Streptomycetes are filamentous microorganisms of high biotechnological relevance, especially for the production of antibiotics. In submerged cultures, the productivity of these microorganisms is closely linked to their growth morphology. Microfluidic lab-on-a-chip cultivation systems, coupled with automated time-lapse imaging, generate spatio-temporal insights into the mycelium development of streptomycetes, therewith extending the biotechnological toolset by spatio-temporal screening under well-controlled and reproducible conditions. However, the analysis of the complex mycelial structure formation is limited by the extent of manual interventions required during processing of the acquired high-volume image data. These interventions typically lead to high evaluation times and, therewith, limit the analytic throughput and exploitation of microfluidic-based screenings.mycelyso (MYCElium anaLYsis SOftware), an image analysis system tailored to fully automated hyphae-level processing of image stacks generated by time-lapse microscopy. With mycelyso, the developing hyphal streptomycete network is automatically segmented and tracked over the cultivation period. Versatile key growth parameters such as mycelium network structure, its development over time, and tip growth rates are extracted. Results are presented in the web-based exploration tool mycelyso Inspector, allowing for user friendly quality control and downstream evaluation of the extracted information. In addition, 2D and 3D visualizations show temporal tracking for detailed inspection of morphological growth behaviors. For ease of getting started with mycelyso, bundled Windows packages as well as Docker images along with tutorial videos are available.We present the tool mycelyso is a well-documented, platform-independent open source toolkit for the automated end-to-end analysis of Streptomyces image stacks. The batch-analysis mode facilitates the rapid and reproducible processing of large microfluidic screenings, and easy extraction of morphological parameters. The objective evaluation of image stacks is possible by reproducible evaluation workflows, useful to unravel correlations between morphological, molecular and process parameters at the hyphae- and mycelium-levels with statistical power.The online version of this article (10.1186/s12859-019-3004-1) contains supplementary material, which is available to authorized users. Streptomyces are important producers of a wide range of pharmaceuticals, such as antibiotics, anticancer agents, and immuno-suppressants [Streptomyces are industrial production hosts for a large variety of enzymes and heterologous proteins [Streptomyces are predominantly performed in liquid (submerged) culture. Bioprocess optimization, although successful on a case-to-case basis, however, has hardly been found to be transferable between strains and target products [Streptomyces begins with spore germination, followed by growth of the initial germ tube, and outgrowth of hyphae which form a branched network, the mycelium logical metrics. To this end, specific software tools have been developed. Two recent approaches to pellet- or mycelium-level analysis of e ImageJ  plugin A[AnaMorf  and a MA[AnaMorf . Althougvailable . While trocesses . In the Particle , 15. TheSummarizing, employing existing tools for hyphae-level feature extraction and tracking from streptomycete time-lapse image sequences involves manual interactions which remain to be laborious and extremely time consuming, since automation aspects are insufficiently addressed by available image software packages. This lack of computational solutions hampers the exploitation of the unique possibilities offered by microfluidic single-cell technologies: the assessment of heterogeneity in a variety of growth measures and the ability to follow the development of exact mycelium structures over time, which later gives rise to pellet formation.mycelyso for analyzing mycelial network development of Streptomyces, with special focus on the extraction of growth metrics at the hyphae-level from time-lapse images. With mycelyso, growth metrics can be automatically extracted, in a reproducible manner, such as the hyphal growth unit , hyphal elongation rates (change in hypha length over time), and whole mycelium elongation rates . A viewer is developed, to provide an effective overview over the (both summary and detailed) results. Analysis results are stored in the HDF5 format to enable flexible and convenient post-processing and analysis, even in cases of large result sets. Diverse growth metrics can be extracted, with minimal manual burden and maximal reproducibility. The utility of mycelyso has been proven in a comprehensive screening study with Streptomyces lividans TK24 [Here we describe the automated image processing suite ans TK24  where wemycelyso analysis suite consists of two independent parts: the core command line tool analysis tool mycelyso for batch processing of phase contrast time-lapse image data, and the interactive web-based results viewer mycelyso Inspector. Both tools are written in the Python programming language, with a bundled package available for Windows, as well as a Docker image for quick and reproducible invocation. Documentation is available from the web page.The mycelyso is a cross-platform command-line tool accepting plain TIFF, OME-TIFF [mycelyso has been developed with fully automated batch processing in mind. Nevertheless, before starting a batch analysis process, adjustment of image analysis parameters might be indicated. To this end, mycelyso can be run in a preview mode, visualizing the binarization and the hyphae network in a per-frame manner. This way, the user can first visually assess and validate the analysis quality before launching batch processing of the image stacks.OME-TIFF , Zeiss COME-TIFF , 19. Datmycelyso, it has to be registered (i.e. drift compensated). Depending on the on-chip growth structures present, it can be beneficial to crop images to a region of interest (ROI) to speed up processing and reduce possible artifacts. In microfluidic setups with rectangular growth chambers, such as described in Grünberger et al. [mycelyso, meaning that image stacks can be analyzed directly, as acquired from the microscope. Otherwise, cropping to a region of interest is to be performed beforehand, e.g. with standard tools like ImageJ [To prepare input data for r et al. , findinge ImageJ . The cromycelyso provides both, common local thresholding functions as well as a custom segmentation approach . Minor The binary image is skeletonized using Zhang-Suen’s approach , i.e., iThe single pixel wide skeleton is then transformed into a graph representation Fig. c by idenStreptomyces grow by extension, thus moving forward during mycelial growth. To robustify the results, various sanity checks are applied while tracking the paths of individual hyphae. For instance, it is asserted that the paths remain connected by assuring they are connected components in the graphs in consecutive time points. Adjacent hyphae that cannot be separated in the binarized image , appear as a single edge in the graph, which might introduce “shortcuts” in the hypha tracks. To not generate artifactual growth metrics, affected hyphae are not tracked further. Once the tracking has been performed per growth chamber of the microfluidic chip (position), this information feeds in associated growth metrics, such as growth rate per position.Tracking the growing mycelium at the hyphae level differs from other single-cell tracking problems, in that the hyphae have to be tracked individually within their mycelial context to produce hyphae-level metrics. Therefore, once the graphs of all images in a time-lapse image stack are