Development of a temperature-controlled phantom for magnetic resonance quality assurance of diffusion, dynamic, and relaxometry measurements.

Purpose Diffusion-weighted (DW) and dynamic contrast-enhanced magnetic resonance imaging (MRI) are increasingly applied for the assessment of functional tissue biomarkers for diagnosis, lesion characterization, or for monitoring of treatment response. However, these techniques are vulnerable to the influence of various factors, so there is a necessity for a standardized MR quality assurance procedure utilizing a phantom to facilitate the reliable estimation of repeatability of these quantitative biomarkers arising from technical factors (e.g., B1 variation) affecting acquisition on scanners of different vendors and field strengths. The purpose of this study is to present a novel phantom designed for use in quality assurance for multicenter trials, and the associated repeatability measurements of functional and quantitative imaging protocols across different MR vendors and field strengths.Methods A cylindrical acrylic phantom was manufactured containing 7 vials of polyvinylpyrrolidone (PVP) solutions of different concentrations, ranging from 0% (distilled water) to 25% w/w, to create a range of different MR contrast parameters. Temperature control was achieved by equilibration with ice-water. Repeated MR imaging measurements of the phantom were performed on four clinical scanners (two at 1.5 T, two at 3.0 T; two vendors) using the same scanning protocol to assess the long-term and short-term repeatability. The scanning protocol consisted of DW measurements, inversion recovery (IR) T1 measurements, multiecho T2 measurement, and dynamic T1-weighted sequence allowing multiple variable flip angle (VFA) estimation of T1 values over time. For each measurement, the corresponding calculated parameter maps were produced. On each calculated map, regions of interest (ROIs) were drawn within each vial and the median value of these voxels was assessed. For the dynamic data, the autocorrelation function and their variance were calculated; for the assessment of the repeatability, the coefficients of variation (CoV) were calculated.Results For both field strengths across the available vendors, the apparent diffusion coefficient (ADC) at 0 °C ranged from (1.12 ± 0.01) × 10(-3) mm(2)/s for pure water to (0.48 ± 0.02) × 10(-3) mm(2)/s for the 25% w/w PVP concentration, presenting a minor variability between the vendors and the field strengths. T2 and IR-T1 relaxation time results demonstrated variability between the field strengths and the vendors across the different acquisitions. Moreover, the T1 values derived from the VFA method exhibited a large variation compared with the IR-T1 values across all the scanners for all repeated measurements, although the calculation of the standard deviation of the VFA-T1 estimate across each ROI and the autocorrelation showed a stability of the signal for three scanners, with autocorrelation of the signal over the dynamic series revealing a periodic variation in one scanner. Finally, the ADC, the T2, and the IR-T1 values exhibited an excellent repeatability across the scanners, whereas for the dynamic data, the CoVs were higher.Conclusions The combination of a novel PVP phantom, with multiple compartments to give a physiologically relevant range of ADC and T1 values, together with ice-water as a temperature-controlled medium, allows reliable quality assurance measurements that can be used to measure agreement between MRI scanners, critical in multicenter functional and quantitative imaging studies

Supervised Machine-Learning Enables Segmentation and Evaluation of Heterogeneous Post-treatment Changes in Multi-Parametric MRI of Soft-Tissue Sarcoma.

Background: Multi-parametric MRI provides non-invasive methods for response assessment of soft-tissue sarcoma (STS) from non-surgical treatments. However, evaluation of MRI parameters over the whole tumor volume may not reveal the full extent of post-treatment changes as STS tumors are often highly heterogeneous, including cellular tumor, fat, necrosis, and cystic tissue compartments. In this pilot study, we investigate the use of machine-learning approaches to automatically delineate tissue compartments in STS, and use this approach to monitor post-radiotherapy changes. Methods: Eighteen patients with retroperitoneal sarcoma were imaged using multi-parametric MRI; 8/18 received a follow-up imaging study 2-4 weeks after pre-operative radiotherapy. Eight commonly-used supervised machine-learning techniques were optimized for classifying pixels into one of five tissue sub-types using an exhaustive cross-validation approach and expert-defined regions of interest as a gold standard. Final pixel classification was smoothed using a Markov Random Field (MRF) prior distribution on the final machine-learning models. Findings: 5/8 machine-learning techniques demonstrated high median cross-validation accuracies (82.2%, range 80.5-82.5%) with no significant difference between these five methods. One technique was selected (Naïve-Bayes) due to its relatively short training and class-prediction times (median 0.73 and 0.69 ms, respectively on a 3.5 GHz personal machine). When combined with the MRF-prior, this approach was successfully applied in all eight post-radiotherapy imaging studies and provided visualization and quantification of changes to independent STS sub-regions following radiotherapy for heterogeneous response assessment. Interpretation: Supervised machine-learning approaches to tissue classification in multi-parametric MRI of soft-tissue sarcomas provide quantitative evaluation of heterogeneous tissue changes following radiotherapy

Utility of Multi-Parametric Quantitative Magnetic Resonance Imaging for Characterization and Radiotherapy Response Assessment in Soft-Tissue Sarcomas and Correlation With Histopathology.

