with economic designs, analyze the cost of kits available at a mean cost of € 132.90 per kit per PRP session^(^ [@B20] ^)^ and US \$450.00 per two sessions^(^ [@B21] ^)^. Both of these studies were conducted outside the Brazilian context, and with a different socioeconomic scenario.
Given the worldwide evidence of PRP use, and the possibility of obtaining and using PRP, this study aims to contribute with evidence on the cost of human resources, specifically nurses, in obtaining blood and preparing the PRP.
Resource allocation effectiveness has been the subject of policy discussions, as with health spending growth and limited resources; decision-making on resource allocation should be based on health technology assessment, as the health economy is based on opportunity cost, that is, resources applied to certain programs and technologies imply that others are not provided, so that their cost is not only represented by the resources spent on that technology, but also on the value of what is no longer being provided^(^ [@B22] ^)^. In this sense, cost analyses and economic assessments can provide decision-making inputs, contributing significantly to health policies^(^ [@B23] ^)^.
Cost analyses are the key steps in providing support for the development of economic assessments^(^ [@B24] ^)^, which involve comparing the cost between one or more alternatives, and the outcomes of interventions.
The objectives were to estimate the direct cost of producing autologous PRP gel, and to compare the cost of PRP production, considering the material cost in units of the Unified Health System (SUS).
Method
======
This was an economic, prospective, longitudinal study, with direct cost estimation, whose setting was the SUS, conducted in a university hospital in the state of Rio de Janeiro, over a period of 12 weeks.
Direct costs are related to the resources consumed directly in treatment, health intervention, or care, such as: material and human resources, products, and services^(^ [@B22] ^)^.
This study is part of a macroproject, "Effectiveness and Cost of PRP as a topical therapy for venous ulcer patients", approved by the Ethics Committee of the School of Medicine, under Opinion No. 1,378,184.
For production of autologous PRP gel, two steps were necessary: Step 1 - participant's blood collection by venipuncture, with vacuum catheter; Step 2 - blood centrifugation to prepare the PRP by means of an adapted single centrifugation technique^(^ [@B25] ^)^ and activation using 10% calcium glyconate in the PRP gel.
To estimate the direct cost of production, the following categories were analyzed: material and human resources.
Data collection was performed from May of 2016 to December of 2017, using direct observation to identify and quantify the cost items (material resources) required to perform the procedure. The length of time required to perform the procedure was timed. The human resources assessed were nurses who had been trained to perform the procedure for at least one year.
For the production of the PRP gel, a fixed angle bench centrifuge with a capacity of 12 tubes (conical bottom) of 15 ml, with an adjustable speed of up to 4000 rpm, and a timer of 1 - 60 minutes was used. For each session, the material resources and the minimum quantity required to perform the procedure are described in [Figure 1](#t3){ref-type="table"}.
###### Material resources required for autologous platelet rich plasma gel production
------------------------------------------------------------------------------ ------------------------------- ------------------------------- ------------------
**Material resources for production of autologous platelet rich plasma gel**
Step 1 - Blood collection Step 2 - Blood centrifugation
Description Minimum quantity Description Minimum quantity
Tubes containing 3.2% sodium citrate (5mL) 04 tubes a Disposable pipette 01 u
Cotton balls 02 u 01 mL syringe 01 u
Alcohol 70% 02 ml 10% Calcium Glyconate Ampoule 01 u
19 or 21 vacuum catheter with disposable adapter 01 u 40x12 needle 01 u
Post-puncture hemostatic adhesive dressing 01 u Single tube 01 tubo
Procedure glove 02 u Procedure glove 02 u
------------------------------------------------------------------------------ ------------------------------- ------------------------------- ------------------
The value assignment was performed through data collection, in July of 2018. With regard to the criterion for value assignment for material resources: Category A was the price established by the University Hospital (HU) electronic trading floor; Category B, human resources, considered the cost of the procedure, represented by the professional's salary per hour, multiplied by the time consumed (in minutes) to accomplish steps 1 and 2, divided by sixty (minutes).
[Figure 2](#t4){ref-type="table"} provides an overview of the steps, cost categories, and sources of information related to value assignment for cost calculation.
###### Summary of steps and cost categories, July of 2018
-------------------------------------------------------------------------------------------------------------------------------------------------------
Step Cost category Measuring technique Information sources
-------------------------- ------------------------- ----------------------- --------------------------------------------------------------------------
1 - Blood collection Nursing human resources Activity time (timed) Ministry of Planning, Development and Management
2 - Blood centrifugation
1 - Blood collection Material resources Direct observation Electronic auction of the University Hospital.\
Permanent Committee on Standardization of Medical and Hospital Materials
2 - Blood centrifugation
-------------------------------------------------------------------------------------------------------------------------------------------------------
The costs corresponded to the remuneration of all levels of staff, with and without additional qualifications, in order to evaluate the minimum and maximum value, respectively and regardless of employment type, provided by the hospital and available on the website of the Ministry of Planning, Development and Management. The mean value was \$ 0.17 per minute; the minimum was \$0.13 and the maximum was \$0.21 per minute.
The costing formula for each PRP gel production session per participant (C) involved the cost of two steps: Step 1 - blood collection, and, Step 2 - blood centrifugation. In each step, the costs of the material resources category (A) and human resources category (B) were summed, as follows:
The cost of the PRP Session = A + B costs (Step 1) + A + B cost (Step 2);
To compare the cost of the session with PRP gel in the HU with other SUS units, material prices were gathered from the price panel platform of the Ministry of Planning, Development and Management, and are presented as mean, minimum, and maximum prices.
Results
=======
The Step 1 cost, in dollars per day per participant was US \$1.23. The human resources and material resources category showed equivalent costs, as it is a procedure that does not require much time to perform. The mean time in minutes for blood collection was 4.0 ± 0.8 minutes. Still, the cost of the human resources category was slightly higher, representing 54.2% of the total cost ([Table 1](#t1){ref-type="table"}).
###### Cost, in US dollars, of Platelet Rich Plasma Gel according to steps and categories: material and human resources. Niteroi, RJ, Brazil, 2018
Cost of Platelet Rich Plasma Gel at University Hospital[\*](#TFN1){ref-type="table-fn"}
----------------------------------------------------------------------------------------- ------- ------- -------- ------- -------- -------- --------
One session per participant 0.57 0.67 1.23 0.69 2.96 3.65 4.88
Six sessions (mean) 3.39 4.02 7.41 4.16 17.73 21.90 29.31
Standard deviation 0.19 0.81 0.95 0.16 1.26 1.31 1.64
Minimal 2.80 3.00 5.97 3.50 14.67 18.87 25.80
Maximal 3.80 6.50 10.30 4.20 20.67 24.87 32.23
Total cost for 18 participants 61.05 72.33 133.38 74.95 319.19 394.14 527.52
Quoted on September 6, 2018, US \$1.00 = R\$ 4.14;
PRP = Platelet Rich Plasma
In step 2, the dollars per day per participant costs were US \$3.65, and the category that most impacted the total cost was human resources (81%), as the time for the supernatant centrifugation and aspiration process, with the addition of 10% Calcium Glyconate, was 17.9 ± 1.3 minutes.
Thus, the cost of producing PRP technology, in dollars, considering material resources (MR) and human resources (HR), was US \$4.88 per PRP application session, totaling US \$29.31 ± 1.63 for six sessions ([Table 1](#t1){ref-type="table"}). The time required from blood collection through PRP delivery was approximately 22 minutes.
Most of the total cost for a PRP session was related to human resources (85.8%). The minimum cost of six sessions was US \$25.80, and the maximum was US \$32.23. The total cost of six sessions for the 18 participants was US \$527.52.
The variation in material costs of the blood collection and PRP preparation steps is due to variation in the amount of materials that was reduced in a participant who performed only five PRP applications, due to healing of an ulcer prior to the 12-week follow-up time. The item of material resources that varied the most was the amount of catheters, as two participants had difficult punctures, which also increased the time required for the procedure to be performed by the nurse.
The estimated cost of one session per PRP gel participant in the HU was US \$4.88. The pricing panel platform is US \$ 0.48 (minimum cost), US \$ 5.16 (mean cost), and US \$ 11.13 (maximum cost). The cost found in the HU is equivalent to the mean cost presented on the platform ([Table 2](#t2){ref-type="table"}).
###### Cost, in dollars, of platelet rich plasma gel in the Unified Health System. Niterói, RJ, Brazil, 2018
Comparative Cost of Autologous Rich Plasma Gel Production[\*](#TFN3){ref-type="table-fn"}
------------------------------------------------------------------------------------------- -------- -------- -------- ---------
One session per participant 4.88 5.16 3.48 11.13
Six sessions (mean) 29.31 30.94 20.87 66.75
Standard deviation 1.64 1.49 0.99 2.75
Minimum 25.80 27.09 18.25 57.58
Maximum 32.23 33.91 22.82 70.98
Total cost for 18 participants 527.52 556.88 375.60 1201.56
Quoted on September 6, 2018, US \$1.00 = R\$ 4.14;
PRP = Platelet Rich Plasma
As shown in [Table 2](#t2){ref-type="table"}, the cost of a six-session PRP protocol at HU (US \$29.31) is close to the mean cost (US \$ 30.94) of the other units that make up the SUS, the minimum cost (US \$20.87) is 29.8% lower than the HU, and the maximum cost (US \$66.75) is 127.7% higher than the HU, according to the Pricing Panel Platform.
Discussion
==========
The PRP production method is considered to be simple, although it requires a speed-regulating centrifuge, and training for careful handling^(^ [@B2] ^)^.
The PRP gel production in this study was autologous, using single centrifugation for topical use. There are several methods and processes for converting whole blood into a product (PRP) for topical application; all include blood centrifuging. Differences in the centrifugation process include velocity, acceleration, deceleration, angulation, and radius, as well as the types of platelet lysis activators that can be: calcium chloride with or without thrombin, batroxobin (a proteolytic enzyme that acts on plasma coagulation), thrombin, and freezing^(^ [@B26] ^)^.
Autologous PRP has high therapeutic potential and can be used in various formulations and in various fields of medicine and bioengineering. In 2012, more than 40 autologous platelet products were already available, with different characteristics in relation to platelet enrichment, presence of leukocytes, activator type, and final volume, making it difficult to compare results between studies^(^ [@B27] ^)^.
The closed system PRP preparation kit with centrifuge and preparation material for up to 100 PRP ranges from US \$811.35 to US \$929.95, on the market.
Commercialized systems enable the fractionation of plasma rich growth factor (PRGF-Endoret), where the vacuum suction is controlled in the fractionation tube. After separation of the PRP occurs, calcium chloride for platelet activation^(^ [@B28] ^)^, or ascorbic acid, thrombin and calcium chloride are added to centrifuged plasma for lysis and activation of PRP (Autologel™) ^(^ [@B28] ^)^.
A cross-sectional study conducted in Malaysia evaluated the cost of single-centrifuged autologous PRP compared to commercial kits. The findings showed that the PRP produced with this technique contained a significantly higher mean platelet and leukocyte count than whole blood, and no difference was found in the mean blood level leukocyte and platelet counts between the single centrifugation technique performed and commercial kits, demonstrating that PRP can be produced with clinically available material resources of similar quality to the commercial kit, at a cost of 29.02RM (Malaysian Ringgit) or (US \$7.02), with a preparation time ranging from 25 to 30 minutes from collection to final product. In the other four commercial kits evaluated, the preparation time ranged from 15 to 20 minutes, and the costs ranged from 400.00 to 1600.00 RM^(^ [@B29] ^)^. The cost of the human resource involved for preparation and the cost of the centrifuge was not evaluated in this study.
Another study, conducted in the United States, shows that it is possible to train professionals to obtain PRP, via a single centrifuge cycle at 3200 RPM (1430G), for 10 minutes, with a material cost of less than US \$10, with a similar time as is suggested by commercial kit manufacturers^(^ [@B30] ^)^. In these studies, although the cost of human resources and the centrifuge has not been accounted for, it is clear that the preparation of PRP without the commercial kit reduces the cost, enabling it to be used in the different types of treatment in which PRP has been shown to be effective, including within the SUS.
Moreover, a barrier remains for comparing cost studies, because many studies use materials and methods that are used with commercial kits, which increases the cost of materials and restricts treatment in the public system^(^ [@B30] ^)^.
In the Brazilian SUS, there is still no record of commercial kit purchases in the Price Panel Platform of the Ministry of Planning, which makes it difficult to compare costs.
Regarding the procedure, it is important to consider that competence in performing the procedure directly influences the cost of a product. Training is an important step, and should be performed by competent professionals, generating an interface of inter-professional education. This is especially true when it is observed that the cost of human resources is higher than the cost of material in the development of processes and products.
In this study, human resources accounted for 85.8% of the total cost of one PRP session per participant. In other studies, the cost of human resources represented the largest percentage of the cost of outpatient procedures, such as the treatment of venous ulcers with Carboxymethylcellulose gel^(^ [@B31] ^)^ and compressive therapy^(^ [@B32] ^)^.
Nursing activities are permanent sources of scientific and technological innovation, requiring skilled labor, and having a direct impact on the basis of organizational productivity, while being "socially committed to the public policies of the Unified Health System (SUS)". This occurs through technological care, when: applying new ideas and adopting best practices; improving processes, care models and protocols, aimed at customer care and Administrative Technology, with management of operational processes which require knowledge and skills; using indicators, administrative processes, and cost management in care^(^ [@B33] ^)^.
Nurses are the professionals who have incorporated the practice of care the most, and their professional development has been permeated by the acquisition of fundamental theoretical and philosophical bases, incorporating technological innovations without losing the values, vision, and mission of their profession which means "giving attention to", treating, respecting, and welcoming the human being and his/her needs". Thus, the professional advancement of nurses is important, considering that this is a human resource which is committed to the sustainability of the health system^(^ [@B34] ^)^.
The lack of studies on the cost of PRP production conducted in the Brazilian context that could be used to compare the findings of this research was a limitation of this study.
Conclusion
==========
The cost of producing autologous PRP gel for topical use involves low-cost material resources for blood collection and blood centrifugation, totaling US \$4.88 per session, and US \$29.31 ± 1.63, for a six-session protocol, considering the nurse as the human resource.
The cost of a six-session PRP protocol at the HU (US \$29.31) is close to the mean cost (US \$30.94) of the other units that make up the SUS; the minimum cost (US \$20.87) is 29.8% lower than the HU cost, and the maximum cost (US \$66.75) is 127.7% higher than at the HU, according to the Pricing Panel Platform.
Therefore, this study provides evidence of the Brazilian cost of producing autologous PRP gel, from the perspective of the SUS. It is also concluded that the production of PRP should be performed by a trained professional, in an appropriate place, as it is a safe and inexpensive technology that can be developed by health professionals, especially nurses, for outpatient application in patients with chronic injuries.
Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - MCTI/CNPq - Universal Notice \# 01/2016 and by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) - Notice \# 15/2015 - Apoio às Instituições de Ensino e Pesquisa Sediadas no Rio de Janeiro, Brazil.
| {
"pile_set_name": "PubMed Central"
} |
Asthma and allergic rhinitis (AR) frequently coexist, with epidemiological data suggesting that most asthma patients also suffer from AR.\[[@CIT1]\] We also find similar trends in asthma and AR prevalence around the world using objective epidemiological instruments.\[[@CIT2]\] Both diseases share similar triggers and similar pathophysiology characterized by similar inflammatory cell infiltrates.\[[@CIT3]\] It has also been found that the degree of inflammation in asthma correlates highly with the level of inflammation in the nose,\[[@CIT4]\] and that allergen challenge of the nose leads to hyperresponsiveness in the lungs\[[@CIT5]\] and allergen challenge in the lung with inflammation in the nose.\[[@CIT6]\] This and other evidence suggests a central mechanism behind the link with eosinophil precursors emanating from the bone marrow in response to triggers migrating not only to the site of stimulation, such as the nasal mucosa, but also to other sites within the one airway, including the lower respiratory tract.
Three areas of research suggest there is a real importance clinically in the 'one-airway' concept. The first of these relates to the possibility of interference in the development of asthma in patients with rhinitis; the second is that the severity of rhinitis appears to be a risk factor for poor asthma control and thirdly that common anti-inflammatory therapy may improve outcomes for both.
Examining the first of these, one study from the United States showed that those students with rhinitis in college were up to three times more likely to develop asthma by the age of 40 than those without rhinitis.\[[@CIT7]\] Intriguingly, Johnstone and Möller observed that young patients with rhinitis receiving subcutaneous immunotherapy for their rhinitis were less likely to develop subsequent asthma suggesting that intervention in patients with rhinitis might reduce the likelihood of developing asthma.\[[@CIT8]\]
Regarding the second of these, it has been found that clinically diagnosed AR is associated with significantly worse asthma control in adults and children. A recent analysis of patients in a General Practice database in the UK found that children with physician-diagnosed AR were more than twice as likely to be hospitalized for asthma as those without and 50% more likely in adults.\[[@CIT9][@CIT10]\] Studies on children hospitalized for asthma in Norway showed that those children with clinically recognized rhinitis had a greater risk of rehospitalization for asthma.\[[@CIT11]\] In addition, a recent evaluation of asthma control in general practice suggested that those with asthma and moderate-to-severe rhinitis were four times more likely to have poor asthma control than those with no rhinitis symptoms.\[[@CIT12]\]
Considering the third one, treatment guidelines have recognized that asthma and AR are linked conditions of 'One Airway' and recommend that patients with asthma be evaluated for AR and vice versa. These guidelines support a combined approach to treating both conditions.\[[@CIT3]\] Such strategies with our current range of therapeutic options include:
**Upper airway treatment options**
Nasal steroids
Antihistamines
**Lower airway treatment options**
Inhaled steroids
**Upper and lower airway treatment options**
Leukotriene receptor antagonists
Anti-IgE
Immunotherapy
What evidence beyond a common-sense approach do we have to support this approach? Many of the studies that have supposedly set out to test this hypothesis have unfortunately been flawed, in that they have often only looked at using treatment for one part of the airway. For example, we have many studies of nasal steroids in patients with rhinitis and asthma but not used in conjunction with inhaled steroids to treat the asthma -- these studies in seasonally triggered rhinitis and asthma have been examined in a recent meta-analysis and showed minimal impact on asthma outcomes -- which is unsurprising and neither refute nor support the one-airway approach.\[[@CIT13]\] Because of this lack of appropriate, randomized, controlled trials we have until recently had to rely on data from observational studies relating to the use of antihistamines and nasal steroids. One of these studies used the MarketScan database in Washington, USA, that examined patients with asthma and rhinitis treated for concomitant rhinitis compared to those only treated for their asthma and found asthma-related events were reduced by 49% in those treated for both conditions.\[[@CIT14]\] Whilst certainly supporting the one-airway approach, randomized trials are needed to confirm these findings.
The advent of treatments that both treat the upper and lower airways provides an opportunity to examine this in a different way. These include drugs that block IgE, cysteinyl leukotrienes and the use of immunotherapy as all three approaches have evidence to support their usage in patients with asthma and AR.\[[@CIT15]--[@CIT19]\] The ideal study design would compare the benefits of a sole lower airway approach with a combined approach in patients with asthma alone and asthma with rhinitis. The only analysis that has set out to do this to date is that of the COMPACT study. The COMPACT study was a randomized, double-blind trial of 889 adults with asthma, whose asthma symptoms persisted despite being prescribed inhaled steroids with groups randomized to an increase in inhaled steroids or addition of a leukotriene receptor antagonist.\[[@CIT20]\] Further analysis examined the response in patients with and without a physician and patient report of rhinitis. This showed that those patients with asthma and no rhinitis had similar improved outcomes irrespective of the type of treatment increase; however, those with rhinitis showed a greater benefit with a one-airway approach of using upper and lower airways treatment with inhaled steroids and a leukotriene antagonist. This benefit was predominantly produced because of a reduced efficacy from increasing inhaled steroids in this subgroup.\[[@CIT21]\]
In conclusion, AR and asthma are inflammatory disorders that have been linked epidemiologically, pathophysiologically and clinically. Allergic rhinitis increases morbidity, therapeutic needs and use of healthcare resources in patients with asthma. It is, therefore, important to recognize co-morbidity in patients with asthma and consider management strategies from this perspective; failure to do so appears to result in adequate asthma control.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Driving under the influence (DUI) is a significant problem. Injury from alcohol-related motor vehicle crashes is a leading cause of premature death and disability \[[@CR1]\]. Even after individuals with a first offense attend required alcohol education programs, rates of recidivism are high \[[@CR2], [@CR3]\]. Despite a decline in recidivism between 1990 and 1996 in California, rates of DUI incidents in the state have remained stable since 2010.
In California, individuals with a first-time DUI conviction must complete a state-licensed DUI program in order to regain their driver's license \[[@CR4]\]. Programs are didactic and lecture-based and provide strategies to reduce drinking and driving, education about alcohol, and presentations by panels of victims whose lives have been affected by a DUI incident \[[@CR5]\]. Unfortunately, DUI programs have had only modest effects on recidivism \[[@CR4]\]. In 2011, there was no significant difference in the rates of 1-year crash and DUI incidents in California between individuals with a DUI conviction who were court-assigned to a DUI program and those who were not \[[@CR6]\]. This finding is consistent with the general literature showing that educational-type lectures do not have any effect on behavior change among individuals with alcohol use disorders \[[@CR7]--[@CR9]\].
A more effective approach towards behavior change may be a counseling method that focuses on exploring an individual's reasons for change and helping them develop a change plan that is meaningful for them. Motivational interviewing (MI) is a collaborative counseling style that strives to strengthen a client's commitment to change \[[@CR10]\]; it is grounded in theories of self-determination \[[@CR11]\] and self-efficacy \[[@CR12]\]. Treatment approaches grounded in these theories empower an individual's motivation to change, reaffirm their autonomy, and guide people toward change if they are ready. MI could be particularly acceptable among individuals with DUI convictions who may vary considerably in their motivation to change. For example, some individuals may be ambivalent about changing their drinking and driving behaviors, whereas others may be very motivated to change because of the adverse event or "teachable moment" they experienced \[[@CR13], [@CR14]\].
MI is flexible and tailors the intervention content based on the individual's readiness to change. Counselors use processes such as engaging (establishing a connection), focusing (establishing goals such as behavior change), evoking (eliciting the client's motivations to change), and planning (committing to change and developing a plan) to individualize sessions \[[@CR10]\]. Within the context of a DUI program, individuals who are not ready to change may benefit most from counselors who spend time engaging the individual and establishing rapport; individuals in later stages or those who are already moving toward change may be better helped by planning when to use problem-solving strategies and in ways to increase their commitment to action \[[@CR10], [@CR15]\].
Although many studies have shown the effectiveness of MI in other settings (e.g., college, primary care, substance abuse treatment) compared to no treatment \[[@CR16]\], in-person interventions are limited by the availability of trained counselors, training costs, and the challenge of implementing the approach uniformly \[[@CR17]\]. For example, research shows that counselors attending MI workshops often lose MI proficiency over time unless they receive ongoing feedback and coaching post-training \[[@CR18], [@CR19]\]. Thus, it is important to think about ways to utilize MI that may be more cost-effective and consistent. Web-based interventions that utilize MI principles (web-MI) may be a promising approach.
Web-MI makes it possible to disseminate evidenced-based approaches with high fidelity because the content is programmed and automated. Web-MI has been shown to be effective in reducing at-risk drinking among college students \[[@CR20], [@CR21]\] and adults in the general population and military \[[@CR22], [@CR23]\]. Web-MI has also been effective in reducing smoking among English and Spanish speakers \[[@CR24]\], as well as drug use among postpartum women \[[@CR25]\]. While some web-MIs are being explored in criminal justice settings \[[@CR26]\], web-MI has not been tested in DUI settings. Few studies have compared the effectiveness of web-MI to active comparison groups (i.e., other interventions that include alcohol content) \[[@CR27]\]. Existing research studies have typically compared the effects of a stand-alone web-MI to an assessment-only group \[[@CR28]\], and they generally show small effect sizes at short-term follow-up \[[@CR29]\]. Although even small effects may be clinically meaningful, more research is needed to improve their comparative effectiveness and determine whether these interventions may serve as an adjunct to more intensive approaches.
The current study evaluated the acceptance and efficacy of web-MI and in-person MI interventions among a diverse sample of individuals with a first-time DUI offense. This Stage 1b trial focused on determining participant acceptance of the intervention and intervention feasibility, and predicting the likely size of intervention effects for future trials \[[@CR30]\]. We randomly assigned individuals enrolled in a DUI program to usual-care (UC-only), in-person MI plus UC, or web-MI plus UC. Given the pilot nature of this work, our primary aims were to evaluate the acceptance and efficacy of these MI interventions on alcohol-related outcomes compared to UC-only. We hypothesized that participants in the in-person and web-MI interventions would have greater acceptance and reduced alcohol consumption and alcohol-related consequences compared to UC-only.
Methods/design {#Sec2}
==============
Setting and design {#Sec3}
------------------
Project REACH (Rethinking Avenues for Change; in Spanish, *REtomondo Avenidas para el Cambio Hoy*) was conducted in collaboration with the Los Angeles County Alcohol and Drug Program Administration (ADPA) and three private DUI programs under ADPA's regulatory authority. All clients received UC. Consenting clients were randomized to one of three conditions: UC-only, UC plus in-person MI, or UC plus web-MI using randomized block sampling with equally-sized blocks of six.
All procedures were approved by the institution's IRB. Because of the sensitivity of collecting data while clients were enrolled in the DUI program, participants were told that our Certificate of Confidentiality protected their privacy from any civil, criminal, administrative, legislative, or other proceeding at the federal, state, or local level and that participation in the study would not affect the services to which they were entitled.
Study conditions {#Sec4}
----------------
### Usual care {#Sec5}
UC consisted of nine 2-h group sessions, twelve 1-h educational classes, and six community-based 12-step meetings. The 2-h group sessions were unstructured support groups that encouraged participants to examine their own personal attitudes and behavior and receive support for their alcohol or drug problems \[[@CR31]\]. In the beginning of the study, we conducted focus groups with UC clients who reported that sessions were focused mainly on the consequences of DUI and heavy drinking \[[@CR32]\].
### Intervention conditions {#Sec6}
We conducted focus groups with DUI program staff and clients to develop the in-person MI intervention for this population. Next, we adapted the in-person intervention for the web, and then conducted individual usability testing interviews with web-MI clients who were already enrolled in the DUI program \[[@CR32]\]. We used the usability feedback to iteratively revise the interventions. We developed the interventions simultaneously in English and Spanish in order to create interventions that were culturally equivalent. The current pilot study evaluates the final revised intervention created from these formative assessment procedures.
Both the in-person MI and web-MI interventions consisted of one 45-min individual session and two 10-min booster sessions that were delivered by the same facilitator. Each session was different. The goals of the MI interventions were to reduce drinking and alcohol-related problems. The content of both the in-person and the web-MI intervention was adapted from earlier MI work \[[@CR33]--[@CR35]\] and covered similar content, but the efficacy of the revised interventions had not been tested until the current pilot study. The first session included normative personalized feedback in the following areas: (1) how their drinking and estimates of others' drinking compared to other men/women their age \[[@CR36]\]; (2) their positive beliefs about drinking and the balanced placebo design experiment, which describes how alcohol expectancies (i.e., actual vs. expected effects) can influence drinking \[[@CR37]\]; (3) their negative consequences from drinking, including their estimated blood alcohol content values; and (4) strategies for avoiding consequences in the future. Participants who were ready to change their drinking were asked to discuss a drinking-related goal they wanted to work on before the next booster session. We then used rulers to assess their confidence and willingness to work on that related goal. If the participant was not ready to change, the facilitator went straight to the rulers.
The in-person and web-MI booster sessions were formatted similarly and included a check-in about the participant's drinking and goals from the previous session (e.g., "Last session, you said that you would try to stop your drinking by not going to happy hour. How did that go for you?"). Booster sesssions provided an opportunity to talk about new strategies to stop drinking, if they were willing, and to discuss their confidence and willingness to change using rulers with a 1--10 scale (e.g., not confident to very confident).
The style of the MI interventions was as important as the content. Both web-MI and in-person MI interventions used core MI skills, such as open-ended questions, affirmations, reflective statements, and summaries to convey a nonjudgmental and non-confrontational style \[[@CR10]\]. For example, the in-person MI manual had examples of open-ended questions and reflective statements that facilitators could use, and these same statements were used by the narrator in the web-MI intervention. Both interventions emphasized the underlying spirit of MI (e.g., collaboration, evocation, acceptance, compassion).
While the web-MI intervention incorporated the same sections of the in-person MI intervention, we used artificial intelligence (e.g., automatic responses tailored to the participant's responses) and personalized feedback to tailor the intervention to the participant so that each session was interactive. For example, we used audio recordings and videos to share personalized feedback from the participant's baseline survey (e.g., "We asked you what your drinking was like and you said you drank 4 days a week."); asked questions that participants could respond to and receive tailored audios/videos based on those responses \[e.g., "What do you think of this information?" (participant clicks "I'm surprised"); "It is very common to be surprised by this information and wonder if the numbers are correct..."\]; used interactive exercises ("Click a number that best describes your mood when you start to drink. What happens to your mood as you continue to drink?"); and elicited change talk (e.g., "For the confidence ruler, why a 4 and not a 0?"). The web-MI intervention was narrated by a female Latina, and text captions were also available at the bottom of the screen. It was programmed at a 5th-grade reading level.
Participants and recruitment {#Sec7}
----------------------------
Participants were individuals 21 and older convicted of a first-time DUI offense and who had entered one of the three participating 3-month DUI programs. Upon enrolling in the program, program staff asked clients if they could be contacted about a research study. Interested clients completed a consent-to-contact form. Consenting clients were contacted and screened for 5th-grade completion (because of the nature of our self-report instruments) and at-risk drinking, using a score of 3 or higher for women or 5 or higher for men on the Alcohol Use Disorders Identification Test consumption questions (AUDIT-C; \[[@CR38]\]). These cut points perform well, with high sensitivity and specificity in screening for at-risk drinking in the general population (men: \>90 %, women: 80 %; \[[@CR39]\]). Approximately 52 percent were ineligible for the study based on low AUDIT-C score and education (see Fig. [1](#Fig1){ref-type="fig"}).Fig. 1CONSORT diagram. \*Denominator is total screened for eligibility (n = 387); \*\*denominator is number eligible (n = 185); \^denominator is number randomized (n = 159); \^\^denominator is number allocated to each group (IP-MI n = 51, web-MI n = 54, UC n = 54)
Participants (N = 159) were randomized to one of the three conditions. Participants assigned to either of the two MI interventions were asked to complete their first individual session by the third week of entering the DUI program, and their booster sessions by their ninth and eleventh week in the DUI program, respectively. The timing of these intervention sessions was meant to correspond with the beginning, middle, and end of a participant's 3-month DUI program. As noted earlier, participants in both MI interventions had the same number of sessions as participants in the UC-only condition because their MI sessions replaced two 12-step meetings. All MI intervention sessions were delivered in a private office at the DUI program where UC groups took place. A total of 138 participants were successfully assessed at follow-up (86.8 %).
Procedures {#Sec8}
----------
### Client data collection {#Sec9}
We used in-person interviews to assess DSM-IV criteria for past-year alcohol abuse and dependence. Participants also completed web-based surveys at baseline, after the first session, and 3 months after baseline or at program termination. Participants received a \$25 gift card for the Alcohol Use Disorder and Associated Disabilities Interview schedule (AUDADIS; \[[@CR40]\]), \$25 for the baseline survey, \$10 for a satisfaction survey immediately after their first session, and \$50 for the 3-month follow-up. All clients were followed in our intent-to-treat analyses, regardless of whether they completed all intervention sessions.
### Facilitator training and supervision {#Sec10}
Three bilingual facilitators received 40 h of MI training that included a one-day MI workshop delivered by authors KCO and EJD, who are clinical psychologists affiliated with the Motivational Interviewing Network of Trainers. In addition, facilitators received additional coaching and feedback after each session from KCO, who listened to audio recordings of sessions.
### Intervention fidelity data collection and coding {#Sec11}
In-person MI sessions were audio recorded. Two independent coders received 40 h of coding training \[[@CR41]\], which included a half-day training on the Motivational Interviewing Treatment Integrity (MITI) scale \[[@CR42]\] and several MITI practice assignments with pre-coded audio recordings \[[@CR43]\]. Raters met weekly to discuss coding discrepancies and reconcile questions to maintain inter-rater agreement. UC sessions were not coded because not all individuals attending these groups were enrolled in the study, so it was not possible to record these sessions.
To monitor web-MI intervention fidelity, our web program measured the number of minutes each participant spent in each session. A facilitator was present in the room to address any problems that might have emerged, which may have also helped to ensure that the participant completed the session.
Measures {#Sec12}
--------
At baseline, we collected demographics and alcohol abuse and dependence information using AUDADIS and AUDIT-C \[[@CR38], [@CR40]\]. We collected client acceptance/satisfaction data immediately following the first session. At baseline and at 3-month follow-up, we collected data on alcohol use.
### Client ratings of quality and satisfaction {#Sec13}
Participants in all three conditions answered questions about the quality of and their satisfaction with the experience. Participants were asked, "How would you rate the quality of your session?" on a 4-point Likert scale, with a higher score representing higher quality. Satisfaction was measured using 22 items that were averaged (e.g., This program was respectful of my background; I felt the program respected where I was at with my alcohol and that any change was up to me; The program valued my opinion). Additional items included questions about the usefulness, quality, impact, and helpfulness of the session. They also were asked nine questions rating their session facilitator \[[@CR44]\].
### MI intervention fidelity {#Sec14}
MITI 3.1 was used to code competency and adherence to in-person MI, and integrity was measured through global scores and behavioral counts \[[@CR42]\]. The MITI 3.1 has five global scales (evocation, collaboration, autonomy/support, direction, and empathy) that are scored from 1 (low) to 5 (high), with a score of 3.5 indicating beginning proficiency and 4 indicating competency. The rater also counts the number of specific behaviors that occur during each coded segment, including the number of open questions and closed-ended questions, MI-adherent and nonadherent statements, and simple and complex reflections. Whereas global scores have a limited range (1--5), behavioral counts utilize a running tally with no upper end on the scale; thus, these scores can vary by session.
Twenty percent of the in-person MI sessions (n = 10) were randomly selected for double-coding. We calculated prevalence-adjusted, bias-adjusted kappa (PABAK; \[[@CR45]\] to assess inter-rater agreement for each global score by dichotomizing the 1--5 scale into 1--3 (MI beginning proficiency) and 4--5 (MI competent). The PABAK scores for the global scores of evocation, collaboration, autonomy/support, direction, and empathy were 0.6, 0.2, 0.6, 1.0, and 0, respectively, while the agreement averaged 72 percent across the global scores. We also calculated intraclass correlations (ICCs) between raters for each behavioral count. These ICCs ranged from 0.30 (MI-adherent) to 0.91 (closed questions), and averaged 0.69 across the behavioral counts. Since the distribution of MI-nonadherent behavioral counts was skewed toward 0 (with only one value among the coders that was neither 0 nor 1), a kappa statistic was computed instead to assess inter-rater agreement for reporting any versus no MI-nonadherent behaviors (PABAK = 0.2).
### Client outcomes {#Sec15}
Outcomes included changes in drinking behaviors and related consequences in the past 3 months. We examined the intensity and frequency of drinking in the past 3 months \[[@CR46]\]. Drinking frequency was measured by asking how often participants drank alcohol in the past 3 months. Reponses ranged from 0 ('Never') to 10 ('Every day'). We converted these response categories to a pseudo-continuous variable to easily interpret the results as the number of days. Drinking frequency ranged from 0 to 90 days (e.g., 'Never' = 0 days, 'Less than Once a Month' = 2 days). Drinking quantity was measured by asking the respondent the typical number of drinks on a given occasion. Days of reported heavy drinking, defined as four or more drinks for women and five or more drinks for men, was also transformed from a categorical variable to a pseudo-continuous variable ranging from 0 to 90 days, as described above for drinking frequency. Drinking and driving in the past 3 months was reported on a categorical scale ranging from 0 ('Never') to 10 ('Every day'). Due to the skewed distribution of this variable, we created a dichotomous version to indicate any drinking while driving in the past 3 months. We assessed negative consequences from alcohol use using the Shortened Inventory of Problems Modified for Alcohol and Drug Use \[[@CR47]\]. Finally, marijuana use frequency was assessed by asking participants how often they used marijuana in the past 3 months. Reponses were transformed to a continuous variable ranging from 0 to 90 days.
Analytic strategy {#Sec16}
-----------------
### Client ratings of session quality and satisfaction {#Sec17}
Client quality and satisfaction data were analyzed for differences across the three conditions using ANOVA and pair-wise *t*-tests.
### Preliminary intervention efficacy {#Sec18}
We first examined whether there was significant change over time in the outcomes within each study condition by conducting Wilcoxon signed-rank tests on the difference scores of continuous outcomes. We conducted a McNemar's test to assess a significant change in the rate of obtaining a DUI and experiencing negative consequences (none vs. any) in the past 3 months between baseline and follow-up, within study condition. Pseudo-continuous variables were treated as continuous, given the assumption that each variable reflected an underlying continuum \[[@CR48], [@CR49]\]. The treatment of these variables as continuous were expected to result in low bias when measuring more than seven categories and the measure had a bell-shape \[[@CR50]\]. When the latter condition was not met, we ran sensitivity analyses with the ordinal outcome to confirm that conclusions did not differ under the two model specifications.
We next conducted analyses to test for a significant intervention effect. All outcomes were analyzed using an intent-to-treat approach. To compare the baseline characteristics of clients assigned to each condition, we used Chi squared tests for categorical variables and one-way ANOVAs for continuous variables. Longitudinally, each outcome was modeled with generalized, linear, mixed-effects regression modeling using the GLIMMIX procedure in SAS software (Version 9.2). Covariates included in the model were those characteristics identified as significant (p \< 0.1) in bivariate analyses with the outcomes. These included days of marijuana use in the last 3 months and average number of drinks \[[@CR46]\]. The baseline value of the outcome was included as a covariate in all models to control for any important differences among conditions and to improve the precision of the intervention-effect estimates \[[@CR51]\]. Dummy variables for web-MI and in-person MI, with the comparison condition as the hold-out category, were included in all models. When the distribution of the pseudo-continuous measures was not bell-shaped, ordinal logistic regressions were conducted on the original ordinal measure as a sensitivity analysis to confirm that analytic conclusions did not differ under the two model specifications.
Results {#Sec19}
=======
Client acceptance {#Sec20}
-----------------
Approximately 57 percent attended all three in-person MI sessions, 14 percent attended two sessions, and 29 percent attended one session. Approximately 65 percent attended all three web-MI sessions, 17 percent attended two sessions, and 18 percent attended one session. We did not have data on the number of UC sessions attended. Participants' ratings of session quality varied significantly across conditions \[*F*(2135) = 6.93, *p* = 0.0014\]. On average, in-person MI participants rated the quality of their session highest compared to web-MI and UC-only, and there were no differences in ratings by facilitator. Quality ratings were next highest for participants in the web-MI, and then the UC-only condition. Participants in the in-person MI intervention rated their satisfaction with the sessions significantly higher than participants from the other two conditions. There were no significant differences in satisfaction between web-MI and UC participants.
Intervention fidelity {#Sec21}
---------------------
Facilitators of the in-person MI scored a mean of 4.2 on the MITI global scores (*SD* = 0.1; range: 3.9--4.5), which indicates MI competency. Behavioral counts ranged between 5.5 (giving information), 9.5 (simple reflections), and 11.1 (MI-adherent statements, complex reflections), suggesting high frequency of MI-consistent behaviors.
Intervention efficacy {#Sec22}
---------------------
### Sample characteristics {#Sec23}
Sixty-five percent of the participants were male, 40 percent were Hispanic/Latino/a, 87 percent were born in the United States (excluding Puerto Rico), 91 percent had at least a high school education, and 64 percent were fully employed. Participants were 30.0 (SD = 9.8) years of age (Table [1](#Tab1){ref-type="table"}).Table 1Baseline characteristics of the study sampleUC (N = 54)In-person MI (N = 51)Web-MI (N = 54)Mean (%)SDMean (%)SDMean (%)SDMale64.8168.6362.96Race Hispanic/Latino38.8941.1840.74 African American9.269.8011.11 White35.1939.2231.48 Asian/PI7.415.8811.11Place of birth US, except Puerto Rico85.1992.1685.19 Other14.817.8414.81Education \<HS/GED3.705.883.70 HS/GED3.703.925.56 \>HS92.5990.2090.74Employment Full/part time66.6764.7159.26 Unemployed9.269.8020.37 Other work situation24.0725.4920.37Age at time of DUI29.568.9629.7510.1830.5610.40Alcohol use measures Negative consequences (SIP)8.708.2610.0611.0810.7610.14Past 12 Months AUDADIS alcohol dependence\^64.1564.7174.07 AUDADIS alcohol abuse\^88.6896.0890.74Past 3 months Alcohol use \# of days25.0324.7331.1828.5625.9923.53 \# Drinks on typical occasion4.352.304.371.915.023.27 Heavy drinking \# of days10.1912.8713.9318.3910.5513.09 Drink and drive past 3 months51.8549.0248.15 \# Drinks on heaviest occasion\*7.984.038.983.9910.5624.07 Marijuana use42.5950.9844.44 Marijuana use in days\*16.1131.2227.7428.7710.5624.07 Any other drug use3.709.8012.96\* p \< 0.05; \^ 1 UC participant did not complete the AUDADIS
Overall, 92 percent of the sample met diagnostic criteria for past-year alcohol abuse. Sixty-seven percent met diagnostic criteria for dependence. Diagnoses of alcohol abuse and dependence were not significantly different across the three conditions (for abuse, Χ^*2*^(2) = 1.52, *p* = 0.468; for dependence, Χ^2^(2) = 2.00, *p* = 0.368).
At baseline, web-MI clients reported more drinks on the occasion they drank the most compared to UC-only or in-person MI clients \[see Table [1](#Tab1){ref-type="table"}; *F*(2156) = 3.39, *p* = 0.036\]. Clients receiving in-person MI also reported more days of marijuana use in the past 3 months compared to UC-only and web-MI clients \[*F*(2156) = 3.97, *p* = 0.021\]. Only one participant in our sample was monolingual Spanish-speaking, and was assigned to the web-MI intervention and included in analyses. There were no significant differences in outcomes by ethnicity.
### Alcohol-related outcomes {#Sec24}
Table [2](#Tab2){ref-type="table"} shows differences in within-group outcomes between baseline and follow-up. Overall, participants from all three conditions reported reduced drinking quantity, alcohol-related consequences, and drinking and driving between baseline and 3-month follow-up (p \< 0.05). Regarding drinking outcomes, all participants drank about one drink less on a typical occasion compared to baseline amounts (p \< 0.05) and reported fewer and less frequent consequences (3- to 4-point reduction roughly translates to experiencing several problems weekly to experiencing problems a few times in the past 3 months). Web-MI participants reported drinking 4.58 fewer days in the past 3 months at follow-up compared to baseline (p = 0.036). UC-only and in-person MI clients did not report a significant decrease in drinking days.Table 2Differences in within-group outcomes between baseline and follow-upVariableUCIn-person MIWeb-MIDifference (SD)Test stat\**d* ^+^*p*Difference (SD)Test stat\**d* ^+^*p*Difference (SD)Test stat\**d* ^+^*p*Negative consequences (SIP)3.82 (8.81)186.500.460.0133.24 (6.10)233.000.290.0013.20 (5.86)326.500.32\<0.0001Alcohol use \# of days2.63 (19.12)35.500.110.4743.54 (17.49)66.500.120.1324.58 (14.30126.500.190.036\# Drinks on typical occasion0.61 (1.97)111.500.270.0490.82 (2.10)115.500.430.0141.08 (3.33)143.000.330.011Heavy drinking \# of days2.03 (15.46)40.000.160.4421.70 (10.90)69.500.090.2180.61 (13.21)24.000.050.742\* Wilcoxon signed rank test for continuous variables^+^Effect size is computed as Cohen's *d*: difference/within-group baseline standard deviation
While there were within-group reductions in alcohol-related consequences, participants in all three groups continued to report alcohol-related consequences at follow-up. The proportions of participants who reported having at least one consequence at baseline and subsequently reported at least one consequence at follow-up were 61 percent of in-person MI, 78 percent of UC-only, and 81 percent of web-MI individuals.
Participants also reported within-group changes in their drinking and driving in the past 3 months (p \< 0.0001). Across the three groups, 40--56 percent of participants reported not drinking and driving at follow-up. We were specifically interested in seeing the proportion of participants who reported drinking and driving behavior at baseline and whether they continued this behavior at follow-up. Across all conditions, about 50 percent of participants reported drinking and driving in the 3 months prior to baseline.[1](#Fn1){ref-type="fn"} Of those participants, about 8 percent of them reported drinking and driving at follow-up. Also important to note, of participants who reported no drinking and driving within 3 months of baseline, 96 percent of them continued to report no drinking and driving at follow-up.
Table [3](#Tab3){ref-type="table"} shows the estimated intervention effect of each MI + UC condition compared to UC-only. After adjusting for baseline levels, there were no significant group differences between the MI conditions and the UC-only condition in alcohol consumption (number of typical and heavy drinking days, average number of drinks) and risk behaviors (alcohol-related negative consequences and drinking and driving). Further, estimates of the effect sizes were small (Cohen's *d* = 0--0.12) for typical and heavy drinking days, average number of drinks, and alcohol-related consequences. For drinking and driving in the past 3 months, the width of the confidence intervals indicates that substantial variability exists in our estimates.Table 3Intervention effect estimates of outcomes compared to usual care at 3 months post-baselineOutcomeEstimateConfidence intervalt statistic (132 df)*p* *valued\**Negative consequences (SIP) Web-MI1.13−1.423.690.880.3820.12 In-person MI1.13−1.473.720.860.3920.12Alcohol use \# of days Web-MI0.03−6.156.200.010.9930.00 In-person MI0.48−5.796.750.150.8800.04\# Drinks on typical occasion Web-MI0.00−0.810.810.000.9980.00 In-person MI−0.01−0.820.81−0.010.9880.00Heavy drinking \# of days Web-MI1.29−3.315.900.560.5800.08 In-person MI1.20−3.475.870.510.6120.09Log-odds ratioConfidence intervalt statistic (132 df)*p* *value*Odds ratioDrink and drive past 3 months Web-MI−1.39−12.7810.00−0.240.8100.25 In-person MI−1.99−13.439.46−0.340.7320.14\* Cohen's *d* = estimate/pooled standard deviation across the two comparison conditions
Discussion {#Sec25}
==========
This pilot study takes an important first look at the acceptance and efficacy of new in-person MI and web-MI interventions added to DUI UC compared to UC-only for a diverse sample of individuals enrolled in a first-time DUI program. Participants in both the in-person and web-MI intervention conditions rated the quality of and satisfaction with their session higher than participants in the UC-only condition, suggesting that clients were more receptive to the MI interventions. Clients viewed the in-person MI more favorably than web-MI, which may be related to the stronger therapeutic alliance often found in the in-person interactions compared to web-based interactions \[[@CR52]\]. However, we did not find statistically significant differences in outcomes between the MI conditions and UC-only condition. In fact, regardless of study condition, participants reported significant reductions in both alcohol consumption and risk behaviors. Thus, at program completion, participants from all three study conditions reported reduced alcohol consumption, DUI, and fewer alcohol-related consequences.
There were at least two unexpected results from our study. First, despite recruiting individuals with a first-time offense into the study, 67 percent of our sample met criteria for alcohol dependence, and the majority of participants in all three study conditions continued to report alcohol-related consequences at follow-up. From our previous discussions with DUI providers, we anticipated a larger percentage of at-risk versus dependent drinkers, and therefore designed the MI intervention for an at-risk population rather than a population with dependence. Our second unexpected result was that participants from each of the MI interventions did not report differences in their outcomes when compared to UC-only. We had hypothesized that because our MI interventions were focused on exploring behavioral change and developing a change plan, we would see significant improvements in individuals who received the MI interventions compared to individuals who only received UC.
There are several possible explanations for these findings. First, the extensiveness of UC services (i.e., 36 program hours) in these DUI programs may have been sufficient to improve outcomes in the short term, and an additional 3-session MI (i.e., about 65 min) may not have had an additive effect on outcomes. Second, our follow-up timeframe was short. We were only able to measure outcomes at the conclusion of clients' 3-month DUI program. Receiving a DUI and having to deal with the numerous financial, emotional, and social consequences (e.g., vehicle impoundment, jail time, probation, injury) related to this type of event may be significant enough to reduce a client's alcohol-related behaviors in the short term, but might not be enough to sustain long-term changes such as reductions in recidivism (D'Amico et al. \[[@CR13]\]). This speaks to the challenge of conducting research in DUI programs that have strong behavioral expectations and high sanctions for failing those expectations. Longer follow-up assessments (e.g., 6 months to one year after program completion) may be needed to better understand whether MI interventions and UC are differentially associated with sustained behavior change after a client completes a DUI program. Finally, MI interventions may not be the best fit for individuals with a first-time DUI offense, given the high levels of dependence that were reported in this study. Future studies should evaluate whether alcohol dependence is as common in other first-time DUI offense programs. If dependence rates are similarly high in other programs, more intensive treatment approaches such as cognitive behavioral therapy or medication-assisted therapy may be more effective \[[@CR53]--[@CR58]\]. MI interventions may still be used to enhance engagement prior to these more intensive and long-term approaches \[[@CR59]\] or they may be a better fit as a preventive intervention with individuals who are at risk for a future DUI but who have not yet been convicted. Determining which therapy or combination of therapies is associated with long-term changes should be the subject of future research.
In conducting community-based work, it is always important to examine lessons learned to help inform future research. Although MI is an evidence-based treatment that has been successful as a brief intervention in a variety of settings, it may be important to have more sessions for this more severe population. The population and providers were very receptive to MI \[[@CR32]\], and it could perhaps be integrated into the lengthy UC treatment and provided in this group setting as with other mandated populations \[[@CR13]\]. Given that clients felt that the MI intervention was of higher quality and were more satisfied with it than UC, integrating MI into UC could help standardize services provided to clients, help make the program more acceptable to them, and perhaps increase attendance, which could lead to better outcomes.
Our sample was recruited from DUI programs in California and may not be representative of clients in DUI programs nationally. Of note, about 52 percent of our sample was excluded mostly due to low AUDIT-C at program entry, which also affects generalizability. In addition, inter-rater agreement on the MITI was low for five measures, suggesting improvements in measure performance and/or the process for coding those items are needed for future studies. It is also important to note that under-reporting of DUI behaviors might be an issue, given the setting. The limitations of self-report data are well-known, although much research has shown that self-report is valid when procedures such as those used in the current study are implemented (e.g., establishing rapport and discussing confidentiality) \[[@CR60]\].
This pilot study addresses an important policy question by examining whether individuals with a first-time DUI offense find MI interventions acceptable in a DUI setting and whether clients who receive an MI intervention have improved outcomes relative to UC. Findings suggest that participants from all three conditions experienced improved outcomes, regardless of study condition. Although it is possible that a longer follow-up may provide insights into whether within-group differences are sustained, we hypothesize based on our effect sizes and confidence intervals that between-group differences in a larger future trial with this population are not supported by the data. Instead, given that individuals with a first-time DUI offense are likely a unique population of individuals who may be experiencing consequences related to alcohol dependence, future research is needed to better understand the potential heterogeneity of this population and to determine the most appropriate level of care for these high-risk individuals to reduce long-term recidivism.
In some cases, individuals might enter the DUI program more than 3 months after their DUI arrest.
KCO, KEW, EJD, and SMP conceptualized the study and obtained funding. KCO, EJD, and KEW designed the interventions. KCO had overall responsibility for executing the MI interventions, data collection, analyses, and reporting. SMP, TJL, and BAE performed quantitative data analyses. All authors read and approved the final manuscript.
Acknowledgements {#d30e1840}
================
This research was supported by grants from NIAAA awarded to Katherine E. Watkins (RC1AA019034) and Susan M. Paddock (R01AA019663). The content is solely the responsibility of the authors and does not necessarily represent the official views of NIAAA or the National Institutes of Health. The authors would like to thank Jeannette and Jim Gilmore for their administrative support, along with DUI program staff Kramer Ruppe, Tarquino Ubidia, and Carlos Ubidia. We would also like to thank our intervention facilitators, Marylou Gilbert, Claudia Diaz Fuentes, Blanca Dominguez, Rob Reaugh, and our data collection staff, Kirsten Becker, Jen Parker, and Jeni Catch. Portions of this paper were presented as a poster at 2014's Research Society on Alcoholism conference in Bellevue, Washington.
Compliance with ethical guidelines {#d30e1845}
==================================
**Competing interests** The authors declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
{#sp1 .156}
| {
"pile_set_name": "PubMed Central"
} |
The knee damage in children may be a sign of many diseases. They include bone and joint infections, neoplastic disorders, trauma and arthritis.
The aim of our study was to detect diseases which coursing with knee damage and to reveal arthroscopic and synovial histological changes.
We included in our study 29 children with chronic knee monoarthritis (CKMA), 15 girls and 14 boys with duration knee monoarthritis symptoms \> 6 weeks. Most of the patients were examined with arthrosonography and MRI. Arthroscopy with synovial biopsy was performed on 10 children (2 with tuberculosis infection and 8 with CKMA unknown origin).
Five CKMA boys transformed in juvenile spondiloarthropathy (3 had psoriatic arthritis). Juvenile rheumatoid arthritis was diagnosed in 6 children (4 F: 2 M), all girls were ANA-positive and developed oligoarthritis and one also had uveitis. There were 3 cases of tuberculosis arthritis without primary lung damage. Another cases included knee osteoarthritis, Borrellia burgdorfery infection, angiodysplasia of knee, posttraumatic arthritis with meniscus damage, osteochondritis dissecans and Hoffa disease. The largest group consisted of children with chronic villous proliferate synovitis (6 F: 2 M) unknown origin was revealed by arthroscopy and synovial biopsy. Chlamydia infection was detected in synovial fluid and membrane in 2 children.
Synovial histology was determined as focal hypertrophy of synovial membrane with mononuclear cell infiltration in half patients as signs of neoangiogenesis in another part. This form of CKMA was torpid.
CKMA is very geterogenous; some of them need in arthroscopy with synovial biopsy.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Discovery of new biological information and knowledge extracted from all kinds of biological entities has been hotspot in recent biomedical researches. These entities have included macromolecules (e.g. DNA, RNA, protein), subcellular structures (e.g., membrane, nucleus, mitochondria), cells, tissues, organs, and so on. Much effort has been made in finding the connections between phenotype and genotype, between function of a biological system (like a cell) and its properties (proteome, transcriptome, metabolome, etc.). Obviously, cell viability is one of the basic properties indicating the physiological state of the cell, thus, has long been one of the major considerations. Recently lots of projects have been carried out on studying mechanisms of cell death \[[@B1]-[@B4]\]. In general, viable cells can be distinguished from dead ones according to either the physical properties, like membrane integrity, or their metabolic activities, such as cellular energy capacity, macromolecule synthesis capacity, or hydrolysis of fluorogenic substrates. Conventional methods for extracting information about cell viability usually need reagents to be applied on the targeted cells, and comprehensive reviews of these methods can be found in Ref \[[@B5]-[@B7]\]. These reagent-based techniques are reliable and versatile, however, some of them might be invasive and even toxic to the target cells.
Much effort has also been made in developing noninvasive, reagent free methods for measuring cell viability, because the latter are more suitable for on-line or *in situ*monitoring of cells, for instance, in bioreactors. Typical on-line instruments are based on, e.g., capacitance (Aber Instruments Ltd, Aberystwyth, UK), infrared sensing (Finesse LLC, California, USA), and turbidity (Aquasant Messtechnik AG, Bubendorf, Switzerland). Recently an *in situ*dark field microscopy probe for online monitoring of cell density and viability in bioreactors has been proposed \[[@B8],[@B9]\]. With the rapid progress in machine learning and pattern recognition, more and more biological research can be carried out via image-based techniques \[[@B10]-[@B13]\]. Wei et al. developed a method to detect cell viability based on evaluation of time series images \[[@B14]\], in which multiple micrographs captured at different time points are needed to extract information for cell classification. Here, a Machine Vision System (MVS) is proposed for automated noninvasive assessment of cell viability. This MVS employs dark field microscopy plus modern image processing. It need not use time series images, but distinguishes live and dead cells by analyzing only micrographs captured at a single time point. In contrast to the system developed by Long et al. \[[@B15]\], which employs iterative training procedures to choose the most representative samples for the decision boundary, our system is focused on selection of the features of cell images that support the best classification of cell viability.
Results and discussion
======================
The implemented microscope uses a dark field condenser with an numerical aperture (NA) of 0.96 and a 40× objective that has an NA of 0.65. The light source is a Halogen reflector lamp. A CCD camera (XCD-X700, Sony Inc., Tokyo, Japan) is installed to capture the micrographs. This camera is working in visible light range. 10 probes are sampled from the all-live culture and imaged with the CCD camera, and the resulting micrographs are analyzed by the MVS. In the cell detection procedure of the MVS, a reading window of 31 × 31 pixels is used to scan the images, and 466 live cells are recognized in these images. Analogously, 491 dead cells are detected in the images of 10 samples of the all-dead culture. From these, 232 live cells and 247 dead cells are used to generate the training set; while the remaining 234 live cells and 244 dead cells are used to generate the test set. Feature selection is performed and the SVM (Support Vector Machine) classifier with a linear kernel is trained with these datasets. The best subsets of wavelet features are selected with the SBFS algorithm, in which a criterion function is defined in the form of Eq. (10). The selection results are shown in Figure [1](#F1){ref-type="fig"}. The optimum is found when the number of features is 16, with a criterion value of -0.01454. It is evident that the decline of the criterion within 10% is tolerable (the shadowed region shown in the inset of Figure [1](#F1){ref-type="fig"}), as a result, the best choice of the feature number should be 12, which leads to a criterion of -0.01516, within the tolerable region. In this 12-featured subset, features 0, 1, 2, 3, 5, 6, 10, 16, 19, 20, 24, 28 are included. When too many features are discarded the criterion declines significantly, especially when the feature number is less than 6.
{#F1}
In order to evaluate the performance of the MVS with cultures of given viability, mixed cultures are prepared. Mixed cultures are obtained by mixing all-live and all-dead cultures at a series of ratios (1:4, 2:3, 3:2, 4:1, 17:3, and 9:1). For each mixture, the reference viability is determined by taking the average of five manual counts based on the FUN 1 stain. The numbers of live and dead cells in these counts and standard deviations are shown in Table [1](#T1){ref-type="table"}. As the cell density of the all-live cultures is slightly different from that of the all-dead cultures, the outcome viability is slightly deviated from the nominal value of the mixing ratio. For instance, the nominal viability of the 2:3 (all-live to all-dead) mixture is 0.4, while the actual value is nevertheless about 0.37. The viability determined by the FUN 1 stain is regarded as the gold standard and compared with that by the MVS. For each mixture, five samples are investigated by the MVS and the viability values are averaged.
The correlation of the results given by the MVS and those by the gold standard is displayed in Figure [2](#F2){ref-type="fig"}. The total number of the cells in the test sets of all mixed cultures for the MVS is 1702. The system performance is evaluated in three different cases. In Figure [2(a)](#F2){ref-type="fig"}, the training set and test set of the classifier are composed of only raw image patches (namely, without feature generation and selection). In Figure [2(b)](#F2){ref-type="fig"} the complete set of 32 wavelet features is used. In Figure [2(c)](#F2){ref-type="fig"} a selected subset of 12 features is used. The effect of feature extraction can be recognized in these figures. Comparing Figure [2(a)](#F2){ref-type="fig"} with [2(b)](#F2){ref-type="fig"}, it can be seen that the use of wavelet features leads not only to stronger correlation with the gold standard, but also to lower variances. By comparing Figure [2(b)](#F2){ref-type="fig"} and [2(c)](#F2){ref-type="fig"}, it is clear that discarding 20 features (from 32 to 12 features) does not impair the system\'s performance. On the contrary, the selected feature set helps not only to increase the accuracy of the measurement, but also to reduce the variance in spite of a slightly increased variance at the viability of 0.85.
######
Statistics of the fluorescent reagent based manual counts for the determination of the reference viability of the mixed cultures
MR\* count 1 count 2 count 3 count 4 count 5 ML\*\*(%) SD\*\*\*
------ --------- --------- --------- --------- --------- ----------- ---------- ----- ----- ----- ------ -------
1:4 34 163 30 147 24 150 36 144 48 141 18.7 0.044
2:3 65 118 72 116 67 118 80 108 77 127 38.1 0.027
3:2 95 86 93 83 110 65 115 69 101 86 56.9 0.052
4:1 154 39 121 37 120 42 130 52 130 38 75.9 0.032
9:1 143 38 158 37 147 26 131 34 135 39 80.4 0.028
17:3 165 19 130 31 156 22 150 16 142 36 85.6 0.050
\* MR: mixture ratio; \*\* ML: mean percentage of live cells; \*\*\* SD: standard deviation
{#F2}
In the course of feature selection, after each backward step a number of forward steps are performed as long as the resulting subsets are better than those previously evaluated at that level. With this so-called floating search mechanism, it will often achieve results close to the optimum, thus, it is valuated as one of the currently best sub-optimal methods for feature selection \[[@B16],[@B17]\]. In this sense, the features most frequently selected at all levels (at different levels, different number of best features are determined at the end of the algorithm) can be considered as carrying the most important information. In our case, features 0, 3, 5, 10, 16, and 19 are the most frequently selected ones. Referring to the definitions in subsection \"Wavelet packet feature analysis\", these features are associated to subimages (0,0), (0,1), (3,0) and (3,3) of the wavelet packet decomposition. The physical significances of them are given in Table [2](#T2){ref-type="table"} according to the principle of wavelet packet decomposition. These subimages contain the most important discriminative information. It is evident that higher order details in horizontal and diagonal direction (subimage (3,0), (3,3)) and vertical details at a low level (subimage (0, 1)) are critical for classifying live and dead cells.
######
The physical significances of the most frequently selected features of the two level complete wavelet packet decomposition (refer to Figure 8)
Feature Subimage Physical Significance
--------- ---------- ----------------------------------------------------------------------------
0, 16 (0,0) approximations of approximations (higher order approximations)
5 (3,0) horizontal details of horizontal details (higher order horizontal details)
3, 19 (0,1) vertical details of approximations (lower order vertical details)
10 (3,3) diagonal details of diagonal details (higher order diagonal details)
This opinion can be supported by a reconstruction of cell images in following steps. Firstly, any of the original cell images is decomposed using FWT. Secondly, a value of zero is assigned to each pixel in subimages (0,1), (3,0), and (3,3). Thirdly, an inverse FWT (IFWT) is used to obtain a reconstructed image, which has lost all the most important discriminative information. The comparison between original and reconstructed images is shown in Figure [3](#F3){ref-type="fig"}. It can be seen that the reconstructed live cells (column 2) exhibit more \"grid effect\" than the reconstructed dead cells (column 4), which leads to a greater difference between the reconstructed and original live cells (column 1) than that between reconstructed and original dead cells (column 3). That may serve as evidence for supporting the assumption that live cells contain more detail information than dead ones. Based on Figure [3](#F3){ref-type="fig"}, it is also clear that with the loss of the information in subimages (0,1), (3,0) and (3,3), which benefits viability classification, live cells are hardly to be distinguished from dead ones.
{#F3}
Following a similar idea, a value of zero is assigned to each pixel in all the subimages except (0, 0), (0,1), (3,0), and (3,3). This attempt discards all information that is of less significance for distinguishing cell viability. The comparison between the original and reconstructed images is shown in Figure [4](#F4){ref-type="fig"}. It is clear that the differences between the original and the reconstructed cell images is not so significant as those shown in Figure [3](#F3){ref-type="fig"}. It is also implied that containing only information in the subimages (0,0), (0,1), (3,0), and (3,3), live cells can still be distinguished from dead ones.
{#F4}
For an extended feature analysis, the distribution of the features for live and dead cells from the training set is displayed in a parallel coordinate plot in Figure [5](#F5){ref-type="fig"}. Each thin, red, solid line represents a live cell, and each thin, black, dashed line represents a dead cell. The mean feature value over all live cells is displayed with a thick, white, solid line, while that over all dead cells is displayed with a thick, white, dashed line. Referring to the definition of features, it is clear that with any energy feature (feature 0 \~15), live cells have a higher mean value. That means, on average live cells look brighter than dead cells (feature 0), and contain more details (feature 1 \~15). It is also clear that with any entropy feature (feature 16 \~31), live cells have a lower mean value (except for feature 16). It implies that live cells contain more inhomogeneous fine structures than dead cells.
{#F5}
In the aforementioned attempts dead cells in both the training sets and test sets have been thermally treated. However, it is yet unclear whether the MVS based measurement is still applicable on cell populations, in which cell death is induced by a different mechanism. In order to validate our methodology, we have tested the MVS on another image set containing cells that have died naturally.
In this scenario the implemented microscope uses the same light source and CCD camera as before, however, it uses a dark field condenser with a numerical aperture (NA) of 0.87 and an 10× objective that has an NA of 0.25. Five probes are sampled from the all-live culture and imaged, and 4131 cell positions are found by the MVS, in which 581 recognized cells are used to generate the positive training set. Analogously, five probes are sampled from the all-dead culture, which is thermally treated, and imaged. 446 out of 1645 recognized cells are selected to generate the negative training set. Besides, the remaining 3550 cells from the all-live culture and 1199 cells from the all-dead culture are added to the test sets for feature selection.
In addition, a naturally grown cell culture *K*~1~with a known viability of 0.799 (determined by the FUN 1 dye) is also sampled and imaged. In *K*~1~five probes are taken and in total 2575 cells are recognized (a reading window of 11 × 11 pixels is used to scan the images) as additional test sets for feature selection. The best subsets of wavelet features are selected also with the SBFS algorithm with a criterion function defined in the form of Eq. (11). The optimum is found by SBFS when the number of features is 14. In this best subset, feature 2, 4, 6, 8, 9, 13, 15, 17, 21, 23, 25, 26, 27 and 31 are included. This subset is different from that one determined in the previous scenario, probably because of the different microscopy settings (e.g. different objective magnification factor and different dark field condenser).
In order to evaluate the performance of the MVS, not only all-live and all-dead cultures, but also *K*~1~and *K*~2~have been used. The numbers of live and dead cells in these counts and standard deviations of *K*~1~and *K*~2~are shown in Table [3](#T3){ref-type="table"}.
######
Statistics of the fluorescent reagent based manual counts for the determination of the reference viability of the cultures that have been kept for a long time
---------- -------- -------- ------- -------- -------- -------
count \# k~1~ k~2~
\#live \#dead %live \#live \#dead %live
1 67 17 79.8 55 14 79.7
2 86 27 76.1 113 27 80.7
3 89 14 86.4 66 13 83.5
4 98 28 77.8 86 21 80.4
5 126 20 86.3 89 14 86.4
6 96 33 74.4 136 27 83.4
7 126 34 78.8 75 21 78.1
8 73 16 82.0 80 7 92.0
9 95 29 76.6 48 11 81.4
10 104 25 80.6 62 13 82.7
mean 79.9 mean 82.8
SD\* 4.1 SD\* 4.0
---------- -------- -------- ------- -------- -------- -------
\*SD: standard Deviation
The correlation of the results given by the MVS and by the gold standard is displayed in Table [4](#T4){ref-type="table"}. It can be seen that not only is MVS reliable for measuring cell viability in all-live (*K*~0~) and all-dead cultures (*K*~3~), but also in *K*~1~and *K*~2~, which contains cells that are naturally dead.
######
Measurement results of the MVS for the cultures *K*~0~, *K*~1~, *K*~2~and *K*~3~, the viability of which are 1, 0,799, 0,828 and 0, respectively
-------- --------- -------- --------- --------- --------- -------- -------- -------- -------- -------- ------- -------
sample count 1 cout 2 count 3 count 4 count 5 mean SD\*
\#live \#dead \#live \#dead \#live \#dead \#live \#dead \#live \#dead %live
*K*~0~ 741 26 283 30 310 6 304 33 303 25 93.5 0.036
*K*~1~ 492 128 474 100 311 69 430 104 412 55 82.5 0.034
*K*~2~ 409 79 401 75 350 70 323 35 255 51 85.0 0.030
*K*~3~ 8 273 39 260 9 279 17 253 10 244 5.8 0.042
-------- --------- -------- --------- --------- --------- -------- -------- -------- -------- -------- ------- -------
\* SD: Standard Deviation
From the results it can be seen that the MVS is reliable in measuring cell viability. Although it learns from examples of dead cells that were thermally killed (as the negative training set consists of only thermally treated cells), it is able to predict accurately the viability of the cells that have not been thermally treated. Despite of the fact that the selected feature subsets in these two scenarios are different -- in this sense, no universal feature subset has been found that is applicable in both cases, it is still evidence that it needs only to train the system with new datasets to have it adapted to a new scenario; however, not a single part of the system framework itself requires any change.
Conclusion
==========
It has been shown that a machine vision system based on dark field microscopy in conjugation with wavelet feature selection has very good performance in cell viability assessment.
Wavelet features are found to be suitable to describe the discriminative properties of the live and dead cells in viability classification. According to the analysis, live cells exhibit morphologically more details and are intracellularly more organized than dead ones, which display more homogeneous and diffuse gray values throughout the cells.
Feature selection increases the system\'s performance. The reason lies probably in the fact that feature selection plays a role of excluding redundant or misleading information that may be contained in the raw data, and leads to better results.
Feature selection also reduces the dimensionality of the datasets. That enables the implementation of SVM classifiers with a linear kernel, which are supposed to be unsuitable for high-dimensionality cases. One of the advantages of using linear kernel is that the choice of the proper parameters of a kernel, like the width of the envelop of a Gaussian kernel, can be avoided.
Methods
=======
Principle of the MVS
--------------------
The main idea of the system is to train the MVS with cell samples, the viability of which are known, in order that the MVS learns from the example images some criterion for distinguishing live cells from dead cells just based on their visual appearance. In this learning process, image features are extracted and selected in order to support the classification.
The MVS is composed of two main modules: a training (Figure [6](#F6){ref-type="fig"} -- a) and a test module (Figure [6](#F6){ref-type="fig"} -- b). In the training module, two special kinds of cell cultures are used to generate the training dataset. The first kind, the all-live culture (Figure [6](#F6){ref-type="fig"} -- a (1)) are cell populations in which each cell is alive; while for the second kind, the all-dead cultures (Figure [6](#F6){ref-type="fig"} -- a (2)), each cell is dead. Micrographs of these cultures are captured with a laboratory microscope under dark field settings, and then a cell detection program as described in \[[@B9]\] is run to find the positions of the cells on the micrographs (Figure [6](#F6){ref-type="fig"} -- a (3)).
{#F6}
After the stage of cell detection, an image patch of each cell is collected within a window of *N*× *N*pixels around the detected cell centre. These cell image patches compose the training set (Figure [6](#F6){ref-type="fig"} -- a (4)). Thereafter, features are computed for the *N*× *N*sized image patches prior to performing feature selection (Figure [6](#F6){ref-type="fig"} -- a (5)), which determines the best subset of these features according to certain criteria so that the best performance can be achieved. A class label *y*= 1 is assigned to the feature vector **x**of each live cell; while a class label *y*= -1 is assigned to that of each dead cell. All of these labelled feature vectors are used to train a classifier (Figure [6](#F6){ref-type="fig"} -- a (6)) based on Support Vector Machine (SVM) technique \[[@B18]-[@B20]\]. Our algorithm is implemented with the SVM-LIGHT (Version date: 02.07.02) \[[@B21]\].
After training the classifier, it can be applied to investigate new cell cultures, in which cell viability is unknown (Figure [6](#F6){ref-type="fig"} -- b (1)). The test images are processed in the similar way as in the training module: capture of micrographs under dark field microscope, cell detection (Figure [6](#F6){ref-type="fig"} -- b (2)) and computation of selected feature (Figure [6](#F6){ref-type="fig"} -- b (3)). The selected subset of features is the same as in the training module. After that, the viability of each tested cell is determined with the SVM classifier (Figure [6](#F6){ref-type="fig"} -- b (4)). To evaluate the system\'s performance, the results of different customized cultures are compared with an experimentally derived gold standard (Figure [6](#F6){ref-type="fig"} -- b (5)) in order to evaluate the system\'s performance.
Gold standard of cell viability assessment
------------------------------------------
In order to evaluate the system\'s performance, the viability determined by the MVS is compared with that assessed by a commonly used standard method. In this work, a commercial fluorescence probe for live/dead yeast viability evaluation (FUN^®^1 cell stain, Invitrogen Ltd, Karlsruhe, Germany) is used. With this stain, only live cells are marked clearly with fluorescent intravacuolar structures, while dead cells exhibit extremely bright, diffuse, green-yellow fluorescence. Therefore, the viability of the any cell cultures can be determined easily by manual counting. This viability value is then taken as gold standard.
In this work, the used fluorescence microscope is Nikon Optiphot-2. The protocol of viability assessment with FUN 1 is:
1\. Add FUN 1 stain to a yeast suspension at a concentration of 0.5 mM.
2\. After incubating yeast for 30 minutes in a dark room, trap 10 *μ*L of the yeast suspension between a microscope slide and coverslip.
3\. Examine the stained yeast cells under the fluorescence microscope (excitation: \~450 nm; emission: \~515 nm) and assess manually the ratio of live to dead cells according to the distinguishing intracellular form and color of the fluorescence.
Strain and medium
-----------------
Brewer\'s yeast, *Saccharomyces cerevisiae*(strain Tokay), is chosen to be the target microorganism. In cultivating yeast, a YM medium (glucose: 10 g/L^-1^, peptone: 5 g/L^-1^, yeast extract 3 g/L^-1^, malt extract 3 g/L^-1^, pH 6.2 ± 0.2) is used.
To generate a training set consisting of examples of the live and dead cell images we have prepared all-live and all-dead cell cultures. In order to obtain the all-live cultures, yeast is precultured with the YM medium at 25°C in a 1 L Erlenmeyer flask (filling volume: 0.1 L) on a rotary shaker at the speed of 300 rpm, and harvested in the middle of the exponential growth phase; in order to obtain the all-dead cultures, yeast cells are killed in a water bath at 70°C for 2 hours. It is convenient to obtain a culture with a specific viability by mixing the broth from the all-live and all-dead cultures at a variety of ratios. This kind of cultures is referred to as mixed cultures.
In addition, a naturally grown culture, *K*~1~, which has been kept for 14 days at 25°C in a 1 L Erlenmeyer flask (filling volume: 0.1 L) on a rotary shaker at the speed of 300 rpm with a final viability of 0.799 (determined via the FUN 1 dye), and another naturally grown culture, *K*~2~, which has been kept for 7 days under the same conditions with a final viability of 0.828, have also been prepared.
Wavelet packet feature analysis
-------------------------------
In the MVS, the discrete wavelet transform is performed for feature computation. Wavelet transform as an approach to multi-scale analysis of signals and images has been widely used in image compression, noise removal, texture segmentation, face recognition, medical image processing \[[@B22]-[@B25]\], and is explained here only briefly for 1-D signals, which can be readily expanded to 2-D signals, i.e., gray value images.
In Wavelet analysis, a 1D continuous signal *f*(*x*) can be expanded into the following form:
$$f(x) = {\sum\limits_{k}{W_{\phi,j_{0}}(k)\phi_{j_{0},k}(x)}} + {\sum\limits_{j = j_{0}}^{\infty}{\sum\limits_{k}{W_{\psi,j}(k)\psi_{j,k}(x)}}}$$
where {*φ*~*j*,*k*~(*x*)} and {*Ψ*~*j*,*k*~(*x*)} are sets of scaling functions and wavelet functions, respectively. This series of functions have two parameters: the width, *j*, and the position, *k*:
The coefficients $W_{\phi,j_{0}}(k)$ and *W*~*Ψ*,\ *j*~(*k*) are determined with following relationships:
$$W_{\phi,j_{0}}(k) = {\int{f(x)\phi_{j_{0},k}(x)dx}}$$
Any of the scaling or wavelet functions can be represented as a weighted sum of scaling functions that have a double frequency:
$$\phi(x) = {\sum\limits_{n}{h_{\phi}(n)\sqrt{2}\phi(2x - n)}}$$
$$\psi(x) = {\sum\limits_{n}{h_{\psi}(n)\sqrt{2}\phi(2x - n)}}$$
in which *h*~*φ*~and *h*~*Ψ*~are called scaling and wavelet vectors.
If *f*(*x*) is a discretized function (*x*= 0, 1, 2, \..., *M*-1), then Eq. (4) and (5) should be modified to:
$$W_{\phi,j_{0}}(k) = \frac{1}{M}{\sum\limits_{x}{f(x)\phi_{j_{0},k}(x)}}$$
$$W_{\psi,j}(k) = \frac{1}{M}{\sum\limits_{x}{f(x)\psi_{j,k}(x)}}$$
Eq. (8) and (9) are called discrete wavelet transform (DWT), which is performed through operating *f*(*x*) with scaling and wavelet functions. In the fast wavelet transform (FWT) algorithm, relationship between DWT coefficients in adjacent levels is discovered, and the operation is performed with scaling and wavelet vectors (*h*~*φ*~and *h*~*Ψ*~):
where \* denotes the convolution operator, ↓2 denotes sub-sampling. It is evident that *h*~*φ*~plays a role as a low-pass filter and *h*~*Ψ*~as a band-pass filter, and the original signals can be split into approximations (*W*~*φ*~) and details (*W*~*Ψ*~). In 2-D cases, such as in classical wavelet decomposition of images, each image is split into approximations and details. The approximations are further split into approximations and details with a 2-D FWT:
$$\begin{array}{l}
{W_{\phi,j - 1}(m,n) = {\lbrack h_{\phi}( - m)*{\lbrack h_{\phi}( - n)*W_{\phi,j}(m,n)\rbrack}_{\downarrow 2(c)}\rbrack}_{\downarrow 2(r)}} \\
{W_{\psi,j - 1}^{H}(m,n) = {\lbrack h_{\psi}( - m)*{\lbrack h_{\phi}( - n)*W_{\phi,j}(m,n)\rbrack}_{\downarrow 2(c)}\rbrack}_{\downarrow 2(r)}} \\
{W_{\psi,j - 1}^{V}(m,n) = {\lbrack h_{\phi}( - m)*{\lbrack h_{\psi}( - n)*W_{\phi,j}(m,n)\rbrack}_{\downarrow 2(c)}\rbrack}_{\downarrow 2(r)}} \\
{W_{\psi,j - 1}^{D}(m,n) = {\lbrack h_{\psi}( - m)*{\lbrack h_{\psi}( - n)*W_{\phi,j}(m,n)\rbrack}_{\downarrow 2(c)}\rbrack}_{\downarrow 2(r)}} \\
\end{array}$$
where ↓2(*c*) (↓2(*r*)) denotes sub-sampling along the columns (rows). If the original level of the signals is *J*, then *W*~*φ*,\ *J*~(*m*, *n*) = *f*(*m*, *n*) is the original image. *W*~*φ*,\ *J*-*i*~(*m*, *n*) denotes the approximations subimage at level *i*, and $W_{\psi,J - i}^{t}(m,n)$ denotes the details subimages at level *i*(*t*= *H*, *V*, *D*for horizontal, vertical and diagonal details information).
In a *wavelet packet decomposition*, both the approximations and details are split, which provides richer information for signal analysis. In the proposed scheme, a two levels wavelet packet decomposition is performed, as shown in Figure [7](#F7){ref-type="fig"}. For simplicity, in this figure *W*~*φ*~and *W*~*Ψ*~are denoted as *V*and *W*, respectively. A Daubechies wavelet with four taps is used for filtering the images. At each level of the decomposition, the frequency space is split into four sub-spaces, which leads to a total of 4^2^sub images at level 2, including one approximation and 15 details. Provided that each sub image has a size of *N*× *N*pixels, its energy (*E*) and entropy (*S*) are computed as follows:
{#F7}
$$\begin{matrix}
{E = \frac{\sum_{i}{\sum_{j}x_{ij}^{2}}}{N^{2}}} \\
{S = - {\sum_{i}{\sum_{j}{p(x_{ij}^{2})\log(p(x_{ij}^{2}))}}}} \\
\end{matrix}$$
in which *x*~*ij*~is the *ij*-th pixel value of the subimage, and *p*(·) denotes the probability of the occurrence of value *x*~*ij*~^2^(here the values *x*~*ij*~^2^are quantized into 50 bins).
Energy and entropy are computed for all subimages, therefore, in total 32 features can be used for each image. An index is assigned to each feature in accordance with a layout shown in Figure [8](#F8){ref-type="fig"}. The meaning of the feature can also be determined in this figure. For instance, feature 2 is the energy of the subimage (1,1), namely, $W_{J - 2}^{D}$; feature 28 is the entropy of the subimage (0,2), namely, $W_{J - 2}^{V,A}$.
{#F8}
Feature selection
-----------------
Feature selection has been one of the focuses in pattern recognition because it discovers the subset of features that carries the most discriminative information and abandons those containing more noise than useful information. The advantages of feature selection can be versatile: for instance, reducing dimensionality, enhancing system robustness, increasing recognition rate, and so on.
A large number of algorithms have been proposed for feature selection. Among them, a sequential floating selection algorithm \[[@B16],[@B26],[@B27]\] has been shown to be superior to others in comparative studies. This algorithm can be carried out in two different directions -- forward (**S**equential **F**orward **F**loating **S**election, or SFFS) and backward (**S**equential **B**ackward **F**loating **S**election, or SBFS). The source code of these algorithms can be found in \[[@B28]\]. In the former case, the program starts with an empty subset, and searches for the optimal solution by iteratively adding features into the subset; while in the latter case, it starts with a complete set of features, and discards features iteratively. A floating selection process has been applied so that previously added or discarded features still own the chance to be discarded or added, which leads to a higher probability of finding the optimum. If a criterion function *f*($\mathbf{\widetilde{X}}$) can be determined for any feature subset $\mathbf{\widetilde{X}}$, then a best subset **X***\**can be found using the SFFS or SBFS algorithm. One essential requirement imposed upon the definition of the criterion function is that the better the subset *S*is, the higher is *f*($W_{\phi,j_{0}}(k) = {\int{f(x)\phi_{j_{0},k}(x)dx}}$).
Feature selection in the MVS is performed based on the performance evaluation of the SVM classifier on determining viability of given cultures. For instance, assume that from the all-live culture *N*samples are taken, and the corresponding viability values determined by the classifier, are denoted as $l_{j}^{\mathbf{\widetilde{X}}}$ (*j*= 1, 2, \... *M*), $\mathbf{\widetilde{X}}$ being the present feature subset. From the all-dead culture also *M*samples are taken, and the viability values determined by the MVS are denoted as $d_{j}^{\mathbf{\widetilde{X}}}$ (*j*= 1, 2, \... *M*).
Suppose the true viability of the all-live culture is *l*~0~, and the true viability of the all-dead culture is *d*~0~, the criterion function *f*with regards to $\mathbf{\widetilde{X}}$ can be constructed in the following form:
$$f(\mathbf{\widetilde{X}}) = - \frac{1}{M}{\sum\limits_{j = 1}^{M}{(l_{0} - l_{j}^{\mathbf{\widetilde{X}}})}^{2}} - \frac{1}{M}{\sum\limits_{j = 1}^{M}{(d_{0} - d_{j}^{\mathbf{\widetilde{X}}})}^{2}}$$
It is obvious that the higher the criterion function value is, the better is the classifier\'s performance, and consequently, the better is the feature subset *S*, which satisfies the aforementioned requirement of the SBFS algorithm.
In some cases, the given cultures can also have arbitrary viability. For instance, there are *P*cultures with known viability *v*~1,0~, \..., *v*~*P*,0~, the criterion function can be extended into the following form:
$$f(\mathbf{\widetilde{X}}) = - \frac{1}{M}{\sum\limits_{j = 1}^{M}{(v_{1,0} - v_{1,j}^{\mathbf{\widetilde{X}}})}^{2}}... - \frac{1}{M}{\sum\limits_{j = 1}^{M}{(v_{P,0} - v_{P,j}^{\mathbf{\widetilde{X}}})}^{2}}$$
in which $v_{P,j}^{\mathbf{\widetilde{X}}}$ is the viability measured by the MVS.
Each *N*× *N*-sized image patch can be depicted as a vector $\mathbf{V}_{\omega} \in \text{R}^{N^{2}}$. If **V**~*ψ*~is a live cell patch, it is added to the positive training set with a class label \"1\". Similarly, a dead cell patch is added to the negative training set with a class label \"-1\". Thus, the whole training set can be interpreted as follows (suppose there are *n*~*train*~training cells):
The whole training set can also be divided into two subsets **Ψ**= **Ψ**^+^∪ **Ψ**^-^, with the positive subset
$$\begin{matrix}
{\Psi^{+} = \left\{ \left( {\mathbf{V}_{\omega^{+}},1} \right) \right\},} & {\omega^{+} \in \left\{ \omega \middle| \mathbf{V}_{\omega}\text{~is~a~live~cell~patch} \right\}} \\
\end{matrix}$$
and the negative subset
$$\begin{matrix}
{\Psi^{-} = \left\{ \left( {\mathbf{V}_{\omega^{-}}, - 1} \right) \right\},} & {\omega^{-} \in \left\{ \omega \middle| \mathbf{V}_{\omega}\text{~is~a~dead~cell~patch} \right\}} \\
\end{matrix}$$
Suppose there are *P*test sets, they are obtained from cultures with known viability *v*~1,0~, \..., *v*~*P*,0~, and from each culture *M*samples are taken. Analogously they can be interpreted as follows:
$$\begin{array}{l}
\begin{array}{ll}
{\Gamma_{l} = \Gamma_{l},_{1} \cup ... \cup \Gamma_{l},_{M},} & {l = 1,...,P} \\
\end{array} \\
\begin{array}{ll}
{\Gamma_{l},_{j} = \left\{ \mathbf{V}_{\varphi} \right\},j = 1,...,M,} & {\varphi = 1,2,...,n_{test,l,j}} \\
\end{array} \\
\end{array}$$
Detailed training procedure is shown as follows:
1\. The SBFS algorithm selects a sub feature set $\mathbf{\widetilde{X}}$.
2\. Compute the wavelet features that correspond to $\mathbf{\widetilde{X}}$ of each input ($\mathbf{V}_{\omega^{+}}$, $\mathbf{V}_{\omega^{-}}$) in the training set **Ψ**:
$$\begin{matrix}
\left. \mathbf{V}_{\omega^{+}}\rightarrow\mathbf{W}_{\omega^{+}}^{\mathbf{\widetilde{X}}}, \right. & \left. \mathbf{V}_{\omega^{-}}\rightarrow\mathbf{W}_{\omega^{-}}^{\mathbf{\widetilde{X}}} \right. \\
\end{matrix}$$
3\. Train the SVM classifier with ($\mathbf{W}_{\omega^{+}}^{\mathbf{\widetilde{X}}}$, 1) and ($\mathbf{W}_{\omega^{-}}^{\mathbf{\widetilde{X}}}$, -1)
4\. Compute the wavelet features that correspond to $\mathbf{\widetilde{X}}$ of each input (**V**~*φ*~) in each of the test sets **Γ**~*l*,*j*~:
$$\left. \mathbf{V}_{\varphi}\rightarrow\mathbf{W}_{\varphi}^{\mathbf{\widetilde{X}}} \right.$$
5\. Used the trained SVM classifier to assign a class label (+1 for live and -1 for dead) to $\mathbf{W}_{\varphi}^{\mathbf{\widetilde{X}}}$ and thereby determine the viability of each test set ($v_{l,j}^{\mathbf{\widetilde{X}}}$).
6\. Compute the criterion function value with regards to $\mathbf{\widetilde{X}}$ according to Eq. (11).
7\. According to the returned criterion function value, the SBFS algorithm determine whether $\mathbf{\widetilde{X}}$ is optimal. If not, go to step 1; otherwise, return **X**\* = $\mathbf{\widetilde{X}}$, and end the program.
Abbreviations
=============
DWT: Discrete Wavelet Transform; FWT: Fast Wavelet Transform; MVS: Machine Vision System; SBFS: Sequential Backward Floating Selection; SFFS: Sequential Forward Floating Selection; SVM: Support Vector Machine
Authors\' contributions
=======================
NW participated in conception, design and test of the system, and drafted the manuscript. TWN contributed to conception and design of the system, and drafted the manuscript. EF and KF participated in design of the system. All authors read and approved the final manuscript.
Acknowledgements
================
Gratitude is shown to the Graduate College of Bioinformatics (Graduiertenkolleg Bioinformatik) of Bielefeld University, Germany and German Research Foundation (Deutsche Forschungsgemeinschaft) for funding this project. The authors thank Axel Saalbach and Thorsten Twellmann for providing the C++ programming library on machine learning, and Sebastian Burgemeister for providing some yeast micrographs that have been used to test our programs.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
Type 2 diabetes (T2DM) is often accompanied with cognitive dysfunction, and has an increased rate for developing dementia \[[@R1]\] and Alzheimer's disease (AD) \[[@R2]--[@R4]\]. Previous studies have reported that T2DM was associated with cognitive deficits in certain domains, including immediate and delayed memory, processing speed, learning, as well as executive function \[[@R5]--[@R7]\]. Yet, the exact mechanisms underlying these decrements of cognitive function in T2DM are still unclear.
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that modulates synaptic transmission \[[@R8]\] and hippocampal neuroplasticity that is correlated with learning and memory \[[@R9]\]. More specifically, BDNF induces long-term potentiation (LTP), which is thought to underlie learning and memory \[[@R10]\]. In BDNF knockout mice, hippocampal LTP is significantly decreased, but reversed by administrating exogenous BDNF or increasing BDNF expression \[[@R8]\]. Furthermore, using gene knockout or antisense RNA to inhibit the production of endogenous BDNF resulted in impairment in spatial learning and memory \[[@R11]\], suggesting the critical role of BDNF in learning and memory in the hippocampus \[[@R12]\]. Indeed, previous studies showed that a functional polymorphism Val66Met in the 'pro' region of *BDNF* was associated with a reduction in hippocampal volume \[[@R13]\] and hippocampal-dependent memory in healthy human subjects \[[@R9], [@R12], [@R13]\]. Moreover, some studies found that this polymorphism could influence vulnerability of the brain structural network \[[@R14]\] and human memory-related hippocampal activity \[[@R15]\] in non-diabetic populations. For example, a previous study showed that individuals with Met alleles displayed episodic memory deficiencies \[[@R9]\]. A subsequent study also showed that the Met allele correlated with poor medial temporal lobe-related memory performance \[[@R16]\]. Our study also demonstrated a correlation between the Met allele and cognitive deficit in visuospatial/ constructional index in both schizophrenia subjects and healthy controls \[[@R17]\]. Interestingly, Met homozygotes had volume deficits in gray matter, such as frontal, temporal, and thalamus areas in cognitive-declined diseases such as AD and depression \[[@R18]--[@R20]\]. Val-allele carriers showed better integrities of fiber tracts in white matter compared to Met homozygotes in the occipital area as well as in frontal temporal lobe \[[@R21]\]. Therefore, we hypothesized that *BDNF* Met-allele carriers would present poorer cognition compared to Val homozygotes in patients with T2DM. However, to the best of our knowledge, the association between Val66Met polymorphism of *BDNF* and the cognitive performance in T2DM has not been investigated.
To date, several studies have showed the alterations of circulating BDNF in T2DM patients, with mixed results. For example, some studies found decreased BDNF levels in T2DM \[[@R22]--[@R25]\]; however, the opposite results were also reported in newly diagnosed T2DM patients \[[@R26], [@R27]\]. The inconsistent results could be attributed to several factors, such as sampling of patients with different complications, different illness courses and clinical profiles, exposure for different medications, or the biological heterogeneity \[[@R9], [@R22]\]. Hence, the peripheral levels of BDNF merit further investigation in T2DM patients.
Previous research indicates that serum BDNF levels are positively associated with memory and general cognitive performance in healthy seniors \[[@R28]\]. This is also supported by a significantly lower level of BDNF in individuals with cognitive decline-associated disorders, such as AD and mild cognitive impairment \[[@R29]\]. Our previous study found both cognitive impairment and decreased BDNF serum levels in T2DM patients, as well as a positive correlation between delayed memory and BDNF levels in these patients \[[@R30]\]. Interestingly, our finding was confirmed by a subsequent study showing significant association between cognitive impairment and low BDNF levels in T2DM patients \[[@R31]\], suggesting that BDNF plays an important role in cognitive impairment in T2DM patients.
However, none have explored the inter-relationships among cognitive function, BDNF serum levels and *BDNF* genotype in T2DM patients versus healthy controls. Thus, the purposes of this study were to explore the inter-relationships among cognitive function, BDNF serum levels and *BDNF* Val66Met gene polymorphism in T2DM as well as healthy subjects. Our main aims were: (1) to confirm the association between peripheral BDNF levels and cognitive performance in both T2DM and healthy subjects; (2) to examine whether cognitive performance may be different in the *BDNF* genotype subgroups; and (3) to investigate whether the *BDNF* genotype impacts the relationship of cognitive performance and BDNF serum levels.
RESULTS {#s2}
=======
Table [1](#T1){ref-type="table"} shows the clinical and demographic data. The patients' average age was 54.9 ± 10.7 years, and their illness duration was 6.0 ± 1.0 years. BMI, serum TC, fasting glucose and TG were all greater in T2DM patients than those in the healthy subjects (all *p* \< 0.01).
###### Demographic characteristics and BDNF allele and genotype distributions in the controls and patients with type 2 diabetes
Type 2 diabetes patients (*n* = 311) Normal controls (*n* = 346) Statistics (*p* value)
----------------------------- -------------------------------------- ----------------------------- ------------------------
Sex (M/F) 136/175 138/208 0.99 (0.32)
Age (years) 54.93 ± 10.73 53.43 ± 9.86 3.5 (0.06)
Education (years) 9.82 ± 3.49 10.01 ± 5.85 0.21 (0.65)
BMI(kg/m^2^) 26.02 ± 3.73 24.86 ± 4.60 12.0 (\<0.01)
WHR 0.92 ± 0.25 0.87 ± 0.07 8.6 (\<0.01)
Fasting glucose (mmmol/l) 8.76 ± 2.96 5.02 ± 1.42 405.7 (\<0.01)
Lipids (mmmol/l)
Total cholesterol 5.77 ± 2.02 4.94 ± 1.16 38.7 (\<0.01)
Triglyceride 4.13 ± 4.92 1.38 ± 0.96 97.0 (\<0.01)
Duration of illness (years) 6.00 ± 1.10 NA
HbA~1~c (%)(mmol/mol) 7.42 ± 2.02 NA
Serum BDNF level (ng/ml) 7.73 ± 2.95 11.56 ± 2.67 295.2 (\<0.01)
Allele frequency
Val 51.0% 51.9% 0.11 (0.74)
Met 49.0% 48.1%
Genotype frequency
Val/Val 76 (24.4%) 88 (25.4 %) 0.12 (0.94)
Val/Met 165 (53.1%) 183 (52.9%)
Met/ Met 70 (22.5%) 75 (21.7%)
Abbreviation: *BMI* body mass index, *WHR* Waist-to-hip ratio, *HbA1c* Hemoglobin A1~C~.
Both the patients and healthy controls' genotype distributions were in HWE (both *p* \> 0.05). No significant differences in the *BDNF* genotype and allele frequencies were observed between the patient and control subjects (*Χ*^*2*^ = 0.12, *df* = 2, *p* \> 0.05 and *Χ*^*2*^ = 0.11, *df* = 1, *p* \> 0.05, respectively) (Table [1](#T1){ref-type="table"}). In addition, there was no significant differences in genotype distributions between obese and non-obese groups (*p* \> 0.05).
BDNF serum levels in the patient and control subjects {#s2_1}
-----------------------------------------------------
BDNF levels were significantly decreased in T2DM patients than that in healthy subjects (7.7 ± 3.0 vs. 11.6 ± 2.7 ng/ml, *F* = 295.2, *df* = 1, 638, *p* \< 0.001) (Table [1](#T1){ref-type="table"}). After controlling for gender, age, education, BMI, fasting glucose, TC and TG, BDNF levels were still lower in patients than that in healthy subjects (*F* = 29.1, *df* = 8, 537, *p* \< 0.001).
Further, BDNF levels were negatively associated with HbA~1~c (*r* = --0.13, *df* = 264, *p* = 0.029) in T2DM patients, and inversely with age (*r* = --0.13, *df* = 332, *p* \< 0.05) in controls. In addition, there was no significant relationship between BDNF levels and any other clinical variables in either T2DM patients or controls (all *p* \> 0.05).
Cognitive function in the patient and control subjects {#s2_2}
------------------------------------------------------
Cognitive test by the RBANS were available for 311 patients and 346 healthy controls (Table [2](#T2){ref-type="table"}). RBANS total score and its three domain indexes, immediate memory, language, delayed memory were significantly lower in patients than in healthy controls (all *p* \< 0.001). These significant differences still existed after controlling for gender, age, education, BMI, fasting glucose, TC and TG in the ANOVA as covariates (Table [2](#T2){ref-type="table"}). Also, these significant differences passed Bonferroni corrections.
###### Total and index scores on the RBANS in patients with type 2 diabetes and normal controls
-----------------------------------------------------------------------------------------------------------------
Type 2 diabetes\ Controls (*n* = 346) *F* *P* Adjusted *F* *P*
(*n* = 311)
----------------------------- ------------------ ---------------------- ------ --------- -------------- ---------
Immediate memory 69.45 ± 14.46 76.70 ± 17.64 32.7 \<0.001 11.8 \<0.01
Visuospatial/constructional 81.83 ± 16.06 81.98 ± 15.41 0.02 \>0.05 0.01 \>0.05
Language 89.05 ± 13.50 94.25 ± 13.03 25.2 \<0.001 14.2 \<0.001
Attention 85.74 ± 16.73 88.17 ± 20.26 2.8 \>0.05 0.08 \>0.05
Delayed memory 78.56 ± 13.56 87.84 ± 14.72 70.0 \<0.001 17.5 \<0.001
Total 75.50 ± 13.42 81.45 ± 15.00 28.4 \<0.001 9.4 \<0.01
-----------------------------------------------------------------------------------------------------------------
Note: Adjusted *F* shows the *F* value controlled for sex, age, education, BMI, TC, TG and fasting glucose.
Association of BDNF with cognitive performance {#s2_3}
----------------------------------------------
Serum BDNF levels were positively correlated with the RBANS total score (*r* = 0.13, *df* = 306, *p* \< 0.05) and delayed memory index score (*r* = 0.22, *df* = 306, *p* \< 0.001) in T2DM patients. However, there was no significant correlation of BDNF with any index or RBANS total scores in the controls (all *p* \> 0.05).
For the controls, multiple regression analysis showed that BDNF did not contribute to any RBANS test scores (all *p* \> 0.05). For the patients, multiple regression analysis demonstrated that age (*β* = −0.23, *t* = −3.71, *p* \< 0.001), education (*β* = 0.48, *t* = 8.04, *p* \< 0.001), and BDNF (*β* = 0.12, *t* = 2.04, *p* = 0.043) contributed to the RBANS total score. Furthermore, education (*β* = 0.30, *t* = 4.55, *p* \< 0.001) and BDNF (*β* = 0.21, *t* = 3.26, *p* \< 0.01) were associated with the RBANS delayed memory.
Effects of BDNF genotype on serum in patients and controls {#s2_4}
----------------------------------------------------------
Table [3](#T3){ref-type="table"} showed that the T2DM patients had significantly lower BDNF levels than healthy subjects when sub-grouped by *BDNF* genotype (all *p* \< 0.01). However, BDNF levels were not significantly different among the three *BDNF* genotype subgroups in either patients or healthy subjects (both *p* \> 0.05), without genotype × diagnosis effect (*p* \> 0.05), suggesting that the *BDNF* genotype does not adjust the BDNF levels in both the patient and healthy subjects. After controlling for gender, age, education, BMI, fasting glucose, TC and TG, we still did not observe significant differences in BDNF levels among the three genotypic subgroups in these two groups (both *p* \> 0.05).
###### Serum BDNF levels (ng/ml) in controls and type 2 diabetes by genotype groupings
Val/Val Met/Val Met/Met
----------------------------- ------------ ------------ ------------
Controls (*n* = 332) 11.5 ± 2.4 11.5 ± 2.9 11.8 ± 2.4
Type 2 diabetes (*n* = 306) 8.2 ± 3.0 7.6 ± 3.0 7.5 ± 2.9
*p* value^a^ \<0.01 \<0.01 \<0.01
^a^Indicates the comparison between patients and controls by genotype groupings. There was no significant effect of genotype on serum BDNF levels (*p* \> 0.05) or genotype × diagnosis effect (*p* \> 0.05).
Effects of BDNF genotypes on cognitive performance in patients and controls {#s2_5}
---------------------------------------------------------------------------
Cognitive test by RBANS were performed in 311 patients and 346 healthy controls. Table [4](#T4){ref-type="table"} showed their RBANS total and index scores, and the effects of the BDNF Val66Met polymorphism on the RBANS total and index scores.
###### Comparisons of total and index scores on the RBANS by diagnostic and genotype groupings
---------------------------------------------------------------------------------------------------------------------------------------------------
Cognitive\ Type 2 diabetes Controls Genotype,\ Genotype×\
index *F* (*p* value) diagnosis,\
*F* (*p* value)
------------------ ----------------- ------------- ----------------- ----------------- ------------- ------------- -------------------- -----------
Immediate memory 72.2 ± 14.2 68.9 ± 14.4 67.9 ± 14.9 79.2 ± 16.9 75.3 ± 18.2 77.1 ± 17.1 2.9 (0.06) 0.4 (0.7)
Visuospatial/\ 80.5 ± 16.3 82.4 ± 16.1 82.0 ± 15.9 82.1 ± 14.9 81.3 ± 15.4 83.5 ± 16.1 0.3 (0.7) 0.6 (0.6)
constructional
Language 91.2 ± 13.0 88.5 ± 12.8 88.0 ± 15.4 95.8 ± 12.2 94.3 ± 13.7 92.3 ± 12.3 2.6 (0.07) 0.2 (0.8)
Attention 87.5 ± 15.4 85.9 ± 17.3 83.5 ± 16.8 91.1 ± 18.6 87.2 ± 21.0 87.1 ± 20.2 2.1 (0.13) 0.3 (0.7)
Delayed memory 82.0 ± 14.1 77.7 ± 13.1 76.8 ± 13.5 89.4 ± 14.5 87.5 ± 15.0 86.8 ± 14.5 **3.4 (0.04)** ^a^ 0.5 (0.6)
Total 77.5 ± 12.7 75.3 ± 13.2 73.9 ± 14.5 83.4 ± 13.4 80.7 ± 15.5 80.9 ± 15.5 2.2 (0.11) 0.2 (0.9)
---------------------------------------------------------------------------------------------------------------------------------------------------
^a^There were significant genotype effects on the delayed memory index score (*F* = 3.4, *p* \< 0.05). In addition, there was also a linear negative correlation between the number of Met 66 alleles and the language, immediate and delayed memory indexes and RBANS total score in patients (all *p* \< 0.05). There was no significant effect of genotype×diagnosis on RBANS total score and all the five subtest indexes (all *p* \> 0.05).
As shown in Table [4](#T4){ref-type="table"}, the delayed memory score was significantly different among the three genotypes (*F* = 3.4, *p* = 0.03) in T2DM patients. The Met/Met group displayed significantly lower delayed memory score than Va/Val group (*p* \< 0.05). However, no significant difference was observed in the RBANS total and index scores among the three genotype groups in the controls (all *p* \> 0.05) (data not shown).
Further, there was a linear negative association of the number of Met 66 alleles (dummy number, 0, 1, or 2) with the language (*β* = --0.14, *t* = --2.23, *p* \< 0.05), immediate (*β* = --0.26, *t* = −4.17, *p* \< 0.001) and delayed memory (*β* = --0.26, *t* = --4.01, *p* \< 0.001) and the RBANS total scores (*β* = --0.17, *t* = --2.90, *p* \< 0.01) in the T2DM patients.
BDNF genotypic effects on associations of BDNF serum levels with cognitive functioning {#s2_6}
--------------------------------------------------------------------------------------
Regression analyses found a significantly positive association between BDNF levels and delayed memory score (*β* = 0.29, *t* = 2.21, *p* = 0.033) in Met homozygote patients, as well as negative associations between BDNF and RBANS total score (*β* = --0.92, *t* = --3.40, *p* = 0.002) and language index (*β* = --1.17, *t* = --3.54, *p* = 0.001). However, among Val/Met heterozygous patients, no significant associations were found between the BDNF levels and any RBANS scores (all *p* \> 0.05).
DISCUSSION {#s3}
==========
In our current study, there was no significant difference in the *BDNF* Val66Met polymorphism between T2DM and the controls, which is consistent with a study in a Caucasian population in Denmark \[[@R22]\] and in a Chinese population \[[@R24]\], suggesting that the *BDNF* Val66Met polymorphism may be not associated with susceptibility to T2DM directly. Interestingly, the *BDNF* Val66Met polymorphism has been studied for possible association with obesity, a key pathogenic factor in development of T2DM, but reports are contradictory \[[@R32]--[@R35]\]. In our present, we did not find correlation of the *BDNF* Val66Met polymorphism with obesity in a Chinese Han population. One possible reason for this inconsistent result may be associated with different ethnicities with variable allele frequency distribution of *BDNF* Val66Met. For example, in our study, the frequency of the Met allele was 48.1% in the controls, which is close to other reports in Chinese population \[[@R24]\], but different from other populations, such as Caucasian subjects \[[@R22]\]. Therefore, the differences in the *BDNF* Val66Met genotype distribution frequencies in different ethnicities may result in inconsistency across the studies from the different populations. In addition, it is worth noting that a SNP (rs4074134) near the *BDNF* gene was recently reported to be associated with T2DM independently of obesity in the Chinese Han population \[[@R36]\]. Hence, the role of the *BDNF* polymorphisms and haplotypes in the development of T2DM deserves further examination in different populations.
BDNF Val66Met variant and cognitive impairments in T2DM patients {#s3_1}
----------------------------------------------------------------
The RBANS total score and three index scores- immediate memory, language and delayed memory, were significantly lower in T2DM patients than in healthy subjects. This supports the notion that T2DM has decrements in many aspects of cognitive function \[[@R1], [@R5]--[@R7]\], and increases the risk for some diseases associated with cognitive decline, such as AD and vascular dementia \[[@R2]--[@R4]\].
We found that delayed memory index score were significantly lower in the *BDNF*-Met homozygous carriers than in the Val allele carriers in T2DM patients. Further regression analysis found that the number of Met66 alleles was positively correlated with the performance on delayed memory in T2DM patients, supporting that Met-allele may contribute to impaired memory function \[[@R9], [@R12]\]. However, the exact mechanisms underlying the impact of the Met allele on these cognitive domains are still unclear. Previous reports demonstrated that the *BDNF* Val66Met variant was associated with reductions in hippocampal-dependent memory and in hippocampal volume \[[@R12], [@R13]\] in control subjects. Moreover, Pezawas *et al.* \[[@R37]\] found that *BDNF* Val66Met polymorphism affected the function of BDNF in neurons, and correlated with several neurological and psychiatric disorders, especially the alteration in human memory. Preclinical studies showed that rats transfected with the *BDNF*-Met allele exhibited a lower secretion of BDNF than those transfected with the Val allele \[[@R38]\] in hippocampal neurons. Accordingly, the *BDNF*-Met variant affects memory and hippocampal function by reducing BDNF transmission within cells and activity-dependent secretion in humans \[[@R9]\]. Furthermore, Met-homozygous carriers show poorer memory function than their Val allele counterparts in humans \[[@R9]\]. In short, these studies indicated that the *BDNF* Met-66 variant may influence memory in humans, with or without T2DM.
Cognitive deficits and low BDNF serum: association with BDNF Val66Met genotype {#s3_2}
------------------------------------------------------------------------------
The present study demonstrated that lower BDNF levels were positively associated with cognitive performance in T2DM patients, consistent with recent studies demonstrating association of BDNF levels with cognitive function in ageing adults \[[@R28]\]. However, the underlying mechanisms for these results are still unclear. One possible reason is that BDNF has neuroprotective effect, which is involved in regulating synaptic transmission \[[@R8]\] and activity-dependent neuroplasticity that is critical for learning and memory in the hippocampus \[[@R9]\]. It can also induce long-term potentiation (LTP), which is regarded as the neurophysiological mechanisms for learning and memory \[[@R10]\]. Preclinical studies show that hippocampal LTP was markedly reduced in *BDNF* knockout mice, and could be reversed by administrating exogenous BDNF or increasing BDNF expression \[[@R9]\]. Taken together, these findings suggest a close correlation between cognitive function and BDNF level.
However, the origin of peripheral BDNF are still unclear. Although BDNF is mainly expressed in central nervous system, it also occurs in the circulatory system, such as in serum and platelets \[[@R39], [@R40]\]. Studies have demonstrated that BDNF can pass the blood-brain barrier \[[@R39]\], and serum BDNF levels were found to be associated with that in the brain \[[@R40]\], suggesting that peripheral BDNF may reflect its levels in the brain. Hence, our finding of decreased BDNF may reflect low BDNF brain levels, which could impact cognitive performance in T2DM patients. Indeed, we found that BDNF levels were positively related with delayed memory score in T2DM patients with Met/Met homozygote, while BDNF levels were negatively correlated with the RBANS total and language scores in those with Val/Val homozygote. However, the clinical significance of these discrepant BDNF-cognitive function associations in different *BDNF* genotypes in T2DM patients are still unknown.
Like other studies, we found decreased BDNF levels in T2DM patients \[[@R23], [@R24]\], which were not associated with the *BDNF* Val66Met genotype in both patients and healthy subjects. However, a recent study found that the Val66Met genotype affected serum BDNF levels, showing that Met-allele carriers had higher levels of BDNF in European populations \[[@R41]\]. Hence the *BDNF* Val66Met polymorphism may correlate with BDNF serum levels in some western population, but not in Asian population. More specifically, a previous study reported that the Met variant was associated with decrease only in the activity-dependent BDNF secretion \[[@R17]\]. The relationship between peripheral BDNF and BDNF gene polymorphisms warrants further investigation.
However, there were several limitations in this study. First, previous studies reported that exercise \[[@R42]\], smoking \[[@R43]\] and alcohol \[[@R44]\] were correlated with cognition. However, unfortunately, we did not collect the data for exercise level, smoking or alcohol drinking in our current study, which will be remedied in future investigation. Second, that Val66Met polymorphism may be related to delayed memory is actually a very important observation in our current study. We should provide another way to double confirm this important finding, such as using MRI test, since some previous studies have shown that Met allele affect human memory-related hippocampal activity in healthy persons \[[@R15]\]. Also Met homozygotes showed decreased gray matter volume \[[@R18]--[@R20]\] and worse integrities of fiber tracts in white matter compared to Val allele carriers \[[@R21]\]. Unfortunately, we did not carry out the MRI test in our current study. However, we are performing the MRI test in our ongoing project. Third, our T2DM patients had old age of average 54.9 years and longer duration of illness of average 6 years and had been on oral hypoglycemic, which limit the generalization of our findings to other studies. Fourth, BDNF levels were measured in serum, but not in central nervous system. It remains unknown whether there is a parallel alteration between peripheral BDNF and the central nervous system.
In summary, this study has provided new evidence to support decreased BDNF serum level in T2DM patients, which was significantly associated with the degree of cognitive impairments in T2DM, suggesting that circulating BDNF level may be considered as biomarker of cognitive function in T2DM patients. Also, we found the *BDNF*-Met allele carrier exhibited a poorer delayed memory index score than their Val counterparts in T2DM patients, suggesting that the *BDNF* Val66Met polymorphism was involved in some domains of cognitive deficits in T2DM patients but not in healthy subjects. Furthermore, the association of serum BDNF level with cognitive impairment in T2DM patients was moderated by the *BDNF-*Val66Met polymorphism. Our study, however, is limited by its moderate sample size, wide age range from 20--70 years and longer duration of illness. Additionally, it should be mentioned that that there are significant differences in allele frequency of *BDNF* Val66Met varies between Asian and Caucasian populations, showing that the frequency of Met allele was 48.1% in our present study, which is almost 2.5 times more than that in Caucasian subjects (around 20%) \[[@R38]\]. Thus, it is likely that the specific role of the *BDNF* Met allele in cognitive deficits, especially delayed memory in T2DM patients may be limited to Chinese or Asian populations, which may not be adapted to the western patients. Hence, the association between cognitive impairment and the *BDNF* Met variant in T2DM will need to be confirmed in the future studies in different ethnicities, for instance, in Caucasian population.
MATERIALS AND METHODS {#s4}
=====================
Subjects {#s4_1}
--------
Three hundred and eleven outpatients (male/female = 136/175) were recruited from the Tangshan Gongren Hospital in TangShan city, 50 miles from Beijing in the period from March 2008 to March 2010. Inclusion criteria included: (1) aged between 20--70 years, Han Chinese; (2) meeting the diagnosis of T2DM according to the World Health Organization 1999 criteria \[[@R45]\]; (3) illness duration \< 10 years; (4) without any history of diagnosed coronary artery disease, cerebrovascular disease, stroke, known central nervous system or neuropsychiatric diseases, and any other complications (nephropathy or retinopathy) of diabetes \[[@R46], [@R47]\]; (5) without any audiovisual or motor coordination impairment affecting the cognitive function tests; (6) be able to complete neurocognitive test. In addition, diabetic nephropathy was defined by increased urinary albumin excretion (UAE) in the absence of other renal diseases, with UAE \>20 μg/min \[[@R48]\]. Diabetic retinopathy was defined as the presence of typical retinal microvascular lesions in an individual with diabetes. High-quality fundus photographs of both eyes from all patients were taken and interpreted by a retinal specialist using the International Clinical Diabetic Retinopathy and Diabetic Macular Edema Disease Severity Scales \[[@R49]\]. Diabetic retinopathy was deemed to be present if characteristic lesions were detected (i.e. microaneurisms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, and new retinal vessels). All of the patients were receiving conventional medical treatment with the most commonly prescribed drugs being oral hypoglycemics such as Metformin and Repaglinide.
Three hundred and forty-six normal controls (male/female = 138/208) were recruited from the community in Beijing at the same period in parallel with T2DM patients, and matched for sex, age, and education. All controls were in good physical and mental health.
We obtained a complete medical history and conducted physical examinations and laboratory tests for both patients and control subjects. Subjects with any other illnesses, or drug or alcohol abuse/dependence were excluded from this study. All subjects were Han Chinese. They gave signed and informed consent to participate in the study, which was approved by the Institutional Review Board of Tangshan Gongren Hospital.
Clinical measures {#s4_2}
-----------------
General information, socio-demographic characteristics, medical and psychological conditions of all subjects were collected by a member of the research staff. Additional information was collected from available medical records.
Cognitive assessment {#s4_3}
--------------------
We individually measured cognitive functioning of all subjects using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A) \[[@R50]\]. The RBANS was previously translated into Chinese by our group and its clinical validity and test-retest reliability established among controls and schizophrenia patients \[[@R51]\]. The subjects were administered with the RBANS test on the same day as the blood sample withdrawal. All the subjects performed the cognitive test at least 1 h after breakfast or lunch. No one had taken the cognitive test after an overnight fast.
Four researchers rated subjects on this scale after simultaneously attending a training session in using the RBANS. After training, repeated assessment indicated that the four raters maintained a correlation coefficient for the RBANS total score greater than 0.8. All the four researchers were blinded to all subjects.
Blood sampling and serum BDNF measurements {#s4_4}
------------------------------------------
We collected serum samples at the same period from patients and normal controls between 7 and 9 a.m. following an overnight fast. The serum was separated, aliquoted, and stored at −70°C before use.
We applied a commercially available, sandwich enzyme linked immunosorbent assay to measure serum BDNF levels, as previously described \[[@R52]\]. All samples were assayed by a research assistant blind to the clinical status. Inter- and intra-assay variation coefficients were 8 and 5%, respectively.
Genotyping {#s4_5}
----------
The genotypes of the *BDNF* Val66Met polymorphism were identified as reported previously \[[@R53]\]. A research assistant who was blind to the clinical status genotyped every subject twice to ensure accuracy.
Statistical analysis {#s4_6}
--------------------
Deviations from the Hardy--Weinberg equilibrium (HWE) were assessed using the x^2^ goodness-of-fit test. Chi-squared tests were used to compare *BDNF* Val66Met allele and genotype frequencies between T2DM patients and healthy controls. Demographic and clinical variables of the T2DM patients and healthy controls were compared using analysis of variance (ANOVA) for the continuous variables, and chi-squared for the categorical variables. Since the BDNF variables were normally distributed in the patients and normal controls (Kolmogorov-mirnov one-sample test; both *p* \> 0.05), the principal outcome analysis consisted of one-way ANOVA. When ANOVA was significant, the effect of sex, age, education, body mass index (BMI), and the clinical variables were tested by adding these variables to the analysis model as covariates. Relationships between variables were assessed with Pearson's product moment correlation coefficients.
For the main models, the *BDNF* genotype and diagnosis (cases vs controls) were entered as fixed effects. Scores for each cognitive domain and the total scores of RBANS were entered as the dependent variables, with sex, age, and education included as covariates as appropriate. In each model, the main effect of diagnostic group, the main effect of genotype, and diagnostic group × genotype interaction were tested. The diagnostic group × genotype interaction term in the model detects the differential effects that alleles might have on cognitive scores between diagnostic groups. Similarly, the main effect of the *BDNF* genotype on serum BDNF levels was also analyzed using ANCOVA. Bonferroni corrections were applied to each test to adjust for multiple testing.
Lastly, we performed exploratory regression analyses to examine whether the relationships between BDNF serum levels and cognitive function were different across *BDNF* genotype groups. Stepwise multiple regression analysis used RBANS total or Index scores as dependent variables, with BDNF levels as the independent variable in each *BDBF* genotype group. Covariates in these stepwise forward entry models included age, sex, education, BMI, serum total cholesterol (TC) and triacylglycerol (TG), fasting glucose in both the patient and control groups, and clinical variables in the patient group, such as duration of illness and hemoglobin A1c (HbA~1~c). SPSS version 16.0 was used for all of the statistical analyses. Statistical significance was defined as *p* \< 0.05.
**CONFLICTS OF INTEREST**
None.
**FUNDING**
Funding for this study was provided by grants from the National Natural Science Foundation of China (81371477), the NARSAD Independent Investigator Grant (20314) and the Natural Science Foundation of Hebei Province (H2015105083).
[^1]: These authors contributed equally to this work
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
ABC-transporters belong to one of the largest superfamilies of transporters in cells of all kingdoms [@pone.0070040-Higgins1]. *abc* genes encode conserved multispan trans-membrane proteins that export cargo molecules while utilizing ATP hydrolysis. They participate in multiple pathways by exporting a wide variety of molecules, including lipids, peptides, amino acids, carbohydrates, ions, and xenobiotics [@pone.0070040-Dean1]. ABC proteins can be found on multiple membranes, including the plasma membranes and membranes of intracellular compartments such as the Golgi, endosome, multi-vesicular bodies, endoplasmic reticulum, peroxisome, and mitochondria.
The *D. discoideum* genome contains 68 *abc* genes, which have been classified into 8 groups, *abcA* through *abcH*, based on their sequence and structural features [@pone.0070040-ABCTransporter1], [@pone.0070040-Anjard1]. A fully functional ABC-transporter contains two nucleotide-binding domains and two 6-pass trans-membrane domains. The ABCA, ABCB, ABCD, and ABCG classes of transporters include a few half-transporters, which contain one nucleotide-binding domain and one 6-pass trans-membrane domain. Such half-transporters may form hetero- or homo-dimers that function as full transporters. ABC-transporters of the classes ABCE, ABCF, and ABCH, do not contain any predicted trans-membrane domains and hence may not function as transporters or may need to associate with other proteins to function as transporters [@pone.0070040-Anjard1]. There is currently no general functional classification of these genes because the majority of transporters has not been characterized genetically or functionally in any given organism.
