Fmr1 knockout (KO)] mice in the ventral cochlear nucleus (VCN), medial nucleus of the trapezoid body (MNTB), and lateral superior olive (LSO). We studied features of the brainstem known to be altered in FXS or Fmr1 KO mice, including cell size and expression of markers for excitatory (VGLUT) and inhibitory (VGAT) synapses. We found that cell size was reduced in the nuclei with different time courses. VCN cell size is normal until after hearing onset, while MNTB and LSO show decreases earlier. VGAT expression was elevated relative to VGLUT in the Fmr1 KO mouse MNTB by P6, before hearing onset. Because glial cells influence development and are altered in FXS, we investigated their emergence in the developing Fmr1 KO brainstem. The number of microglia developed normally in all three nuclei in Fmr1 KO mice, but we found elevated numbers of astrocytes in Fmr1 KO in VCN and LSO at P14. The results indicate that some phenotypes are evident before spontaneous or auditory activity, while others emerge later, and suggest that Fmr1 acts at multiple sites and time points in auditory system development.Fragile X syndrome (FXS), the most common monogenic cause of autism, is often associated with hypersensitivity to sound. Several studies have shown abnormalities in the auditory brainstem in FXS; however, the emergence of these auditory phenotypes during development has not been described. Here, we investigated the development of phenotypes in FXS model [ Fmr1 KO). We found that some of the reductions in cell size and imbalances in inhibitory/excitatory input present in adult Fmr1 KO mice emerged early in postnatal development. This study suggests that even before hearing onset, loss of the Fmr1 gene drives disruptions in the auditory system.Individuals with fragile X syndrome (FXS) are hypersensitive to sound and show enhanced cortical responses to sound stimuli. Recent work suggests that functional and physiologic abnormalities in the auditory brainstem may contribute to the dysfunction found in the auditory system. We investigated the emergence of the FXS auditory brainstem phenotype in the development of auditory brainstem nuclei in FXS model mice  results from transcriptional silencing of the in FMRP; . Individin FMRP; , repetitin FMRP; , and unuin FMRP; ; accordiin FMRP; , especiain FMRP; . Dysfuncin FMRP; , exaggerin FMRP; , and faiin FMRP; , b.In addition to these cortical phenotypes, recent studies suggest that some aspects of auditory dysfunction in FXS arise in the auditory brainstem, particularly in nuclei that comprise the sound localization circuits. Auditory input from the periphery first contacts the cochlear nucleus, and axons from the ventral cochlear nucleus (VCN) send excitatory input to the ipsilateral lateral superior olive (LSO) and the contralateral medial nucleus of the trapezoid body (MNTB). In addition to the large calyx of Held excitatory input from VCN, MNTB also receives glycinergic and GABAergic inhibitory input from the ventral nucleus of the trapezoid body VNTB; . MNTB prFmr1 KO mice. Fmr1 KO mice display auditory cortical hyperexcitability  thresholds, indicating a modest peripheral hearing loss , eight P6, and fifteen P14 FVB strain wild-type mice, and five P1, fourteen P6, and sixteen P14 Mice were perfused transcardially with 4% paraformaldehyde (PFA) in PBS, and brains were dissected. Brainstems were fixed in PFA solution for 2 h, then equilibrated in a 30% sucrose solution in PBS overnight. Brains were cryosectioned at 16 μm in the coronal plane at the level of the auditory brainstem nuclei. Sections were mounted on chrome-alum glass slides in a 1-in-4 (P1) or 1-in-6 (P6 and P14) series. Mounted sections were then surrounded with a Pap Pen hydrophobic barrier and rinsed in PBS for 10 min. For antigen retrieval, sections were incubated with 0.1% SDS in PBS solution for 5 min. Slides were rinsed three times in PBS, then incubated with either normal goat blocking solution , or bovine serum albumin blocking solution  for 1 h in a humid chamber at room temperature. Bovine serum albumin blocking solution was used for synaptic marker immunolabeling, and normal goat blocking solution was used for immunolabeling of glial cells. Primary antibodies (see below) were applied, and slides were incubated overnight in a humid chamber. Slides were washed in PBS and incubated at room temperature for 1 h with appropriate Alexa Fluor (Invitrogen) secondary antibodies diluted 1:500. Slides were rinsed three times in PBS and coverslipped with Glycergel mounting medium (Dako C0563).To detect excitatory synaptic input, we used a guinea pig polyclonal antibody that recognized vesicular glutamate transporter-2 protein (VGLUT2) diluted to 1:2500 (Millipore AB2251). This protein reliably labels calyces of Held and shows stable expression levels during postnatal development in rats . InhibitTo visualize cell bodies and brainstem nuclei, we performed fluorescent Nissl staining using the BrainStain Imaging Kit (Life Technologies B34650). Slides were rinsed in 0.2% Triton X-100 solution in PBS then incubated for 20 min at room temperature with NeuroTrace 530/615 red fluorescent Nissl stain diluted 1:300 in PBS. Slides were then rinsed three times with PBS and coverslipped in Glycergel mounting medium.z-stacks of VCN, medial MNTB, lateral MNTB, and LSO were generated. Ten cells were randomly selected from each image for measurement; only cells in which the nucleus was visible and with a cross-sectional area >45 µm2 were included in the analysis. For brainstem nucleus size, nuclei from both the right and left hemispheres were imaged, with a minimum of two sections imaged per brain. Nuclei were outlined and measured using AxioVision software. The number of neurons in each section within the outlined nucleus was counted using ImageJ cell counter using above inclusion criteria, and a mean number of cells per section was obtained.To evaluate brainstem nucleus metrics, VCN, MNTB, and LSO of fluorescent Nissl stained sections were imaged at 20× using a Zeiss Axioskop-2 microscope. To study cell size, 40× 2, MNTB = 11799.3 μm2, medial MNTB = 6899.5 μm2, lateral MNTB = 11,799.3 μm2, and LSO = 13,239.5 μm2. The red (VGLUT) and green (VGAT) color channels for each ROI were separated, and the threshold of each channel was adjusted to highlight the immunopositive areas within each ROI. ImageJ was used to sum the area of all of the immunolabeled objects within an ROI. Fractional coverage was defined as the summed area of immunolabeling divided by the area of the ROI. To compare the relative amounts of VGLUT and VGAT, we defined a synaptic protein index (ISP) to describe each ROI, where ISP = (VGLUT – VGAT)/(VGLUT + VGAT). ISP values range between –1 and 1, with negative values indicating relatively more VGAT, and higher positive values indicating relatively more VGLUT.High magnification images (63×) of regions within VCN, medial MNTB, lateral MNTB, and LSO of VGLUT- and VGAT-immunolabeled sections were acquired. Regions of interest (ROIs) within each image were selected. ROI size for each brainstem nucleus was consistent across images: VCN = 10138.8 μmBased on fluorescent Nissl staining, VCN, MNTB, and LSO were outlined using AxioVision software. The outlines of these nuclei were then superimposed on images of Iba1- or ALDH1L1-immunolabeled sections. The ImageJ cell counter function was used to quantify Iba1- or ALDH1L1-positive cells. As both microglia and astrocytes are highly ramified, glia were counted only if the somata were present within a section.Fmr1 KO) and differences between ages  using two-way ANOVAs to analyze cell size, nucleus size, number of cells, synaptic protein expression, and number of glia. We also examined medial versus lateral differences in MNTB cell size. We performed separate two-way ANOVAs to test the effects of genotype (wild-type or Fmr1 KO) and location . Statistics supporting data shown in all figures are summarized in We used Sigma Stat and Sigma Plot software for our statistical analyses and graphing. We tested for differences between genotypes . No interaction was seen between the effects of genotype and age. The mean cell size for wild-type mice was 101.9 ± 3.0 μm2 at P1, 118.9 ± 7.4 μm2 at P6, and 193.8 ± 5.1 μm2 at P14. In Fmr1 KO mice, the mean cell size was 94.4 ± 5.3 μm2 at P1, 116.9 ± 8.3 μm2 at P6, and 171.5 ± 7.0 μm2 at P14 . Cells were significantly smaller in Fmr1 KO MNTB . The mean cross-sectional area of wild-type MNTB cells was 108.2 ± 3.5 μm2 at P1, 113.2 ± 2.6 μm2 at P6, and 204.9 ± 6.3 μm2 at P14. In contrast, the mean cross-sectional area of MNTB cells in Fmr1 KO mice was 63.9 ± 3.9 μm2 at P1, 96.8 ± 3.6 μm2 at P6, and 166.5 ± 5.9 μm2 at P14. No significant interactions were seen between age and genotype.s tested A, B. BotFmr1 KO mice in MNTB at P1  and locations  and locations . Medial and lateral MNTB cell sizes were significantly different in wild-type mice, but not different in Fmr1 KO mice. An interaction between location and genotype was seen at P1 and P6. At P14  and cell location  was significantly different, but no interaction was observed at this age. In the medial portion of MNTB, wild-type cells were 112.6 ± 3.5 μm2 at P1, 106.8 ± 1.5 μm2 at P6, and 131.1 ± 2.7 μm2 at P14. In Fmr1 KO medial MNTB, cells were 69.3 ± 4.4 μm2 at P1, 89.5 ± 3.6 μm2 at P6, and 98.9 ± 2.3 μm2 at P14. In wild-type mice, lateral MNTB cells were 136.5 ± 3.3 μm2 at P1, 134.6 ± 5.1 μm2 at P6, and 214.3 ± 8.4 μm2 at P14. In Fmr1 KO mice, lateral MNTB cells were 68.8 ± 2.6 μm2 at P1, 95.5 ± 4.9 μm2 at P6, and 169.0 ± 8.9 μm2 at P14. Thus, while MNTB cells were smaller in Fmr1 KO mice throughout the nucleus at all ages tested, we found that a gradient of cell size emerged early in wild-type mice and was evident by P14 in both Fmr1 KO and wild-type mice.Because FMRP is expressed along a medial-lateral gradient in MNTB  we testeTB at P1 A, P6  for wild-type and Fmr1 KO mice , but the correlation between VCN and LSO in P14 wild-type mice did not reach significance after correction. The statistics are shown in Because cell size reduction was a prominent phenotype in all three nuclei, we tested whether cell size was correlated between nuclei in individual animals. We obtained Pearson correlations among the three nuclei separately for mutant and wild-type animals and for both ages. After Bonferroni correction, we did not find significant correlations at for either genotype at P6. We found that cell size in VCN was positively correlated with cell size in LSO in P14 F2,53 = 74.602, p < 0.001), but genotype (wild-type or Fmr1 KO) did not . Pairwise analysis showed that VCN grew at a consistent rate in both wild-type and Fmr1 KO mice . Wild-type mice gained cells in VCN from P1 (445.6 ± 23.2 cells) to P6 (587.2 ± 24.4 cells), but not from P6 to P14 (581.1 ± 19.7 cells). VCN in Fmr1 KO mice also showed a significant gain in cell numbers between P1 (484.8 ± 38.9 cells) and P6 (545.4 ± 40.1), and between P6 and P14 (567.2 ± 18.2 cells). Both Fmr1 KO and wild-type mice showed a significant increase in cell number between P1 and P14  had a significant effect on nucleus size , but not with genotype  and P6 , and between P6 and P14 . MNTB size in Fmr1 KO mice also showed gains at each age tested; a significant change in MNTB size was found between P1  and P6 , and a significant increase was seen between P6 and P14 , though the number of cells did not differ by genotype . In both wild-type and Fmr1 KO mice, the number of cells in MNTB increased significantly from P1 to P6, but the increase from P6 to P14 did not reach significance in wild-type mice , but did not vary by genotype , but not with genotype  to P6  and from P1 to P14 . This finding is consistent with data showing no significant difference in the LSO cell density of adult Fmr1 KO mice , but no difference in the amount of VGLUT or VGAT was found between genotypes at any age tested  or ages . At P6, the ISP value was 0.12 ± 0.04 in wild-type mice and 0.04 ± 0.04 in Fmr1 KO mice. At P14, the ISP value was –0.05 ± 0.04 in wild-type mice and –0.03 ± 0.01 in Fmr1 KO mice. Thus, as in adult Fmr1 KO mice, synaptic inputs to VCN were not altered.The expression