generated, hyphal development is tracked with the nodes of the graph representations at hand. Here, junction points are tracked by nearest-neighbor matching as they keep their position under planar growth conditions, delivering node correspondences between time frames Fig. e-g. Tip mycelyso provides plenty of growth and tracking metrics on hyphae, hyphae track, and position level . Alternatively, two Jupyter notebooks are included in the source code repository, which show additional ways to analyze the raw data stored in the HDF5 file. One notebook shows how to explore individual hyphae lengths and graph representations. The second notebook exemplifies how length-based growth rates can be determined using custom growth models by utilizing a third-party fitting tool.For visualization and exploration of the data contained in the HDF5 file, the viewer mycelyso Inspector, has been developed  which is essential before reliable interpretation and downstream analysis. To make high-volume data sets easily accessible, the web-based interactive data exploration tool mycelyso Inspector relies on a HTML5-based front end being served by a Python-based back end. The different kinds of data stored in the HDF5 files can be viewed either in a tabular or graphical  manner, and individual results can directly be saved in tab-separated values (TSV) format. Graph data structures and intermediate segmentation results are visualized for checking the processed results. In addition, 3D views visualizing the growing hyphal network are presented for a quick overview of the spatio-temporal hyphal development and the analysis quality  took 52.5 ± 1.2 s on a quad core Intel Core i7 4790 (3.6 GHz) with 32 GB RAM under Linux . Automated analysis directly yields important second-order metrics, such as the total mycelium growth rate, μ=0.45 h−1, derived from the mycelium length in one cultivation chamber  into account was determined to be 0.38±0.11 h−1 [ans TK24 . For theans TK24 . Within 0.11 h−1 , and themycelyso as described before and generated the biotechnological key parameters needed for the study in an automated manner. On this basis, we were able to draw statistically sound comparisons between the employed conditions without manual adaption of the processing. mycelyso contains specific developments for the task of streptomycete analysis, such as an adapted segmentation routine, as well as an individual hyphae-tracking approach for taking the mycelial network (graph) structure into account.Hyphae-level metrics unravel the precise kinetics of growth and response of the organism to the selected cultivation conditions, and provide insights into the formation of macroscopic phenotypes. In the larger study , from whmycelyso is an open source, ready-to-use platform independent analysis software for all steps of the analysis of time-lapse image stacks of growing hyphae networks of microfluidic Streptomyces cultivations. The end-to-end suite is designed for high-throughput hyphae tracking in a fully automated manner, and comes with the dedicated viewer mycelyso Inspector for visualizing the extracted results. mycelyso was successfully applied to evaluate a microfluidic Streptomyces growth study Additional file 2mycelyso Inspector: 2D/3D in-browser visualization. (PDF 464 kb)Figure S2.


Purpose: To develop a model to select appropriate candidates for irradiation stent placement among patients with unresectable pancreatic cancer with malignant biliary obstruction (UPC-MBO).Methods: This retrospective study included 106 patients treated with an irradiation stent for UPC-MBO. These patients were randomly divided into a training group (74 patients) and a validation group (32 patients). A clinical model for predicting restenosis-free survival (RFS) was developed with clinical predictors selected by univariate and multivariate analyses. After integrating the radiomics signature, a combined model was constructed to predict RFS. The predictive performance was evaluated with the concordance index (C-index) in both the training and validation groups. The median risk score of progression in the training group was used to divide patients into high- and low-risk subgroups.Results: Radiomics features were integrated with clinical predictors to develop a combined model. The predictive performance was better in the combined model  than in the clinical model . According to the median risk score of 1.264, the RFS was significantly different between the high- and low-risk groups .Conclusions: The radiomics-based model had good performance for RFS prediction in patients with UPC-MBO who received an irradiation stent. Patients with slow progression should consider undergoing irradiation stent placement for a longer RFS. Pancreatic cancer is one of the leading causes of cancer-related death , and it Currently, different models have been developed to predict survival outcomes in patients with different stages of pancreatic cancer –16. A coIn this study, we proposed a novel model incorporating clinical biomarkers and CT radiomics features to predict restenosis-free survival (RFS) for individual patients with UPC-MBO who undergoing irradiation biliary stent placement. According to our proposed model, irradiation stent placement could be recommended for appropriate candidates with slow progression for a longer RFS.This multicenter retrospective study was approved by the institutional review boards at all participating centers. The need for informed consent was waived due to the study's retrospective nature.Between January 2012 and December 2017, 106 patients  treated with irradiation stent placement for UPC-MBO from four centers were finally included and randomly divided into a training group (74 patients) and a validation group (32 patients). The sample size calculation is shown in The inclusion criteria were as follows: (a) age 18 years or older; (b) clinical or histopathological diagnosis of UPC-MBO; (c) unresectable disease due to extensive lesions, metastases, a poor medical condition, or refusal to undergo surgery; (d) initial percutaneous transhepatic biliary stent placement; and (e) standard contrast-enhanced CT performed <2 weeks before stenting. The exclusion criteria were as follows: (a) history of surgical resection, (b) presence of severe infection or organ failure before stenting, (c) Eastern Cooperative Oncology Group score of 4 before stenting, (d) any other anticancer therapy except supportive treatment after stenting, (e) incomplete clinical or CT imaging data, or (f) loss to follow-up.The following clinical characteristics were recorded: (a) demographics, including sex, age, and body mass index; (b) preprocedural status, including pain as assessed by a visual analog scale score, Eastern Cooperative Oncology Group performance status, prior biliary drainage, history of chemotherapy, history of radiotherapy, and degree of ascites; (c) preprocedural blood biochemical analysis, including total bilirubin, direct bilirubin, direct bilirubin/total bilirubin ratio, carbohydrate antigen (CA) 19-9, CA125, and carcinoembryonic antigen; and (d) parameters related to pancreatic cancer, such as the tumor stage according to the TNM classification system  , liver mA standard percutaneous transhepatic biliary stenting procedure was performed under fluoroscopic guidance with or without ultrasonographic guidance by interventional radiologists with more than 15 years of experience. The irradiation stent consisted of two overlapping parts, an outer 125I seed-loaded stent and an inner conventional uncovered self-expanding metal stent . The two parts were assembled in the biliary tract during the procedure. The 125I seeds  were preloaded into the sheaths that were attached to the outer surface of the stent immediately