Whole genome expression profiles, obtained by RNA-seq or microarrays, can be used as molecular phenotypes for genome-wide functional analysis [@pone.0070040-Hughes1]--[@pone.0070040-Morley1]. Transcriptional phenotypes provide quantitative parameters that can be used to compare between mutants, identify networks of gene interactions, perform functional analysis of genes, and even analyze epistatic relationships [@pone.0070040-Hughes1]--[@pone.0070040-VanDriessche1]. Transcriptional response phenotypes are considered as higher resolution phenotypes than morphological phenotypes, especially where morphological phenotypes are less diverse and hence less informative [@pone.0070040-VanDriessche1]. RNA-seq has recently supplanted microarrays as the method of choice to obtain global transcriptional responses to environmental changes or mutations [@pone.0070040-Parikh1], [@pone.0070040-Shendure1]. The addition of multiplexing resulted in considerable reduction in cost [@pone.0070040-Miranda1], [@pone.0070040-Noonan1]. In this study we used transcriptional phenotyping with RNA-seq to infer functional similarities between different *abc* transporter mutants in *D. discoideum* and to identify genes that are important for development.
*D. discoideum* are social amoebae that grow as solitary cells when food is abundant and embark on multicellular development when food is scarce. Upon starvation, around 10^5^ cells aggregate through a process of chemotaxis to form a mobile structure called a slug, which ultimately becomes a fruiting body. The fruiting body is comprised of 80% spores that can germinate and propagate to the next generation, and 20% stalk cells, which die while supporting the spore cell mass [@pone.0070040-Loomis1]. *Dictyostelium* development involves multiple signaling molecules and many genes that process them [@pone.0070040-Kessin1], including the *abc* transporter genes *tagB*, *tagC* and *tagA* [@pone.0070040-Good1], [@pone.0070040-Shaulsky1].
In this study, we have obtained mutants for 35 of the *abc*-transporter genes in *D. discoideum* and characterized them based on their morphological and transcriptional phenotypes. We observed that most of the mutants exhibited subtle morphological phenotypes, but the higher resolution of their transcriptional phenotypes allowed us to group them into functional groups. We have also utilized the transcriptional phenotypes to identify genes that are important for *D. discoideum* development. We further show that *abcG6* and *abcG18* influence spore differentiation during the final stages of development.
Materials and Methods {#s2}
=====================
Strain construction, growth and development {#s2a}
-------------------------------------------
The *abc* transporter genes were mutated by homologous recombination in the parental strain AX4. We used two methods to construct the DNA vectors for mutating the genes: a) transposon mutagenesis [@pone.0070040-Abe1] and b) direct cloning [@pone.0070040-Good1]. The vectors and the *Dictyostelium* strains are described in [Table S1](#pone.0070040.s002){ref-type="supplementary-material"}. We verified the insertion sites in each mutant by performing PCR on genomic DNA and also validated each strain by RNA-seq, testing that the insertion modified the expression of the respective *abc* gene in each case.
We grew all the strains in liquid suspension in HL5 medium supplemented with streptomycin (50 mg/mL) and penicillin (50 U/mL), shaking at 220 rpm at 22°C [@pone.0070040-Sussman1]. For scoring morphological phenotypes, we harvested the cells at the logarithmic growth phase by centrifugation and washed them once with KK2 buffer (16.3 mM KH~2~PO~4~, 3.7 mM K~2~HPO~4~, pH 6.2). We deposited 5×10^7^ cells onto a 5 cm nitrocellulose membrane placed on a pad saturated with 2 mL PDF buffer (9.2 mM K~2~HPO~4~, 13.2 mM KH~2~PO~4~, 20 mM KCl, 1.2 mM MgSO~4~, pH 6.5). We incubated the cells at 22°C in a dark humid chamber [@pone.0070040-Sussman1]. We recorded the developmental phenotypes at each time point as specified in the text. Terminal phenotypes were recorded 48 hours after the initiation of development.
Sporulation efficiency {#s2b}
----------------------
We grew and developed 2.5×10^7^ cells on a 5 cm nitrocellulose membrane as described above. We harvested the entire population after 48 hours into a 50 mL falcon test tube containing 10 mL of KK2 buffer, 10 mM EDTA, and 0.1% NP40. We passed the cell suspension 10 times through an 18G syringe needle and counted the spores under a microscope. To test whether sporulation defects were cell autonomous, we mixed mutant and GFP-labeled wild-type cells at equal proportions and developed them together as above. After 48 hours of development, we counted the visible non-fluorescent spores (mutant) and green-fluorescent spores (wild type) as described [@pone.0070040-Santorelli1]. Briefly, green fluorescence was normalized to the number of GFP-labeled wild-type spores that were fluorescent when developed in a pure population (usually greater than 90%) and in a 1∶1 mix with unlabeled wild-type cells. Each experiment was repeated twice unless otherwise indicated. Average values or actual values are presented in [Table 1](#pone-0070040-t001){ref-type="table"}.
10.1371/journal.pone.0070040.t001
###### Morphological phenotypes, sporulation efficiencies and sorting preferences of all the *abc* mutant strains during development.
{#pone-0070040-t001-1}
Mutant Doubling time (h) Morphological phenotype Terminal phenotype Sporulation efficiency (%) Sorting preference in chimera with WT Devel. category
-------- ------------------- ------------------------------------- -------------------- ---------------------------- --------------------------------------- --------------------------- ---------------- --------- ----------------- ----------------
A3 9.0 VEG[a](#nt101){ref-type="table-fn"} RIP LAG, MND MND, FNG, SLG MND, FNG, FBS MND, FNG, GNR 73±2.0 none Normal
A4 8.9 VEG RIP TAG, MTAG TAG, MFNG, SLG MND, FNG, ECUL, FBS GNR, FBS 91±4.0 none Normal
A5 10.3 VEG RIP MND FNG, SLG, MND FBS, ASN ASN 32±6.7 prestalk (pstO) Normal
A6 11.5 VEG RIP TAG, MTAG MFNG, SLG, MXH SFBS SFBS ND ND Normal
A7 10.2 VEG RIP TAG FNG, MXH FBS, ASN ASN 50, 70 prespore Normal
A9 8.5 VEG RIP TAG, MTAG FNG, MFNG, SLG FBS, ASN FBS, GNR 45, 60 none Normal
A10 11.4 VEG RIP MND, TAG MFNG, SLG FNG, SLG, ECUL, ASN FBS, ASN 53, 55 prestalk (pstA) Normal
A11 7.7 VEG RIP LAG, MND ECUL, FNG, SLG, MXH FNG, SLG, FBS, ASN FBS 58, 78 prestalk (pstO) Normal
B1 7.2 VEG RIP TAG TAG, SLG, ECUL, MXH ECUL LFB 93±8.6 prestalk (pstO) Normal
B4 8.4 VEG RIP TAG ECUL, MXH, FNG FBS, MFBS EFB, MFNG 38, 48 prestalk (pstO) Normal
B5 15.3 VEG RIP TAG, FNG SLG, FNG ECUL LFB 82, 117 prestalk (pstO) Normal
C2 11.4 VEG LAG MTAG MFNG MFBS, MFNG MFBS, ASN, LFB 79±3.1 prestalk (pstO) Normal
C3 11.8 VEG RIP MTAG, TAG MFNG, FNG, MXH SLG LFB 53, 80 prespore Normal
C6 10.0 VEG RIP LAG, TAG SLG, FNG FBS, ECUL, MND ASN, LFB ND ND Normal
C8 18.9 VEG RIP LAG MFNG, FNG MFBS MFBS 80±1.6 prespore Normal
C12 11.0 VEG RIP TAG ECUL, FNG EFB FBS 66, 81 prestalk (pstA) Normal
C13 9.9 VEG RIP TAG, MTAG TAG, FNG, SLG, ECUL ECUL LFB, ASN 50, 51 prestalk (pstO) Normal
C14 8.4 VEG RIP TAG, MTAG FNG, MXH, SLG SFBS, ASN SFB, ASN 38, 50 prestalk Normal
D2 11.2 VEG RIP MND FNG SFBS SFBS 90, 97 prestalk (pstO) Normal
F1 7.0 VEG RIP TAG TAG, SLG, ECUL ECUL, ASN ASN, LFB 80±6.0 prestalk Normal
F2 8.3 VEG RIP MND TAG, FNG ECUL FBS 37, 47 none Delayed
F4 7.3 VEG RIP TAG, MTAG FNG, SLG, MFNG, ECUL ECUL, FBS, ASN FBS, ASN, GNR 65, 85 prestalk (pstO) Normal
H2 9.7 VEG RIP MTAG MFNG FNG, ECUL, LCUL FBS 0, 0 ND Normal
G2 10.8 VEG RIP LAG, STR TAG, FNG ECUL, SFBS SFBS 50, 88 prestalk (pstA) Delayed
G5 9.9 VEG RIP TAG, FNG SLG ECUL LFB 73±4.3 prestalk (pstO) Normal
G6 8.4 VEG RIP TAG, MTAG FNG, SLG, MFNG FBS FBS 58±12.6 none Normal
G7 10.4 VEG RIP LAG, MND TAG, FNG ECUL, SFBS SFBS 13, 25 prestalk (pstO) Delayed
G10 9.1 VEG RIP STR, RIP TAG, FNG ECUL, LCUL, SFBS SFB 10, 50 prestalk Delayed
G15 8.4 VEG RIP TAG FNG, SLG, TAG FBS, MXH ASN 32, 40 prespore Normal
G16 11.5 VEG RIP TAG FNG, SLG, MXH SLG, MXH LFB 66, 110 none Normal
G17 9.7 VEG RIP TAG SLG, MXH SFBS SFB 19±2.2 prestalk Normal
G18 8.3 VEG RIP MND, LAG TAG, FNG MFBS MFNG 80±8.1 prestalk Normal
G19 8.0 VEG RIP RIP RIP RIP SFBS ND prestalk No development
G22 9.3 VEG RIP STR, RIP TAG, FNG TAG, SLG, ECUL, FBS, ASN, FBS, GLR, ASN 19, 28 none Delayed
G24 8.5 VEG RIP TAG TAG FNGS, ECUL LFB 6, 18 prestalk Normal
WT 8.0 VEG RIP TAG SLG FBS FBS 113±5.0
Abbreviations: ASN = asynchronous; CUD = culmination defective; ECUL = Early culminants; EFB = early fruiting bodies; FBS = fruiting bodies; FNG = fingers; LAG = loose aggregate; LCUL = late culminants; LFB = late fruiting bodies; MFBS = Multiple fruiting bodies from a single base; MFNG = Multiple fingers; MND = mounds; MTAG = Multi tipped aggregate; MXH = Mexican hats; RIP = Ripples; SFBS = small fruiting body; SLG = Slugs; STR = streams; TAG = tipped aggregate; VEG = Vegetative; ND: not determined. The morphologies expected during wild type development are provided in [Table 2](#pone-0070040-t002){ref-type="table"}.
Transcriptional responses of *abc* genes {#s2c}
----------------------------------------
To determine the transcriptional response of *abc* genes to various treatments at the vegetative stage, we grew wild-type cells in 5 mL of shaking culture in the presence of 5 mM cAMP, 100 nM DIF, or 10 µM Cisplatin in HL5 medium for 16 hours. We extracted RNA and prepared cDNA using the Cell-to-cDNA II kit (Ambion, TX, USA) according to manufacturer\'s recommended protocol. We determined the abundance of each *abc* transporter transcript by quantitative RT-PCR using oligonucleotides specific to each *abc* transporter gene ([Table S2](#pone.0070040.s003){ref-type="supplementary-material"}). Treated samples were compared to untreated samples; we considered transcripts that exhibited a twofold change between the treated and untreated samples as differentially responsive. To determine the transcriptional response of *abc* genes to various treatments during development, we developed 1×10^7^ cells on 5 cm petri dishes containing 1.5% agar in KK2 buffer supplemented with 5 mM cAMP or 100 nM DIF-1. We prepared cDNA from cells at the slug stage (around 20 hours of development) and performed quantitative RT-PCR using *abc*-gene-specific oligonucleotides.
Sorting assays {#s2d}
--------------
To determine the position of the mutant cells in chimeric slugs, we mixed unlabeled mutant cells and GFP-labeled wild-type cells [@pone.0070040-Khare1] at equal proportions (1∶1) and developed them together as above on 5 cm petri dishes containing 1.5% agar in KK2 buffer. We recorded the positions of the mutant cells in the slugs by fluorescence microscopy at around 20 hours of development.
RNA purification and cDNA synthesis {#s2e}
-----------------------------------
RNA purification and cDNA synthesis were performed as described [@pone.0070040-Miranda2]. Briefly, we developed 5×10^7^ cells on a 5 cm nitrocellulose membrane as described above for the number of hours specified in the text. At each time point, we harvested the cells directly into 1 mL of Trizol® (life technologies, CA, USA) and extracted total RNA according to the manufacturer\'s recommended protocol. We performed two rounds of poly-A selection and fragmented 100 ng of the resulting mRNA into approximately 200 bases fragments. We prepared cDNA and the second strand as described before [@pone.0070040-Parikh1], [@pone.0070040-Miranda2].
Illumina multiplexed library preparation for sequencing {#s2f}
-------------------------------------------------------
We prepared Illumina multiplexed libraries as described [@pone.0070040-Miranda2]. We prepared 24 individual libraries separately (one from each sample) and added a unique barcode to each library at the final step of PCR amplification. We pooled equal amounts of DNA from each library and sequenced one pool per lane of a flow cell on an Illumina Genome Analyzer II using the manufacturer\'s recommended pipeline (versions 1.2 and 1.3, read length = 50 bases). The resulting FASTQ files were mapped using the short-read alignment software bowtie (version 0.12.7) allowing 2 mismatches. Mapping was performed as described [@pone.0070040-Miranda2]. The RNA-seq data are available at the NCBI GEO database, under accession number GSE45555 <http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xbuzhssekieyyxm&acc=GSE45555>. Access to individual datasets and a complete list of the RNA-seq data are available in [Table S3](#pone.0070040.s004){ref-type="supplementary-material"}. We define the raw abundance level of a transcript as the sum of all the reads that uniquely map to that transcript. In order to compare transcript abundance between different samples, we normalized the raw abundance values to account for differences in the total number of mapable reads obtained with each RNA-seq run and differences in mapable gene length as described [@pone.0070040-Miranda2]. Briefly, we computed Exp~RPKM~ for every gene in the genome as the number of raw reads scaled per kilobase of the uniquely mapable length of exons per million uniquely mapped reads for the polyadenylated gene models in the experiment.Where:
Exp~RAW~ = the number of raw reads for the gene
Exon~MAPPABLE~ = uniquely mapable length of the exons of the gene (nucleotides)
N~UNIQUE~ = total number of all uniquely mapped reads to polyadenylated gene models in the experiment
We used the Exp~RPKM~ values for all of the analyses, unless otherwise specified.
Multidimensional scaling (MDS) {#s2g}
------------------------------
We computed the mean values of the transcript abundance for each gene at each time point and computed the distances between the transcriptional profiles of the different strains as 1 -- Spearman\'s rank correlation coefficient. Multidimensional scaling (MDS), as implemented in Orange [@pone.0070040-Curk1], was used to present the distances between all the *abc* mutants and the wild type.
Differentially-expressed genes {#s2h}
------------------------------
We carried out differential expression analysis using the baySeq Bioconductor R package (<http://www.bioconductor.org/packages/2.11/bioc/html/baySeq.html>) [@pone.0070040-Hardcastle1]. First, we categorized the mutants as described in the text. We then used baySeq to compare genes in each category to the wild type in order to obtain a set of genes that were not differentially expressed (NDE) and a set of genes that were differentially expressed (DE). baySeq was parameterized using a bootstrap value of 3 and a convergence value of 1e-5 and two models were defined, NDE and DE. Genes with an FDR \<0.005 and a Likelihood \>0.90 were considered NDE.
Results and Discussion {#s3}
======================
*abc*-transporter mutants show robust developmental phenotypes compared to the wild type {#s3a}
----------------------------------------------------------------------------------------
We obtained transcriptional profiles for 35 *abc*-transporter mutant strains and for the laboratory wild-type AX4 strain in two biological replicates at four different time points during development. We developed the cells and collected RNA at 0-, 6-, 12- and 18-hours of development. We subjected the samples to RNA sequencing (RNA-seq), computed the distances between the transcriptional profiles and used multidimensional scaling (MDS) to visualize them in two dimensions ([Figure 1](#pone-0070040-g001){ref-type="fig"}). We surmise that close similarities in mutant transcriptomes suggest that the mutated genes have common physiological functions [@pone.0070040-VanDriessche1]. We also monitored the morphological phenotypes and sporulation efficiencies of all the strains during development and measured the transcript abundance of all the *abc*-transporter genes in wild-type cells under various conditions. Loss of one of the *abc* genes (*abcG19*) resulted in a severe delay in morphogenesis. Mutations in 4 other *abc* genes (*abcF2, abcG2, abcG7, and abcG10*) resulted in a delay in forming tipped aggregates. Mutations in 12 *abc* genes reduced the efficiency of sporulation to less than half that of the wild type. These results are summarized in [Table S4](#pone.0070040.s005){ref-type="supplementary-material"} and [Table S5](#pone.0070040.s006){ref-type="supplementary-material"}.
{#pone-0070040-g001}
We observed that most of the mutants showed similar transcription profiles to the wild type, except for the *abcG19^−^* mutant profile that was separated from the rest ([Figure 1A](#pone-0070040-g001){ref-type="fig"}). The *abcG19^−^* cells do not aggregate until 24 hours of development and form tiny fruiting bodies after 48 hours, suggesting that the transcriptional profile provides a valid reflection of the developmental process. In fact, the *abcG19^−^* profile became exceedingly different from the wild type over the course of development ([Figure 1B--E](#pone-0070040-g001){ref-type="fig"}), consistent with its attenuated development.
All the mutants exhibited transcriptional profiles that were very similar to the wild type at 0-hours ([Figure 1B](#pone-0070040-g001){ref-type="fig"}), which is consistent with the observation that their growth rates do not vary significantly ([Table 1](#pone-0070040-t001){ref-type="table"}). As development progressed to 6-hours ([Figure 1C](#pone-0070040-g001){ref-type="fig"}), the similarity was reduced and an even greater dispersion was observed at 12-hours ([Figure 1D](#pone-0070040-g001){ref-type="fig"}). The transcriptional patterns became more similar to the wild type again at 18-hours of development ([Figure 1E](#pone-0070040-g001){ref-type="fig"}). However, we did not observe any clear trends in the dispersion of the morphological phenotypes as a function of time. This observation is consistent with the notion that the resolution obtained with transcriptional profiling is higher than the resolution of morphological phenotyping. The variation seen in the 12-hour time point might reflect greater asynchrony between the cells in the population at that time, but we do not have other data that could further validate that hypothesis. The *abc* genes have been classified according to their predicted amino acid sequences and predicted structures [@pone.0070040-Anjard1]. We colored the symbols in [Figure 1](#pone-0070040-g001){ref-type="fig"} according to that classification, but we did not observe any class dependent similarities among the *abc*-transporter mutants.
These results support the idea that the transcriptional profiles provide a faithful representation of the mutant phenotypes and suggest that the differences observed between the mutants are informative. Moreover, it is curious that mutations in more than half of the *abc*-transporter genes in *D. discoideum* have such small effects on growth and development.
A group of mutants that were similar to each other and were distant from the wild type includes *abcF2^−^*, *abcG2^−^*, *abcG7^−^*, *abcG10^−^* and *abcG22^−^* (tan arrow, [Figure 1A](#pone-0070040-g001){ref-type="fig"}). This divergence from AX4 is not evident at the beginning of development ([Figure 1B](#pone-0070040-g001){ref-type="fig"}). It begins to appear at 6-hours ([Figure 1C](#pone-0070040-g001){ref-type="fig"}) and becomes more evident at 12- and 18-hours of development ([Figure 1D and E](#pone-0070040-g001){ref-type="fig"}, respectively). Examination of the morphological phenotypes revealed that these mutants were delayed during later stages of development ([Table 1](#pone-0070040-t001){ref-type="table"} and [Table 2](#pone-0070040-t002){ref-type="table"}). These results further suggest that the changes in developmental phenotypes are reflected in the transcriptional phenotypes. It is interesting that although 5 of 6 mutants that display delayed developmental phenotypes belong to the *abcG* class, they do not show other phenotypic similarities, suggesting that they do not have common functions.
10.1371/journal.pone.0070040.t002
###### Morphological phenotypes during wild type development.
{#pone-0070040-t002-2}
Time (h) 0 6 8 10 14 16 18 20 22 24
------------------------ ----- ----- ----- ----- ----- ----- ----- ----- ----- -----
WT morphology VEG RIP STR LAG TAG FNG SLG MXH EFB FBS
Alternative morphology CUL
Abbreviations: VEG = Vegetative; RIP = Ripples; STR = streams; LAG = loose aggregate; TAG = tipped aggregate; FNG = fingers; SLG = Slugs; MXH = Mexican hats; CUL = culminant; EFB = early fruiting bodies; FBS = fruiting bodies.
Other similarities in the transcription profiles of the mutants were also correlated to morphological or other phenotypes ([Table 1](#pone-0070040-t001){ref-type="table"} and [Table S4](#pone.0070040.s005){ref-type="supplementary-material"}). Mutants *abcB4^−^ and abcC13^−^* (blue arrow, [Figure 1A](#pone-0070040-g001){ref-type="fig"}) displayed asynchronous development with similar levels of sporulation efficiency. In chimeric organisms, both mutant strains preferentially sorted to the prestalk area and formed most of the pstO cells when co-developed in a mix with GFP-labeled wild-type cells ([Figure 2](#pone-0070040-g002){ref-type="fig"}), although the sorting phenotype of the *abcC13^−^* strain was less penetrant and was observed in fewer than half of the slugs whereas the sorting phenotype of the *abcB4^−^* strain was observed in most of the chimeric slugs. Examination of the morphological phenotypes alone ([Table 1](#pone-0070040-t001){ref-type="table"}) would have not revealed the similarities between *abcB4^−^ and abcC13^−^*.
{#pone-0070040-g002}
The mRNA levels of most *abc*-transporter genes are developmentally regulated ([Figure S1](#pone.0070040.s001){ref-type="supplementary-material"}). We tested the correlation between the time of *abc*-transcript abundance in the wild type and the time of maximal deviation between the respective mutant phenotype and the wild-type phenotype. We found that for 18 mutants the time of peak transcript abundance preceded the time of maximal deviation and it overlapped with the time of maximal deviation for 8 mutants ([Table 3](#pone-0070040-t003){ref-type="table"}). This finding suggests a causative relationship between the time of maximal transcript abundance and the time of maximal deviation from the wild-type phenotype, which is likely to reflect the time at which the respective genes function.
10.1371/journal.pone.0070040.t003
###### Correlations between *abc*-transcript abundance and *abc*-gene function.
{#pone-0070040-t003-3}
Peak transcript abundance relates to maximal deviation in phenotype Precedes Equals Succeeds No correlation
--------------------------------------------------------------------- ------------------------------------------------------------------------------- ------------------------------------ --------------------- -----------------
*abc* mutants A3, A7, A9, B1, C2, C6, C8, C13, C14, D2, F1, F2, F4, G10, G15, G17, G19, G22 A6, A10, A11, B5, C12, G2, G7, G24 A4, A5, B4, G6, G18 C3, G5, G16, H2
Total number of mutants 18 8 5 4
A majority of the *abc*-transporter mutant strains showed decreased fitness levels, as evident from their low sporulation efficiencies and/or other aberrations in their development ([Table 1](#pone-0070040-t001){ref-type="table"}), although all the *abc*-transporter mutants we analyzed were ultimately able to form fruiting bodies. These results suggest that the ABC transporters studied here are necessary for proper development even though the mutant strains do not exhibit extreme developmental defects under the conditions tested.
In higher organisms, mutations in *abc*-transporter genes that encode multi drug resistance (MDR) transporters do not result in overt phenotypes unless the mutant cells are challenged with toxins [@pone.0070040-Jonker1]--[@pone.0070040-Zhou1]. Considering these reports, we speculate that some of the *Dictyostelium abc*-transporter genes, which show peak transcript abundance at 0-hours, such as *abcA7*, *abcA9*, *abcB1*, *abcC2*, *abcC14*, and *abcG15*, may be involved in toxin resistance during cell growth. Few ABC transporters have also been implicated in developmental processes of other organisms [@pone.0070040-Uchida1], [@pone.0070040-Campanale1], [@pone.0070040-Ricardo1]. Most of the *abc*-transporter mutants in our study show no strong developmental defects even though the respective genes are developmentally regulated. It is therefore unlikely that all of the remaining 33 transporters, which we have not characterized in this study, would have developmental defects if mutated. We speculate that the absence of overt developmental phenotypes is due to functional overlaps between ABC transporters. This possibility could be tested experimentally by characterizing the developmental phenotypes of strains in which two or more *abc*-transporter genes are mutated. Our results may be instructive in selecting small groups of *abc*-transporter genes for such analyses.
Identification of developmental genes {#s3b}
-------------------------------------
Because most of the *abc*-mutants showed only subtle developmental defects, we hypothesized that the expression of genes crucial for development was not perturbed much in these mutants. To test this hypothesis and to identify a set of developmental genes, we divided the *abc*-transporter mutants into three categories, based on their phenotypes at the 12- and 18-hour time points. The first category included mutants that showed normal development, the second category included mutants that showed delayed development and the third category consisted of mutants that did not develop (only *abcG19^−^*) ([Table 1](#pone-0070040-t001){ref-type="table"}). We did not consider the transcriptional responses at 0-hours because the mutants were nearly indistinguishable during growth ([Table 1](#pone-0070040-t001){ref-type="table"}). We compared the transcriptomes of the mutants in each category to the wild-type transcriptome at 6-, 12- and 18-hours using baySeq [@pone.0070040-Hardcastle1]. In each comparison we identified sets of transcripts that showed little or no difference in abundance (FDR \<0.005; Likelihood \>0.90). We generated two sets of unperturbed transcripts, A -- transcripts unperturbed in the normal development group and B -- transcripts unperturbed in the delayed development or no development groups. We then excluded the genes in group B from the genes in group A to identify genes that were unperturbed exclusively in strains that developed normally. We named that group gene set C ({Gene set C} = {Gene set A} \\ {Gene set B}) ([Figure 3A](#pone-0070040-g003){ref-type="fig"}). Gene set C includes 1,935 genes ([Table S5](#pone.0070040.s006){ref-type="supplementary-material"}).
![Identification of important developmental genes.\
The method used to identify developmental genes is depicted as a flow chart (A). Blue boxes represent the wild type (AX4) and mutant strains with normal or delayed development as indicated. Red boxes represent transcripts whose abundance was not significantly altered by the mutations. Green boxes represent selected developmental genes. Ellipses represent the genomes of *D. discoideum* (yellow) and *D. purpureum* (purple) and the overlapping orthologs (not to scale). The volcano plot (B) shows the false discovery rate (FDR) of each transcript (y-axis, −log\[FDR\]) as a function of the difference in mRNA abundance between the median of all the normally developing mutants and the wild type (x-axis, log(mutant/WT) at 12-hours, when the gene expression was most variable across mutants. Each dot represents a gene, yellow dots represent genes that were identified as important for development and conserved between *D. discoideum* and *D. purpureum* (Gene set D), red dots represent genes that show little or no perturbation in expression during development (Gene set C -- Gene set D), and black dots represent the rest of the genes. We validated the approach by examining the enrichment of developmental and non-developmental genes in gene sets C and D (C).](pone.0070040.g003){#pone-0070040-g003}
Previously we found that the two evolutionarily divergent dictyostelid species, *D. discoideum* and *Dictyostelium purpureum* have conserved regulation of developmental gene expression. The set of 3,733 orthologous genes that exhibit conserved mRNA abundance patterns between these two species also shows enrichment in genes which, when mutated, confer developmental defects [@pone.0070040-Parikh1]. We found that 668 of the 1,935 genes in Gene set C belong to that group. We named that subset Gene set D ([Table S6](#pone.0070040.s007){ref-type="supplementary-material"}). We hypothesize that Gene sets C and D include genes that are important for *D. discoideum* development with higher enrichment in Gene set D. Comparing global mRNA abundance in mutants that developed normally and mRNA abundance in the wild type revealed the widest variability at 12-hours ([Figure 3B](#pone-0070040-g003){ref-type="fig"}), consistent with the observations made using MDS ([Figure 1B--1D](#pone-0070040-g001){ref-type="fig"}). However, the 668 genes in Gene set D were the least variable at that time (yellow dots, [Figure 3B](#pone-0070040-g003){ref-type="fig"}), suggesting that their regulation is largely unperturbed in the mutants.
To test whether Gene sets C and D are enriched in developmentally important genes, we looked for enrichment of genes with known developmental roles among these sets ([Figure 3C](#pone-0070040-g003){ref-type="fig"}). A list of genes that have essential developmental roles was obtained from dictyBase [@pone.0070040-Kreppel1]. These genes constitute about 8% of the 12,285 genes in the *D. discoideum* genome. We found that Gene set C contained 13% and Gene set D contained 22% developmental genes. Both values were highly statistically significantly enriched compared to the overall level of 8% ([Figure 3C](#pone-0070040-g003){ref-type="fig"}). Gene set D includes genes that have been studied previously and their developmental mutants are well characterized, including *pkaC*, *tagC*, and *culB* [@pone.0070040-Shaulsky1], [@pone.0070040-Firtel1], [@pone.0070040-Wang1]. It also includes 14 transcription factors, including *dimB*, *gbfA*, *crtf*, *warA*, and *sir2E*, which have important roles in development [@pone.0070040-Han1]--[@pone.0070040-Zhukovskaya1].
To test if the enrichment was specific to developmental genes, we also looked for enrichment of genes in which mutations do not confer obvious developmental defects. There are 620 such genes in dictyBase, which constitute 5% of the *D. discoideum* genome. We observed no significant enrichment in Gene set C and a modest enrichment in Gene set D ([Figure 3C](#pone-0070040-g003){ref-type="fig"}), suggesting that our method of differential expression analysis is predictive of developmentally important genes.
In this analysis we used mutants with subtle phenotypes, which are due to mutations in paralogous *abc* genes that may be involved in various pathways. This approach allowed us to find transcripts that show low plasticity despite the genetic perturbations we introduced. Efforts have previously been made to identify genes that are critical in processes such as cancer or other conditions by using meta-data to identify common sets of genes that vary in multiple conditions [@pone.0070040-Cancer1], [@pone.0070040-Kyndt1]. Here, we used a counter approach and looked for genes that show the least variation upon subtle perturbation. The assumption in our analysis is that an alteration in mRNA abundance of developmentally important genes would lead to developmental abnormalities. This idea may explain why Gene set D is not effective in identifying all of the developmentally important genes; it probably contains only a few false positives.
ABC-transporters involved in prestalk/prespore cell differentiation {#s3c}
-------------------------------------------------------------------
*Dictyostelium* development and cell-type differentiation are mediated, in part, by soluble signals such as cAMP, DIF and SDF [@pone.0070040-Anjard2]--[@pone.0070040-Konijn1], but little is known about the mechanisms that export these and other signaling molecules across the plasma membrane. ABC transporters are potential exporters of signaling molecules, so we were interested in identifying *abc*-transporter mRNAs that showed differential abundance between prespore and prestalk cells. We have previously identified genes that are differentially expressed between prespore and prestalk cells during slug migration [@pone.0070040-Parikh1]. Among those, we found 8 *abc*-transporter genes that exhibited strong cell-type enrichment (\>6 fold) and were mutated in this study ([Figure 4](#pone-0070040-g004){ref-type="fig"}). We then looked for mutants whose transcriptomes differed most from the wild type at 12- and 18-hours and identified 4 such genes. We also found that among the *D. purpureum* orthologs of the *abc*-transporter genes, two exhibited similar cell-type preference, *abcG6* and *abcG18* ([Figure 4](#pone-0070040-g004){ref-type="fig"}). We propose that these genes are candidate exporters of developmental signals.
{#pone-0070040-g004}
To test the role of the *abc*-transporter genes in signaling, we examined the sporulation efficiencies of these mutants in pure populations. We found that all of the mutants exhibited significantly lower sporulation efficiencies compared to the wild type, except for *abcA4^−^* ([Figure 4](#pone-0070040-g004){ref-type="fig"}). We then mixed the mutants with GFP-labeled wild-type cells and allowed them to develop in chimeras to test whether the sporulation defects were cell autonomous. We found that the sporulation defect of the *abcG6^−^* mutant is cell autonomous whereas the sporulation defect of *abcG18^−^* is non-cell autonomous, suggesting that the latter is involved in signaling. Furthermore, we performed differential expression analyses on each of these mutants compared to the wild type transcriptomes at 12- and 18-hr [@pone.0070040-Hardcastle1]. We focused on 1,328 previously identified cell-type enriched transcripts [@pone.0070040-Parikh1] and searched for transcripts that were differentially abundant (FDR \<0.005 and Likelihood \>0.9) either at 12-hr or at 18-hr between each mutant and the wild type. We observed that more than one third of the cell-type enriched transcripts were altered in the *abcG6^−^* and *abcG18^−^* mutant transcriptomes (37% and 44% respectively) ([Table S7](#pone.0070040.s008){ref-type="supplementary-material"} and [Table S8](#pone.0070040.s009){ref-type="supplementary-material"}). These genes include cell-type specific markers such as the prespore genes *cotB*, *cotC*, *pspA* and *pspB* [@pone.0070040-Early1], [@pone.0070040-Fosnaugh1] and the prestalk gene *ecmA* and *ecmB* [@pone.0070040-Early1]. Our results show that both *abcG18* and *abcG6* affect cell type proportioning and spore differentiation, possibly through export of signaling molecules.
Genes of the *abcB* family, namely *tagB, tagC and tagA*, play a critical role in development through signal processing and export [@pone.0070040-Good1], [@pone.0070040-Shaulsky1], [@pone.0070040-Cabral1]. *tagC*, which is primarily expressed in prestalk cells, plays a role in SDF-2 signaling [@pone.0070040-Anjard3], [@pone.0070040-Anjard4]. SDF-2 is a spore differentiation inducing peptide which is produced by processing of the Acyl Coenzyme A binding protein, AcbA [@pone.0070040-Anjard5]. SDF-2 and GABA-2 signaling result in presentation of the protease domain of TagC on the cell surface of prestalk cells, where it processes a diffusible AcbA protein, primarily produced in prespore cells, into SDF-2. The ABC transporter activity of *tagC* is necessary for the protease domain presentation [@pone.0070040-Anjard4]. Similarly, *tagB* is involved in processing the SDF-1 precursor to SDF-1 [@pone.0070040-Anjard6] whereas *tagA* and *acbA* have a coordinated role in *D. discoideum* cell differentiation [@pone.0070040-Cabral1]. In this study we used transcriptional and genetic methods to infer a role for *abcG6* and *abcG18* in spore differentiation. We propose that *abcG18*, which is expressed in prestalk cells, may facilitate the secretion of a diffusible signaling molecule that affects sporulation of prespore cells. Examples of potential cargo molecules include cytokinins, which influence sporulation during culmination [@pone.0070040-Anjard7], but we have not tested that possibility.
Others have shown that mutating both *abcG18* and *abcG2* (*abcG18^−^/abcG2^−^*) can suppress the developmental arrest and the aberration in prespore/prestalk cell ratio observed in the *rtoA^−^* mutant [@pone.0070040-Brazill1]. In this report we found that mutating *abcG18* alone leads to compromised spore differentiation. It is interesting that we did not observe other *abc*-transporter mutants that share a physiological function with either *abcG18^−^* or *abcG2^−^*, as no other mutant showed similar phenotypes to these two mutants ([Figure 1](#pone-0070040-g001){ref-type="fig"} and [Table 1](#pone-0070040-t001){ref-type="table"}).
Supporting Information {#s4}
======================
######
**Each panel describes the transcript abundance of an** ***abc*** **-gene (as indicated above the panel) in the wild type (red, normalized read count) and the maximal deviation of the respective** ***abc*** **-mutant phenotype from the wild-type phenotype (blue, 1 -- Spearman correlation between the wild type and the mutant) as a function of time (hours).**
(PDF)
######
Click here for additional data file.
######
**This table contains information regarding the** ***abc*** **-mutant generation.** Column B contains the name of each mutant strain. Column C describes which of the two methods was used to construct the DNA vector for generating the insertional mutant: Transposon mediated mutagenesis (TZ) or direct cloning using the vector DT2-bsr (DT2). The restriction endonucleases used to clone the BSR cassette into the gene fragment in the second method are listed in column D. Columns E and F contain the primers used to amplify a genomic DNA fragment from each gene. Column G lists the insertion site of the BSR cassette within the gene relative to the ORF and the total ORF length is listed in column H.
(XLSX)
######
Click here for additional data file.
######
**This table contains the oligonucleotide sequences used to determine the abundance of each** ***abc*** **-transporter transcript by quantitative RT-PCR.** The first column represents the gene name, the second column represents the forward primer and the third column represents the reverse primer.
(XLSX)
######
Click here for additional data file.
######
**This table contains the GEO Accession Numbers, sample descriptions and hyperlinks of all the RNA-sequencing data deposited in the NCBI GEO public database.** Access to the entire dataset is available at: <http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xbuzhssekieyyxm&acc=GSE45555>
(XLSX)
######
Click here for additional data file.
######
**This table describes the change of all the** ***abc*** **-transporter transcript abundance in wild-type cells under various conditions determined by quantitative RT-PCR.** Treated samples were compared to untreated samples; we considered transcripts that exhibited a two fold change between the treated and untreated samples as differentially responsive. The response is represented as a binary value in the table, which is further explained in column G.
(XLSX)
######
Click here for additional data file.
######
**This table contains the list of predicted developmental genes (gene set C).** The first column header 'ddb_g' is the primary, unique identifier for each gene in dictyBase and the second column header 'genename' is the primary gene name. Each baySeq comparison performed with two pre-defined models (DE-differentially expressed and NDE-not differentially expressed) at each time point (0-, 6-, 12- or 18-hr) produces two output parameters: Likelihood (Lik) and False Discovery Rate (FDR). We performed three kinds of comparisons: 1) regular mutant transcriptomes vs. wild type (reg), 2) delayed mutant transcriptomes vs. wild type (del), and 3) non developing mutant transcriptome vs. wild type (nod) as described in the text. Hence the column header 'Lik.NDEreg00hr' represents the Likelihood of a gene being not differentially expressed when regular mutant transcriptomes were compared to wild type transcriptome at 0-hours. The other column headers follow the same logic.
(XLSX)
######
Click here for additional data file.
######
**This table contains the list of predicted conserved developmental genes (gene set D).** The column headers are similar to the ones given in [Table S6](#pone.0070040.s007){ref-type="supplementary-material"}.
(XLSX)
######
Click here for additional data file.
######
**This table contains differential abundance data of known prespore- and prestalk- enriched transcripts in** ***abcG6^−^*** **.** The column headers are similar to the ones given in [Table S6](#pone.0070040.s007){ref-type="supplementary-material"} except that in this case only *abcG6^−^* was compared with the wild type at 12- and 18-hours. The column header 'cell_type' indicates whether transcripts of a gene are preferentially enriched in either pre-stalk or pre-spore cell types as described in Parikh *et. al.* (2010).
(XLSX)
######
Click here for additional data file.
######
**This table contains differential abundance data of known prespore and prestalk enriched transcripts in** ***abcG18^−^*** **.** The column headers are similar to the ones given in [Table S8](#pone.0070040.s009){ref-type="supplementary-material"}.
(XLSX)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: EM OZ MT BS BZ AK GS. Performed the experiments: EM OZ. Analyzed the data: EM OZ MT BS. Contributed reagents/materials/analysis tools: EM OZ MT BS. Wrote the paper: EM GS.
| {
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{#sp1 .369}
| {
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It is widely believed that an initial overcorrection is required for the surgical management of intermittent exotropia due to a tendency toward postoperative exodrift \[[@B1]\]. The desired surgical goal in the first week after surgery is to create a small angle of 5 to 10 prism diopters (PD) of esotropia in children with intermittent exotropia \[[@B2]\]. Many ophthalmologists have suggested that initial overcorrection after exotropia surgery in children is necessary for satisfactory long-term correction \[[@B3][@B4][@B5][@B6][@B7]\]. In children, postoperative diplopia is used to stimulate the development of fusional vergences and to stabilize postoperative alignment \[[@B2]\]. However, adults cannot overcome intermittent diplopia induced by overcorrection after surgery for intermittent exotropia. In adults with intermittent exotropia, the first week after surgery usually demonstrates slight undercorrection or orthotropia. The long-term surgical outcome of undercorrection vs. overcorrection in adults remains controversial \[[@B8][@B9]\]. The purpose of this study was to evaluate the relationship between initial postoperative overcorrection and long-term surgical success.
Materials and Methods
=====================
The study was approved by Gachon University, Gil Medical Center. The medical records of 46 patients who underwent surgery for intermittent exotropia after 18 years of age at Gachon University Gil Medical Center between January 2007 and October 2014 were enrolled and reviewed. The patients also had a postoperative follow-up period of at least 2 years. The records were analyzed for the following data: sex ratio, age at the time of surgery, cycloplegic refractive errors, preoperative angle of deviation, number of patients with good stereoacuity (≤100 arcsec), preoperative stereoacuity, best-corrected visual acuity, duration of postoperative follow-up, and type of surgery. The records were also analyzed to determine the preoperative deviation with stereoacuity and postoperative deviations with stereoacuity during the follow-up examinations at the following intervals: 1 week; 1, 3, and 6 months; and 1 and 2 years \[[@B10]\].
All study examinations were performed by one ophthalmologist (HJP). Visual acuity was assessed after cycloplegic refraction. Ocular alignment was assessed by the use of cover/uncover and alternate prism cover testing at a distance of 20 feet in primary position. Motor alignment at near distance was assessed at 14 inches. To evaluate the sensory outcomes of surgery for intermittent exotropia, stereoacuity was tested with the Titmus stereoacuity test (Stereo Optical, Chicago, IL, USA) and transformed to log arcsec for the purpose of statistical analysis, since stereoacuity thresholds are not on a linear scale. An arbitrary value of 4,500 arcsec (3.65 log arcsec) was assigned to the non-measurable stereopsis. One hundred arcsec was regarded as a reference point for good stereopsis. The questionnaire regarding diplopia asked whether an object appears as two at both near and far distances in primary gaze without abnormal head posture. In addition, patients were asked about binocular diplopia to exclude unilateral diplopia due to postoperative astigmatism.
Intermittent exotropia was defined as divergent strabismus that alternates between phoric and tropic phases. Out of the 4 types of exotropia- basic, convergence insufficiency, pseudo-divergence excess, and true divergence excess-all patients demonstrated only the basic type. Patients with A or V pattern, oblique muscle overactions, or dissociated vertical deviation were not included. The exclusion criteria were a mechanical or neurological cause of strabismus, constant exotropia, previous extraocular muscle surgery, or other ophthalmic or systemic diseases. In addition, patients with exodeviation more than 10 PD at postoperative week 1 were excluded.
The surgical procedure was performed by a single surgeon (HJP) who used the same surgical Park\'s formula dosage based on the angle of distant deviation for each patient. All patients were examined 1 week after surgery. Postoperative measurements of distance and near deviations were performed at all follow-up examinations in the same manner. On the basis of the initial postoperative deviation at distances measured by prism and alternating-cover test at 1 week, patients were assigned to one of the following groups: group A included patients who showed any esodeviation (−); group B included patients who showed from orthophoria to exodeviation (+) of 10 PD.
Surgical success was defined as an ocular alignment from orthophoria to 10 PD of exodeviation. The rate of exodeviation recurrence, defined as \>10 PD of exotropia at distance, was determined by reviewing patient records for up to 2 years after surgery. Analysis of variance was performed by comparing groups A and B based on patient demographic data and preoperative and postoperative angles of deviation. In order to investigate the influence of overcorrection at 1 week postoperatively on long-term surgical success, a preliminary univariate analysis (Fisher exact test or Mann Whitney *U*-test) was conducted. Statistical analyses were performed using PASW Statistics ver. 18.0 (SPSS Inc., Chicago, IL, USA). A *p*-value less than 0.05 was considered statistically significant.
Results
=======
Forty-six patients with intermittent exotropia were included in this study, 18 (39%) of whom demonstrated some degree of esodeviation (group A), and 28 (73%) showed orthophoria to exodeviation of 10 PD (group B) in the first postoperative week. Of the 46 individuals, 32 were female and 14 were male. The age at the time of surgery ranged from 18 to 64 years, while the average age was 29.6 ± 11.6 years. The mean follow-up period was 28.8 ± 6.1 months. The mean preoperative angle of deviation was 33.4 ± 11.0 PD and ranged from 15 to 50 PD. Fourteen patients underwent recession and resection surgery, 8 patients required unilateral lateral rectus recession; and 24 patients required bilateral lateral rectus recessions ([Table 1](#T1){ref-type="table"}).
Between the 2 groups, there were no statistically significant differences in sex ratio, age at the time of the surgery, spherical equivalent, preoperative angle of deviation, number of patients with good stereoacuity (≤100 arcsec), preoperative stereoacuity, best-corrected visual acuity, duration of postoperative follow-up, or type of surgery ([Table 1](#T1){ref-type="table"}).
The postoperative angle of deviation for distance fixation up to 2 years after surgery showed a statistically significant difference between the two groups (*p* \< 0.003 in all comparisons). The amount of exodrift for up to 2 years after the operation in group A (from −9.7 ± 6.1 to 1.6 ± 3.7) was greater than that observed in group B (from 2.0 ± 2.7 to 6.8 ± 5.6). In group A, the success rate at each postoperative time increased over the 2 years. In group B, the success rate at each postoperative time decreased and the proportions of recurrence increased during the follow-up period. The long-term surgical success rate within 2 years after surgery was 69% in all patients, 89% in group A, and 57% in group B (*p* = 0.027) ([Table 2](#T2){ref-type="table"} and [Fig. 1](#F1){ref-type="fig"}).
Upon questioning, some patients complained of intermittent diplopia. The number of patients with intermittent diplopia and the duration of diplopia were greater in group A than in group B ([Table 3](#T3){ref-type="table"}).
Among 46 patients who had completed the final stereopsis test, 26 (57%) demonstrated good stereopsis of 100 arcsec. In group B, two patients showed improved to good stereoacuity of 100 arcsec after surgery. No patients in group A showed improved to good stereoacuity after surgery. Mean stereoacuity throughout 2 years of follow-up did not show statistically significant differences between the groups. However, the improvement of stereoacuity was better in group B (from 2.66 ± 0.75 to 2.40 ± 0.64 log arcsec) than in group A (from 2.56 ± 0.75 to 2.51 ± 0.72 log arcsec) (*p* = 0.737) ([Table 4](#T4){ref-type="table"}).
Discussion
==========
Although strabismus surgery is still more commonly performed on children, there has been a significant increase in the number of strabismus operations performed on adults \[[@B11]\]. Studies involving adult exotropia, which should be considered from a different point of view than exotropia in childhood, number many fewer than those of childhood exotropia \[[@B9]\].
The medical records of 46 patients with intermittent exotropia were reviewed. To be included in the study, the patients must have fit the following criteria: undergone surgical correction and followed up for at least 2 years postoperatively. The long-term surgical outcomes from this study indicated that 69% of patients achieved good ocular alignment within 10 PD of exodeviation or orthophoria, and the mean stereoacuity after surgery in the two groups was increased.
Numerous studies have reported that initial postoperative deviation was the only factor found to determine the motor outcome of exotropia in children \[[@B5][@B12]\]. Jung et al. \[[@B13]\] showed that overcorrection in adults with exotropia offers a good outcome without side effects. In the present study, the initial postoperative deviation at 1 week after surgery was useful in predicting the overall results of intermittent exotropia surgery for adults. In the overcorrected group 1 week after surgery, 89% of the patients achieved long-term surgical alignment success. For long-term follow-up, the surgical success rate for intermittent exotropia could decrease due to the progression of exodrift, which lasted for 3 to 4 years after surgery \[[@B14]\]. Therefore, overcorrection seems to guarantee a good long-term motor outcome.
There are two aims of strabismus surgery. One aim is to obtain complete balance between the alignment of the two eyes, and the other is to gain binocular vision, including good stereoacuity. Recovery of stereoacuity, which is one factor of binocular vision, improves fine eye movement, maintains the stability of orthophoria, and prevents recurrence \[[@B15][@B16]\]. Orthophoria affects fusion at the stage of binocular vision development. However, Morris and colleagues showed that adults who had not had orthophoria also achieved peripheral fusion after strabismus surgery \[[@B17]\]. Moreover, many investigators have also reported that good stereoacuity could be obtained by other types of strabismus surgery in adults \[[@B18][@B19][@B20][@B21]\]. It seems that success in motor alignment leads to better stereoacuity; however, in our study, good stereoacuity was achieved in group B (from 2.66 ± 0.75 to 2.40 ± 0.64 log arcsec), which showed a larger PD than group A (from 2.56 ± 0.75 to 2.51 ± 0.72 log arcsec), with a PD close to zero. According to our results, stereoacuity is not proportional to eye alignment. Diplopia and stereopsis were observed in the overcorrection group for 3 months after surgery, but all individuals showed resolution at 6 months. Stereopsis deterioration due to overcorrection was transient and recovered after several months. Patients who underwent initial overcorrection had the disadvantage of complaining of diplopia for several months; however, after two years, the angle of deviation was closer to orthophoria. In patients with diplopia, occlusion therapy was performed for 1 month after surgery, and prism glasses were prescribed when diplopia persisted thereafter.
This study is limited by the small number of patients and its retrospective nature. Furthermore, there could be a selection bias since we enrolled only patients who were followed for more than 2 years. However, the results of this study could still be valuable, because of the smaller number of patients in group A than group B. Patients with favorable postoperative ocular alignment deviation might not return to the clinic, and this might influence the duration of follow-up. Imaging of the central nervous system was not routinely employed and, in the absence of any abnormality, was not assured. However, any patient whose history or ophthalmologic examination suggested an unrecognized underlying disorder underwent consultation by a neurologist. Furthermore, none of the participants in this study were known to have had a neurologic deficit during the follow-up period.
In conclusion, long-term surgical success was achieved in 89% of patients who were initially overcorrected. Overcorrection of an average of 10 PD at the first postoperative week was found to be associated with a more favorable long-term surgical outcome than undercorrection.
**Conflict of Interest:** No potential conflict of interest relevant to this article was reported.
{#F1}
###### Patient demographics, preoperative data, and operative data
Values are presented as number or mean ± standard deviation.
D = diopters; PD = prism diopters; BCVA = best-corrected visual acuity.
^\*^Patients with any esodeviation at postoperative week 1; ^†^Patients with orthophoria to exodeviation 10 prism diopters at postoperative week 1; ^‡^Fisher exact test; ^§^Mann-Whitney *U*-test; ^Π^Visual acuity: logarithm of the minimum angle of resolution.
###### Postoperative angle of deviation for distant fixation and success rate at 1 week, 1 month, 3 months, 6 months, 1 year, and 2 years postoperatively in each group
Values are presented as mean ± standard deviation or number (%). Minus value in the angle of deviation means esodeviation.
^\*^Orthophoria to exodeviation 10 prism diopters; ^†^Patients with any esodeviation at postoperative week 1; ^‡^Patients with orthophoria to exodeviation 10 prism diopters at postoperative week 1; ^§^Mann-Whitney *U*-test; ^Π^Fisher exact test.
###### Postoperative intermittent diplopia at 1 week, 1 month, 3 months, 6 months, 1 year, and 2 years postoperatively in each group
Values are presented as number.
^\*^Patients with any esodeviation at postoperative week 1; ^†^Patients with orthophoria to exodeviation 10 prism diopters at postoperative week 1; ^‡^Fisher exact test.
###### Number of patients with good stereoacuity at preoperative, 1 week, 1 month, 3 months, 6 months, 1 year, and 2 years postoperatively in each group
Values are presented as number (%) or mean ± standard deviation.
^\*^Patients with any esodeviation at postoperative week 1; ^†^Patients with orthophoria to exodeviation 10 PD at postoperative week 1; ^‡^Fisher exact test; ^§^Mann-Whitney *U*-test.
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1. Introduction {#sec1-jcm-09-00089}
===============
The growing technological development improved diagnostic imaging techniques allowing early disease detection and diagnosis \[[@B1-jcm-09-00089],[@B2-jcm-09-00089],[@B3-jcm-09-00089],[@B4-jcm-09-00089]\]. Even if different imaging modalities are extensively used in clinical practice such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission tomography (SPECT), each one presents strong points and limits. Nuclear medicine imaging techniques (PET and SPECT) are highly sensitive (pM range) and quantitative but suffer from poor resolution (mm range) \[[@B5-jcm-09-00089],[@B6-jcm-09-00089]\]; CT is widely available and can detect several pathologies through rapid examinations and easy three-dimensional (3D) reconstructions but radiation dose to the patient is a noticeable concern and it is limited in soft-tissue resolution \[[@B7-jcm-09-00089]\]; MRI gives high resolution, anatomical information, and good soft-tissue contrast but has low sensitivity (mM) \[[@B8-jcm-09-00089],[@B9-jcm-09-00089],[@B10-jcm-09-00089]\]. [Table 1](#jcm-09-00089-t001){ref-type="table"} summarizes imaging modalities and related features. Since no single imaging modality allows gathering all the necessary morphological and functional information, the combination of two or more imaging techniques, also called *multimodal imaging* or *hybrid imaging*, can offer synergistic advantages over any modality alone \[[@B11-jcm-09-00089]\], overcoming its drawbacks and strengthening the peculiarities. The traditional approach was directed to the integration of a structural imaging modality (CT, MRI) with a functional highly sensitive imaging modality (PET/SPECT). Thus, firstly, PET/CT and SPECT/CT were introduced in clinical settings. The first PET/CT scanner was developed in 1998 by Townsend and colleagues \[[@B12-jcm-09-00089]\] and was commercialized in 2001. It consists of a PET component independent from CT, and a single bed moves axially into the scanner while the patient sequentially performs CT and PET scans \[[@B13-jcm-09-00089]\]. To date, PET/CT scanners completely replaced standalone PET scanners \[[@B14-jcm-09-00089]\], exploiting anatomical reference and attenuation estimation from CT data. The success of PET/CT scanners inspired the feasibility of a PET/MRI scanner \[[@B15-jcm-09-00089]\]. Three different configuration options were developed over the years \[[@B16-jcm-09-00089]\]: the first consists of a sequential acquisition, similarly to PET/CT, where the patient undergoes firstly a MRI scan and later a PET scan; even if the MRI and PET components must be minimally modified, two consecutive acquisitions are performed without simultaneity. Temporal mismatches between PET metabolic data and MRI morphological information such as patient motion are the main weak points \[[@B7-jcm-09-00089]\]. Nearly 15 years ago, some researchers working in preclinical settings analyzed the possibility of integrating a modified PET scanner into an MRI system. In Tubingen, Germany, an MRI-compatible PET scanner was inserted into a 3T clinical MRI scanner \[[@B8-jcm-09-00089]\]; this system is suitable for preclinical studies or human brain imaging. The third option considers a first fully integrated whole-body PET/MRI system, with MRI-compatible photodiodes (avalanche photodiodes) and MR-based attenuation correction, and it became commercially available in 2010. It is worth noting that, in addition to anatomical information, MRI also provides functional information such as diffusion-weighted imaging (DWI), blood level oxygen-dependent (BOLD) imaging in functional MRI (fMRI), T~1~/T~2~ mapping, perfusion imaging and spectroscopy, and dynamic contrast-enhanced (DCE) imaging. PET and MRI can take reciprocal advantages: MRI anatomical data are useful for correction of the partial volume effect caused by PET \[[@B17-jcm-09-00089]\], enable motion correction, improve arterial input function characterization for PET kinetic modeling, and are used as priors in PET iterative reconstruction; on the other hand, PET provides molecular information and highly sensitive quantification \[[@B18-jcm-09-00089]\]. Furthermore, the simultaneous acquisition of fMRI data and metabolic PET information can investigate the coupling between metabolic demand and functional activity of the brain, since oxygen and glucose metabolism are strongly related to cerebral blood flow that delivers O~2~ and glucose to tissues \[[@B19-jcm-09-00089]\]. High resolution and high tissue contrast, as well as multiparametric, functional, and quantitative imaging, supply complementary information for breast, head and neck \[[@B20-jcm-09-00089]\], liver, musculoskeletal, and brain tumors \[[@B21-jcm-09-00089]\] and heart \[[@B22-jcm-09-00089]\] imaging. Hybrid imaging spread goes hand in hand with molecular imaging development, where molecular imaging stands for "in vivo" visualization, characterization, and measurement of biological processes at the molecular and cellular levels \[[@B23-jcm-09-00089],[@B24-jcm-09-00089]\]. So far, various molecular imaging modalities were exploited not only for disease diagnosis, stratification, and treatment assessment \[[@B25-jcm-09-00089]\] but also for image-guided therapy. Molecular imaging involves administration of imaging probes and detection of signals produced from the probes \[[@B26-jcm-09-00089]\] and plays a key role in understanding important pathophysiological principles of diseases. In this context, personalized medicine aims to identify the adequate treatment and control its therapeutic efficacy. Suitable imaging probes are currently being developed and represent an exciting challenge for chemists and imaging scientists \[[@B27-jcm-09-00089]\]. In this review, we focus on nanoparticle (NP)-based PET/MRI multimodal tracers in oncological imaging. A few of them were broadly tested in preclinical studies and show promising results in tumor detection, staging, and grading.
1.1. MRI Contrast Agents {#sec1dot1-jcm-09-00089}
------------------------
In imaging, the term "contrast" refers to the capability of distinguishing between two adjacent structures; a contrast agent (CA) increases image contrast and highlights organs or blood vessels. The most common CAs used in X-ray or CT are iodinated and produce a direct effect on the image since they attenuate the X-ray beam, thereby increasing the signal intensity \[[@B20-jcm-09-00089]\]; MRI CAs produce an indirect effect as they influence the relaxation times T~1~ and T~2~ of the neighboring water molecules. MRI CAs can act by reducing T~1~ or T~2~: the former are called T~1~-weighted CAs since they reduce the T~1~ relaxation time and brighten the resulting image, the latter are called T~2~-weighted CAs since they reduce T~2~ relaxation time and darken the resulting image \[[@B21-jcm-09-00089]\]. T~1~-weighted CAs are paramagnetic lanthanide compounds like gadolinium (Gd^3+^) and manganese (Mn^2+^) chelates, while T~2~-weighted CAs are superparamagnetic agents like iron-oxide NPs \[[@B22-jcm-09-00089]\]. CAs acting on both T~1~ and T~2~ relaxation times are called "dual mode" and are NP-based. MRI CAs available for the clinical practice are reported in [Table 2](#jcm-09-00089-t002){ref-type="table"}.
The first requirement for a very efficient CA is a high relaxivity; this parameter indicates the efficiency in reducing T~1~ or T~2~ relaxation time of the surrounding water protons. Paramagnetic metal ions like Gd^3+^ cannot be used as CAs in their ionic form since their accumulation in specific tissues, for example, kidneys, liver, spleen, bone marrow, and the lymphatic system \[[@B28-jcm-09-00089]\], causes toxicity. This challenge can be addressed by using chelators which hide the Gd ion through coordination bonds and are less likely to release it, conferring thermodynamic and kinetic stability and, therefore, less likely to induce toxicity. In particular, CAs based on Gd chelates are strongly associated with nephrogenic systemic fibrosis (NSF) in patients with renal impairment; the disease observed seems to be due to Gd release by chelating molecules in renal compartments \[[@B24-jcm-09-00089]\]. In addition, recently, they were demonstrated to also accumulate in brain and kidneys in healthy patients \[[@B29-jcm-09-00089],[@B30-jcm-09-00089]\].
To improve diagnostic efficacy and reduce the nephrotoxic effects, an ideal CA should be stable, biocompatible, not toxic, and specific; it should remain within the system for a sufficient time to produce desired effects, such as tumor accumulation for oncological imaging, but should also be excreted from the body to minimize unwanted effects of foreign materials within body. In addition, higher relaxivity suggests a lower CA dose in patients. Most CAs currently used (typically small Gd^3+^ ion chelates) lack in specificity because they are confined in the vascular space and do not accumulate in a specific tissue. It is not a coincidence that, in the last decade, CAs were refined by optimizing the relaxivity and developing amplification strategies aimed at increasing probe accumulation at the target site \[[@B25-jcm-09-00089]\]. Moreover, the recent development of molecular and cellular imaging led to the recognition of NPs as MRI CAs.
1.2. Nanoparticles {#sec1dot2-jcm-09-00089}
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In its first applications, hybrid PET/MRI was realized through the simultaneous administration of a mixture of MRI and PET probes, resulting in a cocktail of imaging agents causing high risk for the patient \[[@B38-jcm-09-00089]\]. Additionally, this mixture could not guarantee an exact spatial and temporal correlation of the two imaging modalities due to the different biodistribution, pharmacodynamic and pharmacokinetic properties of the imaging agent. To overcome these limitations, nanoparticles (NPs) were proposed as delivery systems for different imaging agents to obtain bimodal probes for the simultaneous monitoring of both modalities. NPs are defined as particles with at least one dimension lying between 1 and 100 nm \[[@B39-jcm-09-00089],[@B40-jcm-09-00089]\]. In recent years, very different NPs such as proteins, polymers, dendrimers, micelles, liposomes, viral capsids, metal oxides (iron-oxide NPs), zeolites, and mesoporous silicas were investigated, and very different shapes, such as spheres, cylinders (nanorods), and tubes were explored \[[@B24-jcm-09-00089]\].
An NP-based PET/MRI bimodal probe constitutes three essential components: a carrier, a PET tracer usually represented by a positron emitter radioisotope characterized by high sensitivity (e.g., ^18^F-fluorodeoxyglucose), and an MRI component (e.g., gadopentetic acid (Gd-DTPA)), providing high tissue contrast and resolution. The MRI component can either work as a carrier itself (iron-oxide or gadolinium-oxide NPs) or can be a moiety bound to or entrapped in the carrier (e.g., Gd ions grafted onto NPs and polymeric matrices or biologically derived nanosized systems like apoferritin cages \[[@B41-jcm-09-00089]\] and low-density lipoprotein (LDL) particles \[[@B42-jcm-09-00089]\], respectively). Some possible configurations are reported in [Figure 1](#jcm-09-00089-f001){ref-type="fig"}.
As carriers, NPs offer a number of different design options, and the tailoring of their properties can be exploited to directly impact the in vivo fate of the resulting probe. Particle size, charge, core and surface properties, shape, and multivalency are the main features to be finely tuned in order to achieve a proper in vivo distribution, confer a targeting ability, and reduce toxicity of the NPs \[[@B43-jcm-09-00089]\]. The hydrodynamic size determines the NP fate in the body, since vectors with a mean diameter smaller than 5 nm are usually eliminated by renal excretion, whereas larger particles (100 nm) are easily taken up by macrophages \[[@B44-jcm-09-00089],[@B45-jcm-09-00089]\]. NP shape influences the internalization into cells that is relevant in cell tracking and labeling; for example, rod-like particles present higher internalization rates compared to spherical particles \[[@B46-jcm-09-00089]\]. This phenomenon can be explained considering its similarity to rod-like bacterium internalization in nonphagocytic cells \[[@B47-jcm-09-00089]\].
After NP injection into the bloodstream, they are rapidly coated by plasma proteins in a process called opsonization. The NPs are then recognized by plasma membrane receptors found on monocytes and macrophages and are, thus, taken up by the body's main defense system, the reticuloendothelial system (RES), also known as the mononuclear phagocyte system (MPS). The liver, spleen, and bone marrow are rich in macrophages, thus becoming the most accessible organs to NPs \[[@B48-jcm-09-00089]\]. For these reasons, NPs should be coated by adequate materials, to avoid nonspecific uptake by the RES \[[@B48-jcm-09-00089],[@B49-jcm-09-00089],[@B50-jcm-09-00089]\] (stealth effect). In general, hydrophilic and neutral surfaces do not tend to interact with blood components (serum proteins); therefore, they are optimal for minimizing opsonization and clearance \[[@B51-jcm-09-00089],[@B52-jcm-09-00089]\]. Since neutral polymers have no functional groups (amine, carboxyl, or hydroxyl) for ligand linkage, a further step of functional group activation is often mandatory. Another coating strategy employs hydrophilic bifunctional materials such as biphosphonate \[[@B53-jcm-09-00089]\] or aluminum hydroxide \[[@B54-jcm-09-00089]\]. NPs with biocompatible coating layers such as polymers (polyethylene glycol (PEG)), dendrimers, polysaccharides (dextran and chitosan), and polypeptides (serum albumin) can have enhanced properties including better stability in terms of agglomeration, biocompatibility, and solubility in water, along with low toxicity. The most used coating polymers are dextran, chitosan and, above all, PEG \[[@B41-jcm-09-00089]\]. PEG is a hydrophilic, water-soluble, biocompatible polymer widely used to reduce opsonization and increase circulation time from seconds or minutes up to hours \[[@B55-jcm-09-00089]\]. It is important to notice that surface modifications may have an impact on the superparamagnetic properties of iron-oxide NPs; for this reason, coating materials must be carefully chosen \[[@B56-jcm-09-00089]\]. In particular, the nature and the thickness of the coating affect relaxivity. A more hydrophilic coating material results in more water molecules being retained for interacting with the magnetic centers; on the other hand, a thicker coating results in more protons being shielded from the magnetic field \[[@B57-jcm-09-00089]\].
NP delivery to malignant cells can be achieved through both passive and active targeting. Passive targeting is due to the enhanced permeability and retention effect (EPR); since tumor vessels have larger fenestrations, the vascular permeability is higher, and NPs can easily extravasate in tumor tissue. Moreover, the inefficient lymphatic drainage contributes to NP retention in the tumor interstitial space \[[@B58-jcm-09-00089]\]. Even though non-targeted NPs can accumulate in the tumor region due to the EPR effect, the lack of efficient lymphatic drainage generates an increase in interstitial pressure and, consequently, a drop in pressure gradient between the vessel and the extracellular space, causing nanoparticle stacking around the vessel wall \[[@B44-jcm-09-00089],[@B59-jcm-09-00089]\]. For these reasons, there is a need for the development of NPs capable of efficiently and specifically targeting tumor cells \[[@B27-jcm-09-00089]\]. The high NP surface-to-volume ratio helps to overcome this limitation since the NP surface can be functionalized through target-specific moieties that allow an active targeting of cancer cells.
Finally, multivalence refers to the ability to bind different imaging probes, targeting ligands, and therapeutic formulations. This feature is very important for multimodal and molecular imaging where a significant number of targeting probes are needed to track a specific biological path.
1.3. Radiolabeled Nanoparticles {#sec1dot3-jcm-09-00089}
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Tracers currently used in clinical practice are labeled using positron emitters with a relatively low half-life time ranging from 2.037 to 109.8 min. Most of the radionuclides used for labeling are produced via a cyclotron. It generates a beam of accelerated protons and deuterons that are used to irradiate a target (e.g., ^14^N~2~ gas, ^20^Ne gas, ^18^O water or gas), thereby giving the desired radioisotope through a nuclear reaction. [Table 3](#jcm-09-00089-t003){ref-type="table"} shows the main radionuclides with related half-life time, the average energy of positron (β^+^), and means of production.
In PET clinical applications, ^18^F is one of the most suitable radionuclides for radiotracer synthesis since 97% of isotope decay is via positron emission \[[@B60-jcm-09-00089]\], with a fairly low energy of positron emission (maximum 0.635 MeV) and an optimal half-life of 109.8 min, which is considered acceptable for chemical syntheses and favorable when investigating biological processes with a time frame longer than 100 min \[[@B61-jcm-09-00089]\]. ^18^F-based radiotracers are essentially synthesized through two reactions: nucleophilic substitution or electrophilic substitution. Frequently, ^18^F is introduced to replace hydrogen in biomolecules. However, in terms of size, the van der Waals radius of ^18^F (1.47 Å) is closer to oxygen (1.52 Å) than that of hydrogen (1.20 Å) \[[@B62-jcm-09-00089]\]; thus, ^18^F is generally obtained starting from water enriched with ^18^O through a nuclear reaction like ^18^O(p, n)^18^F.
PET radiotracers for cancer diagnosis can be grouped based on their target mechanism as follows:Radiotracers for the evaluation of glucose metabolism, such as fluorodeoxyglucose (FDG);Radiotracers for cell proliferation, such as ^18^F-fluorothymidine (FLT);Radiotracers for the evaluation of vascular perfusion, which include ^15^O-water and ^13^N-ammonia;Radiotracers for the evaluation of hypoxia, such as ^18^F-fluoromisonidazole (FMISO).
Other widely used radionuclides are ^68^Ga and ^64^Cu. In particular, ^64^Cu is gaining increasing interest for its theranostic potential \[[@B63-jcm-09-00089]\]; during its decay, it emits both positron and Auger electrons allowing for both PET imaging and internal targeted radiation therapy. Indeed, Auger-emitting radionuclides that localize in the nucleus of tumor cells demonstrate a potential for cancer therapy. However, their biological effect is critically dependent on their sub-cellular (and sub-nuclear) localization \[[@B64-jcm-09-00089]\] and on the DNA topology \[[@B65-jcm-09-00089]\].
The chemical structures of the most common radiotracers are reported in [Figure 2](#jcm-09-00089-f002){ref-type="fig"}.
NP radiolabeling with the abovementioned tracers can be achieved through different techniques. In the literature, the four following main strategies are reported \[[@B66-jcm-09-00089]\]:Complexation reactions of metallic radioisotope ions through coordination chemistry with the use of chelators;Direct NP bombardment;NP synthesis from radioactive and non-radioactive precursors;Post-synthesis NP radiolabeling without the use of chelators.
The coordination chemistry approach is the most used since radioisotopes can be chelated by different molecules that are covalently bound directly to the NP surface. A strong linkage between the chelator coordinating the radioisotope and the NP surface is desired to assure the stability of the radiolabeling. It is worth noting that many exogenous chelators can currently only coordinate with certain radioisotopes, meaning that an effective chelator-based radiolabeling requires the selection of the best chelator for the isotope of interest \[[@B67-jcm-09-00089]\]. In addition, the choice of the chelating agent should be such to minimize in vivo transchelation.
New chelators for metallic radioisotopes were recently synthesized, including tetradentate acyclic chelators such as PTMS, esadentate acyclic chelators such as ethylenediaminetetraacetic acid EDTA or DTPA, and macrocyclic chelators such as 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA \[[@B68-jcm-09-00089]\], whose chemical structures are presented in [Figure 3](#jcm-09-00089-f003){ref-type="fig"}.
Recently, Laverman et al. reported the possibility of ^18^F chelation through an "Al--^18^F" complex, which carries out a coordination bond with the macrocyclic chelator NOTA \[[@B69-jcm-09-00089]\].
Direct bombardment is achieved by direct irradiation of inorganic NPs with protons and neutrons to obtain radiolabeled NPs. Perez-Campana et al. demonstrated the nuclear reaction ^16^O(p, α)^13^N on Al~2~O~3~ NPs, where the radioisotope is incorporated in the inorganic NPs without any modification of the particle surface and morphology. Moreover, they demonstrated the stability of the radiolabeling by monitoring the in vivo signal after NP intravenous (i.v.) injection \[[@B70-jcm-09-00089]\]. The main limitation of this approach is related to its application to functionalized NPs; the irradiation procedure may induce damages to the organic molecules conjugated onto the NP surface, causing the loss of their biological activity.
An alternative approach is the synthesis of radioactive NPs starting from radioactive and non-radioactive precursors. ^64^Cu is the most widely used radioisotope for this strategy thanks to which both organic and inorganic NPs can be obtained as liposomal ^64^Cu, \[^64^Cu\] CuS, or \[^64^Cu\] CuFe~3~O~4~ \[[@B71-jcm-09-00089],[@B72-jcm-09-00089],[@B73-jcm-09-00089]\]. However, high temperatures and elevated incubation times are required for their production; thus, radiocontamination problems may arise.
Finally, post-synthesis NP radiolabeling seems to be a very promising chelator-free approach. However, both NP properties and chemical and physical interactions between NPs and the radioisotope have to be carefully taken into account. As an example, Chakravarty et al. produced a probe for dual MRI/PET imaging by ^69^Ge radiolabeling of superparamagnetic iron-oxide NPs (SPIONs). They were realized by exploiting the unique interaction between the NP surface and the radiotracer contact, overcoming all the limitations associated with the complex ^69^Ge coordination chemistry of traditional chelator-based methods \[[@B74-jcm-09-00089]\].
Moreover, by exploiting the ability of some radiotracers to emit α and β particles, radiolabeled NPs can be used for radiation therapy in theranostic applications. These radiotracers, indeed, generate ionization in the atoms (mostly in water molecules), with the formation of free radicals and consequent damage to cellular DNA. As an example, liposomes containing α-emitters are widely described in the literature for their ability to improve the radionuclide circulation time and mediate its interaction with the biological environment \[[@B75-jcm-09-00089],[@B76-jcm-09-00089],[@B77-jcm-09-00089]\]. Through this approach, it is possible to improve the ratio between radiation dose to tumor and normal tissues. Secondly, because of a better time to circulate, these formulations cause larger concentrations to diffuse within the tumor tissue and may, therefore, provide a less heterogeneous tumor dose \[[@B75-jcm-09-00089]\].
1.4. PET/MRI Nanoparticles and Preclinical Applications {#sec1dot4-jcm-09-00089}
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NPs were extensively studied at a preclinical level as imaging probes for dual MRI/PET tumor imaging. According to the chemical composition of the core, NPs can be classified into inorganic and organic \[[@B78-jcm-09-00089]\]. Inorganic NPs recently gained significant attention due to their unique physical and chemical properties. In particular, their chemical inertness, good stability, and the easiness of surface functionalization make inorganic NPs attractive for imaging of malignant tumors. However, their toxicity remains the main concern; it was demonstrated that iron-oxide NPs entering into cells through endocytosis show high toxicity because of their accumulation in endo-lysosomal compartments \[[@B59-jcm-09-00089]\]. The most used carriers of this category are iron-oxide NPs and silica NPs. Common nanoconstructs are shown in [Figure 4](#jcm-09-00089-f004){ref-type="fig"}.
### 1.4.1. Iron-Oxide Nanoparticles {#sec1dot4dot1-jcm-09-00089}
Magnetic iron-oxide NPs, typically magnetite Fe~3~O~4~ and maghemite, γ Fe~2~O~3~, are broadly employed in MRI imaging especially for the liver, spleen, and bone marrow due, to their ability to shorten T~2~ and T~2~\* relaxation times. According to their size, they can be categorized into *micrometer-sized paramagnetic iron oxide* (MPIO) (several micrometers), *superparamagnetic iron oxide* (SPIO) (hundreds of nanometers), *ultrasmall superparamagnetic iron oxide* (USPIO) (below 50 nm) \[[@B79-jcm-09-00089]\], and *mono crystalline iron oxide* (MION) (representing a subset of USPIO ranging from 10 to 30 nm) \[[@B78-jcm-09-00089],[@B80-jcm-09-00089]\].
The most common method for SPIO and USPIO synthesis is the reduction and coprecipitation reaction of ferrous and ferric salts in a basic aqueous media \[[@B81-jcm-09-00089],[@B82-jcm-09-00089],[@B83-jcm-09-00089]\]. Resulting NPs are generally polydisperse and poorly crystalline; therefore, other preparation methods are often preferred, such as thermal decomposition and microwave synthesis \[[@B17-jcm-09-00089]\]. Bare NPs are prone to agglomeration due to their high surface energy. In order to improve both colloidal and chemical stability, many polymeric coating materials were proposed, such as dextran, carboxymethylated dextran, carboxydextran, chitosan, starch, PEG, heparin, albumin, arabinogalactan, glycosaminoglycan, sulfonated styrene--divinylbenzene, organic siloxane, polyvinyl alcohol, poloxamers, and polyoxamines \[[@B84-jcm-09-00089],[@B85-jcm-09-00089]\]. In addition, the polymeric corona is able to protect iron-oxide NPs, preventing erosion at acidic pH, lowering cytotoxicity \[[@B63-jcm-09-00089]\]. The coating can be performed during the co-precipitation process, with the synthesis of the NPs occurring simultaneously to its coating \[[@B86-jcm-09-00089],[@B87-jcm-09-00089]\] or post-synthesis, with the coating realized after the synthesis of the NPs \[[@B88-jcm-09-00089],[@B89-jcm-09-00089]\]. Surface coating is a key factor for NP bioconjugation to biological ligands such as peptides or antibodies; therefore, it represents clinical potential for cancer imaging. Nevertheless, iron-oxide NPs have some important drawbacks. First of all, they act as negative contrast and, after administration, there is a loss of signal that makes medical evaluation less easy compared to T~1~ CA brightness. Moreover, the high susceptibility causes distortion artefacts and reduces the contrast-to-noise ratio \[[@B79-jcm-09-00089]\]. Gd-based T~1~ agents are the most extensively and clinically used. Alloy materials were investigated to obtain more efficient T~2~ CAs because they are endowed with higher magnetic anisotropy \[[@B69-jcm-09-00089]\], crystallinity, and relaxivity; thus, various bimetallic ferrite NPs named *magnetic engineered iron-oxide NPs*, such as CoFe~2~O~4~, MnFe~2~O~4~, and NiFe~2~O~4~, were tested \[[@B90-jcm-09-00089]\].
There are several commercially available superparamagnetic iron-oxide NP formulations such as Feridex (Berlex,, Hanover, NJ, USA), Endorem (Guerbet, Villepinte, EU), and Resovist (Schering, EU, Japan). They are mostly used for liver and spleen tumors diagnosis \[[@B91-jcm-09-00089]\], and the coating polymers are dextran for Feridex and Endorem, and an alkali-treated low-molecular-weight carboxydextran for Resovist \[[@B92-jcm-09-00089]\]. Many preclinical studies were conducted to assess the iron-oxide NP potential as PET/MRI probes for cancer imaging exploiting both passive targeting (for lymph node mapping) and active targeting strategies (mainly through RGD (Arg--Gly--Asp) conjugation).
Thorek and coworkers \[[@B93-jcm-09-00089]\] prepared ^89^Zr radiolabeled iron-oxide NPs (ferumoxytol) to visualize the axillary and brachial lymph node drainage in healthy wild-type mice. In detail, the iron-oxide core was surrounded by a semisynthetic polysaccharide coating of polyglucose sorbitol carboxymethylether, and desferrioxamine was used as a chelator. In the same study \[[@B93-jcm-09-00089]\], after intraprostatic administration in Hi-Myc transgenic mice bearing invasive prostatic adenocarcinoma, PET/MRI imaging delineated draining nodes in the abdomen and the inguinal region, in addition to prostatic ones.
In 2019, Madru et al. \[[@B94-jcm-09-00089]\] proposed a new, time-efficient, chelator-free conjugation of ^64^Cu on PEGylated SPIONs for PET/MRI detection and localization of sentinel lymph nodes (SLNs) in C57BL/6J mice. The stability of radiolabeling up to 24 h and NP accumulation in the SLN were demonstrated through a biodistribution study. Lymph nodes metastases are important markers for cancer staging and treatment, and their localization can be useful in presurgical planning.
Xie and colleagues \[[@B95-jcm-09-00089]\] encapsulated iron-oxide NPs, after modification with dopamine, into human serum albumin (HSA) matrices and labeled them with Cy5.5 dye and ^64^Cu-DOTA. NPs were injected into a U87MG xenograft mouse model; PET and NIRF imaging showed a higher signal-to-noise ratio compared to MRI because of their higher sensitivity. On the other hand, MRI scans post NP injection showed a clear inhomogeneous distribution thanks to their high spatial resolution. These findings were confirmed by histological studies. The HSA shell conferred prolonged circulation time and lower macrophage uptake rate. Such NPs are suitable for theranostic applications if co-loaded with drug molecules.
An active targeting probe was developed by Lee and coworkers \[[@B96-jcm-09-00089]\] who conjugated RGD to ^64^Cu radiolabeled iron-oxide NPs. As a coating material, polyaspartic acid was chosen since it exposes both carboxyl groups interacting with NPs and amine groups useful for DOTA and RGD conjugation. Imaging was performed on a U87MG mouse model, and both PET and MRI confirmed that the accumulation of NPs was mediated by α~v~β~3~ integrin binding. Kim and colleagues \[[@B97-jcm-09-00089]\] injected ^68^Ga labeled iron-oxide NPs into BALB/c nude mice bearing colon cancer (HT-29) cells, using oleanolic acid as a tumor-targeting molecule. This ligand was shown to inhibit colon cancer cell proliferation, as well as induce apoptosis and cancer cell death. Binding assays and histological studies confirmed the tumor uptake of NPs thanks to oleanolic acid affinity for HT-29 cancer cells. PET/MRI scans provided high-quality images and precise quantification of the tumor area.
### 1.4.2. Silica-Based Nanoparticles {#sec1dot4dot2-jcm-09-00089}
Silica-based NPs are widely applied in drug delivery, bio-imaging, and cell targeting as they are considered an ideal biocompatible matrix to integrate imaging probes. There are two major classes of silica-based NPs: solid (SiNPs) and mesoporous (MSNs). SiNPs are extensively used as optical imaging agents, while MSNs are often used in CT, MRI, PET, molecular, and multimodal imaging. MSNs are synthesized by a surfactant templated sol--gel method \[[@B98-jcm-09-00089]\] and have attractive properties such as an extremely large surface area, a tunable structure in terms of size, morphology, and porosity, and ease of functionalization through synthetic approaches \[[@B99-jcm-09-00089]\]. In bimodal PET/MRI imaging, MSNs are used as a coating material for metallic NPs or as carrier for MRI CAs and PET radioisotopes. CAs can be encapsulated in channels and protected from the environment, together with drugs or genes for theranostic purpose. A porous silica shell improves MRI contrast enhancement since the pores allow intimate contact between water molecules and the iron-oxide NPs \[[@B100-jcm-09-00089]\]. We report two examples of MSNs in PET/MRI for cancer imaging. Burke and colleagues \[[@B101-jcm-09-00089]\] described silica-coated iron-oxide-based nanorods radiolabeled with ^68^Ga. In detail, nanorods were coated with various ratios of a siloxane-terminated tetraazamacrocycle (siloxane-DO3A) and a siloxane PEG derivative. Nanorods offer some advantages over nanospheres such as improved T~2~ MRI contrast and direct uptake in the liver via phagocytosis. Moreover, thanks to the silica coating, a macrocyclic chelator for highly stable radiolabeled nanoconstructs was not required. Huang and coworkers \[[@B102-jcm-09-00089]\] reported a mesoporous silica-based triple modal imaging nanoprobe to map and track tumor metastatic sentinel lymph nodes (T-SLNs). In this system, three imaging probes including near-infrared (NIR) dye ZW800, T~1~ CA Gd-DTTA, and the positron-emitting radionuclide ^64^Cu were integrated into MSNs via different conjugation strategies. PET and MRI imaging probes were located on the surface and in the mesoporous channel of NPs. A faster uptake rate and higher uptake of the multifunctional MSN probes were observed in T-SLNs compared with normal SLNs, confirming the feasibility of these MSN probes as CAs to map SLNs and identify tumor metastasis. Images revealed that NP accumulation in T-SLNs was much higher than in normal controlateral SLNs (N-SLNs), where almost no signal was observed.
### 1.4.3. Organic Nanoparticles {#sec1dot4dot3-jcm-09-00089}
Over the last decade, a number of organic NPs, such as dendrimers, polymeric micelles, liposomes, and proteins were used in various applications for cancer diagnosis. These organic NPs carry imaging moieties such as radionuclides and show potential for tumor diagnosis \[[@B78-jcm-09-00089]\]. Liposomes are spherical phospholipid bilayers similar to a cell membrane. Phospholipids are amphiphilic molecules as they have a hydrophilic head group and two hydrophobic tails; thus, they present an inner aqueous compartment that can encapsulate hydrophilic molecules, while hydrophobic agents can be inserted in the lipid shell. Liposomes can be classified by size or by the number of bilayers. In fact, they can also present more than one bilayer; these multilamellar constructs are characterized by an onion structure where each bilayer of phospholipids is separated from the adjacent by a water layer \[[@B82-jcm-09-00089]\]. Among the various protocols for preparing liposomes with different size and number of layers, the most established are based upon sonication and extrusion \[[@B82-jcm-09-00089]\]. Functional moieties can be attached on the bilayer membrane surface. They are biocompatible, non-toxic, and biodegradable, and they are extensively used for drug delivery. After PEGylation, the blood circulation time of liposomes can be prolonged for sustained release or targeted delivery of imaging and therapeutic agents \[[@B44-jcm-09-00089]\]. Liposomes can exploit the EPR effect or active targeting with antibodies, peptides, and vitamins to reach cancer cells \[[@B103-jcm-09-00089]\]. To date, several liposomal formulations were approved for cancer therapy, mainly loaded with doxorubicin, and treatment of infections such as fungal infections; a few anticancer-loaded liposomes are currently undergoing clinical trials \[[@B104-jcm-09-00089]\].
In MRI imaging, liposomes can be used as a coating material to prevent iron-oxide NPs from aggregating and to target tumor cells. They are an excellent platform for multimodal imaging and theranostic application. As an example, Malinge et al. \[[@B85-jcm-09-00089]\] realized magnetic liposomes by incorporation of iron-oxide NPs in the liposomal aqueous core. Liposomes were radiolabeled through a ^68^Ga-based radiotracer allowing a dual-modality tracking of particle in vivo distribution through MRI and PET imaging; in addition, glucose was grafted onto the NP surface. On U87MG-bearing mice, the magnetic characteristic of the liposomes and the superficial presence of the glucose enabled a dual tumor-targeting mechanism. Through an external magnet, particles were driven in the tumoral region, and the Warburg mechanism allowed their preferential interaction with tumoral cells. In addition, this study confirmed the role of the lipid bilayer in regulating the exchange of water molecules from the external environment to the aqueous core and the consequent increase of the iron-oxide NPs relaxivity r~2~, improving MRI performance. In another study, Li and colleagues \[[@B105-jcm-09-00089]\] constructed a multifunctional theranostic liposomal drug delivery system; liposomes encapsulated doxorubicin and were conjugated with Gd-DOTA for MRI and IRDye for near-infrared fluorescence. Liposomes were also radiolabeled with ^99m^Tc and ^64^Cu for SPECT and PET imaging. After intratumoral injection, MR images displayed, with high resolution, the micro-intratumoral distribution of the liposomes in squamous cell carcinoma of head and neck tumor xenografts in nude rats. NIR fluorescent, SPECT, and PET images confirmed MRI findings. In addition, these multifunctional liposomes have the potential for the accurate monitoring and in vivo delivery of liposomal chemotherapeutic drugs or therapeutic radionuclides such as ^186^Re/^188^Re. Mitchell et al. \[[@B106-jcm-09-00089]\] prepared liposomal formulations with short *n*-ethylene glycol spacers of varying length; multifunctional imaging was gained through a chelator (DOTA) in the head group of lipids, thereby chelating Gd^3+^ for MRI, ^111^In for SPECT, and ^64^Cu for PET. Compared to conventional PEG shielded liposomes (DSPE-PEG2000), this system showed good cellular internalization in tumor cells and similar distribution and blood half-lives. Abou and colleagues \[[@B107-jcm-09-00089]\] radiolabeled preformed paramagnetic (Gd) liposomes with ^89^Zr (positron emitter). The authors used a chelator-free strategy thanks to the radiometal affinity for the lipid phosphate head groups; this dual mode CA was conjugated with octreotide to selectively target neuroendocrine tumors via human somatostatin receptor subtype 2 (SSTr2). MR and PET images revealed significantly greater accumulation of octreotide liposomes to SSTr2-expressing cells compared to control liposomes.
Like liposomes, micelles are also characterized by a core/shell structure but, unlike liposomes, the core can also be hydrophobic while the shell is hydrophilic. Micelles can be made of nonionic surfactants (surfactant micelles) or of amphiphilic block copolymers (polymeric micelles). In polymeric micelles, the length of the hydrophilic block exceeds that of the hydrophobic one, thus resulting in spherical shapes \[[@B88-jcm-09-00089]\]. Polymeric micelles were greatly investigated for delivering hydrophobic drugs; moreover, they have a smaller size compared to liposomes, and the hydrophilic shell reduces interactions with macrophages \[[@B108-jcm-09-00089]\]. Because of the hydrophilic nature of CAs, they can be bound to the hydrophilic blocks or covalently conjugated to the hydrophobic lipid chain in order to be incorporated into micelles \[[@B109-jcm-09-00089]\]. A special group of polymeric micelles can be synthesized by the conjugation of water-soluble copolymers with lipids constituting the hydrophobic blocks (such as polyethylene glycol--phosphatidyl ethanolamine, PEG--PE). The main feature that makes PEG--lipid micelles attractive for diagnostic imaging applications is their size \[[@B88-jcm-09-00089]\]; in fact, due to the lipid bilayer curvature limitation, it is not possible to prepare liposomes that are smaller than a certain minimal diameter (usually, 70--100 nm) \[[@B110-jcm-09-00089]\]. Such a vector was realized by Trubetskoy and colleagues \[[@B111-jcm-09-00089]\]; Gd-DTPA-PE and ^111^In-DTPA-SA were incorporated into 20-nm PEG--PE micelles to visualize lymph nodes during percutaneous lymphography using gamma scintigraphy and MRI imaging in rabbits. A recent study by Starmans et al. \[[@B112-jcm-09-00089]\] provided a PET/MRI dual imaging polymeric micellar system consisting of self-assembling amphiphilic diblock copolymers functionalized with ^89^Zr deferoxamine and Fe^3+^ deferoxamine. In vivo PET and MRI images clarified tumor visualization thanks to the EPR effect. However, both liposomes and micelles are unstable, especially in the presence of serum, and, for this reason, many authors crosslink them to achieve better stability \[[@B94-jcm-09-00089],[@B95-jcm-09-00089]\].
Dendrimers are a group of highly branched spherical polymers with a tree-like internal structure. They are characterized by an inner core surrounded by a number of branches called *generations*. Depending on the number of generations, they vary in size and molecular weight. CAs or drugs can be encapsulated in the inner spaces or anchored on the external terminations \[[@B113-jcm-09-00089]\]. To date, dendrimers as dual modal agents are used for MRI and fluorescence \[[@B114-jcm-09-00089],[@B115-jcm-09-00089]\], optical imaging and nuclear medicine \[[@B116-jcm-09-00089]\], CT, and MRI \[[@B117-jcm-09-00089]\].
However, few studies on positron-emitting radionuclide-labeled dendrimers were reported \[[@B118-jcm-09-00089],[@B119-jcm-09-00089]\], and when such dendrimer platforms are used to develop PET/MRI or SPECT/MRI agents, it is challenging to achieve precise control of radioisotope loading into specific chelating moieties \[[@B120-jcm-09-00089]\]. Indeed, to our knowledge, studies about dendritic formulations for combined PET/MRI remain to be published.
In recent years, the biomimetic approach gained increasing interest in the scientific community, and many scientists are trying to mimic what naturally occurs in the body in order to obtain more biocompatible and biodegradable materials for medical applications. The crucial idea behind the biomimetic approach is that a biopolymer naturally occurring in living organs can be modified for diagnostic and therapeutic purposes, improving probe efficiency and reducing immunogenicity and inflammatory potential. Biological polymers such as alginate, hyaluronic acid, and chitosan, as well as proteins, antibodies, enzymes, lipoproteins, and viral capsids (protein cages), are becoming very attractive for diagnostic and therapeutic applications \[[@B121-jcm-09-00089]\].
Maham et al. synthesized engineered platforms for drug delivery systems of different types and shapes (NPs, microspheres, films, minirods, hydrogels) using gelatin, albumin, collagen, elastin, ferritin/apoferritin, gliadin, casein, zein (corn protein), whey protein, and soy protein \[[@B103-jcm-09-00089]\] highlighting promises and challenges.
Vecchione et al. \[[@B122-jcm-09-00089]\] proposed a fully biocompatible platform for dual MRI/PET imaging with improved relaxometric properties. The core--shell nanocarriers made of chitosan and hyaluronic acid entrapped Gd-DTPA, boosting its relaxometric properties up to five times, and carried the adsorbed ^18^F-FDG without any modification of both FDA-approved CAs.
Fan and coworkers \[[@B123-jcm-09-00089]\] produced a water-soluble melanin NP formulation; this system, after PEGylation, naturally bound ^64^Cu and Fe^3+^ for PET and MRI imaging, and its surface was functionalized with RGD. Shukla et al. \[[@B124-jcm-09-00089]\] proposed a virus-based synthesis, where bacteriophages and plant viruses were used as a scaffold to carry ^18^F and iron oxide or Gd^3+^. These virus-based NPs resulted homogenous and monodisperse, representing a promising delivery system for CAs. However, there are still several open concerns related to their immunogenicity and loading efficiency. In [Table 4](#jcm-09-00089-t004){ref-type="table"}, a comprehensive overview of nanoparticulate constructs used in PET/MRI imaging and related properties is provided.
2. Conclusions and Perspectives {#sec2-jcm-09-00089}
===============================
Molecular imaging and multimodal imaging are current topics extensively investigated by researchers and scientists. Since each diagnostic modality presents advantages and drawbacks, no single technique is able to provide a comprehensive overview of morphological, functional, and metabolic processes underlying tumors. Thus, a deep analysis was conducted on hybrid imaging, and several multimodal scanners are now routinely used in clinical practice among which PET/CT and PET/MRI are the most popular. The complementary information simultaneously obtained by PET and MRI offers new insights into disease diagnosis and treatment. As an example, dynamic contrast-enhanced MRI and PET with perfusion tracers are used to assess the tumor perfusion. A dual CA in a single probe allows a really simultaneous acquisition, and the co-localization of the two CAs guarantees a temporal and spatial correlation of the two imaging modalities. NPs can be used in PET/MRI as CAs for cancer imaging and, in order to gather anatomical and pathological information, their features must be properly adjusted: size, shape, charge, coating, and multivalency. Nevertheless, active targeting opened new pathways through the possibility of NP accumulation at the pathological site and, therefore, quantification is possible also on low-sensitivity techniques such as MRI. The impact of this approach can also be huge in the theranostic field since cancer imaging, diagnosis, and characterization can be used to gather important information about drug release, efficient therapy, and monitoring of response to treatment. Ideal candidates for a specific treatment could be so individuated, and personalized medicine can offer better results and faster healing. Even though, in the last few years, a variety of multimodal probes were produced, only few of them are approved for clinical use. Many challenges must be solved to promote NP clinical translation for both diagnostic and theranostic purposes. A multidisciplinary approach is necessary in order to focus on diagnostic applications and understand biomolecular processes at the basis of several pathologies. We expect that prevention, early diagnosis, patient management, and treatment could be improved such that a single performance can provide a comprehensive examination from which all essential parameters can be derived. In this perspective, multimodality plays a key role, and NPs can display their potential.
E.F., D.F., A.C.d.P., E.T., C.C., and M.A. wrote the manuscript with the support of P.A.N., M.S. All authors have read and agreed to the published version of the manuscript.
This research was supported by "Ricerca Corrente" Grant from Italian Ministry of Health (IRCCS SDN).
The authors declare no conflict of interest.
{#jcm-09-00089-f001}
{#jcm-09-00089-f002}
{#jcm-09-00089-f003}
{#jcm-09-00089-f004}
jcm-09-00089-t001_Table 1
######
Molecular imaging modalities.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Imaging Technique Source of Imaging Spatial Resolution Tissue Penetration Depth Sensitivity Agent Ref.
---------------------------------------------------- -------------------------------- -------------------- -------------------------- ------------------- ---------------------------------------------------------------- ---------------------------------------------
Magnetic resonance imaging (MRI) Radio wave 25--100 µm No limit mM to µM (low) Para-(Gd^3+^) or\ \[[@B31-jcm-09-00089]\]
superparamagnetic (Fe~3~O~4~) materials
Single-photon emission computed tomography (SPECT) γ-ray 6--7 mm No limit pM (high) Radionuclides (^99m^Tc,^201^Tl,^111^In,^131^I, ^123^I, ^67^Ga) \[[@B32-jcm-09-00089]\]
Positron emission tomography (PET) γ-ray 1--2 mm No limit pM (high) Radionuclides (^18^F,^11^C,^13^N,^15^O,^124^I,^64^Cu, ^68^Ga) \[[@B33-jcm-09-00089]\]
Computed tomography (CT) X-ray 50--200 µm No limit n.c. High-atomic-number atoms (iodine, barium sulfate) \[[@B34-jcm-09-00089]\]
Ultrasonography (US) Ultrasounds 50--500 µm mm to cm n.c. Microbubbles \[[@B35-jcm-09-00089]\]
Optical fluorescence imaging Visible or near-infrared light In vivo 2--3 mm\ \<1 cm nM to pM (medium) Fluorescent dyes, quantum dots \[[@B36-jcm-09-00089],[@B37-jcm-09-00089]\]
in vitro µm
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
n.c., not well characterized.
jcm-09-00089-t002_Table 2
######
Magnetic resonance imaging contrast agents.
Brand Name Active Substance Chemical Name Molecular Structure Company Current Status
------------ ------------------- --------------- --------------------- --------------- ----------------------
Omniscan Gadodiamide Gd-DTPA-BMA Linear, non-ionic GE Healthcare Suspended
OptiMARK Gadoversetamide Gd-DTPA-BMEA Linear, non-ionic Mallinckrodt Suspended
Magnevist Gadopentetic acid Gd-DTPA Linear, ionic Bayer Suspended
MultiHance Gadobenic acid Gd-BOPTA Linear, ionic Bracco Only for liver scans
Primovist Gadoxetic acid Gd-EOB-DTPA Linear, ionic Bayer In use
ProHance Gadoteridol Gd-HP-DO3A Cyclic, non-ionic Bracco In use
Gadovist Gadobutrol Gd-BT-Do3A Cyclic, non-ionic Bayer In use
Dotarem Gadoteric acid Gd-DOTA Cyclic, ionic Guerbet In use
jcm-09-00089-t003_Table 3
######
Principal radionuclides and related features.
Radionuclide Half-Life Time \* Electronic Emission Energy β+ Production
-------------- ------------------- ------------------------------- ----------------------
^11^C 20.385 min 386 keV Cyclotron
^13^N 9.965 min 492 keV Cyclotron
^15^O 122.24 s 735 keV Cyclotron
^18^F 109.77 min 250 keV Cyclotron
^64^Cu 12.701 h 655 keV Cyclotron or reactor
^68^Ga 67.629 min 836 and 353 keV \*\* Generator
\* The values were obtained from the database of the National Nuclear Data Center (NNDC) at Brookhaven National Laboratory, Upton NY, USA. \*\* Mean energy of the β spectrum.
jcm-09-00089-t004_Table 4
######
Overview of multimodal PET/MRI nanoparticles.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------
Nanostructure MRI\ PET\ Chelator Other Biological Target Ref.
Component Component
---------------------------------- ------------------- -------------- ------------------- --------------------- ---------------------------- --------------------------
Bacteriophages/plant viruses Iron oxide/Gd^3+^ ^18^F Passive targeting \[[@B124-jcm-09-00089]\]
Hyaluronic acid + chitosan Gd-DTPA ^18^F-FDG No chelator Passive targeting \[[@B122-jcm-09-00089]\]
Iron oxide + ligands\ Iron oxide ^11^C No chelator Passive targeting \[[@B125-jcm-09-00089]\]
(--NH~2~ --COOH)
Iron oxide + micelle + PEG Iron oxide ^64^Cu DOTA Passive targeting \[[@B126-jcm-09-00089]\]
Iron oxide + dextran Iron oxide ^64^Cu DTCBP Passive targeting \[[@B53-jcm-09-00089]\]
Iron oxide + HSA Iron oxide ^64^Cu DOTA Cy5.5 Passive targeting \[[@B95-jcm-09-00089]\]
Iron oxide + mannose Iron oxide ^68^Ga NOTA Passive targeting \[[@B127-jcm-09-00089]\]
Iron oxide + micelle + PEG Iron oxide ^68^Ga NOTA Oleanolic acid \[[@B97-jcm-09-00089]\]
Iron oxide + PASP Iron oxide ^64^Cu DOTA RGD \* \[[@B96-jcm-09-00089]\]
Iron oxide + PEG Iron oxide ^64^Cu NOTA Au Anti EGFR affibody \[[@B128-jcm-09-00089]\]
Iron oxide + PLGA + lipids + PEG Iron oxide ^64^Cu DOTA Passive targeting \[[@B129-jcm-09-00089]\]
Iron oxide + polyglucose Iron oxide ^89^Zr Desferrioxamine Passive targeting \[[@B93-jcm-09-00089]\]
Iron oxide + PEG Iron oxide ^64^Cu No chelator Passive targeting \[[@B94-jcm-09-00089]\]
Iron oxide + silica + PEG Iron oxide ^68^Ga DO3A Passive targeting \[[@B101-jcm-09-00089]\]
Liposome Iron oxide ^68^Ga NODA Glucose External magnetic field +\ \[[@B130-jcm-09-00089]\]
Warburg effect
Liposome Gd-DTPA ^89^Zr no chelator Octreotide \[[@B107-jcm-09-00089]\]
Liposome Gd ^3+^ ^64^Cu DOTA IRDye--doxorubici\ Passive targeting \[[@B105-jcm-09-00089]\]
n--^99m^Tc
Liposome + nEG spacer Gd ^3+^ ^64^Cu DOTA ^111^I--fluorescein Passive targeting \[[@B106-jcm-09-00089]\]
Melanine NP + PEG Fe^3+^ ^64^Cu No chelator RGD \* \[[@B123-jcm-09-00089]\]
Mesoporous silica NP Gd^3+^ ^64^Cu DOTA ZW800 Passive targeting \[[@B102-jcm-09-00089]\]
Micelle Fe^3+^ ^89^Zr Desferrioxamine Passive targeting \[[@B112-jcm-09-00089]\]
MnMEIO/iron oxide + Al(OH)~3~ MnMEIO/iron oxide ^18^F/^64^Cu No chelator/DTCBP Passive targeting \[[@B54-jcm-09-00089]\]
MnMEIO + SA MnMEIO ^124^I No chelator Passive targeting \[[@B131-jcm-09-00089]\]
Silica NP Gd^3+^ ^68^Ga DOTAGA/NODAGA Passive targeting \[[@B132-jcm-09-00089]\]
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------
\* HSA = human serum albumin; RGD = Arg--Gly--Asp; PEG = polyethylene glycol; PLGA = polylactic-*co*-glycolic acid; nEG = *n*-ethylene glycol spacers; MnMEIO = Mn-doped magnetism engineered iron oxide; PASP = polyaspartic acid; DTCBP = dithiocarbamatebisphosphonate; EGFR = epidermal growth factor receptor; NOTA = 1,4,7-triazacyclonane-1,4,7-triacetic acid; DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTAGA = 1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid; NODAGA = 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl) diacetic acid; DO3A = 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide. FDG = fluorodeoxyglucose.
[^1]: Both authors equally contributed to this manuscript.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-nanomaterials-09-01352}
===============
Cellulose is one of the most abundant biopolymers on Earth, and is mainly of plant, wood and bacterial origin. The cellulose of bacterial origin exhibits the highest purity and has thus attracted the interest of many researchers and industrial sectors. Generally, it consists of randomly assembled, \<100 nm wide ribbon-shaped fibrils, composed of 7--8 nm-wide elementary nanofibrils aggregated in bundles. As such, it delivers a combination of exclusive properties, such as flexibility, high water holding capacity, hydrophilicity, crystallinity, mouldability in different shapes, elevated purity with absence of lignin and hemicellulose and biomimetic three-dimensional (3D) network as a hallmark. Because of these features, this type of cellulose attracts interest for different medical applications such as the engineering of artificial skin (particularly in recuperation of burned skin), artificial blood vessels, topical covering for severe wounds, coverings in nerve surgery, dura mater prosthesis, arterial stent coating, wound dressings, hemostatic material, electronic platforms, implants for cartilage and bone repair etc.
For efficient bacterial cellulose (BC) production we need an efficient and stabile bacterial strain with demands for growing that are not too expensive and with ability of being easily scaled up to industrial settings. The produced cellulose is generally easily separated from growth medium and further on modified using different approaches for various medically relevant applications. All these aspects ([Figure 1](#nanomaterials-09-01352-f001){ref-type="fig"}) will be discussed in this review paper.
2. Bacteria Have High Capacity for Cellulose Production {#sec2-nanomaterials-09-01352}
=======================================================
BC is nanofibrillar, extracellular polysaccharide produced by diverse bacteria when they are growing statically, but also when bacteria are submerged in liquid and cultured by shaking. Bacteria produce BC in media with different carbon sources, although the efficiency of BC production differs substantially among various growth substrates. The substrate supplies energy to bacterial metabolism during the exhaustive energy-consuming pathway of cellulose synthesis. Theoretically, every carbon block which the bacterial cell metabolizes into glucose, can be used for cellulose production \[[@B1-nanomaterials-09-01352],[@B2-nanomaterials-09-01352]\].
The capacity of BC production is widespread among bacteria, but the most prominent and well-known BC-producer is species *Komagataeibacter xylinus*, which belongs to the group of acetic acid bacteria (AAB). AAB are strictly aerobic Gram-negative bacteria classified into *α-Proteobacteria* \[[@B3-nanomaterials-09-01352],[@B4-nanomaterials-09-01352],[@B5-nanomaterials-09-01352]\]. The species has been for many years known as *Acetobacter xylinum*, but has been later classified into *Gluconacetobacter xylinus* and due to further taxonomic changes finally reclassified into *Komagataeibacter xylinus*. *K. xylinus* is not the only species among AAB with an immense potential for BC production, since also other species, such as *Komagataeibacter hansenii*, *Komagataeibacter medellinensis*, *Komagataeibacter nataicola*, *Komagataeibacter oboediens*, *Komagataeibacter rhaeticus*, *Komagataeibacter saccharivorans* and *Komagataeibacter pomaceti* have been characterized as strong cellulose producers \[[@B4-nanomaterials-09-01352],[@B6-nanomaterials-09-01352],[@B7-nanomaterials-09-01352],[@B8-nanomaterials-09-01352],[@B9-nanomaterials-09-01352]\]. An important aspect of using AAB for cellulose production is their characteristic of being food-grade or GRAS bacteria (generally recognized as safe).
BC is synthesized in bacterial membrane from nucleotide-activated glucose \[[@B10-nanomaterials-09-01352]\]. Bacteria then channel BC through pores of cell membrane as fibrils composed of D-glucose units which are linked with β-1,4-glycosidic bonds. The chain is linear and extruded from the cell. Then the lateral and unidirectional aligned chains form intra- and inter-chain hydrogen bonding through all available hydroxyl groups. In this way the chains merge into insoluble nanofibrils of up to 25 nm in width and 1 to 9 μm in length which represents 2000 to 18,000 glucose residues \[[@B11-nanomaterials-09-01352]\]. These nanofibrils further aggregate into \<100 nm wide ribbon-shaped fibrils what delivers a combination of exclusive properties to BC such as high water holding capacity, hydrophilicity, crystallinity and mouldability. Although almost all hydroxyl groups of the cellulose polymer are occupied with hydrogen bonds, one end of each cellulose polymer carries an unmodified C4-hydroxyl group and the opposite end a free C1-hydroxyl group, both of them representing possible sites for chemical modifications of cellulose \[[@B12-nanomaterials-09-01352]\].
Synthesis of nucleotide-activated glucose takes place in bacterial cytoplasm. If the starting substrate is glucose, the uridine diphosphate (UDP)-glucose is produced in three steps: phosphorylation of glucose by glucokinase, isomerization of glucose-6-phosphate into glucose-1-phosphate by phosphoglucomutase and synthesis of UDP-glucose by uridylyltransferase (UTP)-glucose-1-phosphate. Finally, cellulose synthase transfers glucosyl residues from UDP-glucose to the nascent β-D-1,4-glucan chain. Cellulose synthase is a membrane-embedded glycosyltransferase composed of two or three subunits \[[@B13-nanomaterials-09-01352]\]. The catalytic subunit of cellulose synthase is a major determinant of chemical and physical properties of BC, meaning that different bacterial species are able to generate cellulose with different lengths \[[@B14-nanomaterials-09-01352]\].
Comparison of AAB genomes revealed that AAB can possess more operons for cellulose production, moreover, the composition of operons differs from each other \[[@B4-nanomaterials-09-01352],[@B15-nanomaterials-09-01352]\]. These differences very likely influence cellulose synthesis, cellulose transport to the cell surface and/or assembly of fibrils into ribbons \[[@B1-nanomaterials-09-01352]\].
The BC is not the only extracellular polysaccharide secreted by AAB. The other two well-known extracellular polysaccharides, acetan and levan are, however, water soluble \[[@B4-nanomaterials-09-01352],[@B16-nanomaterials-09-01352],[@B17-nanomaterials-09-01352]\]. Interestingly, acetan was first described in species *Komagataeibacter xylinus*. In contract to cellulose, acetan is branched acidic heteropolysaccharide \[[@B18-nanomaterials-09-01352]\]. Ishida et al. \[[@B19-nanomaterials-09-01352]\] identified lower cellulose production in a mutant not producing acetan. However, the cellulose production could be recovered by addition of acetan into the medium, meaning that the synthesis of both polymers is not connected at the genetic level.
3. Different Carbon Sources Used for Bacterial Cellulose (BC) Production {#sec3-nanomaterials-09-01352}
========================================================================
The production of BC is extremely expensive, which is mainly a consequence of high costs of synthetic media for its production. The most well-known complex synthetic medium for growing cellulose producing AAB is Hestrin--Schramm medium (HS), composed of 2% (w/v) glucose, 0.5% (w/v) peptone, 0.5% (w/v) yeast extract, 0.27% (w/v) Na~2~HPO~4~ and 1.15 g/L citric acid \[[@B20-nanomaterials-09-01352]\]. During BC production, other by-products, such as gluconic and other acids are formed, that can decrease the BC yield \[[@B8-nanomaterials-09-01352]\]. The composition of HS medium can be further optimized for the highest cellulose yield by replacing glucose with other carbon sources, such as maltose, fructose, cellobiose, mannitol, xylose, sucrose, galactose etc. In most cases glucose turned out to be the best energy source for bacteria, besides, glucose can be directly used as precursor for the assembly of glucose units into cellulose. Wang et al. \[[@B2-nanomaterials-09-01352]\] have recently reported that fructose had in their microbial process higher cellulose yield in comparison to other carbon sources, also to glucose. The process for BC production can be further optimized by adding buffers into medium for keeping pH at optimal value for growing bacterial strains \[[@B6-nanomaterials-09-01352]\].
To reduce the costs for BC production, the alternative natural carbon sources are utilized, such as waste substrate from different sectors of the food industry, sugar cane molasses etc. The BC yield can be improved also by addition of additives into growth medium such as glycerol, agar, xanthan, sodium alginate, ethanol ([Figure 2](#nanomaterials-09-01352-f002){ref-type="fig"}), carboxymethyl cellulose (CMC), etc. Naritomi et al. \[[@B21-nanomaterials-09-01352]\] reported on enhanced cellulose yield during continuous BC production with *K. xylinus* subsp. *sucrofermentans* BPR3OOlA using fructose medium supplemented with 0.1 wt% of ethanol. The production of cellulose in a static culture with strain *K. xylinus* DA increased about 4-fold as a result of adding 2 wt% acetic acid in glucose medium \[[@B22-nanomaterials-09-01352]\]. Lu et al. \[[@B23-nanomaterials-09-01352]\] reported enhanced BC production with *K. xylinus* in chemically defined medium under static cultivation by the addition of pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, and ethanol ([Figure 2](#nanomaterials-09-01352-f002){ref-type="fig"}) in concentrations 0.15%, 0.1%, 0.3%, 0.4%, 0.1%, 0.2%, 4%, respectively. Li et al. \[[@B24-nanomaterials-09-01352]\] improved cellulose production with the strain *K. hansenii* M2010332 by the addition of ethanol and sodium citrate. Lu et al. \[[@B25-nanomaterials-09-01352]\] reported that the addition of 1% of methanol, 0.5% ethylene glycol, 0.5% of n-propanol, 3% of glycerol, 0.5% of n-butanol and 4% of mannitol produced 21.8%, 24.1%, 13.4%, 27.4%, 56% and 47.3% higher yield of cellulose by culturing strain *K. xylinus* 186 statically in glucose medium. The experiments of Matsuoka et al. \[[@B26-nanomaterials-09-01352]\] showed that the addition of lactate and methionine in fructose medium improved cellulose production with *K. xylinus* subsp. sucrofermentas BPR200. However, the BC yield reached 90% of that obtained in corn steep liquor. There is also a report on improved BC production with *K. xylinus* ATCC 10,245 by adding vitamin C in growth medium \[[@B27-nanomaterials-09-01352]\].
The production of BC in synthetic media with different carbon sources and growth factors, which are usually added as yeast extract and peptone, is expensive. The researchers are thus searching for inexpensive raw material containing high levels of sugars as substrates for BC production. To this aim several raw materials have been analyzed for BC production, such as tobacco waste extract \[[@B22-nanomaterials-09-01352]\], sugar beet molasses, cheese whey media \[[@B23-nanomaterials-09-01352]\], distillery effluent \[[@B24-nanomaterials-09-01352]\], corn steep liquor \[[@B25-nanomaterials-09-01352]\], fruit juice \[[@B26-nanomaterials-09-01352]\], corn stalks \[[@B28-nanomaterials-09-01352]\], litchi extract \[[@B29-nanomaterials-09-01352]\], beverage industrial waste \[[@B30-nanomaterials-09-01352]\], corncob acid hydrolysate \[[@B31-nanomaterials-09-01352]\] and waste beer yeast \[[@B32-nanomaterials-09-01352]\]. Another possible natural growth medium would be waste material from wine production. According to recent reports \[[@B33-nanomaterials-09-01352]\], 1.17 kg of grapes are used to produce 750 mL wine, and after the grapes are squeezed, about 20% of that weight remains in the form of grape skins, seeds and stems, counting for \~12 million tons each year. This substrate contains soluble carbohydrates (white grapes), fibers, acids, salts, and phenolic compounds (red grapes) \[[@B34-nanomaterials-09-01352]\] and as such it is often considered as a convenient source of carbon for microbial processes. Moreover, grape waste as carbon source in BC production may contribute to reduce winery residuals, reduce BC production costs, offering new ways to diversify BC production by taking into account also the environmental aspect by diminishing waste products in nature.
The carbon source used for growing BC-producers affects BC properties: water holding capacity, surface area, porosity, polymerization degree, molecular weight, crystallinity index (67%−96%), mean crystallite size (5.7−6.4 nm), intrinsic viscosity, oxygen and water vapor transmission rates, mechanical properties, etc. Molina-Ramírez et al. \[[@B35-nanomaterials-09-01352]\] reported improved BC yield by addition of ethanol and acetic acid in growth medium, however, the crystallinity index, the degree of polymerization and maximum rate of degradation temperatures decreased by 9.2%, 36%, and 4.96%, respectively, by the addition of ethanol and by 7.2%, 27%, and 4.21%, respectively, by the addition of acetic acid. The crystallinity index of BC produced in the presence of ascorbic acid also decreased with remarkable change in d-spacing \[[@B27-nanomaterials-09-01352]\]. However, a recent study of Wang et al. \[[@B2-nanomaterials-09-01352]\] reported similar morphology and microfibrils of BCs from different carbon sources, meaning that these characteristics have to be checked for each bacterial strain before starting BC production at large scale.
The production of BC can be simply performed in vessels with large surface area which support direct supply of oxygen and assembly of large cellulose sheets ([Figure 3](#nanomaterials-09-01352-f003){ref-type="fig"}). To improve the efficiency of BC production and to produce cellulose of desired characteristics, different technological approaches can be used ([Table 1](#nanomaterials-09-01352-t001){ref-type="table"}).
4. BC Modifications with Medical Relevance {#sec4-nanomaterials-09-01352}
==========================================
3D structuring of BC within a translucent, gelatinous, interwoven, nano-fibrous network of linear polysaccharide polymers is formed at static conditions, as displayed within [Figure 4](#nanomaterials-09-01352-f004){ref-type="fig"}. In comparison with vegetal cellulose sources, BC demonstrate remarkable mechanical properties, such as flexibility \[[@B42-nanomaterials-09-01352]\] and soft-tissue resembling stress-strain behavior \[[@B43-nanomaterials-09-01352]\], as well as a high level of crystallinity and water-holding capacity. BC is a very pure material where common cellulose associates, i.e., lignin and hemicellulose, are absent. As such, is considered a non-cytotoxic, non-genotoxic and highly biocompatible material.
However, BC lacks appropriate functionalities to trigger the initial cell attachment and control over the porosity, and it has very slow degradation, etc. To overcome this, BC has been modified by chemical (modification of chemical structure and functionalities) and physical means (change in porosity, crystallinity and fiber density) by applying versatile in situ and ex situ methods. In situ modifications are performed by the variation of culture media, carbon source and addition of other materials, while ex situ modifications are carried out by chemical and physical treatment of formed BC.
Chemical modification rely on inherent chemical reactivity due to the presence of hydroxyl groups, allowing reaction not only at heterogeneous, but also under homogeneous conditions. When compared with plant cellulose, the BC was found to be more reactive towards cynoethylation and carboxymethylation \[[@B44-nanomaterials-09-01352]\]. The homogeneous reaction including dissolving of BC with acetic anhydride and further iodination also reveals the highest reactivity of BC, yet, such a type of modification destroys the nanofibrillar structure.
Variation of water content within BC largely influences its viscoelastic and electrochemical properties. Due to increased resistance of BC to electron transfer, it becomes stiff at 50%--80% of water \[[@B45-nanomaterials-09-01352]\]. Such a finding was particularly important in wound dressing applications, where moisture content is an imperative. Addition of water-soluble polymers, such as CMC, methylcellulose (MC), and poly(vinyl alcohol) (PVA), was found to influence the water content of never dried and re-swollen BC \[[@B46-nanomaterials-09-01352]\]. On the other hand, Bottan et al. \[[@B47-nanomaterials-09-01352]\] introduced the guided assembly-based biolitography as technique to change the BC surface topography what is related to migratory patterns and alignments of human dermal cells, the fibroblasts and keratinocytes.
Some of modifications and resulting properties of BC are summarized within [Table 2](#nanomaterials-09-01352-t002){ref-type="table"}.
4.1. In Situ Modifications {#sec4dot1-nanomaterials-09-01352}
--------------------------
Several studies identify in situ modifications as straightforward approach for introduction of particular functionality to BC by addition of reinforcement material (chitosan, gelatin, poly-3-hydroxybutirate, nanomaterials, clays, silica) to the bacterial culture medium, mostly at the beginning of BC production. The great advantage of such a process is encaging materials that become part of the fibrils, thus enhancing BC by altering mainly the physical--mechanical properties of BC fibrils. Moreover, new functionalities also can be introduced. Recent work of Gao et al. \[[@B65-nanomaterials-09-01352]\] propose in situ introduction of glucose being pre-modified with carboxyfluorescein (6CF), which supplements the BC with green fluorescence signal based on ultraviolet (UV) spectroscopy and confocal microscopy detection as presented by [Figure 5](#nanomaterials-09-01352-f005){ref-type="fig"}.