values of VGLUT and VGAT in VCN are shown in F1,22 = 0.417, p = 0.525; Fmr1 KO mice . At P6, wild-type VGLUT fractional coverage was 0.08 ± 0.01 and VGAT coverage was 0.02 ± 0.001. P6 Fmr1 KO fractional coverage for VGLUT was 0.07 ± 0.003 and for VGAT was 0.03 ± 0.001. At P14, VGLUT coverage in wild-type mice was 0.07 ± 0.01 and in Fmr1 KO mice was 0.08 ± 0.005; VGAT coverage in wild-type mice was 0.02 ± 0.002 and in Fmr1 KO mice was 0.03 ± 0.002. Overall, synaptophysin fractional coverage was significantly greater in Fmr1 KO mice than in wild-type mice . At P6, synaptophysin coverage in wild-type mice was 0.10 ± 0.01 and 0.12 ± 0.02 in Fmr1 KO mice. Wild type coverage at P14 was 0.10 ± 0.005, and Fmr1 KO coverage was 0.12 ± 0.005. Relative levels of VGLUT and VGAT expressed as ISP values , indicative of reduced excitatory input relative to inhibitory input. No changes in ISP values were found with age . ISP values in wild-type mice were 0.59 ± 0.03 at P6 and 0.60 ± 0.06 at P14. In Fmr1 KO mice, ISP values were 0.38 ± 0.03 at P6 and 0.45 ± 0.03 at P14. These results suggest that greater VGAT fractional coverage values drive a decrease in Fmr1 KO ISP values and that this phenotype is present early in postnatal development.We evaluated expression of presynaptic markers in MNTB A–C. VGLUP values G were siFmr1 KO mice, and VGAT coverage increased with age , although there was no difference between genotypes  at both P6 and P14 . In wild-type mice, LSO ISP values were 0.59 ± 0.07 at P6 and 0.21 ± 0.09 at P14. In Fmr1 KO mice, ISP values were 0.53 ± 0.04 at P6 and 0.04 ± 0.03 at P14. ISP values differed between genotypes at P14 , and decreased with age .VGLUT, VGAT, and synaptophysin expression were examined in LSO A–C. VGLU and P14 F. There Fmr1 deletion alters glial populations in the auditory brainstem, we next quantified the number of microglia and astrocytes using immunofluorescence for the markers Iba1 and ALDH1L1, respectively.To determine whether the F2,53 = 24.955, p <0.001) but not with genotype , but did not depend on genotype  and P6 (9.53 ± 1.34), between P6 and P14 (18.94 ± 2.01). Thus, no difference between wild-type mice and Fmr1 KO mice was seen in microglial numbers in these auditory brainstem nuclei. We examined Pearson’s correlation coefficients to determine correlation of the number of microglia with values for VGAT, VGLUT, and synaptophysin in mutants and wild-type animals at P6 and P14 in each nucleus and used Bonferroni correction for these multiple comparisons. A significant positive correlation was found only between Iba1 values and VGLUT values for P6 in MNTB in Fmr1 KO mice  but did not differ between genotypes  and also differed between genotypes at P14 . Wild-type and Fmr1 KO mice gained a significant number of astrocytes between P6 and P14, with significantly more astrocytes present in P14 Fmr1 KO mice than in wild-type mice. Wild-type mice had 3.95 ± 1.12 astrocytes per section at P6 and 17.17 ± 2.58 at P14; Fmr1 KO mice had 6.65 ± 0.91 astrocytes per section at P6 and 31.26 ± 5.96 at P14  caused significant differences in nucleus size, number of cells, cell size, synaptic proteins, or glia number in VCN, MNTB, and LSO. Although any genotype differences found were consistent with previous results, we did not find any significant differences between male and female mice in any measure tested in any nucleus examined  within each genotype (wild-type or examined . We therFmr1 KO mouse brainstem reported both anatomic and functional phenotypes (Fmr1 KO mice had heightened ABR thresholds and reduced peak I, consistent with a modest hearing loss. Central auditory brainstem dysfunction in Fmr1 KO may thus arise secondarily from reduced peripheral input. However, FMRP is expressed throughout the auditory brainstem (Previous studies of the adult enotypes . ABR mearainstem , and thurainstem  to deterFmr1 KO mice were smaller than in wild type at early postnatal ages. In contrast to VCN, these differences were already seen at P1, before hearing onset and before the onset of spontaneous activity (We found broad variability among nuclei in the emergence of differences in cell size. Differences in VCN cell size were not present at any of the ages examined, and thus develop after P14. As in adults, MNTB neurons in activity . FMRP miFmr1 KO mice until after hearing onset. The delay in the formation of a cell size gradient in MNTB suggests a role for FMRP. FMRP is an activity-dependent negative inhibitor of mRNAs that is expressed in a medial-to-lateral gradient within MNTB (Fmr1 results in a loss of this kv3.1b gradient (Our data show that a medial–lateral gradient in cross-sectional cell area was evident to a moderate degree at early ages in wild-type mice and became more pronounced at P14. In contrast, the cell size gradient did not form in hin MNTB  and playhin MNTB . FMRP regradient . It is tgradient . Taken tFmr1 KO mice at all ages examined. Because these differences are not observed in adults (Fmr1 KO mice, synaptic strengthening events are delayed, but are considerably larger than those in wild-type mice (Fmr1 KO LSO receives an exaggerated amount of input (Fmr1 KO mice.In LSO, we found significant reductions in cell size in n adults , our resype mice , and theof input . WorkingThe reduction in cell size that we observed in the early postnatal MNTB and LSO were not accompanied by any differences in the nucleus size or cell number. These observations suggest that the nuclei contain greater intercellular space. The increased number of astrocytes in LSO might fill some of the space. In MNTB, where this issue persists through adulthood, this space might be filled with expanded axon tracts in the ventral acoustic stria, changes in the extracellular matrix, and/or increased neuropil volume.Fmr1 KO mice have shifted sensitivity for interaural level differences (Fmr1 KO mice.Changes in neuronal excitability contribute to neurologic dysfunction in FXS . In the ferences . Enhanceferences ; additioferences . Both ofFmr1 KO mice was seen at P6 and persisted into adulthood. The ISP ratios in MNTB are generally large and positive, reflecting the presence of the large, excitatory input from the calyx of Held. Ratios are reduced in magnitude but remain positive in Fmr1 KO mice relative to wild-type mice. At P14, the ISP ratios for both genotypes were somewhat higher than those previously published in adult mice (Fmr1 KO animals. This finding suggests that the mutation might lead to enhanced branching of the normal population of VNTB GAD67-positive cells to result in more exuberant terminals in MNTB in Fmr1 KO mice. In that study, a decrease in expression of the glycine transporter Glyt2 was seen, but only in the medial, high-frequency region of the MNTB. It is not known whether the shift from GABA to glycine is altered in Fmr1 KO mice. However, our results suggest that altered inhibitory input to MNTB is seen before hearing onset, before inhibition has matured in the auditory brainstem.In MNTB, increased VGAT expression in ult mice , suggestult mice . Howeverult mice  in adultSP in Fmr1 KO mice. There is other evidence, however, that calyx of Held volume may also grow larger in Fmr1 KO mice (Fmr1 KO mice. Interestingly, despite an enlarged presynaptic terminal, cells in the Fmr1 KO MNTB did not show significant differences in firing behavior (Fmr1 KO. While MNTB cells may have larger calyces of Held, work done in Fmr1 KO rats found fewer MNTB neurons received input from VCN globular bushy cells (Our data suggest that increased VGAT expression drives the decrease in I KO mice . Our intbehavior , presenthy cells .Fmr1 KO mice at both P6 and P14. This observation suggests that overall input is increased. The ISP ratio was significantly lower in mutants than in wild-type mice at P14; this difference is not evident in adults (Fmr1 KO mice in adult LSO using GlyT2 as a marker of inhibitory input (Previous studies demonstrated an increase in VGLUT expression in the adult LSO , as welln adults . Interesry input . While Vry input , these fFmr1 KO astrocytes adopted a dendritic morphology similar to that found in Fmr1 KO mice (Microglia have several roles in synaptic maturation, neuronal homeostasis, and immune responses . Astrocy KO mice .Fmr1 KO mice, but that astrocytes are elevated in VCN and LSO at P14. Microglia were positively correlated with VGLUT in the P6 MNTB in Fmr1 KO mice. Astrocytes were strongly positively correlated with both VGLUT and VGAT in P14 in LSO in Fmr1 KO mice, but not in wild-type mice. The findings suggest that glial cells represent an important factor in establishing excitatory and inhibitory synapses and that their role in synaptic development may be enhanced in FXS.Typically, mice steadily gain both microglia and astrocytes in the auditory brainstem between P0 and P14  MicrogliFmr1 inactivation (Fmr1 KO mice used here contain two alleles of the Fmr1 KO, one on each X-chromosome. Male and female Fmr1 KO mice show differences in behavioral measures (FXS is far more common and severe in males, as they lack an additional X-chromosome that may partially compensate for tivation . The femmeasures , vocalizmeasures , and neumeasures , but othmeasures . We founFmr1 KO mice. Our findings show developmental effects at very young ages, implying that early developmental events initiate some of the auditory phenotypes in FXS.In addition to enhanced responses to sensory stimuli, individuals with FXS have a host of symptoms that are confined to childhood (reviewed in . This in


Imaging included 4D PET-CT, 3D PET-CT, endoscopic ultrasound and planning 4D CT. Target volume delineation was performed on 4D CT, 4D CT with co-registered 3D PET and 4D PET-CT. Planning target volumes (PTV) generated with 4D CT . Median DICE similarity coefficients comparing PTV4DCT with PTV3DPET4DCT, PTV4DCT with PTV4DPETCT and PTV3DPET4DCT with PTV4DPETCT were 0.85 (range 0.65–0.9), 0.85 (range 0.69–0.9) and 0.88 (range 0.79–0.9) respectively. The median sensitivity index for overlap comparing PTV4DCT with PTV3DPET4DCT, PTV4DCT with PTV4DPETCT and PTV3DPET4DCT with PTV4DPETCT were 0.78 (range 0.65–0.9), 0.79 (range 0.65–0.9) and 0.89 (range 0.68–0.94) respectively.Mean PTV3DPETCT and PTV4DPETCT are not included in PTV4DCT, leading to under-coverage of target volume and a potential geometric miss. Additionally, differences between PTV3DPET4DCT and PTV4DPETCT suggest a potential benefit for 4D PET-CT.Planning 4D PET-CT is feasible with careful patient selection. PTV generated using 4D CT, 3D PET-CT and 4D PET-CT were of similar volume, however, overlap analysis demonstrated that approximately 20% of PTVNCT02285660 (Registered 21/10/2014).ClinicalTrials.gov Identifier –  Oesophageal cancer is the 8th commonest malignancy worldwide with approximately 456,000 cases diagnosed in 2012 –5. Use o18Fluorine fluorodeoxyglucose (FDG) positron emission tomography – computed tomography (PET-CT) is firmly established in guiding optimal management of radically treatable oesophageal carcinoma [arcinoma , 8. The arcinoma –11. Two arcinoma , 13. Howarcinoma . Hypothearcinoma . There iarcinoma , 17. TheThe purpose of this study was to assess the feasibility and potential impact on target delineation of contrast-enhanced 4D PET-CT acquired in the treatment planning position, for a prospective cohort of patients with lower third oesophageal cancer.ClinicalTrials.gov identifier – NCT02285660, Registered 21/10/2014). Trial participants underwent standard-of-care imaging including 3D FDG PET-CT, endoscopic ultrasound and radiotherapy planning 4D CT. A trial-specific contrast-enhanced 4D FDG PET-CT in the treatment position with limited coverage of the lower thorax and upper abdomen was also performed. Subsequent treatment was not affected by the trial-specific PET-CT and was delivered according to institutional clinical protocols.This was a non-randomised prospective single centre study in patients with distal oesophageal carcinoma suitable for treatment with radiotherapy or concurrent chemo-radiotherapy  performance status 0–2; histologically proven distal oesophageal carcinoma; clinical decision made to proceed with radiotherapy +/− concurrent