Purpose: To evaluate repeatability of quantitative multi-parametric MRI in retroperitoneal sarcomas, assess parameter changes with radiotherapy, and correlate pre-operative values with histopathological findings in the surgical specimens. Materials and Methods: Thirty patients with retroperitoneal sarcoma were imaged at baseline, of whom 27 also underwent a second baseline examination for repeatability assessment. 14/30 patients were treated with pre-operative radiotherapy and were imaged again after completing radiotherapy (50.4 Gy in 28 daily fractions, over 5.5 weeks). The following parameter estimates were assessed in the whole tumor volume at baseline and following radiotherapy: apparent diffusion coefficient (ADC), parameters of the intra-voxel incoherent motion model of diffusion-weighted MRI (D, f, D*), transverse relaxation rate, fat fraction, and enhancing fraction after gadolinium-based contrast injection. Correlation was evaluated between pre-operative quantitative parameters and histopathological assessments of cellularity and fat fraction in post-surgical specimens (ClinicalTrials.gov, registration number NCT01902667). Results: Upper and lower 95% limits of agreement were 7.1 and -6.6%, respectively for median ADC at baseline. Median ADC increased significantly post-radiotherapy. Pre-operative ADC and D were negatively correlated with cellularity (r = -0.42, p = 0.01, 95% confidence interval (CI) -0.22 to -0.59 for ADC; r = -0.45, p = 0.005, 95% CI -0.25 to -0.62 for D), and fat fraction from Dixon MRI showed strong correlation with histopathological assessment of fat fraction (r = 0.79, p = 10-7, 95% CI 0.69-0.86). Conclusion: Fat fraction on MRI corresponded to fat content on histology and therefore contributes to lesion characterization. Measurement repeatability was excellent for ADC; this parameter increased significantly post-radiotherapy even in disease categorized as stable by size criteria, and corresponded to cellularity on histology. ADC can be utilized for characterizing and assessing response in heterogeneous retroperitoneal sarcomas

Extracranial Soft-Tissue Tumors: Repeatability of Apparent Diffusion Coefficient Estimates from Diffusion-weighted MR Imaging.

Purpose To assess the repeatability of apparent diffusion coefficient (ADC) estimates in extracranial soft-tissue diffusion-weighted magnetic resonance imaging across a wide range of imaging protocols and patient populations. Materials and Methods Nine prospective patient studies and one prospective volunteer study, performed between 2006 and 2016 with research ethics committee approval and written informed consent from each subject, were included in this single-institution study. A total of 141 tumors and healthy organs were imaged twice (interval between repeated examinations, 45 minutes to 10 days, depending the on study) to assess the repeatability of median and mean ADC estimates. The Levene test was used to determine whether ADC repeatability differed between studies. The Pearson linear correlation coefficient was used to assess correlation between coefficient of variation (CoV) and the year the study started, study size, and volumes of tumors and healthy organs. The repeatability of ADC estimates from small, medium, and large tumors and healthy organs was assessed irrespective of study, and the Levene test was used to determine whether ADC repeatability differed between these groups. Results CoV aggregated across all studies was 4.1% (range for each study, 1.7%-6.5%). No correlation was observed between CoV and the year the study started or study size. CoV was weakly correlated with volume (r = -0.5, P = .1). Repeatability was significantly different between small, medium, and large tumors (P < .05), with the lowest CoV (2.6%) for large tumors. There was a significant difference in repeatability between studies-a difference that did not persist after the study with the largest tumors was excluded. Conclusion ADC is a robust imaging metric with excellent repeatability in extracranial soft tissues across a wide range of tumor sites, sizes, patient populations, and imaging protocol variations. Online supplemental material is available for this article

Transcriptome Analysis of the Vernalization Response in Barley (Hordeum vulgare) Seedlings

Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals

Genome-wide gene expression analysis supports a developmental model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.)

