Three dimensional (3D) genome spatial organization is critical for numerous cellular functions, including transcription, while certain conformation-driven structural alterations are frequently oncogenic. Genome conformation had been difficult to elucidate but the advent chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin architecture and yielded numerous biological insights. Although most of these findings have flowed from analysis of proximity data produced by these assays, added value in generating 3D reconstructions has been demonstrated, deriving, in part, from superposing genomic features on the reconstruction. However, advantages of 3D structure-based analyses are clearly conditional on the accuracy of the attendant reconstructions, which is difficult to assess. Proponents of competing reconstruction algorithms have evaluated their accuracy by recourse to simulation of toy structures and/or limited fluorescence in situ hybridization (FISH) imaging that features a handful of low resolution probes. Accordingly, new methods of reconstruction accuracy assessment are needed.Here we utilize two recently devised assays to develop methodology for assessing 3D reconstruction accuracy. Multiplex FISH increases the number of probes by an order of magnitude and hence the number of inter-probe distances by two orders, providing sufficient information for structure-level evaluation via mean-squared deviations (MSD). Crucially, underscoring multiplex FISH applications are large numbers of coordinate-system aligned replicates that provide the basis for a referent distribution for MSD statistics. Using this system we show that reconstructions based on Hi-C data for IMR90 cells are accurate for some chromosomes but not others. The second new assay, genome architecture mapping, utilizes large numbers of thin cryosections to obtain a measure of proximity. We exploit the planarity of the cryosections – not used in inferring proximity – to obtain measures of reconstruction accuracy, with referents provided via resampling. Application to mouse embryonic stem cells shows reconstruction accuracies that vary by chromosome.We have developed methods for assessing the accuracy of 3D genome reconstructions that exploit features of recently advanced multiplex FISH and genome architecture mapping assays. These approaches can help overcome the absence of gold standards for making such assessments which are important in view of the considerable uncertainties surrounding 3D genome reconstruction. Many novel conformational-related findings have flowed from direct analysis of such contact level data. But, by converting contact frequencies into distances  are frequently oncogenic. Until recently, genome conformation had been notoriously difficult to interrogate. However, the emergence of the suite of chromatin conformation capture assays, notably Hi-C, has transformed understanding of chromatin architecture and yielded numerous downstream biological insights , 17, 24.ionships , 27, 29)ionships , gene exlciparum , and elulciparum .6) cell populations on which Hi-C assays are performed is highly simplified [single-cell Hi-C assays [Yet, such putative advantages of 3D structure-based analyses are clearly conditional on the accuracy of the corresponding reconstruction and there are many reasons why such accuracy can and has been questioned. First, the very notion of a single genome architecture being representative of the large Here we show how newly devised biotechnologies can dramatically improve 3D genome reconstruction accuracy assessment and demonstrate computational and statistical techniques for realizing this promise. The advent of multiplex FISH  has the https://doi.org/10.1126/science.aaf8084. The numbers of probes, centered at previously defined topologically associated domains  probe missingness pertained over the replicates. We handled this via imputation – simply averaging over non-missing  coordinates of the corresponding probes across replicate chromosomes – and discuss implications of this approach under Accuracy Assessment below. Crucial to this imputation approach was the fact that the chromosome replicates were imaged using a common coordinate system [We obtained multiplex FISH probe coordinates for chromosomes 20, 21, and 22 for IMR90 cells from Figures & Data at e system .While multiplex FISH probe coordinates are also available for active and inactive X chromosomes we are not positioned to evaluate corresponding 3D reconstruction accuracy since the associated Hi-C data does not differentiate between active and inactive X. Given the multiplex FISH data, deconvolving the Hi-C X chromosome data into active and inactive counts would be possible, but then we couldn’t deploy the same multiplex FISH data for accuracy assessment.Hi-C data  for IMR9GAM data for mouse embryonic stem cells (mESC) was obtained from GEO with accession GSE64881. Available data include measures of proximity  which, as demonstrated , are higThe focus of the present work is advancing methods for evaluating the accuracy of 3D genome reconstructions by taking advantage of newly devised assays: multiplex FISH and GAM. While the proposed methods are agnostic with respect to the technique used to effect the reconstruction, we illustrate ideas using the hybrid simulated annealing  algoritn, via Poisson regression: Like other 3D reconstruction algorithms , 29, HSAk indexes track, so that in some of our applications K=2 corresponding to primary and replicate data, and ik,jk. For notational simplicity we will impose that there are n common loci across tracks: ik=1,…,n;jk=1,…n;∀k although the HSA algorithm does not require this. The parameters βk1 correspond to (per-track) power-law indices relating expected counts (μ) to Euclidean distances (d). Provision exists for additional covariate terms  to be included in  parameters constituting the reconstruction. These are subject to constraints owing to the local contiguity of chromatin. Zou et al. [β,X is then where in  k indexementclass2pt{minimu et al.  capture X enters (μ and d from (cf Poisson regression) fitting to obtain estimates to which a penalty term controlling local smoothness is added. Note that (constrained) X enters  through d d from  and 1)  X entersWe note that the the GLM routine used (R function glm) does not require integral (count) data and so can accommodate normalized counts and GAM linkage disequilibrium scores. HSA subsumes several tuning parameters governing simulated annealing search. We have used default values throughout; however, the ability to compare accuracy of competing solutions as developed next provides a means for exploring differing tuning parameter settings.geometric information independent of derived distances and attendant reconstructions, as we detail below.The two recent assays under consideration, multiplex FISH and GAM, provide distinct approaches to evaluating the accuracy of 3D genome reconstructions. Multiplex FISH imaging provides a gold standard from which the closeness of a 3D reconstruction can be measured, with inference making recourse to replicates. By deploying large numbers of thin nuclear cryosections reconstructions based on GAM assays admit accuracy assessment even in the absence of external gold standards. This results from the fact that the sectioning itself provides We take the image-based 3D genomic coordinates furnished from multiplex FISH  scaling ) is stra scaling , and so n common genomic loci. Then the mean square deviation is given by To simplify notation we let We obtain MSD values using the R package vegan  which alviations , our conprotein structure comparisons have promulgated various prescriptions for evaluating agreement adequacy in terms of RMSD Ångström thresholds, although these have been called into question [genome reconstruction comparisons, most of which do not provide configurations with an underlying physical distance. Instead, we seek appropriate (R)MSD referent distributions, developing differing approaches for multiplex FISH and GAM in accordance with data structure and availability.3D question . HoweverLu replicates for chromosome u=1,…,U. Indeed, l values captures experimental variation around the multiplex FISH gold standard. Interpretation of l distribution is demonstrated in the Results.For each chromosome we treat 3D coordinates provided by multiplex FISH as our gold standard X^ using . But, asLu>110 ∀u) so that the contribution of individual chromosome replicates is modest; (ii) the imputation scheme used to handle missing coordinate data necessarily borrows strength across replicates, so even the leave-one-out scheme would not eliminate (complex but slight) dependencies of A fine point is that, by construction, this distribution will exhibit reduced dispersion than its targeted population quantity (based on independently obtained We note that this approach to accuracy assessment relies on the availability of suitably large numbers of multiplex FISH image replicates. Absent such replication assessment of planar nuclear profiles (cryosections) can be used as follows.We obtained 3D genome reconstructions for mESC chromosomes based on applying HSA to GAM linkage disequilibrium scores (at 1Mb resolution) as described above. As there are currently no public multiplex FISH imaging studies for mESCs, reconstruction accuracy assessment based on RMSD is precluded. However, information contained in locus membership in the collection of The number of available nuclear profiles is 408, this number being sufficient to study chromatin architecture at 30kb at the sequencing depths deployed, as formally determined by power analysis [We make this proposal concrete by first using a measure of the degree to which a set of 3D points are planar, namely the proportion of variance explained by the sum of the first two principal components (corresponding to a planar projection), hereafter termed PC1+PC2 as given by the associated eigenvalues. We also consider the second principal component alone (termed PC2) in order to distinguish scenarios where the sum is dominated by the first principal component. Note that these measures are coordinate system-free, essential in view of the nature of 3D reconstruction solutions. Further, as some nuclear profiles may capture few or even zero loci, we restrict determination of PC1+PC2 and PC2 to those profiles with sufficient numbers of loci. By taking this to be approximately the upper quartile of the per chromosome per profile locus count we obtain 100 values of PC1+PC2 and PC2 for each reconstructed chromosome.j (included in the top 100 locus counts) that contains say nj loci, we sample nj chromosomal loci, equivalent to permuting the loci indicator column vector of the segmentation matrix. We then obtain the 3D coordinates in the GAM-based reconstruction corresponding to these sampled loci and compute the PC scores for these. This amounts to assessing the planarity of nj points in 3D that are randomly sampled from the reconstruction, with a view to contrasting such planarity summaries with corresponding summaries from the actual nuclear profiles. The contrasting is effected by performing the re-sampling a large number of times so as to provide a null distribution for the summaries. To confer robustness, we use ranking to summarize how the original PC scores compare to this null distribution which corresponds to computing an empiric p-value. A further level of summarization – over the 100 profiles – is required and, for robustness, we use the median.As was the case with multiplex FISH accuracy assessment we need a reference system for evaluating our PC scores. While there is an extensive body of work on random matrix theory (e.g. ) and attl values obtained using the multiplex FISH replicates as described