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Data are available upon request from Ms. Catherine Hamill (<[email protected]>).
Introduction {#sec007}
============
Global mortality for infants under age 5 y halved from 90 to 43 deaths per 1,000 live births between 1990 and 2015. This is the result of a tremendous global effort to achieve the UN Millennium Development Goals \[[@pmed.1002220.ref001]\] and the goals of the UN Secretary-General's Every Woman Every Child initiative \[[@pmed.1002220.ref002]\]. Neonatal mortality in the first 28 d declined (by 47%) from 5.0 to 2.6 million deaths annually over this period. Unfortunately, inequality between countries persists, with 98% of neonatal deaths occurring in low- and middle-income countries \[[@pmed.1002220.ref003]\]. Importantly, more than 60% of such deaths are associated with low birthweight due to intrauterine growth restriction or preterm birth or both \[[@pmed.1002220.ref004],[@pmed.1002220.ref005]\]. Ultrasound imaging has become an essential tool for assuring correct gestational age and for fetal size assessment, increasingly so even in societies with restricted resources. Correspondingly, evidence is emerging at the population level that use of ultrasound biometry increases the rate of detection of fetal growth restriction and the identification of those at increased risk of neonatal morbidity \[[@pmed.1002220.ref006]\].
Birthweight, closely linked to fetal growth, is also a marker of risks for noncommunicable diseases in adult life, with cardiovascular diseases, type II diabetes, and obesity being the most prominent \[[@pmed.1002220.ref007],[@pmed.1002220.ref008]\]. While the birthweight gradient across the entire population reflects the distribution of degrees of such risk, it is increasingly evident that it is the developing physiology associated with fetal growth, rather than birthweight per se, that conditions cardiovascular, metabolic, endocrine, and neural functions for the life course, and thus long-term health and disease risks \[[@pmed.1002220.ref009]\]. For this reason, fetal growth data and aspects of intrauterine development need to be included as an important part of an early-life noncommunicable disease prevention initiative, as this targets the time when the effect of an intervention is greatest \[[@pmed.1002220.ref010]\].
A meeting of experts convened by WHO in 2002 reviewed current knowledge on birthweight as a health outcome and identified a need for research to develop fetal growth charts for international use \[[@pmed.1002220.ref011]\]. In 2006, WHO published the multicenter WHO Child Growth Standards \[[@pmed.1002220.ref012]\] using a prescriptive concept that assumes that, under optimal socioeconomic and nutritional conditions, all children follow one growth standard, regardless of ethnic background. Some support for this concept was drawn from previous studies \[[@pmed.1002220.ref013],[@pmed.1002220.ref014]\]. Although widely adopted, the applicability of these child growth standards has been questioned on the grounds of lack of fit to some populations \[[@pmed.1002220.ref015],[@pmed.1002220.ref016]\], especially for the head circumference standards \[[@pmed.1002220.ref017]\].
Recently, a large multicenter study, the Fetal Growth Longitudinal Study of the Intergrowth-21st Project \[[@pmed.1002220.ref018]\], applied the same concept and approach to fetal growth. The study presented growth standards using ultrasound biometric measurements but did not estimate fetal weight (EFW), even though this is the single most widely used clinical assessment of fetal growth today. Another large recent study, the NICHD Fetal Growth Studies, showed significant differences in fetal growth with ethnicity, and established ethnic-specific growth charts \[[@pmed.1002220.ref019]\]. This contradicts the prescriptive concept that one standard fits all. The study was, however, restricted to four self-reported ethnic groups of Asian, Hispanic, black, and white women in the US.
The present study is the fetal component of the WHO Multicentre Growth Reference Study, which aimed to establish growth charts for clinical use based on populations recruited from multiple countries \[[@pmed.1002220.ref020]\].
Methods {#sec008}
=======
Design {#sec009}
------
This was a multinational observational study approved by the WHO Research Project Review Panel (RP2) and the WHO Research Ethics Review Committee, secondarily approved by the national or local ethics review committee for each study center, and correspondingly carried out according to the Helsinki declaration on ethical principles for medical research in humans \[[@pmed.1002220.ref020],[@pmed.1002220.ref021]\]. All women were recruited specifically for this study, gave written informed consent at inclusion, and otherwise followed their conventional antenatal care program separately from study sessions. Study measurements were revealed to the clinician when the information was thought to be of importance for the management of the pregnancy. The study protocol was published previously \[[@pmed.1002220.ref020]\], so here we present a condensed account of the methods. The study selected participating centers from a range of ethnic and geographical settings, and intended to recruit 1,400 participants. The sample size calculation procedure was published previously \[[@pmed.1002220.ref020]\].
Setting {#sec010}
-------
The following centers participated in the study based on the proficient use of ultrasonography: Centro Rosarino de Estudios Perinatales, Rosario, Argentina; University of Campinas, Campinas, Brazil; University of Kinshasa, Kinshasa, Democratic Republic of the Congo (D. R. Congo); Rigshospitalet, Copenhagen University, Copenhagen, Denmark; Assiut University, Assiut, Egypt; Hôpital Antoine Béclère, Paris, France; University Medical Center, Hamburg-Eppendorf, Germany; All India Institute of Medical Sciences, New Delhi, India; Haukeland University Hospital, Bergen, Norway; and Khon Kaen University, Khon Kaen, Thailand.
Participants {#sec011}
------------
Participants without known health, environmental, and/or socioeconomic constraints were invited to participate in the study. Further inclusion criteria were used: living at an altitude lower than 1,500 m and near the study area (intended to promote compliance for the duration of the study and any possible follow-up studies); age ≥ 18 y and ≤ 40 y; body mass index (BMI) 18--30 kg/m^2^; singleton pregnancy; gestational age at entry between gestational week 8+0 d and 12+6 d according to reliable information on last menstrual period (LMP) and confirmed by ultrasound measurement of fetal crown--rump length; no history of chronic health problems; no long-term medication (including fertility treatment); no environmental or economic constraints likely to impede fetal growth; not smoking currently or in the previous 6 mo; no history of recurrent miscarriages; no previous preterm delivery (\<37 wk) or birthweight \< 2,500 g; and no evidence in the present pregnancy of congenital disease or fetal anomaly at study entry. Fetal anomalies detected during pregnancy or at birth were noted and verified postnatally. Pregnancies in which small-for-gestation-age fetuses were observed or intrauterine growth restriction was suspected were also noted. All mothers recruited were followed up until the end of the study, apart from those withdrawing consent.
Study Procedures {#sec012}
----------------
Women in the first trimester (before week 12+6 d of gestation) attending antenatal care clinics were approached by members of the study team and asked to participate. They were informed about the study objectives and procedures. Those who signed the consent form were enrolled in the study. After the ultrasound scan to assess agreement between gestational age based on LMP and that based on crown--rump length, they were scheduled for fetal biometry scans at monthly intervals.
All infants had an anthropometric assessment after delivery, including measurement of birthweight. All pregnant women in the study were asked for a 24-h dietary recall at entry into the study (and at 28 and 36 wk of gestation) \[[@pmed.1002220.ref022]\]. Clinically relevant conditions (e.g., hypertension, preeclampsia, and diabetes) occurring during pregnancy and childbirth were noted. Otherwise, no further procedures were added to the routine antenatal care provided at the study centers.
Gestational Age Assessment {#sec013}
--------------------------
Gestational age was confirmed by measuring the crown--rump length between gestational week 8 + 0 d and 12 + 6 d based on LMP and recorded as the average of three measurements. To acquire the crown--rump length, the midline sagittal section of the whole fetus was visualized with the fetus horizontal on the screen at 90 degrees to the angle of insonation. Gestational age was assessed by using the reference charts published by Robinson and Fleming \[[@pmed.1002220.ref023]\]. The woman was eligible for the study provided that gestational age by crown--rump length confirmed LMP-based age within 7 d. The LMP-based age was used for the analyses.
Ultrasound Measurements {#sec014}
-----------------------
The first visit (dating scan) was between 8 + 0 and 12 + 6 wk, and subsequent visits for fetal biometry were scheduled at approximately 4-wk (±1 wk) intervals at 14, 18, 24, 28, 32, 36, and 40 wk. All scanning appointments were arranged at the time of the dating scan and study enrollment. All participants were scanned in the lateral recumbent position.
The compulsory ultrasound measurements obtained at all visits included the following biometric parameters: biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL), and humerus length (HL). At each examination, all measurements were obtained three times from three separately generated ultrasound images and uploaded electronically (with the associated images) to the data management system. The median of the three measurements of each parameter was used in the analyses.
In addition, a full morphological evaluation (anomaly scan) was conducted at 18--24 wk following standard practice at each center. Fetuses diagnosed with any anomaly were managed according to local clinical guidelines. Their ultrasound measurements were included in the study, and the possible effect on the percentiles derived was evaluated. The following measurement techniques were used. BPD was measured as the outer--inner distance of the parietal bones in a cross-sectional view of the fetal head at the level of the thalami and cavum septi pellucidi or cerebral peduncles. The cerebellum was not included in the section. The measurement was obtained from an image with the midline echo as close as possible to the horizontal plane, 90 degrees to the ultrasound beam. HC was obtained from the same image as BPD as follows: calipers were placed on the outer borders of the occipital and frontal edges of the bone at the point of the midline of the skull, and the ellipse facility was used to follow the outer perimeter of the skull to calculate HC. AC was measured in the transverse section of the fetal abdomen that was as close as possible to circular and that included the stomach and the junction of the umbilical vein and portal sinus. The anteroposterior and transverse diameters were then measured with calipers placed on the outer borders of the body outline. The anteroposterior diameter was measured from the spine to the anterior abdominal wall, and the transverse diameter at a right angle to the anteroposterior diameter. The ellipse facility was used to calculate AC as outlined above. FL was measured from an image of the full femoral shaft in a plane close to 90 degrees to the ultrasound beam. The distal femoral epiphysis was excluded. Similarly, HL was measured from an image of the full humeral shaft in a plane close to 90 degrees to the ultrasound beam.
The participating centers used identical ultrasound machines during the project (Voluson Expert E8, General Electric, Kretz Ultrasound, Zipf, Austria) equipped with two curvilinear transabdominal transducers (4--8 MHz and 1--5 MHz) and a transvaginal transducer (6--12 MHz), observing that the energy output was set so that thermal index (TI) was \<1.0. The TI was automatically recorded and transmitted to the web-based data management system by the ultrasound machine.
Measurement results were stored electronically, with the images together with all information collected from the mother and the perinatal outcomes. EFW was calculated by including HC, AC, and FL in Hadlock et al.'s third formula \[[@pmed.1002220.ref024]\]. To facilitate assessment of relative fetal head size and growth, the ratios FL/HC and FL/BPD were established.
Training and Quality Assurance {#sec015}
------------------------------
The choice of participating centers was based on their proficient use of ultrasound by experienced sonographers. The sonographers participating in the study received specific training for the study and were certified as proficient under the supervision of a qualified instructor, according to a standard protocol. All the ultrasound operators had their scans assessed for quality during their early period in the project. Instruments and techniques used in all centers were standardized, i.e., equipment and training were provided to each of the measurement teams.
Maternal Anthropometric and Nutritional Assessment and Birthweight {#sec016}
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Weight wearing light clothing was measured using a beam balance with nondetachable weights and recorded to the nearest 0.1 kg. Height of the mother was measured in the standing position using a stadiometer and recorded to the nearest millimeter. If the reading fell between two values, the lower was recorded.
The 24-h diet recall assessment was carried out by a specifically trained nutritionist or nurse who asked the study participant about food and beverages consumed during the previous 24 h \[[@pmed.1002220.ref022]\]. Further details are available elsewhere \[[@pmed.1002220.ref020]\]. Birthweight was assessed at delivery, and neonatal morphometry carried out within 24 h according to the protocol \[[@pmed.1002220.ref020]\].
Data Management {#sec017}
---------------
Data were collected via a web-based data management system developed by Centro Rosarino de Estudios Perinatales, Rosario, Argentina. All data (clinical, anthropometric, nutritional, and fetal biometry measurements plus 2-D/3-D images) were stored in a central server compliant with good clinical practice. Data transmission was encrypted to assure data integrity and patient confidentiality. Access to the web system was password protected, and only authorized users had access. Data changes were documented by a complete audit trail record kept automatically by the web system (recording when, by whom, and why data were changed). Data entered into the web system were checked by the coordinating unit at Centro Rosarino de Estudios Perinatales for completeness, accuracy, reliability, and consistent intended performance. Different kinds of validation procedures were carried out (checking missing values and outliers, cross-checks, cross-time verifications among scanning appointments, and protocol compliance). Measurements and 2-D/3-D images corresponding to fetal biometry had special processing. In collaboration with General Electric Healthcare, Germany, ViewPoint software was installed at all participating centers, allowing a standard interface/procedure for scans and an automatic transfer of fetal biometry measurements/images to the web-based system. Thus, all fetal biometry measurements considered by the protocol were automatically transferred instead of being entered manually (except for D. R. Congo; there, a complete checking of values was done by the comparison of images and values entered into the web-based system). The above mentioned web-based system and procedures have been used in five previous HRP (UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction)/WHO multicenter studies and are proven to be efficient and compliant with HRP/WHO Standard Operating Procedures as well as with Title 21 CFR Part 11 of the Code of Federal Regulations, which deals with United States Food and Drug Administration guidelines on electronic records.
Adjustments of Analyses Compared with the Protocol and Justifications {#sec018}
---------------------------------------------------------------------
Compared with the original protocol \[[@pmed.1002220.ref020]\], the following aspects of the study were adjusted. Reliable information on LMP (confirmed by a measurement of crown--rump length), rather than ultrasound measured crown--rump length alone, was used as the basis for gestational age calculation for the following reasons: there is no evidence that ultrasound dating more accurately determines gestational age than a reliable LMP confirmed by crown--rump length; reliable LMP is the basis for establishing crown--rump length charts for dating; crown--rump length dating translates natural variation of size into variation of gestational age, which is not desirable for a study of growth; and LMP, not crown--rump length, is the accessible, low-cost method for gestational age assessment for all women in the world, and for the low-income areas usually the only one.
The sample size calculation was based on the assumption of normality for the distribution of ultrasound measurements. However, we used quantile regression, which calculates quantiles (i.e., percentiles) directly from the observed measurements without making assumptions about the distribution.
Maternal and fetal conditions occurring during pregnancy were not excluded from the analysis. The rationale for this was that the reference intervals of this study are intended primarily for clinical use and therefore should reflect the population for which they are intended as closely as possible. The pregnancy conditions (e.g., complications) that the study population experienced are those common to low-risk pregnancies around the world. Likewise, excluding all neonates below the 10th percentile of birthweight, as suggested in the protocol \[[@pmed.1002220.ref020]\], would by definition remove the 10% of the participants at the bottom of the range (the vast majority being healthy in this low-risk cohort) and cause a corresponding distortion of the new growth charts, i.e., a substantial upward shift of all the lowest percentiles (10, 5, 2.5, and 1) in the direction of supernormal.
Given the plethora of measurements, we prioritized clinical usefulness in the analyses and results presented here (e.g., EFW and common biometric measurements) and left the following for secondary studies and publications: transverse cerebellar diameter, fetal foot length, 3-D ultrasound acquisitions, maternal anthropometric measurements except height and weight, the second and third sets of dietary 24-h-recall data (at 28 and 36 wk of gestation), and newborn anthropometric measurements except birthweight.
Data Analysis and Statistical Methods {#sec019}
-------------------------------------
Descriptive statistics were calculated for the women's characteristics at study entry, for mode of delivery, for birth events, and for fetal, neonatal, and maternal conditions, by country and overall. Protocol compliance was evaluated by comparing the dates of the windows of gestational age defined in the protocol with the dates of actual measurements.
The ultrasound measurements were used to estimate reference curves for individual parameters (BPD, HC, AC, FL, HL, FL/HC, FL/BPD) and EFW based on Hadlock et al.'s formula 3 \[[@pmed.1002220.ref024]\]. Reference curves were fitted using quantile regression for reference models, as described by Wei et al. \[[@pmed.1002220.ref025]\] from the work of Koenker \[[@pmed.1002220.ref026],[@pmed.1002220.ref027]\].
The development of reference curves has up to now in general used parametric models, based on assumptions about distribution and on transformation of the observations to normal distributions. Advances brought by computer power and by the work of Koenker and others have made it possible to estimate the distributions directly by estimating their quantiles. Quantile regression is now a well-established technique \[[@pmed.1002220.ref026],[@pmed.1002220.ref027]\], and statistical software is available to fit quantile regression models. Quantile regression fits a function to each chosen quantile using linear programming and has the advantage of not imposing any distributional assumptions. The asymmetry and kurtosis of the fitted distributions may thus assume any form dictated by the data, even changing with gestational age. In addition, quantile regression is more robust against the influence of outliers in the data. The flexibility of the fitting and the fact that any inference drawn is entirely data-driven led us to choose quantile regression as the method for the construction of reference curves.
The estimated quantiles were smoothed by polynomial functions of gestational age. Full models fitted a polynomial on gestational age for each country by including interaction terms between gestational age polynomial and country. Additive terms were included for other covariates.
The models were checked by the residual analysis produced by the software. Hypotheses on the overall importance of covariates were formally tested using likelihood ratio or Wald chi-square tests. In addition, visual inspection of quantile profilers was used to assess the relevance of each covariate in explaining the variation. To compare the distributions of the different countries with the overall distribution, we used quantile--quantile plots. We calculated 95% confidence intervals for the difference between country and global EFW percentiles for particular gestational ages, using the result that the parameter estimates from quantile regression were asymptotically normally distributed \[[@pmed.1002220.ref028]\].
Logarithms of ultrasound parameters and EFW were used for the fitting. This was done only to achieve better numerical accuracy and faster convergence of the fitting algorithm. After the fitting, the results were retransformed to the original scale. To describe growth asymmetry, we used the Bowley coefficient of asymmetry \[[@pmed.1002220.ref029]\], based on differences of semi-quartile ranges relative to the quartile range, for the gestational ages 15 and 40 wk.
Data were analyzed using SAS Software version 9.4 (SAS Institute, Cary, North Carolina, US) and JMP Pro 12 (SAS Institute, Cary, North Carolina, US).
Results {#sec020}
=======
Participants {#sec021}
------------
A total of 1,439 women were enrolled between October 2009 and September 2014, with data collection being completed with the last childbirth in April 2015. Of these, 52 (3.6%) withdrew consent, leaving 1,387 women and their fetuses participating in the study. [Table 1](#pmed.1002220.t001){ref-type="table"} shows the numbers of women recruited, those withdrawing consent, those lost to follow-up, and those having miscarriages or intrauterine deaths, by country. Among women lost to follow-up and with miscarriage or intrauterine death, 10 and 15, respectively, did not contribute ultrasound information. All women other than those withdrawing consent were included in the growth curve analyses if they contributed ultrasound information, with the number in this analysis being 1,362.
10.1371/journal.pmed.1002220.t001
###### Number of women recruited to the study by country, with withdrawals and discontinuations.
{#pmed.1002220.t001g}
Country Number of Women Recruited Consent Withdrawal Discontinuation
------------- --------------------------- -------------------- ----------------- -------- --------- -------- ---------
Argentina 143 0 0.0 2 1.4 1 0.7
Brazil 157 4 2.5 2 1.3 3 1.9
D. R. Congo 157 15 9.6 6 3.8 10 6.4
Denmark 142 2 1.4 3 2.1 1 0.7
Egypt 180 25 13.9 11 6.1 9 5.0
France 109 1 0.9 9 8.3 2 1.8
Germany 141 0 0.0 2 1.4 0 0.0
India 146 0 0.0 7 4.8 3 2.1
Norway 140 2 1.4 1 0.7 1 0.7
Thailand 124 3 2.4 3 2.4 4 3.2
**Total** **1,439** **52** **3.6** **46** **3.2** **34** **2.4**
\*Two medical abortions, 29 miscarriages, and three intrauterine deaths.
D. R. Congo, Democratic Republic of the Congo.
Population Characteristics {#sec022}
--------------------------
Statistics for participating women's characteristics, their daily caloric intake, and ethnicity are presented in [Table 2](#pmed.1002220.t002){ref-type="table"}. Median age at study entry was 28 y but varied between 24 y (Argentina and Egypt) and 32 y (France). Median maternal height ranged from 155 cm (India) to 169 cm (Germany), and weight from 54 kg (Thailand) to 66 kg (Germany). While overall median BMI was 23.1 kg/m^2^, the median by country ranged from 21.6 kg/m^2^ in Thailand to 25.9 kg/m^2^ in Egypt. Median daily caloric intake in the study group was 1,848 calories according to the 24-h dietary recall assessment, with Thailand having the lowest median, 1,232 calories, and Egypt having the highest median, 2,094 calories. The ethnic distribution of the study group was roughly 20% African (including the peri-Mediterranean Egypt), 20% Asian, and 60% white.
10.1371/journal.pmed.1002220.t002
###### Characteristics of the participating women by country at study entry.
{#pmed.1002220.t002g}
Characteristic Statistic Argentina (*N* = 143) Brazil (*N* = 153) D. R. Congo (*N* = 142) Denmark (*N* = 140) Egypt (*N* = 155) France (*N* = 108) Germany (*N* = 141) India (*N* = 146) Norway (*N* = 138) Thailand (*N* = 121) Total (*N* = 1,387)
------------------------------------------- ------------------- ----------------------- -------------------- ------------------------- --------------------- ------------------- -------------------- --------------------- ------------------- -------------------- ---------------------- ---------------------
**Age (y)** **Missing** 0 0 0 0 0 0 0 0 0 0 0
**Q1** 20 27 24 28 22 28 28 25 26 26 25
**Median** 24 30 27 30 24 32 31 27 28 29 28
**Q3** 28 33 31 32.5 28 34 33 30 30 32 31
**Weight (kg)** **Missing** 0 0 0 1 8 0 0 0 1 1 11
**Q1** 52 57 53 58 57 57 60 50 59 50 55
**Median** 58 63 60 62 65 63 66 57 63 54 61
**Q3** 64 69 66 67 75 69 72 62 71 59.5 68
**Height (cm)** **Missing** 0 0 0 1 8 0 0 0 1 1 11
**Q1** 153 160 157 164 155 162 165 152 165 155 157
**Median** 157 163 162 168 159 165 169 155 168 157 163
**Q3** 162 167 165 171 163 170 174 160 173 161 168
**BMI (kg/m**^**2**^**)** **Missing** 0 0 0 1 8 0 0 0 1 1 11
**Q1** 21.2 21.6 20.8 20.8 23.5 21.1 21.1 20.0 20.5 20.0 21.0
**Median** 23.3 23.5 22.9 22.2 25.9 22.9 23.2 23.0 22.2 21.6 23.1
**Q3** 26.3 25.8 25.6 24.1 29.0 24.5 24.9 25.3 24.9 23.9 25.4
**Total calories in 24-h dietary recall** **Missing** 0 0 0 0 4 10 0 28 1 6 49
**Q1** 1,666 1,441 1,460 1,584 1,747 1,489 1,674 1,514 1,558 1,004 1,487
**Median** 1,928 1,709 2,063 1,820 2,094 1,736 1,978 1,831 1,890 1,232 1,848
**Q3** 2,189 2,148 2,605 2,053 2,525 2,053 2,285 2,194 2,314 1,534 2,222
**Ethnicity, *n* (percent)** **White** 143 (100.0) 146 (95.4) 0 (0.0) 140 (100.0) 0 (0.0) 100 (92.6) 136 (96.5) 0 (0.0) 137 (99.3) 0 (0.0) 802 (57.8)
**Asian** 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (1.4) 146 (100.0) 1 (0.7) 121 (100.0) 270 (19.5)
**African** 0 (0.0) 7 (4.6) 142 (100.0) 0 (0.0) 133 (85.8) 8 (7.4) 3 (2.1) 0 (0.0) 0 (0.0) 0 (0.0) 293 (21.1)
**Other** 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 22 (14.2) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 22 (1.6)
**Parity (nulliparous *n*)** ***N*** 137 153 142 139 57 108 141 138 138 121 1,274
**Missing** 6 0 0 1 98 0 0 8 0 0 113
***n* (percent)** 64 (46.7) 108 (70.6) 51 (35.9) 86 (61.9) 21 (36.8) 51 (47.2) 104 (73.8) 115 (83.3) 67 (48.6) 72 (59.5) 739 (58.0)
BMI, body mass index; D. R. Congo, Democratic Republic of the Congo; Q1, first quartile; Q3, third quartile.
Perinatal Outcomes {#sec023}
------------------
[Table 3](#pmed.1002220.t003){ref-type="table"} shows delivery information. The overall rate of spontaneous onset of birth was 67.3%, with a wide range by country: 28.5% in Brazil to 94.5% in D. R. Congo. There was an overall cesarean section rate of 32.1%, with a considerable range from 5.5% in D. R. Congo to 70.1% in Brazil. The occurrence of Apgar score \< 7 at 5 min was similar in all countries, i.e., 0%--2.2%. Most of the countries had a similar distribution between female and male neonates except for Egypt, Germany, and Norway, where about 40% of neonates were female. The incidence of preterm birth varied from 3.6% in Germany to 14.7% in Egypt (*p* = 0.03 for differences among countries). It was lowest in D. R. Congo, Denmark, Germany, and Norway and highest in Egypt and India.
10.1371/journal.pmed.1002220.t003
###### Mode of delivery, gestational age at birth and outcomes.
{#pmed.1002220.t003g}
Characteristic Statistic Argentina (*N* = 140) Brazil (*N* = 150) D. R. Congo (*N* = 127) Denmark (*N* = 137) Egypt (*N* = 140) France (*N* = 97) Germany (*N* = 139) India (*N* = 139) Norway (*N* = 136) Thailand (*N* = 114) All (*N* = 1,319)
---------------------------------------- -------------------- ----------------------- -------------------- ------------------------- --------------------- ------------------- ------------------- --------------------- ------------------- -------------------- ---------------------- -------------------
**Neonatal sex (*n* female)** ***N*** 140 148 127 136 132 97 139 137 131 112 1,299
***n* (percent)** 68 (48.6) 70 (47.3) 67 (52.8) 75 (55.1) 54 (40.9) 45 (46.4) 56 (40.3) 67 (48.9) 52 (39.7) 54 (48.2) 608 (46.8)
**Apgar \< 7 at 5 min** ***N*** 140 147 127 135 136 97 139 138 136 113 1,308
***n* (percent)** 1 (0.7) 1 (0.7) 1 (0.8) 1 (0.7) 3 (2.2) 0 (0.0) 1 (0.7) 1 (0.7) 2 (1.5) 0 (0.0) 11 (0.8)
**Preterm (gestational age \< 37 wk)** ***N*** 140 148 127 137 136 97 139 138 136 114 1,312
***n* (percent)** 12 (8.6) 11 (7.4) 6 (4.7) 8 (5.8) 20 (14.7) 7 (7.2) 5 (3.6) 15 (10.9) 6 (4.4) 9 (7.9) 99 (7.5)
**Birthweight (g)** ***N*** 140 148 127 136 117 97 139 137 136 113 1,290
**Q1** 2,990 2,910 2,850 3,133 3,000 2,965 3,100 2,656 3,348 2,980 2,980
**Median** 3,328 3,290 3,170 3,462 3,100 3,370 3,480 2,975 3,575 3,130 3,300
**Q3** 3,620 3,608 3,500 3,790 3,500 3,600 3,820 3,200 3,900 3,400 3,615
**Gestational age (days)** ***N*** 140 148 127 137 139 97 139 138 136 114 1,315
**Q1** 270 268 270 272 262 273 273 265 276 267 269
**Median** 276 273 277 282 271 279 279 270 283 271 276
**Q3** 281 278 283 287 280 284 285 277 288 278 282
**Mode of delivery, *n* (percent)** **Spontaneous** 91 (67.9) 41 (28.5) 120 (94.5) 105 (83.3) 64 (45.7) 80 (85.1) 82 (73.2) 84 (64.1) 113 (91.1) 58 (50.9) 838 (67.3)
**Intrapartum CS** 30 (22.4) 33 (22.9) 6 (4.7) 7 (5.6) 16 (11.4) 8 (8.5) 24 (21.4) 20 (15.3) 9 (7.3) 26 (22.8) 179 (14.4)
**Elective CS** 13 (9.7) 68 (47.2) 1 (0.8) 13 (10.3) 54 (38.6) 6 (6.4) 6 (5.4) 27 (20.6) 2 (1.6) 30 (26.3) 220 (17.7)
**Vacuum** 0 (0.0) 0 (0.0) 0 (0.0) 11 (8.7) 0 (0.0) 0 (0.0) 25 (22.3) 5 (3.8) 1 (0.8) 0 (0.0) 42 (3.4)
**Forceps** 6 (4.5) 6 (4.2) 0 (0.0) 0 (0.0) 0 (0.0) 3 (3.2) 2 (1.8) 3 (2.3) 11 (8.9) 0 (0.0) 31 (2.5)
**Unknown** 0 (0.0) 2 (1.4) 0 (0.0) 1 (0.8) 6 (4.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 9 (0.7)
CS, cesarean section; D. R. Congo, Democratic Republic of the Congo; Q1, first quartile; Q3, third quartile.
Gestational Age at Birth and Birthweight {#sec024}
----------------------------------------
Gestational age at birth varied between countries from a median of 38 wk 4 d in India to 40 wk 3 d in Norway (*p \<* 0.001 for differences among countries) ([Table 3](#pmed.1002220.t003){ref-type="table"}). Norway had the highest median birthweight (3,575 g), and Denmark and Germany had birthweights approximately 100 g less, while Argentina, Brazil, and France had birthweights 200 g less. There is a group of countries (D. R. Congo, Egypt, and Thailand) with birthweight a median 400 g less than that of Norway, and lastly India, with birthweight 500 g less. The differences in birthweight between countries were highly significant for all percentiles (*p \<* 0.001 for all). When adjusted for gestational age at birth, the differences were still significant for all the percentiles (*p* = 0.0018 for the 5th percentile and *p \<* 0.001 for the 10th, 25th, 50th, 75th, 90th, and 95th percentiles). The estimated birthweight according to neonatal sex and gestational age is shown in [Table 4](#pmed.1002220.t004){ref-type="table"}.
10.1371/journal.pmed.1002220.t004
###### Estimated birthweight percentiles for female and male neonates according to completed gestational week.
{#pmed.1002220.t004g}
Percentile Birthweight (g) by Gestational Age (wk)
------------ ----------------------------------------- ------- ------- ------- ------- ------- ------- ------- ------- ------- ------- -------
**5** 1,968 2,315 2,575 2,748 2,835 2,834 2,062 2,451 2,723 2,880 2,921 2,845
**25** 2,493 2,698 2,891 3,072 3,241 3,398 2,705 2,890 3,061 3,218 3,362 3,491
**50** 2,786 2,990 3,173 3,336 3,479 3,601 2,919 3,153 3,354 3,519 3,650 3,747
**75** 2,951 3,217 3,443 3,631 3,779 3,888 3,143 3,387 3,608 3,806 3,982 4,134
**90** 3,181 3,451 3,682 3,871 4,021 4,130 3,450 3,666 3,871 4,067 4,253 4,428
**95** 3,238 3,593 3,867 4,060 4,171 4,200 3,584 3,813 4,036 4,251 4,459 4,659
Maternal Complications and Perinatal Conditions {#sec025}
-----------------------------------------------
Conditions occurring in the mother during pregnancy are shown in [Table 5](#pmed.1002220.t005){ref-type="table"}, together with fetal malformations and neonatal conditions. In addition to globally experienced maternal complications such as preeclampsia, pregnancy-induced hypertension, gestational diabetes, and anemia, 42 had identified malaria. There was no maternal death. Four small-for-gestational-age fetuses were identified clinically, of which two were examined using Doppler ultrasound; none had abnormal recordings in the umbilical artery or middle cerebral artery, and all were kept in the analysis. It was registered when neonates needed transmission to the neonatal intensive care unit, commonly due to prematurity, respiratory distress syndrome, infections, or jaundice. There were three intrauterine deaths and three neonatal deaths, representing a perinatal mortality of 0.4%.
10.1371/journal.pmed.1002220.t005
###### Maternal complications, fetal malformations, and neonatal conditions by country.
{#pmed.1002220.t005g}
Condition Argentina (*N* = 143) Brazil (*N* = 153) D. R. Congo (*N* = 142) Denmark (*N* = 140) Egypt (*N* = 155) France (*N* = 108) Germany (*N* = 141) India (*N* = 146) Norway (*N* = 138) Thailand (*N* = 121) All (*N* = 1,387)
------------------------------------------------------------------ ----------------------- -------------------- ------------------------- --------------------- ------------------- -------------------- --------------------- ------------------- -------------------- ---------------------- -------------------
**Fetal malformation**[^**§**^](#t005fn002){ref-type="table-fn"} 4 (2.8) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.6) 1 (0.9) 1 (0.7) 0 (0.0) 1 (0.7) 0 (0.0) 8 (0.6)
**Neonatal condition** 19 (13.3) 12 (7.8) 7 (4.9) 10 (7.1) 4 (2.6) 2 (1.9) 9 (6.4) 8 (5.5) 3 (2.2) 9 (7.4) 83 (6.0)
**Maternal complication**[\*](#t005fn003){ref-type="table-fn"} 24 (16.8) 10 (6.5) 42 (29.6) 4 (2.9) 3 (1.9) 8 (7.4) 7 (5.0) 23 (15.8) 6 (4.3) 10 (8.3) 137 (9.9)
Data are given as *n* (percent).
^**§**^One malformation was discovered at birth, here counted as fetal malformation. Sacrococcygeal cyst (1), Jarcho-Levin syndrome (1), clubfoot (1), polycystic kidneys (1), cardiac malformations (3), cleft palate (1).
\*Preeclampsia (22), hypertension (16), gestational diabetes (32), malaria (42), anemia (19), and other (16); some participants had more than one diagnosis.
D. R. Congo, Democratic Republic of the Congo.
Compliance with Ultrasound Scans {#sec026}
--------------------------------
The median number of ultrasound scans (excluding the study entry screening scan) in all women was 6 (range 0--7). Compliance by gestational age window as defined in the protocol is presented in [S1 Table](#pmed.1002220.s008){ref-type="supplementary-material"}, by country and for all countries combined ("Total"). Compliance for all countries combined in each gestational age window was between 89.1% and 100%; 72% of the participants had a complete set of all the scheduled scans. In addition, for each of the measurements BPD, HC, AC, FL, and HL, scans were obtained ≥2 times for at least 95% of participants.
Thermal Index {#sec027}
-------------
Of the 8,372 scan sessions in the project, 115 had no scans stored and 54 belonged to women who withdrew consent, leaving 8,203 for the statistics. The median TI was 0.2, and none had TI ≥ 1.0.
Reference Intervals for Biometric Parameters and Estimated Fetal Weight {#sec028}
-----------------------------------------------------------------------
[Fig 1](#pmed.1002220.g001){ref-type="fig"} presents the overall growth curves for BPD, HC, AC, FL, HL, and EFW, and for the ratios FL/HC and FL/BPD, based on quantile regression. The corresponding reference values are shown in Tables [6](#pmed.1002220.t006){ref-type="table"}--[13](#pmed.1002220.t013){ref-type="table"} and in csv format in [S1 File](#pmed.1002220.s007){ref-type="supplementary-material"}.
{#pmed.1002220.g001}
10.1371/journal.pmed.1002220.t006
###### Growth chart for fetal outer--inner biparietal diameter.
{#pmed.1002220.t006g}
Gestational Age (Weeks) Biparietal Diameter (mm) by Percentile
------------------------- ---------------------------------------- ---- ---- ---- ---- ---- ----- ----- -----
14 23 24 24 26 27 28 29 30 31
15 26 27 27 29 30 31 32 33 34
16 29 30 30 32 33 35 36 37 38
17 32 33 33 35 36 38 39 40 41
18 35 36 37 38 40 41 43 44 45
19 38 39 40 42 43 45 46 47 48
20 41 42 43 45 47 48 50 51 52
21 44 45 46 48 50 52 53 54 55
22 47 48 50 51 53 55 57 58 59
23 50 52 53 55 57 59 60 61 62
24 53 55 56 58 60 62 64 65 66
25 56 58 59 61 63 65 67 68 69
26 59 60 62 64 66 68 70 71 72
27 62 63 65 67 69 71 73 74 75
28 64 66 67 69 72 74 76 77 78
29 67 68 70 72 74 76 78 80 81
30 69 71 72 74 77 79 81 82 83
31 71 73 74 76 79 81 83 85 86
32 73 75 76 79 81 83 86 87 88
33 75 77 78 81 83 86 88 89 90
34 77 79 80 83 85 88 90 91 92
35 79 80 82 84 87 89 92 93 94
36 80 82 84 86 89 91 93 95 96
37 82 84 85 88 90 93 95 96 97
38 84 85 87 90 92 95 97 98 99
39 85 87 89 92 94 96 99 100 101
40 87 88 90 93 96 98 100 101 102
10.1371/journal.pmed.1002220.t007
###### Growth chart for fetal head circumference.
{#pmed.1002220.t007g}
Gestational Age (Weeks) Head Circumference (mm) by Percentile
------------------------- --------------------------------------- ----- ----- ----- ----- ----- ----- ----- -----
14 86 88 91 95 100 104 107 110 112
15 97 99 102 106 111 115 119 122 124
16 108 111 114 118 123 128 132 134 137
17 120 123 126 130 135 140 144 147 149
18 132 135 138 143 148 153 157 160 162
19 145 147 150 155 161 166 170 173 175
20 157 159 163 168 173 179 183 186 188
21 169 172 175 180 186 191 196 199 201
22 181 184 187 193 198 204 209 212 214
23 193 196 199 205 210 216 221 224 227
24 204 207 211 216 222 228 233 236 239
25 215 218 222 227 233 239 245 248 251
26 225 228 232 238 244 250 256 259 262
27 234 238 242 248 254 261 267 270 273
28 243 247 251 257 264 270 277 280 283
29 251 256 260 266 273 280 286 290 293
30 259 264 268 274 281 288 295 299 302
31 266 271 275 282 289 296 303 307 311
32 273 278 282 289 296 304 311 315 318
33 279 284 289 295 303 311 318 322 326
34 285 290 295 302 309 317 324 328 332
35 291 296 300 307 315 323 330 335 338
36 296 301 306 313 321 329 336 340 344
37 302 306 311 318 326 334 341 345 349
38 307 311 315 324 332 339 347 350 354
39 313 316 320 329 337 344 352 355 359
40 319 321 325 334 342 350 357 360 363
10.1371/journal.pmed.1002220.t008
###### Growth chart for fetal abdominal circumference.
{#pmed.1002220.t008g}
Gestational Age (Weeks) Abdominal Circumference (mm) by Percentile
------------------------- -------------------------------------------- ----- ----- ----- ----- ----- ----- ----- -----
14 69 71 73 77 81 86 89 92 95
15 79 81 83 87 92 96 100 103 106
16 89 91 93 98 103 108 112 115 118
17 99 102 104 109 114 119 124 127 130
18 110 113 116 121 126 131 136 139 142
19 121 124 127 132 138 143 148 152 155
20 132 136 139 144 150 155 161 164 167
21 143 147 150 156 162 168 173 177 180
22 154 159 162 167 173 180 186 189 193
23 165 170 173 179 185 192 198 202 205
24 176 181 184 190 197 203 210 214 217
25 186 191 195 201 208 215 222 226 229
26 196 201 205 212 219 226 233 238 241
27 206 211 215 222 230 237 245 249 253
28 215 220 225 232 240 248 256 260 264
29 224 229 234 242 250 258 266 271 276
30 233 238 243 251 260 269 277 282 287
31 241 246 252 260 269 279 287 292 298
32 249 254 260 269 279 288 298 303 308
33 257 262 269 278 288 298 308 313 319
34 265 270 277 287 298 308 318 324 330
35 273 279 286 297 307 318 329 335 342
36 282 287 294 306 317 329 340 346 353
37 290 296 304 316 328 340 352 358 365
38 299 306 313 326 338 351 364 371 378
39 309 316 324 337 350 363 377 384 392
40 319 327 335 349 363 377 391 399 406
10.1371/journal.pmed.1002220.t009
###### Growth chart for fetal femur length.
{#pmed.1002220.t009g}
Gestational Age (Weeks) Femur Length (mm) by Percentile
------------------------- --------------------------------- ---- ---- ---- ---- ---- ---- ---- ----
14 10 10 11 12 13 14 15 16 17
15 12 13 14 15 16 17 18 19 20
16 15 16 17 18 19 20 22 22 23
17 19 19 20 21 22 24 25 26 26
18 22 22 23 24 26 27 28 29 30
19 25 26 26 28 29 30 31 32 33
20 28 29 30 31 32 33 35 35 36
21 31 32 33 34 35 36 38 38 39
22 34 35 35 37 38 39 40 41 42
23 36 37 38 39 41 42 43 44 45
24 39 40 41 42 43 45 46 47 47
25 41 42 43 44 46 47 48 49 50
26 43 44 45 46 48 49 51 51 52
27 46 46 47 49 50 52 53 54 55
28 48 48 49 51 52 54 55 56 57
29 50 50 51 53 54 56 57 58 59
30 51 52 53 55 56 58 60 60 61
31 53 54 55 57 59 60 62 63 64
32 55 56 57 59 61 62 64 65 66
33 57 58 60 61 63 65 66 67 68
34 59 60 61 63 65 67 68 69 70
35 61 62 63 65 67 69 70 71 73
36 63 64 65 67 69 70 72 73 75
37 65 66 67 68 70 72 74 75 76
38 66 67 68 70 72 74 75 77 78
39 67 68 69 70 73 75 76 78 79
40 68 68 69 70 73 75 77 78 79
10.1371/journal.pmed.1002220.t010
###### Growth chart for fetal humerus length.
{#pmed.1002220.t010g}
Gestational Age (Weeks) Humerus Length (mm) by Percentile
------------------------- ----------------------------------- ---- ---- ---- ---- ---- ---- ---- ----
14 10 11 11 12 14 15 16 16 17
15 13 13 14 15 16 18 19 19 20
16 16 16 17 18 19 21 22 22 23
17 19 19 20 21 23 24 25 25 26
18 22 22 23 24 26 27 28 28 29
19 25 25 26 27 28 30 31 31 32
20 27 28 29 30 31 32 33 34 35
21 30 31 31 33 34 35 36 37 38
22 32 33 34 35 36 37 39 39 40
23 34 35 36 37 38 40 41 42 42
24 36 37 38 39 41 42 43 44 45
25 38 39 40 41 42 44 45 46 47
26 40 41 42 43 44 46 47 48 49
27 42 43 43 45 46 47 49 50 51
28 43 44 45 46 48 49 51 52 52
29 45 46 47 48 49 51 52 53 54
30 46 47 48 50 51 53 54 55 56
31 48 49 50 51 53 54 56 57 58
32 49 50 51 53 54 56 57 59 59
33 51 52 53 54 56 58 59 60 61
34 53 53 54 56 58 59 61 62 63
35 54 55 56 57 59 61 62 63 64
36 55 56 57 59 61 62 64 65 66
37 56 57 58 60 62 64 65 66 67
38 57 58 59 61 63 65 66 67 68
39 58 59 60 62 64 65 67 68 69
40 57 58 60 62 64 66 68 69 69
10.1371/journal.pmed.1002220.t011
###### Growth chart for estimated fetal weight regardless of fetal sex.
{#pmed.1002220.t011g}
Gestational Age (Weeks) Estimated Fetal Weight (g) by Percentile
------------------------- ------------------------------------------ ------- ------- ------- ------- ------- ------- ------- -------
14 70 73 78 83 90 98 104 109 113
15 89 93 99 106 114 124 132 138 144
16 113 117 124 133 144 155 166 174 181
17 141 146 155 166 179 193 207 217 225
18 174 181 192 206 222 239 255 268 278
19 214 223 235 252 272 292 313 328 340
20 260 271 286 307 330 355 380 399 413
21 314 327 345 370 398 428 458 481 497
22 375 392 412 443 476 512 548 575 595
23 445 465 489 525 565 608 650 682 705
24 523 548 576 618 665 715 765 803 830
25 611 641 673 723 778 836 894 938 970
26 707 743 780 838 902 971 1,038 1,087 1,125
27 813 855 898 964 1,039 1,118 1,196 1,251 1,295
28 929 977 1,026 1,102 1,189 1,279 1,368 1,429 1,481
29 1,053 1,108 1,165 1,251 1,350 1,453 1,554 1,622 1,682
30 1,185 1,247 1,313 1,410 1,523 1,640 1,753 1,828 1,897
31 1,326 1,394 1,470 1,579 1,707 1,838 1,964 2,046 2,126
32 1,473 1,548 1,635 1,757 1,901 2,047 2,187 2,276 2,367
33 1,626 1,708 1,807 1,942 2,103 2,266 2,419 2,516 2,619
34 1,785 1,872 1,985 2,134 2,312 2,492 2,659 2,764 2,880
35 1,948 2,038 2,167 2,330 2,527 2,723 2,904 3,018 3,148
36 2,113 2,205 2,352 2,531 2,745 2,959 3,153 3,277 3,422
37 2,280 2,372 2,537 2,733 2,966 3,195 3,403 3,538 3,697
38 2,446 2,536 2,723 2,935 3,186 3,432 3,652 3,799 3,973
39 2,612 2,696 2,905 3,135 3,403 3,664 3,897 4,058 4,247
40 2,775 2,849 3,084 3,333 3,617 3,892 4,135 4,312 4,515
10.1371/journal.pmed.1002220.t012
###### Growth chart for fetal femur length/head circumference ratio.
{#pmed.1002220.t012g}
Gestational Age (Weeks) Femur Length/Head Circumference Ratio by Percentile
------------------------- ----------------------------------------------------- ------ ------ ------ ------ ------ ------ ------ ------
14 0.50 0.52 0.53 0.54 0.56 0.57 0.59 0.59 0.60
15 0.54 0.55 0.56 0.57 0.59 0.60 0.61 0.62 0.62
16 0.57 0.58 0.59 0.60 0.61 0.62 0.63 0.64 0.64
17 0.60 0.60 0.61 0.62 0.63 0.64 0.65 0.65 0.66
18 0.62 0.62 0.63 0.64 0.65 0.66 0.66 0.67 0.67
19 0.64 0.64 0.65 0.65 0.66 0.67 0.68 0.68 0.68
20 0.65 0.66 0.66 0.67 0.67 0.68 0.69 0.69 0.69
21 0.66 0.67 0.67 0.68 0.68 0.69 0.69 0.70 0.70
22 0.67 0.67 0.68 0.68 0.69 0.69 0.70 0.70 0.71
23 0.68 0.68 0.68 0.69 0.69 0.70 0.70 0.71 0.71
24 0.68 0.69 0.69 0.69 0.70 0.70 0.71 0.71 0.71
25 0.69 0.69 0.69 0.70 0.70 0.71 0.71 0.71 0.72
26 0.69 0.69 0.69 0.70 0.70 0.71 0.71 0.72 0.72
27 0.69 0.69 0.70 0.70 0.71 0.71 0.72 0.72 0.72
28 0.69 0.70 0.70 0.70 0.71 0.71 0.72 0.72 0.72
29 0.70 0.70 0.70 0.71 0.71 0.72 0.72 0.72 0.73
30 0.70 0.70 0.70 0.71 0.71 0.72 0.72 0.73 0.73
31 0.70 0.70 0.71 0.71 0.72 0.72 0.73 0.73 0.73
32 0.70 0.71 0.71 0.72 0.72 0.73 0.73 0.73 0.74
33 0.71 0.71 0.71 0.72 0.72 0.73 0.73 0.74 0.74
34 0.71 0.71 0.72 0.72 0.73 0.73 0.74 0.74 0.74
35 0.71 0.72 0.72 0.73 0.73 0.74 0.74 0.74 0.75
36 0.72 0.72 0.72 0.73 0.73 0.74 0.74 0.75 0.75
37 0.72 0.72 0.73 0.73 0.74 0.74 0.74 0.75 0.75
38 0.72 0.72 0.73 0.73 0.74 0.74 0.75 0.75 0.75
39 0.72 0.72 0.73 0.73 0.74 0.74 0.75 0.75 0.75
40 0.71 0.72 0.72 0.73 0.73 0.74 0.75 0.75 0.75
10.1371/journal.pmed.1002220.t013
###### Growth chart for fetal femur length/biparietal diameter.
{#pmed.1002220.t013g}
Gestational Age (Weeks) Femur Length/Biparietal Diameter Ratio by Percentile
------------------------- ------------------------------------------------------ ------ ------ ------ ------ ------ ------ ------ ------
14 0.71 0.72 0.74 0.76 0.78 0.80 0.82 0.83 0.84
15 0.75 0.76 0.77 0.79 0.81 0.83 0.84 0.85 0.86
16 0.79 0.80 0.81 0.82 0.84 0.85 0.87 0.88 0.88
17 0.82 0.82 0.83 0.85 0.86 0.87 0.89 0.89 0.90
18 0.84 0.85 0.85 0.87 0.88 0.89 0.90 0.91 0.91
19 0.86 0.86 0.87 0.88 0.89 0.90 0.91 0.92 0.92
20 0.87 0.88 0.88 0.89 0.90 0.91 0.92 0.93 0.93
21 0.88 0.89 0.89 0.90 0.91 0.92 0.93 0.93 0.94
22 0.89 0.89 0.90 0.91 0.92 0.92 0.93 0.94 0.94
23 0.89 0.90 0.90 0.91 0.92 0.93 0.94 0.94 0.95
24 0.90 0.90 0.91 0.91 0.92 0.93 0.94 0.94 0.95
25 0.90 0.90 0.91 0.92 0.92 0.93 0.94 0.94 0.95
26 0.90 0.91 0.91 0.92 0.93 0.93 0.94 0.95 0.95
27 0.90 0.91 0.91 0.92 0.93 0.93 0.94 0.95 0.95
28 0.90 0.91 0.91 0.92 0.93 0.94 0.94 0.95 0.95
29 0.90 0.91 0.91 0.92 0.93 0.94 0.94 0.95 0.95
30 0.91 0.91 0.91 0.92 0.93 0.94 0.94 0.95 0.95
31 0.91 0.91 0.92 0.92 0.93 0.94 0.95 0.95 0.95
32 0.91 0.91 0.92 0.93 0.93 0.94 0.95 0.95 0.96
33 0.91 0.92 0.92 0.93 0.94 0.94 0.95 0.96 0.96
34 0.92 0.92 0.92 0.93 0.94 0.95 0.95 0.96 0.96
35 0.92 0.92 0.93 0.93 0.94 0.95 0.95 0.96 0.96
36 0.92 0.93 0.93 0.94 0.94 0.95 0.96 0.96 0.97
37 0.92 0.93 0.93 0.94 0.94 0.95 0.96 0.96 0.97
38 0.92 0.93 0.93 0.94 0.95 0.95 0.96 0.96 0.97
39 0.92 0.92 0.93 0.94 0.94 0.95 0.96 0.96 0.97
40 0.91 0.92 0.92 0.93 0.94 0.95 0.96 0.96 0.97
The distribution of EFW starts with a slight asymmetry to the left (i.e., lower percentiles) in early pregnancy and ends with a very noticeable right asymmetry (i.e., higher percentiles) in later pregnancy. The Bowley coefficient of asymmetry \[[@pmed.1002220.ref029]\], based on differences of semi-quartile ranges relative to the quartile range, was −0.016 for gestational age 15 wk and +0.111 for 40 wk.
Influence of Covariates on Growth Percentiles {#sec029}
---------------------------------------------
### Fetal sex {#sec030}
Male fetuses were larger than female fetuses as measured by EFW, but the disparity was smaller in the lower quantiles of the distribution (3.5%) and larger in the upper quantiles (4.5%) (Fig [2](#pmed.1002220.g002){ref-type="fig"} and [S2 Table](#pmed.1002220.s009){ref-type="supplementary-material"}, without adjustment for country differences). This difference in size by fetal sex was significant at the 5% level for all percentiles. EFW reference values were also established for female and male fetuses separately (Tables [14](#pmed.1002220.t014){ref-type="table"} and [15](#pmed.1002220.t015){ref-type="table"}) to allow assessment customized according to fetal sex. For example, at gestational week 37, the median EFW of female fetuses is 84 g lower than that of male fetuses.
{#pmed.1002220.g002}
10.1371/journal.pmed.1002220.t014
###### Growth chart for estimated fetal weight for female fetuses.
{#pmed.1002220.t014g}
Gestational Age (Weeks) Female Estimated Fetal Weight (g) by Percentile
------------------------- ------------------------------------------------- ------- ------- ------- ------- ------- -------
14 73 77 82 89 96 102 107
15 92 97 104 113 121 129 135
16 116 122 131 141 152 162 170
17 145 152 164 176 189 202 211
18 180 188 202 217 233 248 261
19 221 231 248 266 285 304 319
20 269 281 302 322 346 369 387
21 324 339 364 388 417 444 466
22 388 405 435 464 499 530 557
23 461 481 516 551 592 629 660
24 542 567 608 649 697 740 776
25 634 663 710 758 815 865 907
26 735 769 823 880 946 1,003 1,051
27 846 886 948 1,014 1,090 1,156 1,210
28 967 1,013 1,083 1,160 1,247 1,323 1,383
29 1,096 1,150 1,230 1,319 1,418 1,505 1,570
30 1,234 1,296 1,386 1,489 1,601 1,699 1,770
31 1,379 1,451 1,553 1,670 1,796 1,907 1,984
32 1,530 1,614 1,728 1,861 2,002 2,127 2,209
33 1,687 1,783 1,911 2,060 2,217 2,358 2,445
34 1,847 1,957 2,101 2,268 2,440 2,598 2,690
35 2,008 2,135 2,296 2,481 2,669 2,846 2,943
36 2,169 2,314 2,494 2,698 2,902 3,099 3,201
37 2,329 2,493 2,695 2,917 3,138 3,357 3,462
38 2,484 2,670 2,896 3,136 3,373 3,616 3,725
39 2,633 2,843 3,096 3,354 3,605 3,875 3,988
40 2,775 3,010 3,294 3,567 3,832 4,131 4,247
10.1371/journal.pmed.1002220.t015
###### Growth chart for estimated fetal weight (EFW) for male fetuses.
{#pmed.1002220.t015g}
Gestational Age (Weeks) Male Estimated Fetal Weight (g) by Percentile
------------------------- ----------------------------------------------- ------- ------- ------- ------- ------- -------
14 75 79 84 92 99 105 109
15 96 100 107 116 126 134 139
16 121 127 136 146 158 169 175
17 152 158 170 183 197 210 219
18 188 196 210 226 243 260 271
19 232 241 258 277 298 320 333
20 282 293 314 337 362 389 405
21 341 354 380 407 436 469 489
22 408 424 454 487 522 561 586
23 484 503 539 578 619 666 695
24 570 592 635 681 730 785 818
25 666 692 742 795 853 917 956
26 772 803 860 923 990 1,063 1,109
27 888 924 989 1,063 1,141 1,224 1,276
28 1,014 1,055 1,129 1,215 1,305 1,399 1,458
29 1,149 1,197 1,281 1,379 1,482 1,587 1,654
30 1,293 1,349 1,442 1,555 1,672 1,788 1,863
31 1,445 1,509 1,613 1,741 1,874 2,000 2,085
32 1,605 1,677 1,793 1,937 2,085 2,224 2,319
33 1,770 1,852 1,980 2,140 2,306 2,456 2,562
34 1,941 2,032 2,174 2,350 2,534 2,694 2,814
35 2,114 2,217 2,372 2,565 2,767 2,938 3,072
36 2,290 2,404 2,574 2,783 3,002 3,185 3,334
37 2,466 2,591 2,777 3,001 3,238 3,432 3,598
38 2,641 2,778 2,981 3,218 3,472 3,676 3,863
39 2,813 2,962 3,183 3,432 3,701 3,916 4,125
40 2,981 3,142 3,382 3,639 3,923 4,149 4,383
### Country {#sec031}
Countries differed in EFW ([Fig 3](#pmed.1002220.g003){ref-type="fig"}). Using country as a covariate in a quantile regression model, including interaction terms with gestational age, showed significance at the 5% level for all percentiles 5th, 10th, 25th, 50th, 75th, 90th, and 95th ([S2](#pmed.1002220.s009){ref-type="supplementary-material"} and [S3](#pmed.1002220.s010){ref-type="supplementary-material"} Tables). This variation due to country was adjusted for maternal characteristics (mother's age, parity, height, and weight, or with BMI substituting the latter two) and sex of the fetus. To assess the relative contribution of these variables to the variation in EFW, the Wald chi-square statistics in [S2](#pmed.1002220.s009){ref-type="supplementary-material"} and [S3](#pmed.1002220.s010){ref-type="supplementary-material"} Tables are informative, e.g., for the 5th percentile (quantile 0.05, first table in [S2 Table](#pmed.1002220.s009){ref-type="supplementary-material"}), as expected, most of the variation (Wald chi-square = 1,797, 1 df) is due to gestational age (linear) as the fetus grows, and there is significant curvature (Wald chi-square = 207, 1 df). Country variation gives Wald chi-square = 36 (9 df); sex of the fetus, 29 (1 df); mother's height, 26 (1 df); and mother's age, 22 (1 df), while the Wald chi-square value for weight is negligible. In the same table, the level of significance is listed for these variables, e.g., *p* \< 0.001 for country, highly significant. It is clear that variation due to country also occurs independently of maternal characteristics and the sex of the fetus. [Fig 3](#pmed.1002220.g003){ref-type="fig"} offers a visualization of country variation for the 10th, 50th, and 90th percentiles for EFW. Country variation in the other ultrasound parameters for the 10th, 50th, and 90th percentiles is presented in [S2](#pmed.1002220.s002){ref-type="supplementary-material"}--[S6](#pmed.1002220.s006){ref-type="supplementary-material"} Figs. Country differences in EFW percentiles and overall EFW percentiles are presented in [S4 Table](#pmed.1002220.s011){ref-type="supplementary-material"}.
{#pmed.1002220.g003}
The clinical relevance of the differences between the country quantiles and the global quantiles can be assessed in quantile--quantile plots ([Fig 4](#pmed.1002220.g004){ref-type="fig"}). These plots are intended to enable the reader to derive the magnitude of difference in grams for any size and country and percentile. For example, consider the quantile--quantile plot for the individual country 0.05 quantile (i.e., the 5th percentile) for EFW versus the global 0.05 quantile: the 5th percentiles at low values of EFW cannot be differentiated because of the relative smallness of EFW at early pregnancy ([Fig 4](#pmed.1002220.g004){ref-type="fig"}). However, at the end of gestation (high values of EFW), the 5th percentile for Norway is 3,200 g, while the overall 5th percentile is 2,800 g; for France it is 2,800 g, and for Egypt, 2,700 g. Similarly, it can be seen that while the 10th percentile for EFW at the end of gestation for Norway is 3,400 g, it is 2,700 g for India (versus about 3,100 g for the global 10th percentile), showing that a fetus weighing 3,200 g would be below the 10th percentile for Norway but well above it for India. The magnitude of the differences among countries can also be appreciated in [Fig 5](#pmed.1002220.g005){ref-type="fig"}, where selected country percentiles are shown with the corresponding global percentile curve.
{#pmed.1002220.g004}
{#pmed.1002220.g005}
### Maternal age and maternal height {#sec032}
Maternal age and height seem to be associated with a positive effect on EFW, especially in the lower tail of the distribution, significant at the 5% level, of the order of 2% to 3% for each additional 10 y of age of the mother and 1% to 2% for each additional 10 cm of height ([S1D and S1F Fig](#pmed.1002220.s001){ref-type="supplementary-material"}, without adjusting for country differences).
### Maternal weight {#sec033}
Maternal weight seems to be associated with a small positive effect on EFW, especially in the higher tail of the distribution, significant at the 5% level, of the order of 1% to 1.5% for each additional 10 kg of weight of the mother ([S1E Fig](#pmed.1002220.s001){ref-type="supplementary-material"}, without adjusting for country differences).
### Parity (0 versus ≥1) {#sec034}
Parous women had heavier fetuses than nulliparous women, with the disparity being much higher in the lower quantiles of the distribution, of the order of 1% to 3%, significant at the 5% level, and subsiding in the upper quantiles ([S1C Fig](#pmed.1002220.s001){ref-type="supplementary-material"}, without adjusting for country differences).
Influence of Clinical Conditions on Growth Percentiles {#sec035}
------------------------------------------------------
Participants for whom clinical conditions occurred during pregnancy and childbirth were retained in the study. We then assessed the effect of excluding them on the parameter estimates of the quantiles. We excluded successively maternal conditions, fetal malformations, and neonatal conditions and assessed the fit for the global EFW percentiles. The parameter estimates obtained were indistinguishable.
In order to illustrate variation of the clinically relevant 10th and 90th percentiles for EFW, we compiled the values (without any formal comparison) for 24, 28, 32, and 36 wk of gestation from the present study, the NICHD Fetal Growth Studies \[[@pmed.1002220.ref019]\], a study from D. R. Congo \[[@pmed.1002220.ref030]\], and another study from Norway \[[@pmed.1002220.ref031]\] ([Table 16](#pmed.1002220.t016){ref-type="table"}). Since the other existing multinational study, the Fetal Growth Longitudinal Study of the Intergrowth-21st Project, did not publish EFW but rather AC, which is a major determinant for EFW, we also compiled 10th and 90th percentiles for AC from relevant studies \[[@pmed.1002220.ref018],[@pmed.1002220.ref019],[@pmed.1002220.ref030],[@pmed.1002220.ref032]--[@pmed.1002220.ref034]\] ([Table 17](#pmed.1002220.t017){ref-type="table"}).
10.1371/journal.pmed.1002220.t016
###### The 10th and 90th percentile for estimated fetal weight in relation to other relevant reference values.
{#pmed.1002220.t016g}
Reference Chart Gestational Week
---------------------------------------------------- ------------------ --------- ----------- ----------- -----------
**10th percentile of EFW (g)**
US, white[^¶^](#t016fn002){ref-type="table-fn"} 289 583 1,045 1,686 2,432
D. R. Congo[^\#^](#t016fn003){ref-type="table-fn"} 288 576 1,023 1,624 2,310
**WHO** **286** **576** **1,026** **1,635** **2,352**
US, black[^¶^](#t016fn002){ref-type="table-fn"} 286 559 985 1,579 2,264
Norway[\*](#t016fn004){ref-type="table-fn"} 283 610 1,102 1,730 2,411
US, Hispanic[^¶^](#t016fn002){ref-type="table-fn"} 279 555 987 1,595 2,298
US, Asian[^¶^](#t016fn002){ref-type="table-fn"} 275 546 978 1,574 2,262
**90th percentile of EFW (g)**
Norway[\*](#t016fn004){ref-type="table-fn"} 408 833 1,472 2,304 3,230
US, white[^¶^](#t016fn002){ref-type="table-fn"} 381 771 1,391 2,276 3,368
**WHO** **380** **765** **1,368** **2,187** **3,153**
US, Hispanic[^¶^](#t016fn002){ref-type="table-fn"} 379 755 1,353 2,209 3,245
US, black[^¶^](#t016fn002){ref-type="table-fn"} 376 742 1,317 2,135 3,115
US, Asian[^¶^](#t016fn002){ref-type="table-fn"} 373 737 1,318 2,129 3,111
D. R. Congo[^\#^](#t016fn003){ref-type="table-fn"} 345 700 1,277 2,083 3,032
Percentiles from the present multinational study (bold), a recent multiethnic national study in the US \[[@pmed.1002220.ref019]\], a study from D. R. Congo \[[@pmed.1002220.ref030]\], and another study from Norway \[[@pmed.1002220.ref031]\] are listed according to descending values at 20 wk, but are not formally compared or ranked.
^¶^Buck Louis et al. \[[@pmed.1002220.ref019]\].
^\#^Landis et al. \[[@pmed.1002220.ref030]\].
\*Johnsen et al. \[[@pmed.1002220.ref031]\].
D. R., Congo, Democratic Republic of the Congo; EFW, estimated fetal weight.
10.1371/journal.pmed.1002220.t017
###### The 10th and 90th percentile for fetal abdominal circumference in relation to relevant reference values.
{#pmed.1002220.t017g}
Reference Chart Gestational Week
---------------------------------------------------------------- ------------------ --------- --------- --------- ---------
**10th percentile AC (mm)**
US, white[^¶^](#t017fn002){ref-type="table-fn"} 141 185 227 268 306
**WHO** **139** **184** **225** **260** **294**
Norway[\*](#t017fn003){ref-type="table-fn"} 139 182 223 262 299
US, Asian[^¶^](#t017fn002){ref-type="table-fn"} 139 182 221 260 295
US, Hispanic[^¶^](#t017fn002){ref-type="table-fn"} 138 181 221 262 299
Intergrowth-21st Project[^§^](#t017fn004){ref-type="table-fn"} 138 179 219 257 291
US, black[^¶^](#t017fn002){ref-type="table-fn"} 137 179 217 267 293
Thailand[^\#^](#t017fn005){ref-type="table-fn"} 135 177 217 254 290
UK[^&^](#t017fn006){ref-type="table-fn"} 135 175 213 249 283
**90th percentile AC (mm)**
Norway[\*](#t017fn003){ref-type="table-fn"} 165 213 259 303 346
US, white[^¶^](#t017fn002){ref-type="table-fn"} 164 212 258 306 353
US, Hispanic[^¶^](#t017fn002){ref-type="table-fn"} 163 210 255 303 349
**WHO** **161** **210** **256** **298** **340**
US, Asian[^¶^](#t017fn002){ref-type="table-fn"} 161 208 252 299 343
Thailand[^\#^](#t017fn005){ref-type="table-fn"} 159 208 256 301 339
US, black[^¶^](#t017fn002){ref-type="table-fn"} 159 205 249 295 340
UK[^&^](#t017fn006){ref-type="table-fn"} 158 204 248 290 330
Intergrowth-21st Project[^§^](#t017fn004){ref-type="table-fn"} 158 203 248 291 335
Percentiles from the present multinational study (bold), a recent multinational study (Intergrowth-21st Project), a recent multiethnic study in the US, and three studies from Norway, Thailand, and the United Kingdom are listed according to descending values at 20 wk, but are not formally compared or ranked.
^¶^Buck Louis et al. \[[@pmed.1002220.ref019]\].
\*Johnsen et al. \[[@pmed.1002220.ref033]\].
^§^Papageorghiou et al. \[[@pmed.1002220.ref018]\].
^\#^Sunsaneevithayakul et al. \[[@pmed.1002220.ref034]\].
^&^Chitty et al. \[[@pmed.1002220.ref032]\].
AC, abdominal circumference; D. R., Congo, Democratic Republic of the Congo.
Discussion {#sec036}
==========
In this paper we present the WHO fetal growth charts for EFW and common ultrasound biometric measurements intended for international use. They reveal a wide range of variation in human fetal growth across different parts of the world. Significant differences in fetal growth between countries are confirmed by differences in birthweight. Furthermore, the study shows that intrauterine growth is influenced by fetal sex and by maternal age, height, weight, and parity, although these influences explain only partially the differences in growth between countries.
The primary motivation for this study, the fetal component of the WHO Multicentre Growth Reference Study \[[@pmed.1002220.ref011]\], was the need for clinical reference intervals applicable internationally, including for areas of the world where perinatal morbidity and mortality are high, hence the multinational design. Driven by the same motivation, we prioritized ultrasound measurements in common clinical use worldwide, the most prominent being EFW ([Fig 1](#pmed.1002220.g001){ref-type="fig"}; [Table 11](#pmed.1002220.t011){ref-type="table"}). The use of estimated weight in grams is simple and intelligible, which enhances clinical management, facilitates communication within the health care system, and is valuable when counselling patients. In addition to the other common measurements in daily use (BPD, HC, AC, and FL) ([Fig 1](#pmed.1002220.g001){ref-type="fig"}; Tables [6](#pmed.1002220.t006){ref-type="table"}--[9](#pmed.1002220.t009){ref-type="table"}), we established reference intervals for the ratios FL/HC and FL/BPD aimed at facilitating the identification and monitoring of disproportionate fetal head development, e.g., hydrocephaly or microcephaly ([Fig 1](#pmed.1002220.g001){ref-type="fig"}; Tables [12](#pmed.1002220.t012){ref-type="table"} and [13](#pmed.1002220.t013){ref-type="table"}). The diagnosis in pregnancies complicated by such conditions is often hampered by uncertainty about gestational age since head size (BPD and HC) is also commonly used for the dating of the pregnancy. FL/HC and particularly FL/BPD are less dependent on gestational age after 20 wk of gestation ([Fig 1](#pmed.1002220.g001){ref-type="fig"}) and may therefore have diagnostic utility.
A strength of the new growth charts provided by the study (Tables [6](#pmed.1002220.t006){ref-type="table"}--[15](#pmed.1002220.t015){ref-type="table"}) is that they are based on multinational data, i.e., ten countries, and therefore are more likely to be applicable internationally than previously published reference intervals for EFW based on single countries. A recent sizeable study found significant variation in fetal growth between Asian, black, Hispanic, and white ethnic groups, with Asian fetuses being the smallest and white fetuses the largest, justifying ethnic-specific growth charts \[[@pmed.1002220.ref019]\]. However, that study was confined to the US. [Table 16](#pmed.1002220.t016){ref-type="table"} demonstrates the relation between studies for the clinically important 10th and 90th percentiles for EFW. The WHO growth chart for all countries lies in the middle of them. Although the present study was not designed to investigate ethnic differences, a limited record of participants' ethnicity showed a distribution largely according to country ([Table 2](#pmed.1002220.t002){ref-type="table"}). Interestingly, there was a significant difference in the growth of EFW between countries that was not explained by maternal factors ([Fig 3](#pmed.1002220.g003){ref-type="fig"}; [S2 Table](#pmed.1002220.s009){ref-type="supplementary-material"}). While ethnic differences may play a role in this variation, as for the US-based study \[[@pmed.1002220.ref019]\], variation could also be due to differences in diet and cultural and socioeconomic factors commonly associated with particular ethnic groups. These may also have played a role in the US-based study.
Another recently published multinational study by the Intergrowth-21st Project presented biometric growth but not EFW data \[[@pmed.1002220.ref018]\]. We therefore present variation in AC, which is closely linked to EFW and is an important predictor of perinatal outcome \[[@pmed.1002220.ref006]\], for the commonly used cutoffs, the 10th and 90th percentiles ([Table 17](#pmed.1002220.t017){ref-type="table"}). Interestingly, the 10th percentile for the Intergrowth-21st Project results seems to fall below that of the WHO study, even though the Intergrowth-21st Project study was carried out according to a strictly "prescriptive" concept to establish so-called optimal fetal growth (low-risk pregnancies with no environmental and nutritional constraints, and excluding all conditions during pregnancy and childbirth that may be associated with effects on fetal growth). The WHO study had a similar recruitment but retained in the analysis pregnancies with maternal, fetal, and neonatal clinical conditions, based on the principle that reference intervals should reflect as closely as possible the population to which they will be applied. Furthermore, we assessed the effect of removing such pregnancies from the dataset and found no identifiable effect on the percentiles. As seen from [Table 17](#pmed.1002220.t017){ref-type="table"}, it is as if rigorous selection and exclusions have limited effect, and other uncontrolled factors are responsible for the variation between studies and countries. Apart from random error, systematic error due to differences in ultrasound measurement techniques could influence the differences between the studies. However, these studies had well-trained ultrasound operators specifically instructed for the research procedure using internationally accepted techniques, and this should minimize such error.
Another strength of the present WHO study is the use of quantile regression to establish the reference intervals. Quantile regression makes an inference about regression coefficients for the conditional quantiles of a variable without making assumptions about its distribution: there is no need to assume a particular distribution and to estimate its moments. In consequence, it provides a more direct representation of the observed measurements. This is nicely demonstrated in a recent large study establishing population-specific fetal growth charts \[[@pmed.1002220.ref035]\]. The technique is especially useful when the quantiles vary differently with a covariate such as, in the present study, gestational age. In addition, the method is robust against the effect of outliers and can capture important features of the data that might be missed by models that average across the conditional distribution \[[@pmed.1002220.ref025]\].
Quantile regression is particularly useful in studying distribution changes, and shows in the present study that fetal growth in the population is not symmetrical with gestation. Starting with a higher distribution towards the lower percentiles, EFW shifts to an expanded distribution among the higher percentiles and ends with a noticeable asymmetry near term. The Bowley coefficient for asymmetry changed from −0.016 to +0.111 during that period. We are not sure of the nature of the small negative asymmetry in early pregnancy, but speculate that regulatory functions, such as the process of maternal constraint of fetal growth, change through gestation, i.e., fetuses in the higher percentiles may be exposed to greater influences, which vary with maternal characteristics. This corroborates the differential effects of covariates across the percentiles shown in [S1 Fig](#pmed.1002220.s001){ref-type="supplementary-material"}. We believe that studying distribution dynamics may yield more information on the control of fetal growth.
The study confirmed the biologically interesting facts that fetal sex and maternal height, weight, parity, and age significantly influence fetal growth \[[@pmed.1002220.ref031],[@pmed.1002220.ref036],[@pmed.1002220.ref037]\]. Together with the country differences, the ethnic differences shown in the US population \[[@pmed.1002220.ref019]\], and, not least, the substantial variation in birthweight among carefully selected low-risk pregnancies, these findings document a diversity and plasticity in human prenatal growth dynamics that is only partially understood. There is increasing evidence linking fetal development, and proxies of development such as birthweight, to postnatal health and life course risk of disease \[[@pmed.1002220.ref007],[@pmed.1002220.ref009]\]. This issue is prioritized by the UN and WHO at a time when noncommunicable diseases are becoming global epidemics \[[@pmed.1002220.ref010],[@pmed.1002220.ref038]\]. For example, in our study, birthweights in India were significantly lower than in the other countries, and Indian participants also had the lowest fetal growth and were the shortest mothers. It is known that body composition in Indian newborns contains relatively more fat \[[@pmed.1002220.ref039]\], a pattern that passes across generations \[[@pmed.1002220.ref040]\] and that is linked to increased risk of subsequent type 2 diabetes \[[@pmed.1002220.ref041]\]. It seems clear that the understanding of "optimal" fetal growth needs to incorporate more than birthweight.
To have a single fetal growth chart that fits all pregnancies across the world would require that all fetuses had the same genetic background for growth, that this genetic background was reliably expressed in the mother, and that influences such as nutrition, physical activity, stress, toxicants, and other environmental conditions had similar effects on the genotype in all embryos and fetuses. This is very unlikely: recent research has revealed a range of interactions between the developmental environment and genetic and epigenetic processes \[[@pmed.1002220.ref009]\]. Even influences on fetal growth classically thought to be primarily genetic, such as maternal and paternal height, are complicated by environmental factors. Altitude, climate, geography, other environmental conditions, and the challenges of daily life and nutrition vary around the world. Humans adapt across generations to local conditions, and fetal development adds an important adaptive refinement for the next generation. Secular changes in birthweight and child growth patterns have been shown to accompany social changes \[[@pmed.1002220.ref042],[@pmed.1002220.ref043]\]. Fetal growth charts may thus need to be adjusted to fit the diversity of individuals and populations if they are to be of the greatest clinical utility.
While including ten countries in the present WHO study was a strength compared to previous studies, it still has limitations. The ten population samples, including two in South-East Asia and two in Africa, were included to increase generalizability, but they are still a very limited sample of the global human population. Africa alone has a greater genetic diversity than has the rest of the world \[[@pmed.1002220.ref044]\], and anthropometric variation on that continent is substantial. The present study showed population differences within the pooled dataset, and so the extent to which the results can be extrapolated to other populations, which possibly have other growth dynamics, is at present unknown.
A limitation of the study is that ultrasound measurements were accompanied by a corresponding gestational age exposed on the screen, which could have led to undue changes in the management of the pregnancy and pregnancy duration. However, it was common practice among the sonographers and midwives doing the examination not to pay attention to this gestational age because the department was using other reference values than the one on the screen. On the other hand, part of the ethical commitment of the study was actually to let the mother be informed of any abnormality or deviation of importance discovered, so that it could be taken into account for the management of the pregnancy, and to refer the case to the managing clinician. However, the reported referrals were few and were found not to influence the statistics.
Pooling data is not ideal in the presence of variation among populations, and a single overall growth chart will only partially reflect the individual populations included. Figs [4](#pmed.1002220.g004){ref-type="fig"} and [5](#pmed.1002220.g005){ref-type="fig"} show the variation of country-specific percentiles compared with the corresponding overall percentiles of the study and provide an opportunity to assess the magnitude and clinical relevance of the observed variation. Tables [16](#pmed.1002220.t016){ref-type="table"} and [17](#pmed.1002220.t017){ref-type="table"} illustrate a similar pattern when compiling the 10th and 90th percentiles for EFW and AC from various relevant high-quality studies available for clinical use. Although no formal statistical comparison was undertaken, the results of these studies illustrate the distribution that can be found around the world. This gives an impression of a wider spread for the 90th percentile than for the 10th. A similar pattern is found within the WHO study itself: a more obvious diversity between the countries for the 90th percentile than for the 10th percentile ([Fig 3](#pmed.1002220.g003){ref-type="fig"}). As seen from these figures, variation between countries may increase to several hundred grams towards the end of pregnancy, and may cause misclassifications when the overall percentile is used. Secondly, it seems that population variation in growth is more reflected in the 90th percentile than in the lowest percentiles. Thus, it is possible that the 10th, 5th, and 2.5th percentiles of a pooled study are more universally applicable, while the upper percentiles---90th, 95th, and 97.5th---vary more according to population characteristics and accordingly will be more in need of adjustment, i.e., customization, for use at the population level \[[@pmed.1002220.ref037]\].
It follows that whenever the WHO growth charts, or any reference intervals, are applied to a population, their performance should be checked or tested in order to ensure appropriate use. It is possible to adjust them by changing cutoffs (e.g., from 10th to 5th percentile) to fit clinical needs better, and it is possible to customize the percentiles to country, maternal characteristics, and fetal sex to improve diagnostic performance \[[@pmed.1002220.ref045]\]. A further refinement would be to introduce conditioning terms when using repeated ultrasound measurements for monitoring growth \[[@pmed.1002220.ref046],[@pmed.1002220.ref047]\], i.e., narrowing the expected reference interval for an assessment by conditioning it using a previous measurement. WHO is working on these methods to make them generally available with the growth chart.
If such adjustments and refinements do not suffice to make the growth charts fit clinical needs appropriately, then it may be necessary to establish new high-quality reference intervals for a population. For example, the WHO growth charts and many others are based on populations living at altitudes \< 1,500 m. However, millions of people live at higher altitudes, and their physiological adaptations include pregnancy and fetal development. It might be that specific charts will be needed for such populations.
The concept of a "standard," whether international or national, is often used for instruments and methods to make procedures uniform and to reduce random and systematic error, rather than to set a standard for a biological parameter such as height or bodyweight for the population globally. We are inclined to the view that, while the methodology to define reference ranges or charts for fetal growth needs to be standardized, fetal growth itself is a biological parameter expected to reflect adaptive processes and to change with development, time, location, and environmental conditions. Variation in fetal growth within and between populations should therefore not be ignored.
To apply any growth chart sensibly requires insight, critical attitude, and pragmatism. We believe that the present WHO fetal growth charts can be used internationally, particularly where no local data exist. However, once they are in use, it will be prudent to test the performance of the charts in a particular setting in case adjustments, customization, or replacement with population-specific high-quality reference intervals is needed. With the currently varying degrees of resources, health, and needs around the world, health care professionals have the responsibility of fitting and refining the use of the fetal growth charts to best serve the population in their care.
Supporting Information {#sec037}
======================
###### Influence of covariates on estimated fetal weight quantiles.
\(A\) Intercept; (B) fetal sex; (C) parity; (D) maternal age; (E) maternal weight; (F) maternal height; (G) gestational age linear component; (H) gestational age quadratic component; (I) gestational age cubic component. Output of quantile profilers from quantile multivariate regression in the logarithmic scale, presented as the effect of covariates with 95% confidence bands. For binary variables (sex of the fetus and parity), the relative change is between the two categories; for continuous variables, the relative change refers to the increment in EFW resulting from a unit increment of the independent variable (year for maternal age, kilogram for maternal weight, and centimeter for maternal height). Gestational age was included in the model with polynomial terms (linear, quadratic, and cubic).
(DOCX)
######
Click here for additional data file.
###### Influence of country on fetal growth expressed as the ultrasound measure biparietal diameter.
Graphs of the 10th, 50th, and 90th percentiles for the ultrasound measure BPD in millimeters for the ten participating countries.
(TIF)
######
Click here for additional data file.
###### Influence of country on fetal growth expressed as the ultrasound measure head circumference.
Graphs of the 10th, 50th, and 90th percentiles for the ultrasound measure HC in millimeters for the ten participating countries.
(TIF)
######
Click here for additional data file.
###### Influence of country on fetal growth expressed as the ultrasound measure abdominal circumference.
Graphs of the 10th, 50th, and 90th percentiles for the ultrasound measure AC in millimeters for the ten participating countries.
(TIF)
######
Click here for additional data file.
###### Influence of country on fetal growth expressed as the ultrasound measure femur length.
Graphs of the 10th, 50th, and 90th percentiles for the ultrasound measure FL in millimeters for the ten participating countries.
(TIF)
######
Click here for additional data file.
###### Influence of country on fetal growth expressed as the ultrasound measure humerus length.
Graphs of the 10th, 50th, and 90th percentiles for the ultrasound measure HL in millimeters for the ten participating countries.
(TIF)
######
Click here for additional data file.
###### Growth charts for the fetal ultrasound measurements biparietal diameter, head circumference, abdominal circumference, femur length, and humerus length; for estimated fetal weight; and for the ratios femur length/head circumference and femur length/biparietal diameter in one Excel file.
(XLSX)
######
Click here for additional data file.
###### Compliance of ultrasound visits with protocol, measured by observed versus expected.
(DOCX)
######
Click here for additional data file.
###### Variation of estimated fetal weight quantiles due to country, maternal characteristics (age, height, weight, and parity), and sex of the fetus.
Output from quantile multivariate regression showing Wald chi-square tests for gestational age; country; the interaction of gestational age and country; sex of the fetus; and maternal characteristics.
(DOCX)
######
Click here for additional data file.
###### Variation of estimated fetal weight quantiles due to country, maternal characteristics (age, BMI, and parity), and sex of the fetus.
Output from quantile multivariate regression showing Wald chi-square tests for gestational age; country; the interaction of gestational age and country; sex of the fetus; and maternal characteristics.
(DOCX)
######
Click here for additional data file.
###### Comparison of country percentiles with overall percentiles.
The 10th, 50th, and 90th percentiles for overall EFW, and the 95% confidence intervals for the difference between each country's percentiles and the overall percentiles at 20, 24, 28, 32, and 36 wk of gestational age. The results should be interpreted with caution (the study was not powered for this analysis; multiplicity of inferences implies that the confidence is much lower than 95%).
(DOCX)
######
Click here for additional data file.
We thank Mario Merialdi and George Bega for their contributions during the first stages of the study. We thank General Electric for loaning the ultrasound machines and for providing technical assistance during the study. We thank José de Souza Ramos for his contributions to the programming of descriptive tables and creation of analysis files. The Argentina team would like to acknowledge ultrasonographers: Sergio Ricci, Débora Montoya, Pablo Cañón, Mario Marchetti; nutritionists: Silvina Tosticarelli, Silvia del Cerro; physicians at the primary care health centers: Malvina Giuliani, Marisa Menighini, Karina Martínez, Virginia Díaz, Ana Paula Bogino, Paola Salina, Fernanda Candio, Duilio Filiberto, María Belen Bosch, Eliana Juan, Guadalupe Morón, Mariela Giraudo, Natalia Blanco, Berenise Macagno; neonatologists at Maternidad Martin: Ofelia Casas, Gabriela Puig, Lorna Andreuzzi, Marcelo Rodríguez, Susana Morales, Silvia Carazzone, Andrea Bobatto, Marina Duarte; obstetricians at Maternidad Martin: Daniel Crosta, Silvia Carbognani; and staff at the Centro Rosarino de Estudios Perinatales: Edgardo Abalos, Liana Campodonico, Cristina Cuesta, Hugo Gamerro, Lucia Darder, Carla Salas, Renata Zanello, Fernando Burgueño. The Brazil team would like to acknowledge Maria Laura Costa, Carla Silveira, Kleber Cursino Andrade, Cristiane Martins Almeida, Ana Gabriela Bortoleto, and Daiane Sofia Paulino. The D. R. Congo team would like to acknowledge sonographers Luyeye M. Mandiangu Godefroid and Lokomba Bolamba Victor, site physicians Kiumbu Nzita and Modeste Luzingu Kinko Joy, study coordinator Bidashimwa Nzabonimpa Dieudonné, nurses Omba Dihandjo Betty and Matondo Lutonadio Hélène, and nutritionists Diba Tshilenge Solange and Bauma Juhudi Mamy. The Egypt team would like to acknowledge Elwany Elsonosy, Mostafa Hussein, Mahmoud A. Abdel-Aleem, and Dina Habib. The France team would like to acknowledge ultrasonographer Catherine Egoroff. The Germany team would like to acknowledge study nurse Gudula Hansen. The India team would like to acknowledge Vatsla Dadhwal. The Norway team would like to acknowledge the local coordinating physician Synnøve Lian Johnsen; midwives Ine Hildershavn Moen, Guro Kyte Børsheim, and Jeanette Aasland; and dietitians Gro Trae and Hilde Mollestad Tveit. The Thailand team would like to acknowledge site physician and ultrasonographer Kiattisak Kongwattanakul and nutritionist Benja Muktabhant.
AC
: abdominal circumference
BMI
: body mass index
BPD
: biparietal diameter
D. R. Congo
: Democratic Republic of the Congo
EFW
: estimated fetal weight
FL
: femur length
HC
: head circumference
HL
: humerus length
IQR
: interquartile range
LMP
: last menstrual period
TI
: thermal index
[^1]: I have read the journal\'s policy and the authors of this manuscript have the following competing interests: GP is a WHO statistical consultant and has a contract to give statistical support to the Fetal Growth Study. GP has worked with WHO for 15 years and has a relationship with WHO, both paid (contracts) and unpaid. GP is also a good friend of many of the investigators involved in this study. LDP is a Board Member of the Perinatal Quality Foundation, a nonprofit organization related to the Society for Maternal Fetal Medicine. LDP has received research support from General Electric Medical Systems unrelated to fetal growth. LDP also lectures 1 or 2 times per year at an educational meeting supported by General Electric Medical Systems unrelated to fetal growth.
[^2]: **Conceptualization:** TK GC JGC HAA SAT AB AD ATK PL AT AK RG KH LDP MAH.**Formal analysis:** JC GP DG.**Investigation:** TK GC JGC HAA AB AD ATK PL AT AK LNJ JT.**Methodology:** TK GC JGC HAA SAT AB AD ATK PL AT AK RG KH LDP JC GP MG MW.**Project administration:** MW MG.**Resources:** MW MG TK GC JGC HAA AB AD ATK PL AT AK LNJ JT.**Software:** DG GP JC.**Supervision:** TK.**Visualization:** GP JC TK.**Writing -- original draft:** TK GP GC MW JC LNJ DG JGC HAA SAT AB AD ATK JT PL AT AK RG KH MAH MG LDP.**Writing -- review & editing:** TK GP GC MW JC LNJ DG JGC HAA SAT AB AD ATK JT PL AT AK RG KH MAH MG LDP.
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A curriculum vitae (CV) serves as an important means of helping you to secure an interview or a job. Although many medical job applications such as the foundation program are submitted online without the need for a CV, it still remains important when applying to surgical training, any consultant posts, general practice training, portfolio assessments, electives, and grants[@R1],[@R2]. It also functions as a way of recording all your achievements, skills, and experiences and keeping them up to date. This will help to identify areas which still need improving or which areas are particularly relevant for the role you are applying to. Therefore, it is important to review your CV regularly for each new role or purpose that you are using it for and tailor it accordingly[@R3].
Length
======
There is no required length for medical CVs, with the general trend to be 2--3 pages of A4 that keeps the information succinct and relevant. An academic CV may tend to be longer, with research and publications being included.
Top tips
========
- Before starting to write the CV, identify exactly what the role/person specification is looking for and ensure that they will be able to identify straightaway from your CV that you are applying for that role.
- A CV has to make an immediate impact, so good presentation is vital---nice paper quality, clear font 12-point Arial or Times, clear layout with enough white space margins, avoid large chunks of text, use bullet points, ensure no spelling mistakes.
- Style of writing---professional short and simple sentences, use active words when referring to skills, focus on positive aspects.
- Do not exaggerate or fabricate any information on the CV. Also do not include everything you have done if it is not relevant to the application. There will be plenty of opportunity at interviews to get this information across if it is important.
The following headings are useful to ensure a good structure to your CV.
Cover letter
============
The first page is usually independent and just contains what role you are applying for, a brief introduction to you and why you want to apply for this role. It serves more of an administrative purpose.
Personal details
================
This should include your full name and abbreviated qualifications (eg, MBBS, BSc). Contact details including address, telephone number, and email address should be provided. Ensure that these are professional contact details and not personal ones. Other details such as date of birth, nationality, and sex are optional and should not make a difference to your application. It is also a good idea to include general medical council number and medical defense numbers once you have been registered.
Career statement
================
This is a useful and quick way to highlight how you are suitable for the role in question by stating your most relevant experience and skills as well as express your professional goals regarding your future career[@R4].
Education and qualifications
============================
This should list the most recent qualification first. For university degrees, include details of any special study modules or electives if they are relevant to the role. Qualifications gained in school such as GCSEs and A-levels can be stated with a summary line that includes the institution, year, and grades.
Present position
================
Highlight your current role and what responsibilities and skills it entails, including any relevant information such as the hospital, supervisor name, and date started.
Career history
==============
Similar to the previous section, include a brief summary of the job description and any other relevant information especially the dates. Order this with the most recent job first. There is no need to include every single job that you have ever had especially if it is not relevant.
This section can be expanded upon with a "clinical skills" section where you can expand on any relevant experiences and skills that are relevant to the new role, for example highlighting any surgical skills gained that are needed for a new surgical job.
Voluntary/work experience
=========================
This is a good section to include any experience gained either medically, such as any specialty experience taster days. Non-medical experience such as charity work and any volunteering are also useful. For both it is useful to highlight what you have gained from it.
Audit and quality improvement work
==================================
Participating in clinical audits or quality improvement works is important in a medical career and will provide points in speciality applications[@R5]. Therefore, you should clearly show any audits that you have been involved in including the dates, topic, your role, any guidelines used, your conclusions, and future outcomes.
Management and leadership
=========================
Management and leadership skills are vital for a doctor, and this can be highlighted through clear examples either medically or nonmedically related[@R6]. Examples include positions in committees, supervising juniors, and organizing events[@R7].
Prizes and awards
=================
This can include awards received both in education and at work. Again, list the most recent one first and highlight the ones that are most relevant to the role in question.
Publications and presentations
==============================
This is important in academic CVs in particular, but is also relevant in any medical CV. State your publications in the same format that it would appear in a journal and include a pubmed ID number if available. This section also includes any posters or presentations that you have produced from any research projects or audits.
Teaching experience
===================
This is another skill that is vital for doctors, so include any teaching experience either formal or informal at any level, the topics and audience taught and what you gained from it. Have some feedback forms within your portfolio as evidence of this.
Training courses and educational symposia
=========================================
Include training courses and symposiums that add value to your CV, for example a suturing course will look good for surgical applications, as will basic and advanced life support courses. Courses related to examination preparation are usually not relevant. The courses can also relate to other aspects such as management, teaching, or research. For each one state the date and title of the course, and can be presented either chronologically or in order of importance. Importance can further be split up into international, national, or regional courses.
Information technology skills
=============================
Many workplaces now require competent information technology skills such as prescribing drugs in hospital, so make sure to include any relevant experience and skills here[@R8]. Examples can include basic software such as the Microsoft packages but also any specialist software such as statistics packages, databases for research.
Professional and society memberships
====================================
This usually applies to ones where you have been elected toward, as opposed to ones you pay a subscription fee to join.
Personal interests
==================
This is designed to present you as a well-balanced individual and to catch the reader's attention. Include any extracurricular activities that you do but rather than listing them, demonstrate why you do them and what you have gained from them especially how they have improved your ability as a doctor. It is important not to fabricate any information this section as you can be quizzed about it during an interview.
Referees
========
You should list at least 2 references, either with their name and contact details present or state "available upon request." However, it is a good idea to ensure that your referees have agreed to act as such and they are aware of what role you are applying for so they can tailor their reference accordingly[@R9].
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
Published online 15 June 2017
Conflict of interest statement
==============================
The authors declare that they have no financial conflict of interest with regard to the content of this report.
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Storch I, Penner J, Asbeck T, et al. Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary, multi‐scale approach. Ecol Evol. 2020;10:1489--1509. 10.1002/ece3.6003
1. INTRODUCTION {#ece36003-sec-0001}
===============
1.1. Forest biodiversity and the need for integrated forestry {#ece36003-sec-0002}
-------------------------------------------------------------
Since the UN Convention on Biological Diversity (CBD) was signed in 1992, the conservation of biodiversity has become a global commitment. Yet, species extinction rates continue to accelerate, as recently documented by the Intergovernmental Science‐Policy Platform on Biodiversity and Ecosystem Services (IPBES, [2019](#ece36003-bib-0044){ref-type="ref"}).
In Europe, forests are the dominant natural vegetation form, and present the primary evolutionary background of today\'s plant and animal species and communities. Forests provide habitat for numerous species and therefore play a key role in the conservation of biodiversity (European Environment Agency, [2016](#ece36003-bib-0025){ref-type="ref"}; Forests Europe, [2015](#ece36003-bib-0030){ref-type="ref"}).
Forests are also valued for safeguarding ecosystem services, such as production of wood, protection against natural disasters, and provision of recreational opportunities. Further, forests and forestry traditionally play an important role in European culture, provide employment, and contribute to local economies. In view of these manifold human demands for wood and other ecosystem goods and services, the vast majority of forests in Europe are managed to serve economic, social, and environmental functions at the same time (Forests Europe, [2015](#ece36003-bib-0030){ref-type="ref"}).
These multiple functions are not always easily reconciled (Niemelä et al., [2005](#ece36003-bib-0068){ref-type="ref"}; Verkerk et al., [2014](#ece36003-bib-0090){ref-type="ref"}). For example, temperate forests managed for high economic benefit are unlikely to be rich in biodiversity, because trees and stands are typically harvested at economic maturity, which can be reached, depending on the tree species, after 60--200 years. This is relatively early in the life span of forests whose trees may live for several hundred years, and long before the development of old and structurally rich forest successional stages with their high level of biodiversity and many unique species (Gustafsson et al., [2012](#ece36003-bib-0040){ref-type="ref"}; Hilmers et al., [2018](#ece36003-bib-0043){ref-type="ref"}; Scherzinger, [1996](#ece36003-bib-0078){ref-type="ref"}). Thus production forests, here understood as "forests available for wood supply" (FAWS) (UNECE/FAO, [2000](#ece36003-bib-0087){ref-type="ref"}) lack in many cases the structural heterogeneity of natural forest ecosystems. Specifically, they lack late successional stages and structural elements (e.g., snags, coarse woody debris, and canopy gaps) created by decay processes or natural disturbances. This is of major hindrance for various groups, such as epiphytic lichens and plants, saproxylic fungi and invertebrates, and cavity‐nesting mammals and birds, which depend on the specific resources available in old forests (Goldberg, Kirby, Hall, & Latham, [2007](#ece36003-bib-0038){ref-type="ref"}; Kraus & Krumm, [2013](#ece36003-bib-0053){ref-type="ref"}; Siitonen, Martikainen, Punttila, & Rauh, [2000](#ece36003-bib-0081){ref-type="ref"}). Forests rich in late successional stages and structural elements are extremely rare in Europe. Indeed, across Europe, only marginal proportions (1.5%) of forest are permanently taken out of production ("no active intervention," Forests Europe, [2015](#ece36003-bib-0030){ref-type="ref"}, p. 160) and set aside in protected areas such as National Parks and other strictly protected forests. Reserves without human intervention are valuable reference areas for research on natural forest ecosystems, and important cornerstones for the conservation of forest biodiversity. However, a reserve‐based segregative approach alone cannot ensure the conservation of forest biodiversity because small reserve sizes and insufficient connectivity limit the effectiveness of these remaining islands of natural forest for biodiversity conservation (Bollmann & Braunisch, [2013](#ece36003-bib-0019){ref-type="ref"}).
Thus, integrative conservation measures in production forests are a crucial complement in safeguarding forest biodiversity not only at the local scale of protected areas, but in particular across entire landscapes. Increasingly, retention‐forestry approaches (Bauhus, Puettmann, & Messier, [2009](#ece36003-bib-0014){ref-type="ref"}) are applied "to provision for continuity in structural, functional, and compositional elements from the preharvest to the postharvest forest" (Gustafsson et al., [2012](#ece36003-bib-0040){ref-type="ref"}). Retention forestry, that is, retaining small patches or structural elements within a production forest matrix, is implemented particularly in public forests, and often related to requirements originating from EU legislation. Integration of biodiversity conservation into forest management is a major policy goal throughout Europe (cf. the EU Forest Strategy, European Commission, [2013](#ece36003-bib-0024){ref-type="ref"}; and the EU Biodiversity Strategy, European Commission, [2011](#ece36003-bib-0023){ref-type="ref"}; the German National Strategy on Biological Diversity, BMU ([2007](#ece36003-bib-0018){ref-type="ref"}); and the German Forest Strategy 2020, BMELV, [2011](#ece36003-bib-0026){ref-type="ref"}). In the federal system of Germany, the responsibility for programme implemention is focused on state level (ForstBW, [2016](#ece36003-bib-0033){ref-type="ref"}).
1.2. State of knowledge and research gaps {#ece36003-sec-0003}
-----------------------------------------
Retention forestry has been practiced and extensively studied in clear‐cutting systems for 30 years (Fedrowitz et al., [2014](#ece36003-bib-0027){ref-type="ref"}; Gustafsson et al., [2012](#ece36003-bib-0040){ref-type="ref"}; Mönkkönnen, Ylisirniö, & Hämäläinen, [2009](#ece36003-bib-0063){ref-type="ref"}). However, there is still a need to extend research on the effectiveness of retaining structural elements for biodiversity conservation from clear‐cutting to the continuous‐cover forestry systems (Gustafsson et al., [2019](#ece36003-bib-0041){ref-type="ref"}), which dominate major parts of the temperate regions in Europe.
Few studies have examined retention approaches in selectively logged forests in the temperate zone (Müller, [2005](#ece36003-bib-0065){ref-type="ref"}); thus, retention guidelines, as they are currently implemented across central Europe, are based on plausibility and expert knowledge rather than on scientific evidence (Vítková, Bače, Kjučukov, & Svoboda, [2018](#ece36003-bib-0091){ref-type="ref"}). For example, in the state forests of Bavaria, Germany, an average of ten habitat trees per hectare has to be retained in near‐natural stands (BaySF, [2009](#ece36003-bib-0016){ref-type="ref"}), whereas in the neighboring state of Baden‐Württemberg, one group of habitat trees consisting of about 15 trees is to be secured per 3 ha (ForstBW, [2016](#ece36003-bib-0033){ref-type="ref"}). Across Europe, all countries use their own, variable prescriptions (Sotirov, [2017](#ece36003-bib-0082){ref-type="ref"}; Winter et al., [2014](#ece36003-bib-0093){ref-type="ref"}). This also holds for FSC and PEFC certification standards in Europe: In national certification standards, quantitative retention targets are either not specified, or they vary greatly (e.g., 1--10 living habitat trees per ha; 1--20 dead trees per ha) among European countries (Gustafsson et al., [2019](#ece36003-bib-0041){ref-type="ref"}).
Trade‐offs between conservation and production objectives may explain different thresholds set in conservation programmes in production forests. Yet, there is obviously also uncertainty about the thresholds required for retention amounts to achieve the desired effects on biodiversity. Recommendations to date, that is, for the required amount of deadwood and habitat trees, have been based mostly on single taxonomic groups such as forest birds, saproxylic insects, lichens, or fungi (Müller & Bütler, [2010](#ece36003-bib-0066){ref-type="ref"}; Sandström et al., [2019](#ece36003-bib-0077){ref-type="ref"}). Studies investigating a wide spectrum of species including multiple taxa and trophic groups in the same study system are rare (Franklin, Macdonald, & Nielsen, [2019](#ece36003-bib-0034){ref-type="ref"}; Müller & Bütler, [2010](#ece36003-bib-0066){ref-type="ref"}; Paillet et al., [2018](#ece36003-bib-0070){ref-type="ref"}; Ranius & Fahrig, [2006](#ece36003-bib-0073){ref-type="ref"}; Vítková et al., [2018](#ece36003-bib-0091){ref-type="ref"}). Further, relationships between forest structure and biodiversity have not been studied comprehensively in a landscape context (Mori, Tatsumi, & Gustafsson, [2017](#ece36003-bib-0064){ref-type="ref"}). Landscape ecology and community ecology theory (Leibold et al., [2004](#ece36003-bib-0056){ref-type="ref"}; Lindenmayer & Franklin, [2002](#ece36003-bib-0058){ref-type="ref"}; Tscharntke et al., [2012](#ece36003-bib-0086){ref-type="ref"}) for instance would predict that a network of stands rich in deadwood may contribute more to biodiversity than uniformly distributed retention of moderate amounts of deadwood across the landscape (Müller & Bütler, [2010](#ece36003-bib-0066){ref-type="ref"}). In other words, the amounts and spatial distribution of structural elements, such as deadwood, required at the landscape scale to allow for functional connectivity of taxa dependent on old forests have not received sufficient attention (Müller & Bütler, [2010](#ece36003-bib-0066){ref-type="ref"}; Percel, Laroche, & Bouget, [2019](#ece36003-bib-0071){ref-type="ref"}; Ranius & Fahrig, [2006](#ece36003-bib-0073){ref-type="ref"}). Increasing the knowledge of the minimum landscape‐scale requirements for biodiversity conservation through retention forestry would help to improve existing retention schemes (Mori et al., [2017](#ece36003-bib-0064){ref-type="ref"}).
Evidence‐based prescriptions for the amount and distribution of retention elements, however, will not guarantee the implementation of recommended measures into forestry practice. Successful biodiversity conservation in multi‐functional forests necessitates that conservation objectives are compatible with the aspirations of landowners and other goals of forest management (BMELV, [2011](#ece36003-bib-0026){ref-type="ref"}), and that potential trade‐offs are known and regulated by respective conservation policies. Conservation measures often represent a loss in income, cause issues of work‐safety, and result in higher management costs for forest owners (Rosenkranz, Seintsch, Wippel, & Dieter, [2014](#ece36003-bib-0074){ref-type="ref"}). Hence, the success of biodiversity conservation approaches such as retention forestry is greatly affected by the socio‐economic context, including local knowledge, traditions, motivations, and practices; yet, little is known about these human dimensions underlying the conservation of forest biodiversity in Central Europe and elsewhere (Bennett et al., [2017](#ece36003-bib-0017){ref-type="ref"}; Gorenflo & Brandon, [2006](#ece36003-bib-0039){ref-type="ref"}; Maier & Winkel, [2017](#ece36003-bib-0060){ref-type="ref"}; Rutte, [2011](#ece36003-bib-0076){ref-type="ref"}).
The inter‐ and transdisciplinary approach and the multi‐scaled design of the Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) explicitly address these two major gaps in forest biodiversity research, namely the influences of the landscape context and the relevance of the socio‐economic context for the effectiveness of retention measures to maintain biodiversity in multi‐functional forests of temperate Europe. To the best of our knowledge, such a broad integrative analysis of the ecological and social preconditions for forest biodiversity conservation has not yet been attempted in Central Europe.
In the following, we first present the rationale and lead questions underlying the ConFoBi research programme, then outline how these ideas were translated into a research design, describe how this design was implemented in the Black Forest, south‐west Germany, characterize the pool of 135 ConFoBi study plots and place the study system in a European context. We then briefly present specific projects, their methods and linkages. Finally, we discuss perspectives of ConFoBi for research and conservation, and encourage scientists from multiple disciplines to join the ConFoBi Research Training Group.
2. RATIONALE OF THE CONFOBI RESEARCH PROGRAMME {#ece36003-sec-0004}
==============================================
2.1. Guiding principles {#ece36003-sec-0005}
-----------------------
The ultimate goal of ConFoBi is to provide an evidence‐based framework for socio‐political decision‐making, which rests on a comprehensive analysis of retention forestry and its consequences for and linkages to ecological and societal systems in the multiple‐use landscapes of temperate Europe. Key principles guiding ConFoBi\'s research toward this goal are (a) the application of a transdisciplinary approach, (b) the development of a consistently interdisciplinary research programme, and (c) the implementation of a real‐world representative study system spanning across multiple spatial scales, from plot (1 ha) to landscape.
### 2.1.1. Transdisciplinarity {#ece36003-sec-0006}
Biodiversity conservation often fails because of a lack of communication and understanding between researchers and practitioners (Mehring, Bernard, Hummel, Liehr, & Lux, [2017](#ece36003-bib-0062){ref-type="ref"}; Pregernig, [2014](#ece36003-bib-0072){ref-type="ref"}; Tinch et al., [2018](#ece36003-bib-0085){ref-type="ref"}). To overcome this deficiency, ConFoBi chose to apply a transdisciplinary approach to maintain a regular dialogue between science and practice. At the outset, we held a workshop with relevant stakeholders, which allowed us to orient research toward the knowledge gaps of forestry and conservation practice. Participants from different administrational levels within the State of Baden‐Württemberg, Germany, suggested that decision makers need (a) quantitative values for minimum amounts and distribution of retention elements required for forest biodiversity conservation at spatial scale extents from plots to landscapes and (b) knowledge related to local implementation practices of biodiversity conservation measures and instruments. Now, that ConFoBi is operational, a number of its research projects are designed and carried out in close cooperation with relevant decision‐making and managing bodies; especially the Ministry of Rural Affairs and Consumer Protection of Baden Württemberg (MLR) and the State Forest Service (ForstBW) are explicitly targeted at pertinent state policies, including the Forest Conservation Strategy (Waldnaturschutzstrategie; ForstBW, [2015](#ece36003-bib-0032){ref-type="ref"}), and the Old and Dead Wood Programme (AuT Programm; ForstBW, [2016](#ece36003-bib-0033){ref-type="ref"}).
### 2.1.2. Interdisciplinarity {#ece36003-sec-0007}
Successful biodiversity conservation in multi‐functional forests requires a solid ecological basis, but lastly depends on the compatibility of conservation measures with the human dimensions of forest management. In response to these requirements, ConFoBi adopted also an explicitly interdisciplinary approach, integrating multi‐scale ecological studies of forest biodiversity with social and economic studies of the preconditions and consequences of biodiversity conservation. To maximize interdisciplinary synergies, ConFoBi researchers of numerous disciplines work in the same set of study plots and surrounding landscapes and hence also in the same socio‐economic environment.
### 2.1.3. A real‐world study system {#ece36003-sec-0008}
Experiments have played an important role in research on the effectiveness of retention forestry (see Gustafsson et al., [2012](#ece36003-bib-0040){ref-type="ref"} for an overview). Experimental results will generate valuable knowledge on the *potential* outcomes of retention approaches. However, whether these results are transferable to the conditions of real landscapes under different types of multi‐functional forest management remains questionable (Mori et al., [2017](#ece36003-bib-0064){ref-type="ref"}). In order to study retention effects in a setting representative of regular forestry practices, and to generate results directly relevant to forest owners and practitioners (see above, Section [2.1.1](#ece36003-sec-0006){ref-type="sec"}), ConFoBi is using plots in state‐owned forests available for wood supply, as a real‐world study system for its research programme. ConFoBi concentrates its research in a specific region, the Black Forest in southern Germany, with its specific forestry and biodiversity conservation structures and practices, to allow for transdisciplinarity. While the study system is regional, the findings are interpreted within a larger framework to achieve relevance for temperate forests with continuous‐cover forestry across Europe.
2.2. Research programme and lead questions {#ece36003-sec-0009}
------------------------------------------
ConFoBi assesses whether and how structural retention measures contribute to the conservation of forest biodiversity and analyses the potential of retention forestry to be adopted by landowners and be supported by stakeholders, using montane forests of the Black Forest, Germany, as a model system. ConFoBi will explicitly concentrate on the influences of the landscape context and the socio‐economic context on the effectiveness of retention measures to maintain biodiversity in uneven‐aged and selectively harvested continuous‐cover forests (Figure [1](#ece36003-fig-0001){ref-type="fig"}).
{#ece36003-fig-0001}
The structure of the collaborative research within ConFoBi consists of four modules A--D (Figures [1](#ece36003-fig-0001){ref-type="fig"}, [2](#ece36003-fig-0002){ref-type="fig"} and 2) with several projects each. Module A provides tools for multi‐scale assessment of structures ranging from trees to landscapes. Module B studies a wide range of taxa (understory vegetation and epiphytes, invertebrates, mammals, birds) and relates biodiversity‐relevant metrics such as species occurrence, richness, and diversity to (a) plot‐scale (1 ha) forest structure, that is, abundance, quality, heterogeneity, and spatial distribution of structural elements within the forest and (b) to the landscape context, that is, abundance, quality, heterogeneity, and spatial distribution of forests in the surrounding landscape. Among structural elements, the focus is on habitat trees (large trees with hollows, cracks, crevices, crown deadwood, and other habitat features sensu, Bütler, Lachat, Larrieu, and Paillet, [2013](#ece36003-bib-0021){ref-type="ref"}) and deadwood, whereas site conditions and tree species compositions will be considered as covariates. Module C uses a subset of the ConFoBi plots selected along social gradients (e.g., ownership, protection status) to assess how forest practitioners in different settings perceive and practice biodiversity conservation, and will assess costs and benefits to model and optimize the economic efficiency of retention measures. Using a translational approach (Musacchio, [2009](#ece36003-bib-0067){ref-type="ref"}; Schlesinger, [2010](#ece36003-bib-0080){ref-type="ref"}), module D focuses on the interface between science and practice to assess how knowledge is generated and evidence is translated into practice, and to provide integration and communication between ConFoBi and forest and conservation managers and policy makers. In summary, ConFoBi: investigates the effects of forest structure including structural elements such as habitat trees and deadwood, and landscape context on multiple taxa including different trophic levels and functional groups (Modules A, B);analyses how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates (Module C); andidentifies how biodiversity conservation can be effectively and efficiently integrated in multi‐functional forest management through a translational approach focusing on epistemologies (how knowledge is generated and transferred) and by developing evidence‐based guidelines for practitioners (Module D).
{ref-type="fig"}) (Illustration: Flimmern DC)](ECE3-10-1489-g002){#ece36003-fig-0002}
3. DESIGN AND IMPLEMENTATION OF THE CONFOBI STUDY SYSTEM {#ece36003-sec-0010}
========================================================
3.1. The Black Forest as a model system {#ece36003-sec-0011}
---------------------------------------
ConFoBi has implemented its research programme in the southern Black Forest, Germany, as a model system for temperate forests. The Black Forest is a forest‐dominated low mountain range within a multiple‐use landscape typical of central Europe. It extends over an area of about 5,000 km^2^ and has a forest cover of 75% (365,000 ha). In the western and southern parts, the geology is dominated by granite and gneiss, whereas in the eastern and northern parts, sandstone prevails. The macroclimate is strongly influenced by the elevational gradient, ranging from 120 to 1,493 m a.s.l., with an average annual temperature between 4°C at the higher elevations and 10.4°C in the lowlands (Gauer & Aldinger, [2005](#ece36003-bib-0037){ref-type="ref"}). Norway spruce (*Picea abies* (L.) H. Karst.) is the most important tree species (42.8%), in particular in the northern and eastern Black Forest, whereas silver fir (*Abies alba* Mill.) (18.5%) and beech (*Fagus sylvatica* L.) (15.3%) maintain a higher share of the tree species composition in southern and western parts. In recent decades, the forests in the Black Forest have become older, more mixed and more structurally diverse (Kändler & Cullmann, [2016](#ece36003-bib-0049){ref-type="ref"}). In 2012, 71.5% of the forested area was covered by mixed stands, 29.5% was stocked with trees older than 100 years, two‐layered and multi‐storeyed forests made up 55% and 26%, respectively (Kändler & Cullmann, [2016](#ece36003-bib-0049){ref-type="ref"}). The average amount of deadwood (\>10 cm diameter, incl. stumps \>20 cm diameter) is 33.4 m^3^/ha, and there are on average five habitat trees per ha; the latter comprise 83% deciduous trees and more than two‐thirds of these trees have a diameter (dbh) \>50 cm (Kändler & Cullmann, [2016](#ece36003-bib-0049){ref-type="ref"}).
The forested landscape is further characterized by a fine‐scale mosaic of ownerships, with some regional differences in the relative share of ownership types. In the Black Forest 39% of forests are privately owned, 27% are state owned, and the remaining 34% are owned by municipalities and public corporations (Kändler & Cullmann, [2016](#ece36003-bib-0049){ref-type="ref"}).
Forests in the region are managed using a variety of silvicultural systems. Owing to strict forest legislation, a prevailing paradigm of close‐to‐nature forest management, and the wide coverage of certifications systems (PEFC and FSC), clear‐cutting is basically not practiced in the Black Forest. Instead, regeneration methods such as shelterwood, group shelterwood ("Femelschlag"), strip cutting, and single‐tree selection ("Plenter" forest) are employed to create and maintain structurally diverse and species‐rich continuous‐cover forests (Bauhus & Pyttel, [2015](#ece36003-bib-0015){ref-type="ref"}), although the selection systems tend to disadvantage light demanding species such as pine and oak (Bauhus, Puettmann, & Kuehne, [2013](#ece36003-bib-0013){ref-type="ref"}).
In the State of Baden‐Württemberg, including the study area, a retention programme ("AuT‐Konzept" \[translated as: old and dead‐wood programme\]; ForstBW, [2016](#ece36003-bib-0033){ref-type="ref"}) stipulates the retention of one group of habitat trees ("Habitatbaumgruppe") consisting of about 15 trees per 3 ha throughout state forests. Moreover, efforts have been taken to assist forest biodiversity on private property through contractual nature conservation, extension services, or subsidies.
3.2. Selection and establishment of study plots {#ece36003-sec-0012}
-----------------------------------------------
The study design of ConFoBi is based on an "all‐measurements‐on‐all‐plots" approach with a common pool of 135 quadratic study plots (1 ha) distributed along two environmental gradients: (a) landscape‐scale forest connectivity (measured by the proportion of forest in the 25 km^2^ surrounding plot centers) and (b) retention‐related forest structure at the plot scale, that is, richness in habitat trees and deadwood per ha. For logistic and authorization reasons, all plots were selected in state forests and outside of wildlife protection areas with restricted access.
Plots were preselected on the basis of a set of criteria (Table [1](#ece36003-tbl-0001){ref-type="table"}) to reduce variation in confounding factors. Major criteria were topography (\<35° slope, \>500 m a.s.l.), stand age (\>60 years), and the absence of waterbodies and human infrastructures. The remaining areas were classified into three forest‐connectivity classes (\<50%, 50%--75%, \>75% forest) based on the amount of forest in the surrounding landscape, as calculated within a circular moving window of 25 km^2^ based on a raster map of 25 × 25 m resolution.
######
Criteria used to identify potentially suitable plots as well as geodata sources used for plot selection. After preselection based on the general criteria, potential plots were classified according the two design gradients, forest structure, and landscape pattern
Selection stage Criterion Feature/definition Source
---------------------------------------------- ------------------------------------------ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Preselection Forest ownership State owned Forest inventory data, Geodata service of the Forest administration of Baden‐Württemberg (FGeo)
Region southern Black Forest, Baar‐Wutach Ecoregions according to Aldinger et al. ([1998](#ece36003-bib-0001){ref-type="ref"}), Forest Research Institute of Baden‐Württemberg
Elevation ≥500 m a.s.l. Digital elevation model (DEM), aggregated to 25 × 25 m resolution; State Agency of spatial information and rural development of Baden‐Württemberg (LGL), <https://www.lgl-bw.de/lgl-internet/opencms/de/05_Geoinformation/Geotopographie/Digitale_Gelaendemodelle/> (30 October 2015)
Steepness of slope ≤35° DEM, State agency of spatial information and rural development of Baden‐Württemberg (LGL), <https://www.lgl-bw.de/lgl-internet/opencms/de/05_Geoinformation/Geotopographie/Digitale_Gelaendemodelle/> (30 October 2015)
Stand age ≥60 years Forest inventory data, Geodata Service of the forest administration of Baden‐Württemberg (FGeo)
Distance between plot centers \>750 m GIS
Infrastructure (buildings, roads) (excluded) ATKIS^®^, State Agency of spatial information and rural development of Baden‐Württemberg (LGL); Amtliches Topographisch‐Kartographisches Informationssystem. <http://www.lgl-bw.de/lgl-internet/opencms/de/05_Geoinformation/AAA/ATKIS/> (30 October 2015)
Waterbodies (excluded) ATKIS^®^, State agency of spatial information and rural development of Baden‐Württemberg (LGL)---Amtliches Topographisch‐Kartographisches Informationssystem. <http://www.lgl-bw.de/lgl-internet/opencms/de/05_Geoinformation/AAA/ATKIS/> (30 October 2015)
Restricted species protection areas (excluded) Geodata service of the Forest Research Institute FVA
Landscape‐scale forest‐connectivity gradient Forest within surrounding 25km^2^ 3 classes: \<50%, 50%--75%, \>75% ATKIS^®^, State agency of spatial information and rural development of Baden‐Württemberg (LGL)---Amtliches Topographisch‐Kartographisches Informationssystem. <http://www.lgl-bw.de/lgl-internet/opencms/de/05_Geoinformation/AAA/ATKIS/> (30 October 2015)
Forest structure gradient *N* standing dead trees within 1‐ha plot 3 classes: 0, 1--9, \>10 Stereo color‐infrared aerial images of 2015, State agency of spatial information and rural development of Baden‐Württemberg (LGL)
John Wiley & Sons, Ltd
To determine the structural gradient, using designated habitat trees of the AuT‐Programme was not an option. At the time of plot selection, in 2016, AuT was still in its initial implementation stage, habitat trees had been selected for only minor parts of the study, and designated habitat trees were not (yet) older than the surrounding stands. Instead other proxies for forest structures that are expected to be enhanced by retention forestry, that is, old or dead trees rich in microhabitats, were used for plot selection. We screened the study area for 1 ha‐plots with low (0), medium (1--9), and high (≥10) numbers of standing dead trees. Trees were visually assessed using stereo aerial color‐infrared imagery, as provided by the state agency of spatial information and rural development of Baden‐Württemberg (LGL), together with a stereo viewer. Fifteen plots from each category (low, medium, and high structure) were then randomly selected for each of the forest‐connectivity classes, ensuring that the centers of two neighboring plots were \>750 m apart, resulting in 135 plots arranged in a stratified design with 3 × 3 = 9 categories.
These 135 candidate plots were cross‐checked with the local forest managers for the possibility to largely exclude forestry operations (e.g., harvesting and road construction) on these plots until the end of the anticipated maximum ConFoBi funding period (2016--2025). Individual plots, for which operations were already scheduled, were randomly replaced by plots of the same category. The final plots were confirmed in agreement with the individual State Forest Offices in charge of the respective forest stands. Of the 135 final ConFoBi plots, 115 are located in multi‐functional forests regularly managed by the State Forest Service; another 20 plots (all belonging to the category richest in deadwood) are in strict forest reserves (without any harvesting of timber). Plots are distributed as shown in Figure [3](#ece36003-fig-0003){ref-type="fig"}.
{#ece36003-fig-0003}
This 3 × 3 (=9 plot categories) ordinal design was used for securing an even distribution of plots along the two design gradients; subsequently, however, plot‐scale forest structure and landscape‐scale forest connectivity were measured using numerous continuous metric variables (see Figures [5](#ece36003-fig-0005){ref-type="fig"} and [6](#ece36003-fig-0006){ref-type="fig"}, Appendix [1](#ece36003-app-0001){ref-type="app"}). The sample size of 135 plots was chosen to compromise between statistical power and logistic limitations.
Plots were established in the field by marking the center with a metal rod and a magnet at ground level. Afterward the borders were identified with a differential GPS and marked. A schematic overview of sampling design on the plots is shown in Figure [4](#ece36003-fig-0004){ref-type="fig"}.
{#ece36003-fig-0004}
All plots were inventoried by measuring the DBH of every tree (above 7 cm DBH) and the amounts of lying and standing deadwood. Because habitat trees are an important structural element in retention approaches, for each study plot we mapped the 15 largest trees (measured by crown area from aerial images) and quantified in great detail their existing microhabitats (such as hollows, cracks, and crevices; Larrieu et al., [2017](#ece36003-bib-0055){ref-type="ref"}). Abiotic measurements in the plots include air temperature at 150 cm height at 1‐hr intervals in the center of every plot and quantification of light levels at the understorey, as well as soil nutrients. Additional abiotic data (e.g., precipitation) are derived from existing sources on a landscape scale (atlas data). Landscape patterns have been described using a range of metrics (measured around plot centers), including amount of forest cover, land cover composition, and edge density (Appendix [1](#ece36003-app-0001){ref-type="app"}).
3.3. The study system in a European context {#ece36003-sec-0013}
-------------------------------------------
The ConFoBi study system is embedded in a typical multiple‐use landscape of Central Europe; its study plots are part of a forest landscape with a variety of silvicultural systems and ownership types. Yet, the selection of study plots ensured that a wide range of the conditions found in European montane forests are represented; this in particular holds for the two design gradients, amount of deadwood at the plot scale, and amount of forest at the landscape scale.
The distribution of the 135 study plots along these two design gradients is shown in Figures [5](#ece36003-fig-0005){ref-type="fig"} and [6](#ece36003-fig-0006){ref-type="fig"}. The landscape gradient ranges from a forest matrix with high connectivity, to highly fragmented forests with an open matrix (Figure [5](#ece36003-fig-0005){ref-type="fig"}). Mean volumes of standing and lying deadwood amount for 14.0 and 43.6 m^3^/ha, respectively (Figure [6](#ece36003-fig-0006){ref-type="fig"}). The plots richest in deadwood with close to 500 m^3^/ha are located in strict forest reserves. According to studies in forest reserves of temperate Europe, natural amounts of \>200 m^3^/ha of standing and downed deadwood can be expected in montane beech‐fir forests (Bujoczek, Szewczyk, & Bujoczek, [2018](#ece36003-bib-0020){ref-type="ref"}). Temperate forests used for timber production typically are poor in deadwood (Vítková et al., [2018](#ece36003-bib-0091){ref-type="ref"}); on average, the volume of total deadwood is around 11.5 m^3^/ha in the forests of European countries, with standing deadwood making up for about one third, and lying deadwood for two‐thirds of the total volume (Forests Europe, [2015](#ece36003-bib-0030){ref-type="ref"}). For Germany, 4.7 and 15.9 m^3^/ha standing and lying deadwood, respectively, are reported (Forests Europe, [2015](#ece36003-bib-0030){ref-type="ref"}); for the State of Baden‐Württemberg, total deadwood amounts average 28.8 m^3^/ha, about half of which (14.1 m^3^/ha) is standing deadwood (Kändler & Cullmann, [2014](#ece36003-bib-0048){ref-type="ref"}); slightly higher amounts are reported for the Black Forest (total deadwood 33.4, lying 17.2 m^3^/ha) (Kändler & Cullmann, [2016](#ece36003-bib-0049){ref-type="ref"}). Thus, the ConFoBi study plots represent the full gradient of the deadwood amounts reported from managed as well as protected mixed montane forests in Central Europe, spanning from close to zero to volumes typical of natural stands. Further variables characterizing the ConFoBi study system at plot and landscape scales are presented in Appendix [1](#ece36003-app-0001){ref-type="app"}.
{#ece36003-fig-0005}
{#ece36003-fig-0006}
4. PROJECTS, METHODS, AND LINKAGES {#ece36003-sec-0014}
==================================
Currently, there are 14 different ConFoBi projects, focussing on the topics and methods sketched in Box [1](#ece36003-fea-0001){ref-type="boxed-text"}. Module A projects provide structural data at plot and landscape levels, which form a basis for analyses of biodiversity responses of various taxa to retention measures (B‐projects), and provide input for the study of economic implications of biodiversity‐oriented forest management, and biodiversity knowledge and conservation practices of forestry practitioners (C‐projects). All projects with an ecological or economic focus work on all 135 plots (Figures [1](#ece36003-fig-0001){ref-type="fig"}, [2](#ece36003-fig-0002){ref-type="fig"} and 2). The remaining social sciences projects do not strictly work on the plots only, because the relevant unit of analysis is not a spatially delimited area, but rather, for example, a specific type of organization (like a forest enterprise, a forest owner association, a forestry training center or a conservation agency) in which biodiversity‐relevant decisions are being made. Since in the landscapes surrounding the 135 plots all types of land tenure and forest ownership (state, communal, and private) are represented, the social sciences projects are tightly linked to and integrated into the ConFoBi study system.
Additional projects with complementary research questions are under development; for example, recently initiated (pilot) studies address effects of forest structure on the abundance of ticks and transmittable diseases, moths, leaf litter‐inhabiting organisms, fungi, and salamanders.
A Structures {#ece36003-sec-0015}
------------
### A1 Remote sensing based methods for the assessment of forest structures {#ece36003-sec-0016}
The project develops remote‐sensing methods to assess abundance, heterogeneity, and spatial distribution of structural elements, as predictors of biodiversity across multiple temporal and spatial scales. Advanced remote‐sensing techniques, such as LiDAR and digital stereophotogrammetry, in conjunction with platforms such as Unmanned Aerial Vehicles and terrestrial laser are used and new algorithms for extraction and unification of data are developed to provide structural information at scales appropriate for other ConFoBi projects.
### A2 Retention of structural elements in selectively used forests {#ece36003-sec-0017}
Live or dead standing trees that provide tree‐related microhabitats (TreMs) such as cavities, large dead branches, loose bark, epiphytes, bracket fungi, cracks, or trunk rot are defined as habitat trees. In natural forests habitat trees and TreMs may be highly clumped and variable, which may affect structure and viability of communities and meta‐communities of species reliant on microhabitats. A2 analyses and predicts TreMs on potential habitat trees, and assesses factors relevant for habitat tree selection, such as distribution, quality and longevity of habitat trees and TreMs, forest management intensity, and landscape context.
B Biodiversity components {#ece36003-sec-0018}
-------------------------
### B1 Epiphyte and microhabitat diversity and function on habitat trees {#ece36003-sec-0019}
The project quantifies the role of habitat trees for conserving forest epiphytes, and assesses how habitat trees differ in their epiphytic lichen and bryophyte diversity from trunk up to the crown and in variation of the species identity of the habitat tree, the main tree species of the stand, the diameter and microhabitat characteristics of the habitat tree, and landscape‐scale forest connectivity.
### B2 Mechanisms of vegetation change and diversity in retention forestry {#ece36003-sec-0020}
The project addresses the role of habitat trees and deadwood, and their landscape context, for plant diversity by disentangling various effects of abiotic changes on plant performance. B2 determines the composition and spatial distribution of understorey higher plants and ground‐dwelling mosses, to assess how heterogeneity in forest structure influences resource heterogeneity and availability of light for understorey vegetation, and hence species diversity and composition, and to quantify the role of forest structure and resource heterogeneity on trait distributions and functional diversity of the understorey.
### B3 Diversity and functions of plant‐insect interactions along a retention gradient {#ece36003-sec-0021}
The project addresses the overall hypothesis that stand‐scale retention measures influence the diversity and trophic interactions of hymenoptera and other arthropods, which are mediated by forest composition and configuration. B3 analyses the relationship between components of insect diversity and environmental variables related to retention (e.g., deadwood, microhabitats). To quantify trophic interactions and food webs plant galls with their gall‐forming communities and cavity‐nesting Hymenoptera are studied.
### B4 Functional connectivity among saproxylic beetles in dead‐wood patches {#ece36003-sec-0022}
The project investigates species and genetic diversity of saproxylic beetles and hymenoptera, and assesses gene flow of dead‐wood specialists with different dispersal ability as a function of abundance, distribution and isolation of deadwood across the landscape. Species are sampled at dead‐wood patches of different size and distance by means of trapping and metabarcoding. Genetic diversity is quantified using metabarcoding for total diversity and species‐specific markers (RADSeq) for genetic distance. Finally, B4 infers thresholds for functional connectivity among dead‐wood patches.
### B5 Landscape‐moderated use of forest structures by bats {#ece36003-sec-0023}
The project assesses linkages between multi‐scale forest structures and bat activity, richness, and diversity, as well as the functional importance of structures, specifically for foraging and commuting. Automated acoustic recorders are deployed to detect bat species(‐groups) and to quantify their activity. Presence, richness, diversity, and activity of bats are then related to measures of forest structure, including LiDAR‐information capturing the 3D‐characteristics of subcanopy space, and landscape‐scale connectivity metrics. Availability of food will be assessed (with B3, B4, and B6) to address inter‐trophic relationships that may explain the observed results.
### B6 Multi‐scale assessment of bird‐forest relationships {#ece36003-sec-0024}
The project quantifies occurrence and abundance of bird species based on repeated aural‐visual point counts in order to assess multi‐scale structural predictors of avian diversity. B6 first focusses on linkages between retention elements, landscape patterns, and birds; thereafter, on this basis, B6 assesses inter‐trophic relationships of birds that may explain the patterns observed. Assessment of food availability (with B3--B5) will allow elucidating functional relationships.
### B7 Soundscapes {#ece36003-sec-0025}
This project examines how acoustic diversity can be quantified to reflect avian diversity, and how these metrics can be combined with other variables to describe a forests potential for biodiversity. To test the hypothesis that acoustic diversity of avian vocalizations varies in response to multi‐scale forest structures B7 collects audio files and analyses acoustic indices to identify and track phenological changes in avian communities. These indices are validated against observed species richness and abundance (B6), then related to additional taxonomic groups (B3--B5) and metrics of forest structure (A1, A2, B2) at the plot and landscape level to model optimal combinations of indices and quantitative benchmarks to predict biodiversity.
### B8 Ungulate‐forest relationships {#ece36003-sec-0026}
The project focusses on the relationship between roe deer *Capreolus capreolus* and forest structure, addressing both bottom‐up and top‐down effects. Local abundance and habitat selection of roe deer, quantified through camera trapping and pellet counts, are related to biomass and composition of understorey vegetation, and to forest structure and landscape pattern. Effects of deer on plant community composition and understory species richness are addressed through browsing surveys and germination experiments based on deer feces. Finally, co‐occurrence and direct and indirect biotic interactions among roe deer and other taxa (with B1‐6) are assessed.
### B9 Effects of forest structures on fungi {#ece36003-sec-0027}
The project quantifies the impact of multi‐scale forest structure and fragmentation on the diversity of fungi with focus on wood‐inhabiting species and interactions between fungi and other organismic groups. It combines standard monitoring techniques with genetic analyses of selected species to determine genetic differentiation among local populations along gradients of forest fragmentation. Taxonomic, functional and genetic diversity measures of fungi are related to various forest structural and landscape metrics, as well as to diversity and composition of plants and animals (B1‐8).
C Human dimensions {#ece36003-sec-0028}
------------------
### C1 Economic valuation of biodiversity‐oriented forest management strategies {#ece36003-sec-0029}
The project hypothesizes that orienting forest management toward biodiversity has economic implications that can be quantified on different spatial scales. First, the value of biodiversity is expressed as an opportunity cost within a forward‐looking simulation‐optimization approach; thereafter, C1 quantifies social benefits of biodiversity, investigating optimal retention levels and spatial allocation of retention under multiple uncertainties including climate change. Finally, C1 aims to propose cost‐effective measures to promote biodiversity. For this, C1 collaborates with the A and B projects for structural and biodiversity data, and with C2 and D1 to find out which role economic parameters play in decision‐making related to biodiversity.
### C2 Local biodiversity knowledge and forest conservation practices {#ece36003-sec-0030}
This project connects three distinct fields of research. It conceptually links socio‐psychological findings of research on forest owners, their attitudes, practices and institutional integration and the work on traditional ecological knowledge and practices in conservation to the literature on forest and biodiversity conservation discourses and professional forest management paradigms among practitioners and policy makers. These questions have never systematically been related to each other in an empirical study. After a systematic review, exploratory interviews, and document analysis, qualitative interviews are combined with participatory observation. Finally, a representative mail survey will quantify the derived patterns.
D Science‐practice interface {#ece36003-sec-0031}
----------------------------
### D1 Professional epistemologies and integration of biodiversity‐related knowledge into socio‐political decision‐making {#ece36003-sec-0032}
The project assesses under which conditions specific stocks of biodiversity‐related knowledge are taken up in different decision‐making contexts. D1 is built on the hypothesis that both within biodiversity science and biodiversity policy and management there are distinguishable professional epistemologies that have an impact on problem definition, agenda setting as well as the formulation and implementation of problem‐solving strategies. The project uses a qualitative‐interpretative approach to investigate the specific "thought styles" of ConFoBi‐relevant scientific disciplines as well as those found in practical decision‐making contexts. Major approaches are document analysis, expert interviews, and participatory observations.
### D2 Evidence‐based biodiversity management of forests {#ece36003-sec-0033}
This project explores the scientific basis of management principles for conservation in forests. One challenge is the plethora of studies of different design and quality, sometimes yielding equivocal conclusions about causal links between management and biodiversity. Experts integrate the information available to them into a belief, which may or may not be a reasonable summary of the state of knowledge. D2 aims to critically evaluate the foundation of such models, both mathematical and mental, and juxtapose them with scientific evidence in the literature. D2 compares three representations of understanding: general causal knowledge with high levels of published evidence; specific causal assumptions represented in mathematical and computer models with biodiversity as a state variable; and intuitive causal belief of biodiversity‐generating processes in scientists and forest managers.
5. PERSPECTIVES FOR RESEARCH AND CONSERVATION {#ece36003-sec-0034}
=============================================
5.1. Complementing other research efforts {#ece36003-sec-0035}
-----------------------------------------
To our knowledge, ConFoBi is the first research programme that investigates how retention of forest structures affects forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe (Gustafsson et al., [2019](#ece36003-bib-0041){ref-type="ref"}). With its explicit focus on retention practices and by integrating the landscape and socio‐economic contexts of forest biodiversity, ConFoBi complements other research efforts on forest biodiversity. Synergies mayarise with the "Biodiversity Exploratories" (Fischer et al., [2010](#ece36003-bib-0028){ref-type="ref"}), "FunDivEUROPE" (Baeten et al., [2013](#ece36003-bib-0010){ref-type="ref"}) and its follow‐up process "SoilForEUROPE" (<http://websie.cefe.cnrs.fr/soilforeurope/>), as well as small FOREST (Valdés et al., [2015](#ece36003-bib-0088){ref-type="ref"}). Other related projects include "L57" (Management of species diversity in integrative forestry, <http://www.waldbau.wzw.tum.de/index.php?xml:id=155>), "BioHolz" (Biodiversity and Ecosystem Services of Forests, <https://www.bioholz-projekt.de>) and "RTG 2300: Enrichment of European beech forests with conifers" (<https://www.uni-goettingen.de/en/574316.html)>. These projects also work on biodiversity in forests, but do not explicitly address effects of retention measures on biodiversity. Finally, ConFoBi is collaborating with the European Forest Institute\'s (EFI) INFORMAR project focussing on socio‐economic driving factors that determine the scope for implementing integrated forest management approaches across Europe, and with regard to science‐policy‐practice interface activities to strengthen the practice and policy impact of its work internationally.
5.2. Knowledge and expertise for integrated forest management {#ece36003-sec-0036}
-------------------------------------------------------------
ConFoBi\'s research results will lead to quantitative target values describing the optimal amount, quality, and distribution of retention sites for different species and taxonomic groups as well as to recommendations how to efficiently integrate these targets into multi‐functional forest management. ConFoBi will provide an extensive case example for both reseach and management of forest biodiversity across the multiple‐use landscapes of the temperate zone.
In its first stage, ConFoBi focusses on describing patterns of biodiversity in relation to forest structures at spatial scales from plot to landscape. In later stages, the focus will shift from biodiversity patterns to functional relationships (e.g., between forest structures and organisms; among taxa and across trophic levels) and processes, which shape the observed patterns. To understand the socio‐economic context of biodiversity conservation in managed forests, ConFoBi will move from describing general knowledge structures to actions and impacts, to assess how practitioners integrate conservation in forest management. Finally, in an interdisciplinary synthesis, ConFoBi will quantify retention targets, and in particular the amounts and distribution of habitat trees and deadwood, required across the landscape for effective biodiversity conservation, identify the trade‐offs between forestry and biodiversity conservation, and will elaborate the socio‐economic prerequisites for their implementation by forest owners and managers. Wider applicability of the results will be ensured in cooperation with research groups elsewhere, for example, in the newly established COST Action BOTTOMS‐UP (<https://www.cost.eu/actions/CA18207/#tabs%7CName:overview>).
ConFoBi emphasizes the ecological foundations and socio‐economic frameworks of retention approaches in continuous‐cover forestry (Gustafsson et al., [2019](#ece36003-bib-0041){ref-type="ref"}). Preliminary results indicate positive overall effects of retention of structural elements for forest biodiversity but also high variability among taxa and landscape settings; further, our findings suggest high potential for optimizing the integration of retention into management practice. The first ConFoBi publications described technical and methodological advances for quantifying forest structures; for example, we found a high potential for optimizing the quality in reconstruction of 3D forest models from aerial images based on drones (Frey, Kovach, Stemmler, & Koch, [2018](#ece36003-bib-0036){ref-type="ref"}), showed how the abundance and diversity of tree‐related microhabitats can be predicted with readily available forest attributes (Asbeck, Pyttel, Frey, & Bauhus, [2019](#ece36003-bib-0002){ref-type="ref"}), recorded a lichen species new for Germany (Wirth, Tønsberg, Reif, & Stevenson, [2018](#ece36003-bib-0094){ref-type="ref"}), optimized the trap design to capture flying arthropods (Knuff, Winiger, Klein, Segelbacher, & Staab, [2019](#ece36003-bib-0051){ref-type="ref"}), and showed that the occurrence of specialist herbivore communities might be best explained by plant species composition rather than the abiotic environment (Knuff, Staab, Frey, Helbach, & Klein, [2019](#ece36003-bib-0050){ref-type="ref"}). Based on meta‐analyses, ConFoBi researchers confirmed that crown‐damaged trees improve nesting opportunities for cavity‐nesting birds (Gutzat & Dormann, [2018](#ece36003-bib-0042){ref-type="ref"}) and that woodpeckers select cavitiesby relative rather than absolute tree size (Basile, Mikusinski, & Storch, [2020](#ece36003-bib-0012){ref-type="ref"}), but found that bird guilds are affected differently by forestry measures including retention, according to their life history, biome, and forest type (Basile, Mikusinski, & Storch, [2019](#ece36003-bib-0011){ref-type="ref"}). A joint study by social and remote‐sensing scientists of ConFoBi found expert ratings of forest structure, despite large individual bias, were on average significantly related to technical structural complexity indices based on terrestrial laser scanning (Frey, Joa, Schraml, & Koch, [2019](#ece36003-bib-0035){ref-type="ref"}), and a review concluded that local ecological knowledge holds significant promise for integrating conservation objectives into forest management under changing environmental conditions (Joa, Winkel, & Primmer, [2018](#ece36003-bib-0046){ref-type="ref"}; Joa & Schraml, [2018](#ece36003-bib-0045){ref-type="ref"}). Analyses of the opportunity costs arising from retention forestry suggest that conservation practices, such as habitat networks of deadwood islands, will only marginally impact profitability when conservation and production goals are balanced through suitable planning tools (Augustynczik, Yousefpour, Rodriguez, & Hanewinkel, [2018](#ece36003-bib-0009){ref-type="ref"}). Interdisciplinary modeling suggested that integration of uncertainty into conservation planning may reduce the trade‐off between production and conservation objectives in forest landscapes (Augustynczik et al., [2017](#ece36003-bib-0005){ref-type="ref"}; Augustynczik, Yousefpour, & Hanewinkel, [2018](#ece36003-bib-0006){ref-type="ref"}), and a diversification of forest management regimes is recommended for securing various model taxa, including saproxylic beetles (Augustynczik, Yousefpour, et al., [2018](#ece36003-bib-0006){ref-type="ref"}), as well as tree microhabitats and birds under climate change (Augustynczik, Asbeck, et al., [2019](#ece36003-bib-0003){ref-type="ref"}; Augustynczik, Yousefpour, et al., [2018](#ece36003-bib-0006){ref-type="ref"}). Overall, ConFoBi\'s analyses suggest that current forest management for biodiversity is inefficient under climate change (Augustynczik et al., [2020](#ece36003-bib-0004){ref-type="ref"}; Augustynczik, Yousefpour, & Hanewinkel, [2019](#ece36003-bib-0008){ref-type="ref"}).
ConFoBi\'s results and recommendations will be translated for practice and political decision makers using the established science‐practice‐policy communication pathways of the collaborating partners Forest Research Institute of Baden Württemberg (FVA) and European Forest Institute (EFI). National and State Agencies directly concerned with forests and conservation and other national‐level and EU‐level decision makers are involved in a transdisciplinary dialogue throughout ConFoBi. ConFoBi will identify the ecological as well as the socio‐economic prerequisites for effective implementation of retention measures for biodiversity by forest owners and managers. ConFoBi\'s results will be seminal for integrating biodiversity conservation into forest management by providing an interdisciplinary evidence base for optimizing the effectiveness of retention approaches.
5.3. Complementing ConFoBi: an invitation {#ece36003-sec-0037}
-----------------------------------------
While ConFoBi is primarily a Research Training Group for doctoral students, we have also set it up to provide a research platform that can be extended in scope, spatial, and temporal scales, as well as interdisciplinarity. Whereas the first set of studies primarily addresses disciplinary questions, later studies will focus on synergies across projects and disciplines. Finally, ConFoBi will synthesize and validate its results on the relationships between forest biodiversity, retention measures, and their landscape and socio‐economic contexts, using a range of approaches from empirical studies to scenario modeling. ConFoBi is open to complementary projects. Scientists from all career levels---doctoral students to senior researchers---and from all disciplines are welcome to propose complementary research ideas within the framework of ConFoBi; PhD students and PostDocs are particularly invited; inter‐ and transdisciplinary studies are strongly encouraged. Further information on ConFoBi as well as information on study sites and datasets generated within ConFoBi will be made available here: <http://confobi.uni-freiburg.de/en> and can be requested from the corresponding author.
CONFLICT OF INTEREST {#ece36003-sec-0039}
====================
Authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS {#ece36003-sec-0040}
====================
All authors (IS, JP, TA, MB, JB, VB, CFD, JF, SG, MH, BK, A‐MK, TK, MP, PP, AR, MS‐L, GS, US, MS, GW, and RY) have contributed to the development of the research programme and study design of ConFoBi, and/or the selection, establishment, and inventory of the study plots. The lead author (IS) has written the manuscript and is the spokesperson of ConFoBi; the second author (JP) collated the plot data, all remaining authors are listed in alphabetical order. All authors contributed to drafts and gave final approval for publication.
This article is part of the Research Training Group ConFoBi (GRK 2123/1 TPX), which is funded by the German Research Association (DFG). The authors thank the Forest Research Institute of Baden Württemberg (FVA) for participating in this research, the Ministry for Rural Affairs and Consumer Protection (MLR) and the State Forests of Baden‐Württemberg for supporting the ConFoBi research programme, and the foresters in charge of the ConFoBi study plots for support in the field.
DATA AVAILABILITY STATEMENT {#ece36003-sec-0042}
===========================
Data sharing is not applicable to this article as no new data were analyzed in this article. The datasets used to illustrate the distribution of the study plots along variables of forest structure and landscape connectivity (Figures [5](#ece36003-fig-0005){ref-type="fig"} and [6](#ece36003-fig-0006){ref-type="fig"}) are available from the corresponding author on reasonable request.
{#ece36003-sec-0043}
Examples of measures of topography, vegetation, and landscape pattern for the 135 ConFoBi study plots. Topography and forest characteristics are mean values and total counts, respectively, referring to 1 ha; landscape metrics were calculated for various scales (see Schindler, Wehrden, Poirazidis, Wrbka, & Kati, [2013](#ece36003-bib-0079){ref-type="ref"}; respective moving window sizes are given in brackets). Definitions of landscape metrics according to McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"}). Note that Landsat forest classes (conifer, broad‐leaved, and mixed) were merged into class "forest," remaining classes to "nonforest."
MeasureUnitDefinitionSourceRange (min--max)Mean*SD*ReferenceTopographyElevationm a.s.l.Mean value derived from 1 m digital terrain modelLGL ([2005](#ece36003-bib-0057){ref-type="ref"})443--1334822±182 SlopeDegreeMean value derived from 1 m digital terrain modelLGL ([2005](#ece36003-bib-0057){ref-type="ref"})1--3415±9 AspectDegreeMean value derived from 1 m digital terrain modelLGL ([2005](#ece36003-bib-0057){ref-type="ref"})3--360172±109 TRI (Terrain ruggedness index)mMean value of the mean difference between a central pixel and its surrounding cells derived (moving window) from 40 cm GSD DSM (Ground Sampling Digital Surface Model) generated from 20 cm aerial images using SfM (Structure from Motion)ConFoBi data0.25--0.970.56±0.16Wilson, O\'Connell, Brown, Guinan, and Grehan ([2007](#ece36003-bib-0092){ref-type="ref"})VegetationNo of trees*N*Inventory of all trees inside 1 ha plot with DBH \>7 cmConFoBi data98--1212425±205 Tree speciesSpeciesInventory of all trees inside 1 ha plot with DBH \>7 cmConFoBi data DBHmmInventory of all trees inside 1 ha plot with DBH \>7 cmConFoBi data70--1268271.0±166.0 Basal area living treesm^2^Inventory of all trees inside 1 ha plot with DBH \>7 cmConFoBi data9.4--73.134.1±9.9 Tree heightmMean value derived from subtraction of digital terrain (DTM) model from calibrated surface heights from UAV‐SfM (Unmanned aerial vehicle‐Structure from Motion) flightsDTM: LGL 2005; UAV: ConFoBi data8.6--40.624.1±5.9Frey et al. ([2018](#ece36003-bib-0036){ref-type="ref"})Standing deadwood*N*Calculated from plot inventoryConFoBi data0--39433.4±53.6 Basal area standing deadwoodm^2^Mean value derived from plot inventory (BA = 0.00007854 × DBH^2^)ConFoBi data0--51.22.2±5 Standing dead‐wood volumem^3^Calculated from plot inventory (*V* = Basal area × height × 0.5 (form factor))ConFoBi data0--2163140±282 Lying dead‐wood volumem^3^Calculated from described *V* transect (*V* = (k/*L*)∑*d* ^2^; k = constant = 1.234 (see Van Wagner, [1982](#ece36003-bib-0089){ref-type="ref"}), *L* = length of transect, *d* = DBH)ConFoBi data2.7--282.943.6±43.7Van Wagner ([1982](#ece36003-bib-0089){ref-type="ref"}), Kahl and Bauhus ([2014](#ece36003-bib-0047){ref-type="ref"})NDVI Normalized Difference Vegetation Index; mean value derived from Sentinel 2 dataESA ([2018](#ece36003-bib-0022){ref-type="ref"})0.61--0.820.72±0.036Rouse, Haas, Schell, and Deering ([1973](#ece36003-bib-0075){ref-type="ref"})LandscapeHeterogeneity as proportion of stands%Derived from stand based local forest inventory of Baden‐WürttembergFoGIS10/InFoGIS (MLR) ([2018](#ece36003-bib-0029){ref-type="ref"})0.001--10054.0±39.5 Distance from plot center to nearest forest edgemValue derived from OpenStreetmap‐DataOpenStreetMap Contributors ([2016](#ece36003-bib-0069){ref-type="ref"})44--1503256±213 Area of surrounding forestkm^2^Total size of the forest patch which contains the plotOpenStreetMap Contributors ([2016](#ece36003-bib-0069){ref-type="ref"})0.14--333.6296.64±112.97 Forest connectivity%Percentage of forest cover in the 25 km^2^ surrounding the plot centerConFoBi data3.0--92.259.9±19.7 Edge density (10 ha surrounding plot center)m/haSum of lengths (m) of all edge segments involving forests per 1‐ha plot; mean value derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})121--350226±61McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Euclidean nearest neighbor distance (20 ha), CVmCoefficient of variation of the distance (m) to the nearest neighboring patch of forest, based on shortest edge‐to‐edge distance; derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0--42.59.8±8.7McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Euclidean nearest neighbor distance (20 ha), meanmArea‐weighted mean distance (m) to the nearest neighboring patch of forest, based on shortest edge‐to‐edge distance; derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0--66.512.7±12.0McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Euclidean nearest neighbor distance (50 ha), meanmArea‐weighted mean distance (m) to the nearest neighboring patch of forest, based on shortest edge‐to‐edge distance; derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})1.56--152.670.735.5McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Euclidean nearest neighbor distance (50 ha), CVmCoefficient of variation derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0--519.4±8.8McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Aggregation index (50 ha)%Mean number of like adjacencies involving forest divided by the maximum possible number of like adjacencies involving forest; multiplied by 100. From landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})64.8--99.583.1±7.4McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Contiguity index (50 ha) The sum of the cells divided by the total number of pixels in the patch minus 1, divided by the sum of the template values minus 1. Area‐weighted mean derived from Landsat TM5 (yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0.00167--0.021560.00875±0.00459McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Landscape shape index (50 ha) 0.25 the sum of entire landscape boundary and edge segments (m) within landscape boundary involving forest, divided by square root of total landscape area (m^2^). Mean derived from Landsat TM5 (yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})1.08--5.253.19±0.91McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Perimeter‐area ratio distribution (50 ha)m/m^2^A simple measure of shape complexity. Area‐weighted mean derived from Landsat TM5 (yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})1.6--27.811.1±5.7McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Percentage of like adjacencies (50 ha)%Percentage of cell adjacencies involving forest that are like adjacencies. Mean derived from Landsat TM5; yrs 2009, 2010LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})58.7--95.076.6±7.9McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Contiguity index (100 ha) The sum of the cells divided by the total number of pixels in the patch minus 1, divided by the sum of the template values minus 1. Area‐weighted mean derived from Landsat TM5 (yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0.0007--0.01490.0057±0.0032McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Core area (100 ha)m^2^Area (m^2^) within the patch that is further than the specified depth‐of‐edge distance from the patch perimeter. Area‐weighted mean derived from landuse map (Landsat TM5; yrs 2009, 2010)LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0.06--5.111.44±0.99McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})Splitting index (100 ha) Total area (m^2^) squared divided by the sum of patch area (m^2^) squared, summed across all patches of forest. Mean derived from Landsat TM5; yrs 2009, 2010LUBW ([2010](#ece36003-bib-0059){ref-type="ref"})0.007--1.350.23±0.25McGarigal ([2015](#ece36003-bib-0061){ref-type="ref"})
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Background {#Sec1}
==========
In clinical practice, an adequate measurement of resting energy expenditure (REE) for adult patients is important for optimal nutritional therapy in order to prevent under- and over nutrition \[[@CR1]\]. REE in adult patients can be measured by indirect calorimetry, based on oxygen consumption and carbon dioxide production \[[@CR2]\]. Indirect calorimetry is considered as the most accurate method \[[@CR3]\] for determining the REE in adult patients \[[@CR4], [@CR5]\]; however, this measurement is time-consuming and not available in most clinical settings. As an alternative, REE is usually calculated with various REE predictive equations, based on healthy subjects \[[@CR1], [@CR6]\].
Only few studies have validated REE predictive equations in hospitalized patients \[[@CR7]--[@CR9]\]. The number of validated predictive equations is small \[[@CR7], [@CR8]\] and studies have small sample sizes \[[@CR7], [@CR9]\]. Therefore, there is no consensus about which equation to use in hospitalized patients. According to Boullata et al. \[[@CR8]\], the Harris & Benedict (1918) (HB1918) \[[@CR10]\] equation is the best equation to predict REE, when using an illness factor of 1.1. It appeared 62% of the patients were predicted accurately using this equation. Anderegg et al. \[[@CR7]\] suggests HB1918 with adjusted bodyweight and a stress factor, which led to 50% accurately predicted patients. Weijs et al. \[[@CR9]\] suggest the WHO and adjusted Harris & Benedict (HB1984) \[[@CR11]\] equations, predicting about 50% of the patients accurately. More recently, Jesus et al. \[[@CR12]\] showed that the original Harris & Benedict equation (HB1918) performed reasonably, but no equation was adequate for extreme BMI groups (\<16 and \>40).
Therefore, it is unclear which REE predictive equation performs most uniform across BMI subgroups for hospital patients. The aim of this study is to examine the validity of REE predictive equations for underweight, normal weight, overweight, and obese patients by comparison with indirect calorimetry.
Methods {#Sec2}
=======
Patients {#Sec3}
--------
Between March 2005 and December 2015, data were collected at the VU University Medical Center Amsterdam. Patients who had an indication for nutritional assessment by the dietitian were included in this study. All measurements were performed according to a standardized operating procedure (SOP), and personal was trained in a standardized manner. Patients were measured as part of patient care. As malnutrition is the main reason for measurement, withholding food for longer than absolutely necessary is questionable and maybe unethical. All patients were restricted from food for at least 2 h before the measurement. None of the patients were restricted from food for 8 h, as the guideline \[[@CR13]\] indicates.
Only adult patients with complete data (height, weight, age, and gender) were included. When repeated REE measurements were available, only the first measurement was included. Exclusion criteria were patients at ICU, pregnant women, and REE measurements shorter than 15 min. All procedures were in accordance with ethical standards of the institution.
Indirect calorimetry and anthropometric measurements {#Sec4}
----------------------------------------------------
Indirect calorimetry measurements were performed by using a metabolic monitor (Deltatrac 2 MBM-200, Datex-Ohmeda, Helsinki, Finland; Vmax Encore n29, Viasys Healthcare, Houten, The Netherlands). Both devices were calibrated every day before use and Vmax also every 5 min during measurement. The Deltatrac was calibrated with one reference gas mixture (95% O~2~, 5% CO~2~), whereas Vmax was calibrated with two standard gases (26% O~2~, 0% CO~2,~ and 16% O~2~, 4% CO~2~). Patients were measured in supine position. Calibration and measurements were performed by a trained dietitian. Oxygen analyser sensitivity was checked yearly by supplier.
Body weight was measured using a calibrated electronic stand-up scale (Seca Alpha, Hamburg, Germany). In case of severe oedema or when weighing was not possible, even weighing in bed, self-reported weight was used. Height of the patient was measured or self-reported. BMI was calculated as weight (kg) divided by the square of height (m^2^).
REE predictive equations {#Sec5}
------------------------
Predictive equations were obtained by a systematic search using PubMed. Mesh-derived keys 'energy metabolism', 'basal metabolism' and 'indirect calorimetry' and additional terms ('predict\*', 'estimat\*', 'equation\*' and 'formula\*') were applied in every possible combination. Applied limitations were 'English language', 'humans' and the age of 18 years and older. Additional publications were checked based on reference lists. Equations were included when based on body weight, height, age, and/or gender.
The Weijs equation for overweight patients \[[@CR14]\] was tested in patients with BMI \> 25. For the BMI \< 25 subgroup, a new REE predictive equation was developed in this subpopulation with BMI \< 25 using regression analysis with measured REE (kcal/day) as dependent and body weight (kg), height (m), age (y), and sex (F = 0, M = 1) as independent variables.
Statistical analysis {#Sec6}
--------------------
An independent samples *T*-test was used for differences in weight, BMI, age, and REE between inpatients and outpatients, as well as between males and females. BMI subgroups were analysed: underweight (BMI \< 18.5 kg/m^2^), normal weight (BMI ≥18.5- \< 25 kg/m^2^), overweight (BMI ≥25- \< 30 kg/m^2^), and obese patients (BMI ≥ 30 kg/m^2^). The difference between the REE predictive equation and REE measured was calculated as percentage. A prediction between 90 and 110% of the REE measured was considered as accurate prediction. A prediction below 90% was considered as under prediction and a prediction over 110% was considered as over prediction. The bias indicates the mean percentage error between REE predictive equation and REE measured. The root-mean-square-error (RMSE), expressed in kcal/day, was used to measure how well the equations fitted the REE measurement.
To check whether in underweight and obese patients adjustment of weight in the REE predictive equation resulted in a better performance of the equation, body weight adjustment was applied (BMI \< 18.5: weight adjusted to BMI = 18.5); BMI \> 30: weight adjusted to BMI = 30). The criterion for improvement of performance was percentage accurate predictions. Statistical significance was reached when *p* \< 0.05. Data was analysed with IBM SPSS Statistics 20.
Results {#Sec7}
=======
Patients {#Sec8}
--------
Table [1](#Tab1){ref-type="table"} shows characteristics of study populations. REE measurements of 593 patients were available. Eighty had incomplete data. In total, 513 general hospital patients were included, (253 F, 260 M), 237 inpatients and 276 outpatients. These patients were often complex patients with multimobidity and were categorised as oncology (29%), gastroenterology (19%, Diabetes/overweight (14%), Nephrology (10%), Lung diseases (7%), Neurology (5%), diagnostics in unintentional weight loss (5%) and a rest group (8%) of cardiology, anorexia nervosa, auto immune disease, spinal cord injury and RA patients.Table 1Patient characteristics for the total group and per BMI groupTotal groupBMI \< 18,5BMI 18,5--25BMI 25--30BMI \> 30N (%)513141 (27%)209 (41%)77 (15%)86 (17%)MeanSDMeanSDMeanSDMeanSDMeanSDAge (y)53.015.651.317.054.115.255.315.250.914.2% Male51%44%58%53%41%Weight (kg)70.122.949.47.364.28.783.211.0106.721.3Height (m)1.730.101.720.101.740.091.740.101.710.12BMI (kg/m^2^)23.47.216.61.521.31.827.31.436.35.4REE (kcal/day)16784081448318169635817303521966488REE in kcal/kg/day (range)25.1 (12--53)6.229.4 (18--43)5.526.6 (14--53)5.320.8 (12--31)3.318.5 (13--29)3.2% inpatients46%57%55%35%17%
REE predictive equations {#Sec9}
------------------------
In total, 15 predictive equations were used. The most used fixed factors (25 kcal/kg/day, 30 kcal/kg/day and 2000 kcal for female and 2500 kcal for male) were added. These fixed factors calculate total energy expenditure and in order to provide REE, they were divided by a physical activity and/or stress factor of 1.3. [Appendix 1](#Sec13){ref-type="sec"} shows the descriptives of the included REE predictive equations.
Accuracy of predictive equations {#Sec10}
--------------------------------
Based on REE data of patients with BMI \< 25 a new equation was developed in the current population: BMI \< 25: REE (kcal/day) = 11.355 × weight (kg) + 7.224 × height (cm) - 4.649 × age (y) + 135.265 × sex (F = 0; M = 1) - 137.475; for BMI ≥ 25 an equation had been developed on healthy overweight and/or obese subjects by Weijs and Vansant \[[@CR14]\]: BMI ≥ 25: REE (kcal/day) = 14.038 × weight (kg) + 4.498 × height (cm) - 0.977 × age (y) + 137.566 × sex (F = 0; M = 1) - 221.631.
Table [2](#Tab2){ref-type="table"} shows statistics of the REE predictive equations for all patients. The percentage of accurate predicted REE was low in all equations, ranging from 8 to 49%. Overall the new equation performed equal to the best performing Korth equation and slightly better than the well-known WHO equation based on weight and height (49% vs 45% accurate).Table 2Statistics of REE prediction equation performance, N = 513REE (kcal/day)SDUnder prediction (%) ^a^Accurate prediction (%) ^b^Over prediction (%) ^c^BIAS ^d^RMSE ^e^REE by calorimetrie1678408New equation16983131949324286Korth \[[@CR18]\]1621344304922−1295WHO-wtht \[[@CR15]\]1540288404514−6321Schofield-wtht \[[@CR19]\]1513282464212−7333Henry-wtht \[[@CR20]\]1489291513910−9344WHO-wt \[[@CR15]\]1504304493913−8345Harris& Benedict 1918 \[[@CR10]\]1490324513811−9350Muller \[[@CR21]\]1493308523711−9347H&B by Roza \[[@CR11]\]1494321533711−9344Schofield-wt \[[@CR19]\]1483293533612−9355Mifflin \[[@CR22]\]144430460328−12369Henry2005-wt \[[@CR20]\]1458320583110−11370MullerBMI \[[@CR21]\]139643560319−1645030 kcal/kg1618527442828−2435Livingston \[[@CR23]\]140528466277−1439925 kcal/kg134844068239−195022000 kcal for female and 2500 kcal for male22532503118741689Bernstein \[[@CR24]\]12082719082−26557^a^ The percentage of subjects predicted by this predictive equation \< 10% of the measured value^b^ The percentage of subjects predicted by this predictive equation within 10% of the measured value^c^ The percentage of subjects predicted by this predictive equation \> 10% of the measured value^d^ Mean percentage error between predictive equation and measured value^e^ Root mean squared prediction error
Table [3](#Tab3){ref-type="table"} shows statistics for the best predictive equations categorized by BMI subgroups. The new equation, Korth and the WHO equation based on weight and height performed best in all categories except from the obese subgroup. HB1918 was best for obese patients.Table 3REE predictive accuracy of prediction equations in BMI subgroupsTotal group (n = 513)BMI \<18.5 (n = 141)BMI 18.5--25 (n = 209)BMI 25--30 (n = 77)BMI \> 30 (n = 86)Under predic-tionAccu-rateOver predic-tionUnder predic-tionAccu-rateOver predic-tionUnder predic-tionAccu-rateOver predic-tionUnder predic-tionAccu-rateOver predic-tionUnder predic-tionAccu-rateOver predic-tion%%%%%%%%%%%%%%%New equation194932214435225127145827144442Korth \[[@CR18]\]304922354024345214175627224830WHO-wtht \[[@CR15]\]40451440451448439275518334423Schofield-wtht \[[@CR19]\]46421243441354407364816404021Henry-wtht \[[@CR20]\]51391050371360354404514384516WHO-wt \[[@CR15]\]49391352351260337305317314523Harris & Benedict 1918 \[[@CR10]\]51381160271363333345313265321Muller \[[@CR21]\]52371159291262335384814285121H&B by Roza \[[@CR11]\]53371157301365333394516295021Schofield-wt \[[@CR19]\]53361254331361327424712404021Mifflin \[[@CR22]\]583386028126630445459484013Henry-wt \[[@CR20]\]603286028126729449438523710MullerBMI \[[@CR21]\]60328632710702734843943431430 kcal/kg58311067231169274404514354322Livingston \[[@CR23]\]6031999105536939471429502125 kcal/kg4428287816651391184052510852000 kcal for female and 2500 kcal for male66277731987323353398503812Bernstein \[[@CR24]\]68239919086122404910144740Accurate prediction: the percentage of subjects predicted by this predictive equation within 10% of the measured valueUnderprediction: the percentage of subjects predicted by this predictive equation \<10% of the measured valueOverprediction: the percentage of subjects predicted by this predictive equation \> 10% of the measured value
Figure [1](#Fig1){ref-type="fig"} shows the percentage of accurately predicted underweight and obese patients with actual as well as adjusted weight using the WHO equation with weight and height \[[@CR15]\] and HB1918 \[[@CR10]\]. Adjusting the weight in the equation in underweight and obese patient did not improve the percentage of patients with an accurate predicted REE.Fig. 1The percentage of accurately predicted underweight and obese patients with actual as well as adjusted weight (BMI \< 18.5: weight adjusted to BMI = 18.5); BMI \> 30: weight adjusted to BMI = 30)
Discussion {#Sec11}
==========
This study shows that for hospital inpatients and outpatients the generally applied WHO \[[@CR15]\] and the original Harris & Benedict equation (HB1918) \[[@CR10]\] can only predict resting energy expenditure accurately in one of two to three patients. The generally used fixed 25 kcal/kg body weight was only accurate in 28% of the patients. The Korth equation also performed well, but not significantly better than the well implemented WHO and H&B equations. The newly developed equation performed equal to the best performing equations but showed no additional value. Generally applied weight adjustments all failed to improve accuracy. Hospital inpatients and outpatients may still benefit from using indirect calorimetry for assessment of energy needs.
Studies by Anderegg et al. \[[@CR7]\] and Boullata et al. \[[@CR8]\] analysed (in part) mechanically ventilated patients and are therefore more difficult to compare to current inpatient and outpatient analysis. However, in general they also showed rather inaccurate estimates using different REE estimating equations. Based on a similar analysis with a much smaller sample size, Weijs et al. \[[@CR9]\] concluded that the WHO equation (1985) \[[@CR15]\] based on weight and height and Harris & Benedict (1984) \[[@CR11]\] were the best predictive equations. The current analysis confirms that the overall accuracy of REE predictive equations is only about 50%, however this study extends this analysis to BMI subgroups for which predictive accuracy may in fact be much worse.
Jesus et al. \[[@CR12]\] showed that the overall accuracy of the Harris & Benedict equation was reasonable for the outpatient sample. The authors stress that predictive accuracy is much worse in extreme BMI subgroups with BMI under 16 and BMI over 40. The current study generally supports these conclusions, however extend these observations in two ways. First, the general accuracy is not that much higher in the normal weight patient group, in fact accuracy increases to highest level in overweight subgroup. Secondly, we agree that the subgroup of patients with BMI less than 16 has a low prediction accuracy, however we have also shown low prediction accuracy for a large cohort of malnourished hospitalized elderly with mean BMI 21 (SD 4) \[[@CR16]\]. Therefore, the suggestion that predictive equations perform well between BMI 16 and BMI 40 is largely false for hospital patients.
According to Frankenfield et al. \[[@CR17]\], adjusting body weight in obese patients leads to underestimation of the energy expenditure. When this is done with a fixed BMI level, the adjustment appears too large and does not result in a higher accuracy of REE prediction. However, accuracy remains low in all predictions.
This study has several strengths and limitations. The sample size of 513 patients was large enough for subgroup analysis, namely BMI subgroups. Furthermore, these data were derived from daily clinical practice and therefore the study population is representative for the inpatient and outpatient population. Another advantage is the exclusion of ICU patients that may not be entirely comparable to the general hospital population. Therefore, this study has a large generalizability to other hospitals and patients.
However, this study has some limitations as well. The measurements were performed in clinical practice and therefore patients were not measured in overnight fasted state. However, since patients were measured because of nutritional problems, the thermic effect of larger meals, if any, were not a problem in this patient sample. This could have been a problem in obese outpatients, however according to the results the estimations are most accurate in this subgroup. Only when the dietitian indicated the patient for nutritional assessment, a measurement was performed. This may have led to selection bias as only patients who were difficult to assess and/or treat were included in this study. This may largely explain the low level of accuracy in the current analysis.
This study population was too small to develop a new equation for the hospital in and outpatients. The variation of REE between patients and probably between disease groups is too large. A possible way forward, is to develop new equations in more homogenous subgroups. For this purpose a very large database would be needed on REE in hospital patients. We propose to develop an REE repository for clinical data, comparable to the Oxford database on REE in healthy subjects. This could be jointly organised within ESPEN and ASPEN.
Conclusions {#Sec12}
===========
In conclusion, REE predictive equations are only accurate in about half the patients. The WHO equation is advised up to BMI 30, and HB1918 equation is advised for obese (over BMI 30). Measuring REE with indirect calorimetry is preferred, and should be used when available and feasible in order to optimize nutritional support in hospital inpatients and outpatients with different degrees of malnutrition.
Appendix 1 {#Sec13}
==========
Table 4Descriptives of included predictive equationsAuthor, year of publication and referred to asStudy population and nAge (mean ± SD or range)REE Equations (kcal/day)Bernstein, 1983 \[[@CR24]\]Obese individuals; patients who enrolled the Weight Control Unit of the Obesity Research Center IC instrument: Beckman\
*n*: 48 M/154 FM: 39 ± 12 yM: 11.02 × WT + 10.23 × HTCM - 5.8 × AGE -- 1032F: 40 ± 13 yF: 7.48 × WT - 0.42 × HTCM - 3.0 × AGE + 844FAO/WHO/UNU, 1985 \[[@CR15]\]\
WHO-wt\
WHO-wtht*n*: 575 M/734 FAll: 30--82yM 18--30: (15.3 × WT) + 679F 18--30: (14.7 × WT) + 496M 30--60: (11.6 × WT) + 879F 30--60: (8.7 × WT) + 829M 60+: (13.5 × WT) + 487F 60+: (10.5 × WT) + 596Equations based on weight and heightM 18--30: (15.4 × WT) -- (27 × HTM) + 717F 18--30: (13.3 × WT) + (334 × HTM) + 35M 30--60: (11.3 × WT) + (16 × HTM) + 901F 30--60: (8.7 × WT) - (25 × HTM) + 865M 60+: (8.8 × WT) + (1128 × HTM) -- 1071F 60+: (9.2 × WT) + (637 × HTM) -- 302Harris & Benedict, 1918 \[[@CR10]\]*n*: 136 M/103 FM: 27 ± 9 (16--63) yM: 66.4730 + (13.7516 × WT) + (5.0033 × HTCM) -- (6.7550 × AGE)F: 31 ± 14 (15--74) yF: 655.0955 + (9.5634 × WT) + (1.8496 × HTCM) -- (4.6756 × AGE)Harris & Benedict, 1984 Roza & Shizgal \[[@CR11]\]\
H&B by RozaData of Harris & Benedict (1918) and data of two further studies by Benedict with data on additional subjects (*n*: 168 M/169 F)M: 30 ± 14 yM: 88.362 + (13.397 × WT) + (4.799 × HTCM) -- (5.677 × AGE)F: 44 ± 22 yF: 447.593 + (9.247 × WT) + (3.098 × HTCM) -- (4.330 × AGE)Korth, 2007 \[[@CR18]\]Healthy euthyroid weight stable subjects who were recruited by local announcements\
*n*: 50 M/54 FM: 39 ± 14 (21--68) yAll: (41.5 × WT) -- (19.1 × AGE) + (35.0 × HTCM) + (1107.4 × SEX) -- 1731.2/4.184F: 35 ± 15 (20--66) yLivingston, 2005 \[[@CR23]\]Institute of Medicine population\
*n*: 299 M/356 FM: 36 ± 15 (18--95) yM: 293 × WT ^0.4330^ -- (5.92 × AGE)F: 39 ± 13 (18--77) yF: 248 × WT ^0.4356^ -- (5.09 × AGE)Mifflin, 1990 \[[@CR22]\]IC instrument: metabolic measurement cart with a canopy hood (Metabolic Measurement Cart Horizons System)\
*n*: 251 M (122 obese)/247 F (112 obese)M: 44 ± 14 (19--78) yM: (9.99 × WT) + (6.25 × HTCM) -- (4.92 × AGE) + 5F: 45 ± 14 (20--76) yF: (9.99 × WT) + (6.25 × HTCM) -- (4.92 × AGE) -- 161Muller, 2004 \[[@CR21]\]Data from seven different research centers in Germany\
IC instruments: Deltatrac, Beckman, Mouthpiece (metabolic chamber)\
*n* BMI \< 18.5: 58\
*n* BMI 18.5--25: 444\
*n* BMI 25--30: 266\
*n* BMI \> 30: 278BMI ≤ 18.5: 32 ± 12 yAll: (0.047 × WT) + (1.009 × SEX) -- (0.01452 × AGE) + 3.21/4.184 ×1000BMI \> 18.5--25: 38 ± 17 yBMI ≤ 18.5: (0.07122 × WT) -- (0.02149 × AGE) + (0.82 × SEX) + 0.731/4.184 ×1000MullerBMI \> 25--30: 53 ± 16 yBMI \> 18.5--25: (0.02219 × WT) + (0.02118 × HTCM) + (0.884 × SEX) -- (0.01191 × AGE) + 1.233/4.184 ×1000MullerBMIBMI ≥ 30: 47 ± 13 yBMI \> 25--30: (0.04507 × WT) + (1.006 × SEX) -- (0.01553 × AGE) + 3.407/4.184 ×1000BMI ≥ 30: (0.05 × WT) + (1.103 × SEX) -- (0.01586 × AGE) + 2.924/4.184 ×1000Henry, 2005 \[[@CR20]\]Worldwide population (excluded\
Italian subjects) from several papers\
M 18--30 y: 2821/2816\
F 18--30 y: 1664/1655\
M 30--60 y: 1010/1006\
F 30--60 y: 1023/1023\
M 60+ y: 534/533\
F 60+ y: 334/324M 18--30: 22 yM 18--30 y: (16 × WT) + 545Henry-wtF 18--30: 22 yF 18--30 y: (13.1 × WT) + 558M 30--60: 40 yM 30--60 y: (14.2 × WT) + 593F 30--60: 41 yF 30--60 y: (9.74 × WT) + 694M 60+: 70 yM 60+ y: (13.5 × WT) + 514F 60+: 69 yF 60+ y: (10.1 × WT) + 569Equations based on weight and heightHenry-wthtM 18--30 y: (14.4 × WT) + (313 × HTM) + 113F 18--30 y: (10.4 × WT) + (615 × HTM) -- 282M 30--60 y: (11.4 × WT) + (541 × HTM) -- 137F 30--60 y: (8.18 × WT) + (502 × HTM) -- 11.6M 60+ y: (11.4 × WT) + (541 × HTM) -- 256F 60+ y: (8.52 × WT) + (421 × HTM) + 10.7Schofield, 1985 \[[@CR19]\]Collection of different authors and papers\
M 18--30 y: 2879\
M 30--60 y: 646\
M 60+ y: 50\
F 18--30 y: 829\
F 30--60 y: 372\
F 60+ y: 38M 18--30: 22 yM 18--30 y: (0.063 × WT) + 2.896/4.184 × 1000Schofield-wtF 18--30: 22 yF 18--30 y: (0.062 × WT) + 2.036/4.184 × 1000M 30--60: 40 yM 30--60 y: (0.048 × WT) + 3.653/4.184 × 1000F 30--60: 40 yF 30--60 y: (0.034 × WT) + 3.538/4.184 × 1000M 60+: 72 yM 60+ y: (0.049 × WT) + 2.459/4.184 × 1000F 60+: 66 yF 60+ y: (0.038 × WT) + 2.755/4.184 × 1000Equations based on weight and heightSchofield-wthtM 18--30 y: (0.063 × WT) -- (0.042 × HTM) + 2.953/4.184 × 1000F 18--30 y: (0.057 × WT) + (1.184 × HTM) + 0.411/4.184 × 1000M 30--60 y: (0.048 × WT) -- (0.011 × HTM) + 3.67/4.184 × 1000F 30--60 y: (0.034 × WT) + (0.006 × HTM) + 3.53/4.184 × 1000M 60+ y: (0.038 × WT) + (4.068 × HTM) -- 3.491/4.184 × 1000F 60+ y: (0.033 × WT) + (1.917 × HTM) + 0.074/4.184 × 1000*M* male, *F* female, *y* years, *WT* weight in kilogram, *HTM* height in meters, *HTCM* height in centimetres; *SEX* (male = 1, female = 0) sex, *REE* resting energy expenditure; *kcal/d* kilocalories a day, *IC* indirect calorimetry
BMI
: Body Mass Index
H&B
: Harris & Benedict
ICU
: Intensive Care Unit
REE
: Resting energy expenditure
WHO
: World Health Organisation
This study was supported by the VU University Medical Center, Amsterdam, the Netherlands. We thank the dietetic team of the VU University Medical Center Amsterdam for indirect calorimetry measurements and students for their support in the project.
Funding {#FPar1}
=======
Not applicable.
Availability of data and materials {#FPar2}
==================================
Please contact author for data requests.
Authors' contributions {#FPar3}
======================
HK, GH and PW designed the study, performed literature search, data analysis, and writing of the manuscript, and confirmed final draft of the manuscript.
Competing interests {#FPar4}
===================
The authors declare that they have no competing interests.
Consent for publication {#FPar5}
=======================
Not applicable.
Ethics approval and consent to participate {#FPar6}
==========================================
All measurements took place in daily clinical practice. Data were anonymised. All procedures were in accordance with ethical standards of the institution.
|
{
"pile_set_name": "PubMed Central"
}
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Background
==========
Unsaturated fatty acids, particularly α-linolenic acid (LNA; *cis*-9, *cis*-12, *cis*-15-18:3) and linoleic acid (LA; *cis*-9, *cis*-12-18:2), are abundant in grass and other ruminant feedstuffs, yet are present at low concentrations in meat and milk. Furthermore, tissue lipids of ruminants have been known for a long time to be more saturated than those of non-ruminants \[[@B1]\]. As the consumption of saturated acids in dairy products and ruminant meats is often associated with an increased incidence of coronary heart disease in man \[[@B2]\], the transformation of unsaturated fatty acids to saturated fatty acids, or biohydrogenation, in ruminants presents a major human health issue. The biohydrogenation process has long been known to occur in the rumen as the result of microbial metabolic activity \[[@B3],[@B4]\]. Thus, if ruminal biohydrogenation of unsaturated fatty acids can be controlled, it may be possible to improve the healthiness of ruminant meats and milk by increasing their unsaturated fatty acids composition in general and the *n*-3 fatty acids in particular \[[@B5]\]. One of the unsaturated fatty acids that appears most desirable is conjugated linoleic acid (CLA; *cis*-9, *trans*-11-18:2) because of its anticarcinogenic and other health-promoting properties \[[@B6],[@B7]\]. Major advances have been made in achieving the desired changes in fatty acid content of meat and milk experimentally, via dietary manipulation in ruminants, generally by adding oils containing unsaturated fatty acids to the diet \[[@B5],[@B8]-[@B10]\]. The inclusion of fish oil in particular seems to alter biohydrogenating activity in the rumen \[[@B11]\].
*Butyrivibrio fibrisolvens*was identified many years ago to undertake biohydrogenation of fatty acids \[[@B12]\] and to form CLA as intermediate in the process \[[@B13]\]. Kim *et al*. \[[@B14]\] noted that LA inhibited growth of *B. fibrisolvens*A38, an effect that depended both on the concentration of LA and the growth status of the bacteria. Growing bacteria were more tolerant of LA. In a study of CLA production in different strains of *B. fibrisolvens*, Fukuda *et al*. \[[@B15]\] found that the most tolerant strain had the highest linoleate isomerase (forming CLA from LA) specific activity. Different members of the *Butyrivibrio*/*Pseudobutyrivibrio*phylogenetic grouping, all of which biohydrogenate PUFA, had different sensitivities to growth inhibition by LA, the most sensitive possessing the butyrate kinase rather than the acyl transferase mechanism of butyrate production \[[@B16]\]. For reasons that were unclear, lactate exacerbated the toxicity of LA to *Clostridium proteoclasticum*\[[@B17]\], now renamed *Butyrivibrio proteoclasticus*\[[@B18]\]. The aim of this work was to understand the nature of the toxic effects of PUFA on *B. fibrisolvens*JW11. Strain JW11 is located in the middle of the numerous *B. fibrisolvens*/*Pseudobutyrivibrio*cluster, members of which share the ability to form CLA and vaccenic acid (VA; *trans*-11-18:1) but which also lack the ability to biohydrogenate VA to stearic acid (SA; 18:0) \[[@B16]\]. Understanding these effects could have important indirect implications for human health by enabling ruminal biohydrogenation of dietary PUFA to be manipulated in order to provide healthier ruminant-derived foods.
Results
=======
Fatty acid metabolism by *B. fibrisolvens*JW11
----------------------------------------------
The metabolism of LA was measured during the growth cycle of *B. fibrisolvens*JW11 (Figure [1](#F1){ref-type="fig"}). No growth occurred until 10 h, but then growth was initiated and bacteria grew at a specific growth rate similar to that found in the absence of added fatty acid (not shown). During the lag phase, LA was very rapidly converted to CLA, but growth was not initiated until all the dienoic acids had been metabolized and converted extensively to vaccenic acid. No SA was formed.
{#F1}
A longer lag phase was seen with LNA (Figure [2](#F2){ref-type="fig"}). LNA was also metabolised rapidly during early lag phase, being converted firstly to the conjugated *cis*-9, *trans*-11-*cis*-15-18:3. A little *trans*-9, *trans*-11, *cis*-15-18:3 was formed as well. The main dienoic acid formed transiently was *trans*-11, *cis*-15-18:2, which was subsequently converted to VA. Variation in the time taken for different replicate tubes to escape the lag phase meant that the average concentration across three tubes gives a misleading impression. For example, at 32 h, replicate tubes contained 0.125, 0.140 and 0.193 mg bacterial protein ml^-1^, indicating that the culture in the third tube had begun to grow sooner than the others. The concentrations of *cis-*9, *trans*-11, *cis*-15-18:3 were 23.0, 21.1 and 0 μg ml^-1^, respectively, while the concentrations of *trans*-11, *cis*-15-18:2 were 0, 0 and 24.5 μg ml^-1^. An analysis comparing bacterial protein concentrations and fatty acid concentrations in the same tubes (not shown) demonstrated that bacterial protein concentration was low while *cis-*9, *trans*-11, *cis*-15-18:3 and *trans-*9, *trans*-11, *cis*-15-18:3 were present. Higher bacterial concentrations occurred only when these fatty acids were removed from individual cultures. High concentrations of VA did not affect growth, while *trans*-11, *cis*-15-18:2 also appeared to permit growth. No SA was formed in any LNA-containing culture.
{#F2}
Comparative effects of fatty acids and methyl esters on growth and metabolism by *B. fibrisolvens*JW11
------------------------------------------------------------------------------------------------------
The effects of various fatty acids and their methyl esters on the growth of *B. fibrisolvens*and the biohydrogenation products in M2 medium were carried out in a similar way, and are summarized in Table [1](#T1){ref-type="table"}. The more unsaturated fatty acids were more toxic, with γ-linolenic acid (γ-LNA; *cis*-6, *cis*-9, *cis*-12-18:3) and the fish oil fatty acids, DHA and EPA, causing lag phases \>72 h. LNA induced a lag phase of 37 h, longer than that found with LA (*P*= 0.001), which in turn was longer than that caused by CLA (*P*= 0.01). VA and SA had little growth-inhibitory activity, while oleic acid (OA; *cis*-9-18:1) caused a short lag of just under 2 h (*P*= 0.005). γ-LNA was metabolized to a trienoic acid, which from its elution time in GC was judged to be conjugated, most likely *cis*-6, *cis*-9, *trans*-11-18:3. OA was not metabolised and was slightly toxic. Neither EPA nor DHA was metabolised. SA did not cause a lag phase and was not metabolised. No methyl esters caused a lag phase, yet they were converted to the same products as the free fatty acids, with the exception of methyl-γ-LNA, which formed a dienoic acid eluting in the same area as CLA. GC analysis of extracted samples before they were methylated indicated that the fatty acid methyl esters had been hydrolysed before the fatty acids were metabolized.
######
Effects of fatty acids and methyl esters (50 μg ml^-1^) on growth of, and metabolism of fatty acids by, *Butyrivibrio fibrisolvens*JW11 in M2 medium.
DHA EPA γ-LNA LNA LA CLA VA OA SA
---------------------- ------- ------ ----------------- ------ ------ ------- ------- ------- ------
**Free fatty acids**
Lag phase (h)
Mean \>72 \>72 \>72 37.0 7.1 4.7 0.01 1.88 0.82
SD 5.25 0.60 0.20 0.13 0.23 0.48
Biohydrogenation No No Yes Yes Yes Yes No No No
End product at 72 h ND^a^ ND Conjugated 18:3 VA VA VA ND ND ND
**Methyl esters**
Lag phase (h)
Mean NA NA NA NA 0.37 -0.17 -0.23 -0.07 1.54
SD 1.12 0.44 0.35 0.09 0.23
Biohydrogenation No No Yes Yes Yes Yes No No No
End product at 72 h ND ND Conjugated 18:2 VA VA VA ND ND ND
^a^ND - None detected
^b^NA - Not analyzed. The first OD reading was made at 16 h, by which time the cultures had grown.
Influence of fatty acids on cell integrity of *B. fibrisolvens*JW11
-------------------------------------------------------------------
The influence of different C-18 fatty acids and their methyl esters on cell integrity was determined using propidium iodide (PI) fluorescence (Figure [3](#F3){ref-type="fig"}). All unsaturated fatty acids, including OA and VA, had a similar effect, although the monoenoic acids tended to cause less disruption (*P =*0.063). The effects of 200 μg ml^-1^fatty acids were only slightly greater than 50 μg ml^-1^(*P*\< 0.001). SA gave a small response in comparison with the unsaturated fatty acids (*P*\< 0.001), and methyl esters caused only about one-tenth of the disruption of the free fatty acids (*P*\< 0.001) (Figure [3](#F3){ref-type="fig"}).
{#F3}
The influence of fatty acids on cell integrity was analysed further by flow cytometry (Figure [4](#F4){ref-type="fig"}). All unsaturated fatty acids transformed the PI signal to one in which the great majority of cells displayed fluorescence, i.e. the fluorescence response profile moved to the right in the flow display. The unsaturated fatty acids caused apparently greater disruption than boiling the cells, suggesting that the fatty acids enhanced access of PI to the bacterial cytoplasm. SA had no effect, the profile following exactly that of untreated cells. Differences between the different unsaturated fatty acids were minor. Even in untreated cell suspensions, some fluorescence was observed at the 10^2^region, consistent with about 25% of the bacteria being non-viable. Very few cells remained unaffected by either boiling or the fatty acids, judging by the low incidence of fluorescence at the \<10^1^region of the traces.
{#F4}
The presence of 70 mM sodium lactate in the growth medium increased the lag phase from 7 to 16 h (not shown) when LA was present. The influence of LA on PI fluorescence and growth was also determined in the presence and absence of sodium lactate (Figure [5](#F5){ref-type="fig"}). As before, LA increased the fluorescence due to PI (*P*\< 0.001), indicating that cell integrity had been disrupted. Sodium lactate did not alter the response significantly (*P*\> 0.05).
{#F5}
Influence of LA on ATP and acyl CoA pools of *B. fibrisolvens*JW11
------------------------------------------------------------------
LA was added to culture that had been mixed with an equal volume of fresh medium, and the pool sizes of ATP (Figure [6](#F6){ref-type="fig"}) and acyl CoA intermediates (Table [2](#T2){ref-type="table"}) were measured. Initially, the ATP pools were similar, at about 2 nmol (mg protein)^-1^. Thereafter, the ATP pool remained similar in the LA culture, while the concentration increased 3-4-fold (*P*\< 0.05 from 40 min onwards) in cultures to which no LA was added. The acyl CoA pools were measured only after 20 min, at which time the ATP pool had not yet changed significantly (*P*\> 0.05). In control cultures, the highest pool sizes of short-chain acyl CoAs were of acetyl CoA and butyryl CoA, followed by propionyl CoA. Crotonyl CoA and acetoacetyl CoA were present at much lower concentrations, 10 pmol (mg protein)^-1^or less. β-Hydroxybutyryl CoA was not determined by the methods used. All CoA pools, except acetoacetyl CoA, were decreased by \>96% (*P*\< 0.001) in LA-containing cultures.
{#F6}
######
Influence of LA on acyl CoA pools of *B. fibrisolvens*JW11 20 min after inoculation into fresh medium.
------------------------------------------------------
Acyl CoA concentration\
(pmol mg protein^-1^)
------------- ------------------------- ----- ---- ---
Acetyl 375 158 17 5
Propionyl 53 14 2 1
Isobutyryl 16 4 0 0
Butyryl 213 77 10 2
Crotonyl 10 6 0 0
Isovaleryl 8 2 0 0
Hexanoyl 2 1 0 0
Acetoacetyl 4 1 7 1
------------------------------------------------------
Results are means and SD from three separate cultures.
Discussion
==========
*B. fibrisolvens*was originally described as a small, Gram-positive bacterium particularly prevalent in the rumen of grazing animals \[[@B19]\]. Many strains are proteolytic and involved in fibre breakdown \[[@B19],[@B20]\]. *B. fibrisolvens*JW11 was originally isolated as a proteolytic strain \[[@B21]\]. It has been many years since the importance of *B. fibrisolvens*in the process of PUFA reduction, or biohydrogenation, was first documented \[[@B12]\]. Although other bacteria have been implicated \[[@B22]\], biohydrogenating activity is high among all members of what is now known to be an extensive *Butyrivibrio*phylogenetic tree \[[@B16]\]. Indeed, in our experience, its activity is many times higher than in other species \[[@B17]\]. \'Type B\' bacteria, which complete the reduction of 18:1 isomers to SA, was identified as *C. proteoclasticum*\[[@B23]\], which has recently been renamed *Butyrivibrio proteoclasticus*\[[@B18]\]. The pattern of metabolism of LA and LNA observed here, and the identity of the intermediates, follows the pathways established first by Kepler *et al*. \[[@B13]\] and confirmed later by others \[[@B24]-[@B26]\]. The observations linking growth and LA metabolism with *B. fibrisolvens*JW11 are consistent with those obtained with *B. fibrisolvens*A38 \[[@B14]\] and *B. fibrisolvens*TH1 \[[@B15]\]. What is novel about the present observations is that they clearly demonstrate that biohydrogenation is a detoxification process, necessary to escape from the bacteriostatic effects of PUFA. Indeed, they explain that the concentration-dependence of LA toxicity and its apparently lower toxicity in growing cultures, observed by Kim *et al*. \[[@B14]\], have to be considered in terms of time required by different biomass concentrations to hydrogenate, and thereby detoxify, different concentrations of fatty acids.
Henderson \[[@B27]\] examined the effects of fatty acids on ruminal bacteria. A *Butyrivibrio*sp. was generally most sensitive to fatty acids, but only saturated and monoenoic acids were included in the study. OA was much more toxic than the saturated fatty acids. Marounek *et al*. \[[@B28]\] found that C-12 and C-14 fatty acids were more toxic to ruminal and rabbit caecal bacteria than other chain lengths, but again the study was of saturated acids and oleic acid. In non-ruminal bacteria, LA and LNA were much more toxic than saturated or monoenoic acids \[[@B29]\]. The present paper describes the effects of the more abundant poly- and monounsaturated fatty acids on *B. fibrisolvens*. The PUFA were found to be much more toxic than more saturated fatty acids.
The present experiments help to resolve the purpose of biohydrogenation in the ruminal bacteria that undertake this reductive metabolism. Our results provide support for the conclusions of Harfoot and Hazlewood\[[@B22]\], Kemp and Lander \[[@B30]\] and Kemp *et al*. \[[@B31]\] that biohydrogenation is a detoxification mechanism rather than a means of disposing of reducing power, as proposed earlier \[[@B32]\]. The reductase which converts CLA to VA in *B. fibrisolvens*comprises 0.5% of the total cell protein \[[@B33]\], a very significant expenditure of cellular resources that signifies a vital function. It should be noted that, although more research emphasis is placed on its metabolism of LA because CLA is an intermediate, biohydrogenation is probably more important for *B. fibrisolvens*to survive high LNA concentrations, as LNA is more toxic than LA and is usually present at higher concentrations than LA in forages (e.g. \[[@B3]\]). Also to be noted is that CLA is almost as toxic as LA, as found before \[[@B14]\].
There are several possible reasons why unsaturated fatty acids are generally more toxic than saturated fatty acids. The double bonds alter the shape of the molecule, such that kinked unsaturated fatty acids disrupt the lipid bilayer structure \[[@B34]\]. The finding that different PUFA isomers, such as LNA and γ-LNA, had different toxicity would be consistent with such an interpretation. However, it is not clear that the toxicity was necessarily a membrane effect. The free carboxyl group was necessary for growth inhibition to take place. Methyl esters, which might be expected to be sufficiently hydrophobic to be incorporated into a membrane just as efficiently as a free fatty acid, were non-toxic. They were metabolized in the same way as the free fatty acids, however, as they were hydrolysed by bacterial esterase activity. The free carboxyl group was also necessary for disruption of cell integrity, as measured by PI ingression. However, PI ingression was affected little by the number of double bonds in the fatty acid molecule, as detected by both fluorescence spectroscopy and flow cytometry, and could not explain why 18:3 fatty acids were more toxic to growth than 18:2 fatty acids, and especially why 18:1 was not toxic to growth. Furthermore, sodium lactate exacerbated growth inhibition by LA, in a similar manner to that observed with *B. proteoclasticus*\[[@B23]\], but had no similar effect on the influence of LA on cell integrity of *B. fibrisolvens*. A similar conclusion was reached by Maia *et al*. \[[@B17]\] when comparing the toxic effects of fatty acids on growth and cell integrity in different species of ruminal bacteria. Thus, although a toxic mechanism involving disruption of the extraordinarily thin cell envelope of *B. fibrisolvens*\[[@B35]\] seems an attractive and logical possibility, the evidence suggests that the primary effect of PUFA lies elsewhere.
An alternative possibility is that the ready diffusion of the free fatty acid across the membrane causes chemiosmotic difficulties, perhaps uncoupling the proton-motive force \[[@B36]\], dissipating the membrane potential by facilitating ion leakage \[[@B37]\] or decoupling intramembrane pathways \[[@B38],[@B39]\]. While this remains a possibility, the different effects on acyl CoA and ATP pools on PUFA toxicity suggest a metabolic effect, specifically in acyl CoA metabolism. Measurement of CoA metabolic pools in bacteria is relatively rare. Here, acetyl CoA and butyryl CoA were present at highest concentration and the butyrate pathway intermediates at much lower concentrations, as found also in *Clostridium acetobutylicum*\[[@B40]\]. All acyl CoAs except acetoacetyl CoA were diminished by \>96% when LA was added to the medium. In contrast, the ATP pool was affected later than acyl CoA pools, and remained at about one-third of the control values, presumably due to the contribution of glycolysis.
The toxicity of PUFA in different species of ruminal bacteria was found to be related partly to whether the bacteria produced butyrate; cellulolytic bacteria were the other most sensitive species \[[@B17]\]. Within the *Butyrivibrio*phylogenetic group, the most sensitive species were those that formed butyrate *via*the butyrate kinase mechanism rather than acyl CoA transferase \[[@B16]\]. Thus, there seems to be a connection between PUFA toxicity and butyrate formation. A metabonomic analysis \[[@B41]\] might help to identify precisely where the PUFA act. It may also be instructive to determine why *trans*-11, *cis*-15-18:2, a product of LNA metabolism, appeared to permit growth while the other dienoic acid investigated here did not.
The influence of sodium lactate in lengthening the lag phase indicates that lactate potentiates the toxic effects of PUFA in *B. fibrisolvens*, as shown previously with *B. proteoclasticus*\[[@B22]\]. Such a high concentration of lactate (70 mM) would only occur in animals suffering acidosis \[[@B42]\]. The toxicity may be an osmotic effect, or due to a leakage of ions across the membrane, or may even be a metabolic effect. Lactate is a major product of glucose metabolism in *B. fibrisolvens*JW11 and many other butyrivibrios \[[@B16]\], and high concentrations may therefore impose metabolic feedback, further stressing bacteria affected by PUFA.
Conclusions
===========
This paper explains the basis of the beneficial effect on meat and milk fatty acid composition of adding oils to the ruminant diet. Ruminal biohydrogenation is modified via differential toxicity to ruminal bacteria of different PUFA, including the fish oil fatty acids, EPA and DHA. If we can understand how selective fatty acid toxicity, or indeed other factors, affects the physiology of biohydrogenating bacteria in the rumen, we may be able to suggest new, rational dietary modifications that will eventually lead to ruminant products that are healthier for human consumption.
Methods
=======
Bacteria and growth conditions
------------------------------
*Butyrivibrio fibrisolvens*JW11 was originally isolated from sheep as a proteolytic species \[[@B21]\], and is held in the culture collection maintained at the Rowett Institute. All transfers and incubations were carried out under O~2~-free CO~2~and at 39°C in Hungate-type tubes \[[@B43]\]. Inoculum volumes were 5% (v/v) of a fresh culture. The media used in these experiments were the liquid form of M2 medium \[[@B44]\]. Fatty acids were prepared as a separate solution, sonicated for 4 min in water and added to the medium before autoclaving. Growth of bacteria was measured from the increase in optical density (OD) at 650 nm of the control tubes, in triplicate, using a Novaspec II spectrophotometer (Amersham Biosciences, UK).
The influence of fatty acids and their methyl esters was determined in two kinds of experiment. In experiments where fatty acid concentrations were measured at the end-point of the growth curve, usually in stationary phase, the tubes were freeze-dried in order to enable fatty acid extraction from the whole culture. The experiment was conducted by inoculating multiple 10-ml tubes. At each sampling time, three tubes were removed, the turbidity was determined, and the tubes were placed in a heating block at 100°C for 5 min, left to cool and frozen. One ml was taken for protein analysis and for fatty acid extraction and derivatization.
Fatty acid extraction and analysis
----------------------------------
Extraction, derivatization of fatty acids and GC analysis of methyl esters were carried out using procedures described by Wąsowska *et al*. \[[@B11]\]. The products from incubations with LNA were identified by comparing elution profiles and mass spectra with those identified previously from analysis of methyl and 4,4-dimethyloxazoline (DMOX) esters \[[@B11]\].
Measurement of cell integrity using propidium iodide
----------------------------------------------------
One ml of overnight culture was inoculated into 10 ml of M2 medium and incubated at 39°C until it reached mid-exponential phase (OD~650~= 0.4, approx. 4 h). The bacterial cultures were centrifuged (3000 ***g***, 10 min, 4°C) and the pellet was washed twice with anaerobic potassium phosphate buffer (100 mM; pH 7.0) containing 1 mM dithiothreitol (DTT). Anaerobic conditions were maintained by carrying out transfers in an anaerobic chamber with a gas phase of 80% N~2~, 10% CO~2~and 10% H~2~and temperature of 39°C. Cells were resuspended in 15 ml of the same buffer, and fatty acids and their respective methyl esters (Sigma, St. Louis, MO, USA) were added to the cell suspension to a final concentration of 50 μg ml^-1^. Stock solutions (1 mg ml^-1^) of fatty acids and methyl esters were prepared immediately before use by sonication for 4 min in anaerobic potassium phosphate buffer (100 mM, pH 7.0, containing 1 mM DTT). Untreated and heat-treated cells (100°C for 20 min) served as control samples.
Following 30 min incubation of cell suspensions with fatty acids, cell integrity was measured using PI. Ten μl of each sample were added to 985 μl of anaerobic potassium phosphate buffer, to which was added 5 μl of 1.5 mM PI (prepared in distilled water and stored at 4°C in the dark). The mixtures were incubated for 15 min at 39°C in the anaerobic chamber, then transferred to an ice-water slurry and kept in the dark for up to 45 min before being analysed for fluorescence using a fluorimeter or by flow cytometry. Fluorimetry measurements were made using a spectrofluorimeter set at λ~EX~= 488 nm and λ~EM~= 650 nm. Flow cytometry was carried out with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, USA) equipped with an air-cooled argon ion laser emitting 15 mW of blue light at 488 nm. The red fluorescence of the PI signal was collected in the FL3 channel (\>600 nm long-pass filter). FACSFlow solution (Becton Dickinson) was used as sheath fluid. The analyses were done using the low rate settings (12 μl/min).
ATP and acyl CoA pools
----------------------
The influence of LA on metabolic pools in *B. fibrisolvens*was measured in cells growing in Roché *et al*. \[[@B45]\] medium in the anaerobic chamber, as follows. Fresh overnight culture (60 ml) of *B. fibrisolvens*JW11 was mixed with 60 ml of uninoculated medium, or uninoculated medium containing 200 μg LA ml^-1^, then samples (3.0 ml) were taken periodically into 1 ml of 30% (w/v) perchloric acid. After 10 min, 4 ml of KOH were added to the acidic solution, forming a precipitate of potassium perchlorate, which was removed by centrifugation (15,000 ***g***, 15 min, 4°C). The supernatant was stored at -80°C, then subsequently thawed and ATP was measured using a luciferase preparation according to the manufacturer\'s (Sigma) instructions.
Acyl CoA measurements were made in parallel 120-ml control or LA-containing cultures after 20 min incubation. Cultures were maintained under CO~2~and centrifuged immediately at 15,000 ***g***for 15 min at 39°C. The pellet was stored in liquid nitrogen. Derivatization, separation, and fluorescence detection of acyl CoAs were carried out as described by Larson and Graham \[[@B46]\]. Identification of acyl CoAs was carried out using mass spectrometric analysis of peaks obtained from a Hypercarb porous graphitic carbon column \[[@B47]\].
Bacterial protein was measured by a modification of the Lowry method \[[@B48]\].
Data analysis
-------------
All data were derived from three separate cultures. Means were compared by a Student\'s *t*-test. Measurement of the lag phase was carried out by fitting a gradient by linear regression to log(*A*~650~) *vs*. time during exponential phase. The lag phase was defined as the time at which the best-fit gradient passed an OD~650~of 0.1, and was compared to the time at which the control cultures passed 0.1. Propidium iodide ingression was determined by 8 fluorescence measurements for each culture.
List of abbreviations
=====================
CLA: conjugated linoleic acid; DHA: docosahexaenoic acid; DMOX: 4,4-dimethyloxazoline; DTT: dithiothreitol; EPA: eicosapentaenoic acid; LA: linoleic acid; LNA: α-linolenic acid; γ-LNA: γ-linolenic acid; OA: oleic acid; OD: optical density; PI: propidium iodide; PUFA: polyunsaturated fatty acids; SA: stearic acid; VA: vaccenic acid.
Authors\' contributions
=======================
MRGM designed and carried out cell integrity studies, some growth experiments, and assisted in drafting the manuscript. LCC carried out growth experiments and fatty acids analysis. CSB participated in the design and implementation of flow cytometry experiments and in discussion of bacterial viability. AJR carried out experiments on metabolic pools, and assisted in drafting the manuscript. NM supervised growth experiments, fatty acids analysis and assisted in drafting the manuscript. TRL and IAG undertook the analysis of acyl CoAs. RJW designed the studies, collated the experimental data and wrote the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
================
The Rowett Research Institute receives funding from the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD). LCC was in receipt of a Wellcome Travelling Fellowship. We thank David Brown and Maureen Annand for their technical help and expertise. MRGM received support from the Marie Curie Training Site, \'Anaerobe\'; we thank Jamie Newbold and Estelle Devillard for their help and advice. MRGM was also supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, with a PhD grant (SFRH/BD/6976/2001).
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{
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}
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In the 1950s, 'moronic' children at the Walter E. Fernald School in Massachusetts were given the opportunity to join a science club. Excited, a number of enthusiastic pupils enrolled unaware that they would be providing the raw human material for a range of institutional experiments involving the endless consumption of oatmeal, isolation and a relentless routine of daily injections and providing urine and faeces samples. Decades later, it transpired that their oatmeal had been mixed with radioactive milk; an initiative knowingly sponsored by Quaker Oats and the US Atomic Energy Commission. *Against their Will: The Secret History of Medical Experimentation on Children in Cold War America* explores how the social value of certain disadvantaged population groups became undermined by early twentieth century eugenics. The authors maintain that this encouraged aspiring, often fame-hungry, medical researchers to use the institutionalised for the study of human illness. In addition, the Cold War created an environment that supported and sanctioned human experimentation. This underpinning meta-narrative fails to fully engage with the inherent complexity of twentieth century medical ethics or to comprehensively situate the experiments investigated against the dramatic shift in bioethical behaviour and regulation that occurred in that century. Nonetheless, Hornblum, Newman and Dober offer a thoroughly researched and well-written account of a highly sensitive and emotionally charged topic that recounts the perspectives of a range of involved actors including experimental victims, parents, institutional staff and medical researchers.
*Against their Will* makes strikingly clear that throughout much of the twentieth century, a significant number of researchers saw the residents of America's overflowing institutions (including asylums, prisons and reformatories) as suitable test subjects. They identified human biomaterial in groups ranging from the severely mentally incapacitated to the shy and stuttering. According to the authors, these groups constituted an 'undesirable' class whose value to the community could only be redeemed if they contributed to society by assisting in scientific advancement. Trained in the 1920s and 1930s at major American universities in a curriculum steeped in eugenic theory, leading Cold War scientists saw no scruples in using those deemed unproductive and socially useless to make scientific and social progress, the authors maintain. The twentieth-century elevation of the brave medical scientist into public hero also played an important role in shaping moral attitudes towards experimenting upon the institutionalised. These explanations could have been more convincingly expanded upon, yet what is made clear is that many researchers, for a variety of reasons, demonstrated a lack of empathy and concern towards the well-being of their experimental subjects that seems morally reprehensible today.
Medical scientists had expressed interest in the institutionalised since at least the nineteenth century when they came to realise that prisoners could be used to undertake investigations into diet and nutrition. They recognised that institutionalised groups could be easily segregated into control groups and that their diet, behaviour, regimen and medicines could be closely monitored and regulated. Yet it was only in the twentieth century that certain institutions were truly transformed into laboratory-like environments where medical conditions that had long evaded the investigative repertoires of vivisectionists could be examined using living human beings as material. While the presence of medical investigators in institutions is, in many ways, unsurprising, the authors convincingly argue that the use of children for experimentation became accepted and implemented across Cold War America at a staggering level.
In penning *Against their Will*, Hornblum, Newman and Dober intend to shock. Their study is replete with accounts of young children being systematically castrated, sterilised, brainwashed, dosed with LSD, injected with radioactive material, subjected to mentally damaging psychological experiments and contaminated with dangerous diseases. In examining these medical excesses, the authors successfully challenge the misconception that eugenics and human experimentation was solely a preoccupation of the Nazis. On the contrary, America had a vibrant research programme that rejected, or chose to ignore, the Nuremberg codes of medical ethics devised shortly after the Second World War. The authors consult an impressive array of sources including medical journals, oral history, official records and personal records. These reveal the varying points of view of medical scientists (ranging from overt enthusiasm for experimentation to unease), occasional public outbursts by family members and individuals concerned about children being experimented upon without parental consent and the permanent physical and psychological damage that seriously impacted on the quality of life of institutionalised children raised in the Cold War era. In addition, the authors do not shy away from engaging with the perspectives of some medical scientists who genuinely believed that their actions were justifiable as they advanced medical knowledge and saved the lives of countless other adults and children reaping the benefits of a therapeutic progress. Having said that, the therapeutic value of many of the experiments remained strikingly uncertain, a problem emphasised by Hornblum, Newman and Dober.
*Against their Will* is an emotive study of how the quest for scientific fame often over-ruled medico-ethical principles. By castigating and reprimanding doctors for misusing their power to select and manipulate the institutionalised, their study persuasively challenges the myth of the rise of the heroic microbe hunter. Problematically, the reader is left with the impression that virtually all Cold War medical scientists in America eagerly experimented on the young. To balance this perspective, it might have been useful to fully assess whether a cadre of medical men existed who rejected the dubious activities of their colleagues and who found alternative ways of gaining information. Certainly, although briefly mentioned, the impact of Henry Beecher's controversial 1966 article which brought important medico-ethical issues to public attention could have been used more effectively to explore how and why child experimentation gradually became deemed less acceptable. The weakness of *Against their Will* is that, serving as an exposé, it mostly seeks to condemn instead of comprehend. Nonetheless, Hornblum, Newman and Dober have produced an important, well-written and meticulously researched account of a controversial period in America's medical past that will undoubtedly serve as a catalyst for future academic research.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Soybean (*Glycine max* (L). Merr.) is an important legume crop used in processed foods and animal feed worldwide. Improvement in yield is one of the most important objectives of soybean breeding programs. Stem growth habit is an important trait affecting seed yield ([@b4-68_17137], [@b28-68_17137]) and is classified into three types: indeterminate, semi-determinate, and determinate growth habit, based on the growth dynamics of the main stem after flowering ([@b2-68_17137]). Stem growth habit is controlled by the combination of two genes, *Dt1* and *Dt2* ([@b2-68_17137]). The *Dt1* gene has been demonstrated to be the TERMINAL FLOWER1 (*GmTFL1*) gene on chromosome 19, determined via transformation and virus-induced gene silencing experiments ([@b24-68_17137], [@b35-68_17137]). The *GmTFL1* transcript accumulates in the shoot apical meristem during early vegetative growth in both the determinate and indeterminate lines, but thereafter is abruptly lost in the determinate line ([@b24-68_17137]). Cultivating recombinant inbred line populations developed from crosses between indeterminate and determinate in northern and southern parts of Japan, [@b20-68_17137] showed that yields of early maturity indeterminate lines were higher than those of determinate ones in the northern part of Japan.
Single seed weight (SSW) is one of the crucial traits affecting the processing quality of soybean used in foods and it determines a soybean's commercial value. Varieties with large SSW (\>300 mg per seed) have been used for boiled, green soybeans (edamame) and boiled beans, while varieties with small SSW (\<150 mg per seed) have been used for fermented, steamed beans, called 'natto'. Furthermore, SSW is an important trait for processed tofu. It has been shown that SSW is positively correlated with fresh tofu yield because the ratio of insoluble seed coat to seed volume of heavy-seeded varieties is lower than the ratio determined from light-seeded varieties ([@b6-68_17137]). Therefore, in Japan, China, and North America, SSW has been one of the most important commercial traits associated with soybean production for indigenous consumption and/or for export. As a consequence, many soybeans of both heavy- and light-seeded varieties have been developed ([@b6-68_17137]). Therefore, soybean genetic resources show a wide variation in SSW, which ranges from less than 50 mg to more than 750 mg ([@b17-68_17137]). In addition, the uniformity of SSW is also a commercially important trait ([@b42-68_17137]). In boiled beans, the uniformity of SSW directly affects the aesthetic quality of the end product, while in tofu, *miso* (a fermented and steamed bean paste), and natto, this trait affects the uniformity of soaking time, steaming, and fermentation. In Japan, the prices of soybean products vary depending on the grade based on the aesthetic quality and seed uniformity is one of the important factors affecting this quality. Therefore, seed uniformity has been considered as a high priority trait in Japanese soybean breeding programs ([@b42-68_17137]). In indeterminate soybean varieties, variations in the timing of flower formation and length of the seed-filling period depends more on flower node position than determinate-growth habit ([@b26-68_17137]); these variations are presumed to affect SSW and its uniformity. [@b2-68_17137] developed near isogenic lines (NILs) with various stem growth habits by introducing the *dt1* gene to varieties with medium-weight soybeans seeds (150--300 mg per seed) grown in the United States (US) (*i.e.*, 'Harosoy' and 'Clark') by backcrossing, demonstrating that there is no significant difference in SSWs between indeterminate and determinate lines. Similarly, using the same NILs, [@b42-68_17137] showed that there was no significant difference in uniformity of SSW between indeterminate and determinate growth characteristics. However, it is not known whether similar results could be obtained if indeterminate traits were introduced into determinate varieties with heavy seeds. It has been known that the seed-filling periods of late-blooming flowers are shorter than those of early-blooming ones, and that seed-filling speed in late-blooming flowers is higher than that in early-blooming ones ([@b9-68_17137]). However, when introducing indeterminate growth habit into determinate varieties with heavy seeds, it is possible that an unattainable rapid seed filling rate is required for forming large seeds in late-blooming flowers due to the limited time available for seed filling before senescence, and that average single seed weight and seed uniformity becomes lighter and worse with some seeds in late-blooming flowers failing to become large enough, respectively.
There are concerns about some agricultural traits (*e.g.*, time to maturation and lodging resistance) when considering whether to use indeterminate varieties in areas where determinate varieties have been exclusively cultivated. Maturation may take longer and lodging may be more severe in indeterminate NILs than in determinate NILs. This difference may be due to the longer main stem length and more numerous main stem nodes in indeterminate varieties ([@b2-68_17137], [@b5-68_17137]). In addition, when [@b28-68_17137], [@b29-68_17137] developed 23 pairs of stem-growth-habit NILs from the crosses between indeterminate and determinate varieties, they showed that the effects of stem growth habit on agricultural characteristics (*e.g.*, yield, yield components, and the number of branches grown), varied depending on the genetic background and growing environment of the crosses. Furthermore, recent analyses of allelic diversity at molecular marker loci revealed that genetic resources from soybean gene pools in Japan differ in genetic background from the varieties cultivated in North America and China ([@b1-68_17137], [@b14-68_17137], [@b17-68_17137], [@b37-68_17137], [@b44-68_17137]). Therefore, the effects of stem growth habit on soybean traits should be evaluated on each genetic background and growing environment.
Because indeterminate soybean varieties are rarely cultivated in Japan, research is limited on the effects that indeterminate varieties have on agricultural characteristics under field conditions in Japan and/or their genetic backgrounds. In this study, we developed stem-growth-habit NILs by introducing indeterminate traits into determinate varieties that have heavy seeds, and evaluated the influence of the introduced traits relative to stem growth patterns on SSW, uniformity of SSW, and other agronomic characteristics.
Materials and Methods
=====================
Plant materials
---------------
Growth habit NILs were developed by backcrossing four times. We used an indeterminate breeding line (donor parent) with medium-weight seeds ('Y1312-2') and three determinate breeding lines (recurrent parents) with heavy-weight seeds (*i.e.*, 'Tohoku 160', 'Tohoku 162', and 'Tohoku 164'). Maturation times for the recurrent parents varied: 'Tohoku 160' (late), 'Tohoku 162' (middle), and 'Tohoku 164' (middle). 'Tohoku 160' and 'Tohoku 164' varieties were registered as 'Satonohohoemi' and 'Akimiyabi', respectively ([@b19-68_17137], [@b22-68_17137]). The donor parent, 'Y1312-2', is a breeding line developed from a cross between the Japanese determinate line 'Tohoku 140' and the US indeterminate variety 'Olympus' ([@b7-68_17137]).
The three recurrent parents (*dt1/dt1*) were crossed with 'Y1312-2' (*Dt1/Dt1*) in 2008, and backcrosses were repeated four times from 2009 to 2010 at the Kariwano Branch of the Daisen Research Station of Tohoku Agricultural Research Center, NARO (hereafter, Kariwano Branch). When the backcrosses were repeated, DNA markers near the *Dt1* locus were genotyped, and *Dt1/dt1* plants were selected as pollen parents. In each backcross combination, one *Dt1/dt1* BC~4~F~1~ plant was grown in 2011, and then in 2012, five *Dt1/Dt1* and five *dt1/dt1* plants were selected (based on the alleles of DNA markers) from a BC~4~F~2~ population. In 2013, the five *Dt1/Dt1* and five *dt1/dt1* BC~4~F~3~ lines were cultivated in the field and the consistency between the phenotype and genotype of *Dt1* of each line was confirmed by observing the growth of their main stem after flowering. BC~4~F~4~ lines, which were obtained by bulk harvesting seeds of BC~4~F~3~ lines, were used as the growth habit NILs in this study.
Maker-assisted selection
------------------------
The physical map positions of DNA markers and genes were based on the soybean genome database Wm82.a1 ([@b33-68_17137]). Since it was difficult to select the *Dt1/dt1* plants morphologically before flowering, SSR markers located near the *Dt1* gene were genotyped (*i.e.*, *GmTFL1* (*Glyma19g37890*, 44,979--44,981 Kb on chromosome 19), which is Sat_286 (44,978 Kb on chromosome 19) and Sat_184 (45,179 Kb)) ([@b24-68_17137], [@b35-68_17137]). In the process of selecting *Dt1/Dt1* and *dt1/dt1* plants from the BC~4~F~2~ population, we used the DNA marker 'Dt1Int', which was designed using the polymorphism within the *GmTFL1* gene ([@b20-68_17137]).
Because the major maturity gene, *E3* (*GmPhyA3*, *Glyma19g41210*, 47,512--47,519 Kb on chromosome 19), is located near *GmTFL1* on the same chromosome, plants which were heterozygous at *Dt1* (and had the alleles of the recurrent parents at the *E3* locus) were selected as pollen parents. Thus, the linkage between *Dt1* and *E3* were broken. The SSR markers near the *E3* gene (*i.e.*, E3at-U113k, FT3SSR4, FT3SSR1dom, and FT3SSR3) were used for genotyping of *E3* ([@b40-68_17137]). Genotypes of 'Dt1Int' were analyzed by using 7.5% polyacrylamide gel electrophoresis, while the other markers were genotyped with a DNA sequencer (3730 Genetic Analyzer DNA sequencer; Applied Biosystems, CA, US), according to [@b31-68_17137].
Because there are six alleles at the *GmTFL1* locus (*i.e.*, *GmTfl1-a* (*Dt1*), *GmTfl1-b* (*Dt1*), *Gmtfl1-ta* (*dt1*), *Gmtfl1-ab* (*dt1*), *Gmtfl1-bb* (*dt1*), and *Gmtfl1-tb* (*dt1*)) ([@b35-68_17137]), the alleles of *GmTFL1* used for the parents ('Tohoku 160', 'Tohoku 162', 'Tohoku 164', and 'Y1312-2') were identified by sequencing this gene before our marker analysis for *GmTFL1*, in accordance with [@b35-68_17137]. Furthermore, four major maturity genes (*i.e.*, *E1*, *E2*, *E3*, and *E4*) were genotyped using seven DNA markers developed by [@b36-68_17137] with some check varieties (*i.e.*, 'Enrei' (JP28862, *E1/e2-ns/e3-tr/E4*), 'Harosoy-*e4*' (PI591435, *e1-as/e2-ns/E3-Ha/e4-SORE1*), 'Fukuyutaka' (JP29668, *E1/E2-dl/E3-Mi/E4*), 'Toyosuzu' (JP27540, *e1-nl/e2-ns/E3-Mi/E4*), 'Sakamotowase' (JP27450, *e1-fs/e2-ns/e3-tr/E4*), 'Harosoy' (PI548573, *e1-as/e2-ns/E3-Ha/E4*), 'Moshidou Gong 503' (JP27603, *e1-as/E2-in/e3-Mo/E4*)) (JP, accession number of NARO Genebank in Japan; PI, accession number from the USDA--ARS National Plant Germplasm System in US).
Evaluation of agricultural characteristics
------------------------------------------
Agricultural characteristics of the NILs were evaluated at Uenodai (39°32′N, 140°22′E) and Ishinazaka (39°32′N, 140°23′E) of the Kariwano Branch. Soils in both experimental fields are andosols. The field at Uenodai was fertilized with 24 kg ha^−1^ of N, 160 kg ha^−1^ of P~2~O~5~, 80 kg ha^−1^ of K~2~O, and 20 t ha^−1^ of compost before sowing, whereas the field at Ishinazaka was fertilized with 24 kg ha^−1^ of N, 120 kg ha^−1^ of P~2~O~5~, 80 kg ha^−1^ of K~2~O, and received no compost before sowing. Each line was planted in plots 1.2--1.6 m in length with a row spacing of 0.75 m and 0.12 m plant separations within each row, except at the Uenodai field in 2014 (0.16 m plant separation within each row). Experiments in all growing environments consisted of three cross combinations (main-plot) and two stem growth habits (sub-plot) in split-plot design with two replications. The planting dates of the fields varied by year: Uenodai (27 May 2014 and 23 May 2016) and Ishinazaka (21 May 2014 and 19 May 2016).
We measured the following fifteen characteristics in our soybean varieties: flowering time, seed-filling period, maturation time, lodging, main stem length, lowest pod height, number of main stem nodes, number of main stem branches, number of pods per plant, number of seeds per pod, seed yield per plant, SSW, coefficient of variation of SSW (SWCV), protein content, and oil content. Flowering time, maturation time, and seed-filling period were defined as the number of days from sowing to flowering, sowing to maturation, and flowering to maturation, respectively. Lodging score was visually recorded in each plot as 0 (no lodging) to 5 (completely lodged) by intervals of one based on the number of lodged plants and its degree of lodging according to [@b21-68_17137]. Three plants were selected randomly in each plot to measure main stem length, lowest pod height, number of main stem nodes, number of main stem branches, number of pods per plant, and number of seeds per pod. Plants were threshed individually and SSW and SWCV were measured by weighing a single seed (with a Quintix213-1SJP electronic force balance, Sartorius, Göttingen, Germany) from 100 seeds randomly selected from each plant. The above metrics for the three selected plants from each plot were averaged and the means were assigned to the appropriate plots. The other plants in each plot (excluding the two plants at the both ends of every row) were threshed in bulk and seed yield was calculated by dividing total seed weight by the number of harvested plants (Seed yields for the three individually threshed plants (above) were included in the yield of the plants threshed in bulk). Protein and oil content were determined for the seed mass threshed using a near-infrared spectrophotometer (Infratec 1241 Grain Analyzer; FOSS Hillerød, Denmark). Moisture content was determined with a grain moisture tester (PM830-2; Kett Electric Laboratory, Tokyo, Japan) and seed yield per plant was adjusted to reflect a water contents of 15%.
Split-plot analysis of variance (ANOVA) was carried out with SAS (SAS, Cary, NC, USA). Cross combination and stem growth habit were considered main-plot and sub-plot, respectively, and location and year were considered fixed effects. Where there was a significant combination by stem growth habit interaction, the comparisons between stem growth habits in each cross combination was carried out with three-way ANOVA with EZR ([@b18-68_17137]), which is a graphical user interface for R (<http://www.r-project.org>). A *P* value of \<0.05 was considered significant.
Results
=======
Genotyping of Dt1 and the four major maturity genes of the recurrent and donor parents
--------------------------------------------------------------------------------------
The *Dt1* alleles of the recurrent and donor parents were identified by nucleotide sequencing of the *GmTFL1* locus from −740 to +1440 (the adenine of the start codon was numbered +1). The *GmTFL1* sequence of 'Y1312-2' was identical to that of *GmTfl1-a* (*Dt1*), while the *GmTFL1* sequences of all three recurrent parents were identical to the *Gmtfl1-ab* (*dt1*) sequence. The *Gmtfl1-ab* allele has a non-synonymous substitution from arginine (*Dt1*) to tryptophan (*dt1*) at 166-th amino acid residue affecting the function of TFL1 protein ([@b35-68_17137]). Because there was a 6-bp length polymorphism between the *GmTfl1-a* and *Gmtfl1-ab* sequences in the 1st intron, the *GmTFL1* genotypes of the recurrent and donor parents were analyzed by distinguishing this polymorphism length of PCR products amplified by a primer pair of Dt1Int. The PCR products of recurrent parents produced bands of 123 bp, while the PCR products of the donor parent produced bands of 129 bp. The genotyping of the seven DNA markers for the four major maturity genes revealed that the 'Y1312-2' variety was *E1/e2-ns/e3-tr/E4*, while all the three recurrent parents were *E1/e2-ns/E3-Mi/E4*.
Development of the stem growth habit NILs
-----------------------------------------
The recurrent parents and 'Y1312-2' were crossed in 2008. Backcrosses to the recurrent parents were repeated four times with marker-assisted selection for the *Dt1* and *E3* genes. For each backcrossed combination, one BC~4~F~1~ plant, whose linkage between *Dt1* and *E3* was broken, was grown in 2011. In 2012, 60 BC~4~F~2~ seeds were planted, and then, *Dt1* genotyping of 38--48 healthy BC~4~F~2~ plants in each cross combination showed that there were 15 *dt1* homozygotes, 14 *Dt1* homozygotes, and 19 heterozygotes in NILs developed using 'Tohoku 160' as a recurrent parent (hereafter named, BC/T160): 13 *dt1* homozygotes, 10 *Dt1* homozygotes, and 17 heterozygotes in NILs using 'Tohoku 162' as a recurrent parent (hereafter, BC/T162); and, 10 *dt1* homozygotes, 13 *Dt1* homozygotes, and 15 heterozygotes in NILs using 'Tohoku 164' as a recurrent parent (hereafter, BC/T164). In 2013, 10 BC~4~F~3~ lines were cultivated in the field; these lines were derived from five indeterminate and five determinate plants in BC~4~F~2~ for each cross combination. The consistency between phenotype and genotype of *Dt1* was confirmed by observing the growth of the main stem after flowering. In addition, the lines did not segregate for flowering or maturity time, or main stem length. In 2014, BC~4~F~4~ lines were obtained by harvesting each BC~4~F~3~ line in bulk.
The effect of stem growth habit on SSW and SWCV
-----------------------------------------------
Comparison of stem growth habit (S) demonstrated that SSW values of indeterminate lines were slightly heavier compared to determinate lines, and that there was no significant difference in SWCV ([Tables 1](#t1-68_17137){ref-type="table"}, [2](#t2-68_17137){ref-type="table"}). The effects of cross combination (C) were significant for SSW and SWCV, while the effect of location (L) and year (Y) was significant only for SSW.
There were significant interaction effects for S × C in SWCV, but no significant interaction effect in SSW ([Table 2](#t2-68_17137){ref-type="table"}). The comparison of mean values between growth habits for SWCV in each cross combination (using factor S) demonstrated that the effects of stem growth habit on SWCV depends on genetic background ([Fig. 1](#f1-68_17137){ref-type="fig"}). However, the mean squares of the interaction for SWCV between stem growth habit and cross combination were 3.5 times lower than that of cross combination effects ([Table 2](#t2-68_17137){ref-type="table"}).
The effect of stem growth habit on the other agricultural characteristics
-------------------------------------------------------------------------
There were significantly more seeds per pod in indeterminate lines than in determinate lines, whereas there was no significant difference between seed yield and the number of pods ([Tables 1](#t1-68_17137){ref-type="table"}, [2](#t2-68_17137){ref-type="table"}). Flowering time, seed-filling period, and time to maturation of indeterminate lines was 0.8 days, 0.5 days, and 1.2 days longer than those parameters in determinate lines, respectively; except for seed-filling period, these differences were significant ([Tables 3](#t3-68_17137){ref-type="table"}, [4](#t4-68_17137){ref-type="table"}). The number of main stem nodes of indeterminate lines was 1.3 times more than in determinate lines, while main stem length was 1.6 times longer ([Table 3](#t3-68_17137){ref-type="table"}). The mean number of branches in indeterminate lines was 0.7 greater than in determinate lines ([Table 3](#t3-68_17137){ref-type="table"}). The lowest pod height of indeterminate lines was 3.1 cm higher, while mean lodging score of indeterminate lines scored 1.1 higher than determinate lines ([Table 3](#t3-68_17137){ref-type="table"}). The protein content of indeterminate lines was 0.3% lower than the protein content in determinate lines, while there was no difference in oil content ([Table 1](#t1-68_17137){ref-type="table"}).
Discussion
==========
Timing of flower formation and the length of seed-filling period in indeterminate varieties tend to vary more than those of determinate varieties, depending on the flower node positions ([@b26-68_17137], [@b42-68_17137]). Variations in timing and duration of seed-filling period are presumed to affect SSW and its uniformity. Although [@b2-68_17137] and [@b42-68_17137] reported that there is no significant difference in SSW and seed uniformity between stem growth habit of NILs with normal SSW, it is unclear what the effect would be on these traits if indeterminate traits were introduced into determinate varieties with heavier seeds. It is possible that an unattainable rapid seed filling rate is required for forming large seeds in late-blooming flowers due to the limited time available for seed filling before senescence. In this study, we developed NILs with genes controlling stem growth habit by introducing indeterminate traits into determinate varieties that have large SSWs, and evaluated the influence of stem growth habit on SSW and SWCV. The cultivation of NILs with a variety of stem growth habits, from multiple genetic backgrounds and grown under two different growing environments, demonstrates that there is no significant difference between the indeterminate and determinate lines in SWCV, and that indeterminate lines produce slightly heavier seeds than do determinate lines. On the other hand, the mean squares of the cross combination effects for SSW and SWCV were 22 and 197 times higher than those of stem termination effects, respectively ([Table 2](#t2-68_17137){ref-type="table"}). These results show that differences between genetic backgrounds were more than the differences between the two growth habit characteristics. For example, seed weight uniformity of NILs in soybeans developed using 'Tohoku 160' as the recurrent parent was higher than any of the other NILs we developed, regardless of stem growth habit. This suggests that some control of seed uniformity works independently of stem growth habit. In determinate soybeans, pod formation ratio of early blooming flowers tends to be higher than that of later ones ([@b11-68_17137], [@b34-68_17137]). In contrast, the ratio of pod formation in indeterminate soybeans is low in flowers that bloom within 10 days after flowering commences, although the ratio of pod formation in the latest-blooming flowers tend to be low in both indeterminate and determinate soybeans ([@b42-68_17137]). These previous studies demonstrated that the length of flowering is not directly related to time required for seed filling. [@b30-68_17137] and [@b43-68_17137] also found that there is a lag period between flowering and pod elongation and that the lag period of late-blooming flowers is shorter than the lag period of early-blooming flowers. The lag period promotes the synchronous development of pods and seeds originating from flowers blooming at different times on the same plant and this duration differs among soybean varieties ([@b43-68_17137]). Therefore, traits for pod setting ratio and duration of lag period other than seed filling speeds, might be more strictly controlled in the 'Tohoku 160' variety. Indeed, we cannot deny the possibility that seeds from the pods on the first to second nodes from the top raceme of indeterminate line may be small by being affected by the considerably short seed filling periods. However, the number of these seeds is few compared to the number of remaining seeds, and, when harvesting all seeds in bulk, these small seeds are not considered to markedly affect seed uniformity strongly. Based on the aforementioned data, we conclude that indeterminate growth habit in soybeans does not much influence the single seed weight or the uniformity of seed weight.
We found significant interaction for seed uniformity between cross combination and stem growth habit. Although we hypothesized that maturations and SSWs were the factors that affect the interaction between seed uniformity and growth habit, consistent tendencies for these interaction among maturations and SSWs were not observed in our results. It is difficult to know what kinds of genetic background have undesirable effects on seed uniformities of indeterminate growth habit from our results. Tests in which seed uniformities are compared between growth habits under more diverse genetic backgrounds are required in order to understand this interaction in detail. However, when comparing the mean squares of cross combination effect with that of the interaction for SWCV between stem growth habit and cross combination, this interaction were relatively low ([Table 2](#t2-68_17137){ref-type="table"}). Therefore, although we cannot deny the possibility that the introduction of *Dt1* may reduce seed uniformities in some genetic backgrounds, it is considered to be possible to solve this uniformity problem due to the introduction of different growth habits to some extent by using determinate varieties with excellent seed uniformity as recurrent parents. On the other hand, there was no significant interaction for seed weight between cross combination and stem growth habits ([Table 2](#t2-68_17137){ref-type="table"}). However, there are some determinate varieties that have particularly heavy seeds (\>600 mg), for example 'Tamafukura' and 'Hyokei-kuro 3' ([@b13-68_17137]). The effect of introducing indeterminate growth habit into super-heavy-seeded determinate varieties still needs to be demonstrated.
Although there was no significant difference in seed yield between growth habits (determinate vs. indeterminate), significantly more seeds per pod were produced in indeterminate lines than in determinate lines. This result corresponds to previous studies ([@b10-68_17137], [@b12-68_17137], [@b20-68_17137]). *Gm-JAG1*, the gene that governs leaf shape (*Ln*), has also been reported to affect the number of seeds per pod and seed weight, and there are more seeds per pod and lighter seeds in soybeans with narrow-leaf alleles (*ln*) than in homozygotes with broad-leaf alleles (*Ln*) ([@b8-68_17137], [@b15-68_17137], [@b16-68_17137], [@b32-68_17137]). *Dt1* and *Ln* genes commonly influence the number of seeds per pod, although it is impossible to compare the effects of these genes in this study, because leaf shapes of all the recurrent and donor parents were broad type (*Ln*). On the other hand, there is no difference in seed weight between indeterminate and determinate soybean lines. All the above results demonstrate that the relationship between the number of seeds per pod and the other yield components related to stem growth habit differs from those caused by the introduction of the *Ln* gene.
Soybean breeders have long been concerned that indeterminate varieties show a severe lodging and the late maturity (caused by an increase of the main stem length and the number of main stem nodes) associated with continued vegetative growth long after flowering ([@b2-68_17137], [@b5-68_17137], [@b38-68_17137]). In this study, we found that the introduction of indeterminate traits increased main stem length and main stem nodes, and that the mean rank of lodging scores of indeterminate lines was higher than determinate lines. Since the effects of lodging seriously and negatively impact yield ([@b27-68_17137]), improvements in lodging resistance will be needed for the introduction of indeterminate traits into determinate varieties. Although, as aforementioned, there was no yield advantage in indeterminate lines, the development of indeterminate varieties with lodging resistance equivalent to that of determinate ones may contribute to breeding new varieties with high yield. On the other hand, although time to maturation in indeterminate varieties was significantly later than in the determinate varieties, the mean squares for the effect for stem growth habits was less than those of location and year ([Table 4](#t4-68_17137){ref-type="table"}), and the difference was only one day on an average, which would probably be acceptable.
In our study, there were no differences in oil content between lines, but protein content of indeterminate lines was slightly lower than determinate lines. The influences of introduction of indeterminate traits into determinate varieties for this balance have been reported to change depending on length to maturity, growing conditions, and genetic background of the plants ([@b2-68_17137], [@b5-68_17137], [@b28-68_17137], [@b39-68_17137]). It is not clear whether the *Dt1* gene has a pleiotropic effect on protein content or whether the various genes that regulate protein content are located close to the *Dt1* gene; however, when introducing indeterminate traits into determinate varieties, the selection of acceptable protein and oil content will be needed in light of the study results described above.
Lowest-pod height is an important trait affecting mechanical harvesting. Planting varieties whose lowest-pod height is high on the plant reduces yield loss when harvesting with a combine ([@b25-68_17137]). Our results show that the lowest-pod height on a plant of indeterminate varieties was significantly higher than that of determinate varieties, which is consistent with the findings of [@b29-68_17137].
In conclusion, the introduction of indeterminate traits into determinate varieties with heavy seeds did not much influence seed weight or the variation in seed weight (uniformity), but it did increase the number of main stem nodes, main stem length, and caused more severe lodging. The QTLs related with lodging resistance ([@b3-68_17137], [@b23-68_17137], [@b41-68_17137]) have been reported to be located in a different chromosome region than the *Dt1* locus. Introduction of these QTLs into soybeans will be required to breed indeterminate varieties.
We thank Dr. Elroy R. Cober, Dr. Bahram Samanfar for critical reading, and Hitoshi Sato, Osamu Fujii, Yuko Sato, Akihiro Takahashi, Kosei Awatsu, Hisaho Takagai and Kenei Takahashi (NARO) for the field management of yield trials. This study was supported by grants from the Ministry of Agriculture, Forestry, and Fisheries of Japan \[Genomics-Based Technology for Agricultural Improvement (SFC-1001)\]. The authors would like to thank Enago ([www.enago.jp](www.enago.jp)) for the English language review.
{#f1-68_17137}
######
Mean values in each level for yield, yield components, and traits related to seeds of near isogenic lines with different growth habits
Factor Level Number of pods Number of seeds per pod Seed yield (g/plant) SSW (mg) SWCV Protein content (%) Oil content (%)
----------------------- --------------- ---------------- ------------------------- ---------------------- ---------- ------- --------------------- -----------------
Stem growth habit (S) Determinate 66.5 2.03 45.9 347 0.110 43.1 19.8
Indeterminate 65.4 2.10 46.0 352 0.110 42.8 19.8
Cross combination (C) BC/Tohoku162 70.8 1.99 46.1 334 0.115 41.6 20.7
BC/Tohoku164 66.2 2.08 45.2 344 0.113 43.6 18.3
BC/Tohoku160 61.0 2.11 46.7 370 0.102 43.6 20.5
Location (L) Ishinazaka 61.3 2.06 41.2 340 0.114 42.6 20.0
Uenodai 70.6 2.06 50.8 359 0.106 43.2 19.6
Year (Y) 2014 72.3 2.02 45.7 362 0.110 43.1 18.9
2016 59.6 2.10 46.2 337 0.110 42.8 20.7
######
Analysis of variance for yield, yield components, and traits related to seeds of near isogenic lines with different growth habits
Sources of variation Degree of freedom Mean of square
----------------------- ------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------------ -------------------------------------------------- ----------------------------------------------------- ------------------------------------------------------- ----------------------------------------------------- -----------------------------------------------------
Location (L) 1 5156.9 [\*](#tfn1-68_17137){ref-type="table-fn"} [a](#tfn1-68_17137){ref-type="table-fn"} 0.0018 NS 5508.5 [\*](#tfn1-68_17137){ref-type="table-fn"} 20314 [\*](#tfn1-68_17137){ref-type="table-fn"} 0.00400 NS 21.2 NS 9.7 [\*](#tfn1-68_17137){ref-type="table-fn"}
Year (Y) 1 9744.8 [\*\*](#tfn1-68_17137){ref-type="table-fn"} 0.3383 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 15.7 NS 38964 [\*](#tfn1-68_17137){ref-type="table-fn"} 0.00001 NS 4.6 NS 191.7 [\*\*](#tfn1-68_17137){ref-type="table-fn"}
L × Y 1 1068.9 NS 0.0002 NS 1810.6 NS 2344 NS 0.00008 NS 1.0 NS 1.6 NS
Cross combination (C) 2 1952.7 [\*\*](#tfn1-68_17137){ref-type="table-fn"} 0.3042 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 45.3 NS 27749 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 0.00394 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 110.4 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 141.6 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"}
Pooled error a 8 104.7 0.0030 41.4 710 0.00018 3.0 0.8
Stem growth habit (S) 1 67.5 NS 0.3132 [\*\*\*](#tfn1-68_17137){ref-type="table-fn"} 0.4 NS 1251 [\*](#tfn1-68_17137){ref-type="table-fn"} 0.00002 NS 6.6 [\*](#tfn1-68_17137){ref-type="table-fn"} 0.0 NS
S × C 2 3.0 NS 0.0320 [\*\*](#tfn1-68_17137){ref-type="table-fn"} 7.8 NS 87 NS 0.00113 [\*\*](#tfn1-68_17137){ref-type="table-fn"} 3.6 NS 0.4 NS
Pooled error b 12 104.0 0.0029 35.8 134 0.00013 1.0 0.2
are significant at 5%, 1%, and 0.1% probability levels, respectively.
NS: not significant.
######
Mean values in each level for duration between growth stages and morphological characteristics of near isogenic lines with different growth habits
Factor Level Flowering time Seed-filling period Maturing time Main stem length (cm) Number of main stem nodes Lodging (0--5) Number of branches Lowest pod height (cm)
----------------------- --------------- ---------------- --------------------- --------------- ----------------------- --------------------------- ---------------- -------------------- ------------------------
Stem growth habit (S) Determinate 65.0 76.9 142.0 69 16.2 1.3 4.9 17.0
Indeterminate 65.8 77.4 143.2 110 21.8 2.4 5.6 20.1
Cross combination (C) BC/Tohoku162 61.7 70.8 132.4 84 17.8 2.1 5.0 14.6
BC/Tohoku164 66.5 77.1 143.6 90 20.1 1.4 5.0 17.6
BC/Tohoku160 68.2 83.7 151.8 95 19.2 2.0 5.8 23.4
Location (L) Ishinazaka 66.8 77.4 144.2 88 18.9 1.8 4.8 18.0
Uenodai 64.0 77.0 141.0 91 19.1 1.9 5.7 19.1
Year (Y) 2014 62.0 81.4 143.3 98 19.7 1.9 5.9 18.3
2016 68.9 73.0 141.9 81 18.4 1.8 4.7 18.8
######
Analysis of variance for duration between growth stages and morphological characteristics of near isogenic lines with different growth habits
Sources of variation Degree of freedom Mean of square
---------------------- ------------------- ---------------------------------------------------------------------------------------------- ------------------------------------------------------ ------------------------------------------------------ -------------------------------------------------------- ------------------------------------------------------ --------------------------------------------------- ---------------------------------------------------- ------------------------------------------------------
L 1 467.6 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} [a](#tfn3-68_17137){ref-type="table-fn"} 7.7 NS 595.4 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 459.5 NS 2.8 NS 2.0NS 46.0 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 64.7 NS
Y 1 2863.5 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 4208.4 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 129.1 [\*](#tfn3-68_17137){ref-type="table-fn"} 17973.0 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 110.4 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 1.7NS 85.8 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 10.2 NS
L × Y 1 22.2 [\*](#tfn3-68_17137){ref-type="table-fn"} 37.6 NS 117.6 [\*](#tfn3-68_17137){ref-type="table-fn"} 44.5 NS 1.1 NS 0.3NS 0.4 NS 21.7 NS
C 2 912.8 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 3335.0 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 7602.7 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 2196.6 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 107.6 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 13.9[\*](#tfn3-68_17137){ref-type="table-fn"} 16.1 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 1594.5 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"}
Pooled error a 8 2.6 30.2 27.2 124.5 1.0 2.2 0.6 8.2
S 1 39.2 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 12.6 NS 96.3 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 102709.3 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 1870.4 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 84.0[\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 34.9 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 564.3 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"}
S × C 2 8.1 [\*\*](#tfn3-68_17137){ref-type="table-fn"} 5.0 NS 19.7 NS 122.9 [\*](#tfn3-68_17137){ref-type="table-fn"} 13.4 [\*\*\*](#tfn3-68_17137){ref-type="table-fn"} 0.4NS 1.4 NS 1.0 NS
Pooled error b 12 0.9 4.7 5.5 24.1 0.8 0.4 0.5 7.4
are significant at 5%, 1%, and 0.1% probability levels, respectively.
NS: not significant.
[^1]: Communicated by Toyoaki Anai
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Several studies have reported on regulation of protein synthesis in skeletal muscles in fasted and fed state indicating considerably elevated synthesis during 2--3 hours postprandially \[[@B1]-[@B6]\]. Usually, such studies are based on estimates of protein synthesis by incorporation of labeled amino acids into newly synthesized proteins \[[@B7]-[@B10]\]; methods that are dependent on complex assumptions, related to distribution of tracers among intra- and extra cellular pools of amino acids \[[@B8],[@B11],[@B12]\], and represent expensive and complex analytical methods. \[[@B13],[@B14]\]. Consequently, alternative methods are needed in clinical studies. Therefore, tracer independent methods, measuring initiation of translational phosphoprotein complexes as well as cellular alterations in transcript concentrations of regulatory and target proteins for synthesis should be of value from several perspectives.
Our previous studies have confirmed that extracellular provision of amino acids activates translation initiation of protein synthesis in skeletal muscle tissue during both oral and intravenous feeding \[[@B12],[@B15],[@B16]\]. Such induction of translation initiation may be triggered by concentration changes of amino acids outside or inside muscle cells through mTOR signaling without a critical presence of insulin or extracellular IGF-1 \[[@B17]-[@B19]\]. However, strictly controlled experiments, based on labeled amino acids, did not provide consistent results on amino acid stimulation of total muscle protein synthesis, due to tissue pool- and tracee uncertainties \[[@B12],[@B20]\]. Therefore, the present study was conducted to evaluate how provision of extracellular amino acids influenced on cellular expressions and content of transcripts of amino acid transporters and myofibrillar MHC2A as well as α-actin as possible markers for the synthesis of contractile proteins in skeletal muscles at feeding relevant for clinical nutrition studies.
Material and methods
====================
Patient studies
---------------
Twelve patients who underwent upper gastrointestinal tract surgery participated \[[@B15]\]. They were randomized to receive overnight constant infusions of either saline or TPN, (Kabiven Perifer) for at least 12 hours prior to surgery as described elsewhere \[[@B15]\]. All infusions continued until muscle biopsies were taken from the rectus abdominis muscle directly after induction of anesthesia. Muscle biopsies with remaining intact RNA from 10 out of 12 randomized patients were used in present analyses. Amino acid concentrations in blood and translation initiation factor analyses from study and control patients have been reported elsewhere \[[@B15]\].
Animal experiments
------------------
Female, weight stable C57 BL/6 mice were used. They were either starved or refed with standard rodent chow (2016 Global Tekland®, Netherlands) and had always free access to water. Starved mice had no access to food overnight for 12 hours before termination, while refed animals were similarly starved overnight for 12 hours, but had then free access to food for 3 hours before termination \[[@B18]\]. Animals were killed by cervical dislocation and mixed hind limb muscles were excised and immediately frozen in liquid nitrogen. Muscle samples were stored at −70°C until RNA extractions were performed. All animal procedures were performed in accordance to national guidelines for animal research and approved by the regional animal research ethics committee in Gothenburg.
Cell cultures
-------------
Rat myoblast L6 cells were seeded in 25 cm^2^ flasks, 48 well- or 6-well dishes and grown to confluence in Dulbecco's modified Eagle's medium, with 4.5 g/l glucose (DMEM), supplemented with 10% foetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine. At day 4, when cells were confluent, medium was changed to standard DMEM supplemented with 2% FBS. At day 5, medium was changed to DMEM with very low amounts of all amino acids (0,14 mM), and without addition of FBS and antibiotics. Cells were cultured for 24 hours and thereafter cells were given new medium with either 0.28 mM amino acids (referred to as low AA) or 9 mM amino acids in total (referred to as high AA), which equals the concentration in standard DMEM. Cells were then cultured from 0.5-18 hours before harvest. In some experiments only groups of amino acids were included at elevated (high) concentrations in the medium while the remaining amino acids were kept at very low amino acid concentration (0.14 mM). Cells used in array experiments were harvested after 18 hours of refeeding. Cells were kept in an incubator with 95% air, 5% CO~2~ environment during the entire experiment.
RNA isolation and cDNA synthesis
--------------------------------
RNA from L6 cells was extracted using RNeasy mini kit (Qiagen) with DNAse step included. Cells were lysed in RLT buffer according to kit instructions by adding lysis buffer directly to cells in the culture dishes. Cell lysates were collected and homogenized by flushing 10 times through a 20G needle. Skeletal muscle tissue was homogenized with an Ultra-Thurrax homogenizer and RNA from human and mouse muscle tissue was extracted by RNeasy fibrous tissue mini kit with DNAse step included. Total RNA concentrations were measured by spectrophotometer (Nanodrop ND-100) and RNA quality was checked using an Agilent 2100 bioanalyser and RNA 6000 Nano kit. One μg of total RNA was reverse transcribed to cDNA using oligo d(T)-primers according to kit instructions (Advantage® RT for PCR kit, Clontech). Positive and negative controls were included in each run of cDNA synthesis.
Real-time PCR
-------------
Commercially available primers from Qiagen were used for analysis of human α-actin/ ACTA 1 (QT00199815), rat α-actin/acta 1 (QT01081374), human myosin heavy chain 2A/MYH2 (QT00082495), human SLC38A2/(Snat 2) (QT 00030499), rat Slc38a2/(Snat 2) (QT00186116) and mouse Slc38a2/(Snat2) (QT00129542). Real time PCR was performed using QuantiTect™SYBR®Green PCR kit (Qiagen) according to kit instructions. 2 μl of cDNA and 2 μl of premixed Quantitect primers were used for each reaction of 20 μl exept for rat acta 1 where 5 μl cDNA were used. For analysis of mouse α-actin/acta 1 (5′-3′ Forward; CCAGAGTCAGAGCAGCAGA, Reverse CACGATGGATGGGAACAC), mouse myosin heavy chain 2A (5′-3′ Forward; TGGAGGGTGAGGTAGAGAGTG, Reverse; TTGGATAGATTTGTGTTGGATTG) primers were used. PCR analysis was performed with the PerfeCTa SYBR Green SuperMix (Quanta Biosciences) with the following settings; 95°-10 sek, 60°-30 sek, 72°-30 sek. 2 μl of cDNA and 3 pmol of each primer were used to a reaction of 10 μl. Real-time PCR was performed on either a LightCycler 1.5 instrument or a LightCycler 480 (Roche). Quantitative results were produced by the relative standard curve method and results are given in arbitrary units. All samples were analyzed in duplicates and negative controls were included in each run. Results from human and mice experiments are related to the expression of GAPDH as housekeeping gene which did not change significantly at starvation - refeeding. Results from cultured cells are reported as expression/ 18S. Levels of 18S RNA expression are provided separately since neither GAPDH nor 18S levels were stable at all experimental conditions in cell culture experiments. Only acta 1 (α-actin) and Slc38a2 (Snat 2) transcripts were measured in cell culture experiments since Mhc 2A transcripts were below detection levels when analyzed by real-time PCR.
Microarray experiments
----------------------
Microarray analysis was performed on RNA from eight samples (4 starved, 4 refed). 500 ng of RNA from starved and refed cells (18 hour) were labeled with Cy-3-dCTP and Cy-5-dCTP respectively (Amersham BioSciences), in a cDNA synthesis reaction with Agilent Flourescent Direct Label. cDNA from starved versus refed cells were then hybridized in competition on Whole Rat Genome Microarrays (4x44K expression oligoarrays, Agilent) during 17 hrs followed by post-hybridization washes according to in situ instructions (Hybridization Kit Plus, Agilent). This provides relative changes in gene expression. Microarrays were quantified on Agilent G2565 AA microarray scanner and data were pre-processed in Feature Extraction 9.1.3.1 software program (Agilent). Dye-normalized, outlier- and background subtracted values were imported into GeneSpring GX 10 software program (Agilent) for further analysis.
Subcellular fractionation and labeling of cells
-----------------------------------------------
Subcellular separation of the L6 cells was performed to check for protein expression of cytoskeletal proteins. L6 cells contained both actin as well as myosin heavy chains (Figure [1](#F1){ref-type="fig"}). Cells were labeled with ^35^S-Methionine as described elsewhere \[[@B12]\]. Both specific radioactivity and tracee concentrations of methionine were held constant in cell cultures when grown in presence of high or low amino acid concentrations. Stepwise subcellular fractionation of cells was performed by using Proteoextract® Subcellular proteome extraction kit (Calbiochem, Merck Biosciences) according to kit instructions. Cell fraction 4, which contains cytoskeletal proteins, was separated by electrophoresis and proteins were either stained by Coomassie brilliant blue or visualized by autoradiography as described elsewhere \[[@B12]\].
![**Electrophoretic separation of cytoskeletal proteins in L6 cells by stepwise purification.** Cytoskeletal proteins were extracted using Calbiochems sub-cellular proteome extraction kit and separated by electrophoresis as described in Material and Methods. Lane 1--2 are autoradiograms of ^35^S-methionine labeled cells from low AA (lane 1) and high AA refed (lane 2) cells. Lane 3--4 are the same samples stained by Coomassie brilliant blue. Lane 5 is purified rabbit muscle actin (Sigma-Aldrich A-2522). Lane 6 is purified rabbit muscle myosin (Sigma-Aldrich M-1636). It has been demonstrated that L6 cells contain myosin \[[@B44]\].](1479-5876-10-238-1){#F1}
Incorporation of L-\[U-^14^C\]-phenylalanine (40 μCi/μmol phe) to cellular proteins was performed as described elsewhere in the presence of low or high medium concentrations of amino acids in the presence of constant concentration of phenylalanine (12μM).
### Statistics
Results are presented as mean ±SE. Group comparisons were performed by factorial ANOVA followed by Fisher PLSD post hoc testing. p\<0.05 was regarded statistically significant in two-tailed tests. Statistics used in the array experiment are described in the results section.
Results
=======
Changes in transcript levels of myofibrillar proteins
-----------------------------------------------------
Skeletal muscle tissue from surgical patients, who received 12 hours continuous infusion of total parenteral nutrition, displayed significantly decreased MHC2A transcript levels compared to muscle tissue from control patients receiving saline only (p\<0.05), while ACTA 1 transcripts were numerically decreased but did not reach statistical significance (Figure [2](#F2){ref-type="fig"}A). Similarly, both Mhc2A and acta 1 transcripts appeared to decline in skeletal muscles from refed mice compared to starved mice, but the difference did not reach statistical significance (Mhc2a p\<0.18, Acta 1 p\<0.10, n=16; Figure [2](#F2){ref-type="fig"}B). Similar findings occurred for Acta 1in confluent L6 cells refed low (0.28 mM) vs. high AA (9 mM) concentrations during 18 hours ( Acta 1, p\<0.3, n=14; Figure [2](#F2){ref-type="fig"}C).
{#F2}
Acta 1 transcripts increased significantly at 60 min of AA refeeding compared to 24 hours starved cells (cultured in presence of very low AA concentrations (0.14 mM) for 24 hours, before start of refeeding) with no difference between low and high AA groups (Figure [3](#F3){ref-type="fig"}A; n=9). Acta 1 levels remained increased at 4 and 8 hours compared to 24 hours starved cells, without any differences between low and high AA groups (4 hours; Low AA 0.818±0.273, High AA 1.141±0.796 , 8 hours; Low AA 1.152±0.740, High AA 0,992±0,330; n=3). Refed confluent L6 cells, based on different groups of AA at high concentrations for 18 hours, did not result in clear-cut significant alterations of Acta 1 levels among the groups (Figure [4](#F4){ref-type="fig"}A).
{#F3}
{#F4}
Changes in transcript levels of amino acid transporter slc38a2/Snat 2
---------------------------------------------------------------------
SLC38A2 (Snat2) levels did not differ between skeletal muscles from TPN and saline infused patients (P\<0,7) as well as in refed mice compared to starved mice (Figure [2](#F2){ref-type="fig"}A,B). In contrast, Slc38a2 (Snat2) levels were significantly lower in L6 cells refed high AA concentrations during 18 hours compared to cells receiving low amino acid concentrations. (p\<0,001) (Figure [2](#F2){ref-type="fig"}C). The discrepancies between slc38a2 transcripts at 60 min (Figure [3](#F3){ref-type="fig"}B) and 18 hours (Figure [4](#F4){ref-type="fig"}B) appeared to emerge beyond 4 hours incubation at high versus low AA concentrations (4 hours; Low AA 14.7±3.2, High AA 10.4±2.5, 8hours; Low AA 19,6±5.1, High AA 5.0±1.3; n=3) Based on these findings we chose 18 hours incubation for comparisons among groups of AA experiments. Cellular Slc38a2 (Snat2) transcripts were thus influenced by refeeding by various combinations of amino acids for 18 hours. Cells refed by branched chain amino acids (leu, ile, val) or by glutamine in the presence of other non-essential amino acids (Arg, Thr, His, Lys) showed decreased levels of Slc38a2 (Snat2) transcripts, while refeeding by aromatics (phe, tyr, trp) or sulphur amino acids (met, cys) in the presence of all amino acids in DMEM at low background concentration (0.14 mM) did not alter Slc38a2 mRNA levels (Figure [4](#F4){ref-type="fig"} B).
Total protein synthesis in cultured L6 cells
--------------------------------------------
Incorporation of ^14^C-phenylalanine into total cellular proteins increased continuously in the presence of amino acids and was significantly higher in the presence of high amino acids in the medium after 20 hours incubation compared to cells incubated with low amino acid concentrations (Figure [5](#F5){ref-type="fig"}).
![**Time course changes in L-\[U-**^**14**^**C\]-phenylalanine incorporation into proteins.** Confluent L6 cells were cultured in medium containing very low amounts of amino acids (0,14 mM) for 24 hours, and then refed in low (0,28 mM) or high (9 mM) concentration of total AA concentrations, with constant specific radioactivity of phenylalanine (40 μCi/μmol phe). Cells were harvested by TCA-precipitation of proteins. (\* p\< 0.01 vs. low AA).](1479-5876-10-238-5){#F5}
Microarray results
------------------
Of 41 090 probes on the Agilent 4 × 44K whole genome rat array, 22 318 entities remained after filtering of flags to remove low expressed genes. Statistical evaluation by *t*-test with Benjamini-Hochberg correction for multiple significance showed that 6675 entities were significantly different between starved and refed cells (p\< 0.05). 745 entities showed at least 2 fold changes and were used for further analysis (399 up-, 346 down-regulated). A search by name (actin, myosin, tropo and slc) among the significantly changed transcripts with a fold change above 2 was performed in order to find mRNAs for amino acid transporters and contractile proteins. Several amino acid transporters demonstrated alterations in expression levels in response to provision of amino acids to cell cultures (Table [1](#T1){ref-type="table"}). A gene ontology (GO) analysis was performed to find categories/pathways with significant enrichment of genes. Ten GO categories were found; all related to lipid, cholesterol and steroid metabolism (Table [2](#T2){ref-type="table"}). Differentially expressed entities in GO cathegories "steroid biosynthethic and metabolic processes" are presented in Table [3](#T3){ref-type="table"}. These results provide evidence that amino acid have profound effects on intermediary and nitrogen metabolism in muscle cells.
######
Altered transcript levels of amino acid transporters and muscle proteins in confluent L6 muscle cells refed high amino acid concentrations versus low amino acid concentration assessed in microarray experiments as described in Material and Methods
**Agilent entity** **Foldchange array** **Foldchange Q-PCR** **Common name** **Gene symbol** **Protein name/Function**
-------------------- ---------------------- ---------------------- -------------------- -------------------- -------------------------------------------
A_44_P463878 **−3.2** **−2.6** NM_181090 Slc38a2 Snat 2/ System A transporter
A_44_P393273 **−3.0** NM_053818 Slc6a9 Glyt1/ System Gly transporter
A_44_P104652 **−2.7** NM_181090 slc38a2 Snat 2/ System A transport
A_44_P510515 **−2.3** NM_017206 Slc6a6 Taut/ System BETA, taurine transport
A_44_P410954 **−2.0** ENSRNOT00000011006 Slc43a1_predicted Lat 3/ System L-like transporter
A_44_P994686 **−3.6** NM_012676 Tnnt2 Troponin T2, cardiac
A_42_P786933 **−2.4** NM_012983 Myo1d Myosin 1D
A_44_P489468 **−2.1** ENSRNOT00000030661 ENSRNOT00000030661 Myosin heavy chain, smooth muscle isoform
######
GO categories with significant enrichment of entities in L6 cells refed high amino acid concentrations
**GO ID** **GO Accession nr** **GO Term** **Corrected p-value**
----------- --------------------- ---------------------------------- -----------------------
8636 GO:0016126 sterol biosynthetic process 1.289 E-12
4323 GO:0006695 cholesterol biosynthetic process 3.812 E-9
4322 GO:0006694 steroid biosynthetic process 9.406 E-8
8635 GO:0016125 sterol metabolic process 9.406 E-8
5578 GO:0008610 lipid biosynthetic process 7.391 E-7
4260 GO:0006629 lipid metabolic process 4.741 E-6
18769 GO:0044255 cellular lipid metabolic process 3.867 E-6
3797 GO:0006066 alcohol metabolic process 2.552 E-6
5262 GO:0008203 cholesterol metabolic process 2.552 E-6
5261 GO:0008202 steroid metabolic process 1.768 E-4
Based on entities selected by fold change ≥2.
######
Altered transcript levels (entities) in GO categories "steroid biosynthetic and metabolic processes" in confluent L6 muscle cells refed high amino acid concentrations
**Agilent entity** **Foldchange array** **Foldchange Q-PCR** **Common name** **Symbol** **Name**
-------------------- ---------------------- ---------------------- ----------------- ------------ -------------------------------------------------------------
A_44_P365580 **6.0** NM_017136 Sqle Squalene epoxidase
A_42_P814765 **5.8** NM_001013071 Tm7sf2 Transmembrane 7 superfamily member 2
A_43_P16774 **5.6** **6.5** NM_001006995 Acat2 Acetyl-Coenzyme A acetyltransferase 2
A_42_P794613 **5.1** **6.5** NM_031062 Mvd Mevalonate (diphospho) decarboxylase
A_44_P251944 **5.0** NM_053539 Idi1 Isopentenyl-diphosphate delta isomerase
A_43_P12843 **4.5** NM_053539 Idi1 Isopentenyl-diphosphate delta isomerase \]
A_44_P512136 **4.5** NM_022389 Dhcr7 7-dehydrocholesterol reductase
A_44_P487240 **3.6** NM_017268 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1
A_44_P168285 **3.5** NM_012941 Cyp51 Cytochrome P450, subfamily 51
A_43_P11890 **3.4** NM_017268 Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1
A_44_P422696 **3.2** **4.1** NM_031541 Scarb1 Scavenger receptor class B, member 1
A_42_P796502 **3.1** NM_017235 Hsd17b7 Hydroxysteroid (17-beta) dehydrogenase 7
A_44_P334218 **3.0** NM_031049 Lss Lanosterol synthase (Lss)
A_44_P379244 **2.9** NM_031541 Scarb1 Scavenger receptor class B, member 1
A_44_P432965 **2.8** NM_031840 Fdps Farensyl diphosphate synthase (Fdps)
A_43_P22542 **2.7** NM_001009399 Nsdhl NAD(P) dependent steroid dehydrogenase-like
A_44_P347250 **2.6** NM_001080148 Dhcr24 24-dehydrocholesterol reductase
A_43_P13043 **2.6** NM_057137 Ebp Phenylalkylamine Ca2+ antagonist (emopamil) binding protein
A_44_P143567 **2.5** NM_031840 Fdps Farensyl diphosphate synthase
A_44_P315661 **2.4** NM_013134 Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase
A_43_P13088 **2.2** NM_080886 Sc4mol Sterol-C4-methyl oxidase-like
A_43_P11729 **2.0** NM_013134 Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase
A_44_P237994 **−2.1** NM_053502 Abcg1 ATP-binding cassette, sub-family G (WHITE), member 1
A_44_P536613 **−2.1** NM_001025415 Ch25h Cholesterol 25-hydroxylase
Discussion
==========
A large number of studies have evaluated rates and translation initiation of total protein synthesis in skeletal muscles in response to feeding during recent decades \[[@B13],[@B21]\]. However, such studies, mainly based on incorporation of labeled amino acids, suffer from uncertainties and complex assumptions for calculation of protein synthesis rate \[[@B10],[@B11],[@B22]-[@B24]\]. Difficulties occur particularly at rapid alterations in bio-dynamics during non-steady state conditions \[[@B10],[@B14]\]. Therefore, alternative and tracer independent methods have recently been applied in both animal and clinical experiments, complementary to tracer based methods. Such techniques are mainly based on assessment of phosphorylation/de-phosphorylation of regulatory proteins or protein complexes related to translation initiation of proteins where advantages are straight forward assessment of protein phosphorylation status in cells and tissues under evaluation without the need of steady state \[[@B6],[@B15]\]. Tissue sampling and processing are comparatively easy and analytical principles are robust at standardized conditions \[[@B15]\]. However, limitations are that results reflect only initiation of overall protein bio-synthesis and do not reflect alterations of defined proteins. Determinations of the amount of a particular protein(s) in skeletal muscle tissue should in part resolve this problem, but is only applicable in long-term experiments, since it would be practically difficult to correctly assess quantitative alterations of defined proteins in skeletal muscle cells during short-term responses. Therefore, it should be possible to obtain relevant information by assessment of tissue transcript levels of defined myofibrillar proteins in response to feeding as applied in studies on orally refed healthy volunteers \[[@B25]\]. Unexpectedly, it was then observed that oral refeeding caused a decline of myofibrillar transcripts in skeletal muscles, at conditions otherwise associated with anabolic metabolism \[[@B16],[@B26]-[@B28]\]. Such transcript information was seen in the light of observations that stimulation of gene transcription is usually reflected by increased tissue levels of transcripts for defined proteins aimed at subsequent translation to meet cellular requirements. \[[@B29]-[@B32]\]. Therefore, expected findings should be that net efflux of amino acids from skeletal muscles, due to increased net protein breakdown, should be associated with postprandial down-regulation in transcription of myofibrillar proteins. Normal oral feeding which is leading to rapid and pronounced activation of skeletal muscle protein synthesis, should then be characterized by increased transcription of required proteins \[[@B33]\]. Based on this simplistic view, we decided to re-evaluate effects on transcripts of myofibrillar proteins as Myosin heavy chain 2A (myosin) and acta 1 (α-actin) in skeletal muscle tissue in response to refeeding, particularly with focus on effects by amino acids in both patient and animal experiments.
Myosin heavy chains contributes to 20-25% of overall muscle protein synthesis in humans \[[@B34],[@B35]\] while actin may display both lower and higher turnover compared to mixed muscle proteins \[[@B35],[@B36]\]. Muscle tissue is however composed of many different proteins where sarcoplasmatic and myofibrillar proteins have different basal turnover and synthesis rates at feeding \[[@B37]\]. Adult human muscle tissue expresses three different isoforms of myosin heavy chain (MHC-I, MHC-IIa and MHC-IIx), where MHC-IIa is highly expressed in humans, while rodents express one additional form (MHC-IIb) \[[@B38]\].The myosin gene family is located in a cluster on chromosome 17 in humans and on chromosome 11 in mice \[[@B39]\]. Studies have indicated that mRNA content of different myosin isoforms correlates to the relative content of various MHC proteins present in skeletal muscle tissue \[[@B40],[@B41]\] Changes in expression patterns of myosin heavy chain proteins exist in skeletal muscles during hypertrophy in the control of net muscle mass subsequent to loading \[[@B42]-[@B46]\], but less is known in response to feeding, although Mhc 2X mRNA is reported to unexpectedly increase after 7 days at reduced oral intake in rats \[[@B47]\]. Our present findings show that that transcripts of myosin heavy chain 2A and actin appeared to decrease during continuous TPN administration in agreement with previous findings showing decreased MHC 2X mRNA levels at 3 hours after oral meal intake \[[@B25]\];conditions that provide increased formation of eIF4G·eIF4E complex and decreased association of 4E-BP1·eIF4E \[[@B15]\].
There may be several reasons why myosin transcripts do not clear-cut reflect transcriptional activities and translational needs in cells during continuous long term nutrition exposure, although Rennie and coworkers \[[@B48]\] have reported transient changes in myofibrillar protein synthesis suggesting that muscle cells become refractory to amino acids in response to oral bolus feeding. However, long term provision of intravenous nutrition to patients leads to both time-proportional increases in muscle mass and continuously increased incorporation of labeled amino acids during the presence of high amino acid provision as seen in our present cell experiment (Figure [5](#F5){ref-type="fig"}). Therefore, it appears that transcript cellular levels of actin and myosin are influenced by a variety of factors that possibly determine absolute levels in both short and long term perspectives at nutrition.
It has never been finally assessed how amino acids signal across cell membranes to elicit triggers for induction of translation initiation, although it is assumed there are extracellular/intracellular amino acid sensors since muscle cells are sensitive to alterations of amino acid concentrations \[[@B49]\]. Recently, amino acid transporter proteins gained increased interest based on their ability to sense amino acid changes and influence intracellular signaling \[[@B50]\]. Regulation of expression of amino acid transporters may thus be an important part of the cell machinery in control of protein synthesis secondary to amino acids availability \[[@B49],[@B51]\]. Therefore, we investigated how transcription of the transporter protein Snat 2 (encoded by the gene slc38a2) was affected by refeeding in our models. Snat 2 is a transporter of neutral amino acids belonging to system A \[[@B52]\]. Several amino acids in the refeeding medium (glutamine, histidine, cysteine, methonine) are transported by Snat 2, while branched chain- and aromatics are transported by system L across cell membranes \[[@B51],[@B52]\]. Amino acid transporting by system A increased following amino acid deprivation \[[@B53]\]. Accordingly, we found that Snat 2 mRNA was lower in refed L6 cells compared to starved cells, although such alterations were not evident in vivo. Concentrations of Snat 2 mRNA were also decreased in refed cells by a group of amino acids (Gln, His, Lys, Arg, Thr). Refeeding L6 cells by branched chain amino acids decreased Snat 2 mRNA, although transported by system L, which operates by 1:1 amino acid exchange, which may couple influx of branched chain amino acids to efflux of cytoplasmatic amino acids such as glutamine \[[@B54]\]. It is possible that refeeding cells with branched chain amino acids caused either efflux or influx of other amino acids, which may alter Snat 2 mRNA levels. If so, Snat 2 should be influenced by extra cellular concentrations or transmembrane fluxes of either Gln or His, since it was not changed by refeeding of cysteine or methione which are Snat 2 substrates.
Our microarray data on cultured cells confirm that amino acids have pronounced effects on steroid and lipid metabolism in skeletal muscle cells. Only GO categories/pathways related to lipid and steroid metabolism showed significant enrichment, although microarray experiments indicated that a large number of individual transcripts (30%) were changed following amino acid provision. It has been reported earlier that skeletal muscle cells are capable of local synthesis of sex steroid hormones \[[@B55]\], and there are several ways for cells to provide cholesterol for use in steroid synthesis, such as the mevalonate pathway, where cholesterol is synthesized through a series of enzyme reactions from Acetyl CoA and HMG-CoA \[[@B56],[@B57]\]. Thus, it was interesting to find that transcripts of all enzymes in this pathway were increased following amino acid provision to L6 cells. The expression of steroids and enzymes increases after exercise and may therefore represent an important part of anabolism following physical training in skeletal muscles \[[@B58]\]. Thus, results in the present study confirm that amino acids have profound metabolic effects upstream to initiation of protein synthesis in cultured isolated skeletal muscle cells, as observed in animal and human skeletal muscle tissue \[[@B12],[@B59]-[@B64]\], in part related to individual groups of amino acids \[[@B65]-[@B69]\], as also observed in human biopsy specimens \[[@B17],[@B61]\].
Conclusion
==========
In conclusion, previous and present studies confirm that skeletal muscle cells are sensitive to alterations in extracellular concentrations of amino acids for translation initiation of protein synthesis, usually indicated by polysome aggregation, increased incorporation of amino acids into cellular proteins and activation of translation initiation \[[@B12],[@B15],[@B19]\]. However, transcripts of myofibrillar proteins and amino acid transporters showed unexpected complex time course changes in response to various conditions of refeeding and should therefore be used only in combination with other indicators of muscle protein synthesis. Thus, tissue levels of actin and myosin transcripts are not suitable as in vivo markers for protein accretion in skeletal muscles in response to feeding.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
BI conducted all experiments and statistical analyses. KL conceived of the study. Both authors participated in design of the study, drafted the manuscript and approved the final version.
Acknowledgements
================
We would like to thank Ph.D. Christina Lönnroth and Marianne Andersson for advice and technical help in performing the microarray analyses.
Supported in parts by grants from the Swedish Cancer Society (2014), the Swedish Research Council (08712), Assar Gabrielsson Foundation (AB Volvo), Jubileumskliniken Foundation, IngaBritt & Arne Lundberg Research Foundation, Swedish and Gothenburg Medical Societies and the Medical Faculty, University of Gothenburg, Sahlgrenska University Hospital Foundation, Swedish Nutrition Foundation.
|
{
"pile_set_name": "PubMed Central"
}
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Chlorophylls are produced from a branched pathway located within plastids that also produces heme, siroheme, and phytochromobilin. In photosynthetic organisms, the universal tetrapyrrole precursor 5-aminolevulinic acid (ALA)[^3^](#FN4){ref-type="fn"} is derived from glutamyl-tRNA and subsequently converted into protoporphyrinogen IX in the chloroplast stroma. Protoporphyrinogen IX is converted to protoporphyrin IX (PPIX) and then ultimately to chlorophylls on plastid membranes. Almost all the genes encoding chlorophyll biosynthetic enzymes have been identified. Transcriptional control provides coarse regulation of this pathway, and the regulation of enzyme activities provides fine regulation ([@B1], [@B2]).
Arabidopsis GUN4 (hereafter referred to as GUN4) was identified from a screen for plastid-to-nucleus signaling mutants ([@B3][@B4]--[@B5]). GUN4 is a major positive regulator of chlorophyll biosynthesis but is not absolutely required for the accumulation of chlorophyll in Arabidopsis ([@B5]). In *Synechocystis*, one of the GUN4 relatives, *sll0558* (hereafter referred to as SynGUN4), was subsequently shown also to be required for the accumulation of chlorophyll ([@B6], [@B7]). The 140-kDa subunit of Mg-chelatase copurifies with the 22-kDa GUN4 from solubilized Arabidopsis thylakoid membranes ([@B5]); similar results were subsequently reported using *Synechocystis* ([@B7]). Mg-chelatase catalyzes the insertion of Mg^2+^ into PPIX, yielding Mg-protoporphyrin IX (Mg-PPIX). This reaction diverts PPIX from heme biosynthesis and commits this porphyrin to chlorophyll biosynthesis. Mg-chelatase requires three subunits *in vitro* and *in vivo*. These three subunits are conserved from prokaryotes to plants and are commonly referred to as BchH or ChlH, BchD or ChlD, and BchI or ChlI. In Arabidopsis, these subunits are 140, 79, and 40 kDa, respectively. ChlH is the porphyrin-binding subunit and is likely the Mg^2+^-binding subunit of Mg-chelatase. ChlI and ChlD are related to AAA-type ATPases and form two associating hexameric rings that interact with ChlH and drive the ATP-dependent metalation of PPIX ([@B8], [@B9]). SynGUN4 stimulates *Synechocystis* Mg-chelatase ([@B5], [@B10], [@B11]). Cyanobacterial relatives of GUN4 bind deuteroporphyrin IX (DPIX) and Mg-deuteroporphyrin IX (Mg-DPIX) ([@B5], [@B10], [@B11]), which are more water-soluble derivatives of PPIX and Mg-PPIX. Crystal structures of SynGUN4 and *Thermosynechococcus elongatus* GUN4 indicate a novel fold that resembles a "cupped hand" that binds DPIX and Mg-DPIX ([@B10], [@B11]). Preincubation experiments indicate that a SynGUN4-DPIX complex stimulates Mg-chelatase more potently than SynGUN4 ([@B5]). SynGUN4 was found to lower the *K~m~*^DPIX^ of *Synechocystis* Mg-chelatase ([@B10]) and to cause a striking increase in the apparent first-order rate constant for DPIX-Mg-chelatase interactions, an effect that is particularly striking at low Mg^2+^ concentrations ([@B11]). The Mg-DPIX binding activity of SynGUN4 was also found to be essential for stimulating Mg-chelatase ([@B10]).
GUN4 and Mg-chelatase subunits have been found in both soluble and membrane-containing fractions of purified chloroplasts ([@B5], [@B12][@B13][@B14]--[@B15]). In contrast, protoporphyrinogen IX oxidase (PO) and Mg-PPIX methyltransferase (Mg-PPIX MT), which function immediately upstream and downstream of Mg-chelatase in the chlorophyll biosynthetic pathway, are found only in the membrane-containing fractions and not in stromal fractions when purified chloroplasts are lysed and fractionated ([@B16][@B17][@B18][@B19]--[@B20]). PPIX and Mg-PPIX accumulate in chloroplast membranes rather than soluble fractions, which provides more evidence that these chlorophyll precursors are synthesized on chloroplast membranes ([@B21]). If GUN4 promotes chlorophyll biosynthesis by not only stimulating Mg-chelatase activity but also promoting the formation of enzyme complexes that channel porphyrins into chlorophyll biosynthesis, GUN4 would be expected to more stably associate with chloroplast membranes by interacting with chloroplast membrane lipids or chlorophyll biosynthetic enzymes after binding porphyrins. In the following, we provide experimental evidence supporting this model.
EXPERIMENTAL PROCEDURES
=======================
###
#### Construction of Plasmids and Strains
For *in vitro* transcription/translation experiments, the entire GUN4 open reading frame (ORF) was amplified from bacterial artificial chromosome clone T1G3 (Arabidopsis Biological Resource Center, Ohio State University, Columbus) using CGGGATCCTATCTTCCCCTGACGTGAC, AACTGCAGAAAGACATCAGAAGCTGTAATTTG, and *PfuTurbo*® DNA polymerase (Stratagene, La Jolla CA). The resulting PCR product was ligated into pCMX-PL1 ([@B22]) between BamHI and PstI. *In vitro* transcription and translation of the control protein translocon at the inner envelope 40 (Tic40), the small subunit of ribulose-bisphosphate carboxylase/oxygenase (SS), and a light-harvesting chlorophyll *a*/*b*-binding protein (LHCP) were as described previously ([@B23], [@B24]). A glutathione *S*-transferase (GST)-tagged GUN4 deletion mutant that lacks the predicted 69-residue transit peptide (GST-GUN4 Δ1--69) was used for the expression and purification of GUN4 from Escherichia coli, as described previously ([@B5]). Site-directed mutagenesis was performed on each of these plasmids using the QuickChange® XL site-directed mutagenesis kit (Stratagene) and oligonucleotides that were designed according to the manufacturer\'s recommendations ([supplemental Table 1](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). All mutations were confirmed by sequencing at the Research Technology Support Facility (Michigan State University, East Lansing, MI).
#### Isolation of Pea Chloroplasts
Intact chloroplasts were isolated from 6- to 8-day-old pea seedlings and purified over a Percoll gradient as described previously ([@B25]). Intact pea chloroplasts were reisolated and resuspended in import buffer (330 m[m]{.smallcaps} sorbitol, 50 m[m]{.smallcaps} HEPES-KOH, pH 8.0) at a chlorophyll concentration of 1 mg/ml. Protein import was performed as described previously ([@B25]).
#### In Vitro Translation of Precursor Protein
All precursor proteins used in this study were either radiolabeled with \[^35^S\]methionine or \[^3^H\]leucine and translated with the T[n]{.smallcaps}T® Coupled Reticulocyte Lysate System (Promega, Madison WI) according to the manufacturer\'s recommendations.
#### Import Assays
Large scale import assays contained 50 m[m]{.smallcaps} HEPES-KOH, pH 8.0, 330 m[m]{.smallcaps} sorbitol, 4 m[m]{.smallcaps} Mg-ATP, 100 μl of chloroplasts with a chlorophyll concentration of 1 mg/ml, and labeled precursor protein at a final volume of 300 μl. After a 30-min incubation at room temperature under white light provided by broad spectrum fluorescent tube lamps at 75 μmol m^−2^ s^−1^, the import assay was divided into two 150-μl aliquots. One aliquot was not further treated. For this aliquot, intact chloroplasts were directly recovered by centrifugation through a 40% Percoll cushion. The remaining aliquot was incubated with trypsin for 30 min on ice as described previously ([@B26]). After stopping the protease treatment with trypsin inhibitor as described previously ([@B26]), we again recovered chloroplasts by centrifugation through a 40% Percoll cushion. Recovered intact chloroplasts were resuspended in 200 μl of lysis buffer (25 m[m]{.smallcaps} HEPES-KOH, pH 8.0, 4 m[m]{.smallcaps} MgCl~2~), incubated on ice for 20 min, and then fractionated into a soluble and membrane-containing pellet fraction by centrifugation at 16,000 × *g* for 5 min. The pellet fraction contains the outer envelope, inner envelope, and thylakoid membranes. All fractions were analyzed using SDS-PAGE and subjected to fluorography. Fluorograms were exposed to x-ray film (Eastman Kodak Co.) for 1--7 days. Import assays were quantitated by scanning developed films with the VersaDoc 4000 MP Imaging System and Quantity One software, as recommended by the manufacturer (Bio-Rad).
#### ALA and Porphyrin Feeding
Prior to import, intact chloroplasts were incubated in import buffer (330 m[m]{.smallcaps} sorbitol, 50 m[m]{.smallcaps} HEPES-KOH, pH 8.0) that contained or lacked 10 m[m]{.smallcaps} ALA (Sigma) for 15 min at 26 °C in the dark, unless indicated otherwise. PPIX, Mg-PPIX, uroporphyrin III, coproporphyrin III, hemin, and pheophorbide *a* were all purchased from Frontier Scientific (Logan, UT). These porphyrins were first dissolved in DMSO, and their concentrations were determined spectrophotometrically as described previously ([@B27][@B28][@B29]--[@B30]). These stock solutions were diluted with import buffer, giving final porphyrin concentrations of 20 μ[m]{.smallcaps} and final DMSO concentrations of 1--2%. Intact chloroplasts were incubated in these solutions exactly as described for ALA.
#### Fractionation of Chloroplasts into Stroma, Thylakoid, and Envelope Fractions
Fractionation of chloroplasts was performed as described previously ([@B31]), with modifications. First, large scale import assays were performed with (+) or without (−) an ALA pretreatment as described above. After import, intact chloroplasts were recovered by centrifugation through a 40% Percoll cushion. The intact chloroplasts were then resuspended in 0.6 [m]{.smallcaps} sucrose containing 25 m[m]{.smallcaps} HEPES-KOH, pH 8.0, 2 m[m]{.smallcaps} MgCl~2~, 8 m[m]{.smallcaps} EDTA. The suspension was placed on ice for 20 min and then placed at −20 °C overnight. Subsequently, the suspension was thawed at room temperature, gently mixed, and then diluted with 2 volumes of dilution buffer (25 m[m]{.smallcaps} HEPES-KOH, pH 8.0, 2 m[m]{.smallcaps} MgCl~2~, 8 m[m]{.smallcaps} EDTA). This suspension was then centrifuged at 1,500 × *g* for 5 min. The resulting pellet predominantly contained the thylakoid fraction and was diluted 2-fold with 2× SDS-PAGE loading buffer ([@B32]). The remaining supernatant was then centrifuged at 100,000 × *g* for 1 h. The resulting pellet fraction predominantly contained envelopes and was diluted 2-fold in 2× SDS-PAGE loading buffer. Cold acetone was added to the supernatant fraction to a final concentration of 80%, incubated on ice for 30 min, and then centrifuged at 15,000 × *g* for 5 min. The precipitated soluble protein fraction was resuspended in 2× SDS-PAGE loading buffer. All fractions were analyzed by SDS-PAGE.
#### Analysis of Porphyrins in Purified Chloroplasts
PPIX and Mg-PPIX levels in purified chloroplasts were quantitated following ALA feeding and mock protein import. 0.1-ml aliquots of chloroplasts were collected by centrifugation at 1,900 × *g* for 5 min at 4 °C through a 40% Percoll cushion. Recovered chloroplasts were lysed by resuspension in 700 μl of acetone, 0.1 [m]{.smallcaps} NH~4~OH (9:1, v/v). These lysates were clarified by centrifugation at 16,000 × *g* for 10 min at 4 °C. Chlorophyll was removed from the resulting supernatants by hexane extraction as described previously ([@B28]). We quantitated the amount of PPIX and Mg-PPIX in these hexane-extracted supernatants using fluorescence spectroscopy with a QuantaMaster^TM^ spectrofluorometer (Photon Technology International, Inc., London Ontario) as described previously ([@B28]). PPIX and Mg-PPIX, purchased from Frontier Scientific, were used to construct standard curves.
#### Quantitative Analysis of Porphyrin Binding
GUN4 Δ1--69 and versions of GUN4 Δ1--69 that contain amino acid substitutions were expressed and purified from E. coli as described previously ([@B5]). Binding constants were measured by quantitating the quenching of tryptophan fluorescence in GUN4 Δ1--69 by bound porphyrins essentially as described for the cyanobacterial relatives of GUN4 ([@B5], [@B10], [@B11]). Binding reactions were in 20 m[m]{.smallcaps} MOPS-KOH, pH 7.9, 1 m[m]{.smallcaps} DTT, 300 m[m]{.smallcaps} glycerol and contained 200 n[m]{.smallcaps} GUN4 Δ1--69 or GUN4 Δ1--69 with the indicated single amino acid changes and variable concentrations of DPIX and Mg-DPIX (Frontier Scientific). We determined binding constants for PPIX, Mg-PPIX, and Mg-PPIX ME, uroporphyrin III, coproporphyrin III, hemin, and pheophorbide *a* using the same conditions except that binding reactions also contained 1% DMSO. Stock solutions of DPIX and Mg-DPIX were prepared as described previously ([@B33]). Stock solutions of all other porphyrins were prepared as described under "ALA and Porphyrin Feeding." We calculated binding constants using DYNAFIT ([@B34]) as described previously ([@B33]). The data fit best with a model that predicts a single binding site.
#### Mg-chelatase Assays
Chloroplasts were purified from pea and subjected to hypotonic lysis as described above, except that lysis buffer also contained 1 m[m]{.smallcaps} DTT and 2 m[m]{.smallcaps} Pefabloc (Sigma). Supernatants were flash-frozen in liquid nitrogen and stored at −80 °C. We assayed pellet fractions for Mg-chelatase activity immediately by resuspending them in a Mg-chelatase assay buffer (50 m[m]{.smallcaps} Tricine-KOH, pH 7.8, 1 m[m]{.smallcaps} EDTA, 9 m[m]{.smallcaps} MgCl~2~, 4 m[m]{.smallcaps} MgATP, 1 m[m]{.smallcaps} DTT, 0.25% bovine serum albumin, 5% glycerol, 60 m[m]{.smallcaps} phosphocreatine, 4 units/ml creatine phosphokinase) and incubating the resuspended pellets for 30 min at 30 °C as recommended previously ([@B14], [@B35]). PPIX dissolved in DMSO was added to particular reactions as indicated in the text. The final concentrations of PPIX and DMSO were 1.5 μ[m]{.smallcaps} and 2%, respectively, as recommended previously ([@B36]). Aliquots of 8 μl were removed at 5-min intervals during a 30-min incubation, diluted into 200 μl of acetone, 0.1 [m]{.smallcaps} NH~4~OH (9:1, v/v), and vortexed to terminate the reaction. The terminated reactions were centrifuged for 10 min at 16,000 × *g* at 4 °C. Mg-PPIX in the resulting supernatants was quantitated as described under "Quantitative Analysis of Porphyrin Binding." We subtracted the amount of Mg-PPIX in the membranes before the reactions were initiated from the amount of Mg-PPIX at the end of each time point. Three replicates were analyzed for each time point. Mg-PPIX accumulated linearly for the entire 30-min assay. To assay supernatants for Mg-chelatase activity, supernatants were rapidly thawed, clarified at 16,000 × *g* at 4 °C for 10 min, and then concentrated nearly 5-fold using an Amicon ultracentrifugal filter device with a nominal molecular weight limit of 10,000 (Millipore, Billerica, MA). The concentrated supernatants were diluted into a concentrated Mg-chelatase assay buffer yielding the same assay conditions described for pellets. Reactions were initiated by adding 1.5 μ[m]{.smallcaps} PPIX.
#### Polyclonal Anti-ChlH, Anti-ChlI, and Anti-ChlD Antibody Development
Poly(A)^+^ mRNA was isolated from *Arabidopsis thaliana* (Columbia-0 ecotype) using the Absolutely mRNA kit (Stratagene). We prepared first-strand cDNA using Superscript II (Invitrogen). A cDNA encoding a 62-kDa fragment of ChlH that lacks the first 823 amino acid residues (ChlH Δ1--823) was amplified from this first-strand cDNA as described for GUN4 Δ1--69, except that CCGGAATTCGCTGTGGCCACACTGGTCAAC and TCGCGTCGACTTATCGATCGATCCCTTCGATCTTGTC were used. To express ChlH Δ1--823 as a His-tagged protein in E. coli, this PCR product was ligated into pHIS8-3 ([@B37]) between EcoRI and SalI. The resulting plasmid was sequenced at the Research Technology Support Facility (Michigan State University) to confirm that no mutations were introduced during PCR. The His-tagged ChlH Δ1--823 protein was expressed from the resulting plasmid in the E. coli strain BL21-CodonPlus® (DE3)-RIL (Stratagene) at 18 °C in Terrific Broth ([@B32]). We induced expression by adding 1 m[m]{.smallcaps} isopropyl β-[d]{.smallcaps}-1-thiogalactopyranoside (Sigma) when the *A*~600~ was 0.8. All subsequent steps were performed at 4 °C, unless indicated otherwise. Cells were harvested by centrifugation at 6,000 × *g* for 10 min and resuspended in 20 ml of buffer A (50 m[m]{.smallcaps} Tris acetate, pH 7.9, 500 m[m]{.smallcaps} potassium acetate, 20 m[m]{.smallcaps} imidazole, 20 m[m]{.smallcaps} β-mercaptoethanol, 20% glycerol, 1% Triton X-100) per g of bacterial pellet. Cells were lysed by sonication. The resulting lysate was clarified by centrifugation at 10,000 × *g* for 20 min. The supernatant was batch-bound to Ni-NTA-agarose (Qiagen, Valencia, CA) equilibrated in buffer A. Bound proteins were batch-washed twice with buffer A and twice with buffer A lacking Triton X-100. Ni-NTA-agarose was poured into an Econo-Pac column (Bio-Rad), and proteins were step-eluted using buffer B (20 m[m]{.smallcaps} Tris acetate, pH 7.9, 500 m[m]{.smallcaps} potassium acetate, 250 m[m]{.smallcaps} imidazole, 20 m[m]{.smallcaps} β-mercaptoethanol, 20% glycerol). Eluted proteins were dialyzed against buffer C (20 m[m]{.smallcaps} Tris acetate, pH 7.9, 150 m[m]{.smallcaps} potassium acetate, 2.5 m[m]{.smallcaps} CaCl~2~, 20 m[m]{.smallcaps} β-mercaptoethanol, 20% glycerol), digested with thrombin (Sigma) at room temperature, and applied to the aforementioned Ni-NTA-agarose column equilibrated in buffer A. Proteins in the flow-through fraction were dialyzed against buffer D (20 m[m]{.smallcaps} Tris acetate, pH 7.9, 100 m[m]{.smallcaps} potassium acetate, 1 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} DTT, 20% glycerol), applied to a HiPrep^TM^ 16/10 Q FF column (GE Healthcare) that was equilibrated in buffer D at a flow rate of 1.0 ml/min, and eluted with a 200-ml linear gradient to buffer E (20 m[m]{.smallcaps} Tris acetate, pH 7.9, 1000 m[m]{.smallcaps} potassium acetate, 1 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} DTT, 20% glycerol) also at a flow rate of 1.0 ml/min. Fractions of 2.5 ml containing ChlH Δ1--823 were pooled, concentrated using an Amicon Ultra-15 centrifugal filter unit with a nominal molecular weight limit of 30,000 (Millipore), dialyzed against storage buffer (50 m[m]{.smallcaps} Tricine-KOH, pH 7.9, 1 m[m]{.smallcaps} DTT, 50% glycerol), flash-frozen with liquid N~2~, and stored in small aliquots at −80 °C. For polyclonal antibody development, purified ChlH Δ1--823 was dialyzed extensively against phosphate-buffered saline, pH 7.4 ([@B32]), and used to develop anti-ChlH Δ1--823 polyclonal antisera in New Zealand White rabbits at Strategic Diagnostics, Inc. (Newark DE). IgGs were purified from these antisera using Affi-Gel protein A (Bio-Rad) as recommended by Harlow and Lane ([@B38]). Anti-ChlH Δ1--823 antibodies were affinity-purified from total IgGs on ChlH Δ1--823 columns that were constructed by linking purified ChlH Δ1--823 to Affi-Gel 15 (Bio-Rad) at ∼15 mg/ml. Antibodies were eluted from the ChlH Δ1--823 columns in buffer F (100 m[m]{.smallcaps} glycine-HCl, pH 2.5, 50% ethylene glycol) and immediately mixed with 1:10 volume of 1 [m]{.smallcaps} Tris-HCl, pH 8.0, as recommended by Harlow and Lane ([@B38]). Protein-containing fractions were pooled, dialyzed against phosphate-buffered saline, concentrated using Amicon Ultra-15 centrifugal filter units as described above, flash-frozen with liquid N~2~, and stored at −80 °C in small aliquots.
For anti-ChlI antibody development, ChlI was expressed and purified as described for ChlH Δ1--823, except that a cDNA encoding a ChlI ORF that lacks the predicted transit peptide (ChlI Δ1--60) was amplified using CCGGAATTCGCTGTGGCCACACTGGTCAAC and TCGCGTCGACTTATCGATCGATCCCTTCGATCTTGTC. ChlI Δ1--60 antibody development and affinity purification were as described for ChlH Δ1--823, except that ChlI Δ1--60, rather than ChlH Δ1--823, was linked to Affi-Gel 15.
For anti-ChlD antibody development, ChlD was expressed and purified as described for ChlH Δ1--823, except that a cDNA encoding a ChlD ORF that lacks the first 516 residues (ChlD Δ1--516) was amplified using GCGGGATCCACCCTTAGAGCAGCTGCACCATAC and TCGCGTCGACTCAAGAATTCTTCAGATCAGATAGTGCATCC and ligated into pHIS8-3 using BamHI and SalI. Another difference was that after elution from Ni-NTA-agarose and thrombin digestion, ChlD Δ1--516 was further purified by fractionating on a HiLoad^TM^ 26/60 Superdex^TM^ 200 prep-grade column equilibrated in buffer G (Tris-HCl, pH 7.9, 500 m[m]{.smallcaps} NaCl, 1 m[m]{.smallcaps} EDTA, 1 m[m]{.smallcaps} DTT, 10% glycerol) at 2 ml/min and at 4 °C. Anti-ChlD Δ1--516 antibodies were developed and purified as for anti-ChlH Δ1--823 and anti-ChlI Δ1--60 except that affinity purification was performed using ChlD Δ1--516 linked to Affi-Gel 10 rather than Affi-Gel 15.
All immunoblotting was done as described previously ([@B5]) using SuperSignal® West Dura extended duration substrate (Pierce). We quantified immunoreactive bands with the VersaDoc 4000 MP and Quantity One software (Bio-Rad).
RESULTS
=======
###
#### In Vitro Import of GUN4 into Pea Chloroplasts
To test whether the porphyrin binding activity of GUN4 affects interactions between GUN4 and chloroplast membranes, we imported GUN4 into purified pea chloroplasts *in vitro*. Because chlorophyll biosynthesis is well conserved among plant species ([@B2], [@B39]), we expected that GUN4 would interact similarly with proteins associated with chloroplast membranes such as ChlH from pea and Arabidopsis. The full-length GUN4 precursor containing the transit peptide was produced by *in vitro* translation. During SDS-PAGE, this translation product migrated like a 30-kDa protein ([Fig. 1](#F1){ref-type="fig"}*A*), which was expected, based on the mass calculated from the derived amino acid sequence of GUN4 containing the predicted transit peptide ([@B5]). We tested whether GUN4 could be imported into pea chloroplasts as judged by the following: (i) a mobility shift that is consistent with the removal of the predicted 69-residue transit peptide ([@B5]), and (ii) resistance to a trypsin protease treatment, which cannot digest proteins that are transported across the inner envelope of pea chloroplasts ([@B26]). After import into the pea chloroplast, GUN4 migrates as a doublet in SDS gels, with the predominant band migrating like a 22-kDa protein, consistent with the removal of the predicted transit peptide ([Fig. 1](#F1){ref-type="fig"}*A*). A similar doublet was previously observed when whole cell and various chloroplast extracts from Arabidopsis were analyzed by immunoblotting with affinity-purified anti-GUN4 antibodies ([@B5]). After GUN4 was imported, chloroplasts were digested with trypsin. Intact chloroplasts were then recovered using Percoll gradients and subjected to hypotonic lysis, and the soluble and membrane-containing fractions of the chloroplast were separated by centrifugation. GUN4 was observed in both the soluble and the membrane-containing pellet fractions ([Fig. 1](#F1){ref-type="fig"}), which indicates that GUN4 was imported into these chloroplasts and not digested by trypsin. Two control proteins, Tic40 and SS, accumulated in membrane and soluble fractions, respectively ([Fig. 1](#F1){ref-type="fig"}, *B* and *C*), as has been demonstrated previously ([@B23]).
{#F1}
#### Subchloroplastic Distribution of GUN4 in ALA-fed Chloroplasts
To test whether porphyrin binding might affect the interactions between GUN4 and chloroplast membranes, we imported GUN4 into pea chloroplasts that were fed with ALA prior to initiating protein import. ALA feeding was previously reported to induce the levels of PPIX and Mg-PPIX in whole plants ([@B40], [@B41]) and to increase the levels of heme efflux from purified chloroplasts ([@B42]). Consistent with these previous reports, we found that PPIX and Mg-PPIX levels increased 20--30-fold when purified chloroplasts were fed ALA under these conditions ([Fig. 2](#F2){ref-type="fig"}*A*) and that these porphyrins accumulated in the membrane-containing pellet fraction ([Fig. 2](#F2){ref-type="fig"}*B*). We found that half of GUN4 associated with the membrane fraction in unfed chloroplasts, and the amount of GUN4 in the membrane fraction increased by 50% in ALA-fed chloroplasts ([Fig. 2](#F2){ref-type="fig"}, *C* and *D*). In contrast, the distribution of Tic40 and SS did not change after ALA feeding ([supplemental Fig. 1, *A* and *B*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). Additionally, ALA feeding did not appear to change the total protein profile of the soluble and pellet fractions ([supplemental Fig. 1*C*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). ALA feeding did not affect the nature of GUN4-chloroplast membrane interactions as judged by extracting chloroplast membranes with either Na~2~CO~3~, pH 11, or Nonidet P-40 ([supplemental Fig. 2, *A* and *B*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). These data are consistent with the following: (i) elevated porphyrin levels causing GUN4 to accumulate in the membrane-containing pellet fraction after GUN4 has been imported into the chloroplast, but also with (ii) the distinct targeting of GUN4 to the stroma and to the chloroplast membranes and ALA feeding inhibiting stromal targeting. To distinguish between these possibilities, we fed ALA to chloroplasts following the import of GUN4. We found that ALA feeding subsequent to the import of GUN4 leads to increased levels of PPIX and Mg-PPIX and causes GUN4 to redistribute to the membrane-containing pellet fraction ([supplemental Fig. 3, *A--C*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). Based on these data, we conclude that inducing a rise in PPIX and Mg-PPIX levels by ALA feeding causes GUN4 to accumulate in the pellet fraction rather than blocking the import of GUN4 into the stroma.
![**Subchloroplastic distribution of PPIX, Mg-PPIX, and GUN4 after ALA feeding.** *A*, quantitation of PPIX and Mg-PPIX levels in ALA-fed chloroplasts. PPIX and Mg-PPIX levels were quantitated in intact chloroplasts that had been treated with 0, 1.0, or 10 m[m]{.smallcaps} ALA. *n* = 3. *Error bars* represent standard error. *B*, distribution of PPIX and Mg-PPIX in fractionated ALA-fed chloroplasts. Chloroplasts were fed with 10 m[m]{.smallcaps} ALA as in *A*. After ALA feeding, chloroplasts were lysed and separated in soluble (*S*) and membrane-containing pellet (*P*) fractions. PPIX and Mg-PPIX levels were determined in each fraction. *n* = 3. *C*, distribution of GUN4 in fractionated chloroplasts after ALA feeding. Chloroplasts were fed with ALA as described in *A*. Import and post-import analysis of GUN4 was performed as described in [Fig. 1](#F1){ref-type="fig"}*A*. Masses of protein standards are indicated at the *left* in kDa. *D*, quantitation of GUN4 in soluble and membrane fractions after ALA feeding. The amounts of radiolabeled GUN4 in soluble (*S*) and membrane-containing pellet (*P*) fractions were quantitated in independent experiments that were performed as in *C*, using different preparations of chloroplasts. The amount of \[^3^H\]GUN4 found either in the soluble (*S*) or pellet (*P*) fraction is presented as a percentage of total imported \[^3^H\]GUN4. Column numbers correspond to lane numbers in *B. n* = 3. *Error bars* are as in *A*.](zbc0410988620002){#F2}
GUN4 was previously detected in stroma, envelope, and thylakoid fractions derived from purified Arabidopsis chloroplasts ([@B5]). To test whether ALA feeding preferentially causes the accumulation of GUN4 in the chloroplast envelope or thylakoid membranes, we imported GUN4 into chloroplasts that were either fed or not fed ALA and then compared the levels of GUN4 in the stromal, envelope, and thylakoid fractions. We found that ALA feeding caused GUN4 levels to increase in the envelope and thylakoid membranes ([Fig. 3](#F3){ref-type="fig"}, *A* and *B*). In this experiment, Tic40, LHCP, and SS accumulated in the envelope ([Fig. 3](#F3){ref-type="fig"}*C*), thylakoids ([Fig. 3](#F3){ref-type="fig"}*D*), and stroma ([Fig. 3](#F3){ref-type="fig"}*E*), respectively, as reported previously ([@B23]). The levels and distributions of Tic40, LHCP, and SS were not different from those previously reported after ALA feeding ([Fig. 3](#F3){ref-type="fig"}, *C--E*).
![**Distribution of GUN4 in the chloroplast envelope, thylakoid, and stroma fractions after ALA feeding.** Chloroplasts were either pretreated without (*A*) or with ALA (*B*) prior to the import of \[^3^H\]GUN4. Control import assays with ALA-fed chloroplasts were likewise performed with ^35^S-prTic40 (*C*), ^3^H-prLHCP (*D*), and ^35^S-prSS (*E*). After import, chloroplasts were lysed and fractionated into soluble (*S*) and membrane-containing pellet fractions (*P*), or chloroplasts were separated into envelope (*Env*), stromal (*Str*), and thylakoid (*Thy*) fractions. All fractions were analyzed by SDS-PAGE as an equal load, using fluorography.](zbc0410988620003){#F3}
To test whether the tendency of ALA to cause accumulation of GUN4 in the membrane-containing fractions was unique to the newly imported radiolabeled GUN4 or whether a similar effect might be observed with the endogenous pea GUN4, we monitored the distribution of pea GUN4 in the soluble and membrane fractions of purified pea chloroplasts after ALA feeding. We detected an immunoreactive band with essentially the same mobility as GUN4 during SDS-PAGE and found a 22% increase in the membrane-containing fraction and the same fold decrease in the soluble fraction after ALA feeding that caused PPIX and Mg-PPIX levels to increase ([Fig. 4](#F4){ref-type="fig"}, *A--C*).
{ref-type="fig"}*A* after ALA feeding as described in [Fig. 3](#F3){ref-type="fig"}, *A* and *B. n* = 5. *Error bars* are as in *B*.](zbc0410988620004){#F4}
#### Quantitation of Porphyrin Binding by GUN4
The above findings are consistent with either porphyrin binding causing GUN4 to accumulate in the membrane-containing pellet fraction or ALA feeding somehow promoting interactions between GUN4 and chloroplast membranes by some other mechanism. To distinguish between these possibilities, we took advantage of a previous structure-function analysis of SynGUN4 ([@B10]). Based on this previous work, we made 11 single amino acid changes in GUN4 using site-directed mutagenesis ([supplemental Table 2](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). Homologous amino acid substitutions in SynGUN4 cause general defects in porphyrin binding or specific defects in binding either DPIX or Mg-DPIX ([@B10]). We also introduced the L88F substitution from the *gun4-1* missense allele. This amino acid substitution causes the GUN4 protein to accumulate at much lower levels *in vivo* compared with the wild type. A Phe substitution at the homologous Leu residue in *T. elongatus* GUN4 and SynGUN4 causes a 6--15-fold increase in the affinities for DPIX and Mg-DPIX without affecting folding in the case of *T. elongatus* GUN4 ([@B11]). We expressed these site-directed mutants as GST fusion proteins without the predicted 69-residue transit peptide, as described previously ([@B5]). Six of these amino acid substitutions, including the L88F, caused GST-GUN4 Δ1--69 to accumulate in the insoluble fraction ([supplemental Table 2](http://www.jbc.org/cgi/content/full/M109.025205/DC1)) and were not analyzed further. The remaining seven amino acid substitutions did not affect the solubility of GST-GUN4 Δ1--69 in E. coli ([supplemental Table 2](http://www.jbc.org/cgi/content/full/M109.025205/DC1)) and were purified.
We determined the *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^ for GUN4 ([Table 1](#T1){ref-type="table"} and [supplemental Fig. 4](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). During the course of these studies, we observed that including 1% DMSO in binding assays does not significantly affect the *K~d~*^DPIX^ or the *K~d~*^Mg-DPIX^ and that including 1% DMSO in binding assays solubilizes PPIX and Mg-PPIX sufficiently for us to perform binding assays with these natural ligands, which has not been reported for GUN4 from any species. We determined that the *K~d~*^PPIX^ was almost 2-fold higher than *K~d~*^DPIX^ and that the *K~d~*^Mg-PPIX^ was 1.5-fold lower than *K~d~*^Mg-DPIX^ ([Table 1](#T1){ref-type="table"}; [supplemental Fig. 5](http://www.jbc.org/cgi/content/full/M109.025205/DC1)).
######
**Quantitation of porphyrin binding by GUN4**
Binding reactions were performed with (+) or without (−) 1% DMSO.
Porphyrin 1% DMSO *K~d~*
-------------------- --------- --------------------
μ[*m*]{.smallcaps}
DPIX − 6.4 ± 0.21
DPIX \+ 6.0 ± 0.39
Mg-DPIX − 2.7 ± 0.29
Mg-DPIX \+ 2.2 ± 0.30
PPIX \+ 11 ± 0.50
Mg-PPIX \+ 1.6 ± 0.17
Mg-PPIX ME \+ 4.0 ± 0.20
Hemin \+ 8.1 ± 0.75
Uroporphyrin III \+ 10 ± 0.57
Coproporphyrin III \+ 15 ± 0.82
Pheophorbide *a* \+ 3.8 ± 0.33
Based on previously published biochemical and genetic data, we suggest that the major function of GUN4 *in vivo* is to stimulate Mg-PPIX biosynthesis ([@B5], [@B10], [@B11]). Nonetheless, we cannot rule out that GUN4 might participate in other reactions. To begin exploring this possibility, we tested whether GUN4 might bind other porphyrins. We performed binding assays with Mg-PPIX ME, which is the next chlorophyll precursor downstream of Mg-PPIX in the chlorophyll biosynthetic pathway. In this preparation of Mg-PPIX ME, either carboxyl group is methylated in a roughly 1:1 ratio. In contrast, only the carboxyl group associated with ring C is methylated in nature ([@B2]). We found that the *K~d~*^Mg-PPIX\ ME^ was more than 2-fold higher than *K~d~*^Mg-PPIX^ ([Table 1](#T1){ref-type="table"}; [supplemental Fig. 5](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). We found that GUN4 also binds uroporphyrin III, coproporphyrin III, hemin, and pheophorbide *a* ([Table 1](#T1){ref-type="table"}; [supplemental Fig. 6](http://www.jbc.org/cgi/content/full/M109.025205/DC1)) and that the affinities of GUN4 for these porphyrins are intermediate between Mg-PPIX ME and PPIX.
The amino acid substitutions F120W, E194A, and Q214E in GUN4 did not affect *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^.[^4^](#FN5){ref-type="fn"} The homologous substitutions (*i.e.* F132W, D199A, and R217E) significantly reduced the affinity of SynGUN4 for porphyrins ([@B10]). Amino acid substitutions V123A, F191A, and R211A caused both *K~d~*^DPIX^ or *K~d~*^Mg-DPIX^ to increase in GUN4 ([Table 2](#T2){ref-type="table"}; [supplemental Fig. 7, *A--C*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)), although the degrees of the porphyrin-binding defects were not exactly as observed for the homologous residues (*i.e.* V135A, F196A, and R214A) in SynGUN4 ([@B10]). The solubilities of PPIX and Mg-PPIX were not sufficient for us to quantitate the affinities of F191A, V123A, and R211A for these natural ligands.^4^
######
**Quantitation of DPIX and Mg-DPIX binding by GUN4 containing the indicated amino acid substitutions**
Substitution *K*~*d*~^DPIX^ *K*~*d*~^Mg-DPIX^
-------------- -------------------- --------------------
μ[*m*]{.smallcaps} μ[*m*]{.smallcaps}
V123A 9.6 ± 0.44 8.0 ± 0.44
F191A 12 ± 0.77 7.7 ± 0.37
R211A 14 ± 0.83 14 ± 0.64
#### Subchloroplastic Distribution of GUN4 Proteins with Porphyrin-binding Defects
To test whether porphyrin-binding defects might affect interactions between GUN4 and chloroplast membranes, we imported F191A, V123A, and R211A into ALA-fed chloroplasts and fractionated these chloroplasts into soluble and membrane-containing pellet fractions. A smaller percentage of V123A associated with the membrane-containing pellet fraction compared with the wild-type GUN4, and barely detectable levels of F191A and R211A were found in the pellet fraction ([Fig. 5](#F5){ref-type="fig"}). Additionally, and in contrast to wild-type GUN4, ALA feeding did not affect the subchloroplastic distribution of F191A, V123A, or R211A ([Fig. 5](#F5){ref-type="fig"}).
{ref-type="fig"}*A*. The position of the GUN4 precursor containing the transit peptide (*prGUN4*), the major form of mature GUN4 generated by proteolytic removal of the transit peptide during import into the chloroplast (*mGUN4*), and a minor form of mature GUN4 (\*) are indicated. Representative fluorograms from three independent experiments are shown for GUN4 containing the amino acid sequence found in wild type and for GUN4 containing the amino acid substitutions V123A, F191A, and R211A. *S*, soluble; *P*, pellet; *TP*, ∼10% of the precursor added to an import assay.](zbc0410988620005){#F5}
#### Mg-chelatase Activity in Chloroplast Membranes of ALA-fed Chloroplasts
Because GUN4 binds ChlH and stimulates Mg-chelatase ([@B5], [@B10], [@B11]), the finding that boosting PPIX and Mg-PPIX levels in purified chloroplasts by ALA feeding causes GUN4 to accumulate in the membrane-containing pellet fraction suggests that ALA feeding might affect the Mg-chelatase activity that was previously reported to associate with pea chloroplast membranes ([@B35]). To test this idea, we assayed chloroplast membranes for Mg-chelatase after ALA feeding. Reactions were initiated by addition of PPIX dissolved in DMSO. When membranes derived from chloroplasts that were not fed ALA were provided or not provided PPIX, they could synthesize 4 or 3 pmol Mg-PPIX/20 min/mg protein, respectively ([Fig. 6](#F6){ref-type="fig"}), which is 10--16-fold less activity than previously reported for pea chloroplast membranes ([@B35]). These differences may be caused by the more dilute hypotonic lysis and the distinct hypotonic lysis buffer used here, compared with that of Walker and Weinstein ([@B35]). We observed up to a 43-fold increase in Mg-chelatase activity in membranes isolated from ALA-fed chloroplasts ([Fig. 6](#F6){ref-type="fig"}). The activity of membranes that were isolated from ALA-fed chloroplasts was reduced by more than 2-fold when exogenous PPIX was included in these assays ([Fig. 6](#F6){ref-type="fig"}). Additionally, we observed no significant difference in activity when membranes were assayed with the following: (i) 2% DMSO containing 1.5 μ[m]{.smallcaps} PPIX or (ii) 2% DMSO not containing PPIX. These data indicate that Mg-chelatase does not utilize exogenous PPIX efficiently and that 2% DMSO has an inhibitory effect that is similar to PPIX dissolved in DMSO. Based on these data, we suggest that PO loads at least a fraction of the Mg-chelatase that associates with chloroplast membranes with PPIX during ALA feeding and that this preformed PPIX-Mg-chelatase complex turns over during the subsequent Mg-chelatase assay.
{#F6}
#### Subchloroplastic Distribution of Mg-chelatase Subunits in ALA-fed Chloroplasts
The elevated levels of Mg-chelatase activity in ALA-fed chloroplasts might not result solely from preloading Mg-chelatase with PPIX during ALA feeding. Porphyrins or a GUN4-porphyrin complex might contribute to this elevated level of Mg-chelatase activity by promoting the redistribution of Mg-chelatase subunits from the soluble into the membrane-containing pellet fraction. For example, a GUN4-porphyrin complex might stabilize interactions between Mg-chelatase and chloroplast membrane lipids or between Mg-chelatase and other chlorophyll biosynthetic enzymes such as PO and Mg-PPIX MT. This type of regulation would be distinct from the stimulation of Mg-chelatase activity that was previously reported for GUN4 ([@B5], [@B10], [@B11]). To begin exploring these possibilities, we monitored the levels of ChlH in these chloroplast fractions using polyclonal antibodies raised against a truncated Arabidopsis ChlH that lacks the first 823 residues and contains all of the remaining 558 carboxyl-terminal residues (hereafter referred to as ChlH Δ1--823). We found that affinity-purified anti-ChlH Δ1--823 antibodies recognize a band that was extracted from chloroplasts purified from Arabidopsis and pea that migrates like a 150-kDa protein during SDS-PAGE ([supplemental Fig. 8, *A* and *B*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). The mass of Arabidopsis ChlH calculated from the derived amino acid sequence lacking the predicted transit peptide is 144 kDa. We also found that there is a striking increase in the level of this 150-kDa protein in the Arabidopsis mutant *cch* ([supplemental Fig. 8, *A* and *B*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)), which is a strong loss-of-function allele of *ChlH* ([@B4], [@B43]). We conclude that this band corresponds to ChlH. In four separate experiments, we observed that 40--70% of pea ChlH was in the soluble fraction of unfed chloroplasts and the remainder was in the pellet fraction. Although the distribution of ChlH in the soluble and pellet fractions was somewhat variable, we observed a statistically significant 15% increase of pea ChlH in pellet fractions after ALA feeding in each experiment ([Fig. 7](#F7){ref-type="fig"}).
{#F7}
Pea ChlI was previously reported to localize mostly within the soluble fraction when purified chloroplasts from pea were lysed and fractionated ([@B14]). If ALA feeding stabilizes not only the association of GUN4 with Mg-chelatase at the site of chlorophyll biosynthesis but also interactions between Mg-chelatase and chloroplast membranes, we would expect that ChlI levels in the pellet fraction would also increase during ALA feeding. To test this idea, we monitored the levels of ChlI in these chloroplast fractions using polyclonal antibodies raised against a truncated Arabidopsis ChlI lacking the predicted 60-residue transit peptide (hereafter referred to as ChlI Δ1--60). We found that affinity-purified anti-ChlI Δ1--60 antibodies recognize a 43-kDa protein in extracts prepared from wild-type Arabidopsis. The mass of Arabidopsis ChlI calculated from the derived amino acid sequence lacking the predicted transit peptide is 40 kDa. We found that this immunoreactive band is present at a lower concentration in the Arabidopsis mutant *cs* ([supplemental Fig. 9, *A--C*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)), which contains a loss-of-function allele for *ChlI* ([@B44]), and that this immunoreactive band was less than 2 kDa larger in *cs* compared with wild type ([supplemental Fig. 9*B*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). This decrease in mobility is consistent with the 0.8-kDa increase in the mass of the derived amino acid sequence reported for the *cs* allele ([@B44]). We conclude that our affinity-purified anti-ChlI antibodies specifically bind ChlI. These antibodies specifically recognize a single 44-kDa band in pea chloroplasts ([supplemental Fig. 9*D*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)), which we conclude is pea ChlI. Using these affinity-purified anti-ChlI Δ1--60 antibodies, we tested the distribution of pea ChlI in the same experiments in which we observed a 15% increase in pea ChlH in the pellet fraction after ALA feeding. We found pea ChlI predominantly in the supernatant, detectable pea ChlI in the pellet, and no effect of ALA feeding on the distribution of ChlI between soluble and pellet fractions ([Fig. 8](#F8){ref-type="fig"}*A*).
{#F8}
Pea ChlD was previously reported to localize in light membranes ([@B15]) that we would not expect in pellet fractions prepared using our experimental conditions. We monitored the levels of pea ChlD in these same chloroplast fractions using affinity-purified antibodies raised against a truncated Arabidopsis ChlD lacking the first 516 residues (hereafter referred to as ChlD Δ1--516). We found that affinity-purified anti-ChlD Δ1--516 antibodies recognize an ∼94-kDa protein in Arabidopsis whole-seedling extracts. The mass of Arabidopsis ChlD calculated from the derived amino acid sequence lacking the predicted transit peptide is 79 kDa. This band is not detectable in an Arabidopsis mutant that contains a tDNA insertion in an exon of *ChlD* ([supplemental Fig. 10, *A--C*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). We conclude that this band is Arabidopsis ChlD. These antibodies also recognize a 93-kDa band extracted from pea chloroplasts ([supplemental Fig. 10*D*](http://www.jbc.org/cgi/content/full/M109.025205/DC1)), which we conclude is pea ChlD. Using these affinity-purified anti-ChlD Δ1--516 antibodies, we tested the distribution of pea ChlD in the same four independent experiments in which we observed a 22% increase in pea GUN4, a 15% increase in pea ChlH in the pellet fraction, and no change in the distribution of pea ChlI after ALA feeding. Similar to our results with pea ChlI, we found pea ChlD predominantly in the supernatant fraction and only small quantities of ChlD in the pellet fraction, regardless of whether chloroplasts were fed ALA ([Fig. 8](#F8){ref-type="fig"}*B*).
Our data are consistent with porphyrins affecting the distribution of only GUN4 and ChlH within pea chloroplasts. Next, we tested whether the redistribution of pea GUN4 and pea ChlH from the supernatant to the pellet fractions during ALA feeding might depend on chloroplast membranes or whether these proteins might accumulate in the pellet fraction for some other reason such as a fraction of a GUN4-ChlH complex denaturing and becoming insoluble during ALA feeding. To distinguish between these possibilities, we lysed pea chloroplasts, depleted the membranes from these lysates by centrifugation, and performed Mg-chelatase assays on the resulting supernatants. We found that supernatants prepared from three independent chloroplast preparations contained from 13 to 41 units of Mg-chelatase activity ([Table 3](#T3){ref-type="table"}), which is similar to the activity previously reported for such supernatants ([@B35]). After performing Mg-chelatase assays with these membrane-depleted supernatants, we centrifuged these reactions using the same conditions that caused pea GUN4 and pea ChlH from lysed chloroplasts to accumulate in the membrane-containing pellet fraction. We found that in contrast to the results that we obtained with lysed chloroplasts, pea GUN4 and pea ChlH remained entirely within the supernatant fraction when Mg-chelatase reactions were performed with membrane-depleted supernatants ([Fig. 9](#F9){ref-type="fig"}).
######
**Quantitation of Mg-chelatase activity in supernatants prepared from lysed chloroplasts**
A unit of Mg-chelatase activity produces 1 pmol of Mg-PPIX/20 min/mg protein.
Preparation Mg-chelatase activity
------------- -----------------------
*units*
1 16
2 13
3 41
{#F9}
#### Subchloroplastic Distribution of GUN4 and Mg-chelatase Subunits in Chloroplasts Fed with Various Porphyrins
We expected that PPIX and Mg-PPIX would stabilize interactions between chloroplast membranes and the GUN4 and ChlH proteins because PPIX and Mg-PPIX are the only porphyrin substrate and product of Mg-chelatase and because ChlH and GUN4 are not known to catalyze other reactions. Nonetheless, our finding that GUN4 can bind a variety of porphyrins implies that GUN4 binding any of these ligands might cause a redistribution of GUN4 from the soluble to the membrane-containing pellet fraction. We expected that chloroplasts would take up PPIX and other porphyrins *in vitro* and that GUN4 might bind these porphyrins after they are taken up because purified chloroplasts were reported previously to synthesize Mg-PPIX from exogenous PPIX ([@B36], [@B45], [@B46]). To test this idea, we fed pea chloroplasts with various porphyrins that GUN4 binds and monitored the distribution of GUN4 in soluble and membrane-containing fractions by immunoblotting. We found that similar to feeding chloroplasts ALA, feeding chloroplasts PPIX, Mg-PPIX, uroporphyrin III, coproporphyrin III, hemin, or pheophorbide *a* caused GUN4 to accumulate in the pellet fraction ([Fig. 10](#F10){ref-type="fig"}).
![**Subchloroplastic distribution of GUN4 after feeding with ALA or various porphyrins.** Pea chloroplasts were incubated in import buffer lacking ALA (−*ALA*), containing ALA (+*ALA*), containing 2% DMSO (*DMSO*), or 20 μ[m]{.smallcaps} of the indicated porphyrin and 1--2% DMSO. Next, the chloroplasts were subjected to a mock import assay that lacked a radiolabeled precursor. Chloroplasts were lysed and fractionated. Soluble (*S*) and membrane-containing pellet (*P*) fractions were analyzed by immunoblotting with affinity-purified anti-GUN4 antibodies. A representative immunoblot is shown (*top*). Immunoreactive bands were quantitated in fractions derived from at least two independent preparations of chloroplasts (*bottom*). *Error bars* represent S.E.](zbc0410988620010){#F10}
DISCUSSION
==========
GUN4 was previously localized to the thylakoid and envelope membranes but was found mostly in the soluble fraction of chloroplasts that were purified from fully expanded rosette leaves of Arabidopsis ([@B5]). Here we report a roughly 50:50 distribution, or that GUN4 predominantly associates with the membrane-containing fraction depending on whether GUN4 was imported or pea GUN4 was monitored in 6--8-day-old pea leaves. Differences in the affinities of GUN4 and pea GUN4 for chloroplast membranes or differences in the chlorophyll biosynthetic capacities of fully expanded and young rapidly growing leaves may account for these differences. Consistent with the relatively higher rate of chlorophyll biosynthesis in young leaves stabilizing interactions between GUN4 and chloroplast membranes, we found that feeding ALA to purified chloroplasts causes a striking increase in the biosynthesis of PPIX and Mg-PPIX and also causes both GUN4 and ChlH to accumulate in the membrane-containing fraction at the expense of the soluble fraction. Although we cannot rule out possibilities such as porphyrin binding causing a rapid redistribution of GUN4, ChlH, or a GUN4-ChlH complex from the chloroplast stroma to the chloroplast membranes, this possibility seems unlikely because of the high viscosity of the chloroplast stroma ([@B47]). The most parsimonious interpretation of our data is as follows: (i) most of GUN4 and ChlH associate with chloroplast membranes; (ii) a fraction of GUN4, ChlH, or a GUN4-ChlH complex dissociates from chloroplast membranes and accumulates in the soluble fraction during chloroplast lysis and fractionation; and (iii) the porphyrin-bound conformations of GUN4 and ChlH have higher affinities for either chloroplast membrane lipids and/or chlorophyll biosynthetic enzymes that more stably associate with chloroplast membranes such as PO and Mg-PPIX MT.
Although GUN4-porphyrin complexes were previously reported to stimulate Mg-chelatase activity ([@B5], [@B10], [@B11]), our findings suggest that GUN4 might regulate chlorophyll biosynthesis on another level. We propose that GUN4 or a GUN4-porphyrin complex helps to stabilize interactions between ChlH and chloroplast membranes (*i.e.* the site of chlorophyll biosynthesis), which was not previously reported for GUN4. Complexes of chlorophyll biosynthetic enzymes that include Mg-chelatase have been reported or suggested because Mg-chelatase and Mg-PPIX MT can convert PPIX to Mg-PPIX ME *in vitro* without appreciable accumulation of Mg-PPIX ([@B48]) and because ChlH stimulates Mg-PPIX MT ([@B49][@B50]--[@B51]). However, all Mg-PPIX does not appear to be efficiently channeled through a complex composed of Mg-chelatase and Mg-PPIX MT because Mg-PPIX accumulates in green Arabidopsis seedlings ([@B52], [@B53]). We propose that GUN4 binds at least a fraction of the Mg-PPIX that accumulates *in vivo* by itself or as part of a GUN4-ChlH complex. GUN4 may be necessary if Mg-chelatase-Mg-PPIX complexes are unstable or if a fraction of active ChlH does not associate with Mg-PPIX MT and might help prevent the photosensitizing chlorophyll precursors from causing photooxidative damage.
These findings may contribute to our understanding of the competition between Mg-chelatase and ferrochelatase for PPIX. By stabilizing complexes of enzymes that contain Mg-chelatase, GUN4 might help divert PPIX from heme biosynthesis and into chlorophyll biosynthesis. Both PO and ferrochelatase stably associate with chloroplast membranes ([@B16][@B17][@B18]--[@B19], [@B54]) and have been predicted to form a complex based on their crystal structures ([@B55]). Porphyrin-bound GUN4 might help Mg-chelatase compete with ferrochelatase for interactions with PO, thereby helping to divert PPIX from heme to chlorophyll biosynthesis. Alternatively, if Mg-chelatase and ferrochelatase utilize separate pools of PPIX, as has been suggested ([@B56]), we would expect that GUN4 would affect only chlorophyll biosynthesis.
Complexes of cooperating enzymes are expected to limit active site access of exogenously supplied substrates ([@B57], [@B58]). Consistent with this idea, we found that exogenously supplied PPIX did not stimulate Mg-chelatase activity in our membrane-containing pellet fraction but that PPIX derived from ALA feeding caused a striking increase in Mg-chelatase activity. Pea chloroplast membranes were previously reported to contain low levels of Mg-chelatase activity when assayed with exogenous PPIX. Reactions lacking exogenous PPIX were not previously reported ([@B35]). The striking increase in Mg-chelatase activity caused by ALA feeding likely results from the 22% increase in GUN4 and the 15% increase in ChlH in the chloroplast membranes, along with the preloading of ChlH or a GUN4-ChlH complex with PPIX. Our finding that, in contrast to GUN4 and ChlH, ALA feeding does not stabilize interactions between chloroplast membranes and either ChlI or ChlD suggests that these Mg-chelatase subunits do not have a higher affinity for the porphyrin-bound form of ChlH or at least that such a difference cannot be detected with the chloroplast lysis and fractionation assay described here. The low levels of ChlI and ChlD that we observed in the pellet fraction likely explain the low Mg-chelatase activity that we and Walker and Weinstein ([@B35]) observed in membrane-containing pellet fractions relative to reactions programmed with both soluble and membrane-containing fractions ([@B35]).
*K~d~*^DPIX^ and *K~d~*^Mg-DPIX^ have been reported for SynGUN4 and *T. elongatus* GUN4 ([@B5], [@B10], [@B11]), but only qualitative porphyrin-binding experiments were previously reported for GUN4 ([@B5]). We found similarities and differences in the porphyrin-binding constants of GUN4 and its cyanobacterial relatives. Like its cyanobacterial relatives ([@B5], [@B10], [@B11]), GUN4 has a higher affinity for Mg-DPIX than DPIX, but GUN4 has a 3--10-fold lower affinity for these porphyrins than its cyanobacterial relatives. *K~d~*^PPIX^ and *K~d~*^Mg-PPIX^ had not been reported previously for GUN4 or any cyanobacterial relatives of GUN4. We found that including 1% DMSO in binding assays did not significantly affect *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^ but promoted the solubility of PPIX and Mg-PPIX, thereby allowing us to determine *K~d~*^PPIX^ and *K~d~*^Mg-PPIX^, which resembled *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^, respectively. GUN4 has a slightly higher affinity for Mg-PPIX than for Mg-DPIX and has an almost 2-fold lower affinity for PPIX than for DPIX. The higher affinity of SynGUN4 for metalated porphyrins was previously attributed to these porphyrins assuming a planar conformation in contrast to unmetalated porphyrins, which assume a more puckered or ruffled conformation ([@B10]). Our findings indicate that this preference for metalated porphyrins is more striking for the natural ligands than for the deuteroporphyrins, and we suggest that the vinyl groups that distinguish PPIX from DPIX contribute to this binding selectivity. This finding may at least partially explain why *K~m~*^DPIX^ is lower than *K~m~*^PPIX^ in lysed pea chloroplasts ([@B14]).
SynGUN4 was previously shown to bind a variety of porphyrins with *K~d~* values that range from 0.26 to 11 μ[m]{.smallcaps} ([@B5], [@B10], [@B11]). Here we report that GUN4 can bind nine different porphyrins with *K~d~* values ranging from 1.6 to 15 μ[m]{.smallcaps}. Most of the porphyrins that we analyzed are found in plants or are oxidized versions of porphyrins and porphyrinogens found in plants. Although the only difference in Mg-PPIX and hemin is a chelated magnesium or ferric ion, we found that GUN4 binds Mg-PPIX with a 5-fold higher affinity than hemin. SynGUN4 was previously reported to bind cobalt(III) PPIX with an almost 2-fold higher affinity than hemin ([@B10]). Thus, like SynGUN4, GUN4 can distinguish between derivatives of PPIX that contain distinct metal ions. Modifications of the porphyrin ring were also reported to affect the affinities of SynGUN4 for porphyrins ([@B10]). Arg-214 of SynGUN4 is conserved as Arg-211 in GUN4. In SynGUN4, Arg-214 resides in the α6/α7 loop that lies loosely across the "palm" region of the cupped hand domain and makes a major contribution to porphyrin binding, presumably by interacting with one of the carboxyl moieties of DPIX and Mg-DPIX ([@B10]). Methylation of a carboxyl moiety in Mg-PPIX ME could lower the affinities of both GUN4 and SynGUN4 for this porphyrin. Indeed, we observed that *K~d~*^Mg-PPIX\ ME^ was 2.5-fold higher than *K~d~*^Mg-PPIX^. However, because either the carboxyl moiety of the commercially available Mg-PPIX ME used here is methylated rather than the carboxyl moiety attached only to ring C, as observed in nature, these data do not unambiguously establish that GUN4 binds Mg-PPIX ME with a lower affinity than Mg-PPIX. GUN4 binding uroporphyrin III, coproporphyrin III, hemin, and pheophorbide *a* indicates that GUN4 is similar to SynGUN4 in that GUN4 can bind a variety of porphyrins with diverse ring substituents ([@B10]). Moreover, we found that feeding uroporphyrin III, coproporphyrin III, hemin, or pheophorbide *a* to chloroplasts causes pea GUN4 to accumulate in the membrane-containing pellet fraction. These data lend further support to a model in which a porphyrin-bound conformation of GUN4 has an elevated affinity for chloroplast membranes. The finding that binding a variety of porphyrins promotes interactions between GUN4 and chloroplast membranes implies that GUN4 could be involved in other reactions besides the Mg-chelatase reaction. However, previous analyses of *gun4* mutants are consistent with GUN4 not having a major role in the metabolism of porphyrinogens and other porphyrins besides PPIX and Mg-PPIX. *gun4* mutants are chlorophyll-deficient ([@B4], [@B5], [@B59]); *gun4* nulls are albino under optimal growth conditions but viable when they are provided sucrose ([@B5]). Although these phenotypes are expected for mutants that cannot synthesize chlorophyll, we expect that mutants with severe defects in plastid heme metabolism would exhibit more severe or lethal phenotypes regardless of whether sucrose is provided. Additionally, *gun4* mutants were not reported to exhibit lesions ([@B4], [@B5], [@B59]) like those present in mutants with reduced levels of uroporphyrinogen III decarboxylase and coproporphyrinogen III oxidase ([@B60][@B61]--[@B62]). Moreover, although we found that GUN4 binds pheophorbide *a*, an early intermediate in chlorophyll catabolism, we observed that *gun4* mutants do not exhibit phenotypes like those with defects in the enzymes that degrade chlorophyll such as the stay-green phenotype ([@B63], [@B64], [@B65]) ([supplemental Fig. 11](http://www.jbc.org/cgi/content/full/M109.025205/DC1)). From these data we conclude that GUN4 does not appear to promote chlorophyll catabolism. In summary, although GUN4 can bind diverse porphyrins *in vitro* and binding any of these porphyrins causes GUN4 to more stably associate with chloroplast membranes, the simplest interpretation of these new data and previous analyses of *gun4* mutants is that the major function of GUN4 *in vivo* is to promote the biosynthesis of Mg-PPIX and to bind Mg-PPIX.
Several amino acid substitutions that do not affect the solubility of His-tagged SynGUN4 expressed in E. coli appear to cause GST-GUN4 Δ1--69 to accumulate as inclusion bodies. Species-specific differences in stability, or technical explanations such as the use of different tags, may explain these apparent differences in solubility. Among the seven amino acid substitutions that did not cause GST-GUN4 Δ1--69 to accumulate in inclusion bodies, only V123A, F191A, and R211A reduced the affinity of GUN4 for porphyrins. The finding that four amino acid substitutions inhibit porphyrin binding in SynGUN4 but not GUN4 is consistent with species specificity in particular binding determinants or in indirect effects on binding. Val-123 in GUN4 is homologous to Val-135 in SynGUN4. In SynGUN4, Val-135 contributes to a concave and hydrophobic surface referred to as the "greasy palm" of the cupped hand domain ([@B10]). Phe-191 in GUN4 is homologous to Phe-196 in SynGUN4. Like Arg-214 of SynGUN4, Phe-196 resides on the α6/α7 loop, which lies across the greasy palm ([@B10]). Because V123A, F191A, and R211A elevate *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^, we expected that these amino acid substitutions would also impair the porphyrin-mediated interactions between GUN4 and chloroplast membranes. Indeed, we found that much less V123A, F191A, and R211A associated with membranes. However, because we found that these amino acid substitutions impair rather than abolish binding, and because ALA feeding causes a striking increase in PPIX and Mg-PPIX, we expected that ALA feeding might stabilize interactions between these proteins and chloroplast membranes, thereby causing more V123A, F191A, and R211A to accumulate in the membrane-containing pellet fraction. However, we found that ALA feeding could not stabilize interactions between chloroplast membranes and V123A, F191A, or R211A. These data are consistent with pea GUN4 competing much more effectively with V123A, F191A, and R211A relative to wild-type GUN4. These data are also consistent with V123A, F191A, and R211A not only affecting porphyrin binding but also directly or indirectly affecting interactions between GUN4 and other molecules that might tether GUN4 to chloroplast membranes such as ChlH.
The L88F substitution derived from the *gun4-1* missense allele causes the GUN4 protein to accumulate at much lower levels than in wild-type Arabidopsis, as judged by immunoblotting ([@B5]). The homologous amino acid substitution in *T. elongatus* GUN4 does not cause misfolding and causes a striking decrease in the *K~d~*^DPIX^ and *K~d~*^Mg-DPIX^ for *T. elongatus* GUN4 and SynGUN4. Together, these data provide evidence that GUN4 might be degraded more rapidly when bound to porphyrins. However, the observation that the L88F substitution promotes insolubility of GST-GUN4 Δ1--69 expressed in E. coli reported here suggests that, in Arabidopsis, less GUN4 accumulates in *gun4-1* because of protein instability caused by misfolding, as suggested previously ([@B5]), rather than from protein instability caused by a striking increase in the affinities for porphyrins.
In summary, our major finding is that porphyrin binding helps stabilize interactions between GUN4 and possibly ChlH with chloroplast membranes. Based on these data, we suggest that porphyrins and/or GUN4-porphyrin complexes might stabilize interactions between ChlH and chloroplast membranes, thereby facilitating the channeling of porphyrins into chlorophyll biosynthesis. These findings support a model in which GUN4-porphyrin complexes promote chlorophyll biosynthesis not only by stimulating Mg-chelatase activity but also by affecting interactions between ChlH and chloroplast membranes.
Supplementary Material
======================
###### Supplemental Data
This work was supported in part by Department of Energy Grant DE-FG02-91ER20021 and National Science Foundation Grants IOB 0517841 (to R. M. L.) and DE-FG02-04ER15540 (to J. C.).
The on-line version of this article (available at <http://www.jbc.org>) contains [supplemental Tables 1 and 2 and Figs. 1--11](http://www.jbc.org/cgi/content/full/M109.025205/DC1).
N. D. Adhikari, unpublished data.
The abbreviations used are: ALA5-aminolevulinic acidDPIXdeuteroporphyrin IXLHCPlight-harvesting chlorophyll *a*/*b*-binding proteinGSTglutathione *S*-transferase, GUN4, GENOMES UNCOUPLED 4Mg-DPIXMg-deuteroporphyrin IXMg-PPIXMg-protoporphyrin IXMg-PPIX MTMg-PPIX methyl transferaseORFopen reading framePOprotoporphyrinogen IX oxidasePPIXprotoporphyrin IXSSthe small subunit of ribulose-bisphosphate carboxylase/oxygenaseTic40Translocon at the inner envelope 40Ni-NTAnickel-nitrilotriacetic acidDTTdithiothreitolMOPS4-morpholinepropanesulfonic acidTricine*N*-\[2-hydroxy-1,1-bis(hydroxymethyl)ethyl\]glycineMg-PPIX MEMg-protoporphyrin IX monomethyl ester.
We thank Ron Evans (Salk Institute for Biological Studies, La Jolla, CA) for providing pCMX-PL1. We thank Rob Last (Michigan State University) for providing seeds from Arabidopsis plants heterozygous for the Salk_150219 TDNA allele.
[^1]: Supported by Department of Energy Grant DE-FG02-91ER20021 (to Ken Keegstra).
|
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"pile_set_name": "PubMed Central"
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1. Introduction {#sec1}
===============
Growing teratoma syndrome (GTS) is thought to be the progression of immature to mature teratoma during or following chemotherapy. GTS requires three criteria: normalization of previously elevated tumor markers (AFP or *β*HCG), enlargement of the primary tumor, or finding of new tumor mass and only mature teratoma elements on pathologic examination. The incidence of GTS is 12% of ovarian germ cell tumors (GCT) occurring in young adults and adolescents \[[@B1]--[@B3]\]. GTS is most likely to occur if mature teratoma elements are found in the primary tumor, and there is no reduction in tumor size following chemotherapy or incomplete resection of the primary tumor \[[@B4]\].
2. Case {#sec2}
=======
A 12-year-old female patient presented from a referring institution in November 2015 for evaluation of 1-month history of an enlarging abdominal mass. The patient reported a 4.5 kg weight loss with increased lethargy. The physical examination was normal except for a large abdominal solid mass. Pelvic CT ([Figure 1](#fig1){ref-type="fig"}) revealed a large pelvic mass. Tumor markers were significant for alpha fetoprotein (AFP) 656 ng/mL (nml ≤ 7 ng/mL), CA-125 401 U/mL (nml \< 35 U/mL), and CEA 6.7 ng/mL (nml ≤ 3.5 ng/mL). Inhibin A/B and *β*-HCG levels were normal. She underwent an exploratory laparotomy, right salpingoophorectomy, omentectomy, peritoneal washings, diaphragmatic/peritoneal/pelvic biopsies, and periaortic lymph node biopsy. Intraoperative findings were significant for a distinct right ovarian mass with evidence of prior rupture. Ascites was noted and aspirated. There was peritoneal seeding along the pelvis and right pericolic gutter and along the right hemidiaphragm. Gross residual disease remained consisting mostly of the peritoneal seeding despite removal of all gross abdominal disease. The cytology examination of the peritoneal fluid demonstrated no malignant cells. Histologically, all the pathology specimens returned with high grade immature teratoma ([Figure 2](#fig2){ref-type="fig"}). She was diagnosed as grade 3, FIGO stage IIIC. Chromosome analysis returned trisomy 3 in four of her autosomal cells, consistent with the diagnosis of immature teratoma. Since there is currently no "standard of care" for pediatric grade immature teratoma FIGO stage IIIC, we elected to follow tumor markers closely. Following tumor markers, her AFP fell by approximately 50% every week almost normalizing over the course of three months from diagnosis (from 656 ng/mL to 9.78 ng/mL). In January 2016, repeat tumor marker labs demonstrated that AFP levels increased to 28.3 ng/mL and CA-125/*β*-HCG were within normal limits (18 units/mL CA-125, \<3 units/mL). Repeat CT of chest, abdomen, and pelvis revealed that, in the abdomen and pelvis, she now had enlarged peritoneal implants, as large as 3.5 × 1.5 cm, but no other masses, and, in the chest, she had a 1.3 × 2 × 0.3 cm left anterior pleural nodule, as well as bilateral diaphragmatic lesions, 2.5 × 2.1 × 1.8 cm on right and 2.1 × 3 × 1.5 cm on left. She subsequently underwent chemotherapy with bleomycin, etoposide, and cisplatin (BEP) between February and May 2016 for high risk malignant ovarian germ cell tumor. After 4 cycles, her AFP was 0.84 ng/mL.
After 4 cycles, she underwent interval radiologic evaluation in preparation for second look surgery. The CT scan of the chest, abdomen, and pelvis in May 2016 revealed a significant increase in the size of the thoracic masses, as well as the size and a number of abdominal and pelvic masses ([Figure 3](#fig3){ref-type="fig"}). The largest mass was 7.9 × 7.7 × 8.7 cm in the rectouterine pouch with evidence of fat and calcification. There was also a 4.4 × 7.2 × 6.6 cm peritoneal implant superior to the liver that also had similar visual findings. There were also findings of bilateral cardiophrenic masses, with the largest measuring 2.1 × 1.3 × 1.1 cm. In June 2016, she underwent a second debulking procedure consisting of complete infracolic omentectomy, para-aortic lymph node dissection, hepatic mass resection, and pelvic tumor removal on the right side. Gross residual disease remained in her abdomen and pelvis, since she had innumerous peritoneal studding, and thus her thoracic tumors were not removed. A total of 5 pelvic masses and 3 liver masses were excised, and pathologic analysis indicated mature teratoma ([Figure 4](#fig4){ref-type="fig"}). At the time of surgery her liver masses were found to compress the parenchyma of the liver and were not invasive.