<p>Abstract</p> <p>Background</p> <p>To identify the genes involved in the development of low temperature (LT) tolerance in hexaploid wheat, we examined the global changes in expression in response to cold of the 55,052 potentially unique genes represented in the Affymetrix Wheat Genome microarray. We compared the expression of genes in winter-habit (winter Norstar and winter Manitou) and spring-habit (spring Manitou and spring Norstar)) cultivars, wherein the locus for the vernalization gene <it>Vrn-A1 </it>was swapped between the parental winter Norstar and spring Manitou in the derived near-isogenic lines winter Manitou and spring Norstar. Global expression of genes in the crowns of 3-leaf stage plants cold-acclimated at 6°C for 0, 2, 14, 21, 38, 42, 56 and 70 days was examined.</p> <p>Results</p> <p>Analysis of variance of gene expression separated the samples by genetic background and by the developmental stage before or after vernalization saturation was reached. Using gene-specific ANOVA we identified 12,901 genes (at <it>p </it>< 0.001) that change in expression with respect to both genotype and the duration of cold-treatment. We examined in more detail a subset of these genes (2,771) where expression was highly influenced by the interaction between these two main factors. Functional assignments using GO annotations showed that genes involved in transport, oxidation-reduction, and stress response were highly represented. Clustering based on the pattern of transcript accumulation identified genes that were up or down-regulated by cold-treatment. Our data indicate that the cold-sensitive lines can up-regulate known cold-responsive genes comparable to that of cold-hardy lines. The levels of expression of these genes were highly influenced by the initial rate and the duration of the gene's response to cold. We show that the <it>Vrn-A1 </it>locus controls the duration of gene expression but not its initial rate of response to cold treatment. Furthermore, we provide evidence that <it>Ta.Vrn-A1 </it>and <it>Ta.Vrt1 </it>originally hypothesized to encode for the same gene showed different patterns of expression and therefore are distinct.</p> <p>Conclusion</p> <p>This study provides novel insight into the underlying mechanisms that regulate the expression of cold-responsive genes in wheat. The results support the developmental model of LT tolerance gene regulation and demonstrate the complex genotype by environment interactions that determine LT adaptation in winter annual cereals.</p

45S rDNA Regions Are Chromosome Fragile Sites Expressed as Gaps In Vitro on Metaphase Chromosomes of Root-Tip Meristematic Cells in Lolium spp

BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. CONCLUSIONS: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification

Interaction of Temperature and Light in the Development of Freezing Tolerance in Plants

Abstract Freezing tolerance is the result of a wide range of physical and biochemical processes, such as the induction of antifreeze proteins, changes in membrane composition, the accumulation of osmoprotectants, and changes in the redox status, which allow plants to function at low temperatures. Even in frost-tolerant species, a certain period of growth at low but nonfreezing temperatures, known as frost or cold hardening, is required for the development of a high level of frost hardiness. It has long been known that frost hardening at low temperature under low light intensity is much less effective than under normal light conditions; it has also been shown that elevated light intensity at normal temperatures may partly replace the cold-hardening period. Earlier results indicated that cold acclimation reflects a response to a chloroplastic redox signal while the effects of excitation pressure extend beyond photosynthetic acclimation, influencing plant morphology and the expression of certain nuclear genes involved in cold acclimation. Recent results have shown that not only are parameters closely linked to the photosynthetic electron transport processes affected by light during hardening at low temperature, but light may also have an influence on the expression level of several other cold-related genes; several cold-acclimation processes can function efficiently only in the presence of light. The present review provides an overview of mechanisms that may explain how light improves the freezing tolerance of plants during the cold-hardening period

Transcriptional responses of winter barley to cold indicate nucleosome remodelling as a specific feature of crown tissues

We report a series of microarray-based comparisons of gene expression in the leaf and crown of the winter barley cultivar Luxor, following the exposure of young plants to various periods of low (above and below zero) temperatures. A transcriptomic analysis identified genes which were either expressed in both the leaf and crown, or specifically in one or the other. Among the former were genes responsible for calcium and abscisic acid signalling, polyamine synthesis, late embryogenesis abundant proteins and dehydrins. In the crown, the key organ for cereal overwintering, cold treatment induced transient changes in the transcription of nucleosome assembly genes, and especially H2A and HTA11, which have been implicated in cold sensing in Arabidopsis thaliana. In the leaf, various heat-shock proteins were induced. Differences in expression pattern between the crown and leaf were frequent for genes involved in certain pathways responsible for osmolyte production (sucrose and starch, raffinose, γ-aminobutyric acid metabolism), sugar signalling (trehalose metabolism) and secondary metabolism (lignin synthesis). The action of proteins with antifreeze activity, which were markedly induced during hardening, was demonstrated by a depression in the ice nucleation temperature