before the procedure. According to the Treatment Planning System , the number, dosage, and distribution of the 125I seeds were calculated. The standards of radiation safety and management were performed after irradiation stent placement .Routine follow-up, including performance status, clinical signs, postprocedural treatment, blood biochemical analysis, and imaging, was performed 1 week after stent placement, monthly for 6 months, and then every 3 months. The endpoint of this study was the occurrence of restenosis or death. RFS was calculated from the date of stenting to the date of the endpoint, which equaled the duration of stent function. Restenosis indicated stent dysfunction, which was defined by clinical signs of recurrent jaundice with elevated bilirubin levels along with biliary reobstruction as evidenced on CT, ultrasound, magnetic resonance cholangiopancreatography, or percutaneous transhepatic cholangiography. RFS was censored at the date of the last follow-up visit for restenosis-free patients.www.itksnap.org) by an experienced radiologist (reader 1). Each two-dimensional CT image covering the visible tumor region was delineated along the tumor boundaries, and the overlap in the delineated areas was selected as the final ROI , including 25 non-texture features, 51 texture features and wavelet features in wavelet images decomposed on different scales. Non-texture features reflect the shape, size and intensity of tumor lesions, and texture features represent the inherent heterogeneity of tumors based on four textural matrices. In addition, a three-dimensional wavelet transform was applied to decouple the first-order statistical features and texture features for each CT image. Finally, we extracted 620 radiomics features from original CT images and wavelet decompositions in each phase from each patient. The details of these features are exhibited in For feature preselection, intra-, and inter-observer coefficients were used to detect the stability of features with a threshold of 0.8. Then, Pearson's correction analysis was applied to identify redundant and collinear features, and features with mutual correlation coefficients >0.9 were excluded. After initial selection, the least absolute shrinkage and selection operator (LASSO)-Cox regression approach was used to identify predictive factors for RFS in the training group . Ten-folClinical and combined models were also built for comparison with the radiomics model. Univariate and multivariate Cox proportional hazard analyses were applied to identify effective clinical predictors. Based on the Cox proportional hazard regression model, a clinical model was constructed with clinical predictors, and the combined model integrated clinical predictors and the radiomics signature. In the combined model, the radiomics signature was calculated as the Rad-score for quantification. The C-index of the clinical and combined models was also calculated to illustrate their performance. The 3-month RFS rate of the combined model was assessed through receiver operating characteristic curve analysis along with the area under the curve. Decision curve analysis was used to compare the net benefit at different threshold probabilities from the clinical and combined nomograms.t-test for continuous variables and by Pearson's chi squared or Fisher's exact test for categorical variables. With the R package , the nomograms were formulated in the training group based on the results of the multivariate analysis and by the Cox proportional hazard regression modeling strategies. Receiver operating characteristic curves were drawn and the area under the curve was calculated to evaluate the discrimination performance for 3-month RFS. Calibration curves were drawn to compare the 3-month RFS between the predicted and actual outcomes using the Hosmer–Lemeshow test. Decision curve analysis was used to evaluate the clinical utility of the nomogram by calculating the net benefit at different threshold probabilities. The combined model generated a risk score for RFS and dichotomized the patients into two groups with different risks of progression using the median risk score in the training group. Kaplan-Meier curves were generated to evaluate the ability of the risk score to stratify the patients, and log-rank tests were applied to assess the statistical significance with p < 0.05.Continuous variable is described as mean ± standard deviation, and categorical variable is described as number and percentage. Baseline characteristics between two groups were compared by Student's p > 0.05, p = 0.926).A total of 106 patients  were included in this study, including 74 patients in the training group and 32 patients in the validation group. The clinical characteristics showed no significant differences between the two groups , M stage , and CA19-9  were ultimately selected as clinical predictors of RFS  was used to divide patients into high- (score ≥1.264) and low-risk (score < 1.264) groups. Kaplan-Meier curves and the log-rank test indicated significant differences in RFS between the high- and low-risk groups , while the radiomics signature showed a better C-index . This result indicated better predictive performance of radiomic biomarkers than of clinical biomarkers. Moreover, the combined model also performed well with C-indexes of 0.791 and 0.779 in the training and validation groups, respectively. The reason may be that radiomics features from the tumor can provide more information on the cancer phenotype and the tumor microenvironment , 40, butp = 0.123). The prognostic importance of CA125 in pancreatic cancer should be further evaluated.As shown in Imaging-based texture analysis is used to quantify intratumoral heterogeneity in patients with pancreatic cancer , 47, 48.This study has several limitations. First, as shown in In conclusion, the proposed model based on radiomics had good performance for RFS prediction in patients with UPC-MBO who underwent irradiation stent placement. Patients with slow progression should consider undergoing irradiation stent placement for a longer RFS. With further sufficient validation and future clinical trials, this model might be an important tool for clinical decision making in interventional oncology.The datasets generated for this study are available on request to the corresponding author.This study was approved by the institutional review boards at all participating centers, including IEC for Clinical Research of Zhongda Hospital, Southeast University, IEC for Clinical Research of Yunnan Tumor Hospital, IEC for Clinical Research of Lishui Central Hospital, and IEC for Clinical Research of Anhui Provincial Hospital.G-JT, H-FZ, Y-QH, JL, J-HG, and H-DZ contributed to the study concept and design. MH, J-SJ, W-FL, and G-YZ contributed to perform stent placement. JL, LC, and Z-CJ contributed to the acquisition of clinical data. J-WW contributed to the statistical analysis. H-FZ, Y-QH, H-DZ, and J-HG contributed to data interpretation. H-FZ wrote the first draft of the manuscript. Y-QH wrote the sections of the manuscript. G-JT, JT, H-DZ, and J-HG supervised and oversaw the study. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plant cells, tissues and organs are composed of various biomolecules arranged as structurally diverse units, which represent heterogeneity at microscopic levels. Molecular knowledge about those constituents with their localization in such complexity is very crucial for both basic and applied plant sciences. In this context, infrared imaging techniques have advantages over conventional methods to investigate heterogeneous plant structures in providing quantitative and qualitative analyses with spatial distribution of the components. Thus, particularly, with the use of proper analytical approaches and sampling methods, these technologies offer significant information for the studies on plant classification, physiology, ecology, genetics, pathology and other related disciplines. This review aims to present a general perspective about near-infrared and mid-infrared imaging/microspectroscopy in plant research. It is addressed to compare potentialities of these methodologies with their advantages and limitations. With regard to the organization of the document, the first section will introduce the respective underlying principles followed by instrumentation, sampling techniques, sample preparations, measurement, and an overview of spectral pre-processing and multivariate analysis. The last section will review selected applications in the literature. Plant structures are composed of primary metabolites: carbohydrates, proteins, lipids, nucleic acids, various secondary metabolites and other compounds . These bIn addition to IR microspectroscopy, a variety of imaging methodologies including Raman microspectroscopy, visible light imaging, fluorescence imaging, 3D imaging, laser imaging, and tomographic imaging (magnetic resonance tomography (MRT), positron emission tomography (PET) and computed tomography (CT)) are also alternatives to analyze plant structures ,12,13,14−1, 780–2500 nm), mid-infrared  and far-infrared  . Th. Th26]. −1), Region II (8500–5500 cm−1) and Region III (5500–4000 cm−1) [Near-IR region is divided into three regions: Region I  . The spe00 cm−1) ,19. It r00 cm−1) . While t00 cm−1) . All mol00 cm−1) . Figure The most characteristic near-IR bands of some primary  and secondary  metabolites are shown in As demonstrated in Light source. A single polychromatic thermal source is heated to 1500–2200 K as light source. Silicon-carbide is used in mid-IR and a tungsten filament in near-IR. Here, it should be mentioned that conventional IR thermal sources only provides a spatial resolution of many tens of micrometers, thus restricting the analysis to tissue level [ue level ,36. In tue level . Hence, ue level ,22.Splitter. Fourier transforms (FT) interferometers, tunable filters, and diffraction grating spectrometers are three main types used in IR imaging. FT interferometers record information from several wavelengths simultaneously [aneously  and offeaneously . The liqaneously . Narrow aneously .Detector. Photon detectors are used to record signal after wavelength separation. In near-IR imaging, lead sulfide, indium antimonide, and uncooled indium gallium arsenide are commonly used, while cadmium telluride and mercury are used in mid-IR imaging due to wide spectral sensitivity [sitivity ,42.Optics. Typically, 6×, 15×, and 32× objectives are implemented in mid-IR or near-IR microscope [croscope .Mid-IR and near-IR imaging systems are composed of four main parts as shown in Mid-IR and near-IR microspectroscopy sampling techniques can be categorized as transmission and reflection. In transmission measurement, the sample is illuminated by source and the detector is placed behind the sample. The sample should be partly transparent. In most cases, this measurement is not possible with thick samples. However, when thin specimens are obtained, the transmission imaging mode provides easy-to-interpret spectra, improves signal-to-noise ratio, decreases the distortion of the spectra, and shows the definite correlation between molecular structures and spectral features. Due to the mentioned reasons, particularly mid-IR imaging has been widely employed in qualitative plant analyses . However−1 and 1700–1500 cm−1 strongly mask the modes belonging to proteins and lipids. In such cases, samples may be also dehydrated or freeze-dried [In mid-IR imaging studies, for most plant samples, thinner sections are usually required to be examined on a calcium or barium fluoride or zinc selenide window. For sample preparation, special care should be given to avoid changes in the chemical composition of the sample. Three sectioning techniques are commonly employed to prepare the samples for imaging. They include Fresh , Cryogenze-dried . Anotherze-dried ,50. Howeze-dried . For exaze-dried . Even thze-dried , there ize-dried . HoweverComparatively, near-IR imaging having high penetration of the specimen often does not require time-consuming procedures for sample preparation. The sample can be examined either directly or as sections mounted onto window as the same in mid-IR imaging. However, again, overlapping and broad near-IR absorption bands may still necessitate chemometrics to take the relevant spectral data ,22.Point mapping. A regular grid of spatial positions on the sample surface is defined and a spectrum is measured at one position; and as the sample moves to the next measurement point on the grid, the next spectrum is recorded, and this continues for all positions in the area defining the image. Thus, different areas of the sample are consecutively analyzed.Line mapping. Spectra are acquired according to predefined spatial positions and the line is moved right to left and up to down to cover the whole area. Subsequently, a series of spectra along one dimension is obtained.Area mapping. Depending on the overall mapping size, the sizes of the individually analyzed areas, the spectral resolution and the number of repeated scans, mappings with single element detectors can be time-consuming. With Focal Plane Array (FPA), detectors which enable obtaining a series of spectra collected in two dimensions [mensions , the reqmensions ,56.Hyperspectral imaging. The images are acquired at wavelengths in the near-IR region. For this measurement, a huge amount of data is collected in a hyper spectral cube where the three axes include two spatial axes and one spectral axis. This can be generated in one of the four ways: a point-to-point spectral scan in a spatial grid pattern; FT imaging; a line-by-line spatial scan ; and wavelength tuning with filters. In this cube, the sample is compartmented into small surface or volume areas (referred to as pixels) each of them representing a full spectrum. These cubes are mostly displayed as a three-dimensional matrix or data cube spanning two spatial dimensions, x and y. The third dimension z corresponds to the individual wavelength/wavenumber , damped harmonic oscillator fitting procedure, generalized second-order vibrational perturbation (GVPT2) and density functional theory (DFT) to real systems represents the essence of computational chemistry. Among such approaches, anharmonic vibrational analysis is commonly used for non-fundamental modes. For example, GVPT2 allows acquiring of wavenumber and intensity parameters for first overtones and binary combination modes. The use of this theoretical calculation on the spectrum gives opportunity for fitting procedure. Thus, the information about the number of modes’ components can be obtained with selecting appropriate non-overlapping modes for further analysis. In addition, DFT data offer interpretation of non-fundamental modes. Even though these mentioned methods are needed to extract relevant information from non-fundamental bands, it should be stated that such modes have potential to affect entire spectral regions including mid-IR, for which fundamental bands dominate. For that reason, such analysis should be also performed for mid-IR region. In addition, it has been reported that DFT analyzing non-fundamental modes provides not only analysis for near-IR spectra and but also accurate reproduction of mid-IR region ,59,60. RTypical imaging experiments often require the co-addition of many scans to improve signal-to-noise ratio while maintaining high spatial resolution, which may result in the production of 100 or more spectra. The data must therefore