above, for chromosomes 20, 21 and 22. Also shown are MSD values, derived using  down to n19=27 (chromosome 19) and correlate (r = 0.97) with chromosome length. These counts represent the minimum numbers of loci per profile over the 100 nuclear profiles we utilize – they range up to maxima of n1=169 to n19=56. In Table For each chromosome, and for each PC measure, the Tables Analogously, in Table Results for the 19 chromosomes reveal some interesting patterns and putative groupings. For several chromosomes  attained PC1+PC2 median rankings are best – the reconstruction profiles have most, or near most, explained variance compared with resampling-based profiles – while some additional chromosomes  are in the top 20%. These results all align with the percent variance explained summaries provided in Table In this paper we have exploited two recent biotechnologies, multiplex FISH with TAD-based probes and genome architecture mapping, in order to assess the accuracy of 3D genome reconstructions. As indicated in the Introduction, such reconstructions can confer added value with respect to downstream biological insight, but clearly any putative insights are conditional on the quality of the reconstruction. Gauging quality has proven exceedingly difficult accentuating the need for methods such as those proposed here. Both these methods, and the new assays themselves, can be refined and deployed in additional ways.As well as being used for accuracy assessment, multiplex FISH can also be used to improve the actual 3D reconstructions via better calibration of the power-law transfer function (e.g. ) for conciencies , 19 to bentclass1pt{minimamultiplex FISH, with the associated substantive increase in probe numbers and resolution, mitigates these concerns. Moreover, we have accommodated variation in multiplex FISH imaging in adopting it as a referent. Additionally, we have relied upon (R)MSD as a measure of configuration closeness. Other metrics are available, with distanceError being frequently used in the context of 3D genome reconstruction [Our use of multiplex FISH to gauge the accuracy of Hi-C based 3D genome reconstructions is predicated on its constituting a suitable gold standard. One limitation to use of multiplex FISH is resolution disparity with Hi-C data being available at much higher resolutions. Another issue is that Hi-C counts can be driven by factors beyond spatial proximity, such as access to the nuclear compartment outside the chromosome territory core  as well truction , 26, 29 truction  and corrtruction , 33 measGAM ostensibly offers several advantages over Hi-C . Whetherhttps://github.com/marksegal/reconstruct-accuracy.We have developed methods for assessing the accuracy of 3D genome reconstructions that exploit features of recently advanced multiplex FISH and genome architecture mapping assays. These approaches can help overcome the absence of gold standards for making such assessments which are important in view of the considerable uncertainties surrounding 3D genome reconstruction. R code implementing multiplex FISH- and GAM-based accuracy assessment is available on github:


Locally advanced pancreatic cancer (LAPC) is characterized by poor prognosis and low response durability with standard-of-care chemotherapy or chemoradiotherapy treatment. Stereotactic body radiation therapy (SBRT), which has a shorter treatment course than conventionally fractionated radiotherapy and allows for better integration with systemic therapy, may confer a survival benefit but is limited by gastrointestinal toxicity. Stereotactic MRI-guided adaptive radiation therapy (SMART) has recently gained attention for its potential to increase treatment precision and thus minimize this toxicity through continuous real-time soft-tissue imaging during radiotherapy. The case presented here illustrates the promising outcome of a 69-year-old male patient with LAPC treated with SMART with daily adaptive planning and respiratory-gated technique. Locally advanced pancreatic cancer (LAPC) has traditionally been treated with first-line single-agent chemotherapy or conventionally fractionated chemoradiotherapy over a period of five to six weeks, with characteristically poor prognosis due to unresectability of a disease in which long-term survival is achieved almost exclusively through curative resection. While newer therapeutic agents such as nab-paclitaxel and FOLFIRINOX have improved the efficacy of these standard-of-care treatments , survivaThe LAP07 trial determined that conventionally fractionated chemoradiotherapy is able to achieve survival outcomes similar to those of chemotherapy alone but introduces additional treatment toxicities . The preStereotactic body radiation therapy (SBRT) is an innovative treatment modality notable for its shorter treatment course (one to two weeks), which is beneficial for systemic therapy integration and allows for minimal interruption of chemotherapy. A review of locally advanced pancreatic cancer patients in the National Cancer Database juxtaposed SBRT with conventionally fractionated radiotherapy (CFRT) and found that SBRT was a viable radiotherapy modality that conferred a significant overall survival (OS) benefit (13.9 months vs 11.6 months for CFRT) . The stuSBRT, however, presents several challenges, whereby toxicity remains an issue at higher doses and treatment efficacy is compromised by lower doses. A phase II multi-institutional trial sought to characterize the gastrointestinal toxicity of SBRT, delivered in five fractions to patients without metastases after up to three cycles of gemcitabine, and reported acute and late grade ≥2 gastrointestinal toxicity rates of 2% and 11%, respectively, for a patient cohort with a median OS of 13.9 months and a one-year local control of 78% . In a laMRI-guided radiation therapy (MRgRT) optimizes the delivery of hypofractionated radiotherapy, allowing SBRT to achieve higher doses while avoiding damage to critical structures. Endoscopic ultrasound-guided placement of fiducial markers in linac-based SBRT allows for target position verification and location tracking during treatment, but this approach fails to account for inter-fractional changes of surrounding radiosensitive tissue. MRgRT can be utilized to spare radiosensitive normal tissue adjacent to the treatment target: MRgRT counters the limitations of poor soft-tissue resolution of available cone-beam computed tomography (CT) daily imaging with superior soft-tissue visualization and addresses stochastic inter-fractional changes with on-board adaptive planning.The following LAPC case is illustrative of the unique capabilities of stereotactic MRI-guided adaptive radiation therapy (SMART), whereby treatment was delivered with respiratory-gating and adaptive plans were implemented for each of the radiotherapy fractions.The patient is a 69-year-old man with no family history of cancer and a prior medical history significant only for hypertension and irritable bowel syndrome. He presented with three months of abdominal pain radiating to his back and a weight loss of 12 pounds; CT imaging demonstrated a 4.7 x 3.1 cm mass within the pancreatic body, with circumferential encasement of the proximal hepatic artery, superior mesenteric artery, and left gastric artery, and occlusion of the superior mesenteric vein , and the patient received definitive SBRT to the pancreatic body over a period of 13 days (9/20/16 – 10/3/16). The gross tumor volume (GTV) was expanded by 3 mm to obtain the planning target volume (PTV), and respiratory gating was performed with a 3 mm margin with deep inspiratory breath-hold. Normal tissue constraints were defined as follows: duodenal loop and stomach V35 Gy < 0.5 cc; spinal cord and left and right kidneys maximum 12.5 Gy; skin maximum 39.5 Gy, V36.5 < 10 cc; liver ≥ 1000 cc < 15 Gy. The prescribed dose constraints were more stringent than reported objectives in similar studies and represent the ideal target and normal tissue dosimetry, while adaptive plan constraints were V35 Gy < 1 cc in the duodenum and stomach and a maximum of 20 Gy for the spinal cord.Prior to each treatment, the initial planned treatment dose was predicted onto the patient’s current anatomy to ascertain whether the treatment plan was optimal for that day’s specific anatomy Figure , Table 1Adaptive planning was deemed necessary for each of the five fractions due to clinically significant changes in the anatomical relationships between the tumor and normal tissue Figure , which rThe patient experienced bilateral shoulder discomfort with the length of treatment during the first fraction but did not suffer any adverse events during or following radiation. At the completion of treatment, his CA 19-9 level had further diminished to 55. His systemic therapy (FOLFIRINOX) was resumed after radiotherapy to achieve a total of 12 cycles, concluding on December 27, 2016. Although there is a trend towards improved survival in resected LAPC patients [The patient continues to undergo follow-up scans every three months and continues to exhibit sustained stability of the radiated pancreatic lesion. Follow-up CT scans on June 27, 2017 demonstrated lung nodules that were suspicious for metastasis but were found to have no avidity on positron emission tomography (PET). PET/CT on November 29, 2017 revealed mildly avid, slowly progressing lung nodules, and the patient opted for surveillance. The patient remains asymptomatic and was most recently evaluated at his 14-month follow-up.The value of MRgRT is demonstrated by the reported patient, who received a BED of 72 delivered through SMART with no significant toxicities and minimal treatment discomfort with the length of treatment. With continued local control at 16 months post radiotherapy and 21 months since diagnosis, the patient is an excellent example of the benefit conferred by on-board imaging and adaptive planning. The patient’s response to treatment points to a favorable outcome in the context of LAPC treatment and emphasizes the value of MRgRT in abdominal soft-tissue radiation.The total treatment length was clinically feasible and well-tolerated by the patient, with the minimal shoulder discomfort during the first fraction in large part attributable to difficulty with inspiratory breath-hold, which diminished with each subsequent fraction. The adaptive planning process, which includes MRI imaging, contouring, re-optimization, and plan evaluation and quality assurance, took between 15 and 20 minutes, while the treatment delivery time ranged from 21 to 35 minutes, depending on the patient’s breath hold duty cycle. Contouring was performed by the physician the day of treatment, and the contouring inconsistencies between fractions  in 2017 determined that patients treated with BED > 70 achieved a higher rate of overall survival than patients with a BED < 70, with 66% of the former cohort treated with adaptive portions that allowed for the safe administration of the higher doses [MRgRT stands at the forefront of efforts to deliver higher doses of radiation more accurately through the use of soft-tissue imaging before and during treatment. Radiation of LAPC is hindered by severe accompanying gastrointestinal toxicity, and SMART may play an important role in achieving local control with minimal toxicity. Currently, there are plans to escalate dose further to 50 Gy in five fractions in a prospective multi-institutional study, and trials are seeking to validate that dose escalation and more robust planning will further aid the efficacy of local therapies as systemic therapies continue to improve in combating metastatic spread. Further investigation is necessary, but early results such as this case have demonstrated the potential of SMART in advancing treatment precision and safety.