chemotherapy; measurable primary tumour +/− loco-regional metastatic lymph nodes on standard-of-care imaging; able to provide fully informed written consent; able to lie flat for 1 h; not pregnant or breast feeding. Female patients of childbearing potential agreed to use effective contraception, were surgically sterile, or were post-menopausal.Exclusion criteria included: poorly controlled diabetes; renal impairment with estimated glomerular filtration rate < 30 mL/min; allergy to iodinated contrast media.The study was approved by the regional Research Ethics Committee . All patients provided informed written consent prior to trial entry. A total of 15 patients were recruited between October 2011 and July 2014. All patients provided informed written consent prior to study entry.18Fluorine-FDG. Patients were positioned on the hard-top couch with laser alignment to skin tattoos marked during standard-of-care radiotherapy planning CT 45 min’ post injection. A non-contrast CT of the treatment area (lower thorax/upper abdomen) was obtained using standard settings: 120 kV, variable mA , tube rotation time 0.4 s, pitch 1.984 with a 2.5 mm slice reconstruction. Static PET acquisition from mid thorax to upper abdomen was then performed scanning in a cranial direction with 23 slices (50% overlap) acquired over 1 min. 60-min after tracer injection a 4D respiratory-gated PET acquisition commenced scanning in a cranial direction over the same volume (20-min acquisition). Breaths per minute (BPM) were monitored during this acquisition and average breathing period in seconds (60/average BPM) was calculated. The cine acquisition parameters for 4D CT were based on average breathing period determined from the 4D PET acquisition. The 4D CT component was obtained 35 s following a bolus of 100 ml of iodinated contrast  injected at 2 ml/s using the following settings; 120 kV, 150 mA, tube rotation 0.4 s per rotation, pitch 1.984, 40 mm detector coverage (centred over the tumour) with a 2.5 mm helical thickness (16 images per rotation). Cine duration varied for individual patients . PET images were reconstructed using a standard ordered subset expectation maximization (OSEM) algorithm with CT for attenuation correction. Both non-attenuation corrected and attenuation corrected datasets were reconstructed.4D PET-CT scans were performed using a 64-slice GE Discovery 690 PET-CT scanner  using Real-time Position Management (RPM) respiratory-gating hardware , a flat couch top and laser alignment. Patients were scanned supine in the treatment position; immobilised with a wing board and both arms raised above their heads. Serum blood glucose was checked routinely and imaging was not performed in patients with a blood glucose level of >10 mmol/L. Patients fasted for at least 6 h prior to intravenous injection of 400 MBq of The 4D CT and 4D PET data was divided into 10 phase bins. Post-processing was used to generate an averaged 3D PET from the 4D PET scan. No co-registration was necessary as the PET and CT components were inherently registered.Contouring was performed using specialized software . PET and CT images were displayed using preset window levels and/or colour scale per a standardized institutional protocol Fig. . Patient. The Inc.Idx is the probability that a voxel of a contour is really a voxel of a reference contour. CGD is the distance between the geometric centres of two contours [Five positional metrics were used to compare target volumes, calculated using ImSimQA software : Dice index (DICE); sensitivity index (Se.Idx), inclusiveness index (Inc.Idx), centre of gravity distance (CGD) and mean distance to conformity (MDC). The Dice index produces output values ranging from 0 and 1 where 0 represents two contours with no overlap and 1 represents two contours that are perfectly overlapping . The Se.contours . MDC is contours .p-value <0.05 was taken as evidence of statistical significance.Descriptive statistics  and Mann-Whitney test were used to assess for statistically significant differences between tumour volumes and lengths. Non-parametric analysis of variance (ANOVA) was used to assess the statistical significance of positional metrics between different imaging techniques (Friedman test). Stastistical analysis was performed using IBM SPSS Statistics . A Fifteen patients were recruited to the study but only 9 underwent 4D PET-CT imaging for various reasons. Of the 6 patients who did not undergo 4D PET-CT, 3 withdrew before scanning; 2 had erratic breathing which made respiratory gating impossible and 1 patient had very low-grade tumour uptake having completed induction chemotherapy 6 weeks earlier. It was decided to discontinue data acquisition following the initial static PET for this patient. A further 2 patients who underwent 4D PET-CT had erratic breathing leading to technical difficulties with acquisition of the 4D CT component. Consequently, only 7 patients had complete datasets suitable for comparative analysis. 6 of these patients were treated with curative intent and 1 patient underwent palliative treatment. Patient characteristics are detailed in Table 3): 4D PET-CT  in comparison to PTV delineated on 4D CT . Despite this there was no statistically significant difference between PTV volumes across the 3 methods (Mann-Whitney test). Tumour length comparison between the different modalities was also not statistically significantly different.Table 4DCT with PTV3DPET4DCT, PTV4DCT with PTV4DPETCT and PTV3DPET4DCT with PTV4DPETCT were 0.85 (range 0.65–0.9), 0.85 (0.69–0.9) and 0.88 (0.79–0.9) respectively. The median sensitivity index for overlap comparing PTV3DPET4DCT and PTV4DPETCT with PTV4DCT was 0.78 (0.7–0.91) and 0.79 (0.65–0.92) respectively. The median sensitivity index for overlap comparing PTV3DPET4DCT with PTV4DPETCT was 0.89 (0.68–0.98). The median centre of gravity distance was 5.19 mm (1.6–27.3 mm) for the PTV4DCT to PTV4DPETCT structures, 3.52 mm (2.9–7.8 mm) for PTV3DPET4DCT to PTV4DPETCT structures and 4.97 mm (1.6–28.2 mm) for the PTV4DCT to PTV3DPET4DCT structures. The median mean distance to conformity was 9.15 mm (5–22.9 mm) for the PTV3DPET4DCT to PTV4DCT structures, 10.68 mm (5.2–22.6 mm) for the PTV4DPETCT to PTV4DCT and 7.41 mm (5.2–10.8 mm) for the PTV3DPET4DCT to PTV4DPETCT.Median DICE similarity coefficients comparing PTVTable Respiratory motion affects imaging quality which is of direct relevance in RTP of potentially mobile tumours . 4D CT gThis study has confirmed that incorporating 4D PET-CT into the RTP pathway of patients with lower oesophageal carcinoma is feasible. However, careful patient selection is important as it may not be possible in patients with an irregular breathing pattern. This prevented or significantly degraded 4D imaging in 4 patients in our study. Low-grade tumour uptake was also a limitation. The only other prospective study of 4D PET-CT in this setting reported 6/18 patients (33.3%) had imaging unsuitable for contouring for similar reasons . Their s3DPET4DCT to PTV4DPETCT structures (3.52 mm) and greatest for PTV4DCT to PT4DPET4DCT structures (5.19 mm). The median mean distance to conformity was lowest (7.41 mm) for the PTV3DPET4DCT to PTV4DPETCT and highest for the PTV4DPETCT to PTV4DCT (10.68 mm). The observed differences between PTVs are potentially clinically important; for example, the median sensitivity index of 0.79 between 4D CT and 4D PET-CT implies 21% of the PTV4DPETCT is not included within the PTV4DCT. The use of 4D PET-CT might minimise the risk of geometric miss but this has not been confirmed and requires further evaluation.In this study 4D CT and PET data acquisitions were acquired at the same attendance with the patient in the radiotherapy treatment position with immobilization to ensure inherent co-registration. Post-processing was used to generate an averaged 3D PET from the 4D PET scan. This methodology should largely obviate any potential error introduced by registration of separately acquired CT and PET data. To the best of our knowledge, this is the first study to report positional metric comparison of tumour volumes delineated on 4D CT and 4D PET-CT in lower oesophageal carcinoma. Concordance between the contoured volumes on different studies were high with no statistical significance reached between PTV measured on each combination for the 5 positional metrics analysed, although the small sample size limits the value of statistical analysis; overlap indices were greatest between 3D PET-CT and 4D PET-CT  and slightly lower when comparing 4D CT with 3D PET-CT  and 4D CT with 4D PET-CT . Median centre of gravity distance was smallest for PTV4DPETCT was not included within PTV3DPET4DCT) suggests that there might be a potential additional role for the use of 4D PET-CT but the cohort is too small to draw conclusions on this. A small retrospective study of 4 patients with lower oesophageal cancer reported that target volumes delineated on inhale and exhale phase 4D PET-CT deformably co-registered with a planning CT were up to 30% smaller in 3 of 4 patients on the exhale plan with potential for dose sparing to organs at risk [A key unanswered question is whether 4D PET-CT provides incremental benefit compared with 4D CT and 3D PET-CT. Although positional metrics comparing PTVs generated using these methods suggested differences were small, the sensitivity index of 0.89  compared to 4D CT (average PTV 528.4 cm3), although the difference between volumes was not statistically significant. 3D CT represents a random snapshot during the breathing cycle whereas 4D CT is a selected series of snapshots at points on the breathing cycle. PET is different; 3D PET is a time-averaged image (showing motion blur covering the full breathing cycle) and 4D PET is a time-averaged image covering a phase bin where the amount of motion blur depends on degree of motion within that bin. Differences between volumes in part reflects this but also that PET and CT are demonstrating different tumour characteristics which are unlikely to inherently have the same volume but should provide complimentary information. 4D PET-CT volumes may have been smallest due to the benefit of PET and CT information but with a reduction in the motion blurring of 3D PET.PTVs delineated using 4D CT are often smaller than with 3D CT and doses to organs at risk in oesophageal cancer  are reduced using 4D CT which may facilitate dose escalation without significant collateral damage . In thisThe study has limitations particularly the small cohort size and absence of histological validation of tumour volumes which is frequently the case in feasibility studies. Despite these limitations this small study has proven the feasibility of the technique and confirmed that tumour volume delineation using 4D PET-CT results in PTV differences compared with either 4D CT alone or 4D CT combined with 3D PET. Our data add to a recent literature review of the potential utility of 4D PET-CT in GTV delineation of intra-thoracic tumours which concluded that this may be the best approach currently available but warrant further investigation in future prospective studies . Further3DPET4DCT and PTV4DPETCT are not included in the PTV4DCT, theoretically leading to under-coverage of the target volume and a potential geometric miss. The current study is too small to draw any conclusions about clinical benefit and further investigation is warranted.Acquisition of a planning 4D PET-CT is feasible with careful patient selection. PTVs generated using 4D CT, 4D CT co-registered with 3D PET-CT, and 4D PET-CT were of similar volume; however, percentage overlap analysis demonstrates that approximately 20% of the PTV