For application in regenerative medicine and tissue engineering, the BC modification emphasis is on extracellular matrix (ECM) recapitulation \[[@B66-nanomaterials-09-01352]\], yet approaches are application-dependent and vastly diverse. Bone tissue engineering requires the presence of a bioactive component like hydroxyapatite Ca~5~(PO~4~)~3~OH (HAp) and tricalcium phosphate (TCP) Ca~3~(PO~4~)~2~ and several research works report on their inclusion within BC culture medium, resulting in BC/hyaluronic acid (HA) composite with high bone regeneration capacity. BC/HA composite prepared in the process of the cellulose biosynthesis with the introduction of aqueous HAp suspension, allows simultaneous formation of microfibrillar stripes and partial texturing of HA crystals onto them \[[@B67-nanomaterials-09-01352]\]. The addition of CMC in growth media modify medium's viscosity and thus positively impacts assembling of calcium-deficient Hap powders formation in post synthetic stage, while not affecting the composite biocompatibility \[[@B68-nanomaterials-09-01352]\]. For vascular tissue engineering applications, the heparin-modified BC was produced by adding heparin to growth media of BC-producers, thus resulting in anticoagulant sulfate groups-enriched BC-heparin hybrid \[[@B69-nanomaterials-09-01352]\]. Other study introduces chitosan to BC trough in situ approach, being further ex situ modified with heparin, ending up with BC/chitosan/heparin composites with antimicrobial and anticoagulant properties \[[@B70-nanomaterials-09-01352]\]. For tissue-regeneration procedures, where porosity is an essential property, the paraffin microspheres were added to BC culture medium, resulting in microporous BC for bone regeneration \[[@B71-nanomaterials-09-01352]\], urinary conduit formation \[[@B72-nanomaterials-09-01352]\], etc. For wound-healing and temporary artificial skin applications, the BC culturing media is supplemented with glucose, dextrin \[[@B73-nanomaterials-09-01352]\], potato starch, cotton gauze, *Aloe vera*, which allows processing of composites, where only morphologies and physical properties are altered and not the chemical composition of BC itself. Addition of deacetylated chitin nanocrystals to BC culture media resulted in composite with bactericidal activity \[[@B74-nanomaterials-09-01352]\], while CMC addition introduced the surface charge, effective for further conjugation to affibody ligands applicable in tubular bio-filtration of blood proteins \[[@B66-nanomaterials-09-01352]\].
Apart from published studies, the critical limitation of the in situ modification approach presents incorporation of reinforcement materials that also have antibacterial activity against BC strains, the insolubility of various materials in culture media, high surface tension towards hydrophobic materials, the lack of structure control of BC nanofibers, and introduction of particles with low suspension stability within BC growing media, etc.
In situ modification of BC porosity is not affected by the aforementioned limitations and several studies demonstrate facile procedure for pore size manipulation. As shown by Lu et al. \[[@B60-nanomaterials-09-01352]\], the addition of potato starch to culture medium increases BC viscosity by interrupting BC assembly during static culture and thus creating more free spaces within the fibrous network. Further culturing of muscle cells onto loose surface of produced scaffolds results in new biomaterials for hollow organ reconstruction. The procedure for processing of macro-porous and foam-like BC was recently reported by Rühs et al. \[[@B75-nanomaterials-09-01352]\]; they cultured *K. xylinus* in mannitol-based media by foaming and then stabilized the product with surfactant Cremodan and viscosified with xanthan to prevent water drainage ([Figure 6](#nanomaterials-09-01352-f006){ref-type="fig"}).
4.2. Ex Situ Modifications {#sec4dot2-nanomaterials-09-01352}
--------------------------
Ex situ modifications are either chemical (e.g. periodate oxidation and grafting \[[@B76-nanomaterials-09-01352]\] or crosslinking reactions) or physical (physical absorption from solutions or particle suspensions, the homogenization or dissolving of BC mixing with additive material \[[@B77-nanomaterials-09-01352]\]). The BC is compounded with bioactive materials for applications such as tracking of tumor cells behavior \[[@B78-nanomaterials-09-01352]\], enhancement of osteoblasts cell growth in bone regeneration, fibroblast/endothelial cells guide in wound healing, etc. For the replacement of small blood vessels and improvement of the adhesion of human endothelial cells, the BC surface was modified with Arg-Gly-Asp (RGD) tripeptide, directly \[[@B79-nanomaterials-09-01352]\] or indirectly through xyloglucan-Gly-Arg-Gly-As-Ser (XG-GRGDS) conjugates \[[@B80-nanomaterials-09-01352]\]. For blood clothes control, the isolated BC from nata di coco was compounded with different fractions of kaolin \[[@B81-nanomaterials-09-01352]\]. To mimic the glycosaminoglycans of cartilage tissue, the surface charge was added to BC by means of chemical phosphorylation and sulfatation \[[@B82-nanomaterials-09-01352]\]. Incorporation of N-containing groups on BC was succeed by nitrogen plasma treatment, which also improved its porosity and enhanced the attachment of neuroblastoma (N1E-115) and human dermal microvascular endothelium (HMEC-1) cells. For application as a wound dressing, the BC was immersed into chitosan solution, forming BC/chitosan composite with high water-retention capacity \[[@B83-nanomaterials-09-01352]\]. For cardiovascular soft tissue replacement applications, the BC suspension was mixed with PVA, which improves the final mechanical performance \[[@B84-nanomaterials-09-01352]\]. Soaking of BC in silk fibroin solution results in nanocomposite with enhanced cell permissiveness, keeping the non-cytotoxicity and non-genotoxicity as in native BC \[[@B76-nanomaterials-09-01352]\]. For introducing antimicrobial activity against *Escherichia coli*, *Staphylococcus aureus* and *Candida albicans* while keeping biocompatibility of BC towards human embryonic kidney cells, the sodium alginate solution with silver sulfadiazine was mixed with BC slurry and further cross-linked with CaCl~2~ \[[@B85-nanomaterials-09-01352]\]. Different type of nanoparticles were simultaneously formed and introduced into BC- the antimicrobial ZnO \[[@B86-nanomaterials-09-01352]\] and Ag nanoparticles \[[@B87-nanomaterials-09-01352]\], where BC was initially impregnated with zinc acetate and silver nitrate, respectively. The bone morphogenetic protein-2 was introduced into BC to promote the bone regeneration \[[@B88-nanomaterials-09-01352]\]. Other reported BC modifications are gentamicin-, RGD-grafted BC \[[@B89-nanomaterials-09-01352]\], the gelatin-grafted BC using procyanidin \[[@B90-nanomaterials-09-01352]\], phosphorylation \[[@B91-nanomaterials-09-01352]\], etc. The periodate oxidation was used for region-selective oxidation of BC and further coupling with gelatin biopolymer ([Figure 7](#nanomaterials-09-01352-f007){ref-type="fig"}). Such composite demonstrate improved physiological degradation (compared to non-degradable, native BC) as well as capacity for accommodation of flake-like apatite minerals in short-term incubation within supersaturated simulated body fluid (SBF) \[[@B92-nanomaterials-09-01352]\].
5. BC in Regenerative Medicine {#sec5-nanomaterials-09-01352}
==============================
Nanocellulose materials attract significant attention in biomedical materials research \[[@B93-nanomaterials-09-01352],[@B94-nanomaterials-09-01352],[@B95-nanomaterials-09-01352]\] devoted to tissue engineering \[[@B96-nanomaterials-09-01352]\], cell \[[@B97-nanomaterials-09-01352]\] and gene therapy \[[@B98-nanomaterials-09-01352]\], diagnostic \[[@B99-nanomaterials-09-01352]\] and controlled delivery \[[@B100-nanomaterials-09-01352]\], mainly related to their nano-features and properties arising from them. For BC, there is also ultra-high purity and net-like morphology similar to (human) collagen as a biomimetic feature, which facilitates applications such as artificial skin ([Figure 8](#nanomaterials-09-01352-f008){ref-type="fig"}a), vascular grafts ([Figure 8](#nanomaterials-09-01352-f008){ref-type="fig"}b), tissue-engineering scaffolds, dental implants, medical pads, artificial bone and cartilage, delivery of drugs, proteins and hormones \[[@B101-nanomaterials-09-01352]\]. Several commercially available products are available on market, applied during skin transplantation, second and third degree ulcer treatment, decubitus, substitution of dura mater in bran, recovery of periodontal tissues, etc. The biocompatibility assessment of BC implant, by means of chronic inflammation, foreign body responses, cell ingrowth, and angiogenesis evidence no macroscopic signs of inflammation around the implants, absence of fibrotic capsule or giant cells and fibroblasts infiltration without chronic inflammatory reaction \[[@B102-nanomaterials-09-01352]\].
BC efficiency in wound healing generally relies on effective cohesion with wound boundaries, preservation of a moist environment (important for re-epithelization) combined with exudates retention capacity, high mechanical strength at wet state, liquid/gasses permeability, very low risk for irritation due to its ultra-high purity, and ease of wound inspection due to its transparency \[[@B103-nanomaterials-09-01352],[@B104-nanomaterials-09-01352],[@B105-nanomaterials-09-01352]\], etc. In case of chronic wound treatment with BC-based wound dressing materials, the reduction of proteolytic enzymes activity, cytokines and production of reactive oxygen species are reported.
![(**a**) BC dressings as produced and when applied on wounded torso, face and hand. Reproduced from \[[@B106-nanomaterials-09-01352]\], with permission from Biomacromolecules, 2007; (**b**) vascular graft and blood vessel tubes with different sizes and shape, produced by fermentation onto a branched silicone tube. Reproduced from \[[@B107-nanomaterials-09-01352],[@B108-nanomaterials-09-01352],[@B109-nanomaterials-09-01352]\], with permission from Frontiers, 2016, European Polymer Journal, 2014 and Biotechnology and Bioengeneering, 2007, respectively.](nanomaterials-09-01352-g008){#nanomaterials-09-01352-f008}
Even though BC possess many insintric features that encourage its use in wound dressing, its commercial dissemination is not exhaustively exploited yet \[[@B104-nanomaterials-09-01352]\]. The first BC-based commercial medical product was Biofill^®^, a thin BC film with a water content of 8.5%. Material is used as a temporary skin substitute and wound dressing in treatment of basal cell carcinoma, severe burns, dermal abrasions, chronic ulcers as well as at donor and receptor sites in skin grafts. Pain relief, close adhesion to the wound bed, spontaneous detachment following reepithelization and reduced treatment times as well as costs, yet limited elasticity, when applied in areas of great mobility, are related to this product \[[@B110-nanomaterials-09-01352]\]. Membracell^®^ is also a temporary skin substitute used in treatment of burns and ulcers, sim providing pain relief, reduced infection, faster healing, etc. Bionext^®^ and Xcell^®^ are wound-dressing materials with similar outcomes \[[@B111-nanomaterials-09-01352]\]. Nanoderm™ is wound treatment product for acute and chronic wounds, allowing a barrier to infections while allowing gaseous exchange, exudate evaporation, and pain alleviation, acting as a regenerative tissue scaffold to affect fibroblast, endothelial and keratinocyte function, enhancing granulation tissue formation and epithelization \[[@B112-nanomaterials-09-01352]\]. The Cellumed^®^ product is used in veterinary medicine for treatment of large surface wounds on horses and \[[@B113-nanomaterials-09-01352]\].
Further incorporation of inorganic (Ag \[[@B114-nanomaterials-09-01352]\], ZnO \[[@B114-nanomaterials-09-01352]\], CuO \[[@B115-nanomaterials-09-01352]\] and TiO~2~ particles \[[@B116-nanomaterials-09-01352]\]) and organic antimicrobial agents (lysozyme \[[@B117-nanomaterials-09-01352]\], ε-poly lysine \[[@B53-nanomaterials-09-01352]\], nisin \[[@B118-nanomaterials-09-01352]\] garlics' allicin \[[@B119-nanomaterials-09-01352]\]), evoke their effectiveness against several bacterial strains (*Staphylococcus aureus* and *Escherichia coli*), as well as fungal strains (*Aspergilus niger* and *Candida albicans*). Abdominal hernia treatment is another application of BC as a dressing material, where better absorption in native tissue with less risk of mesh-related infections, impact and hypersensitivity at the implant site were reported \[[@B120-nanomaterials-09-01352]\].
A recent strategy in treatment of skin injuries is incorporation of mesenchymal stem cells, the adult pluripotent cells that can differentiate more than two cell times \[[@B121-nanomaterials-09-01352]\]. Loh et al. \[[@B122-nanomaterials-09-01352]\] seeded the human epidermal keratinocytes and dermal fibroblasts onto BC/acrylic acid hydrogel and further transferred them to a wound, reporting that the procedure accelerated the healing process.
Porosity, mouldability, foldability, hemocompatibility and good mechanical properties are attributes which position BC also in blood vessel replacement applications \[[@B123-nanomaterials-09-01352]\]. Especially in replacement of small blood vessels (\<5 mm) as alternative to thorax or legs-harvested vessels or synthetic Dacron, extended Polytetrafluoroethylene (ePTFE) and polyurethane (PU) materials \[[@B124-nanomaterials-09-01352]\]. Control over porosity is prime requirement, as proliferation and migration of endothelial cells within the membrane is essential when semi-synthetic products are considered. Composite with graphene oxide \[[@B125-nanomaterials-09-01352]\], functionalization with chimeric proteins (conjugates of cellulose binding module and RGD adhesion peptides) \[[@B126-nanomaterials-09-01352]\], blending with PVA polymer \[[@B127-nanomaterials-09-01352]\] are among reported studies where coagulation issues and hemocompatibility are toughly investigated.
A commercial product used in the area of guided tissue and bone regeneration is Gengiflex®, the two-layer membrane comprised of native and alkali-modified BC, used for treating the osseous deficiency surround TiAl~6~V~4~ (IMZ) dental implant with simultaneous restoration of the aesthetic and mouth function \[[@B128-nanomaterials-09-01352]\]. This product was shown to support recovery of periodontal tissue by reduced inflammatory response, requiring fewer surgical steps. Saska et al. \[[@B129-nanomaterials-09-01352]\] reported a combination of glycine-modified BC and type I collagen with high alkaline phosphatase (ALP) activity for bone tissue regeneration. For same application, the hydroxyapatite-coated BC was investigated by Ahn et al. and new bone formation within rat calvarian defect model in 8 weeks study was defined as highly promising outcome \[[@B130-nanomaterials-09-01352]\]. Complexation capacity of phosphorylated BC towards calcium was utilized in study of augmentation of mineralization yields and migration of bone-forming osteoprogenitor cells \[[@B131-nanomaterials-09-01352],[@B132-nanomaterials-09-01352]\].
In a recent study, Gorgieva et al. \[[@B92-nanomaterials-09-01352]\] combined BC membrane with gelatin utilizing successive periodate oxidation and a freeze-thawing/carbodiimide crosslinking procedure, which forms µ-porous composite membrane. Acting as a barrier for fibroblast penetration, the membrane did not evoke any cytotoxic effects toward human fibroblast (MRC-5) cells, while the same preferentially attached on a gelatin porous site ([Figure 9](#nanomaterials-09-01352-f009){ref-type="fig"}).
In neural tissue engineering, Innala et al. \[[@B133-nanomaterials-09-01352]\] reported that BC adapts to the SH-SY5Y neuroblastoma cells, which adhered, proliferated and differentiated towards mature neurons as measured by electrophysiological data. A study generated a 3D model that can be used for developing in vitro disease models. For example, combining this scaffold with human-induced pluripotent stem cells that have been derived from diseased patients, the 3D model can be used for detailed investigations of neurodegenerative disease mechanisms and in the search for new therapeutics \[[@B133-nanomaterials-09-01352]\].
The absence of suitable polymers and proteins, and the presence of low endotoxin units (according to the U.S. Food and Drug Administration (FDA) legislation), further expands the BC application portfolio towards drug-delivery applications \[[@B134-nanomaterials-09-01352],[@B135-nanomaterials-09-01352]\], especially to tuning the drug release kinetic and optimization of drug concentration. Amin et al. \[[@B136-nanomaterials-09-01352]\] reported pH sensitive hydrogel formulations of BC with polyacrylic acid and bovine serum albumin as a model drug. Another study investigate BC membranes with added photosensitizer, chloroaluminum phthalocyanine for photodynamic therapy in skin cancer treatment \[[@B137-nanomaterials-09-01352]\].
Other cellulosic fibers (nitrocellulose in particular) have a long history as anchoring substrate for antibody conjugation in diagnostic assays \[[@B138-nanomaterials-09-01352]\], where also the BC appear as suitable candidate. Major effort in this area is given to processing on homogenous, 3D films in order to increase the quantity of antibodies to be further anchored. BC combination with PVA was investigated as artificial cornea \[[@B61-nanomaterials-09-01352]\] and aortic heart valve leaflet \[[@B139-nanomaterials-09-01352]\]. Tronser et al. \[[@B140-nanomaterials-09-01352]\] identify BC as convenient material enabling for long-term maintenance of mouse embryonic stem cells, simultaneously facilitating their culturing and handling.
6. Perspectives and Challenges for BC {#sec6-nanomaterials-09-01352}
=====================================
BC offers an inestimable platform for development within the biomedical field, especially towards high-tech products, from nursing and diagnostic to theranostic and highly demanding regenerative, tissue-engineering products. However, more effort needs to be made in initial production steps and the fact that AAB productivity towards BC production varies strongly among different species and strains, as well as the carbon source, opens room for additional basic research input in this area. Traditional carbon sources in BC production are glucose, fructose and glycerol, which significantly increases expenses, presenting \~30% of total BC production costs. The industrial wastes or by-products have recently been proposed as cheap local sources for BC production. Some examples are corn steep liquor (CSL)-fructose medium, which is a fully enriched medium with minerals, inositol, nicotinic acid, thiamine and pantothenic acid. Date syrup and molasses are other alternatives, being highly competitive with traditional Hestrin--Schramm and Yamanaka media in BC production. Alternative carbon sources, (i.e., what straw, fruit juices, rotten fruit, waste from cotton textiles, dairy industries, biodiesel industries are already suggested) may potentially enlarge, speed up and cheapen BC production. As such, they have not been fully explored to the stage of semi-final, biomedical products. This in turn will seek more facile, cost-effective and industry-translatable modifications beyond standard post-synthetic oxidation and grafting pathways. Potential "housing" of selected and suitable biopolymers or particulates within BC during the synthetic procedure while keeping in mind no restricted BC production is one way to tackle the problem.
Silvo Hribernik for SEM images and Nina Jančič for photography of BC product.
Conceptualization: S.G. and J.T.; investigation: S.G. and J.T.; resources: S.G. and J.T.; writing---original draft preparation: S.G. and J.T.; writing---review and editing: S.G. and J.T.; visualization: S.G. and J.T.; supervision: S.G.; project administration: S.G.; funding acquisition: S.G.
The authors acknowledge the project (Z7-7169) and programs Textile chemistry (P2-0118 (B)) and Physico-Chemical Processes on the Surface Layers and Application of Nanoparticles (P2-0006), under financial support from Slovenian Research Agency.
The authors declare no conflict of interest.
{#nanomaterials-09-01352-f001}
![Influence of different organic acids and ethanol on cellulose yield. Reproduced from \[[@B21-nanomaterials-09-01352]\] with permission from Research & Reviews: Journal of Microbiology and Biotechnology, 2016.](nanomaterials-09-01352-g002){#nanomaterials-09-01352-f002}
{#nanomaterials-09-01352-f003}
{#nanomaterials-09-01352-f004}
![Synthesis of 6CF-BC by in situ microbial fermentation method, using glucose (Glc) modified with 6CF as a carbon source for *K. sucrofermentans* fermentation. (**a**) Glc and 6CF-Glc molecules; (**b**) microorganism fermentation; (**c**) the synthesis of 6CF-BC fibers through *K. sucrofermentans*, (**d**) microstructure of 6CF-BC; (**e**) the 6CF-BC pellicle obtained through microorganism fermentation. Reproduced from \[[@B65-nanomaterials-09-01352]\], with permission from Nature Communications, 2019.](nanomaterials-09-01352-g005){#nanomaterials-09-01352-f005}
![Schematic presentation of the BC foam formation process by *K. xylinus* suspension foaming and stabilization by Cremodan and xanthan as a thickener. Reproduced from \[[@B75-nanomaterials-09-01352]\], with permission from npj Biofilms and Microbiomes, 2018.](nanomaterials-09-01352-g006){#nanomaterials-09-01352-f006}
![Scanning electron microscopy images of (**A**) native and post-synthetically modified BC; (**B**) oxidation with NaIO~4~; (**C**) further coupling with gelatin (GEL), carbodiimide crosslinking and freeze-thawing; (**D**) in situ mineralization by incubation in (10× concentrated) simulated body fluid medium. Adapted from \[[@B92-nanomaterials-09-01352]\], with permission from Nanomaterials, 2019.](nanomaterials-09-01352-g007){#nanomaterials-09-01352-f007}
![(**a**) Fluorescent microscopy images of top, bottom and cross-section aspect of BC-gelatin composite membranes; (**b**) their degradation kinetic; (**c**) barrier effect towards MRC-5 cells. Adapted from \[[@B92-nanomaterials-09-01352]\], with permission from Nanomaterials, 2019.](nanomaterials-09-01352-g009){#nanomaterials-09-01352-f009}
nanomaterials-09-01352-t001_Table 1
######
The most common methods for bacterial cellulose (BC) production.
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Method for BC Production Basic Characteristics of The Process and The Cellulose
-------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------
Static production \[[@B36-nanomaterials-09-01352]\] Most commonly used method at the lab scale.\
Duration of the process is up to two weeks.\
Cellulose is in the form of hydrogel sheet.
Production in shaking culture \[[@B37-nanomaterials-09-01352],[@B38-nanomaterials-09-01352]\] Increased delivery of oxygen to bacteria.\
Might result in reduced genetic stability of bacteria and lower BC production.\
Production of cellulose of different particle sizes and various shapes (mainly of spherical structure).\
Suitable for economic scale production.
Production in airlift bioreactor \[[@B38-nanomaterials-09-01352],[@B39-nanomaterials-09-01352]\] Efficient oxygen supply with low power supply.\
Cellulose produced in pellet.
Production in rotating disc bioreactors \[[@B40-nanomaterials-09-01352]\] Production of homogenous cellulose.\
Cellulose yield is compared to the static process.
Production in trickling bed reactor \[[@B41-nanomaterials-09-01352]\] Provides high oxygen concentration and low shear force.\
Produce BC in form of irregular sheets.
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nanomaterials-09-01352-t002_Table 2
######
Modifications of BC and resulting properties.
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Modification Application Resulting Properties
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BC nanocrystals/Regenerated Chitin fibers (BCNC/RC) \[[@B48-nanomaterials-09-01352]\] Suture biomaterials Biocompatible surgical sutures increasing strength of BCNC/RC filaments;\
Enzymatic degradation possible;\
Degradation rate can be tuned by varying concentration of BCNCs in the yarn;\
Chitin can promote cell proliferation (in vivo).
BC with modified topography \[[@B47-nanomaterials-09-01352]\] Wound dressing Improved cell alignment;\
Promotion of fibroblast infiltration and new collagen deposition in the wound bed.
Vaccarin impregnated on BC \[[@B49-nanomaterials-09-01352]\] Neovascularization;\
Stratified squamous epithelium;\
Dense new- born subcutaneous tissue formation of collagen fibers and hyperplastic fibrous connective tissue.
2,2,6,6-Tetramethylpiperidinyloxy (TEMPO)-Oxidized BC with Ag nanoparticles \[[@B50-nanomaterials-09-01352]\] Antimicrobial activity;\
Ag^+^ release with a rate of 12.2%/day at 37 °C in 3 days;\
Biocompatible.
BC/ZnO nanocomposite \[[@B51-nanomaterials-09-01352]\] Antimicrobial activity against *Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus* and *Citrobacter freundii;*\
Significant healing of 66% after 15 days related to day 0.
BC/TiO~2~ nanocomposite \[[@B52-nanomaterials-09-01352]\] Antimicrobial activity against *Escherichia coli* and *Staphylococcus aureus.*
BC/ε -poly-L-Lysine (ε-PLL) nanocomposite \[[@B53-nanomaterials-09-01352]\] Antimicrobial activity (broad-spectrum) without affecting the beneficial structural and mechanical properties;\
Modification with non-toxic biopolymer\
ε-PLL inhibited growth of *S. epidermidis* on the membranes but did not affect the cytocompatibility to cultured human fibroblast.
BC/Ag nanoparticle composite \[[@B54-nanomaterials-09-01352],[@B55-nanomaterials-09-01352]\] Environmentally benign and facile approach;\
Sustained release of Ag;\
Prolonged antibacterial performance against *Staphylococcus aureus.*
Silymarin (SMN)-zein nanoparticle/BC nanocomposite \[[@B56-nanomaterials-09-01352]\] Change of wettability and swelling;\
Antioxidant and antibacterial activity;\
Air-dried SMN-zein/BC nanocomposite slow down the lipid oxidation.
BC/Octenidin/Poloxamer hybrid system \[[@B57-nanomaterials-09-01352]\] Drug deliveryWound treatment Long term controlled release of octenidine; Improved mechanical and antimicrobial properties;\
Ready-to-use system with Poloxamer-loaded BC for advanced treatment of infected wounds;\
Non toxicity in test with shell-less hen's egg model.
BC/CMC/Methotrexate \[[@B58-nanomaterials-09-01352]\] Impact of DS-CMC on methotrexate loading;\
Topical treatment of psoriasis;\
Decrease of the elastic modulus as the degree of substitution (DS) of CMC increased;
BC/PHEMA Hydrogel matrice \[[@B59-nanomaterials-09-01352]\] Biomedical application New modification: in situ ultraviolet (UV) radical polymerization; Tensile strength increased;\
Nontoxic;\
Rat mesenchymal stem cells (rMSCs) proliferation;\
Tissue replacement and wound healing.
BC with tuned porosity \[[@B60-nanomaterials-09-01352]\] Tissue engineering Higher pore size than native BC to allow muscle cell ingrowth;\
Small decrease in mechanical strength.
BC/PVA composite \[[@B61-nanomaterials-09-01352]\]\ Artificial cornea Higher visible light transmittance than plain BC.
BC/Hyaluronic acid (HA) \[[@B62-nanomaterials-09-01352]\]
BC/urinary bladder matrix \[[@B63-nanomaterials-09-01352]\] Retinal pigment epithelium Higher adhesion and proliferation of retinal pigment epithelium cells than uncoated BC;\
Closer recapitulation of the *in vivo* cell phenotype than uncoated BC.
BC/iron oxide nanoparticles \[[@B64-nanomaterials-09-01352]\] Blood vessels Introduction of magnetic domains;\
Young\'s modulus correspond to values for blood vessels.
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| {
"pile_set_name": "PubMed Central"
} |
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Cells routinely alter their transcriptional program in response to a changing internal and external environment. These responses are mediated by the binding of transcription factors to specific sequences within the promoter of each responding gene. A major aim of studies of gene regulation is to ascertain which transcription factors control which genes. Simply identifying consensus binding sites by computerized searching of the genome is insufficient, because many transcription-factor binding sequences will occur at random in genomic sequences with some frequency. These fortuitous sites occur within both intergenic and intragenic regions; typically, the intragenic sites are not bound by their cognate factor, and are not functional.
Changes in gene expression profiles can be simultaneously monitored for every gene of an organism by hybridizing cDNAs to DNA microarrays (see Figure [1a](#F1){ref-type="fig"}) \[[@B1],[@B2]\]. Unfortunately, such gene-expression profiling does not distinguish between direct effects of a transcription factor binding to target genes and indirect effects resulting from one transcription factor inducing the expression of a second. In an effort to measure the binding of transcription factors to their cognate sites, directly and on a genome-wide scale in the yeast *Saccharomyces cerevisiae,* two recent papers \[[@B3],[@B4]\] describe the coupled use of the chromatin immunoprecipitation (ChIP) assay and DNA microarrays.
The ChIP assay involves the use of formaldehyde to covalently crosslink proteins to DNA *in vivo* (see Figure [1b](#F1){ref-type="fig"}) \[[@B5],[@B6],[@B7],[@B8]\]; formaldehyde reacts with the lysine and arginine side chains of proteins and the purine and pyrimidine moieties of DNA. Antibodies against target proteins are used to purify the crosslinked DNA once it has been sheared into small fragments. After amplification of the enriched DNA fragments by PCR and labeling them with the green fluorescent dye Cy5, their identity is revealed by hybridization to DNA probes arrayed at specific locations on a glass slide. Each probe on such a microarray corresponds to a PCR-amplified intergenic region of the yeast chromosome. The study by Iyer *et al*. \[[@B4]\] also included intragenic (or open reading frame, ORF) probes.
Because of the nonuniform deposition of probe DNA during microarray fabrication (among other factors), more reliable results are achieved when a (red) Cy3-labeled reference sample is also included in each microarray hybridization. Using the two samples together provides a two-color readout, where the ratio of changes of the \'test\' sample relative to the \'reference\' sample is determined (expressed as \'-fold\', after local background subtraction). The most appropriate reference material to use is a matter of debate. Unenriched total genomic DNA was used in both of the recent studies \[[@B3],[@B4]\], and this is expected to provide a constant reference level. The study by Iyer *et al.* \[[@B4]\] included additional references, such as DNA immunoprecipitated using an antibody to the Swi4 DNA-binding protein from a *swi4*-deletion strain. In practice, these additional references serve to control for the unavoidable nonuniform enrichment of DNA that nonspecifically affects the immunoprecipitations. In an ideal immunoprecipitation with no genomic DNA contamination, such controls would not be appropriate, because the denominator in the two-color ratio scheme would be zero.
The genome-wide analysis by Ren *et al.* \[[@B3]\] examined binding of the galactose-utilization transcription factor Gal4 in the presence and absence of galactose, and binding of the mating-pathway transcription factor Ste12 in response to the mating pheromone ? factor. Ten out of approximately 6,400 yeast intergenic regions appeared to be bound by Gal4; 29 were bound by Ste12. In the study by Iyer *et al.* \[[@B4]\], 163 regions were bound by Swi4, a subunit of the SBF transcription factor, and 87 by the MBF transcription factor. Both SBF and MBF have been implicated in control of the cell cycle, so it is not surprising that Iyer *et al.* found that half of the regions bound by MBF were also bound by SBF.
Interestingly, genome-wide analysis reveals that SBF and MBF appear to control a number of non-cell-cycle-regulated genes involved in cell-wall biogenesis and DNA metabolism, respectively, so they might also function in distinct pathways separate from the cell cycle. Given that cell-wall biogenesis and DNA replication occur simultaneously under one state (mitotic growth) and separately under others (pseudohyphal or invasive growth and meiotic S phase), it is reasonable to expect that these pathways are regulated by separate transcription factors that function coordinately during the cell cycle.
The intergenic regions identified as bound by a transcription factor using the ChIP assay are likely to be only the strongest binding regions, so they tell only part of the story. The avidity of binding of a transcription factor from the strongest site to the weakest is a continuum. Statistical analysis is essential for determining the confidence level (*p* value) associated with each binding. For example, a binding event that has a *p* value of 0.001 indicates only a 0.1% chance of this level of binding being due to random data fluctuation. Even at this high level of confidence, for 6,400 intergenic regions it is expected that approximately six of the binding events will be \'false positives\' (results obtained by chance). In situations like that of Gal4, for which the number of detected binding events is similar to the number expected by chance at this *p* value, it is critical to incorporate gene-expression information into the analysis, to help filter out false positives. For example, Ren *et al.* \[[@B3]\] established a cut-off of three-fold or greater difference between the test and reference samples to indicate \'real\' binding (with *p* \< 0.001), and a cut-off of two-fold or greater for gene expression. By these criteria, ten genes were determined to be regulated by Gal4. Seven of these genes were known from other studies to be regulated by Gal4, and all ten appear to play a role in galactose utilization. Lowering the binding ratio cut-off to 2.5 (still with *p* \< 0.001) identified another 23 genes, all of which showed less than a 1.8-fold increase in gene expression. Because the biology of these additional genes did not suggest roles in galactose utilization, a gene\'s biological function, if known, could serve as an additional subjective criterion to be used in establishing appropriate cut-off values for binding and expression data.
The relationship between the results obtained in these arrays and the behavior of transcription factors *in vivo* is not a simple one. For example, the use of gene-expression profiles is important when ChIP analysis detects binding of a factor to a region that lacks any discernible consensus binding site for the factor. In the study by Iyer *et al.* \[[@B4]\], approximately half of the Swi4-bound regions lacked a consensus site for binding SBF. Although some of these are likely to be false positives, it is possible that SBF can in fact recognize additional sequences, or that promoter specificity at the consensus site is achieved through interactions with other promoter-bound transcription factors. Also, binding of transcription factors to promoter sites does not necessarily result in transcriptional activation. Genome-wide analysis confirmed previous findings that Gal4 is associated with the promoters of the genes *gal1* and *gal10* under glucose-repressed conditions; but for these promoters, displacement of the Gal80 repressor is necessary to achieve activation by Gal4. Finally, although SBF appears to be bound to many intergenic sites, it is perplexing that deletion of its Swi4 subunit had little effect on the expression of putative SBF target genes \[[@B4]\]. It is possible that additional or redundant transcriptional programs direct the expression of these genes, possibly throughout the cell cycle. If so, the current analysis using asynchronous cells would reveal only a composite expression profile, and studies of synchronized cell populations will be required to resolve this issue.
Genome-wide location analysis offers the promise of being able to identify the complete set of genomic regions to which transcription factors are bound *in vivo.* When coupled with gene-expression profiling and searches for consensus binding sites, it has the potential to identify the direct effectors of complex gene expression programs. Application of these techniques to additional transcription factors as cells respond to changing internal and external environments should lead to a broader understanding of the physical regulatory networks governing cellular behavior.
Figures and Tables
==================
{#F1}
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"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
*Mycobacterium tuberculosis*, the causative agent of tuberculosis (TB), presents a major health-care problem worldwide, with a global incidence of \~100 per 100,000 people and as much as 1/3 of the world's population carrying a latent TB infection that can later reactivate. The success of *M. tuberculosis* is partially owing to the pathogen's ability to evade the host immune system, lying dormant until the host becomes immunocompromised. Understanding the mechanisms of mycobacterial immune system evasion is necessary to combat the disease, which grows increasingly more elusive as multidrug-resistant strains emerge.
Mycobacteria are well recognized for their remarkable ability to sense engulfment by macrophages and then survive the numerous and different forms of macrophage bactericidal attack^[@CR1]^. After macrophage engulfment, a mycobacterium is exposed to antimicrobial redox stresses, including reactive oxygen species (ROS); hydrogen peroxide (H~2~O~2~); superoxide (O~2~^−^); hydroxyl radicals (OH), mainly produced by NADPH oxidase (the main generator of ROS inside host cells), and reactive nitrogen species (RNS), specifically nitric oxide (NO), which is generated mainly by inducible nitric oxide synthase (iNOS)^[@CR1]^. The importance of ROS in controlling mycobacterial infection has been shown by the observation that children with defective NADPH oxidase (NOX2) suffer from chronic granulomatous disease, are susceptible to TB, and are likely to develop serious complications after vaccination with Bacillus Calmette--Guérin^[@CR2]^. A body of evidence indicates that survival of the bacilli to early macrophage redox actions is essential for later mycobacterial replication, persistence, and ultimately the establishment of latent infection^[@CR1],[@CR3],[@CR4]^. The role of NO during mycobacterial infection of human macrophages has until recently been unclear, partly as a result of the low amounts of NO produced by these cells. Increasingly, evidence suggests that iNOS and the production of RNS play a significant role during the mycobacterial infection of human macrophages^[@CR5]^.
Mycobacteria have evolved complex redox-sensing pathways to monitor both the intra- and extracellular redox environment. In this context, ROS and NO can also act as signaling molecules, promoting recognition at the atomic level^[@CR6]^. In mycobacteria, ROS and NO exposure modulate multiple redox-sensing pathways, including the SigH/RshA, DosR/S/T, MosR, and WhiB families, to maintain redox homeostasis^[@CR7]--[@CR11]^. Both ROS and RNS, in a dose-dependent manner, can act as signaling molecules that trigger certain responses that determine mycobacterial intracellular survival and growth. Whether these molecules act synergistically by interfering at independent points within the same signal network or whether they activate completely different signal pathways in mycobacteria is unknown. A 2-dimensional gel-based study indicated that little overlap was observed between the H~2~O~2~- and NO-induced responses in *M. tuberculosis* cell cultures when exposed to either stress^[@CR12]^. In contrast, recent transcriptomic analyses showed that NO exposure initiated much the same transcriptional responses as H~2~O~2~. However, unlike H~2~O~2~ exposure, NO exposure induced dormancy-related genes and caused dose-dependent bacteriostatic activity without killing^[@CR13]^. Although some compelling transcriptomic data^[@CR14]^ exist regarding mycobacterium responses to different concentrations of ROS or RNS, equivalent data at the proteome level still remain scarce. In this context, when comparing the protein fold changes obtained to the mRNA fold changes, Aebersold et al.^[@CR15],[@CR16]^ reported that global correlations between mRNA and protein regulation in *M. tuberculosis* were surprisingly low, with *R*^2^ ranging between 0.06 and 0.52; furthermore, Cortes et al.^[@CR17]^ found that protein expression is delayed compared with expression at the level of the transcriptome. These findings suggest that protein levels are essentially decoupled from gene expression^[@CR13],[@CR15],[@CR16]^. This argues strongly for the need to gain a detailed understanding of ROS- and RNS-induced mycobacterial responses at the proteome level. However, *M. tuberculosis* is a slow-growing pathogen that must be cultured in BSL3 containment facilities, with culture times measured in months; furthermore, strict biosafety considerations now require that all new experimental procedures on *M. tuberculosis* need to be first demonstrated on a nonpathogenic organism in a BSL2 laboratory. The fast-growing, nonpathogenic *M. smegmatis* is therefore often used by researchers in the TB field as a model organism on which a wider range of experiments can be conducted more rapidly, before continuing the studies with *M. tuberculosis*^[@CR18]--[@CR20]^. For example, previous work in our laboratory employed a proteomic approach to investigate the effects of sublethal doses of rifampicin and vitamin C on *M. smegmatis*^[@CR21],[@CR22]^. As a complementary approach to the growing transcriptomic data of mycobacterial response to NO and H~2~O~2~, we have carried out a large-scale, label-free, mass spectrometry-based study to investigate proteome dynamics at three distinct time points of *M. smegmatis* growth in vitro after exposure to sublethal concentrations of NO and H~2~O~2~. In addition, informed by our proteomics findings, we investigated the effect of pre-exposure to sublethal NO and H~2~O~2~ on the survival of *M. smegmatis* to macrophage attack, which to the best of our knowledge, is the first time that prior phenotypic adaption to oxidative stress has been directly associated with increased mycobacterial survival within the macrophage.
Results {#Sec2}
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Determination of sublethal concentrations of diethylenetriamine/nitric oxide (DETA-NO) and H~2~O~2~ {#Sec3}
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### treatment with DETA-NO {#Sec4}
The DETA/NO adduct breaks down in solution and exhibits a half-life of ca. 20 h at pH 7.4, with an NO release duration \> 24 h, to give a low, quasi-steady-state concentration of NO^[@CR23]^. A previous study in *M. tuberculosi*s by Voskuil et al.^[@CR13]^ reported that exposure to 0.05 mM DETA-NO introduced no growth defects but resulted in subsequent specific NO-related transcriptomic perturbation. On the other hand, higher concentrations of DETA-NO-induced general oxidative stress responses. Our results confirm that in *M. smegmatis* 0.05 mM DETA-NO confers no noticeable growth defect (Supplementary Figure [2](#MOESM2){ref-type="media"}), and based on our finding as well as previous observations, 0.05 mM was selected as the sublethal concentration for the current study.
Treatment with H~2~O~2~ {#Sec5}
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*Mycobacterium smegmatis* batch (shake flask) cultures at mid-log phase were treated with concentrations of H~2~O~2~ ranging from 10 mM to 200 mM (Fig. [1a](#Fig1){ref-type="fig"}). When treated with 200 mM H~2~O~2~, *M. smegmatis* culture suffered from an unrecoverable growth defect, whereas cultures treated with 50 and 100 mM H~2~O~2~ showed recovery of growth 120 min after treatment. Cultures treated with 10 mM H~2~O~2~ showed a growth defect within 30 min and had recovered to normal growth by 120 min. The sublethal dose selected for this study was therefore 10 mM H~2~O~2~. Based on growth curves of *M. smegmatis* treated with 10 mM H~2~O~2~, three time points after treatment were selected for cell harvesting and subsequent proteomic analysis (30, 75, and 150 min; T1, T2, and T3, respectively) (Fig. [1b](#Fig1){ref-type="fig"}). These three time points represent the onset of (T1), recovery from (T2), and post-recovery of (T3) H~2~O~2~ exposure. Note that cultures treated with DETA-NO (Supplementary Figure [2](#MOESM2){ref-type="media"}) were harvested at the same time points after treatment to maintain comparability between both conditions.Fig. 1Growth curves for *Mycobacterium smegmatis* cultures treated with various H~2~O~2~ concentrations at mid-log phase.**a** The growth of *M. smegmatis* cultures as measured by absorbance at 600 nm when treated with increasing concentration of H~2~O~2~. Concentrations of 0--200 mM are represented. After treatment with 10 mM H~2~O~2~ cultures showed a slight growth defect, however growth recovered. Cultures treated with 50 mM and 100 mM H~2~O~2~ showed a larger growth defect when compared with cultures treated with 10 mM H~2~O~2~, and recovered growth much later. Cultures treated with 200 mM H~2~O~2~, showed an irreparable growth defect, suggesting in these conditions 200 mM H~2~O~2~ is lethal. **b** Arrows indicate the time points selected for further analysis at when treated with 10 mM H~2~O~2~
Proteome dynamics {#Sec6}
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From a total of 1,357,381 spectra that were submitted, 797,173 were identified (58.7%). This resulted in the identification of 31,270 nonredundant peptides, of which 31,127 were unique to a protein group. The peptide identifications had a false discovery rate of 0.26%. From these peptides, a total of 3336 proteins were inferred, at a false discovery rate of 0.99%. For more details on the search engine parameters, see Supplementary Table [1](#MOESM2){ref-type="media"}. Protein groups were identified by at least one unique peptide, and at least two peptides were taken forward, which resulted in 3135 confident protein group assignments. To insure a reliable label-free quantification, we considered only those protein groups quantified in all experiments within a treatment for further analysis. After applying these filtering criteria, a total of 1713, 1674, and 1713 protein groups for the DETA-NO, H~2~O~2~ and untreated treatment groups, respectfully, remained. For details on the quantifiable proteins and their overlap between treatment conditions, see Supplementary Tables [2](#MOESM1){ref-type="media"}--[5](#MOESM1){ref-type="media"}.
To assess the reproducibility of the MaxQuant label-free quantification, we assessed the Pearson correlation between biological replicates. Multiscatter plots of each biological triplicate were then generated in Perseus^[@CR24]^ (Supplementary Figure [3](#MOESM2){ref-type="media"}). The Pearson correlation scores for each comparison were \>0.97, indicating a highly reproducible label-free quantitation. Principle component analysis (PCA) plots indicated that T3 of the treated samples (panel A and B) form a much more distinct cluster, whereas differences between the T1 and T2 samples seem to be less pronounced (Fig. [2](#Fig2){ref-type="fig"}). In contrast, PCA plots from the untreated group (panel C) did not cluster in a well-defined manner. In the case of the treated conditions, the relatively short time between sampling (45 min) may account for the increased similarity between the first and second time points, whereas the longer time between sampling for the second and third time points (75 min) may explain why the third time points form a more distinct cluster. In the case of the untreated condition, the absence of an introduced stress to drive the bacteria toward a distinct phenotype may account for the lack of clustering observed.Fig. 2All replicates of all time points consisting of nine samples were used to generate a single plot for each treatment condition.Stars, circles, and squares represent samples from T1, T2, and T3, respectively. T1 and T2 are less distinct from each other in both treatment conditions (**a**---DETA-NO and **b**---hydrogen peroxide), and in the untreated condition there is little notable clustering of the first two time points (**c---**untreated). The treatment conditions samples from T3 are more distinct from the other time points. Untreated samples at T3 are notably less distinct from samples from T1 and T2. In the treated conditions, as the time of exposure increases, the proteomic signature becomes more distinct
Statistical analysis {#Sec7}
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Subsequent label-free differential protein abundance analysis was carried out, and proteins with a fold change of one standard deviation greater or less than the median fold change were considered for a *t* test (see Table [1](#Tab1){ref-type="table"} for more details). The fold change cutoff reduced the protein group list further to between 156 and 354 proteins per comparison (for a full list see Supplementary Tables [6](#MOESM1){ref-type="media"}--[11](#MOESM1){ref-type="media"}). Significant differences were assessed using a two-tailed *t* test, and a *p* value of \<0.05 was considered significantly different. For full details, see Supplementary Tables [12](#MOESM1){ref-type="media"} and [13](#MOESM1){ref-type="media"}.Table 1The median fold changes for all comparisons as well as the standard deviation of the fold changeTime point comparisonMedian fold changeFold change standard deviationFold change cutoff(−) median − SDFold change cutoff( + ) median + SDUntreated 1 vs 20.0050.154−0.1480.159 2 vs 30.0080.297−0.2900.305DETA-NO 1 vs 20.0030.167−0.1640.171 2 vs 30.0090.283−0.2730.292Hydrogen peroxide 1 vs 2−0.0110.213−0.2240.202 2 vs 30.0150.227−0.2120.243The standard deviation was used to perform a fold change cutoff around the median fold change. Such that any fold change less than the fold change cutoff(−) was accepted as well as any fold change greater than the fold change cutoff( + ).
Bioinformatics {#Sec8}
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### Differentially expressed proteins unique to DETA-NO or H~2~O~2~ treatment {#Sec9}
Venn diagrams were generated to identify proteins with differential expression that was unique to each treatment. When we compare T1 and T2, of the 95 proteins with differential abundance, 22, 40, and 23 proteins were uniquely different in the DETA-NO, H~2~O~2~, and control conditions, respectively (see Fig. [3](#Fig3){ref-type="fig"} and Supplementary Table [15](#MOESM1){ref-type="media"}). In the comparison between T2 and T3, of the 281 proteins found to have a differential abundance, 81, 35, and 61 proteins were uniquely different in the DETA-NO, H~2~O~2,~ and control conditions, respectively. Notably, between T2 and T3, 39 proteins showed a change in abundance in both the DETA-NO and H~2~O~2~ treatment but not in the untreated cultures (see Fig. [3](#Fig3){ref-type="fig"} and Supplementary Table [16](#MOESM1){ref-type="media"} for full details).Fig. 3Proteins from each treatment condition that were found to have a differential abundance, as well as the overlap of these proteins between all conditions**a** 30, 48, and 29 proteins from DETA-NO, hydrogen peroxide, and untreated, respectively, were assessed for the overlap in protein identity. Of these, 22, 40, and 23 proteins were found to be unique to DETA-NO, H~2~O~2~, and untreated conditions, respectively. **b** 177, 117, and 130 proteins belonging to the DETA-NO, H~2~O~2~, and untreated conditions were assessed for overlap. It was found that 81, 35, and 61 proteins were unique to DETA-NO, H~2~O~2~, and untreated conditions, respectively
Changes in *M. smegmatis* proteome induced by exposure to sublethal concentration of DETA-NO {#Sec10}
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To assess the potential biological implications of the changes seen at the proteome level, we performed more downstream bioinformatics analyses. These included String-db and KEGG pathway mapper, and they revealed that, after DETA-NO treatment from T1 to T2, changes in the proteome were mainly related to acetyl-COA metabolism, including the increased levels of glucose transporters MSMEG_2116 and MSMEG_2117. In addition, the increased abundance of orB and MSMEG_4646 from T2 to T3 suggests a plausible increase in acetyl-CoA synthesis from both pyruvate (a product of glycolysis) and 2-oxoglutarate Figs. [3a](#Fig3){ref-type="fig"} and [4a](#Fig4){ref-type="fig"}. In addition, during T2 to T3, differential levels of proteins involved in glycolysis were observed, e.g., Pgm (up), MSMEG_4646 (up), MSMEG_2597 (up), MSMEG_1543 (down), adhE1 (up), and MSMEG_5287 (up) (Fig. [4a](#Fig4){ref-type="fig"} and Supplementary Figure [4A](#MOESM2){ref-type="media"}). These results point toward a possible increased synthesis of acetyl-CoA at T3. Within this context, lipid metabolism is intimately associated with carbohydrate metabolism, as products of glycolysis such as acetyl-CoA, can be used in the synthesis of lipids. Between T1 and T2, several of the proteins that were differentially regulated play a known role in lipid metabolism; for example, KasA, KasB, and FabD levels increased between the time points, and MSMEG_2536 decreased (Supplementary Figure [6a](#MOESM2){ref-type="media"}). Furthermore, from T2 to T3, after treatment, proteins associated with lipid metabolism, such as Des, Glpk, MSMEG_5242, MSMEG_3580, and MSMEG_2597, showed increased abundance, whereas FadD9 (acyl-CoA synthetase) showed decreased abundance. These findings point to a tentative relationship between carbohydrate metabolism, lipid metabolism, and sublethal DETA-NO treatment.Fig. 4Clusters of associated proteins found to have significant differences in abundances as a result of DETA-NO pre-exposure (as determined by String-db)Clusters were determined by the EAGLE algorithm using ClusterVis via Cytoscape. The shape of the nodes denotes if a protein is a known drug target or virulence factor. Octagons represent known virulence factors, arrowheads represent known drug targets, and ellipses represent proteins that are not known to be either. The color of the nodes denotes the protein expression relative to the previous time point on a gradient of dark blue to deep red, with dark blue indicating the relatively lowest expression and deep red indicating the relatively highest expression. The color of the ring surrounding the nodes denotes when the protein showed differential expression, with black rings indicating that in both comparisons (T1 and T3) the protein showed altered expression, the yellow rings indicate altered expression in only the first comparison (T1), and the gray rings indicate altered expression in only the second comparison (T3). **a** shows proteins associated with carbohydrate metabolism involving pathways such as glycolysis/gluconeogenesis and the citrate cycle. **b** shows ribosomal proteins and some proteins associated with gene expression, most of which are downregulated. **c** shows the DosR regulon proteins
Our analyses indicate that the levels of several proteins involved in signaling were affected between T1 and T2; specifically, the levels of PhoU, which is annotated as a virulence factor, were decreased, and the levels of the TetR transcription factor MSMEG_2553 were increased. The trend continued between T2 to T3, with a change in abundance of several virulence factors, such as RegX3, MtrA, DevR, MSMEG_3240, and MSMEG_5424, which are all members of two-component systems (see Fig. [4a](#Fig4){ref-type="fig"}). In addition to the increase of DevR abundance, other proteins controlled by the DosR regulon also increased in abundance, such as MSMEG_5243, MSMEG_5733, MSMEG_3945, MSMEG_5246, MSMEG_3940, MSMEG_5245, MSMEG_3942, MSMEG_3952, MSMEG_3950, and HspX (see Fig. [4c](#Fig4){ref-type="fig"}).
Among the members of the DosR response proteins, several were annotated as universal stress responders, including MSMEG_3945, MSMEG_3940, MSMEG_3950, MSMEG_5245, and MSMEG_5733. Notably, at T3, the abundance of proteins associated with DNA repair, such as Ku, MSMEG_2778, and MSMEG_5004, increased, suggesting DNA damage is induced by DETA-NO treatment. Evidence of protein expression perturbation from T2 to T3 was observed with the decrease in abundance of 11 ribosomal proteins. Additional accessory proteins involved in gene expression, such as Efp, MSMEG_1930, Rne, Rho, RecA, MSMEG_6892, and RpoC, were all found to decrease in abundance. These data are summarized in Table [2](#Tab2){ref-type="table"}.Table 2Summary of a subset of differentially abundant proteins due to DETA-NO treatment, from all time pointsProcessUniprot accessionGene nameProtein nameEC numberFold changeGlycolysis/ gluconeogenesisA0QU86MSMEG_2116PTS system, glucose-specific IIBC component (EC 2.7.1.-) (EC 2.7.1.69)2.7.1.-; 2.7.1.690.22Glycolysis/ gluconeogenesisA0QU87MSMEG_2117Beta-glucoside-specific EII permease (PTS system sugar phosphotransferase component IIA) (EC 2.7.1.69)2.7.1.690.22Glycolysis/ gluconeogenesisA0R170orBAlpha oxoglutarate ferredoxin oxidoreductase, beta subunitN/A0.27Glycolysis/ gluconeogenesisA0R171MSMEG_4646Pyruvate flavodoxin/ferredoxin oxidoreductase-like protein (EC 1.2.7.3) (pyruvate synthase)1.2.7.30.31Glycolysis/ gluconeogenesisA0QUA6pgmPhosphoglucomutase PgmA (phosphoglucomutase, alpha-[d]{.smallcaps}-glucose phosphate-specific) (EC 5.4.2.2)5.4.2.20.29Glycolysis/ gluconeogenesisA0QVJ6MSMEG_2597Aldehyde dehydrogenase (EC 1.2.1.3) (aldehyde dehydrogenase AldC) (EC 1.2.1.-)1.2.1.3; 1.2.1.-0.37Glycolysis/ gluconeogenesisA0QSN7MSMEG_1543Eptc-inducible aldehyde dehydrogenase (EC 1.2.1.3)1.2.1.3−0.36Glycolysis/ gluconeogenesisA0QNQ5adhE1Oxidoreductase, zinc-binding dehydrogenase family protein (zinc-type alcohol dehydrogenase (E subunit) AdhE) (EC 1.1.1.1)1.1.1.10.30Glycolysis/ gluconeogenesisA0R2Z4MSMEG_5287Alcohol dehydrogenase (EC 1.1.1.1) (dehydrogenase)1.1.1.10.35Lipid metabolismA0R0B4kasA3-oxoacyl-(Acyl-carrier-protein) synthase 1 KasA (3-oxoacyl-\[acyl-carrier-protein\] synthase 1) (EC 2.3.1.41)2.3.1.410.37Lipid metabolismA0R0B5kasB3-oxoacyl-(Acyl-carrier-protein) synthase 1 KasA (3-oxoacyl-\[acyl-carrier-protein\] synthase 2) (EC 2.3.1.41)2.3.1.410.28Lipid metabolismA0R0B2fabDMalonyl CoA-acyl-carrier protein transacylase (MCT) (EC 2.3.1.39)2.3.1.390.57Lipid metabolismA0QVD5MSMEG_25363-oxoacyl-\[acyl-carrier-protein\] reductase (EC 1.1.1.100) (short-chain dehydrogenase/reductase SDR)1.1.1.100−0.28Lipid metabolismA0R4B3desAcyl-acyl-carrier protein desaturase DesA1 (EC 1.14.19.2) (Fatty-acid desaturase)1.14.19.20.68Lipid metabolismA0R726glpKGlycerol kinase (EC 2.7.1.30) (ATP:glycerol 3-phosphotransferase) (glycerokinase) (GK)2.7.1.300.35Lipid metabolismA0R2V0MSMEG_5242Diacylglycerol [o]{.smallcaps}-acyltransferase (EC 2.3.1.20)2.3.1.201.41Lipid metabolismA0QY95MSMEG_3580Antigen 85-C (EC 2.3.1.-)2.3.1.-0.48Lipid metabolismA0QVJ6MSMEG_2597Aldehyde dehydrogenase (EC 1.2.1.3) (Aldehyde dehydrogenase AldC) (EC 1.2.1.-)1.2.1.3; 1.2.1.-0.37Lipid metabolismA0QWI7fadD9Fatty-acid-CoA ligase FadD9 (EC 6.2.1.3) (NAD dependent epimerase/dehydratase family protein)6.2.1.3−0.49Response regulatorsA0R4B7phoUPhosphate-specific transport system accessory protein PhoUN/A−0.22Response regulatorsA0QVF2MSMEG_2553Transcriptional regulator, TetR family proteinN/A0.25Response regulatorsQ9F868regX3Sensory transduction protein regX3N/A0.46Response regulatorsA0QTK2mtrADNA-binding response regulator MtrAN/A0.34Response regulatorsA0R2V2devRLuxR family two-component response regulator (two-component transcriptional regulatory protein devr)N/A0.79DosR regulonA0QXB5MSMEG_3240DNA-binding response regulator, LuxR family proteinN/A0.49DosR regulonA0R3C6MSMEG_5424Transcriptional regulator, TetR family proteinN/A0.31DosR regulonA0R478MSMEG_5733Putative universal stress protein UspA (universal stress protein family protein)N/A0.58DosR regulonA0QZ91MSMEG_3940Universal stress protein family protein (UspA)N/A0.72DosR regulonA0QZ93MSMEG_3942Uncharacterized proteinN/A0.86DosR regulonA0QZ96MSMEG_3945Universal stress protein family proteinN/A0.61DosR regulonA0QZA2MSMEG_3952Uncharacterized proteinN/A0.87DosR regulonA0QZA1MSMEG_3950Universal stress protein MSMEG_3950/MSMEI_3859 (USP MSMEG_3950)N/A0.90DosR regulonA0R2V1MSMEG_5243Helix-turn-helix motif (pyridoxamine 5'-phosphate oxidase-related, FMN-binding protein)N/A0.45DosR regulonA0R2V3MSMEG_5245Universal stress protein family protein (UspA)N/A0.72DosR regulonA0R2V4MSMEG_5246Uncharacterized proteinN/A0.71DosR regulonA0QZ83hspX14 kDa antigen (Heat shock protein hspX)N/A1.05DNA repairA0R3S7kuNon-homologous end joining protein KuN/A0.28DNA repairA0QW21MSMEG_2778Putative ribonuclease D (EC 3.1.-.-) (Ribonuclease D)3.1.-.-0.63DNA repairA0R267MSMEG_5004DNA repair exonuclease (DNA repair exonuclease SbcD)N/A0.43Protein expressionA0QWR4efpElongation factor P (EF-P)N/A−0.32Protein expressionA0QTQ8MSMEG_1930DEAD/DEAH box helicaseN/A−0.38Protein expressionA0R218rhoTranscription termination factor Rho (EC 3.6.4.-) (ATP-dependent helicase Rho)3.6.4.-−0.25Protein expressionA0R152rneRibonuclease E (RNase E) (EC 3.1.26.12)3.1.26.12−0.46Protein expressionQ59560recAProtein RecA (recombinase A)N/A−0.33Protein expressionA0R7F4MSMEG_6892Replicative DNA helicase (EC 3.6.4.12)3.6.4.12−0.58Protein expressionA0QS66rpoCDNA-directed RNA polymerase subunit beta' (RNAP subunit beta') (EC 2.7.7.6) (RNA polymerase subunit beta') (transcriptase subunit beta')2.7.7.6−0.39Protein expressionA0QSD1rplC50 S ribosomal protein L3N/A−0.25Protein expressionA0QSG4rplF50 S ribosomal protein L6N/A−0.25Protein expressionA0QS62rplJ50 S ribosomal protein L10N/A−0.25Protein expressionA0QSG8rplO50 S ribosomal protein L15N/A−0.26Protein expressionA0QSD6rplV50 S ribosomal protein L22N/A−0.25Protein expressionA0QSD3rplW50 S ribosomal protein L23N/A−0.18Protein expressionA0QV03rpmB50 S ribosomal protein L28N/A−0.31Protein expressionA0QSL7rpsD30 S ribosomal protein S4N/A−0.25Protein expressionA0QSG6rpsE30 S ribosomal protein S5N/A−0.27Protein expressionA0QSD0rpsJ30 S ribosomal protein S10N/A−0.30The process the protein is involved in as well as aliases is shown. The EC number, also displayed on the KEGG pathway diagrams, is shown along with the fold change of each protein
Changes in *M. smegmatis* proteome induced by exposure to sublethal concentration of H~2~O~2~ {#Sec11}
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After treatment with H~2~O~2~ between T1 and T2, an increase was observed in the abundance of proteins, such as Alpha oxoglutarate ferredoxin oxidoreductase and beta subunit orB, and a decreased level was observed of MSMEG_1543, a protein also involved in acetyl-CoA metabolism (Supplementary Figure [5A](#MOESM2){ref-type="media"}). Changes in the proteome during T1 to T2 suggest a potential deregulation of alanine, aspartate, and glutamate metabolism. From T2 to T3, an increase was observed in MSMEG_4646, a pyruvate synthase that like orB may suggest an increase in acetyl-CoA synthesis through the reaction 1.2.7.11 (Supplementary Figure [4](#MOESM2){ref-type="media"}). However, MSMEG_6297, which is also involved in acetyl-CoA metabolism, was observed to have a decreased abundance. The increased levels of proteins such as KasB and Des (Supplementary Figure [6B](#MOESM2){ref-type="media"}) suggest that exposure to H~2~O~2~ may also alter lipid metabolism and the cell envelope. H~2~O~2~ also appears to induce signaling responses because, from T1 to T2, a decrease in proteins such as the response regulator MSMEG_6236 (LuxR family transcriptional regulator) and an increased level of two TetR family transcription factors MSMEG_0532 and MSMEG_1611 was observed. From T2 to T3, evidence existed for further signaling perturbation, as indicated by the abundances of two-component system response regulators. Regulators such as RegX3, MtrA, and DevR, which are annotated as virulence factors, showed an increased abundance from T1 to T2, whereas other virulence factors such as LeuD and Mce4B decreased with treatment in the same time frame (Table [3](#Tab3){ref-type="table"}).Table 3Summary of a subset of differentially abundant proteins due to Hydrogen Peroxide treatment, from all time pointsProcessUniprot accessionGene nameProtein nameEC numberFold changeGlycolysis/ gluconeogenesisA0QSN7MSMEG_1543Eptc-inducible aldehyde dehydrogenase (EC 1.2.1.3)1.2.1.3−0.38Glycolysis/ gluconeogenesisA0R171MSMEG_4646Pyruvate flavodoxin/ferredoxin oxidoreductase-like protein (EC 1.2.7.3) (pyruvate synthase)1.2.7.30.28Glycolysis/ gluconeogenesisA0R5S7MSMEG_6297Aldehyde [d]{.smallcaps}ehydrogenase (EC 1.2.-.-) (aldehyde dehydrogenase)1.2.-.-−0.41Glycolysis/ gluconeogenesisA0R170orBAlpha oxoglutarate ferredoxin oxidoreductase, beta subunitN/A0,49Lipid metabolismA0R0B5kasB3-oxoacyl-(Acyl-carrier-protein) synthase 1 KasA (3-oxoacyl-\[acyl-carrier-protein\] synthase 2) (EC 2.3.1.41)2.3.1.410.33Lipid metabolismA0R4B3desAcyl-acyl-carrier protein desaturase DesA1 (EC 1.14.19.2) (fatty-acid desaturase)1.14.19.21.23Response regulatorsA0QPV5MSMEG_0532Transcriptional regulator, TetR family proteinN/A0,27Response regulatorsA0QSV1MSMEG_1611Putative transcriptional regulatory protein (transcriptional regulator, TetR family protein, putative)N/A0.32Response regulatorsA0R5L8MSMEG_6236Response regulator, two-component system (two-component system, regulatory protein)N/A−0.51Response regulatorsA0R2V2devRLuxR family two-component response regulator (two-component transcriptional regulatory protein devr)N/A0.44Response regulatorsA0QUZ0leuD3-isopropylmalate dehydratase small subunit (EC 4.2.1.33) (alpha-IPM isomerase) (IPMI) (Isopropylmalate isomerase)4.2.1.33−0.42Response regulatorsA0R4N7mce4BMCE family protein MCE4b (virulence factor Mce family protein)N/A−0.23Response regulatorsA0QTK2mtrADNA-binding response regulator MtrAN/A0.32Response regulatorsQ9F868regX3Sensory transduction protein regX3N/A0.30DosR regulonA0QZ91MSMEG_3940Universal stress protein family protein (UspA)N/A0.51DosR regulonA0QZ93MSMEG_3942Uncharacterized proteinN/A0.54DosR regulonA0QZ96MSMEG_3945Universal stress protein family proteinN/A0.43DosR regulonA0QZA1MSMEG_3950Universal stress protein MSMEG_3950/MSMEI_3859 (USP MSMEG_3950)N/A0.61DosR regulonA0QZA2MSMEG_3952Uncharacterized proteinN/A0.63DosR regulonA0R2V1MSMEG_5243Helix-turn-helix motif (pyridoxamine 5'-phosphate oxidase-related, FMN-binding protein)N/A0.38DosR regulonA0R2V3MSMEG_5245Universal stress protein family protein (UspA)N/A0.43DosR regulonA0R2V4MSMEG_5246Uncharacterized proteinN/A0.46DosR regulonA0R478MSMEG_5733Putative universal stress protein UspA (Universal stress protein family protein)N/A0.37Redox homeostasisA0R683gltDGlutamate synthase, NADH/NADPH, small subunit (EC 1.4.1.-) (Glutamate synthase, small subunit)1.4.1.-−0.24Redox homeostasisA0QWZ9sufBFeS assembly protein SufBN/A−0.62Redox homeostasisA0QTL3MSMEG_18852Fe-2S iron-sulfur cluster binding domain protein (oxidoreductase FAD-binding domain protein)N/A0.91Protein expressionA0QZ11rbpARNA polymerase-binding protein RbpAN/A−0.26Protein expressionA0QS66rpoCDNA-directed RNA polymerase subunit beta' (RNAP subunit beta') (EC 2.7.7.6) (RNA polymerase subunit beta') (transcriptase subunit beta')2.7.7.6−0.23Protein expressionA0R218rhoTranscription termination factor Rho (EC 3.6.4.-) (ATP-dependent helicase Rho)3.6.4.-−0.21Protein expressionA0QVB8rpsB30 S ribosomal protein S2N/A−0.22Protein expressionA0QSD7rpsC30 S ribosomal protein S3N/A−0.22Protein expressionA0QSP9rpsI30 S ribosomal protein S9N/A−0.22Protein expressionA0QSD0rpsJ30 S ribosomal protein S10N/A−0.21Protein expressionA0QSL5rpsM30 S ribosomal protein S13N/A−0.25Protein expressionA0QS62rplJ50 S ribosomal protein L10N/A−0.23Protein expressionA0QSP8rplM50 S ribosomal protein L13N/A−0.23Protein expressionA0QSG8rplO50 S ribosomal protein L15N/A−0.25Protein expressionA0QSG5rplR50 S ribosomal protein L18N/A−0.28Protein expressionA0QSG0rplX50 S ribosomal protein L24N/A−0.23The processes the protein is involved in as well as aliases are shown. The EC number, also displayed on the KEGG pathway diagrams, is shown along with the fold change of each protein
Similar to the response with the DETA-NO treatment, between T2 and T3, DevR showed an increase with H~2~O~2~ treatment, which was accompanied by increased levels of other proteins controlled by the DevR regulon, including MSMEG_5243, MSMEG_5246, MSMEG_3952 (Putative NAD(P)H nitroreductase), MSMEG_5245 (Usp), MSMEG_3945 (Usp), MSMEG_3940 (UspA), MSMEG_3950 (Usp), and MSMEG_3942 (Fig. [5c](#Fig5){ref-type="fig"} and Table [3](#Tab3){ref-type="table"}). Some differentially expressed members of the DosR regulon were annotated as universal stress responders such as MSMEG_3945, MSMEG_3940, MSMEG_3950, and MSMEG_5245. Another universal stress responder, MSMEG_5733, not annotated as part of the DevR regulon, also increased in abundance. Finally, the increased levels of proteins annotated with oxidoreductase activity were notable. These enzymes may play a role in restoring/maintaining the appropriate redox potential in the cell. Among this group, some proteins (MSMEG_1885, which shows increased abundance, SufB, and GltD, both show decreased abundances) were annotated as an iron-sulfur cluster binding protein. Proteins that modulate transcription and translation such as RpoC, RbpA, and Rho as well as 10 ribosomal proteins, observed as having decreased abundance between T2 and T3 (Fig. [5b](#Fig5){ref-type="fig"} and Table [3](#Tab3){ref-type="table"}).Fig. 5Clusters of associated proteins found to have significant differences in abundances as a result of H~2~O~2~ pre-exposure (as determined by String-db).Clusters were determined by the EAGLE algorithm using ClusterVis via Cytoscape. The shape of the node denotes if a protein is a known drug target or virulence factor. Octagons represent known virulence factors and arrowheads represent known drug targets, whereas ellipses represent proteins that are not known to be either. The color of the nodes denotes the protein expression relative to the previous time point on a gradient of dark blue to deep red, with dark blue indicating the relatively lowest expression and deep red indicating the relatively highest expression. The color of the ring surrounding the nodes denotes when the protein showed differential expression, with black rings indicating that in both comparisons (T1 and T3) the protein showed altered expression, the yellow rings indicate altered expression in only the first comparison (T1), and the gray rings indicate altered expression in only the second comparison (T3). **a** shows proteins associated with carbohydrate metabolism, involving pathways such as; glycolysis/gluconeogenesis and the citrate cycle. **b** shows ribosomal proteins and some proteins associated with gene expression, all of which are downregulated. **c** shows the DosR regulon related proteins, all of which are upregulated
Pre-exposure to DETA-NO or H~2~O~2~ enhanced survival during macrophage attack {#Sec12}
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To evaluate whether pre-exposure to sublethal concentrations of DETA-NO or H~2~O~2~ would confer a survival advantage during macrophage attack, we assessed macrophage uptake and bacterial survival at the three time points and compared the CFUs of pretreated bacteria and untreated bacteria. At T1, no significant difference occurred between treated and untreated bacteria for uptake into macrophages, as determined by a two-tailed student *t* test (see Fig. [6a](#Fig6){ref-type="fig"}). For survival at T1 in the DETA-NO treatment, no significant change was observed in survival despite a fold change increase of 1.88, whereas the H~2~O~2~ treatment showed a significantly higher (*p* = 0.02) rate of survival, with a fold increase of 2.94 (Fig. [6b](#Fig6){ref-type="fig"}). At T2, the bacteria pretreated with DETA-NO showed significantly decreased (*p* = 0.015) uptake into macrophages, with a 1.77-fold decrease. The H~2~O~2~ treatment showed no significant change in uptake (see Fig. [6a](#Fig6){ref-type="fig"}). Both DETA-NO and H~2~O~2~ pretreatments showed significant increases in survival with fold changes of 2.34 and 2.04, respectively (Fig. [6b](#Fig6){ref-type="fig"}). At T3, the bacteria treated with DETA-NO or H~2~O~2~ showed significantly decreased uptake of 1.78- and 1.94-fold decrease (Fig. [6a](#Fig6){ref-type="fig"}). At T3, DETA-NO pretreatment showed the greatest increase in survival, increasing 9.67-fold. H~2~O~2~ treatment resulted in a 2.83-fold increase in surviving bacteria to macrophage attack (Fig. [6b](#Fig6){ref-type="fig"}).Fig. 6**a** CFU's of uptake experiments. For T1, no significant differences between treatment conditions were observed. For T2, uptake following DETA-NO exposure was significantly lower than the untreated condition. At T3, both DETA-NO and H~2~O~2~ pre-exposure resulted in statistically significant lower uptake than the untreated condition. **b** CFU^'^s of survival experiments. For T1, only H~2~O~2~ pre-exposure resulted in a statistically significant increase in survival, with a 2.94-fold increase. For T2, pre-exposure with both DETA-NO and H~2~O~2~ resulted in a significant increase in survival, a 2.34- and 2.04-fold increase in survival, respectively. At T3 both DETA-NO and H~2~O~2~ pre-exposure resulted in a significant increase in survival compared with the untreated condition, with fold increases of 9.66 and 2.83, respectively. In both panels \* represent statistically significant comparisons; \* represents a *p* value \< 0.05, \*\*\* represents a *p* value \< 0.001 and \*\*\*\* represents a *p* value \< 0.0001
Discussion {#Sec13}
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Previously, Li et al. demonstrated that 30 min of exposure to H~2~O~2~ was sufficient to induce widespread transcriptional adaptation in *M. smemgmatis*^[@CR14]^. They also observed that 7 mM H~2~O~2~ was lethal after 3 h of exposure. These observations differ from those reported here. We observed 10 mM H~2~O~2~ as sublethal and able to induce a proteomic response as early as 30 min post exposure. These differences can be explained by the growth media supplements used in both cases. The supplement oleic acid albumin dextrose complex (OADC) used here contains catalase, which rapidly degrades H~2~O~2~. The supplement used by Li et al. was lacking catalase. The proteomic changes we observe here in cultures treated with H~2~O~2~ (Table [3](#Tab3){ref-type="table"}) are the result of brief exposure to H~2~O~2~ and the likely signaling events associated with this brief exposure.
Redox stresses that mycobacteria encounter within hosts, such as ROS, RNS, hypoxia, and starvation, have been shown to trigger important adaptive changes in mycobacterial physiology that ultimately contribute to survival inside the host^[@CR3],[@CR25]^. Understanding the mechanistic connection between the initial stimuli activated by signaling molecules such as H~2~O~2~ or NO and the subsequent events leading to bacterial cell responses requires a global view of the changes occurring at gene expression level^[@CR13],[@CR14]^ as well as those happening at the protein level^[@CR16],[@CR26]^. From this perspective, we aimed to establish a link between the changes induced in the *M. smegmatis* proteome and enhanced survival to macrophage attack. This was performed by exposing of *M. smegmatis* to sublethal concentrations of DETA-NO or H~2~O~2~ in liquid culture. Our results clearly indicate that the pretreatment of *M. smegmatis* to sublethal doses of DETA-NO or H~2~O~2~ conferred resistance to macrophage attack (Fig. [6b](#Fig6){ref-type="fig"}). Specifically, mycobacteria pretreated with H~2~O~2~ showed a higher survival rate at T2 and comparable survival rates between T1 and T3 (Fig. [6b](#Fig6){ref-type="fig"}). In contrast, pretreatment with DETA-NO resulted in an increase of bacterial survival throughout the time course assay, with the greatest improvement in survival occurring with bacteria having been exposed for an extended period to DETA-NO before infection (harvested at T3 for infection). Although H~2~O~2~ is often referred to as a main signaling molecule across species, in liquid culture, this compound degrades over time, either slowly by natural decomposition or more rapidly by the action of H~2~O~2~-scavenging enzymes. In addition, our complementary assays indicated that, under the studied conditions, 10 mM H~2~O~2~ persisted in the media at measurable levels for \~15 min (data not shown). This indicates that changes observed in the proteome throughout the time course are most likely owing to the earlier response signal pathways initiated during the first 30 min (T1). Previous work indicated that 40 min of exposure to sublethal concentrations of H~2~O~2~ (5 and 10 mM) had a strong effect on gene expression of *M. tuberculosis*^[@CR13]^. By comparison, in *M. smegmatis* exposure to 0.2 mM H~2~O~2~ changed the expression of \~10% of the genome, whereas 29% of the genes were significantly changed in response to 7 mM H~2~O~2~^[@CR14]^. Here, our proteomic data revealed that important transcriptional regulatory proteins showed a change in abundance after exposure to H~2~O~2~. For instance, two proteins annotated as TetR transcriptional regulators increased from T1 to T2 after H~2~O~2~ exposure only. TetR family like proteins control genes whose products are associated with several cellular functions including stress response, multidrug resistance, metabolic modulation, efflux pumps, and pathogenesis^[@CR27]^. In addition, the increased abundance of two-component system (TCS) regulatory protein MSMEG_6236 indicates that the initial presence of H~2~O~2~ altered the phosphorelay signal transduction pathways also likely to impact gene expression. We therefore hypothesize that during T1, the H~2~O~2~ triggered cellular responses that promote differential gene expression, preparing *M. smegmatis* for an eventual oxidative stress. In culture, the responses initiated at T1 most likely resulted in some of those protein changes observed later at T2 and T3, including the DevR regulon. Possibly, specific signal pathways initiated by H~2~O~2~ in vitro are then further propagated once the bacterium is in contact with the macrophage. This would, in part, account for higher survival of T1-treated *M. smegmatis* in comparison with the untreated control. Interestingly, when compared with the proteome of *M. smegmatis* treated with sublethal doses of rifampicin^[@CR21]^, 30 of 34 common differentially abundant proteins were found to have opposite changes in abundance. Phenotypically, *M. smegmatis* pretreated with rifampicin resulted in lower macrophage survival rates when compared with the control (data not shown). This further supports the potential role of the above-mentioned proteins in macrophage survival.
Sensors and response regulators of ROS and NO {#Sec14}
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During T2 and T3, evidence was observed of the regulation of redox sensors that are known to drive important protective response in mycobacteria. Herein, our results strongly indicate that the DevR regulon responds to both NO or H~2~O~2~ insult at the protein level. In *M. tuberculosis*, the DevR regulon comprises 48 genes whose expression is altered under hypoxic conditions and exposure to NO^[@CR28],[@CR29]^. In *M. smegmatis*, DevR has a key role in adaptation to the oxygen-starved stationary phase and resistance to environmental stresses via the induction of three ubiquitin proteasome system proteins, nitroreductase, and HspX^[@CR30]^. By comparison, here we observed the induction of 11 DevR regulated proteins in response to H~2~O~2~ or DETA-NO, which is consistent with previous observations^[@CR17]^. This subgroup of proteins included five USPs, one LuxR TC response regulator, nitroreductase like proteins MSMEG_3952, MSMEG_5243, one pyridoxamine 5-phosphate oxidase-related PdxH, and hypoxia-induced HspX. As mentioned earlier, treatment with H~2~O~2~ slowed down the growth rate, whereas DETA-NO had no effect on culture growth, so in *M. smegmatis* the induction of DevR could be part of an early defense mechanism to redox or NO stimuli, whereas mycobacteria are not necessary committed to a dormant state. In this regard, the induction of DevR-regulated defense proteins, such as Rv2623, PdxH, and HspX, certainly contributed to the survival of the pathogen in macrophages^[@CR31],[@CR32]^.
Notably, evidence exists of transcriptional activity of the DevR regulon together with increased levels of other important mycobacterial responses regulators (RR) such as RegX3 and MtrA. In mycobacteria, the response to environmental stimulus is in part mediated by TCSs. These sensor systems consist of a paired histidine kinase coupled to a respective response regulator RR. The sensing of a signal by the kinase triggers autophosphorylation on a histidine residue, which then transfers the phosphate to an aspartate residue of the cognate RR, facilitating binding to its specific DNA sequence resulting in subsequent transcriptomic changes. SenX3--RegX3 is expressed during phosphate starvation, whereas MtrAB is the only essential TCS known so far. Recent evidence showed that RegX3 and MtrA belong to the OmpR family that shares a conserved DNA-binding motif. Of interest, in *M. tuberculosis*, the survival of the DNA-binding mutant RegX3 strain is compromised in macrophages^[@CR33]^. Preliminary work in our laboratory indicates that *M. smegmatis* treated with sublethal doses of rifampicin resulted in a decrease in the abundance of both RegX3 and MtrA^[@CR21]^, as well as lower rates of survival in macrophages (data not shown). Overall, the information gathered here suggest that sublethal concentrations of NO or H~2~O~2~ initiate a concerted network of phosphorylation events that ultimately modulate gene expression responses. During the adaptation of *M. tuberculosis* to the macrophage microenvironment, the DosR regulon is upregulated along with genes associated with fatty-acid metabolism, and in addition, ribosomal genes are downregulated^[@CR34]^. These findings are similar to our own observations and suggest that a sublethal oxidative or nitrosative stress simulates in part the macrophage microenvironment. Although further research is needed for confirmation, possibly the modulated gene expression may offer an initial advantage to the bacteria during macrophage attack.
At T3, protein changes overlap between the two treatments were greater than those unique to either treatment, although the survival rates were considerably higher for bacteria cells pretreated with DETA-NO. This finding not only reinforces the concept that these two compounds initiate different molecular responses in the mycobacteria but also suggest that, in this case, specific responses to DETA-NO or H~2~O~2~ are the basis of the differences seen between survival rates of the treated versus the untreated bacteria once inside the macrophage. The bacteria treated with DETA-NO for 150 min (harvested at T3) before infecting the macrophages were found to have the highest survival rate.
DETA-NO pretreatment results in altered expression of proteins involved in lipid metabolism and, potentially, in altered lipid metabolism {#Sec15}
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A particularly striking observation is that the abundance of several proteins involved in lipid metabolism was altered exclusively at T3 in response to the DETA-NO treatment; these include FabD, KasA, PimA, MSMEG_3580, and MmsA. Both FabD and KasA are involved in the early steps for mycolic acid synthesis^[@CR35]^. In *M. tuberculosis*, the FabD transfers a malonate moiety to an acyl-carrier protein before it enters into the fatty-acid synthase---II (FAS-II) pathway for merochain biosynthesis^[@CR36]^. KasA is a member of the FAS-II biosynthetic pathway, where it is responsible for lengthening the merochain of mycolic acids^[@CR35],[@CR36]^. In addition, it has been shown that *Rhodococcus equi* KasA mutants have shorter mycolic acid chain lengths, and these mutants were attenuated in both macrophage and mouse infection models^[@CR37]^. PimA, an enzyme that transfers a mannosyl residue from GDP-[d]{.smallcaps}-mannose across the plasma membrane to phospho-*myo-*inositol on the inner side of the plasma membrane, has been shown to be essential for growth of *M. tuberculosis* both in vitro and in vivo^[@CR38],[@CR39]^. In addition, depletion of PimA by gene silencing allowed for infections in mouse lungs to be effectively cleared^[@CR38]^. MmsA was shown to be upregulated in a long-term macrophage infection model, coinciding with the increase of *M. tuberculosis* growth, suggesting that MmsA plays a role in the adaptation to the phagosomal environment^[@CR34]^. These results tentatively suggest an important role for lipid metabolism during a mycobacterial infection, possibly mediated through the alteration of membrane lipids such as mycolic acids and phosphatidyl-*myo-*inositol, which can serve to protect the infecting bacterium from macrophage attack. The increased survival rate of DETA-NO pre-exposed bacteria (T3, see Fig. [6b](#Fig6){ref-type="fig"}) could be owing to the increased abundance of various proteins involved in lipid synthesis, causing structural changes at the microbial lipid layer and improving survival rates within the macrophage.
Conclusion {#Sec16}
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Findings presented in this work show a large overlap in the proteome changes in *M. smegmatis* when exposed to DETA-NO and H~2~O~2~, as suggested by transcriptomic data. In both treatment conditions, the abundance of proteins involved in lipid metabolism, as well as the DosR response, were affected, and the survival to subsequent macrophage attack was increased as a result of the phenotypic proteome adaption that is characterized here. DETA-NO treatment resulted in the increased abundance of more proteins related to lipid metabolism when compared to H~2~O~2~ treatment, and the survival rates for the DETA-NO pre-exposed bacteria was higher. These results point to the potential importance of changes in the mycobacterial lipidome that are driven by phenotypic adaption of the proteome in the context of infections. In conjunction with preliminary work in our laboratory, these findings indicate that sublethal concentrations of bactericidal stressors cause different signaling pathways to be activated, leading to varying phenotypic responses.
Materials and methods {#Sec17}
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Bacterial cell culture {#Sec18}
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*M. smegmatis mc (2)155* was grown in shaking liquid batch culture at 37 °C in Middlebrook 7H9 (Beckton Dickinson (BD)) supplemented with 10% (v/v) OADC (BD), 0.5% (v/v) glycerol, and 0.05% (v/v) Tween-20. Liquid cultures were grown from single colonies, cultured on 7H10 (BD) agar plates, supplemented with 0.5% (v/v) glycerol and 10% (v/v) OADC (BD).
Growth curves {#Sec19}
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Mid-log phase *M. smegmatis* cultures (OD~600~ \~ 1.2) were exposed to a single dose of H~2~O~2~ (Sigma Aldrich, St. Louis, USA) or the DETA/NO (Sigma Aldrich, St. Louis, USA). Doses of 0, 10, 50, 100, and 200 mM H~2~O~2~ and 0.05 mM DETA/NO were used. The OD~600~ was monitored after exposure of the cultures to respective H~2~O~2~/DETA concentrations at 15-minute intervals for 2 hours, 30-minute intervals for an additional 2 h, and a final reading 5 h after oxidant exposure.
Protein extraction {#Sec20}
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*M. smegmatis* cultures were grown to the mid-log phase in the conditions described above, in biological triplicate for each condition, and then treated with either H~2~O~2~ or DETA-NO at a concentration of 10 mM and 0.05 mM, respectively. Bacterial pellets were snap frozen in liquid nitrogen and stored at − 80 °C, until lysis. Frozen bacterial pellets were thawed on ice, in lysis buffer (1% m/v sodium dodecyl sulfate, 1.5% m/v sodium deoxycholate, 1× protease inhibitor, and 7.5 µl lysozyme in 0.5 [m]{.smallcaps} Tris-HCL at pH 7). The resulting lysate was centrifuged at 13,000 rpm for 5 min to sediment the cell debris. The supernatant was passed through a 20 µm filter. Protein was precipitated from the supernatant by chloroform/methanol precipitation, and the protein pellet was resuspended in denaturation buffer (6 M urea, 2 M thiourea in 10 mM Tris at pH 8). Protein was quantified using the modified Bradford assay^[@CR40]^. Proteins were digested with trypsin (Promega) at a ratio of trypsin to protein of 1:50 w/w at room temperature for 16 h. Peptides were desalted using C18 solid phase extraction.
Liquid chromatography with tandem mass spectrometry (LC/MS/MS analysis) {#Sec21}
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For label-free quantification, samples were separately injected into a Dionex UltiMate 3500 RSLCnano system (Thermo Scientific, Waltham, Massachusetts) coupled to an Orbitrap Q Exactive mass spectrometer (Thermo Scientific) for analysis. Each sample was loaded onto a 2 cm trap (packed in-house, 5 μm beads, 100 Å pores, Luna beads by Phenomenex) at a flow rate of 300 nL/min and subsequently run on a 20 cm C18-reversed phase analytical column (packed in-house, 5 μ[m]{.smallcaps} beads, 100 Å pores) at 40 °C. Approximately 600 ng of peptides was loaded in each LC/MS run. Elution from the column occurred over a 190 min segmented gradient consisting of an increasing ratio of 2% acetonitrile acidified with 0.1% formic acid (FA) (buffer B), to H~2~O acidified with 0.1% FA (buffer A): 1% B from 0 to 10 min, increasing up to 6% at 12 min, to 35% at 130 min, and to 80% at 135 min and continuing at 80% until 150 min, before decreasing from 80 to 2% at 152 min. Each run had a built-in wash at the end, increasing to 50% B at 167 min and continuing at this concentration until 169 min, at which time the gradient decreased to 2% for the final 20 min. Before entering the mass analyzer, the eluents were subjected to electrospray ionization. Mass spectra were acquired in a data-dependent manner, with automatic switching between MS and MS/MS scans using a top-10 method. MS spectra were acquired at a resolution of 70,000 with a target value of 3 × 10^6^ or a maximum integration time of 250 milliseconds (ms). The scan range at the MS level was limited to 300--1750 *m/z* and high-energy collision dissociation used for peptide fragmentation, with the energy set at normalized collision energy 28. Multiple charge exclusion was used (unassigned, 1, 5--8, \> 8). MS/MS spectra were acquired at a resolution of 17,500, with a target value of 5 × 10^6^ or a maximum integration time of 80 ms. The scan range at the MS/MS level was limited from 200 to 2000 *m/z*. The fixed first *m/z* was 200 to 2000 *m/z*, and the isolation window was 2 *m/z*.
Peptide identification and protein inference {#Sec22}
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Raw spectral data were processed using MaxQuant^[@CR41],[@CR42]^ software package (version 1.5.7.4) for protein and peptide identification and quantitation with the below-described parameters. Trypsin/P was selected as the protease with a maximum of two missed cleavages allowed. Quantitation by MaxLFQ algorithm was selected with a minimum ratio count of two peptides. Protein ratios were calculated from unique and razor peptides. Protein N-terminal acetylation and methionine oxidation were set as variable modifications, and cysteine carbamidomethylation selected as a fixed modification. The Andromeda search engine was used for identification of the proteins and protein groups from the UniProt *M*. *smegmatis* reference proteome (FASTA, downloaded 02/05/2017). Mass tolerance for the precursor ions in the initial search was set at 20 ppm, and in the main search, at 4.5 ppm tolerance. The default protein-level false discovery rate setting of 1% was retained. Only proteins with a minimum of one unique peptide were accepted. The minimum length of acceptable identified peptides was set to seven amino acids. The option to match retention times between runs was selected to maximize identifications. MS/MS spectra were matched against a decoy database and reverse hits (i.e., spectral mapping to the decoy databases) and probable contaminants filtered from the protein list prior to statistical analysis.
Experimental design and statistical rationale {#Sec23}
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In this study, 27 samples were analyzed, corresponding to three biological replicates of nine conditions. In brief, the nine conditions were divided into three time points and three treatment conditions: 10 mM H~2~O~2~, 0.05 mM DETA-NO, and untreated (control). For a graphical representation of the experimental workflow see Supplementary Figure [1](#MOESM2){ref-type="media"}.
An expression fold change cutoff was employed, so protein groups that changed by less than one standard deviation around the median of the fold change between conditions were excluded from the *t* test. These values can be seen in Table [1](#Tab1){ref-type="table"}. Protein groups with a *p* value \< 0.05 were considered to have a statistically significant change in abundance. Samples within each treatment condition were compared between time points to assess the response over time to treatments with the oxidative stressors. Changes found between corresponding time points within the control were subtracted from the treatment conditions, as those were likely to be related to normal growth. Remaining changes were assumed to be specific to the oxidative stressor condition.
Proteins with altered expression between the first and second time points, as well as between the second and third time points, were analyzed simultaneously while the time dependence was still preserved. Proteome changes owing to treatment were analyzed using String-db^[@CR43]^ protein association networks generated in Cytoscape^[@CR44]^ to visualize the data. The networks were clustered using the EAGLE algorithm via ClusterViz^[@CR45]^ and were used to query String-db for a general overview of the proteomic changes. To gain further insight into the dysregulation at the metabolic level, we used the KEGG^[@CR46]^ pathway mapper to visualize and analyze the metabolic changes associated with treatment.
Patients suffering from acute pulmonary tuberculosis expel droplets containing mycobacteria, which are unlikely to be naive to sublethal oxidative and or nitrosative stress^[@CR3],[@CR47]^. These non-naive bacteria can then be inhaled by others and establish a new infection, or dormant bacilli may undergo reactivation to establish an active infection. In both cases understanding the adaptation to sublethal stresses are important. Our study aims to use *M. smegmatis* as a model organism to study how prior adaptation to sublethal oxidative or nitrosative stress influences bacterial survival within macrophages.
Murine macrophage culture {#Sec24}
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Raw 264.7 cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM, Sigma), supplemented with 10% heat inactivated fetal bovine serum (FBS) at 37 ^o^C in an atmosphere of 5% CO~2~ and 95% O~2~. All cells used were between passage 21 and 25. Infection of Raw 264.7 macrophages with H~2~O~2~ or DETA-NO pretreated *M. smegmatis*
For infection assays 25000 cells were seeded onto 24-well plates and incubated for 72 h pre-infection in DMEM, 10% FBS. Murine Interferon-γ (mIFN-γ, 250 U/ml) was added 48 h after seeding. After 72 h cells were washed with pre-warmed phosphate-buffered saline (PBS) and incubated with DMEM, 10% FBS, 250 U/ml mIFN-γ, and *M. smegmatis* at a multiplicity of infection of 4:1, for 3 hours. The cells were washed five times with pre-warmed PBS, to remove extracellular bacteria, and incubated in DMEM, 10% FBS and 250 U/ml mIFN-γ. After a further 21 h cells were washed five times with pre-warmed PBS, to once again remove extracellular bacteria, before cell lysis for colony forming unit (CFU) determination. Before infection *M. smegmatis* cultures were treated with 10 mM H~2~O~2~ or 0.05 mM DETA-NO, or left untreated as described above. At 30, 75, or 150 min post exposure to the respective treatment condition, *M. smegmatis* cultures were harvested for infection.
Macrophage infections {#Sec25}
---------------------
After infection, CFUs were used to assess mycobacterial survival. Raw 264.7 cells were washed after incubation with *M. smegmatis* for either three hours (uptake) or 24 h (survival) as described above. The cells were lysed by incubation in PBS with 0.1% triton X-100 for ten min on a rotary shaker at 200 rpm. The lysate was diluted and cultured for 48 h on 7H10 agar (0.5% glycerol and 10% OADC) at 37 °C in a standing incubator. The inoculum was cultured and used to normalize uptake values. Three independent experiments were performed, each experiments was performed in technical triplicate. CFU counts for uptake were used to normalize CFU counts for survival, according to the equations below:$$\documentclass[12pt]{minimal}
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\begin{document}$$Normalised\,uptake = \frac{{CFU_U \times DF_U}}{{CFU_I \times DF_I}}$$\end{document}$$$$\documentclass[12pt]{minimal}
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\begin{document}$$Normalised\,survival = \frac{{\begin{array}{*{20}{c}} {CFU_S \times DF_S} \end{array}}}{{CFU_U \times DF_U}}$$\end{document}$$
CFU~U~ = colony-forming units from uptake experiments, 3 hours post infection
DF~U~ = dilution factor used for plating uptake experiments, 5000
CFU~I~ = colony-forming units for the inoculum used for infections
DF~I~ *=* dilution factor used for plating the inoculum, 20000
CFU~S~ = colony-forming units for the survival experiments, 24 h post infection
DF~s~ = dilution factor used for plating survival experiments, 80000
Supplementary information
=========================
{#Sec26}
Parameters used for the MaxQuant search and subsequent protein quantitation Supplementary Figures
**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
These authors contributed equally: Naadir Ganief, Jessica Sjouerman
Electronic supplementary material
=================================
**Supplementary Information** accompanies this paper at (10.1038/s41426-018-0210-2).
We thank the NRF for financial support. This work was supported by a research grant from the NRF (grant number 98963 and 95984). JMB thanks the NRF for a South African Research Chair grant. NCS and CH thank the South African Medical Research Council for the Junior Research Fellowship and Postdoc Fellowship respectively. KCN thanks NRF and UCT/CSIR for PhD bursaries. Computations were performed using facilities provided by the University of Cape Town's ICTS High Performance Computing Team: <http://hpc.uct.ac.za>.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE^[@CR48]^ partner repository with the dataset identifier PXD010020.
The authors declare that they have no conflict of interest.
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Introduction {#s1}
============
Microglial phagocytosis of apoptotic cells (efferocytosis) is at the core of the brain regenerative response. Its relevance in maintaining brain tissue homeostasis from development to aging and neurodegenerative diseases is undisputed but, nonetheless, many fundamental questions about the biology of the process remain open: How is phagocytosis efficiency regulated at the cellular and molecular levels? How can it be manipulated? Is it a dead-end road or does it trigger changes in the phagocyte? Is phagocytosis simply a process of garbage removal or does it actively participate in the well-being of the surrounding tissue? We here try to address these questions by collecting answers from microglia\'s cousins, the macrophages that reside in other tissues. In the first part of this review, we will discuss similarities and differences in the identity of microglia and other macrophages, taking into account their developmental origin and the maintenance of the adult populations. In the second part, we will compare the mechanisms that mediate recognition and engulfment and their epigenetic, transcriptional, metabolic, and immunological consequences. We will conclude that phagocytosis has a tremendous potential to impact on brain physiology and pathology.
Lineage and Origins of Microglia and Other Macrophages {#s2}
======================================================
Microglia are brain-resident macrophages. They interact with the brain parenchyma and carry out essential maintenance functions. They are often studied independently from other tissue-resident macrophages, probably because they are unique in some aspects, most notably in their isolation from the rest of the body through the blood brain barrier (BBB). But how different are microglia really from other tissue resident macrophages in terms of origin, lineage, and identity? A close review of the literature shows that microglia are not as coarsely distinct to other macrophages as one may think, yet there are some fine differences in how they behave in their local environment. In the next sections, we will review evidence about the origin, lineage, identity, and population dynamics of microglia compared to other tissue-resident macrophages and highlight commonalities and differences.
Lesson 1. The Monocytic Origin of Macrophages Is More the Exception Than the Rule
---------------------------------------------------------------------------------
Macrophages are widespread and reside in many different organs, where they fulfill different functions. Because it is such a diverse population of cells, a fundamental question is whether they have a common precursor or whether each macrophage population develops from a different precursor. Based on the observation that some macrophages are short-lived and that they are often renewed by circulating monocytes, already in 1972 van Furth et al. proposed the "mononuclear phagocyte system" theory, by which tissue-resident macrophages were assumed to derive from blood-circulating monocytes and to differentiate within the host tissue ([@B1]). However, at that time there was evidence that some macrophage populations can proliferate locally and self-renew ([@B2], [@B3]), which clashed with the idea that macrophages are of monocytic origin. Why would they need to divide and self-renew locally if the main source is in fact a pool of circulating monocytes? Over the last decade, tracing and parabiosis experiments, where the origin and location of specific macrophage populations can be followed over time, have demonstrated that in fact most macrophage populations originate in the embryo prenatally. And it is only some macrophage populations, such as gut ([@B4]), dermis ([@B5]), pancreas ([@B6]), and heart ([@B7]) macrophages that are replaced from circulating monocytes. In contrast, other macrophage populations, most notably microglia and skin macrophages (Langerhans cells), but also others, originate early during development, are long-lived and capable of local self-renewal with no significant involvement of circulating monocytes ([@B8]--[@B12]).
The notion that macrophages have a prenatal and not a monocytic origin caused a paradigm shift in the field and prompted questions about their precise site of origin and about the routes by which they reach target organs. The precise spatial origin of each macrophage pool is still under debate, but there are essentially two accepted sources: the yolk sac and the aorta-gonad mesonephros. From there, macrophages take one of two routes: they migrate directly to their target organ or they go through fetal liver and then on to their target tissue ([Figure 1](#F1){ref-type="fig"}). What percentage of the mature tissue-resident macrophages in fact originate via each of these routes is still being investigated for most macrophage populations. In this respect, the origin of microglia is probably the most undisputable: they originate in the yolk sac and migrate directly to the brain as early as embryonic day E10.5 in the mouse. Other macrophages, such as Langerhans cells seem to have a mixed origin: some derive directly from the yolk sac, whereas others travel through the fetal liver before they reach their destination ([@B9]). The latter route through fetal liver seems to be the standard and more prevalent for most other macrophage populations ([@B13], [@B14]).
 with permission.](fimmu-11-00506-g0001){#F1}
In summary, the original theory that tissue-resident macrophages universally derive from circulating monocytes has now been replaced with a more refined picture, where macrophages in fact originate from an early embryonic precursor, seed their target tissue, and self-renew locally ([@B12]). In this new picture, macrophages that derive from circulating monocytes are more the exception than the rule and microglia probably constitute the paradigmatic example of macrophages of prenatal origin with self-renewal capacity and no exchange with circulating monocytes under physiological conditions.
But if monocytes are not the precursor cells for most macrophages, what is in fact the molecular identity of their precursors and how different is it for different macrophage populations? In the next section, we will summarize what is known about the identity of the embryonic precursors that give rise to different tissue-resident macrophages.
Lesson 2. Microglia Do Not Require the Transcription Factor c-Myb to Develop, but Other Macrophages Do
------------------------------------------------------------------------------------------------------
After it was demonstrated that most macrophages originate prenatally either from the yolk sac or from the aorta-gonad mesonephros, investigators turned their attention to the precise molecular identity of the precursors. The goal was to identify markers and transcription factors that would allow us to identify and classify them.
There is a certain correlation between the physical and temporal origin of macrophages and the markers they express. The most polarizing marker, the one that probably allows the most straightforward classification, is the transcription factor c-Myb. Early (E7.5-8.5 in mice) progenitors from the yolk sac (erythromyeloid precursors or EMPs) do not express or require the transcription factor c-Myb for development and maturation. Accordingly, macrophages that stem from EMPs, such as microglia, Langerhans cells and Kupffer cells (liver macrophages), develop normally in c-Myb^−/−^ animals ([@B10]). In contrast, late (E10.5 in mice) progenitors that originate in the fetal liver (hematopoietic stem cells, HSCs) do express and require c-Myb for proper development ([@B15], [@B16]). This is the case for most tissue-resident macrophages, which fail to develop in the absence of c-Myb ([@B10]). c-Myb dependence is therefore a classifying criterion for macrophage lineage, but it is not the only transcription factor involved in maturation. The overall topic of macrophage lineage markers is beyond the scope of this review and has been covered extensively before ([@B13]). For microglia in particular, key players in their development include the growth factor receptor CSF1R (Colony Stimulating Factor 1 Receptor), the chemokine receptor CX~3~CR1 (CX3C chemokine receptor 1), the calcium binding protein Iba-1 (ionized calcium binding adaptor molecule 1), the G-protein-coupled receptor F4/80 (also known as EMR~1~ - EGF-like module-containing mucin-like hormone receptor-like 1) and the integrin CD11b (cluster of differentiation molecule 11B, also known as Integrin Alpha M - ITGAM) ([@B17], [@B18]).
The lesson that derives from these studies is that ontogeny and molecular identity of different macrophage populations go hand in hand. There is an additional component, longevity, which also shows some level of correlation with macrophage ontogeny. Early, c-Myb-negative progenitors in the yolk sac give rise to populations that tend to be long-lasting (microglia, Langerhans and Kupffer cells), and later, c-Myb-positive cells in the mesonephros and fetal liver give rise to a mix of long- and shorter-lived populations. Microglia are particularly long-lived and have the ability to self-renew several times over a lifetime ([@B19]). A question that derives from this notion is how the population behaves with respect to individual cells. What is the natural "life cycle" of a single microglia and how does it contribute to the overall stability of the population? The next section will examine evidence of how microglia are replenished under homeostatic conditions.
Lesson 3. As Long as the Blood-Brain Barrier Is Intact, Microglia Can Self-Renew Throughout a Lifetime Without a Significant Monocyte Contribution
--------------------------------------------------------------------------------------------------------------------------------------------------
As reviewed in the previous section, under physiological conditions, microglia have a prenatal yolk sac origin. The BBB protects and isolates the brain from exchange with blood circulation, effectively turning it into a very isolated microenvironment. Here, microglia stay stable and self-renew with virtually no contribution from circulating monocytes in physiological conditions ([@B8]). But what is the exact population dynamics of microglia and how do they behave individually? And what happens if microglia are depleted in a healthy brain, how do they replenish under these conditions?
One critical factor in microglial development and survival is CSFR1. Mice devoid of this receptor do not have microglia ([@B8], [@B20]), and mutations that affect the function of the kinase domain of the receptor cause severe neurological syndromes in mice and humans ([@B21]--[@B24]). In line with this, in 2014, Elmore et al. showed that a chemical inhibitor of CSF1R virtually ablated the microglial population in the healthy brain. Strikingly, they also showed that microglia could be repopulated upon withdrawal of the inhibitor ([@B25]). It was then critical to identify which cells microglia repopulated from. The original report suggested the existence of a "hidden" microglial progenitor expressing the intermediate filament nestin ([@B25]). However, more detailed studies have in fact demonstrated that the most likely scenario is one where microglia replenish from the few remaining cells that are not removed during CSF1R inhibition ([@B19], [@B26]). A few questions are left unanswered regarding how this repopulation happens, including why some cells remain resistant to CSF1R inhibition and what signaling mechanisms tell microglia that they need to stop proliferating once the repopulation is completed. In this regard, it is interesting to note that microglia seem to have a very active population dynamics at steady-state, with a balanced ratio of proliferation and apoptosis and several self-renewal cycles during a lifetime ([@B19]).
A similar scenario is observed in Langerhans cells, which also have a predominant yolk sac origin, are long-lived ([@B9]), renew during a lifetime ([@B27]) and most likely proliferate from fully differentiated cells ([@B28]). Indeed, proliferating, self-renewing macrophages can be found in almost all tissues under physiological conditions ([@B11], [@B29]), and often even more so under pathological conditions, although in the latter case the contribution from circulating monocytes is most likely instrumental ([@B30], [@B31]). Only when the BBB integrity is compromised, most commonly after head irradiation, circulating monocytes can give rise to functional microglia \[for a review see ([@B31])\]. Nonetheless, the fact that a population of cells exists that has the potential to differentiate into microglia under certain pathological conditions is highly relevant for therapeutic intervention and merits further research.
Overall, macrophages seem to be in charge of their own local population dynamics and reach a steady state of functional self-renewal with balanced division/death rates that match the functions they carry out in their host tissues.
Lesson 4. Microglia Have Specialized in Homeostatic and Stimulus-Triggered Remodeling of Brain Circuits and Structure
---------------------------------------------------------------------------------------------------------------------
The original notion that macrophages are a uniform pool of phagocytes that reside in all organs and perform immune functions has long been discarded. Instead, the current view is that macrophages are essentially as dissimilar as their target tissues and have specialized in performing tissue homeostasis functions that make sense in their local microenvironment. For instance, whereas lung macrophages are specialized in surfactant clearance ([@B32]) and adipose macrophages participate in lipid and insulin metabolism ([@B33]), spleen macrophages are critical for iron homeostasis ([@B34], [@B35]). In the particular case of microglia, their specialized functions include interactions with neurons, potentially participating in synaptic remodeling ([@B36]), modulating experience-triggered events, such as neurogenesis ([@B37]), and participating in memory acquisition ([@B38], [@B39]). Microglia are also very active in surveilling the brain parenchyma with their highly motile processes ([@B40], [@B41]) and this is believed to be essential for brain maintenance and protection and to be in close connection with neuronal activity ([@B42], [@B43]). However, microglial motility may not be so unique, as late evidence suggests that other macrophages might also perform this type of surveillance in the intersticial space ([@B44]).
It is not surprising that macrophages have specialized roles within their host tissues to aid their homeostasis and maintenance. In this context, microglia need to be in close contact with neurons and be highly sensitive to changes in the microenvironment. In the next section, we will focus on this sensitivity to environmental factors and how that may impact the epigenetic and transcriptional identity of microglia.
Lesson 5. Microglia Have Developed a Unique Transcriptional and Epigenetic Landscape
------------------------------------------------------------------------------------
Given the wide diversity of macrophage tissue-specific phenotypes, a very relevant question to ask is how different macrophages are at the molecular level. How different are their transcription and epigenetic profiles under physiological conditions and how do they respond to changes in their environment? With the advent of "omics" methods and, in particular, with the application of single-cell studies the field has experimented a boom. It is now clear that macrophages are as different at the transcription and epigenetic level as they are at the phenotypic level. Macrophages share a common core transcriptional identity, but each tissue-specific population expresses a unique set of transcripts that is organ-specific ([@B45], [@B46]). In line with this, microglia are transcriptionally distinct to other macrophage populations but, in addition, single-cell studies have shown that there are several subpopulations of microglia that could perform functionally different tasks under basal and inflammatory conditions ([@B47], [@B48]). Similar observations are being made in other macrophage populations, with single-cell omics techniques revealing their underlying richness and diversity ([@B49], [@B50]).
In the particular case of microglia, these studies have revealed that microglia are transcriptionally homogeneous across brain regions but diverge in their transcriptional profiles across their cell division state ([@B48]). Another distinct microglia subpopulation that has earned a fair bit of attention is the so-called Disease-Associated Microglia (DAM), a subgroup of microglia revealed by single-cell sequencing specifically in the brain of an Alzheimer\'s disease mouse model ([@B47]). However, despite the power of these approaches in revealing the heterogeneity of distinct microglia subpopulations, there is debate as to the relevance of these populations in human brain, which seem to have subpopulations of their own (see comment in <https://www.alzforum.org/news/research-news/when-it-comes-alzheimers-disease-do-human-microglia-even-give-dam>). Ultimately, and under the premise that to study human disease one should focus on human microglia as much as possible, these studies reveal that microglia are in fact more diverse than anticipated and encourage further investigation aiming at dissecting their identity.
An ever-expanding source of cell diversity lies in the epigenetic landscape, which is most commonly read in the form of histone modifications (acetylations, methylations), DNA modifications (DNA methylation) and small non-coding RNAs (miRNAs, lncRNAs), and which has also been probed in different macrophage populations. The combinatorial patterns of histone 3 acetylation (at lysine 27) and methylation (at lysine 4) have been profiled in detail, revealing a core epigenetic macrophage signature, as well as a set of specific enhancer marks that are unique to each organ ([@B46]). This study also served to validate the idea that local macrophage identity is shaped by the local microenvironment. Previous experiments had shown that some macrophages populations are highly plastic and can adopt a tissue-specific identity even after ex vivo culture ([@B51], [@B52]). Recent evidence suggests that this phenotypic identity reshaping is accompanied by rewiring of the epigenetic landscape ([@B46]). In this context, microglia proved to be transcriptionally and epigenetically distinct to all other tissue-resident macrophages and clustered systematically separated from other populations for different epigenetic marks ([@B46]).
A follow-up question that emerges from these observations is to which extent epigenetic reprogramming is relevant for macrophage biology, and how it may affect microglia in particular. Microglia depletion studies in diseased brains have shown that after replenishment with "fresh" microglia, animals have improved cognitive scores ([@B53]--[@B55]). These studies have played with the idea that microglia in a diseased brain may have an altered epigenetic signature that is erased at the time of depletion and that the replenishing microglia are reprogrammed and therefore functionally fitter. How much of the original epigenetic identity is retained by the few remaining microglia and what role exactly cellular reprogramming plays in microglia phenotype is still unclear. Nonetheless, microglia and other tissue-resident macrophages appear to have a unique transcriptional and epigenetic signature that is highly plastic and may play a fundamental role in how they interact with their environment.
Since the molecular and phenotypic identities of any cell are in constant interplay, it is expected that different macrophages with different tissue-specific functions and phenotypes would have different transcriptional and epigenetic signatures. A more thorough characterization of the effect of environmental factors on cellular reprogramming and function will shed light on how dynamic these changes are and, most importantly, how relevant under physiological and disease situations. However, in spite of their phenotypic diversity, macrophages do share some core functions, such as phagocytic clearance of dead cells and immune signaling. Yet even those have tissue-specific nuances. The next section will address the similarities and disparities in mechanisms and dynamics of phagocytosis in microglia vs. other macrophages.
Phagocytosis by Macrophages and Microglia {#s3}
=========================================
The efficient phagocytic removal of apoptotic debris (efferocytosis), mostly carried out by resident macrophages, vastly influences our daily physiology. It is estimated that billions of cells are removed everyday ([@B56]): aged erythrocytes and neutrophils in the spleen, liver and bone marrow ([@B57], [@B58]), epithelial cells in the mammary gland after the lactation period ([@B59]), spermatogenic cells in the testis ([@B60]), and the outer segment of light-exposed photoreceptors in the retina ([@B61]), among others. In the brain, microglia remove the excess newborn cells produced during embryonic and postnatal development in the cortex, cerebellum and amygdala ([@B62]--[@B64]) and in adult neurogenic niches in the hippocampus and subventricular zone (SVZ) ([@B37], [@B65]), as well as deceased cells during aging and neurodegenerative diseases ([@B66]). Other cell types, such as astrocytes, neuroblasts or cells of the neural crest may also act as phagocytes but generally with less efficiency and thus microglia are considered the brain "professional" phagocytes ([@B67]).
In this section, we will compare available data on the process of phagocytosis by macrophages and microglia. We will then describe that phagocytes express overlapping recognition mechanisms of apoptotic cells, and that this redundancy ensures that phagocytosis is fast and efficient. We will then describe the functional consequences of ingestion in the phagocyte, focusing on inflammatory responses, and metabolic adaptations. Ultimately, we will conclude that phagocytosis is a powerful mechanism that needs to be firmly controlled to ensure the efficient removal of target cells while preventing the demise of live cells.
Lesson 6. Phagocytosis Is Tightly Coupled to Apoptosis Due to Redundant Recognition Mechanisms
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Phagocytosis and apoptosis are evolutionarily linked together as a mechanism that allows the elimination of excess, dysfunctional, or aged cells without imposing alterations or damage in the surrounding cells ([@B67]). Homeostatic phagocytosis is ensured by the immediate recognition of the apoptotic cell by phagocytes through a redundant plethora of released "find-me" and membrane-bound "eat-me" signals that are recognized by the phagocytes ([Table 1](#T1){ref-type="table"}). Herein we summarize some of the most relevant signals and receptors involved in apoptotic cell recognition, and we prompt the reader to comprehensive reviews on the topic for more details ([@B67], [@B72]).
######
Expression of phagocytosis-related receptors by microglia.
**Phagocyte receptor** **Gene** **Function** **Brain database ([@B68])** **Immunological database (skyline)** **Human database ([@B69])** **DAM microglia ([@B47])** **Aging human signature ([@B70])** **Aging human database ([@B70]) vs. ([@B68])** **Cerebellum vs. Striatum ([@B71])** **PAM microglia ([@B48])**
------------------------ -------------- ----------------------------------------------------------------------- --------------------------------------------- --------------------------------------- ----------------------------- ---------------------------- ------------------------------------ ------------------------------------------------ -------------------------------------- ----------------------------
G2A GPR132 G protein-coupled receptor (GPCR), recognizes lysophosphatidylcholine Microglia T lymphocytes, DCs. Neurotrophils Mo \> Mi =Ma \- HuMi_Aged Up \- \-
CX3CR1 CX3CR1 Fractalkine receptor Microglia Microglia (Monocytes low) Mi \>\> Mo \>\> Ma Down HuMi_Aged Down Striatal Down
P2Y6 P2RY6 Purinergic receptor type Y6 Microglia Microglia (Macrophages low) Mi = Ma \> Mo Down HuMi_Aged Up Striatal=Cerebellar =
P2Y12 P2RY12 Purinergic receptor type Y12 Microglia (OPCs low) Microglia Mi \>\> Mo \>\> Ma Down HuMi_Aged Down Striatal Down
BAI1 BAI1 (ADGRB) adhesion G protein-coupled receptor B1 Astrocytes, neurons, OPCs, oligodendrocytes Epithelial cells, neutrophils, NKs Mi = Ma = Mo \- \- \- \- \-
TIM4 TIMD4 T cell immunoglobulin and mucin domain containing 4 Microglia Macrophages \- \- \- \- Cerebellar \-
Stabilin 1 STAB1 \- Microglia (endothelium low) Microglia, endothelium Mi = Ma \> Mo Down HuMi_Aged Up \- Up
Stabilin 2 STAB2 \- Endothelium Endothelium, macrophages \- \- \- \- \- \-
SIRPa SIRPA Signal regulatory protein alpha Microglia Microglia, macrophages, neutrophils Mi = Ma = Mo Down \- Down Striatal \-
CD300b CD300LB CD300 molecule like family member B Microglia Neurotrophils, macrophages Mo = Ma \>\> Mi \- \- \- \- \-
TREM2 TREM2 Triggering receptor expressed on myeloid cells 2 Microglia Microglia Mi \> Ma \> Mo Up HuMi_Aged Up \- Up
MerTK MERTK MER proto-oncogene, tyrosine kinase Astrocytes, microglia Microglia, macrophages Mi \>Ma \> Mo Down \- Down Striatal \-
Axl AXL AXL receptor tyrosine kinase Astrocytes, OPCs Macrophages Mi \> Mo \> Ma Up HuMi_Aged No change Cerebellar \-
Tyro3 TYRO3 TYRO3 protein tyrosine kinase Oligodendrocytes NKs, T lymphocytes Mi = Ma = Mo \- \- \- \- \-
CD36 CD36 Fatty acid translocase Astrocytes, OPCs, microglia macrophages, endothelium Ma = Mo \>\> Mi \- \- \- \- \-
CD11b ITGAM Integrin subunit alpha M Microglia Neurotrophils, macrophages, microglia Mi = Ma = Mo Down HuMi_Aged No change Striatal \-
CD206 MRC1 Mannose receptor C-Type 1 Microglia Macrophages Ma \> Mi \> Mo \- HuMi_Aged Down Cerebellar \-
Clec7a CLEC7A C-type lectin domain containing 7A Microglia Macrophages, neutrophils, monocytes Mo \> Ma \> Mi Up HuMi_Aged Down \- Up
CD22 CD22 Sialic acid-binding Ig-like lectin 2 Microglia Macrophages Ma \> Mo \> Mi Up \- No change Striatal \-
*The table indicates the name of the receptor, the gene ID, its function and the expression in different cell types found in RNAseq public databases: a brain-specific database of microglia compared to other brain cells ([@B68]); an immunological database of microglia and other immune cells (<http://rstats.immgen.org/Skyline/skyline.html>); a human database of microglia (Mi), macrophages (Ma), and monocytes (Mo) ([@B69]); a database of genes upregulated (Up) and downregulated (Down) in disease-associated microglia (DAM) ([@B47]); a database of genes specific of a human aging signature (HuMi-aged) ([@B70]); a database of genes enriched in cerebellar (non-phagocytic) and striatal (phagocytic) microglia ([@B71]); and a database of genes upregulated (Up) or downregulated (Down) proliferative-region associated microglia (PAM)([@B48])*.
Among "find-me" signals stand out purines (ATP, UTP, and their dephosphorylated derivatives) and the chemokine fractalkine, and the corresponding metabotropic purinergic P2Y receptors and CX3CR1 expressed in phagocytes. Another important "find-me" signal is the lipid lysophosphatidylcholine (LPC), which binds to GPR132 (G2A) receptors in phagocytes. The best known "eat-me" signal is the lipid phosphatidylserine (PS), with its many phagocyte receptors, such as BAI1, TIM4 and stabilins. PS is also indirectly recognized by several bridge-molecules, including MFG-E8 (Milk-fat globule E8), as well as Protein S and Gas6, which bind to members of the TAM (Tyro3, Axl, Mer) family in phagocytes. In addition, other well-known receptors classically involved in immune responses are the inflammation triggering receptors expressed on myeloid cells TREM2 and CD300B; and integrins and complement receptors such as CD11b, which recognize opsonized apoptotic cells both directly and some of them indirectly via MFG-E8. More recently, three sugar-related receptors have been involved in phagocytosis: the mannose receptor CD206 (MRC1), which is upregulated in bone marrow and intestinal phagocytes compared to non-phagocytic macrophages and predicts their phagocytic performance ([@B73]). The sialic acid binding protein CD22, a negative regulator of phagocytosis identified in an *in vitro* screen that is upregulated in microglia in the aging brain ([@B74]). And the beta glucan receptor Clec7a, associated to the phagocytosis of oligodendrocytes by microglia during early postnatal development ([@B48]). Each type of phagocyte expresses their own set of receptors ([@B67]) and microglia is no exception.
The most conspicuous phagocytosis receptors expressed in both mouse and human microglia are CX3CR1, P2Y6, P2Y12, stabilin 1, SIRPα, TREM2, MerTK, and CD11b, based on brain RNASeq databases ([@B68], [@B69]) and the immunological RNASeq database Skyline (<http://rstats.immgen.org/Skyline/skyline.html>) ([Table 1](#T1){ref-type="table"}). However, it is important to note that each of these receptors is dynamically expressed during aging, disease and in different brain areas ([@B47], [@B48], [@B70], [@B71]) ([Table 1](#T1){ref-type="table"}), and each may serve different functions. For instance, in macrophages, MerTK and Axl show opposite regulation of their expression during inflammatory conditions, and specialize in different types of phagocytosis: MerTK in homeostasis, Axl during inflammation ([@B75]). Similarly, CD206, MerTK, and TIM4 (but not other receptors) are upregulated in peripheral macrophages early upon phagocytosis ([@B73]). In microglia, the expression of these receptors is not a proxy of their phagocytic performance either, as some including CX3CR1, P2Y12, and MerTK, have higher expression in striatal than in cerebellar microglia, whereas phagocytosis seems more prominent in the cerebellum than in the striatum ([@B71]). Similarly, their expression is also not correlated with PAM (proliferative-region- associated microglia), which has been involved in phagocytosis of developing oligodendrocytes during early postnatal development ([@B48]). Moreover, the efficiency of phagocytosis does not rely on their expression alone, but also on the motility of the microglial processes, the relative distribution of microglia compared to apoptotic cells, or the lysosomal efficiency, to name a few other factors. Their expression, however, has been linked to functional changes in microglia. For instance, some of these receptors, such as CX3CR1 and P2Y12, are part of the so-called "homeostatic microglia signature," and are downregulated in DAM ([@B47]) but upregulated in aging microglia ([@B70]). Therefore, the expression of phagocytosis receptors is not invariably linked to microglial phagocytosis efficiency. A careful analysis of the subsets of receptors expressed by microglia in different regions across the lifespan and during specific disease will shed light into their functional implication in microglial phagocytosis.
Finally, as microglia is specialized in surveilling the brain parenchyma, several receptor systems involved in the recognition of apoptotic cells have also been "hitchhiked" by other brain-specific cargos. For instance, TREM2 has been deeply involved in the recognition of beta amyloid extracellular deposits in the context of Alzheimer\'s disease (AD) ([@B76]), and is one of the main genetic risk factors for its development ([@B77]). The complement receptor CD11b ([@B78], [@B79]) and the fractalkine receptor CX3CR1 ([@B36]) are used to recognize dendritic spines and are related to synaptic prunning. SIRPα recognizes the "don\'t-eat-me" signal CD47 on myelin debris and on active synapses, inhibiting their phagocytosis ([@B80], [@B81]). CD22 binding to sialic acid inhibits the phagocytosis of extracellular deposits of beta amyloid, myelin and α-synuclein during aging ([@B74]). However, to which extent the phagocytosis of beta amyloid, α-synuclein, spines, and synapses, or myelin debris phenocopies the complete process of efferocytosis, including attraction, engulfment, and degradation, remains to be determined.