Due to the massive growth of tumor in 4 months, as well as recently undergoing 2 major surgical debulkings, and still being unable to completely resect her tumors, medical treatment was discussed with the patient and her parents. In an attempt to prevent regrowth of her tumors, in August 2016, the patient was started on experimental protocol \#009 of the Neuroblastoma and Medulloblastoma Translational Research Consortium (NMTRC). This treatment regimen consists of temozolomide 40 mg/m^2^ PO daily (days 1--28), tretinoin 25 mg/m^2^/day PO BID (days 1--14), and sorafenib 150 mg/m^2^ PO BID (days 1--28). Thalidomide was started during cycle 2. This protocol was chosen as this study tests the feasibility of experimental technologies to determine a tumor\'s molecular makeup. This technology includes a genomic report based on DNA exomes and RNA sequencing that will be used to discover new ways to understand cancers and potentially predict the best treatments for patients with cancer in the future. Prior to the experimental therapeutics, a CT scan was obtained to evaluate disease progression. She is currently without new tumor growths.
3. Discussion {#sec3}
=============
Immature ovarian teratoma is a rare germ cell neoplasm that comprises \<1% of ovarian teratomas \[[@B5]\]. The immature ovarian teratoma must be distinguished from the common benign mature teratoma (dermoid cyst). The difference between the malignant and benign tumors is the presence of immature components most predominantly neuroectodermal, such as neural and glial cells \[[@B6]\]. GCT typically present within the first two decades of life. About 60--70% of malignant germ cell ovarian tumors are diagnosed as FIGO stage I. Stage IA dysgerminoma and grade-I immature teratoma without ascites are treated effectively with surgery alone. In adults, higher staging requires at least 3 courses of BEP (bleomycin, etoposide, and cisplatin) chemotherapy, but children may not need postoperative chemotherapy \[[@B7], [@B8]\]. The Children\'s Cancer Group (CCG) recommends obtaining peritoneal fluid, the removal of tumor without compromising surroundings structures, sparing the fallopian tube if not adherent to the tumor, examination of omentum with removal of suspicious areas, and palpation of pelvic and para-aortic lymph nodes with biopsy in case of abnormality in malignant ovarian tumors. Thus, the relative risk of incomplete staging is higher for pediatric general surgeons \[[@B9]\]. Stratifying grade 3 tumors by stage estimated 5-year event-free survival for patients with grade 3, stage I/II disease was 0.92 (0.72--0.98) for estimated proportion of event-free survival, whereas it was 0.52 (0.22--0.75) for grade 3, stage III patients (*p* = 0.005) \[[@B10]\]. The overall survival for all grade 3 patients, regardless of stage, was 100%. Neither the age at diagnosis nor the AFP level was related significantly to the risk of relapse. The administration of postoperative chemotherapy did not decrease the risk of relapse in the pediatric cohort \[[@B10]\]. Similarly to our study, other pediatric studies have shown no benefit of adjuvant chemotherapy postoperatively in the management of ovarian immature teratomas. In a nonrandomized study by Göbel et al. \[[@B11]\], 76 patients were treated by surgery alone, and 40 patients received adjuvant chemotherapy.
GTS was first introduced by DiSia et al. in 1977 and was called "chemotherapeutic retroconversion" (CR) \[[@B12]\] while Logothetis et al. in 1982 coined the term GTS \[[@B13]\]. Amsalem et al. concluded that these two findings were likely the same event \[[@B6]\]. Currently the pathologic mechanism is thought to arise from either malignant cells differentiation into mature elements or the fact that chemotherapy destroys immature cells leaving mature cells to survive \[[@B14]\].
Ovarian GTS has been described in 101 published English literature cases \[[@B15], [@B16]\]. Most of the patients had abdominal symptoms, such as abdominal pain and distension when they first sought evaluation. The median age for the diagnosis of primary immature teratoma was 22 years (range 4--48 years, *n* = 56) \[[@B15]\]. Many cases of GTS metastasis spread in the peritoneal cavity and tend to occur in the pelvis, peritoneum, or retroperitoneum but have been located in other places such as the liver, pineal gland, and mediastinal/cervical lymph nodes \[[@B7], [@B14], [@B17], [@B18]\]. A single case report described spread via both the lymphogenous and hematogenous route in the same patient \[[@B18]\]. GTS tumors have a growth rate of 0.5 to 0.7 cm/month and a volume increase of 9.2 to 12.9 cm^3^/month \[[@B19], [@B20]\].
Recurrent tumors following chemotherapy for germ cell tumors should be resected to confirm diagnosis, relieve impending/possible obstruction, and prevent future malignant transformation \[[@B6]\]. GTS has a high recurrence rate between 72% and 83% with partial resection versus 0% and 12.7% in those who undergo complete resections \[[@B20], [@B21]\]. Laparoscopy can be utilized as a diagnostic and therapeutic approach in cases of questionable or limited GTS \[[@B16]\].
GTS histology is comprised of mature teratoma elements, but morbidity and mortality arise from its local expansion and the potential for malignant degeneration \[[@B20], [@B22], [@B23]\]. There have been cases of safe IVC resection secondary to production of collateral vasculature \[[@B24]\]. Approximately 3% of GTS cases are associated with malignant transformation including adenocarcinoma, sarcoma \[[@B25], [@B26]\], or primitive neuroectodermal tumors \[[@B4]\]. GTS nodules can appear at any stage during or after chemotherapy, up to 8 years posttreatment, with an average interval of 8 months \[[@B22], [@B27], [@B28]\]. Therefore, regular follow-up contributes to early detection, diagnoses, and treatment.
A rare form of tumor spread can be associated with GTS known as Gliomatosis Peritonei (GP). This tissue is composed of mature glial tissue and can be located in the peritoneal cavity and omentum in patients with ovarian teratoma. GP is often mixed with GTS series despite this being a separate entity \[[@B13]\] because it is defined by pure mature glial tumor tissue in the peritoneum \[[@B29]--[@B31]\] and is encountered in a variety of clinical scenarios \[[@B29], [@B32], [@B33]\]. For example, GP can be found in children with ventriculoperitoneal shunts where it is thought that the cerebrospinal fluid\'s neural growth factor induces glial differentiation \[[@B34], [@B35]\]. GP ultimately acts as a prognostic factor for patients with immature ovarian teratoma as those with GP have a shorter recurrence-free survival (2-year recurrence-free survival rates were 59.3 if with GP versus 96.3% without) \[[@B36]\]. In pediatric patients, larger studies are needed to confirm this and it has not been thought to be true, and the presence of GP does not upstage them.
No standardized management protocol has been established, but one has been set out by Byrd et al. \[[@B1], [@B37]\] ([Figure 5](#fig5){ref-type="fig"}). A key point regarding patients with GTS is regular follow-up as the majority of GTS is found on routine follow-up and presents without symptoms \[[@B21], [@B37]\]. MR imaging and CT are the preferred modalities \[[@B38]\]. CT scan may show a low-density cystic lesion or an increase in the cystic component of the mass suggestive of teratomatous element \[[@B39]\]. CT imaging typically shows radiographic maturation including increased density, better circumscribed margins and onset of internal calcifications, an amalgamation of fat, and solid/cystic components \[[@B21], [@B40]\]. A downside of CT scans is that it underestimates tumor masses smaller than 1-2 cm particularity within the mesentery and omentum when closely related to small bowel loops in the absence of ascites \[[@B39]\]. FDG-PET as a modality for detection of multiple masses associated with GTS has low utility for diagnosis as the GTS lesions do not uniformly appear as hypermetabolic tissue such as brain and thyroid \[[@B41]\]. Ultrasonographic surveillance is made difficult by the variable appearance of these lesions and it is also less sensitive for fat than other modalities \[[@B42]\]; but it contains no radiation for the patient.
Adjuvant chemotherapy with bleomycin, etopside, and cisplatin is recommended for patients when diagnosed with immature teratoma following primary surgery. Palbociclib, a CDK4/6 inhibitor (PD0332991), is reported that it can stabilize the vascularization of the tumor in pediatric patients with an intracranial teratoma \[[@B43]\]. It is a selective reversible inhibitor of cyclin-dependent kinases (CDK) 4 and 6. Inhibition of CDK 4/6 blocks DNA synthesis by prohibiting progression of the cell cycle from G1 to S phase \[[@B43]\]. The main side effect appears to be reversible neutropenia \[[@B44]\]. It has been especially effective in pRB-expressing teratomas (retinoblastoma protein) \[[@B45]\]. While further investigation of the use of Palbociclib in patients with GTS should be carried out \[[@B43]\], the number of major disease-related clinical events decreases with use of Palbociclib as did the median number of progression-free survival months for unresectable mature teratoma \[[@B46]\]. Inoperable metastases have been known to be treated with bevacizumab and cyclin-dependent kinase inhibitors \[[@B47]\], with tumor marker AFP usually returned to within the normal range \[[@B48], [@B49]\]. Interferon-alpha has been successfully used in unresectable tumors to stop their growth for a prolonged period of time based on its antiproliferative and antiangiogenetic abilities and by its immune modulatory effect \[[@B50]--[@B53]\], but tumors while stabilized can stay the same size \[[@B54], [@B55]\].
Surgical approaches stress fertility sparing surgery as recommended for women of childbearing age. Successful pregnancy following development of GTS has been reported indicating necessity of fertility sparing approaches \[[@B56]\]. The treatment of choice during childbearing age for immature teratoma is composed of unilateral oophorectomy and in the case of metastatic disease postoperative chemotherapy. Preservation of fertility is a major concern for young patients undergoing surgical bulking and chemotherapy. Low antimullerian hormone (AMH) levels are associated with a higher incidence of posttreatment amenorrhea \[[@B57]\]. Options for fertility preservation include embryo cryotherapy, oocyte cryopreservation, ovarian autotransplantation, in vitro maturation (IVM), and oophoropexy \[[@B58]\]. New innovations in cryotherapy, such as vitrification, allow for rapid cooling of tissue and decrease the risk of intracellular crystallization and damage.
4. Conclusion {#sec4}
=============
In the setting of immature ovarian teratoma treated with chemotherapy, a rapid growth rate in the presence of normalized serum tumor markers should raise suspicion of GTS \[[@B20]\]. No effective medical treatment of GTS exists due to its unresponsiveness to chemotherapy or radiotherapy. Treatment is currently excision of GTS lesions and close monitoring with imaging and tumor markers. Some trials indicate various treatments such as CDK inhibitors for inoperable disease or stabilizing growth of recurrent lesions. GTS has an overall good prognosis with few reported deaths.
Conflicts of Interest
=====================
The authors have no conflicts of interest to declare.
{#fig1}
{#fig2}
{#fig3}
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{#fig5}
[^1]: Academic Editor: Menelaos Zafrakas
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{
"pile_set_name": "PubMed Central"
}
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Introduction {#s01}
============
Erythropoiesis, the process of RBC production, claims ∼80% of the iron flux in mammals ([@bib17]). When iron delivery falls below a critical threshold, erythroid progenitors promptly enact a lineage-specific response suppressing proliferation and differentiation while retaining viability ([@bib27]; [@bib45]; [@bib8]; [@bib4]; [@bib5]; [@bib39]; [@bib56]). This response preserves limited iron stores for vital functions but also underlies the pathogenesis of iron-restricted anemias: iron deficiency anemia (IDA) and anemia of chronic disease and inflammation.
The principal pathogenetic mechanism in iron-restricted anemias involves development of marrow resistance to the cytokine erythropoietin (Epo). Epo normally acts on early hematopoietic progenitors to promote erythroid lineage commitment and on erythroblasts to mediate survival, proliferation, and differentiation ([@bib32]; [@bib19]; [@bib1]). Patients with IDA strongly increase serum Epo levels, with responses actually exceeding those of iron-replete subjects matched for hypoxia ([@bib16]). However, their marrows fail to undergo compensatory hyperplasia and contain the same number of erythroblasts as marrows from iron-replete subjects ([@bib8]). Furthermore, patient responses to exogenous Epo in anemia of chronic kidney disease critically depend on iron availability ([@bib11]; [@bib12]). Thus, intravenous iron is often administered to abrogate clinical Epo resistance ([@bib49], [@bib50]; [@bib44]).
How iron deprivation mechanistically alters the erythroblastic response to Epo remains unresolved but appears to involve a specialized mechanism. Responsiveness to all cytokines is not impaired, and inflammatory mediators such as TNF-α and IFN-γ actually exert magnified rather than blunted effects on iron-deprived erythroblasts ([@bib42]). In addition, iron deficiency does not uniformly restrict all features of the Epo response but rather restrains proliferation and differentiation while preserving prosurvival signaling ([@bib53]; [@bib4]; [@bib39]; [@bib56]). Iron-sensing molecules implicated in erythropoietic regulation include aconitase enzymes and transferrin receptors. Aconitase conversion of citrate to isocitrate uses an active site 4Fe^2+^-4S cluster, which is highly sensitive to iron restriction in the erythroid lineage ([@bib4]). A functional role for aconitase is suggested by the reversal of the erythroid iron deprivation response with isocitrate treatment and by selective inhibition of erythropoiesis with a targeted enzyme inhibitor ([@bib4]; [@bib51]; [@bib42]; [@bib20]; [@bib26]). The erythroid- and liver-specific transferrin receptor 2 (TfR2) also responds to iron deprivation, shifting from recycling to lysosomal catabolism in response to decreases in its ligand holotransferrin ([@bib24]; [@bib25]). Its contribution to the erythroid iron deprivation response has been demonstrated by the inappropriate erythroblastic expansion observed in iron-deficient mice with erythroid TfR2 knockout ([@bib39]; [@bib43]).
This study delineates a molecular pathway by which iron availability dictates the Epo response in erythroid progenitors. Direct involvement of the Epo receptor (EpoR) is demonstrated by its impaired surface presentation caused by iron deprivation and by the loss of the iron deprivation response in mice with a receptor mutation causing surface trapping. Scribble, a master regulator of receptor trafficking and signaling, is identified as a novel iron response factor that coordinates EpoR surface levels and amplitude of Akt signaling. Specifically, erythroid iron deprivation caused Scribble down-regulation through a cathepsin- and TfR2-dependent mechanism. Scribble deficiency in turn compromised EpoR surface delivery while enhancing Akt activation, thus providing a means for integration of iron sensing with Epo responsiveness.
Results {#s02}
=======
EpoR surface modulation is a critical component of the erythroid iron deprivation response {#s03}
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Because Epo resistance is a critical feature of the erythroid iron deprivation response, we examined whether EpoR is affected by this pathway. To this end, primary human erythroid progenitors were subjected to overnight iron withdrawal or enzymatic aconitase inhibition followed by surface biotinylation, streptavidin pull-down, and immunoblot. We have previously shown in these progenitors that aconitase inhibition with 50 µM sodium fluoroacetate (FA) recapitulates the iron deprivation phenotype and does not impair viability ([@bib51]). With these overnight treatments, total membrane-associated EpoR levels remained unaffected ([Fig. 1 B](#fig1){ref-type="fig"}). In contrast, surface EpoR levels underwent major decreases (approximately threefold) with both treatments, whereas overall levels of surface-biotinylated proteins remained unaffected ([Fig. 1 A](#fig1){ref-type="fig"}). By the same approach, iron withdrawal had no significant effect on surface levels of TfR1 (Fig. S1 A). To determine whether this receptor modulation affected capacity for downstream signaling, iron-replete and -deprived progenitors underwent cytokine starvation followed by Epo stimulation for 0, 10, and 30 min. Consistent with their diminished surface EpoR levels, iron-deprived progenitors showed an approximately threefold decrease in peak phosphorylation of STAT5 at 10 min ([Fig. 1 C](#fig1){ref-type="fig"}).
{#fig1}
To assess whether alterations in EpoR surface expression contribute to the erythroid iron deprivation response, we analyzed mice with the *EpoR-H* knock-in mutation. This mutant allele causes truncation of the distal cytoplasmic domain containing motifs essential for receptor internalization ([@bib47]) but retains the tyrosine 434 residue critical for productive JAK2 signaling ([@bib34]). Thus, EpoR-H signals normally in response to Epo, but fails to execute the subsequent internalization and degradation characteristic of WT EpoR ([@bib3]). Under normal conditions, adult *EpoR-H* mice displayed no evidence of splenic stress erythropoiesis (not depicted) and had a subtle but significant shift toward immaturity in marrow erythroid progenitors (Fig. S1 B). Their steady-state serum Epo levels were diminished approximately threefold compared with WT animals, consistent with enhanced progenitor sensitivity (Fig. S1 C). For assessment of in vivo iron deprivation responses, WT and *EpoR-H* mice were placed on an iron-deficient diet and monitored weekly for blood cell counts and indices. At the start of the study, the *EpoR-H* mice had elevated RBC numbers but normal size mean corpuscular volume. After several weeks of iron deprivation, WT animals manifested an RBC decline, which was highly significant at 42 d. In contrast, the iron-deprived *EpoR-H* animals displayed no decline in RBC throughout the entire period ([Fig. 1 D](#fig1){ref-type="fig"}). Notably, both strains developed significant microcytosis over this period, with similar magnitudes and kinetics. Under conditions of iron deprivation, no discernable differences in marrow erythroid maturation were seen between strains (not depicted); however, a trend toward increased splenic erythroid maturation was observed in *EpoR-H* mice (Fig. S1 D).
To compare ex vivo iron deprivation responses, we isolated Lin^−^ Kit^+^ Ter119^−^ erythroid stress progenitors from spleens of hemolytically challenged WT and *EpoR-H* mice. These progenitors were then subjected to culture in a defined system adapted from iron deprivation studies in human progenitors ([@bib4]). Specifically, this system used serum-free medium with specified TSATs and included an isocitrate treatment, which is known to reverse the erythroid iron deprivation response ([@bib4]). With mouse progenitors, these culture conditions permitted an assessment of early erythroid differentiation, marked by CD71 up-regulation, and provided an iron deprivation response reversible by isocitrate ([Fig. 1 E](#fig1){ref-type="fig"}). Notably, progenitors from the *EpoR-H* mice showed a complete loss of responsiveness to iron deprivation and isocitrate ([Fig. 1, E and F](#fig1){ref-type="fig"}), consistent with a cell-intrinsic role for surface EpoR modulation.
Scribble is regulated by the erythroid iron deprivation response and influences surface EpoR display {#s04}
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The alterations in surface EpoR seen with iron deprivation suggested the potential involvement of Scribble, a large multidomain regulator of receptor trafficking and signaling ([@bib30]; [@bib35]; [@bib41]). Notably, several features of the erythroid iron deprivation response phenocopy changes reported with deficiency of Scribble: hyperactivation of multiple kinase pathways ([@bib4]; [@bib37], [@bib38]; [@bib31]; [@bib13]), enhanced formation of endocytic vesicles ([@bib45]; [@bib23]), and sensitization of cells to TNF-α ([@bib23]; [@bib42]). Furthermore, within the hematopoietic hierarchy in humans and mice, *SCRIB* expression demonstrated lineage- and stage-selective modulation in erythroid progenitors ([@bib2]). Specifically *SCRIB* underwent dramatic up-regulation at the first committed erythroid stage, corresponding to BFU-E, followed by down-regulation in subsequent stages ([Fig. 2 A](#fig2){ref-type="fig"} and Fig. S2 A).
{#fig2}
Immunoblot analysis of human erythroblasts identified Scribble in cytosolic and membrane fractions as three to four species ranging from ∼180 to 250 kD, with the largest form predominating in the cytosolic and whole cell lysate preparations ([Fig. 2 B](#fig2){ref-type="fig"}). Assay specificity was confirmed using multiple independent antibodies and shRNA knockdown (Fig. S2, B and C). The basis for the multiple species appeared to arise, at least in part, from differential ubiquitylation, as treatment of cells with a deubiquitylase inhibitor induced a pattern shift to predominance of the largest form (Fig. S2 D). Importantly, Scribble levels in both fractions underwent robust down-regulation with erythroid iron deprivation and showed restoration with isocitrate treatment ([Fig. 2 B](#fig2){ref-type="fig"}). Immunofluorescence on iron-replete erythroblasts revealed Scribble to be concentrated at the periphery of the cell, but also distributed throughout the cytoplasm in a vesicular pattern ([Fig. 2 C](#fig2){ref-type="fig"}). Scribble displayed a pancellular decrease with iron deprivation, and isocitrate treatment rescued expression particularly at the cell periphery ([Fig. 2 C](#fig2){ref-type="fig"}). In contrast to the protein changes, *SCRIB* transcripts did not decline with iron deprivation and did not respond to isocitrate ([Fig. 2 D](#fig2){ref-type="fig"}). To address the basis for Scribble down-regulation at the protein level, iron-deprived erythroblasts were screened with a series of protease inhibitors. As shown in [Fig. 2 E](#fig2){ref-type="fig"}, treatment of cells with a cell-permeable cathepsin inhibitor CA074me prevented Scribble down-regulation by iron deprivation (Fig. S2 E). In iron-replete cells, cathepsin inhibition minimally affected cytosolic Scribble but did increase membrane levels.
To determine the consequences of Scribble down-regulation on EpoR trafficking, primary progenitors underwent lentiviral shRNA-mediated knockdown. As shown in [Fig. 2 F](#fig2){ref-type="fig"}, Scribble knockdown in these cells markedly diminished surface EpoR expression, but concomitant decrease in total EpoR levels (likely caused by impaired maturation as shown in [Fig. 3 C](#fig3){ref-type="fig"} and Fig. S3 B) complicated the interpretation of these findings. To circumvent this limitation, we also knocked down Scribble in HUDEP-2 cells, a nontransformed human erythroblast cell line ([@bib29]). In these cells, Scribble knockdown strongly decreased surface EpoR while minimally affecting total cellular levels ([Fig. 2 G](#fig2){ref-type="fig"}).
Scribble regulates erythropoiesis, and its deficiency phenocopies characteristics of the erythroid iron deprivation response {#s05}
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Previous ultrastructural comparisons of erythroblasts from iron-deficient and -replete human marrow samples revealed a highly significant (P \< 0.001) increase in empty cytoplasmic vesicles associated with iron deficiency ([@bib45]). This finding correlates with the erythrocyte vesicles specifically found in peripheral blood smears of iron-deficient subjects ([@bib21]). We therefore determined whether deficiency of Scribble, known to regulate vesicle formation in *Drosophila melanogaster* ([@bib23]), affected erythroid ultrastructure. In these studies, electron microscopy (EM) of erythroid progenitors demonstrated an association of Scribble deficiency with increases in peripheral empty vesicles, resembling those that [@bib45] identified in patients with IDA ([Fig. 3, A and B](#fig3){ref-type="fig"}). Cells deficient in Scribble also displayed increases in larger vesicles with intraluminal vesicle formation.
{#fig3}
Prior studies have determined that iron restriction of human progenitors cultured in unilineage erythroid medium for 3--5 d impairs their up-regulation of surface glycophorin A (GPA) on flow cytometry ([@bib4]; [@bib42]). Therefore, we used this assay system to assess the consequences of Scribble deficiency under iron-replete conditions. Strikingly, knockdown of Scribble blocked GPA up-regulation in day 4 erythroid cultures while fully maintaining cell viability ([Fig. 3, C and D](#fig3){ref-type="fig"}; and Fig. S3 A). As previously described for iron restriction ([@bib42]), this blockade preferentially affected the later erythroid marker GPA while permitting up-regulation of the earlier marker CD36 (Fig. S3 B). To assess the role of Scribble in mouse erythropoiesis, *Scrib* floxed (f) animals were mated with a variety of deleter strains. Poor deletion efficiency was obtained with the *EpoR-Cre* and *Vav-Cre* strains. An additional problem with *EpoR-Cre* is that *EpoR* up-regulation lags behind that of *Scrib* during erythroid development. Our studies therefore focused on animals with germline excision (Δ). Because of the neonatal (P0) lethality of *Scrib Δ/Δ* mice ([@bib40]), we analyzed fetal livers at day 13.5 after conception. In these specimens, deletion of *Scrib* was associated with significantly reduced liver cellularity ([Fig. 3 E](#fig3){ref-type="fig"}). Colony-forming assays revealed the frequency of nonerythroid progenitors to be unaffected by *Scrib* status ([Fig. 3 F](#fig3){ref-type="fig"}). In contrast, the frequency of Epo-dependent BFU-E colonies was markedly diminished in *Scrib*-null samples ([Fig. 3 G](#fig3){ref-type="fig"}).
The capacity of iron-deprived erythroid progenitors to maintain survival ([@bib4]; [@bib39]; [@bib56]) despite EpoR down-regulation suggested enhancement in Akt signaling, a pathway previously implicated in Epo-mediated survival ([@bib18]). To examine this possibility, primary erythroblasts cultured overnight in iron-replete or iron-free medium underwent cytokine starvation followed by Epo stimulation. As previously observed ([Fig. 1 B](#fig1){ref-type="fig"}), iron deprivation compromised Epo induction of STAT5 phosphorylation ([Fig. 3 H](#fig3){ref-type="fig"}). In contrast, Epo induction of Akt phosphorylation was unimpaired in iron-deprived cells ([Fig. 3 H](#fig3){ref-type="fig"}). Similar studies in HUDEP-2 erythroblasts showed that iron deprivation actually enhanced the Epo induction of Akt phosphorylation while blunting the induction of STAT5 phosphorylation ([Fig. 3 I](#fig3){ref-type="fig"}). Prior studies have shown that Scribble, through recruitment of the phosphatases PHLPP1 and PTEN, acts as an inhibitor of Akt signaling ([@bib31]; [@bib14]; [@bib7]). Consistent with this role, Scribble knockdown in erythroid progenitors induced Akt hyperphosphorylation, despite the concurrent down-regulation of EpoR levels ([Fig. 3 J](#fig3){ref-type="fig"}). Thus, for Akt signaling, Scribble down-regulation has both negative (decreased surface EpoR) and positive (decreased phosphatase recruitment) consequences that offset one another.
Scribble and TfR2 are regulated by a shared pathway in erythroid iron deprivation {#s06}
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The cathepsin-dependent down-regulation of Scribble in response to iron deprivation ([Fig. 2 E](#fig2){ref-type="fig"} and Fig. S2 E) suggested involvement of lysosomal catabolism. A prior study of TfR2 in hepatoma cells has shown that its destabilization in response to holotransferrin withdrawal occurs as a result of enhanced lysosomal trafficking ([@bib24]). Our analysis of protein decay rates in cycloheximide-treated erythroblasts confirmed the destabilization of TfR2, but not TfR1, by iron deprivation ([Fig. 4 A](#fig4){ref-type="fig"}). Notably, isocitrate treatment completely abrogated the destabilization caused by iron deprivation ([Fig. 4, A and B](#fig4){ref-type="fig"}). As expected for a factor regulated by lysosomal catabolism, treatment of cells with the cathepsin inhibitor CA074me robustly increased TfR2 levels under both iron-replete and iron-deficient conditions ([Fig. 4 C](#fig4){ref-type="fig"}), a finding replicated in three independent experiments (Fig. S4 A). This treatment also reversed the block in GPA up-regulation associated with iron deficiency, implicating lysosomal catabolism as an integral component of the erythroid iron deprivation response ([Fig. 4 D](#fig4){ref-type="fig"}).
{#fig4}
The parallel regulation of TfR2 and Scribble by iron, isocitrate, and cathepsin (Fig. S2 E and Fig. S4 A) raised the possibility of their participation in a protein complex. Supporting this notion, immunoprecipitation (IP) of endogenous TfR2 from K562 erythroleukemic cells efficiently coprecipitated Scribble, whereas IP of TfR1 did not ([Fig. 4 E](#fig4){ref-type="fig"}). This interaction was retained in the presence of iron chelation or aconitase inhibition and was also readily detectable in nontransformed HUDEP-2 erythroblasts ([Fig. 4 F](#fig4){ref-type="fig"}, lanes 4 and 5). Prior findings of TfR2 interacting with EpoR ([@bib15]) also raised the possibility of a Scribble--EpoR interaction. Such an interaction was confirmed by Scribble coprecipitation with endogenous EpoR in HUDEP-2 erythroblast cells ([Fig. 4 F](#fig4){ref-type="fig"}). This Scribble--EpoR interaction showed a minor but reproducible decrease associated with iron deprivation. Immunofluorescence experiments on primary erythroblasts provided evidence of EpoR colocalization with Scribble both at the plasma membrane and in cytoplasmic clusters that likely represent Golgi (Fig. S4 B). These studies further showed loss of membrane localization for both factors occurring with iron deprivation. Because of a lack of antibodies suitable for endogenous Scribble IP, additional experiments were conducted with epitope-tagged factors expressed in HEK293 transfectants. These experiments used bidirectional IPs to confirm the interaction of Scribble with TfR2 but not with TfR1 (Fig. S4 C). These studies also showed that the interaction of TfR2 with Scribble was enhanced by coexpression of EpoR and JAK2, raising the possibility of a multicomponent complex (Fig. S4 D); however, overexpression of these components in a nonerythroid milieu may not recapitulate their endogenous configuration.
TfR2 regulates Scribble in the erythroid iron deprivation pathway {#s07}
-----------------------------------------------------------------
To dissect its role in regulation of the erythroid iron deprivation response, TfR2 underwent lentiviral shRNA--mediated knockdown in primary human progenitors. In iron-replete erythroid cultures, its knockdown induced a major (3.4-fold) increase in total cellular Scribble levels ([Fig. 5, A and B](#fig5){ref-type="fig"}). Because membrane-localized Scribble represents the active form ([@bib14]; [@bib7]), we next focused on the contribution of TfR2 to modulation of membrane Scribble in the erythroid iron deprivation response. Results shown in [Fig. 2](#fig2){ref-type="fig"} demonstrate that membrane Scribble undergoes down-modulation during erythroid iron deprivation but can be rescued by treatment of cells with isocitrate or cathepsin inhibitor. We therefore subjected control and TfR2 knockdown progenitors to iron deprivation alone or with these rescue treatments. In these experiments, TfR2 deficiency completely abrogated Scribble modulation by iron restriction and by cathepsin inhibitor or isocitrate ([Fig. 5, C--F](#fig5){ref-type="fig"}). To determine the phenotypic consequences of these alterations, transduced progenitors were analyzed by flow cytometry, and TfR2 knockdown was found to eliminate GPA modulation by iron restriction and isocitrate ([Fig. 5, G and H](#fig5){ref-type="fig"}). Therefore, TfR2 plays an essential role in the modulation of Scribble and in the phenotypic changes associated with the erythroid iron deprivation response.
{#fig5}
Blockade of isocitrate production impairs TfR2 surface delivery {#s08}
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To further explore the mechanism by which isocitrate affects TfR2 catabolism ([Fig. 4, A and B](#fig4){ref-type="fig"}), erythroid progenitors underwent overnight aconitase inhibition followed by analysis of TfR2 trafficking. Initial experiments examined surface TfR2 turnover rates using pulse-chase analysis of surface-biotinylated proteins. This approach revealed, unexpectedly, no effect of aconitase inhibition on degradation of surface TfR2 ([Fig. 6, A and B](#fig6){ref-type="fig"}). An alternative mechanism for enhanced catabolism could occur through lysosomal shunting of TfR2 export vesicles. To address this possibility, erythroid progenitors underwent a stripping of surface TfR2 by transient, light trypsinization followed by incubation under erythroid culture conditions. At various intervals, recovering surface TfR2 levels were assessed by surface biotinylation followed by streptavidin pull-down and immunoblot as in [Fig. 1 A](#fig1){ref-type="fig"}. This approach revealed that the restoration of surface TfR2 was significantly impaired by aconitase inhibition, with inhibitor-treated cells failing to recover normal surface levels even after long-term cultures ([Fig. 6, C--E](#fig6){ref-type="fig"}). These findings, coupled with the results in [Fig. 4 (A and B)](#fig4){ref-type="fig"}, suggest an influence of isocitrate on trafficking patterns of TfR2, with blocked production potentially rerouting export vesicles from a cell surface destination to lysosomes.
{#fig6}
Discussion {#s09}
==========
A model for erythroid coupling of iron availability with Epo responsiveness is shown in [Fig. 7](#fig7){ref-type="fig"}. The top two panels highlight an iron/isocitrate-regulated vesicle transport pathway in which TfR2 trafficking patterns influence Scribble levels and Scribble levels in turn regulate EpoR surface delivery. This pathway is supported by several key findings: (a) TfR2 undergoes enhanced lysosomal catabolism with iron deprivation, and isocitrate blocks this effect ([Fig. 4](#fig4){ref-type="fig"}); (b) TfR2 surface trafficking relies on the activity of iron-dependent aconitase enzymes ([Fig. 6](#fig6){ref-type="fig"}); (c) TfR2 binds Scribble and mediates its lysosomal catabolism in the iron deprivation response ([Fig. 4](#fig4){ref-type="fig"} and [Fig. 5](#fig5){ref-type="fig"}); (d) Scribble interacts with EpoR and promotes its surface delivery ([Fig. 2](#fig2){ref-type="fig"} and [Fig. 4](#fig4){ref-type="fig"}); (e) Scribble down-regulation decreases surface EpoR display, as is seen in erythroid iron deprivation ([Fig. 1](#fig1){ref-type="fig"} and [Fig. 2](#fig2){ref-type="fig"}). Therefore, TfR2 is proposed to function in this pathway as an upstream sensor, whereas Scribble serves as an intermediary transducing element, and EpoR consists of the effector target. It is possible that Scribble down-regulation may affect delivery of other receptors to the cell surface. However, the levels of most of the surface-biotinylated proteins did not decline with erythroid iron deprivation ([Fig. 1](#fig1){ref-type="fig"} and Fig. S1 A). Although these panels depict separate TfR2--Scribble and Scribble--EpoR complexes, it is possible that complexes may contain all three factors.
{#fig7}
The bottom two panels in [Fig. 7](#fig7){ref-type="fig"} depict the signaling consequences of Scribble down-regulation in the context of iron deprivation. Increased circulating levels of Epo are depicted because of its known up-regulation with IDA. The decrease in erythroid surface EpoR associated with iron deficiency is expected to diminish signaling via downstream pathways including JAK2--STAT5 and Akt. However, Scribble also functions as an inhibitor of Akt signaling. Therefore, iron deprivation--mediated Scribble down-regulation is expected to have both positive and negative consequences for Akt, but only negative consequences for JAK2--STAT5. This notion is supported by our finding that iron deprivation impairs Epo activation of JAK2--STAT5 but preserves or enhances Epo activation of Akt ([Fig. 3](#fig3){ref-type="fig"}).
The iron-sensing pathway depicted in [Fig. 7](#fig7){ref-type="fig"} is proposed to act in conjunction with several previously characterized iron regulatory mechanisms. These mechanisms include the classical binding of iron response proteins to cis-acting iron response elements in transcripts relevant to iron metabolism, such as *FTH*, *FPN*, *TFRC*, and *EPAS1* ([@bib36]). In addition, erythroid sensing of heme iron by several factors including the transcriptional repressor BACH1, the heme-regulated eIF2-α kinase (HRI), and the erythroid transcription factor GATA1 insures that heme and globin production maintain their balance, loss of which impairs differentiation ([@bib6]; [@bib52]; [@bib55]). The modulation of Scribble by iron availability adds a new layer of regulation at the level of cytokine receptor transport.
Morphological studies of human clinical samples have suggested an association of IDA with abnormal vesicular trafficking in erythroid cells. Notably, EM on marrow erythroblasts from subjects with or without IDA identified markedly increased void ropheocytotic--like cytoplasmic vesicles as the principal ultrastructural abnormality associated with IDA ([@bib45]). In peripheral blood smears, abnormal erythrocytes with submembranous vacuoles, called prekeratocytes, represented the most reliable light microscopic feature for distinguishing IDA from other microcytic anemias ([@bib21]). Interestingly, Scribble deficiency in *Drosophila* imaginal disks strongly increased the numbers of Rab5-positive early endosomes ([@bib23]). As the hub in a regulatory network controlling endosomal trafficking and sorting ([@bib48]; [@bib33]), Scribble thus offers a plausible molecular target in the erythroid vesicular pathology of IDA. Importantly, direct Scribble knockdown in human erythroid progenitors sufficed to recapitulate the ultrastructural abnormalities of IDA ([Fig. 3, A and B](#fig3){ref-type="fig"}).
With regard to EpoR expression on the cell surface, prior studies have shown that ∼95% of the receptor molecules in a given cell reside in intracellular pools that fail to undergo constitutive export, a property distinct from other cytokine receptors ([@bib22]; [@bib3]). The manner in which EpoR surface density is regulated has remained obscure, with proposed contributions coming from ligand-dependent and -independent mechanisms ([@bib3]; [@bib46]). Our results reveal iron availability to be a critical influence on EpoR surface density and show that Scribble levels function as a molecular determinant of this parameter. Previous studies have shown TfR2 to bind EpoR and promote its surface export ([@bib15]). Our results suggest that this function could be exerted through the recruitment of Scribble. However, our knockdown studies in human progenitors suggest a more stringent requirement for Scribble than for TfR2 in erythropoiesis. Furthermore, mouse studies on TfR2 loss of function have provided conflicting results, one suggesting increased and another supporting decreased erythroid Epo sensitivity ([@bib15]; [@bib39]). An important feature distinguishing mouse from human erythropoiesis consists of a mouse-specific Epo signaling axis that suppresses lysosomal function through up-regulation of the antiprotease Spi2A ([@bib9]). Thus, the complex interplay between iron regulation of TfR2 lysosomal trafficking and Epo regulation of lysosomal function could contribute to disparate functional results based on subtle differences in model systems. Our findings ultimately support the model of [@bib39] that TfR2 in erythroid cells mediates the Epo resistance associated with iron deficiency. We further propose that Scribble, in its regulation of EpoR, represents a key downstream target in this pathway. However, TfR2 may also directly regulate EpoR, promoting surface delivery in the presence of iron and degradation in its absence.
Epo is the principal cytokine driving erythropoiesis, and one of its primary actions is to promote effective erythropoiesis by preventing apoptosis ([@bib28]). A clinical hallmark of iron-restricted anemias (IDA and anemia of chronic disease and inflammation) consists of resistance to endogenous and exogenous Epo ([@bib11]; [@bib12]). However, despite their Epo insensitivity, iron-deprived erythroid progenitors do not manifest ineffective erythropoiesis and show no increase in apoptosis ([@bib10]; [@bib45]; [@bib53]; [@bib4]; [@bib39]). Our work reconciles this paradox by demonstrating a reconfiguration of EpoR signaling associated with iron deprivation: decreases in surface receptor expression causing diminished STAT5 phosphorylation but unchanged or enhanced Akt phosphorylation. This reconfiguration reflects Scribble's opposing roles in positively regulating cell surface transport of receptors ([@bib30]; [@bib35]; [@bib41]) while negatively affecting downstream survival signaling ([@bib37], [@bib38]; [@bib31]). Thus, the down-regulation of Scribble associated with iron deprivation represents a novel nutrient/metabolic response that concurrently desensitizes growth signaling and sensitizes survival signaling via a common cytokine receptor. Such a response offers an adaptive advantage by maintaining a viable pool of erythroid progenitors poised to reconstitute erythropoiesis upon iron repletion.
Materials and methods {#s10}
=====================
Cell culture {#s11}
------------
Purified normal human donor CD34^+^ progenitors derived from G-CSF--mobilized peripheral blood mononuclear cells were purchased from Fred Hutchinson Cancer Research Center (FHCRC). These cells were obtained through a National Heart, Lung, and Blood Institute--funded core facility (HL66947) that has received approval from the FHCRC Institutional Review Board. After thawing, these cells were cultured for 72 h in prestimulation medium consisting of IMDM (GIBCO BRL) with BIT 9500 supplement (BITS; Stem Cell Technologies) and a cytokine mix comprising 100 ng/ml human stem cell factor (SCF; PeproTech), 100 ng/ml human *FMS*-like tyrosine kinase 3 ligand (PeproTech), 100 ng/ml human thrombopoietin (PeproTech), and 20 ng/ml human IL-3 (PeproTech). After prestimulation, the cells were moved to erythroid medium consisting of IMDM with 2 mM [l]{.smallcaps}-glutamine (Thermo Fisher Scientific), Chelex-100--stripped 0.05% BSA (Sigma-Aldrich), insulin-transferrin-selenium (ITS) supplement (Stem Cell Technologies), 0.0012% 1-thioglycerol (Sigma-Aldrich) recombinant human Epo at 4.5 U/ml (Procrit), and 25 ng/ml human SCF. Where indicated, isocitrate (DL-trisodium salt isocitric acid; Sigma-Aldrich) was included at a concentration of 20 mM. Modulation of TSAT was performed by combining holotransferrin ITS-A (Stem Cell Technologies) and apo-transferrin ITS-B (Stem Cell Technologies) at the appropriate ratios. For most experiments, cells underwent analysis after 3--5 d of erythroid culture.
For analysis of TfR2 turnover rates, primary human CD34^+^ progenitors previously cultured for 3 d in erythroid medium were treated with cycloheximide (Cell Signaling Technology) at 75 µg/ml for the indicated durations followed by immunoblot analysis. For treatment with cathepsin inhibitor, primary human progenitors underwent culture for the indicated durations in erythroid medium supplemented with 5 µM CA074me (PI-126; Enzo Life Sciences) or DMSO only as solvent control. For endogenous IP experiments, K562 cells were treated for 16 h with 50 µM FA (Sigma-Aldrich) or 50 µM desferrioxamine (DFO; Sigma-Aldrich). For analysis of the effects of iron deprivation on Epo signaling, primary human progenitors cultured for 3 d in standard erythroid medium were moved into iron-replete versus iron-deficient (100% vs. 0% TSAT) erythroid medium for 16 h. HUDEP-2 cells were cultured in HUDEP-2 medium (described below) with doxycycline withdrawal for the last 24 h of culture, ± iron deprivation for 16 h. For Epo stimulation, both primary and HUDEP-2 cells underwent 3 h of cytokine deprivation followed by Epo treatment at 4.5 U/ml for the indicated durations and then immunoblot analysis of whole cell lysates.
K562 cells (ATCC) were grown in RPMI medium (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific), 2 mM [l]{.smallcaps}-glutamine, and antibiotic supplement (Anti-Anti; Thermo Fisher Scientific). HEK293T cells (ATCC) were grown in DMEM with 10% FBS, 2 mM [l]{.smallcaps}-glutamine, and antibiotic supplement. HUDEP-2, a nontransformed, immortalized human umbilical cord blood erythroid progenitor cell line, was derived as described previously ([@bib29]). HUDEP-2 cells were cultured in serum-free expansion medium (StemSpan; Stem Cell Technologies) with 50 ng/ml human SCF, 3 U/ml Epo (Procrit), 1 µM dexamethasone (Sigma-Aldrich), and 1 µg/ml doxycycline (Sigma-Aldrich).
Flow cytometry {#s12}
--------------
For flow cytometry, cells were centrifuged, washed, and resuspended in PBS with 1% FBS and conjugated antibody cocktail mix. Antibodies were added at 2 µl per 100 µl of sample, which also contained a 1:100 dilution of violet dye (Zombie; BioLegend). After 30 min of staining on ice, the samples were washed with PBS with 1% FBS and run on an ADP analyzer (CyAn; Beckman Coulter) or a flow cytometer (FACSCalibur; BD). Data analysis used the FlowJo 8.8.7 software package, which was used for compensation and gating of live cells (based on FSC/SSC and Zombie exclusion). Fluorochrome-conjugated monoclonal antibodies to human markers (CD235a, CD36, CD13, and CD34) and mouse markers (Ter119 and CD71) were purchased from BD PharMingen.
Microscopy {#s13}
----------
For immunofluorescence, cells at a density of 10^6^/ml were cytospun onto glass slides (10^5^ per slide) and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Slides were washed in PBS and then permeabilized and blocked in staining buffer (0.06% Triton X-100, 2% BSA, and 2% FBS in PBS) for 1 h at room temperature. Staining was performed using rabbit anti--human Scribble (sc-28737; Santa Cruz Biotechnology) at a dilution of 1:100 in staining buffer overnight in a hybridization chamber at 4°C. Slides were washed three times with staining buffer, and Alexa Fluor 488--conjugated secondary antibody (Thermo Fisher Scientific) was applied at a dilution of 1:500 in staining buffer for 1 h at room temperature. Slides were washed three times in staining buffer and once in PBS, mounted with coverslip and medium (H-1000; Vectashield), and imaged on a confocal microscope (LSM-700; Zeiss) with a 40× oil objective. Images were analyzed by Fiji ImageJ version 2.0.0 (National Institutes of Health). For transmission EM on primary erythroid progenitors ± knockdown of Scribble, cells were washed in PBS and fixed in PBS with 2% paraformaldehyde and 2% glutaraldehyde for 20 min at room temperature. Samples were postfixed in 1% osmium tetroxide for 1 h at room temperature and then embedded and polymerized at 65°C for 24 h. 70-nm sections were placed on 200 mesh grids and stained with uranyl acetate and lead citrate. Grids were carbon coated to minimize conductance and imaged on an electron microscope (1230; JEOL) at 80 kV. Images were assessed in a blinded manner to quantify small circular 50--100-nm vesicles and large intraluminal vesicle-containing multivesicular bodies.
Cell extraction, fractionation, immunoblot, and IP {#s14}
--------------------------------------------------
To produce whole cell lysates for SDS-PAGE, cell pellets were combined with equivalent volumes of 2× Laemmli sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 100 µM dithiothreitol, 10% glycerol, and 0.01% bromophenol blue) supplemented with protease inhibitors (cOmplete; 11836170001; Roche Diagnostics) and phosphatase inhibitors (PhosSTOP; 04906845001; Roche Diagnostics) followed by shearing of DNA and boiling for 5 min. We used a cell fractionation kit (ab109719; Abcam) for isolation of cytosolic and membrane fractions. Kit buffers and detergents were stored as frozen aliquots and supplemented with cOmplete protease inhibitors and PhosSTOP phosphatase inhibitors before cellular extraction. After electrophoresis and transfer to nitrocellulose or polyvinylidene difluoride membranes, the blots were probed overnight at 4°C or for 1 h at room temperature with primary antibodies at a 1:1,000 dilution in Tris-buffered saline with Tween 20 with 1% nonfat dried milk. HRP-conjugated secondary antibodies (Bethyl Labs) were applied for 1 h at room temperature at a 1:5,000 dilution. For global detection of biotinylated proteins, goat polyclonal antibiotin HRP conjugate (ab19221; Abcam) was used for 1 h at room temperature at a 1:5,000 dilution. HRP detection was performed using chemiluminescent SuperSignal West Pico substrate and West Femto Maximum Sensitivity substrate (both from Thermo Fisher Scientific).
Immunoblot primary antibodies consisted of the following: rabbit polyclonal anti-Scribble (4475; Cell Signaling Technology), mouse monoclonal anti--human TfR2 (sc-32271; Santa Cruz Biotechnology), rabbit polyclonal anti-EpoR (sc-695; Santa Cruz Biotechnology), rabbit polyclonal anti-TfR1 (sc-9099; Santa Cruz Biotechnology), rabbit polyclonal anti-ATP1A1 (3010; Cell Signaling Technology), rabbit polyclonal anti-STAT5 (9363S; Cell Signaling Technology), rabbit monoclonal antiphospho-STAT5 (pY694; 4322P; Cell Signaling Technology), rabbit monoclonal antiphospho-Akt (pS473; 4060S; Cell Signaling Technology), rabbit monoclonal anti-Akt (4685S; Cell Signaling Technology), mouse monoclonal antitubulin (clone DM1A; T9026; Sigma-Aldrich), and rabbit polyclonal anti-LDH (sc-33781; Santa Cruz Biotechnology). The specificity of the sc-695 anti-EpoR antibody preparation was validated by [@bib15] and by our experiments using HEK293 transfectants (Fig. S4 D).
For IPs on K562 cells ± DFO or FA treatment, cells were washed in PBS and resuspended in IP lysis buffer (150 mM NaCl, 2 mM MgCl~2~, 10 mM Hepes \[Thermo Fisher Scientific\], 0.5% NP-40, protease inhibitor, and phosphatase inhibitor) at a density of 2 × 10^6^ cells per 100 µl. The same extraction protocol was used for IPs on HUDEP-2 cells subjected to overnight doxycycline withdrawal ± iron deprivation. For IPs on HEK293T transfectants, cells were washed and overlaid with 500 µl IP lysis buffer per semiconfluent 10-cm plate. Suspensions were incubated on ice for 15 min and centrifuged for 10 min at 15,000 rpm at 4°C. Protein content in the supernatant was quantified by bicinchoninic acid. For all IPs, 2 µg of antibody was added per milligram of protein extract in 1 ml lysis buffer followed by overnight rotation at 4°C. The following antibodies were used for IP of K562 and HUDEP-2 endogenous proteins: mouse monoclonal anti--human TfR1 (sc-32272; Santa Cruz Biotechnology), mouse monoclonal anti--human TfR2 (sc-376278; Santa Cruz Biotechnology), mouse monoclonal anti--human EpoR (38409; Thermo Fisher Scientific), and mouse IgG control (sc-2025; Santa Cruz Biotechnology). The following antibody conjugates were used for IP of epitope-tagged recombinant proteins: mouse monoclonal anti-Myc magnetic beads (5698S; Cell Signaling Technology), mouse monoclonal anti-FLAG magnetic beads (M8823; Sigma-Aldrich), and control mouse IgG magnetic beads (5873; Cell Signaling Technology). IPs with unconjugated antibodies were captured with a 1:1 mixture of magnetic beads conjugated to protein A (88846; Thermo Fisher Scientific) and to protein G (88847; Thermo Fisher Scientific), prewashed three times in lysis buffer, and added at a volume of 25 µl of packed beads per 1-ml sample. Bead suspensions were rotated for 2 h at 4°C, quickly washed twice in lysis buffer, slowly washed twice with 15 min of rotation in 4°C lysis buffer, again quickly washed twice in lysis buffer, resuspended in 50 µl Laemmli buffer, boiled, and analyzed by immunoblot as described above.
Immunoblot signals were quantified using a GS-800 calibrated densitometer (Bio-Rad). Signals were normalized using loading controls to derive relative band densities. Ponceau S--stained membranes were imaged with a membrane imager (Fluorchem HD2; Alpha Innotech), and total lane density was quantified by ImageJ. Immunoblots shown are representative of at least three independent experiments.
Cell surface biotinylation and surface TfR2 recovery after trypsinization {#s15}
-------------------------------------------------------------------------
Cells were washed with PBS and resuspended at a density of 10^7^/ml in surface-impermeable biotinylation reagent: 1 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (21335; Thermo Fisher Scientific) in PBS, pH 8.0. The cell suspension was incubated on ice for 30 min and washed twice with PBS, pH 8.0, supplemented with 100 mM glycine. The cells were resuspended at a density of 10^6^ cells per 150 µl in biotin lysis buffer (150 mM NaCl, 5 mM EDTA, and 10 mM Tris-HCl, pH 7.4, with 1% Triton X-100), incubated for 15 min on ice, and centrifuged at 17,000 rpm for 10 min at 4°C. A portion of supernatant was directly analyzed by immunoblot as input, and the remainder was combined with prewashed resin (NeutrAvidin; Thermo Fisher Scientific) at a ratio of 3:1 lysate/bead slurry. Slurry/lysate mixtures were rotated for 1 h at 4°C, washed twice in biotin lysis buffer, and resuspended in Laemmli buffer for immunoblot analysis of eluates. For analysis of surface TfR2 recovery after trypsinization, 10^6^ cells were resuspended in 100 µl of ice-cold PBS with 0.05% trypsin (Sigma-Aldrich). After incubation on ice for 20 min, the cells were washed with ice-cold IMDM and then resuspended in erythroid medium. The cells were cultured at 37°C for the indicated durations and then subjected to surface biotinylation, streptavidin pull-down, and immunoblot as described above.
Transfections and transduction {#s16}
------------------------------
For cotransfections, HEK293T cells were grown in 10-cm plates to 60% confluency and transfected in normal culture medium with 20 µg of plasmid using a CalPhos kit (631312; Clontech). At 16 h after transfection, the medium was exchanged with fresh growth medium for protein extraction or Opti-MEM I (Thermo Fisher Scientific) for lentiviral packaging. Protein extracts for IP were obtained 40 h after initiation of transfection. For lentiviral vector packaging, supernatants were collected 40 h after initiation of transfection.
Lentiviral packaging plasmids pCMV-dR8.74 (GAG POL TAT REV) and pMD2.G (VSV-G) were cotransfected with pLKO.1 shRNA vectors via calcium phosphate precipitation into HEK293T cells as above. A mass ratio of 3:1:4 for pCMV-dR8.74/pMD2.G/pLKO.1 was used for transfection. pLKO.1 vectors expressing shRNAs active against the following human targets were purchased (GE Dharmacon): *SCRIB* (TRCN0000004457 and TRCN0000004458) and *TFR2* (TRCN0000063628 and TRCN0000063630). Viral supernatants were filtered with 0.45-µm syringe filters and stored at −80°C. K562 cells were incubated with viral supernatants for 24 h, followed by puromycin selection at 2 µg/ml in growth medium for 48 h. HUDEP-2 cells were incubated for 24 h in viral supernatants supplemented with 50 ng/ml human SCF, 3 U/ml Epo, 1 µM dexamethasone, and 1 µg/ml doxycycline, followed by puromycin selection at 2 µg/ml in HUDEP-2 media for 72 h. For human primary progenitors, cells were expanded in prestimulation medium for 48 h and transferred to retronectin-coated 12-well plates containing viral supernatants supplemented with prestimulation cytokines. After 2 h at 37°C, the plates were spun at 500 *g* for 90 min and then returned to 37°C. After overnight culture, the cells were transferred to fresh viral supernatants with prestimulation cytokines and subjected to a second round of spinoculation and overnight culture followed by selection with 2 µg/ml puromycin in prestimulation medium. After 48 h of puromycin selection, live cells were enriched by centrifugation on Ficoll-Paque PLUS medium (17-1440-02; GE Healthcare) and washed in IMDM. Viable mononuclear cells were then subjected to erythroid cultures as described above.
The following expression constructs were used for cotransfection and co-IP: pcDNA6/myc-his human and mouse TfR1 and TfR2 from P. Schmidt (Boston Children's Hospital, Boston, MA); pcDNA3-FLAG human Scribble from I. Macara (Vanderbilt University School of Medicine, Nashville, TN); and pcDNA3-Human-EpoR and human-JAK2 from F. Verdier (Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Paris, France) and O. Bernard (Université René Descartes), respectively.
Mouse models {#s17}
------------
All animal experiments were approved by the University of Virginia Institutional Animal Care and Use Committee. The *EpoR-H* mouse strain on a BALB/c background was provided by J. Ihle (St. Jude Children's Research Hospital, Memphis, TN). This strain was subsequently backcrossed onto a C57BL/6J background for 10 generations. WT controls consisted of age-, sex-, and strain-matched mice purchased from Jackson Labs. Serum EPO levels were determined with the Quantikine Mouse EPO ELISA kit (R&D Systems). For dietary induction of IDA, 3-wk-old male weanlings were placed on iron-deficient chow (TD.80396 custom diet; Envigo Teklad Diets) for the duration of the experiment. Weekly retroorbital bleeds into EDTA-coated collection tubes were analyzed for complete blood counts on a hematology system (Hemavet 950; Drew Scientific Group).
For induction of erythroid stress progenitors, mice received phenylhydrazine hydrochloride (P6926; Sigma-Aldrich) at 60 mg/kg/d for two sequential days by intraperitoneal injection. 3 d after the second injection, spleens were isolated, passed through a 70-µm filter mesh, incubated for 5 min at room temperature in ammonium chloride lysis buffer (Thermo Fisher Scientific) to remove RBCs, and washed in PBS. The cells were then incubated with a biotinylated antilineage cocktail (130-090-858; Miltenyi Biotec) and lineage depleted on a column (AutoMACS Pro; Miltenyi Biotec). The resulting cells were resuspended in 100 µl of sterile PBS with 1% FBS plus 2 µl APC-conjugated anti--c-Kit and 2 µl PE-conjugated anti-Ter119 (341096; 555673; BD PharMingen). After a 30-min incubation on ice, the stained cells were washed in PBS with 1% FBS, resuspended in IMDM, and subjected to sorting on a flow cytometer (FACSVantage SE TurboSort DIVA; BD) in the University of Virginia flow cytometry core facility. The isolated Lin^−^ Kit^+^ Ter119^−^ progenitors were directly cultured in erythroid medium as described above, except that 100 ng/ml mouse SCF (PeproTech) was substituted for human SCF. Cells were analyzed by flow cytometry after 4 d of erythroid culture under the indicated conditions.
The *Scrib*-floxed mouse strain on a 129/Sv/CB57BL/6 background was provided by A.E. Griep (University of Wisconsin-Madison, Madison, WI; [@bib54]). Germline deletions were generated by crossing with the *EIIa-Cre* strain (Jackson Labs). E13.5 embryos from Δ/f intercrosses underwent sterile dissection of fetal livers, which were converted to single-cell suspensions via 21-gauge syringe trituration in IMDM. Control f/f samples were derived from littermates of Δ/Δ fetuses. Total cells per fetal liver were counted and resuspended in medium (MethoCult M3534; Stem Cell Technologies) at a density of 20,000 cells per well. Epo (Procrit) was supplemented to generate a final concentration of 0.03 or 1.0 U/ml. Colonies were counted on day 7 after seeding.
RNA analysis {#s18}
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RNA was isolated from human erythroid progenitors using the RNeasy Plus Mini kit (QIAGEN) with DNase treatment of columns before RNA elution. RNA yield and quality were determined on a spectrophotometer (Thermo NanoDrop; Thermo Fisher Scientific). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription kit (4368814; Applied Biosystems). Quantitative PCR was performed using the iQ SYBR green assay on a CFX Connect Real-Time PCR Detection System (1708880; Bio-Rad). For relative quantitation of transcript levels, we used the comparative ΔΔC~t~ formula delineated in the ABI Prism 7700 Sequence Detection System user bulletin no. 2. All samples underwent triplicate analysis with normalization performed by subtraction of the C~t~ value for *GAPDH*. Primers were as follows: human *GAPDH* (forward: 5′-AGCCACATCGCTCAGACA-3′; reverse: 5′-GCCCAATACGACCAAATCC-3′) and human *SCRIB* (forward: 5′-CTGACCCTCACCATCCTG-3′; reverse: 5′-CAGAGCCACACCATTCAC-3′).
Statistics {#s19}
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Individual results shown are representative of at least two independent experiments. Statistical analysis was performed with Prism 6 (GraphPad Software). Graphs are displayed as the mean of independent experiments ± SEM. Data were analyzed either by two-tailed Student's *t* test, one-way ANOVA, or two-way ANOVA on a minimum of three independent experiments. Post-hoc analysis of one-way ANOVA studies was performed using Dunnett's multiple comparisons test. Post-hoc analysis of two-way ANOVA studies was performed using Sidak's multiple comparisons test.
Online supplemental material {#s20}
----------------------------
Fig. S1 shows surface TfR1 levels in erythroid progenitors ± iron withdrawal, erythroid maturation and serum Epo levels in iron-replete *EpoR-H* versus WT mice, and erythroid maturation in iron-deprived *EpoR-H* versus WT mice. Fig. S2 shows a heat map of *Scrib* expression in mouse hematopoiesis, confirmation of three distinct protein species as Scribble, and densitometry for Scribble modulation by iron deprivation and cathepsin inhibition. Fig. S3 shows phenotypic consequences of Scribble knockdown on human erythropoiesis, as assessed by flow cytometry. Fig. S4 shows densitometry for TfR2 modulation by iron deprivation and cathepsin inhibition, immunofluorescence for Scribble and EpoR in erythroid progenitors ± iron deprivation, and interactions of epitope-tagged Scribble in HEK293 transfectants.
Supplementary Material
======================
###### Supplemental Materials (PDF)
We thank Olivier Bernard, Ian Macara, Paul Schmidt, and Frédérique Verdier for providing plasmids; Jim Ihle and Anne Griep for mouse strains; Joanne Lannigan and the University of Virginia Flow Cytometry Core Facility for assistance with flow cytometry and cell sorting; Dave Kashatus and Bettina Winckler for reviewing the manuscript; and Rob Fleming for valued advice and discussions.
This work was supported by grants from the National Institutes of Health (grants R01 DK079924, R01 DK101550, and K08 HL093355), the Leukemia and Lymphoma Society (grant LLS NIA-8988-15), the Farrow Fellowship, the National Cancer Institute Cancer Center support grant P30 CA44579, and the Medical Scientist Training Program at the University of Virginia (grant 5T32GM007267-38).
The authors declare no competing financial interests.
Author contributions: S. Khalil designed and conducted experiments, interpreted data, and wrote the manuscript. L. Delehanty designed and conducted experiments, interpreted data, and edited the manuscript. S. Grado, M. Holy, Z. White III, K. Freeman, and G. Bullock conducted experiments and edited the manuscript. R. Kurita and Y. Nakamura developed the HUDEP-2 system and edited the manuscript. A. Goldarb supervised the project, designed experiments, interpreted data, and wrote the manuscript.
[^1]: S. Khalil and L. Delehanty contributed equally to this paper.
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Background
==========
National and international studies in recent years have revealed that a significant number of physicians working in the field of patient care are not satisfied with their job and the associated working conditions \[[@B1]-[@B3]\]. A survey conducted in the US, for example, demonstrated this dissatisfaction in revealing that up to 40% of the physicians practicing in hospitals would not take up this profession again. Even a higher portion of the questioned physicians stated that they prevented their children from becoming a physician \[[@B4]\]. The workload of physicians proves to be one of the causes for the situation described. This fact is shown by different North American and European studies \[[@B5]-[@B10]\]. Also, the working conditions of clinicians in Germany have changed significantly in recent years. The number of patients in German hospitals has increased from approximately 14 million in 1990 to approximately 17 million in 2004. During the same period, however, the average length of stay has fallen from 14.7 days to 8.7 days \[[@B11]\]. In addition to this development, increasing bureaucracy and mechanisation in daily clinical life, particularly the growing demands relating to documentation and quality control, play a significant role in a physician\'s practice and result in distancing from the patient \[[@B12]\]. This is not only the case in Germany, but can be seen as an international trend \[[@B4],[@B8],[@B13],[@B14]\]. However, in addition to workload, other factors exist. It is reported that growing patients\' needs and economic, organisational, and regulatory factors affect job satisfaction decisively \[[@B15]\]. In particular, many physicians feel their autonomy or their self-conception as physicians are restricted by these factors \[[@B16],[@B17]\]. There exist studies indicating that increasing deprofessionalisation and restriction of professional autonomy evoke job dissatisfaction in physicians \[[@B1],[@B16],[@B18]-[@B20]\]. In addition, advanced vocational training facilities (offered on the job) have to be considered \[[@B15]\]. Finally, a linkage exists between a physician\'s salary and satisfaction with the job \[[@B5]\]. The heavy objective and subjective burdens and the dissatisfaction in practising within the medical profession causes a growing number of physicians, especially young physicians, to change their vocational field \[[@B21]\]. Many studies have demonstrated a link between job satisfaction of physicians and the probability of quitting their job or the frequency of job changes \[[@B22]-[@B26]\]. A growing number of physicians try to avert the possible consequences of the workload felt to be too heavy and the dissatisfaction involved by taking up jobs in non-medical professions at an early stage. In particular, stress and burnout are common consequences of regular overwork, which is not merely due to the pure quantity of work, but also due to the quality to be delivered. Being responsible for appropriate and successful therapy and catering to the demands of different social circles (colleagues, relatives, and health insurance) frequently leads to chronic stress, burnout, and physical and other mental diseases \[[@B7],[@B27]\]. Furthermore, a clear relationship between stress, burnout, and job satisfaction could be shown \[[@B17],[@B28],[@B29]\]. This relationship certainly has to be considered as reciprocal: stress and burnout diminish job satisfaction, low job satisfaction in turn intensifies the symptoms of stress and burnout. Obviously, it appears that high job satisfaction, however, can also act as a protective factor and prevent chronic job stress \[[@B7],[@B17]\]. The particular relevance of job satisfaction is demonstrated further by several studies showing links between job satisfaction of physicians and the quality of medical care \[[@B23],[@B30]-[@B34]\]. Thus, for example, in addition to the impact of physician job satisfaction on patient satisfaction \[[@B23],[@B30]\] and adherence \[[@B23],[@B33]\], links between the occurrence of errors in treatment and job satisfaction have been described \[[@B15]\]. Although physician job satisfaction has been assessed to a large extent in recent years, very little is known about the effects of organizational characteristics, such as a culture of value and trust, which are expressions of social capital, on overall job satisfaction.
General definitions of Job Satisfaction and Social capital in the workplace
---------------------------------------------------------------------------
### Job satisfaction
Job satisfaction is defined as a global attitude that individuals have towards their jobs \[[@B35]\]. It is an extent to which one feels positively or negatively about different facets of the job e.g. job conditions, co-workers and working time \[[@B36]-[@B39]\] and is a complex set of interrelationships of tasks, roles, responsibilities, interactions, incentives and rewards \[[@B40]\].
### Social capital in the workplace
Social capital can be regarded as a resource which helps people and organizations cope with stress and helps foster salutogenic potential. There are two forms of social capital: 1) individual social capital and collective social capital. An individualistic version of social capital has been defined by Bourdieu \[[@B41]\]. In brief, social capital, according to Bourdieu, is the \"aggregate of the actual or potential resources which are linked to possession of a durable network of more or less institutionalized relationships of mutual acquaintance and recognition -- or in other words, to membership in a group -- which provides each of its members with the backing of the collectively owned capital, a credential which entitles them to credit, in the various senses of the word\" \[[@B42]\]. Social epidemiologic research during the last 20 years shows that social relationships, which are experienced as being helpful and positive, promote general well-being and protect against physical harm \[[@B43],[@B44]\]. Coleman \[[@B45]\] described the collective version of the term \"social capital\" as follows: \"Unlike other forms of capital, social capital inheres in the structure of relations between persons and among persons. It is lodged neither in individuals nor in physical implements of production.\" Given this definition, it can be assumed that not only individuals, but also complex organizations, such as hospitals, possess social capital. Its components are, in particular, the existence of collective values and convictions and mutual trust between the members of an organization \[[@B46]\]. Collective social capital can be defined as a feature of social systems that is able to improve the health and the capacity to perform of its members \[[@B47]\]. Research into support and networks has also shown that a person\'s social network has a significant impact on his or her performance capacity, health, and emotional balance. The stability, scope, and functionality of social networks have a modulating effect on cognition, motivation, and emotions \[[@B48]-[@B53]\]. A successfully established atmosphere of trust and a feeling of common values and convictions may help people work together and make it easier for them to assess the conditions of their daily work by reducing insecurity, uncertainty, and disorientation, and to improve their performance. Social capital is generated from internalized, informal standards within an organization and produces cooperation \[[@B54]-[@B57]\]. Putnam and Coleman regard social capital as a way of solving collective problems through a sense of community and trust. The inherent potential for people to exploit others in the production of collective goods is reduced by activity structures that are governed by reciprocity standards \[[@B55]\].
Aim of the study
----------------
Little empirical work has been published using the concept of social capital in the health care industry \[[@B58]\]. However, the studies which have been published on this topic yet refer to the particular importance of social capital in the health care sector, eg. in the inpatient or ambulatory sector. Thus, Waisel demonstrates in his study how social capital improves the operating room working environment and therefore increases efficiency and quality of patient care \[[@B59]\]. DiCicco et al \[[@B60]\] developed a model of social capital to enhance relationships within primary care practices that promote organizational success and improve patient care outcomes. Hoelscher, Hoffman & Dawley \[[@B61]\] reviewed the literature and showed that existing social capital leads to competitive advantage and enhanced medical group performance. Hofmeyer & Marck \[[@B62]\] outlined the role of social capital for organizational integrity, healthy workplace cultures, sustainable resource management, improved nurse retention, effective knowledge translation, and safer patient care. Research into the relationship between social capital in hospitals and job satisfaction of clinicians is however still at an early stage. To our knowledge, there is no existing literature to date that has explicitly examined the relationship between social capital in hospitals and physician job satisfaction. Therefore, the aim of the current study was to examine the effects of collective social capital at the workplace on overall job satisfaction of clinicians. We hypothesized that a significant relationship exists between social capital in the hospital and physician satisfaction, assuming that this relationship can be proved not only in bivariate form, but persists after controlling for socio-demographic factors, such as age, gender, professional experience, and subjective workload.
Methods
=======
The following analysis is based on data from a project entitled \"Corporate Governance Using Biopsychosocial Indicators\" (CoBI) study, funded by the German Federal Ministry of Education and Research. The \"Biopsychosocial Indicators for Employees Questionnaire\" (BIQ) used herein was especially developed for this study. It contains both internationally-established instruments, such as the \"Maslach Burnout Inventory,\" and scales especially developed for this and further studies, among them the social capital scale described below. It consists of valid indicators of how employees regard their work situation and their organization \[[@B63]\].
Ethics
------
This study was approved by the Research Ethics Board at the University of Cologne. All participants provided informed consent for the survey.
Participants and procedure
--------------------------
The CoBI study surveyed clinicians, nursing staff, and administrative and technical employees. In 2002 a total of 2,644 employees representing four German hospitals received an anonymous questionnaire by post. These employees had been working full-time or part-time in one of the four hospitals during the survey period. Further inclusion and exclusion criteria were not defined. Two hospitals in East Germany and two hospitals in West Germany, two of which offer maximum healthcare services and two of which offer basic healthcare services, were included (Table [1](#T1){ref-type="table"}). Of the 2,644 employees, 454 were clinicians. 277 clinicians responded to the poll, for a response rate of 61%. This population made up the sample for the present study.
######
Structures of the selected hospitals
**Hospital 1** **Hospital 2** **Hospital 3** **Hospital 4**
----------------------------- ---------------- ---------------- ---------------- ----------------
**Region** West West East East
**Care level** Max. care Basic care Max. care Basic care
**Specialist departments** 23 6 25 7
**Beds** 1.500 353 1.855 454
**Number of cases in 2001** 47.673 9.437 58.841 15.089
Measures
--------
### Overall Job Satisfaction
Following Scarpello and Campell \[[@B64]\], Wanous et al. \[[@B65]\] and Nagy \[[@B66]\] we decided to measure overall job satisfaction taking a single item approach. The most frequently argued advantages of single item measures in contrast to multi-item, multi-dimensional instruments measuring overall job satisfaction are the following: single item measures are much shorter and take up less space, are more cost-effective, may contain more face validity, appear to be correlated fairly with multi-item measures of overall satisfaction and may be better to measure changes in job satisfaction. Furthermore, the problem to operationalize job satisfaction -- similar to patient satisfaction -- is to integrate all factors influencing job satisfaction in one comprehensive instrument according to their individual weighting. In particular due to the lack of knowledge of the completeness of all potential influence factors and the lack of empirical and theoretical information about their individual weighting, a single item approach seems to be the more appropriate method. Highhouse and Becker \[[@B67]\] e.g. found that facets such as company loyalty, enjoyment of work, and job significance were not captured by a composite facet measure, but were considered in making a global judgment about job satisfaction. Therefore the used variable, \"job satisfaction\" \[[@B68]\], is based on a homonymous item worded as follows: \"If you consider everything that matters in your job (e.g., kind of work, working conditions, colleagues, and working time), how satisfied are you with your job?\" Subjective complaints were assessed with a seven-point Likert-type scale with smiley/sad faces above each point.
### Sociodemographic characteristics and workload
Information about age, gender, and years of professional experience was provided by the respondents. The variable, \"workload,\" based on the scale, \"intensity of labour\" according to Richter et al. \[[@B69]\], was designed to measure the workload of physicians (Cronbach\'s α: .78). The six items of the scale were worded as follows: 1) \"The required pace of work is very fast,\" 2) \"The tasks are often very difficult to cope with,\" 3) \"I often have very much work to be done,\" 4) \"Usually time is too short and I often work under time pressure,\" 5) \"I\'m often exposed to physical strain,\" and 6) \"Too much work has to be done at the same time.\" Answers ranged from \"disagree\" to \"agree,\" with each response category measured by a score from 1--4 points; all item values were summed and divided by the number of items.
### Social capital in hospitals
The variable, \"social capital in hospitals,\" was designed to measure two key features of social capital: 1) common values and 2) perceived trust at the hospital \[[@B64]\]. We used six items to measure this variable \[[@B68]\], e.g., \"Agreement and consent dominate in our hospital\" and \"At our hospital we trust each other.\" The items were developed using basic sociological principles and central statements relating to social capital described by Coleman \[[@B45],[@B54]\], Putnam \[[@B55]\], and Fukuyama \[[@B56]\]. Respondents could choose from four given responses and each response was assigned points ranging from 1--4; the total scores ranged from 6--24 points. The central determinants of this variable were divided into three types: common values, perceived trust, and reciprocity. Agreement and consent (item 1) and the presence of a \"sense of unity\" (item 3) represent a common value base \[[@B57]\]. One of the prerequisites for cooperative action is \"trust\" (item 2). The probability of trust in a partner increases with the expectation of benefits from trust-based action. Putnam \[[@B55]\] speaks of the standard of generalized reciprocity; these standards ensure that hospital employees work together by causing them to behave cooperatively \[[@B54]\]. Item 4 looks at perceptions of the quality of the atmosphere at work. Reciprocal behaviour (item 5) is a form of exchange, commonly referred to as a *quid pro quo*and forms the basis of what is called \"a contingent relationship.\" In other words, workplace relationships lead to staff feeling obliged to the organization to act reciprocally. According to Coleman \[[@B70]\], the decisive prerequisite for solving problems using cooperation is the amount of social capital \[[@B71]\]. The common values within the hospital are emphasized in item 6.
### Statistical methods
We performed stepwise multivariate linear regression using SPSS 15.0. If the proportion of missing values for a variable was \< 25%, a mean value was imputed; variables with \> 25% of missing values were excluded from analysis \[[@B72]\]. Variables with an intercorrelation \> 0.8, which is an indicator for problems of collinearity, would also have been excluded from analysis, but no variable fulfilled this (both) criteria either \[[@B73]\]. The following analysis was conducted in a three step manner, as follows: first, we give an overview of descriptive values of all analysis variables, in the second step, we show Pearson product moment correlation coefficients (PMCC) for correlations between all variables to demonstrate bivariate relationships and to control for multicollinearity; and in the final analysis, we computed a multiple linear stepwise regression model on \"job satisfaction.\"
Results
=======
163 respondents were male (58.8%) and 114 (41.2%) female. The average age was 40.0 years (standard deviation 9.9 years). 89 of the respondents (32.1%) had up to five years of professional experience, 111 (40.1%) had 6--16 years, and 77 (27.8%) had 17 years or more. 82 physicians (29.6%) specialize in internal medicine (e.g., cardiology and oncology), 57 physicians (20,6%) in visceral and vascular surgery, 40 physicians (14.4%) in neurology, psychiatry, and psychosomatics, 29 physicians (10.4%) specialize in other fields (e. g. gynaecology and paediatrics). 69 physicians (25.0%) hadn\'t specified their speciality.
Table [2](#T2){ref-type="table"} describes the constant independent analysis variables and the dependent variable, \"job satisfaction,\" the distribution of which was approximately normal; the Kolmogorov-Smirnov test demonstrated no significant deviation from a normal distribution.
######
Measurements of the variables \"social capital,\" \"workload,\" and \"job satisfaction\"
**Variable** **Measurement** **M** **SD** **Min** **Max**
---------------------------- ----------------------------------------------------------------------------------------- ------- -------- --------- ---------
Social capital in hospital Total score of \"social capital in organizations\" according to Pfaff et al. \[[@B68]\] 13.8 3.7 6 24
Workload Total score of \"intensity of labour\" according to Richter et al. \[[@B69]\] 19.3 3.2 7 24
Job satisfaction Single item according to Pfaff et al. \[[@B68]\] 4.74 1.3 1 7
M = Mean; SD = Standard deviation; Min = Minimum; Max = Maximum
In the second step of the analysis, we want to give an overview of the Pearson product moment correlation (PMCC) of all analysis variables (see table [3](#T3){ref-type="table"}). This step followed two aims: 1) show bivariate relationships between all analysis variables, and 2) control for multicollinearity of variables used in the final regression model.
######
Intercorrelation coefficients for all ordinal and metric analyses variables
1\. Age 2\. Profess. experience 3\. Workload 4\. Social capital 5\. Job satisfaction
------------------------- --------- ------------------------- -------------- -------------------- ----------------------
1\. Age .736\*\* .073 .166\*\* .296\*\*
2\. Profess. experience .055 .114 .285\*\*
3\. Workload -.176\*\* -.277\*\*
4\. Social capital .524\*\*
\*\* = Correlation is significant at the 0.01 level (2-tailed)
The intercorrelations ranged between .055 (workload with professional experience) and .736 (age with professional experience). Seven of 10 relationships were significant at the 0.01 level (2-tailed). The link between social capital and job satisfaction as a main topic of this analysis proved to be highly significant on a bivariate level (.524). All in all, no variable in the present study reached a critical correlation with another variable of \> .8, which is discussed with regard to multicollinearity \[[@B73]\].
Regarding collinearity diagnostics, the magnitude of the intercorrelations among variables, is just one of the indicators for this problem. Therefore, additional indicators such as the tolerance of the variables and the variance inflation factor (VIF) are presented in table [4](#T4){ref-type="table"}. The tolerance value shows the extent to which the independent variable in the corresponding line is predictable by other variables included in the model or whether it correlates with them. Tolerance values not lower than 0,1 or VIF-values up to 10 (VIF = reciprocal of tolerance value) \[[@B73]\] may be accepted. In this regression model no problems of collinearity arise, as all tolerance values are higher than 0,1 and all VIF-values are significantly lower than 10.
######
Results from hierarchical multiple linear regression
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Unstand**.\ **Stand**.\ **Collinearity statistics** **Sign**.\ **Corr**.\ **R^**2**^-changes**
**coefficients** **coefficients** **level** **R**^**2**^
------- ------------------------- ------------------ -------------------- ----------------------------- --------------- -------------- ---------------------- ----------- ----------
**B** **Standard error** **Beta** **Tolerance** **VIF**
**1** (Variable) 3.44 .408 .000\*\*\* **0.089** **.099**
gender -.086 .151 -.033 .971 1.03 .569
**age** .024 .011 .183 .451 2.21 **.033\***
professional experience .242 .140 .146 .458 2.18 .086
**2** (Variable) 5.875 .582 .000\*\*\* **0.180** **.091**
gender -.180 .144 -.069 .958 1.04 .214
**age** .026 .010 .199 .451 2.21 **.015\***
professional experience .243 .133 .147 .458 2.18 .069
**workload** -.124 .022 -.308 .981 1.01 **.000\*\*\***
**3** (Variable) 3.392 .582 .000\*\*\* **0.366** **.186**
gender -.128 .127 -.049 .956 1.04 .314
age .015 .009 .116 .444 2.25 .108
professional experience .255 .117 .155 .458 2.18 **.030\***
workload -.089 .020 -.222 .944 1.05 **.000\*\*\***
**social capital** .155 .017 .445 .935 1.07 **.000\*\*\***
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\*Dependent variable: job satisfaction; \* = p ≤ 0.05; \*\* = p ≤ 0.01; \*\*\* = p ≤ 0.001
The last analysis is computed with a 3-step hierarchical multiple linear regression model with overall job satisfaction as dependent and age, gender, professional experience, workload, and social capital as independent variables (see table [4](#T4){ref-type="table"}). In the first step, only socio-demographic variables (age, gender, and professional experience) are introduced in the model. In the second step, workload is additionally introduced, and in the third step, social capital is added.
In the first step of the regression model, the explained variance of the dependent variable, job satisfaction, accounted for nearly 9%, and only age reached a significance level of p \< .05. In the second step, the explained variance increased up to 18%. Besides workload, age remained significant. In the third step, the explained variance increased up to 37%. Significant information for explaining the dependent variable, job satisfaction, was given by the independent variables, professional experience, workload, and social capital. These variables explained \> 1/3 of the variance of the dependent variable job satisfaction.
In order for a multiple linear regression analysis to be appropriate, it is important to conduct a search focused on residuals to look for evidence that the necessary assumptions -- i. e. normality and homogeneity of variance (homoscedasticity) -- are not violated. Figure [1](#F1){ref-type="fig"} shows the frequency of certain residuals. The value \"0\" indicates that no prediction error occurs. Negative values are corresponding to errors of overestimation, and positive values to errors of underestimation. If the residuals appear at random -- as it is the case in this study -, the distribution of their frequency of occurence should converge to a normal distribution.
{#F1}
Furthermore there should be no relationship between the predicted and residual values in the cumulative probability plot of the residuals. The residuals should be randomly distributed about the horizontal straight line through zero. Figure [2](#F2){ref-type="fig"} shows the cumulative probability plot of the residuals and confirms the assumption of homoscedasticity.
{#F2}
Discussion
==========
To our knowledge, there is no existing literature to date that has explicitly examined the relationship between social capital in hospitals and physician job satisfaction. Therefore, this article extends prior research of social capital in the health care industry by examining the relationship between social capital at the workplace and job satisfaction of clinicians. Our analysis demonstrates that not only subjective workload and professional experience show a statistical significant correlation with job satisfaction, but also social capital in the hospital. Trust, mutual understanding, and shared aims are qualities of social capital, which unify members of social networks and communities and enable them to act cooperatively. Investment in the social capital of an organisation, e.g., a hospital, is a valuable investment in the social system, since the social capital, as shown in this analysis, has a significant impact on job satisfaction. On the basis of the recent literature, it is to be assumed that job satisfaction, in turn, affects well-being and health of an organisation\'s members and therefore the efficiency of the organisation itself. Furthermore, it becomes evident that job satisfaction is significantly associated with professional experience. The reason for this may be partly due to a \"survival\" function, which means that physicians who have found more strategies to maintain their satisfaction are more likely to survive a full career as a physician. It is argued that the higher satisfaction of physicians in later career stages results from \"weeding out\" the less satisfied physicians \[[@B15]\]. It has been further shown that subjective workload is associated with job satisfaction, such that the lower the workload, the higher the job satisfaction. Like many other studies, this analysis confirms again that clinicians consider their workload generally to be high (19.3 points on average of a maximum of 24). In contrast, sociodemographic variables, such as age and gender, did not have a significant impact.
Limitations of the study
------------------------
The current study had methodological limitations that may have affected interpretation of the results. Our cross-sectional design allowed identification of several factors associated with job satisfaction, although causal inferences can hardly be made. Since job satisfaction, as the dependent variable, and all predictor variables were assessed by self-reports, the results might be contaminated by common method variance or self-report bias \[[@B74]\]. Whether the results are applicable to other hospitals is difficult to assess. The selection of the four hospitals intentionally included hospitals in East and West Germany, and hospitals providing maximum and basic healthcare services in an attempt to achieve a form of guided random sampling of German hospitals and hospital-based physicians. On a positive note, a high response rate of 61% was achieved.
Conclusion
==========
Increasing social capital in hospitals requires in-house strategies for reinforcing a culture of trust and willingness to work together. Problems in the hospital or with individuals should be identified using regular, standardized employee polls. We propose regular \"team sessions\" and professional supervision as suitable measures for enhancing the social climate in hospitals and for improving communication structures.
In addition we assume, as often \"the rot starts at the top,\" that the leaders in the middle and upper management levels in hospitals can contribute considerably to strengthen social capital. Management seminars should impart skills of optimising communication structures and processes in the hospital, detecting problems arising in the interaction between staff members and teamwork early, and reacting adequately. Thus, leaders can contribute effectively to improve the working atmosphere significantly in a top-down approach and thus act as role models for the next generation of leaders. This is an important, but too often disregarded task of physicians in leadership positions, especially in hospitals. Furthermore we suggest interventions implemented or designed not only on the individual level (i.e. physicians/leaders) but also on the organizational level. For example, hospital settings should be designed so that there is ample interaction and cooperation among health professionals, emphasizing trust, reciprocity, alliances, bonding, and shared understanding, while promoting organizational justice and conflict resolution. Additionally, we recommend assessing physical and mental symptoms (e.g., burnout, stress) and job satisfaction with standardized instruments within the scope of regular examinations by the hospital medical officer. In this way, first signs of weak social capital or a bad working atmosphere and the potential physical or mental effects on the staff members can be detected early.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
HP designed the study. OO performed the analysis, interpreted the results, and drafted the manuscript. ED interpreted the results, and drafted the manuscript. NE, PS, HP supervised the analysis, helped to interpret the results and participated in the formulation of the discussion. CK, TK, MN helped to interpret the results and contributed to the discussion. All authors reviewed and edited the manuscript for intellectual content.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6963/9/81/prepub>
Acknowledgements
================
The study was funded by the German Federal Ministry of Education and Research. We would like to thank the physicians who participated in the survey and the hospitals supporting this study.
|
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"pile_set_name": "PubMed Central"
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Nowadays, there is growing interest in the development of imaging techniques which are capable of detecting and localizing angiogenesis and neovascularization. These processes induce specific changes in the microvascular structure, represent an established marker for tumours, and also provide indications of tumour aggressiveness[@b1]. In particular, dynamic contrast-enhanced ultrasound (DCE-US) imaging shows promise, with many novel approaches focusing on the direct and/or indirect characterization of the microvasculature. However, when considering the various imaging options, several challenges emerge for imaging the breast.
Typical ultrasound contrast agents (UCAs) are gas-filled microbubbles with diameters ranging between 1 and 10 μm; they are therefore suitable for intravenous injection and can flow through the smallest microvessels. This phenomenon is exploited by super-localization ultrasound techniques which overcome the diffraction limit and are capable of imaging the microvasculature with a spatial-resolution as small as 8--12 μm [@b2][@b3]. Additionally, these techniques provide access to accurate velocity maps, thus offering a powerful tool for the study of microvascular blood flow. However, the relatively long imaging time needed (e.g., \>2 minutes per plane[@b2]), the influence of motion, and the difficulties in imaging and localizing single microbubbles in deep tissue, pose limitations to the use of these modalities in large organs.
Another recently-developed imaging method is acoustic angiography[@b4]. With this technique, high spatial-resolution images (in the order of 100 μm) are obtained using tenfold higher frequencies than with normal DCE-US echo-imaging. Once again, the key lies in the UCAs peculiar response to ultrasound. Because of their highly nonlinear behaviour, UCAs can backscatter high-frequency broadband echo signals (15--35 MHz), which can be used to achieve improved spatial-resolution[@b5]. However, frequency-dependent attenuation practically constrains the applicability of this technique to relatively small depths, such as those required for imaging the peripheral zone of the prostate (1--2 cm).
Other techniques chose a different path rather than targeting high spatial-resolution.
Standard DCE-US imaging (i.e., Harmonic Imaging, Pulse Inversion, and Amplitude Modulation) is an echo graphic technique, which in essence exploits variations in the second harmonic amplitude to generate real-time images of UCA kinetics when flowing through the vasculature[@b6][@b7]. In particular, the analysis of microbubble flow-dynamics through the vessels can be used to reveal changes in the vasculature itself. To this end, several techniques which are based on the quantification of parameters related to UCA perfusion and dispersion have been proposed[@b8][@b9][@b10][@b11][@b12]. Although the typical DCE-US spatial resolution is in the order of 1 mm, hence unsuitable for imaging microvascular changes, these techniques are still able to infer relevant information in relation to the 'angiogenetic switch' (the transition from a pre-vascular to a vascularized tumour phenotype) required for cancer to grow beyond 1--2 mm in diameter[@b13][@b14].
However, performing a handheld DCE-US is particularly challenging for the breast, and makes imaging highly dependent on the skills of the operator. The development of dynamic contrast-specific ultrasound tomography may represent a breakthrough in breast cancer diagnostics, allowing for a more-practical, faster, and less operator-dependent imaging procedure[@b15][@b16]. Moreover, imaging artefacts affect standard DCE-US and limit UCA quantification and localization accuracy[@b17][@b18].
In order to address these shortcomings, and thanks to a newly discovered UCA marker, a contrast-specific imaging modality named cumulative phase delay imaging (CPDI) has recently been proposed for contrast-enhanced ultrasound tomography[@b19][@b20]. CPDI is based on the fact that the diverse physical phenomena behind nonlinear propagation in tissue and UCA are producing a different delay accumulation between the second harmonic (2H) and fundamental (F0) component of the ultrasound field. In particular, a positive delay between 2H and F0 is a marker which is specific to UCAs as opposed to variations in harmonic amplitude (exploited for echo imaging), speed of sound, and attenuation (exploited for ultrasound tomography). CPDI has already proved to be capable of detecting and imaging UCA concentrations when working at pressure regimes (0.05 ≤ MI ≤ 0.2) and frequencies (2.5--3 MHz) of interest for clinical applications. However, although these initial studies demonstrated the feasibility of CPDI, its ability to capture UCA kinetics has never been demonstrated[@b20].
As the achievable spatial resolution for ultrasound tomography is not expected to be comparable with that achievable with acoustic angiography and super-localization techniques, the ability of CPDI to image UCA kinetics is crucial to demonstrate its clinical significance.
Results
=======
This paper reports on the first study to investigate the ability of CPDI to image UCA kinetics. To this end, the passage of repeated UCA boluses through a dedicated gelatin flow-phantom was imaged (see [Fig. 1](#f1){ref-type="fig"}). CPDI and Harmonic Imaging (HI) were simultaneously applied to each bolus passage (in tomography and echo mode, respectively) in order to perform a comparison between the two methods and to analyze the relation between CPD values and harmonic intensity. In this paper, HI specifically refers to the pulse-echo imaging technique which relies on band-pass filters for the extraction of the second harmonic component.
Each bolus resulted from a 5-mL injection with a 240-μL/L UCA dilution. A clinically approved agent, SonoVue® (Bracco, Milan, Italy), was used for this study. To generate and store the ultrasound fields, a ULA-OP[@b21] ultrasound open research platform was employed together with a LA332 linear array probe (Esaote, Firenze, Italy). Insonating frequency, mechanical index (MI), and frame rate were set at 2.5 MHz, 0.07, and 8 Hz, respectively.
The ability of CPDI to capture UCA kinetics and qualitative comparison with HI
------------------------------------------------------------------------------
[Figure 2](#f2){ref-type="fig"} shows, for both CPDI and HI, an example of a time intensity curve (TIC), together with the corresponding images obtained at different time instances. Processed curves (red lines) are obtained using a 0.75-s moving average filter. Various TIC features which are commonly used to quantify UCA kinetics[@b9] are also marked: arrival time (AT), peak time (PT), wash-in time (WIT), and full width half maximum (FWHM). For both imaging modalities, each TIC was obtained by averaging the image values over the surface corresponding to the location of the channel cross-section (indicated by white dashed lines). Qualitatively, the two imaging methods provided similar results.
Reported linear relation between CPD values and 2H intensity and quantitative analysis
--------------------------------------------------------------------------------------
[Figure 3a](#f3){ref-type="fig"} shows the relation between CPD values and second harmonic intensity as a scatterplot of the data-points of all TICs. The blue points refer to the values obtained before the arrival time. A linear relation is observed with a coefficient of determination equal to 0.87. [Figure 3b](#f3){ref-type="fig"} shows a box-plot analysis of the absolute error values calculated for different TIC features when comparing CPDI and HI data. The feature that shows the highest absolute error is FWHM, with a median absolute error value equal to 0.625 s. As for the other features, the median absolute error was 0.25 s, 0.25 s, and 0.0625 s for AT, WIT, and PT, respectively.
The relative standard deviation of the area under the dilution curves, a feature which can be used for perfusion assessment, was also calculated across all the measurements and was found to be equal to 0.18 and 0.17 for CPDI and HI, respectively. Moreover, the mean and standard deviation of the Pearson's correlation coefficient between TICs extracted by CPDI and HI were equal to 0.91 and 0.04, respectively. Overall, the quantitative analysis of TICs obtained with both imaging methods provides equivalent results.
Comparison with speed of sound changes and attenuation due to UCA
-----------------------------------------------------------------
Currently, no contrast-specific modality exists for ultrasound tomography. In fact, speed-of-sound variations and attenuation (normally used to perform ultrasound tomography[@b15][@b16]) due to UCAs can be confused (same range) with those caused by different tissue types[@b20][@b22][@b23][@b24]. Conversely, CPDI is based on a marker specific for UCAs, thus opening the way to dynamic contrast-specific ultrasound tomography.
[Figure 4](#f4){ref-type="fig"} shows the histograms of (a) speed of sound, (b) attenuation (measured at 2.5 MHz), and (c) CPD values (expressed in cycles per m) obtained from our experiment. These histograms were generated by analyzing all the data over the area corresponding to the location of the channel cross-section. Each figure also shows the range of values typically found in breast tissue for each parameter. The data for attenuation and speed of sound in breast tissue were obtained from[@b22] and[@b23], respectively. The tissue range shown in [Fig. 4(c)](#f4){ref-type="fig"} was calculated considering blood, fat, and breast tissue, and based on frequency dispersion as derived from the models described in refs [@b25],[@b26].
When considering speed of sound and attenuation variations, TICs can also be obtained by subtracting the baseline image, i.e., the tomographic image obtained in the absence of contrast, from all subsequent images. For illustrative purposes, [Fig. 5](#f5){ref-type="fig"} shows TICs obtained from speed of sound and attenuation variations corresponding to those shown in [Fig. 2](#f2){ref-type="fig"}. Such an approach would however suffer from motion artefacts; in the presence of motion, the actual baseline image will differ from that measured before contrast enhancement.
Discussion
==========
In this paper the ability of CPDI to image UCA kinetics was investigated for the first time. A qualitative and quantitative comparison with HI was also performed.
Results show that CPDI can be successfully applied to image and quantify UCA kinetics. In particular, when compared to HI, equivalent results were obtained. Variations in speed of sound and attenuation due to UCA were also evaluated, and it was confirmed that they fall within tissue range.
Overall, the speed of sound and attenuation curves are very similar to the curves generated with HI and CPDI. The presence of UCA does in fact alter the speed of the ultrasound wave as well as the attenuation it experiences, the level of the second harmonic amplitude, and the time delay between the second harmonic and fundamental component. However, among all these phenomena, only the accumulation of a positive time delay between the second harmonic and fundamental component is specific to UCA.
When comparing the curves obtained with the different approaches, those based on variations in the attenuation appear to be less affected by amplitude fluctuations. However, as attenuation is not specific to contrast, and attenuation due to contrast is comparable to that exhibited by different tissue types, subtraction techniques will be required in order to exploit attenuation variations to image contrast kinetics. Such an approach is however prone to motion artefacts. Similar problems are encountered in, e.g., diffusion Magnetic Resonance Imaging[@b31], especially with multi-shot acquisition, where several strategies have been developed for motion compensation. However, these strategies add an additional computational layer to the image formation process and are not error free.
CPD values measured during the passage of the UCA boluses were confirmed to be positive. This allows full tissue separation, since CPD values in tissue are inherently negative. The fact that a positive CPD value represents a marker specific to UCA is of particular importance. In principle HI could also be implemented on tomography systems which are capable of reflection tomography. However, this will not avoid typical artefacts common to HI[@b17][@b18], which limit UCA quantification and localization accuracy by HI.
Unlike standard DCE-US in echo-mode, CPDI does not require any particular multi-pulse scheme. In fact, the information required for imaging is contained in the time delay between 2H and F0, which can be extracted from each single pulse[@b19][@b20]. Moreover, with a tomographic approach, only one-way time of flight constrains the pulse repetition frequency. Overall, these aspects allow for a higher time resolution, or faster acquisition time. Furthermore, as opposed to standard uncoded pulse-echo imaging, the pulse-length does not limit the axial-resolution. This allows for the use of longer pulses to enhance penetration and the signal to noise ratio. Moreover, the implementation of CPDI could benefit from existing speed-of-sound reconstruction algorithms which have already been developed for volumetric breast ultrasound scanners[@b27][@b28]; the time-of-flight could simply be replaced with CPD variations. These results are encouraging, and open the way to the development of dynamic contrast-specific ultrasound tomography, which could add important features to the multi-parametric ultrasound tomography of the breast, and improve breast cancer detection.
This paper reports on *in-vitro* results obtained by imaging the passage of UCA boluses through a cylindrical cavity surrounded by a homogeneous medium. In addition, the symmetry of the target was exploited for the tomographic reconstruction, i.e., CPD projections were assumed to be independent on the imaging angle. In real applications these two conditions do not apply and consequently impact on the image quality.
For this reason our future work, thanks to the promising results obtained, will focus on taking new measurements with a breast ultrasound computed tomography scanner on heterogeneous and more complex flow-phantoms, with the ultimate aim of transferring our technology to patients.
Methods
=======
Flow phantom
------------
A dedicated flow phantom was used to perform the experiment. A tissue-mimicking gelatin phantom (as reported in ref. [@b20]) containing a cylindrical cavity with a 6-mm diameter was employed. A solenoid pump E410 (CEME) was used to generate the flow. A calibrated flow value equal to 0.26 L/s was employed. A fixed volume (5 mL) of SonoVue® contrast agent with a concentration equal to 240 μL/L was repeatedly injected (manually) throughout the cavity. The cavity lay at a depth of 5 cm from the location of the probe, and perpendicular to the imaging plane. Further down, at 8 cm, a PVC plate was positioned. The echoes which backscattered from the plate could therefore be recorded by the probe and used to form a tomographic image. At the same time, it was possible to measure the backscattered echoes from the microbubbles and use them to form harmonic images in echo graphic mode. This approach allowed us to compare the two imaging techniques, CPDI and HI, when (simultaneously) imaging the same bolus passage.
Data collection
---------------
An active sub-aperture of 64 elements was used to transmit and receive the ultrasound fields, and linearly shifted over the 192-elements linear array to form a 128-line data set. The field of view was 20 mm wide and 90 mm deep. No focusing was applied in transmission and dynamic receive beamforming was used.
The post beam formed data were stored and used for the analysis. A 10-cycle pulse with its centre frequency at 2.5 MHz, and whose amplitude was modulated by a Hamming envelope, was used as a driving signal. In receive mode, a sampling frequency of 50 MHz was used. A total of 80 frames were consecutively acquired for each bolus, at a frame rate of 8 Hz. A mechanical index (MI) equal to 0.07, which was measured with a hydrophone (HGL-0400 Onda, Sunnyvale, CA) at a depth of 5 cm, was used to minimize bubble disruption. The hydrophone, with a bandwidth ranging from 250 kHz to 20 MHz, was connected to a preamplifier (AH-2010-025 Onda, Sunnyvale, CA) whose bandwidth ranged from 50 kHz to 25 MHz, which in turn was connected to a 100 MHz A/D converter (PCI-5406 National Instruments, Austin, TX). Dedicated Labview® software was implemented and used for data acquisition. The MI was calculated as the ratio of the peak negative pressure in MPa and the square root of the frequency in MHz.
Data analysis
-------------
All data processing and analysis was performed using Matlab® R2014a.
Harmonic Imaging
----------------
The second harmonic (2H) component was extracted by band-pass filtering. A −12 dB bandwidth around 5 MHz was selected. Subsequently, a two-dimensional spatial Gaussian filter (spatial standard deviation *σ*~x~ = 0.5 mm and *σ*~z~ = 0.25 mm) was applied to enhance the signal-to-noise ratio. To reconstruct the harmonic images, an average speed of sound equal to 1510 m/s was assumed.
Cumulative Phase Delay Imaging
------------------------------
In order to measure the cumulative phase delay (CPD) between F0 and 2H, firstly, the pressure fields which had backscattered from the plate were selected by time-windowing (window length equal to 5 μs). Secondly, the two components, i.e., F0 and 2H, were extracted from the data (a −12 dB bandwidth was selected around 2.5 MHz and 5 MHz, respectively) and the corresponding envelopes were obtained by using the Hilbert transform. Thirdly, the time delay between the maxima of the fundamental and second harmonic envelopes was obtained for each line, providing a measure of CPD as a function of the lateral direction, i.e., a projection. For this experiment, the symmetry of the target was exploited for the reconstruction, in other words, CPD projections were assumed to be independent on the imaging angle. Finally, the filtered back-projection (filter type: Shepp-Logan) algorithm[@b29] was used to generate 2D-CPD images.
Time Intensity Curve
--------------------
To obtain the time intensity curves from the HI and CPDI data, the image intensity values were averaged over the region corresponding to the location of the cavity. Following that, a moving average filter (window size equal to 0.75 s) was applied, and the baseline removed from the CPDI and HI TICs. The baseline was calculated as the mean value over the first 0.75 s. The average CPDI baseline was equal to −0.6 cycles/m.
Quantitative analysis
---------------------
The peak time (PT) was calculated as the time when the maximum of a TIC was observed. The arrival time (AT) was estimated as the first time the TIC amplitude exceeded 5% of its value at PT. The wash-in time (WIT) was calculated as the time between AT and PT. The full width half maximum (FWHM) was calculated as the time interval over which the TIC amplitude stayed above 50% of its value at PT. The interval corresponding to the bolus passage is defined as the time between AT and the time showing a drop in TIC-amplitude below 5% of the value at PT. The area under the curve was derived as the sum of the TIC-amplitude values over the entire time window (10 s long) multiplied by the length of the sampling interval (0.125 s).
Speed-of-sound and attenuation estimation
-----------------------------------------
By using the same filtered back-projection algorithm that was applied to perform CPDI, tomographic images based on speed-of-sound and attenuation were obtained from the variations in the time-of-flight and amplitude of the peak of the fundamental envelope, respectively. Subsequently, histograms were obtained by analysing the image values over the region corresponding to the location of the cavity, during the passage of the boluses.
Dispersion evaluation
---------------------
Values of CPD in tissue are determined by the combined effect of tissue nonlinearity and frequency dispersion[@b20]. Both phenomena result in a negative CPD value. However, the contribution due to tissue nonlinearity can be assumed to be negligible as compared to that due to frequency dispersion, especially for highly absorbing tissues. In fact, in line with the Kramers-Kronig relationship[@b30], dispersion and absorption are interlinked. In conclusion, to evaluate CPD values, absorption measures reported in ref. [@b26] for blood, fat, and breast tissue were used in combination with the theoretical model in ref. [@b25].
Additional Information
======================
**How to cite this article**: Demi, L. *et al*. Towards Dynamic Contrast Specific Ultrasound Tomography. *Sci. Rep.* **6**, 34458; doi: 10.1038/srep34458 (2016).
This work was supported by the European Research Council Starting Grant (\#280209) and by the Dutch Technology Foundation (STW) VIDI Grant (\#10769).
**Author Contributions** L.D., R.J.G.V.S. and M.M. conceived the experiment. L.D. and R.J.G.V.S. designed and performed the experiment. L.D., R.J.G.V.S., M.M. and H.W. contributed to data-analysis and writing/revising the manuscript.
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van de Brake HJ, Walter F, Rink FA, Essens PJMD, van der Vegt GS. The dynamic relationship between multiple team membership and individual job performance in knowledge‐intensive work. J Organ Behav. 2018;39:1219--1231. 10.1002/job.2260
1. INTRODUCTION {#job2260-sec-0001}
===============
In an attempt to use scarce human resources as effectively and efficiently as possible, knowledge‐based organizations increasingly rely on flexible project teams in which memberships are frequently shared, shifted, and dissolved (Mortensen, [2014](#job2260-bib-0051){ref-type="ref"}). Within such contexts, many individuals work on more than one project at the same time (O\'Leary, Mortensen, & Woolley, [2011](#job2260-bib-0058){ref-type="ref"}), enabling various teams to concurrently benefit from their expertise. For example, individual research and development employees often work simultaneously on several project teams, with each team utilizing their specific knowledge and contributions (Bertolotti, Mattarelli, Vignoli, & Macrì, [2015](#job2260-bib-0003){ref-type="ref"}). Similarly, many academics are concurrently involved in multiple research and teaching teams. Scholars have estimated that such multiple team membership (MTM) occurs among at least 65% of employees across a wide range of occupations (Mortensen, Woolley, & O\'Leary, [2007](#job2260-bib-0052){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}).
As a result of this development, there is growing scholarly interest in the consequences of MTM (Wageman, Gardner, & Mortensen, [2012](#job2260-bib-0077){ref-type="ref"}). Much of this research has focused on the team level of analysis, illustrating for example that members\' simultaneous involvement in various other teams may shape a focal team\'s performance outcomes (e.g., Bertolotti et al., [2015](#job2260-bib-0003){ref-type="ref"}; Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}). Importantly, however, multi‐teaming may also distinctly influence individual employees\' work experiences and behaviors (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}). Compared with more traditional contexts with clearly defined and delimited team memberships, individuals may face unique opportunities and challenges from their involvement in multiple organizational teams (i.e., individual MTM; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Beyond team‐level performance implications, it therefore seems critical to understand how an individual employee\'s MTM can shape his or her job performance (i.e., an employee\'s overall contributions toward the organization\'s goal achievement across tasks and teams; Borman & Motowidlo, [1997](#job2260-bib-0006){ref-type="ref"}).
The scarce empirical research on individual MTM has generally focused on MTM\'s relatively proximal, psychological, and cognitive consequences (e.g., employees\' project overload and work engagement; Pluut, Flestea, & Curşeu, [2014](#job2260-bib-0062){ref-type="ref"}; Zika‐Viktorsson, Sundström, & Engwall, [2006](#job2260-bib-0079){ref-type="ref"}). These studies have created important insights, and they make it plausible to assume that an individual\'s MTM may also shape his or her job performance as a more distal---yet vitally important---outcome variable. Importantly, however, the existing research has not directly examined MTM\'s role for an employee\'s overall job performance. What is more, theoretical arguments about MTM\'s potential performance consequences have remained ambiguous. Some scholars have suggested that MTM can provide employees with important resources that may enhance their job performance, for example, by increasing their social network or creating unique learning opportunities (Hansen, [1999](#job2260-bib-0027){ref-type="ref"}; Vedres & Stark, [2010](#job2260-bib-0076){ref-type="ref"}). By contrast, other researchers have argued that MTM imposes considerable demands upon employees that may lower their job performance, for example, by forcing employees to regularly relocate and/or to shift between distinct tools, tasks, and technologies (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}; Zika‐Viktorsson et al., [2006](#job2260-bib-0079){ref-type="ref"}). As a result, the performance implications of MTM remain unclear.
Beyond ambiguity about the possible performance benefits and detriments of MTM for individual employees, the current literature cannot answer key questions about the direction of MTM‐performance linkages. Research on team staffing (Hackman & Wageman, [2004](#job2260-bib-0025){ref-type="ref"}) and individuals\' preferred work characteristics (LePine, Podsakoff, & LePine, [2005](#job2260-bib-0038){ref-type="ref"}) suggests that there is a distinct possibility of reciprocal causation, such that the relationship between individual MTM and an employee\'s overall job performance may also flow in the opposite direction. Changes in an employee\'s job performance might shape his or her subsequent MTM, in particular, because (a) managers tend to select high performers when staffing their teams and (b) performance growth may increase an employee\'s confidence and motivation to join additional team settings. Consequently, employees whose performance has improved may experience a subsequent increase in their MTM. The link between an individual\'s MTM and job performance may thus be more intricate than previously believed, with these constructs either amplifying or counteracting each other over time (cf. Maruyama, [1963](#job2260-bib-0046){ref-type="ref"}; Weick, [1979](#job2260-bib-0078){ref-type="ref"}).
The present research uses a novel, dynamic approach to address the above issues. We draw from the notion that increasing an employee\'s MTM may both augment and diminish job‐related resources and build on two resource‐based theories (i.e., social capital and conservation of resources theory; Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}; Lin, [1999](#job2260-bib-0042){ref-type="ref"}) to develop competing hypotheses about the way changes in individual MTM may relate with subsequent changes in employees\' overall job performance. We pit these competing perspectives against each other, using data from 1,875 knowledge workers. Whereas prior work has typically used cross‐sectional, between‐person designs to examine the association between MTM and performance‐related outcomes (e.g., Chan, [2014](#job2260-bib-0010){ref-type="ref"}; Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}; Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}), we adopt a longitudinal, within‐person study design to investigate this linkage over time. This approach enables us to examine whether changes in an employee\'s overall job performance may relate to subsequent changes in his or her MTM (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}; O\'Leary, Woolley, & Mortensen, [2011](#job2260-bib-0059){ref-type="ref"}) and allows us to investigate potentially reciprocal relationships between these variables over time.
Taken together, the present investigation strives to realize several contributions to the MTM literature. Extending previous theory and research on team‐level MTM and on the psychological consequences of individual‐level MTM, we aim to increase our understanding of how individual employees\' engagement and disengagement with multiple concurrent teams relates to their overall job performance over time. More specifically, our goal is to resolve existing ambiguities about the linkage between individuals\' MTM and job performance by clarifying (a) whether MTM\'s performance benefits or drawbacks will prevail, (b) how this relationship unfolds over time, and (c) whether an employee\'s MTM may serve both as an antecedent and as a consequence of his or her job performance. To achieve this goal, we introduce a longitudinal, within‐person perspective to the study of individual MTM that investigates the relationship between changes in employees\' MTM and job performance over time. This dynamic perspective moves beyond the static approaches prevalent in most of the MTM research to date, promoting theory advancement by enabling unique insights into the complex, potentially reciprocal within‐person relationships between individual employees\' MTM and job performance.
2. THEORY AND HYPOTHESES {#job2260-sec-0002}
========================
2.1. Individual employees\' MTM {#job2260-sec-0003}
-------------------------------
Prior studies have typically conceptualized MTM at the team level of analysis, such that MTM represents the extent to which a focal team\'s members are, on average, involved in other teams as well (e.g., Bertolotti et al., [2015](#job2260-bib-0003){ref-type="ref"}; Mortensen, [2014](#job2260-bib-0051){ref-type="ref"}). Importantly, we hold that such team‐level MTM\'s origins are located at the individual level of analysis, denoting the extent to which individual employees simultaneously are members of more than one (project) team (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Empirically, this is reflected in the number of teams to which an individual allocates working time during a specific period (e.g., on a weekly basis; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Recent studies have found that it is rather common for individuals in some occupations, especially in knowledge‐based work, to simultaneously be a member of up to eight or nine teams (Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}; Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}). Moreover, theorists have emphasized the potential relevance of such individual MTM, arguing that it may create unique experiences, demands, and possibilities at work that decisively shape an employee\'s job‐related attitudes, behaviors, and outcomes (e.g., Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). As such, this study examines MTM at the individual level of analysis, defining the concept as an individual employee\'s number of concurrent team memberships.
An employee\'s individual MTM differs from related concepts such as multitasking (Leroy, [2009](#job2260-bib-0040){ref-type="ref"}) and task switching (Monsell, [2003](#job2260-bib-0050){ref-type="ref"}). Multitasking, for instance, refers to a situation in which an employee simultaneously carries out two or more tasks, whereas MTM reflects the number of concurrent teams to which an individual allocates time and attention (Salvucci & Taatgen, [2008](#job2260-bib-0066){ref-type="ref"}). This distinction is important for two reasons. First, MTM does not necessarily involve frequent task switching (and vice versa). Even an employee with high MTM may avoid excessive changes between different tasks, for example, by compartmentalizing his or her working time into predictable sequences (e.g., working for the first team on Monday and Tuesday and the second team on Wednesday and Thursday; Monsell, [2003](#job2260-bib-0050){ref-type="ref"}). Second, both multitasking and task switching usually refer to how employees deal with multiple individual task assignments (Salvucci & Taatgen, [2008](#job2260-bib-0066){ref-type="ref"}). In contrast, MTM is inherently social and interactive, as relevant assignments are carried out interdependently within multiple team contexts (Van Der Vegt, Van De Vliert, & Oosterhof, [2003](#job2260-bib-0075){ref-type="ref"}; Wageman et al., [2012](#job2260-bib-0077){ref-type="ref"}). As such, individual MTM is a unique phenomenon, with causes and consequences that cannot be directly derived from existing knowledge on multitasking and task switching.
2.2. Individual MTM as an antecedent of job performance: A resource‐based perspective {#job2260-sec-0004}
-------------------------------------------------------------------------------------
As noted before, changes in an employee\'s MTM may go along with unique advantages and disadvantages that, ultimately, can enhance or diminish his or her overall job performance (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). In particular, we propose that an increase in MTM may either improve access to, or distract from, key resources required for an employee\'s effective performance at work. Hence, we draw from two prominent theoretical perspectives that both highlight an employee\'s job‐related resources (i.e., valued entities that serve to achieve job‐related ends; Hobfoll, [1989](#job2260-bib-0029){ref-type="ref"}) as key determinants of individual performance. Social capital theory (Kwon & Adler, [2014](#job2260-bib-0037){ref-type="ref"}), on the one hand, points toward possible resource gains that can be achieved through complex interpersonal work arrangements, such as MTM (Lin, [1999](#job2260-bib-0042){ref-type="ref"}). Conservation of resources theory (Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}, [1989](#job2260-bib-0029){ref-type="ref"}), on the other hand, emphasizes possible resource losses that can arise from changes in an employee\'s working conditions (for an overview, see Halbesleben, [2006](#job2260-bib-0026){ref-type="ref"}). Accordingly, the first perspective suggests that an increase in MTMs may increase an employee\'s overall performance levels, whereas the latter perspective suggests that an increase in MTM may decrease an employee\'s job performance. As these conceptual approaches lead to competing hypotheses about the role of MTM changes for subsequent performance developments, they therefore allow us to conceptually disentangle both the positive and negative aspects of an employee\'s concurrent memberships in multiple teams.
### 2.2.1. A social capital perspective on MTM\'s consequences {#job2260-sec-0005}
Social capital theory suggests that an employee\'s social network (i.e., his or her interpersonal connections with coworkers; Borgatti & Foster, [2003](#job2260-bib-0005){ref-type="ref"}) entails valuable interpersonal resources (e.g., knowledge, information, instrumental, and social support) and it defines social capital as an employee\'s capacity to access and utilize these resources (Lin, [1999](#job2260-bib-0042){ref-type="ref"}, [2002](#job2260-bib-0043){ref-type="ref"}). Such social capital is known to be a key factor that can facilitate an employee\'s job performance, because individuals with greater social capital can more easily draw on the resources required to promote their performance outcomes (Kwon & Adler, [2014](#job2260-bib-0037){ref-type="ref"}; Thompson, [2005](#job2260-bib-0071){ref-type="ref"}). Within innovative, nonroutine work contexts, it is particularly useful to establish new connections across distinct organizational subunits (e.g., teams), as these linkages provide access to a greater diversity of perspectives and information, political connections across various parts of the organization, and differing types of expertise (Cross & Cummings, [2004](#job2260-bib-0017){ref-type="ref"}; Lin, [1999](#job2260-bib-0042){ref-type="ref"}).
Based on this notion, it seems plausible to argue that an increase in an employee\'s MTM enables additional productive connections across different teams, thus promoting the social capital needed to achieve higher performance levels. Indeed, by its very definition, MTM requires individuals to cooperate with other employees from multiple distinct teams, often with diverse areas of expertise (O\'Leary, Woolley, et al., [2011](#job2260-bib-0059){ref-type="ref"}). Hence, increasing MTM may enable employees to work with a greater number of previously unfamiliar colleagues, project leaders, and clients, thus providing access to valuable resources that are embedded within different teams and offering the unique opportunity to transfer these resources across team contexts (Choi & Thompson, [2005](#job2260-bib-0013){ref-type="ref"}; Tasselli, Kilduff, & Menges, [2015](#job2260-bib-0070){ref-type="ref"}; Vedres & Stark, [2010](#job2260-bib-0076){ref-type="ref"}). An increase in MTM may, for example, expose an employee to new knowledge sources that spark his or her creativity (Grant, [1996](#job2260-bib-0023){ref-type="ref"}; Perry‐Smith, [2006](#job2260-bib-0061){ref-type="ref"}), familiarize the employee with innovative work practices that could be useful in other team settings as well (e.g., by sharing best practices; Burt, [1992](#job2260-bib-0008){ref-type="ref"}), and create opportunities to establish new and meaningful relationships with coworkers in various parts of the organization (Hansen, [1999](#job2260-bib-0027){ref-type="ref"}; Van der Doef & Maes, [1999](#job2260-bib-0074){ref-type="ref"}). Individuals with stable MTM levels over time, by contrast, have to rely on their existing social resources to a greater extent and, thus, may find it more difficult to realize such opportunities for creativity, learning, and knowledge exchanges.
Taken together, this reasoning suggests that MTM may represent a distinct source of social capital (beyond an employee\'s sheer number of interpersonal connections; Borgatti & Foster, [2003](#job2260-bib-0005){ref-type="ref"}). As such, increasing MTM may provide unique performance advantages for the respective individuals.Hypothesis 1aAn increase in an employee\'s MTM is related to a subsequent increase in his or her overall job performance.
### 2.2.2. A conservation of resources perspective on MTM\'s consequences {#job2260-sec-0006}
Importantly, however, there are also good conceptual reasons to expect a fundamentally different pattern. Conservation of resources theory, in particular, argues that people seek to obtain, retain, and protect valuable resources that help them to perform effectively, and that stress occurs when such resources are threatened or depleted (Brotheridge & Lee, [2002](#job2260-bib-0007){ref-type="ref"}; Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}). In organizational settings, the most widely studied of these resources relate to employees\' perceived ability to control important aspects of their work (Skinner, [1996](#job2260-bib-0067){ref-type="ref"}; Van der Doef & Maes, [1999](#job2260-bib-0074){ref-type="ref"}) and to the time and attention employees are able to direct toward completing their tasks (Hobfoll, [1989](#job2260-bib-0029){ref-type="ref"}; Thompson, [2005](#job2260-bib-0071){ref-type="ref"}). Empirical research has demonstrated that substantial losses of these resources can diminish an employee\'s overall functioning (e.g., by invoking stress and decreasing task efficiency; Halbesleben, [2006](#job2260-bib-0026){ref-type="ref"}; LePine et al., [2005](#job2260-bib-0038){ref-type="ref"}; Rich, Lepine, & Crawford, [2010](#job2260-bib-0063){ref-type="ref"}). As outlined below, we argue that increasing MTM may directly affect an employee\'s perceived control over his or her tasks across various teams and, relatedly, the time he or she has available to meet each team\'s demands. It therefore appears plausible, from this perspective, to suggest that an increase in MTM may decrease an employee\'s subsequent job performance.
First, increasing MTM may reduce an employee\'s ability to control important aspects of the job. An increase in MTM implies that an individual\'s tasks and interdependencies are spread out over a greater number of concurrent teams (Mortensen, [2014](#job2260-bib-0051){ref-type="ref"}; Wageman et al., [2012](#job2260-bib-0077){ref-type="ref"}), such that he or she encounters a greater variety of task requirements and interpersonal expectations from additional colleagues, managers, and clients across diverse team settings (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Accordingly, increases in MTM require an employee to adjust to new team roles and adapt to the unique characteristics of each respective team (Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}; Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}). This may obstruct an employee\'s ability to effectively comprehend the novel procedures, knowledge domains, and social demands relevant for each team\'s task accomplishment, potentially lowering the employee\'s sense of control and, consequently, reducing his or her overall job performance (Hobfoll, [1989](#job2260-bib-0029){ref-type="ref"}; 2002; Kauppila, [2014](#job2260-bib-0034){ref-type="ref"}; O\'Leary, Woolley, et al., [2011](#job2260-bib-0059){ref-type="ref"}).
Second, an increase in MTM decreases the amount of time an employee can spend on a team before having to move on to the next assignment, in a different team context (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}; Rich et al., [2010](#job2260-bib-0063){ref-type="ref"}). This may pose considerable challenges for the effective organization of an employee\'s task routines and time scheduling. Each additional team membership, for example, increases the amount of effort required to catch up with work done in an employee\'s absence, and it decreases his or her available time to adjust to distinct tools, tasks, and technologies used within each specific team (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}; Zika‐Viktorsson et al., [2006](#job2260-bib-0079){ref-type="ref"}).
Together, this reasoning suggests that an increase in MTM may deplete an employee\'s performance potentials (Halbesleben, [2006](#job2260-bib-0026){ref-type="ref"}; Hobfoll, [1989](#job2260-bib-0029){ref-type="ref"}). This rationale is consistent with scholarly arguments pointing to MTM\'s demanding and highly complicated nature as a key source of job strain, lowered satisfaction, and reduced work engagement (Kauppila, [2014](#job2260-bib-0034){ref-type="ref"}; Leroy, [2009](#job2260-bib-0040){ref-type="ref"}; Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}). Consequently, our second hypothesis isHypothesis 1bAn increase in an employee\'s MTM is related to a subsequent decrease in his or her overall job performance.
2.3. Individual MTM as a consequence of job performance {#job2260-sec-0007}
-------------------------------------------------------
So far, we have discussed changes in MTM as an antecedent of an individual employee\'s job performance. Although this reasoning appears theoretically plausible, it seems equally possible that the MTM‐performance linkage follows a reversed direction. Specifically, an increase in an employee\'s performance may associate with an increase in his or her subsequent MTM because increased performance may result in a greater number of requests to join additional teams and increase an employee\'s willingness to accept such requests. [^1]
Research on employee staffing and team member selection suggests that an employee\'s prior job performance may shape his or her attractiveness as a prospective team member (Hinds, Carley, Krackhardt, & Wholey, [2000](#job2260-bib-0028){ref-type="ref"}). When looking for qualified individuals to staff a specific (project) team, it is clear that team leaders and project managers typically strive to attract employees with high potential (Kerzner, [2013](#job2260-bib-0035){ref-type="ref"})---and individual employees\' prior performance trajectories offer an important indication of this potential. Individuals who have exhibited marked performance improvements in the past, in particular, are likely to be motivated and willing to exert effort, and they have demonstrated the ability to learn and adapt (Borman & Motowidlo, [1997](#job2260-bib-0006){ref-type="ref"}). Such individuals implicitly signal, therefore, that they have the potential to develop themselves and, hence, to handle additional task demands and projects (Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}). Similar effects may occur in self‐managing teams, where members themselves take responsibility for staffing decisions (Alper, Tjosvold, & Law, [1998](#job2260-bib-0002){ref-type="ref"}; Chuboda, Wynn, Lu, & Watson‐Manheim, [2005](#job2260-bib-0014){ref-type="ref"}). In these teams, the existing members typically look for new teammates through informal social connections and previous work experiences (Casciaro & Lobo, [2008](#job2260-bib-0009){ref-type="ref"}). In doing so, a candidate\'s prior performance improvements and associated reputation gains may again play a critical role (D\'Souza & Colarelli, [2010](#job2260-bib-0020){ref-type="ref"}; LePine & Van Dyne, [2001](#job2260-bib-0039){ref-type="ref"}).
Another reason why a reversed direction in the MTM‐performance linkage is possible is that employees who have experienced improved overall job performance in the past may be more inclined to proactively seek and accept memberships in additional teams. In this regard, research suggests that positive performance feedback increases an employee\'s confidence in his or her ability to manage complex and demanding working conditions (Jex, Bliese, Buzzell, & Primeau, [2001](#job2260-bib-0033){ref-type="ref"}; Kim & Hamner, [1976](#job2260-bib-0036){ref-type="ref"}). Consequently, such employees may be more open to new challenges that create opportunities for future growth, as compared with employees whose job performance has stagnated or even decreased (LePine et al., [2005](#job2260-bib-0038){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Additional team memberships may provide them with such challenges (e.g., through social network expansion, learning opportunities, and increased task diversity; Bertolotti et al., [2015](#job2260-bib-0003){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Indeed, a qualitative study found that MTM "provides employees with opportunities to shape their careers by joining projects related to expertise they have or want to develop" (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}: 5).
Taken together, this reasoning suggests that employees who have recently increased their performance will receive and accept a disproportionally higher number of invitations for concurrent team memberships. Consequently, we proposeHypothesis 2An increase in an employee\'s overall job performance is related to a subsequent increase in his or her MTM.
2.4. The dynamic relationship between changes in MTM and overall job performance {#job2260-sec-0008}
--------------------------------------------------------------------------------
Whereas our competing Hypotheses [1a](#job2260-mathstat-0001){ref-type="statement"} and [1b](#job2260-mathstat-0002){ref-type="statement"} propose that an increase in an individual employee\'s MTM will either positively or negatively associate with his or her subsequent job performance, Hypothesis [2](#job2260-mathstat-0003){ref-type="statement"} predicts that an employee\'s increasing job performance will positively associate with an increase in his or her subsequent MTM. Taken together, these hypotheses point toward potentially dynamic, reciprocal relationships between changes in an individual\'s MTM and overall job performance. Corroborating Hypotheses [1a](#job2260-mathstat-0001){ref-type="statement"} and [2](#job2260-mathstat-0003){ref-type="statement"}, on the one hand, would suggest that increases in MTM and job performance reinforce each other in a positive, "deviation‐amplifying" feedback loop that spirals both variables toward higher levels over time (Weick, [1979](#job2260-bib-0078){ref-type="ref"}, p. 73). A decrease in individual MTM or job performance, by contrast, would then pose a major risk factor that could trigger a downward‐spiraling relationship.
Corroborating Hypotheses [1b](#job2260-mathstat-0002){ref-type="statement"} and [2](#job2260-mathstat-0003){ref-type="statement"}, on the other hand, would suggest that changes in individual MTM and job performance are dynamically related in a "deviation‐counteracting" feedback loop (Weick, [1979](#job2260-bib-0078){ref-type="ref"}, p. 74), such that increases in one variable would instigate decreases in the other, inducing relative stability (despite minor oscillations) in the long run. This would imply that increasing MTM neutralizes an employee\'s previous performance improvements (and vice versa), thus leading toward stagnating MTM and performance levels.
Clearly, these divergent patterns of reciprocal relationships would carry important implications for our theoretical understanding of the linkage between individual MTM and job performance over time. Hence, we will closely scrutinize these potential dynamics in the following. Given that our competing predictions in Hypotheses [1a](#job2260-mathstat-0001){ref-type="statement"} and [1b](#job2260-mathstat-0002){ref-type="statement"} leave considerable ambiguity about the expected shape of these associations, however, we decided to not develop formal hypotheses in this regard.
3. METHODS {#job2260-sec-0009}
==========
3.1. Sample and data collection {#job2260-sec-0010}
-------------------------------
To test our hypotheses, we used a sample of knowledge workers from an organization of applied research with roughly 3,500 employees, located in the Netherlands. Work within this organization was structured along (contract) research projects, with project managers attracting funding and subsequently staffing temporary teams with suitable employees. In addition, employees had the opportunity to proactively apply for specific team memberships by approaching the respective project managers (who retained final say over staffing decisions). Although the organization did not publicly communicate individuals\' formal performance appraisals, project managers were generally well aware of relevant employees\' performance reputation. In part, this was because work within the organization was highly collaborative and required extensive exchange of information and materials with employees across multiple teams, departments, and knowledge domains. As such, MTM was a relatively common phenomenon within our host organization, offering an ideal setting to examine the linkage between individual employees\' MTM and overall job performance.
We obtained longitudinal data from the organization\'s personnel records, spanning five consecutive years (2008--2012). Specifically, the organization provided weekly work hour registrations for all 3,348 individuals permanently employed with the organization. These employees were obliged to register the number of work hours spent for different project teams in a very detailed manner. Among other things, the organization used this information for billing purposes and to calculate project costs; hence, project managers closely monitored the accuracy of these registrations. Further, the department of human resources supplied us with demographic information and yearly performance evaluations for all employees. Given our study\'s focus, we excluded individuals who, due to the nature of their tasks, were not involved in specific project teams (i.e., lower level administrative personnel and general managers). Finally, an employee\'s inclusion in this study required the availability of complete demographic information as well as data on both MTM and performance for at least one time point each (Li, Fay, Frese, Harms, & Gao, [2014](#job2260-bib-0041){ref-type="ref"}).
Our final sample comprised 1,875 employees that carried out applied research in project teams. These employees were well educated (i.e., they had at least a bachelor\'s degree) and predominantly male (74%); their mean age was 41 years (*SD* = 10.5), and they had been working with the organization for an average of 11 years (*SD* = 9.6) at the beginning of our study period. Moreover, most of the employees in our sample worked on a full‐time basis (81%); they worked on approximately 13 projects per year, with an average of 2.7 projects per week (range = 1--10). Almost all of the sample employees (96%) were, at some point during the 5‐year study period, members of more than one project team at the same time.
As is common in longitudinal research, there were missing data across the different time points. Of the 1,875 sample employees, complete data were available for 1,218 individuals in Year 1; 1,337 individuals in Year 2; 1,452 individuals in Year 3; 1,463 individuals in Year 4; and 1,497 individuals in Year 5. For 947 employees, complete data were available across all study years. The missing data in the present sample predominantly resulted from individuals that, during our study period (a) entered or left the organization, (b) moved to a position within the organization that did not involve work in research projects (e.g., departmental leadership), or (c) were absent for an extended period of time (e.g., due to sickness, pregnancy, or a sabbatical). Following recommendations of Graham ([2009](#job2260-bib-0022){ref-type="ref"}) and Nakai and Ke ([2011](#job2260-bib-0056){ref-type="ref"}), we used maximum likelihood estimation for models with partial missing data when testing the study hypotheses through latent change score models (as outlined below), enabling us to fully utilize all information available in the present sample. We note that the results and conclusions remained virtually unchanged, however, when using a listwise deletion procedure for hypotheses testing. [^2]
3.2. Measures {#job2260-sec-0011}
-------------
We captured employees\' MTM and job performance for each of the 5 years during our study period. Identical procedures were used each year to measure these constructs.
### 3.2.1. MTM {#job2260-sec-0012}
Similar to prior research (e.g., Chan, [2014](#job2260-bib-0010){ref-type="ref"}; Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}), we measured an employee\'s MTM as the number of concurrent project teams in which he or she was actively involved. Contrary to this earlier work, however, we used archival data (rather than survey measures) to obtain a detailed indication of an individual employee\'s number of active team memberships. Specifically, the employees in our sample reported their team‐related working time on a weekly basis through the formal work hour registrations mentioned above, and we used these archival data to capture the number of teams to which an individual allocated working time during a specific week. [^3] To match the annual job performance measure available within personnel records (see below), we subsequently used this information to calculate an employee\'s average MTM within each year of the study period. Our measure therefore represents an individual employee\'s annual average number of teams per week. Conceptually, this implies that MTM increases when an individual becomes actively involved (i.e., spends time) in a greater number of teams (cf. Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}). Mirroring recent reports of increasing MTM across many organizations and occupations (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"})---and corroborating the relevance of our dynamic approach toward examining MTM---our data illustrate a slight trend toward increased multi‐teaming during the study period (see Table [1](#job2260-tbl-0001){ref-type="table"}).
######
Means, standard deviations, and Pearson correlation coefficients
Variable Mean *SD* 1 2 3 4 5 6 7 8 9 10 11 12 13
----------------------------------- ------- ------ ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- ----------------------------------------------
1\. Organizational tenure (years) 11.13 9.61
2\. Gender (F = 0, M = 1) .74 .44 .22[\*\*](#job2260-note-0006){ref-type="fn"}
3\. Salary (standardized) .00 1.00 .49[\*\*](#job2260-note-0006){ref-type="fn"} .24[\*\*](#job2260-note-0006){ref-type="fn"}
4\. FTE .94 .11 .02 .24[\*\*](#job2260-note-0006){ref-type="fn"} --.13[\*\*](#job2260-note-0006){ref-type="fn"}
5\. MTM (Y1) 2.68 1.15 .13[\*\*](#job2260-note-0006){ref-type="fn"} −.02 .10[\*\*](#job2260-note-0006){ref-type="fn"} .05
6\. MTM (Y2) 2.74 1.20 .07[\*\*](#job2260-note-0006){ref-type="fn"} −.02 .06[*\**](#job2260-note-0005){ref-type="fn"} .07[*\**](#job2260-note-0005){ref-type="fn"} .75[\*\*](#job2260-note-0006){ref-type="fn"}
7\. MTM (Y3) 2.77 1.20 .04 −.03 .07[\*\*](#job2260-note-0006){ref-type="fn"} .07[\*\*](#job2260-note-0006){ref-type="fn"} .63[\*\*](#job2260-note-0006){ref-type="fn"} .74[\*\*](#job2260-note-0006){ref-type="fn"}
8\. MTM (Y4) 2.73 1.85 .04 --.06[\*\*](#job2260-note-0006){ref-type="fn"} .05[*\**](#job2260-note-0005){ref-type="fn"} .07[\*\*](#job2260-note-0006){ref-type="fn"} .55[\*\*](#job2260-note-0006){ref-type="fn"} .60[\*\*](#job2260-note-0006){ref-type="fn"} .73[\*\*](#job2260-note-0006){ref-type="fn"}
9\. MTM (Y5) 2.84 1.23 .05[*\**](#job2260-note-0005){ref-type="fn"} --.06[*\**](#job2260-note-0005){ref-type="fn"} .04 .04 .52[\*\*](#job2260-note-0006){ref-type="fn"} .58[\*\*](#job2260-note-0006){ref-type="fn"} .64[\*\*](#job2260-note-0006){ref-type="fn"} .78[\*\*](#job2260-note-0006){ref-type="fn"}
10\. Performance (Y1) 3.30 .58 --.25[\*\*](#job2260-note-0006){ref-type="fn"} --.06[*\**](#job2260-note-0005){ref-type="fn"} --.15[\*\*](#job2260-note-0006){ref-type="fn"} .10[\*\*](#job2260-note-0006){ref-type="fn"} .08[*\**](#job2260-note-0005){ref-type="fn"} .08[\*\*](#job2260-note-0006){ref-type="fn"} .10[\*\*](#job2260-note-0006){ref-type="fn"} .08[*\**](#job2260-note-0005){ref-type="fn"} .08[\*\*](#job2260-note-0006){ref-type="fn"}
11\. Performance (Y2) 3.28 .60 --.24[\*\*](#job2260-note-0006){ref-type="fn"} --.05[*\**](#job2260-note-0005){ref-type="fn"} --.17[\*\*](#job2260-note-0006){ref-type="fn"} .11[\*\*](#job2260-note-0006){ref-type="fn"} −.01 .08[\*\*](#job2260-note-0006){ref-type="fn"} .06[*\**](#job2260-note-0005){ref-type="fn"} .06[*\**](#job2260-note-0005){ref-type="fn"} .02 .44[\*\*](#job2260-note-0006){ref-type="fn"}
12\. Performance (Y3) 3.31 .65 --.25[\*\*](#job2260-note-0006){ref-type="fn"} −.03 --.18[\*\*](#job2260-note-0006){ref-type="fn"} .17[\*\*](#job2260-note-0006){ref-type="fn"} .02 .05 .08[\*\*](#job2260-note-0006){ref-type="fn"} .12[\*\*](#job2260-note-0006){ref-type="fn"} .10[\*\*](#job2260-note-0006){ref-type="fn"} .35[\*\*](#job2260-note-0006){ref-type="fn"} .53[\*\*](#job2260-note-0006){ref-type="fn"}
13\. Performance (Y4) 3.37 .61 --.28[\*\*](#job2260-note-0006){ref-type="fn"} −.02 --.24[\*\*](#job2260-note-0006){ref-type="fn"} .10[\*\*](#job2260-note-0006){ref-type="fn"} .04 .07[*\**](#job2260-note-0005){ref-type="fn"} .07[*\**](#job2260-note-0005){ref-type="fn"} .09[\*\*](#job2260-note-0006){ref-type="fn"} .08[\*\*](#job2260-note-0006){ref-type="fn"} .32[\*\*](#job2260-note-0006){ref-type="fn"} .36[\*\*](#job2260-note-0006){ref-type="fn"} .45[\*\*](#job2260-note-0006){ref-type="fn"}
14\. Performance (Y5) 3.30 .58 --.24[\*\*](#job2260-note-0006){ref-type="fn"} −.04 --.24[\*\*](#job2260-note-0006){ref-type="fn"} .14[\*\*](#job2260-note-0006){ref-type="fn"} −.01 .07[*\**](#job2260-note-0005){ref-type="fn"} .06[*\**](#job2260-note-0005){ref-type="fn"} .06[*\**](#job2260-note-0005){ref-type="fn"} .10[\*\*](#job2260-note-0006){ref-type="fn"} .26[\*\*](#job2260-note-0006){ref-type="fn"} .24[\*\*](#job2260-note-0006){ref-type="fn"} .29[\*\*](#job2260-note-0006){ref-type="fn"} .43[\*\*](#job2260-note-0006){ref-type="fn"}
*Note*. *N* total = 1,875 employees. FTE = full‐time equivalents; MTM = multiple team membership.
*p* \< .05.
*p* \< .01.
### 3.2.2. Overall job performance {#job2260-sec-0013}
At the end of each year, the host organization\'s human resource management system required departmental supervisors to assess each of their direct reports\' overall job performance. These supervisors were responsible for 5--25 employees within their departments (mean = 14), and they typically met with these employees and relevant project leaders on a daily to biweekly basis. As such, supervisors had a relatively detailed and accurate view of their individual employees\' overall performance.
Following prior research (e.g., Bommer, Johnson, Rich, Podsakoff, & MacKenzie, [1995](#job2260-bib-0004){ref-type="ref"}; Cross & Cummings, [2004](#job2260-bib-0017){ref-type="ref"}), we used supervisors\' formal appraisal scores to operationalize individual employees\' yearly overall job performance. Beyond their own assessment of an employee\'s technical proficiency, planning and organizational skills, and research output, supervisors were asked to incorporate into their evaluations (a) feedback provided by project leaders about the quality of an employee\'s performance outcomes and (b) annual peer assessments by direct colleagues and/or customer assessments (if available). Supervisors used a standardized evaluation form to rate each individual employee\'s overall job performance on a 5‐point scale, with 1 representing the *worst* possible evaluation (i.e., substantial need for improvement) and 5 indicating the *best* possible evaluation (i.e., highly effective and well‐functioning). The organization used these formal performance appraisal scores, in part, to determine employees\' salary increases and promotions. As such, appraisal outcomes had direct practical relevance for the employees in our sample.
### 3.2.3. Control variables {#job2260-sec-0014}
We considered a number of covariates that may relate to individual employees\' MTM and/or overall job performance. Previous studies have suggested, in particular, that supervisory performance evaluations may be biased on the basis of employees\' *gender* (Inesi & Cable, [2015](#job2260-bib-0032){ref-type="ref"}), *organizational tenure* (Ng & Feldman, [2010](#job2260-bib-0057){ref-type="ref"}), and *salary* (Cleveland, Murphy, & Williams, [1989](#job2260-bib-0015){ref-type="ref"}). For example, supervisors may expect greater contributions toward organizational goals from employees with longer work experience and higher pay (Sturman, [2003](#job2260-bib-0069){ref-type="ref"}). Hence, even if supervisors have relatively accurate information about an individual employee\'s actual job performance (as was the case in this study context; see above), this information might translate into different performance ratings, depending on an employee\'s organizational tenure or salary. Moreover, organizational tenure may shape an employee\'s MTM, because individuals with higher tenure may develop specific skills that are useful for a greater number of teams (Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}). We therefore included these variables as potential controls. Because the host organization was opposed to publishing detailed salary information, this particular variable was available in *z*‐standardized form only. Finally, we anticipated that full‐time employees had more time available for work in additional teams, as compared with part‐time employees. We therefore incorporated an individual\'s weekly working time (in full‐time equivalents \[FTE\]) as an additional covariate (Pendleton, [2010](#job2260-bib-0060){ref-type="ref"}). [^4] Scores on the control variables were very stable over time (or changed by a fixed amount each year). To reduce the complexity of our models, we therefore created time‐invariant control variables by taking individual means across all available time points (Acemoglu, Johnson, Robinson, & Thaicharoen, [2003](#job2260-bib-0001){ref-type="ref"}).
3.3. Data analysis {#job2260-sec-0015}
------------------
We employed latent change score (LCS) modeling (using Mplus version 7.11; Muthén & Muthén, [1998](#job2260-bib-0055){ref-type="ref"}) to test our hypotheses (McArdle, [2009](#job2260-bib-0048){ref-type="ref"}). Researchers have used this method to examine the potentially reciprocal nature of the relationships between, for example, work characteristics and changes in personality (Li et al., [2014](#job2260-bib-0041){ref-type="ref"}), cognitive training exercises and improvements in critical reasoning (McArdle & Prindle, [2008](#job2260-bib-0049){ref-type="ref"}), and life events and behavioral problems (Malone et al., [2004](#job2260-bib-0045){ref-type="ref"}). In this study, we employed Grimm, An, McArdle, Zonderman, and Resnick\'s ([2012](#job2260-bib-0024){ref-type="ref"}) extension of the LCS framework to examine how within‐person changes in one variable relate to subsequent changes in a second variable. This allowed us to examine whether an increase in an employee\'s MTM was related with a subsequent increase (Hypothesis [1a](#job2260-mathstat-0001){ref-type="statement"}) or decrease (Hypothesis [1b](#job2260-mathstat-0002){ref-type="statement"}) in job performance and, simultaneously, whether an increase in an employee\'s job performance was related with a subsequent increase in his or her MTM (Hypothesis [2](#job2260-mathstat-0003){ref-type="statement"}). In other words, we examined within‐person changes in MTM as both an antecedent and a consequence of within‐person changes in an individual employee\'s overall job performance (Hackman & Wageman, [2004](#job2260-bib-0025){ref-type="ref"}; Hinds et al., [2000](#job2260-bib-0028){ref-type="ref"}), thus testing the dynamic, potentially reciprocal relationship between these variables.
The LCS model used to test our hypotheses is visualized in Figure [1](#job2260-fig-0001){ref-type="fig"} (see Grimm et al., [2012](#job2260-bib-0024){ref-type="ref"}, for a detailed description of each component of the model, as well as the Mplus scripts used to fit the model to the data). A key feature of an LCS model is that it uses a structural equation modeling framework to model change as a latent variable, representing an increase or decrease in the observed scores for each variable between two adjacent time points. These within‐person changes (e.g., ∆ job performance, T2‐T3) are predicted, then, by changes in a second variable at an earlier time point (e.g., ∆ MTM, T1‐T2). In addition, the model controls for changes that occur due to an employee\'s level of MTM or performance (e.g., MTM, T1). Together, this allowed us to examine whether within‐person changes in MTM were indeed related to within‐person changes in an employee\'s overall job performance and vice versa. As is common when using LCS models, all estimates were assumed to be equal across time points (Grimm et al., [2012](#job2260-bib-0024){ref-type="ref"}; McArdle, [2009](#job2260-bib-0048){ref-type="ref"}). Moreover, we controlled for the relationships of gender, organizational tenure, salary, and FTE, on the one hand, with individual differences in MTM and job performance, on the other, when estimating our model. [^5]
{ref-type="ref"}) and Grimm et al. ([2012](#job2260-bib-0024){ref-type="ref"}). Control variables are not shown. MTM = multiple team membership](JOB-39-1219-g001){#job2260-fig-0001}
4. RESULTS {#job2260-sec-0016}
==========
4.1. Descriptive statistics {#job2260-sec-0017}
---------------------------
Table [1](#job2260-tbl-0001){ref-type="table"} reports descriptive statistics and bivariate correlations for all variables across all five time points. As shown, an employee\'s MTM and overall job performance were positively correlated within each year of our study period (*r* range *=* .08 to .10; all *p* \< .01). Moreover, an employee\'s performance was consistently positively correlated with MTM in the subsequent year (*r* range *=* .06 to .12; all *p* \< .05), whereas MTM associated with subsequent performance in only two out of the 5 years (*r* range *= −*.01 to .07). Note, however, that these bivariate correlations reflect between‐person associations. An adequate test of our within‐person hypotheses, in contrast, requires longitudinal techniques of data analysis, as presented below.
Regarding the temporal stability of the variables in our sample, we note that the correlations between MTM across two adjacent years varied between .73 and .78 (all *p* \< .01), suggesting moderate‐to‐high MTM stability over time (which is relatively common in longitudinal studies; see Usami, Hayes, & McArdle, [2016](#job2260-bib-0073){ref-type="ref"}). Similarly, job performance exhibited moderate stability, with correlations across subsequent years ranging from .43 to .53 (all *p* \< .01). These correlations indicate that the study variables were relatively stable at the between‐person level, suggesting that there was little variation between the sample employees in the MTM and performance shifts they experienced. Nevertheless, it is possible that individual employees experienced significant within‐person changes in their MTM and performance during the study period (Grimm et al., [2012](#job2260-bib-0024){ref-type="ref"}). As such, the present stability levels do not prevent further examination of within‐person changes in MTM and performance over time (Li et al., [2014](#job2260-bib-0041){ref-type="ref"}).
Finally, regarding potential covariates, gender was significantly related to both MTM and job performance at two time points (*r* range *=* −.05 to −.06; all *p* \< .05). Moreover, employees\' organizational tenure, salary, and FTE were negatively related to performance across all time points (*r* tenure range *=* −.24 to −.28; *r* salary range *=* −.15 to −.25; *r* FTE range *=* .10 to −.17; all *p* \< .01), suggesting that supervisors\' performance ratings were positively biased toward less experienced employees with lower salaries. Although counterintuitive at first glance, this finding is consistent with prior research that has argued supervisors to hold heightened expectations toward more experienced employees and, thus, to more critically assess their job performance (Cleveland et al., [1989](#job2260-bib-0015){ref-type="ref"}; Sturman, [2003](#job2260-bib-0069){ref-type="ref"}).
4.2. Hypotheses tests {#job2260-sec-0018}
---------------------
Table [2](#job2260-tbl-0002){ref-type="table"} presents the results of an LCS model that tested the relationships between changes in MTM and subsequent changes in overall job performance (ξ~1~), and between changes in performance and MTM changes (ξ~2~). This model (which includes both between‐ and within‐person associations) provided a good fit to the data (χ^2^ = 368.00, *df =* 68, *p* \< .01; RMSEA = .05, CFI = .95, SRMR = .06; cf. Hu & Bentler, [1999](#job2260-bib-0031){ref-type="ref"}), and it fit the data significantly better than a basic LCS model that only included between‐person relationships (χ^2^ ~difference~ = 143.45, *df* ~difference~ = 4, *p* \< .01).
######
Parameter estimates for a latent change score model with multiple team membership and job performance
Parameter Estimate (*SE*)
--------------------------------------------------------- ------------------------------------------------------
Intercepts and slopes
Mean intercept MTM 2.83 (.05)[\*\*](#job2260-note-0011){ref-type="fn"}
Mean slope MTM 5.67 (3.35)
Mean intercept performance 3.20 (.03)[\*\*](#job2260-note-0011){ref-type="fn"}
Mean slope performance −.77 (1.04)
Correlations
Slope MTM with intercept MTM 1.47 (.41)[\*\*](#job2260-note-0011){ref-type="fn"}
Slope performance with intercept performance −.01 (.03)
Slope MTM with intercept performance .15 (.11)
Slope performance with intercept MTM −.34 (.15)[*\**](#job2260-note-0010){ref-type="fn"}
Slope MTM with slope performance −.52(.37)
Intercept MTM with intercept performance .04 (.02)[*\**](#job2260-note-0010){ref-type="fn"}
Controls
Organizational tenure ➔ MTM level .02 (.00)[\*\*](#job2260-note-0011){ref-type="fn"}
Gender ➔ MTM level −.02 (.04)
Salary ➔ MTM level .08 (.02)[\*\*](#job2260-note-0011){ref-type="fn"}
FTE ➔ MTM level −.60 (.06)[\*\*](#job2260-note-0011){ref-type="fn"}
Organizational tenure ➔ performance level −.01 (.00)[\*\*](#job2260-note-0011){ref-type="fn"}
Gender ➔ performance level .02 (.02)
Salary ➔ performance level −.04 (.01)[\*\*](#job2260-note-0011){ref-type="fn"}
FTE ➔ performance level .26 (.04)[\*\*](#job2260-note-0011){ref-type="fn"}
Level MTM ➔ change in performance (γ~1~) .33 (.15)[*\**](#job2260-note-0010){ref-type="fn"}
Level performance ➔ change in MTM (γ~2~) −.58 (.73)
Hypotheses tests
Change in MTM ➔ subsequent change in performance (ξ~1~) −.71 (.31)[*\**](#job2260-note-0010){ref-type="fn"}
Change in performance ➔ subsequent change in MTM (ξ~2~) 9.95 (1.92)[\*\*](#job2260-note-0011){ref-type="fn"}
*Note*. *N* total = 1,875 employees. FTE = full‐time equivalents; MTM = multiple team membership.
*p* \< .05.
*p* \< .01.
Hypothesis [1a](#job2260-mathstat-0001){ref-type="statement"} predicted that an increase in an individual\'s MTM would positively relate with a subsequent change in his or her overall job performance, whereas Hypothesis [1b](#job2260-mathstat-0002){ref-type="statement"} predicted an MTM increase to negatively associate with a subsequent performance change. The results presented in Table [2](#job2260-tbl-0002){ref-type="table"} refute Hypothesis [1a](#job2260-mathstat-0001){ref-type="statement"} but support Hypothesis [1b](#job2260-mathstat-0002){ref-type="statement"}, illustrating that an increase in MTM related to a subsequent decrease in job performance (ξ~1~ = −.71, *p* \< .05). Hypothesis [2](#job2260-mathstat-0003){ref-type="statement"} predicted that an increase in job performance would associate with a subsequent increase in MTM. As shown in Table [2](#job2260-tbl-0002){ref-type="table"}, the respective relationship is indeed positive and significant (ξ~2~ = 9.95, *p* \< .01), thereby supporting Hypothesis [2](#job2260-mathstat-0003){ref-type="statement"}. Together, these results suggest that changes in an individual employee\'s MTM and his or her overall job performance dynamically relate to each other in a negative, deviation‐counteracting feedback loop (cf. Weick, [1979](#job2260-bib-0078){ref-type="ref"}). That is, a within‐person increase in job performance relates to a subsequent increase in MTM, but an increase in MTM relates to a subsequent decrease in job performance.
4.3. Additional findings {#job2260-sec-0019}
------------------------
Beyond the reciprocal relationship between an individual\'s changes in MTM and job performance, our LCS model also assessed (a) whether an employee\'s MTM level was associated with subsequent performance changes and (b) whether an employee\'s performance level was associated with subsequent MTM changes. As shown in Table [2](#job2260-tbl-0002){ref-type="table"}, an employee\'s overall job performance level (i.e., individual performance differences between persons) was not significantly related with changes in his or her MTM (γ~2~ = −.58, *p* \> .10). An employee\'s MTM level, by contrast, was significantly and positively related to subsequent changes in his or her job performance (γ~1~ = .33, *p* \< .05). Hence, employees who worked in more teams at the same time experienced greater increases in performance than employees working in less teams at the same time. Although we did not explicitly formulate hypotheses for such "level‐to‐change" relationships, these additional findings have important implications for the overall pattern of linkages between individual MTM and job performance.
To illustrate the complex interplay of the various parameters in our overall LCS model, Figure [2](#job2260-fig-0002){ref-type="fig"} depicts two exemplary performance trajectories, namely, for an employee that experienced a constant increase in MTM over time (MTM slope = 1; solid line) and for an employee that experienced a constant decrease in MTM (MTM slope = −1; dashed line). With the exception of the respective MTM slope differences, all other parameters used to generate the predicted trajectories were identical, as reported in Table [2](#job2260-tbl-0002){ref-type="table"} (cf. Grimm et al., [2012](#job2260-bib-0024){ref-type="ref"}). As shown, both of these exemplary performance trajectories therefore start at the same level but, subsequently, develop differential patterns over time. Assuming a constant rate of increasing MTM (i.e., MTM increases with 1 at each time point, in addition to the change predicted by other parameters in the model), the solid line indicates that, after a brief period of relative stability, a marked performance decrease ensued. This reflects the detrimental performance effects of a within‐person increase in MTM predicted in Hypothesis [1b](#job2260-mathstat-0002){ref-type="statement"}. Over time, however, constant increases in MTM logically result in a relatively high overall MTM level and, as illustrated by the sharp performance increase between Years 4 and 5, the positive performance effects associated with an individual\'s MTM level eventually prevailed over the negative effects associated with MTM changes. Assuming a constant rate of decreasing MTM, by contrast, the dashed line in Figure [2](#job2260-fig-0002){ref-type="fig"} indicates an initial performance increase, because the observed negative association between changes in MTM and an individual\'s job performance implies that a decrease in MTM improves individual performance. Eventually, however, the relatively low MTM level resulting from continuously decreasing MTM rendered these performance gains untenable, as evidenced by the sharp drop in performance after Year 3.
{#job2260-fig-0002}
5. DISCUSSION {#job2260-sec-0020}
=============
Many of today\'s employees are simultaneously involved in more than one team (Mortensen et al., [2007](#job2260-bib-0052){ref-type="ref"}; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). In the present manuscript, we examined the dynamic relationship between such MTM and individual employees\' overall job performance, drawing on a 5‐year longitudinal sample of knowledge workers. From a within‐person perspective, we found that changes in an employee\'s MTM were negatively associated with subsequent performance changes, whereas changes in an employee\'s job performance were positively related with subsequent changes in his or her MTM. It appears, therefore, that individual MTM and job performance changes are mutually linked with each other in a deviation‐counteracting feedback loop (Maruyama, [1963](#job2260-bib-0046){ref-type="ref"}; Sterman, [2000](#job2260-bib-0068){ref-type="ref"}; Weick, [1979](#job2260-bib-0078){ref-type="ref"}). Moreover, from a between‐person perspective, we found that an employee\'s overall MTM level was positively related with job performance changes, such that employees with a greater number of concurrent team memberships may enjoy performance advantages. Hence, it is clear that the relationship between individual MTM and job performance is highly complex, and an adequate understanding of this linkage requires full consideration of its underlying dynamics. The following sections highlight the implications of such a dynamic perspective, as reflected in the present findings, for both theory and organizational practice.
5.1. Theoretical implications {#job2260-sec-0021}
-----------------------------
The present findings make several important contributions to the literature on teamwork, in general, and MTM, in particular. The overwhelming majority of the empirical research on organizational work teams has focused on stable teams with clear boundaries, with individuals conceptualized as members of one particular team (Wageman et al., [2012](#job2260-bib-0077){ref-type="ref"}). In reality, however, many teams in modern organizations represent unstable, dynamic entities with blurry boundaries (Mortensen, [2014](#job2260-bib-0051){ref-type="ref"}), and individuals often work in multiple teams at the same time (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}). Accordingly, this study addresses repeated calls for research "that analyzes team member time allocation in a more dynamic way, examining how members engage and disengage with particular teams over time" (Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}, p. 338; see also Mathieu, Tannenbaum, Donsbach, & Alliger, [2014](#job2260-bib-0047){ref-type="ref"}; Wageman et al., [2012](#job2260-bib-0077){ref-type="ref"}). Moving beyond prior team‐level performance considerations (e.g., Bertolotti et al., [2015](#job2260-bib-0003){ref-type="ref"}; Mortensen, [2014](#job2260-bib-0051){ref-type="ref"}), our research illustrates the distinct, reciprocal relationships between an individual employee\'s concurrent memberships within different teams, on the one hand, and his or her overall job performance, on the other.
Our findings also help to resolve a puzzle presented by prior theorizing on the consequences of MTM for individual job performance. Indeed, conservation of resources theory (COR) and social capital theory suggest that there are both potential performance benefits (O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}) and detriments (Pluut et al., [2014](#job2260-bib-0062){ref-type="ref"}) associated with MTM. Rather than contradicting each other, our results suggest that COR and social capital theory speak to different aspects of the dynamic linkage between individual MTM and job performance. Consistent with COR theory, the negative association between changes in MTM and subsequent performance changes may reflect the initial depletion of psychological resources (cf. Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}). The positive association between an individual\'s MTM level and performance changes, however, may reflect longer‐term benefits. Indeed, our results suggest that initial resource investments (albeit costly) may eventually strengthen an employee\'s resource base and promote his or her functioning at work (the second principle of COR theory; Brotheridge & Lee, [2002](#job2260-bib-0007){ref-type="ref"}; Chen, Westman, & Hobfoll, [2015](#job2260-bib-0012){ref-type="ref"}). These longer‐term effects are in line with social capital theory, which suggests that employees may benefit from the interpersonal resources embedded within their social networks once they have developed adequate connections with colleagues across various teams (Kwon & Adler, [2014](#job2260-bib-0037){ref-type="ref"}). The present research contributes to the literature by integrating these perspectives using a dynamic perspective on relationship between MTM and individual performance.
Importantly, these complex results highlight a real risk for incorrect conclusions about the MTM‐performance association that may arise when neglecting the underlying dynamics. It appears that MTM, by itself, is neither harmful nor helpful for an employee\'s performance. Rather, the costs and benefits of MTM hinge on the time frame under consideration. Whereas the process of increasing an employee\'s MTM may initially harm his or her job performance, high MTM levels may eventually enable substantive performance improvements. It seems vital, therefore, to consider individual MTM from a longitudinal perspective that combines within‐person and between‐person approaches, considering both the extent to which an employee\'s MTM changes over time and an employee\'s overall level of MTM.
Moreover, our 5‐year longitudinal data allowed us to examine the directionality of the MTM‐performance linkage, thus reconciling previous theoretical claims that have alternatively cast MTM as an antecedent (e.g., Chan, [2014](#job2260-bib-0010){ref-type="ref"}) or as a consequence (e.g., Cummings & Haas, [2012](#job2260-bib-0019){ref-type="ref"}) of an employee\'s job performance. Beyond the complex MTM‐to‐performance linkages discussed before, our within‐person results show that increasing performance may subsequently promote an individuals\' MTM as well. So, paradoxically, increasing performance may carry within itself the potential of both future performance decrements and future performance benefits in MTM settings.
5.2. Limitations and future research directions {#job2260-sec-0022}
-----------------------------------------------
Although our research has several methodological strengths (e.g., 5‐year longitudinal data from a relatively large sample of employees; an MTM measure based on objective data), we acknowledge some limitations that should be considered when interpreting the results. Our sample consisted of employees from a single organization and cultural context, for example, and it therefore seems worthwhile to examine our findings\' generalizability to other organizations, industries, and countries. Furthermore, we used supervisory assessments to capture individual employees\' job performance, based on a scale developed by the host organization, because objective performance information was not available (cf. Bommer et al., [1995](#job2260-bib-0004){ref-type="ref"}; Cross & Cummings, [2004](#job2260-bib-0017){ref-type="ref"}). Although these supervisory assessments had high practical relevance (e.g., providing important inputs for decisions about an employee\'s remuneration and career progress), future research might examine MTM\'s relation with alternative and/or more specific (e.g., objective, customer‐rated, and team‐member rated) performance measures.
Relatedly, our research focused on an employee\'s overall job performance, neglecting the employee\'s differences in performance across teams. It may be worthwhile to control for these performance differences in future research. Moreover, future research might extend the insights from our study by developing a finer‐grained model that explicates the linkage between MTM and specific performance dimensions. It is possible, for example, that MTM\'s performance consequences differ, depending on the job performance dimensions under consideration (e.g., efficiency vs. creativity), or that different performance dimensions differ in how strongly they impact an employee\'s MTM.
Furthermore, despite our longitudinal design, causality claims should be regarded with caution, in particular because we were not able to study individuals\' MTM and job performance from the very beginning of their employment. To more confidently conclude that prior performance changes cause changes in an individual\'s MTM (or that an employee\'s MTM causes performance changes), it would be helpful to more comprehensively track the formation of an individual\'s membership in multiple teams. Certain employees, for example, may be specifically hired (e.g., based on certain skills or personality characteristics; LePine & Van Dyne, [2001](#job2260-bib-0039){ref-type="ref"}) or trained (e.g., through socialization programs; Chao, O\'Leary‐Kelly, Wolf, Klein, & Gardner, [1994](#job2260-bib-0011){ref-type="ref"}) to work in more than one team at the same time. Hence, their work may be characterized by high MTM levels from the outset, rather than by gradual, performance‐induced increases in MTM. Furthermore, we used relatively long, 1‐year intervals to measure both MTM and job performance. As such, we may have primarily captured broad, longer‐term associations between these variables. For a more detailed understanding of the underlying dynamics, future research may benefit from examining MTM‐performance linkages using shorter time intervals.
Future research could also examine potential mediating variables and boundary conditions in the linkage between MTM and individual employees\' performance. Building on COR and social capital theory (Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}; Lin, [1999](#job2260-bib-0042){ref-type="ref"}), we argued that specific social and psychological resources may explicate MTM\'s performance advantages and disadvantages, yet this study context did not allow us to directly capture these mechanisms. The literature could therefore benefit from a further examination of such mediating factors, for example, by directly investigating the potential resource losses (e.g., lower perceived control; Skinner, [1996](#job2260-bib-0067){ref-type="ref"}) and social capital advantages (e.g., increased network centrality; Borgatti & Foster, [2003](#job2260-bib-0005){ref-type="ref"}) associated with increasing MTM.
Similarly, we did not examine potential boundary conditions for the present relationships (beyond the time frame under consideration), and this may be a worthwhile subject for future research as well. Specific features of the work situation, such as support from colleagues and/or supervisors (Cohen & Hoberman, [1983](#job2260-bib-0016){ref-type="ref"}) or personality traits such as proactivity (Li et al., [2014](#job2260-bib-0041){ref-type="ref"}) may enable employees to address the challenges and utilize the opportunities associated with increasing MTM. Relatedly, it may be useful to study the mediating mechanisms that explain how employees respond to increased MTM. Research on employees\' coping with stressful work events points to personal growth and resource accumulation as potentially relevant mediator variables (e.g., Cohen & Hoberman, [1983](#job2260-bib-0016){ref-type="ref"}; Hobfoll, [1988](#job2260-bib-0030){ref-type="ref"}; Updegraff & Taylor, [2000](#job2260-bib-0072){ref-type="ref"}). Investigating such moderating and mediating factors may add more context‐ and person‐specific richness to the longitudinal dynamics examined in our study.
Finally, MTM\'s performance consequences may hinge not only on the number of an employee\'s concurrent team memberships (i.e., MTM quantity, as captured in this study) but also on qualitative differences between teams (i.e., MTM variety; O\'Leary, Mortensen, et al., [2011](#job2260-bib-0058){ref-type="ref"}; O\'Leary, Woolley, et al., 2011). Whereas some employees may be concurrent members in similar teams, others may be immersed in a greater variety of team contexts (e.g., representing diverse organizational areas). The latter employees are exposed to larger variations in social contexts and task procedures, potentially aggravating MTM\'s performance disadvantages by promoting role conflicts and work overload (Kauppila, [2014](#job2260-bib-0034){ref-type="ref"}; Rizzo, House, & Lirtzman, [1970](#job2260-bib-0064){ref-type="ref"}). On the other hand, greater MTM variety might also promote the performance potentials derived from an employee\'s concurrent team memberships, enabling access to a greater variety of knowledge and resources (Vedres & Stark, [2010](#job2260-bib-0076){ref-type="ref"}). Moreover, better performing employees may not only become members of a greater number of teams but might also self‐select into more challenging and varied team contexts (Hackman & Wageman, [2004](#job2260-bib-0025){ref-type="ref"}) or might be assigned to more diverse teams (because these employees may be more attractive for teams across diverse departments; Cummings, [2004](#job2260-bib-0018){ref-type="ref"}). Future empirical research that moves beyond the present focus on quantitative MTM differences could include such variations in MTM quality to further promote theory development in this relatively nascent line of inquiry.
5.3. Practical implications and conclusion {#job2260-sec-0023}
------------------------------------------
The complex, dynamic relationship between individual employees\' MTM and overall job performance uncovered in our study has important practical implications. Based on our findings, managing employees\' MTM to achieve optimal performance appears as a challenging task that requires considerable resource investments from both individual employees and the organization. As illustrated in Figure [2](#job2260-fig-0002){ref-type="fig"}, increasing an employee\'s MTM is likely to initially trigger substantial performance detriments. Consequently, employees and organizations may be tempted to shy away from such changes, retaining or even reducing an employee\'s MTM to yield short‐term performance benefits. Figure [2](#job2260-fig-0002){ref-type="fig"} also shows, however, that increasing MTM may, in the long run, induce pronounced performance gains that may outweigh initial downsides. Hence, it seems worthwhile to consider increasing MTM as a resource investment that, eventually, result in important performance advantages. Both employees and their organization may benefit from accepting the costs of developing an employee to increasingly work in MTM settings, enabling them to reap the longer‐term advantages higher MTM levels entail.
For individual employees, this implies that they should view novel team memberships as resource investments and maintain their involvement in new, additional teams, even if this may initially harm their personal resources and performance potential. Moreover, from an organizational perspective, it implies that managers should not hold immediate performance losses resulting from increasing MTM against an employee, refraining from organizational reprimands and/or premature reduction of the employee\'s MTM. By persisting through the difficulties that increasing MTM entails and by actively supporting employees during this initial phase (e.g., through coaching or mentoring; Cohen & Hoberman, [1983](#job2260-bib-0016){ref-type="ref"}), organizations may eventually achieve a more versatile workforce that can draw from relevant work experiences, insights, and social connections across multiple teams to attain superior performance levels.
Altogether, our study contributes to an integrative understanding of an employee\'s overall performance as both an antecedent and a consequence of his or her MTM, and it underlines the importance of a dynamic, longitudinal approach in examining these linkages. We hope these findings will stimulate further research on the temporal dynamics of contemporary team memberships, and individual‐level MTM in particular, thus helping organizations to effectively manage such complex teamwork arrangements.
**Hendrik Johan van de Brake** is a PhD candidate in Human Resource Management and Organizational Behavior at the University of Groningen Faculty of Economics and Business.
**Frank Walter** is a full professor of Organization and Human Resource Management at the Justus‐Liebig‐University Giessen, Faculty of Economics and Business Studies.
**Floor Rink** is a full professor of Organizational Behavior at the University of Groningen Faculty of Economics and Business.
**Peter Essens** is the director of the Centre of Expertise in Human Resource Management and Organizational Behavior at the University of Groningen Faculty of Economics and Business.
**Gerben van der Vegt** is a full professor of Organizational Behavior at the University of Groningen Faculty of Economics and Business.
[^1]: In contrast to MTM\'s performance consequences, we see little theoretical rationale to expect both positive and negative linkages between an employee\'s overall job performance and subsequent MTM. Hence, we focus on the positive association between performance increases and subsequent MTM changes in the following, and we do not develop competing hypotheses for this association.
[^2]: Scholars have noted that missing data in longitudinal studies can cause biased parameter estimates if it arises from systematic participant attrition (e.g., due to inferior performance evaluations; Graham, [2009](#job2260-bib-0022){ref-type="ref"}). Importantly, however, dismissal of low‐performing employees is unlikely to represent a substantial source of attrition in the present sample. The participants in our sample were employed under permanent (i.e., nontemporary) contracts which, under Dutch labor law, are relatively difficult and costly to terminate and, thus, provide high job security. Hence, lower performance ratings would typically result in improvement interventions and reduced salary increases, rather than layoffs.
[^3]: Because we were interested in individuals\' memberships within multiple teams, work hours for projects with less than three members (less than 1% of all projects) were excluded (Dyer, [1984](#job2260-bib-0021){ref-type="ref"}).
[^4]: To further explore this potential biasing factor, we repeated our hypotheses tests using more restricted samples that only included full‐time employees. The results and conclusions from these supplementary analyses remained virtually unchanged. To preserve statistical power, we therefore report the results based on the full sample in the following.
[^5]: None of the controls predicted within‐person changes in MTM and job performance, and adding these relationships substantially decreased the overall fit of our model (cf. Hu & Bentler, [1999](#job2260-bib-0031){ref-type="ref"}). Hence, we only included the controls to account for individual differences in MTM and job performance.
|
{
"pile_set_name": "PubMed Central"
}
|
**PURPOSE:** Bacterial contamination following implant-based soft tissue reconstruction contributes to significant healthcare costs and patient morbidity. In addition to acute infection, subclinical bacterial colonization is thought to contribute to long-term capsular contracture. The purpose of this study was to design an antibiotic-eluting nanofiber-hydrogel composite sheet for use in implant soft tissue pocket reinforcement. A murine implant infection model was developed to test the impact of the device on implant infection and infection-associated capsule formation.
**METHODS:** Polycaprolactone (PCL) impregnated with linezolid and rifampicin was electrospun into a random-pattern sheet and suspended within a nanofiber-hydrogel composite. Interfacial bonding between the nanofibers and hydrogel matrix was used to improve structural integrity of the material. Mechanical properties and antibiotic release kinetics were assessed in vitro. Silicone disk implants were incubated with a bioluminescent Staphylococcus aureus strain for 24 hours. Biofilm formation was confirmed via crystal violet staining. Thirty-five mice were implanted with either the infected implant alone, infected implant with overlying composite sheet, or infected implant with overlying antibiotic-eluting composite sheet. Bioluminescence imaging was used to assess the in vivo bacterial burden between postoperative days 0 and 15. Postmortem bacterial colony forming unit (CFU) quantification, histology, and immunohistochemistry were performed on harvested tissues.
**RESULTS:** A 1mm-thick nanofiber-hydrogel composite sheet embedded with linezolid and rifampicin was designed and demonstrated favorable mechanical properties, suturability, and antibiotic release kinetics.
Use of the antibiotic-eluting composite sheet device resulted in complete prevention of clinical signs of cellulitis and implant exposure. In vivo bacterial luminescence was reduced in the presence of the antibiotic-eluting composite sheet overlay, returning to background signal levels within the study period.
The post-mortem bacterial burden within the peri-implant soft tissue was reduced 600-fold (1.8e2 +/- 1.8e2 versus 1.1e5 +/- 1.5e5, p=0.03), as was average capsule thickness (79 +/- 35 μm versus 274 +/- 194 μm, p=0.001) and relative collagen density within the peri-implant space (31.2 +/- 14.2 % versus 44.5 +/- 15.6 %, p=0.02).
**CONCLUSION:** An antibiotic-eluting nanofiber-hydrogel composite device was designed to reduce the risk of infection and capsule formation following implant-based soft tissue reconstruction. The device inhibited in vivo bacterial growth following implantation of a contaminated implant in a mouse model. Placement of the antibiotic-eluting sheet overlay led to a reduction in soft tissue cellulitis, implant exposure, and peri-implant capsule formation. The technique permits tailoring of mechanical properties and antibiotic release kinetics of the device to suit a variety of surgical applications. The device provides a platform for local delivery of medication into the peri-implant space combined with soft tissue reinforcement.
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{
"pile_set_name": "PubMed Central"
}
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All relevant data are within the paper.
Introduction {#sec001}
============
By generating electrical current inside the brain, repetitive transcranial magnetic stimulation (rTMS) and transcranial electrical stimulation can modify brain plasticity, in a manner similar to the *in vitro* induction of long-term potentiation (LTP) and depression (LTD) \[[@pone.0203333.ref001],[@pone.0203333.ref002]\]. Depending on various stimulation parameters (frequency, duration, and amplitude) and patterns, such non-invasive brain stimulation (NIBS) is capable of producing long-lasting facilitation or suppression of cortical excitability, with various behavioral and psychological effects. Among various stimulation forms of NIBS, theta burst stimulation (TBS) produces a very powerful and reproducible effect and has thus attracted increasing attention \[[@pone.0203333.ref003]--[@pone.0203333.ref006]\]. By mimicking the coupling of theta and gamma rhythms in rodent electroencephalograms during learning and exploration, electrical TBS was originally applied to the hippocampus and motor cortex to induce LTP in brain slice studies \[[@pone.0203333.ref007],[@pone.0203333.ref008]\]. Using rTMS, TBS protocols can induce LTP- and LTD-like effects in the human motor cortex \[[@pone.0203333.ref004],[@pone.0203333.ref006]\]. The modulating effects of TBS on motor excitability was determined according to the changes in single-pulse TMS-induced electromyographic (EMG) responses, also known as motor-evoked potentials (MEPs). Huang et al. found that MEPs were enhanced after intermittent TBS (iTBS) but suppressed after continuous TBS (cTBS). Related studies confirmed the effectiveness of iTBS and cTBS in modulating motor plasticity in healthy human subjects and rats \[[@pone.0203333.ref009],[@pone.0203333.ref010]\]. ITBS and cTBS patterns determine the direction of change in motor excitability and thus require shorter stimulation duration (192 s for iTBS; 40 s for cTBS) and lower intensity to induce longer-lasting effects compared to those for conventional repetitive stimulation protocols such as low-frequency (≤ 1 Hz) and high-frequency (≥ 5 Hz) stimulation. These properties make iTBS and cTBS more acceptable for human subjects and accessible for non-anesthetized animals.
TBS produces a strong effect and bidirectional plasticity changes, making it a potential option for diagnosis and therapy for many neurological movement disorders, such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke \[[@pone.0203333.ref003]\]. However, the mechanism of the modulation of brain plasticity induced by TBS is still not fully understood. Epidural recordings at a high cervical level suggest that rTMS-iTBS and -cTBS regulate different interneuron networks connected to corticospinal neurons \[[@pone.0203333.ref005],[@pone.0203333.ref011]\]. In the case of modulation of synaptic plasticity, the parameters of the stimulation, such as temporal precision, special localization, and cellular specificity, in a neural network are important \[[@pone.0203333.ref012]\]. According to studies \[[@pone.0203333.ref005],[@pone.0203333.ref011]\] using epidural recordings, it is essential to investigate the *in vivo* mechanisms of iTBS and cTBS by manipulating neural activity in a cell-type-specific way. A previous rodent study demonstrated that rTMS-TBS protocols are applicable for studying impaired motor plasticity in hemiparkinsonian rats \[[@pone.0203333.ref010]\]. Due to the relatively large size of the coil compared to that of the rat brain, the stimulation was not limited to the motor cortex. Cortical electrical stimulation (CES) delivered via microelectrodes has better spatial precision \[[@pone.0203333.ref013]\], but it cannot target specific a synapse, subcellular location, or cell type. Both TMS and CES are quite non-focal and affect neural pathway interactions, which makes their results unpredictable.
The above limitations may be overcome using an emerging technique called optogenetics, which utilizes *in vivo* tissue-specific expression of light-sensitive channels (opsins) and allows specific neuron populations to be selectively activated or inhibited by certain wavelengths of light with millisecond temporal precision \[[@pone.0203333.ref014]--[@pone.0203333.ref016]\]. Because of its specificity and spatiotemporal precision, optogenetics is widely used to study the neural substrates underlying complex animal behavior \[[@pone.0203333.ref017]\]. In the present study, we investigate the role of excitatory neuron in motor plastic modulation by TBS protocols using optogenetic stimulation. CaMKIIα-promoter-driven channelrhodopsin-2 (ChR2) was expressed in glutamatergic pyramidal neurons in the primary motor cortex (M1) of rats. ChR2-mediated neural excitation was induced with blue laser light (473 nm) irradiation \[[@pone.0203333.ref015],[@pone.0203333.ref018]\]. We designed a novel implantable optrode to guide the blue laser light into brain tissue and simultaneously collect local field potentials (LFPs) evoked by optogenetic stimulation to confirm the success of ChR2 expression. CES-induced MEPs were measured before and after TBS treatment to determine the modulating effect of TBS on motor excitability.
Materials and methods {#sec002}
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Animal preparation {#sec003}
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All animal experiments were approved by the National Cheng Kung University Medical College Animal Use Committee (IACUC Approval No.: 104139). Adult male Sprague Dawley rats, weighing 300--350 g, were housed in standard cages at a temperature of 25 ± 1°C with a 12/12-h light/dark cycle and free food and water access.
Lentivirus production {#sec004}
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The lentiviral vector carrying channelrhodopsin-2, pLenti-CaMKIIa-hChR2(H134R)-EYFP-WPRE \[[@pone.0203333.ref019]\], was gifted by Karl Deisseroth from the Optogenetics Resource Center, Stanford University, Stanford, CA, USA (Addgene plasmid \#20944). The construct was packaged in a second-generation lentivirus system via calcium-phosphate co-transfection of 293FT cells with psPAX2 and pMD2.G (gifted by Dr. Didier Trono, Addgene plasmids \#12260 and \#12259). The viral pellet was concentrated and then resuspended in phosphate-buffered saline (PBS) at 1/1000^th^ of the original volume. To determine the infection titer of the viral solution, a neuroblastoma cell line, SH-SY5Y, was infected by the lentivirus at serial dilution concentrations. Seventy-two hours after transduction, the number of eYFP positive cells was counted using fluorescence microscopy. The infection unit (IU) of the viral solution was determined as: \# of eYFP-positive cells/ml of viral solution.
Optrode design {#sec005}
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An integrated optrode was designed to achieve *in vivo* chronic neural recoding, CES, and optical stimulation ([Fig 1A](#pone.0203333.g001){ref-type="fig"}). An optical fiber (BFH22-200, Thorlabs) with a 200-μm-diameter core was inserted into a stainless steel cannula pedestal (C200GS-5/SPC, Plastics One) and glued to it. Both ends of the fiber were polished to avoid light scattering due to a rugged surface. The stainless steel cannula was then wired to a connector to serve as a CES and neural recording electrode. A fiber optic patch cord (core diameter: 200 μm) was fabricated with an FC connector (30230G3, Thorlabs, Inc.) on one end, and a custom-made connector on the other end, which consisted of a fiber housing (18G syringe needle) and a fiber cap (303/OFC, Plastics One). During optical stimulation, the patch cord bridged the 473-nm laser (MBL-III-473-50, Ultralasers Inc.) and the optrode ([Fig 1B](#pone.0203333.g001){ref-type="fig"}). The intensity of the blue laser light emitted from the tip of the optrode was measured (NOVA-II, Ophir Optronics) and plotted versus driving current ([Fig 1C](#pone.0203333.g001){ref-type="fig"}).
{#pone.0203333.g001}
Virus injection and optrode implantation {#sec006}
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Rats were introduced anesthetization with inhalation of 4% isoflurane in O~2~, then placed in a stereotaxic apparatus (Model 902, DAVID KOPF INSTRUMENTS) and maintained anesthetization using 2.5% isoflurane inhalation. The scalp was shaved and sterilized, then an incision was made on the scalp to expose the skull and bregma after the soft tissue had been carefully removed. A burr hole was drilled and 2 μl of concentrated lentivirus solution (10^7^ IU/ml) was injected into the left primary motor cortex of the right forelimb (AP: +1.5 mm; ML: 2.5 mm; DV: 1.5 and 2.5 mm) at a rate of 0.1 μl/min controlled by a syringe pump (KDS 100, KD Scientific Inc.). After injection, the optrode was implanted at the same site for future optical and electrical stimulation and neural recording ([Fig 2A](#pone.0203333.g002){ref-type="fig"}). A reference stainless steel screw electrode (3.0 mm × 1.4 mm) was implanted into the left M1 hindlimb (AP: -1.0 mm and ML: 1.25 mm) according to functional brain mapping of rats \[[@pone.0203333.ref020]\]. Both the optrode and the reference screw were connected to a miniature socket, which was exposed for electrical stimulation and neural recording. After implantation, the electrodes and sockets were covered with dental cement. To relief pain after surgery, buprenorphine (0.1mg/kg) was injected subcutaneously every 12 hours for a week. Experiments began 4 weeks later to allow ChR2 expression.
{#pone.0203333.g002}
Optical stimulation and evoked potential recording {#sec007}
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The rats were anesthetized using i.p. zoletil 50 (50 mg/kg, Virbac) mixed with xylazine (10 mg/kg, Bayer). Thirty minutes after anesthetization, the evoked potential induced by optogenetic stimulation was measured from ChR2+ neurons. A 473-nm diode-pumped laser (MBL-III-473-50, Ultralasers Inc.) was controlled by a pulse generator (Model 2100, A-M Systems). Blue light was guided into M1 optrode through a patch cord fiber. The LFP activity collected by the optrode was amplified (20,000×) and filtered using 60-Hz notch and 10 Hz to 600 Hz bandpass filters with a sampling rate of 10 kHz (MP36, BIOPAC Systems Inc.). During the recording, a marker synchronized with the pulse was sent simultaneously from the pulse generator to the data acquisition unit (MP36, BIOPAC Systems Inc.). The classic waveform of the evoked potential was obtained by applying coherent averaging to a hundred epochs according to the marker. The peak-to-peak amplitude was quantified for representing corticospinal excitability.
Measurement of MEP {#sec008}
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MEP was quantified following the protocol described by Hsieh et al. with several modifications \[[@pone.0203333.ref013]\]. The rats were anesthetized using i.p. zoletil 50 (50 mg/kg, Virbac) mixed with xylazine (10 mg/kg, Bayer). Thirty minutes after anesthetization, EMG activity from the belly of the right brachioradialis muscle was recorded with needle electrodes (Axon Systems, Inc.). A reference electrode was inserted into the right paw and a ground electrode was inserted into the base of the rat's tail. MEP was elicited by applying an electrical pulse (biphasic, amplitude: 5 \~ 8V, pulse duration: 0.6 ms, pulse interval: 10 s; Model 2100, A-M Systems) over M1 via the stainless steel cannula of the optrode. The EMG signal was amplified 1000-fold and filtered using 60-Hz notch and 10 Hz to 1 kHz bandpass filters prior to digitization at a sampling rate of 10 kHz (MP36, BIOPAC Systems Inc.). The minimal intensity of a single biphasic pulse required to induce a MEP of greater than 20 mV was defined as the resting motor threshold (RMT). To quantify MEP, 30 trials at 120% RMT intensity were conducted to obtain the average of peak-to-peak amplitude for each MEP.
Experimental design {#sec009}
-------------------
Before the experiment of MEP modulation by TBS, all of the rats were tested to confirm the successful expression of the ChR2 by measuring the evoked potentials, and successful induction of MEP by determining the RMT. Only the rats with positive expression of ChR2 and MEP were included in the TBS experiment (n = 7 rats).
The basic pattern of TBS consisted of bursts that repeat at 5 Hz, and each burst is composed of three pulses that repeat at 50 Hz. Two types of TBS protocol were used in this study. For the cTBS protocol, the 5-Hz bursts were given in a continuous train and lasted for 40 s (total of 600 pulses). For the iTBS protocol, a 2-s train of TBS followed by 8 s of rest was repeated for 20 cycles (192 s, total of 600 pulses) \[[@pone.0203333.ref004],[@pone.0203333.ref010],[@pone.0203333.ref013]\]. The TBS protocols were generated by the pulse generator and converted into optical pulses (pulse width: 1 ms, intensity: 100 mW/mm^2^) via a transistor-transistor-logic-controlled diode-pumped laser (Opto-iTBS and Opto-cTBS). For sham control intervention, no stimulation was delivered. To study the effect of optogenetic TBS on motor excitability, the MEPs were measured at 10 minute before (baseline) and through 30 minutes after cortical TBS treatment. Every 5 minutes of MEPs (0.1 Hz, 30 trials) were collected for averaging. To compare the effects of TBS intervention on MEPs, all averaged MEP amplitudes were normalized to the baseline. Five types of TBS treatments have been performed on each rat: sham control, CES-iTBS, CES-cTBS, Opto-iTBS, and Opto-cTBS. Three to five days of resting between each experiment allowed elimination of any toxic effect of anesthetizer and accumulated effect of TBS-modulation.
Histological examination {#sec010}
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The intrinsic fluorescence of the ChR2-eYFP fusion protein was monitored to confirm the expression of ChR2. The rats were sacrificed via transcardial perfusion with ice-cold saline followed by 4% paraformaldehyde (PFA) in PBS. Extracted brains were further fixed in 4% PFA/PBS overnight, and then dehydrated in 30% sucrose/PBS for two days. Using a freezing microtome (Shadon Cryotome E, Thermo Electron Corp.), 30-μm sections were cut from optimal cutting temperature (OCT) compound-embedded brain tissue. Fluorescence images (excitation: 460--495 nm, emission: 510--550 nm) of ChR2-eYFP-positive neurons were observed under a microscope (IX-71, Olympus Corp.).
Data analysis {#sec011}
-------------
All data are presented as means ± standard error of the mean (SEM). Data were analyzed using GraphPad Prism (version 5.01, GraphPad Software) with significance set to *p* \< 0.05. Linear regression analysis was performed to test the dose effects of power density (mW/mm^2^, on logarithmic scale) on the amplitude of optogenetic-evoked potential. To compare the effects of various pulse widths of optogenetic stimulation on LFP, the peak-to-peak amplitudes of LFPs were normalized with respect to that amplitude at 1.0 ms. Kruskal-Wallis test with Dunn's post hoc test were applied to compare the differences between various pulse width groups. Non-parametric multiple comparisons were performed to analyze the effects of two factors on MEP activities separately: 1) type of treatment (CES-iTBS/cTBS, Opto-iTBS/cTBS, and sham) and 2) time-course (10 and 5 min before and 5, 10, 15, 20, 25, and 30 min after TBS intervention). The MEPs were normalized to the baseline recorded at 10 min before treatment (-10 min). First, Kruskal-Wallis test was used to analyze the difference between iTBS versus sham and cTBS versus sham at each time-course. Second, Friedman's test was used to exam the difference between each time-courses versus baseline (-10 min) under individual treatments (CES-iTBS/cTBS and Opto-iTBS/cTBS). The significant differences between groups were tested by post hoc Dunn's test, and marked as: ^\#^*p* \< 0.05 versus sham; \**p* \< 0.05 versus baseline (-10 min).
Results {#sec012}
=======
Optogenetic-evoked potentials {#sec013}
-----------------------------
A novel opto-electrical neural interface ([Fig 1](#pone.0203333.g001){ref-type="fig"}) was developed to activate M1 and simultaneously record the neural response. The optrode consisted of an optical fiber inserted in a stainless steel cannula, which was embedded in a mounting pedestal. The interface serves three purposes: (i) optical stimulation via the optical fiber and (ii) CES and (iii) cortical recording via the stainless steel cannula. The optrode was implanted right after lentiviral injection. With the identification of intrinsic fluorescence, expression of the CaMKIIα-promoter-driven ChR2-eYFP was confirmed in the forelimb area of M1 ([Fig 2B and 2C](#pone.0203333.g002){ref-type="fig"}).
To activate ChR2, various optical intensities of the blue laser were emitted into the cortex. Representative LFP traces obtained for various intensities of single-pulse optical stimulation are shown in [Fig 3A](#pone.0203333.g003){ref-type="fig"}. Light-induced artifacts caused by the photoelectric effect appear in the traces for both ChR2-negative (ChR2-) and ChR2-positive (ChR2+) rats. A comparison to ChR2-negative traces allowed the typical waveform of optogenetic-evoked potentials induced by a 1-ms laser pulse to be identified in the traces from ChR2-positive M1. Representative waveforms induced by laser light at three optical intensities (5.61, 52.30, and 181.44 mW/mm^2^) were plotted after coherent averaging. The average latencies of N1 and P1 are 8.92 ± 0.66 ms and 24.38 ± 0.89 ms, respectively. The dose-response curve of the amplitudes elicited by various power densities are shown in [Fig 3B](#pone.0203333.g003){ref-type="fig"}. A linear dependency is observed (R^2^ = 0.91) between the peak-to-peak amplitudes of the evoked potential and optical intensity on the logarithmic scale. The effect of pulse duration on the amplitude of the evoked potential was also tested. A curve of evoked potential versus optical pulse duration is shown in [Fig 3C](#pone.0203333.g003){ref-type="fig"}. Significant differences between the evoked potential for 1.0 ms and those at other stimulator durations were observed in the Kruskal-Wallis test with Dunn's post hoc test (*p* \< 0.05).
{#pone.0203333.g003}
Effects of TBS on MEP activity {#sec014}
------------------------------
The MEP activities were recorded to evaluate the after-effects of sham, iTBS, and cTBS interventions applied in the forms of CES and optogenetic stimulation ([Fig 4](#pone.0203333.g004){ref-type="fig"}). The waveform of evoked potential corresponding to optogenetic theta burst stimulation (Opto-TBS) appears in the averaged LFP trace shown in [Fig 4](#pone.0203333.g004){ref-type="fig"}. This indicates the feasibility of using Opto-TBS intervention for cortical stimulation.
{#pone.0203333.g004}
Representative EMG traces recorded 10 min before and 30 min after various interventions are shown in [Fig 5](#pone.0203333.g005){ref-type="fig"}. Compared to the sham control, enhanced activities were observed in MEP waveforms recorded 30 min after treatment with CES-iTBS, Opto-iTBS, and Opto-cTBS. Decreased activity in MEP waveform was found following CES-cTBS. [Fig 6](#pone.0203333.g006){ref-type="fig"} shows fold changes of MEP activity at each time point following CES-TBS and Opto-TBS interventions. In [Fig 6A](#pone.0203333.g006){ref-type="fig"}, the effects of treatment and time-course were analyzed by Kruskal-Wallis test and Friedman's test separately. First, to analyze the difference between CES-iTBS versus sham and CES-cTBS versus sham at each time-course, Kruskal-Wallis test with post hoc Dunn's test were used. Comparing to sham, MEPs were significantly increased by CES-iTBS at 10 min and 30 min (\#*p* \< 0.05 versus sham). Then, the effect of time-course was analyzed by Friedman's test for each type of treatment. Post hoc Dunn's test revealed that the MEPs significantly increased at 15 min and remained at increased levels for up to 30 min (\**p* \< 0.05 versus -10 min) after CES-iTBS compared to baseline MEP.
{#pone.0203333.g005}
{#pone.0203333.g006}
The TBS protocols were then converted into optogenetic stimulation to test the modulating effects of glutamatergic-specific stimulation on MEP activity. In [Fig 6B](#pone.0203333.g006){ref-type="fig"}, the effects of treatment were first analyzed by Kruskal-Wallis test plus post hoc Dunn's test. Comparing to sham, Opto-iTBS increased MEPs from 5 min through 30 min after the treatment (\#*p* \< 0.05 versus sham). Then, the effect of time-course on MEPs was examined by Friedman's test with post hoc Dunn's test for each type of treatment. Results showed that when comparing to -10 min baseline, the MEPs significantly increased at 15 min and remained at increased levels for up to 30 min (\**p* \< 0.05 versus -10 min) after both Opto-iTBS and Opto-cTBS. No significant after-effect was found on the MEP amplitudes in the sham group comparing to the baseline.
Discussion and conclusion {#sec015}
=========================
We developed a modified optogenetic neural interface, which was integrated with CES and a cortical recording system, to achieve chronic *in vivo* cortical stimulation and monitoring in ChR2-expressed rats. It is highly desirable to simultaneously record electrophysiological responses during optogenetic activation for assessing brain neural networks. In the present study, the modified optogenetic neural interface employed an implanted fiber guide to target the brain region of interest after ChR2 expression (M1 in this case), and a stainless steel fiber cannula, which allowed CES and LFP recording via a dual-function electrode. LFP evoked by optical pulses was recorded using the cannula (contact area = 0.097 mm^2^) from the surface of the cortex simultaneously during optogenetic stimulation. The peak-to-peak amplitude of evoked potentials increased with increasing optical intensity ([Fig 3B](#pone.0203333.g003){ref-type="fig"}). Consistent amplitude and similar morphologies were found compared to those reported in a previous *in vitro* study \[[@pone.0203333.ref021]\].
Using this custom-made neural interface, TBS protocols used for inducing LTP- or LTD-like plasticity in human M1 were converted into an optogenetic animal model, and then optical and electrical control of the rodent's motor activity was achieved. This study showed that single-pulse CES delivered through the custom-made optrode could elicit MEP. The obtained recordings were similar to those obtained using CES delivered through epidural screw electrodes \[[@pone.0203333.ref013]\]. By comparing the MEP amplitudes before and after TBS intervention, modulated neuroplasticity via cortical TBS can be observed. It was found that CES-iTBS induces potentiation whereas CES-cTBS inhibits MEP activity (Figs [5](#pone.0203333.g005){ref-type="fig"} and [6A](#pone.0203333.g006){ref-type="fig"}). These observations are similar to those for TBS-induced LTP- or LTD-like plasticity reported in our previous studies using CES and rTMS \[[@pone.0203333.ref010],[@pone.0203333.ref013]\].
The TBS protocols were converted into a focal optogenetic stimulation scheme to test the after-effects on motor plasticity. A ChR2 (H134R) variant and 1-ms stimulation pulses were used in the TBS protocols to ensure spiking fidelity. Since ChR2 (H134R) can respond to stimulation with frequencies of up to 80 Hz \[[@pone.0203333.ref022],[@pone.0203333.ref023]\], Opto-TBS with a 50-Hz intra-burst frequency could reliably activate evoked potentials in M1 ([Fig 4](#pone.0203333.g004){ref-type="fig"}, upper right). We found that both Opto-iTBS and Opto-cTBS can increase MEP amplitudes. The enhancement of MEP activity by Opto-iTBS was stronger than that by CES-iTBS (Figs [5](#pone.0203333.g005){ref-type="fig"} and [6B](#pone.0203333.g006){ref-type="fig"}). Fold change values of MEP measured at 30 min post-stimulation are: CES-iTBS: 2.57, CES-cTBS: 0.38, Opto-iTBS: 3.77, and Opto-cTBS: 2.21. The potentiating effect of the Opto-iTBS protocol on MEP activity is comparable to that for the CES- and rTMS-iTBS protocols. However, a discrepancy was observed in the case of cTBS treatment: CES- or rTMS-cTBS suppressed MEP activity whereas Opto-cTBS enhanced it. Unlike CES- or rTMS-cTBS, which typically suppressed the MEP response, Opto-cTBS enhanced it at a level similar to that of CES-iTBS.
It is hypothesized that the inconsistency between potentiation caused by Opto-cTBS versus suppression caused by CES/rTMS-cTBS may have arisen from the difference in neural networks targeted by these two TBS modalities. Some studies have indicated that the after-effects of iTBS and cTBS are mediated by different interneuron networks excited by rTMS \[[@pone.0203333.ref005],[@pone.0203333.ref011]\]. Research has also shown that different TMS coil orientations can activate different intra-cortical networks, leading to approximately 50% of the variation in TBS response \[[@pone.0203333.ref024]\]. These results indicate that the response to TBS treatment is strongly influenced by the interneuron networks recruited by the TMS pulse. There is thus a need for a focalized brain stimulation technique to selectively target neural pathways with a specific cell population. In the present study, we tested the after-effects of cell-type-specific TBS using the optogenetic approach. Since ChR2 was expressed specifically in excitatory neurons (such as pyramidal neurons) driven by the CaMKIIα promoter, Opto-TBS should excite primarily the pyramidal neurons in a focalized area, in contrast to CES/rTMS-TBS, which activates all types of neuron, including interneurons. Therefore, it is hypothesized that the opposite modulation of neuroplasticity by Opto-cTBS compared to that of CES-cTBS may be attributed to the priming effects on different neuronal populations recruited by optogenetic and electrical/magnetic pulses. It is known that rTMS and CES approaches excite all types of cell and that optogenetic stimulation targets specific cells. Under rTMS or CES without targeting of specific cells, cTBS may suppress after-effect on cortical excitability through interneuron network. In contrast, Opto-cTBS may enhance cortical excitability when optogenetic stimulation targets excitatory neurons specifically. The major limitation of our study is that other opsin tools, such as GABAergic neurons, were not used to target interneurons in the cortex to investigate the modulating effects of TBS mediated by interneurons.
In conclusion, we developed a custom-made optogenetic neural interface, which can perform cortical recording to test the after-effects of a TBS scheme on motor plasticity in ChR2-expressing rats. Our study showed the feasibility of using the optogenetic approach for focalized and cell-type-specific modulation of neuroplasticity. Using the custom-made neural interface, TBS protocols used for testing LTP- or LTD-like plasticity in the human M1 were converted into an optogenetic animal model. It was confirmed that the potentiating effect of the Opto-iTBS protocol on MEP activity is comparable to that of CES- and rTMS-iTBS protocols. However, a discrepancy was observed in the case of cTBS treatment: CES- or rTMS-cTBS suppressed MEP activity whereas Opto-cTBS enhanced it. The results provide a better understanding of TBS-induced brain modulation and encourage further exploration of cell-type-specific brain stimulation.
We would like to thank Dr. Shaw-Jenq Tsai from the Department of Physiology, National Cheng Kung University, Tainan, Taiwan, for providing the 293FT cell line for lentivirus production. We sincerely thank Dr. Chien-Chung Chen in Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan, for assistance in lentivirus production.
CES
: cortical electrical stimulation
ChR2
: channelrhodopsin-2
cTBS
: continuous theta burst stimulation
iTBS
: intermittent theta burst stimulation
LFP
: local field potential
LTD
: long-term depression
LTP
: long-term potentiation
M1
: primary motor cortex
MEP
: motor-evoked potential
NIBS
: non-invasive brain stimulation
Opto-cTBS
: optogenetic continuous theta burst stimulation
Opto-iTBS
: optogenetic intermittent theta burst stimulation
rTMS
: repetitive transcranial magnetic stimulation
TBS
: theta burst stimulation
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
|
{
"pile_set_name": "PubMed Central"
}
|
Due to privacy regulations, it is not possible to deposit data to a public repository. Interested researchers are kindly asked to directly address Dr. Guangliang with appropriate requests for the data. The authors confirm that any interested reader who contacts Dr. Guangliang will be provided with a de-identified dataset. (Department of Epidemiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China. Email: <[email protected]>).
Introduction {#sec005}
============
Decreased sleep duration has been shown to be associated with increased morbidity and mortality \[[@pone.0117700.ref001]--[@pone.0117700.ref003]\]. Research suggests that factors such as socio-demographic and comorbid health factors \[[@pone.0117700.ref004]\], psychosocial stress \[[@pone.0117700.ref005]\], and lifestyle \[[@pone.0117700.ref006]\] are associated with sleep quality and duration. Hence, sleep duration and quality are not only medicine issue but also a socio-demographic issue. For example, self-reported sleep duration varies by industry and occupation, according to the USA national health interview survey \[[@pone.0117700.ref007]\]. Furthermore, sleep duration varies between countries, races and ethnicities \[[@pone.0117700.ref008]--[@pone.0117700.ref011]\]. For example, according to data from the National Health Interview Survey from 2004--2001, Asians were more likely to report decreased sleep duration compared with Caucasians (33% vs. 28%)\[[@pone.0117700.ref012]\]. Sleep quality also has been found to vary by socioeconomic status (SES) and improves with increasing socioeconomic status \[[@pone.0117700.ref013]\]. Thus, white-collar workers reported the highest quality of sleep, compared with blue-collar workers who had the lowest quality of sleep\[[@pone.0117700.ref014]\]. However, thus far, no studies have investigated the association between occupation and sleep quality and duration.
We conducted a national study comparing sleep performance in Chinese workers in different occupations. We tested three hypotheses: 1) Sleep duration varies amongst different occupations; 2) Sleep quality varies amongst different occupations; 3) Occupation is an independent determinant of sleep duration and quality.
Methods {#sec006}
=======
Participants {#sec007}
------------
The China Sub-optimal Health Survey (CSHS) was created in 2008 to understand the changing health status of China based on a sample population. The CSHS selected individuals from 6 provinces to represent the 1.4 billion individuals in the nation\'s population. A multi-stage, random cluster sampling design was used to designate study subjects. All 31 provinces or municipalities were divided into 6 administrative regions (Northeast, North, East, Central South, Southwest, and Northwest). The regions of Jilin, Beijing, Jiangsu, Hubei, Sichuan and Gansu were randomly selected to represent those six administrative regions. Each of the above randomly selected regions was divided into multiple urban and suburban regions. Then, 1--2 urban regions and 1--2 suburban regions were randomly selected to represent both the urban and suburban populations. Within those selected regions, residents including local college students, government staff, business and farm workers and other non-affiliated local residents were clustered and randomly selected as the sample population.
19,665 participants were selected to participate in the study, of whom 18,631 responded and filled out questionnaires (response rate of 94.7%). We excluded individuals who 1) were less than 18 or greater than 65 years of age; 2) have mental illnesses that could potentially affect sleep patterns; and 3) were employed as shift workers. 18,284 participants were included in the final analysis.
Ethics Statement {#sec008}
----------------
This study was approved by the Institutional Review Board at Peking Union Medical College and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants.
Data collection {#sec009}
---------------
All individuals in each randomly cluster selected unit were asked to completed a self-administered questionnaire. The data on demographic and personal characteristics were collected, including gender, age, marital status, education, smoking, drinking, and health information (medical history, illness and diseases that occurred during the last 12 months). Information on occupation was based on Chinese labor law.
Sleep {#sec010}
-----
### Sleep duration {#sec011}
The number of hours of sleep was assessed by the following questions inserted into a self-reported questionnaire: "On average, when you go to bed at night?" and "On average, when you arise in the morning?".
Sleep duration was calculated according to following formulas: Preferred sleep duration = (preferred arising time + 24) − (preferred going to bed time); Short sleep duration was defined as less than 6 hours, according to the sleep duration. The classification was consistent with the previous studies \[[@pone.0117700.ref015]--[@pone.0117700.ref017]\].
### Sleep quality {#sec012}
The Pittsburgh Sleep Quality Index (PSQI) was used to estimate the participating's sleep quality. The cutoff point of PSQI score was 5 (PSQI \>5 indicate poor sleep quality) which was consistent with the previous studies \[[@pone.0117700.ref018],[@pone.0117700.ref019]\].
Statistic {#sec013}
---------
Statistical analysis was carried out with Windows Statistical Software Package Version 10.0 (SAS Institute, Cary, NC, USA). Occupations were analyzed as a categorical variable. Sleep duration and sleep quality were categorized into two groups (cutoff points were 6 hours and 5 scores, respectively), which were investigated as binary outcome variables. Chi-square tests were used to compare participants' characteristics by sleep duration and sleep quality. We used ANOVA (Analysis of variance) to test the first hypothesis that sleep duration or sleep quality would be varied by occupation. Tukey test was used to compare difference between the groups. Logistic regression model was applied to estimate the odds ratio and 95% CIs of short sleep duration and poor sleep quality by occupations adjusted for potential confounders. Potential confounders considered were sex, age, education, area, marriage, smoking, drinking, body pain, and health status, categorized as in [Table 1](#pone.0117700.t001){ref-type="table"} (A significance level of 0.05 is required to allow the variable enter the model). Health status was assessed based on self-reports of chronic illness including hypertension, diabetes, coronary heart disease, hyperlipidemia, hepatitis, and other diseases. Participants with any of the above chronic disease were labeled as \"unhealthy\". Two sets of potential confounders were used in the adjusted models. Model 1 adjusted for sex and age. Model 2 additional adjusted for education, area, marriage, smoking, drinking, body pain, and health status. All potential confounders were summarized in [Table 1](#pone.0117700.t001){ref-type="table"}. All the tests were two sided and significance level was set at 0.05.
10.1371/journal.pone.0117700.t001
###### Participant characteristics and frequencies (in %) within each sleep duration and quality category.
{#pone.0117700.t001g}
Sleep duration (hours) Sleep quality (score)
------------------------- ------------------------ ----------------------- ------- ------- ------- -------
Sex
Men 50.16 59.55 6.92 51.54 48.47 25.85
Women 49.84 40.45 4.84 48.46 51.53 28.27
Age (years)
18--25 28.71 25.52 5.27 29.67 25.44 24.12
25-≤45 55.08 49.72 5.35 54.08 56.58 27.94
45-≤65 16.21 24.76 8.73 16.25 17.98 29.09
Occupation
Civil 16.74 16.82 5.92 17.00 16.07 25.94
Professional 14.81 15.03 5.98 14.17 16.58 30.25
Worker 26.91 30.62 6.65 25.84 30.61 30.51
Famer 9.38 6.24 4.00 9.70 7.83 23.02
Business man/service 8.77 9.36 6.26 9.22 7.70 23.66
Students 15.86 14.27 5.34 16.35 14.17 24.31
Others 7.52 7.66 5.99 7.71 7.04 25.30
Education
Liberate/primary school 25.76 22.97 5.29 25.68 25.38 26.81
High school 23.09 25.24 6.41 22.9 24.08 28.05
College 51.15 51.8 5.96 51.43 50.55 26.7
Area
Jilin 17.29 17.77 6.05 16.48 19.57 30.56
Gansu 15.5 19.09 7.16 13.49 21.71 37.36
Sichuan 17.48 15.31 5.20 17.51 16.95 26.41
Jiangsu 17.25 5.39 1.92 19.4 8.86 14.47
Hubei 16.92 22.59 7.71 16.12 20.31 31.83
Beijing 15.55 19.85 7.40 16.99 12.61 21.56
Marriage
Single 33.76 31.10 5.45 34.48 31.25 25.15
Married 63.94 64.37 5.93 63.56 65.05 27.5
Devoice/separate/Widow 2.30 4.54 10.98 1.96 3.71 41.19
Smoking
No 76.79 65.5 5.07 76.86 74.13 26.34
yes 23.21 34.5 8.51 23.14 25.87 29.3
Drinking
No 70.07 58.22 4.95 69.82 68.16 26.57
Yes 29.93 41.78 8.04 30.18 31.84 28.11
Body pain
No 32.31 47.45 4.64 25.13 54.97 18.23
Yes 67.69 52.55 8.42 74.87 45.03 44.77
Healthy
Yes 78.75 67.77 5.11 80.42 71.82 24.87
No 21.25 32.23 8.67 19.58 28.18 34.79
Results {#sec014}
=======
The study included a total of 18,316 Chinese adults from 6 provinces with a mean age of 33.1 (SD = 10.6). 50.73% were male and 49.27% were female. Overall, 5.93% of study participants reported a sleep duration of less than 6 hours (male: 6.92%; female: 4.84%) and 26.98% reported poor sleep quality (male: 25.85%; female: 28.87%). The percentage of individuals who reported a sleep duration less than 6 hours (considered a \"short sleep duration\") was varied based on occupation, 5.92% of civil servants, 5.98% professional workers, 6.65% of blue collar workers, 4% of farmers, and 6.26% of business workers, 5.34% of students and 5.99% of other individuals reported short sleep duration. The percentage of individuals who reported a poor sleep quality was varied based on occupation. Based on the cut-off points using the PSQI recommendations, 25.94% of civil servants, 20.25% of professionals, 30.51% of blue collar workers and 23.02% of farmers, 23.66% of business workers, 24.31% of students and 25.30% of other individuals reported poor sleep quality.
[Table 2](#pone.0117700.t002){ref-type="table"} shows the distribution of sleep duration and sleep scores in the study participants. Farmers had the longest sleep duration (mean = 8.22 hours) while civil servants had the shortest sleep duration (mean = 7.85 hours). Farmers also had the best sleep quality (mean score = 3.74) while professional workers had the worst sleep quality (mean score = 4.87).
10.1371/journal.pone.0117700.t002
###### Distribution of sleep duration sleep quality scores by occupation.
{#pone.0117700.t002g}
Sleep duration (hour) Sleep quality (score)
---------------------- ------------------------------------------- ----------------------- ------------------------------------------- ------
Civil 7.85 1.02 4.57 2.37
Professional 7.87 1.08 4.87[\*](#t002fn001){ref-type="table-fn"} 2.41
Worker 8.10[\*](#t002fn001){ref-type="table-fn"} 1.17 4.75[\*](#t002fn001){ref-type="table-fn"} 2.55
Famer 8.22[\*](#t002fn001){ref-type="table-fn"} 1.05 3.74[\*](#t002fn001){ref-type="table-fn"} 2.76
Business man/service 8.20[\*](#t002fn001){ref-type="table-fn"} 1.21 4.31[\*](#t002fn001){ref-type="table-fn"} 2.32
Students 7.88 1.06 4.43 2.13
Others 8.07[\*](#t002fn001){ref-type="table-fn"} 1.14 4.46 2.37
\*Compared with civil, P\<0.05
[Table 3](#pone.0117700.t003){ref-type="table"} shows the results of simple and multiple logistic regression models used to elucidate the effects of different demographic variables on the association of short sleep duration and poor sleep quality.
10.1371/journal.pone.0117700.t003
###### Multiple logistic regression models and the associations between occupations, sleep duration, and sleep quality.
{#pone.0117700.t003g}
Civil Professional Worker Farmer Business Students Others
---------------------- ------- -------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- ------------------- --------------------------------------------------------
Short sleep duration
Model 1 Ref. 1.08 (0.87--1.36) 1.22 (1.01--1.48)[\*](#t003fn002){ref-type="table-fn"} 0.70 (0.52--0.93)[\*](#t003fn002){ref-type="table-fn"} 1.17 (0.90--1.51) 1.10 (0.84--1.45) 1.09 (0.83--1.44)
Model 2 Ref. 1.18 (0.94--1.47) 1.39 (1.11--1.73)[\*](#t003fn002){ref-type="table-fn"} 0.83 (0.59--1.16) 1.41 (1.07--1.85)[\*](#t003fn002){ref-type="table-fn"} 1.25 (0.94--1.67) 1.35 (1.01--1.81)[\*](#t003fn002){ref-type="table-fn"}
Poor sleep quality
Model 1 Ref. 1.24 (1.11--1.40)[\*](#t003fn002){ref-type="table-fn"} 1.28 (1.15--1.42)[\*](#t003fn002){ref-type="table-fn"} 0.87 (0.75--0.99)[\*](#t003fn002){ref-type="table-fn"} 0.93 (0.80--1.07) 1.08 (0.93--1.24) 0.97 (0.85--1.14)
Model 2 Ref. 1.32 (1.17--1.50)[\*](#t003fn002){ref-type="table-fn"} 1.20 (1.06--1.36)[\*](#t003fn002){ref-type="table-fn"} 0.90 (0.75--1.07) 1.15 (0.98--1.34) 1.02 (0.87--1.19) 1.07 (0.90--1.26)
Model 1 adjusted sex, and age; Model 2 adjusted sex, age, education, area, marriage, smoking, drinking, body pain, and health status.
\*P\<0.05
Association between occupation and sleep duration {#sec015}
-------------------------------------------------
The crude OR for short sleep duration is 1.22 (95% CI 1.01--1.48) for blue collar workers and 0.70 (95% CI 0.52--0.93) for farmers compared with civil. Additional adjustments based on education, area, marriage, smoking, drinking, body pain, and health status in model 2 did not attenuate the effect of occupation for civil (OR = 1.39; 95% CI: 1.11--1.73), but there was an attenuation of the effect of occupation for farmers (OR = 0.83; 95% CI: 0.59--1.16). In model 2, the adjusted OR for shortened sleep duration is 1.41 (95% CI: 1.07--1.85) for business compared with civil.
Association between occupation and sleep quality {#sec016}
------------------------------------------------
Similarly, the OR for poor sleep quality was 1.24 (95% CI: 1.11--1.40) for professional, and 1.28 (95% CI: 1.15--1.42) for blue collar workers, and 0.87 (95% CI: 0.75--0.99) for famers compared to that of civil. Additional adjustments based on education, area, marriage, smoking, drinking, body pain, and health status in model 2 did not attenuate the association amongst professional (OR = 1.32; 95% CI: 1.17--1.50) and blue collar workers (OR = 1.20; 95% CI: 1.06--1.36), but there was attenuation of this association found amongst farmers (OR = 0.90; 95% CI: 0.75--1.07).
Discussion {#sec017}
==========
In this nationally representative sample of the Chinese population, we found blue collar workers had a higher prevalence of shortened sleep duration and decreased sleep quality compared with that of individuals in other occupations. In multiple logistic regression analysis, blue collar workers and business workers had shorter sleep duration compared with individuals in other occupations. Professional workers and blue collar workers had decreased quality sleep compared with individuals in other occupations. Our study confirmed the results of a previous study which showed that shortened sleep duration varied based on industry and occupation among US workers \[[@pone.0117700.ref007]\].
There are a few potential explanations for these results. The \"healthy worker\" effect could explain why civil servants are healthier than blue collar workers. In China, all civil servants are required to pass a strict medical examination upon recruitment. Conversely, blue collar worker are only occasionally required to pass a medical examination, and these test requirements are lower compared to civil servants. Hence, civil workers could be healthier and they might have better sleep quality or long duration. However, in our analysis, we adjusted for health status and did not find that this changed the association found with occupation.
Another potential explanation for these results is based on health and sleep disparities due to differences in SES. Anders et al. conducted a cross-sectional study with 3,281 participants in Germany and found that low socio-economic status was associated with poor sleep quality They concluded that SES is one of the determinants of good sleep quality, but it is that not most important determinant and it does not act in isolation \[[@pone.0117700.ref013]\]. Of note, the SES in their study was calculated based on participants\' education, net household income, and occupation. However, occupation was one of the important components in determining SES and highly associated with education level and household income. Occupation was more likely to be determined by education level and affect the household income. Hence, Anders et al. might have over-adjusted for the occupation independent effect of SES.
It is possible that occupation contributes to risk factors associated with shortened sleep duration and poor sleep quality, such as obesity\[[@pone.0117700.ref020]\], and other etiologies.
The strengths of this study include the large sample size and the adjustment for other important cofounders (education, health status, etc.). However, our study has several limitations. Sleep data was self-reported which can be imprecise and subject to reporting biases. This study did not include objective measurements of sleeps. Furthermore, there are no validated tools for assessing self-reported sleep duration. We lacked data on physical activity in subjects, which could be potential confounders. Finally, our study was performed on a relatively homogenous population. Further observational studies focusing on different populations using objective measures of sleep duration may help clarify this association between occupation and sleep.
In conclusion, our results suggest that Chinese workers have a short sleep duration and poor sleep quality compares with civil. occupations that contribute to differences in sleep duration and quality. These differences still need to be identified in the future study.
We wish to thank all the participants for their co-operation in the data collection.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: WS DL GS. Performed the experiments: YY TX GS. Analyzed the data: WS AL JY. Contributed reagents/materials/analysis tools: YY DL TX. Wrote the paper: WS YY CZ JY CL TL. Revised the manuscript: JY CL TL.
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{
"pile_set_name": "PubMed Central"
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It was brought to our attention that, in the original article, inappropriate manipulation of [**Figures 1**](#f1){ref-type="fig"} and [**5**](#f5){ref-type="fig"} took place, specifically, [**Figures 1B, 1C, 1D, 1E, 1F, 1G**](#f1){ref-type="fig"} and [**5B, 5C, 5D, 5E, 5F, 5G**](#f5){ref-type="fig"}. Upon review, we have agreed that these figures be removed from the article. The manipulation of the figures involved an effort to increase the contrast of the images to make them more presentable.
Although this did not change the interpretation of the data, the manipulation was clearly against our lab policy and the policies of the journal. Given that other data in the paper (e.g., qPCR and other experiments) confirm the data presented in these figures, we feel that the conclusions of the paper remain valid even with the figures removed.
To avoid any additional questions or concerns, we have also corrected [**Figures 2D, 2E**](#f2){ref-type="fig"} and [**2F**](#f2){ref-type="fig"}, as well as [**Figures 4D, 4E, 4F**](#f4){ref-type="fig"}, and [**4G**](#f4){ref-type="fig"}, and replaced these figures with original images. The corrected figures appear below. Due to the changes mentioned above, a correction has been made to the **Results**, subsection **Gma-miR171o and Gma-miR171q Exhibit Distinct Expression Patterns in Response to Bacterial Infection**:
"To understand the symbiotic role of gma-miR171o and gma-miR171q in soybean, we measured the relative expression level of these two miRNAs in nodules 3 weeks post-inoculation with *B. japonicum*, as compared to uninfected root tissues ([**Figure 1A**](#f1){ref-type="fig"}). Interestingly, these two miRNAs showed opposite expression patterns in response to *B. japonicum* infection ([**Figure 1A**](#f1){ref-type="fig"}); gma-miR171o expression was suppressed upon bacterial infection, while gma-miR171q was induced."
A correction has also been made to the **Results**, subsection **Gene Expression and Promoter Localization of *GmSCL6-1* and *GmNSP2*.1 Are Inversely Correlated With Gma-miR171o and Gma-miR171q**. Paragraph two has been removed entirely.
{#f1}
{#f2}
{#f4}
{#f5}
The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.
[^1]: Edited and reviewed by: Aardra Kachroo, University of Kentucky, United States
[^2]: This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science
[^3]: †These authors have contributed equally to this work
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Introduction {#sec1}
============
Cancer is one of the most devastating killers of human beings, accounting for millions of deaths around the world each year.[@bib1], [@bib2] Conventional physical and chemical methods, including targeted therapy, chemotherapy, and radiation therapy, remain the principle modes to treat cancer, which focus on killing the diseased cells, but normal cells are also adversely affected.[@bib3], [@bib4] More obviously, these treatments are expensive and inefficient, which means there is an urgent need to develop novel efficient measures to solve this deadly disease.[@bib5] The discovery of anticancer peptides (ACPs), a kind of short peptide generally with a length less than 50 amino acids and most of which are derived from antimicrobial peptides (AMPs), often cationic in nature, has led to the emergence of a novel alternative therapy to treat cancer.
ACPs open a promising perspective for cancer treatment, and they have various attractive advantages,[@bib6], [@bib7] including high specificity, ease of synthesis and modification, low production cost, and so on.[@bib8] ACPs could interact with the anionic cell membrane components of only cancer cells, and, for this reason, they can selectively kill cancer cells with almost no harmful effect on normal cells.[@bib4], [@bib9] In addition, few ACPs, e.g., cell-penetrating peptides or peptide drugs, inhibit the cell cycle or any other functionality. Thus, they are safer than traditional broad-spectrum drugs, which have become the most competitive choice as therapeutics compared to small molecules and antibodies. In recent years, ACP therapeutics have been extensively explored and used to fight various tumor types across different phases of preclinical and clinical trials.[@bib10], [@bib11], [@bib12], [@bib13], [@bib14] However, only a few of them can eventually be employed for clinical treatment. Furthermore, it's time-consuming, expensive, and lab-limited to identify potential new ACPs by experiment.
With the huge therapeutic importance of ACPs, there is an urgent need to develop highly efficient prediction techniques. Some notable research has been reported in the prediction of ACPs.[@bib15] Tyagi et al.[@bib16] developed a support vector machine (SVM) model using amino acid composition (AAC) and dipeptide composition as input features on experimentally confirmed anticancer peptides and random peptides derived from the Swiss-Prot database. Hajisharifi et al.[@bib17] also reported an SVM model using Chou's[@bib18], [@bib19] pseudo AAC (PseAAC) and the local alignment kernel-based method. Vijayakumar and Ptv[@bib20] proposed that, between ACPs and non-ACPs, there was no significant difference in AAC observed. Also, they presented a novel encoding measure, which achieved better predictive performance than AAC-based features, considering both compositional information and centroidal, distributional measures of amino acids. Shortly afterward, based on the optimal g-gap dipeptide components, by exploring the correlation between long-range residues and sequence-order effects, Chen et al.[@bib21] described iACP, which exhibited the best predictive performance at that time. More recently, Wei et al.[@bib22] developed a sequence-based predictor called ACPred-FL, which uses two-step feature selection and seven different feature representation methods.
According to the cognition of the short length of ACPs, it's difficult to exploit the efficient features of many mature feature representation methods, which are widely used on protein sequences.[@bib23] With the rapid growth of the number of ACPs that has been identified experimentally, by machine learning, and by bioinformatics research,[@bib24], [@bib25], [@bib26], [@bib27], [@bib28], [@bib29], [@bib30], [@bib31], [@bib32], [@bib33], [@bib34], [@bib35], [@bib36], [@bib37], [@bib38], [@bib39], [@bib40] the computational prediction methods of ACPs still need further development.
In this study, we proposed a deep learning long short-term memory (LSTM) neural network model to predict anticancer peptides, which we named ACP-DL. The efficient features exploited from peptides sequences are fed as input to train the LSTM model. More specifically, peptide sequences are transformed by *k*-mer sparse matrix of the reduced amino acid alphabet,[@bib41], [@bib42] which is a 2D matrix, and retained almost complete sequence order and amino acid component details. Meanwhile, peptide sequence are also converted by a binary profile feature,[@bib43] which can be regarded as one-hot encoding of categorical variables and has been suggested to be an efficient feature extraction technique.[@bib16], [@bib22] Finally, these features are fed into our LSTM model to predict new anticancer peptides.
To further evaluate the performance of our model, we evaluated the ACP-DL on two novel benchmark datasets. We also compared the purposed ACP-DL with existing state-of-the-art machine-learning models, e.g., SVM,[@bib44], [@bib45] Random Forest (RF),[@bib46]and Naive Bayes (NB).[@bib47] The 5-fold cross-validation experimental results showed that our method is suitable for the anticancer prediction mission with notable prediction performance. The workflow of ACP-DL is show in [Figure 1](#fig1){ref-type="fig"}.Figure 1The Flowchat of Our ACP-DL MethodWe used the *k*-mer sparse matrix and binary profile feature to represent peptide sequences, and the deep LSTM model is trained to predict anticancer peptides.
Results and Discussion {#sec2}
======================
Above all, we compared the different distributions of amino acids in anticancer peptides, non-anticancer peptides, and all peptides in datasets ACP740 and ACP240. The results for ACP740 are shown in [Figure 2](#fig2){ref-type="fig"}, the composition of all 20 amino acids in these peptides were counted and compared. Certain residues, including Cys, Phe, Gly, His, Ile, Asn, Ser, and Tyr, were found to be abundant in anticancer peptides compared to non-anticancer peptides, while Glu, Leu, Met, Gln, Arg, and Trp were abundant in non-anticancer peptides compared to anticancer peptides. Similarly, as shown in [Figure 3](#fig3){ref-type="fig"}, in dataset ACP240, the Phe, His, Ile, and Lys were abundant in anticancer peptides. Since terminal residues play essential roles in biological functions of peptides.Figure 2Comparison of Amino Acid Composition of Anticancer, Non-anticancer, and Total Peptides in Dataset ACP740Figure 3Comparison of Amino Acid Composition of Anticancer, Non-anticancer, and Total Peptides in Dataset ACP240
Evaluation of ACP-DL's Capability to Predict Anticancer Peptides {#sec2.1}
----------------------------------------------------------------
First, we executed our model ACP-DL on the ACP740 and ACP240 datasets to evaluate its ability of predicting anticancer peptides. The 5-fold cross-validation details are offered in [Tables 1](#tbl1){ref-type="table"} and [2](#tbl2){ref-type="table"}.Table 1The 5-Fold Cross-Validation Details in the ACP740 DatasetFold SetAcc (%)Sens (%)Spec (%)Prec (%)MCC (%)179.7381.9477.6381.9459.58283.1185.7180.0086.3066.39381.0879.7584.0078.0862.22485.8186.4985.3386.3071.63577.7079.1776.0079.4555.47Average81.48 ± 3.1282.61 ± 3.3680.59 ± 4.0182.41 ± 3.8163.05 ± 6.23Table 2The 5-Fold Cross-Validation Details in the ACP240 DatasetFold SetAcc (%)Sens (%)Spec (%)Prec (%)MCC (%)193.7589.6699.9986.3687.99281.2577.4292.3168.1863.02387.5088.4688.4686.3674.83483.3390.9176.9290.9167.83581.2576.6792.0069.5763.53Average85.4284.6289.9480.2871.44
The average accuracy of 5-fold cross-validation on ACP740 was 81.48% with 3.12% SD, the average sensitivity (Sens) was 82.61% with 3.36% SD, the average specificity (Spec) was 80.59% with 4.01% SD, the mean precision (Prec) was 82.41% with 3.81% SD, and the Matthews correlation coefficient (MCC) was 63.05% with 6.23% SD. ACP-DL showed an outstanding capability to identify anticancer peptides, performed an area under the receiver operating characteristic (ROC) curve (AUC) of 0.894, as shown in [Figure 4](#fig4){ref-type="fig"}A, and has achieved the best performance on the ACP740 dataset among all comparison methods.Figure 4Performance of the Proposed Model ACP-DL and Comparison Model on Datasets ACP740 and ACP240(A) The performance of the proposed model ACP-DL in dataset ACP740. (B) The performance of the comparison models in dataset ACP740, including SVM, RF, and NB. (C) The performance of the proposed model ACP-DL in dataset ACP240. (D) The performance of the comparison models in dataset ACP240, including SVM, RF, and NB.
The mean accuracy of 5-fold cross-validation on ACP240 was 85.42%, the average Sens was 84.62%, the average Spec was 89.94%, the mean Prec was 80.28%, and the MCC was 71.44%; and, the AUC of ACP-DL was 0.906, as shown in [Figure 4](#fig4){ref-type="fig"}C. In general, the performance of the deep learning model will become better with the increase in the scale of data, and the model that can achieve good results on smaller datasets will also achieve good results on huger data. Actually, the data scale of anticancer peptides is not very large, so we didn't implement a neural network model with very complex architecture; and, to a certain extent, the 5-fold cross-validation is not conducive to the neural network model, because it further reduces the amount of training data. It is noteworthy that, although the dataset ACP240 was smaller than ACP740, our model ACP-DL still performed very well. The experimental results of rigorous cross-validation on benchmark dataset ACP740 and dataset ACP240 confirmed that our model has a good capability to predict anticancer peptides.
Comparison with Three Widely Used Machine-Learning Models {#sec2.2}
---------------------------------------------------------
To evaluate the ability of the purposed method, we further compared ACP-DL with other widely used machine-learning models on the same benchmark datasets, including ACP740 and ACP240. Here we have selected the SVM, RF, and NB models, and we built them using the same cross-validation datasets. The implementation of these three machine-learning models comes from Scikit-learn,[@bib48] and they were tested with default parameters. Since these methods were evaluated using the same evaluation criteria, the comparison model and deep learning model ACP-DL results are shown in [Table 3](#tbl3){ref-type="table"} and [Figures 4](#fig4){ref-type="fig"} and [5](#fig5){ref-type="fig"}. ACP-DL obtained the most significant performance among the contrasted methods.Table 3Actual Performance of Comparison Models and ACP-DL in the Same DatasetDatasetModelAcc (%)Sens (%)Spec (%)Prec (%)MCC (%)AUCACP740SVM64.5962.4390.68[a](#tblfn1){ref-type="table-fn"}37.5733.570.829RF76.3575.1080.3472.2753.060.842NB69.7384.70[a](#tblfn1){ref-type="table-fn"}49.2190.94[a](#tblfn1){ref-type="table-fn"}43.980.825ACP-DL81.48[a](#tblfn1){ref-type="table-fn"}82.6180.5982.4163.05[a](#tblfn1){ref-type="table-fn"}0.894[a](#tblfn1){ref-type="table-fn"}ACP240SVM77.5085.89[a](#tblfn1){ref-type="table-fn"}70.6885.65[a](#tblfn1){ref-type="table-fn"}57.310.855RF72.0873.5376.0967.6344.380.793NB72.5072.2679.9463.9545.440.719ACP-DL85.42[a](#tblfn1){ref-type="table-fn"}84.6289.94[a](#tblfn1){ref-type="table-fn"}80.2871.44[a](#tblfn1){ref-type="table-fn"}0.906[a](#tblfn1){ref-type="table-fn"}[^2]Figure 5Comparison of SVM, Random Forest, Naive Bayes, and ACP-DL in Benchmark Datasets ACP740 and ACP240
[Table 3](#tbl3){ref-type="table"} shows the details of the comparison. In the ACP740 dataset, our method ACP-DL significantly outperformed other methods with an accuracy of 81.48%, a Sens of 82.61%, a Spec of 80.59%, a Prec of 82.41%, an MCC of 63.05%, and an AUC of 0.894. ACP-DL increased the accuracy by over 5%, the MCC by over 10%, and the AUC by more than 5%, respectively. In the dataset ACP240, ACP-DL also performed remarkably with an accuracy of 85.42%, a Sens of 84.62%, a Spec of 89.94%, a Prec of 80.28%, an MCC of 71.44%, and an AUC of 0.906. ACP-DL improved the accuracy by over 8%, the Spec by over 10%, the MCC by over 14%, and the AUC by more than 5%, respectively. Obviously, the deep learning model shows its power, and our model is suitable for anticancer peptide identification and prediction. ACP-DL is a competitive model used to predict anticancer peptides and accelerate related research. The comparison experiment results proved our assumption.
Conclusions {#sec2.3}
-----------
In this study, we proposed a deep learning LSTM model to predict potential anticancer peptides using high-efficiency feature representation. More specifically, we developed an efficient feature representation approach by integrating binary profile feature and *k*-mer sparse matrix of reduced amino acid alphabet feature to fully exploit peptide sequence information. Then we implemented a deep LSTM model to automatically learn how to identify anticancer peptides and non-anticancer peptides. To the best of our knowledge, this is the first time that the deep LSTM model has been applied to predict anticancer peptides.
Meanwhile, to evaluate the capability of the proposed method, we further compared ACP-DL with widely used machine-learning models in the same benchmark datasets, including ACP740 and ACP240; experimental results on the 5-fold cross-validation show that the proposed method achieved outstanding performance compared with existing methods, on benchmark dataset ACP740 with 81.48% accuracy at the AUC of 0.894 and on dataset ACP240 with an accuracy of 85.42% at the Spec of 89.94 and the AUC of 0.906, respectively. The improvement is mainly from the deep LSTM model's model parameter optimization and effective feature representation from original peptide sequences. In addition, we have contributed two novel anticancer peptide benchmark datasets, ACP740 and ACP240, in this work.
It is anticipated that ACP-DL will become a very useful high-throughput and cost-effective tool, being widely used in anticancer peptide prediction as well as cancer research. Further, as demonstrated in a series of recent publications in developing new prediction methods,[@bib49], [@bib50], [@bib51] user-friendly and publicly accessible web servers will significantly enhance their impacts. It is our wish to be able to provide in the future a web server for the prediction method presented in this paper.
Materials and Methods {#sec3}
=====================
In this study, we proposed a novel deep learning LSTM model to predict anticancer peptides, named ACP-DL, using high-efficiency features provided by *k*-mer sparse matrix and the binary profile feature. Furthermore, we evaluated ACP-DL's predictive performance of anticancer peptides in benchmark datasets ACP740 and ACP240. Moreover, we compared ACP-DL with three widely used machine-learning models in the same datasets, including SVM,[@bib44] RF,[@bib46] and NB,[@bib47] to prove the robustness and effectiveness of the proposed method. Eventually, we made a summary, analysis, and discussion of the ACP-DL.
Construction of Datasets {#sec3.1}
------------------------
We constructed two novel benchmark datasets in this work for ACP identification, named ACP740 and ACP240. As previous studies suggested, the new datasets comprised both positive and negative datasets, while positive samples were experimentally validated ACPs and AMPs without anticancer function were collected as negative samples.
The positive anticancer peptide samples can be represented as $P^{+}$, and the negative non-anticancer peptides can be represented as $P^{-}$. So, the whole dataset can be represented as *P*.$$\text{P} = P^{+} \cup P^{-}$$Moreover, there is no overlap between the positive and negative datasets.$$Ø = P^{+} \cap P^{-}$$
### Dataset ACP740 {#sec3.1.1}
We selected 388 samples as the initial positive data on the basis of Chen et al.'s[@bib21] and Wei et al.'s[@bib24] studies, of which 138 were from Chen et al.'s work and 250 were from Wei et al.'s work. Correspondingly, the initial negative data were 456 samples, of which 206 were from Chen et al.'s work and 250 were from Wei et al.'s work, respectively. To avoid the bias of dataset, the widely used tool CD-HIT[@bib52] was further used to remove those peptides sequences with a similarity of more than 90%. As a result, we finally obtained a dataset containing 740 samples, of which 376 were positive samples and 364 were negative samples.
### Dataset ACP240 {#sec3.1.2}
As the same procedure, to validate the generalization ability of the predictive model, we further constructed an additional dataset, named ACP240, which initially included 129 experimentally validated anticancer peptide samples as the positive dataset and 111 AMPs without anticancer functions as the negative dataset, respectively.
Moreover, those sequences with a similarity of more than 90% were removed using the popular tool CD-HIT.[@bib52] The similarity setting was consistent with previous studies.[@bib21], [@bib22] The CD-HIT is available at <http://weizhong-lab.ucsd.edu/cdhit-web-server>. There was no overlap between dataset ACP740 and dataset ACP240, and these two datasets are both non-redundant datasets. The two benchmark datasets are publicly available at <https://github.com/haichengyi/ACP-DL>.
Representation of the Peptide Sequences {#sec3.2}
---------------------------------------
A peptide sequence can be represented as follows:$$\text{P} = p_{1}p_{2}p_{3}p_{\ldots}p_{l},$$where $p_{1}$ represents the first residue in the peptide P, $p_{2}$ denotes the second residue in the peptide P, and so on; *l* represents the length of P. Note that the residue $p_{i}$ is an element of the standard amino acid alphabet to train a machine-learning model; the first step is to convert diverse-length peptides into fixed-length feature vectors. In this study, we exploited two feature representation methods, as described below.
### Binary Profile Feature (BPF) {#sec3.2.1}
As mentioned above, there are 20 different amino acids in the standard amino acid alphabet (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y). Each amino acid type is encoded with the following feature vector composed of 0/1. More specifically, the first amino acid type A in the alphabet is encoded as f(A) = (1,0,...,0), the second amino acid type C is encoded as f(C) = (0,1,...,0), and so on. Subsequently, for a given peptide sequence P, its N terminus with the length of *k* amino acids was encoded as the following feature vector:$$\text{BPF}\left( \text{k} \right) = \left\lbrack {f\left( p_{1} \right),f\left( p_{2} \right),\ldots f\left( p_{k} \right)} \right\rbrack,$$where *k* represents the length of the N terminus of the peptide P.[@bib22] Thus, the dimension of BPF(P) is 1 × 20. Experiments show that the best results can be achieved when *k* is set to 7. So, one given peptide sequence is encoded to a $1 \times 140$ feature vector by binary profile.
### K-mer Sparse Matrix {#sec3.2.2}
We also encoded the peptide sequence by using the *k*-mer sparse matrix previously proposed.[@bib41] In detail, its *k-*1 consecutive nucleotides and *k* consecutive nucleotides are regarded as a unit. 3-mer of peptides is composed of 3 amino acids.[@bib53] First the 20 amino acids were reduced into 7 groups based on their dipole moments and side chain volume: Ala, Gly, and Val; Ile, Leu, Phe, and Pro; Tyr, Met, Thr, and Ser; His, Asn, Gln, and Tpr; Arg and Lys; Asp and Glu; and Cys.[@bib16], [@bib54], [@bib55] So, the peptide sequence was reduced into a 7-letter alphabet. Then we scanned each peptide sequence from left to right, stepping one amino acid at a time, which is considered the characteristics of each amino acid.
Suppose an above-mentioned peptide sequence length is *L*, there would be $7^{k}$ different possible *k*-mer and an $L - k + 1$ step appearing in the RNA sequence.
One peptide sequence is transformed into a $7^{k} \times \left( {L - k + 1} \right)$ *k*-mer sparse matrix *M*. Initialization of all elements is 0. When $m_{j}m_{j + 1}m_{j + 2}$ are just equal to the *i*~th~ *k*-mer among $7^{k}$ different *k*-mer, set the element *a*~*ij*~ *=* 1. The rest can be handled in the same way. Thus, an input peptide sequence is converted into a $7^{k} \times \left( {L - k + 1} \right)$ matrix *M*.
In this study, the value of *k* is set to 3 to process the peptide sequence. The *k*-mer sparse matrix *M* can be defined as follows:$$\text{M} = \left( \text{a}_{\text{ij}} \right)_{7^{\text{k}}} \times \left( {\text{L} - \text{k} + 1} \right)$$$$a_{ij} = \left\{ \begin{matrix}
{1,\ if\ \text{m}_{\text{j}}\text{m}_{\text{j} + 1}\text{m}_{\text{j} + 2} = k - mer\left( i \right)} \\
{0,\mspace{9mu} else} \\
\end{matrix} \right..$$The *k*-mer sparse matrix *M* is a low-rank matrix, which almost retained all the raw information, including sequence frequency, position, and order hidden information. Then, singular value decomposition (SVD)[@bib56] is used to reduce one two-dimensional matrix *M* into a $1 \times 343$ feature vector.
Finally, we conjoined two different feature representation methods' output, each peptide sequence gain $1 \times 483$ conjoined feature vector. Meanwhile, the whole dataset was transformed as a 2D matrix here. The feature matrix was reshaped into a 3D tensor for training the LSTM model, while the feature matrix without being formally reshaped was used to train the comparison model.
Deep LSTM Model Architecture {#sec3.3}
----------------------------
LSTM is an improvement of a recurrent neural network (RNN), which is mainly used in the natural language processing (NLP) and speech recognition field.[@bib57], [@bib58], [@bib59] Different from a traditional neural network, an RNN can take advantage of sequence information. Theoretically, it can utilize the information of arbitrary length sequence; but, because of the problem of vanishing gradient in the network structure, it can only retrospectively utilize the information on time steps that are close to it in practical applications. To solve this problem, LSTM was presented with specially designed network architecture, which can learn long-term dependency information naturally. A general architecture of LSTM is composed of an input gate, a forget gate, an update gate, and a memory block. The improvement of LSTM is mainly from incorporating a memory cell that accepts the network to learn when to forget previous hidden states and when to update hidden states, according to the input information through time. It uses dedicated storage units to store information. To our knowledge, the deep LSTM model was first applied to predict novel anticancer peptides in this work.
LSTM selectively passes information through a gate unit, which mainly is by means of a sigmoid neural layer and a dot multiplication operation. Each element of the sigmoid layer output (a vector) is a real number between 0 and 1, representing the weight (or percentage) that the corresponding information passes through. For example, 0 means no information is allowed, and 1 means let all information pass.
### Forget Gate {#sec3.3.1}
In the information flow processing of LSTM, the first step is to decide what information will discord from the cell state. This decision is accomplished by a way known as forget gate. Forget gate reads $h_{t - 1}$ and $x_{t}$, then outputs a value between 0 and 1 for each digit in cell state $C_{t - 1}$; 1 means reserved absolutely and 0 means discard completely.$$f_{t} = \sigma\left( {W_{f} \cdot \left\lbrack {h_{t - 1},x_{t}} \right\rbrack + b_{f}} \right)$$Here, the $h_{t - 1}$ represents the output of the previous cell, $x_{t}$ represents the current cell input, and $\sigma$ means Sigmoid function.
### Input Gate {#sec3.3.2}
The next step is to decide how much new information will be added to the cell state. To do this, there are two steps: first, a Sigmoid layer called the input gate layer determines which information needs to be updated; and then, a tanh layer generates a vector, which is the alternate content ${\widetilde{C}}_{t}$ to update. We combined the two parts to update the state of cell.$$i_{t} = \sigma\left( {W_{i} \cdot \left\lbrack {h_{t - 1},x_{t}} \right\rbrack + b_{i}} \right)$$$${\widetilde{C}}_{t} = \text{tanh}\left( {W_{c} \cdot \left\lbrack {h_{t - 1},x_{t}} \right\rbrack + b_{c}} \right)$$$$C_{t} = f_{t} \ast C_{t - 1} + i_{t} \ast {\widetilde{C}}_{t}$$We multiply the old state with $f_{t}$ and discard the information we need to discard. Then we add $i_{t} \ast {\widetilde{C}}_{t}$. This is the new candidate value, which is changed according to the degree of each state we decide to update.
### Output Gate {#sec3.3.3}
Ultimately, we need to determine what output is. This output will be based on our cell state, but it is also a filtered version. First, we run a sigmoid layer to determine which part of the cell state will be exported. Then, we process the cell state through a tanh function (to get a value between −1 and 1) and multiply it with the output of the Sigmoid gate, and eventually we just output the portion of the output we determine.$$o_{t} = \sigma\left( {W_{o} \cdot \left\lbrack {h_{t - 1},x_{t}} \right\rbrack + b_{o}} \right)$$$$h_{t} = o_{t} \ast \tanh\left( C_{t} \right)$$In this experiment, considering the limited scale of anticancer peptide samples, we set the parameter of LSTM input layer to 128, and the output of LSTM layers was fed into a dense layer (a fully connected neural network layer) as input to obtain a final prediction result. We also used a sigmoid function as an activation function in the proposed model. The mathematical behaviors of a sigmoid function can be demonstrated as follows:$$\text{σ} = \text{sigmoid}\left( x \right) = \frac{1}{\left( {1 + e^{- x}} \right)}.$$Between them, the dropout layer was applied to reduce over-fitting and enhance the neural network model robustness, and the parameter *dropout* was set to 0.25. Moreover, a loss function can measure the performance of machine-learning models. We selected to use log loss function (binary cross-entropy) corresponding to sigmoid function as loss function, which can be defined as:$$\text{logloss}\left( {t,p} \right) = - \left( {\left( {1 - p} \right) \times \log\left( {1 - p} \right) + t \times \text{log}\left( p \right)} \right),$$where *p* and *t* represent the prediction output of model and true target value, respectively. Finally, the Adam[@bib60] optimizer was used to update the weights of network iteratively, which is popular in the deep learning field and combined the advantage of root-mean-square propagation (RMSProp) and adaptive gradient (AdaGrad) algorithm.
The implementation of the deep learning model is based on the Keras framework, which is capable of running on top of TensorFlow, Theano, or CNTK and is supported on both GPUs and CPUs. It was developed with a focus on enabling fast experimentation.[@bib61]
Performance Evaluation Criteria {#sec3.4}
-------------------------------
In this study, we proposed a novel deep learning LSTM model, ACP-DL, using an efficiency feature to predict potential anticancer peptides. We used 5-fold cross-validation to evaluate the performance of ACP-DL and comparison models. In each validation, all data randomly divide into five equal parts: the 4-fold set data are taken as training data, and the remaining 1-fold data are taken as test data. To guarantee the unbiased comparison, it was confirmed that there was no overlap between training data and test data. The final validation result was the average of 5-fold with SDs. We followed the widely used evaluation criteria,[@bib62], [@bib63] including accuracy (Acc), Sens or recall, Spec, Prec, and MCC, defined as follows:$$\text{Acc} = \frac{TN + TP}{TN + TP + FN + FP}$$$$\text{Sens} = \frac{TP}{TP + FN}$$$$\text{Spec} = \frac{TN}{TN + FP}$$$$\text{Prec} = \frac{TP}{TP + FP}$$$$\text{MCC} = \frac{TP \times TN - FP \times FN}{\sqrt{\left( {TP + FP} \right)\left( {TP + FN} \right)\left( {TN + FP} \right)\left( {TN + FN} \right)}},$$where *TN* indicates the true negative number, *TP* denotes the true positive number, *FN* represents the false negative number, and *FP* stands for the false positive number. Certainly, the ROC curve and the AUC were also adopted to evaluate the performance.
Author Contributions {#sec4}
====================
H.-C.Y. and Z.-H.Y. conceived the algorithm, carried out analyses, prepared the datasets, carried out experiments, and wrote the manuscript. Other authors designed, performed, and analyzed experiments and wrote the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest {#sec5}
=====================
The authors declare no competing interests.
This work was supported in part by the National Natural Science Foundation of China under grants 61572506 and 61722212 and in part by the Pioneer Hundred Talents Program of Chinese Academy of Sciences.
[^1]: These authors contributed equally to this work.
[^2]: This measure of performance is the best among the compared methods.
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"pile_set_name": "PubMed Central"
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Introduction {#s0005}
============
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide [@bb0005], partially due to the lack of effective chemotherapeutic options for patients with advanced-stage disease [@bb0010]. Various molecular profiling approaches have been applied to identify therapeutic targets specifically activated in HCC [@bb0015]. Cancer stem cells (CSCs) are considered a pivotal target for the eradication of HCC [@bb0020]. Some studies have suggested the importance of evaluating stemness in HCC because it reflects the malignant nature of the tumor closely and is related to poor prognosis after surgery [@bb0025], [@bb0030], [@bb0035], [@bb0040]. In HCC, several stem cell markers including CD133, CD90, CD13, epithelial cell adhesion molecule (EpCAM), CD24, and side populations are reportedly enriched in CSC populations [@bb0045].
Recently, we reported that the CSC markers EpCAM and CD90 are expressed independently in primary HCCs and cell lines [@bb0050], and CD90^+^ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor (TGF)-β [@bb0055]. Our previous data suggested that CD105 is not only a vascular endothelial cell marker but also a marker of CSCs with mesenchymal cell features, but the significance of CD105 expression on HCC phenotypes remains to be elucidated. In this study, we evaluated the expression of CD105 in human HCC and found that CD105^+^ HCC cells could be generated from CD105^−^ HCC cells *de novo* after treatment with cytotoxic reagents with activation of the expression of the epithelial-mesenchymal transition (EMT) inducers Snail family zinc finger 1 (*SNAI1*) and *SNAI2*.
Materials and Methods {#s0010}
=====================
Patients {#s0015}
--------
HCC samples were obtained with informed consent from patients who had undergone radical resection at the Department of Gastroenterologic Surgery in Kanazawa University Hospital, Kanazawa, Japan, and tissue acquisition procedures were approved by the Ethics Committee of Kanazawa University. A total of 85 formalin-fixed and paraffin-embedded HCC samples obtained from 2002 to 2008 were used for immunohistochemical analyses.
Cell Culture and Reagents {#s0020}
-------------------------
The human liver cancer cell lines HuH1 and HuH7 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) and routinely cultured with Dulbecco\'s modified Eagle\'s medium supplemented with 10% FBS. Epirubicin and 5-fluorouracil (5-FU) were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and Kyowa Kirin (Tokyo, Japan), respectively. HuH1 and HuH7 cell lines were seeded at 10,000 cells per well in a 6-well plate treated with epirubicin (0.5 μg/ml for HuH1 and 0.1 μg/ml for HuH7) or 5-FU (2.0 μg/ml for HuH1 and 2.5 μg/ml for HuH7) for 5 days based on IC50 data obtained from a cell proliferation assay. These cells were then used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and immunofluorescence analyses, as described previously [@bb0060].
qRT-PCR {#s0025}
-------
Total RNA was extracted using TRIzol (Thermo Fisher Scientific K.K., Yokohama, Japan) according to the manufacturer\'s instructions. The expression of selected genes was determined in triplicate using the 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Each sample was normalized relative to 18S rRNA expression. The following probes were used: ENG, Hs00923996_m1; *KRT19*, Hs00761767_s1; *THY1*, Hs00264235_21; and 18S rRNA, Hs99999901_s1 (Applied Biosystems).
FACS {#s0030}
----
Cultured cells were trypsinized, washed, and resuspended in Hank\'s balanced salt solution (Lonza, Basel, Switzerland) supplemented with 1% HEPES and 2% FBS. The cells were then incubated with antibodies on ice for 30 minutes. Labeled cells were analyzed by FACS using a FACSCalibur (BD Biosciences, San Jose, CA). The following antibodies were used: FITC-conjugated anti-EpCAM monoclonal antibody Clone Ber-EP4 (DAKO, Carpinteria, CA), APC-conjugated anti-CD326 (EpCAM) antibody (Miltenyi Biotec K.K., Tokyo, Japan), FITC-conjugated anti-CD90 monoclonal antibody Clone 5E10 (STEMCELL Technologies, Seattle, WA), APC-conjugated anti-CD133/2 antibody Clone 293C3 (Miltenyi Biotec K.K.), and APC-conjugated anti-CD105 mouse monoclonal antibody (BD Biosciences).
Immunohistochemistry and Immunofluorescence {#s0035}
-------------------------------------------
Immunohistochemistry was performed using Envision^+^ kits (DAKO, Carpinteria, CA) according to the manufacturer\'s instructions. Anti-CD105 rabbit monoclonal antibody EPR10145 (Abcam, Cambridge, UK) was used for detecting CD105. CD105 expression was measured and categorized into two groups, CD105^+^ and CD105^−^, according to the expression status not in vascular endothelial cells but in cancer cells. Immunofluorescence was performed using anti-CD105 mouse monoclonal antibody clone SN6h (DAKO) and Alexa 488 FITC-conjugated anti-mouse IgG as primary and secondary antibodies, respectively. Fluorescence microscopic analysis was performed as described previously [@bb0030].
Statistical Analysis {#s0040}
--------------------
Student\'s *t* test, chi-square test, and unpaired *t* test were performed with GraphPad Prism software 5.0 (GraphPad Software, San Diego, CA) to compare various test groups. Kaplan--Meier survival analysis was also performed with GraphPad Prism software 5.0 (GraphPad Software).
Results {#s0045}
=======
*De Novo* Emergence of CD105^+^ HCC Cells after Treatment with Cytotoxic Reagents {#s0050}
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Previously, we evaluated the expression of the CSC markers EpCAM and CD90 and their tumorigenicity in representative HCC cell lines. We found that the EpCAM^+^ cell lines HuH1 and HuH7 do not express CD90 and show an epithelial cell shape with high tumorigenic capacity, whereas the CD90^+^ cell lines HLE and HLF also do not express EpCAM but show a mesenchymal cell shape with high metastatic capacity. Interestingly, when we explored the expression of CD105 in these cell lines, we identified the abundant expression of CD105 in the CD90^+^ cell lines (89.2% in HLE and 57.2% in HLF) but not in the EpCAM^+^ cell lines (0% in HuH7 and 0.08% in HuH1) ([Figure 1](#f0005){ref-type="fig"}*A*), suggesting that CD105 may be a marker of mesenchymal liver CSCs. Next, we evaluated the expression of CD105 in the EpCAM^+^ cell lines HuH1 and HuH7 treated with the cytotoxic reagents epirubicin and 5-FU. Originally, HuH1 and HuH7 cells contained a population of EpCAM^+^ cells (33.3% in HuH1 and 66.9% in HuH7) but no CD90^+^ and CD105^+^ cells as evaluated by FACS analysis ([Figure 1](#f0005){ref-type="fig"}*B*). Furthermore, we identified the enrichment of the EpCAM^+^ cell population after treatment with epirubicin and 5-FU for 72 hours in HuH1 (39.4% by epirubicin and 50.5% by 5-FU) and HuH7 cells (72% by epirubicin and 82% by 5-FU), consistent with the highly chemoresistant capacity of EpCAM^+^ CSCs. However, surprisingly, we also identified a small population of CD90^+^ and CD105^+^ cells generated *de novo* in HuH1 and HuH7 cells after treatment with these cytotoxic reagents. When we evaluated the nuclear size of HuH1 cells, we identified strong and modest increases of nuclear size following treatment with epirubicin and 5-FU compared with control, respectively ([Figure 2](#f0010){ref-type="fig"}*A*). We confirmed the *de novo* expression of CD105 in HuH1 cells by immunofluorescence ([Figure 2](#f0010){ref-type="fig"}*A*). The induction of genes encoding CD105 (*ENG*), CK19 (*KRT19*), and CD90 (*THY1*) was further confirmed by qRT-PCR analysis in HuH1 and HuH7 cells ([Figure 2](#f0010){ref-type="fig"}*B*). The *de novo* emergence of CD105^+^ cells in HuH1 and HuH7 cells was accompanied by the upregulation of genes encoding the transcription factors SNAI1 and SNAI2, master regulators of genes regulating EMT. These data suggest that CD105, previously recognized as a vascular endothelial marker, was induced *de novo* in EpCAM^+^ HCC cell lines with activation of genes regulating EMT.
CD105^+^ HCCs Correlate with Microvascular Invasion and Poor Prognosis {#s0055}
----------------------------------------------------------------------
To elucidate the expression of CD105 in primary HCC tissues, we immunohistochemically evaluated the expression of CD105 in a total of 85 surgically resected HCC tissue samples. In most cases, CD105 staining was detected in vascular endothelial cells ([Figure 3](#f0015){ref-type="fig"}*A*, *panel a*); however, we also detected CD105 staining in HCC cells with a mesenchymal cell shape ([Figure 3](#f0015){ref-type="fig"}*A*, *panel b*). Most strikingly, CD105^+^ cancer cells were detected in surgically resected HCC patients\' tissues who received transcatheter arterial chemoembolization (TACE) with epirubicin prior to surgery ([Figure 3](#f0015){ref-type="fig"}*A*, *panel c*).
We classified the HCC cases into CD105^+^ and CD105^−^ according to the expression of CD105 in cancer cells, not in vascular endothelial cells. We defined HCC as CD105^+^ even if we could detect only a small subset of cancer cells (\<1%) stained by the CD105 antibody, unless the staining was restricted to vascular endothelial cells. Accordingly, 35 of 85 HCCs were defined as CD105^+^. When we evaluated the clinicopathological characteristics of the CD105^+^ and CD105^−^ HCCs, we could not detect any differences in terms of age; sex; virus infection status; presence of liver cirrhosis; tumor size; serum alpha-fetoprotein values; histological grades; distant organ metastasis; and tumor, node, and metastasis stages (See [Table 1](#t0005){ref-type="table"}). However, we found that CD105^+^ HCC could be characterized by a high frequency of microvascular invasion of the tumor. Consistently, although we could not detect differences in overall survival between CD105^+^ and CD105^−^ HCC cases, we observed the poor recurrence-free survival of CD105^+^ HCCs compared with CD105^−^ HCCs with statistical significance (*P* = .002). These data suggest that the expression of CD105 in HCC tissues may not only correlate with vascular density, as reported previously [@bb0065], but also correlate with the abundance of cancer cells with a mesenchymal cell shape expressing CD105, potentially induced by cytotoxic reagents or activated by the EMT program.
Discussion {#s0060}
==========
Although considered monoclonal in origin, cancer cells are heterogeneous in terms of morphology, proliferation, invasion capacity, and drug resistance. This heterogeneity is currently explained by two models: the clonal evolution model and the CSC model [@bb0020]. Previously, we provided evidence that at least some HCCs follow the CSC model [@bb0035]. We further explored the CSC hypothesis using several CSC markers and found that liver CSCs can be divided into at least two distinct entities: tumorigenic epithelial CSCs expressing EpCAM and metastatic mesenchymal CSCs expressing CD90 [@bb0050]. Besides, morphologically, CD90^+^ CSCs show vascular endothelial cell features, which are closely correlated with the phenotype termed vasculogenic mimicry [@bb0050]. Here, we provided evidence that CD90^+^ CSCs also express the vascular endothelial marker CD105. CD105^+^ HCC is characterized by a high frequency of microvascular invasion and poor prognosis after surgery. CD105^+^ cancer cells were evident in HCC tissues previously treated by TACE, suggesting that CD105^+^ cancer cells may be resistant to chemotherapy and hypoxia. These findings suggest that CD105 may be a good candidate molecule to target HCC cells with a capacity for vascular invasion and resistance to cytotoxic reagents.
CD105 is a type I integral transmembrane glycoprotein that has predominantly been investigated in the vasculature where it is upregulated during angiogenesis [@bb0070]. In endothelial cells, CD105 is an accessory co-receptor for TGF-β and associates at the cell surface with type I and type II TGF-β receptors [@bb0070]. Therefore, CD105 has a key role in modulating downstream signaling molecules such as SMAD family proteins. Because of its role in neovascularization, CD105 is generally considered as one of the vascular endothelial cell markers, such as CD31 and VEGFRs, and reflects the vascular density of a tumor, which may correlate with tumor progression, metastasis, and poor prognosis [@bb0065]. Indeed, the increased expression of CD105 in vascular endothelial cells has been reported as a marker of invasiveness and metastatic potential in cancer, including HCC [@bb0055].
Although CD105 is expressed in vascular endothelial cells and strongly correlates with angiogenesis, recent studies suggested that CD105 is also expressed in tumor epithelial cells of renal cell carcinoma [@bb0075] and ovarian cancer [@bb0080]. Here, we reported that CD105 is also expressed in liver CSCs expressing CD90 with metastatic mesenchymal cell features. Furthermore, we provided evidence that mesenchymal CD105^+^ cells could originate from epithelial CD105^−^ cells *de novo*. It may be possible that CD105 could be a direct target against HCC with chemoresistance and metastatic features. As CD105 is a co-receptor of TGF-β receptors, it is plausible that TGF-β signaling may be activated in CD105^+^ HCC cells. Indeed, we demonstrated the activation of SNAI1 and SNAI2 in CD105^+^ HCC cells, potentially resulting from the activation of TGF-β signaling. Therefore, it could be possible to suppress cancer cell growth by TGF-β receptor signaling inhibitors such as galunisertib (LY2157299) [@bb0085]. Furthermore, a recent phase II study evaluating the effects of TRC105, a chimeric IgG1 anti-CD105 monoclonal antibody, in human HCC patients showed that it was tolerated well [@bb0090]. Future studies are required to evaluate the role of CD105^+^ cells and the therapeutic effects of galunisertib or TRC105 in human HCC.
We would like to thank Ms. Masayo Baba for excellent technical assistance. This study was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (23590967).
This study was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (23590967).
Conflict of Interest: All authors declare that they have no conflicts of interest.
{#f0005}
{#f0010}
![CD105 expression and prognosis in HCC.\
(A) Panel (a) shows CD105 staining in vascular endothelial cells in HCC. Panel (b) shows CD105 staining in HCC cells with a mesenchymal cell shape. Panel (c) shows the enrichment of CD105^+^ cells in HCC tissues surgically resected after treatment with epirubicin TACE.\
(B). Kaplan--Meier survival analysis of CD105^+^ and CD105^−^ HCCs (overall survival \[OS\]).\
(C) Kaplan--Meier survival analysis of CD105^+^ and CD105^−^ HCCs (recurrence-free survival \[RFS\]). CD105^+^ HCCs showed poor RFS compared with CD105^−^ HCCs with statistical significance (*P* = .002).](gr3){#f0015}
######
Clinicopathological Characteristics of CD105^+^ and CD105^−^ HCC
Table 1
Parameter CD105^+^ (*n* = 35) CD105^−^ (*n* = 50) *P* Value
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Age 62.2 ± 11.6 64.8 ± 9.1 .25
Sex (M/F) 26/9 36/14 1
Virus (HBV/HCV/HBV+HCV/NBNC) 14/21/0/0 12/33/2/3
LC (yes/no) 22/13 29/21 .82
Tumor size (\>3 cm, ≦3 cm) 16/19 20/30 .66
AFP (median, \[25%-75%\]) 12.1 (10-418) 16 (10-92.5) .14
Microvascular invasion 16/19 8/42 .0036
Histological grade (well/moderate/poor)[⁎](#tf0005){ref-type="table-fn"} 3/25/7 8/38/4 .2
Distant organ metastasis (yes/no) 6/29 6/44 .54
TNM stages (I-II/III-IV) 25/10 38/12 .8
Edmondson-Steiner.
|
{
"pile_set_name": "PubMed Central"
}
|
Hematopoiesis is regulated by the orchestration of several processes, including cell proliferation, cell differentiation, and cell death. Apoptosis, also referred to as programmed cell death, regulates the survival of progenitor cells and the turnover of mature elements, which are important for maintaining the homeostasis of hematopoietic cells ([@B1]). Growth factors are thought to play important roles in the apoptosis of hematopoietic progenitors. They act as survival factors for hematopoietic precursors, and the hematopoietic progenitors succumb to apoptosis in their absence ([@B2]--[@B4]). On the other hand, the survival of hematopoietic cells is also controlled by the Bcl-2 protein family ([@B5]--[@B8]). Family members such as Bcl-2, Mcl-1, A1, and Bcl-X~L~ are expressed in hematopoietic cells and are considered to function as repressors of apoptotic cell death ([@B9]).
In mouse embryogenesis, erythropoiesis originates in the yolk sac blood islands, beginning at approximately embryonic day 7.5, then migrates to the fetal liver, spleen, and eventually to the bone marrow ([@B10]). Embryonic primitive erythropoiesis in the yolk sac and definitive erythropoiesis in the fetal liver, spleen, and bone marrow produce primitive erythrocytes (EryP)^1^ and definitive erythrocytes (EryD), respectively. Although the glycoprotein hormone erythropoietin (EPO) was initially characterized as stimulating both the proliferation and differentiation of EryD progenitors, it has been shown that EPO maintains the viability of primarily EryD progenitor cells ([@B11]--[@B13]). The dependence of the survival of EryD on EPO is reported in various experimental systems, including Friend virus anemia strain--infected murine splenic erythroid progenitors, the erythroleukemia cell line, and murine fetal liver erythroid progenitors (2, 14-- 16). These studies showed that survival of the late erythroid progenitors (CFU-E) of EryD was dependent on EPO and that the EPO-dependent period lasted from the CFU-E stage to the beginning of hemoglobin synthesis. Gene targeting experiments with EPO and EPO receptor revealed that EPO has important roles in primitive erythropoiesis ([@B12], [@B13]). We found that the survival of immature EryP was also dependent on EPO, as was that of EryD progenitors (our unpublished data). Thus, EPO prevents apoptosis of both EryP and EryD during their immature state.
The protein Bcl-2 prevents apoptosis triggered by various stimuli, including chemotherapeutic drugs, γ irradiation, viral infections, oxidant stress, and notably growth factor deprivation ([@B6]). Bcl-2 and Bcl-X~L~ act as cell death repressors, whereas Bax and the alternatively spliced *bcl-x* gene product, Bcl-X~S~, act as cell death promoters ([@B6], [@B17], [@B18]). A family of *bcl-2* genes participates in the regulation of cell survival in multiple cell lineages, including the hematopoietic lineage. Constitutive overexpression of Bcl-2 suppresses apoptosis in hematopoietic precursors by growth factor withdrawal, and overexpression of Bcl-X~L~ also suppresses apoptosis ([@B19], [@B20]). Both Bcl-2 and Bcl-X~L~ have recently been reported to be involved in regulating erythroid progenitors and survival ([@B21]--[@B25]). However, all of the evidence is circumstantial, and it is uncertain how *bcl-x* functions during erythroid differentiation under physiological conditions. In this study, we analyzed the function of *bcl-x* in erythropoiesis using mouse embryonic stem (ES) cells in which both alleles of *bcl-x* were disrupted ([@B26]--[@B30]). The production of immature EryP and EryD by *bcl-x* ^−/−^ ES cells was normal. Unexpectedly, however, prominent apoptotic cell death of both EryP and EryD occurred at the very end of erythroid maturation. These data clearly show that Bcl-X is essential in the late erythroid maturation stage.
Materials and Methods
=====================
Target Disruption of the bcl-x Gene.
------------------------------------
E14 ES cells derived from strain 129/Ola were used throughout the experiment. ES *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ cell lines were produced as described previously ([@B21]). Genomic DNA containing the *bcl-x* locus was isolated from a library of mouse strain 129/Sv DNA. A 1.8-kb XhoI-BamHI fragment containing most of the *bcl-x* coding region was replaced with either a PGK-*neo* polyadenylated (poly A) cassette or a PGK-*hyg* poly A cassette. Both targeting vectors contain 6.0-kb 5′ and 1.0-kb 3′ regions of homology with the drug-resistance markers and a PGK-*tk* poly A cassette. Transfection and selection were performed as described ([@B31]). DNA prepared from ES cells was digested with EcoRV, transferred to a nylon membrane, and then hybridized with the 0.4-kb KpnI-PstI probe that flanked the 3′ homology region. The expected sizes of wild-type *bcl-x*, mutant *bcl-x* with the *neo* targeting vector, and mutant *bcl-x* with the *hyg* targeting vector were 9.8, 7.0, and 5.5 kb, and were detected in wild-type, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ ES clones, respectively.
Production of Chimeric Mice and Analysis of the Contribution of ES Cells.
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ES *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ cells were injected into the 3.5-d post-coitum blastocysts from C57BL/6 mice to generate chimeric mice. GPI (glucose phosphoisomerase) isozymes were used to analyze the contribution of ES cells in various organs of chimeric mice. The GPI isozyme of B6 mice and of 129/ Ola mice from which E14 ES cells were established are GPI-1A and GPI-1B, respectively. Separation and detection of GPI isoenzymes were performed essentially as described ([@B32]). Chimeric mice were perfuged with PBS to eliminate blood cell contamination, and dissected tissues were kept at --70°C. Frozen tissue samples were thawed and gently homogenized in water, and cells were then lysed by three rounds of freezing and thawing. After centrifugation of the homogenates, the supernatants were diluted with water and then electrophoresed on Titan III Zip Zone cellulose acetate plates (Helena Laboratories) in Tris-glycine buffer (25 mM Tris, 200 mM glycine, pH 8.5) for 4 h at 150 V at 4°C. The stainings were performed by overlaying the mixture consisting of 2 ml of 0.2 M Tris-HCl (pH 8.0), 0.1 ml each of 0.25 M magnesium acetate, 10 mg/ml NADP, and 100 mg/ml fructose 6-phosphate, and 0.2 ml of MTT, 0.05 ml of 2.5 mg/ml phenazine methosulfate, 5 μl glucose 6-phosphate dehydrogenase (140 U/ml; Sigma Chemical Co.), and 5 ml of 2% agarose. The GPI isozyme bands appeared after a few minutes in the dark. Density of the bands was analyzed by densitometer.
The hemoglobin type of B6 mice and 129/Ola mice are single (*Hbb^s^/Hbb^s^*) and diffuse (*Hbb^d^/Hbb^d^*), respectively, and were used to evaluate the contribution of ES cells in circulating erythrocytes of chimeric mice. These two types of hemoglobin can be distinguished by electrophoresis. Cellulose acetate electrophoresis of cystamine-modified hemoglobins was performed essentially as described ([@B33]). Whole blood in PBS containing 50 mM EDTA was layered onto 2 vol of Histopaque-1077 (Sigma Chemical Co.) and centrifuged at 3,500 *g* for 20 min at room temperature. The pellet, enriched for RBCs, was collected. 10 μl purified RBCs was added to 300 μl cystamine lysis buffer (12.5 mg/ml cystamine dihydrochloride, 1 mM dithiothreitol, 0.55% ammonium hydroxide) and agitated to lyse the RBCs. The samples were applied to Titan III cellulose acetate plates and run in TBE buffer (0.18 M Tris, 0.10 M boric acid, 0.002 M EDTA) for 40 min at 300 V. The plates were placed in staining solution (1% Ponceau S, 5% TCA) for 10 min and rinsed in three changes of 5% acetic acid for 10 min each. The percentage contributions of ES cells in adult chimera were examined using the allotype of GPI from various nonhematopoietic organs, such as the liver and kidney. The hemoglobin type analysis data were obtained from the chimera in which the contribution of ES cells to nonhematopoietic organs was \>50%.
ES Cells and Their Differentiation Induction.
---------------------------------------------
ES *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ cells were cultured on embryonic fibroblasts as feeder cells in the presence of a saturated dose of leukemia inhibitory factor using the standard procedure ([@B31]). The culture of OP9 stromal cells and the differentiation induction method were carried out as described ([@B27]). OP9 stromal cells were maintained in α-MEM (Life Technologies, Inc.) supplemented with 20% FCS (Summit) and standard antibiotics ([@B27], [@B34]). 10^5^ ES cells were transferred onto confluent OP9 stromal cells in 10-cm culture dishes (Nunc). After day 3 of the induction, human recombinant EPO (provided by Kirin Brewery Co. Ltd.) was added at a final concentration of 2 U/ml during the differentiation induction. The induced cells were trypsinized at day 5, and 10^6^ cells were transferred onto fresh OP9 cells on a 10-cm plate. Nonadherent cells were harvested on day 6, 7, or 8 to obtain EryP. On day 10, all of the cells on individual 10-cm plates were harvested by vigorous pipetting and transferred to individual 10-cm plates with a fresh OP9 cell layer, and then both adherent and nonadherent cells were harvested to obtain EryD after day 11.
Hematopoietic Colony Formation from Day 8 Hematopoietic Clusters.
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To determine the differentiation capacity of *bcl-x* ^+/+^ and *bcl-x* ^−/−^ ES cells, on day 8 of the differentiation induction 30 hematopoietic clusters were picked and transferred to semisolid culture medium containing IL-3 (50 U/ml) and EPO (2 U/ml), which promote erythroid and myeloid cell growth. As previously reported, the day 8 hematopoietic clusters have a clonal origin and can differentiate into erythroid and various myeloid lineages under these conditions ([@B27]). 5 d after transfer into this myeloid permissive semisolid media, individual colonies were picked, cytospin specimens were stained with May-Grunwald Giemsa, and the emerged blood cells were typed.
Purification and Counting Viability of the Induced Cells.
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More than 75% of the differentiation-induced cells between days 6 and 8 were EryP, and the same proportion of cells between days 11 and 13 were EryD. In some experiments, the purification of EryP and EryD was carried out with metrizamide step gradient centrifugation. The cells were washed once with Tyrode\'s buffer containing 0.1% gelatin. 1--5 × 10^6^ cells in 1 ml of the Tyrode\'s buffer were layered on a step gradient of 2.0 ml of 30% wt/vol metrizamide (Nacalai Tesque) and 2.0 ml of 15% wt/vol metrizamide. The cells were centrifuged at room temperature for 20 min at 400 *g* at the interface between the 15% metrizamide and the 30% metrizamide. The cells remaining at this interface were collected and washed three times with α-MEM with 20% FCS. After the purification, \>98% of the cells were dianisidine-positive erythroid cells, with a viability of 95--98%.
Hemoglobin-containing cells were confirmed with dianisidine staining as reported previously ([@B35]). To examine EPO responsiveness (the experiment shown in Fig. [3](#F3){ref-type="fig"}), 3.0 × 10^5^/ml dianisidine-positive differentiation-induced cells were cultured in 6-well plates containing 20% FCS supplemented with α-MEM in the absence or presence of 2 U/ml EPO without the OP9 cell layer. The viability of the cells was examined using the trypan blue dye exclusion method and calculated by counting \>200 cells. May-Grunwald Giemsa staining of cytospin specimens was also carried out to examine the morphological changes of apoptotic EryP. The number of hemoglobin-containing cells and the percentage of viable cells are reported as mean ± SD. The *t* test was used for statistical analysis, using StatView software.
Electrophoretic Analysis of DNA Fragmentation.
----------------------------------------------
After culture for 18 h in the presence or absence of 2 U/ml EPO, 10^6^ cells were harvested by centrifugation at 200 *g* for 10 min. Low molecular weight DNA was extracted following the method of Sellins and Cohen ([@B36]). One quarter of the extracted DNA was electrophoresed in a 2.0% agarose gel and stained with ethidium bromide.
Results
=======
No Contribution of bcl-x Null ES Cells to Circulating Adult Definitive Erythrocytes.
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ES cells of *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ genotypes were injected into the blastocysts of C57BL/6 mice to assess their ability to differentiate into various organs in vivo. There were no differences in the growth of parental *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ ES cells (data not shown). Chimeric mice of \>80% chimerism by coat color were analyzed for the contribution of the injected ES cells in various organs based on the activity of GPI-1 isozymes. E14 ES cell--derived cells express the GPI-1A isozyme, which is easily distinguishable from the GPI-1B isozyme of the C57BL/6-derived cells ([@B37]). As for heart, kidney, and muscle, there were no differences in the contribution of parental *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ ES cells (Table [I](#TI){ref-type="table"}). On the other hand, the contribution of *bcl-x* ^−/−^ ES cells to lymphoid organs such as spleen and thymus was significantly lower than that of *bcl-x* ^+/+^ or *bcl-x* ^+/−^ ES cells. This result is compatible with a previous report on the shortened life span of *bcl-x* ^−/−^ immature lymphocytes ([@B21]).
Two *bcl-x* ^−/−^ ES cell lines (clones 18 and 3a) were analyzed for the function of the *bcl-x* gene in hematopoiesis. Host blastocysts from the strain C57BL/6 are homozygous for the *Hbb^s^* β-globin haplotype (corresponding to the "single" band in Fig. [1](#F1){ref-type="fig"}, lane 1). In contrast, 129/Ola mice, from which the ES cell line of this study was established, are homozygous for the *Hbb^d^* haplotype (corresponding to the "major" and "minor" bands in Fig. [1](#F1){ref-type="fig"}, lane 2). The proportion of major and minor hemoglobin shows the contribution of the injected ES cells to mature circulating EryD in the chimeric mice. When *bcl-x* ^+/+^ or *bcl-x* ^+/−^ ES cells were used for chimera production, the contribution of the ES cells to the circulating EryD was proportional to the contribution of ES cells to the other organs. However, when *bcl-x* ^−/−^ ES cells were used, no contribution of the ES cells to circulating EryD was detected, despite their significant contribution to the other nonlymphohematopoietic organs (Fig. [1](#F1){ref-type="fig"}). These data clearly show that *bcl-x* has an essential role for the in vivo production of EryD. In addition, the results from the chimeric mice demonstrate that the contribution of *bcl-x* to EryD production is cell autonomous, since the hematopoietic microenvironment in the chimeric animal could not complement the defective EryD production from *bcl-x* ^−/−^ ES cells.
The process of definitive erythroid lineage cell production can be divided into two stages. The earlier stage is commitment and involves differentiation from multipotential progenitor cells to committed erythroid lineage cells. The later stage is proliferation and maturation of the committed EryD progenitors. Two possibilities might account for the failure of EryD production by *bcl-x* ^−/−^ ES cells. One is the commitment failure of the multipotential hematopoietic progenitor cells into erythroid lineage cells, and the other is the proliferation and maturation failure of the committed erythroid cells. To analyze these possibilities, in vitro differentiation induction from *bcl-x* ^−/−^ and *bcl-x* ^+/+^ ES cells was carried out using OP9 stromal cells. The differentiation capacity of day 8 in vitro differentiation--induced hematopoietic progenitor cells was examined. The day 8 hematopoietic clusters were of clonal origin, and most of them could differentiate into multiple hematopoietic lineages, including the definitive erythroid lineage ([@B27]). There were no differences in the number of day 8 hematopoietic clusters induced from *bcl-x* ^+/+^ or *bcl-x* ^−/−^ ES cells (data not shown). As shown in Table [II](#TII){ref-type="table"}, there were also no significant differences in the types of colonies that developed from the day 8 hematopoietic clusters in methylcellulose semisolid media containing IL-3 and EPO as growth factors. These data show that *bcl-x* is not necessary for the differentiation of the definitive erythroid lineage from multipotential hematopoietic progenitors. Since Bcl-X is essential for the production of fully mature EryD, *bcl-x* seemed to play important roles during the maturation of EryD after commitment to the erythroid lineage.
Number and Viability of the In Vitro Differentiation--induced Erythroid Lineage Cells.
--------------------------------------------------------------------------------------
To further analyze the function of *bcl-x* in the production of erythroid lineage cells, in vitro differentiation induction into erythroid lineage cells from *bcl-x* ^−/−^ and *bcl-x* ^+/+^ ES cells was carried out using OP9 stromal cells in the presence of EPO. The number of hemoglobin-containing dianisidine-positive cells was counted between days 6 and 8 and between days 12 and 14. As previously reported, EryP and EryD appear in the former and latter periods, respectively ([@B29]). On day 6, the number of *bcl-x* ^−/−^ EryP was the same as *bcl-x* ^+/+^ EryP. However, on day 8, the difference between the number of *bcl-x* ^−/−^ EryP and *bcl-x* ^+/+^ EryP became pronounced. As shown in Table [III](#TIII){ref-type="table"}, on day 8 the number of *bcl-x* ^−/−^ EryP was only ∼10% that of *bcl-x* ^+/+^ EryP. Moreover, the number of *bcl-x* ^−/−^ EryP on day 8 was ∼10% that of the day 7 *bcl-x* ^−/−^ EryP, suggesting that cell death occurred between these days. Similar results were obtained with EryD. There was no difference in the number of *bcl-x* ^−/−^ EryD and *bcl-x* ^+/+^ EryD on day 12. But, the difference became significant with maturation, and the number of EryD originating from *bcl-x* ^−/−^ ES cells was about one quarter that from *bcl-x* ^+/+^ ES cells on day 14.
The percentage of viable cells was next examined, because apoptotic cell death of *bcl-x* ^−/−^ erythroid cells was suspected (Table [IV](#TIV){ref-type="table"}). Here again, there were no significant differences between the day 7 EryP and the day 12 EryD, but the differences became significant thereafter. The percentages of viable cells mainly reflect viable erythroid cells, because the vast majority of the cells during differentiation induction belong to the erythroid lineage. More than 80% of the cells harvested between days 7 and 8, and \>90% of the cells harvested between days 12 and 14 were EryP and EryD, respectively, when the *bcl-x* ^+/+^ ES cells were induced for differentiation. The percentage of viable *bcl-x* ^−/−^ EryP seems relatively high for the very low number of *bcl-x* ^−/−^ EryP (Table [IV](#TIV){ref-type="table"}). This apparent discrepancy was probably due to the removal of dead *bcl-x* ^−/−^ EryP by adherent macrophages. Electron microscopic features at about day 8 of the differentiation induction showed macrophages with prominently phagocytosed dead EryP (data not shown).
Cell Death of In Vitro Differentiation--induced Erythroid Lineage Cells.
------------------------------------------------------------------------
The morphological features and DNA fragmentation of the induced cells were analyzed to confirm that the decreased number and viability of *bcl-x* ^−/−^ cells were due to apoptosis. Immature EryP and EryD were purified by metrizamide density gradient separation on days 6 and 12 of the differentiation induction, respectively. At these times, no differences in number and viability were detectable between the *bcl-x* ^+/+^ and the *bcl-x* ^−/−^ erythroid cells as shown above. Using this purification method, \>98% of the purified cells were erythroid lineage cells and their viability was 95--98%. These purified EryP and EryD were cultured on OP9 cells for 2 d in the presence of EPO, and the cells were harvested. Their morphological and molecular features were then examined. The *bcl-x* ^+/+^ EryP were viable and had mature morphology on day 8. In contrast, the *bcl-x* ^−/−^ EryP had fragmented nuclei with clumped chromatin, suggestive of apoptosis. On day 14, the vast majority of the cells were enucleated mature EryD when the *bcl-x* ^+/+^ ES cells were induced for differentiation, whereas enucleated EryD were rarely observed when the *bcl-x* ^−/−^ ES cells were induced. The hemoglobinized *bcl-x* ^−/−^ EryD were mainly nucleated erythroblasts. Thus, it was difficult to find viable, fully mature EryP and EryD on days 8 and 14 of the differentiation induction of *bcl-x* ^−/−^ ES cells, respectively, although immature EryP and EryD were equally viable on days 6 and 12, respectively. Low molecular weight DNA was extracted from the cells, and agarose gel electrophoresis was carried out (Fig. [2](#F2){ref-type="fig"}). The nucleosomal DNA ladder, which is characteristic of apoptotic cells, was observed to be significantly more abundant in the *bcl-x* ^−/−^ erythroid lineage cells than in the *bcl-x* ^+/+^ erythroid lineage cells. These data clearly demonstrate that the *bcl-x* ^−/−^ erythroid lineage cells underwent apoptosis during the end stage of maturation.
Effects of Bcl-X and EPO on Apoptotic Cell Death.
-------------------------------------------------
EPO is required by immature erythroid lineage cells to prevent apoptosis. To analyze the roles of EPO and Bcl-X during the maturation of erythroid cells, an EPO deprivation experiment was performed. On days 6.5 and 11.5 of the differentiation induction, immature EryP and EryD were purified. These erythroid cells were not considered to be late-stage erythroid progenitors, but rather immature erythroid cells, because hemoglobinization had already begun but the cells still showed an immature morphology. After purification on days 6.5 and 11.5, the *bcl-x* ^+/+^ and *bcl-x* ^−/−^ erythroid lineage cells were cultured without OP9 stromal cells in the presence or absence of EPO. 1 and 1.5 d after the culture, the viability of the cells was examined (Fig. [3](#F3){ref-type="fig"}). Deprivation of EPO at this stage affected the viability of EryP much more severely than EryD; however, the results of the examination of EryP and EryD were essentially the same. In the presence of EPO, there were significant differences in the viability of *bcl-x* ^+/+^ and *bcl-x* ^−/−^ erythroid cells (*P* \< 0.0005 by *t* test). Even in the absence of EPO, the differences in the viability were significant (*P* \< 0.005 by *t* test). Furthermore, EPO deprivation decreased the viability of both EryP and EryD even in the context of *bcl-x* null (*P* \< 0.0001 by *t* test). Taken together, EPO deprivation and the *bcl-x* null mutation affected cell death additively.
Discussion
==========
*bcl-x*, a member of the *bcl-2* family of apoptosis regulatory genes, can be alternatively spliced to produce two protein isoforms, Bcl-X~L~ and Bcl-X~S~ ([@B6], [@B17], [@B18]). Bcl-X~L~ exhibits remarkable structural homology with Bcl-2 and inhibits apoptotic cell death. Evidence from studies of cell lines and transgenic mice suggests that the *bcl-2* gene family plays a role in the survival of erythroid lineage ([@B22], [@B23], [@B25]). The expression pattern of *bcl-x* obtained from primary human erythroid cells and mouse erythroblasts infected with the anemia-inducing strain of Friend virus (FVA) suggests that *bcl-x* among *bcl-2* gene family members is the principal antiapoptotic regulator during late erythroid differentiation ([@B24]). Bcl-X is strongly increased during the terminal differentiation stages of human and mouse erythroblasts in the presence of EPO, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis. This increase in Bcl-X expression leads to an apparent level ∼50 times greater than the level in proerythroblasts before EPO stimulation. In contrast, neither mouse nor human erythroblasts express Bcl-2 transcript or protein. The levels of other Bcl-2 family members, Bax and Bad proteins, remain relatively constant throughout differentiation, but diminish at the end of terminal differentiation near the time of enucleation. These data on the expression pattern of the *bcl-2* gene family products imply that *bcl-x* is the critical member of the *bcl-*2 family during erythroid differentiation. Furthermore, the increased apoptotic cell death of hematopoietic cells in *bcl-x* ^−/−^ fetal liver and the absence of defects in the fetal liver of *bcl-2* ^−/−^ mice support the hypothesis that Bcl-X, not Bcl-2, is the important factor in erythropoiesis ([@B21], [@B38]--[@B40]). However, there is no direct evidence for the role of *bcl-x* in erythropoiesis, despite this circumstantial evidence.
To examine the critical physiological roles of the *bcl-x* gene on hematopoiesis, chimeric mice production and OP9 in vitro differentiation induction were carried out using *bcl-x* ^−/−^ ES cells. There was no contribution by *bcl-x* ^−/−^ ES cells to the circulating EryD in the chimeric mice, demonstrating that *bcl-x* is indispensable for the full maturation of EryD. Defects in erythropoiesis were analyzed in detail using in vitro differentiation induction from ES cells by coculturing the cells on the macrophage colony-stimulating factor--deficient OP9 stromal cell line (the OP9 system \[28\]). Two waves of erythroid cell production were observed when ES cells were cocultured with OP9 stromal cells. The development of hematopoietic cells in this OP9 system is very similar to that observed in developing mouse embryos ([@B27], [@B29], [@B41]). The first wave of erythropoiesis, appearing between days 6 and 8 of the induction, and the second wave, appearing after day 10 of the induction, correspond to primitive and definitive erythropoiesis, respectively, by morphological and biochemical criteria ([@B29]). Our data clearly show that apoptotic cell death of *bcl-x* ^−/−^ erythroid lineage cells was observed only at the end of maturation in both primitive and definitive erythropoiesis.
*bcl-x*--deficient mice die at about embryonic day 13 ([@B21]). Extensive apoptotic cell death is evident in hematopoietic cells in fetal liver. There is a threefold increase in TUNEL (for terminal deoxynucleotidyl transferase--mediated dUTP nick end labeling)--labeled apoptotic nuclei in histologically identifiable hematopoietic cells in embryonic day 12.5 *bcl-x* ^−/−^ liver compared with wild-type tissue. These data suggest that erythropoiesis in the fetal liver of *bcl-x* ^−/−^ mice is impaired because the vast majority of fetal liver hematopoietic cells at this gestational stage are erythroid lineage cells. The data on defective EryD production by the OP9 system are consistent with these in vivo data. During in vitro differentiation, although significant numbers of *bcl-x* ^−/−^ erythroblasts survived, almost no enucleated erythrocytes could be detected. This result shows that the pivotal function of *bcl-x* is expressed at the late stage of erythroid maturation. The critical role of *bcl-x* seems to be brought about by the remarkable increase of Bcl-X protein at the end of erythroid maturation. In vitro differentiation induction shows that apoptotic cell death of EryP also occurred at the late stage of maturation, which is consistent with primitive erythropoiesis in the *bcl-x* ^−/−^ mice. The effect of *bcl-x* on primitive erythropoiesis was not examined extensively because of the difficulty counting EryP numbers correctly in tiny mouse embryos. However, the following two lines of evidence strongly suggest that EryP production in *bcl-x* ^−/−^ mice was impaired to some extent. First, *bcl-x* ^−/−^ mice were paler than the control mice at day 12.5 of gestation (21; our unpublished data). At this gestational stage, \>95% of the erythrocytes are still EryP, although the relative percentage of EryP begins to decrease ([@B42]). Second, *bcl-x* ^−/−^ mice died at day 13 of gestation, which is earlier than the mutant mice lacking only definitive hematopoiesis by gene targeting of *c-myb* ([@B43]). The *c-myb* targeted mice were severely anemic by day 15; however, the mutant mice appeared normal at day 13 of gestation. On the other hand, EPO signal--deficient mice, which have a partial defect in primitive erythropoiesis and a complete defect in definitive erythropoiesis, die at day 13 of gestation, as early as *bcl-x* ^−/−^ mice ([@B12], [@B13]). Meanwhile, it is reasonable to consider that a similar time course of cell death of *bcl-x* ^−/−^ EryP and *bcl-x* ^−/−^ EryD would reflect a similar underlying molecular mechanism of cell death caused by the null mutation of *bcl-x*. The cause of cell death might be explained by the relationship between massive heme synthesis at the end of maturation of erythroid lineage cells and the antioxidant function of Bcl-X~L~ ([@B24]).
Of the various methods of in vitro hematopoietic differentiation from ES cells, the OP9 system has several remarkable advantages, among which are their potential to differentiate into fully mature blood cells and the feasibility of analyzing the cells quantitatively ([@B26], [@B27]). To analyze the defective erythropoiesis from *bcl-x* ^−/−^ ES cells, quantitative analysis of the fully mature erythroid cells was necessary. However, such analysis is almost impossible by the conventional in vitro differentiation induction method with embryoid body formation. The other substantial advantage of the OP9 system is that hematopoietic microenvironment and hematopoietic cells can be analyzed separately by this method. It is well known that hematopoiesis is maintained by the hematopoietic microenvironment, such as stromal cells. By the conventional embryoid body formation method, both hematopoietic microenvironment and hematopoietic cells are induced from ES cells and are unseparable. But with the OP9 system, hematopoietic cells are induced from ES cells while the hematopoietic microenvironment is provided by OP9 stromal cells. It is concluded from the defective EryD production in the chimeric mice that this defect is cell autonomous. In addition, the defective erythropoiesis of the *bcl-x* ^−/−^ genotype with the OP9 system strongly supports this conclusion.
The production of definitive erythroid lineage cells is controlled by EPO ([@B11]). EPO induces the proliferation and prevents the apoptotic cell death of EryD. The antiapoptotic effect of EPO on EryD was observed from late erythroid progenitors (CFU-E) until the onset of hemoglobinization ([@B2], [@B14]--[@B16]). In other words, EPO-deprived apoptotic cell death is hardly at all observed at the end of maturation when maximal hemoglobin synthesis occurs. On the other hand, massive apoptotic cell death of *bcl-x* ^−/−^ EryD was observed after day 13 of differentiation induction. It is reasonable to consider that the accumulation of Bcl-X (probably Bcl-X~L~) resulting from EPO stimulation prevents the apoptotic cell death of terminally differentiated erythroid cells. However, the accumulation of Bcl-X cannot be the only way to explain the antiapoptotic effect of EPO, because EPO prevents apoptotic cell death to some extent even in the absence of Bcl-X (Fig. [3](#F3){ref-type="fig"}). Taken together, it is likely that EPO has dual roles to prevent apoptotic cell death at different differentiation stages.
The authors thank Dr. Yoshihide Tsujimoto for discussions, and Kirin Brewery Co. Ltd. for their kind gift of human recombinant EPO.
This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, the Research for the Future Program of Japanese Society for Promotion of Sciences (JSPS-RFTF98L01101), the Naito Memorial Foundation, and the Novartis Foundation (Japan) for the Promotion of Science.
N. Motoyama\'s present address is Department of Geriatric Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan.
CFU-E
: colony forming unit-erythroid
EPO
: erythropoietin
EryP
: embryonic primitive erythrocyte(s)
EryD
: adult definitive erythrocyte(s)
ES
: embryonic stem
GPI
: glucose phosphoisomerase
{#F3}
######
Contribution of bcl-x^+/+^, bcl-x^+/−^, and bcl-x^−/−^ ES Cells to Various Organs in Chimeric Mice
Organs Genotype
-------- -- ------------ -- ----------- -- --------------------------------------------
*(n = 12)* *(n = 3)* *(n = 14)*
Heart 62 ± 22 63 ± 15 52 ± 13
Kidney 60 ± 15 60 ± 27 55 ± 18
Muscle 99 ± 2 100 ± 0 99 ± 38
Thymus 38 ± 15 47 ± 21 21 ± 14[\*](#TFI-150){ref-type="table-fn"}
Spleen 71 ± 9 63 ± 15 33 ± 21[\*](#TFI-150){ref-type="table-fn"}
ES cells (GPI-1A) of *bcl-x* ^+/+^, *bcl-x* ^+/−^, and *bcl-x* ^−/−^ genotypes are microinjected into blastocysts of C57BL/6 mice (GPI-1B). Contribution of ES cells is examined by the enzyme activity of GPI-1 isozymes. Percentages of ES cell contribution are shown as mean ± SD. Numbers of the examined chimeric mice are shown in parentheses.
*P* \< 0.05 by *t* test with the groups of +/+ and +/− mice.
{#F1}
######
Differentiation of bcl-x^+/+^ and bcl-x^−/−^ ES Cells into Various Hematopoietic Lineages In Vitro
Colony type Differentiation-induced ES cells
------------- -- ---------------------------------- -- --- -- ----
nmEM 5 4 2
nmE 9 7 4
mEM 1 2 2
mE 11 9 13
E 2 1 1
nm 1 4 3
m 1 3 5
Day 8 hematopoietic clusters were picked and transferred into semisolid media containing IL-3 and EPO. Colonies that appeared 8 d after the transfer were picked and stained with May-Grunwald Giemsa. No significant differences between the groups by χ^2^ test. n, neutrophils; m, macrophages; E, erythroid cells; M, megakaryocytes.
######
Numbers of Dianisidine-positive Cells Induced from bcl-x^+/+^ and bcl-x^−/−^ ES Cells
Day of induction Type of erythroid cells Differentiation-induced ES cells
------------------ -- ------------------------- -- ---------------------------------- -- ----------------------------------------------- -- ------------------------------------------------
Day 6 Primitive 4.0 ± 0.5 4.0 ± 0.4 4.0 ± 0.5
Day 7 Primitive 9.6 ± 0.7 9.5 ± 1.9 7.9 ± 1.2[\*](#TFIII-150){ref-type="table-fn"}
Day 8 Primitive 8.4 ± 1.7 0.7 ± 0.2[‡](#TFIII-152){ref-type="table-fn"} 0.8 ± 0.5[‡](#TFIII-152){ref-type="table-fn"}
Day 12 Definitive 3.2 ± 0.5 3.4 ± 0.4 3.0 ± 0.3
Day 13 Definitive 8.6 ± 0.5 2.8 ± 0.3[‡](#TFIII-152){ref-type="table-fn"} 3.2 ± 0.5[‡](#TFIII-152){ref-type="table-fn"}
Day 14 Definitive 16.8 ± 1.0 4.5 ± 0.9[‡](#TFIII-152){ref-type="table-fn"} 3.8 ± 0.9[‡](#TFIII-152){ref-type="table-fn"}
10^5^ of the day 5 induced cells were cultured on the OP9 cell layer in the presence of EPO (2 U/ml). Data (×10^−4^ of primitive and ×10^−5^ of definitive erythroid cells) are shown as mean ± SD of six dishes. The data shown are representative of three independent experiments.
*P* \< 0.05 by *t* test with the group of +/+ mice;
*P* \< 0.001 by *t* test with the group of +/+ mice.
######
Percentages of Viable Cells during the Differentiation Induction from bcl-x^+/+^ and bcl-x^−/−^ ES Cells
Day of induction Differentiation-induced ES cells
------------------ -- ---------------------------------- -- ------------------------------------------- -- --------------------------------------------
Day 7 90 ± 3 89 ± 2 89 ± 2
Day 7.5 91 ± 3 88 ± 3 84 ± 3[\*](#TFIV-150){ref-type="table-fn"}
Day 8 89 ± 2 75 ± 3[‡](#TFIV-152){ref-type="table-fn"} 78 ± 3[‡](#TFIV-152){ref-type="table-fn"}
Day 12 82 ± 3 81 ± 3 80 ± 1
Day 13 87 ± 3 53 ± 7[‡](#TFIV-152){ref-type="table-fn"} 52 ± 3[‡](#TFIV-152){ref-type="table-fn"}
Day 14 85 ± 2 48 ± 2[‡](#TFIV-152){ref-type="table-fn"} 47 ± 2[‡](#TFIV-152){ref-type="table-fn"}
10^5^ of the day 5 induced cells were cultured on the OP9 cell layer in the presence of EPO (2 U/ml). Data are shown as mean ± SD of six dishes. The data shown are representative of three independent experiments.
*P* \< 0.005 by *t* test with the group of +/+ mice;
*P* \< 0.0001 by *t* test with the group of +/+ mice.
{#F2}
[^1]: Address correspondence to Toru Nakano, Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, Yamada-Oka 3-1, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8361; Fax: 81-6-6879-8362; E-mail: <[email protected]>
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
With diminishing fossil resources and increasing environmental concerns worldwide, searching for alternative fuels has attracted great interest in recent years. As a vital renewable alternative to fossil fuels, cellulose is the most abundant source of biomass and has received considerable attention. Consequently, different conversion routes for cellulose have been explored to achieve high energy efficiency and atom economy^[@CR1]--[@CR3]^. Cellulose has been selectively converted into different chemicals, such as oligomers^[@CR3]^, glucose^[@CR2]^, HMF^[@CR1]^, sorbitol^[@CR4]^, hexitols^[@CR5]^ and C~2~/C~3~ glycols (ethylene glycol and propylene glycol, EG/PG) through a series of chemical reactions, including hydrolysis, dehydration and hydrogenolysis. Among them, the hydrogenolysis of cellulose to C~2~/C~3~ glycols is particularly noteworthy because of the versatile applications of glycols directly and as platform molecules in the synthesis of fuels and value-added products, including polyesters and antifreeze^[@CR6]^.
The catalytic conversion of cellulose is a complex reaction network comprising hydrolysis, retro-aldol condensation, hydrogenation, isomerization, dehydrogenation, thermal side reactions, etc^[@CR7]^. In addition to C~2~/C~3~ glycols, a variety of by-products, such as sorbitol, mannitol, xylitol, 1,2-butanediol, and glycerol, can be coproduced^[@CR7]^. Based on previous studies, three reaction steps are critical for cellulose conversion to EG: hydrolysis, retro-aldol condensation, and hydrogenation. For PG, the isomerization of glucose is also involved. To enhance the above reaction steps and increase the yield of C~2~/C~3~ glycols, a series of effective metals or metallic oxides, such as tungsten derivatives^[@CR8]--[@CR13]^, tin derivatives^[@CR14],[@CR15]^, lanthanides^[@CR6]^, niobium^[@CR16]^ and zinc^[@CR17]^, were employed in the catalytic system. Deng *et al*. modified Pt/Al~2~O~3~^[@CR14]^ and Ni/Al~2~O~3~^[@CR18]^ catalysts by SnO~x~ to enhance the conversion of cellulose to C~2~/C~3~ glycols. Using these two catalysts, carbon selectivity of C~6~ products decreased from 43.3 to 0.8% and 63.3 to 0.2%, respectively, which indicated that SnO~x~ played a significant role in C-C bond cleavage via retro-aldol condensations. In addition, the carbon selectivity for C~3~ products increased from 13.5 to 28.6% and 12.3 to 56.9%, respectively, demonstrating the advantages of SnO~x~ in glucose-fructose isomerization. However, the cellulose conversions obtained with SnO~x~-modified Pt/Al~2~O~3~ and Ni/Al~2~O~3~ catalysts remained below 23% (C%), which was attributed to the fact that these catalysts cannot facilitate cellulose hydrolysis. Sun *et al*. designed Sn powder- and SnO-modified Ni/AC catalysts to enhance the retro-aldol condensation step in cellulose hydrogenolysis. The carbon yield of EG obtained with the Sn powder modification reached 57.6%, and the carbon yield of PG/EG with the SnO-modified catalyst reached 22.9%/32.2%. Our previous studies^[@CR19],[@CR20]^ showed results similar to those obtained in the Sn-enhanced retro-aldol condensation step. In addition to Sn species, W species also facilitated cellulose or glucose hydrogenolysis into C~2~/C~3~ glycols. WO~x~ was introduced onto a Pd/Al~2~O~3~ catalyst by Liu *et al*.^[@CR21]^ to increase the yield of PG from glucose. The results suggested that WO~x~ enhanced glucose-fructose isomerization and achieved PG yields up to 56.1% (C%). They^[@CR22]^ also introduced WO~x~ into a Cu/Al~2~O~3~ catalyst and achieved a PG carbon yield of 38.1%, which was attributed to the glucose-fructose isomerization and retro-aldol condensation steps. In addition, W~2~C/AC, Ni-WP/AC, Ru/C-WO~3~ + C~act~, Ru/AC-H~2~WO~4~ and Cu/CuCr~2~O~3~ provided C~2~/C~3~ glycol carbon yields of 32.8%^[@CR8]^, 52.4%^[@CR9]^, 12.8%^[@CR11]^, 62.0%^[@CR12]^ and 43.9%^[@CR13]^, respectively.
The above studies realized the increase of C~2~/C~3~ glycols yields by enhancing the isomerization or retro-aldol condensation steps. Focusing on the synergy of the hydrolysis, hydrogenation, retro-aldol condensation and hydrogenation steps in C~2~/C~3~ generation is more important than focusing on a single step. In view of this, we will fully consider cellulose hydrolysis, hydrogenation and the synergy of the hydrogenation and retro-aldol condensation in this paper. In order to increase the C~2~/C~3~ generation, the reaction process was expected as below: when cellulose hydrolysis took place, hydrogenation was not expected to occur because the products of the hydrogenation reaction (C~6~ alcohols) were much more stable and more difficult to convert to C~2~/C~3~ species than C~6~ sugars. Then, after the C-C bond cleavage in the sugars via a retro-aldol condensation (C~6~ to C~2~/C~3~), hydrogenation occurs immediately to generate C~2~/C~3~ glycols, avoiding the occurrence of other side reactions. When the reaction reached this point, further C-C bond cleavage should not occur. Otherwise, smaller molecules could be generated from the C~2~/C~3~ glycols. Furthermore, as a more valuable product, PG is more difficult to produce because the cleavage of the C~3~-C~3~ bond in C~6~ sugars should occur, not C~2~--C~4~.
Herein, to balance the hydrolysis, retro-aldol condensation and hydrogenation and to realize the selective conversion of cellulose into C~2~/C~3~ glycols, three aspects will be investigated and discussed: 1) a tailored catalyst will be designed and synthesized, including support selection, support structure modification, control of the Ni-W synergy and co-catalyst screening; 2) the effect of the above factors on the reaction route and the overall mechanism will be studied via a series of catalytic reactions and analyses; 3) the reuse performance and the relationship between Ni-W leaching and product distribution during reuse will be utilized to verify the effect of Ni-W on reaction route selection.
Results and Discussion {#Sec2}
======================
The effect of the acidity of the β-zeolite {#Sec3}
------------------------------------------
Because of the obvious importance of the catalytic support, which not only physically supports and improves the dispersion of the metal but also provides acid sites, different supports (Al~2~O~3~, TiO~2~, SiC, ZSM-5, and raw β-zeolite) were screened to determine the optimal combination for cellulose and glycols based on Ni-W bimetallic synergy. As displayed in Fig. [1](#Fig1){ref-type="fig"}, mannitol (M), sorbitol (S), EG and PG (C~2~/C~3~ glycols) as the major alcohol products were exhibited. There was a significant difference between the total yield of M, S, PG and EG, the yields of C~2~/C~3~ glycols, and the conversions obtained with Ni-W/SiC, Ni-W/TiO~2~, Ni-W/ZSM-5, Ni-W/Al~2~O~3~ and Ni-W/raw-β catalysts. Ni-W/raw-β exhibited the highest reactivity. To understand the reason for the superiority of Ni-W/raw-β, the physicochemical properties of the catalysts were investigated, as displayed in Table [S1](#MOESM1){ref-type="media"} (entries 1 to 6). The total loading of Ni and W remained the same for each case, whereas BET surface area and acidity varied considerably between catalysts. The BET surface areas of Ni-W/SiC, Ni-W/TiO~2~, Ni-W/ZSM-5, Ni-W/Al~2~O~3~ and Ni-W/raw-β were 56, 42, 410, 134, 317 m^2^/g, and the corresponding yields of C~2~/C~3~ glycols were 32.1, 38.2, 32.8, 29.6 and 42.2%, respectively. The total yields of the major alcohol products were 37.9, 40.5, 45.6, 50.1 and 61.2%, respectively. The results suggested that there was no obvious correlation between the BET surface area and the reactivities on different supports, indicating that the surface area was not the primary cause of the superiority of Ni-W/raw-β^[@CR23]^. The NH~3~-TPD curves (Figure [S1(a)](#MOESM1){ref-type="media"}) showed a large difference in the acidity of the catalysts. The raw-β-supported Ni-W catalyst exhibited a significantly larger peak area than the other supports. The desorption peaks of Ni-W/raw-β centred at approximately 200 °C and 450 °C indicated that it had weak and medium-strength acid sites. In contrast, fewer acid sites were seen with other catalysts, such as Ni-W/Al~2~O~3~, which had some weak and medium-strength sites, Ni-W/TiO~2~, which had few weak and strong sites, and Ni-W/SiC, which had no obvious desorption peaks. The quantitative acidity data in Table [S1](#MOESM1){ref-type="media"} (entries 1 to 6) further illustrated that Ni-W/raw-β exhibited significantly more acid sites. As Table [S1](#MOESM1){ref-type="media"} (entries 1 to 6) shows, the acidity of Ni-W/SiC were below the limit suitable for this test and the acidity of Ni-W/TiO~2~, Ni-W/ZSM-5, Ni-W/Al~2~O~3~ and Ni-W/raw-β were 0.15, 0.17, 0.62 and 1.71 mmol/g, respectively, and the total yields of C~2~/C~3~ glycols, mannitol and sorbitol were 37.9%, 40.5%, 45.6%, 50.1% and 61.2%, respectively. The relationship between the acidity and the total yield of C~2~/C~3~ glycols, mannitol and sorbitol further supported that the acidity was an important factor in the reaction performance. The effect of acidity on performance is discussed in the mechanism section. The mechanism of the hydrolysis of cellulose into glucose might be promoted by the abundant acid sites in β-zeolite. This was also demonstrated by Lazaridis, P. A., *et al*.^[@CR24]^ who showed that cellulose can be degraded into glucose and hexitols through conventional acidic hydrolysis and then converted into C~2~--C~6~ alcohols.Figure 1Catalytic reactions on different supports. Reaction conditions: 30 min, 6 MPa H~2~, 245 °C, 50 mL of H~2~O, 0.5 g of cellulose, 0.15 g of catalyst. EG: ethylene glycol; PG: 1,2-propylene glycol; S: sorbitol; and M: mannitol. Other products not listed include methane, methanol, ethanol, 1-propanol, glycerol, 1,2-butanediol, erythritol, 1,2-pentanediol, 1,2-hexanediol, furfural, furan and other unknown products.
To optimize the structure and performance of Ni-W/raw-β, a series of modifications were tested, and of these, removal of the aluminium (denoted Ni-W/β) provided remarkable results. As displayed in Fig. [1](#Fig1){ref-type="fig"}, the yield of C~2~/C~3~ glycols obtained with Ni-W/β increased from 42.2% to 64.6% upon removal of the aluminium. In this process, the aluminium was removed from the β zeolite by the method mentioned in our previous studies^[@CR25]^ to generate empty sites for the loading of other metals (e.g., Ni and W) and redistribution of the acid sites in the catalysts. As Figure [S1(a)](#MOESM1){ref-type="media"} shows, the ammonia desorption peaks of Ni-W/β were shifted from 200 and 450 to 650 °C, indicating that the strength of the acid sites was enhanced by dealumination, although the quantity of sites did not increase. This result suggested that not only the quantity of acid sites but also the strength of the acid sites would influence the performance of Ni-W/β. As Ni-W/β was superior in the hydrogenolysis of cellulose, dealuminated β-zeolite was chosen as the support for the Ni-W catalyst in the following experiments.
The synergistic effect of Ni and W {#Sec4}
----------------------------------
As mentioned above, the acid sites played an important role in the production of glycols, especially in the acidic hydrolysis of cellulose^[@CR20],[@CR25]^. Therefore, we firstly investigated the synergistic effects of Ni and W on the acid sites. As shown in Fig. [2](#Fig2){ref-type="fig"}, although both the Brønsted acidity (1540 cm^−1^) and Lewis acidity (1492 cm^−1^) of raw β-zeolite were removed during the dealumination by HNO~3~ solution, Ni-W/β still showed higher glycol yields than the other catalysts (Fig. [1](#Fig1){ref-type="fig"}), which may be attributed to the function of the Ni and W species in the acid sites. The assumption is confirmed by the data in Fig. [2](#Fig2){ref-type="fig"}; after the loading of Ni and W, great changes occurred in the acidity of the catalyst, suggesting that Ni provided abundant Lewis acid sites in the β-zeolite and that W provided both Brønsted and Lewis acid sites, which could ensure the success of cellulose hydrolysis and hydrogenolysis. Moreover, Fig. [1](#Fig1){ref-type="fig"} shows that Ni-W/β exhibited high catalytic activity, but Ni/β and W/β both exhibited low activity, which indicated that Ni-W bimetallic synergy, and not the individual effects of Ni or W, facilitated the C~2~/C~3~ glycols production. In order to more clearly explain the synergy of Ni and W on acidity, the NH~3~-TPD experiment was showed in Figure [S1(b)](#MOESM1){ref-type="media"}. Similar to Py-IR result, after dealumination, β lost almost all of its acidity. The only small peaks near 150 °C might attributed to the OH groups formed by alumina vacancy, which was similar to the H peaks in Py-IR. After the loading of Ni or W on β (deAl), the catalytic acidity was significantly increased. While Ni and W loaded on β together, the acidity of catalyst increased more significantly. As shown in Table [S1](#MOESM1){ref-type="media"} (entries 6 to 10), the synergy was clearly observed that the acidity of Ni-W/β (1.67 mmol/g) was stronger than the sum of Ni/β and W/β (1.20 mmol/g).Figure 2Py-IR profile of β-based catalysts adsorbed at 150 °C.
To further probe the role of Ni-W in the reaction, different loading of Ni and W were employed in the Ni-W/β catalysts. As displayed in Fig. [3](#Fig3){ref-type="fig"}, the C~2~/C~3~ glycols yield increased from 6.5% to 64.6% when the loading of W remained at 20 wt % and the weight percentage of Ni increased from 1 wt % to 7 wt %. This increase may be attributed to nickel, as the metal responsible for hydrogenation, providing more hydrogenation sites when a greater amount was used, which also increased the yield of hydrogenation products, sorbitol and mannitol^[@CR8]^. With a further increase in the Ni weight percentage from 7 to 15 wt %, the C~2~/C~3~ glycols decreased from 64.6 to 14.8% and the cellulose conversion decreased from 100.0 to 89.0%. These results might be a result of the facile aggregation of the excess nickel, shielding the mesopores and W sites and the decrease of catalytic acidity, which could be verified by the BET (Table [S2](#MOESM1){ref-type="media"}, Entries 6 to 11), TEM (Figure [S2](#MOESM1){ref-type="media"}), XRD results (Figure [S3d](#MOESM1){ref-type="media"}) and NH~3~-TPD results (Table [S3](#MOESM1){ref-type="media"}). Table [S2](#MOESM1){ref-type="media"} (Entries 6 to 11) shows that the BET surface area, as well as the pore volume, decreased with increasing Ni weight percentage. As shown in Figure [S2](#MOESM1){ref-type="media"}, undesired particle aggregation was more apparent on 15Ni-20W/β than it was on 1Ni-, 5Ni-, 7Ni- and 9Ni-20W/β. Furthermore, the poor performance when excess Ni is present in catalytic structure is explained in Figure [S3(d)](#MOESM1){ref-type="media"}, as significant NiWO~4~ diffraction peaks were observed, suggesting that the excess Ni promoted the formation of NiWO~4~, decreasing the number of active Ni and W particles. As shown in Table [S3](#MOESM1){ref-type="media"}, the acidity of 15Ni-20W/β was lower than that of other catalysts, which might attributed to the generation of NiWO~4~ when Ni was excess and the generated NiWO~4~ could not provide acidity sites. The above evidence all confirm that excess nickel has a negative effect on the hydrogenolysis of cellulose into C~2~/C~3~ glycols.Figure 3Catalytic reaction performance over the Ni-W/β catalysts with various Ni-W loading. Reaction conditions: 30 min, 6 MPa H~2~, 245 °C, 50 mL of H~2~O, 0.5 g of cellulose, 0.15 g of catalyst. EG: ethylene glycol; PG: 1,2-propylene glycol; S: sorbitol; M: and mannitol.
When the loading of Ni remained at 7 wt % and the weight percentage of W increased from 5 wt % to 25 wt %, the maximum C~2~/C~3~ glycols yield was obtained by 7Ni-20W/β (Fig. [3](#Fig3){ref-type="fig"}), indicating that the appropriate amounts of W and Ni significantly influenced the C~2~/C~3~ glycols production. As shown in Fig. [3](#Fig3){ref-type="fig"}, the C~2~/C~3~ glycols yield obtained with 7Ni-5W/β was only 25.8%, while the yield of sorbitol and mannitol was up to 44.6%. The formation of a large amount of C~6~ sugar alcohols suggested that the insufficient quantity of W species (5 wt %) impeded the bond cleavage from C~6~ to C~2~/C~3~ products based on the understood mechanism that glycols were formed by the hydrolysis of cellulose into glucose and subsequent bond cleavage of glucose/fructose^[@CR7],[@CR11],[@CR26]^ via a retro-aldol condensation (RAC). Therefore, as Fig. [3](#Fig3){ref-type="fig"} shows, with increasing W content from 5 to 20 wt %, the yield of C~6~ products decreased from 44.6 to 10.3%, and the yield of C~2~/C~3~ glycols increased from 25.8 to 64.6%, which further confirmed that the W species play an important role in bond cleavage in the C~6~ species. However, an especially low yield of glycols was obtained with 7Ni-25W/β, which could be explained by the formation of NiWO~4~ (Figure [S3](#MOESM1){ref-type="media"}), the small BET surface area (Table [S2](#MOESM1){ref-type="media"}) and the lower acidity (Table [S3](#MOESM1){ref-type="media"}).
We could conclude from the above results that excessive amounts of either Ni or W negatively impacted the performance of the catalyst. The cooperation of Ni and W active sites was required for this reaction, and C~2~/C~3~ glycols could be efficiently produced because Ni can promote the hydrogenation while W promotes bond cleavage via a retro-aldol condensation^[@CR8]^. Hence, to obtain a more satisfactory C~2~/C~3~ glycols yield, the 7Ni-20W/β (Ni-W/β for short) system was applied in the following study.
The effect of co-catalysts on product distribution {#Sec5}
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Although the conversion of cellulose into glycols has been widely studied, controlling the C~2~/C~3~ distribution has received less attention, and the increase of PG yield is hard because rigorously controlling the C-C bond cleavage position is important^[@CR6],[@CR8]--[@CR12],[@CR14],[@CR16]^. Hence, to control the position of C-C bond cleavage to increase the selectivity of PG and investigate the influence of these co-catalysts on the C~2~/C~3~ glycols distribution, a series of metal oxides and alkalis were added into the Ni-W/β system, as displayed in Table [1](#Tab1){ref-type="table"}. ZnO, Fe~3~O~4~, MgO and alkalis gave rise to improved yields of C~3~ glycols from 19.3 to 35.8, 24.1, 23.2, 26.8, 29.3 and 32.1%, and the yields of C~2~/C~3~ glycols improved from 64.6 to 70.1 and 65.3% with ZnO and Fe~3~O~4~, respectively. Among these co-catalysts, ZnO significantly influenced the C~2~/C~3~ glycols distribution and PG yield, which was further investigated. First, the effect of the ZnO dosage was tested, as shown in Figure [S4](#MOESM1){ref-type="media"}. Opposite trends in the PG and EG distribution were observed because the PG yield (C~3~) increased significantly with the addition of ZnO, while the EG (C~2~) yield obviously decreased. The balance between the yields of PG and EG indicated that PG was preferentially obtained in the presence of ZnO, i.e., the selective generation of PG and EG was influenced by ZnO^[@CR27]^. Furthermore, LA, another C~3~ product, could not be generated when there was no ZnO in the system. In addition, its yield increased with increasing ZnO dosage (less than 200 mg). Therefore, from the increasing trends in the yields of PG (C~3~) and LA (C~3~) and the decreasing trends in the yields of EG (C~2~) and S (C~6~), we can infer that ZnO selectively promoted the formation of C~3~ products via C~3~-C~3~ bond cleavage from C~6~. Since there are few reports about the specific role of ZnO^[@CR28],[@CR29]^, we further investigated the mechanism of the effect of ZnO in the mechanism section.Table 1The catalytic performance of co-catalysts based on Ni-W/β.Entry CatalystConversion (%)Yield (%)PG/Glycols (%)EGPGSMGlycols1None74.00.00.00.00.00.00.02Ni-W/β100.045.319.311.73.464.629.93Ni-W/β + ZnO100.034.335.86.67.370.151.14Ni-W/β + Fe~3~O~4~100.041.224.117.010.165.336.95Ni-W/β + MgO100.039.823.210.55.663.036.86Ni-W/β + Al~2~O~3~100.043.310.714.73.554.019.87Ni-W/β + TiO~2~100.041.712.610.87.854.323.28Ni-W/β + NaOH100.023.326.84.94.850.150.19Ni-W/β + Ca(OH)~2~100.027.829.36.29.857.151.310Ni-W/β + Ba(OH)~2~100.025.732.14.16.257.855.5Reaction conditions: 30 min, 6 MPa H~2~, 245 °C, 50 mL of H~2~O, 0.5 g of cellulose, 0.15 g of catalyst, 100 mg of co-catalysts. EG: ethylene glycol; PG: 1,2-propylene glycol; S: sorbitol; and M: mannitol.
Reaction mechanism {#Sec6}
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The proposed reaction route for the formation of C~2~/C~3~ glycols from cellulose using Ni-W-ZnO/β is shown in Fig. [4](#Fig4){ref-type="fig"}. Based on this reaction route, the final products, EG, PG, sorbitol and mannitol, were produced by route 1 (R1: hydrolysis, R2: retro-aldol condensation and R3: hydrogenation), route 2 (R1: hydrolysis, R4: isomerization, R2, R4/R3 and R3) and route 3 (R1 and R3), respectively. Figure [1](#Fig1){ref-type="fig"} shows that when β-zeolite served as the support, the maximum total yield of the four final products was obtained. We conjectured in support section above that the excellent performance of β-zeolite might be attributed to the enhancement of cellulose hydrolysis. Here, this hypothesis was further supported. As shown in Table [S4](#MOESM1){ref-type="media"}, when using Ni-W/raw-β as the catalyst, the total yield of the four polyols produced from glucose was 64.9%, while that from cellulose was 61.2%. This result suggested that the pseudo yield of R1 (Fig. [4](#Fig4){ref-type="fig"}) was 94.3, which was obviously higher than the conversion efficiencies obtained with other catalysts. Therefore, it could be concluded that the acidity of Ni-W/raw-β enhanced cellulose hydrolysis. Furthermore, as the strength of the acidity was enhanced by dealumination, Ni-W/β provided a higher pseudo yield of the hydrolysis than that obtained with Ni-W/raw-β.Figure 4Proposed reaction pathway for the transformation of glucose into propylene glycol on Ni-W/β in the presence of ZnO.
The proposed reaction route (Fig. [4](#Fig4){ref-type="fig"}) was also utilized to explain the synergistic effects of Ni and W. As shown in Fig. [3](#Fig3){ref-type="fig"}, a significant difference in the product distribution when using different Ni-W loadings was observed. From the perspective of W, when the loading of W was insufficient (e.g., 7Ni-5W), the yields of sorbitol and mannitol (C~6~ products) were significantly higher than that of C~2~/C~3~ glycols, suggesting that route 3 (Fig. [4](#Fig4){ref-type="fig"}) was favoured over routes 1 and 2. When the W loading was sufficient (e.g., 7Ni-15W), routes 1 and 2 were favoured over route 3. The significant difference between the results with insufficient and sufficient loadings of W indicated that W promoted routes 1 and 2 in which bond cleavage from C~6~ into C~2~/C~3~ (R2 retro-aldol) was the key step. From the perspective of Ni, the yields of the four major polyol products all decreased when the Ni loading was insufficient (e.g., 1Ni-20W), while a high yield of polyols was observed when sufficient Ni was present (e.g., 7Ni-20W). This might be because hydrogenation (R3), as the common formation step of these four products in routes 1, 2 and 3, was disfavoured when Ni was insufficient. In other words, Ni facilitated the hydrogenation step. The fact that Ni facilitated the hydrogenation step could also explain why the yields of sorbitol and mannitol were significantly higher than those of C~2~/C~3~ glycols when the loading of Ni was higher (e.g., 15Ni-20W, Fig. [3](#Fig3){ref-type="fig"}). Excess Ni promoted route 3 because sorbitol and mannitol were readily generated only though glucose hydrogenation^[@CR30],[@CR31]^. In summary, W worked on R2, while Ni acted on R3, and the favourability of routes 1, 2 or 3 was determined by the synergy of Ni and W. Based on the above mechanism, 7Ni-20W/β successfully drove the reaction through routes 1 and 2 and ultimately provided the maximum yield of C~2~/C~3~ glycols.
To further understand the mechanism of the hydrogenolysis of cellulose into C~2~/C~3~ products, the reactions of the possible intermediates (such as glucose, fructose, sorbitol, mannitol, dihydroxyacetone and acetol) were performed, as shown in Table [2](#Tab2){ref-type="table"}. As displayed in Entry 1, sorbitol and mannitol were stable and did not readily undergo further conversion^[@CR27],[@CR32],[@CR33]^, while glucose and fructose (Table [2](#Tab2){ref-type="table"}, entries 3 and 5) were readily converted to EG and PG through hydrogenolysis, indicating that if the target products were C~2~/C~3~, R2 should occur more quickly than R3. This result also supported the above discussion that controlling the synergy of the hydrogenation (Ni) and bond cleavage (W) was important. Furthermore, the reactions of glucose, fructose, dihydroxyacetone and acetol were performed in the presence and absence of ZnO to understand how ZnO influences the hydrogenolysis of cellulose into C~3~ products. As shown in entries 3 to 10, in the absence of ZnO (Entry 3), glucose was converted to EG in a yield of 51% and PG in 14.5%, suggesting that route 1 is favoured over route 2 (Fig. [4](#Fig4){ref-type="fig"}). When ZnO was present (Entry 4), glucose was converted to EG in a yield of 28.9% and PG in 35.7%, suggesting that route 2 is favoured over route 1. The results under different ZnO conditions indicated that ZnO could promote route 2, suggesting ZnO influences a specific step. The reaction results of fructose, dihydroxyacetone and acetol in the presence and absence of ZnO (Entries 5--10) showed that ZnO had no impact on the conversion of fructose to 1,2-PG, dihydroxyacetone to 1,2-PG and acetol to 1,2-PG. As Fig. [4](#Fig4){ref-type="fig"} shows, excluding the above steps, we could conclude that R4 (the isomerization of glucose to fructose) was the major step influenced by ZnO. In addition, the results of dihydroxyacetone (Entries 7 and 8) further demonstrated that ZnO had no significant effect on the isomerization of dihydroxyacetone, indicating that the effect of ZnO on isomerization in this reaction system was specific to the promotion of glucose isomerization.Table 2Conversion of different carbohydrates as probe reactants over Ni-W/β.EntryCatalystReactantYield (%)EGPGSM1Ni-W/βSorbitol0.00.095.00.02Ni-W/βMannose12.38.91.351.53Ni-W/βGlucose51.014.58.81.74Ni-W/β + ZnOGlucose28.935.77.22.35Ni-W/βFructose13.248.710.28.96Ni-W/β + ZnOFructose12.949.29.99.57Ni-W/βDihydroxyacetone0.069.8------8Ni-W/β + ZnODihydroxyacetone0.071.2------9Ni-W/βAcetol0.088.7------10Ni-W/β + ZnOAcetol0.087.4------Reaction conditions: 30 min, 6 MPa H~2~, 245 °C, 50 mL of H~2~O, 0.5 g of reactant, 0.15 g of catalyst, and 100 mg of ZnO. EG: ethylene glycol; PG: 1,2-propylene glycol; S: sorbitol; and M: mannitol.
Reusability {#Sec7}
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Catalyst recycling is an important property for the practical application of catalysts in metal-catalysed liquid-phase reactions for the following two major reasons: 1) the catalyst can readily leach metal, which is attributed to the H^+^ formed in water at high temperature^[@CR34],[@CR35]^, and 2) the complex intermediate components produced by the side reactions can block the pores and poison the catalyst^[@CR36],[@CR37]^. Therefore, the reusability of Ni-W-ZnO/β and the changes in its composition and structure were tested, and the results are displayed in Fig. [5](#Fig5){ref-type="fig"}, Table [S2](#MOESM1){ref-type="media"} and Table [3](#Tab3){ref-type="table"}. As Fig. [5](#Fig5){ref-type="fig"} shows, the yield of total glycols decreased from 70.1% to 68.1%, 64.8% and 59.7% by the second, third and fourth runs, respectively, while the conversion of cellulose remained at 100%, 96% and 92%. The slight decrease could be attributed to structural changes, metal leaching from the catalyst and acidity decrease, which was verified by N~2~-adsorption and desorption (Table [S2](#MOESM1){ref-type="media"}) and ICP results (Table [3](#Tab3){ref-type="table"}) and NH~3~-TPD (Table [S3](#MOESM1){ref-type="media"}), respectively. As shown by Table [S2](#MOESM1){ref-type="media"}, entries 9 and 15--17, with the reuse of the catalyst, the BET surface area of Ni-W/β decreased slightly from 287 to 255 m^2^/g. It could be speculated that deposition of carbon species and collapse of the mesopores occurred during reuse, changing the catalytic structure^[@CR38]^. Nevertheless, differences between the pore volume and size in fresh and used catalysts were not significant, suggesting that the catalytic structure was not badly damaged by four cycles of reuse^[@CR39]^. As displayed in Table [3](#Tab3){ref-type="table"}, the loading of Ni decreased from 7.29 to 6.41, 5.73, 5.64 and 5.58 wt% over four cycles of reuse, while W decreased from 20.87 to 18.11, 15.26, 13.58 and 11.56 wt%. As shown in Table [S3](#MOESM1){ref-type="media"} (Entries 4, 10--12), the acidity of the catalyst decreased during four cycles reaction, which may attributed to the leaching of Ni and W and the decreased BET surface area. What's more, the increasing tendency of Ni/W weight ratio (from 0.35 to 0.41) suggested that the leaching W was faster than Ni, indicating the reaction route might be changed by the synergy of Ni and W. In the first and second cycles (Fresh and Reuse 1), the loadings of Ni and W were relatively balance. In this case, the high C~2~/C~3~ glycol yields (70.1% and 68.1%) could be attributed to the balance loading of Ni and W, and the slight decrease might be caused by the leaching of Ni and W. In the third and fourth cycles (Reuse 2 and 3), the yield of C~2~/C~3~ glycols decreased to 64.8% and 59.7%, respectively, while the yields of mannitol and sorbitol were significantly increased. According to the above mechanism (Fig. [4](#Fig4){ref-type="fig"}), W promoted R2, while Ni promoted R3, and the favourability of routes 1, 2 or 3 was determined by the synergy of Ni and W. Take the fourth cycle (Reuse 3) as an example, the loading of W was insufficient because of the faster leaching of W than Ni. Therefore, R2 in routes 1 and 2 was disfavoured, while R3 in route 3 was favoured, which led to an increase in the mannitol and sorbitol yield and a decrease in EG and 1,2-PG. According to the above results and discussion, the loading of Ni and W and the balance of hydrogenation and RAC are also greatly important during reuse. What's more, because of the leaching of Ni and W was obvious and could not be neglected, the effect of leached Ni and W on cellulose hydrogenolysis into PG/EG was investigated (Table [S5](#MOESM1){ref-type="media"}). The results suggested that when the supported W was sufficient, the leached W did not work significantly on PG/EG production, while there was no supported W, the leached W could play a role in the reaction, which was the evidence that supported W, other than leached W, had a major role on PG/EG production in our catalytic system.Figure 5Reuse of the ZnO and Ni-W/β catalyst system. Reaction conditions: 30 min, 6 Mpa H~2~ 245 °C, 50 mL of H~2~O, 0.5 g of cellulose, 0.15 g of catalyst, and 100 mg of ZnO. EG: ethylene glycol; PG: 1,2-propylene glycol; S: sorbitol; and M: mannitol.Table 3Changes in the nickel, tungsten and zinc contents in the 7Ni-20W/β + ZnO system during four cycles of reuse.CycleNi (wt %)Ni balance (wt %)^c^W (wt %)W balance (wt %)^c^Zinc (%)Zn balance (%)^c^Ni/W weight ratioLiquid^a^Solid^b^LiquidSolidLiquidSolidFresh---7.29------20.87------100.00---0.35Reuse 11.086.41 + 0.202.6218.11−0.143.5296.70 + 0.220.35Reuse 20.625.73−0.063.0115.26 + 0.164.1991.92−0.590.38Reuse 30.345.64 + 0.251.7913.58 + 0.112.9988.65−0.280.41after0.215.58 + 0.152.0311.56 + 0.013.1685.76+0.270.48^a^Liquid (wt% or %) = the weight of Ni, W or Zn in the liquid products after the reaction. The weight of Ni and W in the liquid products was converted into equivalent loadings in the catalyst (wt %).^b^Solid (wt% or %) = the weight of Ni, W or Zn in the solid catalysts after the reaction.^c^Ni, W or Zn balance (wt% or %) = the weight of Ni, W or Zn in the liquid products + the weight of Ni, W or Zn in the solid catalysts -- the weight of Ni, W or Zn before the reaction.
Conclusion {#Sec8}
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To promote the selective transformation of cellulose into C~2~/C~3~ glycols and increase the proportion of C~3~ glycol in the glycol products, we attempted to control the synergy of the retro-aldol condensation, hydrogenation and isomerization reactions. After the optimization of the catalyst support, the Ni-W loading and the co-catalyst, ZnO-modified 7Ni-20W/β was employed, and the influence of these factors on the product distribution was discussed. Compared to supports such as Al~2~O~3~, TiO~2~, SiC and ZSM-5, raw β-zeolite exhibited the best catalytic performance, which was attributed to its ability to promote the hydrolysis of cellulose into small molecules by its abundant acid sites. The synergy of Ni and W was the key to the reaction: on the one hand, the synergy of Ni and W could provide both Lewis and Brønsted acid sites in the catalyst and sequentially promote the formation of C~2~/C~3~ glycols; on the other hand, Ni is effective in the hydrogenation, and W facilitates the bond cleavage (C~6~ to C~2~/C~3~). The optimum loadings of Ni and W were 7 wt% and 20 wt% because an unbalanced loading of Ni and W had a detrimental influence on the synergy of the hydrogenation and retro-aldol condensation. Moreover, with the leaching of the metals during four cycles of reuse, the catalytic performance was also influenced by the synergy of Ni and W primarily through the change in their loading. In addition, by screening a series of metal oxides and alkalis as co-catalysts in the Ni-W/β system, ZnO caused the greatest improvement in the C~2~/C~3~ yield. The increased yield of C~3~ glycol by ZnO was attributed to the promotion of the isomerization of glucose to fructose. After the systematic optimization of the catalyst support, the Ni-W loading and the co-catalyst, the yield of C~2~/C~3~ glycols reached 70.1%, with propylene glycol accounting for 51.1% of the product, under 4 MPa hydrogen pressure at 245 °C for 30 min.
Methods {#Sec9}
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Catalyst Preparation {#Sec10}
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Commercial H-β-zeolite (Catalyst Plant of Nankai University), denoted as "raw β", was firstly dealuminated by refluxing in nitric acid at 80 °C at a solid-to-liquid ratio of 1 g: 20 mL under a stirring speed of 200 r/min for 20 h^[@CR25]^. After dealumination, denoted as "β", the zeolite powder was collected by centrifugation and then washed with deionized water several times until the supernatant was neutral. Finally, the obtained β-zeolite was dried at 150 °C overnight.
The catalysts, including Ni-W/raw-β, Ni-W/β, Ni-W/Al~2~O~3~, Ni-W/SiC, Ni-W/ZSM-5 and Ni-W/TiO~2~, were prepared by incipient wetness impregnation, dried at 120 °C overnight, calcined at 500 °C for 4 h, and reduced under a hydrogen-nitrogen flow (volume ratio = 5%: 95%) at 500 °C for 4 h. In detail, the Ni-W/β catalysts were prepared by co-impregnation with an aqueous solution of ammonium metatungstate hydrate (AMT) and Ni(NO~3~)~2~·6H~2~O. Regarding the nickel and tungsten loadings, the catalyst with 25 wt % of tungsten and 5 wt % of the nickel, for example, is denoted 5Ni--25 W/β. For all of the above catalysts, the nickel and tungsten loadings were 7 wt % and 20 wt %, respectively. All chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd.
Analytical methods {#Sec11}
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The specific surface area, pore volume and pore diameter of the catalysts were tested by N~2~ adsorption--desorption (Micromeritics ASAP 2020)^[@CR40]^. Each sample was purged in a vacuum at 300 °C for 3 h before analysis. The Brunauer-Emmett-Teller (BET) method was used to calculate the specific surface areas of the catalysts, the pore volume was calculated by the Barrett-Joyner-Halenda (BJH) model and the pore diameter was calculated by the BJH method from the desorption branches. Powder X-ray diffraction (XRD) patterns were collected on an X-ray powder diffractometer (Bruker D8 Advance) with Cu Kα radiation (40 kV, 40 mA) over a 2θ range of 5° to 80° at room temperature. Transmission electron microscopy (TEM) images were recorded on a JEOLJEM-1230 instrument operated at 80 kV.
The acidic properties of the catalysts were tested by NH~3~ temperature-programmed desorption (NH~3~-TPD) and pyridine infrared (Py-IR) spectroscopy^[@CR20],[@CR40]^. The NH~3~-TPD was conducted using a Micromeritics AutoChemII 2920 system. The sample was pretreated in helium at 300 °C for 60 min, after which the sample was cooled to 120 °C in a helium flow. Next, 5% NH~3~ in the helium flow was absorbed onto the sample at 120 °C for 120 min. Finally, NH~3~-TPD was carried out from 120 °C to 800 °C at a ramp rate of 10 °C/min in helium flow^[@CR20]^. The Py-IR spectra were recorded on a Perkin Elmer Frontier FT-IR in the range of 1400--1700 cm^−1^ with a spectral resolution of 2 cm^−1^. A 10-mg sample of catalyst was pressed into a wafer with a diameter of 13 mm and then set in a quartz IR cell. The catalysts were dried at 400 °C for 2 h under vacuum. After cooling, pyridine vapor was injected into the cell, and the adsorption period lasted for 30 min. Subsequently, the desorption profiles at 150 °C (1 h), 250 °C (1 h), 350 °C (1 h), and 400 °C (1 h) were recorded^[@CR40],[@CR41]^.
The confirmation of thorough dealumination in β, the metal loadings in the solid catalyst and the leaching of the metals into the liquid were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin Elmer Optima 2100 DV). Prior to the measurements, the solid samples were digested in an acidic mixture (HF:HNO~3~:HClO = 1:1:1), and the liquid samples were digested in HNO~3~ at 150 °C for 12 h.
Catalytic Reaction {#Sec12}
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The catalytic experiments were carried out in a 100-mL stainless steel autoclave with a Teflon insert, and the operating parameters were the same as those in our previous study^[@CR40]^. To the autoclave were added 0.5 g of cellulose, 150 mg of catalyst, and 50 mL of water. Microcrystalline cellulose (Shanghai Chineway Pharm. Tech. Co. Ltd) was dried under vacuum at 105 °C for 12 h before use. After the reactor was sealed, the vessel was purged with nitrogen three times to exclude air and then pressurized with 6 MPa of hydrogen and heated to the desired reaction temperature, which was kept constant throughout the reaction with stirring at 1000 rpm. The zero point of the "reaction time" was defined as the time point at which the required temperature was reached. When the reaction ended, the reactor was immediately quenched to room temperature in an ice-water bath. The products were quantified by gas chromatography (GC, Agilent 7820 A, J&W125--7332, 30 m × 530 μm × 1 μm) with an FID detector and high-performance liquid chromatography (HPLC, Agilent 1200, Shodex SUGAR SH1011) with VWD and RI detectors. The cellulose conversion (%) was determined from the change in the cellulose weight from before to after the reaction, and this value was further verified by measuring the total organic carbon (TOC-VCPH, Shimadzu, Japan). The yield of products (%) was defined as $\documentclass[12pt]{minimal}
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\begin{document}$${\rm{Yield}}\,( \% )=\frac{\,the\,weight\,of\,carbon\,in\,one\,product\,after\,reaction}{the\,weight\,of\,carbon\,in\,cellulose\,before\,reaction}\times 100 \% $$\end{document}$. For the catalyst reuse studies, the solid catalysts were collected by filtration and washed several times with water. The recovered catalysts were used after reduction in a mixed hydrogen-nitrogen flow (volume ratio = 5%: 95%) at 280 °C for 4 h to remove residual cellulose^[@CR42]^ and keep Pt in its metallic state.
Supplementary information
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{#Sec13}
Supplementary figures and tables
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Supplementary information
=========================
**Supplementary information** accompanies this paper at 10.1038/s41598-019-48103-6.
The authors acknowledge sponsorship of the National Natural Science Foundation of China (No. 21676205), the National Science Fund for Distinguished Young Scholars (No. 51625804), the National Key R&D Program of China (No. 2018YFD1100503) and the Fundamental Research Funds for the Central Universities (No. 22120190012, 22120190054).
Minyan Gu, Zheng Shen and Yalei Zhang conceived the idea and designed the experiments. Minyan Gu and Long Yang did the experiments and wrote the paper. Wenjie Dong, Ling Kong, Wei Zhang and Boyu Peng modified the paper. Yalei Zhang and Zheng Shen supervised the whole work.
The authors declare no competing interests.
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}
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CASE PRESENTATION
=================
A 70-year-old male with a history of abdominal aorta endovascular aneurysm repair (EVAR) presented to the emergency department (ED) as a trauma activation after a fall and subsequent lower extremity weakness. The patient reported a near-syncopal episode the night preceding the fall and progressive lower extremity weakness over the course of the morning. On arrival to the ED, he complained of lower extremity weakness with noted initial 2+ palpable, bilateral femoral and dorsalis pedis pulses. Shortly thereafter, he lost palpable femoral pulses bilaterally and had noted cool lower extremities. Computed tomography angiography (CTA) was remarkable for kinking and occlusion of the abdominal aorta endograft below the renal vessels ([Images 1](#f1-cpcem-04-474){ref-type="fig"}--[](#f2-cpcem-04-474){ref-type="fig"}[3](#f3-cpcem-04-474){ref-type="fig"}). The patient was taken to the operating room emergently with successful axillobifemoral bypass revascularization.
DISCUSSION
==========
The patient had undergone EVAR approximately three years prior and had been taking aspirin and clopidogrel but, per the patient, clopidogrel was recently discontinued by his primary care physician to decrease endoleak at aneurysm repair. CTA demonstrated a kinked endograft and thrombus within the graft into the iliac arteries. Kinking of endograft limbs and occlusion has been reported in 2--4% of patients, which can result in acute limb ischemia.[@b1-cpcem-04-474],[@b2-cpcem-04-474] Bilateral leg ischemia due to endograft occlusion is rare with a reported incidence ranging from 0%--0.6%.[@b3-cpcem-04-474]
###### CPC-EM Capsule
What do we already know about this clinical entity?
Kinking and occlusion has been reported as a complication in 2--4% of patients who underwent endovascular aneurysm repair (EVAR) of the abdominal aorta.
What is the major impact of the image(s)?
These images demonstrate kinking and occlusion of the abdominal aorta EVAR endograft with resulting bilateral leg ischemia, which is very rare.
How might this improve emergency medicine practice?
Emergency physicians should consider endograft complications in patients with a history of EVAR presenting with lower extremity neurovascular complaints.
Section Editor: Christopher Sampson, MD
Full text available through open access at <http://escholarship.org/uc/uciem_cpcem>
The authors attest that their institution requires neither Institutional Review Board approval, nor patient consent for publication of this image in emergency medicine. Documentation on file.
*Conflicts of Interest*: By the *CPC-EM* article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The authors disclosed none.
{#f1-cpcem-04-474}
{#f2-cpcem-04-474}
{#f3-cpcem-04-474}
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{
"pile_set_name": "PubMed Central"
}
|
1.. Introduction {#s1}
================
Stereopsis, resulting from the horizontal displacement of the two eyes (i.e. binocular disparity), adds a rich third dimension to the visual world, enabling the observer to discern the relative distances of objects with extraordinary accuracy. Under ideal conditions, stereo thresholds are much finer than the spatial grain of the retinal mosaic and are considered a hyperacuity \[[@RSOS150523C1]\], as small as a few arc seconds of visual angle. The neural computations involved in extracting the binocular disparity information from the two monocular images are largely based on cortical processing at multiple levels \[[@RSOS150523C2],[@RSOS150523C3]\]. It appears that the neuronal mechanisms supporting stereoscopic vision are not hard-wired, but may be modifiable through experience \[[@RSOS150523C2],[@RSOS150523C4],[@RSOS150523C5]\]. There is earlier evidence demonstrating that perceptual learning can enhance stereoacuity in human adults \[[@RSOS150523C6]\], beyond the critical period for visual development \[[@RSOS150523C7],[@RSOS150523C8]\]. Perceptual learning is the long-term improvement in visual function that results from repeated practice of a challenging visual task. Perceptual learning has attracted a great deal of attention in the past several decades, in large part because it has been shown to be highly specific to the orientation and spatial frequency of the trained visual stimuli \[[@RSOS150523C9]--[@RSOS150523C14]\]. There is still much debate about the mechanisms of perceptual learning for stereopsis and where the alternations may occur in the visual brain \[[@RSOS150523C15]--[@RSOS150523C21]\].
Neurons in the primary visual cortex are tuned to encode low-level visual information such as spatial frequency, orientation, retinotopic location, etc. \[[@RSOS150523C22]\]. Thus, a neuron in early visual cortex responds to a limited range of stimulus spatial frequencies, orientations and locations, forming the basis of a multi-channel system analysing the visual image formed on the retina. The properties of visual plasticity in the individual spatial frequency channels \[[@RSOS150523C23],[@RSOS150523C24]\] are not yet clearly understood. We are particularly interested in whether there is 'cross-talk' between these putative channels. Here we ask whether perceptual learning of stereoscopic depth perception generalizes broadly across spatial frequency and orientation. This has important ramifications for clinical applications, because such vision training exercises would not be useful in everyday life if the learning effects do not transfer to other untrained stimuli. We are also interested in making the learning as efficient as possible. In order to determine the most efficient strategy, we compare the transfer of learning from high spatial frequency to low and vice versa. In this study, we systematically quantified the effects of perceptual learning using narrow-band visual stimuli, spanning a large spatial frequency range. Using a multi-stage training protocol, we characterized the magnitude and specificity of learning with respect to low-level, basic visual features, providing new insights into the mechanisms of neural plasticity and importantly, the strategies to optimize visual performance. Our investigations contribute to understanding the hierarchical architecture and functional properties of the network subserving stereovision.
2.. Material and methods {#s2}
========================
2.1. Subject {#s2a}
------------
A total of 31 healthy young adults, ages 20--40 years, participated in three groups. All had normal or corrected-to-normal visual acuity of 20/16^−2^ or better in each eye; the interocular acuity difference was two letters or less on a standard LogMAR letter chart (National Vision Research Institute of Australia, 1978). Inclusion criteria were spherical refractive error in the range of +2D to −5D and astigmatism in the range of 0--0.50D. None of the participants had anisometropia of greater than 1D spherical equivalent difference between the two eyes. They had neither strabismus nor amblyopia. All participants had normal stereoacuity of 40 arcsec or better (Randot^®^ stereotest, Stereo Optical Co., Inc., Chicago, IL, USA) and the heterophoria, if any, was within the normal range at distance and near as examined by alternate cover test. All testing and training was done with the observer wearing best optical correction.
