Each significant SNP was calculated and the SNPs significant associated with kernels, axes, stems, bracts and leaves explained 6.96%–10.56%, 7.19%–15.87%, 7.11%–10.19%, 7.16%–8.71% and 6.91%–9.17% of the phenotypic variation, respectively. Among the significant SNPs, nine co-localised with previously detected quantitative trait loci. This study will aid in the selection of Hg-accumulation inbred lines that satisfy the needs for pollution-safe cultivars and maintaining maize production.Owing to the rapid development of urbanisation and industrialisation, heavy metal pollution has become a widespread environmental problem. Maize planted on mercury (Hg)-polluted soil can absorb and accumulate Hg in its edible parts, posing a potential threat to human health. To understand the genetic mechanism of Hg accumulation in maize, we performed a genome-wide association study using a mixed linear model on an association population consisting of 230 maize inbred lines with abundant genetic variation. The order of relative Hg concentrations in different maize tissues was as follows: leaves > bracts > stems > axes > kernels. Combined two locations, a total of 37 significant single-nucleotide polymorphisms (SNPs) associated with kernels, 12 with axes, 13 with stems, 27 with bracts and 23 with leaves were detected with  Owing to rapid urbanisation and industrialisation, heavy metal pollution has become a widespread environmental problem2. Many processes, such as the application of agrochemicals , sewage irritation and industrial pollution , have emitted heavy metals into soils3. McLaughlin et al.4 reported that metals could be transferred into the roots of plants through the soil pore water in the form of dissolved ions. The heavy metals can then accumulate in the edible part of plants and easily enter the human body through the food chain, which results in an increased risk of disease7. Mercury (Hg) is one of the most toxic heavy metals9. According to the Chinese National Standard for Soil Environmental Quality, the Hg concentration in soils is divided into three classes: in Class I, the Hg concentration is under 0.15 mg kg−1 in soils, which is the natural background value; in Class II, the Hg concentration is under 1 mg kg−1, which is the upper acceptable limit for agricultural soils; and in Class III, the Hg concentration is under 1.5 mg kg−1 in soils, which is the limit for the normal growth of plants10. According to Lin et al.11, the background Hg levels of soils in China range from 0.02 to 0.20 mg kg−1. However, the soil Hg content in many parts of China far exceeds its background value. For example, the soil Hg content at the site of the Wuchuan mine in China is as high as 24 mg kg−1 9.Pollution-safe cultivar, which refers to the use of cultivars that accumulate a very low level of a specific pollutant, have been proposed to be a strategy to ensure the crop remains safe for human consumption, even when grown in contaminated soilOryza sativa seedlings is also inhibited by high Hg concentration12. Hg can cause a large and rapid reduction in the hydraulic conductivity of roots, which results in the inhibition of aquaporin functions14, and higher Hg concentrations have a toxic effect on root growth in alfalfa by inducing oxidative stress15. Additionally, high Hg doses can affect the absorption and evaporation of water, and decrease the chlorophyll content and photosynthetic efficiency18. Importantly, the soil Hg absorbed by crops can also be transported into the human body through the food chain19. Hg, in the form of methylethylmercury, absorbed by the human body can accumulate in the brain, eventually causing neurotoxic effects and nerve-related diseases, such as autism, attention deficit disorder and mental retardation, and death21.A high soil Hg content can impact plant growth and development when absorbed by plants. Seed injury to cereals caused by organomercury can inhibit cell division during seed germination, and the elongation of 22, and inhibiting plant growth and development23. Yu et al. first identified three quantitative trait loci (QTLs) for Hg accumulation and tolerance at the seedling stage in rice using a doubled haploid population24. Wang et al. also detected three QTLs for Hg tolerance at the seedling stage in rice using a recombinant inbred population25. Fu et al. found 23 QTLs for Hg accumulation in five maize tissues using a recombinant inbred population26. In addition to these QTLs, genes closely related to Hg accumulation and tolerance have also been reported. In Arabidopsis thaliana, merApe9 transgenic plants are more tolerant to Hg at the seedling stage and during the flowering period27. The overexpression of HO-1 in algae also resulted in a high tolerance to Hg exposure and a reduced Hg accumulation28. The overexpression of BnHO-1 in Brassica napus resulted in a reduced Hg accumulation in the transgenic plants29. MTH1745 transgenic rice has an enhanced Hg tolerance30.Many studies have investigated the physiological and biochemical responses to Hg intoxication. Hg also inhibits the 5-amino levulinic acid dehydratase activity in the cells of maize leaves, thereby affecting the synthesis of chlorophyll32. For example, the examination of maize oil biosynthesis identified 74 loci significantly associated with kernel oil concentration and fatty acid composition in a GWAS using 1 million single-nucleotide polymorphisms (SNPs) characterised in 368 maize inbred lines33. Furthermore, GWAS and QTL mapping have been found to be complementary and to overcome each other's limitations in Arabidopsis35. In soya bean, the genotyping by sequencing-GWAS approach has been used to identify loci governing eight agronomic traits and was validated by QTL mapping36.Genome-wide association studies (GWASs) are useful for identifying candidate loci associated with traits in animal and plant species12. Thus, maize planted on Hg-polluted soil can absorb and accumulate Hg in its edible parts, posing a potential threat to human health. Although some QTLs and genes regulating the accumulation of, and tolerance to, Hg have been reported previously30, knowledge of the genetic basis for Hg accumulation in maize remains limited. In the present study, an association population consisting of 230 maize inbred lines was evaluated at two locations having different soil Hg concentrations. The main purpose of this study was to detect SNPs that are significantly associated with Hg accumulation in five maize tissues, which may aid in the selection of elite inbred lines with low Hg-accumulation capabilities in the kernels and high accumulation capabilities in the leaves, to maintain maize production.Maize is an important grain and feed crop for humans and animals. Hg accumulation in plants through the plant–soil system can cause toxicity that affects both plant growth and human health through the food chain−1, respectively. At Changge, the average Hg contents in the kernels, axes, stems, bracts and leaves were 1.05, 2.79, 3.84, 6.01 and 26.49 µg kg−1, respectively. The Hg concentrations in the different maize tissues showed the same trend, kernels < axes < stems < bracts < leaves, at the two locations.In the association population, the Hg content in all five of the maize tissues tested was much higher at the Xixian location compared with the corresponding tissues at Changge Table , Fig. 1.−1 at Xixian and from 0.14 to 3.55 µg kg−1 at Changge. The Hg concentrations in axes, stems, bracts and leaves varied from 1.01 to 6.4 µg kg−1, 1.93 to 14.39 µg kg−1, 4.61 to 12.78 µg kg−1 and 20.50 to 46.30 µg kg−1, respectively, at Xixian; and from 1.82 to 4.86 µg kg−1, 1.31 to 10.35 µg kg−1, 2.37 to 13.40 µg kg−1 and 12.26 to 42.43 µg kg−1, respectively, at Changge.The Hg content in each maize tissue varied widely in the association population at the two locations. The Hg concentration in kernels varied from 0.15 to 6.50 µg kgAn analysis of variance indicated that the Hg concentrations in the five measured tissues in the association population were significantly affected by environments, genotypes and genotype × environment interactions Table , indicatThe genome-wide LD was calculated using the 522,744 SNPs , which were used as the input data  were used to control the population structure. A principal component analysis (PCA) showed that the inbred lines used in this study could be separated well by PC1 and PC2 Fig. . A GWAS The overall LD decay for the entire genome in this panel was 100 kb. Given the extent of average LD, we feel confident that a 200 kb window centered on each significant SNP has a good chance to capture the gene of interest.37. In maize, Liu et al.38 reported that the Hg levels in different tissues were different, having the following trend: root > leaf > stalk > grain. Fu et al.26 found a similar distribution of the Hg content, leaves > bracts > stems > axes > kernels, in maize. The same trend for Hg concentrations in different maize tissues was also found in the present study , stems (chr8.S_161469759), bracts (chr8.S_163741736) and leaves (chr8.S_128751246 and chr8.S_162579911), co-localised with the previously reported QTL named qBHC8a. The other significant SNP (PZE-108038334) on chromosome 8 co-localised with the qSHC8b QTL located in bin8.03. On chromosome 9, three significantly associated SNPs observed within the region between 105.527 and 115.321 Mb co-localised with the reported QTL qKHC9b/qBHC9. The other significant SNP (PZE-109058129) on chromosome 9 co-localised with the qKHC9a QTL located in bin9.03.In crop breeding, QTL mapping for important traits is a most common approach, providing the basis for marker-assisted selection. However, QTL detection is a traditional genotyping method and is laborious and time-consuming, and QTLs are low-density markers that map with low resolutions because of the limited recombination numbers. With the advent of next-generation sequencing technologies, GWASs have become a powerful genetics-based strategy to explore allelic variation with a broader scope. GWASs can save time and labour, increase the detectable range of natural variation and improve the resolution of QTL mappingIn the present study, more loci were detected through the GWAS than through linkage mapping, possibly because of the reduced QTL detection efficiency and the smaller number of molecular markers used in linkage mapping. A combination of linkage mapping and GWAS would promote the analysis of complex quantitative traits. The loci identified by the integration of GWAS and QTL mapping would provide useful reference information for studies on the functional verification of Hg accumulation.42, and is also important to ensure the quality of maize grain as worldwide demand for food increases. Heavy metal pollution in soils has become a major issue globally43, and the heavy metals absorbed by crops could affect grain quality. Zhang et al.44 reported that it was necessary and feasible to select and plant low heavy metal accumulation varieties in contaminated soil, which could reduce the heavy metal content of the edible crop parts. The