The objective was to compare thoracic spinal stiffness between healthy participants and participants with chronic thoracic pain and to explore the associations between spinal stiffness, pain and muscle activity. The reliability of spinal stiffness was also evaluated.Spinal stiffness was assessed from T5 to T8 using a mechanical device in 25 healthy participants and 50 participants with chronic thoracic pain . The spinal levels for which spinal stiffness was measured were standardized  to minimize between-individual variations due to the evaluation of different spinal levels. The device load and displacement data were used to calculate the global and terminal spinal stiffness coefficients at each spinal level. Immediately after each assessment, participants were asked to rate their pain intensity during the trial, while thoracic muscle activity was recorded during the load application using surface electromyography electrodes (sEMG). Within- and between-day reliability were evaluated using intraclass correlation coefficients (ICC), while the effects of chronic thoracic pain and spinal levels on spinal stiffness and sEMG activity were assessed using mixed model ANOVAs. Correlations between pain intensity, muscle activity and spinal stiffness were also computed.F1,73 = 4.04, p = 0.048) and terminal  spinal stiffness was observed in participants with thoracic pain. sEMG activity was not significantly different between groups and between spinal levels. Pain intensity was only significantly and "moderately" correlated to spinal stiffness coefficients at one spinal level (-0.29≤r≤-0.51), while sEMG activity and spinal stiffness were not significantly correlated.ICC values for within- and between-day reliability of spinal stiffness ranged from 0.67 to 0.91 and from 0.60 to 0.94 (except at T5), respectively. A significant decrease in the global (The results suggest that spinal stiffness can be reliably assessed using a mechanical device and that this parameter is decreased in participants with chronic thoracic pain. Studies are required to determine the value of instrumented spinal stiffness assessment in the evaluation and management of patients with chronic spine-related pain. Back and neck pain are very common musculoskeletal conditions in the general population, with a one-year prevalence of lumbar, thoracic and neck pain respectively estimated up to 43%, 35% and 32%., 2 The pSMT is often based on the clinician’s capacity to identify mechanical changes in the spine and to select an appropriate treatment option. The identification of a "dysfunctional" spinal level that could benefit from SMT is partly based on segmental motion analysis, which can be assessed with the patient in a static position  or in motion ., 6 In clTo increase the validity and reliability of the assessment of spinal stiffness, mechanical devices allowing assessment standardization have been developed. These devices usually consist of an indenter, a load cell and a displacement sensor, and assess spinal stiffness by gradually applying a specific load over the targeted spinous process with a controlled velocity. In additThe assumption that SMT modulates spinal stiffness and that this latter could be used as both a diagnostic and a prognostic tool in patients with spine-related pain is at least partly supported by the current literature. Indeed, two distinct studies , 16 haveConsidering this evidence, the main objective of the present study was to compare thoracic spinal stiffness between healthy participants and participants with chronic thoracic spinal pain and, in an attempt to better understand the mechanisms by which spinal stiffness could be modulated in participants with chronic thoracic pain, to evaluate the associations between spinal stiffness and both muscle activity and pain intensity during the measurement. Furthermore, since spinal stiffness reliability has rarely been investigated in the thoracic spine, and has yet not been compared between several contiguous spinal levels, the within- and between-day reliability has also been evaluated using a subgroup of the participants.dx.doi.org/10.17504/protocols.io.twwepfe) and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.Twenty-five healthy participants and 50 participants with chronic thoracic pain were recruited in the University community through its web portal and in the general population using a word-of-mouth strategy. The thoracic spine region was defined as the region bounded superiorly by the tip of first thoracic spinous process, inferiorly by the tip of the last thoracic spinous process, and laterally by the most lateral margins of the erector spinae muscles. InclusioTo assess the within- and between-day reliability of spinal stiffness assessment, all healthy participants and the first 25 participants with chronic thoracic pain took part in two identical experimental sessions that were held within 24 to 48 hours. The remaining 25 participants with chronic thoracic pain participated in the first experimental session only. At the