2.2. Visual stimulus {#s2b}
--------------------
The visual stimulus consisted of two horizontally separated black squares. At the centre of each square was a target Gabor patch surrounded by four reference Gabor patches with the same spatial frequency and orientation as the target ([figure 1](#RSOS150523F1){ref-type="fig"}*a*). A custom-built 4-mirror stereoscope was used to present a half monitor screen to each eye (i.e. the left square for the left eye and vice versa). Binocular disparity was introduced by shifting the two target Gabor patches (one in each square) in opposite directions. The position and phase of each Gabor patch, both target and reference, were randomly jittered based on a uniform distribution (vertical and horizontal position range, ±20 screen pixels---more than an order of magnitude larger than the observers' stereo thresholds; phase range, 0--360^°^) to minimize any possible monocular cues, for example, Vernier and bisection cues. The following abbreviations are adopted to describe the Gabor patch parameters: V1, V5, V10, V20 and H5, where the letter describes the carrier orientation (V, vertical; H, horizontal) and the number specifies the carrier spatial frequency expressed in cycles per degree. Figure 1.Stereo stimulus. (*a*) The stereogram consisted of two slightly different pictures---one to each eye. At the centre each square was a target Gabor patch surrounded by four reference Gabor patches. To eliminate any possible monocular cues, the vertical and horizontal coordinates of each Gabor patch and also the patch features, the carrier phase, were randomly jittered according to a uniform distribution. A custom-built mirror stereoscope was used to view the stereo pairs, so that the left eye would see the left square and the right eye would see the right one. Binocular disparity was generated by shifting the two target Gabor patches, one on each side, horizontally in opposite directions (uncrossed disparity, both shifted temporally; crossed disparity, both shifted nasally). (*b*) Binocular fusion of the two monocular images creates a cyclopean image. The visual task was to determine the stereoscopic depth of the target Gabor (in front/behind) relative to the four adjacent references. This schematic diagram illustrates crossed disparity---the target Gabor patch appeared in front of the reference patches.
2.3. Cyclopean view {#s2c}
-------------------
[Figure 1](#RSOS150523F1){ref-type="fig"}*b* illustrates the cyclopean percept of the visual targets in binocular viewing. The outer square served as a fusion lock to ensure proper alignment of the two eyes. The two letter Es at the top right and bottom left corners served as an accommodation lock for stimulating accommodation to bring the visual stimulus in focus. The E at the top left corner and the other E at the bottom right corner served as a binocular indicator ensuring the absence of monocular suppression during testing, and proper alignment (when perceived to be one above the other). Observers were given careful instructions for adjusting the haploscope mirrors in order to avoid stimulating excessive convergence, which could trigger convergence-accommodation and result in blurred vision. The visual task was to determine the stereoscopic depth of the target Gabor (in front or behind) relative to the four reference Gabor patches. Trial-by-trial audio feedback was provided for each response.
2.4. Stimulus scaling {#s2d}
---------------------
The stimulus spatial scale was manipulated by changing the physical stimulus size on the screen and varying the viewing distance, 50 cm (V1) or 2 m (V5, V10, V20 and H5). As for V5 stimuli viewed at 2 m, the s.d. of the Gaussian envelope was 7 arcmin, Gabor centre-to-centre distance was 48 arcmin when positional jittering was off, the letter 'E' size was 25 arcmin and the square was 197.3 arcmin. The carrier spatial frequency and Gaussian envelope s.d. covaried, keeping a constant number of cycles/s.d. All visual stimuli were displayed on a 21-in Sony F520 flat monitor screen at 1800×1440 resolution and 90 Hz refresh rate. The mean luminance of the stimuli was 55 cd m^−2^ and the contrast of each Gabor patch was 99%. Light shielding was used to block stray light from the monitor screen. The inter-pixel distance was 20 arcsec at a viewing distance of 2 m (50 cm, 80 arcsec); sub-pixel accuracy was achieved by contrast manipulation.
2.5. Psychophysical methods {#s2e}
---------------------------
For each trial, the amount of binocular disparity was controlled by two interleaved adaptive staircases to track the stereo threshold: one for crossed disparity (target patch in front of the adjacent reference patches) and the other for uncrossed disparity (target patch behind the references). The trials were divided into triplets: three correct responses decreased the disparity magnitude by one unit step, two correct responses left the disparity unchanged and only one or zero correct response increased the disparity by two unit steps. Stereo threshold was estimated as the disparity at the 84% correct response rate (*d*′=1), obtained by fitting a Probit function. The threshold reported for each observer is the average threshold estimate from two blocks of measurements (training session: 275 trials/block; pre- and post-training sessions: 160 trials/block).
2.6. General experimental design {#s2f}
--------------------------------
The experiment generally consisted of three segments: pre-training measures, training and post-training measures. In the pre-training segment, observers were tested with 3 (Experiment 1) or 4 (Experiment 2) different stimulus conditions, including different spatial frequencies (from 1.25 to 20 cpd) and carrier orientations (horizontal and vertical). In the training segment, observers were trained with a specific stimulus condition for 13 sessions. The post-training segment was identical to the pre-training segment. Each training session consisted of a total of 550 response trials (crossed disparity, 250 trials; uncrossed disparity, 250 trials; catch trials or zero disparity, 50 trials) in about 45 min. The experiment was self-paced, and a break was given whenever the subject requested one. The participants were naive to the purposes of the experiments and none of them had any prior experience in psychophysical experiments. It should be noted that our visual task is easy to comprehend, much like the standard stereopsis screening tests used in clinical settings, and participants were given practice trials (roughly 50 trials) to familiarize them with the stimuli and methods prior to the baseline measurements.
3.. Results {#s3}
===========
3.1. Learning to improve three-dimensional vision {#s3a}
-------------------------------------------------
In the first experiment, we asked whether practicing a stereoscopic depth detection task enhances stereoacuity in adults with normal vision and whether the learning effects, if any, transfer across different stimulus configurations. Ten adults with corrected-to-normal vision participated. The training protocol consisted of three stages, each of 13 sessions ([figure 2](#RSOS150523F2){ref-type="fig"}*a*). In stage 1, participants were trained with targets with a vertical carrier of five cycles per degree (V5: vertical, 5 cpd). In stage 2, they continued to train with the same spatial frequency, but with an orthogonal carrier orientation (H5: horizontal, 5 cpd). They were subsequently required to practice with targets with a vertical carrier at a higher spatial frequency (V10: vertical, 10 cpd) in stage 3. Thresholds for each of the three stimulus configurations were measured before and after each training stage. Each training session consisted of about 500 trials. Figure 2.Experiment 1. Perceptual learning of stereoacuity: specificity for carrier orientation and spatial frequency. (*a*) The training protocol consisted of three training stages: stage 1, V5 (vertical carrier: 5 cpd); stage 2, H5 (horizontal carrier: 5 cpd); stage 3, V10 (vertical carrier: 10 cpd). A 3-parameter exponential function was used to quantify the learning profile. Mean thresholds (*n*=10) for each of the three stimulus configurations were measured before and after each training stage. Error bars indicate the standard error of the mean unless stated otherwise. (*b*) The pre- and post-training threshold data of individual observers (*n*=10) are illustrated in the nine figure panels: 1st row, s15 versus s1; 2nd row, s29 versus s15; 3rd row, s43 versus s29. Arrows indicate the sequence of direct training from stage 1 to 3. White panel area denotes statistically significant. Grey panel area, not statistically significant. Note that the abscissa label for each row is displayed at the right-hand end of all the panels highlighted in dark blue.
We found that practice significantly enhances stereovision. On average, our observers showed almost a factor of three improvement (post/pre, 64%) in mean stereoacuity (red circles, from 27.3 (session 1, s1) to 9.9 arcsec (session 15, s15); paired-*t*=6.510, *p*\<0.001) after practicing with V5 stimuli in stage 1. A 3-parameter exponential function, *Th*=*Th*~i~×e^(−*bx*)^+*Th*~p~, was used to quantify the learning profile, where *Th* is stereo threshold, *Th*~i~ is the initial threshold, *Th*~p~ is the plateau threshold and *x* is the training session. Asymptotic performance was obtained in roughly ten sessions (approx. 5000 responses). The pre- and post-training data of individual observers are displayed in [figure 2](#RSOS150523F2){ref-type="fig"}*b*. The top left panel illustrates the threshold data for V5 direct training (s1 versus s15). Note that the area below the grey 1 : 1 reference line represents improvement in stereoacuity. For clarity, the error bars for all individual training sessions were omitted.
3.2. Specificity to stimulus configurations {#s3b}
-------------------------------------------
We examined the specificity of visual learning; i.e. whether improvement transfers to a different orientation or a different spatial frequency, in order to further explore the possible mechanisms for the plasticity. To test whether the visual learning effects transfer to the untrained stimulus orientation, we compared pre- and post-training measurements of thresholds with horizontal carriers (H5, [figure 2](#RSOS150523F2){ref-type="fig"}*a* inset). To examine the specificity of the visual learning to the trained stimulus spatial frequency, we performed pre- and post-training measurements of thresholds with carriers of higher spatial frequency, by a factor of 2 (V10, [figure 2](#RSOS150523F2){ref-type="fig"}*a* inset).
Learning generalized substantially to the untrained stimuli. Practicing with V5 stimuli resulted in improvements of 46% for H5 (blue squares, from 169.1 to 91.0 arcsec; s1 versus s15: paired-*t*=3.322, *p*=0.009) and 56% for V10 (green circles, from 19.1 to 8.5 arcsec; s1 versus s15: paired-*t*=6.042, *p*\<0.001). The raw training data for individuals are shown in [figure 2](#RSOS150523F2){ref-type="fig"}*b* (first row, middle and right panels).
3.3. Transfer: complete or incomplete? {#s3c}
--------------------------------------
To quantify the transfer of learning, we calculated a Transfer Index (*T*=PI~untrained~/PI~trained~) for individual observers, where PI is the per cent improvement, *T*=1 indicates complete transfer and *T*=0 indicates no transfer. Mean *T*=0.67±s.e. 0.11 and 0.88±s.e. 0.09 for the orthogonal orientation (H5) and higher spatial frequency (V10), respectively. These values suggest nearly complete transfer of learning; however, it is not entirely clear whether the performance on the trained stimulus had reached the respective plateau levels or whether additional direct training could further boost the already sharpened stereoacuity induced by indirect learning.
To answer that question, the participants were asked to engage in subsequent direct training with the two previously untrained stimuli. In stage 2, they continued to train with H5 stimuli for another 13 sessions and interestingly, they continued to improve substantially with practice. The mean improvement relative to session 15 was 29% (s15 versus s29: paired-*t*=2.653, *p*=0.026), resulting in a total improvement (from s1) of 63%. Individual data can be found in [figure 2](#RSOS150523F2){ref-type="fig"}*b* (second row, middle panel). However, no significant change in stereoacuity was observed for V5 or V10 stimuli (second row, left and right panels; s15 versus s29: paired-*t*\<0.481, *p*\>0.642).
In stage 3, the participants were trained with V10 stimuli for 13 sessions. Similar to the results of stage 2, the enhanced performance resulting from indirect learning was further improved following subsequent direct training, with mean acuity improvement of 19% ([figure 2](#RSOS150523F2){ref-type="fig"}*b*, third row, right panel; s29 versus s43: paired-*t*=3.017, *p*=0.015), resulting in a total improvement (from s1) of 64%.
It is worth noting that all participants completed training with V5 stimuli over a large number of sessions and had already given a total of approximately 7000 responses in stage 1, and their performance levels appeared to be very stable at the end of the training course. Surprisingly, practicing V10 stimuli slightly, but significantly boosted the previously 'plateaued' V5 performance, with mean improvement of 18% from 10.2 to 8.4 arcsec ([figure 2](#RSOS150523F2){ref-type="fig"}*b*, third row, left panel; s29 versus s43: paired-*t*=3.928, *p*=0.003). No further significant change was found for the orthogonal H5 stimuli ([figure 2](#RSOS150523F2){ref-type="fig"}*b*, third row, middle panel; s29 versus s43: paired-*t*=0.587, *p*=0.572).
In brief, additional significant improvements obtained with subsequent direct training in stages 2 and 3 evidently suggesting that the transfer of learning observed in stage 1 may not have been complete. Those extra improvements were specific to the difference in stimulus characteristics. We speculate that practice with feedback fine-tunes the internal template to better sample, or learn, the visual stimulus \[[@RSOS150523C25]\], allowing more precise processing of stereoscopic depth information.
3.4. Bandwidth of learning {#s3d}
--------------------------
Here our findings reveal that stereoacuity learning can transfer across the spatial frequency spectrum, from the trained spatial frequency to the untrained 'neighbour' spatial frequency. When trained at a medium spatial frequency (5 cpd), substantial acuity improvement was recorded at one octave higher (10 cpd). However, these two spatial frequencies likely fall within the same spatial frequency selective mechanisms, as these putative channels have a bandwidth of one to two octaves \[[@RSOS150523C26]--[@RSOS150523C29]\]. How broad is the bandwidth of transfer?
To quantify the bandwidth of generalization of learning across spatial frequency, another set of 21 adult participants with normal vision were randomly assigned into two groups (LH, *n*=10; HL, *n*=11). In stage 1, group LH was trained at a low spatial frequency (L, 1.25 cpd) and group HL was trained at a high spatial frequency (H, 20 cpd). In stage 2, observers crossed over and trained at the untrained spatial frequency (group LH, 20 cpd; group HL, 1.25 cpd). Thresholds were measured at each of four spatial frequencies (1.25, 5, 10 and 20 cpd) before and after each training stage. Observers practiced for 16 000 trials over 29 sessions. Trial-by-trial feedback to response was provided.
3.5. 'Coarse-to-fine' spatial frequencies {#s3e}
-----------------------------------------
We found that the relatively coarse stereopsis evident at low spatial frequencies can be substantially enhanced with repetitive practice. The observers in group LH showed a more than threefold (70%) improvement in mean stereoacuity (red circles, from 77.0 to 23.3 arcsec; s1 versus s15: paired-*t*=5.526, *p*\<0.001) after practicing with V1 stimuli in stage 1 ([figure 3](#RSOS150523F3){ref-type="fig"}*a*, top panel); asymptotic performance was achieved in roughly 10 sessions. Interestingly, all three untrained higher spatial frequencies also improved substantially ([figure 3](#RSOS150523F3){ref-type="fig"}*b*, top panel; V5, V10 and V20: paired-*t*\>3.712, *p*\<0.005). The threshold data are replotted as percentage improvement in [figure 3](#RSOS150523F3){ref-type="fig"}*c*. It is important to note that the transfer was progressively reduced as spatial frequency increased (blue symbols); however, a 44% improvement (a *T* of approx. 0.63) was still obtained for V20 stimuli, 4.3 octaves from the trained spatial frequency and quite close to the visual acuity limit. Figure 3.Experiment 2. Bandwidth of generalization across the spatial frequency spectrum. (*a*) Mean stereoacuity as a function of session. In stage 1, group LH (first row, *n*=10) was trained with V1 stimuli and group HL (second row, *n*=11) was trained with V20 stimuli. In stage 2, observers crossed over and trained at the untrained spatial frequency: group LH, V20; group HL, V1. A 3-parameter exponential function was used to quantify the learning profile. (*b*) Mean stereoacuity across spatial frequencies. (*c*) Per cent improvement in mean stereoacuity (*I*) as a function of spatial frequency (*f*). A 3-parameter Gaussian function, *I*=*I*~*t*~×e^−(1/2)(*f*−*f*~*t*~/*σ*)^2^^, was used to quantify the generalization of stereoacuity learning across spatial frequencies, where *I*~*t*~ is the per cent improvement occurring at the trained spatial frequency (*f*~*t*~) and *σ* denotes standard deviation.
We fitted the threshold versus spatial frequency data with a 3-parameter Gaussian function of which the centroid position (mean) was constrained to be at the trained spatial frequency. The bandwidth of transfer, which was defined as the half-width at half height ($B = \sqrt{2\ln 2} \cdot \sigma,$ where *σ* denotes standard deviation) from low frequency (1.25 cpd, approx. Snellen 20/480) to high frequency (20 cpd, approx. Snellen 20/30) was calculated as 23.2 cpd. In other words, the effect of transfer decreased by approximately 40% at 1 s.d. away from the trained spatial frequency. In short, these findings show that practicing with low spatial frequency stimuli enhanced coarse stereopsis and importantly, the learning effects extend broadly towards the high spatial frequency range.
To address the question of whether the transfer was complete or not at high spatial frequencies, the participants continued to train with V20 stimuli in stage 2 ([figure 3](#RSOS150523F3){ref-type="fig"}*a*). A further significant improvement of as much as 63% was observed with subsequent direct training ([figure 3](#RSOS150523F3){ref-type="fig"}*b*, top panel; s15 versus s29: paired-*t*=2.882, *p*=0.018), pointing to an incomplete transfer. Similar to the first experiment, we observed some smaller indirect learning effects for lower spatial frequency stimuli ([figure 3](#RSOS150523F3){ref-type="fig"}*c*, top panel, green symbols)---the improvement was significant for V10 stimuli (s15 versus s29: paired-*t*=2.833, *p*=0.020). Surprisingly, there was also a small improvement for V1 stimuli, 10% beyond the ceiling, although this was not statistically significant (s15 versus s29: paired-*t*=1.610, *p*=0.142). Note the performance appeared to have reached a fairly stable 'plateau' level after a large number of training trials in stage 1.
It is worth pointing out that the transfer of learning in stage 2 ([figure 3](#RSOS150523F3){ref-type="fig"}*c*, blue curve, with the peak at the trained frequency) was in the opposite direction from what we found in stage 1 (green curve) in which the transfer was induced from low to high spatial frequency. Those participants were first trained with V1, leaving little room for improvement, and that is probably why the bandwidth of transfer obtained in stage 2 (11 cpd) was much narrower than that obtained in stage 1 (23.2 cpd).
3.6. 'Fine-to-coarse' spatial frequencies: asymmetric transfer {#s3f}
--------------------------------------------------------------
In order to quantify the characteristics of transfer from high to low spatial frequency, another group of participants (group HL) was first trained with V20 stimuli in stage 1. Direct training resulted in a more than threefold (78%) improvement in mean stereoacuity ([figure 3](#RSOS150523F3){ref-type="fig"}*a*, bottom panel; s1 versus s15: paired-*t*=3.727, *p*=0.004) and the learning effect transferred to all three untrained lower spatial frequencies as well ([figure 3](#RSOS150523F3){ref-type="fig"}*b*, bottom panel; s1 versus s15: paired-*t*\>2.804, *p*\<0.019).
Interestingly, we observed an asymmetric transfer of learning across the spatial frequency spectrum. The bandwidth of transfer induced by training with V20 stimuli appeared to be broader than that induced with V1 stimuli ([figure 3](#RSOS150523F3){ref-type="fig"}*c*, top panel versus bottom panel, blue curve; approximately 35%, 31.1 versus 23.2 cpd), meaning that larger improvements were obtained for the other three untrained spatial frequencies. There was a 59% acuity improvement four octaves away from the trained spatial frequency. By contrast, at the same octave distance the improvement was smaller (44%) for group LH. An additional improvement of 26% was obtained with subsequent direct training with V1 stimuli (s15 versus s29: paired-*t*=4.092, *p*=0.002), with no further significant change observed for the other three higher spatial frequencies. Note that the effect of direct training observed here was substantially weaker when compared with group LH in stage 2 ([figure 3](#RSOS150523F3){ref-type="fig"}*c*, top panel versus bottom panel, grey dashed curve; HL~V\ 1,s29~/HL~V\ 1,s15~ versus LH~V\ 20,s29~/LH~V\ 20,s15~: unpaired-*t*=3.267, *p*=0.004), as a consequence of a stronger transfer effect in stage 1. All these findings clearly indicate that the transfer of learning was stronger when going from high to low spatial frequency than going in the opposite direction.
4.. Discussion {#s4}
==============
We investigated the spatial frequency selectively of perceptual learning of stereopsis, finding approximately 60--70% improvement in stereoacuity across a broad range of frequencies, from as low as 1 cycle per degree to near the resolution acuity limit ([figure 4](#RSOS150523F4){ref-type="fig"}). The spatial frequency bandwidth of stereo perceptual learning (≈4 octaves) is very much broader than the ≈1.4 octave bandwidth of contrast sensitivity learning in normal observers \[[@RSOS150523C30]\]. Figure 4.Enhancing coarse-to-fine stereoacuity with perceptual learning. Mean per cent improvement in stereoacuity resulting from direct training as a function of spatial frequency (stage 1 of the two experiments; V5 from the first experiment; V1 and V20 from the second experiment). There was no significant difference in per cent improvement among the three frequency groups (ANOVA: *F*=0.795, *p*=0.461).
Surprisingly, the improvement also generalized to the orthogonal orientation. With Gabor stimuli, such as those used here, stereoacuity with vertical carrier gratings is highly precise, since the vertical contours provide the necessary horizontal disparity information for making fine stereoscopic judgements. By contrast, with horizontal carrier gratings stereoacuity is coarse---almost a log unit worse than with vertical carriers---because the observer must use the Gaussian envelope to make the depth judgement. Thus, it is remarkable that the learning generalizes across orientations.
This broad transfer of depth discrimination learning contrasts with the highly specific learning that is found in other basic visual functions such as discrimination of contrast, orientation, spatial frequency, direction, depth, Vernier and texture segmentation \[[@RSOS150523C31]\]. The broad transfer reported here is also at odds with the highly orientation-specific learning \[[@RSOS150523C15]\] that has been reported with random dot stereograms. We have no ready explanation for this discrepancy; however, methodological issues are important in perceptual learning \[[@RSOS150523C32]\]. Random-dot stereograms may represent a much more difficult task compared with our contour stereoacuity test, because many observers have difficulties seeing these type of stereograms \[[@RSOS150523C19]\]. As task difficulty increases, learning can become more specific to stimulus features \[[@RSOS150523C13]\]. However, one possible explanation is that our contour stereoacuity test provides two sources of depth information (i.e. envelope and carrier cues), whereas random-dot stereograms provide only a single source of depth information.
Broad transfer occurred even when the visual stimuli differed in appearance from the previously trained ones (for example, see the three stimulus conditions displayed in [figure 2](#RSOS150523F2){ref-type="fig"}). However, the nature of the task remained essentially the same: to determine the stereoscopic depth of the target, relative to the surround. This broad generalization suggests that the learning occurred in the higher level cortical areas, beyond the point of binocular convergence \[[@RSOS150523C33]\].
A close look at the learning curves in [figures 2](#RSOS150523F2){ref-type="fig"} and [3](#RSOS150523F3){ref-type="fig"} reveals that the session-by-session improvements were smooth and gradual, requiring more than a total of 5000 response trials to reach a plateau, suggesting that this was genuine perceptual learning rather than the result of instrumental or cognitive learning (i.e. learning to do the task). Similar learning profiles have been reported for learning contour \[[@RSOS150523C6]\] and random dot stereograms \[[@RSOS150523C34],[@RSOS150523C35]\], with considerable inter-observer variability of learning \[[@RSOS150523C36]\]. Although we cannot completely rule out the possibility that in part the improved stereoacuity might reflect improved vergence, and thus reduced vergence noise, previous work has demonstrated that stereopsis is robust to disjunctive eye movements \[[@RSOS150523C37]\]. We believe that the enhanced visual performance reflects the effects of genuine neural plasticity triggered by perceptual learning.
The issue of specificity/generalization has attracted wide attention in the field of perceptual learning \[[@RSOS150523C38]\], both for theoretical and practical reasons. Perceptual learning would not be practical for clinical use \[[@RSOS150523C39]--[@RSOS150523C42]\] if the learning effects were very specific to the trained stimulus features. In that case, it would be necessary to perform training with a large number of visual targets of different sizes, spatial frequencies, orientations, shapes, etc., in order to reap the benefits of improved stereopsis in the real world.
How can we optimize perceptual learning in order to maximize visual performance? Our findings provide some hints. One surprising finding is that the apparently stable 'learning plateau' for lower frequency stimuli (V5 ([figure 2](#RSOS150523F2){ref-type="fig"}, stage 1) and V1 ([figure 3](#RSOS150523F3){ref-type="fig"}, stage 1)) can be further stretched by subsequent training with higher frequency stimuli (V10 ([figure 2](#RSOS150523F2){ref-type="fig"}, stage 3) and V20 ([figure 3](#RSOS150523F3){ref-type="fig"}, stage 2), respectively). One simple explanation is that the observers had not yet reached a performance plateau in stage 1, but we believe that was not the case. First, in a preliminary study adopting an extended training protocol consisting of 25--30 sessions, we found that 10--12 sessions were sufficient to reach a performance plateau for stereoacuity; therefore, we decided to do 15 sessions in the present experiments. Second, regression analysis (V5: 16 sessions, s1--15 and s29) with an additional plateau parameter (*r*^2^=0.925) provides a better fit than a 2-parameter model with no plateau parameter (*r*^2^=0.891), further supporting the occurrence of the performance plateau during the course of learning. Third, we note that it is not simply the extra number of sessions that matter. When the orientation of the carrier gratings was orthogonal in the subsequent training (H5 ([figure 2](#RSOS150523F2){ref-type="fig"}, stage 2)), no further enhancement beyond the ceiling was obtained for V5.
This 'beyond-the-plateau' learning appears to be specific to stimulus orientation. As shown in [figure 2](#RSOS150523F2){ref-type="fig"}, the subsequent training with finer stimuli (i.e. V10) actually transferred back only to those previously learned coarser gratings with the same carrier orientation (i.e. V5), but not to the orthogonally oriented ones (i.e. H5). Similar orientation-specific 'relearning' was observed for horizontally oriented stimuli (i.e. H5) following generalization from practicing with vertically oriented stimuli (i.e. V5). We also found that despite substantial transfer from low to high spatial frequencies (i.e. [figure 2](#RSOS150523F2){ref-type="fig"}*a*, V5; [figure 3](#RSOS150523F3){ref-type="fig"}*a* top panel, V1), subsequent direct training resulted in a small further improvement in stereoacuity for higher spatial frequencies.
Thus, while there is broad transfer of stereo learning in the first phase, it appears that supplementary direct training may be necessary when optimizing perceptual learning across orientations. Interestingly in most cases, the time needed to achieve plateau performance was quicker than the initial learning ([figures 2](#RSOS150523F2){ref-type="fig"}*a* and [3](#RSOS150523F3){ref-type="fig"}*a*, stage 1). Similarly, earlier studies have reported that considerable practice is necessary when learning new stereo stimuli with different shapes \[[@RSOS150523C17],[@RSOS150523C19],[@RSOS150523C20]\].
We found asymmetric transfer of stereo learning. The bandwidth of transfer was broader when training was at a high spatial frequency than at a low spatial frequency ([figure 3](#RSOS150523F3){ref-type="fig"}*b*). Task difficulty \[[@RSOS150523C13],[@RSOS150523C43]\] and task precision \[[@RSOS150523C44]\] can strongly influence the generalization of learning. Perhaps the higher frequency task with lower thresholds represents an easier task compared with the lower frequency one, thereby facilitating the transfer.
The specificity of perceptual learning for low-level stimulus features has often been taken as reflecting neural alterations at the early stages of cortical processing \[[@RSOS150523C33]\]. Alternatively, with more knowledge about the stimulus configuration acquired during practice, observers can construct a more efficient, better-matched template \[[@RSOS150523C25]\] at the higher cortical levels to more accurately localize the visual target presented to each eye and compute binocular disparity. Previous studies have shown that selective spatial attention mechanisms can explain the learning generalization to untrained retinal locations \[[@RSOS150523C18],[@RSOS150523C21]\]. Notably, a double training technique has been demonstrated to be useful in enabling transfer of learning across retinal locations or orientations through top-down processes \[[@RSOS150523C45],[@RSOS150523C46]\]. In agreement with our results, recent research suggests that perceptual learning can arise from different levels of visual processing: decision rules, attentional learning and physiological changes \[[@RSOS150523C32]\]. Changes in training procedures can cause a preference shift from one of those mechanisms to another.
5.. Conclusion {#s5}
==============
In short, both coarse and fine stereopsis can be enhanced by perceptual learning. Using narrow-band luminance spatial frequency stimuli, we were able to systematically characterize the time-course, magnitude and specificity of stereoacuity learning over a large range of spatial scales, and revealed useful strategies to boost the learning outcomes. The multi-stage training protocol allowed different levels of visual processing to be isolated, providing new insights into the learning mechanisms of stereopsis. For practical purposes, one of the most important findings is the asymmetric transfer of learning across the spatial frequency spectrum. Learning transfers more efficiently from high to low spatial frequencies than from low to high. The ability to generalize efficiently may provide a key principle for triggering neural plasticity and restoring impaired binocular vision in clinical situations.
Ethics {#s6}
======
The experimental procedures were approved by the University Committee for the Protection of Human Subjects, and the research was conducted according to the principles expressed in the Declaration of Helsinki. Informed consent was obtained from each participant.
Data accessibility {#s7}
==================
Data files relevant to this study can be found in the Dryad database (<http://dx.doi.org/10.5061/dryad.3td7f>).
Authors\' contributions {#s8}
=======================
R.W.L. designed the research, performed data processing, analysed data and supervised the research; R.W.L., T.T.T., A.P.C., T.W.L. and S.W.C. performed the experiments. R.W.L. and D.M.L. wrote the paper.
Competing interests {#s9}
===================
We declare we have no competing interests.
Funding {#s10}
=======
This work was supported by a National Eye Institute grant (R01EY01728) from the National Eye Institute. Travel grants were awarded to the authors T.T.T. and A.P.C. from the Association for Research in Vision and Ophthalmology and the American Academy of Optometry for presenting part of the findings at annual meetings.
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Introduction
============
The velvetbean moth, *Anticarsia gemmatalis* Hübner (Lepidoptera: Noctuidae), is one of the main pests of soybean, *Glycine max* L. (Merrill) (Fabales: Fabaece), a legume that provides about half the global demand for vegetable oils and proteins. *A. gemmatalis* causes extremely high levels of defoliation when infestation is heavy and can severely damage axillary meristems ( [@R27] ; [@R22] ).
For the control of pests in storage areas, methyl bromide (MeBr) and phosphine (PH3) are used, but they may cause several problems in stored products ( [@R14] ). In addition, their widespread use has led to some serious problems, including the development of insect strains resistant to insecticides ( [@R6] ; [@R11] ).
The deleterious effects of plant extracts on insects are manifested in several ways, including toxicity, growth retardation, feeding inhibition, oviposition, repellence, suppression of calling behavior, and reduction of fecundity and fertility ( [@R20] ; [@R2] ). Therefore, the objective of this study was evaluate the bioefficacy of an aqueous extract of beech apricot, *Labramia bojeri* A. de Candolle (Sa-potales: Sapotaceae), on *A. gemmatalis* development, nutritional index, digestive proteinase activity, and zymogram analyses of the digestive proteinase activities. In Brazil, it is common to find *L. bojeri* in all regions. The results of this study suggest that small farmers could collect these seeds, make an aquous extract, and use it for control.
Materials and Methods
=====================
Plant material
--------------
*Labramia bojeri* seeds were collected in the city of São João da Barra, in the State of Rio de Janeiro, Brazil.
Insects
-------
The eggs of *A. gemmatalis* were obtained from Dr. J. R. P. Parra (Laboratório de Biologia dos insetos, ESALQ-USP, Piracicaba, SP, Brazil), and a culture was maintained in the Laboratório de Purificação de Proteínas e suas Funções Biológicas, Universidade Federal de Mato Grosso do Sul, Campo Grande, Brazil.
Aqueous extract
---------------
*Labramia bojeri* seeds were triturated, and the finely ground product was mixed with distilled water at concentrations of 10 and 20 g/L. The suspension was kept for 24 hr at room temperature to extract the soluble compounds. The mixture was then filtered through fine fabric (voile), and the aqueous extract was stored at 4º C. A new extract was made every three days and used within two days.
Effects of an aqueous extract on the development of *Anticarsia gemmatalis*
---------------------------------------------------------------------------
Soybean leaves (genotype IAC-19) from the middle third of plants at the R1 and R2 stages (beginning and full bloom, respectively), according to [@R9] were immersed for 2 sec in aqueous extract or distilled water (control) before being offered to larvae (up to the 4 ^th^ stadium). After evaporation of excess water, humidified cotton was placed around petioles to maintain leaf turgor. For each treatment, five neonate larvae were placed in a Petri dish (15.0 x 1.0 cm) lined with paper filter. Each treatment was repeated 20 times for each of the above concentrations (n = 100). Larvae fed on a diet containing 10 g/L *L. bojeri* aqueous extract (LbAE) were used to analyze other biological parameters, such as larval growth, pupal weight, development and mortality, adult longevity, and produced malformations in pupae and adult insects.
Nutritional Parameters
----------------------
A number of nutritional parameters were compared among 4 ^th^ instar larvae exposed to either the LbAE-treated or control diet. The larvae, feces, and remaining uneaten food were separated using a microscope, dried, and weighed. Nutritional indices of consumption, digestion, and utilization of food were calculated, as described by [@R26] and [@R8] . The nutritional indices, namely efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), and approximate digestibility (AD) were calculated as follows:
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}{}$\text{ECI} = (\ddot{\text{A}}\text{B/I})\times 100$\end{document}$$
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}{}$\text{ECI} = [(\ddot{\text{A}}\text{B/I}\times \text{F})]\times 100$\end{document}$$
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}{}$\text{AD} = [(I\times F)/I]\times 100$\end{document}$$
where I = weight of food consumed, ÄB is the change in body weight, and F = weight of feces produced during the feeding period. Metabolic cost (MC) was calculated as:
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}{}$\text{MC} = 100 - \text{ECD}$\end{document}$$
Midgut preparation
------------------
Proteinases were obtained from the midguts of 4 ^th^ instar larvae according to [@R16] . Fourth instar larvae were cold-immobilized, and the midgut, along with its contents, was removed in cold 150 mM NaCl and stored frozen (-20º C) until needed. Guts from the larvae were subsequently homogenized in 150 mM NaCl and centrifuged at 6,000 x g for 5 min, and the supernatants were pooled and kept on ice for enzymatic assays. The protein concentration of the extracts was determined according to [@R3] .
Fecal pellet preparations
-------------------------
Proteinases were obtained from the midguts of 4 ^th^ instar larvae according to [@R17] . Feces of the caterpillars were collected during the experiment and frozen (-20° C). When necessary, feces were macerated, homogenized in 200 mM Tris-HCl buffer (Tris -- hydroxymethyl aminomethane), pH 8.5, and centrifuged at 20,000 g for 30 min at 4º C, and supernatants were used for *in vitro* enzymatic assays.
Enzymatic assays
----------------
The proteolytic activity was estimated using chromogenic substrates such as BApNA (N-benzoyl-DL-arginyl-p-nitroanilide) and SAAPFpNA ( *N-* succinyl-alanine-alanine-proline-phenylalanine p-nitroanilide) at a final concentration of 1 mM. Midgut and feces larvae extracts (50 µg protein) were incubated in 100 mM Tris-HCl buffer, pH 8.0, in a final volume of 0.1 mL for 10 min before the addition of substrate. The reactions were allowed to proceed at 37º C for 20 min and then stopped by adding 0.2 mL of 30% (v/v) acetic acid. The results of *in vitro* enzyme analyses are reported as the means of three independent experiments with appropriate blanks, and absorbance was measured at 410 nm.
Proteinase activity of extracts in polyacrylamide gel containing 0.1% gelatin
-----------------------------------------------------------------------------
Proteins extracted from the midguts and fecal extracts of larvae fed on diets with or without LbAE, and without prior boiling or reduction, were run on SDS-PAGE (10% gels) with some alterations. Midgut and fecal extract proteins from the larvae fed with an artificial diet and fed with 10 g/L LbAE were incubated with 100 µM TLCK (a specific inhibitor of trypsin) for 10 min at 30° C. These mixtures were subjected to SDS-PAGE in gels containing 0.1% gelatin. Following electrophoresis at 5° C, the gels were washed with 2.5% Triton X-100 solution (Sigma-Aldrich, [www.sigmaaldrich.com](http://www.sigmaaldrich.com) ) for 2 hr with shaking to remove the SDS, after which the gels were incubated with 0.1 M Tris--HCl, pH 8.0, for 2--3 hr. The gels were subsequently stained with Coomassie Brilliant Blue R-250.
Statistical analysis
--------------------
All data were examined using the Mann-Whitney test. The *p* \< 0.05 level was considered to be significant.
Results
=======
Effect of LbAE on fourthinstar larvae
-------------------------------------
Larvae fed on diet containing 10 g/L LbAE produced an approximate 50% decrease in weight ( [Figure 1A](#f1){ref-type="fig"} ). LbAE of up to 20 g/L caused \~ 75% decrease in larval weight ( [Figure 1A](#f1){ref-type="fig"} ). Treatment containing aqueous extract at 10 g/L and 20 g/L reduced survival by about 50% ( [Figure 1B](#f1){ref-type="fig"} ). However, 10 g/L LbAE did not significantly affect larval development time ( [Figure 1C](#f1){ref-type="fig"} ).
{#f1}
Effects of LbAE on development stages
-------------------------------------
Pupal viability was significantly reduced by \~ 35% for larvae fed on a diet supplemented with 10 g/L LbAE ( [Table 1](#t1){ref-type="table"} ). Although there was no difference in the duration of pupation, at 24 hr after pupation, the weight of larvae that were fed on the LbAE-containing diet was significantly lower (\~ 18 %) than that of larvae fed on the control diet. Pupal malformation (25%) was also observed, with the pupae of the larvae that were fed with LbAE showing abdominal and thoracic deformations and being smaller in size than the larvae that were fed the control diet ( [Figure 2](#f2){ref-type="fig"} A; [Table 1](#t1){ref-type="table"} ).
######
Effect of *Labramia bojeir* aquous extract (LbAE) on survival and growth of *Anticarsia gemmatalis* . Sample sizes are indicated in parentheses under the mean.
Means followed by the same letter within each column are not significantly different ( *p* \> 0.05; Mann-Whitney U test).
{#f2}
The viability of adult moths was evaluated by counting the number of malformed moths at emergence and by determining their longevity. Adult insects from larvae fed with LbAE were significantly smaller in size by 40% ( [Figure 2](#f2){ref-type="fig"} B; [Table 1](#t1){ref-type="table"} ) than those from larvae fed on the control diet. The longevity of adult insects developed from the larvae fed on the control diet was about 1.4 days longer than that of adults from the larvae fed with LbAE ( [Table 1](#t1){ref-type="table"} ).
Nutritional parameters
----------------------
The larvae reared on an LbAE-containing diet consumed \~ 47% less food than the controls ( [Figure 3](#f3){ref-type="fig"} A). However, when food consumption was expressed as a ratio of body weight, the larvae fed with LbAE consumed 25% more than the controls ( [Figure 3](#f3){ref-type="fig"} B). LbAE had a toxic effect when ingested by larvae ( [Table 2](#t2){ref-type="table"} ). LbAE, when incorporated in an artificial diet at 10 g/L, reduced ECI and ECD and increased the MC and AD for larvae when compared with the control.
{#f3}
######
Nutritional indices of *Anticarsia gemmatalis* fourthinstar larvae on *Labramia bojeir* aquous extract (LbAE) and control diet.
Means followed by the same letter within each row are not significantly different ( *p* \> 0.05; Mann-Whitney U test). AD = approximate digestibility; ECI = efficiency of conversion of ingested food; ECD = efficiency of conversion of digested food; MC = metabolic cost.
Enzymatic activities
--------------------
Extracts of soluble proteins prepared from midguts contained enzymes capable of hydrolyzing the synthetic substrates BApNA and SAAPFpNA, but were unable to hydrolyze BTpNA (data not shown). 10 g/L LbAE-fed larvae showed a significant reduction in trypsin activity (\~ 64%) when compared with control larvae ( [Figure 4](#f4){ref-type="fig"} A). In contrast to the trypsin activity, there was no significant change in the midgut chymotrypsin activity of LbAE-fed larvae (data not shown). Typsin and chymotrypsin activities were also detected in fecal material, with LbAE-fed larvae presenting \~ 4.5 times more trypsin activity than the controls ( [Figure 4](#f4){ref-type="fig"} B), whereas there was no alteration in the chymotrypsin activity of these larvae (data not shown).
{#f4}
Polyacrylamide gels containing 0.1% gelatin
-------------------------------------------
Polyacrylamide gels containing 0.1% gelatin were used to examine the action of LbAE on trypsin activity and to analyze the profile of these enzymes. [Figure 5](#f5){ref-type="fig"} shows a decrease in the trypsin activity of midgut extracts of LbAE-fed larvae when compared with that of the control larvae. The band of approximately 24 kDa had its tryptic activity strongly reduced. There was an increase in the trypsin activity of fecal extracts of LbAE-fed larvae when compared to that of the control larvae.
{#f5}
Discussion
==========
The search for alternative ways of controlling agricultural insect pests has led to the investigation of naturally-occurring compounds from plants that may have toxic, repellant, antifeedant, or anti-hormonal properties ( [@R21] ). LbAE significantly affected larval development, pupal weight, and mortality, and caused malformations. LbAE reduced the survival and longevity of adult moths and produced malformations in 25% of the individuals examined, important parameters with respect to insect control ( [@R21] ). Larvae reared on an LbAE-treated diet consumed 47% less than larvae on the control diet. However, when the consumption was expressed as a ratio of body weight, the LbAE-fed larvae consumed 25% more than the controls. Thus, LbAE apparently did not affect the feeding pattern, as has been previously observed for *A. gemmatalis* ( [@R17] ). The reduction in larval weight, despite the increase in consumption for LbAE-fed larvae, suggests that LbAE inhibits nutrient uptake in *A. gemmatalis* ( [@R7] ).
An index of dietary utilization showed that ECI and ECD decreased when larvae were fed the LbAE diet. In the present study, the AD value for larvae of *A. gemmatalis* was increased throughout the feeding period of the experiment. AD is also evaluated as a function of the time that food is retained in the digestive tract. Thus, an increase in AD leads to a decrease in the ECI. This finding suggests that, during this treatment, the food remained for a greater time in the insect's gut, allowing the detoxification of the protein. A greater AD would help to meet the increased demand for nutrients and compensate for the deficiency in food conversion (reduction in ECI and ECD), perhaps by diverting energy from biomass production into detoxification. This behavior has also been observed by others ( [@R5] ; [@R24] ). A drop in ECI indicates that more food is being metabolized for energy and less is being converted to body mass, i.e., growth of the insect ( [@R15] ). ECD also decreases as the proportion of digested food metabolizes for energy increases. Confirming these results, [Table 2](#t2){ref-type="table"} demonstrates an increase of \~ 80% in MC in *A. gemmatalis* larvae. The reduction in ECD is likely the result of a reduction in the efficiency of converting food into growth, perhaps by a diversion of energy from production of biomass into detoxification of LbAE, i.e., an increase in metabolic costs. The results obtained in our study are in agreement with those obtained by [@R5] and Ramos et al. 2009.
Enzymatic assays were carried out to examine the mechanisms of LbAE toxic action. Trypsin-activity in the midgut was decreased in the larvae that fed on leaves treated with an aqueous extract at 10 g/L. Elevated levels of trypsin activity were also observed in the fecal material of LbAE-fed larvae. A change in the membrane environment and consequent disruption of enzyme recycling mechanisms may provide an explanation for the observed increases in the tryptic activity of fecal extracts collected from both GNA and Con A fed larvae ( [@R10] ). The increase in the trypsin activity of feces from LbAE-fed larvae suggests that LbAE may cause the rupture of the peritrophic membrane of *A. gemmatalis* ( [@R24] ). A polyacrylamide gel containing 0.1% gelatin corroborated these results, where samples of extracts of larvae midgut and feces with and without TLCK confirmed major trypsin-activity and differences between the treatments. No novel enzymes were induced in larvae reared on a diet containing LbAE.
The effects of LbAE on soluble trypsin activity suggest that LbAE significantly decreases trypsin levels in the larval gut and affects the recycling mechanisms of this enzyme, since LbAE increases trypsin activity in the feces more than in the gut ( [@R9] ). These differential trypsin activities can lead to poor nutrient use, retarded development, and eventually death by starvation ( [@R12] ; [@R19] ), as midguts of larval Lepidoptera contain mainly serine proteinases ( [@R25] ; [@R1] ).
The results presented in this study provide further evidence for the potential use of this LbAE for inhibition of the development of *A. gemmatalis* via oral exposure. The use of complex mixtures as pest control agents is advantageous because natural mixtures may act synergistically ( [@R13] ) and may present greater overall bioactivity compared to the individual constituents ( [@R4] ).
Financial support was provided by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FUNDECT (Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e tecnologia), and FINEP (Financiamento de Estudos e Pro-jetos/Ministério da Ciência e Tecnologia).
AD
: approximate digestibility
ECD
: efficiency of conversion of digested food
ECI
: efficiency of conversion of ingested food
LbAE
: L. bojeri seed aqueous extract
MC
: metabolic cost
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We acknowledge the effort of [@r2] in striving to develop a transparent, objective procedure for point of departure (POD) estimation, as encouraged by scien-tific review groups ([@r1]). Although additional charac-teri-za-tion of the statistical properties of the signal-to-noise crossover dose (SNCD) may be warranted, the goal of [@r2] appears consistent with the intent of the POD to charac-terize "the beginning of extrapolation to lower doses" \[U.S. Environmental Protection Agency (EPA) 2005\]. In this letter we respond to the authors' illustration of their approach using cancer bio-assay data to develop reference doses (RfDs) that target a 1/1,000 risk through linear extrapolation from the POD by highlighting opportunities to augment their statistically based approach with biological considerations.
For most carcinogens, the U.S. EPA develops cancer potency estimates as follows (U.S. EPA 2005). A POD associated with a benchmark response level (BMR) is derived and converted to human-equivalent units (incorporating information about cross-species dose scaling). The BMR is then divided by the human-equivalent POD to obtain a potency estimate, under the assumption that risks extrapolate linearly with doses below the BMR. For [@r2], the upper-bound extra risk estimate (UER~SNCD~) is the BMR associated with the SNCD, but we recom-mend expressing SNCDs in human equivalents before deriving potency estimates.
For nonlinear extrapolation resulting in a RfD (which the U.S. EPA uses for non-cancer effects and carcinogens with a threshold mode of action), [@r2] chose to linearly extrapolate to a 1/1,000 risk in the test animal, which they considered analogous to applying a 100-fold uncertainty factor to a BMDL~10~ (lower bound on the benchmark dose corresponding to 10% extra risk). Several aspects of this proposal merit further considera-tion. First, margins of exposure much larger than 100-fold would be typical for cancer. Furthermore, whereas linear extrapo-la-tion involves extrapolation in the same population to a smaller level of effect, the standard uncertainty factor approach involves extrapolation across populations at a fixed level of effect. The alternative we propose separately accounts for these biologically unrelated processes.
Motivating our proposal is the need highlighted by [@r2] to clearly separate statistical factors supporting the level of effect associated with the POD while also fully incorporating biological considerations. We propose specifying "target" effect levels (TELs) associated with different end points based on biological considerations, independent of data set. The TELs could then be compared with the lowest practical BMR for a given data set---the UER~SNCD~ used by Sand et al. The UER~SNCD~/TEL ratio is a diagnostic of the extent of extrapolation to the TEL. If UER~SNCD~ ≤ TEL, then the BMD at the TEL does not involve extrapo-la-tion and can serve as the POD. For a UER~SNCD~ \> TEL, the greater the ratio, the greater the uncertainty in the BMD at the TEL from extrapo-la-tion below the SNCD. In this case, the SNCD could serve as the POD, and the gap between the UER~SNCD~ and the TEL could be bridged by an additional factor (analo-gous to the LOAEL-to-NOAEL factor) or linear extrapolation. Then, inter-species, intra-species, and any other adjustments for deriving RfDs would be applied as usual. Thus, this approach separately takes into account biological considerations related to the severity of the end point (via the TEL), statistical considerations related to the study data (via the UER~SNCD~), and adjustments from the test species to sensitive humans (via uncertainty factors or chemical-specific adjustments).
In sum, the work of [@r2]advances the development of approaches for providing a transparent, objective method to demark where "extrapolation begins." However, for human health risk assessment, we propose augmenting statistically based approaches so that inter- and intra-species adjustments and biological considerations relating to the end points are explicitly addressed. Although consensus on specifying TELs may be challenging, particularly for precursor or toxico-genomic end points, clearly separating biological and statistical considerations will enhance the transparency and consistency of chemical assessments.
The views in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. EPA.
The authors declare they have no actual or potential competing financial interests.
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INTRODUCTION {#sec1-1}
============
Peyronie\'s disease (PD) is a relatively common condition affecting up to 9% of the male population.[@ref1][@ref2] The etiology of PD is not fully understood. Recent studies suggest that repeated trauma to the penis during sexual intercourse may induce localized inflammatory process and aberrant wound healing in tunica albuginea, which is responsible for the plaque development and subsequent penile curvature.[@ref3][@ref4] Despite intensive research into the pathogenesis of PD, the effective nonsurgical or medical treatment options for this entity are relatively lacking.[@ref5][@ref6] Randomized controlled trials revealed that intralesional injection of collagenase *Clostridium histolyticum* and interferon-alpha, or oral administration of pentoxifylline and acetyl-*L*-carnitine induced improvement in penile curvature in PD patients.[@ref7][@ref8][@ref9][@ref10][@ref11] Although an uncontrolled study of verapamil injection demonstrated efficacy in reducing penile curvature,[@ref12] a recent randomized placebo-controlled trial failed to show improvement in penile curvature.[@ref13]
Accumulating evidence suggests that the overexpression of profibrotic cytokines is involved in the pathogenesis of PD.[@ref14] Transforming growth factor-β1 (TGF-β1) is one of the most studied cytokines associated with PD. The expression and activity of TGF-β1 protein as well as their down-stream intracellular signaling mediators involved in tissue fibrosis, such as mothers against decapentaplegic homolog 2 (SMAD2) and SMAD3, were shown to have increased in human PD plaque tissue or fibroblasts derived from human PD plaques.[@ref15][@ref16][@ref17] These findings give us the rationale to use TGF-β blockade for the treatment of PD. We previously observed that inhibition of the TGF-β pathway using small-molecule inhibitor of activin receptor-like kinase 5, a TGF-β type I receptor, accelerated regression of fibrotic plaques in a rat model of PD *in vivo*[@ref18] and suppressed production of extracellular matrix proteins in primary cultured fibroblasts isolated from human PD plaque *in vitro*.[@ref17] Moreover, we observed in human PD fibroblasts that small interfering RNA-mediated silencing of histone deacetylase 2 quashed TGF-β1-induced profibrotic responses by blocking activation of SMAD2 and SMAD3.[@ref19] It was also reported in human PD fibroblasts that pentoxifylline attenuated TGF-β1-induced elastogenesis by enhancing phosphorylation of the inhibitory SMAD6.[@ref20]
Mothers against decapentaplegic homolog 7 (SMAD7) acts as a negative regulator of TGF-β signaling through multiple mechanisms, such as by blocking phosphorylation and recruitment of SMAD2/3 and by inducing degradation of the TGF-β type I receptor.[@ref21][@ref22][@ref23] SMAD7 has been demonstrated to protect TGF-β-induced fibrosis in a variety of organs, including kidney, lung, and liver.[@ref24][@ref25][@ref26] We recently reported that adenovirus-mediated gene transfer of SMAD7 successfully restored erectile function in mice with cavernous nerve injury through an anti-fibrotic effect.[@ref27]
In the present study, we determined the effectiveness of the transient overexpression of the SMAD7 gene on the TGF-β1-induced profibrotic responses in primary fibroblasts derived from human PD plaques.
MATERIALS AND METHODS {#sec1-2}
=====================
{#sec2-1}
### Primary fibroblast culture {#sec3-1}
Fibroblasts were isolated from human PD plaques as previously described.[@ref17][@ref19] Briefly, the plaque tissue was transferred into sterile vials containing Hank\'s balanced salt solution (GIBCO, Carlsbad, CA, USA) and was washed 3 times in phosphate-buffered saline (PBS). Biopsy tissue was minced into 1-mm^2^ segments and incubated in a shaker in 12.5 ml Dulbecco\'s modified Eagle Medium (DMEM) supplemented with 0.06% collagenase A (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. The cells and tissue fragments were collected by centrifugation (400 *g* for 5 min), washed in fresh culture medium, and placed in 100-mm cell culture dishes (Falcon-Becton Dickinson Labware, Franklin Lakes, NJ, USA) under standard cell culture conditions with DMEM supplemented with 10% fetal calf serum, penicillin (100 U ml^−1^), and streptomycin (100 μg ml^−1^). The dishes were incubated in a humidified 37°C incubator with 5% CO~2~. The cells were then characterized as previously described.[@ref17][@ref19] Passages five to eight were used for experimentation. We used four cell lines in this study.
### Transient transfection {#sec3-2}
pCMV5 inserted full-length human SMAD7 gene (GenBank ID: NM_005904) was purchased from Addgene (Cambridge, MA, USA). Transient transfections were performed by using hyperbranched poly (ethyleneimine) (25 kDa, PEI25k; Sigma-Aldrich, St. Louis, MO, USA) and a PEI25k/pCMV5-Smad7 polyplex was prepared at a weight ratio of 1. The fibroblasts were serum-starved for 24 h and transfected with a 1 μg SMAD7 expression vector. In parallel, an empty PEI25k/pCMV5 vector was used as a control. After transfection, cells were plated and cultured for 48 h in DMEM. The fibroblasts were then treated with 10 ng ml^−1^ TGF-β1 (RandD Systems Inc., Minneapolis, MN, USA) for 24 h to detect the protein expression of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen subtypes, smooth muscle α-actin, and HDAC2, or were treated with TGF-β1 for 1 h to detect the protein expression of phospho-Smad2 (P-Smad2), P-Smad3, total Smad2/3, poly (ADP-ribose) polymerase 1 (PARP-1), and cyclin D1 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays.
### Western blot {#sec3-3}
Equal amounts of protein from whole-cell extracts (50 μg per lane) were electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibody against PAI-1 (Abcam, Cambridge, UK; 1:400), fibronectin (Abcam; 1:400), collagen I (Abcam; 1:400), collagen IV (Abcam; 1:400), smooth muscle α-actin (Sigma-Aldrich; 1:400), P-Smad2 (s465/467, Cell signaling, Beverly, MA, USA; 1:400), P-Smad3 (s423/425, Cell signaling; 1:400), Smad2/3 (Cell signaling; 1:400), PARP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:400), cyclin D1 (Santa Cruz Biotechnology; 1:400), or β-actin (Abcam; 1:6000). Results were quantified by densitometry.
### Hydroxyproline assay {#sec3-4}
Collagen protein levels were estimated by hydroxyproline determinations as previously described.[@ref28] Briefly, aliquots of standard hydroxyproline or fibroblasts samples were hydrolyzed in alkali. The hydrolyzed samples were then mixed with a buffered chloramine-T reagent, and oxidation was allowed to proceed for 25 min at room temperature. The chromophore was then developed with the addition of Ehrlich\'s reagent, and the absorbance of the reddish-purple complex was measured at 550 nm using a spectrophotometer. Absorbance values were plotted against the concentration of standard hydroxyproline, and the presence of hydroxyproline in fibroblast lysates was determined from the standard curve.
### Fluorescent immunocytochemistry {#sec3-5}
The fibroblasts were cultured on sterile cover glasses (Marienfeld Laboratory, Lauda-Königshofen, Germany) and grown until nearly confluent. The cells were washed 3 times with PBS and then fixed in 4% paraformaldehyde for 10 min at 4°C and in 100% methanol for 10 min at 4°C. Individual chambers were incubated with an antibody to smooth muscle α-actin (Sigma-Aldrich; 1:300) or Smad2/3 (Cell signaling; 1:200) overnight at 4°C in a moist chamber. After several washes with PBS, the chambers were incubated with fluorescein isothiocyanate-conjugated (Zymed Laboratories, South San Francisco, CA, USA; 1:300) or tetramethylrhodamine isothiocyanate-conjugated (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; 1:300) secondary antibodies for 2 h at room temperature. Mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc., Burlingame, CA, USA) was applied to the chamber and nuclei were labeled. Signals were visualized, and digital images were obtained with a confocal microscope (FV1000, Olympus, Tokyo, Japan) under identical exposure settings. Average fluorescent intensity was measured for every nucleus in the field and averaged for the field with an image analyzer system (National Institutes of Health Image J 1.34; Internet: <http://rsbweb.nih.gov/ij/>).
### Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay {#sec3-6}
The TUNEL assay was performed using an ApopTagR Fluorescein *In Situ* Apoptosis Detection Kit (S7160, Chemicon, Temecula, CA, USA). Nuclei were labeled with DAPI (Vector Laboratories Inc., Burlingame, CA, USA).
### Statistical analysis {#sec3-7}
Results are expressed as mean ± standard error (s.e.) The group comparisons of parametric data were made by a one-way analysis of variance followed by Newman--Keuls *post hoc* tests. We used the Kruskal--Wallis tests for nonparametric data. We performed statistical analysis with SigmaStat 3.5 software (Systat Software Inc., Richmond, CA, USA). We tested data for normality and variance. *P* \< 0.05 were considered significant.
RESULTS {#sec1-3}
=======
{#sec2-2}
### SMAD7 overexpression inhibits extracellular matrix production induced by transforming growth factor-β1 in fibroblasts derived from human Peyronie\'s disease plaques {#sec3-8}
Peyronie\'s disease fibroblasts were transiently transfected with PEI25k/pCMV5-Smad7. Western blot analysis revealed that the treatment of PD fibroblasts with TGF-β1 induced protein expression of extracellular matrix, including PAI-1, fibronectin, collagen I, and collagen IV (*P* \< 0.05 by Kruskal-Wallis tests). The SMAD7 gene profoundly inhibited TGF-β1-induced production of collagen I and collagen IV in PD fibroblasts (*P* \< 0.05 by Kruskal-Wallis tests). Although the SMAD7 gene also decreased production of PAI-1 and fibronectin, it did not yield statistical significance (**[Figure 1](#F1){ref-type="fig"}**).
{#F1}
We also determined total collagen content by measuring the amount of hydroxyproline. The SMAD7 gene reduced TGF-β1-induced collagen production in PD fibroblasts (*P* \< 0.001 by ANOVA), which was comparable to the level found in the untreated control (**[Figure 2](#F2){ref-type="fig"}**).
{#F2}
### SMAD7 overexpression inhibits transforming growth factor-0β1-induced myofibroblastic differentiation in fibroblasts derived from human Peyronie\'s disease plaques {#sec3-9}
The expression of smooth muscle α-actin, a marker for myofibroblasts, was determined with a Western blot analysis. The treatment of PD fibroblasts with TGF-β1 resulted in an increase in smooth muscle α-actin expression (*P* \< 0.05 by ANOVA), which was substantially reduced after treatment with the SMAD7 gene (*P* \< 0.05 by ANOVA; Figure **[3a](#F3){ref-type="fig"} and [3b](#F3){ref-type="fig"}**). Fluorescent immunocytochemistry revealed that the SMAD7 gene inhibited TGF-β1-stimulated α-actin fiber formation (**[Figure 3c](#F3){ref-type="fig"}**).
{#F3}
### SMAD7 overexpression induces apoptosis and blocks cell cycle entry in fibroblasts derived from human Peyronie\'s disease plaques {#sec3-10}
A TUNEL assay showed that the SMAD7 gene profoundly induced apoptosis in PD fibroblasts both in basal and TGF-β1-stimulated conditions (*P* \< 0.01 by ANOVA; **Figure [4a](#F4){ref-type="fig"} and [4b](#F4){ref-type="fig"}**). A Western blot analysis revealed that the treatment of PD fibroblasts with SMAD7 gene decreased expression of cyclin D1, a positive cell cycle regulator, both in basal and TGF-β1-stimulated conditions (*P* \< 0.001 for basal condition and *P* \< 0.01 for stimulated condition by ANOVA; **Figure [4c](#F4){ref-type="fig"} and [4d](#F4){ref-type="fig"}**).
{#F4}
### SMAD7 overexpression inhibits phosphorylation, and nuclear translocation of decapentaplegic homolog 2/3 induced by transforming growth factor-β1 in fibroblasts derived from human Peyronie\'s disease plaques {#sec3-11}
The treatment of PD fibroblasts with TGF-β1 resulted in an increase in phosphorylation of Smad3, which was substantially inhibited by preincubation with the SMAD7 gene (*P* \< 0.05 by ANOVA; **Figure [5a](#F5){ref-type="fig"} and [5b](#F5){ref-type="fig"}**). Although the SMAD7 gene also decreased TGF-β1-stimulated SMAD2 phosphorylation, it did not yield statistical significance (**Figure [5a](#F5){ref-type="fig"} and [5b](#F5){ref-type="fig"}**). In order to evaluate whether SMAD7 is involved for TGF-β1-induced nuclear shuttling of Smad2/3, we performed fluorescent immunocytochemistry with antibody against Smad2/3. The SMAD7 gene reduced TGF-β1-induced nuclear accumulation of the Smad proteins (*P* \< 0.001 by Kruskal-Wallis tests; **Figure [5c](#F5){ref-type="fig"} and [5d](#F5){ref-type="fig"}**).
{#F5}
### SMAD7 overexpression increases poly (ADP-ribose) polymerase 1 expression in fibroblasts derived from human Peyronie\'s disease plaques {#sec3-12}
A Western blot analysis showed that the SMAD7 gene increased the expression of PARP-1, which is known to terminate Smad-mediated transcription, in PD fibroblasts both in basal and TGF-β1-stimulated conditions (*P* \< 0.05 by Kruskal--Wallis tests; [**Figure 6**](#F6){ref-type="fig"}).
{#F6}
DISCUSSION {#sec1-4}
==========
We showed here that the overexpression of the SMAD7 gene decreased the TGF-β1-induced accumulation of extracellular matrix and production of hydroxyproline in human PD fibroblasts by inhibiting TGF-β1-induced myofibroblastic differentiation and by accelerating apoptosis and blocking cell cycle entry. Furthermore, SMAD7 blocked TGF-β signaling through inhibition of the phosphorylation and nuclear shuttling of SMAD3 and/or SMAD2, and up-regulation of PARP-1 that is known to terminate Smad-mediated transcription.
It was first reported in mice that overexpression of SMAD7 prevented bleomycin-induced lung fibrosis.[@ref25] In the present study, SMAD7 overexpression inhibited TGF-β1-induced production of collagen I and collagen IV as well as hydroxyproline in PD fibroblasts *in vitro*. This finding is similar to the results from ours showing that adenovirus-mediated SMAD7 gene transfer into the penis significantly decreased the production of extracellular matrix proteins in mice with cavernous nerve injury *in vivo*.[@ref27] In a tetrachloride-induced liver fibrosis model, overexpression of SMAD7 attenuated a fibrogenic response,[@ref26] whereas down-regulation of SMAD7 accelerated liver fibrosis.[@ref29] These findings suggest a protective role of SMAD7 in suppressing TGF-β-mediated fibrosis in a variety of organs.
Transforming growth factor-β-mediated fibrotic responses are initiated by activation of SMAD2 and SMAD3 upon ligand binding, which in turn induces myofibroblastic differentiation and promotes the synthesis of extracellular matrix proteins.[@ref30] In the present study, overexpression of SMAD7 blocked TGF-β1-induced SMAD3 phosphorylation and the nuclear accumulation of Smad2/3 proteins. Moreover, both Western blot analysis and fluorescent immunocytochemistry revealed that SMAD7 overexpression quashed TGF-β1-induced myofibroblastic differentiation. In the agreement with this finding, overexpression of SMAD7 is known to inhibit SMAD2/3 phosphorylation and protect against activation of hepatic stellate cells into myofibroblasts,[@ref31] whereas repression of SMAD7 promotes SMAD2/3 phosphorylation and activates hepatic stellate cells.[@ref32] It was also reported that SMAD7 gene transfer substantially inhibited SMAD2/3 activation and attenuated myofibroblast accumulation in a rat model of renal fibrosis.[@ref33] These findings suggest that the inhibition of SMAD2/3 activity is the main mechanism responsible for SMAD7-mediated myofibroblastic differentiation and subsequent accumulation of extracellular matrix proteins.
Despite the relatively well-known effect of SMAD7 on transdifferentiation of fibroblasts to myofibroblasts, little data are as yet available in regard to its effect on apoptosis of fibroblasts. In the present study, treatment of PD fibroblast with SMAD7 gene induced apoptosis of fibroblasts in both basal and TGF-β1-stimulated conditions. Mesangial cell proliferation is a key feature of glomerular scarring and end-stage renal disease, and overexpression of SMAD7 is known to increase caspase-3 activity and promote mesangial cell apoptosis.[@ref34] SMAD7 is reported to activate caspase-8 and to induce apoptosis in human breast cancer cell culture *in vitro* and in xenograft model *in vivo*.[@ref35] In the present study, the SMAD7 gene also decreased expression of cyclin D1, a positive cell cycle regulator, suggesting SMAD7 can regulate fibroblast proliferation by modulating cell cycle.
Another mechanism by which overexpression of SMAD7 attenuates fibrotic responses in PD fibroblasts may be attributable to increase in expression of PARP-1. It was reported that PARP-1 mediates poly (ADP-ribosyl) ation (PARylation) and regulates transcription factors.[@ref36] PARP-1 is known to dissociate SMAD complexes from DNA through PARylation of SMAD3 and SMAD4, which terminates Smad-mediated transcription.[@ref37] Additional studies are necessary to document the exact functional roles of PARP-1 in the pathogenesis of PD.
In the present study, we for the first time documented that inhibition of the TGF-β pathway using the SMAD7 gene has an anti-fibrotic effect in human PD fibroblasts *in vitro*, which warrants further studies in PD animal models *in vivo*.
In summary, overexpression of the SMAD7 gene attenuated TGF-β1-induced extracellular matrix production and transdifferentiation of fibroblasts into myofibroblasts by blocking activation of SMAD2/3 pathway and possibly by inhibiting Smad-mediated transcription through an up-regulation of PARP-1. Inhibition of the TGF-β pathway by use of SMAD7 may be a viable therapeutic strategy for the treatment of PD.
AUTHOR CONTRIBUTIONS {#sec1-5}
====================
SHP, DSR, JKR, and JKS participated in the design of the study. MJC and KMS carried out Western blot analysis. JMP carried out hydroxyproline assay. MHK carried out immunocytochemistry. KDK carried out TUNEL assay. MJC performed statistical analysis. SHP and DSR conceived of the study. MJC and JKR drafted manuscript. JKS critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.
COMPETING INTERESTS {#sec1-6}
===================
The authors declare no competing interests.
This study was supported by grant NO. 2011-0015771 (Ji-Kan Ryu) from the Korea Science and Engineering Foundation funded by the Korea government (Ministry for Education, Science, and Technology).
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative syndrome characterised by adult-onset progressive loss of motor neurons with a focal onset of progressive paresis and muscle wasting.[@R1]
Some 5%--10% of patients with ALS self-report a familial predisposition (fALS), and the remaining cases are denoted as sporadic ALS (sALS). Since 1993, mutations in 37 genes have been reported to predispose to ALS. In Caucasians, the most commonly reported mutation is a GGGGCC repeat-expansion in *C9orf72* (*C9orf72HRE*), found in 8%--10% of patients with ALS. Missense, substitution or deletion mutations in the genes encoding superoxide dismutase-1 (*SOD1*), TAR-DNA-binding protein 43 (*TDP-43*), fused in sarcoma (*FUS*) and kinesin heavy chain isoform 5A (KIF5A) are found in 2%--6% of European patients. The remaining gene mutations are rare. ALS is both genetically and clinically heterogeneous and the reported predisposing genes pleiotropic.[@R4] Neuropsychological examinations and imaging studies can show involvement of the frontotemporal lobes,[@R5] though this may remain subclinical.[@R7] In particular, carriers of *C9orf72HRE, FUS, VCP* and *TBK1* mutations may develop frontotemporal dementia (FTD) sometimes without overt ALS. Unfortunately, there have been few comparative neuropathological examinations in patients with different gene defects.
The first ALS-causing gene discovered was *SOD1* [@R8] and since 1993 more than 210 mutations have been found (<http://alsod.iop.kcl.ac.uk/>).[@R9] While some of the mutant SOD1s (eg, C6S, D90A, E100K, L117V) are sufficiently stable and active to yield normal SOD1 activity in human tissues,[@R10] others, for example, A4V, V5M, D76Y, D83G, G127Gfs\*5 (alias 'G127X') are unstable, are rapidly targeted for degradation and can only be detected in minute amounts.[@R11]
Neuronal inclusions containing aggregated SOD1 are recognised pathological hallmarks of ALS caused by *SOD1* mutations in patients and in transgenic (Tg) animal models overexpressing mutant human (h) SOD1s.[@R11] In the Tg models, two structurally different strains have been found.[@R14] When inoculated into the lumbar spinal cord of adult mice, both strains induce cell-to-cell propagating templated hSOD1 aggregation primarily in the motor neurons resulting in rapidly progressing ALS-like fatal diseases.[@R15] Similar effects have been obtained inoculating SOD1-aggregates purified from the spinal cord of a patient with the *SOD1^G127X^* mutation.[@R16] This prion-like action could be the primary pathogenic mechanism of SOD1-provoked ALS, which prompts exploration of SOD1 in ALS caused by other genes.
Previously, we examined spinal cord tissue from 37 patients with sALS and found numerous inclusions containing misfolded wild-type SOD1 (misSOD1^WT^) in the cytoplasm and nuclei of neurons and glial cells.[@R17] The presence of misSOD1^WT^ in motor neurons of patients with ALS was later observed by other groups with the differences that antibodies against other SOD1-epitopes were used and inclusions were not found in all patients studied.[@R19] Supporting that misSOD1^WT^ may cause neurodegeneration, expression of hSOD1^WT^ in Tg mice result in a fatal ALS-like disease with neuronal inclusions containing human misSOD1 aggregates.[@R23] Also, in vitro studies in which motor neurons are cocultured with astrocytes derived from patients with ALS suggests an involvement of misSOD1^WT^ in the pathogenesis.[@R24] Collectively, there is emerging evidence that misSOD1^WT^ is neurotoxic and participate in ALS pathogenesis in general and not only in patients carrying mutations in *SOD1*.
In the present study, we investigated whether inclusions containing misSOD1^WT^ are present in patients carrying mutations in six other ALS-causing genes.
Materials and methods {#s2}
=====================
Human subjects {#s2-1}
--------------
Since 1993, tissue has been collected at autopsy at Umeå University Hospital, Sweden from patients with ALS and FTD. The EFNS Criteria for Managing ALS[@R25] and the Neary Criteria for FTD[@R26] were used to set the diagnosis. Blood leucocyte DNA from autopsied patients (n=97) was analysed for a panel of ALS-linked genes.[@R12] Of these patients, 18 had *C9orf72HRE* (confirmed by Southern blot in CNS tissue),[@R29] 2 had *FUS* mutations, 1 an *ALSIN* mutation (censored, manuscript in preparation), 1 a *VAPB* mutation, 1 a *NEK1* mutation[@R6] and 11 had *SOD1* mutations. Eleven patients carried the coding rs113247976 SNP (P986L) in *KIF5A* recently found to predispose to ALS, three of these being double mutants *C9orf72HRE* plus rs113247976 and one A4V *SOD1* plus rs113247976 SNP.[@R28]
Six additional patients with *SOD1* mutations were obtained from Danish and Swiss pathology units. Similar tissues were obtained from 10 control patients with other neurodegenerative conditions and from 20 patients without neurological diseases ([table 1](#T1){ref-type="table"}, [online supplementary table 1](#SP1){ref-type="supplementary-material"}).
10.1136/jnnp-2018-319386.supp1
######
Summary of clinical features of the 46 patients with ALS/FTD
Case Mutated gene Diagnosis a.o. First symptom d.d. c.i. Sex p.m.
------ ------------------ ----------- ------ --------------- ------ -------- ----- ------
1 C9orf72HRE ALS 64 Hand 25 No M 1.5
2 C9orf72HRE fFTD 60 Disinhibition 168 Yes M 1
3 C9orf72HRE fALS 59 Hand 36 No M 4
4 C9orf72HRE fALS-FTD 51 Disinhibition 43 Yes M 2.5
5 C9orf72HRE sPBP 69 Bulbar 21 No M 3
6 C9orf72HRE ALS 63 Leg 32 No M 1.5
7 C9orf72HRE fALS 58 Leg 18 No M 3
8 C9orf72HRE fALS 49 Arm 36 No M 1
9 C9orf72HRE fALS 51 Arm 130 No M 2
10 C9orf72HRE fALS 66 Arm 29 No M 2
11 C9orf72HRE PBP+FTD 76 Bulbar 60 Yes F 1.5
12 C9orf72HRE fALS 53 Leg 28 No F 0.5
13 C9orf72HRE sPBP 72 Bulbar 35 Yes F 1
14 C9orf72HRE fALS +FTD 67 Leg 35 Yes M 1.5
15 C9orf72HRE fALS 54 Arm 12 No F 2
16 C9orf72HRE+KIF5A fFTD 60 Disinhibition 133 Yes M 3
17 C9orf72HRE+KIF5A fALS 63 Leg 30 No F 2
18 C9orf72HRE+KIF5A ALS 44 Leg 65 n.a. M 0.5
19 KIF5A ALS 61 Arm 13 No F 1.5
20 KIF5A homozygous sALS 61 Leg 61 No F 1
21 KIF5A PBP 67 Bulbar 27 No F 1
22 KIF5A ALS 67 Thoracic 17 No M 1
23 KIF5A PMA 68 Arm 63 n.a. F 1.5
24 KIF5A PBP 65 Bulbar 13 No M 1.5
25 NEK1 R812X fALS 75 Hand 38 Slight F 1.1
26 VAPB S160delS sPBP 55 Bulbar 43 Yes M 0.5
27 FUS R495X fPBP 16 Bulbar 10 No F 4
28 FUS Q23L sPBP 35 Bulbar 175 Yes M 2.5
29 ALSIN+KIF5A sALS 50 Legs 224 Yes M 2
30 SOD1 V5M fALS 71 Leg 40 No M 2.2
31 SOD1 A4V+KIF5A fALS 73 Arm 10 Yes F 1.9
32 SOD1 G127X fALS 72 Thoracic 24 No M 1.1
33 SOD1 G127X fALS 53 Thoracic 6 No M 1.5
34 SOD1 G127X fALS 59 Arm 32 No F n.a.
35 SOD1 G114A fALS 38 Arm 36 No M 1.5
36 SOD1 G114A fALS 29 Leg 14 Yes F 4
37 SOD1 D76Y fALS 49 Leg 204 No M n.a.
38 SOD1 D83G fALS 49 Leg 37 No F 2
39 SOD1 D101G fPMA 48 Arm 6 n.a. M n.a.
40 SOD1 L144F fALS 71 Hand 65 No M 0.2
41 SOD1 D90A fALS 31 Leg 265 No M 2
42 SOD1 D90A fALS 58 Leg 101 No M 2
43 SOD1 D90A fALS 48 Leg 316 No M 1.5
44 SOD1 D90A fALS 53 Leg 131 No F 2.5
45 SOD1 D90A fALS 56 Leg 174 No M 1.5
46 SOD1 D90A fALS 38 Leg 60 No F 2
Cases nr 20, 29 and 41---46 were homozygous, all othersheterozygous for the respective genes.
ALS, amyotrophic lateral sclerosis; a.o, age at onset (years); c.i., cognitive impairment; d.d., disease duration (months); fFTD, familial frontotemporal dementia; F/M, female/male; p.m, postmortem time (days).
All procedures were performed in accordance with the 1964-Helsinki Declaration with amendments and were approved by the Ethical Review Boards for Medical Research at the relevant institutions in Sweden, Denmark and Switzerland. Informed consent for performing autopsies for research purposes was obtained from the patients and/or the next of kin.
Tissue preparation and staining for misSOD1 {#s2-2}
-------------------------------------------
Tissue samples from the spinal cord (cervical, thoracic and lumbar regions), medulla oblongata, motor cortex, frontal lobe and cerebellum were collected and fixed by immersion in 4% paraformaldehyde in 0.1 M Na phosphate buffer, pH 7.4. After fixation, tissues were embedded in paraffin, sectioned (4 µm), and stained with H&E. The antigen retrieval procedure is essential for detecting misSOD1 and should be mild, since heavily denaturing protocols may cause misfolding of the abundant natively folded SOD1. A well-functional retrieval treatment for sections is 10 min in preheated citrate buffer, pH 6.3, without boiling.[@R18]
Immunohistochemical staining (IHC) was initially performed using the ES system and ES reagents and, from 2014, by the more sensitive BenchMark XT automated staining system (both from Ventana Medical System, Illkirch, France) and aminoethylcarbazole or Fast Red was used for development. For immunofluorescent studies, a corresponding fluorescent-labelled secondary antibody was used. Sudan Black diluted to 0.3% was used to avoid autofluorescence. The anti-SOD1 antibodies were diluted to a final concentration of 0.962 µg/mL for 131--153 Ra-ab, 3.7 µg/mL for 57--72 Ra-ab and 1.612 µg/mL for G127X Ra-ab. The sections were examined using a BX53 light microscope (Olympus) and by confocal laser microscopy using a Zeiss LSM 710 confocal microscope and were analysed using the Zen 2011 SP7 software. The presence of intracellular misSOD1 inclusions was rated by two independent examiners according to a 4-point semiquantitative scale.[@R18]
Results {#s3}
=======
Antibodies specifically detect misfolded SOD1 {#s3-1}
---------------------------------------------
SOD1 is abundant in the CNS constituting 0.1% of the tissue protein.[@R11] Carriers of *SOD1* mutations show inclusions containing misfolded aggregated SOD1. It is estimated that ≈1% of the total SOD1 protein is contained in such inclusions.[@R11] Any misSOD1 inclusions occurring in patients with ALS who lack mutations in *SOD1* are likely to be equally scarce. Thus, to detect such species, antibodies that lack reactivity with natively folded SOD1 must be used. Use of Rabbit 1, a Ra-ab raised against native SOD1 with native/denatured reactivity ratio of 0.7/1,[@R31] is found to stain tissues heavily obscuring detection of inclusions containing misSOD1 ([online supplementary figure 1C-D](#SP2 SP3){ref-type="supplementary-material"}). Moreover, the handling of tissue specimen and antigen retrievals have to be lenient to prevent misfolding of native SOD1. We have developed a panel of antibodies, raised against peptides in the human SOD1^WT^ sequence, which only react with disordered or misSOD1 species but show no reactivity against natively folded human SOD1. They were highly specific for SOD1 in western blots of human CNS tissue extracts.[@R17] The two primary antibodies used here were rabbit anti-human antibodies (Ra-ab) to misfolded SOD1, raised against peptides corresponding to amino acids (aa) 57--72 and aa131-153. The 57--72 Ra-ab and 131--153 Ra-ab were chosen because they are well characterised[@R17] and detect misfolded SOD1 in human tissue studies equally well. For detection of mutated SOD1 in patients with the *SOD1* truncating mutation SOD1^G127X^, we developed a mutant-specific peptide antibody, G127X Ra-ab, directed against the neopeptide sequence unique to this mutation (aa 123-127GQRWK-stop). This antibody reacts exclusively with the mutant protein.[@R11] To test the specificity for misSOD1 in histopathological studies, the 131--153 Ra-ab was preincubated with the immunising peptide in increasing concentrations. This resulted in gradual and finally full disappearance of immunoreactivity ([online supplementary figure 1E-H](#SP2 SP3){ref-type="supplementary-material"}).
{#F1}
### Inclusions containing misSOD1^WT^ can be seen in patients carrying *C9orf72HRE* {#s3-1-1}
Different regions of the brain and spinal cord from 18 patients carrying *C9orf72HRE* were stained for misSOD1 and analysed. We found small dot-like granular inclusions of misSOD1^WT^ in spinal cord motor neurons in all 18 patients ([tables 2 and 3](#T2 T3){ref-type="table"}), irrespective of whether the clinical diagnosis was ALS, FTD or both. The inclusions measured 0.5--3 µm were scattered in the cytoplasm of motor neurons and were particularly abundant in the soma ([figure 1A,B,D](#F1){ref-type="fig"} and [figure 2F--H](#F2){ref-type="fig"}) but also present in axons ([figure 1A,B](#F1){ref-type="fig"}). The proportion of spinal motor neurons carrying misSOD1^WT^ inclusions varied between patients; usually, only a few motor neurons per section carried misSOD1^WT^ inclusions. However, in some patients, the proportion was higher. Inclusions were found at all levels of the spinal cord (cervical, thoracic and lumbar) in most patients ([table 3](#T3){ref-type="table"}). MisSOD1^WT^ inclusions were also present in the neurons of Clark's nuclei. Furthermore, small inclusions of misSOD1^WT^ were found in the hypoglossus ([figure 3D](#F3){ref-type="fig"}), vagus, facialis and accessorius nuclei. The inferior olivary nucleus did not show misSOD1^WT^ inclusions and was normal in most patients.
######
Immunohistochemical results of staining for misfolded SOD1 using the misSOD1 peptide antibodies: 131--153 Ra-ab (cases \#1--31, \#35--56) and G127X Ra-ab (cases \#32--34)
Case Mutated gene Spinal cord Medulla oblongata Motor cortex Frontal lobe Cerebellum
------ ---------------- ------------- ------------------- -------------- -------------- ------------ ------ ------ ------ ------ ------
1 C9orf72HRE ++ ++ ++ ++ ++ +++ \+ +++ ++ ++
2 C9orf72HRE ++ ++ -- \+ n.a. n.a. -- \+ -- \+
3 C9orf72HRE ++ ++ -- -- n.a. n.a. -- -- -- --
4 C9orf72HRE ++ +++ +++ +++ \+ +++ \+ ++ \+ ++
5 C9orf72HRE +++ +++ +++ +++ -- +++ ++ +++ \+ +++
6 C9orf72HRE ++ +++ \+ +++ n.a. n.a. n.a. n.a. n.a. n.a.
7 C9orf72HRE ++ ++ ++ ++ -- ++ \+ ++ \+ ++
8 C9orf72HRE ++ ++ ++ ++ n.a. n.a. \+ ++ n.a. n.a.
9 C9orf72HRE ++ ++ n.a. n.a. n.a. n.a. n.a. n.a. -- \+
10 C9orf72HRE ++ +++ n.a. n.a. \+ ++ ++ ++ n.a. n.a.
11 C9orf72HRE +++ ++ n.a. n.a. ++ ++ ++ ++ n.a. n.a.
12 C9orf72HRE ++ ++ n.a. n.a. -- ++ \+ ++ n.a. n.a.
13 C9orf72HRE ++ ++ ++ ++ -- ++ -- ++ -- ++
14 C9orf72HRE \+ \+ \+ ++ \+ ++ -- ++ -- ++
15 C9orf72HRE \+ ++ \+ ++ \+ ++ \+ ++ -- ++
16 C9orf72+KIF5A +++ +++ +++ +++ ++ +++ ++ +++ \+ +++
17 C9orf72+KIF5A ++ ++ n.a. n.a. n.a. n.a. \+ \+ \+ +++
18 C9orf72+KIF5A ++ ++ n.a. n.a. -- ++ ++ ++ n.a. n.a.
19 KIF5A \+ -- \+ -- -- -- -- -- -- --
20 KIF5A homozyg ++ ++ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
21 KIF5A \+ ++ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
22 KIF5A \+ ++ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
23 KIF5A \+ ++ -- ++ -- ++ -- ++ -- ++
24 KIF5A ++ ++ n.a. n.a. -- \+ -- \+ -- ++
25 NEK1 R812X ++ +++ +++ +++ \+ +++ \+ +++ n.a. n.a.
26 VAPB S160delS \+ +++ ++ +++ ++ +++ ++ ++ \+ +++
27 FUS R495X ++ +++ \+ ++ -- \+ -- \+ -- \+
28 FUS Q23L ++ ++ \+ \+ n.a. n.a. -- ++ -- ++
29 ALSIN+KIF5A ++ +++ ++ +++ ++ ++ ++ ++ \+ +++
30 SOD1 V5M ++ \+ n.a. n.a. -- \+ n.a. n.a. -- --
31 SOD1 A4V+KIF5A ++ ++ ++ ++ -- \+ -- \+ -- \+
32 SOD1 G127X \+ +++ ++ \+ ++ \+ -- \+ \+ \+
33 SOD1 G127X \+ \+ \+ \+ n.a. n.a. -- \+ -- --
34 SOD1 G127X \+ \+ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
35 SOD1 G114A \+ \+ n.a. n.a. n.a. n.a. -- --
36 SOD1 G114A ++ ++ \+ \+ \+ \+ \+ ++ -- \+
37 SOD1 D76Y ++ -- ++ -- \+ \+ \+ -- \+ --
38 SOD1 D83G \+ -- n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
39 SOD1 D101G ++ -- n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
40 SOD1 L144F \+ ++ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
41 SOD1 D90A ++ +++ ++ ++ \+ +++ -- \+ -- \+
42 SOD1 D90A ++ \+ -- \+ -- \+ -- \+ -- \+
43 SOD1 D90A ++ ++ ++ ++ -- \+ -- \+ -- --
44 SOD1 D90A ++ +++ \+ ++ -- ++ -- ++ \+ ++
45 SOD1 D90A ++ ++ ++ ++ \+ ++ n.a. n.a. -- \+
46 SOD1 D90A ++ ++ n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
47 N.D. Control -- -- -- -- -- -- -- -- -- --
48 N.D. Control \+ -- -- -- -- -- -- -- -- --
49 N.D. Control \+ -- -- -- -- -- -- -- -- --
50 N.D. Control \+ ++ \+ ++ \+ \+ -- \+ -- ++
51 N.D. Control -- -- -- -- -- -- -- -- -- --
52 N.D. Control \+ -- -- -- n.a. n.a. n.a. n.a. n.a. n.a.
53 N.D. Control -- -- -- -- n.a. n.a. -- -- -- --
54 N.D. Control -- -- -- -- n.a. n.a. n.a. n.a. n.a. n.a.
55 N.D. Control -- -- -- -- n.a. n.a. n.a. n.a. n.a. n.a.
56 N.D. Control -- -- -- -- n.a. n.a. n.a. n.a. n.a. n.a.
Rating for misSOD1 inclusions: -- no inclusion, + \<25% of neurons had inclusions, ++ 25%--75% of neurons had inclusions, +++ \>75% of neurons had inclusions.
GI, glial inclusion; n.a., not available; NCI, neuronal inclusions; N.D. Control, neurodegenerative control.
######
MisSOD1 immunohistochemical results
A Number of patients with misSOD1 inclusions in spinal cord motor neurons
---------------------- ------------------------------------------------------------------------- --------- --------- -------- --------
Cervical spinal cord 17 (17) 17 (18) 11 (11) 1 (10) 0 (14)
Thoracic spinal cord 16 (16) 15 (15) 11 (11) 1 (3) 0 (18)
Lumbar spinal cord 16 (16) 15 (16) 11 (11) 4 (10) 2 (19)
B Proportion of motor neurons with misSOD1 staining
---------------------- --------------------------------------------------- --------------- -------------- ------------- ------------
Cervical spinal cord \+ (+ -- ++) ++ (0 -- +++) ++ (+ -- ++) 0 (0 -- ++) 0 (0)
Thoracic spinal cord \+ (+ -- ++) ++ (+ -- +++) ++ (+ -- ++) 0, 0, ++\* 0 (0)
Lumbar spinal cord \+ (+ -- ++) ++ (0 -- +++) ++ (+ -- ++) 0 (0 -- +) 0 (0 -- +)
Table of findings from sections stained with the131--153 Ra-ab and 57--72 Ra-ab. Data for number of patients with misSOD1^WT^ inclusions show the total number of patients in parenthesis. Data for proportion of misSOD1^WT^ inclusions with staining are shown as median (range) referring to a four-tiered semiquantitative scale (0=no glial or motor neuron with staining; + ≤25% of the motor neurons with staining; ++=25%--75% of the motor neurons with staining; +++ ≥75% of the motor neurons with staining). The total number of patients in each group was as follows: 17 *SOD1*, 18 *C9orf72HRE*, 11 other ALS mutations (2 with *FUS*, 1 *ALSIN+KIF5* *A*, 1 *VAPB*, 1 *NEK1*, 6 *KIF5A*); 10 Controls neurodegenerative, 20 Controls non-neurological. Sections from all levels were not available from all patients. In total, of the 20 non-neurological controls, only two had some staining. \*Since thoracic spinal cord only was available from three patients from the neurodegenerative control group each result is presented individually.
ALS, amyotrophic lateral sclerosis; fALS, familial ALS.
{#F2}
{#F3}
In the motor cortex, degenerated neurons containing numerous small misSOD1^WT^ inclusions were detected in 7 of 12 *C9orf72HRE* patients investigated, similar in size to the ones seen in spinal motor neurons ([figure 3B,C](#F3){ref-type="fig"} and [table 2](#T2){ref-type="table"}) and located both in the cytoplasm and the nucleus. The proportion of motor cortex neurons that carried misSOD1^WT^ inclusions was much smaller than the proportion of spinal motor neurons. In the frontal cortex, 11 out of 16 patients with *C9orf72HRE* carried misSOD1^WT^ inclusions. Noticeably, in some patients, senile plaques were seen in the motor and frontal cortex, these stained heavily for misSOD1^WT^.
In the cerebellum of all 12 patients investigated, loss of Purkinje cells and degeneration of white matter was observed. Staining with anti-misSOD1 antibodies revealed misSOD1^WT^-stained glial cells surrounding either degenerated Purkinje cells or empty baskets. Staining was also found in the neuropil in both grey and white matter as well as in glial cells in the same regions ([online supplementary figure 1A, B](#SP2 SP3){ref-type="supplementary-material"}). In the granular and molecular layers of the cerebellum, p62-positive neuronal cytoplasmic inclusions were observed, a finding proposed to be pathognomonic for this subgroup of ALS.[@R32] No misSOD1^WT^ staining was seen in the granular layer in the patients with *C9orf72HRE*.
Two of the patients with *C9orf72HRE* were siblings, one with FTD and the other with ALS (patients 2 and 3, respectively, [table 2](#T2){ref-type="table"}). The sibling with ALS had a significant loss of spinal motor neurons, whereas the sibling with FTD appeared to have a normal number of spinal motor neurons. However, both had misSOD1^WT^ inclusions of similar morphology in motor neurons at all levels of the spinal cord. No misSOD1^WT^ inclusions were detected in the medulla oblongata. Comparing their frontal cortices, the sibling with FTD had many extracellular senile plaques which stained heavily for misSOD1^WT^ and amyloid-β. This was not seen to the same extent in the ALS sibling. In both, misSOD1^WT^ was rarely observed in cortical glial cell nuclei ([table 2](#T2){ref-type="table"}).
### Patients with *FUS, NEK1, KIF5A, VAPB* and *ALSIN* mutations {#s3-1-2}
We found granular inclusions of misSOD1^WT^ in motor neurons of the spinal cord in all patients with *FUS, NEK1, KIF5A, VAPB* or *ALSIN* mutations ([figures 1C,E--G and 2I](#F1 F2){ref-type="fig"} and [table 2](#T2){ref-type="table"}). The misSOD1^WT^-positive inclusions were morphologically similar to the ones observed in patients with *C9orf72HRE* and in patients with apparently sporadic ALS.[@R17] Moreover, misSOD1^WT^ pathology in the patients with *FUS, NEK1, KIF5A, VAPB* or *ALSIN* mutations included all levels of the spinal cord and brainstem motor nuclei ([figure 3E,G](#F3){ref-type="fig"}), areas of the motor cortex ([figure 3A](#F3){ref-type="fig"}), frontal lobe and the cerebellum. In the patients with *FUS* mutations, large cytoplasmic FUS-positive inclusions were found in the spinal cord and medulla oblongata when stained with an anti-FUS antibody. In the cerebellum, the same type of histopathology was observed as in the patients with *C9orf72HRE*, which included staining of misSOD1^WT^ in glial cells of the molecular and granular layers surrounding degenerated Purkinje cells. The patients with *VAPB* and *ALSIN* mutations in particular had these changes, whereas the cerebellum in the patients with *FUS* mutations was less affected. We conclude that all studied patients with *FUS, NEK1, KIF5A, VAPB* and *ALSIN* mutations had misSOD1^WT^ inclusions in the spinal cord and brainstem motor nuclei and occasionally also in the motor cortex and frontal lobe.
### Comparison with patients carrying different *SOD1* mutations {#s3-1-3}
For comparison, we stained tissue from 17 patients carrying *SOD1* mutations ([tables 2 and 3](#T2 T3){ref-type="table"}). The six patients homozygous for the *SOD1^D90A^* mutation had small granular inclusions ([figure 1l](#F1){ref-type="fig"}) morphologically similar to the inclusions described above in patients carrying mutations in *C9orf72HRE*, *FUS, NEK1, KIF5A, ALSIN* and *VAPB* and previously in sALS.[@R17] This could be expected since the *SOD1^D90A^* mutation produces an essentially fully stable SOD1^D90A^ protein with biophysical properties of SOD1^WT^.[@R33] All patients with the *SOD1^D90A^* mutations had mild cortical atrophy with neuronal loss, reactive gliosis and microvacuolisation of the three superficial cortical laminae. MisSOD1^D90A^ inclusions in the motor cortex and frontal lobe were rarely observed in these patients. In contrast, staining for misSOD1 in patients with ALS heterozygous for the mutations A4V, V5M, D76Y, D83G, D101G, G114A, L144F and G127X - all encoding unstable SOD1 mutants[@R11] revealed larger skein-like and Lewy body-like inclusions in addition to smaller granular misSOD1 inclusions ([figures 1J,K--3H,I](#F1 F2 F3){ref-type="fig"}). Thus, there are (at least) two different morphologies of misSOD1 inclusions present in patients carrying pathogenic mutations in *SOD1*.
The *SOD1^G127X^* mutation results in a shift in the DNA coding frame and a neopeptide sequence of five amino acids 127GQRWK-stop, truncating after codon 132, 21 amino acids short of end of the SOD1^WT^ polypeptide.[@R11] It is therefore suited for studying different misSOD1 inclusions. Staining with the anti-SOD1 antibody raised against the C-terminal end (131--153 Ra-ab) revealed small and dot-like misSOD1 positive inclusions in the motor neurons that resembled the staining seen in patients with sporadic and familial ALS lacking *SOD1* mutations ([figure 2A](#F2){ref-type="fig"}). This was observed in all three patients heterozygous for this mutation. Double staining with the 131--153 Ra-ab and the mutation-specific antibody G127X Ra-ab revealed *separate* inclusions of misSOD1 in motor neurons, without colocalisation ([figure 2A--C](#F2){ref-type="fig"}): the large skein-like inclusions contain the misfolded SOD1^G127X^ mutant protein, the small granular dot inclusions the misSOD1^WT^ generated from the wild-type allele. Both types of inclusions in the same motor neuron were only observed in a few motor neurons per section. Most motor neurons had only one type of SOD1 inclusion and a few neurons lacked inclusions.
### Control patients and reanalysis of spinal cord sections from patients with ALS and controls {#s3-1-4}
To determine whether the misSOD1^WT^ inclusions were specific for patients with ALS and FTD, brain and spinal cord tissue from 10 control patients with other neurodegenerative diseases were stained with the same anti-misSOD1 antibodies. Nine of 10 controls did not show misSOD1^WT^ inclusions anywhere in the spinal cord or brain ([table 2](#T2){ref-type="table"}, [figures 1I and 3F](#F1 F3){ref-type="fig"}). A single patient diagnosed with epilepsy after a cerebral infarction had some misSOD1^WT^ inclusions in the spinal cord and hypoglossal nucleus. Neuronal cytoplasmic misSOD1^WT^ inclusions were also seen to the same extent as in the motor cortex of patients with ALS, but the frontal cortex and cerebellum were blank. We additionally analysed spinal cord sections from cervical, thoracic and lumbar regions of 20 patients with non-neurological diseases ([online supplementary table 1](#SP1){ref-type="supplementary-material"}) with the anti-misSOD1 antibodies. No inclusions of misSOD1^WT^ were observed in any of these ([figure 2E](#F2){ref-type="fig"}).
The above reported IHC investigations were carried out over a period of 14 years. During this time, the staining equipment was changed in our lab. For control purposes, we recently restained and re-examined in parallel lumbar spinal cord specimen from all 30 controls and 38 (of the 46) patients with ALS at the same time (CNS tissues were no longer available from two patients with *C9orf72* and six patients with *SOD1* mutations). Two different antibodies, 57--72 Ra-ab and 131--153 Ra-ab---targeting the active site and the carboxy end of the SOD1 protein, respectively---were used to ascertain the specificity for misSOD1 in the stainings. Identical protocol was used for all specimen. The identities of the restained sections were coded and were independently examined by two neuropathologists (KF, TB).
The results are summarised in [figure 4](#F4){ref-type="fig"}, [online supplementary figure 2 and supplementary table 2](#SP4 SP5 SP6 SP7){ref-type="supplementary-material"}. The two examiners assessed the sections in principle equally, the only differences being a few one step differences in gradings of the SOD1 stainings in patients with ALS, [online supplementary table 2](#SP7){ref-type="supplementary-material"}. In these cases, both gradings are shown. All the patients with ALS without *SOD1* mutations again showed the misSOD1^WT^-positive granular inclusions. They were easily detected and had the same shape and locations as seen in the previous older stainings. The misSOD1^WT^ staining in the neurodegenerative control \#50 was again seen ([online supplementary figure 2 AI](#SP5 SP6){ref-type="supplementary-material"}). However, the principal difference from the previous analyses was that misSOD1^WT^ staining was now seen in three more neurodegenerative control ([online supplementary figure 2 AG, AH, AK](#SP5 SP6){ref-type="supplementary-material"}), which did not appear in the previous stainings ([table 2](#T2){ref-type="table"}). Two of these patients had Parkinson's disease and one had Alzheimer's disease. Noticeably, only a few motor neurons were positive. Moreover, also two of the non-neurological controls were when restained now graded as positive since *a few* motor neurons were positive ([online supplementary figure 2 AQ, AW](#SP5 SP6){ref-type="supplementary-material"}).
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{#F4}
The outcomes of examinations of sections stained with two different anti-SOD1 peptide antibodies (57--72 Ra-ab, 131--153 Ra-ab) were almost identical ([figure 4 and online supplementary table 2](#SP4 SP5 SP6){ref-type="supplementary-material"}). This proves that the component that becomes stained in the granules is indeed misSOD1^WT^. The 131--153 Ra-Ab gives generally less background staining and was therefore chosen for most of the histochemical panels shown.
Discussion {#s4}
==========
We here report the finding of misSOD1^WT^-positive intracytoplasmic inclusions in motor neurons of patients lacking *SOD1* mutations but carrying pathogenic mutations in six other ALS-causing genes *C9orf72HRE*, *FUS, NEK1, KIF5A, ALSIN* and *VAPB*. The inclusions were small, numerous and scattered in the cytoplasm of motor neurons at all levels of the spinal cord, in the motor nuclei of the brain stem and occasionally in the motor and frontal cortex. They were seen in all 18 patients carrying *C9orf72HRE*, regardless of whether the patient had a 'pure' FTD diagnosis, pure ALS diagnosis or both. The morphology and shape of these inclusions resembled that of the inclusions described in sALS.[@R17] Patients with other FTD-associated and ALS-associated mutations had similar inclusions that stained for misSOD1^WT^ in the brain and spinal cord. The finding that misSOD1^WT^ appears in small inclusions in all studied patients with ALS raises the question if these misSOD1^WT^ inclusions are different from the ones formed from mutated SOD1.
In SOD1 Tg murine models overexpressing the G85R, G93A, D90A or G127X human *SOD1* mutations, larger skein-like inclusions are observed when stained with human misSOD1-specific antibodies. The misSOD1 in these SOD1 Tg murine models is aggregated. So far, two strains of aggregates (A and B) have been described which differ with regard to molecular structure.[@R14] Both display prion-like properties and propagate further aggregation and phenotypically different ALS-like diseases when inoculated into the spinal cord of 100-day-old asymptomatic mice Tg for the human *SOD1^G85R^* mutation.[@R15] In patients that carry unstable mutant SOD1s, morphologically similar misSOD1-IR inclusions are found.[@R11] These also appear to contain aggregated SOD1, but the molecular structures have yet to be elucidated. Inoculation of SOD1^G127X^ aggregates purified from the spinal cord of patient no. 32 in this study ([table 1](#T1){ref-type="table"}) into the lumbar spinal cord of 100-day-old mice induced hSOD1-aggregate formation throughout the neuraxis concomitant with onset of ALS.[@R16] Binary epitope mapping revealed that the human misSOD1 aggregates was of the A-type.[@R16] Further inoculation studies of SOD1 aggregates purified from other patients from this study are ongoing.
In patients *homozygous* for the stable wt-like mutant SOD1^D90A^, the motor neurons display small granular misSOD1^D90A^ inclusions. Whether these also have prion-like features as the larger inclusions found in the murine Tg models and in patients that carry unstable mutant SOD1s, remains to be determined. Still, this is the sole conspicuous misSOD1-IR structure found in postmortem tissue from the six SOD1^D90A^ homozygous patients. The differences in morphology compared with patients with other *SOD1* mutations might be related to their different molecular stabilities. SOD1^WT^ and stable SOD1 mutants might associate to form small granular inclusions whereas unstable mutant proteins could either be rapidly degraded or form fewer but larger inclusions. Interesting implications follow from this observation: (1) inclusions of misSOD1 may be pathological hallmarks in all forms of ALS and (2) variation in morphology, distribution and number of aggregates may be used to distinguish different forms and stages of ALS.
Summarising the results of the initial staining and the restaining, 4 of 10 neurodegenerative controls had some inclusions of misSOD1^WT^ ([online supplementary figure 2 AG-AI, AK, and supplementary table 2](#SP5 SP6 SP7){ref-type="supplementary-material"}). One of the positive controls was a patient with epilepsy after cerebral infarction. Here, small granular cytoplasmic inclusions of misSOD1^WT^ were seen in spinal cord motor neurons ([online supplementary figure 2 AI](#SP5 SP6){ref-type="supplementary-material"}), in the hypoglossus nuclei and in the motor cortex. No inclusions were found in the striatum, frontal cortex or mesencephalon. Speculatively, this control individual could have had clinically unrecognised early ALS and/or the cerebral infarction triggered a cascade of events resulting in misfolding of misSOD1^WT^ that might not have prion-competent conformations[@R15] but are still detected by our polyclonal antibodies. However, cerebrovascular injury has been proposed to be a risk factor for ALS.[@R35] Two of the positive controls had Parkinson's disease ([online supplementary figure 2 AG, AK](#SP5 SP6){ref-type="supplementary-material"}). Interestingly, inclusions of misSOD1^WT^ have been found to accumulate in the brains of patients with Parkinson's disease and suggests common mechanisms of misSOD1^WT^ aggregation in both disorders.[@R36] Moreover, neurodegenerative diseases with concomitant copathology is frequent.[@R37]
Two of the non-neurological controls also had a few motor neurons that stained positive for SOD1. One died of a dissection of the aorta and the other of myocardial infarction ([online supplementary figure 2 AQ, AW](#SP5 SP6){ref-type="supplementary-material"}). The first showed small areas of infarction in the spinal cord. As also noted earlier in senile plaques, the state of the tissue might thus affect the immunoreaction found. All other 24 controls stained negative for misSOD1 in all studied tissue specimens, demonstrating major differences between patients with ALS and controls.
The presence of misSOD1^WT^ in postmortem CNS tissue of sALS and fALS without *SOD1* mutations is controversial: some studies support the result of the present study,[@R17] but in other studies misSOD1 could not be detected in sALS[@R38] or there were no differences between sALS and control individuals.[@R41] Reasons for these opposite results were discussed in a recent article[@R22] and includes methodological differences (ie, the use of TRIS/EDTA buffers instead of citrate-based buffers, different preanalytical tissue handling, variable incubation times, different antibody dilutions). Guidelines for staining misSOD1 were proposed.[@R22] These guidelines were adhered to in this work. It should be noted that Ref. [@R21] was a blinded study of sALS cases compared with controls (some of these were the same as in this study) and using some of the anti-misSOD1 antibodies as in the present study.
The present morphological study does not prove that misSOD1^WT^ causes neurodegeneration in patients carrying mutations in *C9orf72HRE*, *FUS, NEK1, KIF5A, ALSIN* or *VAPB*. Nor can we explain how patients with a massive intronic GGGGCC repeat-expansion in *C9orf72* or a R495X-truncation in the mRNA-housekeeping FUS enzyme have inclusions of misSOD1^WT^ in their motor neurons and with a morphology and similar pattern of cellular distribution as in patients with the *SOD1^D90A^* mutation. We speculate that whatever the initial pathogenic events are in patients carrying *C9orf72HRE* or coding mutations in *FUS*, *NEK1, KIF5A, VAPB* or *ALSIN*, eventually they converge to common downstream event(s) involving unfolding and misfolding of SOD1^WT^. For this to happen, the stabilising intrasubunit C57-C146 disulfide bond must be reduced and the stabilising Zn^2+^ cofactor released. If the generated misfolded SOD1 species are pathogenic and form SOD1^WT^ prions, based on injection of SOD1 aggregates,[@R15] this event may only have to occur in a limited number of cells, perhaps only once for the SOD1-prion cascade to be initiated. The finding that overexpression of human SOD1^WT^ in mice elicits a fatal ALS-like disease supports that SOD1^WT^ can cause neurodegeneration.[@R23] The observation that all three patients heterozygous for the *SOD1^G127X^* mutation had inclusions with the truncated mutant SOD1^G127X^ protein (easily recognisable because of its neopeptide sequence) *and* separate inclusions containing only misSOD1^WT^ (detectable by the amino acid sequence that is missing in the mutated SOD1 protein) is interesting. It raises the possibilities that either the misfolding of SOD1^WT^ is a secondary non-pathogenic event in a stressed cell or that misfolded SOD1^WT^ participates and could be pathogenic per se, raising the possibility that there could be more than one kind of SOD1 prions active simultaneously in patients with *SOD1* mutations. Simply targeting a mutant *SOD1* gene with tailored genetic inhibition therapy may therefore not be sufficient to halt disease progression. This observation is important, since new therapies targeting SOD1 are ongoing or are being planned. If SOD1 plays a role in subtypes of ALS without *SOD1* mutations as our results suggest, then many patients may benefit from these new anti-SOD1 therapeutics.
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We are indebted to all the patients who made this study possible. The authors wish to thank Ulla-Stina Spetz, Eva Bern, Helena Alstermark, Ann-Charloth Nilsson, Eva Jonsson, Agneta Öberg and Matthew Marklund for technical assistance.
**Contributors:** Study concept: PA. Designed the study: KF, SM, TB, PA. Collected and evaluated the material: KF, KG, BP, MW, MN, TB, PA. Performed and evaluated the laboratory analysis: KF, MW, MN, SLM, TB. Wrote the manuscript: KF, TB, PA.
**Funding:** This work was supported by grants from the Swedish Brain Foundation (grants nos. 2012-0262, 2012-0305, 2013-0279, 2016-0303), the Swedish Science Council (grants nos. 2012-3167, 2017-03100), the Knut and Alice Wallenberg Foundation (grants nos. 2012.0091, 2014.0305), the Bertil Hållsten Foundation, the Ulla-Carin Lindquist Foundation, the Kempe foundation, the Neuroförbundet Association, the Torsten and Ragnar Söderberg Foundation, Umeå University Insamlingsstiftelsen (223-2808-12, 223-1881-13, 2.1.12-1605-14) and the Västerbotten County Council (grants nr. 56103-7002829).
**Competing interests:** None declared.
**Patient consent for publication:** Not required.
**Ethics approval:** The study was approved by the Regional Medical Ethical Review Board in Umea, Sweden with applications from 1994 (FEK no. 94-135) and 2014 (EPN no. 14-17-31M).
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data sharing statement:** There are additional pictures of misSOD1+ stainings available from the here studied material which we are willing to share.
[^1]: TB and PMA are joint senior authors.
|
{
"pile_set_name": "PubMed Central"
}
|
Related literature {#sec1}
==================
For background on the photochromic behavior of salicylideneanilines, see: Brown (1971[@bb4]); Chemla & Zyss (1987[@bb7]); MacDonald & Whitesides (1994[@bb13]); Cohen *et al.* (1966[@bb8]). For related compounds, see: Ancın *et al.* (2007[@bb1]); Kasumov, Köksal & Köseoĝlu (2004[@bb11]); Kasumov, Medjidov, Yaylı & Zeren (2004[@bb12]); Çelik *et al.* (2007[@bb6], 2009[@bb5]). For graph-set notation, see: Bernstein *et al.* (1995[@bb2]); Etter (1991[@bb9]).
Experimental {#sec2}
============
{#sec2.1}
### Crystal data {#sec2.1.1}
C~21~H~25~F~2~NO*M* *~r~* = 345.42Monoclinic,*a* = 6.423 (5) Å*b* = 17.386 (5) Å*c* = 17.337 (5) Åβ = 90.319 (5)°*V* = 1936.0 (17) Å^3^*Z* = 4Mo *K*α radiationμ = 0.09 mm^−1^*T* = 293 K0.20 × 0.20 × 0.20 mm
### Data collection {#sec2.1.2}
Rigaku RxdiffractometerAbsorption correction: multi-scan (Blessing, 1995[@bb3]) *T* ~min~ = 0.983, *T* ~max~ = 0.98350854 measured reflections5662 independent reflections2715 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.099
### Refinement {#sec2.1.3}
*R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.057*wR*(*F* ^2^) = 0.144*S* = 0.965662 reflections238 parametersH atoms treated by a mixture of independent and constrained refinementΔρ~max~ = 0.11 e Å^−3^Δρ~min~ = −0.13 e Å^−3^
{#d5e478}
Data collection: *CrystalClear* (Rigaku/MSC, 2005[@bb14]); cell refinement: *CrystalClear*; data reduction: *CrystalClear*; program(s) used to solve structure: *SHELXS97* (Sheldrick, 2008[@bb15]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008[@bb15]); molecular graphics: *ORTEP-3* (Farrugia, 1997[@bb10]); software used to prepare material for publication: *SHELXTL* (Sheldrick, 2008[@bb15]) and local programs.
Supplementary Material
======================
Crystal structure: contains datablocks I, global. DOI: [10.1107/S1600536809041099/jh2098sup1.cif](http://dx.doi.org/10.1107/S1600536809041099/jh2098sup1.cif)
Structure factors: contains datablocks I. DOI: [10.1107/S1600536809041099/jh2098Isup2.hkl](http://dx.doi.org/10.1107/S1600536809041099/jh2098Isup2.hkl)
Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?jh2098&file=jh2098sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?jh2098sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?jh2098&checkcif=yes)
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [JH2098](http://scripts.iucr.org/cgi-bin/sendsup?jh2098)).
Comment
=======
Proton tautomerization plays an important role in many fields of chemistry and biochemistry. The tautomerization in salicylideneanilines has been the subject of particular interest, because it is closely related to thermochromisim and photochromisim. While salicylideneanilines are widely used as a precursor compounds for a design of various type new metal complexes they are also a convenient model compounds for studying theoretical aspects of coordination chemistry and photochemistry, as well as for designing molecular architecture by means of molecular motifs capable of H-bond formation. The existence of photochromic behavior suggests the possibility of using these compounds as elements for constructing the optical switches or optical memory devices (Brown, 1971; Chemla *et al.*, 1987; Cohen *et al.*, 1966; Chemla, *et al.*, 1987; MacDonald, *et al.*, 1994). As part of our interest on electron transfer and complexation behaviors of redox-active salicylaldimines, obtained from bulky di-tertbutylated sterically hindered aminophenols, salicylaldehydes and aryl amines and their complexes (Kasumov *et al.*, 2004; Kasumov *et al.*, 2004), we decided to prepare and structurally investigate the bidentate salicylaldimines derived from 3,5-di-tert-butyl-salicylaldehyde and difluorinated anilines.
In the compound, the difluoroaniline atoms (P1) and benzylidene atoms (P2) are plane and the dihedral angle between P1 and P2 planes is 27.90 (5)°. The maximum deviations from the P1 plane of C3 and P2 plane of C11 are -0.008 Å and 0.002 Å, respectivity. The bond between N and C7 atoms is double bond, whose length is 1.282 (2)Å, and the conformation at this double bond is trans with the torsion angle C1-N-C7-C8 is 178.2 (1)°. The bond lengths are as expected. Similar results are were observed in the study of N-\[5- methylisoxazole-amino-3-yl\]-3, 5-di-tert-butylsalicylaldimine amino-3-yl\]-3,5 -di-tert-butylsalicylaldimine(Çelik *et al.*, 2007), N-\[1-(3-Aminopropyl) imidazole\]-3,5-di-tertbutylsalicylaldimine(Çelik *et al.*, 2009) and N,N\'-bis- (5-methylsalicylidene)-2,2\'-diamino-4-4\'-di-(trifloromethyl)-diphenyl disulfide (Ancın, *et al.*, 2007).
In Table 2 is given interactions have types of O-H···N, C-H···O, C-H···N and C-H···F. O-H···N hydrogen bond which are intramolecular interaction causes to reversible proton transfer between imine N atom and the hydroxyl O atom. The three intra-molecular interaction have strong effects for molecule. C-H···O hydrogen bond which are intramolecular interaction causes to reversible proton transfer between methyl C atom and the hydroxyl O atom. Similar Schiff bases usually show photochromism and thermocromism because of the above mentioned intramolecular hydrogen bonds. Similar proton transfer has nor been determined in the molecular structure of our compound. The cause of this results may be explained by a steric effect and are effective for the molecular packing of the compound.
Experimental {#experimental}
============
The pale yellow crystalline the title compound was prepared by using standard procedure involving the condensation of equimolar amount of 3,5-di-*tert*-butyl-2-hydroxybenzaldehyde with 2,5 difluoroaniline in refluxing ethanol in the presence of catalytic amount of formic acid (3--4 drops). To a stirred and heated (60 °C) solution of 3,5-di-*tert*-butyl-2-hydroxybenzaldehyde (0.936 g, 4 mmol) in absolute ethanol (80 ml), a solution of 0.516 g (4 mmol) of 2,5-difluoroaniline in 5 ml methanol was added immediately. Then 4 drops of formic acid was added to this solution and refluxed for 24 h. The volume of the reaction mixture was evaporated to 25 ml and after cooling to 15 °C, the yellow crystals were collected and air dried to yield 1.298 g (94%).
Figures
=======
{#Fap1}
{#Fap2}
Crystal data {#tablewrapcrystaldatalong}
============
------------------------- ---------------------------------------
C~21~H~25~F~2~NO *F*(000) = 736
*M~r~* = 345.42 *D*~x~ = 1.185 Mg m^−3^
Monoclinic, *P*2~1~/*n* Mo *K*α radiation, λ = 0.71073 Å
*a* = 6.423 (5) Å Cell parameters from 5929 reflections
*b* = 17.386 (5) Å θ = 2.6--30.5°
*c* = 17.337 (5) Å µ = 0.09 mm^−1^
β = 90.319 (5)° *T* = 293 K
*V* = 1936.0 (17) Å^3^ Needle, pale yellow
*Z* = 4 0.20 × 0.20 × 0.20 mm
------------------------- ---------------------------------------
Data collection {#tablewrapdatacollectionlong}
===============
---------------------------------------------------- --------------------------------------
Rigaku Rx diffractometer 5662 independent reflections
Radiation source: fine-focus sealed tube 2715 reflections with *I* \> 2σ(*I*)
graphite *R*~int~ = 0.099
Detector resolution: 10.0000 pixels mm^-1^ θ~max~ = 30.5°, θ~min~ = 2.6°
dtprofit.ref scans *h* = −9→7
Absorption correction: multi-scan (Blessing, 1995) *k* = −24→24
*T*~min~ = 0.983, *T*~max~ = 0.983 *l* = −24→24
50854 measured reflections
---------------------------------------------------- --------------------------------------
Refinement {#tablewraprefinementdatalong}
==========
------------------------------------- -------------------------------------------------------------------------------------
Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
*R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.057 Hydrogen site location: mixed
*wR*(*F*^2^) = 0.144 H atoms treated by a mixture of independent and constrained refinement
*S* = 0.96 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0514*P*)^2^\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3
5662 reflections (Δ/σ)~max~ \< 0.001
238 parameters Δρ~max~ = 0.11 e Å^−3^
0 restraints Δρ~min~ = −0.13 e Å^−3^
none constraints
------------------------------------- -------------------------------------------------------------------------------------
Special details {#specialdetails}
===============
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Geometry. All e.s.d.\'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.\'s are taken into account individually in the estimation of e.s.d.\'s in distances, angles and torsion angles; correlations between e.s.d.\'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.\'s is used for estimating e.s.d.\'s involving l.s. planes.
Refinement. Refinement of *F*^2^ against ALL reflections. The weighted *R*-factor *wR* and goodness of fit *S* are based on *F*^2^, conventional *R*-factors *R* are based on *F*, with *F* set to zero for negative *F*^2^. The threshold expression of *F*^2^ \> σ(*F*^2^) is used only for calculating *R*-factors(gt) *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on *F*^2^ are statistically about twice as large as those based on *F*, and *R*- factors based on ALL data will be even larger.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords}
==================================================================================================
------ -------------- --------------- -------------- -------------------- --
*x* *y* *z* *U*~iso~\*/*U*~eq~
F1 1.34032 (17) −0.06161 (7) 0.45693 (7) 0.0928 (4)
F2 0.7822 (2) −0.18169 (7) 0.25642 (7) 0.1106 (5)
O 0.43585 (19) −0.00558 (7) 0.19801 (7) 0.0716 (3)
N 0.7118 (2) −0.03731 (8) 0.30481 (8) 0.0599 (3)
C1 0.8873 (2) −0.07788 (9) 0.33323 (9) 0.0579 (4)
C2 1.0326 (3) −0.04776 (11) 0.38404 (10) 0.0632 (4)
C3 1.2008 (3) −0.09201 (11) 0.40566 (10) 0.0670 (4)
C4 1.2353 (3) −0.16381 (13) 0.37793 (11) 0.0817 (6)
C5 1.0919 (3) −0.19439 (12) 0.32701 (12) 0.0887 (6)
C6 0.9227 (3) −0.15091 (11) 0.30581 (10) 0.0727 (5)
C7 0.6290 (3) 0.01624 (9) 0.34520 (10) 0.0571 (4)
C8 0.4535 (2) 0.06101 (9) 0.31824 (8) 0.0533 (4)
C9 0.3601 (2) 0.04900 (8) 0.24564 (9) 0.0542 (4)
C10 0.1864 (2) 0.09270 (8) 0.22268 (8) 0.0531 (4)
C11 0.1149 (3) 0.14730 (9) 0.27431 (9) 0.0551 (4)
C12 0.2021 (2) 0.16151 (8) 0.34774 (9) 0.0517 (4)
C13 0.3714 (2) 0.11717 (8) 0.36766 (9) 0.0543 (4)
C14 0.0847 (3) 0.08127 (9) 0.14289 (9) 0.0608 (4)
C15 0.2440 (3) 0.10244 (12) 0.07964 (10) 0.0878 (6)
C16 0.0150 (3) −0.00280 (10) 0.13124 (10) 0.0745 (5)
C17 −0.1094 (3) 0.13131 (11) 0.13211 (11) 0.0852 (6)
C18 0.1136 (3) 0.22420 (9) 0.40007 (9) 0.0590 (4)
C19 0.2129 (3) 0.22172 (12) 0.48075 (10) 0.0835 (6)
C20 −0.1214 (3) 0.21370 (11) 0.40956 (11) 0.0767 (5)
C21 0.1544 (3) 0.30292 (10) 0.36414 (12) 0.0909 (6)
H0 0.5354 −0.0269 0.2187 0.107\*
H2 1.018 (3) 0.0018 (10) 0.4049 (10) 0.072 (5)\*
H4 1.3524 −0.1916 0.3929 0.098\*
H5 1.1096 −0.2437 0.3073 0.106\*
H7 0.681 (2) 0.0293 (9) 0.4007 (10) 0.071 (5)\*
H11 0.001 (2) 0.1785 (8) 0.2587 (8) 0.054 (4)\*
H13 0.4337 0.1247 0.4156 0.065\*
H15A 0.3659 0.0709 0.0853 0.132\*
H15B 0.2820 0.1556 0.0846 0.132\*
H15C 0.1830 0.0939 0.0297 0.132\*
H16A 0.1327 −0.0363 0.1374 0.112\*
H16B −0.0422 −0.0088 0.0803 0.112\*
H16C −0.0888 −0.0156 0.1687 0.112\*
H17A −0.1680 0.1223 0.0819 0.128\*
H17B −0.0719 0.1845 0.1369 0.128\*
H17C −0.2100 0.1184 0.1708 0.128\*
H19A 0.1544 0.2617 0.5121 0.125\*
H19B 0.3604 0.2292 0.4766 0.125\*
H19C 0.1858 0.1727 0.5041 0.125\*
H20A −0.1749 0.2537 0.4421 0.115\*
H20B −0.1487 0.1645 0.4326 0.115\*
H20C −0.1881 0.2163 0.3599 0.115\*
H21A 0.0982 0.3423 0.3968 0.136\*
H21B 0.0890 0.3055 0.3143 0.136\*
H21C 0.3017 0.3105 0.3588 0.136\*
------ -------------- --------------- -------------- -------------------- --
Atomic displacement parameters (Å^2^) {#tablewrapadps}
=====================================
----- ------------- ------------- ------------- -------------- -------------- --------------
*U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^
F1 0.0730 (7) 0.1143 (9) 0.0908 (8) −0.0059 (6) −0.0254 (6) 0.0194 (6)
F2 0.1241 (10) 0.0941 (9) 0.1131 (10) 0.0261 (7) −0.0420 (8) −0.0405 (7)
O 0.0734 (8) 0.0773 (8) 0.0639 (7) 0.0170 (6) −0.0125 (6) −0.0166 (6)
N 0.0572 (8) 0.0612 (8) 0.0613 (8) 0.0052 (6) −0.0076 (6) −0.0001 (6)
C1 0.0557 (9) 0.0593 (10) 0.0587 (9) 0.0053 (8) −0.0012 (7) 0.0062 (7)
C2 0.0598 (10) 0.0635 (11) 0.0662 (11) −0.0011 (8) −0.0034 (8) 0.0075 (8)
C3 0.0579 (10) 0.0824 (12) 0.0608 (10) −0.0001 (9) −0.0042 (8) 0.0134 (9)
C4 0.0724 (12) 0.0961 (15) 0.0766 (13) 0.0274 (11) 0.0037 (10) 0.0123 (11)
C5 0.1007 (16) 0.0829 (13) 0.0825 (13) 0.0350 (12) −0.0014 (12) −0.0069 (10)
C6 0.0785 (12) 0.0752 (12) 0.0644 (11) 0.0108 (10) −0.0086 (9) −0.0091 (9)
C7 0.0557 (9) 0.0584 (10) 0.0572 (10) −0.0004 (8) −0.0077 (8) 0.0019 (7)
C8 0.0518 (9) 0.0542 (9) 0.0538 (9) −0.0010 (7) −0.0049 (7) 0.0000 (7)
C9 0.0585 (9) 0.0503 (9) 0.0536 (9) −0.0021 (7) −0.0026 (7) −0.0037 (7)
C10 0.0573 (9) 0.0521 (8) 0.0498 (8) −0.0045 (7) −0.0064 (7) 0.0026 (7)
C11 0.0576 (10) 0.0494 (9) 0.0582 (9) 0.0010 (7) −0.0075 (7) 0.0040 (7)
C12 0.0536 (9) 0.0468 (8) 0.0546 (8) −0.0063 (7) −0.0038 (7) −0.0004 (6)
C13 0.0562 (9) 0.0540 (9) 0.0527 (8) −0.0049 (7) −0.0091 (7) −0.0027 (7)
C14 0.0723 (11) 0.0595 (10) 0.0504 (9) −0.0034 (8) −0.0122 (8) 0.0013 (7)
C15 0.1058 (16) 0.0983 (15) 0.0594 (11) −0.0252 (12) −0.0046 (11) 0.0053 (10)
C16 0.0868 (13) 0.0699 (11) 0.0667 (11) −0.0099 (10) −0.0171 (9) −0.0068 (8)
C17 0.0960 (15) 0.0814 (13) 0.0779 (13) 0.0107 (11) −0.0372 (11) 0.0014 (10)
C18 0.0635 (10) 0.0500 (9) 0.0636 (10) −0.0017 (7) −0.0001 (8) −0.0090 (7)
C19 0.0862 (13) 0.0914 (14) 0.0727 (12) 0.0044 (11) −0.0081 (10) −0.0281 (10)
C20 0.0649 (11) 0.0869 (13) 0.0784 (12) 0.0017 (9) 0.0046 (9) −0.0107 (10)
C21 0.1132 (17) 0.0543 (11) 0.1054 (16) −0.0062 (11) 0.0154 (13) −0.0068 (10)
----- ------------- ------------- ------------- -------------- -------------- --------------
Geometric parameters (Å, °) {#tablewrapgeomlong}
===========================
----------------------- -------------- ----------------------- --------------
F1---C3 1.365 (2) C2---H2 0.940 (17)
F2---C6 1.351 (2) C16---H16A 0.9600
O---C9 1.3503 (18) C16---H16B 0.9600
N---C7 1.282 (2) C16---H16C 0.9600
N---C1 1.416 (2) C19---H19A 0.9600
C1---C2 1.383 (2) C19---H19B 0.9600
C1---C6 1.375 (2) C19---H19C 0.9600
C3---C4 1.356 (3) C17---H17A 0.9600
C3---C2 1.376 (2) C17---H17B 0.9600
C4---C5 1.379 (3) C17---H17C 0.9600
C6---C5 1.372 (3) C20---H20A 0.9600
C8---C13 1.404 (2) C20---H20B 0.9600
C8---C9 1.407 (2) C20---H20C 0.9600
C8---C7 1.445 (2) C4---H4 0.9300
C10---C11 1.385 (2) C15---H15A 0.9600
C10---C9 1.405 (2) C15---H15B 0.9600
C10---C14 1.540 (2) C15---H15C 0.9600
C11---C12 1.410 (2) C5---H5 0.9300
C12---C13 1.375 (2) C21---H21A 0.9600
C12---C18 1.530 (2) C21---H21B 0.9600
C14---C17 1.531 (2) C21---H21C 0.9600
C14---C16 1.542 (2) C11---H11 0.948 (14)
C14---C15 1.548 (3) C13---H13 0.9300
C18---C21 1.527 (2) C7---H7 1.042 (17)
C18---C20 1.530 (3) O---H0 0.8200
C18---C19 1.535 (2)
C7---N---C1 120.24 (14) C14---C16---H16C 109.5
C11---C10---C9 116.96 (14) H16A---C16---H16C 109.5
C11---C10---C14 121.90 (14) H16B---C16---H16C 109.5
C9---C10---C14 121.13 (14) C18---C19---H19A 109.5
C10---C11---C12 124.88 (15) C18---C19---H19B 109.5
C13---C8---C9 119.32 (14) H19A---C19---H19B 109.5
C13---C8---C7 118.16 (14) C18---C19---H19C 109.5
C9---C8---C7 122.50 (14) H19A---C19---H19C 109.5
C13---C12---C11 116.00 (14) H19B---C19---H19C 109.5
C13---C12---C18 123.12 (14) C14---C17---H17A 109.5
C11---C12---C18 120.87 (14) C14---C17---H17B 109.5
C12---C13---C8 122.39 (14) H17A---C17---H17B 109.5
C6---C1---C2 117.22 (16) C14---C17---H17C 109.5
C6---C1---N 118.20 (15) H17A---C17---H17C 109.5
C2---C1---N 124.49 (16) H17B---C17---H17C 109.5
O---C9---C10 119.66 (14) C18---C20---H20A 109.5
O---C9---C8 119.88 (14) C18---C20---H20B 109.5
C10---C9---C8 120.45 (14) H20A---C20---H20B 109.5
C21---C18---C12 109.36 (14) C18---C20---H20C 109.5
C21---C18---C20 108.81 (15) H20A---C20---H20C 109.5
C12---C18---C20 110.42 (13) H20B---C20---H20C 109.5
C21---C18---C19 109.01 (15) C3---C4---H4 120.9
C12---C18---C19 111.50 (14) C5---C4---H4 120.9
C20---C18---C19 107.68 (14) C14---C15---H15A 109.5
N---C7---C8 122.66 (15) C14---C15---H15B 109.5
C17---C14---C10 112.19 (14) H15A---C15---H15B 109.5
C17---C14---C16 106.69 (15) C14---C15---H15C 109.5
C10---C14---C16 111.20 (13) H15A---C15---H15C 109.5
C17---C14---C15 108.64 (15) H15B---C15---H15C 109.5
C10---C14---C15 109.07 (14) C6---C5---H5 120.5
C16---C14---C15 108.97 (15) C4---C5---H5 120.5
C4---C3---F1 118.63 (17) C18---C21---H21A 109.5
C4---C3---C2 123.19 (18) C18---C21---H21B 109.5
F1---C3---C2 118.17 (18) H21A---C21---H21B 109.5
C3---C2---C1 119.19 (18) C18---C21---H21C 109.5
F2---C6---C5 118.48 (17) H21A---C21---H21C 109.5
F2---C6---C1 118.27 (16) H21B---C21---H21C 109.5
C5---C6---C1 123.24 (18) C10---C11---H11 117.7 (9)
C3---C4---C5 118.17 (18) C12---C11---H11 117.4 (9)
C6---C5---C4 118.96 (19) C12---C13---H13 118.8
C3---C2---H2 119.2 (11) C8---C13---H13 118.8
C1---C2---H2 121.6 (11) N---C7---H7 122.0 (9)
C14---C16---H16A 109.5 C8---C7---H7 115.3 (9)
C14---C16---H16B 109.5 C9---O---H0 109.5
H16A---C16---H16B 109.5
C9---C10---C11---C12 −0.5 (2) C11---C12---C18---C19 172.11 (15)
C14---C10---C11---C12 −179.12 (14) C1---N---C7---C8 178.23 (14)
C10---C11---C12---C13 0.3 (2) C13---C8---C7---N 178.59 (15)
C10---C11---C12---C18 178.97 (15) C9---C8---C7---N 0.1 (2)
C11---C12---C13---C8 0.0 (2) C11---C10---C14---C17 −4.9 (2)
C18---C12---C13---C8 −178.57 (14) C9---C10---C14---C17 176.53 (15)
C9---C8---C13---C12 −0.2 (2) C11---C10---C14---C16 −124.32 (17)
C7---C8---C13---C12 −178.68 (14) C9---C10---C14---C16 57.1 (2)
C7---N---C1---C6 155.18 (17) C11---C10---C14---C15 115.49 (18)
C7---N---C1---C2 −28.3 (2) C9---C10---C14---C15 −63.06 (19)
C11---C10---C9---O 179.42 (14) C4---C3---C2---C1 1.8 (3)
C14---C10---C9---O −2.0 (2) F1---C3---C2---C1 −178.84 (14)
C11---C10---C9---C8 0.3 (2) C6---C1---C2---C3 −1.3 (2)
C14---C10---C9---C8 178.94 (14) N---C1---C2---C3 −177.87 (15)
C13---C8---C9---O −179.09 (14) C2---C1---C6---F2 179.66 (16)
C7---C8---C9---O −0.7 (2) N---C1---C6---F2 −3.5 (3)
C13---C8---C9---C10 0.0 (2) C2---C1---C6---C5 0.6 (3)
C7---C8---C9---C10 178.43 (14) N---C1---C6---C5 177.43 (17)
C13---C12---C18---C21 111.27 (18) F1---C3---C4---C5 179.11 (17)
C11---C12---C18---C21 −67.3 (2) C2---C3---C4---C5 −1.6 (3)
C13---C12---C18---C20 −129.03 (17) F2---C6---C5---C4 −179.41 (17)
C11---C12---C18---C20 52.4 (2) C1---C6---C5---C4 −0.4 (3)
C13---C12---C18---C19 −9.4 (2) C3---C4---C5---C6 0.8 (3)
----------------------- -------------- ----------------------- --------------
Hydrogen-bond geometry (Å, °) {#tablewraphbondslong}
=============================
--------------------- --------- --------- ----------- ---------------
*D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A*
O---H0···N 0.82 1.88 2.615 (2) 149
C16---H16A···O 0.96 2.27 2.935 (3) 126
C15---H15A···O 0.96 2.40 3.038 (2) 123
C7---H7···F1^i^ 1.042 2.875 3.010 (2) 87
C16---H16C···N^i^ 0.96 2.72 3.643 (3) 162
C21---H21B···F2^ii^ 0.96 2.68 3.498 (3) 143
--------------------- --------- --------- ----------- ---------------
Symmetry codes: (i) *x*−1, *y*, *z*; (ii) −*x*+1/2, *y*+1/2, −*z*+1/2.
###### Hydrogen-bond geometry (Å, °)
*D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A*
--------------------- --------- ------- ----------- -------------
O---H0⋯N 0.82 1.88 2.615 (2) 149
C16---H16*A*⋯O 0.96 2.27 2.935 (3) 126
C15---H15*A*⋯O 0.96 2.40 3.038 (2) 123
C16---H16*C*⋯N^i^ 0.96 2.72 3.643 (3) 162
C21---H21*B*⋯F2^ii^ 0.96 2.68 3.498 (3) 143
Symmetry codes: (i) ; (ii) .
|
{
"pile_set_name": "PubMed Central"
}
|
{#sp1 .571}
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
Pelvic fracture is an injury associated with high mortality in polytrauma patients \[[@CR1], [@CR2]\]. Its presentation may lead to rapid exanguination despite maximal resuscitative efforts \[[@CR3]\]. It also results in significant morbidity for patients who recover from the initial insult \[[@CR4], [@CR5]\]. The majority of trauma patients are young and in the prime of their lives \[[@CR6]--[@CR9]\]. It is therefore important to understand the epidemiology as well as the predictors for mortality in pelvic fracture so that preventive measures can be undertaken and preemptive treatment can be given. There is a paucity of epidemiological studies related to pelvic fracture in the Asian population. The purpose of this study is to look at the epidemiology of pelvic fracture in an Asian population and to determine factors associated with mortality.
Methods {#Sec2}
=======
This was a retrospective descriptive study in a 1,300-bed acute urban restructured hospital in Singapore. The study hospital has a trauma team on site, and is equipped with interventional angiography facilities and an intensive care unit. From 1 April 2001 to 31 December 2004, data of all consecutive trauma patients with pelvic fracture who presented to the Emergency Department (ED) were analysed. Data that were collected included: (1) the patient's demographic profile, (2) assessment of physiological parameters upon presentation, (3) causes, nature and mechanisms of injury, (4) injury severity based on the Revised Trauma Score (RTS) and Injury Severity Score (ISS), (5) pelvic fracture severity (classified according to the Abbreviated Injury Score AIS), (6) the presence and severity of other concomitant injuries (classified according to AIS), (7) interventions received, (8) length of stay in the hospital including high dependency and intensive care unit and (9) survival outcome upon discharge. Student's t-test and χ^2^ test were used to look for associations between variables where appropriate. Multivariate analysis using logistic regression was performed to evaluate significant predictors (determined on univariate analysis) that were associated with mortality. The significance level was set to less than 0.05. Statistical calculations were performed using STATA version 11.0 (College Station, TX). The Domain-Specific Review Board (DSRB) of the National Healthcare Group (NHG), which is the institution's ethics committee, approved this study.
Results {#Sec3}
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One hundred seventy-nine patients were found to have sustained pelvic fracture during the study period. Table [1](#Tab1){ref-type="table"} shows the demographic characteristics of the patients. They were predominantly male (64%). Seventy-one percent of the patients were aged between 20--49 years old. The most common mechanism of injury was road traffic accidents (52%), followed by falls from height (44%). Motorcyclists (35%) and pedestrians (30%) were the most commonly injured patients. The median ISS was 25 and median RTS was 7. The median total hospital length of stay (inclusive of intensive care and high dependency unit stay) was 9 days. The mortality rate was 37%. Fifty-one percent of deaths occurred in the ED. Pelvic fracture severity, the presence of shock (defined as systolic blood pressure of less than 90 mmHg) and coma (defined as Glasgow Coma Scale of less than 9) at presentation were strong predictors for mortality. The adjusted odds of dying in the presence of shock and coma were 4.44 (95% CI 1.72--11.49, p \< 0.01) and 6.69 (95% CI 2.52--17.74, p \< 0.01), respectively (see Table [2](#Tab2){ref-type="table"}). Elderly age (defined as age ≥65 years old) was associated with higher odds for mortality (adjusted OR 2.52, 95% CI 0.73--8.64, p = 0.142), but this was confounded by the presence of shock and coma at presentation. The presence of concurrent head injuries (adjusted OR 4.57, 95% CI 1.95--10.73, p \< 0.01) and chest injuries (adjusted OR 7.96, 95% CI 2.79--22.68, p \< 0.01) were associated with increased odds of mortality in these patients. Gender (OR 0.79, 95% CI 0.41--1.48, p = 0.455), other mechanisms of injury (OR 0.32, 95% CI 0.04--2.83, p = 0.309) and the presence of other associated injuries were not associated with increased odds for mortality (see Table [3](#Tab3){ref-type="table"}). Table 1Baseline characteristics of patientsDescriptionNo. of patients (%) (n = 179)Gender Male114 (64%) Female65 (36%)Age (years) ≤1911 (6%) 20--2955 (31%) 30--3943 (24%) 40--4929 (16%) 50--5913 (7%) 60--6913 (7%) ≥7015 (8%)Mortality67 (37%) Died in the ED34 (51%) Died in the ward (within 24 h)19 (28%) (Within 48 h)4 (6%) (after 48 h)10 (15%)Injury scores ISS^a^ ≥2590 (50%)Physiological parameters at presentation Shock (\<90 mmHg)65 (36%) Coma (GCS \<9)54 (30%)Mechanism of injuries Road traffic accident94 (52%) Pedestrians28 (30%) Motorcyclists33 (35%) Falls from height79 (44%) Others6 (4%)Pelvic fracture severity (by AIS^b^) 10 (0%) 283 (46%) 372 (40%) 414 (8%) 510 (6%) 60 (0%)Presence of associated injuries Head76 (42%) Face44 (25%) Chest102 (57%) Abdomen90 (50%) Extremities (bones)177 (99%) External (skin, muscle)90 (50%)Interventions Laporotomy28 (16%) Angiography6 (3%) External fixation14 (8%)^a^Injury Severity Score^b^Abbreviated Injury Severity ScoreTable 2Odds ratios for significant predictors of mortality (on univariate and multivariate analysis)PredictorsCrude OR (95% CI)Adjusted OR (95% CI)Shock at presentation10.72 (5.25--21.9)4.44 (1.72--11.49)Coma at presentation19.51 (8.57--44.41)6.69^a^ (2.52--17.74)Age \>65 years3.96 (1.46--10.69)2.52^b^ (0.73--8.64), p = 0.142Presence of head injuries7.58 (3.51--16.34)4.57^c^ (1.95--10.73)Presence of chest injuries17.61 (5.90--52.50)7.96^d^ (2.79--22.68)Note: p-values are \<0.01 unless otherwise stated^a^Adjusted for shock, elderly age and the presence of chest injuries only^b^Adjusted for shock and coma at presentation only^c^Adjusted for shock and presence of chest injuries only. Coma at presentation was not included in the multivariate analysis for head injury because it was on the causal pathway between head injury (exposure) and death (outcome)^d^Adjusted for shock, coma and presence of head injuries onlyTable 3Comparison of characteristics between patients who died and survived and the crude ORs for mortalityDescriptionDied (%)Survived (%)Crude OR95% CIp-valueGender (male)67610.790.41--1.480.455 (NS)Age \>65 years2163.961.51--10.410.005ISS \>25942449.5816.51--148.88\<0.01PF^a^ severityAIS 231551.00 (ref)\--AIS 342391.880.95--3.720.071 (NS)AIS 41537.382.09--26.040.002AIS 512211.812.32--60.070.003Presence of shock691710.725.25--21.90\<0.01Presence of coma66919.518.57--44.41\<0.01Presence of head injury72257.583.83--15.00\<0.01Presence of chest injury913717.617.00--44.29\<0.01Presence of facial injury34201.220.34--3.430.705 (NS)Presence of abdominal injury52482.661.00--7.070.08 (NS)Presence of external/extemities injuries86702.311.00--4.950.06 (NS)^a^Pelvic fracture
Discussion {#Sec4}
==========
Pelvic fracture is a serious injury that is associated with significant morbidity and mortality \[[@CR4], [@CR5]\]. An unstable pelvic fracture results in uncontrolled haemorrhage from the pelvic venous plexus, the tributaries of the common iliac arteries and from the fractured pelvic bones. An incredible amount of force is required to fracture the pelvis, and this can occur in road traffic accidents or from direct crushing due to a fall from height \[[@CR10]\]. The high kinetic energy required to disrupt the pelvis would inevitably result in other significant injuries to the trunk, head and extremities \[[@CR6], [@CR11]\]. Uncontrolled haemorrhage leading to shock is the main cause of mortality \[[@CR1], [@CR2], [@CR12]\]. Despite the advancement of trauma research and management of pelvic fracture, the mortality from pelvic fracture remains high \[[@CR1], [@CR13]\].
In our study, the patients were mostly males (64%) and were aged between 20--49 years old (71%). Forty-eight percent of the male patients were motorcyclists who were involved in road traffic accidents. This demographic trend is similar to studies done in the United States \[[@CR1], [@CR6]--[@CR9]\]. We believe that this is because male and young individuals indulge in more risk-taking behaviour \[[@CR9]\]. The relatively cheap price of motorcycles as compared to other vehicles in Singapore also makes them more attractive financially and readily accessible to young adults. In our data, we did not find any association between gender and the risk of mortality (χ^2^ test = 0.56, p = 0.45). A recent report on motorcycle safety in ASEAN countries has advocated stricter traffic regulations governing the use of motorcycles and education regarding responsible driving as two possible interventions to reduce the mortality rate \[[@CR14]\].
Pelvic fracture severity is an important factor in determining the risk of mortality. In our study, the odds of dying was significantly increased when the fracture severity was at least grade AIS 4 (see Table [3](#Tab3){ref-type="table"}). The odds of dying with severity grade AIS 4 and AIS 5 were 7.38 (95% CI 2.09--26.04) and 11.81 (95% CI 2.32--60.07, p \< 0.01), respectively, compared to severity grade AIS 2 as baseline (there were no patients with fracture severity AIS 1 in our study). Various authors \[[@CR6], [@CR15]\] have used different scales of measurements to ascertain fracture severity (for example, Young-Burgess classification, location of fracture, stability of pelvic ring, vector of injury etc.) and have concluded that fracture severity is associated with overall injury severity, but not with mortality. Poole et al. emphasized the importance of looking for other associated injuries that might ultimately caused the patient's demise \[[@CR6]\]. In our study, shock at presentation was not adjusted as a confounder in the association between pelvic fracture severity and death as it was classified (a priori) as a mediating variable in the causal pathway between pelvic fracture severity and death.
Road traffic accidents and falls from height were the most common mechanisms of injury, accounting for 52% and 44% of the study population, respectively. For patients involved in road traffic accidents, 35% were motorcyclists and 30% were pedestrians (see Table [1](#Tab1){ref-type="table"}). Our result concurs with the trends published in the medical literature \[[@CR4], [@CR16]--[@CR18]\]. This observation is expected as pedestrians and motorcyclists are usually not well protected, and they receive the full impact of the kinetic energy transfer. Thirty percent of motorcyclists and 39% of pedestrians died as a result of their injuries, contributing to two thirds of the overall mortality rate.
Injuries due to falls from height are related to vertical deceleration, where spinal and pelvic fractures as well as retroperitoneal bleeding are more common \[[@CR19]--[@CR21]\]. For this group of patients, at least half of them were males (53%). Of all the patients who sustained pelvic fracture from this mechanism of injury, 44% succumbed to their injuries. Interestingly, trauma due to falls from height is a unique phenomenon to Asian countries. It is an uncommon cause of pelvic fracture in the Western countries compared to road traffic accidents \[[@CR1], [@CR13]\]. There are a few explanations for this observation. Fang et al. discussed the association of falls from height in Taiwan with a positive history of mental disorders such as depression and schizophrenia \[[@CR22]\]. Unfortunately, we did not collect any data about the patients' psychiatric history. The high concentration of high-rise residential and commercial buildings in Singapore likely contributes to the high rate of falls from height. Our analysis revealed that there was a higher proportion of patients with ISS ≥25 in the falls from height group compared to those who sustained injuries from other mechanisms (52% vs. 48%, χ^2^ test = 4.8, p = 0.03). Forty-four percent of fall incidents occurred in public areas, whilst 18% occurred at home. Despite the higher ISS in this group of patients, there was no significant difference in the mean systolic blood pressure or GCS between the falls from height group and those from other mechanisms. Measures that can potentially alleviate the incidence of vertical falls include increasing the awareness of mental disorders such as depression and providing adequate social support. A change in building regulations to reinforce the need for safety barriers in high-rise buildings is also warranted.
Generally, the mortality rate among patients with pelvic fractures ranges from 9--30% \[[@CR8], [@CR16], [@CR23], [@CR24]\]. In our study, the mortality rate was higher, at 37% (see Table [1](#Tab1){ref-type="table"}). Fifty-one percent of deaths occurred in the ED. The mean ISS for our study population was 28, much higher than quoted by some authors \[[@CR7], [@CR8]\]. This could be due to various reasons. Firstly, the method used for capturing data may have been different. In our study, consecutive trauma patients presenting to the ED who were eligible were included in the study, whereas in other studies, patients were included only if they survived to surgical intensive care \[[@CR6]\]. This difference in methodology would have yielded different mortality rates. Secondly, transport time from the scene of the incident to the ED may also play a role. If the transport time was longer, more severely injured patients would have died en route to the hospital. On the other hand, if the transport time was shorter, more severely injured patients might have made it to the ED, but only to die later. The latter setting is applicable to our study, as Singapore's ambulance services adopt the 'scoop and run' policy.
The presence of abnormal physiological parameters is known to predict the risk of mortality \[[@CR15], [@CR23], [@CR25], [@CR26]\]. In our study, shock and coma at presentation were strong predictors of mortality. The adjusted odds ratios for mortality for shock and coma at presentation were 4.44 and 6.69, respectively (see Table [2](#Tab2){ref-type="table"}). As shock and coma at presentation confer very poor prognosis for patients, it is therefore imperative to prevent or delay the progression of such abnormal parameters. Training of the newer generation paramedic staff should include the recognition and management of shock at the scene of the incident \[[@CR27]\].
Various studies have concluded that injuries sustained concurrently with pelvic fracture contributed significantly to mortality \[[@CR6], [@CR11]\]. The results of our study support this observation. Ninety-seven percent of our study population had at least one concurrent injury. Of note, patients who sustained concurrent head and chest injuries had higher odds of dying. Table [2](#Tab2){ref-type="table"} shows the adjusted odds ratios for head and chest injuries after taking into consideration other significant predictors of mortality. Severe chest injuries affect the respiratory mechanics of the patients, impairing gas exchange, which is vital to the body that is already volume depleted. Significant head injuries increase intracranial pressure. This further impairs the body's attempt to cope with hypoxia and hypovolemia. It is therefore vital to adopt a multi-disciplinary approach in managing a polytrauma patient.
Pelvic fracture in the elderly provides a different challenge to the emergency physicians and trauma surgeons. The physiological changes associated with ageing make this population particularly vulnerable to injuries. Elderly patients with pelvic fracture have the worst outcomes, and they succumb to their injuries more easily despite aggressive resuscitation \[[@CR10], [@CR28], [@CR29]\]. Alost et al. observed that exacerbation of the elderly's premorbid condition is a contributory factor to mortality \[[@CR30]\]. In our study, patients aged more than 65 years old had a higher risk of mortality at 67% compared to the younger counterparts (χ^2^ test = 8.68, p = 003). This age group also has a higher proportion with ISS ≥25 (71% vs. 47%, χ^2^ test = 4.26, p = 0.04). The odds of dying for a 65-year-old patient with pelvic fracture was 3.96 (95% CI 1.46--10.69, p \< 0.01). However, this was confounded by the presence of shock and coma at presentation. The adjusted odds ratio was 2.52 (95% CI 0.73--8.64) and was not found to be significant.
Limitations {#Sec5}
===========
Our study is limited by its small sample size, which means that the study may not have enough power to detect small differences between groups. This problem is accentuated when stratification is done to look for the effects of confounding, as evidenced by the wide confidence intervals. The retrospective nature of our study also limits our ability to draw conclusions beyond an association of factors with mortality. Another important factor that was not considered in our study is the timing from arrival to the ED to interventions (such as external pelvic immobilization and angiography), which may behave as a confounder or an effect modifier in the association with mortality.
Conclusions {#Sec6}
===========
Pelvic fracture is a serious injury that is associated with significant morbidity and mortality. This is a result of the high energy transfer to the pelvic region. Road traffic accidents and falls from height are by far the most common mechanisms of injury. Pelvic fracture severity, shock and coma at presentation, and the presence of concurrent head and chest injuries are important predictors of mortality. It can be seen that the prevention of morbidity and mortality of pelvic fractures requires a multifaceted approach in terms of educating the public regarding injury prevention, as well as the need for paramedical and emergency care staff to recognize various prognostic factors and to mobilise resources to manage them.
I would like to thank Associate Professor Eillyne Seow and the staff of the Emergency Department, Tan Tock Seng Hospital, for their support and encouragement in making this research study a reality.
**Conflicts of interest** None.
**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.
The views expressed in this paper are those of the author(s) and not those of the editors, editorial board or publisher.
|
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"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
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Posteromedial fracture of the tibial plateau is a relatively common but overlooked injury, with devastating consequences in knee function and stability \[[@CR1], [@CR2]\]. The fracture mechanism is a combination of axial compression and varus stress with the knee in a semi-flexed position, producing an often grossly displaced condylar split fracture of tibial plateau oriented in the coronal plane \[[@CR1]--[@CR3]\]. Anatomical reduction of the articular surface, restoration of the normal posterior slope of the tibial plateau, and management of the posteromedial corner capsule-ligamentous structures are critical for a painless stable knee joint \[[@CR4], [@CR5]\].
Normally a posteromedially-based approach with direct reduction and rigid fixation using a buttress plate is considered a standard of care to treat an isolated posteromedial tibial plateau fracture \[[@CR6], [@CR7]\]. The optimal placement of the plate should be parallel to the main fracture plane, as described by Kfuri and Schatzker \[[@CR8]\]. However, the choice of approach (either Tscherne-Lobenhoffer or Luo), position of the patient, and type of implant are all variables determined by the surgeon based mainly on the fracture pattern and personal experience and confidence. In a biomechanical in-vitro strength analysis of four different fixation methods, Zeng et al. have shown that a posterior-based buttress technique using a 3.5-mm six-hole T-shaped plate is biomechanically superior than the other methods \[[@CR7]\]. Although not pointed out as a limitation, the authors tested only one fracture model using a posterior buttress plate construction.
To the best of our knowledge, a comparative evaluation between locked and non-locked small fragment straight implants used to buttress the posteromedial shear tibial plateau fragment was not investigated so far. Locking plates were primarily designed to address fragility fractures or high energy periarticular fractures. Despite all theoretical mechanical advantages of these implants, the question is whether they are absolutely needed for the fixation of posteromedial split wedge fractures. The aim of this study is to compare the biomechanical behavior of three different fixation constructions currently used for buttressing the posteromedial shearing tibial plateau fragment. Our hypothesis is that provided the plate is placed parallel to the main fracture plane, locking constructs have no mechanical advantage over conventional ones.
Methods {#Sec2}
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Posteromedial fracture preparation {#Sec3}
----------------------------------
Fifteen left synthetic tibiae (Model 1110, Synbone AG, Swiss) from a single manufacturing batch were used to create a posteromedial shear tibial plateau fracture.
A fracture line was draw based on the model designed for Zeng et al. \[[@CR7]\]. The cut was done with a band saw starting from the articular surface and exiting the posterior side of the synthetic model, making a sagittal angle of 75° (Fig. [1](#Fig1){ref-type="fig"}). Fig. 1A posteromedial fracture line was drawn with the articular fragment angle (α) of approximately −25° and the sagittal angle (β) equal to 75°. Note the orientation of the fracture on the axial view (**a**), internal oblique view (**b**), and coronal posterior view (**c**)
The fracture was directly reduced with a large pointed reduction clamp and fixed with three different posteriorly placed five-hole buttress plate. Five models were fixed with a one-third tubular plate (TTP), five models were fixed with a small fragment dynamic compression plate (DCP), and five models were fixed with a small fragment locking compression plate (LCP). The non-locked plates were from Ortosintese (Jaraguá, Brazil) and the locked plate was from DPS (Paoli, USA). The implants from both DCP and LCP groups were bent to fit the contour of the posteromedial tibial plateau; this was not done for the TTP group.
The first screw in all experimental groups was a non-locked cortical screw inserted 1-mm distal to the apex of the triangular fragment. The other screws were inserted in an alternate manner starting from the second more distal screw-hole. In the LCP group except form the first screw all other screws were locked. In all groups, screws were bicortical and directed for the anterior cortex. Anteroposterior (AP) and lateral fluoroscopic images were obtained for each tibial plateau-implant construct to check for any incongruency on the position of the screws (Fig. [2](#Fig2){ref-type="fig"}). Fig. 2**a** TTP specimen model, **b** DCP specimen model, and **c** LCP specimen model. Note the perfect contouring of the plates to the posteromedial surface of the tibial plateau. Anatomic reduction was warranted both by direct vision and fluoroscopic control
The models were sawed with a length of 170 mm for adjustment to the biomechanical testing machine.
Biomechanical testing {#Sec4}
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The tests were performed on an MTS 810 material testing system Model 318.10 with a maximum force capacity of 100 kN integrated to a FlexTest 40 digital servocontroller (MTS Systems Corporation, Eden Prairie, USA). A load cell with a capacity of 10 kN was used in the tests. The experiment was held on the Mechanical Testing Laboratory, Department of Manufacturing and Materials Engineering, Faculty of Mechanical Engineering -- UNICAMP (Campinas, Brazil).
The specimens were positioned vertically onto a test jig. A polypropylene plate measuring 20 × 15 × 3 mm was clamped on the upper side of the material testing machine and used as an applicator to deliver forces at the point of loading on the posteromedial tibial plateau (Fig. [3](#Fig3){ref-type="fig"}) \[[@CR9]\]. A vertical pre-load of 40 N was applied before the tests were carried out. The vertical load was applied to generate axial compression with a loading speed of 1 mm/minute. Fig. 3**a** The specimens were positioned vertically onto a test jig with a polypropylene plate measuring 20 × 15 × 3 mm clamped on the upper side of the material testing machine to deliver forces at the point of loading on the posteromedial tibial plateau. **b** LCP specimen model before testing
All groups were tested up to 2 mm displacement defined as subsidence of the posteromedial fragment (Stage 1). For the TTP and DCP groups the specimens were tested until failure load defined as bone-plate construction failure or catastrophic failure (Stage 2). Yield load was determined for the TTP and DCP groups at the point where the curve ceased to be linear and suffered an inflection (Fig. [4](#Fig4){ref-type="fig"}). Force versus displacement curves were obtained in the two stages of the experiment. Fig. 4Yield strength (green) vs ultimate strength (pink) of a specimen of DCP group at Stage 2. Yield load was determined for the TTP and DCP groups at the point where the curve ceased to be linear and suffered an inflection. The yield point was determined when there was a crossing between the dashed pink line and the green curve
Statistical analysis {#Sec5}
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All statistical calculations were carried out using SPSS Version 20.0 (SPSS Inc., Chicago, USA). Previous to the beginning of the experiment, the size of the sample was calculated by using the type-II error (beta-error analysis) for the student's t test, with a Cohen's d effect size of 0.2, founding a beta level (two-tailed hypothesis) of 0.967. This was considered adequate in terms of the number of plastic bone models used.
Descriptive statistics was used to determine ranges, means, and standard deviations.
Statistical analysis was performed using one-way analysis of variance (ANOVA one-way) and student's t-test for independent samples to compare stiffness (N / mm), maximum load up to 2 mm (N), energy (J), yield load (N), and maximum load to failure (N) between construction groups \[[@CR10]\]. A *p* value of \< 0.05 was set as the level of significance. Inferential analysis was composed by ANOVA one-way in order to compare stiffness (N / mm), maximum load up to 2 mm (N), and energy (J) between construction groups, and student's t-test for independent samples to compare yield load (N) and maximum load to failure (N) between TTP and DCP groups.
Normality test of Shapiro-Wilk and graphical histogram analysis were used to compare the scores in the sample to a normally distributed set of scores with the same mean and standard deviation \[[@CR11]\]. Data presented a Gaussian distribution by not rejecting the null hypothesis, assuming that the samples were normally distributed.
Results {#Sec6}
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Stage 1 -- subsidence of the posteromedial fragment {#Sec7}
---------------------------------------------------
There was no significant difference in stiffness (*p* = 0.89), maximum load up to 2 mm (*p* = 0.38), and energy (*p* = 0.36) among the three fixation constructions. Mean stiffnesses were 177 (CI 159--195) N/mm for TTP group, 188 (CI 168--208) N/mm for DCP group, and 183 (CI 150--215) N/mm for LCP group. Mean maximum load were 253 (CI 228--279) N for TTP group, 307 (CI 245--369) N for DCP group, and 273 (CI 213--333) N for LCP group (Fig. [5](#Fig5){ref-type="fig"}). Mean energy was 0.25 (CI 0.23--0.28) J for TTP group, 0.31 (CI 0.24--0.37) J for DCP group, and 0.28 (CI 0.22--0.34) J for LCP group. Data is summarized in Table [1](#Tab1){ref-type="table"}. Fig. 5Maximum load up to 2 mm (subsidence) for the three experimental groups (mean and CI 95%) Table 1Subsidence of the posteromedial fragment up to 2 mm (Stage 1 -- TTP vs DCP vs LCP group)Parameter / groupnmeanCI 95%minimummaximum*p value* ^*a*^Stiffness (N/mm) TTP5177159--1951552000.82 DCP5188168--208170224 LCP5183150--215146241Maximum load (N) TTP5253228--2792233010.38 DCP5307245--369211398 LCP5273213--333207377Energy (J) TTP50.250.23--0.280.220.300.36 DCP50.310.24--0.370.210.40 LCP50.280.22--0.340.210.38^a^ANOVA one-way
Stage 2 -- catastrophic failure {#Sec8}
-------------------------------
Yield load revealed significantly less yield strength for the TTP group as compared with the DCP group (*p* = 0.048). Mean yield load was 632 (CI 572--691) N for TTP group and 790 (CI 671--909) N for DCP group. There was no significant difference in maximum load to failure among the TTP and DCP fixation constructions (*p* = 0.16). Mean failure load was 736 (CI 634--838) N for TTP group and 882 (CI 735--1038) N for DCP group (Fig. [6](#Fig6){ref-type="fig"}). Data is summarized in Table [2](#Tab2){ref-type="table"}. Fig. 6Load-to-failure for the TTP and DCP experimental groups (mean and CI 95%) Table 2Catastrophic failure (Stage 2 -- TTP vs DCP group)Parameter / groupnmeanCI 95%minimummaximum*p value* ^*a*^Yield load (N) TTP5**632**572--691569732**0.048** DCP5**790**671--909577944Load-to-failure (N) TTP5736634--8386068750.16 DCP5882725--10386121113^a^Student t test for independent samples01.006
All specimens in the TTP group failed by a combination of overbending of the plate and proximal screws pull-out, whereas in DCP group specimens failed by fragmentation of the posteromedial tibial plateau fracture propagated through the proximal screw shafts. The failure mode of constructs is shown in Fig. [7](#Fig7){ref-type="fig"}. Fig. 7Mode of failure for the TTP and DCP constructs, represented by one specimen of each experimental group
Discussion {#Sec9}
==========
The main findings of the present biomechanical study are that there is no significant difference in stiffness, subsidence up to 2 mm, and energy between non-locked and locked fixation constructions tested for buttressing the posteromedial shearing tibial plateau fragment. Although not reached statistical significance, despite an adequate number of plastic bone models used, as calculated previously to the beginning of the experiment, specimens from the DCP group showed the stiffest bone-implant construction under axial loading, which may result from bigger cross-sectional area of the implant compared to the one-third tubular plate and the locking compression plate. TTP and DCP groups were further tested until catastrophic failure. Yield load revealed significantly less yield strength for the TTP group as compared with the DCP group. Y*ield* strength is a material property defined as the stress at which a material begins to deform plastically, suffering permanent irreversible damage. The yield strength would correspond to the yield load divided by the area of the specimen. Since we cannot define the area, we cannot determine this property. Therefore, instead of presenting the results in terms of strength (MPa), we present them in terms of load (N).
A previous biomechanical study on four different fixation methods for a posteromedial tibial plateau split fracture indicated that a posterior small fragment locked T-shaped buttress plate produced significantly greater stability in controlling the subsidence of the posteromedial fragment under axial loading \[[@CR7]\]. Zeng et al. have argued that osteopenia and fracture comminution should potentially affect the stability provided by non-locked plates, which rely on screw--bone interface. It should be noted, however, that a significant proportion of patients that suffer a posteromedial tibial plateau fracture are young and middle-aged male, with approximately one fifth presenting comminution of the articular surface \[[@CR1], [@CR2]\]. Moreover, as mentioned before the authors tested only one fracture model using a posterior buttress plate construction \[[@CR7]\].
Few studies have specific examined the posteromedial fracture of the tibial plateau, either as an isolated injury or associated with other proximal tibia fracture. Some authors have found this pattern in approximately 18% of isolated fractures and in near one third of bicondylar tibial plateau fractures \[[@CR1], [@CR3]\]. Hohl was the first to describe this injury as a split fracture of the posterior margin of the medial tibial plateau \[[@CR12]\]. More recently some authors have evaluated the morphologic characteristics of the posteromedial fragment in bycondilar injuries \[[@CR1], [@CR2]\]. Barei et al. observed an averaged compromise of approximately 58% of the articular surface of the medial tibial plateau with a mean posteromedial fragment height of 4.2 cm and a mean sagittal fracture angle of 81° \[[@CR1]\]. Higgins et al. noted more than 5 mm of articular displacement in 55% of cases with the posteromedial fragment exhibiting a vertical fracture pattern averaging 73° sagittal angle, highly indicative of shear instability and vertical displacement \[[@CR2]\].
There is a general consensus that the posteromedial fragment should be directly reduced through a posteromedially based approach and fixed with a buttress plate placed to the apex of the triangular fragment \[[@CR5], [@CR8]\]. However, the definition of which plate should be used continues a matter of debate with some authors reporting the placement of different plates in the tibial plateau for buttressing the posteromedial fragment. De Boeck and Opdecam used a posterior large fragment T-shaped anti-glide plate for seven patients, with excellent and good results and no complications \[[@CR13]\]. Bhattacharyya et al. reported on 13 patients with posterior shearing tibial plateau fractures treated through a posterior approach to the knee fixed with a 3.5-mm cloverleaf plate and found eight satisfactory results of nine responding patients \[[@CR14]\]. Brunner et al. presented a series of five patients treated by open reduction and internal fixation with a dorsal 3.5 mm anti-gliding plate as a buttress plate with all patients highly satisfied with the postoperative result \[[@CR15]\].
The present biomechanical study proves that there is no significant difference in stiffness, subsidence up to 2 mm, and energy between non-locked and locked fixation constructions. Although not reached statistical significance, specimens from the DCP group showed the stiffest bone-implant construction under axial loading, which may result from bigger cross-sectional area of the implant compared to the one-third tubular plate and the locking compression plate. TTP and DCP groups were further tested until catastrophic failure. Yield load revealed significantly less yield strength for the TTP group as compared with the DCP group. Y*ield* strength is a material property defined as the stress at which a material begins to deform plastically, suffering permanent irreversible damage. The yield strength would correspond to the yield load divided by the area of the specimen. Since we cannot define the area, we cannot determine this property. Therefore, instead of presenting the results in terms of strength (MPa), we present them in terms of load (N).
Our results show both that subsidence up to 2 mm and catastrophic failure are not influenced by the implant, either locked or non-locked, resulting in a relatively similar load transfer at the bone-plate interface. Previous studies demonstrated the importance of buttressing the medial tibial plateau split fragment with a plate, either isolated or associated to a lateral tibial plateau fracture \[[@CR7], [@CR9], [@CR16]\]. It is suggested that the decreased stress on the medial proximal triangular fragment is likely the consequence of better anchorage and load sharing provided by perfect contouring of the plate \[[@CR16]\]. In order to obtain maximum bone-plate interface, we suggest that the first screw inserted should be a non-locked cortical screw, approximately 1-mm distal to the apex of the triangular fragment.
There are few limitations of the present study. First, synthetic tibiae may not reflect the actual conditions of bone properties. However, similar biomechanical studies have demonstrated good reproducibility using plastic bones to evaluate different assemblies for fixation of medial tibial plateau fractures \[[@CR7], [@CR9]\]. Furthermore, it has been showed that plastic bone models present an advantage over fresh or frozen human bones because the setup variability of natural tibia axial stiffness is unacceptably high, indicating exceptional difficulty in obtaining reproducible bone alignment \[[@CR2], [@CR17]--[@CR19]\]. This, the use of composite tibiae allows small differences to be characterized significantly, even when a small sample is used. Secondly, the synthetic models used reproduced the properties of a normal rather than osteoporotic bone. Therefore, our findings cannot be extrapolated to a clinical situation of poor bone stock. It has been suggested that locking plates appeared less sensitive to bone quality as they rely on the screw-plate interface \[[@CR7]\]. Thirdly, we didn't evaluate the catastrophic failure for the LCP group. This happened due to limited financial resources to obtain a similar number of locked plates to evaluate this group on the second phase of the experiment. Nevertheless, the number of plastic bone models used were considered adequate. In addition, for consistency, due to the reduced sample size, the Kruskal-Wallis ANOVA nonparametric test was applied, which corroborated the conclusions reached by the parametric approach (this analysis was performed for verification purposes only). Finally, stiffness, subsidence, and energy for all three groups and catastrophic failure between non-locked constructions were statistically similar. It can be hypothesized that our findings were underpowered to determine a difference in this experiment and that more specimens would be required to detect a significant difference between groups. It should be noted however, that the differences between data for all variables studied were small and therefore unlikely to be relevant in a statistical scenario.
In conclusion, this study suggests that placement of either a locked or non-locked small fragment straight plate to buttress the posteromedial shear tibial plateau fragment has a similar biomechanical behavior. When the implant is positioned to buttress the shearing fragment it maximizes biomechanical stiffness.
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Conceptualization: VG, AV, ACS, EMS. Data curation: VG, AV, ACS, EMS. Formal analysis: VG, MK, WB. Funding acquisition: nil. Investigation: VG, AV, ACS, EMS. Methodology: VG, MK, WB, REP, HAK. Project administration: VG, MK, WB. Resources: VG, AV, ACS, EMS. Supervision: VG, AV, ACS, EMS. Validation: VG, REP. Visualization: VG. Writing -- original draft: VG. Writing -- review & editing: VG, MK, WB, REP, HAK. All authors read and approved the final manuscript.
The authors did not receive grants or outside funding in support of their research for or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.
This article does not contain any studies with human participants or animals performed by any of the authors.
On behalf of all authors, the corresponding author states that there is no conflict of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1-animals-09-00643}
===============
Indonesia is a major exporter of live reef fishes, especially groupers \[[@B1-animals-09-00643]\]. Various rules and regulations at national and provincial levels have been put in place; however, these regulations are poorly enforced and inadequate to support the sustainable management of these species \[[@B2-animals-09-00643]\]. High profits in the live reef food fish trade and weak surveillance by law enforcement officers motivate Indonesian fishers and traders to avoid compliance, encouraging the continued use of destructive fishing practices \[[@B3-animals-09-00643]\]. Consequently, excessive extraction levels are prevalent and widely reported \[[@B4-animals-09-00643]\]. Negative impacts from overfishing and destructive practices affect grouper stocks directly and indirectly through the degradation of coral reefs and their capacity to provide grouper habitat \[[@B5-animals-09-00643]\].
The leopard coral trout (*Plectropomus leopardus*), squaretail coral grouper (*Plectropomus areolatus*) and camouflage grouper (*Epinephelus polyphekadion*) are three of the 10 fishes most commonly sought by the live reef fish trade (LRFT) in Southeast Asia \[[@B6-animals-09-00643],[@B7-animals-09-00643]\]. These fishes have been intensively exploited throughout Indonesia over the past four decades \[[@B8-animals-09-00643]\]. The leopard coral trout contributes the highest volume to LRFT exports, followed by the squaretail coral grouper and camouflage grouper \[[@B7-animals-09-00643]\]. The squaretail coral grouper and camouflage grouper are relatively fast-growing species \[[@B9-animals-09-00643],[@B10-animals-09-00643]\], maturing early; however, the spawning aggregations of these species are easy to find and usually well known to fishers, making these groupers especially vulnerable to overfishing \[[@B10-animals-09-00643],[@B11-animals-09-00643],[@B12-animals-09-00643],[@B13-animals-09-00643],[@B14-animals-09-00643]\]. The high demand for leopard coral trout was initially prompted by its attractive body coloration (red skin and white meat), making this grouper a menu of choice for Chinese banquets, especially on the eve of the Chinese New Year, as the color red is a symbol of good fortune in Chinese culture \[[@B15-animals-09-00643],[@B16-animals-09-00643],[@B17-animals-09-00643]\]. Heavy exploitation of spawning aggregations can pose a severe threat to marine fishes, affecting reproductive success and the long-term sustainability of capture fisheries, and can eventually lead to the extirpation of fish stocks and cessation of aggregation at overfished sites \[[@B18-animals-09-00643],[@B19-animals-09-00643]\]. Based on the dramatic reduction in fish populations due to excessive extraction, the IUCN Red List assessments designated *P*. *areolatus* and *E. polyphekadion* as Vulnerable (VU A2bd) in 2018 \[[@B20-animals-09-00643],[@B21-animals-09-00643]\]. These assessments state that steps to improve the management of these species are urgently needed. Although the IUCN Red List assessment placed *P*. *leopardus* in the Least Concern (LS) category \[[@B22-animals-09-00643]\], the management of this species needs to be improved in order to prevent it qualifying for Vulnerable (or even Endangered) status. Although a number of studies from several countries have been conducted on the reproductive dynamics of these species \[[@B5-animals-09-00643],[@B9-animals-09-00643],[@B10-animals-09-00643],[@B23-animals-09-00643],[@B24-animals-09-00643]\], data on sexual patterns and spawning seasons in Indonesia are still lacking \[[@B9-animals-09-00643]\].
Size limitation is one of the least complicated fisheries management approaches to dealing with overfishing and is relatively straightforward to implement \[[@B25-animals-09-00643]\]. Most countries with a LRFT fishery (with the exception of Australia) lack regulations for the target species \[[@B9-animals-09-00643]\]. Australia has enacted regulations on allowable catch size, restrictions on fishing gear, recreational bag limits, and spatial--temporal closures for several groupers, including *P. leopardus* \[[@B15-animals-09-00643]\]. In practice, some live grouper traders in Indonesia have set a 500 g minimum weight limit for the live groupers which they purchase for the LRFT (Hj Said, *personal communication*, 18 April 2017). However, this weight limit does not as yet have any legal basis; furthermore, there is some uncertainty as to the likely effectiveness of this size limit (500 g), due to insufficient data on the average size of the fish caught and reproductive dynamics, including size at first maturity and spatial and temporal data on spawning aggregations \[[@B9-animals-09-00643],[@B26-animals-09-00643]\]. Therefore, it is important to understand the reproductive dynamics of the three most commonly traded groupers in the Indonesian LRFT (*P. leopardus*, *E. polyphekadion*, and *P. areolatus*) as a basis for sustainable fisheries management approaches.
This study aimed to determine important reproductive parameters (minimum size at first maturity, evidence of sex change and spawning seasons) to support sustainable fisheries management of three important highly exploited (including two near-threatened) groupers (*P. leopardus*, *E. polyphekadion*, and *P. areolatus*) in Eastern Indonesia. The data on reproductive biology were used to formulate recommendations regarding potential fisheries management approaches for the studied grouper species (leopard coral trout, squaretail coral grouper, and camouflage grouper) in Eastern Indonesia.
2. Materials and Methods {#sec2-animals-09-00643}
========================
Data on the weight (g) of traded groupers were collected from one private company (2015--2016) for leopard coral trout and squaretail coral grouper. Data on the weight of camouflage grouper were collected during 2016 from 5000 fishers in 61 locations within Eastern Indonesia (from East Kalimantan to Papua). Gonads of the three species studied (leopard coral trout, squaretail coral grouper, and camouflage grouper) were collected from groupers caught by hook and line during the reproductive season (between 24 October 2017, and 4 April 2018) in five areas within Eastern Indonesia. These areas were Kapoposang Marine Tourism Park, Takabonerate National Park, Wakatobi National Park, Kei Islands and Karas Islands ([Figure 1](#animals-09-00643-f001){ref-type="fig"}). The reproductive season for the three groupers was estimated based on previous studies. The leopard coral trout is reported to spawn around the new moon during September--November across much of its wide Indo-Pacific distribution, including the Takabonerate Archipelago \[[@B9-animals-09-00643],[@B26-animals-09-00643]\]. Squaretail coral grouper are reported as forming large spawning aggregations during September--February in Selayar \[[@B9-animals-09-00643],[@B26-animals-09-00643]\], as well as February--May \[[@B10-animals-09-00643]\] around the third quarter or new moon phase in Pohnpei \[[@B20-animals-09-00643]\]. Camouflage grouper spawning has been reported during October--November and January--March in Sinjai \[[@B26-animals-09-00643]\].
The gonads of the sampled groupers were extracted, weighed (±0.1 g) and preserved in 10% formalin. Gonad samples were transported by sea and airfreight from the sampling locations to the laboratory of the Balai Besar Veteriner, Maros, in South Sulawesi. Histological slides were prepared as follows. Tissue was excised from a central portion of one gonad lobe of each gonad. The samples were subsequently dehydrated through a series of increasing concentrations of ethanol, cleared in xylene, infiltrated, and embedded in paraffin wax. Transverse sections of 7 µm, made using a hand rotary microtome, were mounted on glass slides using Mayer's egg albumin. The samples were then rehydrated and stained with Harris hematoxylin, and counterstained with eosin. Both ovaries and testes were examined under a light microscope in the School of Biological Science, at The University of Hong Kong.
The histological structure of gonads extracted from the three studied groupers species was similar to that described for the Nassau grouper (*Epinephelus striatus*) ([Table 1](#animals-09-00643-t001){ref-type="table"}) \[[@B27-animals-09-00643]\]. Therefore, assignment of sexual categories and maturity development was based on a modification of the criteria developed for the Nassau grouper (*Epinephelus striatus*) \[[@B27-animals-09-00643]\]. The gonads were then classified as immature, or as one of four stages of sexual maturation, from 'resting' to 'post-spawning' ([Table 1](#animals-09-00643-t001){ref-type="table"}). When gonad tissue contained male (testes or seminiferous tubules) and female (ovarian gonadal structures include lamellae, lumen and numerous stages of oocytes of gonad maturation) characteristics, the gonad was classified as 'bisexual'; however, such characteristics could be unrelated to hermaphroditism \[[@B27-animals-09-00643],[@B28-animals-09-00643],[@B29-animals-09-00643]\]. Data on gonad development was limited to gonad morphology and features providing evidence of 'sexual change' in the samples, and were not related to sexual function \[[@B27-animals-09-00643],[@B29-animals-09-00643]\].
Whenever possible, the total length (TL in cm) and weight (whole body mass, W in g) of the groupers were measured. These data were used to calculate the length--weight relationship (W = *a*L*^b^*) parameters *a* and *b*. In the live reef food fish trade (LRFFT) in Indonesia, fish size (for buying and selling and for reporting) is commonly based on weight (whole body size), not length. The mean size at first maturity (Lm~50~) for female groupers of each species was estimated by fitting a log transformed regression function to the proportion of mature fish in each size cohort \[[@B30-animals-09-00643]\]. Because live groupers are typically measured by weight, while the regression used requires length, the weight data were converted to length using the length-weight relationship equation W = *a*L*^b^*, where W is fish bodyweight (g), L is total length (cm), *a* means a constant obtained from the intercept of the regression model, and *b* is the regression coefficient.
Participatory mapping interviews with the target of 50 fishers per site, for 5 sites, were used to obtain an initial indication of grouper spawning seasons at each site, as suggested by the spawning aggregation sites (SPAGs) monitoring manual from the Society for the Conservation of Reef Fish Aggregation (SCRFA) guidebook \[[@B31-animals-09-00643]\]. The respondents were asked questions regarding any evidence of spawning aggregation sites, types of grouper, spawning periods, water depth at spawning sites, and any changes in spawning aggregations over time. Interviews were conducted with fishers from different age groups and generations ([Appendix A](#app1-animals-09-00643){ref-type="app"}).
Additional data on spawning seasons were obtained from the Wakatobi National Park Authority surveys conducted during spawning seasons from 2005--2011 under a joint monitoring program (2005--2011) by The Nature Conservancy (TNC), the World Wild Fund (WWF) and the Wakatobi National Park Authority. Three spawning aggregation sites of the squaretail coral grouper were monitored using the methods in the SCRFA guidebook \[[@B31-animals-09-00643]\]. Underwater visual census surveys were conducted at least once every two months with each survey lasting one week starting from the waning gibbous moon phase. The surveyors collected data on species identity and fish behavior over the spawning period, such as aggression, courtship, visibly gravid fish, and spawning activity. They also estimated the sizes (total length) of individual fish and number of fish per species in each aggregation.
3. Results {#sec3-animals-09-00643}
==========
3.1. Histological Characteristics of Gonad Developmental Stages {#sec3dot1-animals-09-00643}
---------------------------------------------------------------
The sampled leopard coral trout gonads examined microscopically came from wild grouper caught in the study areas by fishermen, as follows: Kapoposang Marine Tourism Park (MTP) (*n* = 34), Takabonerate National Park (NP) (*n* = 18), Karas Islands (*n* = 31), and Kei Islands (*n* = 5). Squaretail coral trout gonad samples were collected from the Takabonerate National Park (*n* = 9), Wakatobi National Park (*n* = 15), and Karas Islands (*n* = 1). Camouflage grouper samples were also collected from the Takabonerate National Park (*n* = 15) and Wakatobi National Park (*n* = 5) ([Table 2](#animals-09-00643-t002){ref-type="table"}).
### 3.1.1. Leopard Coral Trout (*Plectropomus leopardus*) {#sec3dot1dot1-animals-09-00643}
All stages of leopard coral trout gonad maturation development were observed during this research with the exception of immature males. Reproductively inactive females had previtellogenic oocytes and included immature and resting mature females ([Figure 2](#animals-09-00643-f002){ref-type="fig"}A,B). The cell wall was still somewhat irregular in shape (but less so than oocyte stage I), and the nucleus began to be apparent with few or many pale spots in the lipid vesicles within. In mature active females, the oocyte cells contained vitellin globules, which stained moderately with eosin. The oocytes were double in size in the late mature stage, and the cell walls thickened. The vitellogenic oocytes become hydrated in the post-spawning stage. Male stage features were found in the gonads of larger coral trout specimens, some inactive primary stage oocytes 1/2 were recovered in this stage. Spermatocytes began to fill the lumen in the mature stage. After spawning, there were empty spaces in the lumen of male coral trout gonads ([Figure 3](#animals-09-00643-f003){ref-type="fig"}C,D). The bisexual phase was recorded in both small and large leopard coral trout size classes. Mature bisexual gonads contained stage 2 spermatocytes combined with a high abundance of stage 1/2, 3 oocytes ([Figure 3](#animals-09-00643-f003){ref-type="fig"}F). This condition is likely to be related to the transition stage from female to male, indicating that this species may be protogynous.
Sexual patterns, sex ratio, and approximate mean size of sexual maturation in the leopard coral trout were determined from the limited samples (*n* = *85*) observed in this study ([Figure 4](#animals-09-00643-f004){ref-type="fig"}A). The smallest sampled female with inactive phase gonads measured 24 cm TL and the smallest mature, ripe female measured 27 cm TL. The smallest mature, ripe male measured 36 cm TL. Coral trout in the smaller size classes, but with mature gonad development stages, were mostly from the Kapoposang MTP. For both male and female leopard coral trout, mature active (ripe) gonads were first observed in fish caught during October and continued until December, with the largest proportion of mature active fish observed in November (around 40% of all samples with mature gonads). Inactive females and males (mature, resting) were most common in December (50% of all mature, resting samples). In the leopard coral trout sampled, most inactive female gonads (immature and resting) were found in large individuals (42--51 cm TL). The histological data indicate that males have two pathway developments: direct from juvenile phase to male, or via sex change from adult female. These observations indicate diandric protogyny; however, due to the relatively small sample size, further sampling is needed to confirm whether this is indeed the case.
### 3.1.2. Squaretail Coraltrout (*Plectropomus areolatus*) {#sec3dot1dot2-animals-09-00643}
The number of squaretail coral trout gonad samples (*N* = 24) was low, but covered a wide range of size classes, enabling many stages of gonad development to be observed ([Figure 4](#animals-09-00643-f004){ref-type="fig"}B). Inactive female gonads contained the primary 1/2 and 3 stage oocytes. Mature active ripe female gonads contained a high abundance of vitellogenic oocytes; post-ovulatory follicles were observed at this stage, indicating spawning within the previous 24 h ([Figure 2](#animals-09-00643-f002){ref-type="fig"}C). Based on the interviews with fishermen, this species aggregates to spawn during daylight hours, beginning around noon and peaking in the late afternoon, from around 4 p.m. until dusk. The length of the smallest reproductively inactive female was 21 cm TL, and that of the smallest mature, ripe female was 33 cm TL. The smallest mature, ripe male measured 42 cm TL. There was no evidence of bisexuality at any gonad development stage, even in the larger-sized fish. The peak season based on gonad maturity was in December (50% of all mature gonad samples) for both males and females. The sexual pattern for this species is unresolved. Therefore, further research is urgently needed.
### 3.1.3. Camouflage grouper (*Epinephelus polyphekadion*) {#sec3dot1dot3-animals-09-00643}
Reproductively inactive, but clearly female and male, camouflage grouper were observed (*n* = 22) in the small size class (27--31 cm) TL ([Figure 4](#animals-09-00643-f004){ref-type="fig"}C). The smallest reproductively immature female was 21 cm TL in length, while the smallest mature active female measured 34 cm TL. The smallest immature male was 32 cm TL, and the smallest mature active male was 34 cm TL. This indicates that sexual differentiation tends to occur before the onset of gonad maturation, and sex reversal is unlikely. Although the number of fish sampled in this study was relatively low, the presence of immature males (and females) in the smaller size range (32--39 cm TL) and mature, resting female and males in substantially the same (larger) size range (34--80 cm TL for females and 34--55 cm TL for males) provides supporting evidence for an absence of sex reversal in this species. The highest percentage of mature fish (male and female, 50%) was observed in January.
3.2. Mean Size at Sexual Maturity {#sec3dot2-animals-09-00643}
---------------------------------
Length--frequency histograms of various maturity stages by sex ([Figure 4](#animals-09-00643-f004){ref-type="fig"}.) showed that the mature gonad development phases dominated in the grouper size-classes represented in the study sample. In leopard coral trout and squaretail coral trout, male gonads (testes) were more common in the larger size classes. In contrast, camouflage grouper males were also common in the smaller size classes, with a similar size distribution to females. Based on the fitted regression curve for each of these three species, the total length at 50% sexual maturation (Lm~50~) varied between species ([Table 3](#animals-09-00643-t003){ref-type="table"}).
Based on the fishers' interviews, the history of grouper fishing for the LRFT differs between the sampling sites, with some beginning to be exploited earlier than others. Hong Kong traders have been buying live reef fish caught in the Makassar Strait since the 1980s, including the Kapoposang Marine Tourism Park (MTP) site, followed by expansion to the Takabonerate National Park (NP) area and the Wakatobi (not yet a national park) around 1984. The trade continued to expand eastwards in Indonesia, reaching West Papua (Karas Islands) and the Southeast Moluccas (Kei Islands) in the 1990s. Each area has specific dominant grouper species; for example, the LRFT in Kapoposang and Kei is dominated by leopard coral trout, while in the Wakatobi and Takabonerate areas, the main species fished and traded are the squaretail coral trout and camouflage grouper. [Table 3](#animals-09-00643-t003){ref-type="table"} shows that fish at the most extended places have been extracted, related to a smaller size at maturity.
Based on the fitted regression curve applied to all specimens of each species (all sites pooled), the mean total lengths at 50% sexual maturation were as follows: for leopard coral trout Lm~50~ = 37.7 cm TL; for squaretail coral trout Lm~50~ = 36.6 cm TL; and for camouflage grouper Lm~50~ = 37.0 cm TL ([Figure 5](#animals-09-00643-f005){ref-type="fig"}).
For squaretail coral trout, the length of fish aggregating to spawn during spawning seasons over the period 2005--2011 was confirmed by the underwater census data from known aggregating sites in the Wakatobi NP. In fact, several species of grouper were seen to aggregate at these sites, including *Epinephelus fuscoguttatus*, *E. polyphekadion*, *P. areolatus*, and *Lutjanus bohar*. The only species observed which did not show any evidence of spawning behavior (such as aggression, courtship, and visibly gravid fish) was *Epinephelus polyphekadion*. The mean size of mature squaretail coral trout estimated visually at the spawning sites ranged from 23 TL cm to 73 TL cm ([Figure 6](#animals-09-00643-f006){ref-type="fig"}); the number of individual fish counted in one survey ranged from 2--92 individuals ([Figure 7](#animals-09-00643-f007){ref-type="fig"}) (Data from the Wakatobi National Park Authority, 2017).
3.3. Spawning Seasons {#sec3dot3-animals-09-00643}
---------------------
Based on the gonad development of the fish sampled, spawning appears to begin in late September and continues until early April, for all three grouper species in this study ([Figure 8](#animals-09-00643-f008){ref-type="fig"}). Ripe male and female leopard coral trout were found in the last quarter moon phase in both November and December. Mature ripe female and male squaretail coral trout were found in the last quarter moon phase in January. Mature male and female camouflage grouper were found over several months, and this was the only species studied with indications of reproductive activity in April, around the new moon.
These data were supported by interviews with fishermen (*n* = 228). According to the respondents, leopard coral trout spawn during October to January; squaretail coral trout from September to May, and camouflage grouper from October to April ([Table 4](#animals-09-00643-t004){ref-type="table"}). Data from the Wakatobi National Park Authority monitoring show squaretail coral trout spawning around the new moon, from November--December and March--May ([Figure 8](#animals-09-00643-f008){ref-type="fig"}).
3.4. Live Grouper Market Size {#sec3dot4-animals-09-00643}
-----------------------------
The size of live groupers in the LRFT is commonly recorded as weight rather than length. Data on the weight of commercial catch from 2015--2016 show that weight per fish for *P. leopardus* ranged from 213--6000 g (*n* = 1249) in 2015 and 500--2900 g (*n* = 1226) in 2016. The bodyweight of *P. areolatus* ranged from 500--7086 g per fish in 2015, and 500--3300 g per fish in 2016, with a statistically significant decrease in mean weight from 1083 g (*n* = 1140) in 2015 to 1046 g (*n* = 1042) in 2016 (*T*-test, d.f = 2136, *p* \< 0.025). *Epinephelus polyphekadion* is a relatively large species \[[@B24-animals-09-00643]\], and was on average significantly larger than leopard coral trout and *P. areolatus* (Kruskal--Wallis, d.f. = 2, *p* \< 0.000), with a mean weight of 1170 g per fish (*n* = 799) and a range from 500 g up to 5350 g per fish in 2016 ([Figure 9](#animals-09-00643-f009){ref-type="fig"}).
Based on the length--weight relationship conversion of the first length maturity of groupers, the mean weights at which these three species reach the first maturity are 759 g for *P. leopardus*, 771 g for *P. areolatus*, and 890 g for *E. polyphekadion*. Meanwhile the trade data show that the average sizes of traded fish are 892.3 g for *P. leopardus*; 975.3 g for *P. areolatus*; and 1051.3 g for *E. polyphekadion*. Thus, the mean size at first maturity is lower than the average size of fish in the commercial catch.
4. Discussion {#sec4-animals-09-00643}
=============
4.1. Sexual Patterns {#sec4dot1-animals-09-00643}
--------------------
Evidence of sex transition (presence of male gonad tissue in the late female stage of gonad development) \[[@B29-animals-09-00643]\] was observed in a leopard coral trout, TL = 42 cm ([Figure 5](#animals-09-00643-f005){ref-type="fig"}), but was not found in squaretail coral trout or camouflage grouper. Although the squaretail coral grouper males were in the large size classes, the sample size was not sufficient to draw any strong conclusion; the sexual pattern remains unresolved, but with strong suggestions of *hermaphrodite protogyny* \[[@B10-animals-09-00643]\]. The presences of small immature males in camouflage grouper indicate potential sexual differentiation before gonad maturation; in other words, it seems likely that the camouflage grouper does not undergo sex reversal. The sex pattern of the leopard coral trout is strongly suggested as *diandric protogyny* \[[@B33-animals-09-00643]\], meaning that male leopard coral trout can derive from immature or mature females. Most grouper species are known to change sex; the lower number of males compared to females in this research is consistent with the sex reversal processes. Considering that this is the first such research on these species in Eastern Indonesia, further research appears to be both warranted and urgent.
4.2. Spawning Period {#sec4dot2-animals-09-00643}
--------------------
Most groupers traded in the LRFT are intrinsically vulnerable to over-fishing, due to the large number of fish aggregating at predictable places and times \[[@B18-animals-09-00643]\]. This is known to be a particularly high risk factor for three grouper species (*P. leopardus*, *E. polyphekadion*, and *E. fuscoguttatus*) \[[@B12-animals-09-00643]\]. In this study, the spawning period of leopard coral trout lasted from October to January with a peak season in November and December around the last quarter moon phased, similar to the spawning period in Great Barrier Reef, Australia in September to December \[[@B33-animals-09-00643]\]. The spawning period of squaretail coral grouper lasts from September to May in the last quarter near new moon, with a peak season during January and February. This is similar to spawning patterns for this species reported from Komodo National Park \[[@B13-animals-09-00643]\], Papua New Guinea \[[@B34-animals-09-00643]\] and Pohnpei, Micronesia \[[@B10-animals-09-00643]\]. The camouflage grouper spawns for three consecutive months (February to April) in Pohnpei, Micronesia \[[@B24-animals-09-00643]\]. The gonad development data show that Camouflage grouper seem to have a spawning peak in late January and April during the last quarter moon phase, which has been known as a partial spawner \[[@B21-animals-09-00643],[@B24-animals-09-00643]\]. Based on the interviews with fishers (*n* = 174), camouflage groupers are occasionally seen in shallow water first in the last quarter period, and followed by tiger grouper and squaretail coral trout around the new moon.
4.3. The Minimum Size of Sexual Maturity {#sec4dot3-animals-09-00643}
----------------------------------------
The live reef food fish trade (LRFFT) management in Indonesia needs to achieve sustainable fisheries, preferably using simple management approaches that could be efficiently applied by stakeholders \[[@B25-animals-09-00643]\]. Live fish destined for the LRFFT are generally weighed but only rarely is the length measured. Therefore, the mean length at first maturity (Lm~50~) of the three economically important groupers in this study (leopard coral trout, squaretail coral trout, and camouflage grouper) needed to be converted to weight (using the L-W relationship based on samples from this study). The size of the three studied grouper species was consistent with previous studies on the mean size at first sexual maturity \[[@B10-animals-09-00643],[@B24-animals-09-00643],[@B33-animals-09-00643]\].
In this study in Eastern Indonesia, leopard coral grouper achieve 50% size of sexual maturity (Lm~50~) at 37.7 TL cm, which is comparable to the finding in the Great Barrier Reef, Australia \[[@B33-animals-09-00643]\], where the fish reached first sexual maturity at 35 cm TL. The first size at sexual maturity of squaretail coral trout was the same with the squaretail coral trout found in Pohnpei, Micronesia \[[@B10-animals-09-00643]\] around 36.6 cm TL. Our findings of the first length maturity are for camouflage grouper are bigger for camouflage grouper compare with the conspecifics in Pohnpei, Micronesia \[[@B24-animals-09-00643]\], who reported that the first length of maturity for the species was 27 cm TL smaller than found in Eastern Indonesia up to 36.95 cm TL cm ([Table 5](#animals-09-00643-t005){ref-type="table"}). Based on the length--weight relationship at first length maturity of groupers, the average weight of market size groupers tends to be above the weight at first maturity. Nonetheless, the perception of size limits among live reef fish stakeholders needs to be adjusted to accommodate the data on reproductive biology, with a minimum weight limit of 800 g for the two coral trout species and at least 900 g for the camouflage grouper. An across the board size limit of 500 g is not appropriate for these three grouper species, as at this size very few individuals will have had an opportunity to reproduce. One challenge is that plate-sized (under 1000 g and preferably closer to 500 g) fish are most highly-priced in the LRFFT, especially for groupers \[[@B35-animals-09-00643]\].
The fishery and trade stakeholders would like a single size limitation on wild-caught live groupers in order to make the regulations relatively easy to comply with and also to enforce. However, in Indonesia there are many grouper species, with different life histories and more importantly size at sexual maturity. Some grouper species exploited in Indonesia mature well below the existing 500 g, while others would not mature at 1000 g or more \[[@B9-animals-09-00643]\]. Thus, any legislation will be a compromise, endeavoring to balance the biological requirements of the species exploited with the "art of the possible" in terms of real-world fisheries management in a still developing country.
Lessons learned from the size regulations for the Napoleon wrasse *Cheilinus undulatus* are instructive, and strike a cautionary note. Under Ministerial Regulation Number 37/KEPMEN-KP/2013, the export of Napoleon wrasse is limited to fish in the 1000 g to 3000 g range. To date, this regulation remains poorly, or indeed not, implemented \[[@B36-animals-09-00643]\]. There is a mismatch between the size allowed under the regulation and the available fishery resources (*C. undulatus* population structure and dynamics). Furthermore, size affects the profitability of the trade. These days, consumers in Hong Kong and other LRFFT destination countries prefer plate-sized fish (one per person) rather than a large fish to share. Consequently, for larger fish species, regulations based on fish reproduction will encounter market preference resistance and thus be difficult to implement. For the three groupers in this study, the average size of fish in the LRFFT in Eastern Indonesia is larger than the size at first sexual maturity, which is a positive finding. However, combining the data on LRFFT fisheries history with the Lm~50~ data in [Table 1](#animals-09-00643-t001){ref-type="table"} indicates that the size at first maturity is lower (now) in areas where the LRFFT has been operating for longer. While it is not impossible that this could be a natural phenomenon or a chance result (especially as the sampling size was relatively low), this empirical correlation could be due to fishing pressure, and should be considered as a warning signal.
5. Conclusions {#sec5-animals-09-00643}
==============
The exploitation of LRFFT species in Indonesia needs to be properly recorded, including volume and composition (species and size classes). Such data are vital for sustainable fisheries management, including assessing resources and management effectiveness. In a multi-species multi-scale fishery, this is no easy task. In the meanwhile, as a measure suitable or data-limited fisheries such as the LRFFT in groupers, size or weight limits are an attractive option \[[@B37-animals-09-00643]\]. While the current (unofficial and partial) weight limit of 500 g could have a positive impact, especially for smaller sized grouper species, the minimum weight for the three groupers species studied should be readjusted to 800 g per fish for both dead (fresh/frozen) and live fishes. Such a regulation is urgently needed for grouper fishery and trade sustainability, and would benefit both the wild fish stocks and the people who depend on them for their livelihoods \[[@B4-animals-09-00643]\]. In view of the high value and volume of the trade in *P. leopardus*, as well as the ease of recognizing this fish, it would be possible to begin with a specific regulation for this species. As data on grouper size at first maturity and spawning periods improve in quantity and quality, spatial and temporal closures (at spawning aggregation sites during peak spawning seasons) are recommended. Further research on sex change is urgently needed, especially if there are concerns that the rate of male removal may be too high and could potentially result in sperm limitation during spawning due to low male abundance \[[@B38-animals-09-00643]\]. The change in utilization, from a no-take zone to tourism zone (for dive activity), could potentially be an option for some permanent or temporary closures. Where spawning locations are relatively safe, they could be attractive for visitors, and such a use could contribute to the livelihoods of the surrounding community
We would like to thank Yvonne Sadovy De Mitcheson for helping us to analyse the histological data. Special thanks to Heru Purnomo for useful data. Many thanks also to local fishers, traders, Taka Bonerate National Park Authority, Wakatobi National Park Authority, Kapoposang Marine Tourism Park for their generous help. Thanks to Mala, Marti, Adi Pranata Sopyan, Fajar Izas, and Salim who helped the author collect the gonad samples. This research article is a part of a Ph.D. Thesis and partly financed by PMDSU scholarship program held by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia with Grant Research Contract No: 132/SP2H/LT/DRPM/IV/2017.
M.K. conceived the idea for the manuscript and contributed to writing and editing of the paper and led the article. M.K., J.J. and N.N.K. critically revised the manuscript and added intellectual content. M.K. reviewed and approved the final version of the manuscript to be published.
This research and the APC was funded by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia grant number \[132/SP2H/LT/DRPM/IV/2017\].
The authors declare no conflicts of interest.
**Questionnaire for fishers**
Name:
Address (resident/not resident):
Age:
Last education:When the first time you catch live reef fish?Where you go to fishing live groupers (please mark in this map and tell me what kind of fish you will get there)?Is the place the same as your first time fishing live groupers? If not, please mark where the first time are you fishing live groupers?Did you ever see the fish aggregation? What kind of fish? How depth and when they aggregate? If any, please mark the place?When do you see, the fish came to aggregate? Please tell us the month and moon phase?Is there any missing place (a long time ago were many groupers there, and now the groupers were gone), if any, please mark the place?
Additional information:
{#animals-09-00643-f001}
{#animals-09-00643-f002}
{#animals-09-00643-f003}
{#animals-09-00643-f004}
######
Length at 50% maturity (Lm~50~) estimated for leopard coral trout (**A**), squaretail coral trout (**B**), and camouflage grouper (**C**) from Eastern Indonesia during the spawning season (*n* = 76). Percentages reflect the proportion of mature females (mature resting, ripe, and post-spawning) per size class. The function is: y = 100/1 + e^−1(Xmid−X0.5)/d)^.
{#animals-09-00643-f006}
{#animals-09-00643-f007}
{#animals-09-00643-f008}
{#animals-09-00643-f009}
animals-09-00643-t001_Table 1
######
Descriptive classification of maturity stages in female and male gonads of three grouper species based on histological characteristics, adapted from the classification for Nassau grouper (*Epinephelus striatus*) \[[@B27-animals-09-00643]\].
Gonad Development Histological Description
----------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Immature female The whole gonad was small and compact; the gonads dominated with primary growth stage 1/2 oocytes ([Figure 2](#animals-09-00643-f002){ref-type="fig"}A).
Mature resting female The oocytes stage 1/2 and three presented with occasional early stage 4 oocytes ([Figure 2](#animals-09-00643-f002){ref-type="fig"}B). This stage was mostly found between spawning seasons, but it was not possible to determine whether this stage of ovary had previously ovulated or was maturing for the first time. In this research, the mature resting female was dominated by large species.
Mature ripe female All gonads dominated with oocytes stage 1/2, 3, 4, and vitellogenic oocytes present in various proportions, the ovarian wall may become thin ([Figure 2](#animals-09-00643-f002){ref-type="fig"}C). There are only a few (if any) post-ovulatory follicles visible. At ovulation stage, hydrated eggs released from their follicular developed into the ovary lumen forming post-ovulatory follicles (that were only found around 24 h after ovulation) \[[@B32-animals-09-00643]\].
Post spawning female Gonad contains degeneration of vitellogenic oocytes and 1/2 stages oocytes and lamella wall becomes thick ([Figure 2](#animals-09-00643-f002){ref-type="fig"}D).
Bisexual Gonad contained stages 1/2 oocytes and1/2 spermatocytes (immature bisexual) ([Figure 3](#animals-09-00643-f003){ref-type="fig"}E); 3/4 oocytes and 3/4 spermatocytes (mature bisexual) appear together in similar amount ([Figure 4](#animals-09-00643-f004){ref-type="fig"}F).
Immature male All gonads were small and compact, contained a central lumen, and were dominated by seminiferous tubules. Scattered cysts of spermatocyte stage 1/2 were evident, but not common ([Figure 3](#animals-09-00643-f003){ref-type="fig"}A). An occasional stage 1/2 oocyte was present.
Mature resting male Gonad was dominated by early stages of spermatogenesis (i.e., spermatocyte stages 1 and 2) with scattered cysts of sperm increasingly evident as the spawning season approached. Occasional dispersed phase 1/2 oocytes were present. The testis lumen became smaller than in immature phase ([Figure 3](#animals-09-00643-f003){ref-type="fig"}B).
Mature ripe male Late stages of spermatogenesis were dominant and spread to fill almost all parts of the testis. Sperm occurred in expanded lobules, and spermatocyte stages 1 and 2 were relatively less abundant ([Figure 3](#animals-09-00643-f003){ref-type="fig"}C).
Post spawning male Most of the late stages of spermatogenesis have released. Spermatocyte stages 1 and 2 were relatively less abundant than the mature male phase. The gonad often had a thickened wall; there was space between the muscular wall and sperm ([Figure 3](#animals-09-00643-f003){ref-type="fig"}D).
animals-09-00643-t002_Table 2
######
Total sample of three groupers species per month and species during this study (*n* = 133). F refers to female, M refers to male, and T refers to transitional.
Species Sexual Differentiation 2017 2018 Total
----------------------------- ------------------------ ------ ------ ------- ---- ---- --- --- --- ---- ----
*Plectropomus leopardus* F 0 4 4 21 25 7 9 0 0 70
M 1 0 3 2 2 1 1 0 0 10
T 1 0 0 2 1 1 0 0 0 5
*Plectropomus areolatus* F 0 0 0 7 11 5 0 0 1 24
M 0 0 0 1 1 0 0 0 0 2
T 0 0 0 0 0 0 0 0 0 0
*Epinephelus polyphekadion* F 0 0 3 6 1 7 0 0 1 18
M 0 0 1 1 0 0 0 0 2 4
T 0 0 0 0 0 0 0 0 0 0
Total 133
animals-09-00643-t003_Table 3
######
Mean length at first sexual maturity of three grouper species from different areas in Eastern Indonesia, based on gonad samples collected during this study.
Species Name Sampling Area Lm~50~ (TL, cm) Estimated Weight (g) No. of Samples
----------------------------- ----------------- ----------------- ---------------------- ----------------
*Plectropomus leopardus* Kapoposang MTP 31.56 404.4 34
Takabonerate NP 37.18 670.3 14
Karas Islands 40.35 723.9 31
Kei Island 47.78 1150.3 5
*Plectropomus areolatus* Takabonerate NP 37.80 826.9 9
Wakatobi NP 40.20 948.4 15
*Epinephelus polyphekadion* Takabonerate NP 37.48 885.9 15
Wakatobi NP 40.90 1058.4 5
MTP, Marine; Tourism Park. NP; National Park; **TL,** Total Length.
animals-09-00643-t004_Table 4
######
Spawning aggregation periods according to fisher interviews: leopard coral trout, squaretail coral trout, and camouflage grouper in Eastern Indonesia. The aggregation sites are not listed here to protect the site.
Spawning Month/Location Spawning Month No. of Respondents
------------------------- ---------------- -------------------- ---------- -----
Kapoposang MTP Nov--Jan \- \- 30
Takabonerate NP \- Sept--Feb Jan--Apr 100
Wakatobi NP \- Nov--Dec, Feb--May Oct--Jan 48
Kei Islands Oct--Dec \- \- 24
Karas Islands \- Oct--Feb Nov--Dec 26
animals-09-00643-t005_Table 5
######
Representative of length at first maturity (Lm~50~) of three groupers species.
Species Name Lm~50~ (cm) Australia References
----------------------------- ------------- ----------- ------------ -----------------------------
*Plectropomus leopardus* 37.7 \- 35 \[[@B33-animals-09-00643]\]
*Plectropomus areolatus* 36.6 36.6 \- \[[@B10-animals-09-00643]\]
*Epinephelus polyphekadion* 36.9 27 \- \[[@B24-animals-09-00643]\]
|
{
"pile_set_name": "PubMed Central"
}
|
Background
==========
Quality and safety issues in healthcare continue to receive a growing amount of attention. The role of patient safety culture (PSC) is regarded as important for patient safety. A positive PSC is considered to guide many discretionary behaviours of healthcare professionals towards viewing patient safety as one of their highest priorities \[[@B1]\]. PSC is defined as the product of individual and group values, attitudes, perceptions, competencies, and patterns of behaviour, which determine the commitment to and the style and proficiency of an organization\'s health and safety management \[[@B2]\]. Although several measurement tools were developed, the Hospital Survey on PSC (HSOPS), which was developed by the U.S. Agency for Healthcare Research and Quality (AHRQ), is used widely all over the world \[[@B3]-[@B7]\]. Previous studies that used subjects from a country reported internal consistency and construct validity \[[@B3]-[@B7]\]. HSOPS was also used to assess the effect of interventions for improving patient safety such as encouraging teamwork within hospital units \[[@B8]-[@B11]\]. Although HSOPS subjectively evaluates safety-related responses, it did report on the relationship between clinical outcomes and the PSC values measured by HSOPS \[[@B11]\]. PSC may differ according to professions, units, hospitals, countries, and others. Although multiple studies have referred to PSC results obtained in other studies, few statistical analyses have been conducted \[[@B5]-[@B7],[@B12],[@B13]\]. Chen et al. (2010) used statistical analysis to compare Taiwanese and U.S. PSC, and highlighted some of the characteristics of Taiwanese healthcare workers \[[@B7]\]. However, the subjects originated from teaching hospitals, and the sample size appeared insufficient to understand the characteristics of Taiwanese healthcare workers. For international comparisons of PSC, diverging values should be considered because PSC could be affected by race, religion, healthcare system, and other similar variables. The PSC characteristics from countries with different values have not been explored sufficiently. We hypothesized that PSC characteristics were similar among East Asian countries, whose cultures were closely related, and different from those of western countries, whose cultures include heterogeneous customs such as interpersonal relationships. The aim of this study is to investigate and compare the PSC characteristics of Japan, Taiwan, and the U.S.
Methods
=======
For comparing PSC, we conducted a questionnaire-based, anonymous, and self-administered cross-sectional survey to healthcare workers in Japan and Taiwan. The HSOPS questionnaire was used to measure PSC in each country. Japanese and Taiwanese data were also compared with the U.S. '2010 HSOPS Comparative Database', which was provided by AHRQ. The survey period and recruitment strategy of hospitals and respondents in each country differed from one another; details are listed in Table [1](#T1){ref-type="table"}\[[@B2],[@B14],[@B15]\]. Only acute care hospitals participated in this study because the questionnaire was developed only for this type of hospital.
######
Materials and response rates
**Japan** **Taiwan** **U.S.**
---------------------------------- ---------------------------- ----------------------------------------------- ----------------------------------------------------------------
Recruiting strategy
Hospitals Voluntary participation^§^ Stratified sampling & Voluntary participation Voluntary participation^§^
Respondents in the hospital All healthcare workers A part of the healthcare workers^†^ All healthcare workers or a part of the healthcare workers^††^
Period of survey Jan 2009--Jan 2010 Jul 2007--Aug 2008 Jan 2006--Jun 2009
Number of participated hospitals
\<300 beds 3 23 702
300--499 beds 7 13 117
≥500 beds 4 38 65
Total 14 74 884
Number of the subjects
Distribution 10,466 11,692 827,424
Recovery 8,192 (78.3%) 10,289 (88.0%) 337,862 (40.8%)
Valid data^‡^ 6,963 (66.5%) 10,019 (85.7%) 291,341 (35.2%)
^§^Hospitals were not selected randomly and participation was voluntary.
^¶^Ten hospitals participated voluntarily and 64 were selected randomly.
^†^Depending on the number of beds in each hospital, 10--300 questionnaires were distributed.
^††^The questionnaires were distributed to all staff at 637 hospitals and to some staff at 247 hospitals.
^‡^Incomplete questionnaires were excluded from the analysis, and included surveys with less than an entire section completed, surveys with fewer than half of the total items completed, or surveys with identical responses to every item.
Measures
--------
HSOPS had 51 questions and assessed healthcare worker opinions or attitudes about patient safety issues, errors in hospital settings, or event reporting. It included 42 items that measure 12 sub-dimensions of PSC, 2 items regarding PSC outcome measures, and 6 items regarding background information of the respondent. Each item used a Likert scale of 5-point response options of degree of agreement (1: strongly disagree to 5: strongly agree) or frequency (1: never to 5: always). The questions related to the PSC outcome measures included both a Patient Safety Grade that asked respondents to provide an overall grade on patient safety in their respective departments (1: failing to 5: excellent) and a Number of Events that asked respondents to provide the number of events they reported during the previous 12 months (1: no event to 5: 21 events or more). The requested background information included years in current profession, hours of work per week, and direct interaction with patients \[[@B2],[@B15]\].
The sub-dimensions of PSC consisted of (1) Frequency of Events Reported, (2) Overall Perceptions of Safety, (3) Supervisor/Manager Expectations and Actions Promoting Safety, (4) Organizational Learning/Continuous Improvement, (5) Teamwork within Hospital Units, (6) Communication Openness, (7) Feedback and Communication about Error, (8) Non-punitive Response to Error, (9) Staffing, (10) Hospital Management Support for Patient Safety, (11) Teamwork Across Hospital Units, and (12) Hospital Handoffs and Transitions.
For each sub-dimension, the proportion of positive responses (percent positive score) was calculated for every respondent based on the AHRQ instructions, and it ranged from 0 to 1; higher scores indicated a more positive PSC. The total score of each sub-dimension, which was the sum of the item scores within each sub-dimension, was also calculated and ranged from 3 to 20; higher scores indicated a positive PSC. Although the percent positive scores were recommended by AHRQ for use in analysis, the total scores may have contained more respondent information because total scores reflect the 5-point response to each item. Results with greater accuracy may have been achieved if the total scores were used for the comparisons \[[@B2],[@B15]\].
The HSOPS questionnaire was translated into Japanese and Mandarin. For the Mandarin version, backward translation was performed to confirm that the accuracy of the questionnaire was maintained. In contrast, the Japanese version was translated by a panel comprising a bilingual English--Japanese translators and specialists in patient safety, and backward translation was not used; other experts in safety culture verified the accuracy of the translation \[[@B14]\].
Data analysis
-------------
To estimate internal consistency, we used Cronbach's *α* to calculate values with each sub-dimension of each country. The percent positive scores, total scores, and other items belonging to a particular sub-dimension were compared among Japan, Taiwan, and the U.S. using Tukey's honestly significantly different (HSD) test. In addition, the impact of each effect (Cohen's *d*) was calculated by dividing the difference in score means of 2 countries by the pooled standard deviation. In our study, an absolute value of Cohen's *d* of at least 0.5 indicated that the difference was significant. Chi-square tests were used to compare categorical variables. Statistical analyses were conducted using SPSS 19.0 (SPSS Inc.; Chicago, IL, USA).
Ethical concerns
----------------
According to the Ethical Guidelines for Epidemiological Research, which was drawn up by the Japanese government, the approval of the ethics committee was not required because it was an anonymous and self-administered survey with no intervention or mental anguish \[[@B16]\]. In Taiwan, the survey was approved by Institutional Human subject Ethic Committee of National Chung Cheng University.
Results
=======
The response rates for each country are listed in Table [1](#T1){ref-type="table"}. Respondent characteristics are shown in Table [2](#T2){ref-type="table"}. Among the respondents, a significantly lower proportion of nurses were included in the U.S. (36.6%) compared with Japan (58.1%, P \< 0.01) and Taiwan (57.0%, P \< 0.01). The proportion of U.S. respondents who worked fewer than 40 hours per week (40.4%) was significantly higher than that of either Japan (23.6%, P \< 0.01) or Taiwan (14.1%, P \< 0.01). The proportion in Japan who reported at least 1 adverse event or near-miss event during the previous 12 months (64.0%) was significantly higher than that in Taiwan (48.0%, P \< 0.01) and the U.S. (46.6%, P \< 0.01). With regard to Patient Safety Grade, the proportion of U.S. respondents who answered 'excellent' or 'very good' (70.8%) was significantly higher than that in Japan (44.6%, P \< 0.01) or Taiwan (37.7%, P \< 0.01).
######
Respondent characteristics
**Japan** **Taiwan** **U.S.**
--------------------------------------------------- ----------------------------- ------------ ---------- -------- --------- --------- --------
N (%) N (%) N (%)
Occupation Nurse^‡^ 4,047 (58.1) 5,714 (57.0) 106,710 (36.6)
Patient Care Assistant/Hospital Aide/Care Partner 228 (3.3) 148 (1.5) 16,529 (5.7)
Physician^†^ 597 (8.5) 961 (9.7) 11,881 (4.1)
Pharmacist 171 (2.5) 448 (4.5) 5,203 (1.8)
Dietician 221 (3.2) 31 (0.3) 1,739 (0.6)
Unit Assistant/Clerk/Secretary 237 (3.4) 166 (1.7) 17,982 (6.2)
Respiratory Therapist 0 (0.0) 38 (0.4) 6,710 (2.3)
Physical, Occupational, or Speech Therapist 133 (1.9) 253 (2.5) 8,109 (2.8)
Technician (e.g. EKG, Lab, Radiology) 543 (7.8) 718 (7.2) 32,097 (11.0)
Administration/Management 562 (8.1) 1,029 (10.3) 20,292 (7.0)
Others 187 (2.7) 277 (2.8) 54,028 (18.5)
No answer 37 (0.5) 236 (2.4) 10,061 (3.5)
Years in current profession Less than 1 year 605 (8.7) 896 (8.9) 17,977 (6.2)
1--5 years 2,142 (30.8) 3,687 (36.8) 72,504 (24.9)
6--10 years 1,366 (19.6) 2,643 (26.4) 49,500 (17.0)
11--15 years 891 (12.8) 1,413 (14.1) 36,127 (12.4)
16--20 years 617 (8.9) 695 (6.9) 32,174 (11.0)
21 years or more 1,005 (14.4) 508 (5.1) 68,607 (23.5)
No answer 337 (4.8) 177 (1.8) 14,452 (5.0)
Working hours in hospital Less than 20 hours per week 439 (6.3) 113 (1.1) 12,948 (4.4)
20--39 hours per week 1,206 (17.3) 1,303 (13.0) 104,958 (36.0)
40--59 hours per week 3,936 (56.5) 7,380 (73.7) 141,250 (48.5)
60--79 hours per week 513 (7.4) 728 (7.3) 12,136 (4.2)
80--99 hours per week 80 (1.1) 190 (1.9) 7,445 (2.6)
100 hours per week or more 13 (0.2) 83 (0.8) 343 (0.1)
No answer 776 (11.1) 222 (2.2) 12,261 (4.2)
Number of events reported in the past 12 months No event reports 2,428 (34.9) 4,894 (48.8) 147,892 (50.8)
1--2 event reports 2,609 (37.5) 3,201 (31.9) 80,018 (27.5)
3--5 event reports 1,362 (19.6) 1,091 (10.9) 35,716 (12.3)
6--10 event reports 377 (5.4) 349 (3.5) 12,552 (4.3)
11--20 event reports 73 (1.0) 101 (1.0) 4,587 (1.6)
21 event reports or more 35 (0.5) 71 (0.7) 3,021 (1.0)
No answer 79 (1.1) 312 (3.1) 7,555 (2.6)
Patient safety grade^††^ Failing 66 (0.9) 37 (0.4) 2,139 (0.7)
Poor 479 (6.9) 442 (4.4) 12,614 (4.3)
Acceptable 3,015 (43.3) 4,662 (46.5) 62,801 (21.6)
Very Good 2,803 (40.3) 3,345 (33.4) 130,707 (44.9)
Excellent 299 (4.3) 432 (4.3) 75,348 (25.9)
No answer 301 (4.3) 1,101 (11.0) 7,732 (2.7)
Total 6,963 10,019 291,341
^‡^Registered Nurse, Licensed Vocational Nurse, or Licensed Practical Nurse.
^†^Attending Physician, Staff Physician, Resident Physician, Physician in Training, Physician Assistant, or Nurse Practitioner.
^††^Self-appraisal of overall grade with regard to patient safety in respondent's unit or department.
Cronbach's α for each sub-dimension in Japan, Taiwan, and the U.S. was 0.47--0.88, 0.26--0.83, and 0.61--0.87, respectively (Additional file [1](#S1){ref-type="supplementary-material"}).
The mean total and percent positive scores based on sub-dimension are listed in Table [3](#T3){ref-type="table"}. The mean score differences among Japan, Taiwan, and the U.S. are shown in Table [4](#T4){ref-type="table"}. Although most pairs exhibited significant differences, only some sub-dimensions had pairs with significant differences according to Cohen's d. In Japan, 'Organizational Learning/Continuous Improvement' received the lowest rating among all 3 countries, and 'Hospital Management Support for Patient Safety' received a rating lower than that in the U.S. In Taiwan, 'Frequency of Events Reported' received the lowest rating among all 3 countries, and 'Communication Openness' received a lower rating than that in the U.S. In the U.S., 'Staffing' received the highest rating among all 3 countries.
######
Total scores and percent positive scores in each sub-dimension
**Total Score**^**†**^ **Percent Positive Score**^**††**^
-------------------------------------------------------------- ------- ------------------------ ------------------------------------ ---------------- ------ -------------- --------------
M SD 95% CI M SD 95% CI
Frequency of events reported Japan 11.92 2.97 (11.85--11.99) 0.68 0.41 (0.67--0.69)
Taiwan 9.33 2.50 (9.28--9.38) 0.33 0.39 (0.32--0.33)
U.S. 11.14 2.86 (11.13--11.15) 0.61 0.42 (0.61--0.61)
Overall perceptions of patient safety Japan 13.83 2.51 (13.77--13.89) 0.53 0.35 (0.52--0.54)
Taiwan 13.45 1.93 (13.41--13.49) 0.52 0.28 (0.51--0.52)
U.S. 14.34 3.14 (14.33--14.36) 0.63 0.34 (0.63--0.63)
Supervisor/manager expectations and actions promoting safety Japan 14.68 2.56 (14.62--14.74) 0.62 0.31 (0.62--0.63)
Taiwan 14.57 2.21 (14.52--14.61) 0.65 0.33 (0.64--0.65)
U.S. 15.43 3.19 (15.42--15.44) 0.74 0.33 (0.74--0.74)
Organizational learning/continuous improvement Japan 10.44 1.85 (10.39--10.48) 0.55 0.35 (0.54--0.56)
Taiwan 11.68 1.44 (11.66--11.71) 0.81 0.30 (0.80--0.81)
U.S. 11.32 2.03 (11.31--11.33) 0.72 0.34 (0.72--0.72)
Teamwork within hospital units Japan 14.91 2.65 (14.85--14.97) 0.70 0.34 (0.70--0.71)
Taiwan 15.50 2.27 (15.46--15.55) 0.79 0.30 (0.79--0.80)
U.S. 15.68 3.12 (15.67--15.69) 0.79 0.30 (0.79--0.79)
Communication openness Japan 10.38 2.23 (10.33--10.44) 0.49 0.37 (0.48--0.50)
Taiwan 9.70 1.90 (9.66--9.74) 0.38 0.35 (0.37--0.39)
U.S. 10.96 2.41 (10.95--10.97) 0.61 0.37 (0.61--0.61)
Feedback and communication about error Japan 10.73 2.34 (10.67--10.78) 0.53 0.39 (0.52--0.54)
Taiwan 10.16 1.86 (10.12--10.20) 0.44 0.33 (0.44--0.45)
U.S. 11.20 2.52 (11.19--11.21) 0.63 0.38 (0.63--0.63)
Nonpunitive response to error Japan 9.63 2.34 (9.58--9.69) 0.43 0.37 (0.42--0.44)
Taiwan 8.78 1.92 (8.74--8.82) 0.29 0.31 (0.29--0.30)
U.S. 9.42 2.75 (9.41--9.43) 0.42 0.39 (0.42--0.42)
Staffing Japan 12.16 2.63 (12.10--12.23) 0.37 0.27 (0.36--0.38)
Taiwan 12.15 2.57 (12.10--12.20) 0.36 0.31 (0.36--0.37)
U.S. 13.55 3.04 (13.54--13.56) 0.54 0.33 (0.54--0.54)
Hospital management support for patient safety Japan 10.24 1.98 (10.20--10.29) 0.52 0.37 (0.51--0.52)
Taiwan 10.57 1.81 (10.53--10.61) 0.58 0.36 (0.58--0.59)
U.S. 11.18 2.48 (11.17--11.18) 0.70 0.37 (0.70--0.70)
Teamwork across hospital units Japan 13.12 2.49 (13.06--13.18) 0.44 0.35 (0.43--0.45)
Taiwan 13.69 2.34 (13.64--13.73) 0.51 0.37 (0.50--0.51)
U.S. 13.66 3.04 (13.65--13.67) 0.55 0.37 (0.55--0.55)
Hospital handoffs and transitions Japan 12.61 2.52 (12.55--12.68) 0.35 0.36 (0.35--0.36)
Taiwan 12.67 2.52 (12.62--12.72) 0.39 0.36 (0.39--0.40)
U.S. 12.67 3.21 (12.66--12.68) 0.41 0.39 (0.41--0.41)
M, mean; SD, standard deviation; CI, confidence interval.
^†^Calculated as the sum of scores for each item within the same sub-dimension.
^††^Mean percentage of positive responses calculated according to AHRQ instructions for every respondent.
######
Comparison of total scores and percent positive scores from each sub-dimension across Japan, Taiwan and the U.S.
**Comparisons of Total Scores** **Comparisons of Percent Positive Scores**
-------------------------------------------------------------- --------------------- ---------- --------------------------------- -------------------------------------------- -------- ---------------- -------------------------- -------------- ------- -------- ---------------- ----------------
Country1 Country2 Difference of mean score Cohen\'s *d* *P* 95% CI Difference of mean score Cohen\'s *d* *P* 95% CI
(Country1-Country2) (Country1-Country2)
Frequency of events reported Japan Taiwan 2.59 0.96 ^†^ \<0.01 (2.48--2.69) 0.35 0.88 ^†^ \<0.01 (0.34--0.37)
Japan U.S. 0.78 0.27 \<0.01 (0.70--0.86) 0.07 0.17 \<0.01 (0.06--0.08)
Taiwan U.S. −1.81 −0.63 ^†^ \<0.01 (−1.87--−1.74) −0.28 −0.68 ^†^ \<0.01 (−0.29--−0.27)
Overall perceptions of patient safety Japan Taiwan 0.38 0.17 \<0.01 (0.26--0.49) 0.02 0.05 \<0.01 (0.00--0.03)
Japan U.S. −0.51 −0.16 \<0.01 (−0.61--−0.42) −0.10 −0.29 \<0.01 (−0.11--−0.09)
Taiwan U.S. −0.89 −0.29 \<0.01 (−0.97--−0.82) −0.12 −0.34 \<0.01 (−0.12--−0.11)
Supervisor/manager expectations and actions promoting safety Japan Taiwan 0.11 0.05 0.07 (−0.01--0.23) −0.02 −0.07 \<0.01 (−0.03--−0.01)
Japan U.S. −0.75 −0.24 \<0.01 (−0.84--−0.66) −0.11 −0.35 \<0.01 (−0.12--−0.10)
Taiwan U.S. −0.86 −0.27 \<0.01 (−0.94--−0.79) −0.09 −0.28 \<0.01 (−0.10--−0.08)
Organizational learning---continuous improvement Japan Taiwan −1.25 −0.77 ^†^ \<0.01 (−1.32--−1.17) −0.26 −0.80 ^†^ \<0.01 (−0.27--−0.24)
Japan U.S. −0.88 −0.44 \<0.01 (−0.94--−0.83) −0.17 −0.50 ^†^ \<0.01 (−0.18--−0.16)
Taiwan U.S. 0.36 0.18 \<0.01 (0.32--0.41) 0.09 0.26 \<0.01 (0.08--0.10)
Teamwork within hospital units Japan Taiwan −0.59 −0.24 \<0.01 (−0.71--−0.48) −0.09 −0.27 \<0.01 (−0.10--−0.08)
Japan U.S. −0.77 −0.25 \<0.01 (−0.85--−0.68) −0.09 −0.28 \<0.01 (−0.09--−0.08)
Taiwan U.S. −0.17 −0.06 \<0.01 (−0.25--−0.10) 0.00 0.00 0.99 (−0.01--0.01)
Communication openness Japan Taiwan 0.68 0.33 \<0.01 (0.59--0.77) 0.11 0.32 \<0.01 (0.10--0.13)
Japan U.S. −0.58 −0.24 \<0.01 (−0.65--−0.51) −0.12 −0.32 \<0.01 (−0.13--−0.11)
Taiwan U.S. −1.26 −0.53 ^†^ \<0.01 (−1.32--−1.21) −0.23 −0.63 ^†^ \<0.01 (−0.24--−0.22)
Feedback and communication about error Japan Taiwan 0.57 0.27 \<0.01 (0.47--0.66) 0.09 0.24 \<0.01 (0.07--0.10)
Japan U.S. −0.47 −0.19 \<0.01 (−0.54--−0.40) −0.10 −0.25 \<0.01 (−0.11--−0.09)
Taiwan U.S. −1.03 −0.41 \<0.01 (−1.09--−0.97) −0.18 −0.48 \<0.01 (−0.19--−0.17)
Nonpunitive response to error Japan Taiwan 0.85 0.41 \<0.01 (0.75--0.95) 0.13 0.40 \<0.01 (0.12--0.15)
Japan U.S. 0.21 0.08 \<0.01 (0.13--0.29) 0.00 0.01 0.59 (−0.01--0.02)
Taiwan U.S. −0.64 −0.24 \<0.01 (−0.71--−0.58) −0.13 −0.33 \<0.01 (−0.14--−0.12)
Staffing Japan Taiwan 0.01 0.01 0.96 (−0.10--0.13) 0.01 0.02 0.39 (−0.01--0.02)
Japan U.S. −1.39 −0.46 \<0.01 (−1.48--−1.30) −0.17 −0.52 ^†^ \<0.01 (−0.18--−0.16)
Taiwan U.S. −1.41 −0.46 \<0.01 (−1.48--−1.33) −0.18 −0.54 ^†^ \<0.01 (−0.18--−0.17)
Hospital management support for patient safety Japan Taiwan −0.32 −0.17 \<0.01 (−0.42--−0.23) −0.07 −0.19 \<0.01 (−0.08--−0.06)
Japan U.S. −0.93 −0.38 \<0.01 (−1.00--−0.86) −0.19 −0.51 ^†^ \<0.01 (−0.20--−0.18)
Taiwan U.S. −0.61 −0.25 \<0.01 (−0.67--−0.55) −0.12 −0.32 \<0.01 (−0.13--−0.11)
Teamwork across hospital units Japan Taiwan −0.56 −0.23 \<0.01 (−0.68--−0.45) −0.07 −0.19 \<0.01 (−0.08--−0.05)
Japan U.S. −0.54 −0.18 \<0.01 (−0.63--−0.45) −0.11 −0.29 \<0.01 (−0.12--−0.10)
Taiwan U.S. 0.03 0.01 0.68 (−0.05--0.10) −0.04 −0.11 \<0.01 (−0.05--−0.03)
Hospital handoffs and transitions Japan Taiwan −0.05 −0.02 0.55 (−0.18--0.07) −0.04 −0.11 \<0.01 (−0.05--−0.02)
Japan U.S. −0.06 −0.02 0.35 (−0.15--0.04) −0.05 −0.14 \<0.01 (−0.07--−0.04)
Taiwan U.S. 0.00 0.00 1.00 (−0.08--0.07) −0.02 −0.04 \<0.01 (−0.03--−0.01)
P, according to Tukey\'s honestly significant difference (HSD) test; CI, confidence interval.
^†^Cohen\'s d \> \|0.5\|.
The mean score for each item under each of the 5 sub-dimensions is shown in Figure [1](#F1){ref-type="fig"}, and the mean score differences among Japan, Taiwan, and the U.S. are shown in Additional file [2](#S2){ref-type="supplementary-material"}. In Taiwan, the scores for most items under sub-dimension 'Frequency of Events Reported' were significantly lower than the scores in other countries. In Japan, the mean score of A13, the item on evaluations of improvement effect, received the lowest rating among all 3 countries. In the U.S., the mean score of C2, the item regarding staff capacity to point out poor care of patients by other staff, received the highest rating among all 3 countries.
{#F1}
Discussion
==========
With regard to PSC outcome indicators, Japan had the highest proportion of respondents who had experienced event-reporting during the past 12 months, and the Patient Safety Grade was the highest in the U.S. Regarding PSC characteristics, an attitude of continuous improvement in Japan and the reporting of near-miss events in Taiwan were evaluated low among all 3 countries. In the U.S., staffing received a high rating.
PSC characteristics in each country
-----------------------------------
For continuous improvement of safety and quality, organizations need to construct a Deming Cycle or analyse the course of adverse events to mitigate recurrence \[[@B17]\]. However, in Japan, the sub-dimension 'Organizational Learning/Continuous Improvement' was rated low due to the lack of evaluation on the effectiveness of changes implemented to improve patient safety. In the past, these types of improvement assessments were absent in most U.S. hospitals as well \[[@B18]\]. The evaluation systems used for improvement, such as monitoring or benchmarking, require quality indicators. These evaluations require management and institutional support for patient safety because related activities may be taxing in the work place and may minimize productivity. Therefore, in Japan, the lack of 'Organizational Learning/Continuous Improvement' and 'Hospital Management Support for Patient Safety' may be related.
Under-reporting of adverse events in healthcare settings is a common problem throughout the world \[[@B19]\]. Several studies have revealed that healthcare workers often do not report adverse events for fear of management reaction, blame, or being deemed incompetent. A culture of non-punitive response to error is required in healthcare settings \[[@B19]-[@B21]\]. In Taiwan, the attitude towards reporting near-miss events, which is reflected in the evaluation of sub-dimension 'Frequency of Events Reported', received the lowest rating among all 3 countries. The sub-dimension 'Non-punitive Response to Error' also received a low rating in Taiwan. In Chinese societies, authoritarianism is considered an important feature of leadership; and higher ranking individuals tend to speak poorly of subordinates in an effort to maintain their own dominance \[[@B22]\]. In addition, 'face-saving' is also considered an important tenet of interpersonal interactions in East Asian societies, and healthcare workers in Taiwan may fear to 'lose face' by reporting events \[[@B21],[@B22]\]. This phenomenon may explain the lack of 'Communication Openness' in Taiwan; this sub-dimension includes several items that explore reporting possibilities with regard to a co-worker's faults. Taiwanese individuals may be more likely to avoid reporting the faults of co-workers because intra-group harmony and in-group solidarity are emphasized in East Asian societies, and saving face of a co-worker is important to Taiwanese healthcare workers \[[@B21],[@B22]\]. Those cultures in Taiwan also may affect attitudes toward reporting of near-miss events. Japanese healthcare workers were more likely to report near-miss events, despite Japan being an East Asian country. Japanese healthcare workers may have become familiar with event-reporting systems because nearly a decade has passed since all Japanese hospitals established an in-house, legally bound event-reporting system in 2002.
In the U.S., the sub-dimension 'Staffing' received high ratings because of the higher number of healthcare workers in U.S. hospitals than that in Japan and Taiwan. According to 'OECD Health Data 2010' and national statistics of Taiwan, the number of nurses per bed was 5 times higher (3.4) than that in Japan (0.7), and 4 times higher than that in Taiwan (0.8) \[[@B23],[@B24]\]. U.S. hospitals may encourage job-sharing and part-time schedules because the proportion of U.S. respondents who work fewer than 40 hours per week was significantly higher than that in Japan or Taiwan. By varying the number of part-time workers, U.S. hospitals may be better positioned to adapt the number of healthcare workers to sudden changes in demand of manpower. In U.S. hospitals, temporary nurses, who are identified as 'agency nurses' or 'travel nurses' are hired \[[@B25]\]. In the contrast, most hired nurses in the Japanese and Taiwanese hospitals are permanent staff.
Japanese healthcare workers had more event-reporting experience than Taiwan or the U.S. A possible explanation may lie in the high rating that Japan received for the sub-dimension 'Non-punitive Response to Error'; however, the U.S. exhibited a rating for this sub-dimension similar to that of Japan. Other U.S. factors, such as self-protection against legal action or job loss, may contribute to the smaller proportion of U.S. respondents who noted experiences of event-reporting. The small proportion of nurses in the U.S. respondent pool also may have affected this rating because, as is widely known, most event reports originate from nurses \[[@B26]\]. In addition, the definitions or perceptions of reportable events may differ across the 3 countries.
Challenges associated with PSC comparison
-----------------------------------------
In a cross-cultural study, the differences among societies were shown to be influenced by several factors, such as study design, traditional values, or socioeconomic status \[[@B22]\]. The first problem is translation accuracy of the questionnaire in the context of culture \[[@B22]\]. Most sub-dimensions, except 'Overall Perceptions of Patient Safety' in Taiwan, exhibit sufficient internal consistency. Relatively low levels of internal consistency related to 'Staffing' were common in all 3 countries, and similar problems were pointed out in other studies conducted outside the U.S \[[@B3],[@B6],[@B13]\]. The problem may not be translation but rather the items that comprise the sub-dimension 'Staffing'. Employment system characteristics of each country should be reflected in these items.
The second problem pertains to target population representation \[[@B27]\]. Taiwanese hospitals were selected according to stratified sampling techniques, but Japanese and U.S. hospitals participated voluntarily. The voluntary hospitals may be more aware of PSC, and thereby, the respondents in Japan and the U S may have better PSC compared with respondents in Taiwan. The U.S. response rate was lower than the response rate in Japan and Taiwan, and the characteristics of non-respondents were unknown \[[@B28]\].
The third problem is the central tendency response pattern \[[@B29]\]. For example, with regard to Patient Safety Grade, Japanese and Taiwanese respondents were more likely to choose 'Acceptable', which was the central option of the 5-point Likert scale. This selection pattern may lead to a skewed proportion of 'Excellent' and 'Very Good' responses in Japan and Taiwan that are lower than those in the U.S. In East Asian societies, people may be more inclined to offer ambiguous opinions because they fear that clear or extreme opinions occasionally may yield adverse effects on intra-group harmony and in-group solidarity \[[@B21],[@B22]\]. In this study, the percent positive score results were similar to total score results, which suggests that the effect of this central tendency was small.
The fourth problem is that the comparison was based on subjective evaluations of PSC. Subjective evaluation often is inconsistent with objective evaluations because it is influenced by multiple factors, including internal predispositions of respondents \[[@B30]\]. To identify the impacts of different PSC on objective outcomes of patient safety, we need tools such as Patient Safety Indicators. PSC in each country should be adjusted according to objective data with regard to patient safety.
Conclusions
===========
Healthcare workers in the U.S. were likely to evaluate their PSC as higher than that in Japan or Taiwan. The attitudes towards continuous improvement in Japanese healthcare workers and the reporting of near-miss events in Taiwanese healthcare workers were evaluated as low. The results of this study suggest that PSC varies among different countries, and the effective intervention to improve PSC should be developed with focus on the cultural background of the country. Further investigations with improved methodology and a common protocol will be required for accurate comparison of PSC among countries.
Competing interests
===================
The authors declare no competing interests.
Authors' contributions
======================
SF participated in study design and coordination, collected data, preformed statistical analysis, and drafted the manuscript. KS participated in study design and sequence alignment. SI collected data and performed statistical analysis. YW participated in sequence alignment. HCC provided data from Taiwan. TH conceived the study, participated in its design, 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/1472-6963/13/20/prepub>
Supplementary Material
======================
###### Additional file 1
Internal consistency of each sub-dimension of HSOPS in Japan, Taiwan and the U.S.
######
Click here for file
###### Additional file 2
Comparisons of scores by items in 5 sub-dimensions among Japan, Taiwan and the U.S.
######
Click here for file
Acknowledgements
================
This work was supported partly by the Grant-in-Aid for Exploratory Research (Grant No. 19659132) of the Japan Ministry of Education, Culture, Sports, Science and Technology. We would like to thank the hospitals that contributed to our study.
We obtained U.S. data for this analysis from the AHRQ Hospital Survey on the PSC Comparative Database. The database is funded by AHRQ and managed by Westat under contract number HHSA 290200710024C.
|
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Introduction
============
Pregnancy, childbirth and the transition to motherhood involve complex cognitive, affective, and behavioral changes that require restructuring goals, behaviors, and responsibilities to achieve a new conception of self ([@B57]). In addition to experiencing body changes, new mothers undergo the process of attaining their maternal identity ([@B47]). The transition to parenthood is widely considered a period of increased vulnerability that is often accompanied by stress ([@B60]). A mismatch between parents' perception of the available resources for meeting the demands of parenthood and the perceived demands of the parenting role can cause parental stress ([@B30]).
In particular, [@B3] conceived parenting stress as referring to specific difficulties in adjusting to the parenting role, reflecting parents' conscious perceptions of their child, their relationship with their child and themselves as parents. Parenting stress, as assessed by the Parenting Stress Index (PSI; [@B1]), one of the most widely used instruments, is considered a factor that influences parenting behavior and a determinant of dysfunctional parenting ([@B10]; [@B2]; [@B68]).
Most studies of psychological distress arising from the demands of parenting have investigated the impact of stress on the development of dysfunctional parent--child relationships and on adult and child psychopathology ([@B30]). Studies have largely focused on mothers' postnatal experience (i.e., [@B61]; [@B41]); less attention has been devoted to maternal prenatal characteristics associated with the subsequent onset of parental stress.
Studies of maternal prenatal predictors of postpartum parenting stress levels are few. The majority of studies investigate ante-partum depression and anxiety and postnatal parenting stress. [@B53] in a study aiming to identify risk factors predictive of prenatal depression, postnatal depression, and parenting stress, found that prenatal anxiety, prenatal low self esteem and prenatal depression were related to postnatal parenting stress, although the strongest predictor of parenting stress was postnatal depression. [@B59], in a study with pregnant women prospectively monitored for depression and anxiety during the third trimester and 3 and 6 months postpartum, found that prenatal depression and anxiety directly impact postnatal maternal parenting stress. In a recent longitudinal birth cohort study following sub-Saharan African women from the third trimester in pregnancy to 2 years postnatal and adjusting for confounders, [@B43] found that prenatal depression was associated with parenting stress, while prenatal anxiety was not. Another study focused on the role of a woman's own parenting history in postnatal parenting stress. Specifically, [@B40] examined the associations between perceived parental care and control in childhood assessed during the third trimester of pregnancy and maternal parenting stress at 7 months postpartum. A significant association was found between the maternal perception of parenting as characterized by low care or/and high control and postnatal parenting stress.
Researchers have emphasized the need to examine the relationships among the predictors of parenting stress to develop more comprehensive theoretical models ([@B2]; [@B61]). Furthermore, no studies have examined the association between postnatal PSI and maternal prenatal characteristics exclusively with samples of first-time mothers. The relevance of studies conducted with nulliparous women arises from research showing that first-time mothers have more difficulties coping with life changes than pluriparous women do ([@B65]; [@B27]). The transition from the known reality to a new reality that characterizes the experience of the transition to motherhood in nulliparous mothers brings profound changes affecting the reorganization of the self and of the infant's representations together with transformations in the relationship with one's partner ([@B56]; [@B55]; [@B57]; [@B31]).
The mother's own attachment pattern is considered a powerful predictor of future parenting. The attachment pattern reflects early experiences of handling distress. A secure attachment pattern has been associated with the ability to cope with distress and to adjust to the tasks of parenthood ([@B4]; [@B33]; [@B50]; [@B64]). [@B54] found that in the postnatal period attachment state of mind, along with the current experience between partners, contributed to their adjustment to the task of parenting in terms of parenting stress.
Having a baby is a powerful experience that, according to attachment theory, should activate the attachment system and the related behavioral systems, such as caregiving behavior ([@B13]). The transition to motherhood requires a new organization of mental life that is adapted to the reality of caring for an infant ([@B72]). The development of a tie between the mother and her fetus has been conceptualized and assessed as antenatal attachment. Specifically, [@B19] conceived the term "parent-to-infant attachment", to refer to the emotional bond or tie of affection experienced by the parent toward the infant ([@B21]). So, a key component of the construct seems to be the protection for the fetus, expressed by maternal disposition toward the fetus of knowing, being with, protecting, gratifying needs, and avoiding loss ([@B19]; [@B74]). Recent contributions have proposed that this bond, which involves attending to needs and providing protection, is indicative not of the attachment system but of the caregiving system ([@B38]; [@B14]; [@B74]). During pregnancy, antenatal attachment to the fetus is associated with several maternal characteristics, such as attachment style and the quality of the relationship with one's partner ([@B20]; [@B12]; [@B58]; [@B75]; [@B8]; [@B74]). In the postpartum period, studies have found associations between antenatal attachment and familial and parental functioning, such as the quality of mother--infant interaction and child attachment ([@B70]; [@B15]; [@B5]; [@B24]).
The transition to parenthood also requires adaptive changes in the couple's relationship ([@B45]). As observed by [@B32], if prospective parents feel emotionally distant from and unsupported by their partners, then their adjustment to parenthood is likely to be negatively affected. Studies show that family functioning contributes to parenting satisfaction and perceptions of negative marital quality is associated with higher degrees of parenting stress ([@B46]; [@B61]; [@B69]). In particular, in a longitudinal study with a sample of first-time parents, [@B60] found that poor relationship functioning at mid-pregnancy predicted vulnerability to postnatal distress. The quality of dyadic adjustment during the transition to parenthood has also been associated with attachment patterns ([@B62]; [@B73]; [@B63]).
On the basis of the paucity of studies on maternal prenatal predictors of postnatal parenting stress and the lack of research conducted exclusively with first-time mothers, the aims of the present study were:
1. to investigate the role of certain psychological aspects of maternal functioning during the transition to motherhood in a sample of first-time mothers;
2. to assess their unique role in maternal postnatal adjustment at the very beginning of the relationship with the newborn.
Specifically, using an observational prospective study with two time periods (pre -- and postnatal), this paper explored the following in a group of nulliparous women:
1. the associations between maternal attachment style, maternal antenatal attachment to the fetus (MAAS) and dyadic adjustment in the prenatal period;
2. how maternal parenting stress, as assessed 3 months after delivery, can be predicted by each of the prenatal variables investigated.
On the basis of the reported studies, a relationship among the mother's attachment pattern, the emotional bond with the fetus and the quality of the relationship with the partner was expected. Furthermore, a prediction of each of these prenatal variables of the onset of postnatal maternal parenting stress was hypothesized.
Materials and Methods {#s1}
=====================
Procedure and Participants
--------------------------
An observational prospective study focused on the investigation of certain psychological factors contributing to the construction of the caregiving system in nulliparous women and their power in predicting parenting stress with two time periods, before the child's birth, -- at the 7th months of gestation (32nd weeks) -- and 3 months after the child's birth was used.
The study was approved by the University of Perugia Ethics Committee and was conducted in accordance with the Helsinki Declaration.
Participants were enrolled at the Operative Unit of Obstetrics and Gynecology (OUOG) of a hospital in central Italy. The choice of the hospital resulted from the availability of the unit staff to participate in the study. Pregnant women in this unit received antenatal care from obstetricians. During childbirth classes, obstetricians invited pregnant women to participate in the study, providing a preliminary information session on the aims and two-step methodology of the research. The obstetricians received preliminary training by a study researcher to make them feel confident in responding to any women's questions about the study and its procedures.
For the enrollment, because of the use of questionnaires, the inclusion criteria required the women to be able to read and understand Italian; furthermore, because of the specificity of the questionnaire regarding the couple relationship, the women needed to have a romantic partner and to be in the 32nd weeks of gestation (plus or minus 1 week). This point of pregnancy was chosen because of the evidence of increased expectations regarding the unborn child during this phase of pregnancy as well as because of the increase of fetal movements ([@B55]).
After this presentation, the women were given a packet that included the consent form to participate to the first phase of the study and a consent form to be contacted for the second phase. At approximately the 32nd weeks of gestation, the women who had given written informed consent completed the prenatal measures (including a questionnaire on socio-demographic information) at the hospital before the beginning of a childbirth class. The time needed to complete the questionnaires in the first phase was approximately 50 min. Approximately 3 months after their children's birth, the women who had given consent to participate in the second phase of the study, were invited by phone to return to the hospital to complete the postnatal questionnaire. The time needed for this second phase was approximately 20 min.
A total of 130 packets were distributed; 95 (73%) women agreed to participate in both phases of the study; 5 (5%) questionnaires were not fully complete at the first step and were then excluded; and of the 90 (95%) women who agreed to participate, 20 (22%) refused to participate in the second phase when contacted because they could not reach the hospital. Only women who had completed both the first and second phase measures were included in the data analysis.
Sample
------
The total sample consisted of 70 women recruited at the OUOG of a hospital in central Italy. All the women were nulliparous and were in the 32nd weeks of gestation (plus or minus 1 week). The mean age of the women was 32.75 (*SD* = 4.84); they belonged to a medium socio-economic status (SES) level (Mean = 36.67; *SD* = 12.26). Other socio-demographic information are reported in **Table [1](#T1){ref-type="table"}**.
Measures
========
Prenatal Measures
-----------------
### Socio-Demographic Questionnaire
This series of questions included the participants' age, level of education, employment status, family structure and the duration of the marital/conjugal relationship. Familial SES was calculated using the Hollingshead Index of Social Position (Hollingshead, unpublished manuscript).
### Attachment Style Questionnaire (ASQ; [@B34])
The ASQ is a 40-item Likert-type self-report questionnaire designed to measure five dimensions of adult attachment that are central to [@B44] and [@B9] conceptualizations of attachment: Confidence in Self and Others (eight items), Discomfort with Closeness (10 items), the Need for Approval (seven items), Preoccupation with Relationships (eight items), and Relationships as Secondary (sevan items). Each item is rated on a 6-point scale ranging from 1 ("totally disagree") to 6 ("totally agree"). The ASQ has shown adequate reliability ([@B34]; [@B35]), with Cronbach's alpha coefficients for the five scales ranging from 0.81 to 0.87. According to previous studies ([@B4]; [@B33]), two major factors were computed for these scales.
### Discomfort with Closeness and Relationship Anxiety
The former (16 items) measures the tendency to be uneasy with intimacy and dependency in relationships; the latter (15 items) measures concerns about the attachment other's feelings of love and fears of being rejected. In the present study, the Italian version of this measure was used ([@B36]), with internal consistency in terms of Cronbach's alpha coefficients of 0.80 and 0.85 for the two major factors.
### Maternal Antenatal Attachment Scale (MAAS; [@B19])
Developed to measure the emotional bond between a pregnant woman and her unborn child, the MAAS consists of 19 items focused on the past 2 weeks rated on a 5-point scale. Two scales Attachment Quality (AQ) and Attachment Intensity (AI) are combined to obtain a total score. The two scales, respectively, measure the quality of the emotional bond AQ and the time spent in attachment mode (the intensity of preoccupation). The internal consistency of the instrument is acceptable ([@B19]). In the present study, the Italian version of the MAAS was used ([@B67]), with an internal consistency in terms of Cronbach's alpha of 0.81 for the total score, of 0.80 for AQ scale, and of 0.82 for AI scale.
### Dyadic Adjustment Scale (DAS; [@B71])
Developed to measure conjugal adjustment, the DAS can be used with married or unmarried couples engaged in a dyadic romantic relationship. Consisting of 32 items, the scale has a range of scores from 0 to 151. Lower scores indicate distress and divergence in the dyadic relationship. The DAS has shown adequate psychometric properties and good internal consistency (α = 0.95; [@B16]). In the present study, the Italian validated version of the DAS was used ([@B37]). The internal consistency coefficient in terms of Cronbach's alpha for the total score was 0.85.
Postnatal Measure
-----------------
### Parenting Stress Index: (PSI-SF; [@B1])
This self-report scale was developed to measure stress associated with the parenting role and is used as a screening instrument for dysfunctional parenting. It consists of 36 items rated on a 5-point scale indicating the degree to which each item has been a problem for the parent during the past week. The composite total score range is between 36 and 158, where lower scores indicate lower overall levels of parenting stress. The PSI-SF has shown good overall psychometric properties ([@B1]). The internal consistency reliability for the composite total score was reported by the author to be 0.91. The Italian validated version of the PSI-SF ([@B42]) was used in this study. The internal consistency coefficient for the present sample was 0.89 for the total score.
Data Analysis
=============
Standard descriptive statistics in the form of means and standard deviations or frequencies and percentages were used to summarize the sample's socio-demographic characteristics and used in the measures assessed. A series of one-sample Kolmogorov--Smirnov tests (Z), were conducted and showed that the measures were distributed normally. Pearson's correlation analyses were conducted on the prenatal measures to examine the associations between maternal attachment style, MAAS and dyadic adjustment. *p*-values of 0.05 or less were first identified as statistically significant; Bonferroni's correction was applied, with critical alpha value set to 0.001 (0.005/25 correlations). The effect size of the correlation was classified according to [@B17]: low effect size ≤0.30; medium effect size = 0.31--0.50; and large effect size ≥0.50. To measure the single contribution of each of the prenatal measures to the prediction of postnatal parenting stress, a regression analysis was conducted. Because of the paucity of the studies on maternal prenatal characteristics associated with the subsequent parental stress, the regression analysis was conducted separately for each of the variable investigated, in order to test the existence of each variable's unique effect, as a first investigation of their specific contribution. In the first and the second model two Multivariable Linear Regressions were performed separately in order to test the effect of the two factors of maternal attachment (Discomfort with Closeness and Relationship Anxiety) and of the maternal antenatal attachment AQ and AI. A Univariate Regression was also performed for testing the effect of conjugal adjustment. The data were analyzed using SPSS version 21.0.
Results
=======
Descriptive statistics for the sample socio-demographic characteristics are reported in **Table [1](#T1){ref-type="table"}**.
######
Descriptive statistics for the socio-demographic characteristics.
*M* *SD*
------------------------------------------------ --------- -------
**Socio-demographic variables**
Age 32.75 4.84
SES 36.67 12.26
Time length of the couple relationship (years) 3.15 1.48
**Family situation** ***N*** **%**
Married and cohabiting 45 64.3
Unmarried and cohabiting 25 35.7
Means (*M*) and standard deviations of the pre-natal and post-natal measures are reported in **Table [2](#T2){ref-type="table"}**.
######
Descriptive statistics for the antenatal and postnatal measures.
Pre-natal measures *M* *SD* Range Z *p*
------------------------ -------- ------- --------- ------- -------
ASQ -- DC 48.89 8.41 28--68 0.134 0.200
ASQ -- RA 41.67 9.03 17--61 0.147 0.200
MAAS -- TOT 78.74 4.62 66--88 0.198 0.117
MAAS -- AQ 46.78 2.28 39--50 0.305 0.112
MAAS -- AI 30.28 3.46 23--37 0.305 0.200
DAS TOT 121.84 11.59 75--146 0.186 0.321
**Post-natal measure**
PSI-SF, TOT 60.48 14.54 36--101 0.169 0.212
ASQ -- DC, Attachment Style Questionnaire, Discomfort with Closeness; ASQ -- RA, Attachment Style Questionnaire, Relationship Anxiety; MAAS -- TOT, Maternal Antenatal Attachment, Total Score; MAAS -- AQ, Maternal Antenatal Attachment, Attachment Quality; MAAS -- AI, Maternal Antenatal Attachment, Attachment Intensity; DAS TOT, Dyadic Adjustment Scale Total Score; PSI -- SF, TOT, Parenting Stress Index -- Short Form, Total Score.
Regarding MAAS, data were compared with a previous study conducted with women at 19--23 weeks of gestation in terms of confidence interval. Previous data revealed a slightly lower mean total score (*M* = 75.7, *SD* = 8.15), 95% CI (73.29, 78.11) than that obtained for this sample ([@B67]). This was perhaps due to the specific time of measure in the present study: the last trimester of pregnancy is a time that is particularly rich in emotions and fantasies because of the increase in fetal movements ([@B6]; [@B55]). The dyadic adjustment score for this group had a mean value of 121.84 (*SD* = 11.59). The data were consistent with previous Italian data on nulliparous women ([@B64]). With regard to postnatal parenting stress, the mothers in the sample showed a mean value of 60.48 (*SD* = 14.54). Compared with the normative data for the age subgroup (1 month/2 years and 11 months) extracted from the total sample of the Italian validated version of the scale, the mean score for the present sample showed a lower value than the normative Italian sample (*M* = 69.27, *SD* = 16.91); 95% CI (68.13, 70.42; [@B42]).
Prenatal Measures
-----------------
The correlation analysis revealed the existence of significant associations between the antenatal measures after Bonferroni's correction was applied (**Table [3](#T3){ref-type="table"}**). According with Cohen's criteria ([@B17]), the effect sizes for the significantly correlated measures were generally moderate to large. The correlations between mothers' attachment style (ASQ) and MAAS revealed the existence of a significant small negative association of Relationship Anxiety with the MAAS -- AQ score (*r* = -0.361, *p* = 0.001). Relationship Anxiety was also negatively correlated with dyadic adjustment (DAS TOT; *r* = -0.485, *p* \< 0.001). The total score for dyadic adjustment (DAS TOT) was strongly and positively correlated with MAAS -- TOT (*r* = 0.447, *p* \< 0.001) and with AQ (*r* = 0.528, *p* \< 0.001). The smaller low correlation for the AI subscale (*r* = 0.256, *p* = 0.033) was not significant after Bonferroni's correction.
######
Correlations between antenatal measures.
MAAS -- TOT MAAS -- AQ MAAS -- AI DAS TOT
------------- ------------- ------------ ------------ -------------
ASQ -- DC -0.189 -0.197 -0.127 -0.350^∗^
ASQ -- RA -0.256 -0.361^∗^ -0.104 -0.485^∗^
MAAS -- TOT \- 0.760^∗^ 0.900^∗^ 0.;447^∗^
MAAS -- AQ \- 0.425^∗^ 0.528^∗^
MAAS -- AI \- 0.256
ASQ -- DC, Attachment Style Questionnaire, Discomfort with Closeness; ASQ -- RA, Attachment Style Questionnaire, Relationship Anxiety; MAAS -- TOT, Maternal Antenatal Attachment, Total Score; MAAS -- AQ, Maternal Antenatal Attachment, AQ; MAAS -- AI, Maternal Antenatal Attachment, Attachment Intensity; DAS TOT, Dyadic Adjustment Scale Total Score.
∗
p \< 0.001 \[Bonferroni correction (0.05/25 correlations; N = 70\].
Prediction of Postnatal Stress
------------------------------
Results of the regression analysis showed that mother's attachment style explained 38.1% of the variance (*R*^2^ = 0.381, *F*(2, 68) = 3.699, *p* = 0.047). In detail, Relationship Anxiety (β = 0.516, *SE* = 0.496, *t* = 2.271, *p* = 0.041) significantly predicted postnatal parenting stress, whereas Discomfort with Closeness (β = 0.345, *SE* = 0.351, *t* = 1.519, *p* = 0.155) did not.
Maternal antenatal attachment accounted for 44.5% of the variance in parenting stress (*R*^2^ = 0.445), which was significant \[*F*(2, 68) = 4.807, *p* = 0.029\]. AQ (β = -0.737, SE = 1.898, *t* = -3.094, *p* = 0.009) emerged as a significant predictor for postnatal parenting stress, while AI (β = 0.274, *SE* = 1.142, *t* = 1.149, *p* = 0.879) was not.
Total dyadic adjustment was a significant negative predictor of postnatal maternal parenting stress (β = -0.435, SE = 0.123, *t* = -4.402, *p* \< 0.001) accounting for 18.9% of variance in maternal parenting distress \[*F*(1,69) = 19.380, *p* \< 0.001\]. Results are reported in **Table [4](#T4){ref-type="table"}**.
######
Results of regression analyses for antenatal measures on the Parenting Stress Index, Total Score, at 3 months after childbirth.
*R* *R*^2^ β *SE* *T* *p*
--------- ------------------- ------- -------- ---------- ------- --------- ------------
Model 1 0.618 0.381 -0.14.69 -0.528 0.042^∗^
ASQ -- Discomfort 0.534 0.351 1.519 0.155
ASQ -- Anxiety 1.127 0.496 2.271 0.041^∗^
Model 2 0.667 0.445 3.684 0.023^∗^
MAAS -- AQ -0.737 1.898 -3.094 0.009^∗∗^
MAAS -- AI 0.274 1.142 -0.155 0.879
Model 3 DAS TOT 0.435 0.189 -0.435 0.123 -4.402 0.001^∗∗∗^
ASQ -- DC, Attachment Style Questionnaire, Discomfort with Closeness; ASQ -- RA, Attachment Style Questionnaire, Relationship Anxiety; MAAS -- TOT, Maternal Antenatal Attachment, Total Score; MAAS -- AQ, Maternal Antenatal Attachment, AQ; MAAS -- AI, Maternal Antenatal Attachment, Attachment Intensity; DAS TOT, Dyadic Adjustment Scale Total Score; PSI -- SF, Parenting Stress Index -- Short Form, Total Score (
∗
p
\< 0.05,
∗∗
p \< 0.01,
∗∗∗
p \< 0.001).
Discussion
==========
This study contributes to the research on parenting in first-time mothers by investigating the relationships between some psychological aspects of maternal functioning in late pregnancy and their potential contribution to predict parenting stress during the early period of the mother--child relationship, after childbirth. Because, as noted previously, few empirical papers have addressed this issue, the present paper provides a contribution to the field of research devoted to identifying the contribution of prenatal variables to the onset of postnatal maternal parenting stress. Furthermore, this paper, which focuses on a sample of nulliparous women, contributes new data to a field of research that currently lacks information on this population.
Specifically pertaining to the first aim, this study examined the maternal attachment style, the maternal antenatal bond to the fetus and dyadic adjustment as prenatal factors, and the data showed interesting connections. The literature on parenting states that differences in attachment have implications for the transition to parenthood, with a positive link between attachment security and parenting ([@B66]; [@B58]). Conversely, attachment insecurity has been conceived as a risk factor in the development of parenting. Consistent with previous studies, particularly that of Grant ([@B40]), the findings of this paper showed the importance of attachment to others in adulthood during childhood in the transition to parenthood.
According to [@B33], this study focused on two dimensions of attachment insecurity: discomfort with closeness and relationship anxiety, which, respectively, measure the tendency to be uneasy with intimacy and concerns about the attachment other's feelings of love, alongside fears of being rejected. Attachment anxiety was negatively correlated with maternal antenatal attachment, especially to the quality of the emotional bond, and to the adjustment in the couple relationship. Mothers-to-be who were found to be more anxious and preoccupied in their personal attachment have also less positive feelings about the fetus and were less confident in their relationship with their partner. The results of the present study appear to be consistent with previous studies showing such associations ([@B20]; [@B12]; [@B75]; [@B8]; [@B74]), especially studies devoted to exploring the link between adult attachment patterns and antenatal attachment. Moreover, this research contributes data on the relationship between antenatal attachment to the fetus and couple relationships, a field that has been little investigated by the previous literature ([@B5]). Antenatal attachment, previously explored as aspect connected to attachment system ([@B15]), has recently been viewed as a bond that involves attending to the child's needs and providing protection. Recent studies have suggested considering antenatal attachment to be indicative of the caregiving system instead of (only) the attachment system ([@B14]; [@B74]) and thus to be particularly informative of the parental challenges connected to the transition to parenthood ([@B38]). The couple relationship also faces challenge in the transition to parenthood because of the many re-organizations that this moment brings for the couple: in the partners' relationship quality, in their responsibilities and in their reciprocal routines ([@B7]; [@B45]). Particularly, the birth of the first child has been found to have detrimental effects on partnerships ([@B22]; [@B11]), and relationship quality can decrease as a result of the decline in marital satisfaction and feelings of love that can result from the challenging new task of caring for a newborn.
Based on the need to develop more comprehensive theoretical models to explore the relationships among the predictors of parenting stress ([@B2]), the second aim of this study used a measure at 3 months after the child birth to show that each of the investigated variables was a good predictor of parenting stress 3 months after childbirth. In particular, the mother's adult attachment, specifically the anxiety dimension, contributed to this predictive power. These results are consistent with findings indicating the implications of differences in attachment for parenting ([@B50]). Secure mothers have less difficulty coping with parenthood tasks and are more capable in engaging in supportive parenting behavior. Conversely, in a paper studying the effect of child care on infant development, the authors found that parenting stress was significantly associated with insecure (child) attachment to mothers and fathers ([@B49]). While attachment security can be considered a protective factor against parenting stress, attachment insecurity can be considered a risk factor in predicting parenting stress.
Moreover, maternal antenatal attachment to the fetus significantly predicted parenting stress 3 months postpartum, with the specific contribution of AQ. Previous findings that were largely focused on the association between these dimensions were not consistent, with some studies indicating an inverse correlation between stress and antenatal attachment and others unable to replicate such results ([@B23]; [@B28]; [@B39]). Moreover, findings from the literature suggest that the correlation is also linked to the specificity of the postnatal period investigated. Our data are consistent with the few studies that have investigated this association from a longitudinal perspective ([@B5]). The findings of this study are consistent with those of Mikulincer, who found such a relationship during the first trimester ([@B58]). According to our data, negative AQ during pregnancy seems to be a risk factor in predicting parenting stress during the first 3 months after childbirth. These findings contribute to filling the gap in evidence in the literature ([@B5]).
Regarding dyadic adjustment, findings showed its predictive role. According to the present data, a low level of dyadic adjustment during pregnancy was a risk factor for the onset of parenting stress during the initial period after childbirth. Previous data showed that if a woman lacks adequate social support during pregnancy, the result would be negative outcomes, such as postpartum depression and insensitive parenting behavior ([@B26]; [@B29]). Conversely, women who receive support during pregnancy have more positive physical and mental health outcomes during the postnatal period ([@B18]). Moreover, [@B48] found that women with longer marriages had a significantly lower level of postpartum stress. According to [@B52], being in a marital relationship buffers stress during the postpartum transition. Mothers with a longer length of marriage generally experience more positive qualities in their marriages, with decreased role differentiation and increased role satisfaction in the transition to motherhood ([@B51]).
As noted previously in this paper, parenting stress is a construct that can be affected by different types of stressor. Parenting stress can be considered a function of many different variables ([@B25]). In this paper, the role of some of these variables was considered, but the results need to be interpreted with caution because the relative influence of the different sets of variables were not considered together in the same model. Moreover, the proposed models are not exhaustive. This is a another limitation of the study because other dimensions could affect and mediate the connections observed; for example, everyday tasks specifically associated with parenting as well as the role of children's characteristics (e.g., temperament) should be added to the model to increase understanding of the phenomenon. Notably, this sample of first-time mothers showed lower levels of parenting stress with respect to normative data (thus, low level of parenting stress) and it should be important to further investigate such connection with a larger first-time mothers sample. Moreover, this paper did not consider the specific role played by the women's partner. Another limitation of the study is the exclusive use of a self-report procedure to investigate both prenatal variables and postnatal parenting stress. Further studies should incorporate other psychological aspects, including also observational procedure, to obtain a richer picture of the dimensions that affect the initial emergence of the caregiving system and its first challenges, especially with first-time mothers. In spite of these limitations, findings from this study suggest how important is the role of prenatal characteristics in first-time mothers and underline the need to be considered in order to develop effective prevention plans.
Conflict of Interest Statement
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
[^1]: Edited by: *Silvia Salcuni, Università degli Studi di Padova, Italy*
[^2]: Reviewed by: *Francesca Lionetti, University of Pavia, Italy; Judi Walsh, University of East Anglia, UK*
[^3]: This article was submitted to Psychology for Clinical Settings, a section of the journal Frontiers in Psychology
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{
"pile_set_name": "PubMed Central"
}
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Introduction
============
Following traumatic injuries or restorative procedure, normal dental pulp exposure may occur inadvertently. In this situation, vital pulp therapy (VPT) is performed by placing the direct pulp capping biocompatible materials to maintain the health and vitality of dental pulp \[[@B1]\]. It is obvious that the success of such treatment is related to seal the pathways between the root canal and surrounding tissues \[[@B2]\]. Therefore, the ideal bonding between pulp capping agents and restorative materials is essential \[[@B3]\]. Mineral trioxide aggregate (MTA) is a commonly used material for VPT. After its setting, MTA forms calcium hydroxide in the presence of tissue fluids, which creates the antibacterial environment \[[@B4]\]. It also encourages differentiation and migration of hard tissue cells that release the hydroxyapatite on surface and produce the biological seal \[[@B5], [@B6]\].
Calcium-enriched mixture (CEM) cement is suggested biocompatible pulp capping material produced to overcome the drawbacks of MTA \[[@B7]\]. These biomaterial contains some superior physical and antibacterial properties compared with MTA \[[@B8]\].
After VPT a definite leakage free restoration (composite or amalgam) should be used. Different studies showed that acid etching before composite build up and nature of solvent in the adhesives may influence the mechanical properties and bond strength of pulp covering agent to composite resin \[[@B2], [@B9]\]. Hashem *et al.*\[[@B10]\] also demonstrated that the coronal restorations over pulp capping agents should have low condensation forces. The use of an intermediate restorative materials or liner has been emphasized between the pulp capping agent and final restorative materials. Resin modified glass ionomer (RMGI) and flowable composite (FC) may be suitable materials to be used over the pulp capping agents because of low condensation force required for their placement. The creation of proper bond between intermediate materials and pulp capping agents are essential to seal and guarantee the success of treatment \[[@B11]\]. Oskoee *et al.*\[[@B12]\] and Ajami *et al*. \[[@B13]\] showed that the highest shear bond strength was observed when composite resin was bonded to RMGI than MTA or CEM cement and suggested that it is advisable to cover these pulp capping materials with RMGI before composite buildup.
Vertise Flow (VF; Kerr, orange, CA, USA) is a self-adhering flowable composite which combines an all-in-one adhesive system and a flowable composite for a step-less system \[[@B14]\]. The presence of glycerol phosphate dimethacrylate monomer (GPDM) in this SAFC eliminates a separate adhesive application and save chair time and handling errors. Vichi *et al.*\[[@B15]\] revealed that this composite had lower bond strength to enamel and dentin but better marginal sealing was observed in comparison with the other all-in-one adhesive systems
There is no information on the adhesion of SAFC to MTA or CEM cement. Therefore, the purposes of this study was to determine the shear bond strength of SAFC alone and in conjunction with a self-etch adhesive to MTA and CEM cement and also compare it with RMGI cement. The null hypothesis was that there would be no differences between the bond strength values in different study groups.
Materials and Methods
=====================
In this study, 72 acrylic blocks (Acropars, Tehran, Iran) were with a central hole measuring 4 mm in diameter and 2 mm in depth. The samples were divided into two groups (*n*=36). RootMTA (Tabriz, Iran) and CEM (Bionique Dent, Tehran, Iran) were mixed according to their manufacturers' instructions. The pastes were poured into the holes in the center of acrylic blocks, flattened with a spatula, covered with a moist cotton pellet and temporary filling materials (Cavisol, Golchai Co., Tehran, Iran). Then, the specimens were stored for 72 h at 37^°^C temperature and 100% humidity. After storage, the temporary materials and cotton pellets were removed without interfering with the surfaces of the pulp capping materials. Then, the specimens of each material were divided into the six groups (*n=12*).
In groups 1 and 4, after air drying of the specimen, the SAFC (Vertise Flow, Kerr, orange, CA, USA) was actively applied directly over MTA or CEM with no adhesive. The SAFC was placed into the plastic mold with 3 mm diameter and 2 mm height in one increment. Then the SAFC was light cured with light density 600 mw/cm ^2^(VIP junior; Bisco, Schumburg, USA) for 40 sec.
In groups 2 and 5, after air drying of the specimen, Opti-Bond all-in-one adhesive (Kerr Dental, orange CA, USA) was actively applied on the surfaces with a brush and after air thinning, was light cured for 20sec. Then SAFC was subsequently applied to the conditioned surfaces, similar to the previous groups.
In groups 3 and 6, the surfaces of pulp capping agents were conditioned with 10% poly acrylic acid (GC Corp. Tokyo, Japan) for 20 sec, then rinsed for 30 sec and air dried. After that, the powder and liquid of RMGI (Fuji II Lc; GC Corp.; Tokyo, Japan) was mixed according to the manufacturer's instructions; the paste was placed into the plastic mold and light cured for 40 sec similar to above mentioned groups.
The prepared specimens were kept in 100% relative humidity at 37^°^C temperature for 24 h. After that, the plastic molds were carefully removed from the specimen before the shear bond strength test.
The specimens were mounted in the universal testing machine (Instron; Zwich, Germany) and shear forces were applied at the crosshead speed of 0.5 mm/min until the fracture occurred. The maximum loads at failure were recorded in Newton and were then converted into the Mps.
The fractured specimens were observed under a stereomicroscope under 40× magnification to determine the failure mode. The failure types were categorize as adhesive (two flat surfaces, showing failure of filling materials/pulp capping bond), cohesive (any deficiency in the pulp capping agent surface) and mixed (combination the adhesive residue and deficiency in the pulp capping surfaces).
The two-way ANOVA analysis was applied to determine the interaction effect between the experimental groups and if it was applicable, the post hoc comparison and t-test was used to compare the shear bond strength results among groups. Also, the chi-square and Fissure's exact test were used to compare the fracture surface pattern between groups. The level of significance was set at 0.05.
Results
=======
[Table 1](#T1){ref-type="table"} shows the mean and standard deviations of shear bond strength for all groups. There was a significant interaction effect between the intermediate filling materials (SAFC and RMGI) and the pulp capping agents (Root MTA and CEM cement). The use of an all-in-one adhesive system significantly increased the shear bond strength of SAFC to MTA and CEM cement (*P*=0.008, 0.000, respectively). In both pulp capping agents, RMGI had the lowest shear bond strength values (*P*=0.05). For all intermediate materials in this study, the mean shear bond strength values were significantly higher in MTA than CEM; however, these difference was not obvious in RMGI than other intermediate materials (*P*=0.003). There were a significant difference between fracture patterns between groups (*P*=0.001). Most failures in MTA specimens were adhesive/mix, but in CEM cement specimens, the cohesive failure was the predominant mode of fracture except for RMGI materials which adhesive failures were also observed. All groups showed significant differences with CEM/SAFC with no bonding ([Table 1](#T1){ref-type="table"}).
Discussion
==========
The results of our study showed that SAFC (with or without application of adhesive) had superior bond strength compared to RMGI, either over MTA or CEM cement. The null hypothesis was rejected, because the bond strength changed in relation to adhesive application and filling materials.
More researches have shown that, both MTA and CEM cement can be used effectively as pulp capping agents because they have the ability to stimulate the differentiation of dental pulp stem cells to odontoblast like cells and ultimately initiate the formation of dentinal bridge which is thicker, less porous along with less pulp inflammation than the calcium hydroxide material \[[@B6], [@B16], [@B17]\].
In this study, we used SAFC as an intermediate material before permanent composite restoration over Root MTA or CEM cement. The SAFC used in this experiments a novel flowable resin based composite that eliminates the etching, priming and bonding steps in order to simplify the adhesive procedures to dentin and enamel \[[@B15]\].
The results of our study demonstrated that surface treatment with an all-in-one adhesive before SAFC significantly increased the shear bond strength of SAFC to Root MTA and CEM cement. It was in consistent with the results of the study by Tuloglu *et al.*\[[@B18]\]. Also, the study by Neelakantan *et al*. \[[@B19]\] showed that one-step-self etching adhesive demonstrated the strongest bond to white MTA after 24 h compared to total etching or two-step self-etching adhesives. This can be attributed to the sensitivity of MTA and other calcium silicate cements to acidic environment \[[@B20]\]. However, the study by Bayrak *et al.*\[[@B2]\] indicated that etch-and-rinse adhesives showed stronger bond than self-etch adhesive were adhesive for bonding composite resin to white MTA. It is demonstrated that the different bonding systems (total-etch *vs.* self-etch) did not have a significant effect on the shear bond strength of composite resin to pulp capping biomaterials \[[@B21], [@B22]\]. Kayahan *et al.*\[[@B9]\], evaluated the effect of acid etching on the compressive strength of calcium-silicate based materials and concluded that the acid etching significantly reduced the SBS of Angelus MTA and CEM cement. Several studies have reported that the acidic environment would reduce the several physical properties of MTA \[[@B9], [@B23]\]. In SEM evaluation, the disordered structure, selective dissolution and detachment of filler particles were observed after acid etching process \[[@B24]\]. In our study, the pH value for SAFC and bonding system were reported as 1.9 by the manufacturer; therefore, both acted as a mild self-etch adhesive over the pulp capping agents \[[@B15]\]. Thus, it did not cause the adverse effect of highly acidic pH on MTA or CEM cement.
As mentioned previously, the additional application of self-etching bonding would enhance the shear bond strength of SAFC to MTA and CEM cement. However, the results were in argue with Yesilyurk *et al.*\[[@B25]\] who concluded that the self-etch adhesive significantly reduced the shear bond strength of SAFC to Bioaggregate. However, the surface treatment of Bioaggregate with an etch-and-rinse adhesive significantly increased the shear bond strength after 72 h \[[@B25]\]. In our study, we used Opti Bond all-in-one as a self-etch adhesive system along with SAFC which has the same adhesive technology and chemical composition to eliminate the probable adhesive interaction of two different technology used in Yesilyurk's study. The second possible reason for enhancement of Opti Bond self-etch adhesive over calcium silicate based cements, is that it contains ethanol, acetone and water in its composition as solvents. Hence, smaller contact angle and better wettability of the MTA and CEM cement would be expected \[[@B19]\]. This factor plus the lower viscosity of Opti Bond self-etch adhesive may be responsible for more macromechanical retention than high viscose SAFC, which is the principle adhesion mechanism for bonding of the pulp capping agent/flowable composite \[[@B25]\].
######
Mean (SD) of shear bond strength for experimental groups
**Shear Bond Strength (Mpa)** **Type of failure mode N (%)**
----------------------- ------------------------------- -------------------------------- ---------- ----------
**MTA+SAFC** 7.30 (1.33) 2 (16.7) 3 (25) 7 (58.3)
**MTA+SAFC (bonded)** 8.80 (1.24) 4 (33.3) 2 (16.7) 6 (50)
**MTA+RMGI** 2.25 (0.77) 4 (33.3) 3 (25) 5 (41.7)
**CEM+SAFC** 3.50 (1.25) 0 12 (100) 0
**CEM+SAFC (bonded)** 5.94 (1.09) 1 (8.3) 7 (58.3) 4 (33.3)
**CEM+RMGI** 1.32 (0.61) 5 (41.7) 5 (41.7) 2 (16.7)
In this study, SAFC had significantly higher mean of shear bond strength values than RMGI for both MTA and CEM cement. This results were contrary to the results reported by Ajami *et al.*\[[@B21]\] that showed superior bond of composite compared to RMGI to three pulp capping agents. In this study, 10% polyacrylic acid conditioner was used before applying RMGI. Acidic content and rinsing may be one reason for reducing the shear bond strength of RMGI to pulp capping materials. However, Gulati *et al.*\[[@B26]\] revealed that conditioning MTA with 10% polyacrylic acid could not significantly affect the shear bond strength of RMGI.
According to the manufacturers, SAFC used in this study consists of phosphate functional monomer (GPDM) which may interact with the calcium ions \[[@B27]\]. Both MTA and CEM cement have different calcium compounds which release calcium hydroxide during and after setting \[[@B28], [@B29]\]. The possible chemical bond with GPDM of SAFC and MTA and CEM cement may responsible for the higher shear bond strength than RMGI that should be analyzed in further researches. In the present study, MTA had significantly higher shear bond strength than CEM cement for both filling materials. Sobhnamayan *et al.*\[[@B30]\] revealed that even after 72 h, CEM cement was not completely hardened and had a semi-hard consistency in contrast to MTA. These results confirm the failure mode analysis obtained in the present study, where most of the failures observed in the CEM cement were cohesive, in contrast to MTA and RMGI in which adhesive failures were seen the most. Ajami *et al.*\[[@B21]\] reported that the shear bond strength of resin composite to MTA was weaker than CEM cement and NMTA. The chemical composition and different shape, size and distribution of the hydroxyapatite crystals may explain the different behavior of these two materials \[[@B7], [@B31]\]. In this study a 72-h delay time in 100% humidity was chosen for setting of MTA and CEM cement because many studies suggested that three days are required for displacement resistance and complete setting of MTA \[[@B3], [@B32]\]. Bond strength tests are the most common laboratory test for analyzing the performance of adhesive restoration \[[@B33]\]. Because MTA and CEM cement are brittle materials, the shear bond strength test was chosen and considered as the best method for analysis in our study \[[@B34]\]. Further scanning electron microscope (SEM) and transmission electron microscope (TEM) studies should be planned to analyze the possible bond between the self-etching FC and different pulp capping agents in different time intervals in long-term storage and especially in clinical set-ups.
Conclusion
==========
Within the limitation of this *in vitro* study, it was found that the application of a self-etch adhesive improves the bond quality of Vertise Flow to MTA and CEM cement. Moreover, the bond strength of Vertise Flow was significantly greater than resin-modified-glass ionomer in both materials. All materials had significantly higher bond strength in MTA group than CEM cement after 72 h setting.
The authors thank the Vice Chancellery of Shiraz University of Medical Science for supporting this research (Grant No.: 9379). This article is based on the thesis by Dr. F. Abbasiyan. The authors also thank Dr. Vosough of the Dental Research Development Center, of the School of Dentistry for the statistical analysis and Dr. Hamedani for his editorial assistance.
Conflict of Interest: 'None declared'.
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{
"pile_set_name": "PubMed Central"
}
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Introduction {#S1}
============
Brain-derived neurotrophic factor (BDNF) promotes the neural and behavioral plasticity induced by cocaine or other stimulant drugs of abuse via actions on the mesolimbic dopamine system, which is composed of dopamine neurons in the ventral tegmental area (VTA) of the midbrain and their anterior projections to the nucleus accumbens (NAc) and other forebrain regions^[@R1]--[@R3]^. Previous studies have shown that BDNF-TrkB activity and its downstream signaling cascades are induced in NAc by cocaine exposure^[@R4]--[@R6]^. In addition, manipulations that enhance BDNF signaling in the VTA-NAc circuit increase rewarding and locomotor responses to cocaine, while suppressing BDNF signaling has the opposite effect^[@R5],[@R7]--[@R10]^.
In striking contrast, we showed recently that chronic morphine suppresses *Bdnf* gene expression in mouse VTA and that such blockade enhances rewarding and locomotor responses to morphine by augmenting dopamine neuron activity^[@R11]^. Chronic opiates also induce some unique biochemical and morphological alterations in VTA, such as down-regulation of intracellular neurotrophin signaling cascades and reduced soma size of VTA dopamine neurons, effects not seen with stimulants^[@R12]--[@R15]^. Interestingly, some of these changes are reversed by direct administration of BDNF into this brain region.
Despite this evidence for an inverse relationship between BDNF activity in VTA and morphine action, the transcriptional mechanisms underlying *Bdnf* suppression by morphine are largely unknown. Here, we carried out a uniquely comprehensive analysis of epigenetic regulation at the *Bdnf* gene and demonstrate a series of interacting chromatin mechanisms in mediating morphine's down-regulation of *Bdnf* transcription in rat VTA.. We report that unique binding patterns of RNA polymerase II (Pol II), permissive and repressive histone modifications, their histone modifying enzymes and related regulatory proteins, and key transcription factors at specific *Bdnf* promoters are associated with morphine-induced *Bdnf* suppression in this brain region and with enhanced behavioral responses to opiates.
Results {#S2}
=======
Down-regulation of *Bdnf* expression in VTA by opiates {#S3}
------------------------------------------------------
We first examined postmortem VTA sections of human brain and observed that heroin addicts, compared with matched controls ([Supplementary Table 1](#SD2){ref-type="supplementary-material"}), displayed reduced mRNA levels of *Bdnf* exon IX, which represents the protein-coding region of *Bdnf* mRNA that is common to all *Bdnf* transcripts^[@R16]^ ([Fig. 1a](#F1){ref-type="fig"} and [Supplementary Fig. 1a](#SD1){ref-type="supplementary-material"}). *Bdnf* exon IX mRNA levels were also decreased in VTA of rats that chronically self-administered heroin ([Fig. 1b](#F1){ref-type="fig"} and [Supplementary Fig. 1b](#SD1){ref-type="supplementary-material"}).
To further characterize VTA *Bdnf* gene regulation in opiate action, we used an extensively validated morphine treatment regimen, involving repeated IP injections, which is more amenable to higher throughput analyses. Rats received daily morphine injections (5 mg/kg) for 14 days and were examined 14 days later^[@R17]^. Having confirmed the expected sensitizing behavioral effects of chronic morphine in these rats ([Supplementary Fig. 1c](#SD1){ref-type="supplementary-material"}), we found that *Bdnf* exon IX expression was suppressed in VTA of chronic morphine-treated rats compared to saline controls ([Fig. 1c](#F1){ref-type="fig"}) \[one way ANOVA (*F*~3,32~ = 3.872, *p* = 0.0181) with Fisher's *post hoc* test, *p* \< 0.05\]. In contrast, acute morphine (5 mg/kg, IP) 14 days after chronic (14 days) saline treatment had no effect on *Bdnf* exon IX expression in rat VTA (Fisher's *post hoc* test, *p* = *n.s.*). However, subchronic morphine exposure (15 mg/kg, IP)---during 3 day-training for conditioned place preference (CPP)---was sufficient to decrease *Bdnf* exon IX expression in mouse VTA ([Fig. 1d](#F1){ref-type="fig"} and [Supplementary Fig. 1d](#SD1){ref-type="supplementary-material"}). These findings demonstrate that repeated opiate exposure is required for *Bdnf* mRNA suppression in VTA across species, including human addicts, and supports the relevance of this adaptation to opiate action.
Stalling of Pol II at *Bdnf* promoters in VTA by morphine {#S4}
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Having established opiate suppression of *Bdnf* exon IX expression in VTA, we investigated the underlying mechanisms involved. Initial quantitative PCR (qPCR) analysis showed that *Bdnf* transcripts containing exons III, V, VII, or VIII are expressed at very low levels in rat VTA and show no significant changes after chronic morphine ([Supplementary Fig. 1e](#SD1){ref-type="supplementary-material"}). Thus, we focused on *Bdnf* transcript containing exons I, II, IV, and VI in VTA and observed thatchronic morphine also reduced *Bdnf* transcript levels of exons II, IV, and VI compared to saline controls ([Fig. 2a,b](#F2){ref-type="fig"}). There was no difference in *Bdnf* exon I mRNA expression. These data show that the chronic morphine-induced suppression of *Bdnf* exon IX is attributable to reductions in exons II-, IV-, and VI-containing transcripts. Furthermore, the results demonstrate that such suppression is long-lasting: it persists for at least two weeks after cessation of morphine exposure, features suggestive of an epigenetic mechanism.
To understand the effect of chronic morphine on *Bdnf* gene transcription at the epigenetic level, we performed quantitative chromatin immunoprecipitation (qChIP) with an antibody that recognizes both non-phosphorylated and phosphorylated forms of Pol II (total-Pol II) and with antibodies that specifically recognize Pol II phosphorylated at either Ser2 or Ser5 at its C terminal domain. Ser5 hyper-phosphorylation at gene promoters and Ser2 hypo-phosphorylation in coding regions are thought to be hallmarks of stalled Pol II---associated with suppressed genes^[@R18]^. Total-Pol II binding to *Bdnf* promoter region 2 (*Bdnf*-p2), which corresponds to exon II, was higher in VTA of chronic morphine-treated rats relative to saline controls, with no changes seen at the other promoters ([Fig. 2c](#F2){ref-type="fig"}). Phospho-Ser5-Pol II binding to *Bdnf*-p2, -p4, and -p6 in morphine-treated rats was increased ([Fig. 2d](#F2){ref-type="fig"}). In contrast, phospho-Ser2-Pol II binding in coding regions was decreased at *Bdnf*-exon II (eII), -eIV, and -eVI after chronic morphine ([Fig. 2e](#F2){ref-type="fig"}). These findings suggest sustained suppression of *Bdnf*-p2, -p4, and -p6 via Pol II stalling in VTA in response to chronic morphine.
Histone modifications at *Bdnf* promoters in VTA by morphine {#S5}
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Next, we conducted qChIP for numerous histone modifications at the four *Bdnf* promoters: three histone markers of gene activation, acetylated H3 (acH3), acH4, and trimethylation of Lys4 of H3 (H3K4me3); three markers of gene repression, H3K9me2, H3K9me3, and H3K27me3; and one marker of transcription elongation, H3K36me3. Chronic morphine-exposed animals displayed clear patterns of histone regulation at *Bdnf* promoters in VTA ([Supplementary Fig. 2a--g](#SD1){ref-type="supplementary-material"}), with the most pronounced regulation occurring, as with Pol II, at *Bdnf*-p2. The changes observed in morphine-treated rats represent modifications induced by chronic morphine which persisted despite 2 weeks of abstinence: acH3 was decreased, while H3K4me3 and H3K27me3 were increased, at *Bdnf*-p2 compared to saline controls ([Fig. 3a](#F3){ref-type="fig"}). No changes in acH3, H3K4me3, or H3K27me3 were seen at *Bdnf*-p1, -p4, and -p6 after morphine exposure ([Supplementary Fig. 2a--g](#SD1){ref-type="supplementary-material"}). In addition, we observed no significant alterations in H3K9me2, H3K9me3, or H3K36me3 at any *Bdnf* promoters, whereas elevated acH4 levels at *Bdnf*-p4 were observed in morphine-treated rats.
As follow up, we performed qChIP for key histone modifying enzymes and related regulatory proteins associated with a given histone mark, such as mSIN3a (mammalian SIN3 transcription regulator family member A), ING2 (inhibitor of growth 2), MLL1 (mixed-lineage, leukemia; KMT2A, lysine-specific methyltransferase 2A), G9a (EHMT2, euchromatic histone-lysine N-methyltransferase 2), and Polycomb group proteins. Since mSIN3a is a core component of a prominent co-repressor complex implicated in numerous systems^[@R19]^, we examined the binding of this protein to *Bdnf* promoters. We found that mSIN3a binding to *Bdnf*-p1, -p2, and -p6 was robustly increased in VTA after chronic morphine ([Fig. 3b](#F3){ref-type="fig"} and [Supplementary Fig. 2h](#SD1){ref-type="supplementary-material"}). We examined the binding of ING2 to *Bdnf* promoters, based on our paradoxical observation of increased H3K4me3 levels at *Bdnf*-p2 in VTA of morphine-treated rats. H3K4me3 typically denotes increased gene transcription, however, there is evidence that ING2 represses particular genes by binding to H3K4me3 and recruiting the mSIN3a repressive complex^[@R20],[@R21]^. We found that ING2 binding to *Bdnf*-p1, -p2, -p4, and -p6 was dramatically increased in VTA in response to chronic morphine ([Fig. 3b](#F3){ref-type="fig"} and [Supplementary Fig. 2i](#SD1){ref-type="supplementary-material"}). Such recruitment of ING2 and mSIN3a to *Bdnf* promoters explains why an increase in H3K4me3 is associated with gene repression. Consistent with our H3K4me3 data, morphine-treated rats showed elevated binding of an H3K4 methyltransferase, MLL1, to *Bdnf*-p2 and -p6 in VTA ([Fig. 3c](#F3){ref-type="fig"} and [Supplementary Fig. 2j](#SD1){ref-type="supplementary-material"}). In contrast, an H3K9 methyltransferase, G9a, exhibited no changes in binding to *Bdnf* promoters, consistent with the lack of alterations in H3K9me2 levels ([Fig. 3d](#F3){ref-type="fig"} and [Supplementary Fig. 2k](#SD1){ref-type="supplementary-material"}).
H3K27me3-mediated gene repression is associated with Polycomb group proteins, which reside in two main complexes: PRC1 (Polycomb repressive complex 1) and PCR2. PRC2 contains SUZ12 (zinc finger protein suppressor of zeste 12) and EZH2 (enhancer of zeste homologue 2); the latter catalyzes H3K27me3^[@R22],[@R23]^. PRC1, which contains BMI1, RING1A, and RING1B, contributes to histone H2A monoubiquitination and interactions of Polycomb group proteins with H3K27me3^[@R24],[@R25]^. Consistent with increased levels of H3K27me3 at *Bdnf*-p2, the binding of PRC2 complex proteins---namely, SUZ12 and EZH2---to *Bdnf*-p2 was increased in VTA by chronic morphine ([Fig. 3e](#F3){ref-type="fig"}). High levels of occupancy by EZH2, but not SUZ12, were also found at *Bdnf*-p4 and -p6 ([Supplementary Fig. 2l,m](#SD1){ref-type="supplementary-material"}). In contrast, binding of PRC1 complex proteins such as RING1A to *Bdnf*-p2 was reduced by morphine, whereas no changes were seen at other *Bdnf* promoters or in RING1B and BMI1 binding at any promoter ([Fig. 3f](#F3){ref-type="fig"} and [Supplementary Fig. 2n,o,p](#SD1){ref-type="supplementary-material"}). Together, this comprehensive analysis of epigenetic regulation of the *Bdnf* gene in VTA by chronic morphine demonstrates concerted modifications predominantly at *Bdnf*-p2 that are consistent with transcriptional repression ([Supplementary Fig. 2q](#SD1){ref-type="supplementary-material"}).
To test directly whether alterations in H3K27me3 in VTA influence *Bdnf* expression and morphine-elicited behavioral changes, we generated a Herpes Simplex Virus (HSV) vector to overexpress EZH2 selectively in VTA compared to HSV-GFP controls ([Fig. 3g](#F3){ref-type="fig"} and [Supplementary Figs. 3a and 4a](#SD1){ref-type="supplementary-material"}). Such EZH2 overexpression, which was selective for VTA and not seen in neighboring brain regions ([Supplementary Fig. 4a](#SD1){ref-type="supplementary-material"}), reduced *Bdnf* mRNA levels in VTA ([Fig. 3h](#F3){ref-type="fig"}) and robustly increased morphine CPP (5 mg/kg) in mice ([Fig. 3i](#F3){ref-type="fig"}). In contrast, intra-VTA infusion of a lentiviral vector expressing a short hairpin interfering RNA of *Ezh2* (i.e., LV-shRNA-EZH2), which repressed *Ezh2* mRNA expression ([Supplementary Fig. 3b,c](#SD1){ref-type="supplementary-material"}), blocked the morphine-induced reduction of *Bdnf* expression in VTA ([Supplementary Fig. 3d](#SD1){ref-type="supplementary-material"}).
Regulation of CREB binding to *Bdnf* promoters by morphine {#S6}
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We next examined CREB (cAMP response element-binding protein), which induces *Bdnf* in other systems^[@R26]^. Total-CREB binding to *Bdnf*-p6 was increased in VTA in chronic morphine-treated rats, with no changes seen at the other promoters ([Fig. 4a](#F4){ref-type="fig"}). However, the binding of phospho-CREB (its active form) to *Bdnf*-p1, -p2, and -p4 was robustly decreased under these conditions ([Fig. 4b](#F4){ref-type="fig"}). To confirm the direct connection between CREB activity and *Bdnf* expression in VTA, we infused HSV-CREB into VTA of wildtype mice to overexpress CREB selectively in this region ([Fig. 4c](#F4){ref-type="fig"}). CREB overexpression augmented *Creb1* mRNA and protein levels selectively in VTA ([Supplementary Figs. 4b and 5a](#SD1){ref-type="supplementary-material"}); it also increased *Bdnf* mRNA expression in VTA compared to HSV-tdTomato (TMT) controls ([Fig. 4d](#F4){ref-type="fig"}). Conversely, knocking down CREB by infusing HSV-Cre into the VTA of floxed CREB mice ([Fig. 4e](#F4){ref-type="fig"} and [Supplementary Fig. 5b](#SD1){ref-type="supplementary-material"}) decreased *Bdnf* expression compared to HSV-GFP controls ([Fig. 4f](#F4){ref-type="fig"}).
Next, we addressed how chronic morphine suppresses phospho-CREB binding selectively to *Bdnf* even though chronic morphine increases overall CREB activity in this brain region^[@R27],[@R28]^. We tested the hypothesis that elevated H3K27me3 suppresses phospho-CREB binding to *Bdnf* promoter regions, since a recent study showed that H3K27me3 antagonizes CREB activity and represses expression at certain gene promoters in lymphocytes^[@R29]^. EZH2 overexpression by itself (i.e., without morphine), which increased H3K27me3 levels at all *Bdnf* promoters ([Fig. 4g](#F4){ref-type="fig"}), decreased phospho-CREB binding to all *Bdnf* promoters ([Fig. 4h](#F4){ref-type="fig"}). Moreover, in contrast to the morphine-induced decrease in phospho-CREB binding to the *Bdnf* promoters, phospho-CREB binding was increased by chronic morphine at the promoters of *Th* (tyrosine hydroxylase) and *Gria1* (GluA1 AMPA receptor subunit), two known CREB target genes that are induced in rodent VTA under these conditions^[@R27],[@R28]^ ([Supplementary Fig. 6a,b](#SD1){ref-type="supplementary-material"}). H3K27me3 levels at *Th* and *Gria1* were not affected in VTA by chronic morphine ([Supplementary Fig. 6c](#SD1){ref-type="supplementary-material"}). In contrast to *Bdnf* repression by EZH2 overexpression, *Th* and *Gria1* expression was not altered by EZH2 in mouse VTA ([Supplementary Fig. 3a](#SD1){ref-type="supplementary-material"}). Heroin self-administration also decreased phospho-CREB binding to all *Bdnf* promoters ([Supplementary Fig. 6d,e](#SD1){ref-type="supplementary-material"}) and increased H3K27me3 at *Bdnf*-p1, -p2, and -p4 ([Supplementary Fig. 6f](#SD1){ref-type="supplementary-material"}).
Regulation of NURR1 binding to *Bdnf* promoters by morphine {#S7}
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We also examined the transcription factor NURR1 (nuclear receptor related 1), since it regulates several genes including *Bdnf* in midbrain dopamine neurons^[@R30]--[@R33]^. NURR1 binding to *Bdnf*-p1 and -p2 was decreased in VTA of chronic morphine-treated rats ([Fig. 5a](#F5){ref-type="fig"}). Interestingly, chronic morphine suppressed *Nurr1* mRNA expression in VTA, an effect also seen in heroin self-administering rats ([Fig. 5b,c](#F5){ref-type="fig"}), which could explain the reduced NURR1 binding to *Bdnf* promoters. Consistent with *Nurr1* suppression, H3K27me3 binding at the *Nurr1* promoter in VTA was increased by chronic morphine ([Fig. 5d](#F5){ref-type="fig"}). Furthermore, we examined the relationship between CREB and NURR1 in response to chronic morphine, since *Nurr1* is a known downstream target of CREB in other systems^[@R32],[@R34]^. Chronic morphine decreased phospho-CREB binding to the *Nurr1* promoter in VTA ([Fig. 5e](#F5){ref-type="fig"}), while CREB overexpression in mouse VTA using HSV-CREB, which induced total- and phospho-CREB binding to the *Nurr1* promoter ([Supplementary Fig. 5c,d](#SD1){ref-type="supplementary-material"}), robustly augmented *Nurr1* mRNA expression in this region ([Fig. 5f](#F5){ref-type="fig"}). The induced H3K27me3 and reduced phospho-CREB binding at *Nurr1* in VTA in response to chronic morphine suggest an antagonistic relationship between H3K27me3 and phospho-CREB, as observed for the *Bdnf* promoter under these conditions.
We generated and validated an HSV vector to overexpress NURR1 and examine its functional role selectively in VTA ([Supplementary Figs. 4c and 7a](#SD1){ref-type="supplementary-material"}). We first infused HSV-NURR1 into rat VTA 10 days after the last injection of our standard 14 day chronic morphine paradigm (5 mg/kg, IP). Rats were then re-exposed to morphine (5 mg/kg, IP) 4 days after the HSV-NURR1 infusion ([Fig. 5g](#F5){ref-type="fig"}); such re-exposure (M/M) induces higher locomotor responses compared to a saline challenge (M/S) (see [Supplementary Fig. 1c](#SD1){ref-type="supplementary-material"}). We observed that NURR1 overexpression in VTA blocked this hyper-locomotion by morphine re-exposure ([Fig. 5h](#F5){ref-type="fig"} and [Supplementary Fig. 7b](#SD1){ref-type="supplementary-material"}). We also investigated the influence of NURR1 on morphine reward using the CPP paradigm. We first confirmed that NURR1 overexpression induced *Bdnf* expression in mouse VTA, as observed in rat ([Fig. 5i,j](#F5){ref-type="fig"}), and then demonstrated that morphine reward was also blocked by NURR1 overexpression in mouse VTA ([Fig. 5k](#F5){ref-type="fig"}). Importantly, NURR1 overexpression in VTA of mice lacking BDNF in this brain region (generated by infusing HSV-Cre into VTA of floxed BDNF mice) did not show the suppressive effect of NURR1 overexpression on morphine reward ([Fig. 5l](#F5){ref-type="fig"}), which provides a direct link between NURR1 action and regulation of BDNF activity in this brain region.
Discussion {#S8}
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Consistent with prior reports of opiate regulation of VTA BDNF expression^[@R11],[@R35]^ (but see^[@R36]^), the present study shows that *Bdnf* mRNA levels are robustly reduced in VTA of human heroin addicts, of heroin self-administering rats, and of repeated morphine-treated mice (including those undergoing CPP). We observed that chronic morphine exposure induces a robust and sustained decrease in levels of specific *Bdnf* exon transcripts (i.e., *Bdnf* exons II, IV, and VI) in this brain region in rats. A uniquely comprehensive analysis of epigenetic mechanisms---particularly for a micronucleus such as VTA---revealed that this *Bdnf* suppression by chronic morphine is associated with a series of interacting and sustained transcriptional and chromatin modifications at the corresponding *Bdnf* gene promoters. In particular, we found that chronic morphine decreases binding of the active (phospho-Ser2) form of Pol II within *Bdnf*-eII, -eIV, and -eVI, whereas phospho-Ser5 Pol II binding to *Bdnf*-p2, -p4, and -p6 was augmented in VTA. These findings suggest that chronic morphine induces a sustained Pol II stalling at certain *Bdnf* promoters in concert with the gene's sustained repression.
Such stalling of Pol II is associated with several other key chromatin changes, with the most robust regulation seen at *Bdnf*-p2 in VTA (see [Supplementary Fig. 2q](#SD1){ref-type="supplementary-material"}). Consistent with repression of *Bdnf*-p2, we demonstrated increased binding of H3K27me3, a major form of repressive histone methylation. Occupancy by the PRC2 complex including SUZ12 and EZH2, which mediates H3K27 trimethylation^[@R22],[@R23]^, at *Bdnf*-p2 in VTA is also increased by chronic morphine. We provide a causal connection between H3K27me3 and **s**uppression of the *Bdnf* gene in VTA by showing that EZH2 overexpression in this region decreases *Bdnf* expression, while EZH2 knockdown blocks morphine's *Bdnf* suppression. Together, these data support a scheme whereby PRC2-mediated H3K27me3 reduces *Bdnf* gene expression ([Supplementary Fig. 8](#SD1){ref-type="supplementary-material"}). Interestingly, PRC2 has been shown to interact with other repressor proteins, in particular, with mSIN3a and related proteins^[@R37],[@R38]^, which are also enriched at *Bdnf*-p2 in VTA in response to chronic morphine. Consistent with our previous study, which linked reduced levels of VTA BDNF with increased morphine reward^[@R11]^, we show here that EZH2 overexpression in VTA promotes this morphine-elicited behavior as well.
Repression of *Bdnf*-p2 is associated with increased levels of H3K4me3, which is typically related to gene activation. This observation illustrates the complexity of chromatin regulatory mechanisms *in vivo*. Insight into this complexity is provided by our observation that this paradoxical increase in H3K4me3 is associated with induction of ING2 binding to *Bdnf*-p2 by morphine. ING2, also a subunit of the repressive mSIN3a complex, binds with high specificity to H3K4me3 and consequently represses transcription of certain genes^[@R20],[@R21]^. We show further that this unusual, but precedented, interaction between H3K4me3 and the mSIN3a/ING2 also involves the morphine-induced recruitment of the H3K4-specific methyltransferase, MLL1^[@R39]^, to *Bdnf*-p2 (see [Supplementary Fig. 8](#SD1){ref-type="supplementary-material"}). Interestingly, MLL1, like its H3K4me3 mark, has been shown to interact with repressive complexes at particular genes in other systems^[@R40]^.
The morphine-induced *Bdnf* repression also involves reduced binding of a key transcription factor, phospho-CREB, which has previously been shown to induce *Bdnf* gene expression in other systems^[@R26]^. However, the reduced phospho-CREB binding to *Bdnf* promoters in VTA after chronic morphine is surprising, since chronic morphine increases total levels of phospho-CREB and CREB transcriptional activity in VTA^[@R27],[@R28]^. These findings suggest that there are specific features at *Bdnf* promoters responsible for the exclusion of phospho-CREB after chronic morphine. We show that one mechanism for this exclusion is the induction of H3K27me3, since EZH2 overexpression---which induces H3K27me3---in VTA in the absence of morphine reduces phospho-CREB binding to *Bdnf* promoters. A recent study has reported that PRC2 occupancy and increased H3K27me3 at certain gene promoters antagonizes CREB binding to silence gene expression in lymphocytes^[@R29]^. In contrast, phospho-CREB binding to the promoters of *Th* and *Gria1*, two known targets of phospho-CREB in VTA^[@R28]^, were increased in response to chronic morphine as would be expected. It is notable that chronic morphine did not affect H3K27me3 levels at *Th* and *Gria1* promoters and that *Th* and *Gria1* mRNA expression was not altered by EZH2 overexpression in contrast to *Bdnf* expression. While a caveat of these studies are potential off-target effects of overexpressed transcription factors, our findings together suggest that reduced phospho-CREB binding to *Bdnf* promoters is mediated by the selective recruitment of H3K27me3 to key *Bdnf* promoters during a course of opiate exposure.
*Bdnf* gene regulation is also known to be controlled by NURR1, which is required for maintenance of adult dopamine neurons in VTA^[@R33]^; NURR1 acts downstream of CREB^[@R32],[@R34]^ and upstream of BDNF^[@R31],[@R32]^. This connection between CREB and NURR1 is supported by our data that *Nurr1* mRNA expression is induced in VTA upon CREB overexpression. This finding supports the hypothesis that the morphine-induced reduction in phospho-CREB binding to the *Nurr1* promoter demonstrated in this study mediates the *Nurr1* suppression in VTA by chronic morphine. In parallel, we found that *Nurr1* mRNA levels and NURR1 binding to *Bdnf* promoters are decreased in this region by morphine. Our observation that *Bdnf* mRNA levels are increased in VTA upon NURR1 overexpression is consistent with the scheme that reduced NURR1 binding to *Bdnf* promoters contributes to *Bdnf* suppression in response to morphine. This scheme is also supported by our behavioral data: chronic morphine-induced locomotor responses, as well as morphine-elicited reward, are blocked by NURR1 overexpression in VTA. Since NURR1 overexpression induces *Bdnf*, these behavioral data are consistent with prior reports that elevated VTA BDNF activity is also associated with suppression of morphine's behavioral effects^[@R11]^. Such a connection between NURR1 and BDNF was established by our demonstration that NURR1's ability to suppress morphine-elicited behaviors was lost in mice lacking BDNF selectively in this brain region.
In conclusion, our findings reinforce the complexity of gene and chromatin regulation in adult brain and emphasize the importance of examining numerous chromatin endpoints when investigating epigenetic mechanisms of gene regulation *in vivo* ([Supplementary Fig. 8](#SD1){ref-type="supplementary-material"}). Given that morphine suppression of *Bdnf* in VTA contributes importantly to the drug's behavioral effects^[@R11]^, results of the present study provide fundamentally new insight into the detailed molecular mechanisms underlying morphine-induced neural and behavioral plasticity.
Online materials and methods {#S10}
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General Methods {#S11}
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### Animals {#S12}
Male 8--10 week-old Sprague Dawley rats (225--250 g; Charles River) were used in all experiments unless otherwise noted, given the larger size of their VTA, which makes detailed epigenetic studies possible. In some experiments, male 7--8 week-old c57BL/6 mice (25--30 g, Jackson), and 9--13 week-old floxed CREB and floxed BDNF mice were used. Floxed CREB mice on a c57BL/6 background and floxed BDNF mice on a BL6/sv129 background were generated and maintained, as previously described^[@R41],[@R42]^. These mice were used for key behavioral experiments in order to take advantage of genetic mutants. All animals were housed in groups of 2 rats or 2--5 mice per cage at 22--25°C on a 12-hr light/dark cycle (lights on 7:00 AM) with access to food and water *ad libitum*. Animals were acclimated to vivarium conditions for at least one week before experimentation and assigned randomly to experimental groups. All behavioral experiments, except self-administration, were performed during the light cycle. All animals used were experimentally naïve. All work was in accordance with guidelines of the Society for Neuroscience and the Mount Sinai IACUC.
### Human postmortem subjects {#S13}
Postmortem human brain specimens from heroin users and control subjects were collected within \~24 hr after death under approved protocols at the Department of Forensic Medicine at Semmelweis University, Hungary, or the National Institute of Forensic Medicine, Karolinska Institutet, Stockholm, Sweden. Brains were immediately frozen using dry-ice--cooled isopentane, cryosectioned, and thaw-mounted onto poly-l-lysine--treated slides. Cause and manner of death were determined by a forensic pathologist after evaluating the circumstances of death, toxicology data (blood, urine, and liver), and autopsy results. Information was also evaluated from police reports, family and friends, and medical records as described in [Supplementary Table 1](#SD2){ref-type="supplementary-material"}. Inclusion criteria were death associated with heroin intoxication (verified by toxicology), physical signs of heroin use such as needle tracks, and history of heroin abuse. Exclusion criteria were postmortem interval (PMI) of \>24 hr, age \<20 years, HIV-positive status, and history of alcoholism.
### Heroin self-administration {#S14}
Heroin self-administration in rats was conducted according to published procedures^[@R43]^.
### Morphine CPP {#S15}
An unbiased CPP paradigm was used according to published procedures^[@R11]^.
### Locomotor activity {#S16}
Rats were given morphine (morphine sulfate in saline, 5 mg/kg, IP) or saline daily for 14 days and then challenged with morphine (5 mg/kg, IP) or saline on day 28. On day 0, 14, and 28, morphine or saline was administered after 30 min habituation in the locomotor chamber. Locomotor activity was monitored for 30 min after the administration using the Photobeam Activity System (San Diego Instruments).
### HSV vectors {#S17}
HSV-GFP was generated with the p1005+ HSV amplicon bicistronic plasmid containing a complete CMV-GFP expression cassette as the second cistron. For HSV-NURR1, we designed the forward primer with *Kpn*I restriction sites (5′-GGGGTACCCCATGCCTTGTGTTCAGGCGCAGTATG-3′) and reverse primer with *Xho*I restriction sites (5′-CCGCTCGAGCGGTTAGAAAGGTAAGGTGTCCAGGAAA-3′). The PCR products for *Nurr1* were then digested, purified, and ligated into the first cistron downstream of the HSV IE4/5 promoter of the HSV amplicon at *KpnI*-*Xho*I. For HSV-EZH2, the coding sequences for *Ezh2* from pCMV-HA hEZH2 (Addgene plasmid \#24230) were subcloned into the HSV amplicon at *EcoR*V-*BamH*I. Viral titers were determined using qPCR and \~1×10^9^ particles/site were used. All behavioral experiments were initiated ≥2 days and completed \<5 days after viral injection, the time period when HSV expression is maximal. HSV-CREB and HSV-Cre vectors have been described previously^[@R44],[@R45]^. Viral targeting to VTA was confirmed for all animals; \<3% were excluded for anatomically incorrect placements. For validation of viral-mediated gene transfer, rat or mouse VTA bilateral punches (rat, 14-gauge; mouse, 15-gauge) were dissected under fluorescent stereomicroscope (Leica MZ10F) 4 days after intra-VTA infusion of the HSVs and processed for qPCR as described below ([Supplementary Figs. 3a, 5a,b and 7a](#SD1){ref-type="supplementary-material"}). In addition, rat bilateral 14 gauge punches were collected from substantia nigra and red nucleus (as anatomical controls), and VTA, and processed for Western blotting as described below ([Supplementary Fig. 4](#SD1){ref-type="supplementary-material"}).
### Stereotaxic surgery {#S18}
Mice or rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). 33 gauge needles were used to bilaterally infuse HSVs or LVs into VTA \[for mice, AP = −3.2, ML = ±1.0, DV = −4.6; for rats, AP = −5.5, ML = ±2.0, DV = −7.4 from Bregma (mm), 7° angle\]. An infusion volume of 0.5 μl (mice) or 1.0 μl (rats) was delivered using 5 μl Hamilton syringe over the course of 5 min \[at a rate of 0.1 μl/min (mice) or 0.2 μl/min (rats)\]. The infusion needle remained in place for at least 5 min after the infusions before removal to prevent backflow of the injected material.
### RNA isolation and qPCR {#S19}
Total RNA was isolated from postmortem human brain specimens using Arcturus Pico Pure kit (Life Technologies) and from frozen VTA bilateral punches (rat, 14-gauge; mouse, 15-gauge) using Trizol (Life Technologies) and a micro RNeasy kit (Qiagen) as described^[@R11]^. RNA was reverse transcribed into cDNA using an iScript cDNA synthesis kit (BioRad). Primers were designed to amplify regions of 100--200 bp located within the genes of interest or chosen from previous studies^[@R16],[@R46]^. All reactions were run in triplicate and analyzed using the ΔΔCt method^[@R47]^ with glyceraldehyde-3-phosphate dehydrogenase (*Gapdh*) or 18S ribosomal RNA (*Rn18S*) as a normalization control. qPCR primers are listed in [Supplementary Table 2](#SD2){ref-type="supplementary-material"}.
### Quantitative chromatin immunoprecipitation (qChIP) {#S20}
Freshly dissected rat VTA punches were cross-linked with 1% formaldehyde, quenched with 2 M glycine, and sonicated to \~500 bp at 4°C. For each ChIP for histone modifications, bilateral 14-gauge VTA punches were used; for each ChIP for transcription factors, bilateral 14-gauge punches were pooled from two rats. Separate groups of animals were used for each qChIP experiment. Sheep anti-rabbit or anti-mouse IgG magnetic beads (Invitrogen) were prepared by incubating with antibodies of interest. Chromatin samples were immunoprecipitated with the conjugated bead/antibody mixtures, and then reverse cross-linked. DNA was purified and quantified using qPCR in triplicate from independent groups of animals. Normal mouse or rabbit IgG immunoprecipitations were performed as controls. Primers for most qChIPs, shown in [Supplementary Table 3](#SD2){ref-type="supplementary-material"}, were designed to amplify 150--200 bp products that include CREB binding sites. All ChIP antibodies were validated using qChIP ([Supplementary Fig. 9](#SD1){ref-type="supplementary-material"}) with rat primer sets that were designed, based on previous studies validating ChIP antibodies in other systems or on validation information provided by the manufacturers ([Supplementary Table 4](#SD2){ref-type="supplementary-material"}). The primer pairs for NURR1 CHIP validation (e.g., *Bdnf*, *Pitx3*, and *Th* primers) were designed to amplify 150--200 bp products that contain a NBRE (NGFI-B response element)--like sequence, which are putative NURR1 binding sites^[@R30]^. To further demonstrate the selectivity of anti-NURR1, we performed Western blotting using anti-NOR1 (rabbit polyclonal, Santa Cruz, sc-30154), anti-NUR77 (rabbit polyclonal, Santa Cruz, sc-5569), or anti-NURR1 (rabbit polyclonal, Santa Cruz, sc-991) on samples that were immunoprecipitated with anti-NURR1 ([Supplementary Fig. 7c--e](#SD1){ref-type="supplementary-material"}).
### Immunohistochemistry {#S21}
Immunohistochemistry was conducted according to published procedures^[@R11]^. For GFP/TH and TMT/TH double-labeling, brain sections were incubated in 1:4000 of anti-TH (T1299, Sigma) 1:2000 of rabbit polyclonal anti-GFP (A11122, Invitrogen), or 1:1000 of rabbit anti-dsRed (632496, Clontech), respectively, in block solution overnight at 4°C. The next day, sections were incubated in 1:500 of donkey anti-rabbit Cy2 (Immuno Research) for anti-GFP together with 1:500 of donkey anti-mouse Cy3 for anti-TH or 1:500 of donkey anti-rabbit Cy3 for anti-dsRed together with 1:500 of donkey anti-mouse Cy2 for anti-TH in PBS for 1 hr. For GFP/Cre double-labeling, 1:2000 of rabbit polyclonal anti-GFP (A11122, Invitrogen), 1:1000 mouse anti-Cre recombinase (MAB3120), 1:500 of donkey anti-rabbit Cy2, and 1:500 of donkey anti-mouse Cy3 were used. All sections were imaged on a LSM 710 confocal microscope (Zeiss) at 10× or 20× magnification. All histological procedures were replicated independently at least 2 times.
### Western blotting {#S22}
Western blotting analyses were conducted according to published procedures^[@R15]^. For primary antibody incubation, anti-EZH2 (1:1000; rabbit monoclonal, Cell Signaling, 5246S), anti-total-CREB (1:1000; rabbit monoclonal, Millipore, 17-600), and anti-NURR1 were used. Blots were imaged with the Odyssey Infrared Imaging system (Li-Cor) and quantified by densitometry using NIH ImageJ. The amount of protein blotted onto each lane was normalized to levels of tubulin. Western blot analyses were replicated at least twice.
Specific experiments {#S23}
--------------------
### Experiment 1: *Bdnf* down-regulation by opiates {#S24}
We examined the effect of opiate exposure on *Bdnf* mRNA expression in human, rat, and mosue VTA. Total RNA was isolated from postmortem brain specimens of human heroin addicts (*n* = 9) and controls (*n* = 5) ([Supplementary Table 1](#SD2){ref-type="supplementary-material"}). VTA punches (bilateral, 14-gauge) were collected from previously frozen brains of rats that were killed 24 hr after the final heroin self-administration session (*n* = 24) ([Supplementary Fig. 1b](#SD1){ref-type="supplementary-material"}). We also freshly dissected 14-gauge bilateral rat VTA punches 30 min after completing the last locomotor activity test on day 28 ([Supplementary Fig. 1c](#SD1){ref-type="supplementary-material"}), after 14 days withdrawal from 14 days of prior morphine administration (*n* = 18). Mouse VTA punches (bilateral, 15-gauge) were freshly dissected 1 hr after completing morphine CPP test sessions (*n* = 24) ([Supplementary Fig. 1d](#SD1){ref-type="supplementary-material"}).
### Experiment 2: Stalling of Pol II at *Bdnf* promoters by morphine {#S25}
For each ChIP sample, VTA punches were pooled from two rats (4 punches) that were killed on day 28, after 14 days withdrawal from 14 days of prior morphine administration (*n* = 20 or 24). Anti-total-Pol II (mouse monoclonal, Millipore, 05-623), anti-phospho-Ser2-Pol II (antibody to Pol II phosphorylated at Ser2 of its C terminal domain) (mouse monoclonal, Abcam, ab24758), or anti-phospho-Ser5-Pol II (rabbit polyclonal, Abcam, ab5131) were used for this experiment. Primers for qChIP with anti-total-Pol II and anti-phospho-Ser5-Pol II were designed to amplify 150--200 bp products that include CREB binding sites of *Bdnf* promoters. Primers for qChIP with anti-phospho-Ser2-Pol II were designed to amplify 150--200 bp products within the coding sequence of the *Bdnf* gene ([Supplementary Table 3](#SD2){ref-type="supplementary-material"}).
### Experiment 3: Histone modifications by morphine {#S26}
For each ChIP sample for histone modifications, VTA punches (2 punches) were collected from one control or chronic morphine-treated rat (14 days of morphine administration followed by 14 days of withdrawal) (*n* = 8 -11 for each ChIP). For each ChIP sample for key histone modifying enzymes and related regulatory proteins, VTA punches were pooled from two rats (4 punches) (*n* = 14 -- 22). Anti-acH3 (rabbit polyclonal, Millipore, 06-599), anti-acH4 (rabbit polyclonal, Millipore, 06-598), anti-H3K4me3 (rabbit monoclonal, Millipore, 17-614), anti-H3K9me2 (mouse monoclonal, Abcam, ab1220), anti-H3K9me3 (rabbit polyclonal, Abcam, ab8898), anti-H3K27me3 (mouse monoclonal, Abcam, ab6002), anti-H3K36me3 (rabbit polyclonal, Abcam, ab9050), anti-mSIN3a (rabbit polyclonal, Santa Cruz, sc-994X), anti-ING2 (rabbit polyclonal, Santa Cruz, sc-134973), anti-KMT2A/MLL (rabbit polyclonal, Millipore, ABE240), anti-KMT1C/G9a (rabbit polyclonal, Abcam, ab40542), anti-SUZ12 (rabbit monoclonal, Cell Signaling, 3737S), anti-EZH2, anti-RING1A (rabbit polyclonal, Abcam, ab32644), anti-RING1B (rabbit monoclonal, Cell Signaling, 5694S), and anti-BMI1 (rabbit monoclonal, Cell Signaling, 6964S) were used.
### Experiment 4: Role of EZH2 in *Bdnf* expression and morphine CPP {#S27}
Stereotaxic surgery was performed on adult male c57BL/6 mice (8 weeks) to inject HSV-EZH2-GFP or HSV-GFP into the VTA. The first batch of mice (*n* = 18) was killed for qPCR for validation ([Supplementary Fig. 3a](#SD1){ref-type="supplementary-material"}) and *Bdnf* expression ([Fig. 3h](#F3){ref-type="fig"}) 4 days after stereotaxic surgery. Viral injection sites were confirmed using standard histological methods (see [Fig. 3g](#F3){ref-type="fig"}) using VTA sections from a subset of HSV-EZH2 infused mice (*n* = 3). A second batch of mice (*n* = 24) was used for morphine CPP ([Fig. 3i](#F3){ref-type="fig"}). Two days after the surgery, mice were conditioned in a three-chambered CPP box for three days. The next day, animals were given a final CPP test.
Separate groups of rats were bilaterally injected with LV-shRNA-EZH2 (iV000225, Applied Biological Materials, Canada) or LV-shRNA-scramble (LVP015-G) in VTA. The first batch of rats (*n* = 22) was killed for qPCR for validation ([Supplementary Fig. 3c](#SD1){ref-type="supplementary-material"}) 10 days after stereotaxic surgery. Viral injection sites were confirmed using standard histological methods ([Supplementary Fig. 3b](#SD1){ref-type="supplementary-material"}) using VTA sections from a subset of LV-shRNA-EZH2 infused rats (*n* = 3). A second batch of rats (*n* = 14) was used for qPCR for *Bdnf* expression after chronic morphine treatment ([Supplementary Fig. 3d](#SD1){ref-type="supplementary-material"}). Briefly, we first injected morphine (5 mg/kg, IP) for 14 days and bilateral viral injections with LVs were performed 4 days after the last morphine injection. Rats were killed and VTA punches (bilateral, 14-gauge) were collected 10 days after surgery.
### Experiment 5: Role of CREB in *Bdnf* expression {#S28}
For total- and phospho-CREB ChIP experiments, VTA punches were pooled from two chronic morphine-treated rats (14 days of morphine administration followed by 14 days of withdrawal) (*n* = 16 or 20 for each). Anti-total-CREB and anti-phospho-CREB (rabbit polyclonal, Millipore, 06-519) were used for immunoprecipitation. In separate groups of animals, we performed stereotaxic surgery on male c57BL/6 mice or floxed CREB mice to inject HSV-CREB or HSV-Cre and their controls (HSV-TMT or HSV-GFP, respectively) into the VTA. The HSV-CREB/HSV-TMT infused c57BL/6 mice (*n* = 17) and HSV-Cre/HSV-GFP infused floxed CREB mice (*n* = 20) were killed for qPCR for validation ([Supplementary Fig. 5a,b](#SD1){ref-type="supplementary-material"}) and *Bdnf* expression ([Fig. 4d,f](#F4){ref-type="fig"}) 4 days after stereotaxic surgery. Viral injection sites were confirmed using standard histological methods ([Fig. 4c,e](#F4){ref-type="fig"}) using VTA sections from a subset of HSV-CREB or HSV-Cre infused mice (*n* = 3 each).
### Experiment 6: Epigenetic mechanism of *Bdnf* regulation: interaction between pCREB and H3K27me3 {#S29}
Stereotaxic surgery was performed on adult rats to inject HSV-EZH2 or HSV-GFP into the VTA. Two batches of rats were killed for H3K27me3 ChIP (*n* = 14) and pCREB ChIP (*n* = 22) at *Bdnf* promoters ([Fig. 4g,h](#F4){ref-type="fig"}) 4 days after stereotaxic surgery. For these ChIP experiments, VTA punches were pooled from two rats. For H3K27me3 ChIP experiment, punches from one rat were used. mRNA levels of *Bdnf* exon IX were also measured using cDNA derived from VTA of HSV-EZH2 or -GFP infused mice (from **Experiment 4**) ([Supplementary Fig. 3a](#SD1){ref-type="supplementary-material"}). ChIP experiments with anti-phospho-CREB, anti-total-CREB, and anti-H3K27me3 were also conducted at *Bdnf* promoter regions with VTA punches from heroin self-administered rats (anti-phospho-CREB, *n* = 20; anti-total-CREB, *n* = 14; anti-H3K27me3, *n* = 11) ([Supplementary Fig. 6d--f](#SD1){ref-type="supplementary-material"}).
Another set of ChIP experiments with anti-phospho-CREB, anti-total-CREB, and anti-H3K27me3 was conducted as a control at *Th* and *Gria1* promoter regions with control and chronic morphine-treated rats (14 days of morphine administration followed by 14 days of withdrawal) (anti-phospho-CREB, *n* = 32; anti-total-CREB, *n* = 18; anti-H3K27me3, *n* = 10) ([Supplementary Fig. 6a--c](#SD1){ref-type="supplementary-material"}).
### Experiment 7: Epigenetic mechanism of *Bdnf* regulation: interaction between NURR1 and CREB {#S30}
ChIP experiment with anti-NURR1 was conducted at *Bdnf* promoters with chronic morphine-treated rats (14 days of morphine administration followed by 14 days of withdrawal) (*n* = 18). Another set of ChIP experiments with anti-H3K27me3 and anti-phospho-CREB was conducted at *Nurr1* promoter regions with chronic morphine-treated rats (anti-H3K27me3, *n* = 10; anti-phospho-CREB, *n* = 36). In separate experiments, stereotaxic surgery was performed on rats to inject HSV-CREB or HSV-TMT into the VTA. Two batches of rats were killed for total- (*n* = 20) and phospho- (*n* = 20) CREB ChIP at the *Nurr1* promoter ([Supplementary Fig. 5c,d](#SD1){ref-type="supplementary-material"}). For NURR1 ChIP and total-/phospho-CREB ChIP experiments, VTA punches were pooled from two rats. For H3K27me3 ChIP experiment, bilateral punches from one rat were used. *Nurr1* mRNA levels in VTA were also measured using cDNA derived from chronic morphine-treated rats, from heroin self-administering rats, or from HSV-CREB infused mice generated from prior experiments.
### Experiment 8: Behavioral and molecular effects of NURR1 {#S31}
We performed stereotaxic surgery on rats to inject HSV-NURR1 or HSV-TMT into the VTA. The rats (*n* = 19) were killed for qPCR validation ([Supplementary Fig. 7a](#SD1){ref-type="supplementary-material"}) and *Bdnf* expression ([Fig. 5j](#F5){ref-type="fig"}) 4 days after stereotaxic surgery. Viral injection sites were confirmed using standard histological methods ([Fig. 5i](#F5){ref-type="fig"}) using VTA sections from a subset of HSV-NURR1 infused rats (*n* = 3). Another set of rats was given daily injection of morphine (5 mg/kg, IP; *n* = 27) or saline (*n* = 11) for 14 days. Each morphine-/saline-injected batch was then re-exposed to morphine (5 mg/kg, IP) or saline on day 28. All of these rats underwent locomotor activity tests on day 0, 14, and 28 and stereotaxic surgery with HSV-NURR1 or HSV-TMT into the VTA 10 days after the last injection of chronic morphine or saline (on day 24) ([Fig. 5g,h](#F5){ref-type="fig"} and [Supplementary Fig. 7b](#SD1){ref-type="supplementary-material"}). In a separate experiment, stereotaxic surgery was performed on adult male c57BL/6 mice (8 weeks) or floxed BDNF mice to inject HSV-NURR1, HSV-TMT, and/or HSV-Cre into the VTA. In case of floxed BDNF mice, we infused 0.25 μl of HSV-NURR1 or HSV-TMT together with 0.25 μl of HSV-Cre. Two days after the surgery, the HSV-NURR1/HSV-TMT infused c57BL/6 mice (*n* = 15) and HSV-NURR+HSV-Cre/HSV-TMT+HSV-Cre infused floxed BDNF mice (*n* = 19) were conditioned in a three-chambered CPP box for three days. The next day, animals were given a final CPP test.
Statistical analysis {#S32}
--------------------
Sample sizes were similar to those reported in previous works and based on expected effect sizes and power analyses^[@R1],[@R7],[@R11],[@R15],[@R28],[@R42],[@R44],[@R48]^. Data were collected and processed randomly and analyzed with SigmaPlot 12.5 (Systat) and Prism 5.0 (GraphPad). Data collection and analysis were not performed blind to the conditions of the experiments. Normality (Shapiro-Wilk test) and equal variance (*F*-test) assumptions were confirmed before parametric analysis, unless otherwise indicated. For normally distributed data, Student's *t*-tests were used to assess differences between two experimental groups. For a two-sample comparison of means with unequal variances, Student's *t*-tests with Welch's correction were used. One-way ANOVAs were used for analysis of three or more groups, followed by Fisher's PLSD *post hoc* tests. For all qChIP analyses, two-way ANOVAs with Fisher's PLSD *post hoc* tests were used. For locomotor activity and heroin self-administration data, a repeated-measures two-way ANOVA was used followed by Fisher's PLSD *post hoc* tests. Main and interaction effects were considered significant at *p* \< 0.05. Mann-Whitney *U* tests were performed to compare two columns of non-normally distributed data. Outliers were excluded from analysis when identified by Grubbs' test. Bar graphs show mean ± SEM. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value. Individual statistical values are available in the [Supplementary Methods Checklist](#SD1){ref-type="supplementary-material"}.
Supplementary Material {#S33}
======================
We thank Ted Abel (University of Pennsylvania) for helpful discussions. This work was supported by grants from the National Institute on Drug Abuse (E.J.N., M.M.-R.).
Author contributions: J.W.K., M.S.M.-R., S.J.R., Y.L.H. and E.J.N. designed research; J.W.K., M.S.M.-R., Q.L., G.E., S.A.R.A., D.F., J.F., H.S., K.N.S., D.M.D.-W., E.R., C.J.P., D.W., R.C.B., B.L., G.E.H., H.B., B.C., A.J.R., V.F.V., C.D., Z.L., E.M., M.K.L. and D.M.D. performed the experiments; J.W.K. and R.L.N. generated viral vectors; J.W.K. and Q.L. analyzed data; J.W.K. and E.J.N. wrote the paper.
Competing Financial Interests: The authors declare no competing financial interests.
Note: [Supplementary information](#SD1){ref-type="supplementary-material"} is available in the online version of the paper.
{#F1}
{ref-type="fig"}) relative to saline controls \[two-way analysis of variance (ANOVA), drug effect: *F*~1,64~ = 12.898, *p* \< 0.001; region effect: *F*~3,64~ = 0.923, *p* = 0.435; drug×region effect: *F*~3,64~ = 0.328, *p* = 0.805, *n* = 9 rats\]. (**b**) Schematic diagram depicting the relative position of amplicons (green thick lines) generated by primers used to quantify immunoprecipitated chromatin-DNA. Exons are represented as boxes and the introns as lines. Numbers of the exons are indicated in roman numerals. The positions of CREB binding sites (red circles) at *Bdnf* promoter regions are indicated relative to the transcription start site of exon I (96-84 bp/90-78 bp), exon II (317-307 bp), exon IV (42-33 bp/36-26 bp), and exon VI (84-73 bp). Primer information is provided in [Supplementary Table 3](#SD2){ref-type="supplementary-material"}. (**c**) qChIP showed that binding of total-Pol II to *Bdnf*-p2 was increased in response to chronic morphine (drug effect: *F*~1,39~ =13.279, *p* \< 0.001; region effect: *F*~3,39~ = 1.019, *p* = 0.395; drug×region effect: *F*~3,39~ = 1.096, *p* = 0.362, *n* = 6 rats). (**d**) Binding of phospho-Ser5-Pol II to *Bdnf*-p2, -p4, and -p6 was also increased in VTA of morphine-treated rats (drug effect: *F*~1,31~ = 18.820, *p* \< 0.001; region effect: *F*~3,31~ =6.474, *p* = 0.002; drug×region effect: *F*~3,31~= 2.069, *p* = 0.069, *n* = 5 rats). (**e**) In contrast, binding of phospho-Ser2-Pol II to *Bdnf-*eII, -eIV, and -eVI was decreased after morphine exposure (drug effect: *F*~1,32~ = 19.921, *p* \< 0.001; region effect: *F*~3,32~ = 0.00309, *p* = 1.000; drug×region effect: *F*~3,32~ = 0.165, *p* = 0.919, *n* = 5 rats). Two-way ANOVA with Fisher's protected least significant difference (PLSD) *post hoc* tests, \**p* \< 0.05, \*\**p* \< 0.01, and \*\*\**p* \< 0.001. Bar graphs show mean ± SEM.](nihms651676f2){#F2}
{ref-type="fig"}) selectively altered H3K27me3 (two-way ANOVA, drug effect: *F*~1,22~ = 0.144, *p* = 0.708; region effect: *F*~3,22~ = 6.442, *p* = 0.003; drug×region effect: *F*~3,22~ = 6.178, *p* = 0.003, *n* = 4 rats, [Supplementary Fig. 2f](#SD1){ref-type="supplementary-material"}) at *Bdnf*-p2, particularly. Chronic morphine changed acH4 (drug effect: *F*~1,27~ = 11.509, *p* = 0.002; region effect: *F*~3,27~ = 0.482, *p* = 0.698; drug×region effect: *F*~3,27~ = 0.184, *p* = 0.906, *n* = 5, 4 rats, [Supplementary Fig. 2b](#SD1){ref-type="supplementary-material"}) at *Bdnf*-p4 in rat VTA, with no changes seen in several other histone modifications analyzed. Additional *post hoc* analyses with Student's *t*-tests showed that chronic morphine also changed acH3 (unpaired *t*-test, *t*~6~= 2.581, *p* = 0.0417*, n* = 4 rats) and H3K4me3 (*t*-test, *t*~8~ = 2.312, *p* = 0.0495, *n* = 5 rats) at *Bdnf*-p2 in VTA. Histone modifications by chronic morphine at other *Bdnf* promoters (*Bdnf*-p1, -p4, and -p6) are available in the [Supplementary Fig. 2a--g](#SD1){ref-type="supplementary-material"}. (**b**) Binding of mSIN3a (two-way ANOVA, drug effect: *F*~1,36~ = 25.829, *p* \< 0.001; region effect: *F*~3,36~ = 0.541, *p* = 0.653; drug×region effect: *F*~3,36~ = 0.464, *p* = 0.709, *n* = 6,5 rats) and ING2 (drug effect: *F*~1,28~ = 37.786, *p* \< 0.001; region effect: *F*~3,28~ = 2.450, *p* = 0.614; drug×region effect: *F*~3,28~ = 0.552, *p* = 0.651, *n* = 5,4 rats), core components of a major repressor complex, to *Bdnf*-p2 were increased by chronic morphine. (**c**) Consistent with enhancement in H3K4me3 levels, binding of MLL1 (KMT2A) to *Bdnf*-p2 was increased by chronic morphine (drug effect: *F*~1,23~ = 24.884, *p* \< 0.001; region effect: *F*~3,23~ = 2.285, *p* = 0.106; drug×region effect: *F*~3,23~ = 2.177, *p* = 0.118, *n* = 4 rats). (**d**) There was no morphine-induced alteration in binding of G9a (EHMT2) to *Bdnf*-p2 (drug effect: *F*~1,27~ = 0.00384, *p* = 0.951; region effect: *F*~3,27~ = 0.153, *p* = 0.927; drug×region effect: *F*~3,27~ = 0.153, *p* = 0.927, *n* = 5,4 rats). (**e**) Consistent with enhancement in H3K27me3 levels, binding of SUZ12 (drug effect: *F*~1,24~ = 12.662, *p* = 0.002; region effect: *F*~3,24~ = 1.211, *p* = 0.327; drug×region effect: *F*~3,24~ = 0.526, *p* = 0.668, *n* = 4 rats) and EZH2 (drug effect: *F*~1,20~ =16.872, *p* \< 0.001; region effect: *F*~3,20~ = 1.209, *p* = 0.332; drug×region effect: *F*~3,20~ = 1.111, *p* = 0.368, *n* = 4,3 rats), members of PRC2, to *Bdnf*-p2 was enhanced by chronic morphine. (**f**) In contrast, binding of PRC1 members, RING1A (drug effect: *F*~1,24~ = 13.708, *p* \< 0.001; region effect: *F*~3,24~ = 0.0154, *p* = 0.997; drug×region effect: *F*~3,24~ = 2.713, *p* = 0.067, *n* = 4 rats) and BMI1 (drug effect: *F*~1,32~ = 10.975, *p* \<= 0.002; region effect: *F*~3,32~ = 0.117, *p* = 0.950; drug×region effect: *F*~3,32~ = 0.0345, *p* = 0.991, *n* = 5 rats), to *Bdnf*-p2 were decreased by chronic morphine, but RING1B binding was unaffected (drug effect: *F*~1,23~ =1.372, *p* = 0.253; region effect: *F*~3,23~ = 0.267, *p* = 0.848; drug×region effect: *F*~3,23~ = 0.298, *p* = 0.827, *n* = 4 rats). Morphine-induced epigenetic alterations by key histone modifying enzymes and related regulatory proteins at other *Bdnf* promoters (*Bdnf*- p1, -p4, and -p6) are available in [Supplementary Fig. 2h--p](#SD1){ref-type="supplementary-material"}. (**g**) Representative low-magnification photomicrographs with an inset depicting localized HSV-mediated EZH2 expression (GFP+, green) in dopaminergic neurons (TH+, red) in mouse VTA (scale bar, 50 μm). The results were replicated in three independent experiments. (**h**) Intra-VTA HSV-EZH2 suppressed the expression of *Bdnf* exon IX mRNA in mouse VTA compared to HSV-GFP controls (unpaired *t*-test, *t*~16~ = 2.168, *p* = 0.0456, *n* = 9 mice). (**i**) EZH2 overexpression in mouse VTA dramatically enhances morphine reward (*t*-test, *t*~22~ = 4.134, *p* = 0.000435, *n* = 12 mice). Two-way ANOVA with Fisher's PLSD *post hoc* tests, \**p* \< 0.05, \*\**p* \< 0.01, and \*\*\**p* \< 0.001; *t*-tests, ^\#^*p* \< 0.05 and ^\#\#\#^*p* \< 0.001. Bar graphs show mean ± SEM. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value.](nihms651676f3){#F3}
{ref-type="fig"}) increased total-CREB binding to *Bdnf*-p6 in rat VTA, with no effects seen at other *Bdnf* promoters (two-way ANOVA, drug effect: *F*~1,32~ = 15.053, *p* \< 0.001; region effect: *F*~3,32~ = 0.371, *p* = 0.774; drug×region effect: *F*~3,32~ = 0.412, *p* = 0.745, *n* = 5 rats). (**b**) In contrast, chronic morphine reduced phospho-CREB binding to *Bdnf*-p1, -p2, and -p4 (drug effect: *F*~1,24~ = 32.487, *p* \< 0.001; region effect: *F*~3,24~ = 1.026, *p* = 0.399; drug×region effect: *F*~3,24~ = 0.834, *p* = 0.489, *n* = 4 rats). (**c**) Representative low-magnification photomicrographs with an inset depicting localized HSV-mediated CREB expression \[tdTomato+ (TMT+), red\] in dopaminergic neurons (TH+, green) in VTA of c57BL/6 mice (scale bar, 50 μm). (**d**) HSV-mediated CREB1 overexpression increases the expression of *Bdnf* exon IX in mouse VTA (Mann Whitney *U* test, *U* = 7, *p* = 0.006, *n* = 9,8 mice). (**e**) Representative low-magnification photomicrographs with an inset depicting localized HSV-mediated Cre expression (red) in dopaminergic neurons (TH+, green) in VTA of floxed CREB mice (scale bar, 50 μm). (**c,e**) Histological results were replicated in three independent experiments. (**f**) In contrast, knockdown of *Creb1* in the VTA of floxed CREB mice decreases the expression of *Bdnf* exon IX (unpaired *t*-test, *t*~18~ = 2.506, *p* = 0.0220*, n* = 10 mice). (**g**) HSV-mediated EZH2 overexpression (in the absence of morphine) significantly increased H3K27me3 levels at *Bdnf*-p1, -p2, and -p6 (two-way ANOVA, drug effect: *F*~1,48~ = 35.413, *p* \< 0.001; region effect: *F*~3,48~ = 2.318, *p* = 0.087; drug×region effect: *F*~3,48~ = 2.318, *p* = 0.087, *n* = 8,6 rats). Additional *post hoc* analyses with Mann Whitney *U* tests and Student's t-tests showed that EZH2 overexpression increased H3K27me3 levels at all *Bdnf*-promoters examined (*Bdnf*-p1, *U* test, *U* = 5, *p* = 0.013; *Bdnf*-p2, *t*-test, *t*~12~ = 2.957, *p* = 0.012; *Bdnf*-p4, *t*-test, *t*~12~ = 2.781, *p* = 0.0166; *Bdnf*-p6, *U* test, *U* =2, *p* = 0.003). (**h**) EZH2 overexpression significantly reduced phospho-CREB binding to *Bdnf*-p1, -p2, -p4, and -p6 (two-way ANOVA, drug effect: *F*~1,36~ =25.091, *p* \< 0.001; region effect: *F*~3,36~ = 0.182, *p* = 0.908; drug×region effect: *F*~3,36~ = 0.104, *p* = 0.957, *n* = 5,6 rats). (**a,b,g,h**) Two-way ANOVA with Fisher's PLSD *post hoc* tests, \**p* \< 0.05, \*\**p* \< 0.01, and \*\*\**p* \< 0.001; (**d**) Mann-Whitney *U* test, \*\**p* \< 0.01; (**f**) Student's *t*-test, \**p* \< 0.05; (**g**) Mann-Whitney *U* tests at *Bdnf*-p1 and -p6 and Student's *t*-tests at *Bdnf*-p2 and -p4, ^\#^*p* \< 0.05 and ^\#\#^*p* \< 0.01. Bar graphs show mean ± SEM. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value.](nihms651676f4){#F4}
{ref-type="fig"}) decreased NURR1 binding to *Bdnf*-p1 and -p2 in rat VTA. Two-way ANOVA (drug effect: *F*~1,27~ = 17.990, *p* \< 0.001; region effect: *F*~3,27~ = 0.403, *p* = 0.752; drug×region effect: *F*~3,27~ = 0.990, *p* = 0.412, *n* = 4,5 rats) with Fisher's PLSD *post hoc* tests, \**p* \< 0.05 and \*\**p* \< 0.01. (**b,c**) Chronic morphine (**b**, unpaired *t*-test, *t*~15~ = 2.509, *p* = 0.0241*, n* = 9,8 rats) and self-administered heroin (**c**, *t*-test, *t*~16~ = 2.162, *p* = 0.0461*, n* = 8,10 rats) decreased *Nurr1* mRNA expression in rat VTA. (**d**) H3K27me3 binding at the *Nurr1* gene promoter was increased by chronic morphine in rat VTA (*t*-test, *t*~8~ = 2.733, *p* = 0.0257*, n* = 5 rats). (**e**) Binding of phospho-CREB to the *Nurr1* gene promoter was decreased by chronic morphine in rat VTA (*t*-test, *t*~16~ = 2.285, *p* = 0.0363*, n* = 10,8 rats) (**f**) HSV-mediated CREB1 overexpression induced *Nurr1* expression in mouse VTA (unpaired *t*-test with Welch's correction, *t*~9.496~ = 2.935, *p* = 0.0157*, n* = 9 mice). (**g**) Scheme of the morphine treatment regimen used for locomotor tests in rats. HSV-NURR1 or its control HSV-TMT was infused into VTA of rats that were given chronic morphine (14 days, 5 mg/kg IP, followed by 10 days of withdrawal) and, 4 days later, the rats were challenged with morphine during the locomotor test. (**h**) Morphine-treated rats that were injected with HSV-TMT and then given a morphine challenge (M/M+HSV-TMT) showed higher locomotor activity compared to morphine-treated rats injected with HSV-TMT and then given a saline challenge (M/S+HSV-TMT). HSV-mediated overexpression of NURR1 in VTA blocked the morphine challenge-induced locomotor activation (M/M+HSV-NURR1). One-way ANOVA (*F*~2,24~ = 4.712, *p* = 0.0188, *n* = 10,8,9 rats) with Fisher's PLSD *post hoc* tests, \*\**p* \< 0.01 compared to M/S+HSV-TMT; ^\#^*p* \< 0.05 compared to M/M+HSV-NURR1. (**i**) Representative low-magnification photomicrographs with an inset depicting localized HSV-NURR1 (TMT+, red) in dopaminergic neurons (TH+, green) of rat VTA (scale bar, 50 μm). The results were replicated in two independent experiments. (**j**) HSV-mediated NURR1 overexpression increased the expression of *Bdnf* exon IX mRNA in rat VTA (unpaired *t*-test with Welch's correction, *t*~10.98~ = 2.440, *p* = 0.0328*, n* = 9,10 rats). (**k**) NURR1 overexpression in mouse VTA significantly decreased morphine reward (15 mg/kg, IP) (unpaired *t*-test, *t*~13~ = 2.165, *p* = 0.0496*, n* = 8,7 mice). (**l**) However, there was no effect of NURR1 overexpression on morphine reward in mice with a local knockout of BDNF in VTA (*t*-test, *t*~17~ = 0.4062, *p* = 0.690*, n* = 10,9 mice). (**b--e,k,l**) Student's *t*-tests, \**p* \< 0.05; (**f,j**) Student's *t*-tests with Welch's correction, \**p* \< 0.05 and \*\**p* \< 0.01. Bar graphs show mean ± SEM. Box plots present, in ascending order, minimum sample value, first quartile, median, third quartile, maximum sample value.](nihms651676f5){#F5}
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{
"pile_set_name": "PubMed Central"
}
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Background {#Sec1}
==========
Since the 1970s levothyroxine (LT4) has become the standard of care for thyroid hormone replacement in subjects unable to produce their own thyroid hormones due to congenital, autoimmune or iatrogenic causes. Levothyroxine has become one of the most widely used drugs worldwide, it is the most commonly prescribed drug in the United States (US), the third most in the United Kingdom (UK), and there is evidence that its use is steadily increasing \[[@CR1], [@CR2]\]. Despite this, there remains controversy as to the best way to therapeutically replace thyroid hormones, with a small group of people on LT4 monotherapy not feeling as though they have achieved their premorbid well-being \[[@CR3], [@CR4]\] and hence a significant push for other therapies including combination LT4/liothyronine (LT3) therapy, LT3 alone or extracts from animal thyroids.