This study investigated the impact of temperature and pH on the degradation of the C3G-rich haskap fraction. The effect of the thermal degradation products on the viability of hepatocellular carcinoma HepG2 and breast cancer MDA-MB-231 cells was also studied in vitro. Using column chromatography, the C3G-rich fraction was isolated from acetone extracts of haskap berries. The C3G stability in these fractions was studied under elevated temperatures (70 °C and 90 °C) at three different pH values  by monitoring the concentration of C3G and its major degradation products, protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), using liquid chromatography mass spectrometry. Significant degradation of C3G was observed at elevated temperatures and at neutral pH. Conversely, the PCA and PGA concentration increased at higher pH and temperature. Similar to C3G, neutral pH also has a prominent effect on the degradation of PGA, which is further accelerated by heating. The C3G-rich fraction exhibited dose-dependent inhibitory effects on cell metabolic activity when the HepG2 cells were exposed for 48 h. Interestingly, PGA but not PCA exhibited cytotoxic effects against both MDA-MB-231 and HepG2 cells. The results suggest that thermal food processing of haskap could influence its biological properties due to the degradation of C3G.Cyanidin-3 Lonicera caerulea L.) berries, also known as blue honeysuckle and honeyberry, are a relatively new crop in North America. Berries of this genus have been used for medicinal purposes in Russia and some Asian countries for thousands of years -3--2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) was purchased from Promega .2O):formic acid. For every 1 g of frozen berries, 4 mL of solvent was used. The blended berries and solvent were filtered through six layers of cheese cloth, then vacuum filtered through Whatman # 8 filter paper . Samples were subjected to rotary evaporation to remove the solvent, then frozen at −80 °C and freeze-dried. Crude extracts were then stored at −80 °C.The crude extract (CE) was prepared using frozen haskap berries (500 g) extracted in semi-dark conditions. Berries were ground in a blender  in a solution of 70:28:2 acetone:deionized water  and conditioned with methanol followed by DI H2O acidified to pH 2.5 with hydrochloric acid (HCl). The crude extract was applied to the column and washed with 1 L DI H2O acidified to pH 2.5 with HCl. C3G-rich fraction was eluted with about 500 mL of methanol. Methanol was removed using rotary evaporation and then freeze-dried to prepare dried PE, which was stored at −80 °C.C3G-rich fraction (PE) was prepared in the same manner as crude extracts with additional steps, as described in a previous method . FollowiPE was prepared at a concentration of 40 mg/mL in a citrate–phosphate buffer . Portions of this solution were acidified to pH 2.5, 4, and 7 with citric acid and stored in 2-mL aliquots in 2-mL sealed amber glass vials. Samples were incubated in triplicate, in a water bath at either 70 °C or 90 °C, for periods of 0, 0.5, 1, 2, 4, or 8 h. After incubation, samples were immersed in an ice bath to limit further degradation. Samples were then stored at −80 °C until analysis. Pure PGA solutions  were incubated in a 90 °C water bath for 0, 0.5, 1, 2, 4, and 8 h periods in 2-mL sealed amber glass vials at pH of 2.5, 4, and 7. Each of the heated solutions was diluted with 2 mL MeOH and filtered before HPLC analysis.2) of 375 °C, and a flow rate of 0.35 mL/min. C3G was analyzed using electrospray ionization in positive ion mode (ESI+), with a capillary voltage of 3500 V, a nebulizer gas temperature (N2) of 375 °C, and a flow rate of 0.35 mL/min. Samples were diluted 1:1 with methanol, then filtered through 0.22 μm nylon filters into amber glass UPLC vials.Total monomeric anthocyanin concentration (TAC) was determined using a spectrophotometric pH differential method . The TACp ≤ 0.05) was performed using the statistical analysis system software . The data for C3G incubated at 90 °C was normalized using a log10 transformation.Repeated measures analysis (2O). Degraded extracts were prepared by subjecting aliquots of this PE solution to thermal degradation at 90 °C in a shaking water bath for 2 h (HPE2) or 8 h (HPE8). The crude extract (CE) was prepared by dissolving freeze-dried CE to a concentration of 60 mg/mL in DI H2O.PE was prepared for cell-based assays by dissolving freeze-dried PE to a concentration of 60 mg/mL in deionized water -1-piperazineethanesulfonic acid (HEPES), and 1% penicillin–streptomycin. Hepatocellular carcinoma  cells were also obtained from the ATCC and cultured in EMEM with 2 mM l-glutamine, 10% FBS, and 1% penicillin-streptomycin at 37 °C with 5% CO2.Breast adenocarcinoma  # HTB 26) cells were obtained from the American Type Culture Collection  and cultured at 37 °C with 5% CO® AQueous One Solution Cell Proliferation Assay  was used to determine cell viability. Cells were seeded in 96-well plates at a density of 5 × 103 cells per well and incubated for 24 h to promote cell adhesion. Cells were treated with PE, HPE2, HPE8, or CE. For HepG2 cells, treatments were added at concentrations of 75, 150, and 300 µg/mL with a positive control of sorafenib in DMSO with a final concentration of 10 µg/mL and negative controls consisting of either EMEM with: 0.5% DI H2O for PE, HPE2, HPE8, or CE treatments, or 0.5% DMSO for PCA or PGA treatments. For MDA-MB-231 cells, treatments were added at concentrations of 50, 100, and 200 µg/mL with a positive control of doxorubicin in DMSO at a final concentration of 50 µM, and negative controls of DMEM with either 0.5% DI H2O  or 0.5% DMSO . After either a 24 or 48 h incubation, 20 µL of MTS was added to each well. Plates were incubated for three hours at 37 °C in a 5% CO2 atmosphere before absorbance was measured at 490 nm using a FLUOstar Optima microplate reader . Cell viability calculated with treated cells being expressed as a percent of cells treated with vehicle only.A CellTiter 96v/v Triton X-100, and 4 mg/mL phosphatase substrate , adjusted to a pH of 5.5 with HCl, and stored at 4 °C. HepG2 cells were treated with PE, PCA, and PGA at concentrations of 75, 150, and 300 µg/mL with a positive control of sorafenib in DMSO with a final concentration of 10 µg/mL, and negative controls consisting of EMEM with either: 0.5% DI H2O for PE treatments, or 0.5% DMSO for PCA or PGA treatments. After incubation for a period of 48 h, plates were centrifuged at 2000 rpm for 10 min before the supernatant was removed and 100 µL of PBS added to each well. Plates were again centrifuged before PBS was removed and 100 µL of assay buffer was added to each well. Plates were then incubated for two hours at 37 °C in a 5% CO2 atmosphere. After incubation, 10 µL of 1 N sodium hydroxide was added to each well, and absorbance was measured at 405 nm using a FLUOstar Optima microplate reader . Cell viability was calculated as described in Acid phosphatase buffer was prepared with 0.1 M sodium acetate (NaAc), 0.1% ® Luminescent Cell Viability Assay  was used to determine cell viability and ATP levels. HepG2 cells were treated under the same conditions as those in the acid phosphatase assay. The CellTiter-Glo® reagent was prepared as directed by the manufacturer from CellTiter-Glo® substrate and the CellTiter-Glo® buffer, and 100 µL of reagent was added to each well. Plates were shaken for 2 min at 60 rpm using a VWR orbital shaker  to induce cell lysis followed by a 10-min room-temperature incubation. Luminescence was measured using a FLUOstar Optima microplate reader . Cell viability was calculated as described in The CellTiter-Glon = 3), and all results were expressed as the mean ± standard deviation (SD). Analyses were compared using one-way analysis of variance (ANOVA) and Tukey’s test (p ≤ 0.05). Statistical analyses were carried out using the Minitab v.17 software  package.All experiments were designed using completely randomized design. Assays were performed in triplicate (p ≤ 0.05) by both incubation time and pH.Monomeric anthocyanins’ degradation was more pronounced in extracts incubated at 90 °C than at 70 °C A,E. AlsoAs the pH increased, C3G degraded more readily, with extracts incubated at 90 °C degrading more quickly than those incubated at 70 °C. Extracts incubated for 8 h at pH 2.5, lost 21% of the initial C3G content at 70 °C, and lost 95% of the initial C3G content at 90 °C. Extracts at pH 4.0 followed a similar trend losing 53% of the C3G content at 70 °C versus a 98% loss at 90 °C. At pH 7.0, C3G was almost completely lost at both 70 °C and 90 °C. C3G is the least stable at pH 7 when compared to pH 2.5 and pH 4.0 B,F.p ≤ 0.05) affected the accumulation of PCA as C3G degraded. The highest concentration of accumulated PCA, 50.0 mg/L, was observed over an 8-h incubation at pH 4 and 90 °C. As with C3G degradation, at 70 °C the largest differences in PCA concentrations were found when comparing pH 7.0 to an acidic pH of 2.5 or 4.0. At 90 °C variations were less dramatic, suggesting that the presence of PCA in C3G-rich extracts is an indicator of C3G degradation ; however, PE added at a concentration of 300 μg/mL induced an ATP reduction to 23.5%. PGA induced a strong reduction in detectable ATP, ranging from 35.6% of luminescence at a concentration of 75 μg/mL, to 8.8% at a concentration of 300 μg/mL . PCA induced a slight dose-dependent reduction in luminescence with 150 μg/mL and 300 μg/mL being significantly different than the negative control, but not significantly different from each other.It was determined via an ATP assay that PE-induced a dose-dependent reduction in ATP activity after an incubation of 48 h B. PE addNumerous studies and reviews have investigated and confirmed that anthocyanins undergo first-order kinetic degradation ,32,33,34Along with affecting the rate of C3G degradation, solution pH may also affect the mechanism of degradation. For C3G at a pH of 3.5, it appears that the opening of the pyrylium ring initiates anthocyanin degradation, forming a glycosylated chalcone . For C3GAs with C3G degradation, the widest variation in PCA concentration was observed between measurements taken at pH 2.5 and 7.0 at 70 °C, with there being slightly less variation at 90 °C. This could indicate an inverse relationship, where PCA concentration could potentially represent the level of C3G degradation . The valFor incubation times longer than 2 h, at both 70 °C and 90 °C, PGA accumulation slowed or stabilized. For all samples at pH 7.0, there was a decrease in PGA concentration over time. The decelerating of PGA accumulation could be attributed to PGA degradation. Incubation of pure PGA at 90 °C at pH 7.0 showed its further degradation . This inHaskap extract did not significantly reduce MDA-MB-231 cell viability. This is likely due to differing anthocyanin profiles between haskap berries and blueberries. Interestingly, PGA significantly reduced the MDA-MB-231 viability, which has not previously been reported. PGA has been shown to be anti-proliferative when incubated with Caco-2 cells in vitro ,25. FurtSolution pH, incubation time, and temperature affect anthocyanin stability. As these increase, the anthocyanin concentration decreases. C3G is more stable when treated at lower temperatures over longer incubations when compared to shorter incubations at higher temperatures. Incubation at pH 7.0 promotes rapid C3G degradation; therefore, maintaining an acidic pH during processing is favorable for C3G retention. Haskap juice is naturally acidic in nature, and this acidity should be preserved to maintain anthocyanin stability. Haskap berry extract with high levels of C3G produced a time- and dose-dependent inhibition of HepG2 cell proliferation and PGA generated dose-dependent inhibition of MDA-MB-231 cells. This preliminary in vitro study suggests that haskap berry anthocyanins could potentially have cancer chemopreventive properties. It also indicates that the inhibitory effects of haskap berry extracts are mediated, at least in part, by C3G. Further studies are required to determine the anti-cancer potential and mechanism of action of C3G. One of C3G’s thermal degradation product and in vivo primary metabolites, PGA, also generated significant anti-proliferative effects on HepG2 cells. Processing of haskap berries and their storage could exacerbate anthocyanin degradation, which could lead to a reduction in putative health benefits for the consumer. Since there is growing interest in the use of fruit-derived C3G as a food additive, natural colorant, dietary antioxidant, and nutraceutical, further research is required to determine the toxicity and biological activity of PGA.