Lesson 7. Phagocytosis Is Fast and Apoptosis Is Silent
------------------------------------------------------
A consequence of the tight coupling between apoptosis and phagocytosis is that phagocytes are not easily caught red-handed and, consequently, apoptosis is largely underestimated. Original experiments in the thymus, where T lymphocytes undergo chromosomic rearrangement in their antigen receptor genes, suggested that the negatively selected cells were removed but few apoptotic cells were found. It was only when phagocytosis was analyzed that it became evident that apoptotic T lymphocytes were quickly removed by resident macrophages ([@B82]), leading to the suggestion that phagocytosis lasts minutes ([@B72]). Similarly, taking into account the number of erythrocytes removed daily in the spleen and liver, and the number of resident macrophages in these tissues (pulp macrophages and Kupffer cells, respectively), phagocytosis has been estimated to last under 30 min ([@B67]). Microglia are comparably fast. Using as a model the adult neurogenic cascade of the hippocampus, where newborn cells naturally undergo apoptosis and are phagocytosed by microglia, the estimated average clearance time of an apoptotic cell is 90 min ([@B37]).
A corollary of this data is that phagocytosis efficiency determines the amount of apoptosis visualized. At a given time, the number of apoptotic cells found can be conceived as a black box with doors on each side: an incoming door that represents the cells that enter apoptosis *de novo*; and an outgoing door that represents the cells that are removed via phagocytosis. Using this analogy is easy to understand that the size of the pool of apoptotic cells depends on the relative velocities of the two processes, apoptosis and phagocytosis ([Figure 2](#F2){ref-type="fig"}). Therefore, in physiological conditions apoptotic cells are difficult to observe because microglial phagocytosis is very efficient ([@B37]). In contrast, in pathologies like epilepsy, the increased number of apoptotic cells in early stages is not due to apoptosis induction, but to phagocytosis impairment and accumulation of non-removed apoptotic cells ([@B42]). In conclusion, phagocytosis efficiency determines the dynamics of apoptosis during development and disease.
{#F2}
Lesson 8. Phagocytosis Is Immunomodulatory at the Epigenetic, Transcriptional and Posttranslational Levels
----------------------------------------------------------------------------------------------------------
The tight coupling between apoptosis and phagocytosis has functional relevance, as it prevents the release of intracellular contents in physiological conditions. In contrast, during trauma, uncontrolled and unexpected cell death is usually followed by infection by microorganisms, and the dead cells release damage-associated molecular patterns (DAMPs), such as DNA, RNA, nucleotides, or chromatin proteins such as HMGB1 (high mobility group box 1 protein). These signals are initially recognized by the fluidic branch of the innate immune response (i.e., the complement and the coagulation systems), followed by recognition of specific pattern recognition receptors (PRRs) in several types of leukocytes. This cascade of events initiates a complex immune response that includes chemokine and cytokine release, release of reactive oxygen species and other responses to heal the damaged tissue and kill invading microorganisms ([@B83]). Unlike cell death caused by trauma, programmed cell death during physiological events ensures that DAMPs are contained within membranous blebs (the apoptotic bodies) and do not trigger activation of PRRs ([@B84]). The efficient coupling between apoptosis and phagocytosis avoids the development of secondary necrosis and release of DAMPs as apoptosis progresses, as well as the initiation of an inflammatory response from the immune system ([@B84]). As a result, apoptotic cell removal via phagocytosis is largely anti-inflammatory or at least immunomodulatory ([@B67], [@B85]), although the inflammatory responses of different types of macrophages are indeed heterogeneous ([@B73]).
In microglia, the evidences showing that phagocytosis of apoptotic cells is immunomodulatory are more tenuous than in other macrophage populations. Classic *in vitro* experiments showed that cultured microglia exposed to apoptotic cells express dampened responses to inflammatory stimuli such as bacterial lipopolysaccharides (LPS) ([@B86], [@B87]). *In vivo*, phagocytosis blockade induced by seizures in a mouse model of epilepsy correlated with a pro-inflammatory profile in microglia ([@B42]). Similarly, restoring phagocytosis in the aging brain using an anti-CD22 therapy reduced the microglial expression of inflammatory and disease-associated genes ([@B74]). However, the molecular mechanisms linking efferocytosis and inflammation are still unclear: is inflammation triggered by recognition of surface receptors or by downstream mechanisms related to the cargo degradation? Is it regulated at the epigenetic, transcriptional or the translational levels?
In both macrophages and microglia, these effects are at least partially mediated by apoptotic cell recognition via the complement protein C1q, whose presence turns efferocytosis anti-inflammatory ([@B88], [@B89]). Indeed, some of the transcriptional changes associated to efferocytosis occur while macrophages are still early in the process of phagocytosis, as shown by experiments comparing macrophages containing labeled dead cells and macrophages without apparent cargo (phagocytic and not phagocytic, respectively) ([@B73]). Similarly, posttranslational modifications related to the reduced release of the major pro-inflammatory mediator interleukin 1 beta (IL-1β) are related to the inhibition of the NLRP3 (NLR family pyrin domain containing 3) inflammasome triggered by mere contact with apoptotic cells in the absence of effective engulfment ([@B90]).
In addition to these early changes, it is also likely that the metabolic rewiring associated with apoptotic cell degradation ([@B91]) may trigger a "metabolite storm" in the phagocyte that would further contribute to regulate its function at later time points. For instance, internalization of the apoptotic cell, but not surface-to-surface interaction, triggers in macrophages another set of transcriptional anti-inflammatory changes through a chloride channel, Slc12a2, and chloride-sensing kinases ([@B92]). Time-dependent transcriptional changes have in fact been observed in cultured microglia upon phagocytosis of apoptotic cells ([@B93]). While some genes are transiently regulated at 3 h after engulfment, most are regulated in the post-degradation phase at 24 h after engulfment. This second wave of transcriptional changes is likely related to epigenetic mechanisms, as in fact chromatin remodeling related genes are upregulated in the late phagocytic microglia ([@B93]). In agreement, microglial phagocytosis is related to altered epigenetic and transcriptional profiles *in vivo*, as shown by comparing cerebellar and striatal microglia (i.e., phagocytic and non-phagocytic, respectively) ([@B71]). While this study analyzed epigenetic changes at the whole population level, it is likely that in brain areas with high basal apoptotic cell clearance, such as the cerebellum ([@B71]), the amygdala ([@B64]) or the hippocampus ([@B37]) microglia coexist in different stages related to phagocytosis. Even in these areas, it would be expected that at any given time point there would be non-phagocytic cells as well as cells engaged in different stages of engulfment, early degradation and late post-phagocytic events. This continuum of phagocytosis states is likely reflected on the epigenomic and transcriptional profiles of microglia.
Lesson 9. Phagocytosis Alters the Phagocyte Metabolism and Function
-------------------------------------------------------------------
Along with its epigenetic, transcriptional and immunomodulatory effects, phagocytosis also promotes metabolic adaptations that influence the phagocyte cellular function. In immune cells, the main metabolic pathways are catabolic (related to degradation and energy production) and anabolic (related to synthesis). Catabolic degradation of glucose starts with cytoplasmic glycolisis, which is responsible for glucose oxidation and produces pyruvate and lactate; and continues with the tricarboxylic acid cycle (TCA or Krebs cycle), which oxidizes pyruvate to produce reduced molecules (NADH, reduced nicotinamide adenine dinucleotide). NADH is also produced from the catabolism of fatty acids in the mitochondria through the beta-oxidation pathway. Next, NADH is completely degraded through the mitochondrial electron transport chain (ECT) during mitochondrial oxidative phosphorylation, to finally produce energy as ATP. Major anabolic pathways include the pentose phosphate pathway (PPP), which generates precursors of nucleotides; and the fatty acid and cholesterol synthesis pathway. The connection between metabolism and immune responses in the field of immunometabolism is very complex ([@B94]) and here we will focus on phagocyte\'s metabolic changes after inflammatory and phagocytic challenges.
Inflammatory stimuli trigger different types of metabolic adaptations. In pro-inflammatory conditions, macrophages need to act against infection and microorganisms and they undergo metabolic adaptations that meet the increased energetic demands while assuring cell survival. The most important change is a metabolic shift that potentiates glycolisis and downregulates oxidative phosphorylation, allowing faster, albeit less efficient, ATP production ([@B95]). In addition, several metabolic changes lead to the production of antibactericidal agents ([@B96]), such as reactive oxygen species (ROS) through increased PPP ([@B97]) and fatty acid synthesis ([@B98]); and mitochondrial ROS (mROS), through a disrupted ETC ([@B99]). Moreover, several metabolic pathways, as TCA cycle and fatty-acid synthesis are necessary for pro-inflammatory cytokine production, via HIF1α stabilization and through the fatty-acid synthesis regulator Laccase Domain-Containing Protein 1 (FAMIN), respectively ([@B98], [@B100]). In addition, fatty acid and cholesterol synthesis pathways also contribute to producing inflammatory mediators such as leukotriene B4 (LTB4) and isopropenoids, which bind the nuclear receptors peroxisome proliferator-activated receptors (PPARs) and the liver X receptor LXR (PLXR), respectively ([@B101], [@B102]). On the other hand, metabolic changes after anti-inflammatory stimuli help macrophages to resolve inflammation. In this case, upregulation of glycolisis and a proper TCA cycle contribute to the expression of an anti-inflammatory phenotype ([@B103], [@B104]). In addition, increased oxidative phosphorylation, together with the promotion of fatty-acid oxidation reduces the expression of pro-inflammatory cytokines ([@B105]). Thus, pro- and anti-inflammatory stimulation triggers different metabolic adaptations that contribute to modulate macrophage function.
Phagocytosis-induced metabolic adaptations are less known. In macrophages, phagocytosis of apoptotic cells drives a downregulation of fatty acid oxidation and *de novo* cholesterol, while promoting a metabolic shift that upregulates glycolisis and reduces oxidative phosphorylation ([@B91], [@B106]). In fact, phagocytosis triggers mitochondrial adaptations, such as mitochondrial fission ([@B106]) and decreased mitochondrial membrane potential via mitochondrial uncoupling protein 2 (UCP2) ([@B107]), which together with increased glycolisis ([@B91]) ensure the continued uptake of corpses. In addition, increased lactate release promotes the establishment of an anti-inflammatory environment ([@B91]). However, phagocytosis-induced metabolic adaptations not only have an effect during phagocytosis, but also trigger long-lasting effects. There are several examples of how phagocytosis reprograms the function of macrophages. For instance, during Drosophila early development, naïve macrophages are insensitive to tissue damage or infection. However, upon corpse uptake macrophages become capable of migrating into damaged regions, and phagocytose bacterial pathogens, through the activation of the Jun kinase-signaling pathway and increased the expression of the damage receptor Draper ([@B108]). Similarly, phagocytosis of fungal β-glucan in mammalian macrophages drives a metabolic shift that contributes to an enhanced and nonspecific protection against infections known as trained immunity ([@B109]). In this response, macrophages upregulate glycolisis, glutaminolysis, PPP, and cholesterol synthesis, and decrease oxidative phosphorylation through the activation of the dectin-1--Akt--mTOR--HIF-1α signaling pathway. Metabolic adaptations lead to the increased production of metabolites such as fumarate and mevalonate, key for driving changes in histone acetylation and long-term epigenetic changes, which lead to increased levels of pro-inflammatory cytokines after subsequent exposure to inflammatory challenges ([@B110]--[@B112]). Thus, phagocytosis of apoptotic corpses in macrophages triggers metabolic adaptations that immediately modulate cell function and also drives a long-term reprogramming of the phagocyte ([Figure 3](#F3){ref-type="fig"}).
{#F3}
In microglia, the metabolic adaptations to inflammatory stimuli are well known and similar those of macrophages, although their functional impact is less explored. Pro-inflammatory stimuli cause upregulation of glycolisis, leading to a speedy ATP generation that is crucial for the expression of pro-inflammatory cytokines ([@B113]). Other adaptations include impaired oxidative phosphorylation accompanied by mitochondrial fission ([@B114], [@B115]), increased glutamine entrance to the TCA cycle, suppressed fatty-acid oxidation and synthesis, and contradictory effects on the PPP ([@B116]). On the other hand, metabolic changes induced by anti-inflammatory factors in microglia are less known and show some differences compared to macrophages. Microglia exposed to anti-inflammatory stimuli maintain active both oxidative phosphorylation and PPP, and increase fatty-acid oxidation and synthesis while reducing glycolisis ([@B116], [@B117]). In contrast, little is known about the microglial metabolic adaptations to phagocytosis. Cultured phagocytic microglia upregulate genes related to metabolism and chromatin remodeling ([@B93]), suggesting long-term metabolic and phenotypic changes in microglia upon phagocytosis. Therefore, phagocytosis triggers a complex remodeling of the cell\'s metabolism and a "metabolite storm" that is likely to affect the function of microglia.
Lesson 10. Does Phagocytosis Execute Cell Death?
------------------------------------------------
Since apoptosis and phagocytosis are so closely related, the last question that arises is: when is cell death precisely executed? *in vitro* experiments with apoptosis inducers clearly show that the apoptotic pathways effectively progresses to kill the target cell via proteolytic degradation of intracellular contents by caspases ([@B118]). In this conventional scenario, called-upon macrophages would simply serve to dispose of the garbage. However, the tight coupling between apoptosis and phagocytosis also presumes a second scenario, in which expeditious nearby macrophages detect the first signs of cell distress and execute the latest stages of cell death. In these two cases, canonical efferocytosis/phagocytosis is used as a homeostatic mechanism and blocking engulfment would be expected to have detrimental consequences. In a third scenario, macrophages actually select which cells die and phagocytosis causes neuronal demise, through a process named phagoptosis ([@B119]) ([Figure 4](#F4){ref-type="fig"}).
{#F4}
Discerning between the three scenarios is complicated by the phagocytosis-related downstream transcriptional and metabolic changes, which in turn feedback into the surrounding cells, affecting their survival and proliferation. For instance, liver phagocytic macrophages release VEGF (vascular endothelial growth factor) to support the proliferation of neighboring cells ([@B120]). Similarly, the secretome of phagocytic microglia acutely inhibits proliferation of neural progenitors, allowing their long-term maintenance and the preservation of neurogenesis. These feedback mechanisms are likely related to the compensatory proliferation induced by killer caspases during apoptosis ("apoptosis-induced proliferation," AiP), observed in some cell types and organisms ([@B121]). Because of these loops between death and life processes, the identification of phagoptosis must not be procedural, i.e., phagocytosis blockade increases survival, ergo, phagocytosis must kill cells. Instead, the discrimination between canonical phagocytosis and phagoptosis should be mechanistic and based on direct observation.
In macrophage biology, evidences of phagoptosis are in fact scarce and controversial. For instance, most literature claims that neutrophils die spontaneously by apoptosis after 24 h in circulation and are subsequently phagocytosed by bone marrow macrophages ([@B67], [@B122]). Others in contrast claim that neutrophils die by phagoptosis because phagocytosis blockade leads to more "alive" neutrophils ([@B119]), although in fact they are senescent and have impaired capabilities/migration ([@B123]). In spite of this controversy, macrophages are doubtless capable of activating the apoptosis program by direct contact through the so-called "death receptors," such as Fas and TNFR (tumor necrosis factor alpha receptors) ([@B124]). Similarly, deletion of engulfment genes in C.elegans increases the survival of cells treated with weak apoptotic stimuli, supporting that phagocytes execute death of stressed cells ([@B125]). Microglia execute bona fide canonical phagocytosis of apoptotic newborn cells in the adult hippocampus ([@B37]). They also engage in phagoptosis during inflammatory conditions that lead to dysregulation of the "eat-me" signalization. Dysfunctional and transient expression of PS by stressed neurons leads to their recognition and execution by microglia, and their survival when microglia is not present ([@B126]). Microglia have also been reported to kill neuroprogenitor cells during cortical development, an effect that was exacerbated in mice treated with LPS ([@B62]); and Purkinje cells in the developing cerebellum, through the production of radical oxygen species ([@B127]). However, it is not clear whether in these cases cell death was executed by phagocytosis. In the end, it is likely that canonical phagocytosis and phagoptosis are two sides of a spectrum of ways to die that depends on the fine balance between the different "find-me" and "eat-me" signals in each pair of apoptotic cell/phagocyte.
In summary, phagocytosis is a powerful double-edged sword that must be kept under a tight rein, as is exemplified by two recent papers in zebrafish and Drosophila. Phagocytosis of apoptotic cells is beneficial during brain trauma, as it prevents secondary damage spread in zebrafish larvae ([@B128]). In control larvae, the initial necrotic and apoptotic cells resulting from traumatic brain injury in the optic tectum are cleared by microglia within the first 24 h. When phagocytosis is pharmacologically and genetically disturbed by targeting PS and the zebrafish ortholog of PS receptor BAI1, a larger wave of secondary cell death spread over the brain ([@B128]). In contrast, overexpression of phagocytosis receptors Six-Microns-Under (SIMU) and Draper (Drpr), homologs of Stabilin2 and MEGF10 (Multiple EGF Like Domains 10, a complement receptor), respectively, in adult *Drosophila* leads to phagocytosis of live neurons, motor dysfunction and a shortened lifespan ([@B129]). These recent papers highlight the profound physiological impact of microglial phagocytosis on the survival of their surrounding neurons both in health and in disease.
Given the powerful influence of phagocytosis on tissue homeostasis, it may seem striking that few pathologies have been related to its dysfunction. One may in fact speculate that the redundant apoptotic cell recognition mechanisms are in place to ensure that efferocytosis is effectively executed. Macrophage phagocytosis impairment has been reported mostly in the context of inflammatory and autoimmune diseases ([@B72]). For instance, macrophage efferocytosis is defective in atherosclerotic plaques, in chronic inflammatory lung diseases, and in lymph nodes of systemic lupus erythematosus patients. The involvement of phagocytosis in central nervous system diseases is, however, less explored. Mutations in MERTK lead to retinal diseases possibly linked to deficient phagocytosis of photoreceptors ([@B130]). Similarly, mutations in TREM2 or its bridge protein DAP12 are well known to cause defects in phagocytosis by osteoclasts, causing bone cysts and early dementia (Nasu-Hakola disease) ([@B131]). These mutations have been more recently associated to increased risk of AD ([@B132]) and result in deficient beta amyloid clearance in mouse models of AD ([@B76]) and reduced efferocytosis in cultured human microglia ([@B133]). In addition, mouse models and human biopsy samples have also shown impaired microglial efferocytosis during epilepsy caused by hyperactivity of the neuronal network ([@B42]). Phagocytosis therefore has a strong potential for impinging on the course of neurodegenerative diseases, as has been evidenced by blocking of the phagocytosis inhibitor CD22 in aging mouse brains to restore a microglial homeostatic profile and improve cognitive function ([@B74]).
In closing, we hope to provided enough evidence to support the idea that microglia are to some extent similar to other tissue macrophages and that important lessons can be learnt from them ([Figure 5](#F5){ref-type="fig"}). There are remarkable similarities between microglia and macrophages in many aspects related to their origin, the establishment and maintenance of their identity, and in their dynamic epigenetic and transcriptional landscapes. They are also comparable and at the same time unique in the regulation of their phagocytosis efficiency, the cargos they engulf, and the functional consequences of phagocytosis, including metabolic adaptations, immunomodulation and its impact on the surrounding tissue. Our aspiration is that pointing out the (dys)similarities between microglia and macrophages will help to develop novel tools to harness microglial phagocytosis in the healthy and diseased brain.
{ref-type="fig"}. Some vector graphics were obtained from [Vecteezy.com](https://www.Vecteezy.com) with permission.](fimmu-11-00506-g0005){#F5}
Author Contributions {#s4}
====================
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
Conflict of Interest
--------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
**Funding.** This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities with FEDER funds (BFU2015-66689 and RYC-2013-12817), a Basque Government Department of Education project (PI_2016_1\_0011), and a Fundación Tatiana Pérez de Guzmán el Bueno project (P-048-FTPGB 2018) to AS. In addition, MM-R is recipient of a predoctoral fellowship from the University of the Basque Country UPV/EHU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No funding is available for open access publications from our institution or other sources.
[^1]: Edited by: Esther M. Lafuente, Complutense University of Madrid, Spain
[^2]: Reviewed by: Zsuzsa Szondy, University of Debrecen, Hungary; Raymond B. Birge, Rutgers, The State University of New Jersey, United States
[^3]: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology
[^4]: †These authors have contributed equally to this work
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
As an improvement on SoC (System-on-Chip), NoC is an intercommunication-based network system, which is implemented on an integrated circuit. With the development of IC technology, SoC based on the traditional bus architecture has been unable to meet the increasing requirement of network communication. With the transplantation of network technology from computer systems and the replacement of traditional bus structure with network structure, NoC solves the communication bottleneck issue of SoC and is rather promising. The topology of NoC defines the layout and on-link mode of nodes and lines in the network. As a key technology of NoC, it plays an important role in the performance of the network, such as throughput, delay, fault tolerance, and load balance. At present, total 100 number of research institutions and companies engaged in on-chip network research in which the better-known institutions have Satanford University in \[[@B1]\], Princeton University in \[[@B2]\], the University of Bologna, KTH \[[@B3]\], the SGS-Thomson semiconductor company, Arteriscompany France Curie University \[[@B4]\], and The Royal Swedish Institute of Technology, the Netherlands PHILIP laboratories.
NoC applications can be of a static or dynamic nature. In static applications, the parameters are well defined prior to the design phase, allowing a topology to meet the specific requirements needed by the given application during synthesis. In dynamic workload applications such as those dealt with in MPSoC scenarios, new application PEs may be inserted at any time. Regular topologies such as mesh and torus are often used to overcome such plug-and-play compatibility issues. Regular topologies however result in poor performance due to increases in power usage and hardware area overhead on account of the topological regularity. It has become evident through research that the application specific NoC architecture is superior to regular topologies in terms of power consumption and NoC resources \[[@B5]\]. Regular topologies tend to assume that NoC systems contain homogeneous cores, where in reality, many high performance SoCs make use of heterogeneous processors/cores. As a result, designs containing nonuniform core sizes do not match the standard, tile-based floor plan of these topologies \[[@B6]\]. For the majority of SoCs, it is known that sizes ranging from small to state-of-the-art systems can be designed with static (or semistatic) mapping of tasks to cores, and therefore the communication traffic characteristics of the SoC can also be obtained statically \[[@B7]\]. Given this fact, it is then possible to create a custom design that can cater to the communication characteristics of the system while reducing power and on-chip area and improving performance. This work addresses the static workload scenario, incorporating a number of factors during topology synthesis to generate application specific NoCs which meet the power and performance requirements of the given application.
With the increase of chip integration, the future number of transistors on a single chip will reach billions. On-chip multiprocessor is able to effectively use the massive on-chip transistors resources, which has become the development trend of high-performance processors \[[@B8]--[@B10]\]. Chip network has a more important influence in the scalability and performance of chip multiprocessor, which gradually becomes a research hotspot \[[@B11]--[@B13]\]. In recent years, on-chip network research has focused on low power design \[[@B14], [@B15]\], router design \[[@B16], [@B17]\], implementation and test methods \[[@B18], [@B19]\], and fault tolerance mechanism. Currently, few studies are about the topology and routing algorithm, and most of the existing single-chip multiprocessor uses the classical topology (including Mesh, Torus). As the size of chip network is usually smaller in scale than the large-scale parallel machines, on-chip network design must give full consideration to the difficulty of the physical realization, so the traditional network for parallel machines Mesh, Torus, and so forth does not necessarily apply to the classical topology. Thus according to its characteristics, studying for on-chip network topology is of great significance.
The remainder of this paper is organized as follows. [Section 2](#sec2){ref-type="sec"} provides some introductions on network topology architectures. [Section 3](#sec3){ref-type="sec"} presents the details about the routing algorithm design based on BFC architecture. [Section 4](#sec4){ref-type="sec"} presents the evaluation methodology and simulation results. [Section 5](#sec5){ref-type="sec"} summarizes the paper by presenting our conclusions in this area.
2. Topology Introductions {#sec2}
=========================
2.1. Clos Network Topology {#sec2.1}
--------------------------
Clos network \[[@B20]--[@B22]\] was first proposed by Charles Clos in 1953. Clos network is the most commonly used three Clos network *C*(*m*, *n*, *r*) shown in [Figure 1](#fig1){ref-type="fig"}, where *m* is the number of middle-class switching units; *n* is the input ports of the input stage switch module, which is the output ports of output level switching units; *r* is the number of input stage switching unit, which is the number of output stage switching units. Each switch module is a crossbar structure in accordance with crossbar model to work.
The cache architecture based on three Clos networks can be divided into three categories: full-cache-type Clos network, non-cache-type Clos network, and part-cache-type Clos network.
### 2.1.1. Full-Cache-Type Clos Network (MMM, Memory-Memory-Memory) {#sec2.1.1}
The structure is based on three kinds of shared cache switching fabric. This uses a connection-oriented routing method. When the data packet arrives at the input stage switch modules, switching network based on its source input port and output port for the target opens up a transmission path routing. Because it is connection-oriented routing method, so this kind of transmission is a closed, nonblocking transmission. There are some caches in three-level MMM mode, and the routing can be used to record relevant information. However, this method requires a lot of middle-class switching module, the requirements of the size of the buffer space are very high, the bandwidth allocation and the buffer queue options are needed to solve the problem, and the operation is very complex. Therefore, the application of MMM is currently very narrow, and little has been put into actual business.
### 2.1.2. Non-Cache-Type Clos Network (SSS, Space-Space-Space) {#sec2.1.2}
This kind of structure is a cache of the exchange without any structure. The exchange network design is relatively simple, which does not arrange the cache device. However, because it does not exist cache, the request for a new arrival, such as you need to complete before sending the output port and the use of intermediate-level switching unit selection, requires a more complex scheduling algorithms. Moreover, without caching devices, it is prone to internal blocking.
### 2.1.3. Part-Cache-Type Clos Network (MSM, Memory-Space-Memory) {#sec2.1.3}
This kind of network uses the first- and third-level shared cache, and the exchange unit of the middle class does not use the cached exchange. It uses a packet-oriented routing. In the routing process, the routing operations are completed the input port buffer queue data packets in a cycle, and the selected packets are sent to the cache. In the next cycle it will be sent out, while other groups are rerouting operations experience.
Clos network has a good path diversity, but it may still occur in the internal block. Internal blocking is an effective request for the network, which is not a free intermediate module linked directly, and the so-called effective request is an empty request for input ports and output ports.
There are currently three different methods to ensure nonblocking network work, and the three methods are described as follows.Strict sense nonblocking: for a *C*(*m*, *n*, *r*) network, when *m* ≥ 2*n* − 1 is established, which is called as strict nonblocking network. In a strict nonblocking state of the Internet, for any free port input stage and output stage, there is always a free link linked, which does not adjust to other multilevel network to establish a good path. Regardless of selection strategy will not appear blocked.Wide sense nonblocking: the requirements of the wide sense nonblocking network are relatively low, and it refers to blocking within the network by routing algorithms to help solve the congestion problem and find the right link for the data packet without the need breaking the link for completed routing request.Rearranged nonblocking: rearranged nonblocking network is an improvement for the generalized nonblocking network. It can be rearranged by using a generalized nonblocking mechanism to extend the network implementation, once the blockage occurs in the network by using a specific routing algorithm to break up the current network links based on the routing requests to reestablish the connection. This rearrangement algorithm can make the link to connect to rationalize and improve module utilization greatly. However, the design of corresponding algorithm is more complex which may re-break the link and make the cache delay, energy consumption largely. For a *C*(*m*, *n*, *r*) network, only when *m* ≥ *n*, the network is rearranged.In the actual network, the different types of networks have different roles. Our broad network of nonblocking Clos network routing algorithm provides a more efficient routing algorithm to improve the network performance. Nonblocking network can be rearranged, which can be used to study the reorganization algorithm.
2.2. Butterfly Network Topology {#sec2.2}
-------------------------------
Butterfly network \[[@B23]--[@B25]\] from the hypercube network is a hypercube deformation network.
*n*-Dimensional butterfly network can be written as BF(*n*), and it exists as a vertex set, specifically indicated as shown in ([1](#EEq1){ref-type="disp-formula"}) in \[[@B26]\]: $$\begin{matrix}
{V = \left\{ {\left( {x;i} \right):x \in V\left( {Qn} \right),\,\, 0 \leq i \leq n} \right\},} \\
\end{matrix}$$ where *n* is the number of layers with the butterfly network denoted by BF(*n*) of the butterfly network with *n* + 1 layers. *x* is the horizontal coordinate of the butterfly network. When the value of *n* is determined, the maximum value of *x* will be determined specifically for 2^*n*^. *Qn* is the set of nodes for each layer. [Figure 2](#fig2){ref-type="fig"} shows a three-layer butterfly network.
Known butterfly network layer parameters *n* can determine the width of the network that each node has the number of 2^*n*^, so a BF(*n*) network has (*n* + 1)2^*n*^ vertices. *n* is 3 in [Figure 1](#fig1){ref-type="fig"}, and the number of nodes in the network topology is 32.
If *j* = *i* + 1 and *x* = *y*, or *x* and *y* have exactly *j* different coordinates, (*x*, *i*) and (*y*, *j*) vertices are interconnected by an undirected edge.
When *x* = *y*, the side is called the direct side, and the remaining sides are called cross-edges.
In the butterfly network BF(*n*), each vertex of the 0-layer and *n*-layers is 2, and the remaining vertices are 4, so the entire network has the formula for calculating the number of edges as shown in $$\begin{matrix}
{e\left( {\text{BF}\left( n \right)} \right) = 2 \times n \times 2^{n} = n \times 2^{n + 1}.} \\
\end{matrix}$$
As the butterfly network and hypercube networks are very similar, so they have the following advantages. (1) Butterfly network has a simple recursive structure; that is, BF(*n*) network can be simply divided into two disjoint BF(*n* − 1) networks. Two disjoint BF(*n* − 1) networks will be able to get by removing all the vertices of the *n*th layer BF(*n*). (2) The unique path length *n* exists between (*x*, 0) and (*y*, *n*) vertices, it is just a vertex which passes through each layer, and the use of cross-edges from the *i* to *i* + 1 layer only has exactly *i* + 1 different coordinates. So we can know that BF(*n*) has *n*-order.
The butterfly network structure has good performance, so it can host multiple source nodes, and it can give the full advantages of a high number of routes, and reducing network latency, overhead, and so forth performs well. However, it does not have path diversity, in adversarial networks or high load on the network congestion situation which is more serious.
2.3. BFC Network Topology {#sec2.3}
-------------------------
The analysis from the previous section can know that butterfly network \[[@B27]\] can give the full advantages of a high number of routes, but it does not have path diversity in dealing with congestion in poor performance. Clos network has a good path diversity, which can provide multiple data link between each pair of nodes, so it has a good solution to the problem of network congestion. However, the routing process must use a lot of middle-class switching module, which needs more access lines resulting in much higher routing delay than the butterfly network, but the network overhead is larger than the butterfly network.
This section presents that the BFC topology network has a combination of the above two advantages, while overcoming their shortcomings. It is derived from the butterfly network inheriting the excellent network performance of the butterfly network; you can use a high number of routing equipment; the routing latency is low it also has the advantages of Clos network path diversity.
BFC network layer of the butterfly network with a number of different dimensions of the node modules is integrated into a new module. The exchange of information between different layers uses a two-way link to complete in the new unified network. [Figure 3](#fig3){ref-type="fig"} shows a three-layer butterfly network; each node is a routing node; they can connect a number of resource nodes. Four routing nodes *R* ~0~, *R* ~1~, *R* ~2~, and *R* ~3~ in the leftmost of [Figure 3](#fig3){ref-type="fig"} are integrated into a single routing node, and the remaining nodes are integrated with the same approach, so we can get the routing node map shown in [Figure 4](#fig4){ref-type="fig"}. This transformation forming graph is also known as the planar butterfly network \[[@B28]\]. In the new network topology, the merger of the four routing information transmission between nodes is done directly in the internal nodes, data transfer between nodes using the combined data link transmission. The link is bidirectional, which can satisfy the input and output.
The structure shown in [Figure 4](#fig4){ref-type="fig"} for a certain topology planning can form a network structure shown in [Figure 5](#fig5){ref-type="fig"}.
[Figure 5](#fig5){ref-type="fig"} shows that the routing node *R* ~0~, respectively, interconnects with routing nodes *R* ~1~, *R* ~2~, and *R* ~4~. We make some improvements on the network structure, so that it has symmetry and better path diversity. [Figure 6](#fig6){ref-type="fig"} is the improved network topology-BFC network structure, so butterfly network through several transformations will gradually evolve into BFC network structure.
As can be seen from [Figure 6](#fig6){ref-type="fig"} that the improved routing node *R* ~0~ have existed the channels with *R* ~1~, *R* ~2~, *R* ~3~, *R* ~4~, and *R* ~5~, which greatly increases the network path diversity, which can effectively reduce the network congestion. While the network also has a symmetry, which is more scalable.
2.4. BFC Topology Performance Analysis {#sec2.4}
--------------------------------------
This section is the performance analysis and comparison from the path diversity, scalability, network diameter, and energy consumption in terms of BFC topology and other common topologies.
### 2.4.1. Path Diversity {#sec2.4.1}
Since BFC network topology inherits the characteristics of Clos network, it also has a path diversity. As shown in [Figure 7](#fig7){ref-type="fig"} when a data packet is transported from the routing node *R* ~0~ to *R* ~2~, the transmission path exists as follows: $$\begin{matrix}
\left. R_{0}\longrightarrow R_{1}\longrightarrow R_{2} \right. \\
\end{matrix}$$ Or $$\begin{matrix}
\left. R_{0}\longrightarrow R_{3}\longrightarrow R_{2} \right. \\
\end{matrix}$$
When the transfer process needs not to consider the shortest path, BFC provides network routing path that will be more and more even with the use of non-shortest-path routing algorithm, and it can be circuitous transmission by the nodes of the next level, or it can also be back and forth transmission between layers.
### 2.4.2. Scalability {#sec2.4.2}
From the previous section we can see that butterfly network has a good scalability by the improved Planar butterfly network. Add a new route node, but it lies with its rows and columns of all the nodes connected, but also it will diagonally connect the nodes to complete the expansion of the network. Compared to the butterfly network, BFC network scalability is more favorable. [Figure 8](#fig8){ref-type="fig"} is the BFC network topology expanded from 8 nodes to 12 nodes.
For BFC network that can accommodate more nodes, in addition to the above method for the horizontal expansion of the network, there are several other aspects that may be considered. (1) A single routing node is connected to increase the number of resource nodes, such that the original network routing nodes are connected from 4 resource nodes increased to 8 resource nodes shown in [Figure 9](#fig9){ref-type="fig"}. As the number of resource nodes increases, the corresponding link bandwidth also needs to make appropriate improvements to accommodate increased data traffic. (2) Several BFC networks can be coupled together according to certain structure. There are some limitations using the lateral extension method after the routing nodes increased, because the corresponding link bandwidth between two nodes becomes less and less; then the data transfer rate and the delay will increase. There can be a few same BFC-scale network coupled together using a network topology, such as using Mesh structure coupled together, so as to increase the cost of the average number of hops to reduce the adverse effects of narrow channels, and the 25 specific implementation is shown in Figures [9](#fig9){ref-type="fig"} and [10](#fig10){ref-type="fig"}.
### 2.4.3. Network Diameter {#sec2.4.3}
In the 4 × 4 network topology, the network diameter of Mesh network structure is 6; the network diameter of Torus structure is 3; the network diameter of butterfly network is 3; the network diameter of BFC network structure is 6. Therefore, the network diameter of BFC network topology is greater than the butterfly network.
### 2.4.4. Transmission Delay {#sec2.4.4}
BFC network provides a number of direct lines, the BFC network connection can be routed directly, and the butterfly network may need to jump three times. And because BFC network has the path diversity, and the handling capacity of the network congestion is better than the butterfly network, so its transmission delay is less than the butterfly network. This end-to-end delay in the simulation can demonstrate the advantages of BFC in this regard.
### 2.4.5. Network Throughput {#sec2.4.5}
For a network topology 4 × 4 = 16 nodes, it can be obtained according to formula ([5](#EEq3){ref-type="disp-formula"}). The throughput performance comparison of different topologies is shown in [Table 1](#tab1){ref-type="table"}. The throughput of the Mesh structure and the butterfly network structure is minimum; Octagon and Torus network throughput is followed; the BFC network throughput is the largest. Because the BFC network inherits the path diversity of the Clos network; it has strong processing power for the congestion and very good throughput performance: $$\begin{matrix}
{\text{TH} \leq \frac{2b \times \text{Bc}}{N}.} \\
\end{matrix}$$
### 2.4.6. Chip Power Consumption {#sec2.4.6}
As NoC is based on nanoscale network and the network size is very large, so the network\'s energy consumption has become a major constraining factor in network performance. Because the power not only affects the life of the chip, but also closely relates to the amount of heat networks, such a small amount of heat corresponding to the chip must also be small; otherwise it is easy to accumulate chips burn calories.
Energy consumption of NoC generally consists of three parts. The data sheet needs the energy *P* ~*L*~ from the source node to destination node in the routing chain process. The data sheet needs the energy *P* ~*M*~ on the device forward in the buffer. The data sheet needs the energy *P* ~*R*~ of data routing and switching module.
Specific formula is shown in the below equation: $$\begin{matrix}
{\text{Power} = P_{L} + P_{M} + P_{R}.} \\
\end{matrix}$$
As the BFC network provides the direct link between the same dimension nodes and the cross-links between the different dimension nodes. Compared with the Mesh topology, the required data transmission link length and the power consumption *P* ~*L*~ is smaller. There is only one link between nodes in Mesh structure, but the bandwidth of the same dimension is used by many links simultaneously in the BFC structure, so as to reduce the bandwidth of a single link. Thus, in the routing process, crossbar switch module required for conversion also reduces the amount of data, and the routing process *P* ~*R*~ energy consumption also is decreased. Since, in the BFC structure, each node corresponds to a number of links, so each node corresponds to the number of link input and output buffer more than the Mesh structure. So the energy *P* ~*M*~ required buffer devices higher than Mesh, but due to reduction of bandwidth of each link, you can switch to smaller capacity cache device to indirectly reduce network energy consumption. The comparison of the specific energy consumption of the network is shown in [Figure 11](#fig11){ref-type="fig"}.
3. Routing Algorithm Design {#sec3}
===========================
This section designs a deterministic deadlock-free routing algorithm for BFC network by comparing the size of vertical and horizontal coordinates of the current node and destination node to determine the output port.
In the topology of *N* = 16 routing number, the network will be placed in the coordinate system, and each router has a corresponding coordinate values (*x*, *y*). There are four diagonal nodes and four vertical and horizontal nodes, and the requirements connected with the IP core router port number are 0. The nodes of *X*-axis direction are corresponding to the ports 1, 2, and 3. The port number is 4, 5, 6 in the *Y*-axis direction. Four port numbers of the right diagonal clockwise rotation nodes are 7, 8, 9, and 10 as shown in [Figure 12](#fig12){ref-type="fig"}.
Set the current node coordinates *C*(*cx*, *cy*), the target node coordinates *D*(*dx*, *dy*), and the output port outport.
Routing algorithm is described as pseudocode. When the routing receives a packet, examine the packet header containing the destination node to compute the coordinate difference between the target node and the current node: *X* = *dx* − *cx*, *Y* = *dy* − *cy*. When *X* == 0 and *Y* == 0, it indicates that the data packets reach the destination node, outport = 0. When *Y* == 0 and *X* \> 0, it indicates the packet\'s destination node in the right direction of the current node, and we choose the right direction of the port output. When *Y* == 0 and *X* \< 0, it indicates the packet\'s destination node in the left direction of the current node, and we choose the left direction of the port output. When *Y* \< 0 and *X* == 0, it indicates the packet\'s destination node under the direction of the current node, and we choose the next direction of the port output. When *Y* \< 0 and *X* \> 0, it indicates the packet\'s destination node in the direction of the current node, the choice of the direction of the port output. When *X* \> 0 and *Y* \> 0, it indicates the packet\'s destination node at the top right of the current node to select the top right to the port output. When *Y* \> 0 and *X* \< 0, it indicates the packet\'s destination node at the top left of the current node to select the upper left to the port output. When *Y* \< 0 and *X* \> 0, it indicates the packet\'s destination node in the lower right of the current node to select the port for output to the lower right. When *X* \< 0 and *Y* \< 0, it indicates the packet\'s destination node in the left bottom of the current node to select the lower left to the port output.
Algorithm pseudocode is shown in [Algorithm 1](#alg1){ref-type="fig"}.
The algorithm limits the direction of the routing data packets in the current node and the destination node forms a square area routes, and the direction must always be toward the destination node; then this would limit the generation of the ring; thereby it damages the necessary condition for the formation of a deadlock. Thus, the routing algorithm is deadlock-free.
4. Simulation and Analysis {#sec4}
==========================
4.1. Simulation Setup {#sec4.1}
---------------------
This paper uses simulation software OPNET simulating the performance of the BFC network, which can make us understand the pros and cons of their performance through comparison with the butterfly network.
OPNET has some important features, and a brief introduction is here. OPNET simulation of the entire design process is carried out around the object. Simulation model is based on the object as a unit to build up. OPNET has its own unique C-like language, and its syntax is similar to C language. It also has its own unique core function library, and the library functions begin with "op\_", which is similar to API functions in VC, and it is mainly used in the process model and the transceiver pipeline stage calls. It is divided by function into several different set of functions, and the same function names in a function have the same prefix. In using OPNET simulation process, we can use it for program debugging tool that comes with the test, and you can also call VC for joint commissioning, it is very convenient to people who are familiar with VC debugging environment.
These two concepts of the simulation time and the elapsed time need distinguished in the use.
Simulation time is the event of the system running in a simulated environment. Simulation\'s role in the operation of the core is to maintain the event list, and the table is based on the program listing. Each table represents an event, and that is an interrupt. Each event has its own serial number and the corresponding execution time. When it\'s on an event handler, it may remove it from the list and begin processing the next interrupt after the emulation core. If the simulation time of the events is 00:01, and the simulation time of this incident is 00:05, then the simulation time goes from 01 time to 05 times, but in reality, the process may only take 1 second.
Elapsed time is the actual physical time that the simulation takes in the real world. Executing the event does not require any time, and the simulation time may not be spent between events, but the physical time does not be consumed. Event execution until the event has finished executing, and the simulation time may not consume, but the actual physical time has been consumed.
In the simulation, we compare the performance of the three-tier butterfly network topology and the similar number of nodes in the node 36 BFC network routing. The analyzed network performance parameters are end-to-end delay and throughput. Since the variable rate is still selected into the network. In the simulation, the use of three traffic patterns, namely, uniform flow, hot flow, and matrix flow.
4.2. Simulation Results and Analysis {#sec4.2}
------------------------------------
[Figure 13](#fig13){ref-type="fig"} shows the performance comparison in the uniform flow network corresponding to end-to-end delay and throughput of the butterfly network and BFC network. It can be seen that performance of the BFC network is better than the butterfly network. For end-to-end delay, as shown in [Figure 13(a)](#fig13){ref-type="fig"}, the butterfly network in the injection rate reaches 0.2 when it has begun to rise, while the BFC network began to rise in the injection rate sustaining to 0.35. The number of BFC network links is more, and the advantage of rich path diversity shows up here. Throughput performance is similar to end-to-end delay as shown in [Figure 13(b)](#fig13){ref-type="fig"}, and the throughput and saturation corresponding injection rate of BFC network are much higher than the butterfly network, and the reach saturation is also higher than the butterfly network.
[Figure 14](#fig14){ref-type="fig"} shows the performance comparison in the matrix flow network corresponding to end-to-end delay and throughput of the butterfly network and BFC network. The performance BFC network is still slightly better than the butterfly network, but the performance gap is not large. Two networks in the injection rate reach 0.2; and the latency begins to increase; the increase in the butterfly network trend is more obvious as shown in [Figure 14(a)](#fig14){ref-type="fig"}. Throughput and latency have similar trends, and BFC network reaching the saturation point is higher than the butterfly network as shown in [Figure 14(b)](#fig14){ref-type="fig"}.
[Figure 15](#fig15){ref-type="fig"} shows the performance comparison in the hot spots flow network corresponding to end-to-end delay and throughput of the butterfly network and BFC network. Hot spots traffic patterns are the common processing mode to investigate the ability of commonly used network. In this mode, the weakness of low processing capabilities with congestion of the butterfly network is exposed. For end-to-end delay as shown in [Figure 15(a)](#fig15){ref-type="fig"}, the butterfly network almost reaches 0.17 from the injection rate when it begins to increase, while BFC starts up from the injection rate around 0.3. Throughput performance and end-to-end delay are similar as shown in [Figure 15(b)](#fig15){ref-type="fig"}, and the butterfly network saturation throughput is much lower than the BFC network.
5. Conclusion {#sec5}
=============
This paper in-depth studies the butterfly network and its advantages and disadvantages, and the new network topology, BFC, network is proposed with the combination of the characteristics of the Clos network structure and the butterfly network structure. The network originates in the butterfly network, and it can give full play to the advantages of a high number of routes with the characteristics of the low latency and overhead of the butterfly network, which overcomes the low processing power shortcomings of the butterfly network congestion due to the introduction of the structural characteristics of the Clos network, and it has a better path diversity. In addition, the network absorbs the characteristics of the Clos network, and it provides path diversity, and because it is based on the butterfly network, there is no middle class compared with the Clos network, so it is superior to Clos network in terms of latency or in the overhead area network equipment. The structure of the BFC network has a reasonable change, and the corresponding routing algorithm is proposed for its structural characteristics. Simulation results show that the topology has better latency and throughput than the butterfly network.
This paper is supported by the Scientific Research Fund of Jiangxi Provincial Education Department (GJJ13379).
Conflict of Interests
=====================
The authors declare that there is no conflict of interests regarding the publication of this paper.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
{#fig5}
{#fig6}
{#fig7}
{#fig8}
{#fig9}
{#fig10}
{#fig11}
{#fig12}
{#fig13}
{#fig14}
{#fig15}
{#alg1}
######
Comparison of ideal throughput.
Topology Bc Throughput value
----------- ---- ----------------------
Mesh 8 TH~Mesh~ ≤ *b*
Torus 16 TH~Torus~ ≤ 2*b*
Octagon 12 TH~Octagon~ ≤ 3*b*/2
Butterfly 8 TH~Butterfly~ ≤ *b*
BFC 32 TH~BFC~ ≤ 4*b*
[^1]: Academic Editors: Y. Lu and F. Yu
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S1}
============
A hallmark of the autoimmune disease systemic lupus erythematosus (SLE) is the presence of antinuclear autoantibodies (ANAs) in the serum ([@B1]--[@B6]). These antibodies are directed against histones, DNA, histone--DNA complexes, and various ribonuclear complexes (anti-SM, anti-Ro, and anti-La) ([@B3]--[@B6]) and may be found in immune complexes that play an important role in the pathogenesis of SLE. The disease occurs more frequently in females than males (ratio 10:1) with a peak incidence at 45--65 years. Based on the findings that ANA producing B cells have undergone Ig class switching and carry large numbers of somatic mutations, it is very likely that ANAs arise from B cells participating in T cell dependent antigen responses ([@B3]--[@B6]).
Studies using mouse models spontaneously developing an SLE-like disease have improved our knowledge of the etiology of this disease ([@B7]--[@B9]). In particular, these studies have highlighted the complex genetic contribution to the development of the disease as well as the important role of somatic mutations of antibody genes in the formation of autoantibodies ([@B7]--[@B13]).
The generation of a self-tolerant B cell repertoire is critically dependent upon the processes of clonal deletion, receptor editing, and anergy ([@B14]--[@B19]). Exactly how B cells escape central tolerance is, however, still not completely understood. Ample evidence has been provided indicating that non-autoreactive B cells can become autoreactive through somatic mutations in their variable heavy (Vh) and light (Vl) chain regions ([@B6], [@B10], [@B13], [@B20]). Equally, B cells using germline-encoded Vh and Vl regions escaping central tolerance induction in the bone marrow could also generate autoreactive B cells ([@B21]).
Recently we showed that almost all aging (8--12 months old) C57BL/6 (B6) mice develop several features characteristic of autoimmunity. This included germinal center formations in the spleen, kidney depositions of IgM, lymphocyte infiltrates in the salivary glands, as well as the production of high titers of IgG ANAs. Furthermore, this IgG ANA generation was shown to be T cell dependent ([@B22]). However, aging B6 mice do not develop real signs of disease. Here, we compare the ANA B cell repertoire of such aging B6 mice with that of (B6 × B6.*H-2^bm12^*)F~1~ mice undergoing a chronic graft-versus-host disease (cGVHD) and thus developing an SLE-like disease and death ([@B23]). Results indicate that the ANA B cell repertoire of aging B6 mice is more restricted than that of mice undergoing GVHD and is only partially overlapping. Moreover, we show that the ANA producing B cells in aging mice and in GVHD mice are derived from progenitors expressing B cell receptors (BCRs) either recognizing or not recognizing nuclear antigens. These findings indicate that ANA producing B cells in both aging mice and in GVHD mice are generated by two pathways: by defective tolerance induction in the bone marrow or by hypermutation in the V-regions of B cells responding to a foreign antigen.
Materials and Methods {#S2}
=====================
Mice and Induction of cGVHD {#S2-1}
---------------------------
C57BL/6 and (C57BL/6 × B6(C)-*H2-Ab^bm12^*/KhEgJ)F~1~ mice were bred under specific pathogen free conditions in our animal unit. A cGVHD was induced by i.v. injection of 8 × 10^7^ spleen plus lymph node cells from B6 mice into 8--10 weeks old B6 × bm12 mice, following established protocols ([@B23]).
Generation of Hybridomas {#S2-2}
------------------------
Spleen cells derived from aging B6 mice (aged 8--12 months) or B6 × bm12 mice undergoing a cGVHD were fused to the Sp2/0-Ag14 fusion partner following standard protocols. In brief, 2 × 10^7^ Sp2/0 cells were used for the PEG1500 (Roche Diagnostics)-mediated fusion of all lymphoid cells prepared from an entire spleen. The fused cells were plated into 25 flat-bottom 96-well plates containing 200 µl HAT-medium (2%FBS; GIBCO, 2% IL6, in house, 1× HAT supplement, Sigma) per well and incubated at 37°C in 10% CO~2~ in air. After 10--12 days, supernatants were tested for IgG production by ELISA and for antinuclear reactivity by immunofluorescence (see below). Cells from IgG ANA positive wells were thereafter sub-cloned at limiting dilution. We routinely obtained fusion frequencies between 10^−3^ and 5 × 10^−3^. Thus, for each mouse, we have screened between 5,000 and 20,000 hybrids for IgG ANA production. Since the vast majority of B cell hybrids produce IgM, we tested for the success of sub-cloning by performing simultaneous ELISA for IgM and IgG (see below) in supernatants of growing clones. The IgG containing supernatants were re-tested for ANA reactivity, before being further processed for Ig V-gene analyses (see below).
Determination of IgG Sub-Class and Anti-Histone/DNA/Sm/SS-B/La Reactivity {#S2-3}
-------------------------------------------------------------------------
Determination of IgG sub-class, [l]{.smallcaps}-chain, and detection of anti-DNA antibodies was done by standard ELISA. For determination of anti-histone, anti-Sm or anti-SS-B/La ELISA plates were coated with 2.5 μg/ml of the respective antigens in PBS (all purchased from Immunovision). Alkaline phosphatase labeled goat anti-mouse IgG, goat anti-mouse [l]{.smallcaps}-chain or goat anti-human IgG (Southern Biotech) was used for detection. The ELISA was performed as previously described ([@B24]--[@B26]).
Antinuclear Autoantibody Determination {#S2-4}
--------------------------------------
Kidney cryosections from Rag2^−/−^ mice of homozygous matings were incubated with supernatants or purified antibodies as described in Ref. ([@B27]). For detection, either FITC labeled goat anti-mouse IgG (Jackson ImmunoResearch) or FITC labeled rabbit anti-human IgG (Jackson ImmunoResearch) were used.
Vh and Vl Sequencing Analysis {#S2-5}
-----------------------------
RNA from ANA positive hybridomas was extracted using TRI Reagent (Sigma) followed by cDNA synthesis (GoScript Reverse Transcriptase) according to the manufacturer's protocol and using primers as specified in [Table S5](#SM5){ref-type="supplementary-material"} in Supplementary Material. Amplification of the heavy and light chain V-regions was performed using Vent polymerase (New England Biolabs) and, subsequently, the Vh and Vl regions were ligated into the pJet1.2 blunt end cloning vector (Thermo Fisher Scientific). For sequencing, plasmids were sent to Microsynth (Balgach, Switzerland). Resulting sequences were inspected using DNASTAR and aligned to the germline heavy and light chain sequences of the international ImMunoGeneTics information system^®^ (<http://imgt.org>).
Reversion of Somatic Mutations Back into Germline Configuration {#S2-6}
---------------------------------------------------------------
Double-stranded DNA encoding the variable regions of heavy and light chains obtained from ANA positive hybridomas as well as their germline and mutated versions were ordered as gBlock gene fragments from IDT (Integrated DNA Technologies). These fragments were cloned into heavy and light chain expression vectors driven by the human cytomegalovirus promoter and containing the human IgG~1~ constant region for the heavy chain and the human kappa constant region for the light chain ([@B28]) (a kind gift from Dr. Hedda Wardemann, Deutsches Krebsforschungszentrum, Heidelberg, Germany). Subsequently, antibodies were produced in HEK 293 cells (ATCC, No. CRL-1573) and purified by affinity chromatography on Protein A Sepharose^®^ (GE Health Care, Uppsala, Sweden) as described ([@B28], [@B29]).
Results {#S3}
=======
IgG ANA Producing Hybridomas from Aging B6 Mice {#S3-1}
-----------------------------------------------
Hybridomas were generated independently from spleen cells of seven individual aging B6 mice, which had high titers of serum ANA. The resulting IgG producing hybridomas were then tested for ANA reactivity by immunofluorescence, and positive cultures were sub-cloned. In total, 36 hybridomas producing IgG ANA were generated (Table [1](#T1){ref-type="table"}). Of these, 16 were IgG~2a~, 19 were IgG~2b~, and only one was IgG~1~. This heavy chain selection suggests that IgG ANA formation in aging B6 mice is mainly driven by a T~h~1 response ([@B30]). All ANAs contained a kappa light chain. Sequence analysis of the corresponding Vh and Vk regions used by these hybridomas revealed a restricted repertoire (V, D, and J annotations are according to the IMGT data base). Thus, 25 (69.4%) hybridomas used the Vh1-26 gene and this usage was found among hybridomas of all seven individual mice. Moreover, a very high frequency of Vk4-74 gene usage was also found. Thus, 23 (63.9%) hybridomas, derived from 5 of 7 individual mice, used this particular Vk light chain gene. Strikingly, 21 of the 23 Vk4-74 expressing hybridomas expressed the Vh1-26 heavy chain. Thus, the IgG ANA B cell repertoire of aging B6 mice is dominated by those expressing a Vh1-26 heavy chain gene in combination with a Vk4-74 light chain gene.
######
Characteristics of ANA-reactive mAbs derived from aging B6 mice.[^a^](#tfn1){ref-type="table-fn"}^,^[^b^](#tfn2){ref-type="table-fn"}
Mouse number Hybridoma number IgG isotype Ig-heavy chain v-region Ig-light chain v-region
--------------- ------------------ ------------- ------------------------- ------------------------- --------- -------- -----
Aging B6 \# 1 5.D3.D10 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
5.D3. E12 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
7.E8.B11 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
7.F9.F1 IgG2b Vh1-75 D2-4 J2 Vk?
8G3.E6 IgG2a Vh1-26 D1-1 J4 Vk4-74 Jk4
8.H7.D3 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
15.G10.F10 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
15.H12.E4 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
16.B9.C9 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
16.D7.A9 IgG2a Vh1-26 D1-1 J4 Vk4-74 Jk4
17.A9.E6 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
22.C9.G7 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk4
Aging B6 \# 2 1.G5.B8 IgG1 Vh1-39 D4-1 J1 Vk4-57 Jk5
8.G9.G4 IgG2b Vh1-26 D1-1 J4 Vk6-32 Jk2
Aging B6 \# 3 13.4.5.A IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
13.18.A IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
13.27.B IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
13.31A IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
13.69.B IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
13.85B IgG2a Vh1-26 D2-4 J3 Vk4-74 Jk2
Aging B6 \# 4 1A2.1 IgG2a Vh1-26 D2-2 J4 Vk4-74 Jk2
2F8.1 IgG2b Vh1-26 D1-1 J4 Vk4-74 Jk2
Aging B6 \# 5 12.G3.G7 IgG2a Vh1-26 D1-1 J4 Vk5-43 Jk2
15.D10B3 IgG2a Vh1-26 D1-1 J4 Vk5-43 Jk2
Aging B6 \# 6 3.2.2A IgG2b Vh1-74 D2-4 J3 Vk6-23 Jk5
3.10.1A IgG2b Vh1-26 D1-1 J2 Vk4-61 Jk1
5.13.1A IgG2b Vh1-26 D1-1 J2 Vk4-74 Jk2
6.15.1A IgG2a Vh8-12 D3-1 J1 Vk4-91 Jk2
7.7.3A IgG2b Vh1-22 D2-2 J1 Vk10-96 Jk2
20.15.1A IgG2a Vh1-50 D4-1 J2 Vk4-74 Jk2
23.6.1A IgG2a Vh1-22 D2-2 J1 Vk10-96 Jk2
24.18.1A IgG2b Vh1-22 D2-2 J1 Vk10-96 Jk2
25.8.1A IgG2b Vh3-6 D3-3 J3 Vk4-74 Jk2
Aging B6 \# 7 3.25A IgG2b Vh1-50 D2-4 J2 Vk?
3.36A IgG2a Vh1-80 D2-4 J3 Vk4-58 Jk2
3.73A IgG2a Vh1-26 D1-1 J4 Vk4-74 Jk2
*^a^All the mAbs converted into germline sequences are boxed in orange*.
*^b^All the mAbs using Vh1-26 have been boxed in yellow and those using Vk4-74 in red*.
We also tested these ANAs for their capacity to bind to histones, Sm antigens, SS-B/La antigens, and DNA. By ELISA, 26 of the 36 showed strong and 5 showed weak histone binding, whereas binding to the other nuclear antigens was undetectable (Figure [1](#F1){ref-type="fig"}A).
{ref-type="table"}) or **(B)** young B6 × bm12 mice undergoing a cGVH reaction (Table [2](#T2){ref-type="table"}). Significant binding was defined as an O.D. of five times over background in ELISA. (−), no significant binding at 5 μg/ml; (+), binding at 5 μg/ml; (++), binding at 1.7 µg/ml; (+++), binding at 0.55 µg/ml; and (++++), binding at 0.19 µg/ml or lower.](fimmu-09-00016-g001){#F1}
In Individual Mice, Hybridomas Using Vh1-26/Vk4-74 Were Clonally Related {#S3-2}
------------------------------------------------------------------------
The fact that we obtained 11 hybridomas from mouse 1, and 6 hybridomas from mouse 3 using the Vh1-26/Vk4-74 combination with the same D, Jh, and Jk elements already strongly suggested a clonal relationship among the B cells that had fused to generate these hybridomas (Table [1](#T1){ref-type="table"}). The finding that the amino acid sequences of the IgH and IgL CDR3 regions of these hybridomas were practically identical (Tables [S1](#SM1){ref-type="supplementary-material"} and [S2](#SM2){ref-type="supplementary-material"} in Supplementary Material) also supports this conclusion. Thus, at least in mouse 1 and 3, the ANA production seems to be dominated by a single B cell clone.
IgG ANA from B6 × bm12 Mice Undergoing a Chronic Graft-versus-Host Reaction {#S3-3}
---------------------------------------------------------------------------
We then asked whether the restricted IgG ANA B cell repertoire in aging B6 mice, which do not show obvious signs of disease, was similarly restricted in B6 × bm12 mice undergoing a chronic graft-versus-host reaction and, thus, developing an SLE-like disease. Hybridomas were independently generated from spleen cells of five mice with the highest ANA titers. These fusions resulted in 34 hybridomas with IgG ANA activity (Table [2](#T2){ref-type="table"}). A total of 23 ANAs (68%) reacted to histones but again, none reacted to the other nuclear antigens (Figure [1](#F1){ref-type="fig"}B). IgG constant region usage analysis revealed a distribution rather similar to that in aged mice with 18 IgG~2a~, 13 IgG~2b~, and only 3 IgG~1~, the latter all from mouse 5. Thus, as in aging B6 mice, in B6 × bm12 mice undergoing a cGVHD, the IgG ANA formation appears to be Th1 cell driven. Again, all ANAs contained a kappa light chain.
######
Characteristics of ANA-reactive mAbs derived from graft-versus-host disease (GVHD) mice.[^a^](#tfn3){ref-type="table-fn"}^,^[^b^](#tfn4){ref-type="table-fn"}
Mouse number Hybridoma number IgG isotype Ig-heavy chain v-region Ig-light chain v-region
-------------- ------------------ ------------- ------------------------- ------------------------- ---------- ---------- -----
GVHD \# 1 1.12.1A IgG2b Vh8-8 D2-14 J4 Vk4-74 Jk2
1.28.1 IgG2a Vh1-26 D1-1 J1 Vk4-74 Jk2
1.308.1 IgG2a Vh1-55 D2-4 J3 Vk3-4 Jk1
1.356.1 IgG2a Vh1-55 D2-4 J3 Vk3-4 Jk1
1.741.1 IgG2a Vh1-55 D2-4 J3 Vk3-4 Jk1
1.802.1 IgG2b Vh1-26 D1-1 J1 Vk4-63 Jk5
GVHD \# 2 2.24.0 IgG2a Vh1-52 D1-1 J1 Vk14-111 Jk5
2.53.8 IgG2b Vh14-2 D2-2 J2 Vk14-111 Jk1
2.58.2 IgG2b Vh14-2 D2-2 J2 Vk14-111 Jk1
2.70.8 IgG2a Vh14-4 D1-1 J3 Vk3-10 Jk1
2.80.9 IgG2a Vh14-4 D1-1 J3 Vk3-12 Jk1
2.152.9 IgG2a Vh1-52 D1-1 J1 Vk3-10 Jk1
2.300.2 IgG2b Vh1-55 D2-1 J2 Vk3-10 Jk5
2.344.3 IgG2a Vh1-52 D1-1 J1 Vk14-111 Jk5
2.374.1 IgG2a Vh1-26 D1-1 J1 Vk3-7 Jk1
2.382.2 IgG2a Vh14-4 D2-1 J3 Vk3-10 Jk1
2.388.2 IgG2a Vh1-31 D2-4 J4 Vk3-10 Jk2
2.394.1 IgG2a Vh14-4 D1-1 J3 Vk3-10 Jk1
GVHD \# 3 3.55.1 IgG2a Vh1-26 D1-1 J3 Vk4-74 Jk2
3.69.1 IgG2b Vh1-26 D1-1 J3 Vk4-74 Jk2
3.71.1 IgG2a Vh1-26 D2-5 J1 Vk4-74 Jk2
3.77.1 IgG2a Vh1-26 D1-1 J3 Vk4-74 Jk2
3.152.1 IgG2b Vh1-26 D1-1 J3 Vk4-74 Jk2
GVHD \# 4 63.3.1 IgG2b Vh1-55 D2-4 J2 Vk4-74 Jk2
165.2.1 IgG2a Vh8-12 D2-2 J1 Vk15-103 Jk5
GVHD \# 5 14.11.A IgG2b Vh1-50 D1-1 J2 Vk1-117 Jk2
381.11.A IgG2b Vh1-54 J4 Vk14-111 Jk2
685.9.A IgG2b Vh1-59 D2-5 J3 Vk17-127 Jk5
724.3.A IgG1 Vh1-26 D1-1 J3 Vk4-91 Jk4
738.8.A IgG1 Vh1-26 D1-1 J3 Vk3-7 Jk1
762.5.A IgG2a Vh5-17 D1-2 J4 Vk?
810.4.A IgG1 Vh1-52 D2-12 J2 Vk3-10 Jk1
865.12.A IgG2b Vh1-72 D3-3 J2 Vk4-74 Jk5
1011.39.A IgG2b Vh1-53 D1-1 J2 Vk17-127 Jk5
*^a^All the mAbs converted into germline sequences are boxed in orange*.
*^b^All the mAbs using Vh1-26 are boxed in yellow and those using Vk4-74 in red*.
In aging B6 mice, a dominance of Vh1-26 and Vk4-74 usage by the IgG ANA producing hybridomas was observed. In B6 × bm12-derived hybridomas, the same genes were also found to be used, but at a much lower frequency. Thus, ten (29.4%) of these ANAs used Vh1-26 and nine (26.5%) used Vk4-74. Six of the hybridomas using Vk4-74 used the Vh1-26 heavy chain; however, five of these were derived from one mouse (GVHD mouse 3). These findings show that the IgG ANA B cell repertoires of aging B6 mice and B6 × bm12 mice undergoing a cGVHD are partially overlapping. However, the IgG ANA B cell repertoire seems to be more diverse in the B6 × bm12 mice than in the aging B6 mice. Therefore, the mechanisms underlying the generation of these autoreactive B cells might be different in the two model systems.
Somatic Mutations in the Light Chain Determine the ANA Reactivity of Vh1-26/Vk4-74 Using mAbs {#S3-4}
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Sequence analysis revealed that most mAbs with ANA reactivity carried somatic mutations in their Vh and Vk regions. These results are summarized in Tables [S1](#SM1){ref-type="supplementary-material"}--[S4](#SM4){ref-type="supplementary-material"} in Supplementary Material.
Since in aging B6 mice the ANA reactivity was dominated by mAbs expressing a Vh1-26/Vk4-74 heavy and light chain combination, we tested if somatic mutations in the Vh and/or the Vk regions of these mAbs were required for their autoreactivity. Therefore, the Vh and Vk regions of six (derived from 4 individual mice) mAbs of aging B6 and two mAbs of GVHD mice using the Vh1-26/Vk4-74 combination were reverted to their germline configuration. After expression and purification, the ANA titers of these reverted mAbs were directly compared to their original, mutated forms. As shown in Figures [2](#F2){ref-type="fig"}A,B, the ANA titers of all mAbs, in which the Vh region had been reverted into germline configuration but the Vk regions were still somatically mutated, behaved like the original ANAs, continuing to recognize the histone antigens. However, all mAbs where the Vh was still somatically mutated but the Vk had been reverted to germline configuration lost ANA reactivity. Also, as expected, all mAbs in which both Vh and Vkappa regions had been reverted to germline configuration lost ANA reactivity. Thus, the ANA reactivity of the Vh1-26/Vk4-74 mAbs is due to somatic mutations in the Vk4-74 gene.
{ref-type="table"}) or **(B)** young B6 × bm12 mice undergoing a cGvH reaction (Table [2](#T2){ref-type="table"}). The histograms depict titers of individual mAbs, either with the original SHM, or Vh and/or Vk in germline configuration (GL), or **(C)** having a single mutation in their Vk CDR1 resulting in a serine to arginine conversion. A titer of one was defined as binding at a concentration of 5 µg/ml mAb, followed by 1:2 serial dilution steps and recording the last dilution where binding still was possible. SHM, somatic hypermutation; GL, germline; n.b., no binding detectable at 5 µg/ml of mAb.](fimmu-09-00016-g002){#F2}
Due to the finding that somatic mutations within the Vk4-74 gene determined the ANA reactivity of Vh1-26/Vk4-74 mAbs and in order to identify a common motif that could account for this autoreactivity, we analyzed the sequence of these Vk4-74 genes in more detail. This analysis revealed that 20 of 23 Vh1-26/Vk4-74 mAbs derived from aging B6 mice had a mutation at position 30 (IMGT numbering) in their CDR1 region of the Vk4-74 gene. The germline-encoded serine in these mAbs was mutated into a positively charged arginine residue. Introduction of such a serine to arginine mutation in non-autoreactive germline versions of three different mAbs resulted in a complete gain of ANA reactivity for all of them (Figure [2](#F2){ref-type="fig"}C). Thus, antinuclear reactivity of these mAbs can be induced by a single base pair substitution changing the serine at position 30 in the CDR1 of the Vk4-74 light chain into an arginine.
B Cells Expressing a Germline-Encoded Immunoglobulin Vh Gene with a Negatively Charged CDR2 Region Contribute to the ANA-Reactive B Cell Repertoire {#S3-5}
---------------------------------------------------------------------------------------------------------------------------------------------------
We also tested if non-Vh1-26/Vk4-74 using mAbs require somatic mutations in their Vh and/or Vk regions for ANA reactivity. Therefore, the Vh and Vk region of three mAbs of aging B6 mice and six mAbs of the GVHD mice were reverted to their germline configuration. The 1.G5.B8 mAb derived from an aging B6 mouse (No. 2) used a somatically mutated Vh region and a germline-encoded Vk region. Reversion of the Vh region of this mAb into germline sequences resulted in a complete abrogation of its ANA reactivity (Figure [3](#F3){ref-type="fig"}A). In contrast, upon reversion, the other two mAbs (7.7.3A and 6.15.1A, both from mouse 6) kept their ANA reactivity (Figure [3](#F3){ref-type="fig"}A). Thus, B cells expressing a germline-encoded immunoglobulin Vh can also contribute to the ANA-reactive B cell repertoire in aging B6 mice.
{ref-type="table"} containing V-regions, either with the original somatic hypermutations (SHM), or Vh and/or Vk in germline configuration (GL), or **(C)** containing a Vh CDR2 with conversions of aspartic and glutamic acids to glycine. Titers and legends as defined in Figure [2](#F2){ref-type="fig"}.](fimmu-09-00016-g003){#F3}
From the six GVHD-derived mAbs, one (1.12.1A, mouse 1) lost ANA reactivity upon reversion of the Vk but not the Vh gene into germline configuration (Figure [3](#F3){ref-type="fig"}B). The fact that this mAb uses the Vk4-74 gene indicates that this Vk gene can also give rise to ANA reactivity when paired with a Vh gene other than Vh1-26. The other five mAbs kept their ANA reactivity upon reversion of their Vh and Vk genes to germline configuration (Figure [3](#F3){ref-type="fig"}B). Thus, B cells expressing a germline-encoded immunoglobulin can also contribute to the ANA-reactive repertoire in mice undergoing a cGVHD.
A remarkable observation was that 5 of 7 mAbs (one from aging B6 mice and four from GVHD mice), which kept ANA reactivity upon complete reversion of their Ig genes into germline configuration, had a negatively charged Vh-CDR2 region as defined by having at least two more negatively than positively charged amino acids in this region (see Table [3](#T3){ref-type="table"}). This observation prompted us to test if these negatively charged amino acids are involved in ANA reactivity. Therefore, the negatively charged amino acids in the CDR2 regions of these five mAbs were mutated into neutral glycine. As shown in Figure [3](#F3){ref-type="fig"}C, one mAb derived from an aging B6 mouse (6.15.1A) and two from a GVHD mouse (2.58.2 and 2.394.1), completely lost ANA reactivity upon aspartic acid/glutamic acid conversion into glycine. The ANA titers of the other two GVHD-derived mAbs (2.344.3 and 165.2.1) diminished by a factor of 2 and 3, respectively (Figure [3](#F3){ref-type="fig"}C). Thus, B cells expressing a germline-encoded immunoglobulin with a negatively charged Vh-CDR2 region contribute to the ANA-reactive repertoire. At this point, it is noteworthy that 6 of 11 (54.6%) of the non-Vh1-26 using ANA mAbs from aging B6 mice and 14 of 24 (58.3%) from mice undergoing cGVHD indeed carry a negatively charged Vh-CDR2 (Table [3](#T3){ref-type="table"}).
######
Vh-CDR2 regions of non-Vh1-26 ANA-reactive mAbs of graft-versus-host disease (GVHD) and aging B6 mice.[^a^](#tfn5){ref-type="table-fn"}^,^[^b^](#tfn6){ref-type="table-fn"}
Mouse number Hybridoma number Vh-region Vh-CDR2 region Mouse number Hybridoma number Vh-region Vh-CDR2 region
-------------- ------------------ ----------------------------- ----------------------------- --------------- ------------------------- ------------------------- -------------------
GVHD \# 1 1.12.1A Vh8-8 I W W **D D D** Aging B6 \# 1 7.F9.F1 Vh1-75 I L P G S G S S
1.308.1 Vh1-55 I Y P G S G S T
1.356.1 Vh1-55 I Y P G S G S T Aging B6 \# 2 1.G5.B8 Vh1-39 V N P N Y G T I
1.741.1 Vh1-55 I Y P G S G S T
Aging B6 \# 6 3.2.2A Vh1-74 I H P S **D** S **D** T
GVHD \# 2 2.24.0 Vh1-52 I **D** P S **D** G **E** T Aging B6 \# 6 6.15.1A Vh8-12 I Y W **D D D K**
2.53.8 Vh14-2 I **D** P **E D** G **E** T 7.7.3A Vh1-22 I N P N N G G T
2.58.2 Vh14-2 I **D** P **E D** G **E** S 20.15.1A Vh1-50 I **D** P S **D** T F T
2.70.8 Vh14-4 I **D** P **E** N G **D** T 23.6.1A Vh1-22 I N P N N G **D** T
2.80.9 Vh14-4 I **D** P **E** N G **D** T 24.18.1A Vh1-22 I N P N N **D D** T
2.152.9 Vh1-52 I **D** P S **D** G **E** T 25.8.1A Vh3-6 I S C **D** G S S
2.300.2 Vh1-55 I Y P G S V S T
2.344.3 Vh1-52 I **D** P S **D D E** T Aging B6 \# 7 3.25A Vh1-50 I **D** P S **D** T Y T
2.382.2 Vh14-4 I **D** P **E** N G **D** T Aging B6 \# 7 3.36A Vh1-80 I Y P G **D** G **D** T
2.388.2 Vh1-31 I F P Y N G V S
2.394.1 Vh14-4 I **D** P **E** N G **D** T
GVHD \# 4 63.3.1 Vh1-55 I Y P G S G S T
165.2.1 Vh8-12 I Y W **D D D K**
GVHD \# 5 14.11.A Vh1-50 I **D** P S **D** S Y I
381.11.A Vh1-54 I N P G S G G I
685.9.A Vh1-59 I **D** P S **D** S S S
762.5.A Vh5-17 I S **R** G S G I L
810.4.A Vh1-52 I **D** P S **D** S **E** T
865.12.A Vh1-72 I **D** P S S G G T
1011.39.A Vh1-53 I N P S N **D** G T
1011.39.A Vh1-53 I N P S N **D** G T
*Bold font highlights negatively and positively charged amino acids*.
*^a^All mAbs that were converted into germline sequences or in which the negatively charged amino acids in the CDR2 were converted into glycine are marked with orange boxes*.
*^b^All the negatively charged amino acids in Vh-CDR2 region are marked in yellow and the positively charged amino acids in green*.
Discussion {#S4}
==========
Two Pathways to ANA-Producing B Cells {#S4-1}
-------------------------------------
In the present study, we compared antinuclear autoantibodies (ANAs) from aging B6 mice, which do not show signs of disease, with those derived from mice undergoing a chronic GVH reaction (GVHD mice) and which develop an SLE-like disease ([@B23]). In order to study the V-regions of ANAs, we generated B cell hybridomas from mice with high ANA titers in their serum. Almost all monoclonal ANAs bound to a mixture of histones. As expected from previous studies, ANA production was T cell and antigen dependent ([@B1]--[@B3], [@B22]), since virtually all V-regions of our monoclonal ANAs contained somatic mutations. To determine whether the ANA-producing B cells were derived from progenitors expressing B cell receptors (BCRs) for nuclear antigens, we reverted the mutated V-regions into the corresponding germline sequences and tested the resulting antibodies for ANA reactivity. Based on such analyses, we found that some ANA-producing B cells in both aging B6 mice and GVHD mice must be derived from progenitors expressing germline-encoded genes for BCRs specific for nuclear antigens, while several others were derived from progenitors expressing BCRs that showed no ANA reactivity. The vast majority of the latter were derived from progenitors using Vh1-26 in combination with Vk4-74. Antibodies with the germline sequences of these V-regions had no ANA reactivity. It has been shown previously that non-autoreactive B cells can become autoreactive upon acquiring somatic mutations in their Vh and/or Vl regions ([@B6], [@B10], [@B13], [@B20]). In contrast to the findings with Vh1-26/Vk4-74 expressing ANAs, the reversion of other Vh and Vk regions of several monoclonal ANAs to the corresponding germline sequences did not abolish ANA reactivity. The maintenance of ANA reactivity upon reversion into germline sequences was observed with 2 of 3 monoclonal ANAs from aging B6 mice, and with 5 of 6 monoclonal ANAs from GVHD mice. Thus, two pathways can be envisaged for the generation of ANA specific antibody forming B cells (Figure [4](#F4){ref-type="fig"}). In pathway one, they are derived from B cells undergoing somatic hypermutations in response to a foreign antigen (Figure [4](#F4){ref-type="fig"}, red line); in pathway two, they are derived from B cell progenitors expressing ANA-specific receptors that escape tolerance induction in the bone marrow, possibly by exposure to BCR crosslinking by nuclear antigens (Figure [4](#F4){ref-type="fig"}, blue line). Indeed, we showed previously that anti-dsDNA reactive B cells escaped tolerance induction by the crosslinking of their BCRs by a T cell independent type 2-antigen ([@B24]).
![Two distinct pathways generate ANA producing B cells. \[**(A)** red background\] B cells with a B-cell receptor (BCR) using a germline-encoded Vh1-26 in combination with a germline-encoded Vk4-74 gets stimulated by a non-self (NS) antigen/hapten. Due to somatic hypermutations in the Vk4-74 gene, ANA-reactive memory B cells are generated. In aging B6 mice, these memory cells are then activated by nuclear antigens and Th1 cells to produce ANA. In the GVHD mice, nuclear antigens and alloreactive T cells activate these memory cells, resulting in ANA formation. \[**(B)** blue background\] Immature B cells in the bone marrow with germline-encoded BCRs specific for nuclear antigens escape tolerance induction mechanisms through BCR cross-linking by a T cell independent type 2-antigen (TI-2) antigens or a signal from alloreactive T cells. In the periphery of aging B6 mice, these cells get stimulated by nuclear antigens and Th1 cells to produce ANAs. In the periphery of GVHD mice, nuclear antigens and alloreactive T cells activate these cells to ANA formation.](fimmu-09-00016-g004){#F4}
Restricted Diversity of ANA Specific B Cells {#S4-2}
--------------------------------------------
Inspection of the V-region usage of ANA producing hybridomas shows that many ANAs derived from aging mice (21 of 36) (Table [1](#T1){ref-type="table"}; Figure [4](#F4){ref-type="fig"}) and few from mice undergoing a cGVHD (6 of 34) (Table [2](#T2){ref-type="table"}; Figure [4](#F4){ref-type="fig"}) express Vh1-26 in combination with Vk4-74. Interestingly this Vh/Vk combination is expressed in virtually all ANAs from aging mouse 1 (11 of 12), aging mouse 3 (6 of 6), and from the GVHD mouse 3 (5 of 5). Inspection of the V-regions strongly suggests that these ANAs are generated from single B cell clones with the exception of one clone of the GVHD mouse 3, which differs in the CDR3 compared to the other four. In other mice, ANAs are clearly generated from multiple B cell clones. In aging mouse 6, one ANA expresses the Vh1-26/Vk4-74 combination, one expresses the Vh1-26 with Vk4-61, two express the Vk4-74 with Vh1-50 and Vh3-6, respectively, and five express different Vh/Vk combinations. In GVHD mice, all except one mouse (No. 3) express ANAs derived from multiple B cell clones. Thus, in aging B6 mice, the ANA repertoire tends to be restricted, while in GVHD mice, the ANA repertoire tends to be less restricted. The more important question to be answered is why ANAs are oligoclonal in some mice and polyclonal in others. One reason for this restricted repertoire might be that Vh1-26 and Vk4-74 genes are overrepresented in the B cell repertoire of B6 mice in general. RNA sequence analysis of Vh1 usage by B cells from young and aging B6 mice showed that around 10% of these used Vh1-26 (our unpublished results). Since about 50% of all B cells use a Vh1 family member, Vh1-26 is expressed by 5% of them. We did not attempt to verify this number in the hybrids generated, by, for example, cloning and sequencing the Vh regions of random IgM or IgG antibodies not reacting to nuclear antigens. With respect to Vk4-74 usage, Aoki-Ota et al. ([@B31]) analyzed the kappa light chain repertoire in B6 mice. This analysis revealed that B6 B cells rarely use the Vk4-74 gene. We also have determined the Vk usage in developing B6 B cells and found that Vk4-74 was used less than 1% in single Vk-rearrangements of preBII cells and immature B cells of the bone marrow \[unpublished observation, ([@B32])\]. Thus, the restricted ANA-reactive B cell repertoire does not simply appear to reflect a selective usage of the Vh1-26 and the Vk4-74 genes by the B6 B cells. Instead, and more interestingly, this restricted ANA repertoire may occur through selection by and as yet to be defined T cell dependent antigen.
Many years ago, it was shown that haptens such as NP (4-hydoxy-3-nitrophenyl)acetyl ([@B33], [@B34]), oxazolone (2-phenyl-5-oxazolone) ([@B35], [@B36]), and arsonate (p-azophenyl-arsonate) ([@B37], [@B38]) could elicit an oligoclonal humoral immune response, at least in certain inbred strains of mice. Based on this, one might envisage that a hapten-like structure is responsible for the restricted ANA-reactive B cell repertoire in aging B6 mice. Thus, one could imagine that the frequency of ANA-producing B cells that are derived from hapten-induced memory cells in aging mice (red pathway in Figure [4](#F4){ref-type="fig"}) is higher than the frequency of B cell progenitors with receptors for nuclear antigens that escape tolerance induction in the bone marrow (blue pathway in Figure [4](#F4){ref-type="fig"}).
Activation of ANA-Producing B Cells {#S4-3}
-----------------------------------
In ANA-producing mice, B cells expressing nuclear antigen-specific BCRs must be activated by nuclear antigens and helper T cells. In GVHD mice, the alloreactive T cells act as helper T cells. In their initial report on the "allogeneic effect," Katz et al. reported a drastic enhancement of IgG responses as a result of a GVH reaction ([@B39]). Later, Osborne and Katz showed that a simple non-immunogenic hapten--polypeptide conjugate might elicit a vigorous primary IgG response as a consequence of the allogeneic effect ([@B40]). Likewise, Hamilton and Miller reported that otherwise tolerogenic hapten-conjugated syngeneic mouse erythrocytes would elicit a strong primary antibody response as a result of a GVH reaction ([@B41]). It is possible that this type of general B cell help by alloreactive T cells contributes to the tendency of the ANA response in cGVHD mice to be more diverse.
In aging mice, the specificity and origin of the T helper cells that are required for ANA production is not known. Based on the IgG class of the ANAs produced in these mice, it is conceivable that the helper cells involved in the ANA response are Th1 cells ([@B30]).
Conclusions Regarding the Structure of Anti-Histone Antibodies {#S4-4}
--------------------------------------------------------------
Almost 30 years ago, Weigert and coworkers showed that arginine residues in CDR regions play a crucial role for the specificity of anti-dsDNA autoantibodies ([@B10], [@B11]). In the present study, we find an important role of arginine in the Vk-region of certain ANAs. Site-directed mutagenesis revealed that somatic mutations in the Vk4-74 light chains of the Vh1-26/Vk4-74 mAbs determine ANA reactivity, since conversion into their germline sequence completely abolished ANA reactivity. Interestingly, and highly significant, 20 of 23 Vh1-26/Vk4-74-expressing ANAs derived from aging B6 mice had a serine to arginine mutation at position 30 in the CDR1 region of the Vk4-74 gene. In 1 of these 23 mAbs, this change to arginine was the only mutation present in the entire Vk4-74 gene. The finding that one of these (17A9E6) mAb loses ANA-binding activity upon conversion of this arginine into serine strongly indicates the importance of this mutation for ANA reactivity. Furthermore, the introduction of single serine to arginine mutations in the germline configuration of three different mAbs conferred their antinuclear reactivity. This proves that this single mutation can convert an ANA-negative mAb into a positive one and clearly shows how easily Vh1-26/Vk4-74 using mAbs may become autoantibodies. The ANA-reactive B cell repertoire has also been analyzed in B6 mice congenic for a SLE susceptibility locus. Also this analysis revealed a rather frequent usage of Vk4-74 light chains. Thus, Liang et al. ([@B12]) found that 9 of 30 ANA producing hybridomas used Vk4-74 light chain (herein called *ai4*). Moreover, it was shown that 3 of these 9 had a serine to arginine mutation at position 30 in the CDR1. In a report by Guo et al. ([@B13]), 9 of 33 ANA-producing hybridomas used a Vk4-74 light chain (herein also called *ai4*). At least 5 of these 9 had a serine to arginine mutation at position 30 in the CDR1, like we report herein.
However, since 5 of 6 Vh1-26/Vk4-74 using mAbs derived from the GVHD mice did not have this serine to arginine mutation, other mutations in the Vk4-74 gene can be involved in ANA reactivity.
Yet, another interesting finding reported here is that a high percentage of ANA-reactive mAbs not using the Vh1-26 gene, especially in the repertoire derived from GVHD mice, possess a germline-encoded negatively charged Vh CDR2 region. Thus, 14 of the total 34 monoclonal ANAs (41.2%) derived from the GVHD mice and 6 of 36 (16.7%) derived from the aging B6 mice have such a CDR2 region. Within the total Vh gene repertoire of B6 mice, 11.5% possess such a negatively charged CDR2 ([@B42]). Thus, our findings suggest that B cells with such a charged CDR2 might be positively selected into the ANA-reactive B cell repertoire, especially in GVHD mice. The finding that mutations of these negatively charged amino acids into glycine can result in a complete loss of ANA binding supports this hypothesis and, moreover, indicates the potential importance of this region in ANA reactivity. However, additional structural effects of the amino acid change to glycine cannot be excluded, especially since glycine is known to be a helix breaker.
It is noteworthy that despite high ANA serum titers, aging B6 mice do not show the typical lesions that are observed in multiple organs of SLE patients, whereas the GVHD mice do show this. Therefore, one could envisage that the ANA repertoire difference between aging B6 and GVHD mice is playing a role in the pathogenesis of the SLE-like disease observed in GVHD mice.
Overall, the findings described in the present study highlight various new characteristics of ANA-reactive B cell repertoires and argue for similar mechanisms of ANA generation in SLE patients. This might improve our understanding of the pathogenesis of this disease, thereby opening new concepts and therapies for its control.
Ethics Statement {#S5}
================
The State veterinary authorities of Basel (Kantonales Veterinäramt, Basel-Stadt) had approved all animal experiments under permission numbers 1888 and 2434.
Data Availability {#S6}
=================
Sequencing data can be found in GenBank under accession numbers MG733774---MG733908.
Author Contributions {#S7}
====================
AR conceived the study and designed experiments. All authors performed experiments and analyzed the data. AR, JA, and FK wrote the paper.
Conflict of Interest Statement {#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.
We thank Drs. Rhodri Ceredig, Werner Haas, Fritz Melchers, Michael Parkhouse, and Panagiotis Tsapogas for helpful comments and critical reading of the manuscript. We thank Ricardo Koch, Hannie Rolink, and Mike Rolink for skillful technical assistance. This paper is dedicated to the memory of Antonius (Ton) Rolink, our principal investigator and professor, who unexpectedly died during the preparation of this manuscript.
**Funding.** AR is holder of the chair in Immunology endowed by F. Hoffmann-La Roche Ltd., Basel to the University of Basel. This study was supported by the Swiss National Science Foundation (310030B_160330/1). LA-S was supported by the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program FP7/2007-2013 under Research Executive Agency Grant 315902.
Supplementary Material {#S9}
======================
The Supplementary Material for this article can be found online at <http://www.frontiersin.org/articles/10.3389/fimmu.2018.00016/full#supplementary-material>.
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Nucleotide sequences and their translation into amino acids of Vh- and Vk-regions of all hybridomas described herein, which were derived from aging B6 mice.
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Click here for additional data file.
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Click here for additional data file.
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Nucleotide sequences and their translation into amino acids of Vh- and Vk-regions of all hybridomas described herein, which were derived from young B6 × bm12 mice undergoing a GvH reaction.
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Click here for additional data file.
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Click here for additional data file.
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Sequences of primers used for cDNA synthesis of total mRNA obtained from ANA-reactive hybridomas. The sequences of primers used for sequencing of Vh- and Vk-regions of the obtained cDNA are also shown.
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Click here for additional data file.
[^1]: Edited by: Karsten Kretschmer, Technische Universität Dresden, Germany
[^2]: Reviewed by: Siegfried Weiss, National Research Centre for Biotechnology, Germany; Claudia Berek, Charité Universitätsmedizin Berlin, Germany
[^3]: ^†^Present address: Martin Faderl, Experimental Pathology, University of Bern, Bern, Switzerland; Oliver F. Wirz, Swiss Institute of Allergy and Asthma Research, Davos, Switzerland
[^4]: ^‡^These authors have contributed equally to this work.
[^5]: Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology
| {
"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
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Autism (MIM 209850) is a neurodevelopmental disorder characterized by three core symptom domains: ritualistic-repetitive behaviors, impaired social interaction, and impaired communication and language \[[@CR39]\]. The etiology of autism appears to be primarily genetic, with karyotypically detectable chromosomal abnormalities accounting for \~5--7% of cases \[[@CR22], [@CR23], [@CR36], [@CR49]\]. Given that cytogenetic analysis only detects abnormalities greater than 3--5 megabases, however, it has been postulated that smaller anomalies will also cause some proportion of autism. The emergence of DNA microarray technology has enabled detection of intermediate sized genomic variation, which has since been found to be both common and relevant to human diseases, including autism \[[@CR14], [@CR28], [@CR34], [@CR41], [@CR42]\].
Recent studies have also suggested a significant role for CNVs, both *de novo* and inherited, in the etiology of autism \[[@CR3], [@CR28], [@CR30], [@CR44], [@CR50]\]. *De novo* copy number changes appear to be particularly enriched in sporadic (simplex) autism, though some recurrent CNVs that are primarily *de novo*, such as 16p11.2 deletions and duplications, are also occasionally inherited from an unaffected parent, suggesting incomplete penetrance for some autism-associated CNVs \[[@CR3], [@CR28], [@CR50]\]. Several of the recently highlighted mutations in autism, such as *SHANK3* \[[@CR7]\], *NRXN1* \[[@CR8], [@CR19]\] and contactin 4 \[[@CR38]\] have also been identified in parents, some of whom exhibit features of a broader autism phenotype.
When interrogating a sample for CNVs, it is important to note that enrichment for *de novo* mutations in singleton families is not specific to CNVs and that these families are likely to be enriched for other types of sporadic mutations as well. Chromosomal abnormalities (whether *de novo* or inherited) have historically been associated with syndromic developmental delay such as 15q11-q13 syndrome, or the 22q11 group of deletion disorders. Indeed, one of the first studies to identify copy number variants in mental retardation selected for mental retardation with syndromic presentation \[[@CR15]\].
In order to enrich our sample for potentially pathogenic chromosomal imbalances we applied a positive and negative criteria filter to the study sample. We hypothesized that children with autism who had additional phenotypic features or diagnoses suggestive of a developmental disturbance (positive filter) but who had normal cytogenetic testing (negative filter) would be more likely to harbor novel CNVs. We used this model for two reasons: 1) children with chromosomal abnormalities often have multiple or syndromic developmental anomalies and 2) we recently reported a pathogenic microdeletion in a child with autism and eye abnormalities, stimulating our interest in investigating other individuals with autism and additional developmental disturbances \[[@CR5]\]. While the AGRE sample has been heavily studied, it is important to note that many of the families included in this study have been flagged as "possible non-idiopathic autism" by AGRE due to their associated phenotypes, and therefore have been generally excluded from previous linkage, association, and CNV studies. In addition, we screened a sample of children with autism and no dysmorphology from the AGRE cohort, as well as a sample of unselected controls.
Methods {#Sec2}
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Patient ascertainment and sample {#Sec3}
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Families included in this study came from the Autism Genetic Research Exchange. AGRE houses DNA and phenotypic data on hundreds of simplex and multiplex families of children with autism and makes these materials available to researchers investigating autism. The diagnoses were confirmed in the affected individuals through the Autism Diagnostic Interview (ADI) and in some cases the Autism Diagnostic Observation Schedule---General \[[@CR6], [@CR25], [@CR26]\]. AGRE makes available to researchers data from family medical and psychological history, physical exams, cytogenetic data, and findings from neurological exams.
From the AGRE repository of 814 multiplex and simplex families, we identified a total of 17 children from 15 families with the following features: at least one child with autism who also had a less than common cranio-facial dysmorphology, limb or digit abnormality, or ocular abnormality that were suggestive of a possible chromosomal imbalance. These children were considered the "syndromic autism" sample and are referred to as such throughout this paper. Included among these syndromic features were microopthalmia, iris coloboma, trigonocephaly, Sotos syndrome, cleft lip and palate, alopecia areata, fused baby teeth, fused ribs, metatarsal full syndactyly of toes 2,3 and 4 and adducted thumbs (Table [1](#Tab1){ref-type="table"}). In families AU0590 and AU0720 both affected siblings also showed the same syndromic features. In the majority of cases, only one affected sibling was included in the initial syndromic autism group. In these cases, the affected sibling with syndromic features was analyzed first and additional family members (affected and unaffected) were studied as a follow-up to compelling results. It should be noted that the AGRE physical exam records indicate a number of common and mild birth defects or abnormalities (i.e. Strabismus, simple cutaneous syndactyly). These more common features were not included in our study. We also selected at random 19 subjects with autism and no associated syndromic features from the AGRE cohort as a comparison for the primary study sample. Table 1Children with autism and associated developmental abnormalitiesIndividual IDPhenotype of Affected ChildrenSibling StatusOcular Sub-PhenotypesAU033403Autism, micropthalmia, syndactyly, MRAffected MZ twin, no syndromic featuresAU1334302Broad Spectrum, micropthalmia, MRAffected brother, no syndromic featuresAU027505Autism, micropthalmiaAffected brother, no syndromic featuresAU1376302Autism, Iris colobomaAffected brother, no syndromic featuresCranio-facial Sub-PhenotypesAU021903Autism, Soto Syndrome, MRTwo affected brothers, no syndromic featuresAU005303Autism, alopecia areataAffected brother, no syndromic featuresAU008404Autism, trigonocephalyAffected brother, no syndromic featuresAU028905Autism, fused baby teethAffected brother, no syndromic featuresAU0875301Autism, fused baby teethAffected brother, no syndromic featuresAU1437302Autism, cleft lip and palateAffected brother, no syndromic featuresLimb/Skeletal Sub-PhenotypesAU067703Autism, adducted thumbs, seizures, MRAffected brother, no syndromic featuresAU059003 AU059004Both affected children diagnosed with autism, syndactyly of toes 2,3 and 4AU067208Autism, syndactyly of toes 2,3 and 4Affected brother, no syndromic featuresAU010903Autism Spectrum, syndactyly of toes 2,3 and 4Affected brother, fifth finger clinodactylyAU072004 AU072005Both affected children diagnosed with autism, fused ribs^\*^All samples listed have been tested by Karyotype and Fragile X testing
In addition, we screened an unselected control population of 716 individuals ascertained as part of a study of age-related eye disease at the University of Iowa under the direction of Dr. Edwin Stone. These individuals were analyzed with either the 250K *Nsp*I and 250K *Sty*I or the 5.0 Affymetrix SNP array platforms. The 250K array data was also analyzed with CNAG while the 5.0 array data was analyzed with dChip \[[@CR24]\].
Affymetrix GeneChip® human mapping 250K microarray {#Sec4}
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DNA from each individual was analyzed with one of the Affymetrix 250K GeneChip microarrays (either *Nsp*I or *Sty*I). It is well documented that SNP genotyping arrays can be used successfully for copy number detection \[[@CR11], [@CR31], [@CR48]\]. The DNA was hybridized to the array according to the manufacturer's instructions. Briefly, the assay uses 250ng of genomic DNA digested with *Nsp*I or *Sty*I restriction enzyme (New England Biolabs, Boston, MA), ligated to an adaptor using T4 DNA ligase (New England Biolabs), and amplified by PCR using Titanium Taq (Clonetech). PCR products were then purified from excess primer and salts by a DNA amplification cleanup kit (Clonetech) and a 90 µg aliquot was fragmented using DNaseI. An aliquot of the fragmented DNA was separated and visualized in a 3% agarose gel in 1× TBE buffer to ensure that the bulk of the product had been properly fragmented. The fragmented samples were end-labeled with biotin using terminal deoxynucleotidyl transferase before each sample was hybridized to the *Nsp*I or *Sty*I arrays for 16 h at 49°C. After hybridization, the arrays were washed and stained using an Affymetrix Fluidics Station 450. The most stringent wash was 0.6× SSPE, 0.01% Tween-20 at 45°C, and the samples were stained with R-phycoerythrin (Molecular Probes). Imaging of the microarrays was performed using a GCS3000 (Affymetrix) high-resolution scanner.
Detection of copy number variants {#Sec5}
---------------------------------
To detect changes in copy number we used a publicly available program, Copy Number Analyser for GeneChip® (CNAG) Ver. 2.0, developed at The University of Tokyo \[[@CR33]\]. CNAG was designed specifically for work with high-density oligonucleotide arrays and uses a Hidden Markov Model (HMM) to identify statistically significant deviations in signal intensity between SNPs represented on the array. Instead of using a standard reference panel, CNAG uses a panel of "best fit" references. The reference arrays were drawn from a pool of over 500 arrays of the same type using the above methodology and run on individuals with autism and their family members. Within this pool of reference arrays the signal intensity standard deviation values are ranked and the arrays with the best possible standard deviation values are used for the analysis of each new test array. Each array was referenced to at least 6 other arrays of unrelated individuals. To determine CNV size we relied primarily on boundaries defined by CNAG, though with some deletions (Supplementary Table [1](#AppESM1){ref-type="sec"}) we used loss of heterozygosity (LOH) as determined by genotype data to both validate the CNV and to better define the boundaries. However, it should be noted that LOH is limited by the presence of naturally occurring homozygous SNPs.
Identification and confirmation of CNVs of interest {#Sec6}
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All CNVs identified by CNAG in children with syndromic autism as well as autism alone are described in supplementary materials (control data not included). To identify a CNV of interest for follow-up validation and study, we first ruled out common CNVs by comparison with the Database of Genomic Variants CNV track on the May 2004 UCSC Genome Browser build \[[@CR4], [@CR13], [@CR14], [@CR29], [@CR35], [@CR42], [@CR43], [@CR46]\]. In addition, we used as a local comparison group the 716 eye disorder controls. CNVs with more than one record in DGV or any occurance in our own control sample were not considered for follow-up. The decision to set the follow-up threshold at one CNV occurrence in DGV control populations (instead of zero) was based on previous reports suggesting that autism susceptibility CNVs may be present at a very low frequency in a control population \[[@CR50]\]. Among the CNVs that met these criteria, we identified and validated by qPCR deletions and duplications that influenced brain expressed genes of potential relevance to autism. We then tested for segregation of the CNV with disease in all available members of the proband's family using microarrays or qPCR.
For quantitative real-time PCR (qPCR), we selected an amplicon within the center of the putative CNV. We used an assay targeted for *G6PD* on the X-chromosome as an internal control for gene dosage and an assay targeted for *GAPDH* to normalize signal between DNA samples. As the possibility for copy number variation exists for any given region of the genome, we relied on information obtained from our arrays as well as our gender prediction within the qPCR experiment to support the use of *GAPDH* as a normalization control for validation of copy number variants. The reactions were performed in mixtures containing 12.5 µl of 2x QuantiTect SYBR Green PCR Master Mix (QIAGEN), 12 µl genomic DNA (1ng/µl), 0.25 µl of each primer (10 pmol/µl) in a total volume of 25 µl. The PCR amplification and detections were carried out in an ABI 7700, each with an initial activation step for 15 min at 95ºC followed by 15s at 94ºC, 30s at 55ºC, and 30s at 72ºC for 42 cycles. Each experiment was performed two times with three replicates in each experiment. To exclude the presence of non-specific products, a melting curve analysis was performed. The threshold cycle value was calculated using the comparative *C*~T~ method. *C*~T~ was determined using the thermocycler software and an average of the three replicates was calculated. The fold change from normal samples was set at 1 and the ratio of the normalized fold change in autism compared to that of control samples was calculated.
Direct sequencing {#Sec7}
-----------------
Direct sequencing was used for identification of possible compound heterozygous mutations in one family in which a CNV affecting a single gene (*STXBP5*) was identified. Additionally, direct sequencing was used to rule out a *PAX6* mutation in a child with coloboma of the iris and autism. All *STXBP5* and *PAX6* exons were forward and reverse sequenced in families AU0677 and AU1376, respectively. The sequence data were analyzed using the Sequencher gene analysis computer program (Gene Codes, Ann Arbor, MI).
Results {#Sec8}
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In our sample of 17 children with syndromic autism, we detected an average of 3.1 CNVs per child including 31 deletions and 21 duplications (Table [2](#Tab2){ref-type="table"}), a rate similar to that from previous studies of autism using similar methodologies \[[@CR28]\]. Nine CNVs were novel with five being of particular interest (Table [3](#Tab3){ref-type="table"}). The pedigrees of the families highlighted in the results are shown in Fig. [1](#Fig1){ref-type="fig"}. In the sample of 19 children with non-syndromic autism we detected an average of 3.4 CNVs per child including 33 deletions and 32 duplications (Table [2](#Tab2){ref-type="table"}). Two CNVs were novel and one was of high interest (Table [3](#Tab3){ref-type="table"}). The two groups did not differ in their average number of CNVs per individual. A fisher's exact test of the number of novel CNVs out of the total number of CNVs in each group yields a one-tailed p-value of .04, suggesting that there are significantly more novel CNVs in the sample of children with syndromic autism. However, the number of individual *carriers* of novel CNVs did not differ significantly between the groups as there were two individuals in the syndromic autism group who each carried two novel CNVs (Table [4](#Tab4){ref-type="table"}). Table 2Size of CNVs in children with syndromic autism and children with autism only Syndromic AutismNon-Syndromic AutismDeletionsDuplicationsDeletionsDuplicationsCount31213332Mean Size (bp)285,162515,387506,285539,957Standard Deviation (bp)225,908515,710564,239596,016Median Size (bp)247,667279,487177,559302,457Minimum Size (bp)8,01938,2898,82752,037Maximum Size (bp)1,068,5991,686,1251,896,8641,896,864The descriptive statistics listed include children with syndromic autism and non-syndromic autism that were analyzed with either the *Nsp*I or *Sty*I 250K SNP microarray. These statistics do not include siblings or parentsTable 3Novel CNVs of high interest identified in children with autismAGRE Family IDAGRE Individual IDAdditional PhenotypeDel/DupSize (kb)Chromosome LocationGenes in RegionPresent in Affected SiblingsPresent in Unaffected SiblingsParent of OriginConfirmation TypeAU005303Alopecia AreataDup336 1373p26.2, 3p26.1LRRN1 (NLRR1) Upstream of GRM7YesN/APaternalqPCRAU067703Adducted ThumbsDel2616q24.3STXBP5NoNoMaternalqPCR/LOHAU010904ClinodactylyDup1664q34.3WDR17, SPATA4, ASB5YesNoPaternalqPCRAU1334302MicrophthalmiaDel3171q24.2XCL1, XCL2, DPTYesNoPaternalqPCR/LOHAU038303noneDel1777q35CNTNAP2NoN/APaternalqPCRIndividuals listed (unless otherwise noted) were diagnosed with primary autism and have secondary developmental abnormalities according to physical exams and medical records collected by AGRE. Further information about these families is available in the supplementary materials of this paperFig. 1Pedigrees of families with syndromic autism and copy number variants of high interest. Asterisks indicate parent of CNV origin. Hatched individuals have been diagnosed with an autism spectrum disorder while those in solid shading have been diagnosed with autism. **a** Deletion on chromosome 6q24 was identified in AU067703 and was inherited maternally from AU067701. AU067703 is diagnosed with autism, seizures, mental retardation and adducted thumbs while AU067705 is diagnosed with autism only. Their mother has been diagnosed with bipolar disorder. **b** Deletion on chromosome 1q24.2 was identified in AU1334302 and AU1334303 and was transmitted by AU1334201. Additionally AU1334303 carries another duplication on chromosome 22q11.21 that was not paternally inherited. DNA from AU1334202 was unavailable for testing. **c** A paternally inherited duplication on chromosome 4q34.2 was identified in individuals AU010903 and AU010904. **d** Two paternally inherited duplications on chromosome 3p26.2 and 3p26.1 were present in individuals AU005303 and AU005304. Additionally AU005304 carried a small, apparently *de novo* duplication on chromosome 3p25.1Table 4Statistical Comparisons of CNVs between syndromic autism and non-syndromic autism groupsComparison GroupsNumber in each group out of totalTest of Significance Syndronic AutismAutism Only Novel CNV *carriers* with syndromic features vs. Fisher's ExactNovel CNV *carriers* without syndromic features5/172/19*p* = .15Novel CNVs in syndromic autism group vs. Fisher's ExactNovel CNVs in "autism only" group7/522/65*p* = .04Average number of CNVs in syndromic autism group vs. Student's T-TestAverage number of CNVs in autism only group3.12 (± 1.36)3.42 (± 2.59)*p* = .67
CNVs of interest identified in children with syndromic autism {#Sec9}
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Pedigrees for families with CNVs of interest are provided in Fig. [1](#Fig1){ref-type="fig"} and CNV diagrams are provided in Fig. [2](#Fig2){ref-type="fig"}. We identified a 260 kb deletion on chromosome 6q24 in a family with two affected boys and one unaffected girl. The oldest affected boy, who carried the deletion, was diagnosed with autism, mental retardation and seizures. The deletion was also detected in the mother who has severe bipolar disorder with episodes of psychosis and suicidal ideation. This deletion contained coding sequence from only one gene on chromosome 6q24, syntaxin binding protein 5 (*STXBP5*). This deletion was not present in the mildly affected brother or unaffected sister. *STXPB5* was also sequenced in this family to rule out the possibility of a compound heterozygous mutation, with no new SNPs or mutations being identified. We also detected a 317 kb deletion on chromosome 1q24.2 in two siblings with autism spectrum disorders, one of whom had an additional phenotype of microphthalmia. The deletion was transmitted from the father and was not present in an unaffected paternal half-brother. The father transmitting the deletion has been diagnosed with depression, anxiety disorder, and ADHD. This deletion is novel and contains three genes, *dermatopontin* (*DPT*), *chemokine (C motif) ligand 1, and chemokine (C motif) ligand 2*. A 166 kb deletion on chromosome 4q34.2 was identified in two of three trizygotic triplets. The two children carrying the 4q34.2 deletion have autism and malformations of the limbs. One twin has finger clinodactyly on both hands while the other has metatarsal syndactyly of toes 2, 3 and 4 on both feet. This deletion was paternally inherited and not present in the third unaffected triplet. The father reported learning disabilities as a child and his sister has mild mental retardation. This deletion includes three known genes, *WD repeat domain 17 (WDR17)*, *spermatogenesis associated 4 (SPATA4)*, and *ankyrin repeat and SOX box containing protein 5 (ABS5)*. Deletions in this chromosome region have previously been associated a number of features including mild mental retardation, velo-cardio-facial (VCF) syndrome-like features, and finger clinodactyly \[[@CR16], [@CR45], [@CR47]\]. While there are no clear features of VCF in this family, the affected children do display the 4q34.2 characteristic digit abnormalities. In family AU0053 we identified two duplications in close proximity to each other on chromosome 3p26.2-p26.1 in a family with two affected brothers. This family has been recently published in Christian et al. \[[@CR3]\] and here we add to their findings and further discuss the candidate interval. The father, who transmitted the duplications, endorsed symptoms of obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) and is a self-described loner. The duplication that brought this family to our attention is located on 3p26.1, is 137 kb in length, contains no genes, and is entirely novel. The second duplication on this chromosome lies on 3p26.2 and is approximately 2 Mb telomeric to the first duplication. CNVs overlapping with this duplication on 3p26.2 have been identified in three unselected controls \[[@CR18], [@CR34]\]. The duplication on 3p26.2 is 336 kb in length and contains a portion of only one validated gene, *leucine rich repeat neuronal 1* (*LRRN1*). It is worth noting that according to the microarray analysis, the breakpoints for this CNV, which are novel to DGV, fall within the *LRRN1* gene itself suggesting possible disruption of the transcriptional unit. Finally, a 177 kb deletion on chromosome 7q35 was detected in one male child with autism and no related dysmorphology, but was not identified in his younger affected brother. This deletion encompasses a portion of the first intron of *CNTNAP2* and was previously identified in \[[@CR1]\]. The deletion was paternally inherited and while the father did not report any learning disorders he did report a lack of empathy and considers himself "eccentric and a loner". Fig. 2UCSC Genome Browser May 2004 screen captures of novel structural variants \[[@CR17]\] in individuals with syndromic autism (<http://genome.ucsc.edu>). **a** Deletion identified in two affected siblings (one with microopthalmia), AU1334303 and AU1334302. The deleted region on chromosome 1q24.2 includes the genes *XCL2* and *DPT*. **b** Deletion identified in one affected proband (with microopthalmia) AU027505. As no genes lie within the deletion region, this CNV did not meet our criteria for continued study and additional family members were not screened. The deleted region on chromosome 2p22.1 is non-genic, however, it is notable that region appears to contain conserved elements and is within close proximity of the gene *SLC8A1*. **c** Duplications were identified in two affected siblings (one with alopecia), AU005303 and AU005304. The duplicated regions on chromosome 3p26.2 and 3p26.1 overlap with *LRRN1* and lie close to *CNTN4* and *GRM7*. **d** Duplication identified affected siblings (one with syndactyly, the other with clinodactyly) AU010903 and AU010904. The duplicated region on chromosome 4q34.2 encompasses two genes, *WDR17* and *ABS5*. **e** Deletion identified in one affected sibling (with adducted thumbs), AU067703. The deleted region on chromosome 6q24.3 includes *STXBP5*. **f** Second duplication identified in individual AU1334302. The duplicated region on chromosome 22q11.21 includes a number of genes and overlapping regions of common variation, however, the breakpoints identified in AU1334302 are unique to the Database of Genomic Variants and include genes such as *SNAP29* that do not show evidence of common variation
Discussion {#Sec10}
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This study was designed to identify CNVs that may play a role in the etiology of autism. We applied positive and negative filter criteria to AGRE families in order to enrich our sample for such CNVs. We theorized that children with autism and additional developmental anomalies would be more likely than children with idiopathic autism to harbor pathogenic microdeletions and duplications. We did not find significant enrichment of *de novo* CNVs, however, we did identify significantly more rare inherited CNVs in our sample of syndromic autism compared to a sample of non-syndromic autism. It is worth mentioning that almost all of the rare, inherited, CNVs were transmitted from parents reporting a range of learning disabilities and psychiatric disturbance. We highlight novel deletions and duplications on chromosomes 1q24.2, 3p26.2, 4q34.2, 6q24.3, and 7q35. It is important to note that with increased array density, it is possible that some of the highlighted novel CNVs will be identified in control populations. The "novel" status of CNVs is rather a moving target and even during the preparation of this manuscript, CNV status had to be changed. While this data is preliminary and should be interpreted with caution, several of the deletions and duplications reported here include new and interesting biological candidate genes for autism such as syntaxin binding protein 5 (*STXBP5* also known as *tomosyn*), and leucine rich repeat neuronal 1 (*LRRN1* also known as *NLRR1*).
Perhaps the most compelling of these is *STXBP5*, which plays a key role in neuronal guidance and in regulation of synaptic transmission at the presynaptic cleft \[[@CR51]\]. The protein forms a stable t-SNARE complex with SNAP-25 and a ROCK/Rho phosphorylated form of synatxin-1 (isoforms A and B) at the presynaptic nerve terminal. Its interaction with SNAP-25 and syntaxin-1 ultimately blocks synaptobrevin (part of the v-SNARE machinery) from joining the SNARE complex, resulting in inhibition of vesicle exocytosis \[[@CR10], [@CR12], [@CR51]\]. This synaptic vesicle inhibition is particularly important in early development during extension and retraction of neurites. Phoshorylated syntaxin-1 and STXBP5 have been shown to co-localize to the palm of the growing neurite, thereby inhibiting synaptic formation along the palm and encouraging synaptic vesicle release toward the growth cone of the emerging dendrite or axon \[[@CR40], [@CR51]\]. Ours is the first mutation reported of *STXBP5*, present in a mother with severe bipolar disorder and her son with autism, mental retardation and seizures. A recent paper has suggested syntaxin-1A as a possible autism candidate based on SNP genotype association with autism in a set of AGRE trios as well as mRNA expression data that suggests syntaxin-1A is expressed at a higher level in children with high functioning autism than in age and gender matched controls \[[@CR32]\]. It is also important to note that one child with autism spectrum disorder in this family did not carry the *STXBP5* deletion. This phenomenon has been noted in previous studies of autism susceptibility genes \[[@CR28], [@CR50]\]. It is possible that the *STXBP5* deletion is not causative but influences autism severity, that there is a secondary cause for autism in the sibling, or that *STXBP5* has an additional maternal mRNA contribution during fetal development that resulted in a more severe form of autism in the sibling who was also carrying the deletion.
Duplications flanking the neuronal cell adhesion molecule *LRRN1* were detected in a second family. One of the duplications has unique breakpoints disrupting the only intron of *LRRN1*. It is unclear whether these duplications are functionally connected or arose independently, but the possibility remains that they may act in concert in this family. Leucine rich repeat neuronal genes are heavily expressed during embryonic development of the cortex and are thought to be involved in neuronal outgrowth. However, their role in synaptic cell adhesion is still unclear \[[@CR20], [@CR21]\]. Mutations in *LRRN1* have not been associated with any other disorder, though mutations in the LRRN gene family have been associated with other psychiatric disorders such as Parkinson's disease and schizophrenia \[[@CR9], [@CR27]\]. Additionally, larger deletions and duplications of chromosome 3p26 have been found in children with Prader-Willi syndrome, mental retardation, and social cognition deficits \[[@CR2], [@CR37]\]. Recently, deletions in another cell adhesion molecule, *contactin 4*, were identified in three children with autism. \[[@CR3]\].
One limitation of our study is the possibility that our findings may be associated with the additional phenotype and not autism in these individuals. We believe this to be unlikely, however, as the chromosomal abnormalities that we identified disrupt compelling biological candidates that are heavily expressed in the brain and known to function at the neuronal synapse. We believe it is more likely that these CNVs have a pleiotropic effect based on gene dosage or position of the chromosomal disruption. In addition we were unable to entirely rule out cell line effects; however, as the CNVs highlighted in the results were all inherited, they are unlikely to represent such artifacts.
Lastly, our data suggest that rare microdeletions and duplications may have a statistically higher occurrence in children with autism and additional developmental anomalies and that these children ought not be excluded from studies of copy number variants in autism. Here we present evidence from such a sample, implicating new genes such as *STXBP5* and *LRRN-1* that may play a role in the development of autism. While the results presented here require further investigation in larger samples, it may also be prudent to consider clinical high density microarray testing for children who present with autism and related developmental disturbances.
Electronic supplementary material {#AppESM1}
---------------------------------
Below is the link to the electronic supplementary material.
######
Copy number variants identified in children with autism and developmental abnormalities. This table includes all CNVs identified by CNAG in the children with syndromic autism included in this study. (DOC 161 kb)
######
Copy number variants identified in children with autism and no developmental abnormalities. This table includes all CNVs identified by CNAG in the children with autism and no additional dysmorphology or developmental abnormalities included in this study. Additionally it includes all CNVs identified in affected siblings that were also analyzed on microarray. This table does not include CNV data from parents of affected children. (DOC 213 kb)
We acknowledge support from the Autism Genetic Resource Exchange (AGRE) and Autism Speaks. We gratefully acknowledge the resources provided by the AGRE consortium\* and the participating AGRE families. The Autism Genetic Resource Exchange (AGRE) is a program of Autism Speaks and is supported, in part, by grant 1U24MH081810 from the National Institute of Mental Health to Clara M. Lajonchere (PI). Additionally we would like to thank Dr. Vlad Kustanovitch (AGRE) for his help in providing clinical clarification of families. We also wish to thank Dr. Edwin Stone (University of Iowa) for his assistance in providing control populations for CNV analysis. This work was supported by the Hilibrand Foundation and by NIH Predoctoral Training Grant (2T32GM008629) awarded to Lea Davis.
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Historically periodontal therapy has never been on the priority list of any patient. The reasons could be many.... Lack of knowledge, pain, chronicity of the disease, disease occurrence in older age group, when treatment preferences are for other systemic diseases, economics could be few of them. Other factors which play a role in non-prioritizing periodontal treatment could be myths (may be because of lack of knowledge), hearsay (with quite a bit of reality) about post-treatment effects of sensitivity, increase in mobility, recession, spacing becoming visible and ultimately unaesthetic appearance.
Another major factor which plays a role in patient not giving adequate importance to periodontal therapy requirements is the inability of periodontist to demonstrate in most of the conditions what they have achieved after treatment. For example, in endodontics, patients get relieved of pain after the therapy or gets a new crown which gives him an esthetic look and functional tooth that is so motivating for him to get the treatment. Similarly, in orthodontics, a beautiful smile even after a difficult, costly prolonged treatment of more than one year, is the incentive for which the patient is motivated to take the treatment.
We have nothing much to show as post-operative outcomes in most of the procedures except for few esthetic surgeries. Rather the patients have lot of complaints in this period. And the irony is that the procedures in which we can show results are barely asked for, by patients in Indian clinical practice scenario. The worrying aspect of these esthetic procedures are their unpredictable results. We deal with periodontal diseases in surrogate endpoints like pocket depth, clinical attachment loss, furcation involvement etc., rather than using true endpoints.
We diagnose the periodontal diseases with parameters which are surrogate, we evaluate our treatment success with same surrogate endpoints and in long term we use the same surrogate parameters to determine whether the patient has maintained the oral hygiene or not. The worst part is that we use the same surrogate endpoints even to explain to the patients what has happened at the time of diagnosis, what we plan to do and what can be expected after treatment.
What does patient understand by pocket depth or clinical attachment loss (CAL) or furcation involvement or gingival colour changes at the time of diagnosis. We try to tell them about bone loss which they cannot understand as it is not visible and they are at their imaginative best. How much a radiograph can explain the amount of bone loss to a layman is still a question unanswered. Even after the therapy is over, we evaluate the above mentioned parameters and are very elated if they have improved.
My question is, how does it matter to the patient if pocket depth is reduced or there is a CAL gain or bone gain or improvement in furcation status?
How many patients have we encountered in our practice/departments who have primarily come for the treatment of increased pocket depth or loss of attachment or furcation involvement and want them to be treated. The answer would be none till date.
Patients come with complaint of dirty teeth, bleeding, mobility, recession, halitosis, spacing, inability to masticate and want these to be treated and eventually want to save the tooth. The most important question that is asked by a periodontal patient is "how long will these teeth last if I take the treatment". Ultimately they want all these to be treated because they want the teeth to survive functionally and esthetically for a long time.
So, the fundamental aim of periodontal therapy is to save teeth for the patient to use it essentially for longest period of time.