Possible reasons for some patients not responding to levothyroxine monotherapy and evidence for efficacy of mono- and combination therapy have been well covered in recent systematic reviews by both the American Thyroid Association (ATA) \[[@CR5]\] and European Thyroid Association (ETA) \[[@CR6]\]. Both guidelines suggest that in select cases, a trial of therapy including LT3 can be considered, carefully supervised by an expert in the field. The purpose of this review, therefore, is not to repeat the analysis in these recent reports, but to provide a more practical approach to the use of combination therapy in clinical practice including safety aspects and cost, where a trial is considered appropriate.
The target audience for this review is clinicians who incorporate or would consider incorporating trials of combination LT4/LT3 therapy in their practice, or who have patients who have questions about this therapy. The review was performed by literature search in each area, review of published articles and studies and practical experience.
Thyroid hormone replacement {#Sec2}
===========================
Although the thyroid gland produces T4 and T3, LT4 monotherapy has been the mainstay of thyroid hormone replacement since the 1970s replacing desiccated thyroid extract (DTE) which had been used for many years prior. This is because it is easily administered, well absorbed by the oral route and its long half-life allows for once daily dosing with very stable serum levels. It was also shown to be converted to T3 within the body \[[@CR7]\] alleviating the need to add LT3 which does not have the same stable pharmacological profile. Furthermore, it has also been shown that the majority of circulating T3 comes from peripheral conversion of T4 to T3 and not secretion of T3 from the thyroid \[[@CR8]\], hence a T4:T3 secretion ratio of approximately 14:1 appears average in humans, suggesting only a small role for secreted T3. However, there is a small group of patients who do not feel back to their euthyroid well-being despite having thyroid function tests suggestive of adequate replacement on LT4 \[[@CR3], [@CR9], [@CR10]\]. There are also several studies showing that on LT4 monotherapy serum T3 levels are significantly lower for the same TSH in euthyroid patients \[[@CR11]--[@CR16]\], although the clinical significance of this is unknown. Another study showed it was not possible to normalise serum TSH, T3 and T4 levels or tissue T3 levels in laboratory animals giving them LT4 monotherapy \[[@CR17]\].
Because of this there have now been at least 13 randomized controlled trials (RCT) comparing efficacy of combination LT4/LT3 therapy versus LT4 monotherapy for thyroid hormone replacement \[[@CR18]--[@CR30]\]. There has also been one trial comparing DTE to LT4 monotherapy \[[@CR31]\]. The trials differ significantly in study population size, method of substituting LT3 for LT4, dose, length of study and outcomes measured. There have now been 4 systematic reviews/meta-analyses of these studies attempting to clarify the findings \[[@CR32]--[@CR35]\]. Overall these meta-analyses have suggested there is no significant benefit of combination LT4/LT3 therapy compared to LT4 monotherapy in terms of mood, health-related quality of life or cognitive function. This provides reasonable evidence that at a population level there is no benefit of using combination therapy over monotherapy. However, the different methods of replacing LT4 with LT3 (some resulting in raised TSH suggestive of under replacement) and small size and hence power of some studies should be considered. There was no increase in adverse events in the combination groups in this admittedly short period of follow up. There is one randomized cross-over trial comparing DTE with levothyroxine monotherapy in 70 patients for 16 weeks each arm \[[@CR31]\]. This did not show any significant benefit of DTE over LT4 in symptoms and neurocognitive markers. There was an increase in preference for DTE over LT4 however it is unclear what this signifies given an overall lack of improvement in symptoms. The study was too small to pick up subgroups responding to T3 and future studies in this area may consider having a synthetic T4/T3 arm also to determine if any benefit is due to the addition of T3 or DTE itself.
Whilst this evidence is convincing there remains a possibility that a small subset of hypothyroid patients will do better on combination therapy; this group may get lost in the larger group of patients with no benefit. Practicing clinicians will be able to identify a group of patients not satisfied on LT4 monotherapy which makes up a small subset of all their patients on LT4. There is also one study suggesting that a polymorphism in the DIO2 gene which codes for the deiodinase 2 enzyme (Thr92Ala), important for conversion of T4 to T3 in many tissues including the brain may suggest the group which will respond to therapy (rare homozygotes approximately 12% of the Caucasian population) \[[@CR36]\]. These findings remain controversial however, particularly with inconsistent findings of the functional effect of this polymorphism \[[@CR37]--[@CR39]\].
After extensive review of available literature on this topic guidelines were produced by both the ATA and ETA related to thyroid hormone replacement with LT4 and alternatives. Both guidelines suggest there is insufficient or no strong evidence of superiority of combination LT4/LT3 therapy over LT4 monotherapy in patients with hypothyroidism. However, both acknowledge that there are patients who have persistent symptoms or sub-optimal health despite LT4 therapy guided by normal thyroid biochemistry and suggest possible reasons for this include inadequacy of LT4 monotherapy to normalise serum and tissue T4 and T3 levels. Thus, both suggest that in an appropriate clinical setting (see below) combination therapy may be trialled to determine if it is beneficial for the individual patient \[[@CR5], [@CR6]\]. The remainder of this review will consider practical issues related to such a trial/long term therapy.
As in all clinical practice, clinicians should only offer treatments that they are comfortable using. The experience is, however, that patients wishing for a trial are often frustrated at the lack of specialists willing to provide such, and therefore often obtain combination therapy from health practitioners with little training in the area, who do not monitor for complications, can give incorrect doses/dose ratios and offer ongoing treatment without assessing the benefits. In some instances, patients self-treat with medications obtained online. Until more is known of benefits and risks, LT3 should not be offered routinely. LT3 should only be trialled in pts. who specifically request it, who have marked persisting symptoms, and who fully understand and accept (with written/documented information if possible) the unknown potential for long-term harm. Endocrinologists are clearly best suited to provide this.
Current practice {#Sec3}
================
One of the reasons for this review was that despite recommendations and guidelines from various specialist bodies, use of combination T4/T3 therapy appears significant in most developed countries. In a survey of specialist members of The Endocrine Society, The American Thyroid Association and The American Association of Clinical Endocrinologists spread internationally, but mostly in North America; showed that 3.6% of the 880 respondents would trial adding LT3 to LT4 in the setting of a hypothyroid patient with persistent symptoms despite a TSH within the target range \[[@CR40]\]. De Jong and colleagues from the Netherlands using data from a sample of Dutch pharmacies showed that use of combination T4/T3 was 0.82% of thyroid hormone users in 2005 and this slowly rose to 0.90% by 2011 \[[@CR41]\]. In the TEARS study population sampled from an area in Scotland 0.95% of those on thyroid hormone replacement were taking combination T4/T3 therapy and 0.21% T3 only \[[@CR42]\]. A study by Michaelsson and colleagues from Denmark which elicited survey responses from patients known to be on T4/T3 combinations did not estimate the frequency of combination use, but did show that in the responding population of 293 on combination therapy slightly more patients had received their treatment from their GP (42%) than had from their endocrinologist (39%), 50% were on DTE with 43% on T4/T3 and 28% were adjusting their own dose \[[@CR43]\]. This study suffers from selection bias associated with internet surveys particularly on a topic which has been strongly debated publicly recently in Denmark, however, it does highlight that methods other than LT4 monotherapy for thyroid hormone replacement may frequently not be monitored by specialists."*Practice point: Alternatives to LT4 monotherapy for thyroid hormone replacement are being used in a small percentage of hypothyroid patients and it appears frequently without specialist oversight and appropriate monitoring. If specialists are willing to discuss the available evidence, possible benefits and adverse effects of such therapy with patients, it is likely to make this practice safer.*"
Patient selection {#Sec4}
=================
Patient selection is important to determine pre-trial whether the patient is likely to gain benefit from the treatment and prevent harm to the patient. There are circumstances in which a trial may delay another treatable diagnosis or put the patient at significant risk without possibility of benefit in which it may be inappropriate.
Collect evidence that there is thyroid dysfunction {#Sec5}
--------------------------------------------------
The first important step in this age of accessibility to thyroid function testing is to clarify the diagnosis of hypothyroidism requiring thyroid hormone replacement. There is good evidence from the UK that the median TSH level at which LT4 therapy was commenced is relatively low (Taylor et al. 7.8 mU/L in 2009 \[[@CR2]\], Leese et al. 6.2 mU/L in 2001 \[[@CR44]\]). It is therefore important to clarify the initial diagnosis, if possible with access to diagnosis thyroid function/antibody levels and presenting symptoms. It is also worthwhile considering if there was an initial response to LT4 therapy which was subsequently lost or if there was never a response.
Ensure adequate dose of LT4 has been used {#Sec6}
-----------------------------------------
It is important to ensure that the patient is on adequate T4 dosage prior to a trial of combination therapy. Most guidelines would suggest returning the serum TSH level to the population reference range as an indication of adequate replacement \[[@CR45], [@CR46]\], as clinical symptoms have not been shown to be accurately predict serum levels \[[@CR47]\]. However, even with these guidelines studies have shown that a high percentage of patients on T4 have serum TSH above the reference range, even those on for many years \[[@CR2], [@CR3], [@CR47], [@CR48]\]. Furthermore, many would suggest a serum TSH in the lower part of the reference range is appropriate, given the skewed distribution of TSH values in the reference population \[[@CR49]\], and the concept that individual TSH levels within the reference range vary little in health and therefore each person may have a genetically derived set point \[[@CR50]\]. There are however no trials suggesting this improves any measurable clinical markers compared with TSH within the entire reference range and this practice is not accepted by all \[[@CR51]\]. Furthemore a trial looking at slight alterations in LT4 dose in hypothyroid patients did not show any difference between clinical symptoms or measurable parameters despite clear differences in TSH on the different treatment regimens \[[@CR52]\].
Exclude co-morbidities {#Sec7}
----------------------
Given the generalised nature of hypothyroid symptoms it is possible another condition may be causing them, or that the two conditions may co-exist, given the high prevalence of hypothyroidism in the population. Typical conditions which may lead to a mis-diagnosis are Depression, Chronic Fatigue Syndrome and Fibromyalgia. As the majority of hypothyroidism has an autoimmune origin and these conditions frequently occur with other autoimmune conditions \[[@CR53]--[@CR55]\] it is worthwhile considering whether there may be another diagnosis present with careful history taking, examination and targeted investigations if appropriate. The question of whether thyroid autoimmunity itself can cause symptoms is a more complicated one. A large population study from Norway suggests not \[[@CR56]\], however, two smaller studies suggest an association unrelated to thyroid hormone levels \[[@CR57], [@CR58]\] giving it some plausibility. The answer is therefore not clear. Again, if persisting symptoms are related to thyroid autoimmunity then these would not be expected to resolve with combination therapy but should improve as antibody levels subside. However, due to the tenuous link it would appear inadvisable to be measuring thyroid antibody levels and using this as a deciding factor to initiate a trial of combination therapy.
Be aware of psychological comorbidities {#Sec8}
---------------------------------------
This is important because psychological comorbidities may confuse the diagnosis (depressive symptoms versus hypothyroid symptoms), affect the response to treatment and in some cases be worsened by the addition of LT3 (for example anxiety).
Exclusions {#Sec9}
----------
Although there is little clear evidence most practitioners would exclude patients with significant cardiovascular disease or arrhythmia for a trial, given the potential to cause life-threatening side effects. Furthermore, given the importance of tight control of thyroid hormone levels, particularly T4, for the normal development of the foetus and progression of pregnancy, and lack of data showing LT3 can be safely used in pregnancy, it is not recommended in pregnant women or woman actively trying to conceive. Patients with poorly controlled anxiety and thyroid cancer requiring suppression of serum TSH may also fall into a worrying area. It is unclear whether combination therapy in disseminated thyroid cancer can adequately suppress TSH across a 24 h period, and therefore the individual patients' prognosis and need for TSH suppression needs to be considered and discussed in this setting.
Not currently useful {#Sec10}
--------------------
Is *genetic testing* a useful aid to patient selection? On current evidence there is no clear genetic test which will determine patients who will respond to therapy. Use of an unvalidated test may preclude some patients receiving a trial who may benefit and therefore it is not recommended. Further studies are required to delineate the actual genetic markers which may be useful for patient selection. In a similar way trials have not suggested a biochemical marker (including thyroid hormone levels) which will predict who will respond (Table [1](#Tab1){ref-type="table"}).Table 1Pre-trial assessment of potential patients1. Collect evidence of thyroid dysfunction2. Ensure adequate dosage of levothyroxine has been trialled3. Exclude co-morbidiies4. Be aware of psychological co-morbidities5. ExclusionsNot currently usefulGeneticsBiochemistrySymptom profile
Pharmacology and formulations {#Sec11}
=============================
Studies using both older \[[@CR59]--[@CR61]\] and newer \[[@CR62]\] thyroid hormone assays have suggested a diurnal rhythm of free T3 and TSH in healthy subjects with no thyroid disease. These studies suggest a peak of T3 at around 4 am with a nadir between 3 to 5 pm; this appears to lag behind TSH levels by about 90 mins \[[@CR62]\]. Given its long half-life it is not surprising that in most studies free T4 levels remain very stable throughout the day. Despite a statistically measurable diurnal variation in T3 the actual difference in T3 levels is low (11.2%) and the levels are effectively stable over a 24 h period. It would appear reasonable to mimic these levels if trying to appropriately replace thyroid hormones, particularly as there are few biomarkers which reliably suggest complete thyroid hormone replacement. However, currently this is limited by formulations of T3 which are available (Table [2](#Tab2){ref-type="table"} for a non-exhaustive list). In many countries, the 20μg table of liothyronine is the only available making accurate dosing very difficult. This is reflected in the combination T4/T3 trials which were not uniform in their method of replacing LT4 with T3, either using a 10:1 or 5:1 LT4:LT3 ratio, or replacing an amount of LT4 with from a fifth to a whole dose of LT3.Table 2Available formulations of LT3 and combination LT3/LT4NameT3 doseT4 doseAvailableCytomel5, 25, 50mcgUS, Canada, NetherlandsThybon20, 100mcgUKTertroxin20 mcgAustralia, South AfricaLiotyr5 mcg (soft gel)ItalyProthyroid10 mcg100 mcgGermanyNovothyral5/15/20 mcg25/75/100 mcgSeveral EuropeThyreotom forte10/30 mcg40/120 mcgCzech republic
Studies looking at the pharmacology of LT3 replacement all show a significant peak of serum T3 2--4 h after dose and wearing off after 12 h in those on a single daily dose, these include hypothyroid patients on combination therapy \[[@CR63]\] (Fig. [1](#Fig1){ref-type="fig"}), LT3 monotherapy \[[@CR64], [@CR65]\], and even euthyroid subjects taking LT3 only \[[@CR66]\]. The profiles are very different to those in patient with normal endogenous thyroid function, and depending on the dose of LT3 the peak level is often above the reference range, and/or the serum TSH is raised/suppressed compared to T4 treatment \[[@CR67]\]. Figure [1](#Fig1){ref-type="fig"} suggests that to sample the peak serum T3 level a test taken 2--4 h after ingestion of the T3 would be appropriate. There is a study showing equivalent TSH responsiveness to TRH in patients on either T3 or T4 monotherapy \[[@CR68]\], suggesting the rise in TSH seen in some of these studies is an indication of under replacement. Almost all the combination trials used once daily LT3 dosing with one using twice daily.Fig. 1Mean values for **a** free T3, **b** free T4 and **c** TSH over a 24 h period in 10 patients with hypothyroidism on either LT4/LT3 combination or LT4 alone. From Saravanan et al. \[[@CR63]\], used with permission
To truly mimic the normal production of T3 patients would have to split the dose of T3 and take it two or three times a day, however, the large dose size of the available products may preclude this in patients with a lower requirement for thyroid hormone. Comparison of the figures from Saravanan et al. \[[@CR63]\] and Russell et al. \[[@CR62]\] would suggest that to mimic serum T3 levels in euthyroid individuals the LT3 dose should be split with the second dose given approximately 8 h after the first. This may prevent the insomnia reported by some patients when they take LT3 prior to bed which is presumably secondary to a serum T3 peak whilst trying to sleep. What effect this will have on the steady state of the drug is unclear as there are no studies looking at serum levels in multiple daily dosing, and even the half-life of T3 is debated with a wide range of opinions. Saravanan et al. did not find any difference in cardiovascular parameters (pulse rate and blood pressure) between their groups on T4/T3 and T4 only despite the clear peak in T3 levels at approximately 4 h. This interesting finding reminds us that serum hormone levels do not necessarily reflect action in tissues due to the presence of thyroid hormone transporters and deiodinases in different tissues which may influence the effect of these hormones in individual tissues \[[@CR69]\]. Furthermore, most actions of thyroid hormone take several hours to have effect as they require the synthesis of new mRNA and protein.
There are no commercially available slow release T3 formulations currently. Hennemann and colleagues tested their own slow release T3 preparation in combination with T4 and found that compared to once daily T3 there was a lower peak of T3 and smoother profile \[[@CR70]\]. They suggested all future trials use slow release T3 but a commercially available product has not been forthcoming. Doubts have been raised as to whether this study showed a product that could be used as a once a day dosage and what actual effect it had given no change in TSH levels for either formulation \[[@CR71]\]. Compounding pharmacies will provide compounded formulations of slow release T3, however the ATA do not recommend the use of compounded preparations except in cases of clear allergic reactions to commercial preparations \[[@CR5]\]. This is because there are few studies on these products that meet scientific criteria for rigorous peer review showing that they can be used to provide adequate thyroid hormone replacement, are equivalent to approved preparations or have a long-term safety profile. Furthermore, the preparations need to be used relatively quickly after synthesis to prevent loss of efficacy due to degradation \[[@CR72]\], requiring regular compounding of new products. In addition, compounded preparations are generally higher in cost and not standardised between different pharmacies. There have also been reports of thyrotoxicosis \[[@CR73], [@CR74]\] and hypothyroidism \[[@CR75]\] caused by errors in compounding."*Practice point: be aware of formulations of LT3 and LT4/LT3 available locally and if possible their pharmacokinetics. Errors in the preparations of compounded preparations have been reported.*"
Dosage {#Sec12}
======
Wiersinga et al. in their review suggest 3 different methods for calculating appropriate dosages for combination T4/T3 therapy \[[@CR6]\]. These are based on the assumptions that persisting symptoms are due to LT4 monotherapy being unable to deliver normal serum and tissue T4 and T3 levels in humans as shown in rats \[[@CR17]\] and that mimicking the normal thyroid secretion of T4 and T3 will correct this \[[@CR76]\]. The methods are based on using the dose of LT4 which gives the target TSH in the patient, then replacing a small amount of T4 with T3 using a 3:1 equivalence ratio derived from a study in thyroidectomized patients \[[@CR77]\] to give the appropriate ratio. The three methods give a final dose T4:T3 ratio between 13:1 and 20:1, much closer to normal human thyroid secretion \[[@CR8]\] but in generally lower than those used in the T4/T3 studies, in some cases significantly lower. Furthermore, many of the studies had variable ratios due to a fixed substitution (eg. 10μg T3 for 50μg T4). This dose ratio is also significantly lower than that of animal thyroid extracts in which the T4:T3 ratio is generally around 4:1 (see later). Note also however that for a patient previously on 100μg of T4 a day the T3 dose from these methods is between 4 and 6μg a day which with most current formulations would be difficult to deliver in a split dose."*Practice point: starting dose in a patient on adequate LT4 monotherapy will always require removal of part of the LT4 dose and replacement with LT3. In practice the dose of LT3 will usually be a dose of 5 -- 20 mcg a day in a split dose, by necessity often determined by the availability of low dose formulations of LT3.*"
Length of trial {#Sec13}
===============
Again, there is very little evidence to determine how long a trial of combination therapy should be, the RCTs ranged from 5 weeks to 52 weeks. Most of those who showed a benefit had shown this by 3 months, and it is generally advised that 6 weeks to 3 months are waited until LT4 dose is adjusted to allow a steady state to develop in all tissues. It would therefore be reasonable to give a 3--6 month trial before deciding if it has been beneficial to the patient symptoms. If there is clear benefit it would be reasonable to continue the trial further, however, given the significant placebo effect seen in trials of thyroid hormone replacement \[[@CR26]\] and the fact that LT3 may initially give a feeling of euphoria, clinicians should be encouraged to continue to assess the treatment as the benefit may disappear. The fixed term nature of the trial should be agreed with the patient prior to beginning and there should be agreement that there will be a return to LT4 monotherapy if no significant benefit is seen, given its easier dosing and better evidence of its safety. Figure [2](#Fig2){ref-type="fig"} displays a proposed timeline for a trial."*Practice point: initial trial 6 months, and then confirm benefit is still present at least 1 year before planning long-term therapy*"
Monitoring {#Sec14}
==========
There are no long term studies which link serum levels of T3 to adverse outcomes and therefore are able to direct monitoring of combination T4/T3 therapy. Given evidence for poorer outcomes with raised thyroid hormone levels/suppressed TSH levels in both subjects not on thyroid hormone replacement and on LT4 monotherapy it is reasonable to measure TSH, free T4 and free T3 2--4 h post dose as this is the expected peak of serum T3 post dose. Keeping both the serum T3 and TSH within the reference range at this point would suggest that the patient is at the least risk of developing both short and long term complications. Monitoring of these would include clinical assessment of pulse rate and rhythm, blood pressure, mood (particularly anxiety) and as clinically appropriate (depending on individual patient characteristics): ECG, echocardiogram and Bone Densitometry. Whilst studies in patients on LT4 would suggest that dose changes can be seen in end organ markers such as serum cholesterol and sex hormone binding globulin (SHBG), these changes are so small that they are often contained in the normal variance in the population."*Practice point: Patients should be monitored indefinitely for cardiovascular, psychological and bone adverse effects.*"
Safety {#Sec15}
======
Given the relatively small number of patients using combination T4/T3 therapy safety data has until recently been lacking. None of the 13 RCTs comparing combination therapy to T4 monotherapy showed any increase in adverse events in the combination group, however the follow up was generally short ranging from 5 to 52 weeks. Leese et al. have recently reported safety data in their observational cohort in the TEARS study \[[@CR42]\]. In this study a subgroup of patients on thyroid hormone placement were considered with outcomes for those who had ever been taking LT3 (*n* = 400) compared to those who had only ever taken LT4 (*n* = 33,955). The study showed no increase in cardiovascular disease, atrial fibrillation or fractures (outcomes previously shown to be associated with levothyroxine overtreatment \[[@CR78]\]) with a median follow-up of 9 years. There was an increased risk for new prescriptions of anti-psychotic medications of unclear significance. There was also a possible association with an increased risk of breast cancer in subjects who had taken LT3. Although there is some data on T3 augmentation of breast cancer cell line proliferation and higher endogenous T3 being associated with more aggressive breast cancers, the authors point out that this association was of borderline statistical significance and not related to number of prescriptions of LT3, arguing against a causal relationship. This potential association should be assessed in other large cohorts. The data from Leese et al. are from an observational study and so carry a risk of bias and self-selection; in addition the duration of LT3 use was not quantified. However, this may be the largest and longest duration study of its type for some time and offers some reassurance that the risks of taking T3 are not greater than expected.
Cost {#Sec16}
====
Lack of regulation of the cost of unbranded Liothyronine in the UK has seen the price rise markedly in recent years, currently 28 tablets of 20mcg costs around £258.20, therefore 10mcg bd for a year would cost around £3365 per patient. This represents a greater than 4000% increase from the branded 'Tertroxin' (just over £13 for 100 tablets) which precipitated a 'black listing' of the use of LT3 in several health trusts (Fig. [3](#Fig3){ref-type="fig"}) and has now attracted the attention of the UK regulatory and competition and markets authority. Synthetic T4 as Levothyroxine 100mcg once daily costs approximately £25 per patient per year. Whilst the government looks to close the loophole allowing companies to do this, many patients who had been maintained on LT3/LT4 for many years have been forced to come off LT3 or source it from overseas.Fig. 2Proposed timeline for a trial of combination T4/T3 therapy
In Australia a similar dose of Liothyronine would cost \$142 Australian dollars for a year on a private prescription, it is available on a government subsidized prescription for half that cost, available for patients who 'have a documented intolerance or resistance to thyroxine', which is open to the interpretation of the treating physician. A 100mcg tablet of thyroxine would cost around \$58 Australia dollars a year on the government scheme.Fig. 3Change in costs for Liothyronine compared to Levothyroxine in the United Kingdom since 2009. Data from BNF and drug tariff, graph by British Thyroid Association
In the US for patients not covered by health insurance, hypothyroid treatment typically costs \$15--\$100 per month \-- or \$180--\$1200 per year \-- for the synthetic thyroid hormone typically prescribed. For example, [Drugstore.com](http://drugstore.com) charges about \$15--\$20, depending on the dose, for a one-month supply of the brand-name drug Levothroid, or \$25--\$45 for a one-month supply of the brand-name drug Synthroid. [Drugstore.com](http://drugstore.com) charges up to \$100 or more for a one-month supply, depending on dosage, of the brand-name drug Cytomel (LT3). Therefore in most countries combination therapy is comes at increased cost to the patient (or government) and this needs to be taken into consideration when planning a trial."*Practice point: be aware and make patients aware of costs and availability of formulations prior to prescribing.*"
Dessicated thyroid extract {#Sec17}
==========================
Many patients and alternative physicians prefer to use forms of dessicated thyroid extract (DTE) for thyroid hormone replacement. DTE is described as "the cleaned, dried, and powdered thyroid gland previously deprived of connective tissue and fat. It is obtained from domesticated animals that are used for food by humans" by the United States Pharmacopeia. Extracts of animal thyroids have been used for hypothyroid symptoms for many centuries in different cultures, and as a form similar to what is used today for over 110 years \[[@CR79]\]. Most formulations are from porcine thyroid; however some from bovine thyroid or a combination of the two exist. Initially it was titrated to improvement in clinical symptoms and avoidance of hyperthyroid symptoms. The development of Levothyroxine and ability to assay serum TSH, T4 and T3 levels in the 1980's led to calls for removal of DTE for treatment of hypothyroidism because of supraphysiological levels of T3 post dose, hyperthyroid symptoms and complications, and fluctuating levels of T3 as compared to very stable TSH, T3 and T4 levels with levothyroxine dosing \[[@CR80]--[@CR83]\]. Despite continuing concerns about these issues and also the consistency of the various preparations it continues to be used. Whilst the dramatic cases of thyroxtoxicosis on these preparations are mainly historical, there remain concerns about frequency of adverse events and calls for greater standardization of these preparations \[[@CR84]\].
DTE is often prescribed in grains: 1 grain is typically around 60-65 mg of DTE and most commonly contains 38μg of T4 and 9μg of T3. Bioavailability has been shown to be different to synthetic LT4 and LT3 preparations \[[@CR81]\]. Table [3](#Tab3){ref-type="table"} displays commonly available brands and doses contained. The doses above give a T4:T3 ratio of 4.2:1 significantly more T3 than the 14:1 secreted by the normal thyroid and the doses recommended above. This makes dosing difficult as displayed by several studies which have shown supraphysiological T3 doses post dose, fluctuating T3 levels during the day and more hyperthyroid symptoms in subjects taking DTE compared to LT4 monotherapy \[[@CR80], [@CR82], [@CR85], [@CR86]\]. As with combination T4/T3 therapy, there are no longer term trials or safety data available on long term use of DTE. This is important as there are concerns that regular supraphysiological levels of T3 may induce hyperthyroidism-like complications over the longer term. Given lack of safety data, difference and variability in the various preparations and no studies showing clear benefit over T4 monotherapy, all major endocrine and thyroid societies currently advise against the routine use of DTE for thyroid hormone replacement \[[@CR5], [@CR6]\].Table 3Dessicated thyroid extract formulationsNameT3 doseT4 doseAvailableNature thyroid per 65 mg grain9mcg38mcgUSWesthroid pure per 65 mg grain9mcg38mcgUSNP thyroid per 60 mg grain9mcg38mcgUSThyroid (erfa) per 60 mg grain8mcg35mcgEurope/CanadaArmour thyroid per 60 mg grain9mcg38mcgUS
Amour thyroid is the most commonly used formulation, also the most expensive, and in the US costs around \$1 per 60 mg grain.. Price varies across the UK mostly around £1 per grain. These formulations are not available in Australia and therefore most users import them from the US at similar cost plus postage.
DTE formulations contain unmeasured quantities of diiodothyronine and monoiiodothyronine which many of its supporters believe make it a more suitable replacement for thyroid hormone, although there are no studies suggesting these are required for normal functioning, or that they are secreted in significant quantities from a normal human thyroid. These products also contain other thyroid-related proteins and antigens which could potentially invoke immune responses; these have not been studied to date."*Practice point: whilst all major endocrine and thyroid societies advise against the use of DTE for hypothyroidism, it is clear that its use remains significant. Better regulated formulations and trials are required to determine its role, if any. Until these are available patients who continue to use DTE should be advised on appropriate safety monitoring as for using T4/T3.*"
Conclusion {#Sec18}
==========
Although there is convincing evidence that there is no benefit of combination T4/T3 therapy over T4 monotherapy for management of hypothyroidism at a population level, there remains a population of patients who do not feel well on T4 monotherapy. There are several possible reasons for this, one of which is an inability to use T4 effectively in a group of patients who may respond better to combination T4/T3 therapy. This will remain a possibility until large RCTs in an appropriately targeted population can confirm or refute it, and whilst it does a trial of combination therapy in such patients may be indicated. It is important that this be performed by a clinician with adequate knowledge and experience in the area, with appropriate patient selection, clear explanation of risks and benefits to the patient for consent and careful monitoring and follow-up. Possible ways to do this are covered in this review although it is clear that further research into this area and possible methods of delivering T3 are required.
ATA
: American Thyroid Association
DTE
: Dessicated Thyroid Extract
ECG
: Electrocardiogram
ETA
: European Thyroid Association
LT3
: Liothyronine
LT4
: Levothyroxine
mg
: Milligram
RCT
: Randomised Controlled Trial
SHBG
: Sex Hormone Binding Globulin
T3
: Tri-iodothyronine
T4
: Thyroxine
TRH
: Thyrotropin Releasing Hormone
TSH
: Thyroid Stimulating Hormone
μg
: Micrograms
UK
: United Kingdom
US
: United States (of America)
Not applicable.
Funding {#FPar1}
=======
Not applicable.
Availability of data and materials {#FPar2}
==================================
Not applicable -- no new data.
VP performed literature search and drafted manuscript. CD help draft manuscript, added additional references. Both authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
Not applicable -- review no original study.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#s0005}
===============
In healthy individuals, the internal tissues are devoid of pathogens, whereas the external tissues like oral cavity are exposed to various kinds of microorganisms ([@b0250]). The oral cavity is the starting point of the gastrointestinal tract. Because of the frequent supply of food through oral cavity, it creates a favourable environment for nurturing and harbouring microorganisms. The surfaces of oral cavity are covered with diverse microorganisms, constructing the proverbial bacterial biofilm and maintain a dynamic ecological balance with the host body. Any disruption in this equilibrium causes oral disease ([@b0170]). In healthy oral cavity, there is a distinctive predominant bacterial flora that are not only highly diverse in nature but also subject and site-specific ([@b0005]). There are numerous microorganisms that come under the broader term oral microflora and include bacteria, fungi, most protozoa but rarely viruses. The diversity and counts of microflora vary according to food habits, salivary features and antibiotic regimens used for treating infections ([@b0105]). Oral bacteria have been associated in the pathogenesis of highly prevalent oral diseases such as dental caries ([@b0150], [@b0030], [@b0015]) and periodontal disease ([@b0055], [@b0075]). A clear understanding about the molecular diversity of microflora is important for pre-emption and redemption of associated health conditions. The phenotypic methods of bacterial identification based on microscopy and culture techniques are less sensitive, subjective and time consuming ([@b0215]). On the other hand, polymerase chain reaction (PCR) provides the highest sensitivity for the detection of microorganisms ([@b0190]).
It is important to note that oral bacteria associated with periodontal disease are mainly anaerobic and therefore they are very difficult to cultivate ([@b0040]). Genotyping of microorganisms using 16S rRNA gene sequencing has been recognized as a more sensitive, specific and accurate method for bacterial identification ([@b0050]). By virtue of the well-conserved regions in 16S rRNA gene among different biological species, comparison of these sequences provides a useful tool for studying molecular diversity and evolutionary phylogenetics ([@b0205]). [@b0175] have shown that 16S rRNA gene is highly useful for detection of culture-negative bacteria in patients pre-treated with antibiotic drugs. Molecular methods for species specific identification of oral bacteria can be based on sequence-specific primers ([@b0125]), probes ([@b0185]) or direct sequencing ([@b0110], [@b0180]).
Several studies have reported significantly high prevalence of periodontitis ([@b0100], [@b0105]) and dental caries ([@b0020], [@b0245]) in adults, children, and older population of Saudi Arabia. Moreover, putative role of specific oral bacterial species has also been suggested in many systemic diseases, including diabetes ([@b0225], [@b0195]) and cardiovascular disease ([@b0200], [@b0220]). Poor oral health may adversely affect diabetic glycemic control and subsequent medical complications ([@b0155]). The biofilm built in the oral cavity has been associated with health and disease ([@b0250]). The bacterial diversity of the oral biofilm is variable among individuals and influences by several factors including age, diet, oral hygiene and genetics ([@b0145]). The diversity and pattern of bacterial exposure in early life of an individual play an important role in the gut colonization of bacteria ([@b0010]). A reduced bacterial diversity in the gut has been found to correlate with higher risks of chronic diseases like allergy and asthma ([@b0070]).
Although the implication of molecular methods for identification and diversity analysis of oral microflora is of great concern, there are very few studies undertaken in Saudi Arabia in this particular field. The aim of this study was to compare the diversity of common dental bacteria in non-diabetic and diabetic patients, using the sequence-specific primers. This study is focused on 5 species of periodontal pathogenic bacteria including *Porphyromonas gingivalis* (*P. gingivalis*), *Tannerella forsythia* (*T. forsythia*), *Capnocytophaga ochracea* (*C. ochracea*), *Prevotella intermedia* (*P. intermedia)* and *Aggregatibacter actinomycetemcomitans* (*A. actinomycetemcomitans*).
2. Materials and methods {#s0010}
========================
2.1. Subjects and study design {#s0015}
------------------------------
This study was conducted on 28 unrelated Saudi Arabian female subjects. One group included 14 type-2 diabetic patients with periodontal disease recruited consecutively from Clinic of Dentistry, Section of periodontology, King Saud University, Riyadh, Saudi Arabia. The other group composed of 14 healthy non-diabetic subjects, this group served as control group. All subjects gave an informed written consent before the study, answered a set of questionnaire and underwent clinical and physical examinations. Periodontal examinations were carried out by qualified dentists.
The inclusion criteria were female gender, residents of Riyadh, age range 18--70 years, presence of \> 20 intact natural teeth, features of periodontitis (loss of attachment ≥ 5 mm), and type-2 diabetes. The exclusion criteria were the use of antibiotic therapy during the last 2 months and presence of any systemic disease that may influence the periodontal condition. The study protocol was approved by the Research Ethics Committee of the Faculty of Science, King Saud University, Riyadh, Saudi Arabia.
2.2. Sample collection and bacterial lysis {#s0020}
------------------------------------------
The subgingival samples were collected from four sites (about 5 mm depth) from tooth pockets by a professional dentist in the morning time, using separate sterile paper points (size 40, taper 0.04) from META Company, South Korea. A single vertical stroke was used under quality control procedures for each member of case and control group. All the samples were promptly kept in individual sterile Eppendorf tubes containing Tris-EDTA buffer (pH 7.6). For PCR amplification, 10 μl of lysis buffer was mixed with 90 μl of subgingival sample and the solution was boiled for 5 min. An aliquot of the lysate (4 μl) was used for PCR amplification of each sample.
2.3. Bacterial identification by PCR {#s0025}
------------------------------------
PCR detection for all target bacteria was performed using species specific primers. The primers for the 16S ribosomal DNA sequences were selected and purchased from Eurofins Genomics, Germany**.** The primer pairs used for the PCR amplification and the expected product sizes are given in [Table 1](#t0005){ref-type="table"}.Table 1Sequences of PCR primers.Oral bacteriaPrimer sequenceProduct size (bp)*P. gingivalis*TGT AGA TGA CTG ATG GTG AAA ACC197ACG TCA TCC CCA CCT TCC TC*T. forsythia*GCG TAT GTA ACC TGC CCG CA641TCG TTC AGT GTC AGT TAT ACC T*C. ochracea*AGA GTT TGA TCC TGG CTC AG185GAT GCC GTC CCT ATA TAC CAT TAG G*P. intermedia*TTT GTT GGG GAG TAA AGC GGG575TCA ACA TCT CTG TAT CCT GCG T*A. actinomycetemcomitans*AGA GTT TGA TCC TGG CTC AG\
CAC TTA AAG GTC CGC CTA CGT GCC593
The PCR was carried out in a total volume of 30 μl containing 26 μl of reaction mixture and 4 μl of the lysate sample. The reaction mixture contained 1x PCR buffer (10 mM Tris-HCl pH 8.8, 1.5 mM MgCl~2~, 50 mM KCl, 0.1% Triton X-100), 2 units of Taq DNA polymerase, 0.2 mM dNTPs and 100 pmol of each primer. The cycling conditions were programmed as an initial denaturation step at 95 °C for 5 min followed by the 35 amplification cycles of denaturation at 95 °C for 1 min, primers annealing at 60 °C for 1 min and primer extension at 72 °C for 1.5 min. After completion of all the cycles, a final extension step at 72 °C for 7 min was programmed, followed by storing the tubes at 4 °C. PCR was carried out in a thermal cycler (Applied Biosystems, USA) as reported earlier ([@b0125]).
2.4. Statistics {#s0030}
---------------
The frequency of dental bacteria between the control and diabetic groups was analysed by Fisher's exact test using the CalcFisher program ([@b0120]). P values \< 0.05 were considered as statistically significant.
3. Results {#s0035}
==========
PCR-based identification of dental bacteria using sequences specific primers for each target species resulted intense gel bands of anticipated sizes as follows: *P. gingivalis* (197 bp), *T. forsythia* (641 bp), *C. ochracea* (185 bp), *P. intermedia* (575 bp), and *A. actinomycetemcomitans* (593 bp) ([Fig. 1](#f0005){ref-type="fig"}). The diversity and frequency of dental bacteria among 14 clinically healthy control subjects and 14 diabetic patients are shown in [Table 1](#t0005){ref-type="table"}. In control subjects, the prevalence of *P. gingivalis*, *T. forsythia*, *C. ochracea*, and *P. intermedia* was 35.7%, 14%, 28.5% and 14% respectively, whereas *A. actinomycetemcomitans* was completely absent in this group. In diabetic patients, *C. ochracea* appeared to be highly prevalent (100%) followed by *P. gingivalis* (43%), *A. actinomycetemcomitans* (43%), *P. intermedia* (35.7%) and T. forsythia (28.5%).Fig. 1Representative gel images showing the specific bands for different bacteria. Abbreviations are Pg, *P. gingivalis*; Tf, *T. forsythia*; Co, *C. ochracea*; Pi, *P. intermedia*; Aa, *A. actinomycetemcomitans*.
There were positive amplifications for *P. gingivalis* in 5 out of 14 (35%) healthy subjects and in 6 out of 14 (43%) diabetic samples. PCR amplification using sequences specific primers for *T. forsythia* resulted intense gel bands in only 2 out of 14 (14%) control samples and 4 out of 14 (28.5%) diabetic samples. PCR analysis using 16S rDNA primers confirmed the presence of *C. ochracea* in all subgingival samples from diabetic patients (100%) while only 4 out of 14 (28.5%) control subjects showed the presence of this bacterial species ([Table 1](#t0005){ref-type="table"}). *P. intermedia* were detected in 2 of the 14 control subjects (14%) as compared to 5 of 14 (35.7%) in diabetic group. None of the control subjects showed the occurrence of *A. actinomycetemcomitans* in their subgingival samples whereas 6 of the 14 diabetic patients (42.8%) were found to be positive for *A. actinomycetemcomitans* ([Table 1](#t0005){ref-type="table"}). The statistical analysis showed significantly higher (P \< 0.05) prevalence of *C. ochracea* and *A. actinomycetemcomitans* in diabetic patients as compared to control subjects ([Fig. 2](#f0010){ref-type="fig"}).Fig. 2Comparative prevalence of dental bacteria in controls and diabetic patients. \*P \< 0.05 versus control group using Fisher's exact test. Abbreviations are Pg, *P. gingivalis*; Tf, *T. forsythia*; Co, *C. ochracea*; Pi, *P. intermedia*; Aa, *A. actinomycetemcomitans*.
4. Discussion {#s0040}
=============
The results of this study showed that the occurrence of dental bacteria was more prevalent in subgingival samples from diabetic patients as compared to control subjects ([Table 2](#t0010){ref-type="table"}). Oral bacteria are known to predominantly colonize on different surfaces in oral cavity with the aid of specific adhesins located on the bacterial wall that specifically bind to complementary receptors found on the oral surfaces ([@b0080]). Based on the 16S rDNA sequence data generated from human subgingival plaque samples, the dominant microflora included 347 species that fell into 9 bacterial phyla, while multiple subjects showed the presence of known putative oral bacteria including *P. gingivalis*, *B. forsythus*, and *T. denticola* ([@b0165]). [@b0140] have reported variable trends of 40 cultivable bacteria on the surfaces of oral tissue, gingival plaques and saliva from healthy individuals. In fact, all the patients with periodontal disease do not necessarily harbour the similar subgingival microorganisms, hence, only a limited number of oral bacteria have been suggested as the predictors for the progression of periodontal damage ([@b0235]).Table 2Distribution of oral bacteria in healthy subjects and diabetic patients.SNOral bacteriaControlDiabetesP-Value1*P. gingivalis*5 (35.7%)6 (43.0%)0.7202*T. forsythia*2 (14.0%)4 (28.5%)0.4063*C. ochracea*4 (28.5%)14 (100%)0.000074*P. intermedia*2 (14.0%)5 (35.7%)0.2305*A. actinomycetemcomitans*0 (0.0%)6 (43.0%)0.0079
The prevalence of two bacterial species, *Capnocytophaga ochracea* and *Aggregatibacter actinomycetemcomitans*, was significantly higher in diabetic patients as compared to normal subjects ([Fig. 2](#f0010){ref-type="fig"}). *Capnocytophaga* is a gram-negative bacterium that has been linked in the pathogenesis of periodontal disease ([@b0115]). These microorganisms are able to produce various enzymes with the tendency to periodontal breakdown and therefore considered as opportunistic pathogens. Their high frequency in diabetic group may be attributed to the anaerobic conditions of the subgingival pocket due to high glucose levels that would cause excessive periodontal damage in diabetic patients ([@b0135]). An interesting observation is that all the diabetic patients harboured *C. ochracea* whereas only 4 out of 14 control subjects showed the presence of *C. ochracea* anaerobes in their sub-gingival pockets ([Fig. 2](#f0010){ref-type="fig"}). These results are in agreement with an earlier study reporting that *Capnocytophaga* species are frequently isolated in adult periodontitis subjects while their frequency and counts are significantly higher in the diseased sites of diabetic as compared to non-diabetic periodontitis patients ([@b0045]).
None of the control subjects showed the presence of *A. actinomycetemcomitans* in their subgingival pockets whereas 43% of diabetic patients harboured this pathogen. *A. actinomycetemcomitans* has a vital role in the initiation of periodontal disease and if not treated properly, tooth turns to be mobile and severely damaged because of the progressive bone and attachment loss leading to ultimate tooth extraction ([@b0210], [@b0095]). Interestingly, *A. actinomycetemcomitans* can migrate to and colonize in the heart tissue and has been suggested as a risk factor in the development of cardiac disease ([@b0085], [@b0230])*.* Hence, timely detection of *A. actinomycetemcomitans* in oral samples would have a greater impact on health issues extending beyond the denture. Our results are consistent with previous reports that demonstrated high prevalence of *A. actinomycetemcomitans* in patients with diabetes mellitus ([@b0160])*.* In our study, subgingival samples from 28.5% of diabetic patients were *T. forsythia* positive as compared to 14% healthy controls. *T. forsythia* bacterium is an anaerobic, Gram-negative species of *Cytophaga Bacteroidetes* family, linked with the development of periodontal disease ([@b0035]). [@b0060] failed to observe any association between *T. forsythia* and periodontal status whereas the occurrence of *P. gingivalis* and *A. actinomycetemcomitans* was independently and significantly associated with chronic periodontitis.
In conclusion, the findings of this study showed that dental bacteria are more prevalent in diabetic patients than non-diabetic subjects. Particularly, the prevalence of *C. ochracea* and *A. actinomycetemcomitans* was significantly higher in diabetic patients as compared to healthy individuals. Further studies with larger sample size should be conducted to confirm a more detailed description of bacterial species associated with oral disease in diabetic patients which can throw light on a variety of etiological microorganisms. Recent studies have advocated that patients with periodontal disease are more susceptible to metabolic syndrome ([@b0130], [@b0065]). Severe periodontal disease may also put diabetic patients at increased risk of diabetic complications ([@b0240]). For a long time, it was thought that bacteria were the causative factors for the association between periodontal disease and systemic disease. However, recent researches have demonstrated that inflammation caused by bacterial invasion may be responsible for such association ([@b0090], [@b0025]). Efforts should be made to focus on treating the inflammation besides targeting periodontal bacteria for more efficient management of oral disease.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the Research Group No. RGP-009.
Peer review under responsibility of King Saud University.
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{
"pile_set_name": "PubMed Central"
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Introduction {#S0001}
============
Colorectal cancer (CRC) is the third most common cancer worldwide, and has increased in prevalence in recent years.[@CIT0001] CRC frequently metastasizes to the liver, and liver resection and perioperative chemotherapy are the primary means of therapeutic intervention for these tumors. The median survival time for patients with untreated CRC and liver metastases is 6.9 months, and 5-year survival rates following liver resection range from 30% to 50%. Several recent studies have aimed to evaluate the mechanisms responsible for liver metastasis. However, the mechanisms underlying liver metastasis of CRC have not been characterized, resulting in challenges to development of effective therapies.[@CIT0002]--[@CIT0004]
In 1889, Paget proposed the "seed and soil" theory of metastatic dissemination. Paget suggested that the site of metastasis depended on the affinity of the tumor for the microenvironment.[@CIT0005] To evaluate the hepatic microenvironment, we previously analyzed liver non-parenchymal cells in mice and showed that hepatic stellate cells (HSCs), which store retinol and participate in repair and fibrogenesis during liver injury, play a role in immune regulation.[@CIT0006] Recent studies of HSCs have focused on liver injury, liver fibrosis, and liver regeneration. Several studies have shown that HSCs exhibit immunomodulatory activity and can prolong allograft survival.[@CIT0007],[@CIT0008] Furthermore, HSCs have been shown to promote onset and progression of hepatocellular carcinomas.[@CIT0009],[@CIT0010] We previously showed that quiescent HSCs express low levels of immune surface molecules. Priming HSCs with IFN-γ resulted in marked upregulation of the inhibitory co-stimulatory molecule B7-H1, potentially through activation of the MEK/ERK pathway.[@CIT0006] However, the mechanisms by which HSCs promote metastasis of CRC cells to the liver have not been elucidated.
In this study, we demonstrate that HSCs induce T cell hypo-responsiveness and expand regulatory T (Treg) cells. Moreover, HSCs were shown to play an immunosuppressant role in the hepatic microenvironment and promote CRC metastasis to the liver.
Materials And Methods {#S0002}
=====================
Animals {#S0002-S2001}
-------
BALB/c mice were obtained from the Shanghai SLAC Laboratory Animal Company. All mice were maintained in a specific pathogen-free environment at Huashan Hospital. Animals were fed standard chow ad libitum and subjected to experiments at 7--9 weeks of age. The animal study protocol was approved by the ethics committee of Huashan Hospital. All experiments were performed following the Huashan Hospital Laboratory Animal Centre care guidelines.
Isolation, Culture, And Identification Of HSCs {#S0002-S2002}
----------------------------------------------
HSCs were isolated from murine livers as previously described.[@CIT0011] Briefly, the livers were perfused through the portal vein with collagenase IV (Life Technologies, Grand Island, NY, USA). The smashed cells were filtered through a nylon mesh. HSCs were purified by Percoll density gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA) and cultured in complete medium supplemented with 20% FBS (Gibco, Gaithersburg, MD, USA) for 7 to 14 days, unless otherwise indicated. The purity of HSCs ranged from 90% to 95%, as measured by desmin immunostaining and typical appearance of lipid droplets under a light microscope.
Isolation And Culture Of Dendritic Cells (DCs) {#S0002-S2003}
----------------------------------------------
DCs were generated from bone marrow progenitor cells as previously described.[@CIT0012] Bone marrow cells were extracted from femurs and tibias of BALB/c mice, and erythrocytes were lysed using ammonium chloride. The cells were cultured in 24-well plates (1×10^6^ cells/well) in 1 mL of RPMI 1640 (Gibco) supplemented with 10% FBS and 10 ng/mL recombinant granulocyte-macrophage colony stimulating factor (R&D Systems, Minneapolis, MN, USA). All cultures were incubated at 37°C in 5% humidified CO~2~. Nonadherent granulocytes were removed after 48 hrs of culture. Half of the media was exchanged every 48 hrs. After 6 days of culture, 1 μg/mL lipopolysaccharide (Sigma-Aldrich) was added to the culture media for 18 hrs to allow for maturation. The purity of DC preparations was routinely monitored by flow cytometry using an anti-CD11c monoclonal antibody (mAb) (eBioscience, San Diego, CA, USA). CD11c+ cells were enriched to \>85%.
Tumor Antigen Uptake {#S0002-S2004}
--------------------
The mouse colon carcinoma CT26 cell line was purchased from American Type Culture Collection and cultured in DMEM (Gibco) supplemented with 10% FBS. On day 6 of DC culture, CT26 mouse colon cancer cell lysates were added to the culture medium cultures consisted of a DC-to-CT26 ratio of 1:10 for 18 hrs at 37°C in 5% humidified CO~2~. CT26 mouse colon cancer cell lysates were obtained through six freeze/thaw cycles in PBS (Sigma-Aldrich).
Mixed Lymphocyte Reactions (MLRs) {#S0002-S2005}
---------------------------------
For primary MLRs, nylon wool-eluted spleen T cells (2×10^5^) from BALB/c mice were used as responders, and γ-irradiated (20 Gy) tumor antigen-pulsed DCs derived from BALB/c bone marrow were used as stimulators. Cultures were maintained in complete medium for 3 days at 37°C in 5% humidified CO~2~. \[^3^H\]-TdR (0.5μCi/well) was added for the final 18 hrs of culture. Cells were harvested onto glass fiber disks using an automated system, and incorporation of \[^3^H\]-TdR into DNA was assessed using a Wallac 1450 liquid scintillation counter (PerkinElmer, Boston, MA, USA). Results are expressed as mean counts per minute (cpm)±SD. To examine the effect of HSCs on T cell proliferation, γ-irradiated (50 Gy) HSCs were added at the beginning of culture. T cells were cultured with tumor antigen-pulsed DCs at a ratio of 10:1 for 3 days. HSCs were added into the culture at a HSCs: T cells ratio of 1:20, 1:40, 1:80, or 1:160.
Flow Cytometric Analysis {#S0002-S2006}
------------------------
Expression of cell surface molecules was detected using a FACScan (BD Biosciences, San Jose, CA, USA), and analyzed using CellQuest software (BD Biosciences). Cells were stained with the following monoclonal antibodies: FITC-CD25 (eBioscience), PE-Cy5-CD4, and FITC-CD3 (BD Biosciences). Isotype-matched irrelevant mAbs were used as negative controls. Apoptosis was assessed using PE-Annexin V staining (BD Biosciences). Foxp3 staining was performed using fixation and permeabilization buffers contained in the Foxp3 kit according to manufacturer's instructions (eBioscience).
CCK8 Assay {#S0002-S2007}
----------
CT26 cells were plated in triplicate at 4×10^3^ cells/well in 96-well plates. MLR supernatants were collected and added to the wells. At 24, 48, 72, and 96 hrs, 10 µL of CCK8 solution (Dojindo, Kumamoto, Japan) was added to each well. Absorbance was detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) following incubation at 37°C for 2 hrs.
Transwell Migration Assay {#S0002-S2008}
-------------------------
Transwell migration chambers (Corning Inc., Corning, NY, USA) were used to evaluate the migration of CT26 cells. CT26 cells (5×10^4^) in 200 µL of serum-free medium were added to the upper chamber, and supernatants (800 µL) collected from MLR experiments with or without HSCs were added to the lower chamber. After incubation for 24 hrs, cells on the upper surface of the membrane were removed using a cotton swab. The remaining cells were fixed in methanol, stained using crystal violet, and air-dried. The number of migrating cells on each membrane was counted in five random fields per well using a light microscope.
Cytokine Analysis {#S0002-S2009}
-----------------
IL-2, IL-4, IL-10, and IFN-γ levels in MLR culture supernatants were measured using ELISA kits according to the manufacturer's instructions (Jingmei Biotech Limited Company, Shenzhen, People's Republic of China). The minimum detectable dose of IL-2 is 15.6 pg/mL. The minimum detectable dose of IL-4 is 7.8 pg/mL. The minimum detectable dose of IL-10 is 15.6 pg/mL. The minimum detectable dose of IFN-γ is 9.4 pg/mL. MLR culture supernatants were harvested at the end of the third day of culture.
Animal Model Of CRC Liver Metastasis {#S0002-S2010}
------------------------------------
A murine model of CRC-derived liver metastasis was established as previously described.[@CIT0013] CT26 cells were washed twice with 0.5 M PBS after trypsinization and suspended in PBS. The animal model was established under anesthesia using isoflurane (Abbott Laboratories, Abbott Park, IL, USA). For the laparotomy, a median incision was made and a 30-gauge needle was used to puncture the portal vein. A 0.1 mL cell suspension containing 1×10^6^ CT26 cells, or a mixture of 1×10^6^ CT 26 cells and 5×10^5^ HSCs, was injected to achieve a liver metastasis model.
Immunohistochemistry {#S0002-S2011}
--------------------
Immunohistochemistry was performed in liver metastatic tumor samples from murine model. Tissue paraffin sections were deparaffinized, rehydrated and pre-treated with 10 mM sodium citrate buffer at a sub-boiling temperature for 10 mins to unmask the antigen. The sections were subsequently incubated with 3% H~2~O~2~ for 10 mins at room temperature and dark conditions to block endogenous peroxidase activity, followed by incubation with blocking solution for 1 hr to avoid unspecific binding of the primary antibody. The sections were then incubated overnight at 4ºC with the anti-PCNA antibody (Cell Signaling Technology, Danvers, MA, USA) followed by incubation for 30 mins at room temperature with a biotinylated ECL anti-rabbit IgG (GE Healthcare, Chalfont St. Giles, UK). The color was developed using the diaminobenzidine substrate (Roche Diagnostics, Mannheim, Germany), and the sections were counterstained with hematoxylin. Slides were visualized and photographed using a Leica DM2500 light microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Statistical Analysis {#S0002-S2012}
--------------------
All data analyses were conducted using SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA). Comparative analysis of the data was performed using one-way ANOVA or Student's *t*-test. Differences were considered statistically significant when P \< 0.05.
Results {#S0003}
=======
DCs Effectively Took Up Tumor Antigens {#S0003-S2001}
--------------------------------------
To examine the effects of tumor antigen uptake by DCs, DCs were pulsed with tumor antigens as stimulators in MLRs. On day 6 of DC culture, tumor antigens were added to the culture at a DC:CT26 ratio of 1:10 for 18 hrs at 37°C in 5% humidified CO~2~. The results showed that DCs that did not take up tumor antigens did not promote T cell proliferation. (P\<0.05) ([Figure 1](#F0001){ref-type="fig"}).Figure 1DCs effectively took up tumor antigens. DCs that did not take up tumor antigens did not promote T cell proliferation. DCs able to effectively take up tumor antigens promoted T cell proliferation to a significantly greater extent than DCs that did not take up tumor antigens (P\<0.05). The ratio of T cells to DCs was 20:1.
HSCs Inhibited T cell Proliferation {#S0003-S2002}
-----------------------------------
To evaluate the effects of HSCs on T cell proliferation, HSCs were added to an MLR culture containing splenic T cells stimulated by DCs pulsed with tumor antigens. HSCs inhibited T cell proliferation (P\<0.05) ([Figure 2A](#F0002){ref-type="fig"}) in an HSC:T cell ratio-dependent manner ([Figure 2B](#F0002){ref-type="fig"}).Figure 2(**A**) HSCs inhibited T lcell proliferation. The ratio of T cells to DCs to HSCs was 20:2:1. (**B**) In each group, 2×10^5^ nylon wool-eluted spleen T cells from BALB/c mice were cultured with DCs in the presence or absence of HSCs. The ratio of T cells to DCs (10:1) was the same for all conditions, but the ratio of HSCs to T cells varied. HSCs inhibited T cell proliferation in a dose-dependent manner.
HSCs Induced T Cell Apoptosis {#S0003-S2003}
-----------------------------
We hypothesized that HSC-mediated inhibition of T cell proliferation may have resulted from apoptosis of activated T cells. To test this hypothesis, splenic T cells were cultured for 3 days with tumor antigen-pulsed DCs in the presence or absence of HSCs. The cells were double-stained with anti-Annexin V mAb and anti-CD3 mAb. As shown in [Figure 3](#F0003){ref-type="fig"}, the proportion of cells that were double-positive for Annexin V and CD3 staining was higher in the group cultured with HSCs than that in the group cultured without HSCs (P\<0.05). These results confirmed that HSCs increased T cell apoptosis.Figure 3Nylon wool-eluted spleen T cells from BALB/c mice were cultured with DCs in the presence or absence of HSCs. The ratio of T cells to DCs to HSCs was 20:2:1. Apoptotic cells were double-stained with FITC-anti-CD3 and PE-anti-Annexin V. The number of apoptotic T cells in the group incubated with HSCs was markedly greater than that in the group without HSCs (P\<0.05).
HSCs Promoted Treg Cell Expansion {#S0003-S2004}
---------------------------------
To examine the effects of HSCs on Treg cell activity, Treg cells were quantified in MLRs in which splenic T cells were cultured for 3 days with tumor antigen-pulsed DCs in the presence of HSCs. The percentage of CD4+CD25+ FoxP3+ cells in MLR cultures with HSCs was higher than that in MLR cultures without HSCs (P\<0.05) ([Figure 4B](#F0004){ref-type="fig"}). This result confirmed that HSCs promoted Treg cell expansion.Figure 4HSCs induced expansion of Treg cells. T cells were co-cultured with DCs in the presence or absence of HSCs. (**A**) Gated on the CD4+ cell populations. (**B**) HSCs increased the number of CD4+CD25+FoxP3+ cells, and the percentage of Treg cells in the group with HSCs was higher than that in the group without HSCs (P\<0.05).
Cytokine Levels In Supernatants Of MLRs Were Altered By HSCs {#S0003-S2005}
------------------------------------------------------------
To evaluate the effects of HSCs on cytokine secretion, IL-2, IL-4, IL-10, and IFN-γ levels were quantified in MLR culture supernatants using ELISA. The results showed that the expression levels of IL-2 and IL-4 in supernatants of MLRs with HSCs were significantly lower than those in supernatants of MLRs without HSCs (P\<0.05) ([Figure 5](#F0005){ref-type="fig"}). No differences were observed for IL-10 or IFN-γ expression in MLRs with or without HSCs.Figure 5Supernatants were collected from MLRs and analyzed using ELISA. HSCs reduced the expression of IL-2 and IL-4 in the supernatants (P\<0.05). However, HSCs did not alter the expression of IL-10 or IFN-γ in the supernatants (P\>0.05).
MLR Supernatants Promoted CT26 Cell Proliferation And Migration {#S0003-S2006}
---------------------------------------------------------------
To determine the effects of MLR supernatants on CT26 cells, CCK8 assay was used to evaluate CT26 cell proliferation and transwell migration assay was used to evaluate CT26 cell migration. As shown in [Figure 6](#F0006){ref-type="fig"}, supernatants from MLRs with HSCs promoted proliferation and migration of CT26 cells to a greater extent than MLRs without HSCs (P\<0.05).Figure 6(**A**) CCK8 assay was used to determine whether supernatants of MLRs containing HSCs promoted proliferation of CT26 cells. (**B**) Transwell migration assay was used to determine whether supernatants of MLRs containing HSCs increased CT26 cell migration.
HSCs Promoted Growth Of Liver Metastatic Tumors In Vivo {#S0003-S2007}
-------------------------------------------------------
To determine whether HSCs could promote CRC cell colonization in the liver in vivo, a murine model of CRC-derived liver metastasis was evaluated. In the control group, CT26 cells were injected via the portal vein. In the experimental group, a mixture of CT26 cells and HSCs was injected via the portal vein. As shown in [Figure 7A](#F0007){ref-type="fig"}, the number of liver metastatic tumors in the experimental group was greater than that in the control group (P\<0.05). CT26 cell proliferation was assessed using PCNA immunostaining of liver metastatic tumor samples. The number of PCNA-positive cells was significantly greater in the experimental group than in the control group (P\<0.05) ([Figure 7B](#F0007){ref-type="fig"}).Figure 7Effect of HSCs on liver metastasis in vivo. (**A**) The number of liver metastatic tumors in the experimental group was greater than that in the control group (P\<0.05). (**B**) Immunohistochemical staining for PCNA in liver metastatic tumor samples. The number of PCNA-positive cells in the experimental group was significantly greater than that in the control group (P\<0.05).
Discussion {#S0004}
==========
CRC is a common malignancy of the gastrointestinal tract. The liver, which is nourished by a rich blood supply from both arterial and portal venous systems, is the most common location of CRC metastases. Liver metastasis is the main cause of CRC-related death.[@CIT0014] A large body of evidence has indicated that the liver microenvironment provides autocrine and paracrine signals originating from both parenchymal and non-parenchymal cells that promote development of hepatic metastases.[@CIT0015]
HSCs are the main non-parenchymal cells in the liver. Quiescent HSCs can be activated by cytokines or by liver injury. Recent studies showed that activated HSCs play an important role in liver fibrosis and exhibit immunomodulatory activities. Additionally, HSCs in the liver microenvironment have been shown to promote proliferation and infiltration of hepatoma cells.[@CIT0016],[@CIT0017] Platelet-derived growth factor-C, which is secreted by colon carcinoma cells, induces proliferation of HSCs and promotes tumor growth.[@CIT0018] However, the mechanisms responsible for the effects of HSCs on development of CRC-derived liver metastases remain unclear.
HSCs exert potent immunosuppressive effects via induction of activated T cell apoptosis.[@CIT0019] Furthermore, HSCs can induce T cell hypo-responsiveness and promote Treg cell expansion, allowing hepatocellular carcinoma cells to escape immune detection, resulting in development of liver carcinoma.[@CIT0020] We used MLRs to investigate the effects of HSCs on T cells. Our findings demonstrated that activated HSCs inhibited T cell proliferation and induced T cell apoptosis. In addition, HSCs promoted Treg cell expansion. Treg cells are a subgroup of CD4+ T cells that exhibit immunosuppressive activity. Transforming growth factor β (TGF-β) was one of the first cytokines discovered, and plays an important role in differentiation of Treg cells.[@CIT0021] Activated HSCs secrete TGF-β,[@CIT0011] which may have been responsible for the increased percentage of CD4+CD25+ FoxP3+ cells in MLRs with HSCs. Based on this result, we hypothesized that HSCs may be responsible for creating an immunosuppressive liver microenvironment, resulting in an environment suitable for the growth of CRC metastases.
We also analyzed cytokine levels in MLR supernatants. IL-2 is primarily secreted by CD4+ and CD8+ T cells, resulting in T cell activation and enhanced NK cell activity.[@CIT0022] Consistent with our results, Shimizu et al[@CIT0023] found that IL-2 production was significantly decreased in patients with CRC and hepatic metastasis. IL-4 is primarily secreted by type 2 T helper cells, mast cells, and basophilic granulocytes.[@CIT0024] Recent studies have shown that binding of IL-4 to IL-4R promotes tumor cell proliferation in breast and prostate cancer.[@CIT0025],[@CIT0026] However, Morisaki et al[@CIT0027] found that IL-4/IL-4R prevents gastric cancer cells from entering the G0/G1 phase, resulting in inhibition of cell proliferation. Our results suggest that IL-4 inhibits metastasis of CRC to the liver.
MLRs were used to simulate an immune microenvironment. HSCs, which are liver non-parenchymal cells, were added to MLR cultures to create a hepatic microenvironment in vitro. CCK8 and transwell migration assays showed that supernatants from MLRs containing HSCs promoted CT26 cell proliferation and migration, confirming that HSCs in hepatic microenvironments could induce CRC cell proliferation and migration. Consistent with our in vitro results, we found that the presence of HSCs increased the number of liver metastases and increased proliferation of liver metastatic tumor cells in vivo.
Conclusion {#S0005}
==========
Our study showed that HSCs may contribute significantly to an immunosuppressive hepatic microenvironment, resulting in increased CRC cell proliferation and migration. Furthermore, HSCs promoted CRC cell colonization in the liver. Our findings indicate a potential strategy for treatment of CRC liver metastases.
This study was supported by the "Belt and Road" Young Scientist Communication International Cooperation Project (17410742100).
Disclosure {#S0006}
==========
The authors report no conflicts of interest in this work.
[^1]: These authors contributed equally to this work
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1.. Introduction {#S1}
================
Despite progressively advanced surgical strategy and techniques, surgery for acute type A aortic dissection still has high hospital mortality (12%--22%) and low 5-year survival (59%--66%) \[[@ref001], [@ref002], [@ref003], [@ref004]\] including high incidence of postoperative stroke (10%) \[[@ref002], [@ref003], [@ref004], [@ref005]\].
Recently antegrade cerebral perfusion for brain protection during circulatory arrest has significantly reduced in-hospital mortality after acute type A aortic dissection surgery \[[@ref006]\].
The purpose of this retrospective study was to investigate surgical outcomes associated with simplified surgical approach for acute type A aortic dissection surgery in a center with small number of acute aortic type A dissection operations.
2.. Methods {#S2}
===========
2.1. Patients and preoperative characteristics {#S2.SS1}
----------------------------------------------
From October 2007 to December 2016, 99 consecutive patients (50 men and 49 women; mean age, 59 $\pm$ 14 y) had emergency surgery (within 3 hours after the arrival at the emergency department) for repair of acute type A aortic dissection (Table [1](#T1){ref-type="table"}). In patients suspected of acute dissection from symptoms and signs, acute aortic dissection was diagnosed by contrast computed tomography (CT) angiography, and myocardial dysfunction, pericardial tamponade, extension of aortic dissection into the aortic sinuses, and heart valve function were evaluated by transthoracic echocardiography. Most patients arrived at the emergency department within 24 hours after onset of symptoms. Preoperative characteristics of the patients are listed in Table [1](#T1){ref-type="table"}.
2.2. Surgical procedure {#S2.SS2}
-----------------------
Perioperative findings are listed in Table [2](#T2){ref-type="table"}. Surgical approach was simplified with time and nearly unified by the end of 2013, as listed in Table [3](#T3){ref-type="table"}. Preoperative unresolved shock, which was defined as an unstable preoperative blood pressure less than 90 mmHg with inotropic support and volume replacement, was moved to the operating room immediately after the diagnosis.
Surgery was performed through median sternotomy. Intraoperatively arterial pressure monitoring was performed at three sites of both radial arteries and the left dorsalis pedis artery or left femoral artery. In hemodynamically stable patients, arterial perfusion was achieved through right axillary artery using an 8-mm graft. For retrograde cardioplegia through the purse-string sutured coronary sinus aperture, a small right atriotomy was made with 2 vena cava cannulations. For myocardial protection, retrograde cardioplegia was performed at 20--30-minute intervals. Circulatory arrest for hemi-arch replacement (HAR) or total arch replacement (TAR) was performed at 20$^{\circ}$C bladder temperature.
Table 1Preoperative characteristics of patients who had surgery for acute type A aortic dissection$^{*}$CharacteristicNo. of patients (%)Male sex50 (51)Arrival at emergency department within 24 hr after symptom onset90 (91)Smoking (current or previous)19 (19)Hypertension68 (69)Diabetes mellitus5 (5)Chronic pulmonary disease5 (5)Central neurologic deficit$^{\text{a}}$9 (9)Renal dysfunction$^{\text{b}}$5 (5)Marfan syndrome4 (4)Ventricular dysfunction$^{\text{c}}$5 (5)Aortic regurgitation $\geqslant$ moderate43 (43)Malperfusion$^{\text{d}}$19 (19)Shock (uncorrected hypotension)32 (32)Hemopericardium38 (38)$$ 99 patients. Data reported as number of patients (%). $^{\text{a}}$History of transient ischemic attack or stroke. $^{\text{b}}$Preoperative serum creatinine level $>$ 2.0 mg/dL. $^{\text{c}}$Left ventricular ejection fraction $<$ 50%. $^{\text{d}}$Severe stenosis of cerebral artery, visceral artery, or artery below the bifurcation of the aorta.
Table 2Perioperative findings and operative data in patients who had surgery for acute type A aortic dissection$^{*}$FindingsDataCardiopulmonary bypass time (min)176 $\pm$ 71Hypothermic circulatory arrest time (min)59 $\pm$ 35 Antegrade cerebral perfusion time (min)60 $\pm$ 34 Retrograde cerebral perfusion time (min)52 $\pm$ 39Arterial perfusion Right axillary artery, 8-mm artificial graft61 (62) Right femoral artery, straight cannula38 (38)Aortic valve regurgitation None or trivial39 (39) Mild17 (17) Moderate25 (25) Severe18 (18)Annuloaortic ectasia (5 cm $>$ root dimensions)7 (7)Intimal tear Ascending aorta40 (40) Aortic arch39 (39) Aortic root (sinus)10 (10) Unidentified10 (10)Cerebral perfusion during circulatory arrest Antegrade cerebral perfusion78 (79) Retrograde cerebral perfusion16 (16) No circulatory arrest$^{\dagger}$5 (5)Sinus impending rupture (with bleeding)6 (6)Aortic root replacement or reimplant7 (7)Extents of resection Hemiarch62 (63) Total arch32 (32) Ascending aorta5 (5)$$ 99 patients. Data reported as mean $\pm$ SD or number of patients (%). $^{\dagger}$Ascending aorta only was replaced under moderate hypothermia.
Table 3Simplified surgical approach for acute type A aortic dissection surgeryVariablesThe early periodThe latter periodCardioplegiaAntegradeRetrogradeDissected aortic sinusesPseudomedia (Teflon felt)BioGlueDissected aortic wallsSandwitched with two Teflon stripsCerebral perfusionRetrogradeAntegradeSystemic hypothermia20$^{\circ}$CHead vessel anastomosisCarrol patch
During cooling, the middle portion of the ascending aorta was clamped and opened transversely to locate the intimal tear. Supra-coronary transection was performed to examine the extent of dissection in the sinuses, functional morphology of the aortic valve, and injuries of the coronary ostia. In the early study period, the dissected aortic root was repaired with felt pseudomedia (Teflon felt, Bard PTFE, IMPRA, Tempe, AZ, USA) after resuspension of the aortic commissures \[[@ref007]\]; in the latter period, the dissected aortic root was repaired with BioGlu (CryoLife, Inc., Kennesaw, GA, USA) and the distal end was reinforced with double felt strips \[[@ref008]\].
After systemic circulation was arrested, the ascending aorta was unclamped and the intima and false lumen in the aortic arch were examined. The primary intimal tear was identified in 89 patients (90%) (Table [2](#T2){ref-type="table"}). For hemi-arch replacement (HAR) or total arch replacement (TAR), 78 patients (79%) had bilateral selective antegrade cerebral perfusion and 16 patients (16%) in the early period had retrograde cerebral perfusion. HAR were performed in 67 patients (68%), and TAR in 32 patients (32%). TAR was indicated in cases with intimal tear near head vessels, primary entry located at descending aorta or unknown, or the need for total arch reconstruction dependent on patient age. For HAR, resection of the aortic arch was extended near the subclavian artery and ligamentum arteriosum \[[@ref007]\]. The distal aorta was reinforced with double felt strips using a continuous 4-0 polypropylene mattress suture \[[@ref008]\] and open distal anastomosis was performed with graft-in-aorta telescopic suture technique using a continuous 3-0 polypropylene suture.
In TAR procedure, the three head vessels were mobilized as a Carrel patch \[[@ref010]\] and the brim of the patch was reinforced with double felt strips \[[@ref008]\]. In 4 patients who had intima disruption of the innominate or left subclavian artery, a modified Carrel patch technique with a separate subclavian artery anastomosis was used \[[@ref010]\]. A 4-branch graft was used in 2 patients who had intimal disruption in both innominate and left carotid arteries. After end-to-side anastomosis of the head vessel patch to the graft were performed, systemic antegrade perfusion was restarted through the right axillary artery and a side branch of the graft. The obliquely trimmed proximal end of the graft was anastomosed to the new sino-tubular junction. In all cases, we used a single vascular graft for HAR or TAR.
For treatment of aortic root dilation, 2 patients had aortic root reimplantation (David procedure) \[[@ref011]\] and 5 patients had aortic root replacement (modified Bentall procedure) \[[@ref012]\]. In 14 patients (14%) who had sinus intimal tear, sinus resection and graft replacement ($n =$ 5), sinus plication sutures ($n =$ 5), and sinus patch reinforcement ($n =$ 4) were performed after repair of the dissected aortic root using BioGlue.
2.3. Simplified surgical approach for antegrade systemic perfusion and myocardial and brain protection {#S2.SS3}
------------------------------------------------------------------------------------------------------
Arterial perfusion for cardiopulmonary bypass was performed through right axillary artery in most patients with stable hemodynamics, and another arterial perfusion through a side branch of the aortic graft was added after arch repair. In patients who had initial femoral artery perfusion, after distal aortic anastomosis and arch vessel anastomosis, the arterial perfusion was converted to antegrade systemic perfusion through a side branch of the aortic graft. During the past decade, the surgical procedure has been gradually modified based on common evidences from many documents for the improvement of surgical outcomes. In the last three years, the surgical protocol was the same in most patients, except for some patients who required additional or modified surgery, as described in Table [3](#T3){ref-type="table"}. In most patients who had total arch replacement, Carrel or modified Carrel patch technique was used for repair of the head vessels \[[@ref010]\]. For myocardial protection, retrograde cardioplegia was performed through a 12-Fr balloon catheter directly inserted into coronary sinus with a purse-string suture at the aperture. For brain protection during circulatory arrest, bilateral selective antegrade cerebral perfusion was performed through the axillary artery or the innominate artery and the left carotid artery with balloon occlusion of the left subclavian artery. A left ventricular vent catheter was routinely placed through the right superior pulmonary vein.
2.4. Statistical analysis {#S2.SS4}
-------------------------
All data analyses were performed with statistical software (SPSS, version 18.0, SPSS Inc, Chicago, IL, USA). Continuous variables are reported as mean $\pm$ standard deviation and compared with Mann-Whitney test. Categorical data were compared with chi-square or Fisher exact test. Logistic regression on significant perioperative variables was performed to determine independent risk factors for hospital mortality. Kaplan-Meier curves were constructed to estimate survival. Statistical significance was defined by $P \leqslant$ 0.05.
3.. Results {#S3}
===========
The mean cardiopulmonary bypass time was 234 $\pm$ 86 min, including the mean cerebral perfusion time of 60 $\pm$ 36 min under hypothermic circulatory arrest, and the mean ischemic heart time was 162 $\pm$ 59 min. The mean lowest nasopharyngeal temperature was 15.2 $\pm$ 2.5$^{\circ}$C (Table [2](#T2){ref-type="table"}). In-hospital mortality was 8% (8/99) in all cases; 12% (6/52) during the early 6 years and 4% (2/47) during the last 3 years that had establishment of the simplified surgical strategy ($P =$ 0.27). The causes of the in-hospital death were myocardial infarction or arrhythmia due to coronary ostial injury associated with aortic sinus dissection in 4 patients, uncontrolled bleeding in 2 patients, and mesenteric infarction in 2 patients. Of them, 6 were died within 24 hours after surgery.
Table 4Postoperative findings and outcomes in patients who had surgery for acute type A aortic dissection$^{*}$ParameterResultsRe-exploration due to bleeding4 (4)Ventilator support time (h)92 $\pm$ 85Long ventilator support ($>$ 72 h)38 (38)Postoperative intensive care unit stay (d)8 $\pm$ 6Delayed sternal closure10 (10)New stroke3 (3)Temporary neurologic deficit4 (4)Abdominal organ ischemic injury2 (2)Hospital stay (d)24 $\pm$ 12Early death ($<$ 24 h after surgery)5 (5)Hospital death, including early death8 (8)Late death3 (3)Clinical follow-up (mo)50 $\pm$ 36$$ 99 patients. Data reported as number of patients (%) or mean $\pm$ SD.
Table 5Univariate and multivariate analysis of hospital mortality in patients who had surgery for acute type A aortic dissection$^{*}$ParameterNo. of patients (%)No. of patients whoUnivariateMultivariatedied in Hospital (%)($P$ value)($P$ value)Age (years)0.31 $\leqslant$ 5033 (33)2 (6) 51--7036 (36)5 (14) $\geqslant$ 7130 (30)1 (3)Year range of surgery0.27 2007--201352 (53)6 (12) 2014--201647 (47)2 (4)Male50 (51)4 (8)1.00Hypertension68 (69)6 (9)1.00Smoking19 (19)2 (11)0.65Diabetes mellitus5 (5)0 (0)1.00COPD5 (5)0 (0)0.35Stroke9 (9)1 (10)0.55Renal dysfunction $>$ stage 35 (5)0 (0)1.00Before surgery AR $>$ moderate18 (18)1 (6)1.00 Shock (full inotropics)32 (32)6 (19)0.010.013 Hemopericardium38 (38)5 (13)0.25 Iliac artery involvement51 (52)8 (16)0.010.99 Malperfusion19 (19)4 (21)0.040.14Surgery Arch intima tear40 (40)5 (13)0.26 Retrograde cerebral perfusion16 (16)4 (25)0.030.13 All sinus dissection11 (11)3 (27)0.040.63 Total arch resection32 (32)3 (9)1.00 Femoral artery cannulation38 (38)6 (16)0.06 Sinus repair14 (14)3 (21)0.09 Aortic root surgery7 (7)0 (0)1.00CPB time (minutes)0.010.28 $\leqslant$ 1203 (3)0 (0) 121--18068 (69)3 (4) 180--24018 (18)1 (6) $\geqslant$ 24010 (10)4 (40)HCA time (minutes)0.39 $\leqslant$ 3015 (15)2 (13) 31--6045 (45)2 (4) $\geqslant$ 6133 (33)3 (9)After surgery Delayed sternal closure10 (10)1 (10)1.00 Ventilation time $>$ 72 hours38 (38)2 (4)1.00 ICU stay $>$ 5 days41 (41)3 (7)0.09$$ 99 patients. Data reported as number of patients (%). AR: aortic valve regurgitation; COPD: chronic obstructive pulmonary disease; CPB: cardiopulmonary bypass; HCA: hypothermic circulatory arrest; ICU: intensive care unit. $P <$ 0.05 was considered statically significant.
Postoperative complications included new stroke in 3 patients (3%), aggravation of previous stroke in 3 patients (3%), and prolonged ventilation $>$ 72 hours in 38 patients (38%) (Table [4](#T4){ref-type="table"}). The frequency of postoperative stroke was not different between retrograde and antegrade cerebral perfusions (2/16, 13% vs 4/78, 5%; $P =$ 0.28). There was no paraplegia secondary to spinal cord injury. Postoperative contrast CT angiography was performed in 95% (86/91 survivors) and revealed that 56% (48/86 examiners) had false lumen thrombosis.
Univariate analyses showed that predictive factors for hospital death included preoperative shock, iliac artery involvement, poor preoperative perfusion, retrograde cerebral perfusion, all sinuses' dissection, and longer cardiopulmonary bypass time (Table [4](#T4){ref-type="table"}). Multivariate analysis showed that preoperative shock was an independent predictor of hospital death (relative risk, 9.3; 95% confidence interval 1.6--53.6; $P =$ 0.01) (Table [5](#T5){ref-type="table"}).
Between the patients who had HAR and TAR, the TAR group was significantly younger than the HAR group (53 $\pm$ 11 y vs 61 $\pm$ 14 yr; $P <$ 0.01). Although the TAR group had significantly longer hypothermic circulatory arrest time than the HAR group (92 $\pm$ 41 min vs 43 $\pm$ 15 minutes; $P <$ 0.001), TAR was not a risk factor for operative death (TAR, 3/32 vs HAR, 5/62; $P =$ 1.00) and the incidences of stroke were similar for the 2 procedures (HAR, 5/62 vs TAR, 1/32; $P =$ 0.75). Patients with additional aortic root surgery (5 replacement or 2 reimplantation) had significantly longer bypass time than those without aortic root surgery (299 $\pm$ 130 min vs. 167 $\pm$ 56 min, $P =$ 0.04), but the aortic root surgery was not a risk factor of hospital death (0/7 vs 8/92; $P =$ 1.00). Of the patients with aortic root surgery, 2 patients had a coronary occlusion of the right or left coronary artery caused by ostial intima disruption, respectively. Both received interposition of a short vein graft between the trimmed proximal coronary artery and the replaced aortic root.
Figure 1.Kaplan-Meier curve showing survival after surgery for acute type A aortic dissection.
Between patients that had undergone retrograde and antegrade cerebral perfusions, patients in the two perfusion groups had a similar mean circulatory arrest time (antegrade, 60 $\pm$ 34 min: retrograde, 52 $\pm$ 39 min; $P =$ 0.25), but the retrograde perfusion group had longer ventilation support compared with the antegrade group (146 $\pm$ 125 h vs 74 $\pm$ 69 h; $P =$ 0.004).
The median follow-up time was 44 months (range, 4--124 m; mean, 50 $\pm$ 36 m), with a complete follow-up of 91 survivors. In all patients, the survival rates at 1 and 5 years after surgery were 88% and 77%, respectively (Fig. [1](#thc-26-thc171169-g001){ref-type="fig"}).
After the first surgery, 3 patients (3%) had aortic reinterventions: 1 patient had resection of a noncoronary sinus aneurysm and graft replacement at 35 months after the first operation, and 2 patients had resection of thoracoabdominal aortic aneurysm at 19 months and 24 months each after the dissection surgery and survived.
4.. Discussion {#S4}
==============
During the 9 years of surgery for aortic dissection, we have tried to unify the surgical protocol as one of the efforts to obtain a low surgical mortality in our institute that had a small volume of acute type A aortic dissection operations. Our patient cohort had an overall in-hospital mortality of 8%. In the 3 years since January 2014, in-hospital mortality decreased to 4%, which is similar to the results of a recent study \[[@ref013]\]. These improved outcomes may be secondary to simplified surgical strategy, such as right axillary artery perfusion, sufficient excision of dissected aortic wall, proper approximation of the remaining dissected aortic walls, bilateral selective antegrade cerebral perfusion during circulatory arrest, hemostatic anastomosis, and appropriate use of Carrel or modified Carrel patch technique.
Surgical centers participating in a small volume of aortic dissection operations may show high mortality because of insufficient surgical experiences. During our experience of 9 years, surgical strategy had been unified to improve surgical outcome. The simplified surgical approach has made surgeons more familiar with the operative procedures of acute aortic dissection. Application of different surgical protocols to every case cannot predict the surgical outcome.
We performed ascending aorta clamping and aortic root repair during cooling, confirming absence of malperfusion by monitoring blood pressure in 3 different arterial cannulation sites. With aortic cross-clamping in the middle level of the ascending aorta, we did not see malperfusion sign, even in patients who had the entry site in the ascending aorta.
According to data from the International Registry of Acute Aortic Dissection, independent variables associated with death include advanced age, hypotension, shock, previous heart disease, and preoperative renal, mesenteric or myocardial ischemia \[[@ref001]\]. In our study, preoperative unresolved shock was the only independent risk factor of hospital death, but advanced age was not a risk factor. Recently even in octogenarians, surgical outcome was favorable \[[@ref014]\].
Total arch replacement for acute aortic dissection was reported to cause hospital mortality, and the main causes of death were neurologic deficits and low cardiac output \[[@ref002], [@ref004]\]. The causes of death may be related to inadequate cerebral perfusion and myocardial protection during cardiopulmonary bypass and circulatory arrest. The incidence of temporary and permanent neurologic deficits has been reduced by antegrade cerebral perfusion \[[@ref015], [@ref016]\]. Our brain protection method was bilateral selective antegrade cerebral perfusion. The neurologic complications could be reduced by deep hypothermia and integrated antegrade cerebral perfusion during circulatory arrest \[[@ref015], [@ref017]\]. Recently, moderate hypothermia (23$^{\circ}$C) has showed less operative mortality and neurologic complications than deep hypothermia (17$^{\circ}$C) in emergent and elective aortic arch surgery \[[@ref018]\]. In another study, mild hypothermia (28--30$^{\circ}$C) and antegrade cold blood cerebral perfusion (20$^{\circ}$C) showed low operative mortality without neurologic complications \[[@ref019]\]. We used deep hypothermia, but the addition of the antegrade or retrograde cerebral perfusion showed low neurologic deficit, except delayed wakening in patients who had retrograde cerebral perfusion. In future, we should also consider the proper use of mild to moderate hypothermia. Until November 2013, we frequently used retrograde cerebral perfusion with a mean circulatory arrest time of 52 minutes. Retrograde cerebral perfusion did not affect neurologic deficits and hospital mortality rates, but delayed awakening of the patients caused prolonged mechanical ventilation time. Retrograde cerebral perfusion may be safe in the circulatory arrest time $<$ 45 min \[[@ref020]\], but in cases that require longer circulatory arrest, antegrade cerebral perfusion should be considered. We thought the unique disadvantage of deep hypothermia associated with cerebral perfusion is extended cardiopulmonary bypass time due to cooling and rewarming times.
Postoperative neurologic complications may be associated with retrograde arterial perfusion from the femoral artery \[[@ref015], [@ref016]\]. Compared with femoral artery cannulation for arterial perfusion, right axillary artery cannulation reduced the risk of poor visceral and cerebral perfusion and embolism from the thoraco-abdominal aorta to the brain \[[@ref021], [@ref022]\]. Our practice for arterial perfusion in acute aortic dissection surgery is right axillary artery perfusion using an 8-mm vascular graft. However, in patients who have unresolved hypotension, right femoral artery cannulation is inevitable for emergency cardiopulmonary bypass. In these patients, after distal aortic anastomosis of the graft under antegrade cerebral perfusion, we converted the femoral artery perfusion to the antegrade systemic perfusion through a side branch of the aortic graft.
Myocardial protection during cardiac arrest is an important tool to prevent postoperative low cardiac output. The longer the operation time, the more emphasis is placed on myocardial protection \[[@ref023]\]. In all cases, we performed retrograde cardioplegia every 20--30 minutes through a 12-Fr catheter, which was directly placed in coronary sinus with a purse-string suture through a short right atriotomy. This method does not require operative interruption for the infusion of cardioplegic solution, and it can induce perfusion of the entire myocardium through the middle cardiac vein by positioning the cannula balloon at the aperture of coronary sinus as possible \[[@ref024]\].
In patients who had aortic regurgitation combined with aortic root dilation, aortic root reimplantation or replacement can be aggressively performed according to the pathology of the aortic valve during the initial procedure \[[@ref013], [@ref025]\]. Aortic root surgery associated with acute type A aortic dissection surgery did not affect surgical outcomes of acute dissection surgery \[[@ref013]\].
We had aortic reintervention in 3 patients only: resection of a sinus aneurysm in 1 patient and resection of descending thoracic aortic aneurysm in 2 patients. Such a low reintervention rate may be due to sufficient resection of the dissected aortic tissues and proper approximation of the dissected aortic walls. In TAR for aortic dissections extending to the thoracoabdominal aorta, we did not use elephant trunk technique because it was difficult to insert the elephant graft into the unexpanded intima of the aortic dissection. Instead, a graft length of 3 cm between the left subclavian artery and the distal aortic anastomotic suture line was left as a clamping site for future descending aortic operations.
Survival rates after surgery are similar in most reports \[[@ref005], [@ref026], [@ref027]\], and risk factors for long-term survival included preoperative diabetes, advanced age, and postoperative dialysis \[[@ref027]\]. However, our study population had a high survival curve after discharge, probably due to more younger patients in our study and low incidences of risk factors of late death.
Limitations of the present study included retrospective and nonrandomized design and a small number of the study cases. The small sample size and low mortality and morbidity rates can limit the statistical power for evaluating risk factors for events. Because the various parts of the surgical protocol have been simplified step by step at different times, it is difficult to determine an exact time point to compare early and late surgical outcomes. Although our surgical strategy was not modernized, including levels of hypothermia, we tried to perform the acute dissection surgery with reasonable surgical protocol. We achieved reasonable surgical outcomes with deep hypothermia, but recently the improved results were reported with mild to moderate hypothermia, which we need further studies about.
5.. Conclusion {#S5}
==============
Emergent surgery for acute type A aortic dissection yielded acceptable early and intermediate results. Preoperatively uncorrected severe hypotension was an independent risk factor of in-hospital death in acute aortic type A dissection surgery. The causes of postoperative mortality or morbidity could be reduced with improved brain and myocardial protection. Bilateral selective antegrade cerebral perfusion is a reliable way to protect the brain during circulatory arrest. Our surgical strategy, including antegrade systemic arterial perfusion, effective myocardial protection, deep hypothermia and antegrade bilateral cerebral perfusion has been gradually simplified over a period of 9 years. With the simplified surgical approach, we could obtain satisfactory surgical outcomes in a hospital with a small number of type A aortic dissection operations. The majority of in-hospital deaths occurred because of preoperative acute myocardial injury and mesenteric ischemia, which were not related to surgical procedures during the last 3 years.
This study was supported by funds from the Research Institute of Clinical Medicine of Chonbuk National University and the Biomedical Research Institute of Chonbuk National University Hospital.
Conflict of interest {#sec0100}
====================
None to report.
[^1]: These authors contributed equally to this work.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction
===============
Aleutian mink disease virus (AMDV), also known as *Carnivore amdoparvovirus 1*, is the first-discovered member of the genus *Amdoparvovirus* within the family *Parvoviridae* and subfamily *Parvovirinae*. This genus includes a group of viruses able to infect various terrestrial carnivores ([@vew004-B11]). The primary hosts of AMDV are American mink (*Neovison vison*) and European mink (*Mustela lutreola*) but other mustelids and small animals are also frequently infected ([@vew004-B50]; [@vew004-B21]; [@vew004-B42]). Three other species within the genus *Amdoparvovirus* have been discovered recently and have so far only been identified in foxes and/or raccoon dogs; these are the gray fox amdovirus (GFAV) ([@vew004-B46]), the raccoon dog and fox amdoparvovirus (RFAV) ([@vew004-B78]), and the red fox fecal amdovirus ([@vew004-B5]). However, a wider host range for these viruses is plausible and other AMDV-related but not yet discovered species likely exist ([@vew004-B11]).
Parvovirus virions are composed of a protein capsid encompassing a monosense, single-stranded DNA molecule. The genome contains two main open reading frames (ORFs) flanked by terminal untranslated regions that can fold into hairpins and mediate viral DNA replication ([@vew004-B14]). The ORF located at the 5'-side of the genome encodes the major non-structural protein NS1 and two other smaller proteins, NS2 and NS3, which are generated by alternative splicing ([@vew004-B35]). The 3'-ORF encodes the two capsid proteins, VP1 and VP2, which share partial amino acid (aa) sequence but VP1 has approximately forty additional N-terminal residues, the VP1 unique region (VP1u) ([@vew004-B4]).
In adult mink, AMDV causes an immune complex-associated progressive syndrome, called plasmacytosis or Aleutian disease, characterized by weight loss, hypergammaglobulinemia, necrotizing arteritis, and kidney complications ([@vew004-B69]). Virus-antibody complexes allow viral particles to penetrate into their target cells, circulating macrophages, facilitating viral replication ([@vew004-B40]), and deposit in tissues leading to arteritis and glomerulonephritis ([@vew004-B69]). In kits, AMDV infects cells in the lungs causing a fulminant interstitial pneumonia ([@vew004-B1]).
The infection is widespread in wild as well as in farmed animals ([@vew004-B11]). In farmed mink, AMDV infection is associated with high mortality, reduced pregnancy rates, decreased litter size, and abortion ([@vew004-B9]) and results in severe economic consequences. Vaccination as a preventive measure is not feasible because of the peculiar pathogenic mechanism of AMDV, and the only possible approach to eliminate the virus from an affected farm is to implement eradication measures, which consist of identifying infected animals and culling them ([@vew004-B12]; [@vew004-B13]). However, eradication is difficult because bodily fluids from infected animals contain viral particles that can resist inactivation and persist in the environment ([@vew004-B11]). Furthermore, maintaining a disease-free status can also be challenging because the reintroduction of the virus from the outside, via commercial routes or from the wild where the virus is not eradicable, can quickly reestablish epidemics ([@vew004-B30]). At the same time, farms are also a source of viruses for wild animals, after accidental escape or deliberate release of infected animals, where AMDV has the potential for detrimental effects on wild animal populations ([@vew004-B50]; [@vew004-B26]). Finally, as a consequence of animal trading within the fur industry, the virus, which presumably originated in North America, is currently distributed worldwide ([@vew004-B11]).
Not much is known about the evolutionary dynamics driving genetic changes in amdoparvoviruses, but presumably some of the same processes characterizing the evolution of other parvoviruses are also involved. Single-stranded DNA viruses are characterized by high levels of genetic diversity and evolve at rates approaching those observed in RNA viruses ([@vew004-B17]), and for parvoviruses, this has been estimated to be approximately 10^−4^ substitutions/site/year ([@vew004-B76]; [@vew004-B75]; [@vew004-B86]). Another mechanism that increases genetic variation in parvoviruses is recombination, whereby chimeric genomes are generated after the simultaneous infection of the same cell by two different strains ([@vew004-B77]; [@vew004-B63]; [@vew004-B84]). This evolutionary potential combined with an already existing high viral diversity are likely linked to the emergence of strains with novel characteristics (e.g., increased pathogenicity or virulence) and the ability to replicate in novel hosts ([@vew004-B56]; [@vew004-B17]), as already documented for the emergence of canine parvovirus ([@vew004-B76]).
Viral evolution is also influenced by host population dynamics. Intensive farming practices, where a large number of animals live in very close contact and in fairly restricted areas, create favorable conditions for disease spread, with high local host density offering the perfect opportunity for efficient host-to-host transmission ([@vew004-B55]). Additionally, the high turnover and the shorter lifespan of hosts in these settings favor further viral transmission due to the continual availability of new susceptible individuals. These dynamics are likely to impact the efficiency of viral spread in terms of basic reproductive number *R*~0~ (average number of secondary infections stemming from a single infected host). An *R*~0~ above 1 is needed for substantial transmission and high host densities facilitate the selection for a higher *R*~0~. In these conditions, higher virulence and faster within-host growth rate (i.e., shorter generation time), and therefore a more rapid diversification, are also favored ([@vew004-B19]; [@vew004-B17]; [@vew004-B55]). It is therefore important to study and understand the basic mechanisms of viral evolutionary change in these circumstances.
An outbreak of Aleutian disease involving ten mink farms started in 2007 on the Island of Newfoundland, a large island off the east coast of Canada, and offered an opportunity to study AMDV evolutionary dynamics in the context of intensive farming. We have studied the molecular epidemiology and genetic features of AMDV from farmed and wild animals of Newfoundland over a period of 11 years, from before the beginning of the outbreak until 2014. Furthermore, by comparing these viral sequences to those identified worldwide, we have analyzed the global AMDV epidemic and studied the underlying viral evolutionary dynamics.
2. Materials and Methods
========================
2.1 Sample collections
----------------------
The sequences obtained in this study came from viruses circulating between 2004 and 2014 at various sites on the Island of Newfoundland, other parts of North America, and Europe. Carcasses from suspected AMDV-positive mink from Newfoundland were obtained from ten different farms in 2007--9 and again in 2014 from one of these farms. Samples from farmed mink in Nova Scotia, Ontario, Wisconsin, and Denmark were also included. Samples were collected in 2014 from various wild animals: ten American mink, sixteen ermine (*Mustela erminea*), two Newfoundland lynx (*Lynx canadensis subsolanus*), nineteen red foxes (*Vulpes vulpes deletrix*), and forty coyotes (*Canis latrans*). Additionally, sequences obtained from AMDV-positive wild mink collected in 2004 and in 2007--8 were also included. These sequences originated from two pilot studies showing AMDV-positive levels of 14 per cent (9/64) and 45.2 per cent (51/93) for samples collected from wild mink across Newfoundland before and after the beginning of the farm epidemic, respectively ([@vew004-B8]). During the 2007--8 study samples from six ermine, twenty-nine Newfoundland pine marten (*Martes americana atrata*), and six American red squirrel (*Tamiasciurus hudsonicus*) tested AMDV-negative (H.G. Whitney, unpublished data).
Spleens were used as the source of material for virus characterization. Each spleen collected between 2007--9 was stabbed in several locations with cotton-tipped swabs that were then vortexed in 500 µl tryptose phosphate broth (tryptose 20 g/l, dextrose 5 g/l, NaCl 5 g/l, disodium phosphate 2.5 g/l; pH = 7.3). For the 2014 samples, a 10 mg piece was taken from each spleen.
2.2 Screening and sequencing
----------------------------
Swab suspensions or spleen tissues were used for DNA isolation and molecular screening following two different procedures. DNA was extracted from 200 -µl aliquots of tissue swab suspensions by using the MagNA Pure LC instrument (Roche Diagnostics) and the MagNA Pure LC total Nucleic Acid Isolation Kit (Roche Diagnostics) and screened for AMDV by polymerase chain reaction (PCR) with the primer pair ADV-1207..1239 (5'-KTTGGTTGCTTTACTCC-3')/ADV-1707..1688 (5'-RTCTACTTTTA CATCACCAC-3'). DNA sequences of PCR products were determined at the University of Guelph Laboratory Services sequencing facility by Sanger sequencing with an Applied Biosystems 3730 DNA analyzer.
DNA was extracted from the spleen tissues with the DNeasy Blood and Tissue Kit (Qiagen) and screened for AMDV by PCR with two primer pairs. Three novel members of the genus *Amdoparvovirus* were described between 2011 and 2014 ([@vew004-B46]; [@vew004-B5]; [@vew004-B78]), so the primer sequences were updated to detect all known amdoparvoviruses. Those primers, GF-F (5'-GACAACRAACCAACCAAAG-3')/GF-R (5'-CCHAMSMAACAGTGAATATG-3') and ScF (5'-TGGTTGCT TTACTCC AGAAG-3')/ScR (5'-WCCWCCTCCA GTAATRGC-3'), were designed to amplify two different portions of the NS1 gene (sequence positions 329--597 and 1207--1690, respectively, of the strain AMDV-G, accession number JN040434). Both primer pairs ADV-1207..1239/ADV-1707..1688 and ScF/ScR are able to bind to all known AMDVs. Amplified products from positive ScF-ScR PCRs were purified with AMPure Beads (Beckman Coulter), cloned into pGEM®-T Easy Vector (Promega), and up to ten clones for each positive PCR were sequenced at The Center for Applied Genomics (Toronto, Canada) by Sanger sequencing using an Applied Biosystems 3730XL DNA analyzer.
Coding sequences from a selection of strains containing multiple viral variants (two samples containing putative recombinant viruses and a third randomly chosen sample containing a mixed infection of viruses from the major identified clades) were amplified, cloned, and sequenced in the same way. Complete genome sequencing for a selection of strains (three randomly selected viruses from single infections identified in farmed mink representing every major group and one virus identified in one wild mink) was achieved by amplifying the entire viral coding sequence with two overlapping hemi-nested PCRs that were subjected to direct sequencing using internal primers. Sequences of all primers used in this study are available in the [Supplementary Table S1](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1).
2.3 Phylogenetic analyses
-------------------------
The dataset of sequences obtained for this study included 174 partial NS1 sequences from viruses detected between 2004 and 2014 in Newfoundland, other parts of North America (Nova Scotia, Ontario and Wisconsin), and Europe (Denmark). Sequences from Newfoundland originated from both wild mink and from ten different farms. A subset of 131 sequences was selected by excluding sequences that presented observable double peaks on chromatograms, recombinant sequences (identified using RDP software, see below), and all identical sequences derived from the same year and the same geographical area (same farm for Newfoundland) and used for phylogenetic inference. For each sample collected in 2014, only one representative clonal sequence for every strain was included. Reference sequences included only viruses for which the complete coding sequence of one or both ORFs were available and AMDV-like viruses (RFAV and GFADV) identified in foxes and raccoon dogs ([@vew004-B3]; [@vew004-B28], [@vew004-B29]; [@vew004-B64]; [@vew004-B74]; [@vew004-B46], [@vew004-B47]; [@vew004-B34]; [@vew004-B78]).
Other sequence datasets were built with sequences downloaded from GenBank ([www.ncbi.nlm.nih.gov/genbank](http://www.ncbi.nlm.nih.gov/genbank)) by mapping all available AMDV sequences (*N* = 456) to a reference complete genome to identify the genomic regions most frequently used for molecular epidemiological studies ([@vew004-B65]; [@vew004-B18]; [@vew004-B50]; [@vew004-B41], [@vew004-B42]; [@vew004-B37]; [@vew004-B13]; [@vew004-B38]; [@vew004-B45]). Genomic areas represented by the majority of the sequences were selected, and sequences that mapped to these areas were extracted and used to perform phylogenetic analyses.
Sequences were aligned with ClustalX 2.1 ([@vew004-B44]), alignments were manually edited when needed with BioEdit 7.0.5.3 ([@vew004-B31]) and then used for phylogenetic inference. A model selection was performed for each alignment to identify the best model for distance estimation, and phylogenetic trees were constructed with MEGA 6.06 ([@vew004-B83]) using the maximum-likelihood method ([@vew004-B23]). Information about numbers of sequences, the genomic regions investigated, and models used are provided in the figure legends. Bootstrap analyses ([@vew004-B24]) with 1,000 replicates were performed to test the robustness of the analyses, and only clusters supported by ≥ 70 per cent were considered valid.
Accession numbers of all sequences used in this study are available in the [Supplementary Sequence Details File](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1).
2.4 Amino acid sequence predictions
-----------------------------------
Splicing sites were determined following what was experimentally demonstrated ([@vew004-B71]) and using the AMDV-G virus (accession number JN040434) as a reference. Donor and acceptor splicing sites were also predicted using the splicing prediction algorithm NNSPLICE from fruitfly.org and scores were assigned to each site ([@vew004-B72]). Splicing events were reproduced *in silico* to determine the complete coding sequences of NS1, NS2, NS3, VP1, and VP2, which were then translated into aa sequences.
2.5 Recombination analyses
--------------------------
Alignments of partial NS1 nucleotide sequences and of the complete genomes were screened for the presence of chimeric sequences by all of the different methods included in the RDP 4.55 software package ([@vew004-B52]), specifically RDP (R, [@vew004-B53]), GENECONV (G, [@vew004-B66]), BootScan (B, [@vew004-B51]), MaxChi (M, [@vew004-B82]), Chimera (C, [@vew004-B70]), SiScan (S, [@vew004-B27]), 3Seq (T, [@vew004-B6]), and LARD (L, [@vew004-B33]). Only phylogenetically supported events identified by at least three different methods (*P* \< 0.05) were considered. Each recombination hypothesis was refined following detection order with breakpoint positions manually modified when considered adequate, and events were accepted or discarded based on probability scores, phylogenetic evidence, and sequence analysis. In detail, each identified event characterized by an average probability above the threshold for at least three different methods was evaluated independently. The phylogenetic placements of the putative recombinant strain (together with sequences showing evidence for the same recombination event) and the two putative parental strains (major and minor parents) were evaluated within the RDP framework. Events not associated with clear inconsistencies between different genomic regions were discarded. Inspection by eye of the triplets involved in every detected event allowed assessment of the chimeric nature of the sequences and determination of whether the putative breakpoint was properly located (between the two genetic regions, each derived from a different parental strain). If the position of the breakpoint had to be moved, new associated phylogenetic trees were again evaluated for confirmation. After modifying, accepting, or rejecting an event, the whole alignment was rescanned for new recombination hypotheses. Once all putative recombination breakpoints were identified, separate phylogenetic trees were built with separate genomic regions included between breakpoints using MEGA, as described above.
The analyses included only strains for which the entire genomic coding sequences were available and involved four sequences from Newfoundland (M228, M195, M173, WM25, all from 2014), three sequences from China (LN-1, LN-2 and LN-3, all from 2009) ([@vew004-B47]), two sequences from USA (AMDV-G from the 1970s and UtahI from 1963) ([@vew004-B3]), one sequence from Germany (SL-3, from the 1980s) ([@vew004-B74]), and five outgroup sequences (belonging to RFAV and GFAV) ([@vew004-B46]; [@vew004-B78]). Outgroup sequences were used within the RDP framework only in tree reconstruction and in determining informative sites.
BootScan plots ([@vew004-B73]) were built with SimPlot software 3.5.1 ([@vew004-B48]) using a window of 100 nucleotides and a step of twenty nucleotides with an F84 model ([@vew004-B25]), the maximum-likelihood method ([@vew004-B23]), and 1,000 replicates.
Shimodaira--Hasegawa and Robinson--Foulds recombination matrices ([@vew004-B79]; [@vew004-B81]) were also constructed with the RDP software and used to visualize the impact of recombination on the phylogenetic relationships of the strains. For the construction of both matrices, the default settings were used (window size: 400, step size: 100).
2.6 Selection pressure analysis
-------------------------------
The *Z*-test of selection implemented in MEGA 6.06 ([@vew004-B83]) was used to calculate the overall average synonymous (d*S*) and non-synonymous (d*N*) substitutions for each alignment. The Nei--Gojobori method ([@vew004-B59]) was used to test both hypotheses, d*N* \< d*S* (purifying selection pressure) and d*N* \> d*S* (positive selection pressure), of deviation from strict neutrality (null hypothesis). The variance was estimated with the bootstrap method and 1,000 replicates. Sites under positive and purifying selection were assessed with six different methods: Single-Likelihood Ancestor Counting (SLAC), Fixed Effect Likelihood (FEL), Random Effects Likelihood (REL) ([@vew004-B68]), Internal FEL (IFEL) ([@vew004-B43]), Fast Unconstrained Bayesian Approximation for inferring selection (FUBAR) ([@vew004-B58]), and Mixed Effects Model of Evolution (MEME) ([@vew004-B57]). While SLAC evaluates the deviation of d*N* and d*S* from an expected neutral model, FEL calculates the probability for d*N* to be different from d*S* with a likelihood ratio test and REL uses Bayes Factors to evaluate the posterior probability of rejecting the null hypothesis of neutrality considering the distribution of d*S* and d*N* across the whole alignment. IFEL and MEME are extensions of FEL but FEL assumes d*N*/d*S* ratios apply to all branches, IFEL to interior branches, and MEME allows the ratios to be variable across lineages and sites, allowing the identification of episodic selection, which affects only a subset of lineages. Finally, FUBAR is a hierarchical Bayesian method that uses an MCMC approach; it works similarly to REL but relaxes some of its restrictions. All of these methods are phylogeny based, were used with HKY as the substitution model ([@vew004-B32]), and are available online on the Datamonkey server ([www.datamonkey.org](http://www.datamonkey.org)) ([@vew004-B15]). It is a commonly used practice to accept a site of being under selection only if predicted by more than one method (e.g., [@vew004-B7]; [@vew004-B2]) because some methods, like SLAC, are less sensitive and others, like REL, are more prone to overestimate the number of sites under selection. Furthermore, some methods are based on similar algorithms and likely to produce similar results. Therefore, we accepted a site to be under diversifying or negative selection only if predicted by at least three different methods. Finally, MEME can predict sites under episodic diversifying selection, and therefore, additional sites predicted by this method alone were also considered. Sites under selection were considered acceptable only when statistically significant (*P* \< 0.1 for SLAC, FEL, IFEL and MEME; Bayes Factor \> 50 for REL; posterior probability \>0.9 for FUBAR).
3. Results
==========
3.1 AMDV phylogeography
-----------------------
A total of 131 sequences from the NS1 region (nt 1207--1690) of AMDV genomes that originated from samples collected between 2004 and 2014 from ten different farms and wild animals in Newfoundland (*N* = 97), Nova Scotia (*N* = 13), Ontario (*N* = 9), Wisconsin (*N* = 6), and Denmark (*N* = 6) were obtained and used to study AMDV molecular epidemiology. All sequences originated from viruses of mink because none of the other tested wild species was AMDV-positive. The resulting phylogenetic analysis of these sequences ([Fig. 1](#vew004-F1){ref-type="fig"}) and the calculated identities between and within groups (available as [Supplementary Table S2](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)) were used to define clades and subclades, indicated by arbitrarily assigned numbers that are not meant as a classification but included to allow easier referencing in the text. We have defined clades as bootstrap-supported (\>70%) phylogenetic clusters of sequences sharing \>90 per cent identity with each other and \<90 per cent identity with sequences from other clades. Subclades were defined in the same way but with a cut-off value of 96 per cent identity. Subclade 2A was an exception to this because it was \>90 per cent identical to subclades 1A and 2B. Furthermore, this subclade was possibly responsible for difficulties in resolving the tree structure, resulting in bootstrap values below the threshold for clade 2 (bootstrap: 42) and subclade 1a (bootstrap: 43). Figure 1.Phylogenetic analysis of AMDV partial NS1 sequences obtained during this study and originating from different areas of the world. The evolutionary history of the partial NS1 region (nt 1207--1690) was inferred using the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G = 0.4098). The rate variation model allowed for some sites to be evolutionary invariable (\[+*I*\], 32.514% sites). The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes, and branch lengths are proportional to genetic distances as indicated by the scale bar. Large groups of sequences originating from the same location and falling in the same clade have been collapsed at nodes into a triangle shape. Strains are labelled based on the original name (only for reference sequences, indicated in italics), sampling site (NL: Newfoundland; NS: Nova Scotia; ON: Ontario; WI: Wisconsin; USA: United States of America, state unknown; DK: Denmark; DE: Germany; CN: China) and year. Viral species, clades, and subclades are indicated by square brackets. Tree branches are colored based on sample origin (red: Newfoundland; purple: Nova Scotia; blue: Ontario; orange: USA; pink: Denmark; green: Germany; black: China).
The seven clades resolved within the AMDV sequences were clearly separated from the other two amdoparvoviruses, GFAV and RFAV (≤85% identity between species). Three of the AMDV clades (1, 2, and 6) also included reference sequences, whose complete genomes have been previously sequenced (except for AMDV-K), while the other clades comprised only sequences from this study. Most clades contained viruses from different geographic regions, but viruses collected from the same area tended to cluster together in separate groups within each larger clade. For example, clade 1 contained viruses from Newfoundland, Ontario, Wisconsin, and China and clade 2 included viruses from North America, Europe, and China, but sequences from the same region formed individual clusters within these clades in most cases. The other clades (3--7) were less diverse in terms of geographical origin of the sequences.
To confirm these results, we investigated whether sequences from other regions of the genome that have been used in other epidemiological studies showed a similar pattern. Three genomic regions were identified that included the majority of sequences available in the public databases. These were two regions of the NS1 ORF and one region of the VP1 ORF (nt 602--922, 1859--2208, and 2949--3228 of AMDV-G, respectively). Three separate phylogenetic trees were built with these different regions ([Fig. 2](#vew004-F2){ref-type="fig"}), and we also included some sequences from Newfoundland for which complete coding sequences were obtained. The average identities between sequences used to build these trees were comparable to what was observed for the previously analyzed genomic region (87.8%, range 80.1--100%; 91.6%, range 82.3--100%; 91.7%, range 85--100%; vs. 91.5%, range 82--100%). Similar trends were observed in these trees. For example, viruses from Denmark formed three independent groups within larger groups that included viruses identified in other European regions and Canada ([Fig. 2](#vew004-F2){ref-type="fig"}A). Similarly, the sequences from Newfoundland fell in different clusters but always formed independent groups ([Fig. 2](#vew004-F2){ref-type="fig"}A--C). Lastly, viral sequences close to the reference strain AMDV-K that we identified in Ontario, Wisconsin, and Denmark were also observed interspersed among sequences obtained by others in Ontario, Denmark, Finland, Sweden, and Estonia ([Fig. 2](#vew004-F2){ref-type="fig"}A and B). Figure 2.Phylogenetic analyses of different genomic regions of AMDV strains identified worldwide. Trees were constructed with a 321-nt long portion of the NS1 genomic region (nt 602--922 of AMDV-G) of 179 different viruses (A), a 348-nt long portion of the NS1 genomic region (nt 1859--2208 of AMDV-G) of 56 different viruses (B) and a 280-nt long portion of the VP1 genomic region (nt 2949--3228 of AMDV-G) of 128 different viruses (C). Evolutionary histories were inferred with the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites. The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes, and branch lengths are proportional to genetic distances as indicated by the scale bars. Large groups of sequences originating from the same location and falling in the same clade have been collapsed at nodes into a triangle shape. Collection dates and sites (ON: Ontario; EE: Estonia; SE: Sweden; FI: Finland; NE: Netherlands; DK: Denmark; IE: Ireland; BY: Belarus; CN: China; RU: Russia; NS: Nova Scotia; MT: Montana; ES: Spain) are indicated. Sequences identified in this study from Newfoundland (NL) in 2014 are marked with a black diamond.
3.2 AMDV in Newfoundland
------------------------
Sequences from Newfoundland were found in only two of the seven identified clades ([Fig. 1](#vew004-F1){ref-type="fig"}). Some strains in clade 1 were most similar to a virus from Ontario, while other Newfoundland sequences formed independent clusters that did not show such a close relationship to any other sequences.
We investigated the relationships among virus sequences from Newfoundland according to the year of sampling and the collection source ([Fig. 3](#vew004-F3){ref-type="fig"}). A marked separation of viruses from different farms was found. Closely related sequences were also observed on multiple farms, indicating that viruses in different farms originated from the same recent ancestor or that there was an exchange of viruses or infected animals between farms. In contrast, some farms were characterized by the presence of a distinct clade of viruses. However, with one exception, each farm contained viruses from only one clade. Figure 3.Phylogenetic relationship of AMDV strains from different locations in Newfoundland. The evolutionary history of the partial NS1 region (nt 1207--1690) of AMDV sequences was inferred using the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G = 0.4098). The rate variation model allowed for some sites to be evolutionary invariable (\[+*I*\], 32.514% sites). The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes and only the tree topology is shown. Newfoundland strains are indicated by shapes and colors, where the shapes define the collection year (star: 2004; square: 2007; diamond: 2008; triangle: 2009; circle: 2014) and the colors indicate if the host was wild (light blue) or farmed (all other colors, with each farm represented by a different color). Strains identified in other areas are labelled according to the original name (only for reference sequences), sampling site (NS: Nova Scotia; ON: Ontario; WI: Wisconsin; DK: Denmark), and year.
Temporally, sequences from animals in 2007 were only found in clade 1, while those from samples collected in 2008 and 2009 were identified in both clades. This might indicate that viruses from the two clades were introduced during two separate events.
All viruses from wild animals belonged to clade 1. Approximately half of the wild mink contained viruses that were very close to those identified in farmed animals, as shown by the presence of clusters within lineage 1 that include sequences from both farmed and wild animals ([Fig. 3](#vew004-F3){ref-type="fig"}). The other half of the viruses from wild animals formed independent clusters, where no sequences derived from farms were present and which therefore might represent a separate lineage of viruses unique to wild animals. This observation is further supported by the fact that the vast majority of sequences (7/8) identified in wild animals before the epidemic started (from 2004) are located in these clades. However, one sequence from 2004 could be identified that was closer to strains from 2007 circulating in farms.
3.3 Within-farm AMDV variation
------------------------------
All sequences from 2014 were derived from samples collected on the same farm, for which samples from an earlier time point (2007) were also available, and for each 2014 animal, eight to ten clones were sequenced. Analysis of the relationships of all 127 sequences identified from this farm showed a high level of genetic diversity ([Fig. 4](#vew004-F4){ref-type="fig"}). The overall mean *p* distance was 7.2 per cent, and most of the sequences fell into two distinct clades. Comparison of sequences from the two clades showed an average identity of 89.3 per cent, while sequences within each clade were 95.4 per cent and 97.4 per cent identical for clades 1 and 2, respectively. Only one viral type was identified within the six sequences obtained from samples collected in 2007, while much higher variation was found in 2014 in samples obtained from twelve animals, suggesting that the introduction of viruses belonging to clade 1 into the farm occurred around 2007 and that the clade 2 viruses were introduced later. The presence of subclades within clade 1 that were not detected in 2007 likely reflects viral evolution over the 7-year period between sample collections. Figure 4.Phylogenetic relationships of AMDV strains from different mink within a single farm. The evolutionary history of the partial NS1 region (nt 1207--1690) was inferred using the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]) using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G = 0.1725). The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes, and branch lengths are proportional to genetic distances as indicated by the scale bar. Strains identified in 2007 are indicated in red, while strains from 2014 are in black. Full circles represent viruses found in animals with co-infections, while empty circles represent single infections. Virus sequences with identical symbols and colors were from the same animal.
A high co-infection rate was observed in samples collected from farmed animals in 2014 ([Fig. 4](#vew004-F4){ref-type="fig"}). Remarkably, 41.7 per cent (5 out of 12) of the mink were positive for two or three different viruses. Furthermore, several polymorphic sites in each viral type within the same animal could be identified, revealing substantial intra-host mutation.
Finally, the presence of possible recombinant strains was observed ([Fig. 4](#vew004-F4){ref-type="fig"}) and further investigated. With RDP we detected two possible recombination events with two different putative breakpoints, involving several clonal sequences identified in three animals. For both these events, a BootScan analysis was performed and two separate trees were built with partitions of the original alignment before and after the putative breakpoint ([Fig. 5](#vew004-F5){ref-type="fig"}). Both events were well supported by p values calculated with all methods implemented in RDP (R: 3.5 × 10^−2^; B: 3.4 × 10^−2^; M: 4.0 × 10^−3^; C: 1.1 × 10^−3^; S: 1.2 × 10^−5^; L: 3.5 × 10^−4^; T: 3.4 × 10^−5^ for event in [Fig. 5](#vew004-F5){ref-type="fig"}A--C 1.5 × 10^−2^; B: 2.2 × 10^−2^; M: 2.3 × 10^−5^; C: 6.4 × 10^−5^; S: 1.2 × 10^−6^; L: 4.2 × 10^−2^; T: 7.3 × 10^−8^ for event in [Fig. 5](#vew004-F5){ref-type="fig"}D--F) and the sequences clearly clustered in different clades in the separate trees. In one case ([Fig. 5](#vew004-F5){ref-type="fig"}A--C), one of the two putative parental strains (NL-14_M106_18) was present in the same animal where the recombinant strains were detected. The other recombinants were identified in two different animals ([Fig. 5](#vew004-F5){ref-type="fig"}D--F), and close relatives to the two parental strains (NL-14_M46--12 and NL-14_M10_8) could be identified in the same animals where the recombinants were detected, with each parental strain found in a different animal. We cannot exclude that those chimeric sequences originated artificially during PCR, although the fact that two identical chimeric patterns were identified in two different animals is consistent with the presence of recombinant viruses in the farm. Figure 5.Recombination analysis of clonal AMDV strains from one mink farm. One event is displayed in panels A, B, and C, and the other event in panels D, E, and F. A BootScan analysis is shown for each event (A and D), and involved one of the recombinant sequences as query, sequences from relatives to the two parental strains, and AMDV-K as an outgroup. Trees built with the sequence partitions before the identified breakpoints are shown in panels B (nt 1207--1413) and E (nt 1207--1449), while trees built with the partitions after the breakpoints are shown in panels C (nt 1414--1690) and F (nt 1450--1690). The evolutionary histories were inferred using the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]) using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites. The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes (only values above 50 are reported), and branch lengths are proportional to genetic distances as indicated by the scale bars. Full circles represent viruses found in animals with co-infections, while empty circles represent single infections. Virus sequences with identical symbols and colors were from the same animal. The phylogenetic placements of the recombinant strains are highlighted by shaded areas. Average identities (1−*p*-distance) in percentage values between and within clades (range) are reported in gray on each tree.
3.4 Complete genome sequence analysis
-------------------------------------
Attempts to obtain the complete genome sequences of viruses from samples with evidence of co-infection were not successful. The high within-host strain variability prevented the reliable assembly of cloned fragments. However, complete genome sequences were obtained for viruses from four samples collected in 2014 with single-strain infections. Three viruses were derived from farmed animals (M228, M173, and M195) and one from a wild mink (WM25). Additionally, several partial genomic fragments were obtained for other strains involved in co-infections. These represented one complete and three nearly complete NS1 coding sequences (MC42.1.1 and MC106.1.3, MC106.1.8, and MC46.1.5) from three samples and two complete VP1 sequences (MC42.2.1 and MC42.2.3) from one sample.
The AMDV-G sequence was used as a reference to guide the splicing site search to identify exons, as they have been experimentally verified for that strain ([@vew004-B34]). The splicing pattern was resolved for all NS proteins and for VP1, and all donor and acceptor sites (except for the NS3 acceptor sites) showed high probability scores (scores are provided in the [Supplementary Table S3](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). All AMDV and RFAV NS1 proteins were 641 aa long and the GFAV protein was 653 aa in length. The VP2 protein was 630 aa for the GFAV sequence and varied in length between 633 and 647 aa for AMDV and RFAV sequences. A glycine-rich region at the beginning of VP2, which varied between 3 and 13 aa was identified as the main cause for this variation ([Supplementary Fig. S1](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). A one-aa deletion in VP2 was observed for strains FarEast, Rus17, M195, and WM25 (corresponding to AMDV-G VP2 residue T225), and the same deletion was also present in all RFAV sequences. The VP1u was 44 aa for GFAV and 43 aa for the other viruses. Finally, a 148-nt deletion was identified in one of the viral strains from Newfoundland (MC42.2.3), which most likely resulted in a non-functional capsid protein because of a consequent frameshift.
Phylogenetic analyses performed with all available complete NS1 aa sequences ([Fig. 6](#vew004-F6){ref-type="fig"}A) showed three distinct AMDV clades, where Newfoundland strains formed two separate subclades within the first two main clades. A second analysis involving all available complete VP2 protein sequences ([Fig. 6](#vew004-F6){ref-type="fig"}B) showed only two distinct major AMDV clades, revealed less variation with respect to NS1, and had no bootstrap-supported subclades. Names of clades and subclades are arbitrary and not related to those of [Fig. 1](#vew004-F1){ref-type="fig"}. The AMDV-K strain, which in the NS phylogeny falls outside of the two main clades and forms a separate clade by itself, was originally sequenced from cloned dsDNA fragments obtained after restriction digestion from a viral isolate containing multiple viral strains ([@vew004-B28]). The NS1 and the VP2 sequences originated from two separate clones, and therefore, it is not known whether they belong to the same strain. This prevents us from drawing any conclusions regarding the AMDV-K clade identified in the NS1 tree with respect to the VP2 phylogeny. Figure 6.Analyses of the complete AMDV coding regions. (A) Phylogenetic tree constructed with NS1 protein sequences. The evolutionary history was inferred using the maximum-likelihood method ([@vew004-B23]) based on the JTT model ([@vew004-B39]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G, parameter = 0.6942). The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes and branch lengths are proportional to genetic distances as indicated by the scale bar. (B) Phylogenetic tree based on VP2 protein sequences. The evolutionary history was inferred using the maximum-likelihood method ([@vew004-B23]) based on the General Reverse Transcriptase model ([@vew004-B16]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G = 0.286). The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes and branch lengths are proportional to genetic distances as indicated by the scale bar. (C) Identities (1−*p*-distances) calculated within and between groups considering both NS1 and VP2 protein sequences. Values indicate the range of identities between pairs of sequences and are expressed as percentages. Clades correspond to those indicated on the trees displayed in panels A and B.
Among the AMDV sequences analyzed, there were 289 parsimony-informative sites (PIS) out of 1,923 total sites in the NS1 alignment, which translated into 135 PIS out of 641 positions in the aa alignment. Similarly, we identified 162 PIS out of 1,941 sites in the VP2 alignment, which translated into 47 sites out of 647 aa residues. Analysis of the identities (1−*p*-distances, [Fig. 6](#vew004-F6){ref-type="fig"}C) between groups indicated that AMDV-K, the only member of the third clade for which a complete coding sequence of at least one of the 2 ORFs is available, is distant enough from all other strains to be considered a different species according to the parvovirus classification criteria ([@vew004-B36]) because the NS1 protein sequences need to be ≥85 per cent identical to be considered the same species. However, no appropriate classification can be proposed until the complete sequence is available. The distance analyses reflected what was observed with the phylogenies in that NS1 was less conserved than the structural protein VP2, both within and between species. For example, the RFAV sequences were approximately 72--76 per cent and 86--92 per cent identical to AMDV sequences in NS1 and VP2, respectively.
The clades identified on the NS1 tree did not correspond to those identified in the VP2 phylogeny. For example, the UtahI strain, which formed a 99 per cent bootstrap-supported cluster with strains G and SL-3 in the NS1 tree, was found in a different cluster together with the three Chinese sequences LN-1, LN-2, and LN-3 in the VP2 tree ([Fig. 6](#vew004-F6){ref-type="fig"}A and B). Furthermore, the calculated *p*-distances within and between phylogenetically defined groups were not always coherent, because in some instances the distance between two sequences belonging to different groups was lower than the distance between two other sequences from within the same group ([Fig. 6](#vew004-F6){ref-type="fig"}C). For example, the lowest identity between NS1 sequences in clade 1 was approximately 86 per cent, whereas sequences from clades 1 and 2 were up to 92.3 per cent identical. The same pattern was observed for VP2, where the lowest identity value between sequences within clade 1 was 88.8 per cent and the lowest identity when comparing sequences from clades 1 and 2 was 89.8 per cent. These inconsistencies between the phylogenetic trees and between phylogenetic grouping and *p*-distances suggested the presence of recombinant viruses.
3.5 Recombination breakpoint detection and investigation of chimeric sequences
------------------------------------------------------------------------------
The determination of recombination breakpoints was performed with viruses for which the complete coding sequence was available. Compatibility matrices were built to evaluate the impact of recombination indirectly by determining phylogenetic incongruities across the sequence alignments. The Robinson--Foulds and Shimodaira--Hasegawa compatibility matrices ([Supplementary Fig. S2A](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1) and [B](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)) demonstrated that large variations exist between tree structures over the genome sequences for both adjacent and distant genomic regions, with very limited cold areas. This inconsistency in phylogeny suggested the possible occurrence of recombination during the evolutionary history of AMDV, and therefore, the location of putative recombination breakpoints was studied further with RDP.
Several potential recombination events were detected (a recombination event map and probabilities for each event are provided as [Supplementary Fig. S3](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)) and four putative breakpoints, indicated by letters in [Supplementary Fig. S3](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1), were evaluated. The first considered breakpoint (B) was located approximately 900 nt from the beginning of the NS1 ORF (positions refer to strain AMDV-G), the second (between B and C) was located inside the VP1u region and after the end of NS1 (at approximately nt 2315 of the complete genome), the third (E) and the fourth (F) were located inside the VP1/2 ORF. The alignment was consequently split into five sub-alignments located between the putative breakpoints and new trees were constructed ([Fig. 7](#vew004-F7){ref-type="fig"}). Similar phylogenetic trees based on the same genomic regions were also built including viruses whose genomes have not been completely sequenced to provide a wider view of the AMDV evolutionary history ([Supplementary Fig. S4](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). Phylogenetic trees built with sub-portions of the VP2 ORF (VP2_1, DE: nt 1-732; VP2_2, EF: nt 733--1320; VP2_3, FG: nt 1321--1875) did not strongly support recombination in this area, and therefore, we could not exclude that those phylogenetic incongruities originated from difficulties in resolving the tree structure. However, well-supported phylogenetic incongruities could be observed between trees built with the two portions of the NS1 ORF (NS1_1, AB: nt 1-903; NS1_2, BC: 900-end) and between these and the tree built with the complete VP2 ORF (VP2_total, DG). For example, the two Chinese sequences LN-2 and LN-3 clustered with the other Chinese sequence LN-1 and the Newfoundland strain WM25 in the first and third tree, while clustering with the two USA sequences UtahI and G and the German strain SL-3 in the second tree. A similar pattern in NS1 was observed for the USA strain United ([Supplementary Fig. S4](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). Another example is the Newfoundland sequence M195 that, together with the UtahI strain, clustered with the strains SL-3 and G in the trees built with partial NS1 sequences, while clustering with the Newfoundland WM25 and the Chinese strains in the tree built with the VP2 ORF. However, the inconsistent phylogenies observed between the structural and non-structural genomic regions could also be the result of different evolutionary histories for the two genes (e.g., different evolutionary rates) as also supported by the different amounts of sequence divergence observed among strains in these two regions ([Fig. 6](#vew004-F6){ref-type="fig"}). Figure 7.Phylogenetic reconstruction of complete AMDV genomes based on different genomic regions located between putative recombination breakpoints. Evolutionary histories were inferred with the maximum-likelihood method ([@vew004-B23]) based on the HKY model ([@vew004-B32]), identified as the best fitting model after the model test analysis, using MEGA6 ([@vew004-B83]). A discrete Gamma distribution was used to model evolutionary rate differences among sites. The outcome of the bootstrap analysis ([@vew004-B24]) is shown next to the nodes and branch lengths are proportional to genetic distances as indicated by the scale bars. Trees are based on genomic regions between nucleotides 1--903 (NS1_1), 904--1926 (NS1_2) of the NS1 ORF and between nucleotides 1--1875 (VP2_total), 1--732 (VP2_1), 733--1320 (VP2_2), and 1321--1875 (VP2_3) of the VP2 ORF; all positions refer to the AMDV-G sequence. Viruses are highlighted with the same color throughout all trees.
When the two Newfoundland strains M228 and M195, which consistently segregated into different clusters in these three trees, were used as reference we could identify five different patterns but we did not observe an associated geographical distribution to go along with these patterns, as, for example, sequences identified in Newfoundland and China or in Germany and the USA showed the same genome composition ([Supplementary Fig. S5](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)).
3.6 Overall and site-by-site selection pressure evaluation
----------------------------------------------------------
The non-synonymous/synonymous substitution rate ratios can be overestimated when recombination occurs, leading to the identification of sites falsely recognized as positively selected ([@vew004-B80]). Therefore, each of the five sub-alignments of sequences between breakpoints (the same used for phylogenetic analyses in [Fig. 7](#vew004-F7){ref-type="fig"}) was examined separately. The *Z*-test allowed us to reject the null hypothesis of strict neutral selection in favor of an alternative hypothesis of d*N* \< d*S* in almost all cases. Two exceptions were the clades containing UtahI, SL-3, G, and M195 in NS1_1 and WM25 and M195 in VP2_2, for which the null hypothesis of neutrality could not be rejected. This showed the predominance of negative selection on the analyzed genomic sub-regions of both the entire dataset and separate clades. Mean values for d*N*/d*S* calculated with SLAC ranged between 0.51 and 0.62 for the NS1 ORF and between 0.24 and 0.33 for the VP2 ORF.
Six different methods were used to identify positively and negatively selected sites in both ORFs, and only sites identified by at least three different methods were accepted. In our system, considering the capability of each method to identify accepted sites under selection without overestimating the number of selected sites, FEL was the best to identify positively selected sites, whereas FUBAR was the best to identify sites under negative selection ([Supplementary Table S4](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). Complete lists of all sites identified to be under selection by all methods are available in [Supplementary Tables S5](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1), [S6](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1), [S7](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1), and [S8](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1).
Nine sites under positive selection and forty under negative selection were identified in NS1 ([Supplementary Table S5](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)), while four sites under positive and thirty-four under negative selection pressure were observed in VP2 ([Supplementary Table S6](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)). This represented a similar percentage of sites under purifying selection in both proteins (6.2% and 5.4% for NS1 and VP2, respectively). Only 0.6 per cent of sites in the capsid protein were under diversifying selection, while 1.4 per cent of aa in the non-structural protein were under positive selection. Six (15%) of the sites under purifying selection identified in NS1 were located in the helicase domain, five of which (sites 432, 433, 434, 438, and 439) were localized in the ATP-binding loop ([@vew004-B85]) as illustrated in [@vew004-B10]. Interestingly, MEME predicted eight additional sites to be under episodic diversifying selection, and one of these was also localized in the helicase domain, specifically in the Walker B domain, and involved the cluster formed by three Newfoundland strains (WM25, M173, and M228) in the NS1_2 tree ([Fig. 7](#vew004-F7){ref-type="fig"}). This domain is usually composed of a stretch of four hydrophobic residues, followed by two polar residues (hhhh(D/E)E). The alanine (codon GCT) present at the position of the fourth hydrophobic residue in 80 per cent of the analyzed sequences was replaced by a cysteine (TGT) in M228 and by the polar residue threonine (ACT) in WM25 and represent changes that could have functional consequences for the domain.
Three of the four identified positively selected sites in VP2 ([Supplementary Table S6](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)) were localized in immunogenic loops (one in loop 1: 83; one in loop 2: 227; one in loop 4: 439), and 17.6 per cent (6/34) of sites under negative selection were also localized in immunogenic loops (two sites in loop 1: 80 and 93; two sites in loop 2: 239 and 240; two sites in loop 4: 452 and 481) ([@vew004-B54]). An episodically positively selected residue was identified by MEME and this was located in antigenic loop 3 (residue 308).
Finally, we observed no differences between d*N* and d*S* rates when performing the *Z*-test on the NS2 and NS3 regions after the splicing site (where a frameshift causes the terminal portion of NS2 and NS3 to be different from NS1). The average mean values for d*N*/d*S* calculated with SLAC for these sequences were 1.3 and 3.6, respectively. A site-by-site exploration identified three positively selected sites in the NS2 unique region ([Supplementary Table S7](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)), and no sites under selection were identified in the NS3 unique region ([Supplementary Table S8](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)).
4. Discussion
=============
AMDV is one of the most important pathogens of mink and other mustelids worldwide. It has been recognized for over 50 years as a major problem for farms, where it is often endemic. There is no available vaccine, and affected farms have to implement laborious eradication programs to establish an AMDV-free status ([@vew004-B12]), that is, however, hard to achieve and to maintain ([@vew004-B11]), especially because of the environmental stability of the virus. The virus causes a persistent infection that leads to a slowly progressive wasting syndrome ([@vew004-B69]), which is associated with high mortality, abortion, reduced reproduction rates and litter sizes in farms ([@vew004-B9]). Furthermore, the virus also represents a severe risk for wild animals and poses a serious threat to endangered species ([@vew004-B50]; [@vew004-B26]). Farms represent one source of viruses for wild animals because accidental, or sometimes deliberate, mink release from farms is not a rare event ([@vew004-B62]; [@vew004-B60]). It is therefore important to study the diversity and the molecular epidemiology of this virus to comprehend its evolution and transmission dynamics if we want to be able to contain the disease and prevent further spread.
4.1 Global epidemiology of AMDV
-------------------------------
During this study, we determined 97 AMDV sequences from farmed and wild animals from the Island of Newfoundland to study the molecular epidemiology of AMDV in this region and in a global perspective. Although there was evidence for AMDV in Newfoundland since at least the 1960s--70 s (H.G. Whitney, unpublished data), two studies documented the absence of AMDV from farms during the early 2000s. In 2001, 20 per cent of the farmed mink population of the province was tested and in 2004--5 approximately 50 per cent of the farmed mink population representing all farms in the province was screened again and only one positive animal was found in a small herd (thirty-five animals), which was entirely depopulated ([@vew004-B8]). These studies also showed that AMDV was already circulating in wild mink before the farm epidemic started in 2007. Over a period of 11 years (2004, 2007--2009, and 2014), two of the three main well-characterized AMDV clades ([@vew004-B11]) were identified on the Island of Newfoundland, with viruses belonging to only one of the two clades found in the earliest samples. This suggests that the two variants may have been introduced during at least two separate events, although viruses from the second clade might have gone undetected in the earlier time point, and it is also possible that new introductions keep occurring. The oldest record of mink farming in Newfoundland is from 1934, when mink where imported from Nova Scotia for the establishment of several farms. However, the number of farms started to decline during the 1950s and the last farm pelted out in 1971 ([@vew004-B62]). Mink were then reintroduced for farming purposes in recent years, and mink farming has experienced rapid growth on the Island with farmers actively renovating their farms and importing new animals for stocking. Although it is theoretically possible that AMDV was introduced during the first farming wave, which was also the origin of the local wild mink population, the viruses identified in this study in farms seem to be from a more recent introduction. This is supported by the high genetic similarity of viruses from Newfoundland to viruses circulating in 2007--9 in Wisconsin and Ontario, although we cannot exclude, however unlikely, that viruses moved in those years from Newfoundland to other sites in North America. It is, however, uncertain whether the epidemic in farms originated from a new introduction from outside the island or from viruses already circulating within the wild mink population, because viruses similar to those identified during the epidemic were already present in wild animals. From 2008, the year following the beginning of the outbreak, viruses from a second clade started circulating in farms and these more likely represent a new introduction because we have no evidence for these strains in the wild.
We also obtained sequences from viruses from other locations in North America (Nova Scotia, Ontario, and Wisconsin) and from Europe (Denmark). Among these sequences, we identified several viral clades that are phylogenetically close to a third AMDV clade but for which a complete genome sequence is currently unavailable. These variants were only identified among sequences from Ontario, Wisconsin, and Denmark, and similar clades have been reported previously by others in samples collected in Ontario, Denmark, Finland, Sweden, and Estonia ([@vew004-B65]; [@vew004-B38]; [@vew004-B61]; [@vew004-B42]). The absence of these variants from Newfoundland and Nova Scotia suggests a lower viral diversity in these areas compared to other parts of the world.
Phylogeographic analyses performed on four different genomic regions, involving both original sequences from this study and others from the public databases, and representing viruses circulating all over the world showed the existence of a partial but marked geographic distribution pattern for some strains. Although some viruses from different countries and continents are within the same clades, sequences tended to form independent country-specific subclades. These observations suggest that viruses are actively exchanged between different countries, presumably by trading of infected mink or contaminated equipment, but also that viruses, once reaching a new location, evolve rapidly within that population and produce independent lineages.
Approximately half of the sequences from Newfoundland wild mink were very close to those identified in farmed animals, suggesting a continuing exchange of viruses between wild and farmed populations, as reported in other locations ([@vew004-B60], [@vew004-B61]; [@vew004-B42]). Although viral diversity was higher in farms, we identified a cluster of viruses unique to wild animals and this included the majority of strains circulating in the wild before the epidemic started in farms. However, we cannot exclude the possibility that higher viral diversity remains undetected in farms or in the wild. These observations might indicate that farms represent a source of viruses for wild populations but also that the virus can transmit among wild animals, where it evolves independently, constituting a wild reservoir for farms. The movement of viruses between farms and the wild is also demonstrated by the increase in prevalence of AMDV in the wild around the time the epidemic started in farms (from 14% observed in 2004 to 45% in 2007--8). Furthermore, many AMDV-positive animals were trapped in areas surrounding affected farms, especially in the central part of the Avalon peninsula, where most of the samples collected in 2004 were negative (data not shown). The absence of detection of AMDV in Newfoundland in wild carnivores other than mink suggests that the virus has not transmitted among wild animals in this location as extensively as in other countries ([@vew004-B50]; [@vew004-B26]; [@vew004-B20]). However, the number of animals tested was small and these results are therefore preliminary.
4.2 Evolution of AMDV
---------------------
A second objective of this work was to study the evolutionary dynamics of AMDV, and for this purpose, the genetic variability of viruses within a single farm was analyzed. Intensive farming provides a unique set of conditions that are considerably different from those of a natural environment that favor pathogen diffusion. Pathogen transmission is easier when the local host density is high and a fast turnover of individuals provides viruses the constant presence of a naïve population. In the case of AMDV, its exceptional environmental stability is also likely to favor an increase in *R*~0~ in farmed settings. These conditions facilitate viral spread and overall viral replication is therefore increased by the efficient host-to-host transmission, which are factors that correlate with faster viral evolution and higher diversity ([@vew004-B19]; [@vew004-B55]). Additionally, the continuous movement of animals between affected farms causes the continuous (re)introduction of viruses, which will be co-existing with previously circulating viruses whose evolution has already been shaped by a long period of sustained transmission within the farm, further amplifying viral richness. Our data are consistent with these hypotheses because we observed a very high viral genetic diversity within one farm. This was observed not only at the population level but also within single animals. However, these data need to be validated by the opposite observations in the wild to confirm that frequent co- and super-infections in chronically infected hosts are more frequently observed in high-density host populations.
The evolutionary dynamics of AMDV are further complicated by qualities intrinsic to this virus. The first is the ability of the virus to establish persistent infections, and the second is its exceptionally high environmental resistance ([@vew004-B11]). These two factors favor the occurrence of co-infections that create the condition necessary for viral recombination to take place, a mechanism that serves as positive feedback and further enhances viral diversity. We found the presence of multiple infections by two or three different strains in \>40 per cent of the animals in 2014. Additionally, we identified various recombinant strains and the presence of multiple polymorphic sites in viruses within the same individuals, resulting from point mutations that occurred during chronic infections. This exceptionally high, and possibly continuously increasing, within-host richness is a distinctive feature potentially associated with the increased chance of emergence of new characteristics, for example an extended host-tropism, in evolving pathogens ([@vew004-B76]; [@vew004-B17]). These might pose a threat not only for farmed animals but also for other species should these viruses escape from the farm.
After acquiring the complete genome sequences of several Newfoundland strains and comparing them to reference sequences, we observed that chimeric recombinant AMDV sequences appear to be recurrent. Parvoviruses are particularly recombination-prone because of their genome replication mechanism, which involves the continuous rearrangement of double-stranded intermediate DNA replication forms ([@vew004-B14]), and the presence of recombination within this viral family has been frequently observed ([@vew004-B77]; [@vew004-B63]; [@vew004-B84]). Recombination might be additionally favored in the case of AMDV because of the high rate of co-infection in farms, and its role in viral evolution might be significant in these circumstances. However, we obtained clonal sequences from only one wild mink and our knowledge about AMDV evolutionary dynamics would benefit greatly from more sequences from viruses circulating in wild animals. It would also be very interesting to determine whether similarly high co-infection rates occur and if recombination takes place in farms where other amdoparvoviruses circulate.
Analyses of the direction of selective forces acting on AMDV identified a marked predominance of negative selection pressure on both structural and non-structural proteins. Surprisingly, a site-by-site analysis revealed a higher percentage of positively selected codons within the NS1 protein compared to the capsid protein VP2 (1.4% vs. 0.6%) and we could identify only four residues located in the immunodominant epitopes that were subjected to positive selection pressure. Our data are in agreement with previous literature because the predominance of purifying selection acting on non-structural proteins of parvoviruses has been previously reported ([@vew004-B67]), and the importance of negative selection in shaping the evolution of both ORFs has been documented for different parvoviruses ([@vew004-B49]). When recombination occurs, forces acting on one site can become independent from the action at another site and it is known that recombination can speed up the fixation of beneficial mutations ([@vew004-B22]). Therefore, although we analyzed each genomic area between breakpoints separately, potential mistakes in our site-by-site analysis can be expected because the entire spectrum of AMDV diversity is currently unknown, and therefore, some recombination will not have been documented. However, the low level of diversifying selection acting on the structural proteins is further confirmed by our observation that the degree of genetic variability is much higher in NS1. A possible explanation for this phenomenon can be identified in the context of the pathogenic mechanism of AMDV. The primary replication site for AMDV is in circulating macrophages and viral entry is mediated by cellular Fc receptors recognizing antibody-coated viral particles. Since antibodies enhance viral entry into cells, a phenomenon called antibody-dependent enhancement ([@vew004-B40]), the host immune response contributes to the disease process by being fundamental for viral replication and in these circumstances escaping the immune response might not be beneficial for the virus. The lack of diversifying pressure at the level of the immunoepitopes can also be partially explained by the continuous availability of naïve individuals in farms, as previously reported for the canine parvovirus ([@vew004-B67]). However, the first hypothesis is more compatible with the tendency of AMDV to establish persistent infections.
During our study, we mainly analyzed viruses from farmed animals and it appears many of the wild animals analyzed may have acquired the infection from an original farm source. Therefore, the evolutionary dynamics of AMDV in a more natural environment might be different compared to what we observed. Future studies should focus their attention on viruses identified in areas where mink farms are absent to elucidate how selection pressure shapes the evolution of AMDV in such natural environments. Finally, the acquisition of additional sequence information, especially the complete coding sequences of those variants for which only partial information is currently available, is a priority to obtain a full understanding of AMDV epidemiology, diffusion, and evolution.
In conclusion, the high prevalence of AMDV observed in farms can be explained both by virological factors, such as the ability to establish persistent infections and the high stability of viral particles, and environmental conditions, such as high host density. These conditions facilitate the establishment of co-infections that favor the occurrence of recombination, which enhances the extant AMDV diversity. These viruses can be transmitted to wild animals and exchanged between different farms and countries, where rapidly evolving viruses give rise to many novel parallel lineages.
Data Availability
=================
All sequences obtained in this study were deposited in GenBank under accession numbers GQ895066--GQ895128, GQ981388--GQ981397, and KT878893--KT878968 (strain details are available in the [Supplementary Sequence Details file](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1)).
Supplementary Material
======================
###### Supplementary Table S1
This paper is dedicated to the memory of Bruce Hunter, who passed away suddenly on 19 October 2011. He was an exemplary scientist and friend to many. The authors thank John Reynolds and Dr. Edward Miller for providing ermine samples, Angela Blundon, Joanne Walsh, Hannah Munro, and Jennifer O'Reilly for help with spleen processing, and farmers and trappers from Newfoundland for their generous contribution of samples. This research was supported by the Natural Sciences and Engineering Research Council of Canada (EGP 469208--14), the Newfoundland and Labrador Provincial Agricultural Research and Development Program, and the Newfoundland and Labrador Forestry and Agrifoods Agency.
Supplementary data
==================
[Supplementary data](http://ve.oxfordjournals.org/lookup/suppl/doi:10.1093/ve/vew004/-/DC1) are available at *Virus Evolution* online.
*Conflict of interest*: None declared.
[^1]: ^†^<http://orcid.org/0000-0002-9959-128X>
[^2]: ^‡^Deceased.
|
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"pile_set_name": "PubMed Central"
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|
Introduction
============
Critical illness and intensive care have a profound and traumatic impact on the health and well-being of patients and their loved ones \[[@B1]-[@B3]\]. Previous reports suggest that many patients in the ICU are separated from their families and loved ones by widespread restrictive visitation policies that can negatively impact care and recovery \[[@B3]-[@B6]\]. However, limited data exist about the scope and variability of ICU visitation policies and practices across the United States as well as the hospital factors that influence them \[[@B7]-[@B10]\]. In this study, we conducted a survey of US ICUs to describe the current landscape of ICU visitation policies. We further aimed to evaluate whether key hospital characteristics were associated with visitation restrictions.
Materials and methods
=====================
Based on the American Hospital Association 2008 Hospital Survey Database, we grouped hospitals as either university-affiliated hospitals, federal government (Veterans Health Administration) hospitals, or nonfederal and nonuniversity community hospitals. We aimed to survey all university and government hospitals with an ICU. We then stratified community hospitals (which make up the majority of US hospitals) based on their location in US regions (Midwest, Northeast, South, and West) and aimed to survey an equal percentage of eligible hospitals (25%) within each region to achieve a total of 670 surveyed hospitals (of an estimated 3,228 ICUs in the United States) \[[@B11]\].
For each hospital, we contacted the ICU leadership, if available, or ICU nursing staff familiar with visitation policies to conduct the telephone survey from 2008 to 2009. If a hospital\'s ICU personnel could not be identified or declined survey participation, the next hospital in the randomly generated sample by strata was surveyed. The 17-question survey ascertained hospital characteristics including each hospital\'s self-reported number of beds (total and ICU) and critical care units; if numbers were reported as a range (for example, 25 to 30 beds), we selected the mean value (28 beds). We calculated the percentage of critical care beds within each hospital (ICU bed percentage). We also ascertained the presence or absence of ICU leadership (medical director or clinical nurse specialist). Clinical nurse specialists typically have received training at the level of a master\'s degree and often take a lead role in staff education, protocol development, and standardizing nursing care based on current evidence.
We assessed visitation policies based on whether the hospital or ICU placed restrictions based on a total of five criteria: visiting hours; visit duration; number of visitors; age of visitors; and membership in the patient\'s immediate family. We designated hospitals with zero restrictions as having open visitation policies and those with any restriction as having a restricted policy. We also determined whether exceptions to the visitation policies were allowed within the ICU.
We described data as the number (frequency) and mean ± standard deviation. We used Spearman\'s correlation coefficient to assess the intra-hospital correlation between the number of hospital and ICU visitation restrictions. To determine the association between hospitals\' ICU visitation policies and characteristics, we included key hospital characteristics as predictor variables in univariable and multivariable linear regression where the number of ICU restrictions was the outcome variable. Analyses were conducted using Stata/SE 11.2 (StataCorp. LP, College Station, TX, USA).
Results
=======
Hospital characteristics
------------------------
We contacted 695 hospitals; 87.2% (*n*= 606) completed the survey. Hospitals were located in 50 states and the District of Columbia. More than one-third were from the South (*n*= 222, 36.6%) and most were community hospitals (*n*= 401, 66.2%; Table [1](#T1){ref-type="table"}). The mean hospital bed size was 239 ± 217 (median, 159). The mean ICU bed percentage was 11.6 ± 13.4%; the mean number of ICUs per hospital was 2.1 ± 1.8. A total of 62.2% of ICUs had a medical director and 39.0% had a clinical nurse specialist.
######
Survey hospital characteristics
Hospital region
--------------------------------------- ----------------- ------------- ------------ -------------
Number 103 (17.0) 159 (26.2) 222 (36.6) 122 (20.1)
Hospital type
Community 55 (53.4) 102 (64.2) 154 (69.4) 90 (73.8)
Federal 26 (25.2) 31 (19.5) 40 (18.0) 18 (14.8)
University 22 (21.4) 26 (16.4) 28 (12.6) 14 (11.5)
Hospital bed number
\< 100 16 (15.5) 56 (35.2) 55 (24.8) 39 (32.0)
100 to 299 45 (43.7) 58 (36.5) 101 (45.5) 53 (43.4)
300 to 499 10 (9.7) 26 (16.4) 34 (15.3) 19 (15.6)
\> 500 22 (21.4) 16 (10.1) 28 (12.6) 9 (7.4)
Not reported 10 (9.7) 3 (1.9) 4 (1.8) 2 (1.6)
ICU number 2.4 ± 2.1 2.0 ± 1.8 2.0 ± 1.6 2.0 ± 1.8
ICU bed number
\< 10 16 (15.5) 56 (35.2) 44 (19.8) 26 (21.3)
10 to 15 34 (33.0) 35 (22.0) 88 (39.6) 32 (26.2)
16 to 39 24 (23.3) 40 (25.2) 59 (26.6) 41 (33.6)
≥ 40 27 (26.2) 27 (17.0) 29 (13.1) 23 (18.9)
Not reported 2 (1.9) 1 (0.6) 2 (0.9) 0
ICU bed percentage 11.0 ± 9.9 12.0 ± 20.9 9.9 ± 7.1 15.2 ± 17.6
ICU clinical nurse specialist present 42 (40.8) 63 (39.9) 80 (36.0) 51 (41.8)
ICU medical director present 75 (73.5) 92 (58.6) 121 (54.8) 86 (71.1)
Data presented as number (percentage) or mean ± standard deviation.
The majority of hospitals had restrictive hospital (*n*= 463, 76.4%) and ICU (*n*= 543, 89.6% visitation policies (Table [2](#T2){ref-type="table"}). The mean numbers of restrictions were 1.4 ± 1.2 and 2.8 ± 1.5, respectively. Most ICUs had three or more restrictions (*n*= 375, 61.9%). The most common restrictions were related to visiting hours, followed by visitor number and age. Exceptions to the visitation policies were permitted in 94.8% of the ICUs. Within facilities, the correlation between the number of hospital and ICU visitation restrictions was moderate (correlation coefficient, 0.39). Figure [1](#F1){ref-type="fig"} shows the distribution of ICU visitation restrictions based on hospital bed size and ICU bed percentage. Hospitals with fewer than 150 beds more frequently had open ICU visitation policies when compared with larger hospitals (16.8% vs. 5.1%; χ^2^*P*\< 0.001).
######
Survey hospital visiting hour policies, by hospital location
Hospital location
--------------------------------------- ------------------- ------------
Any restriction present 463 (76.4) 543 (89.6)
Number of restrictions (maximum of 5) 1.4 ± 1.2 2.8 ± 1.5
Restrictions present
Visiting hours 448 (75.2) 487 (80.4)
Visit duration 42 (7.1) 239 (39.4)
Visitor number 134 (22.5) 408 (67.3)
Immediate family members 23 (3.9) 147 (24.3)
Based on visitor age 160 (26.9) 387 (63.9)
Allow exceptions to policy \- 474 (94.8)
Visiting hours \- 410 (82.8)
Visit duration \- 222 (44.9)
Visitor number \- 372 (75.2)
Immediate family members \- 132 (26.7)
Based on visitor age \- 332 (66.8)
Data presented as number (percentage) or mean ± standard deviation.
{#F1}
However, hospital bed size was not significantly associated with the number of ICU restrictions (Table [3](#T3){ref-type="table"}); neither were hospital type, number of critical care units, or the presence of ICU leadership. Only the US region and ICU bed percentage were statistically significant in linear regression analysis; however, the effect size associated with ICU bed percentage was small (-0.03 for each percentage increase; 95% confidence interval, -0.05 to -0.002; *P*= 0.03). On average, hospitals in the Midwest had the least restrictive policies while those in the Northeast had the most restrictive.
######
Variables associated with number of ICU visiting policy restrictions in univariable/multivariable linear regression models
Point estimate from linear regression
----------------------------------- --------------------------------------- ---------------------------
Hospital size, per 100 beds 0.04 (-0.02 to 0.10) 0.01 (-0.10 to 0.11)
ICU bed percentage, per % **-0.02 (-0.04 to -0.01)** **-0.03 (-0.05 to 0.00)**
ICU number, per ICU 0.01 (-0.07 to 0.08) -0.04 (-0.15 to 0.08)
Region
Midwest (reference) \- \-
Northeast **0.91 (0.52 to 1.30)** **0.83 (0.43 to 1.23)**
South **0.84 (0.53 to 1.15)** **0.85 (0.53 to 1.17)**
West **0.53 (0.17 to 0.90)** **0.54 (0.18 to 0.91)**
Hospital type
Community (reference) \- \-
Government 0.13 (-0.20 to 0.46) 0.16 (-0.17to 0.49)
University 0.08 (-0.29 to 0.46) 0.25 (-0.29 to 0.79)
ICU medical director present 0.18 (-0.08 to 0.44) 0.19 (-0.08 to 0.47)
Clinical nurse specialist present 0.06 (-0.20 to 0.32) 0.08 (-0.19 to 0.36)
Data in parentheses are 95% confidence interval. Bold data are statistically significant, *P*\< 0.05. Multivariable linear regression model adjusted for all displayed variables.
Discussion
==========
In this survey of US hospitals, we found that their overwhelming default policy was to restrict ICU visitation. Among ICUs with restrictive policies, there was a high degree of variability in the number of restrictions and no significant association with hospital size or type, number of critical care wards, or leadership roles. The ICU policies were only moderately correlated with hospital-wide policies. In practice, however, nearly all ICUs allowed some exceptions to their visitation restrictions. Only a fraction of ICUs had open visitation policies and these were more common among small hospitals.
Critically ill patients often suffer from life-threatening disease and multisystem organ failure \[[@B12]\]. As a result, the modern ICU has evolved into a highly specialized unit designed to facilitate rapid diagnosis, continuous monitoring, and prompt delivery of multidisciplinary, multimodal, and technologically advanced therapies \[[@B13]\]. The results have been extraordinary, with patient survival steadily improving over time \[[@B14]-[@B16]\]. Because of the complexity of ICU care, prior small studies have raised concerns that open ICU visitation policies could harm patients by increasing physiologic stress, interfering with timely and safe care delivery, infringing on patient privacy, increasing exposure to infection, leading to caregiver exhaustion, and negatively impacting interactions with families \[[@B4],[@B17]-[@B26]\].
Given these concerns, our finding that the majority of ICUs had restrictive and highly variable policies is not surprising. Prior studies have found similar results in US ICUs and international ICUs \[[@B6]-[@B10],[@B21],[@B26]-[@B28]\]. A survey among 171 hospitals in New England found that 32% had unrestricted visiting hours; however, most had restrictions on the age and number of visitors allowed \[[@B10]\]. Another survey of US ICUs, conducted by the American Association of Critical Care Nurses, also found high degrees of variability in visitation practice \[[@B9]\]. Giannini and colleagues reported that only a single Italian ICU, out of 257 surveyed, allowed open visitation \[[@B8]\]. No Belgian ICU, in a study by Spreen and Schuurmans, had an open visitation policy - defined as no restrictions on visiting hours, visit duration, and/or number of visitors \[[@B28]\]. Hunter and colleagues reported that 80% of ICUs in the United Kingdom imposed restricted visiting policies; they also noted wide variations in specific practices \[[@B27]\].
However, while historical practice among ICUs appears to have been to restrict visitation and we have seen concurrent substantial improvements in short-term mortality, new challenges in critical care have arisen. Survivors of critical illness and intensive care can suffer from post-intensive care syndrome - a condition whose sequelae include post-traumatic stress disorder as well as long-term physical and neurocognitive disability \[[@B1],[@B2],[@B29]\]. Furthermore, critical illness not only afflicts ICU patients, it also impacts patients\' families \[[@B3],[@B30]-[@B33]\]. Family members often struggle with decisions about their loved ones\' end-of-life care and can, themselves, experience depression, anxiety, and post-traumatic stress disorder \[[@B3],[@B30]-[@B35]\]. Visitation restrictions can thus further contribute to patients\' and families\' experiences of ICUs as disorienting places that enforce separation during challenging periods of critical illness and recovery \[[@B3],[@B34]-[@B36]\].
As a result, numerous stakeholders and healthcare leaders have recommended liberalizing ICU visitation; Berwick and Kotagal declared restricted visiting practices as \'neither caring, compassionate, nor necessary\' \[[@B3],[@B5],[@B37]\]. In 2010, US President Barack Obama also called on hospitals to foster open visitation policies \[[@B38]\]. Recent data suggest that open visitation policies do not adversely impact patient outcomes and represent only a moderate, and acceptable, intrusion on patient care \[[@B3],[@B39]-[@B45]\]. Furthermore, family-centered care in the ICU is associated with improvements in the long-term psychiatric sequelae of critical illness, the trust between hospital staff and family members, and overall satisfaction with medical care \[[@B2],[@B3],[@B35]\]. Several studies also demonstrate the promise of interventions designed to provide families with a guided tour through the complexities of critical illness and to teach them how they can safely participate in ICU care \[[@B32],[@B46]-[@B48]\].
Despite these reported benefits, we found that few ICUs had open visitation policies and they were more commonly located in small hospitals. Where ICUs had restrictive policies, we found wide variability in practice. Besides broad regional differences in ICU policies, other hospital characteristics were not strongly associated with the number of visitation restrictions. Instead, policies appeared to be distributed among hospitals without a clear pattern and probably reflect historical practices rather than evidence-driven decision-making \[[@B4]\]. Recent studies suggest that ICUs are actively rethinking their visitation policies to move towards more liberal visitation policies - a shift in the United States that has been supported by healthcare accreditation and regulatory agencies including the Joint Commission and the Center for Medicare & Medicaid Services among others \[[@B4],[@B8],[@B49]\].
Our findings should be interpreted in light of the study\'s limitations. First, the survey was conducted in 2008 and 2009. Given the increasing public awareness and unified sentiment that appear to favor open visitation policies since that time, a contemporary assessment of ICUs is urgently needed to determine whether policies have changed and what factors impact such changes. Second, while we sampled a large number of ICUs with high response rates, this survey represents the practices of fewer than 25% of US ICUs. Finally, additional factors that could influence ICU visitation policy (for example, the physical size of each ICU room, the presence of waiting rooms, the duration of visit times allowed) were not evaluated in this study and may offer additional insight into understanding the wide observed variability in practice across centers.
Conclusion
==========
The overwhelming majority of US ICUs in this study had restrictive visitation policies. While there were regional differences in ICU policies and open policies were common in smaller hospitals, other hospital characteristics were not strongly associated with the number of visitation restrictions. Wide variability in visitation policies suggests that further study into the impact of ICU visitations on patients and families are likely to influence and improve future practice.
Key messages
============
• The majority of ICUs in the United States had restrictive visitation policies based on survey results from over 600 hospitals between 2008 and 2009.
• Hospitals in the Midwest region had the most liberal policies while smaller hospitals more frequently had open visitation policies.
• Hospital characteristics - including bed size, number of critical care units, presence of ICU leadership, and hospital type - were not associated with the number of visitation restrictions
• There was wide variability in ICU visitation policies and practices across a broad range of surveyed hospitals.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
VL had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis - he participated in the conduct of the study; analysis and interpretation of the data; and preparation, review, and approval of the manuscript. JLR participated in the design and conduct of the study; collection, management, and interpretation of the data; and preparation, review, and approval of the manuscript. ES participated in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, and approval of the manuscript. EC participated in the design and conduct of the study; collection, management, and interpretation of the data; and preparation, review, and approval of the manuscript. All authors read and approved the final manuscript for publication.
Acknowledgements
================
This work was funded by the Kaiser Foundation Research Institute and The Permanente Medical Group, Inc. The sponsors of the study had no role in the design and conduct of the study; in the collection, management, analysis, and interpretation of the data; and in the preparation, review, or approval of the manuscript. Portions of this work were presented at the Society of Critical Care Medicine Congress in 2010. Carly Skeath, Elena Corona, and Colleen Connell participated in conducting the telephone surveys; and Lue-Yen Tucker participated in the hospital survey sampling design.
|
{
"pile_set_name": "PubMed Central"
}
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\[The copyright line of this article was changed on 18 April 2019 after original online publication.\]
Introduction {#eji4228-sec-0010}
============
Sjögren\'s syndrome (SS) is a complex systemic autoimmune disease characterized by lymphocytic infiltrates of exocrine glands, mainly the salivary and lacrimal glands, leading to dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca) [1](#eji4228-bib-0001){ref-type="ref"}. Hallmarks of SS include the presence of autoantibodies against anti‐Sjögren\'s syndrome A (SSA) and anti‐Sjögren\'s syndrome B (SSB) [2](#eji4228-bib-0002){ref-type="ref"} which may be present in serum decades before clinical disease manifestations [3](#eji4228-bib-0003){ref-type="ref"}, [4](#eji4228-bib-0004){ref-type="ref"}. There is no cure or effective treatment for SS, with management of the disease based on the relief of symptoms. Patients suffer from a significant decrease in quality of life. A lack of effective targeted treatments is linked to the complexity of the disease, with genetic predisposition, hormonal and environmental factors all contributing to disease etiology and pathogenesis. While most SS patients display reduced tear and saliva secretion [5](#eji4228-bib-0005){ref-type="ref"}, there is significant heterogeneity in other disease features, pathology and clinical course. Sjögren\'s syndrome can present a wide range of extraglandular manifestations (EGM) including fatigue, musculoskeletal involvement (arthralgias, myalgias), skin involvement (xerosis, purpura), pulmonary involvement (bronchiectasis, obstructive airway disease), involvement of the liver and kidneys, neuropathy and lymphoma [2](#eji4228-bib-0002){ref-type="ref"}. This heterogeneity has been postulated to reflect distinct patient subsets, driven by unique pathophysiological mechanisms [6](#eji4228-bib-0006){ref-type="ref"}.
Aspects of SS pathogenesis that have gained considerable attention during the past years are features associated with type I interferon (IFN). An activated type I IFN system known as the IFN signature plays an important role in several autoimmune diseases [7](#eji4228-bib-0007){ref-type="ref"}, [8](#eji4228-bib-0008){ref-type="ref"}. In addition, polymorphisms in the genes encoding the transcription factors STAT4 and IRF5, which play a role in type I IFN signalling, have been associated with SS [9](#eji4228-bib-0009){ref-type="ref"}, [10](#eji4228-bib-0010){ref-type="ref"}. These polymorphisms have been speculated to confer a susceptibility favoring a higher IFN response which may play a role in onset or perpetuation of the disease [11](#eji4228-bib-0011){ref-type="ref"}. Gene expression may in addition be altered through changes in responsiveness to a given stimulus. Previous studies of peripheral blood cells from SS patients have found alterations of basal phosphorylation levels of STAT3 and STAT5 [12](#eji4228-bib-0012){ref-type="ref"}, [13](#eji4228-bib-0013){ref-type="ref"}, as well as increased phosphorylation of STAT1 Y701 upon stimulation with IFN‐α, IFN‐γ and IL‐6 [14](#eji4228-bib-0014){ref-type="ref"}.
Since approximately 50% of SS patients have an activated type I IFN system [15](#eji4228-bib-0015){ref-type="ref"}, we here investigated signaling networks upon stimulation with IFN‐α2b. Single cell phospho‐specific flow cytometry (phosphoflow) was used to analyse the phosphorylation status of nine different intracellular phospho‐epitopes in peripheral blood cells from primary SS patients. Moreover, basal and IFN‐α induced phosphorylation of intracellular phospho‐epitopes were correlated to expression levels of IFN responsive genes. Increased phosphorylation of STAT1 Y701 was observed in B cells following stimulation with IFN‐α, which strongly correlated with type I IFN inducible gene expression in PBMC and presence of autoantibodies. In contrast, a negative association was found in T cells for STAT3 S727 with type I IFN inducible gene expression. Signalling pathways involving these transcription factors may be involved in the aberrant induction of type I IFN inducible genes in pSS patients who might benefit from therapies targeting these processes.
Results {#eji4228-sec-0020}
=======
Patient PBMCs display altered phosphorylation levels of proteins involved in signalling pathways {#eji4228-sec-0030}
------------------------------------------------------------------------------------------------
In order to further stratify pSS patients and reveal new treatment targets, we here analysed MAPK/ERK and JAK/STAT signalling networks in peripheral blood cells from female pSS patients and female age‐matched healthy donors in unstimulated and IFN‐α2b stimulated PBMCs. An overview of the results are given in supplementary tables S1 (T cells), S2 (B cells) and S3 (NK cells).
We first compared basal phosphorylation levels between pSS patients and controls. Significantly increased phosphorylation of several epitopes was seen in T and NK cells of pSS patients, while B cells showed no significant differences (Fig. [1](#eji4228-fig-0001){ref-type="fig"}A). Principal component analysis (PCA) separated pSS patients from healthy donors (Fig. [1](#eji4228-fig-0001){ref-type="fig"}C). PCA was further used to concurrently relate multiple basal phosphorylation states to various clinical parameters such as presence of SSA autoantibodies (Fig. [1](#eji4228-fig-0001){ref-type="fig"}D), extraglandular manifestations (EGM) (Fig. [1](#eji4228-fig-0001){ref-type="fig"}E) and effect of prescribed medication (DMARDs and corticosteroids; Fig. [1](#eji4228-fig-0001){ref-type="fig"}F) within the patient cohort. Spatial groupings indicated closer similarities within the pSS and healthy donor cohorts than between the groups. Including clinical parameters in the analysis, patients without autoantibodies against SSA grouped closer to the healthy controls (Fig. [1](#eji4228-fig-0001){ref-type="fig"}D), while patients with EGM (Fig. [1](#eji4228-fig-0001){ref-type="fig"}E) and patients using prescribed medication (Fig. [1](#eji4228-fig-0001){ref-type="fig"}F) grouped throughout the pSS cluster.
{#eji4228-fig-0001}
We next analysed phosphorylation states at different time points after stimulation with IFN‐α2b (Fig. [2](#eji4228-fig-0002){ref-type="fig"}). Reduced NF‐κB, P38, STAT4 Y693, STAT5 Y694 and STAT3 S727 phosphorylation was observed in cells from pSS patients. The phosphorylation profiles of IFN‐α2b stimulated T, NK and B cells were further investigated with PCA (Fig. [3](#eji4228-fig-0003){ref-type="fig"}). Phosphorylation levels after 15 minutes stimulation showed the strongest clustering of subgroups, while extended time course (˃15 min) gave no additional resolution (Supporting Information Fig. 1). We therefore focused all analyses on induced median fluorescence intensity (MdFI) at 15 min (MdFI^15min^ -- MdFI^basal^) after stimulation with IFN‐α2b (Fig. [3](#eji4228-fig-0003){ref-type="fig"}A--E). Principal component analysis showed a positive shift for pSS samples along PC1 and PC2 away from healthy donor samples (Fig. [3](#eji4228-fig-0003){ref-type="fig"}B). PC1 explained 68% of the variation with positive movement along PC1 influenced by changes in phosphorylation of STAT1 Y701 in T cells and to a lesser degree NK and B cells (Fig. [3](#eji4228-fig-0003){ref-type="fig"}A). SSA negative patients and patients prescribed DMARDs or corticosteroids were distributed closer to healthy donors than SSA positive patients and patients without medication (Fig. [3](#eji4228-fig-0003){ref-type="fig"}C and E). Moreover, patients without EGM had little spread along PC1 (Fig. [3](#eji4228-fig-0003){ref-type="fig"}D). Further comparisons of variables used in the final PCA were conducted by Mann‐Whitney U tests (Fig. [3](#eji4228-fig-0003){ref-type="fig"}F). Comparisons were analysed with and without exclusion of patients prescribed DMARDs or corticosteroids. No statistically significant differences were observed between medicated patients and those without medication. However, although not significant, a trend of increased induction of pSTAT3 S727 in T cells was found in patients prescribed DMARDs or corticosteroids compared to patients without medication, making the phosphorylation profile more similar to that of healthy donors. Induction of pSTAT1 Y701 was found to be stronger in B cells from patients with SSA autoantibodies than SSA negative patients. Compared to healthy donors, pSS patients displayed stronger induction of pSTAT1 Y701 in T‐, B‐ and NK cells.
{#eji4228-fig-0002}
{#eji4228-fig-0003}
Patients with an activated IFN system have different phosphorylation profiles upon IFN‐α stimulation {#eji4228-sec-0040}
----------------------------------------------------------------------------------------------------
To investigate whether the phosphorylation profile upon IFN‐α2b stimulation was altered in patients with an activated type I IFN system, we first determined mRNA levels of three type I IFN responsive genes (*MxA, OAS1, IFI44*) and 2 type II IFN responsive genes (*GBP1, GBP2*) in PBMCs from pSS patients (*n* = 19) and healthy donors (*n* = 14). The three type I IFN inducible genes were used to calculate IFN scores. The mean level and SD of each IFN inducible gene in the healthy control group were used to standardize expression levels of each gene for each study subject. The standardized expression levels were subsequently summed for each patient to calculate an IFN score. The threshold was set to 8.8 based on 3 × SD of healthy controls. A type I IFN signature was found in 63% of patients and 0% of controls (Fig. [4](#eji4228-fig-0004){ref-type="fig"}A). The IFN scores of patients prescribed DMARDs or corticosteroids did not differ significantly from those of patients without medication (Fig. [4](#eji4228-fig-0004){ref-type="fig"}B), but tended to be lower (median 12.10 versus 32.36).
{#eji4228-fig-0004}
Interestingly, the phosphorylation pattern upon in‐vitro stimulation with IFN‐α2b differed between the patient groups depending on IFN signature status (Supporting Information Table 4). Increased induction of STAT1 Y701 phosphorylation in B and NK cells was limited to type I IFN signature positive patients, while their T cells displayed reduced induction of STAT3 S727 phosphorylation (Fig. [4](#eji4228-fig-0004){ref-type="fig"}C). We further correlated expression levels of the individual IFN responsive genes to phosphorylation profiles of the pSS patients (Fig. [4](#eji4228-fig-0004){ref-type="fig"}D, Supporting Information Table 5). The observed induced phosphorylation of STAT1 Y701 in B cells correlated positively with increased expression of *MxA*, *OAS1* and *IFI44*. In contrast, no correlation with *GBP1* and *GBP2* expression was observed.
Discussion {#eji4228-sec-0050}
==========
Little is known about the effects of an activated type I IFN system on signalling profiles in primary Sjögren\'s syndrome. Understanding the mechanisms that contribute to these profiles are crucial in both, understanding the pathogenesis and the development of targeted therapies. We here investigated signalling profiles in PBMCs of pSS patients under basal conditions and upon stimulation with IFN‐α2b.
An increased response to IFN‐α through STAT1 Y701 was observed in cells from pSS patients. An increased sensitivity of STAT1 Y701 activating signals in immune cells may in part drive an up‐regulation of IFN induced genes. This is further supported by the association of increased response to IFN‐α2b with upregulated mRNA levels of type I IFN regulated genes and the production of SSA autoantibodies, which have been shown to be positively associated with the upregulation of IFN regulated genes [15](#eji4228-bib-0015){ref-type="ref"}, [16](#eji4228-bib-0016){ref-type="ref"}. Type I IFN could thereby potentially induce B cell autoantibody production through a number of mechanisms including lowering of BCR signalling thresholds, upregulate surface molecules that promote antigen presentation, promoting survival and differentiation and trafficking to germinal centres [17](#eji4228-bib-0017){ref-type="ref"}.
Potentiated phosphorylation of STAT1 Y701 in monocytes, B cells and CD4^+^ T cells from pSS patients has been described previously in response to IFN‐γ and/or IL‐6 [14](#eji4228-bib-0014){ref-type="ref"}. In addition to receptor expression, signal transduction pathways leading to phosphorylation of STAT are mediated through activation of Janus kinases and are negatively regulated by several mechanisms, including suppressor of cytokine signaling (SOCS) family members, ubiquitin carboxy‐terminal hydrolase 18 and various microRNA [18](#eji4228-bib-0018){ref-type="ref"}, [19](#eji4228-bib-0019){ref-type="ref"}. Cross regulation between STATs has also been observed, with negative regulation of STAT1 by STAT3 through competition for common docking sites [20](#eji4228-bib-0020){ref-type="ref"}. Previous studies have described upregulations of STAT1 and STAT3 mRNA in PBMCs from pSS patients [13](#eji4228-bib-0013){ref-type="ref"}, while no differences were found regarding STAT3 protein expression in CD3^+^ and CD19^+^ cells [12](#eji4228-bib-0012){ref-type="ref"}. Increased expression of SOCS1 and ‐3 mRNA have been found in PBMCs from pSS patients [14](#eji4228-bib-0014){ref-type="ref"}, and dysregulation of microRNA expression patterns have been recently observed in B and T cells from pSS patients [21](#eji4228-bib-0021){ref-type="ref"}.
Interestingly, we observed an NK cell specific reduction of STAT4 Y693 phosphorylation in pSS patients upon IFN‐α stimulation. NK cells have previously been shown to display high basal expression of STAT4 and reduced STAT1 compared to other cell subsets [22](#eji4228-bib-0022){ref-type="ref"}. This is thought to predispose the cells to STAT4 activation by type I IFN and IFN‐γ [22](#eji4228-bib-0022){ref-type="ref"}. Our study showed increased basal signalling in NK cells through STAT1 Y701 and STAT1 S727. Upon IFN‐α2b stimulation, pSTAT1 Y701 was greatly increased, while pSTAT4 Y693 was decreased. These results indicate that pSS patients express a NK cell phosphorylation profile represented by low relative phosphorylation ratio of STAT4 to STAT1 in response to IFN‐α, resembling those displayed by individuals with hepatitis C infections receiving IFN‐α therapy [23](#eji4228-bib-0023){ref-type="ref"}. Such a profile might polarize NK cells in pSS towards a low IFN‐γ producing phenotype and increased cell cytotoxicity [22](#eji4228-bib-0022){ref-type="ref"}, [23](#eji4228-bib-0023){ref-type="ref"}, [24](#eji4228-bib-0024){ref-type="ref"}, [25](#eji4228-bib-0025){ref-type="ref"}. However, no increase in NK cell killing ability has been shown on a per cell basis compared to healthy donors in pSS patients [26](#eji4228-bib-0026){ref-type="ref"}. Interestingly, NK cells from pSS patients have been shown to be hyporesponsive to IFN‐α induced cell cytotoxicity [27](#eji4228-bib-0027){ref-type="ref"}. Whether this aberrant profile is associated with polymorphisms in STAT4 commonly associated with pSS [10](#eji4228-bib-0010){ref-type="ref"} is unknown.
Further IFN‐α induced induction of STAT3 S727 phosphorylation in T cells negatively correlated with expression of type I IFN inducible genes. STAT3 is known to negatively regulate type I IFN induced gene expression. Studies with STAT3 knockout or knockdown mouse embryonic fibroblasts have indicated that STAT3 can negatively regulate type I IFN induced antiviral responses and ISRE‐driven genes [28](#eji4228-bib-0028){ref-type="ref"}. Further, overexpression of STAT3 in THP‐1 cells downregulates IFN‐α activated STAT1 dependent genes, and knocking down STAT3 leads to elevated expression of the same genes [20](#eji4228-bib-0020){ref-type="ref"}. Taken together our results indicate that STAT3 S727 responses may play a role in the expression of a type I IFN signature in pSS patients. Further, patients treated with the DMARD hydroxychloroquine (HCQ) or corticosteroids displayed stronger IFN‐α induced STAT3 S727 signalling in T cells, while HCQ had little effect on induction of STAT1 Y701 signalling. In addition, patients treated with HCQ tended to have a lower interferon score. This observation is in accordance with a previous study where patients taking HCQ showed significantly reduced type I IFN scores based on expression of *IFI44L, IFI44, IFIT3, LY6E* and *MxA* in monocytes compared to patients not on HCQ [29](#eji4228-bib-0029){ref-type="ref"}. However, whether the effect on STAT phosphorylation and IFN signature denotes a therapeutic response is unclear.
In T and NK cells of pSS patients, the basal phosphorylation levels were increased for several of the analysed epitopes, while the phosphorylation pattern of B cells was similar to controls. Our results differ from previous studies where B cells from pSS have exhibited increased basal phosphorylation of STAT3 Y705 [12](#eji4228-bib-0012){ref-type="ref"} and STAT5 Y694 [13](#eji4228-bib-0013){ref-type="ref"}, with no difference found for phosphorylation of STAT1 Y701 in T cells (CD4^+^ and CD4^−^) [13](#eji4228-bib-0013){ref-type="ref"}. These discrepancies are likely the result of methodological differences. For example, whole blood [13](#eji4228-bib-0013){ref-type="ref"} or freshly isolated PBMCs [12](#eji4228-bib-0012){ref-type="ref"} were utilized in the previous studies, while our procedure involved cryopreserved cells cultured for 6.5 h.
It should be noted that this study has a number of limitations. The number of patients included is limited, and the small sample size is further affected by the heterogeneity of the patients. Moreover, the analysis was limited to the three main subsets of lymphocytes (T, B and NK cells), and immunophenotyping studies have shown shifts in the relative frequency of cell subsets in the peripheral blood of pSS patients compared to healthy donors [30](#eji4228-bib-0030){ref-type="ref"}, [31](#eji4228-bib-0031){ref-type="ref"}, [32](#eji4228-bib-0032){ref-type="ref"}, [33](#eji4228-bib-0033){ref-type="ref"}. We therefore cannot exclude that the differences in the signalling profiles reflect these changes. The type I IFN signature was assessed in PBMCs, and assessment for each cell type would likely strengthen associations and be more informative in deriving origin of the signature. Further, associations between concentrations of proteins involved in signalling pathways and induction of phosphorylation of the protein would in addition be informative as to the mechanism behind the increased responses.
In conclusion, pSS patients show an increased response to IFN‐α through STAT1 Y701. An increased sensitivity to STAT1‐activating signals in immune cells of pSS patients may in part drive an up‐regulation of IFN induced genes and the production of SSA autoantibodies. However, the lack of an effect of HCQ on this pathway indicates a more complex relationship. Our results further suggest that the IFN signature may also in part be derived from reduced activation of STAT3 S727 which has been shown to inhibit type I IFN inducible gene expression.
Materials and methods {#eji4228-sec-0060}
=====================
Blood sampling {#eji4228-sec-0070}
--------------
Peripheral blood from 24 patients with pSS was collected in Lithium‐heparin tubes (BD diagnostics) at the Department of Rheumatology, Haukeland University Hospital, Bergen, Norway. Blood from 21 healthy age‐ and gender‐matched donors was collected at the blood bank at the Haukeland University Hospital in Bergen, Norway. All blood donors provided written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with lymphoprep^™^ (Axis‐Shield, Oslo, Norway), and cryopreserved as described previously [34](#eji4228-bib-0034){ref-type="ref"}. The cells were stored at −150°C for approximately 12 to 16 months.
All patients fulfilled the pSS American‐European Consensus group criteria (AECC) [35](#eji4228-bib-0035){ref-type="ref"} and displayed no additional autoimmune diseases or lymphoma. An overview of the cohort is shown in Table [1](#eji4228-tbl-0001){ref-type="table"}. The study was approved by the regional ethical committee (\#2009/686).
######
Characteristics of patients and controls used in the study
Cohort characteristics
------------------------------------------------------------------------ ------------- -------------
Females/males 24/0 21/0
Age, median (range) years 57 (33--73) 54 (42--73)
Clinical features
SSA antibodies (%) 18 (75)
SSB antibodies (%) 11 (46)
SSA and SSB antibodies (%) 11 (46)
ANA (%) 18 (75)
Positive Schirmer\'s (tear flow ˂5 mm/5 min) (%); n = 23 15 (65)
Focus score[†](#eji4228-tbl1-note-0001){ref-type="fn"} ≥ 1 (%); n = 14 11 (79)
ESR, high levels[‡](#eji4228-tbl1-note-0001){ref-type="fn"} 6 (25)
CRP high levels (≥5 mg/L) 3 (13)
Extraglandular manifestations (%) 14 (58)
Medication
DMARDs 6 (25)
Corticosteroids 2 (8)
Continuous data are expressed as median. Categorical data is expressed as frequency and percentage. ^†^Focus score indicates the number of inflammatory foci containing more than 50 mononuclear cells per 4 mm^2^ biopsy tissue; ^‡^Age and gender dependent. DMARDs, disease‐modifying anti‐rheumatic drugs; ANA, anti‐nuclear antibodies; ESR, erythrocyte sedimentation rate; CRP, C‐reactive protein
John Wiley & Sons, Ltd.
Routine laboratory assays {#eji4228-sec-0080}
-------------------------
Identification of anti‐Ro/SSA and anti‐La/SSB, other anti‐nuclear antibodies (ANA), erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP) and extraglandular manifestations (EGM) were obtained as part of routine clinical investigation at time of blood sampling. SSA, SSB and ANA were reported as either present or absent, while other serum and blood parameters were reported as continuous values. Extraglandular manifestations were defined as disease features outside surface exocrine glands.
Real‐time quantitative PCR {#eji4228-sec-0090}
--------------------------
Total RNA was isolated from PBMCs of 19 pSS patients and 14 healthy controls using RNeasy plus (Qiagen Nordic, Oslo, Norway). 300 ng RNA each were used in two cDNA reactions with RevertAid reverse transcriptase using Oligo(dT)~18~ and random nonamers, respectively. The cDNA was then pooled, diluted with 1.5 parts DNase and RNase free water (i.e., 40 μl cDNA + 60 μl water), and 5 μl were used in a real‐time PCR reaction using Taqman gene expression assays (Hs00895608_m1 (MxA); Hs00973637_m1 (OAS1); Hs00951349_m1 (IFI44); Hs00977005_m1 (GBP1); Hs00894837_m1 (GBP2); Hs03928990_g1 (18S rRNA); all Thermo Fisher Scientific, Waltham, USA). All real time PCR reactions were run in duplicates on a Light Cycler 480 (Roche Diagnostics, Oslo, Norway). 18S rRNA was used as reference gene, and relative expression levels were calculated as 2^−ΔCt^. The IFN score was calculated according to Feng et al. [36](#eji4228-bib-0036){ref-type="ref"} by standardizing expression levels using mean and SD of the healthy controls for the respective gene and using the following formula: $$\sum\limits_{i}^{3} = \frac{gene\mspace{6mu} i_{pSS} - mean\mspace{6mu} gene\mspace{6mu} i_{Ctr}}{SD\left( {gene\mspace{6mu} i_{Ctr}} \right)}$$where *i =* each of the 3 type I IFN‐inducible genes (*MxA, IFI44, OAS1*), *gene i~pSS~ =* the gene expression level in each pSS patient, and *gene i~Ctr~ =* the gene expression in controls. To set a threshold, 3 × SD of healthy controls was utilized.
Antibodies used for flow cytometry {#eji4228-sec-0100}
----------------------------------
The following phospho‐specific monoclonal antibodies were used in 3 different panels during the flow cytometry protocol as described previously [34](#eji4228-bib-0034){ref-type="ref"}: Alexa Fluor® 647 conjugated anti‐STAT4 (pY693, clone 38/p‐STAT4, panel 1), anti‐STAT1 (pS727, clone K51‐856, panel 2) and anti‐STAT3 (pS727, clone 49/p‐STAT3, panel 3); PerCP‐Cy^™^5.5 conjugated anti‐ERK1/2 (pT202/pY204, clone 20A, panel 1), anti‐STAT1 (pY701, clone 4a, panel 2) and anti‐STAT3 (pY705, clone 4/P‐STAT3, panel 3); and PE‐Cy^™^7 conjugated anti‐NF‐κB p65 (pS529, clone K10‐895.12.50, panel 1), anti‐p38 MAPK (pT180/pY182, clone 36/p38, panel 2) and anti‐STAT5 (pY694, clone 47/ STAT5(pY694), panel 3) (all from BD Biosciences, San Jose, CA, USA). Cell surface markers incorporated in the assays were BV786 conjugated anti‐CD3 (clone SK7, BD Horizon^TM^), Alexa Fluor® 488 conjugated anti‐CD20 (clone H1 (FB1), BD Biosciences) and PE conjugated anti‐CD56 (clone N901, Beckmann Coulter, CA, USA).
Cell culture and stimulation {#eji4228-sec-0110}
----------------------------
Before stimulation, cryopreserved PBMCs were rapidly thawed using a water bath set to 37°C and washed once in prewarmed (37°C) X‐vivo 20^™^ by centrifugation at 300 × *g* for 7 min. The PBMCs were then resuspended in prewarmed X‐vivo 20^™^ and rested at 37°C at 5% CO~2~ for 30 min before the cell concentration was adjusted to 3 × 10^6^ cells/ml in X‐vivo 20^™^. 200 μL of PBMCs were dispensed into 7 wells of a Megablock® 96 well plate (Sarstedt, Nümbrecht, Germany), along with 2 wells of a reference cell sample that was included in every assay. The cells were rested at 37°C with 5% CO~2~ for 2 hours to decrease basal phosphorylation levels. Following, the cells were either left unstimulated or stimulated according to a reverse time course for 15, 30, 60, 120, 180, or 240 min with IFN‐α2b (100 ng/mL; ImmunoTools, Friesoythe, Germany).
Fluorescent cell barcoding and phospho‐epitope staining for flow cytometry {#eji4228-sec-0120}
--------------------------------------------------------------------------
Following stimulation, PBMCs were resuspended by pipetting and immediately fixed at RT for 10 min by adding prewarmed PFA (Electron Microscopy Sciences (Hatfield, PA, USA)) at a final concentration of 1.5%. PBMCs were then centrifuged at 1000 g for 5 min 4°C and resuspended by vortexing in 50 μL PBS before drop wise addition of 1 mL ice cold methanol and incubation on ice for 30 min. The permeabilized cells were kept overnight at −80°C. After washing with PBS, the PBMCs were stained according to a 3 × 3 barcoding grid (9 stimulation conditions) using 3 levels of pacific orange (PO) and pacific blue (PB) succinimidyl ester dyes (PB 100, 25 and 6.3 ng/mL; PO 250, 70 and 0 ng/mL; Life Technologies, Grand Island, NY, USA) for 30 min in the dark at 4°C. Barcoded PBMCs were then washed once with PBS containing 1% BSA, before being combined into one sample. The sample was washed and incubated with 2 μL Fc receptor block (Miltenyi Biotec, Bergisch Gladbach, Germany) per 1 × 10^6^ cells for 10 min on ice. Following, the sample was subdivided into three parts and incubated for 30 min at RT in the dark with the three different antibody staining panels. An aliquot of the barcoded cells was collected before addition of antibody as a barcoding only control. The samples were then washed twice and re‐suspended in PBS containing 1% BSA and 2mM EDTA (Sigma‐Aldrich) prior to analysis.
Data analysis {#eji4228-sec-0130}
-------------
Samples were acquired on an LSRI Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) with BD FACSDiva^TM^ Software (BD Biosciences) at the Bergen Flow Cytometry Core Facility, University of Bergen, Norway. The flow cytometer was equipped with 407, 488, 561 and 635 nm lasers, and emission filters for PerCP‐Cy5.5 (LP: 685, BP: 695/40), Alexa Fluor‐488 (LP: 505, BP: 530/30), PE‐Cy7 (LP: 750, BP: 780/60), PE (LP: ‐, BP: 582/15), APC (LP: ‐, BP: 670‐/‐14), Pacific blue (LP: ‐, BP:450/50), Pacific orange (LP: 570, BP: 585/42) and BV 786 (LP: 750, BP: 780/60). The cytometer was routinely calibrated with BD cytometer setup and tracking beads (BD Biosciences). A minimum of 200 000 events in the intact cell gate was collected for each sample. FACS data were analysed in FlowJo (Tree Star) and Cytobank (<http://www.cytobank.org>). A representative gating strategy and phosphorylation profile for a single barcoded sample is shown in Supporting Information Fig. 2. Cryopreserved PBMCs from a single donor were included in each assay as a positive control, for inter‐assay normalization and assessing assay to assay variability. MdFI of phospho‐epitopes for gated populations were exported to Microsoft excel. The raw flow cytometry data can be found at the flow data repository of the International Society for Advancement of Cytometry [37](#eji4228-bib-0037){ref-type="ref"}, FR‐FCM‐ZYEF. The robustness of the flow cytometry assay has been previously established [34](#eji4228-bib-0034){ref-type="ref"}. Relevant information for repeating the experiments is given in Supporting Information Table 6 following "The minimum information about a Flow Cytometry Experiment (MIFlowCyt)" guidelines [38](#eji4228-bib-0038){ref-type="ref"}.
Statistical analysis {#eji4228-sec-0140}
--------------------
Comparisons between categories, correlations and the production of associated graphs were done using Graphpad Prism (version 6.05). Unpaired Mann--Whitney tests were used in the comparisons between categories. Correlations were assessed by the Spearman\'s rank test, with outliers removed using robust regression and outlier removal (ROUT) method and a ROUT coefficient Q of 1.
Differences were considered statistically significant when *p* ≤ 0.05. The analysis was exploratory in nature hence no correction was made for multiple comparisons. Principle component analysis (PCA) using Unscrambler® × software (Camo software) was used to reduce dimensionality of the dataset and find clusters of patients with similar signaling profile which could be used to differentiate between disease status, presence of SSA autoantibodies, EGM and medication effect. PCA was performed using the algorithm NIPALS, the data was mean centered and run with no weighting for change of MdFI, and weighted for absolute MdFI by dividing by standard deviation. Two methods were used to remove "redundant" variables to simplify interpretation and focusing subsequent analysis. First variables that described less than 50% of the variation were removed from the initial PCA, and where appropriate stepwise reduction of less significant variables with low variable leverage was performed.
Authorship {#eji4228-sec-0150}
==========
P.V., R.J., and S.A. conceived of study and R.D., P.V., and S.A designed the study. R.D. and B.B processed PBMC samples and conducted flow cytometric analysis. S.A. conducted real‐time quantitative PCR. R.D., B.B., S.G., and S.A. analysed and processed the data. D.H. and J.G.B. selected patients and collected patient data. R.D. and S.A wrote the manuscript. All authors read and approved the manuscript.
Conflict of interest {#eji4228-sec-0170}
====================
The authors declare no commercial or financial conflict of interest.
Funding {#eji4228-sec-0180}
=======
The authors' research is supported by the EU H2020 contract HarmonicSS (H2020‐SC1‐2016‐RTD/731944), the Broegelmann Foundation, the Western Norway Regional Health Authorities (grant nr. 912065) and the University of Bergen.
Supporting information
======================
######
Peer review correspondence
######
Click here for additional data file.
######
Supporting Information
######
Click here for additional data file.
We thank all patients and blood donors who participated in this study. We thank Marianne Eidsheim and Kjerstin Jakobsen for excellent technical assistance, and the staff at the laboratory at the Rheumatology clinics for collection of patients' blood samples. The flow cytometry analysis was performed at the Flow Cytometry Core Facility, Department of Clinical Science, University of Bergen. Financial support was obtained from the EU H2020 project HarmonicSS (H2020‐SC1‐2016‐RTD/731944), the Broegelmann Foundation, the Western Norway Regional Health Authorities (grant nr. 912065) and the University of Bergen.
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{
"pile_set_name": "PubMed Central"
}
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INTRODUCTION
============
Following the discovery and isolation of the organism by Marshall and Warren^[@b1-kjim-10-2-125-7]^, the evidence that Helicobacter pylori (H. pylori) causes non-erosive gastritis comes from studies in which eradication has been achieved with antibiotics. In these studies there is usually a marked lessening in the severity of the gastritis^[@b2-kjim-10-2-125-7]--[@b4-kjim-10-2-125-7]^. Probably more than 80% of cases of chronic gastritis are associated with coexisting H. pylori infection, and both the H. pylori and the gastritis are strongly linked with peptic ulcer^[@b5-kjim-10-2-125-7]^. Gastric colonization with H. pylori has been reported in 90 percent of patients with duodenal ulcer (DU) and in 60% of patients with gastric ulcer (GU)^[@b6-kjim-10-2-125-7]^.
Koch's postulates concerning the causal relationship between H. pylori and chronic gastritis seem to be satisfactorily fulfilled^[@b7-kjim-10-2-125-7]^ but the cause of chronic gastritis is various. In general, prevalence of H. pylori in Western countries is low and increases with age but in underdeveloped countries, it is not decidedly checked and most adults may be infected^[@b8-kjim-10-2-125-7]^. Since this is so, there is a possibility that the role of H. pylori in the pathogenesis of chronic gastritis may be different between Western and developing countries, in which the prevalence rate of chronic gastritis is high. Thus, it is still a matter of controversy that H. pylori is really an significant cause in chronic gastritis and associated with the severity of chronic gastritis in developing countries.
The aim of our study was to investigate if H. pylori influences the histological severity of chronic gastritis in Korean peptic ulcer patients. We conducted an analysis of correlation between histological grading of chronic gastritis and the presence of H. pylori infection.
MATERIAL AND METHODS
====================
We studied 80 patients, attending a gastrointestinal clinic of Chung-Ang University Hospital, for investigation of epigastric soreness, hunger pain or dyspepsia with duodenal or benign gastric ulcer as identified from routine gastrofiberscopic examination. The patients who had been taking anti-ulcer treatment in the month before the gastroscopy were excluded from the study. Peptic ulcer staging was reported, according to the Japanese classification of gastrointestinal endoscopy, by two endoscopists.
Peripheral venous blood and gastroscopic antral biopsy specimens were taken from 80 patients. Blood was collected after gastroscopy and kept at 5°C. Serum was separated within two hours and frozen at −20°C until analysis. During endoscopy, four antral biopsy specimens were obtained from the lesser gastric curvature, 2 cm proximal to the pylorus. Two biopsy specimens were used for histological examination(H & E staining, Giemas staining) and two biopsy specimens were taken for the culture of H. pylori. Serum gastritis concentration was measured by the radioimmunoassay technique, using the GammaDab-I^125^ Gastritis RIA kit(INCSTAR Co., UK) and serum pepsinogen concentration was measured by pepsinogen radioimmunoassay kit, Pepsik (Sorin Biomedical, France). Each examination was duplicated.
1.. Assessment of H. pylori
---------------------------
Antral biopsy specimens for histological examination were fixed in formalin and routinely processed, and specimens for culture were embedded in sterile saline and processed by the methods in our previous report^[@b9-kjim-10-2-125-7]^. H. pylori was identified microscopically in sections with Giemsa staining and by culture in blood agar medium. After routine culture, quantitative grading of cultured H. pylori was reported on a scale 0 to 3 (none, few, some, many).
2.. Histological Assessment of Gastritis
----------------------------------------
For histological grading of chronic gastritis, sections were stained with hematoxylin and eosin. The histopathological features of biopsy specimens were reported according to the Sydney classification^[@b10-kjim-10-2-125-7]^ of chronic gastritis (inflammation, activity, atrophy and metaplasia were each graded on a scale 0 to 3) by one histopathologist without knowledge of the clinical and endoscopic findings.
3.. Statistical Analysis
------------------------
Data were expressed in means and standard deviations. X^2^ test was used to determine the significance of differences between means. A value of p\<0.05 was considered to be statistically significant. The Spearman rank correlation test was used to determine whether there was a relationship between increasing grade of gastritis and increasing H. pylori concentration, and we used ANOVA in analysis of multiple groups. All analysis were done with SPSS/PC^+^.
RESULT
======
A total 80 patients were evaluated in the study. Fifty nine patients were male and 21 were female with a mean age of 44.3±13.1 years (range 18--81). Thirty two (40%) had gastric ulcer (30 active or healing stage, 2 scar stage) and 48 (60%) had duodenal ulcer (42 active or healing stage, 6 scar stage) among 80 patients endoscoped. H. pylori was identified in 62.5% (20 of 32 GU, 30 of 48 DU) of the study group. Percentages of gastritis colonization of H. pylori were the same in GU and DU.
There was no significant difference between with and without gastric colonization of H. pylori, with respect to age, sex and underlying type of peptic ulcer ([Table 1](#t1-kjim-10-2-125-7){ref-type="table"}). Gastric colonization rate of H. pylori among patients did not increase with age ([Fig. 1](#f1-kjim-10-2-125-7){ref-type="fig"}).
None of the biopsy specimens showed inflammation grade 0 and so all were chronic gastritis. Seventeen of 80 patients had chronic gastritis without any sign of activity. Forty of 50 biopsy specimens with H. pylori showed active chronic gastritis, whereas 23 of 30 biopsy specimens without H. pylori showed active chronic gastritis. Association of activity of chronic inflammation with H. pylori concentration was not statistically significant ([Fig. 2](#f2-kjim-10-2-125-7){ref-type="fig"}). There was no significant correlation overall between the presence of H. pylori and histological grading of chronic gastritis (p\>0.1), and also no association was found between the quantitative grading of H. pylori and the histological grading of chronic gastritis (p\>0.1) ([Table 2](#t2-kjim-10-2-125-7){ref-type="table"}).
With and without H. pylori, a mean of serum gastritis concentration was 79.4±43.0 pg/ml and 80.2±31.9 pg/ml respectively, and there was no significant difference with respect to the presence of H. pylori. A mean of serum pepsinogen concentration was 87.7±41.6 ng/ml and 119.5±34.4 ng/ml with and without H. pylori, and the statistical analysis, excluding the influence of peptic ulcer, showed significant difference of serum pepsinogen concentrations between the populations with and without H. pylori (p = 0.001) ([Table 3](#t3-kjim-10-2-125-7){ref-type="table"}).
DISCUSSION
==========
The most obvious disease associated with H. pylori is peptic ulcer. The report that more than 90% of duodenal ulcers and about 70% of gastric ulcers were caused by H. pylori is accepted in Western countries^[@b8-kjim-10-2-125-7]^. But in Korea, the infection rates of H. pylori in GU and DU were 62%--81%^[@b11-kjim-10-2-125-7]--[@b13-kjim-10-2-125-7]^ and 62%--83%^[@b9-kjim-10-2-125-7],[@b11-kjim-10-2-125-7]--[@b13-kjim-10-2-125-7]^, respectively, and our study showed 62.5% infection rate of H. pylori, similar to other results of the Korean prevalence study of H. pylori. The role of H. pylori on peptic ulcer is assuredly less in Korea than in Western countries.
In Western countries, H. pylori is uncommon in young children and affects about 20% of persons below the age of 40 yr and 50% of those above the age of 60 yr. Low socio-economic status predicts H. pylori infection^[@b8-kjim-10-2-125-7]^. But in most developing countries like Korea, an endemic of H. pylori goes unchecked, and most adults are infected. In the present study, gastric colonization rate of H. pylori did not increase with age, as suggested in one report dealing with infection rate of H. pylori in Korean children^[@b14-kjim-10-2-125-7]^. The epidemiology reported by Megraud^[@b15-kjim-10-2-125-7]^ that, in countries such as Asia, Africa and Easten Europe, most are infected by their teens may be applicable to Korean cases.
Delineating the normal cellular content of the lamina propria of the gastric mucosa is difficult and disagreement between pathologists on what is an acceptable normal background is the basis of many discrepant reports in the literatures. An even distribution of very small numbers of lymphocytes and plasma cells is acceptable^[@b10-kjim-10-2-125-7]^. In our study, all biopsy specimens showed an increase in lymphocytes and plasma cells within the lamina propria, but 17 of 80 specimens did not show the increased neutrophil polymorphs in the lamina propria, gastric pits and surface epithelium. Endoscopically, many adults in Korea showed chronic gastritis, but the exact prevalence of endoscopic and histological chronic gastritis of Koreans is not available.
There is marked variation between the intensity of inflammation and the number of organisms in a histologic section. When there is intense epithelial change with marked mucus depletion, organisms may be sparse and more easily seen in adjacent areas, where there are more normal mucous cells^[@b16-kjim-10-2-125-7]^. Activity of chronic gastritis is known as a useful measure of response to therapy and can be particularly related to the presence and concentration of H. pylori^[@b2-kjim-10-2-125-7],[@b8-kjim-10-2-125-7],[@b17-kjim-10-2-125-7]--[@b19-kjim-10-2-125-7]^. But, in the present study, H. pylori concentation did not affect the activity of chronic gastritis and overall histological grading of chronic gastritis, and atrophy of chronic gastritis did not affect the presence of H. pylori. There is one possibility that a small number of patients in our study may affect the statistical result, and another possibility that the role of H. pylori on the pathogenesis of chronic gastritis in endemic countries of H. pylori may differ from that in Western countries. Further studies for host reaction to H. pylori and for a causal sequence of H. pylori and chronic gastritis are necessary.
H. pylori causes gastritis and a number of perturbation of gastric and duodenal function. H. pylori-related changes of gastric function are hypergastrinemia, hyperpepsinogenemia and on increase in acid secretion. H. pylori gastritis is associated with a decrease in the number of antral D cells and G cells, although the proportion of G cells to D cells appears to be unchanged, and the exaggerated gastritis release associated with the H. pylori infection appears to be secondary to the production of cytokines^[@b20-kjim-10-2-125-7]^. We reported that eradication of the organism resulted in a significant fall in serum gastritis concentration^[@b21-kjim-10-2-125-7]^ but, in the present study, H. pylori infection did not affect serum gastritis concentration. Because there was a possibility that this outcome may result from the effect of underlying peptic ulcers, we have a plan to study the role of H. pylori on chronic gastritis without peptic ulcer. Asaka M, et al^[@b22-kjim-10-2-125-7]^ reported that pepsinogen I and II levels was increased in association with H. pylori infection and our study showed the same result. But the mechanisms of H. pylori-related hyperpepsinogenemia is uncertain.
On the basis of our observations, we conclude that the influence of H. pylori on the histological grading of chronic gastritis in Korea is less than that in prior studies of Western countries, and further investigation of the pathogenesis of H. pylori in chronic gastritis and peptic ulceration is necessary.
This research was supported by the Chung-Ang University research grants in 1993
{#f1-kjim-10-2-125-7}
{#f2-kjim-10-2-125-7}
######
Characteristics of the Patients
H. pylori negative H. pylori positive
------------------------------------------------------------ -------------------- --------------------
Number of patients 30 50
Age (yr)[^\*^](#tfn1-kjim-10-2-125-7){ref-type="table-fn"} 45.7±14.5 43.5±12.2
Sex (male/female) 19/11 40/10
No. of patients with peptic ulcer (gastric/duodenal) 12/18 20/30
mean±standard deviation
######
Overall Correlation between Histological Grading of Chronic Gastritis and Gastric Colonization of H. pylori
H. pylori status Histological grading of chronic gastritis
------------------------------------------------------------ ------------------------------------------- --- ---- ---- ---- ---- ---- --- ---- ---- --- --- ---- --- --- ---
Negative 0 8 13 9 7 16 6 1 8 16 6 0 17 4 6 3
(No. of patients)
Positive
(No. of patients)
total[^1^](#tfn2-kjim-10-2-125-7){ref-type="table-fn"} 0 9 26 15 10 22 14 4 11 30 9 0 36 7 4 3
few[^2^](#tfn3-kjim-10-2-125-7){ref-type="table-fn"} 0 1 10 4 1 8 4 2 3 10 2 0 11 3 1 0
some[^2^](#tfn3-kjim-10-2-125-7){ref-type="table-fn"} 0 4 5 3 4 6 1 1 2 9 1 0 11 0 0 1
many[^2^](#tfn3-kjim-10-2-125-7){ref-type="table-fn"} 0 4 11 8 5 8 9 1 6 11 6 0 14 4 3 2
Giemsa staining
Culture
######
Concentrations[^\*^](#tfn4-kjim-10-2-125-7){ref-type="table-fn"} of Gastrin and Pepsinogen in Serum from Patients with or without H. pylori Infection
H. pylori status No Gastrin (pg/ml) Pepsinogen (ng/ml)
------------------ ---- ----------------- --------------------------------------------------------------
negative 30 79.4±43.0 87.7±41.6
positive 50 80.2±32.0 119.5±34.5[^\*^](#tfn5-kjim-10-2-125-7){ref-type="table-fn"}
values are mean±standard deviation
Concentration significantly higher in H. pylori positive compared with negative patients: p=0.001
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{
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}
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Adult T cell leukemia/lymphoma (ATL) is an aggressive malignant disease of CD4 positive T lymphocytes caused by infection with human T cell leukemia virus type I (HTLV-I). HTLV-1 causes ATL in 3-5% of infected individuals after a long latent period of 40 to 60 years. The acute and lymphoma types are aggressive ATL characterized by resistance to chemotherapy and a poor prognosis. Leukemia/lymphoma cells and rapidly proliferating cells preferentially accumulate endogenous photosensitizer protoporphyrin IX (PpIX) when supplemented with 5-aminolevulinic acid (ALA). Treatment with 1mM ALA for 48h induced 10 to 100 times accumulation of PpIX in ATL leukemic cell lines and HTLV-I (+) T cell lines than that in healthy PBMCs. Specific induction of apoptosis was observed after 10 min light exposure (28 mW/cm^2^) using Na-Li lamp in ATL leukemic cell lines and HTLV-I (+) T cell lines. ATL patient PBMC specimen showed strong accumulation of PpIX with the treatment of ALA compared to the healthy donor and HTLV-I carrier PBMCs, which could be useful for the diagnostic purposes and monitoring the patient status with high sensitivity. Photodynamic therapy is potentially hopeful treatment especially for lymphoma type ATL as a relatively selective, minimal or no scarring, non-invasive, safe, simultaneous and repeatable multiple lesions treatable modality.
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{
"pile_set_name": "PubMed Central"
}
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INTRODUCTION {#sec1-1}
============
Vitamin D deficiency is common around the world and it is estimated that 1 billion peoples are living with inadequate Vitamin D.\[[@ref1][@ref2]\] Although the main known physiologic function of Vitamin D is the regulation of calcium and phosphorus homeostasis, it has been shown that Vitamin D plays a major role in the extraskeletal metabolic processes such as glucose metabolism, immunomodulation, renin-angiotensin system regulation, cell growth, membrane stabilizing, angiogenesis, anti-proliferation, and apoptosis.\[[@ref2][@ref3][@ref4][@ref5]\]
Vitamin D insufficiency has been reported in more than 80% of critically ill patients, hospitalized in intensive care unit (ICU).
Unexpectedly, up to 17% of this population had undetectable serum level of 25(OH)D.\[[@ref6][@ref7][@ref8]\]
Decrease in the serum Vitamin D level during ICU stay can be explained by the lack of exposure to sunlight and insufficient replacement of this vitamin through parental or enteral metabolic support.\[[@ref9]\] Several studies have evaluated the relationship between serum Vitamin D level and clinical outcome in critically ill patients. In some of the surveys, it has been shown that low serum level of 25(OH)D was associated with adverse clinical outcome such as prolonged length of ICU stay, organ dysfunction, acquiring nosocomial infections, acute kidney injury (AKI), increased health care costs, and mortality.\[[@ref8][@ref10][@ref11][@ref12]\] While in other studies, the correlation between the serum Vitamin D level and worse outcome has not been detected in this population.\[[@ref13][@ref14][@ref15]\]
However, in most of the studies, patient care setting (medical, surgical, or trauma) and also type and severity of baseline diseases have not been considered in the final analysis. Surgical patients are more vulnerable to postoperative sudden cardiovascular events and infections that both of them may be aggravated by Vitamin D deficiency.\[[@ref16][@ref17]\]
There is few data regarding the correlation between serum Vitamin D level and unfavorable ICU outcome in postsurgical patients. In this study, correlation between serum 25(OH)D level and length of ICU stay and in-hospital mortality has been evaluated in surgical, critically ill patients.
METHODS {#sec1-2}
=======
This prospective, cross-sectional, descriptive study was performed during 1-year period from early March 2014 to February 2015 at ICU of the Imam Khomeini Hospital affiliated to Tehran University of Medical Sciences, Tehran, Iran. The Institutional Review Board and the Medical Ethics Committee of the Hospital approved the study and all the patients or their family members signed and approved a written informed consent form.
During the study period, 70 surgical patients admitted to the ICU were recruited. Baseline patients' demographic data, including sex, age, weight, body mass index (BMI), baseline diseases, and type of surgery, were collected from the patients' medical records. Severity of patients' clinical condition at ICU admission was evaluated based on the acute physiology and chronic health evaluation and simplified acute physiologic score criteria.
The patients' laboratory parameters, including serum electrolytes (Na^+^, K^+^, Mg^+2^, and Ca^+2^), renal function tests (serum creatinine and urea), liver enzymes (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase), and liver function indexes (serum albumin and bilirubin, international normalized ratio) also were recorded at the time of recruitment.
From each included patient, 5 ml of venous blood sample was collected at the time of ICU admission. The blood were centrifuged and separated serum samples were stored at −80°C up to the time of measurement of serum 25(OH)D level.
Serum 25(OH)D level was measured by ELISA (Immunodiagnostic Systems, Louvain-la-Neuve, Belgium). Calibration, control, and samples were diluted with biotin-labeled 25(OH)D. The diluted samples were incubated in microtiter wells, which were coated with highly specific sheep 25(OH)D antibody for 2 h at room temperature before aspiration and washing. Enzyme-labeled avidin was added and binded selectively to complexed biotin; then color was developed using a chromogenic substrate. The absorbance of the stopped reaction microtiter were read in a microtiter plate reader; color intensity developed being inversely proportional to the concentration of 25(OH)D. Serum 25(OH)D level \<30 ng/ml was considered as Vitamin D deficiency.\[[@ref2][@ref18]\]
All the statistical analyses were performed by the SPSS (Statistical Package for the Social Sciences) version 18 software (IBM Corp., Armonk, NY, USA). The Kolmogorov--Smirnov test was used to assess the normal distribution of continuous variables. The data were expressed as a mean ± standard deviation. Categorical variables were reported as percentages. Association between serum 25(OH)D category and the main endpoints of study (duration of ICU stay and in-hospital mortality) was evaluated using two-factor analysis of covariance. Multivariable Cox-regression analysis was used for adjusting the effect of season of blood sampling and type of surgery on the main variables. For all the analyses, *P* values \<0.05 were considered as statistical significant.
RESULTS {#sec1-3}
=======
Of the 70 patients who were enrolled in this study, 31 (44.3%) were male and 39 (55.7%) were female. Serum 25(OH)D deficiency was identified in 52 (74.3%) patients. The characteristics of the study population regarding serum 25(OH)D level is shown in [Table 1](#T1){ref-type="table"}. Except for age, there was no significant difference in the baseline characteristics of patients with and without vitamin D deficiency. Vitamin D deficient patients were younger than sufficient ones (*P* = 0.04).
######
Characteristics of the patients regarding serum 25(OH) D levels
The patients' serum 25(OH)D levels regarding season of the ICU admission were shown in [Table 2](#T2){ref-type="table"}. There was a nonsignificant difference between the patients' serum 25(OH)D levels regarding the seasons of sampling (*P* = 0.39).
######
Season of admission of the patients and serum 25(OH) D levels
Regarding baseline disease, 17.1% of patients had a history of hypertension, 8.5% suffered from ischemic heart disease, and 52.8% had gastrointestinal diseases. Gastrointestinal surgery was the most common surgery type (61.5%) in the patients. Serum 25(OH)D level regarding the type of the surgery has been shown in [Table 3](#T3){ref-type="table"}. Considering the possible effect of different types of major surgeries with different stress-inducing properties on serum Vitamin D status, association between serum 25(OH)D level and type of surgery was evaluated. No difference in serum 25(OH)D level based on the type of surgery was detected (*P* = 0.41). Patients with serum Vitamin D levels \<30 ng/ml had longer length of ICU stay than those with serum Vitamin D levels ≥30 ng/ml (7.8 ± 5.1 vs. 4.05 ± 2.12 days, *P* = 0.003).
######
Serum 25(OH) D levels of the patients based on the type of surgery
Following Cox-regression analysis, it was found that serum 25(OH)D status at the time of ICU admission can be a potential predictor for length of ICU stay following adjustment for age, sex, BMI, season, and type of surgery in critically ill surgical patients (*P* = 0.05).
Although hospital mortality was more common in Vitamin D deficient patients than sufficient ones (25% in deficient group vs. 22.2% in sufficient group), there was no significant difference regarding hospital mortality rate between the groups.
DISCUSSION {#sec1-4}
==========
Vitamin D deficiency is common among critically ill medical and surgical patients. There is no uniform definition for Vitamin D deficiency in these populations. In general population, serum 25(OH)D level is defined as the best indicator of body\'s Vitamin D status, since it has longer half-life, more stability in the systemic circulation, and more positive correlation with calcium absorption in comparison with 1,25(OH)2D.\[[@ref19][@ref20]\]
There is no definite cut-off level for 25(OH)D that predict beneficial outcome in critically ill patients. In the previous studies, unfavorable outcome was associated with serum 25(OH)D level of 12 to 20 ng/ml in critically ill medical patients.\[[@ref15][@ref21][@ref22]\] In critically ill surgical patients, serum 25(OH)D level more than 26 ng/ml showed beneficial effects.\[[@ref8]\]
In analysis of the data from 32 studies published between 1966 and 2013, serum 25(OH)D concentration \>30 ng/ml was associated with lower all-cause mortality in critically ill patients compared with concentration ≤30 ng/ml.\[[@ref18]\]
In our study, serum 25(OH)D level \<30 ng/ml was considered as a deficient cut-off in surgical patients.
There are some concerns regarding the accuracy of serum 25(OH)D level as the best indicator of Vitamin D status in critically ill patients. Liver and renal dysfunction, low serum albumin, and Vitamin D binding protein levels that are common in these patients may lead to falsely high serum 25(OH)D level.\[[@ref23][@ref24]\] However, the previous studies have shown association between low serum 25(OH)D and adverse clinical outcomes, including systemic inflammatory response syndrome, nosocomial bloodstream and respiratory tract infections, AKI and metabolic dysfunction as well as longer length of ICU stay, and all-cause mortality in critically ill patients.\[[@ref25][@ref26][@ref27]\]
In this study, 74.3% of the Iranian critically ill surgical patients had Vitamin D deficiency. In a large multicentric study, in a general population of Iran, moderate to severe Vitamin D deficiency was detected in 75.1% and 72.1% of women and men, respectively.\[[@ref28]\] Inadequate sun exposure, lack of Vitamin D food fortification program, clothing habits (especially among women), polymorphism in Vitamin D receptor, and low daily calcium intake introduced as predisposing factors of Vitamin D deficiency in middle-east countries.\[[@ref29][@ref30]\]
Although mechanisms of adverse ICU outcome resulting from hypovitaminosis D in critically ill patients have not been well-defined, several suggestions, including pleiotropic effects of Vitamin D on immunity, mucosal and endothelial functions, and different metabolic effects of Vitamin D have been reported.\[[@ref2]\]
Due to seasonal variations in sunlight, blood sampling in winter and spring is associated with lower 25(OH)D levels compared with summer and fall seasons.\[[@ref10][@ref13]\] In our patients, we did not observe any correlation between serum Vitamin D level and seasonality. This finding might be expected since serum Vitamin D level in critically ill patients is less affected by seasonal changes due to immobility and restricted sun exposure, even before the ICU admission.
In this study, Vitamin D deficiency was associated with younger age. Similar finding was reported in another study in critically ill patients and can be related to the impact of common Vitamin D supplementation in an elderly population in recent years.\[[@ref15]\]
Vitamin D deficiency was associated with increased length of ICU stay in our surgical critically ill patients. This is in line with other studies that showed the correlation between serum level of Vitamin D and length of ICU stay.\[[@ref6][@ref31]\] In Higgins *et al*. study, low serum levels of 25(OH)D was associated with increased time of ICU stay.\[[@ref6]\] Also, in Zittermann *et al*. study, length of ICU stay was significantly higher in Vitamin D deficient cardiac surgery patients compared with adequate ones.\[[@ref31]\] McKinney *et al*. reported similar results in medical critically ill patients.\[[@ref32]\] Correlation between severity of Vitamin D deficiency and length of ICU stay has been reported by Matthews *et al*.\[[@ref8]\] Contrary to the above studies, in Amrein *et al*., Venkatram *et al*. and Aygencel *et al*. studies, the correlation between Vitamin D insufficiency and length of ICU stay was not detected.\[[@ref13][@ref14][@ref15]\]
The second endpoint of our study was evaluating the correlation between serum 25(OH)D level and the in-hospital mortality. Although mortality was more common in Vitamin D deficient critically ill surgical patients than those were sufficient, we did not find a significant correlation between hypovitaminosis D and in-hospital mortality. Similar to this study, in Flynn *et al*. prospective survey, no difference in mortality rate was reported between Vitamin D sufficient and deficient surgical critically ill patients.\[[@ref17]\] In another study, Lucidarme *et al*. showed that mortality rate was unchanged by low serum Vitamin D level in critically ill patients.\[[@ref33]\] Although, Aygencel *et al*. detected a correlation between mortality and Vitamin D insufficiency primarily in univariate analysis, Vitamin D insufficiency was not defined as an independent risk factor for mortality in the later multivariate analysis.\[[@ref14]\]
In contrast to the previous studies, Zhang *et al*. in a meta-analysis of 7 cohort studies suggested that Vitamin D deficiency is associated with an increased risk of hospital mortality.\[[@ref34]\] In addition, higher Vitamin D concentration was associated with a linear reduction of in-hospital mortality in Turan *et al*. study.\[[@ref16]\] Venkatram *et al*. in a retrospective study, in a medical ICU, found a significant correlation between Vitamin D deficiency and mortality, but there was no correlation between Vitamin D insufficiency and hospital mortality. In that study, Vitamin D deficiency and insufficiency were defined as serum 25(OH)D level \<20 ng/ml and 20 to 29 ng/ml, respectively.\[[@ref15]\]
Following a single-center, retrospective, observational study in 655 surgical and nonsurgical critically ill patients, Amrein *et al*. reported the lowest in-hospital mortality at serum 25(OH)D levels between 20 and 40 ng/ml. They also proposed serum 25(OH)D level of 15 ng/ml as the cut-off point for in-hospital mortality.\[[@ref13]\] Ralph *et al*. did not observe any association between low level of serum 25(OH)D at admission and increased risk of mortality in critically ill medical patients, but interestingly, patients with supraphysiologic serum Vitamin D levels at ICU admission had higher mortality rate.\[[@ref35]\] Association between high serum 25(OH)D levels (\>100 nmol/ml or \>40 ng/ml) and adverse clinical outcome has been reported in a recent study of postcardiac surgery patients.\[[@ref31]\] In the second recent study, as a 9 years follow-up, a reverse J-shaped correlation between serum 25(OH)D level and mortality has been shown and serum Vitamin D level more than 120 nmol/ml (or \> 48 ng/ml) was associated with higher rate of mortality.\[[@ref36]\]
No association between low serum levels of Vitamin D and in-hospital mortality in our study may be due to small number of the included patients, different cut-off point values for Vitamin D, deficiency in previous studies (most of the studies that supported correlation between serum 25(OH)D level and mortality used the concentrations below 20 ng/ml), and supraphysiologic serum 25(OH)D level in 72% of the patients in "sufficient" group (13 of 18 patients). The "J-shaped effect theory" should be considered in interpreting mortality rate in the Vitamin D sufficient group.
In our study, critically ill surgical patients were enrolled and so the heterogeneity among medical, surgical, and traumatic critically ill patients that mentioned in the several previous studies was considered. All the patients' blood samples were collected at a definite time, at 5--7 a.m., in 1^st^-day of ICU admission. Also, the effects of potential confounders, including BMI and seasonal variation, were adjusted in the data analysis.
However, some limitations should be considered in our study. The major limitation was small sample size of the study. It was a single-center study and, therefore, the results could not be generalized. Serum Vitamin D levels were assessed only at the time of ICU admission in our patients. Serial Vitamin D assay may result in more reliable judgment about body\'s Vitamin D status. It was not possible to measure serum Vitamin D binding protein in our patients. Concentration of this protein can affect serum Vitamin D level, especially in critically ill patients with malnutrition.
Vitamin D deficiency is common in critically ill surgical patients. Statistically significant association was found between low 25(OH)D level and increased length of ICU stay in critically ill surgical patients. However, there was no correlation between serum Vitamin D level and patients' in-hospital mortality. Further, well-designed, prospective clinical studies with adequate sample size are needed to evaluate the correlation between serum Vitamin D level and mortality in critically ill patients.
AUTHORS' CONTRIBUTION {#sec1-5}
=====================
Alizadeh N: Data gathering and preparation of the article draft. Khalili H: Designing the study, data analysis and final proof of the article. Mohammadi M: Patients' selection and clinical assessment of the patients. Abdollahi A: laboratory measurements.
Financial support and sponsorship {#sec2-1}
---------------------------------
Nil.
Conflicts of interest {#sec2-2}
---------------------
There are no conflicts of interest.
This study was the result of a Ph.D. student\'s thesis and supported by a Vice-Chancellor for the Research of Tehran University of Medical Sciences. The authors expressed sincere gratitude to all the nursing staff of the general ICU of Imam Khomeini Hospital for their kindly support.
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{
"pile_set_name": "PubMed Central"
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Introduction {#s1}
============
Visual perception occurs over time and space. In order to make sense of a continuous flow of information which is frequently occluded by other objects or interrupted by eye movements, our visual system must determine the relationship between successive stimuli. Given the prevalence of occlusion and saccades in visual perception, it is perhaps not surprising that our visual system can spontaneously retrieve features from a previously seen object when it is perceived as a different instantiation of a subsequent object [@pone.0063264-Gordon1]--[@pone.0063264-Mitroff1]. However, so far, there has not been much research on the spontaneous retrieval of features of a recently inhibited object. The present study focuses on two issues: (1) whether features of a previously viewed object can be retrieved spontaneously when that object has recently been inhibited, and (2) whether such retrieval is also contingent upon the perceived object continuity between the two successive stimuli.
An influential theoretical framework that addresses the relationship between object continuity and visual information processing is the object file theory [@pone.0063264-Kahneman1],[@pone.0063264-Kahneman2]. According to this theory, when an object is encountered, the visual system creates an "object file": a temporary episodic representation that contains information about the features of the object. Attention to an object triggers an automatic process of reviewing. When two stimuli appear in close spatiotemporal proximity, depending on their perceived object continuity, the reviewing process will lead to either the updating of a pre-existing object file or the creation of a new one. If an object link is found through a correspondence process between the current object, S2, and an object viewed recently, S1, then the two stimuli are seen as different states of the same object, and the contents of the previous object file are retrieved, and updated if necessary. However, if an object link is not found between S2 and S1, then the two stimuli are seen as belonging to different objects, and a new object file is created for S2. As updating an existing object file requires fewer mental resources than creating a new one, responses to S2 are facilitated when S1 and S2 are perceived as different states of the same object rather than as two different objects.
The object file theory has been supported by many studies [@pone.0063264-Gordon1],[@pone.0063264-Henderson1],[@pone.0063264-Kahneman1],[@pone.0063264-Mitroff1]. For example, in a series of experiments, Kahneman et al. showed participants two successive displays, a preview display with two or more letters, each in an individual frame, and a target display with a single letter in one of the frames. The task was to report the identity of the target letter. The main finding was an object-specific preview effect: responses to the target were reliably faster when it was a previewed letter that appeared in the same frame (absolute or relative) compared with a previewed letter that appeared in a different frame. Furthermore, the benefit of priming, i.e., facilitation due to prior exposure of a stimulus relative to a new stimulus, was small and unreliable. These results suggest that the object-specific preview effect in Kahneman et al.'s paradigm was derived primarily from the retrieval of the features of a previously encountered stimulus by a current object.
More recently, Hommel and his colleagues [@pone.0063264-Hommel1]--[@pone.0063264-Hommel3] extended the object file theory to include response-related information in the episodic representation of an attended object. Hommel coined the term "event file" to emphasize a multi-layered network of bindings among stimulus features, response features, and task context. The general idea of the theory is that the co-occurrence of stimulus features, or the co-occurrence of a stimulus feature and an action (e.g., a left or right response), causes them to bind spontaneously (but see Hommel [@pone.0063264-Hommel1] for evidence of binding between shape and location when shape was task relevant, but not when color was task relevant). Once they are bound, the activation of one leads to the activation of the other. Consequently, a partial match between S1 and S2 delays responses to S2 relative to both a complete match and a complete mismatch between the two stimuli. This partial-repetition cost is presumably caused by the extra time it takes to resolve the conflict induced by a previous binding [@pone.0063264-Hommel2],[@pone.0063264-Hommel3]. Thus, if participants respond to the onset of S1 (i.e., features of S1 are task irrelevant) but to the form of S2, repeating the form of S1 in S2 would produce a cost not only when the colors of S1 and S2 differed (a partial match) relative to when their colors matched (a complete match), but also when the colors of S1 and S2 differed (again a partial match) compared with when both the form and the color of S1 and S2 differed (a complete mismatch). This pattern of data is exactly what Hommel and his colleagues observed in many of their experiments [@pone.0063264-Hommel1],[@pone.0063264-Hommel3],[@pone.0063264-Colzato1].
If we assume that a complete match between the features of S1 and S2 would induce participants to see the two stimuli as different states of the same object, the above studies provide evidence for the spontaneous retrieval of features of S1 when object continuity is perceived between S1 and S2. What is less clear is whether the spontaneous retrieval of features would still occur when a previously viewed object has just been inhibited, and whether such retrieval would also be contingent upon the perceived object continuity between the two stimuli. If participants know that the chances of S1 being the same as S2 are small, will attention to S2 trigger the retrieval of S1 features in a way similar to that observed in previous studies where S1 was not suppressed [@pone.0063264-Hommel3],[@pone.0063264-Colzato1]? Such a mechanism would have the advantage of reducing the processing load of S2 when S1 and S2 are perceived as different instantiations of the same object. However, the same mechanism would not be particularly helpful when S1 and S2 belong to different types of objects, for the exact combination of features in S1 would never repeat in S2.
There is some indication in prior research that the spontaneous retrieval of features from an inhibited object can occur when it is identical to a subsequently presented target. Using a negative priming paradigm [@pone.0063264-Tipper1], Tipper, Weaver, and Houghton [@pone.0063264-Tipper2] manipulated the relationship between a distractor on a prime trial (trial n) and a target on a probe trial (trial n+1). In one experiment (Experiment 1), participants saw displays that consisted of two colored letters in two of four marked locations. One of the letters was a target, and the other was a distractor. The target was defined by color, and the task was to report the target's location. On some trials (the control condition), the stimuli on the probe trial were unrelated to the stimuli on the prime trial. On other trials (the ignored repetition conditions), the target on the probe trial matched the distractor on the prime trial in one or more of its features (i.e., color, location, and/or form). Relative to the control condition, reaction times (RTs) to the probe target were longer in most conditions when the probe target had the same location and/or color as that (or those) of the prime distractor, demonstrating negative priming. Negative priming was not found when the prime and probe matched only in form, which was a task irrelevant feature. Furthermore, positive priming was found when the prime and probe were identical (i.e., a complete match in all the features of the prime and probe) compared with when they were unrelated or when there was only a partial match in their features. These results were interpreted by Tipper et al. [@pone.0063264-Tipper2] in terms of the flexibility of the visual system: a system that can have multiple levels of internal representation and can evoke task-specific inhibition. Their results are also consistent with the notion that the features of an inhibited object can be spontaneously retrieved if the inhibited object can be seen as a different instantiation of a subsequent object (cf [@pone.0063264-Milliken1]--[@pone.0063264-Park1] for non-inhibition interpretations of negative priming).
We were interested in the effect of inhibition on the retrieval of object features when a display consisted of a single object, and participants knew in advance that two successively presented objects were unlikely to be identical. In three experiments, participants saw a task cue, followed by S1 and then S2. Both S1 and S2 consisted of a two-dimensional stimulus that varied in color and orientation. The task was to respond to S2 while ignoring S1. In Experiment 1, S1 and S2 were independent. This experiment was conducted to ensure that spontaneous feature retrieval could occur in our paradigm when inhibition was not evoked. In Experiments 2 and 3, we matched the task relevant features of S1 and S2 on one-third of the trials, and mismatched them on the rest of the trials. We manipulated the identity of S2 so that S1 and S2 were likely to be seen as different instantiations of the same object in Experiment 2 but as different types of objects in Experiment 3. We found evidence for spontaneous feature retrieval in Experiments 1 and 2, but not in Experiment 3. Together, these results suggest that features of a previously inhibited object can be retrieved spontaneously, but such retrieval occurs only when the two stimuli are seen as different instantiations of the same object.
Experiment 1 {#s2}
============
The goal of Experiment 1 was two-fold: to ensure that spontaneous feature retrieval could occur with changing behavioral goals, and to provide a baseline for Experiments 2 and 3. In prior research on feature retrieval, participants typically performed the same task from beginning to end in an experiment [@pone.0063264-Gordon1],[@pone.0063264-Hommel3],[@pone.0063264-Kahneman1]. In the present experiment, they had to switch tasks from trial to trial on the basis of a task cue at the beginning of each trial. In experiments with a single task, the task irrelevant feature may become less salient over time. In contrast, when participants are required to switch between two tasks, the irrelevant feature is likely to be kept salient, and this in turn may influence object-specific feature retrieval. Thus, if evidence for spontaneous feature retrieval was found in Experiment 1, this would generalize the results of prior research to situations where participants' behavioral goals changed constantly across trials.
On each trial, participants saw three displays that consisted of a task cue, followed by S1, and then S2. Both S1 and S2 consisted of a two-dimensional bar that varied in color and orientation. The task was to report the color or the orientation of S2 on the basis of the task cue. Both the task relevant and irrelevant features were independent. This led to S1 and S2 having a complete match in color and orientation on one-fourth of the trials, a partial match on two-fourths of the trials, and a complete mismatch on the rest of the trials. As S1 and S2 were identical on the complete match trials, they were likely to be perceived as different states of the same object, and this, in turn, should encourage spontaneous feature retrieval. If feature retrieval could occur with changing behavioral goals, participants would take longer to respond to S2 when S1 and S2 had a partial match compared to when they had a complete match or a complete mismatch.
Methods {#s2a}
-------
### Ethics statement {#s2a1}
This study received prior ethical approval from The University of Canterbury Human Ethics Committee. The committee approved the consent form and experimental procedure. Written consent was obtained from the participants.
### Participants {#s2a2}
Eighteen undergraduate students from the University of Canterbury volunteered for the experiment either in exchange for course credit or for payment. All of them reported having normal or corrected-to-normal vision.
### Apparatus and stimuli {#s2a3}
All stimuli were displayed against a grey background. They were shown on a Power Macintosh 6100/66 computer with a 13-in. RGB monitor. Participants were tested individually in a dimly lit room. The viewing distance from the monitor was approximately 60 cm. MacProbe [@pone.0063264-Hunt1] was used to generate stimuli and collect responses.
Each trial started with a task cue, which was either a black letter C (for color) or O (for orientation) written in 36-point Geneva font at the center of a computer screen (see [Figure 1](#pone-0063264-g001){ref-type="fig"}). The cue was then followed by two successive displays, each consisting of a red or a green bar with a 45° left or right tilt from vertical. The bar subtended 0.57° of visual angle in length and 0.14° in width. To minimize masking, the two stimuli were shown at different locations. Whereas S1 always appeared at the center, S2 was either 4.12° above or below the center with equal probability.
{#pone-0063264-g001}
### Design and procedure {#s2a4}
The experiment used a within-participants design. The principal manipulations were task (color vs. orientation), the relationship between the task relevant features of S1 and S2 (same vs. different), and the relationship between their task irrelevant features (same vs. different). Altogether, there were eight experimental conditions, four associated with the colour task, and the other four with the orientation task. The four conditions in each task were: the relevant-same- irrelevant-same (SS) condition, where S1 and S2 had the same relevant and irrelevant features; the relevant-same-irrelevant-different (SD) condition, where S1 and S2 had the same relevant but different irrelevant features; the relevant-different-irrelevant-same (DS) condition, where S1 and S2 had different relevant but same irrelevant features; and the relevant-different-irrelevant-different (DD) condition, where S1 and S2 differed in both the relevant and irrelevant features.
Each trial began with the presentation of the task cue for 1,005 ms, with a C referring to color and an O to orientation. The two types of trials were equally likely to appear, and they were randomly mixed within a block. After an inter-stimulus-interval (ISI) of 510 ms, S1 was displayed for 510 ms. Upon its offset, and followed by another ISI of 1,005 ms, S2 was shown for 120 ms. Participants were instructed to respond to S2 as quickly and as accurately as possible.
Participants were informed that S1 did not predict S2 in any way. They pressed one of four designated response keys on each trial, using their right index and middle fingers for the color task (with "." for red and "/" for green) and their left middle and index fingers for the orientation task (with "z" for left and "x" for right). The experiment consisted of 48 practice trials, followed by four blocks of 80 trials. The entire experiment took approximately 40 minutes to complete.
Results and Discussion {#s2b}
----------------------
[Figures 2A and 2B](#pone-0063264-g002){ref-type="fig"} show the mean RTs for the correct responses on the color and orientation trials, respectively. Reaction times greater than 2,000 ms were excluded from analyses. Such times accounted for less than1.2% of the total data both in this and the next two experiments. The mean error rates are shown in [Table 1](#pone-0063264-t001){ref-type="table"}. The data from one participant were excluded from analyses due to long RTs (15% of the data were over the cutoff limit of 2,000 ms). Two separate 2×2×2 repeated-measures analyses of variance (ANOVAs) were conducted, one on accuracy and the other on RTs. The only significant result on accuracy was the main effect of task, *F*(1, 16) = 9.94, *MS~e~* = 75, *p*\<.01. Participants were more accurate in the orientation task (5.8% error) than in the color task (10.5% error), suggesting that orientation discrimination was easier than color discrimination. No other significant effects were found.
{#pone-0063264-g002}
10.1371/journal.pone.0063264.t001
###### Mean Error Rates (Percent Incorrect) for Experiment 1.
{#pone-0063264-t001-1}
SS SD DS DD
------------- ----- ----- ------ ----- ------ ----- ------ -----
Color 9.1 1.2 11.2 1.7 10.0 0.9 11.9 1.2
Orientation 6.0 1.2 5.9 1.0 5.3 1.2 6.1 1.2
Note: SS: the relevant-same-irrelevant-same condition; SD: the relevant-same-irrelevant-different condition; DS: the relevant-different-irrelevant-same condition; and DD: the relevant-different-irrelevant-different condition.
With regard to RTs, ANOVA showed a significant main effect of relevant feature, *F*(1, 16) = 5.32, *MS~e~* = 1986, *p*\<.05, indicating longer RTs when the relevant features of S1 and S2 matched (630 ms) rather than mismatched (612 ms). There was also a significant interaction between the relevant and irrelevant features, *F*(1, 16) = 4.84, *MS~e~* = 1680, *p*\<.05, indicating partial-repetition costs. Specifically, when S1 and S2 had the same relevant feature, RTs were longer when the irrelevant features of the stimuli differed (641 ms) rather than when they matched (619 ms). Similarly, when S1 and S2 had different relevant features, RTs were longer when their irrelevant features matched (617 ms) compared to when they differed (608 ms). No other results were significant, and there was no evidence of speed-accuracy tradeoffs.
It is worth noting that the significant partial-repetition costs were driven primarily by the interaction between the relevant and irrelevant features on the color trials. Although there was no statistically significant 3-way interaction involving the type of task, visual inspection of the data revealed that the hallmark of partial-repetition costs, i.e., slower RTs when S1 and S2 had a partial match rather than a complete mismatch, was more evident in the color task. This suggests that the tightness of the binding between color and orientation may be influenced by which feature is the task relevant feature. We will return to this topic in the discussion of Experiment 2.
Another aspect of data that merits discussion was the finding of a significant main effect of relevant feature, i.e., RT was longer when the relevant features of S1 and S2 differed rather than when they matched. As S1 and S2 were independent, this result was puzzling. Inspection of the data revealed that this effect was caused largely by the shorter RT in the DD condition relative to the other conditions in the color task and the longer RT in the SD condition compared with the other conditions in the orientation task (see [Figures 2A and 2B](#pone-0063264-g002){ref-type="fig"}). A possible way to interpret the pattern of data for the color task is to take the locations of S1 and S2 into consideration even though we did not manipulate that in the experiment. Recall that S1 and S2 were always presented at different locations. If location played a role in the spontaneous feature retrieval between S1 and S2, then the SD and DS conditions, together with the SS condition, should all be considered as partial match conditions, and this would result in the observed longer RTs in these conditions than in the DD condition, where none of the features in S1 and S2 matched. As for why this pattern of data appeared only in the color task, but not in the orientation task, we have no good explanations at present.
The most important finding of the experiment is the partial-repetition costs, especially in the color task. Despite the fact that the task irrelevant feature of S1 and S2 remained relatively salient throughout the experiment, the participants still showed evidence of feature retrieval. These results suggest that spontaneous feature retrieval can occur with changing behavioral goals.
Experiment 2 {#s3}
============
In Experiment 1, the relevant features of S1 and S2 were equally likely to be the same or different, and we found evidence for spontaneous retrieval of S1 features. In Experiment 2, we induced participants to inhibit S1 by matching the task relevant features of S1 and S2 on one-third of the trials, and mismatched them on the rest of the trials. As in Experiment 1, S1 and S2 were two-dimensional bars that varied in color and orientation, and their task irrelevant features were independent. This resulted in S1 and S2 being the same in color and orientation on one-sixth of the trials. Of specific interest was whether participants would again demonstrate partial-repetition costs. If such costs were found, this would indicate the spontaneous retrieval of features of S1 while S2 was being processed.
Methods {#s3a}
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The method was the same as that of Experiment 1 except for two differences. First, the task relevant features of S1 and S2 matched on one-third of the trials, and mismatched on two-thirds of the trials. Second, each of the four experimental blocks consisted of 120 trials, resulting in a total of 480 trials. The entire experiment took approximately 50 minutes to complete.
Results and Discussion {#s3b}
----------------------
[Figures 3A and 3B](#pone-0063264-g003){ref-type="fig"} show the mean RTs of correct responses on the color and orientation trials, respectively. A 2×2×2 repeated-measures ANOVA on mean RTs indicated that RT was longer when the relative features of S1 and S2 were the same (671 ms) than when they were different (599 ms), *F*(1, 23) = 29.52, *MS~e~* = 8417, *p*\<.0001. There was also a significant 2-way interaction between the relevant and irrelevant features, *F*(1, 23) = 4.48, *MS~e~* = 2323, *p*\<.05. A partial-repetition cost was found both when S1 and S2 had the same task relevant feature and when they had different task relevant features. Specifically, when S1 and S2 had the same relevant feature, RT was longer when their irrelevant features differed (i.e., a partial match between S1 and S2; RT = 678 ms) compared to when they matched (i.e., a match in both color and orientation between S1 and S2; RT = 664 ms). Similarly, when S1 and S2 had different relevant features, RTs were shorter when their irrelevant features also differed (i.e., a complete mismatch between S1 and S2; RT = 591 ms) relative to when they matched (i.e., a partial match between S1 and S2; RT = 607 ms). Finally, there was a significant 3-way interaction among task, relevant and irrelevant features, *F*(1, 23) = 5.83, *MS~e~* = 1683, *p*\<.05. The last result suggests that the participants showed different patterns of data in the color and orientation tasks. No other effects reached significance.
{#pone-0063264-g003}
To clarify the 3-way interaction, separate ANOVAs were performed on the color and orientation trials. On the color trials, RTs were faster when the colors of S1 and S2 were different (600 ms) relative to when they were the same (670 ms), *F*(1, 23) = 22.00, *MS~e~* = 5342, *p*\<.001. There was also a significant interaction between the relevant and irrelevant features, *F*(1, 23) = 9.37, *MS~e~* = 2158, *p*\<.01. Repeating the color of S1 in S2 impaired S2 responses when the orientation of S1 was changed (680 ms) relative to when its orientation was repeated (659 ms) in S2, *t*(23) = 1.73, *p*\<.05. Furthermore, changing the color between S1 and S2 prolonged S2 responses when the orientation of S1 was repeated (618 ms) compared with when it was changed (581 ms) in S2, *t*(23) = 3.11, *p*\<.01. The main effect of irrelevant feature was not significant, *F*(1, 23) \<1, *n.s*.
On the orientation trials, the results were different. The only significant effect was the main effect of relevant feature, with faster RTs when the orientations of S1 and S2 were different (599 ms) relative to when they were the same (673 ms), *F*(1, 23) = 32.01, *MS~e~* = 4096, *p*\<.0001. Neither the main effect of irrelevant feature nor the interaction between the relevant and irrelevant features was significant, *F*(1, 23) \<1, *n.s.* in both cases. There was no evidence of partial-repetition costs.
The mean error rates are illustrated in [Table 2](#pone-0063264-t002){ref-type="table"}. A 2×2×2 repeated-measures ANOVA on the accuracy data showed that the main effect of relevant feature was close to significance, *F*(1, 23) = 4.18, *MS~e~* = 14.17, *p* = .052. Consistent with the RT result, the error rate was lower when S1 and S2 had different relevant features (6.7%) than when they had the same relevant features (7.9%). There was also a significant interaction between the task and the relevant feature, *F*(1, 23) = 5.64, *MS~e~* = 13.61, *p*\<.05. Whereas the relevant features of S1 and S2 matched or differed did not influence participants' error rates in the color task (7.8% and 7.9% for the matched and mismatched trials, respectively), it affected the participants' performance in the orientation task, with a higher error rate when the relevant features of S1 and S2 matched (8.0%) rather than differed (5.6%). No other effects reached significance. There was no evidence of speed-accuracy trade-offs.
10.1371/journal.pone.0063264.t002
###### Mean Error Rates (Percent Incorrect) for Experiment 2.
{#pone-0063264-t002-2}
SS SD DS DD
------------- ----- ----- ----- ----- ----- ----- ----- -----
Color 7.6 1.1 7.9 0.9 7.4 0.7 8.3 0.7
Orientation 7.6 0.9 8.3 0.9 5.7 0.7 5.5 0.7
Note: SS: the relevant-same-irrelevant-same condition; SD: the relevant-same-irrelevant-different condition; DS: the relevant-different-irrelevant-same condition; and DD: the relevant-different-irrelevant-different condition.
Before we discuss the partial-repetition costs, it is necessary to establish that inhibition was evoked in Experiment 2. This is especially important given that repeating the relevant feature in S2 also led to longer RT in Experiment 1, where S1 and S2 were independent. One way to examine this issue is to conduct a cross-experiment analysis that compares the magnitude of the relevant feature effect in the two experiments. If inhibition was evoked in Experiment 2, the relevant feature effect should be larger in Experiment 2 than in Experiment 1.
We conducted a combined analysis on the RT data across the two experiments, using a mixed ANOVA with experiment as a between-subjects factor, and task, relevant and irrelevant features as within-subjects factors. For the sake of brevity, we report only the significant interactions with experiment, of which there was one. The interaction between the relevant feature and the experiment was highly significant, *F*(1, 39) = 10.16, *MS~e~* = 58719, *p* = .003. Whereas the difference in RT between the relevant-same and relevant-different trials was 72 ms in Experiment 2, it was 18 ms in Experiment 1. This result confirmed that the participants in the two experiments behaved differently when the relevant features of S1 and S2 matched vs. mismatched. Given the proportion of the relevant-same (one-third) vs. relevant-different (two-thrids) trials in Experiment 2, it seems reasonable to conclude that inhibition was evoked in Experiment 2.
To be cautious, we also examined the pattern of data in Experiment 2 while taking into account the fact that S1 and S2 were presented at different spatial locations. If we treat location as an important feature in Experiment 2, then the SS, SD, and DS conditions should all be considered as partial match conditions. A feature-retrieval-without-inhibition account would predict comparable RTs among these conditions. However, Tukey's Honestly Significant Differences test showed that in both the color and orientation tasks, RT was significantly faster in the DS condition than in both the SS and SD conditions (*p*\<.05 in all cases). This pattern of data is inconsistent with a feature-retrieval-without-inhibition account. Instead, it is consistent with the notion that inhibition was evoked in Experiment 2.
The most important finding of Experiment 2 was the partial-repetition costs in the color task. The participants took longer to respond to S2 when S1 and S2 differed in either color or orientation relative to when both features matched or mismatched. This pattern of data is similar to the results found in previous studies in which S1 was not inhibited ([@pone.0063264-Hommel1],[@pone.0063264-Hommel3],[@pone.0063264-Colzato1] and Experiment 1 in this study), suggesting that a similar mechanism may underlie the retrieval of features from a previously encountered object.
Partial-repetition costs were not found in the orientation task. While the exact nature of this null result was unclear, processing asymmetries between color and orientation have been reported in previous research [@pone.0063264-Colzato1],[@pone.0063264-Chen1],[@pone.0063264-Huang1]. In several experiments, Chen and Cave showed their participants stimulus displays that consisted of a target, a singleton distractor, and a homogenous group of other distractors. The task was to make a speeded color or orientation discrimination of the target on the basis of a task cue at the beginning of each trial. Responses to the target were either compatible or incompatible with a task irrelevant feature of the singleton distractor. The finding most relevant to the present experiment was that the effect of color on orientation differed from the effect of orientation on color. Whereas the orientation of the singleton had a reliable effect on the color of the target, the color of the singleton had negligible influence on the orientation of the target. A similar asymmetry between color and orientation was reported by Colzato et al. ([@pone.0063264-Colzato1] and Experiment 2), who manipulated the frequency of pairing between the different feature values in S2, and found a negligible effect of color on orientation when the conjunction between the specific feature values was infrequent. Interestingly, this null result was found only when S1 and S2 were simple geometric shapes such as bars, but not when they were bananas or strawberries. Huang et al. [@pone.0063264-Huang1] also reported no effect of color on orientation judgment when color was a task irrelevant feature of a preceding target. These results suggest that although features are bound more or less spontaneously, the tightness of the binding and how each feature affects the processing of a subsequent stimulus may depend on a number of factors, including the nature of an individual feature attended and the long-term association between the relevant features in memory. We will return to this topic in the general discussion section.
Experiment 3 {#s4}
============
The results of Experiment 2 show that the features of a recently inhibited object could be retrieved spontaneously when S1 and S2 were likely to be perceived as different instantiations of the same object. In Experiment 3, we changed the identity of S2 from a bar to a letter so that it was unlikely to be seen as a different state of S1. If the results of Experiment 2 were contingent upon the perceived object continuity between S1 and S2, the participants would show a different pattern of data in the present experiment. Moreover, the results of Experiment 3 should resemble the findings typically associated with object-based inhibition reported in previous studies [@pone.0063264-Chao1]--[@pone.0063264-Milliken2].
A number of experiments have shown that when representation of S1 interferes with the processing of S2, participants sometimes demonstrate object-based inhibition, i.e., delayed responses to S2 when it shared features with S1 even though these features were irrelevant to the participants' behavioral goals [@pone.0063264-Chao1]--[@pone.0063264-Milliken2]. Importantly, even though perceived object continuity was not manipulated in these experiments, the specific S1 and S2 that were used were never identical, and were therefore unlikely to be seen as belonging to the same object. For example, Chao and Yeh [@pone.0063264-Chao1],[@pone.0063264-Chao2] investigated the effect of a task irrelevant prime (i.e., S1) on the color naming latencies of a probe (i.e., S2). Both the prime and probe consisted of a single Stroop color word, with the prime written in white ink and the probe written in colored ink incongruent with its meaning. When the meaning of the prime matched the color of the probe on a small proportion of trials within an experiment, probe RTs were longer on these "match" trials relative to the "nonmatch" trials where the two stimuli were unrelated. As meaning was a task irrelevant dimension, these results indicate object-based inhibition.
An important feature in the above-mentioned experiments is their use of the Stroop stimuli in both the prime and probe displays. With Stroop stimuli, the response codes between the relevant feature of the probe target (i.e., color) and the irrelevant feature of the prime distractor (i.e., meaning) were identical, making it unclear whether object-based inhibition would be evoked in experiments that use other visual stimuli.
In Experiment 3, S1 and S2 belonged to different types of stimuli. If features from S1 could only be spontaneously retrieved when object continuity was perceived between successive items, and if object-based inhibition could be generalized to non-Stroop stimuli, we should find slower RTs to S2 not only when S1 and S2 have the same rather than different relevant features, but also when they have the same rather than different irrelevant feature.
Methods {#s4a}
-------
The method was the same as that of Experiment 2 except for the identity of S2. To induce participants to see S1 and S2 as belonging to different objects, we changed the identity of S2 from a bar to a letter, making it a different type of object from S1. Instead of a colored bar, it was a colored letter: a V for some participants (N = 18), and a T for the other participants (N = 20). In both cases, the letter was written in 28-point Geneva font. Since bars and letters are different classes of objects, it was assumed that they would be seen as different entities. As in Experiment 2, the letter was tilted 45° to the left or right from the vertical, and the relevant features of S1 and S2 were the same on one-third of the trials, and different on the rest of the trials. Thirty-eight new volunteers from the same participant pool took part in the experiment.
Results and Discussion {#s4b}
----------------------
The data from two participants in the V group were excluded from analyses due to high error rates (\>50% in multiple conditions). A four-factor (letter×task×relevant feature×irrelevant feature) repeated-measures ANOVA on participants' mean RTs showed no significant effects concerning the specific letter used in the experiment, so the data from the two groups of participants were pooled in the rest of the analyses. The mean RTs for the color and orientation tasks are shown in [Figures 4A and 4B](#pone-0063264-g004){ref-type="fig"}, respectively. As in previous experiments, we performed a 2×2×2 repeated-measures ANOVA on participants' mean RTs. Responses were again faster when the relevant features of S1 and S2 were different (542 ms) compared with when they were the same (586 ms), *F*(1, 35) = 29.97, *MS~e~* = 4617, *p*\<.0001. Moreover, RTs were also faster when the irrelevant features of S1 and S2 were different (561 ms) rather than the same (567 ms), *F*(1, 35) = 4.27, *MS~e~* = 620, *p*\<.05. The three-way interaction among task, relevant, and irrelevant features was also significant, *F*(1, 35) = 4.13, *MS~e~* = 617, *p* = .050. There were no other significant results.
{#pone-0063264-g004}
The color and orientation trials were again analyzed separately. In the color task, both the relevant and irrelevant features affected performance. RTs were slower when S1 and S2 had the same relevant feature (592 ms) compared with different relevant features (549 ms), *F*(1, 35) = 23.13, *MS~e~* = 2895, *p*\<.001. Importantly, unlike the results of Experiment 2, RTs were also slower when S1 and S2 had the same irrelevant feature (577 ms) relative to different irrelevant features (564 ms), *F*(1, 35) = 7.66, *MS~e~* = 762, *p*\<.01. There was no significant interaction between the relevant and irrelevant features, *F*(1, 35) = 2.65, *MS~e~* = 1082, *p*\>.10. These results provide no evidence for feature retrieval. Instead, they suggest that both the relevant and irrelevant features of S1 were inhibited in the color task.
The results in the orientation task were similar to those found in Experiment 2. Participants again showed evidence of inhibiting the relevant feature, with longer RTs when S1 and S2 had the same relevant feature (579 ms) than when they had different relevant features (535 ms), *F*(1, 35) = 26.33, *MS~e~* = 2713, *p*\<.0001. Neither the main effect of the irrelevant feature nor the interaction between the relevant and irrelevant features was significant, *F*(1, 35) \<1 in both cases. These results echoed those found in Experiment 2 in that while orientation appeared to influence color discrimination, color did not affect orientation judgment.
The error rates are shown in [Table 3](#pone-0063264-t003){ref-type="table"}. Consistent with the RT data, error rates were higher when the relevant features of S1 and S2 matched (10.9%) rather than mismatched (8.2%), *F*(1, 35) = 5.78, *MS~e~* = 89, *p*\<.05. However, there were also a significant main effect of irrelevant feature, *F*(1, 35) = 5.0, *MS~e~* = 21, *p*\<.05, and a near significant interaction between task and relevant feature, *F*(1, 35) = 3.9, *MS~e~* = 24, *p* = .055. Subsequent analyses revealed that whereas there were no reliable main effects or an interaction in the color task, there were two significant main effects in the orientation task. Participants made more errors when the task relevant features of S1 and S2 matched (11.2%) rather than mismatched (7.4%), *F*(1, 35) = 6.98, *MS~e~* = 75, *p*\<.05. Surprisingly, they also made more errors when the task irrelevant features of S1 and S2 differed (10.1%) compared with when they matched (8.5%), *F*(1, 35) = 5.46, *MS~e~* = 17, *p*\<.05. The last result was unexpected, and we have no explanation for it. There was no significant interaction between the relevant and irrelevant features, *F*(1, 35) \<.1, *n.s*.
10.1371/journal.pone.0063264.t003
###### Mean Error Rates (Percent Incorrect) for Experiment 3.
{#pone-0063264-t003-3}
SS SD DS DD
------------- ------ ----- ------ ----- ----- ----- ----- -----
Color 10.5 0.9 10.8 0.9 8.4 0.8 9.8 0.8
Orientation 10.4 0.8 12.1 1.4 6.7 0.9 8.1 0.9
Note: SS: the relevant-same-irrelevant-same condition; SD: the relevant-same-irrelevant-different condition; DS: the relevant-different-irrelevant-same condition; and DD: the relevant-different-irrelevant-different condition.
To verify that the pattern of data in Experiments 2 and 3 differed significantly, we did a combined analysis on the RT data across the two experiments with experiment as a between-subjects factor, and task, relevant, and irrelevant features as within-subjects factors. Again, for the sake of brevity, we report only the significant interactions that involve experiment. Two effects were found. One was a significant 3-way interaction among experiment, relevant, and irrelevant features, *F*(1, 58) = 5.57, *MS~e~* = 9099, *p* = .02. The second was a significant 4-way interaction, *F*(1, 58) = 11.38, *MS~e~* = 11711, *p* = .001. Subsequent analyses on the color and orientation trials separately indicated that the 4-way interaction arose primarily from the participants in the two experiments behaving differently in the color task, where a significant 3-way interaction of experiment, relevant, and irrelevant features was found, *F*(1, 58) = 13.74, *MS~e~* = 20728, *p* = .001. A similar 3-way interaction was not found in the orientation tasks, *F*(1, 58) \<1, *ns*. These results confirmed that the pattern of data in Experiments 2 and 3 differed in the color task. Thus, unlike the participants in Experiment 2, who showed a crossover interaction between the relevant and irrelevant features of S1 and S2 on the color trials (i.e., the partial-repetition costs that were indicative of feature retrieval from S1), the participants in Experiment 3 did not show a crossover interaction. Instead, they showed main effects of the relevant and irrelevant features, with slower RTs both when the task relevant features of S1 and S2 matched rather than mismatched, and when the task irrelevant features of S1 and S2 matched rather than mismatched. These results are consistent with the object-based inhibition observed in previous studies where S1 and S2 were also different [@pone.0063264-Chao1]--[@pone.0063264-Milliken2].
However, despite a significant main effect of irrelevant feature in the color task, one may notice that the effect was driven largely by the differential RTs between the SS and SD conditions (a difference of 22 ms) instead of between the DS and DD conditions (a difference of 4 ms). How can we explain this pattern of data?
If we believe that attention is object-based in addition to location-based ([@pone.0063264-Kahneman2],[@pone.0063264-Chen2]--[@pone.0063264-Kahneman3]; also see [@pone.0063264-Chen3] for a review) and that attending to a stimulus causes the integration of the features that belong to the attended stimulus [@pone.0063264-Allport1]--[@pone.0063264-Treisman1], then the above-mentioned different pattern of data can be explained in the following way. Let us suppose that on a given color trial, S1 was a red bar with a left orientation. As "red" and "left" belonged to the same object, the two features were associated and were both inhibited. When the relevant features of S1 and S2 were identical, S2 would be a red letter with a left orientation in the SS condition, and a red letter with a right orientation in the SD condition. Because "red" was associated with "left" but not with "right", responses would be delayed when S2 was red and had a left orientation in the SS condition, compared with a right orientation in the SD condition. Similar reasoning can be applied to those trials in which the relevant features of S1 and S2 were different. If we again suppose that S1 was a red bar with a left orientation, S2 would be a green letter with a left orientation in the DS condition and a green letter with a right orientation in the DD condition. As the inhibited "left" was not associated with "green", responses to S2 (whose color was green) would be independent of its specific orientation, resulting in comparable RTs between the DS and the DD conditions. Thus, our data are consistent with an object-based inhibition of S1 even though there was no appreciative difference in RTs between the DS and DD condition.
The results of Experiment 3 indicate that spontaneous feature retrieval between S1 and S2 is influenced by perceived object continuity. However, a different conclusion was reached in a recent study by van Dam and Hommel [@pone.0063264-vanDam1]. In a series of experiments (Experiments 1 through 4), the participants in van Dam and Hommel were shown two sequentially presented displays (S1 and S2), each consisted of a red or green circular object containing a smaller yellow elliptic object that was either vertically or horizontally orientated. The task was to determine the orientation of the elliptic object in S2. The principal manipulations were the color of the circular object and the orientation of the elliptic object between S1 and S2. S1 and S2 could be completely matched (i.e., same color and orientation), partially matched (i.e., same color but different orientation or vice versa), or completely mismatched (i.e., different color and orientation). In addition to these within-experiment manipulations, the authors also systematically varied the relationship between the circular and elliptic objects so that from Experiment 1 through 4, these objects were increasingly more likely to be perceived as two distinct objects (e.g., an apple partly occluded by a banana in Experiment 4) rather than as a single object (e.g., a colored ball with a horizontal or vertical stripe in Experiment 1). Partial repetition cost, which was calculated as the mean of color repeated and orientation repeated trials minus the mean of the neither repeated and both repeated trials, was found in all the four experiments. Furthermore, the magnitude of the cost was comparable across the experiments. Based on these results, the authors concluded that object cues are unlikely to be relevant for the retrieval of features in visual perception.
It is interesting to note that although the conclusion reached by van Dam and Hommel [@pone.0063264-vanDam1] was different from the conclusion we reached in the present study, a careful examination of their data ([Table 1](#pone-0063264-t001){ref-type="table"}, p. 1189) revealed that their conclusion was based largely on the way the partial repetition cost was calculated. If we compare the reaction times of those trials in which orientation, i.e., the task relevant feature, was repeated from S1 and S2 (a partial match) with those trials in which neither orientation nor color was repeated from S1 and S2 (a complete mismatch), there was a systematic decrease in the magnitude of the partial repetition cost from Experiment 1 to Experiment 4 (the partial repetition costs were: 19 ms, 9 ms, 5 ms, and −6 ms from Experiments 1 to 4, respectively). As the stimuli from Experiment 1 to Experiment 4 were increasingly more likely to be perceived as two distinct objects rather than as a single object, these results suggest that perceived object continuity can influence, at least to some degree, the retrieval of features between two successive stimuli.
General Discussion {#s5}
==================
Previous work has established that when two objects are in close spatiotemporal sequence, attention to an object can trigger the retrieval of features of a previously viewed object [@pone.0063264-Hommel1],[@pone.0063264-Hommel3],[@pone.0063264-Kahneman1]. In the present experiments, we generalized this finding to an object that had recently been actively inhibited. Experiments 1 and 2 showed that when two successively presented stimuli could be seen as different states of the same object, attention to S2 would trigger the retrieval of features from S1, regardless of whether S1 and S2 were independent (Experiment 1), or whether S1 was known to be different from S2 on a majority of trials (Experiment 2). [Experiment 3](#s4){ref-type="sec"} demonstrated that inducing participants to see S1 and S2 as different objects could disrupt the feature retrieval process, resulting in performance consistent with object-based inhibition. Of course, because we used letters as S2 in Experiment 3, and the processing of letters may differ from that of other visual stimuli in non-trivial ways, it is unclear to what degree the present results were caused by the specific stimuli used in our experiments. Future research is needed to explore this issue.
Feature Retrieval and Object-Based Inhibition {#s5a}
---------------------------------------------
Although we explained the results of Experiment 2 in terms of spontaneous feature retrieval and the results of Experiment 3 in terms of object-based inhibition, we do not believe that these two mechanisms are mutually exclusive. In fact, we consider it likely that object-based inhibition was also evoked in Experiment 2, and that the partial-repetition costs found in the color task were the results of competition between the two mechanisms. Evidence for the spontaneous feature retrieval account comes mainly from the slower RT in the SD condition than in the SS condition. However, if reactivating an inhibited object took less time than resolving the conflicting codes associated with S1 and S2, RTs would be faster when S1 and S2 matched in both color and orientation in the SS condition relative to when only one of these features was matched in the SD condition, even though object-based inhibition had been deployed (i.e., both the relevant and irrelevant features of S1 were inhibited). Thus, the lack of behavioral manifestation of object-based inhibition does not necessarily imply that the latter was not evoked. The effect of object-based inhibition could be masked by a fast-acting feature retrieval process. The idea that participants could inhibit an object under some circumstances with no measurable inhibitory effects is consistent with the results of a recent study by Wyatt and Machado [@pone.0063264-Wyatt1]. They manipulated the response compatibility and stimulus-onset-asynchrony (SOA) between the target and distractors (with the onset of the distractors always preceding that of the target), and found evidence for distractor inhibition even though their participants showed no significant negative compatibility effect when the distractor-target SOA was long.
The notion that multiple mechanisms can co-exist within a single paradigm is not new. It has been shown in experiments on object-based attention (see [@pone.0063264-Chen3] for a review) and in experiments that used the inhibition of return (IOR) paradigm [@pone.0063264-Chou1]--[@pone.0063264-Tipper4]. In both types of experiments, attention is found to select both location and object, either in the form of facilitation (in object-based attention studies) or inhibition (in object-based IOR), suggesting that multiple mechanisms can exist within a single task.
The Role of Location in Feature Retrieval {#s5b}
-----------------------------------------
In their seminal paper on the object file theory, Kahneman et al. [@pone.0063264-Kahneman1] stressed the importance of location in the retrieval of features of a previously viewed object. Their participants (in Study 3) showed evidence for feature retrieval when a spatial correspondence could be found between S1 and S2 via apparent motion. In contrast, a similar effect was not found when the locations of S1 and S2 across different frames did not give rise to the perception that S1 and S2 were different states of the same object. These results led the authors to the conclusion that object files are addressed in location instead of in non-spatial object features such as color or form. Evidence that supports the crucial role of location in the retrieval of features has also been reported by Mitroff and Alzarez [@pone.0063264-Mitroff1] and Saiki [@pone.0063264-Saiki1].
The unique role of location proposed in the object file theory is in line with many theories of attention that emphasize the importance of location in selective attention [@pone.0063264-Broadbent1]--[@pone.0063264-Wolfe1]. There is considerable evidence that attending to an object feature results in the selection of its location regardless of participants' behavioural goals ([@pone.0063264-Cave2]--[@pone.0063264-Tsal1], see also [@pone.0063264-Lamy1] for a review). In contrast, attending to an object's location does not lead to the encoding of an object feature such as color, shape, or texture when the feature in question is not task relevant [@pone.0063264-Chen4],[@pone.0063264-Tsal1]. These results suggest the unique role of location in visual attention.
In the present study, our participants showed evidence for episodic feature retrieval in Experiments 1 and 2 despite the fact that S2 was always at a different spatial location from S1. However, this does not mean that our result is contradictory to what was found in prior research. An important methodological difference between the present experiments and prior research [@pone.0063264-Kahneman1],[@pone.0063264-Mitroff1],[@pone.0063264-Saiki1] is the number of objects in the S1 display. Whereas a single object was used in the S1 display in Experiments 1 and 2 of the present study and in the other experiments that found evidence for episodic feature retrieval via non-spatial object features [@pone.0063264-Hommel1],[@pone.0063264-Hommel3],[@pone.0063264-Colzato1], multiple objects were used in the studies that showed the essential role of location in successful feature retrieval [@pone.0063264-Kahneman1],[@pone.0063264-Mitroff1],[@pone.0063264-Saiki1]. When multiple objects are present in the S1 display, there is uncertainty over the relationship between S2 and a specific S1, and spatial correspondence may be essential in establishing perceived object continuity between two successive stimuli. In contrast, when a single object appears in two sequential displays, the visual system may have a natural tendency to link them regardless of the difference in locations, so long as there is a reasonable match in other features. Thus, even though a location match did not appear to be required in some experiments ([@pone.0063264-Hommel1],[@pone.0063264-Hommel3],[@pone.0063264-Colzato1] and Experiments 1 and 2 of the present study), this by no means suggests that a spatiotemporal correspondence was not established in these studies.
Feature Asymmetry and S1 Inhibition {#s5c}
-----------------------------------
In the experiments reported here, there was a feature asymmetry between color and orientation. Whereas the color of S1 had a clear effect on the orientation judgement of S2, the orientation of S1 had a negligible influence on the color discrimination of S2. One possible explanation of this asymmetry is that in comparison with orientation, color may be a relatively difficult feature to inhibit, and may therefore take longer to be suppressed when it is a task irrelevant dimension. After all, under most circumstances, we do not expect an object to change color, but we do expect an object to change orientations. A book placed vertically on a bookshelf is likely to be seen as the same book when it is lying horizontally. However, a book with a red cover is unlikely to be seen as the same book when its cover becomes green.
If color is a relatively difficult irrelevant feature to inhibit when it is paired with orientation, the null result of color on orientation should disappear when facilitation rather than inhibition is applied to S1. This was indeed what was found in two experiments by Chen [@pone.0063264-Chen5], in which the relevant features of S1 and S2 matched on two-thirds of the trials, and mismatched on one-third of the trials. In both experiments, when S1 and S2 had the same relevant features, the irrelevant feature of S1 influenced the processing efficiency of S2 regardless of whether the task was color or orientation. In other words, having the same orientation between S1 and S2 facilitated the color judgment of S2. Similarly, having the same color between S1 and S2 also speeded up the orientation judgment of S2. There was no asymmetry between color and orientation when participants were encouraged to maintain an active representation of S2. Although these results do not address directly the question of the underlying cause of the asymmetry observed in the experiments reported here, they are consistent with our conjecture that the asymmetry could be due to the differential amount of time required to suppress color and orientation when they are features of the same objects. This interpretation is also consistent with the results of Chen and Cave [@pone.0063264-Chen1].
To conclude, the present experiments suggest that the features of a previously inhibited object can be retrieved spontaneously. However, such retrieval and its effect on a subsequent target depend on the perceived object continuity between the two successive stimuli.
We thank Michael Proulx, Liqiang Huang, Sarah Shomstein, and Bernhard Hommel for their helpful comments on an earlier version of this manuscript.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: ZC. Performed the experiments: ZC. Analyzed the data: ZC. Wrote the paper: ZC YYY.
|
{
"pile_set_name": "PubMed Central"
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|
Background
==========
Osteoporosis is the most common metabolic bone disease \[[@B1]\]. It is characterized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk \[[@B2]\]. Osteoporosis is a multi factorial disease and several factors such as, genetics, gender, age, race, weight, medical conditions, medication and life style risk factors are considered to be important determinants of it \[[@B3]\]. The Iranian Multicenter Osteoporosis Study (IMOS) estimated that the prevalence of osteoporosis among women older than 50 years is 6 percent, which is less than other countries such as Canada and Japan \[[@B4]\].
One of the most important modifiable factors in the development and maintenance of bone mass is nutrition \[[@B5]\]. Adequate nutrition plays a major role in the prevention and treatment of osteoporosis \[[@B6]\]. In recent years, there has been a resurgence of interest in studies concerning the role of elements in the development and maintenance of the skeleton \[[@B7]\].
Zinc (Zn) is an essential mineral that is a component of more than 200 enzymes and is known as to be necessary for normal collagen synthesis and mineralization of bone \[[@B8]\]. Copper (Cu), a cofactor for lysyl oxidase, is required in the cross-linking of collagen and elation. Cu deficiency causes inhibition of bone growth and osteoporosis \[[@B9]\]. The other element, Magnesium (Mg) appears to be important in bone cell activity. It is shown to be mitogenic for osteoblasts and its depletion causes cellular growth inhibition, in vitro \[[@B10]\].
The growths of nails, and their matrix components, are influenced by several physiological, pathological, and environmental factors \[[@B11]\]. Because of the slow rate of nail growth, the elemental composition of the nail is also expected to be affected by transient factors controlling serum components \[[@B12]\]. The mineral components of nail clippings may therefore reflect the long-term patterns of mineral metabolism such as Hypercreatinemia, Hyperthyroidism and Iron Deficiency Anemia \[[@B13],[@B14]\].
To understand the status of elements on postmenopausal women with osteoporosis, we have investigated the Zn, Cu and Mg Cu levels in postmenopausal women with osteoporosis and without osteoporosis.
Method
======
In this case--control study (May 2008 to may 2009) forty eight postmenopausal women aged 36--60 years, according to Bone Mineral Density (BMD) divided into two groups 30 Postmenopausal Osteoporotic women (case) (T score \> -2.5) and 18 non-Osteoporotic postmenopausal women (control) (T score \< -1.0) \[[@B11]\]. They were recruited among patients who applied to outpatient osteoporosis clinic of Tehran University of Medical Sciences (Dr Shariati Hospital). All the postmenopausal women who had passed one year of their last menstrual period were included in the study. Exclusion criteria were as follows:
1\. Arthritis rheumatoid, Diabetes mellitus, Systemic lupus erythematosus, Hypo or hyperthyroidism, Hypo or hyperparathyroidism, Hepatic failure, Renal failure, Cirrhosis, Cushing's syndrome, Adrenal failure, Cancers.
2\. Menstrual disorders as initial menstrual cycle after 18 and permanent discontinuation before 40 years old.
3\. Smoking (more than half a packet per day), addiction and the history of alcohol consumption for more than 5 years.
4\. Professional sports, past history of lumbar fractures, fractures because of simple falls, spinal deformity, and seeking admission in the last two weeks or complete best rest for 3 consecutive months.
5\. Previous Usage of Estrogen Progesterone, Furosemide, Antiepileptic drugs, Corticosteroids, Heparin, Thiazide and any trace mineral Supplements.
The study was performed in accordance with the Declaration of Helsinki and subsequent revisions and approved by ethics committee at Endocrinology and Metabolism Research Centre (EMRC). Informed consent was sought and obtained from individuals before enrollment into the study.
Having received the letters of consent, the related questionnaires were completed and clinical examinations such as height and weight were carried out. The weight of all participants with minimum of clothes by using digital scales (Seca) with an accuracy of 0.1 (kg) was measured. Height was measured using a wall stadiometer (precision 5 mm) with the patient standing upright and without shoes. The body mass index (BMI) was calculated by dividing subject's weight (kg) by the square of their height (m^2^). The baseline information of food intake was collected by using a 24*-*hour recall method on three consecutive days.
BMD was measured by DXA using Lunar DPX-MD device (Lunar Corporation, Madison, Wisconsin, 53713. USA). The DXA device was calibrated daily and weekly by using appropriated phantoms methods. To assess BMD, second to fourth lumbar spine and from the femur bone (neck, trochanter and the whole femur), bone density was calculated based on gr/cm^2^.
Blood and morning second void urine samples were collected after an overnight fast; precautions were taken to avoid contamination. Serum PTH, Vit D, Mg, Zn, Cu, ALT, AST, Alb, Cr, Ca, P, and TSH and urine, Zn, Cu and Mg were collected in the metal free plastic tubes. Toe nail clippings from all 10 toes were collected within 8 weeks of inclusion in the study and were stored in small plastic bags at room temperature. Solid sample of nail was washed immediately with distilled water and then alcohol dried and stored. The sufficient amounts of nail samples were weighed carefully and transfer in to a beaker and added 10 ml of concentrated nitric acid. Then, the solutions were heated for complete digestion. The resulted solution diluted in a volumetric flask with distilled water. Certain amount of blood and urine samples were also mixed with concentrated nitric acid to remove interfering of organic compounds, the resulted solution were then centrifuged.
Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES) has become the most appropriate technique for trace element determination \[[@B12]\]. A Varian ICP-AES (model: VISTA-MPX) was used for analysis. The amount of trace elements of each samples (serum, urine and nail) were measured by ICP-AES instrument using calibration method. This method was based on special software to calculate the numerical calculation analyze signal and reduce noise. In current method all the measurement done by continues cycle voltmeter. This means that in specified temperature range of Cyclic voltammeter (CV) wave potential is applied to electrode and provided number frequency that can shows any changes in flow produced with electrode.
There are no any cutoff for trace elements concentration in Iran, therefore we used the cutoffs' which was determined in similar articles. Cutoff for plasma Zn was 75--120 microgram/dl, and for plasma Cu and plasma Mg were considered 70--140 and 19.5-2.33 microgram/dl respectively. Serum chemical estimations were performed using enzymatic method (BioLif 24i, Premium, Tokyo, Boeki medical system, Japan) and Radioimmunoassay for Vit D (IDS kits, UK), PTH (Diasorin kits, USA) and TSH (Automatic Gama Counter, Wizard of Swiss).
The measurement of Zn, Cu and Mg in diets was done by modified software, Food Processing 2 (FP2).
The significance of difference in trace elements level in samples between two groups was tested using independent *t*-test analysis. Association between variables was determined using the Pearson's correlation analysis on Microsoft excel and SPSS software version 13.0 (California Inc.). A two sided P-value \<0.05 was considered statistically significant for the *t*-test and Pearson correlation analysis.
Result
======
Table [1](#T1){ref-type="table"} shows the baseline characteristics and biochemical findings of two groups based on BMD. There was no statistically significant difference between two groups in the characteristics.
######
Comparison of baseline characteristics between osteoporotic and control groups
**Variable** **Osteoporotic group** **Control group** **p- value**
------------------------------ ------------------------ ------------------- --------------
**Age \[years\]** 58.4 (6.4) 57.6 (4.7) NS
**BMI \[kg/m**^**2**^**\]** 28.4 (4.5) 27.7 (4.3) NS
**Menopausal age \[years\]** 51.6 (2.4) 50.8 (3.2) NS
**AST \[Iu/l\]** 22.86 (9.86) 28.18 (22.14) NS
**ALT(Iu/l)** 11.90 (5.67) 15.56 (13.08) NS
**Alp \[Iu/l\]** 176.03(115.20) 187.94 (130.10) NS
**Ca \[mg/dl\]** 8.7 (0.38) 9.09 (0.39) NS
**P \[mg/dl\]** 3.76 (0.40) 3.95 (0.59) NS
**Vit D \[ng/mg\]** **29.89 (31.70)** 12.56 (14.99) P = 0.04
**PTH \[pg/ml\]** 65.57 (35.91) 69.43 (24.55) NS
**Alb \[mg/dl\]** 5.01(0.31) 4.90 (0.38) NS
**Cr \[mg/dl\]** 0.91(0.21) 0.94 (0.10) NS
*BMI* body mass index, *PTH* paratormone hormone, *Alb* albumin, *P* phosphorus, *Ca* calcium, *ALT* alanine amino transpherase, *AST* aspartate amino transpherase, *Vit* vitamine, *Alb* albumin, *Alp* alkaline phosphatase. The level of vitamin D in osteoporotic group was higher than control group.
Table [2](#T2){ref-type="table"} shows the levels of trace elements in serum, urine and nail in two groups. The serum Mg level was significantly less in cases (P = 0.04) than in controls. The urine Zn level between osteoporotic and control group was significantly different (P \< 0.001).
######
Comparison of Cu, Mg, and Zn in plasma, urine, and nail between osteoporotic and control groups
**Trace elements** **Control group** **Osteoporotic** **P - value**
-------------------- ------------------- ------------------ --------------- ----
Serum **Cu \[ppm\]** 1.12 (0.53) 0.98 (0.43) NS
**Mg \[ppm\]** 19.80 (8.23) 15.08 (4.89) P = 0.04
**Zn \[ppm\]** 0.84 (0.43) 0.68 (0.23) NS
Nail **Cu \[ppm\]** 7.84 (10.71) 6.91(8.15) NS
**Mg \[ppm\]** 87.88 (78.21) 90.76 (97.30) NS
**Zn \[ppm\]** 81.60 (86.14) 82.57 (103.07) NS
Urine **Cu \[μg/g\]** 0.049 (0.013) 0.038 (0.024) NS
**Mg \[μg/g\]** 5.80 (1.36) 6.56 (2.24) NS
Zn \[μg/g\] 0.51(0.35) 1.72 (1.49) P\< 0.001
The significant positive correlation between urine Cu and serum TSH (p = 0.02 r =0.37). There was significantly negative correlation between PTH and urine Zn (p = 0.00, r = -0.53), (p = 0.01, r = -0.47).
There was no correlation between case and control groups in the nails' samples.
Discussion
==========
Some studies have shown the effect of nutrition on bone turnover and osteoporosis. In this study we investigate Zn, Cu and Mg in serum, urine and content of nail in post menopausal women that referred to clinic of osteoporosis - Shariati Hospital in Tehran University and divided them in two groups of osteoporotic and non -osteoporotic women.
Zn is involved in bone metabolism is based on observations that osteoporotic patients have increased urinary Zn losses \[[@B15]\]. Urine Zn excretion in osteoporotic patients was significantly lower than control group and there was not any meaningful difference in serum Zn in two groups. Actually bone demineralization in osteoporosis could cause high excretion of trace minerals, although in study group the level of Zn in plasma was higher than control group in our study. Similar to our study, Reginster et al. \[[@B16]\] reported no significant difference in Zn and Cu plasma levels between postmenopausal women with osteoporosis and non-osteoporotic subjects. Relea et al. \[[@B8]\] have reported that the urine clearance of Zn in postmenopausal women was higher than in the normal women. In contrast, several studies found that serum Zn and Cu levels were lower among patients with postmenopausal osteoporosis than controls \[[@B17]-[@B19]\]. A relationship exists between the excretion of Zn in 24-hour urine and the appearance of osteoporosis, and the urinary Zn/creatinine relationship is a factor to take in mind in the osteoporosis \[[@B20],[@B21]\].
Regarding the relationship between serum \[[@B22]-[@B24]\] and urine Zn, it seems that higher serum PTH in osteoporotic group comparing to normal amounts of PTH, rising Zn urinary excretion. The lack of significant difference in serum Zn between case and control groups can be attributed to higher than normal PTH concentration. In present study in consistency with other studies, urinary Zn levels in control group were higher than cases.
The indicators of discrepancy and accuracy of Zincuria are highly indicative of the value of this parameter as biochemical markers of bone resorption for the diagnosis of osteoporosis \[[@B15],[@B20]\].
We found that there is no significant difference in serum level of Zn in two groups and it may be because of having small population size. Several studies reported that serum Zn and Cu levels were lower among patients with postmenopausal osteoporosis than controls \[[@B25]-[@B27]\]. In contrast, Reginster et al. \[[@B16]\] significant difference in Zn and Cu plasma levels between two groups. Two studies \[[@B28],[@B29]\] found that serum Zn level was lower among the postmenopausal osteoporotic women than controls.
We found similar Cu levels in osteoporotic and non-osteoporotic postmenopausal women. Other studies \[[@B13],[@B14]\] have revealed that Cu deficiency resulted in postmenopausal osteoporosis. In a study by Conlan et al. \[[@B15]\] elderly patients with femoral neck fractures were found to have significantly lower serum Cu levels than controls adjusted for age and sex. Although the Cu content of bone was negatively correlated with bone Ca, bone density and collagen content in aging mice \[[@B16]\], bone Cu levels in human subjects with osteoporosis were found to be the same or slightly higher than normal individuals of the same age \[[@B10],[@B17]\].
Tranquilli et al. \[[@B30]\] reported that the uptake of Mg, P, and Cu are low in women with postmenopausal osteoporosis as compared to the control group and that this correlated with bone mineral content. Cross-sectional studies demonstrated a strong correlation between low BMD, low dietary Ca intake, and serum Cu levels in postmenopausal women \[[@B19]\].
Our study showed that the contents of serum Mg in osteoporotic patients is significantly lower than control group. Mg appears to be important in bone cell activity and contributes macro element quantities to bone ash and is essential for appropriate calcium metabolism, affecting calcium balance. Trabecular but not cortical bone was improved with per oral Mg in a small group of postmenopausal osteoporotic women \[[@B31],[@B32]\].
Carpenter \[[@B33]\] demonstrated that Mg deficiency has an effect on osteocalcin synthesis and secretion and that this resulted in decreased osteocalcin synthesis. Two studies \[[@B20],[@B21]\] reported that serum Mg in osteoporotic women were significantly lower than the value in matched normal subjects. Conversely, Steidl et al. \[[@B17]\] found a significant decrease in erythrocyte Mg content in patients with postmenopausal and senile osteoporosis but not in their serum levels.
Gur et al. \[[@B34]\] reported that Mg levels in serum were lower among patients with postmenopausal osteoporosis than the controls.
The level of Cu and Mg secretion in urine did not have any significant difference in two groups. Urine is a clinical specimen often used in medical diagnostics for monitoring of elements concentrations and osteoporosis. Basing on Dlugaszek study, they concluded that BMD and age of postmenopausal osteoporosis may impact greatly on the elemental status of 24-h urine \[[@B35]\]. The other research showed that Mg levels in 24-hour urinary excretion of Mg were increased in the postmenopausal women compared with the premenopausal women and postmenopausal hypermagnesiuria probably originates from increased intestinal magnesium absorption \[[@B36]\].
Schlemmer reported \[[@B37]\] that Ca supplementation influences the urinary excretion of Mg after menopause. Two studies \[[@B38],[@B39]\] found that women with osteoporosis displayed Mg deficiency and a highly significant correlation existed between increased Mg urinary excretion and low serum Ca.
There are a little evidence related to association between the contents of trace elements in nail and bone densitometry in postmenopausal women.
In our research there was no correlation between case and control groups in the nail samples.
Concentration of Zn in fingernails was significantly but negatively correlated with bone mineral density and a significant positive correlation of Ca/Zn ratio of fingernails was observed with bone mineral density. The content of Zn in nails may be affected by alterations in bone Zn status and a potential application of nail Zn level or Ca/Zn ratio as an indicator of postmenopausal and senile osteoporosis.
Three studies \[[@B25]-[@B27]\] in postmenopausal women have been shown lower nail Ca concentrations than in premenopausal women. Their BMD status showed a significant positive correlation with nail Ca content. The demonstration of an inverse relationship between nail Ca and Mg content may contribute to the understanding of the role of these minerals in the development of osteoporosis \[[@B28]\].
An Irish study demonstrated that nail testing as such a method is inconclusive but these preliminary findings suggest that changes in bone proteins seen in osteoporosis may be mirrored by changes related to structural proteins \[[@B40],[@B41]\].
In Table [1](#T1){ref-type="table"} level of vitamin D in osteoporotic group was higher than control group.
We had limitation in our study: 1. this study is a single center study in osteoporosis clinic of Tehran University of Medical Sciences (Dr Shariati Hospital) 1.not all of our studied patients comes in repeated follow ups, 2. Delay in result of laboratory tests 3. It's difficult to find the suitable samples.
Some studies have shown that all patients with osteoporosis treated with vitamin D and calcium are serum levels of calcium and vitamin D than their peers in non-osteoporotic, as shown in our study \[[@B5],[@B40]\].
Conclusion
==========
Our findings indicate that Urine Zn level could be considerable an appropriate marker for bone absorption, usage of Zn supplements in postmenopausal women may result a beneficial reduction in osteoporotic risk.
Abbreviations
=============
EMRI: Endocrinology and Metabolism Research Institute; TUMS: Tehran University of Medical Sciences; BMD: Bone mineral density; ICP-AES: Inductively coupled plasma -atomic emission spectrometry; BMI: Body mass index; DXA: Dual x-ray absorption; Zn: Zinc; Cu: Copper; Mg: Magnesium; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; Alp: Alkaline Phosphatase; Ca: Calcium; Vit D: Vitamin D; TSH: Thyroid - stimulating hormone; PTH: Parathyroid hormone; Alb: Albumin; Cr: Creatinine; P: Phosphorus.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
Conception and study design and coordination: RH, Drafting manuscript and Data analysis: RR, Participation in its sequence alignment: DK-A, Review of manuscript and important intellectual content: EN-E, All authors read and approved the final manuscript.
Acknowledgement
===============
The help of EMRI BMD unit especially patient's osteoporosis clinic Dr. Shariati Hospital is gratefully acknowledged.
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"pile_set_name": "PubMed Central"
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Introduction
============
*Angiostrongylus cantonensis*, a parasitic nematode (roundworm) that causes angiostrongyliasis, has been detected in lemurs, opossums, tamarins, falcons and non-human primates, and may endanger many wildlife species in areas where the infection is uncontrolled ([@B28]; [@B39]). In addition, since the first clinical diagnosis reported in Taiwan in 1945, the disease has been increasingly observed worldwide, including in previously non-epidemic countries such as France, Germany, the Caribbean region (including Jamaica), Brazil, Ecuador and South Africa ([@B30]; [@B3]). Angiostrongyliasis has been considered as an emerging global public health problem mainly because of the increase of international traffic facility which make it endemic previously in many unaffected areas ([@B13]).
*Angiostrongylus cantonensis* has a complicated life cycle. The first-stage larvae (L1) develop to third-stage larvae (L3) in about two weeks in intermediate hosts such as snails and slugs. When intermediate hosts with L3 are swallowed by permissive hosts, which are usually rats, the parasites finally develop to adults and live in the hosts pulmonary arteries and heart ([@B24]). However, if positive snails are eaten by non-permissive hosts such as humans and mice, the course of infection usually terminates at L5 in the host brain. When the parasites develop from L5 to adult in permissive hosts, the most notable physical change during this period is expansion of the magniloquent body size and this is generally considered to be the parasites most vigorous period of growth ([@B16]; [@B45]). Accordingly, energy and material metabolism are greater during this period than in any other life stages. At the same, there is more intense parasite-host interaction that is mediated by greater release of parasite excretion/secretion protein (ESP) and greater uptake of material from the host.
To reach its final parasitic sites, L5 needs to break through the blood-brain barrier when moving out of the brain ([@B6]). Proteolytic enzymes are assumed to be involved in blood-brain barrier disruption. A comprehensive understanding of the special developmental processes requires a comparison of the L5 and adult life stages to identify key factors related to rapid growth, parasite-host interaction and transmigration. In this regard, next generation sequencing (NGS), an increasingly popular and effective technology for genome-wide analysis of transcript sequences ([@B42]), has been successfully applied to transcriptomic profiling and characterization in the Strongylida and many other parasitic helminths ([@B5]).
In this study, the transcriptomes of L5 and female adults of *A. cantonenesis* were sequenced by NGS and assembled *de novo* with Trinity. Subsequently, genes that were differentially expressed between these two life-stages were identified by comparative transcriptomic analysis. Quantitative real-time polymerase chain reaction (qPCR) was used to validate the transcriptomic data. The findings described here provide a general picture of the gene expression profiles of *A. cantonensis* that may shed light on the development of this parasite and its survival in the host.
Material and Methods
====================
Animals
-------
Female Sprague Dawley (SD) rats (6-8 week-old, 120-150 g) and *Pomacea canaliculata* (channeled applesnail) were used to maintain the parasites and allow completion of the whole life cycle. The rats were housed with free access to water and food in the Xiamen University Laboratory Animal Center. This study was done in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (as approved by the State Council of the People\'s Republic of China). Moreover, the protocols involving rats were approved by the Committee for the Care and Ethics of Xiamen University Laboratory Animals (permit no. XMULAC2012-0122). L5 were obtained from the brains of rats infected with 200 L3 15 days previously. Female adult worms were collected as previously described ([@B53]). The worms were washed three times with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na~2~HPO~4~, 1.8 mM KH~2~PO~4~, pH 7.4) and then stored a −80 °C after soaking in RNAhold reagent (Transgene Biotech, Beijing, China) until RNA extraction.
RNA extraction, cDNA library preparation and RNA sequencing
-----------------------------------------------------------
Total RNA from each life stage was extracted with TransZol Up (Transgene Biotech) according to the manufacturer\'s protocols. The RNA concentration was measured using a Qubit^®^ RNA assay kit in a Qubit^®^ 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Two sequencing libraries were generated and 125 bp pair end-sequencing was done on an Illumina HiSeq 2500 platform by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).
Data quality control and transcriptome assembly
-----------------------------------------------
Raw data (raw reads) in fastq format were first processed through in-house perl scripts to obtain clean data (clean reads) by discarding reads containing adapters, reads containing ploy-N and low quality reads. Parent transcriptome (P transcriptome) assembly was done based on clean data from two samples using Trinity software (v.2.0.6), with all parameters set as default ([@B12]). Before annotation, unigenes were picked from the P transcriptome with CD-hit ([@B22]). Intactness of the assembled P transcriptome was assessed with the software tool BUSCO (Benchmarking Universal Single-Copy Orthologs) that is based on evolutionarily informed expectations of gene content, with default settings ([@B38]). The unigenes were then annotated with BLASTx (BLAST + v.2.2.25) by querying these to the following databases: NCBI non-redundant protein sequences (Nr), NCBI non-redundant nucleotide sequences (Nt), the Protein Family database (Pfam), Swiss-Prot, Gene Ontology (GO), the Eukaryotic Orthologous Groups database (KOG) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The E-value cutoff was set as 1 × 10^−5^.
Gene expression quantification and differentially expressed genes (DEGs) screening
----------------------------------------------------------------------------------
The transcriptomes of L5 and female adults were assembled with the clean data and the P transcriptome was set as the reference. Gene expression levels were estimated by RSEM (v.1.2.15) for each sample, as described ([@B21]). First, the read count for each gene was obtained from the result of clean data mapped back onto the assembled P transcriptome. Subsequently, the read count of each sequenced library was adjusted with the edger program package to 'reads per kilobase per million mapped' (RPKM). The differential expression analysis of two samples was done using the DEGseq R package (v.1.12.0) ([@B43]). The P-value was adjusted by the q-value; a q-value \< 0.005 and \|log~2~ (fold change)\| \> 1 were set as the threshold criteria for significant differential expression.
GO and KEGG pathway enrichment analysis of DEGs
-----------------------------------------------
For functional annotation, GO enrichment analysis of DEGs was implemented using the GOseq R packages (v1.10.0) based on a Wallenius non-central hyper-geometric distribution ([@B51]). KEGG pathway analysis of DEGs was then done through the KEGG database (<http://www.genome.jp/kegg/>) ([@B27]; [@B17]).
cDNA synthesis and quantitative real-time PCR (qPCR)
----------------------------------------------------
One microgram of RNA from each stage was converted to first strand cDNA using a First Strand cDNA synthesis kit (TaKaRa, Dalian, China), according to the manufacturer\'s instructions. Fifteen DEGs were significantly enriched in the lysosomal pathway and were randomly chosen for validation by qPCR. β-Actin was selected as an internal control based on previous studies ([@B53]; [@B25]). Primers were designed using Primer 3.0 and the sequences are listed in the supplementary materials (Table S1). Three biological replicates were used for the qPCRs of each gene. The reaction mixture (10 μL) consisted of 5 μL of SYBR Green PCR master mix, 0.5 μL (10 μM) of the forward and reverse primers, 1 μL of cDNA (diluted 10 times with double distilled water -- ddH~2~O) from each developmental stage and 3 μL of ddH~2~O. The cycling conditions involved an initial activation step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s and fluorescence acquisition at 60 °C for 30 s using a CFX96 Real Time system (BioRad, USA).
Statistical methods
-------------------
Statistical analysis of the qPCR data was done with SPSS18.0 (SPSS statistical package for Windows^®^) and Students *t*-test was used to detect significant differences. A p-value \< 0.05 indicated significance.
Results
=======
Summary of the raw sequence reads and assembly
----------------------------------------------
To obtain a global overview of the *A. cantonensis* transcriptome, clean data from each life stage were combined to provide a relatively comprehensive gene pool. Overall, 12.66 Gb of clean data (\~50-fold coverage of the whole genome) were used for *de novo* transcriptome assembly. Raw reads of the transcriptome have been deposited in the NCBI Short Read Archive (SRA, <http://www.ncbi.nlm.nih.gov/sra/>) under accession numbers SRR3199277 and SRR3199278. [Table 1](#t1){ref-type="table"} summarizes the sequence reads and P transcriptome. BUSCO analysis revealed that the P transcriptome was largely complete as we recovered 531 complete single-copy BUSCOs (68.1%) and an additional 71 fragmented BUSCOs (8.4%). Only 5.1% of the BUSCOs were found to be duplicated in the combined transcriptome, indicating that the transcriptome assembly was successful.
###### Summary of the sequence reads and P transcriptome.
Summary of sequence reads
---------------------------------------- ------------- ------------
Sample F L5
Raw reads 55,725,337 50,234,050
Clean reads 51,957,988 49,275,894
Clean reads (%) 93.24 98.09
Clean bases (Gb) 6.5 6.16
Q20 of clean reads (%) 96.09 96.89
Total clean bases (Gb) 12.66
Characteristics of the P transcriptome
Levels Transcripts Unigenes
Total nucleotides 99,597,159 51,401,554
Numbers (length \> 200 bp) (n) 115,369 82,769
Average length (bp) 863 621
N50 1,731 949
Shortest transcript (bp) 201
Longest transcript (bp) 20,809
Annotation of the P transcriptome
---------------------------------
Most (98.69%) of the P transcriptome unigenes were successfully annotated in less than one of the seven public databases indicated above. The homology and species-based distribution for all of proteins were analyzed against the Nr database and 82% of the sequences showed similarity \> 60% with their blast result. *Ancylostoma ceylanicum, Haemonchus contortus* and *Necator americanus* showed the greatest similarity in the species-based distribution ([Figure 1](#f1){ref-type="fig"}).
{#f1}
GO analysis assigned 12,525 unigenes to the corresponding GO terms, the details of which are shown in [Figure 2](#f2){ref-type="fig"}. Most of the biological process (BP) categories were related to cellular processes (GO: 0009987, 56.73%), metabolic processes (GO: 0008152, 49.64%) and single-organism processes (GO: 0044699, 45.54%), while most unigenes were sorted into the cell part (GO: 0044464, 31.67%), cell (GO: 0005623, 31.67%) and organelle (GO: 0043226, 20.77%) in the cell component (CC) category. Binding (GO: 0005488, 50.05%) and catalytic activity (GO: 0003824, 40.3%) were the main GO terms of the molecular function (MF) category.
{#f2}
Blasting to the KOG database for functional prediction and classification assigned 6,251 unigenes to 26 specific pathways ([Figure 3](#f3){ref-type="fig"}). 'Signal transduction mechanisms' (1,163) was the largest group, followed by 'General function prediction only' (1,016), 'Post-translational modification, protein turnover, chaperones' (664), and 'Transcription' (418).
{#f3}
A total of 4,257 unigenes were functionally assigned to the 259 KEGG pathways of five KEGG categories by KEGG pathway analysis. Lysine degradation (ko00310), purine metabolism (ko00230) and ribosome (ko03010) were the top three pathways sorted by the unigenes numbers involved (Table S2). The distribution of these unigenes in 32 KEGG sub-categories is shown in [Figure 4](#f4){ref-type="fig"}. Endocrine system, amino acid metabolism, translation, signal transduction, transport and catabolism were the highest enriched subcategories of each category.
{#f4}
Gene expression quantification and related analyses
---------------------------------------------------
Gene expression quantification was done as described in the Methods section. The mapping rate for L5 and female adults was 87.15 and 82.94, respectively. The percentages of both samples were \> 80%, suggesting that the transcriptome was well-assembled. Of the 1,346 DEGs analyzed (L5 *vs* female adults), 418 were up-regulated and 928 down-regulated ([Figure 5](#f5){ref-type="fig"}). c6135_g1 was the highest up-regulated gene with a log~2~ fold-change of 8.15; annotation information suggested that this gene was involved in nematode cuticle collagen synthesis. GO enrichment analysis of 1,346 DEGs showed that the extracellular region part (GO: 0044421), ion channel activity (GO: 0005216) and substrate-specific channel activity (GO: 0022838) were the top three of the 11 significantly enriched terms ([Figure 6](#f6){ref-type="fig"}).
{#f5}
{#f6}
Nine hundred and twenty-eight down-regulated unigenes were analyzed in the same way and revealed 23 significantly enriched GO terms ([Figure 7](#f7){ref-type="fig"}). Transporter activity and structural molecule activity were the dominant enriched terms of the MF category. Ion transport was the sole term in the BP category, which had 82 unigenes. In the CC category, the extracellular region, extracellular region part and extracellular matrix were the most highly enriched.
{#f7}
DEGs were further subjected to the KEGG database for pathway enrichment analysis. Lysosome was the only highly enriched KEGG pathway ([Figure 8](#f8){ref-type="fig"}). Lysosomes are membrane-delimited organelles that serve as the cell\'s main digestive compartment where various macromolecules are delivered for degradation and recycling. Lysosomes contain \> 60 hydrolases involved in this degradation in an acidic environment (\~pH 5). Seven of these (legumain \[LGMN\], cathepsins, N4-\[β-N-acetylglucosaminyl\]-L-asparaginase \[AGA\], palmitoyl-protein thioesterase, deoxyribonuclease II \[DNase II\], lysosomal α-mannosidase \[LAMAN\] and sphingomyelin phosphodiesterase \[SMPD1\]) were highly expressed in L5. V-type H^+^-transporting ATPase (V-ATPase), battenin (encoded by the CLN3 gene) and Niemann-pick type C (NPC) were the other three proteins associated with the lysosomal membrane and were down-regulated in female adults. The perturbation of these proteins usually leads to marked changes in lysosomal function.
{#f8}
Validation of DEGs by qPCR
--------------------------
Although biological repeats for the RNA-seq data were not done, qPCR (triplicate replicates) was used to check 15 out of 18 DEGs involved in the lysosomal pathway; 14 of the 15 unigenes revealed consistent expression patterns with the RNA-Seq data ([Figure 9](#f9){ref-type="fig"}).
{#f9}
Discussion
==========
The highest growth rate in *A. cantonensis* occurs from the L5 to the female adult stage, during which period body size increases sharply. In permissive hosts, L5 migrates from the brain to the pulmonary artery via the blood-brain barrier. However, the key factors in this important biological process remain largely unknown. To increase our understanding of this phenomenon, we compared the P transcriptome assembled with clean data obtained from the transcriptomes of L5 and female adults, two important life stages of *A. cantonensis*.
Although two genome assemblies have been reported for *A. cantonensis* ([@B30]; [@B50]), we did not use them as references for the following reasons. First, the assembly level for the first of these two studies was 'Conting' and therefore did not satisfy the requirement for a reference genome. The other study reached the 'Scaffold' level, but the Scaffold N50 (43,900) was too small, indicating that the genome was not well assembled. Second, the primary assembly unit did not have any assembled chromosomes or linkage groups so that no effective information could be extracted for downstream analysis. Third, before transcriptome assembly, the clean data for L5 and female adults had been separately mapped onto the genome sequences with Tophat2 (mismatch = 2) and the mapping rates were only 82.5% and 79.4%, respectively ([@B18]). Fourth, many reports have proven that *de novo* transcriptome assembly can also provide satisfactory results ([@B11]; [@B14]; [@B34]; [@B46]).
Although Lian and Wang reported that *A. cantonensis* had the highest homology to *Caenorhabditis* spp. (up to 97.87%) ([@B30]; [@B44]), our Nr annotation species-based distribution revealed that *A. cantonensis* was most similar to *Ancylostoma ceylanicum* (53.4%), followed by *Haemonchus contortus* (26.9%) and *Necator americanus* (10.1%) ([Figure 1B](#f1){ref-type="fig"}). These differences may have been caused by bias in the sequence information of different species stored in public databases. Furthermore, the rapid development of high-throughput sequencing technologies has narrowed the gap between model and non-model species because more information could be retrieved from public databases, even for non-model species such as *A. ceylanicum, H. contortus* and *N. americanus* ([@B2]; [@B35]; [@B40]). On the other hand, since these three species and *A. cantonensis* are parasitic nematodes, it would be more reasonable for *A. cantonensis* to have a higher homology with these species than with free-living nematodes.
Lysine, an essential amino acid that animals must obtain from their diet because they cannot synthesize it, is not only an important nutritional requirement for growth, but can also directly or indirectly regulate the immune system ([@B41]; [@B48]). Lysine-deficient diets result in greater body length in *Ascaridia galli* and a higher worm burden in chickens. A deficiency in dietary lysine limits protein synthesis and compromises antibody responses and cell-mediated immunity in chickens ([@B4]; [@B23]; [@B7]). Consequently, these parasites show greater growth and development in hosts with impaired immune systems. Various unigenes were found to be involved in the lysine degradation pathway (ko00310, 225) in the P transcriptome, indicating active lysine degradation in these life stages. The consumption of a large amount of host lysine or lysine-containing proteins by these parasites could make the host lysine-deficient, thereby indirectly weakening the host immune system. We therefore speculate that lysine insufficiency resulting from lysine degradation may be a neglected strategy of *A. cantonensis* for escaping immunological detection by the host.
The 23 GO terms and one KEGG pathway were significantly enriched with 928 down-regulated DEGs. Among these, eight of 23 enriched GO terms were related to ion channel transport that is essential for normal cell function ([@B9]). When L5 migrate from the brain to the final parasitic site there are significant changes in the physicochemical properties of the external environment of *A. cantonensis*. The concentrations of Na^+^ and Mg^2+^ are higher in cerebrospinal fluid (CSF), whereas the concentrations of K^+^, Ga^2+^, Cl^--^ and HCO~3~ ^--^ are higher in blood. In addition, the protein and glucose content in CSF is lower than in blood ([@B31]). Obviously, the parasite must adapt to different habitats by modulating the transmembrane transport of ions and organic solutes. Based on the results of the bioinformatics analysis, the bioprocess associated with ion channels and transporters will presumably be active in this period.
The cuticle or outer surface of all parasitic nematodes has important roles in locomotion (by providing points of attachment for muscle), growth, osmoregulation and parasite-host interactions ([@B49]). Cuticles often differ in surface protein expression and composition during the developmental stages in parasitic nematodes. Presumably, the worms adjust to changing developmental needs and environmental conditions, including escape from the hosts immunological system, particularly since cuticles are the target of a variety of host immunological responses that may kill the worms ([@B8]). The protective collagenous cuticle of these parasites is required for survival and the metalloproteinase astacin is a key enzyme involved in the collagen synthesis pathway. GO analyses of 928 down-regulated DEGs identified many GO terms related to muscles and cuticle formation. Based on this finding, we suggest that *A. cantonensis* invests considerable effort on cuticle and muscle biosynthesis in L5 to satisfy the rapid body expansion in the transition to female adulthood and to survive in different host tissues.
A further 23 enriched DEGs with metallopeptidase activity also attracted our attention. In addition to being associated with collagen synthesis, the essential role of these proteases in tissue invasion is well-documented. The metallopeptidases of bacteria such as *Candida albicans* and *Pseudomonas aeruginosa* are located in the cell wall and can degrade host extracellular matrix, thereby accelerating bacterial invasion ([@B32]; [@B29]). Similar host penetration functions for related proteases have been detected in many parasitic nematodes, including *Ancylostoma caninum, Necatora mericanus* and *Nippostrongylus brasiliensis* ([@B15]; [@B19]; [@B20]; [@B47]). Moreover, the work of Miyoshi indicated that metalloproteases present in the ESP of *A. cantonensis* infective larvae may suppress the host\'s immune response and allow parasite migration to the host\'s central nervous system by degrading human matrix metallopeptidase 9 (MMP-9) ([@B1]). Based on these considerations, we speculate that the increased expression of unigenes related to metallopeptidase in L5 may be an important strategy used by *A. cantonensis* for cuticle synthesis and blood-brain barrier permeation.
Lysosome was the only pathway that was markedly enriched in the KEGG pathway analysis of the L5 and female adult transcriptomes. Lysosomes contain \> 60 acidic lysosomal hydrolases belonging to different protein families. This enzymatic diversity makes it possible for lysosomes to participate in many important cellular processes, including protein secretion, macromolecular degradation, energy metabolism and pathogen defense ([@B33]; [@B36], [@B37]; [@B52]). Alterations in the expression of genes coding for lysosomal enzymes will have a marked influence on lysosomal activity. The cuticle is a highly structured, collagenous extracellular matrix secreted by the hypodermis surrounding the worms body ([@B10]) and lysosomes may be involved in the metabolism of cuticlar collagen. Lysosomes also degrade and recycle a broad range of macromolecules as an important source of nutrients thereby providing an ingenious method for controlling and equilibrating anabolic and catabolic cellular processes ([@B26]). Thus, during the high-speed growth stage, lysosomes must adapt to meet the demand for new material and more energy during cuticle formation.
In this work, we compared the transcriptomes of *A. cantonensis* L5 and female adults. Our findings help to explain two main events that can impact parasite development and survival during this period. First, to meet the demands of rapid body growth, especially expansion of the body surface, L5 requires a large amount of material to sustain the formation of the stratum corneum and its associated structures. Based on the special role of lysosomes in autophagy and apoptosis, we speculate that these phenomena may play a certain role in altering the old and new cuticular tissues of these parasites. Secondly, metallopeptidases secreted by these parasites may be key molecules in allowing the parasite to break through the blood-brain barrier by degrading the extracellular matrix of host tissues. These findings offer novel insights into parasite development, survival and host-parasite interactions and provide a solid foundation for understanding how these genes participate in these processes.
The authors have no competing interests with the publication of this work. This research was supported by the National Natural Science Foundation of China (grant no. 81171595, 2012) and by the National Parasite Germplasm Sharing Service Platform (grant no. TDRC-2017-22).
*Associate Editor: Ana Tereza Vasconcelos*
The following online material is available for this article:
Table S1- Primers for qPCR used in this study (designed with Primer 3.0).Click here for additional data file.
Table S2- Summary of KEGG pathway annotation results for the P transcriptome.Click here for additional data file.
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The enteric nervous system (ENS) is a complex neural structure devoted to the control of uptake of nutrients, secretion, blood flow, motility, inflammatory, and immunological processes of the alimentary tract[@b1]. The ENS is mainly represented by two cell populations, neurons and enteric glial cells (EGCs), the latter being present in a ratio of approximately 4:1 compared to neurons[@b2]. The old simplistic concept of EGCs as support cells for the ganglia and/or nutritive elements for the neurons has radically changed in the last years. In fact, this cell population plays a pivotal role in the economy of the gut and, in addition to the mechanical support function[@b3], exhibits trophic functions toward enteric neurons[@b4][@b5], is involved in enteric neurotransmission[@b6], has immunological functions[@b7][@b8], and is involved in dysmotility[@b9][@b10] and inflammatory[@b11][@b12] conditions of the gut.
In view of their specialized functions, EGCs require an extensive energy demand that is covered by the mitochondrial oxidative phosphorylation. It is conceivable that pathogens alter mitochondrial functions, with the opportunistic aim to affect the fate of the infected host cell. Indeed, *Clostridium difficile* toxins, as well as other bacterial toxins, induce severe mitochondrial dysfunction in several cell types[@b13][@b14][@b15][@b16]. Therefore, the possible protective role of EGCs during enteric bacterial insult is attracting increasing interest.
*C. difficile* is a normal inhabitant of the gut microbiota of about 1--3% of adults. However, after pretreatment with a broad spectrum of antibiotics, *C. difficile* can take advantage of the lack of commensal bacteria and colonize the large intestine. Here, it produces the enterotoxins TcdA and TcdB, which elicit an inflammatory immune response that allows them to cross the mucosal membrane. One of the most direct events attributed to TcdA and TcdB is the ability to breach the intestinal epithelial barrier and act on other cell types. In this context, the protective role of EGCs is essential. We found previously that TcdB exherts cytopathic and cytotoxic effect on EGCs, producing cell cycle arrest and apoptosis, and increases cell sensitivity to inflammatory cytokines[@b17]. To extend this study, we investigated the molecular mechanisms activated by EGCs as defense strategy in response to TcdB. As an early event, the toxin triggered cytosolic ROS production and a downstream pathway culminating in DNA fragmentation of a EGCs pool. This mechanism did not involve mitochondrial factors, nor deeply altered mitochondrial functionality, but was restricted to the cytosolic compartment, involving a pathway that flows through a NADPH oxidase/ROS/JNK/caspase-3 axis.
Results
=======
TcdB induced apoptosis in EGCs is mediated by ROS
-------------------------------------------------
Several cell types contrast pathogens by activating a respiratory burst. Since we observed previously that TcdB induced apoptosis in EGCs[@b17], and confirmed here apoptosis already at 18-hour treatment ([Supplementary Fig. S1](#S1){ref-type="supplementary-material"}), we investigated whether ROS act as an upstream signal in this process. Intracellular ROS levels were determined in EGCs treated with TcdB for 2--18 hours, by labeling cells with dichlorofluorescein (CM-H~2~DCFDA). A significant increase of total ROS was evidenced already at 2 hours, and persisted up to 18-hour treatment ([Fig. 1A](#f1){ref-type="fig"}). Despite the early ROS production, no significant LDH leakage occurred at 2 and 6 hours TcdB treatment and more than 75% LDH latency was preserved at 18-hour treatment, indicating that the majority of cells maintain plasma membrane integrity ([Fig. 1B](#f1){ref-type="fig"}). MTT reduction assays showed an increase of metabolic activity over time in control EGCs, whereas the activity remained almost constant in TcdB treated cells ([Fig. 1C](#f1){ref-type="fig"}), probably as the balance between cell proliferation arrest and cell damage. Next, we investigated the contribution of the mitochondrial compartment to ROS production by using MitoSox Red and dihydroethidium (DHE) probes. Flow cytometric analysis showed an increase of FL3 channel fluorescence in response to DHE oxidation in treated cells, indicating the early production of total cellular superoxide ion that accumulated within 18 hours ([Fig. 2A](#f2){ref-type="fig"}. Fluorimetric analysis of EGCs labeled with MitoSox Red showed that, compared to control, mitochondria-derived superoxide were unchanged after 2-hour treatment, but increased at 18 hours ([Fig. 2B](#f2){ref-type="fig"}), left and intermediate panels, trace *c*). Since this increase was not significantly detected by the fluoresceine probe ([Fig. 1A](#f1){ref-type="fig"}), it can be deduced that mitochondrial superoxide represents a small fraction of total ROS. The possible involvement of the NADPH oxidase (NOX) complex in TcdB-mediated ROS production[@b18] was investigated by pretreatment of EGCs with the specific NOX inhibitor ML171, followed by 2-hour treatment with TcdB. Total ROS production was reduced by ML171. At the same time, the antioxidant N-acetylcysteine (NAC) was also effective in scavenging ROS ([Fig. 2C](#f2){ref-type="fig"}). Preincubation of EGCs with ML171 did not prevent mitochondrial ROS production ([Fig. 2B](#f2){ref-type="fig"}, right panel, traces *c* and *e*). We further examined if ROS elevation was involved in TcdB-induced apoptosis. The preincubation with ML171 resulted in a significant decrease in apoptotic cell death, demonstrating the role of NOX complex in EGCs apoptosis ([Fig. 2D](#f2){ref-type="fig"}).
TcdB treatment and mitochondrial impairment
-------------------------------------------
Late mitochondrial ROS production prompted us to investigate whether TcdB affects mitochondrial energetics. First, we evaluated mitochondrial membrane potential (Δψ~m~) after incubation with 0.5 and 5 ng/ml TcdB for 2--18 hours. Although long lasting incubation (18 hours) produced in control a low decrease of Δψ~m~, which involves homogeneously the total cell pool, in TcdB-treated EGCs a well distinguished cell population (about 20%) shifted to very low Δψ~m~ values (C4 quarter) at 18-hour incubation ([Fig. 3A](#f3){ref-type="fig"}). Treatment for 24 hours did not impair further Δψ~m~ (data not shown). Respiratory chain inhibitors, used as positive controls, produced complete Δψ~m~ collapse. Accordingly, ATP decreased at 18 hours of TcdB treatment and was strongly reduced by the respiratory chain inhibitors ([Fig. 3B](#f3){ref-type="fig"}). It is worth to note that, in the conditions that caused Δψ~m~ decrease, no cytochrome c (cyt c) and apoptosis inducing factor (AIF) release outside mitochondria occurred ([Fig. 3C](#f3){ref-type="fig"}). However, in the same experimental conditions, TcdB induced DNA fragmentation ([Fig. 3D,E](#f3){ref-type="fig"}), demonstrating a cyt c independent apoptotic cell death.
TcdB regulates the activation of MAPKs and the expression of pro-apoptotic proteins
-----------------------------------------------------------------------------------
ROS have an important role as signal messengers in regulating cellular functions through the activation of MAPKs[@b19]. MAPK activation was analyzed by Western Blotting, using antibodies for both phosphorylated and total MAPK proteins. Phosphorylation of extracellular signal-regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) occurred at 2-hour treatment with TcdB and persisted until 18 hours ([Fig. 4A](#f4){ref-type="fig"}), without any change in total protein expression. p38 was constitutively phosphorylated and modestly activated by TcdB. As expected, the pretreatment of cells with 1 mM NAC blocked ERK and JNK phosphorylation ([Fig. 4B](#f4){ref-type="fig"}), confirming the role of ROS in MAPK activation.
To clarify the mechanism of TcdB-induced apoptotic cell death, we studied the effect of selective MAPK inhibitors. Pretreatment with the JNK inhibitor SP600125 reduced the apoptotic cell death, while the MEK/ERK inhibitor U0126 did not exert any protective effect ([Fig. 4C](#f4){ref-type="fig"}), suggesting that TcdB acts through the JNK pathway. JNK interacts with different pro- and anti-apoptotic proteins[@b20] of Bcl-2 family members. We reported previously that TcdB did not induce changes in the expression/activation of Bax, Bak, Bcl-2, and Bcl-X~L~[@b17]. One potential target of JNK is Bim. In neurons undergoing JNK-dependent apoptosis Bim was found transcriptionally up-regulated[@b21]. In EGCs, Bim increased already at 6-hour TcdB treatment and was elevated at 18 hours, whereas, as already observed, Bcl-X~L~ was not affected ([Fig. 5A](#f5){ref-type="fig"}). The specific JNK inhibitor SP600125 significantly attenuated TcdB-induced Bim expression, whereas the MEK/ERK inhibitor U0126 had no effect ([Fig. 5B](#f5){ref-type="fig"}), indicating that Bim up-regulation occurs through the ROS/JNK signalling pathway.
ROS-dependent caspases activation
---------------------------------
Caspase-3 is the early executor of TcdB-induced EGCs apoptosis[@b17], which occurs in the absence of cyt c release ([Fig. 3C](#f3){ref-type="fig"}). In view of the central role of caspase-3 and caspase-2 in the apoptotic cascade involving upstream ROS, we investigated whether the ROS/JNK axis could act as a signal for caspases activation in EGCs. Caspase activity was evaluated after treatment with TcdB for 2--18 hours, by monitoring AFC fluorescence that follows the cleavage of the fluorogenic peptides Ac-DEVD-AFC and Ac-VDAVD-AFC, substrates of caspase-3 and caspase-2, respectively. As expected, caspase-3 activity was detectable already at 2 hours and increased noticeably at 6 and 18 hours in treated cells respect to control, whereas caspase-2 followed a different time course, being modestly activated at 6 and 18 hours ([Fig. 6](#f6){ref-type="fig"}). Both caspases were completely inhibited by their specific inhibitors. The possible involvement of an extrinsic pathway in caspase-3 activation was excluded by the lack of caspase-8 activation ([Fig. 6](#f6){ref-type="fig"}). TcdB-induced caspase-3 activity was significantly decreased by the NOX inhibitor ML171 and by the JNK inhibitor SP600125, as well as by the ROS scavenger NAC ([Fig. 7A](#f7){ref-type="fig"}), indicating that caspase-3 activation is triggered by NOX through a pathway involving JNK. At the same time, ML171 and, to a much higher extent the specific caspase-3 inhibitor, reduced capase-2 activation ([Fig. 7A](#f7){ref-type="fig"}), suggesting its dependence on caspase-3. Interestingly, whereas the caspase-3 inhibitor reduced significantly DNA fragmentation, the caspase-2 inhibitor did not exhibit any effect ([Fig. 7B](#f7){ref-type="fig"}), confirming caspase-3 as the executor of apoptotic cell death.
Discussion
==========
The present study was aimed to define the molecular mechanisms of EGCs defense to TcdB aggressiveness. We found out the involvement of NOX complex, whose activity triggers a downstream apoptotic cascade program, unexpectedly devoid of a significant impact of the toxin on mitochondria, including cyt c release. Therefore, in these cells TcdB evokes a cytoplasmic response.TcdB affects several parameters of EGCs cellular integrity. We found previously that the toxin produces morphological alterations of cytoskeleton and affects cell viability[@b17], also demonstrated by the loss of cellular reducing power, as detected by the MTT reduction assay. A peculiar feature of EGCs following TcdB treatment is the early formation of the superoxide O~2~^−^ anion, as well as of total ROS ([Figs 1A](#f1){ref-type="fig"} and [2A](#f2){ref-type="fig"}). Indeed, a significant level of ROS was detectable at 2 hours and increased at 18 hours, the radical trap NAC favouring their disposal, while the specific NOX inhibitor ML171 stopping their release ([Fig. 2C](#f2){ref-type="fig"}). Since ML171 prevented DNA fragmentation ([Fig. 2D](#f2){ref-type="fig"}), it can be deduced that the apoptotic pathway triggered by the toxin originates from the NOX activity[@b22], whereas a minor role can be attributed to the late release of mitochondrial ROS ([Fig. 2B](#f2){ref-type="fig"}). The NOX/ROS pathway activates ERK and JNK, which are components of the serine/threonine MAPK family involved in signal transduction from cell membranes to nucleus[@b23]. The activation depends on the modification of amino acid residues of protein caused by ROS, although ROS molecular targets have not been clarified[@b24]. However, it is known that ROS act in thioredoxin oxidation, resulting in the activation of JNK[@b25].The p38, a subgroup of MAPK family, is usually activated by several stimuli[@b26]. In our experimental conditions, p38 is costitutively activated in control EGCs, no apparent effects being exerted by the toxin ([Fig. 4A](#f4){ref-type="fig"}). Therefore, EGCs are in a condition of channelling to apoptotic fate[@b27]. Of interest, using NAC to reduce free radicals and to block the downstream radical propagation, MAPKs were preserved in dephosphorylated form ([Fig. 4B](#f4){ref-type="fig"}). Once the signal of propagation has reached the two branches of the MAPK family, i.e. ERK and JNK, only the JNK pathway continues, as demonstrated by the fact that the specific JNK inhibitor SP600125, but not the MEK/ERK inhibitor U0126, could significantly attenuate TcdB-induced DNA fragmentation ([Fig. 4C](#f4){ref-type="fig"}). These results are in line with the distinguished role of activated ERK and JNK, the former in regulating cell differentiation and growth, the latter oriented towards stress-responsive gene expression and apoptosis[@b27][@b28][@b29].
Components of the Bcl-2 family of proteins are critical death regulators residing upstream of mitochondria[@b30]. Phosphorylated JNK activates proapoptotic factors of the Bcl-2 family, thus favouring the release of cyt c outside mitochondria, and the downstream cascade that leads to DNA fragmentation[@b20]. Interestingly, contrarily to what expected[@b31], JNK-activated up-regulation of Bim expression in TcdB-treated EGCs ([Fig. 5B](#f5){ref-type="fig"}) does not addresses cells to a mitochondrial apoptotic process. Indeed, the expression of the anti-apoptotic Bcl-X~L~ was invariable, and neither cyt c nor AIF were released ([Fig. 3C](#f3){ref-type="fig"}). Mitochondria of EGCs support almost completely the ATP need, as deduced by the complete ATP collapse that follows treatment with respiratory chain inhibitors ([Fig. 3B](#f3){ref-type="fig"}). In addition, considering that the toxin effect on mitochondrial functionality is restricted to a limited EGCs pool and ATP levels remain sustained, it can be deduced that toxin aggressiveness is mainly confined to the cytosolic compartment, being mitochondria preserved or, at most, involved in the late phase of TcdB treatment.
TcdB does not activate the apical caspase-8 in EGCs, thus excluding an extrinsic apoptotic executory program, and bringing into play other intracellular mechanisms. Pathogenic bacteria can induce cell death through caspase-2[@b32][@b33]. However, our results ruled out the direct caspase-2 intervention on TcdB-induced apoptosis, based on the observation that caspase-2 activation follows caspase-3 activation ([Fig. 6](#f6){ref-type="fig"}), and the caspase-2 inhibitor is not able to prevent DNA fragmentation ([Fig. 7B](#f7){ref-type="fig"}). Hence, caspase-2 activation seems to be dependent on caspase-3, which is the major player in this scenario[@b17]. Indeed, caspase-3 activity is detectable as early as at 2 hours and increases over time ([Fig. 6](#f6){ref-type="fig"}), and its inhibition slows DNA fragmentation ([Fig. 7B](#f7){ref-type="fig"}). In addition, the radical trap NAC and inhibitors along the NOX/JNK axis demonstrate the direct involvement of caspase-3 in the process ([Fig. 7A](#f7){ref-type="fig"}), whereas caspase-2 inhibition by the caspase-3 inhibitor indicates the dependence of caspase-2 on caspase-3 ([Fig. 7A](#f7){ref-type="fig"}).
The behaviour of EGCs to contrast enteric bacterial insult should be coerent with the proposed protective role of EGCs. We found that EGCs surviving to TcdB persistently secreted GDNF[@b17], which has been demonstrated to play an important role in maintaining the integrity of the intestinal epithelial barrier in normal and pathological conditions[@b34]. We speculate that the self-protection role of EGCs could also be exerted by activating cytosolic systems as a *buffer* that contrasts the toxin effect, preserving the mitochondrial power plant and sacrificing to death a subpopulation of EGCs. This represent a peculiar EGCs behavior, since in other cellular types, such as CHO cells and human colonocyte, *C. difficile* toxins produce an early extensive mitochondrial dysfunction, resulting in the generation of oxygen radicals and ATP depletion[@b13][@b14], whereas in epithelial cells TcdB produces early hyperpolarization of mitochondria[@b15]. Here we demonstrate that EGCs adopt a cellular defense pathway, typically found in phagocytic cells, in which NOX is activated, producing a respiratory burst.
In conclusion, all our experimental data support the evidence that caspase-3 activity in TcdB-treated EGCs depends on the upstream ROS, through a pathway initiated by NOX and mediated by JNK. These results are in agreement with previous data indicating that caspase-3 may be promoted by ROS, regardless of their mitochondrial or cytoplasmic origin[@b35][@b36]. Although points remain to be elucidated, such as the lack of mitochondrial apoptosis, despite Bim overexpression, a schematic pathway outlining the cascade of events triggered by TcdB in EGCs is proposed in [Fig. 8](#f8){ref-type="fig"}, which evidences caspase-3 as the promoter of DNA degradation, with NOX/ROS as the initiators throughout the activation of JNK.
Material and Methods
====================
Reagents and cells
------------------
Rat-transformed EGCs (EGC/PK060399egfr; ATCC^®^ CRL-2690^™^) were obtained from the ATCC (Manassas, VA, USA). Dulbecco's modified minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin, were from GIBCO Invitrogen. *C. difficile* toxin B (TcdB) was from Enzo Life Sciences. 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), dihydroethidium (DHE), 5-(and 6)-chloromethyl-2′,7′-dichlorohydrofluorescein diacetate (CM-H~2~DCFDA), and MitoSox Red were from Molecular Probes (Invitrogen, Italy). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), *N*-Acetyl-L-cysteine (NAC), staurosporine, ML171, Ac-DEVD-AFC, Ac-DEVD-CHO, Ac-VDVAD-AFC, Ac-VDVAD-CHO, Ac-LETD-AFC, Ac-LETD-CHO, phorbol 12-myristate 13-acetate (PMA), and ATP Bioluminescent Assay Kit were obtained from Sigma-Aldrich. U0126 and SP600125 were from Cell Signaling Technology. Antibodies: actin and *β*-tubulin mouse monoclonal (Sigma-Aldrich); cyt c mouse monoclonal, COX-IV mouse monoclonal, AIF goat polyclonal, goat anti-mouse and mouse anti-goat HRP-conjugated IgGs (Santa Cruz Biotechnology); JNK rabbit monoclonal, phospho-JNK (p-JNK) rabbit monoclonal, p44/42 MAPK (ERK1/2) rabbit monoclonal, phospho-p44/42 MAPK (p-ERK1/2) (Thr202/Tyr204) rabbit monoclonal, Bim rabbit polyclonal, Bcl-X~L~ rabbit monoclonal, p38 MAPK rabbit polyclonal, and phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal (Cell Signaling Technology).
Cell culture and treatments
---------------------------
EGCs were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 U/ml penicillin. Cells were treated with 0.5 or 5 ng/ml TcdB for the indicated times. Staurosporine (1 μM) was a positive control for apoptosis. Where indicated, 1 h prior to the addition of TcdB the following inhibitors were added to cells: 20 μM Ac-DEVD-CHO (caspase-3), 20 μM Ac-VDVAD-CHO (caspase-2), 20 μM Ac-LETD-CHO (caspase-8), 10 μM U0126 (MEK), 10 μM SP600125 (JNK), and 10 μM ML171 (NOX). We verified that these concentrations of each inhibitor did not exhibit any cytotoxic effect in EGCs (data not shown). NAC (1 mM) was added 1 h prior to TcdB as free radical scavenger. In each experiment, controls received treatments, excluding TcdB.
MTT assay
---------
EGCs (5 × 10^3^/well) were seeded in 96-well plates and treated with TcdB for different times, then MTT was added at 0.5 mg/ml and incubation continued for 2 hours at 37 °C. After removal of the medium, 200 μl DMSO was added to each well. Plates were then shaken for 30 min at 37 °C and the absorbance at 550 nm of reduced MTT was measured using an ELISA reader.
LDH assay
---------
EGCs (0.5 × 10^6^/well) were seeded in 6-well plates. Released and latent LDH activity were determined in the culture medium and in the cell pellet solubilized with 1% Triton X-100. The reaction rate was determined following NADH oxidation at 340 nm.
Fluorescent determination of ROS
--------------------------------
Cells were treated with TcdB in serum- and phenol red-free DMEM, and then stained for 30 min at 37 °C with fluorescent probes, prior to harvesting. Cellular superoxide ion was determined by flow cytometry (λ~exc~ 488 nm, λ~em~ 630 nm) as fluorescence intensity of oxidized DHE (5 μM). Total intracellular ROS were determined by CM-H~2~DCFDA (10 μM) (λ~exc~ 485 nm, λ~em~ 535 nm). For the detection of mitochondrial superoxide ion, the mitochondrial sensitive dye MitoSox Red (5 μM) was used (λ~exc~ 510 nm, λ~em~ 600 nm).
Apoptotic cell detection
------------------------
Apoptotic cells were quantified by flow cytometry analysis of fragmented DNA after PI staining (1 μg/ml) in hypotonic solution, using an EPICS XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Data were processed by an Intercomp and analyzed with Expo 32 software Beckman. For each sample, 10,000 events were recorded and cells with a hypoploid DNA content were quantified as apoptotic cells.
DNA fragmentation analysis
--------------------------
EGCs (0.5 × 10^6^/well) were seeded in 6-well plates and treated with TcdB. After harvesting, the cells were incubated in 250 μl digestion buffer (10 mM Tris--HCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 0.5% SDS, and 250 μg/ml proteinase K) at 50 °C overnight, followed by the addition of RNAase A (250 μg/ml). DNA was extracted by phenol--chloroform--isoamyl alcohol (25:24:1), subjected to electrophoresis on a 0.8% agarose gel at 70 V for 1 h, and visualized by ethidium bromide staining.
Cytofluorimetric analysis of mitochondrial membrane potential
-------------------------------------------------------------
Δψ~m~ was determined by using the JC-1 fluorescent probe (7.5 μM), which selectively enters into mitochondria and changes color from green to orange following Δψ~m~ increase. JC-1 fluorescence of mitochondria was detected as described[@b37], and red/green fluorescence emission (FL1/FL2) of particles was reported as a dot plot. The respiratory chain inhibitors rotenone (10 μM), antimycin (30 μM), and the uncoupler CCCP (10 μM) were used to provide complete depletion of Δψ~m~ (positive control). Flow cytometry analysis was performed using an EPICS XL-MCL (Beckman Coulter). Data were analyzed by a data management system (Expo 32 software, Beckman Coulter, UK). Green (FL1) and red (FL2) fluorescence emission of particles are reported.
ATP determination
-----------------
ATP levels were quantified by the ATP Bioluminescent Assay (FL-AA, Sigma) by using a calibrated ATP standard curve.
Cytochrome c release
--------------------
EGCs (5 × 10^6^) were resuspended in PBS and permeabilized with digitonin (0.1 mg/mg protein). After incubation for 10 min at 0 °C, post-digitonin supernatants were recovered by centrifuging (8,000 *g*, 10 min), whereas pellets were resuspended in a proper amount of PBS. Western blot analysis of cyt c and AIF was performed in pellet and supernatant. The efficiency of permeabilization was evaluated by PI staining of DNA.
Western blotting
----------------
Cell lysates were prepared using lysis buffer (1% SDS, 1 mM Na-vanadate, 10 mM Tris-HCl pH 7.4) in the presence of 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Proteins (40 μg) were subjected to SDS-PAGE and electroblotting on nitrocellulose membranes, which were probed with specific primary and HRP-conjugated secondary antibodies. Immunoblots were revealed by enhanced chemiluminescence reagent (Bio-Rad). Images were acquired using the VersaDoc 1000 imaging system and individual band densities were integrated by Quantity One software (BioRad).
Caspase activity
----------------
Caspase activity was assayed as previously described[@b37]. Cell suspensions (2 × 10^6^, about 300 μg protein in 100 μl) were lysed with 100 μL of lysis buffer containing 50 mM HEPES (pH 7.4), 10% sucrose, 0.1% Triton X-100, 5 mM EDTA, and 5 mM EGTA and incubated 40 min in ice. After centrifugation for 10 min at 9,000 *g* pellet was discarded and supernatant incubated 30 min at 30 °C in the presence of 2.5 mM DTT. The caspase-specific fluorogenic substrate Ac-DEVD-AFC (caspase-3), Ac-VDVAD-AFC (caspase-2), and Ac-LETD-AFC (caspase-8) was then added (20 μM) and incubation continued for 30 min. Samples were then added to 1.8 ml of PBS and elicited AFC fluorescence was recorded (λ~exc~ 400 nm and λ~em~ 505 nm), using a Shimazu RF-500 spectrofluorometer equipped with temperature control and magnetic stirrer device.
Statistical analyses
--------------------
The results, expressed as means ± SD of at least three independent experiments, were analyzed for statistical significance by Student's *t*-test. p-values \< 0.05 were considered significant.
Additional Information
======================
**How to cite this article:** Macchioni, L. *et al*. Enteric glial cells counteract *Clostridium difficile* Toxin B through an NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways. *Sci. Rep.* **7**, 45569; doi: 10.1038/srep45569 (2017).
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Material {#S1}
======================
###### Supplementary Figure S1
This research was supported by funds from the Department of Experimental Medicine, University of Perugia and by a grant from Fondazione Cassa di Risparmio di Perugia, Italy (bando 2015-2015.0327.021 Ricerca Scientifica e Tecnologica) to L.C.
The authors declare no competing financial interests.
**Author Contributions** L.C., L.M., and K.F. designed the experiments; L.M., M.D., M.P., and L.G. performed the experiments; K.F. and D.G. contributed to cytofluorimetric analysis; L.C., R.R., and L.M. wrote the manuscript; R.R., P.M., V.V., M.B., G.B., and L.C., contributed to the conception and critical revision of the manuscript.
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Introduction
============
Osteoporosis is a systemic skeletal disease common in older women, which is characterized by low bone mass and micro-architectural deterioration of bone tissue, followed by increasing bone fragility and susceptibility to fracture ([@b1-mmr-20-04-3746]). It is estimated that \>50% of women aged 50 years and older will sustain an osteoporotic fracture during their lifetime ([@b2-mmr-20-04-3746]). However, at present, it is difficult to achieve satisfactory clinical efficacy for the treatment of osteoporosis due to the side effects of drugs and patient compliance ([@b1-mmr-20-04-3746],[@b3-mmr-20-04-3746]). Promoting osteogenic differentiation is an important strategy to enhance bone mineral density and slow the development of osteoporosis ([@b4-mmr-20-04-3746]). Bone marrow mesenchymal stem cells (BMSCs) are mesodermal cells that can be obtained from multiple sources, including adipose tissue, periosteum and bone marrow ([@b5-mmr-20-04-3746]). Due to their unique capacity to differentiate into osteoblasts, chondrocytes and adipocytes, BMSCs are widely used in osteoporosis research worldwide ([@b6-mmr-20-04-3746],[@b7-mmr-20-04-3746]). *Psoralea corylifolia* L., a traditional Chinese herbal medicine, has a long history of clinical efficacy against conditions such as fractures, bone defects and osteoporosis ([@b8-mmr-20-04-3746]). Psoralen, as the main flavonoid active ingredient in *Psoralea corylifolia* L., was demonstrated to promote BMSCs to undergo osteogenic differentiation through activation of the bone morphogenetic protein (BMP) signalling pathway ([@b9-mmr-20-04-3746]). However, the mechanisms underlying the osteogenic differentiation effects of psoralen have not yet been fully elucidated, to the best of the authors\' knowledge, and the epigenetic regulatory mechanisms required investigation.