Scanning electronic microscopy demonstrated that all samples have similar microstructure morphology but differ in the range of grain sizes. X-ray diffraction measurements indicated that these materials crystallize in a (Fm-3m) cubic crystal structure, and also confirmed the tolerance factor. Rietveld analysis revealed that the lattice parameter decreased from 8.11834 to 8.039361 Å when the substitution of Ba2+ with Sr2+ cations increased from zero to 100%. Fourier transform infrared (FTIR) and Raman spectroscopies displayed a symmetric stretching vibration of WO6 octahedra at 825 cm−1, and an anti-symmetric stretching mode of WO6 was observed by FTIR at 620 cm−1. A strong peak at 420 cm−1 was also observed in the Raman spectra and is due to the W–O–W bending vibration modes. UV-Vis diffuse reflectance spectroscopy was carried out for the series, and the band gap energy decreased from 3.27 eV for Ba2ZnWO6 to 3.02 and 3.06 eV for Ba1.75Sr0.25ZnWO6 and Ba1.5Sr0.5ZnWO6, respectively. The excitation and emission photoluminescence properties were investigated at room temperature.The effect of Sr These m6) , ferroelo0.4WO6) , Multifeu0.05O6) , magneto2FeMoO6) , dielectBiAgCl6) . 6 and SrCaNiTeO6 compounds and used X-ray diffraction (XRD) and scanning electron microscopy (SEM) to describe their crystal structure and phase transition. Chufeng Lau et al. [6 compound and studied the possibility for its solar cell applications. Paiva et al. [3WO6, which is used in Bluetooth and mobile system devices for microwave telecommunications through wireless antennae. By utilizing the conventional solid-state ceramic route, BiCu2VO6 and BiCa2VO6 powders, which are used in low-temperature co-fired ceramic applications, were prepared and examined using XRD, SEM, and the TE01δ shielded cavity method with a network analyzer (8720ES) and temperature chamber (Delta 9023) to characterize their structure and microwave dielectric behaviors. Orlandi et al. [2Mn0.6Co0.4WO6, and used XRD with the SQUID MPMS Quantum Design magnetometer in order to investigate its crystal and magnetic structure. In addition, the perovskite compound can be used in biomedical applications. LaNiMnO6 nanoparticles were synthesized using the co-precipitation method and were characterized by XRD using a vibration magnetometer , Transmission electron microscopy (TEM), and UV-VIS spectroscopy to investigate the structure, magnetic, and adsorption of bovine serum albumin applications. The nanoparticles displayed good adsorption performance in the bovine serum albumin proteins. The Double-perovskite La2NiMnO6 (LNMO) nanoparticles are potential carriers for large biomolecules, which have wide use in biomedical applications.A variety of devices and methods have previously been used to find and characterize new double perovskite compounds at high temperatures. Zaraq et al.  synthesiu et al.  used XRDa et al.  used thei et al.  utilized6, and the crystal structure of AA′BB′O6 consists of the exchange sites of BO6 and B′O6 octahedra across the corners of the network connection. The A and A′ atoms exist in the space between the BO6 and B′O6 octahedra, and can be any element from groups 1 and 2 of the periodic table, especially rare earth elements, while the B and B′ cations can be any transition element [The general chemical formula of double perovskite oxide is expressed as AA′BB′O element . 2FeMox1−WxO6 (where 0 ≤ x ≤ 1) [2Mgx1−CaxWO6 (where 0.0 ≤ x ≤ 0.15) [3WO6:Dy3+ [2MWO6  [2Cax1−2EuxNaxMoO6 [x2−SrxZnWO6 double perovskite series  and discuss the effect of Ba2+ cation substitution with Sr2+ cations in series behavior.The double-perovskite oxide compounds have a very high flexibility in crystal structure and chemical composition, where it is possible to vaccinate or replace the A-sites and B-sites cations with the continuation of the octahedra network connection ,11 such ≤ x ≤ 1) , Ba2Mg1− ≤ 0.15) , Ca3WO6:WO6:Dy3+ , Sr2MWO6xNaxMoO6 . In thisx2−SrxZnWO6  series are displayed in 2NiMnO6 double perovskite oxide. Furthermore, the images reveal the presence of fine fragments that are produced during the preparation grinding. Each of the samples has grains of various sizes, i.e., Ba2ZnWO6 has 1–3 μm grains, Ba1.75Sr0.25ZnWO6 has 1–5 μm grains, Ba1.5Sr0.5ZnWO6 has 1.5–4 μm grains, Ba1.25Sr0.75ZnWO6 has 2–8 μm grains, and BaSrZnWO6 has 2–7 μm grains. An Energy-dispersive X-ray spectroscopy (EDX) analysis is conducted with each sample using the SEM images. 6 sample. All EDX graphs confirm that all samples contain elements of the raw material preparation composition and a proportion approximating the input quantities to configure each sample with a small error, which refers to the homogeneity and crystal purity.The ESM images of the Bax2−SrxZnWO6  double perovskite oxide series prepared by the solid-state reaction is shown in 4 and Ba2WO5 phases displayed as minor peaks at low intensity in the XRD pattern shown in x2−SrxZnWO6  structure around 26° and 28° [4 and Ba2WO5, respectively. The XRD data of each sample in the series are refined by the Rietveld method using the FullProf program. 6, which is represented by a (Fm-3m) cubic structure with lattice parameters a = b = c = 8.039361 Å and α = β = γ = 90°. Identical results are obtained for Ba2ZnWO6 using single-crystal X-ray and neutron diffraction [2−xSrxMgTeO6 series was found in a (Fm-3m) cubic crystal structure [x2−SrxZnWO6  double perovskite series using the Scherer equation [D is the crystalline size, λ is the wavelength (1.5405 Å), β is the full width at half maximum, and θ is the diffraction angle. The tolerance factor was found to be between 0.998 and 1.007, which can be calculated byThe X-ray diffraction data of the perovskite oxide compounds are essential in determining the crystalline structure of the samples in terms of the Bravais lattice, atomic position, lattice parameter, and space group. Many studies refer to the importance of the study of material structure since they govern the other properties of the materials . The XRD and 28° . A plus fraction . Furthertructure . The cryequation , which w2−xSrxZnWO6  using the Rietveld method of refinement. The unit cell volume decreases with an increasing substitution as a result of the larger ionic radius of the Ba2+ cation than that of Sr2+.−1 region [6 octahedral displayed at 620 cm−1 is due to the higher charge of the tungsten cations. The symmetric stretching vibration of the WO6 octahedral appears as a high-intensity band at about 825 cm−1. x2−SrxZnWO6 double perovskite series versus wave number, and all the samples confirm the molecular bands on the perovskite oxide structure [The FTIR spectra identify the crystal structure of the perovskite structure materials that have characteristic absorption bands in the 850–400 cm1 region . The strtructure .x2−SrxZnWO2 . For all samples, the Raman modes are classified into two types of lattice vibration—the W–O–W bending vibration in the 200–500 cm−1 region and the W–O stretching mode between 700 and 950 cm−1. This result was observed in many studies on double perovskite oxides [The Raman spectra of the samples are shown in e oxides ,23. Ba hx2−SrxZnWO6 series at room temperature in the 200–800 nm range. The strong absorption band observed at 300–450 nm refers to the absorption edge in tungsten due to the charge transfer transition of 2+ with Sr2+ cations.tungsten ,19,24. Tx2−SrxZnWO6 series from the diffuse reflectance data using the Kubelka–Munk function [α is the absorption coefficient, s is the scattering coefficient, and R is the reflection coefficient. The absorbance (f(R)hυ) in relation to wavelength is shown in 2ZnWO6, Ba1.75Sr0.25ZnWO6, Ba1.5Sr5ZnWO6, Ba1.25Sr0.75ZnWO6, and BaSrZnWO6, respectively. The band gap energy of the series samples was calculated from the absorption edge [gE = 1240/λ F(R∞ion edge  accordinband gap ). In addband gap , as show2+ with Sr2+, the material of Sr2ZnWO6 has a monoclinic (P21/n) crystal structure [In the case of complete substitution of Batructure ,29 with x2−SrxZnWO6  double perovskite oxide series. The excitation spectra shown in emλ = 380 nm for Ba2ZnWO6, emλ = 346 nm for Ba1.75Sr0.25ZnWO6, emλ = 344 nm for Ba1.5Sr0.5ZnWO6, emλ = 343 nm for Ba1.25Sr0.75ZnWO6, and emλ = 349 nm for BaSrZnWO6. A broad band was observed between 260 and 320 nm, resulting from the electronic excitation of the O (2p) orbital-W (5d) orbital in octahedral WO6 [2+ ratio substitution of Sr2+ cations. The photoluminescence emission of the samples was investigated at exλ = 290 nm for Ba2ZnWO6, exλ = 288 nm for Ba1.75Sr0.25ZnWO6, exλ = 287 nm for Ba1.5Sr0.5ZnWO6, and exλ = 386 nm for Ba1.25Sr0.75ZnWO6 and BaSrZnWO6 displayed a spectral emission spread between 320 and 450 nm. Bugaris et al. [2ZnWO6 displays a maximum at 539 nm when λex = 380 nm. In addition, there is a decrease in excitation intensity peaks with an increase in substitution. The photoluminescence (PL) of Ba2ZnWO6 has an emission spectrum peak at 380 nm and a FWHM of 70 nm. Similarly, Ba1.75Sr0.25ZnWO6, Ba1.5Sr0.5ZnWO6, Ba1.25Sr0.75ZnWO6, and BaSrZnWO6 have peaks at 345, 344, 343, and 342 nm with FWHMs of 40, 40, 40, and 50 nm, respectively. From the peaks of photoluminescence emission, the indirect band gap energy was calculated using the E = hc/λ equation for the series that was found to be 3.26, 3.5, 3.6, 3.6 and 3.65 eV for Ba2ZnWO6 Ba1.75Sr0.25ZnWO6, Ba1.5Sr0.5ZnWO6, Ba1.25Sr0.75ZnWO6, and BaSrZnWO6, respectively [dral WO6 ,28. In as et al.  found a ectively .2−xSrxZnWO6  was synthesized using the solid-state interaction method from BaCO3 (barium carbonite), WO3 (tungsten trioxide), ZnO (zinc oxide), and NiO (nickel oxide) powders mixed in stoichiometric proportions according to the following chemical equation.BaInitially, the materials were used as purchased from Alfa Acer with a purity of 99.99%. Several different treatments of the samples were conducted to achieve the crystal structure. The raw materials were mixed and ground in an agate mortar with acetone, kept in crucibles, and subsequently heated in air at 800 °C for 12 h twice. The sample pellets were prepared in a round shape and heated at 1000 °C twice, following which the same procedure was repeated at 1200 °C. Between the steps for the heating treatment, the sample was ground for 2 h with acetone to increase the homogeneity of the sample at a rate of 10 °C per minute during the heating and cooling process.λ = 1.5406 Å) with a nickel filter. At 40 kV and 40 mA, data were collected for 2θ at 0.02-step sizes and 5-s count times in the 20°–80° range. The XRD data were analyzed using the Rietveld refinement method with the FullProf Suite program [D) [−1) [−1. The Raman spectra were collected in Raman HR , using the high resolution Raman spectrometer with a range of 200–2200 cm−1 at 785 nm with a resolution of 4 cm−1. The Raman probe attaches to the laser via FC/APC and the spectrometer via SMA 905, and has integrated Raman filters and optics with a working distance to the sample of 4.5 mm, configured for the 785 nm laser. A UV–Vis spectrophotometer  using BaSO4 as a reference is used to calculate the UV–Vis diffuse reflectance spectrum at room temperature. In addition, the UV–Vis reflectance spectrum is converted to absorbance using the Kubella–Munk method to estimate the edge of absorption and band gap of the Bax2−SrxZnWO6 double perovskite powder series. A Perkin Elmer LS55 fluorescence spectrometer  was used to investigate the emission and excitation of the Bax2−SrxZnWO6 double perovskite series at room temperature.A Jeol JSM-6360  and high-resolution Stereo Scan LEO 440 SEM  were used to investigate the morphology and determine the homogeneity of the samples, as well as to obtain crystal-scale crystallization. At room temperature, the XRD data were recorded with a Bruker: D8 Advance  using CuKα radiation ( program . The crygram [D)  was calc[D) [−1) , where tx2−SrxZnWO6  double perovskite series was prepared by the solid-state reaction technique. In addition, techniques such as X-ray diffraction, scanning electronic microscopy, Fourier transform infrared spectra, Raman spectra, UV-Vis diffuse reflectance, photoluminescence excitation, and emission spectra were investigated. SEM revealed that the prepared Bax2−SrxZnWO6 samples crystallized at micrometer scales, where the crystal structure of the samples was determined by XRD as a cubic Fm-3m space group. The lattice parameter decreased with an increase in the proportion of substitution from x = 0 to 1, where the result of FTIR also confirmed the double perovskite structure. The Raman spectra of W–O bonds indicated a systematic decrease and re-shifting with an increasing Ba substitution. Strong UV-Vis absorption was found between 350 and 410 nm and smaller optical bandgap energy, 3.02 eV, was found for Ba1.75Sr0.25ZnWO6 compared to the Sr-free sample. The excitation photoluminescence spectra displayed broad bands between 260 and 320 nm, which were assigned to the charge transfer band of Bax2−SrxZnWO6. Bax2−SrxZnWO6 displays photoluminescence in the near-UV and visible region. This feature makes Bax2−SrxZnWO6 a potential semiconductor in optoelectronics applications.The Ba