beginning of each session, participants were asked to complete a Visual Analog Scale (VAS) for pain , the QueSurface electromyography (sEMG) electrodes (bipolar electrodes with a 8 mm inter-electrodes distance) were applied bilaterally at approximately 2 cm from the spine (over the belly of the thoracic erector spinae muscles and in line with muscle fibers), at the level of T5 and T8 spinous processes and between T6 and T7 spinous processes . Before applying the electrodes, the skin was shaved, gently abraded with fine-grade sandpaper , and cleaned with alcohol swabs. All sEMG were recorded at 2000 Hz using Trigno Wireless EMG sensors , which contain two differential EMG inputs with two patented stabilizing references and therefore this system does not require an external reference electrode. Participants were then required to relax quietly on the table while sEMG activity was recorded during 4 s using EMGworks 4.2 software . The signal recorded by each electrode during this trial  was subsequently used to normalize the respective electrode signal obtained during the various spinal stiffness assessments.http://www.randomization.com⟩ was used to determine in which order the spinal levels would be assessed for each participant. These spinal levels were selected a priori for all participants due to technical restriction and to minimize between-individual variations due to the evaluation of different spinal levels. The The28] TImmediately following each spinal stiffness assessment, participants were asked to rate the mean pain intensity perceived during the trial on a 101-points VAS . Pain could not be rated during the assessment itself since it would have modified the lung/chest volume and therefore spinal stiffness.nd order Butterworth filter). A low 40 Hz cut-off allowed filtering of the electrocardiogram signal limiting contamination of the sEMG signal. The root mean square (RMS) value was computed for each electrode over a 1 s window during the assessment of spinal stiffness (between 10 and 45 N force application). The RMS value obtained for each electrode was then normalized (later referred to nRMS) to the respective RMS value calculated during the sEMG normalization trial. The mean value of the closest right and left EMG electrodes during the assessment of spinal stiffness  was then used during subsequent analyses.To assess the muscle activity during the assessment of spinal stiffness, the resulting bipolar sEMG signals were first digitally band-pass filtered in the frequency bandwidth 40–400 Hz  script was developed to calculate the global and the terminal spinal stiffness coefficients using the force-displacement data of each spinal stiffness assessment . Global T-test for independent samples or Mann-Whitney U test. Differences in clinical status between the two sessions were also evaluated independently for the healthy participants and for the participants with chronic thoracic pain subset using, when appropriate, Wilcoxon’s matched pairs test or T-tests for dependent samples. For all analyses, statistical significance was set at p ≤ 0.05 and analyses were performed using STATISTICA 8  and SPSS Statistics 21 .Differences in baseline characteristics between healthy participants and participants with chronic thoracic pain (whole group and reliability subgroup) were assessed using, when appropriate, 3,1 and the guideline provided by Cicchetti [The data from all healthy participants (n = 25) and the first 25 participants with chronic thoracic pain were used to assess spinal stiffness measurement reliability. Within- and between-day reliability was determined using ICCicchetti  for strer ≥ 0.70), "good" (0.50 ≤ r < 0.70), "moderate" (0.30 ≤ r < 0.50) or "poor" (r < 0.30).[To investigate differences in spinal stiffness between groups and between spinal levels, as well as possible interaction effects between these variables, mixed model analyses of variance (ANOVAs) were independently computed for the global and the terminal stiffness coefficients. The dependent variables  were subjected to ANOVAs comparing responses across two groups  and four spinal levels . Similarly, between-groups differences regarding muscle activity amplitude (nRMS) during assessment were determined using a 2(groups) x 4 mixed-model ANOVAs. Post hoc tests using Bonferroni correction were computed for significant effects. Between-group comparisons for pain intensity during assessment were performed using simple comparisons of proportions (Fisher’s exact test) and descriptive analyses. Associations between spinal stiffness and both muscle activity (nRMS) and pain intensity during assessment were explored through Pearson’s correlation or its estimated value obtained from Kendall tau rank correlation coefficient (for non-parametric data). The importance of the correlation was evaluated as being "strong" ( < 0.30).p values > 0.05). Participants with chronic thoracic pain reported significantly higher score on the self-reported clinical outcomes compared to healthy participants . Although participants with chronic thoracic pain showed a statistically significant decrease in their QBPDQ score between the two sessions , this decrease was not clinically significant.