In recent decades, it has been demonstrated that short non-coding RNAs, microRNAs (miRNAs/miRs), are involved in many biological processes, including cell survival, proliferation and differentiation, while their aberrant expression can lead to the development of diseases ([@b10-mmr-20-04-3746]). Notably, miRNAs are also the central regulators of BMSCs in regulating osteogenic differentiation. miR-203 and miR-320 negatively regulated BMP-2-induced osteogenic differentiation by suppressing homeobox protein DLX-5 ([@b11-mmr-20-04-3746]). miR-195 inhibited the abnormal activation of osteogenic differentiation in MC3T3-E1 cells by targeting RAF proto-oncogene serine/threonine-protein kinase ([@b12-mmr-20-04-3746]). miR-495 inhibited new bone regeneration by targeting high mobility group at-hook 2 ([@b13-mmr-20-04-3746]). Therefore, the regulation of osteogenic transcription factors by miRNAs was demonstrated to be an effective strategy in regulating osteogenic differentiation. Runt-related transcription factor 2 (Runx2) is a transcription factor that is indispensable for skeletal development, and controls bone formation by acting as a signalling hub and transcriptional regulator to coordinate target gene expression ([@b14-mmr-20-04-3746],[@b15-mmr-20-04-3746]). However, the role of miRNAs in targeting Runx2 requires clarification in the osteogenic differentiation of BMSCs.
In the present study, BMSCs treated with psoralen were used to identify differentially expressed miRNAs and their target genes via miRNA microarray and bioinformatics analysis. Using overexpression or inhibition methods *in vitro*, the underlying mechanisms of miRNAs in psoralen-induced BMSC osteogenic differentiation were examined, providing a potential molecular therapeutic strategy for osteoporosis.
Materials and methods
=====================
### Isolation and culture of BMSCs
20 male Sprague-Dawley rats, 3 weeks old (weighing 140±20 g), obtained from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China), were sacrificed by decollation and were sterilised using 75% ethanol. The marrow from the femur and tibia was mixed with complete medium (low-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.)) and was gradient centrifuged at 900 × g for 30 min at room temperature with Percoll at a density of 1.073 g/ml. The cells were cultivated with complete medium, with fresh media changes every 3 days, and were incubated at 37°C in 5% CO~2~. Primary rat BMSCs were passaged 6--7 days later. As the BMSCs were obtained from rats, there were many other cells in the primary, first-passage and second-passage cells. The purity (\>95%) and cell viability were high in the third-passage BMSCs, and thus were used for subsequent experiments. All the rats received humane care in accordance with the guidelines set by the Care of Experimental Animals Committee of Guangzhou University of Chinese Medicine. Additionally, the present study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine. All rats were housed in a specific-pathogen-free facility (22±2°C, relative humidity 60±10%), under a 12/12 h light/dark cycle (lights on from 6:00 am), without restriction to food and water, and fasted 12 h before sacrifice.
### Cell Counting Kit-8 (CCK-8) assay of cell viability
Third-passage BMSCs were collected and seeded into 96-well plates at a density of 1×10^4^ cell/ml and were co-cultured with 20 µg/ml psoralen continuously (37°C), which was purchased from The National Institute for the Control of Pharmaceutical and Biological Products. Next, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added into each well after cell culture for 12, 24, 36, 48 and 72 h. The cells were incubated at 37°C for 2 h in the dark. The absorbance values at a wavelength of 490 nm were detected using a microplate reader (Bio-Rad Laboratories, Inc.). BMSCs that were not co-cultured with psoralen were considered the control group. Each experiment was repeated three times.
### Alizarin red staining (AR-S) after osteogenic differentiation
Third-passage BMSCs were seeded into 24-well plates at a density of 5×10^4^ cell/ml and then were divided into three groups: Psoralen group, positive control group and blank control group. BMSCs co-cultured with 20 µg/ml psoralen were considered the psoralen group. The positive control group was induced by α-Minimum Essential Medium (Gibco; Thermo Fisher Scientific, Inc.) containing 5×10^−5^ mol/l isobutyl xanthine, 2×10^−4^ mol/l indomethacin, 1×10^−5^ mol/l dexamethasone and 10 mg/l insulin. BMSCs that were not co-cultured with psoralen or osteogenic-inducing fluid were treated as the blank control group. Medium containing psoralen or osteogenic-inducing fluid was changed every 3 days. After 2 weeks, the BMSCs were fixed with 95% ethanol 10 min at room temperature and then were stained with 40 mM AR-S solution for 10 min at pH 4.2 (room temperature). Subsequently, the BMSCs were treated with 10% cetylpyridinium chloride in 10 mM sodium phosphate for 15 min at room temperature and then were washed with PBS for 15 min. Calcium mineral deposition was observed using a light microscope at three different views (magnifications, ×100 and 200) to compare the degree of osteogenic differentiation between the different groups.
### miRNA microarray analysis
Third-passage BMSCs were seeded at a density of 1×105 cell/ml in a 6-well plate. After reaching 80--85% confluence, the BMSCs treated with 20 µg/ml psoralen represented the psoralen group, while the BMSCs that did not receive treatment with psoralen represented the control group. After 72 h, the total RNA of the two groups was extracted using TRIzol^®^ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. miRNA microarray analysis was then performed by Guangzhou RiboBio Co., Ltd. The differentially expressed miRNAs were verified by reverse transcription-quantitative PCR (RT-qPCR).
### RT-qPCR
RT-qPCR was performed using the Prime Script™ RT reagent kit and SYBR Premix EX Taq II kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. The thermocycling conditions for qPCR were as follows: Pre-denatured at 95°C for 10 min, following by 40 cycles of 95°C denaturation for 10 sec, annealing at 60°C for 20 sec and finally extension 70°C for 15 sec. U6 was used as the internal reference for miRNA, and GAPDH was used as the internal reference for osteogenic-specific factors, and the relative expression levels were calculated using the 2^−ΔΔCq^ method ([@b16-mmr-20-04-3746]). The primers were synthesized by Sangon Biotech Co., Ltd. and were as follows: Runx2 (forward: 5′-TCTTAGAACAAATTCTGCCCTTT-3′; reverse: 5′-TGCTTTGGTCTTGAAATCACA-3′); Osterix (forward: 5′-AGAGATCTGAGCTGGGTAGAGG-3′; reverse: 5′-AAGAGAGCCTGGCAAGAGG-3′); alkaline phosphatase (ALP; forward: 5′-CCAACTCTTTTGTGCCAGAGA-3′; reverse: 5′-GGCTACATTGGTGTTGAGCTTTT-3′); GAPDH (forward: 5′-ATTTGGTCGTATTGGGCG-3′; reverse: 5′-TGGAAGATGGTGATGGGATT-3′); miR-122: (forward: 5′-GCGAAAGCATTTGCCAAGAA-3′, reverse: 5′-CATCACAGACCTGTTATTGC-3′); miR-154 (forward: 5′-TAGGTTATCCGTGTTGCCTTCG-3′; reverse: 5′-AAGGCAACACGAUAACCUAUU-3′); miR-488 (forward: 5′-CGGGGCAGCUCAGUACAG-3′; reverse: 5′-CAGTGCGTGTCGTGGAGT-3′); miR-205 (forward: 5′-CCTCCCTAAATCCTCCATCC-3′; reverse: 5′-TCTAGGAAGGACAGCCTCCA-3′); U6 (forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′).
### Bioinformatics analysis
TargetScan (<http://www.targetscan.org>), PicTar (<http://pictar.mdc-berlin.de>) and miRanda (<http://www.microrna.org>) were used to analyse potential miR-488 binding sites on the Runx2 3′-untranslated region (3′UTR). The consistency of the analyses and predictions using these three websites suggests that they are reliable, and the results were further verified by *in vitro* experiments.
### RNA oligoribonucleotide synthesis and transfection
The RNA oligoribonucleotides \[miR-488 mimics (5′-GGGTCTATTACCGTGAGAGTT), miR-488 inhibitor (TTGAGAGTGCCATTATCTGGG-3′), mimics control (5′-TACGTCCAAGGTCGGGCAGGAAGA-3′), inhibitor control (5′-UCCUCCGAACGUGUCACGUTT-3′), pcDNA 3.1-Runx2 and pcDNA 3.1-control\] used in the present study were synthesized by Shanghai GenePharma Co., Ltd. Prior to transfection, BMSCs were isolated and seeded (2×10^6^ cells/l) into 6-well plates and grown until they were 60--80% confluent. The cells were then transfected with 100 nM miR-488 mimics, miR-488 inhibitor, mimics control, inhibitor control, pcDNA 3.1-Runx2 or pcDNA 3.1-control for 6 h, using Lipofectamine^®^ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were then digested with 0.025% trypsin for 24 h and collected for further analyses, including RTq-PCR, western blotting, a luciferase reporter assay and immunocytochemistry.
### Luciferase reporter assay
Third-passage BMSCs were collected following 0.25% trypsin digestion when the BMSCs reached 80% confluence. The cells were transfected with 0.2 µg pLUC-Runx2 plasmid construct (Guangzhou RiboBio Co., Ltd.) and miR-488 mimics or inhibitor for 6 h using Lipofectamine^®^ 2000. The activity in each well was measured using the Dual Luciferase Reporter Assay System (Promega Corporation) to quantify the luminescent signal. Firefly luciferase was selected as the internal reference. The relative luciferase activity was calculated as the value of each group/the value of miR-control group.
### Rescue assays
The open reading frame (ORF) fragments of Runx2 without the 3′UTRs were amplified by PCR as aformentioned using Taq DNA Polymerase (Beijing Solarbio Science & Technology Co., Ltd.), and then Runx2 recombinant vectors were constructed using the pcDNA 3.1 plasmid (Shanghai GenePharma Co., Ltd.). The thermocycling conditions were as follows: Pre-denatured at 94°C for 5 min, followed by 35 cycles at 94°C for 10 sec, 60°C for 45 sec and 72°C for 1 min. Third-passage BMSCs were seeded at a density of 2×10^4^ cell/cm^2^ into 6-well plates and then were transfected with pcDNA 3.1-Runx2 or miR-488 mimics or co-transfected with both constructs. Cells transfected without any plasmid were treated as the control group. RT-qPCR, western blotting, AR-S staining and immunocytochemistry were then used to detect the degree of osteogenic differentiation.
### Western blotting
The protein was extracted from BMSCs by radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS). The supernatant was obtained by centrifugation at 12,000 × g at 4°C for 20 min, and the concentration of protein was determined by a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). Then 20 µg protein was added to 10% SDS-PAGE for electrophoresis at a constant electric current of 25 mA/gel to the bottom of separation gel. The proteins were then transferred onto a PVDF membrane through electrophoresis at normal temperature with constant voltage of 80 V for 100 min, and the membrane was blocked with 5% non-fat milk in PBS with Tween-20 solution at 4°C overnight. The blots were probed with primary antibodies against Runx2 (Abcam, ab114133, 1:2,000), Osterix (Abcam, ab209484, 1:2,000), ALP (Abcam, ab83259, 1:2,000) and β-actin (Abcam, ab8227, 1:2,000) at room temperature for 2 h, followed by incubation with relevant secondary antibodies (Abcam, ab97051, ab150118, 1:2,000) labelled with horseradish peroxidase at room temperature for 1 h and washing with TBS with 0.1% Tween-20. The protein expression was detected using a SuperSignal West Femto Maximum Sensitivity Substrate kit (Roche Applied Science). The relative value of the target protein was calculated by comparing with the corresponding internal reference. Densitometry was conducted using ImageJ (v1.8.0; National Institutes of Health).
### Immunocytochemistry
After transfection, the BMSCs were fixed with 4% polyoxymethylene for 20 min at room temperature, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich; Merck KGaA), and blocked with 0.1% bovine serum albumin (Roche Diagnostics) at room temperature for 30 min. Subsequently, the BMSCs were incubated overnight at 4°C with the following primary antibodies: Runx2 (Abcam, ab114133, 1:500), Osterix (Abcam, ab209484, 1:500), ALP (Abcam, ab83259, 1:500). After washing three times with PBS, the BMSCs were reacted with the appropriate secondary antibody (Bioss, Beijing, PV-0024, PV-0023, 1:100) for 30 min at 37°C. The cell nuclei were stained with haematoxylin (Abcam) at room temperature for 1 min and were visualized by light microscopy (magnification, ×200, Leica Microsystems GmbH). The BMSCs were observed at three different views to compare the degree of osteogenic differentiation between the different groups.
### Statistical analysis
Each experiment was repeated three times. SPSS 20.0 software (IBM Corp.) was used for the statistical analyses. The data are presented as the mean ± standard deviation. Student\'s t-test was used to compare the differences between two groups. One-way ANOVA was used to compare the differences between three or more groups, and Tukey\'s test was used as a post hoc test to determine which groups were significantly different from each other. P\<0.05 was considered to indicate a statistically significant difference. The plots were constructed using GraphPad Prism 6.0 (GraphPad Software, Inc.).
Results
=======
### Psoralen promotes the viability and osteogenic differentiation of BMSCs
The CCK-8 assay showed that cell viability was significantly elevated in BMSCs treated with psoralen at all time points, with the exception of 12 h ([Fig. 1A](#f1-mmr-20-04-3746){ref-type="fig"}). The AR-S showed negligible calcium mineral deposition in the blank control group, while similar calcium mineral deposition was observed in the psoralen and positive control groups ([Fig. 1B](#f1-mmr-20-04-3746){ref-type="fig"}).
### miR-488 is downregulated in BMSCs during psoralen-induced osteogenic differentiation
To study the different expression levels of miRNAs during psoralen-induced osteogenic differentiation, BMSCs were treated with psoralen for 3 days and then the cells were subjected to miRNA microarray analysis. In total, 91 miRNAs were differentially expressed between the control and psoralen groups. Compared with the control group, 55 miRNAs were upregulated and 36 were downregulated in the psoralen group. The data were analysed and illustrated in a heat map ([Fig. 2A](#f2-mmr-20-04-3746){ref-type="fig"}). Among the miRNAs, miR-122, miR-154, miR-488 and miR-205 were randomly selected to confirm the results of the miRNA microarray analysis via RT-qPCR. Compared with the control group, miR-205 was upregulated, while miR-122, miR-154 and miR-488 were downregulated, and miR-488 was the most downregulated ([Fig. 2B](#f2-mmr-20-04-3746){ref-type="fig"}), which was consistent with the results of the miRNA microarray analysis.
### Runx2 is a potential target of miR-488
To gain insight into the molecular mechanisms by which miRNAs regulate the osteogenic differentiation of BMSCs, the potential miRNAs were predicted using TargetScan and it was identified that the osteogenic transcription factor Runx2 has miR-488 binding sites in its 3′UTR ([Fig. 3A](#f3-mmr-20-04-3746){ref-type="fig"}). None of the other miRNAs regulate the expression of Runx2, thus miR-488 was further investigated. RT-qPCR showed that miR-488 was significantly upregulated in BMSCs transfected with miR-488 mimics ([Fig. 3B](#f3-mmr-20-04-3746){ref-type="fig"}), and was downregulated in BMSCs transfected with miR-488 inhibitor ([Fig. 3C and D](#f3-mmr-20-04-3746){ref-type="fig"}). Runx2 was significantly upregulated in BMSCs transfected with pcDNA 3.1-Runx2 ([Fig. 3E](#f3-mmr-20-04-3746){ref-type="fig"}). These results indicated that miR-488 mimics, miR-488 inhibitor and pcDNA 3.1-Runx2 were successfully transfected. To further examine whether miR-488 directly targets Runx2, a luciferase reporter assay was performed and it was demonstrated that the miR-488 mimics significantly inhibited the luciferase activity of the wild-type Runx2 3′UTR but not that of the mutated 3′UTR ([Fig. 3F](#f3-mmr-20-04-3746){ref-type="fig"}). Inversely, the miR-488 inhibitor significantly enhanced the luciferase activity of the wild-type Runx2 3′UTR but not that of the mutated 3′UTR ([Fig. 3F](#f3-mmr-20-04-3746){ref-type="fig"}).
### miR-488 negatively regulates the osteogenic differentiation of BMSCs
To better understand the functional role of miR-488 in the osteogenic differentiation of BMSCs, the expressions of Runx2, Osterix and ALP were detected after the BMSCs were transfected with the miR-488 mimics or inhibitor. As shown in [Fig. 4A-C](#f4-mmr-20-04-3746){ref-type="fig"}, the mRNA and protein expression levels of Runx2, Osterix and ALP were significantly decreased in the miR-488 mimics group but were significantly increased in the miR-488 inhibitor group compared with the control group. The AR-S result also showed that more calcium mineral deposition was found in the miR-488 inhibitor group than in the control and miR-488 mimic groups ([Fig. 4D](#f4-mmr-20-04-3746){ref-type="fig"}). Immunocytochemistry detected the expression levels of Runx2, Osterix and ALP expressed in BMSCs, where the cytoplasm was stained ([Fig. 4E](#f4-mmr-20-04-3746){ref-type="fig"}). The expression levels of Runx2, Osterix and ALP in the miR-488 mimic group were notably lower than those in the miR-488 inhibitor group, indicating that miR-488 suppressed osteogenic differentiation of the BMSCs.
### Runx2 overexpression partially rescues the inhibitory effect of miR-488 on osteogenic differentiation
To further explore the underlying mechanism of miR-488/Runx2 in the osteogenic differentiation of BMSCs, BMSCs were transfected with miR-488 mimics and pcDNA 3.1-Runx2 under treatment with psoralen. As shown in [Fig. 5A](#f5-mmr-20-04-3746){ref-type="fig"}, AR-S indicated that more calcium mineral deposition was found in the psoralen+pcDNA 3.1-Runx2+miR-488 mimics group than in the psoralen+miR-488 mimics group, while the calcium mineral deposition in the psoralen+pcDNA 3.1-Runx2+miR-488 mimics group was less than that in the psoralen+pcDNA 3.1-Runx2 group. Runx2 partially rescued the inhibitory effect of miR-488 during the psoralen-induced osteogenic differentiation of BMSCs. Western blotting ([Fig. 5B and C](#f5-mmr-20-04-3746){ref-type="fig"}) and immunocytochemistry ([Fig. 5D](#f5-mmr-20-04-3746){ref-type="fig"}) detected the protein expression of osteogenic specific factors in BMSCs transfected with miR-488 mimics and pcDNA 3.1-Runx2, and the results were similar to the AR-S results.
Discussion
==========
A previous study has shown that psoralen has a smooth muscle diastolic effect, stimulating bone formation and inducing osteogenic differentiation without affecting cell growth ([@b9-mmr-20-04-3746]), consistent with the present findings. However, little is understood regarding the roles of miRNAs in BMSCs induced by psoralen. In the present study, miRNA microarray analysis was conducted to identify the differently expressed miRNAs during psoralen-induced osteogenic differentiation of BMSCs, indicating that miR-488 was significantly decreased. However, the underlying mechanism of miR-488 in regulating BMSCs to stimulate osteogenic differentiation under psoralen induction remains largely unknown.
Each miRNA can bind to hundreds of different mRNAs and assemble with Argonaute proteins into miRNA-induced silencing complexes to direct post-transcriptional silencing of complementary mRNA targets ([@b17-mmr-20-04-3746]). For example, miR-488 has been identified as a cancer-associated miRNA that participates in various diseases, such as hepatocellular carcinoma ([@b18-mmr-20-04-3746]), ovarian cancer ([@b19-mmr-20-04-3746]) and colorectal cancer ([@b20-mmr-20-04-3746]). This is the first time, to the best of the authors\' knowledge, that miR-488 has been reported as a key regulator in the osteogenic differentiation of BMSCs. An important mechanism revealed by the present study is that Runx2 is directly targeted by miR-488. Runx2 is a transcription factor that is indispensable for skeletal development and controls bone formation by acting as a signalling hub and transcriptional regulator to coordinate target gene expression ([@b13-mmr-20-04-3746],[@b14-mmr-20-04-3746]). According to existing literature, miR-690, a Runx2-targeted miRNA, regulated the osteogenic differentiation of C2C12 myogenic progenitor cells by targeting NF-κB p65 ([@b21-mmr-20-04-3746]). Heparin-binding EGF-like growth factor and miR-1192 exert opposite effects on Runx2-induced osteogenic differentiation ([@b22-mmr-20-04-3746]). However, to the best of the authors\' knowledge, functional evidence of miR-488 targeting Runx2 in the regulation of BMSCs to undergo osteogenic differentiation has not been previously documented.
Therefore, in the present study, Runx2 was examined, and the hypothesis that the osteogenic differentiation of BMSCs may be regulated by miR-488 targeting Runx2 was evaluated. The pLUC-Runx2 plasmid construct, which contained the miR-488 target site in Runx2-3′UTR-wild-type, was constructed to be co-transfected with the miR-488 mimics or inhibitor. The results showed that the miR-488 mimics significantly reduced the luciferase activity of pLUC-Runx2, while the miR-488 inhibitor significantly increased the luciferase activity of pLUC-Runx2.
To further confirm the hypothesis, the mimics and inhibitor of miR-488 were transfected into BMSCs, and the mRNA and protein expression levels of Runx2 and a downstream signalling mediator (Osterix) ([@b14-mmr-20-04-3746],[@b23-mmr-20-04-3746]) and osteogenic-specific marker (ALP) ([@b24-mmr-20-04-3746]) were detected via RT-qPCR, western blotting and immunocytochemistry. The Runx2, Osterix and ALP mRNA and protein expression levels were reduced in the miR-488 mimics group and were enhanced in the miR-488 inhibitor group compared with the control group. The AR-S result also showed that more calcium mineral deposition was found in the miR-488 inhibitor group than the control and miR-488 mimic groups. Further rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR-488 on osteogenic differentiation under psoralen induction. It should be noted that the present study only observed calcium mineral deposition by AR-S and lacked precise quantification. However, despite this limitation, the present study does support the hypothesis that Runx2 is targeted by miR-488 and could be directly responsible for the osteogenic differentiation of BMSCs under psoralen induction.
The results of the present study have two important clinical implications. Using the miRNA-target gene network to examine the mechanism of psoralen promoting the osteogenic differentiation of BMSCs, the present study provided insight for the development and application of psoralen in osteoporosis treatment. Additionally, the present study identified a potential molecular approach to promote the osteogenic differentiation of BMSCs, and miR-488/Runx2 could be therapeutic targets for osteoporosis. miR-488/Runx2 constitutes a system that has not yet been used in osteoporosis treatment. Inhibition of miR-488 or overexpression of Runx2 appears to be an interesting novel therapeutic option for osteoporosis.
The present study suggested that miR-488 is involved in the osteogenic differentiation of BMSCs by targeting Runx2 under psoralen induction. Altering miR-488/Runx2 may be a targeted and mechanism-based therapeutic strategy against osteoporosis.
Not applicable.
Funding
=======
The present study was supported by the National Natural Science Foundation (grant nos. 81473699 and 81804047), Natural Science Foundation of Guangdong Province (grant nos. 2018A030313694 and 2017A030312009), the Honk Kong Scholar Program (grant no. XJ2018059) and Guangdong Provincial Traditional Chinese Medicine Research Project (grant nos. 20181095 and 20182043).
Availability of data and materials
==================================
The datasets used during the present study are available from the corresponding author on reasonable request.
Authors\' contributions
=======================
YH, TJ and YL designed the research. YH, QH, TJ and DC performed the experiments. YH, HS and DC analysed the data. YH and QH wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
==========================================
All animals received humane care in accordance with the guidelines set by the Care of Experimental Animals Committee of Guangzhou University of Chinese Medicine. Additionally, the present study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine.
Patient consent for publication
===============================
Not applicable.
Competing interests
===================
The authors declare that they have no competing interests.
{#f1-mmr-20-04-3746}
{#f2-mmr-20-04-3746}
{#f3-mmr-20-04-3746}
{#f4-mmr-20-04-3746}
{#f5-mmr-20-04-3746}
[^1]: Contributed equally
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Background
----------
The incidence of melanoma has increased in all Western countries over the last 30 years and has increased 3 to 5 times depending on the country \[[@ref1],[@ref2]\], presently affecting 13.8 people in North America, 14.6 in northern Europe and 35.1 out of the 100,000 people in Australia \[[@ref1],[@ref2]\]. Melanoma 5-year survival depends on the stage at the time of diagnosis, decreasing by 84% at a localized stage to 13% at the metastatic stage \[[@ref2]\]. It is therefore essential that clinicians and policy makers concentrate their efforts to ensure early detection of the disease \[[@ref3]\]. Numerous factors associated with a delayed diagnosis are patient-related \[[@ref4]-[@ref12]\]. Other factors are related to the opportunity to consult a dermatologist rather than a general practitioner \[[@ref4],[@ref13]\]. However, various authors have reported difficulties in obtaining an appointment with a dermatologist \[[@ref12],[@ref14]-[@ref17]\].
Many countries have tested the use of telemedicine in dermatology as a way to increase access to health care services when distance is a critical factor \[[@ref18]-[@ref26]\]. Telemedicine in dermatology can be based either on videoconferences or on store-and-forward teledermatology procedures. Videoconferences, which allow a patient and a dermatologist to be connected for a consultation, are time-consuming for the dermatologist and may require an expensive setup \[[@ref20],[@ref27]\]. Store-and-forward teledermatology procedures are based on sending information and photographs to a dermatologist for a deferred medical opinion \[[@ref20]\].
Although smartphones have now revolutionized the daily life of physicians in all Western countries \[[@ref19],[@ref24],[@ref28],[@ref29]\], one issue is to determine whether smartphone use has been assessed for store-and-forward teledermatology procedures. As various apps exist that provide scores, decision aids, and management advice, an alternative both to videoconferences and to store-and-forward teledermatology might be automated smartphone apps with no need of a dermatologist opinion. An issue to be addressed is whether such apps can help in the early diagnosis of melanoma.
Objective
---------
We initiated a review focusing on the use of a smartphone in sustaining melanoma early detection (either store-and-forward teledermatology or automated apps). The aim was to report evidence on (1) the diagnostic performance of the procedures, (2) the impact on the patient's medical-care course and delays before the dermatological consultation, and (3) the limitations of either store-and-forward teledermatology or automated apps.
Methods
=======
This review was conducted according to the key steps required for systematic reviews \[[@ref30]\]. Considering that evidence might be sparse, the literature review was based on a broad scope and was not restricted to randomized controlled trials.
Study Identification and Selection
----------------------------------
We conducted a systematic search of PubMed for the period from January 1, 2007 (launch of the first smartphone) to November 1, 2017. The keywords were as follows: \[smartphone OR cell phone OR remote OR telemedicine\] AND \[dermatology OR skin disease OR melanoma OR skin neoplasm OR skin abnormalities\]. We also searched the reference lists of reviews and studies identified during the initial search by hand. Abstracts and full texts were reviewed independently by 2 reviewers (SH and JR) for inclusion. Any disagreements on inclusion or exclusion were resolved by consensus, and a third reviewer (CR) was consulted to resolve any remaining disagreements.
Inclusion and Exclusion Criteria
--------------------------------
In this manuscript, the term "smartphones" refers to mobile phones that have an internet data communication system and a digital camera (compared to mobile phones that would not have these two specific devices). The inclusion criteria for the studies included in this review were as follows: (1) photographs concerning pigmented suspicious lesions, (2) photographs taken or analyzed using a smartphone, (3) patients older than 18 years, (4) studies written in French or English, and (5) abstracts available.
The exclusion criteria were (1) no use of a smartphone, (2) the research area was not related to melanoma early detection, (3) dermatology teleconsultation in the form of a videoconference, (4) study based on histology, (5) studies consisting of assessing patients or caregivers' preferences through interviews or surveys, and (6) an editorial or a letter to the editor.
Considering that evidence might be sparse, the literature review was based on a broad scope and the inclusion criteria were not restricted to a "Patients Intervention Comparison Outcome" presentation \[[@ref30]\].
Data Extraction and Synthesis
-----------------------------
Studies were critically appraised by 2 reviewers (SH and JR), and discrepancies were resolved by consensus. The studies were first classified by the type of procedure assessed (app or store-and-forward teledermatology). Then the following data were extracted, such as design, population of the sample, whether the study had been conducted in the context of primary care, whether it was a descriptive or comparative study, and main outcome measures ([Tables 1](#table1){ref-type="table"} and [2](#table2){ref-type="table"}).
The main outcomes in studies focusing on store-and-forward teledermatology were either (1) the diagnostic concordance between the teledermatology procedure and the reference (the kappa coefficient of concordance is a measure of agreement between 2 raters, based on the following formula \[(observed probability--expected probability)/(1--expected probability)\]) or (2) the impact on the patient's medical-care course and delays before dermatological consultation. The proportion of uninterpretable photographs was also reported.
The main outcomes in studies focusing on apps were sensitivity (defined as the number of true positive assessments/number of all positive assessments), specificity (defined as the number of true negative assessments/number of all negative assessments), and accuracy (defined as the number of correct assessments/number of all assessments). The proportion of uninterpretable photographs was also reported. When necessary, the authors were contacted to obtain information not reported in the studies.
Analysis of Bias
----------------
We assessed the risk of bias using the quality assessment of diagnostic accuracy 2nd edition (QUADAS-2) \[[@ref55]\]. The reporting of risk of bias focused on patient selection, index test, reference standard, and flow and timing. For each item, signaling questions helped to estimate whether the risk of bias was low or high. Unclear was used if no information was available. The applicability of the study intervention was also assessed using QUADAS-2, focusing on patient selection, index test, and reference standard.
######
Studies based on store-and-forward teledermatology procedures (design, patients, comparison, and outcome). N/A: not applicable. RCT: randomized controlled trial.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Authors Design Patients,\ Population of sample Comparison Main outcomes
n (%)
-------------------------------------------------------------------- ---------------------------------- ------------------- ---------------------- --------------------------------------------- --------------------------------------- -------------------------
**Store-and-forward teledermatology without teledermoscopy** \ \ \
\ Boyce et al, 2011 \[[@ref31]\] Prospective study N/A Patients at an elevated risk of melanoma Face to face Concordance
\ Lamel et al, 2011 \[[@ref32]\] Prospective study 1 (0.7) Patients from a melanoma screening campaign Face to face Concordance
\ Janda et al, 2014 \[[@ref33]\] RCT 1 (1) Patients at an elevated risk of melanoma Face to face Concordance
**Store-and-forward teledermatology that included teledermoscopy** \ \
\ Ford et al, 2015 \[[@ref34]\] Quasi-experiment 22 (11.3) Patients recruited in primary care centers Face to face Secondary care referral
\ Börve et al, 2013 \[[@ref29]\] Prospective study 12 (17) Patients referred for an excision Face to face^a^ Concordance
\ Börve et al, 2015 \[[@ref35]\] Quasi-experiment 55 (3.52) Patients recruited in primary care centers Teledermatology versus paper referral Delays
\ Hue et al, 2016 \[[@ref36]\] Descriptive study 1 (0.3) Patients from a melanoma screening campaign No comparison Feasibility and delays
\ Janda et al, 2013 \[[@ref37]\] Descriptive study N/A Patients at an elevated risk of melanoma No comparison Feasibility
\ Kroemer at al, 2011 \[[@ref38]\] Prospective study 6 (5) Patients referred to the dermatologist Face to face^a^ Concordance
\ Manahan et al, 2015 \[[@ref39]\] RCT 0 (0.0) Patients with a dermatological follow-up Face to face Concordance
\ Markun et al, 2017 \[[@ref40]\] Prospective study 1 (0.05) Patients from a melanoma screening campaign Face to face^a^ Concordance
\ Massone et al, 2007 \[[@ref41]\] Prospective study 2 (11) Patients with a dermatological follow-up Face to face Concordance
\ Wu et al, 2015 \[[@ref42]\] Prospective study N/A Patients with a dermatological follow-up Face to face Concordance
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
^a^For these studies, suspicious lesions were referred for excision and histopathology results were analyzed as a secondary outcome in the study.
######
Studies based on automated smartphone apps (design, photographs and patients, comparison, and outcomes). N/A: not applicable.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Authors Design n (%)^a^ Patients, N (characteristics) Comparison Main outcomes
----------------------------------------------------- ------------------------------------ -------------------- ------------------------------- --------------------------------------- ---------------------- --------------------------
**Photographs issued from a database** \ \ \
\ Do et al, 2014 \[[@ref43]\] Case-control study 29 (36) N/A Histopathology Accuracy^b^
\ Doukas et al, 2012 \[[@ref44]\] Case-control study 800 (26.67) N/A Clinical evaluation Accuracy^b^
\ Ferrero et al, 2013 \[[@ref45]\] Descriptive study 93 (100) N/A Histopathology Sensitivity, Specificity
\ Ramlakhan et al, 2011 \[[@ref46]\] Case-control study 46 (55) N/A Unclear Sensitivity, Specificity
\ Wadhawan et al, 2011a \[[@ref47]\] Case-control study 388 (29.85) N/A Histopathology Sensitivity, Specificity
\ Wadhawan et al, 2011b \[[@ref48]\] Case-control study 110 (31.7) N/A Histopathology Sensitivity, Specificity
\ Wolf et al, 2013 \[[@ref49]\] Case-control study 60 (31.9) N/A Histopathology Sensitivity, Specificity
**Photographs taken of patients in real condition** \ \ \
\ Dorairaj et al, 2017 \[[@ref50]\] Prospective study 9 (28) N/A (referred for an excision) Teledermatologist^c^ Sensitivity, Specificity
\ Maier et al, 2015 \[[@ref51]\] Prospective study 26 (18.1) N/A (with a dermatological follow-up) Face to face^c^ Sensitivity, Specificity
\ Ngoo et al, 2017 \[[@ref52]\] Prospective study 1 (2) 30 (with a dermatological follow-up) Teledermatologist Sensitivity, Specificity
\ Robson et al, 2012 \[[@ref53]\] Prospective study 2 (6) 31 (referred to the dermatologist) Face to face^c^ Sensitivity, Specificity
\ Thissen et al, 2017 \[[@ref54]\] Prospective study 6 (1.8) 256 (referred to the dermatologist) Face to face^c^ Sensitivity, Specificity
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
^a^Included photographs, proportion with melanoma.
^b^Accuracy=(True Negatives+True Positives)/(True Negatives+True Positives+False Negatives+False Positive).
^c^For these studies, suspicious lesions were referred for excision and histopathology results were analyzed as a secondary outcome in the study.
Results
========
Overview
--------
In total, 1450 titles and abstracts were screened for eligibility, utilizing the inclusion and exclusion criteria. A previous review was identified \[[@ref2]\] and the related studies from the references list were included in this review. A total of 25 studies \[[@ref29],[@ref31]-[@ref54],[@ref56]\] were included in the review ([Figure 1](#figure1){ref-type="fig"}). Of these, 15 studies had been published as original papers \[[@ref29],[@ref31],[@ref32],[@ref34]-[@ref36],[@ref38]-[@ref42],[@ref49],[@ref51],[@ref52],[@ref54]\], 5 were conference papers \[[@ref43],[@ref44],[@ref46]-[@ref48]\] and 5 were research letters \[[@ref33],[@ref37],[@ref45],[@ref50],[@ref53]\]. In these, 12 studies had been conducted in European countries, that is, Great Britain \[[@ref34],[@ref53]\], Austria \[[@ref38],[@ref41]\], Sweden \[[@ref29],[@ref35]\], Ireland \[[@ref50]\], Germany \[[@ref51]\], Switzerland \[[@ref40]\], Greece \[[@ref44]\], the Netherlands \[[@ref54]\], and France \[[@ref36]\]; 7 in United States of America \[[@ref32],[@ref42],[@ref45]-[@ref49]\]; 5 in Australia \[[@ref31],[@ref33],[@ref37],[@ref39],[@ref52]\]; and one in Singapore \[[@ref43]\].
Store and Forward Teledermatology
---------------------------------
A total of 13 studies assessed store-and-forward teledermatology \[[@ref29],[@ref31]-[@ref42]\] ([Table 1](#table1){ref-type="table"}). There were 12 studies that specified the smartphone model used, that is, 9 tested iPhones \[[@ref29],[@ref33]-[@ref37],[@ref39],[@ref40],[@ref42]\] and 3 tested other brands of telephones \[[@ref32],[@ref38],[@ref41]\].
The study population were patients recruited in primary care in 9 studies, that is, either in the context of a screening campaign \[[@ref32],[@ref36],[@ref40]\] or during targeted screening focusing on patients at an elevated risk of melanoma \[[@ref31],[@ref33],[@ref37]\] or during opportunistic screening conducted in general practice \[[@ref34],[@ref35],[@ref38]\]. For the other 4 studies, the patients had already consulted a dermatologist \[[@ref29],[@ref39],[@ref41],[@ref42]\]. The prevalence of melanomas in the related populations varied greatly, ranging from 0% \[[@ref39]\] to 17.3% \[[@ref29]\].
Ten studies compared the conclusion of the teledermatologist with the conclusion of a dermatologist who conducted a face-to-face examination ([Table 1](#table1){ref-type="table"}). There were 2 studies that focused on feasibility without providing any comparison \[[@ref36],[@ref37]\]. From the 13 studies conducted, 10 studies used a mobile teledermoscope \[[@ref29],[@ref34]-[@ref42]\], whereas 3 studies only transferred the pictures taken without the teledermoscope \[[@ref31]-[@ref33]\].
There were 7 studies that provided information on diagnostic concordance for store-and-forward teledermatology based solely on clinical photographs. The diagnostic concordance between the conclusions of the teledermatologist and the dermatologist (face-to-face) ranged from 62% \[[@ref32]\] to 89% \[[@ref41]\]. This concordance was analyzed further using the kappa coefficient \[[@ref29],[@ref31],[@ref32],[@ref38]-[@ref40],[@ref42]\], which ranged from .20 \[[@ref40]\] to .84 \[[@ref38]\]. Börve reported 58% concordance between the conclusions of 2 independent teledermatologists \[[@ref29]\]. Focusing on whether the patients could take pictures of their lesions themselves, Boyce et al reported 69% concordance between the conclusion of a dermatologist (face-to-face) and the conclusion of a teledermatologist (κ=.23) \[[@ref31]\].
{#figure1}
For teledermatology based on pictures taken with a teledermoscope, the diagnostic concordance between the teledermatologist's conclusions and the conclusion of a dermatologist (face-to-face) ranged from 51% \[[@ref29]\] to 97% \[[@ref42]\]. The kappa coefficient varied from .29 \[[@ref40]\] to .87 \[[@ref42]\]. Massone reported 94% concordance between the conclusions of 2 independent teledermatologists who analyzed photographs taken using a teledermoscope \[[@ref41]\]. Focusing on whether the patients could take pictures of their lesions themselves, Manahan and Wu reported concordance ranging from 90% \[[@ref39]\] to 97% \[[@ref42]\] when the patient used a teledermoscope.
There were 4 studies that reported an acceleration in the management of patients when malignancy was suspected \[[@ref29],[@ref34],[@ref36],[@ref40]\]. Börve reported reduced delays in obtaining an appointment (delays shorter than 2 days compared with delays longer than 80 days), a reduced delay for surgical management (36 days vs 85 days), and a lower Breslow index at the time of the diagnosis \[[@ref29]\]. Hue reported that patients with a highly suspicious lesion were asked to return within less than 10 days \[[@ref36]\]. There were 3 studies \[[@ref35],[@ref36],[@ref40]\] that reported a decrease (40%, 53%, and 74%, respectively) in the proportion of patients referred to a dermatologist, whereas Ford reported a slight increase in referrals (an increase of 2.11 per 1000 patients) \[[@ref34]\].
The proportion of uninterpretable images due to their poor quality was, on average, less than 20% \[[@ref29],[@ref40],[@ref42]\]. Only Massone et al (2007) reported a higher percentage of poor-quality images of 70% \[[@ref41]\]. However, only a minority of authors provided information on the modalities for picture taking. A total of 4 studies specified the size of the pictures from 1024×766 to 2592×1224 pixels. Following contact with the authors, the following information on the modalities were collected, that is, the number of pictures could vary from 1 to 12 pictures per lesion \[[@ref33],[@ref39]\], pictures were taken at a distance of 10 to 30 cm from the skin \[[@ref29],[@ref40]\], authors reported taking one close-up picture and another of the surrounding area \[[@ref36],[@ref40],[@ref41]\], and specified the lighting conditions ("strong light" \[[@ref39]\], "day light" \[[@ref41]\], "maximal light" \[[@ref29]\]), neutral background \[[@ref29],[@ref40]\], use of the zoom \[[@ref41]\] or macro mode \[[@ref40],[@ref41]\], and use of the autofocus \[[@ref29],[@ref38],[@ref41]\]). There were 4 authors who reported the time required to take the picture that is, ranging from a few seconds to less than 4 min \[[@ref29],[@ref34]-[@ref36]\]. The photographer was a professional in most cases, that is, either a dermatologist \[[@ref29],[@ref38],[@ref41]\], a general practitioner \[[@ref35]\], or another professional \[[@ref32],[@ref40]\]; however, 6 studies reported that the picture was taken by the patient or a family member \[[@ref31],[@ref33],[@ref37],[@ref39],[@ref42]\]. The notion of encrypting data was not approached systematically. Following individual contact, 8 authors reported encryption of the data, either through the app or through anonymity \[[@ref29],[@ref31],[@ref32],[@ref34]-[@ref36],[@ref39],[@ref40],[@ref42]\]. A total of 8 studies used email for transferring the data \[[@ref29],[@ref31],[@ref33]-[@ref35],[@ref37],[@ref39],[@ref42]\], and 7 used an encrypted platform \[[@ref29],[@ref32],[@ref34]-[@ref36],[@ref40],[@ref42]\].
Analysis of bias is provided in [Table 3](#table3){ref-type="table"}. The risk of bias related to patient selection was high, as patients who participated were either volunteers or chosen by doctors in consultation, but no study was based on a random sample. In 2 studies, the same dermatologist participated in both, the store-and-forward teledermatology procedure and the face-to-face clinical evaluation, with insufficient washout, so that the risk of bias related to the test index was high. The final analysis of the studies did not include all the recruited patients so that the risk of bias of flow and timing was high. Applicability was good.
Automated Smartphone Apps
-------------------------
A total of 12 studies assessed the performance of automated smartphone apps \[[@ref43]-[@ref54]\] ([Table 2](#table2){ref-type="table"}), that is, 1 study compared the conclusions of an automated app with the conclusions of a dermatologist who conducted a face-to-face examination \[[@ref51]\], 1 study compared the conclusions of an automated app with the conclusions of a teledermatologist \[[@ref52]\], 7 studies analyzed the images of an already classified data bank \[[@ref43]-[@ref49]\], and the 3 last ones compared the conclusion of an automated app, both with the pathological report (after excision of the lesion) and with the conclusion of teledermatologists \[[@ref50],[@ref53],[@ref54]\].
Among the 5 studies based on taking a photograph \[[@ref51]-[@ref54]\], 4 tested iPhones \[[@ref51]-[@ref54]\], 1 tested other brands of telephones \[[@ref52]\], and the brand of the telephone was not specified in the last study \[[@ref50]\]. The photographer was a dermatologist \[[@ref53],[@ref54]\] or another professional \[[@ref50]-[@ref52]\].
Participants who were recruited in studies assessing automated smartphone apps were highly selected. Among the 8 studies based on photographs issued from a database, the proportion of melanoma ranged from 26.7% \[[@ref44]\] to 100% \[[@ref45]\]. Among the 5 studies based on taking a photograph, 2 studies included patients from a primary-care setting recruited during an opportunistic screening campaign conducted in general practice \[[@ref53],[@ref54]\]. For the other 3 studies that included patients, the patients had already consulted a dermatologist \[[@ref50]-[@ref52]\]. The prevalence of melanomas in the related populations ranged from 1.8% \[[@ref52],[@ref54]\] to 28.1% \[[@ref50]\].
The performance of the automated smartphone apps were assessed by referring either to their capacity to classify the lesions at risk \[[@ref43],[@ref45],[@ref47],[@ref49]-[@ref54]\], or to their diagnostic capacity \[[@ref44],[@ref46],[@ref48]\] ([Table 2](#table2){ref-type="table"}). The references used could be either the histology results in 5 studies \[[@ref43],[@ref45],[@ref47]-[@ref49]\], the teledermatologist's conclusion in 2 studies \[[@ref50],[@ref52]\], or the dermatologist (face-to-face) conclusion in 3 studies \[[@ref51],[@ref53],[@ref54]\]. For 2 studies, the authors referred to histology-based diagnosis without describing how they obtained the histopathological conclusion \[[@ref44],[@ref46]\].
A total of 5 studies assessed the apps used in real conditions \[[@ref50]-[@ref54]\]. In 4 studies, the images came from medical \[[@ref43],[@ref45],[@ref49]\] or commercial \[[@ref47],[@ref48]\] data banks, whereas no details on the photograph data banks were provided for 2 studies \[[@ref44],[@ref46]\]. The sensitivity ranged from 7% to 87% \[[@ref48],[@ref49]\], and the specificity ranged from 9% to 100% \[[@ref50],[@ref52]\]. Only 2 studies described the area under the curve \[[@ref44],[@ref48]\], providing a better comparison of results. One study reported a kappa coefficient of concordance between the opinion of the app and that of a dermatologist \[[@ref52]\].
None of the studies that assessed automated smartphone apps reported an impact on the patient's medical-care course.
Analysis provided by automated smartphone apps were made difficult by ulcerated, blood stained, speckled or tanned areas, the presence of hair, or several lesions on the same photograph. There were 5 studies that had a proportion ranging from 11% to 30% of the lesions that could not be analyzed because of technical problems other than the problems related to the quality of the initial photograph \[[@ref45],[@ref49]-[@ref51],[@ref53]\]. There were 4 studies based on apps which reported that the time required to analyze the pictures was less than 10 seconds \[[@ref44],[@ref46]-[@ref48]\]. The notion of encrypting data was not approached systematically. Following individual contact, 2 authors \[[@ref52]-[@ref54]\] reported encryption of the data, either through the app or through anonymity.
The analysis of bias is provided in [Table 4](#table4){ref-type="table"}. For studies based on photographs from databank, references were unknown and the risks of bias for patient selection were high. Applicability related to the patient selection was highly concerning. Automated apps had no information on the diagnosis so that the risk of bias related to the test index was low. In 3 studies, pictures were modified before intervention so that the applicability related to the index test was not good. All the photographs were not analyzed so that the risk of bias related to flow and timing was high.
######
Studies based on store-and-forward teledermatology procedures. Risk of bias assessment according to quality assessment of diagnostic accuracy 2nd edition (QUADAS-2).
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Authors Risk of bias Applicability concerns
-------------------------------------------------------------------- ---------------------------------- ------------------------ ------ ------ ------ ------ ------ -----
**Store-and-forward teledermatology without teledermoscopy** \ \ \ \
\ Boyce et al, 2011 \[[@ref31]\] High Low Low High Low Low Low
\ Lamel et al, 2011 \[[@ref32]\] High Low Low Low Low Low Low
\ Janda et al, 2014 \[[@ref33]\] High Low High Low Low Low Low
**Store-and-forward teledermatology that included teledermoscopy** \ \ \ \
\ Ford et al, 2015 \[[@ref34]\] Low Low Low High Low Low Low
\ Börve et al, 2013 \[[@ref29]\] High Low Low Low High Low Low
\ Börve et al, 2015 \[[@ref35]\] High Low Low High Low Low Low
\ Hue et al, 2016 \[[@ref36]\] High Low Low High Low Low Low
\ Janda et al, 2013 \[[@ref37]\] High Low Low High Low Low Low
\ Kroemer at al, 2011 \[[@ref38]\] High High Low High Low Low Low
\ Manahan et al, 2015 \[[@ref39]\] High Low Low High Low Low Low
\ Markun et al, 2017 \[[@ref40]\] High High Low High Low Low Low
\ Massone et al, 2007 \[[@ref41]\] High Low Low Low Low Low Low
\ Wu et al, 2015 \[[@ref42]\] High Low Low High Low High Low
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
######
Studies based on automated smartphone apps. Risk of bias assessment according to quality assessment of diagnostic accuracy 2nd edition (QUADAS-2).
---------------------------------------------------------------------------------------------------------------------------------------------------------------------
Authors Risk of bias Applicability concerns
----------------------------------------------------- ------------------------------------ ------------------------ ----- --------- --------- ------ ------ ---------
**Photographs issued from a database** \ \ \ \
\ Doukas et al, 2012 \[[@ref44]\] High Low Unclear Unclear High Low Unclear
\ Do et al, 2014 \[[@ref43]\] High Low Low Unclear High High Low
\ Ferrero et al, 2013 \[[@ref45]\] High Low Low High High Low Low
\ Ramlakhan et al, 2011 \[[@ref46]\] High Low Unclear High High Low Unclear
\ Wadhawan et al, 2011a \[[@ref47]\] High Low Low High High High Low
\ Wadhawan et al, 2011b \[[@ref48]\] High Low Low High High High Low
\ Wolf et al, 2013 \[[@ref49]\] High Low Low High High High Low
**Photographs taken of patients in real condition** \ \ \ \
\ Dorairaj et al, 2017 \[[@ref50]\] High Low Low High High Low Low
\ Maier et al, 2015 \[[@ref51]\] High Low Low High High Low Low
\ Ngoo et al, 2017 \[[@ref52]\] High Low Low High Low Low Low
\ Robson et al, 2012 \[[@ref53]\] High Low Unclear High Low Low Low
\ Thissen et al, 2017 \[[@ref54]\] High Low Unclear Low Low Low Low
---------------------------------------------------------------------------------------------------------------------------------------------------------------------
Discussion
==========
Store-and-forward teledermatology opens several perspectives, that is, it accelerates the care course, and various studies were performed in primary care populations. However, the concordance between the conclusion of a teledermatologist and the conclusion of a dermatologist who conducts a face-to-face examination depended on the study (the kappa coefficient range was .20 to .84, median κ=.60). The use of a dermoscope may improve the concordance (the kappa coefficient range was .29 to .87, median κ=.74). Regarding automated smartphone apps, the major concerns are their low sensitivity, the lack of assessment in clinical practice conditions, and the lack of assessment in primary care populations.
In a study recently published in *Nature*, Esteva et al reported that an artificial neuronal network had a better capacity to recognize melanomas than a dermatologist \[[@ref56]\]. However, the automated apps available on a smartphone in 2017 do not provide such expertise. Our review shows that the existing automated smartphone apps are unreliable. Some apps have a low diagnostic sensitivity that may induce false negatives and erroneously reassure patients who may then not consult a specialist \[[@ref50]\]. Certificates do not guarantee good diagnostic performance \[[@ref54]\]. Greater control by administrative authorities is necessary \[[@ref45],[@ref57],[@ref58]\].
This literature review suggests that teledermatology decreases the delays in the management of melanoma lesions \[[@ref34]-[@ref36],[@ref40],[@ref59]\], reduces the referrals to a dermatologist by avoiding unnecessary consultations \[[@ref35],[@ref36],[@ref40]\] and limits the number of patients lost from follow-up \[[@ref36],[@ref60]\]. Moreno-Ramirez et al confirmed this point during an experimental teledermatology program without a smartphone \[[@ref61]\]. Other authors have not been as optimistic and suggested that the many false negatives and easy access to a dermatologist's opinion may increase the number of secondary consultations \[[@ref34]\].
Large variations in the kappa concordance coefficient might be related to the range of melanoma prevalence (depending on the recruited population). On the one hand, this result should lead to multiple studies in the general population to assess the performance of the procedures in a primary care setting. On the other hand, this review emphasizes that the publication of data related to up-to-date technologies is a challenge in teledermatology \[[@ref20],[@ref62]\]; it is notable that up to 70% of the photographs were of poor quality in a study performed in 2007 \[[@ref41]\], whereas all photographs were interpretable in 2016 \[[@ref54]\].
The importance of training the person taking the photograph has been underlined \[[@ref33],[@ref35]-[@ref37],[@ref40]\], especially when a dermoscope is used \[[@ref63]\]. Today, it is surprising to note that no standards for taking photographs exist \[[@ref62],[@ref64]\]. Our review identified a few characteristics related to the quality of the photographs, such as several views of the same lesion, close-up and distant pictures, use of the autofocus and macro mode, a neutral background, and good lighting. The homogenization of practices based on these quality criteria is required to obtain better study reproducibility.
This review recalls that to give an opinion solely based on photographs, whatever their quality, is a challenge for dermatologists \[[@ref29],[@ref41],[@ref63]\]. For example, the absence of palpation is one of the limitations of teledermatology. Thissens's study noted the need to obtain supplementary clinical information \[[@ref54]\].
General practitioners and patients are likely to omit suspect lesions \[[@ref31],[@ref33],[@ref39]\]. This limitation, which has been described in general practice \[[@ref13],[@ref65]\], exists in teledermatology, that is, general practitioners could miss up to 30% of melanomas \[[@ref66]\]. Another difficulty is the omission of specific areas that are either difficult to access, such as hair, wounds, or the ear \[[@ref51],[@ref54]\], or those considered sensitive (genitalia) \[[@ref67],[@ref68]\].
Store-and-forward teledermatology is well-accepted by patients and caregivers \[[@ref59],[@ref69],[@ref70]\]. Patients report that one limitation of the apps is the difficulty of not having any human contact \[[@ref20]\]. For both procedures, a limitation is the loss of the face-to-face patient-physician relationship, which may be critical since a melanoma is diagnosed early, at a severe stage, or in the aging population \[[@ref59],[@ref69]-[@ref71]\]. Positive perspectives may improve compliance to referrals and possibilities for the patient to participate actively in his or her health \[[@ref24]\].
Confidentiality, security, and traceability of data exchange are the major ethical and legal stakes \[[@ref20],[@ref63],[@ref72]\]. Although the abusive use of clinical photographs has become an increasing preoccupation of health fund organizations \[[@ref73]\], only 30% of patients worry about the future of their photographs \[[@ref74]\], and our review reported that only slightly more than half of the authors encrypted their data. Although 60% of specialists continue to store photographs of their patients in their personal mobile phones \[[@ref72],[@ref74]-[@ref77]\], one perspective could be to develop recourse to encrypt the medical-image libraries \[[@ref62]\].
This review is original because of its specific focus on (1) melanoma early detection (mortality issues are not comparable for other dermatological pathologies) \[[@ref20],[@ref62],[@ref78],[@ref79]\], (2) a primary care perspective (the sensitivity and specificity of a test depend on the prevalence of the disease), and (3) the use of a smartphone, that is, a tool implemented worldwide at low cost (not comparable to other expensive videoconference procedures) without limiting the study selection to apps \[[@ref20],[@ref78]-[@ref80]\]. However, this study had several weaknesses. First, this review had a large scope because we hypothesized that evidence might be sparse---the heterogeneity in study designs, the populations, the end points, the references used, and the presentations of the results made data comparison difficult. Second, the review was only based on MEDLINE. Third, the selection bias was high, and the prevalence of melanoma, which ranged from 0% to 100%, depended on the population studied. Fourth, the material used differed from one study to the next and from year to year, hence introducing a bias in evaluation.
Our review confirmed the absence of evidence of the safety and efficacy of smartphone medical apps. In contrast, our review found evidence that store-and-forward teledermatology using smartphones may affect patients' care courses, delays in obtaining a dermatologist consultation, and patients' referral to secondary care. Further research is required to determine the quality criteria, as there was major variability among the studies.
The authors wish to thank Sasha Mann, who helped draft the manuscript. Financial support was provided by a grant from the Ministry of Health. The funding source had no role in the design or conduct of the study; the collection, management, analysis, or interpretation of the data; the preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication.
Authors\' Contributions: CR conceived the study, was responsible for its design and supervision, and was responsible for drafting the manuscript. SH participated in the design of the study, managed the selection of the included study, participated in data collection, and helped draft the manuscript. JR participated in the design of the study, managed the selection of the included study, participated in data collection, and helped draft the manuscript. GQ and BD provided administrative and technical support and revised the manuscript critically for important intellectual content. JMN obtained the grant for the corresponding research program, was responsible for the research project, and helped draft the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest: None declared.
QUADAS-2
: quality assessment of diagnostic accuracy 2nd edition
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Breast cancer is one of the most common cancers in women and has been a major cause for cancer death of women \[[@b1-crt-2019-217]\]. In clinical work, breast cancer is classified into four molecule subtypes mainly based on status of estrogen receptor (ER), progesterone receptor (PR), Ki67, and human epidermal growth factor receptor 2 (HER2), consisting of luminal A, luminal B, HER2-overexpressing and triple-negative breast cancer (TNBC) \[[@b2-crt-2019-217]\]. Luminal A and B are ER and PR positive whereas the other two are negative. Among of them, luminal subtype accounts for approximately 70% of all breast cancer patients, TNBC accounts for about 19% and HER2-overexpressing accounts for the rest \[[@b2-crt-2019-217]-[@b4-crt-2019-217]\]. The presence of ER means that tumor may respond to endocrine therapy taking ER signaling pathways as targets. So endocrine therapy has been a major method to treat luminal patients, luminal A is much more sensitive to this method than luminal B \[[@b3-crt-2019-217]\] and patients with luminal A need no other treatment such as extra chemotherapy \[[@b4-crt-2019-217]\]. In luminal B, additional chemotherapy combined with endocrine therapy is very important. But the resistance of chemotherapy in this subtype is common, so recurrence rate is much higher than luminal A and new molecular targets should be developed to improve outcome of patients \[[@b5-crt-2019-217]\].
The proteins of human anterior gradient (AGR) family have three members, respectively named AGR1, AGR2, and AGR3. In higher vertebrates, AGR1 is lost due to decreased regeneration ability, but AGR2 and AGR3 still express \[[@b6-crt-2019-217]\]. However, physiological functions of AGR2 and AGR3 in vertebrates are poorly understood. In tumors such as breast \[[@b7-crt-2019-217]-[@b10-crt-2019-217]\], ovarian \[[@b11-crt-2019-217]\], prostate \[[@b12-crt-2019-217]\], esophageal \[[@b13-crt-2019-217]\], lung \[[@b14-crt-2019-217]\], and pancreas cancers \[[@b15-crt-2019-217]\], AGR2 has been acknowledged as an oncogene and a biomarker indicating poor prognosis of patients. Comparing to definite role of AGR2, AGR3's impacts on cancers are still unclear. Reports about AGR3 in cancers are few and contradictory.
In our clinicopathologic study, we performed immunohistochemistry (IHC) analysis to detect AGR3 expression in 51 cases of breast benign lesions, 62 cases of ductal carcinoma *in situ* (DCIS) and 336 cases of invasive ductal carcinoma (IDC). Our samples were randomly selected and covered all molecular subtypes and all histological grades. We confirmed that AGR3 mainly expressed in the IDC patients of luminal subtype of histological grade Ⅰ-Ⅱ and could predict poor prognosis of luminal B. AGR3 highly expressed IDC patients tend to occur bone metastasis and liver metastasis. In breast cancer cell lines, we found that AGR3 could promote proliferation and invasion ability of cancer cells which proved that AGR3 could accelerate progression of breast cancer from the perspective of cell functions. Besides, AGR3 high expression indicated a poor prognosis of breast cancer patients treated with taxane but a favorable prognosis treated with 5-fluoropyrimidines. Performing collagen gel dropletembedded culture drug sensitivity test (CD-DST) and cytotoxic analysis to achieve more results, we found that breast cancer cells with AGR3 high expression were resistant to taxane but sensitive to 5-fluoropyrimidines. All of these results above had never been reported before.
Materials and Methods
=====================
1. Patient selection and clinical information
---------------------------------------------
Paraffin-embedded specimens were randomly selected from 51 cases of breast benign lesions, 62 cases of DCIS and 336 cases of IDC diagnosed between 2004 and 2009. All specimens came from the Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). All diagnosis of histopathology was confirmed according to World Health Organization (WHO) criteria. The median age of IDC patients was 52 years old which ranged from 28 to 89. None of them had received any oncotherapy before operation. Three hundred thirty-six cases of IDC patients included 240 cases of luminal subtype (71.4%), 28 cases of HER2-overexpressing subtype (8.3%), 62 cases of triple-negative subtype (18.5%), and six cases without information about molecular subtype (1.8%).
After operation, the chemotherapy regimens that IDC patients received included 118 cases of TE/TA (taxane+epirubicin/taxane+doxorubicin) regimen, 111 cases of CEF (cyclophosphamide, epirubicin, and 5-fluoropyrimidines)/CAF (cyclophosphamide, doxorubicin, and 5-fluoropyrimidines) regimen, 78 cases of CMF (cyclophosphamide, methotrexate, and 5-fluoropyrimidines) regimen, nine cases of platinum-based regimen and seven cases just treated with taxane.
2. Patients' prognostic information
-----------------------------------
All IDC patients were followed up with 5-146 months. In this period, 14 cases (4.2%) developed tumor recurrence, 58 cases (17.3%) developed distant metastasis, and 25 cases (7.4%) died for cancer. And 56 cases developed cancer progression within 5 years.
In addition, 58 cases of patients with distant metastasis included 43 cases of bone metastasis, 17 cases of lung metastasis, 13 cases of liver metastasis, eight cases of brain metastasis, and 19 cases with multiple organic metastases.
3. Immunohistochemical staining
-------------------------------
AGR3 expression was detected by IHC. All procedures were performed in Benchmark XT (Roche, Basel, Switzerland). Antigen retrieval was performed in citrate buffer at 121°C for 2 minutes 15 seconds. After retrieval, sections were steeped in 3% H~2~O~2~ buffer for 25 minutes and in 10% goat serum buffer for 25 minutes. Then, the sections were steeped in primary antibody against AGR3 (1:100, HPA053942, Sigma, St. Louis, MO) at 4°C for one night. Next, sections were steeped in anti-rabbit secondary antibody for 20 minutes and in horseradish peroxidase--conjugated streptavidin also for 20 minutes. Finally, sections were incubated with 3,3′-diaminobenzidinetetra hydrochloride which was peroxidase substrate.
4. Evaluation of staining
-------------------------
All stained sections were reviewed independently by two pathologists under light microscopy. Evaluation of AGR3 depended on staining intensity and area. Intensity was graded like this: 0 (--) no staining, 1 (+) weak staining, 2 (++) moderate staining, and 3 (+++) strong staining. Then patients were categorized into two groups: group 1 (the grade of intensity was 2-3 and the staining area of tumor cells was 75%-100%) and group 2 (the grade of intensity was 1 or the staining area of tumor cells was 0%-74%). Additionally, group1 was defined as AGR3 high expression and group 2 was defined as AGR3 low expression.
Evaluation of ER and PR was based on the 2010 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guideline. Sections were regarded as positive cases if the staining area of tumor cells with nuclear staining was more than 1%.
5. Cell culture and reagents
----------------------------
MDA-MB-231 and T47D cell lines were bought from American Type Culture Collection (Manassas, VA) which had been checked by DNA profiling. MDA-MB-231 were cultured with Dulbecco\'s modified Eagle\'s medium and T47D were cultured with 1640 medium which were mixed with 10% fetal bovine serum (FBS). Both two cell lines were incubated in a 5% CO~2~ incubator at 37°C.
6. Construction of lentiviral vector expressed with full length of AGR3
-----------------------------------------------------------------------
Full length of human AGR3 gene (GenBank accession No. NM_176813) was amplified in T47D cell line by polymerase chain reaction (PCR). The PCR primers were as follows: forward, 5′-GAGAGCTAGCGCCACCATGATGCTACACTCAGCT-3′ and reverse, 5′-TCTCGGATCCTTAAGCGTAATCTGGAACATCGTATGGGTACATTAGCTCTGACTGAATAAGTCTTAATGCTTT-3′. The production of PCR contained full length of AGR3 gene, 3×Flag label and HA label. Then the production was cloned into pCDH-CMVMCS-EF1-Puro lentiviral vector. The sequences of the inserts were checked by gene sequencing technique. Lentiviral plasmid, packing plasmids ΔR and pVSVg were transfected into HEK-293T cells to make lentiviruses.
Then, MDA-MB-231 cells were infected by lentiviruses and screened by puromycin. After verification by western blot, stable clones were named as 3×Flag-AGR3-HA/MDA-MB-231.
7. Construction of lentiviral vector knocking down AGR3
-------------------------------------------------------
Two different AGR3 specific RNA interference sequences were applied (siAGR3\#1-\#2). The sequences were as follows: siAGR3\#1, 5′-gccttcacttcaaagaagtca-3′; siAGR3\#2, 5′-gatgacatcacttgggtacaa-3′. Lentiviral plasmid, packing plasmids ΔR and pVSVg were transfected into HEK-293T cells to make lentiviruses. T47D cells were infected by lentiviruses and verified by western blot. Cells knocking down AGR3 successfully were named as siAGR3/T47D.
8. Western blot
---------------
Western blot was performed to detect AGR3 expression of breast cancer tissues and cells. The protein was lysed by lysis buffer (sodium dodecyl sulfate) on ice. After separating with sodium dodecyl sulfate polyacrylamide gel electrophoresis, protein was electro-transferred onto nitrocellulose membranes. The nitrocellulose membranes were incubated by primary antibodies. The primary antibodies contained AGR3 (ab201464, Abcam, Cambridge, UK), Flag (AF519--1, Beyotime, Shanghai, China), and β-actin (SC-47778, Santa Cruz Biotechnology, Santa Cruz, CA). Then the nitrocellulose membranes were treated with secondary antibodies.
9. Cell ATP/viability assay and sulforhodamine B assay
------------------------------------------------------
In cell ATP/viability assay, the cell lines were incubated in 24-well plates. Every type of cell line had six replicates. After 5 days, ATP level was measured by Cell Titer-Glo luminescent cell viability assay kit (catalog number G7571, Promega, Madison, WI). In sulforhodamine B (SRB) assay, cells were also incubated in 24-well plates for 5 days. After that, cells were fixed with 10% trichloroacetic acid and then stained with SRB (0.4%) buffer in 1% acetic acid. Lastly, added Tris-base (10 mM) into the plate to dissolve SRB and measured absorbance at 564 nm.
10. Migration assay
-------------------
Migration assays were performed on cell lines using 24-well transwell chambers (Corning, New York, NY). Cells with 200 μL serum-free medium were added into the upper chambers and 600 μL medium containing 5% FBS were added into the lower chambers. The cells were incubated at 37°C for 16 hours. Then, scraped the cells at upper layer of polyethylene membrane and stained the cells at lower layer with Giemsa solution. Lastly, taken photograph under light microscopy and quantified the cell number in randomly selected fields. All experiments were performed in triplicates.
11. Invasion assay
------------------
Invasion assays were also performed on cell lines using 24-well transwell chambers (Corning). But the upper chambers were coated with prediluted extracellular matrix. Then cells with medium containing 0.5% FBS were added into the upper chambers and 600 μL medium containing 10% FBS were added into the lower chambers. After incubating at 37°C for 24 hours, the invading cells were stained, counted and photographed under light microscopy as the procedure of migration assay. All experiments were performed in triplicates.
12. Cytotoxic analysis
----------------------
To measure the cytotoxic effects of 5-fluoropyrimidine (5-FU) and taxane, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed on cell lines. Briefly, cells were seeded at 24-well plates. After incubation to allow cells adhesion, cells were exposed to various concentrations of taxane or 5-FU (MDA-MB-231, taxane: 15 to 5×10^4^ ng/mL, 5-FU: 1 to 1.5×10^4^ μg/mL; T47D, taxane: 2.5 to 2.5×10^4^ ng/mL, 5-FU: 0.1 to 5×10^3^ μg/mL) with 500 μL/well medium for 48 hours. Besides, drug-free medium was added to the control and blank wells. After exposure was finished, 500 μL MTT (5 mg/mL) were added to each well. After 4-hour incubation, MTT solution was removed and 500 μL dimethyl sulfoxide (Sigma) were added into the wells to dissolve the insoluble formazan crystals. After dissolution, 150 μL supernatant of every well was removed into the 96-well plates and the absorbance of the dye was measured at a wave length of 570 nm. All experiments were performed in triplicates and calculated the IC~50~ value.
13. Wound healing assay
-----------------------
To measure the migration ability of the cell lines, wound healing assay was performed. The cells were seeded at 6-well plates. Then, a scratch was performed with a 10 μL pipette tip. After removing cell debris by phosphate-buffered saline, cells were incubated in a medium without FBS at 37℃. The migration distances were measured at 0, 6, 12, 18, and 24 hours. The cell-free area of the wounds was estimate under a microscope. All assays were performed in triplicates.
14. Statistical analyses
------------------------
Using SPSS ver. 17.0 software package (SPSS Inc., Chicago, IL) to perform statistical analysis. Group comparisons were calculated by Mann-Whitney U test, Kruskal-Wallis test, ANOVA test, and chi-square test. Two variables' correlation was calculated by Spearman's rank correlation test. Using Kaplan-Meier method to calculated patients' overall survival (OS) survival and progression-free survival (PFS) time. p-values were computed by log-rank test. The relevant factors for patients' prognosis were identified by Cox proportional hazards regression model. *In vitro* experiments, using two-tailed Student's t test to detect if the difference between two groups had statistical meaning or not. In all statistical analyses, two-sided p \< 0.05 was regarded as statistical significance.
15. Ethical statement
---------------------
All information about patients in this study were obtained with informed consent. And this study was approved by the Ethic Committee of Tianjin Medical University Cancer Institute and Hospital. The approval number of ethic committee was bc2017019.
Results
=======
1. AGR3 expression increased with the development of breast tumor malignancy
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We detected AGR3 expression at proteomic level by IHC in 51 cases of breast benign lesion tissues, 62 cases of DCIS tissues and 336 cases of IDC tissues. The immunohistochemical staining of AGR3 was assessed and representative images of staining were shown in [Fig. 1A](#f1-crt-2019-217){ref-type="fig"}. We found that AGR3 mainly located in cytoplasm of epithelial cells of mammary gland ducts in breast tissues. From benign lesions to DCIS and to IDC, AGR3 expression gradually evaluated ([Fig. 1B](#f1-crt-2019-217){ref-type="fig"}). 7.8% (4/51) of benign lesions, 27.4% (17/62) of DCIS and 38.4% (129/336) of IDC specimens showed high expression of AGR3 (r~s~=0.195, p \< 0.001) ([S1 Table](#SD1-crt-2019-217){ref-type="supplementary-material"}). This indicated that AGR3 expression increased with the development of breast tumor malignancy.
2. AGR3 mainly expressed in luminal subtype of IDC patients of histological grade Ⅰ-Ⅱ
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In IDC, we analyzed the relationship between AGR3 expression and clinicopathological features of patients. We found that high expression of AGR3 was associated with high ER positivity (r~s~=0.336, p \< 0.001), high PR positivity (r~s~=0.225, p \< 0.001), high risk of recurrence or metastasis (r~s~=0.179, p=0.001), and low histological grade (r~s~=--0.160, p=0.004) ([Table 1](#t1-crt-2019-217){ref-type="table"}). The relationships between AGR3 expression and patients' age (r~s~=--0.099, p=0.069), tumor size (r~s~= 0.041, p=0.457), lymph node metastasis (r~s~=--0.007, p=0.901), distant metastasis (r~s~=0.077, p=0.161), HER2 status (r~s~=--0.098, p=0.074), and Ki-67 status (r~s~=--0.009, p=0.874) were not obvious ([Table 1](#t1-crt-2019-217){ref-type="table"}).
Fifty percent (7/14) of grade Ⅰ, 41.7% (105/252) of grade Ⅱ, and 22.0% (13/59) of grade Ⅲ showed high expression of AGR3 (r~s~=--0.160, p=0.004) ([Table 1](#t1-crt-2019-217){ref-type="table"}). Taking grade Ⅰ and Ⅱ as a group of low grade and grade Ⅲ as a group of high grade, we found that 42.1% (112/266) of grade Ⅰ-Ⅱ showed high expression of AGR3 and 22.0% (13/59) of grade Ⅲ showed high expression of AGR3. The difference also had statistical meaning (r~s~=--0.159, p=0.004) ([Table 1](#t1-crt-2019-217){ref-type="table"}). In addition, western blot analysis were performed in fresh tissues of IDC of different grades (6 cases of normal breast tissues, 6 cases of grade Ⅰ, 6 cases of grade Ⅱ, and 6 cases of grade Ⅲ). Results also showed that AGR3 expression in grade Ⅰ and Ⅱ was significantly higher than grade Ⅲ ([Fig. 1C](#f1-crt-2019-217){ref-type="fig"}).
All results above indicated that AGR3 mainly expressed in IDC patients of histological grade Ⅰ-Ⅱ. In grade Ⅰ-Ⅱ, the relationship between AGR3 expression and clinicopathological features were also analyzed. High expression of AGR3 was also associated with high ER positivity (r~s~=0.333, p \< 0.001), high PR positivity (r~s~=0.224, p \< 0.001) and high rate of recurrence or metastasis (r~s~=0.220, p \< 0.001) ([S2 Table](#SD2-crt-2019-217){ref-type="supplementary-material"}). The correlations between AGR3 expression and patients' age (r~s~= --0.099, p=0.105), tumor size (r~s~=0.035, p=0.647), lymph node metastasis (r~s~=--0.029, p=0.756), distant metastasis (r~s~=0.089, p=0.149), HER2 status (r~s~=--0.113, p=0.066), and Ki-67 status (r~s~=0.014, p=0.823) were also not obvious ([S2 Table](#SD2-crt-2019-217){ref-type="supplementary-material"}). But in grade Ⅲ, all clinicopathological features had no correlation with AGR3 expression ([S3 Table](#SD3-crt-2019-217){ref-type="supplementary-material"}).
Then, we detected AGR3, ER, and PR expression in serial paraffin sections by IHC. The results also showed that AGR3 expression was positively correlated with ER and PR status. Representative images of staining were shown in [Fig. 1D](#f1-crt-2019-217){ref-type="fig"}. Based on the important roles of ER and PR in molecular subtypes \[[@b2-crt-2019-217]\], further analysis was performed to confirm the relationship between AGR3 expression and molecular subtypes. We found that AGR3 expression in luminal subtype was much higher than non-luminal subtype in IDC. Forty-five point six percent (31/68) of luminal A, 47.7% (82/172) of luminal B, 10.7% (3/28) of HER2-overexpressing, and 14.5% (9/62) of TNBC showed high expression of AGR3 (r~s~= --0.249, p \< 0.001) ([Table 2](#t2-crt-2019-217){ref-type="table"}).
Moreover, in IDC patients of grade Ⅰ-Ⅱ, AGR3 expression in luminal subtype was also much higher than non-luminal subtype. Forty-seven point four percent (27/57) of luminal A, 50.3% (72/143) of luminal B, 5.3% (1/19) of HER2-overexpressing, and 19.5% (8/41) of TNBC showed high expression of AGR3 (r~s~=--0.218, p \< 0.001) ([S4 Table](#SD4-crt-2019-217){ref-type="supplementary-material"}). But grade Ⅲ had no such relationship (r~s~=--0.241, p=0.136) ([S5 Table](#SD5-crt-2019-217){ref-type="supplementary-material"}). All of these results above indicated that AGR3 mainly expressed in luminal subtype of IDC patients of histological grade Ⅰ-Ⅱ.
3. High expression of AGR3 was a promoting factor for breast cancer patients to develop recurrence or distant metastasis
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[Table 1](#t1-crt-2019-217){ref-type="table"} showed that AGR3 expression was positively correlated with high risk of recurrence or metastasis. The representative images of IHC were shown in [Fig. 1E](#f1-crt-2019-217){ref-type="fig"}. In order to get more accurate results, similar analysis was completed in different molecular subtypes. Additionally, we also analyzed the relationship between AGR3 expression and risk of recurrence or metastasis within 5 years.
In IDC, AGR3 expression of patients occurring recurrence or metastasis was much higher than disease-free patients (p=0.001); AGR3 expression of patients occurring recurrence or metastasis within 5 years was also higher than patients disease-free over 5 years (p=0.031) ([Fig. 2A](#f2-crt-2019-217){ref-type="fig"}). Then, in luminal and luminal B subtype of IDC, AGR3 expression of patients occurring recurrence or distant metastasis was higher than disease-free patients (luminal, p \< 0.001; luminal B, p=0.005); AGR3 expression of patients occurring recurrence or distant metastasis within 5 years was also higher than patients disease-free over 5 years (luminal, p=0.004; luminal B, p=0.023) ([Fig. 2B](#f2-crt-2019-217){ref-type="fig"} and [D](#f2-crt-2019-217){ref-type="fig"}). But in luminal A and non-luminal subtype including HER2-overexpressing and TNBC, AGR3 expression had no relationship with recurrence or metastasis ([Fig. 2C](#f2-crt-2019-217){ref-type="fig"} and [E](#f2-crt-2019-217){ref-type="fig"}-[G](#f2-crt-2019-217){ref-type="fig"}).
In IDC of grade Ⅰ-Ⅱ, we got similar results. AGR3 expression of patients occurring recurrence or distant metastasis was higher than disease-free patients (p \< 0.001); AGR3 expression of patients occurring recurrence or distant metastasis within 5 years was also higher than patients disease-free over 5 years (p=0.014) ([Fig. 2H](#f2-crt-2019-217){ref-type="fig"}). Besides, the relationship between AGR3 expression and recurrence or metastasis still existed in luminal and luminal B subtype ([Fig. 2I](#f2-crt-2019-217){ref-type="fig"} and [K](#f2-crt-2019-217){ref-type="fig"}), but didn't exist in luminal A, non-luminal and TNBC ([Fig. 2J](#f2-crt-2019-217){ref-type="fig"}, [L](#f2-crt-2019-217){ref-type="fig"}, and [M](#f2-crt-2019-217){ref-type="fig"}). The number of HER2-overexpressing specimens was too few, so the investigation could not be performed. And in IDC of grade Ⅲ, AGR3 expression also had no relationship with recurrence or metastasis ([Fig. 2N](#f2-crt-2019-217){ref-type="fig"}). All results above reminded that high expression of AGR3 indicated high risk of recurrence or metastasis in luminal B subtype of IDC patients of grade Ⅰ-Ⅱ.
In addition, we investigated the relationship between AGR3 expression and metastatic organs such as bone, lung, liver, and brain which were common metastatic organs of breast cancer patients. We found that AGR3 expression was correlated with bone metastasis (total IDC patients: r~s~=0.119, p=0.029; grade Ⅰ-Ⅱ patients: r~s~=0.216, p=0.031) and liver metastasis (total IDC patients: r~s~=0.127, p=0.020; grade Ⅰ-Ⅱ patients: r~s~=0.135, p=0.027) in IDC of grade Ⅰ and Ⅱ ([Table 3](#t3-crt-2019-217){ref-type="table"}). But there was no correlation between AGR3 expression and lung metastasis (total IDC patients: r~s~=--0.015, p=0.788; grade Ⅰ-Ⅱ patients: r~s~=0.009, p=1.0) or brain metastasis (total IDC patients: r~s~=--0.003, p=0.958; grade Ⅰ-Ⅱ patients: r~s~=0.020, p=1.0) ([Table 3](#t3-crt-2019-217){ref-type="table"}). In IDC of grade Ⅲ, relationships between AGR3 expression and these four metastatic organs were not obvious ([S6 Table](#SD6-crt-2019-217){ref-type="supplementary-material"}). These results indicated that high expression of AGR3 could lead to high risk of bone and liver metastasis.
4. AGR3 was an independent prognostic factor in luminal B patients of grade Ⅰ-Ⅱ
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Considering relationship between AGR3 expression and high risk of recurrence or metastasis. Kaplan-Meier analysis was performed in 330 cases of IDC with complete follow-up data to explore the value of AGR3 in prognosis.
In IDC, both OS and PFS of AGR3 high expression group were significantly shorter than AGR3 low expression group (OS, p=0.035; PFS, p=0.004) ([Fig. 3A](#f3-crt-2019-217){ref-type="fig"}). With further analysis in different molecular subtypes, we found that OS and PFS of AGR3 high expression group shortened in luminal subtype (OS, p=0.004; PFS, p=0.001) ([Fig. 3B](#f3-crt-2019-217){ref-type="fig"}). Separately, in luminal A, AGR3 high expression could lead to shorter PFS (p=0.014) but had no influence on OS ([Fig. 3C](#f3-crt-2019-217){ref-type="fig"}); in luminal B, both OS and PFS significantly shorten if AGR3 highly expressed (OS, p=0.010; PFS, p=0.014) ([Fig. 3D](#f3-crt-2019-217){ref-type="fig"}). Whereas, in nonluminal and TNBC subtype, AGR3 expression had no influence on either OS or PFS ([Fig. 3E](#f3-crt-2019-217){ref-type="fig"} and [F](#f3-crt-2019-217){ref-type="fig"}). Number of HER2-overexpressing patients was too few, so Kaplan-Meier analysis could not be performed.
In IDC of grade Ⅰ-Ⅱ, we got similar results. OS and PFS also significantly shortened when AGR3 highly expressed (OS, p=0.027; PFS, p=0.001) ([Fig. 3G](#f3-crt-2019-217){ref-type="fig"}). And AGR3's impacts on OS and PFS still existed in luminal and luminal B subtype ([Fig. 3H](#f3-crt-2019-217){ref-type="fig"} and [J](#f3-crt-2019-217){ref-type="fig"}), but didn't exist in luminal A, non-luminal or TNBC ([Fig. 3I](#f3-crt-2019-217){ref-type="fig"}, [K](#f3-crt-2019-217){ref-type="fig"} and [L](#f3-crt-2019-217){ref-type="fig"}). Kaplan-Meier analysis still could not be performed in HER2-overexpressing subtype of grade Ⅰ-Ⅱ due to the small number of specimens.
Results above reminded that high expression of AGR3 indicated poor prognosis in luminal B subtype of IDC patients of grade Ⅰ-Ⅱ. In addition, in multivariate Cox regression analysis, we found that AGR3 expression was an independent predictor for both OS and PFS ([Table 4](#t4-crt-2019-217){ref-type="table"}).
5. AGR3 affected proliferation and invasion ability of breast cancer cells
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After confirming the promoting role of AGR3 in clinical specimens, we detected AGR3 expression in breast cancer cell lines by western blot. In western blot analysis, we took fresh tissues with AGR3 high expression as positive control. We found that AGR3 highly expressed in luminal cell line T47D, but low expressed in TNBC cell line MDA-MB-231 ([Fig. 4A](#f4-crt-2019-217){ref-type="fig"}). We overexpressed full length of AGR3 in MDA-MB-231 and knocked down AGR3 in T47D (named as 3× Flag-AGR3-HA/MDA-MB-231 and siAGR3/T47D, respectively) to make sure the role of AGR3 *in vitro*. The overexpression and interference effects were detected by western blot ([Fig. 4A](#f4-crt-2019-217){ref-type="fig"}). The interference sequence of siAGR3\#1/T47D was used *in vitro* studies.
In ATP and SRB assays, proliferation ability of 3×Flag-AGR3-HA/MDA-MB-231 significantly increased comparing to 3×Flag-vector/MDA-MB-231; proliferation ability of siAGR3/T47D significantly reduced comparing to scr/T47D ([Fig. 4B](#f4-crt-2019-217){ref-type="fig"}). Besides, we performed proliferation assays by using Nikon ECLIPSE Ti microscope to count cell numbers. The results were the same as ATP and SRB assays ([Fig. 4B](#f4-crt-2019-217){ref-type="fig"}).
In Matrigel Boyden chamber assays and wound healing assays, migration and invasion ability of 3×Flag-AGR3-HA/MDA-MB-231 significantly increased comparing to 3×Flag-vector/MDA-MB-231; migration and invasion ability of siAGR3/T47D significantly reduced comparing to scr/T47D ([Fig. 4C](#f4-crt-2019-217){ref-type="fig"} and [D](#f4-crt-2019-217){ref-type="fig"}). All results of cellular functional experiments were consistent with results we got from clinical samples and indicated that AGR3 might improve the development of breast cancer by promoting tumor cells' proliferation and invasion ability.
6. AGR3 high expression indicated a poor prognosis of breast cancer patients treated with taxane but a favorable prognosis treated with 5-fluoropyrimidines
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From AGR3 highly expressed IDC specimens, we choose 19 cases only receiving TE/TA chemotherapy regimen, 29 cases only receiving CEF/CAF chemotherapy regimen and 20 cases only receiving CMF chemotherapy regimen. These regimens were commonly used in the treatment of breast cancer patients. By performing Kaplan-Meier analysis, we found that PFS of TE/TA group were significantly shorter than CEF/CAF and CMF group (TE/TA vs. CEF/CAF, p=0.009; TE/TA vs. CMF, p=0.035). But PFS had no significant difference between CEF/CAF and CMF group ([Fig. 5A](#f5-crt-2019-217){ref-type="fig"}). In addition, from AGR3 low expressed IDC specimens, we choose 57 cases only receiving TE/TA chemotherapy regimen, 48 cases only receiving CEF/CAF chemotherapy regimen and 24 cases only receiving CMF chemotherapy regimen. Kaplan-Meier analysis showed that both OS and PFS had no correlation with chemotherapy regimen in AGR3 low expressed IDC ([Fig. 5B](#f5-crt-2019-217){ref-type="fig"}).
In AGR3 highly expressed IDC patients, CEF/CAF and CMF chemotherapy regimens could lead better prognosis than TE/TA chemotherapy regimen. We speculated that AGR3 might change the sensitivity of one drug in these regimens. We divided all specimens into ten groups based on whether chemotherapy regimen consisted of taxane, epirubicin/doxorubicin, cyclophosphamide, methotrexate or 5-fluoropyrimidines. These groups were respectively named as T/non-T group, E/non-E group, C/non-C group, M/non-M group and F/non-F group. We wanted to know whether presence or absence of one particular drug would change the prognosis of AGR3 highly expressed IDC patients by comparing "X" group to "non-X" group.
We found that, in AGR3 highly expressed IDC patients, PFS of T group was shorter than non-T group (p=0.009) ([Fig. 5C](#f5-crt-2019-217){ref-type="fig"}) and PFS of F group was longer than non-F group (p=0.007) ([Fig. 5D](#f5-crt-2019-217){ref-type="fig"}). But when comparing other groups, epirubicin/doxorubicin, cyclophosphamide, and methotrexate had no impact on both OS and PFS ([S7A](#SD7-crt-2019-217){ref-type="supplementary-material"}, [S7B](#SD7-crt-2019-217){ref-type="supplementary-material"} and [S7C Fig.](#SD7-crt-2019-217){ref-type="supplementary-material"}). In AGR3 low expressed IDC patients, OS and PFS also had no difference during any comparison.
Then, more analysis was performed in different molecular subtypes. In luminal subtype of AGR3 highly expressed IDC patients, PFS of T group was also shorter than non-T group (p=0.006) ([Fig. 5E](#f5-crt-2019-217){ref-type="fig"}) and PFS of F group was also longer than non-F group (p=0.014) ([Fig. 5F](#f5-crt-2019-217){ref-type="fig"}). But in non-luminal subtype, both OS and PFS had no correlation with taxane and 5-fluoropyrimidine ([S8A](#SD8-crt-2019-217){ref-type="supplementary-material"} and [S8B Fig.](#SD8-crt-2019-217){ref-type="supplementary-material"}). And in AGR3 low expressed IDC patients, no matter luminal subtype or non-luminal subtype had no such relationship ([Fig. 5E](#f5-crt-2019-217){ref-type="fig"}, [5F](#f5-crt-2019-217){ref-type="fig"}, [S8A](#SD8-crt-2019-217){ref-type="supplementary-material"} and [S8B Fig.](#SD8-crt-2019-217){ref-type="supplementary-material"}).
Besides, in IDC patients of grade Ⅰ-Ⅱ, PFS of T group was also shorter than non-T group (p=0.035) ([Fig. 5G](#f5-crt-2019-217){ref-type="fig"}) and PFS of F group was also longer than non-F group (p=0.007) ([Fig. 5H](#f5-crt-2019-217){ref-type="fig"}) when AGR3 highly expressed. And the impact of taxane and 5-fluoropyrimidine on PFS also existed in luminal subtype ([Fig. 5I](#f5-crt-2019-217){ref-type="fig"} and [J](#f5-crt-2019-217){ref-type="fig"}) but not in non-luminal subtype ([S8C](#SD8-crt-2019-217){ref-type="supplementary-material"} and [S8D Fig.](#SD8-crt-2019-217){ref-type="supplementary-material"}). In addition, grade Ⅲ patients had no such relationship ([S8E](#SD8-crt-2019-217){ref-type="supplementary-material"} and [S8F Fig.](#SD8-crt-2019-217){ref-type="supplementary-material"}). All of these results above indicated that AGR3 high expression indicated a poor prognosis of breast cancer patients treated with taxane but a favorable prognosis treated with 5-fluoropyrimidines.
7. Breast cancer cells with AGR3 high expression were resistant to taxane but sensitive to 5-fluoropyrimidines
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CD-DST assay had been widely applied in clinical work to test sensitivities of chemotherapy drugs. Based on the results of CD-DST, we chose other 161 cases of IDC specimens consisting of 44 cases sensitive to taxane, 39 cases resistant to taxane, 42 cases sensitive to 5-fluoropyrimidines and 36 cases resistant to 5-fluoropyrimidines. Then, we performed IHC to detect AGR3 expression in these specimens. We found that AGR3 expression of patients sensitive to taxane was much lower than patients resistant to taxane (p \< 0.001) ([Fig. 6A](#f6-crt-2019-217){ref-type="fig"}) but AGR3 expression of patients sensitive to 5-fluoropyrimidines was much higher than patients resistant to 5-fluoropyrimidines (p=0.004) ([Fig. 6A](#f6-crt-2019-217){ref-type="fig"}).
In breast cancer cell lines, 3×Flag-AGR3-HA/MDA-MB-231 and siAGR3/T47D were performed MTT and ATP assay to measure cell viability with various concentrations of taxane and 5-fluoropyrimidines. Moreover, the IC~50~ values of taxane and 5-fluoropyrimidines in different cells were also detected. 3×Flag-vector/MDA-MB-231 and 3×Flag-AGR3-HA/MDA-MB-231 cells were treated with 15 to 5×10^4^ ng/mL of taxane and 1-1.5×10^4^ μg/mL of 5-fluoropyrimidines for 48 hours. Both MTT and ATP assay showed that 3×Flag-AGR3-HA/MDA-MB-231 cells exhibited more resistant to taxane but more sensitive to 5-fluoropyrimidines than 3×Flag-vector/MDA-MB-231 cells ([Fig. 6B](#f6-crt-2019-217){ref-type="fig"}). Scr/T47D and siAGR3/T47D cells were treated with 2.5 to 2.5×10^4^ ng/mL of taxane and 0.1-5×10^3^ μg/mL of 5-fluoropyrimidines for 48 hours. Both MTT and ATP assay showed that siAGR3/T47D cells exhibited more sensitive to taxane but more resistant to 5-fluoropyrimidines than scr/T47D cells ([Fig. 6C](#f6-crt-2019-217){ref-type="fig"}).
All of these results above indicated that breast cancer cells with AGR3 high expression were resistant to taxane but sensitive to 5-fluoropyrimidines. These results explained why AGR3 high expression could indicate a poor prognosis of breast cancer patients treated with taxane but a favorable prognosis treated with 5-fluoropyrimidines.
Discussion
==========
AGR3's impact on cancers is still unclear. Reports about AGR3 in cancers are few and contradictory. In tumors, AGR3 protein was firstly identified in the membrane of breast cancer cell lines by proteomic screening \[[@b16-crt-2019-217]\]. But later, Loader's group confirmed that AGR3 overexpressed in the cytoplasm of breast cancer cells \[[@b8-crt-2019-217]\]. And Garczyk et al. \[[@b17-crt-2019-217]\] found that AGR3's mRNA significantly increased in breast cancer tissues of histological grade Ⅰ-Ⅱ, but not in grade Ⅲ and AGR3 played a role of indicating poor prognosis in grade Ⅰ-Ⅱ which were consistent with our results. But they didn't perform more study about the correlation between AGR3 and molecular subtypes. They also found that combining expression of AGR3 and AGR2 in serum sample of breast cancer patients could diagnosed early breast cancer \[[@b17-crt-2019-217]\]. Besides, it had been demonstrated that AGR3 also overexpressed in ovarian \[[@b18-crt-2019-217]\] and prostate \[[@b19-crt-2019-217]\] cancer. In ovarian cancer, Gray et al. \[[@b20-crt-2019-217]\] found that AGR3 was up-regulated by a hormone (ER)-independent mechanism and could mediate resistance to cisplatin. In prostate cancer, Vaarala et al. \[[@b21-crt-2019-217]\] found that AGR3 highly elevated in prostate cancer tissues and Bu et al. \[[@b22-crt-2019-217]\] found that both AGR2 and AGR3 genes were regulated by androgen and estrogen. Moreover, androgen receptor-binding sites in the promoter region of AGR2 and AGR3 were identified in the report of Bu et al. \[[@b22-crt-2019-217]\]. In these studies, researchers considered that AGR3 could promote cancer development and regarded AGR3 as an indicator of poor prognosis of cancer patients. However, some studies took opposite opinions. In breast cancer, Obacz et al. \[[@b23-crt-2019-217]\] found that AGR3 was associated with differentiation level, weak proliferation and favorable prognosis of patients. In ovarian cancer, AGR3 expression in serous borderline ovarian tumors and low-grade serous ovarian carcinomas was much higher than high-grade serous ovarian carcinomas and AGR3 was associated with longer patient survival \[[@b24-crt-2019-217]\].
The role of AGR3 remained uncertain and researchers even got opposite results in the same kind of tumor tissues. We thought it was due to the limit of sample size. With larger scale of samples, our study confirmed that AGR3 expression significantly increased with the development of breast tumor malignancy and had close relationships with molecular subtypes and histological grades. AGR3 mainly expressed in luminal subtype of IDC patients of histological grade Ⅰ-Ⅱ. Most importantly, in luminal B subtype, high expression of AGR3 was correlated with high risk of recurrence and metastasis and could predict poor prognosis of patients. Therefore, AGR3 might be a potential prognostic indicator of luminal B subtype in which patients were more likely to occur therapeutic resistance and poor outcome than luminal A \[[@b5-crt-2019-217]\]. As for why AGR3 was also overexpressed in luminal A but couldn't indicate prognosis of this subtype, we considered that it was due to the excellent sensitivity to endocrine therapy in luminal A. And we considered that AGR3's overexpression in luminal subtype of IDC was due to the transcription activating function of ERα. Welboren et al. \[[@b25-crt-2019-217]\] had verified several ERα-binding sites existing on the promoter regions of AGR2 and AGR3. But the accurate mechanism needed more proofs. Besides, through further analysis, we found that the majority of luminal patients were grade Ⅰ-Ⅱ (83.33%) but only 16.67% of luminal patients were grade Ⅲ. This result probably explained why AGR3 mainly expressed in grade Ⅰ-Ⅱ rather than grade Ⅲ.
Besides, we firstly found that taxane lead to worse outcome but 5-fluoropyrimidine lead to favorable outcome in AGR3 highly expressed IDC. Then, by performing CD-DST and cytotoxic analysis, we demonstrated that breast cancer cells with AGR3 high expression were resistant to taxane but sensitive to 5-fluoropyrimidines. As we known, in clinic therapy, luminal B patients were routinely treated with additional chemotherapy on the basis of endocrine therapy \[[@b5-crt-2019-217]\]. These indicated that luminal B patients with AGR3 high expression should be treated with chemotherapy regimens consisting of 5-fluoropyrimidines but not taxane.
As we known, taxane could cure cancer patients depending on its antimitotic characteristic. But ER stress had been identified as another mechanism of taxane cytotoxic effects. The ER stress mediated by taxane could induce apoptosis of cancer cells \[[@b26-crt-2019-217]\]. In this process, unfolded protein response (UPR) signaling pathway could weaken ER stress to maintain ER homeostasis and help cells survive \[[@b27-crt-2019-217]\]. Researchers had demonstrated that AGR2 could bind GRP78 which was a central trigger of UPR. The interaction of AGR2 and GRP78 would activate UPR signaling pathway to attenuate cell death induced by ER stress \[[@b28-crt-2019-217]\]. As homologous protein, AGR2 and AGR3 gene shared 71% sequence identity and lay adjacent to one another at chromosomal position 7p21 \[[@b22-crt-2019-217]-[@b24-crt-2019-217]\]. Therefore, in tumor cells, we speculated that AGR3 might also interact with GRP78 to activate UPR signaling pathway. Then, the UPR would atte-nuates ER stress mediated by taxane so that tumor cells could survive from the taxane treatment. Besides, Kim et al. \[[@b29-crt-2019-217]\] found that ER stress induced 5-fluoropyrimidines resistance in human colon cancer cells and Yun et al. \[[@b30-crt-2019-217]\] found that down-regulation of GRP78 could lead to enhanced sensitivity of cancer cells to cytotoxic effect of 5-fluoropyrimidines \[[@b30-crt-2019-217]\]. Therefore, we considered that AGR3 could promote sensitivity of breast cancer cells to 5-fluoropyrimidines also due to the interaction with GRP78. All of these speculations need a large number of experiments to prove.
Migration and invasion ability of tumor cells played important roles in the development of cancer. We reported for the first time that AGR3 could promote the proliferation and invasion ability of tumor cells which might be the biological basis of AGR3's prompting role in cancer.
In conclusion, our research confirmed that, in luminal B subtype of IDC patients of histological grade Ⅰ-Ⅱ, AGR3 high expression indicated poor prognosis and patients should be treated with chemotherapy regimens consisting of 5-fluoropyrimidines but not taxane. AGR3 could promote tumor progression by improving tumor cells' proliferation ability, invasion ability, and chemotherapeutic drug resistance. AGR3 might be a potential prognostic indicator for classifying prognosis and therapy regimens in this population. But all of these conclusions need more experiments and analysis to confirm.
Conflict of interest relevant to this article was not reported.
This work was supported by National Scientific Foundation of China (81572851) from Dr. Yongjie Ma and National Scientific Foundation of China (81672636) from Dr. Feng Gu. And our clinical trial registration number was bc2017019.
Electronic Supplementary Material
=================================
Supplementary materials are available at Cancer Research and Treatment website ([https://www.e-crt.org](http://www.e-crt.org)).
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AGR3 expression in different types of breast tumors
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AGR3 expression and pathological features in IDC of grade Ⅰ-Ⅱ
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AGR3 expression and pathological features in IDC patients of grade Ⅲ
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Relationship between AGR3 and molecular subtypes in IDC of grade Ⅰ-Ⅱ
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Relationship between AGR3 and molecular subtypes in IDC of grade Ⅲ
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Relationship between AGR3 and metastatic organs in IDC of grade Ⅲ
######
Anterior gradient 3 (AGR3) had no indication function for therapeutic response of doxorubicin, cyclophosphamide and methotrexate in breast cancer patients. (A) Overall survival (OS) and progression-free survival (PFS) curves of AGR3 highly expressed invasive ductal carcinoma (IDC) patients with epirubicin or doxorubicin treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed IDC patients with epirubicin or doxorubicin treatment were shown, respectively (lower panel). (B) OS and PFS curves of AGR3 highly expressed IDC patients with cyclophosphamide treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed IDC patients with cyclophosphamide treatment were shown, respectively (lower panel). (C) OS and PFS curves of AGR3 highly expressed IDC patients with methotrexate treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed IDC patients with methotrexate treatment were shown, respectively (lower panel).
######
Anterior gradient 3 (AGR3) had no indication function for therapeutic response of taxane and 5-fluoropyrimidines in non-luminal patients or grade Ⅲ patients. (A) Overall survival (OS) and progression-free survival (PFS) curves of AGR3 highly expressed non-luminal patients with taxane treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed non-luminal patients with taxane treatment were shown, respectively (lower panel). (B) OS and PFS curves of AGR3 highly expressed non-luminal patients with 5-fluoropyrimidines treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed non-luminal patients with 5-fluoropyrimidines treatment were shown, respectively (lower panel). (C) OS and PFS curves of AGR3 highly expressed non-luminal patients of grade Ⅰ-Ⅱ with taxane treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed non-luminal patients of grade Ⅰ-Ⅱ with taxane treatment were shown, respectively (lower panel). (D) OS and PFS curves of AGR3 highly expressed non-luminal patients of grade Ⅰ-Ⅱ with 5-fluoropyrimidines treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed non-luminal patients of grade Ⅰ-Ⅱ with 5-fluoropyrimidines treatment were shown, respectively (lower panel). (E) OS and PFS curves of AGR3 highly expressed IDC patients of grade Ⅲ with taxane treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed IDC patients of grade Ⅲ with taxane treatment were shown, respectively (lower panel). (F) OS and PFS curves of AGR3 highly expressed IDC patients of grade Ⅲ with 5-fluoropyrimidines treatment were shown, respectively (upper panel). OS and PFS curves of AGR3 low expressed IDC patients of grade Ⅲwith 5-fluoropyrimidines treatment were shown, respectively (lower panel).
{#f1-crt-2019-217}
{#f2-crt-2019-217}
{#f3-crt-2019-217}
{#f4-crt-2019-217}
{#f5-crt-2019-217}
{#f6-crt-2019-217}
######
AGR3 expression and pathological features of IDC patients
Pathological feature No. AGR3 expression, n (%) r~s~ p-value
------------------------------------------------------------------------------------- ----- ------------------------ ------------ --------- --------------------------------------------------------
**Age (yr)**
\< 50 156 88 (56.4) 68 (43.6) --0.099 0.069
≥ 50 180 119 (66.1) 61 (33.9)
**Histological grade^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
Ⅰ 14 7 (50.0) 7 (50.0) --0.160 0.004^[\*\*](#tfn1-crt-2019-217){ref-type="table-fn"}^
Ⅱ 252 147 (58.3) 105 (41.7)
Ⅲ 59 46 (78.0) 13 (22.0)
**Histological grade^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
Ⅰ-Ⅱ 266 154 (57.9) 112 (42.1) --0.159 0.004^[\*\*](#tfn1-crt-2019-217){ref-type="table-fn"}^
Ⅲ 59 46 (78.0) 13 (22.0)
**Tumor size (cm)^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
\< 2 79 51 (64.6) 28 (35.4) 0.041 0.457
2-5 231 141 (61.0) 90 (39.0)
\> 5 23 13 (56.5) 10 (43.5)
**Lymph node metastases**
0 122 75 (61.5) 47 (38.5) --0.007 0.901
1-3 100 62 (62.0) 38 (38.0)
4-9 52 30 (57.7) 22 (42.3)
\> 9 62 40 (64.5) 22 (35.5)
**Distant metastasis**
No 278 176 (63.3) 102 (36.7) 0.077 0.161
Yes 58 31 (53.4) 27 (46.6)
**ER status^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
Negative 123 102 (82.9) 21 (17.1) 0.336 \< 0.001
Positive 212 104 (49.1) 108 (50.9)
**PR status^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
Negative 121 92 (76.0) 29 (24.0) 0.225 \< 0.001
Positive 214 114 (53.3) 100 (46.7)
**HER2 status^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
-- to + 265 157 (59.2) 108 (40.8) --0.098 0.074
++ to +++ 69 49 (71.0) 20 (29.0)
**Ki-67 status^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
Negative 96 59 (61.5) 37 (38.5) --0.009 0.874
Positive 234 146 (62.4) 88 (37.6)
**Recurrence or distant metastasis^[a)](#tfn2-crt-2019-217){ref-type="table-fn"}^**
No 261 173 (66.3) 88 (33.7) 0.179 0.001
Yes 69 31 (44.9) 38 (55.1)
p-value was calculated by Spearman's rank correlation test. IDC, invasive ductal carcinoma; AGR3, anterior gradient 3; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2.
p \< 0.01.
Some missing data.
######
Relationship between AGR3 expression and molecular subtypes in IDC
Molecular subtype No. AGR3 expression, n (%) r~s~ p-value
--------------------- ----- ------------------------ ----------- --------- ----------------------------------------------------------
Luminal A 68 37 (54.4) 31 (45.6) --0.249 0.000^[\*\*\*](#tfn3-crt-2019-217){ref-type="table-fn"}^
Luminal B 172 90 (52.3) 82 (47.7)
HER2-overexpressing 28 25 (89.3) 3 (10.7)
TNBC 62 53 (85.5) 9 (14.5)
p-value (luminal A vs. luminal B)=0.770, p-value (HER2-overexpressing vs. TNBC)=0.876, p-value (luminal A and luminal B vs. HER2-overexpressing and TNBC)=0.000; p-value was calculated by Spearman's rank correlation test. AGR3, anterior gradient 3; IDC, invasive ductal carcinoma; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer.
p \< 0.001.
######
Relationship between AGR3 expression and metastatic organs in IDC patients of grade Ⅰ-Ⅱ
Metastatic organs No. (IDC) AGR3 expression of IDC r~s~ p-value No. (grade Ⅰ-Ⅱ) AGR3 expression of grade Ⅰ-Ⅱ r~s~ p-value
---------------------- ----------- ------------------------ ------------ --------- ------------------------------------------------------ ------------------------------ ------------ ------------ ------- ------------------------------------------------------
**Bone metastasis**
No 293 187 (63.8) 106 (36.2) 0.119 0.029^[\*](#tfn4-crt-2019-217){ref-type="table-fn"}^ 234 154 (65.8) 80 (34.2) 0.216 0.031^[\*](#tfn4-crt-2019-217){ref-type="table-fn"}^
Yes 43 20 (46.5) 23 (53.5) 32 14 (43.8) 18 (56.3)
**Lung metastasis**
No 319 196 (61.4) 123 (38.6) --0.015 0.788 257 149 (58.0) 108 (42.0) 0.009 1.000
Yes 17 11 (64.7) 6 (35.3) 9 5 (55.6) 4 (44.4)
**Liver metastasis**
No 323 203 (62.8) 120 (37.2) 0.127 0.020^[\*](#tfn4-crt-2019-217){ref-type="table-fn"}^ 257 152 (59.1) 105 (40.9) 0.135 0.027^[\*](#tfn4-crt-2019-217){ref-type="table-fn"}^
Yes 13 4 (30.8) 9 (69.2) 9 2 (22.2) 7 (77.8)
**Brain metastasis**
No 328 202 (61.6) 126 (38.4) --0.003 0.958 262 152 (58.0) 110 (42.0) 0.020 1.000
Yes 8 5 (62.5) 3 (37.5) 4 2 (50.0) 2 (50.0)
p-value was calculated by Spearman's Rank-Correlation test. AGR3, anterior gradient 3; IDC, invasive ductal carcinoma.
p \< 0.05.
######
Univariate and multivariate analysis for OS and PFS in IDC patients of grade Ⅰ-Ⅱ
Variable OS (univariate) OS (multivariate) PFS (univariate) PFS (multivariate)
----------------------- --------------------- ------------------- ---------------------- -------------------- --------------------- ------- --------------------- ----------
Age 0.911 (0.385-2.156) 0.832 1.071 (0.430-2.664) \- 0.676 (0.386-1.187) 0.173 0.759 (0.422-1.367) \-
Tumor size 1.624 (0.740-3.565) 0.227 1.017 (0.456-2.270) \- 1.852 (1.096-3.130) 0.021 1.139 (0.656-1.976) \-
Lymph node metastasis 1.680 (1.149-2.455) 0.007 1.757 (1.173-2.630) 0.006 1.651 (1.294-2.106) 0.000 1.679 (1.303-2.164) \< 0.001
ER status 0.766 (0.316-1.857) 0.556 0.436 (0.147-1.295) \- 0.739 (0.417-1.309) 0.300 0.559 (0.282-1.109) \-
PR status 0.907 (0.366-2.251) 0.834 0.780 (0.277-2.197) \- 0.645 (0.366-1.137) 0.129 0.498 (0.261-0.950) \-
AGR3 expression 2.695 (1.082-6.713) 0.033 4.161 (1.406-12.312) 0.010 2.531 (1.421-4.511) 0.002 3.856 (1.953-7.613) \< 0.001
OS, overall survival; PFS, progression-free survival; IDC, invasive ductal carcinoma; HR, hazard ratio; CI, confidence interval; ER, estrogen receptor; PR, progesterone receptor; AGR3, anterior gradient 3.
[^1]: Qiao Xu, Ying Shao, and Jinman Zhang contributed equally to this work.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#S1}
============
Increases in fat mass and weight are specifically linked to the menopausal phase \[[@ref1]\]. Conditions such as obesity, especially the amount of abdominal fat, are often associated with cardiovascular diseases and risk factors such as plasma lipids, hyper-insulinemia, and insulin resistance \[[@ref2]\]. Deterioration of physiological activities, associated with aging and inactivity, may also intensify during menopause \[[@ref3]\]. Exercise, which is believed to be associated with improvements in insulin sensitivity, can aid in visceral fat reduction, especially in the central regions of the body \[[@ref4]\]. The underlying molecular and cellular mechanism is not clearly understood, but research tells us that the muscle contractions lead to high secretions of myokines, which are assumed to mediate the positive effects of exercise \[[@ref5]\].
As such, physical exercise stimulates the expression of the fibronectin type III domain containing 5 (FNDC5) gene, which leads to high levels of eponymous protein successors. FNDC5 is the pro-protein of irisin, a type of myokine, which engenders the transformation of white to beige adipocytes via uncoupling protein 1 \[[@ref6]\]. Subsequently, the body's energy expenditure and thermogenesis also rise in response to the secretion of irisin. This event has an enriching effect on metabolic traits (such as decreased insulin sensitivity, high fasting blood sugar, hyperlipidemia, reduced HDL, impaired adipose tissue function) linked to overweight and obesity \[[@ref6], [@ref7]\].
Based on existing studies, exercise type (endurance/resistance), exercise time (acute/chronic), exercise intensity, and personal training prior to participation in studies count as factors affecting the circulating irisin level. Indeed, different studies have reported physical exercise influences are significant in irisin circulation levels \[[@ref8]-[@ref10]\], while other studies show that exercise, acute and/or chronic, has no substantial effect on blood irisin level \[[@ref11]-[@ref13]\]. Therefore, the role of exercise on irisin activity has been a controversial subject\[[@ref14]-[@ref17]\]. Furthermore, inconsistent results have come into view regarding irisin concentrations caused by exercise. In another study, a significant increase in irisin levels after a session of high-intensity interval training (HIIT) was observed, while Pilates seems to have no influence on overweight women \[[@ref18]\]. Likewise, there is very little knowledge about how high-intensity concurrent training might affect metabolic functions in the middle-aged woman's well-being.
Despite conflicting data, the effect of irisin on metabolic traits linked to obesity appears favorable. However, it must be mentioned that post-training irisin measurements in postmenopausal women were not performed. Therefore, the concrete effect of exercise in promoting expression and secretion of irisin in the human body remains uncertain. On the other hand, as hypothesized by the authors, circulating levels of myokines may fluctuate differently under the influence of different types of exercises. The selected protein was chosen because it was newly discovered as a plasma myokine. Irisin and its metabolic functions have not been previously studied in postmenopausal women.
Based on the favorable effects of aerobic and resistance training on health, the current research intends to examine the effects of two concurrent exercise training programs on insulin sensitivity status, serum levels of irisin andabdominal fat distribution (visceral vs. subcutaneous) and total abdominal fat in postmenopausal women.
To achieve more accurate results, two control groups were selected: one for the potential effects of daily exercises and the second to account for any additional factors. The training program lasted for ten weeks. Our hypothesis herein is that high-intensity concurrent interval exercise will have a greater influence on improving most metabolic-related parameters than moderate-intensity concurrent interval exercise.
Material and methods {#S2}
====================
Participants {#S2_1}
------------
One hundred and seventeen postmenopausal participants within the ages of 45-65 years were recruited to the study via notices at Rasht area City Park and clubs. Being at least one year in menopausal stage and no alcohol or drug addiction were the criteria for women to be a part of the study. All participants were matched for age and body mass index (BMI; calculated as weight in kilograms divided by the height in meters squared). Subjects of the study signed written consent, filled out a medical questionnaire, and received a thorough description of the research methods before participation in the study.
Individual levels of physical activity were evaluated via the International Physical Activity Questionnaire (IPAQ). A detailed questionnaire was used to assess physical activity on which participants answered on a scale of 0 (none) to 10 (exercise \> 1 hour four times/week). A random selection of samples was made according to the results and they were divided into four groups: a control sedentary group (\< 5 on the scale) and three active groups (≥ 5 on the scale) \[[@ref19]\]; high-intensity concurrent interval exercise (HCI; *n* = 15), moderate-intensity continuous concurrent exercise (MCC; *n* = 14), control daily active (CDA; *n* = 10), and control sedentary (COS; *n* = 9) were enlisted. Participants of the control sedentary group, although physically active, did not perform any resistance training or any other type of regular exercise for 6 months before the start of the study ([Fig. 1](#F1){ref-type="fig"}). The study followed the Declaration of Helsinki, which was approved by the research committees of the Universities of Guilan, Rasht, Iran.
{#F1}
Research consideration {#S2_2}
----------------------
Participants' physical activity and demographic backgrounds were determined through a series of interviews. Subsequent to gaining consent, four groups visited the clinical research center twice: at baseline, and post-test after 10 weeks. Participants' physical activity and demographic backgrounds were determined through a series of interviews. As the next step, height (cm) and body weight (kg) measurements were carried out by calibrated devices in a standardized manner. Next, BMI was calculated using the equation body mass/height squared. Additionally, a fasting blood sample was collected, and participants performed a 24-hour food recall. Finally, HRmax measurements were carried out by a telemetric heart rate monitor (Polar Electro Oy, Kempele, Finland) and the Brzycki equation was used for the estimation of the one repetition maximum (1RM) \[[@ref20]\]. At baseline and endpoint measurements were performed by physicians and exercise physiologists at the same time of day.
Concurrent exercise training protocol {#S2_3}
-------------------------------------
The training interventions lasted for 10 weeks and the participants exercised three times per week for 50-65 min/session with a minimal interval of 48 hours between them. During the 2 weeks, both groups (HCI and MCC) performed three times resistance and endurance concurrent training (CT) sessions. Endurance and resistance training (RT) were not done on the same days and in the same session. The HCI (RT + HIIT) and MCC (RT + CT) groups received either the HIIT or CT and RT (moderate or high training) protocols as described above; all participants expended energy up to the 500-kcal goal ([Table 1](#T1){ref-type="table"}).
######
Resistance training program
Program variable Training intensity (1RM) Training volume Rest time (min) Energy expenditure (kcal)
------------------ -------------------------- ----------------------- ----------------- ---------------------------
**Weeks** **High training**
1-2 60-70% 2 × 8-10 1 300
2-4 70% 3 × 8-10 30 400
5-10 70-85% 4 × 8-10 1 500
**Moderate training**
1-2 40-60% 3 × 12-15 1 300
2-4 60% 3 × 12-15 30 400
5-10 60-70% 4 × 12-15 1 500
1RM -- one repetition maximum, volume = sets × repetitions
Resistance training program {#S2_4}
---------------------------
The RT protocol was used to complete a resistance circuit containing specific exercises including (upper and lower muscle groups; nine exercises) leg press, chest press, lat pull-down, leg extension, leg curl, cable crossover, biceps curl, triceps extension, and abdominal crunches. The RT program was based on the recommendations of the American College of Sports Medicine (ACSM) for RT in healthy adults including 3-4 sets, 8-15 repetitions, 30-60 second rest interval between 2 sets, and 2-3 minute rest between exercises \[[@ref21]\]. Total training time ranged from 35 to 45 minutes, based on the number of exercises assigned ([Table 1](#T1){ref-type="table"}). The study group's warm-up and cool-down were performed prior to and after each session (both of 5-10 minutes duration, and consisting of walking and light, static stretching \[avoiding muscle pain\] in most muscle groups). In order to involve the major muscle groups, the RT procedure was followed, using a full range of motion that is considered an accurate method for the studies \[[@ref21], [@ref22]\]. To avoid potential risks, as a rule, the following activities were avoided: 1) events associated with Valsalva's mechanism, 2) ballistic and plyometric movements, and 3) positions of extreme muscular tension.
Continuous aerobic training program {#S2_5}
-----------------------------------
The continuous aerobic training (CAT) session included walking/running on a treadmill, a warm-up period of 5-10 minutes at 50% of HR max, followed by 45-50 minutes of walking/running at 60-75% of HR max. Participants then completed the workout with a cooling-off period of 5-10 minutes at 50% of HR max for a total duration of 45-55 minutes ([Fig. 2](#F2){ref-type="fig"}).
{#F2}
High-intensity interval training {#S2_6}
--------------------------------
HIIT sessions began with 5-10 minutes of warm-up. Subsequently, participants were engaged in training sessions of 4 × 4 minutes intervals at 85-95% HR max (with the target zone maintained for at least 2 minutes) interspersed with a 4-minute recovery period at 65% HR max, concluding with a cool-down (5 minutes), with an overall training time ranging between 40 and50 minutes \[[@ref22], [@ref23]\] ([Fig. 2](#F2){ref-type="fig"}).
Study subjects assigned to the control groups avoided any participation in circuit training sessions. They were requested to keep up with their routine exercises and diet throughout the study. Each training session was performed under the supervision of a researcher, to guide, aid, and to ensure the proper performance rate and load. All these sessions were carried out at Guilan Talent Discovery and Profession Athletic Center, Rasht, Iran.
Magnetic resonance imaging {#S2_7}
--------------------------
Visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and total abdominal fat volumes were measured by magnetic resonance imaging (MRI; premenopausal images: 1.5-T whole-body scanner, General Electric, Waukesha, WI; postmenopausal images: 1.5-T whole-body scanner, Philips Intera, Andover, MA). Slice thickness was 1 cm, with approximately 12-24 consecutive slices per scan. An automatic fat separation program was used for image analysis (Slice-O-Matic, Tomovision Inc., Montreal, Canada) \[[@ref24]\]. A thorough description and model of the methods used for various tissues segmentation are shown in a different location \[[@ref28]\]. Every image was analyzed twice with the subject's name concealed from the operator to avoid any potential errors. A single researcher was held responsible for analyzing both pre- and post-test images. The range from the superior part of the head of the femur up to the inferior most part of the kidneys was set as the abdominal fat calculation area. The sum of overall fat contents of all slices was used to evaluate total belly fat. In order to calculate subcutaneous fat, the visceral fat value in each slice was subtracted from total fat.
Blood sampling {#S2_8}
--------------
Blood samples (5 ml at each time) were retrieved from the left antecubital vein in both pre-training, following a 12-hour overnight fast, and post-training, following a 12-hour overnight fast and a 24-hour period of no exercise. For both groups, this procedure was performed at 7:30 A.M. by the laboratory practitioner with the subject in a sitting position.
Thereafter the samples remained at room temperature for clotting purposes for about half an hour. Next, the samples were centrifuged at 3000 rpm (1500 g) for 15 minutes, and plasma was removed and frozen at --80°C plasma levels of the irisin levels.
Serum irisin levels were measured by enzyme immunoassay (BioVendor, USA) following the user's manual. Assay range: 0.001 µg/ml, 5 µg/ml. Irisin levels range in human plasma and serum from 0.2 µg/ml to \> 2 µg/ml. Special kits, utilizing enzymatic colorimetric techniques, were used for glucose level measurements. The insulin serum level measurement was carried out via an enzyme-linked immunosorbent assay (ELISA) kit. The quantitative insulin sensitivity check index (QUICKI) was determined using the formula described by Katz *et al*. \[[@ref25]\]:
QUICK = 1 / (log (fasting insulin µU/ml) + log (fasting glucose mg/dl))
Statistical analyses {#S2_9}
--------------------
The Shapiro-Wilk test was applied to test the data normality, and homogeneity of variance was examined using Levine's test. ANOVA measures were used to compare data of the four groups, using the Bonferroni method to locate means that were significantly different. Additionally, comparisons were made of variances among four groups using the independent *t*-test at a different time of measurements. Spearman's rank correlation test was used to test the relationship between insulin sensitivity, irisin level, weight loss and abdominal fat distribution. Data are expressed as mean ±SD; *p*-values below 0.05 were considered significant. Statistical analyses were performed using the software SPSS version 20.
Results {#S3}
=======
Pre-intervention subjects' characteristics and changes over the intervention period {#S3_1}
-----------------------------------------------------------------------------------
Forty-eight participants (age; 54.8 ±4.17 years, weight; 72.62 ±8.45 kg, BMI; 29.73 ±3.59 kg/m^2^ mean ±SD) completed the 10-week program without any problems. The subjects showed tolerance to both HCI and MCC, and all the exercise sessions ran smoothly without any interruptions due to health issues. Baseline and post-test characteristics of the study group are summarized in [Table 2](#T2){ref-type="table"}. No significant differences were noted in any demographic parameter between the four groups at pre-test.
######
Baseline and post-training anthropometric factors, clinical characteristics and abdominal fat of subjects who under went study
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Group\ HCI (*n* = 15) MCC (*n* = 14) CDA (*n* = 10) COS (*n* = 9)
\\\
Variable
----------------------------------- ---------------- --------------------------------------------------- ----------------------------------------------- -------------------------------------------------- --------------- ---------------------------------------------- --------------- -------------------------------------------------
Age, years 4.13±57.11 -- 54.10 ±5.08 -- 54.10 ±5.08 -- 56.66 ±3.92 --
Weight, kg 70.32 ±6.49 67.38 ±5.87^[\*\*](#TF2-3){ref-type="table-fn"}^ 74.45 ±6.17 72.65 ±6.67^[\*\*](#TF2-3){ref-type="table-fn"}^ 72.45 ±12.36 74.39 ±11.7 73.34 ±9.66 72.88 ±10.88
BMI, kg/m^2^ 28.31 ±2.20 27.00 ±1.80^[\*\*](#TF2-3){ref-type="table-fn"}^ 30.62 ±3.08 30.02 ±3.83^[\*](#TF2-2){ref-type="table-fn"}^ 29.92 ±4.15 29.77 ±3.83 30.48 ±5.15 30.36 ±5.27
WHR 0.91 ±0.04 0.89 ±0.02^[\*\*](#TF2-3){ref-type="table-fn"}^ 0.03±0.92 0.91 ±0.03^[\*](#TF2-2){ref-type="table-fn"}^ 0.91 ±0.02 0.91 ±0.03 0.93 ±0.02 0.93 ±0.02^[a](#TF2-4){ref-type="table-fn"}^
Glucose, mg/dl 97.00 ±7.24 92.73 ±5.93 94.14 ±7.89 93.21 ±8.18 93.60 ±9.89 93.00 ±7.55 99.66 ±10.12 102.33 ±10.28^[a](#TF2-4){ref-type="table-fn"}^
QUICKI 0.35 ±0.02 0.37 ±0.02^[\*](#TF2-2){ref-type="table-fn"}^ 0.35 ±0.02^[\*](#TF2-2){ref-type="table-fn"}^ 0.36 ±0.02 0.36 ±0.02 0.35 ±0.02 0.34 ±0.02 0.34 ±0.02
Irisin, µg/ml 1.33 ±0.36 2.20 ±0.42^[\*\*](#TF2-3){ref-type="table-fn"}^ 1.30 ±0.28 1.56 ±0.51^[a](#TF2-4){ref-type="table-fn"}^ 1.35 ±0.42 1.34 ±0.40^[a](#TF2-4){ref-type="table-fn"}^ 1.38 ±0.45 1.26 ±0.41^[a](#TF2-4){ref-type="table-fn"}^
Subcutaneous abdominal fat, cm^2^ 97.13 ±21.75 93.25 ±20.87^[\*\*](#TF2-3){ref-type="table-fn"}^ 102.43 ±21.75 101.22 ±21.62^[\*](#TF2-2){ref-type="table-fn"}^ 99.44 ±16.33 99.37 ±16.46 108.6 ±31.58 108.68 ±32.43
Visceral abdominal fat, cm^2^ 11.05 ±2.74 10.14 ±2.62^[\*\*](#TF2-3){ref-type="table-fn"}^ 11.90 ±3.05 11.68 ±2.88 11.75 ±1.89 11.93 ±1.84 12.31 ±4.02 12.62 ±4.10
Total abdominal fat, cm^2^ 105.51 ±24.32 94.66 ±19.59^[\*\*](#TF2-3){ref-type="table-fn"}^ 115.62 ±24.78 110.89 ±20.95 111.20 ±17.98 111.30 ±18.03 120.20 ±35.59 121.30 ±36.34
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
All data are presented as means ±standard deviations; HCI -- high-intensity concurrent interval exercise, MCC -- moderate-intensity continuous concurrent exercise, CDA -- control daily active, COD -- control sedentary, BMI -- body mass index, WHR -- waist-to-hip ratio, QUICKI -- quantitative insulin sensitivity check index,
significant difference from pre-test to post-exercise (*p* \< 0.05),
significant difference from pre-test to post-exercise (*p* \< 0.001),
significant difference in COS or CDA or MCC compared to HCI at the same time (*p* \< 0.05)
Baseline changes in circulating irisin over the intervention period {#S3_2}
-------------------------------------------------------------------
Mean baseline irisin concentration was 1.33 ±0.36 µg/ml for HCI, 1.28 ±0.29 µg/ml for MCC, 1.35 ±0.42 µg/ml for COD, and 1.38 ±0.45 µg/ml for COS, with no differences between groups (*p* = 0.61), as shown in [Table 2](#T2){ref-type="table"}. Individual changes in plasma irisin concentration illustrate the analysis of changes in parameters for the HCI or MCC cohorts \[[@ref2]\]. Plasma irisin significantly increases by 2.20 ±0.42 µg/ml (*p*\< 0.001) following HCI training but remains unchanged after MCC training.
Quantitative insulin-sensitivity check index {#S3_3}
--------------------------------------------
The mean baseline insulin sensitivity QUICKI concentration did not differ significantly between different groups (*p* = 0.80). Two-way mixed ANOVA and Bonferroni follow-up test demonstrated a significant increase from pre- to post-exercise for HCI (4.71%, *p*\< 0.05) and MCC (3.83%, *p*\< 0.05), as shown in [Table 2](#T2){ref-type="table"}. There was no significant difference in the control groups' insulin sensitivity level (*p* \> 0.05).
Abdominal fat distribution (visceral vs. subcutaneous) and total abdominal fat {#S3_4}
------------------------------------------------------------------------------
Inter-group changes indicated that subcutaneous abdominal fat was significantly decreased in HCI and MCC groups (3.99%; *p* \< 0.001 and 1.18%; *p* = 0.011, respectively). There was no significant difference in the control groups' subcutaneous abdominal fat (*p* \> 0.05). The inter-group evaluation showed a significant reduction for visceral and total abdominal fat in the HCI group (8.27%, 10.27%; *p* \< 0.001, respectively), as shown in [Table 2](#T2){ref-type="table"}. It should also be noted that there was no significant difference between the MCC, COS and CDA groups in visceral and total abdominal fat.
Discussion {#S4}
==========
The results of the present study suggest that postmenopausal active women can improve most of the metabolic health-related parameters by concurrently undergoing HIIT and RT. Moreover, the extent of the improvements achieved seems to be affected by volume and/or intensity of the endurance training because different gains were observed for the RT + CT (MCC) and RT + HIIT (HCI) groups.
To our knowledge, this study is the first of its kind to make a comparison between the influences of HCI, MCC, along with two control groups in a 10-week training period, on serum irisin level, insulin sensitivity status and abdominal fat in postmenopausal women.
The new findings of the current study include promising effects that HCI promoted increased plasma levels of irisin, and decreased visceral, subcutaneous and total abdominal fat. Additionally, the increased QUICKI decreased with a positive change in body fat, visceral abdominal fat compared to the MCC group.
One must acknowledge that various concurrent training protocols have been examined in this study. Endurance and RT do not have to be executed on the same day and in the same session. The relative improvement in blood irisin observed in the HCL group could be partially due to the training sequence. However, not all studies report considerable changes\[[@ref12], [@ref26], [@ref27]\] or decline\[[@ref28], [@ref29]\] in baseline irisin concentrations, whereas studies on older subjects presented mixed results\[[@ref16], [@ref28]\]. Some studies suggest that the release of irisin as a myokine into the circulation during training may be an endocrine function of the body to control metabolism. Numerous studies have demonstrated that exercise drives irisin secretion in the skeletal muscle that leads to the expression of FNDC5 protein, thus elevating the irisin formation levels\[[@ref30]\]. Once in circulation, this myokine transforms white adipose tissue (WAT) into brite (brown in white)/beige fat cells by upregulating the genes responsible for changing with browning (such as uncoupling protein -- UCP-1, peroxisome proliferator-activated receptor γ -- PPARγ, and PR domain containing 16 -- PRDM16) \[[@ref31]\]. Once converted from white to brown, adipose tissue takes on the function of dispersing heat energy, while white adipose tissue is responsible for storing energy. This event, in turn, causes an increase in total body energy expenditure\[[@ref32]\].
Therefore, a rise in irisin and FNDC5 levels in response to HCL would be rational as it is largely characterized by heightened oxidative capacity and mitochondrial functions. Such influences of irisin are probably facilitated by pathways associated with phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) \[[@ref33]\]. An increase in energy expenditure and glucose homeostasis is a good reflection of irisin's role in metabolism \[[@ref6]\].
The transformed beige adipocytes are specific for heat generation and energy expenditure due to their rich mitochondrial UCP-1 content \[[@ref34]\]. Irisin upregulates lipolysis related genes, such as hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and fatty acid binding protein 4 (FABP4), ultimately increasing glycerol release and reducing lipid accumulation in adipocytes. Furthermore, the glucose uptake capacity of beige fat cells is also boosted as their glucose transporter type 4 (GLUT4) expression increases.
Taken together, our findings support the hypothesis that insulin sensitivity of peripheral tissues could be more closely linked to irisin values, specifically in striated muscles. This is a difference that can be detected even in basal conditions, independently of physical activity. Irisin, as a myokine secreted by striated muscles, has been known to shift glucose for energy expenditure in muscles to maintain glucose homeostasis. Moreover, insulin resistance associated with aging has also been linked to abdominal obesity \[[@ref35]\] and Rice *et al*. \[[@ref36]\] suggested that improved insulin sensitivity observed after resistance exercises may decrease visceral and abdominal subcutaneous adipose tissue or abdominal obesity. In the present study, HCI led to reductions in both abdominal fat distribution (visceral vs. subcutaneous) and total abdominal adipose tissue ([Table 2](#T2){ref-type="table"}). Thus, it can be said that equipped with such extraordinary properties, irisin perhaps is a strong likely candidate to be used against obesity and likewise other metabolic conditions.
A limitation of the present study which should be noted is the small sample size in control and experimental groups at the end of the experiment. Therefore, further studies with a large sample size are needed to clarify the relationship between irisin and insulin resistance in postmenopausal women.
One of the most significant steps in any future investigation is to determine whether irisin has a receptor for characterizing its mode of action. This would aid us in our understanding of irisin's functions as well as the cascading signals involved in its effect. Likewise, it is important to provide a clear explanation of the mechanism of how muscle tissue secretes irisin during exercise. Such information will be crucial in explaining what distinguishes the effects of acute and chronic training on irisin blood levels, and to discover whether there are other stimulants of irisin release.
Conclusions {#S5}
===========
According to the results, the HCI results in an increased irisin level and decreased visceral abdominal fat. Also, the insulin sensitivity status was significantly increased, and subcutaneous abdominal fat significantly decreased in the MCC group. In contrast, the MCC did not produce a significant effect on irisin level, and decreased visceral and total abdominal fat.
Finally, we hypothesized that active participants in the HIC group would experience a greater response to irisin circulation, in addition to visceral, subcutaneous, and total abdominal fat and QUICKI after 10 weeks, compared to the MCC group. This may be of great significance when it comes to designing an optimal training program for women experiencing menopause.
This article has been taken from the first researcher's Ph.D. dissertation at the University of Guilan. We are grateful to the faculties of physical education and sport science as well as the participants in this research.
Disclosure {#S6}
==========
The authors report no conflicts of interest.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
Brain injury and subsequent neurodevelopmental disabilities resulting from premature birth are a major public health concern. Indeed, preterm birth survivors can suffer from long term clinical, educational and social problems: 10--15% of infants surviving from very preterm delivery develop cerebral palsy and 50% show adverse neurodevelopmental outcome at 30 months of age. Due to dramatic improvement in perinatal management of high risk preterm neonates, pathological conditions associated with neurological impairment have changed over the past 10 years. Major focal destructive lesions remain serious but have become less common. In contrast, the most prominent neuropathological lesion is diffuse white matter damage (WMD) showing an association of glial injury together with microglial activation and, ultimately, myelination defects.
Many factors are associated with WMD, including: infection, hypoxia, ischemia, endocrine imbalances, genetic factors and growth restriction [@pone.0007128-Haynes1]--[@pone.0007128-Fontaine1]. In particular, intra uterine growth retardation (IUGR) has been shown to be associated with both white matter lesions and subsequent neurological impairment [@pone.0007128-PadillaGomes1]--[@pone.0007128-Jacobsson1] Based on these potential targets, a number of treatments for neonatal brain injury including melatonin have been investigated [@pone.0007128-Husson1].
The pleiotropic effects of melatonin, combined with its anti-oxidant, NMDA-blocking and anti-inflammatory properties, probably make this molecule an ideal candidate for pre-clinical studies. Moreover, this pharmacological agent has been proven to be safe for neonates when crossing the blood-brain barrier. Several experimental data have shown potent neuroprotective effects of melatonin both in *in vivo* and *in vitro*. In particular, melatonin acts as a direct and indirect antioxidant, specifically as a powerful scavenger of superoxide anion and stimulator of the synthesis of anti-oxidant enzymes. However, the consequences of melatonin treatment on the myelination process remain largely underexplored. Nonetheless, this question is crucial to thoroughly assess the potential impact of melatonin on myelination repair in perinatal and adult demyelinating diseases.
We addressed this question by testing melatonin treatment in a recently described rat model closely mimicking WMD observed in very preterm neonates [@pone.0007128-Olivier1]. Our data show that melatonin has a powerful protective effect on developing white matter subjected to an antenatal hypoxic-ischemic insult. Melatonin acts through its specific receptors by promoting oligodendroglial maturation and myelination repair together with modulation of astrogliosis and microglial activation *in vivo* and *in vitro*. Our results suggest that melatonin could be an effective neuroprotective candidate not only in perinatal white matter damage but also in inflammatory and demyelinating white matter diseases observed in adult.
Materials and Methods {#s2}
=====================
Uterine artery ligation {#s2a}
-----------------------
Unilateral ligation of the intra-uterine artery was performed on pregnant Sprague-Dawley rat (Janvier, Le Genest-St-Isle, France) during embryonic day 17 (E17) [@pone.0007128-Olivier1]. The mean weight of control pups at birth in our animal facilities was 6.24±0.37 g (n = 56). In each litter, the pups were separated into two groups according to birth weight: control pups and severely growth restricted (GR) pups weighting less than 5.50 g (6.34 g--2 S.D.). Only the most severely GR pups were considered as born from the ligated horn to avoid any bias between the two groups. All experiments were carried out in compliance with ethical standards of our institution (French National Institute for Health and Medical Research, INSERM) and with the recommendations of the National Research Council\'s Guide for the Care and Use of Laboratory Animals.
Experimental groups {#s2b}
-------------------
Pups from at least three different litters were used in each experimental group. From birth (P0) to postnatal day 3 (P3), controls and GR pups received a daily i.p. injection with one of the following drugs (or combination of drugs) diluted in a final volume of 5 µl: 0.002 mg/kg/d to 20 mg/kg/d melatonin (Sigma), 5.0 mg/kg luzindole (Sigma) +/− 20 mg/kg/d melatonin. Luzindole blocks melatonin receptors MT~1~ and MT~2~ with comparable affinities (affinity selectivity ratio MT~1~/MT~2~ = 15.5). To ensure that there were no differences in circadian rhythm between experimental groups, all rat pups were injected once a day at noon.
Immunocytochemistry {#s2c}
-------------------
In each experimental group, we studied 6 to 10 pups on P3 and P14 in three separate experiments. Animals were sacrificed under anaesthesia with inhaled isoflurance (Abbott France, Rungis, France). Brain tissues were immersed in formol 4% during 5 days, embedded in paraffin and cut into 10-µm-thick sections.
Immunolabeling with the primary antibody listed in [Table 1](#pone-0007128-t001){ref-type="table"} was visualized using the streptavidin-biotin-peroxydase method, as previously described [@pone.0007128-Olivier1]. Olig2 marker was used to visualize all oligodendrocytes, APC and MBP to detect post-mitotic oligodendrocytes and myelinated fibers, respectively. Most of Olig2 nuclei did not colocalized with GFAP+ cells ([Figure S1](#pone.0007128.s001){ref-type="supplementary-material"}). Tomato-lectin-, Olig2- and Ki67-immunoreactive cells were revealed using streptatividin, anti-rabbit IgG antibody and anti-mouse IgG antibody coupled to the red fluorescent marker cyanine 3 (Jackson Immunoresearch laboratories, West Grove, PA). Double labelings were performed with secondary antibodies coupled to the green fluorescent marker Fluoroprobe S488 (Interchim, Montluçon, France) or red fluorescent marker cyanine 3 (Jackson Immunoresearch laboratories, West Grove, PA) fluorescent markers.
10.1371/journal.pone.0007128.t001
###### Primary antibodies used for immunohistochemistry analyses.
{#pone-0007128-t001-1}
Markers Labeled structures Manufacturer Dilution
--------------- -------------------------------------------------- -------------------------------------------------- ----------
Tomato-lectin Resident, activated microglia and blood vessels. Vector, Burlingame, CA, USA 1/500
GFAP Astrocytes Sigma Biosciences, St Louis, MO, USA 1/1000
Ki67 Proliferating cells BD Biosciences France, Le-Pont-De-Claix, France. 1/100
Olig2 oligodendroglial lineage IBL, Hamburg, Germany 1/200
APC post-mitotic oligodendrocytes Calbiochem, La Jolla, CA, USA 1/500
MBP Myelinated fibers Sigma Biosciences, St Louis, MOUSA 1/1000
MT1 (Mel-1A) Melatonin receptor 1 Santa Cruz, Santa Cruz, CA, USA 1/100
MT2 (Mel-1B) Melatonin receptor 2 Santa Cruz, Santa Cruz, CA, USA 1/100
Quantitative measurements {#s2d}
-------------------------
All quantitative measurements were done by observers who were blinded to the experimental group of the studied animal.
### Immunoreactive cells {#s2d1}
Immunoreactive cells were counted in the white matter underlying the cingular cortex (+2.16 to −0.36 mm from the bregma) on at least 4 non continuous sections (alternatively, in right or left hemisphere) at P3 and P14. Immunoreactive cells were counted within a 0.065 mm^2^ grid (at ×400 magnification), in at least 6 animals per group.
### Optical density of MBP-positive fibers {#s2d2}
The optical density of MBP-immunoreactive fibers was measured in the thickest white matter in rodents, ie cingular white matter assessed in coronal sections (+2.16 to −0.36 mm from the bregma). At least 4 sections of 6 to 10 animals per group were examined on P14. Optical density was measured at ×100 magnification using a computerized image analysis system (ImageJ, NIH, MA, <http://rsb.info.nih.gov/ij/>) that read optical density as grey levels. Nonspecific background densities were measured at each brain level in a region devoid of MBP immunostaining and were substracted from the cingulum values.
TUNEL staining {#s2e}
--------------
In P3 animals, dying cells in the white matter were detected using TUNEL as previously described [@pone.0007128-Fontaine1] and counted in 4 sections each from a single hemisphere of at least 6 animals per treatment group.
Ultrastructural morphology of cingulate white matter by electron microscopy {#s2f}
---------------------------------------------------------------------------
The morphology of melatonin-treated cingulate white matter was studied by electron microscopy. First, brain were sagitally cut in 100 µm-thick vibratome sections to allow for better penetration of the reagent and postfixed for 10 min at room temperature with 1% osmium tetroxide (Heraeus Chemicals, Port Elisabeth, South Africa) in phosphate buffer. After a 10 min wash in a phosphate buffer, sections were dehydrated in graded alcohol baths and successively incubated in alcohol/araldite (v/v) for 1 h at 37°C, araldite (Fluka, Steinheim, Switzerland) for 15 h at 37°C, and araldite supplemented with 2% accelerator (Fluka) for 3 h at 37°C. Sections were then embedded in araldite (2 days at 60 °C). Silver ultrathin sections of the cingulate white matter (about 70 nm-thick) were made using an ultramicrotome (LKB, Bromma, Sweden) and then collected on copper grids. They were counterstained with lead citrate for 10 min at room temperature in a drying chamber and examined using a Leo 912 (Carl Zeiss, Le pecq, France) electron microscope. In order to study the qualitative aspect of the myelin, the number of axons surrounded with an uncompacted myelin sheet was reported as the total number of myelinated axons counted of 20 electromicrographes at magnification ×4000. A qualitative score was applied to each myelinated axon (see [table 2](#pone-0007128-t002){ref-type="table"}).
10.1371/journal.pone.0007128.t002
###### Quantitative analysis of the optical density of myelin sheets in the cingulate white matter of controls and GR pups with or without melatonin 20 mg/kg.
{#pone-0007128-t002-2}
Group Myelin sheet O.D.
------------------- -------------------
Controls 95.2±0.4
GR pups 58.7±0.7\*
GR pups+melatonin 82.5±0.8
Data are expressed mean ± S.E.M. Asterisk represent significant difference with controls (\* p\<0.05 with one way ANOVA and Newman-Keuls multiple comparision test).
Cell cultures and cell treatments {#s2g}
---------------------------------
Primary neuronal cultures (\>98% purity assessed by MAP-2 (microtubule-associated protein-2) immunostaining, data not shown) were prepared using E18 embryonic rats. For oligodendrocyte, astrocyte and microglia primary cultures, cell populations were isolated from P0--P2 newborn rats and subsequently cultured according to published protocols from 6 days to 3 weeks [@pone.0007128-McCarthy1]--[@pone.0007128-Mesples1]. Purity of oligodendroglial cell cultures was assessed using O4, GFAP and tomato-lectin staining: at DIV10, 65--70% of the cells were found O4+, 25--30% GFAP+ and less than 5% Tomato-lectin+ in three separate experiments.
DNA-free total mRNA from cell cultures and cortical plate from rat aged from 1, 7 and 14 day-old rats were extracted from these cells using the previously published protocol [@pone.0007128-Chomczynski1].
Oligodendoglial cells were exposed to melatonin at 1 and 100 µmol diluted in DMSO 20% after 6, 8 and 10 days of culture. As our in vivo model is characterized by early microglial activation within developing white matter we further tested the ability of melatonin to modulate microglial activation *in vitro*. We used a well-established model of microglia subjected to LPS leading to both morphological and immunocytochemical changes and a dramatic increase in cytokines and free radicals production. In microglial cell cultures, the melatonin treatment was performed after 5 days of culture, 12 h before and at the same time as LPS exposure (100 ng/ml LPS) (Sigma, St Louis, MO).
Immunocytochemistry and immunofluorescence microscopy on cell cultures {#s2h}
----------------------------------------------------------------------
After treatments, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS, and blocked with PBST (PBS 1x, pH 7.4, 0.1% Triton X-100) containing 5% goat serum for 1 hr at room temperature. The coverslips were incubated with mouse monoclonal antibodies O~4~ (1∶100 dilution, gift from Dr. Steven E. Pfeiffer, University of Connecticut Health Center, Farmington, CT), tomato lectin (1∶1000, Vector, Burlingame) and MBP (1∶1000, Chemicon, Temicula, CA) overnight at 4°C. The appropriate secondary antibody was conjugated with either Alexia 488 or Alexia 594 (Molecular Probes Inc., Eugene, OR), added to the coverslips and incubated for 1 hr. Nuclei were stained by adding Hoechst 33258 at a final concentration of 2 µg/ml for 1 min. The coverslips were mounted onto glass slides with FluoroMount and kept dark at 4°C. Cell images were captured with a fluorescence microscope equipped with a digital camera (Leica DMRB with Apogee Instruments Inc.). Quantification of labelled cells were based either on the density of immunoreactive cells compared to the total number of nuclei (O4 and MBP, ×400 magnification) or the optical density of tomato-lectin immunoreactivity using ImageJ analysis system at ×200 magnification.
Quantitative real-time PCR {#s2i}
--------------------------
The DNA-free total RNA from control and GR brain cortex including white matter was obtained using a protocol adapted from Chomczynski and Sacchi^12^. The following oligonucleotides 5′-ATTGTCAAGTTAGTGCCTTCC-3′ and 5′-TTGAGACTGTGGCAAATGTAG-3′, 5′-GTCATTGGCTCTGTCTTCAAC-3′ and 5′-GTAGGTCGCACTGTGACAGAT-3′ were used as sense and antisense primers, for MT1 and MT2. The nature of the amplified DNA was confirmed by sequencing. To standardize gene expression across samples, we first compared the expression levels of four well-known housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase \[GAPDH\], β2-microglobulin, hypoxanthine-guanine phosphoribosyltransferase \[HPRT\], and β-glucoronidase) within the samples. For reverse transcription, we used 600 ng of total RNA and the Iscript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR was set up using sybergreen-containing supermix (Bio-Rad) for 50 cycles with a three-step program (25 sec denaturation at 96°C, 30 sec annealing at 60°C, and 30 sec extension at 72°C). Amplification specificity was assessed by melting curve analyses. Each experiment was run twice with a least 6 animals per group, and in both cases, samples were assessed in triplicate.
Statistical analysis {#s2j}
--------------------
All data were reported as means ± S.E.M. Analysis of variance was performed with age and groups (PBS- and melatonin-treated controls and GR pups) as the factors, and the Newman-Keuls post-test was used. Statistical tests were run on GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA).
Results {#s3}
=======
Melatonin attenuates myelination defect in growth restricted pups {#s3a}
-----------------------------------------------------------------
Systemic treatment using 20 mg/kg/d melatonin given within the first 3 postnatal days was associated with a significant 58% attenuation of myelination defects detected in the cingulate white matter in P14 GR pups (p\<0.001) ([Figures 1A--E](#pone-0007128-g001){ref-type="fig"}). Similar results were observed in the genu of the corpus callosum ([Figure 2A--E](#pone-0007128-g002){ref-type="fig"}). This effect was observed in most of GR pups, however, the most severely growth restricted (below 4.5 g, corresponding to birth weight below 5 SD) only showed a minor benefit from the melatonin treatment ([Figure 1F](#pone-0007128-g001){ref-type="fig"}). For birth weights greater than 4.5 g, the melatonin treatment was associated with a remarkably stabilized MBP density regardless the birth weight, in contrast to untreated animals in which myelin content appeared closely correlated with birth weight ([Figure 1F](#pone-0007128-g001){ref-type="fig"}). Administration of melatonin induced a dose-dependant effect on the density of MBP-positive fibers in the cingulate white matter in GR pups ([Figure 3A](#pone-0007128-g003){ref-type="fig"}). A statistically significant effect of melatonin was observed with a minimal administration of 0.2 mg/kg/d melatonin. No effect of melatonin on both body temperature and weight gain during the first 7 days has been reported (data not shown). Similarly, brain weight gain was not found different during the first 14 days with or without melatonin treatment ([table S1](#pone.0007128.s006){ref-type="supplementary-material"}). No statistically significant difference in oligodendroglial cell death (TUNEL staining) was observed between treated and control rat pups at P3 ([Figure S2](#pone.0007128.s002){ref-type="supplementary-material"}). Finally, melatonin treatment during the first days of life (P0--P3) was not associated with any alteration in myelin content in the normal developing brain (both in cingulum and corpus callosum, [Figure S3](#pone.0007128.s003){ref-type="supplementary-material"}).
{#pone-0007128-g001}
{#pone-0007128-g002}
{#pone-0007128-g003}
In melatonin-exposed GR rats, the blockage of melatonin receptors, using luzindole co-treatment, eliminated the neuroprotective effect of melatonin on white matter myelination ([Figure 3B](#pone-0007128-g003){ref-type="fig"}). In the control rat pups, melatonin alone, luzindole alone or co-treatment did not modify MBP-immunoreactivity cingulate white matter. Luzindole alone did not potentiate the myelin defect observed in GR pups (data not shown).
Melatonin improves the ultrastructural myelin aspect in GR pups {#s3b}
---------------------------------------------------------------
Further experiments using electronic microscopy demonstrated ultrastructural quantitative and qualitative effects of melatonin on myelinated axons of rat pups that are subjected to antenatal uterine ligation. In PBS- or melatonin-treated control P14 pups, the developing white matter exhibited a high density of cells and myelinated ovoid axons with well-compacted myelin sheets ([Figures 4A, 4D](#pone-0007128-g004){ref-type="fig"}). In contrast, PBS-treated GR pups showed dramatic cingulate white matter damage including a disorganized structure of the developing white matter with undermyelinated and swollen axons ([Figure 4B](#pone-0007128-g004){ref-type="fig"}). In most of cases, myelinated axons were surrounded with uncompacted myelin sheets ([table 2](#pone-0007128-t002){ref-type="table"}). Melatonin treatment of GR pups was associated with a quantitative and qualitative improvement of the myelin ultrastructural aspect. Indeed, myelin features in treated animals were characterized by a good compaction of the myelin sheet around axons ([table 3](#pone-0007128-t003){ref-type="table"}; [Figures 4C, 4F](#pone-0007128-g004){ref-type="fig"}). Thirty two percent of myelinated axons observed in untreated GR animals were found embedded with compacted myelin sheets compared to 79% in controls and 69% in treated GR animals. Conversely, melatonin treatment was unable to restore cellular and axonal density suggesting a specific effect on myelination process ([Figures 4E, 4F](#pone-0007128-g004){ref-type="fig"}). Similarly, melatonin treatment was not able to change the immunocytochemical aspect of RT-97-positive axons in both control and GR pups (data not shown).
{ref-type="table"}.](pone.0007128.g004){#pone-0007128-g004}
10.1371/journal.pone.0007128.t003
###### Ultrastructural analysis of myelinated fibers in controls and GR pups with electron microscopy.
{#pone-0007128-t003-3}
Group Qualitative myelin aspect score \% of myelinated axons embedded with compacted myelin sheets
------------------- --------------------------------- --------------------------------------------------------------
Controls 2.6±0.1 79.1(±7.5)%
GR pups 1.1±0.5\* 32.2(±7.8)%\*
GR pups+melatonin 2.6±0.1 68.6(±12.2)%
Qualitative myelin aspect score:
0: no myelinated axons found;
1: very thin myelin sheet;
2: uncompacted myelin sheets;
3: normally compacted myelin sheets.
Data are expressed mean ± S.E.M. Asterisk represent significant difference with controls (\* p\<0.05 with one way ANOVA and Newman-Keuls multiple comparision test).
Melatonin treatment promotes oligodendroglial maturation in GR pups {#s3c}
-------------------------------------------------------------------
To further delineate the cellular target of melatonin treatment, we asked the question whether melatonin could impact the oligodendroglial lineage. To answer this question, the effect of melatonin treatment on oligodendrocyte density was assessed using various immunocytochemical markers (see [methods](#s2){ref-type="sec"}). The cingulate white matter of GR pups appeared to be depleted in total (Olig2+) [and]{.ul} mature (APC+) oligodendrocytes at P14 ([Figures 5A, 5B](#pone-0007128-g005){ref-type="fig"}) as well as P3 (data not shown). Melatonin treatment was not able to restore the Olig2 positive-oligodendrocyte density in the cingulate white matter as well as the adjacent corpus callosum in GR pups ([Figure 5A](#pone-0007128-g005){ref-type="fig"}). In contrast, melatonin-treatment significantly (p\<0.05) reduced the decrease of APC+ oligodendrocytes density observed in cingulate white matter of GR rat pups at P14 ([Figures 5B--F](#pone-0007128-g005){ref-type="fig"}). Moreover, using Olig2/APC double-labelling immunocytochemistry, 63% of Olig2+ oligodendrocytes were found APC+ in untreated GR pups compared to more than 92% in melatonin treated P14 GR pups (p\<0.05). Conversely, melatonin was not found to be associated with any change in cell proliferation in the developing white matter as assessed using Ki67 immunolabeling at P3 and P14 ([Figure S4](#pone.0007128.s004){ref-type="supplementary-material"}). Altogether, these data strongly suggest that melatonin treatment promotes oligodendroglial maturation in GR rat pups.
{#pone-0007128-g005}
Melatonin treatment is associated with a decrease of glial activation in GR pups {#s3d}
--------------------------------------------------------------------------------
Growth restriction induced by antenatal hypoxia was associated with a protracted increase of microglial activation in the cingulate white matter [@pone.0007128-Husson1]. Thus, we investigated the consequences of melatonin treatment on microglial activation in antenatal hypoxia-exposed neonatal rats. In cingulum white matter, melatonin-treated GR pups exhibited a significantly decreased density of activated microglial cells compared to PBS-treated GR rat at P3 and P14 ([Figure 6A](#pone-0007128-g006){ref-type="fig"}). During the same developmental stages, melatonin treatment did not modify microglial activity in the cingulate white matter of controls. An increased density of GFAP-positive astrocytes in the cingulate white matter was observed in P14 GR rats compared to controls. Furthermore, reactive astrocytes from GR pups had a protoplasmic profile, with a bigger cell body and more numerous processes compared to control pups. At similar developmental stages, melatonin treatment was associated with a significantly decrease in reactive astrocyte density in GR pups compared to controls (p\<0.01) ([Figure 6B](#pone-0007128-g006){ref-type="fig"}). Melatonin-treated controls did not exhibit any modification of cingulate white matter astroglial profile and density.
{#pone-0007128-g006}
Melatonin receptors are differentially expressed in the white matter glial cells {#s3e}
--------------------------------------------------------------------------------
Because the effect of melatonin is recognized to be mediated through specific G-protein coupled receptors MT1 and MT2 (antagonized by luzindole), we next asked the question whether these two receptors were expressed in the various cell types populating the developing white matter at the time melatonin treatment was given. We tested this using RT-PCR *in vitro* in neurons, microglia, astrocytes and immature oligodendrocytes, all cell types populating the developing white matter. MT1 and MT2 appeared to be strongly expressed in both astrocytes and microglial cells, and to a lesser extent in neurons and immature oligodendrocytes ([Figure 7A](#pone-0007128-g007){ref-type="fig"}). Immunocytofluorescent stainings using MT1 (Mel-1A) and MT2 (Mel-1B) antibodies revealed similar expression pattern ([Figure 7B](#pone-0007128-g007){ref-type="fig"}).
{#pone-0007128-g007}
Melatonin induces oligodendroglial maturation and modulates microglial activation *in vitro* {#s3f}
--------------------------------------------------------------------------------------------
Developing oligodendrocytes and microglia were the two major cell types involved in the main features of white matter damage in our rat model. Since we found melatonin receptors to be expressed in both cell types, we further investigated the impact of melatonin exposure *in vitro*. In the oligodendroglial lineage model we observed that 1 µmol melatonin exposure was associated with an enhancement of oligodendroglial maturation to myelinating oligodendrocytes ([Figures 8A, 8B](#pone-0007128-g008){ref-type="fig"}). Indeed, MBP positive cell density increased more than two-fold at DIV 10 after melatonin treatment compared to DMSO or untreated cells (p\<0.01). In addition, APC and PLP immunostainings were also found increased after melatonin treatment in oligodendrocyte cell cultures ([Figure S5](#pone.0007128.s005){ref-type="supplementary-material"}). Similar results were obtained using 100 µmol melatonin (data not shown).
{#pone-0007128-g008}
Microglial activation was considered as a key feature in our *in vivo* model, likely to be responsible for subsequent myelination deficit. To mimic this feature *in vitro*, we used LPS to activate microglial cells. Because melatonin treatment was associated with a dramatic effect on microglial activation *in vivo*, we tested the ability of melatonin to modulate the effect of LPS-induced microglial activation *in vitro*. Microglial cells were pre-treated with either 1 or 100 µmol melatonin 12 h hours before and then together with LPS exposure. LPS-treated microglial cells exhibited an amyboid shape and a significant increase in tomatolecin immunoreactivity density compared to non-activated cells. In contrast, melatonin exposure completely reversed the LPS effect and prevented activation of microglial cells ([Figures 8C, 8D](#pone-0007128-g008){ref-type="fig"}). A similar effect was observed using two different cellular densities of microglial cells (data not shown).
Discussion {#s4}
==========
In this study we investigated the underlining cellular mechanisms responsible for the neuroprotective effects of melatonin treatment in antenatal hypoxia-induced white matter injury in the developing rat brain. We have demonstrated that melatonin acts in a neuroprotective fashion by promoting oligodendroglial maturation together with decreased microglial activation, both *in vivo* and *in vitro*. We recently reported a rat model inducing IUGR and white matter damage after prenatal hypoxia and deprivation of nutritional and maternal factors [@pone.0007128-Ishimura1]. As previously described, GR pups exhibited white matter damage characterized by increased microglial activation, increased astrogliosis, leading to a protracted oligodendrocytes depletion associated with myelination defects [@pone.0007128-Husson1], [@pone.0007128-Olivier2]. In this study, electron microscopy analysis of the injured white matter refined the abnormal aspect of the developing white matter. The few myelinated axons were mostly embedded with a poorly compacted myelin. MBP proteins have been recognized to have a crucial role in myelin compaction by intercalating between phospholipidic sheets and interacting with lipids and proteolipids [@pone.0007128-Nave1], [@pone.0007128-Riccio1]. Similarly, Shiverer mice, a natural mutant for MBP gene, exhibit hypomyelination and produce poorly compacted myelin around axons [@pone.0007128-Rosenbluth1], [@pone.0007128-Readhead1].
In GR animals, the white matter also appeared with large areas of swollen axons, suggesting protracted oedematous damage. Despite no detectable change in axonal neurofilament distribution, ultrastructural analysis of neonatal white matter clearly demonstrates that growth restriction observed in our model is associated with oligopathy and myelinopathy.
Several experimental studies have highlighted that melatonin has neuroprotective benefits when given as either a prophylactic or curative treatment in several animal models of brain damage. Most of such studies have analyzed the effect of melatonin in traumatic or brain focal ischemia in adult rodent [@pone.0007128-Sun1]--[@pone.0007128-Kilic1]. In such models, melatonin was found to decrease the volume of brain damage, neuronal cell death, oxidative stress, DNA damage, mitochondrial insult and to improve neurobehavioural outcome. Furthermore melatonin exerts its neuroprotective benefits against excitotoxic insult both *in vitro* [@pone.0007128-Floreani1], [@pone.0007128-Pei1], *ex vivo* in hippocampal neurons in adult rats [@pone.0007128-Chung1] or *in vivo* [@pone.0007128-PadillaGomes1]. Melatonin has also been studied in a large animal model of preterm brain injury in fetal sheep subjected to umbilical cord occlusion [@pone.0007128-Welin1]. This study demonstrated that melatonin attenuates cell death in the fetal brain in association with a reduced inflammatory response in the blood and the brain following intrauterine asphyxia in mid-gestation fetal sheep.
In mature melatonin-deficient rats, lesions induced by hypoxia-ischemia were observed to be larger than in controls [@pone.0007128-Manev1] suggesting that endogenous melatonin is neuroprotective. However newborn animals and humans have low levels of melatonin and the melatonin receptor antagonist luzindole does not potentiate excitotoxic cerebral white matter lesions in rodents, suggesting that endogenous melatonin is not sufficient to protect the newborn. For exactly this reason, melatonin treatment could be relevant in a clinical setting in preterm human neonates.
Melatonin has been shown to have no effect in preventing the initial appearance of lesions in a mouse model of excitotoxic-induced white matter damage, but is able to promote axonal regrowth or sprouting [@pone.0007128-PadillaGomes1]. However, the effect of melatonin on myelination and subsequent white matter repair has not yet been studied. Previous studies have shown that white matter damage observed in our model was characterized by a depletion in post-mitotic oligodendrocytes (APC+ cells) rather than pre-oligodendrocytes (O4+ cells) [@pone.0007128-Olivier2]. In the present study, the olig2 immunolabeling suggest that the entire oligodendroglial lineage is defective in GR animals\' white matter. It is relevant to point out that melatonin-induced preservation of myelination is associated with partial recovering of mature APC+ oligodendrocytes (despite a deficit in total oligodendroglial population). Consequently, we speculate that the main target of melatonin treatment could be the maturation process of the oligodendrocyte lineage. This hypothesis is supported by several lines of evidence: melatonin treatment increased oligodendrocyte maturation and MBP-immunoreactivity normalization *in vivo*. Melatonin treatment was associated with a restoration of myelin sheet compaction surrounding axons in GR pups white matter. This result is consistent with MBP-immunoreactivity normalization as MBP is crucial for myelin sheet compaction [@pone.0007128-Nave1], [@pone.0007128-Riccio1]. Finally, melatonin was able to improve oligodendrocytes maturation without any effect on cell proliferation *in vitro* or *in vivo*.
The mechanism(s) behind the neuroprotective benefits of melatonin are not yet fully elucidated. To date, it has been shown that the effect of melatonin include endocrine, autocrine and paracrine actions, decreased inflammation, and antioxidant effects. Melatonin has also some benefits in other "toxic models" of perinatal brain damage based on excitotoxic cascade [@pone.0007128-PadillaGomes1]. However, in most of perinatal brain damage animal models reported, immune cell activation frequently occurs [@pone.0007128-Haynes1] and melatonin effect appears to be closely linked to inflammation modulation. Some of these actions are receptor-mediated, while others are direct. We demonstrate here that the two major subtypes of melatonin-receptors MT1 and MT2 were expressed by microglia, astrocytes and oligodendrocytes, i.e. all cell types populating the immature white matter. Assuming that the cingulate myelination deficit was associated with focal cingulate microglial activation in our model and that immature oligodendrocytes are highly vulnerable in an inflammatory context [@pone.0007128-Tahraoui1], [@pone.0007128-Khwaja1], it is reasonable to hypothesize that melatonin could restore normal oligodendrocyte maturation by reducing white matter inflammation. In fact, in the present study, we found melatonin treatment to be associated with a reduction of microglial activation in GR pups at P3 *in vivo* and LPS-induced microglial activation *in vitro*. This result is in agreement with previous studies [@pone.0007128-Lee1], [@pone.0007128-Zhou1]. The signalling pathways involved in the maturation effect of melatonin on developing oligodendrocytes remain unclear. However, several effects of melatonin through its receptors may account for its ability to prevent oligodendroglial damage: free radical scavenger production by activated microglia [@pone.0007128-Rosenbluth1], [@pone.0007128-Matuszak1], [@pone.0007128-Tan1], improvement of membrane fluidity and reduction of edema and polymorphonuclear cell infiltration into damaged tissue, prevention of translocation of the nuclear factor-kappa-B to the nucleus and the subsequent reduction of pro-inflammatory cytokines expression, which play a relevant role in the inflammatory reaction [@pone.0007128-Mohan1], [@pone.0007128-Mayo1]. In addition, melatonin could modulate astrocyte reactivity or death through an upregulation of astrocytic anti-oxidative defenses [@pone.0007128-Borlongan1], [@pone.0007128-Martin1].
In conclusion, our results demonstrate that melatonin could preserve axonal myelination in adverse conditions. The neuroprotective pathway appears to implicate both melatonin receptors and inflammatory modulation, leading to a promotion of oligodendrocyte maturation. The present study also delineates the cellular mechanisms of melatonin neuroprotective benefits. Furthermore, our data strongly suggest that melatonin could be of great interest not only in perinatal white matter damage but also as a potential neuroprotective strategy for myelinopathy diseases observed in adults.
Supporting Information {#s5}
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Double immunolabeling using GFAP (astrocytes in green, arrows) and Olig2 (oligodendrocytes in red, arrowheads) markers in cingulate white matter. Most of Olig2 nuclei did not colocalized with GFAP+ cells in the developing white matter.
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Click here for additional data file.
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Quantification of TUNEL+ cells detected in the hemispheric white matter at P3 from control (Ctl) and GR rat pups treated or not with Melatonin (Mel).
(2.36 MB TIF)
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Click here for additional data file.
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Quantitative analysis of the MBP-positive fibers optical density in the cingulate white matter of internal controls and sham control pups treated with either PBS or with melatonin 20 mg/kg.
(1.99 MB TIF)
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Click here for additional data file.
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Quantitative analysis of Ki67+ nuclei in the cingulate white matter according to the experimental groups at P3 and P14.
(1.83 MB TIF)
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Click here for additional data file.
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Immuno-labelling of primary oligodendroglial cell cultures using either APC or PLP at DIV6 and DIV10, respectively with or without treatment with 1 µmol melatonin.
(8.15 MB TIF)
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Click here for additional data file.
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Brain weight (mean +/− SD) from delivery to P14 of rat pups in the experimental groups.
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Click here for additional data file.
The authors wish to thank Marie Conrath, PhD and Marie-Jeanne Brisorgeuil (UMR 7101 CNRS -- Université Pierre et Marie Curie, Paris, France) for their excellent scientific and technical electron microscopy support.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This study has been supported by INSERM, INSERM AVENIR Program and Mairie de Paris fundings. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Paul Olivier and Gauthier Loron were financially supported by Ikaria Inc.
[^1]: Conceived and designed the experiments: PO RHF GL CCM CV PG OB. Performed the experiments: PO RHF GL JVS VB VM AMK JD MSA JP OB. Analyzed the data: PO RHF GL JVS VB VM JD MSA JP PG OB. Contributed reagents/materials/analysis tools: PO RHF VM OB. Wrote the paper: PO AMK CCM CV PG OB.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1-viruses-11-01084}
===============
The family of *Retroviridae* is divided into two subfamilies: The *Spumaretrovirinae* consist of five genera of different spuma or foamy viruses with shared and unique features that separate them from the canonical *Orthoretrovirinae*, which comprise all other known exogenous retroviruses ([Figure 1](#viruses-11-01084-f001){ref-type="fig"}) \[[@B1-viruses-11-01084]\]. The number of research groups working on FVs is correspondingly small and even further split by their individual research focus or the FV isolate or host species used in their studies but also due to the sheer difference in numbers and an apparent lack of pathogenicity of foamy viruses (FV).
Most molecular analyses have been conducted on the so-called prototype/primate FV isolate, also initially designated human FV, but subsequently shown to be the end-product of the zoonotic transmission of a chimpanzee FV to an East African naso-pharynx cancer patient \[[@B3-viruses-11-01084]\]. Upon subsequent propagation and passaging in diverse human and non-human cell lines and concomitant severe genetic changes \[[@B4-viruses-11-01084]\], this virus became the best-studied FV isolate and it gained the name prototype FV (PFV). However, its prototypic character might be questioned, since research on highly related simian FVs or more distantly related FVs of feline, bovine, and equine origin is lagging behind and has revealed---at least in selected cases---more or less different data ([Figure 1](#viruses-11-01084-f001){ref-type="fig"} and [Table 1](#viruses-11-01084-t001){ref-type="table"}) \[[@B5-viruses-11-01084]\]. Simian FVs share substantial relatedness and, despite having a long co-evolutionary history with their cognate hosts, inter-species transmission is frequent and well documented among closely related hosts, like Old World monkeys and apes, including humans, but also between New World monkeys and humans \[[@B5-viruses-11-01084],[@B6-viruses-11-01084],[@B7-viruses-11-01084],[@B8-viruses-11-01084]\]. In general---and there are only very few exceptions known---FVs co-speciate with their cognate hosts and more or less closely related species may be susceptible to the same or a highly related FV \[[@B5-viruses-11-01084],[@B9-viruses-11-01084],[@B10-viruses-11-01084],[@B11-viruses-11-01084]\]. This host range is likely due to the co-evolution of the virus, together with its host with FVs being the most ancient retrovirus according to the presence of endogenous viruses in all of the vertebrate groups \[[@B10-viruses-11-01084],[@B12-viruses-11-01084]\].
Although a so-called prototype (and/or primate) FV exists in the literature, conserved, prototypic features, besides those basic characteristics that led to the establishment of an independent subfamily of FVs, are currently only partially known. Here, an unbiased comparison of distant FVs and their replication strategies might be worth trying to discriminate basic from deduced, secondary features. In addition, unique data not available for the other FVs have been generated for bovine FV (BFV, see [Table 1](#viruses-11-01084-t001){ref-type="table"}), and there is the question whether they represent shared or unique features \[[@B5-viruses-11-01084]\]. In this review, we try to use the current data on diverse aspects of the molecular biology of BFV to broaden and complete the overall knowledge of FV biology and indicate avenues of further investigation on BFV biology in vivo and in vitro. In [Table 1](#viruses-11-01084-t001){ref-type="table"}, the biggest achievements and strengths in the BFV system are summarized and this review will cover some of them in more depth.
2. Specific Topics and Highlights in BFV Biology and Virus-Host Interaction {#sec2-viruses-11-01084}
===========================================================================
2.1. Historic View {#sec2dot1-viruses-11-01084}
------------------
While the first FV was already described in 1954 \[[@B29-viruses-11-01084]\], the first FV from cattle was isolated 15 years later and designated Bovine Syncytial Virus \[[@B30-viruses-11-01084]\]. The subsequent isolates were also designated Bovine Spuma Virus and Bovine Spumaretrovirus before the name bovine foamy virus (BFV) was coined and finally acknowledged by the ICTV in 1999 (<https://talk.ictvonline.org//taxonomy/p/taxonomy-history?taxnode_id=20074661>) \[[@B1-viruses-11-01084]\]. Holzschu et al. \[[@B19-viruses-11-01084]\] published the first full-length nucleotide sequence of a BFV isolate from the United States (US) in 1998. These data confirmed the overall genetic structure and coding capacity of BFV as a typical member of the FV genus ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A).
This opened the way for functional and genetic studies on the molecular biology and replication of BFV in cell cultures and experimentally BFV-infected animals. In addition, it allowed for the establishment of tools for high sensitivity and specificity detection and diagnosis, as described in the subsequent chapters and undertaken in the labs of Jacek Kuzmak and Magdalena Materniak-Kornas, Wentao Qiao, Yunqi Geng and Juan Tan, and Martin Löchelt and co-workers ([Figure 3](#viruses-11-01084-f003){ref-type="fig"}).
Almost unrecognized since exclusively publishing in German, the BFV Riems isolate was established and characterized by Dr. Roland Riebe and co-workers in East Germany (Friedrich Löffler-Institute, Riems, Germany) in the early 80s of the last century \[[@B17-viruses-11-01084],[@B18-viruses-11-01084]\]. The original BFV Riems isolate is, to our knowledge, the only FV that has been exclusively propagated in primary cells of its authentic host species and it thus might have not so much "suffered" genetic changes and co-adaptive imprints due to (repeated) host cell changes and prolonged growth in tumor cells displaying highly selected and aberrant features.
2.2. Excellent, Well Established Non-Primate FV Model of Transactivation, Gene Expression and Gene Function {#sec2dot2-viruses-11-01084}
-----------------------------------------------------------------------------------------------------------
Gene expression and transactivation studies have been mainly conducted in the earlier years of PFV and SFV research, in particular between 1990 and 2000. Research regarding the underlying molecular mechanisms of BFV gene expression has only started in 2008 and it is still ongoing in the lab of Wentao Qiao and Juan Tan while using current, state of the art methods and technologies, thus also extending from this perspective our understanding of FV gene expression and replication as reported here by J.T. ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A). Similarly, BFV Bet and Gag have been additionally studied by this group during the last years and are thus included in this review, allowing for a more comprehensive view on structural and non-structural FV proteins ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A).
### 2.2.1. Function of Tas {#sec2dot2dot1-viruses-11-01084}
Unlike PFV Tas, BFV Tas has no classical nuclear localization signal (NLS), but it is mainly present in the nucleus beside some cytoplasmic localization \[[@B31-viruses-11-01084],[@B32-viruses-11-01084],[@B33-viruses-11-01084]\]. Like most typical DNA-binding transcriptional activators, nuclear localization and multimerization are both required for the transactivation activity of Tas \[[@B31-viruses-11-01084],[@B32-viruses-11-01084]\]. It was reported that PFV Tas has three domains that mediate multimer formation in the nuclei of mammalian cells, but the biological function of PFV Tas multimerization has not been defined \[[@B32-viruses-11-01084]\]. In contrast to PFV Tas, BFV Tas has only one domain that mediates dimer formation. The comparison of the multimerization domains of both proteins does not reveal obvious homologies. Deleting the dimerization region abolishes the Tas-induced transactivation of BFV LTR and internal promoter (IP), which suggests that the active form of BFV Tas is a dimer \[[@B31-viruses-11-01084]\].
There are at least four BFV *tas* mRNAs during BFV infection \[[@B34-viruses-11-01084]\]. These four forms of BFV *tas* mRNA transcripts initiate either at BFV LTR (one) or IP (three), are spliced or unspliced and they have a differential ability to activate the BFV promoters (for clarity, only one representative IP-derived *tas* mRNA is shown in [Figure 2](#viruses-11-01084-f002){ref-type="fig"}A) \[[@B34-viruses-11-01084]\]. According to these findings, we propose the following model of Tas-mediated BFV gene expression. Firstly, activator protein 1 (AP-1) and some other unknown cellular transcriptional genes activate the Tas-mediated transactivation of the BFV IP as the early promoter for BFV gene expression, leading to the transcription of BFV IP *tas* mRNAs \[[@B35-viruses-11-01084]\]. In consequence, BFV Tas quickly accumulates to further enhance BFV IP activity. When a defined threshold level of BFV Tas is reached, the early phase of BFV IP-directed *tas/bet* expression is switched to the late phase of structural gene expression directed by LTR ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A). The transcription of LTR-spliced BFV *tas* transcripts with low biological activity can ensure modest levels of Tas, which makes it possible to establish persistent viral gene expression to complete the viral life cycle and maintain a balance between the virus and host cells.
Until now, the molecular mechanisms of transcriptional activation by Tas have remained unclear. Past investigations indicate that co-activators p300 and PCAF physically and functionally interact in vivo with PFV Tas, resulting in the enhancement of Tas-dependent transcriptional activation \[[@B36-viruses-11-01084]\]. Subsequently, PCAF acetylation of feline FV (FFV) Tas was shown to augment promoter-binding affinity and virus transcription \[[@B37-viruses-11-01084]\]. Similar to Tas of PFV and FFV, p300 can specifically interact in vivo with BFV Tas, which results in the enhancement of Tas-dependent transcriptional activation \[[@B38-viruses-11-01084]\]. In addition, the p300-mediated acetylation of BFV Tas can increase its DNA binding affinity, and the K66, K109, and K110 residues are critical for the DNA binding ability of BFV Tas; however, they are not conserved among different FVs \[[@B38-viruses-11-01084]\]. The K→R mutations in full-length BFV infectious clones reduce the expression of viral proteins, and the triple mutant completely abrogates viral replication \[[@B21-viruses-11-01084]\]. These findings suggest that acetylation might be an ubiquitous mechanism adopted by FVs as an effective means to regulate gene expression and animal FVs potentially share similarities with PFV in their need for essential host cell factors, e.g., p300 and PACF, etc. In addition to p300, BFV also engages the cellular RelB protein as a co-activator of BFV Tas to enhance its transactivation function \[[@B39-viruses-11-01084]\]. Furthermore, it was found that BFV infection upregulates cellular RelB expression through BFV Tas-induced NF-κB activation \[[@B39-viruses-11-01084]\]. Thus, it is a positive virus-host feedback circuit, in which BFV utilizes the host's NF-κB pathway through the RelB protein for its efficient transcription \[[@B39-viruses-11-01084],[@B40-viruses-11-01084]\]. There are many other unknown factors that are involved in the transactivation of Tas and some advanced techniques, such as tandem affinity purification and proximity labelling, can be used to discover new co-activators of Tas.
### 2.2.2. Function of Bet {#sec2dot2dot2-viruses-11-01084}
Although the mechanisms of FV Bet expression by splicing-mediated fusion of the N-terminal domain of Tas to the entire *bel2* coding sequence were described almost 25 years ago, its functions are only partly clarified ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A) \[[@B5-viruses-11-01084],[@B41-viruses-11-01084]\]. FV Bet is highly expressed after infection by different FVs \[[@B13-viruses-11-01084],[@B42-viruses-11-01084]\]. Previously, FFV and PFV Bet were shown to serve as antagonists of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family antiretroviral proteins for facilitating PFV and FFV replication \[[@B43-viruses-11-01084],[@B44-viruses-11-01084],[@B45-viruses-11-01084]\]. In addition, Bet might play an important role in the establishment and maintenance of viral persistence in vitro and in vivo \[[@B46-viruses-11-01084],[@B47-viruses-11-01084]\]. Furthermore, Bet has been described as having a negative regulatory effect upon the basal IP activity of PFV and it might limit the expression of the transcriptional transactivator Tas by inhibiting the activation of the IP \[[@B48-viruses-11-01084]\].
BFV Bet consists of 419 aa and it derives from a multiplied spliced mRNA fusing the first N-terminal 35 aa of BFV Tas to the entire Bel2 open reading frame. Although the sequence homology between the Bet proteins of different FVs is very low, some motifs in Bel2 are similar among the different Bet proteins \[[@B49-viruses-11-01084]\]. The Bet proteins of the known BFV isolates \[[@B17-viruses-11-01084],[@B19-viruses-11-01084],[@B50-viruses-11-01084],[@B51-viruses-11-01084],[@B52-viruses-11-01084]\] are highly conserved. In PFV-infected cells, Bet was shown to fuse with Env and form a glycoprotein of \~170 kDa \[[@B53-viruses-11-01084]\], but a corresponding BFV Env--Bet fusion protein could not be detected while using a BFV Bet antiserum.
BFV3026 Bet is present in both the nucleus and cytoplasm (predominantly in the nucleus) of the infected or transfected cells \[[@B54-viruses-11-01084]\]. Analysis of BFV3026 Bet amino acid sequences did not reveal the apparent structural sequence or functional protein motifs, but a nuclear localization signal (NLS) was predicted at the C-terminal end of BFV3026 Bet (392--396 aa) containing the amino acid sequence RRRRR (PSORT II software, \[[@B55-viruses-11-01084]\]) and NLStradamus model, \[[@B56-viruses-11-01084]\]). PFV Bet was reported to have an effective NLS at the C terminus (between 406 and 459 aa), but it does not contribute to nuclear localization of the protein and PFV Bet is located in both the nucleus and cytoplasm \[[@B57-viruses-11-01084]\]. BFV3026 Bet has a similar subcellular localization as PFV Bet, so it will be interesting to determine whether the predicted NLS of BFV Bet is functional. Nuclear pore complexes are known to allow two modes of transport: the passive diffusion of small molecules (\<20--40 kDa) and active transport of larger molecules (50 kDa and more) \[[@B58-viruses-11-01084]\]. As BFV Bet (55 kDa) is slightly too large to freely shuttle between the cytoplasm and nucleus, it might enter the nucleus using the NLS or by a currently unknown mechanism.
The functions of Bet during FV infection and replication are seemingly contradictory. It is required for FFV productive replication, as Bet mutants showed approximately 1,000-fold reduced viral titer in feline kidney cells when compared to the wild-type FFV \[[@B59-viruses-11-01084]\]. This is in contrast to PFV, where different Bet mutations or deletions did not show a defined phenotype or only an approximately 10-fold decreased cell-free viral transmission, which suggests that Bet might play a role in efficient cell-free viral transmission \[[@B60-viruses-11-01084]\]. However, these studies were often conducted in heterologous or genetically altered cells. Similarly, the BFV Bet mutant BFV3026 genome showed a four-fold higher level of replication than the wild-type genome in engineered human 293T cells \[[@B50-viruses-11-01084]\]. In addition, similar to PFV \[[@B61-viruses-11-01084]\], the overexpression of BFV Bet in heterologous canine fetal thymus cells (Cf2Th) reduced BFV3026 replication approximately threefold \[[@B50-viruses-11-01084]\]. Taken together, these data suggest that BFV Bet may serve as a negative regulator for BFV replication; however, analyses in authentic host cells appear to be absolutely mandatory.
In summary, these observations indicate that a biologically relevant FV Bet phenotype might only be detectable in cells expressing the cellular partner or target molecules of the authentic FV Bet protein. This is e.g. exemplified by the controversial finding on the Bet-induced inactivation of APOBEC3-mediated virus restriction: if APOBEC3 is either missing in the host cell used or the FV in question is propagated in different host cells without APOBEC3 expression or expression of heterologous APOBEC3 proteins, the intricate interaction between these partner molecules is lost, resulting in irrelevant phenotypes. Similarly, the repeated shifts of FVs from one to another host cell may have had similar consequences. These different scenarios are a strong case to use in vitro homologous host cells without genetic changes and adaptations often occurring in tumor cells or after extended passages in vitro and/or to conduct animal experiments in the authentic host species.
### 2.2.3. Function and Localization of Gag {#sec2dot2dot3-viruses-11-01084}
The interaction and subsequent self-multimerization of retro- and foamy virus Gag protein cause capsid formation \[[@B62-viruses-11-01084],[@B63-viruses-11-01084]\]. Unlike Gag proteins from Orthoretroviruses, FVs Gag is not processed into separate matrix (MA), capsid (CA), and nucleocapsid (NC) subunits (see [Figure 2](#viruses-11-01084-f002){ref-type="fig"}). In fact, four processing sites have been identified in the PFV Gag protein, which are divided into the optimal C-terminal cleavage site yielding p68/p3 and three suboptimal cleavage sites yielding p33/p39 or p39/p29 \[[@B64-viruses-11-01084],[@B65-viruses-11-01084]\]. In BFV, four Gag cleavage forms (p71, p68, p33, and p29) were also observed, indicating that both the optimal and suboptimal cleavages of Gag protein also occur in BFV; the Gag p68/p3 cleavage is the most efficiently used cleavage site \[[@B66-viruses-11-01084]\]. In contrast to Orthoretroviruses, the C-terminal domain of PFV Gag (the NC domain equivalent) contains three glycine (G) and arginine (R)-rich motifs (GR boxes) or less-defined RG-rich regions instead of the canonical cysteine-histidine repeat motif \[[@B67-viruses-11-01084]\]. Similar to PFV Gag, BFV Gag also has a nuclear location signal (NLS) in GR box II, which causes the nuclear accumulation of overproduced Gag protein \[[@B66-viruses-11-01084],[@B68-viruses-11-01084]\].
Unlike Orthoretroviruses, but similar to the other FVs, BFV Gag is not myristoylated and it cannot produce cell-free Gag-only virus-like particles \[[@B24-viruses-11-01084],[@B25-viruses-11-01084]\]. Similar to hepatitis B virus (HBV), BFV particle budding and release are instead dependent on the co-expression of the cognate viral envelope (Env) protein \[[@B24-viruses-11-01084],[@B25-viruses-11-01084]\], which suggests that Env provides a critical membrane-targeting function inherently lacking in BFV Gag. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to a lack of a functional ER retrieval signal (ERRS) \[[@B68-viruses-11-01084]\]. The addition of a membrane targeting signal to the N-terminus of Gag restores Gag-only budding from the plasma membrane, implying that Myr-membrane targeting substitutes for Env in particle release \[[@B24-viruses-11-01084],[@B68-viruses-11-01084]\].
Unlike PFV, FFV, and SFVs, BFV is highly cell-associated and it can only transmit through cell-to-cell but not via cell-free pathways \[[@B17-viruses-11-01084],[@B24-viruses-11-01084]\]. Interestingly, the Gag protein of BFV-Z1 (an in vitro selected cell-free infectious BFV3026 clone) lost a 14-amino acid sequence as compared to BFV-B (an infectious cell-associated BFV clone). This 14-residue deletion is located in the central and non-conserved region of FV Gag, which strongly contributes to the size differences of simian versus non-simian FVs \[[@B69-viruses-11-01084]\]. This deletion led to a smaller Gag-Z1 that enhanced cell-free infectivity by four- to five-fold \[[@B25-viruses-11-01084]\]. At the same site in Gag in some high titer (HT) cell-free BFV-Riems variants, insertions, and duplications occurred. However, their impact on BFV titers has not been studied \[[@B26-viruses-11-01084]\]. The Gag-Env interaction is very important for the budding and release of FV virions. Yet, the interaction of Gag and Env in BFV-B and BFV-Z1 was almost the same, which suggests that the contribution of Gag-Z1 to enhanced cell-free transmission is not through promoting interaction with Env \[[@B25-viruses-11-01084]\].
Viruses must engage bidirectional cellular transport mechanisms for completing their whole life cycle, and many viruses require microtubules (MTs) during cell entry for efficient nuclear targeting or the cytosolic transport of naked viral particles \[[@B70-viruses-11-01084],[@B71-viruses-11-01084],[@B72-viruses-11-01084]\]. In BFV, co-localization of MTs and assembling viral particles was clearly observed in BFV infected cells, which implied that BFV particles or assembly intermediates may transport along the cellular MTs to the cellular membrane to ultimately egress from the host cell. In fact, the MTs-depolymerizing assay indicated that MTs are required for the efficient replication of BFV \[[@B66-viruses-11-01084]\]. In conclusion, BFV has evolved this mechanism to hijack the cellular cytoskeleton for its replication. Until now, it is not clear which components of the MTs are involved in a uni- and/or bidirectional cellular transport of BFV particles. Thus, investigations on the direct interaction between the Gag and MT components should be a future research topic.
2.3. BFV-Host Interactions: Restriction Factors, Innate Immunity, miRNAs and Tight Cell Association {#sec2dot3-viruses-11-01084}
---------------------------------------------------------------------------------------------------
### 2.3.1. Restriction Factors {#sec2dot3dot1-viruses-11-01084}
The innate immune system constitutes a first line of defense against invading viruses. Cellular restriction factors are key players of innate and/or intrinsic immunity, which interferes with defined steps in the viral life cycle, leading to the attenuation or complete suppression of virus replication mainly acting immediately after virus infection \[[@B73-viruses-11-01084]\]. On the other side, viruses have evolved strategies to circumvent this inhibitory activity by co-evolution with host-encoded restriction factors.
Restriction factors are constitutively expressed and their expression can usually be increased by interferons (IFNs) \[[@B74-viruses-11-01084],[@B75-viruses-11-01084],[@B76-viruses-11-01084],[@B77-viruses-11-01084]\]. Until now, several restriction factors acting on retroviruses have been characterized in detail: APOBEC3, tripartite motif protein 5α (TRIM5α), bone marrow stromal cell antigen 2 (BST2, also called tetherin), SAMHD1, IFITM, MxB, and SERINC \[[@B78-viruses-11-01084],[@B79-viruses-11-01084],[@B80-viruses-11-01084],[@B81-viruses-11-01084],[@B82-viruses-11-01084],[@B83-viruses-11-01084],[@B84-viruses-11-01084]\]. Recently, some restriction factors were found to inhibit the replication of FVs. For instance, TRIM5α is implicated in restricting PFV, SFV, and FFV during viral entry \[[@B85-viruses-11-01084],[@B86-viruses-11-01084]\]; APOBEC3 proteins are known to act during PFV, SFV, and FFV reverse transcription (RT), and introduce lethal mutations in the viral genome \[[@B43-viruses-11-01084],[@B44-viruses-11-01084],[@B45-viruses-11-01084],[@B87-viruses-11-01084]\]; whereas, human BST2 (hBST2) and bBST2A1 (one isoform of bovine BST2) suppress the release of PFV and BFV \[[@B88-viruses-11-01084],[@B89-viruses-11-01084],[@B90-viruses-11-01084]\]. Moreover, unlike hBST2, bBST2A1 displays no inhibitory effect on cell-to-cell transmission of PFV and BFV \[[@B90-viruses-11-01084]\]. Other antiviral proteins include promyelocytic leukemia protein (PML), IFN-induced 35-kDa protein (IFP35), N-Myc interactor (Nmi), and p53-induced RING-H2 protein (Pirh2), which have been recently shown to inhibit FV replication through interacting with Tas \[[@B27-viruses-11-01084],[@B91-viruses-11-01084],[@B92-viruses-11-01084],[@B93-viruses-11-01084]\]. PML directly interacts with PFV Tas and it interferes with its ability to bind the TREs in the PFV LTR and IP \[[@B91-viruses-11-01084]\]. IFP35 might inhibit BFV Tas-induced transactivation by interfering with the interaction of a cellular transcriptional activation factor(s) and BFV Tas \[[@B27-viruses-11-01084]\]. Nmi inhibits the Tas transactivation of the PFV LTR and IP by interacting with Tas and sequestering it in the cytoplasm \[[@B92-viruses-11-01084]\]. Pirh2 negatively influences the Tas-dependent transcriptional activation of the PFV LTR and IP by interacting with the transactivator Tas and down-regulating its expression \[[@B93-viruses-11-01084]\]. These antiviral proteins likely limit or modulate the viral spread in vivo, but other antiviral proteins detected, for instance, in a recent high-throughput screen using PFV might, in addition, lead to FV latency, but are currently mostly unexplored \[[@B94-viruses-11-01084]\].
### 2.3.2. Innate Immunity {#sec2dot3dot2-viruses-11-01084}
An interesting feature of FVs is their ability to infect a diverse range of cell types and cause a characteristic foam-like cytopathic effect in culture system. However, they appear to be non-pathogenic in either naturally or accidentally infected hosts with a currently "emerging" but still ill-defined capacity to affect blood or kidney parameters without overt clinical consequences \[[@B11-viruses-11-01084],[@B95-viruses-11-01084],[@B96-viruses-11-01084]\]. This suggests that the host immune system controls viral infection and/or FV replication in vivo. Some evidence showed that the innate immune system probably plays an important role in limiting FVs replication to superficial epithelial cells of the oral mucosa \[[@B97-viruses-11-01084]\]. It has been suggested in early studies while using human or primate cell lines that FV does not activate an innate response and cannot induce type I IFNs (IFN-I) \[[@B98-viruses-11-01084],[@B99-viruses-11-01084],[@B100-viruses-11-01084]\]. However, only in recent years, it was reported that PFV is efficiently sensed by primary human hematopoietic cells via Toll-like receptor (TLR) 7, which leads to the production of high levels of IFN-I \[[@B101-viruses-11-01084]\]. The PFV-induced IFN-I induces the expression of IFN-stimulated genes in line with the finding that factors restricting FV replication are IFN-I-induced (e.g. TRIM5α and APOBEC3, see above). This activation of the innate immune responses might be a prerequisite for controlling viral replication in zoonotically infected humans or natural animal hosts. In line with this finding, previous studies reported that FV replication is sensitive to IFN-I \[[@B98-viruses-11-01084],[@B100-viruses-11-01084],[@B102-viruses-11-01084]\] due to the induction of several IFN-induced cellular proteins with antiviral activity in culture systems \[[@B27-viruses-11-01084],[@B43-viruses-11-01084],[@B89-viruses-11-01084],[@B90-viruses-11-01084],[@B91-viruses-11-01084],[@B92-viruses-11-01084],[@B93-viruses-11-01084]\]. Besides IFN-I, gamma IFN (IFN-γ) that is produced by activated human peripheral blood lymphocytes has also been found to be a major suppressive factor of PFV \[[@B103-viruses-11-01084]\].
Unfortunately, knowledge regarding the host-cell responses (especially innate immune responses) to BFV infection on the level of gene expression is still limited. In a recent study, changes in the transcriptome of the bovine macrophage cell line BoMac after in vitro BFV infection were examined while using bovine long oligo plus microarray (BLOPlus, Michigan State University, US) technology \[[@B104-viruses-11-01084]\]. In total, 124 genes involved in distinct cellular processes were up- or down-regulated. Among the differentially expressed genes, only five are involved in immune response. Three genes (Hsp90b1, hla-drb1, and Cxorf15) were up-regulated while two genes (CXCL2 and SELENBP1) were down-regulated. However, only the results of all three up-regulated genes (Hsp90b1, hla-drb1, and Cxorf15) were confirmed by subsequent RT-qPCR analyses \[[@B104-viruses-11-01084]\].
The Hsp90b1 protein is essential for the broad tropism of vesicular stomatitis virus (VSV) and for the establishment of infection with VSV and activation of innate immunity via TLRs \[[@B105-viruses-11-01084]\]. Therefore, the Hsp90b1 protein might have an effect on the capacity of FVs to infect a variety of tissues from different organisms. In addition, HLA-DRb1 (major histocompatibility complex class II, DR beta 1), an HLA class II antigen, plays central roles in immunity by presenting peptides derived from foreign, non-self proteins. It was found that specific HLA haplotypes, including HLA-DR, may protect against human immunodeficiency virus (HIV) \[[@B106-viruses-11-01084]\], and MHC class II molecules are up-regulated in several lymphoid cell lines following infection with feline immunodeficiency virus (FIV) \[[@B107-viruses-11-01084]\], as well as in T-lymphocytes of FIV-infected cats \[[@B108-viruses-11-01084]\]. Taken together, the increased level of HLA-DRb1 in BFV-infected BoMac cells might be responsible for the sustained elevation of MHC class II antigen levels. Furthermore, Cxorf15 γ-taxilin, together with α- and β-taxilins, is a member of the taxilin family. β- and γ-taxilin may play a role in intracellular vesicle trafficking \[[@B109-viruses-11-01084]\], and the α-taxilin levels are elevated in hepatitis B virus (HBV)-expressing cells and are essential for the release of HBV particles \[[@B110-viruses-11-01084]\]. One can assume that the upregulation of the Cxorf15 gene following BFV infection suggests a possible role of this protein in virion egress while taking similarities in budding strategies for FVs and HBV into consideration \[[@B111-viruses-11-01084]\]. These data offer a basis for further investigation of the immune response of host cells to FV infection, but the above speculation also needs to be further experimentally confirmed.
The effects of BFV infection on immune gene networks were confirmed in a recent study using experimentally infected calves; however, the differentially expressed genes identified one and three days after infection of the animals were different from those reported for the in vitro study while using BoMac cells \[[@B14-viruses-11-01084],[@B104-viruses-11-01084]\].
### 2.3.3. miRNA Expression as an Additional Layer to Control Host Gene Expression and Innate Immunity {#sec2dot3dot3-viruses-11-01084}
Recently, BFV and simian FV of African green monkey (SFV~agm~) have been shown to encode miRNAs via RNA polymerase III (RNA Pol III)-directed expression of a complex double-hairpin and, thus, dumbbell-shaped primary miRNA (pri-miRNA) precursor ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A,B) \[[@B22-viruses-11-01084],[@B112-viruses-11-01084]\]. The identification of such FV miRNA cassettes of about 120 nt length was stimulated and directed by prior findings in bovine leukemia virus (BLV), which is a close relative of human T cell leukemia/lymphotropic viruses (HTLVs) and used as an animal model for its human counterpart \[[@B113-viruses-11-01084],[@B114-viruses-11-01084]\]. The BLV RNA Pol III-driven miRNA cassettes consist of single hairpins structures and they were identified by an algorithm combining the search for RNA Pol III promoters and terminators and the presence of stable RNA hairpin structures that were flanked by these RNA Pol III-specific features \[[@B113-viruses-11-01084]\].
In BFV-Riems, only a single two-hairpin, dumbbell-shaped pri-miRNA with its RNA Pol III promoter and terminator is present in the non-coding part of the LTR U3 region downstream of the *bet/bel2* open reading frame ([Figure 2](#viruses-11-01084-f002){ref-type="fig"}A) \[[@B22-viruses-11-01084]\]. In contrast, in SFV~agm~, several different miRNAs are encoded by either dumbbell-shaped precursors RNA Pol III cassettes and possibly other pri-miRNAs that have been mapped to corresponding sites in the 3' end of the SFV~agm~ U3 region \[[@B112-viruses-11-01084]\] (see below and [Table 2](#viruses-11-01084-t002){ref-type="table"}). In both studies, the miRNAs were identified and characterized by miRNA sequencing. In BFV, two high level expressed miRNAs comprising about 70% of the total miRNA pool and a third one at modest levels were detected, a potential fourth miRNA from the remaining strand of the second hairpin was undetectable \[[@B22-viruses-11-01084]\]. In contrast, and reflecting the complexity of situation in SFV~agm~, sequencing identified three high-abundant, two intermediate, and at least six low abundant mature miRNAs \[[@B112-viruses-11-01084]\].
The different miRNA expression capacity and underlying mechanisms of BFV versus SFV~agm~ \[[@B22-viruses-11-01084],[@B112-viruses-11-01084]\] encouraged us to conduct bioinformatics while using the online available and further optimized algorithms to study the situation in BFV-Riems, SFV~agm~, and other FVs. By modifying the original algorithm of Kincaid et al. \[[@B112-viruses-11-01084]\] we especially focused on dumbbell structures of about 130 nucleotides in size in the LTR sequences of 38 FVs ([Table 2](#viruses-11-01084-t002){ref-type="table"}). Kincaid et al. also analyzed most of them for miRNA structures (Table S1 in \[[@B112-viruses-11-01084]\]). We predicted for 37 of the 38 FV sequences one or more dumbbell miRNA structures while using a fold energy cutoff of −30 kcal/mol and the existence of a terminator together with a TATA- and/or A/B-Box (as overview, see [Table 2](#viruses-11-01084-t002){ref-type="table"}).
We confirmed the presence of a single miRNA cassette encoding a dumbbell-shaped pri-miRNA \[[@B22-viruses-11-01084]\] in the genome of all known BFV isolates by using the updated algorithm ([Table 2](#viruses-11-01084-t002){ref-type="table"}). Single dumbbell-shaped pri-miRNAs were also predicted for the closely related EFV and several SFVs from different simian hosts as well PFV derived upon zoonotic transmission into humans. Surprisingly, while a single dumbbell-shaped miRNA cassette was detected in SFV~gor~, it was absent in another SFV~gor~ sequence that was derived from a zoonotically infected person \[[@B115-viruses-11-01084]\]. Our algorithm found each two independent RNA Pol III dumbbell-type pri-miRNA cassettes in SFV~agm~ (representing S1/S2 and S6/S7 miRNAs in \[[@B112-viruses-11-01084]\]). For other SFVs, two, three, and even five dumbbell-shaped miRNA cassettes were predicted. While the different FFV isolates from domestic cats contained four miRNA cassettes, the highly related FFV variant that was derived from Puma concolor was predicted to only encode three miRNAs.
In general, the predicted dumbbell miRNA cassettes are located in the non-coding region of the U3 LTR sequence, except the first miRNA cassette of all FFV isolates, which partially overlaps the 3' end of the *bel2/bet* gene. In addition, the fourth miRNA cassette of the domestic cat FFVs is very close to the transcriptional start site of the LTR promoter and it might interfere with RNA Pol II-directed mRNA expression similar to the situation in SFV~Cni~, where the third miRNA cassette even extends into the R region ([Table 2](#viruses-11-01084-t002){ref-type="table"}). The size of the predicted dumbbell-shaped pri-miRNA of the different FVs varies between 111 and 128 nt.
As experimental miRNA sequencing data are currently only available for SFV~agm~ and BFV-Riems, it is currently an open question as to whether these bioinformatics-based predictions presented here properly reflect the expression capacity and strategy of the different FVs and whether there is a huge variability of miRNAs between different, and even closely related, FVs. Additionally, the experimentally detected central SFV~agm~ miRNA and the corresponding stem-loops 3, 4, and 5 \[[@B112-viruses-11-01084]\] were not detected by our dumbbell-specific miRNA detection tool, so that, in certain FVs, there may be a co-existence of single-hairpin and dumbbell-shaped pri-miRNAs. Alternatively, the central SFV~agm~ stem-loops 3, 4, and 5 may be derived from larger, more complex pri-miRNAs, for instance, with terminal stem-loops but separated by unfolded, single-stranded intervening sequences.
The two independent experimental studies \[[@B22-viruses-11-01084],[@B112-viruses-11-01084]\] and our in silico analyses show that probably all FVs of different origin contain at least one RNA Pol III-directed miRNA cassettes of the dumbbell-shaped type. The miRNA repertoire of in SFV~agm~ is clearly more complex than that of BFV and it is currently unknown as to whether other FVs may or may not encode also SFV~agm~/BLV-like single hairpin pri-miRNAs. Thus, further wet biology analyses, high throughput sequencing and bioinformatics are needed to allow for full understanding of this highly important regulatory system of FVs.
The importance of cellular miRNA processing factors dicer and drosha was shown for SFV~agm~ \[[@B112-viruses-11-01084]\], while, for BFV, the impact of the overall shape of the dumbbell-shaped pri-miRNA was demonstrated \[[@B22-viruses-11-01084]\]. In BFV, minor modifications of the pri-miRNA sequence are well tolerated, while the replacement of an authentic stem-loop by heterologous shRNA sequences reduced but did not eliminate reporter gene suppression in dual luciferase assays \[[@B23-viruses-11-01084]\]. This finding, together with high-throughput optimization procedures, as done, for instance, for the BLV RNA Pol III miRNA cassettes \[[@B116-viruses-11-01084]\], may open the way to engineer efficient and specific chimeric FV-based pri-miRNA constructs for therapeutic application, as discussed below (see below, [Section 2.5](#sec2dot5-viruses-11-01084){ref-type="sec"}).
Similarities to host miRNAs were detected for experimentally validated SFV~agm~ and BFV miRNAs \[[@B112-viruses-11-01084],[@B117-viruses-11-01084]\] (see [Table 3](#viruses-11-01084-t003){ref-type="table"}). SFV~agm~ miR-S4-3p shares seed identity and functionality with host miR-155, a noted host oncogenic miRNA (oncomiR). SFV~agm~ miR-S6-3p shares seed identity with the host miRNA family miR-132, which suppresses innate immunity. In contrast, the similarities that were detected for BFV miRNAs comprise the miRNAs bta-miR-125a and the human counterpart of this miRNA has been described as stabilizing the suppressive phenotype of R848-stimulated antigen presenting cells on different levels in a hsa-miR-99b/let-7e/miR-125a cluster \[[@B118-viruses-11-01084]\]. Furthermore, miR-125a and miR-125b are both involved in the progression of cervical cancer \[[@B119-viruses-11-01084]\]. MiR-125b inhibits the PI3K/AKT pathway through the down-regulation of mRNA and protein PIK3CD, while miR-125a is anti-oncogenic by the downregulation of TRIB2 and HOXA1 by the family miR-99 clustered in miR-let-7c\~99a, miR-125a\~let-7e\~99b, and miR-100\~let-7a-2. The members of these clusters are diminished in cervical cancer \[[@B120-viruses-11-01084]\]. Taking all of this together, the miRNAs coded by FVs seem to interfere with immune and proliferation processes in the cells, but in different ways. The transcription by RNA Pol III makes the expression of miRNAs independent from protein expression, while the location in the LTR avoids the restrictions that are imposed by overlapping coding regions, thus enhancing variability and adaptability.
In BFV, three different and closely spaced miRNAs are detectable in chronically or lytically infected cell culture cells and, importantly, also in experimentally BFV-infect calve peripheral blood mononuclear cells (PBMCs), with the latter confirming the relevance of these findings beyond cell culture systems \[[@B22-viruses-11-01084]\]. The three stable miRNAs are generated from both stem-loops of the dumbbell-shaped pri-miRNA \[[@B22-viruses-11-01084]\]. In chronically BFV-infected cells in vitro, two BFV miRNAs make up more than 2/3 of the total cellular miRNA pool pointing to an important role, especially in chronically infected cells \[[@B22-viruses-11-01084]\]. The two high-abundance miRNAs are localized each to the 5'part of the two different stem-loops, while in SFV~agm~, miRNAs from both strands of the S3 stem-loop are easily detectable \[[@B22-viruses-11-01084]\].
In the BFV system, bovine cells and bovine genomics have been used and the outcomes of bioinformatics-guided target gene prediction as well as wet biology-based experimental validation were mostly conducted in bovine cell cultures and finally in BFV-infected cells and experimentally infected cattle, as described by Cao et al. \[[@B14-viruses-11-01084]\]. In brief, target site predictions for the high abundance BF2-5p miRNA yielded several potential targets with very high scores and two of them with relevance for innate immunity and virus replication, ANKDR17 \[[@B121-viruses-11-01084]\] and Bif-1 (SH3GLB1) \[[@B122-viruses-11-01084]\] were experimentally confirmed in independent assays \[[@B14-viruses-11-01084]\]. In addition, even downstream targets of ANKRD17 showed altered expression in response to BFV miRNA cassette deletion and miRNA co-transfection \[[@B14-viruses-11-01084]\]. A small number of calves were infected with MDBK cells expressing the highly cell-associated BFV Riems isolate and high-titer in vitro-selected BFV variants lacking or carrying the miRNA cassette in order to establish conditions for in depth analyses of the importance of the miRNAs in the authentic host \[[@B14-viruses-11-01084]\]. The data show that all BFV variants are replication-competent in calves; however, the deletion of the miRNA cassette caused a drop of viral infectivity. The deletion of miR-BF2-5p probably reduced the replication competence of the virus, as seen by the lower induction of genes involved in the recognition of viruses by the innate immune system when compared to wt BFV-infected calves. It probably also resulted in the lower level of the humoral response to BFV Gag observed, especially in one of the animal infected with the miR-BF2-5p-deficient BFV variant (for details, see \[[@B14-viruses-11-01084]\]).
### 2.3.4. Highly Cell-Associated Spread, at Least in Cell Cultures---What Is Behind This Phenotype? {#sec2dot3dot4-viruses-11-01084}
Viruses have two major transmission strategies: cell-free transmission, involving the release of virus particles into the extracellular space, and cell-to-cell transmission \[[@B123-viruses-11-01084]\]. Retroviruses exhibit different degrees of cell-free and cell-to-cell transmission. Unlike most other retroviruses, such as HIV-1, murine leukemia virus (MLV), PFV, FFV, and SFV, which transmit through both cell-to-cell and cell-free pathways, the transmission of BFV is highly cell-associated, with very low to undetectable cell-free transmission \[[@B17-viruses-11-01084],[@B24-viruses-11-01084]\]. This lack, or only low level, of cell-free transmission appears to be independent of whether the BFV isolates have been exclusively propagated in primary bovine cells (the BFV Riems isolate) or whether immortalized bovine (MDBK cells) and hamster and canine cell lines, like BHK-21 and Cf2Th, have been used for virus propagation. BFV is an excellent model for studying virus adaption to cell-free transmission and identifying the principles of viral transmission by in vitro selection and evolution analyses, since the BFV particle budding machinery is similar to that of other FVs \[[@B24-viruses-11-01084],[@B25-viruses-11-01084]\].
In two independent selections screen using established immortal MDBK and BHK-21 cells, BFV Riems was shown to adapt to cell-free transmission within 80 and 130 cell-free passages reaching titers of more than 10^5^ and 10^6^ FFU/mL, respectively. The resultant HT variants had independently gained the capacity to spread via cell-free particles, but still also use cell-cell transmission \[[@B24-viruses-11-01084]\]. Genetic studies indicate that consistent and cell type-specific, as well as cell-type-independent adaptive changes, occurred in Gag and Env as well as in the LTR regions where larger changes had also been observed \[[@B26-viruses-11-01084]\] (Bao, Stricker, Hotz-Wagenblatt, and Löchelt, to be published). Importantly, cell-free HT BFV-Riems is still neutralized by serum from naturally infected cows \[[@B24-viruses-11-01084]\]. The different selected HT BFV variants will shed light into virus transmission and the potential routes of intervention in the spread of viral infections.
Zhang and colleagues successfully isolated HT cell-free BFV strains from the original cell-to-cell transmissible BFV3026 strain (Chinese isolate) while using in vitro virus evolution and further constructed an infectious cell-free BFV clone, called pBS-BFV-Z1, to independently explore the molecular mechanisms of BFV cell-free transmission \[[@B25-viruses-11-01084]\]. Following sequence comparisons with a cell-associated clone pBS-BFV-B \[[@B50-viruses-11-01084]\], a number of changes in the genome of pBS-BFV-Z1 were identified. Extensive mutagenesis analyses revealed that the C-terminus of Env, especially the K898 residue, controls BFV cell-free transmission by enhancing cell-free virus entry \[[@B25-viruses-11-01084]\]. The authors also claim that virus release of this variant is increased, although this was not experimentally analyzed \[[@B25-viruses-11-01084]\]. It is well-known that lysine (K) can undergo methylation, acetylation, succinylation, ubiquitination, and other modifications, which play an important role in regulating the protein activity and structure adjustment \[[@B124-viruses-11-01084]\]. Interestingly, the equivalent position of the 898 residue in all known BFV isolates (from the United States, NC001831.1. \[[@B19-viruses-11-01084]\], China, AY134750.1 \[[@B50-viruses-11-01084]\], Poland, JX307861 \[[@B51-viruses-11-01084],[@B52-viruses-11-01084]\], and Germany JX307862.1 \[[@B17-viruses-11-01084],[@B52-viruses-11-01084]\], which only spread through cell-to-cell, is not a lysine (K), while the equivalent position is occupied by a lysine in other high titer cell-free FVs, such as SFV, FFV, and PFV. This suggests that the K898 in Env has an important role in FV cell-free transmission. The underlying mechanisms warrant further studies. Interestingly, the Gag protein of BFV-Z1 lost 14 amino acids in the highly divergent sequence between the matrix and capsid regions, which enhanced cell-free infectivity by four- to five-fold \[[@B25-viruses-11-01084]\]. Other changes of the BFV-Z1 genome contributed little to BFV cell-free transmission. Taken together, these data reveal the genetic determinants that regulate cell-to-cell and cell-free transmission of BFV, and suggest the possibility of generating high-titer BFV vectors through engineering viral Env and, in particular, its C-terminal sequence.
2.4. BFV-Host Interactions at the Organismal and Populational Level {#sec2dot4-viruses-11-01084}
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### 2.4.1. BFV Epidemiology and Naturally Occurring Co-Infections {#sec2dot4dot1-viruses-11-01084}
Infections with BFV have been reported worldwide since the first isolation of BFV by Malmquist and co-workers in 1969 \[[@B30-viruses-11-01084]\]; however, sero-epidemiological data are only available from some countries and they show variable rates of BFV infected animals. The highest sero-prevalence was reported in Canada, where it varied between 40 and 50% \[[@B125-viruses-11-01084],[@B126-viruses-11-01084]\]. A slightly lower rate of 39% was observed in Great Britain \[[@B127-viruses-11-01084]\] and Australia \[[@B128-viruses-11-01084]\]. The most recent data come from Germany, where only 7% of tested animals were identified as being BFV positive \[[@B15-viruses-11-01084]\] and from Poland with BFV sero-prevalence of over 30% in dairy cattle \[[@B129-viruses-11-01084]\]. BFV prevalence based on these studies seems to be very diverse. However, these data span a long time-frame; therefore, one of the reasons for these disparities might be the different sensitivity of methods used for serological testing, from agarose gel immuno-diffusion (AGID) and syncytia inhibition assay to ELISA and indirect immuno-fluorescence assays. Additionally, the age of animals tested might be a reason of such diverse BFV prevalence, but, although the age of the animals was not specified in these studies, the importance of this factor was suggested by Jacobs and co-workers, who observed a higher rate of BFV positive status in older animals \[[@B126-viruses-11-01084]\]. This might be due to the persistence of BFV infections and prolonged sero-conversion in animals, but one cannot exclude other factors, like breed and the type of animal rearing. Interestingly, no disease or clear clinical symptoms were ever associated with BFV infection in cows. However a role of BFV as a co-factor of other retroviral infection has been suggested, especially in the context of mixed infections, which are one of the characteristic features of retroviral infections \[[@B130-viruses-11-01084]\]. This has been also suggested for people infected with HIV and HTLV \[[@B131-viruses-11-01084]\], cats infected with FIV, FeLV, and FFV \[[@B132-viruses-11-01084]\], FIV/FeLV \[[@B133-viruses-11-01084]\], and FIV/FFV \[[@B134-viruses-11-01084]\], and monkeys infected with SIV and STLV \[[@B135-viruses-11-01084]\]. Similar studies have been carried out with respect to co-infections with BFV, bovine leukemia virus (BLV), and bovine immunodeficiency virus (BIV) in cattle. Amborski and co-workers published the first report on BFV co-infection with other lymphotropic retroviruses in dairy cows \[[@B136-viruses-11-01084]\]. In the already quoted study by Jacobs and others carried out in Canada, including numerous dairy cattle herds, it was shown that the percentage of animals with antibodies to BIV, BLV, and BFV is 5.5%, 25.7%, and 39.6%, respectively, however with no statistically significant correlation between the individual values \[[@B126-viruses-11-01084]\].
It is assumed that the source of mixed infections might be due to the same route of retroviral transmission, which results in a statistically significant correlation in the occurrence of antibodies, e.g. for BLV and BIV \[[@B137-viruses-11-01084]\] or FIV and FeLV \[[@B138-viruses-11-01084],[@B139-viruses-11-01084]\] and HTLV II and HIV \[[@B140-viruses-11-01084]\]. Mixed infections are particularly important in herds with BLV-infected animals, due to the fact that BLV is the etiological agent of enzootic bovine leukosis and since BFV has been suggested to act as a cofactor in BLV infections \[[@B130-viruses-11-01084]\]. In a recent study, a statistically significant correlation between the occurrence of serologically positive reactions for BLV and BFV at the herd level was shown \[[@B141-viruses-11-01084]\]. Although the results of these studies cannot be related to individual animals, they indicate a certain pattern in the distribution of herds, where BLV and BFV are present. In the study by Jacobs and others, mixed infections of BLV and BFV were recorded in 9.9% of cows \[[@B126-viruses-11-01084]\]. It has been suggested that BFV and BLV co-infections may impair the immune defense capacity of the host \[[@B136-viruses-11-01084],[@B142-viruses-11-01084]\], similarly as proposed for cats co-infected with FIV and FFV \[[@B143-viruses-11-01084]\]. However, it might also be considered that both viruses interact at the molecular level, especially since both of them use the phenomenon of transactivation in the process of viral replication and, even more interesting, they encode miRNAs that interact with genes directly involved in immune defense processes of the host. There is evidence that BLV- and BFV-encoded miRNAs target genes involved in innate and adaptive immunity and, thus, dysregulating their expression levels might facilitate BFV spread, transmission, or persistence. \[[@B14-viruses-11-01084],[@B144-viruses-11-01084],[@B145-viruses-11-01084]\].
BFV is endemic at high prevalence in livestock cattle in different parts of the world, which can be quite easily confirmed by virus isolation in different types of cells (Cf2Th, BoMac, MDBK, KTR, BHK21); however, it is also possible to develop productive infection under experimental in vivo conditions. Only few reports confirmed the possibility of the experimental infection of cattle with BFV \[[@B128-viruses-11-01084]\]. Materniak and co-workers used the experimental BFV inoculation to determine its replication and immunogenicity, not only in its homologous, but also in the heterologous host \[[@B13-viruses-11-01084]\]. Calves and sheep were selected to analyze the infection kinetics in different, but related, species. Although, neither the experimental BFV infection of calves nor sheep resulted in pathology, BFV spread and replicated to similar degrees in both, the homologous and heterologous hosts. Productive BFV infections were established in calves and sheep, as confirmed by virus isolation from leukocytes of all infected animals. BFV was rescued from both infected animal hosts, even in the presence of BFV-specific antibodies, confirming that BFV infection is not cleared by the host immune system \[[@B13-viruses-11-01084]\]. Additional parameters of BFV infection, like humoral immune response to BFV proteins and the presence of BFV DNA in blood cells and organs, also confirmed the persistence of BFV in both hosts \[[@B13-viruses-11-01084]\]. Interestingly, upon long-term replication in sheep, approximately 70% and 40% of the single nucleotide mutations in the sheep-derived BFV *bet* and *env* sequences, respectively, led to changes in the amino acid sequence. As no consistent pattern of adaptive changes was detectable, this proves the utility of sheep as an animal model to study the biology of persistent spumavirus infections \[[@B13-viruses-11-01084]\].
### 2.4.2. BFV Transmission Route {#sec2dot4dot2-viruses-11-01084}
The transmission of BFV is suggested to occur through close contact. While considering cattle behavior, it is assumed that BFV shedding occurs via saliva through non-aggressive contact, like sneezing or licking and via infected milk \[[@B143-viruses-11-01084]\]. The successful recovery of BFV from saliva and milk of naturally infected cattle tends to confirm this mode of virus shedding and transmission \[[@B15-viruses-11-01084],[@B16-viruses-11-01084]\]. Interestingly, older studies that were reported by Johnson and others, as well as Kertayadnya et al., showed that calves being BFV negative at birth or the beginning of the experiment became infected when placed together with infected adults \[[@B128-viruses-11-01084],[@B146-viruses-11-01084]\]. Additionally, Johnson and others studied different routes of transmission using BFV infected culture fluids containing cell debris. This experiment showed that only throat spray and intravenous application resulted in successful BFV infection in calves, while the swabbing of cell culture-derived BFV into the vagina or onto the prepuce did not lead to infection \[[@B128-viruses-11-01084]\]. Kertayadnya and others also excluded insect or airborne transfer of BFV infection \[[@B146-viruses-11-01084]\]. The authors of both reports state that the most possible source of infection under natural conditions is close contact to a single immunologically tolerant individual, which is productively BFV-infected, but has no or only very low levels of BFV-specific antibodies. However, the source of such BFV tolerance is disputable. One hypothesis is that such animals are characterized by a very early viremic stage of infection before the development of neutralizing antibodies occurs. However, as Kertayadnya and others reported, there are animals that are productively infected with BFV, but do not produce antibodies, even after several months of infection \[[@B146-viruses-11-01084]\]. Another explanation of immunological tolerance towards BFV could be via in utero infection. Such a scenario might be supported by the studies of Bouillant, and Ruckerbauer who recovered BFV from the uterus of BFV infected cows \[[@B147-viruses-11-01084]\]. Finally, perinatal transmission of BFV via colostrum or milk has been proposed. This route of BFV spread is supported by our studies showing that BFV can be reproducibly isolated from the cellular fraction of raw milk \[[@B15-viruses-11-01084],[@B16-viruses-11-01084]\].
### 2.4.3. Interspecies and Zoonotic Transmission of BFV as Part of the Human Food Chain {#sec2dot4dot3-viruses-11-01084}
It has been clearly demonstrated that SFVs can be zoonotically transmitted to humans in Africa, South America, and Asia upon exposure or contact with SFV-infected monkeys \[[@B6-viruses-11-01084],[@B8-viruses-11-01084],[@B148-viruses-11-01084]\]. The nature of human exposure to BFV is slightly different, but it seems to be constantly present in the human food chain when considering the routes of virus transmission and replication sites of BFV. Additionally, there are many products of cattle origin used in pharmaceutical and cosmetic industry. However, direct contact, which seems to be the most likely mode of zoonotic transmission, is restricted to a limited part of the human population. So far, two serological studies were performed to screen for BFV in humans. One of them focused on dairy cow caretakers, cattle owners, and veterinarians who were tested for BFV-antibodies and showed an overall sero-prevalence of about 7%; however, none of them was PCR positive for BFV DNA in PBMCs \[[@B149-viruses-11-01084]\]. Another study included three groups of humans, who were serologically tested for BFV antibodies \[[@B150-viruses-11-01084]\]. BFV-specific reaction was found in 7% of immunosuppressed patients, 38% of people claiming contact with cattle, and 2% of the general population with no interaction with cows. In each group, a single BFV PCR positive individual was identified and the sequence of short PCR product showed high homology to BFV isolates that are available in GenBank. The data obtained suggest that BFV zoonotic infection may be possible, however it is not common and, in most of the cases, cleared by the host immune system.
### 2.4.4. BFV Replication in Naturally and Experimentally Infected Animals {#sec2dot4dot4-viruses-11-01084}
In vivo studies play a vital role in virology, since they allow for investigation of the events taking place during the viral infection in the host. Many aspects of infection can be, in fact, only explored by examining naturally infected animals; however, this needs to be done under carefully controlled conditions in the homologous or a heterologous host. In studies on the replication of BFV in vivo, both directions were used; therefore, these data are quite comprehensive. Similar to other FVs, BFV shows a wide tissue tropism. In naturally infected animals, BFV was recovered from peripheral blood leukocytes/lymphocytes, tumors, fetal tissues, placenta, testis, and from fluids used to flush the uterus and oviducts of super-ovulated cows \[[@B17-viruses-11-01084],[@B30-viruses-11-01084],[@B51-viruses-11-01084],[@B151-viruses-11-01084],[@B152-viruses-11-01084],[@B153-viruses-11-01084],[@B154-viruses-11-01084]\]. Studies on naturally and experimentally infected animals using PCR-based virus detection showed that BFV DNA is present in most tissues, like lung, salivary glands, liver, spleen, and bone marrow \[[@B13-viruses-11-01084]\]. Interestingly, some reports from SFVs in monkeys previously showed that, although DNA is present in most animal tissues, SFV RNA, indicative of viral gene expression and replication, is mostly if not exclusively detected in oropharyngeal sites \[[@B155-viruses-11-01084],[@B156-viruses-11-01084],[@B157-viruses-11-01084]\]. In the most recent studies on tissue distribution of BFV DNA and RNA, different organs, as well as blood, bronchoalveolar lavage cells (BALs), and trachea and pharynx epithelium of experimentally infected calves, were analyzed \[[@B28-viruses-11-01084]\]. The highest load of BFV RNA was detected in the lungs, spleen, liver, PBMC, BALs, and trachea epithelium, while in contrast to the previous studies showing BFV isolation from saliva and milk cells of naturally infected cows \[[@B15-viruses-11-01084],[@B16-viruses-11-01084]\], BFV RNA was detected in the saliva of only a single calf. The presence of BFV RNA in such diverse organs seems to be strong proof that active replication of BFV might be not limited to the oral cavity, in contrast to the findings for SFV gene expression in monkeys.
In a recent study, HT BFV Riems, the cell free variant of BFV, and wild type BFV Riems isolate were used for the experimental inoculation of calves \[[@B14-viruses-11-01084],[@B24-viruses-11-01084]\]. The infection pattern was very similar in both groups of calves. The humoral response was comparable in both groups, but BFV viral load measured in PBMCs of infected animals during 16 weeks p.i. was slightly lower in calves that were infected with HT-BFV Riems. However, at the end of experiment, BFV was rescued from PBLs of all, parental and HT BFV-infected, animals. Interestingly, when changes in the expression of selected genes involved in innate immunity were analyzed at day 1 and 3 p.i., the level of induction was clearly lower in the HT-BFV Riems infected calves as compared to wt BFV Riems inoculated animals, which suggests a slightly impaired detection of HT BFV.
Importantly, and confirming the concept that interspecies transmission of FVs is possible to genetically related hosts, sheep have been shown to be permissive to experimental BFV infection while using intravenous inoculation of BFV100-infected Cf2Th cells \[[@B13-viruses-11-01084]\]. Successful inoculation of sheep with BFV as well as its transmission via saliva may support the risk of cross-species infections in mixed farms, where cattle and sheep are kept in close contact. Previous work, in fact, reported the presence of an FV-like virus in sheep \[[@B158-viruses-11-01084]\], but two scenarios are possible since no further characterization of the isolate was performed. One is that the infection was a result of a cross-species transmission of BFV from cattle; alternatively, the isolate was, in fact, a sheep specific foamy virus. Over 500 German sheep serum samples were tested to try to answer this question and 35 sheep sera showed reactivity to BFV Gag antigen in GST ELISA \[[@B149-viruses-11-01084]\]. Unfortunately, further diagnostics of these BFV-cross-reactive animals by virus isolation or PCR amplification were unsuccessful \[[@B159-viruses-11-01084]\]. Recent studies regarding wild ruminants revealed a similar scenario: some sera also reacted with BFV antigens in ELISA test, but PCR mostly failed to identify genetic material of BFV \[[@B160-viruses-11-01084]\]. In fact, the lack of amplification with BFV-specific primers might suggest the infection with novel, species-specific FVs, which generate antibodies that cross-react with BFV-specific antigen. Therefore, the existence of BFV-related FVs in other ruminants still remains open.
2.5. Utilization of BFV as Viral Vector for Translational Applications {#sec2dot5-viruses-11-01084}
----------------------------------------------------------------------
As with other retroviruses, different viral vectors for the expression of therapeutic genes or the delivery of vaccine antigens have been constructed for PFV, few SFVs, and FFV, as review see \[[@B161-viruses-11-01084]\]. These vectors mostly include replication-deficient gene transfer vectors generated in producer cells, but they also include replication-competent engineered viruses, which are mostly intended for life vaccine applications. Some of these vectors have been tested beyond cell cultures in small lab animals (mostly mice), but also in outbred hosts like dog (ex vivo PFV-based gene transfer vectors to treat canine leukocyte adhesion deficiency \[[@B162-viruses-11-01084]\]) and cats (FFV-based replication-competent vaccine vectors \[[@B163-viruses-11-01084],[@B164-viruses-11-01084]\]). The recent cloning of full-length BFV genomes that allows for cell-free transmission \[[@B14-viruses-11-01084],[@B25-viruses-11-01084],[@B26-viruses-11-01084]\] is an important prerequisite for conducting and extending corresponding studies also for BFV.
Cattle are important livestock animals and vector-based approaches are likely to meet the costs imposed by bovine infectious disease or the need to engineer defined traits in the future. Such vector-directed gene transfer and vaccination might be an interesting option with a corresponding market to explore and use BFV as a suited viral vector for treatment of cattle. The availability of CMV-IE-driven BFV genomes and recent data that HT cell-free BFV variants replicate in cattle are important prerequisites for such studies \[[@B14-viruses-11-01084]\]. Furthermore, even an engineered HT cell-free BFV variant lacking the entire miRNA cassette replicated in experimentally infected animals and induced immunity against BFV Gag and Bet \[[@B14-viruses-11-01084]\]. Together with the data on the function and core features of the BFV miRNA cassette and the chimerization of the BFV pri-miRNA \[[@B23-viruses-11-01084]\], the insertion of therapeutic or prophylactic miRNAs into replication-competent BFV vectors or the construction of BFV-based miRNA expression tools appear to be new and interesting future developments \[[@B161-viruses-11-01084]\].
3. Conclusions and Outlook {#sec3-viruses-11-01084}
==========================
As shown here, research regarding diverse aspects of BFV replication and biology in vitro and in vivo has significantly expanded our understanding of the complexity and diversity of FVs. These new findings are in line with the concept that each of the known exogenous FVs has been shaped by a long history of co-adaptation and co-evolution \[[@B10-viruses-11-01084]\]. It remains to be seen whether, for instance, the host dictates the major pathway of FV transmission: Here, strong differences between herbivorous cattle that do not display aggressive intra-species behavior and carni- and omnivorous simians or felines with a substantial amount of aggressive behavior within and between groups and individual animals can be anticipated. Whether such differences in the host's biology not only affect transmission, but also the repertoire of antiviral restriction, remains to be seen, but appears to be possible.
Additional avenues of high-impact research in BFV may be related to development of vaccine vectors based on the BFV genome or genetic elements thereof. The possibility of cell-free BFV infections recently achieved offset the limitations of a tight cell-associated transmission from BFV as a therapeutic and prophylactic vector candidate \[[@B14-viruses-11-01084],[@B24-viruses-11-01084],[@B25-viruses-11-01084]\]. Vaccines that are based on bovine virus-based vectors could be a great alternative in veterinary science and practice, especially in the context of economically important infections that, due to the high prevalence in cattle populations, cannot be eradicated by culling. Furthermore, extending the studies on the BFV miRNAs as modulators of the virus-host interface and evolutionary struggle, as well as their translational application within a BFV-based vector or as an independent cassette, appears to be a promising extension of ongoing work. Finally, it is of high priority to explore the "requirements" of BFV---as part of the human food chain and present in several raw cattle products---to enter the human population. Such studies may be pretty challenging and also---to a certain degree---unpredictable, but of high medical and epidemiological importance. Here, in vitro selection and evolution screens employing either fresh animal-derived BFV or "native" BFV isolates and primary human cells of different tissue/organ origin will be of special value.
M.M.-K. would like to thank Jacek Kuźmak for introduction into the foamy virus field and his continuous support. M.L. thanks his partners and supporters on BFV research, in particular Roland Riebe, Frank Rösl, Jacek Kuźmak, Timo Kehl, Torsten Hechler, Wentao Qiao, Bryan Cullen, Thomas Vahlenkamp, Lutz Gissmann and M.M.-K. The authors thank Martha Krumbach (DKFZ) for critically reading the manuscript.
M.M.-K., J.T., A.H.-W. and M.L. wrote, corrected and approved the article, A.H.-M. provided valuable information and conducted bioinformatics analyses together with A.H.-W., the initial layout and final editing were done by M.L.
Work by M.M.-K. was supported by Polish Ministry of Science and Higher Education and DAAD, gants no. 5 PO6K 043 27, 484/N-DAAD/2009/2010 and KNOW (Leading National Research Centre) scientific Consortium, "Healthy Animal---Safe Food", decision of Ministry of Science and Higher Education 05-/KNOW2/2015. J.T. was funded by the National Natural Science Foundation of China, grant number 31670151 and A.H.-M., A.H.-W and M.L. were supported by the Baden-Württemberg Stiftung, research grant SID 49 and PPP Polish-German DAAD travel grants.
The authors declare no conflict of interest.
![Phylogenetic tree of known exogenous and endogenous foamy viruses (FVs) (blue branches) and members of the *Orthoretrovirinae*. A fasta file with the conserved regions of the Pol proteins (supplement from ref. \[[@B2-viruses-11-01084]\] and prototype FV (PFV, U21247.1) was used for alignment with ClustalW (<http://www.clustal.org/>). From the alignment, an ML tree was created using fastml (<https://fastml.tau.ac.il>, default parameters). The resulting newick tree was displayed by Itol (<https://itol.embl.de/>).](viruses-11-01084-g001){#viruses-11-01084-f001}
![Genetic structure and schematic illustration of bovine foamy virus (BFV) gene expression and the BFV primary miRNA. (**A**) The BFV provirus DNA genome is shown on top schematically and out of scale with the terminal long terminal repeats (LTRs) consisting of the U3, R, and U5 regions. The position of the miRNA cassette in the U3 regions is indicated in color. BFV genes are shown as overlapping open boxes sub-divided into the mature protein domains. Proteolytic processing is marked by dotted lines. The spliced *bet* gene is separately shown below the genome. Broken arrows indicate the transcriptional start sited and direction of LTR- and internal promoter- (IP) directed gene expression and the Tas-mediated transactivation of the 5'LTR and the IP is indicated in red. Below, a selection of the major early and late BFV transcripts starting at the IP and LTR are shown with spliced-out areas indicated by broken lines. Only the major BFV IP-directed Tas mRNA is shown (\*). The shift between early and late transcription is marked by a boxed arrow at the right-hand margin. (**B**) The predicted folding and secondary structure of the BFV dumbbell-shaped miRNA precursor (BFV pri-miRNA) is given, for additional information, and the sequence of the mature and stable miRNA, see below and Whisnant et al., 2014 \[[@B22-viruses-11-01084]\].](viruses-11-01084-g002){#viruses-11-01084-f002}
{#viruses-11-01084-f003}
viruses-11-01084-t001_Table 1
######
Special features and novel insights that were gained from past and current work on Bovine Foamy Virus.
Subject/Topic References
--------------------------------------------------------------------------------------------------------- -----------------------------------------------------------------------------
BFV as a well-established infection model in life-stock animals (cattle and sheep) \[[@B13-viruses-11-01084],[@B14-viruses-11-01084]\]
BFV as the only known FV in the general human food chain (beef and dairy products) \[[@B15-viruses-11-01084],[@B16-viruses-11-01084]\]
BFV Riems as the only FV passaged exclusively on primary and homologous host cells \[[@B17-viruses-11-01084],[@B18-viruses-11-01084]\]
Integrase domain: disrupted HH-CC zinc finger and unique sequence insertion into the extreme C-terminus \[[@B19-viruses-11-01084]\]
Detailed understanding of gene expression and transactivation of a non-simian FV \[[@B20-viruses-11-01084],[@B21-viruses-11-01084]\]
RNA Pol III miRNAs, unique precursor structure and their functions \[[@B14-viruses-11-01084],[@B22-viruses-11-01084],[@B23-viruses-11-01084]\]
Extremely tight cell association and identification of residues critical for this phenotype \[[@B24-viruses-11-01084],[@B25-viruses-11-01084],[@B26-viruses-11-01084]\]
Detailed understanding of new restriction factors against FVs \[[@B27-viruses-11-01084]\]
Broad tissue tropism and gene expression in BFV-infected calves \[[@B5-viruses-11-01084],[@B28-viruses-11-01084]\]
viruses-11-01084-t002_Table 2
######
Results of bioinformatics on dumbbell-type RNA Pol III cassettes in the LTRs of selected FVs flanked by consensus TATA boxes and termination signals.
Virus-Type Virus Isolate \* and Accession Number Number of Dumbbell-Shaped miRNA Cassettes Number of AB or BB Boxes
------------ ---------------------------------------------------- ------------------------------------------- --------------------------
BFV BFV_Riems \[[@B22-viruses-11-01084]\]; JX307862.1 1 0
BFV_100; JX307861.1 1 0
BFV_11; U94514.1 1 0
BFV_3026; AY134750.1 1 0
EFV EFV; AF201902.1 1 0
FFV FFV Chatul-3; AJ564746.1 4 4
FFV F17; U85043.1 4 4
FFV FUV; NC_039242.1 4 4
FFV~Pco~; KC292054.1 3 3
HFV HFV; U21247.1 1 0
HSRV1; Y07723.1 1 0
HSRV2; Y07724.1 1 0
PFV; Y07725.1 1 0
SFV SFV_AG15; JQ867462.1 1 0
SFV~agm~; NC_010820.1 \[[@B112-viruses-11-01084]\] 2 1
SFV_AXX; EU010385.1 5 3
SFV_BAD327; JQ867463.1 1 0
SFV_BAD468; JQ867465.1 1 0
SFV_BAK74; JQ867464.1 0 0
SFV_CAE_FV2014; MF582544.1 2 1
SFV_CAE_LK3; M74895.1 2 1
SFV_CJA; GU356395.1 1 0
SFV_CNI; JQ867466.1 3 1
SFV_CPZ; U04327.1 1 0
SFV_GOR; HM245790.1 1 0
SFV_MAC; X54482.1 1 0
SFV_MCY; KF026286.1 1 0
SFV_MFA; LC094267.1 1 0
SFV_MFU; AB923518.1 1 0
SFV_MMU; MF280817.1 1 0
SFV_OCR; KM233624.1 1 0
SFV_ORA; NC_039085.1 2 1
SFV_PPY; AJ544579.1 3 2
SFV_PSC; KX087159 1 0
SFV_PVE; NC_001364.1 1 0
SFV_SSC; GU356394.1 1 0
SFV_SXA; KP143760.1 1 0
SFV-6; L25422 1 1
\* References are given for those FVs where experimental miRNA data are available.
viruses-11-01084-t003_Table 3
######
Homology of seed sequences of experimentally identified BFV-Riems and simian FV of African green monkey SFV~agm~) miRNAs to known miRNAs of other species.
miRNA Name Human and Bovine miRNA with Seed Identity
---------------------- ---------------------------------------------
SFVagm -S2-5p hsa-miR-28-5p, hsa-miR-3139, hsa-miR-708-5p
SFVagm -S3-5p hsa-miR-4739, hsa-miR-4756-5p, hsa-miR-1321
SFVagm -S4-3p hsa-miR-155-5p
SFVagm -S6-3p hsa-miR-132-3p, hsa-miR-212-3p
SFVagm -S7-5p hsa-miR-3154
BFV Riems miR-BF1-3p bta-miR-125a, bta-miR-125b, bta-miR-670
BFV Riems miR-BF1-5p bta-miR-3957
BFV Riems miR-BF2-5p bta-miR-199a-3p
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Only a few examples of the preparation of diazacarbazoles have been reported \[[@R1]--[@R5]\] and up to date no general method is available in the literature. However, these diaza analogues of carbazoles \[[@R6]\] have shown interesting biological \[[@R7]--[@R10]\] and photophysical \[[@R11]--[@R13]\] properties and have been used as ligands in catalysis \[[@R14]\].
The double palladium-catalyzed N-arylation strategy for the synthesis of carbazoles has been extensively used in the literature \[[@R15]--[@R22]\]. The methodology was further applied for the preparation of dithienopyrroles \[[@R23]\] and dibenzothienopyrroles \[[@R24]\]. One example was found in a patent concerning the synthesis of 3,6-diazacarbazole by using a palladium-catalyzed double N-arylation of 4,4'-dichloro-3,3'-bipyridine, itself obtained after a long reaction sequence \[[@R25]--[@R27]\]. In another patent, an intramolecular Buchwald--Hartwig amination \[[@R28]--[@R29]\] was used to generate a 2,7-diazacarbazole derivative in low yield \[[@R30]\].
Recently, we reported a one-step preparation of tetrahalogenated 4,4'-bipyridines **2** starting from 2,5-dihalopyridines **1** \[[@R31]\]. Herein, we report on the synthesis of functionalized 2,7-diazacarbazoles **3** through the double N-arylation of **2**. Considering compound **2a**, the C--Br over C--Cl regioselectivity was not obvious since under Suzuki conditions with 2 equiv of 4-formylbenzeneboronic acid (**4**), compound **5**, resulting from a C--Cl functionalization, was obtained as the major product from a complex mixture ([Scheme 1](#C1){ref-type="fig"}) \[[@R32]\].
{#C1}
Results and Discussion
======================
The efficiency of the double amination reaction of **2a** with 4-pentylaniline (**6a**) was investigated with several ligands in toluene at 120 °C, and in the presence of Pd~2~(dba)~3~ as the source of metal catalyst ([Scheme 2](#C2){ref-type="fig"}). Ligands such as Xantphos \[[@R33]\], biaryl monophosphines \[[@R34]\] and *t*-Bu~3~P \[[@R35]\] were tested since they were found to be very effective in the selective C-5 amination of 2-chloro-5-bromopyridines \[[@R36]--[@R37]\]. The use of the chelating Xantphos ligand (**L1**) led to a poor yield of **3a**, whereas *t*-Bu~3~P (**L2**) and JohnPhos (**L3**) afforded product **3a** with a moderate yield. The more hindered monophosphine ligands SPhos (**L4**) and XPhos (**L5**) showed good results, with an optimum yield of 61% achieved with XPhos \[[@R38]\]. The bulk of the phosphine ligand enhances the stability and the activity of the catalytic system \[[@R39]\] thus allowing the use of an elevated temperature (120 °C) necessary for the double N-arylation reaction \[[@R20]\].
{#C2}
Encouraged by these results, we further explored the scope and limitations of the Pd~2~(dba)~3~--XPhos catalyst system ([Table 1](#T1){ref-type="table"}). Under the standard conditions summarized in [Table 1](#T1){ref-type="table"} (footnote a), the double N-arylation of **2a** with various aromatic amines **6** furnished 2,7-diazacarbazoles **3** with good chemoselectivities. Moderate to good yields were generally obtained in this reaction ([Table 1](#T1){ref-type="table"}, entries 1--6). Aniline **6g** bearing an electron withdrawing trifluoromethyl group led to a low yield of **3g** ([Table 1](#T1){ref-type="table"}, entry 7) whereas no cyclized product was observed with other electron-deficient anilines **6h**--**j** and carbamate **7** ([Fig. 1](#F1){ref-type="fig"}). Other methods to react **7** with bipyridine **2a** were tested without success, including the use of Cs~2~CO~3~ in dioxane \[[@R40]\] instead of *t*-BuONa in toluene, and the CuI--DMEDA catalyzed reaction \[[@R41]\].
######
Double N-arylation of **2a** with aromatic amines **6** catalyzed by a palladium--XPhos complex.^a^
------------------------------------------- ----------- -------- -------- -----------
entry R **6** **3** yield (%)
1 C~5~H~11~ **6a** **3a** 61
2 OMe **6b** **3b** 55
3 SMe **6c** **3c** 50
4 H **6d** **3d** 61
5 Cl **6e** **3e** 56
6 F **6f** **3f** 49
7 CF~3~ **6g** **3g** 29
------------------------------------------- ----------- -------- -------- -----------
^a^General reaction conditions: **2a** (0.26 mmol), **6** (0.234 mmol), Pd~2~(dba)~3~ (0.026 mmol), XPhos (0.078 mmol), *t*-BuONa (0.78 mmol), toluene (0.2 M), 120 °C, sealed tube.
{#F1}
We then turned our attention to the preparation of functionalized diazacarbazoles **3** by means of cross-coupling reactions. Initial experiments with 2,7-diazacarbazole **3a** showed a rather low reactivity of the C--Cl bonds in the 3- and 6-positions. Indeed, the Stille coupling \[[@R42]\] of **3a** with 2-tributylstannylpyridine (**8**) afforded a mixture of mono- and di-functionalized compounds **9a** and **9b** in 40% and 33% yields, respectively. The Suzuki coupling \[[@R43]\] of **3a** with boronic acid **4** was less efficient giving only the mono-functionalized compound **10** in 42% yield ([Scheme 3](#C3){ref-type="fig"}) while no conversion was observed with 4-methylthiobenzene boronic acid.
{#C3}
Better results were obtained by means of a reverse sequence. First, the C-2- and C-2'-positions of bipyridine **2b** were functionalized by a selective Suzuki coupling to give the new bipyridine derivatives **11a**,**b**, which underwent the double N-arylation to afford 3,6-difunctionalized 2,7-diazacarbazoles **12a**--**c** in good overall yields ([Scheme 4](#C4){ref-type="fig"}).
{#C4}
Single crystals of compounds **3b** and **12c** were obtained from evaporation of *n*-hexane and chloroform solutions, respectively. Whereas **3b** crystallizes with one molecule per asymmetric unit in space group *C*2/*c*, the more functionalized **12c** diazacarbazole is described by the less symmetric *P*−1 space group with two crystallographically independent molecules ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}). In both compounds the diazacarbazole moieties are almost, but not rigorously, planar, as evidenced by the dihedral angles between the pyridyl rings (2.86(9)° for **3b**; 2.09(14)° and 3.15(14)° for **12c**). Due to steric hindrance, the methoxyphenyl groups are twisted from the diazacarbazole mean plane (dihedral angles of 50.90(4)° for **3b**, 59.00(6)° and 48.45(5)° for **12c**). On the opposite, the methylsulfanylphenyl groups in **12c** are almost coplanar with the diazacarbazole moiety (dihedral angles of 2.17(15)°/13.92(11)° and 8.30(14)°/13.86(11)°).
In both compounds the crystal packing is governed by π--π stacking, forming infinite columns in which molecules interact through diazacarbazole moieties (interplanar distances are 3.558 Å for **3b**, 3.270 Å and 3.404 Å for **12c**). The methoxyphenyl group is also involved in this π--π interaction in **3b** (interplanar distance of 3.415 Å) but not in **12c**: In this latter crystal structure, it contributes to the cohesion of the molecular columns through C--H···π hydrogen bonds (H···π = 2.83 Å) ([Fig. 2](#F2){ref-type="fig"}). However, in **12c** the methylsulfanylphenyl groups are implied in the π--π stacking (interplanar distances of 3.261 Å and 3.549 Å). Such neighboring molecular columns interact through C--H···π,N,O,Cl (**3b**) or C--H···π,N,O,S (**12c**) hydrogen bonds and form infinite channels parallel to the \[[@R10]\] and \[100\] directions for **3b** and **12c**, respectively ([Fig. 2](#F2){ref-type="fig"}). The free-diameter apertures of these channels are 5.39 Å × 3.52 Å and 4.87 Å × 2.64 Å for **3b** and **12c** respectively (i.e., van der Waals radii taken into account), large enough to accommodate *n*-hexane and chloroform molecules, which are disordered.
![**(**a) ORTEP views showing the π--π (dashed lines) and selected C--H···π (dotted-dashed line) interactions in **3b** (up) and **12c** (down). Atomic displacement ellipsoids are drawn at the 50% level of probability; all other hydrogen atoms are omitted for clarity. Disordered solvent molecules are not shown. (b) Space-filling views showing the channels along \[010\] in **3b** (up) and \[100\] in **12c** (down). Disordered solvent molecules occupying these channels are not shown.](Beilstein_J_Org_Chem-08-253-g003){#F2}
Conclusion
==========
In summary, we have described an efficient approach for the synthesis of uncommon 2,7-diazacarbazoles from readily available tetrahalogenated 4,4'-bipyridines. By use of the palladium-catalyzed double N-arylation of electron-rich anilines as the key reaction, the diazacarbazoles were regioselectively generated. Crystal structure determination shows that these molecules interact mainly through π--π stacking. The reported synthesis should widen the use of diazacarbazoles for biological and electronic applications; the easy insertion of substituents of different sizes is expected to influence the dominating molecular π--π stacking, which in turn may influence the solid-state properties of the prepared material \[[@R44]--[@R45]\].
Experimental
============
All reactions were performed under an atmosphere of argon in oven-dried glassware. Toluene was distilled over sodium/benzophenone and stored over sodium. Melting points were measured on a Totoli apparatus. Proton and carbon NMR spectra were recorded on Bruker AMX-400, AC-200 or AC-250 Fourier transform spectrometers with an internal deuterium lock. Chemical shifts are quoted in parts per million (ppm) downfield of tetramethylsilane. Coupling constants *J* are quoted in Hz. Mass spectra with electronic impact (MS--EI) were recorded from a Shimadzu QP 2010 apparatus. High resolution mass spectra were recorded from a Bruker micrOTOFQ. All reagents were used as received. TLC was performed on silica gel plates and visualized with an UV lamp (254 nm). Chromatography was performed on silica gel (70--230 mesh).
**General procedure for the preparation of 3,6-dichloro-9-aryl-2,7-diazacarbazole 3a**--**g.** Argon was bubbled into a mixture of **2a** (100 mg, 0.26 mmol), amine **6** (0.234 mmol), Pd~2~(dba)~3~ (24 mg, 0.026 mmol), XPhos (37 mg, 0.078 mmol), and NaO*t*-Bu (75 mg, 0.78 mmol) in toluene (1.5 mL) for 15 min. The mixture was then heated at 120 °C in a sealed tube for 24 h. After cooling, the mixture was filtered through a pad of silica gel (dichloromethane/ethyl acetate 1/1). The filtrate was concentrated to give a residue, which was purified by column chromatography (silica gel: cyclohexane/ethyl acetate) to afford diazacarbazole **3**. The spectral and analytical data are given in [Supporting Information File 1](#SD1){ref-type="supplementary-material"}.
Supporting Information
======================
######
Characterization data and NMR spectra of all compounds, including X-ray structure determination of **3b** and **12c**.
This work was supported by CNRS and Nancy Université. The authors thank Brigitte Fernette for NMR analysis and the Service Commun de Diffraction X (Nancy Université) for providing access to crystallographic experimental facilities.
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Anterior cruciate ligament (ACL) tears are one of the most common injuries in both contact and noncontact sports.^[@bibr5-2325967114548831]^ Although the incidence of ACL tears in football and basketball players has been well identified,^[@bibr14-2325967114548831]^ there have been no studies on the incidence of ACL tears in hockey players at any level. There is currently no reported incidence of ACL tears in athletes playing for the National Hockey League (NHL) in North America. It is estimated that there will be up to 200,000 ACL tears annually in the United States,^[@bibr3-2325967114548831]^ more than half of which will be reconstructed surgically.^[@bibr7-2325967114548831]^ ACL reconstruction is the standard of care in young athletes.
Although many studies report a high success rate following an ACL reconstruction,^[@bibr9-2325967114548831]^ there are no data concerning NHL players and their return to sport (RTS) after ACL tear. Harris et al^[@bibr8-2325967114548831]^ found an 86% RTS rate in NBA players after ACL reconstruction, while Erickson et al^[@bibr6-2325967114548831]^ found a 77% RTS rate in Major League Soccer (MLS) players after ACL reconstruction. However, Ardern et al^[@bibr2-2325967114548831]^ performed a systematic review of all athletes who underwent ACL reconstruction and found that while 82% RTS, only 63% RTS at their preinjury level. McCullough et al^[@bibr10-2325967114548831]^ found similar results in high school-- and college-level football players who underwent ACL reconstruction, in that 63% and 69%, respectively, RTS, while only 43% reported they were able to return to their preinjury level. With approximately 690 active NHL players, the popularity of the NHL has been growing over the past several years. This is evidenced by record total revenue for the NHL in each season from 2007 to 2011, with the highest revenue to date, US\$2.9 billion, in 2011.^[@bibr13-2325967114548831]^
The primary purposes of this study were to determine (1) RTS rate to the NHL following ACL reconstruction, (2) performance on RTS, and (3) the difference in RTS and performance between players who underwent ACL reconstruction and controls. The authors hypothesized that NHL players who sustained an ACL tear and underwent reconstruction will have (1) a \>90% RTS rate; (2) no statistically significant difference in in-game performance parameters; and (3) no difference between age-, sex-, position-, and experience-matched control players.
Methods {#section1-2325967114548831}
=======
Thirty-nine NHL players who tore their ACLs and underwent reconstructive surgery between 1990 and 2013 were identified. These players were discovered through NHL team websites, publicly available Internet-based injury reports, player profiles/biographies, and press releases. This method of player selection has been used in multiple publications in high-level evidence orthopaedic journals.^[@bibr1-2325967114548831],[@bibr4-2325967114548831],[@bibr6-2325967114548831],[@bibr8-2325967114548831],[@bibr11-2325967114548831],[@bibr12-2325967114548831]^ The search was conducted by an orthopaedic surgery resident and a board-eligible orthopaedic surgeon in sports medicine fellowship training. All players who met the inclusion criteria were included in this study as it related to RTS rate. A player was deemed to have RTS if he played in any NHL game after surgery or if he is less than 12 months out from date of injury but is expected to RTS. A player did not RTS if he failed to meet any of the aforementioned criteria.
Only players who returned to the NHL and had played at least 1 full season were included in the pre- and postinjury in-game statistical analysis. After implementation of this criterion, 35 individual players remained whose pre- and postinjury in-game performance could be analyzed and compared ([Figure 1](#fig1-2325967114548831){ref-type="fig"}). Of these 35 players, 1 player tore his reconstructed ACL 4 years after surgery and then returned the season after revision ACL reconstruction. Players were excluded if they tore both cruciate ligaments or if they tore 2 or more major knee ligaments (ACL, posterior cruciate ligament, medial collateral ligament, and lateral collateral ligament). No player was excluded based on concomitant injuries. Therefore, the total number of ACL tears analyzed in this portion of the study was 36.
{#fig1-2325967114548831}
Demographic data (age at injury, body mass index \[BMI\], position, months from injury to return to play, side of injury, period of the game in which the injury occurred, years played before and after the injury, and whether the player returned to the NHL the season after the injury) as well as average in-game performance data per season (games played, goals, assists, points, +/-- goal differential, penalty minutes, even-strength goals, power play goals, short-handed goals, shots, shooting percentage, total time on ice, and average time on ice per game) were collected and analyzed. In-game performance variables were analyzed as an average over the pre- and postinjury course of the players' careers. In addition, in-game performance variables were analyzed separately in each of the first 5 subsequent seasons the player returned to the NHL following the injury.
A control group was selected to compare the data with the case (ACL reconstruction) group. Controls were matched to cases based on sex, age, BMI, years of experience in the NHL, position, and all measured performance variables ([Table 1](#table1-2325967114548831){ref-type="table"}). ACL reconstruction occurred at a mean of 6.61 ± 4.18 years into a player's career. An "index year" was designated for controls, analogous to ACL reconstruction year in cases, as a matched reference year (relative to years experience in NHL) to compare post-ACL reconstruction or post--index year performance. Hence, if a player tore his ACL 4 years into his career, a control was selected with an index year of 4 years into his career. Demographic data (BMI, age, years in the NHL) and in-game performance data were collected and analyzed over the course of their careers before and after the index year (each season analyzed individually and collectively).
######
Comparison of Cases and Controls Before Anterior Cruciate Ligament Reconstruction (Index Year in Controls)*^a^*
Cases Controls *P* Value*^b^* 95% CI*^c^*
---------------------------------------- --------------- ---------------- ---------------- ----------------
Age, y 27.1 ± 4.05 27.1 ± 1.55 .971 --1.5 to 1.5
Experience in NHL, y 6.61 ± 4.18 6.61 ± 4.20 .999 --2.2 to 2.2
Body mass index, kg/m^2^ 27.1 ± 1.75 27.1 ± 1.19 .924 --0.61 to 0.68
Games played per season, n 391 ± 319 364 ± 276 .696 --113 to 168
Goals per season, n 12.9 ± 10.5 8.70 ± 8.70 .068 --0.33 to 8.8
Assists per season, n 17.7 ± 12.2 12.8 ± 11.3 .081 --0.62 to 10
Points scored per season 31.1 ± 22.0 21.6 ± 20.0 .056 --0.24 to 19
+/-- goal differential per season*^d^* 1.05 ± 6.08 --0.236 ± 6.62 .415 --1.9 to 4.4
Penalty time per season, min 41.9 ± 30.4 40.7 ± 29.9 .868 --13 to 15
Even-strength goals per season, n 8.95 ± 6.09 6.88 ± 5.96 .167 --0.89 to 5.0
Power play goals per season, n 4.27 ± 3.83 2.41 ± 2.67 .026 0.23 to 3.5
Short-handed goals per season, n 0.697 ± 0.840 0.640 ± 1.58 .854 --0.56 to 0.68
Shots per season, n 120 ± 63.0 91.4 ± 58.8 .060 --1.2 to 59
Shooting percentage per season, % 10.5 ± 5.50 8.92 ± 3.84 .187 --0.77 to 3.9
Time on ice per season, min 983 ± 421 861 ± 371 .231 --79 to 323
Time on ice per game, min 17.0 ± 3.90 14.5 ± 4.27 .021 0.38 to 4.5
*^a^*Values are expressed as mean ± SD.
*^b^*Paired-samples Student *t* test with Bonferroni correction (statistical significance, *P* \< .01).
*^c^*95% CI of the difference between cases and controls.
*^d^*A measure of how the player's team did when he was on the ice. If the player has a positive number, more goals were scored by his team when he was on the ice than allowed; if he has a negative number, his team allowed more goals than they scored when he was on the ice.
Single-variable analyses for all continuous variables (performance measures) within groups were performed using paired-sample Student *t* tests. One-sample Kolmogorov-Smirnov goodness-of-fit tests for Gaussian data distribution were performed and confirmed normality of all data. Comparisons between case and control groups were made using Student *t* tests. Comparisons were made between cases and controls for survival in the NHL, each individual parameter's overall mean (all years before and all years after ACL reconstruction \[or index year\]), and each individual year (up to 4 years) following ACL reconstruction (or index year in controls). Bonferroni correction was used to determine significance in the setting of multiple comparisons. Two-proportion *Z* tests with equal variance and α = 0.05 were used to compare left- versus right-hand shooters and left- versus right-side ACL tear distributions. All statistical analysis was performed using PASW Statistics student version 18.0.0.
Results {#section2-2325967114548831}
=======
A total of 36 players (37 ACL tears) with ACL tears who underwent reconstruction between 1990 and 2013 met the inclusion criteria and were analyzed ([Figure 1](#fig1-2325967114548831){ref-type="fig"}). Thirty-five players (36 knees) were able to RTS in the NHL (97% rate of RTS). However, the 1 player unable to return to NHL was able to successfully return to professional international hockey in the Kontinental Hockey League. Players returned to the NHL at a mean of 7.8 ± 2.4 months following ACL tear. Length of career in the NHL following ACL reconstruction up to and including the 2012-2013 season was 4.47 ± 3.33 years. One player of 36 underwent revision ACL reconstruction. Nineteen of these players (53%) remained active in the NHL. There were significantly more players in the NHL at years 2 and 3 after ACL reconstruction (vs index year in controls) ([Figure 2](#fig2-2325967114548831){ref-type="fig"}). Left-handed shooters made up 68% of cases with ACL tears (*P* = .002; *Z* = 3.1), and 55% of ACL tears occurred in the right knee (*P* = .39; *Z* = 0.86). ACL tears by year are listed in [Figure 3](#fig3-2325967114548831){ref-type="fig"}.
{#fig2-2325967114548831}
{#fig3-2325967114548831}
Prior to ACL reconstruction (or index year in controls), players who tore their ACLs and controls had no statistically significant differences in any demographic or performance variable ([Table 1](#table1-2325967114548831){ref-type="table"}). After ACL reconstruction, NHL players who tore their ACLs performed significantly better (vs controls) in mean goals and points per season, mean power play and even-strength goals per season, and mean shots and shooting percentage per season ([Table 2](#table2-2325967114548831){ref-type="table"}). Compared with preinjury, there were no significant differences in any performance measure in players who underwent ACL reconstruction ([Table 3](#table3-2325967114548831){ref-type="table"}). Following the index year, controls played fewer games after their index season ([Table 4](#table4-2325967114548831){ref-type="table"}). In years 3 and 4 after ACL reconstruction in cases (or index year in controls), the cases scored significantly more goals and had significantly more points than controls.
######
Comparison of Cases and Controls After Anterior Cruciate Ligament Reconstruction (Index Year in Controls)*^a^*
Cases Controls *P* Value*^b^* 95% CI*^c^*
---------------------------------------- ---------------- ---------------- ---------------- ----------------
Games played per season, n 256 ± 234 217 ± 142 .473 --68 to 145
Goals per season, n 13.9 ± 13.2 6.82 ± 7.00 ***.009*** 1.8 to 12
Assists per season, n 18.1 ± 12.6 11.7 ± 9.47 .035 0.47 to 12
Points scored per season 32.5 ± 25.1 18.3 ± 15.7 ***.009*** 3.7 to 25
+/-- goal differential per season*^d^* --0.265 ± 8.75 --0.815 ± 4.59 .761 --3.1 to 4.2
Penalty time per season, min 41.0 ± 29.7 36.6 ± 24.8 .550 --10 to 19
Even-strength goals per season, n 10.3 ± 8.84 5.20 ± 4.82 ***.007*** 1.5 to 8.8
Power play goals per season, n 4.43 ± 4.23 1.84 ± 2.14 ***.004*** .86 to 4.3
Short-handed goals per season, n 0.487 ± 0.789 0.235 ± 0.308 .104 --0.05 to 0.56
Shots per season, n 138 ± 75.5 87.1 ± 52.0 ***.007*** 14 to 87
Shooting percentage per season, % 9.94 ± 4.64 6.96 ± 3.35 ***.007*** 0.84 to 5.1
Time on ice per season, min 1172 ± 437 860 ± 359 ***.007*** 89 to 535
Time on ice per game, min 17.5 ± 4.11 14.6 ± 4.37 .014 0.63 to 5.3
*^a^*Values are expressed as mean ± SD. Values in bold and italics indicate statistically significant differences between groups.
*^b^*Paired-samples Student *t* test with Bonferroni correction (statistical significance, *P* \< .01).
*^c^*95% CI of the difference between cases and controls.
*^d^*A measure of how the player's team did when he was on the ice. If the player has a positive number, more goals were scored by his team when he was on the ice than allowed; if he has a negative number, his team allowed more goals than they scored when he was on the ice.
######
Performance Comparison Before and After Anterior Cruciate Ligament Reconstruction (Cases)*^a^*
Before After *P* Value*^b^* 95% CI*^c^*
---------------------------------------- --------------- ---------------- ---------------- ----------------
Goals per season, n 12.9 ± 10.5 13.9 ± 13.2 .733 --6.6 to 4.6
Assists per season, n 17.7 ± 12.2 18.1 ± 12.6 .904 --6.2 to 5.5
Points scored per season 31.1 ± 22.0 32.5 ± 25.1 .805 --12 to 9.7
+/-- goal differential per season*^d^* 1.05 ± 6.08 --0.265 ± 8.75 .482 --2.4 to 5.0
Penalty time per season, min 41.9 ± 30.4 41.0 ± 29.7 .899 --13 to 15
Even-strength goals per season, n 8.95 ± 6.09 10.3 ± 8.84 .459 --5.1 to 2.3
Power play goals per season, n 4.27 ± 3.83 4.43 ± 4.23 .875 --2.1 to 1.8
Short-handed goals per season, n 0.697 ± 0.840 0.487 ± 0.789 .298 --0.19 to 0.61
Shots per season, n 120 ± 63.0 138 ± 75.5 .300 --52 to 16
Shooting percentage per season, % 10.5 ± 5.50 9.94 ± 4.64 .667 --2.0 to 3.0
Time on ice per season, min 983 ± 421 1172 ± 437 .088 --407 to 29
Time on ice per game, min 17.0 ± 3.90 17.5 ± 4.11 .599 --2.6 to 1.5
*^a^*Values are expressed as mean ± SD.
*^b^*Paired-samples Student *t* test with Bonferroni correction (statistical significance, *P* \< .01).
*^c^*95% CI of the difference between before and after values.
*^d^*A measure of how the player's team did when he was on the ice. If the player has a positive number, more goals were scored by his team when he was on the ice than allowed; if he has a negative number, his team allowed more goals than they scored when he was on the ice.
######
Performance Comparison Before and After Index Season (Controls)*^a^*
Before After *P* Value*^b^* 95% CI*^c^*
---------------------------------------- ---------------- ---------------- ---------------- ---------------
Mean games played per season 364 ± 276 217 ± 142 ***.009*** 38 to 255
Goals per season, n 8.70 ± 8.70 6.82 ± 7.00 .374 --2.3 to 6.1
Assists per season, n 12.8 ± 11.3 11.7 ± 9.47 .673 --4.3 to 6.7
Points scored per season 21.6 ± 19.6 18.3 ± 15.7 .497 --6.2 to 13
+/-- goal differential per season*^d^* --0.236 ± 6.62 --0.815 ± 4.59 .718 --2.6 to 3.8
Penalty time per season, min 40.7 ± 29.9 36.6 ± 24.8 .577 --10 to 19
Even-strength goals per season, n 6.88 ± 5.96 5.20 ± 4.82 .269 --1.3 to 4.7
Power play goals per season, n 2.41 ± 2.67 1.84 ± 2.14 .400 --0.78 to 1.9
Short-handed goals per season, n 0.640 ± 1.58 0.235 ± 0.308 .231 --0.26 to 1.1
Shots per season, n 91.4 ± 58.8 87.1 ± 52.0 .782 --26 to 35
Shooting percentage per season, % 8.92 ± 3.84 6.96 ± 3.35 .053 --0.03 to 3.9
Time on ice per season, min 861 ± 371 860 ± 359 .995 --200 to 201
Time on ice per game, min 14.5 ± 4.27 14.6 ± 4.37 .966 --2.4 to 2.3
*^a^*Values are expressed as mean ± SD. Values in bold and italics indicate statistically significant differences between groups.
*^b^*Paired-samples Student *t* test with Bonferroni correction (statistical significance, *P* \< .01).
*^c^*95% CI of the difference between before and after values.
*^d^*A measure of how the player's team did when he was on the ice. If the player has a positive number, more goals were scored by his team when he was on the ice than allowed; if he has a negative number, his team allowed more goals than they scored when he was on the ice.
Discussion {#section3-2325967114548831}
==========
The study hypotheses were confirmed, as there was a 97% rate of RTS in NHL players, with no statistically significant difference in in-game performance parameters from preinjury to postsurgery and improvement in several in-game performance measures compared with controls.
Two separate case-control studies performed at the authors' institution^[@bibr6-2325967114548831],[@bibr9-2325967114548831]^ examined RTS and performance in 64 National Basketball Association (NBA) players and 52 MLS players. Those studies demonstrated a lower return to high-level sports (86% to the NBA and all but 1 player \[98%\] returned to any level of competitive professional basketball, while there was a 77% rate of RTS to MLS) than the current study (97% RTS in NHL). It is unclear exactly what factors play a role in the ability or inability of a professional athlete to RTS after an ACL reconstruction. The demands of each sport, as well as the forces seen by the knees, vary, and because of this, future kinetic and kinematic studies comparing the stresses placed on the knee during various professional sports are necessary to determine if there is a way to improve the RTS rate of these athletes.
The current study demonstrated a revision rate of only 2.5% (1/37 ACL reconstructions needed to be revised), while the NBA and MLS studies demonstrated revision rates of 3.1% and 10%, respectively. This is an interesting finding, as many ACL tears in NHL athletes are likely contact injuries because there is no significant jumping involved in hockey and players cleats do not get caught in the ground (in MLS and the NBA, these injuries are often noncontact). This mechanism of injury may predispose these athletes to higher revision rates on RTS, as ACL reconstructions can more reliably withstand contact injuries than noncontact injuries. This begs the question of whether improved proprioceptive training could lead to a decrease in these revision rates in noncontact injuries of MLS and the NBA.
The NBA study showed a significant decline in games played per season versus controls following ACL reconstruction (or index year in controls), while the current study showed no difference in games played between cases and controls. The MLS study, like the current study, did not demonstrate a decline in any in-game performance parameter. Thus, among players in the NBA, MLS, and NHL, those who sustain an ACL tear in the NHL perform the best and have the highest RTS rate after reconstruction. This is a very significant finding for current and future professional NHL players, as an ACL tear does not signify the end of a player's career, and players can expect to return to their preinjury level of competition. Interestingly, there seemed to be a trend toward an increase in the number of ACL tears in NHL players between 2007 and 2010, although this did not reach statistical significance. As there were no rule changes or other explanations that could have accounted for this spike, it is likely due to chance.
Unlike ACL tears in elite football, soccer, and basketball players, which have been extensively studied,^[@bibr6-2325967114548831],[@bibr9-2325967114548831],[@bibr10-2325967114548831]^ the authors could find no studies to date that have looked at the RTS rate or the performance on RTS in the NHL or other elite-level hockey players. Furthermore, there have not been any studies that evaluated the risk of ACL tear in NHL players. The current study identified 40 ACL tears over a 13-year period, giving an incidence of roughly 3 ACL tears per year in NHL players.
The NHL players reviewed in this study who underwent ACL reconstruction did not see either a decline or improvement in their performance after surgery. This is a reassuring finding as surgeons would not expect to find an improvement in performance after surgery but rather hope to prevent a decline in performance. Also, despite the fact that the NHL players who underwent ACL reconstruction performed better than the control group in some performance variables after surgery, this statistic may be construed as players who have ACL reconstruction do better than those who do not. The players who had ACL reconstruction may have been more elite athletes to begin with and had not had a chance to fully develop prior to their injury. Also, in their recovery period after surgery, they are spared the rigors of playing in game situations, and so when they did return from their injury, their bodies were rested and primed for competition.
A unique finding in the current investigation is that a greater number (68%) of ACL tears in NHL players occurred in left-handed shooters (*P* = .002), while the number of tears in right versus left knees did not significantly differ (55% vs 45%; *P* = .39). In the previously reported NBA player case-control study, the side distribution (right vs left) was nearly identical to the current study (51% vs 49%, respectively), while that of the MLS was significantly different, with 68% of tears occurring in the left knee. The exact etiology of this significant increase in tears in left-handed shooters is not clear at this time. It may take origin in shooting mechanics, but without a biomechanical study to verify this, this is simply an observation.
Limitations {#section4-2325967114548831}
-----------
The strengths of this study include its case-matched control comparative design and use of sport-specific performance parameters. Limitations include the use of publicly available data, which can be subject to observer bias because of anticipated media and/or public scrutiny. There is the possibility that some cases were missed and that these players did not do well after their ACL reconstructions, thereby leading to a falsely high RTS rate. However, the opposite is also true, in which players who had a successful ACL reconstruction were missed but went on to play well, which would have falsely lowered the RTS rate. Nevertheless, this method of subject selection has been utilized in multiple high--evidence level studies in sports medicine journals.^[@bibr1-2325967114548831],[@bibr4-2325967114548831],[@bibr6-2325967114548831],[@bibr9-2325967114548831],[@bibr11-2325967114548831],[@bibr12-2325967114548831]^ Another limitation was that no subject patient-reported or clinician-measured outcomes were available; this includes general health (eg, Short Form--36) and joint-specific (eg, Knee Society Score \[KSS\]). Furthermore, postinjury patient satisfaction and personal perception of knee pain, function, and stability versus preinjury were unable to be assessed. These factors may influence RTS performance. Surgical technique information was unobtainable from publicly available sources, although it can be inferred by the treating surgeon based on preferences expressed in publications and/or presentations. Because of the high-profile nature of these athletes, they are unable to be easily contacted to assess these outcome measures. Additionally, the rehabilitation program utilized postoperatively was unavailable. Even though only 1 player was unable to return to play in the NHL following ACL reconstruction, the inability to return cannot solely be attributed to ACL injury and surgery, as other confounders inevitably coexist. Furthermore, although all performance-based measures were used to compare pre- and postoperative outcomes and between cases and controls, other intangibles (team leadership, teammate motivation, etc) cannot be assessed for relationships.
Conclusion {#section5-2325967114548831}
==========
There is a high RTS rate in the NHL after ACL reconstruction. All players who RTS did so the season following surgery. Performance following ACL reconstruction was not significantly different from preinjury. Cases performed better than controls in several performance measures.
One or more of the authors has declared the following potential conflict of interest or source of funding: B.J.C. is a consultant for Zimmer, Arthrex, Carticept, Biomimetic, Allosource, and DePuy; receives speaking fees from Genzyme; receives royalties from Arthex, DJO, and Elsevier; and receives research funding from Regentis, Arthrex, Smith & Nephew, DJO, Zimmer, DePuy, and Johnson & Johnson. B.R.B. receives publishing royalties from Slack Inc. N.N.V. is a consultant for Arthrex and Smith & Nephew; receives speaking fees from Arthrosurface; receives royalties from Smith & Nephew, Vindico Medical-Orthpedics Hyperguide, and *Arthroscopy*; and holds stock in Omeros.
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Introduction
============
Suicide is the second leading cause of death in youth (age 10-24 years) worldwide.[@B01] Brazil has relatively low suicide rates among young people, ranking 93rd out of 195 countries and territories covered by the Global Burden Study 2017 (GBD 2017).[@B02] Nevertheless, suicide is the third leading cause of death among Brazilian youth.[@B03] Furthermore, data from the Brazilian Ministry of Health indicate a 10.5% increase in the suicide rate reported between 2003 and 2013 among individuals aged 9 to 19 years.[@B03]
Previous reviews reported gaps in the current state of global scientific research on suicide-related phenomena, including the time course of suicidal ideation and behavior and the low incorporation of neurobiological measures.[@B04],[@B05] Moreover, risk factors identified by research are still poor predictors of suicide, which points to an urgent need to establish new predictors, as well as to create and validate new algorithms to track behavior more clearly in different populations, including children and adolescents.[@B06] Although a growing body of evidence supports treatments and prevention strategies, no single intervention is clearly superior to others.[@B07]
Worldwide, reported rates of suicide are probably underestimated due to misclassification of deaths.[@B01] In Brazil, reporting of suicide attempts through the official Sistema de Informações de Agravos de Notificação (SINAN) became compulsory only in 2014. The first official report on suicide attempts, published in 2017, revealed gaps in reporting of self-harm in some cities, as well as inadequate and insufficient reporting by health professionals.[@B08] Moreover, there are no nationwide official statistics about other suicidal phenomena, such as ideation and planning. Thus, it is fundamental to understand how the scientific community has studied suicide among children and adolescents in Brazil in order to identify gaps in the current knowledge and priorities for future research.
The aim of this study is to systematically review and describe the scientific output on suicide in children and adolescents in Brazil, as well as to identify gaps and strengths in this literature.
Methods
=======
Literature search
-----------------
This review followed the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)[@B09] statement. We searched PubMed/MEDLINE from the date of database inception to December 31, 2017. No restriction was placed on date or language for the search query. To identify studies of interest to this review, we used the search strategy provided in Appendix A1 (available as online-only supplementary material). Further details are reported in [Figure 1](#f01){ref-type="fig"}.
Records were reviewed independently by JP and PM and selected when both investigators agreed that the abstract met the inclusion criteria. If the abstract did not provide enough information to support this decision, the full text was screened. When the inclusion criteria were unclear even after full-text analysis, we contacted the authors for more information. Disagreements were discussed with a third author (CK).
Inclusion criteria
------------------
The inclusion criteria were:
1. Sample including children (age 0 to 9 years) and/or adolescents (age 10-19 years -- cutoff were based on the World Health Organization (WHO) classification[@B10]);
2. At least one term referring to self-harm and/or suicide;
3. Five or more Brazilian children and/or adolescents in the sample.
Data extraction and classification criteria
-------------------------------------------
Data were extracted from full-text articles independently by JP and PM. Incomplete information and disagreements were addressed as for inclusion criteria analysis. Studies were classified according to the criteria described in [Figure 1](#f01){ref-type="fig"}. Further details about data extraction and classification of studies are reported in Tables S1 to S3, available as online-only supplementary material.
Results
=======
The search strategy retrieved 1,061 records. Of those, 166 (15.6%) were selected after title and abstract screening for eligibility. A further 20 studies were excluded at the full-text screening stage. Reasons for exclusion at this step are shown in [Figure 1](#f01){ref-type="fig"}. The 146 (13.8%) included studies were published between 1966 and 2017 (47.3% published since 2010). Only three (2.1%) of the included studies were published in a journal with an impact factor higher than 5, and 60% had 10 or fewer citations according to the 2017 Journal Citation Reports (JCR). The highest number of citations recorded was 168, for Devries et al.[@B11] The list of reviewed references is available in Appendix A2 as online-only supplementary material.
Most publications (n=134, 90.4%) were original articles; a minority consisted of reviews and letters/editorials/commentaries (n=6, 4.8% each). All original articles were classified as observational epidemiological studies. The most commonly reported study design was ecological (n=66, 45.2%), followed by cross-sectional (n=44, 30.1%). There were no interventional studies among the 146 publications included. Eighty-two articles (56.1%) used secondary data obtained from medical records and/or official databases such as poison control centers (Centro de Informações Toxicológicas \[CIT\]), state medical examiner's offices (Instituto Médico Legal \[IML\]), epidemiological surveillance, and the DATASUS Mortality Information System (Sistema de Informação sobre Mortalidade \[SIM\]). Of the 52 (35.6%) articles that used primary data, 18 (12.3%) evaluated prevalence of suicidal behaviors in population-based samples ([Table 1](#t01){ref-type="table"}) and 34 (23.3%) used selected clinical samples, such as outpatient samples of depressed adolescents.
Regarding suicide spectrum phenomena reported by the original articles, 65 (48.5%) assessed suicide attempt, 27 (20.1%) assessed suicide ideation, and 11 (8.2%) assessed suicide plans. Nine studies (6.7%) reported suicide risk as the outcome, and 70 studies (52.2%) reported data related to death by suicide.
Only 99 studies (67.8%) reported data extraction specific to our age group of interest. Fourteen studies (9.5%) had samples consisting exclusively of children and adolescents. Twenty-two studies (15.1%) included only adolescents, and only one was limited to children. In terms of specific populations, five studies (2.73%) analyzed solely indigenous community samples, one focused on subjects with substance use disorders, and 17 (11.6%) studied females exclusively, of which 11 (7.5%) were limited to pregnancy.
Regarding the geographic distribution of studies (as expressed by the corresponding author's state of residence as a surrogate), the state of São Paulo corresponded to a plurality of studies (31.5%), followed by Rio Grande do Sul (13.7%), Rio de Janeiro (8.9%), and Minas Gerais (8.9%). When the included studies were stratified by region of Brazil, the South and Southeast accounted for 53.5% of publications; conversely, only 3.9% of studies were conducted with samples from the Northern region. Twenty-eight studies (19.9%) evaluated data from more than one region of Brazil.
Discussion
==========
This study aimed to better understand the scientific output on suicide in children and adolescents in Brazil. Our findings demonstrated that the body of evidence on this topic is still limited, despite a clear increase in publications from 2010-2017.
A large number of studies (56.1%) were found to use secondary mortality data, especially from DATASUS. Although this provides important insights for a baseline understanding of the magnitude of the problem, death by suicide is usually underreported worldwide, and other (nonfatal) suicide-related phenomena are also relevant.[@B12] Ideally, an official database on the risk and prevalence of suicidal ideation and planning would help authorities better identify individuals at risk (much like the CDC High School Youth Risk Behavior Survey[@B13]). Currently, the mental health section of the Brazilian National Survey on Student Health does not include questions about suicidality.[@B14]
Considering the relevance of the topic, it is concerning that no interventional studies were identified in our review. Previous studies in other countries[@B15],[@B16] have suggested strategies to reduce suicidal ideation and suicide attempts in young populations. Nevertheless, a prior review suggested that health care providers in low- and middle-income countries are more likely to change their clinical practice when influenced by local research.[@B17] Therefore, future research is needed to test interventional strategies suited for the Brazilian context.
It is important to note that only a portion of the studies (28.1%) were specifically designed for children and/or teenagers. In addition, 40% of the studies with adults only differentiated age ranges from age 19 years onward. The literature points to distinct lifespan suicidality trajectories for children as compared with adolescents and adults who later died by suicide.[@B18]
The geographical distribution of publications included in this review was skewed toward centralization. Approximately one-third of the studies were published in São Paulo, and more than half used data sources from the Southeast and South regions of Brazil. This contrasts with the fact that the Southeast region has the lowest suicide rate in the age range of interest (0.8/100.000).[@B03] While the North of Brazil has the highest rate (1.7/100.000),[@B03] only 3.9% of the studies were conducted in this region. Previous studies indicate that socioeconomic, cultural, and demographic variations play an important role in these differences and promote distinct risk factors for suicidality.[@B19] It has been argued that the currently known risk factors for suicide are poor predictors of outcome,[@B06] which highlights the importance of nationwide studies to elucidate which factors are associated with differences in suicide rates among regions.[@B20]
Our results should be considered in the light of their limitations. We searched only the PubMed/MEDLINE database. Although most relevant publications in the field of mental health are MEDLINE-indexed, it is possible that studies meeting our inclusion criteria are indexed only in regional databases, such as SciELO or LILACS. Furthermore, due to the limitations and heterogeneity of the reviewed studies, we could not perform statistical analyses to summarize their findings; only a qualitative description was feasible.
Although the literature on suicide in children and adolescents in Brazil has expanded in recent years, major gaps remain. There is a predominance of exploratory and descriptive studies using secondary information, the data sources are geographically concentrated in the South and Southeast regions of Brazil, and little is known about the epidemiology of suicidal spectrum phenomena. There is a complete lack of evidence regarding which strategies work to reduce self-harm or prevent suicide among youth in Brazil. Acknowledging these gaps is essential to prompting innovative and promising directions, with the ultimate goal of reducing the burden of suicide and related phenomena among children and adolescents in Brazil.
Disclosure
==========
MAC receives a research grant from Janssen unrelated to this work. The authors report no conflicts of interest.
JP and PHM receive scholarships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). CK is a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) researcher.
**How to cite this article:** Piccin J, Manfro PH, Caldieraro MA, Kieling C. The research output on child and adolescent suicide in Brazil: a systematic review of the literature. Braz J Psychiatry. 2020;42:209-213. <http://dx.doi.org/10.1590/1516-4446-2019-0497>
{#f01}
###### Results from studies on prevalence of suicidal behavior (references available as online-only supplementary material, Appendix A2)
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Study Source population Sample (age of participants) Assessment tool Prevalence estimate (%)
-------------------------------------------- ------------------------------------- ------------------------------------------------------------------------------------------- -------------------------------------------------------- ---------------------------
Anteghini 2001 Public- and private-school students 2,059 (13-17 years) Washington State Survey of Adolescent Health Behaviors Ideation: 27.1\
Attempts: 9.9
Baggio 2009, Bittencourt 2009, Vieira 2008 Students 1,170 (12-18 years)[\*](#TFN02t01){ref-type="table-fn"} GSHS Plans: 6.3I\
deation: 10.8
Barbosa 2014 Urban population sample 469 (14-20 years) MINI Suicide risk: 10.3
Botega 2005, Botega 2009, Stefanello 2008 Urban cluster sample 157 (14-29 years)[\*](#TFN02t01){ref-type="table-fn"} [†](#TFN03t01){ref-type="table-fn"} EPSIS Ideation: 16.0
Carlini-Cotrim 2000 Students 1,675 (12-18 years) Adapted YRBSS questionnaire Attempts: 7.1
Carlini-Marlatt 2003 Students 1,655 (12-18 years) Adapted YRBSS questionnaire Attempts: 7.1
Carvalho 2011 Public-school students 4,127 (14-19 years) GSHS Plans: 7.8
Castro 2011 Public-school students 699 (10-19 years)[\*](#TFN02t01){ref-type="table-fn"} "In the past year, did you attempt suicide" Attempts: 8.6
Feijo 1997 Public-school students 126 (13-20 years) DIS Ideation: 19.0\
Attempts: 8.7
Golfeto 2004 Students 2,867 (7-14 years) CDI Ideation: 24.2\
Plans: 2.0
Silva 2014 Students 2,207 (17-17 years) Instrument designed specifically for the study Ideation: 14.0\
Plans: 9.5\
Attempts: 5.9
Simioni 2017 Students 2,508 (6-14 years) DAWBA Deliberate self-harm: 2.2
Souza 2010 Urban population sample 1,039 (11-15 years) CDI Ideation: 14.1
Veras 2016 Public-school students 1,379 (10-17 years) MINI Suicide risk: 29.6\
Attempts: 6.9
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
CDI = Child Depression Inventory; DAWBA = Development and Well-Being Assessment; DIS = Diagnostic Interview Schedule; EPSIS = European Parasuicide Study Interview Schedule; GSHS = Global School-based Student Health Survey; MINI = Mini International Neuropsychiatric Interview; YRBSS = Youth Risk Behavior Surveillance System.
Sample used by two or more studies.
No stratification according to 10-to-19-year-old cutoff based on the WHO classification.
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{
"pile_set_name": "PubMed Central"
}
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Dear Editor,
We read with interest the article. Linezolid-induced optic neuropathy by Karuppannasamy *et al*.\[[@ref1]\] We congratulate the authors on highlighting a very important ocular complication due to a drug used for multi-drug resistant (MDR) tuberculosis (TB) - a condition which is becoming fairly common in our country.
We also encountered a similar case in our practice recently, and we would like to highlight the similarities and differences in the two cases.
Our patient was a 30-year-old male who was on treatment for MDR chest TB for the last 9 months with linezolid (600 mg/day), kanamycin 750 mg/day, injection streptomycin and (other drugs).
He presented with blurred vision in both eyes for the last 1 month. His vision was counting fingers at 3 m, \< N36 and 20/80, N18 in the right and left eye, respectively. Anterior segment examination showed no abnormality, and there was no relative afferent pupil defect. Fundus examination was normal. Color vision was grossly abnormal with Ishiharas charts. There was no disc edema.
He was under treatment for peripheral neuropathy for the last 2 months (surprisingly linezolid was not stopped -- even by the neurologist). He had significant gait abnormalities and sensory ataxia.
Perimetry showed generalized field loss in right eye with a prominent field loss superiorly \[[Fig. 1](#F1){ref-type="fig"}\]. Reliability was poor. Left eye perimetry showed a superior field loss also involving the center \[[Fig. 2](#F2){ref-type="fig"}\]. The field defects are very similar to the ones reported by the authors. Are we seeing a pattern to the field loss due to linezolid? Toxic neuritis usually results in central or centro cecal field defects.\[[@ref2]\] Is it possible that linezolid induced toxic neuritis produces superior field defects in addition to central or centro cecal defects?
{#F1}
{#F2}
Linezolid was stopped. The vision gradually improved to 20/80, N18 and 20/30, N8 in 3 months. Perimetry also showed dramatic improvement (as shown by the authors) but similar to their case -- superior defects remained \[Figs. [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}\].
{#F3}
{#F4}
Surprisingly -- there was a dramatic improvement in the peripheral neuropathy as well. There was a marked improvement in the gait and sensory ataxia, which is quite contrary to the reports in the literature.\[[@ref3]\]
Comparing the two cases -- both showed striking similarities in superior field defects, which improved dramatically after stopping the drug, but the superior defects persisted even at 3 months follow-up. This leads us to hypothesize that linezolid induced optic neuropathy produces superior field defects besides central ones although several more cases will have to be studied.
Our patient showed improvement in peripheral neuropathy, which is contrary to what has been reported in literature, which encourages us to state that peripheral neuropathy, just like optic neuropathy is reversible -- though at varying levels, which is not surprising as the cause of the two condition is one and the same.\[[@ref4]\]
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{
"pile_set_name": "PubMed Central"
}
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1.. Introduction {#s1}
================
Understanding the mechanisms that drive speciation remains a challenge of evolutionary research \[[@RSTB20190532C1]--[@RSTB20190532C4]\]. Recently, parapatric speciation---where incipient species are spatially separated, but still exchange migrants---has received considerable attention, in both empirical and theoretical research \[[@RSTB20190532C3],[@RSTB20190532C5]--[@RSTB20190532C7]\]. In particular, several studies have analysed the potential for the evolution of a postzygotic isolation barrier in the presence of gene flow \[[@RSTB20190532C5],[@RSTB20190532C8],[@RSTB20190532C9]\]. Whereas such barriers can easily arise in strict allopatry, even small amounts of gene flow can impede their buildup. This is due to two main problems. First, persistent gene flow acts to swamp divergent alleles between populations \[[@RSTB20190532C5]\]. Second, gene flow creates a permanent fitness cost for any genetic incompatibility that contributes to a genetic barrier owing to production of unfit hybrids \[[@RSTB20190532C9]\]. Local adaptation can be a potent mechanism to protect divergent alleles from swamping. Indeed, there are indications that at least some local adaptation is necessary for parapatric speciation \[[@RSTB20190532C5],[@RSTB20190532C10]\]. Consequently, some authors \[[@RSTB20190532C11]\] have suggested mechanisms purely based on divergent selection to explain how speciation can happen in parapatry. They assumed that each new mutation contributes to local adaptation. Barrier genes are additive without epistasis between single mutations. This corresponds to a scenario of pure ecological speciation. With such unlimited potential for ecological differentiation, evolution can easily build a genetic barrier to gene flow. However, this is not necessarily a realistic mechanism in natural populations. Whereas immigrants from a genetically closely related sister population may often have fitness deficits, they are rarely 'dead on arrival'. Especially early during divergence, environments need to be similar enough for the ancestral population to survive in both habitats. This limits the selection differential generated by local adaptation. For example, Via & West \[[@RSTB20190532C12]\] showed in pea aphids that residents have a fitness that is 3.3--20 times larger than the fitness of migrants. Furthermore, genetic barriers that are based uniquely on ecological differences can only be temporary, since they are maintained only as long as their causal environment persists. The dissociation between local adaptation and the strength of a genetic barrier to gene flow is thus key for the evolution of strong reproductive isolation and for completing the speciation process.
In this manuscript, we address when and how strong reproductive isolation can evolve between two parapatric populations with limited ecological differentiation. To this end, we first define measures that characterize the strength of a genetic barrier and compare this with the amount of ecological differentiation that is available between the two populations. We then focus on the role of epistasis and the pattern of incompatibilities among genes building the genetic barrier. Our results show that, for a broad range of conditions, the potential for ecological differentiation is indeed an upper limit for the strength of the genetic barrier that can be formed. However, we also show that this constraint can be broken and that particular patterns of strong epistasis enable the evolution of strong reproductive isolation in parapatry. A barrier of this type must involve at least three interacting loci: two interacting barrier loci and one locus that changes their genetic background. A strong genetic barrier can thus evolve parapatrically in (minimally) three steps from an undifferentiated initial state.
2.. Model {#s2}
=========
(a). General definition {#s2a}
-----------------------
We consider a migration--selection model in a continent--island framework \[[@RSTB20190532C5],[@RSTB20190532C9]\]. The model consists of two panmictic populations, one on an island and the other on a continent, each of sufficient size that we can ignore genetic drift. We consider the population genetic dynamics of the island population, which receives unidirectional migration from the continental population at (backward) rate *m* per individual and generation. In the main part of this article, we consider a three-locus model, with diallelic loci *A*, *B* and *C*. Ancestral alleles are denoted by lowercase letters and derived alleles by uppercase letters. Allele *A* (resp. *B* and *C*) has a selection coefficient *α* (resp. *β* and *γ*) compared with the ancestral allele *a* (resp. *b* and *c*). We derive extended results for models with more than three loci in the electronic supplementary material. Below and in the electronic supplementary material, for multiple loci *A*~*i*~ and *B*~*j*~, we use the following notation: allele *A*~*i*~ has a selective advantage *α*~*i*~ over allele *a*~*i*~ and its epistatic interaction with allele *B*~*j*~ is given by $\epsilon_{A_{i}B_{j}}$.
Epistasis can occur between any combination of derived alleles and is denoted by *ε*, with the involved alleles indicated as subscript. For example, *ε*~*ABC*~ denotes three-way epistasis between alleles *A*, *B* and *C*. The fitness of each haplotype (*wi*) is given in [table 1](#RSTB20190532TB1){ref-type="table"}. Table 1.Notation of frequencies *x*~*i*~ and fitness values *w*~*i*~ of the eight different haplotypes for haploid populations in the 3-locus model.haplotype*abcAbcaBcabCABcAbCaBCABCx*~*i*~*x*~1~*x*~2~*x*~3~*x*~4~*x*~5~*x*~6~*x*~7~*x*~8~*w*~*i*~0*αβγα* + *βα* + *γβ* + *γα* + *β* + *γ*+*ε*~*AB*~+ *ε*~*AC*~+ *ε*~*BC*~+ *ε*~*AB*~ + *ε*~*AC*~ + *ε*~*BC*~ + *ε*~*ABC*~
We assume that the continent is always monomorphic. When evolution happens on the continent, each substitution is assumed to be instantaneous. That is because we are only interested in the (potential) polymorphic equilibrium state on the island, where individuals from both populations meet and mix. The haplotype frequency dynamics of an arbitrary haplotype *X* on the island (e.g. *X* = *abC*) under the continuous-time weak-selection approximation is:$$\overset{˙}{x} = (w_{X} - \overline{w} - m)x + f_{R}(x) + m_{C},$$where the migration rate *m*~C~ = *m* if *X* is the continental haplotype and *m*~C~ = 0 otherwise. Here, *f*~R~(*X*) describes the change in frequency of haplotype *X* due to recombination. The detailed ordinary differential equation system with an explicit expression of the complicated function *f*~R~(*X*) is given in the electronic supplementary material (electronic supplementary material, equation (S1)). Our analytical results focus on two special cases that have been shown to capture most of the important behaviour \[[@RSTB20190532C5],[@RSTB20190532C9]\]: tight linkage (defined as the limit *r* → 0 for all recombination rates, *f*~R~(*x*) = 0) and loose linkage (defined as the limit *r* → ∞; dynamics are given in electronic supplementary material, equation (S4)). The second scenario corresponds to the assumption of linkage equilibrium between all loci, which approximately holds true when the recombination rates are much larger than the selection coefficients and migration rates \[[@RSTB20190532C5],[@RSTB20190532C9]\] (confirmed in Results, [figure 3](#RSTB20190532F3){ref-type="fig"}). We complement our analytical approach with numerical simulations for intermediate recombination rates. The *Mathematica* \[[@RSTB20190532C13]\] notebook detailing our analysis and the code for the simulations can be found at <https://gitlab.com/evoldyn/strong.ri>.
We study both haploid and diploid populations. For diploid populations, we assume that all direct effects of derived alleles are codominant \[[@RSTB20190532C5],[@RSTB20190532C9],[@RSTB20190532C14]\]. Regarding epistasis, we consider two scenarios: codominance and recessivity of the epistatic interaction. The two scenarios differ in the expression of epistasis in double and triple heterozygotes (cf. \[[@RSTB20190532C5]\]).
With the continuous-time approach employed here, all selection and migration parameters are rates. For the study of equilibria, only relative rates matter and we can thus scale all parameters by the selective advantage of the *A* allele on the island, *α* (note that we always assume *α* ≠ 0).
(b). Measures of the genetic barrier to gene flow and the maximum amount of local adaptation {#s2b}
--------------------------------------------------------------------------------------------
Our aim here is to assess scenarios in which a strong barrier to gene flow can evolve despite limited potential for (extrinsically driven) local adaptation. To this end, we need to define measures for both the barrier strength and the amount of local adaptation.
Following Bank *et al.* \[[@RSTB20190532C5]\] and Blanckaert & Hermisson \[[@RSTB20190532C9]\], we define the barrier strength as the maximum migration rate $m_{\max}$ that can be sustained while maintaining the polymorphism at the barrier loci. Note that $m_{\max}$, defined this way, is specific to a set of barrier loci in a specific genetic background. We reflect this in our notation by adding labels to $m_{\max}$ to indicate the island alleles that are maintained polymorphic. For example, consider a two-locus barrier with derived alleles *A* and *B* at the barrier loci, with allele *A* appearing on the island and *B* on the continent. To maintain both loci polymorphic, alleles *A* and *b* must persist on the island in migration--selection balance, because *aB* is the immigrating haplotype from the continent. $m_{\max}^{Ab}$ is the maximum migration rate for the maintenance of this stable equilibrium; above this value either *A* or *b* (or both) are lost. The barrier strength can also depend on the genetic background. We will include reference to this background in our notation whenever necessary by writing $m_{\max}^{Ab|c}$ or $m_{\max}^{Ab|C}$ for the strength of the (*Ab*) barrier in the background of the ancestral *c* or derived *C* allele, respectively, where either of these alleles at locus *C* is fixed on both the continent and the island. While other measures exist (e.g. introgression probability of a linked neutral allele \[[@RSTB20190532C15]\]), we focus on this measure, which assesses the maintenance of divergence at the barrier itself.
To measure local adaptation, we define two parameters that capture either the current state or the overall fitness landscape of the system. The first one, *Λ*, depends on a subset of model parameters and the time of observation; the second, *Λ*~max~, depends on the whole set of model parameters.
For any state of the population, we measure the *current amount of local adaptation* on the island, *Λ*, as the fitness advantage of the fittest segregating genotype on the island over a continental migrant. Recall that the continent is always monomorphic in our model. (With a polymorphic continent, the genotype with the largest fitness on the continent would provide the reference.) This measure is consistent with the verbal notion of local adaptation by Kawecki & Ebert \[[@RSTB20190532C16]\] and illustrated in [figure 1](#RSTB20190532F1){ref-type="fig"}. Using the two-locus barrier example mentioned above, the current amount of local adaptation, after the first mutational step, is given by $\mathit{\Lambda}_{ab}^{Ab} = \alpha$ if *A* appeared first and $\mathit{\Lambda}_{aB}^{ab} = - \beta$ if *B* appeared first. After the second mutational step, the current amount of local adaptation is given by $\mathit{\Lambda}_{aB}^{Ab} = \alpha - \beta$. Figure 1.Measures of local adaptation. We define two measures of environmental heterogeneity between the continent and the island, the 'current amount of local adaptation' and the 'maximum amount of local adaptation'. (*a*) The schematic shows an example in which six haplotypes are segregating on the island. The current amount of local adaptation of the population, $\mathit{\Lambda}_{{cont}.\,{hap}.}^{{isl}.\,{hap}.}$, corresponds to the difference in fitness, evaluated on the island, between the fittest segregating possible haplotype on the island (in blue) and the fittest possible haplotype on the continent (in magenta). (*b*) Fitness graph and fitness landscapes for a two-locus model with a DMI. The arrows correspond to the fitness comparison between the continental haplotype (base of the arrow) and island haplotype (tip of the arrow), with the number corresponding to the evolutionary step in panel (*c*). The fitness landscape shows a case in which *β* \< 0, meaning that *B* is a local adaptation to the continent. In our general model, *β* can take both positive and negative values, which means that *B* can also be beneficial on both the island and the continent. (*c*) Potential evolutionary histories leading to the formation of a genetic barrier in a two-locus model. For each possible evolutionary step, we compute the current amount of local adaptation of the island population as $\mathit{\Lambda}_{{cont}.\,{hap}.}^{{{polymorphic}\,{island}\,{alleles}}|{{fixed}\,{alleles}}}$. The magenta numbers match the ones in panel (*b*) and correspond to the fitness difference between two haplotypes. The maximum amount of local adaptation, $\mathit{\Lambda}_{\max}^{Ab}$, generated by the fitness graph given in panel (*b*), is the maximum of these values. If we use the fitness landscape depicted in panel (*b*), we obtain $\mathit{\Lambda}_{\max}^{Ab} = \alpha - \beta$. (Online version in colour.)
In addition, we define the *maximum amount of local adaptation* that can occur in the model over the course of the differentiation process that results in a given genetic barrier as *Λ*~max~. Note that *Λ*~max~ does not depend on the current state, but is a property of the full fitness landscape. It captures all states that could have occurred (i.e. that are allowed by the fitness landscape) during the adaptive process from a given ancestral state. We thus need to consider all possible evolutionary histories to determine *Λ*~max~. Using the two-locus barrier example mentioned above, the maximum amount of local adaptation, $\mathit{\Lambda}_{\max}^{Ab}$, is given by: $\mathit{\Lambda}_{\max}^{Ab} = \text{max}(\mathit{\Lambda}_{ab}^{Ab},\mathit{\Lambda}_{aB}^{ab},\mathit{\Lambda}_{aB}^{Ab})$. To match the genetic barrier notation, we will use $\mathit{\Lambda}_{\max}^{Ab|C}$ if we need to mention that the genetic barrier depends on the genetic background (here a fixed allele *C*).
From this definition, we see, in particular, that the maximum amount of local adaptation for a large barrier that includes many loci is always greater than or equal to the value of *Λ*~max~ for any smaller barrier that involves only a subset of these loci. For diploids, we consider the fitness differences between genotypes scaled by the ploidy of the individual. Using this definition allows us to maintain a consistent notation for haploid and diploid populations: for a single locus *A*, we always have $m_{\max}^{A} = \mathit{\Lambda}_{\max}^{A}$. We include a limit to local adaptation into our model by assuming that *Λ*~max~ is bounded by the ecology of the system. However, the fitness difference between the optimal island genotype and a hybrid (or any maladapted genotype) may be much larger, since these genotypes are not part of any evolutionary trajectories.
3.. Results {#s3}
===========
(a). Maximum amount of local adaptation as a limit to barrier strength {#s3a}
----------------------------------------------------------------------
If the external environment sets a limit to the amount of local adaptation, does this also imply a limit on the strength of the genetic barrier that can evolve in the presence of gene flow? We address this question by asking whether the former restricts the latter, i.e. whether the maximum amount of local adaptation during the differentiation process $\mathit{\Lambda}_{\max}$, limits the barrier strength $m_{\max}$. For simplicity, we will refer to genetic barriers as strong if $m_{\max} > \mathit{\Lambda}_{\max}$ and as weak otherwise. Indeed, we find that for many types of fitness landscapes and linkage architectures, genetic barriers can only be weak in this sense.
For a single-locus barrier in a haploid population, it is straightforward to see that *m*~max~ = *Λ*~max~ since local adaptation (direct selection against migrants) is the only mechanism that can maintain a polymorphism. This result holds independently of whether a locally adaptive allele appears on the island or whether a maladaptive allele immigrates from the continent. This result readily generalizes to the case of *n* diallelic loci in tight linkage, which acts like a single-locus model with 2^*n*^ alleles. Only two haplotypes can be maintained at equilibrium \[[@RSTB20190532C17]\]: the best one on the island (verifying electronic supplementary material, equation (S12)) and the immigrating one, regardless of epistasis. This result extends to diploid populations as long as there is no under- or overdominance. If gene flow exceeds the temporary amount of local adaptation, *m* \> *Λ*, the continental haplotype replaces the island haplotype. Since *Λ* ≤ *Λ*~max~, the maximum amount of local adaptation *Λ*~max~ is always an upper bound for the strength of the genetic barrier, *m*~max~ ≤ *Λ*~max~. For weak, but non-zero recombination *r*, this result remains valid as long as *r* is small relative to the selection coefficients and migration rates (electronic supplementary material, figure S7).
In the absence of epistasis and for multiple loosely linked loci, the temporary amount of local adaptation *Λ* is simply the sum of the selection coefficients *α*~*i*~ \> 0 of segregating island alleles relative to the immigrating continental alleles at the same locus (where island alleles can be ancestral or derived). During the differentiation process, this value is maximized in the final state when all mutations that contribute to the barrier under consideration have appeared, $\mathit{\Lambda}_{\max}^{\cdots A_{i}\cdots} = \sum\limits_{i = 1}^{n}\alpha_{i}$. By contrast, the strength of the genetic barrier maintaining all loci *A*~*i*~ polymorphic is given by the smallest selection coefficient: $m_{\max}^{\cdots A_{i}\cdots} = \min_{1 \leq i \leq n}(\alpha_{i})$. For given *Λ*~max~, this barrier is therefore maximized when all loci share the same selection strength: $\alpha_{i} = \mathit{\Lambda}_{\max}^{\cdots A_{i}\cdots}/n$. Clearly, we have *m*~max~ \< *Λ*~max~ for more than a single locus, i.e. the maximum amount of local adaptation *Λ*~max~ is again an upper bound for the strength of the genetic barrier. This result (*m*~max~ \< *Λ*~max~) readily extends to intermediate recombination rates as recombination ends up breaking the best haplotype (once formed) without any additional benefits.
Having shown that it is impossible to form a strong genetic barrier in the absence of epistasis or if all loci are in tight linkage, we now turn to the case with loose linkage and epistasis. This introduces the possibility of selection against recombinant hybrids. Since fitness differences between the optimal types and maladaptive hybrids can be much larger than the strength of local adaptation *Λ*~max~, selection is not constrained by the ecology and can potentially result in a strong barrier. For two loci and negative epistasis (i.e. a DMI), the barrier strength under a combination of local adaptation and selection against hybrids has previously been analysed by Bank *et al*. \[[@RSTB20190532C5]\]. The authors focused on the case of an allele *A* appearing on the island and *B* appearing on the continent, with negative epistasis between the two derived alleles. From their result for $m_{\max}^{Ab}$ \[[@RSTB20190532C5], eqn 11\], we can deduce the maximum strength for the corresponding genetic barrier:$$\left\{ \begin{matrix}
{\text{if~}\,\beta \leq - \alpha} & {\text{max}(m_{\max}^{Ab}) = \alpha\, < \alpha - \beta\, = \mathit{\Lambda}_{\max}^{Ab}} \\
{\text{if~}\, - \alpha \leq \beta \leq 0} & {\text{~max}(m_{\max}^{Ab}) = {{(\alpha - \beta)}^{2}/4\alpha}\, < \alpha - \beta\, = \mathit{\Lambda}_{\max}^{Ab}} \\
{\text{if~}\,\beta \geq 0} & {\text{max}(m_{\max}^{Ab}) = {\alpha/4}\, < \alpha\, = \mathit{\Lambda}_{\max}^{Ab}.} \\
\end{matrix} \right.$$
From equation ([3.1](#RSTB20190532M3x1){ref-type="disp-formula"}), it is clear that the maximum amount of local adaptation is again an upper bound for the strength of the genetic barrier.
We extend this model to allow for positive epistasis and derive the expression for *m*~max~ (given in electronic supplementary material, equation (S6)). With positive epistasis, a genetic barrier can exist only if allele *B* is deleterious on the island, and the maximum of this barrier is given by $\text{max}(m_{\max}^{Ab}) = - \beta$. We therefore always obtain *m*~max~ ≤ *Λ*~max~ = *α* − *β*. However, in contrast to the negative epistasis case, it is possible for a genetic barrier to reach $m_{\max}^{Ab} = \mathit{\Lambda}_{\max}^{Ab}$, when *A* is neutral (*α* = 0) on the island, and *B* is extremely deleterious ($\beta = - \mathit{\Lambda}_{\max}^{Ab}$) on the island when associated with allele *a* but neutral when associated with allele *A*. This corresponds to a scenario in which allele *A* compensates the deleterious effect of allele *B*. Here, immigration of *B* boosts the marginal fitness of allele *A* and therefore counteracts the swamping effect of immigration of *a*. This result also holds if the roles of *A* and *B* are reversed and if both alleles *A* and *B* appear on the island or on the continent.
For two diallelic loci, there is only a single epistasis parameter. In particular, interactions among derived alleles must be either negative or positive. This severely limits the complexity of the fitness landscape. We identify further, more complex, classes of epistasis patterns, where the maximum amount of local adaptation is an upper bound for the strength of the genetic barrier, as illustrated below for three loci and with general results presented in the electronic supplementary material. These patterns include: (1) any barrier that contains either an island allele that is not involved in positive interactions, or a continental allele that is not involved in negative interactions (see electronic supplementary material, §S2.3 and §S2.4); (2) any barrier where all derived alleles originate on the island or all on the continent (see electronic supplementary material, §S2.5 and §S2.6); (3) any barrier with only positive or only negative epistatic interactions between derived alleles (this directly follows from points 1 and 2) (electronic supplementary material, §S2.7); (4) any barrier where derived alleles on the continent and the island do not interact, or interact only through negative epistasis (see electronic supplementary material, §S2.8).
This suggests that only more complex epistasis, with a combination of positive and negative interactions, can result in a strong genetic barrier. We thus consider a diallelic three-locus model in the rest of the manuscript, which is fully parameterized with three direct selection coefficients and four epistatic parameters, allowing for complex interactions.
(b). Three-locus model and the role of cryptic epistasis in the formation of strong genetic barriers {#s3b}
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### (i). Haploid populations {#s3b1}
We first consider a case of with two pairwise epistatic interactions. First, we focus on a case with two island adaptations *A* and *C*, which appear on the island, and a continental adaptation *B*. The different possible cases illustrate the general rules above for the impossibility of a strong barrier. ---Negative pairwise epistasis between *A* and *B* and *B* and *C* cannot result in a strong barrier. Indeed, in the absence of allele *B*, we have $m_{\max}^{AC|b} = \text{min}(\alpha,\gamma)$, which is smaller than $\mathit{\Lambda}_{\max}^{AC|b} = \alpha + \gamma$. Once allele *B* is introduced on the continent, the marginal fitness of alleles *A* and *C* decreases, leading to $m_{\max}^{AbC} < m_{\max}^{AC|b}$. Since the two-locus barrier with *A* and *C* is a subset of the three-locus barrier, $\mathit{\Lambda}_{\max}^{AC|b} \leq \mathit{\Lambda}_{\max}^{AbC}$, and therefore $m_{\max}^{AbC} < \mathit{\Lambda}_{\max}^{AbC}$. The corresponding fitness graph for this case is given in [figure 2](#RSTB20190532F2){ref-type="fig"}*a*.---Similarly, pairwise positive interaction between *A* and *B* and *B* and *C* is not sufficient for a strong barrier. The genetic barrier formed by allele *A* and *C*, assuming *B* is fixed on the island, corresponds to a case described above (i.e. two non-interacting loci), therefore $m_{\max}^{AC|B} = \text{min}(\alpha + \epsilon_{AB},\gamma + \epsilon_{BC})$, while $\mathit{\Lambda}_{\max}^{AC|B} = \text{max}(\alpha + \epsilon_{AB},\gamma + \epsilon_{BC},\alpha + \epsilon_{AB} + \gamma + \epsilon_{BC}) \leq \mathit{\Lambda}_{\max}^{AbC}$. If locus *B* is polymorphic on the island, then the marginal fitness of both allele *A* and allele *C* is reduced, leading to $m_{\max}^{AbC} \leq m_{\max}^{AC|B}$ and therefore, $m_{\max}^{AbC} \leq \mathit{\Lambda}_{\max}^{AbC}$.---We now consider that one pairwise epistatic interaction is positive and the other negative: we assume that alleles *A* and *B* interact negatively and alleles *B* and *C* interact positively. In the absence of allele *C*, this corresponds to the two-locus case mentioned above and therefore $m_{\max}^{Ab|c} \leq \mathit{\Lambda}_{\max}^{Ab|c}$. If allele *C* appears on the island, it directly increases the marginal fitness of allele *B* on the island, facilitating its fixation on the island. In addition, through this effect on *B* (leading to a higher equilibrium frequency for *B*), it also indirectly and negatively affects the marginal fitness of allele *A*, facilitating its loss. As a consequence of its effect on the marginal fitness on alleles *A* and *B*, we obtain $m_{\max}^{AbC} \leq m_{\max}^{Ab|c}$ and $\mathit{\Lambda}_{\max}^{Ab|c} < \mathit{\Lambda}_{\max}^{AbC}$, since the '*Ab*\|*c*' barrier is a subcase of the 'AbC' barrier, leading to $m_{\max}^{AbC} \leq \mathit{\Lambda}_{\max}^{AbC}$.---Finally, we consider that *A* and *B* interact negatively and *A* and *C* positively. In the absence of allele *B*, the genetic barrier obtained in loose linkage is smaller than its equivalent in tight linkage since recombination breaks the association between *A* and *C*. Or in tight linkage, the genetic barrier is equal to $\mathit{\Lambda}_{\max}^{AC|b}$. Therefore, in the loose linkage case, $m_{\max}^{AC|b} < \mathit{\Lambda}_{\max}^{AC|b}$. Once the *B* mutation is introduced, the marginal fitness of allele *A* and *C* decreases owing to the direct (for *A*) and indirect (for *C*) interaction with allele *B*. We therefore obtain $m_{\max}^{AbC} < m_{\max}^{AC|b}$; a strong genetic barrier is thus impossible. Figure 2.With three loci and cryptic epistasis, the high-fitness ridge of a parapatric two-locus DMI can be turned into a deep fitness valley. (*a*) Fitness graph for a model with negative pairwise epistasis between *A* and *B*, and *B* and *C*, which does not allow for parapatric evolution of a strong genetic barrier. Red octagons correspond to low-fitness haplotypes and blue octagons to high-fitness haplotypes. (*b*) Fitness graph for a model with negative epistasis between *A* and *B* and a strongly deleterious allele *C*. Both alleles *A* and *B* can compensate for the deleterious effect of *C* but the compensation is not cumulative. This fitness landscape can allow for the parapatric evolution of a strong genetic barrier, because it contains a two-locus fitness graph with two fitness peaks isolated from each other by a deep valley, if allele *C* is fixed. (*c*) Three possible evolutionary histories and the temporary underlying fitness graphs (subgraphs of (*b*)) can lead to the formation of a fitness landscape in which the two fitness peaks (*AbC* and *aBC*) are separated by an unsurpassable fitness valley. This strong genetic barrier can evolve via single-step mutations in the presence of gene flow, owing to the existence of a high-fitness ridge that disappears through fixation of allele *C*. (Online version in colour.)
Similar arguments show that any barrier must be weak for two derived barrier alleles on the continent and one on the island (see electronic supplementary material, §S1.3). The fitness landscapes of all scenarios described so far share a crucial property ([figure 2](#RSTB20190532F2){ref-type="fig"}*a*) and electronic supplementary material, S4): the continental haplotype and the fittest island haplotype are connected by a fitness ridge (e.g. *AbC* and *aBc* in [figure 2](#RSTB20190532F2){ref-type="fig"}*a*), and all genotypes on this fitness ridge can be reconstructed from the (fittest) island and continental haplotypes by recombination (recombination of *AbC* and *aBc* in [figure 2](#RSTB20190532F2){ref-type="fig"}*a*).
We now consider a case in which a genetic barrier with two barrier loci is combined with a change in the genetic background (through a derived allele at a third locus that fixes on both the continent and the island). We assume (as above) that there is an incompatibility between an adaptation on the island at locus *A* and a continental adaptation at locus *B* (i.e. *α* \> 0, *ε*~*AB*~ \< 0 and *β* \< −*ε*~*AB*~). In addition, we assume that a mutation can occur at a third locus (the *C* locus). We assume that the derived allele *C* is deleterious in the ancestral genetic background (*γ* \< 0), but beneficial in the presence of either the *A* or the *B* allele (*ε*~*AC*~ \> 0, *ε*~*BC*~ \> 0 and *ε*~*ABC*~ ≤ 0; below we assume *ε*~*AC*~ = *ε*~*BC*~ = −*ε*~*ABC*~). If *C* originates on the continent, it can fix on both the continent and the island (electronic supplementary material, equations (S22)--(S29); see [figure 2](#RSTB20190532F2){ref-type="fig"}*c* for the three potential evolutionary histories). We then obtain a two-locus barrier (loci *A* and *B*), but the derived alleles at this barrier interact with a fixed derived allele in its genetic background. We refer to this type of interaction as 'cryptic epistasis' since it will not be detected in a study that focuses on divergent alleles between both populations. Notably, the corresponding fitness (sub-)graph, illustrated in [figure 2](#RSTB20190532F2){ref-type="fig"}*c* (last row; the whole fitness graph is illustrated in [figure 2](#RSTB20190532F2){ref-type="fig"}*b*), is characterized by the existence of two haplotypes (*AbC* and *aBC*) whose recombinants (*abC* and *ABC*) have very low fitness. Fixation of *C* thus deepens the observed fitness valley between *Ab* and *aB*.
To simplify the notation, we define *γ*′ as the effect of the mutation *C* in the background of at least one other derived allele: *γ*′ = *γ* + *ε*~*AC*~. Notably, this system is equivalent to a *C* mutation that appears on the continent, which is advantageous on the island while generating strong negative epistasis with the ancestral background *ab*, *ε*~*abC*~ = −*ε*~*AC*~. For the rest of the manuscript, we will use the alternative notation (*ε*~*abC*~ and *γ*′) as it is more convenient.
For a haploid population and loose linkage, the dynamics simplify to the classical two-locus model \[[@RSTB20190532C5]\] and are therefore identical to the diploid model (up to some reparameterization, electronic supplementary material, equation (S15)). The expression for the maximum amount of local adaptation, generated in this model, is$$\mathit{\Lambda}_{\max}^{Ab|C} = \text{max}(\alpha,{- \beta,}\,\alpha - \beta,\alpha - \beta - \gamma^{\,\prime}, - \gamma^{\,\prime}).$$
This equation has a simple form because the *abC* haplotype is deleterious and therefore no longer a potential step for evolution. When *C* is advantageous (*γ*′ \> 0) on the island (and since *α* \> 0), equation ([3.2](#RSTB20190532M3x2){ref-type="disp-formula"}) can be reduced to$$\mathit{\Lambda}_{\max}^{Ab|C} = \text{max}(\alpha,\alpha - \beta) = \mathit{\Lambda}_{\max}^{Ab|c}.$$
The maximum amount of local adaptation, which characterizes the ecological differentiation in the model, is unaffected by the new mutation; *C* modifies the genetic background of both populations but is not directly involved in the divergence process. Since we assume that the new mutation *C* fixes, its position in the genome is irrelevant for the polymorphic equilibrium state. (For conditions of fixation of allele *C* on the island, see electronic supplementary material, §S3.3.)
We investigate the impact of this change of the genetic background for two cases analytically: loci *A* and *B* are in tight linkage or in loose linkage. Our analysis is complemented with simulations for intermediate recombination rates ([figure 3](#RSTB20190532F3){ref-type="fig"}). With tight linkage, the barrier remains unchanged in comparison with the original background (at equilibrium, haplotype *abC* does not occur anyway). The barrier is therefore again limited by the maximum amount of local adaptation available, $\mathit{\Lambda}_{\max}^{Ab|C}$. With loose linkage, we calculate when the genetic barrier can exceed $\mathit{\Lambda}_{\max}^{Ab|C}$ (using the analytical expressions electronic supplementary material, equations (S5) and ([3.3](#RSTB20190532M3x3){ref-type="disp-formula"})). Strong genetic barriers can form when selection against hybrids, via the strength of epistasis, is strong enough:$$\begin{matrix}
& {m_{\max}^{Ab|C} > \mathit{\Lambda}_{\max}^{Ab|C}\,\text{if~}} \\
& {\quad\left\{ \begin{matrix}
{\beta < 0\,\text{and}\,\epsilon_{abC} < \frac{( - \epsilon_{AB}(3\alpha - 4\beta) + \alpha\beta)}{\epsilon_{AB} + 4\alpha - 3\beta}\,\text{and}\,\epsilon_{AB} < - 4\alpha + 3\beta} & & \\
& & \\
{\beta > 0\,\text{and}\,\epsilon_{abC} < \frac{\alpha(\beta - 3\epsilon_{AB})}{4\alpha + \beta + \epsilon_{AB}}\,\text{and}\,\epsilon_{AB} < - (4\alpha + \beta).} & & \\
\end{matrix} \right.} \\
\end{matrix}$$
From the previous section, we know that a strong single-locus barrier can never form. However, the existence of a single-locus (unstable) equilibrium is a necessary condition for the two-locus genetic barrier to be globally stable (electronic supplementary material, §S3.1.1). Therefore, if $m_{\max}^{Ab} > \mathit{\Lambda}_{\max}^{Ab|C}$, then the two-locus genetic barrier can only be locally stable, emphasizing the important role of selection against hybrids. When compared with the old barrier ($m_{\max}^{Ab|c}$), the genetic barrier is strengthened if −*ε*~*AB*~ \> *α*, i.e., when the incompatibility between *A* and *B* is stronger than the direct selective advantage of *A*; this is therefore a necessary condition for $m_{\max}^{Ab|C} > \mathit{\Lambda}_{\max}^{Ab|C}$. We calculate the genetic barrier numerically for an arbitrary genetic distance between *A* and *B* ([figure 3](#RSTB20190532F3){ref-type="fig"}): as soon as recombination is strong enough (as selection against hybrids depends both on their formation and their fitness effect), we recover the results from loose linkage, independently of the selective advantage of allele *B* on the island. Finally, we also investigate, in the case of loose linkage, the possibility of locus *C* becoming polymorphic instead of being always fixed for allele *C* and show that strong barriers can also form in these conditions (electronic supplementary material, figure S10). Figure 3.Strong genetic barriers form through global fixation of allele *C* if there is sufficient recombination. The relative maximum amount of local adaptation $\mathit{\Lambda}_{\max}^{Ab|C}/\alpha$ is given by the dotted red line. The graph shows the relative strength of the genetic barrier as a function of recombination for the two-locus DMI before the *C* mutation appears, $m_{\max}^{Ab|c}/\alpha$ in green, and after *C* has fixed on the island, $m_{\max}^{Ab|C}/\alpha$ in blue. Their corresponding limits for loose linkage are represented by the dashed lines, in green for $m_{\max}^{Ab|c}/\alpha$ and in blue for $m_{\max}^{Ab|C}/\alpha$. The limits for tight linkage are, for panel (*a*), $m_{\max}^{Ab|c}/\alpha = m_{\max}^{Ab|C}/\alpha = \mathit{\Lambda}_{\max}^{Ab|C}/\alpha$ (red dotted line) and, for panel (*b*), $m_{\max}^{Ab|c}/\alpha = m_{\max}^{Ab|C}/\alpha = 0$. All parameters are scaled by the direct selective advantage of allele *A*. (*a*) *B* is deleterious on the island: *β*/*α* = −0.2, *ε*~*AB*~/*α* = −10, *ε*~*abC*~/*α* = −20; (*b*) *B* is advantageous on the island: *β*/*α* = 1.2, *ε*~*AB*~/*α* = −10, *ε*~*abC*~/*α* = −20. (Online version in colour.)
Our assumptions of loose linkage and the continuous-time approximation both implicitly rely on weak selection. We therefore derive the equivalent of $m_{\max}^{Ab|C}$ in the discrete-time model, assuming that both *abC* and *ABC* are inviable haplotypes and that *A* and *B* are located on different chromosomes. The results are qualitatively similar, i.e. for a range of parameters, a genetic barrier can be stronger than the maximum amount of local adaptation (electronic supplementary material, equation (S34), figure S11). Finally, if we assume that the *abC* haplotype is inviable (*ε*~*abC*~ → −∞), the genetic barrier is given by $m_{\max}^{Ab|C}\rightarrow - ({\epsilon_{AB} + \beta})/4$. Whereas in the simple two-locus model before, the barrier strength was limited by local adaptation (which is limited), the formula here shows that the limit is now set by the hybrid fitness deficit (which is not limited).
### (ii). Diploid populations {#s3b2}
In the diploid model we assume that the direct effects of the mutations (*α*, *β*, *γ*′) are additive and that epistatic interactions (*ε*~*AB*~, *ε*~*abC*~) can be either recessive or codominant (see electronic supplementary material, §S3.2.1). Both the recessive and codominant model simplify to their equivalent dynamics presented in \[[@RSTB20190532C5]\], with the same substitutions as in the haploid model (electronic supplementary material, equation (S15)).
We have established above that the maximum amount of local adaptation, *Λ*~max~, is not a limit to the strength of a genetic barrier for haploid populations, if epistatic interactions are complex and include interactions with the genetic background. For the equivalent model in diploids, the strength of the genetic barrier exceeds the maximum amount of local adaptation when negative epistasis is strong enough (area below the line in [figure 4](#RSTB20190532F4){ref-type="fig"}). More precisely, the maximum amount of local adaptation is not a limit to the strength of the genetic barrier as long as the incompatibilities are strong and expressed in the F~1~ generation. They can be expressed either through recombination (*A* and *B* in loose linkage) or through the codominance of the interactions. When *A* and *B* are in tight linkage and epistasis is recessive, the genetic barrier is given by $m_{\max}^{Ab|C} = \alpha - \beta$ and is therefore at best equal to the maximum amount of local adaptation. Figure 4.Parameter region for strong genetic barriers in diploids. The *x*-axis corresponds to the epistasis between *A* and *B*, *ε*~*AB*~/*α*. The *y*-axis corresponds to the epistasis between *C* and the ancestral background, *ε*~*abC*~/*α*. The area below each curve (strong negative epistasis) indicates the parameter region with a strong genetic barrier, $m_{\max}^{Ab|C} > \mathit{\Lambda}_{\max}^{Ab|C}$. Each colour corresponds to a different value of *β*/*α*, ranging from locally maladapted alleles *B* for *β*/*α* = −0.5 to strongly beneficial alleles *B* on the island for *β*/*α* = 1.2. The left panel corresponds to the codominant diploid model, with the dashed lines corresponding to tight linkage (electronic supplementary material, equation (S21)) and the solid lines to loose linkage (equation ([3.4](#RSTB20190532M3x4){ref-type="disp-formula"})). The right panel corresponds to the recessive model. For the recessive model, a strong barrier cannot form if the loci are in tight linkage (no dashed lines) and the solid lines are obtained from numerical solution of the dynamical equations. In both panels, the squares correspond to results for the equivalent discrete-time model that allows for strong selection, assuming that *A* and *B* are on different chromosomes.
For the codominant model in loose linkage, we proved that a neutral continental adaptation, *β* = 0, is the easiest condition to form a barrier that exceeds $\mathit{\Lambda}_{\max}^{Ab|C}$; by 'easiest condition' we mean that it requires the least amount of negative epistasis, as it maximizes equation ([3.4](#RSTB20190532M3x4){ref-type="disp-formula"}). A neutral *B* allele does not contribute to the maximum amount of local adaptation; therefore all local adaptation can be captured by the *A* adaptation ($\alpha\rightarrow\mathit{\Lambda}_{\max}^{Ab|C}$). At the same time, if *B* is not advantageous on the island, direct selection is not acting against the maintenance of the DMI and does not reduce the strength of the genetic barrier.
For the codominant model, having *A* and *B* in tight linkage requires less epistasis to form a genetic barrier that exceeds $\mathit{\Lambda}_{\max}^{Ab|C}$ than in loose linkage. That is because selection against hybrids is expressed for both linkage architectures, but the migration cost is paid only once if *A* and *B* are in tight linkage, but twice if in loose linkage. Therefore, it is easier to form a strong barrier in tight linkage. For the recessive model, $m_{\max}^{Ab|C} > \mathit{\Lambda}_{\max}^{Ab|C}$ is possible only if there is recombination between the two loci; otherwise the incompatibilities are never expressed and selection against hybrids is inactive.
The discrete-time model is qualitatively similar to the continuous-time model as illustrated in [figure 4](#RSTB20190532F4){ref-type="fig"}*a*: the different-coloured squares correspond to the minimal conditions on the strength of epistasis to observe a genetic barrier stronger than the maximum amount of local adaptation in the discrete-time codominant model. The fact that both the continuous and discrete time model are qualitatively similar is crucial, since the formation of strong barriers requires strong epistatic interactions, for which the equivalence between continuous- and discrete-time model is no longer ensured.
4.. Discussion {#s4}
==============
We here show that interactions between three loci can be sufficient to confer strong reproductive isolation between two populations in parapatry, and the evolution of this barrier is possible in the presence of ongoing gene flow. We first establish that in the absence of epistasis or under a large number of 'simple' epistasis schemes (as described above), the amount of local adaptation between well-adapted types in both populations is a hard limit for the strength of a genetic barrier. We then describe a simple three-locus scenario in which a much stronger barrier can evolve. Crucially, the scenario relies on cryptic epistasis, i.e. epistasis between the divergent alleles and a derived background allele that fixes in both populations. In this case, a strong barrier is possible if a classical two-locus DMI is stabilized by positive epistasis of both interacting partners with such a background allele. Since the strength of the genetic barrier relies on strong selection against hybrids, this phenomenon requires sufficiently strong recombination between the interacting loci to be observable in haploid populations. In diploids, where hybrid genotypes also form without recombination, codominance of the incompatibilities and tight linkage between the loci involved in the initial DMI provide the best conditions for the evolution of strong reproductive isolation.
(a). Postzygotic reproductive isolation and ecological speciation {#s4a}
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The accumulation of genetic incompatibilities due to selection or drift is a standard mechanism to explain the evolution of reproductive isolation between two allopatric populations \[[@RSTB20190532C2]\]. In the presence of gene flow, however, each new incompatible mutation faces a fitness deficit. Theoretically, a contribution to local adaptation by each of these mutations can make up for this deficit. Indeed, it has been shown that the accumulation of locally adaptive mutations between two parapatric populations can result in genetic barriers to gene flow of arbitrary strength \[[@RSTB20190532C9],[@RSTB20190532C11]\]. Realistically, however, the maximum amount of local adaptation that is available (as a function of the differences in the external environment) between two populations will often be limited: while migrants from nearby habitats often have a fitness deficit relative to locals, they are usually not entirely lethal or infertile. Imposing such an upper bound immediately renders an upper bound for the strength of a genetic barrier. In the presence of epistasis and genetic incompatibilities, fitness deficits of hybrids may be much larger than those of migrants, opening up the potential for a stronger barrier. Nevertheless, our results show that for most models with simple epistasis, local adaptation is still a limit for the amount of gene flow that a barrier, built in parapatry, can sustain: $m_{\max} \leq \mathit{\Lambda}_{\max}$. This limit holds (1) for all one- and two-locus models, (2) for all models in which all loci are tightly linked, (3) for models with only island adaptations or deleterious continental mutations, and (4) for models with only negative epistasis between continental and island mutations.
(b). Cryptic epistasis enables the formation of a genetic barrier stronger than the maximum amount of local adaptation {#s4b}
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Conceptually, speciation in the presence of gene flow requires a fitness landscape in which (at least) two peaks are connected via a high-fitness ridge of single-step mutations. Yet, to exceed the limit imposed by the maximum amount of local adaptation, any recombinants between the peak genotypes have to be strongly deleterious. This can be achieved by what we term 'cryptic' epistasis, i.e, when the interaction with (at least) a third derived allele turns the high-fitness ridge that allowed for the evolution of an initial DMI into a fitness valley. Importantly, this third allele must fix in the population, or otherwise the high-fitness ridge is not fully interrupted.
In a minimal model, three loci are necessary to form the required underlying fitness landscape. In this landscape, the first mutational step corresponds to the establishment of initial differentiation between the two populations, which requires some local adaptation (either on the continent or on the island) at the respective locus. The second mutation generates a derived--derived incompatibility with the first adaptation (for the equivalence with other types, see \[[@RSTB20190532C5]\]). At this point, two fitness peaks correspond to the two derived haplotypes, one of which is fixed on the continent, whereas the other dominates on the island. These peaks are still connected via a high-fitness recombinant, namely the ancestral haplotype, which is always segregating owing to migration and recombination of the two derived haplotypes. Finally, a third mutation occurs on the continent; this adaptation is deleterious in the background of the ancestral haplotype, but advantageous in the presence of both previous mutations. If this third mutation fixes on both the continent and the island, recombinants between the prevalent haplotypes on the continent and the island (each of which inhabits a fitness peak) are always unfit. (Note that for simplicity, we discuss here the case when *C* appears last. However, it is only required that *C* appears after the fixation of *B* on the continent and fixes after *A* appears on the island, leading to the three possible scenarios detailed in [figure 2](#RSTB20190532F2){ref-type="fig"}.) As a consequence, the resident island genotype can now withstand much stronger gene flow than suggested by the fitness differences between the two derived haplotypes.
For a hypothetical example of cryptic epistasis, assume that mutations at loci *A* and *B* correspond to adaptations leading to specialization for the prevalent food source on island and continent, respectively. Both come with a (large) cost of catching/exploiting the other one, such that *AB* individuals are not good at catching/exploiting either. Mutation *C* makes individuals stick to a single foraging pattern, which is bad for the *ab* generalists, but good for both specialists, and may thus fix in both populations.
DMIs have been investigated mainly with respect to negative pairwise epistasis \[[@RSTB20190532C5],[@RSTB20190532C14],[@RSTB20190532C18]--[@RSTB20190532C20]\]. Here, we showed that more complex epistasis can significantly alter the potential for the evolution of reproductive isolation in parapatry. A key player in our minimal model of strong reproductive isolation is an allele that becomes fixed across both diverging populations during the course of the speciation process. The possibility that globally fixed mutations are involved in the speciation process complicates the challenge of inferring speciation genes and reconstructing the evolutionary trajectory of the incipient species. Specifically, these fixed mutations, responsible for what we term cryptic epistasis, will only be detected as diergent with a sister-clade and they will not appear in F~1~ and F~2~ hybrid viability analysis \[[@RSTB20190532C21],[@RSTB20190532C22]\]; thus their role in the speciation process may easily be overlooked.
The importance of complex (non-negative pairwise) epistatic interactions in speciation has been stressed in several studies. Fraïsse *et al.* \[[@RSTB20190532C23]\] compiled a list of studies with DMIs of higher order than pairwise interactions and, using the framework of Fisher's geometric model, showed that complex DMIs are likely to play an important role in the speciation process. In a model of secondary contact \[[@RSTB20190532C24]\], divergent gene clusters with complex incompatibilities, but without any local adaptation (neutral gene networks), can be maintained in the face of secondary gene flow. The less connected the neutral network is, the easier it is to maintain the divergence. Since all steps on the network are neutral, however, divergence can never evolve in the presence of gene flow and an allopatric phase is always necessary.
(c). Scope and limits of our model {#s4c}
----------------------------------
The results presented here were derived using an analytical framework, complemented with some numerical calculations. To do so, we used a continuous-time approximation, which has the disadvantage of having parameters that are meaningful only in relationship with each other. We confirmed that we observe a qualitatively similar pattern in a discrete time scenario, where parameters can be transposed to natural cases. Furthermore, we investigated this question under an infinite population size model. Adding genetic drift to the model is of great interest as temporal dynamics, as well as drift, may impact the final outcome. Adding drift may weaken the genetic barrier since the island population will be smaller. However, it may favour the introgression of background mutations from the continent to the island and therefore accelerate the formation of strong genetic barriers. Similarly, we focused mainly on cases of linkage equilibrium. Feder *et al*. \[[@RSTB20190532C25]\] showed that strong linkage disequilibrium between many loci may trigger a genome-wide reduction in gene flow, 'genome congealing' (sensu Turner \[[@RSTB20190532C26]--[@RSTB20190532C28]\]). It will be interesting to see how these two mechanisms may combine during the speciation process. Finally, we only observed the evolution of these strong genetic barriers when the *C* mutation fixed on the island, but could not exclude the possibility that strong barriers can evolve even if the *C* allele remains polymorphic on the island.
In our minimal model, a lot of deleterious hybrids will be generated, which comes at a cost for the island population. Co-existence of the 'island species' and the 'continental species' in this case thus relies on a sufficiently large population size on the island, such that the 'island types' are always in the majority relative to the continental migrants (electronic supplementary material, figure S8). In this case, the continental migrants suffer more from matings with the island types (since continental types will mainly produce inviable hybrid offspring). The dynamics may change if subsequent evolution of prezygotic isolation strengthens the genetic barrier without requiring any further local adaptation. Indeed, our model should provide a favourable scenario for such reinforcement \[[@RSTB20190532C29]--[@RSTB20190532C31]\]. However, even if all types avoid matings with the opposite type, the continental type may eventually still swamp the island owing to migration pressure. This would depend on the details of the assortment mechanism and may be precluded if mate choice comes at a cost.
(d). A route to parapatric speciation? {#s4d}
--------------------------------------
Hybrid incompatibilities have been proposed as an engine of speciation in allopatry, where simple accumulation of individually neutral but negatively interacting mutations 'almost necessarily' leads to a 'snowball effect' and eventual reproductive isolation \[[@RSTB20190532C18]\], a process that is impeded in the presence of any amount of gene flow \[[@RSTB20190532C5]\]. In a similar vein, the accumulation of locally adapted alleles was proposed as a natural engine of speciation in parapatry \[[@RSTB20190532C11]\]. By studying the interaction of local adaptation and hybrid incompatibilities in the presence of gene flow, our previous \[[@RSTB20190532C5],[@RSTB20190532C9]\] and current work challenges the view of parapatric speciation as a gradual and monotonous process that is mainly driven by local adaptation.
We have previously shown that some local adaptation is indeed a necessary ingredient for the evolution of a genetic barrier in the presence of gene flow \[[@RSTB20190532C5]\], and that this barrier can either grow or shrink as additional mutations appear \[[@RSTB20190532C9]\]. Here, we show that in a large class of models with simple fitness landscapes, ecological differentiation is an upper bound for the strength of a genetic barrier that can evolve in the presence of gene flow. Thus, if local adaptation is limited (which it realistically is), also the potential for the evolution of reproductive isolation in parapatry is usually limited.
Importantly, we also discovered specific fitness landscapes that combine locally adapted alleles with specific epistatic interactions, which enable the evolution of much stronger genetic barriers and even complete isolation in the presence of gene flow. Whether strong reproductive isolation between parapatric populations might indeed evolve through the combination of local adaptation and epistasis described here is thus dependent on the existence of the necessary fitness landscapes in nature. If they exist, the route to strong reproductive isolation could require only a small number of mutational steps. If such fitness landscapes do not exist, strong postzygotic reproductive isolation in the presence of gene flow may never be reached even after a very long time. An important conclusion from our work is thus a strong dependence of the feasibility of parapatric speciation on the underlying genetics, which makes it difficult to infer and predict.
Supplementary Material
======================
###### Mathematica notebook
Supplementary Material
======================
###### rapid_adaptation.nb
Supplementary Material
======================
###### Supplementary information
We thank R. Bürger, M. Servedio, C. Vogl, S. Mousset, I. Fragata, I. Höllinger and the Biomathematics Group at the University of Vienna for helpful discussion and comments on the manuscript, and the editor and the two anonymous reviewers for their valuable suggestions that have improved this manuscript.
Data accessibility {#s5}
==================
This article has no additional data.
Authors\' contributions {#s6}
=======================
A.B. and J.H. conceived the study. A.B. carried out the analysis. A.B., J.H. and C.B. wrote the initial draft and edited it. J.H. supervised the study.
Competing interests {#s7}
===================
We declare we have no competing interest.
Funding {#s8}
=======
A.B. was supported by the Marie Curie Initial Training Network INTERCROSSING. C.B. is grateful for support by EMBO Installation grant no. IG4152. A.B. and C.B. were supported by ERC Starting grant no. 804569-FIT2GO.
[^1]: One contribution of 19 to a theme issue '[Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers](http://dx.doi.org/10.1098/rstb/375/1806)'.
[^2]: Electronic supplementary material is available online at <https://doi.org/10.6084/m9.figshare.c.5018471>.
|
{
"pile_set_name": "PubMed Central"
}
|
SERPINA3K, a serine proteinase inhibitor (serpin), is expressed in the liver, kidney, pancreas, and retina ([@B1][@B2]--[@B3]). SERPINA3K specifically binds to tissue kallikrein to form a covalent complex and inhibits proteolytic activities of tissue kallikrein ([@B3]) and is believed to participate in the regulation of vasodilation and local blood flow via interactions with the kallikrein-kinin system ([@B4]). Later studies suggest that SERPINA3K has other functions independent of inhibition of tissue kallikrein. For example, SERPINA3K has been found to inhibit retinal neovascularization in ischemia-induced retinopathy, which is not dependent on its interactions with the kallikrein-kinin system ([@B5]). Further, in a diabetic rat model, SERPINA3K levels have been shown to decrease in retinas, suggesting that decreased SERPINA3K levels may contribute to diabetic retinopathy ([@B6]).
Diabetic retinopathy is one of the leading causes of blindness ([@B7]). In advanced stages of diabetic retinopathy, retinal fibrosis occurs and fibrovascular contraction can cause hemorrhages and retinal detachment ([@B7],[@B8]). Connective tissue growth factor (CTGF) is a profibrogenic factor that stimulates fibroblast proliferation, cell adhesion, and extracellular matrix production ([@B9],[@B10]). The potential role of CTGF in pathological fibrosis has been established ([@B11]), and CTGF has been suggested to be an attractive therapeutic target in some fibrotic diseases ([@B12]). The protein and mRNA levels of CTGF were found to be elevated in retinas with diabetic retinopathy ([@B13]), and the roles of CTGF in fibrovascular proliferation and thickening of capillary basement membrane were also demonstrated in proliferative diabetic retinopathy ([@B13][@B14][@B15]--[@B16]). All of these previous findings suggest a therapeutic potential for anti-CTGF therapy in diabetic retinopathy.
Wnts are a group of secreted, cysteine-rich glycoproteins ([@B17]). As shown in online appendix Figure S1 (available at <http://diabetes.diabetesjournals.org/cgi/content/full/db09-1056/DC1>), in the absence of Wnt ligands, transcription factor β-catenin, a downstream effector of the canonical Wnt pathway, is phosphorylated by a protein complex containing glycogen synthase kinase (GSK)-3 in the cytosol and constantly degraded to prevent its accumulation ([@B18],[@B19]). Upon binding of certain Wnt ligands, the Frizzled (Fz) receptor dimerizes with the coreceptor, LDL receptor--related protein (LRP) 5 or 6, forming a receptor/coreceptor complex ([@B17]). As a result, the downstream signaling is stimulated, including phosphorylation of LRP5/6 and stabilization of β-catenin ([@B20],[@B21]). β-Catenin is subsequently translocated into the nucleus, associates with T-cell factor (TCF) for DNA binding, and regulates expression of target genes including CTGF ([@B17]).
The Wnt signaling pathway is involved in multiple physiological and pathological processes. It has been well studied in embryogenesis and carcinogenesis ([@B22]). Recent evidence suggests that the Wnt pathway is also important in ocular diseases; for example, mutations in the Fz receptor and LRP coreceptor have been shown to associate with the vascular developmental defects ([@B23]). Furthermore, it has been revealed that Wnt signaling is responsible for pathological fibrosis in the lung, suggesting that inhibition of Wnt signaling, such as Wnt antagonists, may represent a therapeutic option ([@B24][@B25][@B26]--[@B27]). As a profibrogenic factor, CTGF was also found to be regulated by Wnt signaling in osteoblast differentiation ([@B28],[@B29]). However, there is little previous evidence to implicate Wnt signaling in fibrosis in the retina with diabetic retinopathy.
In the present study, we have investigated the inhibitory effect of SERPINA3K on the hyperglycemia-induced CTGF overexpression and Wnt pathway activation and further determined if the beneficial effects of SERPINA3K in diabetic retinopathy are through the Wnt antagonistic activity.
RESEARCH DESIGN AND METHODS
===========================
Cell culture.
-------------
A cell line derived from rat retinal Müller cells (rMCs) (rMC-1; a kind gift from Dr. Vijay Sarthy at Northwestern University), were cultured in Dulbecco\'s modified Eagle\'s medium (DMEM; Cellgro, Manassas, VA) containing 10% FBS (Invitrogen, Carlsbad, CA) ([@B30]). Human telomerase reverse transcriptase (HTERT)-immortalized retinal pigment epithelial (RPE) cell line (HTERT-RPE), a cell line derived from human RPE cells, was purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in DMEM containing 10% FBS following ATCC recommendations. L-cells and L-cells stably expressing Wnt3A ([l]{.smallcaps}-Wnt3a) were purchased from the ATCC and cultured in DMEM containing 10% FBS and 0.4 mg/ml G-418 (Invitrogen). The cells and conditioned media (1 g/l glucose, 1% FBS) were harvested following the procedure recommended by the ATCC. The cultured cells were starved in 1 g/l glucose (5 mmol/l) DMEM containing 1% FBS overnight before treatment. For the high-glucose treatment, the cells were exposed to 30 mmol/l [d]{.smallcaps}-glucose (Sigma, St. Louis, MO), and the low-glucose control included 5 mmol/l [d]{.smallcaps}-glucose and 25 mmol/l [l]{.smallcaps}-glucose (Sigma) in the culture medium.