strategy of breeding pollution-safe cultivars, in which heavy metals in contaminated soil could accumulate at sufficiently low levels in the edible parts of crops for safe consumption1, has been applied. Hg usually accumulates to a significantly higher level in leaves than in kernels40, and this was corroborated in the present study. For example, the mean Hg concentrations in leaves were 29.46 µg kg−1 at Xixian and 26.49 µg kg−1 at Changge, which far exceeds that in kernels (1.39 µg kg−1 at Xixian and 1.05 µg kg−1 at Changge). In addition, the Pearson correlation coefficient indicates that there is no significant relationship between the Hg contents in leaves and kernels. The results imply that inbred lines with high accumulations of Hg in leaves and low accumulations of Hg in kernels could be used as parents for selecting elite hybrids, which would be helpful both for soil remediation and for the assurance of food safety.Because maize is a food, feed and industrial crop, increasing its grain yield is an important breeding goal45. The selected inbred lines were genotyped in this study using two genotyping platforms (RNA sequencing and a SNP array) containing 556,809 SNPs according to the method described by Yang et al.45 The SNP data is available from http://www.maizego.org/Resources.html.A total of 298 maize inbred lines, which came from temperate, tropical and subtropical zones, were used in this study. After removing materials with higher heterozygosity and loss rates, 230 inbred lines were selected to constitute the association population, among which 151 inbred lines came from the temperate zone, and 79 came from tropical and subtropical zones  was much higher than at Changge . The soil Hg concentrations at the two locations were higher than in Class I, according to the Chinese national standards related to Hg soil concentration rankings10.The field trials were conducted in 2012 in Xixian  and Changge  Counties, which are located in northern China, with average temperatures of 15.2 and 14.3 °C, respectively, and rainfalls of 873.8 and 462.8 mm, respectively. The maize association population was grown in a randomised complete block design with three replicates at each location. Each plot included 15 plants planted 0.67 m apart in a single row 4 m long, allowing a final plant density of 67,500 plants per hectare. At the Xixian location, because of irrigation with Hg-rich surface water, the soil Hg concentration (457.57 ± 31.30 µg kg3/HClO4 (80/20 v/v) in polypropylene tubes using a heating block . Then, the Hg concentrations in the different plant materials were determined using atomic fluorescence spectrometry   were digested with 5 mL HNO2 using TASSEL 5.047. The PCs and the kinship matrix were also determined using TASSEL 5.0. A mixed linear model with the obtained SNPs, PCs, kinship and the means of the Hg contents was established for the GWAS. The relative distribution of −log10p-values was observed for each SNP association and compared individually with the expected distribution using a quantile–quantile and manhattan plot. The adjusted p-value threshold of significance for each trait was corrected. SNP loci in significant LD regions were identified by revealing the significant contributions to phenotypic variation of the agronomic traits with the highest magnitude of marker trait-association and lowest adjusted p-values (threshold p < 1 × 10−4).SNPs with more than 12% missing data and a minor allele frequency < 5% were excluded, leaving 522,744 SNPs for further analyses. The LD between SNPs on each chromosome was estimated with ret al.48, probes of ~120 bp containing the SNPs associated with Hg accumulation in different maize tissues were used for comparisons with the maize B73 genome. Based on the LD decay, a 200-kb window for the significant SNPs (100 kb upstream and downstream of the lead SNP) was selected to identify the candidate genes. Genes within the region were identified based on the positions of the closest flanking significant SNPs (p < 1 × 10−4). The candidate genes were obtained using the BLASTN algorithm (http://blast.ncbi.nlm.nih.gov/) and annotated against the Gene Ontology database (http://www.geneontology.org/) for functional annotations.The reported genome sequence of maize B73 provides a useful reference database for candidate gene analyses. According to Lawrence Supplementary information file


Data were acquired using double‐gated spoiled gradient echo images and pulse‐acquire spectra. For the in vivo study, the temporal stability of ISO resonances was quantified using coefficient of variability (CV) (5 min) estimates.Objectives are demonstrated using (a) in vitro ISO tests, (b) in vivo temporal accumulation/spatial localization C57BL/6 studies (n = 3), and (c) through injections of perfluoro‐crown‐ether (PFCE) labeled cardiac progenitor cells into femoral muscle areas of the murine hindlimb post‐mortem (n = 1) using 3 and ‐OCHF2 moieties. CV values ranged between 3.2 and 6.4% (‐CF3) and 6.4 and 11.2% (‐OCHF2). Reductions of the ISO dose (2.0 to 1.7%) at 80 min postinduction had insignificant effects on ISO signals . PFCE‐labeled cells exhibited a resonance at −16.25 ppm in vitro that did not overlap with the ISO resonances, a finding that is confirmed with MRS post‐mortem using injected, labeled cells. Based on 19F MRI, similar in vivo/post‐mortem ISO compartmentalization was also confirmed in peripheral and thoracic skeletal muscles.Two ISO resonances were observed in vivo that correspond to the ‐CF19F MRS in vivo with temporally stable signals over 90 min postinduction. ISO effects on PFCE labels are anticipated to be minimal but may be more prominent for perfluoropolyether or perfluorooctyl bromide labels.Significant ISO accumulation was observed by Level of Evidence: 1Technical Efficacy: Stage 1J. MAGN. RESON. IMAGING 2017;45:1659–1667 These peaks may interfere with the detection of fluorinated agents in vivo  due to potential spectral overlaps. Furthermore, despite the existence of numerous prior 19F MRI studies on the uptake and clearance characteristics of ISO in the brain in vivo,19F labels has not been investigated in detail. Additionally, only a few reported cardiovascular applications of fluorinated agents exist,Most preclinical studies are now routinely conducted using inhalational isoflurane (ISO),19F MR imaging in the mouse thorax following injection of PFC emulsions into the venous circulation. While Flögel et al.19F emulsions, van Heeswijk et al.19F MR images of PFC agents, and have suggested the use of selective radiofrequency (RF) excitation to ameliorate any potential issues; however, their discussion is speculative and lacks direct experimental evidence.Specifically, Flögel et al.19F MRS/MRI publications that focused on cardiovascular applications,19F labeled, cardiac progenitor stem cell administrations.The scope of this study is generalized on the accumulation of ISO . It extends beyond the two prominent prior 19F labels, and assess their potential spectral overlaps and interference with respect to ISO, including the extensively used perfluoro‐crown‐ether (PFCE) label, using 19F MRS/MRI.Therefore, the objectives of this study are to: (a) determine the spatial localization of ISO in the thoracic and peripheral skeletal muscle areas of the C57BL/6 mouse, and quantitatively assess its uptake, accumulation, and temporal stability within a period of 90 min postinduction, including its potential accumulation in the ventricular blood pool, and myocardium, (b) define the spectral characteristics of the ISO moieties in vivo, (c) identify the spectral characteristics of extensively used All procedures were approved by the local ethical review committee (U. Oxford), and conformed to the Animal (Scientific Procedures) Act 1986 (UK) incorporating European Directive 2010/63/EU.All experiments were conducted on a 9.4Tesla (T) Agilent scanner equipped with a DirectDrive console and a 1 mT/m actively shielded gradient set  .1H/19F MRS/MRI. The broad frequency response of the coil permitted imaging on both the 1H and 19F nuclei. Post‐mortem imaging was conducted using a home‐built 80 × 40 mm2 butterfly coil.An eight‐rung, low‐pass quadrature birdcage coil , was constructed in‐house, tuned and matched at 375.8 MHz, and used for 1H resonance, as described previously.1H and 19F MR images were acquired in mice (thoracic area) using a segmented k‐space, prospectively electrocardiograph‐ (ECG‐) and respiratory‐gated (double‐gated) spoiled gradient echo (SPGR) sequence. A three‐dimensional (3D) SPGR sequence (ungated) was used for the post‐mortem imaging .An autoshim method was used for in vivo mouse shimming on the For MRS, the temporal evolution of ISO accumulation was monitored using nonlocalized, fully relaxed, pulse‐acquire spectra.1 = 1564 ms; T2 = 1257 ms) were used as reference markers in ISO studies for calibration and quantification. b) Pulse‐acquire spectra were collected  using phantoms containing: (1) liquid ISO , (2) PFC NPs (3.8–10 mg/mL) emulsified in deionized water and mixed in cell media solution , and (3) 25 mM TFA, separately or in combination with NPs and PFCE‐labeled cells .a) Aqueous sodium fluoride (NaF) phantoms at 50 mM  .Particles were synthesized in accordance to previously published methodologies.Cardiac progenitor cells comprising either collagenase‐trypsin (CT) or cardiosphere‐derived cells (CDC)] were isolated from adult, C57BL/6, GFP positive, mouse atria, using standard protocols.Cells were subsequently incubated with PFCE‐containing NPs (loading concentration of 10 mg/ml/million cells) for approximately 24 h before trypsinization, isolation, and washing. Cell pellets were suspended and maintained in IMDM media and were used for MRI, flow cytometry, or confocal microscopy . Successful labeling was confirmed with a CyAn ADP flow cytometer  using control and labeled cell samples.To ascertain the temporal effects of anesthesia, ISO was administered in three C57BL/6 mice . The mice were induced using 4% ISO, and were maintained for up to 95 min with 1.5–2.0% ISO, mixed in 100% oxygen.www.civm.duhs.duke.edu), and were allowed to breathe freely throughout the study. A homeostatically controlled hot‐air system was used to maintain mouse body temperature at approximately 37 °C. ECG and breathing rates were monitored using a gating system.All animals were placed in a specially constructed cradle  was euthanized with an overdose of ISO (5%) and was subsequently injected in the femoral area of the right hindlimb with labeled CDC cells, transferred to a butterfly coil, and then re‐imaged post‐mortem.1H images  = 1 mm; matrix = 128 × 128; acquisition time = 3.25 min; eight phase encoding steps per segment) were acquired with a 2D segmented k‐space pulse sequence. Correspondingly, post‐mortem skeletal muscle images  were acquired with a 3D SPGR sequence.Axial cardiac 19F images of the mouse thorax were acquired . Spectral selection of the ISO and NP label resonant peaks was achieved using broadband excitations and narrowband acquisitions centered on the resonant