[Participants’ baseline characteristics are presented in nificant.Reliability analyses showed ICC values suggesting an overall "good" to "excellent" within- and between-day reliability in participants with and without chronic thoracic pain in exception of T5 between-day reliability in healthy participants . Most ofF1,73 = 4.04, p = 0.048, F1,73 = 4.93, p = 0.03, F3,219 = 3.42, p = 0.02, p = 0.08). No groups by spinal levels interaction effect was observed for the two spinal stiffness coefficients (p > 0.05). Box plots of spinal stiffness are shown on A significant main effect of groups for both the global .p values < 0.001) of participants with chronic thoracic pain reported pain during spinal stiffness assessment (-6.50 < z < -5.10). Indeed, 13%, 24%, 13% and 10% of healthy participants reported pain during the assessment of spinal stiffness at T5, T6, T7 and T8, while these numbers were respectively 89%, 90%, 82% and 73% in participants with chronic thoracic pain. In healthy participants, pain intensity was significantly correlated with the global  and the terminal  spinal stiffness coefficient but only at T6. In participants with chronic thoracic pain, this correlation was only significant, and of lower strength, at T5  and only for the terminal spinal stiffness.All participants could tolerate the 45 N load. A statistically significant higher proportion  was observed in both participants with and without chronic thoracic pain. To our knowledge, only one previous study aimed to compare instrumented spinal stiffness assessment reliability between participants with and without spine-related pain. Indeed, Wong et al. 2013) reported within- and between-day ICC values over 0.98 in both healthy participants and participants with chronic LBP. The high reportedThe minimal difference representing a clinically meaningful change in spinal stiffness has yet to be established and, therefore, it is not known if the modulation in spinal stiffness observed in the participants with chronic thoracic pain is clinically relevant. Although no relationship between muscle activity and spinal stiffness was observed in the current study, it cannot be excluded that deeper muscles, such as multifidus muscles, might have increased their myoelectric activity in response to pain during the assessment of spinal stiffness. Researchers should consider using intramuscular electromyography electrodes to evaluate the potential activation of deeper muscles. Moreover, comfort during spinal stiffness assessment should be optimized to alleviate muscle contraction due to pain during evaluation. This issue can be achieved by increasing the size of the indenter and/or modifying the indenter padding. Another Clinicians should be aware that perception of a variation in spinal stiffness between adjacent spinal levels or between individuals might not represent true variations and can simply be due to human factors, such as anthropometric , 41 and This study is the first to measure spinal stiffness at multiple thoracic spinal levels. The results demonstrated a decrease in thoracic spinal stiffness in participants with chronic thoracic pain compared to healthy participants. The data does not support the hypothesis of an association between spinal stiffness and a muscle contraction due to pain provocation during the measurement. More research is thus required to determine the value of instrumented spinal stiffness assessment in the evaluation and management of patients with chronic spine-related pain.S1 File(XLS)Click here for additional data file.


BEA was the predominant toxin detected, followed by ENB, ENA1, ENA, and ENB1. Corn and corn-based samples were more easily contaminated by BEA with an average concentration of 65.26 µg/kg, compared with that in wheat and wheat-based samples (average = 0.41 µg/kg). Concentrations of BEA, ENA, and ENB1 in corn kernels, flours, and flakes were significantly different , as well as for BEA, ENA, ENB, and ENB1 in wheat kernels, flours, and noodles . Furthermore, 59.5% (94/158) and 59.8% (174/291) corn- and wheat-based samples were co-contaminated by at least two mycotoxins, respectively. Positive correlations in concentrations were observed in corn between levels of ENA and ENB1, ENA and ENB, ENA1 and ENB1, as well as in wheat between BEA and ENA, BEA and ENA1, BEA and ENB, BEA and ENB1, ENA and ENA1, ENA and ENB, ENA and ENB1, ENA1 and ENB, ENA1 and ENB1, and ENB and ENB1. These results demonstrate that co-contamination of BEA and enniatins (ENNs) in corn- and wheat-based samples from Shandong, China is very common. More data on the contamination of five mycotoxins in cereal and cereal-based samples nationwide are needed.Totals of 158 corn and corn-based samples and 291 wheat and wheat-based samples from Shandong province, China in 2017 were analyzed for five mycotoxins including beauvericin (BEA), enniatin