Experimental animals.
---------------------
Brown Norway (BN) rats were purchased from Charles River Laboratories (Wilmington, MA). Care, use, and treatment of all animals in this study were in strict agreement with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines in the care and use of laboratory animals set forth by the University of Oklahoma.
Induction of experimental diabetes.
-----------------------------------
Experimental diabetes was induced as described previously ([@B31]). Briefly, BN rats (8 week of age) were given a single intraperitoneal injection of streptozotocin (STZ; 50 mg/kg in 10 mmol/l citrate buffer, pH 4.5) after an overnight fast. Serum glucose levels were monitored 48 h after the STZ injection and every 2 weeks thereafter, and only the animals with blood glucose levels \>350 mg/dl were used as STZ-induced diabetic rats.
Recombinant proteins, adenovirus, plasmids, transfection, and reporter Assay.
-----------------------------------------------------------------------------
The SERPINA3K cDNA was cloned into the pET28 vector (Novagen, Madison, WI), and the construct was transformed into *E. coli* strain BL-21/DE3 (Novagen). The expression and purification followed the protocol described previously ([@B5]). Endotoxin levels were measured using a limulus amebocyte kit (Biowhittaker, Walkersville, MD). BSA (Sigma) was used as protein control for SERPINA3K. Recombinant Dickkopf (DKK)-1 protein was purchased from R&D Systems (Minneapolis, MN). To clone the SERPINA3K cDNA, total RNA was extracted from the liver and was reverse transcribed to cDNA. The full-length sequence of SERPINA3K containing the signal peptide was cloned into the shuttle vector. The adenoviral vector used in the study is human adenovirus serotype 5 (Ad5). Adenoviruses expressing SERPINA3K and LacZ were generated using AdEasy systems from Qbiogene (Irvine, CA) following manufacturer\'s protocol. These adenoviruses were purified using Adeno-X Virus Purification Kits from BD Biosciences (San Jose, CA). TOPFLASH vector was constructed as described ([@B32]). Fugene 6 (Roche Applied Science, Indianapolis, IN) was used for transfection following manufacturer\'s protocol. Luciferase reporter assays were performed in 12-well plates. The TOPFLASH construct and renilla luciferase pRL-TK vector were cotransfected into the cells. TOPFLASH activity was measured using the dual-luciferase reporter system (Promega, Madison, WI) and normalized by renilla luciferase activity.
Real-time RT-PCR.
-----------------
Total RNA was isolated using an RNeasy Mini Kit (Qiagen Sciences, Germantown, MD). mRNA was reverse transcribed to cDNA using a TaqMan kit from Roche. This cDNA was then used for specific real-time PCR. The specific primers for CTGF (5′-AAGACCTGTGGGATGGGC-3′ and 5′-TGGTGCAGCCAGAAAGCTC-3′) were synthesized from Integrated DNA Technologies (San Diego, CA). To normalize the variation of the amount of mRNA in each reaction, 18S rRNA (primers: 5′-TTTGTTGGTTTTCGGAACTGA-3′ and 5′-CGTTTATGGTCGGAACTACGA-3′) was simultaneously processed in the same sample as an internal control. iQ SYBR Green Supermix from BioRad (Hercules, CA) was used for real-time PCR following the manufacturer\'s procedure.
Standard curves for CTGF primers and 18S primers were constructed using serial 1--10 dilutions of the cDNA product from RT reaction (online appendix Fig. S6). The efficiency of CTGF primers was 99.46% and the efficiency of 18S primers was 97.43%. Dilutions of 1:1,000, 1:100, and 1:10 were used in the assay, and all of the samples were diluted by 1:100 for the real-time PCR. To calculate relative changes in gene expression, we used the δ-δ method following the BioRad\'s introduction.
Western blot analysis.
----------------------
The same amounts of proteins from the cytosolic fraction, total cell lysates, and retinal homogenates were resolved by SDS-PAGE (8 or 10%) and analyzed by Western blotting using specific antibodies. For cytosolic β-catenin measurement, cells were lysed by three freeze/thaw cycles in PBS with a protease inhibitor cocktail (Roche Applied Science) followed by centrifugation, and the supernatants were isolated for Western blot analysis. For total cell lysates, harvested cells were sonicated in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA) containing 1% SDS. For retinal homogenate preparation, retinas were homogenized in PBS with a protease inhibitor cocktail using a soft-tissue pestle (Fisher Scientific, Pittsburgh, PA). Antibodies for CTGF, LRP6, and β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:400, 1:500, and 1:2,500 dilutions, respectively. Antibody for β-actin (1:3,000) was purchased from Invitrogen. Antibodies for phosphorylated LRP6 (S1490), phosphorylated β-catenin (S33/37/T41), phosphorylated GSK3β (S9), GSK3β, and histone H3 were purchased from Cell Signaling Technology and used at 1:500 dilution for the first two antibodies and 1:2,500 dilution for the last three antibodies. The monoclonal antibody for SERPINA3K (1:1,000) was generated using the recombinant SERPINA3K through contracted service with Proteintech Group (Chicago, IL).
Because of the posttranslational modifications, CTGF can display multiple bands with different molecular weights, which has been reported in the literature ([@B33]). Typically, a 36- or 38-kD band, or double bands at these molecular weights, can be detected, dependent on the tissue, cell type, and treatment. In the present study, CTGF showed double bands (36/38 kD) in the Western blotting data using the HTERT--RPE-1 cell lysate.
Enzyme-linked immunosorbent assay for fibronectin.
--------------------------------------------------
Fibronectin concentrations in the retina homogenates were measured using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Assaypro (Winfield, MO), according to manufacturer\'s instruction. The working range of the fibronectin ELISA kit used here is 4--1,000 ng/ml. All of the samples were measured in the linear part of the working range. The assay coefficients of variation are \<2%. The total protein concentration was measured by Bradford protein assay.
Statistical analysis.
---------------------
Student *t* test (two tailed) was used for comparisons between two groups. ANOVA was used for comparisons between groups in [Table 1](#T1){ref-type="table"}. Statistical significance was accepted when the *P* value was \<0.05.
######
Physiological parameters of diabetic rats
No Ad-IVT Diabetes with Ad-IVT
------------------------------ ----------- ---------------------- --------- ---------- ---------- -------
Blood glucose (mg/dl)
Time after STZ injection
−1 day 97 ± 8 99 ± 6 0.955 107 ± 11 103 ± 12 0.789
3 days --- 544 ± 55 --- 515 ± 79 528 ± 59 0.916
1 month 94 ± 8 524 ± 59 \<0.001 531 ± 66 530 ± 72 1.000
2 months 98 ± 4 521 ± 67 \<0.001 533 ± 88 517 ± 77 0.965
3 months 108 ± 22 543 ± 37 \<0.001 534 ± 61 530 ± 50 0.992
Body weight (g)
Time after STZ injection
−1 day 151 ± 13 145 ± 9 0.790 148 ± 15 150 ± 9 0.993
3 days --- 143 ± 12 --- 146 ± 15 145 ± 9 0.979
1 month 176 ± 15 146 ± 13 0.002 152 ± 17 149 ± 13 0.983
2 months 183 ± 11 147 ± 17 \<0.001 150 ± 19 151 ± 15 1.000
3 months 187 ± 16 149 ± 16 0.001 149 ± 20 152 ± 17 0.976
Data are means ± SD. *n* = 8--10.
RESULTS
=======
Clinical characteristics of diabetic animals.
---------------------------------------------
Two months after the induction of diabetes, the ATZ-induced diabetic rats received an intravitreal injection of adenovirus expressing SERPINA3K (Ad-SA3K, 5 × 10^7^ pfu/eye), and the same titer of adenovirus expressing LacZ (Ad-LacZ) was injected for control. The rats were separated into four groups: normal rats without STZ-induced diabetes (control group); rats with STZ-induced diabetes (DM group); STZ-induced diabetic rats with injection of Ad-SA3K (DM-SA3K group), and STZ-induced diabetic rats with injection of Ad-LacZ (DM-LacZ group). There were 8--10 rats in each group. Before the STZ injection, all of these rats had similar blood glucose levels (∼100 mg/dl) and similar body weights (∼150 g). Three months after STZ injection, average body weight of the DM group (149 ± 16 g) was significantly lower than in nondiabetic control group (187 ± 16 g) (*P* \< 0.05). At each time point, there was no significant blood glucose or body weight difference between the DM group, the DM-LacZ group, and the DM-SA3K group ([Table 1](#T1){ref-type="table"}), suggesting that the ocular adenovirus injection had no systemic effect in the diabetic animals.
A novel antifibrogenic activity of SERPINA3K in the retina with diabetic retinopathy.
-------------------------------------------------------------------------------------
To investigate the effect of SERPINA3K on fibrosis in the retina with diabetic retinopathy, we measured the retinal levels of fibronectin in the STZ-induced diabetic rats. Fibronectin is an extracellular matrix protein, and overproduction of fibronectin has been shown to contribute to capillary basement membrane thickening in diabetic retinopathy ([@B34]). Consistent with previous studies, our ELISA results showed that fibronectin levels were significantly higher in the retinas of the diabetic rats compared with that in nondiabetic control subjects ([Fig. 1](#F1){ref-type="fig"}). Ad-SA3K blocked retinal fibronectin overexpression in diabetic rats ([Fig. 1](#F1){ref-type="fig"}), suggesting an antifibrogenic activity of SERPINA3K.
{#F1}
Retinal endothelial cells and pericytes are two major vascular cell types that are profoundly affected in diabetic retinopathy, and their dysfunctions contribute to the blood-retinal barrier breakdown in diabetic retinopathy ([@B35][@B36]--[@B37]). Since these cells are also the sources of fibronectin production responsible for thickening of the basement membrane, we measured the concentration of fibronectin in primary retinal pericytes and endothelial cells by ELISA. The high-glucose--induced overproduction of fibronectin was attenuated by SERPINA3K in both of the cell types (Fig. S2).
Inhibitory effects of SERPINA3K on CTGF overexpression.
-------------------------------------------------------
The inhibitory effect of SERPINA3K on CTGF was evaluated in the STZ-induced diabetic rats. Four weeks after the injection of Ad-SA3K or Ad-LacZ, the retinas were dissected following a thorough perfusion to remove the blood in the retinal vasculature. As measured by Western blot analysis, retinal levels of SERPINA3K were decreased in the untreated diabetic rats, compared with that in nondiabetic rats ([Fig. 2](#F2){ref-type="fig"}*A* and *B*). The Ad-SA3K injection resulted in an increase of SERPINA3K levels in the retinas of the diabetic rats ([Fig. 2](#F2){ref-type="fig"}*A* and *B*). Retinal levels of CTGF were significantly increased in the retinas of untreated diabetic rats, compared with nondiabetic rats at the same age. The injection of Ad-SA3K mitigated the overexpression of CTGF in the retinas compared with the control Ad-LacZ virus ([Fig. 2](#F2){ref-type="fig"}*A* and *B*).
![SERPINA3K attenuated overexpression of CTGF in the retinas with diabetic retinopathy and in retinal cells treated with high glucose. *A*: STZ-induced diabetic rats (DM) received an intravitreal injection of Ad-SA3K, with Ad-LacZ as control. Four weeks after the injection, retinal levels of SERPINA3K and CTGF were measured by Western blot analysis using 100 μg of retinal proteins from each rat. *B*: Retinal levels of SERPINA3K and CTGF were quantified by densitometry from three independent experiments and normalized by β-actin levels (\**P* \< 0.05, means ± SD, *n* = 6). *C* and *D*: rMC-1 cells (*C*) and HTERT RPE-1 cells (*D*) were exposed to low-glucose (LG, 5 mmol/l [d]{.smallcaps}-glucose + 25 mmol/l [l]{.smallcaps}-glucose) and high-glucose (HG, 30 mmol/l [d]{.smallcaps}-glucose) media with various concentrations of SERPINA3K for 24 h. Cellular CTGF levels were measured by Western blot analysis and normalized by β-actin levels.](zdb0061061490002){#F2}
It has been shown that RPE cells and rMCs are two major cell types expressing CTGF in the proliferative vitreoretinopathy ([@B38]). The diabetes-induced blood-retinal barrier breakdown occurs predominantly at the level of the retinal blood vessels ([@B39]). However, the failure of the RPE barrier also occurs at an lower level, suggesting that the pathological change of the RPE is involved in diabetes ([@B39]). To evaluate the direct effect of SERPINA3K on hyperglycemia-mediated CTGF overexpression in vitro, HTERT--RPE-1 cells (human RPE cell line) and rMC-1 (rat rMC line) were exposed to media containing 30 mmol/l [d]{.smallcaps}-glucose (high glucose). CTGF expression was significantly elevated by high glucose exposure, when compared with low glucose control (5 mmol/l [d]{.smallcaps}-glucose and 25 mmol/l [l]{.smallcaps}-glucose, low glucose). SERPINA3K blocked the high-glucose--induced CTGF overexpression in a dose-dependent manner in both of the cell lines ([Fig. 2](#F2){ref-type="fig"}*C* and *D*).
To further study the mechanism for the regulation of CTGF by SERPINA3K, real-time RT-PCR was performed to measure mRNA levels of CTGF in the retinas and cultured retinal cells. The mRNA levels of CTGF were increased in retinas with diabetic retinopathy and decreased by Ad-SA3K ([Fig. 3](#F3){ref-type="fig"}*A*). Similarly, exposure to high-glucose media for 16 h significantly elevated CTGF mRNA levels in both HTERT--RPE-1 cells and rMC-1 cells. In a time course experiment, the cells were exposed to high-glucose medium for 0--24 h. The high-glucose treatment continuously increased the CTGF mRNA level after 4 h of exposure (online appendix Fig. S3). The high-glucose--induced CTGF mRNA overexpression was attenuated by 100 nmol/l SERPINA3K ([Fig. 3](#F3){ref-type="fig"}*B* and *C*). Taken together, these results indicate that SERPINA3K blocks the hyperglycemia-induced CTGF expression at the transcription level.
{#F3}
The role of the high-glucose--induced Wnt/β-catenin signaling in CTGF overexpression.
-------------------------------------------------------------------------------------
To explore the signaling pathway mediating the inhibitory effect of SERPINA3K on CTGF transcription, we first investigated cell signaling responsible for CTGF overexpression induced by high glucose. As the β-catenin--TCF/LEF-binding (TBE) site was identified in the promoter region of the CTGF gene, and CTGF has been shown to be a target gene of β-catenin-TCF/LEF transcription factor ([@B33]), a downstream effector of the canonical Wnt pathway, we investigated the role of the Wnt pathway in the CTGF overexpression in diabetes. A number of Wnt ligands and Fz receptors and both LRP5 and -6 were found to express in the cultured HTERT--RPE-1 cells by RT-PCR, suggesting that this cell line is a suitable model for Wnt signaling studies (online appendix Fig. S4). As phosphorylation of LRP6 is a critical step in the canonical Wnt pathway activation ([@B40]), we measured phosphorylated LRP6 (p-LRP6) levels. As shown by Western blot analysis using an antibody specific for p-LRP6, phosphorylation of the endogenous LRP6 was increased in the RPE cells exposed to 30 mmol/l glucose, compared with that in control cells exposed to the low-glucose medium ([Fig. 4](#F4){ref-type="fig"}*A*). In the same cell line, cytosolic β-catenin levels were also elevated by the high-glucose medium in an exposure time-dependent manner ([Fig. 4](#F4){ref-type="fig"}*A*). Further, under high-glucose conditions, phosphorylated β-catenin levels were decreased, compared with that in low-glucose control (online appendix Fig. S5*A*), while nuclear β-catenin levels were elevated (online appendix Fig. S5*B*). Consistently, GSK3β phosphorylation (inactive form) was increased (online appendix Fig. S5*A*). In vivo, cytosolic β-catenin levels in the retina homogenates were also significantly elevated in the diabetic rats, indicating an activation of the canonical Wnt pathway in retinas with diabetic retinopathy ([Fig. 4](#F4){ref-type="fig"}*C* and *D*). Further, when the Wnt pathway was blocked by DKK-1, a specific inhibitor of the canonical Wnt pathway, the high-glucose--induced CTGF overexpression was attenuated ([Fig. 4](#F4){ref-type="fig"}*B*), suggesting that the Wnt pathway activation induced by high glucose and diabetes is responsible for the CTGF overexpression.
{#F4}
Inhibitory effects of SERPINA3K on Wnt/β-catenin signaling in diabetic retinopathy and high-glucose--treated retinal cells.
---------------------------------------------------------------------------------------------------------------------------
To further investigate whether SERPINA3K inhibits the Wnt pathway in high-glucose--treated cells and in diabetic retinas, SERPINA3K was delivered into the RPE cell culture medium containing high glucose and into the vitreous humor of diabetic rats. After a 6-h exposure, SERPINA3K decreased the high-glucose--induced phosphorylation of LRP6 and accumulation of β-catenin in a concentration-dependent manner ([Fig. 5](#F5){ref-type="fig"}*A*). SERPINA3K at 100 nmol/l decreased p-LRP6 and cytosolic β-catenin to levels similar to that in the low-glucose control ([Fig. 5](#F5){ref-type="fig"}*A*). Similarly, 100 nmol/l SERPINA3K completely reversed the high-glucose--induced changes of phosphorylated β-catenin levels, nuclear β-catenin levels, and phosphorylated GSK3β levels (online appendix Fig. S5*A* and *B*). In STZ-induced diabetic rats, the injection of Ad-SA3K also blocked the accumulation of cytosolic β-catenin in the retina, compared with the control virus Ad-LacZ, suggesting an inhibitory effect of SERPINA3K on the canonical Wnt pathway in diabetes ([Fig. 5](#F5){ref-type="fig"}*B* and *C*).
{#F5}
The Wnt inhibitory effect of SERPINA3K was responsible for CTGF regulation.
---------------------------------------------------------------------------
To determine whether SERPINA3K also blocks CTGF expression induced by Wnt signaling, the RPE cells were exposed to a 50% Wnt3a conditioned medium, with the 50% L-cell medium as control. The Wnt3a-conditioned medium elevated p-LRP6 and cytosolic β-catenin levels in the RPE cells ([Fig. 6](#F6){ref-type="fig"}*A* and *B*). SERPINA3K at 100 nmol/l blocked the increase of p-LRP6 and β-catenin levels induced by Wnt3a ([Fig. 6](#F6){ref-type="fig"}*A* and *B*). TOPFLASH is a luciferase reporter construct under the control of a promoter containing the TCF/LEF-binding sites. To measure β-catenin--dependent reporter gene transcription, TOPFLASH activity assay was performed to further confirm the inhibitory effect of SERPINA3K on Wnt signaling. The RPE cells were transfected with the TOPFLASH construct and then incubated with the 50% Wnt3a-conditioned medium. TOPFLASH assay showed that Wnt3a induced an approximately threefold increase in TOPFLASH reporter (TCF/LEF) activity, which was attenuated by SERPINA3K ([Fig. 6](#F6){ref-type="fig"}*C*). These results indicated that SERPINA3K is a Wnt inhibitor.
{#F6}
To determine the effect of SERPINA3K on the Wnt-induced CTGF expression, the 50% Wnt3a-conditioned medium was used to treat the cultured RPE cells. Wnt3a, as well as high glucose, induced CTGF overexpression in the RPE cells ([Fig. 6](#F6){ref-type="fig"}*D*). Correlating with its inhibitory effect on Wnt signaling, SERPINA3K also inhibited the Wnt3a-induced CTGF upregulation, similar to that in the high-glucose model ([Fig. 6](#F6){ref-type="fig"}*D*).
DISCUSSION
==========
SERPINA3K is an extracellular serpin and has been found to function as an antiangiogenic factor ([@B5]) and an anti-inflammatory factor ([@B41]). Our previous studies showed that SERPINA3K levels are decreased in the retina of diabetic rats ([@B6]). The present study revealed a novel antifibrogenic activity of this serpin, as it inhibits CTGF overexpression and reduces the production of extracellular matrix in retina with diabetic retinopathy. These findings suggest that decreased SERPINA3K levels in the diabetic retina may contribute to pathological fibrosis in diabetic retinopathy. Further, our results demonstrate that the inhibitory effect of SERPINA3K on CTGF overexpression is through mitigating the Wnt signaling activation induced by diabetes.
Fibrosis is an important pathological feature of diabetic retinopathy ([@B7],[@B8]). At early stages of diabetic retinopathy, increased production of extracellular matrix has been shown to contribute to the capillary basement membrane thickening ([@B42]). At advanced stages of diabetic retinopathy, fibrosis can result in contraction and retinal detachment, a major cause of blindness in diabetic retinopathy ([@B7],[@B8]). The molecular mechanism for retinal fibrosis in diabetic retinopathy is not clear. CTGF is a major fibrogenic factor, and its overexpression has been found to play a key role in the basement membrane thickening in diabetic retinopathy models ([@B14],[@B16]). CTGF upregulates production of extracellular matrix proteins such as fibronectin ([@B43],[@B44]). Therefore, CTGF is considered a promising target for treating retinal fibrosis in diabetic retinopathy. The present study identified SERPINA3K as an endogenous inhibitor of CTGF and, thus, an antifibrogenic factor in the retina. On the other hand, CTGF has been reported to bind to Wnt receptor/coreceptor ([@B45]). However, the function of CTGF in canonical Wnt signaling is still controversial ([@B33],[@B45]). Despite the possible feedback regulation by CTGF, blockade of Wnt signaling by SERPINA3K should result in a decrease of CTGF levels and inhibition of fibrogenesis, which supports our conclusion (i.e., the antifibrogenic effect of SERPINA3K is mediated, at least in part, through the Wnt pathway).
Our previous studies have shown that retinal levels of SERPINA3K are decreased in STZ-induced diabetic rats after 1, 2, and 4 months of diabetes ([@B6]). In the same animal model, CTGF overexpression was found in the diabetic rat retinas 3 months after STZ injection ([@B13]), correlating with the decrease of SERPINA3K. The disturbed balance between profibrogenic factor CTGF and anti-fibrogenic factor SERPINA3K may represent a new pathogenic mechanism for the basement membrane thickening in diabetic retinopathy.
Wnt signaling is involved in ocular diseases, such as vasculature disorders in the retina ([@B23]). Our recent study showed that activation of the Wnt pathway also plays a pathogenic role in subretinal neovascularization in an animal model of wet age-related macular degeneration (AMD) ([@B46]). The role of Wnt signaling in pathological fibrosis has been revealed in some tissues (e.g., the lung) ([@B24]). However, in most ocular diseases such as diabetic retinopathy, the role of Wnt signaling, especially the association between Wnt signaling and retinal fibrosis remains obscure. Here, our results showed that cytosolic β-catenin, an essential effector of the canonical Wnt pathway, is accumulated in both diabetic retinas and in the high-glucose--treated retinal cells. Phosphorylation of LRP6 is an early, yet essential, step in activation of the canonical Wnt pathway, as the phosphorylation sites in the LRP6 intracellular domain are known to create inducible docking sites for Axin, leading to stabilization of β-catenin in the cytosol and transduction of the extracellular Wnt signal into intracellular compartments ([@B40],[@B47]). Our results showed that phosphorylated LRP6 levels were increased under high-glucose conditions. In the hyperglycemia models, the induction of Wnt signaling activity was accompanied by CTGF overexpression. Same as the high-glucose exposure, Wnt3a ligand also induced Wnt signaling activation and upregulated CTGF expression, while DKK1, a specific inhibitor of the Wnt pathway, blocked the Wnt pathway activation. Therefore, our results revealed that Wnt signaling is activated in diabetic retinopathy, which may be responsible for CTGF overexpression and retinal fibrosis.
Since DKK-1 is a commonly accepted, specific inhibitor of the canonical Wnt pathway, DKK-1 was used to specifically attenuate the Wnt signaling activation and further to reveal the role of Wnt signaling in high-glucose--induced CTGF overexpression. DKK-1 has been reported to induce the internalization of LRP6 ([@B48]). To investigate the mechanism for the SERPINA3K effect on the Wnt pathway, we have measured several components of the Wnt pathway at different levels of the cascade. Further, we measured the cell surface level of LRP6 to determine the internalization of LRP6 using extracellular biotin labeling. Similar to DKK-1, preincubation of the cells with SERPINA3K decreased the cell surface level of LRP6 (online appendix Fig. S5*C*). This result suggests that induction of LRP6 internalization may represent a mechanism responsible, at least partially, for the inhibition of LRP6 phosphorylation by SERPINA3K.
There are 19 Wnt ligands, many of which function as agonists of the Fz/LRP5/6 receptor complex and, thus, activate Wnt/β-catenin signaling ([@B49]). On the other hand, some natural inhibitors of the Wnt pathway such as DKK family members and IGFbinding protein-4 have been identified ([@B50][@B51]--[@B52]). Here, we showed that SERPINA3K blocks the Wnt pathway activation induced by high glucose and by Wnt ligands, suggesting that SERPINA3K is a novel endogenous inhibitor of the Wnt pathway. SERPINA3K is known to specifically bind to tissue kallikrein, forming a covalent complex and inhibiting proteolytic activities of tissue kallikrein ([@B3]). Through interactions with the kallikrein-kinin system, SERPINA3K participates in the regulation of blood pressure and local blood flow ([@B4]). However, the kallikrein-binding activity cannot explain the broad functions of SERPINA3K, such as antiangiogenic and anti-inflammatory activities ([@B5],[@B41]). In addition, some of these functions have proven to be independent of interactions with tissue kallikrein ([@B5]). The present study demonstrates that SERPINA3K has a potent Wnt antagonist activity. Since the Wnt pathway regulates multiple pathological processes, including angiogenesis, inflammation, and fibrosis, the Wnt antagonizing activity of SERPINA3K may represent a unifying mechanism responsible for the broad beneficial effects of this serpin. As an endogenous inhibitor of the Wnt pathway, this serpin molecule may have therapeutic potential.
Supplementary Material
======================
###### Online Appendix
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This study was supported by National Institutes of Health Grants EY012231, EY018659, EY019390, and P20RR024215 from the National Center for Research Resources and grants from the Oklahoma Center for the Advancement of Science and Technology and the American Diabetes Association.
No potential conflicts of interest relevant to this article were reported.
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INTRODUCTION
============
Motivation
----------
Bighead (*Hypophthalmichthys nobilis*) and silver (*H. molitrix*) carp are cyprinid fishes native to eastern Asia and introduced in the early 1970s to the United States as biocontrol agents for nuisance algae in freshwater ponds and lakes ([@b16]). Since these introductions, Asian carp have escaped into natural systems and caused unwanted ecological and economic impacts ([@b19]; [@b17]). Ongoing efforts to prevent the introduction of these species to the Great Lakes have incurred high costs in research, monitoring, and surveillance actions ([@b1]). Restoration of the natural hydrological separation of the Great Lakes and Mississippi River basins (to prevent the passage of Asian carp and other nonnative species) has been proposed with an estimated cost of \$4 to \$10 billion ([@b1]). As yet, there is no evidence that Asian carp populations have established self-sustaining populations in any of the Great Lakes. One important question relevant to future decisions about hydrological separation or other management strategies is: Will Asian carp successfully establish and cause ecological or economic damage in the Great Lakes? Structured expert judgment (SEJ) is used to address these questions.
Among the Great Lakes, Lake Erie is considered the most vulnerable to Asian carp invasion because of its close proximity to established Asian carp populations, habitat, and high value fisheries species under threat. Results relating to peak and equilibrium biomass of invasive Asian carp and native species are published in [@b33]. Results relating to effectiveness of deterrent strategies are published in [@b32]. The present study focuses on the SEJ methodology, with particular emphasis on cross validation.
Structured expert judgment is an established technique for probabilistic risk assessment ([@b2]; [@b7]; [@b3]) and consequence analysis ([@b11]), and it has previously been used for several environmental applications including assessments of the likelihood of natural disasters (volcanic eruption, dam failure) (Aspinall et al. 2003; [@b18]), consequences of nuclear accidents ([@b11]), drivers of climate change ([@b24]; [@b20]), ice sheet dynamics ([@b4]) fisheries and ecosystems ([@b5]; [@b27], [@b28]; [@b29]; [@b23]) and increases in human mortality attributable to air pollution ([@b14]; [@b30]; [@b26]).
The "classical model" ([@b7]) for combining expert judgment was used in many of the above references, and is used here. This model is distinguished from other methods of combining expert judgments in that it treats experts\' judgments as statistical hypotheses and measures performance in terms of statistical accuracy and informativeness (see below), based on assessments of calibration or "seed" variables. Calibration variables are taken from the experts\' field, and their true values are known post hoc although unknown to the experts at the time of assessment. These measures (statistical accuracy and informativeness) are used to construct an optimal performance-based combination of the experts\' assessments. The use of calibration variables serves the triple purpose of 1) validating expert performance, 2) enabling performance-based combination of experts\' distributions, and 3) evaluating performance of various combinations of experts\' judgments. These combinations are referred to as Decision Makers (DMs). This article extends the evaluation of DM performance by including "out-of-sample" cross-validation (e.g., validating model performance using variables not used to initialize the model).
Why out-of-sample validation for ecological SEJ studies?
--------------------------------------------------------
Evaluating forecasting models, using "out-of-sample" variables is preferable to using "in-sample" variables (e.g., those that also serve to initialize the model). A forecasting method based on calibration variables assumes that performance on calibration variables predicts performance on the variables of interest, for which true values are not known. For applications with short temporal scales, such as leading economic indicators, true values of estimated variables of interest can be observed, enabling true out-of-sample validation ([@b31]; [@b25]). However, in many applications, including ecological forecasting, out-of-sample validation based on observing the variables of interest is simply not possible due to temporal or spatial limitations associated with observing true outcomes of these variables. When variables of interest cannot be observed, out-of-sample validation resorts to cross validation: that is, on splitting calibration variable sets into complementary subsets, and predicting values in one subset (the test set) with a model initialized on the other subset (the training set). In ecological applications, out-of-sample validation must be attained through cross validation.
Summary
-------
The focus of this article is to present and evaluate new out-of-sample validation methods to assess the performance of SEJ with ecologically motivated calibration variables. The following sections describe the expert judgment application to Asian carp invasion of Lake Erie, explores out-of-sample validation, and compares performance-based weighting and equal weighting on the variables of interest. A final section provides conclusions and suggestions for further work. Supplemental Data (SD) presents details of the expert judgment performance measures and the analysis of expert data in this study. SD_A gives details of the mathematical model and the analysis of the expert judgment data in this study, SD_B consists of the elicitation protocol, and SD_C is the briefing booklet sent to all experts before the elicitation.
Expert judgment application to Asian carp invasion
--------------------------------------------------
Eleven experts participated in this study (Table [1](#tbl1){ref-type="table"}) and were offered compensation of \$1000 (although some did not choose to receive compensation). A briefing booklet was prepared (SD_C) describing the scientific background of Asian carp biology in North America and the status of Lake Erie fisheries. This briefing booklet and the elicitation instrument were sent to the experts beforehand, and experts were encouraged to use all accessible sources of information to estimate their responses.
######
Scoring of individual experts, PW, and EW combinations
Expert *p* Value[a](#tf1-2){ref-type="table-fn"} Mean relative information caliber variables[b](#tf1-3){ref-type="table-fn"} Assessed caliber variables (*n*) Unnormalized weight[c](#tf1-4){ref-type="table-fn"}
-------- ------------------------------------------- ----------------------------------------------------------------------------- ---------------------------------- -----------------------------------------------------
1 0.1815 0.6121 15 0
2 0.1227 0.6648 15 0
3 0.005634 1.47 15 0
4 0.7606 0.8562 15 0.6513
5 0.666 0.84 15 0
6 1.93E-06 1.381 15 0
7 0.05946 1.158 15 0
8 0.615 1.086 11 0
9 0.5276 1.288 15 0
10 0.2587 0.8282 15 0
11 0.5276 0.8071 15 0
PW 0.7606 0.8562 15 0.6513
EW 0.3126 0.2943 15 0.09197
EW = equal-weighted; PW = performance-weighted.
The *p* value of falsely rejecting the hypothesis that the realizations are independently drawn from a distribution complying with the expert\'s percentiles.
Informativeness score.
Combined score of the weighted experts used in forming the performance-based DM.
The variables of interest divide into 3 categories. First are quantities that concern the biomass of Asian carp both at peak and equilibrium conditions after establishment in Lake Erie, production and consumption of Asian carp at equilibrium conditions, and predation on Asian carp by other fish species currently present in Lake Erie at the predicted equilibrium condition. Second, some questions concerned equilibrium condition biomass of other fishes in Lake Erie (walleye, yellow perch, gizzard shad, rainbow smelt) following bighead and/or silver carp establishment. Finally, questions regarding the efficacy of different types of Asian carp deterrent strategies proposed for use to prevent Asian carp passage into Lake Michigan were asked.
There were 15 calibration variables. They included observed whole lake biomass measurements and average annual dietary fractions of Lake Erie fishes. These values are estimated annually by Task Groups of the Lake Erie Committee of the binational Great Lake Fisheries Commission and reported to the public in annual reports released each spring. The calibration variables were chosen for their relevance to the variables of interest. Elicitations were conducted with each expert individually. Experts gave 5, 50, and 95 percentiles of their subjective probability distribution for all uncertain quantities. At least 2 elicitors were present, one engaging the expert and encouraging him/her to verbalize his reasoning, and the other taking notes to record the rationale behind his/her uncertainty. A typical elicitation lasted 4 hours. The calibration variables and participating experts are listed in SD_A.
In the classical model, expert performance is measured in 2 dimensions: statistical accuracy and informativeness. Statistical accuracy (also called calibration) is the *p* value of falsely rejecting the hypothesis that the realizations are independently drawn from a distribution complying with the expert\'s stated percentiles. The words "calibration," "*p* value," and "statistical accuracy" are used interchangeably. Informativeness is defined as the Shannon relative information in the expert\'s distribution relative to a background measure chosen by the analyst. The information score does not depend on the realizations, and an expert can give him/herself a high information score by choosing percentiles very close together. The theory of strictly proper scoring rules is invoked to combine these measures as a "product with cutoff," whereby an expert is unweighted if statistical accuracy falls beneath a threshold value. This insures that, in the long run, an expert receives his/her highest expected weight by and only by stating percentiles corresponding to his/her true beliefs. The combined scores for each expert are normalized to provide weights for the performance weight (PW) DM. That is, the PW DM\'s distributions are weighted combinations of the experts\' distributions. The threshold is chosen to optimize the combined score of the DM. The Shannon relative information score is a slow function, whereas the likelihood of observing realizations outside the 90% central confidence band goes decreases very quickly. The product of the calibration and information scores is thus dominated by the calibration score, and informativeness modulates between more or less equally accurate experts. Details on scoring are found in SD_A.
The performance of the individual experts and of the PW and EW DMs are compared in Table [1](#tbl1){ref-type="table"}. PW is statistically more accurate and more informative than EW. In this case, expert 4 received weight 1, and in approximately one-third of all applications, one expert receives all of the weight. Expert 9 also shows very good statistical accuracy and a higher information score than expert 4. The combined score of expert 9 (0.6797) is higher than that of expert 4 (0.6513). However, because the optimization is based on the *p* value, ensuring the strictly proper scoring rule property, it is not possible to give weight 1 to expert 9 and weight zero to expert 4. We can either include expert 4 alone, or, by assigning the cutoff equal to the *p* value of expert 9 (0.5276), we combine experts 4, 5, 8, 9, and 11 using weights equal to their normalized combined scores. This would result in a *p* value of 0.7104 and an information score on calibration variables of 0.5115, for a combined score of 0.3634 (these numbers are not retrievable from Table [1](#tbl1){ref-type="table"} but require recalculation). This demonstrates the strong influence of the strictly proper scoring rule constraint.
It is significant that only 2 experts (3 and 6) have low statistical accuracy, whereby the corresponding statistical hypotheses would be rejected at the 5% level. This is among the best performing expert panels in this regard. Note that the statistical accuracy scores vary over 5 orders of magnitude whereas the informativeness scores vary within a factor 3. Robustness analysis of these results is found in SD_A.
IN- AND OUT-OF-SAMPLE VALIDATION
================================
Background
----------
Table [1](#tbl1){ref-type="table"} compares performance of the PW DM and EW DM on the calibration variables. Because these calibration variables are also used to derive the weights for performance-based weighting, this is "in-sample" validation. That PW should outperform EW in-sample is certainly not a mathematical theorem, and EW does occasionally outperform PW in-sample. Were there not a strong in-sample preference for PW over EW, there would be little motive for considering performance-based weighting at all. [@b12] summarized a TU Delft expert judgment database comprised of 45 studies completed by 2006, in which experts assessed calibration variables and showed that PW DM strongly outperforms the EW DM in-sample. Since then, the number of studies has nearly doubled.
Researchers have used the TU Delft database to explore new models and to study whether good performance on the calibration variables is linked with good performance on the variables of interest. In a few studies, variables of interest were later observed, enabling true out-of-sample validation. In most cases, like the present case, the variables of interest are not observable on time scales relevant for the decision problem. Therefore, various forms of cross validation have been applied. [@b6] proposed a remove-one-at-a-time (ROAT) method according to which the calibration variables were removed one at a time and predicted by the model initialized on the remaining calibration variables. [@b6] pooled these predictions, though originating from different DMs, and compared the resulting synthetic DM with the EQ DM. Of the 14 studies analyzed by [@b6], the PW DM was superior to the equal weight DM on 9, which was not statistically significant in a sample size of 14.
The ROAT method is biased against the PW DM, because each calibration variable was predicted by a DM in which experts who assessed that particular item badly were up-weighted, and all variables were assessed in this manner. In many studies, removing 1 calibration variable can influence an individual expert\'s *p* value by a factor 3 or more ([@b8]), a feature explained by the fact that statistical accuracy is a very fast function. It is easy to under appreciate this effect, and [@b9], [@b10] gave a detailed example making this conspicuous. Other types of cross validation have been carried out by [@b21], [@b22]) and [@b15]. The most extensive study is the [@b13] analysis of 62 cases, which initialized the PW DM on all subsets of calibration variables and, in each case, predicted the complementary subset. In 45 of the 62 cases (73%) the PW DM outperformed the EW DM, and when PW DM was better, it tended to be much better. This provides an indication of the ROAT bias. Details may be found in SD_A.
Some researchers have applied other scoring rules to measure performance that apply to single variables rather than sets of variables, such as the quadratic or logarithmic rule. An extensive discussion in [@b7] discourages this practice. A simple example clarifies the issue. Suppose an expert assess the probability of heads as 1/2 with a coin of unknown composition. On each toss with the coin, the score is the same for heads and tails. If these individual scores are added, then the sum score after 100 tosses is also independent of the actual sequence of outcomes, 50 heads and 50 tails gets the same score as 100 heads. A general conclusion of all this work is that the performance-based DM is degraded on out-of-sample prediction, but is superior to the equal weight DM.
When a cross validation study initializes the PW DM on a training set containing K of the N calibration variables, and measures performance on the remaining N--K variables, the following issues arise. First, if K is close to N, then the small number of out-of-sample predictions have low statistical power and are subject to the bias noted above. Second, if K is small, then the ability to distinguish experts\' high and low statistical accuracy is low. If the experts\' calibration scores on the K variables are similar, then weighting is driven by informativeness that is often negatively correlated with statistical accuracy ([@b7]). For intermediate values of K, statistical power is lost at both ends, though this may be partially compensated by averaging the scores over all training sets of size K. How much statistical power is "recovered" in this way is difficult to judge, as the training sets overlap. In short, we do not know the best way for performing out-of-sample validation at present.
In lieu of an optimal solution, the exhaustive approach of [@b13] at least balances the known biases. Its disadvantage is that it is cumbersome, especially for more than 10 calibration variables. [@b13] noted that with a small training set, the scores on the test set did not predict the scores on the larger set of calibration variables but did confirm the superiority of performance weighting against equal weighting. Unfortunately, this does not mean that future studies can make do with 1 or 2 calibration variables, as this result is attained by averaging over many sparse training sets. The present data set allows us to gain further insights in "sparsely trained PW DM\'s."
Out-of-sample validation for Asian carp study
---------------------------------------------
For this exercise, expert 8 was removed, as he assessed only 11 calibration variables. All scores are now based on 15 calibration variables, and the scores are somewhat different than those in Table [1](#tbl1){ref-type="table"}, where the statistical power of 11 calibration variables is used. Focusing first on training sets of size K = 1 and K = 2, [Figure 2](#fig02){ref-type="fig"} compares the calibration and information scores of these very sparsely trained decision makers. Echoing results of [@b13] this shows 1) that the statistical accuracy and informativeness scores of the PW DM do not predict those in the original study, but 2) do outperform the EW DM, and 3) show considerable scatter. The Asian carp expert panel involved a relatively large number of high scoring experts; it remains to be seen if similar cross validation results emerge from other expert panels. To enable the comparison in [Figure 1](#fig01){ref-type="fig"}, the cross validation comparisons are also based on all 15 seed variables, not just 14 (initialized on 1 variable) or 13 (initialized on 2 variables).
{#fig01}
{#fig02}
[Figure 3](#fig03){ref-type="fig"} shows the PW and EW combined scores averaged over each training set size from 1 to 6. There are 14 training sets of size 1, there are 15-choose-2 = 105 training sets of size 2, and so forth, going up to 5005 training sets of size 6.
{#fig03}
Both the PW and EW scores increase with the training set size, reflecting the diminishing power of the test set. The EW DM is the same in all cases, as the weights do not depend on the training set. The rise in scores is purely a result of decreasing statistical power of the test set. However, PW increases faster than EW, suggesting a gain in performance from increased power of the training set over and above the power reduction of the test set. [Figure 4](#fig04){ref-type="fig"} compares the PW and EW differences, for each of the 9949 training sets of size 1 to 6.
{#fig04}
The comparisons are indexed by size of the training set; with size 1 corresponding to indices 1 to 15 and size 6 corresponding to indices 4944 to 9949. Near the origin, values of PW-EW tend to be smaller, as is expected. The superiority of PW over EW in out-of-sample cross validation is evident. However, the differences in the cross validation comparisons tend to be smaller than the in-sample differences shown in Table [1](#tbl1){ref-type="table"}, where the difference PW-EW is 0.56.
VARIABLES OF INTEREST
=====================
Variables relating to the biomass of Asian carps following establishment in Lake Erie are treated in depth in ([@b33]). Variables relating to the effectiveness of deterrent strategies are discussed in ([@b32]). Present purposes are served by comparing the PW DM and EW DM for these 2 classes of variables of interest.
[Figure 5](#fig05){ref-type="fig"} gives a schematic picture of the EW DM and PW DM. The variable numbering is the same as in the elicitation protocol in SD_B, from which the precise definitions may be retrieved. The scaling on the vertical axes is artificial in the sense that some variables are percentages, others are kilograms per square meter, etc. However, variables with the same number are scaled in the same way.
{#fig05}
[Figure 5](#fig05){ref-type="fig"} shows that the PW DM is usually---not always---more informative than the EW DM. Moreover, their median assessments can also vary substantially.
[Figure 6](#fig06){ref-type="fig"} from [@b32] shows similar information for the 17 Asian carp deterrent strategies assessment. As with [Figure 5](#fig05){ref-type="fig"}, the PW DM is substantially more informative than the EW DM. For the most promising strategies, physical separation (SEP) and electric barrier (ELE), the median effectiveness assessments are nearly identical. For others, this is not the case. Pheromone attractant and/or repellant appears promising to the PW DM but not to the EW DM. For physical block net the reverse holds, the PW DM is highly skeptical whereas EW DM is not. The very large confidence bounds of the EW DM indicate that this DM has no pronounced opinion on any of these strategies except the first two.
![Equally weighted (EW; left) and performance-based (PW; right) expert assessments of the percentage of Asian carp prevented access to the Great Lakes as a result of implementing 17 proposed fish deterrent strategies in the Chicago Area Waterway System. Dots indicate median; lines denote the 5% and 95% intervals. For definitions of acronyms see SD_B or ([@b32]).](ieam0010-0522-f6){#fig06}
CONCLUSIONS
===========
This expert judgment study has demonstrated the experts\' skill in performing probabilistic assessments and demonstrated the superiority of performance-based combinations of expert\'s judgments over equal weighting in this case. Although both performance-based weights and equal weights returned acceptable statistical performance, the performance-based combination was significantly more informative on both the calibration variables and on the variables of interest. Highly informative assessments are valuable only if these assessments are statistically accurate such that the narrower confidence bands are statistically defensible. The differences in the PW and EW DMs highlights the importance of validation.
Informative uncertainty distributions are very useful in making practical choices. For example, [Figure 6](#fig06){ref-type="fig"} shows that the PW DM has a clear preference for 3 strategies (phys, elec, and acousstrobebulb), and this preference is based on the 90% confidence bands as well as on the median value. With the exception of SEP, the EW DM\'s confidence bands are so wide that they provide no practical value. Needless to say, narrow confidence bands are defensible only if their statistical accuracy is affirmed. In this case, based both on in-sample and out-of-sample validation, the statistical accuracy of the PW DM is actually better than that of the EW DM. Both forms of validation are important when structured expert judgment is used to quantify uncertainty.
A key question is whether the experts\' performance on calibration variables will carry over to the variables of interest. These latter variables are not directly observable---otherwise we would not need expert judgment in the first place. Out-of-sample cross validation can be undertaken, whereby the performance weights are computed based on a subset of calibration variables (training set), and performance measured on the complementary set (test set). Using all nonempty subsets of calibration variables yields a super set of the relevant comparisons, but might be a very large set. With 25 calibration variables (not an unrealistic number) there are 33 554 431 training sets; computational advantages are achieved with smaller training sets. A further advantage would result if small training sets were generally sufficient to attest out-of-sample validity, as this would justify reducing the numbers of calibration variables. The present study provides some evidence for that conclusion but further assessments of other SEJ study outcomes will serve to more fully understand this issue.
Supporting Information
======================
All Supplemental Data may be found in the online version of this article.
######
SD_A gives mathematical details.
######
SD_B is the elicitation protocol.
######
SD_C is the briefing booklet.
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M. Regner, A. Bartuce, D. Padmakshan, J. Ralph, S. D. Karlen, *ChemSusChem* **2018**, *11*, 1600.
As a major component of biomass recalcitrant to depolymerization, lignin continues to impede efforts toward less energy‐intensive processes in the pulping and lignocellulosic biofuel industries.[1](#cssc201800617-bib-0001){ref-type="ref"}, [2](#cssc201800617-bib-0002){ref-type="ref"} Formed in a combinatorial fashion via consecutive oxidative radical‐coupling steps,[3](#cssc201800617-bib-0003){ref-type="ref"}, [4](#cssc201800617-bib-0004){ref-type="ref"} lignin contains a variety of phenylpropanoid subunits resulting from linking monomers to the growing polymer. β‐O‐4 ethers (hereafter termed simply β‐ethers), which account for ≈60 % of the interunit linkages, are among the most labile bonds in the polymer, but even they require harsh conditions and high temperatures to cleave.[3](#cssc201800617-bib-0003){ref-type="ref"}, [5](#cssc201800617-bib-0005){ref-type="ref"} In recent years, research efforts have focused on reducing the need for severe conditions through manipulation of the lignin biosynthetic pathway to incorporate novel monomers or upregulate production of more labile subunits (e.g., esters). Results have demonstrated that plants exhibit a high degree of metabolic plasticity for in vivo lignification,[6](#cssc201800617-bib-0006){ref-type="ref"}, [7](#cssc201800617-bib-0007){ref-type="ref"}, [8](#cssc201800617-bib-0008){ref-type="ref"}, [9](#cssc201800617-bib-0009){ref-type="ref"}, [10](#cssc201800617-bib-0010){ref-type="ref"} thus setting the stage for advantageous plant bioengineering. To keep up with these advances, the methodologies for characterizing lignin need to likewise evolve.[11](#cssc201800617-bib-0011){ref-type="ref"}
For many years, NMR spectroscopy has offered excellent, non‐degradative structural information.[12](#cssc201800617-bib-0012){ref-type="ref"}, [13](#cssc201800617-bib-0013){ref-type="ref"}, [14](#cssc201800617-bib-0014){ref-type="ref"} However, although peak integration allows for reasonably accurate quantification in 1 D NMR, the more useful 2 D methods have serious quantitative limitations, especially for rapidly relaxing samples, and cannot provide these data on an absolute scale. Thioacidolysis has served as a powerful degradative tool,[15](#cssc201800617-bib-0015){ref-type="ref"}, [16](#cssc201800617-bib-0016){ref-type="ref"}, [17](#cssc201800617-bib-0017){ref-type="ref"} although it yields a complicated portfolio of products for analysis.[18](#cssc201800617-bib-0018){ref-type="ref"} For monolignol (ML) conjugate analysis, its utility has suffered from reaction conditions that neither leave esters intact nor cleave them completely;[19](#cssc201800617-bib-0019){ref-type="ref"} recent efforts have addressed this issue, as evidenced by the detection of labile ester conjugates in lignin from transgenic *Arabidopsis thaliana*.[20](#cssc201800617-bib-0020){ref-type="ref"}
In unique fashion, derivatization followed by reductive cleavage (DFRC), a degradative method similar to thioacidolysis in that it cleaves β‐ether units in lignins to release analyzable monomers and dimers, uses reductive cleavage instead of hydrolysis to provide useful structural information.[21](#cssc201800617-bib-0021){ref-type="ref"}, [22](#cssc201800617-bib-0022){ref-type="ref"} DFRC offers several features: it selectively cleaves β‐ether bonds while retaining ester linkages, thus providing conclusive evidence of monolignol conjugate incorporation into lignins; a final acetylation step simplifies data interpretation by reducing the number of analytes; and two modified versions of the protocol use common reagents to distinguish between freephenolic and etherified units in the polymer, as well as natural γ‐acetates and those introduced by the method itself (Scheme [1](#cssc201800617-fig-5001){ref-type="fig"}). In recent years, DFRC has been an indispensable tool in confirming the incorporation of monolignol acetate (Ac),[23](#cssc201800617-bib-0023){ref-type="ref"}, [24](#cssc201800617-bib-0024){ref-type="ref"} *p*‐coumarate (*p*CA),[25](#cssc201800617-bib-0025){ref-type="ref"}, [26](#cssc201800617-bib-0026){ref-type="ref"} benzoate (BA),[27](#cssc201800617-bib-0027){ref-type="ref"} *p*‐hydroxybenzoate (*p*BA),[27](#cssc201800617-bib-0027){ref-type="ref"}, [28](#cssc201800617-bib-0028){ref-type="ref"} vanillate (VA),[27](#cssc201800617-bib-0027){ref-type="ref"} and ferulate (FA)[9](#cssc201800617-bib-0009){ref-type="ref"}, [29](#cssc201800617-bib-0029){ref-type="ref"}, [30](#cssc201800617-bib-0030){ref-type="ref"} conjugates into the lignin polymer.
{#cssc201800617-fig-5001}
In spite of its usefulness, however, DFRC in its current form has some shortcomings. "Theoretical" yields based on synthetic models have diverged from those obtained using real plant samples.[21](#cssc201800617-bib-0021){ref-type="ref"}, [31](#cssc201800617-bib-0031){ref-type="ref"} Furthermore, reproducibility has presented a challenge, partly owing to the variety of internal standards that have been used over the years.[9](#cssc201800617-bib-0009){ref-type="ref"}, [21](#cssc201800617-bib-0021){ref-type="ref"}, [32](#cssc201800617-bib-0032){ref-type="ref"} Finally, the discovery of new conjugates in the plant cell wall has required improvements to the original method. Herein, we detail our reconstruction of the DFRC protocol to yield an accurate and reproducible method for the quantification of releasable monolignols and monolignol conjugates from plant biomass.
We report the reaction yields for β‐ether bond cleavage using a family of model compounds designed to represent the different β‐ether crosslinking permutations that exist between coniferyl alcohol, sinapyl alcohol, and the variously γ‐acylated monolignol conjugates (Scheme S1). The model compounds are abbreviated using an AB‐C nomenclature scheme, where "A" is either the coniferyl (G) or sinapyl (S) alcohol base unit, "B" is the β‐ether ring, either guaiacol (G) or syringol (S), and C is the γ‐acylating unit, acetate (Ac), *p*‐hydroxybenzoate (*p*BA), *p*‐coumarate (*p*CA), ferulate (FA), or sinapate (SA). As shown in Scheme [1](#cssc201800617-fig-5001){ref-type="fig"}, loss of the etherified "B" ring (either guaiacol or syringol moieties in our models) through reductive cleavage regenerates the vinyl group, whereas loss of bromide on the ester gives a saturated sidechain; this pattern is diagnostic for the cleavage of β‐ethers by the DFRC method (Figure [1](#cssc201800617-fig-0001){ref-type="fig"}). To quantitate these analytes more accurately, deuterated standards were synthesized. Deuteroacetylation has proven useful in this regard,[33](#cssc201800617-bib-0033){ref-type="ref"} but ketene loss upon electrospray ionization (ESI) and coelution with other products complicates mass spectrometric (MS) peak assignment of the standards versus the natural‐abundance isotopes (^13^C and ^2^H) of the lignin products. Therefore, in addition to deuteroacetylating the phenols and free γ‐hydroxyls, two to four additional deuterium labels were incorporated on either side of the ester (Scheme [2](#cssc201800617-fig-5002){ref-type="fig"}), providing multiple unique MS and multiple reaction monitoring (MRM) fragments between the isotopically labeled internal standards and the DFRC products. In conjunction with a natural abundance "dihydro" conjugate calibration curve, we used this new set of internal standards to quantify DFRC products.
{#cssc201800617-fig-0001}
{#cssc201800617-fig-5002}
Additionally, we optimized each DFRC step to obtain the maximum detectable release of monolignol conjugates from extractive‐free corn stover, using *p*‐coumarate as a proxy for all conjugates. We obtained essentially the same conditions for bromination as in the original DFRC paper (2.5 h instead of 3 h)[21](#cssc201800617-bib-0021){ref-type="ref"} and we discovered that acetylation of the phenols in the product mixture, formed during the reductive cleavage step, occurs almost immediately after addition of the reagents (i.e., acetic anhydride and pyridine). The most important result gleaned from these efforts was our observation that zinc morphology had a significant impact on reaction yield. Zinc nanopowder (Sigma--Aldrich, P/N: 578 002, particle size \<50 nm) greatly increased the yield over that of standard zinc dust for a given reduction time, while simultaneously decreasing variability between samples. As for differences between the rates of cleavage for MLs and ML conjugates, in both cases, zinc reductive cleavage was complete within 30 min; longer reduction times did not diminish the reaction yield (Figure S1 c).
Following optimization of each step, we have used synthetic model compounds to demonstrate that the DFRC procedure cleaves β‐ethers with approximately 80 % efficiency for monolignols and ML‐*p*BAs, and 40--50 % for ML‐*p*CAs, ML‐FAs, and ML‐SAs (sinapates), based solely upon quantification of the expected saturated conjugate, as discussed below (Figure [2](#cssc201800617-fig-0002){ref-type="fig"}). All compounds synthesized for the determination of "theoretical" DFRC reaction yields, for authentication of products, and for use as internal standards, along with details regarding reaction kinetics can be found in the Supporting Information. The detailed procedure can be found in the Experimental Section below, as well as in several recent papers that have applied this method to the analysis of monolignol conjugates.[29](#cssc201800617-bib-0029){ref-type="ref"}, [30](#cssc201800617-bib-0030){ref-type="ref"}, [34](#cssc201800617-bib-0034){ref-type="ref"}
{#cssc201800617-fig-0002}
In the course of our model compound studies, we found monolignol conjugate yields to be unusually low in comparison to general yields published in the original DFRC method paper, although monomer yields were as expected.[35](#cssc201800617-bib-0035){ref-type="ref"} As the data demonstrate (Figure [2](#cssc201800617-fig-0002){ref-type="fig"}), yield varied as a function of the moiety acylating the γ‐hydroxyl unit. In particular, when moving from the *p*BAs to the *p*CAs, yields dropped off markedly. Efforts to identify potential byproducts by GC--MS/MS were unsuccessful owing to poor peak resolution and MS responsiveness, but NMR analysis provided important insight; ^1^H spectra collected immediately following bromination and final acetylation showed residual vinyl peaks in both cases (albeit with different chemical shifts). Because the yield of ML‐*p*CAs obtained from corn stover varied little as a function of bromination time beyond 3 h (Figure S1 b), the presence of these residual vinyl peaks suggested that the ester had established an equilibrium between its brominated and unbrominated form, only the former of which would yield the expected saturated product.
To test this hypothesis, we ran DFRC on a larger scale (≈100 mg), using three permutations of model compound 7 (Figure S1): GG‐*p*CA, GG‐FA, and GG‐SA. Comparing the ^1^H NMR spectra of the product mixtures with those of the appropriate standards \[e.g., G‐*p*CA and G‐DH*p*CA\], we were able to quantify the ratio of unsaturated products relative to saturated ones by integrating the peaks corresponding to the *α* and *β* protons. We discovered that, as electron density increased on the aromatic ring of the γ‐acylating group owing to the presence of methoxyl units, the relative abundance of the unsaturated conjugate decreased, suggesting that the reaction equilibrium in the bromination step had moved more toward the brominated product. Indeed, others have demonstrated that bromination of electron‐rich vinyl groups proceeds rapidly and efficiently,[36](#cssc201800617-bib-0036){ref-type="ref"} and the observed trend in our own data set (≈35 % unsaturated conjugate arising from nonbrominated material for *p*CA, ≈25 % for FA, and ≈20 % for SA) further confirms this reactivity pattern. After incorporating quantitative data for the residual unsaturated conjugates based on our NMR‐determined ratios, the total yields for each conjugate are acceptable (Figure [2](#cssc201800617-fig-0002){ref-type="fig"}). Figures comparing the ^1^H NMR spectra of the crude DFRC reaction mixture with the appropriate standards can be found in the Supporting Information (Figures S2--S4).
In addition to optimizing yield and reaction kinetics, we also evaluated various internal standards and analytical methods for optimal performance. We compared chromatograms obtained from DFRC analysis of Alaskan cedar, poplar, and corn stover under scan mode (100--600 *m*/*z*) and MRM mode (Figure [3](#cssc201800617-fig-0003){ref-type="fig"}, Table [1](#cssc201800617-tbl-0001){ref-type="table-wrap"}). By using MRM technology, we observed significant reduction in background noise, thus allowing for greater accuracy in quantitation of low‐abundance monolignol conjugates (e.g., released ML‐DHFA esters). Yet, even with these improvements, the residual unsaturated conjugates (as described above), such as ML‐FA and ML‐SAs (the later of which has never been found in planta), were not detected in plant samples. Previous work has, however, successfully quantified both S‐DH*p*CA and S‐*p*CA by GC--MS in plants that contain ML‐*p*CAs;[37](#cssc201800617-bib-0037){ref-type="ref"} as such, our analysis of corn stover included quantitative data for both the saturated and unsaturated isomers (Figure [3](#cssc201800617-fig-0003){ref-type="fig"}). As a demonstration of the improvements offered by these new analytics, we then quantified several analytes using selected ion monitoring (SIM) and MRM methods and compared the results (Table [2](#cssc201800617-tbl-0002){ref-type="table-wrap"}). For abundant compounds (e.g., G in all species, S‐DH*p*CA in corn stover, and S‐*p*BA in poplar), we observed comparable performance between SIM and MRM scans. On average, we observed a difference of approximately 20 % between the methods for abundant monolignols and monolignol conjugates. The key advantage of MRM over SIM became apparent in the quantitation of low‐abundance monolignol conjugates (e.g., G‐*p*CA and G‐DH*p*CA in corn stover and G‐DHFA in poplar); whereas MRM allowed for accurate quantitation, these conjugates were not detectable by SIM.
{ref-type="table-wrap"}.](CSSC-11-1600-g003){#cssc201800617-fig-0003}
######
Quantitative data of released target compounds as determined under SIM and MRM modes for Alaskan cedar, poplar, and corn stover. The corresponding GC--MS chromatograms are shown in Figure [3](#cssc201800617-fig-0003){ref-type="fig"}.
Source Method Value \[μmol (g whole cell wall)^−1^\]
--------------- -------- ---------------------------------------- ----------- ----------- ------------- ---------- ------------------ ----------------- ------------- -------------
Alaskan cedar SIM 1.46±0.10 228.7±8.1
MRM 1.76±0.04 181.4±7.2
Poplar SIM 1.11±0.08 91.0±2.8 232.0±6.2 ND 14.9±1.3 ND
MRM 1.12±0.01 75.0±2.4 199.4±7.0 0.038±0.002 19.4±0.6 0.051±0.003
Corn stover SIM 1.79±0.07 31.9±0.6 8.4±0.4 ND^\[a\]^ 11.6±0.0^\[a\]^ ND
MRM 2.24±0.06 32.5±0.9 11.5±0.4 1.21±0.02^\[a\]^ 18.8±0.5^\[a\]^ 0.060±0.003
\[a\] These values include quantitative data for both the saturated and unsaturated conjugates.
Wiley‐VCH Verlag GmbH & Co. KGaA
######
Heatmap depicting the relative suitability of internal standards for various DFRC analytes.^\[a\]^
Analytes Difference in value compared to the deuterated analog \[%\]
----------- ------------------------------------------------------------- ------ ----- ---- ---- ---- ---- ----- ---- ----- ----- -----
H‐OH/Ac 30 47 43 0 2 2 17 40 22 26 24 56
G‐OH/Ac 35 41 145 4 0 4 91 146 95 124 141 158
S‐OH/Ac 31 45 124 4 2 0 63 102 73 88 98 114
G‐*p*BA 3517 4580 54 29 14 19 0 43 15 17 3 62
S‐*p*BA 53 60 64 50 37 41 30 0 40 18 32 13
G‐DH*p*CA 308 308 9 44 50 47 27 34 0 17 6 46
S‐DH*p*CA 11 25 7 25 35 32 9 14 14 0 9 24
G‐DHFA 2123 2378 39 26 4 2 4 47 12 20 0 66
S‐DHFA 2133 2351 4 12 71 67 17 11 36 24 30 0
\[a\] ▪=deuterated analogue, ▪=\<10 %, ▪=10--25 %, ▪=25--50 %, ▪=\>50 % difference.
Wiley‐VCH Verlag GmbH & Co. KGaA
To determine the reliability of various standards, we compared the accuracy of commercially available (or easily prepared) internal standards to isotopically labeled conjugates (using the latter as the "ideal" standard and basis for all calculations) to determine whether a less‐specialized standard could afford comparable performance. After calculating the percentage difference between the "ideal" conjugate and all others that were used in the course of this project, we created a heatmap as a visual aid for evaluating performance of a given analyte (Table [2](#cssc201800617-tbl-0002){ref-type="table-wrap"}). In almost every case, the traditional standards \[tetracosane, 1,1‐bis‐(4‐hydroxyphenyl)ethane (BPO), which is detected as 1,1‐bis‐(4‐acetoxyphenyl)ethane (BPA), and diethyl 5,5′‐diferulate diacetate (DEDF)\] showed poor performance. Furthermore, no single standard offered exceptional performance across the board, thus highlighting the differing MS response factors of each analyte and the necessity of using appropriate standards in MS methods.
Taken together, we contend that the various facets of our work have deconstructed a valuable but increasingly dated tool and fully rebuilt it into a robust method for lignin structural analysis. Yield data based on our expansive array of synthetic model compounds have offered insight into mechanistic details (e.g., the presence of unsaturated ester conjugates), as well as reasonable expectations for how much the method can release from biomass. Our use of isotopically labeled internal standards and cutting‐edge analytics have improved sensitivity such that we can detect cinnamate ester products present at low concentrations in the plant cell wall, which has major implications for plant breeding programs.[9](#cssc201800617-bib-0009){ref-type="ref"}, [29](#cssc201800617-bib-0029){ref-type="ref"}, [30](#cssc201800617-bib-0030){ref-type="ref"} As more researchers direct these programs toward lignin degradation and valorization, mature analytical methods will only grow in importance. By effectively redesigning DFRC from the ground up, we have given it the unique capability of detecting and quantifying low‐abundance monomers and monolignol conjugates, data which will provide critical insight into the chemical structure of engineered lignins and how best to utilize them.
Experimental Section {#cssc201800617-sec-0002}
====================
In detail, the optimized DFRC method (run on synthetic model compounds) is as follows. In a two‐dram vial containing a magnetic stir bar, the β‐ether model compound (1 mg) was dissolved in acetyl bromide‐acetic acid solution (1:4 *v*/*v*, 1.0 mL). The vial was sealed with a polytetrafluoroethylene (PTFE)‐lined cap and heated at 50 °C for 2.5 h, after which the solvent was removed under reduced pressure on a SpeedVac (Savant SPD131DDA concentrator with a RVT5105 refrigerated vapor trap and a OFP400 pump, Thermo Scientific, 50 °C, 25 min, 35 torr min^−1^, 1.0 torr). The resulting film was treated with absolute ethanol (0.5 mL), followed by removal of the solvent on the SpeedVac (50 °C, 30 min, 35 torr min^−1^, 1.0 torr). Ethanol quenches any residual acetyl bromide and, when running whole cell wall or lignin samples, the subsequent removal of ethanol in vacuo affords the brominated biomass as a film. In the next step, these brominated model compounds were dissolved in dioxane/acetic acid/water (5:4:1 *v*/*v*, 2 mL), and zinc nanopowder (50 mg) was charged to the vial with continuous stirring of the suspension at room temperature. After 15 min, the mixture was transferred to a separatory funnel containing saturated ammonium chloride (15 mL) and the corresponding deuterated internal standard (ISTD) using dichloromethane to rinse the reaction vessel (3×1 mL). The organic phase was separated, and the aqueous phase was extracted with dichloromethane (3×10 mL). The combined organic fractions were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Reaction with pyridine/acetic anhydride (1:1 *v*/*v*, 3 mL) for 1 h assured full acetylation of the product. Removal of these solvents under reduced pressure yielded the final oil for analysis. Each sample was analyzed on a Shimadzu GCMS‐TQ8030 operating in MRM mode. The GC--MS/MS program and acquisition parameters are listed in the Supporting Information Tables S1--S3.
Conflict of interest {#cssc201800617-sec-0004}
====================
*Patent submitted for deuterated monolignol conjugates*.
Supporting information
======================
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
######
Supplementary
######
Click here for additional data file.
This work was partially funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE‐FC02‐07ER64494 and DE‐SC0018409), and by Stanford\'s Global Climate and Energy Program (GCEP). We also thank the UW‐Madison Chemistry Department Mass Spectrometry Lab for acquisition of HRMS data. The purchase of the Thermo Q Exactive™ Plus in 2015 was funded by NIH Award 1S10 OD020022‐1 to the Department of Chemistry.
|
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"pile_set_name": "PubMed Central"
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Cells produce a palette of lipid bioregulators, *e.g.* steroid hormones, eicosanoids or endocannabinoids, to mention only a few. Known molecules may be identified by probing MS/MS spectra against a reference spectra database[@b1] or by their direct interpretation relying upon common fragmentation mechanisms and lipid-class specific compositional constraints[@b2]. In principle, these approaches could be extended to the identification of putative lipid structures by relaxing spectra matching requirements[@b3][@b4]. However, in crude extracts the abundance of new molecules is often too low compared to other lipids. Therefore their precursor ions are not selected for fragmentation in data-dependent LC-MS/MS experiments and in shotgun experiments they render non-interpretable MS/MS spectra dominated by product ions of co-fragmented lipids and chemical noise. This constitutes a major bottleneck in a systematic discovery of novel lipids.
Despite their compositional diversity, lipids are composed from a large, yet finite compendium of common structural modules such as fatty acids, amino acids, glycerol, ethanolamine, carbohydrates, *etc.*, which associate in numerous combinations[@b5]. Consistent with the modular organization, MS/MS fragmentation of lipid precursors produces signature ions specific for each structural module and their grouping defines the lipid classes and individual species[@b2][@b6]. To recognize and group the signature ions it might not be necessary to mass-select each precursor individually. Instead, all precursors co-eluted during LC-MS/MS experiment may be fragmented at once and all fragments simultaneously detected in a highly convoluted spectrum -- this approach was termed as MS^E^ and also as all-ions fragmentation LC-MS/MS (AIF LC-MS/MS)[@b7][@b8][@b9][@b10]. Fragment ions could be associated with corresponding precursors by retroactive *in silico* alignment of their peaks in extracted ion chromatograms (XIC). We implemented AIF LC-MS/MS in an unbiased screening approach and showed that it could systematically identify novel lipid molecules and lipid classes at the low picogram level.
Results
=======
We reasoned that novel lipid structures may arise from yet unknown combinations of already known modules and both known and novel combinations may be recognized in the same AIF LC-MS/MS analysis ([Fig. 1](#f1){ref-type="fig"}). For presentation clarity, let us assume that anticipated lipid molecules may consist of two structural modules: a polar head group (H) and a fatty acid (F). To screen for new molecules, we will first design plausible structures by combining all fatty acid moieties *f*~*j*~ with all head groups *h*~*i*~ from the H and F lists, respectively. For each compound *f*~*j*~*h*~*i*~ we compute *m/z* of its precursor ion \[*f*~*j*~*h*~*i*~\]^+^ and characteristic polar head group fragment \[*h*~*i*~\]^+^ which would be most likely detectable in tandem mass spectra. AIF LC-MS/MS of a total extract detects all ionisable precursors (in MS spectra) and all fragments (in AIF MS/MS spectra). Chromatographic peaks of anticipated precursors (*XIC* \[*f*~*j*~*h*~*i*~\]^+^) and fragments (*XIC* \[*h*~*i*~\]^+^) are then aligned by the in-house developed Arcadiate software. They should neatly overlap if \[*h*~*i*~\]^+^ is produced from \[*f*~*j*~*h*~*i*~\]^+^ (see *aligned XIC* trace in [Fig. 1A](#f1){ref-type="fig"}), while no or partial match indicates that this pair of ions is unrelated. The same scheme also applies for more complex fragmentation patterns since peaks alignment specificity increases if XICs of several fragments are required to match. A dataset produced by a single AIF LC-MS/MS experiment suffices for *in silico* screening for an unrestricted number of combinations of modules. If the structure of candidate molecules remains ambiguous, AIF LC-MS/MS should be followed by the targeted acquisition of full high resolution MS/MS spectra of their precursors, chemical derivatization[@b11] and, ultimately, by chemical synthesis.
We applied AIF LC-MS/MS to profile endogenous endocannabinoid-related compounds (ERC) in a total extract of rat kidney ([Fig. 1A](#f1){ref-type="fig"}). ERC consist of two structural modules: a fatty acid or fatty alcohol linked to a polar head group (which determines its class), such as ethanolamine, glycerol, dopamine or amino acids (such as Ser or Gly). *Bona fide* endocannabinoids are physiological ligands of type 1 and type 2 cannabinoid receptors (CB~1~R and CB~2~R) and implicated in neurological, metabolic and cardiovascular diseases (reviewed in)[@b12]. ERC are widespread across phyla[@b13][@b14] and have a broad spectrum of activities[@b15][@b16]. They are present in fluids and tissues at the nM or picogram per mg range[@b17][@b18], respectively, and therefore common LC-MS/MS analyses only target a few key molecules. ERC are structurally diverse, however no method afforded a comprehensive overview of their composition: although in total more than 80 ERC have been identified this list is certainly not exhaustive[@b16][@b19][@b20].
We first benchmarked our method by comparing the number of *N*-acylethanolamines (NAE) identified by AIF and, independently, by the two targeted LC-MS/MS methods[@b17][@b21] -- multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer and by parallel reaction monitoring (PRM) on a hybrid high resolution tandem mass spectrometer Q Exactive ([Fig. 1B](#f1){ref-type="fig"}). Both MRM and PRM rely upon an inclusion list of targeted precursors compiled prior the analysis. Contrary, AIF profiling is unbiased since a full dataset of MS and MS/MS spectra is acquired first and then XIC traces for any desired combination of precursor and fragment masses are retrieved and aligned. For PRM and MRM analyses we compiled a list of *m/z* of 19 candidate NAE molecules that included species with common C16-C20 fatty acids with 0 to 6 double bonds; we also added two shorter saturated C12 and C14 fatty acids and three polyunsaturated C22 fatty acids with 4 to 6 double bonds ([Supplementary Table S1](#S1){ref-type="supplementary-material"}). This list comprised all currently known NAE species and a few plausible candidates comprising common fatty acid moieties. Considering even larger number of candidate molecules could compromise the sensitivity of both reference methods, while having no impact on the sensitivity of AIF. In all three experiments NAE were identified by detecting the ethanolamine head group fragment (*m/z* 62.060) produced by MS/MS of 19 expected precursor ions ([Supplementary Table S1](#S1){ref-type="supplementary-material"}). We note that, although MRM and PRM relied on the same mass transitions, they were performed independently on different instruments and were complementary. MRM was expected to have higher sensitivity, yet lower specificity in selecting precursor and fragment *m/z*, while PRM was performed at much higher mass resolution and therefore should be detecting precursors and fragments with higher specificity albeit lower sensitivity.
In a rat kidney extract AIF LC-MS/MS identified 15 NAE molecules, including 12 out of 13 molecules found by both MRM and PRM ([Fig. 1C](#f1){ref-type="fig"}). In total, it recognized the same number of NAE molecules (15) as the method of MRM. It failed to detect NAE 22:6 because of background interference; this, however, was unrelated to the high degree of unsaturation of its fatty acid moiety. Hence the identification of endogenous NAE by the untargeted AIF and both targeted (MRM, PRM) LC-MS/MS methods was consistent.
We further hypothesized that ERC might feature a broader selection of polar head groups and fatty acids/alcohols, yet sharing the same modular organization. In principle, amino- and hydroxyl groups of any endogenous small molecule are plausible targets for *N*-acylation, *O*-esterification or -etherification, respectively. We therefore selected 52 polar head groups including amino acids, nucleobases and biogenic amines, each of which was conjugated *in silico* to 39 fatty acid/fatty alcohol moieties ([Supplementary Dataset 1](#S1){ref-type="supplementary-material"}), altogether representing over 2000 putative molecules. A single AIF LC-MS/MS run of rat kidney extract recognized 132 ERC of 18 individual classes, out of which 74 molecules were novel ([Supplementary Table S2](#S1){ref-type="supplementary-material"}). Furthermore, we identified 3 molecules of a novel class of ERC: *N*-acylaspartates (NAAsp) having 16:0, 18:2 and 20:4 fatty acid moieties attached to the amino group of aspartic acid ([Fig. 2](#f2){ref-type="fig"}). Considering an extended list of uncommon fatty acids (*e.g.* having odd number of carbon atoms or a hydroxyl group) produced no further hits.
To further validate the identification these three NAAsp were synthesized. We found that their retention times and ratio of abundances of precursor and fragment peaks were the same as of endogenous molecules ([Fig. 2A,B](#f2){ref-type="fig"}).
How abundant were NAAsp molecules compared to other endogenous ERC? We quantified NAAsp and four major ERC classes (1-acylglycerols, 2-acylglycerols, NAE and *N*-acylglycines)[@b22] in the same extract. NAAsp was 2-fold less abundant than the least abundant endocannabinoid class of NAE (including anandamide) and 100-fold less abundant than 1-acylglycerols ([Supplementary Table S3](#S1){ref-type="supplementary-material"}), which underscores the superior sensitivity of AIF LC-MS/MS.
We recently reported that anandamide (NAE 20:4) inhibits Hedgehog (Hh) signalling by binding the 7-pass transmembrane protein Smoothened[@b15]. To ask whether NAAsp may also interfere with Hh, we compared NAE 20:4 with NAAsp for their capacity to block both Sonic Hedgehog (Shh) and SAG-induced Smoothened activation ([Fig. 2C](#f2){ref-type="fig"}). NAAsp 20:4 and NAAsp 18:2 were as active as anandamide in both assays. In contrast to anandamide, NAAsp do not bind CB~2~R, only weakly bind CB~1~R, and are resistant to major anandamide-degrading enzyme FAAH[@b23] ([Fig. 2D](#f2){ref-type="fig"}). This makes NAAsp promising candidates for pharmacological targeting of the Hedgehog signalling pathway.
Discussion
==========
We developed a method to rapidly screen for novel endogenous lipid molecules in complex biological extracts featuring notably improved sensitivity compared to "gold standard" targeted mass spectrometric analyses by MRM and PRM. Our method is unbiased and extendable to any known or plausible combination of lipid structural modules. AIF LC-MS/MS provides a rich dataset that comprises all fragment ions produced from all ionisable precursors, including fragments originating *via* novel or unexpected fragmentation pathways and intermolecular rearrangements. Conceivably, it might be possible to implement a multi-layer datamining strategy that considers alternative combinations of fragments linked by *boolean* logic operations[@b24].
We underscore that the major bottleneck of the identification of picogram amount of lipids is not the acquisition of informative MS/MS spectra or database searches, but rather in recognizing and selecting the appropriate precursor ion for the subsequent fragmentation. We demonstrated that it is circumvented by untargeted fragmentation of all ionised precursors and the complexity of highly convoluted all-fragments tandem mass spectra is compensated by high mass resolution, spectra acquisition rate and dynamic range of the Orbitrap mass analyzer. AIF LC-MS/MS outperformed targeted analytical approaches and identified all known and also a new class of ECR at the concentration levels 2-fold below the least abundant genuine EC.
We also speculate that we might have acquired traces of many more novel compounds, whose identification would require better datamining that could automatically produce and evaluate a large number of putative structures. Alternatively, it could identify candidate molecules by unbiased alignment of XICs of unspecified fragments. Altogether, it paves the way for a comprehensive and systematic characterization of endogenous lipid bioregulators in tissues and fluids and understanding their role in health and disease[@b25].
Materials and Methods
=====================
Chemicals and standards
-----------------------
LC grade solvents and common chemicals (ACS grade) were purchased from Sigma-Aldrich (Munich, Germany) and Fisher Scientific (Schwerte, Germany). Standards of endogenous and deuterium-labeled endocannabinoids, synthetic blockers used in endocannabinoid activity assays ([Fig. 2](#f2){ref-type="fig"}) were purchased from Cayman Chemical Company (Ann Arbor, MI) and Tocris Bioscience (Bristol, UK).
Extraction and quantification of ERC
------------------------------------
By LC-MS/MS on a triple quadrupole mass spectrometer TSQ Vantage (Thermo Fisher Scientific, San Jose CA) was performed as described[@b22] with minor modifications; MRM transitions and instrument settings are in [Supplementary Table S1](#S1){ref-type="supplementary-material"}. NAAsp were quantified by external calibration using unlabeled synthetic compounds. To compensate matrix effects standards were spiked into a rat tissue extract in which NAAsp were undetectable. Comparative profiling of NAE by the method of PRM on a hybrid quadrupole -- Orbitrap tandem mass spectrometer Q Exactive (Thermo Fisher Scientific) was performed as described[@b22] using inclusion list of precursor *m/z* ([Supplementary Table S1](#S1){ref-type="supplementary-material"}). Rat tissues were obtained from licensed Biomedical Services Facility (MPI of Molecular Cell Biology and Genetics, Dresden). Rat tissues were provided by the animal facility of the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany in compliance with German animal welfare legislation and in strict pathogen-free conditions. Animal handling protocols were approved by the Institutional Animal Welfare Officer (Tierschutzbeauftragter), and necessary licenses were obtained from the regional Ethical Commission for Animal Experimentation of Dresden, Germany (Tierversuchskommission Landesdirektion Sachsen).
Software for AIF LC-MS/MS discovery screening
---------------------------------------------
AIF LC-MS/MS spectra were processed by Arcadiate 2.0 software, a tool for visualization and analysis of LC-MS/MS data. Arcadiate is available for download at: <https://itunes.apple.com/de/app/arcadiate/id585258468?mt=12&uo=4>
Arcadiate operates under a Macintosh OS v.10.8 or higher and requires a desktop PC with minimum of 4-core processor and 4 GB memory. LC-MS/MS (.raw) files should be converted to mzML or mzXML format prior processing. In the benchmarking experiment, importing and generating a chromatographic map from 520 MB mzXML file containing 1015 MS and 1016 MS/MS spectra required *ca* two minutes on a desktop PC (16 GB 1600 MHz RAM; 2.0 GHz Quad-Core Intel i7 processor). Importing a .csv library of *ca* 4000 masses of precursor and fragment ions of compound candidates and associating them with the chromatographic map took a few seconds.
Identification of ERC by AIF LC-MS/MS
-------------------------------------
The acquisition cycle consisted of FT MS and FT-MS/MS scans in positive ion mode. FT MS spectra were acquired in the *m/z* range of 225 to 725 at the mass resolution of R~*m/z*\ 200~ = 140,000; automated gain control (AGC) value of 1 × 10^6^; maximum injection time of 1024 ms. All-ions FT MS/MS were acquired from the same *m/z* range of precursors. Fragments were detected within *m/z* 50 to 725 at R~*m/z*\ 200~ = 17,500 with AGC value 3 × 10^6^; maximum injection time of 1024 ms; nCE was step increased at 20, 25 and 35%. Spectra in .raw format were converted to mzML and interpreted by Arcadiate software. It aligned XIC peaks of putative precursors and fragments ([Supplementary Dataset S1](#S1){ref-type="supplementary-material"}) and sorted the alignments according to their quality (see [Supplementary Text](#S1){ref-type="supplementary-material"} for algorithm details). Hits with the alignment score below 50% were discarded.
Chemical synthesis of NAAsp
---------------------------
NAAsp were synthesized in a two-step procedure. In a first step, the respective fatty acids were coupled to bis-*tert*-butyl protected aspartic acid via EDC/DMAP mediated amide bond formation. The *tert*-butyl groups were subsequently removed by treatment with TFA thus yielding the respective NAAsp. For experimental details, please see the [Supplementary Information](#S1){ref-type="supplementary-material"}.
NAAsp binding to CB~1~R and CB~2~R, and FAAH assay
--------------------------------------------------
Binding to CB~1~ and CB~2~ receptors was quantified by displacement of \[^3^H\]CP55.940 from membrane fractions isolated from mouse brain (as a source of CB~1~R) or mouse spleen (as a source of CB~2~R). FAAH activity was assayed in brain homogenates with 10 μM \[ethanolamine-^14^C\]anandamide and the release of \[ethanolamine-^14^C\] in the aqueous phase was measured. Details of experimental protocols are in [Supplementary Materials](#S1){ref-type="supplementary-material"}.
Inhibition of Hedgehog signalling by NAAsp
------------------------------------------
The effect of NAAsp on Hh signalling pathway was tested in the Shh-LIGHT2 reporter assay as described[@b15]. The tested compounds were added together with non-sterol-modified Sonic Hedgehog (Shh) or Smoothened agonist (SAG) to LIGHT2 cells. Luciferase activity was measured in cell lysates and Hh pathway activity was estimated as a ratio between Firefly: Renilla luciferase (see [Supplementary Materials](#S1){ref-type="supplementary-material"} for details).
Additional Information
======================
**How to cite this article**: Bilgin, M. *et al*. Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS. *Sci. Rep.* **6**, 27920; doi: 10.1038/srep27920 (2016).
Supplementary Material {#S1}
======================
###### Supplementary Information
###### Supplementary Dataset 1
MM was partly supported by PRIN 2010--2011 grant from the Italian Ministry of Education, University and Research. Work in AS, CS and SE laboratories was supported by TRR83 grant (Projects A17, A2 and A19, respectively) from Deutsche Forschungsgemeinschaft (DFG). AS, SE, and AN are members of the Lipid Center program of Max Planck Gesellschaft.
**Author Contributions** M.B., M.W. and A.S. designed the study; P.B. and S.E. performed Hedgehog signalling inhibition assay; F.F., N.M. and M.M. performed endocannabinoid activities assays; M.H., C.S., N.W. and A.N. synthesized *N*-acylaspartates; M.W. developed Arcadiate software for new lipids identification; M.B., C.S., M.M., A.N., M.W. and A.S. wrote the manuscript.
![Design and validation of the lipid discovery workflow.\
(**A**) AIF LC-MS/MS screen for molecules consisting of a polar head group H and fatty acid moiety F. A putative compound *f*~*j*~*h*~*i*~ is identified if XIC peaks of its precursor ion \[*f*~*j*~*h*~*i*~\]^+^ (in blue) and fragment \[*h*~*i*~\]^+^ spectra (in red) align (two-colour arrow). (**B**) Molecular ion and the head group fragment of *N*-acylethanolamines (NAE). (**C**) Venn diagram of endogenous NAE independently identified by all-ions fragmentation (AIF) and parallel reaction monitoring (PRM) on a Q Exactive and by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer.](srep27920-f1){#f1}
![Identification and biological activity of N-acylaspartates.\
(**A**) Alignment of XIC peaks of the \[M+H\]^+^ precursor and head group fragment (upper panel) identified NAAsp 20:4, consistently with the structure of its molecular ion (**B**) and independent analysis of the synthesised molecule (lower panel). (**C**) Shh-LIGHT2 cell reporter assay showing that NAAsp inhibit Hedgehog (Hh) signalling. Hh activation after stimulation with Smoothened agonist (SAG) or non-sterol-modified Shh was reduced by 15 μM of NAAsp 16:0; NAAsp 18:2; NAAsp 20:4; NAE 20:4 (positive control) and *O*-acylguanosine 18:1 (negative control) (*n* = 3). (**D**) NAAsp (10 μM) did not interact with CB~2~ and very weakly with CB~1~ receptors; they also did not inhibit FAAH. Ctrl stands for negative control; SR141716A (1 μM); SR144528 (1 μM) and URB597 (0.1 μM) are synthetic blockers serving as positive controls. (*n* = 4; ^\*^p \< 0.05; ^\*\*^p \< 0.001 and ^\*\*\*^p \< 0.0005 *vs* control).](srep27920-f2){#f2}
[^1]: Present address: Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100, Copenhagen, Denmark.
|
{
"pile_set_name": "PubMed Central"
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|
Introduction
============
Human prion diseases include those inherited as autosomal dominant traits, those acquired because of prion-contaminated food, medical products or instruments, and sporadic forms. Although dementia is a core clinical feature, most studies have focused on the neurological and psychiatric, rather than the specifically cognitive, signs and symptoms. Many patients are only diagnosed relatively late in the disease course, a function both of very rapid progression and its relative rarity. The question of whether there is any consistency to the cognitive profile has rarely been addressed.
Phenotypic heterogeneity is regarded as the norm with variability in presentation mainly reflecting the relative timing of cognitive to neurological and psychiatric features. Variant Creutzfeldt-Jakob disease (vCJD) predominantly affects a younger age group, with prominent early psychiatric and sensory symptoms, such as limb pain and dysesthesia.[@b1] Iatrogenic CJD is generally a cerebellar syndrome followed by cognitive change at a relatively late stage of the illness.[@b2],[@b3] There are also differences amongst the inherited forms. For instance, patients with the P102L mutation typically experience cerebellar ataxia well before cognitive dysfunction emerges[@b4],[@b5] while in 6-OPRI (octapeptide repeat insertion, an inherited prion disease \[IPD\] mutation in *PRNP*) patients cognitive impairment is a prominent early sign,[@b6],[@b7] with milder or absent cerebellar signs initially.
While cognitive impairment in prion disease is usually considered to be generalized, some features have recurred in previous reports. Executive deficits have been reported in a number of studies.[@b8]--[@b10] A second feature, often remarked but rarely investigated, is progressive loss of speech.[@b10]--[@b13] Prominent visual symptoms -- the "Heidenhain variant" of sporadic CJD (sCJD) -- have sometimes been identified.[@b14],[@b15] Memory impairment has figured more significantly in some studies than others[@b9],[@b10] but is a less prominent feature than in other dementing illnesses. Finally, patients, even with rapidly progressive sCJD, sometimes present with focal cognitive deficits including hemispatial neglect,[@b16] or language disturbance.[@b12],[@b17]
The view that this is a generalized dementia without distinctive cognitive features has been challenged in one study.[@b10] Notwithstanding heterogeneity of presentation in six patients, common qualitative features were observed including periods of unresponsiveness, intrusion errors from both auditory and visual stimuli, perseveration in the context of preserved self-reflection, and preservation of awareness of illness. They suggested these features might be characteristic of the disease as such, reflecting a fundamental impairment in the activation and regulation of cortical activity from subcortical structures.[@b18],[@b19]
In the current study, comprising near comprehensive nationwide recruitment of patients with all types of prion disease[@b20] we had a unique opportunity to document for the first time the cognitive profile of a large cohort of prion disease patients including the refinement of an appropriate battery of tests. We analyzed performance on cognitive tests in comparison with matched controls, grouped by brain region, ranked by commonly used cognitive and functionally orientated rating scales, by statistical techniques used to reduce complex data sets, and by correlation with demographic and clinical variables, investigations and molecular factors known to be determinants of phenotypic heterogeneity. The opportunity to characterize such a profile offers the possibility of improved operational criteria for diagnosis of the disease.
Methods
=======
Two cognitive batteries were used: a specially devised Short Cognitive Examination (SCE) which could be administered even to patients with moderately advanced disease in their homes, and a comprehensive neuropsychological examination for administration only to mildly affected patients. Using both of these batteries we aimed to detect a broad pattern of performance in the larger patient group, which could then be investigated in more detail in the smaller, less affected group.
Participants
------------
Patients were recruited through the NHS National Prion Clinic (NPC) at the National Hospital for Neurology & Neurosurgery, UCLH NHS Foundation Trust, London, U.K. Ethics approval for the study was granted by the Eastern Multicentre Research Ethics Committee and informed consent for participation was given either by the patient or their next of kin. A total of 456 patients with suspected or confirmed prion disease were recruited to the National Prion Cohort Monitoring Study or MRC PRION-1 trial from 2004 to May 2013. Of these, 81 participants deemed to be symptomatic and able to complete the SCE, were included in the study. Participants were excluded if they were too impaired at the time of the initial assessment to complete the SCE (139 cases); if they were at risk of either genetic or iatrogenic disease but not symptomatic (21 cases); or if they were eventually found to have another neurological disorder (37 cases). Thirty patients were well enough to travel and undergo comprehensive neuropsychological assessment all of which were conducted by D. C., usually in the Neuropsychology Department of the National Hospital for Neurology and Neurosurgery. Definitive diagnosis of prion disease was made either by genetic testing in the case of inherited disease or by postmortem neuropathology. A matched control group of 36 healthy individuals recruited from amongst the patients\' families, to control for possible confounding factors such as education and IQ, was also recruited to the study. This included participants at risk of IPD but who on testing were gene mutation negative. Thirty-three subjects from the original control group underwent neuropsychological testing in addition to the SCE.
Clinical testing
----------------
All participants underwent systematic neurological examination in addition to cognitive examination. The neurological assessment included the NPC-devised MRC Prion Disease Rating Scale which includes neurological, cognitive, and functional components and provides a measure of overall disease severity.[@b20]
Cognitive investigation of the patients comprised two components:
The SCE included the Mini Mental State Examination (MMSE) and a battery of tasks devised to target the cognitive domains reported to be vulnerable in prion disease.[@b8],[@b21] It included brief tests of the following (with maximum number of items in brackets): recognition memory (words \[/12\] and faces \[/12\]), attention (digit span\[/5\]), parietal lobe function (spelling\[/6\], calculation\[/4\], praxis\[/10\]), language (object naming\[/12\], reading\[/5\]), executive function (letter fluency \[number of words in 60 sec\]), perception (incomplete letter recognition\[/3\]), and processing speed (letter cancellation \[time taken\]).*Neuropsychological examination* which include a comprehensive battery of standardized tests: Current intellectual functioning (WAIS-III \[Wechsler 1997\]; seven sub-tests: vocabulary, similarities, digit span, arithmetic, picture completion, picture arrangement and block design); premorbid optimal level of function (National Adult Reading Test[@b22]); Visual and verbal recognition memory (Recognition Memory Test[@b23]); visual (AMIPB complex figure[@b24]) and verbal recall (Paired Associate Learning[@b25]) recall; Language including nonword repetition,[@b26] category ("Animal") fluency; object naming (Graded Naming Test (GNT)[@b27]) synonym matching; and sentence comprehension (Test for Reception of Grammar \[TROG\][@b28]); Visual perception and visuospatial function (Visual object and space processing battery \[VOSP\] Object Decision, Cube Analysis[@b29]; visuoconstruction[@b24]); limb praxis (meaningful[@b30]) and meaningless[@b31] gesture; spelling (Graded Difficulty Spelling Test[@b32]); and calculation (Graded Difficulty Calculation Test (GDCT)[@b33]); Executive function (Modified Card Sorting Test[@b34]; Stroop Test[@b35]; Verbal fluency (FAS)[@b36]; Trail Making Test \[TMT\] Part B[@b37]); and Information processing speed (TMT Part A[@b37]).
The neuropsychological assessment was carried out at the same time as the neurological and clinical investigations, or as close to that as possible.
Statistical analysis
--------------------
We used analysis of variance (ANOVA) and independent *t*-test or its nonparametric equivalent to compare patients\' and controls\' scores on individual components of the two batteries. Multiple comparisons were done between different components, however, these were not independent tests and therefore *P* values uncorrected for multiple testing are presented. SCE scores were also subjected to a principal components analysis (PCA) with orthogonal varimax rotation to identify any clustering of individual measures. The PCA also generated axes (termed Axes 1, 2, etc. in rank order of declining proportion of variance explained) which were used to investigate possible correlation with demographic, clinical category, genetic (*PRNP* mutation and polymorphic codon 129 genotype), and investigation variables (MRI brain signal change in cortex, basal ganglia or thalamus, cerebrospinal fluid (CSF) 14-3-3 protein and electroencephalography \[EEG\]). To address the relative sensitivity of individual tasks comprising each of the two batteries, we calculated the proportion of patients whose performance was impaired on each test. Missing data were treated with a missing at random approach. Statistical analyses were performed using the statistical package for the social sciences V.11.5 (SPSS, IBM, New York).
MRI studies
-----------
Diagnostic MR brain images performed at multiple sites in the U.K. were acquired and re-reported by H. H. and categorized according to clinical normality/abnormality in cerebral cortex (two areas involved and excluding areas known to generate false-positive signal), thalamus, and basal ganglia.
For the subgroup of patients who attended National Hospital for Neurology and Neurosurgery (NHNN) for detailed neuropsychological assessment, 3 T MRI was also acquired. Spatial processing for voxel based morphometry (VBM) was performed for structural T1-weighted data using SPM Version 8 software (SPM8, <http://www.fil.ion.ucl.ac.uk/spm>) as follows: (1) SPM8\'s unified segmentation approach, which combines segmentation, bias correction, and normalization to the MNI (Montreal Neurological Institute) space into a single generative model.[@b38] The rigid component of the normalization transformation was used to produce approximately aligned images for the following step. (2) Generation of a cohort-specific template for gray matter (GM) and white matter (WM) segments using DARTEL.[@b21] (3) Warping and resampling of individual GM and WM segments to the cohort-specific template. Local intensities were *modulated* to account for volume changes associated with the normalization. (4) An isotropic 6-mm full-width-at-half-maximum (FWHM) Gaussian kernel was applied to the gray and WM data sets. (5) An "objective" masking strategy[@b39] was employed to define the voxels for subsequent statistical analysis on GM and WM segments separately. For statistical analysis a group level random effect model Analysis of Covariance (ANCOVA) consisting of diagnostic grouping (controls, symptomatic patients) with individual age and total intracranial volume (GM + WM + CSF segments) as covariates, was performed. In the symptomatic patients, we also assessed the correlation between PCA Axis 1 and Axis 2 scores with GM and WM separately, with individual age and total intracranial volume as covariates. For multiple comparison correction we used voxel-wise false discovery rate (FDR) with *P* \< 0.05. SPM-t maps were produced using a *P* \< 0.05 level of significance after multiple comparison correction using FDR. After results were computed, they were affine transformed to MNI, by affine registering the Dartel template to the MNI space tissue prior probability maps. Results are displayed overlaid on the average of the warped T1 volumes, transformed to MNI space. To illustrate the actual change in GM fraction, a region of interest (ROI) was chosen in the area of the largest cluster of significant voxels. The ROI was manually drawn on the average warped and smoothed T1 volumes by an experienced neuroradiologist and verified on the averaged smoothed data sets to ensure the smoothing did not cause CSF contamination. The correlation between angular gyrus GM fraction and neuropsychology was assessed with the Spearman-rank correlation.
Results
=======
Patient diagnosis
-----------------
Demographic information and MMSE scores for the patients who completed the SCE are reported in Table[1](#tbl1){ref-type="table"}(1). The diagnoses were: sCJD (*n* = 40/81, 49.5%); IPD (*n* = 28/81, 34.5%); iatrogenic CJD (human pituitary growth hormone) (*n* = 8/81, 10%); or vCJD (*n* = 5/81, 6%). The sCJD group included patients will all three genotypes at polymorphic codon 129 of *PRNP* (129MM = 7, 129MV = 20, 129VV = 12, 1 not tested). The IPD group were made up of patients with nine different genetic mutations (P102L \[*n* = 7\], Y163X \[*n* = 2\], 5-OPRI \[*n* = 4\], 6-OPRI \[*n* = 4\], E200K \[*n* = 4\], E196K \[*n* = 1\], D178N \[*n* = 2\], Q212P \[*n* = 1\], A117V \[*n* = 3\]). Sixty-two patients subsequently died, 46 of whom had an autopsy; the clinical diagnosis of prion disease was confirmed in all these. The control group was slightly younger on average than the patients (*P = *0.020) and, unsurprisingly, their MMSE scores were significantly higher than those of the patients (*P \< *0.001).
######
Demographic information (1) with MMSE, for patients assessed on the SCE; and (2) with estimated IQ, for patients assessed on the neuropsychological examination
Patients Controls
----------- ---------- ------------- ------------- ---- ------------- -------------
\(1\) SCE
Male 48 54.0 (14.0) 20.7 (6. 6) 18 51.0 (12.3) 29 (1.0)
Female 33 56.8 (11.6) 21.8 (5.0) 18 48.0 (13.6) 30 (0.70)
Total 81 55.4 (13.7) 21.2 (4.4) 36 49.3 (12.9) 29.5 (0.90)
Patients Controls
-------------------------------------- ---------- ------------- -------------- ---- ------------- ------------
\(2\) Neuropsychological examination
Male 16 49.7 (10.2) 105.1 (15.8) 17 50.0 (12.5) 104 (11.2)
Female 11 52.0 (12.4) 99.9 (9.5) 16 47.0 (14.5) 108 (15.9)
Total 30 50.6 (11.0) 103.0 (13.6) 33 49.0 (13.4) 106 (13.6)
SCE, short cognitive examination; NART, national adult reading test.
Short cognitive examination
---------------------------
Disease severity, early signs, and symptoms and their relative distribution can be seen in Table[2](#tbl2){ref-type="table"}(1). As expected, there was a highly significant difference in mean score between patients and healthy controls on all components of the SCE (see Table S1). Comparison of the proportion of patients with possible or probable impairment on each test showed highly significant differences between tests (ANOVA, *P *\<* *0.001, Fig.[1](#fig01){ref-type="fig"}). These results raised the possibility that some cognitive domains may be more vulnerable than others in this disease. Subgroups, including disease category, age of onset, gender, *PRNP* codon 129, and imaging variables, showed highly consistent test sensitivities (see Table S3). Post hoc analyses also raised the possibility of homogenous subgroups of tests (e.g., Letter fluency, calculation, naming, letter cancelling, spelling, praxis vs. all others, *P* = 0.05, Student--Newman--Keuls method).
######
Clinical features at the time of (1) SCE and (2) neuropsychological examination. The two most dominant clinical features are shown in bold
Clinical feature \(1\) SCE (*n* = 81) *N* (%) patients affected \(2\) Neuropsychological examination (*n* = 30) *N* (%) patients affected
---------------------------------------------------- ------------------------------------------------ ---------------------------------------------------------------------------
**Cognitive complaint** **72 (89)** **22 (73)**
**Ataxia** **63 (78)** **16 (53)**
Anxiety/depression 32 (39) 7 (23)
Speech difficulty 28 (35) 6 (20)
Personality change[1](#tf2-2){ref-type="table-fn"} 28 (35) 10 (33)
Apraxia 28 (35) 8 (27)
Myoclonus 26 (32) 6 (20)
Extra-pyramidal signs 19 (23) 5 (17)
Hallucinations/delusions 19 (23) 4 (13)
Pyramidal signs 17 (21) 1 (3)
Sensory Disturbance 17 (21) 1 (3)
Diarrhea 2 (2) 1 (3)
SCE, short cognitive examination.
Aggressivity/irritability; withdrawal/loss of drive; emotional lability.
{#fig01}
We went on to use PCA as a hypothesis free method to identify key structures in the psychological data set. The components can be conceptualized as a single variable derived from combinations of test scores that account for a maximal proportion of overall variance. The first component (Axis 1) explained 42.1% of the variance in the patient group. The second component (Axis 2) accounted for just 15.4% of the variance (Table[3](#tbl3){ref-type="table"}). Axis 1 was most strongly correlated with the following tasks: spelling, calculation, naming, digit span, reading, praxis and letter fluency, very similar to the homogeneous subgroup suggested by post hoc studies above. No significant correlations were found between Axis 1 and diagnosis, mutation, age, gender, or *PRNP* codon 129.
######
Principal components analysis axis loadings. Bold tests are the strongest correlates of each axis
1 2
-------------------- ----------- ------------
Spelling **0.828** 0.226
Calculation **0.792** 0.194
Naming **0.761** 0.243
Digit span **0.758** 0.089
Reading **0.693** −0.157
Praxis **0.594** 0.376
Letter fluency **0.552** 0.441
Fragmented letters 0.409 0.367
MRC scale −0.010 **0.795**
Memory -- visual 0.310 **0.785**
Letter cancel −0.054 −**0.773**
Memory -- verbal 0.226 **0.693**
MRI analysis
------------
From 30 patients attending for detailed neuropsychological examination at NHNN, 23 patients had 3 T research MRI. Estimated GM partial volume fraction significantly correlated with Axis 1 (reduced GM content was associated with reduced Axis 1 score) in numerous frontal and parietal regions including the superior parietal lobule, supramarginal gyrus, inferior temporal gyrus, middle frontal gyrus, inferior frontal gyrus and pars triangularis, more on the left than on the right (Fig.[2](#fig02){ref-type="fig"}). There were no significant correlations between Axis 2 with either GM or WM, nor between Axis 1 and WM. Angular gyrus GM partial volume fraction correlated significantly with Axis 1 (Fig.[2](#fig02){ref-type="fig"}C) with a Spearman rank correlation coefficient of 0.602 (*P *=* *0.004).
{#fig02}
Detailed neuropsychological examination
---------------------------------------
There was considerable heterogeneity in clinical presentation of the 30 patients undergoing detailed neuropsychological assessment (see Tables[1](#tbl1){ref-type="table"}(2) and [2](#tbl2){ref-type="table"}(2) for clinical and demographic information). There was no age-difference between the patients and the healthy controls who also underwent neuropsychological examination (*t*\[61\] = 0.546, *P *=* *0.590). In estimating IQ, based on the national adult reading test (NART) reading test, three patients with dyslexia were removed from the analysis. Estimated IQ was very slightly higher amongst the healthy controls (mean = 109.30, SD = 11.70) than patients (mean = 103.0, SD = 13.6, *P = *0.059).
Comparison of the difference between patients\' and controls\' optimal full-scale IQ (FSIQ) as estimated on the NART and current FSIQ as measured on the WAIS-III showed a significant change for the patients (Wilcoxon signed-rank test, *P \< *0.001) but not for the controls (*P = *0.062), confirming a marked decline in general intellectual function in this disease. A significant difference between patients and healthy controls was found on all tasks, as was the case with the SCE (see Table S2). Thus, here again the group analysis was not as helpful as interrogation of individual patients\' scores in terms of elucidating patterns of performance.
Based on our findings from analysis of the SCE we predicted that eight tasks would be most impaired on detailed neuropsychological assessment (Stroop Test, TMT Part A, TMT Part B, Praxis, GDCT, Animal fluency, FAS, and GNT) compared with the 17 other tasks (see Table[4](#tbl4){ref-type="table"}). Considering only those impaired (\>2 SD difference from the mean of controls), 152/240 patient-tests were impaired from those tests which were a priori expected to be most abnormal; 207/510 patient-tests were impaired from the remainder (*P *\<* *0.0001, Fisher\'s exact test).
######
Summary of the neuropsychological data for *n* = 30 prion patients
--
--
Table[4](#tbl4){ref-type="table"} shows for each patient whether performance was impaired (\>1 SD or \>2 SD outside the mean for healthy controls) on each test. The left-most group of columns (gray headings) include the cognitive domains reflected in Axis 1 and representing tests of executive function, language, and parietal lobe function, and praxis. The MRC Scale score can be seen alongside the patients\' MMSE score. The tests are arranged in each domain in order of the decreasing percentage of patients liable to be affected in that domain. Table[4](#tbl4){ref-type="table"} also demonstrates that patients with more severe cognitive deficits as identified on the MMSE were impaired in all domains, and sometimes on all or almost all the tasks in each domain. What is of interest here is domains in which the more mildly affected patients were also shown to have deficits, thus offering the possibility of eliciting a more subtle pattern of cognitive decline.
All of the patients were impaired on at least one executive task, with a majority (23/30; 77%) performing below healthy controls on three of the four tests of executive function. A significant proportion was also impaired on tests of language (24/30; 80%). This included not only category fluency (24/30; 80%) and object naming (GNT: 20/30; 67%), but also sentence comprehension (TROG: 21/30; 70%). In contrast only 50% (15/30) had difficulty with a nonspoken test of semantic knowledge (Concrete Synonym Matching), and only 47% (14/30) on each of two tests of repetition. In terms of parietal function both calculation GDC: 23/30; 77%) and praxis (21/30; 70%) were impaired even in more mildly affected patients. While performance on the other parietal tests individually were less liable to be affected as many as 83% (25/30) of patients experienced parietal lobe dysfunction of one kind or another. Thus, confirming the outcome of the PCA for the SCE, the most prominent cognitive symptom, even in the context of mild disease, was executive function with performance also poor on tests of both language (27/30; 90%) and, to a slightly lesser extent, parietal function (25/30; 83%).
Turning to the other cognitive domains, of the memory tasks, nonverbal recall was the most liable to be affected. Of the patients who performed poorly at figure recall, however, 75% (18/24) also scored poorly on the copy condition of the task, with which scores on the memory task were strongly correlated (*r* = 0.563, *P \< *0.001). This likely reflects the significant visuospatial demand of this task. Fewer patients were impaired on the other memory tasks (13/30; 43%--19/30; 63%), consistent with the fact that memory did not load significantly on the first component of the PCA. A large proportion of patients (25/30; 83%) were impaired on a test of psychomotor processing speed (TMTA), a task with strong visuoperceptual and motor demands. Fewer were affected on two less perceptually demanding tests of speed and attention (Reading time on the Stroop Test: 15/30; 50%; Forward Digits: 14/30; 47%). In summary, consistent with the findings on the SCE, the individual neuropsychology patient data revealed a profile comprising prominent executive, language and parietal deficits with memory, speed and attention relatively spared. This was most evident in the ten most mildly affected patients (Table[4](#tbl4){ref-type="table"}, cases 1--10, MMSE ≥29).
Discussion
==========
We have studied a large group of mildly affected prion disease patients by comprehensive, systematic cognitive investigation, and correlated these measures with clinical and molecular investigation. Consistent with the view that prion disease gives rise to pervasive cognitive decline, most patients were impaired in all or most cognitive domains. Nevertheless, principle component analysis revealed an axis comprising tests of frontal executive function, language and parietal functions, which accounted for almost half the variance in the sample. This axis also correlated strongly with GM atrophy in frontal and parietal areas detected on MRI. When patients were ranked by MMSE score, the implicated tests were found to be impaired in incipient disease. Taken together, these findings indicate that a coherent constellation of cognitive variables associated with fronto-parietal function can be considered the leading cognitive features in prion disease, irrespective of etiology.
Executive dysfunction was shown to be a leading cognitive symptom, with all patients impaired in this domain. Executive deficits are often a feature of dementia syndromes[@b40] but they are usually not the leading sign, although PSP may be an exception in this regard.[@b41] Executive deficits were accompanied by personality change -- irritability, aggressiveness, emotional lability -- in about half of patients undergoing either the SCE or full neuropsychological assessment. There is little suggestion in prion disease, however, of the disorder of social cognition with disinhibition seen in behavioral variant frontotemporal dementia (bvFTD).[@b42]
Even mildly affected patients were impaired on some language tasks: letter fluency, animal fluency, sentence comprehension, and object naming. Fewer were impaired on tests of repetition or semantic knowledge. Unlike the logopaenia associated with repetition deficits seen in Alzheimer\'s disease,[@b43] prion patients have reduced output and poor sentence comprehension without repetition deficits, suggesting an executive rather than a phonological underpinning to the language disorder, the precise nature of which is yet to be elucidated.
Although memory complaints are common, memory contributed only to the second axis of the PCA. Just half the sample performed poorly on all or even most of the memory tasks. Many patients were impaired on the adult memory and information processing battery (AMIPB) test of delayed figure recall although, as suggested earlier, this was partly due to impaired visuospatial function, evident in a poor figure copy. Clinically, prion patients are not repetitive in conversation, do not characteristically fail to recognize clinicians and others, and do not seem bewildered in their forgetfulness in the way that patients with Alzheimer\'s disease (AD) do. These findings confirm the impression that, unlike typical AD or even bvFTD,[@b44] an amnesic syndrome per se is not a particularly prominent feature. This may reflect the distribution of pathological changes, implicating the thalamus and basal ganglia in prion disease rather than the frontal, temporal, and posterior cortical regions known to be differentially affected in AD and FTD.[@b45]
Although the first component of the PCA included digit span, calculation, and reading, detailed neuropsychological assessment of these functions showed only calculation to be vulnerable in the majority of patients. On the other hand taking all parietal tasks into account, many patients were impaired in this domain (83%). Apraxia was present in more than two-thirds of cases. Thus, although there is evidence of significant parietal compromise bilaterally, the specific symptomatology is somewhat variable from case to case.
The cognitive signs in mild prion disease thus comprise executive deficits, a largely expressive language disorder, and a constellation of parietal signs including visuospatial impairment and apraxia. Memory is less markedly affected as are semantic knowledge, processing speed and attention. The cognitive deficits arise in the context of a movement disorder in the form of ataxia with other neurological signs including myoclonus and apraxia affecting a smaller proportion of patients. From the point of view of differential diagnosis, prion disease thus resembles movement disorders with associated dementia syndromes including corticobasal degeneration (CBD), PSP, Amyotrophic lateral sclerosis (ALS), and perhaps Lewy body disease. A review of the CBD literature[@b46] yielded a very similar result to that reported here: heterogeneity of presentation but with characteristic features including limb apraxia, constructional and visuospatial difficulties, acalculia, frontal dysfunction, and a nonfluent aphasia. Episodic memory was variable, but when present impairment tended to be milder than in Alzheimer\'s disease. Semantic memory is relatively preserved but a nonfluent speech disturbance is common, and may be the presenting feature.[@b46] A similar neuropsychological profile has also been reported in PSP[@b47] without the prominent language disorder and with a different constellation of neurological signs. Language and executive deficits have been found to be the most prominent cognitive features in ALS, together with changes in behavior and social cognition. Parietal signs are less frequent and the neurological concomitants are also very different from those seen in prion disease.[@b48],[@b49] Dementia with Lewy bodies also falls within the constellation with a characteristic profile of deficits in visuospatial ability and frontal executive function accompanied by mild-to-moderate Parkinsonism.[@b50] Language disturbance is not a prominent feature. Prion disease is thus most similar to CBD but with both a language disorder and motor features that are distinctly different from that condition in the majority of patients.
The results of this study give strong indications for an appropriate test battery for early diagnosis of prion disease. In our view, this should comprise tests of: (1) executive function including response inhibition (Stroop) and generativity (verbal fluency); (2) tests of parietal function including higher order visuospatial function (complex figure copy), calculation and praxis; (3) tests of language including language production (category fluency, nonword repetition) and sentence comprehension; (4) tests of speed of information processing. Tests of memory, visual processing, reading and attention should also be included to avoid false positive findings.
In summary, this is the only large study of the neuropsychology of prion disease ever undertaken. Overall, the results confirm that all patients ultimately develop a global cognitive impairment. However, our data clearly show that frontal and parietal functions are particularly vulnerable in the context of mild disease, even allowing for differences in the overall pattern of symptomatology, including neurological and psychiatric features, in some forms of the disease. This neuropsychological profile taken together with the characteristic neurological features of the disease constitutes a signature that should lead to more straightforward and rapid differential diagnosis of incipient cases in a clinical setting. Given the prospect of further clinical trials for prion disease, we have recommended that functionally orientated scales should be used in rapidly progressive patients.[@b51] Asymptomatic at-risk individuals and early symptomatic patients, such as those studied in this paper, represent an alternative and attractive group to target with an experimental therapy, assuming an adequate safety profile, prior to extensive neuronal damage. Future work building on this study will be directed toward operationalization of a neuropsychological test battery and natural history database to enable timing of disease onset and document cognitive progression in these patient groups. This may be facilitated by further characterization and differentiation of the language disorder in prion disease, in comparison with those found in frontotemporal lobar degeneration and Alzheimer\'s disease.
We thank the *National Prion Disease Monitoring Cohort steering committee*- C. Kennard, P. Mills, V. Farewell, P. Chinnery, A. Mackay, E. Riboli, R. Knight. We thank all the patients, their carers and families, who took part in the Cohort study and U.K. neurologists and the National CJD Research and Surveillance Unit for referring patients. We thank officials at the Department of Health, Medical Research Council Research Management Group staff, co-chairs of the PRION-1 and Cohort trial steering committee, and our colleagues at the National CJD Research Surveillance Unit for establishing the National CJD referral arrangements, without which these studies would not have been possible. We thank all past and present colleagues at the NPC (formerly at St Mary\'s Hospital, London and now at the National Hospital for Neurology and Neurosurgery, Queen Square, London) in particular Kizzy Alner, Sylvia Gamazo- Navarro, Durre Siddique, Suvankar Pal, Thomas Webb, Diego Kaski, Dillip Gallujipali, Nina Bjurstrom Kathryn Prout, Nora Heard, Clare Morris, Rita Wilkinson, Chris Rhymes, Suzanne Hampson, Claire Petersen, Chris Carswell, Joanna Field, Elisabeth Morgan, Imogen Eastwood, Liz Ford, Jane Owen, Veronica O\'Donnell, Michele Gorham, Ekaterina Kassidi, and Colm Treacy and at the Medical Research Council Clinical Trials Unit, in particular Michael Ranopa, Geraldine Keogh, Moira Spyer, Debbie Johnson, Liz Brodnicki, and Patrick Kelleher. The Cohort study was funded by the Department of Health (England) and the National Institute of Health Research\'s Biomedical Research Centre at University College London Hospitals NHS Foundation Trust.
Conflict of Interest
====================
None declared.
Supporting Information
======================
Additional Supporting Information may be found in the online version of this article:
######
**Table S1.** Comparison of patients\' and controls\' mean scores on all components of the SCE using the Mann--Whitney *U*-Test.
**Table S2.** Comparison of patients\' and controls\' mean scores on all components of the Neuropsychological Examination using the Mann--Whitney *U*-Test.
**Table S3.** Consistent impairments in prion disease subgroups. Ranking of proportion of subjects impaired or possibly impaired (\>1 SD below mean, or imperfect score if all controls scored perfectly) from most to least proportion impaired. The proportion impaired in each neuropsychological test was remarkably consistent in the known subgroups of disease, early age of onset, gender, *PRNP* codon 129 genotype and imaging findings. Note that too few subjects had normal CSF or EEG examinations to allow for meaningful comparisons of these diagnostic tests.
[^1]: **Funding Information** The Cohort study was funded by the Department of Health (England) and the UCLH/UCL Biomedical Research Centre with additional support from the Medical Research Council.
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"pile_set_name": "PubMed Central"
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1. Introduction {#sec1}
===============
Cerebral ischemia is defined as a condition where the brain or its parts do not receive enough blood flow to maintain normal neurological function. This causes metabolic changes and possibly cellular death. Restoration of blood flow, although necessary for brain survival, could lead to excessive reactive oxygen species (ROS) formation and nitric oxide synthase (NOS) activation with resulting oxidative/nitrosative stress. Thus, cerebral ischemia and reperfusion can produce neuronal damage triggering a complex series of biochemical events that affect structure and function of brain.
Mechanisms implicated in neuronal death are complex and multifactorial, also involving intracellular Ca^++^ overload with consequent activation of nitric oxide synthase (NOS), the enzyme that catalyses the synthesis of nitric oxide (NO), a small reactive gas acting both as a signalling molecule and a neurotoxin \[[@B1]--[@B4]\]. Several experimental evidence demonstrated a dual role for NO following an ischemic episode \[[@B5]--[@B7]\]; in fact, it can exert detrimental and beneficial effects depending on several factors such as the isoform of NOS involved, the amount of NO released, the time and the site of NO production. To this regard, NO produced by endothelial NOS (eNOS) immediately after ischemic attack, promoting vasodilatation, may play a protective role. Later, when overactivation of neuronal NOS (nNOS) and *de novo* expression of inducible NOS (iNOS) occur, the contribution of NO to brain damage becomes relevant.
An increasing number of reports in the literature indicate that endogenously produced inhibitors of NOS, particularly asymmetric dimethylarginine (ADMA), regulate NO generation, and then may be implicated in the pathophysiology of several disorders \[[@B8]--[@B12]\]. The major pathways for ADMA elimination are via renal clearance and via metabolism to L-citrulline by the intracellular enzymes dimethylarginine dimethylaminohydrolase (DDAH 1 and DDAH 2) and it is increasingly apparent that metabolism of ADMA is highly regulated \[[@B13]\]. Thus, regulation of DDAHs might represent a novel mechanism by which ADMA levels can be modulated to regulate NO generation and to evaluate possible therapeutical options \[[@B14]\].
Although the pathobiological mechanisms of ischemia/reperfusion injury are multifactorial, oxidative stress seems to represent the common final path \[[@B15]\]. Recently, intense interest has focused on the antioxidant properties of natural products. In particular, natural products may act by preventing the free radical generation, neutralizing free radicals by nonenzymatic mechanisms, and/or by enhancing the activity of endogenous antioxidants \[[@B16]\] such as stress-inducible proteins.
Heme oxygenase (HO) (EC 1.14.99.3) is a microsomal enzyme that oxidatively cleaves heme and produces biliverdin, carbon monoxide (CO) and iron \[[@B17]\]. To date, two isoforms of HO have been identified: HO-1, or inducible enzyme, and HO-2 or constitutive isoform \[[@B17]--[@B21]\]. A substantial body of evidence demonstrates that HO-1 induction represents an essential step in cellular adaptation to stress subsequent to pathological events \[[@B13], [@B22]--[@B25]\]; then HO-1 hyper-expression can be considered both a marker of cellular stress and also regarded as a potential therapeutic target in a variety of oxidant-mediated diseases \[[@B26]\].
Recently it has been reported that polyphenolic natural compounds are able to induce potently HO-1 expression, exercising protective effects \[[@B27]--[@B29]\]. As a consequence, the beneficial actions attributed to several natural substances could be also due to their intrinsic ability to activate the HO-1 pathway \[[@B27]--[@B29]\]. The list of natural compounds acting as antioxidants includes anthocyanins, a widespread group of water-soluble plant constituents collectively known as flavonoids.
Cyanidin-3-O-*β*-D-glucoside (C3G) is a natural compound whose antioxidant, anti-inflammatory, and iron-chelating properties have been demonstrated in numerous studies using several methods, both *in vivo* and *in vitro* \[[@B30]--[@B33]\].
The present *in vivo* study was performed to verify whether the treatment with C3G is able to counteract oxidative stress induced by postischemic reperfusion and if its effect may be mediated by HO-1. In addition, the possibility of an interference of C3G on DDAH/NOS pathway was also tested.
2. Material and Methods {#sec2}
=======================
2.1. Animals {#sec2.1}
------------
Male Wistar rats (100--120 g b.w.) were fed a certified balanced diet and kept in temperature (20 [+]{.ul} 1°C) and humidity (50%) controlled rooms, caged with raised floors of wide mesh. The animals were deprived of food for 12 hours before experiment but allowed free access to water. All the experimental procedures reported in this study were approved by the Animal Care and Use Committee of University of Catania, Italy (approval number 037, prot. 37394 TIT cc VIII/2).
2.2. Experimental Protocols {#sec2.2}
---------------------------
For experiments, animals were anaesthetized by ethyl urethane (1.2 g/kg b.w., i.p.); cerebral ischemia was induced by bilateral clamping of common carotid arteries for 20 min. The induction of ischemia was confirmed by measuring lactate levels. A lot of untreated, sham-operated animals was used as control. C3G-pretreated and post-treated sham-operated rats were also included in the experimental protocol. Sham-operated animals did not undergo ischemia and reperfusion: they were anesthetized, their skin was incised, and the carotid arteries were exposed, but not occluded. All the animals were sacrificed by injection of an overdose of anaesthetic. Rats were randomly divided into 3 groups: (a) saline-treated animals, (b) C3G-pretreated rats, and (c) C3G posttreated rats. C3G-pretreated rats were injected with 10 mg/Kg intraperitoneal (i.p.) 1 h before the induction of cerebral ischemia; in C3G post-treated rats the same dosage of C3G was injected during reperfusion (30 min after restoring blood flow). These times were chosen according to data reported in literature about plasma concentrations of C3G after i.p. administration \[[@B34]\]. Ischemic rats were sacrificed immediately after 20 min of bilateral clamping of carotids; animals subjected to postischemic reperfusion were sacrificed after 3 or 24 h restoring blood flow. Since ischemic rats were sacrificed immediately after 20 min ischemia, we could not administer the cyanidin 30 min after restoring blood flow.
2.3. Survival Rate {#sec2.3}
------------------
Percentage of survival was determined by keeping 30 animals, submitted to experimental procedure of 20 min partial cerebral ischemia, under observation for 24 hours. A group of saline-treated, ischemic rats were used as a reference. A lot of sham-operated (both saline- and C3G-pre and posttreated) animals were regarded as control group.
All brains were rapidly removedin a cold room, frozen at −80°C and processed for biochemical analysis within 3 days. Brain tissue was homogenized in 9 volumes of the cold proper buffer. Aliquots of homogenate of each sample were used for determining brain levels of lactate, non proteic thiol groups (RSH) and lipid peroxide (LOOH), for the evaluation of heme oxygenase (HO-1) by specific enzyme-linked immunosorbent assay (ELISA) kit, for expression of *γ*-glutamyl cysteine synthase (*γ*-GCS), endothelial, neuronal, and inducible nitric oxide synthetases (eNOS, nNOS, and iNOS, resp.), DDAH-1 and DDAH-2 by western blot (WB), and for determination of DDAH activity. Results of WB were normalized to *β*-actin and expressed as Arbitrary Units (AU). Protein content was determined by Lowry\'s method \[[@B35]\].
2.4. Lactate Level Determination {#sec2.4}
--------------------------------
For the determination of the lactate levels the tissue was homogenized in 20 mM glycylglycine buffer, pH 10, containing 70 mM glutamate; the homogenate was deproteinized by 4% HClO~4~ (final concentration); the spectrophotometric assay was performed following NADH formation at *λ* = 340 nm using Noll\'s method \[[@B36]\].
2.5. Nonproteic Thiol Group Determination {#sec2.5}
-----------------------------------------
Cerebral levels of non proteic thiol groups (RSH) were measured in 200 *μ*L of brain homogenate using a spectrophotometric assay based on the reaction of thiol groups with 2,2-dithio-bis-nitrobenzoic acid (DTNB) at *λ* = 412 nm (*εM* = 13,600) \[[@B37]\]. Results are expressed as nmoles/mg proteins [+]{.ul} S.D.
2.6. Determination of Lipid Hydroperoxide Levels {#sec2.6}
------------------------------------------------
The levels of lipid hydroperoxides were evaluated following the oxidation of Fe^+2^ to Fe^+3^ in the presence of xylenol orange at *λ* = 560 nm \[[@B38]\]. The assay mixture contained, in a total volume of 1 mL: 100 *μ*L of brain homogenate, 100 *μ*M xylenol orange, 250 *μ*M ammonium ferrous sulfate, 90% methanol, 4 mM butylated hydroxytoluene, 25 mM H~2~SO~4~. After 30 min incubation at room temperature, the absorbance at *λ* = 560 nm was measured using a U2000 Hitachi spectrophotometer (Tokyo, Japan). Calibration was obtained using hydrogen peroxide (0.2--20 *μ*M). Results are expressed as nmoles/mg proteins [+]{.ul} S.D.
2.7. Determination of HO-1 by ELISA {#sec2.7}
-----------------------------------
For the determination of HO-1 protein content, 100 *μ*L of brain homogenates were assayed by enzyme-linked immunosorbent assay (ELISA) \[[@B39]\] (Stressgen, VIC, Canada), according to manufacture\'s instructions. Results are expressed as pg/mg protein [+]{.ul} S.D.
2.8. Western Blotting {#sec2.8}
---------------------
Brain homogenates were collected for western blot analysis and protein levels were visualized by immunoblotting with antibodies against *γ*-GCS, nNOS, eNOS, iNOS, DDAH-1, or DDAH-2. Briefly, aliquots of homogenate containing 50 *μ*g of proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. In order to block nonspecific binding sites, the membranes were incubated overnight with 5% nonfat dry milk in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20 (TBST) buffer at 4°C. After washing with TBST, the membranes were incubated with a 1 : 1000 dilution of anti-DDAH-1, DDAH-2, or anti *γ*-GCS antibodies, and with 1 : 500 dilution of anti-eNOS, iNOS or n-NOS for 12 hours at 4°C with constant shaking. The filters were then washed and subsequently probed with horseradish peroxidase-conjugated anti-rabbit for *γ*-GCS, eNOS, nNOS, and iNOS at a dilution of 1 : 20000, anti-goat for DDAH-1, and DDAH-2 at a dilution of 1 : 10000. Detection was performed using an Enhanced Chemiluminescence Detection kit according to the manufacturer\'s instructions. Results are expressed as Arbitrary Units (AU) normalized with *β*-actin.
2.9. DDAH Activity Assay {#sec2.9}
------------------------
Brain homogenates were centrifuged at 5000 xg for 60 min at 4°C and supernatants were collected for DDAH activity assay, performed by determining L-citrulline formation in 96-well microtiter plate, according to Knipp\'s method \[[@B40]\]. Results are expressed as units/mg protein [+]{.ul} S.D. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of one mmol L-citrulline/min at 37°C.
2.10. Protein Assay {#sec2.10}
-------------------
Protein content was evaluated according to the method of Lowry \[[@B35]\].
3. Statistical Analysis {#sec3}
=======================
One-way analysis of variance (ANOVA) followed by Bonferroni\'s *t*-test was performed in order to estimate significant differences among groups. Data were reported as mean values ± S.D. and differences between groups were considered to be significant at *P* \< 0.005.
4. Results {#sec4}
==========
As already reported in our previous studies, this experimental procedure allowed the induction of cerebral ischemia \[[@B41]--[@B48]\]; in fact a significant increase in lactate levels was found in ischemic animals compared to sham-operated controls (sham-operated: 22.84 ± 3,42 nmol/mg prot, ischemic animals: 72.15 ± 2.89 nmole/mg prot. Each value represents the mean ± SD of 10 animals; *P* \< 0.001).
[Figure 1](#fig1){ref-type="fig"} reports RSH levels in brain tissues of rats submitted to our experimental conditions of partial cerebral ischemia and subsequent reperfusion, both in saline-treated animals and in C3G pre- or posttreated rats. No significant change was observed in saline-treated rats after 20 min ischemia or after 3 h postischemic reperfusion respect to sham-operated animals; after 24 h postischemic reperfusion, brain RSH levels underwent to a significant 23% reduction with respect to sham-operated animals. The same figure also shows results obtained in C3G-treated animals; in sham operated animals, C3G administration induces a significant increase in RSH levels with respect to untreated rats. However, C3G treatment was not able to induce significant modifications in RSH levels in ischemic rats or after 3 h postischemic reperfusion. Significant differences in brain RSH levels are evident after prolonged postischemic reperfusion; in fact, after 24 h postischemic reperfusion, C3G-pretreated rats showed higher RSH levels with respect to untreated animals; however C3G resulted less efficient in maintaining RSH levels when administered after ischemic event (posttreatment).
[Figure 2](#fig2){ref-type="fig"} reports LOOH levels; in saline-treated animals a significant decrease of such oxidative stress marker is evident after 20 min ischemia and after 3 h postischemic reperfusion. These results can be explained considering the need of oxygen for LOOH formation. Following 24 h postischemic reperfusion LOOH levels are similar to those observed in sham-operated rats. C3G treatment significantly reduces LOOH levels in sham-operated animals; however, after 20 min ischemia or after 3 h postischemic reperfusion, neither pretreatment nor posttreatment with C3G induced significant differences with respect to untreated rats. By contrast after 24 h postischemic reperfusion, C3G efficiently contrasted lipid peroxidation; in fact, LOOH levels resulted significantly decreased in C3G-treated animals when compared to untreated rats.
Results regarding HO-1 content are reported in [Figure 3](#fig3){ref-type="fig"}. In untreated animals a significant increase in HO-1 is evident after 24 h postischemic reperfusion. C3G treatment caused a significant increase in HO-1 levels in sham-operated rats, whilst no significant difference was observed between untreated and C3G-treated animals, neither after 20 min ischemia nor after 3 h postischemic reperfusion. The HO-1 inducing effect of C3G is evident in brains of rats pretreated with C3G and submitted to 24 h postischemic reperfusion; in fact, as shown in [Figure 3](#fig3){ref-type="fig"}, when C3G is injected before ischemic insult, it increased HO-1 expression following 24 h reperfusion. This effect was not evident when C3G was administered after the ischemic injury.
No significant change in *γ*-GCS was observed in the brain of saline-treated rats submitted to our experimental conditions of cerebral ischemia and reperfusion ([Figure 4](#fig4){ref-type="fig"}). The same figure also shows the effect of C3G treatment: in sham operated C3G-injected animals a significant decrease in *γ*-GCS expression was observed with respect to untreated animals; however the same pretreatment resulted in a gradual and significant increase following ischemia and subsequent reperfusion particularly. The posttreatment with C3G induced a significant increase in enzyme expression after 3 h of reperfusion; following 24 h postischemic reperfusion the enzyme expression was similar to that observed in saline-treated animals at the same reperfusion time.
Results regarding iNOS expression in brains of rats underwent our experimental conditions of partial and transient cerebral ischemia are reported in [Figure 5](#fig5){ref-type="fig"}; iNOS is expressed in untreated, both sham-operated and ischemic animals. After 3 h and 24 h postischemic reperfusion a significant increase in iNOS expression was observed. The same figure also shows results concerning the effects of C3G treatment. As it can be seen, C3G pretreatment caused a significant reduction of iNOS expression which is evident both in sham-operated and in ischemic or postischemic reperfused rats. Similar results were obtained when C3G was injected after ischemic insult.
The expression of the neuronal isoform of nitric oxide synthase is reported in [Figure 6](#fig6){ref-type="fig"}. In brains of untreated animals the expression of this protein is significantly augmented following 24 h reoxygenation compared to sham-operated rats. The same figure also reports the effect of C3G treatment on the expression of nNOS: no significant difference was observed between saline- and C3G-treated animals either after 20 min ischemia or after 3 h reperfusion. More relevant changes in nNOS expression were observed after 24 h postischemic reperfusion, when both pre- and posttreatment with C3G significantly decreased nNOS expression.
The pattern of eNOS expression, reported in [Figure 7](#fig7){ref-type="fig"}, showed a significant decrease in untreated animals following 20 min ischemia and after 3 or 24 h reperfusion. C3G treatment induced a significant increase in eNOS expression in sham-operated animals; however, only the pretreatment was able to maintain high levels of expression of eNOS after ischemia and 3 or 24 h reperfusion; in fact, C3G posttreated rats showed eNOS levels similar to those observed in untreated animals.
Western blot analysis of cerebral contents in DDAH-1 evidenced that this protein was poorly modified under our experimental conditions of cerebral postischemic reperfusion. In fact, as shown in [Figure 8](#fig8){ref-type="fig"}, no significant change in DDAH-1 levels was observed in saline-treated animals between sham-operated and ischemic, 3 h or 24 h postischemic reperfused rats. C3G both pre- and posttreatment induced a significant increase in DDAH-1 expression with respect to saline-treated rats. Highest levels of DDAH-1 were observed in brains of rats C3G-pretreated and reperfused for 24 hs.
Very relevant resulted the changes observed in DDAH-2 expression and reported in [Figure 9](#fig9){ref-type="fig"}. In untreated rats, DDAH-2 expression significantly increased after 3 h reperfusion and then trended to reduce again to control values. C3G treatment caused a significant DDAH-2 reduction in sham-operated animals and this trend was also kept under ischemia/reperfusion conditions, both following pretreatment and post-injection with C3G.
Results regarding the determination of DDAH activity are reported in [Figure 10](#fig10){ref-type="fig"}: the pattern resembles DDAH-2 expression, both in untreated and in C3G-treated animals.
The administration of C3G significantly increased the survival rate of rats, both if administered before and after ischemic injury ([Figure 11](#fig11){ref-type="fig"}). In sham-operated rats, both untreated and C3G-treated the survival rate was 100% ([Figure 11](#fig11){ref-type="fig"}).
5. Discussion {#sec5}
=============
Stroke represents one of the major causes of death or invalidity in developed countries. In most cases, stroke results from the obstruction of blood flow in a major cerebral vessel. Understanding biochemical mechanisms involved in brain damage subsequent to ischemic injury is crucial for developing new therapies. After ischemic insult, neuronal cell death proceeds through a mixture of mechanisms including excitoxicity, apoptosis, inflammation and oxidative stress \[[@B15]\]. The occurrence of this cascade of events was also demonstrated under *in vivo* experimental conditions of partial and transient cerebral ischemia in rats and was indirectly confirmed by protective effects observed following treatment with drugs acting with different molecular mechanisms \[[@B41]--[@B46]\]. Over recent years, neuroscientists have acquired a considerable body of evidence to support the fact that the mammalian brain can adapt to injurious insults such cerebral ischemia, thus increasing the chances of survival \[[@B47], [@B48]\]. So, sublethal ischemic insults may protect tissues from subsequent insults. This phenomenon is known as *preconditioning* or i*schemic tolerance* (also defined as a short, sublethal ischemic episode that activates endogenous mechanisms able to protect organs or tissues from further longer and more severe episodes of ischemia); while it would be dangerous and impractical to precondition at-risk patients with ischemia, elucidation of the endogenous cell survival pathways involved in ischemic tolerance has many clinical implications and may lead to new therapeutic strategies. HO-1 (also known as stress protein HSP32) can be over-expressed in many tissues following stressful stimuli including hypoxia, hyperoxia, ischemia-reperfusion, and a wide range of conditions characterized by alteration of the cellular redox state \[[@B22], [@B28], [@B49]--[@B55]\]. HO-1 expression might represent an important protective endogenous mechanism; in this regard, induction of this enzyme has shown beneficial effects in several pathological conditions \[[@B56], [@B57]\]. In this context, pharmacologic modulation of HO-1 system may represent an effective strategy to intervene in several pathologic conditions but it is important to induce HO-1 expression without causing cell damages. Recently the ability of several natural antioxidants to induce HO-1 has been reported \[[@B27], [@B58]--[@B62]\]. The anthocyanin are part of the widespread group of plant constituents, collectively known as flavonoids. Cyanidin-3-O-*β*-glucoside, also known as kuromanin, is probably the best known and most investigated cyanidin-glycoside. There are several reports mentioning beneficial effects of C3G, such as prevention of LDL oxidation, cardiovascular diseases, inflammation and obesity, vascular failure, and myocardium damage, besides the well-known free radical-scavenging activity \[[@B32], [@B63]--[@B71]\]. Results obtained in the present study confirmed antioxidant properties of C3G and also suggested that it was not merely attributable to its antioxidant activity.
Interestingly, the effect of C3G treatment on *γ*-GCS expression seemed to be related to cellular needs; in fact, the significant reduction in *γ*-GCS expression observed in C3G-treated sham-operated animals suggested that under physiological condition the antioxidant activity of C3G made cells adequately protected from oxidant; however, the same treatment was able to induce significant increases in *γ*-GCS in animals underwent to ischemic/reperfusion damage thus enhancing glutathione (GSH) levels and their defences against oxidative stress.
Our previous studies, carried out on the same experimental model of partial and transient cerebral ischemia, indicated that the environment of injured neurons seemed to determine the ability of axons to regenerate after injury \[[@B47], [@B72]\]. To this regard, endogenous mediators affecting vasculature such as endothelium-derived NO, inducing smooth muscle relaxation and then vasodilatation of brain vessels, might improve tissue perfusion, attenuating the ischemic insult and promoting functional recovery of the infarcted brain area. Consequently, upregulation of eNOS may serve a protective role by facilitating the maintenance of cerebral blood flow and promoting revascularisation after an ischemic insult. Here reported data demonstrated that C3G treatment was able to induce e-NOS expression both in sham-operated animals and in ischemic/reperfused rats (although pretreatment had more efficient results). In addition it has to be noted that C3G treatment evoked other very important effects on NO pathway. Large amounts of NO produced by nNOS and iNOS contribute to metabolic deterioration and adversely affect the ischemic brain. Results obtained in the present study demonstrated the ability of C3G administration to significantly decrease iNOS and nNOS expressions. One of the more relevant effects of C3G treatment in postischemic reperfusion brain damage was probably due just to its ability to discriminate between endothelial or inducible/neuronal NOS isoforms. Numerous reports in the literature indicated that endogenously produced inhibitors of NOS, particularly ADMA, regulate NO generation \[[@B14], [@B73], [@B74]\]; ADMA levels, in turn, are highly regulated by DDAH enzymes, which are responsible for ADMA metabolism. Thus, activation or hyperexpression of DDAHs, resulting in enhanced catabolism of the endogenous NOS inhibitor ADMA, lead to increased amounts of NO. Conversely, specific inhibitors of DDAH activity or expression induce an accumulation of the inhibitor and then smaller amounts of NO.
Results obtained in the present study evidenced no significant change in DDAH-1 expression in untreated, ischemic/reperfused rats. In the same experimental group the observed changes in enzymatic activity appeared to be mainly due to DDAH-2isoform. In fact enzyme activity showed the same trend of DDAH-2 expression, both in untreated and in C3G-injected rats. In this regard, particularly interesting was the significant decrease observed both in DDAH-2 expression and DDAH activity of C3G-treated rats. Given the pleiotropic effect of NO during cerebral ischemia, DDAH has the potential to regulate all the effects of NO through modulation of ADMA levels. Another equally relevant result was that cyanidin was able to produce its effects even when administered after ischemic insult, not only when given before ischemia.
The ability of C3G to inhibit DDAH activity and expression, along with the capacity to selectively affect different NOS isoforms strongly suggested an important therapeutic role of this anthocyanin also confirmed by the significant increase in survival rate, observed both in C3G -pretreated and post-treated rats. The multiplicity of effects observed in C3G-treated rats indicated a complex mode of action of the natural molecule involving several pathways. All these features, according to reports of Min et al. \[[@B75]\], suggested that it might be very useful both in prevention and therapy of postischemic reperfusion brain damage.
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[^1]: Academic Editor: Yukihiro Shoyama
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Background
==========
Fibromyalgia syndrome (FMS) is a non-inflammatory rheumatologic disease characterized by widespread musculoskeletal pain, lethargy and tenderness without a definite cause \[[@B1]\]. FMS diagnosis is used for heterogeneous pathological states including anxiety disorders, depression, lethargy, sleep disorders, and gastrointestinal system symptoms with widespread pain \[[@B2]\]. One of the reasons that FMS is thought to be such a heterogeneous disease may be due to concurrent psychological disorders. Indeed, psychiatric symptoms are very common in this syndrome, and they influence the course of the disease \[[@B3],[@B4]\]. FMS patients are reported to have increased depression, anxiety and bipolar disorder comorbidities \[[@B3],[@B5]\]. Several studies determined disorders in personality inventory profiles based on the thought that some personality and mood disorders might predispose to FMS \[[@B6]-[@B8]\].
Dopamine is an important mediator in both psychopathological incidents and pain conduction. Dopamine D2 receptor sensitivity and density are increased in FMS patients \[[@B9]\]. Also, dopamine D4 receptor gene polymorphism is found to be relevant to FMS personality profile \[[@B10]\].
SNAP-25 protein is contributory to plasma membrane and synaptic vesicle \[[@B11]\]. SNAP-25 forms complexes with synaptobrevin in synaptic vesicles and with syntaxin in the plasma membrane \[[@B11]\]. In simple terms, SNAP-25 protein is critical in neurotransmission for fusion of plasma membrane and synaptic vesicle. Several studies investigated the relationship between SNAP-25 gene polymorphism and personality disorders, schizophrenia, and attention deficit and hyperactivity disorder; these studies reported that the SNAP-25 gene might influence development of these disorders \[[@B12]-[@B17]\]. Furthermore, SNAP-25 protein might be relevant to other different neurotransmitters due to its involvement in vesicle membrane transition and fusion. This is important because it can be the main reason behind neurological, cognitive, psychological disorders in FMS. If such a relationship exists, it will be enlightening for etiopathogenesis of FMS and treatment approaches. We aimed to evaluate the SNAP-25 gene (MnlI = rs3746544 and DdeI = rs1051312) polymorphism, which is related to many psychiatric diseases, and FMS association in this prospective study.
Patients and methods
====================
Patients and evaluation
-----------------------
We included 71 female patients diagnosed with ACR 2010 fibromyalgia diagnosis criteria and 57 age-matched healthy females in the study. Patients and healthy volunteers were informed about the genetic evaluation and informed consent was taken. Blood (10 cc) was taken into EDTA tubes from both the patient and control groups and stored at -20°C. FMS new diagnosis criteria scoring (ACR 2010), VAS, SF-36, and Beck depression scale were applied to the patient group. SNAP-25 gene polymorphism prevalence and SNAP-25 polymorphism with disease activation association were compared. The Local ethics council of Pamukkale University Clinical Research Ethics Committee approved the study. Patient consent was obtained for this study.
Tools
-----
A sociodemographic information form was developed by the researchers and includes patient's age, sex, education, socioeconomic status, settlement, marital status and disease period. The Beck depression scale (BDS) was used for determining the risk for depression, levels of depressive symptoms and difference of intensity. It was developed by Beck et al. and adapted to Turkish by Hisli (Hisli 1989). The VAS consists of 3 parts for measuring pain. The scale was adapted to Turkish and used in numerous studies. The SF-36 scale to determine quality of life was subjected to validity and reliability testing for its use in Turkish.
Molecular analysis
------------------
Genetic evaluation was done in our university's Medical Genetics Department. Patient and control genomic DNA was isolated from peripheral blood by a DNA Extraction Kit. The SNAP-25 genes MnlI (rs3746544) and DdeI (rs1051312) polymorphisms are on the 8^th^ exon (Forward 5′- TTC TCC TCC AAA TGC TGT CG-3′ and Reverse 5′- CCA CCG AGG AGA GAA AAT G-3′ primary series were used to replicate the UTR region).
In addition to these primer series, we also used 10X PCR Buffer, 5 μl dNTP mix consisting of 0.2 mM of every nucleotide and Taq polymerase enzyme. PCR reaction conditions were 95 degrees C for 2 minutes of denaturation followed by 95 degrees C for 45 seconds, 58 degrees C for 1 min, 72 degrees C for 2 minutes of 35 cycles and final elongation at 72 degrees C for 7 minutes.
10 U Ddel and 10 U MnlI enzymes were added separately to obtained 261 bp PCR products and this was incubated for 14 hours at 37°C for cutting. 3.5% Ultra pure agorose jelly was prepared for separating fragments after cutting. Later, PCR products were subjected to 40--50 minutes of electrophoresis and fragmented. After electrophoresis, the allele band series expected for Ddel polymorphism: for T allele: 261 bp cut band, for C allele: 228 bp and 33 bp two separate bands. The allele band series expected for MnlI polymorphism: for T allele: 256 bp and 5 bp two separate bands, for G allele: 210 bp, 46 bp and 5 bp three separate bands.
Statistical analysis
--------------------
For statistical analysis SPSS version 20 was used. Descriptive statistics were given as mean, standard deviation and percentage. Out study's confidence interval was 95%. Intergroup significance was evaluated with Chi square test for qualitative data and Mann--Whitney U test for quantitative data. Quantitative data relationships were evaluated with Spearman correlation test. Non parametric variance analysis was done between genetic polymorphisms. p \< 0.05 was considered as significant.
Results
=======
Mean age was 38±5,196 and 38.12±4.939 in patient and control groups, respectively (p=0.542). No significant difference was found between the groups regarding age, height, weight, BMI, education, settlement, marital or occupational status (p \> 0.05) (Table [1](#T1){ref-type="table"}). We determined MnlI gene polymorphism for TT genotype in 30 (42.3%), TG genotype in 34 (47.9%) and GG genotype in 7 (9.9%) patients. Control group had TT genotype in 27 (47.4%), TG genotype in 19 (33.3%) and GG genotype in 11 (19.3) patients. Dde1 genotype variance in patient group was as follows: TT genotype in 29 (40.8%), TC genotype in 40 (56.3%), TC genotype in 17 (29.8%) and CC genotype in 4 (7%). Intergroup SNAP-25 Mnl1 polymorphism variance was not significantly different (Table [2](#T2){ref-type="table"}). Dde1 polymorphism variance between groups showed significant difference (p = 0.009). To determine the reason behind this significance, separate match evaluation was applied and TC genotype was discovered to be the reason. Patient and control groups Mnl1 and Dde1 polymorphism variance is given in Table [2](#T2){ref-type="table"}.
######
Demographic findings of patient and control groups
**Parameter** **Patient (n=71)** **Control (n=57)** **p**
-------------------------- -------------------- -------------------- -------
**Age** 38±5,196 38.12±4.939 0.542
**Number of labor** 2.01±0.902 1.96±0.566 0.721
**Number of children** 1.97±0.828 1.91±0.576 0.646
**Settlement n (%)**
Urban 57 (80.3) 45 (75) 0.536
Rural 14 (19.7) 15 (25)
**Marital status n (%)**
Married 69 (97.2) 56 (98.25) 0.398
Single 2 (2.8) 1 (1.75)
**Occupation n (%)**
Working 21 (29.57) 17 (29.82) 0.703
Not working 50 (70.43) 40 (70.18)
######
Mnl1 and Dde1 polymorphism variance in patient and control groups
**Patient n (%)** **Control n (%)** **p**
--------------- ------------------- ------------------- -----------
TT 30 (42.3) 27 (47.4) 0.149
TG 34 (47.9) 19 (33.3)
GG 7 (9.9) 11 (19.3)
**Dde1 gene**
TT 29 (40.8) 36 (63.2) **0.009**
TC 40 (56.3) 17 (29.8)
CC 2 (2.8) 4 (7)
We evaluated fibromyalgia new diagnosis criteria and their subscores, Beck depression scale, visual analogue scale (VAS), short form-36 (SF-36) subparameter scores (Table [3](#T3){ref-type="table"}). Non parametric variance analyses between genetic polymorphisms were applied. When evaluated for Mnl1 polymorphism individuals for all three (TT, TG, GG) genotypes, VAS, BDS and SF-36 did not show significant differences (p \> 0.05). When evaluated for Dde1 polymorphism individuals for all three (TT, TC, CC) genotypes, no significant difference was found for TT and CC individuals; however, TC genotype individuals were significantly higher for BDS (p = 0.045) and VAS scores (p = 0.033) (Table [4](#T4){ref-type="table"}). This difference was significant after Bonferoni correction between the groups.
######
Fibromyalgia new diagnostic criteria, VAS, Beck depression scale, SF-36 scores for patients
**Parameter** **Score**
-------------------------- --------------
WPI1 15.27±1.80
Lethargy 2.28±0.565
Not rested 2.20±0.624
Cognition 1.82±0.661
Part 2a 6.35±1.43
Part 2b 1.99±0.521
**BDS** 16.77±10.638
**VAS** 85.04±12.629
**SF-36**
Physical function 20.08±4.09
Physical role difficulty 4.97±1.25
Pain 5.33±1.33
General Health 11.25±4.01
Vitality 12.83±3.56
Social function 6.423±2.05
Emotional role 3.704±1.0197
Mental health 18.60±4.95
######
VAS and BDI scores according to the presence TC genotypes
**Parameter** **All patients** **TC (+)** **TC (-)** **p**
--------------- ------------------ ------------- ------------- -----------
BDS 16.77±10.6 17.6±11 15.18±10 **0.045**
VAS 85.04±12.629 86.41±12.14 83.14±11.83 **0.033**
Discussion
==========
The etiopathogenesis of FMS, which is characterized by widespread musculoskeletal pain and tenderness, is not clearly understood. FMS, chronic lethargy syndrome, irritable intestine syndrome, tension type headache, and myofacial pain syndrome are parts of central sensitization syndromes. Every one of these syndromes overlaps with FMS. One common feature of these syndromes is increased central neuron stimulation through various synaptic and neurotransmitter/neurochemical activities without structural pathology. This, however, is characterized by increased sensitivity to pressure and touch. Many factors contribute to central sensitization syndrome. These include pain, lethargy, sleep disorder, sensitivity to several stimuli, and psychosocial problems.
For the past several years, environmental as well as genetic conditions are blamed for FMS and related functional somatic disorders such as irritable intestine syndrome, migraine, and chronic lethargy syndrome \[[@B18]-[@B20]\]. Several studies tried to explain the etiopathogenesis of fibromyalgia syndrome. These studies showed that serum and central nervous system serotonin and its metabolite levels are low, and serotonin transport velocity in cerebrospinal fluid is slow. For this reason, the serotonin transport gene region has been brought to attention. Offenbaecher et al. reported that the S/S genotype is increased in FMS patients when compared to healthy subjects \[[@B21]\].
Dopamine is another molecule that is generating research interest, which has a role in both psychopathologic incidents and pain transport. Studies determined both dopamine levels and dopamine receptor gene polymorphisms and FMS \[[@B9],[@B10],[@B22],[@B23]\].
It is known that psychiatric disorders, especially depression and anxiety disorder are quite common in FMS patients \[[@B3]\]. Several studies reported depression prevalence in FMS between 20% and 80% \[[@B3],[@B4],[@B7]\]. It is reported that FMS patients have difficulty in both understanding their own feelings and coping with emotions. Several studies determined disorders in personality inventory profiles based on the thought that some personality and mood disorders might predispose to FMS. When the personality inventory of patients was evaluated, hypochondriasis, hysteria, paranoia, and depression scales were found to be higher than control groups \[[@B6]-[@B8]\]. Studies indicated that low ability to cope with everyday problems can trigger FMS or can increase symptom intensity \[[@B6],[@B8]\]. As a result, some psychiatric disorders are more prevalent in FMS, and some personality and mood features can predispose to FMS development.
Nevertheless, the relationship between these situations and FMS is not clearly known. Psychiatric disorders are increased in FMS and are relevant to symptom intensity \[[@B4],[@B7]\]. Some personality and mood features can predispose to FMS, in addition to these features revealing themselves as a result of stress for coping with pain \[[@B24],[@B25]\].
There can be common pathophysiological features between FMS and psychiatric disorders and personalities. Dopamine and serotonin mediated neurotransmitter transport can be among these common biological factors \[[@B23]\]. However, these disorders have some common risks such as exposure to difficult experience. These factors cause stress in the patient, and prolonged stress leads to cytokine secretion. Increased cytokines precipitate both psychiatric diseases and increased pain perception \[[@B26],[@B27]\].
It is impossible to explain only one cause for FMS and the etiopathogenesis of these disorders. In addition, the relationship between concurrent disorders and FMS is not clearly known.
SNARE (**s**oluble **N**-ethylmaleimide-sensitive factor **a**ctivating protein **re**ceptor) proteins have a role in fusion between organelles, and organelles with plasma membrane in eukaryotic cells. SNAP-25 is a SNARE protein found in the plasma membrane. These proteins have important roles in electrical conduction in nerve cells. Nerve transporters are found in synaptic vesicles and sent by exocytosis to the other synapse. For this reason, SNAP-25 affects dopamine and other neurotransmitter secretions, and thus adds to FMS pathogenesis. Therefore, SNARE like proteins should be studied carefully, leading to better understanding of etiopathogenesis and treatment approaches. Also, the relationship between FMS and concurrent psychiatric disorders and personality features can be explained. For this purpose, we found increased SNAP-25 Dde1 TC gene polymorphism in FMS patients in this prospective study. In individuals with the TC gene genotype, BDS and VAS scores were found to be significantly higher than in individuals without TC genotype. FMS is one of the central sensitization syndromes. One mutual property of these diseases is that neurons have increased stimulus mediated by several synaptic and neurotransmitter/neurochemical activities without structural pathology. This results in increased reaction to basic stimuli. Patients who have the TC genotype have increased BSD and VAS scores in our study, which can result from different affected neurotransmitter functions both from this polymorphism and in pain transport. There are no studies done in this matter, and our results should be compared with similar studies.
The primary limitation of this study is trying to explain a complex disease such as FMS with only one mutation with a small number of patients. With more patients and other polymorphisms related to the disease, it will help us understand FMS better in the future.
Conclusions
===========
FMS etiopathogenesis is not clearly known. Numerous neurologic, cognitive and psychological disorders were found during studies attempting to identify cause. Our study showed increased SNAP-25 Ddel T/C genotype in patients compared to controls, which is related with behavioral symptoms, personality and psychological disorders in FMS patients.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
AB: Study planning, organization, data collection, evaluation of results and article writing. CS: Evaluation of results, article writing and approval. HB: Creation of patient and control groups and obtaining informed consent, data collection and statistical analysis. ET: Molecular analysis of patient and control groups. VC (senior author): Article writing and approval. 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/1471-2474/15/191/prepub>
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The essays contained within this collection derive from sessions sponsored by AVISTA and the History of Science Society at the 2003 International Congress for Medieval Studies in Kalamazoo, and by the International Congress of Medieval Art at the 2003 Annual Meeting of the College Art Association. The volume brings together research stemming from a current vibrant interest in the history of medical and scientific illustration. The editors introduce the collection as, "a conversation among scholars in fields at the intersection of the history of art, science, and medicine" (p. xvii), and indeed it is rare to come across a collected volume which sustains such consistent quality and coherent discussion within such breadth of theme. This is in part because of two recurrent topics in the book, the representation of plants (whether in herbal or botanical compilations) in the chapters by Alain Touwaide, Jean Givens, Karen Reeds and Claudia Swan, and the contribution of Leonardo da Vinci, in a trio of essays by Monica Azzolini, Piers Britton and Karen Reeds. This sense of an ongoing conversation is enhanced by the recurrence of certain manuscripts, the reiteration of shared historical concerns throughout the book, and the successful evocation of continuities which extend from the medieval to the early modern period.
This is a beautiful, intriguing and thought-provoking collection of essays. Every one has been written elegantly and with clarity, an impressive feat given the complex nature of many of the manuscript transmissions discussed. The book is also generously illustrated (though it is a shame not to have colour illustrations at some pertinent points, references are given, wherever possible, to help the reader access colour reproductions). All the essays weave together their pictorial evidence carefully in order to reach some important new conclusions. I would highlight in particular the contributions of Alain Touwaide---who suggests possibilities for the exchange of learning between Byzantines and Latins during the thirteenth-century occupation of Constantinople---and Monica Azzolini---who counteracts the traditional image of Leonardo da Vinci as an isolated genius by situating him firmly in the context of a vibrant Milanese medical community.
If the first strength of this collection lies in the detail of each case study, the second is in its constant engagement with a set of theoretical and methodological problems critical to this interdisciplinary study of the scientific image. The tone is set by Peter Murray Jones's opening essay, which demands that we, "consider the relationship of image, word, and medicine afresh" (p. 1). Common themes and questions which span the book include the practical utility or function of these images; their transmission, adaptation and creation in different contexts and for different audiences; the relationship between the textual and the visual, the image and reality. Above all the collection causes the reader to ask how these manuscripts and images would have been made and how they might have been read. In Karen Reeds's words: "For any given image, we always have to ask: utility to whom? Fidelity to what end?" (p. 236). Claudia Swan\'s final essay acts neatly as an epilogue, returning to the questions raised at the start by Peter Murray Jones, and in turn posing a fundamental question: why were these images produced at all?
The book will of course attract scholars of medieval and early modern medicine and natural history. In the broader questions raised by this collection, however, there lies significance for a much wider readership, for those interested in the history of the book as much as those concerned with the history of the image.
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Introduction {#Sec1}
============
Influenza infection can result in significant morbidity and mortality following allogeneic haematopoietic stem cell transplantation (HSCT) \[[@CR1]--[@CR3]\]. In children, the incidence of influenza following allogeneic HSCT ranges between 0.9 and 9.9% \[[@CR4]--[@CR8]\]. Administration of one dose of the seasonal inactivated influenza vaccine is recommended for children commencing 6 months following HSCT. Children aged 6 months to 8 years who have never previously been vaccinated should receive two doses, 4 or more weeks apart \[[@CR9], [@CR10]\]. However, these recommendations are based on adult studies \[[@CR10]\], with a distinct paucity of paediatric-specific data assessing the immunogenicity of the inactivated influenza vaccine following HSCT \[[@CR11]\]. Given the lack of evidence, we undertook a prospective multicentre study to evaluate the immunogenicity of the seasonal inactivated influenza vaccine in children who have undergone allogeneic HSCT compared with healthy matched controls, and to identify risk factors that predict vaccine response in this population.
Methods {#Sec2}
=======
Patient selection {#Sec3}
-----------------
Children between the ages of 6 months and 18 years who were ≥6 months and ≤2 years post allogeneic HSCT were eligible. Recruitment was undertaken during the Southern Hemisphere influenza seasons of 2013--2016 (March to September) from four tertiary paediatric haematology, oncology and bone marrow transplant units in Australia (Princess Margaret Hospital for Children, Perth; Queensland Children's Hospital, Brisbane; Women's and Children's Hospital, Adelaide; The Royal Children's Hospital, Melbourne). Healthy siblings were recruited as age-matched controls. Exclusion criteria included anaphylaxis to previous doses of any influenza vaccine, a history of egg anaphylaxis, receipt of intravenous immunoglobulin within the last 3 months, a history of Guillain-Barre syndrome or current medical condition that would be compromised by inclusion in the study. Informed consent was obtained from the parents of each child prior to recruitment.
Study design {#Sec4}
------------
Participants were vaccinated with inactivated influenza vaccine according to national Australian guidelines \[[@CR12]\]. Children receiving influenza vaccine for the first time post allogeneic HSCT were given two doses of the vaccine 4 weeks apart, with one dose given to vaccine-experienced children. For healthy age-matched controls, children less than 10 years of age receiving influenza vaccine for the first time, were given two doses of the influenza vaccine 4 weeks apart; children less than 10 years of age who had previously received the influenza vaccine and children who were 10 or older were given a single dose of the vaccine. A 0.25 mL dose was administered to children less than 3 years of age and 0.5 mL to children older than 3. The inactivated trivalent vaccine was given for the 2013--2015 seasons and the inactivated quadrivalent vaccine for the 2016 season. The specific strains included in each annual influenza vaccine were A/Victoria/362/2011 (H3N2), A/California/7/2009 (H1N1), B/Hubei-Wujiagang/158/2009 (B) for 2013; A/Texas/50/2012 (H3N2), A/California/7/2009 (H1N1), B/Massachusetts/2/2012 (B) for 2014; A/South Australia/55/2014 (H3N2), A/California/7/2009 (H1N1), B/Phuket/3073/2013 (B) for 2015; and A/Hong Kong/480/2014 (H3N2), A/California/7/2009 (H1N1), B/Phuket/3073/2013 (B), B/Brisbane/60/2008 (B) for 2016.
Blood was taken prior to each vaccination and 4 weeks following the final vaccination to assess influenza-specific immune responses. Following collection, blood samples were centrifuged and sera stored at −20 °C. At the end of each influenza season, the samples were sent to the World Health Organization (WHO) Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory where standardised hemagglutinin inhibition (HI) assays were performed to determine specific influenza antibody titres towards each virus in the vaccine \[[@CR13]\]. Viruses used for HI analysis were egg propagated; specifically for A(H1N1)pdm NYMC-X-179A (A/California/7/2009) was used for seasons 2013--2016. For A(H3N2) IVR-165 (A/Victoria/361/2011), NYMC X-223A (hy A/Texas/50/2012), IVR-175 (A/South Australia/55/2014) and NYMC X-263 (hy A/Hong Kong/4801/2014) were used for the 2013, 2014, 2015 and 2016 seasons, respectively. Split B/Yamagata viruses B/Hubei-Wujiagang/158/2009, NYMC BX-51B (B/Massachusetts/02/2012) and B/Phuket/3073/2013 were used for the 2013, 2014 and 2015 seasons, respectively. Split viruses B/Yamagata (B/Phuket/3073/2013) and B/Victoria (B/Brisbane/60/2008) were both used for the 2016 season.
Susceptibility to a vaccine-like strain was defined as a prevaccination HI titre of \<40. Seroprotection in an individual was defined as a post vaccination HI titre of ≥40. Seroconversion was defined as either a fourfold increase in HI antibody titre if the prevaccination titre was ≥10 or a rise in HI titre from \<10 to ≥40 following vaccination \[[@CR14]\]. The percentage (95% confidence interval \[CI\]) of patients who individually met the criteria for seroprotection and seroconversion to each strain of the vaccine was calculated.
Criteria as established by the Committee for Proprietary Medicinal Products (CPMP) were used to determine whether the vaccine was considered effective as to elicit an overall immunogenic response \[[@CR15]\]. According to these criteria the influenza vaccine is considered effective if it meets one or more of the following three criteria: seroprotection in \>70% of patients; seroconversion in \>40% of patients; or a geometric mean fold increase (GMFI) of \>2.5. These criteria were used to calculate one-sided *p* values in relation to the null hypothesis for overall seroprotection and seroconversion to each strain of the vaccine. GMFI was calculated for each strain as the geometric mean of the fold increase in antibody level after vaccination, with CI and one-sided *p* values estimated using a log-normal approximation for the distribution of antibody levels pre- and post-vaccination and the CPMP defined threshold of GMFI \>2.5.
Linear regression models, with change in log antibody level as the dependent variable, were used to assess the influence of clinically relevant predictors on vaccine response to each strain. The predictor variables included within the models were age at receipt of first vaccination, the number of vaccination doses received, time since transplant, and the absolute lymphocyte count at the time of first vaccination. Lower than normal limits for absolute lymphocyte counts according to age were defined as 1.7 × 10^9^/L for children \<5 years of age, 1.1 × 10^9^/L for 5 to ≤10 years, and 1.0 × 10^9^/L for ≥10 years \[[@CR16]\].
All patients enroled on the study that developed influenza-like illness were instructed to present for clinical review. Influenza detection was performed on a nasopharyngeal aspirate using polymerase chain reaction. Influenza-like illness was defined as an elevated temperature (≥37.5 °C) or a clear history of fever (e.g. chills, rigors); the presence of at least one constitutional symptom from irritability, myalgia, headache, vomiting, diarrhoea or malaise; and the presence of at least one respiratory symptom from cough, sore throat or rhinorrhoea; with the onset of symptoms occurring greater than 72 h after vaccine administration. Clinical features of all children with laboratory-proven influenza infection were documented.
This study was approved by the Child and Adolescent Health Service Ethics Committee (Ethics Approval Number 1988/EP), with ethical approval granted at all sites under the National Mutual Acceptance agreement. It conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Tokyo, 2004) and the National Statement on Ethical Conduct in Human Research, Australian National Health and Medical Research Council. The study was registered on the Australian New Zealand Clinical Trials Registry (ACTRN12614000240640).
Results {#Sec5}
=======
There were 86 children enroled in the study; 43 children who had undergone allogeneic HSCT and 43 healthy controls. The groups were frequency matched according to age (mean age: 7.7 versus 8.2 years, *p* = 0.65) and sex (females: 37.2% versus 41.9%, *p* = 0.66) Participant characteristics are listed in Table [1](#Tab1){ref-type="table"}, with individualised characteristics listed in Supplementary Table [1](#MOESM1){ref-type="media"}. The percentage of patients in the allogeneic HSCT group that were susceptible to each strain of the vaccine prior to the first dose was 65% to H3N2, 88% to H1N1 and 91% to the B strain. Susceptibility of the healthy age-matched controls was 37% to H3N2, 70% to H1N1 and 79% to the B strain. Seroprotection occurred in 81.4% to H3N2, 41.9% to H1N1 and 44.2% to B strain in children who received allogeneic HSCT compared with 97.7%, 81.4% and 69.8% for healthy age-matched controls (Table [2](#Tab2){ref-type="table"}). Seroconversion occurred in 60.5% for H3N2, 32.6% for H1N1 and 39.5% for B strain in children who received allogeneic HSCT, while 72.1%, 65.1% and 58.1% of healthy age-matched controls seroconverted to the respective strains (Table [2](#Tab2){ref-type="table"}). For children in the HSCT group who received two doses of the vaccine and seroconverted, the second dose of the vaccine was required for seroconversion to occur in 47% of patients to H3N2, 70% to H1N1 and 81% to B strain.Table 1Patient demographicsCharacteristicAllogeneic HSCT(*n* = 43)Healthy controls(*n* = 43)Age at vaccination 6 months to \<3 years96 3 to \<10 years1824 10 to \<18 years1613Number of vaccine doses One dose1123 Two doses3220Diagnosis Malignant26 Non-malignant17Donor type Matched sibling12 Matched unrelated28 Haploidentical3Interval between allogeneic HSCT and influenza vaccine 6 to \<12 months32 ≥12 months11History of graft versus host disease Yes23 No20On immunosuppressive therapy at time of vaccination Yes13 No30Table 2Overall immunogenicity to inactivated influenza vaccine in children who have undergone allogeneic HSCT and age-matched controlsStrainGMFI (95% CI)*P* value^a^Seroprotection % (95% CI)*P* value^a^Seroconversion % (95% CI)*P* value^a^Allogeneic HSCT (*n* = 43) H3N2 (A)5.80 (3.68--9.14)\<0.00181.4 (69.8--93.0)0.05160.5 (45.9--75.1)0.003 H1N1 (A)3.75 (2.20--6.40)0.06941.9 (27.1--56.6)\>0.9932.6 (18.6--46.6)0.84 B3.44 (2.36--5.00)0.04844.2 (29.3--59.0)\>0.9939.5 (24.9--54.1)0.53 B (2016)1.46 (0.79--2.71)0.9644.4 (12.0--76.9)0.9533.3 (2.5--64.1)0.66Controls (*n* = 43) H3N2 (A)8.96 (5.96--13.45)\<0.00197.7 (93.2--99.9)\<0.00172.1 (58.7--85.5)\<0.001 H1N1 (A)6.49 (4.40--9.56)\<0.00181.4 (69.8--93.0)0.05165.1 (50.9--79.4)\<0.001 B6.81 (4.41--10.53)\<0.00169.8 (56.0--83.5)0.5158.1 (43.4--72.9)0.008 B (2016)5.88 (2.24--15.41)0.04166.7 (35.9--97.5)0.5944.4 (12.0--76.9)0.39^a^One-sided *P* values in relation to CPMP criteria
According to CPMP criteria, children who had undergone allogeneic HSCT demonstrated a significant response to the H3N2 (GMFI 5.80, 95% CI 3.68--9.14, *p* \< 0.001 and seroconversion 60.5%, 95% CI 45.9--75.1, *p* = 0.003) and B (GMFI 3.44, 95% CI 2.36--5.00, *p* = 0.048) strains (Table [2](#Tab2){ref-type="table"}). Healthy age-matched controls demonstrated significant response to all vaccine strains: H3N2 (GMFI 8.96, 95% CI 5.96--13.45, *p* \< 0.001; seroprotection 97.7%, 95% CI 93.2--99.9, *p* \< 0.001 and seroconversion 72.1%, 95% CI 58.7--85.5, *p* \< 0.001), H1N1 (GMFI 6.49, 95% CI 4.40--9.56, *p* \< 0.001 and seroconversion 65.1%, 95% CI 50.9--79.4, *p* \< 0.001) and B (GMFI 6.81, 95% CI 4.41--10.53, *p* \< 0.001 and seroconversion 58.1%, 95% CI 43.4--72.9, *p* = 0.008) according to CPMP criteria (Table [2](#Tab2){ref-type="table"}).
The multivariate analysis of predictive variables revealed a significantly higher GMFI to the B strain for children who received two doses of the inactivated influenza vaccine (Two doses GMFI 4.23, 95% CI 2.74--6.53 versus One dose GMFI 1.88, 95% CI 0.90--3.94, *p* = 0.019) and who had a normal lymphocyte count at the time of vaccination (Normal range GMFI 4.22, 95% CI 2.78--6.39 versus Low GMFI 1.59, 95% CI 0.71--3.57, *p* = 0.07). Table [3](#Tab3){ref-type="table"} shows the multivariate analysis of factors predicting GMFI to each strain.Table 3Multivariate analysis of factors predicting GMFI to inactivated influenza vaccine in children who have undergone allogeneic HSCTVariableInfluenza strainH3N2 (A)H1N1 (A)BGMFI (95% CI)*P* valueGMFI (95% CI)*P* valueGMFI (95% CI)*P* valueTime since transplant\<12 months4.86 (2.84--8.33)0.422.65 (1.44--4.87)0.153.12 (1.99--4.89)0.24≥12 months9.66 (3.85--24.24)10.29 (3.64--29.08)4.54 (2.11--9.76)Age at vaccination\<10 years5.73 (3.15--10.43)0.975.31 (2.69--10.48)0.103.52 (2.15--5.76)0.58≥10 years5.91 (2.71--12.86)2.09 (0.86--5.05)3.30 (1.74--6.27)Lymphocyte count at time of vaccinationLow3.17 (1.15--8.76)0.341.71 (0.52--5.62)0.301.59 (0.71--3.57)0.07Normal range6.80 (4.03--11.46)4.61 (2.51--8.49)4.22 (2.78--6.39)Doses of vaccine receivedOne7.05 (2.77--17.98)0.937.51 (2.57--21.96)0.541.88 (0.90--3.94)0.019Two5.42 (3.13--9.38)2.95 (1.57--5.54)4.23 (2.74--6.53)GMFIs are raw subgroup means; *P* values relate to a multivariate comparison of GMFIs between subgroup
There were no adverse effects following vaccination in either the allogeneic HSCT or healthy age-matched control group. There was a single case of laboratory-proven influenza infection in the allogeneic HSCT group (2.3%). This patient was typed as having H1N1pdm09 strain in 2014, with infection occurring 50 days following completion of a two dose immunisation schedule and absence of a serological response to any vaccine strain. The patient received a 5-day course of oseltamivir following confirmation of influenza infection and did not experience any significant influenza associated complications. There were no cases of laboratory confirmed influenza in the healthy age-matched control group.
Discussion {#Sec6}
==========
Influenza infection can result in significant morbidity and mortality following allogeneic HSCT \[[@CR1]--[@CR3]\]. Vaccination with inactivated influenza vaccine represents the main strategy to prevent infection. Several studies have been conducted to determine immunogenicity of the inactivated influenza vaccine in adults who have undergone HSCT, however, there is a distinct lack of paediatric-specific data \[[@CR11]\]. Our study demonstrates that the inactivated influenza vaccine is safe and elicits a statistically significant serological response to H3N2 and B strains in children who have undergone allogeneic HSCT. However, serological response was limited in comparison with healthy age-matched controls. The only study from which meaningful paediatric-specific data can be extrapolated for comparison between children who have undergone allogeneic HSCT is from a mixed paediatric-adult study \[[@CR17]\]. In this study, seroconversion rates in 27 of the study participants under the age of 18 years were 59.3% for H3N2, 55.6% for H1N1 and 40.7% for B strain. Our current study shows comparable seroconversion rates to the H3N2 and B strains, but showed an inferior seroconversion rate to H1N1. There were notable differences between the H1N1 viruses tested in both of these studies as the Yalcin et al. \[[@CR17]\] study was conducted over the 2007--2008 influenza season, when the previous seasonal influenza H1N1 vaccine was used, in comparison with our study, which used a post-2009 H1N1 pandemic influenza virus.
The CPMP have defined criteria to assess whether influenza vaccines are effective within a population. Using these criteria, our study shows that the inactivated influenza vaccine can be considered immunologically effective against the H3N2 and B strains in children following allogeneic HSCT. For the H1N1 strain, statistical significance was not achieved according to GMFI criteria and the numerical thresholds of \>70% for seroprotection and \>40% for seroconversion were not attained. However, interpretation of these findings should also take into consideration that the CPMP criteria were defined based on applicability to immunocompetent populations, further emphasised by satisfaction of the criteria against all three strains in our age-matched healthy control population. This highlights the need for revised definitions according to different populations, such as immunocompromised children.
Previously, only two small studies assessing responses to influenza vaccination following HSCT have been exclusively performed in children \[[@CR18], [@CR19]\]. Haining et al. identified an increase in influenza A-specific CD4+ T-cell proliferation but no increase in influenza A-specific serum antibody levels in four children following vaccination with trivalent inactivated influenza vaccine 4--22 months following allogeneic HSCT \[[@CR18]\]. Guerin-El Khourouj et al. identified a cellular proliferative response to H1N1 strain in 11 of 14 children following vaccination 76--336 days post allogeneic HSCT. H1N1 antibody titres were also measured in eight participants, with seroconversion only occurring in one patient following vaccination \[[@CR19]\]. Although small numbers, both studies are important as they demonstrate that despite the absence of an influenza-specific antibody response, there was generation of an influenza-specific T-cell response. This finding is supported by an adult study, which identified a strong cell-mediated immune response to a single dose of the trivalent inactivated influenza vaccine in 14 patients following allogeneic HSCT, despite a poor influenza-specific antibody response \[[@CR20]\]. This further highlights the potential to consider alternative measures to determine vaccine efficacy in immunocompromised populations, in addition to those defined by the CPMP.
Receipt of two doses of the inactivated influenza vaccine and a normal lymphocyte count at the time of vaccination were identified as significant predictors of vaccine response to B strain. Two studies with combined adult and paediatric populations have identified time from transplantation to vaccination as one of the strongest predictors of response to trivalent inactivated influenza vaccine following HSCT \[[@CR21], [@CR22]\], with one of the studies also identifying absolute CD19^+^ cell counts as being predictive of response \[[@CR22]\]. Although both studies were not able to identify a significant benefit for administration of two doses of the trivalent inactivated influenza vaccine compared with one dose \[[@CR21], [@CR22]\], the benefit of a second vaccine dose has been demonstrated in several studies which have assessed response to the AS03-adjuvanted 2009 H1N1 vaccine in adults post HSCT \[[@CR23]--[@CR25]\], while another study identified comparable response to two doses of the AS03-adjuvanted 2009 H1N1 vaccine in adults post allogeneic HSCT to a single dose received by healthy controls \[[@CR26]\]. Transplant to vaccination interval \[[@CR24], [@CR26]\], presence of graft versus host disease \[[@CR26]\], donor type \[[@CR23]\] and receipt of immunosuppressive therapy \[[@CR25]\] have all been shown to be predictive of response to the AS03-adjuvanted 2009 H1N1 vaccine within these studies. Although this is the largest paediatric study to assess immunogenicity of the inactivated influenza vaccine following allogeneic HSCT, greater patient numbers are required to further validate the variables that have been assessed and to identify whether additional variables, such as presence of graft versus host disease, receipt of immunosuppressive therapy, donor type and stem cell source, predict response to vaccination.
To determine whether a vaccine is clinically effective traditionally relies on comparison of infection rate between unvaccinated and vaccinated subjects. In a previous retrospective study, the clinical efficacy of influenza vaccination in HSCT recipients was identified as 80% \[[@CR27]\]. However, in the modern era, all patients who have undergone allogeneic HSCT are recommended to receive inactivated influenza vaccination \[[@CR10]\], thus precluding assessment of vaccine efficacy due to the low number of unvaccinated patients. This highlights the need for alternative measures to determine the clinical efficacy of influenza vaccine for patients receiving HSCT. Incidence of laboratory-proven influenza infection was defined as the clinical endpoint for this study, limited to one case occurring in the HSCT cohort (2.3%). This patient was typed as having H1N1pdm09 infection in 2014, occurring 50 days following completion of a two dose immunisation schedule, and did not mount protective antibody titres to any strain. The published data regarding clinical effectiveness of the influenza vaccine in HSCT recipients is limited, with reported incidence rates of 10.5% following one dose of the trivalent inactivated influenza vaccine and 4.6% following administration of two doses of AS03-adjuvanted 2009 H1N1 vaccine post allogeneic HSCT \[[@CR26], [@CR27]\]. The low incidence of influenza infection in our study may also be influenced by minimising risk of exposure to infection, which is considered standard practice for children who have undergone HSCT \[[@CR28]\].
In summary, our data demonstrate that the inactivated influenza vaccine is safe and provides immunogenicity to the H3N2 and B strains in children who have received allogeneic HSCT. We identify the need for further research to improve overall vaccine response and immunogenicity to the H1N1 pandemic strain. Receipt of two doses of the inactivated influenza vaccine and a normal lymphocyte count at the time of vaccination were both predictive of response to the B strain, however larger global collaborative studies are required to validate these findings and investigate a greater number of predictive variables. A high proportion of children who received two vaccine doses and seroconverted, required the second dose of the vaccine for seroconversion to each strain. In conclusion, our study provides evidence to support the recommendation for annual administration of inactivated influenza vaccine to children following allogeneic HSCT.
Supplementary information
=========================
{#Sec7}
Supplementary Table 1
**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
The online version of this article (10.1038/s41409-019-0728-5) contains supplementary material, which is available to authorized users.
Funding {#FPar1}
=======
RSK (NHMRC APP1142627) and CCB (NHMRC APP1111596) are supported by Fellowships from the National Health and Medical Research Council of Australia. This study was funded by a Perth Children's Hospital Foundation Project Grant and a Pfizer Cancer Competitive Research Grant. The WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, is supported by the Australian Government Department of Health.
UDW, PCR and RSK conceived and designed the study. ALR, FK CF, HT, FM and RSK recruited patients onto the study. LAC, KLL and IGB performed serological analysis. PJ performed statistical analysis. ALR, PJ and RSK analysed and interpreted the results. ALR and RSK wrote the paper. All authors edited and approved the final version of the paper for submission.
The authors declare that they have no conflict of interest.
|
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"pile_set_name": "PubMed Central"
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Background {#Sec1}
==========
Oestrosis is a nasal myiasis, caused by the infestation of larvae of flies belonging to the genus *Oestrus* (Diptera: Oestridae) and is considered a severe parasitosis in sheep and goats and occasionally in other species of animals \[[@CR1]\]. The adult female fly (*Oestrus ovis*) is commonly known as the sheep nasal bot fly which swarms around the heads of animals. The females are viviparous, and deposit previously hatched larvae (\~ 500) directly to the nostril of host animals \[[@CR2]\]. These larvae are obligatory parasites of nasal cavities and sinuses. The newly deposited first-stage larvae actively migrate to the nasal passage and attach to the mucous membranes \[[@CR3]\]. Later they grow and undergo two moults to become third-stage larvae. During their migration and development, the larvae cause irritation and mechanical damage to the host nasal sinuses. The damaging effect to nasal sinuses can implicate acute clinical problems such as breathing difficulties, profuse nasal discharge and restlessness, which severely impairs the health of the affected animal \[[@CR4]\]. Moreover, biomolecules secreted and excreted by larvae induce a local and systemic immune reaction that can exacerbate the situation \[[@CR5], [@CR6]\]. Mild infestation is typically asymptomatic but may induce signs of generalised disease including emaciation, which may lead to impaired animal production and consequently economic losses \[[@CR7]\]. The infestation period is generally 25--30 days, but it can be longer (up to 10 months) depending on climatic conditions and other variables \[[@CR3], [@CR8]\]. The third-stage larvae are later expelled by the host sneezing onto the ground where they pupate and turn into adult flies. However, in some cases, the third-stage larvae fail to eject from the nasal sinuses and die. This may lead to septic sinusitis resulting in the death of animal \[[@CR9]\]. The host response to oestrosis and larval burden are related to several factors such as susceptibility of host species, chronobiology of *O. ovis* at a particular geographical region and routine animal management practices \[[@CR10]\]. Adult flies cause disturbances in flocks and substantial losses in animal production are particularly associated with the larval development of the parasite \[[@CR11]\]. Adding to these issues, this parasite is difficult to control in the environment and there is a significant disparity in the therapeutic response of sheep and goats \[[@CR12]\].
Oestrosis is primarily a myiasis of sheep and goats; however, there have been widespread reports of human infestation \[[@CR13]--[@CR15]\]. It is the most common cause of human ophthalmomyiasis and is typically occurs in shepherds and farmers \[[@CR16]\]. Although it has also been reported in patients who have no association with animal husbandry and are far from any farming zone, the number of cases is limited \[[@CR17], [@CR18]\]. A high prevalence of oestrosis in sheep and goats in a geographical region may potentially increase the risk of zoonosis and may influence the occurrences of human infection.
Understanding the distribution of oestrosis and associated risk factors is essential to improving animal health. The disease is distributed worldwide and widespread in many regions of the world. An almost full-scale of prevalence estimates ranging between 5.88--100% of oestrosis in sheep and goats has been reported worldwide \[[@CR19]--[@CR23]\]. This inconsistent estimate in various geographical locations is plausible due to an aggregated distribution of the parasite in particular geographical areas and climatic conditions, methods for identifying the disease, the origin of the sample and sampling strategy, study duration and studied species of animal. An overview of knowledge on the geographical distribution and burden of oestrosis in sheep and goats will offer a better understanding of its impacts on animal production and prevent the spread of disease to humans. Therefore, this study aimed to estimate the global prevalence of oestrosis in sheep and goats and to assess the potential factors that contribute to the variability in the prevalence and distribution of the disease using a systematic approach.
Methods {#Sec2}
=======
The study was conducted according to the guidelines provided by PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) for systemic review and meta-analysis. The PRISMA 2009 checklist (Additional file [1](#MOESM1){ref-type="media"}: Table S1) was followed to ensure the inclusion of relevant information and maintain study standard.
Literature search {#Sec3}
-----------------
A systemic search strategy was used to identify all published articles reporting the prevalence of *O. ovis* in sheep and goats. Published works of literature were searched in nine electronic databases: PubMed, CAB Abstracts, Web of Science, Scopus, UCB library, Medline, Biosis Citation Index, Indian journals and Google Scholar, published between January 1970 and March 2018. Searches of the first seven listed databases were undertaken on 30th of March 2018, and the last two on 31st of March 2018. The search terms were categorised into descriptive, population and outcome as described before by Islam et al. \[[@CR24]\]. The modified search terms are presented in Table [1](#Tab1){ref-type="table"}. Search field option was selected as "All Fields". The "descriptive term", "population term" and "outcome term" were combined using the Boolean operator "AND". Search terms were adjusted as per specification and minor differences in syntax rules of individual databases.Table 1Algorithm for electronic database search to find published reports on the prevalence of oestrosis in sheep and goatsSearch termBoolean keywordsDescriptive termPrevalence OR incidence OR frequency OR occurrence OR detection OR identification OR isolation OR characterisation OR investigation OR survey OR ratePopulation term*Oestrus ovis* OR *O. ovis* OR sheep nasal bot OR sheep botfly OR bot fly larva OR nasal myiasis OR oestrosis OR estrosis OR oestrus myiasis OR *O. ovis* myiasis OR botfly encephalitisOutcome termGoat OR doe OR buck OR caprine OR ovine OR sheep OR ram OR ewe OR small ruminant
The search optimisation was performed for articles published in the English language. The reference list of all retrieved items was searched manually in triplicate to identify all eligible studies and to ensure that databases searches have missed no reports.
Selection of studies {#Sec4}
--------------------
Articles were considered eligible for meta-analysis based on the following criteria: published between January 1970 and March 2018; full-text article; English language; any country of the world; reported as animal level prevalence data; studied population is sheep or goat or both; cross-sectional, case-control, longitudinal and cohort studies. Articles were excluded if prevalence data were not reported, species other than sheep or goat, case study, experimental trial and materials other than the English language.
Quality of the studies {#Sec5}
----------------------
Selected studies were evaluated for quality of reporting and selection for bias using a quality appraisal checklist \[[@CR25], [@CR26]\] (Additional file [2](#MOESM2){ref-type="media"}: Text S1, Figure S1).
Data extraction {#Sec6}
---------------
The following data were extracted on a spreadsheet where possible from each eligible article: author, year of publication, country, region/province, continent, study duration, host, breed, origin of sample, method of detection/diagnosis, population, positive, prevalence, season when the prevalence was highest, ambient temperature during the peak prevalence season, specific risk (sex, age, coat colour), mean larval burden and the highest number of larvae per head. Overall, data from 59,086 animals (sheep and goats) of various geographical locations were analysed (Table [2](#Tab2){ref-type="table"}).Table 2Characteristics of 66 studies included in this meta-analysis to investigate the pooled prevalence of oestrosis in sheep and goatsReferenceYearCountryStudy duration (months)HostSample originDetection methodNo. of samplesPositive *n* (%)Benakhla et al. \[[@CR20]\]2004Algeria12SheepAbattoirNecropsy313211 (67.41)Attindehou et al. \[[@CR21]\]2012Benin6SheepAbattoirNecropsy25690 (35.16)GoatAbattoirNecropsy22443 (19.20)Amin et al. \[[@CR22]\]1997EgyptnaSheepAbattoirNecropsy1200104 (8.67)Osman \[[@CR29]\]2010Egypt12SheepAbattoirNecropsy623217 (34.83)GoatAbattoirNecropsy35783 (23.25)Ramadan et al. \[[@CR43]\]2013Egypt12SheepAbattoirNecropsy3132360 (11.49)Alem et al. \[[@CR30]\]2010Ethiopia5SheepAbattoirNecropsy369349 (94.58)GoatAbattoirNecropsy431381 (88.40)Bekele et al. \[[@CR44]\]1995Ethiopia16SheepFarmNecropsy37623 (6.12)Gebremedhin \[[@CR31]\]2011Ethiopia6SheepAbattoirNecropsy311217 (69.77)GoatAbattoirNecropsy243115 (47.33)Yilma & Genet \[[@CR32]\]2000Ethiopia12SheepAbattoirNecropsy248192 (77.42)GoatAbattoirNecropsy258188 (72.87)Gabaj et al. \[[@CR33]\]1993Libya5SheepAbattoirNecropsy1489336 (22.57)GoatAbattoirNecropsy32059 (18.44)Negm-Eldin \[[@CR34]\]2015Libya12SheepAbattoirNecropsy18093 (51.67)GoatAbattoirNecropsy12034 (28.33)Pandey & Ouhelli \[[@CR45]\]1984Morocco12SheepAbattoirNecropsy12083 (69.17)Oniye \[[@CR46]\]2006Nigeria6SheepAbattoirNecropsy11672 (62.07)Horak \[[@CR47]\]1977South Africa24SheepAbattoirNecropsy542398 (73.43)Horak \[[@CR1]\]2005South Africa12SheepFarmNecropsy193103 (53.37)GoatFarmNecropsy9658 (60.42)Pandey \[[@CR48]\]1989Zimbabwe13SheepAbattoirNecropsy507111 (21.89)Biu & Nwosu \[[@CR79]\]1999Nigeria12GoatAbattoirNecropsy40002150 (53.75)Horak & Butt \[[@CR80]\]1977South Africa13GoatAbattoirNecropsy13096 (73.85)Saleem et al. \[[@CR49]\]2017India12SheepAbattoirNecropsy120119 (99.17)Sharma et al. \[[@CR50]\]2012IndianaSheepAbattoirNecropsy12825 (19.53)Dhishonin et al. \[[@CR51]\]2017India12SheepAbattoirNecropsy1439 (6.29)Jagannath et al. \[[@CR35]\]1989India12SheepAbattoirNecropsy520464 (89.23)GoatAbattoirNecropsy263219 (83.27)Pathak \[[@CR36]\]1992India12SheepAbattoirNecropsy384312 (81.25)GoatAbattoirNecropsy466249 (53.43)Dehghani et al. \[[@CR37]\]2012Iran6SheepAbattoirNecropsy59341347 (22.70)GoatAbattoirNecropsy1802409 (22.70)Shoorijeh et al. \[[@CR38]\]2010Iran13SheepAbattoirNecropsy2002994 (49.65)GoatAbattoirNecropsy1998261 (13.06)Shoorijeh et al. \[[@CR52]\]2009Iran13SheepAbattoirNecropsy2002995 (49.70)Tavassoli et al. \[[@CR53]\]2012Iran12SheepAbattoirNecropsy402122 (30.35)AL-Ubeidi et al. \[[@CR54]\]2017Iraq3SheepAbattoirNecropsy13372 (54.14)Abo-Shehada et al. \[[@CR55]\]2000Jordan17SheepAbattoirNecropsy417242 (58.03)Othman \[[@CR56]\]2009Palestine12SheepAbattoirNecropsy335181 (54.03)Alahmed \[[@CR19]\]2000Saudi Arabia12SheepAbattoirNecropsy54432 (5.88)Alikhan et al. \[[@CR57]\]2018Saudi ArabianaSheepAbattoirNecropsy1334400 (29.99)Hanan \[[@CR58]\]2013Saudi Arabia12SheepAbattoirNecropsy480257 (53.54)Arslan et al. \[[@CR59]\]2009Turkey12SheepAbattoirNecropsy387156 (40.31)Ipek & Altan \[[@CR39]\]2017Turkey2SheepAbattoirSemi-nested PCR158133 (84.18)GoatAbattoir2610 (38.46)SheepAbattoirRhinoscopy158104 (65.82)GoatAbattoir2610 (38.46)Karatepe et al. \[[@CR60]\]2014Turkey12SheepAbattoirNecropsy36482 (22.53)Özdal et al. \[[@CR61]\]2016Turkey12SheepAbattoirNecropsy328127 (38.72)Uslu & Dik \[[@CR62]\]2006Turkey13SheepAbattoirNecropsy624368 (58.97)Rahman & Karim \[[@CR81]\]1989Bangladesh12GoatAbattoirNecropsy705175 (24.82)Huq \[[@CR82]\]1983Bangladesh15GoatAbattoirNecropsy600114 (19.00)Jumde & Dixit \[[@CR83]\]2012IndianaGoatAbattoirNecropsy247194 (78.54)Shoorijeh et al. \[[@CR84]\]2011Iran13GoatAbattoirNecropsy1998261 (13.06)Abo-Shehada et al. \[[@CR85]\]2003Jordan13GoatAbattoirNecropsy520126 (24.23)Dorchies et al. \[[@CR40]\]2000France12SheepAbattoirNecropsy631274 (43.42)GoatAbattoirNecropsy672191 (28.42)Yilma & Dorchies \[[@CR63]\]1991France12SheepAbattoirNecropsy555361 (65.05)Bauer et al. \[[@CR64]\]2002Germany24SheepFarmELISA1497753 (50.30)Papadopoulos et al. \[[@CR7]\]2006GreecenaSheepMixed flockELISA397193 (48.61)GoatMixed flockELISA33558 (17.31)Papadopoulos et al. \[[@CR23]\]2001Greece12SheepMixed flockELISA300300 (100)GoatMixed flockELISA500212 (42.40)Papadopoulos et al. \[[@CR41]\]2010Greece12SheepAbattoirNecropsy292126 (43.15)GoatAbattoirNecropsy158120 (75.95)Caracappa et al. \[[@CR65]\]2000Italy24SheepAbattoirNecropsy841469 (55.77)Scala et al. \[[@CR66]\]2001Italy12SheepFree-ranging flocksNecropsy566514 (90.81)Scala et al. \[[@CR3]\]2002Italy12SheepAbattoirELISA and necropsy443327 (73.81)Cozma et al. \[[@CR67]\]2010Romania6SheepFarmNecropsy and skin sensitivity test280140 (50.00)Daniela \[[@CR42]\]2008Romania9SheepMixed flockNecropsy8457 (67.86)GoatMixed flockNecropsy5122 (43.14)Alcaide et al. \[[@CR68]\]2005Spain9SheepFarmELISA and necropsy276218 (78.99)Alcaide et al. \[[@CR69]\]2005Spain23SheepFarmELISA58784070 (69.24)Gracia et al. \[[@CR70]\]2010Spain12SheepAbattoirNecropsy120101 (84.17)Gracia et al. \[[@CR71]\]2006SpainnaSheepPasture and indoorNecropsy2014 (70.00)Paredes-Esquivel et al. \[[@CR72]\]2009Spain2SheepFree ranging flocksNecropsy206173 (83.98)Paredes-Esquivel et al. \[[@CR73]\]2012Spain13SheepAbattoirNecropsy554255 (46.03)Alcaide et al. \[[@CR86]\]2005Spain23GoatFarmELISA1590717 (45.09)9GoatFarmNecropsy8028 (35)Carvalho et al. \[[@CR74]\]2015Brazil80SheepFarmNecropsy7112 (16.90)Silva et al. \[[@CR75]\]2013Brazil8SheepAbattoirNecropsy13919 (13.67)Silva et al. \[[@CR76]\]2012Brazil36SheepFarmNecropsy7236 (50.00)Hidalgo et al. \[[@CR77]\]2015Chile4SheepAbattoirNecropsy8753 (60.92)Murguía et al. \[[@CR78]\]2000MexiconaSheepFarmThin layer immune assay test689229 (33.24)*Abbreviations*: ELISA, enzyme-linked immunosorbent assay; na, not mentioned
Data analysis {#Sec7}
-------------
All obtained data were entered and sorted in a Microsoft Excel spreadsheet. Prevalence was estimated by the number of positive animals divided by the total number of animals. Only the crude estimate of prevalence was used and their 95% confidence interval (CI). The CI was calculated using the standard formula for a proportion (p): $\documentclass[12pt]{minimal}
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\begin{document}$$ {\text{p}}\, \pm \, 1.96\sqrt {\left[ {{\text{p}}\, \times \,\left( {100\, - \,{\text{p}}} \right)\, \div \,n} \right]} $$\end{document}$, where *n* is the studied population size \[[@CR24]\]. In circumstances where the higher limit of CI exceeded 100, the value was settled to 100 to avoid \> 100% prevalence. Data were analysed using STATA v.11.0 (StataCorp LP, College Station, TX, USA). The meta-analysis was performed using the STATA command "metan". The percentage of heterogeneity across studies that is due to variation rather than chance was estimated by interpreting the *I*^2^ statistic value and Cochran's *Q* (represented as *χ*^2^ and *P*-values) \[[@CR27]\]. The *I*^2^ values of 25, 50 and 75% were considered as low, moderate and high heterogeneity, respectively \[[@CR27]\]. Due to a high degree of heterogeneity between studies the random effect model was selected for summary statistics. Furthermore, the potential sources of heterogeneity were investigated by subgroup analysis. Five potential sources of heterogeneity were examined: continent, country, species, origin of the sample and the method of detection. For constructing a forest plot, data of each continent were analysed separately with the stratified command "by" for the variables. The results are presented as prevalence percentage with 95% CI. Assessment of small study effects was determined using two funnel plots, and the sources of funnel plots asymmetry were also tested to identify the publication bias by Egger's test \[[@CR28]\].
Additionally, analysis of climatic data was performed by extracting the ambient temperature data during the peak prevalence season directly from the prospective articles or, where the peak prevalence season was reported but temperature was not mentioned, from the national weather databases. Only the crude estimate of average temperature was used and their minimum temperature (Tmin) and maximum temperature (Tmax). Average ambient temperature was calculated using the formula: (Tmax + Tmin)/2. Similar to other variables, due to a high degree of heterogeneity the random effect model was selected for summary statistics. In circumstances where the average ambient temperature value was a proportion, the value was rounded to the nearest number using the ceiling and floor functions of Microsoft Excel 2016. Three potential sources of heterogeneity were examined: continent, country and temperature range. For constricting the forest plot, data of continent, country and temperature sub-group were analysed separately. The results are presented as degree Celsius (°C) with 95% CI of the mean.
Results {#Sec8}
=======
Search results and eligible studies {#Sec9}
-----------------------------------
Figure [1](#Fig1){ref-type="fig"} shows the search results. In the initial search on selected databases, 2423 potential articles were identified. After screening, a total of 87 eligible articles were found of which 27 articles were excluded due to following reasons: case report (*n* = 7); individual prevalence data not available (*n* = 7); experimental trial (*n* = 8); article other than the English language (*n* = 1); and others (*n* = 4). The list of excluded articles along with reasons for their exclusion is provided in Additional file [3](#MOESM3){ref-type="media"}: Text S2. A total of 66 eligible articles were used for meta-analysis (Additional file [4](#MOESM4){ref-type="media"}: Text S3). Among the selected articles, 18 articles reported the prevalence of oestrosis in both sheep and goats \[[@CR1], [@CR7], [@CR21], [@CR23], [@CR29]--[@CR42]\], 40 articles in sheep \[[@CR3], [@CR19], [@CR20], [@CR22], [@CR43]--[@CR78]\] and 8 articles reported only in goats \[[@CR79]--[@CR86]\]. Based on the origin of samples, 50 studies were from abattoirs \[[@CR3], [@CR19]--[@CR22], [@CR29]--[@CR41], [@CR43], [@CR45]--[@CR63], [@CR65], [@CR70], [@CR73], [@CR75], [@CR77], [@CR79]--[@CR85]\], 10 from farms \[[@CR1], [@CR44], [@CR64], [@CR67]--[@CR69], [@CR74], [@CR76], [@CR78], [@CR86]\], 3 from free ranging flocks \[[@CR66], [@CR71], [@CR72]\] and 3 from mixed flocks \[[@CR7], [@CR23], [@CR42]\]. Based on the method of diagnosis, 56 studies used necropsy \[[@CR1], [@CR19]--[@CR22], [@CR29]--[@CR38], [@CR40]--[@CR63], [@CR65], [@CR66], [@CR70]--[@CR77], [@CR79]--[@CR85]\], 4 used ELISA \[[@CR7], [@CR23], [@CR64], [@CR69]\] and 6 used combined or other methods \[[@CR3], [@CR39], [@CR67], [@CR68], [@CR78], [@CR86]\]. A description of the characteristics of each included study is shown in Table [2](#Tab2){ref-type="table"}.Fig. 1Flow diagram of the selection of eligible studies for inclusion in the meta-analysis
Continents and countries {#Sec10}
------------------------
All included studies on sheep and goats represent data from 5 continents, covering 26 countries of the world. The highest number of articles (*n* = 25) were from Asia covering 8 countries: India (*n* = 6), Iran (*n* = 5), Turkey (*n* = 5), Saudi Arabia (*n* = 3), Bangladesh (*n* = 2), Jordan (*n* = 2), Iraq (*n* = 1) and Palestine (*n* = 1). The second highest was from both Africa and Europe, with the same number of articles from each continent (*n* = 18). Countries within Africa contributed as follows: Ethiopia (*n* = 4), Egypt (*n* = 3), South Africa (*n* = 3), Libya (*n* = 2), Nigeria (*n* = 2), Algeria (*n *= 1), Benin (*n* = 1), Morocco (*n* = 1) and Zimbabwe (*n* = 1). European countries reported as follows: Spain (*n* = 7), Greece (*n* = 3), Italy (*n* = 3), France (*n* = 2), Romania (*n* = 2) and Germany (*n* = 1). Four articles were from South America: Brazil (*n* = 3) and Chile (*n* = 1). One article was from North America: Mexico (*n* = 1).
Prevalence estimates {#Sec11}
--------------------
On the basis of the global burden of oestrosis in sheep, the estimated prevalence ranged from 34.45% (95% CI: 19.90--49.01%) to 63.69% (95% CI: 56.08--71.30%) with considerable heterogeneity (*I*^2^= 96.7%, *P* \< 0.0001). The random effect estimated global prevalence in sheep was 51.15% (95% CI: 42.80--59.51%) (Table [3](#Tab3){ref-type="table"}). Likewise, the estimated prevalence of oestrosis in goats ranged from 37.01% (95% CI: 25.91--48.11%) to 48.56% (95% CI: 33.04--64.09%) with high heterogeneity (*I*^2^= 99.4%, *P* \< 0.0001). The random effect estimated global prevalence in goats was 42.19% (95% CI: 33.43--50.95%) (Table [4](#Tab4){ref-type="table"}). Overall, the global pooled estimated prevalence of oestrosis in sheep and goats was 48.25% (95% CI: 41.82--54.67%) with substantial heterogeneity (*I*^2^= 99.7%, *P* \< 0.0001) (Table [5](#Tab5){ref-type="table"}). The global estimated pooled prevalence of oestrosis in sheep and goats by country are shown in Fig. [2](#Fig2){ref-type="fig"}.Table 3Estimated pooled prevalence of oestrosis in sheep by world regionWorld regionNo. of studiesNo. of sheep sampledNo. of positive sheepPooled estimate %95% CIHeterogeneity (*χ*^2^)*I*^2^ (%)*P*-valueGlobal estimate5840,87018,19451.1542.80--59.5126,703.2199.8\< 0.0001Africa169975295947.4132.16--62.656591.5899.8\< 0.0001Asia2016,897654148.2833.04--63.5312,879.0699.8\< 0.0001Europe1712,940834563.6956.08--71.301108.7798.6\< 0.0001North and South America5105834934.4519.90--49.0176.8094.8\< 0.0001*Abbreviations*: CI, confidence interval; *χ*^2^, Cochran's *Q* Chi square; *I*^2^, inverse variance index Table 4Estimated pooled prevalence of oestrosis in goats by world regionWorld regionNo. of studiesNo. of goats sampledNo. of positive goatsPooled estimate %95% CIHeterogeneity (*χ*^2^)*I*^2^ (%)*P*-valueGlobal estimate2618,216658342.1933.43--50.955277.6299.5\< 0.0001Africa106179320748.5633.04--64.091247.1899.3\< 0.0001Asia108651202837.0125.91--48.111656.0899.4\< 0.0001Europe63386134840.9328.93--52.92291.8397.9\< 0.0001North and South America^a^0--------------^a^No records of goat oestrosis from North or South America*Abbreviations*: CI, confidence interval; *χ*^2^, Cochran's *Q* Chi square; *I*^2^, inverse variance index Table 5Pooled prevalences and estimated sources of heterogeneity in the prevalence of oestrosis in sheep and goatsVariablePopulationPooled estimate prevalence (%)95% CIHeterogeneity (*χ*^2^)*I*^2^ (%)*P*-valueWorld region Global estimate59,08648.2541.82--54.6733,292.8699.7\< 0.0001 Africa16,15447.8536.04--59.669371.5899.7\< 0.0001 Asia25,54844.4833.09--55.8717,688.6299.8\< 0.0001 Europe16,32656.8348.92--64.742380.4599.1\< 0.0001 North and South America105834.4619.90--49.0189.8095.5\< 0.0001Age Young (≤ 1 year)18,18838.3027.21--49.388266.6799.8\< 0.0001 Adult (\> 1 year)18,18849.5338.02--61.037006.9199.7\< 0.0001Sex Male12,53339.5527.73--51.362288.9899.6\< 0.0001 Female12,53348.7431.16--66.336151.1899.8\< 0.0001Origin of sample Abattoir49,12447.7240.53--44.9029,650.4599.8\< 0.0001 Farm996250.7836.49--65.072658.5599.5\< 0.0001Method of detection Necropsy46,39847.1639.81--54.5129,999.0399.8\< 0.0001 Serology11,07550.8739.09--62.65995.7399.2\< 0.0001 Other161356.1837.80--74.57360.6998.3\< 0.0001Study duration (months) ≤ 612,20049.1937.73--60.643046.9999.4\< 0.0001 \> 6 to ≤ 1219,90852.2340.57--63.8918,701.7999.8\< 0.0001 \> 1222,21941.7830.37--53.196898.5299.7\< 0.0001*Abbreviations*: CI, confidence interval; *χ*^2^, Cochran's *Q* Chi square; *I*^2^, inverse variance index Fig. 2Estimated prevalence of oestrosis caused by *Oestrus ovis* in sheep and goats in different countries of the world from 1970 to 2018. The prevalence estimate is based on a meta-analysis of 66 studies comprising 59,086 sheep and goats. The map was produced using ArcGIS v.10.3.1 (Esri, Redlands, CA, USA)
Prevalence estimates from individual contributing studies according to world region are outlined in Figs. [3](#Fig3){ref-type="fig"}, [4](#Fig4){ref-type="fig"}, [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"} and Table [2](#Tab2){ref-type="table"}. The lowest individual prevalence of oestrosis in sheep was reported as 5.88% (95% CI: 3.91--7.86%) in Saudi Arabia \[[@CR19]\] and the highest individual prevalence was reported as 100% (95% CI: 100--100%) in Greece \[[@CR23]\]. In goats, the lowest individual prevalence was reported as 13.06% (95% CI: 11.59--14.54%) in Iran \[[@CR84]\] and the highest individual prevalence was reported as 88.40% (95% CI: 85.38--91.42%) in Ethiopia \[[@CR30]\]. The longest study duration was 80 months \[[@CR74]\] while the shortest study duration was 2 months \[[@CR39]\].Fig. 3Forest plot of the prevalence estimates of oestrosis caused by *Oestrus ovis* in sheep and goats amongst studies conducted in Africa Fig. 4Forest plot of the prevalence estimates of oestrosis caused by *Oestrus ovis* in sheep and goats amongst studies conducted in Asia Fig. 5Forest plot of the prevalence estimates of oestrosis caused by *Oestrus ovis* in sheep and goats amongst studies conducted in Europe Fig. 6Forest plot of the prevalence estimates of oestrosis caused by *Oestrus ovis* in sheep amongst studies conducted in North and South America. Note that there are no data on oestrus in goats between 1970 and 2018 in both North and South America
Age and sex-related prevalence are summarised in Table [5](#Tab5){ref-type="table"}. For determining the age effect, two age groups were selected. The estimated pooled prevalence of oestrosis was 49.53% (95% CI: 38.02--61.03%) in adult animals and 38.30% (95% CI: 27.21--49.38%) in young animals. The overall estimated pooled prevalence of oestrosis was 48.74% (95% CI: 31.16--66.33%) in female animals and 39.55% (95% CI: 27.73--51.36%) in male animals.
Pooled prevalence based on the origin of samples, methods of detection and study duration are shown in Table [5](#Tab5){ref-type="table"}. The overall estimated pooled prevalence of oestrosis in sheep and goats slaughtered in abattoirs was 47.72% (95% CI: 40.53--44.90%) while the prevalence in farmed animals was 50.78% (95% CI: 36.49--65.07%). Prevalence was 47.16% (95% CI: 39.81--54.51%), 50.87% (95% CI: 39.09--62.65%) and 56.18% (95% CI: 37.80--74.57%) by necropsy, serology and others methods of diagnosis, respectively, to detect oestrosis in sheep and goats. Studies conducted for \> 6 to ≤ 12 months had higher prevalence 52.23% (95% CI: 40.57--63.89%) than the study conducted for ≤ 6 months \[49.19% (95% CI: 37.73--60.64%)\] and \> 12 months \[41.78% (95% CI: 30.37--53.19%)\].
Effect of ambient temperature {#Sec12}
-----------------------------
Peak prevalence season of oestrosis in sheep and goats and ambient environmental temperature during that particular period of the year was obtained from 50 articles representing 23 countries of the world. The average ambient temperature during the peak infestation period was 22 °C (95% CI: 20--24 °C) in Africa, 18 °C (95% CI: 15--22 °C) in Asia, 17 °C (95% CI: 14--20 °C) in Europe and 20 °C (95% CI: 15--25 °C) in South America. Overall, the random effect estimated pooled global ambient temperature was 19 °C (95% CI: 18--21 °C) with significant heterogeneity (*I*^2^= 83.8%, *P* \< 0.0001). The lowest temperature at which the peak prevalence of oestrosis observed was 10 °C (95% CI: 6--14 °C) in Iran while the highest temperature at which the peak prevalence found was 27 °C (95% CI: 22--32 °C) in Nigeria. The ambient temperature at which the peak prevalence was observed are shown in Fig. [7](#Fig7){ref-type="fig"} and country-wise results in Table [6](#Tab6){ref-type="table"}. Results of sub-group analysis of peak prevalence estimates based on ambient temperature are shown in Additional file [5](#MOESM5){ref-type="media"}: Figure S2.Fig. 7Ambient environmental temperature at which the peak prevalence of oestrosis found in sheep and goats at different continents and countries of the world Table 6Estimated pooled temperature at which the peak prevalence of oestrosis reported in sheep and goats in different world regionsCountryT (°C)95% CIHeterogeneity (*χ*^2^)*I*^2^ (%)*P*-valueAlgeria2619--320----Bangladesh2013--260----Benin2622--300----Brazil2220--250.420.00.516Chile147--200----Egypt2218--260.920.00.632Ethiopia2014--2717.2982.60.001France126--183.3770.40.066Greece2317--280.470.00.494India2317--297.1672.10.028Iran106--1415.0580.10.002Italy1811--2619.6789.8\< 0.0001Jordan2319--2700.01.000Libya2015--261.8746.50.172Morocco2116--250----Nigeria2722--320.490.00.485Palestine2519--300----Romania126--180----Saudi Arabia2217--270.040.00.843South Africa2014--265.1561.20.076Spain1812--239.9369.80.019Turkey1815--211.220.00.749Zimbabwe2115--280----*Abbreviations*: CI, confidence interval; *χ*^2^, Cochran's *Q* Chi square; *I*^2^, inverse variance index; --, no interaction due to having single study; T, pooled average temperature
Source of heterogeneity {#Sec13}
-----------------------
Six sources of heterogeneity in prevalence of oestrosis in sheep and goats were observed. These were: world region (*P* \< 0.0001); age (*P* \< 0.0001); sex (*P* \< 0.0001); origin of the sample (*P* \< 0.0001); method of detection (*P* \< 0.0001); and study duration (*P* \< 0.0001) (Table [5](#Tab5){ref-type="table"}).
Overall, there was a high level of heterogeneity in most pooled prevalence estimates (*I*^2^ \> 80%). The expanse of publication bias in the selected studies was measured separately for sheep (Fig. [8](#Fig8){ref-type="fig"}a) and goats (Fig. [8](#Fig8){ref-type="fig"}b). Both funnel plots appeared with the asymmetrical appearance with a gap in the right bottom side of the graph and many points fall outside of the funnels in the left side, indicating publication bias. The estimated bias co-efficient in sheep was 4.56 (95% CI: 4.45--4.67) with a standard error 0.055 providing a *P*-value of \< 0.0001 while the estimated bias coefficient in goats was 4.34 (95% CI: 4.07--4.61) with a standard error 0.129 and a *P*-value of \< 0.0001. Bias assessment checklist and scores of individual studies are shown in Additional file [2](#MOESM2){ref-type="media"}: Table S2.Fig. 8Funnel plot for examination of publication bias of the prevalence estimates of oestrosis in sheep (**a**) and goat (**b**). *Abbreviation*: s.e., standard error
Discussion {#Sec14}
==========
This study summarises the prevalence of oestrosis in sheep and goats in global and regional levels based on a large population (*n* = 59,086; sheep: 40,870 and goats: 18,216) derived from 26 countries of five continents of the world that enabled the assessment of reliable prevalence estimates according to the study objectives. To the best of author's knowledge, this is the first meta-analysis of the global prevalence of oestrosis in sheep and goats.
The global prevalence of oestrosis in sheep and goats is high with an estimated pooled prevalence of 48.25% (95% CI: 41.82--54.67%) across 66 published reports. Around the globe, Europe had the highest disease burden 56.83% (95% CI: 48.92--64.74%) and North and South America had the lowest disease burden at 34.46% (95% CI: 19.90--49.01%). Mexico was the only country of North America that reported oestrosis in sheep; therefore, data for Mexico were compiled with the data from South American countries to estimate the pooled prevalence for meaningful analysis. There could be several reasons for the high prevalence of oestrosis in sheep and goats such as a pasture-based farming system, the presence of favourable climatic conditions for flies and a limited level of flock/herd monitoring as farmers usually provide less attention to individual sheep and goats as they do for other farm animals. Moreover, many studies were conducted in slaughterhouses in different countries which quickly revealed the infestation of *O. ovis* larvae from the nasal sinuses of sheep and goats \[[@CR20]--[@CR22]\].
The reported ambient environmental temperature at which the peak prevalence of oestrosis observed in sheep and goats varied dramatically across regions and countries \[[@CR51], [@CR52]\]. This variation could be due to a variable life-cycle of *O. ovis*, which may differ from a couple of weeks to several months in different geographical regions based on climatic and environmental conditions. An earlier study reported that flies became active when the environmental temperatures were above 12--18 °C and larvae showed quick movement and dynamic foraging behaviour at 19--22 °C \[[@CR87]\]; this is in agreement with the finding of this meta-analysis. On the other hand, Cepeda-Palacios et al. \[[@CR87]\] also reported that larvae underwent hypobiosis when the temperature was around 5 °C, and in this meta-analysis, no peak prevalence was observed under a temperature of 8 °C \[[@CR38], [@CR52]\]. However, it is known that *O. ovis* larvae are capable of adjusting their biology according to ambient environmental conditions and that range is quite large \[[@CR88]\].
The estimated pooled prevalence indicates that oestrosis is more prevalent in sheep than in goats. These results are in agreement with other studies that reported a higher prevalence of oestrosis in sheep than in goats \[[@CR40], [@CR42], [@CR51]\]. The higher prevalence in sheep could be due to more host specificity of *O. ovis* to sheep. Another speculation could be the moistness of muzzle. It is known that goats consume less water than sheep; therefore, their noses are usually less humid than in sheep. This higher humidity may help the larvae to survive more easily in sheep \[[@CR89]\]. Additionally, sheep and goats also differ in respect to immune response and may have different responsiveness to an adult fly strike. The host-related odour difference between sheep and goats may also play a significant role in oestrosis prevalence \[[@CR90]\].
The estimated pooled prevalence of oestrosis was higher in adults than in young animals. This finding agrees with the results of many studies \[[@CR21], [@CR29], [@CR34], [@CR54], [@CR79]\], but not with several others \[[@CR30], [@CR59], [@CR60]\]. A possible reason for the high prevalence in adult animals may be that adult animals are more attractive to the female flies and the surface area in the nasal orifice of adult animals are much broader than of young animals. Furthermore, the respiration rates of adult animals are slower than of young that may offer assistance the female fly to oviposit and to larvae to crawl into the nasal sinuses. Moreover, a young animal may have maternally derived antibodies against oestrosis \[[@CR91]\]. Conversely, several reports observed that age of the animal (\> 13 weeks) positively influenced the immune response (humoral or cellular) development against *O. ovis* in sheep, which tends to vary with infestation load and other factors \[[@CR92]--[@CR94]\]. Likewise, another study reported that lamb could have a higher infestation and larval burden and are significantly responsible for maintaining oestrosis due to less developed immune competency \[[@CR95]\].
Oestrosis was higher in female animals than in male animals based on estimated pooled prevalence. This is plausibly due to an increased density of females to males in flocks or to the physiological differences between males and females or a particular habit of female animals which facilitates their infestation by *O. ovis* larvae \[[@CR34]\]. Conversely, the effect of sex on the prevalence of oestrosis was not significant in one study \[[@CR31]\], and a higher prevalence in males was observed in another study \[[@CR34]\].
The difference in prevalence considering the origin of the sample, the method of detection and the duration of the study was also significant. Prevalence was higher in farm-based studies compared to abattoirs; this may be due to the method of detection, as farm-based studies mostly rely upon serology \[[@CR7]\] and there is a high chance that the fly can attack many animals of a flock in a particular farm in an endemic area. On the other hand, animals slaughtered in an abattoir usually come from different regions and different farms; therefore, the pooled prevalence can be lower. However, year-round surveillance can give an actual prevalence estimate.
Funnel plot asymmetry reveals strong evidence of the presence of publications bias. However, there are many other reasons for funnel plot asymmetry like true heterogeneity, location, data irregularity and artefacts, or even by chance \[[@CR28]\].
Limitations {#Sec15}
-----------
This study has several limitations. First, no report on oestrosis of sheep and goats was found in the continent of Australia within the range of this meta-analysis. Thus a reflection of the prevalence from these regions could not be obtained. Moreover, only four studies from South America and one study from North America were obtained, and all five articles reported the prevalence of sheep oestrosis, so the prevalence of goat oestrosis could not be estimated. Secondly, most of the studies were conducted in head samples obtained directly from abattoirs, so in some cases it could be difficult to determine the actual age and sex of the animals and exact prevalence estimation. Thirdly, non-English articles, unpublished articles, case reports and results of experimental trials were not included in this meta-analysis. Fourthly, due to unavailability of data regarding age, sex and peak prevalence season in every article, all articles could not be covered for pooled prevalence estimations. Finally, the data displayed a significant heterogeneity between studies even within a particular region.
Conclusions {#Sec16}
===========
Results of the global meta-analysis show a very high burden of oestrosis reported in many regions, especially in northern Africa and southern Europe. The results also indicate that the disease is more prevalent in sheep than in goats. The main implication of these results is that screening tests for *O. ovis* and treatment should be routinely carried out in sheep and goat flocks in high disease burden regions to improve animal productivity and minimise the potential zoonotic risk to humans. Measures should also be implemented to take adequate preventive measures against *O. ovis* infestation. As oestrosis is found more prevalently in adult animals, vaccine development and immunisation at the young stage of life may prevent the disease. Moreover, reports on the prevalence of oestrosis in sheep and goats are still not available from many regions; therefore, epidemiological surveillance is needed for estimating the disease burden and for controlling the disease. Additionally, factors that contribute to the prevalence estimate should be handled appropriately in any survey to estimate the true prevalence of oestrosis.
Additional files
================
{#Sec17}
**Additional file 1: Table S1.** PRISMA 2009 checklist. **Additional file 2: Text S1.** Quality assessment checklist of individual studies. **Table S2.** Quality score of studies included in the meta-analysis. **Figure S1.** Frequency distribution of eligible studies characteristics. **Additional file 3: Text S2.** List of the articles excluded in the present meta-analysis with justification. **Additional file 4: Text S3.** List of the articles included in the present meta-analysis. **Additional file 5: Figure S2.** Sub-group analysis of ambient environmental temperature at which the peak prevalence of oestrosis was found in sheep and goats in different continents and countries of the world.
**Publisher\'s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The author is grateful to Drs Maurice Zeegers (Maastricht University) for his courses on meta-analysis. The author would like to acknowledge the Dixson Library of the University of New England, Australia which enabled access to literature databases.
MA conceived the idea, designed, analysed, wrote and revised the manuscript. The author read and approved the final manuscript.
Not applicable.
Important datasets that support the conclusions of this article are included within the article and in additional files.
Not applicable.
Not applicable.
The author declares that he has no competing interests.
|
{
"pile_set_name": "PubMed Central"
}
|
All relevant data are within the paper and its Supporting Information file.
Introduction {#sec001}
============
Uncontrolled growth of cells, leading to cancer, constitutes a major cause of death worldwide. The Global Burden of Disease Cancer Collaboration have published that during 2013, cancer caused 8.2 million deaths and 14.9 million of new cancer were diagnosed worldwide \[[@pone.0162007.ref001]\]. The new therapies developed, during last decades, to be less harmful for the patients, as surgery and chemotherapy, were found to have, in too many cases, a relatively low successful rate and a high risk of reoccurrence \[[@pone.0162007.ref002]\]. The main problem with therapeutic molecules, including those from natural sources, concerns their insufficient selectivity and consequently their deleterious effects towards healthy cells \[[@pone.0162007.ref003], [@pone.0162007.ref004]\]. Moreover, cancerous cells develop frequently mechanisms of resistance and particularly through the pumping of anticancer drugs outside their cytosol \[[@pone.0162007.ref005]\].
As a consequence, anticancer peptides appear as promising candidates for cancer therapy. Indeed, these small molecules are expected to become efficient anticancer drugs because of their high selectivity for cancerous cells \[[@pone.0162007.ref006]\]. Moreover, most of these bioactive peptides interact directly with the cell membrane of the target cells, thus are supposed to induce less resistance mechanisms \[[@pone.0162007.ref006]\].
During the last two decades, a growing number of studies reported the cytotoxic activity against cancer cells of antimicrobial peptides (for reviews see \[[@pone.0162007.ref006]--[@pone.0162007.ref009]\]). In 2016, 198 peptides displaying anticancer or antitumor activity were listed in the Antimicrobial Peptide Database \[[@pone.0162007.ref010]\] available on <http://aps.unmc.edu/AP/database/antiC.php>. These multifunction antimicrobial peptides are used to fight against microbial invaders and constitute the first level of immune defense \[[@pone.0162007.ref011]\] that can be found in numerous eukaryotic organisms (plants, insects, reptiles, mammals,...) \[[@pone.0162007.ref012]\]. Anticancer peptides were divided in two classes \[[@pone.0162007.ref009]\]. The first group corresponds to peptides active against cancer cells while not being active against healthy mammalian cells, such as insect cecropins \[[@pone.0162007.ref013]\] and amphibian magainins \[[@pone.0162007.ref014], [@pone.0162007.ref015]\]. The second one corresponds to the cytotoxic molecules exerting the same activity towards healthy as well as cancerous cells. Bee venom mellitin \[[@pone.0162007.ref016]\], human neutrophil defensins \[[@pone.0162007.ref017], [@pone.0162007.ref018]\] and LL-37 \[[@pone.0162007.ref019]\] belong to this class with very low therapeutic potential.
There were only ten (from 198) of the antimicrobial peptides produced by bacteria which were described to display anticancer activities \[[@pone.0162007.ref010]\]. Firstly, microcinE492, a post-translational modified channel-forming bacteriocin produced by *Klebsiellapneumoniae*, was described to induce apoptosis in some cancer cell lines \[[@pone.0162007.ref020]\]. A hydrophobic analogue of Pep27, a *Streptococcus* signal peptide which was described to initiate the cell death program in *S*. *pneumonia* through signal transduction, was shown to induce apoptosis in various cancer cell lines \[[@pone.0162007.ref021]\]. Plantaricin A is a pheromone and antimicrobial linear peptide produced by *Lactobacillus plantarum* \[[@pone.0162007.ref022]\]. Its natural PlnA-22 analogue was shown to be toxic for cancerous GH~4~ cells but not for normal rat anterior pituitary cells \[[@pone.0162007.ref023]\]. More recently, baceridin, a new cyclic hexapeptide non ribosomal synthetized by a plant associated *Bacillus*, was described to induce apoptosis in tumor cells \[[@pone.0162007.ref024]\]. Three small lipopeptides, gageostatins A, B and C, produced by a marine *Bacillus subtilis*, were characterized and found cytotoxic for six human solid cancer cell lines \[[@pone.0162007.ref025]\]. In 2015, it was shown that two lasso peptides produced by *Streptomyces* species, sungsapin and chaxapeptin were both able to inhibit *in vitro* the cell invasion of human lung cancer cell line A549 \[[@pone.0162007.ref026], [@pone.0162007.ref027]\]. Finally, food preservative peptides nisin A and nisin Z were found to induce apoptosis of head and neck squamous cell carcinoma cells. Interestingly, nisin Z reduceedd tumorigenesis and extended survival of oral cancer floor-of-mouth mice \[[@pone.0162007.ref028]\].
The first anti-*Legionella* peptide, warnericin RK (WRK) was characterized in 2008 \[[@pone.0162007.ref029]\]. The anti-*Legionella* mode of action of WRK was described as detergent-like \[[@pone.0162007.ref030]\] and shown to be modulated by the lipid composition of the bacterial membrane \[[@pone.0162007.ref031]\]. Moreover, it was shown that WRK displayed a high hemolytic activity \[[@pone.0162007.ref029]\]. *Legionella* membrane is characterized by a high amount of phosphatidyl-choline (about 30%), which is known to be an eukaryotic phospholipid (present in red cell membranes for example), while only few bacterial species synthesize this phospholipid \[[@pone.0162007.ref032]\]. These data suggest that WRK could be cytotoxic for various mammalian cells, including malignant cells.
A collection of 12 anti-*Legionella* peptides produced by various species of *Staphylococci* were previously isolated and characterized \[[@pone.0162007.ref033]\]. These peptides were mostly already described for their hemolytic activity but were not known to be anti-*Legionella* \[[@pone.0162007.ref034], [@pone.0162007.ref035]\]. It was proposed, on the basis of their biological activities and mode of action, to separate these compounds in two classes \[[@pone.0162007.ref033]\]. The first class corresponds to peptides, resembling WRK, which are bactericidal and highly hemolytic. The peptides from the second group, with PSMα (Phenol-soluble modulin α) from *S*. *epidermidis* as an archetype, display a bacteriostatic mode of action and are poorly hemolytic \[[@pone.0162007.ref033], [@pone.0162007.ref036]\]. The latter peptide is a member of the PSM complex, composed of three peptides (PSMα, γ and δ), which was demonstrated to be implicated in the *S*. *epidermidis* biofilm detachment as well as displaying a pro-inflammatory activity \[[@pone.0162007.ref037]--[@pone.0162007.ref039]\]. Synthetic peptides were designed to display high anti-leukemic activity as WRK; but poor hemolytic effect as PSMα \[[@pone.0162007.ref040]\]. Two peptides were promising: WarnG20D in which the glycine residue in position 20 was replaced by an aspartic acid, and WarnF14V corresponding to the phenylalanine/valine substitution at position 14. Indeed, these peptides displayed an anti-*Legionella* activity close to the one of WRK; but were found less hemolytic than PSMα \[[@pone.0162007.ref040]\].
Confocal Raman spectroscopy is a non-invasive method to establish chemical cell imaging; it does not require chemical or biological markers. In 1990, Puppels *et al*. were the pioneers in introducing confocal Raman microscopy for studying single living cell \[[@pone.0162007.ref041]\]. Up to now, many studies have been done on different type of cells fixed or in solution. Confocal Raman microscopy combined with principal component analysis or clusters analysis allows also to localize compounds and compartments in cell \[[@pone.0162007.ref042], [@pone.0162007.ref043]\].
In this study, we showed that, the anti-*Legionella* bactericidal peptide, WRK displayed a cytotoxic activity towards cancerous cells from glioma, prostatic carcinoma and leukemia cell lines. However, the peptide was also found toxic for healthy glial and prostatic cells. Interestingly, the healthy mononuclear cells were unaffected by WRK. Low hemolytic variants of WRK, WarnG20D and WarnF14V also displayed anti-leukemia activity. Raman imaging allowed to point out the large damages induced by the peptides on the integrity of membranes of the leukemia cancerous cells (Jurkat), suggesting a membranolytic mode of action. In contrast, no modification was observed on healthy mononuclear cells in presence of the peptides.
Materials and Methods {#sec002}
=====================
Peptides {#sec003}
--------
The peptides were purchased from GenScript (Piscataway, USA). Peptide concentration was determined by the bicinchoninic acid assay as described by the supplier (Sigma) with Bovine Serum Albumin as a standard. 25 μL of each sample were mixed with 200 μL of a solution of bicinchoninic acid and copper sulfate 50:1 (v/v). The preparation was incubated at 37°C for 30 minutes and the absorbance at 595 nm was measured by a microtiter plate reader OPSYS MR (ThermoLabsystems).
Cells, culture media and growth conditions {#sec004}
------------------------------------------
Leukemia cell lines, Jurkat, K562 and KG1 were obtained from ATCC (American Type Culture Collection, VA, USA) and were cultured in RPMI 1640 (Roswell Park Memorial Institute), supplemented with fetal bovine serum (FBS) 10% for Jurkat and K562 cells and 20% for KG1 cells. The human prostate carcinoma cells, LNCap, were obtained from ATCC (American Type Culture Collection, VA, USA) and were cultured in DMEM (Dulbecco's modified eagle medium, Invitrogen, CergyPontoise, France) with high glucose 4.5 g/L supplemented with 10% FBS (Lonza, Levallois-Perret, France), 100 IU/mL penicillin and 100 μg/mL streptomycin (Lonza). The adherent cancer cell lines rat glioma C6 cells are a generous gift of Pr. Naus (University of British Columbia) \[[@pone.0162007.ref044]\]. RWPE cells are immortalized prostatic cells and were obtained from ATCC. They were grown in keratinocyte serum free medium supplemented with 0,2 ng/ml epithelial growth factor and 30 μl/mL of bovine pituitary extract. Primary cultures of astrocytes were prepared from cortex and spinal cord of 1-day-old mice as previously described \[[@pone.0162007.ref045]\]. All experimental procedures involving animals were carried out in accordance with the guidelines of the French Agriculture and Forestry Ministry (decree 87849) and of the European Communities Council Directive (2010/63/UE). Mice were euthanized in accordance with the guidelines of the European Communities Council Directive (2010/63/UE). The study and protocol were approved by the local Committee on the ethics of animal experiments (ComEthEA--Region Poitou-Charentes) and does not need a particular permit. Since neonatal rodents are resistant to the hypoxia-inducing effects of CO~2~ and require longer exposure times to the agent, decapitation was used. Astrocytes were grown in the above-mentioned culture medium supplemented with glucose. They were purified by repetition of trypsinisation (0.25% trypsin and 0.02% EDTA, both from Sigma-Aldrich) and re-plating. The cells used in these experiments were obtained after the third passage. Human mononuclear cells (MNC) were isolated from fresh blood of 2 healthy individuals. The fresh whole blood was half diluted in RPMI 1640, supplemented with Ficoll (2:1 v/v) and centrifuged for 20 minutes at 1200*g* at 4°C. The cells from the interphase were extracted with a Pasteur pipette, transferred to RPMI 1640 medium supplemented with 10% FBS. Cells were washed three times, and then maintained in the medium at 4°C. All cells were cultured and maintained at 37°C in a humidified 5% CO~2~ incubator.
Hemolytic activity assays {#sec005}
-------------------------
Hemolytic activity of the peptides was determined by measuring the released hemoglobin from human erythrocytes. Prior to the assay, the fresh human blood was centrifuged (2000 *g*, 3 min, 4°C) to collect red blood cells. Erythrocytes were washed in phosphate buffered saline (PBS) until the supernatant was clear (5 times). Reactions were performed in 1 mL mixtures containing 1% erythrocytes (v/v) and variable amount of peptides or an equivalent volume of PBS buffer. Serial two-fold dilutions of peptides were performed in PBS buffer and added to 1% human erythrocytes at a starting concentration of 50 μg/mL. The reaction mixtures were incubated at 37°C for 30 min. Erythrocytes were removed by centrifugation (2000 g, 3 min, at 4°C) and the A~576nm~ of the supernatant was determined with a spectrophotometer ThermoSpectronicBiomate 3 (Avantec). 100% hemolysis was measured by adding 0.1% Triton X-100 to the reaction mixture instead of peptides solution.
Human blood was obtained from the French national blood transfusion organization, \"Etablissement Français du Sang---Centre Atlantique\", under the agreement number CA-PLER-2014 089. Donors gave their written and informed consent for the use of blood samples in research.
Membrane permeabilization assays {#sec006}
--------------------------------
Cells were washed and resuspended in phosphate buffer saline (PBS) at a concentration of 10^6^ cells /mL. The cells suspension was treated with various concentrations (25, 12.5, 6, 3, 1 and 0 μg/mL), of peptides during 45 min at 37°C. The cells were then treated during 15 min with 5 μl of propidium iodide (PI), a fluorescent dye which only penetrates the cells with damaged cell membrane, before flow cytometry analysis. Flow cytometric measurements were performed on a FACSConto II flow cytometer (BD Bosciences) with a 488 nm argon excitation laser. A total of 100 000 events were analyzed in each sample, using BD FACSDiVa 6 software (BD Biosciences) for data acquisition and analysis. LD~50~ were calculated from the linear regression of the rate of permeabilized cells as a function of the peptide concentration expressed in μM. 100% permeabilization was measured by adding 0.1% Triton X-100 to the reaction mixture instead of peptides solution.
Confocal Raman microscopy {#sec007}
-------------------------
To achieve the Confocal Raman spectroscopy, Jurkat and MNC cells, the cells were centrifuged at 1000*g* for 3 minutes to replace the RPMI medium and diluted to reach 3.10^5^ cells/mL in phosphate buffer saline (PBS) at pH 7.4. The cells were transferred in a Petri dish with a glass microscope slide at the bottom to avoid the signal of the plastic from the dish. The peptides WRK, WarnG20D, WarnF14V and PSMα were added at 10μM on cell suspensions for 1 hour. Raman spectra were recorded on single cell before the peptide addition, and one hour after the addition of the peptide at room temperature.
Raman spectra were recorded using a WITec (Ulm, Germany) Alpha300RS confocal Raman microscope. The excitation wavelength of 532 nm was provided by a frequency-doubled Nd:YAG laser. The beam was focused on the sample using a water immersion Olympus objective (63X/1 NA). The power at the sample was close to 10mW. The sample was located on a piezoelectrically driven microscope scanning stage. The spatial resolution for the Raman scattering is 300 nm for x and y and 500 nm for z due to the diffraction limit. The integration time for each spectrum was 50 ms. The measure was performed on an area of 20 μm x 20 μm with 100 lines and 100 points by line. Thus the time of a single cell scan was about 10 minutes and allowed the cells to stay intact. The spectra were collected and analyzed using the WITec Control and WITec Project Manager softwares (Ulm, Germany). The Hierarchical Cluster Analysis (HCA) was used to identify regions of the sample that have similar spectral response by clustering the spectra into groups or clusters such that differences in the intracluster spectral responses are minimized while simultaneously maximizing the intercluster differences between spectral responses \[[@pone.0162007.ref046], [@pone.0162007.ref047]\].
Results and Discussion {#sec008}
======================
Anticancer activity of WRK {#sec009}
--------------------------
In order to detect the anticancer activity of WRK, various concentrations of the peptide were tested on suspensions of different cancer cell lines as following: one derived from prostatic tumor (LNCaP), one glioma cell line (C6) and the Jurkat leukemia cell line. The cytotoxicity of the peptide was followed by flow cytometry using propidium iodide (PI) staining which penetrates the cells with degraded cytoplasmic membrane. In parallel, same experiments were conducted on the corresponding healthy prostatic cells (RWPE), glial cells (Astrocytes) and peripheral blood mononuclear cells (MNC). [Fig 1](#pone.0162007.g001){ref-type="fig"} presents the rate of permeabilized cancerous and healthy tested cells. All the cancerous cells appeared sensitive to low concentration of the peptide since a 6 μg/mL dose induced about 20% of permeabilization for the solid cancer cells (C6 and LNCaP, [Fig 1A and 1B](#pone.0162007.g001){ref-type="fig"}) and near 60% for the leukemia cells (Jurkat, [Fig 1C](#pone.0162007.g001){ref-type="fig"}). Moreover, addition of peptide at 25 μg/mL induced the permeabilization of more than 90% of all the cells. [Table 1](#pone.0162007.t001){ref-type="table"} listed the median lethal dose (LD~50~) for each cell lines. The high toxicity of WRK towards all the tested cancer cells, from solid tumor as leukemia was confirmed. Jurkat cells seem about twice sensitive (LD~50~ = 2.11 μM) to the peptide than the other cancer cells (LD~50~ = 4.14 and 3.98 μM for C6 and LNCaP respectively). Considering the healthy cells WRK was found toxic for prostatic and glial cells ([Fig 1](#pone.0162007.g001){ref-type="fig"}). WRK applied at 25μg/mL induced more than 70% and 90% of permeabilization for RWPE cells ([Fig 1B](#pone.0162007.g001){ref-type="fig"}) and astrocytes ([Fig 1A](#pone.0162007.g001){ref-type="fig"}), respectively. On the contrary, the MNC were found completely insensitive to WRK. The percentage of permeabilized cells were identical for cells treated by 25 μg/mL of WRK or for untreated cells ([Fig 1C](#pone.0162007.g001){ref-type="fig"}). The LD~50~ for these healthy cells were of the same order as the cancerous cells ([Table 1](#pone.0162007.t001){ref-type="table"}) except for MNC for which appeared totally insensitive to WRK at the tested doses.
{#pone.0162007.g001}
10.1371/journal.pone.0162007.t001
###### Median lethal dose (LD~50~) of WRK on cancer cell lines tested.
{#pone.0162007.t001g}
Glial cells Prostatic cells Leukocytes
------------- ------------- ----------------- ------------ ------ ------ ------ ------ ----
LD~50~ (μM) 4.14 3.36 3.98 6.37 2.11 2.18 4.22 ND
PSMα is an other anti-*Legionella* peptide produced by *S*. *epidermidis* which displayed an MIC similar to the WRK one. However, it differs from WRK because of its bacteriostatic mode of action and its hemolytic activity which is about three times lower than WRK \[[@pone.0162007.ref030]\]. Thus, PSMα was also assayed against the same three types of cancerous cells and corresponding healthy cells (see supplementary information [S1 Fig](#pone.0162007.s001){ref-type="supplementary-material"}). Interestingly, the rate of permeabilized cells treated by PSMα was found very low whatever the cells, indicating that PSMα displayed no toxic activity on healthy or cancer cells.
The non-toxicity of WRK for MNC indicates that this peptide could be a good candidate for leukemia therapy. However, Jurkat are T-lymphocytes which originated from an acute T-cell leukemia. We tested the WRK toxicity on two other different leukocytes cell lines KG1 and K562. Both are macrophages cell types, corresponding to an acute myelogenous leukemia and a chronic myelogenous leukemia respectively. Indeed, the bacterial anticancer peptide, microcin E492, was found to be cytotoxic for Jurkat cells but inactive against KG1 cells \[[@pone.0162007.ref020]\]. The rates of permeabilized leukemic cells by WRK are presented in [Fig 2](#pone.0162007.g002){ref-type="fig"}. Even if the Jurkat cells appeared the most sensitive to the peptide, with 95% of permeabilized cells at 25 μg/mL, compared to about 80% and 65% for KG1 and K562 cells respectively, all the leukemia cell lines were sensitive, in a dose-response manner to WRK. KG1 and K562 cells were found insensitive to PSMα (data not shown).
{#pone.0162007.g002}
Taken together, these results suggest that the mammalian cells toxicity of WRK could be related to its bactericidal mode of action which was demonstrated to be membranolytic (detergent-like) and dependent on the membrane lipids of *Legionella pneumophila* \[[@pone.0162007.ref030], [@pone.0162007.ref035]\], whereas the non-cytotoxic PSMα, is bacteriostatic.
This study clearly demonstrates that WRK has strong activity against cancer cells. It has to be classified in the second group of anticancer peptides, cytotoxic for both healthy and cancerous cells, for prostatic and glial cells. But more interestingly, WRK has to be classified in the first class, active towards leukemia cancer cells but not healthy cells (MNC), that may be very promising for leukemia anticancer therapy. Nevertheless, WRK was described to be highly hemolytic \[[@pone.0162007.ref029]\], which disqualifies it for possible anticancer therapy application.
Anti-leukemia activity of low hemolytic WRK variants {#sec010}
----------------------------------------------------
In a previous study \[[@pone.0162007.ref040]\], we designed and selected synthetic variants of WRK which were as active as WRK against Legionella with a decreased hemolytic activity. The most interesting variants, WarnG20D and WarnF14V, were tested for their anti-leukemia against Jurkat cells. The results, obtained by flow cytometry, are presented in [Fig 3](#pone.0162007.g003){ref-type="fig"}. Even if WRK (black bars) remains the most active peptide against Jurkat cells, WarnF14V (white bars) and WarnG20D (grey bars) displayed also cytotoxicity. WarnG20D and WarnF14V induced, at 25 μg/mL, rates of 88% and 60% of permeabilized cells, respectively. The deduced LD~50~ for these peptides ([Table 2](#pone.0162007.t002){ref-type="table"}), 5.39 and 8.79 μM for WarnG20D and WarnF14V respectively, were found higher than the one of WRK (2.11 μM) but were inferior to 10 μM. Considering their hemolytic activity, the minimal concentration responsible for the lysis of 10% of red blood cells was around 12.5 μM for both variants, whereas it was measured at 2.78 μM for WRK ([Table 2](#pone.0162007.t002){ref-type="table"}). An anti-leukemia index, analog to the therapeutic index, was calculated by divided the 10% hemolysis concentration by the LD~50~ for each peptide ([Table 2](#pone.0162007.t002){ref-type="table"}). The highest value (2.293) was obtained for WarnG20D, by comparison with those of WarnF14V and WRK (1.422 and 1.317 respectively). Moreover, both WRK variants were found inactive against MNC cells (data not shown), which highlights the potential of WarnG20D for leukemia therapy.
{#pone.0162007.g003}
10.1371/journal.pone.0162007.t002
###### Primary sequences and biological activities of WRK and analogs WarnG20D and WarnF14V.
{#pone.0162007.t002g}
Name Sequence LD~50~[^a^](#t002fn001){ref-type="table-fn"} (μM) 10% Hemolysis concentration[^b^](#t002fn002){ref-type="table-fn"} (μM) Anti-leukemia index[^c^](#t002fn003){ref-type="table-fn"}
---------- ------------------------------- --------------------------------------------------- ------------------------------------------------------------------------ -----------------------------------------------------------
WRK MQFITDLIKKAVDFFKGLFGNK 2.11 2.78 1.317
WarnG20D MQFITDLIKKAVDFFKGLF[D]{.ul}NK 5.39 12.36 2.293
WarnF14V MQFITDLIKKAVD[V]{.ul}FKGLFGNK 8.79 12.5 1.422
^a^ Cytotoxic activity was determined on Jurkat cells.
^b^ 10% Hemolysis concentration corresponds to the peptide concentration which induces the lysis of 10% of the red cells in the sample.
^c^ Anti-leukemia index corresponds to the ratio: 10% Hemolysis concentration / LD~50~.
Mode of action of WRK and low hemolytic variants on Jurkat cells by confocal Raman microscopy and imaging {#sec011}
---------------------------------------------------------------------------------------------------------
Jurkat cells incubated with WRK, with low hemolytic variants (WarnG20D and WarnF14V) or PSMα were analyzed by microscopy and confocal Raman microspectroscopy. The effect of each peptide, before and 1 hour after peptide addition at 10 μg/mL, on the Jurkat cell population was firstly estimated by optical microscopy (data not shown). WRK was found to induce lysis of the Jurkat cells. Indeed, about 50% of the cells were still observable after 1 hour. A small decrease (less than 10%) in the Jurkat cell population was observed in presence of WarnG20D and WarnF14V. In contrast, PSMα had no effect on the Jurkat population, which is in agreement with the results presented above. It has to be noticed that the cells are not fixed thus they floated in the buffer; the population count under the microscope objective was only an estimation. These results are in good agreement with the higher permeabilization of Jurkat cell by WRK than variants presented in [Fig 3](#pone.0162007.g003){ref-type="fig"}. Indeed, at 6 μg/mL, about 55% of cells were permeabilized by WRK but only 6 to 10% by WarnF14V and WarnG20D respectively.
Confocal Raman spectroscopy on unique cell was performed, to image the different compartments of the cell before peptide addition. [Fig 4A](#pone.0162007.g004){ref-type="fig"} shows the optical image of the Jurkat cell, the contrast observed in the middle of cell reveals the presence of the nucleus. Raman spectra were recorded on the same zone and hierarchical cluster analysis (HCA) process was performed with six cluster areas in the mapping ([Fig 4B](#pone.0162007.g004){ref-type="fig"}). Shie et al. \[[@pone.0162007.ref041]\] studied the effect of doxorubicin on Jurkat cell by Confocal Raman spectroscopy. The Raman spectra of both Jurkat cells (before treatment) reported by Shie et al. or in our study are very similar. The fingerprints of the classical molecules present in cells, as lipids, proteins and nucleic acids were thus detected. The main bands are labeled on the Raman spectra ([Fig 4B](#pone.0162007.g004){ref-type="fig"}). The band assignments are listed in [Table 3](#pone.0162007.t003){ref-type="table"}.
{#pone.0162007.g004}
10.1371/journal.pone.0162007.t003
###### Raman band assignments according to references \[[@pone.0162007.ref047]--[@pone.0162007.ref049]\].
{#pone.0162007.t003g}
Band (cm^-1^) Assignment
--------------- -------------------------------------------------------------------------------------------
725 DNA/RNA (adenine ring breathing)
755 Proteins (tryptophan symmetric ring breathing)
788 DNA/RNA (thymine, uracil, cytosine ring breathing) and DNA backbone (O-P-O stretching)
1010 Proteins (phenylalanine symmetric ring breathing)
1100 Lipids, Proteins (C-C stretching)
1137 Lipids, Proteins (C-C stretching)
1263 Proteins (Amide III)
1311 Lipids, Proteins, DNA/RNA
1348 Lipids, Proteins, DNA/RNA
1457 Lipids (CH~2~ CH~3~ binding)
1583 DNA/RNA (guanine, adenine)
1665 Proteins (Amide I)
2850--2950 Lipids, proteins (CH~2~, CH~3~, symmetric and antisymmetric stretching and CH stretching)
3080 Lipids (C = C-H, CH stretching)
Cell chemical imaging was reconstructed using the HCA process classification ([Fig 4C](#pone.0162007.g004){ref-type="fig"}). The different colors represent the average of all identical spectra recorded in probed area. The cyan domain (corresponding to cyan spectrum) shows mainly the signature of water with strong band at 3300 cm^-1^ and small band at 1640 cm^-1^ assigned to νOH and δOH, respectively.
A thin band is observed at 1003 cm^-1^; it seems to be a signature of phenylalanine amino acid. It seems to be a residual component of the cell culture medium. The other spectra exhibit signature of lipids, and proteins. The range above 2800 cm^-1^ is dominated by CH stretching vibrations of lipids and proteins. Proteins are revealed by the characteristic amide I and amide III bands at 1665 and 1263 cm^-1^ respectively. HCA analysis points out differences in the low wavenumber range, between 800 and 650 cm^-1^ (see zoom in [Fig 4B](#pone.0162007.g004){ref-type="fig"}). This domain is characteristic of the DNA or RNA bases vibration modes and of particular amino acid residue (tryptophan) ([Table 3](#pone.0162007.t003){ref-type="table"}) \[[@pone.0162007.ref049]--[@pone.0162007.ref051]\]. The two green domains correspond to the localization of the membrane and the cytoplasm. The lipid and protein contributions are small related to weak bands observed and the contribution of water increases, as expected. The orange cluster is specific spectrum of the nucleus. The chemical image of the nucleus is well correlated to the optical image (dashed line [Fig 4A](#pone.0162007.g004){ref-type="fig"}). The additional band at 788 cm^-1^ is associated with Thymine, Uracil and Cytosine ring breathing modes of DNA and RNA bases, and also contains contributions from O-P-O DNA backbone. The blue and purple spectra are similar to the other in the high wavenumber range. The difference with the other compartments is mainly due to the enhancement of the band at 755 cm^-1^ assigned to tryptophan symmetric ring breathing mode. This indicates an increase in the protein content. A weak increase of the amide I and amide III bands is also observed. Lipids contribution is also more important in this range, as revealed by the high intensity of the CH~2~ and CH~3~ stretching modes between 2800 and 3000 cm^-1^. This domain close to the nuclei could be assigned to the endoplasmic reticulum, where takes place the lipid metabolism and protein synthesis.
[Fig 5](#pone.0162007.g005){ref-type="fig"} shows chemical images of the Jurkat cell before and 1 hour after injection of peptides. For all analyzed cells the same chemical images were obtained. The different compartments are still represented in orange for the nucleus, blue and purple for endoplasmic reticulum and green for membrane and cytoplasm. Addition of WRK created large Jurkat cell damages and the image of the cell still present in the buffer was modified ([Fig 5](#pone.0162007.g005){ref-type="fig"}). We repeated the observation for at least three remaining cells in the medium. The recorded spectra are only characteristic of cytoplasm. The arrow [Fig 5](#pone.0162007.g005){ref-type="fig"} pointed out the loss of inner compartments of the cell. The nucleus and the endoplasmic reticulum were both not detected, leading to conclude that WRK is an effective membrane active peptide, as demonstrated for its activity on *Legionnella* \[[@pone.0162007.ref030]\]. Derivative peptides lead to less drastic effect on cells. Deformations of cells were mainly observed after addition of WarnG20D and WarnF14V. The nucleus and endoplasmic reticulum compartment are still observed. Small modification of the relative intensity of the band in the low wavenumber range (725, 755, 785 cm^-1^) are observed, leading to the modification of the color on the chemical images for the reticulum endoplasmic domain ([Fig 5](#pone.0162007.g005){ref-type="fig"}). However, the outline of the compartments is still observable. The deformed areas are related to the cytoplasm and cell membrane domain and could correspond to intracellular material leakage (see white arrow in [Fig 5](#pone.0162007.g005){ref-type="fig"} for both peptides WarnG20D and WarnF14V). PSMα injection has no effect on Jurkat cell morphology, in agreement with results described previously. The compartment integrity was still observed with no cell deformation ([Fig 5](#pone.0162007.g005){ref-type="fig"}). Finally, WRK is the most active peptide on Jurkat cells allowing to large destruction of cell. The other active peptides (WarnG20D and WarnF14V) induce first deformation of the cell that can lead after longer time to the cell lysis, explaining their toxicity.
{#pone.0162007.g005}
The peptides were also added in the medium containing the healthy MNC cells to evaluate their selectivity against cancerous cells. The MNC cells are smaller than Jurkat cells. After addition of the peptides the cell number remains similar even after one hour following the peptide injection (data not shown). [Fig 6](#pone.0162007.g006){ref-type="fig"} shows the chemical image of a unique MNC cell before and 1 hour after peptide addition. Similar Raman spectra were observed with similar peaks assigned to lipids, proteins and nucleic acids, as described for Jurkat cells ([Fig 4](#pone.0162007.g004){ref-type="fig"} and [Table 3](#pone.0162007.t003){ref-type="table"}). Whatever the peptide the integrity of the MNC cells is conserved. The difference in the images can be due to orientation of the cell under the laser beam. Indeed, the cell is floating in the medium, if it rotates the repartition of the various compartments can be modified. But from these images we can conclude that these peptides, added at similar concentration (10 μg/mL), have no activity on the healthy cells (MNC) in contrast to the results obtained on leukemic cells.
{#pone.0162007.g006}
Conclusions {#sec012}
-----------
WRK is cytotoxic for all the tested cells, leukemic cells (Jurkat, KG1, KS62) prostatic and glial cancer cells, prostatic healthy cells and astrocytes, but not for healthy mononuclear cells (MNC).
Low hemolytic WRK derivative peptides, WarnG20D or WarnF14V, were also found to be active against Jurkat cells but not toxic for MNC. Their mode of action was studied using chemical Raman imaging without any labeling of cells. The membranes of the cancer cells, treated by the peptides, are strongly affected, the cell compartments were disorganized and Raman signature of cytoplasm was only observed. This is in agreement with the postulated detergent like mechanism of action of WRK on *Legionella* \[[@pone.0162007.ref027]\]. At similar peptides concentration, no modification of the MNC cells was observed. Low hemolytic WRK variant, WarnG20D, according to its anti-leukemia activity could be good candidates for treatment of leukemia. However, because of the putative cytotoxicity of the peptide for cells other than MNC, it would be interesting to test the efficacy for leukemia therapy of WarnG20D immobilized in an extracorporeal shunt system as proposed by Qiao et al. \[[@pone.0162007.ref052]\] for the anticancer enzyme L-Asnase.
Supporting Information {#sec013}
======================
###### Effect of various concentrations of PSMα on glial cancerous (C6), and healthy (Astrocytes) cells; prostatic cancerous (LNCap) and healthy (RWPE) cells; peripheral mononuclear cancerous (Jurkat) and healthy (MNC) cells.
(TIF)
######
Click here for additional data file.
Authors are grateful to A.C. Balandre and I. Petit (University of Poitiers) for the technical assistance for cells cultures.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** JV SL JMB.**Investigation:** CL JA AM JV.**Methodology:** EH MG.**Project administration:** JMB.**Resources:** MM.**Supervision:** MM SL JMB.**Writing -- original draft:** CL MM SL JMB.**Writing -- review & editing:** SL JMB.
|
{
"pile_set_name": "PubMed Central"
}
|
Related literature {#sec1}
==================
For the antitumor activity of heterocycles containing oxazine, see: Benameur *et al.* (1996[@bb1]). For the treatment of Parkinson's disease with naphthoxazines, see: Millan *et al.* (2004[@bb6]); Joyce *et al.* (2003[@bb3]). For the psychostimulating and antidepressant activity of oxazines, see: Nozulak & Giger (1987[@bb7]). For their analgesic, anticonvulsant, antitubercular, antibacterial and anticancer activity, see: Kurz (2005[@bb4]); Turgut *et al.* (2007[@bb15]). For the range of their biological applications, see: Ohnacker & Scheffler (1960[@bb8]). For synthetic possibilities, see: Szatmari *et al.* (2003[@bb13], 2004[@bb14]). For anticancer derivatives, see: Zhang & Li (2003[@bb18]). For related structures, see: Li *et al.* (2008[@bb5]); Sarojini *et al.* (2007[@bb9]); Şen *et al.* (2008[@bb10]); Yang *et al.* (2008[@bb16]); Zhang *et al.* (2009[@bb17]).
Experimental {#sec2}
============
{#sec2.1}
### Crystal data {#sec2.1.1}
C~24~H~18~BrNO*M* *~r~* = 416.30Monoclinic,*a* = 7.7617 (11) Å*b* = 20.092 (3) Å*c* = 11.5094 (16) Åβ = 91.893 (2)°*V* = 1793.9 (4) Å^3^*Z* = 4Mo *K*α radiationμ = 2.31 mm^−1^*T* = 100 K0.55 × 0.50 × 0.35 mm
### Data collection {#sec2.1.2}
Bruker APEXII CCD diffractometerAbsorption correction: multi-scan (*SADABS*; Bruker, 2008[@bb2]) *T* ~min~ = 0.364, *T* ~max~ = 0.49914693 measured reflections5341 independent reflections4426 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.025
### Refinement {#sec2.1.3}
*R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.040*wR*(*F* ^2^) = 0.105*S* = 1.035341 reflections244 parametersH-atom parameters constrainedΔρ~max~ = 1.64 e Å^−3^Δρ~min~ = −0.84 e Å^−3^
{#d5e557}
Data collection: *APEX2* (Bruker, 2008[@bb2]); cell refinement: *SAINT* (Bruker, 2008[@bb2]); data reduction: *SAINT*; program(s) used to solve structure: *SHELXS97* (Sheldrick, 2008[@bb11]); program(s) used to refine structure: *SHELXTL* (Sheldrick, 2008[@bb11]); molecular graphics: *SHELXTL*; software used to prepare material for publication: *SHELXTL* and *PLATON* (Spek, 2009[@bb12]).
Supplementary Material
======================
Crystal structure: contains datablocks global, I. DOI: [10.1107/S1600536810026553/tk2687sup1.cif](http://dx.doi.org/10.1107/S1600536810026553/tk2687sup1.cif)
Structure factors: contains datablocks I. DOI: [10.1107/S1600536810026553/tk2687Isup2.hkl](http://dx.doi.org/10.1107/S1600536810026553/tk2687Isup2.hkl)
Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?tk2687&file=tk2687sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?tk2687sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?tk2687&checkcif=yes)
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [TK2687](http://scripts.iucr.org/cgi-bin/sendsup?tk2687)).
JPJ thanks Dr Matthias Zeller and the YSU Department of Chemistry for their assistance with the data collection. The diffractometer was funded by NSF grant 0087210, by Ohio Board of Regents grant CAP-491, and by YSU. ANM thanks the University of Mysore and SeQuent Scientific Ltd for research facilities and HSY thanks the University of Mysore for sabbatical leave.
Comment
=======
Heterocycles containing the oxazine nucleus are found to possess a wide range of biological applications (Ohnacker & Scheffler *et al.*, 1960). 1,3-Oxazine heterocycles are of interest because they constitute an important class of natural and non-natural products. Many of them exhibit biological activity such as analgesic, anticonvulsant, antitubercular, antibacterial and anticancer (Kurz *et al.*, 2005; Turgut *et al.*, 2007). 1,3-Oxazine derivatives that display anticancer activity are also known as progesterone receptor agonists (Zhang *et al.*, 2003). Oxazine derivatives with a naphthalene ring, termed naphthoxazines, are used in the treatment of Parkinson\'s disease (Millan *et al.*, 2004; Joyce *et al.*, 2003). Naphthoxazines are also known for their psychostimulating and antidepressant activity (Nozulak & Giger *et al.*, 1987). Dihydrofuronaphth\[1,3\]oxazines have shown anti-tumor activity (Benameur *et al.*, 1996). In addition, naphthoxazines can be used as intermediates in the synthesis of *N*-substituted amino alcohols or in enantioselective synthesis of chiral amines. The tautomeric character of the 1,3-*O*,*N*-heterocycles offers a great number of synthetic possibilities (Szatmari *et al.*, 2003; Szatmari *et al.*, 2004). The crystal structures of a few naphthoxazines *viz*., 6-bromo-2,4-bis(3-methoxyphenyl)-3,4-dihydro-2*H*-1,3-naphthoxazine (Sarojini *et al.*, 2007), 3-(1,3 -benzodioxol-5-yl)-1-phenyl-2, 3-dihydro-1*H*-naphtho\[1,2-*e*\]\[1,3\]oxazine (Yang *et al.*, 2008), 2-butyl-1,3-diphenyl-2,3-dihydro-1*H*-naphtho\[1,2-*e*\]\[1,3\]oxazine (Li *et al.*, 2008), 1,3-di-3-pyridyl-2, 3-dihydro-1*H*-naphth-\[1,2-*e*\]\[1,3\]oxazine (Şen *et al.*, 2008) and 2-benzyl-1,3-diphenyl-2,3-dihydro-1*H*-naphtho\[1,2-*e*\]\[1,3\]oxazine (Zhang *et al.*, 2009) have been reported. In view of the importance of naphthoxazines, this paper reports the synthesis and crystal structure of the title compound, (I).
Compound (I) consists of an envelope configured oxazine (C8/C7/C11/N2/C12/O1) ring with a fused 8-bromo-1,3-diphenyl group and two bonded benzene rings (at C11 and C12) \[puckering parameters Q, θ and φ = 0.460 (6) Å, 54.2 (7) °, and 259.144 (8) °, respectively\] (Fig 1.); for an ideal envelope θ has a value of 54.7°. The dihedral angles between the mean planes of the 8-bromo-1,3-diphenyl (C1---C10) and the benzene rings (C13---C18 and C19---C24) are 54.5 (6) and 87.4 (8) °, respectively. The oxazine ring (C7/C8/O1/C12/N1) is essentially co-planar (dihedral angle = 9.4 (1)°) to the 8-bromo-1,3-diphenyl ring. Weak C--H···π interactions (Table 1) (Spek, 2003) are observed which contribute to crystal stability (Fig. 2).
Experimental {#experimental}
============
Benzaldehyde (2.12 g, 0.02 mol) and 25--30% methanolic ammonia (10 ml) were added to 6-bromo-2-naphthol (2.23 g, 0.01 mol) in methanol (10 ml). The mixture was left to stand at ambient temperature for 3 days, during which the crystalline product separated out. The crude product was filtered off and washed with cold methanol. Crystals suitable for X-ray diffraction studies were grown by the slow evaporation of the acetonitrile solution (m.pt. 423--425 K).
Refinement {#refinement}
==========
All of the H atoms were placed in their calculated positions and then refined using the riding model with C---H = 0.93--0.98 Å, and with *U*~iso~(H) = 1.16--1.22*U*~eq~(C). The maximum and minimum residual electron density peaks of 1.64 and 0.84 eÅ^-3^, respectively, were located 1.01 Å and 0.06 Å from the C12 and H2 atoms, respectively.
Figures
=======
{#Fap1}
{#Fap2}
Crystal data {#tablewrapcrystaldatalong}
============
------------------------- ---------------------------------------
C~24~H~18~BrNO *F*(000) = 848
*M~r~* = 416.30 *D*~x~ = 1.541 Mg m^−3^
Monoclinic, *P*2~1~/*c* Mo *K*α radiation, λ = 0.71073 Å
Hall symbol: -P 2ybc Cell parameters from 5049 reflections
*a* = 7.7617 (11) Å θ = 2.6--31.2°
*b* = 20.092 (3) Å µ = 2.31 mm^−1^
*c* = 11.5094 (16) Å *T* = 100 K
β = 91.893 (2)° Block, colourless
*V* = 1793.9 (4) Å^3^ 0.55 × 0.50 × 0.35 mm
*Z* = 4
------------------------- ---------------------------------------
Data collection {#tablewrapdatacollectionlong}
===============
------------------------------------------------------------ --------------------------------------
Bruker APEXII CCD diffractometer 5341 independent reflections
Radiation source: fine-focus sealed tube 4426 reflections with *I* \> 2σ(*I*)
graphite *R*~int~ = 0.025
ω scans θ~max~ = 31.3°, θ~min~ = 2.0°
Absorption correction: multi-scan (*SADABS*; Bruker, 2008) *h* = −11→10
*T*~min~ = 0.364, *T*~max~ = 0.499 *k* = −22→28
14693 measured reflections *l* = −16→16
------------------------------------------------------------ --------------------------------------
Refinement {#tablewraprefinementdatalong}
==========
------------------------------------- ------------------------------------------------------------------------------------------------
Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
*R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.040 Hydrogen site location: inferred from neighbouring sites
*wR*(*F*^2^) = 0.105 H-atom parameters constrained
*S* = 1.03 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0531*P*)^2^ + 1.779*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3
5341 reflections (Δ/σ)~max~ = 0.001
244 parameters Δρ~max~ = 1.64 e Å^−3^
0 restraints Δρ~min~ = −0.83 e Å^−3^
------------------------------------- ------------------------------------------------------------------------------------------------
Special details {#specialdetails}
===============
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Geometry. All e.s.d.\'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.\'s are taken into account individually in the estimation of e.s.d.\'s in distances, angles and torsion angles; correlations between e.s.d.\'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.\'s is used for estimating e.s.d.\'s involving l.s. planes.
Refinement. Refinement of *F*^2^ against ALL reflections. The weighted *R*-factor *wR* and goodness of fit *S* are based on *F*^2^, conventional *R*-factors *R* are based on *F*, with *F* set to zero for negative *F*^2^. The threshold expression of *F*^2^ \> σ(*F*^2^) is used only for calculating *R*-factors(gt) *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on *F*^2^ are statistically about twice as large as those based on *F*, and *R*- factors based on ALL data will be even larger.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords}
==================================================================================================
----- ------------- --------------- --------------- -------------------- --
*x* *y* *z* *U*~iso~\*/*U*~eq~
Br1 0.26837 (3) 0.385264 (10) 0.377762 (19) 0.02407 (8)
C11 0.1161 (2) 0.04955 (10) 0.22310 (16) 0.0149 (4)
H16 0.1739 0.0708 0.1584 0.018\*
C7 0.1858 (2) 0.08276 (10) 0.33364 (16) 0.0137 (3)
C5 0.2088 (2) 0.15294 (10) 0.34152 (16) 0.0144 (3)
C8 0.2413 (3) 0.04374 (10) 0.42586 (16) 0.0149 (4)
C4 0.2910 (2) 0.18121 (10) 0.44282 (16) 0.0153 (4)
C9 0.3260 (3) 0.07139 (10) 0.52570 (17) 0.0172 (4)
H9 0.3645 0.0437 0.5860 0.021\*
C10 0.3511 (3) 0.13830 (11) 0.53356 (17) 0.0177 (4)
H10 0.4081 0.1561 0.5988 0.021\*
C3 0.3107 (3) 0.25095 (10) 0.45246 (17) 0.0169 (4)
H3 0.3651 0.2693 0.5181 0.020\*
C2 0.2494 (3) 0.29135 (11) 0.36489 (18) 0.0185 (4)
C1 0.1708 (3) 0.26470 (11) 0.26294 (18) 0.0193 (4)
H1 0.1321 0.2928 0.2034 0.023\*
C6 0.1517 (3) 0.19715 (10) 0.25210 (17) 0.0170 (4)
H6 0.1000 0.1799 0.1846 0.020\*
N1 0.1653 (3) −0.02094 (9) 0.22166 (16) 0.0231 (4)
H2 0.2104 −0.0405 0.1636 0.028\*
C12 0.1297 (3) −0.05220 (11) 0.32674 (19) 0.0220 (4)
H13 0.0061 −0.0483 0.3402 0.026\*
O1 0.2269 (2) −0.02379 (7) 0.42750 (12) 0.0207 (3)
C13 −0.0762 (2) 0.05797 (9) 0.19911 (16) 0.0131 (3)
C14 −0.1404 (3) 0.05611 (10) 0.08416 (17) 0.0154 (4)
H27 −0.0651 0.0515 0.0235 0.018\*
C19 0.1801 (3) −0.12560 (11) 0.33218 (19) 0.0232 (4)
C24 0.1387 (3) −0.16039 (11) 0.43087 (18) 0.0215 (4)
H18 0.0831 −0.1385 0.4901 0.026\*
C22 0.2638 (3) −0.26054 (12) 0.3568 (2) 0.0261 (5)
H21 0.2919 −0.3053 0.3654 0.031\*
C23 0.1781 (3) −0.22716 (11) 0.44351 (19) 0.0233 (4)
H22 0.1473 −0.2498 0.5102 0.028\*
C20 0.2633 (3) −0.15863 (12) 0.2441 (2) 0.0279 (5)
H19 0.2902 −0.1361 0.1765 0.033\*
C21 0.3069 (3) −0.22641 (13) 0.2574 (2) 0.0284 (5)
H20 0.3648 −0.2483 0.1991 0.034\*
C15 −0.3168 (3) 0.06123 (11) 0.06044 (18) 0.0196 (4)
H26 −0.3592 0.0590 −0.0160 0.023\*
C18 −0.1906 (3) 0.06585 (10) 0.28794 (17) 0.0167 (4)
H23 −0.1490 0.0669 0.3646 0.020\*
C17 −0.3669 (3) 0.07221 (11) 0.26411 (19) 0.0213 (4)
H24 −0.4421 0.0781 0.3245 0.026\*
C16 −0.4300 (3) 0.06968 (11) 0.1503 (2) 0.0222 (4)
H25 −0.5477 0.0736 0.1340 0.027\*
----- ------------- --------------- --------------- -------------------- --
Atomic displacement parameters (Å^2^) {#tablewrapadps}
=====================================
----- -------------- -------------- -------------- -------------- -------------- --------------
*U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^
Br1 0.03207 (13) 0.01420 (11) 0.02574 (12) 0.00133 (8) −0.00214 (8) 0.00038 (8)
C11 0.0146 (8) 0.0182 (9) 0.0117 (8) 0.0015 (7) 0.0003 (6) −0.0008 (7)
C7 0.0106 (8) 0.0179 (9) 0.0127 (8) −0.0001 (7) 0.0007 (6) 0.0006 (7)
C5 0.0114 (8) 0.0183 (9) 0.0136 (8) 0.0001 (7) −0.0003 (6) 0.0010 (7)
C8 0.0179 (9) 0.0130 (9) 0.0138 (8) −0.0031 (7) 0.0021 (7) 0.0006 (7)
C4 0.0147 (8) 0.0167 (9) 0.0144 (8) 0.0003 (7) 0.0011 (6) −0.0002 (7)
C9 0.0229 (10) 0.0164 (9) 0.0122 (8) −0.0002 (7) −0.0018 (7) 0.0020 (7)
C10 0.0228 (10) 0.0171 (9) 0.0130 (8) −0.0006 (7) −0.0030 (7) −0.0005 (7)
C3 0.0181 (9) 0.0148 (9) 0.0177 (9) 0.0003 (7) −0.0002 (7) −0.0014 (7)
C2 0.0196 (9) 0.0150 (9) 0.0209 (9) 0.0007 (7) 0.0017 (7) 0.0014 (7)
C1 0.0184 (9) 0.0198 (10) 0.0194 (9) 0.0017 (7) −0.0024 (7) 0.0034 (8)
C6 0.0161 (9) 0.0190 (9) 0.0158 (8) 0.0005 (7) −0.0018 (7) 0.0016 (7)
N1 0.0328 (10) 0.0190 (9) 0.0171 (8) 0.0099 (7) −0.0040 (7) −0.0050 (7)
C12 0.0231 (10) 0.0217 (10) 0.0210 (10) −0.0012 (8) −0.0007 (8) −0.0004 (8)
O1 0.0352 (8) 0.0141 (7) 0.0127 (6) −0.0047 (6) −0.0025 (6) 0.0015 (5)
C13 0.0133 (8) 0.0110 (8) 0.0151 (8) −0.0006 (6) 0.0003 (6) 0.0008 (6)
C14 0.0172 (9) 0.0139 (9) 0.0150 (8) 0.0003 (7) −0.0006 (7) −0.0001 (7)
C19 0.0342 (12) 0.0167 (10) 0.0184 (9) −0.0058 (8) −0.0035 (8) −0.0003 (7)
C24 0.0310 (11) 0.0160 (10) 0.0172 (9) −0.0010 (8) −0.0044 (8) −0.0007 (7)
C22 0.0271 (11) 0.0185 (10) 0.0322 (12) 0.0030 (8) −0.0083 (9) −0.0003 (9)
C23 0.0323 (11) 0.0170 (10) 0.0202 (9) −0.0038 (8) −0.0049 (8) 0.0040 (8)
C20 0.0375 (13) 0.0260 (12) 0.0202 (10) −0.0105 (10) 0.0029 (9) −0.0002 (9)
C21 0.0252 (11) 0.0316 (13) 0.0285 (11) −0.0040 (9) 0.0024 (9) −0.0091 (10)
C15 0.0167 (9) 0.0214 (10) 0.0202 (9) −0.0025 (7) −0.0060 (7) 0.0015 (8)
C18 0.0183 (9) 0.0178 (9) 0.0140 (8) −0.0001 (7) 0.0015 (7) 0.0026 (7)
C17 0.0156 (9) 0.0228 (10) 0.0259 (10) 0.0000 (8) 0.0055 (8) 0.0060 (8)
C16 0.0125 (9) 0.0226 (10) 0.0313 (11) −0.0033 (7) −0.0018 (8) 0.0084 (9)
----- -------------- -------------- -------------- -------------- -------------- --------------
Geometric parameters (Å, °) {#tablewrapgeomlong}
===========================
---------------------- -------------- ----------------------- --------------
Br1---C2 1.898 (2) C12---C19 1.527 (3)
C11---N1 1.467 (3) C12---H13 0.9800
C11---C13 1.518 (3) C13---C18 1.385 (3)
C11---C7 1.520 (3) C13---C14 1.399 (3)
C11---H16 0.9800 C14---C15 1.391 (3)
C7---C8 1.377 (3) C14---H27 0.9300
C7---C5 1.424 (3) C19---C24 1.381 (3)
C5---C6 1.420 (3) C19---C20 1.388 (3)
C5---C4 1.428 (3) C24---C23 1.383 (3)
C8---O1 1.361 (2) C24---H18 0.9300
C8---C9 1.418 (3) C22---C21 1.384 (4)
C4---C3 1.414 (3) C22---C23 1.390 (3)
C4---C10 1.421 (3) C22---H21 0.9300
C9---C10 1.361 (3) C23---H22 0.9300
C9---H9 0.9300 C20---C21 1.410 (4)
C10---H10 0.9300 C20---H19 0.9300
C3---C2 1.367 (3) C21---H20 0.9300
C3---H3 0.9300 C15---C16 1.389 (3)
C2---C1 1.410 (3) C15---H26 0.9300
C1---C6 1.370 (3) C18---C17 1.393 (3)
C1---H1 0.9300 C18---H23 0.9300
C6---H6 0.9300 C17---C16 1.384 (3)
N1---C12 1.399 (3) C17---H24 0.9300
N1---H2 0.8600 C16---H25 0.9300
C12---O1 1.477 (3)
N1---C11---C13 111.12 (16) O1---C12---C19 102.55 (16)
N1---C11---C7 110.33 (15) N1---C12---H13 108.8
C13---C11---C7 115.09 (16) O1---C12---H13 108.8
N1---C11---H16 106.6 C19---C12---H13 108.8
C13---C11---H16 106.6 C8---O1---C12 114.47 (15)
C7---C11---H16 106.6 C18---C13---C14 118.95 (17)
C8---C7---C5 118.64 (17) C18---C13---C11 121.92 (17)
C8---C7---C11 119.26 (18) C14---C13---C11 119.11 (17)
C5---C7---C11 121.90 (16) C15---C14---C13 120.09 (19)
C6---C5---C7 122.55 (17) C15---C14---H27 120.0
C6---C5---C4 117.61 (18) C13---C14---H27 120.0
C7---C5---C4 119.84 (17) C24---C19---C20 119.0 (2)
O1---C8---C7 123.66 (17) C24---C19---C12 117.2 (2)
O1---C8---C9 114.54 (17) C20---C19---C12 123.8 (2)
C7---C8---C9 121.75 (18) C19---C24---C23 121.4 (2)
C3---C4---C10 120.77 (18) C19---C24---H18 119.3
C3---C4---C5 120.17 (18) C23---C24---H18 119.3
C10---C4---C5 119.06 (18) C21---C22---C23 119.2 (2)
C10---C9---C8 120.16 (18) C21---C22---H21 120.4
C10---C9---H9 119.9 C23---C22---H21 120.4
C8---C9---H9 119.9 C24---C23---C22 120.1 (2)
C9---C10---C4 120.50 (18) C24---C23---H22 119.9
C9---C10---H10 119.8 C22---C23---H22 119.9
C4---C10---H10 119.8 C19---C20---C21 119.9 (2)
C2---C3---C4 119.77 (18) C19---C20---H19 120.1
C2---C3---H3 120.1 C21---C20---H19 120.1
C4---C3---H3 120.1 C22---C21---C20 120.3 (2)
C3---C2---C1 121.2 (2) C22---C21---H20 119.8
C3---C2---Br1 120.56 (16) C20---C21---H20 119.8
C1---C2---Br1 118.21 (15) C16---C15---C14 120.37 (19)
C6---C1---C2 119.62 (19) C16---C15---H26 119.8
C6---C1---H1 120.2 C14---C15---H26 119.8
C2---C1---H1 120.2 C13---C18---C17 120.99 (18)
C1---C6---C5 121.57 (18) C13---C18---H23 119.5
C1---C6---H6 119.2 C17---C18---H23 119.5
C5---C6---H6 119.2 C16---C17---C18 119.8 (2)
C12---N1---C11 111.40 (17) C16---C17---H24 120.1
C12---N1---H2 124.3 C18---C17---H24 120.1
C11---N1---H2 124.3 C17---C16---C15 119.76 (19)
N1---C12---O1 113.24 (18) C17---C16---H25 120.1
N1---C12---C19 114.31 (19) C15---C16---H25 120.1
N1---C11---C7---C8 −16.2 (2) C7---C11---N1---C12 48.5 (2)
C13---C11---C7---C8 110.5 (2) C11---N1---C12---O1 −62.2 (2)
N1---C11---C7---C5 158.55 (18) C11---N1---C12---C19 −179.14 (17)
C13---C11---C7---C5 −74.7 (2) C7---C8---O1---C12 −6.1 (3)
C8---C7---C5---C6 −178.32 (19) C9---C8---O1---C12 176.55 (18)
C11---C7---C5---C6 6.9 (3) N1---C12---O1---C8 39.9 (2)
C8---C7---C5---C4 1.2 (3) C19---C12---O1---C8 163.56 (18)
C11---C7---C5---C4 −173.63 (17) N1---C11---C13---C18 97.4 (2)
C5---C7---C8---O1 −179.57 (18) C7---C11---C13---C18 −28.9 (3)
C11---C7---C8---O1 −4.6 (3) N1---C11---C13---C14 −81.0 (2)
C5---C7---C8---C9 −2.4 (3) C7---C11---C13---C14 152.64 (18)
C11---C7---C8---C9 172.48 (18) C18---C13---C14---C15 −0.9 (3)
C6---C5---C4---C3 1.0 (3) C11---C13---C14---C15 177.60 (19)
C7---C5---C4---C3 −178.53 (18) N1---C12---C19---C24 −176.1 (2)
C6---C5---C4---C10 −179.44 (18) O1---C12---C19---C24 60.9 (2)
C7---C5---C4---C10 1.1 (3) N1---C12---C19---C20 3.5 (3)
O1---C8---C9---C10 178.84 (19) O1---C12---C19---C20 −119.5 (2)
C7---C8---C9---C10 1.5 (3) C20---C19---C24---C23 0.0 (3)
C8---C9---C10---C4 0.8 (3) C12---C19---C24---C23 179.6 (2)
C3---C4---C10---C9 177.5 (2) C19---C24---C23---C22 1.1 (3)
C5---C4---C10---C9 −2.1 (3) C21---C22---C23---C24 −0.8 (3)
C10---C4---C3---C2 −179.0 (2) C24---C19---C20---C21 −1.2 (3)
C5---C4---C3---C2 0.6 (3) C12---C19---C20---C21 179.2 (2)
C4---C3---C2---C1 −1.9 (3) C23---C22---C21---C20 −0.4 (3)
C4---C3---C2---Br1 178.18 (15) C19---C20---C21---C22 1.4 (4)
C3---C2---C1---C6 1.5 (3) C13---C14---C15---C16 1.4 (3)
Br1---C2---C1---C6 −178.55 (16) C14---C13---C18---C17 −0.3 (3)
C2---C1---C6---C5 0.2 (3) C11---C13---C18---C17 −178.75 (19)
C7---C5---C6---C1 178.12 (19) C13---C18---C17---C16 1.0 (3)
C4---C5---C6---C1 −1.4 (3) C18---C17---C16---C15 −0.4 (3)
C13---C11---N1---C12 −80.4 (2) C14---C15---C16---C17 −0.8 (3)
---------------------- -------------- ----------------------- --------------
Table 1 C---H···π interactions (Å) {#d1e2848}
==================================
Cg3, Cg4 and Cg5 are the centroids of the C4/C5/C7--C16, C13--C18 and C19--C24 rings, respectively.
--------------------- ------------ ---------- ----------
*X*---H···*Cg* *X*···*Cg* H···*Cg* H···Perp
C1--H1···Cg5^i^ 3.357 (8) 2.80 2.67
C24--H18···Cg3^ii^ 3.692 (9) 2.93 2.90
C22--H21···Cg4^iii^ 3.547 (3) 2.68 2.61
C17--H24···Cg3^iv^ 3.587 (8) 2.70 2.67
--------------------- ------------ ---------- ----------
Symmetry codes: (i) -x, 1/2+y, 1/2-z ; (ii) -x, -y, 1-z ; (iii) -x, -1/2+y, 1/2-z ; (iv) -1+x, y, z.
###### C---H⋯π interactions (Å)
*Cg*3, *Cg*4 and *Cg*5 are the centroids of the C4/C5/C7--C16, C13--C18 and C19--C24 rings, respectively.
*X*---H⋯*Cg* *X*⋯*Cg* H⋯*Cg* H⋯Perp
--------------------- ----------- -------- --------
C1--H1⋯*Cg*5^i^ 3.357 (8) 2.80 2.67
C24--H18⋯*Cg*3^ii^ 3.692 (9) 2.93 2.90
C22--H21⋯*Cg*4^iii^ 3.547 (3) 2.68 2.61
C17--H24⋯*Cg*3^iv^ 3.587 (8) 2.70 2.67
Symmetry codes: (i) −*x*, + *y*, − *z*; (ii) −*x*, −*y*, 1 − *z*; (iii) −*x*, − + *y*, − *z*; (iv) −1 + *x*, *y*, *z*.
|
{
"pile_set_name": "PubMed Central"
}
|
1. Introduction {#sec1-nutrients-11-01672}
===============
The relationship between fat intake and heart disease is not entirely clear, particularly among postmenopausal women with relatively low fat consumption \[[@B1-nutrients-11-01672],[@B2-nutrients-11-01672]\]. Some meta-analyses of observational studies and randomized controlled trials found no association between saturated fatty acids (SFA) and risk of coronary heart disease (CHD) \[[@B3-nutrients-11-01672],[@B4-nutrients-11-01672]\]. However, these studies lack a consideration of the nutrient substitution framework, under which significant associations have been observed \[[@B5-nutrients-11-01672],[@B6-nutrients-11-01672],[@B7-nutrients-11-01672]\]. Dietary intervention trials have demonstrated that SFA elevate total cholesterol and low-density lipoprotein cholesterol (LDL-C), and substituting SFA with polyunsaturated fatty acids (PUFA) is associated with lower CHD risk \[[@B8-nutrients-11-01672],[@B9-nutrients-11-01672]\]. Based on the above evidence, several professional organizations have recommended diets limiting SFA and increasing PUFA n-3 \[[@B10-nutrients-11-01672],[@B11-nutrients-11-01672]\].
Until recently, all SFA were viewed as unhealthy as refined carbohydrates when considering their impact on increasing LDL-C and total cholesterol. Although not all SFA have the same cholesterol-raising effect, no recommendations have been made for specific SFA due to insufficient evidence. Evidence supporting the associations of monounsaturated fatty acids (MUFA) and PUFA n-6 with CHD risk is also less definitive \[[@B4-nutrients-11-01672],[@B12-nutrients-11-01672]\]. A recent meta-analysis of prospective observational studies and randomized trials concluded that there was no significant association of dietary MUFA and PUFA n-6 with CHD risk, but a higher CHD risk of *trans* fatty acids (TFA) (pooled RR (95% Cis): 1.16 (1.06--1.27)) and a lower CHD risk of long-chain PUFA n-3 (0.87 (0.78--0.97)) \[[@B4-nutrients-11-01672]\]. Several investigators have questioned the interpretation of these results \[[@B9-nutrients-11-01672],[@B13-nutrients-11-01672]\]. One major concern was that the macronutrient for replacement, which could have had an independent association with CHD outcome, was not identified or accounted for \[[@B9-nutrients-11-01672],[@B13-nutrients-11-01672]\]. An additional concern was that the dietary assessment methods in these reports were via self-report instruments, which is valid when ranking individuals according to the intake levels, but contributes substantial systematic measurement error when the absolute intake levels are of interest.
To address these concerns, the Multi-Ethnic Study of Atherosclerosis \[[@B12-nutrients-11-01672],[@B14-nutrients-11-01672]\] and the Women's Health Initiative Observational Study (WHI-OS) \[[@B15-nutrients-11-01672]\] have measured plasma phospholipid fatty acids (PL-FA) as indicators of medium-term dietary FA intake \[[@B16-nutrients-11-01672],[@B17-nutrients-11-01672]\], and have shown associations between PL-FA and CHD risk. Specifically, one interquartile range increase in PL long-chain PUFA n-3 or SFA 15:0 were associated with a lower CHD risk (HR (95%CIs), 0.40 (0.23--0.69) and 0.76 (0.61--0.93), respectively) \[[@B12-nutrients-11-01672],[@B14-nutrients-11-01672]\]; 1 moL % increase in PL SFA was associated with a 20% higher CHD risk (95% CIs 1.08--1.32); and 1 moL % increase in PL PUFA n-3 was associated with a 11% lower CHD risk (95% CIs 0.83--0.97) \[[@B15-nutrients-11-01672]\]. While plasma PL-FA offer an objective biomarker of dietary FA that could potentially reduce the measurement error from questionnaires or diet recalls, plasma PL-FA reflect both dietary intake and endogenous synthesis. Therefore, caution is needed when interpreting the association with disease outcome.
Large observational studies and dietary intervention trials have demonstrated that PL-FA profiles are good biomarkers of dietary fat, especially for those FA with limited in vivo synthesis---PUFA n-3, n-6, and TFA \[[@B16-nutrients-11-01672],[@B18-nutrients-11-01672]\]. In addition, randomized trials have shown that substituting dietary PUFA n-6 with PUFA n-3 at 10% of total energy led to a 2--3% plasma substitution \[[@B19-nutrients-11-01672]\]. Thus, the substitution of plasma PL-FA by changes in dietary fat types has a potential for evaluating the association of specific dietary FA with CHD risk, with less measurement error. However, the substitution of plasma PL-FA has not been examined so far, and we are still lack evidence from postmenopausal women.
Based upon the previously reported associations between plasma PL-FA and CHD, we sought to examine the following: (1) the association of plasma PL-FA levels, specifically long-chain and very-long-chain plasma PL SFA, with CHD risk; (2) the effect of substituting plasma PUFA n-6 and TFA with PUFA n-3 on CHD risk; and (3) potential food groups that are correlated with plasma PL-FA, using data from the WHI study.
2. Materials and Methods {#sec2-nutrients-11-01672}
========================
2.1. Study Population {#sec2dot1-nutrients-11-01672}
---------------------
The WHI-OS is a prospective cohort study that enrolled 93,676 postmenopausal women between the ages of 50 and 79 years in the United States from 1994 to 1998. It is designed to assess the biological, lifestyle, and genetic factors for CHD and other major health events among postmenopausal women. A detailed description of the WHI study design has been published elsewhere \[[@B20-nutrients-11-01672],[@B21-nutrients-11-01672]\].
A matched case-control design was used for the current study. All CHD cases, including hospitalized myocardial infarction (MI), definite silent MI, and coronary death, were confirmed based on medical records and death certificates. The cases included in the current study were a random sample of all CHD cases identified based on the September 2005 database \[[@B22-nutrients-11-01672]\]. A total of 2468 cases were initially selected and those who met the following criteria were excluded: (1) lack of sufficient baseline plasma sample (*N* = 28) (2) missing baseline dietary measurement (*N* = 126), and (3) self-reported baseline CVD, which includes angina, MI, coronary artery bypass graft (CABG) surgery, percutaneous transluminal coronary angioplasty (PTCA), carotid artery disease, congestive heart failure, stroke or peripheral vascular disease (*N* = 765). Potential controls were women from the entire WHI-OS who did not develop CVD during the follow-up (a mean of 4.5 years) and were excluded if meeting the same exclusion criteria as the cases. Cases and controls were matched on age at screening, enrollment date, race/ethnicity (White, Black, Hispanic, other), and hysterectomy status. The sample size for PL-FA assays included 1224 matched case-control pairs and 10% blind duplicates for quality control. We additionally excluded 20 participants due to the lack of plasma PL-FA profile results (*N* = 11) or missing matched pairs (*N* = 9), and came up with a final sample size of 1214 matched pairs. A separate approval for using de-identified samples and data for this study was obtained from the Tufts University/Tufts Medical Center Institutional Review Board \[[@B15-nutrients-11-01672]\].
2.2. Plasma PL-FA Profiles {#sec2dot2-nutrients-11-01672}
--------------------------
Blood samples were collected at baseline and a minimum of 12 h fasting before blood draw was required. All blood samples were maintained at 4 °C for up to an hour until plasma was separated from cells, frozen at −20 °C, and then sent to the central repository stored at −80 °C. Plasma PL-FA profiles were measured by an established gas chromatography method \[[@B23-nutrients-11-01672]\] at Tufts University. Peaks of interest were identified by comparison with authentic FA standards (National Institute of Health Fatty Acid Standards A, B, and C, Nu-Check-Prep, Elysian, MN, USA), and expressed as molar percentage (moL %) proportions of FA relative to the internal standard (heptadecanoic acid). Internal and external quality controls were performed to guarantee the validity of the measurements, and detailed information has been published previously \[[@B15-nutrients-11-01672]\]. A total of 28 individual plasma PL-FA were measured. We classified these FA into groups based on the number of double bonds---specifically SFA, MUFA, PUFA n-3, PUFA n-6, and TFA. We further classified FA by the length of FA chains: long-chain FA are those with 12--19 carbons and very-long-chain FA are those with 20 or more carbons. [Table 1](#nutrients-11-01672-t001){ref-type="table"} shows the lipid names, common names, categories, and mean (SD) levels of individual plasma PL-FA that were included in this study.
2.3. Covariates and Dietary Data {#sec2dot3-nutrients-11-01672}
--------------------------------
Standard questionnaires following the same protocol were utilized throughout the study to collect information related to socio-demographics, lifestyle factors, and CHD risk factors \[[@B20-nutrients-11-01672]\]. We initially considered the following variables as potential confounders: (1) socio-demographic variables (including age, U.S. region, race/ethnicity, education, and income); (2) lifestyle factors such as recreational physical activity, body mass index (BMI), waist circumference, waist-to-hip ratio, and smoking; and (3) CHD risk factors (family history of MI/diabetes/stroke, anticoagulant/anti-diabetic/lipid lowering medication use, postmenopausal hormone use, and self-reported hypertension/diabetes/hypercholesterolemia/hysterectomy status at baseline).
Information on nutrient intake and food consumption was assessed by food frequency questionnaire at baseline \[[@B20-nutrients-11-01672]\]. Potential dietary confounders included alcohol intake (g/day), percent calories from protein/carbohydrates, and total energy intake (kcal/day) \[[@B24-nutrients-11-01672]\]. Foods high in fats or that have the potential to influence fat metabolism were assessed for further correlation analysis with plasma PL-FA. For this analysis, we considered the following nine food groups: fish, dairy products, butter, margarine, olive/canola oil, other vegetable oils, red meat, alcohol, and carbohydrates \[[@B25-nutrients-11-01672],[@B26-nutrients-11-01672],[@B27-nutrients-11-01672],[@B28-nutrients-11-01672]\].
Age and BMI were treated as continuous variables. Physical activity, measured by recreational physical activity score (MET-h/week) based on a series of questions related to exercise intensity levels \[[@B29-nutrients-11-01672]\], was treated as continuous or categorical dichotomized at median. Education was categorized as ≤high school, some college, or postgraduate. Income was categorized as \<\$20,000, \$20,000--74,999, or ≥\$75,000 per year. Smoking was categorized as current, past, or never smoker. Family history was defined as first-degree relatives having MI, diabetes, or stroke. Postmenopausal hormone use was categorized into current estrogen + progesterone, current estrogen alone, past users, or never used. Hypertension was defined as self-reported hypertension/taking antihypertensive medication, or systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg. Baseline diabetes and hypercholesterolemia were defined as taking anti-diabetic or cholesterol-lowering medications, respectively.
2.4. Statistical Analysis {#sec2dot4-nutrients-11-01672}
-------------------------
We initially examined the baseline distribution of socio-demographics, lifestyle factors, CHD risk factors, and dietary factors by CHD status, as well as by five subtypes of plasma PL-FA in tertiles ([Appendix A](#app1-nutrients-11-01672){ref-type="app"}). Descriptive statistics such as median, mean, standard deviation, frequency, and proportion were used to summarize the aforementioned variables. Depending on the distribution of the variables, we used the paired *t*-test, Wilcoxon signed rank test, or McNemar test for the comparison between cases and controls.
We employed multivariable conditional logistic regression models to estimate odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for CHD risk in association with a 1 moL % increase in plasma PL-FA. The covariates in the multivariable model were selected based on a hypothesized causal diagram ([Appendix B](#app2-nutrients-11-01672){ref-type="app"}) to adjust for potential confounding, and a backward selection method was used to generate a parsimonious model with the best model fit \[[@B30-nutrients-11-01672]\]. The final multivariable model adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income, physical activity, smoking, family history of MI/diabetes, postmenopausal hormone use, self-reported hypertension/diabetes, percent calories from protein/carbohydrates, and total energy intake. We have performed model diagnosis and examined model assumptions. No outlier was observed and all model assumptions held.
Linoleic, α-linolenic, and *trans* FA cannot be synthesized in vivo. In addition, the elongase and desaturase enzymes in human livers have low activity when regulating the synthesis of long-chain PUFA from their precursors \[[@B31-nutrients-11-01672]\]. Therefore, plasma PL PUFA n-3, n-6, and TFA are good biomarkers of corresponding dietary FA, and substitutions of dietary FA can be estimated by plasma substitutions. To estimate the theoretical effect of substituting 1 moL % of plasma PL PUFA n-6 with the same proportion of PUFA n-3, we left out PUFA n-6 in the multivariable model. The relation can be expressed as follows: where $\beta_{1}$ to $\beta_{4}$ are regression coefficients. The total of all PL-FA is 100 moL %, so that the coefficient $\beta_{1}$ can be interpreted as the effect of substituting 1 moL % of PUFA n-6 with the same proportion of PUFA n-3 while holding other FA constant \[[@B24-nutrients-11-01672]\]. This substitution model was similarly applied to TFA substitution analysis.
To further examine potential food sources that related to plasma PL-FA levels, we calculated Spearman's rank correlation coefficients between plasma PL-FA levels and the consumption of nine selected food groups. Considering the cases might have altered metabolic status, thus not representing the overall population, the correlation coefficients were calculated among controls only, adjusting for matching factors and the aforementioned non-dietary confounders.
Multiple imputation (five times) by chained equations \[[@B32-nutrients-11-01672]\] was used to impute missing values on the following covariates: income (*N* = 118), physical activity (*N* = 28), smoking (*N* = 28), family history (*N* = 209, among which 119 were missing MI and 122 were missing diabetes), and self-reported hypertension (*N* = 47) and diabetes (*N* = 2).
We conducted the following sensitivity analyses to assess the robustness of findings: (1) comparing the association analysis results from different regression models; (2) examining the association between plasma PL-FA groups and CHD additionally adjusting for anthropometric measures (BMI, waist circumference, waist-to-hip ratio) and chronic weight cycling (three-year BMI change); (3) performing substitution analysis stratified by physical activity levels; and (4) performing the association and substitution analyses among participants with complete information (*N* = 2181). All analyses were performed using Statistics Analysis Systems software package (version 9.4; SAS Institute, Inc., Cary, NC, USA).
3. Results {#sec3-nutrients-11-01672}
==========
The characteristics of the cases and controls can be found in [Table 2](#nutrients-11-01672-t002){ref-type="table"}. The mean (SD) age was 67.8 (6.8) years, and the median time from baseline plasma PL-FA measures to CHD event among cases was 4.5 years. Compared with controls, cases had significantly lower education and income levels, higher BMI (26.9 vs. 25.9 kg/m^2^) and lower physical activity level (8.3 vs. 10.8 MET-h/week). More cases were smokers, had hypertension or diabetes, or reported a family history of MI and medication use, while fewer were currently using postmenopausal hormones.
3.1. Associations between Increased Plasma PL-FA and CHD Risk {#sec3dot1-nutrients-11-01672}
-------------------------------------------------------------
To examine the relationships between plasma PL-FA and CHD risk, we calculated the ORs (95% CIs) of CHD in association with 1 moL % increase in plasma PL-FA ([Table 3](#nutrients-11-01672-t003){ref-type="table"}). In the adjusted multivariable model, we observed higher CHD risk for increased plasma total PL SFA (OR = 1.20 (1.10--1.30)) and long-chain SFA (OR = 1.18 (1.09--1.28)), but not for very-long-chain SFA (OR = 1.00 (0.77--1.30)). We also observed a lower CHD risk associated with plasma PL PUFA n-3 (OR = 0.93 (0.88--0.99)). However, no significant associations were observed for plasma PL MUFA, PUFA n-6, and TFA. The associations between individual plasma PL-FA and CHD have been published elsewhere \[[@B15-nutrients-11-01672]\].
3.2. Plasma PL-FA Substitutions {#sec3dot2-nutrients-11-01672}
-------------------------------
To further estimate the effect of substituting dietary PUFA n-6 or TFA with PUFA n-3 on CHD risk, we calculated the ORs (95%CIs) of CHD risk from plasma PL-FA substitutions ([Table 4](#nutrients-11-01672-t004){ref-type="table"}). In the initial models, lower CHD risk was observed when 1 moL % of plasma PL PUFA n-6 or TFA were substituted with the same proportion of PUFA n-3. Although these associations were attenuated on adjustment for covariates (model 2), results were still statistically significant at the 0.05 significance level (OR = 0.90 (0.84--0.96) and 0.74 (0.56--0.99), respectively). However, in all models, substituting 1 moL % PL TFA with PUFA n-6 was not associated with CHD risk.
3.3. Correlations between Plasma PL-FA and Select Food Groups {#sec3dot3-nutrients-11-01672}
-------------------------------------------------------------
Correlations between food groups and plasma PL-FA levels have the potential to provide information regarding food sources that may influence plasma PL-FA concentrations ([Appendix C](#app3-nutrients-11-01672){ref-type="app"}). The strongest correlations that we found were in the PUFA n-3 group: fish and olive/canola oil intakes were positively correlated with plasma PL PUFA n-3 (*r* = 0.34 and 0.12, respectively; *p* \< 0.0001). We also observed a positive correlation between alcohol intake and plasma PL long-chain SFA (*r* = 0.13; *p* \< 0.0001). Margarine and red meat intakes were positively correlated with plasma PL PUFA n-6 and TFA (*r* ranges between 0.11 to 0.15; *p* \< 0.0001).
3.4. Sensitivity Analyses {#sec3dot4-nutrients-11-01672}
-------------------------
In the sensitivity analysis of comparing difference regression models, our results were consistent across models thus supporting the robustness of findings ([Appendix D](#app4-nutrients-11-01672){ref-type="app"}). When examining the association between plasma PL-FA and CHD while adjusting for different anthropometric measures, we found very similar results when adjusting for BMI, waist circumference, waist-to-hip ratio, or a three-year BMI change ([Appendix E](#app5-nutrients-11-01672){ref-type="app"}). In the substitution analysis stratified by physical activity levels, we classified participants into two groups: physically active (those with physical activity levels above the median, 9.5 MET-h/week) and physically inactive (those with physical activity levels ≤ 9.5 MET-h/week). As the point estimates varied to a small extent between the physically active and inactive, physical activity is less likely to be an effect modifier than a confounder ([Appendix F](#app6-nutrients-11-01672){ref-type="app"}). When comparing the association and substitution analyses using complete cases versus multiple imputation, we observed very similar results, thus suggesting the validity of the multiple imputation approach ([Appendix G](#app7-nutrients-11-01672){ref-type="app"} and [Appendix H](#app8-nutrients-11-01672){ref-type="app"}).
4. Discussion {#sec4-nutrients-11-01672}
=============
This prospective matched case-control study nested in the WHI-OS assessed the association of plasma PL-FA profile with CHD risk among 2428 postmenopausal women. In the adjusted analysis, we found that higher PL SFA, especially long-chain SFA, were associated with increased CHD risk, while higher PL PUFA n-3 were associated with lower CHD risk. No significant associations were found for PL very-long-chain SFA, MUFA, PUFA n-6 and TFA. In the substitution analysis, we found that substituting 1 moL % of plasma PL PUFA n-6 or TFA with the same proportion of PUFA n-3 were associated with lower CHD risk.
4.1. Plasma PL SFA Profiles and CHD Risk {#sec4dot1-nutrients-11-01672}
----------------------------------------
Individual SFA have diverse biological functions determined by the chain length \[[@B33-nutrients-11-01672]\]. For example, the effect of raising LDL-C decreases as the chain length increases \[[@B33-nutrients-11-01672]\]. Long-chain SFA, especially palmitic (16:0) and stearic (18:0) acids, are the primary dietary FA. Within the human body, long-chain SFA are a major component of cell membranes, and endogenous synthesis contributes a significant portion of SFA in the circulation with palmitic and stearic acids being the primary product \[[@B34-nutrients-11-01672]\]. Accumulating evidence has supported the relationship of the aforementioned long-chain FA metabolism with potential CHD risk \[[@B33-nutrients-11-01672]\]. The modest positive associations of total and long-chain PL SFA with CHD risk from our study were consistent with some \[[@B26-nutrients-11-01672],[@B35-nutrients-11-01672]\], but not all \[[@B36-nutrients-11-01672],[@B37-nutrients-11-01672]\], cohort studies that have assessed either total or individual SFA. The discordance may come from differences in age distributions, sources of blood SFA, and specific SFA included in analyses.
We did not observe significant associations between very-long-chain plasma PL SFA and CHD risk, which differs from a few recent population-based studies showing blood concentrations of very-long-chain SFA were associated with lower risk of cardiometabolic conditions, including CHD \[[@B38-nutrients-11-01672]\] and diabetes \[[@B39-nutrients-11-01672]\]. The mechanisms underlying these observations are not well established. Compared with long-chain SFA, very-long-chain SFA have lower water solubility and oxidation susceptibility, and they are major components of ceramides and sphingomyelins that affect liver homeostasis, myelin maintenance, and anti-inflammatory response through ceramide synthase expression, therefore showing potential beneficial effects on CHD \[[@B40-nutrients-11-01672],[@B41-nutrients-11-01672]\]. The discrepancies between our results and other studies may be explained by the following: (1) differences in study populations: participants in other studies were younger and had lower BMI, and research has shown that the association of very-long-chain SFA with lower cardiometabolic risks appeared strongest in participants with normal BMI \[[@B39-nutrients-11-01672]\]; and (2) different sources of SFA: most beneficial effects were identified from circulating very-long-chain SFA, while no significant results were found from plasma PL or erythrocyte membranes.
4.2. Plasma PL-FA Substitution in Groups {#sec4dot2-nutrients-11-01672}
----------------------------------------
We observed a lower risk of CHD when substituting plasma PL PUFA n-6 with n-3. The beneficial effects of PUFA n-3 on CHD have been reported in previous studies \[[@B12-nutrients-11-01672],[@B14-nutrients-11-01672],[@B15-nutrients-11-01672]\]. PUFA n-3 have been shown to lower plasma triglyceride levels, to have anti-thrombotic and anti-arrhythmic properties, to reduce macrophage infiltration into the vessel wall, and to reduce the proatherogenic secretion of growth factors and cytokines by monocytes \[[@B42-nutrients-11-01672]\].
Although dietary TFA have been shown to increase CHD risk \[[@B43-nutrients-11-01672]\], we only observed a significant change in CHD risk when substituting PL TFA with PUFA n-3, while no significant result was observed when TFA were substituted with PUFA n-6. This discrepancy may be explained by the limitation in the gas chromatography methodology we used to measure plasma PL-FA, which did not distinguish TFA isomers elaidic acid (18:1 n-9 t) and vaccenic acid (18:1 n-11 t) \[[@B15-nutrients-11-01672]\]. It has been suggested that 18:1 n-11 t, as the predominant TFA in dairy products, may have a weaker association with CHD compared with other TFA resulting from partial hydrogenated vegetable oils, mainly 18:1 n-9 t \[[@B44-nutrients-11-01672]\]. Another possibility is that the relatively small proportion of PL TFA, reflecting the dietary characteristics of the cohort of older women, lead to a lack of power to detect an association.
The substitution analysis between different plasma PL-FA may be more informative than the measured plasma PL-FA profiles themselves when examining the association of one type of plasma PL-FA with CHD. The plasma PL-FA were measured in moL % with a summation of 100% for all PL-FA, and 1 moL % increase in one type of plasma PL-FA is accompanied by reciprocal decrease in 1 moL % for another. The plasma PL-FA substitution analysis is also more informative than the substitution analysis of dietary FA and CHD risk, given that there is a lack of accuracy for each type of dietary fat using any diet assessment method \[[@B45-nutrients-11-01672]\].
4.3. Potential Food Sources of Plasma PL-FA and Dietary Recommendations {#sec4dot3-nutrients-11-01672}
-----------------------------------------------------------------------
The Spearman correlations we observed between plasma PL-FA and selected food groups were moderate; however, they were consistent and comparable to previous evidence from other observational studies using food frequency questionnaire as dietary measurement \[[@B18-nutrients-11-01672],[@B46-nutrients-11-01672]\]. In short-term dietary intervention trials and observational studies using multiple dietary measurements, the correlations were stronger, therefore showing the measurement error in food frequency questionnaire might be a cause of our moderate correlations \[[@B47-nutrients-11-01672],[@B48-nutrients-11-01672]\].
The strongest correlation that we found was between plasma PL PUFA n-3 and fish intake. This result is consistent with both observational study and dietary intervention trials \[[@B18-nutrients-11-01672],[@B46-nutrients-11-01672]\], showing fatty fish and fish oils are the predominant sources of PUFA n-3. We found a positive correlation between plasma PL long-chain SFA and alcohol intake. This may be because alcohol can increase the activity of acetyl-CoA carboxylase and FA synthase---the key enzymes in SFA 16:0 synthesis \[[@B49-nutrients-11-01672]\]. Our analysis also showed that margarine and red meat intake was positively associated with plasma PL TFA levels. Because humans do not synthesize TFA, diet contributes to the occurrence of these FA isomers in the plasma. TFA can be found naturally from ruminant-animal meat (mainly 18:1 n-7 t), dairy fat (mainly 18:1 n-11 t), and unnaturally from industrially hydrogenated vegetable oils (mainly 18:1 n-9 t), such as margarine \[[@B50-nutrients-11-01672]\].
Our findings are consistent with the recommendation made in the Scientific Report of the 2015 Dietary Guidelines Advisory Committee on food sources of nutrients among U.S. adults \[[@B51-nutrients-11-01672]\]. Foods associated with an increase of plasma PL long-chain SFA and TFA (including alcohol, red meat, and margarine) should be limited. Dietary PUFA n-3, which can be found in fatty fish and canola oil, should be recommended. However, caution is required when attempting to apply dietary recommendations extrapolated from plasma levels of nutrients, which are only modestly correlated with dietary intake.
There are several strengths of this study compared with previous ones. The matched case-control design nested in a prospective cohort study allowed us to assess exposure in cases before the diagnosis of CHD and select controls based on incidence density sampling, thus addressing the temporality between exposure and disease onset. In addition, our study is novel due to the focus on the association of theoretical plasma PL-FA substitutions on CHD risk, which reflect in vivo FA metabolism as well as dietary changes with less measurement error. However, our study had several limitations. We did not detect all plasma PL-FA (such as short- and medium-chain SFA with carbons less than 12 and isomers of TFA), thus limiting our analyses to a selective set of FA. However, these unmeasured FA are present in very small amounts. Additional limitations include: (1) confounding bias due to unmeasured CHD risk factors, such as blood lipids; and (2) limited evidence when extrapolating plasma PL-FA findings to dietary changes.
5. Conclusions {#sec5-nutrients-11-01672}
==============
This plasma PL-FA analysis suggests that long-chain SFA may be associated with increased risk of CHD, and substituting PUFA n-6 or TFA with PUFA n-3 may be associated with lower risk of CHD. Further work is needed on distinguishing the specific dietary factors that influence plasma PL-FA levels within the context of the other covariates that may likewise impact outcomes.
WHI investigators. Program Office (National Heart, Lung, and Blood Institute, Bethesda, MD, USA): Jacques Rossouw, Shari Ludlam, Joan McGowan, Leslie Ford and Nancy Geller. Clinical Coordinating Center (Fred Hutchinson Cancer Research Center, Seattle, WA, USA): Garnet Anderson, Ross Prentice, Andrea LaCroix and Charles Kooperberg. Investigators and Academic Centers (Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA): JoAnn E. Manson; (MedStar Health Research Institute/Howard University, Washington, DC, USA) Barbara V. Howard; (Stanford Prevention Research Center, Stanford, CA, USA) Marcia L. Stefanick; (The Ohio State University, Columbus, OH, USA) Rebecca Jackson; (University of Arizona, Tucson/Phoenix, AZ, USA) Cynthia A. Thomson; (University at Buffalo, Buffalo, NY, USA) Jean Wactawski-Wende; (University of Florida, Gainesville/Jacksonville, FL, USA) Marian Limacher; (University of Iowa, Iowa City/Davenport, IA, USA) Jennifer Robinson; (University of Pittsburgh, Pittsburgh, PA, USA) Lewis Kuller; (Wake Forest University School of Medicine, Winston-Salem, NC, USA) Sally Shumaker; (University of Nevada, Reno, NV, USA) Robert Brunner. Women's Health Initiative Memory Study: (Wake Forest University School of Medicine, Winston-Salem, NC, USA) Mark Espeland.
Q.L. contributed to the study conceptualization, analysis, interpretation of data, and drafting of the manuscript. C.B.E. supervised the project, contributed to the study conceptualization, design, interpretation of data, revising the manuscript, and final approval of the manuscript. A.H.L. designed the study, provided the specimen data, and participated in revising the manuscript. N.R.M., J.E.M., B.V.H., L.F.T., M.L.N., L.V.V.H., J.E.R., M.A.A., L.W.M., W.L., L.G.S., and L.W. participated in revising the manuscript, providing critical comments related to model fitting, and data interpretation.
The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through contracts HHSN268201600018C, HHSN268201600001C, HHSN268201600002C, HHSN268201600003C, and HHSN268201600004C.
There is no disclosure for potential conflict of interests.
AA
arachidonic acid
CHD
coronary heart disease
DHA
docosahexaenoic acid
HDL-C
High-density lipoprotein cholesterol
LA
linoleic acid
LDL-C
low-density lipoprotein cholesterol
MI
myocardial infarction
MUFA
monounsaturated fatty acids
PL-FA
phospholipid fatty acids
PUFA
polyunsaturated fatty acids
SFA
saturated fatty acids
TFA
trans
fatty acids
WHI-OS
Women's Health Initiative Observational Study
nutrients-11-01672-t0A1_Table A1
######
Relationship of plasma phospholipid fatty acids ^a^ and socio-demographic, lifestyle, and CHD risk factors in the matched case-control study of the Women's Health Initiative Observational Study (1994--2005) (*N* = 2428).
SFA MUFA PUFA n-3 PUFA n-6 TFA
------------------------ ------ -------- ---------- ---------- ------ -------- ------ ---------- ------ -------- ------ ---------- ------ -------- ------ ---------- ------ ---------- ------ ----------
Median, moL % 44.8 46.0 47.3 10.4 11.6 13.3 3.9 4.9 6.5 34.0 36.5 38.5 0.4 0.6 1.0
**Socio-demographics**
Age, y ^c^ 67.8 68.1 67.5 *0.34* 67.4 68.1 67.9 *0.16* 67.4 67.7 68.3 *\<0.01* 68.0 68.0 67.4 *0.09* 67.6 68.0 67.8 *0.64*
White, % 92 89 88 *0.41* 86 91 92 *\<0.01* 93 89 86 *\<0.01* 89 88 91 *0.04* 87 91 90 *0.02*
Income, % *0.94* *0.24* *\<0.01* *\<0.01* *\<0.01*
\<\$20,000 19 18 18 17 20 18 23 18 14 16 19 20 15 16 25
\$20,000--\$74,999 63 63 64 65 63 62 66 65 59 59 66 65 62 66 62
[\>]{.ul}\$75,000 18 18 18 18 17 20 11 17 27 25 16 14 23 18 14
**Lifestyle factors**
PA, MET-h/week ^d^ 10.5 8.3 9.8 *\<0.01* 8.6 9.5 10.5 *\<0.01* 7.5 9.0 11.3 *\<0.01* 11.1 9.0 7.8 *\<0.01* 11.3 9.5 7.5 *\<0.01*
BMI, kg/m^2^ ^d^ 25.2 26.2 27.8 *\<0.01* 27.0 26.7 25.3 *\<0.01* 27.1 26.5 25.8 *\<0.01* 26.1 26.6 26.5 *\<0.01* 26.0 26.6 26.6 *\<0.01*
Smoking, % *0.24* *0.06* *\<0.01* *0.04* *\<0.01*
Never 54 49 49 54 51 47 51 51 51 47 50 54 46 52 54
Past 40 43 44 40 43 45 39 43 46 46 43 39 46 423 39
Current 6 8 6 6 7 8 11 6 3 6 7 7 8 5 7
**CHD risk factors**
Family history, % yes
MI 57 56 58 *0.75* 59 53 55 *0.37* 56 59 56 *0.41* 58 56 57 *0.75* 57 57 57 *0.93*
Diabetes 31 32 38 *0.01* 36 31 33 *0.24* 35 36 30 *0.03* 33 36 34 *0.30* 33 38 13 *0.02*
Hormone usage *0.10* *0.33* *0.02* *0.16* *\<0.01*
Current E + P 14 13 17 13 16 15 13 14 17 17 13 14 17 14 13
Current E alone 20 23 24 23 25 21 20 25 23 23 24 21 25 25 18
Past Users 17 15 14 16 15 17 17 16 13 14 15 18 15 15 16
Never Used 49 48 45 49 45 48 50 45 47 46 48 47 44 45 52
Hypertension *\<0.01* *\<0.01* *0.85* *0.90* *0.01*
Never 67 54 55 55 57 63 59 57 57 58 58 59 53 59 62
Untreated 8 11 10 9 10 11 10 10 10 11 9 10 10 10 9
Treated 25 36 35 36 34 27 31 33 33 32 33 32 37 31 29
Diabetes, % 6 7 10 *0.01* 11 8 5 *\<0.01* 10 8 6 *0.01* 6 8 10 *0.01* 8 9 7 *0.62*
Hysterectomy, % 39 41 43 *0.19* 43 42 38 *0.13* 42 43 39 *0.28* 39 41 43 *0.25* 40 45 38 *0.01*
**Dietary Factors**
Alc, g/d ^d^ 0.5 1.0 1.0 *0.02* 0.5 1.0 1.2 *\<0.01* 0.4 1.0 1.0 *\<0.01* 1.1 1.0 0.2 *\<0.01* 1.9 0.9 0.1 *\<0.01*
Carb % cal ^d^ 52.3 51.2 52.6 *0.55* 50.9 52.0 53.4 *\<0.01* 50.0 52.0 55.0 *\<0.01* 54.7 52.3 49.7 *\<0.01* 52.4 52.1 52.1 *0.96*
Protein % cal ^d^ 16.7 16.6 17.0 *0.06* 16.9 16.7 16.6 *0.10* 16.3 16.8 17.2 *\<0.01* 17.2 16.6 16.5 *\<0.01* 17.0 17.0 16.3 *\<0.01*
Energy, kcal/d ^d^ 1481 1500 1541 *0.03* 1499 1495 1518 *0.72* 1558 1535 1426 *\<0.01* 1524 1471 1533 *0.15* 1483 1542 1501 *0.52*
^a^ Tertiles of plasma phospholipid fatty acids (moL %) ^b^ *p* trend: Test for linear trends were conducted by treating the median value for each quartile of fatty acids as a continuous variable. ^c^ The mean (SD) of continuous variables. ^d^ The median (IQR) of continuous variables. Abbreviations: Alc (alcohol), BMI (body mass index). Cal (calories), Carb (carbohydrates), CHD (coronary heart disease), E (Estrogen), MI (myocardial infarction), MET-h (metabolic equivalent-hours), MUFA (mono-unsaturated fatty acids), P (Progesterone), PA (physical activity), PUFA (poly-unsaturated fatty acids), SFA (saturated fatty acids), TFA (*trans* fatty acids), SD (standard deviation), IQR (interquartile range).
{#nutrients-11-01672-f0A1}
######
The adjusted Spearman correlations ^a^ between plasma phospholipid fatty acids and select food groups. (**A**) Correlations between selected food groups and plasma PL long-chain SFA (**B**) Correlations between selected food groups and plasma PL PUFA n-3 (**C**) Correlations between selected food groups and plasma PL PUFA n-6 (**D**) Correlations between selected food groups and plasma PL TFA. ^a^ Correlations were adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income, lifestyle factors (physical activity and smoking), and CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes). Solid line---reference line of 0 Spearman correlations. Dashed line---Spearman correlations among controls. \* Significant Spearman correlations with *p* value \< 0.0001. Abbreviations: CHD (coronary heart disease), PL (phospholipid), PUFA (polyunsaturated fatty acids), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t0A2_Table A2
######
Associations between plasma phospholipid fatty acids (1 moL %) and CHD risk adjusting for different confounders in the matched case-control study (*N* = 2428).
Plasma Phospholipid Fatty Acids Crude Model ^a^ Fully Adjusted Model ^b^ Parsimonious Model ^c^ Adjusted Model from a Prior Study \[[@B15-nutrients-11-01672]\] ^d^
--------------------------------- ------------------- -------------------------- ------------------------ ---------------------------------------------------------------------
SFA 1.19 (1.11, 1.28) 1.23 (1.13, 1.34) 1.20 (1.10, 1.30) 1.20 (1.08, 1.32)
Long-chain SFA 1.17 (1.09, 1.25) 1.21 (1.12, 1.32) 1.18 (1.09, 1.28) NR
Very-long-chain SFA 1.00 (0.80, 1.26) 1.00 (0.76, 1.30) 1.00 (0.77, 1.30) NR
MUFA 0.96 (0.91, 1.01) 0.99 (0.93, 1.05) 0.98 (0.93, 1.04) 0.97 (0.91, 1.04)
PUFA n-3 0.89 (0.84, 0.94) 0.94 (0.88, 0.99) 0.93 (0.88, 0.99) 0.89 (0.83, 0.97)
PUFA n-6 1.03 (0.99, 1.06) 0.98 (0.95, 1.03) 1.00 (0.96, 1.03) 1.02 (0.97, 1.07)
TFA 1.06 (0.84, 1.33) 0.97 (0.74, 1.27) 1.01 (0.78, 1.31) 1.00 (0.81, 1.24)
^a^ Crude model adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status). ^b^ Fully adjusted model additionally included socio-demographic variables (region, education, and income), lifestyle factors (physical activity, BMI, smoking), and CHD risk factors (family history of MI/diabetes/stroke, medication use, postmenopausal hormone use, and self-reported hypertension/diabetes/hypercholesterolemia), and dietary factors (dietary alcohol intake, percent calories from protein/carbohydrates, and total energy intake). ^c^ Parsimonious model included matching factors, income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates, and total energy intake). ^d^ Adjusted model from the prior study included matching factors, education, BMI, lifestyle factors (physical activity, BMI, smoking), CHD risk factors (systolic blood pressure, family history of cardiovascular disease/stroke/MI/diabetes, medication use, and postmenopausal hormone use), and dietary factors (carbohydrate, protein, and alcohol intake). Abbreviations: BMI (body mass index), CHD (coronary heart disease), MI (myocardial infarction), MUFA (monounsaturated fatty acids), NR (not reported), PUFA (polyunsaturated fatty acids), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t0A3_Table A3
######
Associations (1 moL %) between plasma phospholipid fatty acids and CHD risk when adjusting for different anthropometric measures in the matched case-control study (*N* = 2428).
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Plasma Phospholipid Fatty Acids BMI Adjusted Model ^a^\ Waist Circumference Adjusted Model ^a^\ Waist: Hip Adjusted Model ^a^\ BMI Change Adjusted Model ^a,b^\
OR (95% CIs) OR (95% CIs) OR (95% CIs) OR (95% CIs)
--------------------------------- ------------------------- ----------------------------------------- -------------------------------- ----------------------------------
SFA 1.20 (1.10, 1.30) 1.17 (1.08, 1.28) 1.17 (1.08, 1.28) 1.26 (1.13, 1.39)
Long-chain SFA 1.18 (1.09, 1.28) 1.16 (1.07, 1.26) 1.16 (1.07, 1.26) 1.23 (1.12, 1.36)
Very-long-chain SFA 1.00 (0.77, 1.30) 1.01 (0.78, 1.32) 0.99 (0.76, 1.29) 1.04 (0.75, 1.44)
MUFA 0.99 (0.93, 1.05) 0.99 (0.94, 1.05) 1.00 (0.94, 1.05) 0.97 (0.90, 1.04)
PUFA n-3 0.93 (0.88, 0.99) 0.93 (0.88, 0.99) 0.94 (0.88, 1.00) 0.93 (0.87, 1.00)
PUFA n-6 1.00 (0.96, 1.03) 1.00 (0.96, 1.04) 0.99 (0.96, 1.03) 0.99 (0.95, 1.04)
TFA 1.00 (0.78, 1.30) 1.04 (0.80, 1.35) 1.00 (0.77, 1.31) 1.01 (0.74, 1.39)
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
^a^ Multivariable model included matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income), lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction /diabetes, postmenopausal hormone use, and self-reported hypertension /diabetes), and dietary factors (percent calories from protein/carbohydrates, and total energy intake). ^b^ BMI change indicated the difference between two BMI measures, one at baseline and one at year 3. Abbreviations: BMI (body mass index), CHD (coronary heart disease), CIs (confidence intervals), MUFA (mono-unsaturated fatty acids), PUFA (polyunsaturated fatty acids), OR (odds ratio), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t0A4_Table A4
######
Odds ratios ^a^ (95% CIs) of CHD associated with 1 moL % substitutions between plasma phospholipid fatty acid groups stratified by physical activity levels in the matched case-control study (*N* = 2428).
Plasma Phospholipid Fatty Acids Overall Physically Active Physically Inactive
--------------------------------- ------------------- ------------------- ---------------------
PUFA n-6 ↓ PUFA n-3 ↑ (1 moL %) 0.90 (0.84, 0.96) 0.95 (0.84, 1.06) 0.88 (0.76, 1.02)
TFA ↓ PUFA n-3 ↑ (1 moL %) 0.74 (0.56, 0.99) 0.88 (0.50, 1.54) 1.04 (0.60, 1.82)
TFA ↓ PUFA n-6↑ (1 moL %) 0.82 (0.61, 1.11) 0.92 (0.52, 1.65) 1.19 (0.67, 2.11)
^a^ Multivariable model adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates and total energy intake). Abbreviations: CHD (coronary heart disease), CIs (confidence intervals), PUFA (polyunsaturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t0A5_Table A5
######
Multivariable adjusted associations ^a^ (1 moL %) between plasma phospholipid fatty acids and CHD risk among participants with complete information versus using multiple imputation in the matched case-control study.
Plasma Phospholipid Fatty Acids Complete Analysis (*N* = 2181) Multiple Imputation (*N* = 2428)
--------------------------------- -------------------------------- ----------------------------------
SFA 1.21 (1.08, 1.35) 1.20 (1.10, 1.30)
Long-chain SFA ^b^ 1.19 (1.07, 1.33) 1.18 (1.09, 1.28)
Very-long-chain SFA ^c^ 0.99 (0.70, 1.41) 1.00 (0.77, 1.30)
MUFA 0.98 (0.91, 1.06) 0.98 (0.93, 1.04)
PUFA n-3 0.93 (0.86, 1.01) 0.93 (0.88, 0.99)
PUFA n-6 1.00 (0.95, 1.05) 1.00 (0.96, 1.03)
TFA 0.97 (0.69, 1.38) 1.01 (0.78, 1.31)
^a^ Multivariable model adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates and total energy intake). ^b^ Long-chain SFA included lauric acid (12:0), myristic acid (14:0), pentadecylic acid (15:0), palmitic acid (16:0), and stearic acid (18:0). ^c^ Very-long-chain SFA included arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0). Abbreviations: CHD (coronary heart disease), CIs (confidence intervals), MUFA (mono-unsaturated fatty acids), PUFA (polyunsaturated fatty acids), OR (odds ratio), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t0A6_Table A6
######
Odds ratios ^a^ (95% CIs) of CHD associated with 1 moL % substitutions between plasma phospholipid fatty acid groups among participants with complete information versus using multiple imputation in the matched case-control study.
Plasma Phospholipid Fatty Acids Complete Analysis (*N* = 2181) Multiple Imputation (*N* = 2428)
--------------------------------- -------------------------------- ----------------------------------
PUFA n-6 ↓ PUFA n-3 ↑ (1 moL %) 0.90 (0.85, 0.97) 0.90 (0.84, 0.96)
TFA ↓ PUFA n-3 ↑ (1 moL %) 0.75 (0.57, 1.00) 0.74 (0.56, 0.99)
TFA ↓ PUFA n-6↑ (1 moL %) 0.85 (0.62, 1.15) 0.82 (0.61, 1.11)
^a^ Multivariable model adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status), income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates and total energy intake).Abbreviations: CHD (coronary heart disease), CIs (confidence intervals), PUFA (polyunsaturated fatty acids), OR (odds ratio), TFA (*trans* fatty acids).
nutrients-11-01672-t001_Table 1
######
The lipid names, common names, categories, and mean (SD) levels (moL %) of plasma phospholipid fatty acid profiles measured in the matched case-control study of the Women's Health Initiative (1994--2005) (*N* = 2428).
Lipid Names ^a^ Common Names Category Mean (SD) Levels (moL %)
----------------- ------------------------------------------- ----------------- --------------------------
SFA 46.09 (1.29)
12:0 Lauric acid Long-chain 0.07 (0.04)
14:0 Myristic acid Long-chain 0.69 (0.21)
15:0 Pentadecylic acid Long-chain 0.23 (0.05)
16:0 Palmitic acid Long-chain 30.57 (2.02)
18:0 Stearic acid Long-chain 13.19 (1.42)
20:0 Arachidic acid Very-long-chain 0.24 (0.07)
22:0 Behenic acid Very-long-chain 0.64 (0.23)
24:0 Lignoceric acid Very-long-chain 0.46 (0.18)
MUFA 11.77 (1.64)
14:1 5-myristoleic acid Long-chain 0.12 (0.10)
16:1 n-9 7-palmitoleic acid Long-chain 0.84 (0.32)
16:1 n-7 cis-9-palmitoleic acid Long-chain 0.12 (0.05)
18:1 n-9 Oleic acid Long chain 1.37 (0.28)
18:1 n-7 Vaccenic acid Long-chain 8.48 (1.37)
20:1 n-9 cis-gondoic acid Very-long-chain 0.08 (0.03)
24:1 n-9 Nervonic acid Very-long-chain 0.77 (0.30)
PUFA 41.60 (2.06)
PUFA n-6 36.31 (2.46)
18:2 n-6 Linoleic acid (LA) Long-chain 20.79 (3.00)
18:3 n-6 γ-linoleic acid Long-chain 0.10 (0.05)
20:2 n-6 Eicosadienoic acid Very-long-chain 0.40 (0.16)
20:3 n-6 Eicosatrienoic acid Very-long-chain 3.32 (0.79)
20:4 n-6 Arachidonic acid (AA) Very-long-chain 10.93 (2.07)
22:4 n-6 Docosatetraenoic acid Very-long-chain 0.42 (0.11)
22:5 n-6 4,7,10,13,16-Docosapentaenoic acid Very-long-chain 0.35 (0.13)
PUFA n-3 5.14 (1.58)
18:3 n-3 α-linolenic acid (ALA) Long-chain 0.21 (0.08)
20:5 n-3 Eicosapentaenoic acid (EPA) Very-long-chain 0.81 (0.51)
22:5 n-3 7,10,13,16,19-Docosapentaenoic acid (DPA) Very-long-chain 0.83 (0.19)
22:6 n-3 Docosahexaenoic acid (DHA) Very-long-chain 3.29 (1.13)
TFA 0.69 (0.34)
18:1t All 18:1 *trans* Long-chain 0.54 (0.30)
18:2t All 18:2 *trans* Long-chain 0.14 (0.06)
^a^ Lipid names are presented in the form of C:D, where C is the number of carbon atoms and D is the number of double bonds in the fatty acid. Abbreviations: MUFA (mono-unsaturated fatty acids), PUFA (polyunsaturated fatty acids), SD (standard deviation), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t002_Table 2
######
Baseline characteristics by cases and controls in the Women's Health Initiative study (1994--2005) (*N* = 2428).
Variables Overall CHD Status
----------------------------------------- ---------------- ---------------- ---------------- -----------
**Socio-demographics**
Age, years ^b^ 67.8 (6.8) 67.8 (6.8) 67.8 (6.8) *Matched*
Race/ethnicity, *n* (%) *Matched*
Black 136 (6) 68 (6) 68 (6)
Hispanic 32 (1) 16 (1) 16 (1)
White 2172 (90) 1086 (90) 1086 (90)
Other 88 (4) 44 (4) 44 (4)
Region, *n* (%) *0.40*
Northeast 610 (25) 303 (25) 307 (25)
South 579 (24) 299 (25) 280 (23)
Midwest 547 (23) 264 (22) 283 (23)
West 692 (29) 348 (29) 344 (28)
Education, *n* (%) *\<0.01*
≤High school 845 (35) 380 (31) 465 (38)
Some college and college graduate 907 (37) 471 (39) 435 (36)
Postgraduate 676 (28) 363 (30) 314 (26)
Income, *n* (%) *\<0.01*
\<\$20,000 448 (18) 193 (16) 255 (21)
\$20,000--\$74,999 1541 (64) 776 (64) 765 (63)
≥\$75,000 439 (18) 245 (20) 194 (16)
**Lifestyle factors**
Physical activity, MET-h/week ^c^ 9.5 (16.1) 10.8 (17.3) 8.3 (15.4) *\<0.01*
BMI, Kg/m^2^ ^c^ 26.4 (6.8) 25.9 (6.4) 26.9 (7.4) *\<0.01*
Waist circumference, cm ^c^ 84.2 (13.6) 83.0 (12.3) 86.5 (14.4) *\<0.01*
Waist-to-hip ratio ^c^ 0.8 (0.1) 0.8 (0.1) 0.8 (0.1) *\<0.01*
Smoking, *n* (%) *\<0.01*
Never-smoker 1232 (51) 650 (54) 582 (48)
Past smoker 1033 (43) 501 (41) 532 (44)
Current smoker 163 (7) 63 (5) 100 (8)
**CHD risk factors**
Family history, *n* (% yes)
Myocardial infarction 1383 (57) 647 (53) 736 (61) *\<0.01*
Diabetes 829 (34) 441 (36) 388 (32) *0.03*
Stroke 986 (41) 473 (39) 513 (42) *0.11*
Medication use, *n* (%) ^d^ 364 (15) 123 (10) 241 (20) *\<0.01*
Hormone usage, *n* (%) *\<0.01*
Current Estrogen + Progesterone 355 (15) 205 (17) 150 (12)
Current Estrogen alone 555 (23) 295 (24) 260 (21)
Past Users 375 (15) 175 (14) 200 (17)
Never Used 1143 (47) 539 (44) 604 (50)
Hypertension, *n* (%) *\<0.01*
Never hypertensive 1409 (58) 824 (68) 585 (48)
Untreated hypertensive 237 (10) 96 (8) 141 (12)
Treated hypertensive 782 (32) 294 (24) 488 (40)
Diabetes, *n* (% yes) 197 (8) 46 (4) 151 (12) *\<0.01*
Hypercholesterolemia, *n* (% yes) 380 (16) 176 (14) 204 (17) *0.14*
Hysterectomy, *n* (% yes) 1000 (41) 500 (40) 500 (40) *Matched*
**Dietary factors**
Alcohol, g/day ^c^ 0.9 (6.5) 1.0 (7.0) 0.6 (6.2) *0.02*
Percent calories from carbohydrates ^c^ 52.2 (13.4) 52.8 (9.4) 51.2 (10.0) *\<0.01*
Percent calories from protein ^c^ 16.8 (4.2) 16.9 (4.2) 16.8 (4.2) *0.63*
Total energy, Kcal/day ^c^ 1506.4 (728.3) 1531.3 (702.4) 1482.2 (752.6) *0.84*
Fish, servings/day ^b^ 0.3 (0.2) 0.3 (0.2) 0.2 (0.2) *0.28*
Dairy products, servings/day ^b^ 1.9 (1.5) 1.9 (1.4) 1.9 (1.5) *0.53*
Butter, teaspoons/day ^b^ 0.2 (0.5) 0.2 (0.5) 0.3 (0.6) *\<0.01*
Margarine, teaspoons/day ^b^ 0.2 (0.5) 0.2 (0.5) 0.2 (0.5) *0.41*
Olive/Canola oil, teaspoons/day ^b^ 0.1 (0.2) 0.1 (0.2) 0.1 (0.3) *0.57*
Other vegetable oils, teaspoons/day ^b^ 0.1 (0.2) 0.0 (0.1) 0.1 (0.2) *0.28*
Red meat, servings/day ^b^ 0.6 (0.5) 0.6 (0.5) 0.7 (0.6) *\<0.01*
Carbohydrates, g/day ^b^ 202.5 (76.1) 204.2 (72.3) 200.8 (79.7) *0.27*
^a^*p* values were derived using paired *t* test (continuous and normally distributed variables), Wilcoxon signed rank test (continuous non-normally distributed variables), or McNemar test (categorical variables). ^b^ The mean (SD) of normally distributed continuous variables. ^c^ The median (IQR) of continuous non-normally distributed variables. ^d^ Medications included anticoagulant, anti-diabetic, and lipid lowering medications. Abbreviations: BMI (body mass index), CHD (coronary heart disease), IQR (interquartile range), MET-h (metabolic equivalent-hours), SD (standard deviation).
nutrients-11-01672-t003_Table 3
######
Multivariable adjusted associations (1 moL %) between plasma phospholipid fatty acids and CHD risk in the matched case-control study (*N* = 2428).
Plasma Phospholipid Fatty Acids Mean (SD) Model 1 ^a^ Model 2 ^b^
--------------------------------- -------------- ------------------- ------------------- -------------------
SFA 46.09 (1.29) 1.19 (1.11, 1.28) 1.20 (1.10, 1.30) 1.20 (1.08, 1.34)
Long-chain SFA ^c^ 44.74 (1.37) 1.17 (1.09, 1.25) 1.18 (1.09, 1.28) 1.18 (1.07, 1.31)
Very-long-chain SFA ^d^ 1.35 (0.46) 1.00 (0.80, 1.26) 1.00 (0.77, 1.30) 1.00 (0.71, 1.41)
MUFA 11.77 (1.64) 0.96 (0.91, 1.01) 0.98 (0.93, 1.04) 0.98 (0.91, 1.06)
PUFA n-3 5.14 (1.58) 0.89 (0.84, 0.94) 0.93 (0.88, 0.99) 0.93 (0.86, 1.01)
PUFA n-6 36.31 (2.46) 1.03 (0.99, 1.06) 1.00 (0.96, 1.03) 1.00 (0.95, 1.05)
TFA 0.69 (0.34) 1.06 (0.84, 1.33) 1.01 (0.78, 1.31) 1.01 (0.72, 1.42)
^a^ Model 1 adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status). ^b^ Model 2 additionally adjusted for income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates and total energy intake). ^c^ Long-chain SFA included lauric acid (12:0), myristic acid (14:0), pentadecylic acid (15:0), palmitic acid (16:0), and stearic acid (18:0). ^d^ Very-long-chain SFA included arachidic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0). Abbreviations: CHD (coronary heart disease), CIs (confidence intervals), MUFA (mono-unsaturated fatty acids), PUFA (polyunsaturated fatty acids), OR (odds ratio), SD (standard deviation), SFA (saturated fatty acids), TFA (*trans* fatty acids).
nutrients-11-01672-t004_Table 4
######
Odds ratios (95% CIs) of CHD associated with 1 moL % substitutions between plasma phospholipid fatty acid groups among participants in the matched case-control study (*N* = 2428).
Plasma Phospholipid Fatty Acids Model 1 ^a^ Model 2 ^b^
--------------------------------- ------------------- ------------------- -------------------
PUFA n-6↓ PUFA n-3 ↑ (1 moL %) 0.85 (0.80, 0.90) 0.90 (0.84, 0.96) 0.90 (0.83, 0.98)
TFA↓ PUFA n-3 ↑ (1 moL %) 0.72 (0.55, 0.94) 0.74 (0.56, 0.99) 0.74 (0.51, 1.09)
TFA↓ PUFA n-6 ↑ (1 moL %) 0.84 (0.64, 1.11) 0.82 (0.61, 1.11) 0.82 (0.56, 1.22)
^a^ Model 1 adjusted for matching factors (age, race/ethnicity, enrollment date, and hysterectomy status). ^b^ Model 2 additionally adjusted for income, lifestyle factors (physical activity and smoking), CHD risk factors (family history of myocardial infarction/diabetes, postmenopausal hormone use, and self-reported hypertension/diabetes), and dietary factors (percent calories from protein/carbohydrates and total energy intake). Abbreviations: CHD (coronary heart disease), CIs (confidence intervals), PUFA (polyunsaturated fatty acids), OR (odds ratio), TFA (*trans* fatty acids).
|
{
"pile_set_name": "PubMed Central"
}
|
Legg-Calve-Perthes disease (LCPD) is a common juvenile form of ischemic osteonecrosis of the femoral head (INFH) that can lead to permanent femoral head deformity and end-stage osteoarthritis, even in young adults[@b1]. INFH remains one of the most challenging conditions to treat because little is understood regarding the biology of the disease. Both vascular disruption and defective bone repair are key elements of INFH pathogenesis[@b2]. Because joint replacement arthroplasty is unsuitable for young people, biological treatments to preserve the femoral head structure more effectively are needed.
Obesity is a clinical risk factor for LCPD and poor treatment outcomes are associated with high body mass index (BMI)[@b3]. Leptin, the product of the *ob* gene, is an adipocyte-produced 146 amino acid polypeptide that regulates adipose tissue mass and body weight[@b4][@b5]. Most obese humans have very high plasma leptin concentrations, suggesting they are resistant to its anorectic and metabolic effects[@b6]. In obese, leptin-deficient mice (*ob/ob*), administering exogenous leptin effectively reduces hyperphagia and obesity[@b7][@b8]. Recently, we measured circulating leptin levels, soluble leptin receptor levels, and the free leptin index (FLI) in LCPD patients and healthy controls[@b3]. In LCPD patients, circulating leptin levels were significantly higher and soluble leptin receptor levels were significantly lower than in controls. In addition, FLI was higher in the LCPD group than in controls.
Recent studies have shown that leptin increased in bone formation in leptin deficient mice[@b9][@b10][@b11]. To our knowledge, the therapeutic potential of leptin to increase bone formation and angiogenesis after ischemic osteonecrosis has not been studied. The purpose of this study was to determine if systemic leptin administration stimulated bone formation and preserved femoral head shape in INFH in rats with induced obesity.
Results
=======
Rats returned to normal activity within 24 h after surgery. Five rats died during the postoperative period: one each from the NCD-AVN, HFD-AVN and HFD-AVN-Leptin groups and two from the HFD-AVN-LacZ group. The remaining 115 animals experienced an uncomplicated postoperative course until six weeks post-surgery. For induction of AVN, the femoral heads were dislocated from the acetabulum and then the dislocated femoral heads were re-located to the acetabulum. However, since the rat\'s acetabulum is extremely shallow, all the hips became re-dislocated, except in the sham group, as evidenced by all the rats walked with a limp on their dislocated hips.
Consumption of a HFD for 16 weeks produced a significant (P \< 0.05) increase in body weight compared to the consumption of a NCD. The mean plasma leptin level at the time of necropsy in NCD-Sham, HFD-Sham, NCD-AVN, HFD-AVN, and HFD-AVN-LacZ animals averaged 1.5 ± 0.4, 28.3 ± 1.2, 1.3 ± 0.1, 27.3 ± 2.1, and 26.8 ± 1.6 ng/ml, respectively. In HFD-AVN-leptin rats, the mean plasma leptin level rose within 2 days to 73.5 ± 3.4 ng/ml and remained elevated for 2 weeks after Ad-leptin injection. After the surgery, a reduction in body weight was not observed in NCD-Sham, HFD-Sham, NCD-AVN, HFD-AVN, and HFD-AVN-LacZ groups. In contrast, the HFD rats treated with Ad-Leptin exhibited a 41% reduction in body weight over the 6-weeks period of observation after surgery ([Table 1](#t1){ref-type="table"} and [Supplementary Fig. 1](#s1){ref-type="supplementary-material"}).
Micro-CT assessment of ischemic osteonecrosis
---------------------------------------------
Structural and quantitative assessment of mineralized skeletal tissue formation was performed by micro-CT at 6 weeks after surgery-induced INFH. Femoral heads in the NCD-sham and HFD-sham group animals did not develop osteonecrosis ([Fig. 1](#f1){ref-type="fig"}). However, femoral heads in HFD-AVN-group animals showed significant loss of trabecular bone and reduced preservation of femoral head architecture compared with the NCD-AVN group. Femoral heads in HFD-AVN-LacZ-group animals also showed a prominent area of bone resorption. In contrast, the trabecular network and femoral head architecture were relatively well preserved in the HFD-AVN-Leptin group compared to the HFD-AVN or HFD-AVN-LacZ group. Micro-CT findings were used to measure bone volume, trabecular numbers, and trabecular thicknesses ([Table 2](#t2){ref-type="table"}). Bone volume (BV) in the HFD-AVN-Leptin group in areas undergoing repair 6 weeks after surgery was significantly higher than in the HFD-AVN and HFD-AVN-LacZ groups. Mean BV/TV values and trabecular numbers were significantly lower in femoral heads in the HFD-AVN and HFD-AVN-LacZ groups compared to the NCD-AVN group, whereas the HFD-AVN-Leptin group had bone masses and microarchitectures similar to the sham group. In addition, trabecular number and thickness were significantly higher and trabecular separation was significantly lower in the HFD-AVN-Leptin group compared to the HFD-AVN or HFD-AVN-LacZ group.