The potential uniformity between differentiation and therapeutic potential of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) remains debatable. We studied the gene expression profiles, pathways analysis and the ability to differentiated into neural progenitor cells (NPCs) and motor neurons (MNs) of genetically unmatched integration-free hiPSC versus hESC to highlight possible differences/similarities between them at the molecular level. We also provided the functional information of the neurons derived from the different hESCs and hiPSCs lines using the Neural Muscular Junction (NMJ) Assay. The hiPSC line was generated by transfecting human epidermal fibroblasts (HEF) with episomal DNAs expressing Oct4, Sox2, Klf4, Nanog, L-Myc and shRNA against p53. For the hESCs line, we used the NIH-approved H9 cell line. Using unsupervised clustering both hESCs and hiPSCs were clustered together implying homogeneous genetic states. The genetic profiles of hiPSCs and hESCs were clearly similar but not identical. Collectively, our data indicate close molecular similarities between genetically unmatched hESCs and hiPS in term of gene expression, and signaling pathways. Moreover, both cell types exhibited similar cholinergic motor neurons differentiation potential with marked ability of the differentiated hESCs and hiPSCs-derived MNs to induce contraction of myotubes after 4 days of co-culture. Although human embryonic stem cells (hESCs) are considered to be the “gold standard” of pluripotent cell lines, ethical concerns regarding the way in which they are generated limit their clinical applications. Successful generation of human induced pluripotent stem cells (hiPSCs) by overexpression of the transcription factors Oct4, Klf4, Sox2 and c-Myc could provide promising substitute for hESCs. These substitutes would overcome ethical and moral issues associated with the use of hESCs in the preclinical and clinical settings.1. Using larger numbers of samples, both cell lines have been shown to be nearly identical3. The use of recent platforms for studying the gene expression profiles of both cell types has revealed small numbers of differentially modulated genes4. Several confounding factors have been suggested as causal factors generating the differences in gene expression profiles of hiPSCs and hESCs: these are reprogramming methods5, host genetic background6, clonal origin7, gene silencing8 and sex of cell lines9. One possible way to decipher the effects of different host genetic backgrounds on gene expression, and neuronal differentiation potential of hiPSCs and hESCs is to eliminate major confounding factors such as reprogramming method by comparing genetically unmatched ESCs and integration-free iPSCs.Having two well-established sources of pluripotent cell lines (hESCs and hiPSC), it is critically important to elucidate whether or not they are identical/equivalent. The presence of fundamental differences between the transcriptomes of hiPSCs and hESCs have been reported11. Such an approach may produce inaccurate findings due to instabilities within inherent pluripotent cell lines11. It has been reported that the OCT4 biomarker, a reliable marker to differentiate ESCs from somatic cells, is not consistently expressed in older ESCs. Moreover, low abundance marker protein signals are difficult to detect using current antibody based biomarkers due to low sensitivity of this technique11.Traditionally, somatic and pluripotent cells are distinguished from each other using PCR and immunostaining to detect specific well-characterized biomarkers12, a distinction between hESCs from hiPSCs based only on gene expression and cluster analysis is challenging since the gene signature is not consistent across different cell lines14. Furthermore, in most cases cluster analysis is associated with a low level of sensitivity in the presence of different cellular sources14. There is therefore still a need to design an approach that overcomes these limitations so as to be able to accurately discriminate between the closely similar pluripotent hiPSCs and hESCs.Although a combined array of linear models, cluster analysis and gene expression profiling can be used to distinguish hiPSCs from hESCsLinking gene expression with the ability to differentiate into specific cell lines  as well as providing functional information of the neurons differentiated from different iPSCs and hESCs may be a promising approach to discriminate between hiPSCs and hESCs. We explored the potential differences in gene expression profiles between genetically unmatched, integration-free hiPSCs and NIH-approved H9 hESCs using cluster analysis, gene ontology, biological pathways, their ability to differentiate into NPCs and NMJ Function Assay (Contractility Assay) of the generated iPSCs and hESCs-derived MNs, to elucidate the degree of gene expression uniformity, potential neuronal differentiation potential, and functional information of the neurons differentiated from the two cell populations. Such an approach would help to eliminate major confounding factor related to the presence of integrating DNA viruses during the generation of iPSCs. Provision of a proof of principle determining potential molecular uniformity between genetically unmatched hESCs and hiPSCs would ensure similar differentiation potential and functional outcomes which in turn could expedite the application of hiPSCs in the clinical setting.http://www.ixcellsbiotech.com). The experimental protocol had obtained an IRB waiver from Qatar University Inistitutional Review Board (IBR) due to the use of commercial cell line with no involvement of any sample collections from human.All cell lines including hESC and iPSC were obtained commercially from IXtechnologies, USA . Both hESCs and iPSCs were maintained in mTeSR1 media (Stem Cell Technologies) on Matrigel-coated plates.The iPSC clones were maintained on MEF feeder plates for one passage and switched to mTeSR1 media without MEF beginning with the second passage. The cells were passaged every 5–7 days until cryopreservation and cell pellet preparation .For pathogen screening, the cells were screened to detect any risk of bacterial, fungal and/or mycoplasmal contamination. For purity testing, antibody staining (ICC) and flow cytometry (IF) using cell-type specific antibodies were used. For an episomal DNA integration assay, Q-PCR allowed the quantitative determination of the copy number of episomal DNA integrated into the cells. A cell was considered integration-free if less than 0.01 copy of episomal DNA was detected. For karyology, metaphases were examined for chromosome number and banding pattern. Twenty cells were analyzed.6 cells/1 mL TRIZOL. 200 µl chloroform was added to each tube for 5–8 minutes of incubation. Samples were centrifuged at 12,000 g for 15 minutes at 2–8 °C followed by RNA precipitation using isopropanol. RNA pellets were washed and treated with Dnase before loading on an Rneasy column (Qiagen) for final purification. RNA quantification and quality control were conducted using Gel electrophoresis based on OD values. The RIN software algorithm was used accurately classify the samples. All samples with a RIN higher than the threshold value pass the QC test, while samples below the threshold value are discarded. The cut-off or threshold for RNA quality was adjusted to RIN 8%15.hiPSCs and hESCs were pelleted, and resuspended in TRIZOL at a volume of 5 × 10The RNA quantity was analyzed using the Nano Drop ND1000 (SOP Nu TAL009) and the RNA quality is checked using the Bioanalyzer 2100 (Agilent). Sample amplification was performed with 200 ng of total RNA using Agilent’s Quick Amp Labeling Kit OnoColor to generate complementary RNA (cRNA) for oligo microarrays. cRNA was processed for microarray analysis on a Whole Human Genome Oligonucleotide Microarray , according to the manufacturer’s instructions.The arrays were hybridized at 60 °C for 17 h with the Tecan HS Pro hybridization station in the hybridization buffer containing fluorescence-labeled cRNA. The microarrays were washed once with 63 SSPE buffer containing 0.005% N-laurylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.066 SSPE buffer (37 °C) containing 0.005% N-laurylsarcosine for 1 min. The last washing stage was performed with acetonitrile for 30 sec.Fluorescence signals of the hybridized microarrays were detected using Agilent’s and Illumina DNA microarray scanner (Agilent and Illumina Technologies) with a resolution of 5 lM. Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities and normalized ratios by linear LOWESS with background subtraction, rejects outliers and calculates statistical confidences . A quality control was performed at this stage. Genes with a P-value smaller than 0.001 were considered significant. Only genes differentially expressed in all experiments were considered as relevant genes.16. Probes returning a p-value < 0.05(t-test) and fold change(FC) > 2 in comparisons of the control and test classes were considered to be detecting differential expression. To avoid distortion of the results by noise, we removed probes returning signals that were highly likely to be due to non-specific background signal rather than specific probe-target hybridization. The specificity of individual probe signals was estimated using the detection p-value, which is the probability of seeing a certain signal level without probe-target hybridization. All probes returning a detection p-value > 0.01 in both the control and test classed were eliminated from further analysis16. Genes with significant difference in expression levels were uniquely retrieved from all probes.Signal intensities were normalized by Cubic Spline strategy in the GenomeStudio software to reduce differences due to technical variation , while conserving true biological effects All significantly expressed genes  were clustered using a hierarchical clustering method. Data from each sample were normalized in log-space to have a mean of zero using Cluster 3.0 software.19. We considered a GO category significantly differentially regulated if either significance level was less than 0.01.Gene ontology (GO) analysis was conducted according to published methods23. Gene ontology (GO) and KEGG molecular pathway analysis were performed to identify possible enrichment of genes with specific biological themes using both the data set as a whole and then in the individual K-means clusters.Functional annotation of significant genes identified by microarray analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 2009 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (david.abcc.ncifcrf.gov)The iPSC and ESC were differentiated to generate ESC/iPSC derived NPC and MN and were subjected to transcriptomic analysis by qPCR on top 10 induced and suppressed genes identified in by DNA microarray. A list of primers used for the top 10 induced/repressed genes is provided in Supplementary Table ESCs (H9) and iPSCs were cultured in neural progenitor differentiation medium . The neural progenitor cells (NPCs) were characterized by immunofluorescence staining (Nestin) and flow analysis (Nestin), and also collected for RNA extraction.NPCs derived from ESCs or iPSCs were further differentiated into mature motor neurons using the proprietary protocol (iXCells Biotechnologies). The motor neurons (MNs) were characterized by immunofluorescence staining  and flow analysis (ChAT), and also collected for RNA extraction.C2C12 mouse myofibroblasts were seeded on 96-well plates in DMEM with 10% FBS, and myotube differentiation were initiated by switching to DMEM with 2% FBS for 3–4 days. The iPSC-MNs or ESC-MNs were seeded on top of the differentiated myotubes with Motor Neuron Maintenance Medium . The MN-dependent contraction of myotubes was observed after 4 days of co-culture.To confirm the DNA microarray results, we studied the gene expression profile for the top ten induced/repressed genes between hiPSCs/hESCs. The RNA was extracted using Trizol Reagent. For each sample, 1 µg RNA was used for cDNA synthesis using Transcriptor Universal cDNA Master . The real-time PCR was performed using FastStart Essential DNA Green Master  with triplicates derived from different cell lines.hiPSCs/hESCs-derived cell NPCs/MN were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-100 (this last step was not performed for NG2 and O4 immunostaining) and subsequently processed for immunolabeling. The following antibodies were used: α-hTRA-1–81 ; α-hCD90 ; α-OCT4 ; α-PAX6 ; α-SOX1 ; PAX6 ; GAPDH ; β-actin .Statistical analysis was performed using Two-way ANOVA and Bonferroni’s posttests. Data are expressed as mean ± standard error of mean (S.E.M). P values between < 0.05 (*) and < 0.001 (***) were considered statistically significant.An overview of the experimental design is given in Fig. Analysis of global gene expression using DNA microarray revealed a total of 3515 and 3513 dysregulated genes in iPSCs and hESCs, respectively Fig. . In hiPSUsing hierarchical clustering, we compared the expression profiles of up-and down regulated genes in hESCs, hiPSCs, and HDF. A clear segregation was noticed between the pluripotent cells and differentiated fibroblasts Fig. , indicatDistinctive sets of 278, and 221 induced genes are exclusive to hiPSC and hESC, respectively Fig. . LikewisAlthough both hiPSCs and hESCs were separately clustered from HDFs, analysis of gene expression profile between the two pluripotent population suggests the presence of subtle but consistent gene expression profile differences Fig. .