frequency of interest. For skeletal muscle imaging, the acquisition parameters were: TR/TE = 17/8.4 ms; flip angle = 50 °; NEX = 512; FOV = 40 × 40 mm2; 1 slice ; ST = 40 mm; matrix = 32 × 32; BW = 2 kHz; and acquisition time = 4.5 min.Double‐gated axial NP‐labeled cells  and the temporal evolution of ISO accumulation  were monitored using nonlocalized MR spectra  within a temporal window of 35–90 min postinduction.19F images were imported and interpolated in ImageJ  using bicubic splines to match the 1H matrix size. Thoracic 1H and 19F MRI were overlaid in ImageJ (opacity = 70%). For skeletal MRI, the outer hind limb boundaries were segmented in ImageJ by an expert user, and the segmented outline was overlaid on the 19F images. Spectra were processed in CSX  or using custom‐written IDL tools . For temporal ISO comparisons, peak integrals were estimated from gated, magnitude spectra.All results are reported as mean ± SD. The temporal constancy of the spectral areas for both ISO resonances is quantified using the coefficient of variability (CV = 100 × SD/Mean). Repeated‐measures, nonparametric, two‐tailed Student t‐tests, were also used  to determine whether the ISO dose reduction (2.0% to 1.7%) caused significantly different changes in the areas of the spectral peaks (α = 5%).19F spectra of: (a) an ISO phantom, (b) a 75 mM TFA reference standard, (c) a 25 mM TFA phantom in the presence of an NP‐labeled cell phantom, and (d) ISO accumulation in thoracic areas in the in vivo mouse, are, respectively, shown in Figure 3, and ‐OCHF2 moieties.3 peak to 500 Hz. The spectral separation of the two ISO resonances in vivo is ∼6.3 ppm  at 5 min intervals from a typical mouse. To assess any changes in the in vivo 19F spectrum of ISO with time or dose, the areas of both ISO peaks were quantified using CSX, and the ISO dosage was decreased from 2.0% to 1.7% at t = 80 min postinduction , and (b) 11.2, 6, 8.5, and 6.4% (‐OCHF2).The CV of the ISO peaks for each of the studied mice and for the entire cohort (during the time period of 50–90 min postinduction) were: (a) 6.4, 3.9, 3.2, and 3.9%  were observed pre‐ and postreduction of the ISO dose (t = 60–80 min versus t = 80–90 min).Furthermore, no statistically significant changes in mean spectral areas of the ISO peaks  of the two ISO moieties acquired using narrowband acquisitions are shown along with corresponding 1H images . To better demonstrate the anatomical localization of the ISO signal, merged 1H/19F images are also shown  is the resonance corresponding to the PFCE label, which does not overlap with the ISO resonances. Shown also are coronal 19F MR images corresponding to each of the two ISO resonances, and the corresponding 1H images. An axial 19F MR image corresponding to the 19F label is also shown where no observable contribution from ISO is observed.Figure This study demonstrates ISO accumulation in thoracic areas of the mouse. The results also demonstrate that the presence of ISO does not constitute a problem for the detection and quantification of PFCE in cardiac labeled cells.3) and ‐10.3 ppm (‐OCHF2) in C57BL/6 mice in vivo with respect to the TFA resonance at 0 ppm, confirming previous reports in rabbits, and C57BL/6 and BALB/c mice.3 resonance is either co‐resonant with (in vitro) or close to (in vivo) the TFA resonance. Consequently, TFA reference phantoms cannot be used in ISO/NP label studies because the signal overlap can lead to inaccuracies in calibration and quantification. Instead, NaF (‐44.95 ppm) or potassium fluoride (‐46.75 ppm) phantoms may be preferred because their chemical shifts do not overlap with those of the ISO moieties.ISO exhibited chemical shifts of ‐4.0 (CF19F MRS. Prior work in vivo reports that the uptake and equilibration of ISO and sevoflurane in the brain is extremely fast (of the order of minutes),Substantial accumulation of ISO was observed in the live mouse as documented by The results presented in this study show constancy and temporal stability of the evoked ISO spectra from anesthetized mice within time periods spanning 35–90 min postinduction. These findings are reinforced by the lack of statistically significant differences in the ISO signal amplitude following dose decreases. Additionally, the post‐mortem study confirmed persistence of ISO in thoracic and skeletal muscle areas for at least 7 h post‐euthanasia, consistent with prior literature.2 entities. From the technical standpoint, primary effects may be attributed to: (a) shimming, (b) B0/B1 effects (leading to flip‐angle dependencies and off‐resonance effects), and (c) the inability to resolve multiplet patterns from the respective spectral splitting of the two ISO moieties, ultimately leading to two distinct, broadened, peaks. Certainly, the in vivo ISO peak intensities are substantially larger than that of the PFCE spectral intensity arising from the labeled cells.Noted also were significant increases in the linewidths of both ISO peaks attributed to either temporal, or physiologically dependent factors. From the physiological standpoint, the acquired spectra represent nonlocalized, spatiotemporal averaged signals that may be prone to: (a) beat‐to‐beat variations, (b) overall effects from ISO distribution , and (c) heterogeneity of signal contribution arising from multiple organs and multiple T2 or T2* values, or to poor B0 homogeneity over the entire thorax or skeletal muscle areas. The adopted MRS approach and inferences drawn are certainly based on nonlocalized spectra, spanning the entire FOV of the RF coil. Correspondingly, T2 reductions are expected to be most likely an end‐result of ISO binding/complexation. The broad  spectral peaks observed in vivo  in the brain in rats as a result of intracranial skeletal muscle/fat accumulations (14), despite the lack of significant lipid accumulations/contributions as reported in a recent study by van Heeswijk et al.From the biophysical standpoint, it is expected that the observed linewidth changes are attributed primarily to shortened T2 moiety with respect to the PFCE resonance of cardiac progenitor‐labeled cells, and in conjunction with the selective acquisition bandwidths used in 19F MRI, we have shown negligible ISO effects on the imaging of 19F signals arising from PFCE labels in vitro, and in post‐mortem skeletal muscle imaging in the mouse. Combined selective excitation/suppression schemes can be used to excite the PFC label and minimize baseline drifts due to possible contaminating effects from ‐OCHF2. This is particularly important given the multifold 19F signal difference of labels in cells compared with the ISO moieties, particularly considering the expected submillimolar label concentrations in cells.2 responses in the range of 50–600 ms.Given the chemical shift difference between the ISO resonance of the ‐OCHF19F labels, such as perfluoropolyether (PFPE), perfluorodecalin (PFD), trans‐bis‐perfluorobutyl ethylene (F‐44E), or perfluorooctyl bromide (PFOB),However, with the use of other commonly used 1 relaxation values for both anesthetic moieties, and the selective inversion of the undesired spectral peaks in conjunction with the selective excitation of the peaks corresponding to the labels.Despite the technical complexity of this work, there are several limitations in the study design. For example, despite the consistency of the effects of ISO observed in the male mice of this study, and in preliminary studies in female mice (results not shown), the sample sizes were small. Therefore, possible sex differences in the ISO effects need to be accounted for in large‐sample studies. Furthermore, the reported effects of temporal accumulation of ISO and its biodistribution, as well as the spectral response of the PFCE NPs must be assessed in models of pathology, including myocardial infarction and skeletal myopathies. Of more importance may be also the generalizability of the study and its extension to larger animal models  in larger bore, lower‐field magnets. However, migration of the work may impose further challenges in terms of the separability of the ISO and PFCE NP spectra. Thus, a more appropriate study design may also include the characterization of the T19F MRS. The temporal persistence of ISO spans multiple hours. Given the spectral separation of the ISO peaks from the PFCE resonance, and the selective acquisition bandwidths often used in 19F MRI, the anticipated signal overlap/contamination effects of ISO on PFCE labels are minimal, but may be more prominent for PFPE or PFOB labels. Correspondingly, ISO still remains the primary choice for 1H and multinuclear imaging studies.In conclusion, accumulation of ISO was observed in the mouse with signals that are temporally stable over 90 min post‐induction, as documented by


Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment. Liver fibrosis, consequent to chronic liver injuries such as viral infections, alcohol and drug intoxications, metabolic disorders and cholestasis, is a major cause of morbidity and mortality worldwide e.g. fibronectin and collagen 1A1) and α‐smooth muscle actin (α‐SMA), conferring myofibroblasts with the ability to migrate to the site of injury Investigations mostly performed in rat In the animal model of common bile duct ligation (BDL) applied in this study, which mimics obstruction to bile flow, such as that found in primary biliary cirrhosis and primary sclerosing cholangitis, PF play an important role in early ECM deposition around portal tracts Furthermore, growing experimental evidence has shown that ductular epithelial cells Fig.  also conFinally, LSEC Fig.  represenThe study of the main fibrogenic cells contributing to liver injury and the mechanism(s) of activation in different fibrotic injury etiologies could provide new important targets to develop an effective antifibrotic therapy. The therapeutic options now under investigation include molecules that suppress/reduce reactive oxygen species (such as vitamin E), inflammatory mediators  or that block angiotensin II (such as losartan), all of which are involved in HSC activation 4 intoxication Among the proposed antifibrotic approaches, cell‐based therapies have been shown to reduce liver fibrosis in different animal models. We and others have demonstrated that the amniotic membrane and cells derived thereof, namely human amniotic mesenchymal stromal cells (hAMSC) and amniotic epithelial cells (hAEC), decrease hepatic fibrosis induced by BDL Until now studies have been focused on the ability of amniotic cells either to favour hepatocyte proliferation/regeneration However, studies aimed to examine the activity of hAEC on the resident hepatic cells that contribute to myofibroblast generation are lacking. Therefore, the aim of this study was to investigate the effects of hAEC on HSC, PF, epithelial ductular cells and LSEC in order to identify the specific target of hAEC treatment.Comitato Etico Provinciale).Human term placentas were collected after obtaining written informed consent according to the guidelines of the Ethical Committee of the Province of Brescia  and by the Committee