SENP2 mutants. Our data reveal a key genetic pathway, SENP2–Mdm2–p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G–S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53–Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the  SENP2 mutants, all three trophoblast layers were substantially defective, with the layer containing mainly the polyploid cells most severely affected and diminished. This study reveals a key genetic pathway, SENP2–Mdm2–p53, which is pivotal for the genome replication underlying trophoblast cell proliferation and differentiation.Genome replication is essential for both expansion of stem cell numbers through mitosis and their maturation into certain specialized cell types through endoreduplication, a unique mechanism for multiplying chromosomes without dividing the cell. An important function of p53 as a guardian of the genome ensures that the genetic information is properly propagated during these processes. In this study, we discovered that mice with disruption of SENP2, an enzyme that removes small molecular signals  that modify a protein's behavior and stability, are unable to form a healthy placenta as a result of deficiencies in the formation of various trophoblast cell types that give rise to the placenta. In the mutants, SUMO modification of Mdm2, a protein that monitors the cellular levels of p53, is deregulated. The loss of SENP2 causes dislocation of Mdm2, leading to aberrant stimulation of p53. The precursor cells known as trophoblast stem cells rely on p53 to proliferate and differentiate into specialized polyploid cells, which contain multiple copies of chromosomes. In  Targeted disruption of SUMO-specific protease 2 in mice reveals that SUMO modulation of the p53/Mdm2 pathway is pivotal for G/S transition of mitosis and endoreduplication during trophoblast development. The first two distinct lineages to form in the mammalian embryos are the outer trophectoderm and the inner cell mass (ICM) of the blastocyst . The troSENP2 belongs to a family of proteases that remove a small ubiquitin-related modifier (SUMO) from protein substrates. SUMO , which regulates posttranslational modification of proteins, is a member of the ubiquitin-like modifier family . This coWe previously discovered an interaction of SENP2 with Axin ,18, a keSENP2 was observed in extraembryonic tissues, including extraembryonic ectoderm, chorion and ectoplacental cone, at embryonic day (E)7 7 A. In extday (E)7 B. In addday (E)7 C. At E8.tal cone D–1F. By  and TGC H and 1L. at E9.5 I. In the at E9.5 M. SyncytSENP2-null allele was created by the targeted insertion of a lacZ reporter with pgk-neo cassette into exon 2 and the deletion of exons 3 to 5 to inactivate all different forms of the SENP2 gene product , as confirmed by PCR genotyping analysis  mice were viable and fertile without any noticeable abnormalities. However, we were unable to find SENP2-null homozygous (hereafter referred to as –/–SENP2) newborns, implying that they died prematurely. These results prompted us to investigate whether the loss of SENP2 causes embryonic lethality. The –/–SENP2 embryos appeared to be morphologically indistinguishable from their +/+SENP2 and +/–SENP2 littermates at E9.5 . Histological analyses revealed a reduction of the TGC layer by E9.5  and earlier in their precursors at the ectoplacental cone . Consistent with our in vivo findings, these data suggest that TGC differentiation is severely affected by the loss of SENP2. The results suggest an essential role for SENP2 in TGC development during early placentation.The abnormal development of TGC caused by SENP2 deficiency was further tested using an in vitro differentiation analysis. The hatching A and 5B.hatching C and 5D.fter 6 d E–5H. The2) among actively cycling cells decreased 18 units in the mutants  in the stem cell niches  . Taken together, these results suggest that cell cycle progression was defective in all stem cell niches, and the spongiotrophoblast and TGC layers, of SENP2 mutants. The SENP2 mutant cells were trapped or arrested in the cell cycle.Next, we determined whether similar deficiencies also affect development of the spongiotrophoblast layer. We found that this layer expanded rapidly in the wild-type placenta, but not in the mutants, between E9.5 and E10.5 (data not shown). A portion of the at E10.5 I, whereapositive K. In E10ng cells J and 6L., n = 3) N. To exa, n = 3) O. The ce–/–SENP2 TS cell lines from blastocysts. Immunostaining analyses of Oct4 (an ES cell marker) [p = 0.013, n = 3)   and Cdx2 marker)  confirme, n = 3) A. Althou+/+SENP2 and –/–SENP2 TS cells by flow cytometry analysis of PI (propidium iodide) stained cells. There was no significant difference in the cell population of G2–M between +/+SENP2 and –/–SENP2 cells  but the percentage in S was decreased   of the –/–SENP2 TS cells remained in G0–G1  B and 7C.fter 6 h D. Howevein G0–G1 D. Even a–/–SENP2 TGCs were significantly smaller (+/+SENP2) to 7% (–/–SENP2)   H. Theref–/–SENP2 TGCs at E8.5–E10.5  of TGC development . Therefore, Mdm2 appeared to be aberrantly localized in the stem cell niches and all three major layers of trophoblast during early embryogenesis. The data suggest that SENP2 is required for proper localization of Mdm2 and degradation of p53. Disturbance of SUMO modification by the SENP2 deletion thus causes deregulation of the p53–Mdm2 pathway, leading to deficiencies in mitotic and endoreduplication cell cycle progression and abnormal trophoblast development.The cell cycle defects led us to investigate potential downstream targets involved in trophoblast development. We specifically focused on those regulators shown to be conjugated by SUMO. Previous reports showed that SENP2  modulates the canonical Wnt pathway by interacting with its signaling molecules ,31. Even.5–E10.5 D–9F. In e stages A–9C. Theelopment G and 9H.elopment I. The dielopment J–9L. Theelopment J and 9K.controls M and 9N.–/–SENP2 TS cells, Mdm2 accumulated in the SUMO conjugated state . Therefore, the SENP2 mediated SUMO modification of Mdm2 appears to be crucial for its subcellular trafficking.In the SENP2 mutants, the dislocation of Mdm2 implied that its distribution is regulated by the SUMO pathway. We therefore investigated whether Mdm2 localization is affected by SUMO. First, immunoblot analysis after cell fractionation showed that sumoylated Mdm2 is found preferentially in the nuclear fraction of /– cells R. Next, /– cells S and 9V,/– cells T. Howeve/– cells U, with e/– cells W or a nu/– cells X stainin+/+SENP2 cells could elevate p53 in a dosage-dependent manner, but, most importantly, to reach the level detected in the –/–SENP2 TS cells . These results support the hypothesis that stimulation of p53 by alteration in Mdm2 activity induces phenotypic defects in trophoblast proliferation and differentiation, resembling those observed in the SENP2 mutants.To address the importance of p53 in mediating the SENP2-null phenotype, we tested whether p53 activation is necessary and sufficient to affect trophoblast proliferation and differentiation. We used both gain-of-function and loss-of-function analyses. Nutlin-3 is a potent small-molecule antagonist of Mdm2, which binds to the p53-binding pocket of Mdm2 and prevents its interaction, thereby stabilizing p53. We first determined that the Nutlin-3 treatment of the TS cells A. To exaTS cells C. Next,  p450scc E, and pr p450scc F–10K. Th–/–SENP2 TS cells  approach. First, immunoblot analysis showed that the p53 RNAi treatment successfully diminished its levels in the TS cells B. The p5in G0–G1 D. Furthe p450scc E. These This study demonstrates an essential role of SENP2 in trophoblast lineage development during placentation. All three major trophoblast layers were affected by SENP2 deficiency. Our data provide an important connection between SENP2 and the p53–Mdm2 pathway in trophoblast development. The loss of SENP2 caused a deficiency in the G–S transition, which is required for both the mitotic cell cycle  and the endocycle (containing only the G and S phases) during trophoblast proliferation and differentiation, respectively. The cell cycle regulators p53 and Mdm2 appear to be critical for SENP2-dependent trophoblast mitosis and polyploidy. We propose that the SENP2–Mdm2–p53 pathway has a dual role in the G–S checkpoint of mitotic division and endoreduplication A. AlthouThis study provides evidence to support an important function of p53, as a guardian of the genome to control polyploidy. An endoreduplication deficiency was previously observed in embryos lacking cyclin E proteins . In contSENP2 is also known as Axam, which has been shown to modulate Wnt signaling by interacting with Axin, a scaffold protein involved in targeting β-catenin for degradation ,17. AlthThe loss of SENP2 disturbs the balance of SUMO modification. Although sumoylation of Mdm2 has been described , it was Using a mouse model with disruption of SENP2, this study suggests a novel role of SUMO modification in cell cycle progression and induction of polyploidy. Sumoylation, which dictates Mdm2 trafficking, is crucial for modulation of the p53–Mdm2 circuit. Further studies focusing on the detailed mechanistic switch of the SENP2–Mdm2–p53 pathway and its implications in other developmental and pathogenic processes promise important insights into the role of SUMO modification in mammalian development and disease.SENP2 gene (Accession number NC_000082) were isolated by PCR and cloned into the pGEM vector. The 5′ arm contained sequences from the first coding exon to the beginning of the second coding exon, which encodes the first 49 amino acids of SENP2. The 3′ arm included parts of the fifth intron and the sixth coding exon. A β-galactosidase cDNA was fused in-frame to the second coding exon of SENP2. The lacZSENP2 /+ mutant ES cell lines were generated by electroporation of the targeting vector into CSL3 ES cells [SENP2 locus was confirmed by Southern blotting  24 h prior to the transfection procedure. The transfected cells were harvested after 48 or 72 h for further analyses. Total RNA, isolated using Trizol (Invitrogen), was used to produce cDNA according to the manufacturer's instructions . The reverse transcription products were subject to PCR amplifications of the SENP2-lacZ fusion transcript using primers 5′-cagtctctacaatgctgcc-3′ and 5′-ctgtcactctgatctttgg-3′ (exons 3–5), primers 5′-gtgagctgatgagttctgg-3′ and 5′-gtcgctccaataactttcg-3′ (exons 4–6), primers 5′-ggaggagcagaatcatgg-3′ and 5′-ctcaaaatctcatctggtgg-3′ (exons 8–11) and primers 5′-cattaccagttggtctggtg-3′ and 5′-gctgcaataaacaagttccg-3′ (lacZ). The PCR reaction was performed by denaturation at 94 °C for 5 min and 30 cycles of amplification , followed by a 7-min extension at 72 °C.The pCS2-SENP2 clone, containing the Myc-tagged 2 vector . The GFP2 vector  and GFP 2 vector  into the2 vector ,21,23,562 incubator at 37 °C. Cultured embryos were hatched and attached to dishes after 24–36 h. The differentiated trophoblasts became identifiable in a few days. For genotyping, cultured cells were incubated in 10 μl buffer containing 25 mM NaOH and 0.2 mM EDTA, pH 12 for 1 h at 95 °C, followed by the addition of 10 μl buffer containing 40 mM Tris-HCl, pH 5.0. Lysates were subject to PCR analysis. The SENP2 wild-type allele was detected by a nested PCR assay. Primers 5′- ctgttttctactgcagtggacac-3′ and 5′-gctgcctggagtttatctactgtag-3′ were used for the first PCR reaction, performed with 35 cycles of amplification , followed by a 7-min extension at 72 °C. Subsequently, the first PCR products were subject to a second PCR reaction using the method described for genotyping the SENP2 wild-type mouse strain. For genotyping the SENP2 mutant culture, the same method for the SENP2 mutant mouse strain was used.Mouse blastocysts were recovered and cultured in DMEM medium containing 15% FBS, 100 μM β-mercaptoethanol, 100 μM non-essential amino acid, and 100 μg/ml penicillin-streptomycin, in a humidified 5% CO2 incubator at 37 °C. The blastocysts were hatched and attached to the dishes in 24–36 h. After 48 h, a small outgrowth from a blastocyst was formed and cultured in TS medium containing 25 ng/ml FGF4 and 1 ng/ml heparin. After 72–96 h, the outgrowths were ready to be disaggregated by the addition of 0.25% trypsin/EDTA and incubation for 3 min at 37 °C. The disaggregated cells were continuously cultured in TS medium with the presence of FGF4 and heparin. The TS cell colonies began to appear after days 6 to 10, and continued to be cultured until they were about 50% confluent. After expanding the cultures on the feeders for one or two passages, MEF-free TS cells were obtained and maintained in media containing 70% MEF-conditioned medium, 30% TS medium, 37.5 ng/ml FGF4, and 1.5 ng/ml heparin. To differentiate TS cells into TGC, cells were cultured in TS medium with no additions [5 (for mitotic cell cycle) or 105 (for endoreduplication cycle) TS cells were cultured in 6 cm dishes in TS media plus FGF4, heparin, and MEF-conditioned medium (undifferentiated medium) for 2 d, and TS media only (differentiated medium) for 6 d, respectively. Cells were then harvested by trypsinization and fixed in 70% ethanol at 4 °C for at least 24 h. Cells were then treated with RNase (1 mg/ml) for 30 min, followed by PI staining (20 μg/ml) for 10 min at room temperature. Samples were analyzed by an Epics Elite ESP (Coulter Electronics) set to collect 10,000 events. The percentage of cells in G0–G1, S, G2–M or with polyploidy were determined using ModFit LT software. For synchronizing cells in M phase, 3 μM nocodazole was added to the media. Nuclear and cytoplasmic fractionations of TS cells were extracted using an NE-PER extraction kit according to the manufacturer's protocol (PIERCE).To establish the TS cell lines , blastocParaffin sections were treated with buffer containing 0.1 M Tris-HCl and 0.1 M EDTA (pH 8.0) plus 1 μg/ml proteinase K for 30 min, and washed with the same buffer without proteinase K for 5 min at 37 °C. Samples were then incubated with buffer containing 0.2 M Tris-HCl (pH 8.0) and 0.1 M glycine for 10 min at room temp, followed by post-fixing with 4% paraformaldehyde in PBS buffer for 20 min and a 20-min wash in PBS buffer at room temperature. The sections were incubated in buffer containing 0.1 M triethanolamine (pH 8.0) for 10 min, followed by 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine (pH 8.0) buffer for 10 min and by 2× SSC  buffer for 10 min. After dehydration through ethanol gradients and air drying for 2 h, sections were incubated with digoxygenin-labeled probes (1 μg/ml) in 5× SSC buffer containing 50% formamide, 50 μg/ml yeast tRNA and 1% SDS overnight at 70 °C. Samples were then washed three times with 5× SSC buffer for 15 min at 70 °C and 2× SSC buffer containing 50% formamide for 10 min at 45 °C before incubating with buffer containing 20 μg/ml RNase A, 5 U/ml RNase T1, 0.5 M sodium chloride, 10 mM Tris (pH 8.0) and 1 mM EDTA (pH 8.0) for 30 min at 37 °C. After washing with 2× SSC for 10 min at 37 °C and 0.1× SSC for 10 min at 45 °C, samples were incubated in MBST buffer containing 60 mM maleic acid, 0.15 M sodium chloride, and 0.1% Tween-20, pH 7.5 for 10 min and blocked with 10% goat serum in MBST for 2 h at room temperature. After incubating with anti-digoxygenin antibody (Roche) in the blocking buffer for overnight at 4 °C, sections were washed with NTMT buffer  and incubated in NTMT plus 2 mM levamisole overnight at 4 °C. To visualize the bound signals, samples were incubated with BM-purple (Roche) for 2 h to several days. The reaction was stopped by incubating in PBS buffer, followed by counterstaining with nuclear fast red.Samples were fixed, paraffin embedded, sectioned, and stained with hematoxylin/eosin for histological evaluation as described . Tissue Figure S1SENP2 and a pgk-neo gene, flanked by loxP sites, for positive selection. Diphtheria toxin (DTA) was used for negative selection. The pgk-neo was removed by Cre-mediated recombination to generate the null allele as described in Materials and Methods. Southern (A) and PCR analyses  examined the targeted and null alleles. (A) Using a 5′ external probe, the EcoRV-digested wild-type  and knock-in  bands were detected in the targeted ES cells by Southern blotting. Mice carrying either the targeted (B) or the null (C) allele were analyzed by PCR for the WT and KI alleles and the neo and lacZ genes as indicated.(A–C) The targeted locus contains an in-frame insertion of lacZ into the second exon of SENP2 and lacZ in the control (+/+) and homozygous (–/–) E10.5 embryos, respectively.(D) RT-PCR analyses detected the transcripts of (692 KB TIF)Click here for additional data file.Figure S2+/+SENP2  and –/–SENP2  extraembryonic structures were analyzed for terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) staining using fluorescent (green) or immunohistochemical (brown) assays at E7.5  and E9.5 (C–H). No significant differences between the +/+SENP2 and –/–SENP2 were found at the trophoblast stem cell niches , labyrinth , spongiotrophoblast , or TGC layers (n = 2).Sections of the +/+SENP2 (I) and –/–SENP2 (J) TS cell cultures. TUNEL staining identifies cell death in the  TS (K) and mesenchymal (L) cell cultures induced for apoptosis with 50 μM dexamethasone for 24 h were also analyzed as positive controls. Fluorescently and immunohistochemically labeled samples were counterstained with DAPI and hematoxylin, respectively.+/+SENP2 and –/–SENP2 samples (n = 2), and the percentage of apoptotic cells in TS and mesenchymal (MSC) controls.(M) The graph represents the average percentage of apoptotic cells in the (3.15 MB TIF)Click here for additional data file.Figure S3+/+SENP2 ES (first column), +/+SENP2 TS (second column), and –/–SENP2 TS (third column) cells. Cells were counterstained by DAPI (third row). Scale bar, 50 μm.The TS cell lines were derived from blastocysts isolated at E3.5. Immunostaining analyses of Oct4 (first row) and Cdx2 (second row) were performed on (1.73 MB TIF)Click here for additional data file.


Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten.A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation.We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized.TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease. Celiac disease is an intestinal chronic inflammatory disorder elicited by dietary wheat gluten and related proteins from barley and rye. The disease is characterized by malabsorption, flattening of mucosa and the presence of autoantibodies. The large majority of the celiac disease patients express the HLA-DQ2.5 and/or HLA-DQ8 molecules TG2 is a calcium-dependent enzyme which targets specific glutamine residues in peptides and proteins for either transamidation (crosslinking) or deamidation During the recent years several T-cell epitopes of wheat gluten have been identified Several recent studies have pointed towards a role of TG2 in the selection of T-cell epitopes in celiac disease. Using a set of synthetic overlapping peptides covering the whole sequence of a γ-gliadin protein, those peptides that were quickly deamidated by TG2 were also recognized by T-cell lines of celiac disease patients In this study we aimed to shed further light on the role of TG2 in the selection of gluten T-cell epitopes. Gluten is an extreme complex mixture of diverse proteins. Several hundred distinct gluten proteins are belonging to the gliadin and glutenin fractions of a single wheat variety, and therefore tens of thousands different peptides will be present in a digest of wheat flour. To mimic the selection of T-cell epitopes from this vast sea of peptides that is present in the gut after gluten ingestion, we have developed a method to identify the preferred peptide substrates for TG2 in a proteolytic digest of whole gluten. Interestingly, we found that the great majority of preferred substrates for TG2 from this heterogeneous mixture are indeed peptides that harbor known gluten T-cell epitopes. We also discovered two peptides that harbor novel candidate T-cell epitopes.Escherichia coli with an N-terminal hexa-histidine tag and purified as described previously Synthetic peptides were purchased from GL Biochem Ltd . Recombinant human TG2 was expressed in Whole gluten  digested with pepsin, trypsin, chymotrypsin, elastase and carboxypeptidase (PTCEC gluten) was a gift from M. Bethune (Stanford University) and was prepared as follows: A solution of 15 mg/ml gluten was dissolved in 0.01 N HCL (pH∼2.1) and was incubated with 0.6 mg/ml pepsin (American Laboratories) for 60 min at 37°C. The digest was adjusted to pH 6.5 with 50 mM sodium phosphate buffer and incubated with 0.375 mg/ml trypsin (Sigma), 0.375 mg/ml chymotrypsin (Sigma), 0.075 mg/ml elastase (Sigma) and 0.075 mg/ml carboxypeptidase A (Sigma) for 120 min at 37°C. The sample was heated to 95°C for 5 min to stop proteolysis followed by centrifugation and filtration of the supernatants.QLQPFPQPQLPY) and DQ2-γ-II (GIIQPQQPAQL) was separately mixed with 20 µM 5-biotinamido-pentylamine (5-BP) in 10 µl of 100 mM Tris/HCl pH 7.4 supplemented with 2 mM CaCl2. Both samples were incubated with 0.1 µg/µl TG2 at 37°C for 60 min before TG2 was inactivated by adding 1 µl iodoacetamide to a final concentration of 25 mM. A volume of 1 µl of each of the samples was added to 20 µl of 1 mg/ml PTCEC gluten digest. The sample was incubated with 200 µg Dynabeads M270 streptavidin (capacity: 200 pmol biotinylated peptides/1 mg beads)  for 30 min in PBS/0.1% SDS followed by three washing steps each with 100 µl PBS/0.1% SDS and 100 µl 20% acetonitrile/water. For elution, beads were incubated with 15 µl 70% acetonitrile/2% formic acid/0.2 mM biotin for 30 min at 37°C and the eluate was analyzed by MALDI-TOF mass spectrometry .To test the specificity of the enrichment procedure, 20 µM of the synthetic peptides DQ2-α-I (PQPQLPYPQPQLPY), DQ2-γ-II (GIIQPQQPAQL), DQ2-γ-III (FPQQPQQPYPQQP) and DQ2-γ-IV (FSQPQQQFPQPQ) was incubated with 0.1 µg/µl TG2 and 20 µM 5-BP in 20 µl 100 mM Tris/HCl pH 7.4 with 2 mM CaCl2 at 37°C. Aliquots of 5 µl were removed after 1, 5 and 15 min