−37), and M phase  were the most enriched ones (Supplementary Table −13), and protein localization  were significantly enriched (Supplementary Table −31), and M phase  (Supplementary Table −12), and establishment of protein localization   enrichment of hiPSCs and hESCs in comparison to HDFs. We detected a number of Biological Processes (BPs) enriched by the genes upregulated in hiPSCs of which the DNA metabolic process , and M phase  were the most significant.GO analysis (BP) of the genes induced in both iPSCs/hESCs lines revealed significant enrichment of several GO terms (Supplementary Table −12), and establishment of protein localization  , and Spliceosome . GO analysis (Pathway) of downregulated genes in hiPSCs revealed enrichment in several KEGG pathways (Supplementary Table −13), and Focal adhesion  were the most significant.Several KEGG pathways were enriched by the genes upregulated in hiPSCs (Supplementary Table −16), and Spliceosome . Similar KEGG pathways were enriched with genes downregulated in hESCs as in hiPSCs  and Spliceosome  were the most significant. KEGG pathways significantly enriched by repressed genes common to hiPSCs and hESCs included Focal adhesion  and Lysosome   in iPSCs and ESCs. We focused on induced/repressed genes characteristic for each cell type: 278 induced and 191 repressed genes for hiPSCs, and 221 induced and 264 repressed genes for hESCs. We used DAVID to analyse enriched/suppressed pathways for induced/repressed gene list for each cell type (iPSCs and ESCs). KEGG pathway analysis for 278 hiPSCs induced genes revealed the enrichment of five pathways: Cell cycle (6 genes), Butanoate metabolism (3 genes), Base excision repair (3 genes), and Synthesis and degradation of ketone bodies (2 genes). In comparison, the KEGG pathway analysis for 221 repressed hiPSC genes revealed the enrichment of T cell receptor signaling pathway (2 genes).Notably, pathway analysis for the 191 and 264 repressed genes in hiPSCs and hESCs respectively disclosed the enrichment of the same KEGG pathways that included Focal adhesion (16 genes), ECM-receptor interaction (8 genes), p53 signaling pathway (5 genes), Complement and coagulation cascades (4 genes), and Lysosome (5 genes).24. Our data did not reveal any modulation in the gene expression level of LDHA and SLC2A1 in hiPSCs/hESCs.Next we focused on a group of highly modulated genes of both iPSCs and hESCs as a means to explore potential functional differences between the two cell populations. We focused on two of the genes involved in energy metabolism; LDHA and SLC2A1 , because of their previously reported strong basal expression in hESCs and reduced expression in all hiPSCs25. So we studied the potential differences in the gene expression profiles of pluripotent markers between hiPSCs and hESCs. We focused on key pluripotency regulators . No induction/repression was demonstrated in the expression levels in any of the selected pluripotency regulators, a finding that might indicate their stable expression and low variability in the strongly self-renewing fraction of hiPSCs and hESCs.Low variability and high connectivity provide stability to the network, contributing to highly conserved core processes common to all members of a pluripotent cell populationin vitro. Each differentiation step were confirmed by immunostaining and flow cytometry . mRNA extracted from iPSC/ESC, NPCs, MNs were subjected to RT-PCR for the profiling of the top 10 induced/repressed genes in iPSC and ESC. ESCs (H9) and iPSCs were expanded using mTeSR™1 medium (STEMCELL Technologies) without feeder cells  derived from iPSC and H9 embryonic stem cells were confirmed by immunostaining and flow cytometry using anti-ChAT antibody Fig. . 93.39% Based on the microarray analysis, the expression profile the top ten “Induced/upregulated” or “Repressed/downregulated” genes between iPSC and ESC were grouped. Primers are designed to amplify coding region of the listed genes , similar pattern of gene expression profile were identified during ESC and iPSC differentiation into NPC and MNs. The expression level of NNAT was significantly high in NPC, and MNs as compared to ESC, and iPSC. Also, the expression level of TACSTD1 was significant high in MNs as compared to ESC, and NPC Fig. . For theFor NMJ Function Assay (Contractility Assay), the C2C12 mouse myofibroblasts were differentiated into myotube and the iPSC-MNs or ESC-MNs were seeded on top of the differentiated myotubes. In both iPSC and H9 ESC, the MN-dependent contraction of myotubes was observed after 4 days of co-culture. The videos were recorded using Leica DMi8 microscope  were transfected with episomal DNAs expressing Oct4, Sox2, Klf4, Nanog, L-Myc and shRNA against p5329. By using a moderate number of samples (n = 11), we identified subtle molecular differences between hiPSCs and hESCs. Although using a higher number of samples might have minimized most of the previously mentioned variables, it might also contribute to loss of small but vital pathway differences8.In differential expression analysis and clustering, hiPSCs and hESCs were clearly segregated from HDFs indicating their distinct genetic background. Variables such as genetic background, and generation of iPSCs from a single donor have been reported to contribute to the magnification of genetic and epigenetic differences between hESCs and hiPSCsThe great majority of modulated (induced/repressed) genes between undifferentiated hiPSC and hESC lines displayed a similar expression profile that were obviously linked to common biological processes and pathways. A set of common induced , and repressed  genes were identified in hiPSCs and hESCs lines. Such observation could indicate the close similarities between the hiPSCs and hESCs lines where supervised clustering could not distinguish hESCs from hiPSCs. Collectively, these data support the view that the genetic background didn’t account for the previously reported transcriptional differences between hESCs and hiPSCs. The common genetic pool of induced/repressed genes between hiPSCs and hESCs could highlight the existence of a large degree of genetic matching between the two pluripotent cell lines. Moreover, the present results suggest that hESC and hiPSC lines are equivalent, and the remaining differentially expressed genes identified might represent transcriptional noise induced by other confounding factors but not the genetic background.30. In genetically matched hiPSCs and hESCs, a smaller number of differentially expressed genes were demonstrated24. These findings indicate that most of differentially expressed genes identified in the present study could be due to the use of genetically unmatched hiPSCs and hESCs. Moreover, the distinct overlap of the vast majority of induced/repressed genes between our examined hiPSCs and hESCs might provide a further support to our notion regarding potential equivalency and genetic matching between them.Our findings agree with previous studies which have identified similar sets of differentially expressed genes when comparing genetically unmatched hiPSCs and hESCs using a similar cutoff24. The undifferentiated nature of hiPSCs and hESCs makes it difficult to determine whether the absence of any functional outcome for induced/repressed genes is due to aberrant expression or defective post-transcriptional mechanisms, the latter appear to be the case with LDHA24.To further support our findings, no evidence for differences in functional outcome was identified after testing the potential functional outcomes of a group of deferentially expressed genes  These genes are known to play crucial roles in energy production or differentiation potential in neural cells or embryoid bodies, between genetically matched hiPSCs and hESCsWe have demonstrated that the Gene Ontogeny (GO) analysis of the top induced genes in hiPSCs and hESCs shows a significant enrichment of DNA metabolic process and M phase for both cell lines. Likewise, GO analysis (BP) of the top downregulated genes in both stem cell lines revealed significant enrichment of vesicle-mediated transport and establishment of protein localization.GO analysis (Pathway) of the upregulated genes in iPSCs revealed enrichment in hsa03030:DNA replication (24 genes), and hsa03040:Spliceosome (44 genes). GO analysis (Pathway) of top 20% of downregulated genes in hESCs revealed enrichment in s hsa04142:Lysosome (37 genes), and hsa04510:Focal adhesion (49 genes).24 further supports our interpretation regarding the similar genetic status for hiPSCs, and hESCs.Notably, the KEGG pathway analysis for repressed genes in hiPSCs, and hESCs, respectively disclosed the enrichment of the same KEGG pathways. These findings further supports our notion regarding the gene expression profiles similarity between hiPSCs, and hESCs. The fact that genetically unmatched hESC and hiPSC lines exhibit equivalent differentiation potentials using either a directed or spontaneous differentiation paradigmet al.31 who demonstrated that hiPSC lines generated with integrating viruses exhibit dramatic differences in expression, methylation and differentiation potential compared to hiPSC lines generated with non-integrating reprogramming systems31. Moreover, the genetic signature introduced by SeV infection did not separate hESCs from hiPSCs previously generated with retroviral or episomal vectors24.Our study results suggest that the use of non-integrating vectors for reprogramming of HDF into iPSCs may prevent the introduction of tumorigenic transcriptional alterations into iPSCs. These findings are in agreement with those of Ohnuki 24. Our data did not reveal any modulation in the gene expression level of LDHA and SLC2A1 in the examined hiPSCs/hESCs.Next we focused on a group of highly modulated genes between the iPSCs, and hESCs as a mean to explore potential functional differences between the two cell population. We focused on two of the genes involved in energy metabolism; LDHA and SLC2A1 , because of their previously reported strong basal expression in hESCs and reduced expression in all hiPSCs25. The potential differences in the gene we examined in the expression profiles of pluripotent markers between hiPSCs and hESCs focused on key pluripotency regulators . No induction/repression was demonstrated in the expression levels in any of these; a finding that might indicate their stable expression, and low variability in the strongly self-renewing fraction of hiPSCs and hESCs.Low variability and high connectivity contribute to highly conserved core processes common to all members of pluripotent cell populations32.The great majority of modulated (induced/repressed) genes between undifferentiated hiPSC and hESC lines displayed a similar expression profile that were obviously linked to common biological processes and pathways. A large set of common induced and repressed genes were identified in hiPSCs and hESCs lines. Such observation could indicate the close similarities between the hiPSCs and hESCs lines. Taken together, the present study suggests that differences in the gene expression between genetically unrelated hESC and hiPSC lines are primarily driven by other factors than the genetic background. Collectively, these data support the view that other parameters, such as reprogramming method, or gender might account for the majority of previously reported differences in the gene expression profile between hESCs and hiPSCs33. Whereas, the expression level of TACSTD1 was significant in MNs as compared to ESC, iPSC, and NPC. Tumor-associated calcium signal transducer (TACSTD1) is a stem/progenitor cell marker, which is also upregulated in several human carcinomas34. For the downregulated genes , all of them were significantly expressed in MNs as compared to iPSC, ESC, and NPC. The similar gene expression pattern during the differentiation of iPSC and ESC into NPC, and MNs as well as their similar functional assays as demonstrated by NMJ formation provide another evidence for their potential equivalence.The ability of iPSC and ESC to differentiate into NESTIN-NPC and ChAT-MNs was comparable; 97.36% vs 88.79% for NPC, and 85.29% vs. 93.39% for MNs, respectively. This might indicate similar neuronal differentiation potential. Moreover, similar pattern of gene expression profile were identified during ESC and iPSC differentiation into NPC and MNs. For the ten upregulated genes , the expression level of NNAT was significant in NPC as compared to ESC, iPSC, and MNs. Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Ca2 + signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNATAltogether, our findings prove that there is close similarity between genetically unmatched hiPSCs derived from HDFs and the “golden standard” pluripotent hESCs. Although the detailed molecular mechanisms underpinning such similarity are not fully understood, our work points out the importance of reprograming methods and their effects on the genetic background of the parental cells. Moreover, we identified similar pattern of gene expression profile during their differentiation into NPC and MNs. Such findings are crucial for the potential use of iPSCs in cellular based therapy of different neurological and neurodegenerative diseases, and disease modeling in which generation of hiPSCs using non-integrating programming methods from genetically unmatched iPSCs could be used for cellular based therapy; and in understanding the underpinning mechanisms associated with monogenic and polygenic diseases using genetically unmatched healthy and affected parental cell lines.We studied the differences between gene expression, molecular pathways, and motor cholinergic neurons differentiation potential between genetically unmatched hESC and hiPSC in order to highlight potential similarities/equivalence between the two cell lineages. At the gene expression and pathway level, hESC and hiPSC were very similar, but not identical. Pathway analysis of differentially expressed genes and assessment of cholinergic motor neurons differentiation potential disclosed not only enrichment of the same molecular pathways but also similar potential for differentiation into motor cholinergic neurons. Collectively, our data indicate that hESCs and hiPSCs are equivalent at the gene expression level, and their ability to differentiate into NPSCs and MNs, and that different genetic background of hiPSCs and hESCs does not confound their gene expression and neural differentiation potential.Video 1Video 2Supplementary Information


Triticum turgidum var dicoccoides) were found to store and release upon hydration active hydrolases including nucleases and chitinases. High nuclease activity was released upon hydration from glumes derived from wild strains of wheat including Triticum urartu and wild emmer wheat, while very low nuclease activity was detected in glumes derived from domesticated, free-threshing wheat cultivars . Germination from the intact dispersal unit of wild emmer wheat was delayed, but post germination growth was better than those of separated caryopses. Most notable was a significant increase in lateral root production on seedlings germinated from the intact dispersal unit. Proteome analysis of wild emmer wheat glumes revealed many proteins stored and released upon hydration including S1-type nucleases, peptidases, antifungal hydrolases such as chitinases and β-1,3-glucanase as well as pectin acetylesterase, a protein involved in cell wall degradation and remodeling. Also, reactive oxygen species (ROS)-detoxifying enzymes such as superoxide dismutase and ascorbate peroxidase were overrepresented in dead glumes of wild emmer wheat. Thus our study highlighted previously unknown features of the dispersal unit in wild wheat in which the dead, hardened floral bracts enclosing the caryopsis store active hydrolases and nutritional elements and probably growth promoting substances that facilitate seed longevity and germination and increase seedling vigor.It is commonly assumed that the dead, hardened floral bracts of the dispersal unit of grasses have been evolved to protect seeds from predation and / or assist in fruit/caryopsis dispersal. While these structures have important agronomical and economical implications, their adaptive value has not been fully explored. We investigated the hypothesis that the maternally derived hardened floral bracts have been evolved not just as a means for caryopsis protection and dispersal, but also as storage for substances that might affect seed germination and seedling vigor. Dead glumes as well as lemmas and paleas of wild emmer wheat ( Flowering plants (angiosperms) represent the most diverse group of land plants. They have evolved a plethora of strategies to increase fitness through extensive modification of their reproductive organs, influorescence and flower parts . SpeciesTriticum urartu is the progenitor of the AA genome of wild emmer wheat  as well as of the domesticated durum wheat , a tetraploid wheat derived from wild emmer wheat [Triticum aestivum AABBDD) was originated via hybridization of hulled tetraploid emmer wheat with Aegilops tauschii (genomes DD) resulting in hexaploid spelt wheat , from which free-threshing wheat evolved by mutations [Ae. tauschii, glumes are tenaciously enclosed the caryopses and their liberation often requires a strong mechanical force.Because the structures enclosing the caryopsis have important agronomical and economical implications, the domestication of wheat has occurred via genetic mutations that resulted in types of wheats with non-fragile rachises, soft glumes, and free-threshing caryopses ,4. Tritier wheat . It is wutations . In spelAe. kotschyi were shown to contain germination inhibitors that appear to function via controlling GA (gibberelin) metabolism [The dispersal unit of wild wheat is composed of the spikelet whereby the caryopsis is enclosed by dead, hardened parts of glumes, lemmas that carry owns and paleas. Tenacious glumes and other hardened floral bracts  of grasses are believed to function in seed dispersal and provide a physical shield protecting the seed from predation . The awntabolism . Yet, thWe investigated the hypothesis that the maternally-derived dead, hardened floral bracts of wild emmer wheat dispersal unit have been evolved not just as a means for caryopsis protection and dispersal but also as a storage for substances that might affect seed germination and seedling vigor. Our study provide novel insight on the function of the dead, hardened floral bracts enclosing the caryopsis showing it serves as a storage for active hydrolases and growth promoting substances that can increase seedling vigor and consequently establishment and survival in the niche.Triticum dicoccoides Köern.) and winter oat Avena sterilis were collected from their natural habitat in Israel, namely, Tabigha, Sea of Galilee on basalt soil (35°53’53” N 35°33’14” E) and road sides near Sede Boqer, in central Negev (34°47’20” N 34°51’01” E), respectively. Spikes of cultivated and wild strains of wheat grown in a net house were donated by Peleg Z. including T. aurartu, T. monococcum, T. timopheevii, T. aestivum and T. durum. All spikes were collected after they matured and stored at room temp for 1–2 years until used.Dispersal units of wild emmer wheat  for 75 min at 37°C. The gel was stained for 60–80 minutes with 10 mM Tris HCl pH 7.5 containing 2 μg/ml ethidium bromide and inspected under UV light.In gel nuclease assay was performed essentially as described  in polyaN-Glycosidase F  reactions were performed at 37°C for 2h in G7 Reaction Buffer according to the manufacturers’ protocol except that treatment with glycoprotein denaturing buffer was omitted.Peptide -4 (pH = 6) at 4°C for 8–12 h after which samples were centrifuged  and the supernatant was collected and used immediately or stored at -20°C until used. For in gel chitinase assay, 20 μl of the supernatant derived from caryopses or various floral bracts were first incubated in chitin sample buffer  for 1 h at 37°C and followed by separation on 12% SDS/PAGE containing 0.01% glycol chitin. The gel was incubated in buffer containing 100 mM sodium acetate (pH = 5.2) and 1% triton x-100 for 2 h at 37°C followed by staining for 5 min with 0.01% calcofluor white in 500 mM Tris-HCl (pH = 8.9). The gel was washed with distilled water for 1 h and visualized by UV transillumination.In gel chitinase assay was performed essentially as described . Brieflyhttps://www.graphpad.com/quickcalcs/).The germination assay was based on the ‘cigar roll’ method . Ten uniFor nutrient analysis, four dispersal units of wild emmer wheat were dissected into caryopses and floral bracts, namely, glumes, lemmas and paleas (GLPs) followed by incubation in 600 μl of ultrapure water (Milli-Q) for 12 hours on a cooled orbital shaker (4°C). Samples were centrifuged  and the supernatant was filtered (0.22 μm spin filter) and diluted with 4.8 ml of Milli-Q water and subjected to nutrient analysis. Three measurements were performed for each chemical element. The content of K, P, Ca, Mg, S and Zn released upon hydration from wild emmer wheat dispersal unit was determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) using ICP-720-ES . Soluble anions were detected and identified by Ion chromatography (IC). IC was performed by ICS-5000 instrument . Obtained data were analyzed by Chromeleon 6.8 chromatography data system .Ground glumes (10 mg) derived from different dispersal units  of wild emmer wheat grown in a greenhouse at were incubated in 100 μl of PBS at 4°C for 12 h followed by centrifugation . The supernatant containing proteins released from glumes was collected and subjected to proteomic analysis at The Smoler Protein Research Center at the Technion, Israel. Proteins released from glumes were digested with trypsin followed by separation and mass measurement by LC-MS/MS on Q-Exactive (Thermo) and identification by Discoverer 1.4  against Triticum from NCBI and Triticum from Uniprot databases and against Uniprot unspecific.Triticum and none from fungi. All the identified peptides were filtered with high confidence, top rank, mass accuracy, and a minimum of 2 peptides. High confidence peptides were passed the 1% FDR threshold . Semi-quantitation was done by calculating the peak area of each peptide. The area of the protein was calculated from the average of the three most intense peptides from each protein. Three replicates were performed for the analysis of wild emmer wheat glume proteins.In the Uniprot unspecific search we identified only proteins from A more stringent filtering was applied to the proteins, which are considered present in each plant. Considering that the average amino acid length of the proteins in the dataset was nearly 400 and assuming that 2 peptides are the minimum requirement for an average protein, we extrapolated this cutoff to consider protein length as the following: The minimum number of peptides per protein is the protein length divided by 200, but not less than 2 peptides. Additionally, the protein coverage by peptides from all samples must be higher than 30%. A protein is regarded as “present” in a plant if it is “present” in at least two replicates.T. aestivum protein names, their InterPro domains and gene ontology (GO) annotations were retrieved from UniProt. GO, GO slim and