on the Ethics of Animal Experiments of the University of Milano .All animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86‐23 revised 1985).n = 5) as previously described g for 10 min. Cell suspensions were then filtered through a 70‐μm cell strainer , centrifuged and counted. Isolated cells were cryopreserved in 10% DMSO (Sigma‐Aldrich) supplemented with 90% FBS until use.hAEC were isolated from term placentas  and maintained at 2.5% during the surgical procedure. Before BDL, 4 mg/Kg Carprofen  and 5 mg/Kg Enrofloxacin  were administered by subcutaneous injection to decrease pain and prevent infection. The common bile duct was exposed through a whole thickness laparoscopic midline incision, then ligated and sectioned to produce a permanent biliary obstruction. After BDL, 5 mg/kg vitamin K  was administered weekly to reduce haemorrhagic diathesis.All experimental procedures were performed on anaesthetized animals. Biliary fibrosis was induced by BDL in female Wistar rats weighing 220–250 g , as previously described 6 hAEC in 400 μl PBS (BDL + hAEC group). All animals were killed 6 weeks post‐surgery with an excess of isoflurane followed by puncture of the right ventricle and exsanguination.Immediately after BDL, animals were randomly treated through intratail vein (iv) injections, either with 400 μl PBS (BDL + PBS group) or with 3 × 10Total bilirubin concentrations were determined in serum collected from animals 7 days after BDL, by the diazo method using Bilirubina totale, metodo colorimetrico (DMSO) Kit  according to the manufacturer's recommendations.At sacrifice, a portion of the hepatic median lobe from each animal was immediately fixed in 4% formalin for 48 hrs and paraffin‐embedded.To assess the degree of liver fibrosis, 4‐μm‐thick sections were stained with haematoxylin/eosin and Goldner's modified Masson trichrome, according to the manufacturer's instructions . Histological grading of liver fibrosis was evaluated on microscopic fields centred on hepatic lobules, under a bright field microscope  at 100× magnification, by the semiquantitative Knodell's scoring system https://imagej.nih.gov/ij).To evaluate the extent of liver collagen deposition, sections were incubated in PicroSirius Red  for 80 min. and washed for 3 min. in tap water. Twelve non‐overlapping fields were acquired (200× magnification), images digitized and PSR‐positive area measured by computer‐assisted morphometric analysis using FiJi software ; fibronectin ; desmin ; collagen 15A1 ; cytokeratin 19 (CK19) ; αvβ6 integrin ; S100A4, also called fibroblast‐specific protein‐1 (FSP‐1) ; phosphorylated Smad 2/3 (p‐Smad2/3) ; CD32b  and CD34 .Briefly, dewaxing and antigen retrieval was performed at 98°C for 20–30 min. in a citrate buffer solution pH 6 (BiOptica); a further digestion of 10 min. in trypsin solution  at 37°C was necessary for CK19 detection.Endogenous peroxidase activity was quenched by 10‐min. incubation with Dako REAL Peroxidase‐Blocking Solution (Dako), and unspecific labelling was minimized by a 30‐min. incubation with 2.5% normal horse serum  or with 10% normal goat serum (Invitrogen Corporation).Sections, incubated overnight at 4°C with one of the primary antibodies, were then washed and incubated with an horseradish peroxidase (HRP)‐conjugated secondary antibody  or with a biotinylated secondary antibody (Vector Laboratories) . In this last case, antibody binding was detected by Streptavidin‐HRP (Vector Laboratories). Diaminobenzidine  was used as the chromogen.Slides were counterstained with haematoxylin and mounted. Substitution of primary antibody with an irrelevant IgG served as a negative control.https://imagej.nih.gov/ij).For each marker, 10–18 single images at 200× magnification were taken from each tissue section with a digital camera (Olympus Camedia C‐4040 ZOOM), yielding homogeneous 24 bit colour images with a 1024 × 768 pixel size and 200 dpi resolution. The % of area positive for each marker was calculated by computer‐assisted morphometric analysis using FiJi software  following manufacturer's instructions. One hundred nanograms (2 μl) of total RNA was retrotranscribed to cDNA using the ImProm‐II™ ReverseTranscription System  and amplified by polymerase chain reaction (PCR) in a final volume of 50 μl, using Go Taq Polymerase (Promega). The nucleotide sequences of primers used in the PCR are listed in Table Housekeeping genes (β‐actin or 18S rRNA) were used as internal controls to calculate relative quantification of target gene expression. Target and housekeeping genes were amplified in the same reaction.http://rsb.info.nih.gov/ij/docs/menus/analyze.html#gels) to calculate the intensity values of strips.The amplified PCR products were stained with ethidium bromide on a 1.5% agarose gel and visualized by UV light and photographed using UV transilluminator GelDoc 2000 system . Gels were analysed by ImageJ software .Data are expressed as median values and the relative interquartile range (IQR). Differences between experimental groups (PBS and hAEC groups) were assessed by nonparametric Mann–Whitney test. A Liver fibrosis can be defined as a result of the progressive accumulation and remodelling of the ECM, which disrupts the normal hepatic architecture.In this study, liver fibrosis was induced in rats by BDL. The presence of jaundice (2 days post‐ligation) and high serum concentrations of total bilirubin  indicated a successful obstruction.ECM presence in healthy livers was very low. Fibronectin was observed within the portal area and around the central vein ; collagen was present in very low amounts in the periportal area as a thin rim around blood vessels.On the other hand, livers extracted from control animals (BDL + PBS group) 6 weeks after BDL showed a large amount of ECM. Fibronectin was arranged around biliary ducts and along hepatic sinusoids Fig. A. CollagTo evaluate the effects of hAEC treatment, the degree of fibrosis in livers from control and treated animals was determined by analysing fibronectin deposition, collagen accumulation (through quantitation of collagen‐positive area on PSR‐stained liver sections) and collagen distribution in hepatic lobules .P < 0.05] , % liver area occupied by PSR‐positive staining]  score units in treated and control rats, respectively]  to 6.88 IQR: 5.82) % liver‐positive area,  % liver‐versus 2.33 (IQR: 3.06) % liver‐positive area for α‐SMA, P < 0.01]  As myofibroblasts can derive from the activation of PF and HSC, we examined whether hAEC can impact the activation of these cells. We therefore investigated the phenotype of myofibroblasts arising from PF activation as collagen 15A1‐positive cells In liver sections of healthy rats, very rare collagen 15A1‐positive cells were present in the periportal areas. Similarly, desmin staining was very scant, and rare positive cells  were restricted to arterial walls and along sinusoids.The presence of these markers increased in BDL‐injured rats.versus 4.13 (IQR: 1.11) % collagen 15A1‐positive area, P < 0.05]  % desmin‐positive area in hAEC‐treated and control groups, respectively; P = 0.1203]  Data collected from PCR analysis were in line with those obtained from IHC analysis. Fig. A–C.We then addressed the hypothesis that hAEC may reduce myofibroblast generation by acting on ductular epithelial cells. In healthy livers, ductular structures were limited and present only in the portal areas . In response to biliary obstruction, ductular epithelial cells intensively proliferate and give rise to increased numbers of biliary structures (‘ductular reaction’).versus 6.47 (IQR: 7.79), P < 0.05] , P < 0.05] with respect to control animals.We first assessed whether hAEC treatment could limit the ductular reaction by detecting CK19‐positive cells, a marker which is normally expressed by ductular epithelial cells Fig. A. hAEC‐tversus 70.7 (IQR: 25.9)]  counted in control livers (P = 0.1014).We further investigated whether hAEC could inhibit the possible transition of ductular epithelial cells towards a fibroblast phenotype. For this purpose, we assessed hepatic cells expressing S100A4 , a marker of epithelial–mesenchymal transition via inhibition of ductular expression of αvβ6 integrin. We first observed that αvβ6 integrin expression was lower in livers of hAEC‐treated animals   % liver‐positive area for p‐Smad2/3; P < 0.01]  % liver‐positive area for CD32b, respectively; P = 0.593]  characterized by a strong ductular proliferation and acquisition of an inflammatory phenotype leading to the secretion of inflammatory mediators promoting the activation of periductular fibroblasts towards myofibroblasts. Our data demonstrate that hAEC strongly decrease ductular proliferation and thus possibly contribute in reducing the inflammatory milieu generated by the reactive ductular cells and consequently their pro‐fibrogenic activity. Under BDL‐induced inflammatory conditions, αvβ6 integrin is up‐regulated on the ductular surface Importantly, we observed that hAEC treatment effectively decreases ductular expression of αvβ6 integrin, and this action is associated to a significant reduction in TGF‐β activation.Interestingly, given the involvement of fibronectin in TGF‐β activation mediated by αvβ6 integrin Furthermore, as αvβ6 integrin has been shown to induce the proliferation of different cells In addition to ‘ductular reaction’, after liver injury, ductular epithelial cells may undergo epithelial‐to‐mesenchymal transition giving rising to EMT‐derived fibroblasts which potentially differentiate towards ECM‐producing myofibroblasts. We found that hAEC treatment reduces the number of EMT‐derived periductular fibroblasts (S100A4‐positive cells) in vitro culture Finally, on the basis of the ability of the amniotic membrane to maintain liver sinusoidal cell fenestrations during In conclusion, our data show that hAEC reduce PF activation towards myofibroblasts and the ability of reactive ductular cells to generate periductular myofibroblasts. Thus, our results suggest that hAEC act on pro‐fibrogenic cells located in portal area and directly involved in biliary injury, which display an early, proliferative activity and an inflammatory phenotype. Finally, these data suggest the application of hAEC as a possible therapy not only in biliary‐associated fibrosis, but also in any liver injury which involves the expansion and activation of the ductular epithelial compartment.A.C. participated in the design of the study, performed the experiments, analysed the data and drafted the manuscript; S.E.F. and L.B.S. carried out microscopy analysis; E.C.P. and P.R. processed placental amniotic membranes to collect hAEC; P.B.S. and E.C.P. performed molecular analysis; G.V. performed animal surgery; I.T. and V.C. were responsible for animal care; M.M. and A.S. participated in the drafting of the manuscript; O.P. conceived of the study and participated in its design, supervised the research, participated in drafting and critically revised the manuscript. All authors read and approved the final version of the manuscript.The authors confirm that there is no conflict of interests.