A (ENA), enniatin A Fusarium genus such as F. oxysporum, F. avenaceum, F. equiseti, and F. poae. They are structurally related to cyclic hexadepsipeptides, consisting of three alternating hydroxyisovaleryl and N-methylamino acid residues [1 (ENA1), B (ENB), and B1 (ENB1) are frequently detected in various foods and feeds [Beauvericin (BEA) and enniatins (ENNs) are emerging mycotoxins mainly produced by fungi of the residues ,2. In tond feeds .1 such as 0.51 mg/kg to 11.78 mg/kg for BEA and 33.38 mg/kg to 814.42 mg/kg for ENA1 were found in cereals from Spain [BEA and ENNs have been shown to exhibit a variety of biological properties including antibiotic, anti-insect, antifungal, and cytotoxic activities ,5,6,7,8.1 with a positive rate of 56.3% (89/158) and the levels ranging from 0.07 μg/kg to 3.33 μg/kg . Of all the 158 samples analyzed, one was contaminated with BEA at levels higher than 1000 μg/kg, two between 500 μg/kg and 1000 μg/kg, 20 between 100 μg/kg and 500 μg/kg, and 135 below 100 μg/kg. ENA1 and ENB were present at much lower frequencies than those detected for BEA, ENA, and ENB1 in all 158 analyzed samples, with a positive rate of 8.2% (13/158) and the levels ranging from 0.02 μg/kg to 6.29 μg/kg  for ENA1, and a positive rate of 3.8% (6/158) and the levels ranging from 0.05 μg/kg to 3.21 μg/kg  for ENB, respectively. In addition, the concentrations of BEA, ENA, and ENB1 were significantly different in corn kernels, flours, and flakes ; however, no significant difference was found for ENA1  or ENB  in corn kernels, flours, and flakes. In terms of 71 corn kernels, the positive rates of five mycotoxins from highest to lowest were BEA > ENA1 and ENB1 > ENA > ENB. BEA was detected in 85.9% (61/71) samples with the levels ranging from 0.06 μg/kg to 1006.56 μg/kg , which was higher than those found for ENB1, ENA1, ENA, and ENB in corn kernels. In detail, the positive rate of ENB1 in 71 corn kernels was 4.2% (3/71) with the levels ranging from 0.10 μg/kg to 0.32 μg/kg , the positive rate of ENA1 was 4.2% (3/71) with the levels ranging from 0.09 μg/kg to 0.16 μg/kg , and the positive rate of ENA was 2.8% (2/71) with the levels ranging from 0.09 μg/kg to 0.17 μg/kg  in corn kernels. No ENB was found in 71 corn kernels. For 68 corn flour samples, the frequencies and concentrations of five mycotoxins in 68 corn flours were higher than those found in 71 corn kernels. The prevalence of five mycotoxins in 68 corn flour samples studied from highest to lowest was ENB1 > ENA > BEA > ENA1 > ENB, but the average concentration of BEA (86.45 µg/kg) was higher than those of the other four mycotoxins ENA, ENA1, ENB, and ENB1. ENB1 was present with the highest frequency of 98.5% (67/68) with the levels ranging from 0.08 μg/kg to 3.33 μg/kg, followed by ENA with a positive rate of 97.1% (66/68) and the levels ranging from 0.06 μg/kg to 16.61 μg/kg, and BEA with a positive rate of 95.6% (65/68) and the levels ranging from 0.04 μg/kg to 860.16 μg/kg. Furthermore, the positive rates of ENA1 and ENB in 68 corn flour samples were quite low with a positive rate of 14.7% (10/68) for ENA1 and 4.4% (3/68) for ENB. For 19 corn flakes, no ENA1 and ENB were detected and only four (21.1%) samples were positive for BEA; however, all 19 samples were positive for ENA and ENB1. It was found that the levels of BEA, ENA, and ENB1 in the 19 corn flakes studied were all very low with a maximum of 0.2 μg/kg for BEA, 0.07 μg/kg for ENA, and 0.13 μg/kg for ENB1, respectively.1 > ENA > ENA1. The detailed positive rate from highest to lowest for 291 wheat and wheat-based samples was 58.1% for ENB with the levels ranging from 0.02 μg/kg to 56.31 μg/kg , 56.4% for BEA with the levels ranging from 0.08 μg/kg to 11.1 μg/kg , 54.6% for ENB1 with the levels ranging from 0.10 μg/kg to 61.77 μg/kg , 37.5% for ENA with the levels ranging from 0.09 μg/kg to 3.30 μg/kg , and 22.0% for ENA1 with the levels ranging from 0.10 μg/kg to 20.91 μg/kg . It was also found that the contamination of BEA, ENA, ENB, and ENB1 in wheat and wheat-based samples varied by type , but not for ENA1 . For the 75 wheat kernels studied, the positive rate of five mycotoxins from highest to lowest was BEA (48.0%) > ENB (16.0%) > ENB1 (13.3%) > ENA (12.0%) and ENA1 (12.0%). It should be noted that there were three samples with ENB, three samples with ENB1, and one sample with ENA1 at levels higher than 20 μg/kg, with the maximum of 56.31 μg/kg  for ENB, 61.77 μg/kg  for ENB1, and 20.91 μg/kg  for ENA1, respectively. Of the 67 wheat flour samples analyzed, no ENA1 was found in 67 samples, and the maximum levels of the five mycotoxins were all lower than 20 μg/kg. BEA was found in 52 out of 67 samples at levels ranging from 0.12 μg/kg to 11.1 μg/kg , followed by ENB with the positive rate of 56.7% and the levels ranging from 0.02 μg/kg to 0.59 μg/kg , ENA with the positive rate of 52.2% and the levels ranging from 0.09 μg/kg to 0.19 μg/kg , and ENB1 with the positive rate of 49.3% and the levels ranging from 0.14 μg/kg to 1.31 μg/kg . Whereas, the concentrations of the five mycotoxins in 149 noodle samples were all lower than 20 μg/kg with the highest frequency of 79.9% for ENB.For 291 wheat and wheat-based samples including 75 wheat kernels, 67 wheat flours, and 149 noodles, the positive rate of five mycotoxins from highest to lowest was ENB > BEA > ENB1 with a frequency of 4.23% (3/71), BEA-ENA1-ENB with a frequency of 2.81% (2/71), and both BEA-ENA-ENA1 and BEA-ENA-ENB with a frequency of 1.41% (1/71), respectively. In terms of the 68 corn flours, BEA-ENA-ENA1 was the most predominant combination with a frequency of 76.47% (52/68), followed by the combination of four mycotoxins BEA-ENA-ENA1-ENB1 , two mycotoxins combination  including BEA-ENA1 , BEA-ENA  and ENA-ENA1 , and five mycotoxins combination . For 19 corn flakes, there was no co-occurrence of four mycotoxins and five mycotoxins. ENA-ENA1 and BEA-ENA-ENA1 combinations were found in 15 (78.95%) and four (21.05%) samples, respectively. Moreover, in corn and corn-based samples, significant correlations were observed between concentrations of ENA and ENB1 , ENA and ENB , and ENA1 and ENB1 .The co-contamination of five mycotoxins in 158 corn and corn-based samples collected from Shandong province is given in 1-ENB-ENB1 was the most frequent mycotoxin combination with a frequency of 8% (6/75), followed by BEA-ENA1-ENB-ENB1 , ENA-ENA1-ENB-ENB1 , BEA-ENB-ENB1 , ENA1-ENB-ENB1 , BEA-ENA  and BEA-ENB  in 75 wheat kernels. In terms of 67 wheat flour samples, there were 10 mycotoxin combinations, as shown in 1 was the most frequent mycotoxin combination type with a frequency of 25.37% (17/67), followed by BEA-ENB-ENB1 with a frequency of 14.93% (10/67), and BEA-ENA with a frequency of 11.94% (8/133). For 149 noodles samples analyzed, there were 21.48% (32/149), 20.13% (30/149), 18.79% (28/149), and 18.12% (27/149) samples co-contaminated with five, four, two, and three mycotoxins, respectively. BEA-ENA-ENA1-ENB-ENB1 was the most frequent combination with a frequency of 21.48% (32/149) followed by ENB-ENB1 , BEA-ENB-ENB1 , and BEA-ENA-ENB-ENB1 . The significant linear correlations in the concentrations were observed between BEA and ENA , BEA and ENA1 , BEA and ENB , BEA and ENB1 , ENA and ENA1 , ENA and ENB , ENA and ENB1 , ENA1 and ENB , ENA1 and ENB1 , and ENB and ENB1  in wheat and wheat-based samples, respectively.The co-occurrence of five cyclic depsipeptide mycotoxins in 291 wheat and wheat-based samples is listed in 1, ENB, and ENB1 in corn- and wheat-based samples from China. The maximum concentrations of BEA in corn kernel, flour, and flake samples in our study were far higher than those of four ENNs, which were different from those reported by Sørensen et al. for 73 corn samples collected from Demark in 2005 and 2006; ENB (2598 μg/kg) was much higher than BEA (73 μg/kg) [1, 3.8% for ENB, and 56.3% for ENB1) in all corn and corn-based samples in our study were lower than those reported by Uhlig et al. in 2000 and 2002 [1, 100% for ENB, 94% for ENB1 in Norwegian wheat and wheat-based samples, respectively. Furthermore, a similar trend was also found in the wheat and wheat-based samples studied, and the levels of five mycotoxins in wheat kernels, flours, and noodles were quite low with the maximum levels ranging from 11.1 μg/kg to 61.77 μg/kg. These levels were lower than those found in Norwegian grain samples from 2001 and 2002 ranging from 58 μg/kg to 7400 μg/kg [Fusarium head blight (FHB) species were F. culmorum, F. avenaceum, Microdochium species, while F. asiaticum had its main distribution in Asia  [F. culmorum and F. avenaceum, and not F. asiaticum. To the best of our knowledge, this is the first report on the natural co-occurrence of BEA, ENA, ENA3 μg/kg) . In addi3 μg/kg) . Howeverand 2002 , which w00 μg/kg . The read Korea) ,17,18. IFusarium species with different cyclic hexadepsipeptides toxin-producing abilities [We found that concentrations of BEA in corn and corn-based samples were far higher than those in wheat and wheat-based samples. The reason might be that corn- and wheat-based samples can be infected by different bilities ,23,24,25bilities ,27.Fusarium species and contaminations of the five mycotoxins in cereal and cereal-based samples. However, the limitation of the present study was that the samples analyzed were only collected from Shandong province, China. To provide accurate data regarding mycotoxin contamination and the toxin-producing ability of Fusarium species and their biosynthetic genetic differences, there is a need for more samples to be collected nationwide across China.As far as we know, this is the first report concerning BEA and ENNs co-contamination in Chinese cereal and cereal-based samples. The European Food Safety Authority (EFSA) has given a preliminary scientific opinion on the risks to animal and public health related to dietary exposure to five cyclic depsipeptide mycotoxins due to lack of sufficient data regarding the toxigenic ability of 1, ENB, and ENB1 with purities ≥97% were obtained from