I would put across a hypothetical situation; how we, while using surrogate parameters, tend to forget the actual goals we should look at. An example of case of treated periodontitis where periodontally excellent results have been obtained. There is normal pocket depth, no bleeding on probing or any other signs of periodontal disease on reduced periodontium. The reduced periodontium has resulted in post-operative recession, which is a common after-effect of periodontal surgeries. After some time, the exposed root surface encounters root caries and pulp involvement. The tooth had to extracted. The reason may seem to be caries but why did caries occur.... because of exposed root surface. Did the patient get periodontal treatment for reduction of pocket, recession, CAL gain or to lose the teeth because of caries? The real question is "Did we succeed in achieving our ultimate goal of periodontal therapy"? The answer is a big No. We might have treated periodontal disease effectively in this case, got our surrogate parameters in right proportions post-treatment but loss of tooth (even because of caries) has resulted in overall failure as long term survival of tooth was not possible. The patient was ultimately not benefited from the treatment as loss of tooth resulted in loss of function and esthetics.
There is no doubt that the surrogate parameters have been proved with lot of studies that they indirectly infer about the long term survival of teeth but in a practical sense they are of no use to the patient.
Patients will understand the importance of periodontal therapy if we could explain in quantitative terms how much bleeding has reduced, how much is the improvement in mobility, improvement in halitosis, recession, spacing etc., And ultimately how long the patient, who has undergone periodontal therapy, can effectively use the teeth for function.
Till we do not explain the patient our treatment modalities in the terms the patient can understand (patient centric outcomes) and can approximately tell the patient how long is he going to benefit with the therapy (the life span of the tooth) till then it will always be a very hard task to motivate the patient to undertake treatment for periodontitis.
We should look at using patient centric outcomes to diagnose, evaluate and explain to the patient his periodontal condition for him to be motivated and remain interested in periodontal therapy. The use of patient centric outcomes provides patient centric tangible benefits and not clinician centric results. The periodontal therapy becomes more meaningful and of relevance to the patient.
At this moment, we may not know how to effectively use these patient centric parameters for tangible patient benefits, but we will never have an answer to this question till we start working on it and try to find answers. We need to get over with our fascination with surrogate parameters.
As someone rightly said "*Problems are mere absence of ideas*."
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Nutrition status assessment is important in the detection of protein energy malnutrition (PEM), dietary requirements, and the development of the alternative nutritional therapies in chronic kidney disease patients.\[[@ref1]\] Assessment of nutrition status has assumed greater importance because of the association of malnutrition with increasing morbidity and mortality.\[[@ref2][@ref3][@ref4]\] Malnutrition is a strong predictor of mortality and morbidity in maintenance peritoneal dialysis (PD) patients.\[[@ref5][@ref6][@ref7]\] Studies have shown that prevalence of mild to moderate malnutrition varies from 26% to 68% and severe malnutrition from 2% to 8% in PD patients in different parts of the world with high prevalence in developing countries than that of the developed countries.\[[@ref5][@ref6][@ref7][@ref8]\] We have previously shown that the prevalence of malnutrition is high at initiation of PD, and periodic assessment of nutrition status and dietary counseling help in improving the nutrition status of PD patients.\[[@ref8]\]
There are many tools for the assessment of nutrition status. Studies have consistently revealed the inadequacy of any single assessment method or tool to assess the nutrition status of patients. Although subjective global assessment (SGA) is a valid estimate of nutrition status of PD patients;\[[@ref1]\] individual measurements of parameters often have limited its value in accurately determining the nutrition risk of these patients.\[[@ref9]\] Nutrition risk index (NRI), developed by veterans affairs total parenteral nutrition cooperative study group was found to be sensitive, specific and positive predictor for identifying patients with risk of complications after surgery.\[[@ref10]\] We have used NRI for estimating nutrition status of PD patients in one of our studies.\[[@ref11]\] Recently, Szeto *et al*.,\[[@ref12]\] have used a modified NRI formula for nutrition assessment of geriatric PD patients. However, original NRI formula has never been used and validated for the assessment of nutrition status in large cohort of nongeriatric PD patients. We undertook this study to compare NRI and SGA in large cohort of PD patients, and to determine the sensitivity, specificity and predictive values of NRI compared to SGA and validate its utility as screening tool for nutrition status.
Patients and Methods {#sec1-2}
====================
During the study period (January 2009 to July 2012), a total of 323 end-stage renal disease (ESRD) patients were started on PD at our institute. Patients \<12 years (*n* = 8) and \>65 years (*n* = 22) in age, those who did not continue PD for 3 months (*n* = 4), and those who did not consent for the inclusion in the study (*n* = 6) were excluded. Thus, 283 PD patients remained for analysis. All patients were subjected to detailed history and clinical examination. Nutritional indices were assessed by anthropometry, 72-h dietary diary, body mass index, serum albumin, NRI and SGA. Anthropometric measures and nutrition indices were assessed after 1-month of start of PD.
Assessment of subjective global assessment {#sec2-1}
------------------------------------------
All patients were subjected to SGA.\[[@ref8]\] We used a 7-point Likert-type scale of four items: weight loss, anorexia, subcutaneous fat and muscle mass. Each item was given scores to produce a global assessment score. Scores of 1--2 represented severe malnutrition; 3--5 mild-moderate malnutrition; and 6--7, normal nutrition. Patients were subdivided into three groups based on SGA score: normal nutrition, mild-moderate malnutrition, and severe malnutrition as previously reported in CANUSA PD Study Group.\[[@ref7]\]
Assessment of nutrition risk index {#sec2-2}
----------------------------------
Nutrition risk index was calculated as follows: NRI = (1.519 × serum albumin (g/L) +41.7× (present weight/usual weight). The patients with NRI score of \>100 was considered in no risk group, 97.5--100 mild risk, 83.5--97.5 moderate risk, and \< 83.5 has severe risk groups.\[[@ref10]\] The usual body weight was defined as stable body weight for last 6 months, and the patient\'s weight was obtained through history or previous measurements, considered to be stable over time.
The sensitivity, specificity, and predictive values of NRI considering SGA as the gold standard were calculated. The patient survival and number of hospitalization of the patients were compared in three risk groups based on NRI and SGA. The receiver operating characteristic (ROC) curves were generated for NRI for our patient population with the use of the SGA as the reference standard. The area under the ROC curve indicated the probability of discriminating a nutritional risk. The cutoff risk point of nutrition for the reference standard was then defined from the highest sensitivity (1-specificity) value in the ROC curve.
The patients were followed until the death of the patient, technique failure, and shift on hemodialysis or, the end of the study period. The study was approved from the ethics committee of Institute.
Statistical analysis {#sec2-3}
--------------------
Data are expressed as mean ± standard deviation. Chi-square test was used to compare the proportion between two groups. Student\'s test was used to compare the means between different groups. A contingency table was used to determine the sensitivity, specificity, predictive values and accuracy of the NRI as a malnutrition screening tool for PD patients compared to SGA. Kaplan Meier survival analysis was used to analyze the survival of patients in three risk groups based on SGA and NRI, and log rank test was used to test the significance. Simple correlations are reported as the Pearson correlations. Statistical significance was reported at *P* \< 0.05. Data were analyzed using SPSS.11 statistical software (SPSS Inc., Chicago, Illinois, USA).
Results {#sec1-3}
=======
Demographic profile and prevalence of malnutrition {#sec2-4}
--------------------------------------------------
The demographic profile of the patients at the initiation of dialysis is given in [Table 1](#T1){ref-type="table"}. The mean age of patients was 50.02 ± 13.76 years. The patients were followed up to 32.44 ± 8.9 (range 3--45) patient-months. Of the 283 patients, 150 patients were diabetics and 204 male. On evaluation of peritoneal equilibration tests (PET), 11 (3.9%) had low, 83 (29.3%) of patients low average, 147 (51.9%) high average and 42 (14.8%) patients high (H) transport characteristics. Out of 283 PD patients, 132 (46.6%) patients were vegetarians, 67 (23.7%) were ovo-vegetarians and 84 (29.7%) were nonvegetarians. The mean normalized protein catabolic rate at time of PET was 0.92 ± 0.28 (range 0.41--1.71). The prevalence of malnutrition based on SGA and NRI has been shown in [Table 2](#T2){ref-type="table"}. Based on SGA, 71/283 (25.08%) had normal nutritional status, 192/283 (67.84%) had mild-moderate malnutrition, and 20/283 (7.07%) had severe malnutrition. On classifying the patients based on NRI, 38/283 (13.43%) patients had normal nutrition status, 193/283 (68.20%) had mild-moderate malnutrition, and 52/283 (18.37%) had severe malnutrition.
######
Basic demographic profile of patients at initiation of PD
######
Prevalence of malnutrition based on SGA and NRI
The significantly greater numbers of patients with diabetics were detected malnourished based on NRI compared to patients detected malnourished based on SGA. Of the 150 diabetic patients, 122 patients were malnourished and 28 had normal nutrition status based on SGA (*P* = 0.006) while 136 patients were malnourished and only 14 had normal nutrition status based on NRI (*P* = 0.024).
Nutrient Intake in different nutrition status groups based on subjective global assessment and nutrition risk index {#sec2-5}
-------------------------------------------------------------------------------------------------------------------
The mean daily calorie (Kcal/kg/d) intake in patients with normal nutrition status versus mild-moderate malnutrition versus severe malnutrition was 24.7 ± 5.7 versus 17.9 ± 5.4 versus 13.04 ± 4.18, *P* \< 0.001, respectively; and the protein intake (g/Kg/day) was 1.0 ± 0.64 versus 0.8 ± 0.2 versus 0.6 ± 0.6 *P* \< 0.001, respectively based on SGA. Similarly based on NRI, the mean calorie intake (Kcal/kg/d) in patients with normal nutrition status versus mild-moderate malnutrition versus severe malnutrition was 22.8 ± 5.7 versus 19.9 ± 6.5 versus 14.8 ± 4.4, respectively; and the protein intake (g/kg/d) was 0.90 ± 0.3 versus 0.8 ± 0.3 versus 0.6 ± 0.2, respectively. The mean serum albumin level (g/dl) was significantly lower in malnourished patients compared to patients with normal nutrition based on SGA (3.2 ± 0.5 vs. 3.5 ± 0.5 *P* \< 0.001) and NRI (3.1 ± 0.3 vs. 4.0 ± 0.3 *P* \< 0.001) as well.
Risk of hospitalization based on subjective global assessment and nutrition risk index {#sec2-6}
--------------------------------------------------------------------------------------
Of the 212 malnourished patients based on SGA, 138 (65.1%) required hospitalization during follow-up and 74 (34.9%) patients did not require any hospitalization. Of the 138 patients who required hospitalization, 73 (34.4%) patients had multiple hospitalization two or, more times while only 24 (33.8%) patients with normal nutrition based on SGA required hospitalization and 47 (66.2%) did not require any hospitalization. Of the 24 patients who required hospitalization, 13 had two or more than 2 hospitalizations. The relative risk of hospitalization was higher (Relative risk \[RR\] =3.65; 95% confidence interval \[CI\] =2.07--6.43; *P* \< 0.001) in malnourished patients compared to patients with normal nutrition status based on SGA.
However, of the 245 malnourished patients based on NRI, 145 (59.2%) required hospitalization and 100 (40.8%) did not required hospitalization while 17 (44.7%) patients with normal nutrition status (*n* = 37) also required hospitalization and 21 (55.3%) did not required hospitalization. The number of patients who required hospitalization was numerically high in malnourished patients but statistically not significant. The relative risk of hospitalization based on NRI (RR = 1.8, 95% CI = 0.9--3.6, *P* = 0.068).
Survival of the patients based on nutrition status on subjective global assessment and nutrition risk index {#sec2-7}
-----------------------------------------------------------------------------------------------------------
### Based on subjective global assessment {#sec3-1}
The mean survival of the patients with normal nutritional status (33.2 patient-months) was superior to patients with mild-moderate malnutrition (29.3 patient-months) and severe malnutrition (17.8 patient months) based on SGA, *P* = 0.001. The estimated 1, 2 and 3 years survival rate in these groups was 95%, 77.8%, and 50.1% respectively in patients with normal nutrition status, 89%, 64.9%and 50% in mild--moderate malnourished patients, and 58%, 52.4% and 0% in patients with severe malnutrition. None of the patients with severe malnutrition survived for 3 years based on SGA \[[Figure 1](#F1){ref-type="fig"}\].
{#F1}
### Based on nutrition risk index {#sec3-2}
The mean survival of the patients with normal nutritional status (32 patient-months) was superior to patients with mild-moderate malnutrition (30 patient-months) and severe malnutrition (24.4 patient-months) *P* = 0.024 based on NRI. The estimated 1, 2 and 3 years survival rate in these groups was 97.1%, 71.6% and 62.7% respectively in patients with normal nutrition status, 89.6%, 71.2%and 50% respectively in mild--moderate malnourished patients and 77.4%, 49.3% and 37% respectively in patients with severe malnutrition \[[Figure 2](#F2){ref-type="fig"}\].
{#F2}
### Validity of nutrition risk index {#sec3-3}
The ability of NRI as a nutrition screening tool to predict nutrition status is shown in [Table 3](#T3){ref-type="table"}. Totally, 23/283 (8.1%) were correctly classified as being normal/well nourished by NRI screening tool (true negative) and 197/283 (69.6%) of patients were correctly classified as being malnourished (true positive), 15/283 (5.3%) false negative, 48/283 (16.96%) patients were misclassified as being malnourished (false Positive). NRI has a high sensitivity of 92.9% and a low specificity of 32.39%. Positive predictive value of NRI is 80.41%, and negative predictive value (NPV) is 60.53%. Accuracy of the test is 78%. SGA is positively correlated with NRI (*r* = 0.451, *P* = 0.01). The ROC curve (sensitivity vs. 1-specificity) of NRI was 0.63 \[[Figure 3](#F3){ref-type="fig"}\].
######
Validity of NRI as a screening tool for malnutrition in PD patients as compared to SGA
![The receiver operating characteristic (ROC) curve of nutrition risk index (NRI) compared to subjective global assessment (The ROC curve \[sensitivity vs. 1-specificity\] of NRI 1 s 0.63)](IJN-26-27-g006){#F3}
Discussion {#sec1-4}
==========
Bundle of tests varying from simple anthropometry, biochemical parameters, SGA to dual-energy x-ray absorptiometry (DEXA) bio-impedance analysis, and infrared technique based method are used to determine the nutrition status of dialysis patients. SGA has been validated for estimating nutrition status in PD patients and stood the test of time with certain limitations of subjectivity in the test. NRI appears to be simple scoring system for screening malnourished PD patients which can be applied easily and rapidly in a large population with more objectivity compared to SGA.
In this study on the validity of NRI as nutrition status screening tool, we observed that NRI has high sensitivity but low specificity with consideration of SGA as the gold standard for the assessment of nutrition status in PD patients. The NRI has been used to define nutritional risk in a number of recent studies where the effects of under nutrition\[[@ref13]\] or nutritional intervention were investigated.\[[@ref14][@ref15]\] The NRI relies on serum albumin concentration and percentage usual weight. The formulae based calculation of NRI provides some objectivity in the assessment of nutrition status. The formula for NRI also contains serum albumin level which is considered to be one of the important biochemical parameters to assess the nutrition status of PD patients.
Similar to SGA, NRI has been used as a nutrition status tool for the surgical and cancer patients, and it was found to be a sensitive and positive predictor of malnutrition in these patients. However, it has never been used for the ESRD patients on PD. Szeto *et al*.,\[[@ref12]\] have used a modified NRI formulae for the assessment of nutrition status in elderly PD patients. This is the first report on validating the original NRI formula as a malnutrition screening tool compared to SGA in ESRD patients on PD. We observed that NRI underestimated the patients with normal nutrition status (13.43% vs. 25.08%) and over-estimated the severe grades of malnutrition compared to SGA (18.37% vs. 7.07%). The percentage of patients with mild to moderate degree of malnutrition was almost similar by both methods NRI (68.02%) and SGA (67.84%). NRI has the sensitivity of 92.9% and specificity of 32.39%. The ROC curves generated for NRI for our patient population with the use of the SGA as the reference standard, (sensitivity vs. 1-specificity) of NRI was 0.63.
Szeto *et al*., studied a modified NRI formula, Geriatric Nutrition Risk Index (GNRI) in 314 adult PD\[[@ref12]\] patients against their comprehensive malnutrition-inflammation scores (MIS) and 7-point SGA scores. However, they have not found GNRI as a sensitive tool for screening malnutrition and detecting the changes in nutrition status in PD patients. The reason could be the modification from the original formula of NRI. The GNRI was developed by modifying the nutritional risk index for elderly patients. This index is calculated based on serum albumin and body weight, using the following equation: GNRI = (1:4893 × serum albumin \[g/dl/d\]) + (41:7× \[body weight/ideal body weight\]). Body-weight to ideal-body-weight ratio was set to 1 when a patient\'s body weight exceeded the ideal body weight. GNRI was originally meant for the identification of malnutrition in a geriatric population. However, their results remained similar when only patients aged over 65 years were analyzed. However, Yamada *et al*.\[[@ref16]\] found that the GNRI was the simplest and most accurate index for identifying patients on hemodialysis at nutritional risk compared to MIS.
Nutrition risk index predicted hospitalization and survival of PD patients similar to SGA in our study. The NRI has been used to define nutritional risk in a number of recent studies where the effects of undernutrition\[[@ref13]\] or nutritional intervention were investigated.\[[@ref14][@ref15]\] Clugston *et al*.,\[[@ref17]\] found NRI as simple to use and defines a high-risk sub-group of patients with obstructive jaundice. NRI \< 83.5 was significantly associated with mortality and longer duration of hospital admission but not complication rate.
As a screening tool, a drawback of the NRI is the reliance on measurements of current and previous body weight, limiting its usefulness where there is a relative increase in body weight due to increase in total body water. The NRI is open to further criticism as a nutrition screening tool for including serum albumin in its formula.\[[@ref18]\] Despite these problems and limitations, NRI on admission was shown to predict postoperative complications in surgical patients.\[[@ref10]\] Assessing specificity is important in preventing well-nourished patients from being incorrectly identified as malnourished.\[[@ref19]\] Finding malnourished patients in need of nutritional intervention will definitely improve the outcome. High sensitivity is the desirable characteristic, and there is no need for very high specificity to screen out malnourished patients.
Conclusion {#sec1-5}
==========
Nutrition risk index can be used as screening tool for assessment of nutrition status with high sensitivity. However, it cannot be used as a diagnostic tool for assessment of nutritional status in PD patients because of its low specificity and NPV. Further research and multicenter studies are needed using NRI against a broader array of objective and subjective nutritional parameters to confirm its validity as a screening tool for malnutrition in PD patients.
We would like to thank Mr. Santosh Kumar Verma for the secretarial assistance and technicians of Renal Laboratory for the support.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
| {
"pile_set_name": "PubMed Central"
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The North Europe brachytherapy user meeting organized by Elekta was held on October 10-11, 2019 in London, UK. More than 80 radiation and clinical oncologists, medical physicists, and radiotherapy technologists from the UK, Ireland, The Netherlands, Denmark, Sweden, Norway, and Germany attended this meeting. Nineteen brachytherapy users from the UK, Ireland and Germany shared their experience in gynecological, prostate, skin, liver, and esophageal brachytherapy. The user meeting included a practical workshop, where users were given the opportunity to obtain hands-on experience with both treatment planning software and applicators.
Gynecological brachytherapy {#S1}
===========================
Most of the presentations were dedicated to gynecological brachytherapy. Pauline Humphrey from the Bristol Hematology and Oncology Centre reported results of a UK survey with the goal to obtain insight in the brachytherapy practice for locally advanced cervical cancer. The online survey consisted of questions about brachytherapy scheduling, inpatient/day case treatment, anesthetic/analgesic protocols, and non-pharmacological support. Responses were received from 39/43 eligible centers. Brachytherapy was predominantly given on an inpatient basis in 65% and as day case treatment in at 35% of centers. Eleven different brachytherapy scheduling regimes were reported. The typical duration of brachytherapy at each center (number of hours with applicators in place per insertion) varied from 3 to 53 hours. Generally, the respondents were positive about the level of care and assistance offered and provided many examples of good practice but also suggestions on how to improve the patient pathway. Free text answers were given by 33 respondents to the question "What works well in your department?". Three main categories were identified and included "continuity of experienced staff", "good information and support", and "trust and rapport". The question "What could be improved?" was answered by 32 respondents, and three main categories were identified such as "follow up provision", "pain relief", and "care in hospital wards".
Results from another UK high-dose-rate (HDR) cervix brachytherapy survey were presented by **Claire Fletcher** from the University Hospitals Coventry and Warwickshire NHS Trust. In 2018, the HDR cervix brachytherapy service at Coventry was being reviewed due to the number of sessions where treatments occurred out of hours. In June 2018, a questionnaire was sent out to the medical physics e-mail forum to determine HDR service provision in UK centers. 19 centers responded with a wide range of different activity levels. In September 2019, centers were contacted to see if any aspect of their service had changed.
The main findings of the survey were as follows: 79% of all centers who responded were planning cervix brachytherapy conformally and almost all centers had at least some access to MRI. Nearly 60% of centers were using interstitial brachytherapy with most of insertions being performed under ultrasound guidance. The majority of centers were using 3 fractions in a treatment, with each fraction given on a different insertion. However, 47% of centers who responded were applying multiple fractions on a single insertion, and these centers were more likely to give 4 fractions (e.g. 2 insertions with two fractions each). Most centers inserted applicators under general anesthesia, but using a spinal block could speed up the procedures' time and finish the treatment before 6 p.m. One third of all responding centers, routinely or regularly were treating patients outside the usual working hours, but there was a large variation in finishing times even within one center. The greater the complexity of treatment, the higher the chance that the procedure days will run late. However, there were also many centers that used conformal planning and did not routinely proceed with treatment out of routine hours. 63% of all centers had more than one dedicated planning computer, but it did not seem to have a large correlation with finishing time. When the questionnaire was reviewed in 2019, two centers were finishing earlier than previously reported. The reasons suggested included staff availability and training as well as implementation of automated checking procedures.
**Jennifer Cannon** from the James Cook University Hospital shared preliminary results of a UK audit of cervix brachytherapy treatments. Computed tomography (CT) and magnetic resonance (MR) images of two patients previously treated with brachytherapy were distributed to eleven UK radiotherapy centers. The patients were selected to represent a typical patient cohort: an intracavitary treatment only, and a combined intracavitary and interstitial treatment. Centers were asked to delineate target volumes and produce treatment plans on a standard set of delineations, following their local protocol. The variation in target volume delineation was assessed in terms of absolute volume variation, and dose optimization was evaluated with regard to dose-volume histogram (DVH) statistics and total EQD~2~. A further assessment was made by estimating the parameters based on all centers' submissions and comparing them to submissions from centers that specifically followed the EMBRACE II protocol. A variation in interstitial techniques was also assessed by comparing parameters such as total TRAK, needle TRAK, and maximum needle dwell time. This work is currently under Journals' review and results from this study will be published soon.
A presentation of **Rhydian Caines** from the Clatterbridge Cancer Centre NHS Foundation Trust was dedicated to prescription to protocol for HDR cervix brachytherapy. HDR image-guided brachytherapy for cervix cancer is administered in this center following a three-fraction day case model as per the EMBRACE II protocol. CT/MR image fusion is used for each fraction individually planned on Oncentra planning workstations, and delivered on the same day with a Flexitron HDR afterloader. With over 150 treatments delivered per year, the local team is very experienced, but clinician availability at the end of the process for final plan approval is often limited due to multiple competing responsibilities, resulting in additional wait time for patients. A preliminary audit of 92 fractions indicated that a mean interval between plan check and clinician approval was around 14 minutes; however, in 22% of cases, the time was exceeded by 20 minutes and in a few cases, even by 70-100 minutes. Prescription to protocol (PP) is a framework, under which a treatment plan with pre-agreed dose constraints is authorized for a treatment by an appropriately trained and experienced medical physics expert (MPE), instead of a clinician. Justification of the exposure is established by a clinician through authorization of the protocol. A preliminary review of 69 previous fractions suggested that the EMBRACE II optimal planning aims, when radio-biologically transformed into single fraction equivalents, provided an appropriate basis for this protocol, with around 20% of plans meeting all criteria ([Table 1](#T1){ref-type="table"}).
######
The EMBRACE II single fraction constraints adopted for prescription to protocol (PP) implementation for IGBT of cervix cancer D~90~
Parameter Constraint (Gy)
--------------------- -----------------
HR-CTV D~90~ \< 10.1
HR-CTV D~90~ \> 9.4
HR-CTV D~98~ \> 7.2
GTV res D~98~ \> 9.4
Point A mean dose \> 5.4
Rectum D~2cc~ \< 4.6
Bladder D~2cc~ \< 6.4
Sigmoid D~2cc~ \< 5.2
Bowel D~2cc~ \< 5.2
Recto-vaginal point \< 4.6
In six months following implementation, 12 of 87 patents were authorized for treatment under PP, with 8 of 29 patients having at least one MPE authorization. The main failure mode was point A, which among the 75 ineligible plans had failed 43 times (under local policy, more weight is usually given to the HR-CTV D~90~ upper limit). Median time from plan check to plan approval was 3 (0-13) minutes for MPE compared with 11 (1-48) minutes for a clinician (*p* \< 0.01, Mann-Whitney); however, this did not statistically influence the total patient wait time.
In conclusion, early experience with PP has been encouraging, with fewer handovers, streamlined workflow, and a reduced clinician burden. A significantly reduced wait time for plan approval was demonstrated. Low A point dose was the most common failure mode due to the local tendency to favor a lower HR-CTV D~90~ (\< 10.1 Gy).
**Nancy Lewis** from the University Hospital Southampton NHS Foundation Trust reported experience with Elekta's advanced gynecological applicator Venezia. This hybrid applicator allows the combination of intracavitary brachytherapy with interstitial brachytherapy. The local cervix cancer treatment protocol included EBRT of 45 Gy in 25 fractions and HDR brachytherapy consisting of 26 Gy in 4 fractions, with brachytherapy given using two applicator insertions, 1 week apart. All insertions were performed under spinal anesthetic, with interstitial patients receiving an epidural + PCA. Patients selected for possible interstitial treatments received an MRI in week 5 of external beam treatment. From the operating room, patients were taken for MRI and CT imaging.
Since March 2018, seven patients were treated with the hybrid Venezia applicator: 3 patients with the standard tandem and ovoids configuration in combination with a template and needles, 3 with the standard configuration in combination with oblique needles, and one patient with the standard combination using a 70 mm intra-uterine tube. In case of interstitial treatment only sharp needles were used. The sizes of available ovoids were 26 mm and 30 mm. Vaginal caps were not used. Three patients treated with a template and needles had an anterior vaginal extension. Oblique needles were used when the high-risk clinical target volume (HR-CTV) was too wide, to be adequately covered with parallel needles. Insertion of oblique needles was similar to the insertion of parallel needles, only slightly more pressure was required. Positions of needles were changed between implants in only one patient. Needle could be positioned in the expected positions as confirmed using CT imaging. Needle positions were reproducible. Conclusion was that the Venezia applicator allows for treatment of tumors that would otherwise be referred elsewhere. Careful consideration should be given to vaginal packing and wise choice of needles as well as to emergency procedures.
Similarly, **Bettany Cushing** from the Mater Private Hospital in Dublin (department led by Dr. Michael Maher) shared her experience with the advanced gynecological applicator Venezia. Patients that were selected for treatment with the Venezia applicator usually receive EBRT consisting of 50.4 Gy in 28 fractions. They have a pre-brachytherapy MRI after 20 EBRT fractions (36 Gy) to determine the size of applicator to be inserted for optimal fit. HDR brachytherapy fractionation consists of 21 Gy in 3 separate insertions. The brachytherapy procedure is as follows: the patient is admitted into the department the day before the treatment, fasting from midnight. A radiation therapist (RT) delivers the brachytherapy equipment to an operating theatre the night before the procedure. This brachytherapy equipment includes a cervix set (two ovoid tubes that together form a ring, and an intrauterine (IU) tube), a rectal retractor, 2 rubber bungs, guiding tubes, ProGuide needles, and color-coded cable ties. All patients receive an epidural anesthesia, and have a urinary catheter placed. The brachytherapy applicators are inserted under general anesthesia. The insertion is ultrasound-guided or by feel through the consultant's experience. The cervical ring is preloaded with needles, which are secured in position with guiding tubes. The IU tube is inserted into the intrauterine canal and the ring is positioned at the neck of the cervix. The rectal retractor pushes the rectum away and the needles are advanced through the ring using an insertion tool. Packing such as wet gauze is used to secure the entire application in position. The position of each ProGuide needle using color-coded cable ties is recorded, so that the exact position of each needle can be tracked, and each needle can be connected to the Flexitron in the proper order before treatment delivery ([Figures 1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}). Adherence to this protocol eliminates the risk of needles being connected to the incorrect channels of the Flexitron.
{#F1}
{#F2}
When interstitial needles are used, the patient requires both an MRI and CT-scan for planning purposes. This is in contrast with implants without needles, where only an MRI is necessary. As for the MRI, marking of the needle positions with a permanent marker is performed as well as removing the steel obturators from the ProGuide needles. The function of the MRI is to ensure the IU tube is in correct position. With respect to the CT, gold marker wires are placed in the ProGuide needles to verify their position, and specific marker wires are placed into the ring and the IU tube.
The physics team carries out the planning, which is then assessed and signed off by the radiation oncologist. The pretreatment record is then presented to the brachytherapy radiation therapy team and verified with the Oncentra brachytherapy plan. This is to ensure the compatibility of the prescription, indexer length, dwell positions, and treatment times as well as the immobility of ProGuide needles. Transfer tubes 1 + 3 are placed in channels 1 + 5. ProGuide needles are connected as per the diagram on the treatment sheet. Once the treatment is delivered and completed, before exiting the room, it is insured that the source has retracted completely and the wall monitor outside and inside the room show "zero" on a Geiger counter. Check time on post-treatment record should be equal to the time on pretreatment record. The consultant can subsequently remove the urinary catheter and applicators. There is limited number of research papers to serve as a comparison for future studies including patients with two brachytherapy treatment plans created, one with needles and one without to demonstrate the advantages of using interstitial needles. It is expected that the outcome of such a comparison would demonstrate higher dose delivery to CTV, while maintaining dose-volume constraints for organs at risk.
**Claudia Hill** from the University Hospital Southampton NHS Foundation Trust elaborated on the use of inverse planning for multi-channel vaginal HDR brachytherapy. The purpose of this project was to compare the current protocol, using manual and graphical optimization and two established inverse planning methods: inverse planning simulated annealing (IPSA) and hybrid inverse treatment planning and optimization (HIPO). For 13 gynecological cancer patients previously treated with HDR VMC brachytherapy, a planner retrospectively reoptimized the dose distributions using three different methods: manual and graphical optimization, IPSA, and HIPO. Relevant dose-volume histogram (DVH) parameters (HDvol V~100~ and D~90~, LDvol V~100~ and D~90~, rectum D~2cc~, bladder D~2cc~) were recorded in addition to the dose homogeneity index, central catheter loading index, and treatment parameters such as the number of dwell positions and total dwell times. Also, treatment planning time was evaluated. Both inverse methods resulted in shorter planning times as compared to manual and graphical optimization. In terms of DVH parameters, inversely optimized plans were comparable to the manually optimized plans. There was no statistical significance between the quality of differently optimized plans. In conclusion, inverse planning methods decrease the planning time as compared to a combination of manual and graphical optimization, and the DVH statistics are comparable with all methods. HIPO is the most suitable inverse optimizer for VMC brachytherapy due to the ability of the operator to control activated dwells.
**Michael Niekamp** from Elekta presented advanced brachytherapy planning tools for interstitial cervix applications: IPSA, HIPO, and implant modeling. He emphasized that the treatment possibilities with the new hybrid gynecological applicators are increasing. Nowadays, the variation of standard-shaped applicators in combinations with additional interstitial needles create real 3D volume implants. Furthermore, the number of combinations of dwell times and their positions is uncountable. He presented intelligent tools, which help to establish new adapted brachytherapy workflows: Predefined protocols for contouring, prescription, dose optimization, and evaluation lead to standards procedures.Applicator models with 3D source paths will accelerate dose planning, resulting in more reproducible and accurate doses.Intelligent tools for dose optimization and real-time inverse planning help to decide the best adapted implant geometry and optimal dose distribution for treatment. The automation will reduce the overall planning procedure with full control by the user at any time.
Specific workflows with the right utilization of the presented advanced brachytherapy tools will lead to an improvement in treatments.
In the second presentation, **Michael Niekamp** shared his thoughts about the integration of Elekta Brachytherapy in the Oncology Information System (OIS) for radiation oncology. In medicine, there is no doubt that brachytherapy as a complementary treatment, improves tumor control, increases overall survival, reduces side effects, and improves the quality of life after treatment. Brachytherapy needs to be an integral part of cancer treatment, especially in radiation therapy. The presentation showed how an open architecture of various digital tools and their special tasks are adapted for the user (e.g., patient management, diagnostics, targeting, treatment planning and delivery), which optimizes the patient's way through a radiation therapy department. The idea presented in this session is not limited to the integration of brachytherapy treatment execution alone. With a clever assessment and analysis of existing tasks and workflows, one can optimize workplaces by using recent excellent, well-implemented, and accepted functions across all treatment modalities that are required to cure patients. Possibilities and solutions for different platforms provider were discussed.
**Christopher Lee** from the Clatterbridge Cancer Centre NHS Foundation Trust reported results of paperless integration of Elekta's brachytherapy planning and treatment delivery system within a 3^rd^ party record and verification procedure for HDR image-guided brachytherapy of the cervix. At the Clatterbridge Cancer Centre, paperless workflows through the considered use of various management tools available within ARIA 13.6 (Varian Medical Systems, CA) were introduced for Image-Guided Brachytherapy (IGBT) for cervical cancer. This enabled integration of brachytherapy treatment planning (Oncentra Brachy) and treatment delivery system (Flexitron HDR) within ARIA.
The department delivers approximately 150 fractions per year, in two treatment sessions per week. Each fraction uses CT/MRI image fusion with treatments individually planned on an Oncentra Brachy workstation and delivered the same day using a Flexitron HDR afterloader (Elekta, Sweden). The work is complex, very time-sensitive, with several handover points, and critically depending on good communication between staff groups within the team.
Following a review of the previous paper-based process, a new paperless workflow was designed by a multidisciplinary brachytherapy team involving radiographers, physicists, and clinicians. The new paperless workflow clearly emphasizes the safety and efficiency of treatment, with clear communication of salient clinical information. Several ARIA's key workspaces namely electronic prescription, care path workflow management, and documents are utilized. Sign off and approval functions within ARIA serve as definitive legal signatures and provide an audit trail for the different tasks and responsibilities within the procedure. Following implementation, a timing audit was carried out to compare the duration between imaging and treatment for both the paper and paperless procedures. This showed that the overall time was similar for the two processes, but more patients on average were treated with the paperless procedure.
The implementation of paperless working within IGBT for cervix cancer had a positive effect on efficiency for this highly time-sensitive clinical process. Despite treating significantly more patients, waiting times have not increased and all treatments were concluded comfortably within clinical working hours. The brachytherapy team has embraced the changes, which benefited the patients. Electronic workflow management allows easy collection of rich audit data to leverage and measure efficiencies of the pathway and determine areas for future optimization.
**Robert Dacey** from the Northern Centre for Cancer Care (NCCC), Freeman Hospital Newcastle upon Tyne presented benefits of automated brachytherapy planning checks using the DICOM RT plan file. With increasingly complex means of producing and delivering dose distributions, the number of parameters having an impact on treatment delivery is continually expanding. Independent checking of plan parameters to avoid errors in treatment delivery is a well-established practice in radiotherapy; however, the focus has primarily been on external beam deliveries with brachytherapy checks concentrating mainly on dosimetric aspects alone.
Until recently, 1-2 patients were treated at the NCCC each week. Before introducing the use of interstitial needles into routine clinical practice (approximately 18 months ago), a timing study was undertaken to assess the possibility of increasing overall efficiency while maintaining safety in anticipation of a greater workload (3 cases per week are now a routine). Checking plan was identified as one part of the process taking a significant period of time, with the previous "tick box" check-form requiring visual assessment of 10-100 s of parameters, making it both time-consuming and prone to error. It was therefore decided to investigate the possibility of automating the extraction and manipulation of data directly from the DICOM RT plan file (as exported from Oncentra Brachy) to provide a rapid, accurate, and consistent method of checking.
A template was constructed in Microsoft Excel and a series of scripts written in Microsoft Visual Basic, where scripts guide the user through the process and prompt for details where necessary. Before data can be extracted from the DICOM RT plan file (in plain text), it must be filtered to remove extraneous DICOM formatting. The majority of parameters may be identified by unique DICOM tags (e.g., patient name, ring diameter, plan ID); however, some are associated with a common tag to multiple DICOM items (e.g., indexer lengths, channel ID) that must be dealt systematically. Several parameters of interest are not explicitly declared and must be determined via calculation involving some individual DICOM items (e.g., dwell times) including assessments of geometry. The latter is best performed in the applicator frame of reference requiring a linear transformation of the patient frame of reference co-ordinates from the DICOM RT plan file. Extracted values are transformed to the Excel template, where they are applied in calculations or compared directly to reference values.
Therefore, a method has been identified, where with minimal user input, a significant number of checks of the clinical brachytherapy plan are performed in a matter of seconds, thus significantly increasing the speed of checking without compromising on consistency or accuracy.
Skin brachytherapy {#S2}
==================
Skin brachytherapy was one of the main focus areas of the meeting. **Agata Rembielak** from The Christie NHS Foundation Trust and The University of Manchester shared an overview of recently published GEC-ESTRO ACROP guidelines and recommendations for skin brachytherapy. The guidelines constitute a unique publication in skin brachytherapy and provide eight main conclusions with main remark stating that "no standard schedule can be recommended, and total doses are based on experience". It was also advised that "the dose on the skin surface should be recorded to correlate the outcome with late side effects". The full document is available as free access text at <https://www.thegreenjournal.com/article/S0167-8140(18)30035-5/pdf> (source: Guinot *et al*. GEC-ESTRO ACROP recommendations in skin brachytherapy. Radiother Oncol 2018; 126: 377-385, DOI: <https://doi.org/10.1016/j.radonc.2018.01.013>).
In the second part, **Agata Rembielak** elaborated on the history of skin brachytherapy at The Christie, where clinical applications started shortly after radium discovery in 1898, and has been provided as an uninterrupted clinical service until present days. Agata's talk was supported by pictures from The Christie archive illustrating radium applicators and applications in patients. She described the evolution of skin brachytherapy from radium and manual positioning to automated remote afterloading technique. The main part of her talk was dedicated to the experience of The Christie team, with the so-called Christie method of skin brachytherapy. In this method, skin brachytherapy is delivered using a handmade mould or flap applicator, but with an extra layer of a bolus of various thickness, depending on clinical situations. The talk was supported by clinical examples demonstrating excellent cosmetic outcomes and low toxicity from skin brachytherapy, particularly in challenging locations such as face, hands, fingers, legs and feet.
**Orla Brosnan** from the Mater Private Hospital in Dublin, reported results of a treatment of skin lesions on the head using thermoplastic moulds in conjunction with the Freiburg flap. Due to spherical shape of the target volume, it is hard to achieve a homogenous dose distribution using EBRT. It is also difficult to obtain a flush finish using the Freiburg flap and to eliminate air gaps on the surface of the head if lesions are raised or irregularly shaped. This led to investigating customized mould-based surface brachytherapy, with an HDR afterloading procedure frequently used in other parts of the body. A silicone rubber applicator material (the Freiburg flap) is attached to a customized thermoplastic mould shaped to the patient's head. When non-invasive topical therapies failed or the lesion returned, treatment options for large lesions, multiple lesions, and lesions at difficult sites like the scalp are limited. Surgery for these patients results in poor wound healing rates and can be cosmetically unacceptable. Radiation therapy is a valid treatment option for these patients, with good local control and good cosmetic outcome.
The inclusion criteria for mould-based brachytherapy involve superficial small and large-sized lesions as well as angiosarcoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC). It is particularly useful for elderly, frail patients who may not be able to lie flat. It is also suitable for patients using blood thinners, or in sites at risk for delayed healing with surgery. It has a potential use for melanomas and Merkel Cell tumors, depending on clinical assessment. Exclusion criteria involve lesions on the inner and outer canthus, lesions with perineural extension, and recurrent disease with possible deep extension to the bone.
Pretreatment moulding procedure: the consultant defines the treatment area on the patient's skin using a marker pen and the radiation therapist draws the treatment area ([Figure 3](#F3){ref-type="fig"}) -- angiosarcoma.
{#F3}
Fiducial marker wire is placed on the skin outline ([Figure 4A](#F4){ref-type="fig"}). The tape is placed over the fiducial marker and the fiducials are traced with a permanent marker, so that it is visible under the thermoplastic material. The water bath is turned on approximately 1.5 hours before the procedure. The thermoplastic material is cut to the required size. The patient is in a supine position with the head in an appropriate headrest. The thermoplastic mould is placed in the water bath for 2.5 minutes to soften the plastic. It is then molded onto the patient head.
{#F4}
The thermoplastic material takes a minimum of 10 minutes to harden. Before it is removed, a tape is placed over visible fiducial markers and the treatment area is drawn on the mould (Figure 4B). Fiducial markers on the patient's skin should correspond to the marks placed on the thermoplastic material. The patient goes home and returns at a later date for CT planning.
Pretreatment attachment of the Freiburg flap: the appropriate size and number of Freiburg flaps channels are selected. One treatment position is determined for all areas. The optimal configuration of the flap is determined to ensure it is as flush as possible to the thermoplastic material. The flap is sutured roughly in place using dental floss. Sutures should never be running across the catheter, but rather running lengthways down the beads to avoid obstruction of the source.
Planning CT procedure: the treatment area was drawn on the patient's skin and the fiducial marker was placed onto the area. The thermoplastic material was fitted on the patient, ensuring that it was flush with the skin. CT markers were inserted into appropriate number of channels on the flap (i.e., equal to the width of field, which was marked on the skin). Sandbags were positioned on top of the flap to ensure that there were no air gaps between the thermoplastic material and the skin. CT scan (3 mm slices) was performed and the scans were sent to Prosoma for planning.
The planning image ([Figure 5](#F5){ref-type="fig"}) shows a 100% dose distribution to the skin surface. The prescribed dose was 36-40 Gy in 10 fractions, delivered twice per week, over 5 consecutive weeks. The patient's treatment position should be determined during first fraction. The patient can sit up if necessary. Planned number of transfer tubes was attached to the Flexitron. The thermoplastic material was placed on the patient and transfer tubes were connected to the Freiburg flap. Sandbags should be used to eliminate air gaps. The radiation therapists completed the pretreatment checks and the treatment was started.
{#F5}
To date, four patients have been treated, all with good cosmetic outcomes ([Figure 6](#F6){ref-type="fig"}). After treatment all patients had increased social interactions, resulting from a more positive body image and less self-consciousness.
{#F6}
**Satiavani Ramasamy** from the St. James University Hospital reported their initial clinical outcomes obtained with basal cell carcinoma treatment with the Elekta's Valencia applicator. The Leeds Cancer Centre established a skin brachytherapy service using Valencia applicators in September 2017. They audited and performed quality assurance of initial experience and effectiveness of outcomes; all patients were treated between September 2017 and August 2019. The dose used was 42 Gy in 6-7 fractions twice a week (biologically effective dose \[BED\] equivalent 70 Gy). For quality assurance, a multi-disciplinary team consisting of physicists, clinician-scientists, oncologists, mould room technicians, and radiographers analyzes each brachytherapy case before treatment initiation. Patients were followed up and reviewed in 8-10 weeks after treatment completion. 41 patients were included in this study, with a median follow-up of 4.7 months (range, 1.1-24.4 months). The median age was 73 years (range, 55-92 years). WHO performance status was 0 (88%, *n* = 36), 1 (10%, *n* = 1), and 2 (10%, *n* = 1). A total of 43 lesions were treated with a mean size of 1 cm (range, 0.2-2 cm). All were basal cell carcinoma. Sites of lesions were cheek (14%, *n* = 6), chin (2%, *n* = 1), ear (2%, *n* = 1), nose (37%, *n* = 16), lips (14%, *n* = 6), scalp (7%, *n* = 3), forehead (21%, *n* = 9), and extremities (2%, *n* = 1). The reason for choosing skin brachytherapy was driven by patient preference in 36 patients.
All patients completed treatment without any delay. 5 patients (6 lesions) have not had 2 months follow-up so far. Out of the 36 patients (37 lesions), with a minimum of 23 months follow-up, 100% had a complete clinical response at 8-10 weeks post-treatment. Treatment was well tolerated with no unexpected toxicities. No recurrences were identified in the cohort until now.
HDR brachytherapy for NMSC is effective, safe, and very well tolerated. Support from the multidisciplinary team was essential for the successful implementation of the service. The Valencia brachytherapy applicator allows the delivery of hypofractionated radical radiotherapy as an option for skin lesions up to 20 mm in size and 3 mm in depth.
Other indications {#S3}
=================
**Mark Long** from the St. Luke's Cancer Centre in the Royal Surrey County Hospital reported results of implementation and clinical experience of HDR prostate brachytherapy in Royal Surrey. The hospital provides a broad range of brachytherapy services, including Papillon rectal electronic brachytherapy, image-guided gynecological HDR brachytherapy treatments, and a well-established "4D brachytherapy" LDR prostate technique. The center wanted to expand the service to be able to offer T3 prostate cancer patients with an option of HDR prostate treatments. Oncentra Prostate was purchased and commissioned for this purpose. The US-guided planning and treatment process was developed to align with the experience from the center's other brachytherapy services. Blending the process used from LDR treatments allowed to sidestep the virtual contouring phase and develop own pseudo-virtual planning phase with the intent to reduce the time of procedure.
Since May 2018, 21 patients have been treated. It is hoped that with more experience, close communication with the brachytherapy community, and with auditing, the technique will be further developed and become more efficient.
Another presentation about prostate brachytherapy was given by **Katie McHugh** from the Cambridge University Hospitals NHS Foundation Trust who shared clinical data from Addenbrooke's Hospital, where over 750 patients have been treated using LDR prostate seed implants using the Nucletron/Elekta system since 2006 (Oncentra for planning, and SeedSelectron for delivery). All patients have a post-CT scan at 6 weeks after the treatment, and their post-implant dosimetry is calculated and compared with the published RCR guidelines for a satisfactory implant. Towards the end of 2018, the hospital was given an end of life notice for the supply of seeds that fit the Seed Selectron for May 2019, so they had just over 5 months to set up and commission a new system while maintaining a clinical service. They opted to keep the current TPS and found a new seed supplier who provided seeds to fit in the Mick applicator, which also had to be purchased and commissioned. Commissioning involved updating TPS seed model, setting up a method of seed QA, revising contingency plans and risk assessments, and staff training. Up to September 2019, 26 patients were treated, with no break in service delivery and with no significant changes in the hospitals' live plan stats or post-implant dosimetry. The hospital made several service improvements and can now treat patients with larger prostates, reduce operating time, and have a cost saving of up to £35k per year.
**Stefanie Corradini** from the Hospital of the Ludwig-Maximilians University (LMU) in Munich shared her experience in CT-guided interstitial brachytherapy of liver malignancies. Brachytherapy (BT) is an option for the treatment of liver-malignancies in patients with primary liver malignancies (e.g., hepatocellular carcinoma, cholangiocellular carcinoma) or patients with oligometastatic disease. Although brachytherapy is a long-standing, minimally invasive treatment method, it has not been widely implemented for an ablation of liver lesions. Nevertheless, there is emerging evidence for the effectiveness of this method of treatment, and the technique has recently been added to the "toolbox of ablative treatment options" of the European Society for Medical Oncology (ESMO) guidelines for metastatic colorectal carcinoma and hepatocellular carcinomas. Brachytherapy is known to achieve comparable results to SBRT and radiofrequency ablation (RFA) in the treatment of liver malignancies, with excellent local tumor control rates. Brachytherapy is preferred over RFA for large lesions (\> 3 cm), in the proximity of large vessels (no cooling effect) or irregular tumor shape, or as an alternative to SBRT in the proximity of organs at risk (e.g., stomach, bowel), and for very large or multiple lesions.
At LMU Munich, the procedure is performed in close collaboration with the department of interventional radiology. The workflow is as follows: first, diagnostic imaging with hepatospecific contrast-enhanced MRI (Primovist) is acquired for staging and all cases are discussed in a multidisciplinary tumor board. The placement of the brachytherapy catheters is performed by the interventional radiologist under fluoroscopy-CT guidance and local anesthesia. Midazolam and fentanyl are given for sedation and analgesia as individually required by the patients. Hollow 17-gauge needles are placed into the lesions. Thereafter, an angiography sheath with a 6F diameter is inserted over a stiff angiography guidewire, and the 6F brachytherapy catheters are consecutively placed in the angiography sheaths. In case of large lesions, multiple catheters are inserted. After the placement of catheters, a contrast-enhanced planning CT is acquired using a breath-hold technique and a slice thickness of 3 mm, which is used for treatment planning. The patient is then transferred to the brachytherapy unit. Treatment planning is performed using the Oncentra^®^Brachy treatment planning system (Elekta AB, Stockholm, Sweden). Target delineation consist of gross tumor volume (GTV), with an additional margin of 3-5 mm for the clinical target volume (CTV) depending on visualization quality of the GTV. Usually, there is no additional target volume planning (CTV = PTV). Moreover, organs at risk (OAR) such as liver, stomach, duodenum, colon, small intestines, and heart are delineated. After catheter reconstruction, treatment planning and dose optimization are performed. The aimed prescription dose (D~100~) depends on histology and varies between 1 × 15 Gy (HCC) and 1 × 20 Gy (metastases), or 1 × 25 Gy (colorectal metastases), taking into account organ at risk constraints. Moreover, the entire needle track is treated up to a dose of \~5 Gy to prevent needle track seeding.
Brachytherapy is applied using an HDR afterloading system (Flexitron, Nucletron, Elekta AB, Stockholm, Sweden) with an iridium-192 (^192^Ir) source. The treatment time depends on the size of the lesion, number of catheters, dwell positions, and the activity of the source, and can last up to 60 min or even longer in very large lesions. For catheter removal, a gel foam is introduced through each angiography sheath during removal to prevent bleeding.
Taken together, BT allows to treat also very large lesions with single doses of 15-25 Gy and results in excellent local control rates (\> 90%) in large primary or secondary hepatic lesions of up to 12-15 cm. Moreover, in contrast to thermoablative treatment approaches (RFA), it provides an effective treatment option for centrally located liver lesions, close to large vessels. When compared to most SBRT techniques, BT is less affected by uncertainties related to respiratory breathing motion, as the tumor is fixed by the implanted catheters. Another advantage of BT is the possibility of a repetitive approach, with the application of BT using hypofractionated fractionation schedules, with two or three fractions to spare OARs or treat very large tumors. Regarding adverse events in BT, significant bleedings following the interventional catheter implantation occurred in 4% in HCC and 2.5% in colorectal liver metastases. Additionally, the risk for radiation-induced liver disease (RILD) seems to be very low, even after the treatment of very large liver tumors or in livers with underlying cirrhosis.
**Joshua Mason** from the St. James University Hospital presented results of a study of his colleague **Luke Eason** about the evaluation of a collapsed-cone convolution algorithm for ^192^Ir brachytherapy treatment planning for esophageal and surface mould brachytherapy treatments. The purpose of the study: TG43 does not account for a lack of scatter and tissue and applicator heterogeneities. The advanced collapsed-cone engine (ACE) algorithm available in the Oncentra Brachy (OCB) treatment planning system (Elekta AB, Stockholm, Sweden) can model these conditions more accurately and is evaluated for esophageal and surface mould brachytherapy treatments.
ACE was commissioned for use and compared against TG43 for five esophageal and five surface mould treatment plans. Dosimetric differences between each algorithm were assessed using dose difference maps, side-by-side comparisons, and DVH statistics.
Results of the study:
Esophagus (6 Gy per fraction): Compared to TG43, ACE demonstrated up to a 0.63% and 0.05 Gy reduction in PTV V~100~ and PTV D~98~, respectively. Lung D~2cc~ and bone D~2cc~ deviated by up to 0.09 Gy and 0.03 Gy, respectively. Lung D~0.1cc~ and bone D~0.1cc~ both deviated by up to 0.12 Gy. Surface mould (4.5 Gy per fraction): Compared to TG43, ACE demonstrated up to a 12.5% and 0.18 Gy reduction in PTV V~80~ and PTV D~98~, respectively. Bone D~2cc~ and D~0.1cc~ both reduced by up to 0.2 Gy when modeled with ACE. Increasing mould size laterally increased the dosimetric differences between TG43 and ACE.
TG43 generally overestimates the dose delivered to the target volume and OARs for the clinical sites investigated. Dosimetric differences observed for esophageal treatments were minimal; however, treatments involving larger surface moulds would benefit from increased dosimetric accuracy offered by ACE. Implementation should be considered for surface mould ^192^Ir treatment planning, but increased calculation time, additional contouring, and mass density assignment requirements should be scrutinized with regards to their potentially negative impact on current clinical practice.
{#UF1}
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Several lines of evidence suggest that inflammation plays a role in the pathophysiology of major depression and that anti-inflammatory drugs have antidepressant-like effects.[@B1],[@B2],[@B3],[@B4] The administration of the bacterial endotoxin lipopolysaccharide (LPS) induces inflammation and subsequent depression-like behavior in rodents.[@B1],[@B5] Furthermore, the depression-like behavior and altered serum pro-inflammatory cytokine levels, such as tumor necrosis factor-α (TNF-α), induced by LPS are blocked by antidepressants, including selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs).[@B6] A meta-analysis found higher blood TNF-α levels in drug-free depressive patients compared with healthy controls.[@B7] A postmortem brain study showed elevated pro-inflammatory cytokine gene expression in the frontal cortex of people with a history of major depression.[@B8] Therefore, it is likely that both peripheral and central inflammation are associated with depressive symptoms and that anti-inflammatory drugs could ameliorate these symptoms in patients with major depression.
Adenosine triphosphate (ATP) has been implicated in acute and chronic inflammation.[@B9],[@B10] Among the ATP-sensitive purinergic receptors, the P2X7 receptor (P2X7R) has an important role in the post-translational processing of the biologically active pro-inflammatory cytokine interleukin-1β (IL-1β).[@B11] P2X7R is abundantly expressed in microglia[@B12] and to a lesser extent in astrocytes,[@B13] oligodendrocytes,[@B14] and the presynaptic terminals of neurons.[@B15] Studies using P2X7R knock-out (KO) mice showed that the absence of P2X7R leads to reduced immobility time in the tail-suspension test (TST) and forced swimming test (FST),[@B16],[@B17] suggesting a role of P2X7R in depression-like behavior. Therefore, P2X7R antagonists are potential therapeutic drugs for major depression.[@B18],[@B19]
The dye brilliant blue G (BBG), also known as Coomassie blue, is the best-known P2X7R antagonist and has nanomolar potency,[@B20] although it also inhibits voltage-gated sodium currents at higher concentrations.[@B21] This study examined whether BBG shows anti-inflammatory and antidepressant effects in mice after LPS administration.
METHODS
=======
Animals
-------
All experiments used 8-week-old male adult C57BL/6N mice (body weight 20-25 g; Japan SLC, Hamamatsu, Japan). The animals were housed in controlled temperatures under a 12-h light/dark cycle (lights on from 07:00-19:00), with food and water *ad libitum*. All experiments were carried out in accordance with the Guide for Animal Experimentation of Chiba University. The experimental procedure was approved by the Animal Care and Use Committee of Chiba University (permission number: 25-270).
Drug Administration
-------------------
On the day of injection, fresh solutions were prepared by dissolving compounds in sterile endotoxin-free isotonic saline. LPS (0.5 mg/kg; L-4130, serotype 0111:B4, Sigma-Aldrich, St. Louis, MO, USA) was administered intraperitoneally (i.p.). BBG was purchased from Sigma-Aldrich. The dose of BBG was reported previously.[@B17],[@B22]
Enzyme-linked Immunosorbent Assay (ELISA)
-----------------------------------------
Vehicle (10 ml/kg, i.p.) or BBG (12.5, 25, or 50 mg/kg, i.p.) was administered to mice 30 minutes before LPS injection. Under sodium pentobarbital, blood samples were taken via cardiac puncture 90 minutes after LPS administration. Blood was centrifuged at 2,000 g for 20 minutes to generate serum samples, as reported previously.[@B6] The serum samples were diluted 20-fold with ELISA diluent solution (eBioscience, San Diego, CA, USA). The serum TNF-α concentrations were measured using a Ready-SET-Go ELISA kit (eBioscience) according to the manufacturer\'s instructions.
Behavioral Tests
----------------
On day 1, vehicle (10 ml/kg, i.p.) or BBG (50 mg/kg, i.p.) was administered to mice 30 minutes before the i.p. administration of LPS (0.5 mg/kg) or saline (10 ml/kg). On day 2, all behavioral tests were performed in the following order: locomotion at 9:00 h (24 hours after LPS injection), TST at 14:00 h (27 hours after LPS injection), FST at 16:00 h (29 hours after LPS injection). The mice were put in the test room 30 minutes before the behavioral tests. All tests were performed in a quiet room between 9:00 and 17:00 h. After the tests, the mice were returned to their home cages, which were returned to the breeding room.
### Locomotion
The mice were placed in 560×560×330-mm cages (length×width×height). The cage was cleaned between testing sessions. The locomotor activity of the mice was counted using a SCANET MV-40 (Melquest, Toyama, Japan), and the cumulative amount of exercise was recorded for 60 minutes.
### TST
The mice were taken from their home cage, and a small piece of adhesive tape was placed approximately 2 cm from the tip of the tail. A single hole was punched in the tape and the mice were hung individually on a hook. The immobility time of each mouse was recorded for 10 minute. Mice were considered immobile only when they hung passively and were completely motionless.
### FST
The mice were placed individually in a cylinder (diameter 23 cm, height 31 cm) containing 15 cm of water maintained at 23±1℃. The animals were tested in automated forced-swim apparatus using a SCANET MV-40. The immobility time was calculated from the activity time as (total time)-(active time) using software built into the apparatus. The cumulative immobility time was scored for 6 minutes during the test.
Statistical Analysis
--------------------
The data are shown as the mean±standard error of the mean (SEM). The data were analyzed using PASW Statistics 20 (formerly SPSS statistics; SPSS, Tokyo, Japan). All data, including the locomotion, TST, and FST test results, were analyzed using one-way analysis of variance (ANOVA), followed by the *post hoc* Bonferroni/Dunn test. *p*-values\<0.05 were considered statistically significant.
RESULTS
=======
Effects of BBG on Serum TNF-α Levels
------------------------------------
In the vehicle-treated mice, serum TNF-α levels were very low ([Fig. 1](#F1){ref-type="fig"}), consistent with a previous report.[@B6] The serum TNF-α levels were increased significantly after a single dose of LPS (0.5 mg/kg) ([Fig. 1](#F1){ref-type="fig"}). BBG (12.5, 25, or 50 mg/kg) was given 30 minutes before the LPS injection, and blood was collected 90 minutes after the LPS injection. Pretreatment with BBG (12.5, 25, or 50 mg/kg) significantly attenuated the LPS-induced increases in serum TNF-α ([Fig. 1](#F1){ref-type="fig"}).
Antidepressant Effects of BBG on LPS-induced Depression-like Behavior in Mice
-----------------------------------------------------------------------------
In mice, the FST and TST are the behavioral assays used most widely for detecting potential antidepressant-like activity[@B23],[@B24],[@B25] To examine the antidepressant effects of BBG on LPS-induced depression-like behavior, BBG (50 mg/kg) was given 30 minutes before the LPS injection. Behavioral evaluations were performed 24 hours after the LPS injection.
One-way ANOVA of the locomotion data revealed no significant differences among the four groups (F\[3,43\]=2.709, *p*=0.057). Pretreatment with BBG did not affect the spontaneous locomotion in the vehicle- and LPS-treated mice ([Fig. 2A](#F2){ref-type="fig"}). One-way ANOVA on the TST data revealed significant differences among the four groups (F\[3, 50\]=5.766, *p*=0.002). The *post hoc* analysis showed that BBG (50 mg/kg) significantly (*p*=0.036) attenuated the immobility time compared with the LPS-treated group ([Fig. 2B](#F2){ref-type="fig"}). One-way ANOVA of the FST data revealed significant differences among the four groups (F\[3, 50\]=6.737, *p*=0.001). *Post hoc* analysis showed that BBG (50 mg/kg) significantly (*p*=0.035) attenuated the immobility time compared with the LPS-treated group ([Fig. 2C](#F2){ref-type="fig"}). However, BBG (50 mg/kg) alone did not affect the immobility time (TST and FST) of control mice ([Fig. 2B, 2C](#F2){ref-type="fig"}).
DISCUSSION
==========
This study found that BBG, a potent P2X7R antagonist, showed anti-inflammatory effects on the serum TNF-α levels after LPS injection and antidepressant effects in the TST and FST. The anti-inflammatory effects are consistent with the effects of antidepressants (SSRIs and SNRIs) on serum TNF-α levels.[@B6] Furthermore, pretreatment with BBG significantly attenuated the increase in immobility time in the TST and FST after LPS injection. Recently, Pereira et al.[@B26] reported that two P2XR antagonists (PPADS and iso-PPADS) decreased the immobility time in the FST and that the antidepressant-like effect of iso-PPADS was associated with a decrease in nitric oxide levels in the prefrontal cortex. Therefore, P2X7R antagonists such as BBG are potential therapy for inflammation-induced depression.
The peripheral administration of LPS induces sickness behavior that peaks 2-6 hours later and wanes gradually.[@B1] This behavior requires the activation of pro-inflammatory cytokine signaling in the brain in response to peripheral LPS injection, and the depression-like behavior peaks 24 hours post-LPS injection.[@B1] In this study, we measured the serum TNF-α levels in mice 90 minutes after LPS injection and performed the behavioral evaluations 24 hours after LPS injection. BBG showed an anti-inflammatory effect on the serum TNF-α levels after LPS injection and antidepressant effects on LPS-induced depressive behavior in mice. Interestingly, it was reported that genetic deletion of P2X7R leads to an antidepressant-like phenotype in the TST and FST.[@B17] Additionally, BBG did not show antidepressant-effects in P2X7R KO mice.[@B17] These findings suggest that BBG exerts its antidepressant-effects via P2X7R antagonism. A recent study using P2X7R KO mice suggested that P2X7R is involved in the adaptive mechanisms elicited by exposure to repeated environmental stressors that lead to the development of depression-like behavior.[@B27] A study using bone marrow chimeric mice showed that P2X7R expressed on peripheral immune cells is unlikely to mediate the impact of P2X7R on depression-like behaviors in naïve mice,[@B17] showing that the depression-like behavior in P2X7R KO mice was not transferred to wild-type mice recipients of P2X7R KO bone marrow cells. Therefore, P2X7R may play a role in the pathophysiology of major depression associated with inflammation.
Microglial activation is associated with the pathogenesis of major depression.[@B3],[@B28] Although the precise molecular mechanisms underlying microglial activation are largely unknown, P2X7R plays an important role in microglial activation in the brain.[@B29],[@B30] The LPS-induced release of IL-1β was prevented by the P2X7R antagonist A-438079 and was absent in spinal cord slices taken from P2X7R KO mice.[@B31] Furthermore, the TNF-α and IL-1β mRNA levels in the brain were elevated less in P2X7R KO mice than in wild-type mice in response to systemic LPS administration.[@B32] Interestingly, P2X7R was reported to play a role in the altered cytokine levels after LPS injection, whereas it did not play a role in the basal cytokine levels in the brain.[@B32],[@B33] Therefore, P2X7R appears to play a key role in the brain cytokine response to immune stimuli that might be involved in the pathophysiology of major depression.
Recent linkage studies have found a susceptibility locus for mood disorders such as major depression and bipolar disorder on chromosome 12q24.[@B34],[@B35],[@B36] Among the genes on 12q24, a polymorphism (rs2230912) of the gene encoding P2X7R is associated with both major depression and bipolar disorder,[@B37],[@B38],[@B39],[@B40],[@B41] although some studies have reported negative findings.[@B42],[@B43],[@B44] Furthermore, a polymorphism (rs2230912) had a genetic effect on depressive symptom severity in patients with bipolar disorder.[@B44] Therefore, the *P2X7R* gene may be involved in the pathogenesis of mood disorders, such as major depression and bipolar disorder.
In conclusion, this study showed that the P2X7R antagonist BBG has anti-inflammatory and antidepressant effects in mice after LPS-induced inflammation. Therefore, P2X7R antagonists are potential therapy for inflammation-related depression.
This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas of the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K.H.).
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| {
"pile_set_name": "PubMed Central"
} |
After fertilization, mammalian embryos travel along the oviduct and make their way to the uterus before implantation. During this transit, the pre-implantation embryo undergoes cleavage, an important process producing more blastomere to enable differentiation, blastocyst formation, hatching and implantation[@b1][@b2]. Thus, preimplantation embryo development is greatly influenced by oviductal and uterine environments. Although evidences have indicated that pregnancy loss is a multi-factorial phenomenon, the biochemical composition of the embryo and maternal environment are the main players to determine pregnancy outcome[@b3]. While much attention has been paid to understand hormonal influences on embryo development[@b4][@b5], much less studies are reported focusing on factors derived from the embryo-maternal environment.
It has been known for decades that female reproductive tract, including the oviduct and uterus, contains high concentrations of HCO~3~^−^ (up to 90 mM), which is much higher than that in most other tissues[@b6][@b7][@b8]. Impaired HCO~3~^−^ secretion by oviduct epithelium inhibits embryo cleavage and blastocyst formation[@b9], indicating an essential role of oviductal and uterine HCO~3~^−^ in embryo development. In preimplantation embryo, HCO~3~^−^ entry has been shown to activate soluble adenylate cyclase in the cytoplasm and triggers in a series of events required for embryo cleavage[@b9]. However, how HCO~3~^−^ is transported into the embryo remains largely unknown.
Our recent study has demonstrated a crucial role of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel known to conduct both Cl^−^ and HCO~3~^−^ [@b10][@b11], in the process of embryo cleavage and differentiation[@b1]. Disrupting CFTR function by inhibitors or knockdown decreases intracellular pH (pH~i~), suppresses the sAC/PKA cascade and thus embryo cleavage. CFTR knockout embryo also showed reduced cleavage capacity and blastocyst formation *in vitro* and *in vivo*. These results indicate an important role of CFTR in mediating HCO~3~^−^ transport essential for preimplantation embryo development[@b1]. However, it remains to be resolved whether CFTR directly conducts HCO~3~^−^ or acts as a Cl^−^ channel working in parallel with a Cl^−^/HCO~3~^−^ exchanger, thereby providing a recycling pathway for the Cl^−^ that is necessary to operate the anion exchanger[@b12][@b13][@b14].
The SLC26 gene family encodes for anion transporters that transport a variety of monovalent and divalent anions[@b15]. Among the 11 members identified, SLC26A3 and SLC26A6 are the two main Cl^−^/HCO~3~^−^ exchangers expressed in various epithelial tissues[@b16]. Although it has been postulated that Cl^−^/HCO~3~^−^ exchange regulates intracellular pH of embryos[@b17], the identity of Cl^−^/HCO~3~^−^ exchanger(s) involved and its exact role in embryo development remain unknown. We undertook the present study to examine the possible involvement of Cl^−^ and Cl^−^/HCO~3~^−^ exchangers, particularly the two SLC26A family members, in preimplantation embryo development.
Results
=======
Embryo cleavage requires both Cl^−^ and HCO~3~ ^−^
--------------------------------------------------
To test the possible involvement of Cl^−^/HCO~3~^−^ exchangers, we first examined the requirement of Cl^−^ in cleavage and blastocyst formation. When 2-cell mouse embryos were incubated in Cl^−^ deficient TALP for 12 h, there was a significant decrease in the percentage of 4-cell embryo compared to that in the control with complete TALP ([Fig. 1A,B](#f1){ref-type="fig"}). The inhibiting effect was similar to that observed in HCO~3~^−^-deficient TALP medium ([Fig. 1A,B](#f1){ref-type="fig"}). Next, we examined the effect of varying Cl^−^ concentrations while maintaining constant HCO~3~^−^ concentration, or *vice versa*, on embryo cleavage. In the first set of experiment, TALP medium with different HCO~3~^−^ concentrations and a constant Cl^−^ concentration (115 mM) was used. Consistent with previous study, embryo cleavage was reduced as the concentration of HCO~3~^−^ in the TALP decreased, with less than 10% embryos develop into 4-cell stage when HCO~3~^−^-deficient TALP was used ([Supplemental Fig. S1](#S1){ref-type="supplementary-material"}), confirming an essential role of HCO~3~^−^ in the process of cleavage. The second set of experiments was performed using TALP medium with varying Cl^−^ concentrations and constant HCO~3~^−^ concentration (25 mM). The results showed that embryo cleavage decreased with decreasing Cl^−^ concentrations in the medium, despite the presence of sufficient HCO~3~^−^ ([Fig. 1C,D](#f1){ref-type="fig"}). These results indicate that embryo cleavage requires both Cl^−^ and HCO~3~^−^. To further discern whether the Cl^−^ and HCO~3~^−^ act synergistically or dependently on embryo cleavage, we examined the percentage of embryo cleavage in medium containing half effective concentration of both HCO~3~^−^ (12.5 mM) and Cl^−^ (56 mM) ([Fig. 1E](#f1){ref-type="fig"}). The result showed that the percentage of embryo cleavage in medium containing half effective concentration of both ions was similar to that containing half effective concentration of either Cl^−^ ([Fig. 1D](#f1){ref-type="fig"}) or HCO~3~^−^ ion ([Supplementary Fig. S1](#S1){ref-type="supplementary-material"}), suggesting that the function of Cl^−^ on embryo cleavage were largely, if not all, dependent on HCO~3~^−^.
Effect of Cl^−^ and HCO~3~ ^−^ on increased pH~i~ during embryo cleavage
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Next, we examined if Cl^−^ is required for the transport of HCO~3~^−^ into the embryo. The entry of HCO~3~^−^ has been reported to lead to an increase in pH~i~ of blastomere of 2-cell embryo[@b18][@b19], an early hallmark event in the process of embryo cleavage. Therefore, we used pH~i~ as an indicator of HCO~3~^−^ entry. Indeed, as shown in [Fig. 2A](#f2){ref-type="fig"}, the pH~i~ increased with time when 2-cell embryos were incubated in complete TALP, while remained almost unchanged in those incubated in HCO~3~^−^-deficient TALP. Interstingly, pH~i~ also remained unchanged when the embryos were incubated in Cl^−^-deficient TALP, suggesting that Cl^−^ deficiency impairs HCO~3~^−^ entry into the embryos.
Effect of Cl^−^ on cleavage-related gene expression
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In preimplantation embryos, HCO~3~^−^ influx has been shown to act as an environmental cue in activating the expression of miR125b, an effector of HCO~3~^−^-dependent signaling essential for embryo development[@b1]. High level of miR125b expression suppresses the expression of p53, the latency of which is required for normal embryo development[@b20]. To assess the involvement of Cl^−^ in mediating these HCO~3~^−^-dependent gene expressions and embryo development, 2-cell embryos were separately incubated in Cl^−^/HCO~3~^−^ complete TALP, HCO~3~^−^-deficient TALP or in Cl^−^-deficient TALP for 6 h and the gene expression was analysed by realtime PCR. The results showed that embryos incubated in Cl^−^/HCO~3~^−^ complete TALP were accompanied by a relatively high level of miR-125b expression, consistent with that normally observed during cleavage[@b1]. However, embryos incubated in HCO~3~^−^-deficient or Cl^−^-deficient TALP showed significantly decrease in miR-125b expression ([Fig. 2B](#f2){ref-type="fig"}) with elevation of its target p53[@b21] ([Fig. 2C](#f2){ref-type="fig"}). The expression of p21, a downstream target of p53[@b1], was also significantly increased ([Fig. 2D](#f2){ref-type="fig"}). Consistent with the realtime PCR results, p53 and p21 proteins were markedly increased in embryos cultured under HCO~3~^−^ -deficient or Cl^−^-deficient TALP ([Fig. 2E](#f2){ref-type="fig"}). These results suggest that Cl^−^ regulates HCO~3~^−^-dependent events essential for embryo development.
Expression and localization of SLC26A3 and SLC26A6 in preimplantation embryo
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We have previously demonstrated an important role of CFTR in transporting HCO~3~^−^ necessary for embryo cleavage[@b1]. The observed Cl^−^-dependent cleavage and its regulation of HCO~3~^−^-dependent cellular alkalinzation and gene expressions suggest the involvement of Cl^−^/HCO~3~^−^exchanger(s) working in concert with CFTR in transporting HCO~3~^−^. SLC26 mutations have been associated with subfertility in human[@b22] and a SLC26A member had been shown to work in concert with CFTR in sperm[@b23]. Therefore, we screened the expression of SLC family members in preimplantation embryo. The results showed that the expression of SLC26A3 and SLC26A6 mRNA were significantly higher than other SLC members, which were reported to be expressed in early stage embryo[@b24] ([Supplemental Fig. S2](#S1){ref-type="supplementary-material"}). Thus, we focused on SLC26A3 and SLC26A6 and examined their potential role in embryo development. We characterized the expression of SLC26A3 and SLC26A6 by indirect immunofluorescence staining and realtime PCR in different stages of embryo. As shown in [Fig. 3A](#f3){ref-type="fig"}, SLC26A3 and SLC26A6 protein expression was hardly detected in zygote stage but markedly increased at 2-cell stage and continued to express at blastocyst stage in both human and mouse preimplantation embryos. Consistent with the immunofluorescence staining, both SLC26A3 and SLC26A6 mRNA showed a marked increase in 2-cell embryo compared to the zygote stage. Their expression continues to increase until 4-cell stage and tailed off at blastocyst stage ([Fig. 3B--E](#f3){ref-type="fig"}). The observed temporal changes in the expression of SLC26A3 and SLC26A6 in the early stages of embryo cleavage suggest their potential involvements in embryo cleavage.
Effects of inhibitors of SLC26A3 and SLC26A6 on HCO~3~ ^−^-dependent embryo cleavage
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To test the possible involvement of SLC26A3 and SLC26A6 in embryo cleavage, 2-cell embryos were treated with niflumate (inhibitor of SLC26A3) or DIDS (inhibitor of SLC26A6) for 12 h and the formation of 4-cell embryo was counted. As shown in [Fig. 4A,B](#f4){ref-type="fig"}, 20 μM of niflumate or DIDS treatment significantly decreased the percentage of 4-cell embryo formed compared to the control. The inhibiting effects of niflumate or DIDS were dose-dependent ([Supplementary Fig. S3](#S1){ref-type="supplementary-material"}). The degree of inhibition was comparable in niflumate-treated group and CFTR inhibitor-treated group, which was significantly greater than that of the DIDS-treated group. Of note, the inhibiting effects of niflumate, DIDS and CFTR inhibitor were not observed when embryos were incubated in HCO~3~^−^-deficient TALP ([Fig. 4C,D](#f4){ref-type="fig"}) or in Cl^−^-deficient TALP ([Fig. 4E,F](#f4){ref-type="fig"}), suggesting that the effect of these inhibitors were attributed to the blockage of Cl^−^ and HCO~3~^−^ transport. Further, blocking SLC26A3 and SLC26A6 suppressed the expression of miR-125b ([Fig. 4G](#f4){ref-type="fig"}) and elevated the expression of p53 and p21 ([Fig. 4H,I](#f4){ref-type="fig"}). The effect of blocking SLC26A3 on HCO~3~^−^-dependent gene expression was similar to that of blocking CFTR ([Fig. 4G--I](#f4){ref-type="fig"}). These results suggest possible involvement of SLC26A3 and SLC26A6 working in concert with CFTR in regulating HCO~3~^−^-dependent embryo cleavage.
Effects of SLC26A3 and SLC26A6 knockdown on embryo cleavage
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We further knocked down SLC26A3 and/or SLC26A6 by microinjecting siRNAs into blastomere of 2-cell embryo to confirm their effect on embryo cleavage. Quantitative real-time PCR results showed that SLC26A3 and SLC26A6 could be effectively knockdown individually or simultaneously by injecting siRNA targeting one subtype or co-injecting two siRNAs ([Fig. 5A,B](#f5){ref-type="fig"}). As shown in [Fig. 5C,D](#f5){ref-type="fig"}, SLC26A3 or SLC26A6 knockdown both markedly reduced the formation of 4-cell embryos with SLC26A3 knockdown showing a significantly more potent effect compared to that of SLC26A6 knockdown. Double knockdown of SLC26A3 and SLC26A6 further inhibited the formation of 4-cell embryos compared to the individual knockdown group, suggesting that SLC26A3 and SLC26A6 might each contribute to embryo cleavage. Consistent with the phenotypic data, individual knockdown of SLC26A3 or SLC26A6 suppressed miR-125b expression and increased p53 and p21 mRNA expression, while double knockdown showed an additive effect ([Fig. 5E](#f5){ref-type="fig"}--G). These results indicate that both SLC26A3 and SLC26A6 play important, yet independent role during embryo cleavage.
Discussion
==========
Although it is established that HCO~3~^−^ plays an essential role in embryo development before implantation[@b1][@b9][@b25][@b26], the present study has demonstrated an equally important role of Cl^−^ in the process by mediating the entry of HCO~3~^−^ that is required for embryo cleavage. To initiate cleavage, HCO~3~^−^ has to first enter into blastomere of embryo and act on an HCO~3~^−^ sensor, sAC, which in turn triggers the PKA/NFkB signaling cascade. Activation of NFkB elevates the expression of miR-125b, which suppresses the expression of p53 and p21, leading to embryo cleavage[@b1]. In the present study, we showed that some of the key events in this cascade such as increased pH~i~, activation of miR-125b and downregulation of p53, which have been previously shown to be dependent on HCO~3~^−^, were also Cl^−^-dependent. The dependence of the cleavage-associated events on Cl^−^, in addition to HCO~3~^−^, suggests the involvement of Cl^−^/HCO~3~^−^ exchangers in mediating the entry of HCO~3~^−^. Using pharmacological and genetic approaches, we showed that SLC26A3 and SLC26A6 were two Cl^−^/HCO~3~^−^ exchangers required for HCO~3~^−^ entry and HCO~3~^−^-dependent signalling cascades. The operation of these Cl^−^/HCO~3~^−^ exchangers requires CFTR acting as a Cl^−^ channel to provide a recycling pathway for Cl^−^. Defects in either the Cl^−^/HCO~3~^−^ exchange or the CFTR-mediated Cl^−^ recycling pathway would impair HCO~3~^−^ entry, disrupt HCO~3~^−^-dependent signalling cascade and suppress embryo cleavage ([Fig. 6](#f6){ref-type="fig"}).
Despite their similar role as Cl^−^/HCO~3~^−^ exchanger, the stoichiometry of Cl^−^/HCO~3~^−^ exchange by SLC26A3 and SLC26A6 is different. It has been shown that SLC26A3 mediates 2 Cl^−^/1 HCO~3~^−^ exchange while SLC26A6 mediates 1 Cl^−^/2 HCO~3~^−^ exchange[@b27]. Interestingly, despite similar knockdown efficiency, knockdown of SLC26A3 has a more potent inhibiting effect on embryo cleavage compared to SLC26A6 knockdown ([Fig. 5](#f5){ref-type="fig"}), suggesting a more critical role of SLC26A3 in embryo cleavage. Moreover, double knockdown of SLC26A3 and SLC26A6 showed an additive effect compared to individual knockdown. These results suggest that both SLC26A3 and SLC26A6 may each contribute to HCO~3~^−^ entry required for embryo cleavage while the contribution from SLC26A3 appears to be predominant. The reason behind the preference for a slow kinetic HCO~3~^−^ entry path through SLC26A3 may be accounted for the observed gradual increase in the intracellular alkalinzation in hours in preimplantation embryos ([Fig. 2A](#f2){ref-type="fig"}). SLC26A6 may play a minor or alternative role, which requires further investigation.
The presently demonstrated role of SLC26A3 and SLC26A6 in mediating HCO~3~^−^ entry required for embryo cleavage may have implications in fertility diagnosis or prediction of pregnancy outcome. It has been demonstrated that patients carrying mutations in SLC26A3 gene suffered congenital chloride diarrhea[@b28]. SLC26A3 knockout mice also exhibit subfertility, however, whether SLC26A3 is associated with other forms of defects in fertility other than male infertility remains unexplored. Our results have clearly shown an important role of SLC26A3 in embryo cleavage and thus preimplantation embryo development. Therefore, we propose that SLC26A3 could be a potential biomarker for predicting pregnancy failure associated with embryo development blockage.
In conclusion, the present results have demonstrated the importance of a maternal environmental factor, Cl^−^, in addition to the previously demonstrated HCO~3~^−^, in preimplantation embryo development. Cl^−^ is required for the operation of Cl^−^/HCO~3~^−^ exchangers, SLC26A3 and/or SLC26A6, for the transport of HCO~3~^−^ into embryos required for the activation of miR-125 and subsequent inactivation of p53/p21 during embryo cleavage. Therefore, in addition to the previously demonstrated important role of CFTR, these anion exchangers, particularly SLC26A3, may play equally important role in regulating HCO~3~^−^ entry and thus embryo cleavage, defect or mutation of which may lead to pregnancy loss due to disrupted embryo development.
Materials and Methods
=====================
Human embryo samples
--------------------
Human preimplantation embryos were donated by patients who had a successful pregnancy from an *in vitro* fertilization program at Women's Hospital, School of Medicine, Zhejiang University. Donation was voluntary and informed consent was given. All human-related procedures were carried out in accordance with guidelines approved by the Ethics Committee for Research on Human Subjects of Zhejiang University.
Animals
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All animal-related procedures were carried out in accordance with the Institutional Guide for Laboratory Animals established by the Animal Care and Use Committee (ACUC), and were approved by the ACUC of the School of Medicine, Zhejiang University (Approval number: ZJU2015-415-05). The mice were housed under a 12/12-h light/dark cycle at 25 ± 0.5 °C and 50--60% humidity, and were fed ad libitum with a standard diet and water.
Embryo recovery and culture
---------------------------
Female ICR mice (8-week-old) were superovulated by intraperitoneal injections of 10 IU pregnant mares' serum gonadotrophin (PMSG, Hangzhou Animal Pharmaceutical Factory, Hangzhou, Zhejiang, China), followed by 10 IU human chorionic gonadotrophin (hCG, Hangzhou Animal Pharmaceutical Factory) at 48 h after PMSG injection. Females were caged with ICR males (10-week-old) immediately following hCG injection. Embryos at different stages were obtained by sacrificing the mice at indicated time points after hCG injection; zygote-18 h, 2-cell-44 h, 4-cell-56 h, morula-80 h, blastocyst-92 h. Blastocysts were collected by flushing the uterus with TALP-HEPES medium; embryos at other stages were collected by flushing the oviducts with TALP-HEPES from the infundibular end. The embryos were transferred to TALP medium and cultured under 5% CO~2~ at 37 °C. Unless otherwise specified, all embryos were cultured in the presence of 25 mM HCO~3~^−^ and 115 mM Cl^−^. For development assessment, 2-cell embryos were cultured for 12 h untill 4-cell stage and numbers of blastomere were counted and recorded at that time. For analysis of miR-125b, p53, p21, embryos were collected after various treatments for 6 h.
Measurement of pH~i~ in embryos
-------------------------------
The level of pH~i~ was determined at 2-cell embryos stage by loading with 2′, 7-bis-2 (carbosyethyl)-5-(and-b)-carboxyfluorescence, acetoxymethyl ester (BCECF, B8806, Sigma-Aldrich, St. Louis, MO, USA) as previously described (Xu *et al.*, 2007), with minor modifications. Briefly, 2-cell embryos were obtained and were cultured in complete TALP, Cl^−^-deficient TALP and in HCO~3~^−^-deficient TALP in 5% CO~2~ incubator at 37 °C. When needed, 5 μM BCECF was added in the medium and incubated with embryos for an additional 30 min. Following this, the embryos were washed twice to remove free dye before pH~i~ measurement. Fluorescence was detected by an excitation ratio of 490:440 nm (emission, 520 nm) using a fluorescence microscope photometry systems (FMPS, Nikon, Tokyo, Japan). Calibration was performed according to the method previously used (Fraire-Zamora and Gonzalez-Martinez, 2004), with modification. Briefly, at the end of each trace, 10 μl of 5 mM nigericin was added to permeabilize the blastomeres, the pH was then acidified with 10 μl of HCl, and the measured pH values (determined with a conventional pH meter) were compared with the corresponding ratio values. Usually, we make three additions before ending the experiment. These data were analyzed using Prism 5.0 software (GraphPad Software, San Diego, CA) to convert ratios to pH values.
SLC26A3/A6 siRNA microinjection to mouse 2-cell embryos
-------------------------------------------------------
The siRNA negative control (siRNA NC, sc-37007, Santa Cruz Biotechnology, CA, USA), SLC26A3 siRNA (sc-45544, Santa Cruz Biotechnology) or SLC26A6 siRNA (sc-108024, Santa Cruz Biotechnology) was dissolved in RNase-free water. Each blastomere of two-cell embryos was injected with 50 fmol of siRNA solution at the two-cell stage using the micromanipulation system. Formation rate of 4-cell embryo was accessed 12 h after injection.
Quantitative real-time PCR
--------------------------
Total RNA extraction and reverse transcription (RT) from 100 embryos for each group were performed using Cells-to-cDNA^TM^ II Kit as described (Ambion, Austin, TX, USA). Quantitative real-time PCR was performed by using the ABI Prism 7900HT (Applied Biosystems, Carlsbad, CA, USA). The specific primers were provided by Sangon, Shanghai, China. The full list of primer sequences (mouse p53, p21, SLC26A3, SLC26A6 and GAPDH; human SLC26A3, SLC26A6 and GAPDH) are shown in [Supplemental Table 1](#S1){ref-type="supplementary-material"}. Quantitative real-time PCR for miR-125b was carried out with microRNA assay kit (Assay ID: 000449, Applied Biosystems, Carlsbad, CA, USA). snoRNA202 was used as miRNA control (Assay ID: 001232, Applied Biosystems, Carlsbad, CA, USA).
Immunofluorescence staining and confocal microscopy
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The embryos were fixed with 4% paraformaldehyde for 1 h and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 30 min. After incubation with 10% goat serum for 1 h to block the nonspecific antigen, the embryos were incubated with primary antibodies: polyclonal goat anti-SLC26A3 primary antibody (1:200 dilution) (sc-34939, Santa Cruz Biotechnology) or goat anti-SLC26A6 primary antibody (1:200 dilution) (sc-26728, Santa Cruz Biotechnology) or goat anti-p53 polyclonal antibody (sc-1312, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-p21 polyclonal antibody (sc-397, 1:200, Santa Cruz Biotechnology) at 4 °C overnight. After incubation, the embryos were washed with PBS, and then incubated with secondary antibodies: FITC-conjugated donkey anti-goat antibody (1:500, sc-3853, Santa Cruz Biotechnology) or Alexa 488 rabbit anti-goat IgG (A27012, 1:500, invitrogen, Rockford, IL, USA) or Alexa 568 goat anti-rabbit IgG (A11011, 1:500, invitrogen) at room temperature for 45 min, followed by nuclear staining with 4′, 6-diamidino-2-phenylindole (DAPI, 1:1000, D9542, Sigma-Aldrich) for 20 min. For the negative controls, we incubated the embryos in PBS without the addition of the primary antibody to determine the levels of non-specific fluorescence. Finally, the fluorescence images were analysed using a Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA).
Statistical analysis
--------------------
Each experiment was repeated at least three times. Results were presented as means ± standard error (SEM). Student's unpaired *t*-test was used for two-group comparison. One-way analysis of variance (ANOVA) followed by Tukey's *post-hoc* test was used when comparing three or more groups. A probability of *P* \< 0.05 was considered to be statistically significant.
Additional Information
======================
**How to cite this article**: Lu, Y. C. *et al.* Involvement of Cl^−^/HCO~3~^−^ exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage. *Sci. Rep.* **6**, 28402; doi: 10.1038/srep28402 (2016).
Supplementary Material {#S1}
======================
###### Supplementary Information
We would like to thank Dr. Ruan Yechun for the artwork. This work was supported by the National Basic Research Program of China (2012CB944900, 2012CB944903, 2013CB967401) and the National Natural Science Foundation of China (No. 81370684).
**Author Contributions** Y.-C.L., H.-F.H. and H.-C.C. conceived of and designed the study; Y.-C.L., J.Y., Y.-H.Y., L.J., Z.-Y.C. and X.-M.Z. performed experiments; Y.-C.L. and J.Y. analyzed data; Y.-C.L. and K.-L.F wrote the article with contributions from H.-C.C.
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![SLC26A3 and SLC26A6 work in concert with CFTR in regulating HCO~3~^−^ transport in preimplantation embryo.\
Working model for the regulation of early embryo cleavage by SLC26A3 and SLC26A6 in CFTR/HCO~3~^−^-dependent activation of miR-125b. The HCO~3~^−^ influx is mediated by SLC26A3 and SLC26A6 with an exchange of 2Cl^−^/ HCO~3~^−^. Apart from its reported role in conducting HCO~3~^−^ directly, CFTR act as a Cl^−^ channel to provide a recycling pathway for Cl^−^ that is required for SLC26A3 and SLC26A6 function. The sites of action for inhibitors CFTRinh172, niflumate, and DIDS, as well as the intracellular HCO~3~^−^-dependent events, are also shown. HCO~3~^−^ influx mediated by the cooperative action of SLC26A3, SLC26A6 and CFTR activates soluble adenylyl cyclase (sAC) which increase the level of cAMP. This in turn activates PKA/NFkB signaling cascade which increases the expression of miR125b. Expression of miR125b is required for embryo cleavage through suppressing the expression of p53 and p21. Vm, membrane potential; \[pH\]i, intracellular pH.](srep28402-f6){#f6}
[^1]: These authors contributed equally to this work.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
*O*-GlcNAcylation (*O*-linked β-*N*-acetylglucosaminylation) is a post-translational modification that reversibly modifies proteins present in the cytosol, the nucleus and the mitochondria.^[@bib1],\ [@bib2],\ [@bib3]^ Two enzymes control the *O*-GlcNAcylation cycle. First, the *O*-GlcNAc transferase (OGT, uridine diphospho-*N*-acetylglucosamine:polypeptide β-*N*-acetylglucosaminyltransferase or *O*-GlcNAc transferase) transfers the *N*-acetylglucosamine residue onto target proteins. Second, the *O*-GlcNAcase (OGA, *N*-acetyl β-glucosaminidase or *O*-GlcNAcase) removes the GlcNAc residue.^[@bib4]^ OGT (EC. 2.4.1.255) is assigned to the GT41 family in the CAZY (carbohydrate-active enzyme) database^[@bib5]^ and is found in all living beings (animals, plants, protists, bacteria, viruses) except in yeasts, in which its expression remains controversial. OGT participates in many fundamental cellular processes including cell cycle.^[@bib2]^ Our group previously showed that hormonal stimulation of physiologically G2-blocked *Xenopus laevis* oocytes triggered a quick increase in *O*-GlcNAcylation levels and that inhibition of OGT impaired G2/M transition.^[@bib6],\ [@bib7]^ In parallel, Slawson *et al.*^[@bib8]^ observed that OGT localized to the mitotic spindle and midbody during mitosis and that its overexpression resulted in supernumerary chromosomes. Later, the same authors showed that both OGT and OGA physically interact with Aurora B and PP1 to regulate the stability of the midbody and the phosphorylation and/or *O*-GlcNAcylation of vimentin at M-phase.^[@bib9],\ [@bib10]^ Finally, it was observed that OGT expression and *O*-GlcNAcylation reached a maximum level at the M-phase of the cell cycle.^[@bib11]^ Taken together, these studies show that OGT and *O*-GlcNAcylation in general are needed for germ cells meiosis and somatic cell mitosis. However, to our knowledge, there is no report focusing on the expression and activity of OGT during cell cycle entry (G0/G1). Here, we describe that following serum stimulation, OGT is significantly overexpressed. Blockade of OGT delays serum-stimulated cyclin D1 synthesis and cell proliferation. OGT silencing also prevents cyclin D1 expression and diminishes PI3K and MAPK activation. These are the first results demonstrating that OGT is indispensable for G0/G1 transition.
Results and Discussion
======================
OGT and OGA is a couple of non-redundant enzymes that controls *O*-GlcNAc cycling. Although much attention has been paid to OGT and although it has been described that this enzyme interferes with many crucial intracellular processes including cell cycle,^[@bib2],\ [@bib6],\ [@bib7],\ [@bib8],\ [@bib9],\ [@bib10],\ [@bib11]^ no study has focused on its expression and function during cell cycle entry.
OGT is upregulated upon stimulation of MCF7 cells
-------------------------------------------------
As described previously,^[@bib11]^ G0/G1 arrested MCF7 cells were stimulated by addition of fetal calf serum (FCS) and samples were collected at different times to assess OGT expression using western blot ([Figures 1a and b](#fig1){ref-type="fig"}). We observed a significant OGT increase (2.5-fold) 30 min after stimulation. FCS stimulation activated PI3K and MAPK pathways as attested by the use of antiphospho-Akt and antiphospho-Erk1/2 antibodies ([Figure 1a](#fig1){ref-type="fig"}). Cells treated with the protein synthesis inhibitor cycloheximide before stimulation did not show OGT increase in response to FCS ([Figure 1c](#fig1){ref-type="fig"}), indicating that protein translation is required to enhance OGT level. In parallel, OGT mRNA level, assessed by real-time PCR, remained unchanged 60 min post FCS treatment ([Figure 1d](#fig1){ref-type="fig"}). Thus, we conclude that OGT is regulated at the translational level shortly after addition of FCS. This is the first time that OGT induction is reported so rapidly after FCS treatment. Yang *et al.*^[@bib11]^ recently showed that, upon FCS stimulation, OGT levels increased during all the cell cycle, suggesting that OGT might have some oncogenic properties. This hypothesis was first proposed by Caldwell *et al.*^[@bib12]^ who reported higher hexosamine biosynthetic pathway activity and OGT levels in breast cancer cells, and demonstrated that interfering with the glycosyltransferase expression reduced tumor growth. Thus, OGT synthesis and activity may regulate fundamental factors involved in the control of the cell cycle.
β-catenin and OGT interact rapidly upon G1 phase entry
------------------------------------------------------
We and others have previously reported that the oncoprotein β-catenin, a major cell cycle factor, can be *O*-GlcNAcylated by OGT.^[@bib13],\ [@bib14],\ [@bib15],\ [@bib16]^ However, the role of this modification remained poorly understood. Studies performed on plakoglobin,^[@bib17]^ a member of the catenin family, showed that mutation of Thr14, an *O*-GlcNAcylated site close to the D-box that controls plakoglobin degradation, into alanine slightly increased plakoglobin stability. We also reported that β-catenin stability is regulated by the glucose status, the hexosamine biosynthetic pathway (which provides UDP-GlcNAc, the substrate for *O*-GlcNAcylation processes) flux and *O*-GlcNAcylation.^[@bib14],\ [@bib18],\ [@bib19]^ At last, RNA interference of OGA in the colorectal cancer metastatic cell line SW620 resulted in β-catenin overexpression.^[@bib20]^ Our hypothesis is that *O*-GlcNAcylation may serve as a protective signal for short half-life proteins such as β-catenin. Upon cell cycle entry, the expression of β-catenin continuously increases during G1 phase until it reaches a maximal level at G2/M transition.^[@bib21],\ [@bib22],\ [@bib23]^ To establish that this increase is due to *O*-GlcNAcylation, we investigated to measure the OGT interaction with β-catenin after addition of FCS to quiescent cells. First, we observed a concomitant increase of OGT and β-catenin as soon as 15 min after stimulation with FCS ([Figure 2a](#fig2){ref-type="fig"}, left panel). Co-immunoprecipitations performed under the same conditions showed that OGT and β-catenin interacted within minutes ([Figure 2a](#fig2){ref-type="fig"}, right panel) as soon as G0/G1 transition was triggered, as confirmed by phosphorylation of Erk and according to the β-catenin target gene cyclin D1 profile ([Figure 2a](#fig2){ref-type="fig"}, left panel). In this way, note that β-catenin transcriptional activity doubled upon FCS stimulation ([Figure 2b](#fig2){ref-type="fig"}) as attested by reporter gene analysis using TOP/FOP Flash constructs.
β-catenin is highly expressed and *O*-GlcNAcylated in response to FCS
---------------------------------------------------------------------
Total levels of exogenous β-catenin and its *O*-GlcNAcylated fraction were evaluated by western blotting in β-catenin-2XFLAG overexpressing HeLa cells that were either FCS-starved or starved and reincubated with FCS for 1 h ([Figure 2c](#fig2){ref-type="fig"}, left panel). As expected, the total amount of β-catenin was found higher in FCS-stimulated cells. Moreover, and similar to MCF7 cells ([Figure 1](#fig1){ref-type="fig"}), an increased OGT expression was observed in response to FCS. In accordance, we also showed that modification of β-catenin by *O*-GlcNAc was remarkably enhanced concomitantly to OGT induction ([Figure 2c](#fig2){ref-type="fig"}, right panel). This is consistent with our previous report demonstrating that endogenous β-catenin was stabilized by *O*-GlcNAcylation in a model of oocyte maturation (*X. laevis*).^[@bib13]^
Furthermore, β-catenin expression and *O*-GlcNAcylation levels were also compared in confluent and exponentially growing HeLa cells. [Figure 2d](#fig2){ref-type="fig"} shows that β-catenin protein was more abundant in proliferating cells than in quiescent cells. Strikingly, β-catenin was found *O*-GlcNAcylated in proliferating cells but not in quiescent one, suggesting that high level of β-catenin expression is dependent on *O*-GlcNAcylation.
Inhibition or downexpression of OGT impairs cell cycle entry
------------------------------------------------------------
Because FCS stimulation induced OGT expression, we then tested the effect of OGT deficiency on cyclin D1 expression. To this end, we inhibited OGT either at the transferase activity level, using the recently described compound Ac-5SGlcNAc,^[@bib24]^ or at the expression level, using RNA interference (siOGT). Treatment with Ac-5SGlcNAc resulted in a marked decrease of overall *O*-GlcNAcylation in response to FCS ([Figure 3a](#fig3){ref-type="fig"}). Furthermore, cyclin D1 did not accumulated following FCS addition as in control condition ([Figure 3a](#fig3){ref-type="fig"}). To know whether OGT controls the expression of other cyclins, and consequently is crucial for all steps of the cell cycle, we depleted asynchronous MCF7 of OGT through the use of siRNA ([Figure 3b](#fig3){ref-type="fig"}). We observed lower amounts of cyclin D1 and cyclin B1, corroborating previous findings,^[@bib6],\ [@bib7]^ in MCF7 transfected with siOGT when compared with siCtrl (universal negative control), whereas expression of cyclin E and cyclin A were unaffected. The same result was achieved by inhibiting OGT at the catalytic level ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}). Moreover, OGT silencing decreased the phosphorylated forms (active) of Akt and Erk1/2, the downstream effectors of the PI3K and MAPK pathways respectively, in asynchronous cells. In this way, longer time-course experiments showed that Ac-5SGlcNAc slightly delayed cell proliferation ([Figure 3c](#fig3){ref-type="fig"}). RNA interference of OGT also resulted in inhibition of FCS-induced cyclin D1 expression and in inhibition of PI3K/MAPK activation as observed above for asynchronous cells ([Figure 3d](#fig3){ref-type="fig"}). β-catenin expression remained similar in control and siOGT-treated cells. We next tested wortmannin, a PI3K inhibitor in FCS-stimulated cells ([Figure 3e](#fig3){ref-type="fig"}). We observed that reciprocally, inhibiting the PI3K pathway decreased FCS-induced OGT, and cyclin D1, expression, supposing a feedback loop between PI3K and OGT. To evaluate the impact of OGT silencing on the transactivation of genes responsive to the β-catenin/TCF/LEF complex, we performed a reporter gene analysis using the TOP/FOP Flash system in HEK293T cells ([Figure 3f](#fig3){ref-type="fig"}). Silencing OGT expression decreased by 50% the transcription activity of the β-catenin/TCF/LEF complex in this model. Analysis of β-catenin expression by western blot indicates that the transcriptional activity decrease correlated with a loss of β-catenin expression in FCS-induced HEK293T cells.
All together, our results indicate that OGT is a potent regulator of the cell cycle entry essential for FCS-stimulated cells to activate mitogenic pathways and to express cyclin D1. Thus, we propose that abnormal increase of OGT activity, which may occur in a context of nutrient excess that directly modulates the levels of the OGT substrate UDP-GlcNAc, would affect the cell cycle, as recently discussed by others.^[@bib12]^ In such a context, OGT could arguably exert some oncogenic effect, by allowing the cell cycle of the affected cells to bolt off.
This work has been supported by the 'Ligue Contre le Cancer/Comité du Nord\', the 'Association pour la Recherche sur le Cancer\', the University of Lille 1 and the 'Centre National de la Recherche Scientifique\'. SOVS is a recipient of a fellowship from the 'Ministère de l′Enseignement Supérieur et de la Recherche. We are indebted to Dr David Vocadlo (Simon Fraser University), Dr Chunming Liu (University of Kentucky), Dr Randall T Moon (University of Washington) who provided us, respectively, Ac-5SGlcNAc, pCS2+β-catenin-2XFlag and TOPflash/FOPflash reporter plasmids. We also thank Pr. Ralph Schwarz (Marburg University) and Dr Sylvain Julien (King\'s College/Lille 1 University) for the final reading of the manuscript and for the grammatical corrections.
[Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies the paper on the Oncogenesis website (http://www.nature.com/oncsis).
The authors declare no conflict of interest.
Supplementary Material {#sup1}
======================
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Click here for additional data file.
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Click here for additional data file.
![Stimulation of starved MCF7 cells with FCS increases OGT level. (**a**) MCF7 cells were maintained in a Dulbecco\'s modified Eagle\'s medium supplemented with 10% (v/v) FCS, 2 mℳℒ-glutamine, 5 IU/ml penicillin and 50 μg/ml streptomycin at 37 °C in a 5% (v/v) CO~2~-enriched humidified atmosphere. Cells were stopped at G0/G1 using the FCS-starvation method.^[@bib11]^ Cells were FCS-starved for 48 h and then cell cycle was released by FCS addition. FCS-induced cells were collected at the indicated times after FCS addition. Cells were washed with 10 ml of cold phosphate-buffered saline and lysed directly on ice with lysis buffer (10 mℳ Tris/HCl, 150 mℳ NaCl, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/v), 0.1% sodium dodecyl sulfate (w/v) and proteases inhibitors, pH 7.4). Cell lysates were centrifuged (20 000 *g*, 10 min, 4 °C), pellets were discarded and supernatants boiled for 10 min in Laemmli buffer. Proteins were separated by 10% SDS--polyacrylamide gel electrophoresis and electroblotted on a nitrocellulose sheet (GE Healthcare, Orsay, France). Equal loading was verified using Ponceau red staining. Membranes were saturated for 45 min with 5% non-fatty acid milk in (TBS)-Tween buffer (15 mℳ Tris/HCl, 140 mℳ NaCl and 0.05% Tween20 (v/v), pH 8.0). Proteins were immunodetected using the following primary antibodies; OGT: rabbit polyclonal TI14, 1/2000 (Sigma-Aldrich, Saint-Quentin Fallavier, France); α-tubulin: mouse monoclonal B-5-1-2, 1/5000 (Santa Cruz Biotechnology, Heidelberg, Germany); Erk2: D-2, 1/5000 (Santa Cruz Biotechnology); phospho-Erk1/2: rabbit polyclonal, 1/1000 (Cell Signaling, Danvers, MA, USA); phospho-AKT: rabbit polyclonal, 1/1000 (Cell Signaling) and AKT: mouse monoclonal, 1/2000 (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4 °C, washed three times (TBS--Tween, 10 min) and incubated with appropriate horseradish peroxidase-labelled secondary antibodies at a dilution of 1/10 000 for 1 h. After three more washes, detection was performed with enhanced chemiluminescence (GE Healthcare). (**b**) Histograms represent densitometric analyses of western blots (WBs). Results correspond to the mean value ±s.d. of three experiments (\**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001 respectively, NS not significant). (**c**) Starved MCF7 cells were stimulated with FCS with or without the protein synthesis inhibitor cycloheximide (CHX) at a concentration of 15 μg/ml. OGT expression was analyzed by WB and protein loading was verified using alpha-tubulin. (**d**) FCS-starved and 1 h FCS-stimulated MCF7 cells OGT mRNA levels were determined by real-time PCR. Quantitative reverse transcriptase--PCR: Total RNA was reverse transcribed using random primers and MultiScribe reverse transcriptase (Applied Biosystems, Villebon sur Yvette, France). Real-time PCR analysis was performed by Power SYBR Green (Applied Biosystems) in a MX3005P fluorescence temperature cycler (Stratagene, Paris, France) according to the manufacturer\'s instructions. Results were normalized with respect to *RPLP0* RNAs used as internal control. The primers used are as follows: *OGT* sense 5′-TGGCTTCAGGAAGGCTATTG-3′ and antisense 5′-CAAGTCTTTTGGATGTTCATATG-3′, and *RPLP0* sense 5′-GTGATGTGCAGCTGATCAAGA-3′ and antisense 5′-GATGACCAGCCCAAAGGAGA-3′. Results correspond to the mean value ±s.d. of three experiments (NS not significant). Molecular mass markers are indicated at the left.](oncsis201236f1){#fig1}
{ref-type="fig"}). Cells were transfected with pCS2+β-catenin-2XFlag (or the empty vector) in 2.5 ml of optiMEM by the polyethylenimine (PEI, Euromedex) method (10 μl) in 100 mm diameter dishes with 2.5 μg of DNA for 6 h and then incubated for 48 h in 10 ml of fresh complete medium. One day later, cells were FCS-starved and reincubated with FCS for 1 h. The activity of the MAPK pathway was checked by evaluating phosphorylation of Erk. β-catenin was probed using the mouse monoclonal anti-Flag (M2 from Sigma) at a dilution of 1/5000. (left panel). Expression of cyclin D1 was also evaluated. β-catenin was immunopurified using the anti-FLAG antibody and the immunoprecipates were stained either with a mouse monoclonal anti-*O*-GlcNAc antibody (RL2, Ozyme, Saint-Quentin en Yvelines, France) at a dilution of 1/1000 or with the anti-FLAG antibody (right panel). β-catenin is heavily O-GlcNAcylated in exponentially HeLa growing cells (**d**). After transfection with pCS2+β-catenin-2XFlag, HeLa cells were cultured and collected either when in the exponential growth phase or when cell confluence was reached. Cells were cultured either without any drug or with 1 μℳ MG132 (*N*-carbobenzoxyl-Leu-Leu-leucinal, Sigma) or 20 mℳ glucosamine. β-catenin (anti-Flag) expression was measured by WB. Rabbit polyclonal anti-actin (I--19 from Santa Cruz Biotechnology) was used at a dilution of 1/10 000. β-catenin was also immunopurified using the anti-FLAG and immunoprecipates were stained either with anti-*O*-GlcNAc antibody (RL2) or with anti-FLAG antibody (M2). Light microscopy pictures (right) were taken just before cell collection. IP, immunoprecipitation; rIgG, rabbit non-immune immunoglobulin G; AS, asynchronous cells. Molecular mass markers are indicated at the left.](oncsis201236f2){#fig2}
![Inhibiting OGT catalytic activity or interfering with its expression hinders FCS-stimulated cell cycle entry. (**a**) Following FCS starvation, MCF7 cells were incubated with FCS for the indicated time periods with or without the OGT inhibitor Ac-5SGlcNAc at a final concentration of 100 μℳ. Cell lysates were then analyzed by western blot (WB) according to their *O*-GlcNAc and cyclin D1 content. Equal loading was checked by using a rabbit polyclonal anti-GAPDH (Abcam, Paris, France) at a dilution of 1/5000. (**b**) Asynchronous MCF7 cells were reverse-transfected with Lipofectamine RNAiMax (Life Technologies, Carlsbad, CA, USA) according to manufacturer\'s instructions using 10 nℳ small interfering RNA targeting OGT^[@bib25]^ or a control siRNA (MISSION siRNA universal negative control \#1, Sigma). Cell lysates were analyzed by WB according to their cyclin D1, cyclin E, cyclin A, cyclin B1, phospho-Akt, Akt, phospho-Erk1/2, Erk1/2 and tubulin contents. (**c**) MCF7 cells (2 × 10^3^) were cultured in 96-wells plates using Dulbecco\'s modified Eagle\'s medium with or without 100 μℳ Ac-5SGlcNAc over 5 days. Each day, cell growth was determined using the MTS reagent method (Promega, Madison, WI, USA) according to the manufacturer\'s directions at 490 nm (*n*=6). (**d**) Starved MCF7 cells were reverse-transfected with Lipofectamine using siRNA targeting OGT or a control siRNA as described in **b**. 24 h later, cells were FCS-deprived for 48 h and then stimulated for the indicated time periods. Cell lysates were analyzed by WB using anti-OGT, anti-*O*-GlcNAc, anti-β-catenin, anti-phospho-Akt, anti-Akt, anti-phospho-Erk1/2, anti-Erk1/2, anti-cyclin D1 and anti-tubulin antibodies. (**e**) Starved MCF7 cells were stimulated with FCS for the indicated time periods in conjunction with 10 nℳ wortmannin, an inhibitor of the PI3K pathway. Cell lysates were analyzed by WB according to their OGT and cyclin D1 content. Activation or inhibition of the PI3K pathway was assessed using an anti-P-Akt antibody. Equal loading was checked by using an anti-tubulin antibody. (**f**) HEK293T cells were cultured under the same conditions as described for MCF7 and HeLa cells ([Figure 1](#fig1){ref-type="fig"}). Following OGT silencing (see above for details), HEK293T cells were transfected with β-galactosidase, TOP Flash, FOP Flash and β-catenin2-XFlag vector or an empty vector by the Lipofectamine2000 reagent ([Figure 2b](#fig2){ref-type="fig"}) for 24 h to perform TOP/FOP Flash reporter assay. Histogram represents the relative luciferase activity (RLA). Results correspond to the mean value ±s.d. of three experiments (\**P*\<0.05, \*\*\**P*\<0.001, respectively; NS not significant). OGT and β-catenin expression corresponding to the luciferase activity assay were measured by WB. GAPDH was used to attest equal loading.](oncsis201236f3){#fig3}
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family also known as Pou5f1 (Pou domain 5 transcriptor factor 1) \[[@b1],[@b2]\]. It is well-established that Oct4 is responsible for the pluripotency of embryonic stem cells (ESCs) and for their maintenance in undifferentiated state \[[@b3]--[@b6]\]. Oct4 down-regulation causes the differentiation of ESCs in the three germ layers: endoderm, mesoderm and ectoderm which give rise to different organs and tissues in the body \[[@b3]\]. Oct4 has also been detected in adult tissues cells such as human peripheral blood mononuclear cells, bone marrow-derived mesenchymal stem cells and human cancer stem cells \[[@b7]--[@b10]\]. Several studies suggested a role of Oct4 in maintaining self-renewal in somatic stem cells \[[@b11],[@b12]\]. Although Oct4 gene ablation in somatic stem cells revealed no abnormalities in tissue homoeostasis or regenerative capacity, there is increasing evidence that Oct4, even if it is expressed at low levels in somatic cells, is dispensable for the self-renewal and maintenance of somatic stem cells \[[@b13],[@b14]\].
Recently, Oct4 was found to be expressed in telocytes (TCs) in adult human skeletal muscle cell niche \[[@b15]\]. Telocytes are a novel described interstitial (stromal) cell type which display distinct structural features, with long extensions up to 100 μm called telopodes \[[@b16],[@b17]\]. The telopodes comprise thin fragments called podomers and more enlarged fragments called podoms \[[@b18]\]. The TCs form a 3-dimensional network with intercellular junctions \[[@b19]\]. These cells were found in various organs including pleura, epicardium, myocardium, endocardium, intestine, uterus, pancreas, mammary gland and in several others organs (for review see: \[[@b17]\]). Previous electron microscopic studies of the adult mouse lung have shown that the TCs are located in the perivascular and peribronchial spaces and extend to the alveolar compartment \[[@b20]\].
Telocytes express several markers including CD34, alpha-smooth muscle actin (α-SMA), Sca-1, platelet-derived growth factor receptor-beta (PDGFR-β), S-100, C-kit, vimentin and VEGF in human epicardium, human term placenta or skeletal muscle cell niche \[[@b15],[@b21]\]. A role of TCs in inter-cellular signalling *via* paracrine secretion as well as by shed vesicles and exosomes has been suggested because of their distinguished architecture with thin and long telopodes \[[@b22]\]. Their presence in the microenvironment of stem cell niches as well as the expression of stem cell markers suggests a role of these cells in tissue regeneration \[[@b23]\].
In this study, we have identified Oct4 expressing cells in the adult mouse lung. These cells are present in the perivascular and peribronchial spaces corresponding to the localization of TCs identified by electron microscopy. In addition, Oct4-positive cells were found to express several described markers of TCs, such as vimentin, Sca-1, PDGFR-β, C-kit and VEGF. These results were supported by qRT-PCR with mRNA isolated from cell picking by using laser capture microdissection technique. By using Oct4-GFP reporter mice, we were able to isolate Oct4-positive cells that in long-term culture conspicuously displayed morphological and phenotypical features of TCs. In addition, given that Oct4-positive cells in adult mouse lung are EpCAM^neg^/CD45^neg^/Oct4-GFP^pos^ cells and on the basis of negative selection with EpCAM, we demonstrate that Oct4-positive cells are not epithelial cells.
Materials and methods
=====================
Animal for experimentations
---------------------------
For this study, adult 8 week-old wild-type mice and Oct4-GFP transgenic mice were used. Adult wild-type mice were bred in our animal facility and Oct4-GFP mice were purchased from Jackson Laboratory, Bar Harbor, ME USA bred and maintained in our facility.
Western blot analysis
---------------------
Proteins were isolated from lung tissue lysates. In brief, 20--30 mg of tissues were transferred into bead tubes and homogenized by using a precellys homogenizer (PEQLAB Biotechnologie GmbH, Erlangen, Germany) at 6500 r.p.m. for 1 min. in RIPA lysis buffer (cat\# 89901; Thermo Scientific, Rockford, IL USA) with protease and phosphatase inhibitor cocktail (cat\# 18161284; Thermo Scientific). After homogenization, the samples were centrifuged at 15,000 × g at 4°C for 30 min. The supernatant was collected in new tubes and a colorimetric protein assay kit (Bio-Rad protein assay kit: cat\# 210003399, Munich, Germany) was used to measure the levels of proteins. Before electrophoresis, the samples were mixed with Laemmli buffer \[375 mmol, SDS 10% (w/v), glycerol 50% (v/v), β-mercaptoethanol 12.5% (v/v), bromophenol blue 0.02% (v/v)\] and heated at 95°C for 5 min. followed by centrifugation with a speed of 15,000 × g for 12 sec. before loading. The samples were run on 10% SDS-PAGE (375 mmol Tris/Cl, pH 8.9, 10% acrylamide, 0.20% SDS, 0.05% and 0.10% TEMED) for 75 min. with 80--100 V and transferred on nitrocellulose membrane (cat\# S80209; Pall Corporation, Dreieich, Germany) for 75 min. with 100 V. The membranes were rinsed with TBS/T (tris buffered saline with 1% Tween 20), blocked in 5% milk (cat\# M740; Sigma-Aldrich, St. Louis, MO, USA) and then incubated with mouse monoclonal Oct-3/4 (sc-5279; Santa Cruz, Dallas, TX, USA) antibody in 1:1000 dilution overnight at 4°C. After washing with TBS/T, the membranes were incubated 1 hr with a goat antimouse secondary antibody (1:3000). A Super Signal reagent (cat\# 34096; Thermo Scientific) was used for developing the membrane. The bands were detected by the luminescent image analyser LAS-4000 mini (Fujifilm, Tokyo, Japan). After exposure, the membranes were washed in TBS/T, submerged in 25 ml of stripping buffer (cat\# 21059; Thermo Scientific) and incubated for 1 hr at room temperature (RT) on a shaker plate. Then the membranes were blocked in the milk and the immunodetection protocol was repeated with a mouse monoclonal anti-β-actin antibody 1:5000 (cat\# A5316; Sigma-Aldrich) and the appropriate secondary antibody.
Lung isolation and tissue preparation
-------------------------------------
Mice were anaesthetized with isofluran. A large incision was made up to the neck of the mouse. Ten millilitre PBS was perfused through the right ventricle until the lungs were cleared of blood. After pulling back the skin above the head of the mouse to expose the throat, the rest of the rib cage and other surrounding tissues were carefully removed to expose the trachea. A solution of 4% paraformaldehyde (PFA) was injected into the trachea until the lungs inflate. The lungs were dissected and dropped into a conical tube filled with 15 ml of 4% PFA at 4°C overnight. Thereafter, the lungs were processed for paraffin embedding in a tissue processor (ASP200S; Leica Microsystems, Nussloch GmbH, Germany).
Immunohistochemistry
--------------------
Ten micrometre-thick paraffin-embedded tissue sections were placed at 65°C for 20 min., followed by routine deparaffinization and dehydration steps. To block endogenous peroxidase, the tissue sections were incubated with 2% bovine serum albumin (BSA) containing 0.5% NP-40 (Sigma-Aldrich) and 3% normal goat serum. The slices were stained overnight with mouse monoclonal Oct4 primary antibody diluted 1:50 in 2% BSA with 0.5% NP-40 (Sigma-Aldrich). The tissues were rinsed in PBS and incubated for 1 hr at RT with the appropriate biotin-labelled secondary antibody (M.O.M kit, cat\# PK-2000; Vector Laboratories, Burlingame, ON, Canada) at 1:1000 dilution. After repeated washing steps in PBS, the tissue slides were incubated for 1 hr at RT with the Vecstain® ABC substrate (M.O.M kit). The slides were then washed three times in PBS and incubated with 10 mg 3,3′-diaminobenzidine (cat\# D4168; Sigma-Aldrich) diluted in PBS solution with 30% H~2~O~2~ for 15--30 min. at RT. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Darmstadt, Germany), the slides were mounted with Mowiol (cat\# 475904; Merck Chemical Ltd., Darmstadt, Germany).
Immunofluorescence and confocal microscopy
------------------------------------------
After routine deparaffinization and dehydration, the sections were incubated for 10 min. at 37°C with 1% trypsin (Digest All, cat\# 003008; Invitrogen, Darmstadt, Germany) for antigen unmasking. After washing with PBS, the tissues were blocked with 5% BSA for 1 hr at RT and incubated with the appropriate primary antibodies diluted in 3% BSA with 0.2% TritonX-100 in PBS overnight at 4°C. Mouse monoclonal Oct-3/4 (C-10), dilution 1:50 (sc5279; Santa Cruz), mouse monoclonal anti-vimentin, 1:500 (C 9080; Sigma-Aldrich), monoclonal anti-α-actin smooth muscle conjugated with Cy3 (α-SMA), 1:500 (C6198; Sigma-Aldrich), anti-PDGFR-α, 1:100 (ab90967; Abcam, Cambridge, UK), rabbit polyclonal PDGFR-β, 1: 100 (sc-432; Santa Cruz), rat antimouse Ly-6A/E/Sca-1, 1:100 (cat \# 553333; BD Biosciences, Heidelberg, Germany), rabbit polyclonal C-kit, 1:100 (sc-168; Santa Cruz), rabbit polyclonal VEGF, 1:100 (sc-152; Santa Cruz) were used for the staining. The sections where then incubated with the appropriate secondary antibodies (1:500--1000) for 1 hr at RT, washed with PBS and counterstained with DAPI (1:1000) for 10 min. The slices were mounted with Mowiol and investigated with a Zeiss LSM 710 laser scanning confocal microscope.