Recent studies have shown that breast cancer subtype can change from the primary tumour to the recurrence. Discordance between primary and recurrent breast cancer has implications for further treatment and ultimately prognosis. The aim of the study was to determine the rate of change between primary and recurrence of breast cancer and to assess the impact of these changes on survival and potential treatment options.Patient demographics were collected on those who underwent surgery for breast cancer between 2001 and 2014 and had a recurrence with biopsy results and pathology scoring of both the primary and recurrence.n = 20), followed by triple negative (n = 4), luminal B (n = 3) and HER2 (n = 3). Patients who changed from luminal A to triple negative (n = 18) had a significantly worse post-recurrence survival (p < 0.05) with overall survival approaching significance (p = 0.064) compared to concordant luminal A cases (n = 46). Overall receptor discordance rates were: estrogen receptor 20.4% (n = 27), progesterone receptor 37.7% (n = 50) and HER2 3% (n = 4). Loss of estrogen receptor and progesterone receptor was more common than gain (21 vs. 6 (p = 0.04) and 44 vs. 6 (p = 0.01) respectively). Nine patients (6.8%) gained receptor status potentially impacting treatment options.One hundred thirty two consecutive patients were included. There were 31 (23.5%) changes in subtype. Discordance occurred most frequently in luminal A breast cancer  contains supplementary material, which is available to authorized users. Breast cancer is the second most common cancer worldwide and the most common cancer among women with an estimated 1.67 million women diagnosed annually, and the fifth leading cause of death from cancer overall . Breast The aim of our study was to identify subtype change in recurrent breast cancer at our institution, to assess the impact of discordance on patient outcomes, and to identify any potential changes in treatment due to a subtype change and if in reality patients who changed subtype experienced a change in treatment strategy.Data was collected on patients who had a recurrence of breast cancer following surgery +/− chemotherapy/hormonal therapy/radiotherapy at the Galway Hospitals group between 2001 and 2014. Loco-regional recurrence after surgery was defined as the appearance of tumour in the ipsilateral chest wall or axillary, internal mammary or supraclavicular lymph nodes while distant recurrence was defined as recurrence to distant organs, confirmed by pathologists report. Only patients who had clinical pathology scoring of receptor status of both the primary and recurrent cancer were included. Exclusion criteria included presentation with bilateral tumours, biopsy results that were incomplete, and pathologist report of the recurrence as a new primary tumour. PAS software was used to access pathology records with MOSAIQ software used to determine patient pathways and treatment.Analysis of all samples was performed at the Pathology Laboratory, University Hospital Galway independently by clinical pathologists. Samples were obtained following surgery and at recurrence, with sufficient slides taken to perform all necessary immunohistochemical and pathological analysis. Samples were reviewed by a minimum of two pathologists, with an initial assessment from at least one primary reporting pathologist and a subsequent review performed by a pathologist at a multi-disciplinary meeting. The ER and PR receptor status were determined independently by clinical pathologists using immunohistochemistry  as per Ap < 0.05.Data analysis was performed using SPSS Version 21 . Overall survival and post-recurrence survival were estimated using the Kaplan-Meier product limit method. The log rank was used to determine any statistically significant differences in survival between the indicated groups. Comparative analyses were performed between groups using Chi-squared and T-tests. Statistical significance was accepted for This study was conducted in accordance with the granted National University of Ireland Galway and University College Hospital Galway ethical approval. All patients had histologically confirmed breast cancer and all relevant clinic-pathological and demographic data were obtained from a prospectively maintained breast cancer database. This study used retrospectively collected, de-identified data, and no patients were involved.n = 27), followed by liver (n = 22) and lung (n = 16) (Table One hundred thirty two patients were included. Mean age at diagnosis was 53.3 year range 21–84). 58 patients (44%) had a loco-regional recurrence while 74 (56%) had a distant recurrence (Table –84. 58 pn = 27), 37.8% (n = 50), and 3% (n = 4) respectively (Table n = 27) and the ER concordant group (n = 105), (60.2 vs. 59.3 months), while post-recurrence survival (PRS) was shorter in the discordant group, but this was not statistically significant . There was no statistically significant difference in OS or PRS between the PR discordant (n = 50) and concordant (n = 82) groups . In terms of HER2 receptor, there was a significant difference between the discordant (n = 4) and concordant (n = 128) groups in OS and PRS . However, the very low numbers in the discordant group limit the value of this result. There was a statistically significant loss compared to gain of both ER and PR receptor status (ER loss n = 21 (15.9%) vs. gain n = 6 (4.5%), p = 0.04; PR n = 44 (33.2%) vs. n = 6 (4.5%), p = 0.01). Of the four HER2 receptor discordant cases, two gained and two lost receptor status, however these numbers are too low to draw statistical significance.Rates of single receptor discordance for ER, PR and HER2 receptors were 20.4% (n = 31) had a longer mean time to recurrence compared to the concordant group (n = 101)    subtype resulted in poorer 10 year OS versus the concordant luminal A group (n = 46) which approached statistical significance    to the concordant triple negative group (n = 35), there was no significant difference in 10 year OS  or 5 year PRS   who had a different subtype on recurrence, 17 were loco-regional recurrences and 14 were distant  Fig. . Importa05) Fig. . CompariIn terms of changes in subtype that could potentially lead to a change in treatment, nine patients (6.8%) gained receptor status on recurrence. Seven patients went from HR negative to positive, with 6 patients going from ER negative to positive (ALLRED score 0 in the primary to > 2 in the recurrence). One patient went from PR negative to positive. Of these seven patients, five had a loco-regional recurrence and two had distant recurrences . None of these patients received additional endocrine therapy following the biopsy results of the recurrence. All nine patients are deceased with a mean OS of 52 months and a mean PRS of 21 months.Two patients gained HER2 receptor status, both going from HER2 score of 0 on Herceptest of the primary to 1 of the recurrence, with both subsequently testing positive on FISH. One patient had a distant recurrence in bone, and was enrolled in the TRIO 022 trial , subsequIn summary, only one patient in our study of nine who gained receptor status ultimately received additional targeted therapy.In our single-centre analysis the rate of subtype change of was 23.5%, supporting previously published figs. , 16. In p = 0.04) and PR (p = 0.01), in line with published data [Single receptor discordance was 20.4%, 37.8%, and 3% for ER, PR and HER2 receptor respectively, similar to that reported in a recent meta-analysis examining 48 papers, which reported pooled discordance rates of 20%, 33% and 8% for ER, PR and HER2 receptor . HER2 rehed data .There are a number of possible aetiologies for receptor discordance. Firstly, variability exists in the reproducibility and accuracy of immunohistochemical staining . There iIn terms of potential alterations to treatment and survival benefits of performing a recurrence biopsy, there is conflicting data with much of the literature being retrospective and examining small populations with variability in assay used, site of metastasis and definition of recurrence , 34, 35.In terms of the effect that changing management had on patient outcomes, the results were unclear and only the DESTINY trial looked at overall survival. There was no significant association between overall survival and discordance (median OS 27.6 vs. 30.2 months in the concordant and discordant groups respectively). Other retrospective studies have identified a change in management plan in 12–20% of patients where there was a gain in receptor status , 35, 38.Current guidelines by the American Society of Clinical Oncology (ASCO)  advise oLimitations of our study include the relatively small sample size. The retrospective nature of the study made it difficult to accurately collate data on patient’s precise treatment regimes. Furthermore, as discussed above technical misclassification is a significant contributor to receptor discordance. Gain in receptor status may be attributable to this misclassification as opposed to a genuine change in tumour biology , 42. It In summary, our study demonstrates the discordance of receptor and subtype between primary and recurrent breast cancer at our institution. It highlights the importance of performing a biopsy of recurrent breast cancer, due to the implications that change in subtype has on survival. Further research is required to investigate the aetiology and biology of subtype discordance and the optimal strategy for treatment change based on this discordance. Our results highlight the need for a prospective, multicentre trial collecting data on patients who experience recurrence (including routine biopsy’s of recurrence), to establish if all recurrent patients should be biopsied, or only a subset of patients most likely to benefit from additional treatment options.Additional file 1:Table S1. Impact of Location/Neoadjuvant chemotherapy/Surgery on discordance. Quantifying: Recurrence location, Neoadjuvant Chemo Rx, Surgery, Change subtype, Gain of Receptor. (DOCX 24 kb)Additional file 2:Figure S1. Triple negative to Luminal A (n = 4) vs. triple negative concordant (n = 35). A 10 year overall survival (p = 0.378). B 5 year post recurrence survival (p = 0.919). (TIFF 542 kb)


RHA) and Low‐Avoidance (RLA) rats for, respectively, rapid versus poor acquisition of the active avoidance response has generated two distinct phenotypes differing in many behavioral traits, including coping strategies to aversive conditions. Thus, RLA rats are considered as a genetic model of vulnerability to stress‐induced depression whereas RHA rats are a model of resilience to that trait. Besides the monoamine hypothesis of depression, there is evidence that alterations in neuronal plasticity in the hippocampus and other brain areas are critically involved in the pathophysiology of mood disorders.The selective breeding of Roman High‐ (WB) and immunohistochemistry were used to investigate the basal immunochemical occurrence of brain‐derived neurotrophic factor (BDNF) and its high‐affinity tyrosine‐kinase receptor trkB in the dorsal and ventral hippocampus of adult RHA and RLA rats.Western blot  in brain sections showed that BDNF‐LI is 24% to 34% lower in the different sectors of the Ammon's horn of RLA versus RHA rats, whereas line‐related differences are observed in the dentate gyrus (DG) only in the ventral hippocampus. As for trkB‐LI, significant differences are observed only in the dorsal hippocampus, where density is 23% lower in the DG of RLA versus RHA rats, while no differences across lines occur in the Ammon's horn.BDNF/trkB signaling in the hippocampus of RLA versus RHA rats may contribute to their more pronounced vulnerability to stress‐induced depression.These findings support the hypothesis that a reduced  For immunohistochemistry, brains were fixed by immersion in freshly prepared 4% phosphate‐buffered paraformaldehyde, pH 7.3, for 4–6 hr at 4°C, and then rinsed overnight in 0.1 m phosphate buffer (PB), pH 7.3, containing 20% sucrose.Immediately after sacrifice by guillotine, the brains were rapidly dissected and processed for either WB or immunohistochemistry. For WB, brains were cooled in dry ice for 15 s, placed in a brain matrix and cut in 2 mm thick coronal slices using the stereotaxic coordinates of the rat brain atlas of Paxinos and Watson  as a ref2.3m Tris base and 137 mm sodium chloride (TBS) containing 0.1% Tween 20 (TBS‐T) and 5% milk powder for 60 min at room temperature. The primary antibodies were rabbit polyclonal antibodies against BDNF  and trkB , both diluted 1:1000 in TBS containing 5% milk powder and 0.02% sodium azide. Incubations with primary antiserum were carried out for two nights at 4°C. After TBS/T rinse, blots were incubated for 60 min, at room temperature, with a peroxidase‐conjugated goat anti‐rabbit serum , diluted 1:10,000 in TBS/T. Controls for equal loading of the wells were obtained by immunostaining the membranes as above, using a mouse monoclonal antibody against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) , diluted 1:1,000, as primary antiserum, and a peroxidase‐conjugated goat anti‐mouse serum , diluted 1:5,000, as secondary antiserum. In order to control for nonspecific staining, blots were stripped and incubated with the relevant secondary antiserum. After TBS/T rinse, protein bands were developed using the Western Lightning Plus ECL , according to the protocol provided by the company, and visualized by means of ImageQuant LAS‐4000 . Approximate molecular weight (mw) and relative optical density (O.D.) of labeled protein bands were evaluated by a blinded examiner. The ratio of the intensity of BDNF‐ and trkB‐positive bands to the intensity of GAPDH‐positive ones was used to compare relative expression levels of these proteins in the RHA and RLA lines. The O.D. was quantified by Image Studio Lite Software .Total protein concentrations were determined as described by Lowry, Rosebrough, Farr, & Randall  using bo2.4RRID:AB_2313606, Vector, Burlingame, CA, USA), diluted 1:400, was used as secondary antiserum. The reaction product was revealed with the ABC , diluted 1:250, followed by incubation with a solution of 0.1 m PB, pH 7.3, containing 0.05% 3,3'‐diaminobenzidine , 0.04% nickel ammonium sulfate and 0.01% hydrogen peroxide. All antisera and the ABC were diluted in PBS/T. Incubation with primary antibodies was carried out overnight at 4°C. Incubations with secondary antiserum and ABC lasted 60 min and 40 min, respectively, and were performed at room temperature. Negative control preparations were obtained by incubating tissue sections in parallel with either PBS/T alone or with the relevant primary antiserum preabsorbed with an excess of the corresponding peptide antigen . Slides were observed with an Olympus BX61 microscope and digital images were captured with a Leica DFC450C camera.Coronal sections of RLA and RHA rat brains were examined in pairs on the same slide. Semiconsecutive cryostat sections (14 μm thick) were collected on chrome alum‐gelatin coated slides and processed by the avidin–biotin–peroxidase complex (ABC) immunohistochemical technique. The endogenous peroxidase activity was blocked with 0.1% phenylhydrazine  in phosphate buffered saline (PBS) containing 0.2% Triton X‐100 (PBS/T), followed by incubation with 20% of normal goat serum . The same rabbit polyclonal antibodies against BDNF and trkB  used for WB, both diluted 1:500, were used as primary antibody. A biotin‐conjugated goat anti‐rabbit serum  to calculate the density of immunoreactivity per μm2. Mean gray values from unstained areas were subtracted from the gray values of the immunostained regions to exclude background staining.For the quantitative evaluation of BDNF and trkB immunohistochemical labeling, representative 10x magnification microscopic fields, taken from sections of six animals for each line, were blindly analyzed with ImageJ , −31% in CA1  and −24% in CA2 , while in the ventral hippocampus were −28.5% in CA3  and −27.5% in the DG .BDNF‐immunostaining labeled extensive nerve fiber networks that were mostly distributed in the Ammon's horn, where labeled filamentous elements occurred in between the cell bodies of the pyramidal layer and in the molecular layer of the CA1 Figure a, b, CA2p < .0001), while no significant differences were found in the Ammon's horn (see Table TrkB‐immunostaining also labeled extensive nerve fiber systems, mostly appearing as filaments, short hollow tubules and coarse punctate elements. Positive elements occurred in both the Ammon's horn Figure a–f and t4The main finding of the present study is that, under baseline conditions, the relative protein levels of BDNF and its selective receptor trkB, measured by WB, are significantly lower in the dorsal hippocampus of depression‐vulnerable RLA rats versus depression‐resistant RHA rats. Moreover, the densitometric analysis of immunostained brain slices revealed that the BDNF‐LI is lower in the Ammon's horn whereas trkB‐LI is lower in the dentate gyrus of RLA rats as compared to their RHA counterparts. These findings add experimental support to the neurotrophic hypothesis of depression, which postulates that decreased levels of BDNF in the hippocampus play a critical role in the pathophysiology of this mental disorder (Duman & Monteggia, Interestingly, it has been shown that different antidepressant treatments, such as physical activity (Baj et al., The lower levels of BDNF‐like immunoreactivity in the Ammon's horn of RLA versus RHA rats, especially in the CA3 sector, are consistent with the hypothesis that, in the RLA line, CA3 neurons may have a slower synthesis rate of BDNF. In the CA3 sector BDNF is known to be both locally produced and anterogradely transported along the mossy fiber axons of the granule cells in the dentate gyrus. Thus, the possible slower production of BDNF protein in RLA versus RHA rats, as also suggested by the lower density of BDNF‐LI in the dentate gyrus of the ventral hippocampus, may in turn lead to a reduced target‐derived support to promote the synaptic contacts with the mossy fibers. Interestingly, a selective increase in both BDNF mRNA and protein in the CA3 and CA2 sectors has been reported upon repeated antidepressant treatment (De Foubert et al., The immunolabeling of both BDNF and trkB in the dentate gyrus is localized to cell bodies and nerve fiber networks. The occurrence of BDNF‐containing granule cells is in line with the reported synthesis of BDNF mRNA in this hippocampal region (Phillips et al., Studies on the BDNF targeting on hippocampal CA3 dendrites (Baj, Pinhero, Vaghi, & Tongiorgi, As mentioned above see , RLA andThe 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.OG, MGC, MQ: Designed the study and wrote the manuscript; all authors contributed to experimental design; OG, MGC, MQ, and MPS: Interpreted the findings and revised the article; MGC, MAP, FS, and OG: Selected and bred the Roman rats; MPS, LP, MB, and MAP: Performed the sampling; MPS, LP, MB, and MQ: Performed the immunochemical experiments, image and statistical analyses; MQ: Performed the artwork. All authors read and approved the final manuscript.


QT prolongation and/or torsade de pointes (TdP) associated with the use of hydroxyzine, a first generation sedating antihistamine. We have used a combined methodological approach to re‐evaluate the cardiac safety profile of hydroxyzine, including: (1) a full review of the sponsor pharmacovigilance safety database to examine real‐world data on the risk of QT prolongation and/or TdP associated with hydroxyzine use and (2) nonclinical electrophysiological studies to examine concentration‐dependent effects of hydroxyzine on a range of human cardiac ion channels. Based on a review of pharmacovigilance data between 14th December 1955 and 1st August 2016, we identified 59 reports of QT prolongation and/or TdP potentially linked to hydroxyzine use. Aside from intentional overdose, all cases involved underlying medical conditions or concomitant medications that constituted at least 1 additional risk factor for such events. The combination of cardiovascular disorders plus concomitant treatment of drugs known to induce arrhythmia was identified as the greatest combined risk factor. Parallel patch‐clamp studies demonstrated hydroxyzine concentration‐dependent inhibition of several human cardiac ion channels, including the ether‐a‐go‐go‐related gene (hERG) potassium ion channels. Results from this analysis support the listing of hydroxyzine as a drug with “conditional risk of TdP” and are in line with recommendations to limit hydroxyzine use in patients with known underlying risk factors for QT prolongation and/or TdP.Several noncardiac drugs have been linked to cardiac safety concerns, highlighting the importance of post‐marketing surveillance and continued evaluation of the benefit‐risk of long‐established drugs. Here, we examine the risk of  CSIcardiac safety indexDDDdefined daily doseECGelectrocardiogramHCPhealthcare professionalhERGether‐a‐go‐go related geneIBDInternational Birth DateICHInternational Conference on HarmonizationMedDRAMedical Dictionary for Regulatory ActivitiesPTspreferred termsSMQstandard MedDRA queryTTOtime to onsetWHOWorld Health OrganizationA growing number of noncardiac drugs have been found to delay cardiac repolarization, causing QT interval prolongationIn past years, concerns have been raised regarding the cardiac safety of hydroxyzine, a first generation sedating antihistamine used for the management of anxiety and pruritus .The sponsor pharmacovigilance database provides spontaneous adverse event reports from worldwide sources  for all sponsor marketed products, as well as serious adverse event reports from clinical trials of any sponsor compounds in development. For this study we conducted a review of all adverse event reports occurring between 14th December 1955  and 1st August 2016, for formulations containing hydroxyzine as the active compound.In brief, the sponsor database was searched for hydroxyzine and Medical Dictionary for Regulatory Activities (MedDRA) preferred terms (PTs) for TdP and QT prolongation. Six narrow standard MedDRA query (SMQ) PTs, considered highly likely to represent adverse events associated with QT prolongation and/or TdP, were used: electrocardiogram (ECG) QT prolonged, ECG QT interval abnormal, long QT syndrome, long QT syndrome congenital, TdP, and ventricular tachycardia. An additional 14 broad SMQ PTs were used to identify all possible related cases: cardiac arrest, cardiac death, cardiac fibrillation, cardio‐respiratory arrest, ECG U‐wave abnormality, ECG repolarization abnormality, loss of consciousness, sudden cardiac death, sudden death, syncope, ventricular arrhythmia, ventricular fibrillation, ventricular flutter, and ventricular tachyarrhythmia. MedDRA version 19.1 was used for the coding of adverse events.®   was set at 7 days to include all cases potentially linked to hydroxyzine intake. In summary, the cases were assessed using the following strategy: TTO (<7 days); daily dose (mg); presence of risk factors/underlying medical conditions; and positive dechallenge/rechallenge.Exposure data for hydroxyzine was estimated based on sales data from 1st January 2007 to 31st July 2016 (these data were not available prior to 2007). The defined daily dose (DDD) was 75 mg, in accordance with the World Health Organization (WHO), and patient‐years of exposure was calculated using the following formula:μmol/L), all within the clinically relevant range, were tested at room temperature (22 ± 2°C) and at near‐physiological temperature (35 ± 1°C).In vitro whole‐cell patch‐clamp experiments were performed using human embryonic kidney 293 (HEK293) cells to assess the concentration‐dependent effects of hydroxyzine on the peak amplitude of tail currents mediated by stably expressed hERG potassium channels. A conventional manual patch‐clamp assay technique was used , tested at room temperature, covered and exceeded the estimated therapeutic exposure range.In a second nonclinical study, patch‐clamp techniques  / [(kn) + (Xn)] where y denotes percentage inhibition of the current of interest at hydroxyzine concentration X; Vmax, k and n correspond to the maximal current inhibition of the hERG tail current (data fixed to 100), IC50 and Hill coefficient, respectively.For nonclinical data, the IC50 value for inhibition of the hERG tail current by the Cmax,free of hydroxyzine at a dose of 50 mg (0.01 μmol/L). CSI evaluation at hydroxyzine doses >50 mg remains theoretical and incomplete, as pharmacokinetics for higher concentrations of hydroxyzine in humans are unknown.The cardiac safety index (CSI) of hydroxyzine was calculated by dividing the estimated ICBased on marketing experience between 14th December 1955 and 