Histological assessments
------------------------
Histologically, femoral heads of NCD-sham and HFD-sham animals were intact and articular surface and secondary ossification centers were well preserved. However, femoral heads in the HFD-AVN and HFD-AVN-LacZ groups were damaged. Articular surfaces and metaphyseal physes were also damaged and secondary ossification centers were collapsed and replaced with nonhematopoietic fibrovascular tissue ([Fig. 2a](#f2){ref-type="fig"}). Although femoral heads in the NCD-AVN group were also damaged, the degree of deformity was less than in the HFD-AVN or HFD-AVN-LacZ groups. Damage scores for the HFD-AVN or HFD-AVN-LacZ groups were significantly higher than the NCD-AVN group (p \< 0.05) ([Fig. 2b, c](#f2){ref-type="fig"}). In contrast, femoral heads in the HFD-AVN-Leptin group were relatively well preserved compared to those in the HFD-AVN or HFD-AVN-LacZ groups. Although articular surfaces were slightly irregular and showed detachment of the articular cartilage, the general contour of the articular surface was maintained. Secondary ossification centers were intact and hematopoietic. Bone in secondary ossification centers was rimmed with osteoblasts and well-formed blood vessels in the bone marrow ([Fig. 2a](#f2){ref-type="fig"}). Damage scores were significantly lower in the HFD-AVN-Leptin group compared with the HFD-AVN or HFD-AVN-LacZ groups (p \< 0.01) ([Fig. 2b, c](#f2){ref-type="fig"}).
Enhanced vascularity induced by leptin
--------------------------------------
To evaluate the effects of leptin on femoral head vascularity, femoral heads were immunostained for factor VIII-related antigen. Immunostaining for factor VIII-related antigen revealed more blood vessels in sham-operated and the HFD-AVN-Leptin groups compared with the HFD-AVN or HFD-AVN-LacZ groups ([Fig. 3a](#f3){ref-type="fig"}). Elevated vessel density in the HFD-AVN-Leptin group was confirmed by morphometric analysis for vessels in the secondary ossification centers of infarcted femoral heads. Vascular densities in the HFD-AVN and HFD-AVN-LacZ groups were significantly lower than in the sham-operated groups (p \< 0.01). However, vascular density of the HFD-AVN-Leptin group was significantly higher than in the HFD-AVN or HFD-AVN-LacZ groups (p \< 0.01) ([Fig. 3b](#f3){ref-type="fig"}). Vascularization of the femoral head represented by vascular density paralleled radiological and histological findings.
Leptin induces increases in VEGF
--------------------------------
Serum leptin levels peaked two days after Ad-Leptin injection and slowly declined over 10 days, maintaining a concentration of approximately 40 ng/mL until 14 days ([Fig. 4a](#f4){ref-type="fig"}). In experimental groups, serum leptin levels were significantly higher in the HFD-AVN-Leptin group compared to the HFD-AVN or HFD-AVN-LacZ groups ([Fig. 4b](#f4){ref-type="fig"}). Serum VEGF levels were consistently higher with increasing serum leptin after Ad-Leptin treatment in the HFD-AVN-Leptin group compared to the HFD-AVN or HFD-AVN-LacZ groups ([Fig. 4c](#f4){ref-type="fig"}).
Leptin expression associates with HIF1α -related pathways
---------------------------------------------------------
Western blots were used to determine the impact of leptin treatment. Leptin treatment was associated with increase in STAT3 phosphorylation, increase expression of the leptin canonic signaling kinases MAPK/ERK 1/2 and PI-3K/AKT1 and noncanonical JNK and P38. In addition, HIF-1α expression also increased in the HFD-AVN-Leptin group compared to the HFD-AVN and HFD-AVN-LacZ groups ([Fig. 5a](#f5){ref-type="fig"}, [Supplementary Fig. 2](#s1){ref-type="supplementary-material"}) and the immunohistochemical expression of p-STAT3 and HIF-1α was similar to the results of western blots ([Fig. 5b](#f5){ref-type="fig"}). Expression of p-STAT3 and HIF-1α were seen in stromal cells on bone surfaces (which are likely either osteoblasts or bone lining cells) and was stronger in the HFD-AVN-Leptin group than the HFD-AVN or HFD-AVN-LacZ groups ([Supplementary Fig. 3](#s1){ref-type="supplementary-material"}). Expression of phosphorylated STAT3 and HIF-1α in the NCD-AVN group was intermediate between its expression levels in the HFD-AVN-Leptin and HFD-AVN groups. Real-time RT-PCR was performed to determine the mRNA levels for HIF-1α and VEGF. Leptin treatment increased HIF-1α and VEGF mRNA in the HFD-AVN-Leptin group compared to the HFD-AVN and HFD-AVN-LacZ groups ([Figure 5c](#f5){ref-type="fig"}). These results suggest that Ad-Leptin treatment causes an increase in angiogenesis and that might be related to the canonical (MAPK and PI-3K) and noncanonical (JNK and p38 kinase) leptin signaling pathways in INFH.
The effect of leptin for the genes related to osteogenesis
----------------------------------------------------------
mRNA was isolated from necrosis areas of the femoral heads and assayed for the expression of several osteogenic genes: runt-related transcription factor 2 (Runx2), bone sialoprotein, type 1 collagen, osteopontin, and osterix. Expressions of the osteogenic genes were significantly lower in the HFD-sham groups compared to the NCD-sham group. The NCD-AVN group showed higher levels of osteogenic genes compared to NCD-sham group, whereas, there were no differences between the HFD-sham group and the HFD-AVN group. Expressions of the osteogenic genes were significantly lower in the HFD-AVN and HFD-AVN-LacZ groups compared to the HFD-AVN-Leptin group. The HFD-AVN-Leptin group showed a higher level of expression of osteogenic genes than the other groups ([Fig. 5c](#f5){ref-type="fig"}).
Discussion
==========
In this study, we found that INFH in rats with diet-induced obesity was strongly associated with severe destruction of bone architecture and reduced potential for bone regeneration. We examined the effects of leptin on revascularization and repair of the femoral head in rats with diet-induced obesity and INFH. Intravenously injected Ad-leptin effectively facilitated repair of the ischemic femoral head and enhanced angiogenesis and bone regeneration in rats with induced obesity. This study showed that leptin might be useful for the treatment of INFH in obese patient and indicated factors that might predict poor treatment outcome.
The role of leptin in the pathogenesis of obesity can be inferred by measuring plasma leptin[@b12]. The increase in leptin is a result of obesity as adipocytes secrete leptin. In general, obese animals have higher leptin levels than controls, indicating that these forms of animal obesity are associated with leptin resistance[@b13]. In our previous study, circulating leptin levels in LCPD patients were significantly higher and levels of soluble leptin receptor were significantly lower than in controls. In addition, the FLI was higher in the LCPD group. Furthermore, the circulating leptin and soluble leptin receptor levels and FLI correlated with LCPD disease severity and treatment outcomes[@b3]. Therefore, leptin resistance could be relevant to pathogenesis in LCPD, the childhood form of INFH. We suggest that leptin resistance in obesity deteriorates the bone repair process in INFH. In present study, AVN of HFD animals was associated with poor outcome that appear to be improved by intravenous Ad-leptin injection.
Leptin contributes to the formation of a relationship between fat mass and bone[@b14] and circulating leptin concentration is related to bone mass. Although Ducy et al. reported that leptin inhibits bone formation *in viv*[@b15], other studies found that leptin promotes differentiation into an osteoblast phenotype and increases synthesis of bone matrix proteins such as type I collagen and osteocalcin[@b16][@b17]. Steppan *et al*. reported that *in vivo* administration of leptin to *ob*/*ob* mice results in increased bone size and mass[@b18]. The skeletal abnormalities caused by leptin deficiency are markedly attenuated in morbidly obese *ob/ob* mice[@b14]. Insulin might be involved in the effect of leptin on bone mass since central administration of leptin dramatically decreases circulating insulin concentration[@b19][@b20][@b21][@b22]. Systemic administration of leptin is associated with reduced insulin concentration[@b20][@b23][@b24][@b25], so the positive skeletal effects found in our *in vivo* study emphasize the direct effects of leptin on bone. Both intracerebroventricular and subcutaneous leptin treatment of *ob/ob* mice stimulated bone growth[@b10]. Recent study reported that peripheral leptin is essential for normal bone resorption and enhances bone formation[@b9]. Hamrick *et al*. reported peripheral delivery of leptin for 2 weeks increased bone formation in ob/ob mice[@b26]. We have also shown that peripherally administered leptin in HFD rats resulted in increased osteogenesis and preserved infarcted femoral head although our adenoviral delivery system provides transient leptin exposure during the first two weeks following induction of AVN. However, for clinical application of leptin therapy, a continuous delivery system should be developed because of the long duration of disease of INFH.
Angiogenesis is an early and essential component of the bone repair process[@b27]. Leptin, initially identified as a pro-angiogenic factor, is also a positive VEGF regulator[@b28]. Leptin can directly interact with the OB-R receptor in endothelial cells and activate the STAT3 pathway to enhance its DNA binding activity[@b28]. Leptin also acts as a modulator of other angiogenic factors and has indirect effects on vascular permeability[@b29][@b30][@b31]. Kim et al. reported that upregulated VEGF in the epiphyseal cartilage in INFH might stimulate vascular invasion into the necrotic area of the epiphyseal cartilage and restore the femoral head[@b32]. Our results suggest that leptin might induce angiogenesis by enhancing VEGF expression. Furthermore, capillary densities were greater at sites of new bone formation, demonstrating the importance of angiogenesis during new bone formation after administration of Ad-leptin in the HFD-AVN group. The positive relationship between angiogenesis and new bone formation was consistent with results by Ma *et al*., who showed that VEGF upregulation induces new bone formation and promotes the repair process[@b33]. The mechanisms involved in VEGF upregulation involved both canonical and noncanonical signaling pathways. Normally, leptin increases VEGF by MAPK/ERK 1/2 and PI-3K/AKT1 and it activates a series of transcription factors such as HIF-1α[@b34][@b35]. INFH is a well-known hypoxic condition implicated in the transcriptional upregulation of VEG[@b32]. The VEGF promoter contains distal enhancer sites that bind HIF-1α. Leptin induces canonic and noncanonic signaling pathways to activate HIF-1α in INFH. Therefore, we suggest that increased angiogenesis after leptin treatment enhances bone regeneration in ischemic femoral heads.
Although leptin treatment in the HFD-AVN group increased the mRNA osteogenic genes in our study, we could not determine if leptin directly stimulated osteoblastic differentiation. However, our findings were consistent with previous results that leptin enhances osteoblast differentiation and proliferation[@b16]. Although we did not conclusively demonstrate direct action of leptin, our results suggest that leptin acts on bone by influencing osteoblasts and osteogenic growth factors and newly formed blood vessels that supply oxygen. Turner *et al*. provided strong evidence that leptin acts primarily through peripheral pathways to enhance bone growth and maturation by increasing osteoblast number and activity, and osteoclast activity[@b9].
A limitation of this study was that we did not include a leptin treated sham and AVN treated group. Evaluation of leptin treatment in the absence of obesity provides clinically relevant data due to the lack of leptin resistance as a confounder. Another limitation of this study is the occurrence of dislocation after surgery, which could cause changes in bone loading; nevertheless all rats walked with a limp on the dislocated hips. Another limitation of this study was that we did not investigate leptin signaling in the hypothalamus and in sympathetic regulation of bone formation. Further studies on sympathetic regulation in INFH are needed to investigate the specific mechanism of leptin regulation. In summary, the results of this study demonstrated that systemic administration of leptin enhanced bone regeneration and preserved osteonecrotic femoral heads in rats with INFH and induced obesity through increasing vascularity and bone formation. These results suggested that the administration of leptin could be a new therapeutic strategy to improve INFH treatment outcomes.
Methods
=======
Animals and surgical procedures
-------------------------------
Male Sprague-Dawley rats (eight weeks old, 170--190 g) were used. Rats had free access to water and standard rat chow pellets and were housed under controlled temperature (22 ± 1°C) and humidity (50% to 60%) with a 12-hour light-dark cycle from 7 AM to 7 PM. After acclimatization for 1 week, animals were fed either a NCD or HFD. NCD (by weight) was 21% protein, 4.5% fat, and 52% carbohydrate (3.94 Kcal/g). HFD was 26.5% protein, 1% cholesterol, 0.4% sodium cholate, 35.4% saturated fat (lard), and 26.6% carbohydrate (5.44 Kcal/g). After 16 weeks of NCD or HFD feeding, AVN was induced by surgical application of a tight ligature around the femoral neck, as previously described[@b2]. Adenovirus-mediated introduction of the leptin gene (Ad-Leptin) was by intravenous injection 2 days before surgery-induced AVN ([Supplementary Fig. 4](#s1){ref-type="supplementary-material"}). Animals were divided into six groups: 1) sham-operated, fed NCD (NCD-sham); 2) sham operated, fed HFD (HFD-sham); 3) AVN, fed NCD (NCD-AVN); 4) AVN, fed HFD (HFD-AVN); 5) Ad-LacZ-injected AVN, fed HFD (HFD-AVN-LacZ); and 6) Ad-Leptin-injected AVN, fed HFD (HFD-AVN-Leptin) (n = 20/group). To minimize the unnecessary use of animals two groups that were Ad-LacZ-injected AVN fed NCD and Ad-Leptin-injected AVN fed NCD, were not used in this study as these animals do not develope leptin resistance.
Animals were euthanized by exsanguination under sodium pentobarbital anesthesia at 6 weeks after inducing INFH. Animals were cared for in accordance with the National Institutes of Health Guidelines for Animal Care. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Chonbuk National University (Approval number: CUH 2012-12-008).
Intravenous administration procedures and groups
------------------------------------------------
At 2 days before surgery, rats were given 100 μL recombinant adenoviruses (1 × 10^12^ plaque-forming units) by intravenous injection to the tail vein. Adenoviruses contained either leptin cDNA (Ad-Leptin) or, as an inactive control, β-galactosidase cDNA (Ad-LacZ) under control of the cytomegalovirus promoter[@b36]. Ad-Leptin and Ad-LacZ were generously provided by R.H. Unger (University of Texas Southwestern Medical Center, Dallas, TX, USA).
Assessment of bone destruction by MicroCT
-----------------------------------------
A SKYSCAN 1076 Micro-CT unit (Skyscan, Kontich, Belgium) was used to assess bone volume within defect sites. The X-ray source was set at 75 kV and 100 μA, with a pixel size of 8.8 μm and 400 projections were acquired over an angular range of 180° (angular step of 0.45°). The area included in CT scans was from the upper margin of the femoral head epiphysis to the femoral neck. A global thresholding algorithm was applied at a constant threshold for all specimens. The threshold was the intensity (gray value) that corresponded to \~45% of the average intensity of the intact cortical bone in specimens. Voxels with intensities exceeding the threshold were considered to contain mineralized tissue. All system aspects were operated using Dataviewer software (SkyScan). On stacked, reconstructed micro-CT cross-section images, manual regions of interest (ROIs) of irregular anatomical contour were drawn on transverse images at the middle of the femoral head ([Figure 1](#f1){ref-type="fig"}). ROIs excluded cortical bone. The volume of interest (VOI) was a stack of ROIs drawn over 52 cross-sections, resulting in a height of 0.45 mm. Tb.Th was the trabeculae mean thickness, Tb.Sp was the mean distance between trabeculae and Tb.N was the average number of trabeculae present per unit length. Tb.Th and Tb.Sp were assessed using direct 3D methods and Tb.N was calculated using the formula Tb.N = (BV/TV)/Tb.Th.
Histological methods
--------------------
Resected femurs were fixed in 10% neutral buffered formalin and decalcified in 10% EDTA for 10 days or in rapid decalcifying solution (Calci-Clear Rapid, National Diagnostics, Atlanta, GA, USA) for 12 h. Tissue sections were from the femoral head midline as the most representative area. To evaluate histologic findings, paraffin-embedded tissue sections were stained with hematoxylin and eosin or Safranin-O staining (Sigma-Aldrich, MA, USA). Femoral head damage was graded from 0 to 5 as: 0, no damage; 1, mild damage with maintaining femoral head articular cartilage and architecture; 2, destruction of the articular cartilage and collapse of the secondary ossification center but with less than one-third of the femoral head showing destruction; 3; destruction of between one-third and two-thirds of the femoral head; 4, destruction of more than two-thirds of the femoral head; and 5, complete or near-complete femoral head destruction.
Immunohistochemical staining and vascular density measurements
--------------------------------------------------------------
Tissue sections were deparaffinized. Antigen retrieval was in a pressure cooker with sodium citrate buffer for 12 min. Tissue sections were incubated with antibody for factor VIII-related antigen (1:50, Chemicon, Temecula, CA, USA), Ob-R (1:200, Santa Cruz Biotech, Santa Cruz, CA, USA), phospho-STAT3 (1:200, Cell Signaling, Beverly, MA, USA), or HIF-1α (1:200, Santa Cruz Biotech) overnight at 4°C. For negative controls, sections were incubated in normal mouse IgG (1:100, Santa Cruz Biotech) instead of specific primary antibody. Peroxidase activity was detected using the enzyme substrate 3-amino-9-ethyl carbazole and counterstained with hematoxylin.
Vascular density was evaluated by measuring factor VIII-related antigen-stained areas in ×200 magnification images obtained using a Nikon ECLIPSE E600 microscope with a 20× objective lens (Plan Fluor20/0.50NA, Nikon) and a digital camera (Nikon DXm1200F). Each image was 0.33 mm^2^. Factor VIII-related antigen-stained images were acquired at the most vascularized area of the femoral head from each rat without knowledge of the experimental group. Six images were taken from each of the six rats per group. Vascular densities were measured using an image analysis system (analySIS, Soft Imaging Systems, Germany) and calculated as (factor VIII-stained vascular area/total image area in ×200 magnification) × 100 (%).
RNA preparation
---------------
A subset of rats was euthanized four weeks after surgery. Five rats from each group were evaluated. Total RNA was prepared from the entire mass of regenerated bone using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Tissue harvesting was performed with a distractor for precision and accuracy. All extraneous soft tissue was cleaned and tissues were snap frozen in liquid nitrogen.
Real time RT-PCR
----------------
RNA from extracted tissues was precipitated with isopropanol and dissolved in DEPC-treated distilled water. Total RNA (500 ng) was treated with RNase-free DNase (Invitrogen) and first-strand cDNA was generated using random hexamer primers from first-strand cDNA synthesis kits according the manufacturer\'s protocol (Applied Biosystems, Foster City, CA, USA). Specific primers were designed ([Table 3](#t3){ref-type="table"}) using Primer Express software (Applied Biosystems). GAPDH sequence was used as an invariant control. Real-time RT-PCR reaction mixtures were 10 ng reverse-transcribed total RNA, 2 nM forward and reverse primers, and 5 × PCR master mixture in 10 μL. Reactions were performed in 384-well plates using the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Samples from 5 rats were analyzed from each group and RNA from individual rats was analyzed separately.
Enzyme-linked immunosorbent assay (ELISA)
-----------------------------------------
Approximately 600 μL blood was collected from tail veins into heparinized capillary tubes at indicated times for 5 rats per group. Serum was obtained by centrifugation and stored at −80°C until measurement by enzyme-linked immunosorbent assay (ELISA). Leptin and VEGF levels were determined using Quantikine Kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer\'s instructions. Samples were assayed in duplicate.
Western blot analysis
---------------------
Whole femoral heads were harvested from five rats per group and decalcified in rapid decalcifying solution (Calci-Clear Rapid) for 12 h. Soft tissue was trimmed from specimens, which was washed with phosphate buffered saline and ground to a fine powder using a manual biopulverizer cooled in liquid nitrogen. Total cell extracts were generated using lysis buffer (Cell Signaling) and centrifugation at 12,000 × *g* for 15 min at 4°C. Quantification of total protein was performed with BCA protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking in 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST), membranes were incubated with primary antibodies for phospho-STAT3, total STAT3, ERK, p38, Akt (Cell Signaling), Osteocalcin (Santa Cruz Biotech), Collagen I (Abcam, Kendall Square, MA, USA) or HIF-1α (Santa Cruz Biotech). After washing, blots were incubated with secondary antibody diluted 1:5000 in TBS-T at room temperature for 1 h. Signals were detected by an enhanced chemiluminescence reagent (Santa Cruz Biotech), according to the manufacturer\'s instructions. Densitometric analysis was conducted directly on the membrane using an LAS-3000 luminoimage analyzer system (Fujifilm, Tokyo, Japan). Relative phosphorylation of proteins was calculated as the ratio of phosphorylated protein to total protein.
Statistical analysis
--------------------
Statistical analyses were by one-way ANOVA followed by post-hoc test. Total bone volumes were compared across groups using a Kruskal-Wallis test (ANOVA by ranks). Data are expressed as mean ± SEM. Differences with p values \< 0.05 were considered statistically significant.
Author Contributions
====================
Conceived and designed the experiments: K.Y.J., B.H.P. and J.R.K. Performed the experiments: L.Z., K.Y.J.,Y.J.M., S.W., K.M.K., K.B.L. and J.R.K. Analyzed the data: L.Z., K.M.K. and J.R.K. Wrote the paper: Lu Zhou, K.Y.J. and J.R.K. J.R.K. takes responsibility for the integrity of the data analysis.
Supplementary Material {#s1}
======================
###### Supplementary Information
supplementary information
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea. (HI12C1044).
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###### Body weight, BMI, serum insulin and leptin levels at the time of necropsy
NCD-Sham HFD-Sham NCD-AVN HFD-AVN HFD-AVN-LacZ HFD-AVN-Leptin
------------------------- ------------- ----------------------------------------- ------------- -------------- -------------- -------------------------------------------
Initial body weight (g) 184.8 ± 4.3 180.1 ± 4.7 180.2 ± 2 184.1 ± 1.1 181.8 ± 1.7 180.2 ± 4.1
Final body weight (g) 276.4 ± 6.8 496.6 ± 11.5[a](#t1-fn1){ref-type="fn"} 253.4 ± 9.6 514.8 ± 11.1 519.2 ± 7.4 308.5 ± 14.6[b,c](#t1-fn1){ref-type="fn"}
BMI (g/cm2) 5.9 ± 0.2 7.2 ± 0.8[a](#t1-fn1){ref-type="fn"} 5.20 ± 0.57 7.2 ± 0.3 7.2 ± 2.6 5.4 ± 0.6[b,c](#t1-fn1){ref-type="fn"}
Leptin (ng/ml) 1.5 ± 0.4 28.3 ± 1.2[a](#t1-fn1){ref-type="fn"} 1.3 ± 0.1 27.3 ± 2.1 26.8 ± 1. 6 28.4 ± 4.5[b,c](#t1-fn1){ref-type="fn"}
All measurements are mean ± SEM.^a^ p \< 0.01 vs. NCD-Sham;^b^ p \< 0.01 vs. HFD-AVN;^c^ p \< 0.05 vs. HFD-AVN-LacZ. Values represent mean ± SEM for five animals in each group.
###### Effect of obesity and leptin on ischemic osteonecrosis in femoral heads as assessed by Micro-CT
NCD-Sham HFD-Sham NCD-AVN HFD-AVN HFD-AVN-LacZ HFD-AVN-Leptin
--------------- -------------- -------------- ----------------------------------------- ----------------------------------------- -------------- -------------------------------------------
BV (mm^3^) 13.37 ± 3.25 14.47 ± 2.14 8.21 ± 1.98[a](#t2-fn2){ref-type="fn"} 4.91 ± 1.02[b](#t2-fn2){ref-type="fn"} 4.78 ± 1.72 10.21 ± 4.02[c,d](#t2-fn2){ref-type="fn"}
BV/TV (%) 48.08 ± 6.48 49.48 ± 3.13 39.84 ± 7.60[a](#t2-fn2){ref-type="fn"} 32.48 ± 3.13[b](#t2-fn2){ref-type="fn"} 28.18 ± 7.40 47.95 ± 5.67[c,d](#t2-fn2){ref-type="fn"}
Tb.Th (mm) 0.24 ± 0.05 0.25 ± 0.05 0.20 ± 0.06[a](#t2-fn2){ref-type="fn"} 0.18 ± 0.03[b](#t2-fn2){ref-type="fn"} 0.17 ± 0.07 0.24 ± 0.06[c,d](#t2-fn2){ref-type="fn"}
Tb.N (mm^−1^) 2.79 ± 0.32 3.31 ± 0.34 2.28 ± 0.51[a](#t2-fn2){ref-type="fn"} 1.92 ± 0.94[b](#t2-fn2){ref-type="fn"} 1.87 ± 0.58 2.42 ± 0. 45[c,d](#t2-fn2){ref-type="fn"}
Tb.Sp (mm) 0.22 ± 0.08 0.23 ± 0.05 0.26 ± 0.06[a](#t2-fn2){ref-type="fn"} 0.31 ± 0.07[b](#t2-fn2){ref-type="fn"} 0.32 ± 0.07 0.24 ± 0.08[c,d](#t2-fn2){ref-type="fn"}
Values represent mean ± SEM for five animals in each group. BV, bone volume; TV, tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.
^a^p \< 0.01 vs. NCD-Sham;^b^ p \< 0.01 vs. HFD-Sham;^c^ p \< 0.05 vs. NCD-AVN;^d^ p \< 0.01 vs. HFD-AVN-LacZ.
###### Sequences and accession numbers for forward (FOR) and reverse (REV) primers used in real-time RT-PCR
Gene Sequences for primers Accession No.
-------------------------------- -------------------------------- ---------------------------
Runx2 FOR: TCCCCGGGAACCAAGAAG **[NM_053470.1]{.ul}**
REV: GGTCAGAGAACAAACTAGGTTTAGA
BSP FOR: CCGGCCACGCTACTTTCTT **[J04215.1]{.ul}**
REV: TGGACTGGAAACCGTTTCAGA
Osteopontin FOR: CTGGCAGTGGTTTGCTTTTG **[AB001382]{.ul}**
REV: CCACTTTCACCGGGAGACA
Type 1 collagen FOR: CGATGGCGTGCTATGCAA **[Z78279]{.ul}**
REV: TCGCCCTCCCGTTTTTG
Osterix FOR: CATCTAACAGGAGGATTTTGGTTTG **[NM_053470]{.ul}**
REV: AAGCCTTTGCCCACCTACTTTT
Osteocalcin FOR: AAGCCCAGCGACTCTGAGTCT **[NM_013414.1]{.ul}**
REV: AGGTAGCGCCGGAGTCTATTC
HIF-1α FOR: GAACAAAACACACAGCGAAGCT **[XM_006240197.2]{.ul}**
REV: TGCAGTGCAATACCTTCCATGT
VEGF FOR: TGTGCGGGCTGCTGCAATGAT **[AY702972.1]{.ul}**
REV: TGTGCTGGCTTTGGTGAGGTTTGA
GAPDH FOR: AATGAAGGGGTCATTGATGG **[NM_017008.4]{.ul}**
REV: AAGGTGAAGGTCGGAGTCAA
|
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INTRODUCTION
============
High-dose chemotherapy followed by autologous haematopoietic stem cell transplantation (HSCT), aiming to reset the dysregulated immune system, is becoming an established means of treating refractory autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (Saccardi et al, [@b20]). However, few data are available regarding the specific changes in the immune system caused by HSCT, especially concerning the survival and re-expansion of T cell clones, which survive chemotherapy.
Several studies to characterize the changes in T cell receptor (TCR) repertoire after HSCT have been performed. The results of most reports indicate complete or nearly complete T cell repertoire 'renovation' and depletion of autoreactive clones (Alexander et al, [@b2]; Muraro et al, [@b17]), although some studies have indicated that pre-existing T cells can survive the conditioning regimen (Sun et al, [@b22]). However, in-depth comparative analysis of TCR repertoires before and after HSCT could not be performed, mainly because of the methodological limitations of the spectratyping and sequencing approaches employed for these studies (Rufer, [@b19]).
RESULTS
=======
To overcome these limitations, we have developed an optimized TCR beta amplification technique and employed massive sequencing using the 454 Genome Sequencer FLX (Roche). The Genome Sequencer is preferable for this task since it generates a large number of reads of approximately 300--500 bp in length that is sufficient to acquire a barcode, TCR J beta segment, CDR3 region, and a significant portion of the TCR V beta segment sequences, allowing us to distinguish even highly homologous V beta genes. The latter is crucial for the rational analysis of TCR repertoires, since the V beta gene segment determines the complementarity determining region 1 (CDR1) and CDR2, which contribute to the TCR specificity.
To provide unbiased and representative TCR beta amplification without distorting the natural abundance of TCR clonal sequences that closely reflects the abundance of the corresponding T cell clones, we have developed an optimized technique (Supporting Information [Fig 1](#SD1){ref-type="supplementary-material"}), which:
Starts from RNA to exclude rearranged but non-functional TCR beta genes that are strongly downregulated by the nonsense-mediated mRNA decay mechanism (Bhalla et al, [@b4]; Wang et al, [@b24]).Uses optimized TCR C beta-specific priming for cDNA synthesis, dramatically improving further amplification compared to oligo-dT or random hexamer priming.Exploits the reverse transcriptase template switching effect (Douek et al, [@b9]; Matz et al, [@b15]; Zhu et al, [@b25]) to generate a universal primer at the 5′ end of the TCR beta chain and thus allows unbiased amplification using a single pair of universal primers.Uses nested PCR to increase amplification specificity and step-out PCR (Matz et al, [@b15]) accompanied by the PCR-suppression effect (Siebert et al, [@b21]) to suppress products of non-specific primer annealing.Introduces oligonucleotides for subsequent sequencing and DNA barcodes during amplification to avoid inefficient ligation of DNA adapters (Supporting Information [Fig 1](#SD1){ref-type="supplementary-material"}).
The combination of these technical solutions allowed us to specifically and uniformly amplify a ready-for-sequencing, rearranged TCR beta gene fragment library within 23 PCR cycles, when starting with approximately 2 µg of total RNA obtained from peripheral blood mononuclear cells (PBMC) purified from a 2 ml blood sample, containing approximately 2 million T cells (see Supporting Information [Figs 1--3](#SD1){ref-type="supplementary-material"}, Supporting Information [Tables 1 and 2](#SD1){ref-type="supplementary-material"}, and Supporting Information 'Notes for details concerning technology development and verification').
To analyse massive sequence data, we have developed a special software, named TCRbase, that has several capabilities including: extraction of the CDR3 regions; correction of typical Genome Sequencer errors; identification of TCR V beta and J beta gene families; clustering and ranging identical sequences; *in silico* spectratyping and clonotyping within specific TCR V beta and/or J beta gene families; and, importantly, cross analysis of multiple datasets to search for identical or homologous nucleotide or amino acid CDR3 sequences (see Supporting Information [Figs 4 and 5](#SD1){ref-type="supplementary-material"} and Supporting Information 'Notes for software details').
This arsenal of tools was employed to perform the first deep, quantitative tracking of the fate of T cell clones in a 46-year-old patient with ankylosing spondylitis (a form of chronic, inflammatory arthritis) before and after autologous HSCT (see Supporting Information 'Notes for the clinical details'). Samples of peripheral blood were collected 1 week before as well as 4 months and 10 months after the procedure and TCR beta amplification performed as described above. From these samples, TCRbase extracted approximately 11,500, 14,500, and 19,500 valid TCR beta sequences from the two small sequence runs performed (1/16 of a PicoTiterPlate) (see Supporting Information Data 1 for the list of clustered clonal sequences, raw data are available at NCBI SRA database, study accession number SRP005664). The three sequence sets were further analysed using TCRbase, which revealed the following:
At least 250 T cell clones survived HSCT, and we believe that deeper analysis would reveal many more clones that survived the procedure. This striking result demonstrates that effectiveness of HSCT therapy is based on reprogramming of re-expanding T cells, but not on the physical elimination of the 'last autoimmune clone' (de Kleer et al, [@b7]) (Supporting Information [Table 3](#SD1){ref-type="supplementary-material"}).HSCT decreased overall diversity of T cell clones (Supporting Information [Fig 6](#SD1){ref-type="supplementary-material"}), while the number of 'hyper-expanded' clones (comprising \>1% of all TCR beta sequences) increased, leading to the propagation of specific TCR V beta gene families (Supporting Information [Fig 7](#SD1){ref-type="supplementary-material"}). The cumulative contribution of 'hyper-expanded' clones increased from 3% before to 26% after HSCT and remained at this high level for at least 10 months after HSCT ([Fig 1A](#fig01){ref-type="fig"}).A significant number of the clones, which survived the conditioning regimen, expanded after transplantation. Hence, most of the prominent expansions (Supporting Information [Table 3](#SD1){ref-type="supplementary-material"}) and as much as 40% of all T cells found after HSCT ([Fig 1B](#fig01){ref-type="fig"}) originated from the clones found before HSCT.Clones, which survived, established a new balance that was unexpectedly stable, as revealed by *in silico* analysis, which demonstrates that the spectratypes and clonotypes skewed by HSCT remained intact over the next 6 months ([Fig 2](#fig02){ref-type="fig"}). Restoration of a full TCR beta repertoire which would re-produce the previous diversity and the clonal expansions normally present in healthy subjects, would probably take years, especially given the low abundance of naive T cells observed in the patient blood after HSCT (Supporting Information [Fig 8](#SD1){ref-type="supplementary-material"}).At least some clones, which survived, expanded to provide specific immune defence early after transplantation. Using FACS sorting of T cells stained with an HLA-A2 MHC tetramer loaded with the immunodominant cytomegalovirus (CMV) peptide NLVPMVATV, we have identified two CMV-specific T cell clones that dramatically expanded after HSCT as shown by both mass sequencing ([Fig 2](#fig02){ref-type="fig"} and Supporting Information [Table 3](#SD1){ref-type="supplementary-material"}) and flow cytometry (Supporting Information [Fig 9](#SD1){ref-type="supplementary-material"}) analysis. Both clones displayed the same TCR beta amino acid sequence identical to the CMV-specific sequences previously reported in other patients (V beta 7-6, J beta 1-4, CDR3: CASSLAPGATNEKLFF) (Price et al, [@b18]; Venturi et al, [@b23]). Expansion of these CMV-specific clones is consistent with clinical studies reporting the activation of persistent viral infections after HSCT (Afessa & Peters, [@b1]).Stably expanded T cell clones have a high chance of surviving transplantation. Earlier, we described 11 T cell clones that were stably expanded in the peripheral blood of this patient for at least 3 years (Chkalina et al, [@b5]; Mamedov et al, [@b14]). Remarkably, all of these clones survived HSCT, although expansion of most of them was significantly suppressed (Supporting Information [Tables 3 and 4](#SD1){ref-type="supplementary-material"}).Strikingly, more than 100 of the TCR beta CDR3 amino acid sequences from this one patient are 100% identical to CDR3 variants already referenced in the NCBI protein database (see Supporting Information Data 2 for the list of identified matches). Thus, despite the huge potential diversity of TCRs in humans (\>10^15^, Davis & Bjorkman, [@b6]), the TCR sequence pool recognizing key antigenic peptides can appear to be rather restricted, at least in subjects sharing the same Class I MHC alleles.
{#fig01}
{#fig02}
DISCUSSION
==========
This work is the first deep analysis of the fate of clonal T cell populations after HSCT. It provides objective data to clarify the consequences of HSCT, which could previously only be assumed, and suggests the directions for further longitudinal studies of T cell repertoire and optimization of HSCT protocols. Importantly, intensive myeloablative regimens preceding HSCT were initially developed to eradicate tumour cells, which is critical for the successful treatment of malignant diseases (Dingli & Michor, [@b8]). However, satisfactory therapeutic effects using milder pre-transplant regimens were demonstrated for the treatment of autoimmune diseases (Hugle & van Laar, [@b12]). In our case, complete remission was observed for at least one year in spite of the fact that multiple T cell clones survived the procedure. On the other hand, although specific clones required to resist persistent infections and activated following immunosuppressive chemotherapy may successfully expand, as shown by the example of CMV-specific clones, the long term efficiency of adaptive immune response decreases (Kunisaki & Janoff, [@b13]). Thus, our data support the concept that chemotherapy regimens for the treatment of autoimmune diseases should be significantly moderated, both to reduce the toxicity of treatment and to preserve the clonal diversity of immune cells, which is otherwise dramatically altered.
The evolution of HSCT therapy protocols will require further studies of TCR repertoires before and after HSCT in order to understand the fate and the role of T cell clones in more detail. In particular, the fate of various subpopulations of T cells, including regulatory T and Th17 cells, is of significant interest. In addition, it is important to study whether changes in the T cell repertoire are dependent on different graft manipulation protocols, such as CD34^+^-positive selection or negative purging of lymphocyte subsets by monoclonal antibodies (Farge et al, [@b11]; Moore et al, [@b16]). While it was shown that CD34^+^-selected grafts do not contain large amounts of T cell clones after HSCT (Dubinsky et al, [@b10]), CD34^+^-positive selection was not performed in our case, and thus re-infusion of T cell clones with the autograft cannot be excluded.
Mass sequencing comparing TCR repertoires from a large number of healthy and diseased individuals, along with the progress in the analysis of TCR specificity (Bakker & Schumacher, [@b3]), should lead to the identification of common CDR3s that are generated in response to particular pathogens or to peptides from autoantigens. Along with the rapidly increasing cost efficiency of mass sequencing, these approaches might allow single-step diagnosis of particular infectious and autoimmune diseases, as well as deciphering an individual\'s history of diseases and vaccinations.
The paper explained
-------------------
PROBLEM:
Autologous haematopoietic stem cell transplantation (HSCT) is successfully used to treat severe autoimmune diseases, but the mechanisms, by which it resets the dysregulated immune system, remain poorly understood.
RESULTS:
In this work, we report an optimized TCR profiling technology based on 454 massive sequencing and use it to track the fate of T cell clones at three time points: before, 4 months after and 10 months after high-dose chemotherapy and HSCT. We directly demonstrate that multiple T cell clones survive transplantation and that some of them expand and fight infection early after HSCT. We observe that, by 4 months after HSCT, a new balance is formed between the clonal T cell populations, which then remains stable at least for the next 6 months. Interestingly, we show that multiple TCR beta CDR3 amino acid sequences from one patient can be identical to CDR3 variants found in other patients, indicating that the actual TCR sequence pool recognizing key antigenic peptides can be rather restricted, in spite of their huge potential diversity.
IMPACT:
We anticipate that our work will intensify studies of human TCR repertoires and optimization of HSCT regimens aimed to reduce their toxicity and to improve long-term outcomes.
MATERIALS AND METHODS
=====================
HSCT
----
The study was approved by local ethical committee. Patient gave written informed consent prior to enrolling in the study. Stem cells were mobilized with G-CSF (10 µg/kg b wt.). A 'Haemonetics MCS' instrument was used for autologous stem cells aphaeresis. CD34^+^-positive selection was not performed. The patient received 200 mg/kg of cyclophosphamide over 4 days with autologous blood stem cells for rescue and infusion of antithymocyte globulin (ATG) for *in vivo* T cell depletion. On day 0 (June 6, 2009), the patient received 2.4 × 10^6^ kg b wt. CD34^+^ stem cells, the number of which was determined by counting total number of cells and FACS analysis of an aliquot of cells using a CD34-specific antibody. No life-threatening events occurred during transplantation. Post-transplant toxicity included neutropenia with neutrophil count \<0.5 × 10^9^/L (from day +2 to day +9); thrombocytopenia, with thrombocyte count \<50 × 10^9^/L (from day +2 to day +12); fever (from day +4 to day +5); and stomatitis.
Flow cytometry
--------------
PBMCs were isolated from the peripheral blood samples by Ficoll-Paque (Paneco, Russia) density gradient centrifugation. Cells were washed twice with PBS and resuspended to a density of approximately 10^6^ cells/ml. Cells were incubated with antibodies or MHC tetramer for 20 min at room temperature or at +8°C and washed twice with PBS. Phenotypic cell analysis was performed using a Cytomics FC 500 (Beckman Coulter) flow cytometer. Data were analysed using the program Cytomics RXP Analysis (Beckman Coulter).
Antibodies
----------
Cells were stained with the following mouse anti-human antibodies: CD3-PC5 (clone UCHT1, Invitrogen), CD4-PC5 (clone S3.5, Invitrogen), CD8-FITC (clone YTC 182.20, Abcam, UK). The remaining antibodies were purchased from Beckman Coulter (USA): CD45RA-FITC (clone 2H4LDH11LDB9), CD27-PC5 (clone 1A4CD27), TCR-VB1(VB9)-PE (clone BL 37.2), TCR-VB7(VB4-1)-FITC (clone ZOE), TCR-VB16(VB14)-FITC (clone TAMAYA 1.2), CD8-PC7 (clone SFCI21Thy2D3).
Identification of CMV-specific T cell clones
--------------------------------------------
PBMCs were incubated with CD8-specific antibody and MHC tetramer for 20 min at room temperature in PBS Ca^2+^, Mg^2+^ free/1 mM EDTA/1% BSA with added DNaseI (Promega, USA) and sorted on a MoFlo cell sorter (DakoCytomation, USA). RNA was extracted using TRIZOL reagent (Invitrogen). Fifty cloned TCR beta chains were sequenced.
Preparation of sample for mass sequencing
-----------------------------------------
PBMCs were isolated by Ficoll-Paque (Paneco, Russia) density gradient centrifugation from fresh peripheral blood samples. RNA was isolated using Trizol reagent (Invitrogen, USA) according to manufacturer protocol. First strand cDNA was synthesized over a period of 2 h using Mint cDNA synthesis kit (Evrogen, Russia) and the specific primer BC1R, which binds to both constant regions of the human TCR beta transcript. Plug oligo (Evrogen) was added after the first 30 min of synthesis. The PCR amplification protocol was as follows: 94°C for 20 s; 65°C for 20 s; and 72°C for 50 s, 16 cycles. The PCR mixture (25 µl) contained 1× PCR buffer for Encyclo polymerase (Evrogen), 0.125 mM of each deoxyribonucleotide triphosphate (dNTP), 10 pmol of primers M1 and BC2R, 0.5 µl of Encyclo polymerase mix, and 1 µl of undiluted first-strand cDNA. After the first PCR step, the product was diluted 20× and 1 µl was used in the second 25-µl PCR reaction. This PCR reaction contained 1× PCR buffer, 0.125 mM of each dNTP, 10 pmol of each of the primers B-M1 and A-MID1-BC3R for the pre-HSCT sample, primer A-MID2-BC3R for post-HSCT sample, and B-MID3-BC3R, B-MID5-BC3R, B-MID6-BC3R for the 10 months post-HSCT sample along with 0.5 µl of Encyclo polymerase mix (Evrogen). The amplification protocol was as follows: 94°C for 20 s; 68°C for 20 s; and 72°C for 50 s, 12 cycles. After the second PCR, the amplicons were purified by agarose gel electrophoresis using a DNA gel extraction kit (Cytokin, Russia). The purified amplicons were collected, re-amplified for additional two cycles to polish the ends, and finally purified with QIAquick PCR purification kit (Qiagen, USA). Sequencing was performed using a Genome Sequencer FLX using a GS Em PCR Kit II (Roche Applied Science). For the oligonucleotides used, see Supporting Information [Table 2](#SD1){ref-type="supplementary-material"}.
We are grateful to Prof. J.S.H. Gaston for the valuable suggestions and comments on the manuscript. This work was supported by Molecular and Cell Biology program RAS, Rosnauka 02.512.12.2053, RFBR 10-04-01771-a, and Basic Research for Medicine RAS.
Supporting information is available at EMBO Molecular Medicine online.
The authors declare that they have no conflict of interest.
Author contributions
====================
IZM, DAB, AVC, AAK, MAT, and IVZ performed DNA-technique optimization and prepared the samples for pyrosequencing. DAF and AAN performed HSCT. OVB, DBS, AAK, and GVS worked with blood samples and performed flow cytomerty analysis and sorting. DAB and DMC developed software. SL, YBL, and DMC contributed to the overall idea, organization and manuscript preparation.
For more information
====================
Dmitriy Chudakov lab website:
<http://www.ibch.ru/en/structure/groups/fluortools>
Pirogov National Medical Surgical Center, Department of Haematology and Cellular Therapy:
<http://www.gemclinic.ru/english.php>
IMGT Repertoire, TCR and Ig database:
<http://www.imgt.org/textes/IMGTrepertoire/>
454 Sequencing:
<http://www.454.com/>
Supplementary material
======================
Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors.
[^1]: These authors contributed equally to this work.
|
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###### Strengths and limitations of this study
A strength of this study is that it included a wide range of participants and so gained a variety of perspectives.Several barriers to shoe-wearing not previously identified emerged from this study.All participants were from rural northern Ethiopia, so the findings may not be generalisable beyond this.
Introduction {#s1}
============
Footwear can provide considerable health benefits in reducing the incidence or progression of a range of neglected tropical diseases (NTDs).[@R1] Walking barefoot has long been considered an important risk factor for podoconiosis, a non-filarial, geochemical form of lymphoedema and elephantiasis that results in bilateral swelling of the lower legs.[@R2] Podoconiosis imposes huge economic burdens that worsen the prevailing poverty, and results in considerable social stigmatisation associated with the belief that the condition is familial and incurable.[@R3] [@R4] Available evidence indicates that podoconiosis is preventable if individuals consistently use footwear and begin doing so early in life.[@R5] Footwear is also associated with low prevalence of other soil-transmitted diseases,[@R8] and has been shown to prevent the recurrence of attacks of adenolymphangitis among patients with lymphoedema[@R9] and to reduce foot complications in other diseases such as diabetes.[@R10]
Nevertheless, several studies have documented that a considerable proportion of rural communities do not use footwear, or use it only during specific seasons such as the rainy or hot seasons.[@R5] [@R7] Inconsistent use of footwear is associated with increased risk of acquiring soil-transmitted and foot-related disease.[@R1] [@R8] [@R11]
People living in areas endemic for podoconiosis hold misconceptions about the causes of podoconiosis. Individuals believe, for example, that one may acquire podoconiosis through bad fortune, evil spirits or stepping on goat\'s blood.[@R4] [@R16] Many people, studied in southern Ethiopia, believed that heredity made podoconiosis inevitable, and considered the presence of an inherited gene to be an absolute guarantee of podoconiosis occurrence. These beliefs were associated with decreased likelihood of preventive behaviours such as footwear use.[@R17] [@R19]
A study conducted in southern Ethiopia identified financial constraints, unsuitability of shoes for specific activities and low perception of risk, as barriers to footwear use.[@R5] However, little is known about the personal, cultural and socioeconomic contexts of footwear use in northern Ethiopia, where podoconiosis is highly endemic in a population group with different cultural norms to those in southern Ethiopia. This study, therefore, aimed to explore the influence of personal, cultural and socioeconomic factors related to footwear use and non-use in northern Ethiopia.
Methods {#s2}
=======
Study area {#s2a}
----------
The study was conducted in East and West Gojjam Zones, Amhara region, northwest Ethiopia. According to the 2007 census, East and West Gojjam Zones have a population of 4 260 533 people with an annual growth rate of 2.5%.[@R20] The main economic activity in the zones is subsistence agriculture. The point prevalence of podoconiosis in East and West Gojjam Zones was estimated to be 3.4% in 2012.[@R18]
The International Orthodox Christian Charities (IOCC), a non-government organisation, launched the first podoconiosis prevention and treatment programme in East Gojjam Zone, Amhara Regional state, in 2010. The prevention and treatment package includes foot care and hygiene, raising awareness about the disease and rehabilitation of patients. Shoes donated by TOMS (a US-based shoe company that donates one pair of shoes for each pair sold) have been distributed to rural school children to reduce the risk of acquiring podoconiosis.
Sampling and data collection {#s2b}
----------------------------
A qualitative study was conducted in August and September, 2014. Six of thirteen IOCC treatment sites were selected purposively based on their geographic representation and history of treatment services. A total of 91 individuals from four target groups participated: (1) non-affected community leaders: *Idir* (a form of social insurance) leaders, school principals, *kebele* (the lowest administrative unit) officials, health professionals, teachers, merchants and religious leaders; (2) affected men and women; (3) non-affected men and women not in leadership positions; and (4) school children (both male and female). A total of six focus group discussions (FGDs) were carried out: women\'s and men\'s group discussions were held separately for affected and unaffected people, whereas all community leaders joined one group. In each group, 8--12 individuals participated while 17 individual in-depth interviews (IDIs) were held with affected and unaffected individuals.
Semistructured interview guides adapted from those used in a similar study in southern Ethiopia[@R6] were used to collect data, and additional items were included as data collection progressed. Data collection continued until saturation was reached. All interviews were conducted in *Amharic*, the native language of the local people, and recorded with consent. Interviews lasted on average 1 h for IDIs and 2 h for FGDs.
Data coding and analysis {#s2c}
------------------------
AA (Amharic language expert and native speaker) transcribed the data and collaborated with AT during translation. Three of the team members (AT, AA and AK) coded the data by themes predefined in the interview guides and emerging during analysis, using a grounded theory approach, a qualitative research methodology in which a researcher systematically identifies themes and concepts emerging from a chunk of text data and theorises, as coding is being carried out, about how each concept identified is related to a larger, inclusive concept.[@R21] This was followed by reconciliation of coding by three members through frequent discussions on deviations and common themes. All team members were involved in draft organisation of codes and corresponding quotes to identify consistencies and contradictions in the data and interpretation. NVivo software for qualitative data analysis was used along with a manual approach.
Ethical statement {#s2d}
-----------------
Introductory letters were obtained from East and West Gojam Zonal Health Departments and Woreda Health Offices. Oral informed consent was obtained from each study participant: participation in the study was voluntary and any information provided was kept confidential. Quoted information was anonymised during the analysis and reporting.
Results {#s3}
=======
The following major themes and subthemes were identified as barriers related to footwear: (1) misconceptions about podoconiosis and inaccurate risk perceptions; (2) the barefoot tradition; (3) the perceived importance of shoes for special occasions; (4) gender inequality; (5) perceived poverty; and (6) the perceived inaccessibility of shoe markets.
Misconceptions about podoconiosis and inaccurate risk perceptions {#s3a}
-----------------------------------------------------------------
Affected and unaffected participants were asked whether they knew what causes podoconiosis and how to prevent it. This was to explore whether shoe wearing as a preventive health behaviour was central to community members' conceptions of podoconiosis cause and prevention. Many had no idea what caused the disease and few associated it with prolonged exposure to irritant soil particles. Lack of shoe wearing was rarely mentioned as a cause of the disease in response to such questions. Speculations regarding podoconiosis causes were categorised under three subdomains and are discussed below.
### Spiritual factors as a cause {#s3a1}
Beliefs that evil spirits cause podoconiosis are very common. For the majority, stepping on things thrown down by someone who is jealous or hostile was believed to be a major cause of podoconiosis. This is a major source of fear, and stems from the suspicion that an acquaintance thinks or does evil things to damage one\'s health either physically or psychologically."I got the disease when I was crossing the river. It was after four years of marriage. When I was crossing the river, I stepped on tetracycline that was put in a small scarf and tied together with 'injera' \[a kind of pancake made from a grain, 'teff'\]. My feet began swelling after that and gradually advanced. (Affected FGD female, age 35 years)"
### Overlooking barefoot exposure to soil as a cause {#s3a2}
Barefoot exposure to soil was rarely mentioned as the cause of podoconiosis. However, participants often mentioned environmental and other risk factors such as cold weather, 'mich' (wounds believed to be caused by taking off shoes during burning sun), 'yekola mujele' (lowland jiggers), using second-hand shoes, sharing water with affected people and stepping on waste such as cattle dung or farm residues."I know some people with swollen feet. But, I don\'t exactly know the cause. They usually mention exposure to cold weather as a cause. (Unaffected IDI Male, age 21 years)""I got the disease because of 'chamma-mich'. This open plastic shoe exposes to 'mich'. The disease is caused by 'mich' related to wearing shoes. (Affected IDI male, age 56 years)"
The habit of eating goat meat was the most commonly perceived and feared cause of podoconiosis among the participants."I have never been worried about the disease as those who eat goat meat are believed to get the disease in our community. I have never eaten goat meat. There is an affected woman in our neighborhood. When she was asked why her feet swelled, she said, "I have never eaten goat meat. Unknowingly, I ate goat meat in my friend\'s house". Since I heard this, I never eat goat meat. (Unaffected Student FGD female, age 15 years)"
Since exposure to soil is not commonly considered to be linked to podoconiosis, few participants consider that footwear might prevent it. Many indicated their efforts to prevent podoconiosis in avoiding the environmental risk factors discussed above:"I try to protect my children by advising them not to wear the shoes or socks I have worn. I throw away my old shoes into the toilet fearing they may use them and get the disease. (Affected IDI male, age 38 years)""Though I am not sure, I think I got the disease because of contact with sweat from my affected husband. But now, to prevent my children, we don\'t share water for washing feet. I dump the water I used for washing my feet and wash the container carefully before my children use it for washing their feet. (Affected FGD female, age 40 years)"
### Inaccurate perception of hereditary risk of podoconiosis {#s3a3}
Associating podoconiosis with heredity was common. Amharic terms, such as 'endezer hono', 'be zer' and 'keziriya', were used to explain the role of heredity in the local language. However, participants had little knowledge of what was inherited through the blood line. Some participants confused heredity and contagion, either of which could lead to podoconiosis clustering within affected families. Even in families in which two or more members or blood relatives were affected by podoconiosis, affected participants associated the cause of their foot condition to sharing of beds, water, shoes or tools used by other affected people in the family. Footwear was often seen as the culprit rather than as a way of preventing podoconiosis:"It was at the time of DERG regime. I bought 'Keskis chamma' from someone. I started wearing the shoes. Just after a month, my feet began swelling. I was shocked and said 'wa, what is this?' My parents said, 'please throw away this shoes into the river'. I didn\'t throw it away, but left it in the house. My younger brother wore the shoes unknowingly. As a result, his feet also started to swell. (Affected FGD male, age 60 years)"
Barefoot tradition {#s3b}
------------------
Participants indicated that owning shoes does not guarantee they are worn, because of the established barefoot tradition. They said that, for the majority of community members, barefoot walking was easier than using shoes. The barefoot tradition is manifested in various ways: in perceptions that shoes are heavy, or that they weaken the feet or fall outside 'standard' dress and are therefore not prioritised."People feel at ease when they walk barefoot. Some consider shoes to be heavy particularly in the mud. They think walking barefoot speeds up performance of any activity including running. (Affected, FGD male, age 55 years)"
Most adult community members find going barefoot more efficient in use of time and energy. In slippery places, many participants thought that walking barefoot prevented them from slipping. Under conditions where fast running was required (eg, being chased by an enemy or an animal), they said that those with shoes could not escape. Going barefoot was perceived to have many advantages in rural areas:"People usually avoid shoe wearing as they are not well aware of its importance. They think that their feet would go weaker and weaker and can\'t resist challenges for the future.(Affected man, age 75 years)""There is an old story. Two persons were talking each other. One was wearing shoes while the other was not. The one with shoes asked the other one, "my brother, why didn\'t you wear shoes?' The other one replied "why should I weaken my foot which will serve me in bad days?" You know, wearing shoes was not common in our tradition. (Affected, male FGD participant, age 60 years)"
Shoes are commonly 'forgotten', because they are not considered part of 'standard' dress. The subconscious self-monitoring of shoe use may be affected by the lack of a shoe-wearing tradition in the community. When we challenged a person who claimed to wear shoes regularly but was without shoes at the time of interview, he said"I forgot to wear them as I was rushing to come here. (Unaffected IDI male, age 25 years)"
The barefoot tradition results in shoes being given lower priority than clothes. Many community members would prefer to be without shoes than without clothes. They fear being considered 'mad' if they do not wear clothes, but the same is not a consequence of not wearing shoes."I give priority to clothes. It depends on where you live. It is rare to see people using shoes in our community. People decorate themselves with clothes not shoes. Hence, if you want to be equal with others, you need to have clothes. It does not matter if you don\'t have shoes. And, if you appear with shoes all the time, people say 'he is boastful'. People will laugh at you if they see you working in the farm with shoes. I never saw a person wearing shoes while plowing or cultivating the land. (Unaffected IDI male, age 21 years)"
Perceived importance of shoes for special occasions {#s3c}
---------------------------------------------------
The term 'shoes' is universally known among community members and it is rare to find a person who has never seen or heard of 'shoes' these days in rural areas in northern Ethiopia. Several local terms are available for different types of shoes. The most common Amharic word used is 'chamma'. Participants could list various kinds of footwear available in their community, each given different names. For instance, open shoes made locally from tires are called 'barbasso', 'yegebere chama' (farmer\'s shoes), 'ekedeke', 'gelet', etc. 'Gomma chamma' is a common term for dry rubber plastic shoes while 'kofkuafie' refers to foam-type rubber plastic shoes. 'Shera chamma' refers to canvas shoes, while 'koda chamma' are shoes made of leather. 'Bot chamma' is a common name for both dry and foam rubber plastic boots while 'keskis chamma' is used to refer to boots made from leather, particularly for soldiers.
Participants relate different types of shoes to different activities and seasons. They adapt their use of shoes according to the situation."I have three types of shoes. I use 'yegebere chamma' \[shoes made from tyres\] whenever I travel far away or for working on the farm in the dry season, to protect my feet from injuries. Compared to other shoes, they withstand hardships and can be used for about 7 to 8 years. (Unaffected IDI male, age 21 years)"
The types of farming activities influence the pattern of shoe use in several ways. For instance, during threshing and clearing weeds from the farm, shoes are not worn. The major reason for not wearing shoes while clearing weeds is so as not to damage crops:"when we enter into the farm with shoes, it damages the plants. (Unaffected IDI male age 21 years)"
The reason for not wearing shoes during threshing is harder to rationalise, but is deep set in community traditions."it is taboo to wear shoe at 'beray' \[threshing season\] time...you know why?...it is a sign of respect to the crops. (Unaffected IDI male, age 28 years)"
If an individual lacks appropriate shoes for a task or season, he or she is likely to go barefoot. For instance, 'barbasso' shoes are impractical during 'kiremt' (the rainy season)."In the rainy seasons, 'barbasso' is less functional in muddy and slippery places as it potentially exposes to injuries related to slipping, and it does not protect the feet from cold weather since it does not cover them entirely. I have 'eke-deke'. But, I didn\'t wear them today as it is so muddy. So, I went out barefoot because I cannot walk with shoes in the mud. I use 'eke-deke' in dry time. As it does not fully cover the foot, mud enters into it. (Unaffected FGD male, age 53 years)"
Similarly, closed rubber plastic footwear, whether short shoes or boots, are commonly worn in the rainy season as they are thought to protect the feet from the cold. In the hot season, plastic boots are avoided because of the discomfort and bad smell associated with them. Participants also felt that plastic boots were not comfortable for long journeys."It is difficult to use plastic boots for long journeys. When it is hot, they collect blood from our body and make it clot around our feet. Also, when it\'s hot, plastic boots cause a bad smell. But they are helpful for cold weather and mud since they are light. (Unaffected FGD male, age 35 years)"
Canvas shoes are preferred during the dry and cold seasons and by young people. Overall, community members are less likely to own or wear them as they become dirty and are easily damaged as they trap dust and mud."I like canvas shoes. Others of my age also prefer canvas or sneaker shoes. I feel discomfort wearing plastic boots or leather shoes because they are heavy. Whenever I go to market or for holidays, I use canvas shoes. I wear plastic sandals when I am around home. But, the problem is, you cannot wear canvas shoes in the rainy season because of dirt and mud. (Unaffected IDI male, age 21 years)"
Leather shoes, particularly those that are closed, seem to be the least owned or worn type of shoes in rural areas for several reasons. First, the perceived cost of leather shoes is very high. Second, they are thought to be very heavy. Third, they are suitable for the hot or rainy seasons."I don\'t have leather shoes, but my father has. My foot size and his are similar. When I check his shoes, even the soles are heavy. You can wear canvas shoes without socks. But you cannot comfortably wear leather shoes without socks. When socks are added, they become heavier. If I need to walk about two to three hours on foot to go to 'Merawi' \[name of nearby town\], it is really heavy. As a result I don\'t like to buy leather shoes. But, they are also so expensive even when you need it. (FGD student male, age 18 years)"
Some participants thought that, unlike other types of shoes, leather shoes require socks and washing of the feet whenever they are worn. Washing the feet and socks every time before and after using closed leather shoes is impossible for rural farmers, whose lives are associated with mud and dust every day."I bought new leather shoes. When I wore them without socks, they hurt the skin around the toes. I then started using socks. It increased the comfort. But, it brought a bad smell to my feet. I took off the socks and washed my feet. And I started using 'kongo' plastic shoes. If you wear leather shoes, it requires washing feet and socks immediately you use them. (Unaffected FGD male, age 35 years)"
Some participants linked the difficulties of wearing shoes that require socks and foot washing with chronic shortages of water in their locality:"In the dry season, we face serious shortages of water, even for drinking. There is no tap water. There is one spring where everyone goes there. We wait overnight to get it. (Unaffected FGD male, age 59 years)."
Gender inequality {#s3d}
-----------------
Discussions held with all categories of participants revealed that both the pattern of shoe use and the types of preferred shoes differed between women and men. Men are relatively advantaged in terms of availability of shoes appropriate for various activities, and have a lighter burden of day-to-day tasks. In addition to farm work, women shoulder the burdens of carrying out domestic chores and trading in the market. As a result, it is common to observe men wearing shoes while they go to market, while most women are barefoot and burdened by many other items."If the road is muddy, men take off their shoes, hang them on their stick and carry them across their shoulders. But, as women already carry things on their back, they cannot handle shoes. They are physically weak compared to males. While carrying things on their back, they cannot walk wearing shoes in the muddy and bumpy roads. The shoe adds weights to the heavy things they carry on their back. (Affected FGD male, age 55 years)""It is men who frequently wear shoes. We carry many things when we go to market or other places. Men do not carry anything when they go to distant places. We carry 'tela' \[local beverage\], bread, 'injera', etc. Since shoes are heavy, we take them off and walk barefoot. (Unaffected FGD female, age 50 years)"
The types of shoes that are locally available also favour men. Boots (both leather and plastic) and 'barabasso' are more commonly worn by men. Though some women wear plastic boots, many consider that doing so would expose them to ridicule. It is rare for women to wear 'barabasso' simply because the community is accustomed to seeing men wearing them. If a woman wears shoes and neat clothes all the time, she may be pointed at or gossiped over---people will speculate that she is looking for another partner or has a foot problem like 'mujale' (jiggers) or leprosy. It is not only men who ridicule women who wear shoes regularly; women also take part."For instance, if the girl appears with shoes during a wedding ceremony, people say "her feet are deformed due to 'mujale'" or suspect other problems. They say, "Why does she wear shoes if her feet are clean?" There are also other conditions; those women who wear shoes regularly are demeaned. (Affected IDI female, age 25 years)"
Shoes were thought not to be a priority for many women, compared with clothes and 'timtam' (a scarf tied around the head). Compared with men, many women own shoes but do not wear them, except for special occasions. Wedding gifts are a good example: the groom is expected to buy a 'kemis' (skirt), 'ambar' (bracelet) and 'timtam' for the bride, while the groom\'s family buys him special shoes."My brother is married. After he participated in the Community Conversation conducted by IOCC, he wears shoes all the time. Though he bought two pairs of shoes for his wife, she does not like to wear them. When I told her to wear shoes when she goes somewhere, she said, "why should I worry since I have healthy feet". She left the shoes in her house. (Affected IDI female, age 25 years)"
Perceived poverty {#s3e}
-----------------
Most participants said that poverty was a major deterrent to ownership and use of footwear. They admitted that shortage of money meant they did not always own or wear shoes, often saving them for special occasions rather than wearing them out in everyday activities."In fact, shoes are very expensive for poor people. Those who don\'t have assets, find it so difficult to buy shoes. These days, the cost of shoes is 300 to 400 \[Birr, US\$15--20\] even for canvas shoes, let alone leather shoes. Life is very expensive nowadays. (Affected FGD male, age 52 years)"
Inaccessibility of shoe market {#s3f}
------------------------------
Shoes are often unavailable in smaller village markets. Traders supply better quality shoes to the larger markets, usually in towns. Rural residents, particularly those in remote villages, may have to either walk for several hours on foot or use a vehicle to get to market, which may also limit their motivation to purchase shoes."We go to Debremarkos \[the Zonal capital\] to buy shoes. There is a small market in Robit, very near, but shoes are not supplied there. We usually walk on foot to go to Debremarkos. We find a vehicle once in a week that is on Saturday. Saturday is the largest market day so that many people go to Debremarkos. Other times, we walk on foot. On average, it takes 3 to 4 hours to walk. (Unaffected IDI male, age 21 years)"
Discussion {#s4}
==========
This study explored the barriers to footwear use in an area endemic for podoconiosis and several NTDs. We identified individual behaviours and structural factors that influence individuals' decisions on footwear ownership and use. We found that misconceptions about podoconiosis and inaccurate risk perceptions, barefoot traditions, the perceived importance of shoes for special occasions, gender inequality, perceived poverty and perceived inaccessibility of shoe markets, are the most important barriers to the ownership and use of footwear among rural community members in northern Ethiopia.
Underestimating the importance of barefoot exposure (due to misconceptions about podoconiosis and inaccurate risk perceptions) was found to be an important barrier to the use of footwear. This was echoed in previous studies in podoconiosis-endemic settings.[@R1] [@R2] [@R5] Existing barefoot traditions were other reasons that individuals avoided wearing shoes. Beliefs that being barefoot is advantageous and that wearing shoes is potentially dangerous still prevail in the community. The barefoot tradition also results in shoes being afforded lower priority than clothes. This finding is congruent with a study in southern Ethiopia,[@R5] but quite different to those of studies investigating other health commodities such as bed nets. Utilisation of bed nets was found in 73% and 68.6% of households in malaria-endemic areas (Arba Minch Town and Raya Alamata district, respectively[@R22] [@R23]), suggesting considerably greater priority given to bed nets in those areas than shoes in this area.
Though community members report that they own certain types of shoes, the perception that shoes are only for special occasions has been a major barrier to their use. The use of shoes also varies with the type of shoe owned, the activities being undertaken and the season. As shoes are believed to damage the crops, people do not wear shoes while entering into the field. It is also considered taboo to wear shoes while threshing crops. Choices of shoe type are made according to the weather, for instance, 'Barbasso' shoes are avoided during 'kiremt' (rainy season). However, closed rubber plastic footwear, whether short shoes or boots, are commonly worn in the rainy season, as they are believed to protect the feet from cold weather. During the hot season, plastic boots are avoided due to the discomfort and the bad smell associated with them. Interestingly, heat was also given as a reason for 15.7% of households not using bed nets in Raya Alamata.[@R22]
Gender inequality is the most important determinant of optimum use of shoes. In many instances, men are relatively advantaged due to a lower burden of day-to-day tasks, meaning that they are able to wear shoes more frequently than women. Consequently, unlike males, females usually go to market barefoot, since they think that shoes will be difficult to carry in addition to their other burdens. The types of shoes that are locally available are most appropriate for men. Most shoe types are worn by men, and if a woman appears in shoes she is likely to be ridiculed, pointed at or gossiped over by the community (both males and females). An earlier study in the same study setting demonstrated more men than women to be wearing shoes at the time of interview,[@R18] as did a study in western Ethiopia.[@R15] Recent national podoconiosis mapping showed that more men than women were wearing shoes at the time of interview.[@R24] Overall gender mainstreaming and women\'s empowerment might address this challenge in the long term. In the short term, however, it will be important to address this gap during free shoe distribution for the prevention of podoconiosis. As for communities in southern Ethiopia,[@R5] participants in this study confirmed that poverty was another barrier to the ownership and use of footwear, as is the inaccessibility of shoe markets.
Conclusion {#s5}
==========
Although the nature of this qualitative study may limit its generalisability to other contexts, it reveals information that is likely to be helpful in guiding further research and interventions to prevent podoconiosis. Using footwear optimally to prevent multiple NTDs is contingent on addressing these barriers. Increasing community awareness about the causes of NTDs, social transformation to diminish barefoot traditions and to create favourable attitudes towards footwear, increasing access to affordable footwear and addressing broader gender inequalities, are recommended to increase the wider acceptance and utilisation of footwear as an important public health intervention. The Ethiopian government may also consider adopting a policy of footwear use in schools, as is now the case in certain other African countries.
The authors would like to thank the study participants for their cooperation during field work. They would also like to thank Mr Gharib Murbe and Mr Henok Nigussie for editing the manuscript.
**Contributors:** AK, AT, GD, KD and TA conceived the study design. AK, AT and GA carried out the data collection. AA transcribed and translated the data. AT and AK performed the data analysis. AA, AK, AT, TA, KD and GD interpreted the results. AK drafted the manuscript. All the authors read and approved the final manuscript.
**Funding:** This research was funded by IOCC Podoconiosis research. KD is supported by a Wellcome Trust Fellowship in Public Health and Tropical Medicine (grant number 099876). GD is supported by a Wellcome Trust University award (grant number 091956) to carry out work in podoconiosis.
**Competing interests:** None declared.
**Ethics approval:** Amhara Regional Health Bureau Institutional Review Board.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data sharing statement:** No additional data are available.
|
{
"pile_set_name": "PubMed Central"
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|
1. Introduction {#sec1-micromachines-07-00150}
===============
Mixing microliter volumes of liquids is a challenging task, as inertia effects are negligibly small compared to friction. At low Reynolds numbers, as they occur in most microfluidic applications, mixing is diffusion-limited and hence very slow. To overcome this limitation, several different types of micromixers were presented previously. In general, micromixers can be classified into two groups, passive and active ones \[[@B1-micromachines-07-00150]\]. For passive micromixers, there exist rather simple geometries like Y- or T-shapes \[[@B2-micromachines-07-00150]\] but also more complex ones, e.g., the herringbone mixer \[[@B1-micromachines-07-00150]\]. All passive methods have the problem of long mixing times and channel lengths in common. In contrast, we here present an active micromixer using surface acoustic waves (SAW).
In our group, acoustic streaming and its application for mixing and application to biophysical questions has a long standing tradition, starting with the demonstration of the ability of SAW to actuate and process the smallest amounts of fluids on the planar surface of a piezoelectric chip in 2003 \[[@B3-micromachines-07-00150],[@B4-micromachines-07-00150]\]. There, we already demonstrated the possibility for lithographical surface modifications to create virtual walls and vessels and presented a variety of assays on a chip that have already been realized. The flow profile in a capillary gap and the pumping efficiency of an acoustic micropump employing propagative SAW and Rayleigh streaming \[[@B5-micromachines-07-00150],[@B6-micromachines-07-00150],[@B7-micromachines-07-00150]\] was investigated both experimentally and theoretically \[[@B8-micromachines-07-00150]\]. The streaming velocity was found to depend on the applied linear power and to decrease quickly with the distance from the chip surface. Later in a short letter \[[@B9-micromachines-07-00150]\], we demonstrated the application of fast acoustic mixing at low Reynold's numbers by combining simple Y-shaped channels with a SAW-chip. This early principle was taken up by other groups \[[@B10-micromachines-07-00150]\] and is the basis of the in-depth studies presented in the present manuscript. Since then, the number of applications of SAW microfluidics has continuously increased \[[@B11-micromachines-07-00150],[@B12-micromachines-07-00150],[@B13-micromachines-07-00150],[@B14-micromachines-07-00150]\].
Our work on acoustic mixing was complemented by cooperative, theoretical and modeling investigations with the Hänggi group demonstrating a striking similarity to the experimental results with model calculations of the flow patterns and the advective transport by applying a raytracing algorithm. The presented concept can be transferred to acoustic streaming systems with different sound sources, like bulk acoustic waves \[[@B15-micromachines-07-00150]\]. We furthermore demonstrated the feasibility of this technique in two different modes of operation. For both modes, optimal frequencies characterizing the relevant stretching-folding duty cycles causing the chaotic advection were identified in experimentally accessible frequency regimes \[[@B16-micromachines-07-00150]\]. That work presents an approach for determining the streaming patterns that are generated by Rayleigh SAW in arbitrary 3-D geometries by finite element method (FEM) simulations. An efficient raytracing algorithm was applied to the acoustic problem while the acoustic streaming interaction was modeled by a body force term in the Stokes equation. Detailed theoretical investigation of leaky surface acoustic wave-induced streaming was reported by Vanneste et al. \[[@B17-micromachines-07-00150]\].
Moreover, we demonstrated the broad applicability of SAW streaming for various biological and chemical applications. First, we developed a microfluidic device on a planar surface using the techniques mentioned above. Here, we combined the SAW technique with thin film resistance heaters for a biological analysis chip with integrated DNA amplification by polymerase chain reaction (PCR) and hybridization. The necessary volume for this was as low as 200 nL \[[@B18-micromachines-07-00150],[@B19-micromachines-07-00150]\]. Moreover, within the last ten years or so, our group did not only employ surface acoustic waves for mixing or handling of small volumes but also to elucidate biophysical questions. One of the very exciting applications was the investigation of the highly non-linear shear stress dependent unfolding of the "von Willebrand factor" (VWF), a protein being omnipresent in our circulatory system and necessary to start primary hemostasis. By mimicking a wide range of blood flow conditions with direct visualization, the conformational dynamics of this protein in the presence of any adsorbing surface were shown to be of a reversible nature \[[@B20-micromachines-07-00150]\]. Along the same lines, employing the combination of a SAW based microfluidic reactor with an atomic force microscope, we studied the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, from which the slowest one is dominated by the internal conformations and effective friction within the bundle. These findings on VWF-VWF-interaction under shear became possible due to broad range of tunable shear forces with such a hybrid reactor \[[@B21-micromachines-07-00150]\]. Moreover, for the interaction of VWF with melanoma cells and the matrix protein collagen type I, we also applied the open system based on hydrophilic tracks and SAW as a nanopump \[[@B22-micromachines-07-00150]\]. Within the last few years, we have developed a miniaturized (\~100 μL) lab-on-a-chip hybrid system which allows for the quantification of cell adhesion under dynamic conditions which are comparable to those of physiological relevance. Amongst others, we investigated an osseointegration model with Saos-2 cells \[[@B23-micromachines-07-00150]\]. Just recently, we also demonstrated the positive effects of very low amplitude SAW on the stimulation of cell migration \[[@B24-micromachines-07-00150]\]. Our data clearly exhibits the SAW induced, dynamic mechanical and electrical stimulation and directly promotes the cell growth. Thus, we firmly believe that this SAW based cell stimulation method offers a powerful platform for future medical treatment.
The many different SAW based application examples as summarized above, are quite far and advanced from simple mixing at low Reynolds numbers. Nevertheless, this apparently simpler SAW task, which was demonstrated almost 10 years ago is still of high interest, as for example reproducible and parallelizable systems are needed for, e.g., the synthesis of nanoparticles (NP). For some particle systems SAW atomization has been demonstrated before \[[@B25-micromachines-07-00150]\].
Here, we now focus on controlled and reproducible mixing for NP production based on SAW induced chaotic advection \[[@B9-micromachines-07-00150],[@B15-micromachines-07-00150]\], based on propagative waves and Rayleigh streaming with a typical decay length at the solid-fluid interface of about 10 wave lengths \[[@B16-micromachines-07-00150]\]. To combine the microfluidic NP production reactor and SAW mixing, we use a polydimethylsiloxan (PDMS) Y-shaped channel, which is mounted directly on a SAW chip. This hybrid approach gives us the possibility of inducing acoustic streaming in any desired direction with respect to the mean flow in the microfluidic channel. We are thus able to mix two fluids in a highly controllable and fast way by relying on chaotic advection. In contrast to previous works \[[@B9-micromachines-07-00150],[@B26-micromachines-07-00150],[@B27-micromachines-07-00150],[@B28-micromachines-07-00150]\] we investigate the mixing quality as a function of tunable system parameters, such as applied SAW power, volume flux and fluid viscosity.
Finally, we demonstrate the advantages of our hybrid approach for the production of therapeutic nanoparticles. The basic mechanism for particle formation is the formation of polyplexes by mixing of cationic polymers in one solution and negatively charged nucleic acid in the second \[[@B29-micromachines-07-00150],[@B30-micromachines-07-00150]\]. The microfluidic technology, due to the ability of rapid mixing of fluids on the nanoliter scale, is highly beneficial for obtaining well defined samples of organic nanoparticles. Fast mixing provides homogenous reaction environments, which in the case of macroscopic hand mixing is affected by mixing kinetics and results in spatial and temporal inhomogeneities of particle formation. Furthermore, homogenous reaction environments can lead to a more reliable formation of particles with decreased size and polydispersity compared to particles generated by conventional methods. Here, mixing is achieved by diffusion alone due to the low Reynolds number regime. In our approach, we are using SAW driven mixing to increase the speed of the mixing process by folding streamlines and thus reducing the distance for diffusion in order to decouple the process of particle formation and the mixing kinetics even further. Two different cationic systems for a potential transfection use were tested. The systems are chosen to represent different types of transfection agents (cationic polymers and lipids) and different synthesis methods (mixing of aqueous solutions and solvent exchange approach). The first system is a commonly used branched polyethylenimine (bPEI)/plasmid DNA (pDNA) particle system, where the relatively huge polymer bPEI (25 kDa) with high nitrogen content and multiple positive charges at neutral pH, forms polyplexes by ionic interaction with the negatively charged nucleic acid after mixing \[[@B31-micromachines-07-00150],[@B32-micromachines-07-00150],[@B33-micromachines-07-00150]\]. The second system are the stable nucleic-acid-lipid particles (SNALPs) \[[@B34-micromachines-07-00150],[@B35-micromachines-07-00150],[@B36-micromachines-07-00150]\]. Those small particles are dedicated for short siRNA oligonucleotide encapsulation and are synthesized by a solvent exchange method \[[@B37-micromachines-07-00150]\]. An additional polyethylene glycol shielding layer on the mono-nucleic acid/lipid particles (mNALPs) surface results in an increased particle stability under biomimetic conditions and prevents unspecific interactions with the cell membrane. Here we will focus on a particular formulation protocol leading to mNALPs that were shown to consist of a single siRNA duplex covered by a single, highly curved lipid bilayer \[[@B38-micromachines-07-00150]\]. mNALPs by design are already limited in size (\~30--38 nm) but the production does not always lead to monodisperse particle distributions due to poorly controlled kinetics during manual hand mixing. The later leads to formation of clustered structures and hence more reliable mixing methods for synthesis by self-assembly are demanded.
2. Materials and Methods {#sec2-micromachines-07-00150}
========================
2.1. SAW-Chip and Microchannel Fabrication {#sec2dot1-micromachines-07-00150}
------------------------------------------
For the generation of the microfluidic flow, we fabricated so called "tapered" Inter-Digital Transducers (IDT) \[[@B39-micromachines-07-00150]\] of Ti-Au-Ti (5 nm-50 nm-5 nm height) on a LiNbO~3~ (128° rot Y-cut) substrate. The tapering of the IDT results in a band of excitable SAW frequencies ranging between *f* \~79.5--82.5 MHz on the same chip. Electronically, such a tapered transducer acts as a passband filter. A nice side effect for research purposes is, however, that by variation of the applied radio frequency, SAW are generated in the form of a narrow jet at different positions along the IDT aperture. Hence, this spatial variation of the sound path allows us to also control the position of the acoustically induced fluid jet. Signal generators (CellEvator, Advalytix, München, Germany) with customized Lab-View-based control software and standard SMA-connectors were used. Typical voltages are 5.6 V (peak) according to *P* = 25 dBm. We decided to design the setup in a way that can be easily reproduced and installed in multiple labs. Thus, we chose a frequency in the frequency range typically exploited for SAW-mixing \[[@B40-micromachines-07-00150]\]. Lower frequencies and thus a higher wave length could lead to undesired standing waves or reduced mixing efficiency. In contrast, to employ higher frequencies requires more sophisticated techniques, which contradicts our intention for accessibility for labs without decent ultra-high-frequency equipment. To protect the multi-finger electrodes, a SiO~2~ coating was sputter-deposited on top of the IDT structures. At the same time the LiNbO~3~ substrate acts as the bottom of our microchannel.
The Y-shaped elastomer microchannel consists of a simple polydimethylsiloxane (PDMS) single layer and is fabricated by standard soft lithography \[[@B41-micromachines-07-00150]\]. Two equally sized inlets with a cross section of 100 µm × 100 µm converge in a rectangular main channel of 200 µm width. The two solvents are injected into the channel at identical flow rates F/2. In the 3-in-1 channel, three inlets with cross sections of 50 µm × 50 µm and 20 µm × 50 µm for the outer inlets and the middle inlet, respectively, converge in a main channel with a cross section of 120 µm × 50 µm. The PDMS block was placed carefully on top of the chip with the designated IDT fitted precisely in the intended cavity. The distance between the first finger of the IDT and the channel wall was approximately 200 µm. The SAW coupled perpendicularly into the channel approximately 450 µm and 330 µm downstream from the junction for the Y-shaped channel and the 3-in-1 channel respectively. The block was pressed with about 0.5 mNm on the chip with an aluminum plate fastened to the bottom plate with four screws.
2.2. Particle Image Velocimetry Experiments {#sec2dot2-micromachines-07-00150}
-------------------------------------------
The generated flow field was characterized as described earlier \[[@B42-micromachines-07-00150]\]. In short, latex beads (Polystyrene, diameter 3 μm, Polybead^®^, Polysciences, Inc., Warrington, PA, USA) were added to the fluid in the channel. These particles were then used as tracers to follow the streamlines and make the fluid motion visible. For the analysis, the flow was recorded by a high speed camera (FASTCAM 1024PCI, Photron, Ottobrunn, Germany). At each position along the fluidic channel, 50 frames at a rate of 1000 fps were captured. A MATLAB (7.11.0.548 (R2010b), The MathWorks, Inc., Natick, MA, USA) script based on the open source PIVlab (version 1.35) toolkit \[[@B42-micromachines-07-00150],[@B43-micromachines-07-00150],[@B44-micromachines-07-00150]\] was employed to extract the three-dimensional velocity profile and will be described subsequently. The captured videos were analyzed in a PIVlab batch process in order to determine the local velocity profiles. The results at different positions were then stitched and the missing data points eventually recovered by a linear interpolation, ending up with layered *x-y* velocity profiles for the whole region of interest.
2.3. Mixing Experiments {#sec2dot3-micromachines-07-00150}
-----------------------
To verify the SAW induced mixing quality, we used light microscopy in combination with a high speed camera (Photron). For the analysis process, one of the two fluids to be mixed was dyed with a food coloring (Patent Blue V calcium salt, 1 mM, Sigma Aldrich, St. Louis, MO, USA). To vary the fluid viscosities, different water-glycerol-mixtures were used \[[@B45-micromachines-07-00150]\]. The videos were analyzed with the public domain software package ImageJ (1.48v, National Institutes of Health, Bethesda, MD, USA) \[[@B46-micromachines-07-00150]\]. In [Figure 1](#micromachines-07-00150-f001){ref-type="fig"}, we show typical micrographs illustrating the mixing process. The area highlighted by the red box indicates the analyzed region. The red box of size 200 µm × 580 µm is defined to start at the right end of the window in the PDMS channel above the IDT through all measurements. These cavities define the IDT position for all experiments. For the Y-shaped channel this results in a distance between the junction and the fluid jet of 450 µm. For the latter introduced 3-in-1 channel this distance is 330 µm. [Figure 1](#micromachines-07-00150-f001){ref-type="fig"}a exhibits the typical laminar flow pattern of a microfluidic channel without any applied SAW, while [Figure 1](#micromachines-07-00150-f001){ref-type="fig"}b shows a mixing pattern generated by SAW. The 8 bit gray scale videos yields discrete values ranging from 0 (black) to 255 (white). To quantify the mixing quality, we introduce a mixing parameter $\widetilde{M}$, given by the mean gray scale value of the analyzed region divided by the standard deviation: $$\widetilde{M} = \frac{\overline{X}}{\mathsf{\sigma}} = \ \frac{\overline{X}}{\sqrt{\frac{1}{n - 1}\sum_{i = 1}^{n}\left( {X_{i} - \overline{X}} \right)^{2}}}\ $$
Here, $\overline{X}$ is the arithmetic mean grey value, σ is the standard deviation, *n* is the number of pixels and $X_{i}$ is the gray scale value at the position *i*. A homogenous distribution of the gray scale values results in small values of σ, which in turn leads to large values of σ^−1^. To compensate the influence of slightly different illumination for the different images and to ensure comparability, σ^−1^ is scaled with $\overline{X}$. To ensure comparability and intuitive understanding of the according results, we normalize the difference of the mixing parameter $\widetilde{M}$ for SAW-Mixing and diffusive mixing (SAW off) to the interval \[0, 1\] named "mixing efficiency": $$M = \frac{\widetilde{M} - {\widetilde{M}}_{\min}}{{\widetilde{M}}_{\max} - {\widetilde{M}}_{\min}},$$ where ${\widetilde{M}}_{\min}$ is the value of the unmixed and ${\widetilde{M}}_{\max}$ the value of the mixed state.
2.4. Nanoparticle Synthesis and Sample Evaluation {#sec2dot4-micromachines-07-00150}
-------------------------------------------------
For the production of bPEI polyplexes all solutions have been degassed for 15 min at approximate 8 mbar. Purified water (evoqua water technologies) was filled into three 1 mL syringes (Norm-Ject^®^ Tuberkulin, Henke Sass Wolf, Dudley, MA, USA) equipped with Hamilton needles (ga27/90mm/pst4, Hamilton, Bonaduz, Switzerland) and placed into a syringe pump (LA-160, Landgraf Laborsysteme HLL GmbH, Langenhagen, Germany). The needles were inserted into the tubes and the whole system was washed with purified water for 15 min to stabilize the flow and to remove any unwanted substances from the channels at a flow rate of *F* = 600 µL/h. Then, the two syringes connected to the outer inlets were replaced carefully to avoid bringing gas into the system. The solutions in these syringes contained 0.12 mg/mL branched polyethylenimine (bPEI) (PEI25, Sigma Aldrich, St. Louis, MO, USA) and 0.156 mg/mL plasmid DNA (pDNA) (pCMVLuc, Plasmid Factory, Bielefeld, Germany) in purified water respectively. Using a flow rate of *F* = 150 µL/h, a reference mixed diffusively and a fraction mixed by SAW streaming was collected. Each sample was stirred for 8 min. In addition, a third sample entitled "hand mixing" was prepared manually by adding 7 µL of the above mentioned pDNA and bPEI solutions to 7 µL of purified water by vigorous pipetting. All samples have been incubated for 45 min and were measured afterwards by Dynamic Light Scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) using DTS1070 folded capillaries.
The chloroform solutions of lipids used for mNALP synthesis, namely: DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt); DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPC, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine; DSPE-PEG(2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-\[methoxy(polyethylene glycol)-2000\] (ammonium salt) and DSPE-PEG(2000)-FolA,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-\[folate(polyethylene glycol)-2000\] (ammonium salt) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Since DNA is less prone to nuclease degradation, the 21 bp double stranded DNA was used as physicochemical model of siRNA. The single-stranded DNA oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany). Oligos were lyophilised and HPLC-purified by the company.
Two 21 base complementary sequences, strand 1: 5′-CCA-ACA-GTA-AAA-GGA-ATA-TCC-3′ and strand 2: 5′-GGA-TAT-TCC-TTT-TAC-TGT-TGG-3′ were used. The 5′ end of strand 1 was conjugated with Cy3 dye to facilitate the further sample evaluation by fluorescence correlation spectroscopy (FCS, Zeiss, Jena, Germany). The oligonucleotides were annealed in equimolar solution of 20 µM in 30 mM HEPES + KOH, 100 mM KCl, 2 mM MgCl~2~, 50 mM NH~4~Ac, pH = 7.5 annealing buffer. Annealing was done by 2 min incubation at 96 °C and subsequent slow cooling at room temperature. The 21 bp dsDNA product was dialysed against 20 mM TRIS + HCl, 2mM EDTA (2X TE) pH = 8 buffer and stored at −20 °C.
The mNALP particles were formed by a ten-fold dilution of molecular solutions of all components (lipids and dsDNA; 50% (*v/v*) IPA/H~2~O) in de-ionised water, as described in detail in \[[@B24-micromachines-07-00150]\]. Firstly, the required amount of all lipids (chloroform suspensions) in the molar ratios 1:5:6:1.2 DOTAP:DOPE:DOPC:DSPE-PEG(2000) were placed in glass vials, then dried under nitrogen flow and finally placed in a vacuumized exsiccator overnight. Then, the lipids were resuspended in an isopropanol/deionised water mixture (60% (*v/v*) IPA/H~2~O) to the final DOTAP 300 µM concentration. The lipid solution was sonicated and gently mixed with 21 bp dsDNA stock solution (20 µM), IPA and H~2~O to a final 400 nM concentration of dsDNA, 84 µM of DOTAP and 50% (*v/v*) IPA/H~2~O in another glass vial. This final solution was diluted ten times in de-ionised water which finally led to the particle formation. For comparison reasons, the actual samples were prepared employing three different mixing methods: Hand mixing by dropwise dilution and vortexing; and SAW mixing on a microfluidic chip (3-in-1-geometry); and diffusive mixing in the channel of the same geometry. The flow rates were controlled with TSE systems syringe pumps (model 540200, TSE Systems, Bad Homburg vor der Höhe, Germany) and kept at *F* = 0.05 mL/h and *F* = 0.45 mL/h for the molecular solution and water, respectively. For the acoustic mixing case, the SAW was generated at an IDT frequency of *f*~SAW~ = 81.2 MHz and a constant power of *P*~SAW~ = 27 dBm. All solutions and liquids used for microfluidic synthesis were degased in a vacuumized exsiccator.
Fluorescence Correlation Spectroscopy (FCS) was used to evaluate the quality, the size and the encapsulation efficiency of the lipid-DNA complexes. For the FCS experiments, a Zeiss LSM510 ConfoCor2 confocal microscope (Zeiss, Jena, Germany) with a C-Apochromat 40×/1.2 NA water immersion objective was used. The measurements were performed using a λ = 543 nm HeNe laser excitation and a bp 560--615 nm emission filter on Cy3-labelled samples. 21 bp DNA duplex was taken as a size reference. For both mNALP samples, the residual, freely diffusing dsDNA was seen in the system. Due to this, the model with the two 3D diffusion components (one for mNALP one for free dsDNA) and one triplet component was used. The model is described by following equation: $$G\left( \mathsf{\tau} \right) = \ A_{0} + \frac{1}{n\left( {\widetilde{F} + \mathsf{\alpha}{({1 - \widetilde{F}})}} \right)^{2}}\left( {1 + \frac{Te^{- \mathsf{\tau}/\mathsf{\tau}_{trip}}}{1 - T}} \right)\left\lbrack {\frac{1}{\left( {1 + \mathsf{\tau}/\mathsf{\tau}_{1}} \right)}\frac{1}{\sqrt{1 + \mathsf{\tau}/\left( {{SP}^{2}\mathsf{\tau}_{1}} \right)}} + \mathsf{\alpha}^{2}\frac{1 - \widetilde{F}}{\left( {1 + \mathsf{\tau}/\mathsf{\tau}_{2}} \right)}\frac{1}{\sqrt{1 + \mathsf{\tau}/\left( {{SP}^{2}\mathsf{\tau}_{2}} \right)}}} \right\rbrack$$
Here, *A*~0~ denotes an offset, *n* the effective number of particles in the confocal volume (*n* = *n*~1~ + *n*~2~), τ~1~ the diffusion time (correlation time) of particle species 1; τ~2~ the diffusion time (correlation time) of particle species 2. $\widetilde{F}$ represents the fraction of molecules of species 1 ($\widetilde{F}~$= *n*~1~ /(*n*~1~ + *n*~2~), α the relative molecular brightness of particles 1 and 2 (α = *q*~2~/*q*~1~). SP is a structural parameter, *T* the fraction of particles in triplet state and τ~trip~ the characteristic residence time in the triplet state. The particle fraction $\widetilde{F}$, as being obtained directly from the fit was taken as a measure of the encapsulation efficiency.
3. Results and Discussions {#sec3-micromachines-07-00150}
==========================
The structure of this section is as follows: First, we characterize the mixing process of aqueous solutions using a Y-shaped channel in general and estimate the mixing time. Second, we elucidate the role of flow rate and applied SAW power and their importance and influence for an optimal mixing effect. Next, we study the role of viscosity: Based on the findings of the role of flow velocities, we therefore measure the acoustic streaming velocity as a function of the viscosity. For the production of particles with a lipid shell and a DNA core, often mixing of different solvents is necessary. Thus, in the following step, we characterize the SAW induced mixing of both isopropanol and ethanol with water. Finally, we demonstrate the potential of the SAW mixing technique for the production of therapeutic nanoparticles using established systems. Here, we discuss hurdles like unintended complex formation at the interface of the two liquids and how to overcome those by using an additional separation layer in a 3-in-1 channel with three inlets. Finally, we apply the SAW-mixing to the fabrication of nanosized polyplexes composed of bPEI and pDNA, as well as to the mNALP system.
3.1. Mixing Process {#sec3dot1-micromachines-07-00150}
-------------------
In [Figure 2](#micromachines-07-00150-f002){ref-type="fig"}a, we sketch the mixing process in a Y-shaped channel employing acoustic streaming. As described earlier \[[@B15-micromachines-07-00150],[@B16-micromachines-07-00150]\], the mixing is based on chaotic advection, by generating a fluid jet perpendicular to the mean flow direction. Under the influence of the SAW induced acoustic streaming, the streamlines become folded which in turn contributes to an increase of the diffusive mixing efficacy. In [Figure 2](#micromachines-07-00150-f002){ref-type="fig"}b, a time sequence of the mixing process after switching on the SAW is shown. At *t* = 0, the SAW is not yet coupled into the fluid and the picture is the same as for laminar flow of the two fluids from left to right. In this case, only diffusive mixing takes place at the fluid-fluid interface. At *t* = 5 ms, the SAW already generates acoustic streaming in the fluid as can be seen by the colored fluid being pushed into the transparent water. During the ongoing process, two vortexes are formed with opposite sense of rotation. The last micrograph, finally shows the stationary state, where the two solutions are completely mixed.
Due to the complexity of the streamline geometries and the two vortexes of opposite sense of rotation, it is difficult to define a specific value for the mixing time of the fluids. Hence, we conservatively estimate an upper limit for this mixing time, by determining the time until the two volume elements of the fluids are completely mixed under stationary conditions. To do so, we measure the distance that the volume elements travel before they are completely mixed and divide this distance by the mean flow velocity. [Figure 2](#micromachines-07-00150-f002){ref-type="fig"}c shows the mixing parameter *M*, determined from the region indicated by the red box as function of the position along the channel until it reaches a constant value at a certain position *s*. Given the mean flow velocity *v* in the channel, the mixing time *t*~M~ can now be estimated: $$t_{M} = \ \frac{s}{v}$$
For our chip geometry, the result is a mixing time *t*~M~ = 189 ms for two aqueous solutions at a flow rate of 0.2 mL/h. As mentioned, this time depends on the flow rate, as a conservative upper limit and can still be reduced, as will be shown in [Section 3.4](#sec3dot4-micromachines-07-00150){ref-type="sec"} and [Section 3.5](#sec3dot5-micromachines-07-00150){ref-type="sec"}.
3.2. Role of the Flow Rate and RF-Power {#sec3dot2-micromachines-07-00150}
---------------------------------------
The two most important system parameters having a large influence on the mixing quality *M*, are the flow rate *F* and the RF-power *P*. We measured the maximal flow velocity in the center of the channel as a function of flow rate *F* (see [Figure A1](#micromachines-07-00150-f010){ref-type="fig"}a). The blue squares are taken from particle image velocimetry (PIV) measurements, the black dots are the results of theoretical calculations \[[@B47-micromachines-07-00150]\]. As expected, we clearly see a linear dependence of the flow velocity on the flow rate in our experiments as well. The systematically smaller values for the experimental data are due to the fact that in the model, only the exact center of the channel is considered, whereas experimentally, the evaluation of the velocity also includes regions slightly away from the center and hence regions of slightly smaller velocities. [Figure 3](#micromachines-07-00150-f003){ref-type="fig"}a shows *M* as a function of the flow rate at a constant power of *P* = 25 dBm. In the regime 0.01 mL/h \< *F* \< 0.6 mL/h, *M* decreases linearly, whereas for higher flow rates *F* \> 0.6mL/h, almost no mixing is further observed.
Moreover, [Figure 3](#micromachines-07-00150-f003){ref-type="fig"}b shows *M* as a function of the RF-power *P* at the constant flow rate of *F* = 0.2 mL/h. For *P* \< 19 dBm, almost no mixing is observed. For 18 dBm \< *P* \< 22 dBm, the mixing quality *M* increases and eventually saturates for higher values *P* \> 22 dBm, indicating that the fluids are completely mixed. To understand this, we recall the dependency of acoustic streaming velocity and SAW power *P*. [Figure A1](#micromachines-07-00150-f010){ref-type="fig"}b shows the induced SAW-streaming velocity *v*~SAW~ in bulk parallel to the chip surface as function of *P*. In accordance to previous work \[[@B16-micromachines-07-00150]\] *v*~SAW~ increases linearly with increasing *P*.
The data in [Figure 3](#micromachines-07-00150-f003){ref-type="fig"} allows us to explain the upper limit for the flow rate where suffiecient mixing is achieved: a constant SAW-power, e.g., *P* = 25 dBm, results in a constant velocity *v*~SAW~, e.g., *v*~SAW~ = 7 mm/s, as determined by PIV measurements in the channel. As it turns out, optimum mixing occurs if the main flow velocity *v~F~* and *v*~SAW~ are equal. A further increase of *v*~SAW~ does of course not yield better than optimum results. For $r ≔ \frac{v_{SAW}}{v_{F}}~$\<\< 1, no sufficient mixing is achieved.
3.3. Role of Viscosity {#sec3dot3-micromachines-07-00150}
----------------------
Here, we elucidate the role of the fluid viscosity on the SAW induced mixing process. To do so, we measure both the SAW velocity *v*~SAW~ as well as the mixing quality *M* as function of viscosity η. For the SAW velocity experiments, a PDMS wall was placed on a SAW-chip and filled with water-glycerol mixtures of different dilution and thus different viscosities as illustrated in [Figure 4](#micromachines-07-00150-f004){ref-type="fig"}a. The red cuboid next to the IDT marks the volume, which is evaluated by PIV measurements. Typical flow fields obtained from such measurements are shown in [Figure 4](#micromachines-07-00150-f004){ref-type="fig"}b. Obviously, the lateral velocity field in the fluid is a function of the height above the chip. Along the channel, the position of the region of highest velocities shifts to positions further "downstream" with increasing height. This is a consequence of the existence of the Rayleigh diffraction angle, when the SAW couples into the medium. For LiNbO~3~ and water, this angle between the jet and the vertical is approximately 22°. From the measurements, we determine the magnitude of the speed as a function of height as being depicted in [Figure 4](#micromachines-07-00150-f004){ref-type="fig"}. Here, we show the extracted maximum speed of the fluid as a function of the position along the channel for various fluid viscosities η. With increasing viscosity, the maximum speed decreases. For a viscosity of η = 1.0 mPas, which corresponds to water at 20 °C, the fluid velocity reaches almost *v*~fl~ = 10 mm/s. For viscosities higher than η = 25 mPas, the velocity drops below *v*~fl~ = 1 mm/s. The maximal velocity as function of viscosity is described best by a bi-exponential decay, as can be seen in [Figure 4](#micromachines-07-00150-f004){ref-type="fig"}d. Theoretically an exponential decay with only one decay constant is expected \[[@B48-micromachines-07-00150]\]. We furthermore compare our data with a very recent publication on simulations of acoustic streaming in droplets \[[@B49-micromachines-07-00150]\]. If plotted in a double logarithmic scale, our data for *v*~SAW~ (η) match the values in this publication extremely well (compare [Figure A2](#micromachines-07-00150-f011){ref-type="fig"}), though Riaud et al. claim a power law to fit the data best. Alternatively, the deviations from an exponential decay here could be attributed to be caused by a second force which becomes dominant for low viscosities and thus low friction. This contribution strongly depends on the boundary conditions like microchannel dimensions and materials and has to be determined for each designated setup separately. An increased temperature due to increased dissipation may lead to a decreased viscosity, which could explain the appearance of a second decay constant. However, in the most relevant viscosity range for SAW mixing of solutions in microchannels (η = 40 mPas) *v*~SAW~ is sufficiently described by a single exponential decay.
To investigate the mixing of solvents with higher viscosities, we used the same setup, the same parameters and the same procedures as described above and characterized the mixing quality *M* as function of η. [Figure 5](#micromachines-07-00150-f005){ref-type="fig"} shows these mixing results for water-glycerol-mixtures of different viscosities. First, in [Figure 5](#micromachines-07-00150-f005){ref-type="fig"}a, we show *M* as a function of the viscosity η for a constant flow rate of *F* = 0.2 mL/h and *P* = 25 dBm in a Y-shaped channel of the same geometry. *M* decreases rapidly with increasing η. For η \> 5 mPas, no sufficient mixing can be detected. This can be understood by a reduced SAW-velocity (see [Figure 4](#micromachines-07-00150-f004){ref-type="fig"}d) in combination with a reduced diffusion coefficient, being indirectly proportional to η. Typical micrographs of the according situations point up this behavior. For η = 1 mPas, a good mixing quality can be achieved and the distribution is homogeneous. For higher η, the distribution of the dye is not homogenous but rather, exhibits a striped pattern. Here, due to the low *v*~SAW~ and the large attenuation, the fluid jet only "squeezes" and thus folds the streamlines, as can be seen in the insets in [Figure 5](#micromachines-07-00150-f005){ref-type="fig"}a.
Consequently, for η \> 5 mPas and to create a sufficiently low ratio *r*, the main flow velocity *v~F~* has to be significantly reduced. In [Figure 5](#micromachines-07-00150-f005){ref-type="fig"}b proof is shown that in fact, good values of *M* are achieved by reducing the main flow by a factor of ten to *F* = 0.02 mL/h.
3.4. Mixing Aqueous Solutions with Ethanol and Isopropanol {#sec3dot4-micromachines-07-00150}
----------------------------------------------------------
The fabrication of therapeutic nanoparticles (TNP) with a lipid shell and nucleic acid core in aqueous solutions requires effective mixing of nonpolar solvents with aqueous solutions. To characterize the mixing efficiency as function of *F* and *P* for such systems, we here characterize the mixing of water with ethanol (1.2 mPas at 20 °C) and isopropanol (2.4 mPas at 20 °C) respectively, in the same Y-channel setup as described above. [Figure 6](#micromachines-07-00150-f006){ref-type="fig"} shows the mixing quality *M* as function of the flow rate *F* for applied SAW power *P* = 25 dBm and *P* = 29 dBm.
Applying a power *P* = 25 dBm allows sufficient mixing of aqueous solutions for flow rates up to 0.3 mL/h. While this upper flow rate is comparable to the one for mixing of isopropanol and water, it is shifted to about 0.6 mL/h for ethanol and water. In contrast to the mixing of aqueous solutions, here, increasing the SAW power to *P* = 29 dBm allows for sufficient mixing at flow rates up to 1.4 mL/h. For ethanol-water-mixtures the better mixing efficiency is in accordance with the finding of Orsi et al. \[[@B30-micromachines-07-00150]\]. They attribute this effect to the increased residence time of the fluid occupying the interfacial region in the water-ethanol case, due to an increased viscosity of the mixture at the interface compared to the pure solvents. The difference in mixing quality for water-isopropanol mixtures compared to water-ethanol mixtures for *P* = 25 dBm is in accordance with a higher viscosity of isopropanol compared to ethanol. Moreover, the lower surface tension of ethanol and isopropanol compared to water may contribute significantly to the mixing \[[@B31-micromachines-07-00150]\]. However, we did not study the role of surface tension systematically here, as the knowledge about the applicable flow rate range seems absolutely sufficient to us from a pragmatic point of view.
3.5. Formation and Characterization of Therapeutic Nanoparticles {#sec3dot5-micromachines-07-00150}
----------------------------------------------------------------
In this last section, we demonstrate the applicability and the potential of SAW-mixing for the production of therapeutic nanoparticles. First, we concentrate on an unwanted and unintended complex formation at the interface of the two fluids that turned out to be very counterproductive for the NP production process. Secondly, we apply the optimized acoustic mixing technique to a commonly used polyplex system of bPEI and pDNA. Finally, we investigate the application of SAW mixing to a more sophisticated system of lipid-based mono-nucleic acid lipid particles (mNALPs) yielding very good results. To benchmark the results we here compare mixing by hand following a strict protocol, diffusive mixing using a channel as shown in [Figure 7](#micromachines-07-00150-f007){ref-type="fig"}d and SAW-mixing in the same channel.
In a first step, to investigate the formation of polyplexes from cationic polymers and plasmid DNA, we mix 80 µg/mL bPEI with 100 µg/mL pDNA in the above characterized Y-shaped channel: The flow rate was set to *F* = 0.2 mL/h. Since no SAW is applied, only diffusion is thus acting on the fluids. As can be seen in [Figure 7](#micromachines-07-00150-f007){ref-type="fig"}, at the interface of the two solutions close to the junction of the inlets, a thin solid structure is formed within about 150 s. The thickness of this layer increases with increasing distance downstream from the inlets. [Figure 7](#micromachines-07-00150-f007){ref-type="fig"}c shows the same channel after 24 h of incubation at rest, i.e., without flow. Obviously, a thin wall had been formed by a complex formation due to the rapid and firm ionic interaction of charged bPEI and pDNA at the fluid--fluid interface. This layer of course deteriorates the ability to actively mix, employing a SAW. To minimize the area where this unintended complex formation appears, the relative distance of the IDT to the inlet in principle could be reduced. However, if the IDT is too close to the junction, the pressure increases in the inlets and the according changes of the flow profile lead again to suboptimal mixing. A more elegant and controllable resolution, is to use a 3-in-1 channel with a third inlet. The latter one is used to create a thin separation streamline of pure solvent (see [Figure 7](#micromachines-07-00150-f007){ref-type="fig"}e). Thus, the first contact between cationic polymers and anionic pDNA occurs at the position where the fluid jet breaks the separation layer (see [Figure 7](#micromachines-07-00150-f007){ref-type="fig"}f).
Using such a 3-in-1 channel, we mix bPEI and pDNA at a total flow rate of *F* = 150 µL/h and an applied power of *P* = 27 dBm. As reference measurements, we fabricate particles by hand mixing as described in the materials and methods section and by diffusive mixing without applied SAW. [Figure 8](#micromachines-07-00150-f008){ref-type="fig"} shows the according size distributions as measured by DLS, the mean hydrodynamic radius *R*~h~ and the polydispersity index PDI. Hand mixing results in particle radii of *R*~h~ = 54.1 ± 0.84 nm with a PDI = 0.292 ± 0.026. Microfluidic mixing increases reproducibility in terms of the decreased PDI = 0.232 ± 0.017 and its standard deviation and results in particles with a radius of *R*~h~ = 81.8 ± 1.5 nm. Here, we like to point out the difference between the Y-shaped channel and the 3-in-1 channel: While the Y-shaped channel does not lead to the desired TNP formation by diffusive mixing, interestingly the use of the 3-in-1 channel does. This may be a consequence of the low concentrations and less steep concentration gradients within the separation layer compared to the Y-shaped channel. Finally, SAW-mixing combines the advantages of both other methods resulting in automated particle fabrication with higher reproducibility compared to hand mixing; and smaller particles compared to microfluidic mixing with radii of *R*~h~ = 55.4 ± 0.65 nm with a PDI = 0.283 ± 0.004. However, hand mixing strongly depends on handling protocols and can vary from person-to-person or lab-to-lab. Thus, SAW-mixing definitely bears the potential to improve reproducibility. Finally, we like to mention another hurdle that could appear: using SAW-mixing for very high values of power *P* can result in heating of the chip \[[@B24-micromachines-07-00150]\]. We did not optimize the setup concerning this but a cooling circuit integrated in the PDMS could easily allay heating effects here.
As microfluidic polyplex formation using SAW-mixing has been demonstrated for the classic bPEI/pDNA composition, feasibility was also tested with a different more sophisticated type of TNP. Lipid-based mNALP \[[@B38-micromachines-07-00150]\] particles are synthesized in a solvent exchange method by 10-fold dilution of 50% (*v/v*) Isopropanol/water solution of lipid/nucleic acids in water. Here the interactions between molecular components, namely lipids and nucleic acids, occur when triggered by the change in the solvent quality. When the non-polar solvent (e.g., alcohol) is replaced with the polar water the hydrophobic interactions and electrostatic interactions leads to particle formation in the process of self-assembly. The kinetics of solvent exchange, thus the kinetics of mixing, is particularly crucial, as the slow alcohol/water mixing rates may interfere with particle formation and lead to formation of undesirable structures. For the purpose of our study, the same setup (3-in-1 channel) and mixing parameters as above were employed. To evaluate the quality of SAW assisted mNALP synthesis, comparative studies of both "bulk" and SAW assisted mNALP samples are performed. We use the FCS technique to investigate the basic colloidal properties, like the hydrodynamic radius *R*~h~ of the formed particles for both samples. Moreover, FCS allows us to measure the encapsulation efficiency as well. To monitor the particle formation, Cy3-labelled 21 bp dsDNA was used as physicochemical model of siRNA. It has a significant practical advantage, as DNA is less prone for nuclease degradation when compared with less stable siRNA. The naked, non-complexed dsDNA is taken as reference for monitoring the relative changes in diffusive behavior of particles. All mNALP samples are showing a complicated shape of autocorrelation curves reflecting the multiple fluorescent particle species present in the sample. All measurements which were significantly influenced by the occurrence of bright clusters were discarded from the calculations of averages. By applying the correlation model function consisting of two 3D diffusion components and one non-diffusive triplet component, we obtained the best results. One of the fitted components corresponds with the uncoated dsDNA as the diffusion time (thus the particle sizes, *R*~h~) correlates with the values obtained for reference dsDNA measurement. The second component corresponds to 3D diffusion of the particles with hydrodynamic radius *R*~h~ \~ 19 nm. It correlates well with the expected sizes of mNALP. Additionally, no significant changes in concentration of diffusing particles and counts per particle were seen. It leads to the conclusion that every diffusing particle contains a single dsDNA particle as expected for the mNALP system. For this particular binary system, the fraction of particles $\widetilde{F}$ derived directly from fitting the model function to the experimental correlation function corresponds directly to the encapsulation efficiency of dsDNA in the mNALPs. The results of FCS analysis are shown in [Figure 9](#micromachines-07-00150-f009){ref-type="fig"}. The normalized autocorrelation curves for samples prepared in all mixing modes and as a reference naked dsDNA sample (which corresponds to 0% encapsulation efficiency) are shown in [Figure 9](#micromachines-07-00150-f009){ref-type="fig"}a. An additional theoretical correlation curve generated for the single component mNALP system (particles of hydrodynamic radius *R*~h~ = 19.2 nm; 100% encapsulation efficiency) is shown as a guide. A noticeable shift in autocorrelation towards higher diffusion times for SAW mixed samples compared to hand and diffusive mixing can be seen. This corresponds to an increase in encapsulation efficiency, as the sizes of particles are comparable for both mNALP samples ([Figure 9](#micromachines-07-00150-f009){ref-type="fig"}). Moreover, the narrowing in size and encapsulation efficiency variances reflected in decreased standard deviations by a factor of about 2 (error bars in [Figure 9](#micromachines-07-00150-f009){ref-type="fig"}), shows that the samples prepared by microfluidic SAW mixing are more reproducible in terms of those two parameters. The statistical significance of the results from [Figure 9](#micromachines-07-00150-f009){ref-type="fig"}b was tested---the standard Student's *t*-test was used \[[@B50-micromachines-07-00150]\]. The *t*-values are given in [Table 1](#micromachines-07-00150-t001){ref-type="table"}. We expect this benefit to increase further for more sensitive multicomponent TNP systems, as we have already reached higher reproducibility and encapsulation, even without further optimizations e.g., in terms of concentrations, concentration ratios or acoustic wave length. A direct comparison of the approach presented here: based on chaotic advection with more conventional ones reported earlier \[[@B51-micromachines-07-00150]\], based on sonication or turbulent microfluidic mixing would be highly interesting \[[@B40-micromachines-07-00150]\]. Such a systematic study could elucidate the question which mixing approach (laminar, chaotic advection or turbulent) is favorable for which particle formation mechanism.
4. Conclusions {#sec4-micromachines-07-00150}
==============
We demonstrated the applicability and benefits of SAW-assisted fabrication of TNP. We elucidated the role of controllable parameters for the mixing process of aqueous solutions as well as other solvents like ethanol and isopropanol. We found the ratio of main flow velocity along the channels axis and the SAW-induced streaming velocity perpendicular to it, to be of highest importance. Consequently, for higher solvent viscosities compared to water, this ratio should be set to 1, either by increasing SAW-power or by decreasing the flow rate. Mixing of aqueous solutions with ethanol or isopropanol even allows for higher flow rates and thus higher throughput. For the formation of polyplexes from bPEI and pDNA, we introduced a separation layer to avoid an undesired complex formation prior to mixing. In contrast to Y-shaped channels, using such a 3-in-1 channel, even without SAW-mixing, allows the production of TNP by diffusive mixing. However, this leads to bigger particles compared to hand mixing, while SAW-assisted particle formation results in particles of the desired size comparable to hand mixing. Moreover, the technique enhances a more sophisticated formation of mNALPs regarding increased encapsulation as well as increased reproducibility. Summing up, we here presented a unique, promising and precisely controllable technique of SAW-assisted fabrication of therapeutic nanoparticles that opens up new possibilities for the nanomedicine community, especially for particle systems that are very sensitive to preparation conditions.
The authors like to acknowledge funding by Nanosystems Initiative Munich (NIM) and the Center for NanoScience (CeNS) for financial support for this project. The authors thank Adrian Mainka for chip fabrication and Andreas Hörner, Christian Dohmen and Christian Plank for inspiring discussions.
U.L., J.O.R., A.W., C.W. and E.W. designed the study. R.K., U.L., L.G.S., C.W. and D.W. carried out the experiments and analyzed the data. R.K., U.L., L.G.S., A.W. and C.W. wrote the manuscript.
The authors declare no conflict of interest.
{ref-type="fig"}a) parallel to the chip surface as a function of SAW power.](micromachines-07-00150-g010){#micromachines-07-00150-f010}
{ref-type="fig"}d) in a double logarithmic scale.](micromachines-07-00150-g011){#micromachines-07-00150-f011}
{#micromachines-07-00150-f001}
{#micromachines-07-00150-f002}
{#micromachines-07-00150-f003}
{#micromachines-07-00150-f004}
{#micromachines-07-00150-f005}
{#micromachines-07-00150-f006}
{#micromachines-07-00150-f007}
{#micromachines-07-00150-f008}
{#micromachines-07-00150-f009}
micromachines-07-00150-t001_Table 1
######
Student's *t*-test results for data in [Figure 9](#micromachines-07-00150-f009){ref-type="fig"} (critical value of Student's *t*-test for significance level *α* = 0.05 and *ν* = 10 degrees of freedom; *t~0.95,\ 10~* = 1.812. If the test value exceed the critical value the difference in experimental values are statistically significant).
Student's *t*-Test Results *R*~h~ Encapsulation *R*~h~ Encapsulation
---------------------------- --------------------- --------------------- -------------------------- --------------------------
Comparison SAW vs. hand mixing SAW vs. hand mixing SAW vs. diffusive mixing SAW vs. diffusive mixing
*t*-value 1.263 4.483 1.922 14.101
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Introduction {#Sec1}
============
In December 2019, the Wuhan city of Hubei province in China became the epicenter of an outbreak of local pneumonia in some patients with an unknown cause (Bherwani et al. [@CR1]; Gautam [@CR6]). By January 7, 2020, the Chinese research community isolated a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the virus-infected pneumonia patients (Phelan et al. [@CR16]; Gorbalenya et al. [@CR9]). On January 30, 2020, World Health Organization (WHO) declared the SARS-CoV-2 epidemic a Public Health Emergency of International Concern, which was later designated as a COVID-19 in February 2020 (Muhammad et al. [@CR15]; Gautam and Hens [@CR5], [@CR7]; WHO [@CR24]). Although the outbreak is plausible to be associated with the large seafood market which has triggered a zoonotic transmission event, but soon it became clear that an efficient human-to-human transmission is also taking place (Li et al. [@CR13]; Wu et al. [@CR28]; Sarkodie and Owusu [@CR18]). Due to the potential of COVID-19 to generate widespread outbreaks in confined settings and cross border following the mobility of human patterns, it was regarded as a pandemic by the WHO on March 11, 2020 (Cucinotta and Vanelli [@CR3]).
Coronavirus is a current novel and highly infectious contagion disease that belongs to the family of "Nidovirus" which includes "Roiniviridae," "Artieviridae" and "Coronavirdae" family, causing respiratory illness in humans from the common cold to more severe diseases such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (De Wit et al. [@CR4]; Gautam and Hens [@CR5]). Coronavirus is enveloped positive-sense RNA virus mainly comprising of almost 30,000 nucleotides in its genome, and its replication and transcription are dependent on the replicase gene which encodes two overlapping polyproteins, pp1a and pp1ab (Zhou et al. [@CR29], [@CR30]). The diameter of coronavirus ranges from 60 to 140 nm with club-shaped spikes on its surface and making a crown-like structure as evident from an electron microscopy, hence the name given coronavirus (Richman et al. [@CR17]). Coronavirus is a "communicable" disease and its spread all over the world is entirely associated with human-to-human transmission rather than transmission through the air (Wang et al. [@CR21], [@CR22]). The transmission of COVID-19 through airborne spread has not been reported yet and it is not believed to be the source of major transmission as per the evidence (WHO, [@CR23]). Due to its small size (about 0.1 micron), it can enter into the body via droplets from mouth or nose during close and unprotected contact between an infector and infectee. It has also been observed that coronavirus infected people generally develop symptoms and signs, including mild respiratory illness, fever, dry cough, and tiredness, with a mean incubation period of 5 to 6 days which can also range up to 1 to 14 days (WHO [@CR24]). Predominantly, the presentation of disease can also range from asymptomatic (with no symptoms) to severe pneumonia, and even death (WHO [@CR23]). Recent studies showed that the number of infected individuals with the novel COVOD-19 may get double every 7 days and each person could spread this virus to 2.2 to 3.58 persons on an average (Chan et al. [@CR2]). Additionally, the individuals ≥ 60 years of age and individual with chronic underlying health conditions are in a greater risk of being infected with COVID-19 in comparison to the children who might have less probability to get infected or, if so, these children may show mild symptoms or may have asymptomatic infection (Li et al. [@CR13]; Sun et al. [@CR20]). Besides, cleaning surfaces with an alcohol-based disinfectant is also important to practice to stop the spread as COVID-19 can survive on surfaces such as copper (up to 4 h), plastic and stainless steel (up to 72 h), and cardboard (up to 24 h) (WHO [@CR24]). Some of the countries have adopted the early isolation of infected patients and quarantine policies to prevent the transmission from the travelers who have returned from the COVID-19 infected countries (Hellewell et al. [@CR10]). Practicing frequent hand hygiene (with alcohol-based gel or sanitizer), maintaining respiratory hygiene, and a physical distance of at least 1 m (3 feet) is the best possible way to protect yourself and others from the novel COVID-19 (WHO [@CR23]). In addition, staying in home quarantine and seeking medical care early if person is allied with the acute symptoms fever, cough, or difficulty breathing, not going outside unnecessarily, avoid touching your eyes, nose or mouth, avoid eating raw meat and unnecessary contact with wild animals, etc. are some other precautionary measure for protection against the coronavirus disease. Nevertheless, a clear representation of the epidemiology of this novel coronavirus and vaccination is still being elucidated (Zhou et al. [@CR31]).
With the two months of the outbreak, the pandemic spread throughout the world at an alarming speed. As of March 1, 2020, a total of 87,137 confirmed cases and 2997 deaths had been reported globally by the WHO (WHO [@CR23]), and 58 countries were affected (WHO [@CR23]). While the cases continue to decline in China, the COVID-19 cases have been reported in more than 100 countries outside of China, including South Korea, Iran, Italy, Japan, France, and Germany by the end of March 2020 (WHO [@CR25]; Shim et al. [@CR19]; Gautam and Trivedi [@CR8]). The first case in South Korea was reported on January 20, 2020, followed by a rapid increase in the number of cases in subsequent days (Shim et al. [@CR19]). In particular, South Korea quickly became the hardest-hit country affected by the COVID-19 virus, with a steady increase in the number of cases over the month of February 2020. By the end of March 2020, South Korea was successfully able to control the outbreak of the novel COVID-19, and since its peak on March 11, 2020, the daily number of cases has been decreasing. This paper analyzes the trend and data of COVID-19 on certain criteria using "Exploratory Data Analysis." It is a revelatory step to any kind of analysis based on the date sets. In this paper, we reviewed the COVID-19 spread in South Korea with time by considering the different factors during the analysis. This will also enable us to know how South Korea managed to flatten the curve of COVID-19 cases with time.
COVID-19 South Korea's data analysis {#Sec2}
====================================
Current and widespread outbreak of novel coronavirus motivates to perform Exploratory Data Analysis (EDA) on the datasets, which was collected from the different sources such as, Korea Centers for Disease Control and Prevention (KCDC, South Korea [@CR12]), the Ministry of Health and Welfare of South Korea (MOHW, South Korea [@CR14]), John Hopkins GitHub repository (JHGR, USA [@CR11]) and Coronavirus Worldometer (Worldometer [@CR27]). The data was used to analyze the trend and spread pattern of COVID-19 cases in South Korea and compared with a pattern of cases in the most affected countries in the world. The data collected, organized, and processed in the form of graphical representation for the better visualization of results using OriginPro 2016 software (Version 9.3.226, OriginLab Corporation, USA). The total number of confirmed cases, active cases, deaths, and recovered cases as of July 8, 2020 (05:00 GMT), as well as the fatality rate distribution by province-wise was also presented.
COVID-19 Spread in South Korea over time {#Sec3}
----------------------------------------
From the first detected patient on January 20, 2020, to March 1, 2020, when the third patient was reported, confirmed cases were mostly inbound travelers from overseas and their contacts. Following February 18, an exponential surge with a short period was observed, starting with cluster infections mainly related to the Shincheonji Church and Cheongdo Daenam Hospital in Daegu city or South Korea. At that time, South Korea was on the verge of facing a continued surge in the COVID-19 outbreak with new confirmed cases per day amounting to 909 and total cases under isolated treatment at 7470 (MOHW, South Korea). However, the situation has been successfully contained as of now with new confirmed cases per day reduced to a single digit. Figure [1](#Fig1){ref-type="fig"} shows the trend of confirmed, recovered, active and deceased cases of COVID-19 in South Korea till July 8, 2020 (GMT 05:00).Fig. 1Trend of COVID-19 in South Korea over time as of July 8, 2020.(*Source*: MOHW, South Korea and John Hopkins GitHub repository, USA)
As of July 8, 2020, the total number of confirmed cases in South Korea reported to be 13,244, with the largest cluster of infected patients from Daegu and Gyeongsangbuk-do accounting for 62.8% of the total cases. According to the MOHW, among the total cases of 10,806 as on May 5, 2020, females account for 40.5% and males for 59.5% (Fig. [2](#Fig2){ref-type="fig"}a). Figure [2](#Fig2){ref-type="fig"}b shows the age distribution of infected persons. The number of deceased reaches 255 with 92.2% of the deaths from the age group of ≥ 60 and a fatality rate of 2.36%. The average reproduction number of COVID-19 in South Korea was estimated to be 1.5 (Shim et al. [@CR19]).Fig. 2**a** Gender-wise cases and **b** age-wise distribution of COVID-19 in South Korea as of May 5, 2020.(*Source*: MOHW, South Korea)
History of imported cases of COVID-19 spread in South Korea {#Sec4}
-----------------------------------------------------------
As of July 8, 2020, there were 1714 of total confirmed cases that had a travel history to different countries. Among them, the USA and Europe together contribute to more than 65.2% of the total confirmed cases in South Korea as shown in Fig. S2 (supplementary material). Out of the 1714 cases, 1286 (75.1%) cases were of Korean nationality and 428 (24.9%) cases were of foreign nationality.
COVID-19 spread in South Korea versus other countries {#Sec5}
-----------------------------------------------------
When the COVID-19 started spreading across the globe, the initial phase saw South Korea becoming the second most affected country after China (the epicenter of the outbreak). But soon within a span of 2--3 weeks, South Korea managed to control this virus from spreading through its rigorous and effective approach. As of now on July 8, 2020 as shown in Fig. [3](#Fig3){ref-type="fig"}, South Korea has witnessed a total of 0.11% of the COVID-19 cases being reported worldwide, with USA reporting one-fourth of the total number of cases. The number of causalities stands at 0.05%, attributing to the advanced health care system of South Korea. The world is now witnessing the biggest pandemic, with the total death exceeding 550,000 till date. As of July 8, 2020, the total confirmed, deceased, recovered and active cases of COVID-19 were 11,954,944, 546,720, 6,846,897, and 4,206,920, respectively. Table S1 (supplementary material) provides detailed information on the COVID-19 cases around the world with USA and Europe being affected the most in terms of causalities and the number of infected patients.Fig. 3Comparison of confirmed COVID-19 cases and deaths in South Korea versus other countries.(*Source*: MOHW, South Korea, As of July 8, 2020, GMT 05:00)
Compared to the worst hit countries with COVDD-19, South Korea has reported the lowest mortality rate of 2.2, when compared to USA, UK, Spain, Mexico, and Italy with mortality rate of 4.4, 15.5, 11.3, 11.9, and 14.4, respectively (MOHW, South Korea). Thus, the mortality per lakh population in India is 6 deaths per 1 million populations, which is lowest in comparison to the global case mortality rate of 69.7 deaths per 1 million populations (MOHW, South Korea). The relatively low death rate, in terms of case mortality rate, signifies timely clinical management and identification of the COVID-19 cases in South Korea.
Testing of COVID-19 in South Korea {#Sec6}
----------------------------------
Owing to its early detection and testing capabilities which included extensive use of ICTs and drive-thru testing stations, South Korea was able to contain the spread of this deadly contagion. South Korea is capable of conducting 23,000 diagnostic tests per day, and almost a total of 1,359,735 tests have been conducted until July 8, 2020 (05:00 GMT). Besides, the healthcare professionals in South Korea were allowed to conduct a COVID-19 diagnostic test on any suspected individual without any charge. The efforts made by the South Korean Government for carrying the extensive and free of charge diagnosis of COVID-19 was key to minimize the outspread and damage of this virus at an early stage. As of April 27, there are 347 of National Safe Hospitals designed by the government to provide the services for non-respiratory patients and ensuring the safety of general patients. Table S2 (supplementary information) provides the detail of the total numbers to tests conducted, in progress, and the concluded results.
Province wise spread of COVID-19 in South Korea {#Sec7}
-----------------------------------------------
As of July 8, 2020 the total number of confirmed cases stands at 13,244, which is concentrated mainly in Daegu and Gyeongsangbuk-do province (62.8% of all confirmed cases) and the specific religious sector (Shincheonji) as the major epidemiological link (47%). Figure [4](#Fig4){ref-type="fig"} provides the province wise data related to the spread of COVID-19 in South Korea. Table S3 (supplementary information) provides the province wise detail on the total number of confirmed and active cases along with the recovered and deceased patients. The death toll in South Korea stands at 285 to date with Daegu having the largest share of causalities of 189 deceased.Fig. 4Province wise spread of COVID-19 in South Korea.(*Source*: MOHW, South Korea, As of July 8, 2020 (05:00 GMT))
South Korea's model response and flattening of its COVID-19 curve {#Sec8}
=================================================================
The world is facing an unprecedented economic, social, and political crisis with the spread of COVID-19. Global leaders and experts regard this pandemic as "the gravest challenge since World War II." South Korea too is no exception in this global pandemic. South Korea had a surge of COVID-19 outbreak and virus started spreading at the local and community level since the 31^st^ patient linked to Shincheonji Church was reported. The number of new coronavirus cases increased exponentially peaking at 909 new infections on February 29. But since then, new cases have dropped significantly. South Korea is being applauded worldwide for being able to successfully flatten the curve on COVID-19 in only 20 days without enforcing extreme draconian measures that restrict the freedom and movement of people. To begin with, South Korea adopted its rigorous quarantine measures for incoming passengers from abroad (both symptomatic and asymptomatic). Figure [5](#Fig5){ref-type="fig"} and [6](#Fig6){ref-type="fig"} provide details on the Quarantine Inspection Procedures (QIP) adopted by South Korea for symptomatic and asymptomatic individuals, respectively.Fig. 5Flowchart of quarantine inspection procedures for incoming passengers from abroad (from 00:00, April 1)---Symptomatic.(*Source*: MOHW, South Korea)Fig. 6Flowchart of quarantine inspection procedures for incoming passengers from abroad (from 00:00, April 1)---Asymptomatic.(*Source*: MOHW, South Korea)
One of the key aspects which played a significant role in South Korea's effective response was its use of ICTs and the state-of-art devices and applications including, Self-Isolation Safety Protection App, Self-Diagnosis App and Epidemiological Investigation Support System which were able to tract the routes of confirmed individuals. Mobile devices, CCTV recordings and credit card usage data were used extensively for contact tracing to boost early testing. Advanced ICTs were particularly useful in spreading key emergency information on the novel virus and help to maintain extensive "social distancing." The testing results and latest information on COVID-19 was made available via national and local government websites. Culturally and legally, South Korea is more tolerant of personal data-sharing, and its success has been heavily dependent on its citizens that followed the government guidelines and took responsibility on themselves to self-quarantine in case of suspected illness. The government provided free smartphone apps indicating infection hotspots along with text alerts on testing. Municipalities throughout South Korea made every effort into curbing the contagion as they used the Cellular Broadcasting Service (CBS) to inform the public of the movement paths taken by confirmed patients and other related information. Residents who received COVID-19 emergency texts can quickly check if they have been anywhere that overlaps with the movement of a confirmed patient, allowing them to get tested quickly if necessary. This formed a strong basis for South Korea's early detection and testing capabilities. Figure S2 (supplementary material) shows one such website providing the travel history of the confirmed cases. The image shows the track of COVID-19 patient at a particular location in South Korea using government data available online.
Besides, a walk-thru testing station named K-Walk-Thru testing facilities was adopted by South Korea for the first time in the world to safely and quickly collect the samples of COVID-19 with minimal contact. Since the 2015 outbreak of the Middle East Respiratory Syndrome (MERS), South Korea has learned key aspects, remained vigilant, and continued to refine its response system to better address potential outbreaks of large-scale epidemics, supporting hospitals to set up negative pressure rooms in 2018. The swift actions South Korea has lately taken against COVID-19, including its innovative "drive-thru" testing stations that help minimize the risk of cross infections at the testing centers while maximizing daily testing capacity amid fast rates of new cases, have received international attention and commendations. In addition, the government efficaciously explored various ways to balance the demand and supply of infection control supplies such as hand sanitizers and face masks from the early stage of the outbreak.
Future prospects and conclusion {#Sec9}
===============================
South Korea\'s success with widespread testing and its approach to slowing the spread of COVID-19 offers a way out for most countries that are stuck in lockdown and it also acts as a model response to faces the future outbreak of such diseases as well. From the experience of the MERS outbreak, the country learned the importance of diagnostic testing and basic infection prevention measures, which all came into play during the current pandemic. After the MERS outbreak, when KCDC was not able to trace the movements of the virus, South Korea changed the law allowing the government to collect a patient\'s data and security footage during an outbreak. All their steps are logged and then shared to alert people to stay away from the path of infection and if a confirmed case is found near a person\'s whereabouts, authorities send a text to let the person know if he or she has crossed paths with an infected person. While tracing people\'s every move can be controversial, many in South Korea prioritize public health over privacy in an outbreak. As a result, South Korea was able to test hundreds of thousands of people, more than any other country at the time, and this made it easier for authorities to track and contain the spread of the virus. This ability to find and treat infected people has allowed Korea to avoid aggressive lockdowns and helped to bend the curve of the outbreak that started out dangerously steep. With just 30 cases reported, South Korean Health authorities had already started working with biotech companies to develop a test kit for the novel coronavirus, making available thousands of test kits. Because the government had already equipped hospitals with coronavirus tests, doctors were able to test (patients right away), and contact tracing, in which all people an infected patient has had contact with are traced and tested, helped break the chain of infections and prevent mass outbreaks. At this time, when almost every country is fighting against the same enemy, an invisible micro-pathogen. The success or failure of this fight will naturally be a measure of the overall capacity of a country and will have great implications for the global community and history. Through the key outcome reported in the study it is believed that a small but significant contribution could be made to the policy guidelines for the international community by sharing Korea\'s experiences and countermeasures against COVID-19 over the past six months. Although COVID-19 countermeasures taken by the South Korean government may not be conclusive or universal for all but its exemplary approach to COVID-19 can aid countries across the globe to strengthen their response system for the future outbreak of such contagious disease. For the successful resolution of COVID-19 spread, there is a need for a robust response system powered by the combination of state-of-art techniques and the public's voluntary cooperation along with the sharing of information transparently and promptly in society.
Electronic supplementary material
=================================
{#Sec10}
Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 360 kb)
**Publisher\'s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
No funding or sponsorship was received for this study or publication of this article.
The authors declare that they have no conflict of interest.
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INTRODUCTION {#s1}
============
The terms pseudoaneurysm, false aneurysm and pulsating haematoma all describe the same phenomenon, that is a defect in the vascular wall leading to an extra-vascular haematoma that freely communicates with the intravascular space \[[@RJT069C1]\]. Once in its fully developed state, it may have a cavity with an endothelial lining in continuity with that of the artery \[[@RJT069C2]\]. Isolated internal iliac aneurysms are an uncommon entity and isolated pseudoaneurysms of the internal iliac artery even more so. Here we present an unusual aetiology of massive per-rectal bleeding attributable to pseudoaneurysm of the internal iliac artery, leading to arterio-colic fistula.
CASE REPORT {#s2}
===========
An 83-year-old male presented to the emergency department with a history of passing a cupful of bright red blood with his morning bowel motion. His past medical history was significant for diverticular disease, which had resulted in a perforated sigmoid colon 23 years before, requiring Hartmann\'s procedure which was subsequently reversed.
He arrived with a blood pressure of 130/75 mmHg and a tachycardia of 104 beats per minute. The haemoglobin level was 13.7 g/l on arrival. Rigid sigmoidoscopy in the emergency department revealed an empty rectum with copious amounts of stool and blood up to 10 cm. The patient was presumptively diagnosed with a diverticular bleed and admitted to the general surgical unit for observation. The following day a further significant per-rectal bleed occurred on the ward, associated with chest pain and ECG changes. Blood tests revealed a haemoglobin drop to 11.3 g/l and an elevated serum troponin level of 0.19 μg/l. Myocardial infarction was diagnosed and he was placed on telemetry in a cardiac monitoring unit. A further massive per-rectal bleed was reported on the ward that evening, with a subsequent drop in haemoglobin to 7.7 g/l. Resuscitation was commenced with three units of packed red blood cells and a mesenteric CT angiogram arranged.
An arterial phase CT abdomen with IV contrast was performed. This revealed a contrast blush within the descending colon closely associated with an aneurysm of the internal iliac artery. The possibility of an arterial fistula to the colon was raised, and a vascular surgery consult was sought. Angiography confirmed a false aneurysm arising from the proximal aspect of the left internal iliac artery along its medial and inferior wall. Endovascular coil embolization was performed with occlusion of the vessel at the level of the aneurysm.
No further episodes of per-rectal bleeding have been reported in 3 months following embolization. Follow-up colonoscopy revealed multiple diverticulae in the descending colon, but the site of the previous bleed was not evident.
DISCUSSION {#s3}
==========
This patient had an isolated pseudoaneurysm of the internal iliac artery resulting in an arterio-colic fistula (Fig. [1](#RJT069F1){ref-type="fig"}). Isolated aneurysms of the internal iliac artery themselves are a rare phenomenon, estimated to account for only 0.3--0.4% of all intra-abdominal aneurysms. Forty per cent of patients with isolated internal iliac aneurysms will present with a rupture, with mortality estimated to exceed 50% in such patient groups \[[@RJT069C3]\]. Figure 1:CT angiogram: a saggital view showing pseudoaneurysm of the left internal iliac artery (white arrow).
Pseudoaneurysms are usually secondary to trauma, but may be iatrogenic following intravascular catheterization. They have also been reported as a rare complication of pelvic surgery \[[@RJT069C4]\]. Over the last few decades there has been an increase in the incidence of pseudoaneurysms in general, due to an increase in interventional vascular access \[[@RJT069C5]\]. Infections, connective tissue disorders, vasculitis and erosion secondary to malignancy may also be implicated in the formation of pseudoaneurysms \[[@RJT069C6]\].
In a review of the literature on isolated internal iliac aneurysms, Dix *et al*. \[[@RJT069C7]\] found ∼6% of patients with such aneurysms will present with rectal bleeding. Cases described have included iliac-rectal fistula following coil occlusion of an iliac aneurysm and fistulous connection between aneurysm and caecal carcinoma \[[@RJT069C8]\].
The aetiology of this patient\'s pseudoaneurysm is unclear. Diverticulitis in and of itself is not known as a cause for pseudoaneurysm formation; however, pelvic infections have been implicated. This patient had an episode of diverticular disease 23 years previously which required a Hartman\'s procedure, this was reversed 6months later. Whether the cause of the pseudoaneurysm was mycotic or traumatic (secondary to either the first or second stage procedure) is a matter of conjecture. Anastamotic leak post-colostomy reversal could have been implicated, however, on imaging the pseudoaneurysm communicates with a large bowel ∼5 cm proximal to the anastamotic suture line.
Regardless of the aetiology, if the pseudoaneurysm occurred around the time of his original operation, it remained quiescent for 23 years before causing symptoms. The reported mortality rate for ruptured internal iliac aneurysms is ∼50%---fortunately in this case the diagnosis was made rapidly with imaging and immediate treatment led to a good outcome. The efficacy of expedient angiography in per-rectal bleeding has been shown previously, with mesenteric embolization clinically successful in 76% of cases of lower gastrointestinal bleeding with active bleeding on angiography \[[@RJT069C9]\]. Aggressive management of internal iliac aneurysms, regardless of the mode of presentation, is required to avoid the high morbidity and mortality associated with rupture. They can be managed with open or endovascular techniques, with a tendency towards the latter over the previous years. Occlusion of the internal iliac artery can result in multiple sequelae, including lumbosacral plexus ischaemia, buttock necrosis, colorectal infarction, impotence and buttock claudication. Buttock claudication, the most common of these complications, is estimated to occur in 28--40% of patients, with severe debilitating claudication in 9% \[[@RJT069C10]\]. At follow-up, no symptomology suggestive of claudication was elicited in our patient.
Per-rectal bleeding is a common presentation with most cases of massive bleeding caused by diverticular disease and angiodysplasia. The above case will hopefully serve as a reminder that per-rectal bleeding can have a less common aetiology. Identification of an iliac aneurysm on imaging for rectal bleeding should alert the clinician to the possibility of an arterial fistula to the colon.
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Introduction {#sec1}
============
Health care providers are coming under greater financial pressure, especially from more value-based and accountable payment methods. To cope, they need to understand their costs accurately over the full course of treatment by medical condition. Previous efforts to understand cost variation across providers have used measurement approaches that did not reflect the actual cost of the people and equipment used to deliver care. For example, studies of abdominal and congenital heart surgery [@bib1], [@bib2] used the ratio of cost-to-charges (RCC) method, a widely-used but inaccurate health care costing method. The RCC method assumes that the cost to perform each service is the same percentage of the charge for each service. For instance, if total costs in a unit are \$1.2 million and total charges are \$2 million, the RCC method estimates the cost to perform each service as 60% of the service\'s charge. While the RCC approach is simple to understand and easy to implement, it is inaccurate because the charges have not been established based on actual costs of resources used to deliver each service.
We applied time-driven activity-based costing (TDABC) to study the cost variation for performing total joint arthroplasties (TJAs) at 29 high-volume hospitals. TDABC, a bottoms-up approach, uses process mapping to identify the types and costs of all personnel utilized in each step of a complete cycle of care [@bib3], [@bib4], [@bib5], [@bib6]. TDABC has previously been used to examine the costs of TJAs at individual sites [@bib7], [@bib8].
TJAs are one of the most commonly performed major surgeries in the country. Currently, more than 1 M joint arthroplasties are performed per year in the United States, and the number is projected to reach 4.05 M per year by 2030 [@bib9], [@bib10]. Also, motivating this study is the increased use of bundled payments for joint arthroplasties, which causes hospitals to become more financially accountable for their costs of care. The Centers for Medicare and Medicaid Services introduced bundled payments in 2013 through a Bundled Payments for Care Improvement initiative, comprised of 4 models of bundled payments across a range of medical and surgical conditions, including TJAs. In April 2016, Centers for Medicare and Medicaid Services introduced the Comprehensive Care for Joint Replacements model for primary TJAs. This payment initiative holds hospitals financially accountable for all related Medicare Part A and B expenditures from the time of hospital admission through 90 days post discharge [@bib11]. Beyond these public payer programs, a national alliance of leading providers has been established to offer orthopaedic surgeries at a fixed bundled price directly to large employers and employer groups [@bib12].
The present study brought together 29 US-based sites to facilitate the measurement of their direct costs of providing a total knee arthroplasty (TKA) and help them to understand the primary drivers of cost variation across providers. Overall, Medicare data indicate that 95%-97% of hospitals were not statistically different from one another on risk-adjusted complication and readmission rates for TKAs ([Table 1](#tbl1){ref-type="table"}) [@bib13]. While these quality measures are not comprehensive, the little variation among them indicates large opportunities to reduce the variation in costs of TKAs without adversely impacting outcomes. Previous research has documented wide variation in reimbursement rates and hospital and physician charges [@bib14], [@bib15]. A recent study showed a 380% variation in reimbursement rates across the country for TKAs, including as much as a 2.67× difference within a single geographic market [@bib16]. These studies, however, have not compared providers\' actual costs for delivering TKAs over an entire cycle of care.
Material and methods {#sec2}
====================
The article authors and the Institute of Healthcare Improvement organized a Joint Replacement Learning Community during calendar year 2014 to measure the value and improve the performance of the participating hospitals ("sites"). Hospitals that performed at least 200 primary TJAs in the prior year were eligible to participate. Thirty-two hospitals enrolled in the program, 29 were from the United States. The participating hospitals performed an average of 800 TJAs in 2013.
Nine of the US-based hospitals were academic medical centers; 20 were community hospitals. Their TJA patients had similar average age (65 years) and body mass index of 31 [@bib17]. We excluded cases with major complicating conditions by studying only cases equivalent to a Medicare Severity-Diagnosis Related Group (MS-DRG) of 470. Twenty-four of the providers had risk-adjusted readmission and complications rates for TJAs that were no different than average based on Medicare Hospital Compare data. Two organizations had rates that were statistically better than average, and 3 organizations were worse on one or both outcome dimensions [@bib13].
The analysis included costs over a care cycle that started with the initial office visit at which the decision for surgery was made and concluded 90 days post discharge. It included the costs of care delivered by physicians, whether employed at the hospital or not, and for postacute care, such as for rehabilitation and therapy. Except for the postacute care costs, the analysis focused on direct personnel and consumable supply costs. We excluded space and equipment costs since our previous research projects found these costs to be less than 10% of personnel and supply costs [@bib18]. We also excluded indirect costs, such as information technology, human resources, and billing, as assigning these costs accurately would have required extensive cost modeling and analysis for every indirect and overhead cost category in the hospital.
We used TDABC to measure the direct personnel costs and trained the sites on the approach [@bib4], [@bib5], [@bib6]. TDABC involves identifying the clinical and administrative activities performed over the care cycle, including the types of clinicians and staff members that perform each activity and the amount of time each personnel type spends on each activity. The second step in the TDABC process calculates the cost per minute for each of the personnel types used in the care cycle. The cost per minute divides the fully loaded cost for each type of personnel (compensation plus benefits) by the quantity of time that each personnel type has available for performing productive work per year on average. The employee activity time is then multiplied by the employee\'s cost per minute and summed across all employees to calculate total personnel costs. For example, if a nurse spends 20 minutes with a patient during an office visit (inclusive of prep and follow-up time), and the fully loaded cost for that nurse is \$1.50/minute, then the cost of nursing time for the visit is \$30. The cost of all of the resources utilized as part of a particular service (eg, a clinic visit, a rehabilitation session, or a surgical intervention) can then be summed together to determine the total cost of that service [@bib3].
The organizations used actual purchase prices, the price the organization paid to the manufacturer or distributor, as the cost for consumable supplies. For postacute care costs, the organizations used internal cost estimates when they provided the service and external claims data when internal cost data were not available.
The unit of analysis for the study was the average cost to deliver the care for primary TKAs at each site. The project teams reported their cost data in a common format with a web-based system, developed by the study authors, which then calculated the TDABC costs for each site. The authors analyzed the consolidated data to compare costs across sites for the complete care cycle, as well as for important segments within the care cycle, such as preoperative, day of surgery, postacute care, and for different categories of expense, principally personnel and purchased supplies. A unique capability of the TDABC costing approach enabled the authors to use accounting variance analysis to adjust for differences in labor cost rates across the sites [@bib19]. The authors could also identify the variation in postacute care spending caused by differences in payments for different types of postacute care services (eg, home health care vs skilled nursing facility) across sites.
The sites had the option to either report costs for the site on average or to provide a breakdown of costs based on the practices of individual physicians. In the latter situation, we used an average cost across physicians for those sites.
The authors performed a separate analysis of the costs of the prosthetic implant, the largest single cost component for TKAs [@bib20]. The sites replied to a survey asking them to report the average purchase price for prosthetic implants used in primary TKA procedures for DRG 470 over the 12 months ending September 30, 2013. They reported their average purchase price with in \$500 ranges. For statistical analysis, we used the midpoint of each price point range [@bib21].
Results {#sec3}
=======
[Table 2](#tbl2){ref-type="table"} summarizes the variation in costs across the 29 sites. Due to nondisclosure agreements with the participating hospitals, we show the cost comparisons in indexed form, rather than the actual cost data. We report the median cost in each cost comparison as 1.00; values greater than 1 represent higher costs and values below 1 represent lower costs. Higher percentiles represent higher costs (eg, for a site at the 90th percentile, 90% of sites had costs lower than it). The total personnel and consumable supply costs for the site at the 90th total cost percentile were 1.6× (60%) greater than the costs of the site at the 10th percentile. A site at the 90th percentile for total personnel and supply costs that improved to the 75th percentile would save 12%; a site at the 75th percentile that improved to the median would save 8%; a site at the median that improved to the 25th percentile would save 12%; and a site at the 25th percentile that improved to the 10th percentile would save 13%.
On average, consumables represented 59% and personnel 41% of personnel and supply costs. There was a 2.3× range in consumable costs between the 90th and 10th percentiles. Prosthetic implants, the largest consumables expense, had a 2.1× range between the 90th and 10th percentiles [@bib20]. Other types of consumables had a much larger range. The cost of bone cement, for example, was 17× higher for the 90th percentile site than for the site at the 10th percentile. While we are not able to quantitatively explain all the variation in bone cement costs, anecdotally, the variation was caused by having different purchase prices for the same type of cement, using different types of cement (eg, premixed antibiotic cement is more expensive than hand-mixed or plain bone cement) and using different quantities of cement during the actual procedure.
Total personnel costs had a 2.3× range between the 90th and 10th percentiles, for example, the site at the 90th percentile spent 130% more on personnel than the site at the 10th percentile. Some of the cost variation across the hospitals could be attributed to the different compensation paid by each site to similar types of comparable employees (called a price variance by accountants). We controlled for this source of variation by also calculating costs using an average wage and benefit rate for each personnel type at all sites, for example, if we were doing a comparison across only 2 sites (instead of the 29 actually used) and site 1\'s nurses cost \$1/minute while site 2\'s cost \$1.5/minute, we would use an average rate of \$1.25/minute for the cost of nursing time at each site when calculating standardized personnel costs. Total personnel costs after standardization still had a 1.9× difference between the 90th and 10th percentiles. This variation, called a quantity or productivity variance, is exclusively due to differences in the mix of personnel utilized and the quantity of time each type spent over the episode of care, factors that are much easier to change than local, and site-specific compensation rates.
We analyzed personnel cost variation at each stage of the care cycle: before day of surgery, day of surgery before the operation, surgical operation, postanesthesia care unit stay, inpatient stay, and postoperative orthopaedic follow-up visits. The greatest variation in personnel costs occurred during the postoperative follow-up visits, with a 5.9× range in costs from the 90th to the 10th percentiles. Since most sites had 2 follow-up visits within 90 days of discharge from the operation, the variation in follow-up visit personnel costs was driven by which employees saw patients and the time each spent with them during each follow-up visit, rather than by the number of visits.
The inpatient floor hospitalization costs were driven primarily by nursing costs. After standardizing for differences in wage rates among sites, the 2 largest drivers of nursing expenses were the length of the patient\'s hospital stay and the patient-to-nurse staffing ratio (ie, how many patients each nurse was expected to care for).
Cost estimates for patients discharged to the various types of postacute care (inpatient rehabilitation, skilled nursing facility, home with home health care, and home with outpatient therapy) varied by at least 6× between the 90th and 10th percentiles. We again used variance analysis by calculating an average cost for each type of postacute care. This enabled us to focus our analysis on the mix of postacute services used across hospitals. A 2.2× variation between the 90th and 10th percentiles for postacute care costs still remained. The variation was due to variation in discharge disposition (the type of setting patients were discharged to) and readmission rates, with most of the variation due to discharge disposition. We found no correlation between performing statistically better or worse than average on the Medicare Hospital Compare metrics and standardized personnel costs. We also found no correlation between standardized personnel costs up through discharge and standardized postacute care costs.
Discussion {#sec4}
==========
Personnel costs were highest for the surgical operation and the inpatient hospitalization stay. The primary drivers of surgical operation costs were the duration of the operation, including time spent prepping and cleaning the room, and the mix of staff members used at each stage of the operation. Surgeons are by far the most expensive personnel type. Surgeons at the sites with the lowest surgical personnel costs per patient (after standardizing for differences in labor cost rates across sites, which varied by over 60%) had shorter surgery times either because they were faster or, more commonly, were present for fewer process steps of the surgery. At the low surgical personnel cost sites, the surgeon\'s time lasted only from first incision to the time closure began, at which point the surgeon left to prepare for the next case in a second operating room. Lower cost (but still qualified) staff members performed the remaining functions, including closure. From a productivity perspective, most of the surgeons who performed at least 300 TJAs per year had access to two operating rooms, half the surgeons who performed between 100 and 300 cases per year used 2 operating rooms, while all of the surgeons who performed under 100 cases per year had access to only 1 operating room for their procedures.
The cost of a surgeon with access to only 1 operating room is much higher, as the surgeon\'s time includes the actual operating time plus the time spent waiting for the current case to be finished, for the room to be cleaned and prepared, and for the next patient to be transferred to the room. A conflict can therefore arise between hospitals that want high utilization of their expensive operating rooms and surgeons who want high utilization of their time. Accurate cost accounting helps to resolve this conflict. Representative numbers based on our prior (unpublished) work have shown that, on average, a fully equipped operating room costs about \$0.50 per minute of the available time. A fully staffed surgical team, including the surgeon, costs \$10-\$20 per minute. Even excluding the surgeon from the analysis, the cost per minute for the rest of the operating room team is typically above \$5 per minute, an order of magnitude higher than the space cost. A hospital striving to get maximum utilization of its most expensive resources should learn how to keep its skilled surgical teams as productive as possible.
The other principal source of variation in day of surgery costs was the 2.1 to 1 variation (90th to 10th percentiles) due to implant purchase prices. A statistical analysis revealed that the most statistically and economically significant variable to explain this large variation was whether a joint committee of hospital administrators and surgeons made the purchase decision and negotiated with vendors [@bib20]. Sites that used such a joint committee paid, on average, 17% less for implants than those without a joint purchasing committee. Volume played a statistically significant but small-in-magnitude role (3% reduction per 100 incremental surgeries).
The cost of bone cement also varied widely across sites, largely caused by variation in the type of cement used. For example, premixed antibiotic cement is much more expensive than other types. Research has shown, however, that hospitals do not necessarily need to purchase more expensive bone cement. A recent recommendation made by the American Academy of Orthopaedic Surgeons noted that in most cases, antibiotic cement is unnecessary, providing an actionable way for organizations to reduce costs quickly without compromising patient outcomes [@bib22].
Postacute care was also costly. [Table 3](#tbl3){ref-type="table"} shows that the major explanation of the variation in costs of postacute care arises from the discharge disposition for the patient. Sites in the lowest quartile of standardized postacute costs discharged 86% of their patients to their homes. Sites in the highest quartile discharged less than 50% of their patients to home. Home rehabilitation costs were 20% of the costs of skilled nursing facilities and 15% of the costs for extended inpatient facilities. There was no systematic relationship across sites between the average patient age and the standardized postacute care costs at each site. Several of the low postacute cost care sites reported that they set an expectation for discharge to home rehabilitation in presurgical visits with patients and their families and subsequently reinforced this communication during the hospital stay. During the hospital stay, the sites also designed their pain management and physical therapy to increase a patient\'s comfort level with being discharged to home. For example, one site used a prototype vehicle for the patient to practice entering and leaving and a 12-step staircase with rails on either side to practice going up and down stairs. While the extra time spent talking with patients and their families and the specialized therapy approaches did involve higher costs, these extra costs were less than \$100 per patient, a savings of thousands of dollars when compared to discharging patients to skilled nursing and specialized rehab facilities. Sites that started physical therapy earlier in the postsurgical stage also discharged a higher percentage of patients to home health care.
Our data and methods are subject to a number of limitations. First is the nonrandomized sample of hospitals in the analysis. The 29 hospitals in the study each performed at least 200 primary TJAs per year, and all chose to participate in the Joint Replacement Learning Community program in 2014. A second limitation is that each hospital self-reported the data. While we continually questioned and challenged the hospitals to confirm that the reported data were correct, we were not able to independently verify the data ourselves. It is thus possible that some of the variation in cost is due to differences in how well and how accurately the hospitals captured information used to calculate the TDABC costs at their institution. A third limitation is that while hospitals had data on the discharge disposition of the patient, they did not have comprehensive data on the postacute care costs for the patients. The variation in postacute care costs is therefore based on the average cost for the discharge disposition used for patients, not the actual full cost for the 90 days post discharge. A fourth limitation, as noted in the [Material and methods](#sec2){ref-type="sec"} section, is that we did not include space and equipment costs in the analysis based on our prior experience that these do not typically represent a substantial cost on a per patient basis. It is possible though that varying use of advanced technology, such as computer navigation, advanced imaging, robotics, and custom jigs contributes to variation in cost across hospitals.
Conclusions {#sec5}
===========
Hospitals with similar patient demographics and similar patient volumes for TKAs have wide variation in their costs over the TKA care cycle. This large variation suggests a great opportunity to lower costs while maintaining quality and patient outcomes by identifying and transferring the best practices of sites in the lowest percentiles of cost. These best practices improve the efficiency of care and productivity of employees and facilities, enabling them to handle much of the expected growth in TKA volume without adding costly new capacity. Several sites in the program made immediate and significant improvements based on what they learned from the analysis, such as optimizing the discharge disposition of the patients from the hospital and changing pain management to enable physical therapy to start earlier. Looking forward, policies such as bundled payments will lead to greater price (payment rate) pressure on hospitals, increasing the benefits from proactive management of actual costs over complete episodes of care. TDABC provides both accurate estimates of care cycle costs, as well as greater transparency into the drivers of those costs.
With almost all hospitals publically reporting similar outcomes on complication and readmission rates despite large variation in costs, such efficiency and productivity improvements should be possible while maintaining or improving outcomes. Further efforts, however, should strive to encourage hospitals and clinicians to adopt and report condition-specific patient-reported outcome metrics, which would allow a better comparison and improvement of quality and costs across providers.
Appendix A. Supplementary data {#appsec1}
==============================
Conflict of Interest Statement for HaasConflict of Interest Statement for HaasConflict of Interest Statement for KaplanConflict of Interest Statement for Kaplan
The authors acknowledge Dr. Anthony DiGioia of Magee-Womens Hospital of UPMC, Dr. Kevin Bozic of Dell Medical School, as well as Katherine Luther, Jill Duncan, Kayla DeVincentis, and Kevin Little from the Institute for Healthcare Improvement (IHI) for their significant contributions during the Joint Replacement Learning Community, which was the source for the data analyzed in the article. The authors would also like to acknowledge Lila Kelso for her valuable support in preparing the article for publication. The authors received compensation from IHI for their work during the JRLC.
One or more of the authors of this paper have disclosed potential or pertinent conflicts of interest, which may include receipt of payment, either direct or indirect, institutional support, or association with an entity in the biomedical field which may be perceived to have potential conflict of interest with this work. For full disclosure statements refer to [http://dx.doi.org/10.1016/j.artd.2016.08.001](10.1016/j.artd.2016.08.001){#intref0010}.
######
Medicare risk-adjusted complication and readmission rates [@bib10].
Table 1
Comparison Risk-adjusted complication rate (%) Risk-adjusted readmission rate (%)
------------------------------------ ------------------------------------- ------------------------------------
Better than US national rate 3 2
No different than US national rate 95 97
Worse than US national rate 2 1
Source/notes: Hospital Compare data. 2015 \[cited 2015 April 12\]. Available from:
https://data.medicare.gov/
######
Distribution in indexed costs for primary total knee arthroplasties.
Table 2
Measure Higher percentile is a higher cost. Unit of analysis is a hospital and affiliated surgeons.
--------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------- ------ ------ ------ ------ ------
Total personnel and consumable supply costs for hospital stay and orthopaedic office visits 0.77 0.88 1.00 1.09 1.24 1.6
Total consumable supply costs 0.62 0.90 1.00 1.08 1.41 2.3
Prosthetic implant cost 0.58 0.79 1.00 1.11 1.23 2.1
Bone cement costs 0.22 0.52 1.00 1.68 3.81 17.4
Total personnel costs 0.59 0.77 1.00 1.13 1.37 2.3
Total standardized personnel costs 0.68 0.82 1.00 1.14 1.31 1.9
Standardized personnel costs before day of surgery 0.57 0.84 1.00 1.62 2.10 3.7
Standardized personnel costs day of surgery before surgery 0.59 0.69 1.00 1.48 1.85 3.1
Standardized personnel costs during surgery 0.61 0.78 1.00 1.33 1.64 2.7
Standardized personnel costs in the postanesthesia care unit (PACU) 0.59 0.85 1.00 1.38 1.63 2.8
Standardized personnel costs during inpatient stay 0.74 0.84 1.00 1.14 1.35 1.8
Standardized personnel costs during postdischarge follow-up visits 0.34 0.53 1.00 1.43 2.00 5.9
Readmission cost 0.53 0.73 1.00 1.36 1.58 3.0
Standardized postacute care costs 0.74 0.88 1.00 1.22 1.63 2.2
Source/notes: Data from 29 JRLC partner sites.
######
Quartiles of postacute care spend and discharge disposition.
Table 3
Quartiles of postacute care spend Home with outpatient (%) Home health (%) Inpatient rehabilitation (%) Skilled nursing facility (%) Readmissions (%)
----------------------------------- -------------------------- ----------------- ------------------------------ ------------------------------ ------------------
Highest 4 42 24 29 3
2nd highest 17 50 4 29 3
2nd lowest 23 57 7 13 3
Lowest 44 42 4 10 2
Source/notes: Data from 29 JRLC partner sites.
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Introduction {#s0001}
============
Medical reasons for an ultrasound scan during early pregnancy are to estimate the date of delivery, diagnose multiple pregnancies, detect aberrations in fetal anatomy, identify the location of the placenta, and measure the quantity of amniotic fluid ([@C1]). With more sophisticated ultrasound techniques it has become possible to identify fetal sex during an ultrasound scan with high accuracy ([@C2],[@C3]). Clients' reasons have been shown to be somewhat different---to confirm fetal viability, to become informed of any fetal abnormalities, and an opportunity to get to know the child's sex ([@C4]). In Sweden all pregnant women are offered a routine ultrasound scan in pregnancy week 15--22 that is free of charge, and almost all (97%) attend this examination ([@C7]); 80% of these ultrasound scans are carried out by registered nurse-midwives who are specially trained in the obstetric ultrasound technology.
Internationally, many parents wish to know the sex of the expected child ([@C8]). According to Shipp et al. ([@C8]), 58% of pregnant women in a US study desired to find out the sex prenatally, while studies from Nigeria show that over 90% wanted to know the sex ([@C11],[@C12]). There are several reasons for this wish: to plan for the baby's arrival, acquire appropriate things for the child, personal and partner's curiosity, and to make sure sex determination at birth is correct ([@C12],[@C14],[@C15]).
Few studies from the Nordic countries have investigated prenatal sex determination or sex selection. However, in a Swedish study the majority of pregnant women considered it unimportant to know the sex, yet 57% of study participants chose to find out ([@C16]). In another study ([@C17]), 13% of women and 17% of men believed that a reason to do an ultrasound was to reveal the sex of the fetus. According to the National Board of Health and Welfare, prenatal testing for sex determination should not be offered without medical indication, and the sex of a fetus should be disclosed only if the woman requests it ([@C18]).
Prenatal attachment is a theory about the relationship between expecting parents and their unborn children, and the parents' behavior in relation to the fetus ([@C19]). Prenatal attachment can be divided into dimensions that differ depending on the trimester of the pregnancy. Prenatal attachment plays a major role during pregnancy, and in the postnatal period it affects the mothers' ability to give good child care ([@C20]). The ultrasound examination may contribute to the prenatal attachment ([@C20]). One study shows that health-care professionals consider sex determination as a key part of the attachment to the fetus ([@C23]).
Factors associated with the desire to know the sex of the baby were: unplanned pregnancy, previous experience of knowing the sex of an expected child, not planning to breastfeed, low socioeconomic status, being unmarried ([@C9],[@C10]), being younger than 22 or older than 40 years ([@C8],[@C9]), and considering that the sex of the baby affects family planning. Women who had a perfectionist view of parenting were more likely to have found out the baby's sex ([@C10]). It was more common to have prenatal sex determination among women with prior deliveries compared to first-time mothers ([@C8],[@C24]), and those who previously had a boy and a girl more often chose to find out the sex ([@C8]). Shipp et al. ([@C8]) showed that couples who had chosen prenatal sex determination agreed on the decision.
Sex determination in early pregnancy has been feared to be a risk for selective abortion ([@C25]), especially in countries with a son-preference such as China and India ([@C24],[@C26]). People with low education, low socioeconomic status, and women who already have children are more likely to prefer a certain sex of the expected child. In Western countries preference for boys or girls is not as clear, and sex selection would probably not affect the ratio between the percentage of girls and boys ([@C27]). In the US, the UK, and Germany, people have more negative attitudes toward sex selection compared to China and India ([@C28]).
No recent Swedish study has investigated the wish to find out the sex of the fetus at the routine ultrasound scan. The main aim of the present study was to investigate if pregnant women and their partners wanted to know the sex of the expected child, if this had been discussed with the midwife during the examination, and if there were any differences related to age, education, having previous children, planned/unplanned pregnancy, and importance of religion. Another aim was also to explore any interest in sex selection. We hypothesized that women with a very planned pregnancy would be more interested in knowing the sex of the fetus compared to women with less planned pregnancies.
Method {#s0002}
======
This cross-sectional study is based on selected items from a Swedish longitudinal study on health and lifestyle before, during, and after pregnancy---The Swedish Pregnancy Planning Study (SWEPP). The study procedure has been described by Stern et al. ([@C29]) and Bodin et al. ([@C30]). A total of 215 antenatal clinics were invited to participate in the data collection. Of these, 71% (*n* = 153) accepted, and recruitment took place between September 2012 and July 2013.
Participants gave their written consent, and the midwives kept logs over the enrollment process. Women answered three questionnaires; the first (Q1) in early pregnancy, the second (Q2) in late pregnancy, and the third (Q3) one year post partum. Variables regarding background and level of pregnancy planning were retrieved from Q1, and items regarding the desire to find out the fetal sex were collected in late pregnancy (Q2).
In total 5796 women visited the clinics during the study period. Of these, 303 were not invited to participate in the study for various practical reasons. Thus, 5493 women were invited, and 62% (*n* = 3389) participated. We sent a postal questionnaire (Q2) to 3215 women who had responded to Q1 and had revealed their contact details, and of these 77% (*n* = 2583) returned Q2. An analysis of those who dropped out showed no differences with respect to age, education, religion, or previous children. In total, 2405 of the women responded that they were still pregnant, and, among those, 2393 had attended the routine ultrasound scan in early pregnancy. These women thus formed the sample for the present study.
The questionnaire consisted of 148 questions, most of which were multiple choice questions. The questionnaire was designed by researchers and clinicians, and some items were adjusted after a pilot study ([@C31]).
Questions regarding the demographic background used in this study covered the woman's age, origin, highest completed education, monthly income, number of previous children, and the importance of religion. Pregnancy planning in this study refers to whether the pregnancy was planned before conception and was measured with the 5-grade Swedish Pregnancy Planning Scale (SPPS): very planned, fairly planned, neither planned nor unplanned, fairly unplanned, and very unplanned. This SPPS has been used in previous studies ([@C29],[@C30],[@C32]).
Items presented and analyzed in the present paper were phrased as follows: From where did you and/or partner look for information about pregnancy? (Eleven pre-defined options/One open option/Have not looked for any information)Before the ultrasound examination, did you and your partner discuss if you wanted to know the child's sex? (Yes/No)Did you want to know the child's sex? (Yes/No)Did your partner want to know the child's sex? (Yes/No)Did you and the midwife discuss the opportunity to identify the child's sex? (Yes, on my/my partner's initiative/Yes on the midwife's initiative/No/Do not remember)Do you know the child's sex today? (Yes, a boy/Yes, a girl/No, did not want to know/No, was not allowed to know)If it was possible, would you take advantage of the possibility to select the sex of your baby? (Yes/No/Do not know)Did you go through any other fetal diagnostic method in addition to the routine ultrasound? (Yes/No)
The regional ethical review board in Uppsala, Sweden, approved the study (Dnr: 2010/085).
Data analysis {#s0003}
-------------
The participants were divided into three age-groups: ≤24 years, 25--34 years, and ≥35 years. The variable regarding importance of religion had five response alternatives, which were collapsed into three: great importance/indifferent/little importance. The highest completed education was collapsed into low/high education, and the monthly income was collapsed into low/high income.
To analyze differences between those who wanted to find out the sex of the fetus and those who did not, and between those potentially interested in sex selection and those who were not, we used the chi-square test and Fisher's exact test for nominally and ordinally scaled variables. The *t* test was used to analyze any age difference. A *p* value of *p* \< 0.05 was chosen.
Results {#s0004}
=======
A majority of the participants belonged to the age-group 25--34 years (mean age 29.4 years), and more than 90% had a Nordic origin. Almost half (48%) had a university education, and 54.7% already had one or more children. Most pregnancies (75.5%) were very or fairly planned. A minority (11.5%) stated that religion was of great importance to them ([Table 1](#TB1){ref-type="table"}).
######
Characteristics of the respondents (*n* = 2393). Missing values excluded from the analysis.
*n* \%
-------------------------------------------------- ------------------- ------
Mean age (range), years^a^ 29.4 (17**--**47)
Age-group
≤24 years 377 16.1
25--34 years 1625 69.4
≥35 years 341 14.6
Origin
Nordic 2203 92.2
Non-Nordic 186 7.8
Income
High 1110 47.5
Low 1227 52.5
Previous children
No previous children 1085 45.3
Has previous children 1308 54.7
Gone through another method of fetal diagnostics 984 41.1
Education
Primary school (9 years) 128 5.4
Secondary school (12 years) 927 39
Vocational education 181 7.6
University \<2.5 years 108 4.5
University \>2.5 years 1035 43.5
Pregnancy planning
Very planned 1126 47.5
Fairly planned 665 28.0
Netiher planned nor unplanned 319 13.4
Fairly unplanned 77 3.2
Very unplanned 186 7.8
Importance of religion
Great importance 273 11.5
Indifferent 530 22.3
Little importance 1575 66.2
Age range: Minimum age of respondents was 17 years and maximum age was 47 years.
Most women had searched for information in early pregnancy, and the most cited sources of information were different Internet fora, followed by the antenatal clinic ([Table 2](#TB2){ref-type="table"}). Before the routine ultrasound scan 95.8% (*n* = 2289) of the women had discussed with their partner whether they wanted to know the sex of the fetus, and an equal number of women (56.9%, *n* = 1356) and partners (56.9%, *n* = 1359) wanted to find out their baby's sex. After the ultrasound scan, 48.1% (*n* = 1152) responded that they did not know the sex of the expected child ([Table 3](#TB3){ref-type="table"}).
######
Sources of information used by the participants in early pregnancy (*n* = 2393). Missing values excluded from the analysis.
Sources of information[^a^](#TF2){ref-type="table-fn"} *n* (%)
-------------------------------------------------------- ------------
Internet fora targeting expectant parents 967 (40.4)
Internet site for health-care information 679 (28.4)
Antenatal clinic 613 (25.6)
Family and friends 510 (21.3)
The National Food Agency 416 (17.4)
Mobile applications 466 (19.5)
Other Internet sources 316 (13.2)
Books 244 (10.2)
Newspapers 223 (9.3)
Blogs 176 (7.4)
Other health services 66 (2.8)
National Public Health Agency 18 (0.8)
Did not look for any information 846 (35.4)
Several alternatives could be chosen.
######
Interest in sex determination and sex selection (*n* = 2393). Missing values excluded from the analysis.
Item *n* (%)
---------------------------------------------------------------------------------------- -------------
Discussed if they wanted to know the sex of the baby before the ultrasound examination 2289 (95.8)
Pregnant woman wanted to know the sex of the baby 1356 (56.9)
Partner wanted to know the sex of the baby 1359 (56.9)
Discussed sex determination with the midwife
** **On own/partner's initiative 1088 (45.5)
** **On the midwife's initiative 229 (9.6)
Knows the sex of the baby 1241 (51.9)
Interested in sex selection 118 (4.9)
During the ultrasound scan 45.5% (*n* = 1088) of the couples and 9.6% (*n* = 229) of the midwives initiated a discussion about sex determination. Four out of 10 (39.5%, *n* = 943) did not discuss this, and 5.4% (*n* = 130) did not remember whether or not the possibility of sex determination was discussed. Most women (84%, *n* = 2007) responded that they would not use the possibility to select the sex of a baby if it was possible; 5% expressed such an interest, and 11% were unsure.
Women who wanted to find out the sex of the baby did not differ from those who did not, in any of the analyzed characteristics---age, origin, highest completed education, monthly income, previous children, importance of religion, or level of pregnancy planning ([Table 4](#TB4){ref-type="table"}). Neither did women potentially interested in sex selection differ from women who were not, in any of the above-mentioned variables (data not shown). However, women who also had gone through another method of fetal diagnostics were more interested in finding out the sex of the fetus (*p* \< 0.01) and in a potential possibility to select the sex of a child (6.2% versus 4.1%, *p* \< 0.01).
######
Comparisons between women who wanted to know the sex of the baby versus those who did not (*n* = 2393). Missing values excluded from the analysis.
Item Wanted to know the sex of the baby Did not want to know the sex of the baby *p* value
-------------------------------------------------- ------------------------------------ ------------------------------------------ -----------
Mean age, years 29.4 29.4 0.80
*n* (%) *n* (%)
Age groups 0.99
** **17--24 years 214 (16.1) 160 (15.9)
** **25--34 years 923 (69.4) 697 (69.4)
** **35--47 years 193 (14.5) 148 (14.6)
Origin 0.45
** **Nordic 1245 (92.1) 950 (92.3)
** **Non-Nordic 107 (7.9) 79 (7.7)
Education 0.32
** **Low 584 (43.3) 465 (45.5)
** **High 764 (56.7) 558 (54.5)
Previous children 0.43
** **No previous childen 624 (46.0) 456 (44.3)
** **Has previous children 732 (54.0) 573 (55.7)
Income 0.53
** **Low 620 (46.9) 485 (48.2)
** **High 703 (53.1) 521 (51.8)
Pregnancy planning 0.34
** **Very/fairly planned 1004 (74.7) 781 (76.4)
** **Neither planned nor unplanned 180 (13.4) 139 (13.6)
** **Very/fairly unplanned 160 (11.9) 102 (10.0)
Gone through another method of fetal diagnostics 594 (43.8) 388 (37.7) \<0.001
Importance of religion 0.44
** **Great importance 146 (10.9) 127 (12.4)
** **Neither important nor unimportant 296 (22.0) 231 (22.5)
** **Little importance 903 (67.1) 667 (65.1)
Discussion {#s0005}
==========
An interesting finding was that almost all women (96%) had discussed with their partner before attending the ultrasound scan whether they wanted to find out the fetal sex, and slightly more than half of the women (57%) and a similar proportion of their partners desired to find out the fetal sex. In almost half of the cases it was the couple who brought up the possibility of sex determination with the midwife. Equally interesting in an international perspective is the fact that almost half of the respondents had not, or did not remember if they had, discussed this issue during the ultrasound examination.
The proportion of women who wanted to find out the fetal sex during routine ultrasound is in line with Sjögren ([@C16]). This suggests that the desire to know the sex in Sweden is as common today as it was 29 years ago, even though the ultrasound technique has developed over the years and private clinics offering this service are available at least in some cities. The proportion of women who wished to know the child's sex is also consistent with what Shipp et al. ([@C8]) showed in the study from the US. It was, however, less common compared to the studies from Nigeria ([@C11],[@C12]). This may be due to a greater similarity between Sweden and the US compared with Nigeria. The sampling procedure may also have been different.
The proportion of partners who wanted to know the child's sex during routine ultrasound was the same as the proportion of the pregnant women, which indicates a common decision-making, which also a previous study noted ([@C8]). Almost all had discussed with their partner about sex determination before the routine ultrasound scan. This demonstrates that expecting parents in Sweden are aware of the possibility to identify the sex of the expected child during the ultrasound scan. It also indicates that a pregnancy is regarded as a joint project, which was also shown by Bodin and co-workers ([@C30],[@C33]).
Most discussions about sex determination were initiated by the couple, but in some cases the midwife raised the issue. This is not in accordance with national guidelines, but these midwives may see the ultrasound scan as an important part of prenatal attachment, as demonstrated in a previous study ([@C23]). However, from the midwife's perspective, the intention to identify the sex during a routine ultrasound is secondary to the medical examination. Determining the sex is sometimes difficult, depending on the weight of the woman and the position of the fetus, and the examination may therefore demand more time ([@C34]). Many parents want to know the sex, but the caregiver is only allowed to disclose the sex of the expected child if the pregnant woman expresses such a wish. Recall bias, regarding who initiated the discussion, cannot be ruled out, since the women responded to the question in the third trimester and the actual examination took place in the second trimester.
In our study there was no difference in the desire to know the baby's sex with respect to age. This is in contrast to previous studies showing that age is important for prenatal sex determination ([@C8]). The importance of religion had also no bearing on the desire to know the baby's sex, but our question about the importance of religion was not linked to a specific religion. According to Shipp et al. ([@C8]) Catholics were less inclined to find out the baby's sex compared to people belonging to another religion. Neither did the level of education influence the desire to know the sex, which is in contrast to previous studies showing that low education was associated with higher levels of prenatal sex determination ([@C8],[@C10],[@C24]).
In the present study there was no difference in the level of pregnancy planning between those who wanted to know the sex of the baby and those who did not. This is in contrast to other studies showing that women with an unplanned pregnancy were more keen to find out the sex of the expected child ([@C9],[@C10]). We can only speculate about the reason for this difference. One possible explanation is that the proportion of unplanned pregnancy in our sample was low, and even though some pregnancies were unplanned they were probably not unwanted since the woman/couple had decided to carry the pregnancy to term.
In addition, we could not demonstrate any difference regarding previous children or not between those who wanted to know the sex of the baby and those who did not. In the study from India more women with prior deliveries wished to find out the baby's sex, compared with first-time mothers ([@C24]). In India sex determination is illegal. Nonetheless, most pregnant women know the sex of their baby, and it has been estimated that around seven million girls are aborted every year ([@C35]). In Western countries, there is not such an out-spoken preference for male offspring ([@C27]), which can explain that there was no difference between first-time pregnant women and those who already had children. However, an American study also showed that it was more common among women with prior deliveries to find out the sex prenatally, especially if they only had one previous child ([@C8]).
The finding that women who had gone through another method of fetal diagnostics were more interested both in sex determination and sex selection did not come as a surprise. In a subsample of this cohort and their partners, an association between an interest in preconception genetic carrier screening (PCS), experiences of prenatal diagnostics, and wanting to find out or select the sex of their child was found ([@C36]).
This study focused on the desire to find out the sex during a routine ultrasound scan. Another non-invasive way to perform prenatal sex determination is by non-invasive prenatal diagnosis (NIPD). This means that fetal DNA is analyzed in a maternal blood sample, and the test can be performed already in pregnancy week seven. The method is not yet used for sex determination of the fetus in Sweden, but it has created an ethical discussion on prenatal sex determination. This new technique is expected to lead to increased sex selection in developed countries where this test is available ([@C25]). In Sweden, any woman can choose to terminate a pregnancy until the end of the eighteenth week of gestation without having to disclose any reason for the abortion. According to the National Board of Health ([@C37]) 94% of all abortions in 2016 in Sweden were performed before 12 weeks, and 53% before pregnancy week seven. These high numbers of very early induced abortions indicate that an abortion decision is taken very early in pregnancy and in most cases because the pregnancy is unwanted or mistimed ([@C38]). More studies on the effects of early sex determination in Sweden are needed to rule out the potential risk that abortions can be carried out because of the sex of the fetus.
Wanting to know their child's sex is not the same as having a preference for a particular sex. However, according to Shipp et al. ([@C8]), prenatal sex determination is much more common among those who have a preference for one sex compared with those who have no such preference. In our study, very few wanted to be able to decide on the sex of a future child. However, prenatal sex determination is a prerequisite for sex selection ([@C39]). This reluctance toward sex selection is in line with the study about potential interest in PCS, showing that both women and men had relatively high uncertainty toward PCS ([@C35]). Around half of the women were opposed to such selection of a child, and they were also concerned about negative consequences. Other ethical aspects, such as justice and autonomy, have also been raised in relation to PCS ([@C40]). The expanding use of fetal diagnostics technology worldwide calls for increased access to high-quality counseling already before pregnancy---so-called preconception care ([@C41]). Such counseling can provide future parents with unbiased information so they can make well-informed decisions once a pregnancy has occurred.
Strengths and limitations {#s0006}
-------------------------
The strength of the study is the high number of participants and the broad recruitment from many different ANCs in the country. Since almost 100% of pregnant women attend ANCs in Sweden, our sample is most probably representative for the entire Swedish-speaking population. However, we have no information about women who were not invited to participate or declined participation. Another limitation was that only the pregnant women responded, also on behalf of their partners' wishes.
We have no information about the sex of the women's previous children so we could not analyze the wish for sex determination against the background of the sex of previous children. Only Swedish-speaking women could respond to Q2, which limits the possibility to generalize the results to all women living in Sweden.
Conclusions {#s0007}
===========
More than half of all pregnant women and their partners wanted to find out the sex of the expected child during the routine ultrasound scan. This percentage is low from an international perspective, but is still a challenge, since present national guidelines do not include sex determination as an option. We need more national and international studies on sex determination during pregnancy for further knowledge of how the desire to find out the sex of the baby is shaped and what this development might lead to in the future.
We thank Jenny Stern, Jennifer Drevin, and Maja Bodin for distribution of questionnaires and data collection.
Disclosure statement {#s0009}
====================
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Notes on contributors
=====================
***Margareta Larsson***, RN, RM, PhD, is a Senior Lecturer and Associate Professor at the Department of Women's and Children's Health, Uppsala University.
***Minna Berglund***, RN, RM, MMSc, Department of Women\'s and Children's Health, Uppsala University.
***Emelie Jarl***, RN, RM, MMSC, Department of Women's and Children's Health, Uppsala University.
***Tanja Tydén***, RN,RM, PhD, is a Senior Professor at the Department of Women's and Children's Health, Uppsala University.
|
{
"pile_set_name": "PubMed Central"
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|
Introduction {#Sec1}
============
Chronic obstructive lung disease, a collective term that includes asthma, and chronic obstructive pulmonary disease (COPD), are highly prevalent conditions that produce significant morbidity. Approximately 26.5 million adults and children suffer from asthma^[@CR1]^, and chronic lower respiratory disease, comprised mainly of COPD, is the third leading cause of death in the United States. Although etiologies of these diseases differ, they are commonly associated with inflammation-driven remodeling of structural elements of the lung. Pulmonary fibrosis is a common pathology of these lung diseases. Fibrosis results from an imbalance of synthesis and proteolytic degradation of the extracellular matrix. Depending on the environmental stimuli involved and structural cells affected, airway fibrosis can assume a variety of tissue distribution patterns^[@CR2]^. In allergen-driven asthma, T lymphocytes and epithelial cells produce a characteristic pattern of sub-epithelial fibrosis in the lamina reticularis; an early and persistent finding^[@CR3]^. Sub-epithelial fibrosis is produced by fibrillar collagen types I and III, downregulation of matrix metalloproteinases (MMPs), and proteoglycan deposition^[@CR4]^. Moreover, the amount of lamina reticularis fibrosis correlates with increased obstruction and disease severity^[@CR5]^. By contrast, intimal thickening and outer adventitial extracellular matrix deposition in COPD is associated with reduced expiratory airflow and exercise capacity. The molecular mechanisms, patterns of fibrosis, and relationships with other cellular events in remodeling are not completely understood. While a number of animal models have provided much of the current understanding of pulmonary fibrosis^[@CR6]^, there is a lack of approach for direct study of fibrosis in the intact lung, particularly those that do not require tissue sectioning. Robust imaging approaches to identify patterns and characteristics of fibrosis throughout the full intact lung would greatly advance our understanding of this pathology, help assess disease risk factors, and contribute to identifying therapeutic targets.
Current approaches for preclinical study of lung fibrosis include whole-body imaging methods, histological approaches, and molecular biology methods. Whole-body imaging methods, including X-ray radiography, computed tomography (microCT), positron emission tomography (microPET), magnetic resonance imaging (MRI), and whole body bioluminescence/fluorescence by IVIS imaging provide *in vivo* indication of lung pathology at millimeter level resolution (50--100 µm for microCT) but do not provide direct assessment of the elements of fibrosis, namely collagen, and cannot delineate between lung inflammation and fibrosis^[@CR7]--[@CR10]^. Histology and immunohistology, used commonly in studies of fibrosis, provide subcellular visualization of microstructure including contrast of fibrillar collagen with staining. However, sampling is limited to 4--10 µm thick sections comprising small areas and is prone to regional sampling bias. This limited sampling of histology prohibits patterns of fibrosis to be characterized over large volumes and whole lung assessment is improbable as it would require sectioning across an entire organ^[@CR11]^. Finally, molecular biology methods sample molecules directly associated with fibrosis, but are limited to fluids or digested tissue and thus lack spatial localization.
The nonlinear optical microscopy methods of multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) provide imaging depths of hundreds of micrometers and in the lung have provided insights into the processes of inflammation and cancer both in *ex vivo* and intravital lungs^[@CR12]--[@CR20]^. MPM may be performed label-free with contrast for imaging three-dimensional lung structure based on intrinsic autofluorescence from cells and the extracellular matrix^[@CR13]--[@CR15]^. SHGM provides specific contrast to noncentrosymmetric molecules, of which fibrillar collagen is the primary source in lung^[@CR16]^. Thus, with collagen overproduction and remodeling being central to fibrosis, SHGM provides a powerful option for fibrosis studies^[@CR17],[@CR18]^. MPM and SHGM combine naturally and are often used together. With depths of imaging in the native lung to \~200 µm^[@CR16],[@CR19],[@CR20]^ and the potential for image tiling or mosaicking (in which neighboring image stacks are acquired over large lateral areas) imaged volumes can far surpass volumes assessed by histology but are not sufficient for whole lung imaging due to depth limitations. However, recent developments in tissue optical clearing (OC) in which *ex vivo* tissues are rendered transparent through immersion in chemical agents, have made large scale microscopic imaging of full organs possible^[@CR21]--[@CR24]^.
OC includes a variety of chemical immersion approaches to reduce light scattering in tissue by index matching of tissue and interstitial components^[@CR25]^. The method termed CLARITY which combines electrophoretic removal of lipids with immersion in clearing solvents was first introduced for full murine brain microscopy of neurons in fluorescent transgenic mice^[@CR26]^. CLARITY was recently adapted and shown to render the lung transparent but was not applied for whole lung microscopy^[@CR27],[@CR28]^. Lung CLARITY is a lengthy process, taking 13--17 days and leads to swelling with considerable expansion of organ volume^[@CR28]^. A few studies have applied OC by direct solvent immersion with OC processing occurring in as little as a few days to a few hours, and imaged lung structures by confocal, MPM, or SHGM microscopy^[@CR21]--[@CR23],[@CR29],[@CR30]^. One limitation of solvent immersion methods is potential organ shrinkage \>20--25%^[@CR25]^. Three studies have demonstrated whole lung lobe microscopy, two employing confocal microscopy through intact lungs labeled with fluorophores and cleared with solutions of benzyl alcohol: benzyl benzoate or dibenzyl ether^[@CR23],[@CR30]^, with Scott *et al*., first demonstrating imaging of the full lobe airway structure and visceral pleural nerves labeled with fluorescent antibodies^[@CR23]^. Limitations in objective working distance limited imaging depths to 2 mm, thus lobes were separated and compressed between slides for imaging (details of depth were not discussed in Erturk *et al*.). In a recent study full lung fluorescence lightsheet microscopy with OC by immersion in tetrahydrofuran and dibenzyl ether (DBE) was demonstrated with the study focus on bronchus-associated lymphoid tissue^[@CR24]^.
Thus far, whole lung microscopy of collagen for study of lung fibrosis has not been demonstrated. SHGM for imaging collagen has been performed on cleared lung, applied for regional lung imaging - with one study demonstrating 2 × 2 × 2 mm volume imaging of normal lung by MPM/SHGM^[@CR22]^ while another achieved lateral lobe imaging but was limited by light attenuation to imaging depths of 397 µm^[@CR21]^. Imaging depths needed to image uncompressed lung are 3--5 mm. Here, to address the gap in whole lung collagen imaging, which will enable studies of lung fibrosis, we have combined an optimized OC approach based on benzyl alcohol: benzyl benzoate (BABB) immersion that results in minimal change in tissue volume with whole-organ large scale microscopy by MPM/SHG mosaic imaging and image stitching. This combination of approaches (optimized OC sample processing, 3D MPM/SHGM mosaic imaging and stitching, and quantification) results in the ability to clear and image whole fixed lungs and reveal the global spatial distribution of fibrillar collagen throughout the organ at high resolution. The ability to identify and quantify fibrosis at the airway level and the volume of the lung is demonstrated.
Methods {#Sec2}
=======
Mouse model of lung fibrosis {#Sec3}
----------------------------
A repetitive poly(I:C) challenge murine model of airway remodeling and subepithelial fibrosis served as the lung fibrosis model^[@CR31]^. Animal experiments were performed according to the NIH Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee (approval no. 1312058A). Male C57BL6/J mice (18 weeks old) purchased from The Jackson Laboratory (Bar Harbor, ME) were housed under pathogen-free conditions with food and water *ad libitum*. Mice were placed under light anesthesia \[(ketamine (15 mg/kg bodyweight)/diazepam (1.5 mg/kg body weight)\] for intranasal delivery of poly(I:C) to induce fibrosis. Intranasal poly(I:C) (500 μg/dose in 50 μL PBS) or an equivalent volume of PBS (50 μL) was administered every other day for a total of 15 doses (n = 4/group). Twelve days (12 d) after the last treatment at age 22 weeks, mice were anesthetized and lungs were harvested for OC and imaging or histology.
Preparation of Lung extraction for OC {#Sec4}
-------------------------------------
Mice were anesthetized with 5% isoflurane using a nose cone for 3--5 minutes and reduced to 1.5% isofluroane. A transcardial perfusion was performed by opening the thoracic cavity exposing the heart and lungs. A blunt tip needle (Thermo Fisher Scientific, NC) was inserted in the right ventricle and a small incision was made in the left atrium (Fig. [1](#Fig1){ref-type="fig"}, lower leftmost panel). Transcardial perfusion was initiated with \~4 °C PBS (30--40 mL) followed by \~4 °C 4% formaldehyde (20--30 mL). While still attached *in situ*, lungs were then directly perfused via the trachea in order to avoid collapsing of the airways and maintain an expanded lung. Specifically, a blunt tip needle was inserted in the most superior position of the trachea, with the needle secured using a small bulldog clamp (World Precision Instruments, FL). Lungs were filled with 4% formaldehyde and the needle was removed leaving the bulldog clamp on the trachea. The lungs were removed and immersed in 4% formaldehyde (40 mL) overnight at 4 °C then subsequently placed in PBS (40 mL).Figure 1Whole Lung Optical Clearing and Imaging Workflow. 1. A murine poly(I:C) fibrosis model was used. Animals were treated with intranasal poly(I:C) to induce fibrosis or PBS as a control. 2. Lungs were extracted following cardiac perfusion in \~4 °C with PBS followed by 4% formaldehyde and were expanded post-extraction. 3. Lungs were treated with graded concentrations of methanol (second panel, bottom) for dehydration. Lungs were optically cleared by treatment with BABB for refractive index matching. Air microbubbles were removed by placing lungs in a vacuum chamber (third panel, bottom). 4. Samples were mounted (rightmost bottom panel) in an image chamber between a cover glass and a microscope slide. MPM/SHG mosaic imaging was performed by taking sequential z-stacks through the full depth of the lung (4 × 0.16 N.A. air objective, z interval: 5 µm n = 600--800 planes) across the entire organ with intrinsic tissue auto Fluorescence (MPM) and collagen SHG providing image contrast for the 3D volumetric acquisitions. 5--6. Image stack stitching was done by Fourier Transform Phase Correlation and quantification of collagen deposition (SHG) using 3D image analysis software.
Optical Clearing (OC) {#Sec5}
---------------------
An approach incorporating methanol for tissue dehydration followed by benzyl alcohol: benzyl benzoate (BABB) was employed for whole lung optical clearing, the protocol modified from previous lung OC studies^[@CR22],[@CR23]^ to avoid significant shrinkage observed in such studies and to eliminate potential sources of scatter from air bubbles caught in the airway. For the dehydration step, graded concentrations of methanol starting with 10% methanol in PBS (v/v) in increments of 10% up to 100% methanol (room temperature) were infused into the lung via the cannulated trachea (Fig. [1](#Fig1){ref-type="fig"}, lower panels). Specifically, following overnight fixation in formaldehyde, described above, 10% methanol (5 ml) was slowly delivered via the trachea into the lung airways using a cannula tubing (10-gauge polyethylene cannula, Warner Instruments) attached to a 21-gauge needle on a syringe. The 5 ml solution was delivered manually over a period of approximately 1 minute. Next 20% methanol was delivered in the same manner (delay time of \~2 minutes), and so on in increments of 10% until 100% methanol was delivered. The 100% methanol infusion was repeated a second time and the lungs were placed in a conical tube containing fresh 100% methanol for 1 hour while placed on a shaker (Fisher Scientific CAT. NO. 22--363152). The purpose of this gradual increase in infusion solution was to avoid rapid volume changes in the lung during the methanol dehydration process. By slowly infusing the graded methanol concentrations through the trachea, large osmotic shifts were avoided. In pilot trials, we applied 50% methanol immersion as a first step as in two previous studies employing BABB clearing in lung^[@CR22],[@CR23]^, but observed lung collapse and visible shrinkage thus we modified and optimized the dehydration step as described. Next, to render the lung transparent, it was infused with BABB (1:2 vol/vol mixture of benzyl alcohol \[305197\] and benzyl benzoate \[B6630\], Sigma-Aldrich) solution at room temperature as described above. Partial clearing of the lung became evident during this infusion with BABB delivered in approximately 1 minute. Following the 5 ml infusion with BABB, the lungs were immersed in 10 ml fresh BABB in a glass dish (QorPak, Bridgeville, PA) placed with a loose cap into a vacuum chamber overnight for removal of air bubbles seen in the transparent lungs. This step removed potential sources of light scatter from air bubbles trapped in the lung and allowed for complete BABB diffusion into tissue overnight. In preparation for imaging, samples were then mounted in a glass bottom dish (Willco Wells, UK) between a \#1.5 coverglass on the imaging side and a 1 mm thick glass microscope slide on the opposing side (Fig. [1](#Fig1){ref-type="fig"}, lower rightmost panel), the two separated by moldable putty (Grainger, 5DLD8 or Bostik, 30840350) which served to seal the chamber and provided a spacing the thickness of the lung without compression. The chamber spacing was filled with fresh BABB immersion solution.
Multiphoton and Second Harmonic Generation Microscopy (MPM/SHGM) {#Sec6}
----------------------------------------------------------------
Multiphoton microscopy and second harmonic microscopy were performed on a Prairie Technologies Ultima IV laser scanning nonlinear optical microscope (Bruker, Middleton, WI) using a 4X 0.16 N.A. air objective (UPLSAPO 4X, Olympus) having a 2.2 µm lateral resolution and working distance of 13 mm and field of view (F.O.V.) of 2327 × 2327 µm as well as a 25 × 1.0 N.A. super-objective (XLSLPLN25XGMP, Olympus) with 0.4 µm lateral resolution and working distance of 8 mm and F.O.V. of 486 × 486 µm. The 4x objective was used for whole lung acquisitions while the 25X was used to collect regional areas. The sample holder was placed on the upright microscope stage below the objective with the coverglass facing the objective as shown in Fig. [1](#Fig1){ref-type="fig"} (lower rightmost panel). Illumination for excitation of MPM autofluorescence and SHG was provided by a femtosecond laser (Mai Tai, Spectra Physics, Santa Clara, CA) tuned to 880 nm. Autofluorescence emission was collected using a 500--650 nm bandpass filter while SHG was collected at 440 nm using a notch filter with a bandwidth of 50 nm. To further increase the remitted nonlinear signal, a broadband metallic mirror (10D20ER.2, Newport Corp., Irvine CA) was placed under the sample chamber to reflect forward propagating light toward the objective, an approach shown to maximize SHG^[@CR32]^.
To acquire whole lung image acquisitions (large scale microscopy), the 4X objective was used to acquire tiled image stacks through the full depth of the lung, acquiring neighboring image stacks in a tile pattern from left to right, row by row to form a mosaic tile pattern with the final number of tiles dependent on size of the lung. The increment between planes in depth for image stacks was 5 µm for the 4x acquisitions and total imaging depth was variable and ranged from 3--4 mm depending on the thickness of the lung. Acquisitions produced \~28,000--30,000 images each with each image stored as a 16-bit raw image (\~10--15 GB of raw data per sample). Image stacks using the 25X objective were acquired with 2 µm step size and while image depths comprising full lung thickness were possible, regional imaging was generally limited to \<1 mm due to time of acquisitions (typically 6 µs dwell time, 512 × 512).
Image Processing and Analysis {#Sec7}
-----------------------------
Image processing and stitching of tiled acquisitions to create full lung mosaics were performed using FIJI/ImageJ^[@CR33]^. Raw images acquired from the 12-bit sensor were stored as 16-bit tiffs (a standard storage protocol that incorporates zero padding of histogram values above 4095 (2^12-1^) when using a 12-bit sensor). Images were contrast stretched to remove zero padding prior to being converted to 8 bit images. A 2 × 2 median filter was applied to the image stacks. Stitching was performed with a 10% overlap of tiles having field of view of 2327.3 × 2327.3 µm, providing 232.73 µm of co-registration in both X and Y coordinates. The Fourier Transform phase correlation stitching method was applied using the ImageJ plugin *Grid/Collection Stitching*^[@CR34]^. Quantification and 3D rendering were performed with Imaris software (Bitplane USA, Concord MA). After opening the mosaic stack, a 3D reconstruction was created and the *Surfaces* tool in Imaris was used to define the volume comprising SHGM. The *Surfaces* tool provides a threshold preview overlaid on the original image to allow for visual setting of a threshold value to match collagen structures visible in the SHG channel, then provides surface rendering. A threshold was determined visually for multiple stacks representing the dataset then kept the same between all lungs. The total SHG volume, representing the full lung fibrillar collagen content, was defined by surface rendering and calculated for the full lung using the volume analyzer in this tool. The total SHG volume was normalized to the total volume occupied by lung tissue, indicated from the volume of the MPM autofluorescence signal which was also processed with the "Surfaces" tool.
Volumetric analysis on individual airway types (primary bronchus, secondary bronchi, terminal bronchioles) was performed using ImageJ software (NIH). Specifically, for each airway type, non-branching segments of the airway conduit were identified visually and regions of interest chosen manually in the whole lung MPM/SHGM mosaic stack to isolate an image cube comprising the segment of airway conduit. Specifically, the *Rotated Rectangle* selection tool allowed for selection of laterally angled regions of interests for angled airways and the z dimension of the ROI cube was specified by choosing a plane immediately above the topmost surface and bottommost surface having SHG signal (the ImageJ *Slice Remover* tool was used to remove planes above and below stack of interest). The ROI cube lateral borders running parallel to the airway direction were also chosen immediately beyond termination of SHG signal. MPM (red) and SHGM (green) channels of the selected ROI image cube were separated and SHGM ROI stacks were manually thresholded (kept constant between samples) and binarized. The *Voxel Counter* plugin in ImageJ^[@CR33]^ was used to calculate the volume of SHG-positive signal normalized to the ROI image cube. In this analysis, 6 airway segments were measured per each of the three airway types in each animal, and an average obtained per animal with a final average obtained per treatment group. As a complementary qualitative examination of collagen deposition in distal bronchioles, a visual count was made on the 3D lung view of the number of distal bronchioles surrounded by collagen in poly(I:C) vs control lungs seen using the *3D viewer* tool.
All processing was on a custom-built computer with Dual Quad-Core Xeon E5-2623 processors (10 MB Cache; 3.0 GHz; 128 GB of ECC RAM) with AMD Firepro W7100 graphics card.
Histology {#Sec8}
---------
For histological examination, whole lungs were extracted without PBS perfusion. Extracted lungs were inflated under 25 cm H~2~O pressure with 10% (v/v) neutral buffered formalin through the tracheal cannula and immersed in 10% formalin solution (20X volume over the size of lungs) overnight. The next day, the whole lungs were replaced in fresh 10% formalin solution for another 48 hours and sent to UTMB Histopathology core for histological processing, where they were embedded into paraffin blocks, and cut into 5-μm sections. Slides were stained with Masson's Trichrome to assess fibrotic changes. The histological images were taken on a standard microscope (Olympus IX71 using a Nikon Ds-Fi1 5-megapixel CCD (2560 × 1920 pixels).
Statistics {#Sec9}
----------
Statistical analysis of total lung collagen volume between two groups (PBS vs. poly poly(I:C)) was performed using Student's t-test (two-tailed) with p \< 0.05 considered statistically significant. ANOVA Two Way Factor with Replication was performed for analysis of the three airway types (main stem bronchus, segmented bronchus, and terminal bronchioles) between PBS and poly(I:C) treated lungs. P values of \<0.05 were considered statistically significant.
Results {#Sec10}
=======
Optimized Whole Lung OC with MPM/SHGM Mosaic Imaging {#Sec11}
----------------------------------------------------
Figure [1](#Fig1){ref-type="fig"} depicts the workflow of whole lung OC and large-scale microscopy. Optimized OC involved full lung inflation, a graded concentration dehydration step to avoid major size deviations, infusion of the lung with methanol and BABB to allow contact with the tissue from both the external and the internal airway surfaces, and the use of a vacuum chamber to remove air microbubbles which could contribute to light scattering in the lung. Tissue autofluorescence in MPM provided the contrast signal for microstructural assessment, while SHG provided contrast from fibrillar collagen in the extra-alveolar space.
Representative images of a murine lung before and after OC are shown in Fig. [2](#Fig2){ref-type="fig"}. Freshly extracted native lung is highly turbid with typical color shown (Fig. [2a](#Fig2){ref-type="fig"}). Following infusion and immersion in BABB, the murine lung becomes fully transparent (Fig. [2b,c](#Fig2){ref-type="fig"}). In Fig. [2b](#Fig2){ref-type="fig"}, the transparent lung is held in air following perfusion and immersion in BABB. In this image, a cannula inserted into the trachea and used to infuse the lung with fluid of a slightly different refractive index to that of BABB allows the airways of the lung to appear with slight contrast relative to the lung parenchyma. However, once the lung is infused and fully immersed again in BABB (Fig. [2c](#Fig2){ref-type="fig"}) the airways are no longer distinguishable and the whole lung appears virtually invisible to the naked eye (a dotted line border delineates the edges of the lung against the solution in Fig. [2c](#Fig2){ref-type="fig"}). Here, only the cardiac structures that remain attached to the lung are evident, indicated by the arrow in Figs [2c](#Fig2){ref-type="fig"}. Fig. [2d](#Fig2){ref-type="fig"} shows the full depth three-dimensional MPM-SHGM reconstruction of the intact lung following mosaic imaging and tiling, with parenchymal autofluorescence in red and fibrillar collagen SHG in green. The right lung shows a multi-lobe structure as expected (arrow in Fig. [2d](#Fig2){ref-type="fig"}) with a single lobe in the left lung. A cutout of the left lung (removing a volume of the red channel) is made to reveal the intricate airway structure surrounded by fibrillar collagen. Select, high resolution (25x) regions of interest of multiphoton autofluorescence, shown in false color, illustrate airway morphology. A large airway (likely secondary bronchus) is shown lined with epithelial cells denoted by (\*). Alveoli (\^), and vasculature (+), is denoted by symbols (Fig. [2e,f](#Fig2){ref-type="fig"}). Lateral measurements in still images and photographs made on lungs that were dehydrated using the graded concentration application of methanol indicted +2.8 ± 1.7% change size, versus -16.9 ± 1.7% measured in lungs dehydrated with 100% methanol dehydration (5% shrinkage for 50% methanol). In the current study, lungs experienced a horizontal size change of approximately +6% (6.1% ± 1.5% for the lung shown in Fig. [1](#Fig1){ref-type="fig"}) after the full process of dehydration and clearing.Figure 2Optical Clearing and Large Scale Microscopy of Whole Lung. (**a**) Native murine lung following extraction (**b**) Intact lung following whole lung optical clearing. In this photograph, the lung is held in air following perfusion and immersion in BABB. To capture the contrast of the airway against the parenchyma, a cannula is used to perfuse the lung with fluid of slightly different refractive index to the BABB OC fluid. (**c**) Optically cleared lung infused with and immersed in BABB in a sample imaging chamber. A dotted line outlines the borders of the lung and the arrow points to area of cardiac structures that appear dark. (**d**) Volumetric (3D) representation of the entire murine lung obtained by mosaicking and stitching of MPM-SHGM image stacks using the 4 × 0.16 NA objective. Imaging was through the full depth of the lung with mosaicking (r x r arrangement) to acquire the entire organ, with MPM tissue autofluorescence shown in red and collagen SHG shown in green. The right lung shows multi- lobe structure (arrow). To create the cutout shown in (**d**), cropped areas of the red channel were removed from the left lung to reveal underlying sub epithelial collagen network surrounding airways. 3D reconstruction was by Imaris 3D analysis software. Reconstructed volume represents n = \~25,000 images. (**e**) Representative high resolution 25x MPM of lung regions showing a segment of a pulmonary bronchus (shown in false color). (**f**) MPM (25x) showing a segment of lung tissue with a vessel (+) near two converging bronchi (\*) and surrounded by alveoli (\^). (Epithelial cells (\*), alveoli (\^), and blood vasculature distribution (+)).
Imaging Capabilities of MPM/SHGM in Optically Cleared Lungs {#Sec12}
-----------------------------------------------------------
The capabilities of large scale MPM/SHGM mosaics with image stitching combined with optically cleared whole lungs are demonstrated in Fig. [3](#Fig3){ref-type="fig"}. Figure [3a--d](#Fig3){ref-type="fig"} show the ability to define the full lung structure including airways lined by subepithelial collagen, and the increase in collagen deposition found in the fibrosis model. 3D volumes of the SHG channel representing fibrillar collagen are shown in Fig. [3b,f](#Fig3){ref-type="fig"} for PBS control and poly(I:C) fibrosis model lungs. Transverse cut (Fig. [3d,h](#Fig3){ref-type="fig"}) renderings highlight and define the localization of fibrosis surrounding the airway to the subepithelial space.Figure 3Large Scale MPM/SHG Microscopy Reveals Whole Lung Airway Structure and Airway Subepithelial Collagen Throughout the Full Lung. MPM/SHG representative 3D reconstructions of PBS and poly(I:C) treated lungs. PBS (**a**,**c**) and poly(I:C) (**e**,**g**) MPM/SHG 3D coronal cross-section of full lung. MPM autofluorescence is shown in red and fibrillar collagen SHG is shown in green. Representative 3D rendering of whole lung collagen network in PBS (**b**) and poly(I:C) (**f**) treated lungs. Close up of coronal (**c**) and transverse (**d**) cross-sections of PBS treated lungs. Close up of coronal (**g**) and transverse (**h**) cross-sections of poly(I:C) treated lung. Images show fibrotic collagen deposition surrounds airways in the subepithelial space (**g**,**h**). Primary bronchus (PB), Secondary bronchus (SB), Terminal bronchioles (TB) are designated in (**a**,**b**).
Comparison of MPM/SHGM with Histology {#Sec13}
-------------------------------------
The distribution of collagen near airways and vessels are further validated and appreciated in high resolution MPM/SHGM micrographs of Fig. [4](#Fig4){ref-type="fig"} in PBS and poly(I:C) treated lungs. Airways (labeled, A) are defined by the presence of an epithelial lining visible due to MPM autofluorescence in red. Airway, vessel (V) and collagen morphology resembles that seen in histology in the lower panels shown at comparable scales (Fig. [4c,d](#Fig4){ref-type="fig"}). Vessels (labeled, V) are identified by the presence of thin endothelium. Airway subepithelial collagen is significantly increased in the poly(I:C) treated lung, as shown in MPM/SHGM micrographs (Fig. [4b](#Fig4){ref-type="fig"} vs. [4a](#Fig4){ref-type="fig"}), corroborated in histology (Fig. [4d](#Fig4){ref-type="fig"} vs. [4c](#Fig4){ref-type="fig"}) through Masson's trichrome staining on tissue sections from comparable areas of PBS and poly(I:C) treated lungs.Figure 4Collagen Deposition Revealed by MPM/SHG is Reflective of Deposition Seen in Histology. (**a**) MPM/ SHGM micrographs of PBS and (**b**) poly(I:C) treated lung showing airways and vessels surrounded by alveoli. (**c**) Histology with Masson's Chrome staining for collagen shown at 20x for PBS and (**d**) poly(I:C). Enhanced collagen deposition is visible in the fibrosis model both in panels (**b**) MPM/SHG and (**d**) histology, with similarities in distribution and structure. Note the epithelial structure of airways in MPM micrographs (**b**) can be seen and similar to that of histology (arrows); Vessel endothelium(en).
Whole Lung Fibrosis Assessment {#Sec14}
------------------------------
Volumetric (3D) reconstructions (coronal cross-sections are shown so SHG within the organ can be visualized easily) of PBS- (Fig. [5a,c](#Fig5){ref-type="fig"}) and poly(I:C)-treated (Fig. [5b,d](#Fig5){ref-type="fig"}) lungs reveal full organ structural differences between the groups, particularly differences in collagen deposition. Co-registration of MPM with SHGM defines the distribution of SHG throughout the lung structures (Fig. [5a,b](#Fig5){ref-type="fig"}), while single green channel (SHGM) volumes (Fig. [5c,d](#Fig5){ref-type="fig"}) reveal the extent of fibrosis compared to PBS controls. To quantitatively describe the degree of fibrosis in the poly(I:C) model, whole lung collagen content was quantified by calculating the volume occupied by full lung SHG and normalized to whole lung as described above. SHG signal was substantially greater throughout poly(I:C) treated lungs than in the PBS group (Fig. [5e](#Fig5){ref-type="fig"}), providing a quantitative indication of fibrosis.Figure 5Quantification of Whole Lung Fibrosis Induced by poly(I:C) Treatment and Imaged by MPM/SHGM Mice administered intranasal doses of poly(I:C) or PBS every other day for a total of 15 administrations were imaged following the final dose. (**a**) High resolution MPM (red) and SHGM (green) in the PBS treatment group consists primarily of MPM autofluorescence which delineates the structures of airways and vessels with a component arising from SHG from fibrillar collagen surrounding airways and potentially vessels. (**b**) In poly(I:C) lungs SHGM is significantly increased throughout in both lobes compared to PBS treated with similar MPM. Images (**a**,**b**) are 3D coronal cross-sections with proximal planes removed to highlight the SHG component of the airways. 3D SHG reconstructions depicting fibrillar collagen in PBS (**c**) and poly(I:C) (**d**) treated lungs. Quantification of whole collagen distribution revealed significantly higher collagen deposition in poly(I:C) treated lungs compared to PBS treated (**e**).
Airway Specific Collagen deposition {#Sec15}
-----------------------------------
While it is of value to explore the volume of collagen throughout the full lung as a measure of fibrosis, a measure not provided by traditional tools to study fibrosis, it is also of interest to examine differences within finer structures. Thus, the amount of collagen was evaluated for specific airway types throughout the lungs (primary bronchi, secondary bronchi, terminal bronchioles) as shown in Fig. [6](#Fig6){ref-type="fig"}. Figure [6a,b](#Fig6){ref-type="fig"} show close-up views from full lung reconstructions (coronal cross-sections are shown) demonstrating higher collagen is associated with airways in the fibrosis model (Fig. [6b](#Fig6){ref-type="fig"}) than the control (Fig. [6a](#Fig6){ref-type="fig"}) acquired and displayed with the same gain/contrast parameters. In a qualitative assessment, a visual count (Fig. [6c](#Fig6){ref-type="fig"}) was made of the number of distal bronchioles seen in rotations of a 3D volume that were surrounded by collagen as a percentage of total number of distal bronchioles in each lung. Approximately 60% of distal bronchioles were associated with collagen deposition in the poly(I:C) group, while 16% were associated with collagen in the PBS group (Fig. [6c](#Fig6){ref-type="fig"}, n = 4/group; p \< 0.001, two-tailed t-test). A separate quantitative analysis of SHG volume fraction per sampled segment was performed of isolated airway segments (primary bronchus, secondary bronchi, and terminal bronchioles) comparing airways of same type/ diameters with examples shown (Fig. [6d--f](#Fig6){ref-type="fig"}). The panels shown in Fig. [6d,e](#Fig6){ref-type="fig"} are representative SHG 3D volumes segment regions of interest (for each group, the top row shows the segment longitudinal view and the bottom row a transverse view showing the airway opening). These SHG volumes represent the airway subepithelial collagen, so openings in transverse views are not the full airway lumen. Poly(I:C) treated airways of all three types showed higher collagen deposition compared to that of PBS, all with similar degree increase in volume fraction (Fig. [6f](#Fig6){ref-type="fig"}). Finally, for each treatment group, we evaluated whether there were differences in fibrosis around airways between the left and right lungs. Results showed no significant difference between airways of a given type when comparing airways from right and left lobes within each treatment group (Supplementary Table [I](#MOESM1){ref-type="media"}).Figure 6Airway Specific Quantitative Analysis of Subepithelial fibrosis. Coronal cross-sections of lobe segments in PBS treated lungs (**a**) and poly(I:C) treated lungs (**b**). (C) Poly(I:C) lungs showed a higher percentage of distal bronchioles (denoted by \*) with visible collagen deposition than PBS controls. (**d**--**e**) 3D reconstructions of the collagen surrounding airway segments. Airway segments comprised non-branching sections of primary bronchi, secondary bronchi, and terminal bronchioles. Alveoli are seen as small circular structures in the MPM channel as denoted by "a" and an example of secondary bronchi segment is shown by the box in (**b**) which were isolated as described in the methods section. (**f**) Volume analysis of airway segments show significantly higher volume fraction of collagen surrounding conduits of poly(I:C) lungs, but no difference between airway type per treatment group. ANOVA: \*\*p \< 0.01.
An example of a 25x acquisition in the poly(I:C) fibrosis model is shown in Fig. [7](#Fig7){ref-type="fig"}. The top panel represents the 3D reconstruction (Imaris) of a region of the lung acquired within the lung volume starting at the surface of a primary bronchus and proceeding in depth to 450 µm. The same volume is shown with cuts made at various depths to reveal underlying structures of airway, vasculature, and collagen. Inflammatory infiltrates shown by puncta surrounding alveoli in the red MPM channel can also be appreciated, though cannot be specifically subtyped due to the nonspecific autofluorescence signal.Figure 7A representative 25x volume of poly(I:C) treated lung. Top row: 3D reconstruction of a volume at the primary bronchus surface extending 450um in depth with a step size of 2 um and shown with cuts at various depths to reveal airway and vascular structures. Bottom row: single plane 25X (1.0 NA) micrographs at various depths. Structures are denoted as Airway(A), Vessel (V), Alveoli (a), Infiltrates (\*) are seen as small bright puncta; Arrowhead denotes bronchi epithelium with individual epithelial cells visible on the surface.
Discussion {#Sec16}
==========
There remains a lack of understanding of the pathogenesis and molecular mechanisms that drive pulmonary fibrosis. A gap in the field has been lack of methods for direct mapping of collagen deposition throughout the murine lung. This study demonstrates the ability to visualize and evaluate fibrillar collagen throughout the entirety of the murine lung with microscopic resolution applied for study of whole lung fibrosis. No known previous imaging studies have provided the ability to specifically evaluate the deposition of fibrillar collagen within the entirety of the murine lung or quantitatively evaluated fibrosis. Traditional imaging methods that have been applied for the study of fibrosis either do not provide specific contrast for fibrillar collagen comprising fibrotic areas (e.g. xray, microCT, microPET, IVIS) or require sectioning or destruction of tissue to evaluate collagen content (histology, molecular biology techniques such as western blot analysis). To achieve full adult murine lung collagen and fibrosis imaging our study combined and optimized approaches in lung optical clearing with large scale, label-free MPM/SHGM. OC has previously been shown to result in full lung transparency, allowing imaging of entire lobes labeled with a fluorescent contrast agent^[@CR23],[@CR30]^ or segments of lung based on endogenous signals^[@CR21],[@CR22]^, with no known OC study imaging and quantifying collagen deposition across the entire lung, shown here in an adult mouse model of lung fibrosis.
The approach for lung OC was optimized to include several important steps to maintain lung shape and to avoid major size deviations of tissue reported in previous OC studies, including those of the lung while achieving full transparency and full lung microscopy without compression of the tissue. OC was based on methanol dehydration with clearing by BABB, chosen due to simplicity of the protocol and rapid clearing which occurred within minutes after treatment with BABB. To maintain the shape and avoid collapsing of the lungs, perfusion with PBS and the fixative solution with lung inflation was employed (Fig. [1](#Fig1){ref-type="fig"}). An important area of optimization was a modified dehydration procedure that resulted in minimal change in lung size in contrast to other OC studies of lung, in which lungs shrink up to 20--25% with solvent based methods including methanol/BABB^[@CR23],[@CR24],[@CR30]^ or expand with CLARITY methods^[@CR27],[@CR28]^. We did this with a gradual graded concentration infusion of lungs with methanol which resulted in gradual dehydration of the tissue, and found minimal tissue size change (+1% increase after graded methanol and 6% after BABB along the horizontal dimension). The methanol dehydrating agent and BABB OC solution were infused into the airway through the cannulated trachea to ensure direct exposure of the internal airway surfaces to dehydration and clearing solutions (Fig. [1](#Fig1){ref-type="fig"}). Finally, care was taken to remove all air microbubbles which could contribute to light scattering in the lung -- this was done by placing the sample in a vacuum chamber^[@CR21]^. Physical compression of the sample accomplished bubble removal in another study, but was not used here as we avoided compressing the tissue. We note that in our early trials, extracted adult lungs that were simply immersed in methanol/BABB without infusion^[@CR23]^ did not clear throughout the entire lung, possibly because lungs in this study were from 18 week mice versus 6--10 week mice. Also in early pilot trials, we began the dehydration step with 50% methanol, followed by 100% methanol as in previous studies using BABB for clearing^[@CR23],[@CR30]^ but we observed significant shrinkage of lungs. These observations in early pilot trials prompted the improvements described.
As indicated, the choice of methanol dehydration with BABB clearing was influenced by the simplicity and low cost of solvent based clearing and rapid clearing which largely occurred within minutes after treatment with BABB. The full lung processing took one day following fixation an advantage over other OC methods of CLARITY which may take up to two weeks^[@CR28]^ and reported by one study to result in suboptimal clearing^[@CR24]^, or resin based approaches^[@CR21]^ which are more complex. Other immersion methods applied in the clearing of lungs, such as DBE^[@CR24],[@CR30]^, do match similar refractive indices to BABB, however, a reagent (Tetrahydrofuran, THF) used in the dehydration step of DBE optical clearing may form explosive crystals. We therefore choose BABB because of potential safety concerns.
Complete transparency of lungs (Fig. [2b,c](#Fig2){ref-type="fig"}), combined with the use of several millimeter long working distance objectives, enabled imaging without the need for further tissue processing such as cutting/sectioning, deflation, or flattening. This is in contrast to previous studies in which the infusion steps outlined above were not used (though clearing by BABB was a similarity) and in which confocal microscopy required separation of lobes and trachea with further sample flattening to accommodate the 2 mm working distance^[@CR23]^. It is noted that while previous efforts in full lung microscopy facilitated by OC used young mice aged 5--10 weeks old^[@CR23],[@CR29]^ or 12 weeks old in optical tomographic imaging^[@CR21]^, in the current study adult mice aged 22 weeks at the time of optical clearing were used, demonstrating that full lung optical clearing for studies of fibrosis and other disease characteristics are applicable to adult mice with larger organs.
Scott *et al*. first demonstrated whole lobe microscopic imaging and used BABB clearing, with confocal imaging using fluorescent casting of airways and fluorescent antibody labeling of nerve structures^[@CR23]^. In the label-free imaging used in this study, signal contrast was provided solely from the endogenous optical characteristics of autofluorescence (MPM) and collagen second harmonic generation (SHG) (e.g. Figs [2d](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}). These endogenous signals revealed the complex airway structure as well as the subepithelial collagen matrix throughout the entire volume of the lung. An enhancement in collected signals was likely aided by a mirror placed below the sample, particularly highly forward propagating SHG, a technique adapted from the literature^[@CR32]^. Results using a 4x objective demonstrate the ability to delineate fine structures of the airway from alveoli seen in MPM micrographs to subepithelial collagen along with small airways and, through the use of mosaicking and image stack stitching, provide a global view of these key structures. Individual alveoli and subepithelial collagen deposition were revealed at 4X (e.g Figs [2d](#Fig2){ref-type="fig"}, [6](#Fig6){ref-type="fig"}) which provided 2.2 µm resolution and in greater detail at 25x providing 0.4 µm resolution which also provided close detail of airway epithelial lining and vascular endothelial lining (Figs [4a,b](#Fig4){ref-type="fig"} and [7](#Fig7){ref-type="fig"}). Label-free MPM/SHGM have been applied to native lung and a variety of other tissues to reveal structures several hundred microns in depth^[@CR17],[@CR19],[@CR35],[@CR36]^ and include studies in lung fibrosis of uncleared lungs^[@CR18]^. A variety of approaches to evaluate collagen revealed by SHGM exist and include algorithms for evaluating organization and content^[@CR17]--[@CR20]^. We did not note collagen orientation differences between treatment groups and with an interest in deposition, we chose volumetric analysis to provide an indication of collagen deposition. It may be of interest to explore more complex organization analyses in future studies. It may also be of interest to explore the combination of SHGM with fluorophores associated with key structures or macromolecules imaged with multiphoton fluorescence. Possibilities for whole lung labeling include vessels, airways, and nerves as introduced by Scott *et al*., or targeted fluorophores for immune cells as explored by Cuccarese *et al*. who used CUBIC tissue clearing in lung carcinoma^[@CR29]^.
For this study, we used a fibrosis model driven by chronic TLR3 activation induced by repetitive administration of the poly(I:C) agonist, the model representative of a viral-induced fibrosis. This innate epithelial activation model has been shown to cause significant, chronic airway fibrosis and remodeling within a 30 day period^[@CR31]^. Previous assessment of this model involved histology of regional areas of the lung. As discussed above histology provides a highly limited sampling of tissue areas relative to the full organ. Thus, while previous investigation indicated fibrosis in this model, the nature of this fibrosis including volumetric distribution throughout the lung was unknown. The methods employed here allowed evaluation of collagen deposition throughout the lungs in their entirety, with ultra-deep imaging up to the full thickness of intact murine lungs (up to 4 mm in depth). Results indicated widespread subepithelial fibrosis throughout the volume of the lung of the poly(I:C)-treated mice compared to the PBS group, as observed in Figs [3](#Fig3){ref-type="fig"} and [5](#Fig5){ref-type="fig"} with the differences quantified in (Fig. [5](#Fig5){ref-type="fig"}). Masson's trichrome staining confirmed collagen deposition (Fig. [4](#Fig4){ref-type="fig"}), indicated by MPM/SHG imaging.
Beyond full volumetric analysis we evaluated subepithelial collagen deposition in airways according to type, with both macroscopic and microscopic assessment made possible in the same acquisitions (Fig. [6](#Fig6){ref-type="fig"}). In a qualitative assessment in which we graded the most distal bronchioles for visible collagen deposition, 60% of the distal bronchioles showed a positive SHG (collagen) signal in the fibrosis model vs. only 16% with visible SHG in the PBS treated group. This provided an indication that fibrosis reached the most distal bronchioles in this model. We then quantified subepithelial collagen volume in the main bronchi in lobes, secondary bronchi, and bronchioles separately. We found that these three airway types experienced fibrosis in the poly(I:C) group, and increases in collagen deposition were of comparable percentage increase (Fig. [6f](#Fig6){ref-type="fig"}). Finally, quantitation indicated no significant difference in collagen deposition between lobes within each group (any appearance of increased collagen deposition in right lungs in 3D reconstructions or z-projections are due to the right lung being multilobed (Fig. [2d](#Fig2){ref-type="fig"}). Overall, these findings indicate that distribution of pathology in this model extends to the distal airways, a sign of distribution of the intranasal-delivered poly(I:C) beyond proximal airways. A major controversy in asthma and COPD has been the relative contributions of proximal versus distal airway involvement and their relationship to functional impairment of the lung. Because the methods here enables quantitation of fibrosis throughout the entire murine lungs, they can be used study patterns of fibrosis. This includes study of distribution patterns in the variety of models of airway remodeling such as the bleomycin-induced lung injury model mediated by direct epithelial injury, or allergen-induced remodeling mediated by immune cell-secreted fibrogenic cytokines.
In conclusion, this study presents a new method by which fibrosis may be studied across the whole murine lung, addressing a significant gap in the field for visualizing fibrosis in this organ. This capability will be a powerful tool for preclinical studies of fibrosis that can be coupled with other traditional approaches such as whole body imaging by xray, CT, PET or IVIS which do not provide direct measure of collagen but which can provide *in vivo* functional measures prior to lung extraction for methods described here. OC in combination with whole lung MPM/SHGM provided global assessment of lung fibrosis, avoiding regional bias. To our knowledge, this is the first study to quantify fibrillar collagen in the bronchial subepithelial space throughout the entirety of the lung with microscopy. Micro- and macro- structural anatomical components can be studied with this application, making it a useful method to investigate the effects of pulmonary diseases.
Ethical Approval {#Sec17}
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Animal experiments were performed according to the NIH Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee (approval no. 1312058A).
Electronic supplementary material
=================================
{#Sec18}
Supplementary Material
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
=================================
**Supplementary information** accompanies this paper at 10.1038/s41598-018-31182-2.
The authors acknowledge Heather Lander and Olivia Solomon for technical as well as editorial assistance. We would also like to thank Jinping Yang for support on animal handling and tissue preparation. Core laboratory support was provided by the UTMB Immunohistochemistry Core and the Optical Imaging Cores. This work was supported, in part, by NIH grants NIAID AI062885 (ARB), UL1TR001439 (ARB, GM, MM), NIEHS T32ES007254 (BTA), NIEHS ES006676 (ARB, BTA), the Institute for Human Infections and Immunity (AF, GV, MM), the Sealy Center for Molecular Medicine, the Sealy Center for Environmental Health and Medicine, and the Brown Foundation.
L.F.O., R.P., A.F., B.T., A.R.B., M.M. and G.V. designed research; L.F.O. and A.K. conducted research; L.F.O., R.P., P.V., and G.V. analyzed data; L.F.O., R.P., P.V., and G.V. wrote the manuscript, all other authors critically revised the manuscript; all authors approved of the submitted version of the manuscript.
Data will be made available upon reasonable request.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
|
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1. Introduction {#sec1}
===============
Recent decade has witnessed the great progress of neural stem cell- (NSC-)based cancer gene therapy. NSCs are multipotent cells that give rise to the three principal neural lineages, neurons, astrocytes and oligodendrocytes, throughout the nervous system. When brain tumors are present, NSCs are capable of migrating through the brain parenchyma to home in on tumor foci by using cytokines, chemokines, and growth factors released from the tumor sites as candidate migration stimulatory signals. In animal tumor models, the innate tropic behavior of NSCs for tumors has been exploited extensively for targeted delivery of therapeutic genes not only to brain tumors, but also to peripheral nerve tumors and solid tumors of a nonneural origin. In these studies, intratumorally or intravenously injected NSC vectors expressing a therapeutic transgene, such as the suicide gene thymidine kinase (TK), are demonstrated to significantly suppress the growth of various solid malignant tumors via strong bystander effects \[[@B1]--[@B4]\]. However, further biodistribution experiments using mouse breast cancer models showed that the majority of intravenously injected NSCs became trapped in some nontarget organs, such as lung, liver, spleen, and kidney, probably due to the narrow diameters of capillaries in these organs \[[@B1], [@B5], [@B6]\]. Moreover, constant expression of the suicide gene TK from these off-target NSCs caused significant cytotoxic effects on liver and kidney \[[@B1]\]. Thus, it raises a safety concern that the use of these NSC vectors may deteriorate the situations of the patients. To harness the cytotoxic effect of these NSC vectors, an expression cassette that triggers tumor site-specific transgene expression is desired.
In view of the recent finding that hypoxia plays a key role in the directed migration of NSCs towards tumors \[[@B7]\], hypoxia-targeting approach can be employed to regulate transgene expression in NSC vectors. Hypoxia is a hallmark of the tumor microenvironment. In most human tissues, the physiological oxygen tensions range from 2% to 9%. However, due to the high metabolic rate, the oxygen tensions in tumor sites can fall far below the normal physiological levels, even down to 0.1% in necrotic regions \[[@B8]\]. Hypoxia correlates positively with tumor aggressiveness and has been implicated in inducing secretion of angiogenic factors, activating metabolic shift to anaerobic glycolysis, promoting epithelial-to-mesenchymal transition, remodeling extracellular matrix, and providing a selective survival advantage to cancer stem cells \[[@B9]--[@B11]\]. The dominant regulator of cellular response to hypoxia stress is hypoxia-inducible factors (HIFs), which bind to hypoxia-responsive elements (HREs) (5′-A/GCGTG-3′). HREs are minimal cis-regulatory elements that mediate transcriptional activation of more than 60 genes crucial to systemic hypoxia responses \[[@B12]\]. Some of these hypoxia-responsive genes, such as CXCR4 and VEGF, are closely correlated to the regulation of tumor tropism of NSCs \[[@B7]\]. Therefore, HREs can be used as a means for transcriptional targeting of tumor hypoxia.
MicroRNAs (miRNA) are alternative regulators of the cellular response to hypoxia stress. MiRNAs are endogenous small noncoding RNAs of 22\~23 nucleotides that suppress mRNA translation by targeting complementary sequences in 3′-untranslated region (3′UTR). It has been described that hypoxia can cause miRNA expression alternations in cells. For instance, miRNA-210 is stimulated under hypoxic condition and regulates E2F3, an important protein in cell cycle, in breast and ovarian cancers \[[@B13], [@B14]\]. On the contrary, miRNA-199a-5p is reported to be acutely downregulated and derepress its target HIF1A in cardiac myocytes upon hypoxia treatment \[[@B15]\]. Recently, a series of studies have demonstrated that by incorporating properly engineered targeting sequences for a highly expressed endogenous miRNA into the 3′UTR of the expression cassette, the transgene expression can be suppressed up to 100-fold in a cell type-specific manner \[[@B16]--[@B18]\]. Thus, artificial miRNA targeting sequences have emerged as a powerful new tool for regulation of transgene expression.
To realize the full potential of NSC-based cancer therapy, it is desirable to develop approaches that enable precise control of transgene expression. We reason that the use of a hypoxia-responsive promoter could be a means to direct transgene expression preferentially to hypoxic tumor sites. Furthermore, the use of miRNA regulation, adjunct to the use of the hypoxia-responsive promoter, would provide the second layer of control to differentiate transgene expression between tumor sites and healthy organs. To test the hypothesis, we constructed an NSC vector platform that couples hypoxia-responsive promoter with miRNA regulation for tumor hypoxia-targeted gene therapy. The goal was to drive transgene expression in hypoxic tumor site, while sparing other healthy organs in case of local or systemic leakage of intratumorally injected vectors. We show that this combinatorial control using transcriptional targeting and miRNA regulation resulted in negligible off-target transgene expression in a mouse tumor model.
2. Materials and Methods {#sec2}
========================
2.1. iPS Cell Generation and Neural Differentiation {#sec2.1}
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An induced pluripotent stem (iPS) cell line was derived from the skin fibroblasts of a healthy person as described previously \[[@B19]\]. Briefly, skin fibroblasts were infected with viral supernatants generated by transfection of HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with retroviral pMXs vector (AddGene, Cambridge, MA, USA) containing the cDNAs of human OCT4, SOX2, KLF4, and c-MYC. Two rounds of infection were performed successively (12 h each). Polybrene (4 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to increase infection efficiency. After the second round of infection, cells were trypsinized and seeded onto a layer of mouse embryonic fibroblasts cell (MEF) feeders in a 10 cm culture dish using hES cell medium. The medium was changed every day. From day 12 to day 18, those colonies, which were large enough and identifiable as hES cell-like, were picked mechanically and expanded in hES medium on feeders. The iPS clones were characterized by alkaline phosphatase (AP) activity staining and immunostaining for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Then the iPS cells were routinely passed every 4-5 days, and the medium was changed every day.
NSCs were derived from human iPS cells using an adherent monoculture differentiation method as described previously \[[@B5]\]. In brief, iPS cell colonies were detached from the 6-well cell-culture plate 7 days after plating by mechanical cutting. Then, iPS cells were dissociated using TrypLE express dissociation enzyme (Invitrogen) and plated onto a 0.1% gelatin-coated 6-well cell-culture plate at a density of 10^6^ per well and cultured in NSC medium, which was a 1 : 1 mixture of DMEM/F12 (Invitrogen) supplemented with 2% B27 (Invitrogen), 2 mM L-glutamine, 50 U/mL penicillin, 50 *μ*g/mL streptomycin, 20 ng/mL EGF (Sigma-Aldrich), and 20 ng/mL bFGF (Invitrogen). Half of the cell-culture medium was changed every 2 days. After 7 days of differentiation, the cells reached 90% confluence and were split at ratio of 1 : 2. After 1 month of expansion, NSCs were derived from iPS cells. NSCs were digested using TrypLE for cell passage and subcultured at ratio of 1 : 2 twice weekly.
2.2. Cell Culture {#sec2.2}
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MCF-7 breast cancer cell line was maintained in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin, and 50 *μ*g/mL streptomycin. For hypoxia treatment, cell cultures were incubated in a hypoxia chamber (STEMCELL Technologies) filled with an anaerobic gas mixture of 94% N~2~, 5% CO~2~, and 1% O~2~.
2.3. In Vitro Boyden Chamber Cell Migration Assay {#sec2.3}
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In vitro migration assays used the MCF-7 breast cancer cell line as an attractant and was performed in Boyden chambers with the BD Falcon HTS FluoroBlok 24-well Multiwell Insert System with 8 mm pore size (BD Falcon). One day before the assays, MCF-7 cells were suspended in serum-free Opti-MEM (Invitrogen) and seeded into a 24-well companion plates at a cell density of 2.5 × 10^5^ per well. NSCs were labeled with Calcein-AM (Invitrogen). The labeled NSCs were suspended in serum-free Opti-MEM and seeded into the Boyden chamber Transwell inserts at a cell density of 5 × 10^4^ per insert. After 24 h of culture at 37°C in 5% CO~2~, the fluorescent cells on the bottom side of the inserts (corresponding to migrating cells) were counted and the migration rate was calculated. All experiments were conducted in 6 replicates.
2.4. Plasmid Constructs {#sec2.4}
-----------------------
pGL4.11 (Promega) carrying the luc2P reporter gene was used as a starting backbone to construct the combinatorial transgene expression cassettes through multistep subcloning. Firstly, all promoters used in this study were placed upstream of the reporter gene. Secondly, 4× miRNA targeting sequences (mirT) were designed to be perfectly complementary to the respective miRNA (in lowercase in [Table 1](#tab1){ref-type="table"}) with 3 different linkers spacing each targeting sequence. The respective sense and antisense strands of the 4× mirT oligonucleotides were phosphorylated, annealed, and then inserted downstream of the reporter gene. A control scramble targeting sequence (ScrT) of the same size was designed based on the lack of significant similarity to any known miRNA and subcloned into the same region ([Table 1](#tab1){ref-type="table"}).
2.5. Luciferase Assay {#sec2.5}
---------------------
Subconfluent cells in 48-well plate were transfected with plasmids encoding luciferase reporter gene at 400 ng per well, using 1.2 *μ*L Fugene 6 (Roche) according to the manufacturer\'s protocol. After 24 h, the cells were lysed by freeze-thaw method and the supernatants were measured for luciferase activity using Luciferase Assay System (Promega) according to the manufacturer\'s instructions. All samples were assayed 3 times in triplicate.
2.6. qPCR {#sec2.6}
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For mRNA qRCR, total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer\'s protocol. First-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). 1 *μ*L of cDNA reaction mix was subjected to PCR amplification using PCR SuperMix (Invitrogen). The forward and reverse primers for qPCR analysis were listed in [Table 2](#tab2){ref-type="table"}. GAPDH was selected as the internal reference gene for PCR quantification.
For miRNA qPCR, small RNA was isolated using PureLink microRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion). PolyA tailing and cDNA synthesis of the DNase-treated small RNA were performed using Ncode VILO microRNA cDNA Synthesis Kit (Invitrogen) according to the manufacturer\'s protocol. The forward primers for qPCR analysis were designed based on entire known mature miRNA sequence, with additional 3 "A"s at the 3′ end to improve amplification specificity ([Table 2](#tab2){ref-type="table"}). The reverse primer used was the universal primer in the EXPRESS SYBR GreenER microRNA qRT-PCR Kit (Invitrogen). 5S rRNA was selected as the internal reference gene for PCR quantification. To determine absolute copy number, a standard curve was generated using a synthetic LIN-4 RNA oligonucleotide.
qPCR was performed on iQ5 RT-PCR detection system (BioRad). All reactions were run in triplicate.
2.7. Animal Experiment {#sec2.7}
----------------------
Three adult female BALB/c athymic, immunoincompetent nude mice (weight 20 g; aged 6--8 weeks) were used. To establish an orthotopic mouse model of breast tumor, 1 × 10^6^ MCF-7 cells were injected into the right mammary fat pad and sham injection was given on the contralateral side. After 1 week, the tumor developed and 1 × 10^6^ NSC vectors were injected into the tumor site and into sham injection site, respectively. After 24 h, in vivo transgene expression levels were measured by the Xenogen IVIS-100 bioimaging system (Caliper).
2.8. Statistical Analysis {#sec2.8}
-------------------------
All data are represented as mean ± s.d. The statistical significance of differences was determined by Student\'s *t*-test or the two-factor analysis of variance (ANOVA). A *P* value of \<0.05 was considered to be statistically significant.
3. Results {#sec3}
==========
3.1. Generation of iPS-Derived Tumor-Tropic NSCs {#sec3.1}
------------------------------------------------
For the derivation of NSCs, an induced pluripotent stem (iPS) cell line was created from skin fibroblasts by retroviral transduction of the four Yamanaka\'s factors (Oct4, Sox2, Klf4, and c-Myc) \[[@B20]\] and characterized by AP staining and immunostaining for the pluripotency markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 ([Figure 1(a)](#fig1){ref-type="fig"}). Then NSCs were derived from the above iPS cells by a simple adherent monolayer culture method and characterized by immunostaining for nestin, an early-stage marker of NSCs ([Figure 1(b)](#fig1){ref-type="fig"}). After sequential withdrawal of bFGF and EGF, they generated a mixed population of glial fibrillary acidic protein- (GFAP-)positive glial cells and *β*-III tubulin-positive neurons ([Figure 1(b)](#fig1){ref-type="fig"}). Thus, the iPS-derived NSCs we obtained possessed the potential to differentiate into glial cells and neurons.
We then further characterized the tumor-tropic capacities of these iPS-derived NSCs under hypoxic condition. Since it is reported that the tumor tropism of NSCs is regulated by hypoxia-related signaling pathways \[[@B7]\], the mRNA expression alternation of hypoxia-related signal receptors, c-Met, CXCR4, c-Kit, and VEGFR2, in NSCs upon hypoxia treatment was measured by qPCR ([Figure 2(a)](#fig2){ref-type="fig"}). The results showed that the expression levels of all the above receptors were upregulated by 3-4 folds in NSCs after hypoxia treatment. Next, the in vitro migration ratio of NSCs towards MCF-7, a human breast cancer cell line, was measured by Boyden chamber cell migration assays ([Figure 2(b)](#fig2){ref-type="fig"}). The results displayed that under hypoxic condition, the percentage of migrated NSCs towards MCF-7 increased from 20% to 50%. Taken together, our results demonstrated that the iPS-derived NSCs maintain the tumor-tropic migratory capacity which is probably stimulated by the hypoxia-related cytokines released from tumor cells.
3.2. OptHRP Is the Optimal Hypoxia-Responsive Promoter in NSCs {#sec3.2}
--------------------------------------------------------------
To explore the optimal hypoxia-responsive promoter for transcriptional targeting in the NSC vectors, we chose three candidate HRE-containing promoters based on literatures: (1) a 300 bp CXCR4 promoter \[[@B21]\]; (2) a 2 kb artificial promoter (VEGF) composed of 5× VEGF HREs followed by a miniCMV promoter \[[@B22]\]; (3) a 64 bp artificial promoter (optHRP) composed of 4× optimized HREs followed by a TATA box \[[@B23]\]. We cloned these promoter constructs into the pGL4.11 luciferase reporter plasmids and then transfected them into NSCs to test their activities under normoxic and hypoxic conditions, respectively. Another one driven by EF1*α* promoter, a strong promoter widely used for constitutive transgene expression in most human cell types, was included as a positive control. The promoterless basic pGL4.11 plasmid was used as a reference for minimal transgene expression.
The luciferase assay results ([Figure 3(a)](#fig3){ref-type="fig"}) showed that all the three hypoxia-responsive promoters displayed lower activities under normoxic condition and could be induced under hypoxia treatment. Moreover, their hypoxia-induced activities were comparable to the EF1*α* promoter, which indicated that their activities were strong enough to drive robust transgene expression for therapeutic purpose. Among these promoters, optHRP exhibited the highest differential expression levels between normoxic and hypoxic conditions, up to 34 folds. Therefore, optHRP was selected as the optimal hypoxia-responsive promoter for NSC vectors. However, its basal activity in NSC vectors under normoxic condition was still 100 folds higher than that of the promoterless reference, which implied a promoter leakage at off-target sites. Since prolonged, though low, off-target transgene expression can result in cytotoxicity to surrounding healthy tissues, it is necessary to add a second layer of control to shut down the leaky transgene expression.
3.3. miR-199a-5p Is Enriched in NSCs and Downregulated upon Hypoxia Induction {#sec3.3}
-----------------------------------------------------------------------------
In order to solve the problem of promoter leakage, we next screened for endogenous miRNAs that selectively express under normoxic condition to block off-target transgene expression. According to literature, miR-199a-5p is an inhibitor of HIF-1*α* and decreases in response to hypoxia in cardiac myocytes \[[@B15]\]. Hence, we investigated whether it would be the same case in NSCs. Small RNA samples were extracted from NSCs under normoxic and hypoxic conditions, respectively. And the absolute copy number per pg small RNA of miR-199a-5p was quantified using qPCR. The results ([Figure 3(b)](#fig3){ref-type="fig"}) showed that miR-199a-5p was highly expressed in NSCs under normoxic condition, up to 2130 copies per pg small RNA. Based on previous report, an expression level above 100 copies per pg small RNA is sufficient for the endogenous miRNA to yield suppression effect on exogenous transgene expression \[[@B17]\]. In addition, the miR-199a-5p expression level in NSCs was dramatically downregulated by about 73% upon hypoxia treatment. Therefore, miR-199a-5p holds the potential to block transgene expression in a normoxia-specific manner.
3.4. Combinatorial Regulation of Transgene Expression In Vitro {#sec3.4}
--------------------------------------------------------------
To employ both hypoxia-responsive promoter and miRNA regulation to restrict transgene expression to hypoxic condition, we constructed a combinatorial expression cassette ([Figure 4(a)](#fig4){ref-type="fig"}). We used the optHRP-luc plasmid generated in the previous promoter activity experiment and introduced perfectly complementary miRNA target sequences of miR-199a-5p into the 3′UTR of the luciferase reporter gene (optHRP-luc-mir199a5pT). To rule out the possibility of less favorable transcription caused by introduction of a long repeat sequence into the 3′UTR, we constructed a control plasmid optHRP-luc-ScrT by replacing the miR-199a-5p target sequences with a scramble target sequence of the same length, which was designed based on the lack of significant similarity to any known miRNA ([Table 1](#tab1){ref-type="table"}).
After including the above regulatory elements into the luciferase reporter plasmids, we tested the ability of miRNA target sequences to repress luciferase gene expression in NSC vectors before and 24 h after hypoxia treatment. The promoterless reference plasmid and the EF1*α* positive control plasmid were included as previously mentioned. The results ([Figure 4(b)](#fig4){ref-type="fig"}) showed that the optHRP-miRNA199a5pT expression cassette obtained significantly higher hypoxic selectivity than that of the optHRP-luc expression cassette, from 34 folds up to 176 folds. Remarkably, its expression level at normoxic condition is suppressed to be as low as that of the promoterless reference plasmid. As a comparison, the insertion of scramble targeting sequence did not repress transgene expression under normoxic condition significantly. Taken together, our findings suggest that the optHRP-luc-mir199a5pT expression cassette is a hypoxia-inducible, nonleaky transgene expression cassette for NSC vectors. And the inhibition of transgene expression under normoxic condition is probably due to the binding of the artificially introduced target sequences to corresponding endogenous miRNAs but not due to transcriptional suppression.
3.5. Tumor Site-Specific Transgene Expression In Vivo {#sec3.5}
-----------------------------------------------------
We next investigated whether the combinatorial NSC vectors can mediate tumor site-specific transgene expression in vivo. An orthotopic mouse model of breast cancer was established by inoculating MCF-7 breast cancer cells into the right mammary fat pad and sham into the left ([Figure 5(a)](#fig5){ref-type="fig"}). After the tumor developed, NSCs transfected with the optHRP-luc-mir199a5pT plasmid were injected into the tumor sites and sham sites, respectively. The next day, luciferase reporter gene expression levels were monitored by a live animal bioimaging system ([Figure 5(b)](#fig5){ref-type="fig"}). Quantitative results from all the three mice were summarized in [Figure 5(c)](#fig5){ref-type="fig"}, showing that NSC vectors displayed induced luciferase gene expressions in tumor sites by averagely 30 folds higher than those in sham sites. Thus, the combinatorial NSC vectors are demonstrated to be capable of mediating tumor site-specific transgene expression in vivo.
4. Discussion {#sec4}
=============
In this study, we report the successful construction of a novel expression cassette which triggers transgene expression within NSC vectors in a hypoxia-inducible manner. To the best of our knowledge, this is the first combinatorial transgene expression cassette designed for NSC vectors.
The great potential of NSCs in cancer therapy underlines the need for reliable and stable sources for the large scale production of human NSCs suitable for clinical applications. The use of human iPS cells has provided a robust and accessible source to produce unlimited amounts of human NSCs for cancer gene therapy \[[@B24]\]. iPS cell-based approach also helps bypass the sensitive ethical issue surrounding the use of human embryonic stem (hES) cells and the safety concern of immune rejection by allogeneic transplantation. In this study, an iPS cell line derived from skin fibroblasts was created and characterized for derivation of NSCs. The iPS-derived NSCs possessed the potential to differentiate into multiple neural lineages and could be genetically manipulated for the test of our system. More importantly, our in vitro Boyden chamber cell migration assays demonstrated that these NSCs maintained the tumor-tropic capacity as previously described, which indicated the feasibility of translating the iPS cell techniques into personalized therapies. Intriguingly, our NSCs displayed a significantly higher migratory rate towards tumor cells under hypoxic condition. Based on the recent findings that hypoxia plays an essential role in the maintenance and growth of cancer stem cells \[[@B10], [@B25]--[@B27]\], there has been an emergent hypothesis that cancer stem cells are concentrated in the more alkaline hypoxic regions of a tumor mass \[[@B28]\]. Therefore, the use of hypoxia-inducible transgene expression cassette in NSC vectors may offer an additional advantage in targeting cancer stem cells.
Previous studies have extensively exploited hypoxia-responsive promoters for transcriptional targeting of tumor hypoxia. In this study, we tested the CXCR4 promoter \[[@B21]\], an engineered VEGF promoter \[[@B22]\] and an artificially optimized promoter optHRP \[[@B23]\] based on literatures. Although optHRP displayed the highest hypoxic selectivity (34 folds) among these promoters in our NSCs, its basal activity under normoxic condition was still detectable, approximately 100 folds higher than the promoterless reference. This finding indicates that even after careful design and artificial engineering, "promoter leakage" is still hard to avoid. Since NSC vectors trapped in off-target organs may survive for up to 14 days even in immunocompetent mice \[[@B5]\], the prolonged, though low, off-target transgene expression may cause decent damages in the these organs. Hence, a second layer of transgene control on top of transcriptional targeting is necessary.
Substantial studies from different groups have demonstrated the feasibility of employing endogenous miRNA to inhibit transgene expression in a cell type-specific manner \[[@B16]--[@B18], [@B29]\]. However, our study is the first to demonstrate that endogenous miRNA can also be exploited to block transgene expression in a condition-specific manner, as miRNA expression level alters in response to cell stress. In this study, we showed that incorporation of miR-199a-5p targeting sequences into hypoxia-inducible expression cassette successfully enhanced the specificity from 34 folds to 176 folds in NSCs. Remarkably, the transgene expression level under normoxic condition was suppressed to a minimal level as low as the promoterless reference, which implied the effectiveness of using endogenous miRNA regulation to diminish the off-target transgene expression. Therefore, this combinatorially regulated transgene expression cassette represents a valuable tool for the NSC-mediated gene delivery, as it provides a feasible solution for the safety concern on the off-target transgene expression and makes the NSC vectors more translatable for clinical application.
Besides the transcriptional and post-transcriptional regulations, other additional approaches can also be employed to enhance the hypoxic selectivity of the transgene expression cassette. For example, the oxygen-dependent degradation (ODD) domain can be employed as a post-translational regulator to suppress transgene expression under normoxic condition \[[@B30]\]. The ODD domain derived from HIF1A gene can be inserted downstream of the transgene and facilitate degradation of the fusion protein product in cells under physiological oxygen concentration, thus adding an extra barrier on off-target transgene expression.
5. Conclusion {#sec5}
=============
In summary, this report demonstrates an inducible, nonleaky expression cassette which functions within NSC-based gene delivery vectors for tumor site-specific transgene expression. Our data exhibit that it is able to selectively trigger reporter gene expression under hypoxic condition in vitro and at tumor sites in vivo. Most importantly, a negligible transgene expression level in nontarget region is observed, indicating the high safety of applying this regulatory system for NSC-mediated gene delivery. Further refinement of this system may lead to the development of optimal cell-based gene delivery vector to target malignant tumors.
This work was supported by China Postdoctoral Science Foundation General Programs the 55th batch the 1st class (no. 2014M550434), Academic Research Grants of Guangzhou Medical University (nos. 2012A09, 2013C55) and Academic Research Grants of the Third Affiliated Hospital of Guangzhou Medical University (no. 2013Y08).
Conflict of Interests
=====================
All authors have no conflict of interests.
{#fig1}
{#fig2}
{#fig3}
{ref-type="table"} were inserted into the 3′UTR; pA, polyA signal. (b) Transgene expression levels of different expression cassettes within NSCs under normoxic and hypoxic conditions are quantified by luciferase assays. Abbreviation: RLU, relative luminescence unit. Error bars: s.d. \**P* \< 0.05, \*\**P* \< 0.01.](BMRI2014-751397.004){#fig4}
{#fig5}
######
MicroRNA targeting (mirT) sequences.
mirT Sequence
------------- ------------------------------------------------------------------------- ----------------------------------------------------------------------
miR-199a-5p S1 5′-CTAGATAAgaacaggtagtctgaacactgggCGATgaacaggtagtctgaacactggg-3′
S2 5′-ACGCGTgaacaggtagtctgaacactgggTCACgaacaggtagtctgaacactgggGCATG-3′
AS1 5′-ACGCGTcccagtgttcagactacctgttcATCGcccagtgttcagactacctgttcTTAT-3′
AS2 5′-CcccagtgttcagactacctgttcGTGAcccagtgttcagactacctgttc-3′
ScrT S1 5′-CTAGAtaatttatgatctgcgcgtggagacgcccgattttatgatctgcgcgtggagacgcc-3′
S2 5′-acgcgttttatgatctgcgcgtggagacgcctcactttatgatctgcgcgtggagacgccGCATG-3′
AS1 5′-acgcgtggcgtctccacgcgcagatcataaaatcgggcgtctccacgcgcagatcataaattaT-3′
AS2 5′-Cggcgtctccacgcgcagatcataaagtgaggcgtctccacgcgcagatcataaa-3′
######
qPCR primers.
Gene symbol Primer sequence
---------------------------------- -------------------------------------
MET F: 5′-TGATGATGAGGTGGACACA-3′
R: 5′-ATTTTGGCAAGAGCAAAGA-3′
CXCR4 F: 5′-CAAGGCCCTCAAGACCACA-3′
R: 5′-CCCAATGTAGTAAGGCAGCCAA-3′
KIT F: 5′-CGCCTGGGATTTTCTCTGC-3′
R: 5′-TCACAGATGGTTGAGAAGAGCCT-3′
VEGFR2 F: 5′-CGGCTCTTTCGCTTACTGTTCT-3′
R: 5′-AGCATGGAAGAGGATTCTGGACT-3′
miR-199a-5p F: 5′-CCCAGTGTTCAGACTACCTGTTCAAA-3′
F: forward primer; R: reverse primer.
[^1]: Academic Editor: Shinsuke Yuasa
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that are activated by growth factors or oncogenes,[@b1-ijn-8-2689] following which they phosphorylate the 3′-hydroxyl group of membrane phosphatidylinositols. Subsequent activation of the AKT (protein kinase B, PKB) downstream pathway by PI3Ks promotes cell survival.[@b2-ijn-8-2689] Aberrations in PI3K signaling commonly occur in many cancers; for example, *PIK3CA*, which encodes the p110 catalytic subunit of PI3K, is probably the most commonly (15%) mutated kinase in all cancers. In contrast, phosphatase and tensin homolog (*PTEN*), which encodes the phosphatase opposing PI3K, is known to be mutationally or post-translationally inactivated in tumors.[@b3-ijn-8-2689],[@b4-ijn-8-2689] The activation of PI3K signaling in cancer also occurs via receptor tyrosine kinases (RTKs), AKT, and RAS.[@b5-ijn-8-2689] It is believed that the many variations in PI3K signaling present important therapeutic targets for cancer treatment.
LY294002 (LY) is a potent inhibitor of PI3K activity and acts as a competitive and reversible inhibitor for the ATP-binding site of the enzyme.[@b6-ijn-8-2689] It has also been demonstrated to have antitumor activity in vitro.[@b7-ijn-8-2689]--[@b10-ijn-8-2689] Moreover, LY significantly inhibits growth and ascites formation associated with ovarian carcinoma and induces apoptosis of nasopharyngeal carcinoma cells in animal xenograft models.[@b11-ijn-8-2689]--[@b13-ijn-8-2689] However, LY has poor solubility in water and poor pharmacokinetics in vivo.[@b14-ijn-8-2689] Improvement of the physical and pharmacokinetic properties of LY is therefore desirable for therapeutic application.
Nanotechnology is considered to be a feasible approach for targeted drug delivery by minimizing potential side effects.[@b15-ijn-8-2689] To date, biocompatible nanoparticles (NPs) comprising various materials such as metals, polymers, and nano-magnets have been employed as nanodrug delivery systems to target cancer cells.[@b16-ijn-8-2689]--[@b19-ijn-8-2689] Among the nanomaterials available for drug delivery, poly(lactic-co-glycolic acid) (PLGA) has been approved by the US Food and Drug Administration for use in humans.[@b20-ijn-8-2689] It has been demonstrated to be an ideal carrier of drug delivery systems owing to its unique properties, including biodegradable hydrolysis to metabolite monomers (lactic and glycolic acids); easy formulation for hydrophobic, poorly soluble drugs; easy surface modification or bioconjugation for targeting; and high biochemical and physicochemical stability.[@b21-ijn-8-2689]--[@b23-ijn-8-2689]
A previous study reported the use of a surfactant (polyvinyl alcohol; PVA) to formulate LY-loaded, PVA-containing, PLGA NPs (PVA-LY NPs) for improving LY solubility.[@b24-ijn-8-2689] Although PVA-LY NPs could cause cell death resulting from the sustained release of LY inside cells, the conjugates inhibited cell proliferation to a lesser extent compared with free LY.[@b24-ijn-8-2689] A possible explanation is that the residual P VA associated with PLGA prevented cellular uptake of NPs.[@b25-ijn-8-2689] Accordingly, we developed a novel surfactant-free PLGA copolymer[@b26-ijn-8-2689],[@b27-ijn-8-2689] to carry LY. We demonstrate that in the absence of surfactant, LY-loaded, surfactant-free NPs (SF-LY NPs) enhanced the incorporation efficacy of NPs, caused sustained inhibition of AKT phosphorylation, targeted LY to the endoplasmic reticulum (ER) lumen, and effectively decreased cancer cell growth in vitro and in vivo.
Material and methods
====================
Cell culture and reagents
-------------------------
The cell lines H460, H157, H1650, and NL20 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). H460, H157, and H1650 cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA) and the NL20 cells were grown in Ham's F12 medium supplemented with glucose (2.7 g/L), L-glutamine (2 mM), nonessential amino acids (0.1 mM), insulin (0.005 mg/mL), epidermal growth factor (EGF, 10 ng/mL), transferrin (0.001 mg/mL), hydrocortisone (500 ng/mL), and FBS (4%). The parental PC14PE6 cell line was generously provided by Dr Fidler (MD Anderson Cancer Center, Houston, TX, USA), and its derivative PC14PE6/AS2 (AS2) cell line was subsequently established in our laboratory.[@b28-ijn-8-2689] The AS2 cells were maintained in Minimum Essential Medium Alpha Modification (α-MEM; Life Technologies) supplemented with 10% FBS. Copolymers of PLGA (molecular weight: 35,000--65,000 Da; lactide:glycolide, 50:50) were purchased from Bio Invigor Corporation (Taipei, Taiwan). PVA, 3-\[4,5-dimethylthiazol-2-yl\]-2, 5-diphenyltetrazolium bromide (MTT), Nile red, 3-methyladenine (3-MA), bafilomycin A1, dimethyl sulfoxide (DMSO), Tris-HCl, NP-40, ethylene diamine tetra-acetic acid (EDTA), sodium dodecyl sulfate (SDS), ethanol, acetone, sodium chloride, disodium hydrogen phosphate, potassium chloride, trifluoroacetic acid (TFA), and dimethyl fumarate (DMF) were purchased from Sigma-Aldrich (St Louis, MO, USA). ER-Tracker™ Green was purchased from Molecular Probes (Life Technologies) and LY was purchased from Biomol (Plymouth, PA, USA). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Preparation of PLGA NPs
-----------------------
The method for preparation of surfactant (PVA)-containing PLGA NPs was described previously,[@b24-ijn-8-2689] while the method for preparation of surfactant-free PLGA NPs (SF NPs) was modified from a previous study.[@b26-ijn-8-2689] In brief, to prepare LY or Nile red-loaded PLGA NPs (SF-LY NPs or SF-Nile red NPs), 50 mg of PLGA polymers was dissolved in 5 mL of acetone and 1 mg of LY or Nile red, respectively, was added. Next, an ethanol/H~2~O (50/50, % v/v) solution was added dropwise (1 mL/minute) into the PLGA solution using a tubing pump. The solution was stirred at 240 rpm until the mixture was turbid. The suspension was transferred into 20 mL of deionized water and stirred at 300 rpm for 15 minutes. The organic solvent was then removed under reduced pressure for 30 minutes. To prevent PLGA from aggregating, the solution was filtered using 90 mm filter paper. To measure encapsulation capacity, PLGA NPs were collected and dissolved in acetone and the content of LY was determined using an ultraviolet-visible (UV--Vis) spectrometer (Hewlett-Packard Model 8453; Hewlett Packard, Palo Alto, CA, USA) at a wavelength of 300 nm. Encapsulation capacity (%) was determined by comparing the amount of encapsulated LY with the initial LY concentration.
Transmission electron microscopy (TEM) of NPs
---------------------------------------------
Electron micrographs of PLGA NPs were acquired by placing a drop of the sample onto a copper mesh coated with amorphous carbon film (79725; Ted Pella Inc, Redding, CA, USA) and dried in a vacuum desiccator (Thermo Scientific Nalgene Vacuum Desiccator, EW-06520-05; Thermo Fisher Scientific, Waltham, MA, USA). PLGA NPs were stained with phosphotungstic acid before TEM (CM-200; Philips, Amsterdam, Netherlands) at 80 KV The mean diameter and morphology of PLGA NP images were recorded.
In vitro PI3K inhibitor release studies
---------------------------------------
NPs (50 mg) were added directly to a 1.5-mL Eppendorf tube containing 1 mL of phosphate-buffered saline (PBS; 10 mM, pH 7.4) with 0.1 mL of FBS. The supernatant containing LY released from the PLGA NPs was collected after 10 minutes of centrifugation at 13,000 rpm at various time intervals after the addition of NPs. The total amount of LY in NPs (50 mg) was determined by the addition of acetone to dissolve the PLGA NPs and collection of the supernatant for assays. The amount of LY in the supernatant was measured by high-performance liquid chromatography (HPLC; Agilent 1100 series; Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of water (0.1% TFA) and methanol (0.1% TFA; 40:60 v/v). The reverse-phase column used was a CC 125/4 Nucleod UR 100-5 C18 (Macherey-Nagel, Düren, Germany). The column temperature was maintained at 25°C. The flow rate was set at 1.0 mL/minute and the detection wavelength was 300 nm. Sample solution was injected in a volume of 10 μL. The release pattern was defined as the ratio of measured to initial amount of LY encapsulated in the NPs.
MTT cytotoxicity assay
----------------------
Cells were seeded (5×10^3^ per well) in a 96-well culture plate and incubated overnight at 37°C in humidified air containing 5% CO~2~. Concentration-effect and time-course studies were performed in the presence of free LY, SF NPs, and SF-LY NPs. After the required incubation periods, 20 μL of MTT reagent stock solution (5 mg/mL in PBS) was added to each well and incubated for 4 hours at 37°C. Plates were centrifuged at 1200 rpm for 5 minutes. After the supernatant was discarded DMSO (200 μL) was added to each well followed by incubation for 5 minutes. The supernatants were transferred into a new enzyme-linked immunosorbent assay (ELISA) plate and absorbance was measured at 490 nm with an ELISA reader (Varioskan; Thermo Fisher Scientific).
Cytochrome c detection
----------------------
Cells (5×10^6^) were trypsinized and resuspended in ice-cold preparation buffer (10 mM Tris-HCl, pH 7.5, containing 300 mM sucrose supplemented with a protease inhibitor cocktail) on ice for 20 minutes. After incubation, cells were homogenized with Dounce homogenizer and centrifuged at 800 *g* for 10 minutes at 4°C. The resulting supernatant was centrifuged at 8000 *g* for 20 minutes at 4°C to yield mitochondria-enriched pellet and cytosolic supernatant fractions. These fractions were used to monitor cytochrome c release from mitochondria to cytosol by Western blotting.
Caspase 3/7 and caspase 8 activity assay
----------------------------------------
Cells (1×10^4^) were lysed in caspase assay buffer, mixed gently, and incubated at room temperature for 3 hours according to the Caspase-Glo™ 3/7 Assay or Caspase-Glo^®^ 8 Assay (Promega Corporation, Fitchburg, WI, USA) protocol. Caspase activity was quantified in a Veritas™ Microplate Luminometer (Promega).
Intracellular LY uptake
-----------------------
Cells (5×10^3^ per well) were seeded onto 6 cm dishes and incubated overnight at 37°C in humidified air containing 5% CO~2~. Free LY and NPs (SF-LY and PVA-LY) with equal concentrations of LY (50 μM) were added to the medium. Cells were harvested at the indicated time periods for further analyses. For LY detection in the ER organelle, isolation of the ER was performed with a Sigma-Aldrich ER isolation kit (catalog number ER0100--1 KT) according to the supplier's instructions. The levels of intracellular LY were measured according to a previously published protocol[@b24-ijn-8-2689] with a slight modification. Briefly, lysed cells were lyophilized to remove water before suspension in 100 μL of DMF and shaken for 1 hour to dissolve internalized LY. The samples were then centrifuged at 14,000 rpm for 1 hour to obtain supernatants for LY quantification by measurement of absorbance on a UV-Vis spectrometer at 300 nm against a standard concentration-absorbance curve.
Intracellular localization by confocal laser microscopy
-------------------------------------------------------
Viable cells (1×10^5^) were plated onto 3.5 cm dishes with 3 mL of culture medium. After overnight incubation, cells were treated with free Nile red or PLGA NPs loaded with Nile red (SF-Nile red NPs) for 3 hours of incubation and stained with ER tracker™ Green dye according to the manufacturer's protocol (Molecular Probes^®^; Life Technologies) and 4′,6-diamidino-2-phenylindole (DAPI). After replacement of original medium with particle-free, fresh medium, cells were immediately imaged with a scanning confocal microscope (Fluoview^®^ FV-1000; Olympus Corporation, Tokyo, Japan) set to detect fluorophores by A laser (excitation 405, 488, or 546 nm).
Western blot analysis
---------------------
Cells were lysed on ice for 30 minutes with whole-cell extract lysis buffer (50 mM Tris-HCl, pH, 7.2--7.8; 1% NP-40; 2 mM EDTA; 100 mM NaCl; 0.1% SDS supplemented with protease inhibitor cocktail; Roche Applied Sciences, Indianapolis, IN, USA). Lysates were collected by centrifugation at 14,000 rpm for 10 minutes and protein concentration was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Protein extracts (20--50 μg) were boiled for 5 minutes in sample buffer before separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 400 mA for 1.5 hours using an electroblotter (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA). Membranes were blocked with a mixture of Tris-buffered saline and Tween 20 (TBST) containing 5% nonfat milk for 1 hour at room temperature and incubated overnight at 4°C with pAKT-S473, AKT, CHOP, p-JNK, JNK, GRP78, and actin antibodies. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies using an enhanced chemiluminescence (ECL) kit (Amersham) according to the manufacturer's instructions.
Design and transfection of small interfering RNA (siRNA)
--------------------------------------------------------
The 21-nucleotide CHOP siRNA, 19-nucleotide JNK siRNA, and negative control duplexes were purchased from Dharmacon Research (Thermo Fisher Scientific). The CHOP siRNA sequence used was 5′-AAGAACCAGCAGAGGUCACAA-3′ and the siRNA JNK sequence for targeting both JNK isoforms (JNK1 and JNK2) was 5′-GAAAGAATGTCCTACCTTC-3′. The transfection of siRNA oligonucleotides was performed with LipofectAMINE 2000 (Life Technologies) according to the manufacturer's recommendations. In brief, 16 μL of LipofectAMINE 2000 reagent was mixed with 400 μL of Opti-MEM (Life Technologies) at room temperature for 5 minutes and incubated with a mixture of 12 μL of siRNA duplex and 400 μL of Opti-MEM for an additional 20 minutes at room temperature. The complexes were then applied to cultured cells at 70% confluence on a 60 mm plate. After incubating for 6 hours, the medium was replaced with fresh medium supplemented with 10% FBS and 1% penicillin/streptomycin.
Tumor xenograft experiments
---------------------------
Female athymic nude mice (6--8 weeks) were obtained from the National Laboratory Animal Center in Taiwan. All animal studies were approved by the Animal Welfare Committee at National Cheng Kung University for the same environment with controlled temperature, humidity, and a 12-hour light/ dark cycle. Mice were subcutaneously inoculated in the flank with PC14PE6/AS2 cells (1×10^6^ cells/mouse in 100 μL of PBS) and randomized at approximately 14 days after implantation, when the tumor size reached 50--60 mm^3^. The following intratumoral injections were administered every other day for a total of six times to the xenograft mice: normal saline (n = 8), SF NPs only (n = 8), LY (1 mg/kg; n = 8), or SF-LY NPs (LY, 1 mg/kg; n = 10). The treated mice were monitored for signs of distress and tumor size was measured with calipers. Tumor volume was calculated according to the following formula: (volume = long axis × short axis[@b2-ijn-8-2689] ×0.5). For ethical reasons, mice were sacrificed when tumor size reached 4000 mm^3^.
Statistical analysis
--------------------
All experiments were repeated at least three times. The data are presented as means ± standard error of the mean. Statistical analyses were performed using one-way and two-way analysis of variance (ANOVA). Differences in the mean values for apoptosis, tumor growth, and body weight response to treatment were compared between groups using two-way ANOVA with Bonferroni post-testing. All other data for statistical differences between experimental groups were analyzed using one-way ANOVA. A *P*-value of \< 0.05 was considered statistically significant.
Results
=======
Characteristics of surfactant-free PLGA NPs
-------------------------------------------
The particle size and polydispersity index (PDI) of SF NPs and SF-LY NPs were analyzed by dynamic light scattering (DLS; [Table 1](#t1-ijn-8-2689){ref-type="table"}). The mean diameter of SF NPs was 74.5 ± 1.74 nm, while that of SF-LY NPs was 98.9 ± 2.64 nm. Representative transmission electron micrographs illustrate that SF-LY NPs were spherical in shape and monodispersed. The mean diameter of SF-LY NPs was 80 nm as measured by TEM ([Figure 1A](#f1-ijn-8-2689){ref-type="fig"}). The encapsulation effciency and loading capacity of LY in SF NPs was 37% and 7.4 μg/mg, respectively. The release of LY from the SF-LY NPs was measured in vitro in serum-containing PBS. After incubation, there was a burst of initial release of LY (approximately 27% of total loaded LY) within the first 6 hours, followed by a slow release of LY (57% of the total LY amount) from 6 to 12 hours and a sustained release of approximately 73% of the total loaded LY over the next 7 days ([Figure 1B](#f1-ijn-8-2689){ref-type="fig"}).
In vitro cytotoxicity assay
---------------------------
To evaluate the dose-dependent cytotoxicity of SF NPs, free LY, PVA-LY NPs, and SF-LY, we conducted MTT assays in AS2 (*PTEN*-null), H157 (*PIK3CA* mutation, *PTEN*-null), H460 (*PIK3CA* mutation), and H1650 (EGFRΔE747-A750 mutation, *PTEN*-null) cells ([Figure 2A](#f2-ijn-8-2689){ref-type="fig"}). No suppression of cell growth was evident after AS2, H460, and H157 cells were treated with SF NPs and PVA-LY NPs for 48 hours or after H1650 cells were treated with SF NPs and PVA-LY NPs for 24 hours. Treatment with free LY induced modest cytotoxicity in all four cell lines, whereas treatment with a low concentration of LY (0.25--1.00 μM) in SF-LY NPs induced marked cellular death in the H460, H157, and H1650 cell lines. A relatively high concentration of LY (5 μM) was required in SF-LY NPs to induce similar cytotoxicity in AS2 cells. A time-course study of cytotoxicity was conducted with a fixed concentration of LY compounds ([Figure 2B](#f2-ijn-8-2689){ref-type="fig"}). Treatment with SF-LY NPs again resulted in a significantly greater suppression of cell growth in these lung cancer cell lines compared with treatment with free LY and PVA-LY NPs. The half maximal inhibitory concentration (IC~50~) value for free LY in the four cancer cell lines was 25--50 μM; however, the IC~50~ values for PVA-LY NPs in the four cell lines were greater than 50 μM ([Figure 2C](#f2-ijn-8-2689){ref-type="fig"}). These results are consistent with an earlier report[@b24-ijn-8-2689] that PVA-LY NPs are less effective than free LY in the inhibition of cancer cell growth. Taken together, these findings suggest that the cytotoxicity of SF-LY NPs is greater than that of PVA-LY NPs and free LY and that a relatively low concentration of LY in SF-LY NPs is sufficient to induce cancer cell death. Next, we examined the cytotoxicity of SF-LY NPs on an NL20 cell line; an immortalized, nontumorigenic, human, bronchial epithelial cell line, in a dose-dependent manner ([Figure 2D](#f2-ijn-8-2689){ref-type="fig"}). Even after treatment with SF-LY NPs containing relatively high concentrations of LY (0.25 to 5 μM), the growth suppression rate was approximately 15%. The toxicities from SF NPs, free LY, PVA-LY NPs, and SF-LY NPs to NL20 cells were comparable. The data show that there is only modest toxicity of SF-LY NPs to NL-20 cells, suggesting that the therapeutic window of SF-LY NPs is promising.
The mechanisms of cell death induced by SF-LY NPs
-------------------------------------------------
First, we analyzed the mechanism of cell death[@b29-ijn-8-2689] induced by SF-LY NPs using flow cytometric analysis (annexin V/propidium iodide \[PI\] double staining method) and found that SF-LY NPs, but not free LY, induced significant cellular apoptosis in the four cell lines ([Figure S1](#SD1-ijn-8-2689){ref-type="supplementary-material"}). The phenomenon of apoptosis was further supported by DAPI staining, which showed an increase in chromatin condensation in SF-LY NP-treated cells ([Figure S2](#SD2-ijn-8-2689){ref-type="supplementary-material"}). We next examined the release of cytochrome c from mitochondria into the cytosol. Treatment with SF-LY NPs resulted in higher levels of cytosolic cytochrome c in four lung cancer cells compared with free LY ([Figure 2E](#f2-ijn-8-2689){ref-type="fig"}). To further characterize the type of apoptosis activated, we measured the activities of caspase 3/7 and caspase 8 using a luminescent assay kit ([Figure 2F](#f2-ijn-8-2689){ref-type="fig"}). Although SF-LY NPs induced significantly high caspase 3/7 activities, they had little effect on caspase 8 activities in the four cells ([Figure 2F](#f2-ijn-8-2689){ref-type="fig"}). With regard to the free LY, it only modestly induced both caspase 3/7 and 8 activities in the four cells. We conclude that SF-LY NP-induced cell death occurs primarily via the intrinsic apoptotic pathway.
The effects of SF-LY NPs on phosphorylation of AKT in cells
-----------------------------------------------------------
The phosphorylation of serine 473 on AKT in response to different concentrations of LY, PVA-LY NPs, or SF-LY NPs was measured in AS2, H157, H460, and H1650 cell lines by Western blotting ([Figure 3A](#f3-ijn-8-2689){ref-type="fig"}). The ratio of pAKT/AKT was calculated for comparison of the effects of the test agent effects on the cells ([Figure 3A](#f3-ijn-8-2689){ref-type="fig"}). At the indicated concentrations, only SF-LY NPs effectively suppressed the levels of constitutively phosphorylated AKT in the four cancer cell lines. A sustained (\>24 hours) suppression of AKT phosphorylation was observed in H460, H157, and H1650 cells in response to a fixed concentration of SF-LY NPs. The same concentration of free LY had no effect ([Figure 3B](#f3-ijn-8-2689){ref-type="fig"}). In AS2 cells, free LY at a concentration of 5 μM induced a transient suppression of AKT phosphorylation at 8 hours, whereas the suppression of AKT phosphorylation by SF-LY NPs began after 8 hours and lasted up to 48 hours. In contrast to free LY, PVA-LY NPs with 25 μM LY were effective in decreasing AKT phosphorylation only in H1650 cells ([Figure 3C](#f3-ijn-8-2689){ref-type="fig"}). The difference between the phosphorylation levels in the four cell lines suggested that refractoriness to PVA-LY NPs is independent of the activation status of the target protein.[@b24-ijn-8-2689] Therefore, SF-LY NPs, but not free LY or PVA-LY NPs, can induce sustained suppression of AKT phosphorylation with relatively low concentrations of LY in cancer cells.
Internalization and subcellular accumulation of NPs
---------------------------------------------------
Analysis of the intracellular levels of LY in these lung cancer cell lines after exposure to equivalent concentrations of free LY, SF-LY NPs, or PVA-LY NPs for various time periods ([Figure 4A](#f4-ijn-8-2689){ref-type="fig"}) revealed that the intracellular LY levels of cells were saturated within 8 hours and declined gradually thereafter in response to free LY, SF-LY NPs, and PVA-LY NPs. The intracellular LY levels were highest in cells treated with SF-LY NPs, whereas the intracellular levels of LY after treatment with PVA-LY NPs were similar to those of free LY following incubation for the same time intervals, confirming the results of the previous report[@b24-ijn-8-2689] that the intracellular LY concentration in LY-loaded PLGA NPs with P VA was similar to that in free LY. Therefore, our results showed that intracellular effects are greatly enhanced when induced by SF NPs in all lung cancer cell lines. Next, the subcellular localization of SF NPs was studied using confocal microscopy. Nuclei stained with DAPI (blue) were observed by confocal microscopy, and the ER, marked by ER tracker in green, was disturbed in the perinuclear region. Following treatment with native Nile red or SF-Nile red NPs for 3 hours, Nile red carried by SF NPs tended to colocalize with ER (yellow spot). This colocalization was not observed when cells were treated with native Nile red ([Figure 4B](#f4-ijn-8-2689){ref-type="fig"}) or PVA-Nile red NPs ([Figure S3](#SD3-ijn-8-2689){ref-type="supplementary-material"}), where the Nile red staining is separated from green staining. The data suggest that SF-Nile red NPs target Nile red to ER. To provide more evidence to show the colocalization, we isolated the ER organelle from the cytosol and separately measured the concentration of LY in ER and cytosol after treatment with free LY or SF-LY NPs in the four cell lines. The amount of LY in the ER was significantly higher after SF-LY NP treatment than after free LY treatment ([Figure 4C](#f4-ijn-8-2689){ref-type="fig"}).
SF-LY NPs induced ER stress and ER stress-related apoptosis in cancer cells
---------------------------------------------------------------------------
Following the observation that SF NPs may target their payloads to ER, we investigated whether ER stress could be induced by SF-LY NPs. We assayed the expression of three important ER stress markers: CHOP, phosphorylated JNK, and GRP78. In H460, H157, and H1650 cells, the SF-LY NP-induced elevation of CHOP and GRP78 began at 3 hours and persisted up to 24 hours, whereas the CHOP and GRP78 expression induced in AS2 cells began 24--48 hours after treatment with SF-LY NPs ([Figure 5A](#f5-ijn-8-2689){ref-type="fig"}). The levels of phosphorylated JNK increased rapidly within 3 hours in H157, H460, and H1650 cells after SF-LY NP treatment, whereas the elevation of phosphorylated JNK occurred 24 hours after SF-LY NP treatment in the AS2 cell line. In the four cell lines, treatment with free LY at a dose equivalent to that in SF-LY NPs had very little effect on the expression of CHOP, GRP78, and phosphorylated JNK ([Figure 5A](#f5-ijn-8-2689){ref-type="fig"}). Treatment with SF NPs alone did not induce expression of CHOP ([Figure S4](#SD4-ijn-8-2689){ref-type="supplementary-material"}). These findings suggest that SF-LY NPs, but not free LY or SF NPs, can induce ER stress. Because CHOP has been implicated in ER stress-induced cell death[@b30-ijn-8-2689] and the activation of JNK by unfolding protein response (UPR) may lead to apoptotic cell death,[@b31-ijn-8-2689] we investigated whether SF-LY NP-induced ER stress contributed to apoptotic death. We used salubrinal to block ER stress and SP600125 to inhibit JNK function in the four cancer cell lines and observed that pretreatment with salubrinal or SP600125 alone slightly rescued SF-LY-NP-induced cell death in the four cell lines. Again, the combination of salubrinal and SP600125 significantly rescued cell death induced by SF-LY NPs ([Figure 5B](#f5-ijn-8-2689){ref-type="fig"}). The data suggested that both JNK and CHOP are critical, but redundant, in mediating cell death induced by SF-LY NPs via ER stress. We attempted to silence endogenous CHOP expression using siRNA. After transfection of the siRNA into the four cell lines, CHOP expression was suppressed in H460 and H157 cells, but not in the AS2 and H1650 cells. Accordingly, we selected H460 and H157 for further studies. We treated the two cell lines with free LY or SF-LY NPs after silencing CHOP or JNK with siRNA. The silencing of CHOP or JNK alone partially abolished the antiproliferative activity of SF-LY NPs, whereas the combination of CHOP and JNK siRNA to silence both proteins together restored cell death induced by SF-LY NPs ([Figure 5C](#f5-ijn-8-2689){ref-type="fig"}). ER stress in cells was reported to induce autophagy.[@b32-ijn-8-2689] It is intriguing to ask whether SF-LY NP treatment induces autophagy in cancer cells. We found that the LC3 protein converted from Type I to Type II, a hallmark of autophagy, in cells treated with SF-LY NPs but not free LY ([Figure S5A](#SD5-ijn-8-2689){ref-type="supplementary-material"}). We used autophagy inhibitors, including 3-MA (inhibition of autophagosome formation) and bafilomycin A1 (inhibition of autophagosome and lysosome fusion), to characterize the impact of SF-LY NP-induced autophagy on cell viability. Pretreatment with 3-MA or bafilomycin A1 further enhanced cell death induced by SF-LY NPs ([Figure S5B](#SD5-ijn-8-2689){ref-type="supplementary-material"}), indicating that the activation of autophagy by SF-LY NP treatment is essential for cell survival.
In vivo efficacy in an AS2 xenograft mouse model
------------------------------------------------
To investigate whether LY in the SF NP formulation was active in vivo, we injected it intratumorally into mice bearing tumor xenografts. A previous study suggested that localized drug delivery through intratumoral administration is an attractive approach, given that direct application permits higher concentrations of the drug to access the tumor tissue and avoid entering the systemic circulation.[@b33-ijn-8-2689] An AS2 tumor model was developed in athymic mice. After the average tumor volume reached 50--60 mm^3^ (approximately 14 days), the following regimens were administered by intratumoral injections every other day for a total of six times: (1) saline, (2) LY (1 mg/kg), (3) SF NPs (1 mg/kg), and (4) SF-LY NPs (1 mg/kg). The change in tumor volume from the initial volume (50--60 mm^3^) was monitored until the average tumor size reached 3000--4000 mm^3^ after intratumoral injection. The results showed that tumor volume steadily increased with time in mice injected with saline, SF NPs, and free LY, reaching an approximately 40-fold increase from the size of the original tumor after 12--14 days. In contrast, administration of SF-LY NPs (1 mg/kg) effectively slowed tumor growth, restricting the total increase in tumor volume to approximately 2.5--3 times the original tumor size ([Figure 6A](#f6-ijn-8-2689){ref-type="fig"}). No significant difference in body weight was observed within or among each group of mice ([Figure 6B](#f6-ijn-8-2689){ref-type="fig"}), indicating that SF NPs, free LY, and SF-LY NPs were not toxic to mice. Collectively, these results indicated that intratumoral injection of SF-LY NPs induced long-lasting inhibition of tumor growth.
Discussion
==========
In this study, we formulated a surfactant-free PLGA copolymer[@b26-ijn-8-2689],[@b27-ijn-8-2689] as a carrier for LY and demonstrated that in the absence of surfactant, SF-LY NPs could enhance the incorporation efficacy of NPs, cause sustained inhibition of AKT phosphorylation, target LY to the ER lumen, and effectively decrease cancer cell growth in vitro and in vivo.
The size (diameter) of SF-LY NPs was in the range of 80--100 nm. NPs of this size have the potential to avoid retention in the reticuloendothelial system (RES).[@b34-ijn-8-2689] In a previous study, we found that surfactant-free PLGA copolymers could be easily and directly conjugated on their surface.[@b26-ijn-8-2689] This advantage was particular to surfactant-free, but not surfactant-containing, PLGA NPs.[@b26-ijn-8-2689] Because the hydrophilicity of surfactant P VA may decrease intracellular uptake and lysosomal escape of NPs,[@b25-ijn-8-2689],[@b35-ijn-8-2689],[@b36-ijn-8-2689] we attempted to formulate PVA-LY NPs and examine their activities in the four cell lines ([Figure 2C](#f2-ijn-8-2689){ref-type="fig"}). Similar to the findings in a previous study,[@b24-ijn-8-2689] PVA-LY NPs showed inferior antiproliferative activity compared with SF-LY NPs. According to the release profile of SF-LY NPs, LY is released from NPs in a sustained manner. The results of in vitro experiments showed a sustained inhibition of AKT phosphorylation for up to 48 hours in cells treated with SF-LY NPs, but not in cells treated with free LY ([Figure 3](#f3-ijn-8-2689){ref-type="fig"}). The sustained inhibition of AKT activity by SF-LY NPs may contribute substantially to its prominent proapoptotic property, given that constitutive activation of AKT may promote cellular survival in lung cancer cells.[@b37-ijn-8-2689] Among the four cell lines incubated with SF-LY NPs, the ratio of LY uptake in AS2 cells was the lowest ([Figure 4A](#f4-ijn-8-2689){ref-type="fig"}). The effect of suppressing AKT phosphorylation by SF-LY NPs in AS2 cells was also inferior to that observed in the other three lung cancer cell lines. Consequently, SF-LY NPs induced less cell death in the AS2 cell line than in the other cell lines. Therefore, the ratio of cellular uptake of NPs may influence the final therapeutic effects. Our data demonstrated that cells take up a greater amount of NPs synthesized without surfactant compared with surfactant-containing NPs. The higher intracellular concentration of LY delivered by SF-LY NPs contributed to the greater cytotoxicity of SF-LY NPs compared with that of free LY and PVA-LY NPs in cancer cells.
The four cancer cell lines used in this study harbor genetic alterations in the PI3K pathway. Both AS2 and H1650 cells are *PTEN*-null, whereas H460 cells harbor a PIK3CA mutation and H157 cells are *PTEN*-null and harbor a mutation in PIK3CA. Although a low concentration of free LY induced modest cellular death in the four cell lines, treatment with SF-LY NPs over the same concentration range resulted in significantly increased cellular death. In contrast, in a normal bronchial cell line called NL-20, we found only modest toxicity of SF-LY NPs to NL-20 cells. Toxicities of SF NPs, free LY, PVA-LY NPs, and SF-LY NPs to normal cells are comparable, suggesting that SF-LY NPs offer a promising therapeutic window for cancer therapy.
A study of the subcellular localization of SF NPs revealed that SF NPs targeted Nile red to the perinuclear region and colocalized with an ER marker ([Figure 4B](#f4-ijn-8-2689){ref-type="fig"}). A previous study reported that PLGA NPs could localize with specific organ-elles such as ER and the Golgi apparatus,[@b38-ijn-8-2689] suggesting that SF NPs in this study delivered their payloads to ER and induced specific cellular responses. We examined the changes in ER stress-related proteins and found that treatment with SF-LY NPs, but not free LY or SF NPs, enhanced the expression of CHOP, phosphorylated JNK, and GRP78 in the four lung cancer cell lines ([Figure 5A](#f5-ijn-8-2689){ref-type="fig"} and [Figure S4](#SD4-ijn-8-2689){ref-type="supplementary-material"}). We further demonstrated that the induced ER stress facilitates cellular apoptosis. The inhibition of CHOP or JNK alone by either siRNA or inhibitors partially abolished the antiproliferative activity of SF-LY NPs, whereas combinational inhibition of CHOP and JNK significantly rescued the cell death induced by SF-LY NPs ([Figure 5B](#f5-ijn-8-2689){ref-type="fig"} and C). The data suggested that both JNK and CHOP are critical but redundant in mediating cell death induced by SF-LY NPs via ER stress. Interestingly, the induction of ER stress signals by SF-LY NPs is less obvious in AS2 cells than in the other three cell lines. These observations are consistent with those for SF-LY NPs, which produce the least cytotoxic effects on AS2 cells.
Although SF-LY NPs induced significantly high caspase 3/7 activities, they exerted little effect on caspase 8 activities in the four cancer cell lines ([Figure 2F](#f2-ijn-8-2689){ref-type="fig"}). Free LY only modestly induced both caspase 3/7 and 8 activities in the four cell lines. We conclude that SF-LY NP-induced cell death is primarily through the intrinsic apoptotic pathway. We found that after SF-LY NP treatment, autophagy was induced in the four cancer cell lines ([Figure S5A](#SD5-ijn-8-2689){ref-type="supplementary-material"}). Because treatment with autophagy inhibitors 3-MA and bafilomycin A1 further enhanced cell death induced by SF-LY NPs ([Figure S5B](#SD5-ijn-8-2689){ref-type="supplementary-material"}), we considered the activation of autophagy by SF-LY NPs treatment to have protective effects on cell survival.
Although the AS2 cells are relatively insensitive to treatment with SF-LY NPs in vitro ([Figure 2](#f2-ijn-8-2689){ref-type="fig"}), they exhibit rapid xenograft tumor growth in nude mice. For this reason, the AS2 xenograft model was chosen to test the in vivo antitumor activity of SF-LY NPs. Treatment with SF-LY NPs significantly suppressed growth of AS2 xenograft tumors in mice, suggesting that they have strong in vivo antiproliferative activities ([Figure 6](#f6-ijn-8-2689){ref-type="fig"}), even against tumors generated from a less sensitive cell line.
Conclusion
==========
We successfully synthesized SF-LY NPs and demonstrated their potential application in biomedical engineering. The particle size, encapsulation effciency, and the controlled release kinetics of SF NPs were particularly suited for drug delivery. It was demonstrated that SF-LY NPs induced significantly increased cell death through the apoptotic pathway in lung cancer cells compared with free LY and PVA-LY NPs. Moreover, surfactant-free PLGA NPs were able to enhance intracellular uptake of LY and subsequently deliver their payloads to ER, inducing ER stress. Finally, we demonstrated that SF-LY NPs have impressive in vivo antitumor activities. We conclude that surfactant-free PLGA is an ideal nanocarrier for the LY compound.
Supplementary materials
=======================
Materials and methods
---------------------
### Flow cytometric analysis
Asynchronous cultures were set for experiments (2×10^5^ cells in 6-well plates). Individual wells were treated with free LY or SF-LY NPs for 24 hours. Cells were stained according to the manufacturer's instructions with Annexin V-fluorescein isothiocyanate and propidium iodide staining assay kit (Life Technologies) and measured by flow cytometry (BD FACS-Calibur System; BD Biosciences, San Jose, CA, USA).
### DAPI staining for chromosome condensation
Chromosome condensation due to free LY and SF-LY NPs in cell lines was observed by DAPI staining. DAPI solution was used to stain the exposed cells in chamber slides, which were incubated for 10 minutes in the dark at 37°C. Images of the nucleus were captured using a fluorescence microscope (Nikon Corporation, Tokyo, Japan) at an excitation wavelength of 330 nm and an emission wavelength of 420 nm.
### MTT cytotoxicity assay
Cells were seeded (5×10^3^ per well) in a 96-well culture plate and incubated overnight at 37°C in humidified air containing 5% CO~2~. Concentration-effect and time-course studies were performed in the presence of free LY, SF NPs, and SF-LY NPs. After the required incubation periods, 20 μL of MTT reagent stock solution (5 mg/mL in PBS) was added to each well and incubated for 4 hours at 37°C. Plates were centrifuged at 1200 rpm for 5 minutes. After discarding the supernatant, DMSO (200 μL) was added into each well and incubated for 5 minutes. Supernatants were transferred into a new ELISA plate and absorbance was measured at 490 nm with an ELISA reader (Varioskan; Thermo Fisher Scientific).
### Intracellular localization by confocal laser microscopy
Viable cells (1×10^5^) were plated onto 3.5 cm dishes with 3 mL of culture medium. After overnight incubation, cells were treated with free Nile red or PLGA NPs loaded with Nile red (PVA-Nile red NPs) for 3 hours of incubation and stained with ER tracker™ Green dye according to the manufacturer's protocol (Molecular Probes^®^, Life Technologies) and DAPI. After replacement of original medium with particle-free, fresh medium, cells were immediately imaged with a scanning confocal microscope (Fluoview^®^ FV-1000; Olympus Corporation) with settings to detect fluorophores by A laser (excitation 405, 488, or 546 nm).
### Western blot analysis
Cells were lysed on ice for 30 minutes with whole-cell extract lysis buffer (50 mM Tris-HCl, pH 7.2--7.8; 1% NP-40; 2 mM EDTA; 100 mM NaCl; 0.1% SDS supplemented with protease inhibitor cocktail; Roche Applied Sciences). Lysates were collected by centrifugation at 14,000 rpm for 10 minutes, and protein concentration was determined using the Bradford assay (Bio-Rad). Protein extracts (20--50 μg) were boiled for 5 minutes in sample buffer before separation by SDS-PAGE. The proteins were transferred to PVDF membranes (Millipore) at 400 mA for 1.5 hours using an electroblotter (Amersham Pharmacia Biotech Inc). Membranes were blocked with TBST containing 5% nonfat milk for 1 hour at room temperature and incubated overnight at 4°C with LC3 or CHOP antibodies. Proteins were detected with horseradish peroxidase-conjugated secondary antibodies using an ECL kit (Amersham) according to the manufacturer's instructions.
######
The level of apoptosis after exposure to free LY or SF-LY NPs.
**Notes:** The graphs present flow analysis of four cell lines treated with free LY or SF-LY NPs for 24 hours. The scatter plot of FL1-H (X-scale) versus FL2-H (Y-scale) indicates annexin V staining and propidium iodide staining.
**Abbreviations:** LY, LY294002; NPs, nanoparticles; SF-LY NPs, surfactant-free NPs loaded with LY.
######
The level of chromatin condensation after exposure to free LY or SF-LY NPs.
**Notes:** The graphs show chromatin stained by DAPI after exposure to LY and SF-LY NPs at a concentration equivalent to that of LY for 24 hours in the four cell lines. The arrow indicates chromatin condensation.
**Abbreviations:** DAPI, 4′,6-diamidino-2-phenylindole; LY, LY294002; NPs, nanoparticles; SF-LY NPs, surfactant-free NPs loaded with LY.
######
Intracellular localization of PVA NPs in cells.
**Notes:** Confocal microscopic analysis of PVA-Nile red NPs. The cells were incubated with PVA-Nile red NPs for 3 hours at 37°C, stained with ER tracker and DAPI, extensively washed, and imaged for living cells under a confocal microscope.
**Abbreviations:** DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; NPs, nanoparticles; PVA, polyvinyl alcohol; PVA-Nile red NPs, PVA NPs loaded with Nile red.
######
The level of CHOP after exposure to SF NPs or SF-LY NPs.
**Notes:** Western blots showing levels of CHOP in the four cell lines following treatment with SF NPs or SF-LY NPs for a time-course study.
**Abbreviations:** LY, LY294002; NPs, nanoparticles; SF NPs, surfactant-free NPs; SF-LY NPs, surfactant-free NPs loaded with LY; h, hours.
######
The regulation of autophagy after treatment with free LY or SF-LY NPs.
**Notes:** (**A**) Western blots showing levels of LC3 in four cell lines following treatment with LY or SF-LY NPs for a time-course study. (**B**) The analysis of cytotoxicity by MTT assay after treatment with 3-MA (5 mM) or bafilomycin A1 (1 nM). Each experiment was repeated three times. Data are mean ± SEM. \**P* \< 0.05; \*\**P* \< 0.005.
**Abbreviations:** 3-MA, 3-methyladenine; baf, bafilomycin; A1LY, LY294002; MTT, 3-\[4,5-dimethylthiazol-2-yl\]-2, 5-diphenyltetrazolium bromide; NPs, nanoparticles; SEM, standard error of the mean; SF-LY NPs, surfactant-free NPs loaded with LY; h, hours.
This research was supported in part by the Ministry of Education, Taiwan, ROC, and the Aim for the Top University Project to the National Cheng Kung University (NCKU). The authors also thank the Department of Health and National Science Council of Taiwan for financial support of this research under contracts DOH99-TD-B-111-002, DOH99-TD-C-111-003, DOH100-TD-PB-111TM014, NSC 100-2120-M-006-002, and NSC-100-2314-B-006-032-MY3.
**Disclosure**
The authors report no conflicts of interest in this work.
{#f1-ijn-8-2689}
![Cytotoxic effects of SF-LY NPs determined by MTT assay.\
**Notes:** (**A**) The concentration-dependent effect on viability of four lung cancer cell lines. (**B**) Time-course study of cytotoxicity by MTT assay following treatment with LY or SF-LY NPs. (**C**) The analysis of cytotoxicity by MTT assay following treatment with LY or PVA-LY NPs. (**D**) The effect on viability of normal lung cells (NL20) by MTT assay following treatment with LY or PVA-LY NPs. (**E**) Cytochrome c was monitored by Western blotting after treatment with free LY and SF-LY NPs. (**F**) Caspase 3/7 or caspase 8 activity was detected after treatment with free LY and SF-LY NPs. The concentrations of free LY and SF-LY NPs were 5 μM for the AS2 cell line, 0.5 μM for the H157 cell line, and 0.25 μM for the H460 and H1650 cell lines. The concentration of TNF-α was 50 ng/mL. Each experiment was repeated three times. Data are expressed as mean ± SEM. \**P* \< 0.05; \*\**P* \< 0.005; \*\*\**P* \< 0.001 (free LY versus SF-LY NPs). TNF-α is positive control for caspase 8-dependent cell death.\
**Abbreviations:** cyto c, cytochrome c; LY, LY294002; MTT, 3-\[4,5-dimethylthiazol-2-yl\]-2, 5-diphenyltetrazolium bromide; NPs, nanoparticles; PVA, polyvinyl alcohol; PVA-LY NPs, PVA NPs loaded with LY; SEM, standard error of the mean; SF NPs, surfactant-free NPs; SF-LY NPs, surfactant-free NPs loaded with LY; TNF-α, tumor necrosis factor alpha.](ijn-8-2689f2){#f2-ijn-8-2689}
{#f3-ijn-8-2689}
{#f4-ijn-8-2689}
![The regulation of ER status after treatment with free LY or SF-LY NPs.\
**Notes:** (**A**) The change in CHOP, activated JNK expression, and GRP78 status after treatment with free LY or SF-LY NPs in four lung cancer cell lines. (**B**) Analysis of cytotoxicity by MTT assay after treatment with salubrinal, SP600125, or both. Each experiment was repeated three times. Data are mean ± SEM. \*\**P* \< 0.005; \*\*\**P* \< 0.001. (**C**) Analysis of cytotoxicity by MTT assay and immunoblotting after siRNA treatment for CHOP, JNK, or both. Data are mean ± SEM from three independent experiments. \*\**P* \< 0.005; \*\*\**P* \< 0.001.\
**Abbreviations:** ER, endoplasmic reticulum; LY, LY294002; MTT, 3-\[4,5-dimethylthiazol-2-yl\]-2, 5-diphenyltetrazolium bromide; NPs, nanoparticles; sal, salubrinal; SEM, standard error of the mean; SF NPs, surfactant-free NPs; SF-LY NPs, surfactant-free NPs loaded with LY; SP, SP600125.](ijn-8-2689f5){#f5-ijn-8-2689}
{#f6-ijn-8-2689}
######
Particle size, polydispersity index (PDI), entrapment effciency, and drug-loading capacity of SF NPs and SF-LY NPs as detected by dynamic light scattering (DLS)
Particle size (nm)[a](#tfn1-ijn-8-2689){ref-type="table-fn"} Polydispersity index Encapsulation effciency (%)[b](#tfn2-ijn-8-2689){ref-type="table-fn"} Loading capacity (μg/mg)
----------- -------------------------------------------------------------- ---------------------- ----------------------------------------------------------------------- --------------------------
SF NPs 74.5 ± 1.74 0.19 ± 0.01 N/A N/A
SF-LY NPs 98.9 ± 2.64 0.134 ± 0.03 37% ± 2.1% 7.4 ± 0.42
**Notes:**
Data represent mean ± SEM from three independent experiments;
effciency expressed as a percentage mean of three determinations ± standard deviation of drugs recovered in NPs compared with theoretical drugs loaded in the generated formulations.
**Abbreviations:** LY, LY294002; N/A, not applicable; NPs, nanoparticles; SEM, standard error of the mean; SF NPs, surfactant-free NPs; SF-LY NPs, surfactant-free NPs loaded with LY.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Low back pain (LBP) is the most common pain problem in the general population[@b1-jpr-8-523]--[@b6-jpr-8-523] and is one of the most common reasons for seeking health care and for use of avoidable care.[@b7-jpr-8-523],[@b8-jpr-8-523] Guidelines recommend watchful waiting, maintaining activities, and use of non-steroidal anti-inflammatory drugs, among other conservative actions.[@b9-jpr-8-523] Avoidable care generally occurs because of overdiagnosis, which leads to "subsequent overtreatment, diagnostic creep, shifting thresholds, and disease mongering, processes that ultimately reclassify a healthy low risk patient with mild to moderate problems as a sick patient".[@b10-jpr-8-523] Overdiagnosis manifests as inappropriate use of imaging, treatments (ie, multiple infusions and long-term opioid prescription use), surgery, and other care[@b9-jpr-8-523],[@b11-jpr-8-523]--[@b15-jpr-8-523] that is also related to pain severity, persistence, and recurrence, depression, female sex, neuropathic pain, and narcotics use.[@b4-jpr-8-523]--[@b6-jpr-8-523],[@b12-jpr-8-523],[@b13-jpr-8-523],[@b16-jpr-8-523] A minority of LBP patients account for a dominant share of overall medical care costs, but little is known about how the use of this care unfolds.[@b17-jpr-8-523]--[@b23-jpr-8-523]
While considerable research has focused on describing overall utilization of care and predicting costs, relatively little is known about how this care is used for LBP after an initial diagnosis. In this study, we used electronic health record (EHR) data on primary care patients in the 12-month period following an incident LBP encounter to understand how care was used. We first determined how patients clustered by their care utilization patterns. We then examined how care was used within defined patient clusters or groups and then compared these groups on demographics, health behaviors, disease burden, and physician ordering practices.
Materials and methods
=====================
This study involved a retrospective analysis of utilization of care among primary care patients with at least one ambulatory encounter for LBP. We used EHR, not claims data, because of its potential practical utility. EHR data can be evaluated in real time and used at the point of care to assess and determine the course of LBP care management in a way that claims data cannot be used. Also, claims data heavily reflect coverage decisions and utilization management practices that are often idiosyncratic to a given insurer. EHR data, on the other hand, reflects physician orders and treatment decisions. For the purposes of this study, data were extracted from the Geisinger Clinic EHR for the period from January 1, 2007 to December 20, 2011. In this section, we describe the source and patient inclusion and exclusion criteria.
In this study, we defined potentially avoidable care as high end diagnostics, selected treatments (ie, 3+ injections, or 3--5 to 5+ opioid prescriptions in a 12-month period), urgent or inpatient care without surgery, and surgeries. We emphasize "potentially avoidable" care, because we are unable to determine appropriateness from our data. Nevertheless, guideline-based evidence does not support the use of these potentially avoidable cares for improving diagnostic accuracy in most cases or for improving patient outcomes.[@b6-jpr-8-523]--[@b9-jpr-8-523] Evidence indicates that some, if not a majority, of these types of encounters are avoidable with more accurate diagnosis and symptom management.[@b10-jpr-8-523]
Source of population and data
-----------------------------
The Geisinger Health System (GHS) is an integrated delivery system offering health care services to residents in central and northeastern Pennsylvania. The GHS includes the Geisinger Clinic, a multi-specialty group medical practice with 850 physicians and physician's assistants and more than 200 primary care physicians in 41 community practice sites and two ambulatory surgery centers as of the time of this study. Adult (ie, 18+ years of age) primary care patients were the source population for this study. Data for this study have been extracted from the Geisinger Clinic EHR and include patient demographics, smoking history, use of alcohol, and all relevant clinical encounter information such as vitals, encounter type (for example primary, specialty, or emergency department (ED) visit, inpatient admissions, etc.), International Classification of Diseases, Ninth Revision (ICD-9) diagnosis codes, and orders for prescriptions, laboratory, and procedures for LBP at any Geisinger facilities.
Eligibility, follow-up, and utilization of care
-----------------------------------------------
Eligible individuals were 18+ years of age and assigned to a Geisinger Clinic primary care physician before January 1, 2007 but at least 12 months before December 20, 2010 (end of follow-up) and had at least 12 months of care with their primary care physician before their first recognized encounter for LBP. Patients were excluded if they had less than 12 months of follow-up, and were censored or excluded if they had a diagnosis of malignant cancer, sickle cell anemia, hemophilia, HIV/AIDS, or end-stage renal disease, an organ transplant, a prior long-term care facility admission, or when pregnant. Eligible patients had to have an incident LBP encounter, defined by an encounter diagnosis for LBP with no LBP diagnosis in the prior 12 months.
Data were extracted on all patient encounters in the 12 months before and the 12 months following the incident LBP encounter. Utilization of health care for LBP was defined by the type of encounter (ie, primary care, specialty care, ED or inpatient encounter without surgery, outpatient surgery, inpatient surgery) and by the orders (ie, prescription for opioids, other prescriptions, laboratory, infusion, magnetic resonance imaging \[MRI\], other imaging, surgery) that were specific to LBP. Providers are required to document one or more ICD-9 codes for the encounter or for any order. An encounter was defined as specific to LBP if an ICD-9 diagnosis, as defined by Cherkin et al[@b24-jpr-8-523] and modified by Vogt et al,[@b15-jpr-8-523] was documented in the patient record for the encounter or order. The LBP diagnosis was assigned to one of the following diagnostic subgroups based on the first encounter: group 1, back pain with no neurologic findings; group 2, back pain with neurologic findings; group 3a, congenital lumbar spine structural disorders; group 3b, acquired lumbar spine structural disorders; and group 4, acquired lumbar spine structural disorders[@b15-jpr-8-523],[@b24-jpr-8-523] (see [Table S1](#SD1-jpr-8-523){ref-type="supplementary-material"} for a complete list of ICD-9 codes and categorizations).
We excluded telephone encounters unless it was specifically to order an opioid prescription. Telephone calls are frequently documented for prescription renewals or for questions but do not usually constitute an encounter where care decisions are made. On the other hand, because opioid prescriptions are legally controlled, such prescription renewals were considered an actionable decision point. As such, encounters involving opioid prescriptions were included in the analysis.
Analysis
--------
Principal component analysis was completed to determine how care encounters and orders within encounters clustered together. Encounter types and orders were represented as individual binary variables for each LBP encounter, where three binary variables were included to represent as many as three serial encounters or orders of each type. For instance, three binary variables (ie, surgery_1, surgery_2, and surgery_3) were created to describe situations in which a patient had received one, two, or three or more surgeries during the 12-month period following their incident LBP encounter. If the patient had no surgery, then this would be represented as the following configuration of the binary variables: surgery_1=0, surgery_2=0, and surgery_3=0. Alternatively, if the patient had three or more surgeries, it would be denoted by the following: surgery_1=0, surgery_2=0, and surgery_3=1. The same coding scheme was applied to primary care, specialty care, emergency room, and inpatient encounters without surgery, non-MRI, and MRI. This method of capturing different configurations of the utilization patterns made it possible for factors to form around patients who had multiple encounters of the same type versus none or only a single encounter for a given type.
Incident LBP patients who only had one or two LBP encounters without an MRI, inpatient or surgery care encounter were excluded from the factor analysis and defined as a separate group for all other analyses. Polychoric correlations were estimated and used in the factor analysis given that a categorical variable format was used. Varimax rotation with Kaiser normalization was used to aid in interpretation.[@b25-jpr-8-523] Six factors emerged with an eigenvalue of 1.5 or greater. These factors were used to define patient groups that were then compared on utilization of care and on demographic, health behavior, clinical, diagnosis, and other measures. Groups were compared on sex, age, body mass index, diagnosis of selected cardiometabolic diseases (eg, diabetes and hypertension), chronic episodic conditions (ie, migraine, gastroesophageal reflux disease, asthma), depression, anxiety disorders, sleep disorders, and fibromyalgia. Presence of these conditions was defined by the appearance of the corresponding ICD-9 codes in EHR at least twice, as either one of the encounter diagnoses, or with medication orders on at least two separate encounters, within 12 months of each other. Groups were also compared on the Charlson Comorbidity Index[@b26-jpr-8-523]--[@b28-jpr-8-523] and on a physician efficiency index (PEI) for LBP care that was derived as: $$PEI_{i_{j}} = \left\lbrack {\left( {\sum\limits_{i = 1}^{N}{Cost_{i}}} \right) - Cost_{i}} \right\rbrack \div (N - 1)$$
PEI represents the physician efficiency index of care for patient *i* who was treated by provider *j* during a calendar year. N represents the total number of patients (including patient *i*) treated by provider *j* during the same calendar year. Therefore, the PEI captures the average per-year cost of care across all patients treated by provider *j*, except for patient *i*, during the given calendar year. Put differently, the PEI captures how the physician treats all patients under his or her care during the year other than the patient under consideration. A relatively high PEI value is therefore indicative of a physician whose practice pattern involves more expensive types of care in general.
The Mantel--Haenszel chi-square test was used for statistical comparisons of ordinal categorical variables, such as age group, depression, total number of encounters in the year before the first LBP encounter, total opioid prescriptions in the year before the first LBP encounter, and the chi-square test was used for nominal categorical variables. For continuous variables, the non-parametric Kruskal--Wallis test[@b29-jpr-8-523] was used to account for non-normality and test median. We used analysis of covariance to compare six subgroups on prior diagnoses for chronic progressive, symptomatic, and psychiatric diagnoses, utilization of care variables, and other factors. We adjusted for age and sex to account for differences among the groups, using the low utilization care group (ie, 1--2 LBP encounters in 12 months) as the reference group. We used pairwise comparisons to specifically identify the subgroups that accounted for overall differences.[@b30-jpr-8-523] All analyses were conducted using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA).
Cost of care for LBP-related services was not directly available from the EHR. As a substitute, we implemented a cost imputation method based on the subsample of the LBP patient cohort who had Geisinger Health Plan claims data.[@b31-jpr-8-523] Approximately one-third of the incident LBP patients had a Geisinger Health Plan as their primary payer type. These claims data were extrapolated to the remaining two-thirds of the patient population who did not have Geisinger Health Plan coverage. For a more detailed description of this methodology, see the Supplementary materials section.
Results
=======
From January 1, 2007 to December 20, 2011, there were 306,839 eligible primary care patients with 1,264,766 person-years of follow-up. Thirty-four percent (n=104,963) of these patients had at least one LBP encounter. Of these, the following were excluded: 37,645 prevalent LBP cases actively using care for LBP at the time of cohort inception (ie, January 1, 2007), 17,900 who met previously noted exclusions, and 15,841 who had less than 12 months of follow-up from their incident LBP encounter. The remaining 10.9% (n=33,577) met the inclusion criteria as an incident LBP patient for a rate of 37.3/1,000 person-years. In the 12-month period following the incident LBP encounter, there were 27,421 ambulatory LBP visits. Approximately 67% (n=22,645) of the included patients had only one or two LBP encounters with no MRI, inpatient care, or surgery care. These patients were excluded from the factor analysis and instead were clustered separately as another subgroup.
Factor analysis
---------------
In the varimax rotation with Kaiser normalization, six factors had an eigenvalue of 1.5 or greater ([Table 1](#t1-jpr-8-523){ref-type="table"}). These six factors explained 71.2% of the variance among the variables. The dominant factor ([Table 1](#t1-jpr-8-523){ref-type="table"}) represented diverse utilization, with high items weights for specialty care, high negative item weights for primary care, and modest weighting for non-surgery inpatient care. Surgical care followed second with loading for the 1, 2, and 3+ surgery groups. However, the group with only a single surgery also loaded separately on the sixth factor. The remaining three factors were represented by item loading for labs with indications for LBP, 3+ opioid prescription orders, and 3+ inpatient encounters. The factors results and other considerations were used to define seven utilization groups as having: 1--2 LBP encounters with no MRI, inpatient encounters, or surgery encounters; 2+ surgeries; one surgery; at least one specialty care visit without a primary care visits for LBP; 3+ opioid prescriptions; one or more laboratory orders specific to a LBP indication but with no inpatient care; and patients who did not meet the criteria for the other six groups. The last group included 40 patients with 3+ inpatient visits, which was too small to form a separate group.
Utilization of care
-------------------
[Table 2](#t2-jpr-8-523){ref-type="table"} shows the relative intensity of each type of care used by the seven subgroups. Approximately two-thirds of all ambulatory care was used by the 1--2 LBP encounter subgroup and the "other" subgroups. MRIs were most heavily used by surgery patients as well as by the 3+ opioid prescription and specialty patients. While these subgroups of patients comprised approximately 14% of the LBP patients in the sample, they accounted for more than half of all the MRI ordered.
Use of pain control therapies varied. More than half of all opioid prescriptions were prescribed to the 3+ opioid prescription group. Patients with a single surgery were prescribed considerably more opioids than patients with two or more surgeries. Injectables orders were primarily for patients with 2+ surgeries (65.1%) or a single surgery (13.7%). The ED was most heavily used by patients with 3+ opioid prescriptions (25.8%) and patients with 2+ surgeries (15.2%), followed by those with a single surgery or who used specialty care. Inpatient care was most heavily used by the specialty care subgroup and the surgery subgroups, as well as the 3+ opioid prescription subgroup. The 2.3% of patients with 2+ surgeries accounted for 80.1% of surgeries.
[Table 3](#t3-jpr-8-523){ref-type="table"} compares the proportions of patients within each subgroup who had only one versus two or more of each type of care. The ratio of these two proportions (ie, \[% with only one\]/\[% with 2+\]) reveals how use of care is concentrated within a smaller number of patients. The lower the ratio, the more utilization of care is concentrated in a smaller number of patients within the defined group. Concentration of care, as indicated by the ratios, varied most for laboratories and ED use.
Profile of utilization subgroups
--------------------------------
In the pairwise comparisons ([Tables 4](#t4-jpr-8-523){ref-type="table"} and [5](#t5-jpr-8-523){ref-type="table"}) subgroups differed in one or more two-way comparisons for all variables examined. With only a few exceptions (eg, proportion with a diagnosis of fibromyalgia), the specialty care subgroup did not differ from the reference subgroup (ie, 1--2 LBP encounters in 12 months), but both of these subgroups differed from one or more of the other five subgroups on all variables. In contrast, the 3+ opioid prescription subgroup was considerably more likely than the reference subgroup to be current cigarette smokers and to have a diagnosis for all other conditions listed in [Table 4](#t4-jpr-8-523){ref-type="table"}. The remaining four subgroups were older, but did not have many other features in common. Patients in the laboratory dominant care subgroup were more likely to have hypertension and heart failure and there were substantially higher proportions with chronic episodic disease, severe depression, anxiety and sleep disorders, and fibromyalgia.
Patients with 1--2 LBP encounters had the lowest proportion with a body mass index of 30+ kg/m^2^. Compared with the reference subgroup, both surgery subgroups had high proportions with diabetes and vascular diseases, elevated proportions only for migraine (one surgery group only), arthritis, depression, and sleep disorders, and very high proportions with a diagnosis of fibromyalgia. The "other" subgroup had higher proportions with chronic progressive diseases like the one surgery subgroup and higher proportions with chronic episodic diseases and other disorders like the laboratory dominant subgroup.
In the year before the LBP diagnosis ([Table 5](#t5-jpr-8-523){ref-type="table"}), utilization of ambulatory care was in general considerably lower for the 1--2 LBP encounter subgroup and the specialty care subgroup, especially compared with the 3+ opioid prescription and laboratory dominant subgroups (as shown in the \>15 category of the rows labeled "Percent by total number of encounters in year before first LBP encounter" in [Table 5](#t5-jpr-8-523){ref-type="table"}). The 1--2 LBP encounter and the laboratory dominant subgroups had the highest proportions with a group 1 or 2 LBP diagnosis followed by the 3+ opioid prescription and other patient subgroups. The specialty care and the 2+ surgery subgroups had the lowest proportion of patients with a group 1 or 2 diagnosis. Finally, the PEI was relatively high for the two surgery subgroups and elevated for patients in the specialty care subgroup.
Cost of LBP care
----------------
The average estimated cost of care per patient for the first 12 months from the incident LBP encounter was \$2,380. The 2+ surgeries subgroup accounted for 40.4% of the total cost of LBP care as represented in the EHR, while the one surgery subgroup accounted for another 19.2%. For the 2+ surgery subgroup, the median 1-year cost was \$39,504 (mean \$47,542) with an interquartile range of \$30,497 and \$76,080. This suggests that this 2+ surgery group is the main drivers of the total cost of LBP care incurred by this cohort.
Discussion
==========
Two-thirds of LBP patients have only one or two encounters during an incident LBP episode and use very little care. The remaining one-third of patients has very heterogeneous patterns of utilization that appear to fall into six defined groups, four of which account for 13.8% of patients and most of the MRI, injectable, ED, and inpatient care, with a heavy concentration of utilization in surgery patients. However, use of potentially avoidable care was pervasive across groups. Physician ordering practices and a number of patient health factors differed among the LBP patient groups.
Patients who had 2+ surgeries had a somewhat unique profile. As expected, these patients were less likely to have benign diagnoses. However, this alone does not explain the use of surgery; 64.1% had an initial diagnosis that fell into group 1 or group 2 diagnosis as defined by Vogt et al,[@b15-jpr-8-523] a proportion that is similar to that in the specialty care subgroup. In contrast, 71.6% of patients with a single surgery had a group 1 or 2 diagnosis. Fibromyalgia diagnosis was considerably more common among the 2+ surgery subgroup followed by the one surgery group, but did not differ significantly across the 1--4 LBP diagnostic groups.
We explored EHR data to identify potential reasons to explain differences among the LBP patient groups. We used the proportion with diagnoses of chronic episodic conditions as an indicator of susceptibility to symptomatic diseases, including polysymptomatic disorders. Relative to other patient subgroups, surgery patients did not have a high burden of these symptomatic conditions, including depression, anxiety disorders, or sleep disorders. In general, anxiety disorder diagnoses were relatively low among the surgery patients. However, the proportion of surgery patients with diagnoses of depression and sleep disorders was consistently higher than that of patients in the specialty care subgroup.
Previous studies have indicated that having a depression or anxiety disorder is more common among LBP patients who are heavy utilizers of care. While the findings from our study are generally consistent with this finding, the elevated prevalence (ie, compared with the 1--2 LBP encounter subgroup) of a diagnosis of depression or anxiety disorder seems largely confined to patients in the 3+ opioid prescription and laboratory dominant care subgroups, not the specialty care or surgery subgroups. It is possible that the difference between our findings and those of previous studies is that we relied on a physician diagnosis of these conditions versus a direct assessment of depression and anxiety by questionnaire.
The specialty care subgroup appears similar to the surgery subgroups in the use of MRI, ED, and inpatient care and contained the heaviest users of inpatient care. However, this subgroup does not seem to contain heavy users of injectables in general, and certainly not when compared with those with surgery. The specialty care subgroup may be averse to these types of intrusive interventions, including surgery. Alternative explanations include a lower susceptibility to symptomatic conditions or younger age. These patients may simply have had fewer repeat incident LBP episodes (ie, more than a year between the last LBP encounter and a subsequent encounter) which explains, in part, why they did not have surgery.
[Table 5](#t5-jpr-8-523){ref-type="table"} suggests that surgery patients had primary care physicians with the highest (ie, worst) efficiency index, by far, when compared with other patient groups. This score indicates that physician practice explains, in part, the differences in use of care for these two patient groups. Historically, efficiency scores have largely been confined to insurance companies[@b32-jpr-8-523] that derive such measures from claims data to identify providers who are outliers in the volume of care they either provide or order. The rapid adoption of EHRs opens opportunities to derive such scores in real time and to use these scores as decision support aids in the clinical practice setting, a capability that could serve to foster accountable care relationships between providers and payers.
Approximately 5% of the LBP patients have been defined as moderate to heavy users of opioid prescription medication. Use of opioids has been previously reported to be associated with a higher prevalence of comorbidities and anxiety disorders.[@b33-jpr-8-523] This finding is consistent with our study, as this subgroup of patients consistently contained higher proportions with symptomatic disorder diagnoses and had the highest proportion with severe depression, anxiety disorders, and sleep disorders. This same subgroup also appears to have the highest proportion of current smokers and the heaviest use of opioids in the year before the LBP diagnosis. However, the results suggest that use of opioids was not common among patients with multiple surgeries but was elevated among patients with one surgery. Our findings indicate that these previously identified patient features may be associated with utilization of care, but not necessarily with patients who eventually have LBP surgery.
It is well known that patients with unexplained somatic complaints are relatively high utilizers of health care.[@b34-jpr-8-523],[@b35-jpr-8-523] In our study, symptomatic disorders appeared to be more common among the 3+ opioid prescription and the laboratory dominant subgroup. However, these two subgroups also have the highest proportion of patients with diagnoses of severe depression and anxiety and sleep disorders. Further, the heavy use of care in these subgroups appears to be selective and primarily confined to the 3+ opioid prescription group heavily using MRI and ED care when compared with other patient subgroups. The combination of psychiatric diagnoses and diagnoses of other symptomatic conditions including benign LBP, and heavy use of care, in general, may signal that the patient has unmet needs that are not expressed during an encounter or not understood.[@b36-jpr-8-523]
There are several potential limitations to our study findings. First, our notion of "incident LBP encounter" is that the sample is comprised of patients who had their first ever LBP encounter and patients with a history of LBP who were experiencing a recurrent episode after a long quiescent period. Identification of a true incident LBP encounter cohort would be helpful in understanding how use of LBP care emerges among first time users versus patients with repeated episodes of LBP over longer periods of time. Second, EHR data offer an incomplete profile of total use of care, in general, and for LBP care. While the Geisinger Clinic provides comprehensive care, it is likely that some care was obtained outside of the clinic and was not documented. As a consequence, patients were likely to be systematically misclassified by utilization group, where the most common error is that use of potentially avoidable care and LBP surgery was underestimated. Lastly, not all LBP patients will seek care, so our finding is restricted to those patients who had access to care and chose to obtain care from care providers.
Supplementary materials
=======================
Cost imputation algorithm
-------------------------
As is typically the case, electronic health record (EHR) data at Geisinger Clinic lack cost information. To circumvent this problem, we developed a regression-based cost imputation method based on Geisinger Health Plan claims data as outlined below: Start by applying the same inclusion and exclusion criteria to the claims data as done for the EHR data to select the eligible patient population from the claims data.Categorize all encounter types in EHR and claims into a set of mutually exclusive major categories. In this study, we use the following major categories: inpatient visit, outpatient visit, emergency department, diagnostic imaging (ie, X-rays, computed tomography, and magnetic resonance imaging), and all prescription drugs. Professional charges, which are typically available as separate claim types, are assumed to have been incurred in every encounter in EHR.In the claims data, estimate the following multivariate regression model using a generalized linear model with log link and gamma distribution function: $$\text{Mean~cost} = \beta_{0} + \beta_{1}\left( \text{encounter~type} \right) + \beta_{2}\left( \text{Medicare} \right) + \beta_{3}\left( \text{age} \right) + \beta_{4}\left( \text{sex} \right)$$"Encounter type" denotes a set of binary indicator variables that represents each major encounter type category (eg, inpatient, outpatient, emergency department); "Medicare" is a binary indicator variable that equals 1 if the patient has Medicare coverage and 0 otherwise; "age" is a continuous variable capturing the patient's age at the time of the study; and "sex" captures the patient's sex.Take the beta coefficient estimates obtained in 3) and apply them to similarly structured EHR data to obtain the estimated mean cost in the EHR.
The above method can be modified by introducing interaction effects between the encounter type variables and age or sex, for instance. In our estimates, the results were not sensitive to such alternative specifications. The resulting cost estimates can be interpreted as "imputed cost" under the hypothetical scenario that the patient had been covered by Geisinger Health Plan. The advantage of this cost imputation method is that it is not necessary that those patients who are included in the claims data be also included in the EHR data; as long as the structure of the EHR data can be modified to accommodate the above regression model, the estimated cost can be obtained for that patient. The disadvantage of this method is that its accuracy may depend on the potentially subjective categorization of claim and encounter types.
######
Low back pain categories and diagnostic codes
Category ICD-9 diagnosis code Diagnosis name
-------------------------------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Group 1: back pain with no neurologic findings 724.2 Lumbago
724.5 Backache, unspecified
846.0--846.9 Sprains and strains of sacroiliac region
847.2,3,9 Sprains and strains of back (lumbar, sacrum, unspecified)
Group 2 back pain with neurologic findings 721.42 Spondylogenic compression of lumbar spinal cord
721.91 Spondylosis of unspecified site, with myelopathy
722.73 Lumbar disc disorder with myelopathy
722.80 Post-laminectomy syndrome of unspecified region
724.3 Sciatica
724.4 Thoracic or lumbosacral neuritis or radiculitis, unspecified
Group 3a: congenital lumbar spine structural disorders 737.1 Kyphosis (acquired)
737.20 Lordosis (acquired) (postural)
737.3 Kyphoscoliosis and scoliosis
739.3 Nonallopathic lesions, lumbar region
739.4 Nonallopathic lesions, sacral region
756.13--756.19 Anomalies of spine
Group 3b: acquired lumbar spine structural disorders 721.5--721.90 Kissing spine; ankylosing vertebral hyperostosis; traumatic spondylopathy; other allied disorders of spine; spondylosis of unspecified site without mention of myelopathy
722.10 Lumbar intervertebral disc without myelopathy
722.2 Displacement of intervertebral disc, site unspecified, without myelopathy
722.30 Schmorl's nodes, unspecified region
722.32 Lumbar schmorl's nodes
722.52 Degeneration of lumbar or lumbosacral intervertebral disc
722.6 Degeneration of intervertebral disc, site unspecified
722.90 Other and unspecified disc disorder of unspecified region
722.93 Other and unspecified lumbar disc disorder
724.00 Spinal stenosis, unspecified region
724.02 Lumbar stenosis
724.09 Other
738.4 Acquired spondylolisthesis
756.12 Congenital spondylolisthesis
Group 4: other 307.89 Pain disorders related to psychological factors, other
722.83 Post-laminectomy syndrome, lumbar
724.6 Disorders of sacrum, including lumbosacral joint instability
724.8 Other symptoms referable to back
724.9 Other unspecified back disorders
756.10 Anomaly of spine, unspecified
805.4 Closed fracture of lumbar vertebrae without mention of spinal cord injury
805.6 Closed fracture of sacrum or coccyx without mention of spinal cord injury
805.8 Fracture of vertebral column without mention of spinal cord injury
996.4 Mechanical complication of internal orthopedic device, implant, and graft
**Note:** Vogt et al[@b37-jpr-8-523].
**Abbreviation:** ICD-9, International Classification of Diseases, Ninth Revision.
**Author contributions**
WFS, DDM, XY, JAB, and MRVK were responsible for conception and study design. XY, WFS, and DDM acquired the data. WFS, DDM, XY, JAB, JM, RJS, and MRVK were responsible for the analysis and interpretation of the data. WFS, DDM, XY, JAB, and JM drafted the paper and DDM, XY, JM, RJS, and MRVK critically revised the final version. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
**Disclosure**
Geisinger Health System (GHS) was paid by Pfizer Inc. in connection with development of this paper. RJS was an employee of Pfizer at the time the study was conducted and the paper was initiated. DDM and JAB are employees of GHS. XY and WFS were employees of GHS at the time the study was conducted and were paid in connection with development of this paper. MRVK and WFS were paid consultants to Pfizer Inc., in connection with this study and development of this paper. The authors report no other conflicts of interest in this work.
This study was sponsored by Pfizer Inc., New York, NY, USA.
######
Factor loading for LBP-related care among an incident LBP patient cohort[\*](#tfn1-jpr-8-523){ref-type="table-fn"} from Geisinger Clinic, 2012
Number of each type of care Variable Specialty without primary care Surgical care Laboratory dominant care Opioid prescription 3+ inpatient encounters One surgery
----------------------------- --------------- -------------------------------- --------------- -------------------------- --------------------- ------------------------- -------------
Only one PCP encounter −0.85
Specialty encounter 0.85
ED encounter −0.81
Inpatient encounter −0.44 −0.72
Surgery 0.65 0.52
Non-MR image 0.41
MR image
Laboratory order 0.76
Opioid order 0.93
Injection for pain 0.88
Only two PCP encounter −0.95
Specialty encounter 0.95
ED encounter 0.54 −0.7
Inpatient encounter 0.48 −0.41
Surgery 0.77
Non-MR image 0.42 0.57
MR image
Laboratory order 0.83 0.41
Opioid order 0.97
Injection for pain 0.89
Three or more PCP encounter −0.8
Specialty encounter 0.95
ED encounter −0.41 −0.5
Inpatient encounter 0.46 −0.42 0.66
Surgery 0.82
Non-MR image 0.56
MR image −0.43
Laboratory order 0.79
Opioid order 0.97
Eigenvalues 7.07 5.79 2.47 2.35 2 1.68
**Note:**
Excluding patients who only had 1--2 LBP encounters without MR imaging, inpatient care, or surgery.
**Abbreviations:** ED, emergency department; MR, magnetic resonance; PCP, primary care physician; LBP, low back pain.
######
Comparison of LBP-related care utilization by subgroup, Geisinger Clinic, 2012
Type of care Utilization of care subgroup
--------------------------------------------------------------------- ------------------------------ ------ ------ ------ ------ ------ ------ ---------------------------------------------------------
Ambulatory care visits (%)[a](#tfn3-jpr-8-523){ref-type="table-fn"} 42.8 6.7 8.9 5.1 4.4 7.2 24.9 100%(n=27,42l[b](#tfn4-jpr-8-523){ref-type="table-fn"})
MR imaging (%) 0 10.8 17.5 9.4 10.4 17.1 35 100%(n=5,803[b](#tfn4-jpr-8-523){ref-type="table-fn"})
Laboratory (%) 45.9 5.7 7.5 26.1 7.4 7.4 0 100%(n=7,042[b](#tfn4-jpr-8-523){ref-type="table-fn"})
Opioids (%) 15 7.5 53.4 3 5.4 2.2 13.5 100%(n=20,879[b](#tfn4-jpr-8-523){ref-type="table-fn"})
Injectable (%) 7.3 1.1 3.1 2 13.7 65.1 7.8 100%(n=4,304[b](#tfn4-jpr-8-523){ref-type="table-fn"})
ED use (%) 0 10.6 25.8 5.7 6.1 15.2 36.7 100%(n=652[b](#tfn4-jpr-8-523){ref-type="table-fn"})
Inpatient without surgery (% total) 0 22.6 16.7 9.8 13 12 25.9 100% (n=1,127[b](#tfn4-jpr-8-523){ref-type="table-fn"})
Surgery (% total) 19.9 80.1 0 100%(n=396[b](#tfn4-jpr-8-523){ref-type="table-fn"})
**Notes:**
Includes face to face visits with a primary care physician, specialist, and chiropractor but excludes medication orders only without a visit;
refers to the number of encounters or orders for the specific type of care.
**Abbreviations:** ED, emergency department; LBP, low back pain; MR, magnetic resonance; PCP, primary care physician.
######
Comparison of LBP-related care utilization concentration within each subgroup, Geisinger Clinic, 2012
Type of care Number Utilization of care subgroup
------------------------------------------------- -------- ------------------------------ ------ ------ ------ ------ ------ ------ ------
MR imaging 1 0 27.6 25.4 21.6 39.5 36.8 21.4 8.3
2+ 0 9.5 11.4 6.9 13.2 17.9 6.2 2.9
Ratio[\*](#tfn5-jpr-8-523){ref-type="table-fn"} -- 2.9 2.2 3.1 3.0 2.1 3.5 2.9
Laboratory 1 12.2 19.1 14.4 63.5 26.2 16.4 0 13.1
2+ 1.0 7.6 6.8 36.5 16.4 12.8 0 3.4
Ratio[\*](#tfn5-jpr-8-523){ref-type="table-fn"} 12.1 2.5 2.1 1.7 1.6 1.6 -- 3.9
Injectable 1--2 1.4 3.1 5.3 4.2 57.2 41.8 4.2 4.7
3+ 0 0.3 0.5 0.6 2.4 49.7 0.4 1.7
Ratio[\*](#tfn5-jpr-8-523){ref-type="table-fn"} -- 10.3 10.6 7.0 23.8 0.8 10.5 2.8
ED use 1 0 4.3 3.7 2.8 4.2 4.7 3.1 1.2
2+ 0 0.4 2.3 0.1 0.4 1.3 0.7 0.3
Ratio[\*](#tfn5-jpr-8-523){ref-type="table-fn"} -- 10.8 1.6 28.0 10.5 3.6 4.4 4.0
Inpatient but not surgery 1 0 14.6 7.1 5.8 12.9 8.8 3.1 2.1
2+ 0 4.0 1.9 1.2 2.3 1.7 0.8 0.5
Ratio[\*](#tfn5-jpr-8-523){ref-type="table-fn"} -- 3.7 3.7 4.8 5.6 5.2 3.9 4.2
**Notes:**
Obtained by dividing the top row (1) by the second row (2+) in each care type category. Values in the table are presented as %, except for the rows already stated as being measured as a ratio.
**Abbreviations:** ED, emergency department; LBP, low back pain; MR, magnetic resonance; PCP, primary care physician.
######
Demographic and health profile of the incident LBP patient cohort, Geisinger Clinic, 2012
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Variable Category/statistics 1--2 LBP visits\ Specialty and no PCP\ 3+ opioid prescriptions\ Laboratory dominant care\ One surgery\ 2+ surgeries\ Other\
n=22,645\ n=1,095\ n=1,656\ n= 1,238\ n=788\ n=1,060\ n=5,095\
(67.4%) (3.3%) (4.9%) (3.7%) (2.4%) (3.2%) (15.2%)
----------------------------------------------------------- ----------------------------------------------- ----------------------------------------------- ----------------------------------------------- ----------------------------------------------- ----------------------------------------------- ----------------------------------------------- ----------------------------------------------- -----------------------------------------------
Percent by female sex status Percent by age range (years) 62.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 59.8 58.9 64.5[a](#tfn7-jpr-8-523){ref-type="table-fn"} 53.6[b](#tfn8-jpr-8-523){ref-type="table-fn"} 59.7 60.4
\<30 11.5[a](#tfn7-jpr-8-523){ref-type="table-fn"} 8.3[a](#tfn7-jpr-8-523){ref-type="table-fn"} I3.I[a](#tfn7-jpr-8-523){ref-type="table-fn"} 8.2 6.0 6.2 8.7
30--39 I5.I[a](#tfn7-jpr-8-523){ref-type="table-fn"} I3.3[a](#tfn7-jpr-8-523){ref-type="table-fn"} I7.0[a](#tfn7-jpr-8-523){ref-type="table-fn"} 12.9 13.5 10.7 12.9
40--49 2I.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 25.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 22.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 17.6 20.6 20.3 20.0
50--59 2I.5[a](#tfn7-jpr-8-523){ref-type="table-fn"} 2I.9[a](#tfn7-jpr-8-523){ref-type="table-fn"} I8.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 22.6 22.6 23.2 21.8
60+ 30.7[a](#tfn7-jpr-8-523){ref-type="table-fn"} 3I.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 29.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 38.6 37.4 39.6 36.6
Percent by smoking status Current smoking I8.8[a](#tfn7-jpr-8-523){ref-type="table-fn"} 21.0 34.2[a](#tfn7-jpr-8-523){ref-type="table-fn"} 20.0 23.6 20.2 20.9
Quit 21.1[a](#tfn7-jpr-8-523){ref-type="table-fn"} 21.4 2I.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 24.6 23.6 23.6 23.7
Never 38.4[a](#tfn7-jpr-8-523){ref-type="table-fn"} 30.8 25.1[a](#tfn7-jpr-8-523){ref-type="table-fn"} 33.4 29.8 30.9 33.9
Missing 2I.7[a](#tfn7-jpr-8-523){ref-type="table-fn"} 26.9 I9.I[a](#tfn7-jpr-8-523){ref-type="table-fn"} 22.0 23.0 25.3 21.6
Percent with BMI of 30+ (kg/m^2^) Yes 45.6 46.8 53.0 50.4 48.0 50.4 50.9
Missing 3.3 1.8 1.6 1.3 1.0 0.9 1.9
Percent with a specific chronic progressive diagnosis Type 2 diabetes I6.6[b](#tfn8-jpr-8-523){ref-type="table-fn"} 16.9 24.0[b](#tfn8-jpr-8-523){ref-type="table-fn"} 20.6 19.3 20.0 22.0
Hypertension 42.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 40.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 52.4 52.0 53.4 48.9 53.6
Stroke-hemorrhage 0.3 0.6 0.4 0.7 0.5 0.4 0.5
CAD 6.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 8.7 9.9 8.6 10.3 10.3 10.2
Heart failure 3.3 3.7 6.6 5.8 5.5 4.0 5.4
Percent with chronic episodic diagnosis Migraine 24.4 22.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 30.4 31.0 27.8 24.3 28.1
Arthritis 32.5[a](#tfn7-jpr-8-523){ref-type="table-fn"} 38.1[a](#tfn7-jpr-8-523){ref-type="table-fn"} 42.2 46.0 45.9 44.1 42.3
IBS 5.9 4.8 6.6 7.4 5.6 4.2 7.6
GERD 32.9 27.1[a](#tfn7-jpr-8-523){ref-type="table-fn"} 38.5 43.1 35.3 34.3 39.5[b](#tfn8-jpr-8-523){ref-type="table-fn"}
Asthma 14.3 I0.4[b](#tfn8-jpr-8-523){ref-type="table-fn"} I9.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 18.1 12.6 14.6 16.3
Allergic rhinitis 23.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 16.9 25.8[a](#tfn7-jpr-8-523){ref-type="table-fn"} 33.0[a](#tfn7-jpr-8-523){ref-type="table-fn"} 21.2 19.2 28.6[a](#tfn7-jpr-8-523){ref-type="table-fn"}
Percent by diagnosed depression severity None 68.8[a](#tfn7-jpr-8-523){ref-type="table-fn"} 67.5 47.7[a](#tfn7-jpr-8-523){ref-type="table-fn"} 58.6 62.8 62.2 59.1
Mild-moderate 20.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 21.8 28.9[a](#tfn7-jpr-8-523){ref-type="table-fn"} 24.8 23.0 25.2 25.1
Severe I0.6[a](#tfn7-jpr-8-523){ref-type="table-fn"} 10.7 23.4[a](#tfn7-jpr-8-523){ref-type="table-fn"} 16.6 14.2 12.6 15.8
Percent with anxiety disorder diagnosis 10.1 9.3 I9.4[a](#tfn7-jpr-8-523){ref-type="table-fn"} 13.8 11.4 7.9 13.1
Percent with sleep disorder diagnosis I8.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 20.9[b](#tfn8-jpr-8-523){ref-type="table-fn"} 33.0[@b1-jpr-8-523] 27.5 27.0 21.9 26.8
Percent with fibromyalgia diagnosis I5.I[a](#tfn7-jpr-8-523){ref-type="table-fn"} 25.3 22.3 24.6[@b1-jpr-8-523] 32.4[a](#tfn7-jpr-8-523){ref-type="table-fn"} 39.6 21.I[b](#tfn8-jpr-8-523){ref-type="table-fn"}
Mean Charleston Comorbidity Index before first encounter 0.6 0.6 0.9 0.8 0.6 0.7 0.7
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Notes:**
Pairwise comparison differences between corresponding group and all other groups (*P*\<0.05);
pairwise comparison differences between corresponding group with at least five other groups (*P*\<0.05).
**Abbreviations:** BMI, body mass index; CAD, coronary artery disease; GERD, gastroesophageal reflux disease; IBS, irritable bowel syndrome; PCP, primary care physician; LBP, low back pain.