GO slim plant ontology files (in.obo format) were downloaded from the Gene Ontology web site (http://geneontology.org/page/download-ontology). GO enrichment analysis was carried out using BiNGO (v3.0.3) in Cytoscape (v3.4.0). All T. aestivum proteins from UniProt were used as the reference set. Over-representation analysis was performed using the hyper-geometric test, and the Benjamini-Hochberg FDR method was used for multiple testing correction. Proteome parameter definition is given in Functional annotation and enrichment analysis were performed as follows: Dispersal units of wild emmer wheat collected in the field  were sepTriticum species may have been lost in the course of domestication. Glumes and caryopses were obtained from spikelets collected from a net house-grown wild and cultivated varieties of wheats including diploid wheat Triticum urartu (AAu) and T. monococcum (AAm), tetraploid wild emmer wheat (BBAAu) and T. timopheevii (GGAAm), the domesticated tetraploid durum wheat  and two cultivated hexaploid wheat, T. aestivum (spelta) and T. aestivum (compactum). Intact caryopses and chopped glumes (derived from 3 spikelets) were incubated with PBS at 4°C and equal volume of extracts were analyzed for nuclease activities using in gel assays. In gel nuclease assays showed that all examined wheat lines possess various nuclease activities at positions of about 23, 35 and 37 kDa, which were mainly released from both caryopses and glumes in wild wheat species; in cultivated varieties nucleases were released mainly from glumes showing significant reduction in activity of 35 and 37 kDa nucleases compared to wild wheat varieties  whose dispersal unit is composed of florets (the rachilla disarticulate below the lowermost floret only), namely, caryopses enclosed by lemmas and paleas , Brachypodium distachyon (XP_003567956), Hordeum vulgare (BAJ95618), Oryza sativa (XP_015614478) and Zea mays (ACG43533). The S1/P1 type endonucleases were shown to undergo N-glycosylation that may affect their subcellular localization and activity [A 36 kDa nuclease activity was recovered in glume extracts by in gel nuclease assays using either denatured salmon sperm DNA or yeast torula RNAs (not shown) leading to the hypothesis that this activity may be related to S1/P1 type endonucleases. First, S1/P1 type endonucleases are bifunctional capable of cleaving both RNA and single stranded DNA and second the 36 kDa nuclease released from glumes has nearly the predicted molecular mass of S1-type endonucleases identified in various monocots species including a XP_0156478 and Zactivity  resultedAspegillus S1 nuclease and mung bean nuclease are widely used in molecular biology applications and although their preferred substrate is single stranded nucleic acids they are capable of introducing nicks and double strand DNA breaks (DSBs) into supercoiled plasmid DNA converting it to relaxed and/or linear forms—a well established method for monitoring single-stranded DNA endonuclease activity [2+/Mg2+ cations as cofactors. Indeed nucleases released from glumes of emmer wheat were capable of converting supercoiled plasmid DNA (SC) into relaxed (R) and linear (L) forms  forms . Thus, nTo assess the biological significance of germination from the dispersal unit, we germinated wild emmer wheat either as a naked caryopsis where lemmas, paleas and glumes were removed or as intact dispersal unit. We used the ‘cigar roll’ system on standard germination papers soaked in distilled water and placed in a growth room. Notably, the caryopsis alone was germinated faster than the intact dispersal unit with a delay of about 4 days. However, after germination, seedlings that emerged from the dispersal unit grew and developed better than seedlings that emerged from the caryopsis alone. Accordingly, DU seedlings had higher average length of the first leaf  and of t42-) and chloride (Cl-) but also nitrate (NO3-), while very low levels were released from caryopses following hydration.The finding that seedlings derived from intact DU perform better than seedlings derived from the caryopsis suggests that the dead bracts enclosing the caryopsis  contribute substances such as nutrients that promote seedling vigor. Nutrient composition and content released from dead bracts of the DU were compared with those released from the caryopsis. Accordingly, four DUs of wild emmer wheat were separated into caryopses (Caryo) and glumes, lemmas and paleas (GLPs) and incubated in 0.8 ml of PBS at 4°C for 12 h, the sup was collected and analyzed by ICP-OES for detection of micro- and macro-elements. The results showed  that GLPTriticum from Uniprot databases  followed by separation on LC-MS/MS on LTQ-Orbitrap and identification by Discoverer software against the atabases . Notablyentified . Functioentified  that amoentified . Moleculentified  showed tEurotia lanata, Chenopodiaceae), the fruit  or the fruit with attached structures, such as the dispersal unit of various grasses, which are composed of the whole spikelet (wild emmer wheat) or the florets (Avena sterilis). Although the term dispersal unit highlights an entity that is specialized for dispersal it has been previously shown that the dispersal unit has evolved to fulfill multiple functions that contribute to seedling establishment including dispersal, embryo protection, seed positioning and orientation, seed fixation, water uptake and respiration [Triticum and Avena species, which have not been recognized previously. These include the finding that dead floral bracts of the dispersal unit  function as storage for active hydrolases that are released upon hydration to the ‘seedsphere’ and that seedling vigor is enhanced when germinated from the whole dispersal unit compared to the thrashed seed. In addition, out data showed that the dead floral bracts of the dispersal unit of wild emmer wheat function as a nutritional storage that might provide immediate supply of necessary nutrients at early stages of germination.Angiosperm evolution has led to the development of diverse fruit morphologies, which is believed to assist seed dispersal . In angipiration . Data prClavibacter michiganensis subsp. michiganensis, a Solanaceae species-pathogenic Gram-positive actinomycete contains two plasmids, designated pCM1 and pCM2, which are important for its pathogenic activity, inasmuch as plasmid-free derivative CMM100 can colonize tomato, but showing no disease symptoms [Phytophthora parasitica [It is commonly believed that during programmed cell death (PCD), macromolecules such as proteins, DNA and RNA are degraded and their constituents remobilized into other parts of the plant . This hysymptoms . Targetirasitica ,27, a oorasitica . In-gel Avena sterilis and in domesticated, free-threshing durum wheat.In gel assays also revealed active chitinases that could potentially act against pathogens. Chitinases are enzymes that degrade chitin an abundant polysaccharide found in a variety of organisms including insects, fungi, yeast, and algae. Chitinase and glucanase genes were often over-expressed in plants to confer resistance against fungal pathogens –31. Thus2−) radical into either molecular oxygen (O2) or hydrogen peroxide (H2O2), as well as catalase and ascorbate peroxidase or thiol-based proxidases (PRXs) that detoxify H2O2 [Overrepresentation of proteins involved in oxidation-reduction processes may demonstrate their importance in seed biology ,33. Geneify H2O2 .Of particular interest was the biological effect of the dead hardened floral bracts on seedling vigor. In all germination experiments, the intact dispersal unit (DU) showed delay in germination time, that is, protrusion of roots, compared to the naked caryopsis. This is probably because the dead hardened floral bracts enclosing the caryopsis pose a physical barrier for root emergence. Indeed, the study of germination of various Brassicaceae species having indehiscent fruits suggested that the pericarp plays a dominant role in seed dormancy by mechanically restricting embryo growth . Thus, tHordeum vulgare) showed that local supply of nitrate or phosphate stimulated lateral root growth [via changing auxin distribution [Notably, post germination growth and development were enhanced in seedlings derived from the intact DU. Particularly, seedlings derived from the DU have significantly higher number and higher length of lateral roots than seedlings derived from naked caryopsis. Similar effects were reported for germination of the winterfat dispersal unit, which consists of hairy bracts enveloping utricle (fruit) and the seed, which is composed of testa, embryo and perisperm (nutritive tissue made from the nucellus). Accordingly, the removal of the hairy bracts significantly reduced seedling establishment and vigor ,41. Enhat growth . Multiplt growth ,47. Alsoribution .The dispersal units of Poaceae species often composed of the maternally-derived dead, hardened floral bracts that enclose the caryopsis have been evolved not only for protecting caryopses from predation or as a means for seed dispersal and placement in the ground but also as an active entity that provide defense, growth regulatory factors and probably nourishing substances that assist germination and promote seedling vigor and consequently seedling survival in the niche. This appears to be a general phenomenon in plant seed biology whereby dead organs enclosing embryos including glumes in grasses and seed coats in dicotyledonous species such as Brassicaceae and Fabaceae species  functionS1 File(PDF)Click here for additional data file.S1 TableProteome raw data of all proteins released from dead glumes of wild emmer wheat.(XLSX)Click here for additional data file.S2 TableList of proteins released from dead glumes of wild emmer wheat after filtering.(XLSX)Click here for additional data file.S3 TableGO term classification.(XLSX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors.Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A Traumatic injury to the spinal cord, as well as congenital conditions such as spina bifida, can result in diminished urinary tract function. In such patients, the bladder commonly displays reduced capacity, poor compliance and overactivity. In addition detrusor-sphincter dyssynergia (DSD) – a loss of coordination between detrusor smooth muscle contraction and urethral sphincter relaxation – leads to high storage and voiding pressures, and subsequent bladder wall remodelling. Management of neurogenic detrusor overactivity (NDO) in patients with spinal cord injury (SCI) emphasises catheterisation to overcome DSD and achieve bladder emptying, together with medication to reduce non-voiding contractions and improve compliance . Taken together, these findings suggest that inosine can act locally within the bladder via the adenosine A2B receptor to inhibit non-voiding contractions and spontaneous activity, and represents a novel treatment for neurogenic detrusor overactivity.The aim of this study was to investigate the role of adenosine receptor signalling in the ability of inosine to attenuate detrusor overactivity in the context of SCI, and to explore local action of inosine in the bladder. Intravesical application of anti-muscarinic treatment with oxybutynin has recently demonstrated benefit in patients due to fewer adverse reactions compared to systemic administration13Our previous study demonstrated significant improvements in NDO in SCI rats receiving chronic systemic treatment with inosineNext, we determined the impact of inosine on spontaneous activity (SA) of isolated bladder tissues. The amplitude and frequency of SA that occurred at low and high frequency was determined as illustrated in We employed a panel of pharmacological antagonists to determine the role of adenosine receptors in inosine-induced inhibition of SA. For these experiments inosine at a dose of 1 mM was used, and we confirmed that solvents used to prepare antagonists had no effect on SA prior to testing . As abov1 receptor-specific antagonist PSB36 (0.1 μM) did not prevent the ability of inosine to attenuate the amplitude of either low frequency SA  or high frequency SA . Similarly, MRS3777 (0.1 μM), a specific antagonist for the A3 receptor, did not prevent the effect of inosine on low frequency SA  inhibited the ability of