1st August 2016, a total of 59 case reports of QT prolongation and/or TdP were retrieved from the safety database using the narrow SMQ PTs for QT prolongation/TdP  of 1 day following hydroxyzine administration. Among cases that described the TTO, 15 were reported following an overdose of hydroxyzine and patients experienced adverse events within 1 day. Excluding these 15 reports of overdose, the median TTO was 3 days. Overall, 14 out of the 59 cases (excluding cases with overdose) had a TTO of <7 days; considered a sufficiently long period to capture all cases potentially linked to hydroxyzine intake. Of these cases, the most reported risk factor (50% of cases) was the combination of cardiovascular disorders with concomitant administration of drugs known to induce QT prolongation/TdP. Positive dechallenge was reported in 16/59 cases, with the adverse event subsiding or stopping upon discontinuation of drug therapy. In the majority of cases multiple medications were discontinued, including 12 cases in which other drugs linked to QT interval prolongation and/or TdP had also been discontinued.Among the 59 case reports, a total of 69 events were identified . This included 41 events of ECG QT prolonged, 18 events of TdP, and 10 events of ventricular tachycardia Table . There wAll 27 case reports of QT prolongation and/or TdP in patients exposed to hydroxyzine ≤100 mg (maximum recommended dosage) had at least one underlying independent risk factor for the onset of the QT prolongation/TdP Table . The mosOf the 59 case reports, 15 reports were in elderly patients (≥65 years of age) and 39 reports were in females Tables , S2. TheFrom the broad SMQ search, 226 cases were retrieved; excluding the 59 cases identified using the narrow SMQ PTs, 167 cases were reported. Of these, 24 cases were reported with a TTO <7 days, including 13 reports of loss of consciousness and syncope (symptoms that may describe conditions associated with QT prolongation and TdP), 10 cardiac and/or cardiorespiratory arrests , and one case of ECG repolarization abnormality. This latter case was particularly supportive for our analysis because it described a suggestive event for the development of QT prolongation/TdP, and also reported concomitant medications known to induce QT prolongation and TdP.50 of 0.39 μmol/L at near‐physiological temperature .All of the 59 cases reported involved either underlying medical conditions or concomitant medications that constituted at least one baseline risk factor (and often multiple risk factors) for such events, or were associated with an intentional overdose. Potential risk factors included a history of cardiac disorders , metabolic abnormalities, genetic disorders, renal disease, and concomitant medications associated with QT prolongation and/or TdP.The results presented here are aligned with the listing of hydroxyzine as having a “conditional risk of TdP”; with QT prolongation and/or TdP only observed in patients with underlying risk factors or following intentional overdose  was observed among different dose, age and gender groups indicating a possible causal relationship between hydroxyzine administration and the event of interest, and is similar to the TTO observed in recently published case reports , the elderly (who may have a decreased clearance), and those exposed to interactions of hydroxyzine with concomitant drugs. In line with previous reports that hydroxyzine can inhibit hERG potassium channels with IC50 values ranging from 0.62 μmol/L down to 0.16 μmol/L, , assuming no other risk factors. This is above the recommended CSI ratio of 30 and suggests that hydroxyzine has an acceptable safety margin for proarrhythmia risk (Redfern et al. Since the CSI decreases with higher Limitations of this study include the use of a post‐marketing database, which may contain incomplete reports, and the well‐described under‐reporting in these databases. Although measures were taken to perform appropriate control experiments and manage experimental conditions for the patch‐clamp studies, (Gintant et al. When taken at a maximum recommended dosage of 100 mg/day, hydroxyzine can provide important clinical benefits for many patients across indications. To ensure that the benefit‐risk ratio is positive for all patients receiving hydroxyzine, precautions should be taken to limit its use in those with known risk factors for QT prolongation and TdP regardless of patient age and the daily dose taken. This includes, but is not limited to, pre‐existing cardiac conditions, metabolic abnormalities, and/or exposure to concomitant medications associated with QT prolongation and/or TdP. The use of hydroxyzine is not recommended in the elderly due to a prolonged elimination half‐life, but if unavoidable, a maximum dose of 50 mg/day should be used.In summary, based on an overall assessment of data from the literature, non‐clinical studies, and pharmacovigilance data we have re‐evaluated the risk of QT prolongation and/or TdP associated with exposure to hydroxyzine, a drug used in clinical practice since the 1950s. There are several implications of our findings. Firstly, they confirm that hydroxyzine can be listed as a drug with “conditional risk of TdP”. Secondly, they demonstrate that the combination of cardiovascular disorders with concomitant treatment of drugs known to induce arrhythmia is the greatest risk factor for the onset of QT prolongation/TdP after hydroxyzine administration. Lastly, they emphasize the importance of continual re‐evaluation of potential safety issues of older drugs using modern knowledge and standards. Taking into account the full critical evaluation of information presented here, and the implementation of appropriate measures to ensure that hydroxyzine use is carefully considered in those patients with an underlying risk of QT prolongation and/or TdP, the benefit‐risk balance of hydroxyzine remains positive for the recommended doses and indications of anxiety and pruritus.Substantial contributions to study conception/design, or acquisition/analysis/interpretation of data: A‐FS, AD, AC, BC, LC, RB, J‐PV, CP, VSS, JB; Drafting of publication, or revising it critically for important intellectual content: A‐FS, AD, AC, BC, LC, RB, J‐PV, CP, VSS, JB; Final approval of the Publication: A‐FS, AD, AC, BC, LC, RB, J‐PV, CP, VSS, JB.A‐FS, AD, RB, J‐PV, VSS and JB are employees and shareholders of UCB Pharma; AC and CP are former employees of UCB Pharma; BC and LC are contract employees for UCB Pharma.Supplemental Methodology. Non‐clinical electrophysiological studies.Table S1. Most reported risk factors among the 15 cases in elderly patients.Table S2. Most reported risk factors among the 39 cases in female patients.Click here for additional data file.


Salvadora persica is commonly used chewing sticks in many parts of the world as an oral hygiene tool. This study measured the amount of benzyl isothiocyanate (BITC) released into the mouth and assessed its retention time in saliva. The study also tested if the released amount of BITC could potentially be antibacterial or cytotoxic. Twelve subjects brushed their teeth with fresh Miswak once, twice, and four times. The amount of BITC in the saliva and in the used brushes was quantified using gas chromatography-mass spectrometry. The antibacterial effect of BITC and Miswak essential oil (MEO) was tested against Haemophilus influenzae, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The cytotoxic effect on gingival fibroblasts and keratinocytes was tested using MTT. The highest amount of the active compounds was detected in saliva after using the Miswak tip for once and immediately. It significantly decreased when the Miswak tip was used more than once and thus after 10 min. The growth of the tested bacteria was inhibited by MEO and BITC in a dose dependent manner, P. gingivalis being the most sensitive. MTT assay showed that BITC and MEO were cytotoxic towards gingival fibroblasts while oral keratinocytes showed resistance. This study suggests that the Miswak tip should be cut before each use to ensure the maximum effect. Humans have been interested in maintaining clean and healthy teeth since ancient time . The use Salvadora persica are commonly used in major parts of Middle East, Africa, South Asia, and many parts of Europe and America [ S. persica is shrub like plant belonging to the family Salvadoraceae. It is widespread in large parts of Africa and Asia. In Middle East, S. persica or Arak tree is also known by several other names such as Siwak, Miswak, Mustard tree, and natural toothbrush tree. The use of chewing sticks (Miswak) made from S. persica is an old pre-Islamic custom adhered to by the ancient Arabs in order to make their teeth white and shiny [The chewing sticks made from the roots of America , 5. S. pnd shiny . When Isnd shiny . In this S. persica root and essential oil exhibit a strong antibacterial effect, especially on Gram-negative bacteria [ S. persica extract [A number of studies have shown that chewing sticks are effective in reducing plaque and gingival inflammation , 8. Whenbacteria –22. Othe extract , 24.There seems to be a discrepancy between a very clear antibacterial effect in vitro and a less obvious effect in vivo. Thus the aim of this study was to establish how much of the BITC is contained inside the fresh chewing stick that could be released into the mouth and how long it is retained in the saliva. Furthermore, we wanted to know if the released amount of BITC could be antibacterial and/or cell toxic.This study consists of three main parts, (i) measurements of the amounts of BITC released from the Miswak chewing sticks into the saliva during use, (ii) measurement of cytotoxicity, and (iii) measurement of antibacterial effect of Miswak oil and BITC. S. persica were purchased directly from a farm in Jizan, Saudi Arabia and shipped to Karolinska Institutet, Stockholm, Sweden, by FedEx within 3 days after harvest. During the transportation, they were sealed by paper wrap and plastic bag as an outer layer. On reaching destination, they were manually cleaned, sorted, placed in airtight polyethylene bags, and stored at −80°C till used for essential oil extraction or using as chewing stick (Miswak) in different studies.Fresh roots of S. persica roots (1.4 kg) were cut into 20–30 mm long pieces and mixed with 700 mL double-distilled water. The resulting mixture was subjected to hydrodistillation for 4-5 h in a glass distillation apparatus. The collected distillate (oil/water mixture) was extracted three times with HPLC grade hexane  with the help of a separating funnel. Anhydrous MgSO4  was added to the hexane extract to remove any trace of water. After filtration, hexane was evaporated using a rotary evaporator  at 20°C under reduced pressure. The essential oil obtained was then weighed and the yield calculated as percentage (w/w) of the total plant material. The essential oil was stored at −20°C in a freezer till further experiments and analyses on GC-MS.The essential oil was extracted by adopting methodology described by El-Seedi et al., 2012 . Briefly S. persica root that was 150 mm long and about 10 mm diameter. This twig was to be used as chewing stick throughout the entire study period. In order to expose fresh twig, each volunteer cut off roughly a 2 mm long piece from one side of the twig. From the remaining twig, approximately 7 mm long piece was cut; this piece was collected in a labelled Eppendorf tube so that the quantity of compounds originally present in each twig could be determined. After removal of the “baseline” piece, the 5–7 mm length of the twig was chewed and the resulting fibrous brush was used to clean the teeth. During the cleaning process saliva collected in mouth was transferred in a 15 mL Falcon tube. The used brush was then cut and placed in a labelled Eppendorf tube. The saliva and used Miswaks brushes were collected after the first, second, and fourth use of the twig. All the Eppendorf tubes and Falcon tubes were collected from the participants and stored in a freezer at −80°C pending extraction and analysis of volatile compounds using gas chromatography-mass spectrometry (GC-MS).Twelve adults, five men and seven women, who reported good oral health were chosen for this study. The purpose of the study was fully explained and informed consent was obtained from all the participants before the study was commenced. The study was approved by the regional Research Ethics Committee in Stockholm (#2012/609-31/1). Each of the twelve participants was assigned with a fresh chewing twig ofIn order to check the retention of BITC and other compounds in saliva, the volunteers using freshly cut Miswak brush were also asked to collect saliva before brushing, immediately after brushing, and 5, 10, and 30 min after brushing. The saliva samples were treated as described above until analysed for the quantification of volatiles by GC-MS.