be reduced by several image planes and spectral parameters. For this, the first step is pre-processing. It is performed to enhance information and to reduce irrelevant information such as the scattering effects contained in the spectra. These mathematical pre-treatments cover baseline corrections, normalizations, derivatives and smoothing ,65. The This section provides some reports in which mid-IR and near-IR imaging have been employed to investigate phenotypic properties of plant structures for basic plant sciences and agricultural purposes. In order to present a variety of the subjects on which two techniques were used, different applications for each method are described.In comparison to near-IR imaging, mid-IR imaging was developed earlier with conventional light sources to study chemical constituents within plant cell walls  and whol−1 region related to cell wall components [Triticum aestivum) endosperm textures, and found the presence of higher amounts of a water-extractable arabinoxylan in the peripheral endosperm of soft grains. In another study, the changes in similar molecules during cell-elongation were monitored to characterize maize (Zea mays) coleoptile growth by application of neural networks on mid-IR spectra [−1 and a decrease in pectins denoted by modes at 1014, 1094, 1152, 1238, and 1741 cm−1 were obtained during Arabidopsis embryo germination [Luffa cylindrical multiple pollen tubes formation from a single pollen grain has revealed lower amounts of pectins and accumulation of abnormal cell wall components related to lignin, pectin, cellulose, callose and overall cell wall carbohydrate content [Zinnia elegans by monitoring 1510 and 1595 cm−1 [Populus tremula) and to monitor changes in the cell walls. In this study, in fiber cells, lignin predominated in early wood and hemicelluloses/cellulose in late wood during the growing season. Additionally, xylem ray cells were found to contain more aromatic compounds (lignin and monolignols) in early wood and more pectins and/or hemicelluloses in late wood [−1 attributed to hydrocinnamic acid in (H type) lignin. A similar technique was conducted to identify the acetyl esterification of the cell walls of the black cotton-wood (Populus trichocarpa). According to the results, p-coumarate accumulated in young leaves and declined in mature leaves, while ferulate and acetate were predominantly found in stems. Over the course of stem development, the amount of ferulate increased, whereas the initial amount of p-coumarate diminished [Petunia hybrida from wild-type and from two transgenic lines in which the PhEXPA1 expansin gene expression was down-regulated and up-regulated to determine the role of expansin in the rearrangement of the cell wall polymer network. Within the scope of the study, the changes in cell wall composites absorption bands assigned to pectin were as follows: 1740 cm−1, 1595 cm−1, 1440 cm−1, 1150 cm−1, 1105 cm−1 and 975 cm−1; hemicellulose: 1260 cm−1, 1230 cm−1 and 1075 cm−1; and cellulose: 1025 cm−1. The data revealed that compared to wildtype there was a decrease in down-regulated samples for pectin and cellulose but in up-regulated samples for hemicellulose [Gloeophyllum trabeum or Poria placenta) by transmission FT-IR imaging microscopy and multivariate analysis. The findings showed that brown-rot starts to become significant in the outer cell wall regions . Most significant during incipient decay was the cleavage of glycosidic bonds, i.e., depolymerization of wood polysaccharides and the degradation of pectic substances. Accordingly, intramolecular hydrogen bonding within cellulose was reduced, while the presence of phenolic groups increased [The chemical specificity of mid-IR region allows the identification of certain peaks in 1200–950 cmmponents . Taking mponents  compared spectra . Similarmination . Likewis595 cm−1 . Recentlate wood . Furtherate wood  employedminished . Again, ellulose . Lastly,ncreased .Lc-transgenic and non-transgenic alfalfa using Gaussian and Lorentzian methods of multi-component peak modeling. HCA and PCA as well as curve fitting analysis revealed that transgenic alfalfa contained a relatively lower percentage of alpha helices and beta-sheets and a higher percentage of turns and random coils. On the other hand, transgenic Lc-alfalfa leaves had similar proteins with non-transgenic alfalfa obtained by no difference in cluster and PCA analysis [Arabidopsis thaliana cv. by Withana-Gamage et al. (2013) [In mid-IR spectra, amide I and amide II modes are attributed to proteins. Since the height of amide I is very sensitive to changes in proteins structures, the changes in secondary structure elements can be predicted . Relatedanalysis . Similaranalysis , on seed. (2013)  and on f. (2013) . Identic. (2013) .k-means clustering, fuzzy cmeans clustering and HCA) to differentiate morphological and molecular patters of different tissues of St. John’s wort (Hypericum perforatum) by monitoring lipids (1740 cm−1), phospholipids (1240 cm−1), proteins , carbohydrates (between 1185 and 930 cm−1), and nucleic acids (1080 cm−1). Depending on the contents of the components such as epidermis, phloem, protoxylem, sclerenchyma, and xylem tissues were successfully discriminated. Parallel distinction had been previously done on barley tissues such as percarp, seed coat, aleurone, and endosperm by detecting lignin, protein, carbohydrates, cellulosic materials and lipids [−1 (protein), 1650–950 cm−1  and 1185–800 cm−1 . The findings revealed that effective discrimination of three samples could be achieved based on protein mode but not the other modes [−1. In this study, HCA provided reproducibility of the FT-IR spectra of the same taxon. In supervised method, best results were obtained from a K-nearest neighbors’ classifier and the leave-one-out cross validation procedure on the dataset composed of single pollen grain spectra .Huck-Pezzei et al. (2012)  used simd lipids . The caper modes . In addiIllicium verum (Chinese star anise) and Illicium anisatum (Japanese star anise) could be distinguished by short-wave infrared hyperspectral push-broom imaging (920–2514 nm) system and multivariate analysis. In this study, a classification model with four principal components and an R2X cum of 0.84 and R2Y cum of 0.81 was developed for the species using Partial Least Squares Discriminant Analysis (PLS-DA). The model was used to predict the identity of I. anisatum (98.42%) and I. verum (97.85%) [K-nearest neighbor algorithm (KNN) and support vector machine (SVM), and random forest (RF) was also applied to distinguish rice seed samples based on 1039 nm and 1612 nm spectral range. For classification, PLS-DA and KNN models represented over 80% accuracy, and SIMCA, SVM and RF models produced 100% accuracy in both the calibration sample sets [Even though near-IR imaging is widely applied for quantitative analyses with the use of chemometrics, it has also been utilized for discrimination of plant samples. For example, dried fruits of two different species (97.85%) . The sample sets . Similarple sets  employedR = 0.8712). Distribution of chlorophyll concentrations on the nitrogen deficient and control cucumber leaves were obtained. Thus, the results concluded that hyperspectral imaging exhibits considerable promise for non-destructive diagnostics of nitrogen deficiency in cucumber plant [2P) = 0.938 and 0.965, residual predictive deviation (RPD) = 4.590 and 9.335 for lycopene and total phenolics content prediction, respectively.One of the studies measured content of biomolecules in plant samples by using near-IR imaging was performed by Yu et al. 2014)  to deter  to deteer plant . Similarer plant  to measuF-value. The optimal waveband ratio for the discrimination of bruises was obtained