Flow cytometry and cell sorting
-------------------------------
The lungs were isolated and filled up with 2 ml dispase (cat\# 354235; BD Bioscience). After mincing with a sterile scalpel, the lung tissue was incubated with 2 μg/ml collagenase B (cat\# 11088815001; Life Sciences, Darmstadt, Germany) and 0.001% DNAse (cat\#18535; Serva Electrophoresis, Heidelberg, Germany) in DMEM for 15 min. at 37°C. The resulting cell suspension was filtered through a 100 μm and 40 μm cell strainers (cat\# 352340; BD Falcon, Heidelberg, Germany), harvested by centrifugation and resuspended in cell blood lysis buffer (0.15 mol NH~4~Cl, 10 mmol KHCO~3~, 0.1 mmol EDTA). After washes in PBS, the cells were resuspended in FACS-buffer (PBS, 2 mmol EDTA, 25 mmol HEPES) at 1 million cells per 100 μl buffer and incubated for 20 min. on ice with a mixture of anti-EpCAM and anti-CD45 antibodies (e-Biosciences, Frankfurt, Germany). The cells were analysed with a BD LSR II flow cytometer by using flowJo7.6.4 software, or were sorted with a BD FACSAria III cell sorter.
Cell culture
------------
Oct4-positive cells were sorted based on GFP fluorescence. The GFP-positive cells were collected in FACS-tubes filled with α-MEM medium (cat\# 41061; Invitrogen) containing 10% foetal calf serum supplemented with penicillin/streptomycin, insulin/transferrin/selenium (cat\# 51500-56; Invitrogen), 2 mmol l-glutamine (cat\# P04-80100; PAN Biotech, Aidenbach, Germany) and 0.0002% heparin (cat\# H3149; Sigma-Aldrich). After centrifugation for 5 min. at 400 × g, the cells were resuspended in MEM medium and kept in culture chamber slides for 5 days in normoxic conditions.
Embryonic stem cells
--------------------
Embryonic stem cell line E14 was derived from inbred mouse strain 129/Ola. The cells were plated in culture chambers coated with 0.2% gelatin and were kept in basal medium supplemented with 20% foetal calf serum, 1.25% sodium puryvate, 1.25% non-essential amino acids, 1.25% penicillin/streptomycin, 0.0088% beta-mercaptoethanol, 0.01% leukaemia inhibitory factor (Gibco, Darmstadt, Germany) and co-cultured with feeders cells.
Laser capture microdissection and quantitative PCR
--------------------------------------------------
Frozen, 10 μm-thick tissues sections were used for the microdissection. The tissues sections were placed in 70% ethanol for 60 sec. and directly passaged in sterile distilled water for 10 sec. The slides were then shortly stained with haematoxylin (cat\# MHS16; Sigma-Aldrich), washed in sterile water and tap water for 10 sec. each. Rapid steps of rehydration with increasing concentrations of ethanol were done to prevent the slides to dry and to maintain the integrity of the RNA. The microdissected samples were collected in tube filled with 350 μl of RLT buffer (Qiagen, Hilden, Germany), vortexed for 30 sec. and RNA were isolated by using the RNeasy Plus Micro Kit (Qiagen® Kit, cat\# 74034). The reverse transcription PCR (RT-PCR) was performed with a cDNA kit from Bio-Rad (iScript™ Select cDNA Synthesis Kit, cat\# 170-8896). Primers used for amplification are shown in Table [1](#tbl1){ref-type="table"}. mRNA levels of different genes were measured by qRT-PCR during 40 cycles of amplification and were expressed as ΔCt values compared to GAPDH housekeeping gene control. Similar PCR protocols were employed for isolated Oct4-positive cells and ESCs.
######
Primers used for determination of mRNA expression levels by qRT-PCR
Primer names Foward primer Reverse primer
-------------- ------------------------------- ---------------------------------
Oct-4 5′-caagttggcgtggagactttgc-3′ 5′-ccccaaggtgatcctcttctgc-3′
Klf4 5′-tgtgactatgcaggctgtggc-3′ 5′-ggccctgtcacacttctggc-3′
Nanog 5′-gaacgcctcatcaatgcctgc-3′ 5′-tgttctcctcctcctcagggc-3
Sox2 5′-gggctctgtggtcaagtccg-3′ 5′-cgctctggtagtgctgggc-3′
Vimentin 5′-gagatcgccacctacaggaa-3′ 5′-tccatctctggtctcaaccg-3′
PDGFR-β 5′-agggggcgtgatgactagc-3′ 5′-ttccaggagtgataccagctt-3′
Sca-1 5′-gcctgcaaccttgtctgag-3′ 5′-cagactccatcagggtaggg-3′
VEGF 5′-ggagatccttcgaggagcactt-3′ 5′-ggcgatttagcagcagatataagaa-3′
C-kit 5′-tgtaaggcctccaacgatgt-3′ 5′-cagagtgtgggcctggattt-3′
GAPDH 5′-tgaggccggtgctgagtatgtcg-3′ 5′-ccacagtcttctgggtggcagtg-3′
Transmission electron microscopy
--------------------------------
Transmission electron microscopy (TEM) was performed on small (1 mm^3^) lung tissue samples, which were processed according to a routine Epon-embedding procedure as previously described \[[@b20],[@b24]--[@b26]\]. Briefly, mouse lung samples were fixed by immersion in 4% glutaraldehyde, post-fixed in 1% OsO~4~ and further processed for Epon embedding. Thin sections (60 nm) were double-stained with uranyl acetate and lead citrate and examined under a Morgagni 268 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) at 80 kV. Digital electron micrographs were recorded with a MegaView III CCD using iTEM-SIS software (Olympus, Soft Imaging System GmbH, Münster, Germany).
Results
=======
Oct4 is expressed in adult mouse lung tissues
---------------------------------------------
Western blot analyses were performed to detect the expression of Oct4 in whole cell lung lysate. Samples from adult wild-type and Oct4-GFP transgenic mice were used. A band of 50 kD was detected by using the C-10 anti-Oct4 antibody in wild-type cell lysates (Fig. [1](#fig01){ref-type="fig"}A) and a band of 27 kD was detected with a GFP antibody in transgenic cell lysates (Fig. [1](#fig01){ref-type="fig"}B).
{#fig01}
Oct4 is expressed in peribronchial and perivascular cells and display TC features
---------------------------------------------------------------------------------
DAB staining and immunofluorescence on paraffin-embedded tissues sections were carried out to determine the localization and the labelling pattern of the Oct4 signal. Both, immunohistochemical and immunofluorescent analyses revealed the presence of Oct4-positive cells in the peribronchial (Fig. [2](#fig02){ref-type="fig"}A) and in the perivascular areas (Fig. [2](#fig02){ref-type="fig"}B). The intracellular distribution of Oct4 was observed in cytoplasmic pattern. Similar pattern of Oct4-positivity by using the C-10 anti-Oct4 antibody has previously been observed in adult stem cells \[[@b27]\].
{#fig02}
Immunoconfocal analysis of lung tissues in Oct4-GFP transgenic mice showed that the cells expressing Oct4 display TC features. Oct4-GFP^pos^ cells showed long extensions and exhibited a strong tendency to create cell-to-cell contacts between them (Fig. [3](#fig03){ref-type="fig"}).
{#fig03}
Oct4-positive cells display TC features in culture
--------------------------------------------------
Oct4-positive cells were sorted based on GFP fluorescence, and the cell population EpCAM^neg^/CD45^neg^/Oct4-GFP^pos^ was identified (Fig. [4](#fig04){ref-type="fig"}). Dead cells were excluded with DAPI (Fig. [4](#fig04){ref-type="fig"}A). Hematopoietic CD45^pos^ cells were excluded from the analysis as well (Fig. [4](#fig04){ref-type="fig"}B). EpCAM^pos^ epithelial cells and EpCAM^neg^ cells were separately analysed (Fig. [4](#fig04){ref-type="fig"}C). A small population (1.3%) of Oct4-GFP^pos^ cells was identified in the EpCAM^neg^ cell fraction (Fig. [4](#fig04){ref-type="fig"}D through F, panel 2). Conversely, Oct4-GFP^pos^ cells were almost absent in the EpCAM^pos^ cell fraction (Fig. [4](#fig04){ref-type="fig"}G through I, panel 3).
{#fig04}
After cell sorting, the Oct4/GFP^+^ cells were plated and kept in culture under normoxic conditions. After 5 days in culture, the cells showed a fusiform shape resembling that of TCs with one (Fig. [5](#fig05){ref-type="fig"}A), two (Fig. [5](#fig05){ref-type="fig"}B) or three (Fig. [5](#fig05){ref-type="fig"}C) extensions. In addition, cells with moniliform extensions were conspicuously observed (Fig. [5](#fig05){ref-type="fig"}C). The immunofluorescence staining of Oct4-positive cells showed that these cells were positive for vimentin (Fig. [5](#fig05){ref-type="fig"}D) and negative for PDGFR-α (Fig. [5](#fig05){ref-type="fig"}E).
{#fig05}
Oct4-positive cells express several TC markers mainly in the perivascular areas
-------------------------------------------------------------------------------
Immunofluorescent confocal analyses carried out on paraffin-embedded tissues showed that Oct4-positive cells expressed several described markers of TCs including vimentin, C-kit, PDGFR-β, Sca-1 and VEGF. Our data showed that Oct4 co-localized with vimentin in both, peribronchial (Fig. [6](#fig06){ref-type="fig"}) and perivascular regions (Fig. [7](#fig07){ref-type="fig"}). In the peribronchial/peribronchiolar areas we found very little co-localization of Oct4 with C-kit (Fig. [8](#fig08){ref-type="fig"}), PDGFR-β (Fig. [9](#fig09){ref-type="fig"}), Sca-1 (Fig. [10](#fig10){ref-type="fig"}) and VEGF (Fig. [11](#fig11){ref-type="fig"}). In contrast, the adventitia of blood vessels showed a marked co-localization of Oct4 with C-kit, PDGFR-β, Sca-1 and VEGF (Figs [12](#fig12){ref-type="fig"}--[15](#fig15){ref-type="fig"}).
{#fig06}
{#fig07}
{#fig08}
{#fig09}
{#fig10}
{#fig11}
{#fig12}
{#fig13}
{#fig14}
{#fig15}
Gene expression of TC markers in adult mouse lung tissues
---------------------------------------------------------
Single cell picking was performed with laser capture microdissection technique at the TC locations. After the dissection (Fig. [16](#fig16){ref-type="fig"}A--C), the cells were collected (Fig. [16](#fig16){ref-type="fig"}D) and the RNA was then isolated. The gene expression levels of the TC markers were determined by real-time quantitative PCR. The data showed that the Oct4 mRNA levels, albeit very low, were similar in vessels, bronchi, and septa. The vimentin, C-kit and Sca-1 mRNA levels were higher in vessels and alveolar septa than in bronchi. The VEGF mRNA expression was the highest in septa, whereas the mRNA levels of PDGFR-β were similar in all three compartments (Fig. [17](#fig17){ref-type="fig"}).
{#fig16}
{#fig17}
Pluripotency-related genes in Oct4-positive cells and ESCs
----------------------------------------------------------
Mounting evidence indicates that maintenance, self-renewal and pluripotency of embryonic or adult stem cells is regulated by several transcription factors such as Nanog, Sox2 and Klf4 \[[@b28]--[@b32]\]. To investigate whether Oct4-positive cells possess a regulatory network similar to that of ESCs we have conducted qRT-PCR analysis of Oct4, Sox2, Nanog and Klf4 transcripts in Oct4-positive cells and mouse ESCs. The results presented in Figure [18](#fig18){ref-type="fig"} show that Oct4 and Sox2 transcripts in Oct4-positive cells is lower than in ESCs. However, the mRNA levels of Nanog and Klf4 was found to be higher in Oct4-positive cells than in ESCs. These data indicate that Oct4-positive cells express a subset of pluripotency-related genes that are also expressed in pluripotent ESCs \[[@b33],[@b34]\].
{#fig18}
Ultrastructure of lung TCs
--------------------------
Transmission electron microscopy showed a network of TCs located beneath bronchiolar epithelium (Fig. [19](#fig19){ref-type="fig"}A), surrounding bronchiolar muscle cells (Fig. [19](#fig19){ref-type="fig"}A and B), and blood vessels (Fig. [19](#fig19){ref-type="fig"}B). Small adherens junctions were found to connect the TCs in this network (Fig. [19](#fig19){ref-type="fig"}A inset). Telocytes showed characteristic ultrastructural features: a thin layer of cytoplasm around nucleus and long telopodes, and cellular prolongations with a narrow emergence from the cellular body (Fig. [19](#fig19){ref-type="fig"}A and B). Telopodes showed very long slender segments (50--150 nm) and dilation (about 500 nm) containing mitochondria and endoplasmic reticulum (Fig. [19](#fig19){ref-type="fig"}B, inset). Telocytes were found to have close contacts with undifferentiated putative stem cells in the perivascular space (Fig. [19](#fig19){ref-type="fig"}C).
{#fig19}
Discussion
==========
The purpose of this study was to characterize and trace Oct4-positive cells in adult mouse lung. The expression of Oct4 in adult tissues is indeed a controversial topic in the field of adult stem cells research \[[@b27],[@b29],[@b30],[@b35]\]. The presence of Oct4 in adult tissue has been indeed related to either PCR product artefacts, Oct4 pseudogenes or to other members of Oct family \[[@b13],[@b35],[@b36]\]. In the present study, we have demonstrated that Oct4 was expressed in adult mouse lung tissues. Oct4 expression was first detected by western blot and immunohistochemical examination of lung tissue sections and revealed that Oct4-positive cells were located in the peribronchial/peribronchiolar and perivascular spaces. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal with that of DAB. The localization of Oct4-positive TCs detected in wild-type or Oct4-GFP transgenic mice were confirmed by electron microscopy. In fact, several recent studies also revealed that TCs are present in the murine pulmonary tissues lining blood vessels and bronchi \[[@b20]\].
In this study, we were able to isolate Oct4-positive cells and maintain them in culture. It is worthy to note that Oct4-positive cells *in vitro* displayed similar structural features with already described characteristics of TCs, isolated from various organs \[[@b16],[@b21],[@b37]\]. In particular, we found similar numbers and shapes of telopodes growing and extending from Oct4-positive cells. These results are in accordance with the observations in isolated and cultured Sca-1 TCs \[[@b21],[@b38]\]. Moreover, we observed that Oct4-positive cells exhibit moniliform prolongations originating from the cell body. These features are also typical for TCs \[[@b38]\].
We have also identified Oct4-positive cells in the peripheral mononuclear blood cell population but we did not focused on this cell type. They were discriminated during the isolation with an anti-CD45 antibody and therefore, only the EpCAM^neg^/CD45^neg^/Oct4-GFP^pos^ cell fraction was characterized. Moreover, immunostaining of isolated and cultured Oct4-GFP^pos^ cells showed that these cells were positive for vimentin and negative for PDGFR-α, a marker that is more representative of fibroblast cell lineages. Thus, these data provide another evidence that Oct4-positive cells are TCs having mesenchymal origin by expressing vimentin but they are not fibroblasts because of the lack of PDGFR-α expression. Our results are in agreement with studies comparing the gene expression profile of TCs to that of fibroblasts in murine lung and clearly demonstrate that TCs are not fibroblasts \[[@b39]\]. In addition, numerous studies have demonstrated that TCs have different ultrastructure and phenotype than fibroblasts (for review see: \[[@b17],[@b21]\]). Moreover, the present study based on negative selection with EpCAM, provide additional evidence that EpCAM^neg^/CD45^neg^/Oct4-GFP^pos^ cells are not epithelial cells but represent a subtype of Oct4-positive TCs.
By immunofluorescence, we confirmed that Oct4-positive cells share several markers with TCs including C-kit, PDGFR-β, Sca-1 and VEGF. It is important to mention that such a co-localization was mainly observed in the perivascular areas, while in the peribronchial/peribronchiolar spaces showed little co-localization. These observations suggest that TCs in the lung represent, at least immunophenotypically, a heterogenous cell type. The expression of the different above mentioned markers were confirmed on the mRNA level by qRT-PCR of cells captured with laser microdissection from different locations including blood vessels, bronchi and alveolar septa. It is obvious that the mRNA levels of the above mentioned TC markers do not fully concur with the immunohistochemical analyses. This especially applies to the peribronchial TCs. However, it is possible that such a difference in the expression profile might be because of the contamination with other Oct4-negative cells which might have a different expression profile.
In summary, we were able to show that Oct4 is expressed in adult mouse lung. In addition, we have demonstrated that Oct4-positive cells closely resemble the morphology of TCs seen by electron microscopy. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration.
Conflicts of interest
=====================
The authors declare that there are no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Mammography using two views (craniocaudal in addition to the standard mediolateral oblique view) became standard practice in the National Health Service Breast Screening Programme (NHSBSP) at prevalent (first) screens in 1995, with single-view mammography used at incident (second and subsequent) screens. This followed the UKCCCR randomised controlled trial comparing the cancer detection rate achieved when using one *vs* two-view mammography during routine breast screening. Results showed a 24% increase in cancer detection with two-view mammography ([@bib16]).
Further research was undertaken, including a number of observational studies, which found that programmes using two-view mammography saw an 8.9% increase in sensitivity (true cancer detection) for invasive cancer detection compared with those using a single view only ([@bib4]). This led to the decision that two-view mammography would be undertaken at all incident screens as well as prevalent screens. The aim was for all units in the United Kingdom to be using two-view mammography at all screening rounds by December 2003 ([@bib13]) but in practice, only 81% of units (74 out of 91 units) had achieved this target. All screening units in England, Wales and Northern Ireland were using two-view mammography at incident screens by 1 December 2004. The programme in Scotland adopted two-view mammography in 2008 ([@bib12]). It should be noted that screening units in Northern Ireland used two-view mammography at both prevalent and incident screens when the breast screening programme was introduced in 1989.
Assessment of the impact of the introduction of two-view *vs* one-view mammography has been carried out in relation to screen-detected cancer and recall rates. [@bib3] found the overall rate of invasive and *in situ* cancers detected at screen increased by 0.65 per 1 000 women screened following the introduction of two-view mammography with recall rates falling by 0.27 per 1 000 women screened .To date, there has been no assessment on the impact of the introduction of two views on interval cancer rates in the UK screening programme; therefore, this is what we explore in this study. We focus on the UK breast screening programme as the screening regimes in other countries differ to that in the United Kingdom. In particular, the interval between routine screening episodes varies; in the United Kingdom, it is 36 months whereas many other European countries with national screening programmes have a screening interval of 24 months ([@bib8]) and thus, it would be difficult to compare changes in interval cancer rates. Also, we were particularly keen to assess whether the recent policy change in the United Kingdom, in relation to the use of two-view mammography at incident screens, has been of equal benefit for interval cancer rates as it has been for screen-detected cancer rates.
Interval cancers are those that occur symptomatically in the interval between routine screening episodes. These can be cancers which were missed by screening, cancers which became screen detectable after the screen and then became symptomatic, and cancers not detectable by mammography ([@bib17]). Interval cancer rates provide a good indicator of the performance of a screening programme; therefore, assessment of the change in interval cancer rates following the introduction of two-view mammography is of interest.
Materials and Methods
=====================
The NHSBSP currently invites women aged 50--70 to attend breast screening every 3 years. Screening years, and subsequent reporting, run from 1st April to 31st March the following year. Expansion of the age range to 47--73 is being introduced, in a randomised fashion, to enable full evaluation of the benefit of expanding the screening age. We collated annual screening and interval cancer data for all UK breast screening units for the screening years 2003/04 and 2004/05 from the NHS Information Centre and the Breast Screening Quality Assurance Reference Centres (QARCs), respectively. Accurate assessment of interval cancer rates is often hampered by the poor availability of necessary data. The NHSBSP, however, has a rigorous system in place to ensure all screening and interval cancer data are correctly identified and recorded. Cancer registries are responsible for the identification of all breast cancers diagnosed in their catchment area, including interval cancers. Data are then collated on a regional level by QARCs, who assign a screening history to each case of breast cancer diagnosed in women eligible for screening, from which the cancer status can be identified. The QARCs assess these data against numerous quality indictors to ensure ascertainment is good before being made available for analysis ([@bib10]). This is a process that can take some time, particularly in the identification of interval cancers, as they can occur up to 3 years after a women\'s previous screen. The most recent data available for analysis, during which both one- and two-view mammography was in use, were for screening years 2003/04 and 2004/05. Also, over 50% of screening units introduced two-view mammography at incident screen in screening year 2002/03; therefore, any analysis undertaken prior to 2003/04 would not have had a sufficient number of units which had fully implemented two-view mammography at incident screen for a complete screening year.
Only interval cancers occurring within 36 months of a woman\'s last negative screen were included, in line with the NHSBSP screening guidelines for analysing interval cancers.
Only women aged 50--64 at last screen were included in the analysis as not all units had extended the screening age to 70 years during the study period. Data relating to routine screening were available for all 91 screening units in the United Kingdom for both screening years and included details of the number of women screened and the number of cancers detected. Interval cancer data were available for all screening units for both screening years, apart from the six screening units in Scotland, who were only able to provide data for screening year 2003/04.
As two-view mammography for incident screens was in the process of being introduced during our study period, we categorised units as follows: Full two view: the screening unit carried out two-view mammography for the whole screening year of interest;Partial two view: the screening unit introduced two-view mammography at some stage during the screening year of interest;One view only: the screening unit carried out one-view mammography only during the screening year of interest.
Interval cancers were defined as cancers diagnosed following a normal screening result during the interval beginning with the closure of the previous NHS screening episode and ending when the next screening episode was due to commence ([@bib10]). The dates to be used for the screening episodes were the date when the last screening mammogram was taken and the date of the next routine screening appointment, where this was less than 36 months. If the next appointment was more than 36 months after the last mammogram, only interval cancers up to 36 months after the latter were included. The way in which round length is defined means that the achievement of the national standard is not affected by women choosing to alter their screening appointment -- a woman\'s round length is determined by the date of first offered appointment (DOFOA) ([@bib11]). A screening episode is closed when the screening outcome is known or 180 days after DOFOA in the case of non-attenders.
The aim of this analysis was to assess the effect of the introduction of two-view mammography on interval cancer rates in the NHSBSP. Confidentiality regulations restricted the availability of individual data, so we were unable to conduct analyses on a per-patient level. We calculated the rates of interval cancers per 1 000 women screened by the two-view mammography status of the unit as defined above. We assessed the rates of invasive interval cancers only and of all interval cancers; and for each screening year separately and the two screening years combined. Poisson regression was used to assess whether the rates varied by two-view status. Analyses were performed using STATA Version 11.2 (College Station, TX, USA; [@bib15]).
As interval length, that is, the length of time elapsing between two consecutive screens, varied between units, we repeated the analysis restricting it to interval cancers that occurred within 24 months following a negative screen.
Where regional boundaries changed during the period under scrutiny, the configuration of regions and responsibilities for screening units was taken to be that which existed in screening year 2009/10, when the interval cancer data were first compiled. Five pairs of screening units merged prior to this but as the introduction of two-view mammography occurred in the same screening year for each merged pair, the change in configuration will not have had an impact on the results of this study. One new screening unit was opened in the South West region during screening year 2004/05. This unit was excluded. Therefore, data from 91 screening units in the United Kingdom were included in the study.
Results
=======
A total of 1 400 613 women were screened across 91 screening units in 2003/04, giving an average of 15 391 women screened per unit (range 3 835--45 632). A total of 9 957 cancers were detected as a result of screening (unit range 22--309) giving an average rate of 7.11 cancers detected per 1 000 women screened. As data for Scotland were unavailable for screening year 2004/05, data on 1 278 989 women screened across 85 units (unit range 3 979--33 521) were available for analysis. This resulted in 9 192 cancers being detected (unit range 33--251) leading to an average rate of 7.19 cancers detected per 1 000 women screened. A total of 4 518 interval cancers were identified following screens in 2003/04 and 3 915 following screens in 2004/05, of which 4 212 (93.2%) and 3 665 (93.6%) respectively, were invasive cancers.
[Table 1](#tbl1){ref-type="table"} shows the number of units by two-view mammography status for each screening year. In 2003/04, 70.3% units had fully implemented two-view mammography; this had risen to 87.9% in 2004/05.
The interval cancer rate decreased upon the implementation of two-view mammography in screening year 2003/04 ([Table 2](#tbl2){ref-type="table"}). There was a 17% reduction (RR: 0.83 (95% CI 0.77--0.90, *P*\<0.001)) in interval cancers occurring following screening using two-view mammography compared with single-view mammography. A slightly smaller but significant reduction was also seen when comparing interval cancer rates in units which introduced two views at some stage during the screening year compared with those units that used one-view mammography only (RR: 0.86 (95% CI 0.78--0.95, *P*=0.004)).
By 2004/05, all screening units had introduced two-view mammography either before the start of, or at some stage during, the screening year. There was a significant reduction in interval cancers of 13% (RR: 0.87 (95% CI 0.76--0.99, *P*=0.035)) in units which used two-view mammography throughout the screening year compared with units who introduced it part way through.
Combining the data from both screening years produced similar results to those seen when analysing 2003/04 data only. There was an 18% reduction (RR: 0.82 (95% CI 0.76--0.88, *P*\<0.001)) in interval cancers occurring following a screen using two-view mammography for the whole screening period compared with units which used single view only.
We repeated the analysis to assess the effect of the introduction of two-view mammography on invasive interval cancers only ([Table 3](#tbl3){ref-type="table"}). There was no significant difference in interval cancer rates following screening in year 2003/04 between units which had introduced two-view mammography part way through the year compared with units using one-view mammography only. There was, however, a 10% reduction (RR: 0.90 (95% CI 0.83--0.98, *P*=0.017)) of invasive interval cancers occurring in units using two-view mammography for the whole screening year compared with one-view mammography only.
When analysing women screened in 2004/05, there was a 14% reduction (RR: 0.86 (95% CI 0.75--0.98, *P*=0.027)) in the number of women who developed invasive interval cancers following screening at units that had fully implemented two-view mammography compared with units that introduced it part way through the year. This is similar to the reduction seen for all interval cancers.
For both screening years combined, there was no significant difference in interval cancer rates between units that used one-view mammography only and units that introduced two-view mammography part way through the screening year. However, there was a significant 12% reduction (RR: 0.88 (95% CI 0.81--0.96, *P*=0.003)) in invasive interval cancer rates for women screened at units using two-view mammography compared with those using one-view mammography only.
Repeating the analysis restricting the diagnosis of interval cancer to within 24 months following a negative routine screen produced similar results to those shown in [Tables 2](#tbl2){ref-type="table"} and [3](#tbl3){ref-type="table"}. However, ∼44% of interval cancers were diagnosed in the third year following a negative routine screen in both screening years 2003/04 and 2004/05 (1 958/4 518 and 1 728/3 915, respectively) and thus the reduction in sample size led to wider confidence intervals. Results are shown in [Tables 4](#tbl4){ref-type="table"} and [5](#tbl5){ref-type="table"}.
Discussion
==========
The introduction of two-view mammography has led to a reduction in interval cancers occurring following a negative routine screen in comparison with one-view mammography. This is consistent with a corresponding rise in the number of cancers detected at screening ([@bib3]). The reduction in all interval cancer rates associated with two-view mammography was 0.68 per 1 000 screened ([Table 2](#tbl2){ref-type="table"}, both screening years combined), very close to the 0.65 per 1 000 increase from 5.87 to 6.52 in screen-detection rates in the Study Group of Blanks and colleagues ([@bib3]). This indicates that the increase in screen-detection rates with two-view mammography is not likely to be attributable to overdiagnosis. Overdiagnosis is the detection, by screening, of cancers that would never have come to clinical attention had screening not taken place ([@bib7]). If the decrease in interval cancer rates was smaller than the increase in screen-detected cancers, then the excess in screen-detected cancers following the introduction of two-view mammography may be as a result of overdiagnosis. However, given that the increase in screen-detected cancers is consistent with the decrease in interval cancers, the number of additional screen-detected cancers using two-view mammography, which are attributable to overdiagnosis, is likely to be minimal. There is always room for argument about overdiagnosis, as the phenomenon cannot be observed at an individual level. However, the results here suggest that at least there is no cause for alarm in this respect over the change to two-view mammography. Further observation of both screen-detected and interval cancer rates in the future will be useful in terms of likely overdiagnosis due to other innovations such as digital mammography.
A study in France found a larger proportional effect of changing from single- to two-view mammography on two-year interval cancer rates, finding a reduction of more than 30% ([@bib14]). However, this study compared universal two-view with universal single view rather than a change for incident screens only, as in our case.
Interval cancers tend to have poorer stage and poorer prognosis as one would expect ([@bib2]; [@bib9]; [@bib6]). Interval cancer rates are in the order of 2--3 per 1 000 screened in the UK programme ([@bib1]). This is somewhat less than 50% of tumours diagnosed in women attending the programme, but still a non-negligible number. Results from other countries with more frequent screening are consistent with this ([@bib5]). Clearly, reduction in interval cancer incidence is a suitable target for screening programmes.
The benefit seen following the introduction of two-view mammography was larger when analysing all interval cancers than when restricting to invasive interval cancers only ([Tables 2](#tbl2){ref-type="table"} and [3](#tbl3){ref-type="table"}). This was not simply a feature of greater statistical power with larger numbers, which would lead to smaller standard errors (s.e.) and thus more precise estimates of rates. The absolute and relative reductions in interval cancer rates were also larger when non-invasive cancers are included. This is consistent with the observation that compared with contemporaneous changes in a reference group not using two-view mammography, the proportional increase in detection of *in situ* tumours with two-view mammography was greater than for invasive ([@bib3]).
This study uses data on interval cancers from almost all units in the United Kingdom over a 2-year period and thus benefits from large number of women screened and subsequent interval cancers recorded. Also, as the breast screening programme operates on a national level and has a rigorous call/recall system in place, we can be confident that the interval cancer rates calculated accurately reflect the true rates occurring in the population eligible for screening. Many other countries do not have similarly organised population-based screening programmes in place, thus making it difficult to clearly distinguish interval cancers from those detected at screening or in non-attenders.
Assessment of potential confounding factors, such as age at screening, was not possible due to confidentiality restrictions meaning we were unable to obtain data at an individual patient level. Comparison of interval cancer rates by unit may therefore have introduced potential confounders at a geographical level, such as variation in underlying incidence or the recording of interval cancers, or the possibility that units which were quicker to change to two-view mammography are more effective in identifying interval cancers *a priori* than those still using one view. However, the NHSBSP has a robust quality assurance system in place to ensure that all cancers that occur in women eligible for screening are identified and classified appropriately as well as having strict screening guidelines in place to ensure that all women receive the same high quality level of care when attending screening. Incidence does vary geographically within the United Kingdom, but it is uniformly high. In any case, as interval cancers following subsequent years of screening become available, it will be possible to track interval cancer rates over time by individual screening unit, and assess whether changes in rates are consistent with the time of the move to two-view mammography. By considering changes within rather than between units, geographical/unit level confounders can be controlled for.
In conclusion, this study found that the introduction of two-view mammography at incident screens was accompanied by a 15--20% reduction in interval cancer rates in the NHSBSP. This, therefore, supports the decision at national level to change to two-view mammography at incident screens. In Europe, there are a number of programmes which still use single-view mammography at incident screens ([@bib8]). These results will have relevance for those programmes.
We thank the staff at the screening units and QARCs whose hard work provided the data for this paper. SWD, AD and DP contributed to this work as part of the programme of the Policy Research Unit in Cancer Awareness, Screening and Early Diagnosis. The Policy Research Unit in Cancer Awareness, Screening and Early Diagnosis receives funding for a research programme from the Department of Health Policy Research Programme. It is a collaboration between researchers from seven institutions (Queen Mary University of London, UCL, King\'s College London, London School of Hygiene and Tropical Medicine, Hull York Medical School, Durham University and Peninsula Medical School).
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 3.0 Unported License.
The authors declare no conflict of interest.
###### Two-view mammography status of screening units by screening year
**Mammography Status** **1 April 2003 to 31 March 2004** **1 April 2004 to 31 March 2005**
------------------------ ----------------------------------- -----------------------------------
One view only 11 6[a](#t1-fn1){ref-type="fn"}
Partial two view 16 5
Full two view 64 80
Scottish units for whom we do not have interval cancer data following screening year 2004/05.
###### Interval cancer rates by screening year
**Mammography in use** **Number of women screened** **Number of interval cancers** **Rate per 1 000 women screened** **Rate ratio (95% CI)**
----------------------------------- -------------------------------- -------------------------------- ----------------------------------- --------------------------------
**1 April 2003 to 31 March 2004**
One view only 195 224 732 3.75 Baseline
Partial two view 249 865 808 3.23 0.86 (0.78--0.95)
Full two view 955 524 2 978 3.12 0.83 (0.77--0.90)
**1 April 2004 to 31 March 2005**
One view only N/A[a](#t2-fn2){ref-type="fn"} N/A[a](#t2-fn2){ref-type="fn"} N/A[a](#t2-fn2){ref-type="fn"} N/A[a](#t2-fn2){ref-type="fn"}
Partial two view 68 379 239 3.5 Baseline
Full two view 1 210 610 3 676 3.04 0.87 (0.76--0.99)
**1 April 2003 to 31 March 2005**
One view only 195 224 732 3.75 Baseline
Partial two view 318 244 1 047 3.29 0.88 (0.80--0.96)
Full two view 2 166 134 6 654 3.07 0.82 (0.76--0.88)
Abbreviation: CI=confidence interval.
Only six units in Scotland used one view only in 2004/05, and for these units data were not available.
###### Invasive interval cancer rates by screening year
**Mammography in use** **Number of women screened** **Number of interval cancers** **Rate per 1 000 women screened** **Rate ratio (95% CI)**
----------------------------------- -------------------------------- -------------------------------- ----------------------------------- --------------------------------
**1 April 2003 to 31 March 2004**
One view only 195 224 638 3.27 Baseline
Partial two view 249 865 761 3.05 0.93 (0.84--1.04)
Full two view 955 524 2 813 2.94 0.90 (0.83--0.98)
**1 April 2004 to 31 March 2005**
One view only N/A[a](#t3-fn2){ref-type="fn"} N/A[a](#t3-fn2){ref-type="fn"} N/A[a](#t3-fn2){ref-type="fn"} N/A[a](#t3-fn2){ref-type="fn"}
Partial two view 68 379 226 3.31 Baseline
Full two view 1 210 610 3 439 2.84 0.86 (0.75--0.98)
**1 April 2003 to 31 March 2005**
One view only 195 224 638 3.27 Baseline
Partial two view 318 244 987 3.10 0.95 (0.86--1.05)
Full two view 2 166 134 6 252 2.89 0.88 (0.81--0.96)
Abbreviations: CI=confidence interval.
Only six units in Scotland used one view only in 2004/05, and for these units data were not available.
###### Interval cancer rates by screening year for cancers diagnosed within 24 months of last routine screen
**Mammography in use** **Number of women screened** **Number of interval cancers** **Rate per 1 000 women screened** **Rate ratio (95% CI)**
----------------------------------- -------------------------------- -------------------------------- ----------------------------------- --------------------------------
**1 April 2003 to 31 March 2004**
One view only 195 224 399 2.04 Baseline
Partial two view 249 865 465 1.86 0.91 (0.80--1.04)
Full two view 955 524 1 696 1.78 0.87 (0.78--0.97)
**1 April 2004 to 31 March 2005**
One view only N/A[a](#t4-fn2){ref-type="fn"} N/A[a](#t4-fn2){ref-type="fn"} N/A[a](#t4-fn2){ref-type="fn"} N/A[a](#t4-fn2){ref-type="fn"}
Partial two view 68 379 127 1.89 Baseline
Full two view 1 210 610 2 060 1.7 0.92 (0.77--1.10)
**1 April 2003 to 31 March 2005**
One view only 195 224 399 2.04 Baseline
Partial two view 318 244 592 1.86 0.91 (0.80--1.03)
Full two view 2 166 134 3 756 1.73 0.85 (0.77--0.94)
Abbreviation: CI=confidence interval.
Only six units in Scotland used one view only in 2004/05, and for these units data were not available.
###### Invasive interval cancer rates by screening year for cancers diagnosed within 24 months of last routine screen
**Mammography in use** **Number of women screened** **Number of interval cancers** **Rate per 1 000 women screened** **Rate ratio (95% CI)**
----------------------------------- -------------------------------- -------------------------------- ----------------------------------- --------------------------------
**1 April 2003 to 31 March 2004**
One view only 195 224 355 1.82 Baseline
Partial two view 249 865 435 1.74 0.96 (0.83--1.10)
Full two view 955 524 1 589 1.66 0.91 (0.82--1.03)
**1 April 2004 to 31 March 2005**
One view only N/A[a](#t5-fn2){ref-type="fn"} N/A[a](#t5-fn2){ref-type="fn"} N/A[a](#t5-fn2){ref-type="fn"} N/A[a](#t5-fn2){ref-type="fn"}
Partial two view 68 379 120 1.76 Baseline
Full two view 1 210 610 1 914 1.58 0.90 (0.75--1.08)
**1 April 2003 to 31 March 2005**
One view only 195 224 355 1.82 Baseline
Partial two view 318 244 555 1.74 0.96 (0.84--1.10)
Full two view 2 166 134 3 503 1.62 0.89 (0.80--1.00)
Abbreviation: CI=confidence interval.
Only six units in Scotland used one view only in 2004/05, and for these units data were not available.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
The effective exchange of oxygen and carbon-dioxide between air and blood within the lungs is arguably the most important process for sustaining mammalian life. In order to transport oxygen from fresh air to the gas exchange surfaces, and to remove carbon dioxide from within the lungs to the atmosphere, the incoming gas must be mixed with the resident gas that remains within the lungs. Understanding the mechanisms behind this transport is critical for understanding the effects of lung disease on lung function, for designing optimum mechanical ventilation (MV) techniques, and for optimizing inhaled drug delivery.
Airflow in the lungs is typically thought of as being generated by the diaphragm and chest wall, yet pressure oscillations caused by the beating heart, termed cardiogenic oscillations (CO), can also generate significant airflows^[@CR1]^. These oscillations can be observed from birth^[@CR2]^, and have been discussed as a potential mechanism for apneic respiration (i.e. respiration during breath-holding) in crocodilians^[@CR3]^ and hibernating mammals^[@CR4],[@CR5]^. Cardiogenic oscillations have also been shown to influence aerosol deposition in humans^[@CR6]--[@CR8]^.
Cardiogenic oscillating flows were first measured directly in the lobar airways by West and Hugh-Jones^[@CR1]^, who postulated that the phase shifts between these oscillations may cause internal flows with the potential to enhance gas mixing in the lungs.
Hyperpolarized He^[@CR3]^ MRI has been used to investigate cardiogenic flows in the lungs of humans^[@CR9],[@CR10]^. These studies found significant cardiogenic flows in the lungs that resulted in flow of air between the left and right lungs^[@CR9]^ and also between the upper and lower regions of the left lung^[@CR10]^. Due to the spatial resolution of the MRI technique used, these investigations were limited to measuring flow only at the main bronchi. In fact, Collier *et al*.^[@CR10]^ concluded that quantitative measurement of cardiogenic flow at the lobar scale or finer is necessary to fully assess the magnitude and consequence of cardiogenic effects in the lungs.
Recently, we have developed functional lung imaging using synchrotron-based 4D-CT, and have demonstrated the capability to measure airflow throughout the entire airway tree in mice^[@CR11]^. This method has been applied to models of disease, revealing the regional patterns of disease^[@CR12]^. Additionally, a two-dimensional variant of this method has revealed patterns of lung tissue oscillation under high-frequency ventilation^[@CR13]^ and forced oscillation technique for lung function measurement^[@CR14]^.
Using these advances, we present here, for the first-time, detailed measurement of tissue displacement, tissue expansion and flow in the airway tree due to cardiogenic oscillations within the mouse lung. These quantitative measurements allowed us to digitally isolate the effects of mechanical ventilation and cardiogenic oscillations, without the need to alter normal physiological conditions. These measurements allowed us to establish the pattern and magnitude of cardiogenic flows within the lung. Using a numerical modelling analysis based on virtual tracers, we use this new data to investigate the contribution of cardiogenic oscillations to the enhancement of gas mixing.
To investigate the relative contributions of each type of flow (MV and CO), analysis was performed on each of three mice under 4 states: (1) DIFF: zero flow (molecular diffusion only), (2) CARD: cardiogenic oscillations only (no mechanical ventilation) (3) VENT: mechanical ventilation (no cardiogenic oscillations) (4) BOTH: cardiogenic oscillations plus mechanical ventilation. The magnitude and distribution of the resulting tissue displacement, expansion and airway flow due to MV and CO was investigated. The contribution of MV and CO to gas mixing for each state was deduced through a novel gas tracer technique, whereby massless tracers allow visualization and analysis of the gas transport within the lung (see Methods for details).
Results {#Sec2}
=======
Magnitude and distribution of cardiogenic oscillations {#Sec3}
------------------------------------------------------
The dynamic tissue motion due to mechanical ventilation and cardiogenic oscillations are visualized in Supplementary Movie [S1](#MOESM2){ref-type="media"}. The influence of the heart on the lung tissue is clearly apparent throughout both the left and right lungs.
The local displacement and tidal volume of lung tissue resulting from MV and CO for Mouse 2 are shown in Fig. [1](#Fig1){ref-type="fig"}. Displacement was calculated as the total translation of each region of tissue over the relevant cycle (either mechanical ventilation cycle or heart beat). The fractional tidal volume is defined as the change in volume of each region of tissue over the cycle (maximum volume minus minimum volume) divided by the initial volume of the region. MV caused large displacements of the lung tissue, with a distinct cranio-caudal gradient. The tidal volume under MV is more evenly distributed throughout the lung. The CO causes significant displacement and expansions of the lung tissue, of similar maximum magnitude to the MV in some areas adjacent to the heart. The effects of CO on the lung tissue appear highly localized to the vicinity of the heart (predominantly in the left lobe and cardiac lobe).Figure 1Regional tissue displacement (top) and tidal volume (bottom) for mechanical ventilation (left) and cardiogenic oscillations (right) for Mouse 2. The displacement and tidal volume generated by the cardiogenic oscillations of the heart are localized to the vicinity of the heart.
Total lung volume vs. time for CARD, VENT and BOTH are shown in Fig. [2A](#Fig2){ref-type="fig"}. The BOTH and VENT curves are very similar, although some differences are apparent due to the addition of CO. The relative magnitude of CARD is clearly lower than for VENT and BOTH, but the frequency of the cycle is increased, reflecting the frequency of the heartrate relative to the ventilation cycle. The faster cycle of the heart (\~3× the ventilation cycle) leads to a relative increase in minute volume (compared to tidal volume), as is apparent in Fig. [2B,C](#Fig2){ref-type="fig"}, resulting in minute volumes generated by CO in isolation to be between 13% and 20% of the minute volume generated by MV.Figure 2Lung volume (**A**), tidal volume (**B**) and minute volume (**C**) generated by mechanical ventilation and cardiogenic oscillations. The oscillations in global lung volume measured at the trachea are smaller in amplitude than the tidal ventilation, but occur at a higher frequency.
Flow in the airways {#Sec4}
-------------------
Gas flow through any bifurcation in the airway tree can be classified into one of three regimes (Fig. [3](#Fig3){ref-type="fig"}). Bulk flow is the unidirectional flow from parent to siblings (or vice versa) that transports gas between the trachea to the alveoli. Pendelluft is flow between two sibling airways that transports gas internally between adjoining lung regions, which is thought to occur during the transition between inspiration and expiration due to differences in compliance between lung units^[@CR15]^. A combination of these two flows may also be present (mixed flow).Figure 3Flow regimes at a bifurcation in the airway tree. Bulk flow transports gas between the trachea and alveoli, while pendelluft acts to redistribute gas between different lung regions.
A novel gas tracer modelling analysis was developed to visualize the gas transport within the lungs due to MV and CO. The airway tree was numerically seeded with virtual massless tracers (representing gas molecules) that follow the measured flow through the airways (see Methods for details). The tracer paths were visualised to reveal the transport of gas within the airway tree.
The dynamic tracer visualizations for MV and CO are shown in Fig. [4](#Fig4){ref-type="fig"} and Supplementary Movies [2](#MOESM3){ref-type="media"} and [3](#MOESM4){ref-type="media"}. During MV inspiration, the gas flows are predominantly direct from the trachea to the periphery. During expiration, gas flows are predominantly from the periphery to the trachea via a similar path but reversed pathway as MV inspiration. However, during CO, the gas oscillates along these pathways, and also exhibits pendelluft flow during much of the cycle.Figure 4Single frames from Supplementary Movie [S2](#MOESM3){ref-type="media"} (left) and Supplementary Movie [S3](#MOESM4){ref-type="media"} (right), showing tracer paths in mechanical ventilation during inspiration (left) and cardiogenic flows (right). Red paths indicate flow travelling in the direction of ascending generations (ie from trachea to the periphery) and blue indicates flow travelling through descending generations. The ventilation flows demonstrate a single serial path of tracers from the trachea to the periphery. In contrast, the cardiogenic flows show pendelluft and redistribution of flows between lung regions, with both blue and red tracer paths apparent at this time-point.
The change in total lung volume, as shown in Fig. [2A](#Fig2){ref-type="fig"}, may underestimate the effects of CO, as any redistribution of gas internally to the lung due to pendelluft will not incur a change in total volume. To more accurately assess the contribution of CO to gas transport in the lungs, we defined Total Internal Flow: Q~tot~ = \|Q~A~\| + \|Q~B~\| + \|Q~C~\|, where Q~A~, Q~B~ and Q~C~ denote the flow through airways A, B and C respectively, as labeled in Fig. [3](#Fig3){ref-type="fig"}. The bulk flow can be determined as Q~bulk~ = \|Q~A~ + Q~B~ + Q~C~\| and pendelluft as the difference Q~pend~ = Q~tot~ − Q~bulk~. Note that although Fig. [3](#Fig3){ref-type="fig"} shows a single bifurcation, these calculations can be performed across the entire airway tree.
Figure [5](#Fig5){ref-type="fig"} shows Q~pend~, Q~bulk~, and Q~tot~ for CARD, VENT, and BOTH for each mouse, integrated over time and expressed as ml/s. Q~tot~ is significantly higher for VENT and BOTH when compared to CARD. However, a large proportion of Q~tot~ for CARD is made up of pendelluft. (35--41%). In fact, Q~pend~ for CARD is between 14 and 21 times larger than for VENT, and around 5 times larger than for BOTH.Figure 5Pendelluft and bulk flow for CARD, VENT and BOTH. Q~tot~ is the sum of Q~pend~ and Q~bulk~, represented as the total height of the columns in the figure. Although Q~tot~ is far greater in VENT and BOTH, Q~pend~ is greater for CARD.
Gas mixing {#Sec5}
----------
The contribution of cardiogenic oscillations to gas mixing was investigated using the gas tracer analysis. The lung was initially seeded with particles (Type A), representing resident gas. Tracers incoming to the trachea were assigned to Type B, representing fresh gas. As the incoming tracers mixed with the initial tracers, and the initial tracers are expelled through the trachea opening, the concentration of Type A tracers within the lung is reduced. This is analogous to an inert gas washout. Enhanced gas mixing within the lung will increase the proportion of resident gas within the expired air, thus increasing the rate at which the Type A particle concentration is reduced within the lung. Figure [6](#Fig6){ref-type="fig"} shows concentration through time of Type A particles, calculated at end inspiration, for each mouse under the 4 states: DIFF, CARD, VENT and BOTH. Compared to DIFF, the introduction of CO in the CARD state increased mixing in the lung considerably. However, with MV present, the addition of CO provided only a small enhancement to the gas mixing within the lung.Figure 6Concentration profiles for gas mixing. Four cases are shown: DIFF, CARD, VENT, and BOTH. Cardiogenic flows produce a significant increase in gas mixing over the diffusion only case. However, the addition of cardiogenic flows provides only a minor enhancement to mixing under mechanical ventilation.
The mixing enhancement due to CO is shown in Fig. [7](#Fig7){ref-type="fig"}. This enhancement was calculated as the ratio of the concentrations of fresh gas in the system (Type B tracers) with cardiogenic oscillations compared to without (CARD:DIFF and BOTH:VENT) over time. CO provided up to 4 times greater mixing in the absence of mechanical ventilation (Fig. [7](#Fig7){ref-type="fig"}, left). However, only minimal enhancement is seen with MV present (Fig. [7](#Fig7){ref-type="fig"}, right).Figure 7Mixing ratio CARD:DIFF (left) and BOTH:VENT (right). Cardiogenic oscillations produce up to 4 times mixing compared to diffusion only. However, in the presence of mechanical ventilation, mixing enhancement due to cardiogenic oscillations is approximately 2%. Note the vertical axis scales are different for the two plots for clarity.
Discussion {#Sec6}
==========
Using high-speed dynamic synchrotron-based imaging we have, for the first time, acquired detailed measurements of the effects of cardiogenic oscillations on lung tissue displacement, expansion and flow in the lungs. Additionally, we used a novel gas tracer modelling analysis to investigate the effects of cardiogenic oscillations on gas mixing. Tissue displacement and tidal volume maps showed large perturbations due to CO that were most prominent near the heart. Qualitative and quantitative analysis of the airflow within the airway tree showed significant pendelluft due to CO, but this was reduced in the presence of mechanical ventilation. Likewise, CO produced enhanced mixing during the breath-hold state (up to 4 times), but only limited enhancement was seen due to CO in the presence of MV (\~2%). This suggests that pendelluft present within the lung may be an effective gas mixing mechanism, albeit only in the presence of reduced tidal flows.
The effect of cardiogenic oscillations on gas mixing have been previously studied experimentally using gas washout or bolus dispersion measurements^[@CR16]--[@CR21]^. The use of gas washout or bolus dispersion can provide an overall inference of the gas mixing enhancement due to cardiogenic effects. However, these techniques are limited to either global lung measurements, or at best measurements in only a small number of airways. These studies produced conflicting results on the significance of cardiogenic mixing. For example, Engel *et al*.^[@CR16]^ found up to 5 times increase in mixing due to cardiogenic oscillations, whilst Schell *et al*.^[@CR20]^ concluded that there was a limited effect of cardiac action on gas mixing.
A potential contributing factor to these conflicting results is the varied experimental conditions used that may have significantly affected the resulting measurements, such as using open-chest post-mortem measurements^[@CR16]--[@CR18]^, temporary cardiac arrest to stop the heart^[@CR20],[@CR21]^, or exercise to alter the cardiac output^[@CR19]^. Additionally, these studies have performed measurements either under breath-hold conditions^[@CR16],[@CR18],[@CR21]^ or in the presence of tidal breathing^[@CR19],[@CR20]^.
Importantly, our study has shown the increase in mixing when adding CO in apnea was much larger when compared to its addition to MV. The flow patterns shown in Supplementary Movies [S2](#MOESM3){ref-type="media"} & [S3](#MOESM4){ref-type="media"} and Fig. [4](#Fig4){ref-type="fig"} demonstrate the mechanism behind this. During apnea, CO can produce significant pendelluft. This is not possible during MV as the bulk flow cannot be overcome by the CO to drive the flow between the sibling branches during most of the ventilation cycle. This results in a weaker mixing effect in the presence of MV. This may explain inconsistencies in the literature regarding the contribution of CO to enhanced gas mixing, which used measurements acquired either in the presence or absence of breathing.
The results were consistent with previous direct flow measurements and imaging studies on humans and dogs^[@CR1],[@CR9],[@CR10]^, conducted under breath-hold conditions, which showed out-of-phase cardiogenic flows that result in pendelluft. Our results showed that, in the mouse, internal pendelluft flows contribute up to 41% of the total flow due to CO during breath-holding. Measurements of the flow or pressure at the mouth will therefore greatly underestimate the effects of CO and cannot be used for assessing the magnitude of internal cardiogenic flows.
Although the measurements taken in this study were performed on mice, the conclusions are likely to be relevant for humans. Collier *et al*. measured tidal volumes due to CO in humans of around 53 mL, or approximately 9% of total lung capacity (\~6 L). Similarly, our measurements showed tidal volumes of between 5.5% and 7.1% of total lung capacity in the mouse (\~1 mL). Therefore, the relative magnitude of the cardiogenic flows is comparable between humans and mice, and the relative effects may also be comparable. A notable difference is that Reynolds number and Womersley number in humans is much greater than in mice. This can lead to turbulent flows in the trachea and main bronchii, and a highly skewed velocity profile with significant secondary flows at the bifurcations. This increases gas mixing at these locations. A more comprehensive flow model that removes the parabolic flow profile assumption would be required to apply the tracer modelling analysis in human lungs to fully quantify the effects of CO in humans.
Our results show that in the absence of tidal ventilation, the cardiogenic oscillations may assist in respiration and mixing within the lung. There are situations in which this may be relevant for humans. For example, humans exhibit respiratory pauses during normal activities, such as swallowing and talking. Long breath-holds are often performed during free diving, swimming, and inhaled drug delivery. Apneic events during sleep are common, with an estimated range of 9% to 38% of the population exhibiting more than 5 apneic events (\>10 s) per hour during sleep^[@CR22]^ and respiratory pauses of greater than 3 s are common during sleep in normal infants^[@CR23]^.
It should also be noted that normal breathing in humans typically includes an expiratory pause of around 2 s. These pauses were not present in the mechanical ventilation in this study. CO may therefore provide greater enhancement of mixing in normal breathing in humans than was apparent in the mice in this study.
Although the results showed greater gas mixing due to CO in the absence of tidal flows, this mixing was still much less than that generated by the mechanical ventilation. As such, the mixing caused by cardiogenic oscillations may not be sufficient to significantly affect gas exchange. Further modelling that includes gas exchange across the air/blood barrier in the terminal compartments is necessary to investigate the physiological significance of the mixing induced by cardiogenic oscillations.
In conclusion, this study has presented the first detailed measurement of cardiogenic oscillations across the whole lung, revealing the influence that cardiogenic oscillations have on ventilation in the mammalian respiratory system. The effects of cardiogenic oscillations on gas flow and mixing were shown to be greatest during periods where the tidal ventilation is absent, as this allows the existence of pendelluft. The results highlight the importance of considering this often-disregarded phenomenon when investigating lung function, particularly in conditions where tidal ventilation is reduced or absent.
Methods {#Sec7}
=======
Synchrotron imaging projection data from baseline scans of control mice acquired as part of a previous experiment^[@CR24]^ were used for analysis. The animal procedures and imaging used are detailed in that study, and briefly outlined here for convenience.
Animal Procedure {#Sec8}
----------------
All animal procedures were approved by the SPring-8 Animal Care Committee and Monash University's School of Biomedical Science's Animal Ethics Committee, and all methods were performed in accordance with the relevant guidelines and regulations. All studies were conducted in experimental hutch 3 of beamline 20B2 in the Biomedical Imaging Centre at the SPring-8 synchrotron in Japan.
Adult male Balb/C mice were anaesthetised using sodium pentobarbitone (i.p.; 70 mg/kg) and tracheostomised, Anaesthesia was maintained throughout the experiment with top-up of sodium pentobarbitone every 30 minutes (i.p.; 30 mg/kg). Positive pressure ventilation was delivered through a custom designed ventilator (based on that described in Kitchen *et al*.^[@CR25]^ with 120 ms inspiration time, 280 ms expiration time, 10 cmH~2~O inflation pressure and 2 cmH~2~O positive end expiratory pressure (PEEP), consistent with the recommendations of Glaab *et al*.^[@CR26]^: PEEP is required to maintain functional residual capacity, as active inspiratory muscle tone is reduced in anesthetized mice^[@CR26]^. Electrocardiography (ECG) was attached to record the precise timing of both the ventilation cycle and the cardiac cycle, and airway pressure was measured at the entrance to the tracheal tube. Each mouse was ventilated for at least 5 minutes prior to imaging to allow it to stabilize after anaesthetization and surgery.
Imaging {#Sec9}
-------
Imaging was conducted using a modification of the dynamic computed tomography method described in Dubsky *et al*.^[@CR11]^. Briefly, phase-contrast images were acquired at the SPring-8 synchrotron, Japan, at the BL20B2 beam-line. Images were acquired at 50 fps using a PCO.edge sCMOS detector (PCO AG, Germany), optically coupled with a scintillator crystal. During imaging, the animal was placed upright in a custom-built holder, which was mounted on a 5-axis motor controller to provide stable rotation during the 3 minute scan. The custom designed ventilator provided stable, pressure-controlled ventilation, and provided triggering to the imaging system for synchronization with the ventilation cycle. Single-image phase retrieval^[@CR27]^ and simultaneous algebraic reconstruction technique^[@CR28]^ was used for CT reconstruction. Imaging parameters resulted in high-resolution CT with an isotropic voxel size of 15 μm.
Double-gated CT reconstruction {#Sec10}
------------------------------
A retrospective double-gating procedure was developed to separately bin projection images into 19 ventilation phases and into 8 cardiac phases. The images were binned into 19 ventilation phases based on the image acquisition triggered by the mechanical ventilation. Separately, the images were binned into 8 cardiac phases based on the ECG trace. Motion blur due to ventilation on the cardiac phase CTs was reduced by only utilizing images acquired at an airway pressure of \<4 cmH~2~O (as measured by the ventilator), thus effectively producing cardiac phase data at approximately expiratory pressure.
Subsequent CT reconstruction resulted in two 4DCT movies, one showing lung motion due to ventilation, and the other showing lung motion due to the beating heart (see Supplementary Movie [S1](#MOESM2){ref-type="media"}). This data isolates the effects of ventilation and heart action without requiring physical intervention (ie. by pausing ventilation or stopping the heartbeat), which would disrupt the physiological processes occurring within the system.
Airway tree segmentation {#Sec11}
------------------------
The airway tree was segmented according to the process described in Dubsky *et al*.^[@CR24]^. This process utilizes the vesselness filter described by Frangi *et al*.^[@CR29]^. This Hessian-based filter uses analysis of the eigenvalues of the image intensity Hessian-matrix at different spatial scales (*σ*) in order to assign a probability value to each voxel. This value describes the probability of a cylinder (in our case an airway) being present at each voxel. The spatial scale that yields the maximum vessel probability at any point may be used to estimate the vessel diameter. The 4DCT images were processed using the vesselness filter to yield a vessel probability field. This was segmented using a flood-fill, providing a binary image of the airway tree. Auto-skeletonisation (Avizo, FEI software, USA) was then used to find the centerline of the airways. The scale of the vesselness filter that yielded the highest vessel probability at each centerline point of the airway tree was used as an estimate of the diameter of the airway at that point. These estimates were averaged across each airway segment to yield the average diameter of each airway segment. This method provides robust, unsupervised diameter estimation across the entire airway tree.
Tissue expansion and airway flow measurement {#Sec12}
--------------------------------------------
Tissue displacement, expansion and airway flow was calculated using the cross-correlation velocimetry procedure described previously^[@CR11],[@CR12]^. Briefly, three-dimensional cross-correlation was performed between two successive interrogation regions, with the maximum correlation value representing the modal displacement of the lung tissue within that region. The cross-correlation analysis was performed with interrogation regions of 32 × 32 × 32 voxels (representing a 480 μm^3^ region) with a regular spacing of 16 voxels between the centres of adjacent interrogation regions. The expansion field of lung tissue was calculated from the local gradients in the lung displacement.
The segmented airway tree was associated with the lung expansion maps to calculate the time-varying airflow throughout the bronchial tree. The method utilises the local ventilation values of lung tissue to directly infer the local airflow through each airway supplying a given region of tissue. The supplying airways are defined as the terminal airways of the airway segmentation, that is the airways with a parent but no daughter branches. Each interrogation region is associated with its closest supplying airway. The expansion of the regions supplied by an airway (occurring due to gas flowing into/out of that region) equals the flow through that airway. Assuming negligible compressibility effects, the principle of continuity dictates that at each bifurcation, the flow through a parent segment must equal the sum of the flow through its daughter segments. The flow through the entire tree can therefore be calculated by recursively summing the airflows in daughter segments at each bifurcation to calculate the airflow through the parent segment.
Gas tracers and mixing calculation {#Sec13}
----------------------------------
A numerical gas tracer method was developed to visualize the internal flows and deduce the effects of flow on gas mixing within the lungs. To investigate the relative contributions of each type of flow (diaphragmatic flow, cardiogenic flow), the tracer analysis was performed on each data set under 4 conditions: (1) DIFF: zero flow (molecular diffusion only), (2) CARD: cardiogenic oscillations, (3) VENT: mechanical ventilation (4) BOTH: cardiogenic oscillations + mechanical ventilation. The concentration of initial (Type A) particles in the system was plotted against time for each condition. The method consisted of the following steps:
### Geometry simplification and peripheral airway modelling {#Sec14}
To increase efficiency and reduce the effects of segmentation errors, the airway geometries were first simplified into straight cylinders with a single radius. This allows each airway to be completely specified by its two end-points and its radius. The segmentations defined airways down to approximately 85--100 µm, corresponding to generation 14 in the Oldham & Robinson model^[@CR30]^. To model the peripheral airways and the acini, each segmented distal airway was replaced with a trumpet model, according to the geometry specified by Oldham and Robinson. An acinar compartment was placed at the end of each trumpet with equivalent cross-sectional area and volume as generation 23 in the Oldham & Robinson model.
### Tracer advection {#Sec15}
The geometry was numerically seeded with massless tracers. Each airway and terminal compartment is initially seeded with a number of tracers proportional to its volume, so that the tracer density throughout the domain is constant. Each tracer is defined by its containing airway, its location within that airway in cylindrical co-ordinates. The initial tracers were designated as Type A (representing the resident gas) and new tracers entering via the trachea opening were designated Type B (representing fresh gas from the atmosphere).
During each time-step, tracers were advected according to the gas velocity at its location, using a 4^th^ order Runge-Kutta time integration. The image analysis produces a single bulk-flow for each airway over time. A Poiseuille (parabolic) velocity profile is assumed for all airways at each time-point. As the Womersley number in the system is \<1 throughout the system, the effects of oscillations on the velocity profile are negligible. Linear interpolation in time was used to oversample the flow data obtained from the imaging.
As tracers pass through a bifurcation, to preserve mass continuity they are randomly assigned to one of the other two airways based on the ratio of flow between the two candidate airways. The tracer maintained its relative radius location in its new containing airway.
The volume flux into the trachea was integrated over time, and at each timestep the integer value of this integral (multiplied by the initial volumetric tracer concentration) was used to insert tracers (Type B) at the trachea opening at a randomly assigned cross-sectional position, with the remaining decimal value being conserved in the integrated volume flux for subsequent time-steps. Tracers that traverse out of the trachea at the proximal boundary (i.e. leave through the trachea opening due to flow out of the system, typically during expiration) are removed from the simulation.
Tracers that enter the terminal compartments from the terminal airways were added to a tally of tracers within that compartment, and no longer subjected to advection or diffusion updates. The volume flux from the terminal compartments into the terminal 'trumpet' airways was monitored, and tracers introduced into the terminal airways from the terminal compartments according to the accumulated magnitude of this flux. The type of tracer that was introduced was randomly assigned in proportion to the concentration of Type A and Type B tracers contained in the compartment.
### Diffusion {#Sec16}
Molecular diffusion was modelled using the mathematical technique known as random walks. At every time-step, each tracer is randomly displaced according to a normal distribution with zero mean and standard deviation of *6Dt*, where *D* is the diffusion coefficient, and *t* is the timestep, and in a random direction in 3D. The diffusion coefficient was chosen was 0.23 cm^2^/s which is the self-diffusivity coefficient of nitrogen at body temperature^[@CR31]^. This was chosen in order to model the intrinsic mixing of air within the lungs, which is predominantly nitrogen. This displacement is sub-stepped by 100 times. If a tracer crosses an airway wall during a sub-step, the direction is randomly reset to simulate interaction with a solid boundary. If the displacement enters a bifurcation, the tracer is randomly assigned to one of the three airways of the bifurcation according to the ratio of cross-sectional areas, thus maintaining mass continuity. Tracers that cross the system boundary (ie. pass out of the trachea entrance or into a terminal compartment) are dealt with as in the advection step.
Data availability {#Sec17}
-----------------
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Electronic supplementary material
=================================
{#Sec18}
Supplementary Information Supplementary Movie S1 Supplementary Movie S2 Supplementary Movie S3
**Electronic supplementary material**
**Supplementary information** accompanies this paper at 10.1038/s41598-018-23193-w.
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We acknowledge the support of the Japan Synchrotron Radiation Research Institute (JASRI; 2012B1100), the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE; <https://www.massive.org.au/>), Australian Research Council (DP150102240), and the Monash Immersive Visualization Platform (MIVP; <http://www.monash.edu/mivp/>).
S.D., J.T. and A.F. conceived and designed the experiments. S.D., J.T. and G.S., conceived and developed the analysis protocols. J.T. and S.D. analysed the data. S.D., B.T., and G.S. interpreted the data. S.D. wrote the manuscript. S.D., J.T., G.S., and B.T. revised the manuscript.
Competing Interests {#FPar1}
===================
S.D., J.T., and A.F. hold beneficial interests in 4Dx Ltd., which commercializes respiratory imaging technology related to that used in this study.
| {
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One of the main technological and fundamental challenges of our days is the design of electromagnetic architectures that allow for an efficient manipulation of the amplitude, phase, polarization, or direction of electromagnetic signals. Management of these features can lead to many diverse applications ranging from optical[@b1] and microwave communications[@b2], sensors and power limiters[@b3], to energy harvesting, switching, and optical computing[@b4]. In this endeavor, the enhancement and control of the interaction between electromagnetic radiation and matter is of utmost importance.
An efficient way to achieve this enhancement is via localized modes supported by defect layers embedded in a layered photonic structure. Such localized modes develop nodal points where the amplitude of the oscillating electric field is very small. Placing a thin metallic nanolayer at such positions will have nearly no effect on the localized mode and the resonance transmission associated with this mode. This is the well-known phenomenon of induced transmission (see, for example[@b5][@b6][@b7][@b8], and references therein). By comparison a stand-alone metallic nanolayer of the same thickness is totally opaque at the same frequency range which explains the term "induced transmission"[@b5]. Here we argue that a small perturbation in the permittivity of a layer(s) nearby to the metallic nanolayer, can drastically affect the localized mode and resonance transmission associated with it. Depending on the nodal point symmetry, there are three possible scenarios: (a) the nodal point (with the metallic nanolayer) coincide with the mirror plane of the layered structure before and after the perturbation; (b) the nodal point coincide with the mirror plane in the original configuration, but the perturbation destroys this symmetry; and (c) the nodal point of the localized mode with the metallic nanolayer is not a symmetry point, neither before nor after the perturbation, in which case the coincidence of the metallic nanolayer and the node of the unperturbed localized mode can be viewed as *accidental spatial degeneracy* (ASD). In the case (a), the symmetric alteration of the layered structure results simply in a shift of the localized mode frequency. The metallic nanolayer still coincides with the nodal point of the localized mode at the shifted frequency and hence does not affect the resonant transmission at that frequency. In the cases (b) and (c), the nodal point of the localized mode shifts away from the metallic nanolayer, which can result in a dramatic suppression of the localized mode, along with the resonant transmission. In either case, the layered structure becomes opaque at any frequency. Due to the presence of the metallic nanolayer, the abrupt transition from resonant transmission to *broadband opacity* can be caused by just a tiny change (few percentile point) of the permittivity of one of the dielectric layers of the defect cavity, which justifies the use of the term *hypersensitivity*. The above feature equally applies to the cases (b) and (c), but with one important exception, when the permittivity alteration is self-induced by the localized mode. Typically, a self-induced change in the permittivity is associated with nonlinear effects, heating, etc. If the permittivity change is indeed self-induced, the transition from resonant transmission to broadband opacity is very pronounced and abrupt in the case (c) of accidental spatial degeneracy as compared to the case (b), where the unperturbed layered structure is symmetric.
In most applications of metallo-dielectric layered structures (see for example[@b5][@b9]) the abovementioned hypersensitive transport characteristics of asymmetric configurations to a self-induced alteration of the refractive index would be undesirable and counterproductive. In this paper we take an alternative viewpoint. We demonstrate how such hypersensitive transport can be used in microwave (and optical) limiters, and we show that it can dramatically enhance their performance. As an example, we consider a microwave limiter based on an asymmetric metal-dielectric layered structure supporting a localized mode with *ASD*. We show that even a small self-induced alteration of the refractive index at the neighborhood of the maxima of the localized mode produces an abrupt transition from resonant transmission for low-level radiation to high broadband reflectivity for high-level radiation. On the other hand, if the asymmetric permittivity alteration is caused by external physical action, such as, asymmetric mechanical stress, electric field etc., rather than being self-induced by the localized mode, the abovementioned hypersensitivity will not be related to the ASD, and it will be equally strong in the setting (b) and (c). This effect can be used in switches, modulators and sensors.
It is important that the induced transmission and its hypersensitivity to the incident electromagnetic wave intensity in asymmetric metal-dielectric photonic structures are significant only in cases where the imaginary permittivity of the metallic nano-layer is large. This critical condition is satisfied at frequencies starting from microwave and up to the mid infrared.
Consider a 1D photonic crystal (PC) consisting of two lossless Bragg gratings (BG), with constitutive components different for each grating, as shown in [Fig. 1](#f1){ref-type="fig"}. The refraction indices and thicknesses of the bilayers of the left BG (LBG) layers are and and those of the right BG (RBG) are ; and respectively. The interface between the two gratings constitutes an asymmetric cavity. The periodic modulation of the index of refraction of each grating is engineered in a way that both of them have the same band-gap structure, which is just a matter of convenience. The cavity consists of two different quarter-wave layers with and and a thin metallic nanolayer between them with thickness and permittivity . Under typical circumstances the permittivity of each of the two layers of the cavity is affected differently by an external perturbation. For example the left layer can be more sensitive to high-level radiation than the right layer. The defect cavity supports a localized mode with a frequency *f*~*r*~ located in the middle of the photonic band-gap and whose nodal point coincide with the metallic nanolayer. Evidently, this cavity is asymmetric, which corresponds to the case (c) described above.
The transmission , reflection , and absorption are calculated via the transfer matrix approach. The latter connects the amplitudes of forward and backward propagating waves on the left and the right domains outside of the PC. At the layer inside the structure, and also outside of the PC, a time-harmonic field of frequency ***ω*** satisfies the Helmholtz equation:
where ^2^ is the permittivity of the *j*-th layer (*ε* = 1 for the vacuum). At the *j*-th layer, Eq. [(1)](#eq18){ref-type="disp-formula"} admits solutions of the form , where is the wavevector at the vacuum. Outside the PC, Eq. [(1)](#eq18){ref-type="disp-formula"} admits the solution . The continuity of the field and its derivative at the interface between two layers (or a layer and the vacuum) can be expressed in terms of the total transfer matrix which connects the forward and backward amplitudes on the left (L) and right (R) of the PC:
where *N* is the total number of layers. The single-layer transfer matrix connects the field amplitudes of the *j*-th and the layers i.e. . Thus the transfer matrix approach allows us also to construct the field at each layer, provided that appropriate scattering boundary conditions are imposed. The latter, for a left incident wave, take the form . It is easy to show that , , and [@b10][@b11].
We start our analysis with the investigation of the transmission spectra of each of the two mirrors. Their dispersion relation is calculated using the transfer matrix of one bilayer [@b11]
where the indices a and b indicate the layers 1 and 2 (3 and 4) associated with the LBG (RBG).
Propagating waves in each grating correspond to frequencies for which[@b11]
where the total width of the bilayer defines the periodicity of the LBG (for or the RBG (for . Direct inspection of Eq. [(4)](#eq37){ref-type="disp-formula"} indicates that the dispersion relations and are identical as long as and . Once turned to finite photonic structures, both LBG and RBG will share the same band-gap structure of the transmission spectrum, as long as these conditions are satisfied. Below we consider that each BG consists of five (quarter-wavelength) bilayers.
In [Fig. 1b](#f1){ref-type="fig"} we show the transmission spectrum of our PC for . The position of the band-edges is nicely described by Eq. [(4)](#eq37){ref-type="disp-formula"}. Moreover a resonant mode with at resonance frequency , in the middle of the band-gap, has been created. The resonant mode is localized at the vicinity of the defect cavity and decays exponentially inside the two mirrors due to destructive interferences from the layers (blue profile at [Fig. 1](#f1){ref-type="fig"}). The electric field has a nodal point at the position of the metallic layer (blue profile at [Fig. 1a](#f1){ref-type="fig"}). Thus the resonant localized mode is unaffected by the presence of the lossy layer and the entire PC is completely transparent at (see [Fig. 1b](#f1){ref-type="fig"}). Furthermore, the lack of mirror symmetry ensures that the ASD occurs only for the resonance mode *f*~*r*~. For all other (Fabry-Perot) resonances with frequencies the electric field distribution has finite amplitude at the position of the metallic layer leading to large reflection (see discussion below) and vanishing transmission .
Moreover, any small perturbation (say, due to heating), which will change the permittivity of any of the two layers of the defect cavity (say the left one) by , will engage immediately the metallic nanolayer and lift the ASD of the resonance localized mode. In other words, the electric field will no longer have a nodal point at the position of the metallic layer (see red profile at [Fig. 1a](#f1){ref-type="fig"}). This will trigger various competing mechanisms. On the one hand, it will increase the impendence mismatch and thus it will enhance the reflection. This mechanism is present whenever the electric field interacts with the metallic layer, even for . On the other hand, it will lead to an increase of absorption. One can estimate the effect of these two competing mechanisms by analyzing the transport from a single lossy *δ*-like defect with permittivity . In this case, we have that:
which indicates that a lossy defect is a source of increased absorption but at the same time a way to enhance reflection. Also note that the absorption is not a monotonic function of the tangent loss parameter *γ*. Rather it takes its maximum value at
The second mechanism applies only for resonant transport. In this case, the bulk losses due to the strong interaction of the electric field with the metallic layer compete with the losses due to the leakage from the boundaries of the structure. The former are proportional to the field intensity at the position of the lossy defect, while the latter depend on the coupling of the resonant mode to the free space via the boundary of the PC. As increases, the bulk losses overrun the losses due to the boundary leakage and eventually spoil the resonance (see red electric field in [Fig. 1](#f1){ref-type="fig"}). Thus, photons do not dwell in the resonant mode and therefore cannot be absorbed by the metallic layer i.e. the absorption diminishes while , and .
In [Fig. 2](#f2){ref-type="fig"} we report the transmission , reflection , and absorption of our PC, versus frequency and versus the relative permittivity change occurring at a single (left of the metallic nanolayer) layer. For small , ([Fig. 2a](#f2){ref-type="fig"}) while respectively ([Fig. 2b,c](#f2){ref-type="fig"}). As increases, the absorption is initially increasing (see peak at but for it starts decreasing reaching values as low as −40 dB. For even larger , the structure becomes completely reflective (see [Fig. 2b](#f2){ref-type="fig"}).
The hypersensitivity of the transport characteristics of our composite structure to small permittivity changes can find various applications including sensors, switches, power modulators, etc. Here, however, we will discuss the advantages to implement our PC as an efficient energy limiter. These are devices that protect electromagnetic sensors from high-energy radiation, while at the same time they are transparent to low energy radiation[@b12][@b13][@b14][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24]. Typically this protection is achieved via the absorption of the incident energy from the limiter, which turns opaque. At the same time this excessive energy overheats the limiter and leads to its self-destruction. Recently, however, the concept of reflective limiters has been introduced[@b25][@b26]. These structures consist of a BG with one lossy defect layer, which undergoes a uniform self-induced permittivity change. The proposal for limiting action was based on the phenomenon of resonant transmission via a localized (defect) mode. The defect mode is transmissive at low-energy incident pulses, while it becomes highly reflective (and not absorbing) at high-energy pulses. Nevertheless, this proposal suffers from one drawback; the limiting action requires several orders of magnitude change of the permittivity of the lossy defect. Instead, our design requires changes of only a few percentage points in the permittivity of one composite layer in order to provide limiting action. Importantly, the high reflectivity for the high intensity input persists within a broad frequency range -- not just within a photonic band-gap is was the case in refs [@b25],[@b26].
We consider the PC of [Fig. 1](#f1){ref-type="fig"}. We further assume, for the sake of the discussion, that the left layer of the defect cavity has a permittivity which depends on temperature (*T*) variations as where for simplicity we consider that . Since the layer on the right is composed of a different material, in general, we expect a different variation of its permittivity with the temperature. For simplicity, we assume that the right layer is much more resilient to the changes in temperature and thus we will keep its permittivity constant . There are various physical mechanisms that can lead to the heating of the dielectric layer. For example, it can originate from the heating of the nearby metallic nanolayer or from the presence of a small at the permittivity of the dielectric layer itself (which is usually the case in practical situations) or from a combination of these two physical mechanisms.
The rate equation that determines the temporal behavior of the temperature at the cavity is[@b26]:
where is the incident pulse intensity, which is a given function of time, *C* is the heat capacity, and is the temperature dependent absorption coefficient of the asymmetric cavity. A numerical integration of Eq. [(6)](#eq57){ref-type="disp-formula"} (for a given pulse profile allows us to evaluate the temperature and from there the permittivity variations which are reported in [Fig. 3a](#f3){ref-type="fig"} as a percentage change . Then , and are calculated using transfer matrices as a function of pulse duration *t* (see [Fig. 3b--d](#f3){ref-type="fig"}). We find that ([Fig. 3b](#f3){ref-type="fig"}) initially increases and reaches some maximum value around corresponding to very small permittivity changes . Further increase of leads to an abrupt decay of for resonance frequencies to values smaller than −30 dB. The off-resonance values already have absorption that is below −60 dBs. At the same time the transmission ([Fig. 3c](#f3){ref-type="fig"}) decays while the reflection ([Fig. 3d](#f3){ref-type="fig"}) reaches unity. Therefore our photonic structure acts as a hypersensitive reflective microwave limiter- it will turn highly reflective within a broad frequency range for very small relative permittivity changes \~0.5%. This behavior has to be contrasted with the proposal of ref. [@b25] where a limiting action is triggered only when the variation (due to heating) of the refraction index of a defect lossy layer, which is embedded in a Bragg grating, is many orders of magnitude. The outcome of these calculations is also reported in the inset of [Fig. 3a](#f3){ref-type="fig"} by referring to , and at resonance frequency versus the relative change of the permittivity. For these simulations we have used a BG with the same constitutive layers as the LBG of our PC. The lossy defect layer is placed in the middle of the grating and has . We see that the reflective limiting action occurs when the permittivity changes of the lossy defect layer are more than seven orders of magnitude.
In conclusion, we have introduced a photonic layered structure design with hypersensitive transport characteristics. This layered structure consists of an asymmetric dielectric cavity incorporating a metallic nanolayer and sandwiched between two Bragg mirrors. When the metallic nano-layer coincides with the nodal point of the localized mode, the system develops the phenomenon of induced transmission. However, even a small change in and/or in one of the dielectric layers of the asymmetric cavity abruptly suppresses the localized mode and renders the layered structure highly reflective at all frequencies -- not just at frequencies of the photonic band gap. Furthermore, we have shown that these metal-dielectric structures can be used as hypersensitive microwave (or optical) limiters. Specifically, at low incident wave intensity, these structures support a narrow-band transmission. If the electromagnetic wave intensity and/or fluence exceed certain level, even a small self-induced change (due to non-linearities or heating effects) in the refractive index of the asymmetric layer causes an abrupt transition to a broadband reflectivity. The proposed design can be adjusted to different frequency ranges starting from microwave frequencies and up to the mid infrared. The main physical requirement is that the imaginary part of the metallic nanolayer is large. The concept of hypersensitive layered structures can be applied not only to electromagnetic waves but also to acoustic waves, matter-waves etc.
Additional Information
======================
**How to cite this article**: Makri, E. *et al.* Hypersensitive Transport in Photonic Crystals with Accidental Spatial Degeneracies. *Sci. Rep.* **6**, 22169; doi: 10.1038/srep22169 (2016).
We acknowledge AFOSR support from the portfolio of Dr. A. Nachman via LRIR09RY04COR Grant (I.V.) and MURI No. FA9550-14-1-0037 (E.M., T.K.). (A.C.) acknowledges AFOSR support from the portfolio of Dr. A. Sayir via FA9550-16-1-0058 Grant.
**Author Contributions** E.M. and K.S. carried out most of the calculations. A.C., I.V. and T.K. formulated the problem and wrote the manuscript with input from the rest of the team. All the authors have contributed to discussion and analysis of the results.
{#f1}
{#f2}
![(**a**) A density plot of the change of permittivity of the left layer of the defect cavity in the vicinity of transmission resonance as a function of the pulse duration. Inset: the transmission T, reflection R and absorption A at resonance frequency versus for a reflective limiter of refs [@b25],[@b26]. (**b**) The absorption; (**c**) the transmission; and (**d**) the reflection versus pulse duration, for a frequency window around the resonance mode.](srep22169-f3){#f3}
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Introduction {#S1}
============
The purpose of this paper is to distinguish between sadness and depression. Current descriptions suggest that depression is caused mainly by exposure to stress and the ups and downs of life ([@B1], [@B2]). If depression were simply extreme sadness, then patients would have more control over symptoms, and it would be less stigmatizing as a consequence ([@B3], [@B4]). Freud provided a clinical foundation for this idea by concluding that mourning and melancholia were comparable because the symptoms are similar, they are both precipitated by loss and improve with time ([@B5]). He noted that people with melancholia could become over-talkative and manic but did not adequately explain why this is so ([@B5]).
Other influential writers in psychiatry and psychology have endorsed similar notions. Bowlby compared negative emotions in adults to those that occur during separation from the attachment figure in infants ([@B6]). Beck attributed depression to dysfunctional negative thoughts of defeat, failure, and rejection ([@B7]). He dismissed mood-swings as a normal phenomenon ([@B7]). Although Watson paid more attention to mood instability (MI), he also dismissed negative mood variability as mundane and of little importance ([@B8]).
Kraepelin was a notable exception in that he emphasized brief moods swings and rapidly alternating mood symptoms in patients with manic--depressive illness ([@B9]). Consistent with his work, recent studies have shown that patients with mood disorders report increased affective lability and emotional instability; and this also occurs during euthymic periods in bipolar patients ([@B10]--[@B12]).
Depression could be understood as a consequence of loss or stress, but if high moods do occur during a depressive episode, they tend to be ignored ([@B2], [@B6]), dismissed ([@B7], [@B8]), or isolated to separate categories with low prevalences of about 1% such as bipolar mood disorder ([@B13]) and borderline personality disorder ([@B14]). Recently, DSM-5 has introduced the specifier "with mixed features" to acknowledge manic/hypomanic symptoms, but three out of seven symptoms are required ([@B13]). Any attempt to expand the bipolar spectrum to account for brief periods of high mood ([@B15]--[@B17]) has been vigorously criticized ([@B18]--[@B21]).
The reliance on retrospective methods for clinical and research interviewing ([@B22]) has made it easier to dismiss brief high moods because retrospective recall in depressed people is biased toward the negative. Recalling mood over the previous 2 weeks is more likely to result in smoothing away mood variation ([@B23]--[@B25]). If so, the raw data for clinically diagnosing major depression are systematically distorted ([@B26]). Ecological momentary assessment, which asks patients to record affect at a given moment over a specified duration, provides a fuller picture of mood by capturing variation in addition to severity. As early as 2006, the US Food and Drug Administration recommended that pharmaceutical companies make use of real-time data instead of patient recollection ([@B26]). Increasingly, smartphones are being used as a tool for ecological momentary assessment in psychological and clinical studies ([@B27], [@B28]).
Another way by which recalled moods are distorted is the "common-sense" view that low mood and high mood are mutually exclusive or negatively correlated ([@B8], [@B29]). Since negative affect predominates in depressed people, brief episodes of positive affect tend to be subsumed under overall gloom in patient recollections. There is evidence, however, that positive and negative affect are independent of each other ([@B30]) and that people can feel both happy and sad at the same time ([@B31]).
Among clinical samples, when low and high moods are measured prospectively on separate axes, the rapid cyclic recurrence of high moods with low moods becomes apparent, a phenomenon known as mood instability ([@B11], [@B32]--[@B34]). MI has been shown to exist in up to 13.9% of the adult population ([@B34]). MI seems to be the essential component of neuroticism ([@B35], [@B36]) and is an antecedent to major depression ([@B37]), psychotic symptoms ([@B38]), severity of distress ([@B39]), suicidal thoughts ([@B35]), and self-harm behavior ([@B40]). In this study, we investigated how MI might characterize the experience of people who were depressed as compared with those who were not depressed.
Hypothesis {#S1-1}
----------
People who are depressed (distressed with negative mood and symptoms) are more likely to have more strongly correlated low and high unstable moods than people who are not depressed.
Materials and Methods {#S2}
=====================
Participants {#S2-1}
------------
We used data from participants in four controlled studies (*n* = 168) that we have published ([@B32], [@B41]--[@B43]). All patients had been referred by family physicians for treatment and were all under treatment at the time of the study. In three of the studies, males and females had been referred to general outpatient practices, in one study women had been referred for alcohol abuse, but all had been alcohol free for 3 weeks. All patients completed mood diaries (described in the next section) over a week. All of the studies received approval from the university ethics board. The patient group (*n* = 104) was assessed with the Mini-International Neuropsychiatric Interview (MINI English Version 5.0.0 for DSM-IV), and 49 met criteria for major depression ([@B44]). There was high comorbidity with anxiety disorders. Twenty-two of those with major depression (45%) reported hyperthymic symptoms in the past, which did not meet criteria for hypomania ([@B45]). The controls (*n* = 64) included health-care personnel and 17 graduate students.
Procedure {#S2-2}
---------
Participants completed the Beck Depression Inventory-IA ([@B46], [@B47]), which is a 21-item retrospective self-report questionnaire. Statements are presented in the first-person ("I feel sad"), and subjects select the item that best reflects their recent state from a choice of four items. It is reliable and correlates well with other measures of depression but may in part assess a general distress or neuroticism factor ([@B48]--[@B50]). In a study with undergraduate students, the BDI showed strong latent dimensional structure that is there was no evidence that a cut-score defined a latent class of depression ([@B50]).
Participants completed separate visual analog scales (VAS) ([@B8]) for low mood and high mood. They rated their moods in the morning after awakening and at night before bedtime, for 7 consecutive days ([@B41], [@B51]). The anchor points were "not at all" and "very much so."
For clarity, we define "low mood" as participant ratings for "sad/blue" or "depressed" over 1 week (twice daily ratings for a total of 14 ratings). Similarly, "high mood" is defined as participant ratings for "enthusiastic/interested" or "high mood." "Low MI" refers to the fluctuation of low mood, and "high MI" refers to the fluctuation of high mood over the same period. MI is operationalized as the mean square successive difference (MSSD) statistic across 14 ratings ([@B52]). One can think of MSSD as the SD of ratings, taking temporal sequence into account.
Analysis {#S2-3}
--------
We used the BDI scores to divide the participants into two distinct groups: those who were within a "normal" range of BDI scores (BDI ≤10) (*n* = 59) ([@B47]) and those with BDI scores ≥18 or who were likely clinically depressed (*n* = 78) ([@B46], [@B47]). The remaining participants (*n* = 31) fell outside of these two groups and were excluded from the sample. We used the conventional terms "non-depressed" (BDI ≤10) and "depressed" (≥18) to designate the two groups without assuming that the cut-score of BDI ≥18 defined a latent class of major depression or any particular form of depression. The mean BDI scores of the non-depressed and depressed groups were 4.51 (SD: 2.59) and 20.05 (SD: 13.89), respectively.
We used Pearson's correlation as a measure of association between low and high moods. We then tested whether the correlations between (a) mean low and high mood and (b) low and high MI were different between the non-depressed and depressed groups. This comparison of correlation magnitudes is, in principle, similar to Meehl's MAXCOV procedure in which the correlation of two variables is compared along successive cuts in a third variable ([@B22]).
Results {#S3}
=======
In the original group of referred patient participants (*N* = 104), mean low mood was correlated with low MI; as were mean high mood and high MI (*p* \< 0.001). In the original group of controls (*N* = 64), mean low mood was correlated with low MI (*p* \< 0.001); as were mean high mood and high MI (*p* \< 0.01).
Sample and Group Demographics {#S3-1}
-----------------------------
The participants (*n* = 137) ranged in age from 15 to 64 years (mean age = 30.0 years, SD = 10.9), and 104 (75.9%) were females. The "depressed" group (*n* = 78) (mean age = 30.62, SD = 11.60; 80.8% females) was composed of 51 patients, 24 volunteers as "controls" in the original studies, and 3 graduate students. The "non-depressed group" (*n* = 59) (mean age = 29.15 years, SD = 9.66; 69.5% females) included 40 people who volunteered as controls from the original studies, 14 graduate students, and 5 people referred as patients. There was no difference in age or sex distribution between the two groups.
Mean Mood {#S3-2}
---------
Table [1](#T1){ref-type="table"} shows mood scores for both groups. Compared to the non-depressed group, the depressed group experienced more severe low mood (*t* = −4.73, df = 129, *p* \< 0.001) and less severe high mood (*t* = 5.41, df = 135, *p* \< 0.001), consistent with the selection into non-depressed and depressed groups. Table [2](#T2){ref-type="table"} shows mean low and mean high mood correlations for both groups. Notably, in the non-depressed group, the correlation between mean low and mean high moods was not significant (*r* = −0.01), but in the depressed group, mean low and high moods were positively correlated (*r* = 0.38). The Fisher *r*-to-*z* transformation indicated that the difference between these correlations was significant (*z* = −2.30, *p* = 0.02, two-tailed).
######
**Mood and mood instability (MI) in depressed and non-depressed groups**.
Non-depressed Depressed *t* df Sig (two-tailed)
----------- --------------- ----------- ------ ---- ------------------ ------ ------- -------- --------
Low mood 59 1.50 1.34 78 2.95 2.22 −4.73 129.22 \<0.00
High mood 59 4.81 1.69 78 3.05 2.13 5.41 134.68 \<0.00
Low MI 59 1.72 1.30 78 2.40 1.49 −2.81 135 \<0.01
High MI 59 2.84 1.06 78 2.44 1.60 1.77 132.94 0.08
*Low, high mood: mean visual analog scale mood ratings for low and high moods*.
*Low, high MI: mean square successive difference statistic for low mood and high mood*.
######
**Correlations**.[^a^](#tfn1){ref-type="table-fn"}
Non-depressed Depressed
----------------------------------------------------------------------------------------------------- --------------- --------------------------------------- ------------- ---- --------------------------------------- ------------ ------- ------
Correlation mean low mood and high mood 59 −0.01 −0.27--0.25 78 0.38[^b^](#tfn2){ref-type="table-fn"} 0.17--0.55 −2.3 0.02
Correlation mean square successive difference (MSSD) (mood instability) low mood and MSSD high mood 59 0.31[^a^](#tfn1){ref-type="table-fn"} 0.06--0.53 78 0.61[^a^](#tfn1){ref-type="table-fn"} 0.45--0.73 −2.18 0.03
*^a^Correlations between mean low and mean high moods for the non-depressed and depressed groups and MSSD low and MSSD high moods for the non-depressed and depressed groups*.
*^b^Correlation is significant at the 0.05 level (two-tailed)*.
Mood Instability {#S3-3}
----------------
The depressed group experienced more severe low MI than the non-depressed group, as consistent with the findings of the original individual studies. The difference in high MI between the depressed and non-depressed groups was not significant. Table [2](#T2){ref-type="table"} shows the important finding that low MI and high MI were correlated in both the depressed and the non-depressed groups, but the magnitude of correlation for the depressed group (*r* = 0.61) was almost twice that for the non-depressed group (*r* = 0.31). The difference between these correlations was significant (*z* = −2.18, *p* = 0.03, two-tailed).
Discussion {#S4}
==========
On the VAS ratings, the depressed group experienced more severe low moods and less severe high moods than the non-depressed group, as would be expected given the selection criteria. This is consistent with reports of more severe negative emotions and variable positive emotions in ecological momentary assessment studies of patients with major depression ([@B12], [@B33], [@B53]).
In the non-depressed group, the overall means of low mood and high mood were uncorrelated. This supports the observation that in normal people low and high moods are not strongly related and easily distinguished ([@B8]). In the depressed group, however, mean low mood and mean high mood were moderately positively correlated. This indicates that the depressed group experienced high moods concurrent with low moods ([@B54], [@B55]). This is contrary to the common-sense view that low mood should not be associated with high moods.
Low MI and high MI were weakly correlated in the non-depressed group. In the depressed group, the correlation was moderate to large, and the difference between these correlations was significant. In other words, in the depressed group, the fluctuations of low moods and high moods are more closely related (Table [2](#T2){ref-type="table"}).
Taken together, these results suggest that in people with depression, mood is a complex combination of rapidly fluctuating seemingly polar opposite emotions. This distinction is more easily understood if MI is considered along with stable low mood ([@B56]). Other studies have shown complex emotional patterns in anxiety and mood disorders ([@B12], [@B56]--[@B58]). Two clinical applications are (a) that the usual semi-structured retrospective assessment might provide a limited appreciation of "nuanced" mood symptoms ([@B12]) and (b) that attention to mood stabilization might add an extra dimension to treatment ([@B12], [@B59]). In other words, since MI reflects neuroticism ([@B36]) that is an antecedent of depression ([@B60]), attention to the assessment and treatment of MI in addition to specific symptoms of depression ([@B61], [@B62]) might increase the treatment efficacy.
Our study had several methodological limitations. First, the number of participants was relatively small and from one center. Second, the wording of the questions for low mood and high mood varied slightly between studies, although the words were similar. This might be an advantage by reflecting real-world interviewing conditions. Third, paper and pencil diaries were used, raising the possibility of people retrospectively filling in data. Participants understood that they were to record momentary mood ratings, and the method of calculating MI was not intuitively apparent to the participant ([@B52]). Furthermore, all of the individual study results produced clear differences suggesting that the participants understood the instructions. Fourth, choosing graduate students as controls in one of the studies was a convenience sample, but we considered that graduate students would be generally more stable than undergraduates and that they would be older and closer in age to patients. Fifth, for simplicity we considered only low and high moods. Moods in broadly defined depression would likely appear even more complex and distressing if anxiety, irritability, and psychotic symptoms were included ([@B63]). Sixth, five people in the "non-depressed" group had been referred as patients. These people may have improved while waiting for treatment or may have had symptoms that were not detected by the way that they completed the BDI. Finally, during the diagnostic interview we did not probe sufficiently for hypomanic symptoms during the depressive episode, so we cannot say how many of the participants would have met criteria for the DSM-5 category of major depression with mixed features ([@B13]).
There are also limitations to the conclusions that can be drawn. The correlation between low and high MI might not be the best representation of complex emotions. The data do not address the question of the relative merits of continuous or categorical approaches to classification in psychiatry. Recent taxometric analyses indicate that in the mood and anxiety domains, dimensional distributions are much more likely ([@B64], [@B65]). We studied MI as a phenomenon, similar to instability in physical parameters such as blood pressure ([@B66]) and blood sugar ([@B67]) and without assumptions as to cause. MI is considered to be a simpler concept than "affective instability due to a marked reactivity of mood" ([@B13]) or emotional dysregulation ([@B68]). One question for further studies would be whether MI leads to unstable interpersonal relationships or *vice versa*. The relationship of MI to interpersonal and social--environmental events is a matter for further research. Instability of low moods is not entirely accounted for by reactivity to negative events ([@B12]). Finally, associating MI with neuroticism does not detract from evidence that certain kinds of stress can affect depression ([@B69], [@B70]). The main advantage to this study is the longitudinal collection of data, which is likely to give a more accurate depiction of moods than retrospective studies ([@B23]).
The results show that low and high moods, and low and high MI, are highly correlated in people with depression compared with those who are not depressed. Current psychiatric practice does not assess or treat MI or brief high mood episodes in patients with depression. New models of mood that also focus on MI will need to be developed to address the pattern of mood disturbance in people with depression.
Ethics Statement {#S5}
================
This study was carried out in accordance with the recommendations of the Canadian Tri-Council Policy Statement for Ethical Conduct in research involving humans with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Saskatchewan Behavioural Ethics Board.
Author Contributions {#S6}
====================
RB: study conception, drafting the article, revised the article after peer review, and responded to reviewer comments. EP: contributing to the concepts as well as reading and adding to the final version. SM and MB: contributing to the concepts as well as reading and providing approval to the final version. LB: conception of the work, analysis of the data, and revised the article after peer review.
Conflict of Interest Statement {#S7}
==============================
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 {#S8}
=======
This research did not receive funding from any sources.
[^1]: Edited by: Renerio Fraguas, University of São Paulo, Brazil
[^2]: Reviewed by: Casimiro Cabrera Abreu, Queen's University and Providence Care, Canada; Angela Marie Lachowski, Ryerson University, Canada; Serafim Carvalho, Hospital de Magalhães Lemos, Portugal
[^3]: Specialty section: This article was submitted to Mood and Anxiety Disorders, a section of the journal Frontiers in Psychiatry
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Repeated sequences are the main component of plant genomes, especially those with large C-value. In bread wheat, barley, and maize, more than 80% of the sequenced DNA is classified into mobile elements, so called transposable elements (TEs) ([@evv001-B28]; [@evv001-B34]). TEs were traditionally classified into two main classes according to their lifestyle cycle: Class I, or retrotransposons, for TEs moving through an RNA intermediate, which use a so called "copy and paste" mechanism, and Class II, or transposons, for TEs moving through a DNA intermediate, which use a so called "Cut and Paste" mechanisms ([@evv001-B33]). Long terminal repeat (LTR)-retrotransposons, that pertain to Class I, are the most abundant TEs identified in plant genomes. The activity of TEs has a deep influence on the evolution and function of plant genes and genomes and so contributes to the implementation of molecular diversification and genetic diversity. Their activity is controlled at the transcriptional and posttranscriptional levels by the host. However, the high activity of LTR-retrotransposons overtakes occasionally these mechanisms that control TE proliferation leading to sudden accumulation of LTR-retrotransposon copies (so called "burst") and, consequently to a rapid genome size increase ([@evv001-B21]).
With the advent of large-scale plant genome sequencing and the advances in TE bioinformatics analysis ([@evv001-B7]), it became clear that most of the TEs identified so far were not able to synthesize the full enzymatic machinery and all the molecules involved in their own mobility and to accomplish their multiplication cycle, disabling their coding capacities, that lead to their inactivation and so counteract their impact on genome size increase ([@evv001-B6]; [@evv001-B18]; [@evv001-B32]). In some cases, homologous recombination mechanisms occurring between LTR sequences in the same LTR-retrotransposon element lead to solo LTR formation implicating the removal of a large internal portion of elements. These altered elements are usually considered as dead elements, which are no longer capable of transcription and mobility.
However, there are reports where elements carrying a defective transposition machinery can get "back to life" and meet again the ability to move and to multiply their copy numbers in the host genome ([@evv001-B35]; [@evv001-B11]; [@evv001-B30]). Such elements, often called nonautonomous elements, are supposed to mobilize through a cross activation (in trans) with autonomous and functional partners. This interaction requires that nonautonomous elements still carry recognition domains for proteins encoded by autonomous partners ([@evv001-B33]; [@evv001-B36]). Two groups of Class I nonautonomous LTR-retrotransposons were identified in numerous plant genomes: TRIM (terminal-repeat retrotransposons in miniature) ([@evv001-B35]), and LARD (large retrotransposon derivative) ([@evv001-B11]) ([fig. 1](#evv001-F1){ref-type="fig"}). TRIMs and LARDs are, respectively, short (\<2 kb) and long (\>4 kb) elements that although they have lost their internal coding regions, they are involved in restructuring plant genomes ([@evv001-B35]; [@evv001-B12]). BARE-2 is another type of active nonautonomous elements found in Barley ([@evv001-B30]). BARE-2, which lacks the GAG domain, involved in the packaging of the element into the virus-like particle, remains mobile using the functional GAG capsid protein encoded by the BARE-1 autonomous elements ([@evv001-B30]). BARE-2 elements represent the unique described case of *cis*-parasitims of an LTR-retrotransposons in plants. However, the BARE-2 nonautonomous structure was investigated only in *Triticeae* genomes ([@evv001-B31]). The profusion of LTR-retrotransposons within plant genomes, the abundance of structural variation of defective elements, and the recent discovery of nonautonomous elements raise the question to know whether the whole structural variety of nonautonomous LTR-retrotransposons has been really identified or whether novel structures remain to be characterized. F[ig]{.smallcaps}. 1.---Conserved structures of nonautonomous LTR-retrotransposons documented in plant genomes. Autonomous refers to the structure of complete LTR-retrotransposons (here *Copia*-like): The coding regions are in gray; the PBS motif is represented as a black triangle and the PPT is represented as a white triangle; GAG, capsid; AP, aspartic protease; INT, integrase; RNAse, RNAse H. BARE-2 refers to the BARE-2 nonautonomous found in barley ([@evv001-B30]).
In an attempt to characterize the whole set of mobile elements within the *Coffea* genomes, especially in *Coffea canephora* ([@evv001-B5]), we report here a new group of nonautonomous LTR-retrotransposons, called TR-GAG (terminal-repeat retrotransposons with GAG domain) in plants. TR-GAG elements are short LTR-retrotransposons (\<4 kb) carrying a unique open reading frame (ORF) coding for a GAG capsid protein. In *C. canephora* genome, five families of TR-GAG elements were described. These elements are expressed and their evolutionary dynamics in the *Coffea* genus indicated different pathways in the copy number variations. Similar structures were found in numerous available sequenced eudicotyledoneous, monocotyledoneous, and algae genomes, indicating that TR-GAG elements could be ubiquitous TEs in plants.
Materials and Methods
=====================
Plant Material, DNA, and RNA Preparation
----------------------------------------
Three coffee species were used in our analyses: *Coffea arabica* (accessions AR52 and ET39), *Coffea eugenioides* (accession DA71), and *C. canephora* (accessions BA58, BB60, BD69, and DH 200-94). All plants were growing in the greenhouses at the IRD center, Montpellier (France). Leaves were harvested and stored at −80 °C prior to DNA extraction, using Qiagen DNeasy Plant Mini extraction kits. Quantity and quality of DNA were measured using a Nanodrop (ND-1000). RNA preparations were obtained from leaves of *C. arabica* (accession ET39), *C. eugenioides* (accession DA71), and *C. canephora* (accession DH 200-94), using the SV Total RNA Isolation System (Promega).
Identification, Classification, and Annotation of LTR-Retrotransposons
----------------------------------------------------------------------
A manual annotation procedure was undertaken on 17 publicly available *C. canephora* and *C. arabica* bacterial artificial chromosome sequences (accounting for 3,023,472 bp) and from the ten largest *C. canephora* scaffolds (accounting for 65,698,623 bp, from the *C. canephora* draft genome generated by the Coffee Genome Consortium) to build an initial database. A total of 948 elements were finally annotated as follows and classified according to the universal classification of TEs ([@evv001-B33]): 516 transposons (DTX), 7 helitrons (DHX), 14 LINE (RIX), 330 LTR-retrotransposons (RLX), 1 Retrovirus (RTX), 61 SINE (RSX), and 19 Unclassified (XXX, noCat). This manually curated database was enriched by a de novo detection of LTR-retrotransposons using the LTR_STRUC algorithm ([@evv001-B19]) against 568 Mb of the *C. canephora* draft genome (Coffee genome project; <http://coffee-genome.org>; [@evv001-B5]). A total of 1,799 full-length LTR-retrotransposons were detected from *C. canephora* scaffolds with a size larger than 5 kb. This data set was classified into *Gypsy* (RLG) and *Copia* (RLC) according to their similarity matches against the GyDB domain libraries (<http://www.gydb.org/index.php/Main_Page>) ([@evv001-B15]). Sequences were classified into the RXX (Unclassified retrotransposon) category if no conserved domains were found or if only a GAG domain was identified. The LTR_STRUC data set was composed of 745 RXX (41%), 580 RLG (32%), and 474 RLC (26%).
In Silico Characterization of Nonautonomous Elements
----------------------------------------------------
The identification of complete, and fragmented copies of elements was done using Censor ([@evv001-B13]) against the 568 Mb of the *C. canephora* draft genome. A complete copy is considered if it covers a minimum of 80% of the reference sequence with a minimum of 80% of nucleotide identity, a distantly complete copy is considered if it covers a minimum of 70% of the reference sequence with a minimum of 70% of nucleotide identity. The genomic distribution of elements was plotted using CIRCOS (<http://circos.ca>). The insertion sites of complete copies were identified using the best-conserved sequence considered as reference to extract complete copies with 100% of coverage against the reference sequences. Sequence of 10 bp downstream and upstream the insertion sites were extracted and analyzed using WebLogo (<http://weblogo.berkeley.edu/logo.cgi>).
Characterization of TR-GAG Families in *C. canephora* draft Genome
------------------------------------------------------------------
Raw results from LTR_STRUC were filtered to retrieve putative TR-GAG families, according to the following parameters: 1) A maximum length of 4 kb for each predicted element, 2) similarity (*e* value \< 10e^−4^ on BLASTx) with only the GAG capsid domains downloaded from the GyDB database (<http://www.gydb.org/index.php/Main_Page>), and 3) a redundancy of a minimum of two copies within the genome. Sequence of TR-GAGs was submitted to GenBank: TR-GAG1: KM360147, TR-GAG2: KM371274, TR-GAG3: KM371276, TR-GAG4: KM371277, TR-GAG5: KM371275.
Estimation of TR-GAG Copy Number Using 454 Sequencing Survey
------------------------------------------------------------
One plate of 454 Pyrosequencing (GS Junior System Roche) was performed for each *Coffea* species classified early by [@evv001-B2] into Eucoffea such as: Two *C. canephora* Pierre ex A.Froehner accessions (DH200-94 from Congo Democratic Republic and BUD15 from Uganda), *Coffea heterocalyx* Stoff. (JC62) from Cameroon, *C. arabica* L. (ET39) from Ethiopia, *C. eugenioides* S. Moore (DA59) from Kenya, Mozambicoffea such as *Coffea pseudozanguebarie* Bridson (H52) from Kenya, *Coffea racemosa* Lour. (IA56) from Mozambique, Mascarocoffea such as *Coffea humblotiana* Baill. (A230) from Comoro Islands, *Coffea tetragona* Jum. & H.Perrier (A252) and *Coffea dolichophylla* J.-F.Leroy (A206) from Madagascar ([supplementary data S1](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online) and *Coffea horsfieldiana* (Miq.) J.-F. Leroy from Indonesia, formerly classified as *Psilanthus* and recently placed into *Coffea* ([@evv001-B37]), and *Craterispermum* Sp. *Novo kribi* (Rubiaceae) from Cameroon. The cultivars and accessions used grow in the IRD greenhouses (Montpellier, France) and FOFIFA research station (Kianjavato, Madagascar).
Total genomic DNA was extracted from young leaves using the Qiagen DNeasy Plant Mini Kit following the manufacturer protocol. The library construction and NGS sequencing were performed at Nestlé R&D laboratory according to the Roche/454 Life Sciences Sequencing protocol. In total, 1,624,178 sequences were generated accounting for 678 Mb. Data were submitted to GenBank, BioProject PRJNA242989.
BLASTN searches were carried out with the five TR-GAG families found previously in the *C. canephora* genome. Reads with more than 80% of nucleotide identity with the reference sequence over a minimum 80% of the read lengths were considered as potential fragments of the element. Cumulative lengths of aligned reads were used to extrapolate the contribution of the element to each genome size investigated. For each element family, the potential number of full-length copies is estimated by the division of the estimated size of total members of the element in the genome by the reference sequence length.
Characterization of TR-GAG Families in 33 Plant Genomes
-------------------------------------------------------
LTR_STRUC ([@evv001-B19]) was used to predict LTR-retrotranposons in 33 available plant genomes retrieved from specific sites and the Phytozome web site (<http://www.phytozome.net>; [supplementary data S2](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online) as follows: 24 dicotyledonous genomes---*Nicotiana sylvestris*, *Solanum lycopersicum* (tomato), *Solanum tuberosum* (potato), *Mimulus guttatus*, *Uticularia gibba* (bladderwort), *Vitis vinifera* (grape), *Cucumis sativus, Citrullus lanatus* (watermelon), *Fragaria vesca* (strawberry), *Prunus persica* (peach), *Malus domestica* (apple), *Medicago truncatula, Cicer arietinum* (chickpea), *Lotus japonicus, Glycine max* (soybean), *Phaseolus vulgaris* (common bean) *Populus trichocarpa* (poplar), *Manihot esculenta* (cassava), *Ricinus communis, Theobroma cacao* (cacao), *Carica papaya* (papaya), *Arabidopsis thaliana, Brassica rapa* (rapeseed), and *Citrus clementina* (clementine); seven monocotyledonous genomes---*Phoenix dactylifera* (date palm), *Elaeis oleifera* (oil palm), *Musa acuminata* (banana), *Zea mays* (maize), *Sorghum bicolor* (sorghum), *Brachypodium distachyon* (false brome), and *Oryza sativa* (rice), and two other genomes: *Amborella trichopoda* (angiosperm) and *Selaginella moellendorffii* (nonangiosperm). A total of 18.9 Gb of sequence was downloaded, processed with LTR_STRUC, and filtered out as described above.
Search for TR-GAG Pattern in Genomes
------------------------------------
We developed an algorithm to automatically detect TR-GAG elements in genomes. The algorithm consists in translating the six frames for every "pseudomolecule" present in the target genome, followed by a search for HMM (Hidden Markov Models) motifs using the hmmer package (<http://hmmer.org>). The Retrotrans_gag, UBN2, UBN2_2, and UBN2_3 motifs were used to detect GAG protein signatures. Flanking regions of 5 kb are extracted for all hits with *e* value \< 1e^−5^ and direct repeats greater than 200 bases are searched by dividing the sequence in two and using BLASTN alignment. The region including the direct repeats and the GAG motif is extracted, translated, and searched for reverse transcriptase motifs and only the candidates that present no *Copia* or *Gypsy* reverse transcriptase motifs are retained. These candidates are further filtered by size, keeping those sequences between 1 and 6 kb, whereas redundant candidates are eliminated.
Transcriptional Analysis of the TRIM-1-S and TR-GAG1 Elements by Reverse Transcription Polymerase Chain Reaction
----------------------------------------------------------------------------------------------------------------
Reverse transcription polymerase chain reaction (RT-PCR) was done using cDNA from *C. arabica* (ET39), *C. eugenioides* (DA71), and *C. canephora* (DH 200-94). cDNA was synthetized from 250 ng of total RNA using the ImProm-II Reverse transcription System Kit (Promega). Primers were selected using Primer3 (<http://frodo.wi.mit.edu>) on TR-GAG1 and TRIM-1-S sequences ([table 1](#evv001-T1){ref-type="table"}). PCR was performed in a final volume of 20 μl as follows: 0.5 μl of dNTP (10 nM), 1 μl of each primer (10 mM), 0.2 μl of Taq polymerase (GoTaq, Promega), 4 μl of buffer, and 2 μl of cDNA. We used the following PCR amplification cycle: 98 °C 5 min; and three steps (98 °C 30 s, 55 °C 30 s, 72 °C 30 s) repeated 35 times followed by a final elongation step (72 °C 5 min). Table 1List of Primers Used for RT-PCR AnalysisPrimersSequences (5′--3′)Product Size (bp)TRIM-1-S-FCACCTCCAACGGTTGATTCT361TRIM-1-S-RATGTGTAGTTGCCCCGAGTCTR-GAG1-FGCAGCAGACCTCTGGAAAAA328TR-GAG1-RTGGTTTGCCTTCCTTTGTTTG3-FACGAGTGGGTTTCCTGAGTG---[^a^](#evv001-TF1){ref-type="table-fn"}G3-RTGGGTCTCTGGAACTTACCG[^4]
Transcriptional Analysis of TR-GAG Elements Using RNA Sequencing
----------------------------------------------------------------
RNA sequencing (RNA-seq) data generated under the *C. canephora* genome project (coffee-genome.org) from leaves, roots (*C. canephora* accession T3518), stamen, and pistil (*C. canephora* accession BP961) were used to identify the transcriptional pattern of reference sequences (<http://coffee-genome.org>; [@evv001-B5]). Nearly 130 million of Illumina reads (2 × 100 bp) were cleaned using prinseq ([@evv001-B27]) and mapped against reference TR-GAG sequences using bowtie 2 ([@evv001-B14]). Number of mapped reads per reference sequence was processed and RPKM (reads per kilo base per million) was calculated. Differential expression among RNA-seq libraries was detected from variation of mapped reads and all sequenced reads using Winflat ([@evv001-B1]).
Phylogenetic Analyses and TR-GAG Insertion Times
------------------------------------------------
The classification of GAG domains from TR-GAG elements found in the Coffee genome was confirmed by phylogenetic analyses. GAG domains were first identified by similarity against the GAG domains from the Gypsy Database 2.0 (290 domains as in August 2014), extracted from the nucleotide sequence of TR-GAG, and translated into amino acids. Amino acid sequences (with a minimum of 200 residues) were aligned (ClustalW) to construct a bootstrapped neighbor-joining tree, edited with FigTree (<http://tree.bio.ed.ac.uk/software/figtree/>).
The insertion times of full-length copies, as defined by a minimum of 80% of nucleotide identity over 100% of the reference element length, were dated. Timing of insertion was based on the divergence of the 5′- and 3′-LTR sequences of each copy. The two LTRs were aligned using stretcher (EMBOSS), and the divergence was calculated using the Kimura 2-parameter method implemented in distmat (EMBOSS). The insertion dates were estimated using an average base substitution rate of 1.3 E-8 ([@evv001-B17]).
Results
=======
Annotation and Identification of the Nonautonomous LTR-Retrotransposons TRIM-1 Family in the *C. canephora* Genome
------------------------------------------------------------------------------------------------------------------
We used the draft genome sequence of the *C. canephora* accession DH 200-94 to annotate TEs ([@evv001-B5]; <http://coffee-genome.org>). We first performed a manual annotation of TEs using the ten largest scaffolds from *C. canephora* genome sequencing project (accounting for 65,698,623 bp, scaffold1--10), and an initial database of 948 TEs was produced (Guyot R, unpublished data). Among the 948 elements, 11 conserved short elements (\<3 kb) harboring a typical LTR-retrotransposon structure (two duplicated regions starting by TG, and finishing by CA, flanked by a target site duplication \[TSD\] of 5 bp, and a polypurine tract \[PPT\] located upstream the 3′-duplicated region) were identified using similarity searches (BLASTN). After initial analyses, we found two sequence groups with different lengths. Short sequences (∼1,700 bp) were called TRIM-1-S have the typical structure of TRIM ([@evv001-B35]), whereas long sequences (∼2,500 bp) were called TR-GAG1 (terminal repeat with GAG domain). They are similar to the TRIM but carry an internal region similar to LTR-retrotransposons GAG capsid domain ([fig. 2](#evv001-F2){ref-type="fig"}*A* and *B*). The last structure was not previously described in plant genomes. The two groups of sequences are conserved except for the presence of the GAG domain in TR-GAG1 ([fig. 2](#evv001-F2){ref-type="fig"}*C* and [supplementary data S3](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). Multiple alignment of the LTR sequences from the TRIM-1-S and TR-GAG1 elements shows an overall strong conservation between the two groups as well the presence of a putative TATA box that could intervene in the initiation of the elements' transcriptions ([supplementary data S3](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). An exhaustive search against the *C. canephora* draft genome (568 Mb) indicated the presence of 71 and 60 complete copies of TRIM-1-S and TR-GAG1, respectively. All complete dispersed copies within the chromosomes with conserved LTR extremities, showed different insertion sites ([supplementary data S4](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). The complete elements are flanked by 5-bp direct repeats usually generated during the LTR-retrotransposon insertions, suggesting that they are originated from different replications events. Using BLASTN algorithm, we searched in the *C. canephora* genome for autonomous elements sharing high nucleotide conservation with TRIM-1-S and TR-GAG1, but we did not find any autonomous full-length elements in the available genomic sequences. F[ig]{.smallcaps}. 2.---Structure and graphical alignments of the nonautonomous LTR-retrotransposons TRIM-1 family. (*A*) Schematic representation of the TRIM-1-S element and alignment of five different *C. canephora* TRIM-1-S genomic copies against themselves using Dotter ([@evv001-B29]). (*B*) Schematic representation of the TR-GAG1 element and alignment of five different *C. canephora* TR-GAG1 genomic copies against themselves using Dotter. (*C*) Dotter alignment between TR-GAG1 (horizontal sequence) and TRIM-1-S (vertical sequence).
Detailed Analysis of the TR-GAG1 Elements
-----------------------------------------
We detailed the structure of the TR_GAG1 elements (Copy found in *C. canephora* draft genome located on "Chr 0," positions 113020990--113023502, accession KM360147), as such conserved structure of nonautonomous LTR-retrotransposon was not described yet. TR-GAG1 elements have LTR lengths of approximately 485 bp. The 5′-LTR is flanked downstream by a primer binding site (PBS) complementary to the Leucine transfer RNA and the 3′-LTR is flanked upstream by a PPT 5′-AAAAGGCAAATGGAG-3′ ([fig. 3](#evv001-F3){ref-type="fig"}). Beside LTR regions, no internal duplicated region was found in the TR-GAG1 sequence. The inner region is composed of an ORF of 433 amino acids with strong similarities with GAG (group-specific antigens) and more particularly with the UBN2 family domain from Pfam (gag-polypeptide of LTR *Copia* superfamily). The small structural motif of Zinc finger (Zf-C2HC) is also found at the amino-acid residue 275 the ORF (position 1245--1286 along the full-length TR-GAG nucleotide sequence). At the C terminal part, few similarities were observed with aspartic proteases from the GyDB but no motif was conserved in Pfam database ([@evv001-B22]). The UBN2 Pfam domain (PF14223) from TR-GAG1 is described as associated with *Copia* Superfamily of complete LTR-retrotransposons (<http://pfam.xfam.org>). No significant RNA secondary structure was found with the putative leader sequence of TR-GAG1, suggesting either absent or labile PSI (Packaging Signal) and DIS (Dimerization Signal) motifs. These motifs were identified in Retroviruses and are involved in the packaging and RNA dimerization ([@evv001-B30]). F[ig]{.smallcaps}. 3.---Schematic representation of the TR-GAG1 structure. The TR-GAG1 element contains the following sequence characteristics: LTR, PBS, PPT, and an ORF harboring known GAG motifs (here UBN2 and Zf-C2HC motifs). The element shown is located on "Chr. 0" positions 113020990--113023502 from the *C. canephora* draft genome (<http://coffee-genome.org>).
Transcriptional Responses of the TRIM1/TR-GAG Family
----------------------------------------------------
We analyzed the transcriptional pattern of TRIM-1-S and TR-GAG1 elements in three coffee species. Specific primers were selected in TRIM-1-S and TR-GAG1 to amplify the inner regions. For TR-GAG1, primers amplify a 328-bp product from the GAG precursor. RT-PCR analyses indicate the presence of transcripts for TRIM-1-S and TR-GAG1 originating from mRNA leaves, suggesting that elements are expressed in *C. canephora*, *C. eugenioides,* and *C. arabica* ([supplementary data S4*A*](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) and [*B*](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). RNA-seq analysis using 130 million of Illumina reads shows that 38 complete copies of TR-GAG1 are expressed at low level in vegetative tissues (leaves and roots) whereas no or few expression was detected in reproductive tissues (pistil and stamen) ([supplementary data S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online).