μm stable flax fibre coated with polydimethylsiloxane/divinylbenzene . Prior to its first use the SPME fibre was conditioned at 250°C for 30 min as advised by the manufacturer.The frozen saliva samples were thawed by being kept at room temperature for 1.5 h and then for another 30 min after covering with aluminum foil so that the headspace (HS) air in the tubes could be equilibrated with volatile compounds in saliva. The volatiles from the saliva samples were extracted by using HS solid phase microextraction (SPME) method –29. AfteμL pure hexane and the extracts were pooled in the collection vials. Following the chemical extraction, the twig pieces were dried in an oven at 80°C for 15 h. Their dry mass was measured after keeping the oven dried pieces in desiccator to obtain constant mass. The dry mass of Miswak brushes was used to calculate the absolute amount of volatiles compounds present in the Miswak brushes. Hexane extract of twig pieces was injected (1 μL) in GC-MS via liquid injection using a syringe.The extraction of chemicals from the fresh and used Miswak brushes and the quantification was made by using previously reported method by Zhao et al., 2011 . Pieces μm film thickness, J & W Agilent, Santa Clara, CA, USA). The temperature of the GC oven was maintained at 45°C for 30 sec which was gradually raised up to 235°C at a rate of 8°C min−1 and maintained at 235°C for 5.75 min. The injector temperature was isothermally set at 230°C and the transfer line connecting the GC to the MS was isothermally set at 235°C. The MS ion source temperature was 150°C; mass spectra were obtained at 70 eV with a mass range from 30 m/z to 400 m/z in positive centroid mode. Separated compounds were initially identified by comparing their mass spectra to the NIST-08  MS library. The final authentication was made by analysing pure standard compounds on GC-MS using the same parameters used for the chew twigs and saliva samples. Quantification of identified compounds was made by running different concentrations of standard compounds on GC-MS followed by producing standard curve.Volatile compounds collected on a SPME fibre and extracted in hexane were separated and identified in GC-MS, using a Varian 3400 gas chromatograph (GC)  connected to a Finnigan SSQ 7000  quadruple mass spectrometer with electron ionisation. The GC was equipped with a split/split less injector ; the carrier gas was helium , with a constant pressure of 10 psi. The GC was fitted with a DB-Wax capillary column . Gingival fibroblasts were established and cultured in the manner previously described . Gingivaμg/mL gentamicin, 0.25 μg/mL amphotericin B, and defined growth serum (EDGS)  in a humidified atmosphere of 5% CO2 at 37°C.Oral keratinocytes (OKF6/TERT-2)  were cul S. persica roots essential oil (EO) or pure compounds BITC, benzaldehyde (BA), and benzyl cyanide (BC) were prepared in absolute ethanol. The solutions were sterilized by filtering them through a 0.22 μm filter before the working concentrations of EO or pure compounds were made in Dulbecco's modified Eagle's medium (DMEM) by diluting the respective stock solutions.The stock solutions (1 mg/mL) of4) were seeded in 96-well plates in tetraplicate manner in DMEM supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL), and fetal calf serum (FCS) (5%) and cultured at 37°C for 24 h. The cell layers were rinsed twice with serum-free DMEM followed by the addition of 10 μL serum-free medium containing different concentrations (0.4 μg/mL–3 μg/mL) of EO, BITC, BC, and BA. Control cells were treated with corresponding amounts of ethanol, added in serum-free DMEM to test the effect of ethanol on cells. After an incubation period of 24 h, the colorimetric assay, 3--2,5-diphenyltetrazolium bromide (MTT) kit , was used to assess the viability of gingival fibroblast according to the manufacturer's instructions. Fibroblasts were incubated with MTT reagent for 4 h at 37°C and solubilizing solution was added. The resulting mixture was shaken for 1 h at room temperature and absorbance was measured at λmax 507 nm on an ELISA reader.The fibroblasts (1 × 10μL of medium with increasing concentrations of EO or BITC (5–100 μg/mL) and incubated for 24 hrs. Cell viability was detected by adding MTT to the medium, incubating it for 4 hrs, and then adding DMSO to dissolve the formazan product. After the plate was shaken at low speed for 10 minutes absorbance was measured at 540 nm using an ELISA reader.Oral keratinocytes were seeded in 96-well plates until reaching 80% confluence; the medium was then removed and replaced with 100 μg/mL to 100 μg/mL, whereas for BC and BA it ranged from 0.5 μg/mL to 20 μg/mL. After an exposure time of 10 min, the cells were washed twice with a serum-free medium. The cells were then incubated for another 24 hrs after adding 100 μL of complete medium. The cell-recovery assessment was carried out using the MTT kit described above. Each cell viability assay was performed using four replicates of all tested dilutions and the experiments were repeated three times.Human gingival fibroblast cells were seeded in a 96-well microtiter plate as previously mentioned, incubated for 24 h, washed with a serum-free medium, and then treated with EO, BITC, BC, or BA. The concentration of EO and BITC ranged from 1 S. persica EO and its pure constituents were tested for antibacterial activity against 3 oral pathogenic bacteria: Aggregatibacter actinomycetemcomitans KH 1519, Porphyromonas gingivalis, ATCC 33277, and, Haemophilus influenzae, ATCC 49247 as a well characterized reference. H. influenzae was propagated for 16 h in chocolate agar plates in a 5% CO2 incubator. A. actinomycetemcomitans was propagated in blood agar plates for 40 h in 5% CO2 atmosphere. P. gingivalis was propagated for 136 h on Brucella agar plates supplemented with Hemin (0.05 mg/mL), vitamin K (0.01 mg/mL), and citrated horse blood (5%) in an anaerobic atmosphere created with gas-packs . All agar plates were prepared and retrieved from the substrate unit, at the laboratory of clinical microbiology, Karolinska Hospital, Huddinge, Sweden. H. influenza, A. actinomycetemcomitans, and P. gingivalis were suspended in 2 mL of 0.9% NaCl solution and optical density (absorbance) of resultant suspension was measured at the wavelength of 590 nm by using a double beam spectrophotometer. The bacterial suspensions were then diluted to get optical density of 0.5; the number of colony forming units (CFU) was in the range of 107–108 per mL of suspension depending upon the bacteria. Prior to the antibacterial assay, the bacterial suspensions were further diluted 10,000 times in fresh saline media. The 495 μL of the diluted bacterial suspension (103–104 CFU/mL) was poured in each Eppendorf tube to which 5 μL of saline solution or dimethyl sulfoxide (DMSO) or test oil/compound solution was added. The test solutions of EO, BITC, BA, and BC were prepared by dissolving them in DMSO. The final concentration of the DMSO (solvent) was never more than 1% of the bacterial suspension in sample or control tubes. The final concentration of the test compounds or EO was in the range of 11.7 μg/mL to 11700 μg/mL of bacterial suspension and that of chlorhexidine was 500 μg/mL. DMSO worked as a negative control whereas chlorhexidine was used as positive control for all tested bacteria. The contents of Eppendorf tubes were vortexed and incubated in a shaker for 10 min after that 100 μL of suspension mixture from each Eppendorf tube was evenly spread on an agar plate with a respective growth medium. At least two replicates were run for each test or control sample and two replicates were plated by taking sample from each Eppendorf tube. The plates of H. influenza were incubated for 16 h, A. actinomycetemcomitans for 40 h, and P. gingivalis for 136 h. The bacterial colonies on each plate were counted by using a colony counter. The antibacterial bioassay of each test sample was repeated in triplicate manner.The antibacterial activity was performed by broth dilution assay according to previously described method , 33, 34  t-test was used to examine the significance differences between control and test samples. One way ANOVA with Bonferroni post hoc test was used to find difference between bacterial growth when they were treated with same concentration of Miswak essential oil or pure compounds. The statistical analyses were carried out using SPSS 20 computer software .All data are presented as mean values and standard deviation of three separate experiments with two to three replicates. Two-tailed paired S. persica roots twigs produced 1.4 g of essential oil giving rise 0.001% yield. GC-MS analysis of the oil revealed the presence of three major antibacterial compounds constituting 92% of the oil , (CID: 2346), 74.4% followed by benzyl cyanide (BC) (CID: 8794), 16.3%, and benzaldehyde (BA) (CID: 240), 0.7% (The hydrodistillation of the oil . The mai0), 0.7% .p < 0.05) from the amount of BITC found in Miswak brushes after one-, two-, and four-time use (p < 0.05) than Miswak-2 and Miswak-4 brushes; however, Miswak-2 and Miswak-4 exhibited similar amount (p > 0.05). The amount of benzyl cyanide decreased significantly after 2 and 4 uses (p < 0.05) while benzaldehyde contents remained the same (The amount of BITC found in unused Miswak pieces (base line) was statistically different  when fresh Miswak was used to clean the teeth  found in the saliva, and it disappeared completely after 10 min  (μg/mL chlorhexidine showed similar viability as control cells (p > 0.05). The most cytotoxic compound was benzaldehyde that decreased the cell viability even at lower concentration 0.5 μg/mL (μg/mL of benzyl cyanide except for 10–20 μg/mL (p < 0.05) (A 10 min exposure to 1–5  < 0.05) . The cel.5 μg/mL . However < 0.05) .μg/mL or higher (p < 0.05) and to BITC at concentrations ≥1 μg/mL (p < 0.05) (μg/mL) showed no significant effect on cell viability whereas benzaldehyde showed significant cell toxicity at concentration of 0.8 μg/mL or higher (A significant decrease of cell viability was noted after a 24-hour exposure to Miswak oil at concentration 1.4  < 0.05) . Twenty-r higher .μg/mL; however, at higher concentration they show better cell viability (p < 0.05).The Miswak oil and BITC did not show any bad effect on the viability of keratinocytes when they were stimulated with Miswak oil or BITC for 24 h at the concentrations 5–100 iability  than con H. influenzae and P. gingivalis were more susceptible towards Miswak essential oil; thus their growth was significantly inhibited (p < 0.01) at lowest concentration (11.7 μg/mL) tested in experiments (p < 0.05) from each other  showed 100% mortality of the cells , 41. Thintration . S. persica Miswak extract was toxic to mice at doses of up to 1200 mg/kg.The oral keratinocytes proved to be a much more resistant cell type. There was no decrease in cell viability when the keratinocytes were exposed to the same concentrations as the gingival fibroblasts. These results are in agreement with a previous study where oral keratinocytes were found more resistant towards long-chain bases compared with oral fibroblasts . MoreoveThis study shows that BITC is released from the Miswak into the oral cavity but that retention time is very short. The amount of BITC decreases gradually if the same piece of Miswak is used several times. These observations strongly suggest that the Miswak should be cut before each use to ensure that fresh, unexposed Miswak is used to get the maximum effect.


Scientific Data 4:170032 doi: 10.1038/sdata.2017.32 (2017); Published 14 March 2017; Updated 9 May 2017The original HTML version of this Data Descriptor incorrectly included Oliver Speck’s name in the Data Citations as ‘Speck, C.’ instead of ‘Speck, O.’ This has now been corrected in the HTML version. The PDF version was correct at the time of publication, and has not been altered.