BioAustralis . Acetonitrile (ACN) and methanol (MeOH), both of LC-MS grade, were obtained from Fisher Scientific . Ammonium acetate purchased from Sigma-Aldrich  was of MS grade. Water was purified successively by a Millipore Milli-Q system  with a conductivity ≥18.2 MΩ.cm at 25 °C.Standard powders of BEA, ENA, ENAA total of 158 corn-based samples including 71 kernels, 68 flours, and 19 flakes, and 291 wheat-based samples including 75 kernels, 67 flours, and 149 noodles were randomly collected in different districts of Shandong provinces, an area which was the most important grain planting and consuming province in China. All these corn- and wheat-based samples were intended for human consumption and collected from a supermarket, farmer’s market, or local resident’s home. Three subsamples were taken from the top, middle, and bottom layers of the container (five sampling sites for each layer). For wheat flour and corn flour samples, 5 g of each were weighed for toxin extraction directly after mixing. For corn kernel, corn flake, wheat kernel, and noodles, the subsamples were pooled and mixed thoroughly using a grinder , and approximately 200 g of the mixture was finely ground to a powder and kept in Ziploc plastic bags, sealed, and stored at −20 °C prior to analysis. A 5 g finely ground portion of each sample was weighed for analysis.v/v) by blending for 30s to 60s using a vortex and then shaking for 30 min. The extract was transferred to a new 50 mL centrifuge tube and allowed to stand for 15–20 min under room temperature. Each aliquot of 10 mL of supernatant was diluted with 20 mL of distilled water followed by homogenization using a vortex. A quantity of 4 mL of the diluted extract was applied to a Sep-Pak Vac 3cc (200 mg) C18 Cartridge , which was pre-equilibrated with 3 mL of methanol and 3 mL of water. The cartridge was washed with 3 mL of 10% acetonitrile in water followed by a wash with 3 mL of 50% acetonitrile in water. Toxins were eluted using 2 mL of 90% acetonitrile in water.All five mycotoxins were extracted according to the method reported previously . A totalTM 5500 MS/MS system  and a MultiQuantTM Version 3.0.2 software  for data acquisition and analysis was used to quantify 5 mycotoxins in positive mode. Chromatographic separation was achieved using a C18 column . Temperatures of the UPLC column and autosampler were set at 35 °C and 15 °C, respectively. The mobile phase A was water containing 2 mmol/L ammonium acetate, and the mobile phase B was acetonitrile. The program was starting from B/A  in the first 2 min, reached B/A  in 2 min to 3 min, and continued to decrease B/A  in 3 min to 19 min. Afterward, A was linearly increased to 100%  within 2 min and maintained at this composition of the mobile phase for 0.1 min.UPLC/ESI-MS/MS system equipped with ExionLC , QTRAP+) mode. The curtain gas was set to 30 psi, the collision gas to medium, Gas 1 to 80 psi, and Gas 2 to 80 psi. The source desolvation temperature was set at 550°C. The ion spray voltage was set to +5500V. Parent and fragment ions  for each analyte were chosen based on the best signal-to-noise ratios in a blank spiked sample. Detailed information for the parent and fragment ions is shown in Data acquisition was performed by applying multiple reaction monitoring (MRM) with an optimized dwell time of 200 ms in order to obtain the optimal sensitivity and selectivity of MS conditions in positive electrospray ionization (ESIn = 6 repeats each level) were extracted and analyzed for recovery calculation. Mean recoveries, in which the matrix effect was compensated, were in the ranges of 91.6–129.0% (BEA), 95.8–140.5% (ENA), 97.1–139.3% (ENA1), 106.5–149.7% (ENB), and 102.7–142.2% (ENB1) as determined from six parallel analyses of blank samples spiked with 50 to 250 µg/kg for BEA, from 7.5 to 37.5 µg/kg for ENA, from 20 to 100 µg/kg for ENA1, from 20-100 µg/kg for ENB, and from 50 to 250 µg/kg for ENB1 with the relative standard deviations (RSD) of 1.5–7.7% for BEA, 0.4–12.9% for ENA, 1.4–6.9% for ENA1, 1.7–13.8% for ENB, and 1.2–12.6% for ENB1, respectively. The limits of detection and limits of quantification for BEA, ENA, ENA1, ENB, and ENB1 in different types of samples are presented in To evaluate the method proficiency, BEA- and ENNs-free corn- and wheat-based samples were spiked with five mycotoxins at three concentrations. The spiked samples . The Kruskal–Wallis test was employed for statistical analysis by comparing the mycotoxin concentrations in different samples.