Characterization of TR-GAG Families in *C. canephora*
-----------------------------------------------------
We searched the presence of other TR-GAG families in the draft genome of *C. canephora*. We used first the results of LTR_STRUC prediction of LTR-retrotransposons. The 1,799 putative LTR-retrotransposons predicted by LTR_STRUC were filtered out according to the features identified for TR-GAG1. Beside an overall structure of elements, such as presence of LTR, PBS, and PPT regions, sequences with a maximum length of 4 kb, a minimum redundancy of two copies, and with similarities for GAG Capsid proteins but not with aspartic protease, integrase, reverse transcriptase, and RNAse H domains were selected for further analysis. On 1,799 predicted elements, 130 were retained. Sequences were compared against themselves using dot-plot alignments ([fig. 4](#evv001-F4){ref-type="fig"}*A*). Sequences were clustered into five groups of sequences according to their similarities and classified into five different families (called TR-GAG1 to TR-GAG5). One family called TR-GAG2, which exhibited a large number of conserved predicted structures (110 elements) as observed in dot-plot and alignment analysis ([supplementary data S6](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online), was analyzed further ([fig. 4](#evv001-F4){ref-type="fig"}*A* and *B*). Among the 110 conserved predicted elements, we selected one copy for detailed analysis (located on pseudochromosome 4 21003142--21006851). This element presented an overall similar structure to TR-GAG1 ([fig. 4](#evv001-F4){ref-type="fig"}*C*). TR-GAG3, TR-GAG4, and TR-GAG5 families were analyzed and also shown a typical structure of TR-GAG nonautonomous elements ([supplementary data S8](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). Although TR-GAG2 shares similarities with the same *Copia* GAG Pfam domain family (UBN2) with TR-GAG1, TR-GAG3 and TR-GAG4 contain the Retrotrans_gag motif (Pfam PF03732) that appears associated with annotated *Copia* and *Gypsy* polyproteins in Uniprot database (<http://www.uniprot.org>). Phylogenetic analysis with reference GAG domains from GyDB confirmed the similarity of TR-GAG1 and TR-GAG2 GAG domains with *Copia* and TR-GAG4 with *Gypsy* subfamily GAG domains ([supplementary data S7](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). All five TR-GAG families were analyzed using RNA-seq. We observed different pattern of expression according to the four tissues analyzed: Leaf, root, pistil, and stamen ([supplementary data S5](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). F[ig]{.smallcaps}. 4.---Characterization of TR-GAG families in the *C. canephora* draft genome. (*A*) Dot-plot of 130 predicted TR-GAG sequences against themselves. TR-GAGs were predicted by LTR_STRUC and filter out according to features described for TR-GAG1. Sequences were clustered by similarity. (*B*) Detailed structure of one copy (Chr. 4, positions 21003142--21006851) of the TR-GAG2 family.
Distribution and Copy Number Estimation of TR-GAG Elements in the *Coffea* Genus
--------------------------------------------------------------------------------
We first investigate the copy number of the five identified TR-GAG families in the *C. canephora* sequenced genome ([supplementary data S9](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). Complete copies of TR-GAG1 and TR-GAG2, as defined by 80% of nucleotide identity over 100% of the reference element length, were used to estimate their insertion times ([supplementary data S10](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). Our analysis indicates a relatively recent increase of TR-GAG2 elements (highest peak at 0.5--1 Ma).
The distribution of the five identified TR-GAG families along the reconstructed pseudochromosomes in *C. canephora* was also studied. Copies (with two level of conservation: 80--80 and 70--70), solo LTRs, and fragmented copies were identified from the *C. canephora* draft genome sequence ([supplementary data S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online).
In order to investigate the evolution of TR-GAG families, we used in silico approaches to search for its presence in the *Coffea* genus. Nine additional *Coffea* species (including *C. horsfieldiana* \[ex-*Psilanthus horsfieldiana*\]) and an outgroup in the Rubiaceae family: *Craterispermum kribi* from Cameroon, were surveyed using a high-throughput 454 sequencing analysis. The *Craterispermum* genus, belonging to the Rubioideae subfamily, diverged early from the *Coffea* genus (Ixoroideae sub-family), about 80 Ma ([@evv001-B38]).
The 454 sequences ([table 2](#evv001-T2){ref-type="table"}) were first used to survey the presence of highly conserved reads of TR-GAG, using the criteria of 80% minimum nucleotide identity with over 80% of the read length. Sequences fitting these criteria show a large variation of reads for the TR-GAG2 family in *Coffea* and *Cr. kribi* genomes. Additionally, with this approach we could estimate the copy number of TR-GAG elements in several genomes. Using these conserved reads, TR-GAG was estimated to range from 0 to 696.7 copies in diploid species and from 10.2 to 1,168.7 copies in *C. arabica*. However, in almost all cases (at the exception of *Craterispermum* and *C. tetragona*), the highest copy numbers were obtained for TR-GAG-2. Only few copies (respectively, 5 and 7 copies) of TR-GAG-2 and TR-GAG-1 were detected for the *Craterispermum* outgroup (Rubiaceae) ([supplementary data S11](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). The TR-GAG-2 family contributes to the genome size of diploid species, but with a relatively weak intensity ([supplementary data S12](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). However the genome size contribution of TR-GAG-2 appears to decrease in species going from West to East in species belonging to Eucoffea (*C. canephora, C. heterocalyx, C. eugenioides*, and *C. arabica*), Mozambicoffea (*C. pseudozanguebariae* and *C. racemosa*), and Mascarocoffea (*C. humblotiana,Coffea millotii ex-dolichophylla,* and *C. tetragona*). The Indonesian *C. horsfieldiana* appears intermediate between Eucoffea and Mozambicoffea or Mascarocoffea botanical groups. Only traces of TR-GAG2 and TR-GAG1 were detected in *Craterispermum* (Rubiaceae). Table 2Estimation of the TR-GAG Family's Copy Number in *Coffea* Genomes Using 454 Sequencing SurveySpeciesPloidy LevelEstimated Genome Size (Mb)\#454 SequencesProduced Bases (Mb)Genome Coverage (%)TR-GAG1 CopiesTR-GAG2 CopiesTR-GAG3 CopiesTR-GAG4 CopiesTR-GAG5 Copies*Coffea canephora (HD94-200)*2n700106,45945.056.40172,48563,076,748,1827,28*Coffea canephora (BUD15)*2n700149,19667.089.5869,61390,6214,8522,2044,88*Coffea arabica*4n1,240122,25854.54.39111,551168,7255,4010,2135,21*Coffea eugenioides*2n645101,30942.16.5262,56659,4428,6426,1422,42*Coffea heterocalyx*2n863194,360.5112.2597,94696,7113,979,0024,68*Coffea racemosa*2n50688,49834.195.754,02103,022,960,0016,04*Coffea pseudozanguebariae*2n593215,11791.715.459,76157,791,127,3413,67*Coffea humblotiana*2n469160,47967.9914.4926,7780,000,000,0013,64*Coffea tetragona*2n513160,10772.6614.1048,4534,350,920,0021,63*Coffea dolichophylla*2n682163,87376.6511.2361,91144,930,000,0018,40*Psilanthus horsfieldiana*2n593112,79346.257.843,56336,741,350,0024,50*Craterispermum kribi*2n74849,78919.442.945,076,960,000,000,00[^5]
Characterization of TR-GAG Families in Genomes Using LTR_STRUC Algorithm
------------------------------------------------------------------------
We searched TR-GAG element structures in 33 available plant genomes. In total, more than 18 Gb of genomic sequences were processed with LTR_STRUC and a total of 38,772 predicted LTR-retrotransposons were found ([supplementary data S13](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). After filtering, a total of 373 candidates were found distributed among 23 different monocotyledonous and dicotyledonous plant genomes ([fig. 5](#evv001-F5){ref-type="fig"}). Detailed analysis of candidates TR-GAG elements confirmed the structures previously discovered in the *C. canephora* genome. F[ig]{.smallcaps}. 5.---Identification of TR-GAG families in available plant genomes. (*A*) Dot-plot of predicted TR-GAG sequences from 23 plant genomes against themselves. TR-GAGs were predicted by LTR_STRUC and filter out according to features described for TR-GAG1. Sequences were clustered by plant genomes. (*B*) Detailed structure of one TR-GAG family for seven different plant genomes.
Detection of TR-GAG Families in Genomes Using HMM
-------------------------------------------------
In order to validate the detection of TR-GAG by LTR_STRUC, we developed HMM to recognize GAG motifs (retrotrans_gag, UBN2, UBN22, UBN23) surrounded by direct repeats. The new model was used in Banana (*Musa acuminata*, angiosperm, monocots), *Cacao* (*Theobroma cacao*, angiosperm, dicots), coffee (*C. canephora*, angiosperm, dicots), *Ectocarpus* (*Ectocarpus siliculosis*, brown algae; [@evv001-B3]), *Chondrus* (*Chondrus crispus*, red algae; [@evv001-B4]), and *Drosophila* (*Drosophila melanogaster*, insect) genomes. Although TR-GAG elements were found in all angiosperm and brown algae genomes, no potential candidate was predicted in red algae and *Drosophila* genomes. Twenty-five TR-GAG families were detected for Banana and one of them shows a high copy number (∼700 copies, [supplementary data S14](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). In brown algae (*Ectocarpus*), the presence of one TR-GAG-like sequence was previously reported ([@evv001-B3], in [supplementary material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online). Using our detection approach, four TR-GAG families were finally predicted in this genome ([@evv001-B3], in [supplementary material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1), [Supplementary Material](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) online).
Discussion
==========
The identification and classification of the whole spectrum of LTR-retrotransposon structures is particularly a complex process in plant genomes due to the huge number and variety of defective LTR-retrotransposon structures. Although most of the defective structures, deriving from a wide variety of rearrangement mechanisms, lead to inactive elements, some of them remain mobile like TRIM, LARD, and BARE2 elements ([@evv001-B35]; [@evv001-B11]; [@evv001-B30]). These known nonautonomous LTR-retrotransposon structures redefined our view of the definition of what is really an active element in genomes, and raised new questions about their precise classification and their mechanisms of mobility. The discovery of such exceptional diversity of nonautonomous structures opened the door to the large-scale in silico exploitation of plant genome sequences to seek novel nonautonomous structures.
The novel element called TR-GAG belongs to such type of nonautonomous structures and brings new insight on TE and genome evolution. TR-GAG elements clearly belong to LTR-retrotransposons order of TEs ([@evv001-B33]). TR-GAG can be identified using de novo LTR-retrotransposons finding programs like LTR_STRUC ([@evv001-B19]), as they share key structural features with them, like LTR domains, PPT and PBS motifs and a 5-bp TSD at their insertion sites in the host genome. TR-GAGs appear generally smaller (\<4 kb) than typical full-length *Copia* and *Gypsy* LTR-Retrotransposons (5--20 kb) in plants. Several signs suggest that TR-GAGs are active elements in *Coffea* species in spite of the absence of an internal polyprotein domain: 1) RT-PCR and RNA-seq data show the transcription of TR-GAG families. Although TR-GAG1 is mainly expressed at a low level in vegetative tissues, other families (TR-GAG2 and TR-GAG3) show a significant expression in reproductive tissues suggesting that new insertions could be vertically transmitted to the progeny; 2) the copy number of TR-GAG elements in *C. canephora* and the different TSD motifs found for each copy suggests an amplification mechanism that can be achieved by the lifestyle cycle of mobile LTR-retrotransposons; 3) the high conservation of sequence and structure between each TR-GAG copy in the *C. canephora* genome; and 4) their insertion time patterns.
TR-GAG elements lack a polyprotein domain involved in the mobility, but carry a GAG precursor, which is usually processes by protease into protein subunits (matrix, capsid, and nucleocapsid) ([@evv001-B9]). This structure is the strict opposite of the described BARE-2 nonautonomous elements in barley, lacking only the GAG domain. It remains mobile as a two-component system: A nonautonomous elements (BARE2) and an autonomous counterpart (BARE-1) providing by complementation-like a functional GAG precursor ([@evv001-B30]). For TR-GAG1 elements, no full-length autonomous element similar to the TR-GAG1 sequence was found in the draft genome sequence of *C. canephora*, suggesting that either the mobility of TR-GAG1 is driven in trans by a compatible but different full-length autonomous elements, or the complete element appears as absent due to incompleteness of the sequenced genome or it has been specifically lost in the studied and sequenced genotype. The presence of a functional GAG precursor in TR-GAG elements also raises the question to know their potential role in the cycle of other LTR-retrotransposon elements. The capsid (CA) and nucleocapsid (NC) protein subunits of GAG precursors are, respectively, implicated in the transposition and in the assembly packaging, reverse transcription, and integration mechanisms. More generally GAG proteins appear to be able to engage interactions with a wide spectrum of molecules such as proteins, DNA, RNA, and lipids ([@evv001-B9]).
The GAG peptides encoded by TR-GAG elements may drive in trans the mobility of a variety of other LTR-retrotransposons that lack functional GAG domain similarly to the BARE2. Additional molecular experimental data will be required to precisely understand the function of GAG domain in TR-GAG elements.
Five different families of TR-GAG were identified in *C. canephora.* They carry GAG domains that show similarities with both *Copia* and *Gypsy* superfamily related GAG domains suggesting that TR-GAG structures have been generated with a frequent and common mechanism for all LTR-retrotransposon superfamilies certainly involving unequal recombination events ([@evv001-B18]). In *C. canephora*, all five TR-GAG families show different complete, fragmented and solo LTR copy numbers, suggesting distinct levels of proliferation control by the host genome. Interestingly, TR-GAG2 that shows the highest copy number is nonrandomly distributed along the *C. canephora* pseudomolecules and targets preferentially TE -rich regions.
The TR-GAG2 family shows high variation in copy number among the ten *Coffea* species we analyzed. These variations are in agreement with the three botanical sections (or groups) defined by [@evv001-B2], strongly suggesting that TR-GAG2 copy number proliferation is associated with the evolution of botanical groups of *Coffea*. These botanical sections correspond also to genetically differentiated groups as obvious from fertility of FI interspecific hybrids ([@evv001-B16]), mean genome sizes ([@evv001-B20]; [@evv001-B23]), and from genetic diversity revealed by simple sequence repeat markers ([@evv001-B24]).
Finally, the presence of TR-GAG structures in 23 different plant genomes from dicotyledonous and monocotyledonous species, as well as in basal Angiosperms (*Amborella*) and one algae species, indicates that these elements are ubiquitous mobile elements. Comparisons between all predicted TR-GAG elements in plants ([fig. 5](#evv001-F5){ref-type="fig"}) show the absence of conservation between species suggesting that TR-GAG elements were originated from distinct pool of full-length autonomous LTR-retrotransposons. The notable exception is the conservation of one TR-GAG family between *Cicer arietinum* and *Lotus japonicus* genomes ([fig. 5](#evv001-F5){ref-type="fig"}). Such significant conservation of TEs over different plant families suggests that TR-GAG elements could also be subjected to events of horizontal transfer like LTR-retrotransposons ([@evv001-B8]; [@evv001-B26], [@evv001-B25]).
Conclusions
===========
In conclusion, TR-GAG elements are a new nonautonomous element ubiquitous in plant genomes. TR-GAG elements are potentially active indicating that they are associated to functional full-length LTR-retrotransposons to achieve their life cycle. Considering their significant copy numbers TR-GAG elements could play an important role in chromosome structure, alteration of coding region expression, and genome evolution in plants.
Supplementary Material
======================
[Supplementary data S1--S14](http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evv001/-/DC1) are available at *Genome Biology and Evolution* online (<http://www.gbe.oxfordjournals.org/>).
###### Supplementary Data
This research was supported by Agropolis Fondation through the "Investissement d'avenir" program (ANR-10-LABX-0001-01) under the reference ID 1102-006 (Retro-cof). CAPES, through the "CAPES/Agropolis" program, partially funded the work, supported RFS post doc fellowships and ALLV and DSD working missions. R.G. is also supported by a Special Visiting Scientist grant from the Ciência sem Fronteiras program under the reference ID 84/2013 (Cnpq/CAPES). The authors thank Philippe Lashermes and the Coffee Genome Consortium for the availability of the *Coffea canephora* genome sequence and the South Green Bioinformatics Platform ([www.southgreen.fr](http://www.southgreen.fr)), for providing computational resources.
[^1]: ^†^These authors contributed equally to this work.
[^2]: **Associate editor:** Emmanuelle Lerat
[^3]: **Data deposition:**PRJNA242989, KM360147, KM371274, KM371276, KM371277, KM371275.
[^4]: ^a^Control primers used as in [@evv001-B10].
[^5]: N[ote]{.smallcaps}.---Only 454 reads with a minimum of 80% of nucleotide identity over 80% of the read length were considered. Genome sizes were listed in [@evv001-B20] and [@evv001-B23].
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Introduction {#S0001}
============
Le cancer du sein localement avancé est une hantise dans notre pratique quotidienne. Les étiologies qui poussent les femmes à consulter tardivement sont généralement en rapport avec la pauvreté intellectuelle et économique, mais de nos jours on assiste à une novelle cause du retard diagnostic émanant de la mauvaise compréhension des sujets médicaux traités sur le net.
Patient et observation {#S0002}
======================
Patiente âgée de 45 ans bibliothécaire de fonction, adepte à l\'automédication orientée par certains sites internet. Elle est multipare ayant allaitée cinq enfants, non fumeuse, sans notion de cancer dans la famille et n\'ayant jamais utilisée de contraception hormonale. Elle rapporte l\'apparition six mois auparavant d\'un nodule de petite taille au niveau du quadrant supero-externe du sein droit douloureux à l\'autopalpation dans un contexte d\'amaigrissement qui est mis sur le compte d\'un auto-régime hypocalorique entrepris par la patiente à cause de son surpoids. La patiente a faussement éliminé le diagnostic de cancer du sein en se basant sur l\'absence des facteurs de risque et la douleur du nodule. L\'apparition des ulcérations de la peau faisant penser la patiente au zona et traité entant que tel ce qui a retardé d\'avantage le diagnostic. L'évolution de la lésion s\'est faite d\'une façon spectaculaire et rapide dans le temps vers l\'extension à la peau avec un amaigrissement cadavérique ce qui a motivé une consultation au service de gynécologie de l\'hôpital militaire de la ville de Meknès. L\'examen à l\'admission objective une patiente d\'un teint cireux, cachectique, déshydraté dont l'état général est altéré; pesant 40 kilogrammes avec un amaigrissement chiffré à 36 kilogrammes en six mois. L\'examen mammaire révèle un ensemble de lésions nodulaires du sein droit, confluentes ulcérées par endroit, occupant presque la totalité de l\'hémi-thorax, auquel elle adhère intimement ([Figure 1](#F0001){ref-type="fig"}). L\'aire axillaire homolatérale est comblée par un paquet ganglionnaire de six centimètre de grand axe. Le sein controlatéral ne présente pas de lésion, la palpation abdominale met en évidence une hépatomégalie nodulaire. Une macro-biopsie du sein réalisée objective un carcinome canalaire infiltrant, grade III SBR avec emboles vasculaires; les récepteurs hormonaux sont positifs aussi bien pour les œstrogènes que la progestérone et une surexpression du HER2. Le bilan d\'extension découvre une métastase osseuse au niveau du col fémorale droit et plusieurs nodules métastatiques hépatiques. Un traitement palliatif à base de radio-chimiothérapie plus le trastuzumab est décidé par le staff multidisciplinaire d\'oncologie de l\'hôpital, mais malheureusement la patiente est décédée juste après la deuxième cure de chimiothérapie dans un tableau de défaillance multi viscérale.
{#F0001}
Discussion {#S0003}
==========
Le cancer du sein vient au premier rang des cancers de la femme au Maroc. Selon le registre des cancers de la ville de Rabat son Incidence est de 20,5 pour 100 000 habitants et il est responsable de 300 000 décès annuel, en partie en rapport avec un diagnostic tardif. 63,07% des patientes consultent à un stade localement avancé et 13,84% aux stades de métastases \[[@CIT0001]\]. Ce retard diagnostic est en rapport selon la littérature avec l\'ignorance, la pauvreté et les habitudes socioculturelles auxquelles s\'ajoute ces dernières années l\'auto-prise en charge médicale basée sur l\'information apportée par le net \[[@CIT0001]--[@CIT0003]\]. On soulève ainsi d\'après une étude en 2012 du Haut-Commissariat au plan, que près de 30% des Marocaines de 15 ans et plus sont analphabètes et donc incapables d\'interagir avec toutes sortes d\'informations médicale. C\'est une tranche de population qui ne peut émerger que par des programmes gouvernementaux et communautaires pour lutter contre l\'analphabétisme ainsi que l'élaboration d\'une information audio-visuelle simple à assimiler quant à la nécessité du dépistage précoce du cancer du sein \[[@CIT0001], [@CIT0003]--[@CIT0005]\]. L\'absence d\'une carte sanitaire au Maroc est responsable d\'une disparité de répartition des formations médicales avec une concentration des hôpitaux et des praticiens dans les grandes villes laissant un vide ailleurs. L\'accès géographique au dépistage du cancer du sein se trouve ainsi compromis pour une partie de la population. La solution réside dans le rapprochement des structures sanitaires des citoyens avec la création d\'une carte sanitaire faisant profiter toute la population des soins médicaux \[[@CIT0001], [@CIT0004]--[@CIT0008]\]. Le manque de moyens financiers et l\'absence de couverture sociale est un élément capitale dans le retard diagnostic du cancer du sein, une mammographie coûte près de 50 euros ce qui représente le quart du Salaire minimum interprofessionnel garanti au Maroc (SMIG en 2014 est 209 euros). Le Maroc vient de se doter depuis 2012 d\'un système de couverture médicale gratuite appelé RAMED (Régime d\'Assistance Médicale aux populations démunis) mais il n\'intéresse que 10% de la population, une généralisation de la couverture sociale pourrait contribuer au diagnostic précoce du cancer du sein \[[@CIT0001]\]. Le traitement traditionnel est ancré dans les habitudes socioculturelles marocaines ainsi que d\'autres pays africains et asiatiques. Ce traitement passe en premier plan dans un groupe de population particulière causant un retard diagnostic du cancer du sein et même une source de complications infectieuses voire toxique \[[@CIT0001], [@CIT0003]\]. Au Maroc on compte 56% d\'internautes en 2013, ce qui parait bien comme outil d\'information, mais l\'interprétation ambiguë des informations médicales peut être néfaste et causer un retard diagnostic. C\'est le cas de notre patiente qui a substitué l\'internet au médecin pour écarter l'éventualité du cancer du sein en interprétant mal la notion d\'absence des facteurs de risque du cancer du sein. La mondialisation de l\'information médicale à travers internet est une arme à double tranchant, elle apporte certes un éclaircissement important dans le domaine médical mais peut entraîner une confusion si l\'utilisateur n\'a pas le niveau intellectuel nécessaire pour bien assimiler l\'information. Cette situation pose un défi aux responsables de la santé publique qui devraient éveiller toutes les classes sociale pour mieux utiliser le net \[[@CIT0009], [@CIT0010]\].
Conclusion {#S0004}
==========
Le retard diagnostic du cancer du sein au Maroc est lié plusieurs facteurs entremêlés on en connaissait trois, l\'ignorance, la pauvreté, et les habitudes socio-culturelles auxquels s\'ajoute actuellement la mauvaise interprétation des informations médicales du net. La précocité du diagnostic du cancer du sein passe donc par l'éducation, la généralisation de la couverture sociale médicale, la lutte contre la pauvreté et les traitements traditionnels et le développement d\'un esprit critique quant aux informations médicales sur le net.
Conflits d\'intérêts {#S0005}
====================
L\'auteur ne déclare aucun conflit d\'intérêts.
Contributions des auteurs {#S0006}
=========================
Tous les auteurs ont contribué à la conduite de ce travail. Tous les auteurs déclarent également avoir lu et approuvé la version finale du manuscrit.
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Researchers from a wide array of disciplines have investigated ancient "plagues", from fields as diverse as microbiology, ancient history, epidemiology, zoology, palaeopathology and archaeology, both independently from each other or in collaborative multidisciplinary teams. This paper aims to discuss the role of archaeology within such investigations and, perhaps more significantly, to highlight some of the limitations of the archaeological data on which many such studies are based. Non-archaeologists may not fully appreciate these limitations, as archaeology invariably operates under the constraints of an incomplete, biased, poorly preserved and often problematic sample. Only once these limitations are taken into account can the archaeological record be used to its "full" potential. Applying scientific methods to, and developing elaborate theories from, archaeological material without taking such shortcomings into account can seriously affect academic validity. Nonetheless, archaeology has a role to play in the study of ancient "plagues". Archaeological methods from a wide range of sub-disciplines can offer additional or alternative avenues of research that may help us identify and understand the diseases behind ancient "plagues". The Black Death burial site at East Smithfield, London, will provide a background to the discussion.[1](#fn1){ref-type="fn"}
The Black Death in London
=========================
The Black Death is believed to have arrived in Europe from the Crimea during October 1347. It rapidly spread across Europe, arriving in Weymouth on the south coast of England in June/July 1348, and by November it had reached London.[2](#fn2){ref-type="fn"} In the capital, the death rate rose rapidly from December 1348, reaching a peak in April 1349 and falling significantly by June 1349. The impact of the Black Death epidemic was catastrophic, and at its height, a contemporary observer stated that approximately 200 bodies a day were being buried.[3](#fn3){ref-type="fn"} The disposal of so many dead ought to have left its mark in the archaeological record and, in 1986, a large cemetery was located on the site of the Royal Mint near the Tower of London, in East Smithfield. It was excavated by archaeologists from the Museum of London and, as shown in [Figure 1](#fig1){ref-type="fig"}, it contained two mass burial trenches and a mass burial pit, densely filled with several hundred articulated skeletons, as well as many individual graves.[4](#fn4){ref-type="fn"} Written evidence confirms that, with West Smithfield, the East Smithfield Royal Mint cemetery was one of two emergency burial grounds created to cope with the Black Death epidemic.[5](#fn5){ref-type="fn"} It is currently the largest and most comprehensively excavated Black Death cemetery in England, and the majority of an estimated 2400 people buried there are believed to have died from the Black Death, of which over 600 were recovered by the Museum of London.[6](#fn6){ref-type="fn"} Opened in late 1348 or early 1349 by Ralph Stratford, the Bishop of London, it provides researchers and archaeologists with a unique assemblage of people who died at the peak of the Black Death epidemic in London.[7](#fn7){ref-type="fn"} The cause of the epidemic has recently come under scrutiny and the established view that the Black Death was caused by the bacterium *Yersinia pestis* has been challenged.
{#fig1}
Plague and *Yersinia pestis*
============================
From the Latin "plaga", meaning "blow", the word "plague" can be used in its broad sense to describe any large-scale calamity, such as an insect infestation or an epidemic disease with a high mortality rate. It is also often employed to describe a specific disease, the acute epidemic disease of rats and other wild rodents caused by the bacterium *Yersinia pestis*. In modern epidemics, the bacterium is usually transmitted to man by fleas---in particular by the rat flea *Xenopsylla cheopis*---resulting in the most common clinical form of the disease: bubonic plague. Although an uncommon occurrence in modern outbreaks, the bacterium *Yersinia pestis* can also enter the blood-stream (septicaemic plague) or the lungs (pneumonic plague), leading to highly fatal forms of the disease. Patients with bubonic plague are likely to suffer from high fever, delirium, aching limbs and painful swelling of the lymph nodes called buboes. In some patients, the buboes burst after a few days, releasing pus and then healing. In others, subcutaneous bleeding may occur, producing black patches that may lead to fatal ulcers. Until recently, it was widely believed that bubonic plague was behind the 1347--49 epidemic, and the presence of black subcutaneous lesions on some of the plague victims may have explained the origin of the term "Black Death".[8](#fn8){ref-type="fn"} However, some researchers have highlighted interesting differences between the epidemic of 1347--49 and more recent outbreaks of bubonic plague, raising the possibility that the bacterium *Yersinia pestis* was not behind the Black Death epidemic. A good review of the main arguments levelled against bubonic plague---as well as the more contagious pneumonic plague---can be found in Stephen Porter's 'An historical whodunit'.[9](#fn9){ref-type="fn"} Indeed, works by Graham Twigg---*The Black Death*---and more recently by Susan Scott and Christopher Duncan---*Biology of plagues*and *Return of the Black Death*---as well as Samuel Cohn---*The Black Death transformed*[10](#fn10){ref-type="fn"}---have challenged the established view that *Yersinia pestis* and bubonic plague were the cause of the fourteenth-century epidemic.
The Black Death had a devastating impact on Europe. In contrast with the epidemiology of recent outbreaks of plague, the disease appears to have killed a large proportion of the population and to have swept rapidly across the continent. Europeans are unlikely to have had any prior exposure to *Yersinia pestis*, which could explain the unusually high overall mortality of the Black Death epidemic.[11](#fn11){ref-type="fn"} As discussed by Porter, the role that rats---and their fleas---may have played in the spread of plague has come under some scrutiny by what some people have described as "plague-revisionists".[12](#fn12){ref-type="fn"} For example, the cool climatic conditions of northern Europe would have made it difficult for the rat fleas to reproduce, particularly during the winter months.[13](#fn13){ref-type="fn"} This ought to have hampered, if not stopped, the spread of the disease during the winter period but there appears to be no evidence of this occurring. Plague outbreaks also tend to be preceded by the presence of many dead rats, as they are themselves susceptible to the plague, but there seems to be a lack of contemporary accounts mentioning dead rats in the medieval literature.[14](#fn14){ref-type="fn"} In Europe, unlike Asia, there are no known plague-resistant wild rodents that would have acted as a natural reservoir for *Yersinia pestis*. It is difficult to explain how the bacterium continued to affect Europe for another three centuries without the presence of a local reservoir. Interestingly, despite two well-documented fifteenth-century plague epidemics in Iceland, rats are not believed to have settled on the island until much later.[15](#fn15){ref-type="fn"}
Most of the discrepancies highlighted in the literature rely on an absence of evidence, which---as all archaeologists know---is not evidence of absence. Nonetheless, such inconsistencies certainly warrant further investigation and *Yersinia pestis* is no longer regarded as the only suspect. Scott and Duncan have suggested that a highly lethal and contagious virus, similar to the filoviruses responsible for haemorrhagic fevers such as Ebola, was behind the Black Death epidemic.[16](#fn16){ref-type="fn"}
The Role of Archaeology
=======================
The investigation of ancient "plagues" has always relied upon a multidisciplinary approach, involving fields as diverse as ancient history and zoology. Archaeology's contribution to the investigation has the potential to be multifaceted, as it encompasses numerous sub-disciplines, many of which may be relevant to such studies.
- *Vertebrate and invertebrate zooarchaeology*: to investigate possible disease hosts or vectors.
- *Archaeobotany, geoarchaeology and palaeoecology*: to reconstruct the climatic conditions during plague epidemics, and the impact this may have had on potential disease hosts or vectors.
- *Urban archaeology*: to establish the "environmental" conditions and their impact on disease biology and disease transmission such as housing, population density, town size, water supply, transport, food supply and general facilities.
- *Material culture, burial archaeology and archaeological dating*: to ascertain the date of burial sites through scientific dating and/or by the presence of artefacts, as well as any cultural information found in the burial (such as the care with which the bodies were buried or the quality and quantity of any offerings).
- *Bioarchaeology and palaeopathology*: to analyse the physical remains of the victims of the plague.
Archaeology, Climate and Vectors of Transmission
================================================
During the Black Death epidemic, *Yersinia pestis* is believed to have been carried primarily by rats and transmitted to humans by their fleas. As discussed above, some authors have argued against this vector of transmission, and archaeology could help researchers explore alternative hosts and vectors. Vertebrate zooarchaeology may allow us to identify the presence or availability of animal hosts, and (in the case of *Yersinia pestis*) the existence of any wild rodents that may act as a reservoir for the disease. The identification of rodent vectors may prove critical in the interpretation of such epidemics.[17](#fn17){ref-type="fn"} The skeletal remains of many rodents can "easily" be identified through the study of their complex and characteristic dentitions.[18](#fn18){ref-type="fn"} It has been argued that the rat played a key role in the appearance, spread and, in particular, the disappearance of plague in Europe. The black rat (*Rattus rattus*) is regarded by some as the dominant reservoir of *Yersinia pestis* and this species may have eventually been displaced and succeeded throughout Europe by the bigger brown rat (*Rattus norvegicus*). As the brown rat is believed to be less likely to transmit the fleas to humans, this might have resulted in the eventual disappearance of the disease. Changes in rodent ecology may thus explain the complex patterns of eruption, disappearance and re-emergence of plague that continued in Europe over many centuries.[19](#fn19){ref-type="fn"} Archaeologically, it is possible to distinguish between *Rattus rattus* and *Rattus norvegicus* using differences in cranial morphology.[20](#fn20){ref-type="fn"} Unfortunately, the patchy nature of the archaeological evidence and the contexts in which such remains may be preserved (i.e. plague pits, refuse pits and domestic habitations of the right period) make it unlikely that we will ever be able to determine any temporal and geographical changes in European rodent populations. With regards to ascertaining the origin of *Rattus rattus*, Anton Ervynck points out:
> Generally, the dataset ... is rather limited. This must be partly caused by unfavourable preservation conditions for small mammal bones at many archaeological sites, but is, without doubt, also linked to poor sampling and recovery methodology on previous excavations (i.e. lack of sieving).[21](#fn21){ref-type="fn"}
Similarly, the poor preservation of invertebrates, including fleas, limits the usefulness of invertebrate zooarchaeology in the study of ancient plague.[22](#fn22){ref-type="fn"} Nonetheless, some invertebrates---and molluscs in particular---are well preserved, can be used to investigate past climates, and have been employed to reconstruct sea temperatures through the analysis of changes in the ratio of oxygen isotopes.[23](#fn23){ref-type="fn"} As climatic and environmental conditions are likely to be key factors in determining the efficiency of disease vectors (such as the ability of fleas to reproduce and survive), it may be wise to invest greater efforts in determining the climatic conditions prevalent during the plague epidemics. Indeed, the year 1348 is believed to have been unusually warm and this may have had an impact on the virulence and spread of the Black Death.[24](#fn24){ref-type="fn"} Palaeoclimatology employs a wide range of techniques to reconstruct past climates, including the analysis of ice cores (pollen and isotopic sampling), tree rings, lake and oceanic sediment layers (vegetation, insect, animal, pollen and isotopic sampling), molluscs (isotopic analysis) and historical documents. The climate between [ad]{.smallcaps} 1000 and the early 1900s appears to show periods---several decades in length---when the temperature seems to have been warmer or colder than the overall trend. Interestingly, mild episodes seem to have occurred in the early and late fourteenth century and cooler episodes in the fifteenth century and the late seventeenth century. Unfortunately, the record is poor in many areas before the seventeenth century and various uncertainties remain, including the precise nature of the climate in medieval times.[25](#fn25){ref-type="fn"}
Archaeology and the Epidemiological Environment
===============================================
Archaeology may also play a part in establishing the "epidemiological environment" by providing researchers with an understanding of the urban environment and the social conditions prevalent at the time of a plague epidemic. This could help us determine whether past living conditions---including housing, population density, town size, water supply, transport infrastructure, food supply and general facilities---may have had an impact on the emergence and spread of a disease. Urban centres such as London have experienced periods of enormous change and growth over the past 1000 years. During the thirteenth century, the period immediately preceding the Black Death, multi-storey timber-framed buildings appeared in the streets of London in order to cope with the need for space.[26](#fn26){ref-type="fn"} This must have led to overcrowding and increased demand on London's infrastructures, such as water supply and the disposal of sewage, all of which are likely to have facilitated the spread of infectious disease. The London Bills of Mortality (seventeenth to nineteenth centuries) offer particular insights into diseases that cannot be detected in archaeological skeletons, such as cholera, smallpox, measles and plague.[27](#fn27){ref-type="fn"} The nineteenth-century population increase, for example, was accompanied by several outbreaks of typhus and cholera.[28](#fn28){ref-type="fn"} The creation of the London Granary in 1440 and changes in travel, trade and transport with the colonization of the New World (fifteenth-century onwards), the expansion of the British empire (mid-eighteenth century) and the development of the railways in the nineteenth century, may have improved food availability, but must have also introduced new pathogens and vectors of disease.[29](#fn29){ref-type="fn"} One of the greatest improvements in health was the construction of London's modern sewage system during the 1860s, which had a fundamental impact on diseases such as cholera. With regard to the Black Death, the urban environment and social conditions prevalent at the time of the epidemic may have played an important role in the biology of the disease. Has the epidemiological environment been established with sufficient detail? How would the living conditions in major urban centres, such as the use of multi-storey timber-framed buildings in London, have affected the ecology of humans, rodents and other potential vectors of the plague? Indeed, would the houses and storage rooms have been "suitable" to *Rattus rattus* and *Rattus norvegicus*, or sufficiently warm for *Xenopsylla cheopis* to reproduce or live during the winter months? Experimental archaeology, by reconstructing a fourteenth-century house, or ethnoarchaeological research, may help establish whether medieval houses and towns provided the "appropriate" epidemiological environment for *Yersinia pestis*.
Archaeology and the Burial Environment
======================================
Burial sites, such as plague pits, offer archaeologists the unique opportunity of studying the remains of people who died from the plague. As discussed above, one such plague pit was excavated by the Museum of London in 1986. The site contained two mass burial trenches, a mass burial pit and numerous individual graves. The interpretation of such archaeological sites, including the distribution of the skeletal remains, can be fraught with dangers. Indeed, when dealing with any archaeological site, it is important to appreciate the nature of the evidence, as non-randomness and systematic bias in an assemblage can lead to interpretation errors.[30](#fn30){ref-type="fn"} An assemblage is the result of culture, preservation or a mixture of both. Many factors can have an impact on the number and/or distribution of skeletal remains. The study of the processes that cause sampling bias or differential preservation is known as taphonomy, and can be highly variable on a local scale.[31](#fn31){ref-type="fn"} The acidity of the soil, for example, may differ across a site due to variations in underground water movements, damaging and destroying skeletons in specific areas, but not others. Out of the "total dead population", only a proportion is likely to have been buried at one site. There is also a progressive loss of information during and after burial, with only a percentage of the original cemetery population ending up in front of the archaeologist. This is mostly due to decay and decomposition but is also affected by the amount of post-burial disturbance, and the extent of the excavation.[32](#fn32){ref-type="fn"} Indeed, only a percentage of the remains will be preserved, of which only some will be discovered and recovered. Sites are often only partially excavated and material can be lost during the excavation process, resulting in an incomplete and therefore biased assemblage. At the Royal Mint for example, out of an estimated 2400 buried at the site, approximately 600 skeletons were recovered. This makes it difficult to interpret the significance of any organization within the Black Death site, such as any biological or burial-goods differences between the people buried in the individual graves, the mass burial trenches and the pit. This is especially relevant when comparing assemblages from different sites, particularly when the proportions of total buried population discovered or excavated are not the same.[33](#fn33){ref-type="fn"} Differences in the number of dead individuals and in mortality profiles (age and sex distribution) may be used to assess the impact of plague on different populations, but any biases not associated with the presence of plague need to be taken into account.[34](#fn34){ref-type="fn"}
In order fully to interpret any archaeological site and, in the case of plague, determine whether it was used during an epidemic, it is important to establish the period during which the burial site was in use. As plague outbreaks tend to be catastrophic in nature, burial sites such as plague pits are used over a short period of time, as in the Royal Mint site, and determining when the site was in use may be beyond most archaeological scientific dating methods (such as radiocarbon dating). Changes in grave goods (material culture) can be used to provide a "relative" date. This approach was recently employed by researchers examining a pit containing approximately 150 skeletons believed to have died during the plague of Athens.[35](#fn35){ref-type="fn"} The remains of approximately thirty small vases scattered among the bodies of the lower layers of the pit were recovered. Most of the vases were dated at around 430 [bc]{.smallcaps}, some within the decade of 420 [bc]{.smallcaps}, and a few from the last quarter of the fifth century [bc]{.smallcaps}. The authors suggest that the poor quality and quantity of the offerings, the hurried manner of burial, and the chronology of the burial offerings, link this site with the outbreak of the plague of Athens during the early part of the Peloponnesian War, between 430 and 426 [bc.]{.smallcaps}[36](#fn36){ref-type="fn"} In this case, the knowledge of Greek pottery chronology is fairly well established and this method offers a good alternative to conventional dating. However, this relative level of accuracy is rare and may not be adequate. As in the case of the Royal Mint site, as discussed above, historical records are usually required to identify or confirm the site as a plague burial.
Archaeology and Skeletal Remains
================================
Burial environment and embalming practices affect the overall preservation of human remains and the information that can be derived from these remains. In most archaeological sites, the skeleton is all that survives. Soft tissues are rarely preserved, especially in Europe.
Skeletal Analysis
-----------------
In order to determine the mortality profile of an assemblage, archaeologists need to establish the age and sex of each skeleton. Sex determination based on skeletal features can be problematic in some adults, and is not possible in non-adult skeletons.[37](#fn37){ref-type="fn"} The ageing of a non-adult skeleton is fairly accurate and is based on the development of bones and teeth. The ageing of an adult skeleton is usually based on the degenerative age-related changes that occur on parts of the skeleton where bones come into contact (i.e. joint surfaces). The most reliable areas are on the hip bone, and the changes are assigned scores. Scores are combined to provide an approximate age-at-death within a five or ten year range.[38](#fn38){ref-type="fn"} However, the methods employed tend to overestimate young adults and underestimate older adults, thus providing flawed mortality profiles.[39](#fn39){ref-type="fn"} Adults tend to be grouped into broad age categories (in years): 15--24, 25--34, 35--44, 45+, adults or unknown. The problems inherent in these methods have created different interpretations of the mortality profile of the Royal Mint assemblage:[40](#fn40){ref-type="fn"} in particular, whether or not such a "catastrophic assemblage" reflects the once living population---this assumes that the disease responsible for the epidemic killed indiscriminately, irrespective of age or sex---and how the Royal Mint assemblage ought to differ from an "attritional assemblage" that accumulates over a long period (i.e. a cemetery that is used over several years, decades or centuries).
Regrettably, only a limited number of diseases affect the skeleton, and bone can only react in a limited number of ways. It can be removed, added, or a combination of both. To make matters more complex, a skeleton can be affected by more than one disease. All these factors make diagnosis difficult and, when possible, it is based on the location, distribution and nature of the changes. Unfortunately, plague (or any of the alternative candidates) does not appear to affect bone, and cannot be identified skeletally.[41](#fn41){ref-type="fn"} Remains with preserved soft tissue (such as natural mummification) may help diagnosis but no such specimens appear to have been found.[42](#fn42){ref-type="fn"} Instead, researchers have focused on trying to extract *Yersinia pestis* DNA from skeletal remains.
Biomolecular Analysis
---------------------
Recent developments in biomolecular methods, such as polymerase chain reaction (PCR), have allowed researchers to amplify and analyse very small fragments of ancient DNA preserved within the bones and teeth of our ancestors. The publication of several "unrepeatable" high-profile studies and the technical difficulties of ancient DNA research (i.e. detecting small amounts of degraded DNA and the high risk of contamination) lead Alan Cooper and Hendrik Poinar to propose the following "criteria of authenticity":[43](#fn43){ref-type="fn"}
1. *Physically isolated work area*: a laboratory in which the target DNA is not routinely amplified should be used to avoid contamination problems.
2. *Control amplifications*: several extraction and PCR controls should be carried out to detect sporadic or low-copy number contamination.
3. *Appropriate molecular behaviour*: the strength of PCR amplifications should be inversely related to product size and, when single-copy nuclear or pathogen DNA is detected, reproducible mitochondrial DNA results ought to be included.
4. *Reproducibility*: researchers ought to be able to repeat their results and include the use of different overlapping primer pairs to help detect numts (nuclear mitochondrial-like sequences or pseudogenes) or contamination by a PCR product.
5. *Cloning*: researchers should check the direct PCR sequences by cloning the amplified products.
6. *Independent replication*: separate samples (of the same specimen) should be extracted and sequenced by independent laboratories.
7. *Biochemical preservation*: researchers should assess the overall preservation of amino acids and other residues to provide indirect evidence of DNA survival.
8. *Quantitation*: in order to eliminate the risk of sporadic contamination, the copy number of the DNA target needs to be determined using competitive PCR.
9. *Associated remains*: when targeting human DNA, researchers should demonstrate that similar DNA targets have survived in any associated faunal remains.
Unfortunately, these guidelines add both time and expense to any ancient DNA project and are not always fully adhered to by researchers. The uncertainties surrounding some of the published studies, the high risk of contamination and the possibility of false positive results certainly warrant the use of independent replication. If the analysis of ancient DNA is going to help us identify the disease responsible for ancient "plagues", researchers also need to agree on:
- What constitutes a sufficient number of specimens/individuals/sites to ascertain whether a pathogen (identified by ancient DNA analysis) was responsible for a particular epidemic.
- What constitutes adequate control samples to test for DNA contamination.
- Whether soil bacteria are a source of false positive results.
- If we should be collecting soil samples from the burial sites to include as control samples to test for false positive reactions.
- Whether two independent laboratories should *always* be used to confirm the results.
- Whether we should be using the same approach for all "plague" epidemics so that results may be comparable.
With regards to detecting "plague" DNA, these methods have provided mixed results. Molecular identification by "suicide PCR" of *Yersinia pestis* in the pulp tissue of teeth (one child, two adults) from a large fourteenth-century plague cemetery (approximately 800 skeletons) in Montpellier, southern France, tested positive.[44](#fn44){ref-type="fn"} These results have generated a lot of controversy as DNA samples were not recovered and the results have never been duplicated by another laboratory.[45](#fn45){ref-type="fn"} Thomas Gilbert and his co-researchers tried to confirm the French research and tested 108 teeth belonging to sixty-one individuals from different European plague burial sites (thirteenth to seventeenth century).[46](#fn46){ref-type="fn"} They failed to amplify any *Yersinia pestis* DNA from the samples extracted and believe that the results proposed by Didier Raoult, Gérard Aboudharam and colleagues should be independently corroborated before confirming *Yersinia pestis* as the aetiological agent of the Black Death. This led to a response by Michel Drancourt and Raoult, and further comments by Gilbert and his co-researchers,[47](#fn47){ref-type="fn"} but many now regard the research by Raoult, Aboudharam, *et al*. as unsatisfactory and the jury is still out. Using the same "suicide PCR" approach as Raoult, Aboudharam, *et al.*, Ingrid Wiechmann and Gisela Grupe also claim to have identified *Yersinia pestis*-specific DNA sequences in two sixth-century skeletons from Upper Bavaria.[48](#fn48){ref-type="fn"} Drancourt and Raoult have also published their own authentication criteria[49](#fn49){ref-type="fn"} and have alleged that the *Yersinia pestis* Orientalis genotype was involved in all three pandemics.[50](#fn50){ref-type="fn"} Recently, Drancourt, Linda Houhamdi and Raoult have proposed a new model to explain the persistence and periodic re-emergence of plague in Europe.[51](#fn51){ref-type="fn"} In this yet unproven model, they suggest soil as the reservoir of *Yersinia pestis*, burrowing rodents as a first link, and human ectoparasites as the driving force behind the pandemics. No one has attempted to extract ancient *Yersinia pestis* DNA from the remains of possible hosts, such as rodents, but there is no reason why the DNA should not be present in the pulp chambers of plague-infected rat teeth. Testing the method on the teeth of modern rats infected with *Yersinia pestis* ought to be straightforward but finding archaeological specimens of the right period, or context, may be more difficult.
Recent work investigating the plague of Athens (430--426 [BC]{.smallcaps}) tested three human teeth from a mass burial pit containing at least 150 individuals.[52](#fn52){ref-type="fn"} Interestingly, the results appear to incriminate typhoid fever as the probable cause of the epidemic. Six DNA amplifications targeted at genomic parts of the agents of plague (*Yersinia pestis*), typhus (*Rickettsia prowazekii*), anthrax (*Bacillus anthracis*), tuberculosis (*Mycobacterium tuberculosis*), cowpox (*cowpox virus*) and "cat-scratch" disease (*Bartonella henselae*) failed to yield any product in "suicide" reactions from DNA samples isolated from the three teeth. On the seventh such attempt, DNA sequences of *Salmonella enterica* serovar Typhi were identified. They argue that this provides clear evidence for the presence of that micro-organism in the dental pulp of the teeth recovered from the Kerameikos mass grave. These results are very interesting, but, as previously suggested, it would be useful if another laboratory repeated this work on a larger sample from a Greek site of the same period before typhoid fever is attributed as the sole "cause" of the plague of Athens, and thus eliminated the possibility of an isolated outbreak of typhoid fever.[53](#fn53){ref-type="fn"} Manolis Papagrigorakis and his co-researchers admit they did not follow all of Cooper and Poinar's recommendations (see criteria 7 and 8 above)[54](#fn54){ref-type="fn"} and, using phylogenetic analysis, Beth Shapiro and her colleagues have also questioned whether the DNA sequence amplified was that of typhoid. They suggest the sequence may be that of a free-living soil bacterium and, as discussed above, recommend sampling the surrounding soil as a negative control.[55](#fn55){ref-type="fn"} In their response, Papagrigorakis, *et al.* refute any environmental contamination, as they tested the soil washed off the ancient teeth, and argue that the phylogenetic analysis of a single gene may be insufficient and misleading. Nonetheless, they agree that there is, as yet, no definite proof that the plague of Athens was caused by typhoid fever.[56](#fn56){ref-type="fn"}
Conclusion: The Black Death and the Epidemiological Context
===========================================================
Over three decades before the Black Death epidemic, the Great Famine of 1315--17 had devastated northern Europe, causing great suffering and high mortality. This prolonged period of famine is regarded as one of the greatest calamities of the medieval period, but little is known about its impact on the people who survived it and how it altered the lives of Europe's inhabitants. Can we look at the Black Death epidemic without considering the effect of the Great Famine on previous generations? Did years of famine and high mortality alter the genetic makeup of northern Europeans and affect their susceptibility to an epidemic disease? Could this explain why the Black Death appears to have had a different impact on northern Europe?
Between 1315 and 1317, a series of unusually cold winters and wet summers appears to have caused a succession of failed harvests across northern Europe.[57](#fn57){ref-type="fn"} Records of wheat prices have been used to monitor the impact of the famine.[58](#fn58){ref-type="fn"} In London, for example, the price of wheat achieved unparalleled heights in July 1315, rose to its highest level between April and July 1316 and peaked once more during May 1317.[59](#fn59){ref-type="fn"} The moderately wealthy could not afford sufficient food and many people are believed to have died in the streets.[60](#fn60){ref-type="fn"} This period is also likely to have had a profound effect on the health of people who survived the famine, particularly the children growing up at the time. Recent research is investigating the impact of the Great Famine on the skeletal and dental development of individuals who grew up during the famine years, using skeletons aged between thirty and forty years at death, from the Royal Mint site.[61](#fn61){ref-type="fn"} The Black Death epidemic took place some thirty years after the Great Famine and any plague victim from the Royal Mint site that died in their thirties would have lived through the famine as a child. The results of this research should allow us to determine the impact of the Great Famine on the development of individuals who were children during the famine years and give us an insight into the overall health of Londoners in the fourteenth century.[62](#fn62){ref-type="fn"}
Many archaeological sub-disciplines have a role to play in the study of ancient plagues, particularly in the investigation of the transmission and epidemiological environment of diseases, and the burial and physical remains of the victims. Understanding how ancient calamities, such as plague and famine, may have affected continents like Europe, and how pathogens may have adapted to northern conditions and changed over the past 600 years, could become more relevant than ever, particularly with the emergence of antibiotic resistant strains of bubonic plague.[63](#fn63){ref-type="fn"}
I would like to thank the organizers for inviting me to speak at the conference and Prof. Charlotte Roberts for her useful comments. I wish to thank the Museum of London, and in particular Bill White of the Museum\'s Centre for Human Bioarchaeology, for arranging access to the Royal Mint skeletal assemblage. I would also like to thank the other participants in the Royal Mint research: Prof. Simon Hillson (Institute of Archaeology, UCL), Prof. Tony Waldron (Institute of Archaeology, UCL), Prof. Derek Keene (Institute of Historical Research, University of London), Gustav Milne (Institute of Archaeology, UCL) and Prof. Chris Dean (Department of Anatomy and Developmental Biology, UCL). Our research was supported by the Wellcome Trust (2002--2005) and is currently funded by the Leverhulme Trust (2006--2009).
^1^ Daniel Antoine and Simon Hillson, 'Famine, Black Death and health in fourteenth-century London', *Archaeol. Int.*, 2004/2005, **8**: 26--8.
^2^ Philip Ziegler, *The Black Death*, Harmondsworth, Penguin, 1970, pp. 123--4, 161; Duncan Hawkins, 'The Black Death and the new London cemeteries of 1348', *Antiquity*, 1990, **64** (244): 637--42.
^3^ Rosemary Horrox (trans. and ed.), *The Black Death*, Manchester University Press, 1994, pp. 64--5; see also Antoine and Hillson, op. cit., note 1 above, p. 26.
^4^ Antoine and Hillson, op. cit., note 1 above, pp. 26--8.
^5^ Ziegler, op. cit., note 2 above, p. 162; Hawkins, op. cit., note 2 above, pp. 637--8.
^6^ Antoine and Hillson, op. cit., note 1 above, p. 26.
^7^ Ziegler, op. cit., note 2 above, p. 162; Hawkins, op. cit., note 2 above, pp. 637--8; Antoine and Hillson, op. cit., note 1 above, pp. 26--8.
^8^ Antoine and Hillson, op. cit., note 1 above, p. 26.
^9^ Stephen Porter, 'An historical whodunit', *Biologist*, 2004, **51** (2): 109--13.
^10^ Graham Twigg, *The Black Death: a biological reappraisal*, London, Batsford, 1984; Susan Scott and Christopher Duncan, *Biology of plagues: evidence from historical populations*, Cambridge University Press, 2001; Susan Scott and Christopher Duncan, *Return of the Black Death: the world's greatest serial killer*, Chichester, Wiley, 2004; Samuel K Cohn Jr, *The Black Death transformed: disease and culture in early Renaissance Europe*, London, Arnold, 2002.
^11^ Cohn, op. cit., note 10 above, pp. 26--8, 100--1, 111--13.
^12^ Porter, op. cit., note 9 above, pp. 109--13; see also Antoine and Hillson, op. cit., note 1 above, p. 26.
^13^ Gunnar Karlsson, 'Plague without rats: the case of fifteenth-century Iceland', *J. Mediev. Hist.*, 1996, **22** (3): 263--84.
^14^ David Herlihy, *The Black Death and the transformation of the west*, ed. Samuel K Cohn, London, Harvard University Press, 1997, p. 26; Antoine and Hillson, op. cit., note 1 above, pp. 26--7.
^15^ Karlsson, op. cit., note 13 above, p. 265.
^16^ Scott and Duncan, *Return of the Black Death*, op. cit., note 10 above, p. 225.
^17^ Michael McCormick, 'Rats, communications, and plague: toward an ecological history', *J. Interdiscip. Hist.*, 2003, **34**: 1--25.
^18^ Simon Hillson, *Teeth*, Cambridge University Press, 2005.
^19^ Andrew B Appleby, 'The disappearance of plague: a continuing puzzle', *Econ. Hist. Rev.*, 1980, **33** (2): 161--73; Paul Slack, 'The disappearance of the plague: an alternative view', *Econ. Hist. Rev.*, 1981, **34** (3): 469--76.
^20^ H R Hunt, S Rosen, and C A Hoppert, 'Morphology of molar teeth and occlusion in young rats', *J. Dent. Res.*, 1970, **49**: 508--14; M J Lawrence and R W Brown, *Mammals of Britain: their tracks, trails and signs*, London, Blandford Press, 1973, pp. 194--9.
^21^ Anton Ervynck, 'Sedentism or urbanism? On the origin of the commensal black rat (*Rattus rattus*)', in Keith Dobney and Terry O'Connor (eds), *Bones and the man: studies in honour of Don Brothwell*, Oxford, Oxbow Books, 2002, pp. 95--109, on p. 95.
^22^ Several examples of fleas from the archaeological record are discussed in Paul C Buckland and Jon P Sadler, 'A biogeography of the human flea, *Pulex irritans* L. (Siphonaptera: Pulicidae)', *Journal of Biogeography*, 1989, **16** (2): 115--120.
^23^ Marcello A Mannino, Baruch F Spiro, and Kenneth D Thomas, 'Sampling shells for seasonality: oxygen isotope analysis on shell carbonates of the inter-tidal gastropod *Monodonta lineata* (da Costa) from populations across its modern range and from a Mesolithic site in southern Britain', *J. Archaeol. Sci.*, 2003, **30**(6): 667--79.
^24^ Charlotte Roberts and Margaret Cox, *Health and disease in Britain: from prehistory to the present day*, Stroud, Sutton Publishing, 2003, p. 227.
^25^ R S Bradley, K R Briffa, J E Cole, M K Hughes, and T J Osborn, 'The climate of the last millennium', in Keith D Alverson, Raymond S Bradley, and Thomas F Pedersen (eds), *Paleoclimate, global change and the future*, Berlin and New York, Springer, 2003, pp. 105--41.
^26^ John Schofield, *Medieval London houses*, New Haven and London, Yale University Press, 1995.
^27^ Roberts and Cox, op. cit., note 24 above, pp. 287, 290--3.
^28^ Ibid., pp. 337--8; Hugh Clout (ed.), *The Times history of London*, London, Times Books, HarperCollins, 2004, pp. 10--11, 88--89, 96--97.
^29^ M Samuel and Gustav Milne, 'The "Ledene Hall" and medieval market', in Gustav Milne (ed.), *From Roman basilica to medieval market: archaeology in action in the City of London*', London, HMSO, 1992, pp. 39--50; Clout (ed.), op. cit., note 28 above, pp. 82, 88--91; Roberts and Cox, op. cit., note 24 above, pp. 368--9.
^30^ T Waldron, *Counting the dead: the epidemiology of skeletal populations*, Chichester, Wiley, 1994, pp. 10--27; see also T Waldron, *Shadows in the soil: human bones and archaeology*, Stroud, Tempus, 2001, pp. 44--48.
^31^ S P Nawrocki, 'Taphonomic processes in historic cemeteries', in Anne L Grauer (ed.), *Bodies of evidence: reconstructing history through skeletal analysis*, New York, Wiley-Liss, 1995, pp. 49--66.
^32^ Waldron, *Shadows in the soil*, op. cit., note 30 above, pp. 41--53.
^33^ Ibid.
^34^ Charlotte Roberts and Anne Grauer, 'Commentary: Bones, bodies and representivity in the archaeological record', *Int. J. Epidemiol.*, 2001, **30** (1): 109--10.
^35^ Manolis J Papagrigorakis, Christos Yapijakis, Philippos N Synodinos, and Effie Baziotopoulou-Valavani, 'DNA examination of ancient dental pulp incriminates typhoid fever as a probable cause of the plague of Athens', *Int. J. Infect. Dis.*, 2006, **10** (3): 206--14.
^36^ Ibid.
^37^ Roberts and Grauer, op. cit., note 34 above.
^38^ Jane E Buikstra and Douglas H Ubelaker, *Standards for data collection from human skeletal remains*, Arkansas Archeological Survey Research Series No. 44, Fayetteville, AR, Arkansas Archaeological Survey, 1994.
^39^ Theya Molleson and Margaret Cox, *The Spitalfields project. Volume 2: the anthropology: the middling sort*, Research Report 86, York, Council for British Archaeology, 1993, pp. 145--155, 167--179; Roberts and Grauer, op. cit., note 34 above.
^40^ H A Waldron, 'Are plague pits of particular use to palaeoepidemiologists?', *Int. J. Epidemiol.*, 2001, **30** (1): 104--8; Beverley J Margerison and Christopher J Knüsel, 'Paleodemographic comparison of a catastrophic and an attritional death assemblage', *Am. J. Physical Anthropol.*, 2002, **119** (2): 134--43.
^41^ Arthur C Aufderheide and Conrado Rodríguez-Martín, *The Cambridge encyclopedia of human paleopathology*, Cambridge University Press, 1998, pp. 195--198; Roberts and Grauer, op. cit., note 34 above.
^42^ Aufderheide and Rodríguez-Martín, op. cit., note 41 above, p. 198.
^43^ See publication for full guidelines: A Cooper and H N Poinar, 'Ancient DNA: do it right or not at all', *Science*, 2000, **289**: 1139.
^44^ Didier Raoult, Gérard Aboudharam, Eric Crubézy, Georges Larrouy, Bertrand Ludes, and Michel Drancourt, 'Molecular identification by "suicide PCR" of *Yersinia pestis* as the agent of medieval Black Death', *Proc. Natl. Acad. Sci. USA*, 2000, **97**: 12800--803.
^45^ James Wood and Sharon DeWitte-Aviña, 'Was the Black Death yersinial plague?', *Lancet Infectious Diseases*, 2003, **3** (6): 327--8; Michael B Prentice, Tom Gilbert and Alan Cooper, 'Was the Black Death caused by *Yersinia pestis*?', *Lancet Infectious Diseases*, 2004, **4** (2): 72.
^46^ M Thomas P Gilbert, Jon Cuccui, William White, Niels Lynnerup, Richard W Titball, Alan Cooper, and Michael B Prentice, 'Absence of *Yersinia pestis*-specific DNA in human teeth from five European excavations of putative plague victims', *Microbiology*, 2004, **150**; 341--54.
^47^ Michel Drancourt and Didier Raoult, 'Molecular detection of *Yersinia pestis* in dental pulp', *Microbiology*, 2004, **150**: 263--4; M Thomas P Gilbert, Jon Cuccui, William White, Niels Lynnerup, Richard W Titball, Alan Cooper and Michael B Prentice, 'Response to Drancourt and Raoult', *Microbiology*, 2004, **150**: 264--5.
^48^ Ingrid Wiechmann and Gisela Grupe, 'Detection of *Yersinia pestis* DNA in two early medieval skeletal finds from Aschheim (Upper Bavaria, 6th century A.D.)', *Am. J. Physical Anthropol.*, 2005, **126**: 48--55.
^49^ Michel Drancourt and Didier Raoult, 'Paleomicrobiology: current issues and perspectives', *Nat. Rev. Microbiol.*, 2005, **3**: 23--35.
^50^ Michel Drancourt, Véronique Roux, La Vu Dang, Lam Tran-Hung, Dominique Castex, Viviane Chenal-Francisque, Hiroyaki Ogata, Pierre-Edouard Fournier, Eric Crubézy, Didier Raoult, 'Genotyping, Orientalis-like *Yersinia pestis*, and plague pandemics', *Emerg. Infect. Dis.*, 2004, **10** (9): 1585--92; Michel Drancourt, Michel Signoli, La Vu Dang, Bruno Bizot, Véronique Roux, Stéfan Tzortzis, Didier Raoult, '*Yersinia pestis* Orientalis in remains of ancient plague patients', *Emerg. Infect. Dis.*, 2007, **13**: Available from http://www.cdc.gov/EID/content/13/2/332.htm; see criticism by Gilles Vergnaud, '*Yersinia pestis* genotyping' \[letter\], *Emerg. Infect. Dis*., Aug. 2005, **11** (8); available from http://www.cdc.gov/ncidod/EID/vol11no08/04-0942_05-0568.htm.
^51^ Michel Drancourt, Linda Houhamdi, and Didier Raoult, '*Yersinia pestis* as a telluric, human ectoparasite-borne organism', *Lancet Infectious Diseases*, 2006, **6** (4): 234--41.
^52^ Papagrigorakis, *et al.*, op. cit., note 35 above.
^53^ Daniel Antoine, comment on 'Typhoid caused fall of Athens', BBC News, 2006, http://news.bbc.co.uk/1/hi/health/4639840.stm.
^54^ Papagrigorakis, *et al.*, op. cit., note 35 above, p. 212; Cooper and Poinar, op. cit., note 43 above.
^55^ Beth Shapiro, Andrew Rambaut and M Thomas P Gilbert, 'No proof that typhoid caused the plague of Athens (a reply to Papagrigorakis et al.)', *Int. J. Infect. Dis.*, 2006, **10** (4): 334--5.
^56^ Manolis J Papagrigorakis, Christos Yapijakis, Philippos N Synodinos, and Effie Baziotopoulou-Valavani, 'Insufficient phylogenetic analysis may not exclude candidacy of typhoid fever as a probable cause of the plague of Athens', *Int. J. Infect. Dis.*, 2006, **10** (4): 335--6; see also Michael P Cummings and Axel Meyer, 'Magic bullets and golden rules: data sampling in molecular phylogenetics', *Zoology*, 2005, **108** (4): 329--36.
^57^ Antoine and Hillson, op. cit., note 1 above, p. 27.
^58^ Derek Keene, personal communication; Bruce M S Campbell, James A Galloway, Derek Keene, and M Murphy, *A medieval capital and its grain supply: agrarian production and distribution in the London region c. 1300*, Historical Geography Research Series No. 30, London, Institute of British Geographers, 1993.
^59^ Antoine and Hillson, op. cit., note 1 above, p. 27.
^60^ William C Jordan, *The great famine: northern Europe in the early fourteenth century*, Princeton University Press, 1996.
^61^ Antoine and Hillson, op. cit note 1 above, p. 27.
^62^ Ibid., pp. 26--28; Daniel Antoine, Simon Hillson, Derek Keene, M Christopher Dean and Gustav Milne, forthcoming.
^63^ Timothy J Welch, W Florian Fricke, Patrick F McDermott, David G White, Marie-Laure Rosso, *et al.*, 'Multiple antimicrobial resistance in plague: an emerging public health risk', *PLoS ONE*, 2007, **2** (3): e309.
| {
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} |
Although there are numerous ways children with asthma and allergies can reduce attacks and live a more normal life, researchers at the 2004 annual meeting of the American Academy of Allergy, Asthma, and Immunology (AAAAI) in San Francisco said cigarette smoking in the home virtually negates those interventions. "The data are clearly there," said Robert Holzhauer, a clinical assistant professor of pediatrics at the University of Rochester School of Medicine and Dentistry. "We have unequivocal data to show that sidestream smoke is dangerous to people with asthma."
In one study presented at the March meeting, Holzhauer and colleagues identified Rochester schoolchildren aged 3--7 who had mild persistent to severe persistent asthma. Children were assigned randomly to school-based care groups. One group received daily inhaled corticosteroids---a proven, effective treatment to prevent asthma attacks---at school, while the other did not.
Children in the treated group had fewer attacks and school absences; their parents reported fewer worries about their children's health, work absences, and unexpected changes in plans. But if there was smoking in the home, those advantages were almost completely nullified. These findings have since been published in the May 2004 issue of *Archives of Pediatrics and Adolescent Medicine*.
In another presentation, Dennis Ownby, chief of allergy and immunology at the Medical College of Georgia, described his examination of the relationship between exposure to cats and dogs during the first year of life and the risk of allergy at age 6--7 years. He selected a birth cohort of 474 children, who were classified as having no exposure to cats or dogs during the first year of life, exposure to 1 cat or dog, or exposure to 2 or more cats or dogs.
Children of nonsmoking parents were significantly less likely to have allergies if they were exposed to 2 or more cats or dogs; about 14% of these children were allergic, compared to 37.5% of children with 1 pet and 36.8% of children with no pets. But this benefit was not seen in children of smoking parents. "This research shows that cigarette smoking is not innocuous to young children," Ownby said. "We see evidence that it's affecting their immune system."
Holzhauer said parents must be convinced of how important it is to stop smoking in the home if they have children with asthma. However the doctors stopped short of advocating persuasion through legislation. "I think that if we were to report these parents to the authorities for child abuse, we would lose the children as patients," Holzhauer said.
Rather, Kathleen Sheerin, a private-practice allergy specialist and chair of the AAAAI Public Education Committee, suggested that pediatricians and other health care professionals more strongly emphasize to parents the link between asthma, allergies, and smoking. She said, "We counsel parents to go to another room to smoke or to go outside if there is a child in the house."
| {
"pile_set_name": "PubMed Central"
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
The development of a plant from the germinating seed to the mature plant setting seeds of its own is governed by the activity of a vast number of different transcription factors (TFs). In higher plants 58 different TF gene families are known, comprising from only a few to, in rare cases, more than 200 genes \[[@pone.0209769.ref001]\]. One of the largest TF families depict the plant specific NAC ([N]{.ul}AM-[A]{.ul}TAF1/2-[C]{.ul}UC2) TFs, with often more than 100 members in both monocot and dicot species \[[@pone.0209769.ref002]--[@pone.0209769.ref011]\].
NAC TFs are defined through their highly conserved N-terminal part, the NAC domain, which consists of five sub-domains, designated A to E \[[@pone.0209769.ref004]\]. The E sub-domain can, however, be missing. The NAC domain mediates homo- or heterodimerization, a requirement for the DNA binding properties of the NAC TFs \[[@pone.0209769.ref012]\]. The C-terminal part of the protein by contrast is in general highly divergent, both in length and structure. It, however, contains short motifs conserved within subfamilies, which are believed to be of importance for the trans-activating function of the NAC TFs \[[@pone.0209769.ref013],[@pone.0209769.ref014]\].
NAC TFs are involved in the regulation of a range of different developmental processes in plants, of which two have particularly been in focus: Secondary cell wall formation during development of vascular tissues (reviewed by \[[@pone.0209769.ref015]\]), and the senescence and nutrient remobilization processes taking place in vegetative tissues prior to whole plant senescence in monocarpic plants (reviewed by \[[@pone.0209769.ref016]\]). In addition, a plethora of reports on the association of NAC TFs with both abiotic and biotic stress responses in plants is available (reviewed by \[[@pone.0209769.ref017]\]). One striking common denominator observed across the developmental processes associated with NAC TFs, is the occurrence of programmed cell death (PCD), e.g. during the formation of tracheary elements and, rather obviously, during senescence processes \[[@pone.0209769.ref018]\].
Phylogenetic relationships of the NAC TF family across plant species have been described in several reports \[[@pone.0209769.ref004],[@pone.0209769.ref008],[@pone.0209769.ref010],[@pone.0209769.ref014],[@pone.0209769.ref019]\]. These show the occurrence of lineage specific expansions and sometimes extinctions of certain subfamilies \[[@pone.0209769.ref019]\], suggesting that NAC TFs may have played an important role in the evolutionary diversification of plants. It has, for instance, been hypothesized that the group of NAC TFs involved in formation of water conducting xylem vessels depict the basis for the development of land plants \[[@pone.0209769.ref020]\]. This example illustrates that it is relevant to characterize the NAC TF family at species or genera level to observe evolutionary specializations. This is certainly facilitated by the continuously increasing number of fully sequenced even complex plant genomes, wheat and barley being recent examples \[[@pone.0209769.ref021],[@pone.0209769.ref022]\]. The characterization of the NAC TF genes encoded in the barley genome, which we report in this study, prompted us to investigate a specific specialization event, the emergence of a NAC TF subclade only present in the Poaceae family of monocotyledonous plants. These TFs are strongly and exclusively expressed in the specialized fruit of the Poaceae, the caryopsis, commonly referred to as grain. Hence, we designate these TFs as Grain-NACs. Notably, the Poaceae family comprises a large number of cereal crops of particular importance for human nutrition, such as rice, wheat, maize and barley, which are cultivated for their starchy edible grains. We will further discuss the association of the Grain-NACs with another process associated with the expression of NAC TFs: the PCD of the endosperm occurring during grain maturation. Based on the hypothesis that NAC TFs have played an important role in evolutionary diversification of plants, we propose that the emergence of Grain-NAC TFs in the Poaceae was involved in the evolution of the cereal grain.
Material and methods {#sec002}
====================
Identification of barley NAC TFs {#sec003}
--------------------------------
The recently published new barley genome assembly, along with annotations and 333,571 protein sequences (for all gene models) \[[@pone.0209769.ref022]\], was used to identify barley NAC (HvNAC) TFs. HvNACs were identified by (1) an annotation search of the human readable description available for the barley gene models using the keywords "NAC", "NAM" and "PF02365", (2) a BLASTp \[[@pone.0209769.ref023]\] search using all known protein sequences from rice (*Oryza sativa* 'Japonica'), *Brachypodium distachyon*, *Arabidopsis thaliana* NAC TFs as well as the barley NAC TFs, which had been identified by the annotation search as query, (3) a HMMER \[[@pone.0209769.ref024]\] search of the profile hidden Markov model for PF02365 (extracted from the Pfam database release 30 \[[@pone.0209769.ref025]\]) with hmmsearch version 3.1b3 (<http://hmmer.org/>) run against all 333,571 barley protein sequences with all heuristics turned off and using the gathering bit scores in the model as thresholds and (4) tBLASTn (evalue cutoff: 1e-25) using the sequences from the HvNACs, which had already been identified by the attempts previously listed as query \[[@pone.0209769.ref023],[@pone.0209769.ref026]\]. The protein sequences from rice, *B*. *distachyon* and *A*. *thaliana* NAC TFs were collected from publicly available databases and publications \[[@pone.0209769.ref006],[@pone.0209769.ref009],[@pone.0209769.ref027]--[@pone.0209769.ref033]\].
The presence of the NAM domain PF02365 was verified in all identified NAC TFs with hmmsearch and peptide sequences for the domains were extracted. Some proteins had more than one NAC domain and in such cases, they were all kept. Sequences that could not be verified, were excluded. For the tBLASTn hits, flanking sequences of 5kb were added, and EMBOSS getorf v. 6.5.7.0 \[[@pone.0209769.ref034]\] was used to predict open reading frames used as input for hmmsearch.
When several protein sequences (corresponding to different gene models) were identified for one gene locus, the most complete one was manually selected, based on whether the gene model comprised a start codon and on sequence length (the longest sequence was preferred). Some identified protein sequences however still appeared incomplete, possibly due to inaccurate gene prediction or sequence gaps. In such cases, the sequences were used as query for a BLASTp search against the database of predicted proteins from the barley WGS Morex Assembly v3 \[[@pone.0209769.ref035]\] to retrieve full length sequences where possible.
DLN and LVFY motifs in the NAC domain sequences were identified by motif search applying default settings in CLC Main Workbench 8.0 (QIAGEN Aarhus, Denmark; <https://www.qiagenbioinformatics.com/>).
Identification of segmental and tandem duplications {#sec004}
---------------------------------------------------
Segmental or tandem duplications were identified using BLASTp with full protein sequences and BLASTn with coding sequences as queries on the IPK Barley BLAST Server (<http://webblast.ipk-gatersleben.de/barley_ibsc/>) with the default settings. Sequence pairs with more than 90% identity were considered as duplicated sequences. In some cases, it was not possible to evaluate duplication events due to incomplete sequences. Those cases were assessed individually.
Phylogenetic analysis of NAC TFs in *H*. *vulgare*, *O*. *sativa*, *B*. *distachyon and A*. *thaliana* {#sec005}
------------------------------------------------------------------------------------------------------
NAC domain (Pfam ID: NAM; PF02365) peptide sequences from barley (167), rice (139), *B*. *distachyon* (136) and *A*. *thaliana* NAC TFs (116) were aligned using MAFFT ver.7 with iterative refinement (G-INS-i) with"leave gappy regions" set to "Unalignlevel 0.8" \[[@pone.0209769.ref036],[@pone.0209769.ref037]\]. A phylogenetic tree was constructed using the maximum likelihood method with 100 bootstrap repeats in MEGA 7.0 \[[@pone.0209769.ref038]\].
Further, the NAC domain peptide sequences from barley, rice, *B*. *distachyon* and *A*. *thaliana* were used for phylogenetic tree construction together with those of the Triticeae species common wheat (*Triticum aestivum*), *Aegilops tauschii*, wild emmer (*Triticum turgidum*), wild einkorn (*Triticum urartu*) and rye (*Secale cereale*), the Panicoideae species *Sorghum bicolor*, foxtail millet (*Setaria italica*), switchgrass (*Panicum virgatum*) and maize (*Zea mays*); the Chloridoideae species *Zoysia pacifica* the Bromelioideae species pineapple (*Ananas comosus*); the Zingiberales species banana (*Musa acuminate*); and the Commelinids species oil palm (*Elaeis guineensis*) (compare [Table 1](#pone.0209769.t001){ref-type="table"}). The sequences were acquired from the Plant Transcription Factor Database v4.0 (\[[@pone.0209769.ref001],[@pone.0209769.ref039]\]; <http://planttfdb.cbi.pku.edu.cn/>) for *A*. *tauschii*, *S*. *bicolor*, foxtail millet, switchgrass, maize, *Z*. *pacifica*, pineapple, banana and oil palm. For wild emmer, wild einkorn, wheat and rye, the protein sequences were obtained from the respective genome databases \[[@pone.0209769.ref040]--[@pone.0209769.ref042]\]. NAC domains were identified by HMMER search with the same setting as described above. An alignment of the NAC domains was carried out using MAFFT ver.7 with the "L-INS-i" algorithm. Due to the large size of the alignment, an approximately-maximum-likelihood phylogenetic tree was built from the alignment with FastTree version 2.1.10 run with options "-pseudo -spr 4 -mlacc 2 -slownni" for increased accuracy \[[@pone.0209769.ref043],[@pone.0209769.ref044]\].
10.1371/journal.pone.0209769.t001
###### Overview of the taxonomy and the number of NAC TFs in the species used in this study.
{#pone.0209769.t001g}
Species Superorder Order Family Subfamily Tribe Ploidy Genome designation in allopolyploids Total NAC TFs NAC-d NAC d-9 Grain NACs References
------------------------------------ ------------------------------- ---------------- ---------------- ----------------- ----------------- -------- -------------------------------------- --------------- ------- --------- ------------ -----------------------------------------------------------------------------------------------------------------------------------------------
*Arabidopsis thaliana* *Eudicotyledons* *Brassicales* *Brassicaceae* *Brassicaceae* *Camelineae* 2 117 14 2 0 \[[@pone.0209769.ref001],[@pone.0209769.ref028]--[@pone.0209769.ref030],[@pone.0209769.ref039]\]
*Elaeis guineensis (Oil palm)* *Monocotyledons (Liliopsida)* *Arecales* *Arecaceae* * Arecoideae* *Cocoseae* 2 170 34 5 0 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Musa acuminata (Banana)* *Monocotyledons (Liliopsida)* *Zingiberales* *Musaceae* * * * * 2 168 41 10 0 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Ananas comosus (Pineapple)* *Monocotyledons (Liliopsida)* Poales *Bromeliaceae* *Bromelioideae* * * 2 73 15 4 0 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Zoysia pacifica* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Chloridoideae* *Zoysieae* 4 All 200 49 16 5 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
A (Odd) 89 19 6 3
B (Even) 94 26 7 1
U 17 4 3 1
*Panicum virgatum (switchgrass)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Panicoideae* *Paniceae* 4 All 297 50 13 7 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
K (a) 132 23 6 3
N (b) 146 25 7 4
U 19 2 0 0
*Setaria italica (Foxtail millet)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Panicoideae* *Paniceae* 2 134 23 7 3 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Sorghum bicolor* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Panicoideae* *Andropogoneae* 2 127 24 8 4 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Zea Mays (Mays)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Panicoideae* *Andropogoneae* 2 131 24 7 2 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Oryza sativa (Rice)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Oryzoideae* *Oryzeae* 2 151 27 8 2 \[[@pone.0209769.ref001],[@pone.0209769.ref009],[@pone.0209769.ref027],[@pone.0209769.ref028],[@pone.0209769.ref032],[@pone.0209769.ref039]\]
*Brachypodium distachyon* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Brachypodieae* 2 136 20 7 4 \[[@pone.0209769.ref001],[@pone.0209769.ref006],[@pone.0209769.ref031],[@pone.0209769.ref032],[@pone.0209769.ref039]\]
*Hordeum vulgare (Barley)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 2 167 29 16 6 This study, \[[@pone.0209769.ref001],[@pone.0209769.ref035],[@pone.0209769.ref039],[@pone.0209769.ref045],[@pone.0209769.ref046]\]
*Secale cereale (Rye)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 2 109 26 16 9 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Triticum aestivum (Bread wheat)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 6 All 436 74 35 19 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
A 145 24 12 6
B 134 23 10 6
D 142 22 11 6
U 15 5 2 1
*Aegilops tauschii* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 2 114 28 14 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
*Triticum turgidum (Wild emmer)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 4 All 229 54 28 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
A 115 26 14
B 110 27 13
U 4 1 1
*Triticum urartu (Wild einkorn)* *Monocotyledons (Liliopsida)* Poales *Poaceae* *Pooideae* *Triticeae* 2 72 10 2 \[[@pone.0209769.ref001],[@pone.0209769.ref039]\]
In this tree, the NAC-d-9 subfamily TFs were identified following the nomenclature introduced by \[[@pone.0209769.ref014]\] and subjected to a refined analysis. Only sequences containing at least subdomains A to D were considered. Wild einkorn, *A*. *tauschii* and wild emmer were not included in the refined analysis, as common wheat was represented. NAC domain peptide sequences were aligned using MAFFT ver.7 with iterative refinement (G-INS-i) set to "leave gappy regions" with "Unalignlevel 0.8" \[[@pone.0209769.ref036],[@pone.0209769.ref037],[@pone.0209769.ref047]\]. The phylogenetic tree was constructed using maximum likelihood with 100 bootstrap repeats in MEGA 7.0 \[[@pone.0209769.ref038]\].
Gene expression, promoter region and C-terminal sequence analysis {#sec006}
-----------------------------------------------------------------
Gene expression analysis for all the barley NAC genes across 15 tissues/developmental stages was based on gene expression data (FPKM values from RNAseq experiments) \[[@pone.0209769.ref022],[@pone.0209769.ref035]\] obtained from the Barlex genome explorer, and heatmaps were constructed using the heatmap.2 function of the R package gplots \[[@pone.0209769.ref045],[@pone.0209769.ref048]\]. Eleven genes with no expression across all samples were removed before heatmap construction. Values of other individual samples with expression value = 0 were changed to the minimum positive values of all samples before log transformation. Genes were either ordered in the heatmap according to their NAC subfamily (a to h), and within each subfamily ordered by descending total expression values across all samples, or they were ordered according to a hierarchical clustering of the log2 FPKM values, using a Pearson correlation distance function. The number of basic clusters was determined by the Nbclust R package \[[@pone.0209769.ref049]\]. An additional heatmap for wheat NAC-d-9 genes across 25 different samples/tissues types was also constructed, using RNA-seq data compiled by \[[@pone.0209769.ref050]\].
All the putative NAC promoter sequences were obtained from Ensembl Plants gene annotation (\[[@pone.0209769.ref033]\]; [https://plants.ensembl.org](https://plants.ensembl.org/), accessed 24 April 2018), confined to the 1 kb upstream region from ATG start codon of each gene. The cis-regulatory analysis of Grain-HvNAC promoters (HORVU4Hr1G089450, HORVU3Hr1G014090, HORVU7Hr1G039700, HORVU3Hr1G014100, HORVU7Hr1G031260, HORVU7Hr1G122680), senescence-associated HvNAC promoters (HORVU5Hr1G045640, HORVU2Hr1G017400, HORVU2Hr1G017380, HORVU5Hr1G074810, HORVU4Hr1G051360, HORVU2Hr1G080460, HORVU7Hr1G082420), and four house-keeping genes (HORVU7Hr1G074690, HORVU3Hr1G079700, HORVU1Hr1G081280, and HORVU1Hr1G002840) were determined using the PLACE database of motifs found in plant cis-acting regulatory DNA elements (\[[@pone.0209769.ref051]\]; <https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace>). Also, the orthologous Grain-NAC promoters in wheat (TRIAECS42_7DS_TGACv1_623144_AA2049980, TRIAECS42_3DS_TGACv1_273115_AA0928510, TRIAECS42_7DS_TGACv1_623437_AA2053190, TRIAECS42_7DS_TGACv1_623146_AA2050060, TRIAECS42_3AS_TGACv1_210879_AA0680650, TRIAECS42_7DL_TGACv1_602807_AA1969110), maize (GRMZM2G154182, GRMZM2G062650) and rice (ONAC20; Os01g0104500 and ONAC26; Os01g0393100) were included. Only identical boxes were considered in the search of motif for the promoter sequences. EIN3 motifs were found in the PlantPAN 2.0 database (\[[@pone.0209769.ref052]\]; <http://plantpan2.itps.ncku.edu.tw/promoter.php>).
The encoded C-terminal protein sequences (defined to start at pos. 6 after the conserved cysteine residue of the NAC E-subdomain) of the barley, rice and maize Grain-NACs and senescence-associated HvNACs mentioned above were analyzed using the MEME software (\[[@pone.0209769.ref053]\]; <http://meme-suite.org/>, number of motifs=6, max. motif length=12, accessed April 24, 2018) for the search of conserved motifs. Alignment of NAC promoter and C-terminal sequences was done in CLC Main Workbench 7.9.1 using the "very accurate" algorithm (QIAGEN Aarhus, Denmark; <https://www.qiagenbioinformatics.com/>).
Results and discussions {#sec007}
=======================
Identification of HvNACs in the barley reference genome assembly {#sec008}
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Recently, the NAC TF family of hexaploid wheat was thoroughly categorized \[[@pone.0209769.ref007]\], based on the recent update of the wheat genome assembly \[[@pone.0209769.ref021]\]. The latest study of the barley NAC TF gene family however dates back to 2011 and identified only 48 different NAC TF encoding genes \[[@pone.0209769.ref046]\]. Due to the limited sequence resources available at that time, this list was however bound to be incomplete. The recently published barley reference genome assembly \[[@pone.0209769.ref022]\] now enabled us to characterize the full set of NAC TF genes present in barley. In total, 167 barley NAC TFs ([S1 Table](#pone.0209769.s009){ref-type="supplementary-material"}) were identified. One hundred thirty sequences were found by keyword search; hence they were annotated as NAC TFs. Subsequent BLASTp analysis and a HMMER search based on the Hidden Markov model for the Pfam domain PF02365 characterizing the conserved N-terminus of the NAC TFs identified 11 and 2 additional HvNACs respectively in the peptide sequences representing annotated gene models. Furthermore, tBLASTn against the genomic sequence was done using the protein sequences of all HvNACs identified so far as query. This approach identified 24 additional genomic regions. While 11 of those were overlapping gene annotations, 13 genomic regions were not. One of the HvNACs, which was annotated in the previous genome assembly \[[@pone.0209769.ref035]\] and described as HvNAC026 by Christiansen et al. (2011) \[[@pone.0209769.ref046]\] was, however not identified by any of our searches in the new genome assembly \[[@pone.0209769.ref022]\].
[Fig 1](#pone.0209769.g001){ref-type="fig"} shows the distribution of NACs in the barley genome. More than 30 NAC TF genes were found on chromosomes 2H and 7H, while chromosome 1H carries the lowest number of only 9 genes. However, no specific pattern of distribution was observed. Among the 167 loci, 10 tandem duplications and 8 segmental duplications were identified ([Fig 1](#pone.0209769.g001){ref-type="fig"}). Most of the tandem duplications were found on chromosomes 2H and 7H, which also bear the highest number of NAC TF genes. Segmental duplications were found on all chromosomes.
{#pone.0209769.g001}
The NAC TF encoding genes identified in this study tend to be located in the distal regions of the chromosomes, where also many of the duplication events were observed ([Fig 1](#pone.0209769.g001){ref-type="fig"}). This unequal localization of NAC TF genes across chromosomes is not surprising as the gene density in the distal regions of the chromosomes is increased \[[@pone.0209769.ref022]\]. In wheat, it has further been shown that - while single loci are more frequent in proximal parts of the chromosomes, gene duplications often occur in the distal regions \[[@pone.0209769.ref054]\].
Barley NAC TFs can be classified into different subfamilies {#sec009}
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Shen et al. (2009) \[[@pone.0209769.ref014]\] classified NAC TFs into eight subfamilies, designated as NAC-a to NAC-h, and suggested that these subfamilies might have distinct functions. For instance, many NAC-a subfamily members function in stress responses and hormone signalling \[[@pone.0209769.ref055]--[@pone.0209769.ref059]\], while the NAC-b classified factors are mainly involved in ER stress regulation and cell cycling \[[@pone.0209769.ref060]\]. The NAC-c subfamily contains NACs involved in secondary cell wall biosynthesis and PCD \[[@pone.0209769.ref014],[@pone.0209769.ref061]--[@pone.0209769.ref063]\]. NAC-d TFs appear to have a role in organ initiation and differentiation like shoot apical meristem development or the formation of lateral roots and flowers \[[@pone.0209769.ref014],[@pone.0209769.ref064]--[@pone.0209769.ref066]\]. Several NAC-d subfamily members are further involved in the senescing process both under normal and stress conditions \[[@pone.0209769.ref067]--[@pone.0209769.ref073]\].
In order to classify the newly identified barley NAC TFs according to the nomenclature introduced by Shen et al. (2009) \[[@pone.0209769.ref014]\], the peptide sequences spanning the NAC domains of the 167 HvNACs identified were used together with the NAC domains from *O*. *sativa*, *A*. *thaliana* and *B*. *distachyon* to construct a phylogenetic tree ([Fig 2](#pone.0209769.g002){ref-type="fig"}; [S2 Fig](#pone.0209769.s002){ref-type="supplementary-material"}; [S2 Table](#pone.0209769.s010){ref-type="supplementary-material"}). Sequences from barley, rice and *B*. *distachyon* were found to group into clusters of orthologues, while *A*. *thaliana* NAC TFs often formed their own subclades reflecting the larger distance between monocot and dicot NAC domains. The TF subfamilies corresponding to NAC-c, NAC-d and NAC-f could be identified unequivocally, meaning that all sequences clustered into the expected subfamilies following Shen et al. \[[@pone.0209769.ref014]\]. Minor discrepancies were, however, found within the other subfamilies. Notably, five members of the NAC-b subfamily were found to possess transmembrane domains, an observation that has been described before \[[@pone.0209769.ref060],[@pone.0209769.ref074]\]. In the current study, sequences failed to cluster unambiguously into subfamilies NAC-f and NAC-g \[[@pone.0209769.ref014]\], as some sequences previously shown to belong into these subfamilies \[[@pone.0209769.ref014]\], were found elsewhere in the current study ([Fig 2](#pone.0209769.g002){ref-type="fig"}), which might be due to differences in the sequences used as well as in alignment and tree construction settings.
![Phylogenetic analysis of the NAC TFs from *H*. *vulgare*, *O*. *sativa*, *B*. *distachyon* and *A*. *thaliana*.\
(A) Phylogenetic tree of the NAC domain peptide sequences from *H*. *vulgare*, *O*. *sativa*, *B*. *distachyon* and *A*. *thaliana*. The tree was constructed using the Maximum Likelihood method with 100 bootstrap repetitions. The subfamilies NAC-a to NAC-h were assigned using the nomenclature introduced by Shen et al. (2009) \[[@pone.0209769.ref014]\]. Each species is represented by symbols with different colours. (B) Subtree of the NAC-d subfamily. The subtrees of the other subfamilies are shown in [S2 Fig](#pone.0209769.s002){ref-type="supplementary-material"}.](pone.0209769.g002){#pone.0209769.g002}
Expression patterns of barley NAC TF genes {#sec010}
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Apart from phylogenetic relationships, gene expression patterns can hint at the function of specific genes as well. Therefore, a heatmap of expression levels for all barley NAC TF genes, for which gene models can be found in the barley reference assembly, was constructed. The heatmap was based on publicly available RNAseq data from 15 samples representing different tissues and developmental stages \[[@pone.0209769.ref022],[@pone.0209769.ref035]\] and is shown in [Fig 3](#pone.0209769.g003){ref-type="fig"}. As also observed in wheat \[[@pone.0209769.ref007]\], we overall found similar expression patterns within the different subfamilies. The NAC-d subfamily however depicts a striking exception, as its members clearly have two different patterns of expression. While most genes in the NAC-d-9 subfamily are expressed over a wide range of different samples, six genes (HORVU4Hr1G089450.1, HORVU7Hr1G039700.1, HORVU7Hr1G122680.1, HORVU3Hr1G014090.1, HORVU7Hr1G031260.1, HORVU3Hr1G014100.1) show a very strong and almost exclusive expression in developing grains ([Fig 3](#pone.0209769.g003){ref-type="fig"}).
![Heatmap of gene expression values (log2 FPMK values) for HvNAC genes across 15 samples representing different tissues and developmental stages.\
The RNA-seq expression data are taken from Mascher et al. (2017) \[[@pone.0209769.ref022]\], omitting one sample from the inflorescence (referred to as INF1) as this sample was characterized by very low expression levels. The NAC genes are ordered according to their respective subfamily classification (designated as a to h \[[@pone.0209769.ref014]\]), and within each subfamily, genes are ordered by descending total expression values across all samples. The last gene, HORVU0Hr1G000700.1, could not be associated to any of the subfamilies. Alternating colouring of gene names delineates subfamilies. On the right, filled circles mark the NAC-d-9 subgroup, and red filled circles further mark the six Grain-NAC genes within the NAC-d-9 subgroup.](pone.0209769.g003){#pone.0209769.g003}
Notably, five of these six genes depict the five most highly expressed genes across all samples. This observation prompted us to explore the group of highly expressed grain-specific NAC-d genes further. A heatmap based on hierarchical clustering of expression levels across the 15 different samples was constructed at the overall expression level to detect NAC genes from other subfamilies with grain-specific expression ([S1 Fig](#pone.0209769.s001){ref-type="supplementary-material"}). This effort divided the NAC gene expression patterns into 14 clusters. The NAC-d genes with grain specific expression clustered in clade \#14, which consists of genes that have their highest expression levels in the developing caryopsis. Indeed, there was one additional gene, HORVU2Hr1G082320.2, that showed close to exclusive expression in the caryopsis, but at a much lower level in both of the two caryopsis samples included in the analysis than the NAC-d genes with grain specific expression. Phylogenetically, this gene falls into the NAC-h subfamily ([Fig 2](#pone.0209769.g002){ref-type="fig"}; [S2 Fig](#pone.0209769.s002){ref-type="supplementary-material"}; [S2 Table](#pone.0209769.s010){ref-type="supplementary-material"}), the members of which are characterized by diverging, weaker NAC domains \[[@pone.0209769.ref014]\]. The other genes in this cluster however did not show an exclusive expression in the grain. Hence, the six NAC-d genes referred to above stood out as an exceptional set of genes with very high and grain-specific expression, making a detailed analysis of the NAC-d subfamily relevant.
Evolution of the NAC-d subfamily in monocots {#sec011}
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Next, a comprehensive analysis of the evolution of the NAC-d subfamily in monocots was performed. To this end, a phylogenetic tree using the NAC domain sequences from 17 monocot species, for which fully sequenced genomes are available, and *A*. *thaliana* was constructed ([S3 Fig](#pone.0209769.s003){ref-type="supplementary-material"}). The monocotyledonous species included in this analysis were barley (*H*. *vulgare*), common wheat (*T*. *aestivum*), *A*. *tauschii*, wild emmer (*T*. *turgidum*), wild einkorn (*T*. *urartu*), rye (*S*. *cereale*), rice (*O*. *sativa*), *B*. *distachyon*, *S*. *bicolor*, foxtail millet (*S*. *italica*), switchgrass (*P*. *virgatum*), maize (*Z*. *mays*), *Z*. *pacifica*, pineapple (*A*. *comosus*), banana (*M*. *acuminata*) and oil palm (*E*. *guineensis*). The systematic classification of the species can be found in [Table 1](#pone.0209769.t001){ref-type="table"}. The phylogenetic analysis enabled the identification of the NAC-d subfamily ([S3 Fig](#pone.0209769.s003){ref-type="supplementary-material"}) in all species mentioned and it became apparent that the NAC-d-9 subgroup \[[@pone.0209769.ref014]\] has expanded during the evolution of monocots ([Fig 4](#pone.0209769.g004){ref-type="fig"}). In particular, the Triticeae species were found to have nearly twice as many NAC TFs in this subgroup as the other monocot species.
{#pone.0209769.g004}
For further analysis, a phylogenetic tree based on the peptide sequences of the NAC domains of the d-9 NAC TFs was constructed. *A*. *tauschii*, wild emmer and wild einkorn were omitted from the analysis, as common hexaploid wheat was included. The tree branched into three clades designated as I, II and III ([Fig 4](#pone.0209769.g004){ref-type="fig"}; [S3 Table](#pone.0209769.s011){ref-type="supplementary-material"}). Clade I contains the six barley NAC-d-9 genes with almost exclusive expression in the grain. Both clade I and II were found to be specific to Poaceae, while clade III contained NAC TFs from monocotyledonous species not belonging to the Poaceae family and from *A*. *thaliana*. Notably, several of the NAC-TFs in clade II and III are expressed and up-regulated in senescing leaves \[[@pone.0209769.ref016],[@pone.0209769.ref069],[@pone.0209769.ref073],[@pone.0209769.ref075]\] and are further referred to as senescence associated NAC TFs in this study. The observation of two Poaceae specific subclades within NAC-d-9 hints to a diversification of this specific subgroup after the separation of the Poaceae family from other families in the order of Poales. Furthermore, an increased number of NAC TFs in the d-9 subgroup of the Triticeae implies an expansion of NAC TFs in this specific subgroup during the evolution of Triticeae ([Table 1](#pone.0209769.t001){ref-type="table"}).
A subclade of NAC-d-9 TFs shows grain specific expression {#sec012}
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Notably, the six barley d-9 NACs with almost exclusive expression in the developing grain fell into the same clade (Figs [3](#pone.0209769.g003){ref-type="fig"} and [4](#pone.0209769.g004){ref-type="fig"}; [S3 Table](#pone.0209769.s011){ref-type="supplementary-material"}) as a number of genes from other Poaceae species for which high expression in the grain has been observed previously; however, without making clear associations with a Poaceae-specific phylogenetic clade as we describe it here (e.g. \[[@pone.0209769.ref046],[@pone.0209769.ref076]--[@pone.0209769.ref078]\]). The wheat homologs in this clade for instance showed a strong and exclusive expression patterns in wheat grain tissues ([S4 Fig](#pone.0209769.s004){ref-type="supplementary-material"}; \[[@pone.0209769.ref007],[@pone.0209769.ref021],[@pone.0209769.ref050]\]. The two rice NAC TFs, ONAC020 and ONAC026, that fell into clade I of the NAC-d-9 subgroup as well, are known to be highly expressed specifically in grains during maturation. Notably, they are associated with grain size and weight phenotypes \[[@pone.0209769.ref079]\]. Further, two maize NACs, which also fell into clade I, have previously been characterized as grain specific: NRP1 (GRMZM2G062650) and ZmNAC4 (GRMZM2G154182) \[[@pone.0209769.ref065],[@pone.0209769.ref080]\]. We hence show here that a group of NAC TFs, that are highly and specifically expressed in grains, forms a distinct clade within the NAC-d-9 subgroup. We therefore propose to designate these TFs as Grain-NACs. Grain-NACs are specific for the Poaceae family of monocotyledonous plants, as they were found to be absent from the Bromeliaceae species *A*. *comosus* and from the Arecales and Zingiberales species included in this study as well as from the dicotyledonous *A*. *thaliana*.
Due to their organ specific expression and as NAC-d TFs are often involved in organ formation and differentiation, Grain-NACs possibly have a role in the development of the caryopsis, the fruit typical for the Poaceae family of grasses, and more specifically its storage tissue, the starchy endosperm. Notably, it is particularly the endosperm that depicts the value of such important cereal crops as wheat, rice, maize, or barley for human nutrition and for countless industrial applications. Its main function is to provide nutrients to the developing and later germinating embryo. The Poaceae endosperm is, in contrast to many species, including *A*. *thaliana*, a persistent seed structure. Endosperm development includes several distinct phases: Upon double fertilization, syncytium formation and subsequently cellularization occurs. This is followed by cell differentiation and the periods of mitosis, endoreplication and storage compound accumulation and finally maturation, including PCD, dormancy and desiccation (summarized in \[[@pone.0209769.ref081]\]).
Notably, several reports on gene expression in developing grains have included Grain-NAC genes, still without characterizing them as a Poaceae-specific clade of NAC genes. Retrospectively, this data can be used to relate the Grain-NAC gene expression profiles to the specific stages of endosperm development. Thus, expression of the Grain-NAC genes appears to be initiated 7-10 days after flowering in both barley and wheat \[[@pone.0209769.ref076]--[@pone.0209769.ref078]\] just before the start of storage compound accumulation \[[@pone.0209769.ref082]\]. Hence, there is high expression of the Grain-NAC genes throughout most of the grain filling period, paralleling the accumulation of starch and storage proteins. Maximal expression of Grain-NACs in barley and wheat occurred 15-25 days after flowering. Similar patterns were observed for the two maize Grain-NAC genes \[[@pone.0209769.ref065],[@pone.0209769.ref080]\]. Notably, in maize and wheat the earliest occurrence of PCD in the endosperm is around 16 DAP \[[@pone.0209769.ref083]--[@pone.0209769.ref085]\]. It has been suggested that ethylene is involved in PCD during endosperm development \[[@pone.0209769.ref086]\] and NACs are known to play a role in ethylene signalling \[[@pone.0209769.ref087]--[@pone.0209769.ref089]\].
Cis-regulatory elements leading to grain-specific expression in Grain-NACs {#sec013}
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As Grain-NAC genes are exclusively and strongly expressed during seed development the question about which cis-regulatory elements in promoter regions are involved in grain specificity is obvious. To explore this, we compared the occurrence of specific promoter motifs involved in seed development both in Grain-NAC and senescence-associated NAC promoter sequences. Although these are phylogenetically closely related, their expression patterns differ considerably ([Fig 3](#pone.0209769.g003){ref-type="fig"}). Seventeen distinct cis-regulatory elements involved in seed development were found ([S4 Table](#pone.0209769.s012){ref-type="supplementary-material"}). Amongst them, P-BOX2, RYREPEAT, EBOXBNAPA, DPBFCORE, AACA motif 2, MYB1AT and MYBCORE elements were conserved and exhibited similar patterns across all six Grain-HvNAC promoters ([Fig 5A](#pone.0209769.g005){ref-type="fig"}). In total, 197 motifs were found in the six Grain-HvNAC promoters, an average of about 33 motifs per sequence, while only 138 of such motifs were found in the seven senescence-associated HvNAC promoters, an average of about 20 motifs per sequence ([S4 Table](#pone.0209769.s012){ref-type="supplementary-material"}). A statistical test showed that promoter motifs associated with seed development were significantly over-represented in Grain-HvNAC promoter sequences compared with senescence-associated HvNAC promoters and also with a group of four unrelated house-keeping genes ([Fig 5A and 5B](#pone.0209769.g005){ref-type="fig"}, [S4 Table](#pone.0209769.s012){ref-type="supplementary-material"}).
{#pone.0209769.g005}
The P-BOX2 element or prolamin box was found to be exclusively present in Grain-HvNAC promoters while absent from senescence-associated HvNAC promoters ([Fig 5A and 5B](#pone.0209769.g005){ref-type="fig"}). The P-BOX2 element occurred only once per Grain-HvNAC promoter. This box is essential for regulating the expression of seed storage proteins (SSPs) both in barley and wheat \[[@pone.0209769.ref090]\]. Further, an alignment of Grain-HvNAC and senescence-associated HvNAC promoter sequences identified a fully conserved motif of eleven nucleotides ([Fig 5C](#pone.0209769.g005){ref-type="fig"}), representing overlapping P-BOX2 and MYBCORE motifs, in the Grain-HvNAC promoters, which is absent in the promoters of the senescence-associated HvNAC genes. The MYBCORE, as well as the AACA motif 2 and MYB1AT elements are specific binding sites for the R2R3-MYB TFs \[[@pone.0209769.ref091],[@pone.0209769.ref092]\]. Among these, GAMYB, MCB1 and MYBS3 have an important role in activating endosperm specific genes during seed development \[[@pone.0209769.ref093]--[@pone.0209769.ref096]\]. The conserved eleven nucleotide core may hence be essential for the regulation of the grain specific expression of the Grain-NAC genes in barley. Only 54 nucleotides further downstream of this region another conserved region was found, corresponding to the EBOXBNAPA, AACA motif 2 and DPBFCOREDCDC3 ([S5 Fig](#pone.0209769.s005){ref-type="supplementary-material"}). EBOXBNAPA, conserved in SSP promoters, is critical for directing seed specific expression in *Brassica napus* \[[@pone.0209769.ref097]\]. DPBFCOREDCDC3 is an embryo specific element that interact with bZIP-TFs \[[@pone.0209769.ref086]\]. Finally, the AACA motif 2 is required for the high level of glutenin expression in the starchy endosperm \[[@pone.0209769.ref098]\]. Ravel et al. (2014) \[[@pone.0209769.ref099]\] observed a common regulatory framework of cis-elements in high molecular weight glutenin subunits (HMW-GS) gene promoters that regulate the transcription of SSP genes and hence partially explain their high expression level in several wheat varieties. Potentially the P-BOX2, RYREPEAT, EBOXBNAPA, AACA motif 2 and MYB1AT motifs depict a similar framework for Grain-HvNAC promoters, resulting in the high grain-specific expression levels shown in [Fig 3](#pone.0209769.g003){ref-type="fig"}. Notably, the same cis-elements described above for barley were further found with similar numbers in wheat, rice and maize Grain-NAC promoters ([S6](#pone.0209769.s006){ref-type="supplementary-material"}--[S8](#pone.0209769.s008){ref-type="supplementary-material"} Figs, [S5 Table](#pone.0209769.s013){ref-type="supplementary-material"}). As discussed above, grain-specific expression of wheat and maize Grain-NAC genes has been observed, and Mathew et al. (2016) \[[@pone.0209769.ref079]\] showed that OsNAC20 and OsNAC26 are expressed specifically during rice seed development at extremely high levels.
Yet another motif conserved across the Grain-HvNAC promoters was the RYREPEAT motif ([S5 Fig](#pone.0209769.s005){ref-type="supplementary-material"}). This motif was found to be positioned distantly from the transcription initiation site when compared to the conserved motifs described above and it was present in multiple copies in some of the Grain-HvNAC promoters ([Fig 5A](#pone.0209769.g005){ref-type="fig"}). This motif is known to participate in the transcriptional activation of the endosperm specific genes *Hor2* and *Itr1* through binding of the FUS3 protein to RYREPEAT element in barley \[[@pone.0209769.ref100]\].
Notably, the RYREPEAT motif (`CATGCA`) is very similar to the EIN3 motif (`ATGCAT`). These DNA binding domains are often found together in the same promoter region. The EIN3 motif, as its name indicates, serves as a binding site for EIN3 and EIN3-like transcription factors \[[@pone.0209769.ref101],[@pone.0209769.ref102]\], which are positive regulators in ethylene signaling \[[@pone.0209769.ref103]\]. Ethylene is a key regulator of plant senescence \[[@pone.0209769.ref104]\] and is also a mediator of PCD in the cereal endosperm \[[@pone.0209769.ref085]\]. The promoters of both the Grain-HvNAC and senescence-associated HvNAC genes characterized in this study display a high number of RYREPEAT/EIN3 motifs, however, with a higher number in the Grain-HvNAC genes ([S4 Table](#pone.0209769.s012){ref-type="supplementary-material"}). It hence appears plausible that the multiple copies of RYREPEAT/EIN3 motifs add to the differential expression levels between Grain-NAC and senescence-associated NAC genes.
Overall, our analysis of the Grain-HvNAC promoters showed that those likely are unrelated to promoters of the phylogenetically most closely related senescence-associated HvNAC genes (based on the NAC A-E subdomains). There is no apparent conservation of promoter motifs between these two groups ([Fig 5](#pone.0209769.g005){ref-type="fig"}), and upon alignment they did not show higher similarities to each other than to NAC promoters from other HvNAC subfamilies (data not shown). Hence, we conclude that the origin of the Grain-HvNAC promoters is different from that of the senescence-associated HvNAC genes, which apparently is associated with the evolutionary acquisition of seed specific motifs in the Grain-HvNAC promoters.
DLN and NARD motifs are conserved in Grain-NACs as well as in senescence-associated NACs whereas conserved motifs in C-terminal parts differ considerably {#sec014}
---------------------------------------------------------------------------------------------------------------------------------------------------------
The two rice NAC TFs, ONAC020 and ONAC026, that fell into the Grain-NAC subclade have been shown to comprise a DLN repressor motif in the subdomain B of their NAC domain, which is an EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif functioning as repressors in various signalling pathway including ethylene \[[@pone.0209769.ref079],[@pone.0209769.ref105],[@pone.0209769.ref106]\], and a NAC repression domain (NARD) in their subdomain D \[[@pone.0209769.ref079],[@pone.0209769.ref105],[@pone.0209769.ref107]\]. Transactivation and transrepression assays in yeast demonstrated that these motifs act as repressors of the transactivation function \[[@pone.0209769.ref079],[@pone.0209769.ref107]\]. In this study, we found that among the 141 NAC-d-9 sequences included in the analysis ([Fig 4](#pone.0209769.g004){ref-type="fig"}; [S3 Table](#pone.0209769.s011){ref-type="supplementary-material"}), 102 (72%) had a DLN motif and 135 (96%) a LVFY motif, which is the most conserved motif in the NARD domain ([S6 Table](#pone.0209769.s014){ref-type="supplementary-material"}). Notably, all NACs with a DLN motif also comprised a LVFY motif. This is contrasted by the fact that a NARD motif was found only in about half and a DLN motif only in one fifth of the total population of NACs when all the subfamilies were included. The observation that more than half of all NAC-d-9 TFs comprise DLN motifs hints to a function involved in ethylene signalling and regulation. Furthermore, it has been suggested that the overall activity of NAC TFs is determined through interaction between the NARD repressor domain and an activation domain at C-terminus \[[@pone.0209769.ref107]\]. Since the NARD domain is present in most of the members of the NAC-d-9 subgroup, these might share similar regulative mechanisms.
In contrast to the similarities observed in the N-terminus, the C-terminal parts of the NAC-d-9 members differ considerably: Apart from HORVU3Hr1G014090, which has a short, apparently truncated C-terminus of only 38 aa, the encoded Grain-HvNAC protein sequences have relatively conserved C-terminal parts, around 175 aa long, with 51.5-64.5% aa identity among each other. When comparing the five long Grain-HvNACs with the senescence-associated NAC-d-9 members, the intergroup amino acid identity range was 13.6-25.0% for the C-terminal parts, whereas this range for the NAC domain (A-E subdomains) was 51.3-66.8%. For both regions of the sequence, the Grain-HvNACs showed the highest level of similarity with the senescence-associated gene HORVU4Hr1G051360 (HvNAC013 in \[[@pone.0209769.ref073]\]). The intragroup conservation of the Grain-HvNAC C-termini probably reflects relatively recent duplication events giving rise to the expanded Triticeae clade of Grain-NACs. It was shown previously that the intrinsically disordered C-termini of NAC TFs contain short conserved motifs of possible importance for the interaction characteristics of the TFs \[[@pone.0209769.ref108]\]. Accordingly, an analysis for conserved motifs across the five long Grain-HvNAC C-terminal sequences using the MEME software (\[[@pone.0209769.ref053]\]; number of motifs=6, max. motif length=12) showed high conservation of at least five short motifs ([Fig 6](#pone.0209769.g006){ref-type="fig"}), and three of these motifs (a proximal and two distal) were conserved across to the rice and maize Grain-NACs. When including the senescence-associated HvNACs of the NAC-d-9 subgroup, only one motif showed conservation across the two subclades with respect to both sequence and relative position. Hence, in contrast to the N-terminal NAC domain, the C-termini are rather diversified, which is a typical characteristic of NAC TFs \[[@pone.0209769.ref014]\]. Albeit diverse, the C-terminal part appears to function as a transcription regulatory domain and, hence, it is important for fine-tuning of the transcriptional activity of the NAC TFs \[[@pone.0209769.ref030]\].
![Positions of conserved short motifs in the C-terminal parts of NAC-d-9 protein sequences, for 13 barley, 2 rice, and 2 maize proteins.\
The C-terminal part was defined to start at position 6 after the conserved cysteine residue of the E subdomain. Conserved motifs were identified by the MEME software (\[[@pone.0209769.ref053]\]; [http://www.meme-suite.org](http://www.meme-suite.org/)) with the settings: number of motifs -- 6, max. length of motif -- 12. WebLogos of the identified motifs are shown. On the left, full lines indicate Grain-NAC sequences, and the dotted line indicates senescence-associated HvNAC sequences. Rice Grain-NAC TFs: ONAC20 (LOC_Os01g01470.1), ONAC26 (LOC_Os01g29840.1). Maize Grain-NAC TFs: ZmNAC128 (GRMZM2G062650), ZmNAC130 (GRMZM2G154182).](pone.0209769.g006){#pone.0209769.g006}
Possible roles of the Grain-NAC TFs {#sec015}
-----------------------------------
From our analysis of the Grain-NAC TFs of the Poaceae, and particularly the Triticeae, an interesting picture arises with respect to evolution of the Poaceae and the processes involved in grain filling and maturation of the caryopsis. A model of this is presented in [Fig 7](#pone.0209769.g007){ref-type="fig"}. First, the divergence of the Grain-NACs within the NAC-d-9 subclade of NAC genes appears to have taken place soon after, or during, the formation of the Poaceae, since they occur in all Poaceae species included in the analysis, but not outside the Poaceae. Our analysis of Grain-NAC promoter sequences revealed that an event must have taken place in which a copy of a NAC-d-9 coding sequence was combined with a promoter sequence mediating strong seed/endosperm specific expression. We hypothesize that this event could have contributed specifically to the evolution of the caryopsis, typically characterized by a starchy and dry endosperm at maturity \[[@pone.0209769.ref081]\]. Furthermore, we observed an expansion of the Grain-NAC clade within the Triticeae compared with other Poaceae species, e.g. rice and maize. A major radiation of Triticeae species (including barley and wheat) appears to have taken place 6.1-9.2 MYA in the Mediterranean area under a climate with cool winters and dry summers \[[@pone.0209769.ref109]\]. The action of the highly expressed Grain-NAC genes, enhanced by the gene duplications, might have been important for proper, i.e. fast, maturation in these dry environments, similar to contemporary Mediterranean environments.
{#pone.0209769.g007}
Second, our analysis shows that there is a close phylogenetic relationship between the Grain-NACs and senescence-associated NACs of the NAC-d subfamily, based on a high degree of similarity among their respective conserved NAC domains. Since the NAC domains confer the DNA binding specificity \[[@pone.0209769.ref012]\], this similarity indicates that the Grain-NAC TFs and the senescence-associated NACd TFs also target similar down-stream genes. The identity of these genes is not firmly established, but there are many indications (see \[[@pone.0209769.ref016]\]) that they belong to the battery of genes encoding degradation factors involved in the senescence processes, e.g. proteases. The senescence process depicts a PCD event, in which a controlled degradation of contents in affected cells is completed \[[@pone.0209769.ref018]\]. Notably, the grain filling and maturation process in the cereal caryopsis is also characterized by a controlled PCD process \[[@pone.0209769.ref085]\]. Hence, we see a parallel between the leaf senescence process and the grain maturation process in the strong upregulation of NAC TFs accompanying PCD. The latter takes place in the endosperm towards the end of development while the machinery for starch and storage protein biosynthesis is still running to fill up the endosperm with storage compounds. Ethylene appears to be a strong regulator of the endosperm PCD \[[@pone.0209769.ref085]\], and the occurrence of binding signatures for EIN3, a central factor in ethylene signalling \[[@pone.0209769.ref110]\], within the many RYREPEAT motifs of Grain-HvNAC promoters ([Fig 5A](#pone.0209769.g005){ref-type="fig"}) might indicate an involvement of the Grain-NAC TFs in the ethylene regulation of endosperm PCD. Interestingly, EIN3 is also suggested to be a central up-stream regulator of senescence-associated NAC TFs in leaves \[[@pone.0209769.ref111]\], supported by the occurrence of a considerable number of RYREPEAT elements also in promoters of the senescence-associated NAC-d-9 genes, although at a lower number than for the Grain-NAC genes.
Further experiments are required in order to firmly establish the down-stream target genes of the Grain-NAC TFs. Even though the Grain-NAC and senescence-associated NAC-d TFs presumably recognize similar target promoter sequences, the actual outcome in terms of trans-activated genes might be considerably influenced by transcriptional interaction partners, possibly involving the interaction motifs of the diversified C-terminal parts of the NAC TFs \[[@pone.0209769.ref112]\]. Furthermore, the occurrence of the repressive DLN and NARD motifs in the NAC domain probably adds to the complexity of the regulatory role of Grain-NAC TFs during grain development. Hence, the degree of neo-functionalization of the Grain-NAC TFs compared with the senescence-associated NAC-d-9 members remains to be firmly established.
Conclusions {#sec016}
===========
The update of the barley NAC TF family performed in this work resulted in a total number of 167 members, i.e. in a range similar to that of other cereals such as rice. Most of the members could be allocated to the eight subfamilies a to h, as defined by Shen et al. (2009) \[[@pone.0209769.ref014]\]. This also reflects the quality of the new assembly of the barley genome \[[@pone.0209769.ref022]\], even though a number of the gene models still showed partial or truncated NAC TFs. Starting out from gene expression profiles, our analysis directed us towards a subclade of genes in the NAC-d subfamily deviating strongly from other NAC-d gene with respect to their expression patterns, as they showed strong and exclusive expression in the developing caryopsis coinciding with grain filling and the occurrence of PCD in the endosperm. We propose to designate this subclade of NAC-d TFs as Grain-NACs. Promoter analysis of the Grain-NAC genes revealed the occurrence of a number of cis-elements that are known to drive seed-specific expression of genes. Furthermore, phylogenetic analyses showed that the Grain-NAC subclade is specific for the Poaceae species. Based on this, we propose that the encoded Grain-NAC TFs might have played a role in the evolution of the caryopsis, i.e. the specialized fruit of the Poaceae with its typical dry and starchy endosperm.
Supporting information {#sec017}
======================
###### Heatmap of gene expression values (log2 FPMK values) for HvNAC genes across 15 samples representing different tissues and developmental stages.
The RNA-seq expression data are taken from Mascher et al. (2017) \[[@pone.0209769.ref022]\], omitting one sample from the inflorescence (referred to as INF1) as this sample was characterized by very low expression levels. Grain-NACs are marked in red. A Pearson correlation distance function was used in the hierarchical clustering of log2 FPKM values. This divided the NAC gene expression patterns into 14 clusters, indicated by numbers on the left.
(PDF)
######
Click here for additional data file.
###### Detailed subtrees of NAC subfamilies -a,b,c,e,f,g and h from the phylogenetic tree shown in [Fig 2](#pone.0209769.g002){ref-type="fig"}.
(PDF)
######
Click here for additional data file.
###### Global phylogenetic analysis of NAC TFs.
The NAC domain peptide sequences of barley (*H*. *vulg*are), common wheat (*T*. *aestivum*), *A*. *tauschii*, wild emmer (*T*. *turgidum*), wild einkorn (*T*. *urartu*) rye (*S*. *cereale*), rice (*O*. *sativa*), *B*. *distachyon*, *S*. *bicolor*, foxtail millet (*S*. *italica*), switchgrass (*P*. *virgatum*), maize (*Z*. *mays*), *Z*. *pacifica*, pineapple (*A*. *comosus*), banana (*M*. *acuminata*) and oil palm (*E*. *guineensis*) were used to construct an approximately-Maximum Likelihood tree with 1000 resamples. The NAC d-9 subgroup, was identified following Borrill et al. (2017) \[[@pone.0209769.ref007]\] and Pereira-Santana et al. (2015) \[[@pone.0209769.ref019]\]. The respective sequences are coloured in red.
(EPS)
######
Click here for additional data file.
###### Heatmap showing gene expression of wheat *Grain-NAC*s.
Heatmap of gene expression values (log2 tpm values) for Grain-TaNAC and senescence-associated TaNAC orthologous genes across 25 different samples/tissue types. RNA-seq expression data are from Borrill et al.(2016) \[[@pone.0209769.ref050]\]. Grain-TaNACs are clustered at the bottom of the figure and mainly expressed in tissues of the developing grain [S5 Fig](#pone.0209769.s005){ref-type="supplementary-material"}. Alignment of Grain and senescence-associated HvNAC promoter sequences. The identical and similar conserved P-BOX2, MYBCORE, AACA motif-2, MYB1AT, RYEREPEAT, EBOXBNAPA and DPBFCORE motifs are indicated in squares.
(PDF)
######
Click here for additional data file.
###### Alignment of Grain and senescence-associated HvNAC promoter sequences.
**The** identical and similar conserved P-BOX2, MYBCORE, AACA motif-2, MYB1AT, RYEREPEAT, EBOXBNAPA and DPBFCORE motifs are indicated in squares.
(PDF)
######
Click here for additional data file.
###### Cis-regulatory element annotations of wheat, rice and maize Grain-NAC promoters.
The positions of seven conserved cis-elements involved in seed development are shown in wheat (A) rice (B) and maize (C) NAC promoters. Identical seed-specific motifs are represented by triangles fully coloured, while similar motifs (element with a SNP variation) are represented by triangle stroke paint. Triangles on the upper and lower side represent the orientation on positive and negative strands, respectively.
(PDF)
######
Click here for additional data file.
###### Alignment of promoter sequences from Grain-HvNAC and Grain-TaNAC homologous genes.
The identical and similar conserved P-BOX2, MYBCORE, AACA motif-2, MYB1AT, RYEREPEAT, EBOXBNAPA and DPBFCORE motifs are indicated in squares.