incubation time and 1 µl iodoacetamide  was added to inactivate TG2. For enrichment, 1 µl of each of the samples was incubated with 150 µg Dynabeads M270 streptavidin for 30 min in PBS/0.1% SDS. Washing of the beads and elution of the enriched biotinylated peptides was performed as described above. MALDI-TOF spectra were acquired for the eluates of enriched transamidated peptides.In order to rank the peptides according to their level of transamidation, a sample containing 20 µM of each of the peptide epitopes DQ2-α-II , the eluate was vacuum-dried, and the peptides were redissolved in 0.1% formic acid. Next, 300 fmol of the sample was analyzed by nano-LC coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer (settings as described below). Similarly, 42 µM of a chymotryptic digest of α2-gliadin was incubated under identical conditions in the presence of 2 µM 5-BP. Transamidated peptides were enriched by incubating with 150 µg Dynabeads M270 streptavidin for 30 min in PBS/0.1% SDS followed by washing of the beads as described above. Enriched peptides were eluted by adding 10 µl of 70% acetonitrile/2% formic acid/0.2 mM biotin and incubating for 30 min at 37°C. Finally, MALDI-TOF spectra were acquired.Further, 30 µM of a chymotryptic digest of α2-gliadin was incubated with 0.1 µg/µl TG2 in 25 µl 100 mM Tris/HCl pH 7.4 supplemented with 2 mM CaCl2  was incubated at 37°C for 30 min. After inactivation of TG2 by adding iodoacetamide , the sample was incubated with 1 mg Dynabeads M270 streptavidin for 30 min in PBS/0.1% SDS and the beads were washed as described above. Enriched transamidated and biotinylated peptides were eluted from the beads by adding 25 µl of 70% acetonitrile/2% formic acid/0.2 mM biotin and incubating for 30 min at 37°C. Finally, the eluate was vacuum-dried.A sample containing 1 mg/ml PTCEC gluten digest, 0.1 µg/µl TG2 and 200 pmol 5-BP in 100 mM Tris/HCl pH 7.4 supplemented with 2 mM CaCl18 ZipTips , vacuum-dried and redissolved in 10 µl of 1% formic acid, and then 3 µl were injected into an Ultimate 3000 nanoLC system . For separation of peptides, an Acclaim PepMap 100 column (120 mm×0.075 mm) packed with 100 Å C18 3 µm particles (Dionex) was used. A flow rate of 300 nL/min was employed with a solvent gradient of 7–35% B in 77 min and then from 35% to 50% B in 10 min . The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Data were acquired using Xcalibur v2.5.The transamidated peptides enriched from PTCEC gluten were analyzed either by a Q-TOF hybrid mass spectrometer  or an Orbitrap mass spectrometer , both coupled to a nano-LC system. For nano-LC-Q-TOF analysis, dried samples were dissolved in 8 µl 0.1% trifluoroacetic acid and a volume of 6 µl was injected into an Agilent 1100 series nano-LC system . Peptides were separated on an analytical column (150 mm×0.075 mm) packed with 100 Å C18 3.5 µm particles . A linear gradient of 2–60% solvent B in 60 min was applied with a flow rate of 300 nL/min . The Q-TOF was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Collision energies were set between 30–50 eV depending on the charge state of the precursor ions. Data were acquired using microTOFControl v2.0 and processed using DataAnalysis v3.4. For nano-LC-Orbitrap analysis, the peptides were purified by CTriticum aestivum was built by extracting entries (n = 4654) from the UniprotKB database (release 15.0) at the European Bioinformatics Institute (EBI) using the SRS server. The LC-MS/MS data were searched against this database using the Mascot search engine and Proteome Discoverer software version 1.0 . In Mascot the glutamine modification by 5-BP linkage (mass increase of 311.17 Da) was implemented and selected as variable modification together with deamidation of glutamine and aspargine and pyro-glutamate formation. For analysis of LTQ-Orbitrap data, the mass tolerance was set as 10 ppm for the precursor ion and 0.6 Da for the fragment ions, whereas for analysis of Q-TOF data the mass tolerance was set as 0.5 Da for the precursor ion and 0.2 Da for the fragments ions. All reported hits were in addition manually interpreted. If the glutamine residues targeted by TG2 could not be unambiguously assigned based on the MS/MS spectrum, also the established rules governing the targeting of glutamine residues by TG2 were considered First, a database consisting of all known protein sequences derived from VPVPQLQPQNPSQQQPQEQVPL), #19 (PHQPQQQVPQPQQPQQPF), #25 (SHQQQPFPQQPYPQQPYPS), and #30 (SFPQPQPQQPQQPS) were tested in T-cell proliferation assays after TG2 treatment done by incubating 100 µM peptide with 0.1 µg/µl TG2 in 100 mM Tris/HCl pH 7.4 buffer supplemented with 2 mM CaCl2 for 120 min at 37°C. A panel of synthetic peptides representing already known T-cell epitopes was included in the same assay as reference. Sixty thousand HLA-DQ2 homozygous EBV-transformed B lymphoblastoid cells were irradiated (75 Gy) and incubated overnight with different concentrations of the TG2-treated peptides. On day one, 40 000 T cells were added. On day three, 1 µCi 3H-thymidine was added per well and T-cell proliferation was measured on day 4 as thymidine incorporation. The HLA-restriction of the T-cell lines TCL.422.02.2.4 and TCL.497.C.1.3 was assessed by blocking recognition of TG2-treated peptide #25 (10 µM) using 5 µg/ml anti-DR (B8.11), anti-DQ (SPV-L3), or anti-DP (B7/21) specific monoclonal antibodies during B cell and antigen co-incubation.Twenty polyclonal T-cell lines derived from intestinal biopsies of celiac disease patients as previously described Incubation of a PTCEC digest of whole wheat gluten with small amounts of the primary amine 5-BP and TG2 will result in transamidation of a limited number of gluten peptides. In order to detect and identify these TG2 peptide substrates in such a heterogeneous peptide mixture, transamidated and hence biotinylated peptides were enriched using magnetic streptavidin beads . First, VRVPVPQLQPQNPSQQQPQEQVPL were identified as substrates. Thus we concluded that enrichment of gluten peptides by means of crosslinking with 5-BP can be used to provide information as to which peptides in a heterogeneous mixture are preferred also for the deamidation reaction. The method can also determine which glutamine residues within the preferred substrates are targeted by the enzyme.We next tested whether the deamidation and transamidation reactions catalyzed by TG2 prefer the same gluten peptides. Previously we have shown that epitope peptides DQ2-α-II and DQ2-γ-II undergo rapid deamidation by TG2, whereas epitope peptides DQ2-γ-III and DQ2-γ-IV are poorly deamidated In order to identify optimal TG2 gluten peptide substrates, a PTCEC digest of whole gluten was incubated with TG2 in the presence of limiting amounts of 5-BP for 30 minutes. After specific enrichment of transamidated gluten peptides by magnetic streptavidin beads, eluted peptides were identified by LC-MS/MS analysis and subsequent database searching . AltogetTen peptides derived from γ-gliadin were identified, and eight of these peptides contained parts of the DQ2-γ-II epitope (peptides #10–17). Notably, these peptides shared the same peptide core region but differed in their N- and C-terminal extensions. The DQ2-γ-VIIb/DQ8-γ-I epitope was present in one of the identified γ-derived peptides (peptide #18).Twelve identified peptides derived from glutenin proteins. The complete DQ2-restricted glutenin-17 epitope, Glt-17, was present in three of these peptides  while two of the peptides (peptides #20 and #23) harbored truncated versions of the epitope. Interestingly, several of the other identified TG2 peptide substrates of glutenin proteins have sequences similar to known epitopes.QQQPQEQPSVPLVPVPQLQPQN) which also contains peptide #8, peptide #19 (QQPQQVPQPQPFPHQPQQ), peptide #25 (QQPYQQPFPPQQPYPSSHQ) and peptide #30 . Peptides were treated with TG2 and tested together with a panel of known gluten epitopes as reference in a T-cell proliferation assay. While none of the T-cell lines responded to peptides #9 and #30, the three T-cell lines TCL.BW.CD-E, TCL.497.C.1.3 and TCL.422.02.2.4 responded to peptide #25 in a TG2-dependent and dose-dependent fashion , transamidation and deamidation sites could be assigned to Q11 and Q4 (underlined), respectively. A transamidated and deamidated derivative was also observed for an elongated version of peptide #1. In this 25mer α-gliadin peptide , it could however not be unambiguously determined which of the two targeted glutamine residues (Q9 and Q16) was transamidated or deamidated. For peptide #8 (QQPQEQVPLLQPQNPSQ), Q14 was found to be transamidated whereas Q9 was deamidated. These modifications were observed for the same glutamine residues in peptide #9 which represents an N-terminally extended derivative of peptide #8. The γ-gliadin derived peptide QGIIQPQQPAVQG (peptide #13) was identified to be transamidated at the Q4 residue and deamidated at the Q10 residue.Four α-gliadin and one γ-gliadin derived TG2 peptide substrates were observed as derivatives carrying both a transamidated and a deamidated glutamine residue. For peptide #1  were represented by their intact 9mer sequences whereas the T-cell epitopes that are infrequently recognized  were represented by partial 9mer sequences. This was particularly striking for the DQ2-γ-II epitope which was never represented by a complete 9mer, but appeared in C-terminally truncated forms in 8 distinct peptides. In keeping with our results, Shan Among the peptides selected as substrates by TG2 we identified sequences that are likely to contain novel T-cell epitopes. Five peptides that did not harbor already known T-cell epitopes were suspected to be T-cell stimulatory as their sequences indicated potential HLA-DQ2.5 binding. We tested synthetic peptides representing these five sequences for recognition by twenty polyclonal T-cell lines of HLA-DQ2.5-positive celiac disease patients. Two of them, peptide #19 and peptide #25, elicited dose-dependent T-cell responses in one or more T-cell lines. Peptides #8 and #9, which are found in the well-investigated α2-gliadin protein, have also been reported as substrates for TG2 Triticum aestivum. Thus, the α33mer peptide was likely present in a low amount in the PTCEC gluten digest we used which could explain its absence among the identified TG2 substrates. We observed, however, several variants of the α33mer peptide (peptides #3 and #5) and as well as truncated versions of these peptides . Notably, it has been demonstrated that such shorter versions can be as potent as the α33mer peptide in stimulating intestinal T-cell responses The immunostimulatory α-gliadin derived 33mer fragment, which is a very good TG2 substrate and which is resistant to proteolysis Among the identified TG2 peptide substrates a few peptides were observed as transamidated and deamidated at the same time. These findings may have implications for the generation of TG2-specific autoantibodies in celiac disease whose appearance is strictly dependent on the dietary intake of gluten In a heterogeneous gluten digest, TG2 is not targeting all the different gluten peptides, but shows a clear preference for those which are found as T-cell epitopes in celiac disease patients. This finding argues for TG2 exerting a strong pressure in the selection of T-cell epitopes in celiac disease. Our data also demonstrate the important role of proteolysis in the selection of T-cell epitopes as the T-cell epitopes that are frequently recognized by T cells of celiac disease patients more often remain intact than the epitopes that are infrequently recognized. Together, the selective forces exerted by TG2 and gastrointestinal proteolysis are key factors to determine the repertoire of gluten epitopes in celiac disease.