To identify cross-border international registries for rare endocrine conditions that are led from Europe and to understand the extent of engagement with these registries within a network of reference centres (RCs) for rare endocrine conditions.Database search of international registries and a survey of RCs in the European Reference Network for rare endocrine conditions (Endo-ERN) with an overall response rate of 82%.Of the 42 conditions with orphacodes currently covered within Endo-ERN, international registries exist for 32 (76%). Of 27 registries identified in the Orphanet and RD-Connect databases, Endo-ERN RCs were aware of 11 (41%). Of 21 registries identified by the RC, RD-Connect and Orphanet did not have a record of 10 (48%). Of the 29 glucose RCs, the awareness and participation rate in an international registry was highest for rare diabetes at 75 and 56% respectively. Of the 37 sex development RCs, the corresponding rates were highest for disorders of sex development at 70 and 52%. Of the 33 adrenal RCs, the rates were highest for adrenocortical tumours at 68 and 43%. Of the 43 pituitary RCs, the rates were highest for pituitary adenomas at 43 and 29%. Of the 31 genetic tumour RCs, the rates were highest for MEN1 at 26 and 9%. For the remaining conditions, awareness and participation in registries was less than 25%.Although there is a need to develop new registries for rare endocrine conditions, there is a more immediate need to improve the awareness and participation in existing registries. Multicentre collaboration between centres of expertise within registries also plays a vital role in the development of clinical benchmarks, thereby acting as a platform for quality improvement.Rare diseases or rare conditions are defined by the European Union (EU) as life-threatening or chronic debilitating conditions with a prevalence rate of less than 5 per 10 000 (European Commission (EC) Regulation # 141/2000). The management of these conditions requires coalesced efforts over the life time of the patient to reduce morbidity and mortality in affected individuals. Rare endocrine conditions pose a particular challenge due to current knowledge gaps regarding long-term outcome, lack of expert and evidence-based multidisciplinary care resulting in substantial variation in care. Registries have the potential to improve patient care and healthcare planning in those with rare conditions; they enable pooling of data for research activities and surveillance; by creating a virtual environment of collaboration, they can also facilitate communication between professionals and affected individuals   and RD-Connect   ; Europea 1 2018)  have attEuropean Reference Networks (ERNs) are networks of reference centres (RCs) across Europe that aim to manage rare medical conditions that require specialist attention and a concentration of knowledge and resources , 6. The Although registries are regarded as essential for Endo-ERN, knowledge on the range of registries that exist for cross-border sharing of information on rare endocrine conditions in Europe is scarce. Furthermore, the extent of awareness, participation and availability of registries for the wide range of conditions within Endo-ERN is unclear. The development of Endo-ERN provided a perfect opportunity to identify registries that already exist in Europe for the rare endocrine conditions that have been included in the network and to also determine the extent of engagement in registries of health professionals within an expert network such as Endo-ERN.https://www.orpha.net/consor/cgi-bin/index.php; last accessed August 1 2018) and RD-Connect  projects using Orphacodes for all conditions and, in addition, surveyed Endo-ERN members between October and November 2016. For the purpose of this mapping exercise, an international registry was defined as a patient registry that collected uniform information on individual patients  was used to collate and analyse numerical data. Median and range values were obtained for the dataset.A response rate of 82% (58/71 RC) was obtained. Twenty-five (35%) RCs were involved in six or more themes and 26 (37%) RCs were involved in one theme only. The average response rate across all eight themes was 80% .Figure 1Of the 33 centres surveyed within the adrenal theme, awareness of an international registry was 68% for adrenocortical tumours, 57% for phaeochromocytoma, 54% for congenital adrenal hyperplasia (CAH), 46% for bilateral macro- and micro-nodular adrenal hyperplasia and 29% for adrenal insufficiency . InternaOf the 29 centres within the calcium/phosphate theme, international registry awareness was 17% for disorders of phosphate disturbances, 13% for hypercalcaemia and 4% for hypocalcaemia; participation was 8, 8 and 0%, respectively. Awareness and participation in local registries was higher for all conditions . HypocalOf the 29 centres within the glucose theme, participants reported the highest rates of awareness and participation in an international registry for rare diabetes  . Median Of the 31 centres reporting on the rare genetic tumour theme, 26% reported an international registry awareness for MEN1 and 9% reported participation . SeventyOf the 30 centres within the growth theme, local and national registry awareness and participation for all conditions was greater than that for international registries. International registry awareness was highest for Prader–Willi syndrome at 17% with a participation level of 10% . ParticiOf the 43 centres within the pituitary theme, awareness and participation in an