(PDF)
######
Click here for additional data file.
###### Alignment of promoter sequences from Grain-HvNAC, Grain-OsNAC and Grain-ZmNAC homologous genes.
The identical and similar conserved P-BOX2, MYBCORE, AACA motif-2, MYB1AT, RYEREPEAT, EBOXBNAPA and DPBFCORE motifs are indicated in squares.
(PDF)
######
Click here for additional data file.
###### 167 barley NAC TFs identified in this study.
(XLSX)
######
Click here for additional data file.
###### Listing of corresponding gene IDs for the sequences used in the phylogenetic tree in [Fig 2](#pone.0209769.g002){ref-type="fig"}.
(XLSX)
######
Click here for additional data file.
###### Listing of corresponding gene IDs for the sequences used in the phylogenetic tree in [Fig 4](#pone.0209769.g004){ref-type="fig"}.
(XLSX)
######
Click here for additional data file.
###### Number of identical motifs known to be involved in seed development of both Grain- and senescence-associated HvNAC promoters.
(XLSX)
######
Click here for additional data file.
###### Number of identical motifs known to be involved in seed development of maize, rice and wheat Grain-NAC promoters.
(XLSX)
######
Click here for additional data file.
###### Number of DLN and LVFY motifs identified in NAC TFs.
(XLSX)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S0001}
============
The most common causes of zonular insufficiency are pseudoexfoliation syndrome, trauma, previous ocular surgery (eg, filtering surgery or vitrectomy), hypermature cataracts, and increased axial length. Less common causes include Marfan's syndrome, homocystinuria, Weill--Marchesani syndrome, microspherophakia, retinitis pigmentosa and intraocular neoplasms.[@CIT0001]
The Cionni's modified capsule rings (MCTRs) has an open ring design with one (model I-L or I-R) or two (model 2-L) fixation eyelets attached to the main ring which allows the ring to be fixated to the sclera. This eyelet protrudes 0.25mm forward from the ring and then lays anterior to the anterior capsular rim.[@CIT0001],[@CIT0002]
Modified capsule tension rings are sutured to the sclera to give a long-term stability for the capsular bag with severe (more than 180 degree) zonular dialysis. Posterior chamber IOLs can be implanted in the bag after being stabilized and centered.[@CIT0001],[@CIT0002]
After passing the sutures through the eyelets, the sutures are externalized, and tied under a scleral flap, tied under a corneoscleral pocket, or tied over episclera according to surgeon preference, as no technique has been definitively proven superior over the other.[@CIT0004]--[@CIT0006]
This work describes a novel technique to fix the MCTRs to the sclera using a 6/0 polypropylene sutures with double flanges.
Methods {#S0002}
=======
This is a prospective interventional case series which included 7 cases with severe (more than 180 degrees) zonular dialysis. Cases with previous ocular surgeries, glaucoma, posterior segment pathology or corneal opacity were excluded. This work was done according the Declaration of Helsinki and was approved by Alpha Vision center research ethics committee. A written informed consent was obtained from all patients or the parents after explanation of the type of the procedure and the follow up. All cases were done by the same surgeon (A.S).
Surgical technique: (As shown in the [[supplementary video](http://youtu.be/rWVsORmKKJs)]{.ul}).
After I/A flexible, iris retractors placed through limbal stab incisions were used to hook the capsulorhexis edge and support the bag during insertion of the MCTR.
The Cionni ring was inserted manually with a McPherson forceps and Sinsky hook was used to guide the trailing end to be placed in the bag.
The Cionni ring was placed so that the eyelet for scleral fixation was placed at the maximum point of subluxation.
A 6/0 polypropylene suture (Prolene^®^; Ethicon, Somerville, NJ) was introduced through a paracentesis the passes through the eyelet of the ring. A 27-gauge needle was inserted 2 mm behind the limbus transconjunctivally to the sulcus. A microforceps wasused to thread the prolene suture into the lumen of the needle. The needle was withdrawn outside the eye carrying the prolene suture. A hand held ophthalmic cautery (Accu-Temp Cautery; Beaver Visitec, Waltham, MA) wasused to create a flange at the end of the prolene suture at the paracentesis side. To create an adequate sized flange that prevents the suture from passage through the eyelet, more than 5 mm of the suture should be cauterized. The other end of the prolene suture at the scleral side was pulled till the ringwas centered. The suture outside the eye is pulled to the maximum with McPherson forceps in flush with the sclera and the cautery was used to create another bulb then the suture was released till the bulb was totally covered by the conjunctiva and rest in the outer end of the scleral track created by the 27-gauge needle ([Figure 1](#F0001){ref-type="fig"}).Figure 1(**A--H**) Shows surgical steps.
After the bag was well centered by the scleral-fixated MCTR, a single piece IOL was implanted in the bag.
All patients were treated with topical broad spectrum antibiotic drops four times per day and topical prednisolone acetate four times per day. The antibiotic eye drop was stopped after two weeks while the steroid eye drop was tapered over four weeks.
The cases were followed up for at least three months for the best corrected visual acuity, IOP, IOL decentration, and presence of complications as endophthalmitis or intraocular hemorrhage.
Results {#S0003}
=======
The technique was used in 7 cases with severe (more than 180 degrees) zonular dialysis.
Three cases were hereditary lens subluxation (Marfan syndrome), 2 cases with traumatic subluxation and 2 cases with pseudo-exfoliation syndrome.
All cases achieved a good postoperative stable and centered IOL with good visual results. No postoperative complications were recorded apart from PCO in 3 cases and mild bleeding during needle passage in one case ([Table 1](#T0001){ref-type="table"}). Table 1Preoperative and Postoperative BCVA, Etiology of Lens Subluxation and Postoperative ComplicationsPatientSexAgeCausePreop BCVAPostop BCVAComplication1M69Pseudoexfoliation20/7020/20NA2M31Traumatic20/15020/40Bleeding during needle passage3F6Marfan20/20020/30PCO4M34Traumatic20/8020/20NA5F9Marfan20/10020/40PCO6M72Pseudoexfoliation20/6020/20NA7M8Marfan20/15020/30PCO
Discussion {#S0004}
==========
Capsular tension rings (CTRs) and other endocapsular devices have become well-established tool that allow surgeons to deal with zonular weakness with increased safety. The choice of capsular tension device in a particular surgery depends on the amount of zonular dehiscence as well as the likelihood of progression. CTRs can re-center a mildly subluxated capsular bag, but it will not recenter a severely subluxated capsular bag nor will it cure a progressive zonulopathy. In these situations, a modified CTR (MCTR) or a capsular tension segment (CTS) provides a stable long-term solution through scleral-fixation.[@CIT0002]
The standard method of Trans- scleral fixation of Cionni ring for stabilization of capsular bag in cases of zonular dialysis,involves conjunctival peritomy, caurety of subconjunctival vessels, scleral flaps groove or pocket creation which is time consuming and needs along learning curve.[@CIT0005],[@CIT0006]
Suture material should be permanent. Polypropylene 10--0 is commonly used but it will hydrolyze over time with a roughly 5- to 10-year survival time. Polypropylene 9--0 should have a longer survival time. The interval before degradation of this suture gauge has not been reported. A polytetrafluoroethylene CV-8 suture has been used for scleral fixation off label and to date has had excellent longevity.[@CIT0003]
This described technique is a simple, time sparing and minimally invasive method for achieving good bag centration. It eliminates the need of conjunctival peritomy, subconjunctival vessels cautery and scleral fashioning of a flap, pocket or a groove. The use of 6/0 prolene theoretically can achieve better longevity. However longer period of follow up is required.
Disclosure {#S0005}
==========
The authors report no conflicts of interest in this work.
| {
"pile_set_name": "PubMed Central"
} |
Huang W, Huang C, Ding H, et al. Involvement of miR‐145 in the development of aortic dissection via inducing proliferation, migration, and apoptosis of vascular smooth muscle cells. J Clin Lab Anal. 2020;34:e23028 10.1002/jcla.23028
Wenhui Huang and Cheng Huang contributed equally to this work.
**Funding information**
This work was financially supported by the National Natural Science Foundation of China (81300230), and High‐level Hospital Construction Project (DFJH201807).
1. INTRODUCTION {#jcla23028-sec-0005}
===============
Thoracic aortic dissection (AD) is a fatal disease, including approximately 3 cases per 100 000 individuals per year. Nearly 40% of patients with AD do not have sufficient time to reach a hospital and die immediately. The molecular mechanism underlying AD is not well understood. Hence, its prevention is not possible pharmacologically.[1](#jcla23028-bib-0001){ref-type="ref"}, [2](#jcla23028-bib-0002){ref-type="ref"}, [3](#jcla23028-bib-0003){ref-type="ref"}, [4](#jcla23028-bib-0004){ref-type="ref"} Thoracic AD is associated with laceration and degeneration of aortic media.[5](#jcla23028-bib-0005){ref-type="ref"} Vascular smooth muscle cells (VSMCs) constitute the major cells in the aortic media layer. They are crucial in maintaining aortic wall homeostasis.[1](#jcla23028-bib-0001){ref-type="ref"}, [6](#jcla23028-bib-0006){ref-type="ref"}, [7](#jcla23028-bib-0007){ref-type="ref"} Vascular smooth muscle cells undergo apoptosis and abnormal proliferation in vascular diseases.[8](#jcla23028-bib-0008){ref-type="ref"}, [9](#jcla23028-bib-0009){ref-type="ref"}
MicroRNAs (miRNAs) represent highly conserved short noncoding RNAs. They primarily bind to the 3′‐untranslated regions (3′‐UTRs) of mRNAs, thus serving as posttranscriptional regulators. They are closely associated with diverse cardiovascular diseases. In 2009, a series of studies emphasized the importance of miR143/145 cluster in the phenotypic modulation of smooth muscle cells.[10](#jcla23028-bib-0010){ref-type="ref"}, [11](#jcla23028-bib-0011){ref-type="ref"}, [12](#jcla23028-bib-0012){ref-type="ref"}, [13](#jcla23028-bib-0013){ref-type="ref"}, [14](#jcla23028-bib-0014){ref-type="ref"} The findings demonstrated a reduction in the miR143/145 levels in proliferative conditions. Ning et al showed that miR‐145 modulated the expression of TGF‐β receptor II (TGFBR2) and downstream matrix genes in smooth muscle cells.[15](#jcla23028-bib-0015){ref-type="ref"} However, the causal mechanism of miR‐145 in developing vascular diseases, especially AD, is yet to be identified and has gained immense attention of researchers.
The current work aimed to confirm miR‐145 downregulation in serum and aortic specimens from patients with AD. The study revealed that miR‐145 contributed to the proliferation and apoptosis of VSMCs. Furthermore, the expression of SMAD3 was modulated by transfection with miR‐145 inhibitors or mimics in vitro, indicating the involvement of miR‐145 in the development of AD through targeting SMAD3. Moreover, circulating miR‐145 amounts were remarkably reduced in the AD group in comparison with the non‐AD hypertensive control group. These findings might provide insights into the role of miRNAs in regulating the function of VSMC in AD at the molecular level.
2. MATERIALS AND METHODS {#jcla23028-sec-0006}
========================
2.1. Recruitment of participants and collection of samples {#jcla23028-sec-0007}
----------------------------------------------------------
This study was performed on Stanford type A AD cases (total of 80) and healthy control patients (total of 71) After 12‐14 hours of fasting, 5‐10 mL of whole venous blood was extracted from the elbow vein and centrifuged (3000 *g*, 10 minutes; ambient conditions). The resulting plasma was kept at −80°C until further use. Aortic samples were collected from 6 patients undergoing surgical treatment. The exclusion criteria were as follows: (a) aortic aneurysm, congenital bicuspid aortic valves, Ehlers--Danlos syndrome, Loeys--Dietz syndrome, Marfan syndrome, traumatic dissection, Turner syndrome, or other connective tissue disorders; and (b) age \<18 years. Normal aortic samples were obtained from four deceased organ donors whose cause of death was nonvascular diseases. The samples were collected within 30 minutes following excision. The tissue specimens were rinsed with precooled saline for at least 5 times, followed by the quick removal of thrombus and adventitia. The specimens were cut into 2‐mm‐thick pieces and snap frozen in liquid nitrogen for further use. The time taken to complete this whole procedure was 10 minutes. No additional samples were collected. Table [1](#jcla23028-tbl-0001){ref-type="table"} presents the clinical and demographic characteristics of participants.
######
Clinico‐demographic features of tissue sample donors
+-----------------------+----------------------+----------------+----------------+-----------+
| | Non‐AD control group | AD group | Total | *P‐*value |
| | | | | |
| | (N = 4) | (N = 6) | (N = 10) | |
+=======================+======================+================+================+===========+
| Gender (male), n (%) | 3 (75.0) | 5 (83.3) | 8 (80.0) | .747 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Age (year), mean (SD) | 46.75 (7.50) | 52.67 (10.50) | 50.30 (9.45) | .362 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Hypertension, n (%) | 0 (0.0) | 6 (100.0) | 6 (60.0) | .002 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Dyslipidemia, n (%) | 1 (25.0) | 0 (0.0) | 1 (10.0) | .197 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Diabetes, n (%) | 0 (0.0) | 1 (16.7) | 1 (10.0) | .389 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Smoking, n (%) | 0 (0.0) | 2 (33.3) | 2 (20.0) | .197 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Drinking, n (%) | 0 (0.0) | 1 (16.7) | 1 (10.0) | .389 |
+-----------------------+----------------------+----------------+----------------+-----------+
| AST (U/L), mean (SD) | 71.00 (14.18) | 43.67 (26.82) | 52.78 (26.20) | .150 |
+-----------------------+----------------------+----------------+----------------+-----------+
| ALT (U/L), mean (SD) | 32.75 (17.15) | 32.17 (21.00) | 32.40 (18.52) | .964 |
+-----------------------+----------------------+----------------+----------------+-----------+
| Cr(mmol/L), mean (SD) | 157.24 (102.26) | 106.05 (25.27) | 126.52 (67.38) | .262 |
+-----------------------+----------------------+----------------+----------------+-----------+
John Wiley & Sons, Ltd
This study complied with the Declaration of Helsinki and had approval from the Medical Ethics Committee of Guangdong Provincial People\'s Hospital. Each participant signed an informed consent form before the initiation of the study.
2.2. Hematoxylin and eosin staining {#jcla23028-sec-0008}
-----------------------------------
The aortic samples, after fixation with 10% formalin (procured from Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for 24 hours followed by dehydration in ethyl alcohol series, underwent xylene clearing and deparaffinization. The samples were then submitted to paraffin embedding to produce 4 μm‐thick sections. Upon hematoxylin staining (5 minutes) and differentiation in acid alcohol (1%), the specimens were subsequently stained with eosin (3 minutes), dehydrated with ethyl alcohol series, and washed twice with xylene (10 minutes). Gum Arabic was employed for mounting before observation under an optical microscope (CX‐31 optical microscope).
2.3. Fluorescence in situ hybridization {#jcla23028-sec-0009}
---------------------------------------
After fixation with 4% formaldehyde (10 minutes) and three phosphate‐buffered saline (PBS) washes (5 minutes), aortic specimens underwent permeabilization with 0.5% Triton X‐100 in PBS (10 minutes) at 4°C and further washing with PBS 3 times (5 minutes). The FITC‐labeled miR‐145 probes were incubated with prehybridization buffer (1:100) at 88°C (5 minutes) and 4°C (3 minutes) in a polymerase chain reaction (PCR; Bio‐Rad) instrument. miR‐145 probes labeled with FITC were used to incubate the specimens at 37°C overnight. Subsequently, the specimens were washed (2 × SSC 6 times for 3 minutes) and then stained with DAPI. Following these, the specimens were washed again (2 × SSC 3 times for 5 minutes) and scanned under a laser scanning confocal microscope (Leica TCS‐SP2) as described previously.[16](#jcla23028-bib-0016){ref-type="ref"}
2.4. Isolation of RNA and quantitative reverse transcription--PCR {#jcla23028-sec-0010}
-----------------------------------------------------------------
TRIzol (Invitrogen) was employed for total RNA (comprising small RNAs) extraction from the aortic tissue or cultured VSMCs. The Geneseed II First Strand cDNA Synthesis Kit purchased from Geneseed Biotech was applied to reverse transcribe the RNA thus obtained into complementary DNA (cDNA) following the manufacturer\'s protocols.[9](#jcla23028-bib-0009){ref-type="ref"} Then, reverse transcription (500 ng of total RNA) was carried out with PrimeScript reverse transcription (RT) reagent kit obtained from TaKaRa. Next, quantitative PCR (qPCR) was performed with SYBR Green Master Mix (Geneseed Biotech) on an ABI 7500 real‐time PCR system (ABI) as described previously.[17](#jcla23028-bib-0017){ref-type="ref"}, [18](#jcla23028-bib-0018){ref-type="ref"}
The Primer 5.0 software was used to construct primers in line with gene sequences in GenBank. The primers were validated using Oligo 7 and NCBI BLAST (Molecular Biology Insights) (Table [2](#jcla23028-tbl-0002){ref-type="table"}). Each step involved the use of negative controls with no template. Further, 20 μL of SYBR Green reaction mixture comprised a template (2 μL of cDNA) and primers (10 μmol/L of each). The RT reaction comprised the following steps: 25°C for 10 minutes, 42°C for 15 minutes, and 85°C for 5 minutes; incubation at 95°C (5 minutes); and 40 cycles at 95°C (10 seconds), 60°C (34 seconds) and 60°C (60 seconds). Next, the heat dissociation protocol was applied to the samples, and qRT‐PCR products were assessed with SYBR Green. Whether qRT‐PCR was reliable was evaluated by melting curve analysis using β‐actin as a reference gene. Relative miRNA expression was determined by the 2^−△Cq^ method. This was followed by calculating the mean Cq values and standard deviations in duplicate assays.
######
Sequences of primers
Sequence (5′--3′)
------------------------- -------------------------------------------------------------
SMAD3 F‐1: AGAAGGCTGGGGCTCATTTG
SMAD3 RT‐1: CCTCTTCCGATGTGTCTCCG
Product length: 115 bp
miR145‐5p F‐1: ATGGTTCGTGGGGTCCAGTTTTCCA
miR145‐5p RT‐1: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGGTATTC
Com R: GTGCAGGGTCCGAGGT
Product length: 68 bp
John Wiley & Sons, Ltd
2.5. Western immunoblotting {#jcla23028-sec-0011}
---------------------------
Aortic tissues or VSMCs were lysed using RIPA lysis buffer containing protease inhibitors and Western blotting along with quantification of images were performed as described previously.[18](#jcla23028-bib-0018){ref-type="ref"} RIPA buffer (100 mg/1000 mL) was used to extract total protein from aortic specimens. Phosphate buffered Saline and RIPA buffer (100 μL per well) (Cell Signaling Technology) were used to wash VSMCs 3 times. A cell lifter obtained from Corning Costar was employed for lysing the specimens, with protein concentrations adjusted to 1.0 mg/mL. The lysate (20 μL, or 20 μg) was separated by 6%‐12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and bands were electro‐transferred onto a polyvinylidene difluoride membrane. Nonfat milk (5%) (Becton Dickinson) was used to block the membranes, followed by washing, probing with rat anti‐human SMAD3 (1:1000; Abcam) and anti‐GAPDH (1:500; Abcam) primary antibodies, and incubation with a horseradish peroxidase (HRP) conjugated goat anti‐rat secondary antibodies (1:2000; Sigma) for 60 minutes. Further, an enhanced chemiluminescence kit was used to wash and expose the membrane following the manufacturer\'s protocols (Thermo Fisher). The immunoblots obtained from three rounds of Western blot analyses were assessed on a flatbed scanner, and the intensities were calculated with Image‐Pro Plus (v6.0; Media Cybernetics).
2.6. Luciferase assay {#jcla23028-sec-0012}
---------------------
PCR was used to magnify a specific sequence of SMAD3 gene---3′‐untranslated region (UTR)---harboring miR‐145‐binding sites. For the cloning of the magnified sequence, the pMIR‐REPORT luciferase vector (psiCHECK2‐SMAD3‐wild‐type) was used. The binding site in the aforementioned region of SMAD3 was mutated and inserted into the plasmid psiCHECK2‐SMAD3‐mutant. Lipofectamine 2000 obtained from Invitrogen (Thermo Fisher Scientific) was applied to transfect 293T cells with miR‐145 mimics and luciferase plasmid psiCHECK2‐SMAD3 (wild‐type/mutant, WT/Mut) as directed by the manufacturer. The reporter assay was performed as described previously.[19](#jcla23028-bib-0019){ref-type="ref"}
The cells were cultivated for 48 hours, harvested, and lysed. TransDetect Double‐Luciferase Reporter Assay Kit obtained from TransGen Biotech was applied to assess and standardize the firefly luciferase activity to Renilla luciferase activity. The whole procedure was repeated thrice.
2.7. Culture and transfection of cells {#jcla23028-sec-0013}
--------------------------------------
Primary human arterial smooth muscle cells (HASMCs) isolated from aortic tissues were procured from Geneseed Biotech. They were cultivated in smooth muscle cell medium (Cat. No. 1101; ScienCell) containing fetal bovine serum (FBS; 10%), streptomycin (100 μg/mL), and penicillin (100 U/mL) (Invitrogen) at 37°C in a humid environment with 5% CO~2~. Passage 3‐6 cells were assessed in subsequent experiments. miR‐145 mimics (50nM) or inhibitors (50 nmol/L) (Geneseed Biotech) were employed to transfect HASMCs for 48 hours with Lipofectamine 2000 (Invitrogen) following the manufacturer\'s instructions. All transfections and assays on HASMCs were conducted in low serum medium (1% FBS).
2.8. Transwell HASMC migration assays {#jcla23028-sec-0014}
-------------------------------------
Human arterial smooth muscle cells at 2 × 10^4^ cells/well were seeded in 24‐well plates. After overnight incubation of HASMCs, miR‐145 mimics or inhibitors were used to transfect the cells for 24 hours. Trypsin (2.5%) was used to detach the cells, which were resuspended serum‐free medium at 10^6^/mL. Further, 200 μL of cell suspension and 600 μL of growth medium were placed in the upper and lower chambers of the Transwell and incubated at 37°C for 48 hours. For the migration assay, the 8.0 μm pore filter of the Transwell chamber underwent pre‐treatment with 50 μL Matrigel obtained from BD Biosciences. The cells migrating to the lower side underwent fixation with 10% formalin and crystal violet (0.5%) staining. The cell count was determined for five randomly selected high‐power fields (×200) by light microscopy.
2.9. Cell proliferation assay {#jcla23028-sec-0015}
-----------------------------
Cell Counting Kit‐8 (CCK‐8; Dojindo) was employed for assessing cell proliferation as directed by the manufacturer. The cells were plated in 96‐well plates in M199 medium containing 1% FBS at 5 × 10^3^/well followed by incubation for 24 hours. Further, the cells were treated with adenovirus at the MOI of 120. After every 24 hours, the medium was refreshed. After 0, 12, 24, 36, and 48 hours of transfection, respectively, 10 μL CCK‐8 reagent was supplemented to individual wells, for further incubation at 37°C for 4 hours. Optical density was obtained on a plate reader at 450 nm. Vascular smooth muscle cells serving as negative controls were transfected with adenovirus lacking the miR‐145‐interfering effect. Colonies were counted and analyzed using the Prism software (version 6.00).[20](#jcla23028-bib-0020){ref-type="ref"}
2.10. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay for HASMC viability {#jcla23028-sec-0016}
--------------------------------------------------------------------------------------------------
A total of 5 × 10^4^ HASMCs per well were plated in 96‐well plates. They were grown the whole night and transfected with miR‐145 mimics or inhibitors with Lipofectamine 2000 (Invitrogen) for 24 hours following the manufacturer\'s protocols. Then, cells were cultured for various time points (24, 48, and 72 hours) after replacing the growth medium. MTT (Beyotime) was supplemented at the end of each experiment for 4 hours, following the manufacturer\'s instructions, followed by the replacement of the cell culture medium with 150 μL dimethyl sulfoxide. Upon thorough shaking, optical density at 490 nm was assessed on a microplate reader (Bio‐Rad 680). The data thus obtained were used to plot cell growth curves. The whole procedure was carried out in three sets and repeated at least 3 times.
2.11. Apoptosis assay by flow cytometry {#jcla23028-sec-0017}
---------------------------------------
The adhering HASMCs were trypsinized and submitted to staining with Annexin V‐FITC and propidium iodide (Annexin V--FITC Apoptosis Detection Kit), and screened on a FACScan flow cytometer (DB Biosciences). The experiment was conducted in triplicate. The CellQuest software (BD Biosciences) was used to determine the apoptotic rate.
2.12. Statistical analysis {#jcla23028-sec-0018}
--------------------------
Data are mean ± standard deviation (SD) and were assessed with SPSS 17.0 (SPSS). Independent‐samples *t* test and the Kolmogorov--Smirnov test were used for intergroup comparison and normality assessment, respectively. One‐way analysis of variance (ANOVA) was employed to evaluate differences in continuous numerical parameters with normal distribution between groups. Post‐hoc analyses were carried out by the Tukey\'s test. *P* \< .05 indicated statistical significance.
3. RESULTS {#jcla23028-sec-0019}
==========
3.1. miR‐145 and SMAD3 were largely located in vascular smooth muscle {#jcla23028-sec-0020}
---------------------------------------------------------------------
Vascular smooth muscle cells were less abundant but continuously and normally arranged and medial laceration was observed in aortic specimens from the non‐AD control group (Figure [S1](#jcla23028-sup-0001){ref-type="supplementary-material"}). Next, the localization and expression of SMAD3 and miR‐145 in aortic tissue were examined using immunochemical staining and fluorescence in situ hybridization (FISH), respectively. The findings showed that SMAD3 was mainly located in VSMCs (Figure [1](#jcla23028-fig-0001){ref-type="fig"}A), while miR‐145 was abundantly distributed in the cytoplasm of VSMCs (Figure [1](#jcla23028-fig-0001){ref-type="fig"}B and Figure [S2](#jcla23028-sup-0002){ref-type="supplementary-material"}).
{#jcla23028-fig-0001}
3.2. Specimens of dissected and normal aortic tissues exhibiting differential expression of miR‐145 and SMAD3 {#jcla23028-sec-0021}
-------------------------------------------------------------------------------------------------------------
SMAD3 protein amounts in the human aorta were detected using the Western blot analysis. Figure [2](#jcla23028-fig-0002){ref-type="fig"}A,B show the significantly increased expression level of SMAD3 in AD samples than in NA specimens. The mRNA amounts of miR‐145 and SMAD3 in AD and their matched NA samples were determined using qRT‐PCR to assess miR‐145 expression (Figure [2](#jcla23028-fig-0002){ref-type="fig"}C,D) and SMAD3 in the human aorta, which revealed marked downregulation of miR‐145 and significant SMAD3 upregulation in the AD group. The results suggested the AD group had increased expression levels of SMAD3 in comparison with the non‐AD control group, as opposed to miR‐145.
{#jcla23028-fig-0002}
3.3. Overexpressed miR‐145 promoted the proliferation, migration, and apoptosis of HASMCs {#jcla23028-sec-0022}
-----------------------------------------------------------------------------------------
Human arterial smooth muscle cells underwent transfection with miR‐145 mimics or inhibitors to investigate the involvement of miR‐145 in regulating the biological functions of VSMCs (Figure [S3](#jcla23028-sup-0003){ref-type="supplementary-material"}). The MTT assay revealed miR‐145 downregulation in HASMCs increased the proliferation of HASMCS, while its upregulation significantly suppressed cell proliferation (Figure [3](#jcla23028-fig-0003){ref-type="fig"}A). The involvement of miR‐145 in the migration of HASMCs was determined using the Transwell test. miR‐145 mimics markedly promoted the migratory response of cultured HASMCs. However, cell migration was not affected by miR‐145 downregulation (Figure [3](#jcla23028-fig-0003){ref-type="fig"}B,D). Apoptosis in HASMCs was obviously affected upon transfection with miR‐145 mimics, as detected by flow cytometry (Figure [3](#jcla23028-fig-0003){ref-type="fig"}C,E). These in vitro observations revealed the participation of miR‐145 in HASMC proliferation, migration, and apoptosis.
{#jcla23028-fig-0003}
3.4. miR‐145 regulated the expression of SMAD3 {#jcla23028-sec-0023}
----------------------------------------------
The targets of miR‐145 were predicted using TargetScan and miRanda, aiming to determine the effects of miR‐145 on HASMC proliferation, migration, and apoptosis. The data demonstrated that one of the predicted target genes for miR‐145 was SMAD3. Two putative binding sites were confirmed at 56‐69 nt and 1397‐1403 nt. They were highly conserved across species (Figure [4](#jcla23028-fig-0004){ref-type="fig"}A). Hence, the WT and Mut 3′‐UTRs of SMAD3 were cloned into a luciferase vector, respectively.
{#jcla23028-fig-0004}
The predictions were confirmed using the luciferase reporter assay. The WT and Mut 3′‐UTRs of SMAD3 luciferase reporter vectors were developed. Further, miR‐145 mimics and WT/Mut luciferase vector were used to cotransfect 293T cells, revealing a remarkable impairment of the relative luciferase activity (Figure [4](#jcla23028-fig-0004){ref-type="fig"}B). However, the activity was not altered in 293T cells transfected with Mut luciferase vector (Figure [4](#jcla23028-fig-0004){ref-type="fig"}C). The findings indicated miR‐145 directly bound to the 3′‐UTR of SMAD3.
Human arterial smooth muscle cells underwent transfection with miR‐145 mimics and inhibitors to confirm the direct regulatory activity of miR‐145 on SMAD3. qPCR (Figure [5](#jcla23028-fig-0005){ref-type="fig"}A) and immunoblot (Figure [5](#jcla23028-fig-0005){ref-type="fig"}B,C) were employed to determine SMAD3 mRNA and protein levels, respectively, indicating a reduction in the expression of SMAD3 with the overexpression of miR‐145 in HASMCs. However, SMAD3 mRNA and protein amounts in HASMCs were elevated by miR‐145 inhibitor.
{#jcla23028-fig-0005}
3.5. Circulating miR‐145 amounts are reduced in the AD population {#jcla23028-sec-0024}
-----------------------------------------------------------------
Circulating miR‐145 amounts were determined in 80 AD cases and 71 non‐AD controls, whose clinic‐demographic features demonstrated marked differences between the two groups in gender (66.2% and 94.4%), age (65.12% and 74.4%), smoking rate(18.8% vs 46.5%), as well as incidence of hypertension (75.0% vs 85.9%), dyslipidemia (97.5% vs 18.8%), diabetes (37.5% vs 8.5%) (*P* \< .001, respectively) (Table [3](#jcla23028-tbl-0003){ref-type="table"}). Notably, circulating miRNA‐145 amounts were significantly lower in the AD group in comparison with the non‐AD control group, as expected (Figure [6](#jcla23028-fig-0006){ref-type="fig"}).
######
Clinico‐demographic features of blood sample donors
+------------------------+-----------------+---------------+-----------+
| | Non‐AD group | AD group | *P*‐value |
| | | | |
| | (N = 71) | (N = 80) | |
+========================+=================+===============+===========+
| Gender (male), n (%) | 67 (94.4) | 53 (66.2) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Age (y), mean (SD) | 74.41 (3.53) | 65.12 (10.75) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Hypertension, n (%) | 61 (85.9) | 60 (75.0) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Dyslipidemia, n (%) | 13 (18.8) | 78 (97.5) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Diabetes, n (%) | 6 (8.5) | 30 (37.5) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Smoking, n (%) | 33 (46.5) | 15 (18.8) | \<.001 |
+------------------------+-----------------+---------------+-----------+
| Drinking, n (%) | 2 (2.8) | 4 (5.0) | .493 |
+------------------------+-----------------+---------------+-----------+
| AST (U/L), mean (SD) | 73.14 (393.26) | 30.50 (31.43) | .338 |
+------------------------+-----------------+---------------+-----------+
| ALT (U/L), mean (SD) | 56.32 (270.22) | 35.67 (54.53) | .510 |
+------------------------+-----------------+---------------+-----------+
| Cr (mmol/L), mean (SD) | 123.37 (123.13) | 91.46 (51.74) | .036 |
+------------------------+-----------------+---------------+-----------+
John Wiley & Sons, Ltd
{#jcla23028-fig-0006}
4. DISCUSSION {#jcla23028-sec-0025}
=============
Previous findings indicate a vital role for VSMCs in degenerating the aortic wall, which marks the initiation of pathological remodeling in AD and aneurysms.[21](#jcla23028-bib-0021){ref-type="ref"} The present study revealed prominent signs of aortic medial laceration in AD and emphasized the participation of VSMCs in the development of AD. Importantly, several miRNAs were involved in regulating VSMC phenotypes. Previous studies indicated a role of miR‐145 in modulating smooth muscle differentiation.[22](#jcla23028-bib-0022){ref-type="ref"} Smooth muscle cells received miR‐145 from exosomes excreted by endothelial cells.[23](#jcla23028-bib-0023){ref-type="ref"} Further, miR‐145 was found to have an atheroprotective role.[21](#jcla23028-bib-0021){ref-type="ref"}
However, a few studies identified a function for miR‐145 in AD pathogenesis. Hence, this study intended to determine the pattern of expression of miR‐145 in AD and also its participation in the biological functions of VSMCs. The findings indicated the location of miR‐145 in VSMCs and significant downregulation of its expression levels in AD settings. The study further investigated the significance and potential impact of miR‐145 dysregulation on proliferation, migration, and apoptosis in HASMCs using functional assays. The results showed that miR‐145 depletion induced proliferation and migration in VSMCs. However, the overexpression of miR‐145 using specific mimics suppressed the proliferation and apoptosis of VSMCs. The levels of circulating miRNA‐145 also markedly reduced in patients with AD compared with non‐AD hypertensive controls, further substantiating the association of miR‐145 with AD, and indicating that miRNA‐145 could be novel serum biomarkers for AD.
Abnormal proliferation and migration of VSMCs are the major contributing factors for pathological vascular remodeling through undermining vasculature stability.[24](#jcla23028-bib-0024){ref-type="ref"} Müller et al and Wang et al demonstrated the more rapid proliferation of VSMCs from dissected aorta compared with VSMCs from normal aortic tissues. Moreover, the expression levels of genes involved in proliferation were elevated.[25](#jcla23028-bib-0025){ref-type="ref"}, [26](#jcla23028-bib-0026){ref-type="ref"} On the contrary, VSMCs showed signs of apoptosis in AD settings.[27](#jcla23028-bib-0027){ref-type="ref"}, [28](#jcla23028-bib-0028){ref-type="ref"}, [29](#jcla23028-bib-0029){ref-type="ref"}, [30](#jcla23028-bib-0030){ref-type="ref"}
The above in vitro findings provided valid evidence that miR‐145 knockdown promotes the proliferation, migration, and apoptosis of VSMCs, which implied that miR‐145 is vital in the pathogenesis and development of AD.
Further, the results confirmed SMAD3 as a direct miR‐145 target in VSMCs. SMADs are pivotal elements among the intracellular effectors of TGF‐β1. They regulate various biological activities, including cell proliferation, apoptosis, migration, differentiation, and adhesion.[31](#jcla23028-bib-0031){ref-type="ref"} Dysregulated TGF‐β signaling is crucial in the pathogenesis of AD.[32](#jcla23028-bib-0032){ref-type="ref"} Mutations in genes encoding the TGF‐β‐signaling components, including FBN1, TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, and SMAD3, have been detected in thoracic AD.[33](#jcla23028-bib-0033){ref-type="ref"} Of note, SMAD3 mutations are found in families with TAAD in an autosomal dominant manner.[34](#jcla23028-bib-0034){ref-type="ref"} The findings indicated the dissected aorta had upregulated expression of SMAD3 in comparison with non‐AD tissues. Therefore, in vitro assays were carried out to better elucidate the relationship between miR‐145 and SMAD3 in VSMCs. The findings revealed an altered expression of SMAD3 due to the abnormal expression of miR‐145 in VSMCs, implying a direct regulatory effect of miR‐145 on SMAD3.
This study had several limitations. First, the sample size was relatively small. Therefore, large‐scale studies should be performed on tissue and plasma specimens of AD to further validate the findings. Second, the association of miRNA‐145 with VSMCs in aortic wall degeneration deserves further investigation. Third, the present study shows that SMAD3 is a target of miR‐145, hence in vitro studies will be needed to perform to address whether overexpression or knockdown of SMAD3 will affect the effect of miR‐145 on proliferation, migration, and apoptosis of VSMCs. Finally and notably, the similar expression pattern of miR‐145 has been observed in aortic tissues and circulating‐cell free miRNAs are relatively stable in blood due to the protection by their inclusion in exosomes, therefore, the interaction between miR‐145 and exosomes and the detailed mechanisms remains unclear. All mentioned above limited the role of miR‐145 as a biomarker in the prognosis of AD. These shortcomings should be addressed by performing prospective AD cohort and by using molecular, cell, animal, as well as human approaches.
Overall, the study showed the downregulation of miR‐145 and the upregulation of SMAD3 in AD tissues. The miR‐145 ectopic expression promoted the proliferation and apoptosis of VSMCs. However, miR‐145 suppressed the expression of SMAD3 through targeting the 3′‐UTR of SMAD3. The findings of this study indicated the involvement of miR‐145 in AD pathogenesis through direct targeting of SMAD3, thereby providing novel insights into the effective clinical management of AD.
CONFLICT OF INTEREST {#jcla23028-sec-0026}
====================
The authors do not have a conflict of interest.
Supporting information
======================
######
######
Click here for additional data file.
######
######
Click here for additional data file.
######
######
Click here for additional data file.
The authors acknowledge the efforts and contributions of all the participating volunteers.
| {
"pile_set_name": "PubMed Central"
} |
Editor: Michael Grabe.
Introduction {#sec1}
============
Much of biology depends on the voltage across cell membranes. The voltage across the membrane must be sensed before it can be used by proteins. Permanent charges move in the strong electric fields within membranes, so carriers of sensing charge were proposed as voltage sensors even before membrane proteins were known to span lipid membranes ([@bib1]). The movement of permanent charges of the voltage sensor is gating current, and the movement is the voltage-sensing mechanism. Permanent charge is our name for a charge or charge density independent of the local electric field (for example, the charge and charge distribution of Na^+^ but not the charge in a highly polarizable anion like Br^−^ or the nonuniform charge distribution of H~2~O in the liquid state with its complex time dependent (and perhaps nonlinear) polarization response to the local electric field).
Knowledge of membrane protein structure has allowed us to identify and look at the atoms that make up the voltage sensor. Protein structures do not include the membrane potentials and macroscopic concentrations that power gating currents, and therefore, simulations are needed. Atomic-level simulations like molecular dynamics (MD) do not provide an easy extension from the atomic timescale ∼10^−15^ s to the biological timescale of gating currents that starts at ∼10^−6^ s and reaches ∼10^−2^ s. Calculations of gating currents from simulations must average the trajectories (lasting ∼10^−1^ s sampled every 10^−15^ s) of ∼10^6^ atoms, all of which interact through the electric field to conserve charge and current while conserving mass. It is difficult to enforce continuity of current flow in simulations of atomic dynamics because simulations compute only local behavior, whereas continuity of current is global, involving current flow far from the atoms that control the local behavior. It is impossible to enforce continuity of current flow in calculations that assume equilibrium (zero net flow) under all conditions.
A hybrid approach is needed, starting with the essential knowledge of structure but computing only those parts of the structure used by biology to sense voltage. In close-packed ("condensed") systems like the voltage sensor or ionic solutions, "everything interacts with everything else" because electric fields are long ranged as well as exceedingly strong ([@bib2]). In ionic solutions, ion channels, even enzyme active sites, steric interactions that prevent the overfilling of space in well-defined protein structures are also of great importance because they produce short-range correlations ([@bib3]).
Closely packed charged systems are well handled mathematically by energy variational methods. Energy variational methods guarantee that all variables satisfy all equations (and boundary conditions) at all times and under all conditions and are thus always consistent. We use the energy variational approach developed in ([@bib4]) and ([@bib5]) to derive a consistent model of gating charge movement, based on the basic features of the structure of crystallized voltage-sensitive channels. A schematic of the model is shown below. The continuum model we use simulates the mechanical dynamics in a single voltage sensor, although the experimental data is from many independent voltage sensors. Ensemble averages of recordings of individual independent voltage sensors are equivalent to macroscopic continuum modeling in a single voltage sensor if correlations are captured correctly in the model of the single voltage sensor.
Materials and Methods {#sec2}
=====================
Theory: Mathematical model {#sec2.1}
--------------------------
The reduced mechanical model for a voltage sensor is shown in [Fig. 1](#fig1){ref-type="fig"} *a* with four arginines (*R*~*i*~, *i* = 1, 2, 3, 4), each attached to the S4 helix by identical springs with the same spring constant *K*. The electric field will drag these four arginines because each arginine carries +1 charge. The charged arginines can also move as a group. S4 connects to S3 and S5 at its two ends by identical springs with spring constant *K*~S4~/2.Figure 1(*a*) Geometric configuration of gating pore in this model, including the attachments of arginines to the S4 segment. (*b*) Following (*a*), an axisymmetric three-zone domain shape is designated in *r*-*z* coordinate for the current 1D model. Here, the diameter of the hydrophobic plug is 0.3 nm (arginine's diameter); *L* = 0.7 nm; *L*~*R*~ = 1.5 nm; and the radius of the vestibule is *R* = 1 nm. BC means boundary condition. To see this figure in color, go online.
Once the membrane is depolarized from, for example, −90 mV inside negative to +10 mV inside positive, arginines together with S4 will be driven toward the extracellular side. A repolarization from +10 to −90 mV moves the arginines back to the intracellular side. This movement is the basic voltage-sensing mechanism. The movement of S4 triggers the opening or closing of the lower gate---consisting mainly of S6 forming the ion permeation channel---by a mechanism widely assumed to be mechanical, although electrical aspects of the linker motion are likely to be involved as well.
When arginines are driven by an electric field, they are forced to move through a hydrophobic plug composed of several nonpolar amino acids from S1, S2, to S3 ([@bib6]). Arginines reside initially in the hydrated lumen of the intracellular vestibule. They then move though the hydrophobic plug and wind up in the vestibule on the extracellular side. This movement involves dehydration when the arginines move through the hydrophobic plug, in which the arginines encounter a barrier in the potential of mean force (PMF), mainly dominated by the difference of the solvation energy in bulk situation and in the hydrophobic plug ([@bib7]). Note that Na^+^ and Cl^−^ (which are the only ions in the bulk solution in this article for simplicity) are found only in vestibules and are not allowed into the hydrophobic plug in our model. The ends of the two vestibules on each side of the hydrophobic plug act as impermeable walls for Na^+^ and Cl^−^ in our model. When the voltage is turned on and off, these two walls store/release charge (carried by ions) in their electric double layers (EDL) that have many of the properties of capacitors.
In this continuum model, the four arginines (*R*~*i*~, *i* = 1, 2, 3, 4) are described by their individual density distributions (concentrations) (*c*~*i*~, *i* = 1, 2, 3, 4), allowing the arginines to interact with Na^+^ and Cl^−^ in vestibules. The density (i.e., concentration) distributions represent probability density functions as shown explicitly in the theory of stochastic processes used to derive such equations in ([@bib8]) using the general methods of ([@bib9]). The important issue here is how well the correlations are captured in the continuum model. Some are more likely to be faithfully captured in molecular or coarse-grained dynamics simulations (e.g., more or less local hard sphere interactions) ([@bib10], [@bib11], [@bib12], [@bib13], [@bib14]) and others in continuum models (e.g., correlations induced by far-field boundary conditions like the potentials imposed by bath electrodes to maintain a voltage clamp) ([@bib15], [@bib16], [@bib17], [@bib18]).
Here, we treat the S4 itself as a rigid body, so we can capture the basic mechanism of a voltage sensor without considering the full details of structure, which might lead to a three-dimensional model difficult to compute in reasonable time. We construct an axisymmetric one-dimensional (1D) model with a three-zone geometric configuration illustrated in [Fig. 1](#fig1){ref-type="fig"} *b*, following [Fig. 1](#fig1){ref-type="fig"} *a*. Zone 1 with *z* ∈ \[0, *L*~*R*~\] is the intracellular vestibule; zone 2 with *z* ∈ \[*L*~*R*~, *L*~*R*~ + *L*\] is the hydrophobic plug; zone 3 with *z* ∈ \[*L~R~* + *L*, 2*L*~*R*~ + *L*\] is the extracellular vestibule. Arginines, Na^+^, and Cl^−^ can all reside in zone 1 and 3. Zone 2 only allows the residence of arginines, albeit with a severe hydrophobic penalty because of their permanent charge, in a region of low dielectric coefficient, hence called hydrophobic.
Based on [Fig. 1](#fig1){ref-type="fig"} *b*, the governing 1D dimensionless Poisson-Nernst-Planck (PNP)-steric equations are expressed below with the detailed nondimensionalization process shown in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S1. The first one is a Poisson equation that shows how charge creates potential:$$- \frac{1}{A}\frac{d}{dz}\left( {\mathit{\Gamma}A\frac{d\phi}{dz}} \right) = \sum\limits_{i = 1}^{N}{q_{i}c_{i}},\ \ \ i = \text{Na},\text{Cl},1,2,3,4,$$where *ϕ* is electric potential; *c*~*i*~ is concentration of species *i* with valence *q*~*Na*~ = 1, *q*~*Cl*~ = −1, *q*~*i*~ = *q*~*arg*~ = 1, *i* = 1, 2, 3, 4; $\mathit{\Gamma} = {\lambda_{D}^{2}/R^{2}}$ with $\lambda_{D} = \sqrt{{\varepsilon_{r}\varepsilon_{0}k_{B}T}/{c_{0}e^{2}}}$ being the Debye length, and the characteristic length (radius of vestibule) *R* = 1 nm here. *A*(*z*) is the channel cross-sectional area at position *z*. For zones 1 and 3, *Γ* = 1 by setting NaCl bulk concentration *c*~*0*~ = 184 mM and *ε*~*r*~ = 80. For zone 2, we assume a hydrophobic environment with *ε*~*r*~ = 8 and therefore *Γ* = 0.1. The value of the dielectric constant inside the hydrophobic plug (zone 2) is not experimentally available; however, the computational result is not sensitive to this value based on our sensitivity analysis.
The second equation is the species transport equation based on conservation laws:$$\frac{\partial c_{i}}{\partial t} + \frac{1}{A}\frac{\partial}{\partial z}\left( {AJ_{i}} \right) = 0,\ \ \ \ i = \text{Na},\text{Cl},1,2\ ,3,4,$$with the content of flux *J*~*i*~ expressed below based on the Nernst-Planck equation for Na^+^ and Cl^−^:$$J_{i} = - D_{i}\left( {\frac{\partial c_{i}}{\partial z} + c_{i}q_{i}\frac{\partial\phi}{\partial z}} \right),\ i = \text{Na},\text{Cl},\ z\ \text{in}\ {zone}\ 1\ {and}\ 3,$$and for four arginines *c*~*i*~, *i* = 1, 2, 3 and 4 based on the Nernst-Planck equation with steric effect and some imposed potentials:$$J_{i} = - D_{i}\left( {\frac{\partial C_{i}}{\partial z} + q_{arg}c_{i}\frac{\partial\phi}{\partial z} + c_{i}\left( {\frac{\partial V_{i}}{\partial z} + \frac{\partial V_{b}}{\partial z}} \right) + gc_{i}\sum\limits_{j \neq i}\frac{\partial c_{j}}{\partial z}} \right),\ z\ {in}\ \text{all}\ \text{zones},$$where *D*~*i*~ is the diffusion coefficient for species *i*.
The first and second terms in [Eqs. 3](#fd3){ref-type="disp-formula"} and [4](#fd4){ref-type="disp-formula"} describe diffusion and electromigration, respectively. The third terms in [Eq. 4](#fd4){ref-type="disp-formula"} are external potential terms with *V*~*i*~, *i* = 1, 2, 3, and 4 being the constraint potential for the four arginines *c*~*i*~ to S4, represented here by a spring connecting each arginine *c*~*i*~ to S4, as shown in [Fig. 1](#fig1){ref-type="fig"} *a*. Governing equations [Eqs. 1](#fd1){ref-type="disp-formula"}, [2](#fd2){ref-type="disp-formula"}, [3](#fd3){ref-type="disp-formula"}, and [4](#fd4){ref-type="disp-formula"} were derived by energy variational methods, which is further shown in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S3.
The elastic system is described by$$V_{i}\left( {z,t} \right) = K\left( {z - \left( {z_{i} + Z_{S4}\left( t \right)} \right)} \right)^{2},$$where *K* is the spring constant, *z*~*i*~ is the fixed anchoring position of the spring for each arginine *c*~*i*~ on S4, and *Z*~*S*4~(*t*) is the center-of-mass *z* position of S4 by treating S4 as a rigid body. Here, we set *z*~1~ = 0.6, *z*~2~ = 0.2, *z*~3~ = −0.2, and *z*~4~ = −0.6 using structural information that gives the arginine anchoring interval on S4 as 0.4 nm. *Z*~*S*4~(*t*) follows the motion of equation based on the spring-mass system:$$m_{S4}\frac{d^{2}Z_{S4}}{dt^{2}} + b_{S4}\frac{dZ_{S4}}{dt} + K_{S4}\left( {Z_{S4} - Z_{S4,0}} \right) = \sum\limits_{i = 1}^{4}{K\left( {z_{i,CM} - \left( {z_{i} + Z_{S4}} \right)} \right)},$$where *m*~*S*4~, *b*~*S*4~, and *K*~*S*4~ are the mass, damping coefficient, and restraining spring constants for S4. *Z*~*S*4,0~ is the resting position of *Z*~*S*4~(*t*). Here, *z*~*i,CM*~ is the center of mass for the set of arginines *c*~*i*~, which can be calculated by$$z_{i,CM} = \frac{\int_{0}^{L + 2LR}{A\left( z \right)zc_{i}dz}}{\int_{0}^{L + 2LR}{A\left( z \right)c_{i}dz}},i = 1,\ 2,\ 3,\ 4.$$
We assume that the spring-mass system for S4 is overdamped, which means the inertia term in [Eq. 6](#fd6){ref-type="disp-formula"} can be neglected.
The energy barrier *V*~*b*~ in [Eq. 4](#fd4){ref-type="disp-formula"} is nonzero only in zone 2, which mainly represents the difference in solvation energy, chiefly characterized by the difference of dielectric constants, in the hydrophobic plug and bulk solution. The structure of the energy barrier is actually very complicated. Here, we simply assume a hump shape for PMF (see more in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S2), although we will seek greater realism in later work.
The last term in [Eq. 4](#fd4){ref-type="disp-formula"} is the steric term that accounts for steric interaction among arginines ([@bib5], [@bib19]). Here, we set *g* = 0.5, a reasonable value. Though there is actually no experimental measurement available for *g*, the computation results have been verified to be insensitive to its value.
Here, we assume quasisteady state for Na^+^ and Cl^−^, which means $\partial c_{i}/\partial t = 0,\ i = \text{Na},\text{Cl},$ in [Eq. 2](#fd2){ref-type="disp-formula"}, and the reasons are elaborated in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S4. The formulation of boundary and interface conditions is also shown in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S5.
Besides the main input parameter *V*, which is the applied voltage bias (corresponding to the command potential in voltage-clamp experiments), other parameters like *D*~*i*~ (*i =* 1, 2, 3, 4), *K*, *K*~*S4*~, and *b*~*S4*~ are also required. Results are especially sensitive to the values of *K*, *K*~*S4*~, and *b*~*S4*~. We have tried and found *D*~*i*~ = 50; *i =* 1, 2, 3, and 4; *K* = 3; *K*~*S4*~ = 3; and *b*~*S4*~ = 1.5 provide the best fit to the experimental Q(charge)-voltage (QV) curve reported in ([@bib20]). Some additional explanation on fitting these parameter values is described in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S6.
Usually, the electric current in the ion channel is treated simply as the flux of charge and is uniform in the *z* direction when steady in time. This is not so in this nonsteady dynamic situation because the storing and releasing of charge in vestibules is involved. Here, the flux of charge at the middle of hydrophobic plug, *z* = *L*~*R*~ + *L*/2, was computed to estimate the experimentally observed gating current. However, it is actually impossible (so far) to experimentally measure the current at the middle of the hydrophobic plug. In experiments, the voltage-clamp technique is used, and on/off gating current through the membrane is measured, which should be equal to the flux of charge at *z* = 0 in this framework, as shown in [Fig. 1](#fig1){ref-type="fig"} *b*. The flux of charges at any *z* position *I*(*z*, *t*) can be related to the flux of charges at *z* = 0, *I*(*0*, *t*), simply by charge conservation:$$\frac{\partial}{\partial t}Q_{net}\left( {z,t} \right) = I\left( {0,t} \right) - I\left( {z,t} \right),$$where$$Q_{net}\left( {z,t} \right) = {\int\limits_{0}^{z}{A\left( \xi \right)\sum\limits_{all\ i}{q_{i}c_{i}d\xi}}},$$and flux of charges at any *z* position *I*(*z*, *t*) is defined by$$I\left( {z,t} \right) = A\left( z \right)\sum\limits_{all\ i}{q_{i}J_{i}\left( {z,t} \right)}.$$
We identify ${\partial/{\partial t}}Q_{net}\left( {z,t} \right)$ as the displacement current and denote it as *I*~*disp*~ (*z*, *t*) because [Eq. 8](#fd8){ref-type="disp-formula"} is equivalent to Ampere's law in Maxwell's equations, and $\partial/\partial t\left( {Q_{net}\left( {z,t} \right)} \right)$ is exactly the displacement current in Ampere's law. The proof is elaborated on in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S7. A general discussion about displacement current can be found in ([@bib21], [@bib22], [@bib23]), which does not involve assumptions concerning the dielectric coefficient *ε*~*r*~ or polarization properties of matter at all. Hence, [Eq. 8](#fd8){ref-type="disp-formula"} can be simply rewritten as$$I_{tot}\left( {z,t} \right) = I\left( {z,t} \right) + I_{disp}\left( {z,t} \right) = I\left( {0,t} \right),$$where we define the sum of displacement current and flux of charges as the total current *I*~*tot*~ (*z*, *t*). The *z* distribution of the total current should be uniform by Kirchhoff's law, and we verify this by computations shown in the section under heading [Flux of Charges at Different Locations](#sec3.3){ref-type="sec"}. Note the ionic current *I*(*z*, *t*) changes a great deal with location. The displacement current *I*~*disp*~(*z*, *t*) varies a great deal with location. The total current, the sum *I*~*tot*~(*z*, *t*), does not vary at all with location, although of course it varies a great deal with time. For example, calculations of current in the baths (which are not reported here) would show only ionic current in the time range considered here, but it would equal the total current that flows anywhere in our 1D model of the voltage sensor domain.
We are also interested in observing the net charge at vestibules. Consider, for example, the net charge at the intracellular vestibule, *Q*~*net*~(*L*~*R*~, *t*). The net charge consists of arginine charges and their countercharges formed by the EDL of ionic solution in that location. Electroneutrality is approximate but will not be exact there. Flux of charge, displacement current, and net charge at vestibules will be discussed further in the section under heading [Flux of Charges at Different Locations](#sec3.3){ref-type="sec"}.
To evaluate the current theoretical model, it is important to compare our computational results with experimental measurements ([@bib20]) in the curves of gating current and amount of gating charge moved versus applied voltage (I(current)-voltage \[IV\] and QV curves). To construct the QV curve, we calculate $Q_{1} = {\int_{0}^{L_{R}}{A\left( z \right)\sum_{i = 1}^{4}{c_{i}dz}}},\ $ $Q_{2} = {\int_{L_{R}}^{L_{R} + L}{A\left( z \right)\sum_{i = 1}^{4}{c_{i}dz}}}$, $Q_{3} = {\int_{L_{R} + L}^{2L_{R} + L}{A\left( z \right)\sum_{i = 1}^{4}{c_{i}dz}}}$, which are the amounts of arginine found in zone 1, 2, and 3, respectively. Usually *Q*~2~ ≈ 0 is due to the energy barrier *V*~*b*~ in zone 2. Arginines tend to jump across zone 2 when driven from zone 1 to zone 3 as the voltage *V* is turned on. The number of arginines that move and settle at zone 3 depends on the magnitude of *V*. Besides IV and QV curves, the time course of the movement of arginines and S4, *z*~*i,CM*~(*t*) and *Z*~*S*4~(*t*), is important to report here because recording these movements in experiments is becoming feasible nowadays by optical methods. Many qualitative models accounting for the movement of S4 and conformation change of the voltage sensor have been proposed. Readers are referred to review articles ([@bib24], [@bib25]) for more details.
Numerical method {#sec2.2}
----------------
[Equations 1](#fd1){ref-type="disp-formula"}, [2](#fd2){ref-type="disp-formula"}, [3](#fd3){ref-type="disp-formula"}, and [4](#fd4){ref-type="disp-formula"} are first discretized in space by high-order multiblock Chebyshev pseudospectral methods and then integrated in time under the framework of method of lines. The details of the numerical method are referred to [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S8.
Results and Discussion {#sec3}
======================
Here, numerical results based on the mathematical model described above were calculated and compared with experimental measurements ([@bib20]). Our 1D continuum model has advantages and disadvantages. The lack of three-dimensional structural detail means that some details of the gating current and charge cannot be reproduced. It should be noted, however, that to reproduce those, one needs more than just static structural detail. One must also know how the structures (particularly their permanent and polarization charge) move and change after a command potential is applied in the experimental ionic conditions. The 1D model has advantages because it computes the actual experimental results on the actual experimental timescale in realistic ionic solutions and with far-field boundary conditions actually used in voltage-clamp experiments. It also conserves total current, as we will demonstrate later. Conservation of current needs to be there and verified in theories and simulations because it is a universal property of the Maxwell equations ([@bib21], [@bib22], [@bib23]).
QV curve {#sec3.1}
--------
When the membrane and voltage sensor is held at a large inside negative potential (e.g., hyperpolarized to −90 mV), S4 is in a resting potential position, and all arginines stay in the intracellular vestibule. When the potential is made more positive (e.g., depolarized to +10 mV), S4 is in the active potential position, and all arginines are at the extracellular vestibule.
The voltage dependence of the charge (arginines) transferred from intracellular vestibule to extracellular vestibule is characterized as a QV curve in experimental papers, and it is sigmoidal in shape ([@bib20]). [Fig. 2](#fig2){ref-type="fig"} *a* shows that our computed QV curve---the dependence of *Q*~3~ on *V*---is in very good agreement with the experiment ([@bib20]). This good agreement comes from the fact that our resultant QV curve is also a sigmoidal curve, and, most important of all, the slope of QV curve can be tuned, mainly by the adjustment of *K*, *K*~*S4*~, and *b*~*S4*~, to agree with experiment. Not many theoretical models can achieve this agreement, especially for the slope. Models in ([@bib15], [@bib16]) show good agreement with experiments, whereas a mismatch of slope was reported in ([@bib17], [@bib18]). The voltage dependence of activation has been considered a crucial property of the sodium conductance since it was defined ([@bib1]). [Fig. 2](#fig2){ref-type="fig"} *b* shows the steady-state distributions of Na^+^, Cl^−^, and arginines in the inside negative, hyperpolarized situation (*V* = −90 mV). As we can see, all the arginines stay in the intracellular vestibule, and none of the arginines move to the extracellular vestibule (*Q*~3~ ≈ 0).Figure 2(*a*) QV curve and comparison with ([@bib20]). Steady-state distributions for Na^+^, Cl^−^, and arginines are shown at (*b*) *V* = −90 mV, (*c*) *V* = −48 mV, and (*d*) *V* = −8 mV. Note that the experimental data in ([@bib20]) were scaled to 4e. To see this figure in color, go online.
[Fig. 2](#fig2){ref-type="fig"} *c* shows the situation at *V* = −48 mV, which is the midpoint of the QV curve. As we can see, each vestibule has distributions of *c*~*i*~ (*i* = 1, 2, 3, and 4), resulting in half of the arginines staying in it (*Q*~3~ = 2). The center-of-mass position for each arginine, presented later in [Fig. 6](#fig6){ref-type="fig"}, shows that R1 and R2 are in the extracellular vestibule, and R3 and R4 are in the intracellular vestibule. There are almost no arginines in zone 2 (hydrophobic plug) because of the energy barrier in it. Note that this represents an average because in a single molecule interpretation, half of the sensors will be with all R's inside and the other half with all R's outside. The midpoint of −48 mV from ([@bib20]) requires the resting position of S4, *Z*~*S*4,0~, to be biased from *L*~*R*~ + 0.5*L* to *Z*~*S*4,0~ = *L*~*R*~ + 0.5*L* + 1.591 nm; otherwise, the midpoint would be 0 mV. [Fig. 2](#fig2){ref-type="fig"} *d* shows the situation at full depolarization (*V* = −8 mV), at which time all arginines move to the extracellular vestibule (*Q*~3~ ≈ 4) in the fully depolarized, activated state.
Gating current {#sec3.2}
--------------
[Fig. 3](#fig3){ref-type="fig"} shows the time course of gating currents, observed as flux of charge at the middle of hydrophobic plug *I*(*L*~*R*~ + *L*/2, *t*) because of the movement of arginines when the membrane depolarization is large and when the depolarization is small. In the case of large depolarization, *V* rises from −90 mV at *t* = 10 to −8 mV and drops back to −90 mV at *t* = 150 ([Fig. 3](#fig3){ref-type="fig"} *a*). The time course of gating current and contributions of individual arginines are shown in [Fig. 3](#fig3){ref-type="fig"} *b*. As expected, the rising order of each current component follows the moving order of R1, R2, R3, and R4 when depolarized and that order is reversed when repolarized. The area under the gating current is the amount of charge moved. Because arginines move forward and backward in this depolarization/repolarization scenario, the areas under the ON current and the OFF current are same. The areas are equal for each component of current as well. The equality of area is an important signature of gating current that contrasts markedly with the properties of ionic current ([@bib26], [@bib27]). In the case of small depolarization (*V* rises from −90 to −50 mV at *t* = 10 to and drops back to −90 mV at *t* = 150, [Fig. 3](#fig3){ref-type="fig"} *c*), the time course of gating current and its four components contributed by each arginine for this situation is shown in [Fig. 3](#fig3){ref-type="fig"} *d*. Under this small depolarization, not all arginines move past the middle of the hydrophobic plug because of the weaker driving force in the small depolarization compared with the large depolarization case. This can be inferred because the areas under each component current are different ([Fig. 3](#fig3){ref-type="fig"} *d*).Figure 3(*a*) The time course of *V* rising from −90 to −8 mV at *t* = 10 holds on until *t* = 150 and then drops back to −90 mV. (*b*) The time course of gating current, *I*(*L*~*R*~ + *L*/2, *t*), and its components corresponding to (*a*) are shown. (*c*) The time course of *V* rising from −90 to −50 mV at *t* = 10 holds on until *t* = 150 and then drops back to −90 mV. (*d*) The time course of gating current, *I*(*L*~*R*~ + *L*/2, *t*), and its components corresponding to (*c*) are shown. To see this figure in color, go online.
The gating currents can be better understood by looking at a sequence of snapshots showing the spatial distribution of electric potential, species concentration, and electric current. The distributions at several times are shown in [Fig. 4](#fig4){ref-type="fig"} *a* for the case of sudden change in command voltage to a more positive value and a large depolarization, and the distributions are shown in [Fig. 4](#fig4){ref-type="fig"} *b* for the case of a small depolarization. The electric potential profiles at *t* = 13 and *t* = 148 show that the profile of electric potential changes as arginines move from left to right even though the voltage is maintained constant across the sensor. Slight bulges in electric potential profile exist wherever arginines are dense. This can be easily explained by understanding the effect of [Eq. 1](#fd1){ref-type="disp-formula"} on a concave spatial distribution of electric potential.Figure 4(*a*) The top row shows dimensionless species concentration distributions at *t* = 0, 13 (right after depolarization), and 148 (right before repolarization) for the case of large depolarization with *V* from −90 mV at *t* = 10 to −8 mV and dropping back to −90 mV at *t* = 150. The middle row shows concurrent electric potential profiles. The bottom row shows concurrent electric current profiles with the components of flux of charge, displacement current, and total current. (*b*) The same as (*a*) is shown except with *V* depolarized from −90 to −50 mV. To see this figure in color, go online.
In [Fig. 4](#fig4){ref-type="fig"}, the total current defined in [Eq. 11](#fd11){ref-type="disp-formula"}, though changing with time, is always constant in *z* at all times, satisfying Kirchhoff's law (i.e., conservation of current). At *t* = 13, when gating current is substantial, as seen from *t* = 13 in [Fig. 3](#fig3){ref-type="fig"}, *b* and *d*, we can visualize the *z* distributions of flux of charges *I*(*z*, *t*), displacement of current *I*~*disp*~(*z*, *t*), and total current *I*~*tot*~(*z*, *t*) individually in [Fig. 4](#fig4){ref-type="fig"}.
Flux of charges at different locations {#sec3.3}
--------------------------------------
Flux of charges *I*(*z*, *t*), together with displacement current *I*~*disp*~(*z*, *t*) and total current *I*~*tot*~(*z*, *t*), depicted in [Fig. 4](#fig4){ref-type="fig"}, deserve more discussion here. Though *I*(*z*, *t*), *I*~*disp*~(*z*, *t*), and *I*~*tot*~(*z*, *t*) are well defined in [Eqs. 8](#fd8){ref-type="disp-formula"}, [9](#fd9){ref-type="disp-formula"}, [10](#fd10){ref-type="disp-formula"}, and [11](#fd11){ref-type="disp-formula"}, the actual computation of them takes an indirect path because of the assumption of quasisteady state for Na^+^ and Cl^−^ in [Eq. 2](#fd2){ref-type="disp-formula"}. The details are presented in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S9. The computed total current *I*~*tot*~(*z*, *t*) does indeed satisfy Kirchhoff's law by its uniformity in *z*. This verification is shown in [Fig. 4](#fig4){ref-type="fig"} at several times, and we have checked that this is in fact true at any time.
In the bottom rows of [Fig. 4](#fig4){ref-type="fig"} at *t* = 13, we observe that *I*(*z*, *t*) is generally nonuniform in *z* and is accompanied by congestion/decongestion of arginines in between. However, *I*(*z*, *t*) is almost uniform in zone 2 (hydrophobic plug), which means almost no congestion/decongestion of arginines occurs there, and therefore, there is no contribution to the displacement current ${d/{dt}}Q_{net}\left( {z,t} \right)$ from zone 2. This is because arginines can hardly reside in zone 2 because of the energy barrier in it.
Several things are worth noting in the time courses of $I\left( {L_{R} + {L/2},t} \right)$ and *I*(0, *t*) (equal to uniformly distributed *I*~*tot*~ as depicted by [Eq. 11](#fd11){ref-type="disp-formula"}) illustrated in [Fig. 5](#fig5){ref-type="fig"} *a* under the case of large depolarization. First, $I\left( {L_{R} + {L/2},t} \right)$ is noticeably larger than *I*(0, *t*) in the ON period. This is because their difference, exactly the displacement current *I*~*disp*~, is always negative at zone 2 when depolarized because arginines are leaving zone 1 and make ${d/{dt}}Q_{net}$ \< 0 for zone 2. It is expected that the area under the time course of $I\left( {L_{R} + {L/2},t} \right)$ would be very close to 4e, as verified by the time courses of *Q*~3~ in [Fig. 5](#fig5){ref-type="fig"} *b*. We use *I*(0, *t*) to estimate the experimentally measured voltage-clamp current, whereas the counterpart area of experimentally measurable *I*(0, *t*) would be less than 4e because of its smaller magnitude compared with $I\left( {L_{R} + {L/2},t} \right)$. This may partly explain the experimental observations that at most 13e ([@bib25], [@bib28], [@bib29]), instead of 16e, are moved during full depolarization in four voltage sensors (for a single ion channel) based on computing the area under voltage-clamp gating current. Therefore, flux of charge at any location of zone 2, though impossible to measure in experiments so far, will give us the amount of arginines moved during depolarization more reliably than the measurable *I*(0, *t*).Figure 5(*a*) The time courses of $I\left( {L_{R} + {L/2},t} \right)$, *I*(0, *t*), and despiked *I*(0, *t*) for the case of large depolarization with *V* rising from −90 to −8 mV at *t* = 10, holding on till *t* = 150, and then dropping back to −90 mV. The inset plot is a magnification of the ON current to visualize the difference of *I*(0, *t*) and despiked *I*(0, *t*) more clearly. (*b*) The time courses of $Q_{1},Q_{3},{\int_{0}^{L_{R}}{\left( {c_{Na} - c_{Cl}} \right)dz}}$, and $\int_{L_{R} + L}^{2L_{R} + L}{\left( {c_{Na} - c_{Cl}} \right)dz}$ are under the same depolarization scenario as (*a*). To see this figure in color, go online.
Second, we see in [Fig. 5](#fig5){ref-type="fig"} *a* with magnification in its inset plot that, as in experiments, *I*(0, *t*), but not $I\left( {L_{R} + {L/2},t} \right)$, has contaminating leading spikes in ON and OFF parts of the current. These spikes are capacitive currents from solution EDL of vestibules caused by the sudden rising and dropping of command potential. These spikes need to be removed in voltage-clamp experiments to get rid of the contribution from vestibule solution EDL (and membrane) to the transport of gating charges (arginines) when computing the area under gating current. The technical details of removing these spikes are shown in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S10, and more details about spikes can be found in [Supporting Materials and Methods](#mmc1){ref-type="supplementary-material"}, Section S11.
Third, in [Fig. 5](#fig5){ref-type="fig"} *b*, as arginines move from one vestibule to another, the concentrations of Na^+^ and Cl^−^ also correspondingly change with time at the vestibules. They form countercharges through EDL and balance arginine charges at vestibules. However, these EDL changes only maintain an approximate, not exact, charge balance, as shown in [Fig. 5](#fig5){ref-type="fig"} *b*. The violation of electroneutrality causes the displacement current, which is not negligible. This further causes the underestimate of arginines that move when the voltage sensor is depolarized if the estimate is made by measuring the area under *I*(0, *t*).
As in the previous section, we used flux of charges at the middle of the hydrophobic plug, *I*(*L*~*R*~ + *L*/2, *t*), instead of experimentally measurable *I*(0, *t*) to represent the gating current in discussions. We may as well name *I*(*L*~*R*~ + *L*/2, *t*) as the arginine current to avoid the confusion with the actual gating current *I*(0, *t*) here. This arginine current leaves out its associated displacement current *I*~*disp*~(*L*~*R*~ + *L*/2, *t*) and serves to represent gating current better for two reasons:1)The area under the time course of *I*(*L*~*R*~ + *L*/2, *t*) gives us the amount of arginines moved during depolarization more faithfully than *I*(0, *t*). The fluxes of charge for each arginine shown in [Fig. 3](#fig3){ref-type="fig"}, *b* and *d* carry important information about how each arginine is moved by the electric field that will be further illustrated in [Fig. 6](#fig6){ref-type="fig"}. All these will not be easy to display and comprehend if we use *I*(0, *t*) instead.Figure 6(*a*) and (*c*) are the time courses of the amount of arginines moved to the extracellular vestibule. (*b*) and (*d*) are center-of-mass trajectories of individual arginines and S4. (*a*) and (*b*) are the case of large depolarization with *V* rising from −90 to −8 mV at *t* = 10, holding on till *t* = 150, and then dropping back to −90 mV. (*c*) and (*d*) are the case of small depolarization with *V* rising from −90 to −50 mV at *t* = 10, holding on till *t* = 150, and then dropping back to −90 mV. To see this figure in color, go online.2)Using *I*(0, *t*) as a definition of gating current would require a decontamination by removing the leading spikes, which is computationally costly. Removing spikes would especially pose a heavy numerical burden when doing parameter fitting in which numerous repeated computations are done.
Time course of arginine and S4 translocation {#sec3.4}
--------------------------------------------
[Fig. 6](#fig6){ref-type="fig"} shows the time course of *Q* (amount of arginines moved to extracellular vestibule, equal to *Q*~3~ here) and center-of-mass trajectories of individual arginines (*z*~*i,CM*~, *i* = 1, 2, 3, and 4) and S4 segment (*Z*~*S*4~). [Fig. 6](#fig6){ref-type="fig"}, *a* and *b* show the case of large depolarization, and [Fig. 6](#fig6){ref-type="fig"}, *c* and *d* show the case of small depolarization.
In the case of large depolarization ([Fig. 6](#fig6){ref-type="fig"} *b*), the arginines and S4 *z* positions quickly reach individual steady states, with almost all arginines transferred to the extracellular vestibule as previously shown in [Fig. 4](#fig4){ref-type="fig"} *a*. Therefore, *Q* is close to its saturated value 4 as shown in [Fig. 6](#fig6){ref-type="fig"} *a*. Arginines and S4 move back to the intracellular vestibule once the voltage drops back to −90 mV. From [Fig. 6](#fig6){ref-type="fig"} *b*, the forward-moving order of arginines is R1, R2, R3, and R4, and the backward-moving order is the opposite R4, R3, R2, and R1 with agreement with the structure. This agreement might look trivial in molecular dynamics simulations but is not a trivial check here because this model describes arginines not by particles, as in molecular dynamics, but by concentrations. Note that an incorrect order and pace of the movement of arginines would cause disagreement with experiments in the shape of IV curve as well. S4 is initially farthest to the right but lags behind R1 and R2 during movement in depolarization, as shown in [Fig. 6](#fig6){ref-type="fig"} *b*. This is certainly because S4 is finally relaxed to an almost unforced situation close to its resting position *Z*~*S*4,0~ during this large depolarization. We can further calculate the displacements of each arginine and S4 during this full-saturating depolarization and find *Δz*~1,*CM*~ ≈ *Δz*~2,*CM*~ ≈ *Δz*~3,*CM*~ ≈ 1.93 nm, *Δz*~4,*CM*~ = 1.76 nm, and *ΔZ*~*S*4~ = 1.51 nm. Besides almost the same displacements for R1, R2, and R3, their average moving velocities are also very close to each other. This seems to suggest a synchronized movement among R1, R2, and R3 that we have not imposed on the arginines in our model. Also, we can see the movements of arginines contribute significantly to the movement of the S4 segment. This can be seen from the steady-state *z* position of S4 derived from [Eq. 6](#fd6){ref-type="disp-formula"},$$Z_{S4} = \frac{K}{K_{S4} + 4K}\sum\limits_{i = 1}^{4}{\left( {z_{i,CM} - z_{i}} \right) + \frac{K_{S4}}{K_{S4} + 4K}Z_{S4,0}} = \frac{1}{5}\left\lbrack {Z_{S4,0} + \sum\limits_{i = 1}^{4}z_{i,CM}} \right\rbrack.$$
Experimental estimates of S4 displacement during full depolarization range from 2 to 20 Å ([@bib24], [@bib30]), depending on the model of the voltage sensor and its motion, including the transporter model, the helical screw, and the paddle model ([@bib24]). Our *ΔZ*~*S*4~ = 1.51 nm here is large and seems to agree better with experimental estimates requiring large displacements, such as the paddle model. In contrast, the helical screw model, which is supported by most of the recent data, is known to have shorter displacements. A plausible explanation for our overestimate of *ΔZ*~*S*4~ is that our 1D model uses a straight line perpendicular to the hydrophobic-plug path for the movement of the arginines. In reality, the S4 segment is significantly tilted with respect to the membrane, and the arginines follow a spiral along the helix. Therefore, if the S4 segment rotates and changes its tilt during activation, the total vertical translation needed to cross the hydrophobic plug is significantly reduced, as was shown by Vargas et al. ([@bib31]). The value obtained in ([@bib31]) was between 0.7 and 1 nm when comparing the displacement perpendicular to the membrane of the open-relaxed state crystal structure of Kv1.2 ([@bib32]) and the closed structure that has been derived by consensus from experimental measurements ([@bib31]).
In the case of small depolarization, the driving force is weaker than in a large-saturating depolarization, so their *z* positions do not have a chance to reach steady states as they do during a full-saturating depolarization. Rather, in a small depolarization, the motion of the arginines and S4 are aborted. They return to the intracellular vestibule because the depolarization drops (i.e., decreases in magnitude, and the membrane potential becomes more negative) before arginines and S4 have a chance to reach their steady-state positions. This detailed atomic interpretation likely overreaches the resolution of our model. At the single-sensor level, we do not expect partial movements; instead, some sensors will have moved all the way and others not at all, but the distribution of sensors in the two extreme positions should follow what we predict with this model, which is an ensemble average. We look forward to measurements of movements of probes that mimic arginine in its environment that require improvements in the resolution and structural realism of our model.
[Fig. 6](#fig6){ref-type="fig"} *c* illustrates these aborted motions. *Q* reaches 1.57 at most, which should be 2 instead if steady-state was reached as it is if time is long enough. See the steady-state behavior shown in the QV curve of [Fig. 2](#fig2){ref-type="fig"} *a*. [Fig. 6](#fig6){ref-type="fig"} *d* shows that the S4 segment is initially farthest to the right, lags behind R1 during movement, and is almost caught up by R2. The maximal displacements of arginines and S4 calculated from [Fig. 6](#fig6){ref-type="fig"} *d* are *Δz*~1,*CM*~ = 1.36 nm, *Δz*~2,*CM*~ = 0.966 nm, *Δz*~3,*CM*~ = 0.459 nm, *Δz*~4,*CM*~ = 0.316 nm, and *ΔZ*~4,*CM*~ = 0.616 nm. The significant difference between *Δz*~1,*CM*~, *Δz*~2,*CM*~, *Δz*~3,*CM*~, and *Δz*~4,*CM*~ may imply that R1 and R2 have jumped across the hydrophobic plug and entered the extracellular vestibule, whereas R3 and R4 still stay at the intracellular vestibule during this small depolarization. This is consistent with the observation from individual gating-current components of arginines in [Fig. 3](#fig3){ref-type="fig"} *d*.
Family of gating currents for a range of voltages {#sec3.5}
-------------------------------------------------
Though we prefer $\ I\left( {L_{R} + {L/2},t} \right)$ to *I*(0, *t*) for representing gating current as explained in the section under heading [Flux of Charges at Different Locations](#sec3.3){ref-type="sec"}, we here use the actual gating current, despiked *I*(0, *t*), to compare with experiment ([@bib20]). [Fig. 7](#fig7){ref-type="fig"} *a* shows the time courses of a subtracted gating current (despiked *I*(0, *t*)) for a range of voltages *V* ranging from −62 to −8 mV. The area under gating current, for both ON and OFF parts, increases with *V* because more arginines are transferred to the extracellular vestibule as *V* increases. The shapes of this family of gating currents agree well with experiment ([@bib20]) in both magnitude and time course.Figure 7(*a*) The time courses of subtracted gating current, despiked *I*(0, *t*), with the voltage rising from −90 to *V* mV at *t* = 10, holds on till *t* = 150, and then drops back to −90 mV, where *V* = −62, −50, ..., −8 mV. (*b*) *τ*~2~ versus *V* compared with experiment ([@bib20]) is shown. To see this figure in color, go online.
We can characterize the time course by fitting the decay part of a subtracted gating current by $ae^{{- t}/\tau_{1}} + be^{{- t}/\tau_{2}}$, *τ*~1~ \< *τ*~2~ as generally done in experiments ([@bib20]) in which *τ*~1~ is the fast time constant and *τ*~2~ is the slow time constant. Usually, the movement of arginines is dominated by *τ*~2~. Here, *τ*~2~ was calculated from simulation and compared with experiment ([@bib20]) as shown in [Fig. 7](#fig7){ref-type="fig"} *b*. Because in our computation the time is in arbitrary units, we have scaled the time to have the maximal *τ*~2~ to fit with its counterpart in experiment ([@bib20]). Overall, the trend of *τ*~2~ versus *V* in our result, though not the whole curve, agrees well with experiment ([@bib20]). To the left of the maximal point in [Fig. 7](#fig7){ref-type="fig"} *b*, simulation results fit rather well with the experiment compared with the values to the right of the maximal point, at which it overestimates *τ*~2~ compared with the experiment. This overestimate is consistent with the observation that the amount of transferred charges *Q* saturates slightly faster in experimental data than in this simulation as *V* increases (see QV curve of [Fig. 2](#fig2){ref-type="fig"} *a*). This phenomenon is related to the cooperativity of movement among arginines, which will be further discussed below.
Effect of voltage pulse duration {#sec3.6}
--------------------------------
[Fig. 8](#fig8){ref-type="fig"} shows the effect of voltage pulse duration with [Fig. 8](#fig8){ref-type="fig"} *a* for the case of small depolarization and [Fig. 8](#fig8){ref-type="fig"} *b* for the case of large depolarization. The magnitude and time span of subtracted gating current (despiked *I*(0, *t*)) are changed by pulse duration in both cases, but the shape will asymptotically approach the same curve as pulse duration increases, no matter the size of the depolarization. This behavior occurs because it takes time for the command pulse to drive the arginines toward the extracellular vestibule. If the pulse duration is long enough, the time course of *Q* will approach its steady state for large depolarization as in [Fig. 6](#fig6){ref-type="fig"} *a*. Small depolarization takes a longer time to reach its steady state, as demonstrated in [Fig. 6](#fig6){ref-type="fig"} *c*. The shapes of gating currents in [Fig. 8](#fig8){ref-type="fig"} compare favorably with experiment ([@bib20]) in which the OFF subtracted gating currents for short pulses have very fast decays, whereas for long pulses, the OFF subtracted gating currents have larger rising amplitude and slower decay because of a larger amount of arginines moved.Figure 8Subtracted gating currents, despiked *I*(0, *t*), showing the effect of voltage pulse duration. (*a*) *V* increases from −90 to −35 mV at *t* = 10 and drops back to −90 mV at various times. (*b*) *V* increases from −90 to 0 mV at *t* = 10 and drops back to −90 mV at various times. To see this figure in color, go online.
Conclusions {#sec4}
===========
Previous work with molecular and coarse-grained simulations have captured some interactions, but they have not yet reproduced the time course and voltage dependence of macroscopic gating currents ([@bib10], [@bib11], [@bib12], [@bib13], [@bib14]), and previous continuum models have captured only the steady-state properties of charge movement ([@bib15], [@bib16], [@bib17], [@bib18]).
This 1D continuum mechanical model of the voltage sensor tries to capture the essential structural details of the movement of mass and charge that are necessary to reproduce the basic features of experimentally recorded gating currents. After finding appropriate parameters, we find that the general kinetic and steady-state properties are well represented by the simulations. The good agreement of our numerical results with salient features of gating current measured experimentally would be impossible by simply tuning of parameters if our model had not captured the essence of physics for the voltage sensor. The continuum approach seems to be a good model of voltage sensors, provided that it 1) takes into account all interactions crucial to the movement of gating charges and S4; 2) computes their correlations consistently, so all variables satisfy all equations under all conditions with one set of parameters; and 3) satisfies conservation of current. This last point gave us a new insight: what is measured experimentally does not correspond to the transfer of the arginines because the total current, containing a displacement current, is smaller than the arginine current. It should be noted, however, that the total energy provided by the voltage clamp is *qV*, where *q* is the time integral of the measured gating current and *V* is the applied voltage. This is the total energy that explains the correspondence of charge per channel with the charge estimated by the limiting slope method ([@bib33], [@bib34], [@bib35]).
We have simplified the profile of the energy barrier in the hydrophobic plug because the PMF in that region, and its variation with potential and conditions, is unknown. There is plenty of detailed information on the amino acid side chains in the plug and how each one of them changes the kinetics and steady-state properties of gating charge movement ([@bib6]). Therefore, the next step is to model the details of interactions of the moving arginines with the wall of the hydrophobic plug and the contributions from other surrounding charged protein components. Some of the effects to be included are as follows:1)Steric and dielectric interactions of the arginines that this model does not include. These include the interaction of arginines with negative charges of the S2 and S3 segments and the negative phospholipids as well as the hydrophobic residues in the plug. These interactions may be responsible for the simultaneous movement of two to three arginines across the plug, which is an experimental result that this model does not reproduce ([@bib36], [@bib37]).2)Time dependence of the plug energy barrier *V*~*b*~*.* Once the first arginine enters the hydrophobic plug by carrying some water with it, this partial wetting of the hydrophobic plug will lower *V*~*b*~, chiefly consisting of solvation energy, and enable the next arginine to enter the plug with less difficulty. This might explain the cooperativity of movement among arginines when they jump through the plug. The addition of details in the plug may also produce intermediate states that have been measured experimentally. In this situation, arginines may transiently dwell within the plug.3)A very strong electric field might affect the hydration equilibrium of the hydrophobic plug and would lower its hydration energy barrier as well ([@bib38]). This cooperativity of movement may help explain the quick saturation in the upper right branch of the QV curve (and smaller *τ*~2~). It may also explain the experimentally observed translocation of two to three arginines simultaneously ([@bib36], [@bib37]).
The power of this mathematical modeling is precisely the implementation of interactions and the various effects in a consistent manner. Implementing the various effects listed above is likely to lead to a better prediction of the currents and to the design of experiments to further test and extend the model.
Further work must address the mechanism of coupling between the voltage sensor movements and the conduction pore. For example, the spring constant of the two sides of S4 have been made equal, which does not take into account the structural reality that one side has a linker to S3, whereas the other links to the pore opening. It seems likely that the classical mechanical models of coupling will need to be extended to include coupling through the electrical field. The charges involved are large. The distances are small, so the changes in electric forces that accompany movements of charged mass (and flows of displacement current) are likely to be large and important. It is possible that the voltage sensor modifies the stability of a fundamentally stochastically unstable, nearly bistable, conduction current (of single channels) by triggering sudden transitions from closed to open state in a controlled process reminiscent of Coulomb blockade in a noisy environment ([@bib39]).
Author Contributions {#sec5}
====================
All authors conceived the research, T-L. H. wrote the code and carried out the computations, and all authors contributed to the interpretation and writing of the manuscript.
Supporting Citations {#app3}
====================
References ([@bib40], [@bib41], [@bib42], [@bib43], [@bib44], [@bib45], [@bib46], [@bib47], [@bib48], [@bib49], [@bib50], [@bib51]) appear in the [Supporting Material](#app2){ref-type="sec"}.
Supporting Material {#app2}
===================
Document S1. Supporting Materials and Methods and Fig. 1Data S1. MATLAB Code Based on Mathematical Model Described in this ArticleDocument S2. Article plus Supporting Material
Dr. Horng thanks the support of National Center for Theoretical Sciences Mathematical Division of Taiwan and Dr. Ren-Shiang Chen for the long-term helpful discussions.
This research was sponsored in part by the National Institutes of Health grant R01GM030376 (F.B.), National Science Foundation Division of Mathematical Sciences grant 1759535 (C.L.), National Science Foundation Division of Mathematical Sciences grant 1759536 (C.L.), and Ministry of Science and Technology grant 106-2115-M-035-001-MY2 (T.-L.H.).
Supporting Materials and Methods, one figure, and one data file are available at <http://www.biophysj.org/biophysj/supplemental/S0006-3495(18)34501-6>.
| {
"pile_set_name": "PubMed Central"
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1. Background {#sec1}
=============
Team leadership in emergencies is reported as being important for the quality of the technical performance of teams \[[@bib1], [@bib2], [@bib3], [@bib4], [@bib5], [@bib6], [@bib7], [@bib8], [@bib9], [@bib10], [@bib11], [@bib12]\], for patient outcome, patient safety and patient care \[[@bib10], [@bib11], [@bib13], [@bib14], [@bib15], [@bib16], [@bib17], [@bib18], [@bib19], [@bib20], [@bib21], [@bib22], [@bib23]\]. The quality of team leadership may even influence patient mortality and survival rates \[[@bib7], [@bib11], [@bib15], [@bib20]\].
A systematic review (conducted in 2016) searching the literature identifying training in empowering leadership for the team leader in emergencies, concluded that no focused leadership training has taken place [@bib24]. Medical scholars have over many years interpreted leadership from a measurable perspective (simulation training and repeatedly developing taxonomy-based feedback). However, leadership in emergencies is, in the authors\' opinion, basically -- isolated from professional ability -- a complex and multifaceted *social* competence. Analysing social interaction or training social competencies, including dissemination, requires an interpretative approach rather than the positivist\'s need for measurability and testing.
As previously described in the mentioned systematic review [@bib24], leadership is acquired through practice, and therefore leadership competencies should be acquired not through theoretical learning, but through practice as described by Mintzberg [@bib25]. In accordance with this, surgeons learn to sew a wound together by doing it in practice. In order to learn to ride a bike, theoretical knowledge, as well as feedback from educators and experts, is definitely valid but only by getting up on the bike and experiencing the feeling it is possible to acquire this competence. Experience obtained by practice is prerequisite for acquiring learning in certain areas. The authors of this study do not consider that feedback based on behavioural indicators (leadership measurements tools) is equivalent to workable training of practicing leadership in emergencies, it is necessary to exercise *practicing* leadership in order to fully acquire leadership competence.
Measuring time to perform tasks and giving feedback according to objectives defined in taxonomies mixes professionalism and attempts at leadership training but without taking into account the fact that more and very different mind systems are challenged, described as Systems 1 and 2 by Kahneman [@bib26]. In accordance with Kahneman, it is possible to argue that training in clinical skills and memorizing algorithms, addresses the effortful, slow and rational operations of system 2 while interaction between people (leadership) takes place in the automatic, fast and intuitive system 1. It is difficult to operate in both systems simultaneously and errors can occur if the two systems conflict with each other. Consequently, this brings a focus on the role of the medical expert (medical competence) in this training and draws attention away from actually practising training of leadership.
Medical scholars have sought help in other high-risk professions of military and aviation. With the help of these professions, the medical scholars have theoretically developed "what to measure". Leadership is implicitly defined in the Leadership Behaviour Description Questionnaire (LBDQ) checklist \[[@bib27], [@bib28], [@bib29]\] (adapted from military, created in 1945), as well as in Crew Resource Management [@bib30] (CRM) and the Non-Technical Skills checklist (NOTECHS) (adapted from aviation to healthcare for the first time in 2003, Anaesthetists\' non-technical skills [@bib31] (ANTS)). This is accepted by the medical world without distinction to the differences between captains, officers and the young doctor (the resident). Military education and aviation education are aimed at professionals with different prerequisites/conditions than doctors. Officers and pilots in command of aircraft are leaders in their professions. It could be argued that their situation is essentially different to that of newly qualified young doctors who have not yet been introduced to leadership. In addition, it can be noted that doctors have to cope with several other roles in their profession, in accordance with CanMEDS definition of the seven roles of the doctor [@bib32].
The previously mentioned systematic review suggested in addition that it is necessary to work with the personal challenges associated with stressful situations. As a consequence, it is necessary to work with the resident\'s courage, self-confidence and authority when he or she acts as a team leader [@bib24]. This is further supported by three studies, Hunziker et al. describes the negative emotions caused by stress in a simulated CPR situation [@bib33], Petrosoniak et al. suggest it is advantageous to work with stress inoculation alongside with CRM training and simulation [@bib34], and Mantha et al. who designed a curriculum for paramedic trainees in order to address stress and anxiety [@bib35].
There are many training programmes for leaders in different contexts. We focus our review on *real* and *operational* leadership training for physicians in emergency situations in healthcare.
The quantitative research was effective in answering questions such as "Time to start cardiopulmonary resuscitation", "Time to perform critical tasks" or "time to call for help during bleeding, operative time, and path length of laparoscopic instruments", it would be less useful if we want to know the actual need for the training of empowering team leadership in emergencies that are sought in practice, that is, in the clinic. Furthermore, it will not provide the actual and personal challenges, which the residents are facing when performing their team leader function in emergencies. Qualitative research helps describe and find the reasons for needs, behaviour, emotions and its social context and therefore the reason for the methodology used in this study. It should be noted that in this study, articles with a qualitative approach were identified: Sadideen et al. [@bib36], Leenstra et al. [@bib10], Kolehmainen et al. [@bib20], Jacobsson et al. [@bib18], Hjortdahl et al. [@bib4], and Yule et al. [@bib37].
This study has three questions for the literature. The first question; does the literature itself have statements about the importance of leadership training and leadership in emergencies? Secondly, does the literature define any actual barriers, challenges and learning objectives that should be addressed in terms of training of the leadership function in emergencies? And finally, depending on the findings, the study will reveal if there is consistency between the actual focus in literature on leadership training versus the need described in the literature based on opinions, experience and knowledge.
2. Methods {#sec2}
==========
2.1. Data sources {#sec2.1}
-----------------
A search of the databases of PubMed, Psycinfo (via Ovid), and ERIC was carried out in June--December 2016 with the aid of a research librarian. Keywords in the search were: gesture, mimic, eye-contact, NTS Skills, NOTSS, communicat\*, non-verbal, task management, authority, lead, teamleader, teach\*, educat\*, train\*, learn\*, advanced life support, cardiopulmonary resuscitation \[MeSH Terms\], cardiopulmonary \[All Fields\], resuscitation\" \[All Fields\], leadership \[Mesh\], Education, Medical \[Mesh\], urgency, leadership, "cardiac arrest", human factors), where \* indicates wildcard. No time limit or language restrictions were applied. A thorough review of the search strategy has been described previously [@bib24].
40 articles fulfilled the inclusion criteria, which were expanded to cover experience, perceptions and emotions relating to leadership training in emergency situations, and therefore primary as well as secondary articles became relevant in this study.
The research papers were treated as documents and primary sources in data collection, the method is inspired by a study conducted by Marston et al. [@bib38] All 40 articles were grouped together in a single PDF document (a total of 299 pages) and searched several times for keywords, their synonyms and if available, their meaningful similar positive and negative analogies, phrases and sentences. In both analyses, keywords were selected by TL, and relevance to inclusion and exclusion was discussed until TL and RBH reached agreement on the decision and then grouped them into categories. These categories helped to inform the development of patterns and finally themes seen across the selected studies.
The relevance of the identified keywords, phrases and sentences was discussed between TL and RBH. There was a high degree of overall agreement between the two authors (94.45%). Inter-coder reliability was very good (Cohen\'s κ coefficient = 0,852).
2.2. First qualitative content analysis searching leadership {#sec2.2}
------------------------------------------------------------
Basically, this analysis stems from the wish to find an explanation for why the systematic review did not reveal any training in leadership. The source of 40 articles was examined for answers to 5 questions as expressed in [Table 1](#tbl1){ref-type="table"}.Table 1QCA-1, team leadership in emergencies.Table 1QCA-1: Team leadership in emergenciesIs importantIs lackingTraining is importantTraining is ignoredTraining is required**Total3111282230**1986: Iserson [@bib42]XXXXX1986: McCue, Magrinat et al. [@bib43]XXX1999: Cooper, Wakelam [@bib27]XXXX2001: Cooper [@bib49]XXX2003: Wisborg, Ronning et al. [@bib1]XXXX2004: Itani, Liscum et al. [@bib50]XXXXX2004: Marsch, Muller et al. [@bib2]XXXX2006: Yule, Flin, Paterson-Brown et al. [@bib37]XX2006: Yule, Flin et al. [@bib13]XXX2007: Flin, Yule et al. [@bib16]XXXX2007: Gilfoyle, Gottesman et al. [@bib14]XXXX2007: Hayes, Rhee et al. [@bib15]XXXXX2007: Makinen, Aune et al. [@bib3]XXX2009: Carlson, Min et al. [@bib51]X2009: Hjortdahl, Ringen et al. [@bib4]XXXXX2010: Georgiou, Lockey et al. [@bib6]X2010: Hunziker, Buhlmann et al. [@bib5]XXX2010: Hunziker, Tschan et al. [@bib7]XXXX2011: Hunziker, Johansson et al. [@bib8]XXX2011: Ringen, Hjortdahl et al. [@bib17]XXXX2012: Jacobsson, Hargestam et al. [@bib18]X2013: Fond, Ducasse et al. [@bib52]XXX2013: Hunziker, Tschan et al. [@bib19]XXXX2013: Stoller, Taylor et al. [@bib53]XXXXX2013: Willems, Waxman et al. [@bib54]XXX2014: Kolehmainen, Brennan et al. [@bib20]X2014: Krage, Tjon Soei Len et al. [@bib55]XXX2014: Mercer, Arul et al. [@bib45]X2014: Roberts, Williams et al. [@bib56]XX2014: Sommer [@bib57]2015: Briggs, Raja et al. [@bib9]XXX2015: Fernandez Castelao, Boos et al. [@bib21]XXX2015: Nicksa, Anderson et al. [@bib44]X2015: Yule, Parker et al. [@bib22]XXX2016: Ford, Menchine et al. [@bib12]XXXX2016: Hargestam, Hultin et al. [@bib23]XXX2016: Leenstra, Jung et al. [@bib10]XXXX2016: Mantha, Coggins et al. [@bib35]XXX2016: Robinson, Shall et al. [@bib11]XXXX2016: Sadideen, Weldon et al. [@bib36]X
First, the source was examined in an abductive [@bib39] -- inductive Qualitative Content Analysis (QCA-1). An *inductive approach* (conventional, inductive [@bib40]) is appropriate when prior knowledge regarding the phenomenon under investigation is limited or fragmented [@bib41]. Using an inductive approach, codes, categories, or themes are directly drawn from the data [@bib41] without imposing preconceived categories or theoretical perspectives [@bib40]. By adding 'latent content analysis' [@bib41] it is possible to interpret the underlying meaning of the words. This investigation was conducted with the purpose of identifying opinions about leadership in emergencies and leadership training. The quotes were grouped into themes in an iterative process.
The purpose of the search was to investigate whether the literature expressed that leadership was important, whether the training was adequate or if further action was required in the training of leadership in emergencies. The analysis was conducted across the articles in the assembled pdf document to determine patterns. First, the document was searched for the words 'important, importance, adequate, sufficient'. When relevant phrases and sentences were identified new keywords emerged and the search started over again. The identified sentences were grouped into categories, and then into the themes as presented in [Table 1](#tbl1){ref-type="table"}.
Among relevant keywords in QCA-1were: Important, importance, optimal, essential, adherence, correct, recognize, favourable, comfortable, sufficient, good, better, adequate, improve, enhance, critical, negative, poor, absence, lack, eclipsing, deviation, error, delay, uncomfortable, failure, fail, problem, deficit, variable, variety, varies, obstacle, insufficient, neglect, safe, safety, outcome, quality, performance, goal, need, coordinate, ability, role, ambiguity, demand, and require.
2.3. Second qualitative content analysis searching barriers, challenges and learning objectives {#sec2.3}
-----------------------------------------------------------------------------------------------
Secondly, a directed [@bib40] -- deductive QCA (QCA-2) was carried out to determine whether the literature reveals any opinions, attitudes, experiences or views to identify any *actual* challenges or barriers which leadership training for residents in emergencies needs to address. *Deductive* QCA starts with preconceived codes or categories derived from prior relevant theory, research, or literature [@bib41]. The preconceived categories in the search were directed by the content of the eldest article found (published in 1986) written by Kenneth Iserson [@bib42]. The article was identified as very relevant as it focused on the actual experience of physicians when performing as team leader in an emergency. Additionally, we decided that using the oldest of the 40 articles as the basic opinion on leadership would give an overview of developments in opinions or knowledge over the 30-year period, the articles represented.
The source was searched in order to establish whether the key issues identified by Iserson are still relevant today in the themes presented in [Table 2](#tbl2){ref-type="table"}. The analysis was conducted across the articles in the assembled pdf document to determine patterns. Keywords were selected from Iserson\'s article. As in QCA-1, when relevant phrases and sentences were identified new keywords emerged and the search started over again. Sentences were included if they contained anything about other perspectives on team leadership, teamwork, or personal challenges and feelings related to this, and grouped to themes demonstrated in [Table 3](#tbl3){ref-type="table"}.Table 2Isersons statements.Table 2Isersons statements -- QCA-2 (directed, deductive)Theme*The situation* itself, which may be *"stressful"*, *"chaotic"* and *"confusing"****Stress****Negative feelings*, such as being *"uncomfortable"*, as an *"overwhelming burden"*, and perceive it with *"fear"* and *"anxiety"*.***Anxiety****Self-confidence* is prerequisite for good leadership***Self-confidence****Capacity as leader/appears as authoritative* if he is able to show *"responsibility"*, *"knowledge"*, *"power and authority"*.***The perception of the leader****Assuming leadership*, which involves the mental decision (mental preparedness) to assume *"the mantle of the group\'s possible failure"****Mental aspect***Table 3QCA-2, challenges and learning goals for the team leader in the emergency situation.Table 3QCA-2: Challenges and learning goals for the team leader in the emergency situationThe scenario is complex/stressful/unpredictable/chaoticPerceived with anxiety or panic/is disturbing/worrying/overwhelmingThe leaders confidence is important for the teamNon-verbal communication is important for the situation/for the perception of the leaderAssuming the leadership: Mental process/risk taking/managing oneself/mental preparation**Total278171316**1986: Iserson [@bib42]Stress, chaosAnxietyConfidenceCalm, quiet toneDecision to assume the role1986: McCue, Magrinat et al. [@bib43]ThreatConfidenceAssume the responsibility1999: Cooper, Wakelam [@bib27]Stress, chaosUnpleasant, disturbing, fight a losing battleConfidence, trustNon-verbal behaviourTaking risks2001: Cooper [@bib49]Confidence2003: Wisborg, Ronning et al. [@bib1]Confidence2004: Itani, Liscum et al. [@bib50]Stressors2004: Marsch, Muller et al. [@bib2]Emotional, concern, fail2006: Yule, Flin et al. [@bib13]Complex, stressSelf-beliefexpertise is difficult to verbalizePersonal vulnerability, mental readiness2007: Flin, Yule et al. [@bib16]Stress2007: Gilfoyle, Gottesman et al. [@bib14]Calm, clear voiceAssume the leadership2007: Hayes, Rhee et al. [@bib15]StressAnxiety, unprepared, worrying, overwhelmedConfidence2009: Hjortdahl, Ringen et al. [@bib4]Complex, stressAnxietyConfidence, trustworthy, calmnessCommunicate clear and distinctresponsibility2010: Georgiou, Lockey et al. [@bib6]Comfortable directing2010: Hunziker, Tschan et al. [@bib7]Complex2011: Hunziker, Johansson et al. [@bib8]Complex, stressAssume responsibility2011: Ringen, Hjortdahl et al. [@bib17]Complex, stressRadiate confidenceCommunicate clearly2012: Jacobsson, Hargestam et al. [@bib18]Complex, time pressureConfidence, ethos, trustPosition and how they speak2013: Fond, Ducasse et al. [@bib52]ConfidenceApperance, charismal, non-verbalIntrospection/Assume responsibility2013: Hunziker, Tschan et al. [@bib19]Stress2013: Stoller, Taylor et al. [@bib53]StressConfidence, trustManage oneself2013: Willems, Waxman et al. [@bib54]ChaosSelf-care2014: Kolehmainen, Brennan et al. [@bib20]Feverish, chaosDiscomfort, panic, anxietyCalm people down, confidencePowerful posture, non-verbal, speak clearlyMentally prepare, assume leadership2014: Krage, Tjon Soei Len et al. [@bib55]Stress2014: Mercer, Arul et al. [@bib45]ComplexCrowd controlMentally rehearse2014: Sommer [@bib57]Stress2015: Briggs, Raja et al. [@bib9]Complex2015: Fernandez Castelao, Boos et al. [@bib21]Complex, time pressureGesture, eye contact2015: Nicksa, Anderson et al. [@bib44]ComplexConfidence2015: Yule, Parker et al. [@bib22]Stress2016: Ford, Menchine et al. [@bib12]Complex, chaosAssume the role2016: Hargestam, Hultin et al. [@bib23]ComplexUse their bodies, fluent speech2016: Leenstra, Jung et al. [@bib10]Complex, pressure2016: Mantha, Coggins et al. [@bib35]Complex, stressConfidenceBody language, speech pace2016: Robinson, Shall et al. [@bib11]StressAnxietyConfidenceAnxiety management2016: Sadideen, Weldon et al. [@bib36]ComplexNon-verbal behaviourMental strain
Among relevant keywords in QCA-2 were: Theme 1: stress, pressure, chaotic, confusing, unpredictable, unanticipated. Theme 2: uncomfortable, overwhelming, fear, anxiety, panic, disturbing, worrying, unpleasant, discomfort, calm. Theme 3: confidence, personality, relax, competent, self-image, self-belief, risk, performance, clear, communicate, charisma. Theme 4: responsible, knowledge, power, authority, planning, organization, command, trustworthy, credible, ideal, clear, distinct, influence, body, posture, pose, position, verbal, talk, speak, non-verbal, voice, gaze, eye, gesture, ethos. Theme 5: assume, failure, responsible, risk, vulnerable, mental, ready, directing, introspection, oneself, prepare, rehearse, role, strain, manage.
The keywords in each QCA were grouped in five different themes and the themes and quotes containing the words were sorted chronologically. Comprehensive data presentation is shown in the Appendix.
3. Results {#sec3}
==========
Ten themes emerged: QCA-1 demonstrated five themes related to leadership and QCA-2 showed five themes related to barriers, challenges and learning goals for the team leader in emergencies. The results (keywords) of QCA-1 are presented in [Table 1](#tbl1){ref-type="table"} and the results (keywords selected from Iserson) of QCA-2 are presented in [Table 3](#tbl3){ref-type="table"}.
3.1. QCA-1 on leadership {#sec3.1}
------------------------
### 3.1.1. Theme 1, leadership is important {#sec3.1.1}
""... clinical leadership is the ultimate synthesis of medical knowledge and one of the most important aspects of critical medical care"Iserson, 1986 [@bib42]"""Leadership skills directly correlate with the quality of technical performance of cardiopulmonary resuscitation (CPR) and clinical outcomes".Robinson, 2016 [@bib11]"
Thirty one studies repeatedly stressed the importance of the teamleader in emergencies. This function is important for the 'quality of team performance', 'crowd control', 'task performance', 'adherence to algorithms', 'guidelines', 'established protocols', 'errors', 'clear communication', 'success of operations', 'medical performance', 'patient safety', and 'patient outcome'. In particular, the importance of leadership in specialties such as paramedics, anaesthesia, ALS, resuscitation teams, trauma teams, and surgical performance were identified. Thus, there is consensus in 77.5% of the articles that leadership is important for the team\'s work and the clinical outcome.
### 3.1.2. Theme 2: leadership is lacking {#sec3.1.2}
""The results suggest that residents perceive deficits in their training and supervision to care for critically ill patients as cardiac arrest team leaders".Hayes, 2007 [@bib15]"""Level of experience among team leaders was highly variable and their educational background insufficient according to international and proposed national standards".Ringen, 2011 [@bib17]"
Eleven studies explicitly state that leadership is 'lacking', 'residents lack competence', 'failed to treat', 'perceive deficits', 'have not linked the necessity of leading', 'is insufficient', 'residents feel unprepared', and 'eclipsing attention to leadership development'. It is thus suggested that leadership is missing in clinical emergencies in 27.5% of the studies covering a 30-year period.
### 3.1.3. Theme 3: leadership training is important {#sec3.1.3}
""An emergency leadership training programme is essential to enhance the performance of leaders and their teams".Cooper, 1999 [@bib27]"""Despite its importance, dedicated leadership education is rarely part of physician training programs"Ford, 2016 [@bib12]"
Twenty-eight studies repeatedly express that leadership training is important to 'improve performance' and 'effectiveness' of the leader. A high level of consensus was identified in 70% of the articles stating that leadership training is important.
### 3.1.4. Theme 4: leadership training has been ignored {#sec3.1.4}
""Leadership has been neglected as a part of the education and training of physicians".McCue, 1986 [@bib43]"""Traditionally, surgical education has not formally taught leadership skills, effective communication, professionalism, or team management; instead, these skills are learned on the job during surgical residency... residents often take years to gain the confidence and expertise to master the nontechnical and technical skills needed to address high-risk clinical emergencies".Nicksa, 2015 [@bib44]"
Twenty-two studies point out that leadership training has been 'ignored', or 'neglected' and that this is a serious deficiency in medical training. As 55% of the studies thus states that training has been ignored, this finding complies with theme 2: Leadership is lacking. But it is surprising in relation to theme 3: Leadership training is important. If 70% of the studies states that training is important, and 55% expresses that the training has been ignored, then a problem has been identified.
### 3.1.5. Theme 5: leadership training is required {#sec3.1.5}
""... physicians are never taught clinical leadership".Iserson, 1986 [@bib42]"""... practical guidance needed for the deliberate practice of leadership skills".Leenstra, 2016 [@bib10]"
Thirty studies repeatedly stress that training is 'required'. The authors find that it is surprisingly strong as 75% of the studies -- over three decades -- suggests there is an urgent need. This is a strong indication that a problem remains, in spite of the existing CRM and simulation training, NOTECHS and LBDQ.
3.2. QCA-2 on barriers, challenges and learning goals {#sec3.2}
-----------------------------------------------------
### 3.2.1. Theme 6: stress {#sec3.2.1}
""In fact, in a medical crisis, even with limited time and resources, sparse information, rushed and somewhat stressed personnel, the patient should be the only one with an emergency."Iserson, 1986 [@bib42]"""Participants explained that the code leader should be the "chaos control factor" and project a composed demeanor to "help calm other people down." They also described often being "panicked" but trying to appear as the "calmest person in the room." One likened his behavior to a duck, saying: "If you\'ve ever watched a duck on a pond, it looks as though it\'s floating effortlessly across the lake. But if you\'ve ever looked underneath at a duck\'s feet, they\'re paddling feverishly."Kolehmainen, 2014 [@bib20]"
Leadership in emergencies as well as the emergency scenarios have -- for a variety of reasons -- been described as 'complex'. The situation is furthermore described as 'stressful', 'under time pressure', 'unpredictable', 'unanticipated', 'urgent', and 'chaotic'. A consensus was identified in 67,5% of the studies thus describing the emergency itself as stressful.
### 3.2.2. Theme 7: anxiety {#sec3.2.2}
""Interns felt that the combined simulation-debriefing sessions would reduce their anxiety and improve their confidence and skills in participating in real cardiac arrests." ... "The perceived lack of adequate training seemed to be responsible, at least in part, to feelings of being unprepared, overwhelmed, and of worrying about committing errors during cardiac arrests in a significant number of residents".Hayes, 2007 [@bib15]"""Many residents spoke about increased anxiety before codes, a numb feeling during codes, and anxiety afterwards that some described as "PTSD" like".Kolehmainen, 2014 [@bib20]"
Leadership in emergencies has been perceived with negative feelings and emotions as stated in 20% of the studies. These emotions range from 'unpleasant', 'disturbing', and 'worrying' to 'overwhelming', 'fighting a losing battle' perceived with 'anxiety', and 'panic'.
### 3.2.3. Theme 8: confidence {#sec3.2.3}
""... mental preparation in the operating theater, including commitment, self-belief, positive imagery, mental readiness, and distraction control".Yule, 2006 [@bib13]"""One nurse said that if the leader seems confident she feels confident too"Hjortdal, 2009 [@bib4]"
Self-confidence is important for the team leader\'s ability to lead, but the team leader\'s confidence is as important for the team to engender trust in the leader and allow the team to assess whether he or she is 'competent' and 'credible.' Having self-confidence, being trustworthy and being credible is important in order to appear competent was emphasized in 42.5% of the studies.
### 3.2.4. Theme 9: the perception of the leader {#sec3.2.4}
""The leader needs to be flexible, but at the same time engender trust and respect from his team"Cooper, 1999 [@bib27]"""Team members were silent, mainly listening, and did not question leaders\' knowledge and priorities. This could be explained in situations where the leader had a strong ethos and expressed competence"Jacobsson, 2012 [@bib18]"
The teamleader\'s non-verbal communication was stated as important for the team\'s perception of the leader. Nineteen of the studies (47,5%) emphasised that the leader could deliberately work with 'body language', 'voice' and pace when speaking, his or hers\' 'positioning', 'physical gesture', 'eye-contact' and 'charisma' in order to appear 'powerful' and strengthen his or her 'credibility' and 'authority'.
### 3.2.5. Theme 10: mental aspect {#sec3.2.5}
""In accepting the leadership role, the individual assumes the mantle of the group\'s possible failure. Any individual\'s faltering can be laid at the leader\'s feet. This is the law of total responsibility".Iserson, 1986 [@bib42]"""... allows time for the team leader to brief the team, determine names, roles and competencies, prepare contingency plans, check equipment and mentally rehearse any expected mental models"Mercer, 2014 [@bib45]"
Sixteen studies (40%) emphasised the 'mental preparation' when assuming the leadership. It is important to 'manage oneself' in the process, demonstrate 'mental preparedness' and 'recognize personal vulnerability', 'introspection', 'rehearse expected mental models', and exercise 'psychological self-care', and 'anxiety management'.
4. Discussion {#sec4}
=============
Our review of research suggests that there is consensus about an urgent need for workable training of the practise of the teamleader function in emergencies and that leadership training has been neglected. Despite that the majority of the medical scholars request workable leadership training, the objective of the scholar\'s research has focused on developing or adapting measurement-based (taxonomy-based) feedback.
The question is if the research, -- as stated by Psychologist Kahneman -- has been 'blind to the obvious' and 'blind to our blindness' in the past 30 years [@bib26]. However, medical scholars are beginning to acknowledge the problem. Obviously, there is in literature identified a need for 'something' not identified in the learning goals as defined in the current method of training (feedback), this 'something' should be investigated and defined.
The objective of the training should be to make the residents rise to the occasion when called upon to act as leaders of emergency teams. The mental challenge of assuming leadership in emergencies is -- in the identified literature -- described in 40% of the studies. It is necessary in the training to pay attention to the personal mental preparation for the team leader including considering the primary deliberate decision *to assume* leadership and thereby the risks connected to the function. To 'assume leadership' is a decision that has profound consequences for a person\'s self-understanding, and a deliberate but important choice to be made *before* experiencing the challenging situation.
The emergency situation is described as stressful and chaotic and these conditions are contributing to how some of the residents respond with very strong negative feelings. A particularly strong feeling like 'anxiety' is highlighted in several studies and 'panic' has been mentioned by a single study. We believe it is important to respond to anxiety because anxiety can block learning and performance (amygdala hijack), 'paralyses or causes errors, it can interfere with what we want to do' as stated by Prof. Williams [@bib46]. In addition, when the resident is asked to rise to the occasion adopting the role as leader in an emergency situation, he or she is at the same time tested on professional knowledge -- life and death depends on it. In addition, in this stressful situation, he or she will be tested as to whether he or she can gain their colleagues\' respect when performing as team leader. Those factors may influence his or her career and is a contributing stress factor to an already challenging situation.
We didn\'t find any training describing how to address the fear that residents can experience in a stressful situation. This challenge should be focused in future research if we want to prepare the residents for these stressful situations.
In addition, it is disturbing to learn that all Iserson\'s statements written 30 years ago are still valid today. Despite many initiatives, research projects and man hours spent over these years of intense work, the literature seems to have failed to address what the literature itself has pointed out were important learning objectives during this period.
Over the past 30 years the literature has stated that these negative emotions, feelings and perceptions are highly involved when a team leader needs to rise to the occasion in front of an emergency team, but the literature has not as yet described a method to accommodate this. This has been the case whether the reason has been lack of knowledge, lack of leadership definition, lack of focus or lack of ability to handle the simple but difficult question: to find "something 'that works' in terms of leadership training in emergencies" [@bib24] in order to prepare residents for this.
The necessity of giving the resident the confidence and thus the courage in the situation is emphasised in sixteen studies stating that the team leader\'s degree of self-confidence is important for the quality of the leadership. However, we must keep in mind that simulations and feedback are very different from the real clinical world. We need to find a way to bring what is taught in the simulations to the real world.
4.1. Learning goals, the five Cs {#sec4.1}
--------------------------------
The results of the QCA-2 suggest barriers, challenges and learning goals to be addressed in future training.
We have summarized the results of QCA-2 in [Table 4](#tbl4){ref-type="table"}: The Team-Leader\'s Five Cs.Table 4The Team-Leader\'s five Cs.Table 4The Team-Leader\'s five CsCommandDemonstrate authority and powerCredibleBe trustworthyCompetentDemonstrate ability to the situation at stakeCalmCreate calmness for the teamCommunicateAbility to communicate with the team
**Command:** It is necessary that the team leader is able to demonstrate immediately that he or she is the one in command. By deliberately working with body language and nonverbal communication, it is possible to appear convincing in the role of a leader radiating authority.
**Credible:** It is important the leader appears trustworthy, has a strong ethos, and has charisma*.* It is especially emphasised that gestures and positioning in the room are powerful tools in order to radiate power and gain credibility.
**Competent:** It is important that the teamleader is able to appear competent (show expertise). Jacobsson states in 2012:""In health care, knowledge is strongly linked to power, and power in this context is further linked to the profession. In emergency praxis, the designated formal leader in the team is often a physician (surgeon) who is considered to have expert knowledge".Jacobsson, 2012 [@bib18]"
**Calm:** It has been repeatedly emphasized that the acute situation is stressful, complex and even chaotic and therefore it is important for the team that the teamleader is calm in order to calm people down and demonstrate crowd control.
**Communicate:** It is emphasised that it is important that the leader is able to communicate with his team. In order to do so, he or she must be aware of the use of voice (distinct, clear, calm) and especially eye contact is especially emphasised.
The authors of this study believe that these are important topics which should be explored and developed in future studies. It would be appropriate to relate to the fact that the situation is even extremely challenging for future and current team leaders. This should not be ignored, but on the contrary highlighted and explicitly addressed in the training. The necessity of giving the doctor the courage and confidence in the situation is strongly emphasised stating that the team leader\'s degree of self-confidence is important for the quality of the leadership.
In order to simulate an emergency situation and to optimize leadership, a training course creating a framework with stress, anxiety, and discomfort in a harmless, non-clinical, and safe setting should be developed. It would be important to provide psychological safety [@bib47] where the participants feel able to accept being uncomfortable and feel that they would be viewed positively even if they make mistakes [@bib48].""No matter how much training you have or how many books you have read, nothing can fully prepare you for the challenge. In this respect, leadership isn\'t easy; it's difficult, necessarily difficult. And the most essential things about it cannot really be taught --- although, in the end, they can be learned".(Dov Frohman et al. Leadership the hard way)"
5. Conclusions {#sec5}
==============
Leadership in emergencies is considered very important for patient safety and can affect mortality. However, literature reported that many residents feel unprepared for the leadership role in emergencies. Barriers such as strong negative emotions for acquiring leadership competencies and learning goals were identified.
It is necessary for residents to address and handle anxiety and panic in stressful and complex situations. Therefore, in order to provide good and convincing leadership, residents must learn to be confident and calm when assuming the role of team leader.
5.1. Perspective {#sec5.1}
----------------
There is no systematic leadership training that takes this aspect into account. Addressing residents\' negative feelings is rarely part of existing programs, despite being mentioned in the literature \[[@bib33], [@bib34], [@bib35]\]. Leadership during an emergency situation involves knowledge and skills, but also implies dealing with the pressure of being the decision-maker and the pressure of bearing the ultimate responsibility for what happens (or does not).
5.2. Limitations {#sec5.2}
----------------
Residents in emergency situations will often face complex, stressful, unpredictable and sometime chaotic situations, and one could argue that those who cannot cope with these situations should not become emergency physicians or even physicians. But it has nevertheless been demonstrated that for many residents this is a challenge that should be addressed. Therefore, a way to help those residents who are nonetheless overwhelmed by discomfort in this situation would be a great improvement when training empowering team leadership in emergencies.
Declarations {#sec6}
============
Author contribution statement {#sec6.1}
-----------------------------
Ture Larsen, Randi Beier-Holgersen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Doris Østergaard, Peter Dieckmann: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Funding statement {#sec6.2}
-----------------
Ture Larsen was supported by grants from Tryg Foundation, Laerdal Foundation and Nordsjællands Hospital, Denmark.
Competing interest statement {#sec6.3}
----------------------------
The authors declare no conflict of interest.
Additional information {#sec6.4}
----------------------
No additional information is available for this paper.
Appendix. ASupplementary data {#appsec1}
=============================
The following is the supplementary data related to this article:AppendixAppendix
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