as 1074 nm and 1016 nm: R1074/R1016 with the accuracy of 92% [Penicillium digitatum in citrus fruit [Hyperspectral imaging has been also utilized to identify bruises and damages on different agricultural products. For instance, bruise damages on “Shingo” pears were detected by application of a classification algorithm based on y of 92% . The samy of 92%  and on wy of 92% . Additioy of 92%  and fungus fruit  were effLu et al. (2005)  had earlR2) for the y data (kernel hardness category) for the training set of over 85%. To evaluate endosperm texture of hard, intermediate, or soft whole yellow maize kernels a linescan (push-broom) instrument in the wavelength from 1000 to 2498 nm was tested . After mGermination periods of muskmelon seed were predicted by near-IR hyperspectral imaging by push-broom system in the spectral range of 948–2494 nm. The spectra from seeds on Germination Days 3 and 5 and non-germinated seeds were studied for development of PLS-DA. Most effective wavelengths were selected using Variable Important in Projection (VIP), Selectivity Ratio (SR), and Significance Multivariate Correlation (sMC). The PLS-DA model was constructed using individual VIP, SR, or sMC variables. The results demonstrated that the PLS-DA model, which was developed with the selected optimal variables from the different methods, provided comparable results for the calibration set; however, the PLS-DA-SR method afforded the highest classification accuracy (94.6%) for a validation set used to determine the viability and vigor of muskmelon seeds . More exGinkgo biloba leaves. In order to interpret ATR imaging spectra, PCA, HCA, alternating least squares (ALS), and PLS were used. Near-IR transmission microspectroscopic imaging was applied to explore the primary chemical structures of whole leaf samples and the C–H and O–H bonds in secretory cavities, veins, and mesophylls were found to be different due to overlapped modes. Instead of this, mid-IR images indicated that secretory cavities and mesophylls contained more resin-like compounds and proteins, whereas veins contained more cellulose and calcium oxalate. In addition, within the scope of the study, the surface chemical composition leaf samples were also studied by ATR microspectroscopic imaging. The spectra and distribution images of cuticle, protein, cellulose, ginkgolic acids, and calcium oxalate on the adaxial surface were obtained using the PHAP procedure, which is the combination of PCA, HCA, ALS and PLS. As a result, chemical compositions of secretory cavities, veins, and mesophylls were analyzed in detail, and the distributions of some compounds on the surface layers of Ginkgo biloba leaves were revealed [For imaging measurement, the transmission imaging mode presents easy-to-interpret spectra with high signal-to-noise ratio. However, the native chemical compositions and physical structures of plant samples may be altered during microtome process as stated above. To overcome this direct measurement of thick plant samples by the combination of near-IR and mid-IR imaging can be proposed. Chen et al. recently took advantage of near-IR transmission and ATR microspectroscopic imaging to characterize non-microtomed revealed . In another experiment, the chemical morphology of the Areca nut section was directly characterized by the reflection near-IR imaging and the attenuated total reflectance mid-infrared (ATR-MIR) imaging methods. According to NIR spectra, there are three types of pixels corresponding to the endosperm, perisperm, and testa, respectively. In addition to these data, the endosperm and perisperm contain a lot of carbohydrates and lipids as well as a few protein particles, while the testa mainly consists of tannins obtained by ATR imaging results. Thus, in the content of the study, first, the reflection near-IR imaging explored the sample to find out typical regions in small sizes. Then, ATR-MIR imaging method measured the typical regions and revealed the molecular structures and spatial distributions of compounds of interest .Mid-IR and near-IR imaging are non-destructive versatile analytical techniques in plant analysis providing qualitative and quantitative data with spatial distribution patterns of components in heterogeneous cells, tissues and organs. Two technologies have different advantages and constraints. The mid-IR has the practical limitation of thin specimen and difficult sample preparation. In contrast, near-IR data are more readily obtained with minimal sample preparation while offering deeper sample penetration. The appearance of mid-IR absorptions from vibrations of different functional groups in characteristic frequencies of IR facilitates easy band assignment and interpretation. Thus, plant composites can be qualitatively analyzed with wide and common use of this technique. Comparatively, near-IR interpretation usually requires statistical multivariate data treatment due to broader overlapping modes. However, once calibration method is developed, quantitative information about chemical composition of plants can be obtained, which is an ignored advantage of this technology over other conventional methods. Thus, taking the advantages of these methods several characteristics and properties of plant structures have been analyzed. Even though the benefits of the techniques over other analytical methods are sensitivity, analytical speed, and minimal sample preparation and easy operation, they still require development. Particularly, the use of multivariate data analysis methods and sampling techniques to interpret the data generated may bring some restrictions their use. As future perspectives, the development of the technologies in terms of methodologies and instrumentation will facilitate their applicability for a wide range of plant sciences. The developed theory covers several factors such as higher performing instrumentation, algorithms and software for signal processing. In particular, even though they are well-established as laboratory systems, there is still need for miniaturization. This will enable on-site and in-field analysis, which offers great opportunity to study the plant in their natural environments. Additionally, advances in instrumentation like improvement in resolution will increase their sensitivity, and thus efficacy of investigation. Further developments in chemometrics and quantum chemical methods will obviously contribute to easy spectral analysis. In sum, the integration of mid-IR and near-IR imaging with basic and applied plant research can overcome bottleneck issues due to receiving the full benefit of the available molecular data. In this way, reliable, automatic, multifunctional, and high-throughput plant phenotyping platforms may be developed more, and thus, plant scientists may gain new insights into all the aspects of plants.


Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) is widely used in studying chromatin biology, but a comprehensive review of the analysis tools has not been completed yet. Here, we discuss the major steps in ATAC-seq data analysis, including pre-analysis , core analysis , and advanced analysis . We also review the reconstruction of transcriptional regulatory networks with multiomics data and highlight the current challenges of each step. Finally, we describe the potential of single-cell ATAC-seq and highlight the necessity of developing ATAC-seq specific analysis tools to obtain biologically meaningful insights.Supplementary information accompanies this paper at 10.1186/s13059-020-1929-3. Mammalian DNA is highly condensed through three major hierarchical scales; the first is the nucleosome which then wraps into chromatin leading to the third hierarchy, the chromosome –6. ChromRecent gene regulation studies have focused on epigenetics, and through the advances of high-throughput sequencing technologies, various assays have been developed to decipher the epigenetic landscape. These include Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) , 10, DNaAmong assays designed for detecting chromatin accessibility, ATAC-seq has gained particular popularity since first described in 2013. An exponential increase of curated ATAC-seq datasets and publications indicates its value in a wide spectrum of biological questions  < 100 bp) and mono-, di-, and tri-nucleosomes   and is the basis for advanced analysis. A similar process has been comprehensively reviewed for ChIP-seq , 59 and Unlike in ChIP-seq, input controls for ATAC-seq, in which Tn5 transposase randomly cleaves protein-free DNA, are often unavailable due to high sequencing costs to obtain comparable coverage. Thus, peak callers which require input controls are impractical for ATAC-seq. Moreover, the direct pile-up of paired-end fragments from ATAC-seq represents both nucleosome-free and nucleosome-bound regions Fig. a. Open cPopular peak callers for ATAC-seq can be divided into two major categories: count-based or shaped-based. The count-based peak callers employ different statistical methods to compare read distribution shape in a candidate region to a random background. MACS2 , HOMER , and SICShape-based peak callers are not currently used in ATAC-seq, but they utilize read density profile information directly or indirectly and are believed to improve peak calling in ChIP-seq . PICS 7 models fCurrently, HMMRATAC is the only peak caller that is exclusive for ATAC-seq . It emplOther considerations should include whether the peak caller accounts for Tn5 cleavage bias and how it deals with biological replicates. Similar to DNase-seq, the enzymatic cut by Tn5 will introduce bias due to binding preference , 31, 77,Peak tracks generated by these tools can be visualized in Fig. Because by its nature ATAC-seq reveals multiple aspects of transcriptional regulation, the third major step involves interpretation at four different levels: peak, motif, nucleosome, and TF footprint. However, only a few tools are designed specifically for ATAC-seq.Currently, no differential peak analysis tools have been specifically developed for ATAC-seq data analysis. A straightforward approach would be to find the candidate regions (consensus peaks or binned genome), normalize, and count the fragments in these regions and compare with other conditions statistically . This coP values and more false positives [Among the consensus peak-based tools, HOMER, DBChIP , and Difositives .Sliding window approaches do not require pre-generated peak sets. Instead, they evaluate all windows along the binned genome and tend to yield more false positives and require stringent filtering and false discovery rate (FDR) control. PePr  and DiffCurrently, most studies assume that ATAC-seq reads in peak regions follow a NB distribution, as is the case for RNA-seq data. However, no shape-based differential analysis tools exist for ATAC-seq data. The peaks contain not only read count information, but also the distribution shape profile. It is especially important for broad peaks, as broad peaks can contain multiple local maxima, and those shifts can indicate biologically relevant perturbations, which could be detected in sliding window or shape-based methods. Although not systematically studied, we believe incorporating shape information will improve differential peak analysis. Nevertheless, considering replicate handling, external peak caller dependency and backend statistical methods, csaw is worth a first try due to its easily explainable edgeR framework.cis-regulatory elements including enhancers and promoters [After obtaining peak sets, annotation of peaks can associate chromatin accessibility with gene regulation. Normally, peaks are annotated by the nearest genes or regulatory elements. HOMER, ChIPseeker , and ChIromoters . After oromoters , KEGG [9romoters , and Rearomoters . In geneAlthough peak annotation provides functional interpretation, it does not directly explain the underlying mechanism. Open chromatin can affect transcription through TFs, which facilitate transcription by recognizing and binding to specific sequences on DNA. This sequence is known as a motif and the binding positions are called TF binding sites (TFBS). There are approximately 1600 TFs in human, and more than half have motifs obtained experimentally or computationally . Most TFE. coli. However, there is no central database, which contains comprehensive and consistent motif information, and the discrepancies can arise from differences of original ChIP-seq experiments and the software used to perform de novo motif discovery.In order to exploit motif information, great efforts have been made to compile databases of motif sequences from either experimental methods or computational predictions. Popular databases such as JASPAR  contain The motif information is mainly stored in text format, for example, as a position weight matrix (PWM). HOMER and Bioconductor packages TFBSTools  and motiZ-test. These methods employ different statistical tests to compare the motif frequency in peaks and the background regions.Based on aforementioned motif search tools, the position and frequency of motifs in each peak region can be obtained and compared to a random background or another condition. HOMER uses the hypergeometric test, while MEME-AME  uses theZ-score and is adjusted for known technical bias . It is specifically designed for scATAC-seq data with a large number of cells that could be considered as replicates. However, its performance in bulk ATAC-seq has not been evaluated yet. DiffTF generates a distribution of accessibility fold changes for all TFBSs, adjusted for GC content for each motif and is then compared to a permutated null background to evaluate significance [Apart from an overrepresentation test, accessibility at each putative TFBS is assumed to associate with TF activity and can be measured by fragment counts. ChromVAR  calculatificance , 124. InAll tools mentioned so far predict putative TFBSs indirectly from sequences found within peak regions. Such TFBSs can contain a significant fraction of false positives and are likely to be incomplete and confounded. This is because not all TFs have identified motifs and TFs from the same family can share very similar motifs . MoreoveAnother way to decipher the TF regulation is to use footprints. A footprint in ATAC-seq refers to a pattern where an active TF binds to DNA and prevents Tn5 cleavage within the binding site. This leaves a relative depletion within the open chromatin region Fig. a 47, 12, 127. ThHowever, there are hurdles for ATAC-seq footprinting analysis. First, it is important to shift the raw reads in the pre-processing step to account for the 9-bp duplication for accurate footprint detection . Second,Footprinting analysis tools mainly fall into two categories: de novo and motif-centric methods. De novo methods predict all footprint sites across peaks, according to features of a typical footprint pattern (peak-dip-peak). Then these putative footprint sites are used to match known motifs or identify novel motifs. Instead, motif-centric methods require the input of a priori TFBSs and discriminate these sites as bound or unbound using supervised or unsupervised methods Table .Table 1For de novo methods, it is important to mathematically define what is a footprint and denoise the footprint pattern from Tn5 cleavage bias , 134. BoMotif-centric methods focus on a priori TFBSs and consider TF-specific footprint profiles compared to de novo methods. The challenge is to avoid ascertainment bias where TFs with high-quality motifs