######
Care utilization profile of the incident LBP patient cohort, Geisinger Clinic, 2012
Variable Category/statistics Utilization of care subgroup
--------------------------------------------------------------------------------- ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ ---------- ------------------------------------------------ ------------------------------------------------
First LBP encounter with PCP 77.4 24.9[a](#tfn9-jpr-8-523){ref-type="table-fn"} 75.9 79.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 63.6 52.3 77.8
Percent by total number of encounters in year before first LBP encounter, count 0--2 I4.4[b](#tfn10-jpr-8-523){ref-type="table-fn"} 22.0[b](#tfn10-jpr-8-523){ref-type="table-fn"} 16.7 I2.l[b](#tfn10-jpr-8-523){ref-type="table-fn"} 17.9 17.2 I2.l[b](#tfn10-jpr-8-523){ref-type="table-fn"}
3--6 28.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 26.6[b](#tfn10-jpr-8-523){ref-type="table-fn"} 22.9 20.I[b](#tfn10-jpr-8-523){ref-type="table-fn"} 19.7 24.7 24.8[b](#tfn10-jpr-8-523){ref-type="table-fn"}
7--10 20.4[b](#tfn10-jpr-8-523){ref-type="table-fn"} I7.0[b](#tfn10-jpr-8-523){ref-type="table-fn"} 16.8 20.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 19.5 14.4 I9.2[b](#tfn10-jpr-8-523){ref-type="table-fn"}
11--15 I6.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 13.I[b](#tfn10-jpr-8-523){ref-type="table-fn"} 14.6 I8.2[b](#tfn10-jpr-8-523){ref-type="table-fn"} 15.4 19.5 I7.0[b](#tfn10-jpr-8-523){ref-type="table-fn"}
\>I5 20.7[b](#tfn10-jpr-8-523){ref-type="table-fn"} 21.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 29.1 29.3[b](#tfn10-jpr-8-523){ref-type="table-fn"} 27.5 24.2 26.9[b](#tfn10-jpr-8-523){ref-type="table-fn"}
Total opioid prescriptions in the year before the first LBP encounter, count Non-user 93.5[a](#tfn9-jpr-8-523){ref-type="table-fn"} 91.0 79.2[b](#tfn10-jpr-8-523){ref-type="table-fn"} 90.7 86.9 87.9 90.5
2.2[a](#tfn9-jpr-8-523){ref-type="table-fn"} 2.5 4.6[b](#tfn10-jpr-8-523){ref-type="table-fn"} 2.5 4.8 3.6 2.8
2--4 2.4[a](#tfn9-jpr-8-523){ref-type="table-fn"} 3.4 7.4[b](#tfn10-jpr-8-523){ref-type="table-fn"} 3.6 5.1 4.6 3.5
5+ I.9[a](#tfn9-jpr-8-523){ref-type="table-fn"} 3.2 8.8 3.2 3.2 3.9 3.2
LBP diagnosis group[c](#tfn11-jpr-8-523){ref-type="table-fn"} I 74.3 49.8 63.4 73.5 52.3 44.2 60.5
II 10.8 14.6 13.5 9.6 19.3 19.9 15.2
III 12.1 31.1 19.6 14.6 24.0 29.1 21.4
IV 0.8 2.5 0.6 0.7 1.1 1.9 0.9
Other 2.0 2.1 2.9 1.5 3.3 5.0 2.0
PEI Mean 1.2 1.9 1.4 1.4 3.1 4.7 1.4
Median 0.8 0.9 0.9 0.9 1.6 1.8 0.9
IQR 0.6--1.3 0.6--2.5 0.6--1.4 0.6--1.5 0.8--3.6 0.8--3.7 0.6--1.4
**Notes:**
Pairwise comparison differences between corresponding groups and all other groups (*P*\<0.05);
pairwise comparison differences between corresponding group with at least five other groups (*P*\<0.05);
pairwise comparison differences between all groups (*P*\<0.05).
**Abbreviations:** IQR, interquartile range; LBP, low back pain; PCP, primary care physician; PEI, physician efficiency index
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#s1}
============
The weight and composition of oilseed grains at harvest are complex traits that depend on the dynamics of many processes occurring earlier at both the plant and organ levels. Their response to the genotype and the environment results from several linked processes controlled at different levels of organization, from sub-cellular to crop (Martre et al., [@B28]). The sensitivity of these traits to multiple factors changes during grain development (Aguirrezábal et al., [@B1]; Rondanini et al., [@B38]; Echarte et al., [@B11]). Therefore, understanding how grain weight and composition are determined demands a deep and integrated knowledge of grain filling dynamics.
Most of the photoassimilates supplied to the sunflower grains during their filling period are contemporaneously synthesized by the leaves and thus, leaves are considered the main source of substrate for grain growth (Hall et al., [@B17]; López Pereira et al., [@B22]; Echarte et al., [@B10]). Since grains are the main sink of photoassimilates during this period, changes in assimilate production at the source can be interpreted as changes in carbon availability in the developing grains (Hall et al., [@B16]), sucrose being the major carbohydrate and the main phloem-transported sugar in sunflower plants (Alkio et al., [@B2]). Once in the grain, part of this carbon is directed to the pool of acetyl-CoA, the precursor of fatty acids, which are the main components of sunflower oil.
The biosynthesis of fatty acids involves several enzymes placed in different sub-cellular compartments of the grains (Garcés and Mancha, [@B13]; Gray and Kekwick, [@B15]; Harwood, [@B18]). The enzyme acetyl-CoA carboxylase (ACCase) catalyzes the first reaction of this pathway. After successive elongation reactions, the main products of the intraplastidial *de novo* fatty acid biosynthesis are palmitoyl-ACP (Pp), stearoyl-ACP (Sp), and oleoyl-ACP (Op). These acyl-ACPs are hydrolyzed to free fatty acids, activated to the corresponding acyl-CoAs, and exported to the cytosol to be incorporated into glycerolipids. Finally, oleic acid can be transformed into linoleic acid by the action of oleoylphosphatidylcholine desaturase (FAD2), an enzyme located in the endoplasmic reticulum (E.R), in a step that represents a key point in the regulation of fatty acid composition (Garcés and Mancha, [@B13]).
Progress in understanding and modeling grain weight and composition dynamics can be achieved with two different main approaches: empirical or mechanistic. While empirical models typically describe the data but do not explain them, mechanistic models are reductionist and explain data based on knowledge of processes at the lower levels of biological organization (Loomis et al., [@B21]; Thornley and Johnson, [@B43]). Mechanistic models of physiological processes are often based on biochemical principles such as enzyme kinetics and reaction stoichiometry (Amthor, [@B3]). For instance, a biochemical model of fatty acids biosynthesis has been proposed by Martínez-Force and Garcés ([@B26]). This model is a structured kinetic one based on every known enzymatic step of the pathway. Although useful to understand the fatty acid biosynthetic pathway, this model does not consider the supply of substrate by the mother plant (which changes along grain filling), nor the synthesis of other grain components that contribute to grain weight and composition. The formulations of this kind of model require extensive knowledge of metabolic pathways, often not available in enough detail, and complex computer programming to carry out the calculations involved (Martínez-Force and Garcés, [@B26]). Furthermore, the concentrations of intermediate compounds and enzymatic activities are often difficult to measure, hindering validation of state variables, and making them less attractive for practical applications (Durruty et al., [@B9]).
Mathematical models with a more empirical approach have been developed to predict sunflower development, yield, and yield components (Chapman et al., [@B8]; Steer et al., [@B42]; Villalobos et al., [@B47]; Yeatts, [@B48]). These crop simulation models are useful tools for evaluating different agronomic management strategies (Villalobos et al., [@B47]). Based on a certain crop physiology background, they often adequately address the crop growth and development and their interaction with the environment. However, most of these models describe grain filling with insufficient detail, fail to take into account processes occurring inside the grain and rarely consider the accumulation of the main grain components. Some published sunflower models predict oil yield and quality but they are mainly based on empirical relationships between many traits and environmental factors and, moreover, the simulation of grain components accumulation is not dynamic (Pereyra-Irujo and Aguirrezábal, [@B32]; Casadebaig et al., [@B6]). To the best of our knowledge, a model describing the dynamics of sunflower grain filling and the accumulation of the main grain components in detail (oil and its fatty acid composition) has not been developed so far.
Non-structured models provide a trade-off between the realism of the biological processes and the relative simplicity required by modeling (Tolla et al., [@B44]). They are useful tools when access to the data is limited or the complexity of reactions in the pathway hinders the modeling process (Steer et al., [@B42]; Tolla et al., [@B44]; Durruty et al., [@B9]). The aim of the present work was to develop a non-structured mechanistic kinetic model of grain growth and oil and fatty acids biosynthesis, as a tool to describe the dynamics of grain filling in a comprehensive and quantitative way. Such a kinetic model can predict the dynamics of weight and components of the sunflower grain, and would contribute to understand the underlying mechanisms and the response of grain weight and composition to different growing conditions.
Materials and methods {#s2}
=====================
Model development
-----------------
### General
Our model was built on the basis of biochemical reactions engineering principles. In this first version, the effect of temperature in plant development was taken into account by means of thermal time calculations. Other effects of temperature and effects of variations in daily incident radiation were not considered. Water and nutrients available are considered non-limiting for all processes. The following main assumptions were made:
1. \- the temporal variable *t* is expressed as thermal time after flowering (accumulated degree days);
2. \- grain is considered as a control volume that changes as the grain grows;
3. \- grain filling is treated as a fed batch system where the grain grows in batch mode with an external carbon source;
4. \- the external carbon source changes with time as a consequence of leaf senescence;
5. \- once in the grain, carbon has two possible fates: (i) it turns into substrate for growth (i.e., contributes to grain weight), or (ii) it is used for maintenance.
### Grain growth and maintenance
Logistic functions have been reportedly useful to predict the sigmoid behavior (S-shape) of sunflower grain weight dynamics (Yeatts, [@B48]). In the present work, a logistic equation driven by time expressed in degree days (1) was fitted to experimental data to simulate grain growth. Equation (1) was first presented by M\'Kendrick and Pai ([@B23]). $$\text{W } = \,\frac{\text{W}_{0}.\text{e}^{\mu^{\prime}.\text{t}}}{1\, - \,\frac{\text{W}_{0}}{\text{W}_{\text{max}}}\,\left( {1 - \text{e}^{\mu^{\prime}.\text{t}}} \right)}$$ In this equation, *W* is the dry weight of an individual grain, W~0~ is the initial amount of W at which the model starts to work and *W*~max~ is the maximum potential grain weight. The parameter μ′ represents the specific grain growth rate (i.e., the biomass production rate per unit of biomass).
The rate at which the grain grows (*r*~*W*~) can be written as: $$\begin{array}{l}
{r_{W} = \mu^{\text{'}}.\frac{W_{max}\ - W}{W_{max}}.W\ } \\
\end{array}$$ The grain is not a closed system as it continuously receives the substrate assimilated by the mother plant. Once in the grain, *C* substrate rapidly reacts to form the different grain components. Thus, in a *C* mass balance, the *C* that enters the grain per unit time is equal to the consumption rate (*r*~*C*~), while the accumulation rate can be neglected. Thus *r*~*C*~ represents the rate of carbon allocation to the grains (or feed rate), here expressed as carbohydrate equivalents per degree days (Vertregt and Penning De Vries, [@B46]; Echarte et al., [@B10]).
However, not all the substrate reaching the grain is used for growth (defined as increase in grain weight). Part of this *C* is used to provide the energy needed for diverse processes that do not result in a net increase of dry weight (e.g., turnover of structures, activity of transport and movement, maintenance of concentration gradients and defense systems). The *C* costs of some of these processes are considered in calculations of carbohydrate equivalents (e.g., maintenance of the tools for biosynthesis, Vertregt and Penning De Vries, [@B46]). The Pirt\'s maintenance equation (Pirt, [@B33]) allows us to separate the *C* consumed by these processes from the *C* consumed for growth as follows: $$\begin{array}{l}
{\text{r}_{\text{C}} = \frac{\text{r}_{\text{W}}}{\text{Y}_{\text{G}}}\ + \text{~m}.\text{W}} \\
\end{array}$$ where Y~*G*~ and m are the actual growth yield and maintenance coefficients, respectively. The coefficient Y~*G*~ represents the biochemical efficiency of transformation of glucose into new plant material (Van Iersel and Seymour, [@B45]) and m, the C expended in processes that do not result in a net increase in grain dry matter. The coefficient m differs from those previously reported (Ploschuk and Hall, [@B34]; Van Iersel and Seymour, [@B45]) in that it does not consider all processes related to maintenance respiration (e.g., cell structure maintenance; Vertregt and Penning De Vries, [@B46]) since they were not included in carbohydrate equivalents calculations.
The first term of Equation (3) represents the substrate used for grain growth (*r*~*CG*~) and the second one the substrate used for maintenance purposes (*r*~*Cm*~). Considering total grain weight *W* as the sum of oil (W~O~) and non-oil (W~NO~) fractions, each production rate can be obtained by defining *Y*~*GO*~ and *Y*~*GNO*~ as the actual oil and non-oil fraction yield coefficients, respectively (see Supplementary Material).
### Solar radiation interception
As the plant life progresses, the capacity of the source to feed the grain decreases because of leaf senescence, and then, *r*~*C*~ also decreases. The model takes into account this phenomenon by considering the feed rate as a function of the proportion of the photosynthetically active radiation (*p*~*PAR*~) intercepted by the crop. The value of *p*~*PAR*~ is equal to one at the initial time (beginning of grain filling) and decreases as plant leaves senesce. To solve the model, a mathematical expression of *p*~*PAR*~ is necessary. The radiation interception has been previously calculated as a function of the leaf area index, in agreement with the Lambert-Beer law, which in turn depends on thermal time (Gardner et al., [@B14]; Pereyra-Irujo and Aguirrezábal, [@B32]). *p*~*PAR*~ can be expressed as: $$\begin{array}{l}
{p_{PAR} = 1\ - \ Kp.e^{k_{\lambda}.t}} \\
\end{array}$$ where *k*~λ~ is an empirical parameter that considers the Lambert-Beer law constant and the relationship between the leaf area index and thermal time, and *Kp* is a proportionality constant between *p*~*PAR*~ and radiation interception. In the present work, both parameters were obtained by fitting Equation (4) to experimental data.
### Grain filling
Thus, according to the previous section and in order to take into account the contribution of *C* assimilated by the mother plant to grain filling, the theoretical accumulation of carbohydrate equivalents, i.e., the cumulative amount of substrate that enters the grain expressed by Equation (3) must be modified to: $$\begin{array}{l}
{\frac{\text{dC}}{\text{dt}} = \left( {\frac{\text{r}_{\text{W}}}{\text{Y}_{\text{G}}}\ + \text{~m}.\text{W}} \right){.\text{p}}_{\text{PAR}}} \\
\end{array}$$ On the same way, the production rate of *W* must be expressed as: $$\begin{array}{l}
{\frac{dW}{dt} = Y_{G}{.r}_{CG}{.p}_{PAR} = r_{W}{.p}_{PAR}} \\
\end{array}$$ Finally, according to partitioning of *C* into the oil and non-oil fractions, the production rate of each component can be depicted as: $$\begin{array}{l}
{\frac{dW_{O}}{dt} = Y_{GO}{.r}_{CG}{.p}_{PAR} = \frac{Y_{GO}}{Y_{G}}r_{W}{.p}_{PAR}} \\
\end{array}$$ $$\begin{array}{l}
{\frac{dW_{NO}}{dt} = Y_{GNO}{.r}_{CG}{.p}_{PAR} = \frac{Y_{GNO}}{Y_{G}}r_{W}{.p}_{PAR}} \\
\end{array}$$ Equations (5--8) are ordinary differential equations (ODE) and must be simultaneously solved to predict the profiles of *C* and *W* during grain filling. The production rate and the substrate used for each component growth and maintenance (*r*~*WO*,~ *r*~*WNO*~, *r*~*CNO*,~ *r*~*CO*~, and *r*~*Cm*~), can also be individually predicted by coupling and solving their respective differential equations (see Supplementary Material).
### Fatty acids biosynthesis
A simplified model of fatty acids biosynthesis is shown in Figure [1](#F1){ref-type="fig"}. Compounds that react in the plastid are grouped in a global variable *C*~*F*~, which represents the precursor for all the fatty acids produced and stored. This is a simplification of the model presented by Martínez-Force and Garcés ([@B26]). Lumping several intermediates into a global variable *C*~*F*~ allows considering the serial-parallel nature of the synthesis pathway without the need for a complex segregated model. Furthermore, the global variable *C*~*F*~ implicitly considers the dynamic channeling model presented by these authors.
![**(A)** Cartoon representing the steps proposed by the model and their localization. Once carbon (C) has been allocated to the grain it can be used for maintenance or growth. Grain weight (W) is formed by oil (W~*O*~) and non-oil (W~*NO*~) components. Compounds that react in the plastid \[palmitoyl-ACP (Pp), stearoyl-ACP (Sp), and oleoyl-ACP (Op)\] are grouped in a global variable C~*F*.~ Fatty acids forming glycerolipidis are translocated to the endoplasmic reticulum (E.R), where the microsomal oleoylphosphatidylcholine desaturase transforms oleic acid (O) into linoleic acid (L) **(B)** Simplified reaction scheme used in this work. The parameters r~*i*~ represent the rates of production of "i" compounds. Compounds inside the dashed line form the oil fraction of grain weight.](fpls-07-00586-g0001){#F1}
Figure [1](#F1){ref-type="fig"} shows the proposed kinetic model and the simplified reactions involved in the synthesis of fatty acids. In previous sections, substrate consumption was divided into maintenance and growth, and the grain growth was fractionated into oil and non-oil components. Since the oil fraction is composed of several fatty acids and intermediates, the rate depicted in Equation (7) pertains to the first step of fatty acids biosynthesis, i.e., the production of intermediary *C*~*F*~. The intermediate *C*~*F*~ accumulated is equal to the difference between its production from C allocated to the grain and its consumption to produce P, S, and O, and storage. In agreement with the proposed model, the following net intermediate production rate results: $$\begin{array}{l}
{\frac{dC_{F}}{dt}\ = \ Y_{GO}r_{CG}p_{PAR}\ - \ r_{P}\ - \ r_{O}\ - \ r_{S}} \\
\end{array}$$ Assuming the production and active transport of P, S, and O follows a specific Michaelis-Menten kinetics (i.e., they depend on grain weight, the heavier the grain, the higher the rate of synthesis of fatty acids), the production rate can be written as: $$r_{i}\, = \,\frac{v_{max}{}_{i}\left( \frac{C_{F}}{W} \right)}{Ks_{i}\, + \,\frac{C_{F}}{W}}W$$ where *v*~*max*~ and *Ks* are the maximum specific rate and half saturation constants, respectively. The subscript *i* represents P, S, or O. The fatty acid production rate is expressed here as a function of intermediate concentration (i.e., the amount of *C*~*F*~ per unit weight).
As stated above, linoleic acid is produced from oleic acid inside the endoplasmic reticulum. Then: $$r_{L}\, = \,\frac{v_{max}{}_{L}\left( \frac{O}{W} \right)}{Ks_{L}\, + \,\frac{O}{W}}W\, - \,\frac{v_{max}{}_{\, - \, L}\left( \frac{L}{W} \right)}{Ks_{\, - \, L}\, + \,\frac{L}{W}}W$$ Finally, the kinetics of *C*~*F*~ and the different fatty acids in the oleosome can be calculated via their respective mass balances. $$\frac{dC_{F}}{\text{dt}}\, = \, Y_{GO}r_{CG}p_{PAR}\, - \,{\sum_{i = P,S,O}{\frac{v_{max}{}_{i}\left( \frac{C_{F}}{W} \right)}{Ks_{i}\, + \,\frac{C_{F}}{W}}W}}$$ $$\frac{\text{dP}}{dt} = \frac{v_{max}{}_{P}.\text{ }\left( \frac{C_{F}}{W} \right)}{\text{Ks}_{\text{P}}\, + \frac{\text{C}_{\text{F}}}{\text{W}}}.\text{W}$$ $$\frac{\text{dS}}{\text{dt}} = \frac{\text{v}_{\text{max}}{}_{\text{S}}.\,\left( \frac{\text{C}_{\text{F}}}{\text{W}} \right)}{\text{Ks}_{\text{S}}\, + \,\frac{\text{C}_{\text{F}}}{\text{W}}}.\text{W}$$ $$\begin{array}{l}
{\frac{\text{dO}}{\text{dt}} = \frac{\text{v}_{\text{max}}{}_{\text{O}}.\left( \frac{\text{C}_{\text{F}}}{\text{W}} \right)}{\text{Ks}_{\text{O}}\text{+}\frac{\text{C}_{\text{F}}}{\text{W}}}.\text{W } - \left( {\frac{\text{v}_{\text{max}}{}_{\text{L}}.\left( \frac{\text{O}}{\text{W}} \right)}{\text{Ks}_{\text{L}}\text{+}\frac{\text{O}}{\text{W}}}\text{W}} \right.} \\
{\text{ }\left. {- \frac{\text{v}_{\text{max}}{}_{- \text{L}}.\left( \frac{\text{L}}{\text{W}} \right)}{\text{Ks}_{- \text{L}}\, + \,\frac{\text{L}}{\text{W}}}\text{W}} \right)} \\
\end{array}$$ $$\frac{\text{dL}}{\text{dt}}\, = \,\frac{\text{v}_{\text{max}}{}_{\text{L}}.\,\left( \frac{\text{O}}{\text{W}} \right)}{\text{Ks}_{\text{L}}\, + \,\frac{\text{O}}{\text{w}}}\text{W } - \,\frac{\text{v}_{\text{max}}{}_{\, - \, L}.\,\left( \frac{\text{L}}{\text{W}} \right)}{\text{Ks}_{\, - \, L}\, + \,\frac{\text{L}}{\text{w}}}\text{W}$$ Since *W* grows with *C*~*F*~, P, S, O, and L, the ODE Block (12--16) and differential Equation (6) must be solved simultaneously in order to predict the biosynthesis of fatty acids during grain filling.
Experimental data
-----------------
Sunflower (*Helianthus annuus* L.) was grown in the field at Balcarce Experimental Station (Unidad Integrada Balcarce INTA-FCA; 37°S, 58°W), in the Buenos Aires Province, Argentina. The model was initially calibrated with hybrid ACA885, and later evaluated with experimental data from hybrids MG2 and DK3820. The soil was a Typic Argiudoll. Experiments were performed during growing seasons 2007--2008 and 2012--2013. Each experimental unit consisted of six rows 6 m long spaced at 0.7 m. Plant population density at sowing was 6.5 plants m^−2^. The crops were grown under optimal nutrient and water conditions. Soil fertility in all experiments was adequate to attain maximum yields for sunflower crops grown under non-limiting water conditions---yield \>5000 kg ha^−1^ (Sosa et al., [@B41]; Andrade et al., [@B4]). Pests, diseases and weeds were successfully controlled. Flowering of a plant was defined by the appearance of stamens in all florets from the outer whorl of the capitulum---R5.1 stage, (Schneiter and Miller, [@B40]).
Sampling and chemical analysis
------------------------------
Sampling and chemical analysis were performed as in Echarte et al. ([@B11]). Briefly, 12 grains of rows 6--8 were excised from the same plant as long as the total removal did not exceed 5% of the average final capitulum grain number. The number of sampling dates varied between 8 and 12, depending on the experiment. Grains were oven-dried at 60°C and weighed. Lipids were extracted with 5 ml of hexane:isopropanol (7:2, v/v) and 2.5 ml Na~2~SO~4~ (67 g l^−1^) in the presence of 0.2 ml of 1, 2, 3 triheptadecanoyl-glycerol (50 mg ml^−1^) as internal standard. The lipidic phases from the extracted samples were evaporated to dryness under a nitrogen stream. The residue was dissolved in 0.5 ml of hexane and incubated for 1 h at 80°C in the presence of the methylation mixture methanol:toluene:H~2~SO~4~ (88:10:2) and 1 ml of heptane. After samples had cooled down to room temperature, the upper phase containing fatty acids methyl esters was separated. The fatty acid composition of the extracts was determined by gas chromatography (GLC) with a Shimadzu GC-2014 chromatograph (Kyoto, Japan). The fatty acid content and total lipids extracted were calculated with the internal standard method. The oil content was assumed equal to the total extracted lipids given that they reportedly represent more than 96% of the oil (Robertson et al., [@B36]).
Measurements
------------
Global daily incident radiation was measured with pyranometers (LI-200SB, LI-COR, Lincoln, NE) from a meteorological station located \~400 m away from the experimental units. The proportion of photosynthetically active radiation (*pPAR*) intercepted by the crop at noon (±1 h) was calculated according to Gallo and Daughtry ([@B12]) as (1 − Rb/Ro), where Rb is the radiation measured below the oldest green leaf, and Ro is the radiation measured above the canopy. Rb was measured weekly with a line quantum sensor (LI-191SB, LI-COR, Lincoln, NE, USA) positioned across the rows (the length of the sensor was modified according to the distance between rows, 0.7 m). Three measurements were taken per plot. Air temperature was measured using shielded thermistors (Cavadevices, Buenos Aires, Argentina) next to the capitulum every 60 s and averaged hourly. Measurements began after flowering and finished at physiological maturity and were recorded by data loggers (Cavadevices, Buenos Aires, Argentina).
The amount of assimilates effectively allocated to the grains (*C*) was assumed to be represented by carbon equivalents for grain biomass production (Vertregt and Penning De Vries, [@B46]). For this, carbon and nitrogen in the grains were determined with a TruSpec CN equipment (Leco Corporation, St. Joseph, MI), and the ash content was measured according to AOAC recommendations (Aoac, [@B5]). Carbohydrate equivalents for grain biomass production were calculated as described by Vertregt and Penning De Vries ([@B46]).
Model determinations
--------------------
### Thermal time
The temporal variable *t* is expressed as cumulative degree days, with the aim of expressing time and rates in a temperature compensated way to make temporal effects independent of temperature fluctuations (Kiniry et al., [@B20]; Parent and Tardieu, [@B30]). Cumulative degree days are calculated from daily data for mean temperature (*Tm*) and a base temperature (*Tb*) of 6°C (Kiniry et al., [@B20]) as follows: $$\begin{array}{l}
{t = \sum{({Tm\ - \ Tb})}} \\
\end{array}$$
### *C~F~* intermediate
Values of *C*~*F*~ were calculated for every "*j*" thermal time from experimental data using a box mass balance Equation (18). *Y*~*WO*∕*C*~ represents the apparent oil yield coefficient, it was obtained from the linear regression of oil vs. C experimental data. $$\begin{array}{l}
{{C_{F}}_{t = j} = Y_{WO \slash C}\ C_{t = j}\ - \ P_{t = j}\ - \ S_{t = j}{\ - \ O}_{t = j}\ - \ L_{t = j}} \\
\end{array}$$
### Actual yield coefficients
The actual growth yield coefficient (*Y*~*G*~) and the maintenance coefficient (*m*) were calculated by linear fitting of Pirt\'s Equation (Pirt, [@B33]) to experimental data: $$\begin{array}{l}
{\frac{1}{Y} = \frac{1}{Y_{G}}\ + \ \frac{m}{\mu}} \\
\end{array}$$ where *Y* is the instantaneous growth yield (Pirt, [@B33]). Values of *Y* and μ were calculated for every "*j*" time interval as central differences of the experimental data in agreement with Equations (20) and (21), respectively. $$\begin{array}{l}
{Y_{t = j} = \frac{W_{t = j\ + \ 1}\ - \ W_{t = j\ - 1\ }}{C_{t = j\ + \ 1}\ - \ C_{t = j\ - 1}}} \\
\end{array}$$ $$\begin{array}{l}
{\mu_{t = j} = \frac{dW}{dt} = \frac{W_{t = j + 1} - W_{t = j - 1}}{t_{j + 1} - t_{j - 1}}} \\
\end{array}$$ Subsequently, the actual non-oil fraction yield coefficient (*Y*~*GNO*~) and actual oil fraction yield coefficient (*Y*~*GO*~) were determined using a general non-linear regression.
### Kinetic parameters
Growth kinetic parameters *W*~0~, *W*~*Max*~, and μ′ were calculated by non-linear fitting of Equation (1). The kinetic parameters for fatty acids production *v*~*max*~ and *Ks* were obtained by non-linear fitting of Equations (10) and (11). The values of production rates were obtained by finite differences (central differences) according to Equation (22): $$\begin{array}{l}
{{r_{i}}_{t = j}\ = \ \frac{di}{dt}\ = \ \frac{i_{t = j\ + \ 1}\ - \ i_{t = j - 1}}{t_{j\ + \ 1}\ - \ t_{j - 1}}} \\
\end{array}$$ where *i* represent P, S, O, or L. Once all the parameters were obtained, a further step of model optimization was performed using a general non-linear regression framework.
Sensitivity analysis
--------------------
A sensitivity analysis was performed as in Villalobos et al. ([@B47]). The effects of ±25% variation in every kinetic parameter of the model on the main variables output (C, W, P, S, O, and L) were analyzed. The sensitive coefficient (*SC*) was calculated as in Equation (23), being *V* the output variable and *P* the kinetic parameter. $$\begin{array}{l}
{SC = \frac{\left. \Delta V\slash V \right.}{\left. \Delta P\slash P \right.}} \\
\end{array}$$
Informatics tools and statistics
--------------------------------
Linear and non-linear regressions were performed with Origin 8.0® (OriginPro, v. 8.0724; OriginLabCorporation, Northampton, MA 01060, USA). Once the kinetic parameters were obtained, profiles were modeled using a fourth-order Runge-Kutta algorithm coupled to the regression in order to integrate the differential equations simultaneously (MathCad 14.0.0.163, Parametric Technology Corporation). The goodness-of-fit of the model was evaluated using the regression coefficient (*R*^2^) and Reduced Chi-square (χ^2^) test via the Prob (χ^2^ \> *F*) with α \< 0.05. Significant differences among parameters were evaluated by Student *t*-test (95% confidence interval).
Results {#s3}
=======
Solar radiation interception
----------------------------
Figure [2](#F2){ref-type="fig"} shows the experimental values of *p*~*PAR*~ as a function of degree days after flowering for several experiments. The line represents the result of fitting Equation (4) to the experimental data (*R*^2^ = 0.7633, *P* \< 0.001). The values obtained for the empirical parameter *k*~λ~ and the proportionality constant *Kp* resulted 4.04 × 10^−3^ ± 7.12 × 10^−4^ °Cd af^−1^ and 0.0434 ± 0.0199, respectively.
{#F2}
Grain growth and filling
------------------------
Grains grow with thermal time following a sigmoid curve (Figure [3A](#F3){ref-type="fig"}). After fitting Equation (1) to the experimental data, the values obtained for *W*~0~, *W*~*max*~ and μ′ were 0.4299 ± 0.1634 mg, 34.5396 ± 0.9131 mg, and 0.0145 ± 0.0013 °Cdaf^−1^, respectively \[*R*^2^ = 0.94547, Prob (χ^2^ \> *F*) = 0\]. Figure [3B](#F3){ref-type="fig"} shows the experimental values of C vs. thermal time. The values obtained for *Y*~*G*~ and *m* by fitting Equation (19) to the experimental data were 0.671 ± 0.036 mg $\text{mg}_{\text{C}}^{- 1}$ and 4.301 × 10^−3^ ± 4.28 × 10^−4^ mg~C~ mg^−1°^Cdaf^−1^, respectively. The fitted values for *Y*~*GNO*~ and *Y*~*GO*~ were 0.368 ± 0.021 g $\text{g}_{\text{C}}^{- 1}$ and 0.303 ± 0.016 g $\text{g}_{\text{C}}^{- 1}$, respectively \[*R*^2^ = 0.9552, Prob(χ^2^ \> *F*) = 0\].
{ref-type="fig"} represent carbohydrate equivalents consumed for maintenance purposes (Cm), to produce grain (CG), non-oil fraction (CNO), and oil fraction (CO).](fpls-07-00586-g0003){#F3}
Grain weight and cumulative carbohydrate equivalents were simulated by solving simultaneously ODE Block (5--8) with the previously fitted parameters. The values predicted by the model are presented in Figures [3A,B](#F3){ref-type="fig"} as solid lines. The figures indicate the model successfully describes the experimental grain growth kinetics and it is able to predict the theoretical cumulative amount of carbohydrates that is allocated to the grain during the filling period. The amount of substrate spent in growth (CG), maintenance (Cm), oil (CO), and non-oil (CNO) grain fractions were also predicted and plotted as a function of thermal time (dashed lines in Figure [3B](#F3){ref-type="fig"}). Different dynamics of carbon investment were observed: at first, most of the substrate is used to produce grain mass, later, the maintenance requirements increase and thus less substrate is used for growth.
Values of W~NO~ and W~O~ were also obtained by solving ODE Block (5--8) (Figures [3C,D](#F3){ref-type="fig"}). The simulations seem to overestimate the experimental *W*~*O*~-values before 200°Cdaf.
Grain weight was plotted as a function of carbohydrate equivalents in Figure [4](#F4){ref-type="fig"}. The solid line shows the values predicted by the model; the instantaneous growth yield is represented by the slope of this line. It can be observed that the model acceptably simulates the non-linear behavior of experimental data. At the beginning of grain filling, when grains are small, they present lower maintenance requirements, which results in a higher apparent growth yield. As grains grow in size, more substrate is used for maintenance and the apparent yield coefficient decreases.
{#F4}
Fatty acids biosynthesis
------------------------
Table [1](#T1){ref-type="table"} shows the kinetic parameters obtained from fitting Equation (10) and (11). The values predicted by the model are presented in Figure [5](#F5){ref-type="fig"} as solid lines. The model successfully describes the experimental behavior of all fatty acids. The rates of production of every fatty acid increase early during grain filling together with grain weight, later they decrease until they completely stop. The model predicts the amount of oleic acid increases at the early stages of grain filling, reaches a maximum at 400°Cdaf and then decreases. Linoleic acid production follows an end-product saturating specific kinetics, like palmitic, and stearic acid, but smoothed and delayed by a reversible reaction and by the fact that linoleic acid is the final product of three serial steps (C→C~F~ →O→L).
######
**Kinetic parameters of fatty acids biosynthesis for ACA885 sunflower hybrid**.
**Product** **Parameter** **Value ± *SD*** **Prob(χ^2^ \< *F*)**
--------------- --------------- --------------------------------- -----------------------
Palmitic acid v~max~ 2.025 × 10^−4^± 6.769 × 10^−5^ 4.71E-4
Ks 0.0139 ± 0.022
Stearic acid v~max~ 7.783 × 10^−5^ ± 2.814 × 10^−5^ 2.04E-4
Ks 0.0214 ± 0.0029
Oleic acid v~max~ 3.157 × 10^−3^ ± 8.463 × 10^−4^ 2.76E-5
Ks 0.0199 ± 0.0025
Linoleic acid v~max~ 0.0197 ± 2.646 × 10^−3^ 3.15E-8
Ks 0.0202 ± 0.0322
v~max−1~ 0.0190 ± 6.837 × 10^−4^
Ks~−1~ 0.0159 ± 0.00221
{#F5}
Once the profile of each fatty acid was obtained, the concentration of oil was calculated as the sum of all fatty acids and plotted in Figure [5E](#F5){ref-type="fig"} as a solid line. The dotted line in the figure represents the oil grain weight predicted in a first approach (Figure [3D](#F3){ref-type="fig"}), where a single step production process was considered (see Section Grain Growth and Filling). A better performance of the model was achieved when the serial nature of fatty acids biosynthesis was considered, especially at short times (\<300°Cdaf) when *C*~*F*~ is accumulated.
Sensitivity analysis
--------------------
A sensitivity analysis was performed on the proposed model. The effect of ±25% variation in every kinetic input parameter on the model output was observed at three different times during grain filling. The results, presented in Table [2](#T2){ref-type="table"}, show that the relative influence of the parameters on both grain weight and composition varies along the grain development. Specific growth (μ′) has significant influence on all the variables at the beginning of grain filling (*SC* \> 0.5) but its effect decays later on (*SC* \< 0.5), when C is used in maintenance processes. On the other hand, W~0~, W~max~, Y~G~, and *m* exert low influence (*SC* \< 0.3) on most of the variables. However, palmitic, stearic, and linoleic acids were sensitive to grain growth and oil yield (*Y*~*G*~ and *Y*~*GO*~), showing the influence of the partitioning of carbon to the oil or non-oil fraction on fatty acid composition. Saturated fatty acids were sensitive to their own maximum specific rate of synthesis (*v*~*maxS*~ and *v*~*maxP*~) and to the oleic acid maximum specific rate (*v*~*maxO*~), while oleic acid was mostly influenced by parameters driving the synthesis of linoleic acid.
######
**Sensitivity coefficients of parameters for ACA885 sunflower hybrid dynamics**.
**Positive change** **Negative change**
---------------------------------- --------------------- --------------------- ------- -------- -------- -------- -------- -------- ------- -------- -------- --------- --------
**SENSITIVITY COEFFICIENT (SC)**
μ′ 300 0.54 0.522 0.564 0.58 0.57 0.569 0.594 0.593 0.549 0.557 0.579 0.54
600 0.125 0.046 0.034 0.055 0.008 0.059 0.344 0.27 0.274 0.285 0.148 0.311
800 0.103 0.023 0.004 0.029 0.024 0.025 0.282 0.184 0.162 0.193 0.185 0.188
W~0~ 300 0.125 0.119 0.158 0.159 0.144 0.164 0.203 0.197 0.237 0.228 0.224 0.245
600 0.03 0.011 0.01 0.008 -- 0.01 0.055 0.024 0.024 0.018 0.001 0.025
800 0.024 0.005 0.003 0.003 0.005 0.002 0.044 0.012 0.008 0.011 0.011 0.008
W~max~ 300 0.07 0.075 0.041 0.047 0.055 0.039 0.129 0.138 0.084 0.089 0.104 0.076
600 0.179 0.201 0.201 0.205 0.217 0.2 0.256 0.28 0.281 0.285 0.294 0.281
800 0.186 0.209 0.211 0.211 0.21 0.212 0.263 0.287 0.291 0.293 0.288 0.292
Y~G~ 300 −0.138 -- −0.045 −0.056 −0.071 −0.054 −0.312 -- −0.042 −0.063 −0.068 −0.051
600 −0.109 -- −0.152 −0.180 −0.069 −0.198 −0.245 -- −0.338 −0.407 −0.145 −0.449
800 −0.101 -- −0.153 −0.180 −0.055 −0.199 −0.228 -- −0.339 −0.408 −0.080 −0.458
m 300 0.044 -- -- -- -- -- 0.06 -- -- -- -- --
600 0.081 -- -- -- -- -- 0.11 -- -- -- -- --
800 0.091 -- -- -- -- -- 0.123 -- -- -- -- --
Y~GO~ 300 -- -- 0.024 0.037 0.042 0.031 -- -- 0.084 0.114 0.134 0.102
600 -- -- 0.188 0.227 0.076 0.25 -- -- 0.258 0.303 0.121 0.334
800 -- -- 0.188 0.225 0.047 0.253 -- -- 0.26 0.304 0.098 0.336
v~maxP~ 300 -- -- 0.217 -- −0.002 −0.001 -- -- 0.293 −0.013 −0.003 −0.002
600 -- -- 0.202 −0.014 −0.005 −0.016 -- -- 0.281 −0.022 −0.008 −0.022
800 -- -- 0.202 −0.016 −0.004 −0.016 -- -- 0.281 −0.018 −0.005 −0.023
Ks~P~ 300 -- -- −0.017 -- −0.012 0.007 -- -- −0.023 -- −0.017 0.01
600 -- -- −0.052 0.003 −0.007 0.006 -- -- −0.091 0.007 −0.009 0.01
800 -- -- −0.057 0.003 −0.008 0.006 -- -- −0.101 0.007 −0.010 0.011
v~maxS~ 300 -- -- -- 0.224 −0.001 -- -- -- -- 0.291 −0.002 −0.001
600 -- -- −0.004 0.211 −0.002 −0.005 -- -- −0.006 0.289 −0.003 −0.007
800 -- -- −0.004 0.211 −0.002 −0.006 -- -- −0.006 0.29 −0.002 −0.007
Ks~S~ 300 -- -- -- −0.019 −0.019 0.011 -- -- -- −0.038 −0.027 0.015
600 -- -- 0.002 −0.060 −0.013 0.004 -- -- 0.003 −0.107 −0.018 0.007
800 -- -- 0.001 −0.063 −0.013 0.004 -- -- 0.002 −0.114 −0.018 0.007
v~maxO~ 300 -- -- −0.027 −0.037 0.194 0.162 -- -- −0.023 −0.038 0.274 0.27
600 -- -- −0.142 −0.167 0.004 0.017 -- -- −0.306 −0.359 0.005 0.038
800 -- -- −0.143 −0.167 0.003 0.016 -- -- −0.309 −0.358 0.008 0.035
Ks~O~ 300 -- -- 0.003 0.009 −0.039 −0.009 -- -- 0.005 -- −0.058 −0.013
600 -- -- 0.055 0.057 −0.015 −0.004 -- -- 0.089 0.089 −0.021 −0.007
800 -- -- 0.061 0.061 −0.014 −0.004 -- -- 0.097 0.097 −0.019 −0.007
v~maxL~ 300 -- -- -- -- −0.45 0.249 -- -- -- -- --1.235 0.682
600 -- -- -- -- −0.453 0.096 -- -- -- -- --3.642 0.775
800 -- -- -- -- −0.443 0.088 -- -- -- -- --3.859 0.764
Ks~L~ 300 -- -- -- -- 0.13 −0.072 -- -- -- -- 0.192 −0.107
600 -- -- -- -- 0.214 −0.046 -- -- -- -- 0.277 −0.059
800 -- -- -- -- 0.215 −0.043 -- -- -- -- 0.278 −0.055
v~max−L~ 300 -- -- -- -- 0.701 −0.387 -- -- -- -- 0.642 −0.355
600 -- -- -- -- 2.003 −0.426 -- -- -- -- 0.694 −0.148
800 -- -- -- -- 2.056 −0.407 -- -- -- -- 0.687 −0.136
Ks~−L~ 300 -- -- -- -- −0.063 0.035 -- -- -- -- −0.103 0.057
600 -- -- -- -- −0.047 0.01 -- -- -- -- −0.073 0.016
800 -- -- -- -- −0.045 0.009 -- -- -- -- −0.068 0.014
*Positive and negative change refers to the fitted kinetic parameter ±25% variation, respectively*.
Model extrapolation to different hybrids
----------------------------------------
The ability of the model to predict the behavior of two independent hybrids that were not used for model calibration (MG2 and DK3820) was evaluated. Parameter values and their comparison among hybrids are provided as Supplementary Material (Table [SM1](#SM1){ref-type="supplementary-material"}). In order to define the smallest set of values necessary to obtain an appropriate simulation, the model was run by: (i) freely fitting all the parameters, or (ii) fitting the minimum amount of parameters that would ensure a good predictive quality \[Prob(χ2 \> *F*) \< 0.01\]. For this, parameters of low sensitivity (W~0~, W~max~, *Y*~*G*~, *m*, Ks~i~) and μ′ (sensitive only at the beginning of grain filling and low genetic variability) were fixed and *v*~*maxi*~ parameters were refitted. In (ii), the ACA885 parameter values were used instead of each hybrid\'s own parameters (bold values in Table [3](#T3){ref-type="table"}). Table [3](#T3){ref-type="table"} summarizes the kinetic parameters obtained by fitting the model to the experimental values measured for all hybrids.
######
**Kinetic parameters for different hybrids (MG2 and DK3820)**.
**Parameter** **MG2 (i)** **MG2 (ii)** **DK3820 (i)** **DK3820 (ii)**
-------------------------------------- ---------------------------------- ------------------------------------------- --------------------------------- -------------------------------------------
*μ′* \[°Cdaf^−1^\] 0.0126 ± 0.0017 **0.0145 ± 0.0013** 0.018 ± 0.003 **0.0145 ± 0.0013**
W~0~ \[mg\] 0.5862 ± 0.1906 **0.4299± 0.1634** 0.2400 ± 0.2273 **0.4299± 0.1634**
W~max~ \[mg\] 37.5079 ± 1.2389 **34.5396 ± 0.9131** 39.35186 ± 1.67339 **34.5396 ± 0.9131**
Y~G~ \[mg.mg${}_{\text{C}}^{- 1}$\] 0.950 ± 0.082 **0.671 ± 0.036** 0.638 ± 0.145 **0.671 ± 0.036**
m \[mg~C~.mg^−1^.°Cdaf^−1^\] 4.25 × 10^−3^ ± 7.69 × 10^−4^ **4.31** × **10^−3^ ± 4.28** × **10^−4^** 2.43 × 10^−3^ ± 1.37 × 10^−4^ **4.31** × **10^−3^ ± 4.28** × **10^−4^**
Y~GO~ \[mg.mg${}_{\text{C}}^{- 1}$\] 0.406 ± 0.035 **0.303 ± 0.016** 0.208 ± 0.048 **0.303 ± 0.016**
v~maxP~ \[mg.°Cdaf-1\] 1.455 × 10^−4^ ± 6.815 × 10^−5^ 2.531 × 10^−4^ ± 9.856 × 10^−5^ 3.581 × 10^−4^ ± 9.157 × 10^−5^ 1.758 × 10^−4^ ± 7.492 × 10^−5^
Ks~P~ \[mg.mgW-1\] 0.0106 ± 0.00137 **0.0139 ± 0.0022** 0.0755 ± 0.01855 **0.0139 ± 0.0022**
v~maxS~ \[mg.°Cdaf-1\] 1.6004 × 10^−4^ ± 1.107 × 10^−5^ 2.058 × 10^−4^ ± 1.925 × 10^−5^ 3.315 × 10^−4^ ± 9.216 × 10^−5^ 1.414 × 10^−4^ ± 7.890 × 10^−5^
Ks~S~ \[mg.mgW-1\] 0.04537 ± 0.00114 **0.0214 ± 0.0029** 0.1336 ± 0.04891 **0.0214 ± 0.0029**
v~maxO~ \[mg.°Cdaf-1\] 3.410 × 10^−3^ ± 1.683 × 10^−4^ 4.095 × 10^−3^ ± 2.021 × 10^−4^ 3.912 × 10^−3^ ± 1.345 × 10^−4^ 4.116 × 10^−3^ ± 1.452 × 10^−4^
Ks~O~ \[mg.mgW-1\] 0.0415 ± 0.0012 **0.0199 ± 0.0025** 0.03778 ± 0.00301 **0.0199 ± 0.0025**
v~maxL~ \[mg.°Cdaf-1\] 0.0367 ± 5.049 × 10^−3^ 0.0405 ± 5.571 × 10^−3^ 0.0440 ± 7.093 × 10^−3^ 0.0357 ± 8.742 × 10^−3^
Ks~L~ \[mg.mgW-1\] 0.0595 ± 0.0017 **0.0202 ± 0.0322** 0.0423 ± 0.0052 **0.0202 ± 0.0322**
v~max−L~ \[mg.°Cdaf-1\] 0.0291 ± 4.032 × 10^−4^ 0.0381 ± 5.241 × 10^−4^ 0.0349 ± 6.121 × 10^−4^ 0.0295 ± 5.221 × 10^−4^
Ks~−L~ \[mg.mgW-1\] 0.0391 ± 0.0021 **0.0159 ± 0.0022** 0.0723 ± 0.0036 **0.0159 ± 0.0022**
*The model was run by: (i) freely fitting all the parameters or (ii) fitting the minimum amount of parameters that would ensure a good predictive quality (Prob(χ2 \> F) \< 0.01). Highlighted (bold) values represent parameters obtained for ACA885*.
Growth parameter values are similar for all hybrids, with the exception of *Y*~*G*~ and *Y*~*GO*~ of MG2. Parameter μ′ showed high sensitivity early during grain filling but did not differ among hybrids, while *v*~*maxO*~ showed both low sensitivity and low genetic variability. When the set of parameters (i) was used, the model successfully predicted the kinetics of every trait explored in this research (data not shown). Grain weight, theoretical accumulated carbohydrates, and fatty acid composition dynamics simulated by fitting the minimal amount of parameters (case ii in Table [3](#T3){ref-type="table"}) are shown in Figure [6](#F6){ref-type="fig"}. In this case, simulated values of C and W adequately described the experimental behavior of both MG2 and DK3820 hybrids (Figures [6A,B](#F6){ref-type="fig"}). On the other hand, the extrapolation of all fatty acids biosynthesis parameters was not possible due to the high sensitivity of P, S, O, and L to *v*~*maxi*~. The model adequately predicted fatty acids dynamics during grain filling when five out of the 16 parameters were refitted.
{ref-type="table"}: **(A)** grain growth **(B)** carbohydrate equivalents; **(C)** palmitic acid; **(D)** stearic acid; **(E)** oleic acid; **(F)** linoleic acid. ACA885, closed circles; MG2, open circles; DK3820, triangles. Solid lines represent the model predictions.](fpls-07-00586-g0006){#F6}
Discussion {#s4}
==========
In the present work, a non-structured mechanistic kinetic model of grain growth and oil and fatty acids biosynthesis has been developed. By setting initial conditions --*W*~0~ for grain weight-- and calculating carbon assimilated by leaves and allocated to the grains as the substrate, the oil, and non-oil weight and oil composition dynamics have been successfully simulated for different sunflower hybrids. To the best of our knowledge, a model with the ability of describing the grain filling dynamics in such detail has not been previously developed for sunflower, or any other crop species.
Carbon partitioning to growth and maintenance
---------------------------------------------
The model considered that the carbon substrate was destined to both growth and maintenance. The amount of substrate that was actually transformed into grain biomass (*Y*~*G*~) was in the range of previously reported values for conversion efficiency (0.6 to 0.8 mg mg^−1^ Mccree, [@B29]; Van Iersel and Seymour, [@B45]). The maintenance coefficient (*m*) was as well in the range of reported values \[0.003 to 0.050 mg mg^−1^ d^−1^ (Hesketh et al., [@B19])\], despite in the present work it did not consider the carbon costs of cell structure maintenance (Penning De Vries et al., [@B31]). High variability of *m* has been associated to the dependence of this coefficient on the age of the plant and the environmental conditions during grain filling (Van Iersel and Seymour, [@B45]).
The results show that if maintenance processes were negligible, 45% of the carbohydrates destined for growth would be transformed into oil. When maintenance processes were considered, 45% of the carbohydrates allocated were destined to grain growth and only 40% of them were converted into oil. Therefore, the results of the model indicate that maintenance processes not only reduce the grain growth, but also the selectivity to oil. In light of these findings, further research might help to understand the physiological processes underlying the relationship between maintenance and grain composition.
Carbon partitioning to maintenance or growth changed with ontogeny. As the grain grew, the substrate destined to maintenance increased (Figure [3C](#F3){ref-type="fig"}) and the apparent yield coefficient decreased, in agreement with Pirt\'s law, (Equation 3; Pirt, [@B33]). Van Iersel and Seymour ([@B45]) found that *r*~*Cm*~ depends on both the age of the plant and the biomass dry weight. A similar behavior was found when analyzing the consumption rate of substrate for maintenance (*r*~*Cm*~) or grain growth (*r*~*CG*~) as a function of thermal time (Figure [7A](#F7){ref-type="fig"}) or grain weight (Figure [7B](#F7){ref-type="fig"}; see Supplementary Material for *r*~*Cm*~ and *r*~*CG*~ calculation). The value of *r*~*CG*~ presents a maximum at 300°Cdaf, in concordance with the inflection point of the sigmoid growth curve (Figure [3A](#F3){ref-type="fig"}). The value of *r*~*Cm*~ reaches its maximum later and at higher grain weight than *r*~*CG*~ (Figure [7B](#F7){ref-type="fig"}). The earlier decrease of *r*~*CG*~ indicates the system is more selective to maintenance when the grain is bigger. In this sense, Van Iersel and Seymour ([@B45]) propose that younger plants do not show substrate limitations and maintenance increases as the grain grows. The amount of substrate consumed for growth (*r*~*CG*~) increases with *W* due to higher carbon use efficiency as the plants become bigger. As the plant life progresses (later in the plant cycle and higher *W*), the substrate available diminishes together with the substrate destined for both, growth and maintenance. According with Figure [1A](#F1){ref-type="fig"} when *r*~*C*~ falls due to *p*~*par*~ effect, both *r*~*Cm*~ and *r*~*CG*~ fall because they are also limited by carbon allocation (Equation 5).
{#F7}
### Simulation of grain weight and composition dynamics
Grain growth (total weight, oil, and non-oil components) followed sigmoid functions with time, in agreement with many reports in the literature (Aguirrezábal et al., [@B1]; Mantese et al., [@B25]; Rondanini et al., [@B37]; Echarte et al., [@B11]). In a previous work, Echarte et al. ([@B10]) reported that the grain weight and oil content linearly increased with the amount of carbon allocated to the grains. In this research, a model with a more mechanistic approach was able to predict the theoretical cumulative amount of carbohydrates that was allocated to the grain during the filling period, and successfully described the grain growth kinetics.
The kinetic parameters of fatty acid biosynthesis were first obtained for sunflower hybrid ACA885. Given a parallel reaction scheme, the system is considered more selective toward the reaction with higher rate. A higher maximum rate of production for oleic acid (*v*~*maxO*~) than for palmitic and stearic acids made the system more selective toward oleic acid. Similar values of Ks for P, S, and O, which represent the affinity of enzymes involved in their active transport out of the plastid, suggest that these enzymes have similar affinity for the three fatty acids. According to the model predictions, palmitic, and stearic acids followed typical end-product saturating specific kinetics (Figures [5A,B](#F5){ref-type="fig"}), while oleic acid increased at early stages of grain filling up to a maximum. These predictions are in agreement with previous studies (Martínez-Force et al., [@B27]; Santonoceto et al., [@B39]; Echarte et al., [@B11]). One possible explanation for the behavior of oleic acid is that as grain filling progresses, oleate desaturase activity increases (Gray and Kekwick, [@B15]), but carbon accumulates in the grain faster than the increment of this catalytic activity. Therefore, between 150 and 300°Cdaf, oleic acid begins to accumulate. Between 300 and 350°Cdaf, a high desaturation activity produces a decrease of oleic acid with a concomitant increase of linoleic acid, being more evident when carbon is scarce (Echarte et al., [@B11]). The accumulation of linoleic acid responds to higher values of *v*~*maxL*~ than ~*Vmax*−*L*~. Furthermore, *v*~*maxL*~ was the highest *v*~*max*~ value of all, explaining the high productivity of this compound in agreement with previously reported data (Martínez-Force et al., [@B27]; Santonoceto et al., [@B39]; Echarte et al., [@B11]).
In a first approach, the oil fraction was predicted as if it were produced in a single reaction step (Figure [3D](#F3){ref-type="fig"}), although it is the final product of a more complex reaction pathway. The low oil fraction values estimated this way suggest an accumulation of intermediate compounds, that were assigned to the non-oil fraction in this first approach. However, when total fatty acids (oil) were estimated as the sum of every fatty acid predicted (Figure [5E](#F5){ref-type="fig"}) a better performance was achieved. Thus, the model depicted in Figure [1](#F1){ref-type="fig"}, which considers the serial nature of fatty acids biosynthesis, was able to better represent the experimental behavior, especially at short times (\<300°Cdaf) when *C*~*F*~ is accumulated.
### Finding key genotype parameters of the model
Complex models are not suitable for the characterization of the dynamics of multiple genotypes, since once the model has been built, finding the kinetic parameters of a new hybrid might be laborious, expensive, and time consuming. Thus, many models rely on a limited number of genetic parameters that appropriately describe one particular genotype behavior, while assuming that the rest of the parameters do not significantly influence the model output for any genotype (Quilot et al., [@B35]; Makowski et al., [@B24]). Given a small set of parameters, re-parameterizing many growing models may not require new dynamic measurements, and parameters can be estimated from the final values by optimization methods. The model developed here for sunflower hybrid ACA855 used 16 input parameters to simulate grain weight and component dynamics. Combining the results of sensitivity analysis and the genetic variability of parameters of two other hybrids (MG2 and DK3820), the number of model input parameters was reduced to five. Whether this is sufficient to simulate the behavior of the universe of commercial hybrids should be further tested working with a bigger pool of hybrids than the one explored in this research.
A sensitivity analysis showed that the influence of parameters changed with ontogeny. Although the specific growth rate (μ′) was the most influential grain growth parameter on model output at the beginning of grain filling, similar values among hybrids indicate low genetic variability and thus, a unique value of μ′ could satisfactorily simulate grain filling dynamics for any hybrid. Although they are parameters of low sensitivity, higher values of conversion efficiencies (*Y*~*G*~ and *Y*~*GO*~) for MG2 suggest this hybrid devotes more substrate to grain weight than the other hybrids.
Fitting of parameters related to fatty acids biosynthesis, more specifically the maximum specific rates (*v*~*maxP*~, *v*~*maxS*~, *v*~*maxO*,~ *v*~*maxL*~, *v*~*max*−*L*~), is needed to re-calibrate the model for every hybrid to adequately predict the fatty acid composition. Traditional commercial hybrids have been improved to obtain maximal grain weight and oil content, but not targeting their fatty acid composition. The latter could have been modified or unintentionally selected when domesticating or breeding other characters (Chapman and Burke, [@B7]). Hybrids with different potential fatty acid composition have been obtained by mutagenesis (e.g., high oleic hybrids) and oils with certain fatty acid composition receive a prime over the regular price in the market. This model could help to understand the dynamics of oil and fatty acid biosynthesis in sunflower hybrids with modified potential fatty acid composition (high oleic, high stearic, high oleic-high stearic, etc.).
### Potential uses of the model
The model presented in this paper has been mainly targeted at simulating genetic effects on grain filling and composition dynamics. The identification of key genotypic parameters could guide future research on physiological processes and guide breeding programs. In addition, it is a promising tool to model the effects of biotic and abiotic factors on these dynamics. In this first version, the assimilates availability was estimated based on available data of canopy interception. However, the model could be linked to crop models capable of simulating it by considering different environmental input variables and calculating intermediate phenological or plant structure ones (e.g., Pereyra-Irujo and Aguirrezábal, [@B32]). In addition, using the present model as a platform and making the necessary modifications, it will be possible to explore the dynamics of other oilseed species (like soybean or rape), where enzymes and pathways are known to significantly differ from those in sunflower.
Conclusion {#s5}
==========
In this work, a kinetic model of sunflower grain filling and fatty acids biosynthesis has been developed. The ability of the model to predict the experimental values was successfully evaluated and validated in different hybrids. The combination of sensitivity analysis and the genetic variability of parameters allowed minimizing the number of input parameters required to appropriately simulate the dynamics of grain filling and component accumulation in different hybrids. The growth model considered a simple effect of carbon source dynamics, maintenance requirements, and a simplified serial-parallel reaction system to describe the fatty acids biosynthetic pathway. The model developed represents a useful tool for future research to evaluate the effects of different factors on grain weight and composition, in a comprehensive and a quantitative way.
Author contributions {#s6}
====================
In this work the skills from two different knowledge areas have been integrated. The experience of ID on kinetic modeling based on chemical reaction engineering basis was used to modelate experimental data from agricultural field. The experimental data have been obtained in INTA-BAlcarce by ME and LA during several years. ME provided the theoretical base and ID developed and programmed the kinetic model, aiming to represent the metabolic behavior of sunflower grain filling and fatty acids biosynthesis. All the authors wrote the manuscript.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP0362), Universidad Nacional de Mar del Plata (UNMdP) and PNCYO 1127042-Programa Nacional de Cereales y Oleaginosas-INTA.
Supplementary material {#s7}
======================
The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fpls.2016.00586>
######
Click here for additional data file.
######
Click here for additional data file.
μ′
: defined specific growth parameter \[◦Cdaf^−1^\]
C
: carbohydrate equivalents \[mgC\]
C~*F*~
: fatty acids synthesis intermediate \[mg\]
k~λ~
: Lambert-Beer law adjusted constant \[◦Cdaf^−1^\]
k~max~
: maximum specific rate \[mg ◦Cdaf^−1^\]
Ks
: half saturation constant \[mg mgW^−1^\]
Kp
: proportionality constant \[dimensionless\]
L
: linoleic acid \[mg\]
m
: maintenance coefficient \[mgC ◦Cdaf^−1^ mgW^−1^\]
pPAR
: fraction of photosynthetic active radiation intercepted by the crop \[unitless\]
O
: oleic acid \[mg\]
P
: palmitic acid \[mg\]
r~i~
: production/degradation rate of component i \[mgi ◦Cdaf^−1^\]
S
: stearic acid \[mg\]
t
: thermal time after flowering \[◦Cd af\]
W
: dry weight of grain \[mg\]
W~0~
: initial grain dry weight \[mg\]
W~max~
: maximum theoretical grain dry weight \[mg\]
W~NO~
: non-oil grain fraction dry weight \[mg\]
W~O~
: oil grain fraction dry weight \[mg\]
Y~G~
: actual growth yield coefficient or conversion efficiency \[mg mgC^−1^\]
Y~GNO~
: actual non-oil fraction yield coefficient \[mg mgC^−1^\]
Y~GO~
: actual oil fraction yield coefficient \[mg mgC^−1^\]
```{=html}
<!-- -->
```
− (minus sign)
: represents the inverse reaction
C
: relative to substrate as carbohydrate equivalent
G
: relative to growth
L
: relative to linoleic acid
m
: relative to maintenance
O
: relative to oleic acid
P
: relative to palmitic acid
S
: relative to stearic acid
W
: relative to grain weight
[^1]: Edited by: Hartmut Stützel, Leibniz Universität Hannover, Germany
[^2]: Reviewed by: Jinkee Lee, Sungkyunkwan University, South Korea; Michel Genard, Institut National de la Recherche Agronomique, France
[^3]: This article was submitted to Plant Biophysics and Modeling, a section of the journal Frontiers in Plant Science
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
Zika virus (ZIKV; family Flaviviridae, genus *Flavivirus*) is a single-stranded positive-sense enveloped RNA virus. The 10.8 kb ZIKV genome encodes a single polyprotein that is processed into three structural proteins (C, PrM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral and host proteases^[@CR1]^. Phylogenetic studies have shown that all ZIKV strains characterized to date belong to two distinct lineages (African and Asian) based on the initial geographic distribution of this virus^[@CR2]^. ZIKV is a mosquito-borne flavivirus transmitted primarily by *Aedes* spp. mosquitoes^[@CR3]^.
Long considered to cause mild disease in humans, this arbovirus remained relatively unstudied until 2007, when it provoked a large outbreak in Micronesia^[@CR4]^. Subsequently, several outbreaks occurred in different Pacific Ocean islands, including French Polynesia in 2013, where it was associated with an increased incidence of Guillain--Barré syndrome^[@CR5]^. ZIKV then spread to the American continent, causing major outbreaks in Central/South America and the Caribbean and was linked with an increase of congenital neurological complications. Sexual transmission of ZIKV was also reported^[@CR6]^. There is currently no commercial antiviral drug or vaccine for this virus^[@CR7]^.
Several approaches are now available with which to develop inactivated^[@CR8]^ and recombinant (DNA-^[@CR9]^ or RNA-based^[@CR10]^) ZIKV vaccines. However, live-attenuated vaccines have several advantages, including reduced costs and single-dose induction of long-term immunity^[@CR11]^. Several groups developed live ZIKV vaccine candidates by making deletions in the 3′ untranslated region of the viral genome^[@CR12],[@CR13]^. More recently, a chimeric ZIKV vaccine candidate based on the Japanese encephalitis virus live-attenuated strain SA14-14-2 was reported^[@CR14]^. The chimeric approach had been used since the late 1990s to develop vaccine candidates against several health-threatening flaviviruses, including West-Nile virus, Japanese encephalitis virus, and all serotypes of the dengue virus^[@CR15]--[@CR17]^. This approach consists of incorporating prM/E of a pathogenic flavivirus in a backbone of a licensed live-attenuated vaccine strain. Indeed, E protein is prominently exposed at the surface of viral particles and is the de facto the major determinant of viral antigenicity^[@CR1]^. In almost all cases, the well-characterized live-attenuated 17-D strain used to prevent yellow fever virus (YFV) infections has been used as the genetic backbone. Some of these live-attenuated vaccines are currently commercially available^[@CR17],[@CR18]^.
In this study, we describe the development of a chimeric virus harboring the prM/E of an epidemic ZIKV (H/PF/2013) strain and the 17-D vaccine strain as the genetic backbone. The user-friendly and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method was used to generate the chimeric virus^[@CR19]^. Finally, in cellulo and in vivo characterization of this strain demonstrated its potential as a live-attenuated vaccine candidate.
Results {#Sec2}
=======
Design and rescue of chimeric viruses {#Sec3}
-------------------------------------
The chimeric viruses were constructed using the yellow fever 17-D vaccine strain as a genetic backbone and by replacing prM/E from this vaccine strain with those of the Asian ZIKV PF epidemic strains. Three different constructs, designated A, B, and C, were constructed using variable sites flanking the ZIKV prM/E coding sequences (Fig. [1](#Fig1){ref-type="fig"}). Construct A harbored the pre-peptide and cleavage site before prM from the 17-D vaccine strain. Construct B harbored the pre-peptide from the 17-D vaccine strain and had a cleavage site before prM from ZIKV. Construct C harbored the pre-peptide and had a cleavage site before the prM of ZIKV. All the constructs contained the cleavage site from the 17-D vaccine strain between the E and NS1 proteins.Fig. 1Schematic representation of the design and recovery strategies used to generate chimeric viruses. We recovered infectious virus only with construct C.The two cleavage sites are enlarged in boxes, with the amino acid alignment shown with separations between different proteins
The ISA procedure was used to rescue the viruses. Three overlapping amplicons, which encompassed the complete genome flanked at its 5′ and 3′ extremities by the human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) respectively, were transfected into a mix of HEK-293/BHK-21 cells. Because the first amplicon contained the entire structural coding region, it was only necessary to exchange the first amplicon in our previously designed and functional yellow fever 17-D vaccine strain reverse genetic system to attempt replicative virus production.
For each construct, we performed two independent cell transfection experiments with five replicates. After incubating for 6 days, the cell supernatant medium was passaged four times in Vero-E6 cells. Virus replication was assessed in cell supernatant medium from the last passage (passage \#4) using a real-time quantitative reverse transcription PCR (qRT-PCR), and no viral replication was detected for constructs A and B. In contrast, for construct C, we detected virus replication in one well (1/5) in both independent transfection experiments. These results highlighted that the choice of the nature of the pre-peptide and cleavage site between the capsid and prM proteins is a crucial parameter when designing chimeric flaviviruses. During the first cell transfection experiment, we obtained high amounts of viral genome copies at passage \#4 (1.78 e+9 viral RNA copies/ml). This virus was designated CH-17-D/ZIKV and used for in cellulo and in vivo characterizations. Surprisingly, during the second experiment, we detected very low quantities of the viral genome at passage \#4 (3.57 e+3 viral RNA copies/ml), and this virus was designated as CH-17-D/ZIKV\*. We next performed four additional passages using the same procedure. The quantities of viral genomes in cell supernatant medium was assessed from passage \#1 to \#8 and compared with that of CH-17-D/ZIKV (Fig. [2](#Fig2){ref-type="fig"}). We observed that the amount of viral genome for CH-17-D/ZIKV reached a plateau at passage \#2, whereas an increase in the production of CH-17-D/ZIKV\* was observed from passage \#6 to passage \#8, reaching viral genome values similar to those observed with CH-17-D/ZIKV (2.67e+9 viral RNA copies/ml) (Fig. [2](#Fig2){ref-type="fig"}).Fig. 2Evolution of viral production of chimeric viruses during serial passage that followed cell transfection.A mix of BHK-21/HEK-293 cells was transfected. Cell supernatant medium was subsequently passaged 4--8 times in Vero-E6 cells. Viral production in cell supernatant medium was assessed using a real-time quantitative RT-PCR assay
CH-17-D/ZIKV genotypic characterization {#Sec4}
---------------------------------------
To identify the genotypic determinants associated with the difference in viral replication observed between CH-17-D/ZIKV and CH-17-D/ZIKV\*, the complete genome of CH-17-D/ZIKV was obtained at passages \#2 and \#4 and compared with the sequence of the original construct. Only five substitutions were detected at passage \#2, of which two were non-synonymous, confirming the genome integrity of this strain (Table [1](#Tab1){ref-type="table"}). In addition, four substitutions were already fixed or almost fixed. At passage \#4, all these mutations were fixed, and no additional mutations were observed. Interestingly, both non-synonymous mutations are located in domain II of the E protein at residues E255 and E285^[@CR20]^. Subsequently, we determined the sequence of the 5′ region of the CH-17-D/ZIKV\* viral genome (until the NS1 coding region) at passage \#4 and the complete genome sequence of CH-17-D/ZIKV\* at passage \#8 (Table [1](#Tab1){ref-type="table"}). While only one transitory substitution was detected at passage \#4, all the mutations that were detected in CH-17-D/ZIKV were detected at passage \#8, including the two non-synonymous mutations located in the E coding region. This high level of parallel evolution associated with the observed chronology of events strongly suggests that these five mutations are associated with the increase in replicative fitness observed for both viruses.Table 1Mutations detected during the passages that followed cell transfection of chimeric virusesChimeric virusNucleotide positionFrequency at \#P2Frequency at \#P4Frequency at \#P8Frequency at \#P10RegionNucleotide changeaa change**CH-17-D/ZIKV**291100%100%100%100%CA\>G--162566%90%92%94%ET\>CV\>A1706100%100%100%100%EG\>TG\>V2514100%100%100%79%NS1A\>G--4482100%100%100%100%NS2BA\>G--**CH-17-D/ZIKV\***291n.an.d70%n.aCA\>G--1303n.a100%n.dn.aEC\>TH\>Y1625n.an.d68%n.aET\>CV\>A1706n.an.d69%n.aEG\>TG\>V2514n.an.d69%n.aNS1A\>G--4482n.an.a56%n.aNS2BA\>GOnly consensus mutations (frequency \>50%) are shown. *n.a.* not available, *n.d.* not detected
CH-17-D/ZIKV initial characterization {#Sec5}
-------------------------------------
To confirm the presence of the ZIKV E protein in Vero-E6 cells infected by the chimeric virus, we performed an indirect immunofluorescence assay using a specific ZIKV immune serum as the primary antibody (Fig. [3a](#Fig3){ref-type="fig"}). ZIKV PF and the 17-D vaccine strains were used as positive and negative controls, respectively. As expected, no fluorescence was observed with the 17-D vaccine strain, and positive cells were observed at day 2 and 5 post-infection with both the chimeric and ZIKV strains, confirming that the ZIKV E protein was expressed in infected cells. At day 2 post-infection, the number of cells that were positive for ZIKV was greater than that observed for CH-17-D/ZIKV, in agreement with the observed growth replication kinetics in Vero-E6 cells. Since a cytopathic effect was observed with the ZIKV strain at day 5, the number of positive cells was lower using this virus. Viability assays in Vero-E6 cells confirmed this observation; the CH-17-D/ZIKV virus is less cytopathic (mean value: 73% of cell viability) at day 5 post-infection than the ZIKV (mean value: 49% of cell viability) (Supplemental Fig. [1](#MOESM1){ref-type="media"}). We next performed comparative growth kinetics of these viruses in three different cell lines (HUH7.5, HEK-293, and Vero-E6). Cell supernatant medium was harvested at different time points after infection to assess the amount of viral RNA (Fig. [3b--d](#Fig3){ref-type="fig"}). Similar growth kinetics curves were observed for all viruses in HUH7.5 cells. In Vero-E6 cells, higher amounts of viral genome were not observed in cell supernatants until day 5 post-infection with the chimeric virus. In HEK-293 cells, the chimeric virus had a similar behavior to that of the 17-D vaccine strain.Fig. 3CH-17-D/ZIKV in cellulo characterization.**a** Expression of the ZIKV E protein in Vero-E6 was confirmed at day 2 and 5 post-infection using an indirect immunofluorescence assay with a specific ZIKV immune serum as the primary antibody. Uninfected cells (mock) and cells infected by ZIKV and the 17-D vaccine strain were used as controls. **b**--**d** Comparative growth kinetics of the CH-17-D/ZIKV and ZIKV 17-D vaccine strains in HUH7.5 (**b**), HEK-293 (**c**), and Vero-E6 cells (**d**). **e,** **f** Comparative growth kinetics of the CH-17-D/ZIKV and ZIKV 17-D vaccine strains in Vero cells. Cell supernatant medium was harvested at different time points after infection to assess the amount of viral RNA present using a real-time quantitative RT-PCR assay (**e**; expressed as the means ± SD) and the infectious titers using a TCID~50~ assay (**f**; expressed as the means ± SD)
CH-17-D/ZIKV characterization in Vero cells {#Sec6}
-------------------------------------------
Since Vero cells are widely used for vaccine production^[@CR21]^, we characterized CH-17-D/ZIKV in this cell line. Because CH-17-D/ZIKV was already adapted at passage \#4 (see above), we used cell supernatant from this passage to perform growth kinetics in Vero cells. Cell supernatant medium was harvested at different time points after infection to assess infectious titers (via a TCID~50~ assay) and the amount of viral RNA (Fig. [3e, f](#Fig3){ref-type="fig"}). The results showed that these cells enabled the production of highly infectious viral particles at day 6 post-infection. We also studied the genetic stability of CH-17-D/ZIKV by performing six additional passages in Vero cells, and the complete genome sequence was obtained at passages \#8 and \#10 (Table [1](#Tab1){ref-type="table"}). Our findings revealed a remarkable genetic stability since all mutations at passage \#4 remained stable and no additional mutations were detected.
CH-17-D/ZIKV in vivo characterization {#Sec7}
-------------------------------------
Because ZIKV and the 17-D vaccine strain do not replicate in immunocompetent mice, we used immunocompromised mice as a model to study the chimeric virus in vivo. Each time animals were immunized or infected; they were transiently immunocompromised following a two-step inoculation with an anti-IFNAR antibody^[@CR22]--[@CR24]^ as described in the Methods section.
Six groups of four mice were inoculated with two different dosages of CH-17-D/ZIKV, ZIKV, or the 17-D vaccine strain to assess antibody production (Fig. [4a](#Fig4){ref-type="fig"}). A control group (mock) of four mice were inoculated with phosphate-buffered saline (PBS). Twenty-one days after immunization, mice were sacrificed and their sera were tested for the presence of antibodies to ZIKV and YFV (Fig. [4b, c](#Fig4){ref-type="fig"}) using a viral RNA Yield Reduction Neutralization Test (YRNT; see Methods). The results demonstrated that immunization with the chimeric virus induced the production of neutralizing antibodies against ZIKV, confirming the initial hypothesis of this study. We detected a slightly higher level of neutralizing antibodies when mice were infected with ZIKV. In all cases, both dosages used induced comparable neutralizing titers. Consistent with previous studies, mice immunized with the 17-D vaccine strain did not produce antibodies against ZIKV. Indeed, based on the amino acid sequence divergence of antigenic proteins, it is well established that no cross-neutralizing activity exists between these two distant flaviviruses^[@CR25]^. As expected, mice immunized with the 17-D vaccine strain produced high levels of neutralizing antibodies against YFV, while those infected with ZIKV did not produce any antibodies against YFV. Interestingly, immunization with CH-17-D/ZIKV induced the production of neutralizing antibodies against YFV. This result demonstrated the immunogenicity of the viral proteins encoded by the 17-D vaccine strain backbone. We also attempted to isolate chimeric virus from animal blood samples to assess the ability of the chimeric virus to replicate in vivo. To avoid the possibility of isolating residual virus from the immunization, at days 2 and 3 post immunization we collected a blood drop from the tails of mice and found two positive samples (one from each day) (Supplemental Fig. [2](#MOESM1){ref-type="media"}). These findings suggest that CH-17-D/ZIKV is able to replicate in mice, since comparable neutralizing titers were measured with all mice immunized.Fig. 4Neutralizing antibody titers in transitory immunocompromised mice at day 21 post-immunization.**a** Experimental timeline. **b**, **c** Groups of four mice were immunized with two doses of CH-17-D/ZIKV, ZIKV, and the 17-D vaccine strain (from 10e4 to 10e6 TCID~50~). Twenty-one days later, sera from mice were tested for the presence of antibodies to ZIKV and YFV using a viral RNA Yield Reduction Neutralization Test. The results are expressed as individual log of YRNT50 (**b**) and YRNT90 titers (**c**) with mean values ±SD represented by black lines with error bars, respectively
In another experiment, we assessed protection against subsequent infection by wild-type ZIKV following immunization with CH-17-D/ZIKV or the 17-D vaccine strain (Fig. [5a](#Fig5){ref-type="fig"}). Groups of mice were immunized with two dosages of CH-17-D/ZIKV and the 17-D vaccine strain 21 days prior to challenge with a ZIKV African strain (Dak84). Three control groups were also used, one that was immunized with PBS and then challenged (the unvaccinated group), one that was immunized with ZIKV PF and then challenged (the ZIKV PF group), and one that was immunized and challenged with PBS (the mock group). Since 100% of the mice of the ZIKV PF control group were viremic at days 2 and 3 post-challenge, this criterion was not used to assess protection (Supplemental Fig. [3](#MOESM1){ref-type="media"} and Supplemental Table [1](#MOESM1){ref-type="media"}). Therefore, the protection was evaluated by determining the proportion of mice with organs (brain and spleen) that tested positive for the presence of ZIKV at day 10 post-challenge. We observed that 10% of the spleens and brains from mice immunized with the chimeric virus (both groups) were positively tested positive for ZIKV (Table [2](#Tab2){ref-type="table"}). In contrast, 100% and 87.5% of the spleens and brains from mice immunized with the 17-D vaccine strain (both groups) tested positive for ZIKV, respectively (*p*-value = 0.0004 for spleens and 0.0029 for brains; Fisher exact test). As expected 100% and 0% of the organs from mice in the unvaccinated group and from the ZIKV PF group were positive for ZIKV, respectively. Viral RNA yields from the organs were highly variable in all positive samples (Fig. [5b](#Fig5){ref-type="fig"}). These results demonstrated that immunization with the chimeric virus significantly protected mice against the systemic and brain infection induced by a heterologous ZIKV strain.Fig. 5Amounts of viral RNA detected in brain and spleen samples collected during challenge experiments.**a** Experimental timeline. **b** Amounts of viral RNA in brain and spleen samples collected during challenge experiments (cf. Table [2](#Tab2){ref-type="table"}) measured using a real-time quantitative RT-PCR assay. Mean values ± SD are represented by black lines and error bars, respectively. The results from both doses of viruses are pooledTable 2Protection of transitory immunocompromised mice challenged with a heterologous strain of ZIKVViral strainSpleensBrainsCH-17D/ZIKV (both doses)10% (1/10)10% (1/10)17-D vaccine strain (both doses)100% (8/8)87.5% (7/8)Unvaccinated100% (4/4)100% (4/4)ZIKV PF0% (0/4)0% (0/4)Groups of mice were immunized with two doses (10e4 and 10e5 TCID~50~) of CH-17-D/ZIKV, the 17-D vaccine strain or PBS (unvaccinated). Twenty-one days later, mice were challenged with 10e6 TCID~50~ of an African ZIKV strain. The proportion of mice testing positive for ZIKV in spleen/brain samples at day 10 post-challenge was expressed as a percentage. The results from both doses of viruses are pooled (the results for individual groups are provided in Supplemental Table [2](#MOESM1){ref-type="media"}). Viral RNA was detected using a real-time RT-PCR assay. The amounts of viral RNA detected in samples are shown in Fig. [5](#Fig5){ref-type="fig"}
Discussion {#Sec8}
==========
We present here the initial development of a chimeric ZIKV live-attenuated vaccine candidate based on a yellow fever attenuated 17-D genetic backbone. Using the ISA reverse genetics method, we were able to rapidly test several combinations of subgenomic amplicons starting from a pre-existing 17-D vaccine strain reverse genetics system. This method was recently applied to Asian and African strains of ZIKV^[@CR26]^.
Three different designs were tested to incorporate the prM/E of ZIKV into the 17-D vaccine genetic backbone. Our results highlighted the necessity of modifying the cleavage site between the pre-peptide and the prM protein for the construction of chimeric viruses, as was previously described during development of chimeric ZIKV/DENV and DENV/ZIKV strains^[@CR27]^.
Nevertheless, we also demonstrated that chimeric viruses are needed to acquire adaptive mutations to properly replicate in mammalian cells. Indeed, we observed a low percentage of virus recovery during cell transfection experiments, and both replicative viruses rescued shared five substitutions, of which two were non-synonymous and located in domain II of the E protein. Interestingly, mutations located in this particular domain of the E protein were previously described in cellulo with 17-D vaccine strain-based chimeric flaviviruses (DENV type 1/2 and Japanese encephalitis virus)^[@CR28],[@CR29]^. These findings suggest that the emergence of compensatory mutations in the E protein is probably necessary to restore the replicative fitness of the virus following the exchange of two of its structural proteins.
By comparing the growth properties of our chimeric virus with its two parental strains in different mammalian cells, we observed that this new synthetic virus had its own biological properties, probably due to the nature of this new combination of viral proteins. In fact, we observed than this strain is fitter than parental strains in Vero-E6 cells and is close to the fitness of the 17-D vaccine strain in HEK-293 cells.
Genetic stability is a major concern when designing future live-attenuated vaccine candidates. Using Vero cells, which are widely used for vaccine production^[@CR21]^, and the adapted chimeric virus, we performed serial passages to assess this essential criterion. We demonstrated that once initial adaptation was achieved, the chimeric virus remained genetically stable.
We used transitory immunocompromised mice as an animal model system to characterize the chimeric virus in vivo. We demonstrated that mice infected with this virus produced levels of neutralizing antibodies that were close to those observed following infection by ZIKV. Our results also showed that immunization using the chimeric strain significantly protected 90% of mice against brain and spleen invasion induced by a heterologous strain of ZIKV. This incomplete protection (i.e., one mouse out of ten was unprotected) could result from the incomplete CD8+ T cell immunity induced by structural proteins alone^[@CR30]--[@CR32]^. This explanation has been recently proposed to explain the failure of the CYD-TDV dengue chimeric vaccine^[@CR33]^. Altogether, these results provide evidence that this chimeric strain has all the prerequisites needed to be tested in a more relevant animal model, such as the microcephalic-sensitive mouse model^[@CR34]^.
The strategy used in the present study to develop a live ZIKV vaccine candidate has several advantages, including that the 17-D vaccine strain has long history of use in hundreds of millions of persons^[@CR35]^ and is considered as the safest live-attenuated vaccine^[@CR36]^. Moreover, compared with targeted attenuation strategies, such as local modification of genomic regions, our approach eliminates the risk of phenotype reversion by potential homologous recombination. Finally, although the potential occurrence of the antibody-dependent enhancement phenomenon has to be considered with chimeric vaccines, there is currently no epidemiological data supporting this hypothesis in areas where several flaviviruses co-circulate^[@CR37]^.
In conclusion, our data provide a sound basis for the future development of this vaccine candidate. Furthermore, the approach used in this study to rescue the chimeric virus showed that significant advances in the development of reverse genetics methods now offer the possibility of drastically reducing the time frame between the emergence of a novel viral pathogen and the availability of a live-attenuated vaccine candidate.
Materials and methods {#Sec9}
=====================
Cell lines {#Sec10}
----------
All cells were grown at 37 °C with 5 % CO~2~ with 1 % penicillin/streptomycin (PS; 5000 U/ml and 5000 µg/ml, respectively; Life Technologies) and supplemented with 1% non-essential amino acids (Life Technologies) in media as specified below.
Baby hamster kidney (BHK-21; ATCC number CCL-10), human hepatocellular carcinoma (HUH7.5^[@CR38]^; RRID CVCL_7927) and human embryonic kidney (HEK-293; ATCC number CCL-1573) cells were grown in Dulbecco's modified Eagle's medium high glucose (4500 mg/l) (Life Technologies) with 7 .5% heat-inactivated fetal bovine serum (FBS; Life Technologies). Vero (ATCC number CCL-81) and Vero-E6 (ATCC number CRL-1586) cells were grown in minimal essential medium (Life Technologies) with 7 .5% FBS.
Viruses {#Sec11}
-------
ZIKV Asian lineage strains PF (H/PF/2013, GenBank accession number: KJ776791) and Mart2015 (MRS_OPY_Martinique_PaRi_2015, GenBank accession number: KU647676), ZIKV African lineage strain Dak84 (A.taylori-tc/SEN/1984/41662-DAK, GenBank accession number: KU955592), YFV 17-D strain (produced by reverse genetics as described below; GenBank accession number: EU074025), and YFV strain BOL 88/1999 (isolated in 2009 from human serum and kindly provided by the National Center of Tropical Diseases (CENETROP), Santa-Cruz, Bolivia, GenBank accession number: KF907504) were used in this study. All these viral strains are available for the scientific community via the European Virus Archive goes Global (EVAg) project, a non-profit organization (<https://www.european-virus-archive.com>).
For each viral strain, we prepared a stock solution of clarified cell culture medium that was subsequently used for all analyses. Briefly, a 25 cm^2^ culture flask of confluent Vero-E6 cells containing 667 µl of medium with 2.5% FBS (Life Technologies) was inoculated with 333 µl of clarified infectious medium, incubated for 6 h, washed once with Hank's Balanced Salt Solution (HBSS, Life Technologies), and then incubated for 3 days with 7 ml of fresh medium. Cell supernatant medium was subsequently harvested and clarified by centrifugation, supplemented with HEPES buffer (final concentration of 25 mM; Sigma), and then aliquoted and stored at −80 °C.
All experiments using replicating viruses were performed in BSL3 facilities.
ISA procedure {#Sec12}
-------------
Chimeric viruses and the YFV 17-D vaccine strain were rescued using the ISA (Infectious Subgenomic Amplicons) reverse genetics method as previously described^[@CR19],[@CR26],[@CR39],[@CR40]^.
### Preparation of subgenomic DNA fragments {#Sec13}
The complete viral genome was amplified by PCR as three overlapping DNA fragments. The first and last fragments were flanked by the 5′ and 3′ termini, which included the human cytomegalovirus promoter (pCMV) and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. We started by using a reverse genetics system designed for the YFV 17-D strain (described in the Supplemental Material). Because the first DNA fragment contained all the regions encoding structural genes, only this fragment was modified to design chimeric viruses (the primers are listed in Supplemental Table [3](#MOESM1){ref-type="media"}).
DNA fragments were generated by PCR using de novo synthesized genes (Genscript) as templates. The sequences of the primers used are listed in Supplemental Table [2](#MOESM1){ref-type="media"}. PCR mixes were prepared using a Platinum PCR SuperMix High Fidelity kit (Life Technologies) following the manufacturer's instructions. PCR amplifications were performed using an ABI 2720 thermal cycler (Applied Biosytems) with the following conditions: 94 °C for 2 min followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s, 68 °C for 5 min, with a 10 min final elongation at 68 °C. PCR product sizes and quality were controlled by running gel electrophoresis and DNA fragments were purified using a High Pure PCR Product Purification kit (Roche).
### Cell transfection {#Sec14}
Mixtures of BHK-21 and HEK-293 cells were seeded into PureCoat amine six-well cell culture plates (Corning) 1 day prior to transfection. Cells were transfected with 2 µg of an equimolar mix of the three DNA fragments using lipofectamine 3000 (Life Technologies) following the manufacturer's instructions. Each transfection was performed in five replicates. After incubating for 24 h, the cell supernatant medium was removed and replaced by fresh cell culture medium. Six days post-transfection, cell supernatant medium was passaged four times using six-well cell culture plates of confluent Vero-E6 cells. Cells were subsequently inoculated with 100 µl of diluted (1/3) cell supernatant media, incubated 2 h, washed with HBSS, and incubated 6 days with 3 ml of medium. Remaining cell supernatant medium was stored at −80 °C and samples were referred to as passages \#1, \#2, \#3, and \#4. To ensure the complete removal of DNA used during the transfection, passage \#4 was used to assess viral replication, where 100 µl of cell supernatant medium was collected to detect viral RNA using a qRT-PCR assay (see below). Passage \#3 was used to produce virus stock solutions of YFV 17-D and chimeric viruses.
RNA extraction and real-time quantitative PCR assays {#Sec15}
----------------------------------------------------
RNA extraction was performed using the Qiacube HT and the Cador pathogen extraction kits (both from Qiagen) following the manufacturer's instructions. Briefly, 100 µl of cell supernatant medium was transferred into an S-block containing the recommended quantities of VXL, proteinase K and RNA carrier. A DNAse digestion step (Qiagen) was performed to remove the DNA used during cell transfection. The quantity of viral RNA was quantified by real-time quantitative RT-PCR (qRT-PCR; EXPRESS One-Step Superscript™ qRT-PCR Kit, universal; Life Technologies). The sequences of the primers used to detect ZIKVs, YFV 17-D, and chimeric viruses are listed in Supplemental Table [4](#MOESM1){ref-type="media"}. For each reaction, 3.5 µl of RNA was used (final volume of 10 µl) and amplifications were performed using a QuantStudio 12 K Flex Real-Time PCR System (Applied Biosytems) with the following conditions: 10 min at 50 °C and 2 min at 95 °C, followed by 40 amplification cycles (95 °C for 3 s followed by 30 s at 60 °C). The amounts of viral RNA present were calculated from standard curves (quantified T7-generated synthetic RNA standards were used).
Complete genome sequencing {#Sec16}
--------------------------
Complete and partial genome sequencing of chimeric viruses were performed as previously described^[@CR40]^. Viral RNA extraction was performed as described above. A set of specific primer pairs (Supplemental Table [5](#MOESM1){ref-type="media"}) was used to generate amplicons by RT-PCR using a Superscript III One-Step RT-PCR Platinum TaqHifi kit (Life Technologies). For each passage sequenced, purified PCR products were pooled and analyzed using an Ion PGM Sequencer (Life Technologies) according to the manufacturer's instructions. The resulting reads were analyzed using CLC Genomics Workbench 6 (CLC Bio). The sequences were trimmed based on quality scores by removing the primer sequences at their termini and systematically removing 20 nt at the 5′ and 3′ termini. The remaining reads with lengths greater than 99 nt were mapped using the designed sequence of the chimeric virus as a reference to obtain a consensus sequence. The mutation frequency for each position was calculated as the number of mutated reads divided by the total number of reads at that site.
Tissue culture infectious dose 50 (TCID~50~) assay {#Sec17}
--------------------------------------------------
A 96-well cell culture plate containing confluent Vero-E6 cells with 100 µl/well of media were inoculated with 10-fold serial dilutions of centrifugation-clarified cell culture supernatant medium (50 µl/well). Each dilution was repeated six times. The plate was incubated for 7 days and read to assess the absence or presence of CPE in each well. TCID~50~ titers were subsequently calculated using the Reed--Muench method^[@CR41]^.
Cell viability assay {#Sec18}
--------------------
Confluent cells were inoculated at an MOI (multiplicity of infection) of 0.01 in a 96-well cell culture plate in triplicate for each measurement. Every day for a period of 5 days we performed the cell titer blue viability assay (Promega) following the manufacturer's instructions.
Virus growth kinetics {#Sec19}
---------------------
Confluent cells were inoculated at an MOI of 0.01 in a six-well cell culture plates in triplicate. Every day for a period of 7 days, 100 µl of cell supernatant medium was collected to measure the amount of viral RNA by qRT-PCR (see above) and 200 µl was collected to assess TCID~50~ values.
Indirect immunofluorescence assay {#Sec20}
---------------------------------
Confluent Vero-E6 cells were inoculated at an MOI of 0.01 in an eight-well cell culture Lab-Tek II Chamber Slide System in duplicate. At 2 and 5 days post-infection, cells were washed twice with HBSS and fixed with 4% paraformaldehyde for 2 h. Viral antigens were detected as previously described^[@CR19],[@CR42]^ using a specific ZIKV immune serum as the primary antibody (dilution: 1/50) collected from a Syrian Hamster immunized with the ZIKV strain Mart2015 (see below). This serum was shown to neutralize more than 90% of ZIKV PF replication up to a 1/3000 dilution (data not shown). The secondary antibody used was a goat anti-hamster Alexa 488 antibody (Invitrogen), which was used at a 1/500 dilution. Slides were observed using an Eurostar II fluorescence microscope with the Europicture software (Euroimmune).
Viral RNA YRNT {#Sec21}
--------------
Vero-E6 cells were seeded into a 96-well cell culture plate 1 day prior to infection (5 × 10^4^ cells in 100 µl of medium containing 2.5% FBS per well). The next day, two-fold serial dilutions of sera (from 1/20 to 1/2560; diluted with medium containing 2.5% FBS) were mixed (50:50; v/v) with appropriate amounts of viral stock (diluted in medium containing 2.5% FBS), incubated for 1.5 h at 37 °C under a 5% CO~2~ atmosphere, and then were added to cells (50 µl/well). The amount of virus added had been calibrated to ensure that virus production in the cell supernatant medium did not reach a plateau at the readout time^[@CR43]^. Cells were incubated for 3 days, after which 100 µl of cell supernatant medium was harvested to perform nucleic acid extraction and to quantify amounts of viral RNA using a qRT-PCR assay (see above). Each serum dilution was tested in triplicate and duplicate for the control group. For each experiment, a virus replication control (VC) was performed in quadruplicate to assess viral replication. For each serum dilution, viral RNA yield reduction (% of viral inhibition) was calculated using the mean amount of viral RNA obtained with VC as a reference. The 50% and 90% viral inhibition cut-offs were used to estimate viral RNA Yield Reduction Neutralization 50% and 90% (YRNT50; YRNT90) titers using the method of Reed and Muench^[@CR41]^.
In vivo experiments {#Sec22}
-------------------
### Animal handling {#Sec23}
Animals were maintained in an ISOcage P Bioexclusion System (Techniplast) with unlimited access to food and water and 12 h light/12 h dark cycle. Animals were individually monitored every day to detect the appearance of any clinical sign of illness/suffering. Virus/Antibody inoculation, blood collection, and euthanasia (cervical dislocation) were performed under general anesthesia (isofluorane).
### Golden hamster immunization {#Sec24}
One 4-week-old female Syrian Hamster (Janvier) was intraperitoneally immunized with 100 μl containing 10^5^ TCID~50~ of ZIKV strain Mart2015. After 21 days, the Hamster was re-injected with the same dose. The hamster did not show any sign of illness or weight loss. After an additional 15 days, the hamster was euthanized and a blood sample (intracardiac puncture) was collected. After centrifugation, the serum was stored at −80 °C.
### Administration of anti-IFNAR antibody {#Sec25}
All the mice used were immunocompromised following a two-step inoculation of an anti-IFNAR antibody (clone MAR1-5A3; Interchim; intraperitoneal injection; 120 µl)^[@CR22],[@CR24]^, with 1 mg administered 1 day prior and 1 day after each infection/immunization (i.e., the mice challenged were immunocompromised twice with this two-step procedure).
### Mouse immunization {#Sec26}
Six groups of four 3-week-old female C57/bl6 mice (Charles River) were intraperitoneally inoculated with 100 µl of virus: two groups were immunized with the YFV 17-D strain (two dosages: 10^4^ and 10^5^ TCID~50~), two groups were immunized with the ZIKV PF strain (two dosages: 10^5^ and 10^6^ TCID~50~), and two groups were immunized with the CH-17-D/ZIKV strain (two dosages: 10^4^ and 10^5^ TCID~50~). A control group of four mice was used as a negative control group (non-immunized mice).
Blood collection (10 µl) from the tail vein was performed at days 2 and 3 post-immunization to detect infectious virus by cell culture isolation. Immediately after collection, all the blood was inoculated into a 12-well cell culture plate containing confluent Vero-E6 cells and 150 µl of medium/well. After incubating for 2 h, 100 µl of the inoculum was harvested. The cells were washed with HBSS and then 1.5 ml/well of fresh medium was added to the cells, which were incubated for 6 days. Finally, 100 µl of cell supernatant medium was harvested to perform nucleic acid extraction and to quantify amounts of viral RNA using a real-time qRT-PCR assay as described above.
At day 21 post-infection, all animals were euthanized and blood samples were collected via intracardiac puncture. After the blood samples were centrifuged, sera were stored at −80 °C before being used to perform the neutralization tests.
### Challenge experiments {#Sec27}
Five groups of four 3-week-old female C57/bl6 mice (Charles River) were intraperitoneally inoculated with 100 µl of virus: two groups were immunized with the YFV 17-D strain (two dosages: 10^4^ and 10^5^ TCID~50~), two groups were immunized with the CH-17-D/ZIKV strain (two dosages: 10^4^ and 10^5^ TCID~50~), and one group was immunized with the ZIKV PF strain (10^5^ TCID~50~). Two control groups of four mice were used as a (i) mock control group (non-immunized/non-challenged mice) and (ii) a negative control group (non-immunized mice; challenged).
All animals (except the mock control group) were then challenged with 10^6^ TCID~50~ of ZIKV Dak84. Blood collection (10 µl) from the tail vein was performed at days 2 and 3 post-challenge to assess viremia by qRT-PCR. At day 10 post-challenge, all the animals were euthanized. Organs (spleen and brain) were then collected in 1 ml of HBSS supplemented with 10% of FBS and crushed for 10 min at 30 cycles per second with tungsten beads using a Tissue Lyser machine (Retsch MM400). After centrifugation at 5000 *g* for 10 min, the supernatant medium was collected and then centrifuged again at 10,000 *g* for 10 min. Fifty microliters of the supernatant medium was used to perform nucleic acid extraction and to quantify the amount of viral RNA using a real-time qRT-PCR assay (see above).
Statistical analysis {#Sec28}
--------------------
All data obtained were analyzed using Graphpad Prism 7 (Graphpad software), which was also used for all graphical representations and statistical analyses.
Electronic supplementary material
=================================
{#Sec29}
Supplemental material
**Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
=================================
**Supplementary Information** accompanies this paper at (10.1038/s41426-018-0161-7).
We thank Ernest A. Gould from the UMR UVE (Marseille, France) for his valuable review of the manuscript. We also thank Geraldine Piorkowski and Karine Barthelemy from the UMR UVE (Marseille, France) for performing viral sequencing. This work was supported by the French "Agence Nationale de la Recherche" (grant agreement no. ANR-14-CE14-0001) and the European Virus Archive goes global project (EVAg; European Union---Horizon 2020 programme under grant agreement no. 653316; <http://www.european-virus-archive.com/>).
F.T., X.D.L., and A.N. conceived the experiments. X.D.L. obtained the funding for this study. F.T., M.G., F.A., and R.K. performed the experiments. F.T., M.G., F.A., R.K., and A.N. analyzed the results. F.T., M.G., and A.N. wrote the manuscript. F.A., R.K., and X.D.L. reviewed and edited the manuscript.
The authors declare that they have no conflict of interest.
Animal protocols were approved by the local ethics committee (Comité d'éthique en expérimentation animale de Marseille---C2EA-14; protocol number \#9327). All in vivo experiments were performed in accordance with the European legislation covering the use of animals for scientific purposes (Directive 210/63/EU) and French national guidelines.
|
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Introduction {#S1}
============
Understanding the dynamic nature of G protein-coupled receptors (GPCRs) is critical given their capacity to modulate numerous biological responses in health and disease. Largely localized to the plasma membrane, GPCRs respond to an array of extracellular stimuli including photons, odors, hormones, peptides, lipids, and sugars ([@B1]). With over 800 genes expressed in the human genome, they are found in nearly every organ of the body ([@B2], [@B3]). The β~2~-adrenergic receptor (β~2~AR) is one of the most studied GPCRs and is tightly regulated as part of elaborate multicomponent signaling networks. Upon ligand binding, the receptor undergoes a conformational change that stimulates the exchange of guanine diphosphate for guanine triphosphate in the Gαs subunit leading to the functional dissociation of the Gβγ dimer from Gα ([@B1]). These G proteins then independently act on downstream effector molecules in a number of signaling cascades. This simplified notion of receptor activation provides only a glimpse into the complex processes of signal transduction, of which we have much to learn.
Understanding GPCR function involves determining how agonist binding translates into receptor activation. The traditional view of receptor activation has evolved from where it was initially thought of as a switch from a single inactive state to a single active state. Now it is widely accepted that the receptor pool in any given cell can occupy a number of different inactive and active conformations ([@B4]--[@B6]). At equilibrium, there are numerous conformations within the receptor population and different orthosteric and allosteric ligands can stabilize diverse receptor states. The fundamental mechanisms of GPCR activation have been investigated by several groups using diverse techniques, including but not limited to nuclear magnetic resonance, double electron--electron resonance, and fluorescence spectroscopy ([@B4], [@B7]--[@B9]). Both fluorescence and bioluminescence resonance energy transfer (FRET and BRET) approaches have also been used to explore the conformational dynamics of GPCRs ([@B9]--[@B14]). The site specific introduction of the short tetracysteine motif CCPGCC within the coding frame of a receptor when labeled with a fluorescein derivative can be used in resonance energy transfer (RET) appli-cations to report on conformations adopted by the receptor upon ligand binding in living cells ([@B15], [@B16]).
We have explored the use of FlAsH BRET in the conformational profiling of the prostaglandin F2α receptor \[FP; ([@B11])\], and the angiotensin II type I receptor \[AT1R; ([@B17])\]. Here, we introduced this tetracysteine tag at various locations within the coding sequence of the β~2~AR in order to report on conformational changes upon agonist stimulation. Eight such biosensors were constructed; two within the second intracellular loop, three in the third intracellular loop, and three in the carboxyl terminus of the receptor. In a previous study, the β~2~AR was tagged using FlAsH FRET ([@B18]). In that work, the third intracellular loop was tagged with the FlAsH motif and the carboxyl terminus with CFP after having truncated the C-tail at amino acid 343 ([@B18]). Upon agonist stimulation, an increase in the FRET ratio was observed suggesting that the third intracellular loop approaches the C-terminus ([@B18]). Other groups have also attempted to understand the conformational dynamics of the β~2~AR while using fluorescence-based probes as indicators of conformational changes occurring in real-time. Lohse and colleagues have generated FRET-based biosensors incorporating YFP in the third loop and CFP in the C-terminus of the β~2~AR ([@B19]). Again, the receptor was truncated at amino acid 369. To our knowledge, our study is the first report of using the full-length β~2~AR tagged with reporter proteins to monitor conformations adopted by the receptor upon agonist stimulation. Further, we compare and contrast three distinct GPCRs and show that even though they share a similar seven transmembrane architecture, they behave very differently in regards to the magnitude and kinetics of their BRET responses.
Materials and Methods {#S2}
=====================
Materials {#S2-1}
---------
### Primers {#S2-1-1}
All primers were synthesized and purchased by Integrated DNA Technologies (Coralville, IA, USA, see Table [1](#T1){ref-type="table"}).
######
**List of primers used for the generation of the β~2~-adrenergic receptor (β~2~AR) FlAsH-BRET-based recombinant biosensors**.
Position Sequence (5′ → 3′)
------------------------------------------- -----------------------------------------
*ICL2 p1* F: TGCTGCCCCGGCTGCTGCAGCCTGCTGA
R: GCAGCAGCCGGGGCAGCACTGGTACTTG
*ICL2 p2* F: TGCTGCCCCGGCTGCTGCCCTTTCAAGTACCAGAGC
R: GCAGCAGCCGGGGCAGCATGAAGTAATGGCAAAGTAGC
*ICL3 p1* F: TGCTGCCCCGGCTGCTGCCATGTCCAGA
R: GCAGCAGCCGGGGCAGCAGAAGCGGCC
*ICL3 p2* F: TGCTGCCCCGGCTGCTGCGAGCAGGATG
R: GCAGCAGCCGGGGCAGCACACCTGGCT
*ICL3 p3* F: TGCTGCCCCGGCTGCTGCGGACTCCGCA
R: GCAGCAGCCGGGGCAGCAATGCCCCGT
*C-tail p1* F: TGCTGCCCCGGCTGCTGCGCCTATGGGA
R: GCAGCAGCCGGGGCAGCACTTCAAAGA
*C-tail p2* F: TGCTGCCCCGGCTGCTGCAATAAACTGC
R: GCAGCAGCCGGGGCAGCATTCTTTCTCC
*C-tail p3* F: TGCTGCCCCGGCTGCTGCCATCAAGGTA
R: GCAGCAGCCGGGGCAGCAGCCCACAAA
Name Sequence (5′ → 3′)
*Bam*HI β~2~AR F: CAGTGGATCCATGGGGCAACCCGGGAAC
β~2~AR *Eco*RI *Bam*HI R: CTCCGGATCCGAATTCCAGCAGTGAGTC
*Nhe*I *Xho*I Kozak SP F: CCTAGCTAGCTCGAGGCCACCATGAA
### Constructs {#S2-1-2}
The recombinant receptors used in this paper are as follows: SP-FLAG-hAT1R-CCPGCC-ICL3-p3-RlucII or SP-FLAG-hAT1-R-CCPGCC-C-tail-p1-RlucII in a pIRESH plasmid backbone ([@B17]) along with SP-HA-hFP-CCPGCC-ICL3-p4-RlucII in a pcDNA3.1(−) backbone ([@B11]), in addition to the panel of eight β~2~AR biosensors expressed in a pIRESpuro3 plasmid backbone.
Generation of FlAsH-BRET-Based Biosensors {#S2-2}
-----------------------------------------
The intramolecular biosensors were designed to harbor the tet-racysteine tag positioned at various locations within the intracellular surface of the receptor in addition to a C-terminally fused *Renilla* luciferase. More precisely, the CCPGCC tag was inserted in two positions within the second intracellular loop, three within the third, and three within the carboxyl terminus domain of the receptor. For ease of cloning, compatible restriction sites were introduced by polymerase chain reaction (PCR) at the 5′ and 3′ ends of the receptor to facilitate its insertion into its corresponding mammalian expression vector. Briefly, HA-tagged hβ~2~AR ([@B20]) in a pcDNA3.1(−) backbone vector was used as a template and amplified by PCR using the *Bam*HI-β~2~AR forward and the β~2~AR-*Eco*RI-*Bam*HI reverse primers. The resulting PCR product was cloned into an accepting vector; pIRESpuro3-signal peptide-HA-RlucII using *Bam*HI. We screened for correct orientation using *Pst*I. The introduction of the CCPGCC motif was accomplished by overlapping PCR where the wild-type receptor was flanked by the appropriate primers (Table [1](#T1){ref-type="table"}) in order to introduce the desired TC tag within the coding sequence ([@B11]). In the first round, fragment one was generated using *Nhe*I-*Xho*I-forward primer and the appropriate FlAsH internal reverse primer. Fragment 2 was generated using the appropriate FlAsH internal forward primer and β~2~AR-*Eco*RI-*Bam*HI reverse primer. Both fragments were then combined in equal portions and used as templates for the second round of PCR using *Nhe*I-*Xho*I-Kozak-β~2~AR forward and β~2~AR-*Eco*RI-*Bam*HI reverse primer. This product was cloned into pIRESpuro3-SP-HA-RlucII backbone using *Nhe*I and *Eco*RI. All constructs were confirmed by bidirectional sequencing (Génome Québec).
Cell Culture {#S2-3}
------------
HEK 293 SL cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% vol/vol fetal bovine serum and 1% w/v penicillin--streptomycin from Wisent. The cells were maintained in a controlled environment, 37°C in a humidified atmosphere at 95% air and 5% CO~2~.
Transient Transfection {#S2-4}
----------------------
HEK 293 SL cells were plated at a density of 2.0 × 10^5^ cells per well in clear 6-well plates (Thermo Scientific, 140675) prior to transfection. On the following day, cells were transfected with 1 µg of each of the eight β~2~AR FlAsH biosensors along with pcDNA3.1(−) for a total of 1.5 µg per well using Lipofectamine 2000 (Invit-rogen) following the manufacturer's instructions. Alternatively, 1 µg of AT1R-ICL3-p3-RlucII or AT1R-C-tail-p1-RlucII and 500 ng of the FP-ICL3-p4-RlucII biosensor was also used.
Immunofluorescence {#S2-5}
------------------
The day following transfection, cells were detached with 0.25% Trypsin--EDTA (Wisent) and 2.0 × 10^4^ cells were re-plated onto a poly-[l]{.smallcaps}-ornithine (Sigma-Aldrich) treated clear bottom black 96-well plate (Thermo Scientific, 165305). The next day, the cells were washed once with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Successively, the cells were blocked with a 1% bovine serum albumin (Fisher Scientific) PBS solution for 1 h at room temperature to prevent non-specific interactions of the antibodies. Cells were then incubated with a monoclonal mouse anti-HA primary antibody for 1 h (BioLegend, 1:200, previously Covance). Afterward, the cells were washed three times with PBS and an Alexa fluor-488 goat anti-mouse IgG secondary antibody (Life Technologies, 1:1,000) was used to label cells. To confirm the ability of recombinant receptors to localize to the cell surface, the Operetta High Content Imaging system (Perkin Elmer) with a 20× WD objective was used. The excitation filter was set at 475/15 nm and its corresponding emission filter at 525/25 which permitted to capture the signal produced by Alexa fluor-488.
Gαs Coupling and Downstream cAMP Production {#S2-6}
-------------------------------------------
HEK 293 SL cells were transfected with 1 µg of each of the eight β~2~AR FlAsH biosensors and the β~2~AR-WT-RlucII construct supplemented with 0.5 µg of the previously described H188 EPAC FRET sensor ([@B21]) using Lipofectamine 2000 (Invitrogen) follow-ing the manufacturer's instructions. The day following transfection, cells were detached with 0.25% Trypsin--EDTA (Wisent) and 4.0 × 10^4^ cells were re-plated onto a poly-[l]{.smallcaps}-ornithine (Sigma-Aldrich) treated black flat bottom 96-well plate (Costar, 3916). The day of the experiment, cells were washed once in 150 µL of Krebs buffer and the cells then sat in 90 µL of Krebs at 37°C prior to the start of the assay. A Synergy 2 plate reader (Biotek) was used to assay coupling of the β~2~AR FlAsH biosensors to Gαs by investigating accumulation of cAMP. The temperature of the instrument was set at 37°C and kinetic measurements were taken. The 420/50 excitation filter was used to excite the donor molecule, mTurquoise2, and light was captured by the emission filters 485/20 (mTurquoise2) and 528/20 (Venus). Basal FRET was measured continuously every 5 s for a total of 20 s. Cells were then treated with either the vehicle (ascorbic acid) or the full agonist, 10 µM isoproterenol (in ascorbic acid) using the injector module. Stimulated FRET readings were then captured every 5 s for a total time of 2 min. FRET ratios were computed by dividing the Venus emission channel by the mTurquoise2 emission channel. ΔFRET ratios were calculated by subtracting the averaged isoproterenol stimulated FRET ratio by the averaged basal FRET ratio, as shown; ΔFRET = (avgFRET~stimulated~ − avgFRET~basal~).
ERK1/2 MAP Kinase Activation {#S2-7}
----------------------------
Twenty-four hours post-transfection, cells were detached with 0.25% Trypsin--EDTA (Wisent) and 400 µL of cell suspension was re-plated onto a clear 12-well plate (Costar, 3513). On the day of the experiment, the cells were starved in DMEM without serum supplementation for 5 h. Afterward, cells were stimulated with either vehicle or 10 µM isoproterenol for 5 min at 37°C. The plate was then placed on ice, where the cells were washed once with an ice-cold PBS solution. The cells were lysed in 200 µL of 4× Laemmli buffer (2% SDS, 10% glycerol, 60 mM Tris pH 6.8, 0.02% bromophenol blue, 5% β-mercaptoethanol). In order to shear the genomic DNA, lysates were sonicated three times, each repetition for 5 s at 3 W using a Sonicatior 3000 (Misonix). Lysates were then heated at 65°C for 15 min.
MAP kinase activation was measured by western blot. Correspondingly, 30 µL of cell lysate was loaded and proteins were, respectively, separated by SDS-PAGE and then transferred onto a PVDF membrane *via* a wet transfer technique. To prevent non-specific binding of the primary antibody, the membrane was blocked in a 5% non-fat milk solution in Tris-buffered saline and 0.0005% Tween20 solution. An anti-phospho-ERK1/2 rabbit primary antibody was used (Cell Signalling Technologies, 1:1,000) followed by an anti-rabbit polyclonal IgG peroxidase secondary antibody (Santa Cruz Biotechnology, 1:20,000). Immuno-detection was accomplished *via* chemiluminescence using Western Lightning plus-ECL (Perkin Elmer) or ECL-Select western blotting detection reagent (GE Healthcare) given that the secondary antibody was conjugated to the horseradish peroxidase enzyme.
FlAsH Labeling {#S2-8}
--------------
Twenty-four hours post-transfection, cells were detached with 0.25% Trypsin--EDTA (Wisent) and 4.0 × 10^4^ cells were re-plated onto a poly-[l]{.smallcaps}-ornithine (Sigma-Aldrich) treated white 96-well plate (Thermo Scientific, 236105). The next morning, a 25 mM solution of 1,2-ethanedithiol (EDT) was prepared by diluting it in dimethyl sulfoxide. Then, one volume of FlAsH reagent (2 mM) was added to two volumes of EDT to make a 667 µM FlAsH (Invitrogen) solution, which was incubated for 10 min at room temperature. Following the incubation, 100 µL of Hank's balan-ced salt solution (HBSS) without phenyl red, with sodium bicarbonate, calcium, and magnesium was added to the 667 µM FlAsH solution and further incubated for 5 min at room temperature (Wisent). Then, HBSS was added to make a solution with final concentration of 750 nM FlAsH-EDT~2~. In parallel, cells were washed in 150 µL of HBSS prior to the FlAsH labeling. Subsequently, 60 µL of the 750 nM FlAsH-EDT~2~ solution was added to the cells and incubated for 1 h at 37°C, protected from any source of direct light. Following the incubation, cells were washed once with 100 µL of l M 2,3-dimercapto-1-propanol (BAL, Invitrogen) diluted in HBSS buffer and then incubated for 10 min at 37°C. The cells were washed once again with BAL without incubation. Afterward, cells were washed once with 150 µL of the assay buffer: Krebs (146 mM NaCl, 4.2 mM KCl, 0.5 mM MgCl~2~, 1 mM CaCl~2~, 10 mM HEPES pH 7.4, 0.1% glucose). The cells then sat in 80 µL of Krebs for 2 h at room temperature, in an environment protected from light, prior to the BRET assay. The FlAsH labeling procedure has been previously described elsewhere ([@B11]).
### BRET Measurements {#S2-8-1}
A TriStar^2^ LB 942 multimode plate reader from Berthold Technologies was used to measure BRET using the pre-determined BRET^1^ filter pair F485 and F530. Light was produced *via* enzymatic catalysis of the luciferase substrate coelenterazine h by the donor RlucII. Accordingly, 10 µL of a 2 µM coelenterazine h solution (NanoLight Technologies) was added to the cells and incubated for 5 min whereafter the luminescence was measured. Basal BRET corrected from spectral overlap of the donor and acceptor channels were calculated by subtracting the BRET value obtained from unlabeled cells expressing solely the donor from the corresponding BRET value obtained from the labeled FlAsH recombinant receptors. Additionally, ligand-induced changes were investigated and kinetic readings were reported. Correspondingly, the counting time of the two filters was analyzed continuously every 0.2 s for a total of 50 repeats. Subsequently, either vehicle or a saturating concentration of the agonist, 10 µM isoproterenol, was injected using the injector module. For the AT1R, 1 µM angiotensin II was used and 1 µM PGF2α for FP. Thereafter, the luminescence was again captured every 0.2 s and a total of 100 repeats. The change in BRET, as a response to the addition of agonist or the ΔBRET, as referred to in this paper, was computed by subtracting the average BRET across all reads pre-injection from the average BRET across all reads post-injection: ΔBRET = (avgBRET~post-injection~) −(avgBRET~pre-injection~).
Statistical Analysis {#S2-9}
--------------------
All statistical analysis was performed using GraphPad Prism 7.0 software. Data are reported as mean ± SE. The Prism software performed a Brown--Forsythe test to determine if parame-tric or non-parametric statistics should be performed. The degree of Gαs coupling was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test comparing the various FlAsH positions to the wild type (Figures [3](#F3){ref-type="fig"}A,B). When determining the basal BRET exhibited by each of the recombinant β~2~AR biosensors, a one-way ANOVA was performed. A Dunnett's *post hoc* test was successively completed with the purpose of comparing the basal BRET of the eight recombinant constructs to the wild-type receptor (Figure [4](#F4){ref-type="fig"}A). When evaluating the agonist-induced BRET response, a two-way ANOVA was carried out followed by a Bonferroni corrected Student's *t*-test aimed at comparing the response of the vehicle to the response of the agonist for each individual sensor position (Figure [4](#F4){ref-type="fig"}B).
Results {#S3}
=======
Biosensor Validation {#S3-1}
--------------------
We constructed a number of FlAsH-BRET biosensors in the β~2~AR with a FlAsH-binding site engineered into various intra-cellular sites and *Renilla* luciferase placed on the carboxy terminus (Figure [1](#F1){ref-type="fig"}). If the biosensor components are positioned at appropriate sites within the receptor then this would allow profiling of conformational changes in the receptor upon ligand stimulation. In order for our intramolecular BRET constructs to be meaningful tools for the study of receptor conformational dynamics, recombinant receptors must maintain their native function. If they do not function in a manner similar to the wildtype receptor, then conformational analysis will be meaningless. Immunofluorescence was first used to verify the surface localization of the recombinant receptors generated. An anti-HA antibody was used to label the recombinant receptors, followed by an Alexa fluor-488 conjugated secondary antibody. As illustrated in Figure [2](#F2){ref-type="fig"}, almost all the FlAsH-tagged β~2~AR constructs trafficked to the cell surface. Receptors tagged within the second intracellular loop were less robustly expressed compared to the wild type. However, the fluorescence intensity for all other positions was similar to the wild type providing us with at least six positions to carry forward.
{#F1}
{#F2}
Next, to further validate the functionality of each construct, we measured isoproterenol-mediated cAMP accumulation as well as ERK1/2 MAPK activation. We studied the relative accumulation of cAMP as an indication of Gαs activation using the H188 FRET-based EPAC sensor. FRET was used as BRET-based EPAC biosensors could not be used with the BRET-based conformational biosensors (as both would be activated). As shown in Figures [3](#F3){ref-type="fig"}A,B, the majority of constructs displayed similar levels of cAMP accumulation (measured as a decrease in FRET) as compared to the untagged wild-type receptor. As the β~2~AR shows agonist-independent basal activity, we examined both basal (Figure [3](#F3){ref-type="fig"}A) and agonist-stimulated FRET (Figure [3](#F3){ref-type="fig"}B). We observed that certain sensor positions exhibited high basal activity as shown as the reduced FRET ratio at baseline. This may have been as a result of higher expression levels of these biosensors. For example, the C-tail P3 position may seem as though there is less cAMP production than the wild type in response to agonist; however, the lack of a robust decrease in FRET as a response to agonist is probably due to having attained the threshold of detection at basal levels.
{#F3}
We then examined a more distal readout of receptor functionality; the β~2~AR-mediated MAPK (Raf/Ras/MEK/ERK) signaling pathway that has been previously characterized by various groups ([@B22], [@B23]). As demonstrated by Figure [3](#F3){ref-type="fig"}C, all the recombinant β~2~AR constructs exhibited MAPK activation at similar intensities as the wild-type receptor. As a result, the third intracellular loop sensors as well as the C-tail sensors passed the validation stage although some caution again must be taken when interpreting results using the sensors engineered into the second intracellular loop. It must be noted here that all our transfections were transient and no attempt was made to normalize levels of expression *per se*.
BRET Measurements {#S3-2}
-----------------
Next, we measured basal BRET between the FlAsH-labeled receptors and the C-tail luciferase. Basal BRET or the BRET ratio after it has been corrected for spectral overlap of the donor and acceptor channels was determined by subtracting BRET where cells were not labeled with FlAsH. All receptor biosensors showed basal BRET to varying degrees (Figure [4](#F4){ref-type="fig"}A). The larger the basal BRET, the closer the donor--acceptor pair was at the outset. As a result, there is a greater dynamic range to capture relative changes in receptor conformation. The β~2~AR biosensor with the greatest basal BRET was the third position within the C-tail. There was a position-dependent increase in the basal BRET, as one moves farther down the tail of the receptor, as acceptor and donor moieties get closer together. As for the third loop, the second position showed the largest basal BRET which is in accordance to its position in the middle of the loop (Figure [4](#F4){ref-type="fig"}A).
{#F4}
ΔBRET in response to ligand was measured by subtracting the averaged post-injection BRET from the averaged pre-injection BRET readings. BRET ratios could potentially increase or decrease depending on the ligand used and the subsequent conforma-tion adopted by the receptor. It was hoped that our biosensors would differentially respond to ligands and provide a conformational fingerprint to better understand the dynamic nature of the receptor which could be exploited for validating new drugs in early phases of development. Of all the biosensors tested, only the C-tail positions P2 and P3 showed a robust conformational change upon isoproterenol stimulation (Figure [4](#F4){ref-type="fig"}B). The lack of response in ICL3 was somewhat of a surprise but the functional data (Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}) suggested that ICL2 sensors may not be correctly folded.
In order to make a comprehensive assessment of the isopro-terenol induced responses of the β~2~AR biosensors, we also examined the underlying kinetics. As mentioned, neither the second or third loop positions captured a sustained conformational change in response to isoproterenol (Figures [4](#F4){ref-type="fig"}B and [5](#F5){ref-type="fig"}). Oddly, a small spike was a consistent feature of the ligand-induced response in these sensors with the exception of ICL3 P1 (Figures [5](#F5){ref-type="fig"}D--F). The presence of this spike was not an artifact originating from the sampling instrument as no such spikes were seen when vehicle was similarly injected and it was also absent from kinetic traces of the wild-type receptor expressing RlucII with no FlAsH-binding sequences (Figure [5](#F5){ref-type="fig"}A). The C-tail P1 sensor displayed similar features as the second and third loop positions (Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}A). However, the responses in the other C-tail sensors were much more robust and sustained (Figures [6](#F6){ref-type="fig"}B,C). We have previously analyzed responses to ligand in both FP ([@B11]) and in the AT1R ([@B17]). Responses in ICL3 in AT1R and FP were both robust and sustained (Figures [6](#F6){ref-type="fig"}D,E) compared to the β~2~AR. Further, robust sustained responses have also been detected in both ICL2 and the C-terminus of AT1R \[([@B17]); Figure [6](#F6){ref-type="fig"}F\]. Interestingly, no responses were detected in similar constructs built into either ICL2 or the C-tail of FP (data not shown). Taken together, our data paint a picture which highlights the conformational heterogeneity of different GPCRs in response to ligand stimulation.
{#F5}
{#F6}
Discussion {#S4}
==========
Crystal structures offer snapshot images of receptor structure that can be complemented using more dynamic measures such as RET approaches. Kobilka and coworkers reported that transmembrane domain VI experiences a 14 Å outward movement when comparing the inactive carazolol bound β~2~AR versus the active-Gαs bound crystal structure ([@B24]). We show here that three different GPCRs show distinct patterns of BRET in response to ligand even when biosensors are placed in similar positions (Figure [7](#F7){ref-type="fig"}).
![**Homology-based representation of the positioning of the FlAsH tag in three class A G protein-coupled receptors**. **(A)** Homology model of the hβ~2~AR (P07550-1) based on PDB identifier: 2rh1A with truncated C-tail. Positions highlighted in orange correspond to the first position, in blue the second position, and in green the third position within each respective loop structure. **(B)** Homology model of the human angiotensin II type 1 receptor (P30556-1) modeled upon the existing crystal structure with PBD accession 4yayA, the C-tail was then truncated ([@B25]). The ICL3 p3 biosensor is shown in green. **(C)** Homology model of the human FP (P43088-1) based on the PBD ID: 3emlA. Insertion of the TC tag in ICL3 position 4 is shown in red. **(D)** Superimposing the models of the hβ~2~AR, AT1R, and FP. Overlay of three receptors reveals the relative similarities in the transmembrane domains and differences in the cytoplasmic regions. Approximately 20 residues were removed from the N-terminus and the C-terminus was truncated to facilitate the visualization of the overall structure. *Inset* shows expanded versions of ICL2 (left) or ICL3 (right). The I-TASSER models ([@B26], [@B27]) were exported into PyMOL where the CCPGCC motifs were inserted at their respective positions and color coded to facilitate the visualization of the positioning of the FlAsH tags.](fendo-08-00061-g007){#F7}
For the β~2~AR, our data showed that ICL2 and ICL3 did not respond to the full agonist isoproterenol, whereas two of our C-tail biosensors exhibited sustained BRET responses. As the acceptor was progressively walked down the C-terminus, resonance energy was more efficiently transferred from donor to acceptor under basal conditions and this may explain why BRET was not detected in biosensors with acceptor and donor farther apart. This pattern was distinct in the AT1R and in FP receptor. The AT1R exhibits the most conformational heterogeneity in that sensors engineered into ICL2, ICL3, or the C-tail all reported robustly on conformational changes in response to either canonical (Ang II or Ang III) or biased (SI) ligands ([@B17]). Further, only ICL3 biosensors reported responses upon stimulation with PGF2α in FP (11, data not shown for ICL2 or the C-tail). This may suggest that the movement of the intracellular loops in the β~2~AR or FP is constrained by a protein within the vicinity of the fifth, sixth or seventh transmembrane domain. Even if this constrained con-formation does not allow us to use these biosensors in this cellular background, it does highlight the advantage of using a six amino acid tag since this reduced size allows us to probe receptor conformation. For instance, if GFP or one of its variants were used instead of the FlAsH tag, perhaps the 238 amino acid (27 kDa) insertion would have significantly distorted receptor structure.
G protein-coupled receptors have many associated interacting partners that may pose conformational constraints on the receptor which translates into distinct conformational profiles. One of the major differences between the three receptors is that both the AT1R and FP couple to Gαq whereas the β~2~AR couples to Gαs. The β~2~AR has also been reported to differentially couple to Gαi ([@B28]--[@B30]). It may be interesting to explore the propensity of the receptor to couple to different G proteins in a particular biological context. Such differential coupling may lead to distinct confor-mations adopted by the receptor. Alternatively, it is well known that all three GPCRs form oligomers ([@B31]--[@B35]). Homo-and heterodimers or larger oligomers are not fully characterized and their physiological roles are not fully understood. Perhaps the formation of such larger arrays imposes additional conformational constraints on the receptor. These effects must be considered as early as events occurring in receptor biosynthesis ([@B36], [@B37]). Further exploring the lifecycle of a receptor is merited since oligomerization can alter several aspects of receptor function ([@B37]). Likewise, the β~2~AR experiences a high level of basal activity which some believe is due to the higher availability of G proteins and other effectors; proteins that might restrict receptor movement ([@B38]).
The length of intracellular loops in each receptor may also be related to measured conformational flexibility. The β~2~AR has a much longer third loop than the other two receptors. Taking this into account, we could imagine that the β~2~AR might be more free to adopt a larger range of conformations compared to the AT1R and FP (Figure [7](#F7){ref-type="fig"}). This may be a contributing factor explaining the different conformational patterns exhibited by all three receptors. These receptors are all classed into the same family of class A GPCRs, yet, they show different conformational behaviors. It must also be noted that this method is limited by the orientation of the reporter proteins. If the receptor folds in such a way where the enzymatic pocket of *RlucII* orients itself facing away from the FlAsH tag, the transfer of resonance energy will be less efficient with respect to RET.
In conclusion, we have demonstrated that the β~2~AR, AT1R, and FP display distinct conformational signatures when assayed in HEK 293 cells. Certainly cell context will matter in such experiments. The introduction of these BRET-based biosensors into diverse cell types may result in the detection of multiple different conformations adopted by the receptor depending on the cellular and subcellular contexts. Such receptor-based biosensors will be portable in this regard. Combined with genome editing approaches, these sensors are simple tools that could be used to uncover the complex mechanisms of GPCR activation and function.
Author Contributions {#S5}
====================
KB, DD, RS, and TH designed the study, and wrote and edited the paper. KB, DD, RS, DP, and AZ performed experiments. KB, DD, and RS analyzed the data. KB, DD, RS, and DP generated figures.
Conflict of Interest Statement {#S6}
==============================
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.
This work was supported by a grant from the Consortium Québécois sur la Découverte du Médicament and a grant from the Canadian Institutes of Health Research (CIHR, MOP-130309, TEH). RS was supported by a scholarship from the McGill-CIHR Drug Development Training Program. The authors thank Dr. Kees Jalink at the Netherlands Cancer Institute for the generous gift of the H188 EPAC FRET sensor and Mr. Jace Jones-Tabah for assistance in interpreting the data from these experiments. The authors thank Dr. Wolfgang Reitsch for assistance with high content microscopy. The authors thank Phan Trieu for technical and logistical assistance. The authors thank Dr. Jean-François Trempe and Mr. Andrew Bayne for assistance with PyMOL software.
[^1]: Edited by: Hubert Vaudry, University of Rouen, France
[^2]: Reviewed by: Xavier Iturrioz, Institut National de la Santé et de la Recherche Médicale (INSERM), France; Kazuhiro Takahashi, Tohoku University, Japan; David Chatenet, Institut National de la Recherche Scientifique, Canada
[^3]: Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology
|
{
"pile_set_name": "PubMed Central"
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|
Lépine A, Lagarde M, Le Nestour A. How effective and fair is user fee removal? Evidence from Zambia using a pooled synthetic control. Health Economics. 2018;27:493--508. <https://doi.org/10.1002/hec.3589> 29034537
The work was funded by the Medical Research Council UK. For any conflict of interest disclosures, see Section [5](#hec3589-sec-0011){ref-type="sec"}.
1. INTRODUCTION {#hec3589-sec-0001}
===============
In the past two decades, several countries have taken steps to removing user charges for some or all curative care services (Yates, [2009](#hec3589-bib-0043){ref-type="ref"}), embracing the idea that user fees "deter people from using health services and cause financial stress" (World Health Organisation, [2010](#hec3589-bib-0041){ref-type="ref"}). Such decisions were motivated by the observation that user fees can reduce utilisation of care (Burnham, Pariyo, Galiwango, & Wabwire‐Mange, [2004](#hec3589-bib-0003){ref-type="ref"}; Deininger & Mpuga, [2005](#hec3589-bib-0010){ref-type="ref"}), in particular for poorer population groups whose demand for care is more price elastic (Gertler, Locay, & Sanderson, [1987](#hec3589-bib-0014){ref-type="ref"}; Gilson, [1997](#hec3589-bib-0015){ref-type="ref"}; Sauerborn, Nougtara, & Latimer, [1994](#hec3589-bib-0039){ref-type="ref"}). In Zambia, user fee removal was justified on the grounds that user fees appeared to decrease equity of access to health care and increase poverty (Kahenya & Lake, [1994](#hec3589-bib-0024){ref-type="ref"}; Masiye, Seshamani, Cheelo, Mphuka, & Odegaard, [2005](#hec3589-bib-0032){ref-type="ref"}; Sukwa & Chabot, [1997](#hec3589-bib-0040){ref-type="ref"}).
Some researchers have pointed that removing fees may not necessarily have the beneficial effects one could hope for (Gilson & McIntyre, [2005](#hec3589-bib-0016){ref-type="ref"}; McPake, Brikci, Cometto, Schmidt, & Araujo, [2011](#hec3589-bib-0033){ref-type="ref"}). Although removing fees has the potential to improve service coverage and access, hasty politically driven decisions with no prior preparation can lead to unintended effects, including quality deterioration due to lack of funds, excessive demands on health workers, and depletion of drug stocks (Gilson & McIntyre, [2005](#hec3589-bib-0016){ref-type="ref"}). In addition, the positive effects of removing user fees depend on the determinants of the demand for health care. Economic theory indicates that removing fees increase utilisation if fees represent a significant financial hurdle for households to access care. However, if other factors such as distance to facilities or limited perceived benefits of health care are the main drivers behind low utilisation, removing financial barriers may have a more limited impact.
Some early evidence of the effects of user fee removal in several sub‐Saharan countries suggested that utilisation of health care services would grow after fees were removed (Lagarde & Palmer, [2008](#hec3589-bib-0027){ref-type="ref"}). A more recent review of the evidence on maternal services reached similar optimistic conclusions (Hatt, Makinen, Madhavan, & Conlon, [2013](#hec3589-bib-0021){ref-type="ref"}).
However, both reviews underline the weakness of existing studies, which relied mostly on poor quality routine data and failed to provide a robust identification of the causal impact of the policy (Lagarde & Palmer, [2008](#hec3589-bib-0027){ref-type="ref"}). Recent studies have provided more robust evidence of the effects of free curative care through the (quasi) randomised introduction of health insurance, and their conclusions are less optimistic. [^1] Using the phased randomised implementation of a social health insurance in Mexico, King et al. ([2009](#hec3589-bib-0025){ref-type="ref"}) found no increase in the use of health care services by insured individuals, even though free care drastically reduced their medical expenses. In Gujarat, a programme offering free deliveries to poor women in private facilities was not associated with a change in the probability of institutional delivery (Mohanan et al., [2014](#hec3589-bib-0035){ref-type="ref"}). Slightly more positive results emerged from a randomised controlled trial in Ghana (Powell‐Jackson, Hanson, Whitty, & Ansah, [2014](#hec3589-bib-0037){ref-type="ref"}), where free care resulting from the introduction of health insurance led to a small increase of utilisation of service (3.7 percentage points).
In this paper, we contribute to this debate by presenting new evidence from Zambia where fees were removed in primary care facilities in 54 of the 72 districts in 2006. Using the synthetic control method (Abadie & Gardeazabal, [2003](#hec3589-bib-0002){ref-type="ref"}; Cavallo, Galiani, Noy, & Pantano, [2013](#hec3589-bib-0006){ref-type="ref"}), we estimate the causal impact of user fee removal on health‐seeking behaviours, provider choice, and medical out‐of‐pocket (OOP) expenditures in the general population and explore some heterogeneous effects depending on income level.
The remainder of the paper is organised as follows. In Section [2](#hec3589-sec-0002){ref-type="sec"}, we describe the background and study setting. Section [3](#hec3589-sec-0007){ref-type="sec"} describes the data, and Section [4](#hec3589-sec-0008){ref-type="sec"} the empirical approach adopted. Section [5](#hec3589-sec-0011){ref-type="sec"} presents the results and Section [6](#hec3589-sec-0017){ref-type="sec"} the robustness checks. Section [7](#hec3589-sec-0021){ref-type="sec"} discusses the findings and concludes.
2. BACKGROUND {#hec3589-sec-0002}
=============
2.1. Health‐seeking behaviours and user charges in Zambia before 2006 {#hec3589-sec-0003}
---------------------------------------------------------------------
From 1964 to 1991, the government of Zambia provided health care services for free. In 1991, user fees were introduced to raise additional income to improve quality of services (avoid drug stock‐outs and increase staff motivation thanks to salary top‐ups) and greater accountability to the local communities. [^2] User fees in primary care consisted of a flat consultation fee covering consultation and drugs, set by each district according to the ability to pay of the population (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}). The typical level of fees at primary care level could be considered as relatively low (McPake et al., [2011](#hec3589-bib-0033){ref-type="ref"}), typically between 500 to 1,000 Zambian Kwachas (about US\$ 0.14 to US\$ 0.27 in 2006; Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}) or 5% to 10% of the equivalent of a day\'s average GDP per capita in 2006. Several categories of individuals were exempted from paying fees: patients under 5 and over 65 years old, pregnant women, those suffering from certain diseases (e.g., HIV/AIDS and TB), and indigents (identified by local communities). In practice, children and elderly people made up the majority of exemptions (respectively, 66% and 7% of all exemptions in 1998).
With rapid economic growth and the development of its public health care system, Zambia experienced a sharp increase in the proportion of sick individuals seeking modern care, from 35.89% in 1998 (Central Statistical Office, [1999](#hec3589-bib-0007){ref-type="ref"}) to 57.31% in 2004 (Central Statistical Office, [2005](#hec3589-bib-0008){ref-type="ref"}). In 2004, 56% of individuals reporting an episode of illness sought modern care, 17% did not do anything, and 27% chose self‐medication, usually meaning that they went to drug stores to obtain over‐the‐counter medicines. Among those who sought care, 82% of individuals went to a government facility, 8% to mission providers, 6% to private providers, 1% to traditional practitioners, and 3% to other providers.
2.2. The 2006 policy change {#hec3589-sec-0004}
---------------------------
On January 13, 2006, the Zambian president announced that user fees in primary health care were to be removed in rural areas as a first step towards universal access for all (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}). The policy would apply to publicly funded facilities, which included both government‐run as well as mission facilities. Facilities could still charge two categories of patients: those coming from outside of the catchment area and foreigners. [^3]
Following the presidential announcement, a directive was sent to all districts in March 2006 stating that the policy would apply to all primary health care facilities located in rural areas, everywhere in the country. However, due to multiple challenges to clearly define rural areas, the government changed the definition of the policy at the last moment, and on April 1, 2006 all primary care facilities located in the 54 districts designated as "rural" according to the local government classification were asked to remove user fees. Some confusion ensued at the beginning of the policy change, with local authorities not always clear about the remits of the policy (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}).
To help prevent potential negative effects due to the loss of user fee revenue at facility level, facilities would be compensated through an earmarked monthly grant, to be paid by each district. These compensation grants were loosely linked to actual utilisation of health care services because they were based on projected income loss calculated by each district, based on 2005 routine data (Government of the Republic of Zambia, [2006](#hec3589-bib-0017){ref-type="ref"}). The payment of these grants, funded by a bilateral donor, ended up being compromised by several factors. Essentially, the funds were released to the Zambia Treasury only in August 2006, and they only reached rural districts between December 2006 and March 2007. Furthermore, in the absence of clear guidelines on how to use this additional funding, district authorities followed different approaches: some facilities received monthly payments, whereas others received lump sum payments or no grant at all (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}).
In addition to the lack of replacement funds, the 2006 policy change was introduced in a particularly challenging year for funding to primary health care facilities, as there was a 40% reduction in funding to district primary health care (Government of the Republic of Zambia, [2007](#hec3589-bib-0018){ref-type="ref"}). In effect, this meant that primary care facilities experienced a double loss in revenue, from the district basket as well as from user fees.
Due to these implementation challenges, user fees were not effectively abolished for everyone in rural districts. According to national household survey data, 6 months after the official introduction of the free care policy, 29% of patients aged between 5 and 65 years living in rural districts were still paying for receiving care in a government‐run or mission health centre. Yet, this still represented a sharp decrease compared to 2004, where 64% of the same population would be charged for health care services.
2.3. Anticipated effects {#hec3589-sec-0005}
------------------------
Basic economic theory suggests that a decrease in price will increase the demand for health care services through an income effect and a substitution effect. In the Zambian context, the income effect would allow more people to use public health care services, and the change in the relative prices of different care‐seeking options would lead to a substitution away from private health providers (Gertler & Gaag, [1990](#hec3589-bib-0013){ref-type="ref"}). However, these effects would only occur if the demand for public health care services is sensitive to price and if the perceived quality of care remains unchanged. Both assumptions are potentially problematic in the case of Zambia. There are reasons to believe that changes in prices happened in conjunction with changes in quality of care and that the perceived quality of care diminished in the public sector. Indeed, in Zambia, the health financing reform took place against the backdrop of a critical shortage of health workers, which affected particularly remote and rural areas (Carasso, Lagarde, Cheelo, Chansa, & Palmer, [2012](#hec3589-bib-0005){ref-type="ref"}; McPake et al., [2013](#hec3589-bib-0034){ref-type="ref"}). As a result, it is possible that populations in rural areas would expect relatively low quality of services in the public sector after fees were removed, mitigating their valuation of these free services.
2.4. Existing evidence {#hec3589-sec-0006}
----------------------
There have been a few studies looking at the impact of removing user fees in Zambia. Several have been descriptive, documenting the implementation of the policy and the way health care providers or community members perceived it (Carasso et al., [2012](#hec3589-bib-0005){ref-type="ref"}; Hadley, [2011](#hec3589-bib-0020){ref-type="ref"}; Masiye, Chitah, Chanda, & Simeo, [2008](#hec3589-bib-0029){ref-type="ref"}).
Three studies used routine facility data to investigate the policy impact (Chama‐Chiliba & Koch, [2016](#hec3589-bib-0009){ref-type="ref"}; Lagarde, Barroy, & Palmer, [2012](#hec3589-bib-0026){ref-type="ref"}; Masiye, Chitah, & Mcintyre, [2010](#hec3589-bib-0028){ref-type="ref"}). Although their findings are not directly comparable due to the different scope and type of data and methods used, those studies find mixed evidence regarding the effect of user fee removal on the demand for health services. Using an interrupted time series approach, Lagarde et al. ([2012](#hec3589-bib-0026){ref-type="ref"}) estimate a 40% increase in the volume of outpatient visits 6 months after the policy change in a subset of 17 rural districts, with that effect flattening out over time. Masiye et al. ([2010](#hec3589-bib-0028){ref-type="ref"}) compare trends in the volume of quarterly visits before and after the policy change and find that they increased in rural districts but not in urban districts, or for children under 5. Meanwhile, Chama‐Chiliba & Koch, [2016](#hec3589-bib-0009){ref-type="ref"} use birth histories obtained from household data and find that the policy did not have any significant increase on deliveries in public health facilities.
These studies may not have detected the causal impact of the policy for two reasons. First, the identification strategies used are problematic. Masiye et al. ([2010](#hec3589-bib-0028){ref-type="ref"}) caution that they cannot evaluate the causal impact of the policy, but rather some changes in trends. The validity of the interrupted time series approach used by Lagarde et al. ([2012](#hec3589-bib-0026){ref-type="ref"}) hinges on the assumption that no other concurrent factor may have affected the outcome of interest over the study period. Based on reports and anecdotal evidence of the policy change and the human resources crisis over the period, this assumption is debatable. Finally, Chama‐Chiliba & Koch ([2016](#hec3589-bib-0009){ref-type="ref"}) rely on a difference‐in‐difference identification strategy, but fail to provide evidence of the parallel trend assumption.
Second, two of these studies rely on facility register data that have at least two limitations. First, they suffer from measurement errors, including not at random missing data and obvious inaccuracies, as indicated by Lagarde et al. ([2012](#hec3589-bib-0026){ref-type="ref"}). Second, these routine data report the volume of outpatient consultations, which do not differentiate between unique visits of different patients and multiple visits by the same patients. In other words, the increase in the volume of consultations could be explained by an increase in the frequency of visits of "current" users and not by an increase in utilisation by new users.
3. DATA {#hec3589-sec-0007}
=======
Data on the study outcomes come from the Living Conditions and Monitoring Surveys (LCMS), a repeated cross‐sectional household survey designed to provide the basis for comparison of poverty estimates in Zambia over time. Each survey includes detailed information about health‐seeking behaviours, as well as a variety of socio‐economic variables on a nationally representative sample of the population.
We make use of all surveys we could pretreatment and posttreatment: three waves of the survey pretreatment (1998, 2002, and 2004) [^4] and one survey posttreatment (2006). For that last survey, the data collection occurred over October--November 2006, meaning that we observe the variables of interest 6 months after the policy change. Summary statistics on the outcome measures used before and after the removal of user fees are shown in Panel A of Table [1](#hec3589-tbl-0001){ref-type="table-wrap"}. In addition to information on study outcomes, we exploited data on a broad range of socio‐demographic characteristics as detailed in Panel B of Table [1](#hec3589-tbl-0001){ref-type="table-wrap"}.
######
Mean in outcomes and covariates
1998 (*n* = 72) 2002 (*n* = 71) 2004 (*n* = 72) 2006 (*n* = 72)
------------------------------------------------------------------------------------ ----------------- ----------------- ----------------- ----------------- -------- -------- -------- --------
**Panel A**
\% seeking modern care when ill 0.358 0.310 0.505 0.520 0.565 0.548 0.564 0.584
\% choosing a government or mission facility[a](#hec3589-note-0007){ref-type="fn"} 0.778 0.898 0.829 0.910 0.862 0.905 0.832 0.957
\% buying drugs in the private sector[a](#hec3589-note-0007){ref-type="fn"} 0.129 0.019 0.114 0.022 0.090 0.033
Log of deflated OOP[a](#hec3589-note-0007){ref-type="fn"} 6.703 6.522 5.967 4.547 6.149 5.374 6.088 3.111
**Panel B**
Proportion of male 0.496 0.492 0.489 0.491 0.498 0.494 0.490 0.487
Median age 20.060 19.761 21.162 20.448 21.213 20.488 21.051 19.966
Median log of total expenditures in adult equivalent 13.781 13.268 13.487 13.202 13.483 13.069 12.904 11.752
Median household size 7.039 6.565 6.287 6.496 6.737 6.874 6.107 5.829
Proportion of rural 0.311 0.885 0.303 0.905 0.299 0.807 0.260 0.798
Median distance to health facility (km) 1.580 5.183 1.723 5.784 1.853 4.289 1.104 5.573
*Note*. All values are representative at the national level as the district panel data were constructed based on sampling weights. In 2002, there were only 71 districts because there was no one who sought care when reporting illness in one district. OOP = out‐of‐pocket.
Proportion of individuals seeking modern care.
We investigate the effect of the policy on four outcomes. First, we look at health care utilisation, defined as whether an individual who reported an illness episode the last 2 weeks sought modern formal care---this excludes consultation of traditional or church healers and self‐medication but includes private, government, and mission facilities. Second, we consider the choice of provider of individuals seeking modern care. Specifically, we look at the proportion who went to any government‐funded primary care facility [^5] to test whether the policy led to a substitution away from private‐for‐profit providers. Third, to estimate the effect of the policy on financial protection, we consider health care expenditures incurred by individuals at the point of care, defined as the amount of OOP medical expenses (deflated and expressed in Kwachas 2006). Finally, we consider a potential unintended consequence of the policy, understood as something that was not meant by the policy change. Specifically, we look at the proportion of individuals who sought care and had to purchase drugs from a private pharmacist. This is meant to detect whether increased utilisation of public facilities led to drug stock‐outs, a sign of poor quality of care, and a cause for additional expenditures incurred elsewhere.
4. EMPIRICAL STRATEGY {#hec3589-sec-0008}
=====================
4.1. The synthetic control method {#hec3589-sec-0009}
---------------------------------
The staggered implementation of the policy change creates a natural experiment to analyse the effects of user fee removal. The 54 districts implementing the policy change in April 2006 are treatment units whereas the remaining 18 urban districts where fees are still charged constitute the donor pool (i.e., the comparison group). We exploit the fact that district classification was highly arbitrary because districts containing a city or a municipality were classified urban, whereas the other districts were classified rural. Nevertheless, looking at the distribution of the proportion living in rural areas (see [Appendix S1](#hec3589-supitem-0001){ref-type="supplementary-material"}), we see that on average, 40% of households living in an urban district live in a rural area and that there exist some highly rural areas among the urban classified districts, which provides reassurance about the choice of urban districts as reasonable controls for rural districts.
However, as suggested by the graphs in Figure [1](#hec3589-fig-0001){ref-type="fig"}, we ruled out a simple difference‐in‐differences (DiD) approach because the pre‐intervention outcome trends in the control and treatment groups are not parallel for most outcomes (specifically health care utilisation and health provider choice). [^6]
![Trends in outcomes \[Colour figure can be viewed at <http://wileyonlinelibrary.com>\]](HEC-27-493-g001){#hec3589-fig-0001}
An alternative method to estimate causal effects of a policy affecting one or more units is the synthetic control method (Abadie, Diamond, & Hainmueller, [2010](#hec3589-bib-0001){ref-type="ref"}; Abadie & Gardeazabal, [2003](#hec3589-bib-0002){ref-type="ref"}; Cavallo et al., [2013](#hec3589-bib-0006){ref-type="ref"}). This method involves constructing a counterfactual for the treated group (rural districts) by taking a weighted average of the available control units (urban districts), where a higher weight is given to control units that are more similar to the treated unit. This synthetic twin is created to follow the same pattern than the treated unit in the pretreatment period so that it can be used as a counterfactual after the policy implementation.
Additionally, the synthetic control is built by using the observable characteristics in all the pretreatment years. Unlike matching estimators, the idea behind the synthetic control is that a combination of control units provides a better comparison for the treated unit than a single unit alone. Additionally, the synthetic control is built by using the observable characteristics in all the pretreatment year allowing the effects of the unobserved heterogeneity in the outcome to vary with time (Abadie et al., [2010](#hec3589-bib-0001){ref-type="ref"}).
Using survey sampling weights, we estimate mean values for all outcomes and independent variables at the district level. Because they already benefited from free care before 2006, we exclude from the analysis individuals aged less than 5 and more than 65 years old in each survey wave. Based on available data, it is not possible to exclude other exempted groups but they represent a small proportion of all exemptions, and the exemption rules remain the same over the period of interest. We obtain a panel of 72 districts observed over three pretreatment periods (1998, 2002, and 2004) and one posttreatment period (2006).
Following Abadie et al. ([2010](#hec3589-bib-0001){ref-type="ref"}), let $Y_{\mathit{it}}^{N}$ be the outcome observed for district *i* at time *t* in the absence of intervention, for districts *j* = 1, ..., *J* and time periods *t =* 1, ..., *T*; and let the treated district *i =* 1 be the only one exposed to the intervention only after *T* ~0~ (with 1 ≤ *T* ~0~ \< *T*). Let $Y_{\mathit{it}}^{I}$ be the outcome that would be observed for district *i* at time *t* if district *i* is exposed to the intervention. The effect of the intervention for district *i* at time *t* \> *T* ~0~ can be defined as $\alpha_{\mathit{it}} = Y_{\mathit{it}}^{I} - Y_{\mathit{it}}^{N}$. Because $Y_{1t}^{I}$ is observed for a treated district *i* = 1, to estimate , we just need to estimate $Y_{1t}^{N}$.
To construct the counterfactual outcome in the treated district in the absence of the intervention $\ Y_{1t}^{N}$, the synthetic control method seeks an optimal vector of weights W\* $= \left( \omega_{2}^{*},\ldots,\omega_{j + 1}^{*} \right)^{\prime}$ chosen to minimise the distance between pre‐intervention characteristics and outcomes for the treated districts () and for the control districts (; Abadie et al., [2010](#hec3589-bib-0001){ref-type="ref"}). Using these weights, the synthetic control for unit *i* is given by $${\hat{Y}}_{1t}^{N} = \sum_{j = 2}^{j = j + 1}w_{j}^{*}Y_{\mathit{jt}}{for}\ t > T_{0}.$$
And therefore, the effect of the intervention for district *i* = 1 is ${\hat{\alpha}}_{1t} = Y_{1t} - {\hat{Y}}_{1t}^{N}$.
Formally, if is a vector of weights such that for *J* = 2, ..., *J* + 1 and , then the optimal vector W\* is chosen to minimise the distance between and , measured thanks to the following: $$\left\| {X_{1} - X_{0}W} \right\| v = \sqrt{\left. X_{1} - X_{0}W \right){}^{\prime}V\left( {X_{1} - X_{0}W} \right)},$$where *V* is a diagonal positive semidefinite identity matrix of dimension (K × K) that minimises the root mean squared prediction error (RMSPE), that is, the average of the squared discrepancy between the level of outcomes in the treated units and in their synthetic control counterpart in the pretreatment periods.
In addition to the pre‐intervention outcomes levels, here, we include the following district level covariates in both and [^7]: proportion of male, proportion of households living in a rural area, median age, median household income, median household size, and median distance to the health facility.
Unlike the seminal case presented by Abadie et al. ([2010](#hec3589-bib-0001){ref-type="ref"}) where there was only one treated unit, here, the policy change affected 54 rural districts. This implies that we compute ${\hat{\alpha}}_{\mathit{it}}$ for each of the 54 treated districts. Then, to obtain a national‐level estimate of the policy effect, we take the average of 54 ${\hat{\alpha}}_{\mathit{it}}$, weighted by the district population size (alternative approaches are used as robustness checks).
Statistical significance of the estimated effect is determined by running placebo tests (Abadie et al., [2010](#hec3589-bib-0001){ref-type="ref"}; Abadie & Gardeazabal, [2003](#hec3589-bib-0002){ref-type="ref"}). Specifically, we apply the synthetic control method to every untreated district (urban districts) in our sample. This allows us to assess whether the effect estimated by the synthetic control for the treated districts is large relative to the effect estimated in untreated districts. The idea is that if the distribution of placebo effects yields many effects as large as the estimated effect, then it is likely that the estimated effect was observed by chance.
4.2. Choice of control districts {#hec3589-sec-0010}
--------------------------------
The selection of the units to include in the donor pool (i.e., the technical term for all potential control units in the synthetic method approach) is crucial in the synthetic control method. We should consider discarding districts from the donor pool whose outcomes may be affected by the policy change because this could lead to underestimating its effect for two reasons. First, because the counterfactual outcomes for each synthetic treated district will be constructed as a weighted average of the outcomes of control districts, if some control districts are contaminated by the policy change (and have outcome levels comparable to those in treated districts), the policy effect will be underestimated if these contaminated control districts are given a nonzero weight. Second, because the statistical significance of the policy effect is evaluated against the distribution of placebo effects, if control districts are somehow contaminated by the policy, this will compromise our ability to detect that the change in the treated districts was not obtained by chance.
To choose the pool of donor districts, we have to consider two potential problems. First, due to the confusion around the implementation rules of the policy, some facilities located in the rural areas of urban districts may have wrongly decided to scrap user charges. Figure [2](#hec3589-fig-0002){ref-type="fig"}a shows a map of the proportion of patients from urban districts aged between 5 and 65 years who declared to have received free care. In two districts (Kasama and Mongu), more than half of the respondents received free care.
![(a) Implementation in urban districts. (b) Proportion of urban populations who sought care in a rural district \[Colour figure can be viewed at <http://wileyonlinelibrary.com>\]](HEC-27-493-g002){#hec3589-fig-0002}
Second, although we assume that individuals seek care in the district where they live, people from urban districts could seek care in rural districts because facilities are closer or cheaper. On the basis of 1998 LCMS data, [^8] we find that this issue is generally limited (less than 4% of the population of urban districts seeking care in rural districts---see Figure [2](#hec3589-fig-0002){ref-type="fig"}b), except in three districts (Mongu, Mazabuka, and Kasama) where respectively 25%, 18%, and 12% of the population sought care in rural districts.
On the basis of this, we construct a synthetic control for each of the 54 treated districts by using three alternative pools of control districts: (1) all 18 urban districts; (2) all but two districts (Kasama and Mongu) excluded because a significant proportion of the population received free care in 2006; and (3) all but three urban districts (Kasama, Mongu, and Mazabuka) where more than 10% of the population was declaring seeking care in a rural district in 1998.
5. RESULTS {#hec3589-sec-0011}
==========
5.1. Effects on access to modern care {#hec3589-sec-0012}
-------------------------------------
Table [2](#hec3589-tbl-0002){ref-type="table-wrap"} presents the estimated effects of the policy on the use of modern care, with the three alternative choices of control units.
######
Effect of user fee removal on access to modern care
\(1\) \(2\) \(3\)
--------------------------------------------------- ----------------- ----------------- -----------------
**Panel A**
Seeking care **0.007** **0.016** **0.014**
90% CI \[−0.16, 0.13\] \[−0.16, 0.13\] \[−0.16, 0.13\]
*N* treated 51 51 48
*N* placebo 18 16 15
**Panel B**
Chose government or mission primary care provider **0.016** **0.087** **0.080**
CI \[−0.19, 0.12\] \[−0.19, 0.12\] \[−0.17, 0.12\]
*N* treated 53 53 53
*N* placebo 18 16 15
*Note*. Effects of the policy are reported in bold. The 90% CI in brackets reports the 5th and 95th percentile of the placebo test distribution. Specification (1) is estimated using all 18 urban districts as control districts. Specification (2) excludes two districts where more than 50% of the population was reported to have benefited from free care in 2006. Specification (3) excludes the previous two districts and a third one where more than 10% of the population sought care in a rural district in 1998.
The findings in Panel A show no evidence that the policy increased health care use. The estimated effects suggest that there was an increase in utilisation of modern care by 0.7 to 1.6 percentage points (Columns 1 and 2), which is not statistically different from zero. [^9]
Panel B presents the estimated impact of the policy on the choice of provider, conditional on using modern care. The policy seems to have led to some substitution away from the private sector, as once we account for possible contamination, we find that the proportion of individuals who went to a public health facility increased by up to 8.7 percentage points. However, this effect is not statistically significant at the 10% level because it lies inside the placebo effect distribution. [^10]
5.2. Effects on OOP expenditures {#hec3589-sec-0013}
--------------------------------
Turning to OOP expenditures (Table [3](#hec3589-tbl-0003){ref-type="table-wrap"}), the results indicate a significant and important impact of the policy, a reduction of OOP medical expenditures by 2.18 logarithm points, which is a 89% decrease [^11] compared to 2004 expenditures (Column 1). This effect lies outside the "90% confidence interval" provided by the placebo effect distribution, meaning that the policy effect on financial protection is statistically significant at the 10% level. The reduction in OOP health expenses represents a saving during the last medical contact of US\$2.3 (in 2006 US\$) or 7% of the monthly adult equivalent expenditure. [^12]
######
Effect of user fee removal on out‐of‐pocket health expenses
\(1\) \(2\) \(3\)
---------------------------- ----------------- -------------- --------------
Ln(oop) **−2.177** **−2.258** **−2.279**
\% change compared to 2004 ***−89%*** ***−90%*** ***−90%***
CI \[−1.99, 1.56\] \[−1, 1.56\] \[−1, 1.56\]
*N* treated 48 49 47
*N* placebo 18 16 15
*Note*. Effects of the policy are reported in bold and percentage change are reported in italics. The 90% CI in brackets reports the 5th and 95th percentile of the placebo test distribution. Specification (1) is estimated using all 18 urban districts as control districts. Specification (2) excludes two districts where more than 50% of the population was reported to have benefited from free care in 2006. Specification (3) excludes the previous two districts and a third one where more than 10% of the population sought care in a rural district in 1998.
5.3. Unintended effects {#hec3589-sec-0014}
-----------------------
We now consider whether removing fees led to drug stock‐outs government and mission facilities, leading more patients to buy drugs from private facilities
The results (Table [4](#hec3589-tbl-0004){ref-type="table-wrap"}) show that the policy had a negative effect on the likelihood of buying drugs from the private sector; we estimate a decrease in the probability of buying drugs from the private sector between 1.7 and 6 points depending on the specification (Columns 3 and 1). However, these effects are not statistically significant and are consistent with the fact that drug shortages did not occur more in intervention districts.
######
Effect on the proportion of individuals who bought drugs in private pharmacies
\(1\) \(2\) \(3\)
------------------------------------- ----------------- ----------------- -----------------
Bought from a private drug provider **−0.060** **−0.043** **−0.017**
CI \[−0.24, 0.19\] \[−0.24, 0.19\] \[−0.24, 0.19\]
*N* treated 51 49 50
*N* placebo 18 16 15
*Note*. Effects of the policy are reported in bold. The 90% CI in brackets reports the 5th and 95th percentile of the placebo test distribution. Specification (1) is estimated using all 18 urban districts as control districts. Specification (2) excludes two districts where more than 50% of the population was reported to have benefited from free care in 2006. Specification (3) excludes the previous two districts and a third one where more than 10% of the population sought care in a rural district in 1998.
5.4. Factors affecting the impact of the policy {#hec3589-sec-0015}
-----------------------------------------------
We now investigate whether chaotic implementation of the policy led to its lack of documented impact on health‐seeking behaviours. Figure [3](#hec3589-fig-0003){ref-type="fig"} shows, for the four outcomes, the estimated effect against the degree of implementation of the policy in each district defined as the proportion of individuals not paying for primary care. These graphs suggest that although a better implementation of the policy is correlated with a greater use of publicly funded health facilities and lower OOP medical expenses, there is no association between the degree of full implementation of the policy and its effect on health care use.
![Relationship between the impact of the policy and its degree of implementation \[Colour figure can be viewed at <http://wileyonlinelibrary.com>\]](HEC-27-493-g003){#hec3589-fig-0003}
We investigated this issue formally in a regression framework ([Appendix S3](#hec3589-supitem-0001){ref-type="supplementary-material"}) and found no significant relationship between the degree of implementation of the policy and its effect on health care utilisation once accounting for other elements affecting policy implementation.
5.5. Heterogeneous effects {#hec3589-sec-0016}
--------------------------
We now explore the policy impact among the 50% poorest and the 50% richest [^13] households (Table [5](#hec3589-tbl-0005){ref-type="table-wrap"}). [^14] We find that there was no increase in health care use for either group (Panel A). However, we find that removing fees in government and mission facilities led the 50% richest away from private providers, with an increase in the probability of using a government or mission facility by about 18 percentage points (Panel B). Finally, the policy resulted in a similar relative decrease in OOP expenses for the rich and the poor (Panel C). Because the rich use and spend more on health, for any episode of illness, the policy resulted in a higher reduction of OOP medical expenses for the 50% richest in absolute terms (US\$3.21) compared to the 50% poorest (US\$1.07). [^15] Once adjusting for the fact that the fraction of the population [^16] that has an episode of illness and that seeks care is different between the two income groups, we find that, on average, the policy change represented a yearly government transfer worth about US\$4.47 and US\$1.13 to each individual of the richest and poorest group, respectively.
######
Effect of the policy by income groups
50% poorest 50% richest
---------------------------------------------------- ----------------- ----------------- ----------------- ------------------- ------------------- -------------------
Panel A: % seeking care
Estimated effect **−0.011** **0.031** **0.035** **−0.022** **0.013** **0.009**
CI \[−0.31, 0.32\] \[−0.31, 0.32\] \[−0.31, 0.32\] \[−0.182, 0.149\] \[−0.182, 0.149\] \[−0.182, 0.149\]
*N* treated 53 54 51 51 48 51
*N* placebo 18 16 15 18 16 15
Panel B: % choosing government or mission provider
Estimated effect **0.007** **0.059** **0.064** **0.034** **0.181** **0.153**
CI \[−0.11, 0.08\] \[−0.11, 0.05\] \[−0.10, 0.05\] \[−0.22, 0.16\] \[−0.22, 0.15\] \[−0.16, 0.15\]
*N* treated 47 47 47 50 50 49
*N* placebo 17 15 14 18 16 15
Panel C: Ln(oop)
Estimated effect **−1.573** **−1.694** **−1.720** **−2.298** **−2.413** **−2.624**
*% change* *−79%* *−82%* *−82%* *−90%* *−91%* *−93%*
CI \[−2.59, 3.08\] \[−2.05, 3.08\] \[−2.05, 3.08\] \[−2.01, 1.79\] \[−2.01, 1.79\] \[−2.01, 1.79\]
*N* treated 49 50 50 50 50 51
*N* placebo 18 16 15 17 15 14
Panel D: % buying drugs in the private sector
Estimated effect **0.007** **0.009** **0.010** **−0.261** **−0.271** **−0.120**
CI \[−0.08, 0.12\] \[−0.08, 0.12\] \[−0.08, 0.12\] \[−0.32, 0.16\] \[−0.31, 0.16\] \[−0.31, 0.11\]
*N* treated 52 49 47 45 49 45
*N* placebo 18 16 15 18 16 15
*Note*. Effects of the policy are reported in bold and percentage change are reported in italics. The 90% CI in brackets reports the 5th and 95th percentile of the placebo test distribution. Specifications (1) and (4) are estimated using all 18 urban districts as control districts. Specifications (2) and (5) exclude two districts where more than 50% of the population was reported to have benefited from free care in 2006. Specifications (3) and (6) exclude the previous two districts and a third one where more than 10% of the population sought care in a rural district in 1998.
6. ROBUSTNESS CHECKS {#hec3589-sec-0017}
====================
6.1. Match quality {#hec3589-sec-0018}
------------------
The validity of the synthetic control method partly relies on the quality of the pretreatment match and the extent to which the synthetic districts are able to reproduce the pre‐intervention outcomes. [Appendix S5](#hec3589-supitem-0001){ref-type="supplementary-material"}, which shows the 54 plots presenting the pre‐intervention outcomes for each district and their twin, suggests that some synthetic controls were not able to reproduce the pretrend outcomes perfectly. In turn, this could lead to poor estimates of the policy effect. To account for quality match in the estimation of the national policy effect, we weighted each district effect by the inverse of the logarithm of the RMSPE, effectively giving a higher weight to the closely matching synthetic districts (see Panel A of Table [6](#hec3589-tbl-0006){ref-type="table-wrap"}). We find the same results as before.
######
Effects accounting for pretreatment match quality
Seek care Public facility OOP medical expenses Private drug seller
------------------------------------------------------------------------ ----------- ----------------- ---------------------- ---------------------
Panel A: Weighted average by the logarithm of the inverse of the RMSPE
Estimated effect 0.007 0.016 −2.177 −0.062
Panel B: Restricting the sample to high‐quality matches
Number of perfect matches 9 19 13 28
National effect weighted by district size 0.042 0.024 −2.085 −0.080
*Note*. Perfect‐quality matches means that RMSPE \< 0.01 for binary outcomes and \<0.001 for ln(OOP). RMSE = root mean squared prediction error.
Next, we estimate the national effect by including only districts with high‐quality matches. We find very similar results except for health care use, where, based on only nine high‐quality district twins, the estimated policy effect seems slightly higher.
6.2. Micro data‐level analysis {#hec3589-sec-0019}
------------------------------
As an alternative estimation approach, we use micro‐level data and combine DiD with propensity score matching (Heckman, Ichimura, & Todd, [1998](#hec3589-bib-0022){ref-type="ref"}; Imbens, [2004](#hec3589-bib-0023){ref-type="ref"}) to account for the nonparallel pre‐intervention trends and perform a kernel matching over three groups: the treated and control at baseline t~0~ and the nontreated at follow up t~1~. Following Blundell and Dias ([2009](#hec3589-bib-0093){ref-type="ref"}), the matching estimator combined with DiD (MDiD), noted , is given by $$\alpha^{\textit{MDiD}} = \sum\limits_{\mathit{i\epsilon T}1}\left\{ {\left\lbrack {y_{\mathit{it}1} - \sum\limits_{\mathit{j\epsilon T}0}{\overset{\sim}{w}}_{\mathit{ijt}0}^{T} y_{\mathit{jt}0}} \right\rbrack - \left\lbrack {\sum\limits_{\mathit{j\epsilon C}1}{\overset{\sim}{w}}_{\mathit{ijt}1}^{C} y_{\mathit{jt}1} - \sum\limits_{\mathit{j\epsilon C}0}{\overset{\sim}{w}}_{\mathit{ijt}0}^{C} y_{\mathit{jt}0}} \right\rbrack\ } \right\}\ \omega_{i},$$where *T* ~0~, *T* ~1~, *C* ~0~, and *C* ~1~ stand for the treatment and comparison group before and after user fee removal, and ${\overset{\sim}{w}}_{\mathit{ijt}}^{G}$ represents the weight attributed to individual *j* in group *G* (treatment or control) and time *t* (*t* ~0~, *t* ~1~) when comparing with treated individual *i*.
The results obtained using DiD with matching, reported in Table [7](#hec3589-tbl-0007){ref-type="table-wrap"}, are similar to the ones obtained using the synthetic control method. Specifically, we find that there was no impact of user fee removal on the use of modern care. However, here, the small substitution effect (4.5 percentage points) away from the private sector is found significant at the 10% level (see Panel B, Column 6). The results also confirm the large and significant decrease in OOP expenditures because the policy led to a decrease in OOP health expenditures by 86%. Finally, the results suggest that there was no impact on the likelihood of buying drug from the private sector.
######
Effects of user fee removal using individual‐level data
\(1\) Simple DiD model \(2\) Matching and DiD (MDiD)
---------------------------------------------------- --------------------------------------------------- --------------------------------------------------- -------------------------------------------------------- ---------- --------------------------------------------------- --------------------------------------------------------
Panel A: % seeking care
Estimated effect −0.020 0.026 **0.046** 0.023 0.059[\*\*\*](#hec3589-note-0028){ref-type="fn"} **0.036**
*SE* (0.025) (0.034) (0.034) (0.023) (0.04) (0.045)
*N* 10,295 7,841 18,136 9,711 6,859 16,570
Panel B: % choosing government or mission provider
Estimated effect 0.036 0.110[\*\*\*](#hec3589-note-0028){ref-type="fn"} **0.075** [\*\*\*](#hec3589-note-0028){ref-type="fn"} 0.026 0.072[\*\*\*](#hec3589-note-0028){ref-type="fn"} **0.045** [\*](#hec3589-note-0026){ref-type="fn"}
*SE* (0.024) (0.026) (0.024) (0.023) (0.016) (0.026)
*N* 5,975 4,817 10,792 5,685 4,259 9,944
Panel C: Ln(oop)
Estimated effect −0.771[\*\*](#hec3589-note-0027){ref-type="fn"} −2.755[\*\*\*](#hec3589-note-0028){ref-type="fn"} **−1.985** [\*\*\*](#hec3589-note-0028){ref-type="fn"} −0.195 −2.141[\*\*\*](#hec3589-note-0028){ref-type="fn"} **−1.946** [\*\*\*](#hec3589-note-0028){ref-type="fn"}
*% change* *−53.8%* *−93.7%* *−86.3%* *−17.7%* *−88.2%* *−85.7%*
*SE* (0.370) (0.467) (0.367) (0.161) (0.165) (0.374)
*N* 8,620 6,806 15,426 8,144 6,016 14,170
Panel D: % buying drugs in the private sector
Estimated effect −0.139[\*\*\*](#hec3589-note-0028){ref-type="fn"} −0.168[\*\*\*](#hec3589-note-0028){ref-type="fn"} **−0.029** −0.052 −0.077[\*\*](#hec3589-note-0027){ref-type="fn"} **−0.025**
*SE* (0.052) (0.050) (0.038) (0.046) (0.036) (0.043)
*N* 8,589 6,791 15,380 8,127 6,003 14,130
*Note*. Effects of the policy are reported in bold and percentage change are reported in italics. The propensity score was estimated using the same covariates at the individual level than the ones used in the synthetic control. Survey sampling weights are used. SE clustered at the district level in bracket. Estimated presented in (2) are based on propensity score matching using Epanechnikov kernel weights. DiD = difference‐in‐differences
Statistically significant at the 1% statistical significance level.
Statistically significant at the 5% significance level.
Statistically significant at the 10% significance level.
6.3. Alternative approach to apply the synthetic control method with multiple treated units {#hec3589-sec-0020}
-------------------------------------------------------------------------------------------
Here, we use an alternative method to estimate a unique national effect from multiple treated units. More specifically, we aggregated outcomes and covariates from the 54 treated units to create a single treated unit and then create a synthetic rural unit with the 18 urban districts. We can see from Figure [4](#hec3589-fig-0004){ref-type="fig"} that the pre‐intervention trend of the synthetic rural unit perfectly overlaps the one from the single treated unit. The results we find (Table [8](#hec3589-tbl-0008){ref-type="table-wrap"}) are very similar to the ones based on the 54 treated units. Specifically, we find that the policy had no effect on health care use or on the health provider chosen. However, the result confirms that the policy reduced OOP medical spending by at least 85%. Note that although collapsing all 54 treated units into one average treated unit reduces sampling error and hence leads to a lower RMSPE (see Figure [4](#hec3589-fig-0004){ref-type="fig"}), it does not account for the fact that each rural district has its own specificity and that taking the average outcomes in those districts could prevent from creating a counterfactual that closely accounts for time variant and invariant unobserved characteristics.
![Synthetic control and treated unit trends by outcome and donor pools. The graphs are based on Specification (2) but graphs for Specifications (1) and (3) lead to similar pre‐intervention trends for the synthetic control and treated units \[Colour figure can be viewed at <http://wileyonlinelibrary.com>\]](HEC-27-493-g004){#hec3589-fig-0004}
######
Results from the synthetic control method with a single treated unit
\(1\) \(2\) \(3\)
-------------------------------------- ----------------- ----------------- -----------------
Seek care −0.018 −0.010 −0.013
\[−0.16, 0.13\] \[−0.16, 0.13\] \[−0.16, 0.13\]
Chose government or mission provider 0.029 0.094 0.088
\[−0.19, 0.12\] \[−0.19, 0.12\] \[−0.17, 0.12\]
OOP medical expenses −1.895 −2.078 −2.086
\[−1.99, 1.56\] \[−1.00, 1.56\] \[−1.00, 1.56\]
*% change* *−85.0%* *−87.5%* *−87.6%*
Purchase of private drugs −0.036 0.012 0.014
\[−0.24, 0.19\] \[−0.24, 0.19\] \[−0.24, 0.19\]
*Note*. 90% CI in brackets. Specification (1) is estimated using all 18 urban districts as control districts. Specification (2) excludes two districts where more than 50% of the population was reported to have benefited from free care in 2006. Specification (3) excludes the previous two districts and a third one where more than 10% of the population sought care in a rural district in 1998. OOP = out‐of‐pocket.
7. DISCUSSION AND CONCLUSIONS {#hec3589-sec-0021}
=============================
Our findings indicate that the abolition of user fees in rural Zambia in 2006 did not change the probability of seeking modern care in the population. This seems to contradict the conclusions of previous studies that suggested substantial increases in the volume of outpatient visits recorded in routine data (Lagarde et al., [2012](#hec3589-bib-0026){ref-type="ref"}; Masiye et al., [2010](#hec3589-bib-0028){ref-type="ref"}). Setting aside the methodological problems associated with these past studies, [^17] our results suggest that part of this increase may have come from richer patients previously seeking care in the private sector. Moreover we find that there was a large and positive effect of the policy on OOP expenditures, which decreased by nearly 90% in the population, indicating that there was a positive effect of the policy on financial protection. Due to unequal medical spending between richer and poorer groups, we find that although the reduction was similar in relative terms for both groups, in absolute terms, the policy change benefited the richest, through an income transfer per medical visit of US\$3.2 for them versus US\$1.1 for the poorest. Finally, we find that despite its relative lack of preparation, the policy change did not lead to drug stock‐outs in intervention areas. This result may be linked to reports of widespread shortage of drugs across the country in 2006, before the policy took place (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}).
Our results echo other recent robust empirical studies of the effects of abolishing user fees on the demand for curative care in other low‐income settings (King et al., [2009](#hec3589-bib-0025){ref-type="ref"}; Mohanan et al., [2014](#hec3589-bib-0035){ref-type="ref"}), although one study from Ghana found a slightly more encouraging increase of the demand by 3.7 percentage points (Powell‐Jackson et al., [2014](#hec3589-bib-0037){ref-type="ref"}). These results are at odds with the recent experimental literature on the price effects of the demand for preventive health products and services (Dupas & Miguel, [2016](#hec3589-bib-0012){ref-type="ref"}). There are three main explanations for the lack of effectiveness of the policy change on the demand for curative health care services.
A first explanation could be that the scrapping of official charges was replaced by the introduction (or increase) of informal payments, as suggested to have been the case in Uganda (Xu et al., [2006](#hec3589-bib-0042){ref-type="ref"}). However, to the extent that individuals report informal charges, our results reject this explanation, because we see an important reduction in OOP medical expenses for those visiting publicly funded health facilities.
Another potential explanation is that the demand for curative health care is price inelastic. This might have been possible because the level of fees was particularly low, and because the demand was primarily determined by other factors, such as indirect financial costs. This is supported by an analysis of the determinants of health‐seeking behaviours before 2006 (see [Appendix S6](#hec3589-supitem-0001){ref-type="supplementary-material"}). Although the results show a positive association between income and the probability to seek care, the magnitude of this effect is quite small, [^18] even in rural districts and among the 50% poorest households. In addition, data from LCMS 1998 suggest that indirect costs to accessing care were as important as OOP expenses. Even 7 years after fees were abolished nationally, 11% of patients in rural areas reported catastrophic health expenditures [^19] mostly because of transportation costs, which represented 73% of these costs (Masiye, Kaonga, & Kirigia, [2016](#hec3589-bib-0031){ref-type="ref"}).
A final explanation is that changes occurred over the period of study, which changed individuals\' valuation of health care services. The main suspicion is that quality of care in rural areas deteriorated over the period, possibly partly as a result of the policy change. First, lack of planning and delays in providing adequate funding may have led to deterioration of the quality of services, as suggested before. The uncompensated loss of revenue from the removal of user fees can have important consequences at facility level where a significant share of fee revenue is retained to finance a proportion of staff income (Carasso et al., [2010](#hec3589-bib-0004){ref-type="ref"}). The policy change may also have exacerbated problems of motivation and shortages of staff in rural areas (Picazo & Zhao, [2009](#hec3589-bib-0036){ref-type="ref"}). Because of the substitution away from private‐for‐profit providers and a possible increase in the intensity of health care use, waiting times in health facilities may have increased. Unfortunately, in the absence of data on the quality of care, we cannot disentangle whether the absence of effect on utilisation is mostly explained by the price inelasticity of the demand or a combination of an increase in demand due to the price effect and a downward shift in demand resulting from a poorer quality of care.
In addition to the challenge to identify clearly the reasons behind the lack of effect found on the demand for health care, this study suffers from several limitations.
A first limitation relates to the small number of control units. As a result, the confidence intervals estimated from the placebo effects distribution are relatively large and might preclude a more precise estimation of effects. Still, the upper bound confidence interval from the placebo tests is close to the one obtained with MDiD presented as a robustness check. [^20] Besides, the synthetic control method remains superior to MDiD estimator because, unlike the propensity score matching conducted on 2004 data only, the synthetic districts are created using multiple pre‐intervention periods, allowing us to account for unobserved heterogeneity that varies over time.
Another limitation relates to the messy definition and implementation of the policy, resulting in the potential contamination of the control units in our dataset, which may have led to problems in estimating the effects of the policy (see Section [4.2](#hec3589-sec-0010){ref-type="sec"}). We investigated this issue further in [Appendix S7](#hec3589-supitem-0001){ref-type="supplementary-material"}. We found additional support for the idea that the effect on health provider choice could be underestimated given that, for this outcome, contaminated placebo districts were given a higher weight in the synthetic control. However, this was not the case for the other outcomes. Besides, [Appendix S7](#hec3589-supitem-0001){ref-type="supplementary-material"} also shows that the contamination issue was not the reason for our larger confidence intervals because the effect of the policy was not stronger in contaminated urban districts.
Finally, the challenges to implement the policy could provide an explanation of the absence of short‐term effect on utilisation. However, as highlighted before, a study using longitudinal health facility routine data (Lagarde et al., [2012](#hec3589-bib-0026){ref-type="ref"}) showed that utilisation peaked 6 months after the implementation. This suggests that the timeline we use is probably ideal to capture an effect on utilisation. [^21]
In January 2007, user fees were removed in all the facilities located in the peri‐urban areas of the 18 districts where fees had not yet been abolished. [^22] Later, in January 2012, the policy was extended to all remaining areas. A study looking at the financial protection conferred by the free care policy found that 29.9% and 45% of patients in public rural and urban health centres respectively incurred some expenditures (Masiye et al., [2016](#hec3589-bib-0031){ref-type="ref"}). In a follow‐up study using the same data, Masiye and Kaonga ([2016](#hec3589-bib-0030){ref-type="ref"}) conclude that in 2014, "despite the removal of user fees in public primary healthcare in Zambia, access to healthcare is highly dependent on an individual\'s socio‐economic status, illness type and region of residence." Together with our findings, this suggests that user fee removal may not necessarily be the silver bullet to move towards greater access for the poorer population and quicker move towards universal coverage. Evidence from several settings also point to the potential disruptive effects of free care policies on health systems (e.g., drug shortages, staff dissatisfaction, and insufficient funding) and therefore the need to prepare, plan, and introduce complementary measures to ensure a more positive outcome (Ridde, Robert, & Meessen, [2012](#hec3589-bib-0038){ref-type="ref"})
The debate over whether low‐ and middle‐income countries should charge their populations for using health care services has been highly contentious for several decades. This study suggests important and maybe counter‐intuitive lessons for policy‐makers, with regards to the immediate equity effects of removing user charges. If removing fees does not increase utilisation of services, in particular of the poorest, but effectively reduces OOP medical expenses of those using the services, then the beneficiaries of the policy change are those individuals already using services. The conclusion that user fee removal in Zambia was primarily benefiting the richer groups echoes a typical problem of policies promoting universal access to services in settings where initial inequalities are large and there are multiple barriers to accessing services for the poor (Gwatkin & Ergo, [2011](#hec3589-bib-0019){ref-type="ref"}). It would be interesting to know whether removing these other barriers (e.g., through financial or nonfinancial incentives) while maintaining user fees would be more effective than removing fees.
Supporting information
======================
######
Appendix S1: Proportion living in rural areas in urban and rural districts
Appendix S2: Placebo effects
Appendix S3: Determinants of effects
Appendix S4: Descriptive statistics for 50% poorest and 50% richest in 2004 (*n* = 72)
Appendix S5: Pre‐intervention match quality
Appendix S6: Determinants of health seeking behaviours prior user fee removal
Appendix S7: Potential effects of contamination on estimated impact and confidence intervals
######
Click here for additional data file.
[^1]: Although from a theoretical perspective the two interventions are equivalent (no direct cost for using health care services if the insurance provides a full third‐party reimbursement), in practice, they have important differences. Insurance schemes often limit the number and type of health care providers that can be chosen by members, in a way that user fee removal does not. And there is also evidence that even when they are insured, more disadvantaged groups are likely to claim their benefits and use health insurance less (Devadasan, Criel, Van Damme, Ranson, & van der Stuyft, [2007](#hec3589-bib-0011){ref-type="ref"}) whereas user fee removal does not present any administrative obstacle to anyone.
[^2]: Two reports showed that user fee\'s introduction led to a decrease in utilisation (Kahenya & Lake, [1994](#hec3589-bib-0024){ref-type="ref"}; Sukwa & Chabot, [1997](#hec3589-bib-0040){ref-type="ref"}).
[^3]: The policy change was later scaled up to other areas. In January 2007, user fees were removed in all public health facilities located in the peri‐urban areas of the remaining 18 urban districts. Finally, in January 2012, user fees were removed everywhere else.
[^4]: Due to a change in administrative definition of districts, we could not use surveys that took place before 1998.
[^5]: This is defined as an individual declaring they went to seek care in a government or a mission facility.
[^6]: Results are presented as a robustness check.
[^7]: Note that those covariates were chosen to be included because the RMSPE is minimised under this specification. Given the nonparametric nature of the synthetic control method, we have run a sensitivity analysis on the covariates to include and the model associated with the smallest RMSPE was selected. More specifically, we estimated a model with no covariate as well as another model that includes province dummies and district population size. Although the coefficients obtained were very close, those models lead to a higher RMSPE and higher confidence intervals and hence are not presented in the paper but are available from authors upon request.
[^8]: The only survey wave for which the information is available.
[^9]: Placebo test graphs are presented in [Appendix S2](#hec3589-supitem-0001){ref-type="supplementary-material"}.
[^10]: Placebo effects are presented in [Appendix S2](#hec3589-supitem-0001){ref-type="supplementary-material"}.
[^11]: 1−(exp(−2.177)) = 0.89
[^12]: OOP\'s level in 2004 was Kwatchas 9481 or 2006 US\$2.58.
[^13]: It was not possible to split the sample by income groups more finely given the limited number of household in some districts (e.g., some districts do not include individuals of all quartiles, because the latter are defined at the national level).
[^14]: Note that yearly deflated total household expenditure per adult equivalent was 4.1 times greater among the 50% richest (US\$ 377) than for the 50% poorest (US\$ 92) in 2004 and that health care use was 54% for the 50% poorest and 59% among the 50% richest in 2004 ([Appendix S4](#hec3589-supitem-0001){ref-type="supplementary-material"}).
[^15]: Calculations based on applying the % reduction in OOP estimated in Table [5](#hec3589-tbl-0005){ref-type="table-wrap"} to the deflated OOP medical expenses incurred in 2004.
[^16]: When accounting for the likelihood of being sick (equal to about 10% in the two groups), we find that about 6% and 5% of individuals in the richest and poorest group, respectively, sought care in 2004 in the past 2 weeks.
[^17]: These earlier studies did not address the biases associated with reporting inaccuracies by facilities or any changes in health‐seeking behaviours over time that were unrelated to the policy change.
[^18]: An increase in one point in the logarithm of deflated total household expenditures in adult equivalent (similar to an increase in one tercile in the income distribution) is associated with an increase in health care use by only 4.4 percentage points and a decrease in the likelihood of going to a public health facility by 3.3 percentage points.
[^19]: Defined as 40% of household nonfood expenditures.
[^20]: MDiD 90% upper bound CI = 0.11.
[^21]: Unfortunately, the later scale‐up of the policy does not allow the identification of medium‐ or long‐term effects.
[^22]: The remit of this scale‐up, which could not be well identified in the household survey data due to the absence of geographic coordinates, prevented the use of the 2010 LCMS wave to assess the longer term consequences of the policy.
|
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INTRODUCTION
============
Thrombophilia refers to a group of inherited or acquired coagulation disorders, leading to venous and/or arterial thrombosis. The most frequent clinical manifestations of venous thrombosis are deep vein thrombosis (DVT), pulmonary embolism (PE) and in case of obstruction of placental circulation, fetal loss (FL). Arterial thrombosis can manifest as myocardial infarction (MI), ischaemic stroke or arterial embolism \[[@b1-bjmg-17-02-43]\].
Thrombophilia is a multifactorial disorder that arises from the interaction of acquired and genetic risk factors \[[@b2-bjmg-17-02-43]\]. The most common acquired risk factors include surgery, immobilization, fractures, puerperium, paralysis, prolonged bed rest and use of oral contraceptives \[[@b3-bjmg-17-02-43]\] The most important genetic risk factors contributing to thrombophilia are deficiencies of natural inhibitors of clotting factors (antithrombin, protein C, protein S), Factor V Leiden (FVL) mutation and prothrombin (FII) G20210A mutation. Deficiencies of natural inhibitors occur sparsely, but represent significant risk factor \[[@b4-bjmg-17-02-43]--[@b9-bjmg-17-02-43]\]. The FVL mutation is present 3.0--7.0% in Caucasians and 15.0--50.0% in patients with thrombosis, while the incidence of FII G20210A is 1.0--2.0% in Caucasian populations and 18.0--20.0% in patients with thrombosis \[[@b8-bjmg-17-02-43]\].
The FVL mutation yields an amino acid substitution within the cleavage site of its inhibitor-activated protein C that results in impaired inhibition of FV and increased thrombin generation leading to hyper-coagubility \[[@b10-bjmg-17-02-43]--[@b12-bjmg-17-02-43]\].
The *FII G20210A* gene variant is located in the 3′ untranslated region (3′UTR) of the *FII* gene and it has been associated with elevated plasma FII levels. This is a gain-of-function mutation, causing increased cleavage site recognition and 3′ end mRNA processing, which leads to an increased FII synthesis \[[@b13-bjmg-17-02-43]--[@b16-bjmg-17-02-43]\]. Apart from the *FII G20210A*, several variants have been detected in the 3′ end of the *FII* gene, such as: *A19911G*, *C20211T*, *T20219A*, *A20218G* and *C20209T* \[[@b17-bjmg-17-02-43]--[@b25-bjmg-17-02-43]\].
The 3′ end of the *FII* gene is a dynamic region because of its non canonical architecture, therefore, it could be a potential region for finding new variants which might contribute to thrombophilia \[[@b13-bjmg-17-02-43]--[@b15-bjmg-17-02-43]\]. Despite the fact that several genetic risk factors have been associated with thrombophilia, there are still a certain number of patients suffering from idiopathic thrombophilia, defined as unequivocal clinical picture of thrombosis with unknown risk factors. Additionally, there are no data regarding the frequency of *FII* 3′ end gene variants in patients with idiopathic thrombophilia in Serbia. The aim of this study was to screen 3′ end of *FII* gene in population of patients with idiopathic thrombophilia originating from the geographic area of Serbia.
MATERIALS AND METHODS
=====================
Patients
--------
The study group was formed by searching the database of over 4000 patients who were referred to the Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia for genetic testing of thrombophilia during the period from 2000 to 2013. Anamnestic data were gathered from all the participants and the following subjects were excluded from further investigation: the patients who developed the first thrombotic event at an age above 50 years, the patients with malignancies, diabetes, antiphospholipid antibodies, deficiency of natural inhibitors (antithrombin, protein C, protein S), and the carriers of FVL and *FII G20210A* gene mutations \[[@b26-bjmg-17-02-43]\]. Finally, the study group included 100 patients (age range 16--63, median 38 years; male/female: 45/55) with a clinical picture of recurrent thrombotic events (DVT, PE, FL) or the combination of two or three thrombotic events (DVT, PE, FL, MI and stroke).
The control group comprised 100 healthy subjects (age range: 20--66 years, median 39; male/female: 82/18) with no history of thrombotic events. Informed consent was obtained from all participants and the study protocol was approved by the local research ethics committee.
Laboratory Methods
------------------
Peripheral blood was taken on 3.8% sodium citrate as anticoagulant. Genomic DNA was purified from 200 μL of human whole blood using the QIAamp DNA blood mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. The blood and DNA samples were stored at −20 °C until further use.
The 715 bp fragment that includes the last intron and exon, 3′UTR and the flanking region of the *FII* gene (primers: 5′-GGA AAC GAG GGG ATG CCT GT-3′ and 5′-CCT GCC ATC TTT CCT CTC AC-3′), was amplified by polymerase chain reaction (PCR). The PCR reactions were performed in a 25 μL final volume: 1 × Kapa Buffer B; 2.5 mM MgCl~2~; 200 μM dNTPs; 1U Kapa Taq polymerase (Kapa Biosystems, Boston, MA, USA), 10 pmol of forward and reverse primers and 200 ng of DNA. The thermal cycle profile was: initial denaturation at 95 °C for 5 min. and 37 cycles consisting of denaturation at 95 °C for 1 min., annealing at 61 °C for 1 min. and polymerization at 72 °C for 1 min. were applied. Final extension of the PCR products was at 72 °C for 10 min.
Sequencing of the amplified 715 bp fragments was performed according to the manufacturer's protocol, using the BigDye™ Terminator Version 3.1 Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on a 3130 Genetic Analyzer (Applied Biosystems). Two sequencing reactions for the fragment of interest were performed for each sample using the forward primer (5′-TCT AGA AAC AGT TGC CTG GC-3′) or reverse primer (5′-GAA TAG CAC TGG GAG CAT TGA-3′).
Statistical Analysis
--------------------
Statistical analysis was performed using MedCalc 12.2.1.0 statistical software (MedCalc Software bvba, Ostend, Belgium). The prevalence of detected gene variants was compared between patients and controls with the use of Fisher's exact test. The odds ratio (OR) and 95% CI (95% confidence interval) were also estimated. A *p* value of \<0.05 was considered to be statistically significant. Deviations of genotype distributions from Hardy-Weinberg equilibrium were assessed by the c^2^-test.
RESULTS
=======
In this study, we included 100 patients with idiopathic thrombophilia and 100 healthy controls. The clinical data of the study group are shown in [Table 1](#t1-bjmg-17-02-43){ref-type="table"}. In our study, we detected the presence of A19911G and C20068T variants in the 3′ end of the *FII* gene ([Figure 1](#f1-bjmg-17-02-43){ref-type="fig"}).
In the patient group, there were 51.0% heterozygous and 20.0% homozygous carriers of the A19911G variant, which was similar to the frequency in the control group (51.0% heterozygous and 19.0% homozygous) ([Table 2](#t2-bjmg-17-02-43){ref-type="table"}). The results of our study showed that the difference between patients and controls was not statistically significant (*p* = 0.88) and that A19911G does not represent a risk factor for idiopathic thrombophilia in our study (OR = 1.06; 95% CI 0.53--2.13). Regarding this variant, both groups were in Hardy-Weinberg equilibrium (c^2^ = 0.08; *p* = 0.77).
The C20068T variant was detected only in the heterozygous state, with frequencies of 4.0% in patients and 1.0% in the control group ([Table 2](#t2-bjmg-17-02-43){ref-type="table"}). Even though this difference between patients and controls was not statistically significant (*p* = 0.21), it could represent a risk factor in idiopathic thrombophilia (OR = 4.12; 95% CI 0.45--37.52). The study groups were in Hardy-Weinberg equilibrium for this variant (c^2^ = 0.04; *p* = 0.83).
In our study, 4.0% of the patients were carriers of both A19911G and C20068T mutations. This subset of patients was distinguished by more severe thrombotic manifestations. In one, DVT reoccurred six times and three patients suffered from the combination of more than two thrombotic events (MI, DVT, PE, FL, and stroke).
DISCUSSION
==========
In order to identify potential thrombophilic risk factors in these patients, we chose to sequence the 3′ end of the *FII* gene, since it has been shown that this region is very susceptible to gain-of-function mutations \[[@b13-bjmg-17-02-43]--[@b15-bjmg-17-02-43]\]. We detected two variants in the 3′ end of the *FII* gene, *A19911G* and *C20068T*, in both the patients and control groups.
The *A19911G* gene mutation is located in the last intron of the *FII* gene and functional analyses showed that the presence of this variant enhances splicing efficiency by altering a known functional pentamer motif \[[@b24-bjmg-17-02-43]\]. In our study, the frequency of the *A19911G* gene variant in patients was similar to the frequencies in the control group. The frequency of the G allele in our study was 0.44, which is very similar to the frequencies that were reported for thrombophilia patients in other populations: Italian (0.51), Spanish (0.52) and Dutch (0.48) \[[@b22-bjmg-17-02-43]--[@b23-bjmg-17-02-43]\]. According to our results, A19911G does not represent a risk factor for idiopathic thrombophilia ([Table 2](#t2-bjmg-17-02-43){ref-type="table"}). A recent study by Martinelli *et al.* \[[@b23-bjmg-17-02-43]\] that investigated the risk of cerebral sinus-venous thrombosis in patients with A19911G, also showed that this variant does not represent a significant risk factor for this thrombotic disorder. However, the MEGA study \[[@b25-bjmg-17-02-43]\], which included over 5000 patients and 5000 controls showed that the presence of the 19911GG genotype represents 1.43-fold greater risk for thrombotic occurrence (95% CI 1.27--1.67) compared to the 19911AA genotype.
The *C20068T* gene variant is located in the last exon of the *FII* gene. As it does not result in a change of the amino acid sequence of a protein, it is considered as a synonymous variant. *In vitro* functional assays revealed that constructs with the *C20068T* gene variant increased expression level 1.64 times compared to the wild type construct \[[@b27-bjmg-17-02-43]\]. However, further studies are needed in order to clarify the exact mechanism of overexpression. In our study, we detected this variant only in the heterozygous state, with a higher frequency in patients than in controls. Moreover, further studies in a larger group of subjects are needed in order to elucidate the importance of this gene variant for etiology of idiopathic thrombophilia. To the best of our knowledge, there are no studies considering the prevalence of C20068T in patients with thrombophilia or any other disease.
In our study, 4.0% of the patients were carriers of a combined *20068CT/19911AG* genotype. These patients experienced more severe clinical manifestations of throm-bophilia. This result alludes to the possible role of the association of C20068T and A19911G in pathogenesis of thrombophilia. Nevertheless, functional studies are needed to yield more precise conclusions.
Other variants in the 3′ end of the *FII* gene (*C20211T*, *T20219A*, *A20218G* and *C20209T*) that were reported in previous studies were not detected in our study. Our data indicate that the prevalence of these variants is less than 1.0% in the patients with idiopathic thrombophilia in a population originating from the geographic area of Serbia.
In order to obtain a well-defined group of patients with idiopathic thrombophilia, we applied very strict selection criteria as described in the Materials and Methods section, which led to a relatively small size of the patient group. Also, the results that we obtained can be attributed to the specific criteria we used.
In conclusion, our study showed the first results for the prevalence of variants at the 3′ end of the *FII* gene in patients with idiopathic thrombophilia. These results indicate that the *A19911G* gene variant is not a significant risk factor in our study group. On the other hand, the C20068T polymorphism could be a potential risk factor in idiopathic thrombophilia and therefore, further studies in a larger group of patients should be conducted.
**Declaration of Interest.** This study was supported by grant No. 173008 from The Ministry of Education, Science and Technological Development of the Republic of Serbia, Belgrade, Serbia. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
{#f1-bjmg-17-02-43}
######
Data of the Patients.
**Thrombotic Event** **Number of Patients** **Male/Female** **Age Range (median)**
------------------------------------------------------------ ------------------------ ----------------- ------------------------
Recurrent DVT 50 26/24 16--63 (39)
Recurrent PE 6 3/3 25--41 (36)
Recurrent FL 4 0/4 29--41 (35.5)
Combination of thrombotic events (DVT, PE, FL, MI, stroke) 40 16/24 19--54 (38)
DVT: deep vein thrombosis; PE: pulmonary embolism; FL: fetal loss; MI: myocardial infarction.
######
The frequencies of detected gene variants in the studied population.
**Gene Variant** **Patients/Controls** **OR; 95% CI** ***p* Value**
------------------ ----------------------- ------------------- ---------------
*A19911G*
AG 51/51 − −
GG 20/19 1.06; 0.53--2.13 0.858
*C20068T*
CT 4/1 4.12; 0.45--37.37 0.208
TT 0/0 − −
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction {#Sec1}
============
The Netherlands is known for its varied labour market pattern among women, although, Dutch women, especially mothers, typically work part-time (OECD [@CR160]). In 2010, 32.4 % of Dutch mothers were not in paid work, 42.5 % worked between 12 and 24 h a week, 13.8 % worked 25--35 h a week, and 11.3 % worked 36 h or more (CBS [@CR110]). This variation makes the Netherlands an interesting case to study the origins of women's varied employment choices. To understand this variation most contemporary researchers recognize the importance of incorporating gender role attitudes (Cunningham et al. [@CR17]; Hakim [@CR30], [@CR31]; Hoffnung [@CR35]). Especially differences in *personal* gender role attitudes contribute to the explanation of why women employ different employment patterns (Cloïn [@CR14]; Marks and Houston [@CR47]; Risman et al. [@CR56]). Personal gender role attitudes refer to the ideal of how people prefer to share work and kin care tasks with their own partner. The main contribution of this study is to achieve a better understanding of differences in the social origins of mothers' personal gender role attitudes and to explore possible continuities and interruptions of these attitudes by diverse social influences, and hence get a better understanding of their diverse labour market behaviour. The study's particular interest is to explore the character of transitions from primary to secondary socialization. Is this process characterized by reproduction and re-enforcement, or by recreation and flexibility?
Labour market activity is in this study understood as an outcome of a process that consists of consecutive decision-making moments, like dependent paths: each successive step depends on previously taken decisions (Vespa [@CR67]). Attending school, whether as a high achiever or not, taking erroneously or correctly chosen continuation courses, and then the ensuing steps taking into professional work, are all not facts that can easily adjusted or reversed. Besides being based on personal characteristics, life experiences and external structural factors, like the availability of financial support, studies and jobs, these important steps forward are all partly based on peoples' attitudes. People have developed their attitudes through the exposure to and internalization of significant others' behaviour, norms, values and attitudes, during childhood and later in life (Bandura [@CR2]; Berger and Luckmann [@CR5]; Handel [@CR32]). This interpretation of personal attitudes corresponds with the socialization or exposure-based approach (Blunsdon and Reed [@CR9]; Bolzendahl and Meyers [@CR10]) which assumes that attitudes develop by experiences in childhood, during the school period (young adulthood) and in early work experiences, and are relatively, but not completely, resistant to change after that time. This study focuses on the part of mother's attitudes that possibly lingers.
The investigation of the origins and continuity of Dutch mother's attitudes is employed with a qualitative in-depth research of 39 interviews (conducted in 2010) with mothers living in the vicinity of Amsterdam. Some considerations of the qualitative analysis are in place, and the study must be characterised as an explorative investigation. Mothers were interviewed in their adult-life, and were asked retrospectively to look back at their parental background when they were 12 years old, and to their supportive relationships in the past. This way of questioning recognises a risk of the selectiveness of memory, and the fact that mothers' current situations and attitudes may have influenced their answers. It is possible that people may adjust their memories to justify their present behaviour (Kroska and Elman [@CR45]). A longitudinal qualitative study, at different points in the course of one's life, allows this risk to be avoided.
Moreover, this study is certainly not aimed at denying the interplay between cause and effect of attitudes and behaviour. Various studies have convincingly shown that women are able to shift their attitudes through work and life experiences (Cunningham et al. [@CR17]; Kan [@CR41]; Steiber and Haas [@CR61]). Jansen and Kalmijn ([@CR39]) for example showed that the relationship between labour market activity and attitudes is reciprocal. The effect of emancipatory attitudes on employment is as strong as the effect of employment on these attitudes. The focus of this study is to possibly reveal the presence of a relatively perpetual part within their attitudes. In other words: which attitudes influenced mother's behaviour while she entered the labour market for the first time, and still shape their new social relational contexts and behaviour?
Background of The Netherlands {#Sec2}
=============================
For a long time, Dutch female labour participation, especially among married women, was very low. In 1960, 25 % of women (Tijdens [@CR63]), and 7 % of married women were employed, compared to 30 % of English and 33 % of French married women (Kloek [@CR42]). Since the 1960s, as in many other Western countries, Dutch society changed radically through processes of secularization, increased educational attainment of women, and the greater acceptance of non-familial roles for women and familial roles for men (Sullivan [@CR62]). This rise continued throughout the following decades, resulting in one of the highest levels of female participation among the Western countries, at almost 70 % in 2009 (OECD [@CR160]). However, most of the rise of female participation was in part-time work.
Specific national characteristics can explain the heterogeneous employment---yet dominantly part-time---pattern of Dutch women (Plantenga [@CR52]; Tijdens [@CR63]; Van Doorne-Huiskes and Schippers [@CR64]). In the 1970s and 1980s, when female labour participation started to rise, there were few public childcare facilities, which forced mothers who entered the labour market to combine their paid job with care tasks at home (Plantenga [@CR51]). In the same period, employers wanted to increase the flexibility of their workforce, for which the use of part-time work was one solution. Although at first the trade unions were reluctant to support part-time work, over the course of the 1980s they became more positive, negotiating clauses on the equal treatment of part-time workers in collective labour agreements (Kremer [@CR44]; Visser and Hemerijck [@CR68]). Many branches started to offer family--friendly arrangements which benefitted part-time worker for men and women (Tijdens [@CR63]). The Working Hours Adjustment Act of 2000 served to provide an employee with the right to request a reduction in the number of work hours, which could not be dismissed by the employer unless they could prove that such a move would be detrimental to the company's interests (Plantenga [@CR52]; Van Doorne-Huiskes and Schippers [@CR64]).
At the same time, Dutch institutional care arrangements, such as primary school timetables, and the quality, cost and availability of formal childcare, did/do not facilitate full-time work for mothers (Plantenga [@CR52]). And although there is political and societal support for the dual carer/worker model---50 % of Dutch parents endorse the egalitarian ideal of parental sharing (Merens et al. [@CR49])---contradictory social norms are also apparent. Compared to other European countries, relatively few Dutch couples agree that both partners should contribute to the household income (Haas et al. [@CR130]). Moreover, 63 % of Dutch people consider a working week of 2 days or less to be the ideal for mothers with children up to the age of four, and only 10 % endorse the ideal of working 4 or 5 days per week (Merens et al. [@CR49], p. 130).
Contrary to the era of rapid social change between 1970 and 1980, the last two decades of the Netherlands are characterized by a relatively stable cultural climate as it concerns to the ideas and daily practices of gender roles (Cloïn [@CR15]; Portegijs [@CR53]). The share of Dutch husbands partaking in household tasks and care has shown little progress since 1995 (Bucx [@CR100]), and part-time work remains over the years an accommodating option for most Dutch mothers (Ruitenberg [@CR170]).
Theoretical Framework {#Sec3}
=====================
Primary Socialization {#Sec4}
---------------------
Socialization theory focuses on the social relational context in which specific normative standards and expectations are socially transmitted. Childhood is viewed as the most important formative period in life, in which the basic structure of the individual's social world (base-world) is built, with which it will compare all later situations (Berger and Luckmann [@CR5]; Mannheim (1959) in Everingham et al. [@CR23]). Berger and Luckmann ([@CR5]) explained how primary socialization continues to influence people's life and how it takes biographical shocks to disintegrate the individual perspective of social reality which is internalized in early childhood (Berger and Luckmann [@CR5], p. 154). They argue that during the process of primary socialization there is no problem of identification, because there are no significant others apart from the parents. It is the parents (or caretakers) who set the rules of the game."The child can play the game with enthusiasm or with sullen resistance. But, alas, there is no other game around. \[...\] He internalizes it as the world, the only existent and only conceivable world, the world tout court\[...\]By comparison with it, all later realities are 'artificial' (Berger and Luckmann [@CR5], p. 154)."According to Bandura ([@CR2]) it is important to understand that much 'appropriate' behaviour is learned observationally and symbolically, since often it cannot be readily established by overt enactment, since children may face social prohibitions, lack of opportunity or physical ability. Therefore, most modelled behaviour is acquired and retained through the medium of either imaginable (in the form of visual imagery or mental associations with for example the person who was modelling the behaviour) or verbal coding, referred to as mental and verbal symbols (Bandura [@CR2], p. 33). After modelled activities have been transformed into images, becoming easily assessable and functioning mental and verbal symbols, these memory codes will serve as guides for performance, arising automatically and outside of the subject's own awareness (Bandura [@CR2]). Possible mental and verbal symbols associated with attitudes towards paid work that parents have intentionally or unintentionally 'taught' their children include, for example, money, joy, obligation, status, independence, social life, creativity, boredom, fatigue or paternity.
Not many studies involve the impact of childhood socialization on the impact of attitudes of adults. Some studies note that the saliency of family of origin effects on ideology constructions might diminish during adolescence as the influence of adolescents' peers and their own life experiences becomes stronger (Davis [@CR18]). Other studies emphasize that intrafamilial continuity is likely to become more apparent as the younger generation moves into full adult status, which includes major life transitions such as marriage, parenthood and employment (Inman-Amos et al. [@CR37], p. 460; Biddle et al. [@CR7], p. 1072).
Empirical studies have shown evidence for both bearings. Children of parents with 'modern' values appear toe have a more egalitarian perspective on work and family roles than children of parents with more traditional values (De Valk [@CR20]; Van Wel and Knijn [@CR66]). Glass et al. ([@CR27]) demonstrated that parental gender ideology, such as "Womens' lib makes sense" and "Women should have the main say in marriage" continue to significantly predict children's orientations after childhood. Cunningham's ([@CR16]) analysis found that children's ideal division of labour at home is predicted by maternal gender role attitudes. Moen et al. ([@CR50]) concluded that socialization processes for women operate mainly through verbal persuasion from their mothers, rather than through role modelling (p. 291). A recent quantitative Dutch study has showed that a mother endorses a more egalitarian ideal family life if she recalls the gender role specific parental message 'you should work in order to be financially independent of mothers', and a positively work orientated mother at the age of 12 years (Ruitenberg and de Beer [@CR180], Ruitenberg [@CR190]). The paternal influence should not be underestimated either. A study of Davis and Wills ([@CR19]), showed that it is fathers who are particularly influential in creating the family context in which gender attitudes are transmitted from one generation to the next. As they construct the context, while being more egalitarian (direct influence) or being traditional (moderating influence via the mother), of the socialization process.
Nonetheless, Gerson ([@CR26]) argues, based on her qualitative study, that early childhood experiences are underdetermining: generally they have a weak relationship with children's later life choices or orientations. The interviewees of her study dealt with the mixed messages of their childhoods in a variety of ways; ambivalence, confusion, and the postponement of final decisions were among the more common responses (Gerson [@CR26], p. 66). Starrels ([@CR60]) demonstrates with a longitudinal study that although children's values and opinions are shaped by their mothers, he/she was unable to identify the mechanisms how it occurs. Factors such as the effect of the time spent together or feelings of trust, understanding and respect (associational and affective solidarity) did not increase consensus (p. 101).
Clearly, the time context of last mentioned studies is different to that of the mothers addressed in this study. Over the last half of the twentieth century social research documented wide spread changes in attitudes about the appropriate social roles for men and women (Sullivan [@CR62]), uniformly toward more gender egalitarian attitudes (Axinn et al. [@CR1]). The parents (especially the mothers) of the respondents of this study to a certain extent already paved the way for their daughters, while opening alternative scripts of behaviour to the traditional stay-at-home option once their daughter became a mother herself. This clearly differed from the parent's, especially their mothers, own upbringings during the 1950s and 1960s, which were still deeply implicated within the ideal of women as housewives and men as breadwinners (Kloek [@CR42]). For the mothers of the respondents who themselves were raised during this period, the withering away of the ideal of being a housewife had not yet led to new ideals or guidance for behaviour. And thus their trials of new behaviour would have been more easily and more often acting against their parents' norms, values and attitudes, such as was found by Gerson ([@CR26]). It is interesting to examine whether socialization processes have a distinct character in the current more stable cultural time period of the Netherlands. Even more because the baselines of the socialization process, the nature of the relationship and the division of labour among the respondents' parents, have been certainly more diverse---with an increasing group of mothers being employed and fathers taking up family roles in various ways---compared to those in 1950s and 1960s.
The first research question of this qualitative study is to examine whether the diversity of Dutch mothers' gender role attitudes can be understood by the internalisation of different parental mental and/or verbal symbols.
Secondary Socialization {#Sec5}
-----------------------
As a person lives on, one must learn to function in any new group or organisation (sub-world) that she or he enters. People learn not only new practices, but also new values and norms, new vocabulary, and new ways of interacting with others. The formal processes of secondary socialization persist with an essential problem: they are always determined by an earlier process of primary socialization. As such, they must deal with a pre-formed self and an already internalized world (Handel [@CR32]; Wallace and Wolf [@CR70], p. 290). Compared to the internalized base-world, there will be differences and disagreements about values, norms, vocabulary and ways of interacting within and among the different sub-worlds. At many levels, contradictions between and within the disparate settings exist (Bandura [@CR2], p. 44; Handel [@CR32], p. 17). Berger and Luckmanns' principle assumption is that the individual likes their identity being confirmed, and significant others are vital for this ongoing substantiation of their identity (Berger and Luckmann [@CR5], p. 170). Other scholars rather argued that throughout their lives people establish an acceptable position for themselves out of all these contradictions (Eagle [@CR120]; Gerson [@CR26]; Handel [@CR32]). "Women's orientations toward decisions about, and capacities for working and parenting are thus emergent, developmental and subject to change over time (Gerson [@CR26], p. 199)."Some recent studies have explored how life course events and changes in women's labor market participation are associated with gender role attitude change. They find increases in educational attainment and full-time employment result in more egalitarian attitudes, while marriage, parenthood and reductions in paid work are associated with more traditional attitudes (Cunningham et al. [@CR17]; Himmelweit and Sigala [@CR33]; Kan [@CR41]; Vespa [@CR67]). Berrington et al. ([@CR6]) suggest that it is not entry into parenthood as such, but the change in women's economic activity as a consequence of parenthood, which is associated with attitude change. Himmelweit and Sigala ([@CR33]) show that changes in attitudes or behavior are more likely when mothers' labour market status is inconsistent with their attitudes towards women's employment. Some qualitative studies also provide evidence of a complex interplay of economic and identity-driven motivations for mothers' labor market participation (Himmelweit and Sigala [@CR33]).
Berger and Luckmann ([@CR5]) already acknowledged that partial transformations of identity are common, especially in relation to individuals' social mobility and occupational training (Berger and Luckmann [@CR5], p. 181). Nonetheless, they also emphasised that even within partial transformations there is a continuing association with symbols, persons and groups who were previously significant. Due to the fact that prior associations continue to linger in people's minds (often also physically in their lives), they are likely to protest at overly fanciful re-interpretations of people's new identities. (Berger and Luckmann [@CR5], p. 181).
The second question of this study concerns whether mothers tend to sustain their (acquired) attitudes through secondary social relations, by creating and recreating the familiar, or whether they reset their attitudes if confronted with different situations including possibilities or constraints, and new models of behaviour and attitudes. Within the limited context of this paper, I only explore the agreement or disagreement with a mother's gender role attitudes and those of her partner (or ex-partner) and peers.
### Partner {#Sec6}
Previous research has shown that gender-role attitudes between partners are often similar, based on homogamy in mate selections: people seek marital partners with similar (gender) values and attitudes (Inman-Amos et al. [@CR37]). Regarding employment decisions of mothers, Vlasblom and Schippers ([@CR69]) showed that these are made within the family, and that the views of both partners concerning the division of care play an important role (also Geist [@CR25]). Hoffnung ([@CR35]) revealed with her longitudinal study (1994--2009) among approximately 200 women living in the US, that career-oriented, 'have it all' women often have found partners who supported their full-time work. Their 'husbands' appeared more family-oriented and less career-oriented than the partners of traditional women, or they had a lower educational level and thus earning capacity (Hoffnung and Williams [@CR140]; also Geist [@CR25]).
According to the psychological theory of interdependence (Kelley and Thibaut [@CR200]), partners will sometimes modify attitudes or behaviours to bring them in line with their spouses' preferences, rather than their own (in Hochschild and Machung [@CR34]). Empirical studies among American and Dutch couples provide contradictory evidence for which partner's attitudes are more influential than the wife's or the husband's original attitudes (Johnson and Huston [@CR150]; Kalmijn [@CR40]). Kalmijn showed that especially women's prenatal gender role attitudes have an indirect effect on men's attitudes through her change in employment after the birth of their child (Kalmijn [@CR40]). Decisions about how to combine working and caring are thus still seen as women's business. In addition, Schober ([@CR58]) found that that higher absolute wages and more egalitarian attitudes of women before motherhood reduce the shift towards a more traditional division of labour after couples have their first child. Putting it differently, women's prenatal attitudes linger not only in her own postnatal attitudes but of also in those of her partner. However, the relationship depends on the socio-cultural context (Schober [@CR58]). The conclusion that women's egalitarianism reduces the change towards a more traditional division of labor after the birth of a child is consistent with other recent research employed in Australia and Britain. Nonetheless, earlier American studies found smaller effects and suggest that fathers' attitudes are more important. "These variations may suggest that more generous family--friendly provisions for mothers in the United Kingdom compared to the United States provide women with more choice to follow their attitudes or historically embedded gender norms, which maintain that mother care is best for young children (Schober [@CR58])."Continuing on previous empirical research, this research explores how the dynamics between the gender role attitudes of mother's with her (ex) partner resemble the parental origins of a mother's own gender role attitudes?
### Peers {#Sec7}
Socialization among peers is conceptualised by Ryan ([@CR57]) as a process that occurs through frequent interactions, shared experiences and exchanged information among a relatively intimate group of friends who interact with each other on a regular basis (Ryan [@CR57], p. 1138). Much relevant research has been done on adolescents peer groups (e.g. Biddle et al. [@CR7]; Grusec and Hastings [@CR29]), and it is widely recognised that adolescent peer relationships or peer group pressures have consequences for emotional adjustment, school achievement, and risk-taking behaviours (Biddle et al. [@CR7]; Windle [@CR72]). Concerning employment behaviour among adults, several studies have shown that labour market behaviour can be modified by the behavioural example of other people in the environment, and that other people can act as role models (Sealy and Singh [@CR59]). Some social-psychological studies have shown that someone's labour market behaviour can be modified by the behaviour of important people in his or her environment. Woittiez and Kapteyn ([@CR73]), for example, demonstrated the existence of a so called 'bandwagon effect' among Dutch women in a social group. This effect refers to the fact that if a member of a social group enters the labour market, his or her entrance motivates other members of the same social group to join the labour market as well. This theory corresponds with sociological notions of role models (Bandura [@CR2]). A role model can be an inspiring and motivating person, someone from whom one can learn, providing a script for behaviour in particular contexts (Sealy and Singh [@CR59]). Portegijs et al. ([@CR54]) has found a significant impact on the participation level of mothers if other mothers in their environment work or make use of formal childcare. Also Blaffer Hrdy ([@CR8]) concluded that the expectations that mothers have of their own lives are based on their own ideas and on the ideas of how it should be by others.
However, one should realise that in general people choose friends with similar ideas, attitudes, interests or characteristics as themselves. Or, as Brown et al. ([@CR12]) argue, people do not haphazardly fall into one crowd or another; similarities are prevalent a priori to relationships. The continuum of mother's own gender role attitudes and those of her friends is of particular interest within this paper.
Research Method {#Sec8}
===============
In order to answer the research questions, semi-structured face-to-face interviews have been conducted with 39 mothers, all of whom having at least one child younger than 12 years old living at home, and all living in the vicinity of Amsterdam, The Netherlands. The age at which most parents deem their children old enough to be left on their own is 11 (Duncan [@CR22]). The interviews took place between April 2010 and November 2010. The interviews took on average one and a half hours to complete, and full transcripts of the interviews were made. In order to select the interviewees, four categories of mothers were differentiated according to their employment patterns: mothers who work 0 h (referred to as stay-at-home mothers or full-time homemakers), 12--24 h a week (mothers with a small part-time job), 25--35 h (mothers with a large part-time job) and 36 h or more (full-time working mothers).
As is well known, higher educational levels lead to higher levels of labour participation, especially among mothers (Merens et al. [@CR49]). Sufficient education is then understood as a precondition for labour market participation. For example, higher educated women work more, because their higher wage allows them to pay for child-care facilities. They may also have been exposed more to critical ideas and formed career-oriented networks (Cunningham et al. [@CR17]; Doorewaard et al. [@CR21], p. 11). In 2009, 37 % of Dutch higher-educated mothers worked more than 35 h per week, compared to only 18 % of lower-educated mothers. 52 % of lower-educated mothers did not participate in the labour market at all, as compared to 12 % of higher-educated mothers (Central Bureau of Statistics 2011). In each of the four employment categories, there were approximately equal numbers of lower- (intermediate vocational level and lower) and higher-educated mothers (higher vocational level and university).
For theoretical reasons, the sample of interviewed mothers was drawn largely within one urban area, Amsterdam. In this way, differences in employment behaviour and attitudes among respondents do not differ with respect to the influence of structural and cultural factors that may diverge between urban and rural areas, such as the availability of childcare provisions, jobs and religiousness, which could also affect potential differences in gender and work attitudes.
In order to achieve good correspondence between research questions and sampling, a strategy of purposeful sampling had to be followed (Bryman [@CR13], pp. 458--459). To be able to fill all eight categories (four along employment patterns and two along educational levels) of mothers within one area equally, the respondents were found using the snowball method. Firstly, a small group of mothers in the social environment of the researcher, the so-called weak ties (Granovetter [@CR28]), was approached, especially at a primary school in Amsterdam (Old West Quarter). Subsequently, the other respondents were approached on the advice of the first group of respondents. The collection of material ended when theoretical saturation was reached, and new interviewees did not bring more diversity. Quite clearly this research method cannot produce a statistically representative sample, since it relies upon the social contacts between individuals to trace additional contacts. The research method, however, does permit revealing the reciprocal character between primary and secondary socialization, and is able to highlight what mothers consider as being relevant when describing their childhood and further social relational contexts.
Research Group {#Sec9}
--------------
The interviewed mothers were born between 1962 and 1980. Their average age was 39.3 years. Seven interviewees (18 %) had a non-Dutch background (at least one parent born outside the Netherlands). Within the research group, 23 mothers were highly educated (higher vocational education and university), and 16 lower educated (intermediate vocational education and lower). Ten mothers were full-time homemakers, 8 mothers had a small part-time job (12--24 h), 11 mothers had a large part-time job (25--35 h) and 10 mothers worked full-time. Appendix [1](#Sec17){ref-type="sec"} provides an overview of the backgrounds of the respondents. All names in this paper are anonymised.
Interview Questions {#Sec10}
-------------------
The interviews can be characterised as oral life history interviews (Bryman [@CR13]). The interviewees were invited to look back at specific moments in their lives, especially during childhood, while also concentrating on the behavioural steps of later social life, from finishing high school, choosing a continuation course, entering their first job and giving birth to their first child. Several open questions were asked in order to discover how and with which words women refer to these themes themselves.
*Personal gender role attitudes* refer to a mother's desired division of labour with her own spouse. This personal ideal was more closely examined by mothers' satisfaction with her current division of labour. A traditional personal gender attitude means a desire to have the main responsibility at home, while her partner is in paid work. An egalitarian personal gender attitude implies a wish to share paid and unpaid work equally. Adaptive attitudes are here defined as the personal desires to combine paid work and family tasks, with consent to the idea that mothers have more responsibilities at home and fathers may work full-time. (Do you have ideas about the ideal division of labour with your spouse? Are you satisfied with your own current division of labour? What would you like to change?). Also some questions were asked about her general ideas about the ideal division of labour between men and women. Questions towards a mother's general gender attitude were for example: How do you perceive differences between men and women? Do you have an opinion about full-time working mothers or mothers who are not employed?
*Work attitudes* are defined as mother's personal motivations to pursue paid work. (What are the most important reasons for you to work? Did you have ideas about your future profession at a young age?). A strong personal work attitude means that someone was already as a young adult strongly oriented to pursue paid work and is intrinsically motivated to work.
Following this, the mothers were asked several questions about their family backgrounds, looking back to when they were 12 years old. In particular, this included questions addressed the gender division of labour of their parents (Did your parents work? Did your father help with household chores and childcare? Were your parents happy about their division of tasks?), parental attitudes (What were the important norms, values, and (implicit and explicit) messages that were transmitted by your parents?). In addition, several questions were asked on upbringing matters, like whether the parents were strict or encouraged their daughters to fulfil their full potential at school or at work.
Finally, several questions were asked about how mothers perceived their attitudes and behaviours to have been influenced by their partners and close friends in fulfilling their full professional potential.
Interview Analysis {#Sec11}
------------------
The research was specifically sensitive to perceiving the lives of the respondents in terms of continuity and process, especially referring to the theoretically assumed continuity throughout the course of life between primary and secondary socialization processes. Therefore, the interview transcripts of each respondent were not cut into different codes, but kept as close as possible to each story told by the respondents.
The main part of the analysis consisted of searching for sensitizing concepts that could be used as pegs to describe the central narratives of, and the similarities within, the different groups. The sensitizing concepts were: acceptance towards the marital division of labour of her own mother, the nature of memories of her mother (or the mother-figure), message importance of economic independence, parental support towards their daughter's professional life, presence or absence of stimulating partner or friends towards mother's professional life. Following this, the transcripts were reread while focusing on these sensitizing concepts, and memos were written throughout the process. Below, the findings are described along the three research questions, while attention is given to the sensitizing concepts. The narratives of mothers' attitudes could be patterned along two groups: traditional-adaptive attitudes versus egalitarian attitudes. The differences between these groups are specifically addressed.
Findings {#Sec12}
========
How Can the Diversity of Dutch Mothers' Gender Role and Work Attitudes be Understood by the Internalisation of Different Parental Mental and/or Verbal Symbols? {#Sec13}
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In this study, the interviewed mothers with more traditional attitudes had a few common characteristics, although there were also often exceptions, which are addressed if relevant. Firstly the relatively traditional mothers have no job, or else, work small part-time jobs. They tend to perceive it as their natural role to execute most of the unpaid family tasks, and do not put much value on their economic independence. They also appear mostly satisfied about the division of the work at home with their partners, which is often around 80 % of the tasks for the mothers and 20 % for the fathers. Contrary, mothers with more egalitarian gender attitudes, who often have large part-time or full-time jobs, cannot imagine not working themselves; moreover they consider it as unwise, citing the necessity of economic independence. They also expect a more equal share from their husbands in the unpaid tasks at home, which full-time working mothers have greater success in achieving.
Mothers with traditional or adaptive gender attitudes often emphasise that they come from warm families, and often have many happy memories from youth. Consistently, the relationship with their parents is still close. On the question of who is a shoulder to lean on when they must make a difficult decision, traditional or adaptive mothers often recall their parents. The parental division of work used to be traditional, not because their mothers did not work, but especially in the sense that their mothers unquestioningly did most of the unpaid family work. The presence of a caring mother, who carried out her unpaid duties without complaint, is recollected as a natural and self-evident situation by their traditional or adaptive adult daughters. Often, the interviewees have no clear memories of their mother during primary school. Yet, mostly they presume that their mothers liked their role of child carer and housewife, although they admit they never really asked their mother: "I actually don't know. I don't have the impression that she missed anything." Respondents recall that their fathers pursued full-time jobs, and often also at home were also the boss."My mother did everything. I found that normal. I cannot remember her complaining about it, or that she found it too much work or too busy. I think she enjoyed it, I have never asked her, to be honest. My father was just an authority (Astrid)."Almost half of the mothers of relatively traditional daughters worked, mostly part-time, often to assist their husbands in their store or family company, which not always was their own 'choice' but rather occurred from necessity. Often these mothers worked during school hours, and were at home when their children came back from school."My mother was always there after school. She worked in the mornings. I never questioned what my mother did, when I was at school. She was always there, yet I know she had a job (Esther).When we were older my mother had two jobs, as a general practitioner. Yet, she did most tasks at home, she was always there, she was the one who cared for us, which was so normal (Duke)."Sometimes these mothers' return to work meant an essential change of family life, which was not always liked by their daughters. Especially if their mothers became too occupied with their jobs, their daughters could come to feel neglected. As a consequence, two daughters tend to show the opposite behaviour of their mothers, now they are mothers themselves. This is illustrated by Nora who temporarily gave up her job as a judge to care for her four children:"I thought it's constantly about you and it's constantly about your job. And for her, it was a huge part of her confidence; she got a lot of self-esteem from her work. I found that really stupid... I wouldn't say I come from a warm nest, my mother was too preoccupied with her own business."In addition a remarried stay-at-home mother, Mireille, who knows she can stand on her own legs, which she did the period after her divorce, is now happy she can afford to stay at home because her husband earnings suffice. She remembers how she pitied her mother, "after I was sixteen, I went always straight home from school, because I found it sad for my mother, because I knew she was not happy." Nonetheless, before puberty she really liked it that her mother was there for her after school, and therefore she wants to be there for her own children when they are still young.
The parental work ethic received by daughters with now traditional or adaptive gender attitudes was: follow a good education to be able to contribute to society. The narratives of stay-at-home mothers in particular reveal that after they finished high school, their parents were not particularly helpful in assisting their daughter's choice of continuation course or profession: "They never asked me, 'what do you want to be, what is important for you?'".
The family backgrounds of the interviewed mothers with *egalitarian* attitudes appear more diverse. Remarkably, the interviewees were often raised in non-standard families. Roughly one-third of the mothers with egalitarian attitudes were solely raised by their mothers, as a result of divorce or through alcoholism, disability or death of the father. Sophie, a mother of 48 years old with one child, who works 32 h as a copywriter, describes her youth with her alcoholic father."He read nothing, didn't have one friend, no contact with the neighbors, or anything. When we came home, he just sat there, sloshed in his chair, and every day a lot of fuss, you know, shouting in the house."Yvette, a 42 years old carpenter with two children who grew up with 6 brothers and sisters and a single mom, describes her childhood after her father had deceased when she was 8 years old:"It was just natural that everyone did something. You saw that mom did everything and that was not right, so we helped. We got the groceries; my mother was not a very domestic mother. So we actually grew up like this: we had to take care of ourselves and of mom."Other stories of egalitarian mothers reveal that their parents did not give them much attention when they were young. One daughter went to a boarding school in England, another daughter experienced traumatic family happenings at a young age, which preoccupied her parents, and there were parents who always fought. Additionally, the mothers describe upbringing matters that made them independent women, sometimes reluctantly. For example, some mothers were only 11 or 12 years old when they were made responsible for taking care of their parents' shop while they were away on vacation, or for baby-sitting much younger brothers and sisters. One respondent was 'pushed' onto the train to Amsterdam alone (while living in Groningen, 220 km away) so that she could go and purchase her desired rucksack.
Also recognized from the narratives is the role of respondents' own mothers, who generally were not described as self-evident and consenting mother-figures, as is the picture that emerges in the chronicles of traditional/adaptive mothers. Regularly, the respondents asserted that if their mothers had lived in the present, they certainly would have worked, or would have had a different job. The daughters often describe their mothers as being clever, assertive, full of initiative and reluctant to fulfil the mother role. Two egalitarian mothers saw their mothers as anti-examples as well, in the sense that their mothers behaved as victims of their era and complained about not having had the chance to do the profession they would have liked. It seems that such mothers' reluctant attitudes towards the traditional mother role, and subsequent feelings of regret, has stimulated their daughters to fulfil their own work potential.
Another pattern among mothers with egalitarian attitudes is that they have explicitly or implicitly received the message (verbal symbol): "Make sure you can stand on your own two feet", or "You must not rely on a man". As mentioned before, it was not always necessary to spell out the message, but obvious because their mothers were sole providers."Particularly my mother used to encourage me a lot, and I feel it is nice to have a lot of encouragement. Yet, maybe my mother encouraged me a little bit too much (Michelle).Straight after finishing high school, I went to university, and that was really because my mother was pushing me, like 'you should not spill a year, then you will never start studying. Later, I have regretted the fact that I went along with her, because it was not my own feeling' (Olga)."Additionally, some mothers with egalitarian attitudes describe how they received contradictory messages, like was found in Gerson's study (1995). They were stimulated to finish school and pursue careers, yet, they were also expected to marry at the age of 24 or have children and work 3 days and, preferably to live nearby their parents. Maybe, when mothers have received conflicting parental messages, mother's own gender role attitudes are more receptive for the attitudes of other people, such as those of her partner?
How Do the Gender Role Attitudes of Mother's (Ex) Partner Resemble a Mother's 'Own' Gender Role Attitudes? {#Sec14}
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Firstly, the findings indicate, as is previously found, that generally there is agreement among the partners about their marital division of labour. The narratives of mothers with rather traditional/adaptive gender attitudes reveal that the number of hours they work and kin care responsibilities are not key subjects of their marital discussion, but rather something often taken-for granted. For example, a mother's decision about how much she works, and subsequently how the children are taken care of, appears to be mainly her own decision, with partners tending to 'leave it' up to their wives. This is illustrated by Leontien, a 42-year-old a highly educated stay-at-home mother with four children:"And what did your husband think about the fact that you wanted to give up your job?*I think for him it was easy.*He was quite happy with it?*Ithink hethoughtitwas comfortable, yet, healways left itto me.*Did you talk about it together?*Yes*, *Ithink so*.Did it feel like a mutual decision or like your decision?*It was my decision, but shared or agreed by him. He said: If that is what you want, it*'*s fine. If I had said: I want to bring our son three days to the crèche, then he would also have said: that*'*s fine.*"Stay at-home mothers describe their partners quite often as egalitarian husbands. "He always said, and I believe him: 'You must do what you want. If you want to work then we can arrange an au pair or bring the children to the day care. If you don't want to work, it is fine as well' (Nora)."
This marital decision-making process vis-à-vis mothers' employment activity expresses two rather opposite ideologies. On the one hand, it expresses the modern view that work is something personal to decide upon, but on the other hand, it might also reveal a rather traditional attitude that work for mothers is not a self-evident matter, and that not working is a viable option. Whatever attitude prevails, the partner's apparent tolerant attitude leads to the situation that how children are taken care of mostly depends on their mother's decisions in relation to work.
Nevertheless, the stories of stay-at-home mothers disclose that their decisions to give up work was not always such a pre-planned or positive choice for motherhood, but often the result of a sequence of unfavourable happenings. In this light, partners' tolerant or phlegmatic attitudes allow a mother to slip into a non-working situation that does not necessarily make her life easier or happier. In addition, there are examples of husbands or partners who did not comply with earlier plans that they would work less. But this has not led to an apparent conflict between the partners, but rather mothers dealt with and adapted to the situation. Moreover, some interviewees emphasise that if their partners clearly and regularly show their appreciation of the way they execute their household tasks and kin care responsibility, they are fully content to perform their 'duties'. "We have divided the tasks fine, he is the full-time worker. He leaves home in the morning between half past seven and a quarter to eight, and 11 h later he returns. But Walter doesn't hit the sofa, as soon as he returns, he keeps working \[...\] Walter will put the garbage out and Walter manages all the business stuff \[...\] I do everything with love, although, I need to hear from him, 'You did that really well,' or 'Hey, that's done---that's wonderful!' (Nel)."The help from their husbands or partners is not taken-for-granted by mothers with more traditional attitudes, but is appreciated largely. The unpaid tasks are divided along recognisable gender lines, but the inequalities that come with that are unquestioned."I think I do more, it just doesn't feel like he's is not the type that hits the sofa. It's more a consequence of the fact that I am home more than he is (Carien)."Egalitarian mothers, especially those in full-time work, seem to have 'found' partners who are more encouraging towards their wife's work ambitions. Moreover, often they are proud of their wife's career and would not appreciate if she wasn't working. These partners take up a share of the unpaid, domestic work more automatically and without much resistance, which is illustrated by the following quote."I talk a lot with my husband about work and about my aspirations. And he encourages me, for example with my idea to go back to university \[...\] He is also someone who always says, 'hey, if you need to work longer, then I'll take the kids home today' (Annemiek)."Most mothers within egalitarian attitudes seem to practise their ideal, especially if their husbands work 4 days per week as well. These mothers realise their rather exceptional gender division of labour, and describe their partners as unmacho men, as gentlemen, or as caring fathers. The greatest contrast with the more traditional women is that for mothers in this group, it is certainly not a self-evident matter to take the lion's share of the household work: "Preferably, I do nothing in the household" (Cathy). The mothers often emphasise that they would never be able to work that many hours without their partner. In some cases, their husbands perform the majority of the unpaid tasks. If gender roles are reversed, these are emphasised and remarked upon.
How Do the Gender Role Attitudes of a Mother's Peers Resemble Her Own Gender Role Attitudes? {#Sec15}
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In the stories of the interviewees, it emerges that the resemblance of close friends on the gender role attitudes of mothers seems less than expected based on research among adolescents (Ryan [@CR57]). Mothers even hesitate to describe the gender role attitudes of their best friends. Sometimes, this was because they were reluctant to speak for other people, but other times this was because they simply did not know. This is revealed through the following quote from Sheila:"I suspect that sometimes they think 'gee, why does she work so much?' I think that, yes (Sheila)."A reason for this lack of knowledge is that most women identify their best friends as the ones they have known since high school or later educational years. Since their youth, mothers and their close friends have experienced a great deal together, such as graduations, weddings, funerals, and the birth of their children. At present, these old and dearest friends often do not pursue the same lifestyles as the mothers themselves, for example because these friends are without partners or children. In addition, most mothers confess that they do not see their best friends much, due to the time commitments of work and children, and sometimes because their friends live in other areas of the country or abroad. Often mothers have also made new friends, for example those they have met at their child's school, or else at work. Although mothers are careful when describing these new friends as 'close', these new friends tend to have more similarities with their own current lifestyles, and 'everyday' contact makes it easier to discuss more everyday subjects, such as household quarrels and grievances.
The social pressure of other people in their social environment, other mothers in particular, is also discussed by the respondents. It appears that if mothers behave in line with the current Dutch norm, which implies a working week of about 3 days (Portegijs et al. [@CR54]), then mothers' narratives reveal how much their life style is socially accepted, as is illustrated by Nel psychologist, working 2 days and mom of a 2 year-old:"My sister in law says that I have it perfect, because I still develop myself, I am not completely out of the labour market, and at home I can also be very much present too."But in the case of mothers who do not suit the current Dutch norm---either due to being a stay-at-home mother or working full-time---the social atmosphere is less accepting. This affects in particular mothers who do not work."The general hip and trendy working women, you know, I feel they have their opinion ready. I often want to shout: 'You don't know what it's like, woman, to have twins!' (Janne).""Sometimes it is very difficult because you feel the pressure from outside. People ask me: 'So what do you do all day?' They think I drink coffee the whole day (Mireille)."Mothers with egalitarian gender attitudes seem to be less affected by the weaker social circle:"You know when you work, you are not concerned with the mothers in the schoolyard. What people think of me there has never interested me at all, really. I don't care. No, I even refuse to be sensitive to that (Claire)."Nonetheless, also among egalitarian mothers one can perceive a pattern that they rather live in social environments that match their own norms and attitudes. If norms and values of the neighbourhood differ too much from their own, mothers (and their families) seem impelled to move away, because they did not feel at ease, of which Cathy, a mother who received contradictory parental gender role messages, who lives now with a (in her own words) very 'unmacho' man, working 4 days and mom of an 11-year old son, gives an example:"Yes, when we had our son, we decided to live outside Amsterdam. But there, I felt the worst mother ever. He was the only one from school who went to after-school care. He was picked up in a little van and went to another village. All the kids went home for lunch... lunch! Drama---I felt terrible (Cathy and her partner subsequently decided to move back to Amsterdam)."Hence, mothers do seem sensitive to real and supposed expectations, and to the approval of other people. Nevertheless, it is difficult to perceive whether and to what extent these social influences and subsequent feelings affect mothers' attitudes. It seems that the ability of mothers to "resist" influences in their environment also varies according to the 'power' of their position. For example, working mothers felt able to ignore the judgements of mothers in the playground, or even physically move away from disapproving others. By contrast, stay-at-home mothers felt less able to resist what they perceived to be negative judgements of others. Nonetheless, the narratives do reveal that people do not haphazardly end up in matching social environments, and childhood originated origins of gender role attitudes do have a traceable resilience.
Conclusions {#Sec16}
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Most Western mothers prefer part-time work in order to achieve a work-life balance (Jacob [@CR38]; Fagan [@CR24]; Reynolds [@CR55]). Therefore the availability of relatively 'sophisticated' part-time work in the Netherlands, enabled by Dutch laws, policies and collective agreements at industry level, is mainly perceived as a privilege in the Netherlands as well as by other Western Countries (Wiesmann et al. [@CR71]). Presumably, more than in other countries, Dutch women can choose whether they want to stay at home with their children, to work at a small or large part-time job, or to continue working full-time. For that reason the Netherlands seems a suitable case to explore the social origins of mothers' diverse labour market decisions.
Various empirical Dutch studies have shown that the diversity of female employment in the Netherlands is related to women's diverse gender and work attitudes (Beets et al.[@CR4]; Cloïn [@CR14]; Hooghiemstra [@CR36]; Kraaykamp [@CR43]; Portegijs et al. [@CR54]). This qualitative and explorative study sheds light on the question of whether an explanation for this diversity lies in prior socialization processes.
Firstly, the findings showed how differences in primary socialization are relevant to an understanding of Dutch mothers' current diverse gender role attitudes. Various mechanisms and patterns emerge. It appeared that intergenerational influence mainly occurs via the transmission of mental symbols, both intentionally and unintentionally, and especially diffused by the respondent's own mother, as demonstrated by earlier research (Moen et al. [@CR50]). Children do not automatically mimic parental behaviour; it is a much more subtle affair (Gerson [@CR26]; Mason [@CR48], p. 240; Moen et al. [@CR50]). Parents' implicit messages, attitudes and feelings concerning their own division of labour are picked up by their offspring, such as feelings of unfairness about the actual division of labour or regrets about missed opportunities.
Examples of the transmission of mental codes are the respondents' mothers' own attitudes towards the traditional mother role, like their being consenting or reluctant with this role. The interviewees with traditional/adaptive gender attitudes seemed almost unquestionably familiarized with the silent presence and submissive, receptive performance of unpaid tasks by their own mothers. Now being mothers themselves, they naturally and automatically identify with the traditional 'mother role'. The narratives of mothers with egalitarian attitudes stood out due to their strong memories of their own mother's presence. She could be the dominant sole provider, or else could be unsatisfied with the mother-role. There were also few anti examples where the maternal codes were too penetrating and in cases even irritated their daughters, causing their daughters to go on and develop opposite attitudes.
The findings further illustrate that it is relevant to notice those thing that were absent from the narratives of mothers' childhoods. For example, there may have lacked a specific message relating to their daughter's future profession. When mothers have not been stimulated (either verbally or mentally) to consider their professional life seriously, it seemed difficult to overcome this arrearage later in life. As expected, a clear parental message towards work and financial autonomy appeared more present in the childhood stories of mothers with egalitarian attitudes, and more absent in the recollections of traditional/adaptive mothers, playing a lucid self-determining role later in life. Additionally, some specific and unusual family situations called upon daughters to become responsible at a very young age. The interviewees who now support symmetrical gender roles often seemed in different ways more or less 'forced' to grow up as independent young women very quickly---a childhood characteristic resulting in the appearance that they are well-equipped for the labour market.
With respect to processes of secondary socialization, it seems unlikely that the part of personal gender and work attitudes with origins in childhood may easily adjust to changing circumstances. This study confirms that in general, people seek and marry partners with a similar sex-role ideology as their own (Inman-Amos et al. [@CR37]). Egalitarian mothers often have found 'fitting' partners who are stimulating towards their career ambitions. This is contrary to the conjugal discussions within traditional/adaptive couples, where it appeared not automatically evident that mothers work (also Moen et al. [@CR50], p. 587)."Guided by one's sympathies and antipathies, affections and aversions, tastes and distastes, one makes for oneself an environment in which one feels 'at home'... And we do indeed observe... a striking agreement between the characteristics of agent's dispositions... and those of the objects with which they surround themselves (Bourdieu [@CR11], p. 150)."The impact of peer groups on mothers' gender and work attitudes remained underdetermined. The interviewed mothers generally did not belong to homogenous peer groups. Nonetheless, anonymous people 'out there' seemed, at least in the mind of some interviewees, able to pressurise mothers' feelings, especially by stay-at-home mothers who generally feel undervalued by society for the work they are doing (also Zimmerman [@CR74]). Some stories of egalitarian mothers of which most of the have large jobs, symbolising a more powerful position in society, disclosed that 'traditional' social environments were reasons for them to move to other neighbourhoods, where they were more surrounded by people with similar gender attitudes. This mechanism illustrates that people do not easily adjust their attitudes, but rather find ways to reconcile them.
Recollecting the central question of this study, the findings revealed that Dutch mothers' diverse current gender and work attitudes did not arise from nowhere, but are (at least partly) grounded in childhood experiences. Later situations and social interactions can modify the intensity of these attitudes, yet some important aspects of attitudes that are originated in childhood---transmitted by parental mental and verbal symbols---seem resistant to change, and rather---often automatically and unintentionally---appear to be re-enforced.
Obviously, this study has an explorative character, and more qualitative research on the micro-interactional processes would help to understand and reveal the construction, reconstruction and consolidation of people's social realities. Moreover, there are good reasons to believe that if we want to understand the way people shape their networks and environments in order to match their attitudes, research on the 'enduring' influence of parents on their adult children's attitudes remains relevant. Several researchers of intergenerational ties argue that family-ties between parents and their offspring have not been weakened but strengthened in the past decades. This increased interdependence is explained by increasing longevity, augmented the number of years of shared lives between generations, the drop in fertility rates which has increased the bonds across generations (Van Gaalen and Dykstra [@CR65]). Moreover, under the influence of individualization, family relationships are becoming more like achieved ties. Given these socio-demographic changes, Bengston (2001) predicts a larger significance of intergenerational bonds in the twenty-oneth century in all Western societies (in Van Gaalen and Dykstra [@CR65], p. 947). Consequently, transmissions of attitudes and behaviour across generations remain relevant in studies on labour market behaviour.
Appendix 1: Overview of Respondents {#Sec17}
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NameAgeWork--hoursWork hours partnerNumber of childrenAge of childrenEducation(Former) professionGender attitudeJanne38040+34, 4, 9Higher polytechnicPhysiotherapistTraditional/adaptiveLeontien42040+46, 8, 10, 12UniversityShop employeeTraditional/adaptiveNora4005042, 5, 5, 7UniversityJudgeTraditional/adaptiveBarbara3904043, 5, 8, 10UniversityRecruiterTraditional/adaptiveAstrid4303638, 5, 3Higher polytechnicControllerAdaptiveMarieke420--413, 10, 9, 6Intermediate polytechnicCashierTraditional/adaptiveMireille35040313, 7, 5Secondary schoolPersonal assistantTraditional/adaptiveNienke4704018Secondary schoolManagerEgalitarianMeriam (Iraq)480Unemployed because of disability325, 22, 8Primary schoolNoneTraditional/adaptiveSaida (Turkey)3303038, 4, 2Primary schoolNoneTraditional/adaptiveHeleen34244037, 4, 1UniversityTeacherTraditional/adaptiveNel32164012UniversityPsychologistTraditional/adaptiveTineke382432213, 3Intermediate polytechnicProfessional child careTraditional/adaptiveYoussra3518Unemployed310, 8, 6Primary schoolCleanerTraditional/adaptiveBrigitte35244025, 1Intermediate polytechnicProfessional child carerTraditional/adaptiveCarien35244011Intermediate polytechnicProject assistantEgalitarianWillemien4724Unemployed because of disability18Intermediate polytechnicCookTraditional/adaptiveEsmé34244024, 2Secundary schoolPersonal assistantTraditional/adaptiveJuul40323222, 10 MonthsUniversityPublisherEgalitarianMedina38323235, 4, 11 MonthsUniversityProsecutorEgalitarianAlice473040213, 10Higher polytechnicJournalistAdaptive/egalitarianMarloes433040211, 8UniversityTranslatorAdaptive/egalitarianCathy443232111UniversityManagerEgalitarianDiana4230--210, 7UniversityPsychologistAdaptive/egalitarianSietske433240311, 8, 8UniversityDirectorAdaptive/egalitarianOlga302832211 MonthsUniversitySocial workerEgalitarianYvette42304027, 6Intermediate polytechnicCabinetAdaptiveSophie48303619Intermediate polytechnicCopy writerEgalitarianAnnemiek37343427, 5UniversityPolicy makerEgalitarianLotte47404027, 9UniversityManagerEgalitarianMichelle4740--213, 9UniversityManaging directorEgalitarianIlse4340+40312, 10, 5UniversityPublisherEgalitarianMarlieke4736Variable211, 8UniversityPolicy makerEgalitarianClaire473636212, 10UniversityManaging directorEgalitarianEbru (Turkey)40404024, 1Higher polytechnicBank employeeEgalitarianAnnelies423632311, 9, 7UniversityBank employeeEgalitarianAlisha (Surinam)43364015Secondary schoolProject assistantEgalitarianSheila (Chinese)37363027, 5Intermediate polytechnicPersonal assistantEgalitarianRosa3236--212, 10Intermediate polytechnicPersonal assistantEgalitarian39
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{
"pile_set_name": "PubMed Central"
}
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Introduction
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Patients with rheumatoid arthritis (RA) and other chronic inflammatory diseases are often subject to prolonged treatment with immunosuppressive drugs which modify the immunologic pathways involved in the pathogenesis of RA. Tumor necrosis factor alpha (TNFα) is among the cytokines that play a major role in the inflammatory process of rheumatic diseases. Its inhibition leads to substantial improvement in clinical signs and symptoms in a majority of patients. To date three different agents are available as monoclonal antibodies or receptor fusion antagonists of TNFα. The finding that TNFα is able to induce tumor cell apoptosis led it to be named TNF before its role in the inflammatory process was revealed \[[@B1]\]. TNFα or rather its nuclear factor-kappa B pathway acts as an early tumor suppressor \[[@B2]\]. This property led to concerns about a possibly increased risk of malignancies when drugs blocking TNFα will be used for long-term treatment.
These concerns were supported by two meta-analyses of randomized controlled trial data. In their first aggregate data meta-analysis of nine randomized controlled trials (RCTs) of anti-TNFα antibody therapies (infliximab and adalimumab) versus placebo in patients with rheumatoid arthritis, Bongartz et al. \[[@B3]\] found a significantly increased risk for malignancies in anti-TNFα versus placebo treated patients with a pooled odds ratio of 3.3 (95% CI: 1.2 to 9.1). In their second meta-analysis Bongartz et al. \[[@B4]\] found a higher malignancy risk also in patients treated with etanercept as compared to the control group, although the relative risk estimate did not achieve statistical significance (Hazard ratio (HR) of 1.84 \[95% CI: 0.79 to 4.28\]).
Considering the strict criteria for the inclusion of patients and the thorough monitoring process preceding controlled trials there might be an even higher risk when unselected RA patients are treated with anti-TNFα agents in daily rheumatologic care. Therefore, real-world data from studies systematically observing patients treated with these agents for long periods are of high importance.
Patients with prior malignancy are usually excluded from participation in RCTs and most clinical recommendations do not encourage treating these patients with anti-TNFα. However, this treatment might be the best therapeutic option for their inflammatory disease. Information regarding the safety of biologic agents prescribed to patients with prior malignancies is available only from two abstracts from the British Society of Rheumatology Biologics Register (BSRBR) \[[@B5],[@B6]\], one of them indicating a possibly increased recurrence risk for melanoma \[[@B6]\].
According to the national recommendations of the German Society of Rheumatology biologic agents should be prescribed after failure of at least six months of treatment with two conventional DMARDs (including methotrexate (MTX)) alone or in combination \[[@B7]\].
The German biologics register RABBIT is an ongoing, nationwide prospective cohort study started in 2001 with the approval of the first biologic agents in Germany. It was established with the aim to assess the long-term safety of biologic agents including TNFα blockers. Time points of follow-up and assessments are identical for patients treated with biologic agents and for those under therapy with conventional DMARDs.
We used the data from RABBIT to investigate the frequency of developing a first malignancy in patients treated with anti-TNFα agents compared to those treated with conventional DMARDs and to study the risk of patients with a history of malignancy receiving anti-TNFα therapy.
Materials and methods
=====================
Patients
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Patients aged 18 to 75 years meeting the American College of Rheumatology (ACR) criteria for RA are eligible to be enrolled in RABBIT at the start of treatment with a biologic agent or a conventional DMARD after failure of at least one other DMARD. Prior to enrollment all patients gave their informed consent. Patients enrolled between 01 May 2001 and 31 December 2006 (end of recruitment to this cohort) were included in the following analyses provided at least one follow-up visit and the baseline status regarding co-morbid conditions were available. Patients were followed up independent of any change in their treatment regimes. Information about patients who missed two or more consecutive follow-up visits was obtained by contacting the treating physicians, and if necessary the patients themselves, their relatives or the local health authorities to determine the patient\'s vital status. The reasons for dropout and the causes of death were ascertained. Details of inclusion criteria for RABBIT were previously reported \[[@B8],[@B9]\]. The ethics committee of the Charité University School of Medicine, Berlin, approved the study protocol.
Assessments
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At baseline and at predefined time points of follow-up (3, 6, 12, 18, 24, 30, 36, 48, 60 months) rheumatologists assessed the clinical status including the components of the disease activity score based on 28 joint counts (DAS28) and reported treatment details and serious and non-serious adverse events according to the International Conference on Harmonization E2A guidelines \[[@B10]\]. All adverse events were coded using the Medical Dictionary for Regulatory Affairs (MedDRA) \[[@B11]\] by one of the authors (AS). Reported malignancies were considered as *events of interest*, and an additional query asking for diagnostic and treatment details and cancer history was sent to the reporting rheumatologist. Only in five cases we did not receive any further information. In 50% of the cases hospital discharge letters with the exact histopathologic results were sent to us.
At study entry rheumatologists reported co-morbidities for every patient on a list of 23 diseases which include among others: prior malignancy or lymphoma, chronic obstructive pulmonary disease (COPD), other chronic lung disease, chronic renal disease, chronic gastrointestinal disease, chronic liver disease, psoriasis, and chronic viral disease. Patients assessed their pain, general health, disability and socioeconomic status. The Hannover Functional Status Questionnaire (Funktionsfragebogen Hannover, FFbH) was used to assess disability. Scores are expressed as percentage of full function (range 0 to 100) and can be transformed into Health Assessment Questionnaire (HAQ) values \[[@B12]\]. Smoking habits were not assessed at baseline but only after 24, 48, 60 months. Since this resulted in a high percentage of missing smoking information we did not include smoking in the multivariate analyses. Nevertheless, in the nested case control study we were able to include smoking by using the missing information as one matching criterion.
Statistical analysis
--------------------
Prior malignancies and tumor recurrence. All patients meeting the study inclusion criteria were stratified by their prior malignancy status. For all patients prior malignancies were reported by the rheumatologist at study entry. Patients with or without prior malignancy were compared with respect to patient characteristics and treatment. In patients with a recurrent malignancy during the observation in RABBIT we analysed whether treatment was associated with recurrence. We defined recurrency as development of any cancer after a history of a prior malignancy, irrespective of the type of the recurrent tumor.
### Tumor incidence
We analysed the tumor incidence during the observation period in all patients without prior malignancy. We included all types of cancer except for basal cell carcinomas. One M. Bowen was reported and included. There was no report of other carcinomas in situ. The observed number of incident cancers was compared with the expected number calculated from population data \[[@B13]\]. Cox regression was used to analyse the effects of treatment and to adjust for demographic and clinical data. Only exposure times after enrollment in the RABBIT register were taken into account. Patients were considered to be exposed to anti-TNFα treatment for the time period from the start of anti-TNFα treatment to the end of follow-up (*ever exposed*-approach). The same definition was used for anakinra exposure. Because of the applied *ever exposed*-approach patients could have been exposed to both, anti-TNFα agents and anakinra. Patients (or follow-up time of patients) not (yet) exposed to anti-TNFα or anakinra were considered as exposed to DMARDs only.
The following baseline characteristics were included in the risk assessment: age, gender, disease duration, rheumatoid factor, functional capacity (measured by the FFbH), selected previous treatment exposures (cyclosporine or azathioprine) \[[@B14]\], and co-morbid conditions.
Since nearly all (98.3%) of our patients were ever exposed to methotrexate and only a small minority (0.6%) to cyclosphosphamide we were not able to investigate a specific cancer risk associated with these agents. A preliminary analysis blinded for treatment assignment revealed associations between the frequency of cancer and COPD, chronic gastrointestinal diseases and chronic renal diseases. Other co-morbidities, such as chronic lung diseases in general, are known to be associated with increased cancer risk. Therefore, the following comorbid conditions were included in the Cox regression analysis: COPD, other chronic lung disease, chronic renal disease, chronic gastrointestinal disease, chronic liver disease, and psoriasis. Furthermore, we investigated the impact of exposure to anti-TNFα agents as well as the impact of long-term high disease activity (measured by time-averaged DAS28 scores) on the risk of cancer. For this analysis, the mean of all DAS28 scores measured more than six months before an event were included as time-dependent co-variables into the Cox regression analysis. Disease activity during the six months prior to a malignancy diagnosis was not considered since it may have been influenced by the carcinogenesis. For malignancies that developed within the first six months of observation, the DAS28 measured at study entry was used. On average 7% of the DAS28 values at follow-up were missing. To minimize possible bias missing values were imputed before the time-averaged scores were calculated. The expectation-maximisation (EM) algorithm most appropriate for approximately normally distributed variables such as the DAS28 was applied for estimation and imputation \[[@B15]\]. Calculations were performed using the SAS procedures MI and PHREG. A test based on the analysis of Schoenfeld residuals of Cox regression was used to investigate the invariance of the HR over time \[[@B16]\].
The control for confounding factors by Cox regression analysis may be insufficient since smoking could not be included and two possible risk factors found in our preliminary analysis (see above) were observed in less than 5% of the patients. Our statistical analysis plan therefore stipulated to perform a nested case control study as our main analysis of the risk of incident cancer. For each case with an incident cancer, a cancer-free control patient was selected who was compatible with the following matching criteria: gender, smoking status, and six co-morbid conditions (same as those used in the Cox regression). Cases with valid data of smoking status were matched to controls with the same smoking status and patients with missing information regarding smoking status were matched to controls who also had no smoking status data. After matching for eight categorical variables, a control patient was selected who fitted best to the case concerning age, follow-up time and DAS28 at baseline. Standardized Mahalanobis metric was used for measuring similarity. The availability of 4,923 possible controls permitted use of this detailed matching algorithm. Mc Nemar test was used to compare the numbers of patients exposed to biologics (anti-TNFα agents or anakinra) between patients and matched controls. For further comparisons within the nested case control study, paired t-test and Wilcoxon test were applied as appropriate. Chi-square test, t-test and Mann-Whitney test were used for statistical comparisons of patient\'s characteristics at baseline. *P*-values \< 0.05 were considered statistically significant.
Results
=======
Patient characteristics and treatment status at study entry
-----------------------------------------------------------
Between 01 May 2001 and 31 December 2006, 5,279 patients were enrolled in RABBIT. One hundred fifty-nine patients were excluded from this analysis because of missing follow-up information or missing co-morbid condition status (Figure [1](#F1){ref-type="fig"}). Their baseline characteristics (age, DAS28, function, co-morbidity status) were not statistically different from the remaining 5,120 patients. Those were stratified according to their prior malignancy status, and both groups were analysed separately. A total of 124 prior malignancies were found in 122 patients: 6 lymphomas (DMARDs: 2, anti-TNFα: 4), and 118 solid tumors (DMARDs: 54, anakinra: 9, anti-TNFα: 55)\]. Patients with prior malignancies were significantly older (P \< 0.001), had a lower functional capacity (56% of full function vs. 60% of full function) and a higher frequency of chronic gastrointestinal disease than patients without prior malignancy (Table [1](#T1){ref-type="table"}). Within both strata, we found that patients receiving biologics had significantly more active disease, and were more limited in activities of daily living (FFbH). As reported previously, there were no significant differences in the clinical characteristics of patients receiving etanercept, adalimumab, or infliximab \[[@B17]\], whereas anakinra patients had more treatment failures with DMARDs and a lower functional capacity (FFbH) than anti-TNFα patients. Because of the differing modes of action and the differences in the clinical characteristics, separate results are provided in the following analyses for patients receiving anti-TNFα agents and patients receiving anakinra.
{#F1}
######
Baseline characteristics of patients
Patients with prior malignancy Patients without prior malignancy
--------------------------------------------------------------- -------------------------------- ---------------- ----------------------------------- ----------- ---------------- ---------------- ---------------- ----------- -------------
**Biologic** **Control** **Total** ***P*\#** **Biologic** **Control** **Total** ***P*\#** ***P*\#\#**
**n** 67 55 122 3279 1719 4998
**Female n (%)** 45 (67.2) 41 (74.5) 86 (70.5) 0.43 2564 (78.2) 1353 (78.7) 3917 (78.4) 0.671 0.047
**Age (mean, SD)** 64.0 (9.0) 63.2 (7.7) 63.7 (8.4) 0.62 53.6 (12.3) 55.9 (11.5) 54.4 (12.1) \< 0.0001 \< 0.001
**Disease duration (yrs), median (IQR)** 10 (6, 16.5) 7 (3, 13) 9 (4, 16) 0.02 9 (5, 17) 6 (2.5, 12) 8 (4, 15) \< 0.0001 0.286
**Time from prior cancer to study entry (yrs), median (IQR)** 5 (2, 9) 5 (3, 11) 5 (2, 10) 0.77
**Follow-up time (yrs), median (IQR)** 2.1 (1.4, 3.0) 2.5 (1.0, 4.0) 2.1 (1.1, 3.1) 0.43 2.4 (1.4, 3.1) 2.5 (1.3, 3.3) 2.4 (1.3, 3.1) 0.081 0.611
**Rheumatoid factor positive n (%)** 53 (79.1) 46 (83.6) 99 (81.1) 0.64 2629 (80.2) 1225 (71.3) 3854 (77.1) \< 0.0001 0.327
**DAS28 (mean, SD)** 5.7 (1.3) 5.4 (1.1) 5.6 (1.2) 0.04 5.8 (1.3) 5.0 (1.3) 5.5 (1.3) \< 0.0001 0.282
**ESR (mm/h), median (IQR)** 38 (18, 51) 26 (15, 42) 32 (17, 50) 0.12 30 (16, 48) 22 (12, 38) 27 (14, 44) \< 0.0001 0.080
**CRP (mg/L), median (IQR)** 25 (10, 46) 15 (8, 30) 19 (9, 43) 0.07 17 (8, 38) 12 (5, 27) 15 (7, 34) \< 0.0001 0.146
**FFbH (mean, SD)** 52.1 (21.3) 59.9 (23.5) 55.7 (22.5) 0.02 57.0 (23.0) 66.8 (21.4) 60.4 (22.9) \< 0.0001 0.025
**Smoking ever n (%)** 23 (57.5) 15 (55.6) 38 (56.7) 1.00 878 (46.9) 473 (46.4) 1351 (46.7) 0.328 0.13
**No. of previous DMARDs (mean, SD)** 3.7 (1.5) 1.9 (1.0) 2.9 (1.6) \< 0.001 3.6 (1.4) 1.9 (1.1) 2.9 (1.5) \< 0.0001 0.656
**COPD n (%)** 3 (4.4) 6 (10.9) 9 (7.3) 0.30 163 (5) 81 (4.7) 244 (4.9) 0.685 0.205
**Chronic renal disease n (%)** 4 (5.9) 1 (1.8) 5 (4.1) 0.38 134 (4.1) 29 (1.7) 163 (3.3) \< 0.0001 0.603
**Chronic lung disease n (%)** 5 (7.4) 1 (1.8) 6 (4.9) 0.22 93 (2.8) 29 (1.7) 122 (2.4) 0.012 0.129
**Chronic gastrointest. disease n (%)** **12 (17.6)** **5 (9.1)** **17 (13.9)** **0.2** **281 (8.6)** **138 (8.0)** **419 (8.4)** **0.510** **0.047**
Values are means and standard deviations if not otherwise specified. IQR = inter quartile range; FFbH = Hannover Functional Status Questionnaire measuring functional capacity in percent of full function; DAS28: disease activity score based on 28 joint counts;
\# = *P*-value for comparison between biologic and control group within strata according to prior malignancy status, \#\# = comparison between strata
Patients with a prior malignancy were insignificantly less frequently treated with anti-TNFα agents or anakinra at inclusion than patients without prior malignancy (Figure [1](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). The adjusted OR to receive biologics (adjusted for age, sex, disability, disease activity) for patients with prior malignancies compared to those without was 0.7 (95%CI: 0.5 to 1.1). Detailed information including the exact type of malignancy was reported in 54 of the 124 prior malignancies. We found some differences regarding the spectrum of those malignancies in anti-TNFα vs. DMARD treated patients: At study entry all nine cases with prior prostatic cancer were treated with biologics (seven with anti-TNFα and two with anakinra) whereas three patients with prior bladder cancer were found in the DMARD treated group and one patient was treated with anakinra. Patients with prior breast cancer were less frequently treated with biologics (n = 11) than with DMARDs (n = 14) at inclusion. The time between onset of the prior malignancy and study entry did not differ between the treatment groups. The median time was five years (IQR: 2 to 9) for patients receiving biologics (anti-TNFα: four years (2 to 10); anakinra: six years (5 to 9)) and five years (3 to 11) for patients receiving conventional DMARDs (*P*= 0.77). In 28 (45.9%) of the patients treated with biologics (27 with anti-TNFα and 1 with anakinra) and in 22 (40.7%) patients in the DMARD group the time since the last tumor diagnosis was less than five years when treatment with the respective agent started.
Recurrence of a prior malignancy
--------------------------------
During follow-up 15 recurrent cancers were observed in 14 patients including 14 recurrences of the same type and site as the prior tumor and one metastasis of unknown origin (Table [2](#T2){ref-type="table"}). Nine recurrences were seen in eight patients under treatment with anti-TNFα agents, one in an anakinra patient and five in patients exposed to DMARDs only. The corresponding crude incidence rates were 45.5 (95%CI: 20.8 to 86.3)/1,000 patient years (pyrs) for patients receiving anti-TNFα agents, 32.3 (95%CI: 0.8 to 179.7)/1,000 pyrs for patients treated with anakinra and 31.4 (95%CI: 10.2 to 73.4)/1,000 pyrs for DMARD treated patients (incidence rate ratio anti-TNFα agents vs. DMARDs: 1.4 (95% CI: 0.5 to 5.5) *P*= 0.63).
######
Recurrence of prior malignancy by type and treatment
Ever exposed to
------------------------------------ ----------- ----------------- -------------- -----------------------------
**Total** **Anti-TNFα** **Anakinra** **Conventional DMARD only**
**N with prior malignancy** 122 72 11 43
**Patient-years of follow-up** 379 198 31 159
**Recurrent malignancies** 15 9 (5 f, 4 m) 1 (m) 5 (4 f, 1 m)
**Breast cancer** 5 4 (f) \- 1(f)
**Lung cancer** 3 1 (m) 1 (m) 1 (f)
**Bladder cancer** 2 1 (m)^\#^ \- 1 (f)
**Liposarcoma** 1 1 (m) \- \-
**Melanoma** 1 1 (f)
**Signet-ring cell carcinoma** 1 \- \- 1 (f)
**Testicular cancer** 1 1 (m)^\#^ \- \-
**Metastasis of unknown origin** 1 1 (m)
M = male, f = female, ^\#^testicular cancer and bladder cancer in one patient
The mean time span between the prior tumor and the diagnosis of the new tumor was 9.5 (SD: 7.8), 9.1, and 9.2 (8.8.) years for patients exposed to anti-TNFα agents, anakinra, or conventional DMARDs, respectively. Three patients developed a recurrent cancer less than five years after the previous cancer (two in the anti-TNFα treated group, one in the DMARD group).
Four of the five patients who were treated with conventional DMARDs only and experienced a recurrence of their prior malignancy died (signet-ring-cell carcinoma, metastasis of unknown origin, breast cancer, lung cancer). One out of the eight patients under treatment with anti-TNFα died (breast cancer), and the one patient under treatment with anakinra (lung cancer) died. All other patients with recurrences were still alive at the time of the analysis.
Incidence of tumors in patients without a prior malignancy
----------------------------------------------------------
### Comparison of the tumor incidence with the general population
Seventy-four patients among the 4,998 patients who did not have a prior malignancy developed an incident tumor. This is an overall incidence rate of 6.0 per 1,000 pyrs \[95% CI: 4.7 to 7.6\]. The figures were 5.1 per 1,000 pyrs (95% CI: 3.7 to 6.9) for patients exposed to anti-TNFα, 7.2 per 1,000 pyrs (95% CI: 2.4 to 16.9) for patients exposed to anakinra and 8.4 per 1,000 pyrs (95% CI: 5.7 to 12.0) for patients exposed to conventional DMARDs. For some of the cancer sites the observed incidence rates in both groups were lower than the age and sex adjusted rates as expected from the general population (for example, breast, male and female reproductive organs and colon cancer) \[[@B13]\] (Figure [2](#F2){ref-type="fig"}). Higher rates were observed for non-Hodgkin\'s lymphoma in patients exposed to biologics and for pancreatic cancer in the group not exposed to biologics. None of the site specific differences were statistically significant when the *P*-values were adjusted for repeated significance testing. Taking into account all malignancies, the number of observed cancers in patients exposed to anti-TNFα agents was non-significantly lower than the expected number from the general population (standardized incidence rate ratio: 0.75, 95% CI: 0.54 to 1.01). No difference was found for patients not exposed to biologics.
{#F2}
### Comparison of patients with and without incident tumors in patients with no prior cancer
Overall, patients who developed malignancies during the study period had more co-morbidities than those who did not (mean = 2.5 (SD = 2.1) vs. 1.7(1.9)). Higher rates were observed for COPD (11/74 (14.9%) vs. 223/4924 (4.5%), *P*\< 0.0001), chronic gastrointestinal diseases ((13/74 (17.6%) vs. 406/4924 (8.2%), *P*= 0.008), and chronic renal diseases (4/74 (5.4%) vs. 159/4924 (3.2%), *P*= 0.22). Furthermore, site specific associations were observed for gastric/colorectal cancer in 2/419 patients with a chronic gastrointestinal disease vs. 3/4579 in the remaining patients (*P*= 0.06), and for bladder cancer/renal cancer in 1/163 patients with chronic renal diseases vs. 3/4835 in the remaining patients (*P*= 0.12).
Crude cancer incidence rates were therefore higher in patients with specific comorbid conditions but also in those with a highly active disease (Table [3](#T3){ref-type="table"}).
######
Crude incidence rates of malignancies
n of CA n pyrs IR
---------------------------------- --------- ------------ -------- ------
**Characteristics at baseline**
Age (per 10 yrs increase)
Female 52 3,917 9,687 5.4
Male 22 1,081 2,554 8.6
Co-morbid conditions:
COPD
No 63 4,754 11,677 5.4
Yes 11 244 564 19.5
Gastrointestinal disease
No 61 4,579 11,153 5.5
Yes 13 419 1,088 11.9
Renal disease
No 70 4,835 11,884 5.9
Yes 4 163 357 11.2
**Characteristics at follow-up**
DAS28
\<3.2 6 787 2,165 2.8
3.2 to 5.1 34 2,823 7,341 4.6
\>5.1 34 1,388 2,735 12.4
**Ever exposed to**
Conventional DMARDs only 30 1,684 ^\#^ 3,561 8.4
Anti-TNF agents 44 3,651 \* 8,558 5.1
Anakinra 5 247 ^\$^ 690 7.2
CA: malignancies, n = number of cases, pyrs = follow-up time in patient years, IR = crude incidence rate per 1,000 pyrs.
^\#^35 of the patients in the DMARD cohort were exposed to anti-TNF agents before inclusion in the study. According to the ever exposed approach their follow-up time was assigned to the anti-TNF treated group.
\* Patients included in this group are patients who were included in the study with the start of an anti-TNF agent (n = 3,201) and patients who changed during follow-up from DMARD or anakinra treatment to anti-TNF treatment
^\$^223 of the 247 pts were exposed to anakinra and anti-TNF agents, they experienced five malignancies.
The univariate analysis showed that patients with a very active disease (DAS28 \>5.1, mean: 5.93) during follow-up had a two times higher cancer risk than those with low disease activity (DAS28 \<3.2, mean: 2.75) (Table [4](#T4){ref-type="table"}).
######
Hazard ratios of developing a malignancy
Univariate Cox regression Multivariate analysis
---------------------------------- --------------------------- ----------------------- ----------- ------------ -------------- -----------
**Characteristics at baseline**
Age (per 10 yrs increase) 1.82 1.44 to 2.31 \< 0.0001 1.71 1.35 to 2.17 \< 0.0001
Male vs. female 1.61 0.98 to 2.65 0.062 1.47 0.89 to 2.43 0.13
Co-morbid conditions:
COPD 3.64 1.92 to 6.91 \< 0.0001 2.63 1.37 to 5.04 0.004
Gastrointestinal disease 2.19 1.20 to 3.98 0.010 1.81 0.99 to 3.30 0.0534
Renal disease 1.93 0.70 to 5.28 0.20
**Characteristics at follow-up**
DAS28 (per unit increase) 1.24 1.02 to 1.50 0.034
DAS28
\< 3.2 *Referent*
3.2 to 5.1 1.28 0.54 to 3.06 0.58
\> 5.1 2.00 0.82 to 4.86 0.13
**Ever exposed to**
Conventional DMARDs only *Referent* *Referent*
Anti-TNF agents 0.61 0.39 to 0.97 0.039 0.70 0.44 to 1.12 0.133
Anakinra 1.16 0.47 to 2.89 0.75 1.39 0.56 to 3.48 0.480
In the multivariate analysis the development of an incident tumor was strongly associated with age (HR = 1.71; 95% CI: 1.3 to 2.2 per 10 years increase in age, *P*\< 0.0001) and COPD (HR = 2.63; 95% CI: 1.4 to 5.0, *P*= 0.004) (Table [4](#T4){ref-type="table"}). A higher cancer risk was also observed for patients with chronic gastrointestinal diseases whereas no significant associations were found for other co-morbid conditions (other chronic lung diseases, psoriasis, chronic liver disease) or for gender (HR (males vs. females) = 1.46; 95% CI: 0.9 to 2.4, *P*= 0.14). Likewise, associations for patients ever exposed to cyclosporine (n = 582, *P*= 0.24) or azathioprine (n = 599, *P*= 0.32) were not statistically significant.
In patients exposed to anti-TNFα agents we observed a non-significantly decreased risk for developing a malignancy compared to patients treated with conventional DMARDs (adjusted HR = 0.70; 95% CI: 0.44 to 1.12, *P*= 0.13).
Time dependency of the hazard risk in patients without prior cancer
-------------------------------------------------------------------
The mean time until the onset of the malignancy was 25.0 (16.8) months for patients exposed to anti-TNFα agents, 14.8 (9.1) for anakinra and 17.4 (15.7) months for patients not exposed to biologics. Ten of the 44 malignancies of anti-TNFα-exposed patients developed in the first year compared to 15 of 30 malignancies in non-exposed patients. This corresponds to adjusted hazard ratios of 0.4 for the first and 1.0 for the second to fourth year. We analysed this possible time trend in the hazard ratio by means of an analysis of standardized Schoenfeld residuals of the Cox regression. The trend was, however, not statistically significant (*P*= 0.13).
Nested case-control study
-------------------------
The main analysis to assess the risk of developing an incident tumor under treatment with anti-TNFα agents was conducted as a nested case control study. Each case with an incident malignancy was matched to one control patient without malignancy (see Methods). Due to the high number of possible matching partners, matching was successful for all parameters involved. Cases with incident malignancy had lower baseline functional capacity than those without cancer (Table [5](#T5){ref-type="table"}). There was no significant difference concerning treatment exposure: Forty-four (59.5%) of the cases and 45 (60.8%) of the controls had ever been exposed to biologics. The numbers of cases ever exposed to etanercept, adalimumab, infliximab, or anakinra (n = 22; n = 20; n = 16; n = 5, respectively) did not differ significantly from the numbers of controls ever exposed to these therapies (n = 27; n = 24; n = 10; n = 5, respectively). A separate analysis of malignancies observed in the first year (anti-TNFα exposed 10/25 cases vs. 11/25 controls) and in the second to fourth year (anti-TNFα exposed: 34/49 cases vs. 33/49 controls) did not show any significant difference between the groups or a significant time trend.
######
Patient characteristics of cases and matched controls
-----------------------------------------------------------------------------------------------------------
Cases\ Matched controls P
(with incident malignancy)
------------------------------------------------ ---------------------------- ------------------ ----------
N 74 74
**Characteristics at study entry**
Females^\#^n (%) 52 (70.3) 52 (70.3) n.a.
Age^\#^(mean, SD) 61.3 (8.9) 61.4 (8.5) 0.97
Observation time^\#^(years) (median, IQR) 2.9 (1.8, 4.0) 2.9 (1.6, 3.9) 0.25
DAS28^\#^(mean, SD) 5.6 (1.0) 5.7 (1.0) 0.31
Smoking status^\#^
Nonsmoker 16 (21.6) 16 (21.6) n.a.
Smoker n (%) 19 (25.7) 19 (25.7)
Unknown status n (%) 39 (52.7) 39 (52.7)
Disease duration (years) (median, IQR) 7 (3,14) 9 (5,16) 0.22
Functional status, FFbH (mean, SD) 57.1 (22.3) 63.2 (22.6) 0.058
**Characteristics at follow-up**
DAS28 (mean, SD)^\$^ 5.0 (1.2) 4.9 (1.2) 0.41
Ever exposed to biologics n (%) 44 (59.5) 45 (60.8) 1.0
Ever exposed to anti-TNFα agents ^&^ 44 (59.5) 44 (59.5) 1.0
Ever exposed to anakinra^&^ 5 (6.8) 5 (6.8) 1.0
**Among them**
Cases with solid tumors (n = 68)
Ever exposed to biologics 39 (57.4) 43 (63.2) 0.56
Ever exposed to anti-TNFα agents 39 (57.4) 42 (61.8) 0.70
Ever exposed to anakinra 5 (7.4) 5 (7.4) **1.0**
**Cases with non-Hodgkin\'s Lymphoma (n = 6)**
**Characteristics at study entry**
**Females^\#^n(%)** 5 (83.3) 5 (83.3) **n.a.**
**Age^\#^(mean, SD)** 65.2 (6.5) 66.8 (8.0) **0.13**
**Disease duration** 5.5 (4,13) 7 (5,11) **0.53**
**Functional status, FFbH (mean, SD)** 52.8 (31.4) 54.2 (27.5) **0.90**
**DAS28^\#^(mean, SD)** 5.9 (0.6) 6.2 (0.5) **0.29**
**Characteristics at follow-up**
**DAS28 (mean, SD)^\$^** 5.2 (0.7) 5.5 (0.7) **0.14**
**Ever exposed to anti-TNFα agents** **5 (83.3)** **2 (33.3)** **0.38**
-----------------------------------------------------------------------------------------------------------
^\#^matching criteria (further matching criteria not shown in the table = COPD, other chronic lung disease, chronic renal disease, chronic gastrointestinal disease, chronic liver disease, and psoriasis)
^&^five cases and four controls received anti-TNFα agents and anakinra (at different points in time)
^\$^cases = mean of DAS28 values over time points until six months prior to the cancer diagnosis, matched controls: mean of DAS28 values over time points of the corresponding case
An insignificantly higher rate of exposure to anti-TNFα agents was found in patients who developed non-Hodgkin\'s lymphoma.
Discussion
==========
First, in patients with prior malignancy we did not find a significant increase in the risk of recurrent tumors under treatment with anti-TNFα agents compared to conventional DMARDs, even though there was a higher recurrence rate under anti-TNFα treatment (IRR = 1.4, *P*= 0.6).
Second, patients without prior malignancy did not have higher rates of incident tumors when they were exposed to biologics compared to unexposed patients.
The strength of our study is that all data on incident and recurrent tumours originate from a prospective, closely monitored observational cohort study established for the purpose of pharmacovigilance. Data were collected in an identical manner and by the same physicians for patients treated with biologic agents or with conventional DMARDs. Due to stringent and close monitoring, drop-out rates were less than 5% per year. Additionally, for all patients lost to follow-up the vital status was ascertained or, if appropriate, the cause of death.
To investigate the occurrence of incident malignancies, we followed three different methodological approaches: a nested case-control study, a multivariate Cox regression and a comparison with population data.
Of note, we excluded basal cell carcinoma from all types of our analysis since no age and sex specific population rates were available.
The design of the nested case control study allowed us to adjust for differences in clinical and demographic parameters (for example, selected co-morbidities, smoking status) which are related to treatment assignment but which are also associated with the risk of cancer. We therefore consider the nested-case control analysis our central assessment of the risk of incident malignancies under treatment with anti-TNFα agents. However, since only a very small proportion of the patients were included in the nested case control analysis and therefore information of a large number of patients was not used, we decided to apply a multivariate Cox regression analysis in addition.
A potential weakness of this study is that we only investigated the overall cancer risk. The risk for site-specific cancers has not been analyzed due to the limited numbers of events. In addition, due to the relatively short time of observation no conclusions can be drawn beyond the scope of four years of exposure.
Limitations of the analysis of tumor recurrence include the relatively small sample size and the fact that physicians were less likely to prescribe cytokine inhibitors for patients with prior malignancy than for those without such a history. Furthermore, our data indicate that physicians might have made different treatment decisions for patients with different prior tumors. We therefore must exhibit caution in drawing firm conclusions.
The same applies for the interpretation of the data for anakinra treated patients: only a few patients in the cohort were ever treated with anakinra, most of them were also exposed to anti-TNFα agents. Therefore, malignancies occurring in this group must be interpreted carefully, taking patient selection (also seen in differing baseline characteristics) into account.
Our results differ from those reported by the British Society for Rheumatology Biologics Registry (BSRBR) \[[@B6]\]. Dixon and co-workers analyzed 177 patients with prior malignancies treated with anti-TNFα agents with a median follow-up of three years and found no increased risk for recurrent malignant diseases compared to 118 patients with prior malignancies treated with conventional DMARDs and followed-up for 1.9 years. Their crude incidence rates were 25.3 and 41.9 per 1,000 patient-years for patients treated with anti-TNFα agents and conventional DMARDs, respectively, compared to 45.5 and 31.4 per 1,000 patients-years in our study. In an earlier analysis of the BSRBR \[[@B5]\] a total of 154 patients in the anti-TNFα cohort had a previous malignancy and six (4%) developed a new malignancy. However, of these malignancies only one was a local recurrence which is in contrast to our findings. In our study, in 14 out of the 15 recurrences the observed malignancies were *true*recurrences of the prior tumor with the same type and site. Only in one of our patients with pulmonary and bone metastases the origin of the malignancy remained unknown. This patient had a history of testicular cancer 20 years before.
Our investigations regarding the risk of developing an incident malignancy were motivated by two meta-analyses of randomized controlled trials which suggested an increased risk of malignancies associated with the treatment with one of the TNFα blocking agents: adalimumab, infliximab, or etanercept \[[@B3],[@B4]\]. The results of these meta-analyses are in contrast with those from observational cohort studies or national cancer registries, in which such an increased risk was not observed \[[@B18],[@B19]\]. The methodological weaknesses of the first meta-analysis investigating the risk for treatment with adalimumab or infliximab have already been discussed elsewhere \[[@B20]\]. However, the repeated finding of an increased cancer risk also in patients treated with etanercept \[[@B3],[@B4]\] requires further research, even if it did not achieve statistical significance. These meta-analyses support the *early mobilization hypothesis*implying a high risk within the first months of treatment with anti-TNFα agents.
However, our results for the first year are in contrast to the meta-analyses reported by Bongartz et al. \[[@B3],[@B4]\]. In the first year of treatment patients receiving biologics had a lower risk for developing an incident malignancy than those receiving conventional DMARDs. This may be due to a selection bias evoked by the screening process for malignancies following the physician\'s decision that a patient should receive biologic treatment. Screening for latent TB by chest x-ray as recommended in guidelines \[[@B21]\] may reveal asymptomatic lung cancers and only those patients who screen negative for current malignancies will receive biologic treatment and be included in the biologics group in the RABBIT register. No such general screening occurs in patients who will receive a new DMARD therapy. Therefore the rate for malignancies in the DMARD group represents the *true*unselected rate that can be expected for RA patients under conventional DMARD treatment. Indeed this concept is supported by our findings that the observed number of cancer cases in the DMARD treated group (n = 30) was equal to the expected number (n = 30.8), whereas the observed number in the biologics group (n = 44) was lower than what would have been expected based on the rates from the general population (n = 64.3).
In contrast to an increased risk, it is also possible that inhibition of TNFα has beneficial or even preventive effects regarding cancer. TNFα is important in all steps of cancer development, for example, initiation, promotion, and survival. Elevated levels of TNFα are linked to a poor prognosis and increased invasiveness in certain human cancers \[[@B22],[@B23]\]. However, the results of first trials to treat breast or ovarian cancer with TNFα inhibitors have been, so far, inconclusive \[[@B24],[@B25]\]. The increased risk of non-Hodgkin\'s lymphoma in RA patients treated with biologic agents is well established \[[@B26],[@B27]\] and has been shown to be strongly associated with long-term high disease activity \[[@B28]\] which is more likely in the history of patients subsequently treated with biologic agents. Nevertheless, in our nested case control study where we controlled for disease activity and duration we still found a higher proportion of anti-TNFα exposure in patients with incident non-Hodgkin\'s lymphomas vs matched controls. This difference was not found for solid tumors.
Conclusions
===========
Our data add to the growing evidence of no overall increased cancer risk for patients treated with anti-TNFα agents during the first years of treatment. This does not preclude that there may be an increased risk for specific cancer types such as lymphoma or skin cancer.
Taking the limitations of the currently available evidence into account there is a need for further large-scale prospective studies investigating risk modifications for different cancer sites as well as investigating the cancer risk after long-term exposure to biologic agents above four years.
Further, this study provides first but limited evidence regarding the risk of patients with a prior malignancy treated with anti-TNFα agents. The finding of an insignificantly increased risk of recurrence under anti-TNFα treatment supports the current practice of carefully balancing treatment decisions in these patients.
Abbreviations
=============
ACR: American College of Rheumatology; BSRBR: British Society of Rheumatology Biologics Register; CI: confidence interval; COPD: chronic obstructive pulmonary disease; DAS28: disease activity score based on 28 joint counts; DMARDs: disease modifying anti-rheumatic drugs; FFbH: Hannover Functional Status Questionnaire (Funktionsfragebogen Hannover); HAQ: Health Assessment Questionnaire; HR: hazard ratio; IQR: interquartile range; IR: incidence rate; IRR: incidence rate ratio; MedDRA: Medical Dictionary for Regulatory Affairs; OR: odds ratio; pyrs: patient-years; RA: rheumatoid arthritis; RABBIT: (German biologics register) acronym for: rheumatoid arthritis observation of biologic therapies; RCT: randomized controlled trial; SD: standard deviation; TB: tuberculosis; TNFα: tumor necrosis factor alpha.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
AS, JL and AZ had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. AZ, JL and AS determined the study concept and design. AS, GRB, BK-L and WD acquired the data. AS, JL and AZ analysed and interpreted the data. AS, JL and AZ drafted the manuscript. RR, GRB, BK-L, WD and FH critically revised the manuscript for important intellectual content and final approval. FH and JL did the statistical analysis. AZ obtained the funding and supervision. AS supervised adverse events reporting, verifications and MedDRA coding. All authors gave final approval of the version to be published.
Acknowledgements
================
RABBIT has been supported by an unconditional, joint grant from Essex Pharma (since 2001), Wyeth pharma (since 2001), Amgen (2003 to 2008)/Biovitrum (since 2009), and Abbott (since 9/2003). Since 2007, it has also been supported by Bristol-Myers Squibb and Roche, and since 2009 by UCB.
The principal investigators and their team have full academic freedom in study design and conduct, data analysis and publication of results. This is laid down in the contract with the sponsors. However, for the purposes of information all six funding companies receive each manuscript 30 days prior to submission.
The authors acknowledge the invaluable contributions of all participating consultant rheumatologists and would like to thank in particular those who enrolled at least 25 patients each: Ulrich von Hinüber, MD, Hildesheim; Andreas Krause, MD, Immanuel hospital Berlin; Maria Stoyanova-Scholz, MD, Wedau Kliniken, Duisburg; Karin Babinsky, MD, Halle; Thilo Klopsch, MD, Neubrandenburg; Arnold Bussmann, MD, Geilenkirchen; Hans Peter Tony, MD, Medizinische Poliklinik der Universität Würzburg; Katja Richter, MD, Universitätsklinikum Carl Gustav Carus Dresden; Anett Grässler, MD, Pirna; Elke Wilden, MD, Köln; Michael Hammer, MD, St. Josef-Stift Sendenhorst; Edmund Edelmann, MD, Bad Aibling; Christina Eisterhues, MD, Braunschweig; Wolfgang Ochs, MD, Bayreuth; Thomas Karger, MD, Eduardus-Krankenhaus Köln-Deutz; Michael Bäuerle, MD, Universität Erlangen, Erlangen; Herbert Kellner, MD, München; Silke Zinke, MD, Berlin; Angela Gause, MD, Elmshorn; Lothar Meier, MD, Hofheim; Karl Alliger, MD, Zwiesel; Martin Bohl-Bühler, Brandenburg; Carsten Stille, MD, Hannover; Susanna Späthling-Mestekemper, MD, and Thomas Dexel, MD, München; Harald Tremel, MD, Hamburg; Stefan Schewe, MD, Medizinische Poliklinik der Ludwig-Maximilians-Universität München; Helmut Sörensen, MD, Krankenhaus Waldfriede Berlin; Florian Schuch, MD, Erlangen; Klaus Krüger, MD, München; Andreas Teipel, MD, Leverkusen; Kirsten Karberg, MD, Berlin; Gisela Maerker-Alzer, MD, and Dorothea Pick, MD, Holzweiler; Volker Petersen, MD, Hamburg; Kerstin Weiss, MD, Lichtenstein; Werner Liman, MD, Ev. Krankenhaus Hagen-Haspe; Kurt Gräfenstein, MD, Johanniter-Krankenhaus im Fläming, Treuenbrietzen; Jochen Walter, MD, Rendsburg; Werner A. Biewer, MD, Saarbrücken; Roland Haux, MD, Berlin; Gross Lübeck; Michael Zänker, MD, Evangelisches Freikirchliches Krankenhaus Eberswalde; Gerhard Fliedner, MD, Osnabrück; Thomas Grebe, MD, Ev. Krankenhaus Kredenbach; Karin Leumann, MD, Riesa; Jörg-Andres Rump, MD, Freiburg; Joachim Gutfleisch, MD, Biberbach; Michael Schwarz-Eywill, MD, Evangelisches Krankenhaus Oldenburg; Kathrin Fischer, MD, Greifswald; Monika Antons, MD, Köln.
We also acknowledge the significant contributions to the conception of RABBIT of Matthias Schneider, MD, University of Duesseldorf, Jörn Kekow, MD, University of Magdeburg, and Peter Herzer, MD, Munich in their function as members of the advisory board. The work in the advisory board of RABBIT is honorary without any financial compensation.
The substantial contribution of Christina Bungartz, Ulrike Kamenz, and Susanna Zernicke, all employees of the German Rheumatism Research Center, Berlin, in the study monitoring and support of the data analyses is gratefully recognized.
|
{
"pile_set_name": "PubMed Central"
}
|
Introduction
============
Interactions of chronic lymphocytic leukemia (CLL) cells with bystander cells in tumor microenvironments, such as the lymph node (LN), provide them with essential survival signals. Upregulation of pro-survival B-Cell Lymphoma-2 family members occurs on stimulation with T cells or with monocyte-derived cells such as macrophages or nurse-like cells (NLCs).^[@bib1]^ Although the tumor necrosis factor (TNF) receptor ligand family member CD40L can account largely for the survival effect by T cells,^[@bib2]^ several factors have been described to have a role in the CLL cell survival effect governed by monocyte-derived cells.^[@bib3],[@bib4]^ A prominent factor in this context is the TNF family member a proliferation-inducing ligand (APRIL).^[@bib4]^
Under physiological conditions, APRIL has diverse roles in the development of B cells. It binds to its cognate receptors transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA) after which TNF receptor associated factors are recruited to the receptor that relay the signal intracellularly. APRIL has furthermore been shown to signal via binding to heparan sulfate proteoglycans on the cell surface of its target cell.^[@bib5]^ In healthy B cells, APRIL signaling has a role in the induction of CD40L-independent class-switch recombination,^[@bib6]^ proliferation,^[@bib7]^ and sustained survival of plasmablasts.^[@bib8]^ APRIL has been reported to be expressed by several cell types including macrophages,^[@bib9]^ stromal cells,^[@bib10]^ CLL cells,^[@bib11]^ and NLCs,^[@bib12]^ which are CLL cell-differentiated monocytes that have been shown to induce survival of CLL cells.^[@bib3]^ APRIL is produced as either a membrane bound or soluble factor, depending on which alternative transcript(s) is/are expressed by the cell.^[@bib13]^ Furthermore, APRIL can be synthesized as part of a hybrid transcript called TWEPRIL (TWEAK-APRIL) together with TNF-related weak inducer of apoptosis (TWEAK), after which it is anchored to the cell membrane by virtue of the TWEAK domain.^[@bib14]^ Both TWEPRIL and the secreted alpha transcript variant of APRIL can be cleaved by furin in the Golgi apparatus or at the cell membrane, respectively.^[@bib15]^ In contrast, the membrane-bound delta variant lacks the furin cleavage domain.^[@bib13]^
In its ability to support cells, APRIL contributes to the growth of several malignancies,^[@bib16]^ and serum APRIL levels are correlated with worse prognosis,^[@bib17]^ which was also shown for CLL.^[@bib18]^ Furthermore, APRIL overexpression by transgenesis in the T-cell leukemia/lymphoma 1A (Tcl-1) CLL mouse model is associated with enhanced disease severity^[@bib19]^ and APRIL transgenic mice show an enhanced proliferation of peritoneal B-1 cells,^[@bib20]^ which are considered to be the precursor cells for CLL in mice.^[@bib21]^ These effects are thought to result from induction of CLL cell survival by APRIL via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-*κ*B).^[@bib4],[@bib12]^ Altogether, these data suggest a role for APRIL in CLL biology. These findings have, however, been questioned by other reports, in which no survival effect on CLL cells was found.^[@bib22],[@bib23]^
To mechanistically dissect the role of APRIL, we used several complementary approaches to study its effects on CLL survival, activation, proliferation, and to investigate its role in macrophage-mediated survival. Surprisingly, we could not detect a direct effect of APRIL on CLL cells. Furthermore, although macrophages induce CLL survival, this effect appears to be independent of APRIL.
Results
=======
APRIL is expressed by macrophages in the CLL LN and CLL cells express APRIL receptors
-------------------------------------------------------------------------------------
We first addressed whether APRIL is expressed in the CLL LN by performing qPCR on total RNA lysates from CLL LNs. These results show that APRIL expression in CLL LNs was \~4 times higher compared with a control systemic lupus erythematosus (SLE) LN extract. As negative control, NIH-3T3 mouse embryofibroblasts (3T3) had no APRIL expression ([Figure 1a](#fig1){ref-type="fig"}).
Next, we verified this finding on protein level using immunohistochemistry by staining for APRIL and macrophage marker CD68. As APRIL has been described to induce cell proliferation,^[@bib7]^ we also stained for proliferation marker Ki67. APRIL was expressed by the large majority of CD68+ cells in both CLL and SLE LNs, but there was no spatial association with Ki67+ lymphocytes in the CLL LNs (CLL LN, [Figure 1b](#fig1){ref-type="fig"} and SLE LN, [Supplementary Figure S1](#xob1){ref-type="supplementary-material"}). Furthermore, expression of APRIL receptors BCMA and TACI was clearly detectable on CLL cells isolated from peripheral blood (PB; [Figure 1c](#fig1){ref-type="fig"}).
In summary, APRIL is expressed in the CLL LN by macrophages and APRIL receptors are present on CLL cells.
No survival effect on CLL cells by *in vitro* APRIL stimulation
---------------------------------------------------------------
To explore direct functional effects of APRIL on CLL cells, we transduced NIH-3T3 cells (DSMZ, Braunschweig, Germany) with three different membrane-docked APRIL constructs ([Figure 2a](#fig2){ref-type="fig"}). We thus generated a system similar to the widely used TNF family member CD40L overexpressing NIH-3T3 line (3T40),^[@bib24; @bib25; @bib26]^ thereby ensuring trimerization of APRIL and expression on the cell membrane. The first cell-line expresses the membrane-bound TWEPRIL hybrid mRNA, with mutated furin consensus sites to render it uncleavable (3TA). In the second and third constructs (3T4A and 3T4sA), the intracellular and transmembrane regions of CD40L were fused to the extracellular domain of APRIL, without or with an interposed spacer ('s') region. The 3T40 cell line^[@bib24; @bib25; @bib26]^ was used as a control.
APRIL expression in these cell lines was then verified by qPCR ([Figure 2b](#fig2){ref-type="fig"}) and western blot ([Figure 2c](#fig2){ref-type="fig"}), and signaling competence was tested using Jurkat-TACI:FAS (JTF) reporter cells^[@bib27]^ ([Figure 2d](#fig2){ref-type="fig"}). These JTF cells undergo apoptosis on TACI signaling as a result of intracellular FAS domains, and provide a sensitive read-out for APRIL binding to its cognate receptor ([Figure 2a](#fig2){ref-type="fig"}). Conditioned medium from APRIL overexpressing HEK293T cells (rhA med) and recombinant human APRIL (data not shown) were included as controls ([Figure 2d](#fig2){ref-type="fig"}). These data showed that all cell lines from our *in vitro* co-culture system express APRIL and that the expressed APRIL is able to signal via TACI.
These APRIL expressing 3T3 cells were subsequently used to test whether APRIL induced CLL cell survival. In contrast to 3T40 cells, we found no survival effect by any of the APRIL constructs or by rhA after 72 h co-culture ([Figure 2e](#fig2){ref-type="fig"}). Similarly, we could not detect a survival effect of conditioned supernatant from APRIL transfected HEK293T cells compared with supernatant from mock transfected cells (data not shown and [Supplementary Figure S2](#xob2){ref-type="supplementary-material"}). Using the same APRIL stimuli, survival of CLL cells was measured at later time points (3, 6 and 10 days). In accordance with the results obtained at *T*=72 h, APRIL did not significantly increase CLL survival, although a minor effect could be observed at day 10 for rhA ([Supplementary Figure S2](#xob2){ref-type="supplementary-material"}), as reported before.^[@bib28]^
No NF-*κ*B activation, activation marker expression, or cell division in CLL cells exposed to APRIL
---------------------------------------------------------------------------------------------------
As several TNF family members can induce NF-*κ*B,^[@bib29]^ we investigated if APRIL is able to induce NF-*κ*B activation by performing an NF-*κ*B DNA-binding enzyme-linked immunosorbent assay and found that, as expected, 3T40 cells induced both the canonical (p65) and non-canonical (p52) pathway in CLL cells. In contrast, no NF-*κ*B activation could be detected after stimulation with various APRIL constructs or rhA ([Figure 3a](#fig3){ref-type="fig"}), and known NF-ĸB target transcripts were not induced (data not shown).
Strong CD40 stimulation via cell-bound CD40L induces high-level NF-*κ*B activation in CLL cells. We have previously found that a CD40 stimulating antibody that induces moderate stimulation is able to upregulate activation markers CD58, CD80 and also CD95 (data not shown), indicating a higher sensitivity of this read-out. We therefore tested the potential of APRIL in this context, but in contrast to CD40L stimulation, APRIL stimulations did not upregulate the indicated markers ([Figure 3b](#fig3){ref-type="fig"}).
To study APRIL's potential involvement in CLL cell proliferation,^[@bib16]^ cell division was traced using carboxyfluorescein succinimidyl ester (CFSE) labeling, and the division index was calculated after various proliferation stimuli in the presence or absence of rhA. In line with a previous report,^[@bib2]^ we found an increased proliferation of CLL cells after stimulation with CpG+interleukin (IL)2, 3T40+CpG, and 3T40+IL21, but no effect of rhA either as a single agent or in combination with these stimuli ([Figure 3c](#fig3){ref-type="fig"}).
Summarizing, although we found that CD40L is able to induce NF-*κ*B activation in CLL cells, activation marker expression and cell proliferation in combination with CpG or IL21, similar effects were not detectable after APRIL stimulation.
Macrophage-mediated CLL survival is independent of APRIL
--------------------------------------------------------
We^[@bib30]^ and others^[@bib3]^ have previously found that monocyte-derived cells such as macrophages are able to induce survival of CLL cells, and it was suggested that survival by monocyte-derived cells is dependent on APRIL.^[@bib12]^ Although we did not observe a survival effect of stimulation with APRIL as a single stimulus ([Figure 2e](#fig2){ref-type="fig"}), the effects of APRIL could be dependent on other macrophage-expressed cytokines.
We, therefore, first generated M1 macrophages *in vitro* by differentiating healthy donor-isolated monocytes with interferon gamma (IFN-Y; R&D systems, Minneapolis, MN, USA). We then tested whether APRIL was expressed by these macrophages on western blot and found high expression in differentiated macrophages compared with low expression in monocytes and no expression in control 3T3 cells ([Figure 4a](#fig4){ref-type="fig"} inset and [Supplementary Figure S3](#xob3){ref-type="supplementary-material"}). The APRIL signaling capacity of these macrophages was then tested by comparing cell-death induced by macrophages in JTF reporter cells with the JTF death-to-rhA dose-response curve. The APRIL signaling capacity of macrophages was between that of 0 and 3.13 ng/ml rhA ([Figure 4a](#fig4){ref-type="fig"}).
To inhibit potential APRIL signaling during macrophage stimulation, we used TACI-Fc (R&D systems), a chimeric decoy receptor for APRIL.^[@bib31]^ We tested the activity of TACI-Fc by its ability to inhibit macrophage-induced cell death of JTF reporter cells cultured on macrophages. We found that TACI-Fc dose-dependently reduced APRIL signaling from macrophages ([Figure 4b](#fig4){ref-type="fig"}).
We then cultured CLL cells on macrophages and measured CLL survival in the absence or presence of 2.5 *μ*g/ml TACI-Fc, the concentration at which macrophage-induced APRIL signaling was completely reverted. These data show that abrogation of APRIL signaling did not decrease the survival effect conveyed by macrophages ([Figure 4c](#fig4){ref-type="fig"}), suggesting no direct role for APRIL in macrophage-mediated CLL survival. Similarly, when culturing CLL cells on NLCs^[@bib12]^ generated by 10 days stimulation of monocytes with CLL cells, inhibition of APRIL signaling by TACI-Fc did not affect CLL survival ([Figure 4d](#fig4){ref-type="fig"}).
Discussion
==========
We studied potential effects of TNF-family member APRIL on CLL cells, using complementary approaches and JTF reporter cells to verify the functionality of recombinantly expressed APRIL and the TACI-Fc decoy receptor. In contrast to our initial expectations, we could not detect an effect of APRIL on either CLL cell survival, cell activation, NF-*κ*B activation or cell proliferation. In addition, we could not detect a direct role of APRIL in macrophage-mediated CLL cell survival.
Various studies reported on the effects of APRIL on CLL. Although some publications show an increased *in vitro* survival of CLL cells by rhA when used at a concentration of 500 ng/ml,^[@bib4],[@bib12]^ our experiments using 200 ng/ml rhA ([Figure 2e](#fig2){ref-type="fig"}) are in line with the data of several other groups that were unable to find effects of recombinant APRIL, either alone^[@bib22]^ or in combination with B-cell activating factor and chemokine (C-X-C motif) ligand 1 (CXCL)12.^[@bib23]^ Also, we established that the amount of APRIL produced by macrophages is \>100 orders of magnitude lower compared with concentrations used in the reports that detect survival by APRIL. Although APRIL may induce survival at high concentrations,^[@bib4],[@bib12]^ this effect might be supraphysiological. Furthermore, concerning the survival effect of APRIL on non-malignant B cells, several groups have shown that APRIL is also dispensable in this context.^[@bib32]^
In the Tcl-1 mouse model for CLL, we found that overexpression of human APRIL results in enhanced disease progression and shorter survival.^[@bib19]^ In light of these results, our current *in vitro* findings were also unexpected. In the APRIL overexpressing Tcl-1 model, the construct encoding human APRIL is under control of the Lck promoter. APRIL is thus predominantly expressed by T cells, and is present in the serum at a concentration comparable to our *in vitro* systems (data not shown). As T cells not only interact with other lymphoid cells including (leukemic) B cells but also with myeloid-derived immune cells, it cannot be ruled out that the observed effects occur indirectly via other cells in the tumor microenvironment. Theoretically, the differences could also be due to distinct APRIL effects in the mouse compared with human situation.
We found no evidence that CLL proliferation is enhanced either *in vitro* or *ex vivo* by APRIL. These data are in line with another publication in which no significant proliferative effect of APRIL medium in the presence of CpG was found.^[@bib28]^ Studies on the effects of APRIL on proliferation of healthy B cells have been inconclusive; APRIL knockout mice for instance show normal B-cell proliferation *in vitro* ^[@bib33]^ and mice deficient for the APRIL receptor TACI paradoxically show increased B-cell proliferation,^[@bib34]^ whereas BCMA knockout mice show no overt phenotype.^[@bib35]^
In conclusion, our data indicate that APRIL does not directly mediate survival and proliferation of CLL cells. Consequently, APRIL signaling as therapeutic target in CLL might be beneficial in consideration that potential effects might be indirect.
Materials and methods
=====================
Patient samples
---------------
Patient material was obtained from CLL patients, after written informed consent and approval by our Ethical Review Board in agreement with the Helsinki Declaration of 1975, revised in 1983, as described before.^[@bib36]^ All samples contained at least 90% CD5^+^/CD19^+^ cells ([Supplementary Table S1](#xob4){ref-type="supplementary-material"}). In all experiments, CLL cells were used at a final concentration of 1.5x10^6^ cells/ml.
APRIL overexpression cell lines and other APRIL stimulations
------------------------------------------------------------
Mouse embryofibroblasts NIH-3T3 cells were transduced for stable overexpression with pBABE vectors expressing (1) TWEPRIL (NM_172089.3) with mutated furin cleavage sites (92RR→AA and 104RR→AA), (2) the transmembrane domain of CD40L (amino acids 1--112 of NM_000074.2) fused to the extracellular part of APRIL (amino acids 105-252 of NM_003808.3) without a linker region, or (3) with a linker region (PAAAAAASAAAAAAWVPVAT; [Figure 2a](#fig2){ref-type="fig"}), (4) CD40L,^[@bib37]^ or (5) empty vector, and all transduced cells were selected using puromycin. All constructs were sequence verified before transduction. Cells were cultured in IMDM supplemented with (IMDM+/+): 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 u/ml Penicillin-100 *μ*g/ml Streptomycin (Life Technologies, Austin, TX, USA), 2 mM [L]{.smallcaps}-glutamine (Life Technologies) and 0.00036% *β*-mercaptoethanol (Sigma, St. Louis, MO, USA). When used as adherent feeder layer, fibroblasts were irradiated (30 Gy) to stop proliferation before being seeded. After feeder cell adhering, CLL cells were plated on the respective cells. Where indicated (rhA) 200 ng/ml rhA was added to the culture medium, or culture medium conditioned on rhA-overexpressing HEK293T cells was added to the CLL cells at 80% final volume (rhA med).
Immunohistochemistry
--------------------
Paraffin embedded CLL LN tissue was obtained from our institute's pathology department. Four-micron sections were de-waxed by immersion in xylene and hydrated by serial immersion in ethanol and TBS. Antigen retrieval was performed by heating sections for 20 min in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween20, pH 6.0). Sections were washed with TBS (2×10 min) and blocked with Ultra V block (Thermo Scientific, Waltham, MA, USA) for 10 min at T~room~. Sections were incubated with primary antibody APRIL-y2 (1:1000 Enzo LifeSciences, Farmingdale, NY, USA) in normal antibody diluent (ImmunoLogic, Duiven, The Netherlands) overnight at 4 °C. After washing, sections were incubated with post-antibody block (ImmunoLogic) and subsequently incubated with secondary Polymer a-Rb/AP antibody (ImmunoLogic) followed by visualization by Vector Red (Vector Laboratories, Burlingame, CA, USA). A second antigen retrieval was performed for 10 min at 98 °C in TRIS-EDTA (pH=9.0) and after Ultra V block (Thermo Scientific), sections were incubated with a combination of primary antibodies directed against CD68 (PG-M1, Dako, Carpinteria, CA) and Ki67 (SP6, Klinipath, Duiven, The Netherlands), both 1:2000 in normal antibody diluent for 1 h at T~room~. After washing, a combination of Polymer a-Rb/AP and Polymer a-Ms/HRP (both Immunologic) was added for 30 min at T~room~ and antibody binding was visualized by Vector Blue (Vector Laboratories) and subsequent DAB (ImmunoLogic) staining after which slides were counterstained with methyl green and mounted with vectamount. Slides were visualized using a Leica DMLB microscope (Leica Microsystems, Buffalo Grove, IL, USA) equipped with a Leica DFC420 camera and cropped using Adobe Illustrator CS5 software (Adobe, San Jose, CA, USA).
APRIL reporter cell assays
--------------------------
To measure APRIL signaling, JTF reporter cells, provided as a kind gift by P. Schneider,^[@bib27]^ were cultured with different APRIL stimuli for 24 h, after which cell death of JTF cells was measured by Dioc6-PI staining as described before.^[@bib36]^
Flow cytometry and cell viability
---------------------------------
Cell viability was measured by Dioc6-PI staining as described before.^[@bib36]^ Flow cytometrical staining for APRIL receptors was performed using the TACI-PE (BD Bioscience, San Jose, CA, USA) and BCMA-FITC (Enzo) antibodies as described previously^[@bib2]^ and stained cells were analyzed on a FACS Canto II (BD). Data was then analyzed using FlowJo 9.9 (FlowJo LLC, Ashland, OR, USA).
Western blot
------------
Western blotting was performed as described previously,^[@bib38]^ using the *α*-human APRIL-y1 antibody (Abcam, Cambridge, MA, USA) and *β*-actin (Santa Cruz, Dallas, TX, USA) as a loading control. IRDye 680 donkey anti-rabbit IgG and IRDye 800 donkey anti-goat IgG (Westburg, Leusden, The Netherlands) were used as secondary antibodies.
Real-time polymerase chain reaction (qPCR)
------------------------------------------
Total RNA was isolated from paraffin-embedded CLL LN material or from APRIL overexpressing 3T3 cells using the GeneElute Mammalian Total RNA Miniprep kit (Sigma) and cDNA was created by reverse transcriptase reaction according to manufacturer's instructions (Promega, Madison, WI, USA). APRIL and household gene hypoxanthine phosphoribosyltransferase 1 (HPRT) were amplified using exon--exon boundary overlapping probes (APRIL 5ʹ-CTGCTATAGCGCAGGTGTCTT-3ʹ and 5ʹ-GGAAGGTTCCATGTGGAGAG-3ʹ; HPRT 5ʹ-CCTGGCGTCGTGATTAGTGA-3ʹ and 5ʹ-CGAGCAAGACGTTCAGTCCT-3ʹ) in a SYBR green (Life Technologies, Austin, TX, USA) reaction (40 cycles of 3 s at 95 °C followed by 30 s at 60 °C). The expression of APRIL was then calculated per sample as the difference in Ct values between the APRIL signal and HPRT signal using the formula 1000x2^-(Ct\ APRIL\ -\ Ct\ HPRT)^.
Cell proliferation assays
-------------------------
Cell proliferation was assessed by CFSE cell tracing as described before,^[@bib2]^ using 200 ng/ml rhAPRIL (rhA; Peprotech, Rocky Hill, NJ, USA) and other reagents as described before.^[@bib2]^ Division indices were calculated using FlowJo 9.9 (FlowJo LLC).
Macrophage and NLC experiments
------------------------------
Monocyte-derived macrophages and NLCs were obtained by differentiating monocytes isolated from healthy donor buffy coats obtained from the central blood bank after obtaining written informed consent. To this end, PBMCs were isolated using ficoll gradient purification (Lucron, Dieren, The Netherlands), after which monocytes were separated from PB lymphocytes using percoll gradient purification (GE healthcare, Milwaukee, WI, USA), both according to manufacturer's instructions. Next, monocytes were incubated to adhere at 37 °C in 5% CO~2~ for 40 min at a concentration of 0.75×10^6^ cells/ml in 6-well plates (3 ml) in IMDM/1% FBS and washed to remove non-adherent cells. The monocytes were then differentiated towards M1 macrophages using 10ng/ml IFN-Y in IMDM+/+ for 72 h or to NLCs by differentiating them using CLL cells for 10 days.^[@bib3]^ After washing the macrophages or NLCs twice, they were pre-incubated for 30 min with TACI-Fc or an equimolar concentration of control IgG (R&D systems) to obtain a final concentration of 2.5 *μ*g/ml of TACI-Fc. Next, thawed CLL cells were added. After 72 h, cell viability was measured as described before.^[@bib36]^
Statistical analysis
--------------------
One-way analysis of variance (ANOVA) with Tukey *post hoc* tests (comparing all groups to each other) or Dunnet's *post hoc* test (comparing all groups to one group) were performed to test for significant differences between multiple groups. When applicable, tests were adjusted for repeated measures. A two-way ANOVA with Bonferroni *post hoc* tests was used when testing for differences between groups with two independent variables. When testing for differences between two groups, a *t*-test was used. *P*-values\<0.05 (\*), *P*\<0.01 (\*\*), and *P*\<0.001 (\*\*\*) were considered statistically significant, non-significance is not indicated except in [Figures 4c and d](#fig4){ref-type="fig"}.
We would like to thank Pascal Schneider for providing the JTF reporter cells, Jan-Paul Medema, and Kate Cameron for the TWEPRIL construct and APRIL conditioned medium, and Steven Pals for the CLL LN material. APK is a Dutch Cancer Foundation fellow. This work was supported by the Dutch Cancer Foundation (Dutch Cancer Society Clinical Fellowship UVA 2011-5097).
APRIL
: a proliferation-inducing ligand
BCMA
: B-cell maturation antigen
CLL
: chronic lymphocytic leukemia
IFN-Y
: interferon gamma
IL
: interleukin
JTF
: Jurkat-TACI:FAS (reporter cells)
LN
: lymph node
NF-*κ*B
: nuclear factor kappa-light-chain-enhancer of activated B cells
PB
: peripheral blood
rhA
: recombinant human APRIL
SLE
: systemic lupus Erythematodes
TACI
: transmembrane activator and CAML interactor
TNF
: tumor necrosis factor
Tcl-1
: T-cell leukemia/lymphoma 1A
ANOVA
: one-way analysis of variance
HPRT
: hypoxanthine phosphoribosyltransferase 1
CFSE
: carboxyfluorescein succinimidyl ester
NLCs
: nurse-like cells.
Supplemental Information accompanies the paper on the *Cell Death and Discovery* website (http://www.nature.com/cddiscovery).
Edited by R Aqeilan
The authors declare no conflict of interest.
######
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{#fig1}
![APRIL does not induce CLL cell survival. (**a**) Depiction of APRIL overexpressing cell lines, control cell lines, and reporter cells used in co-culture experiments. NIH-3T3 cell lines overexpressing three different membrane-bound APRIL constructs were created (Methods section). Apoptosis in APRIL reporter JTF cells is induced on APRIL signaling, as TACI signaling triggers the FAS cell-death pathway. Full-length CD40L overexpressing 3T3 cells (3T40) and empty-vector transduced 3T3 cells (3Te.v.) are used as controls. Mutated furin sites are indicated by 'fm', the spacer region is depicted by a green line. All constructs are drawn to scale. (**b**) APRIL mRNA expression levels of the different APRIL overexpressing cell lines were tested by qPCR and compared with cells overexpressing 3Te.v. The qPCR was performed in triplo and bars show mean±S.E.M., a.u. denotes arbitrary units. (**c**) APRIL protein expression levels of the different APRIL overexpressing cell lines were tested by western blot and compared with cells overexpressing 3Te.v. The predicted molecular weights of the APRIL fusion proteins are indicated in Figure 2a. (**d**) Cell lines described in Figure 2a were seeded as feeder layers, and JTF reporter cells^[@bib27]^ were plated on top. Concurrently, JTF reporter cells were cultured in conditioned medium from APRIL (rhA med) or mock (empty med) transfected HEK293T cells. After 24 h co-culture, the percentage of dead (Dioc6 negative) JTF reporter cells was determined by Dioc6-PI staining. (**e**) CLL cells were cultured for 72 h without stimulation (--) or with 200 ng/ml rhA. Likewise, CLL cells were co-cultured on the indicated APRIL expressing or control cell lines. Next, survival was determined by Dioc6-PI staining. Viable cells were defined as Dioc6-positive cells. CD40L overexpressing feeder cells (3T40) were used as a positive control for CLL cell survival. Bars show mean±S.E.M. for *N*⩾8 for each condition. \**P*\<0.05 in a ANOVA with Tukey *post hoc* tests. When testing for significant differences, rhA was compared with unstimulated cells and 3T3 overexpression cell lines to 3Te.v.](cddiscovery201620-f2){#fig2}
{ref-type="fig"} and nuclear extracts were prepared after 24 h. The binding of activated canonical p65 and non-canonical p52 NF-*κ*B subunits to consensus sequence oligonucleotides was then determined using enzyme-linked immunosorbent assay (ELISA). CD40L overexpressing feeder cells (3T40) were used as a positive control for NF-*κ*B activation. Bars show mean±S.E.M. for *N*=3 for 3TA and 3T4sA and *N*=5 for the other conditions, respectively. \*\*\**P*\<0.001 in an ANOVA test with Tukey *post hoc* analysis. When testing for significant differences, rhA was compared with unstimulated cells and 3T3 overexpression cell lines to empty-vector transduced 3T3 cells (3Te.v.). (**b**) CLL cells were cultured as in [Figure 2e](#fig2){ref-type="fig"} for 72 h and expression levels of activation markers CD58, CD80 and of CD95 were determined using flow cytometry. CD40L overexpressing feeder cells (3T40) were used as a positive control for activation marker induction. Bars show mean±S.E.M. for *N*=3. \*\*\**P*\<0.001 in an ANOVA test for repeated measures with Tukey *post hoc* analysis. When testing for significant differences, rhA was compared with unstimulated cells and 3T3 overexpression cell lines to 3Te.v. (**c**) CFSE-stained CLL cells were cultured with various stimulations as indicated and as described,^[@bib2]^ each time with or without rhA. After 4 days, the CFSE dilution was visualized by flow cytometry and division indices were calculated. Bars show mean±S.E.M. for *N*=6. \**P*\<0.05; \*\**P*\<0.01; \*\*\**P*\<0.001; Each stimulation without rhA (light colored bars) was compared with the unstimulated condition in an ANOVA test for repeated measures with Dunnet's *post hoc* analysis and the differences between − and +rhA for each condition were determined using a two-way ANOVA with Bonferroni correction.](cddiscovery201620-f3){#fig3}
{ref-type="fig"} and the macrophage-induced cell death was plotted alongside of the rhA titration curve. All conditions were performed in triplo and mean±S.E.M. are shown. Inset: APRIL expression was determined in these macrophages (Mφ) by western blot and compared with monocytes (Mo) and untransduced 3T3 cells as negative control. (**b**) JTF reporter cells were stimulated with M1-differentiated macrophages as in Figure 4a in the presence of increasing concentration of the APRIL decoy receptor TACI-Fc (from 0.25 *μ*g/ml to 2.5 *μ*g/ml) or control IgG after which cell viability of the JTF cells was measured as in [Figure 2d](#fig2){ref-type="fig"}. (**c**) Confluent feeder layers of macrophages (Mφ) were generated as in [Figure 4a](#fig4){ref-type="fig"} and 3T40 feeder layers as in [Figure 2e](#fig2){ref-type="fig"}. These feeder layers or empty wells (Ctr) were then pre-incubated for 30 min with TACI-Fc to suppress APRIL signaling or control IgG after which CLL cells were added on these feeder layers and co-cultured for 72 h. Next, survival of the CLL cells was determined as in [Figure 2e](#fig2){ref-type="fig"}. Each point is one CLL sample (*N*=15) cultured in the indicated condition and mean±S.E.M. are indicated. \*\*\**P*\<0.001; NS, not significant, in an ANOVA test for repeated measures with Dunnett's *post hoc* analysis. (**d**) Confluent feeder layers of NLCs were generated by differentiating monocytes for 10 days using CLL cells. After washing, their survival effect on CLL cells in the presence of absence of TACI-Fc was determined as in [Figure 4c](#fig4){ref-type="fig"}. Each point is one CLL sample (*N*=12) cultured in the indicated condition and mean±S.E.M. are indicated. \*\*\**P*\<0.001; NS, not significant, in an ANOVA test for repeated measures with Dunnett's *post hoc* analysis.](cddiscovery201620-f4){#fig4}
[^1]: Shared senior authorship.
[^2]: MHAvA designed and performed the experiments, analyzed the data and wrote the manuscript; ST and ER designed and performed experiments, and analyzed the data; APK and EE designed the experiments and wrote the manuscript.
|
{
"pile_set_name": "PubMed Central"
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|
1. Introduction {#sec1}
===============
Pathological image classification is important in various biomedical applications, such as for lesion detection \[[@B1]\], cancer subtype identification \[[@B2]\], and pathological grading \[[@B3]\]. However, it is difficult for human eyes to recognize subtle differences in the tissue; there are thus different interpretations among medical experts. Moreover, pathologists generally perform the pathological image classification by the microscopic examination, which is time-consuming, operator intensive and subjective. Therefore, computer-aided diagnosis (CAD) is indispensable for pathological image analysis.
Traditionally, machine learning (ML) techniques have been widely used for different medical image processing tasks, including image detection \[[@B4]\], segmentation \[[@B5]\], and recognition \[[@B6]\]. Specifically, many researchers applied them to the field of pathological image analysis, which has achieved a great performance. For example, an automated system \[[@B7]\] was used to extract a set of texture features by multiwavelets, Gabor-filters, gray level cooccurrence matrix (GLCM), and fractal dimensions (FD) for grading pathological images of prostatic carcinoma. Experimental results showed that FD-based features set had very good performance and provided useful information for pathological image classification. If FD-based features were included in the feature set and optimized, the classification accuracy can be increased to 95.6%. Jia et al. \[[@B8]\] proposed an unsupervised network for image segmentation by learning the nonlinear distribution of medical data without prior knowledge. Experimental results demonstrated that the unsupervised one-class support vector machine (SVM) had better segmentation results than a supervised two-class SVM. To sum up, conventional machine learning methods are generally performed based on discriminative hand-craft features from manual features, but the abstract level of the manual features is relatively low, which brings difficulties for subsequent experiments.
Recently, several CAD models based on deep learning (DL) strategies have been developed for pathological image processing, which achieve the automatic extraction of features and improve the accuracy of classification. Hence, CAD using deep learning significantly reduces the subjective misjudgment of doctors. For instance, Sirinukunwattana et al. \[[@B9]\] explored the effect of the convolutional network depth on its accuracy in the localization and classification track respectively. Xu et al. \[[@B10]\] employed a deep convolutional neural network (CNN) to learn the high-level representations of histopathology images based on the transfer learning technology. In other words, initial weights of the model were determined using the optimal parameters pretrained on ImageNet. Lei et al. \[[@B11]\] accomplished a work with respect to a weakly supervised classification and disease localization using pretrained deep convolutional network. Gu et al. \[[@B12]\] employed the classical AlexNet to analyze the corresponding classification problems. The classification accuracy obtained by the proposed model can achieve to 69.9% in the task of cancer classification. Maga$\overset{\sim}{n}$a-Tellez et al. \[[@B13]\] proposed a spatially constrained convolutional neural network (SC-CNN) to perform nucleus detection and develop a novel neighboring ensemble predictor for classification of nuclei. Mercan et al. \[[@B14]\] proposed a deep learning method, which used multiple processing layers to learn representations of data through multiple levels of abstraction. Compared to traditional ML methods, DL can learn low-level features as well as high-level semantic information. Moreover, the extracted features have a stronger generalization performance than the specific settings and can be applied to multiple fields.
Generally, DL methods require a large number of training data. However, one challenge is how to deal with the lack of data samples in the field of medical images. For example, Shang et al. \[[@B15]\] proposed a novel algorithm that can preserve geometric structure information based on the feature selection framework of subspace learning and then used the *L*~2,1~-norm to ensure the sparsity of the feature array and avoid complicated solutions. However, it was expensive and impossible to train an effective model using a small amount of labeled data. To address this issue, the transfer learning technique had been used in this framework, which improved model performance. Gupta et al. \[[@B16]\] proposed a heterogeneous transfer learning framework, which extracted textual information to achieve the image classification. Experimental results showed that this framework performed better than other methods under the premise of using the less labeled data.
As previously mentioned, features extracted by DL and transfer learning are beneficial for pathological image classification. However, different features represent different image characteristics; there are thus many literatures to obtain more comprehensive information via image feature fusion. For instance, Li et al. \[[@B17]\] proposed a multimodal feature fusion-based framework to achieve representations of geographic images by leveraging a low-to-high learning flow for both the deep and shallow modality features. Banerjee et al. \[[@B18]\] presented deep-learning-based CADs for the diagnosis of subtypes of rhabdomyosarcoma (RMS) by analyzing multiparametric MR images. They achieved creating a comprehensive representation of tumor by a fusion method. Finally, they used a pretrained deep convolutional neural network to perform classification of two RMS subtypes, which achieved a fast, efficient, and reproducible diagnosis for RMS subtypes.
In this study, we propose a pathological image classification framework based on a multispace image reconstruction method and the transfer learning technology. The contributions of this paper are summarized as follows. First, the reconstructed image is a new generated image containing three channels composed of gradient, gray level cooccurrence matrix (GLCM) and local binary pattern (LBP) spaces. Specifically, the gradient image is sensitive to the boundaries and GLCM image is sensitive to regions of nuclei. The LBP image mainly highlights the center of each nucleus. Second, high-level semantic features from RGB and pseudocolor images are extracted via the pretrained VGG-16 net and the long short-term memory (LSTM) layer is used to reduce the feature dimension while increasing its discrimination capability. Our framework is evaluated using a publicly available microscopy image dataset of IICBU malignant lymphoma \[[@B19]\]. Experimental results demonstrate the performance advantages of our proposed classification framework by comparing with the related works.
The remainder of this paper is organized as follows. In [Section 2](#sec2){ref-type="sec"}, we introduce NHL pathological image dataset and our classification framework. [Section 3](#sec3){ref-type="sec"} shows the classification results and a comprehensive comparison with some other methods. Finally, discussions and conclusions are summarized in Sections [4](#sec4){ref-type="sec"} and [5](#sec5){ref-type="sec"}, respectively.
2. Materials and Methods {#sec2}
========================
2.1. Dataset Description {#sec2.1}
------------------------
The IICBU lymphoma dataset contains 374 hematoxylin and eosin (H&E) stained non-Hodgkin lymphoma (NHL) images, which are divided into three classes: chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). They are the major types of malignant small B-cell lymphoma \[[@B19]\]. The dataset sections are captured using the bright field microscopy and each image is 1388 × 1040 pixels. In our experiments, each image is divided into 336 nonoverlapped patches with 64 × 64 pixels (21 × 16); we perform the classification tasks in the patch level and image level, and the image classification results are determined via the classification results of the corresponding patches.
2.2. Pathological Image Classification {#sec2.2}
--------------------------------------
As shown in [Figure 1](#fig1){ref-type="fig"}, the proposed process of the pathological image classification is described, which is summarized as four stages, including multispace image reconstruction, feature extraction, feature selection, and image classification. First, a multispace image reconstruction method is developed to generate new color images, which contain the gradient, GLCM, and LBP information adequately. Second, the VGG-16 net pretrained on ImageNet is applied to original images and reconstructed images for feature extraction. Third, the long short-term memory (LSTM) layer is used for feature selection. Finally, the softmax classifier is utilized to perform the classification task.
### 2.2.1. Multispace Image Reconstruction {#sec2.2.1}
Generally, H&E images vary significantly in color, because of many factors, including specimen preparation and staining protocol inconsistencies (e.g., temperature of solutions); variations in fixation characteristics and interpatient variation and the scanner are used to digitize the slides. The classification performance could be hampered by color and intensity variations. Many classification tasks implemented by DL strategies not only considered RGB inputs but also took the advantage of other spaces\' inputs, such as HSV, Lab, and YUV \[[@B20]\]. Inspired by these works, we first use the white balance method to alleviate color influence and then convert RGB images into grayscale images. Besides, in order to consider more beneficial information, we propose a novel multispace reconstruction method to generate a new image which is composed of the gradient, GLCM and LBP spaces\' images. In other words, the R, G, and B channels of a reconstructed image are represented as the gradient, GLCM, and LBP spaces\' images, respectively. [Figure 2](#fig2){ref-type="fig"} presents the generative process of a reconstructed image. [Figure 2(a)](#fig2){ref-type="fig"} is an original H&E pathological image. Figures [2(b)](#fig2){ref-type="fig"}--[2(e)](#fig2){ref-type="fig"} represent the grayscale, gradient, GLCM, and LBP images. [Figure 2(f)](#fig2){ref-type="fig"} is the generated reconstructed image. Visually, the gradient image is sensitive to boundaries and the GLCM image is sensitive to the regions of nuclei. The LBP image mainly emphasizes the center of each nucleus. The following experiments demonstrate that these three kinds of information are effective for the pathological image classification.
### 2.2.2. Feature Extraction via VGG-16 Net {#sec2.2.2}
As is well known, the effective classification greatly depends on the discriminative representations of samples. Some literatures \[[@B21]\] treated the feature extraction and classifier design as two separated processes; they could not work together to maximally extract and retain the most discriminative information. Recently, deep learning strategies are widely applied to the feature extraction on different image classification tasks. Particularly, conventional convolutional neural network (CNN) \[[@B22]\] is consisted of alternating convolution and subsampling operations. Flatten operation was used on the feature maps of the last convolution layer to obtain the feature vectors. The next several fully connected layers were applied one by one on the feature vectors. These series of operations were regarded as a process of feature extraction. In addition, the transfer learning in \[[@B23]\] used the parameters of trained models (via natural images) to extract medical images\' features or initialized the parameters of a particular model. Motivated by \[[@B24]\], our strategy uses the pretrained VGG-16 \[[@B25]\] (Visual Geometry Group) net for performing the feature extraction on lymphoma patches. VGG-16 is a simple and common deep convolutional neural network and can obtain the competitive performances with other networks. The proposed feature extraction process using VGG-16 net is described in [Figure 3](#fig3){ref-type="fig"}. The network is trained on the entire ImageNet \[[@B26]\] dataset and our input images are all resized to 64×64 pixels. The corresponding outputs are 2048-dimensional features. The specific network structure for our classification task is presented in [Table 1](#tab1){ref-type="table"}. Note that the pretrained VGG-16 net is used for the feature extraction on RGB images and the reconstructed patches simultaneously.
### 2.2.3. Feature Selection by LSTM {#sec2.2.3}
Due to the great differences between natural images and pathological images, the features extracted by pretrained VGG-16 net may not obtain the satisfactory classification results. A common method for addressing this issue is to initialize the model\'s parameters using the pretrained net and then train our images to adjust the parameters. However, there is no essential difference compared with training own data directly, and it also could lead to the overfitting. To this end, we propose a "feature selection strategy" to find the discriminative features and remove the redundant features using the long short-term memory (LSTM) layer. The LSTM \[[@B27]\] is an efficient network for text classification tasks, which considers the relationship between the text and time information. For our case, the relationship between original RGB images and corresponding reconstructed images is similar to temporal text data. Specifically, redundant features are removed via the forget gate, then beneficial features are added to the input gate and the optimal feature vector is obtained by the output gate. [Figure 1](#fig1){ref-type="fig"} shows the feature selection process using the LSTM layer. The VGG-16 features of RGB images are regarded as the original content information (text information in LSTM), while the VGG-16 features of the corresponding reconstructed images are considered as the auxiliary information (temporal information in LSTM). There are 32-dimension features selected via the LSTM layer. Afterwards, we cascade the 32-dimension features of RGB images and the reconstructed images into a 64-dimension feature vector. Finally, the softmax classifier is used to perform the pathological image classification task. Note that the integral loss function is defined as follows in ([1](#EEq1){ref-type="disp-formula"})-([3](#EEq3){ref-type="disp-formula"}).$$\begin{matrix}
{Loss = {Loss}_{original} + {Loss}_{reconstruction}} \\
\\
\end{matrix}$$$$\begin{matrix}
{{Loss}_{original} = - {\sum_{i}y_{i}}{\log a_{i}}} \\
\\
\end{matrix}$$$$\begin{matrix}
{{Loss}_{reconstruction} = - {\sum_{i}y_{i}}{\log b_{i}}} \\
\\
\end{matrix}$$where *y*~*i*~ represents the label of each sample. *a*~*i*~ and *b*~*i*~ are the output probabilities obtained via the softmax function *e*^*z*~*i*~^/∑~*k*~*e*^*z*~*k*~^, *z*~*i*~ = ∑~*j*~*w*~*ij*~*x*~*ij*~ + *b* is the output of the neuron, *x*~*ij*~ represents the feature vectors of the RGB and reconstructed inputs, respectively, and *w*~*ij*~ and *b* are the corresponding weights and bias. We can observe that if one loss function (*Loss*~*original*~ or *Loss*~*reconstruction*~) leads to the vanishing gradient, the feedback information from another loss function can also train the LSTM layer. In order to remove redundant information in the features, the LSTM layer for text classification tasks is used in this paper by analyzing each row of the feature matrix recursively, and it has produced good results.
3. Results and Comparisons {#sec3}
==========================
3.1. Experimental Results {#sec3.1}
-------------------------
In order to verify the effectiveness of the proposed framework, three commonly used evaluative criteria are considered as follows in ([4](#EEq4){ref-type="disp-formula"})-([6](#EEq6){ref-type="disp-formula"}).$$\begin{matrix}
{ACC = \frac{1}{n}{\sum\limits_{i = 1}^{n}I}\left( { f\left( { x_{i}} \right) = y_{i}} \right)} \\
\\
\end{matrix}$$$$\begin{matrix}
{SEN\left( { c_{i}} \right) = \frac{Num\left( {PT\left( c_{i} \right)} \right)}{Num\left( {GT\left( c_{i} \right)} \right)} \times 100\%} \\
\\
\end{matrix}$$$$\begin{matrix}
{SPE\left( { c_{i}} \right) = \frac{Num\left( {PT\left( {- c_{i}} \right)} \right)}{Num\left( {GT\left( {- c_{i}} \right)} \right)} \times 100\%} \\
\\
\end{matrix}$$where ACC means the overall grading accuracy. SEN(*c*~*i*~) and SPE(*c*~*i*~) are sensitive and specificity for each class (*c*~*i*~). I(*f*(*x*~*i*~) = *y*~*i*~) is the indicative function. It defines that if *f*(*x*~*i*~) = *y*, I(*f*(*x*~*i*~) = *y*~*i*~) = 1; otherwise I(*f*(*x*~*i*~) = *y*~*i*~) = 0. −*c*~*i*~ denote all the other classes except (*c*~*i*~). Num(PT(*c*~*i*~)) and Num(GT(*c*~*i*~)) denote the number of correctly predicted class (*c*~*i*~) and the total number of (*c*~*i*~) in the ground truth, respectively.
In our experiments, first, we perform the NHL classification tasks using the VGG-16 features ([Section 2.2](#sec2.2){ref-type="sec"}). Our experiments adopt 10-cross validation method to determine the optimal parameters of each model. [Table 2](#tab2){ref-type="table"} presents the different classification results with the VGG-16 features extracted on the original and reconstructed images. We can observe that the feature combination (4096-dimension) is beneficial for our classification task. It can be also proved that reconstructed images can provide complementary information with respect to RGB images. As is well known, more features could not denote that they can achieve a better result. This is because that there are redundant features which can result in the worse classification results. To address this issue, the feature selection strategy is widely used for the more accurate classification result while reducing the computational cost. [Table 3](#tab3){ref-type="table"} shows different classification results using different features selected by the LSTM layer. It can be observed that the LSTM layer is effective for feature selection and improving classification accuracy. Following these experimental results, our proposed framework for NHL image classification is determined. In addition, [Figure 4](#fig4){ref-type="fig"} shows the classification results according to different iteration times and the best time is 250.
3.2. Image-Level Classification {#sec3.2}
-------------------------------
As mentioned before, our objective of this paper is to classify 374 NHL pathological images, which include CLL, FL, and MCL. Our proposed classification framework is performed on the image patches with 64×64×3; the image-level classification is thus accomplished via the label of each patch inside a whole pathological image. Specifically, a pathological image is divided into 336 nonoverlapped patches and the label of each patch is obtained via the proposed classification framework. We can thus determine the pathological image\'s label using the majority voting strategy via the number of each category\'s patches. To visualize the classification results of the whole pathological images, [Figure 5](#fig5){ref-type="fig"} shows the probability atlases of three examples with different classes. Figures [5(a)](#fig5){ref-type="fig"}--[5(c)](#fig5){ref-type="fig"} are CLL, FL, and MCL pathological images. Figures [5(d)](#fig5){ref-type="fig"}--[5(f)](#fig5){ref-type="fig"} are corresponding probability atlases. The colors in the probability atlases represent the probability that each patch is predicted to be a label of the image categories. The colors from bottom to top is ranged from \[0,1\]. According to the probability atlases, we can see that majority of patches inside a pathological image are correctly classified as the corresponding image\'s label.
3.3. Comparisons {#sec3.3}
----------------
To further evaluate our proposed classification framework with other models, there are three popular methods \[[@B28]--[@B30]\] used for comparison in our experiments. Shamir et al. \[[@B28]\] developed CAD software for biological image analysis. This software worked by first extracting image content descriptors from the raw images, image transforms, and compound image transforms. Then, the most informative features were selected and then used for classification and similarity measurement. Meng et al. \[[@B29]\] proposed a framework using the novel and robust collateral representative subspace projection modeling (CRSPM) supervised classification model for general histology image classification. Codella et al. \[[@B30]\] first created additional 5 images for each H&E pathological image, which can emphasize unique aspects of the original image, such as dominant staining and staining segmentations. Then a pretrained CNN model was used to extract 4096-dimension visual features. Finally, nonlinear SVMs were utilized to perform the classification. Similarly, all methods are performed on NHL dataset and tested using 10-cross validation method. The average comparison results are presented in [Figure 6](#fig6){ref-type="fig"}. We can observe that our method performs the best in terms of ACC.
4. Discussions {#sec4}
==============
According to the classification results in [Section 3.1](#sec3.1){ref-type="sec"} (Tables [2](#tab2){ref-type="table"} and [3](#tab3){ref-type="table"}), there are two important characteristics with respect to our proposed classification framework. First, besides RGB image information, we also consider the other classes of information via a "multispace image reconstruction" strategy. As [Figure 2](#fig2){ref-type="fig"} is shown, the gradient image is sensitive to boundaries and GLCM image is sensitive to regions of nuclei. The LBP image mainly highlights the center of each nucleus. The results indicate that this auxiliary information facilitates the improvement of classification accuracy significantly. Second, in order to remove redundant features, the pretrained LSTM layer is used to perform a "feature selection" process. Further, our classification framework obtains the ACC of 98.94% in patch-level classification.
Comparing results from our classification framework with other methods, the software proposed in \[[@B28]\] only used hand-craft features to perform classification tasks and considered no high-level semantic features (extracted via deep learning model). Our method and \[[@B29]\] both converted the image-level classification into the patch-level classification. A difference from \[[@B29]\] was that it divided the image into 25 overlapped patches with a larger size. This, however, may lose more subtle local information. Finally, the method in \[[@B30]\] utilized a pretrained CNN to extract the visual features from different images. Our improvement related to \[[@B30]\] is that we adopt a feature selection method to increase its discrimination capability. The results in [Figure 6](#fig6){ref-type="fig"} demonstrate the advantages of our proposed classification framework.
5. Conclusions {#sec5}
==============
In this paper, we proposed a novel classification framework based on a multispace image reconstruction method and the transfer learning technology. The multispace image reconstruction mapping method can convert original RGB images into gradient, gray level cooccurrence matrix (GLCM), and local binary pattern (LBP) spaces. This auxiliary information is beneficial for more accurate classification results. Then, the pretrained VGG-16 net was utilized to extract the high-level semantic features of RGB and reconstructed images. Subsequently, the LSTM layer was used for feature selection and refinement while increasing its discrimination capability. Experimental results demonstrated the performance advantages of our proposed classification framework by comparing with related works. Future work may be considered based on our proposed framework. For feature extraction, we plan to establish a complete feature set, which includes hand-craft features and high-level representations. Another important work will consider the feature extraction on more other spaces\' images.
This work was supported by the National Natural Science Foundation of China (No. 61872075).
Data Availability
=================
\(i\) The dataset includes three types of malignant lymphoma. (ii) The data can be accessed from <https://ome.grc.nia.nih.gov/iicbu2008/lymphoma/index.html>. In addition, there are no restrictions on data access.
Conflicts of Interest
=====================
The authors declare that they have no conflicts of interest.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
{#fig5}
{#fig6}
######
The network structure for our classification task.
Layer Filter number Kernel size Dimension
----------- --------------- ------------- ---------------
Input \- \- 64 × 64 × 3
Conv1 64 3 × 3 64 × 64 × 64
Conv2 64 3 × 3 64 × 64 × 64
Max-Pool1 \- 2 × 2 32 × 32 × 64
Conv3 128 3 × 3 32 × 32 × 128
Conv4 128 3 × 3 32 × 32 × 128
Max-Pool2 \- 2 × 2 16 × 16 × 128
Conv5 256 3 × 3 16 × 16 × 256
Conv6 256 3 × 3 16 × 16 × 256
Conv7 256 3 × 3 16 × 16 × 256
Max-Pool3 \- 2 × 2 8 × 8 × 256
Conv8 512 3 × 3 8 × 8 × 512
Conv9 512 3 × 3 8 × 8 × 512
Conv10 512 3 × 3 8 × 8 × 256
Max-Pool4 \- 2 × 2 4 × 4 × 256
Conv11 512 3 × 3 4 × 4 × 512
Conv12 512 3 × 3 4 × 4 × 512
Conv13 512 3 × 3 4 × 4 × 512
Max-Pool5 \- 2 × 2 2 × 2 × 512
######
Different classification results using RGB (2048-dimension), reconstruction (2048-dimension), and combination features (4096-dimension).
Index Classes RGB Reconstruction Combination
------- --------- ------------------ ------------------ ------------------
ACC Overall (0.5317 ± 0.016) (0.4423 ± 0.016) (0.6667 ± 0.008)
CLL (0.5205 ± 0.011) (0.4214 ± 0.010) (0.6414 ± 0.010)
SEN FL (0.5179 ± 0.010) (0.4187 ± 0.013) (0.6396 ± 0.018)
MCL (0.5224 ± 0.009) (0.4226 ± 0.009) (0.6428 ± 0.009)
CLL (0.5515 ± 0.008) (0.4602 ± 0.004) (0.6806 ± 0.005)
SPE FL (0.5469 ± 0.012) (0.4584 ± 0.011) (0.6789 ± 0.007)
MCL (0.5524 ± 0.011) (0.4628 ± 0.014) (0.6833 ± 0.009)
######
Different classification results using RGB (32-dimension), reconstruction (32-dimension), and combination features selected by the LSTM layer (64-dimension).
Index Classes RGB-LSTM Reconstruction-LSTM Combination-LSTM
------- --------- ------------------ --------------------- ------------------
ACC Overall (0.8453 ± 0.018) (0.7637 ± 0.012) (0.9894 ± 0.011)
CLL (0.8243 ± 0.014) (0.7459 ± 0.019) (0.9666 ± 0.012)
SEN FL (0.8168 ± 0.010) (0.7321 ± 0.009) (0.9662 ± 0.011)
MCL (0.8251 ± 0.019) (0.7482 ± 0.015) (0.9685 ± 0.013)
CLL (0.8721 ± 0.014) (0.7832 ± 0.008) (0.9931 ± 0.007)
SPE FL (0.8659 ± 0.011) (0.7779 ± 0.007) (0.9912 ± 0.005)
MCL (0.8718 ± 0.012) (0.7834 ± 0.011) (0.9938 ± 0.013)
[^1]: Academic Editor: Ruijin Huang
|
{
"pile_set_name": "PubMed Central"
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