international registry was greatest for pituitary adenomas, with 43% awareness and 29% participation . PituitaOf the 37 centres within the sex development theme, 70% reported an awareness and 52% reported participation in international registries for 46, XX and 46, XY disorders or differences of sex development (DSD) . DespiteOf the 35 centres within the thyroid theme, international registry awareness was greatest for thyroid signalling disorders (21%), however, participation was only 7% . Out of For endocrine conditions within the main thematic groups of Endo-ERN, 33 registries were identified through the above survey of Endo-ERN and the search of the Orphanet and RD-Connect databases . Endo-ERThis contemporary survey of registries has clearly revealed that whilst there are several registries for a range of rare endocrine conditions, there are also several gaps in coverage of conditions. In addition, there are gaps in the level of awareness and use as well as the international profile of existing international registries.Clear patterns were observed for awareness and participation of international registries. For conditions including disorders of calcium and phosphate, endocrine tumour syndromes, growth syndromes and pituitary and thyroid disease, there was greater awareness of local or national registries rather than international registries. On the other hand, for other groups of conditions such as adrenal disorders, insulin resistance syndrome, rare diabetes mellitus and DSD, there was a high level of awareness and participation in international registries.The increased awareness of international registries amongst the Endo-ERN participants for particular subgroups of conditions may be attributed to the presence of already established secure Web-based registries such as those listed within Orphanet and RD-Connect and/or to large international collaboration of RC within a given theme prior to the establishment of Endo-ERN, for example, the glucose, adrenal and DSD themes. The SWEET registry is an example of a registry that was initially developed to cover type 1 diabetes mellitus; however, in doing so, it has also become a powerful resource for rare forms of diabetes . Althoughttp://www.ema.europa.eu/docs/en_GB/document_library/Report/2017/02/WC500221618.pdf; last accessed September 13 2018) and is necessary for effective use of limited resources.Databases such as Orphanet and RD-Connect have been developed to facilitate greater awareness of registries for rare conditions and may also pave the way towards improving the quality of registries. However, it is important to note that current databases of rare disease registries such as Orphanet and RD-Connect did not have a record of several international registries that are currently led from Europe. This was noted particularly for rare disease registries led by industry. Given that rare disease registries have to enter their details on these databases, the discordance suggests a mutual lack of awareness between these databases and the rare disease registries of the respective platforms. It is possible that registries led by industry have been designed specifically for post-marketing surveillance of specific drugs for a fixed period with limited applicability to the wider community and long-term sustainability may not be a priority, and this may explain their lack of visibility on Orphanet and RD-Connect. On the other hand, these registries are generally well resourced with a clear objective, and it is possible that the data within them are of high quality. In the field of rare conditions, greater partnership with industry is generally supported by all stakeholders (European Medicines Agency Report on the Patient Registries Workshop (28 October 2016) Whilst the current survey shows that many centres are collecting data in local registries, participation in international registries is often considered to be the gold standard for improving the quality of research, improving patient care and the external validity of registries for rare conditions , 12, 13.With the recent development of Endo-ERN, consideration should also be given to effective collation of available resources and development of a virtual environment that serves the objectives of this ERN and one that acts as a model for the wider endocrine community as well as other rare disease networks . The EurIn summary, whilst there is a clear need to develop new detailed disease registries, there is also a need to improve the awareness and signposting of existing registries. A common platform that can be used by the whole endocrine community and which directs the user to high-quality detailed disease registries has the potential to achieve this objective.Olaf Dekkers is an editor of the European Journal of Endocrinology. There are no other conflicts of interest that could be perceived as prejudicing the impartiality of this study.This publication is part of the project ‘777215/EuRRECa’ which has received funding from the European Union’s Health Programme (2014–2020).