inosine to attenuate the amplitude of low frequency SA  (2B antagonist PSB603 (0.1 μM) was able to reverse the inhibitory effect of inosine on the amplitude of both low frequency SA  . Similar, n = 7) . The fre, n = 7) . The A2B3, n = 7) and high3, n = 7). The fre3, n = 7).2 agonists could mimic the effect of inosine on SA. The adenosine receptor agonist NECA reduced the tone  suggesting that these channels do not mediate the effect of inosine on SA. In contrast, IBTX (100 nM), an inhibitor of the large conductance BK channel blocked the inhibitory effect of inosine on the amplitude of low frequency SA  identification of BK channels as effectors of inosine-induced inhibition of SA. Together, these findings suggest that acute administration of inosine attenuates neurogenic DO via an A2B adenosine receptor–BK channel-mediated pathway.In this study, we investigated the effects of inosine delivery to the bladder on NDO and explored the underlying signalling events. Our findings demonstrate (i) an improvement in neurogenic detrusor overactivity in SCI rats following intravesical inosine administration as evidenced by a significant reduction in the frequency and amplitude of non-voiding contractions; (ii) reduced spontaneous activity in detrusor muscle strips following acute exposure to inosine; (iii) identification of the Ain vitro, that is likely to be relevant in vivo. Comparison of the activity of inosine on muscle strips with and without mucosa revealed a comparable reduction of spontaneous activity over a similar dose range. While we cannot rule out the possibility that inosine promotes the release of urothelial factors that influence spontaneous activity, our observations from muscle strips without mucosa demonstrate that inosine acts directly on the detrusor to reduce overactivity. Thus, inosine can act through distinct mechanisms to improve NDO under both chronic and acute treatment regimens.Our previous study showed that chronic treatment with inosine led to attenuation of detrusor overactivity, and was associated with alteration in mediators of sensory neurotransmissionin vitro is thought to underlie spontaneous contractile activity that occurs during the filling phase in various species, including humans  family2B adenosine receptors that impinges on large conductance BK channels. These findings suggest that inosine may be a novel intravesical therapeutic agent in the setting of neurogenic detrusor overactivity.In summary, this study demonstrates an important role for acute, local action of inosine in attenuation of neurogenic detrusor overactivity. Inosine inhibits spontaneous activity in bladder tissue from SCI rats through a mechanism mediated by AThese studies and methods were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental protocols were approved by the Animal Care and Use Committee of Boston Children’s Hospital (protocol #13-09-2501 R/16-08-3256 R).TM) was placed between the two cut ends of the spinal cord to aid in haemostasis; the dura was not closed. The paraspinal muscles and skin were closed in two separate layers. Post-operative pain was managed with meloxicam analgesia . every 24 h for 3 d). Rats received Baytril antibiotic prophylaxis  during the post-operative period. During the period characterised by bladder areflexia or spinal shock, bladders were emptied every 12 h by manual expression until reflex voiding returned. This period lasted for 10 d to 2 wk. Rats were monitored and weighed on a daily basis. At 6.5 weeks, cohorts of rats were subjected to either in vivo cystometry or bladder contractility testing. Selected tissues were also used for molecular analysis.Male Sprague Dawley rats  were subjected to mid-thoracic spinal cord transection essentially as described2O  that did not result in a void. Bladder capacity was defined as the sum of the voided volume and post void residual volume. Voiding efficiency was defined as the percentage of the bladder capacity that was voided. Data from 2–3 voiding cycles per condition were averaged from each animal and included analysis from both the storage and emptying phases. The amplitude and frequency of non-voiding contractions, compliance, micturition interval, voided volumes, voiding pressure (mid-void), and maximum micturition pressure were determined. At the end of analysis, rats were euthanised via CO2 inhalation.The storage and emptying abilities of the bladder were assessed 6.5 wk after SCI in a subset of rats (n = 6) using conscious cystometry, essentially as described2 inhalation and preserved in ice-cold Kreb’s buffer  for ex vivo contractility analyses. The bladder was opened longitudinally and its base removed by cutting above the ureteral orifices. Tissue strips with intact mucosa were cut from the remaining bladder segment, mounted in tissue chambers maintained at 37 °C and bubbled with a mixture of 95% O2 and 5% CO2. Bladder tissue was attached to a force transducer (Grass Instruments) and stretched to 1.5 grams. Following an equilibration period of at least 45 min, the functional effects of pharmacological agents  on spontaneous activity were measured. Concentrations of pharmacological agents were either determined empirically or derived from the literature. Data were expressed as force (mN) normalised by tissue cross-sectional area  and presented as mean ± SEM. SA after SCI is characterised by low amplitude, high frequency contractions superimposed on large amplitude phasic contractions that occur with low frequency  were harvested following euthanasia via COrequency . These t2A and A2B, and relative mRNA levels were determined following normalisation to GAPDH.RNA was isolated from mucosa alone and from bladder strips without mucosa  using TRIzol reagent along with the RNeasy MiniKit (Qiagen). RNA was reverse-transcribed using a high-capacity cDNA synthesis kit  according to the manufacturer’s instructions. cDNAs were amplified using gene-specific assays for Ain vitro analysis of SA, differences in the amplitude of low and high frequency SA were compared between baseline conditions and after exposure to inosine, and between inhibitor treatment and inosine in the presence of inhibitor, using paired Student’s t-test, with p < 0.05 considered signficant. The effect of inosine on untreated and antagonist-treated tissues was determined in separate tissue strips as the effects of inosine are not completely reversible. For experiments in which pharmacological agents had little effect on baseline, data were expressed as stress (force (mN)/tissue cross-sectional area) normalised by baseline responses and presented as mean ± SEM. For experiments using potassium channel inhibitors, which had a profound effect on baseline responses, data were normalised to responses with drug alone. For dose response curves, repeated measures ANOVA test was used followed by a Tukey post-hoc test.For cystometry, parameters from each animal were averaged over 3 voiding cycles at baseline and in the presence of inosine. These included the amplitude and frequency of non-voiding contractions, compliance, micturition interval, voided volumes, voiding pressure, and maximum pressure. Means ± SEM calculated from all animals under baseline conditions were compared to those following inosine treatment using a paired t-test. p < 0.05 was considered significant. For 2A (Rn00583935_m1), A2B (Rn00567697_m1) and GAPDH (Rn01775763_g1) were from Life Technologies .Reagents were purchased from suppliers as indicated: Inosine ; CGS15943, MRS3777, PSB36, PSB603, ZM241385, PSB0777, NECA, BAY60-6583, repaglinide, iberiotoxin and apamin . Inosine was dissolved in distilled water. CGS15943, MRS3777, PSB36, PSB603, ZM241385, PSB0777, NECA, BAY60-6583, iberiotoxin and apamin were dissolved in DMSO and repaglinide was dissolved in ethanol. Exposure of tissues to vehicle alone did not alter spontaneous activity . AntagonHow to cite this article: Doyle, C. et al. Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway. Sci. Rep.7, 44416; doi: 10.1038/srep44416 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) are cytokines of particular interest in oncology from the perspective of neutropenia management (Mehta et al., 2015 The data presented here is among the first to show a relationship between pre-existing use of injectable insulin in women diagnosed with breast cancer and type 2 diabetes mellitus, hematopoietic cytokine profiles at time of breast cancer diagnosis, and subsequent cancer outcomes. A Pearson correlation analysis evaluating the relationship between G-CSF, GM-CSF, and IL-7 stratified by insulin use, controls, as well as by estrogen and progesterone receptor status is also provided. Specifications TableValue of the data•This dataset represents the observed relationship between injectable insulin use, hormone receptor status, circulating hematopoietic cytokines at breast cancer diagnosis, and outcomes•Reported data has the potential to guide future studies evaluating insulin-regulated hematopoiesis modulation in breast cancer as well as studies investigating the mechanism responsible to hormone receptor phenotype•Our observations can assist further research clarifying the role of insulin in hematopoietic differentiation and biomarker signaling related to hormone receptor status•This evidence builds on the existing cross-talk exploring the interaction between hematopoiesis and cancer1Reported data represents the observed association between use of injectable insulin preceding breast cancer and the hematopoietic cytokine profiles at the time of cancer diagnosis in women with diabetes mellitus . Data in2Evaluation of hematopoietic cytokine profile association with injectable insulin use and BC outcomes was carried out under two protocols approved by both Roswell Park Cancer Institute (EDR154409 and NHR009010) and the State University of New York at Buffalo (PHP0840409E). Demographic and clinical patient information was linked with cancer outcomes and hematopoietic cytokine profiles of corresponding plasma specimen harvested at BC diagnosis and banked in the Roswell Park Cancer Institute Data Bank and Bio-Repository.2.1All incident breast cancer cases diagnosed at Roswell Park Cancer Institute (01/01/2003-12/31/2009) were considered for inclusion (n=2194). Medical and pharmacotherapy history were used to determine the baseline presence of diabetes.2.2All adult women with pre-existing diabetes at breast cancer diagnosis having available banked treatment-naïve plasma specimens  in the Institute׳s Data Bank and Bio-Repository were included.Subjects were excluded if they had prior cancer history or unclear date of diagnosis, incomplete clinical records, type 1 or unclear diabetes status. For a specific breakdown of excluded subjects, please see the original research article by Wintrob et al. A total of 97 female subjects with breast cancer and baseline diabetes mellitus were eligible for inclusion in this analysis.2.3Each of the 97 adult female subjects with breast cancer and diabetes mellitus (defined as “cases”) was matched with two other female subjects diagnosed with breast cancer, but without baseline diabetes mellitus (defined as “controls”). The following matching criteria were used: age at diagnosis, body mass index category, ethnicity, menopausal status and tumor stage (as per the American Joint Committee on Cancer). Some matching limitations applied 2.4Clinical and treatment history was documented as previously described 2.5All the plasma specimens retrieved from long-term storage were individually aliquoted in color coded vials labeled with unique, subject specific barcodes. Overall duration of freezing time was accounted for all matched controls ensuring that the case and matched control specimens had similar overall storage conditions. Only two instances of freeze-thaw were allowed between biobank retrieval and biomarker analyses: aliquoting procedure step and actual assay.2.6A total of 3 biomarkers  were quantified according to the manufacturer protocol. The HCYTOMAG-60K Luminex® biomarker panel  was utilized in this study. Interferon α2 and γ determinations were done according to the manufacturer protocol as reported in our dataset focusing on Th1/Th2 cytokines׳ determinations 2.7Biomarker cut-point optimization was performed for each analyzed biomarker. Biomarker levels constituted the continuous independent variable that was subdivided into two groups that optimized the log rank test among all possible cut-point selections yielding a minimum of 10 patients in any resulting group. Quartiles were also constructed. The resultant biomarker categories were then tested for association with type 2 diabetes mellitus therapy and controls by Fisher׳s exact test. The continuous biomarker levels were also tested for association with diabetes therapy and controls across groups by the Kruskall-Wallis test and pairwise by the Wilcoxon rank sum. Multivariate adjustments were performed accounting for age, tumor stage, body mass index, estrogen receptor status, and cumulative comorbidity. The biomarker analysis was performed using R Version 2.15.3. Please see the original article for an illustration of the analysis workflow Correlations between biomarkers stratified by type 2 diabetes mellitus pharmacotherapy, controls, and hormone receptor status were assessed by the Pearson method. Correlation models were constructed both with and without adjustment for age, body mass index, and the combined comorbidity index. The correlation stratification by hormone receptor status excluded 3 subjects that were estrogen receptor negative but progesterone receptor positive due to insufficient numbers to compute confidence intervals and correlation significance. Correlation analyses were performed using SAS Version 9.4.Wadsworth Foundation Peter Rowley Breast Cancer Grant awarded to A.C.C. .This research was funded by the following grant awards:


The aim of this study was to evaluate the validity and the inter- and intra-examiner reliability of panoramic-radiograph-driven findings of different maxillary sinus anatomic variations and pathologies, which had initially been prediagnosed by cone beam computed tomography (CBCT).After pairs of two-dimensional (2D) panoramic and three-dimensional (3D) CBCT images of patients having received treatment at the outpatient department had been screened, the predefinition of 54 selected maxillary sinus conditions was initially performed on CBCT images by two blinded consultants individually using a questionnaire that defined ten different clinically relevant findings. Using the identic questionnaire, these consultants performed the evaluation of the panoramic radiographs at a later time point. The results were analyzed for inter-imaging differences in the evaluation of the maxillary sinus between 2D and 3D imaging methods. Additionally, two resident groups (first year and last year of training) performed two diagnostic runs of the panoramic radiographs and results were analyzed for inter- and intra-observer reliability.There is a moderate risk for false diagnosis of findings of the maxillary sinus if only panoramic radiography is used. Based on the ten predefined conditions, solely maxillary bone cysts penetrating into the sinus were frequently detected differently comparing 2D to 3D diagnostics. Additionally, on panoramic radiographs, the inter-observer comparison demonstrated that basal septa were significantly often rated differently and the intra-observer comparison showed a significant lack in reliability in detecting maxillary bone cysts penetrating into the sinus.Panoramic radiography provides the most information on the maxillary sinus, and it may be an adequate imaging method. However, particular findings of the maxillary sinus in panoramic imaging may be based on a rather examiner-dependent assessment. Therefore, a persistent and precise evaluation of specific conditions of the maxillary sinus may only be possible using CBCT because it provides additional information compared to panoramic radiography. This might be relevant for consecutive surgical procedures; consequently, we recommend CBCT if a precise preoperative evaluation is mandatory. However, higher radiation dose and costs of 3D imaging need to be considered. The development of two-dimensional 2D) panoramic imaging techniques began in the first half of the 20th century, but the first device applying this technology was only described in 1959 [D panoramThe advantages of three-dimensional 3D) CBCT over 2D conventional panoramic tomography include an excellent imaging quality of high-contrast structures like the maxillofacial bone anatomy, no geometric distortion, and no superimposition of surrounding anatomic structures D CBCT ov. HoweverPrecise assessment of the maxillary sinus is mandatory when planning a lateral or internal sinus floor elevation , 8. It hTherefore, the present study had three aims: the evaluation of the validity (1), the inter- (2), and the intra-examiner (3) reliability of panoramic-radiograph-driven findings of different maxillary sinus conditions which had initially been prediagnosed in CBCT images.The study protocol was approved by the Eidgenössische Expertenkommission für das Berufsgeheimnis in der medizinischen Forschung . All patients have consented to provide their data for research and publication. Prior to this study, a statistical study design was performed, and in a retrospective approach, radiographic images of patients having received treatment at the outpatient department of the Clinic of Cranio-Maxillofacial and Oral Surgery at the University of Zurich were screened. Inclusion criteria were patients having received 2D panoramic radiographs  and 3D CBCT  showing the maxillary sinus without any surgical procedure in between the two examinations . Subsequently, possible clinically relevant anatomic variations and pathologies of the maxillary sinus were predefined   and a monitor with a display resolution of 1680 × 1050 pixel. Image manipulation through change of brightness, zoom in and out, and rotation was used when needed. No time limit was appropriated, and all observations were performed in the same room under comparable light conditions. Findings of the maxillary sinus were purely based on radiographic appearance without using any other additional clinical or histological information. A questionnaire served for recording the diagnoses  were used: voxel edge length 0.4 × 0.4 × 0.4 mm, slice thickness 1 mm. The two assessors were blinded—disagreement was solved by discussion.The CBCT assessment served as a reference group for later 2D panoramic image diagnostics which were performed separately by the same two consultants more than 6 months after predefinition on CBCT images. An identical questionnaire was used for both 3D and later 2D diagnostics. Inter-imaging technique differences in evaluating the maxillary sinus with 2D or 3D imaging method were analyzed with regard to false-positive and false-negative decisions.Using the same questionnaire, four blinded residents separately performed the evaluation of the 2D panoramic radiographs—two in the first year and two in the last year of training. Agreement between these two rating groups in detecting findings in the maxillary sinus was analyzed for calculating inter-observer reliability.After more than 4 weeks, the same residents newly evaluated all maxillary sinuses on 2D panoramic images. Reassessment served for the calculation of the intra-observer reliability, which means individual agreement between the two elevation runs.The results were further evaluated with regard to the prevalence of radiographic findings in the maxillary sinus.p values were calculated using a chi-squared test. Kappa coefficient and McNemar’s test were used for evaluating the reliability between the two consultants analyzing the CBCT scans. Calculated p values were considered significant for values <0.05. Descriptive statistics computed means and standard deviation for quantitative variables as well as absolute and relative frequencies for qualitative variables.Data was recorded using Excel 2013 (Microsoft) and analyzed using IBM SPSS Statistics for Macintosh, Verion 22.0 . Collected data was analyzed to demonstrate degree of agreement using the following statistical tests: logistic regression was used to estimate odds ratios. This study analyzed comparative and descriptive data for the evaluation of the maxillary sinus. Significant differences in the detection of ten predefined findings between 2D and 3D imaging methods were calculated. Furthermore, the degree of agreement in detecting the ten conditions on the panoramic radiographs was measured between different observers and within the same observer. The radiographic images of 28 patients  were examined. One patient had to be excluded from the study as the radiographic finding on the CBCT did not clearly match any of the predefined ten conditions.p = 0.803, odds ratio (OR) = 1.220). The difference between the two imaging methods was significant solely for maxillary bone cyst penetrating into the sinus (p = 0.032). The estimated OR of this specific finding was significantly lower than 1 (OR = 0.275). No significant differences between 2D and 3D imaging methods were found for the detection of a complete opacity (p = 0.998), a basal opacity (p = 0.714), a foreign body (p = 0.571), an oro-antral communication (p = 0.998), a basal septum (p = 0.911), a polypoid mucosal thickening (0.123), a fluid level (p = 0.253), and a status post sinus lift (p = 0.998) (Table As illustrated by Table p > 0.05).Good inter-observer reliability  between p = 0.004, OR = 0.542). Further, it was shown that there is a significant good inter-observer agreement in detecting complete opacities   examining 2D panoramic images showed that basal septa were significantly often rated differently by these two groups  in diagnosing maxillary bone cysts penetrating into the sinus , followed by basal opacities 43 %), and foreign bodies (15 %) , and 3D CT in the diagnosis of mucosal thickening, mucous cysts, or complete opacity when using implant-planning software and showed that panoramic radiography was comparatively inferior . MaestreThe inter-observer disagreement between the two resident groups (first-year vs. last-year residents) examining 2D panoramic images was significant in the detection of the basal septa. As mentioned above, Maestre-Ferrin et al. have already indicated that panoramic radiography is insufficient for the detection of sinus septa , which cThere was only little intra-observer variation. The literature shows that the intra- and inter-examiner variation in the interpretation of radiographs may exceed the variation of imaging techniques and diagnostic yield , 18, 19.One limitation of the latter and also of the present study may be the relatively small number of raters. Another limitation of this study may be the prevalence imbalance of different findings in the maxillary sinus resulting in a negative impact on our statistical calculation. This might include not only the low prevalence of maxillary bone cysts penetrating into the sinus but also the high prevalence of basal septa, an imbalance former studies have already demonstrated , 21, 22.An explanation for our reported findings may be that, due to the superimposition of different structures, low spatial resolution and visual loss of cortical plates or undulating concavities, precise evaluation of a maxillary sinus finding is difficult in 2D panoramic radiography , 11. Moras low as reasonably achievable) [Undiagnosed sinus conditions may be associated with chronic orofacial pain that is one of the most common reasons why patients consult physicians . Moreoveievable) . This stThe results of this study emphasize that panoramic radiography visualizes relevant findings of the maxillary sinus. In comparison to panoramic radiography, CBCT facilitates diagnosis of special conditions like penetrating cysts. The inter-observer comparison on panoramic radiographs demonstrated that basal septa were significantly often rated differently, and panoramic imaging may be based on a rather examiner-dependent assessment. Supplementary, the detection of maxillary bone cysts penetrating into the sinus with panoramic radiography showed a significant lack in reliability in the intra-observer comparison. Therefore, precise preoperative evaluation of the maxillary sinus on panoramic radiographs may be difficult. This could be relevant for consecutive surgical procedures; however, higher radiation dose and costs of three-dimensional imaging need to be considered.