are enriched.The unsupervised motif-centric methods classify putative TFBSs as bound or unbound, based on features extracted from genomic regions, e.g., distance to TSS, PWM match score, and sequence conservation score , 146, asIn contrast, supervised motif-centric tools require high-quality ChIP-seq to annotate true TFBSs as training data. MILLIPEDE and BinDNase both use logistic regression , 143, whIf global TF footprint pattern changes between conditions is of interest, BaGFoot  can be eThere are several caveats for footprinting analysis. First, supervised motif-centric footprinting tools generally outperform unsupervised counterparts and de novo methods, with the trade-off of less generalizability , 131. ThThe nucleosome consists of a histone octamer complexed with approximately 147 bp of DNA Fig. a and affSoftware developed for MNase-seq, such as DANPOS2, PuFFIN, iNPS, and NucTools, can be applied to ATAC-seq data after filtering for nucleosome-associated fragments –153, whiHowever, all these tools suffer from the same underlying drawbacks of a typical ATAC-seq experiment, which is low coverage beyond open chromatin. In the future, new experimental protocols coupled with bioinformatic methods for ATAC-seq will be required to more efficiently and precisely capture nucleosome occupancy. Here, we believe HMMRATAC and NucleoATAC are two useful and specific tools for ATAC-seq nucleosome detection.Along with the specified requirements for ATAC-seq data analysis we have articulated so far, the integration of ATAC-seq with other high-throughput sequencing technologies such as RNA-seq and ChIP-seq is gaining increasing interest to understand gene regulation.Because open chromatin is the pre-requisite for most TFs to bind, ATAC-seq peaks generally overlap with TF ChIP-seq peaks but are often broader. Thus, TF ChIP-seq and ATAC-seq can mutually validate the quality and reliability of each other within the same experimental system . Unique Researchers are also interested in qualitatively or quantitatively associating changes in chromatin accessibility with changes in gene expression by RNA-seq. Intuitively, researchers can discover whether DE genes also have significantly differential chromatin accessibility surrounding the respective TSS . MoreoveWhile ATAC-seq can simultaneously detect hundreds of TF motif occurrences or footprints, it is possible to reconstruct cell-specific regulatory networks by linking footprints/motifs with downstream genes. Similar approaches have been demonstrated in DNase-seq Fig. c 104, 1. HoweverAlthough it is possible to reconstruct undirected TF-gene regulatory networks with ATAC-seq alone, the directional regulation can be further inferred as activation or repression when RNA-seq is integrated. Duren et al. proposed a model with paired gene expression and chromatin accessibility (PECA) data to predict the target gene expression as a function of TF expression, chromatin remodeller expression and chromatin accessibility . MiraldiIn order to further improve network reconstruction, publicly available ChIP-seq datasets can be integrated to improve the accuracy of footprinting. Incorporating known enhancer-promoter interactions from chromatin conformation data would also be helpful. With the surge of deep learning, it would require more work on feature construction and selection in order to build effective algorithms to predict transcriptional regulatory networks. In summary, integrating ATAC-seq with multiomics data yields biologically meaningful results, which can uncover underlying mechanisms of gene regulation.There is growing need for integrated pipelines to process ATAC-seq data. Several have been developed but have different focus for downstream analysis by stitching together previously discussed tools.To name a few, esATAC  and CIPHThese pipelines will provide a helpful and convenient entry for researchers with minimal programming skills to explore ATAC-seq data. However, a general problem for these pipelines is the lack of flexibility for parameter tuning. Most parameters are hard coded empirically because the combination of them increases exponentially with the number of tools, which makes pipelines difficult to modify for any given context. Overall, a pipeline with visualization and user interface will be more appropriate for nonprogrammers to explore the data.Chen et al. about clustering methods in scATAC-seq showed that SnapATAC, Cusanovich2018 and cisTopic outperformed other methods [Enabled by microfluidic, nano-well, and combinatorial indexing technologies, scATAC-seq is now able to measure the chromatin accessibility for thousands of cells with easy protocol at a low cost –35. The  methods , 173–175New techniques such as scNMT-seq, sci-CAR, and Pi-ATAC were recently developed to measure chromatin accessibility, transcriptome, and proteome simultaneously from exactly the same cell , 177. DaATAC-seq has developed rapidly over recent years and has become a method of choice to investigate chromatin accessibility. There are now optimized protocols that work with single cells, blood samples, and frozen tissue with improved signal-to-noise ratio –35, 178.In this review, we have systematically discussed all major steps in an ATAC-seq analysis pipeline for the reader to consider, starting with raw sequencing reads to the endpoint of biological meaningful interpretation. Here, we offer a guide of available tools and suggested steps of analysis to consider to facilitate proper biological interpretation of ATAC-seq data. The alignment and QC steps are similar to RNA-seq and ChIP-seq. As for the peak calling, most ChIP-seq derived tools are compatible with ATAC-seq data. However, a comprehensive benchmarking would help to select appropriate tools and to guide future development of ATAC-seq-specific peak callers. There is growing evidence that improvement or parametrization of current tools can be applied to fit ATAC-seq data.For downstream interpretation, differential peak analysis can give an overview of the changes of chromatin accessibility. Nevertheless, these changes can arise from both read numbers and the shapes of peaks and can be detected by count-based or sliding window approaches. The performance of these two approaches still requires further evaluation in ATAC-seq and could be specific to particular contexts. In order to infer biological function and related TFs, peak annotation and motif enrichment analysis is a good first pass analysis for initial insight.Motifs and footprints are direct and indirect indicators of regulatory events respectively. The difficulty in detecting footprints comes from both enzymatic cutting bias and weak signals from transient TFs. Instead of defining footprints with a mathematical formula, recent publications made a good first attempt to embrace the fast development of machine learning algorithms with supervised learning , 144. MoAnother consideration for analysis is on reconstructing gene regulatory networks from ATAC-seq data alone or integrating with multiomics data. This is particularly tempting because ATAC-seq can work with as low as 500 cells and allow the study of well-defined subpopulations especially in developmental biology and clinical samples. ScATAC-seq provides another option to study chromatin biology in heterogeneous cell populations.In summary, ATAC-seq, an information rich assay, is in great demand for specific bioinformatic analysis tools for further exploitation in analyzing chromatin state, TF footprint, nucleosome position, and regulatory network reconstruction. As a starting point, we suggest researchers can build an effective workflow, by combining FastQC, trimmomatic, and BWA-MEM for pre-analysis, and MACS2 for peak calling. For advanced analysis, we suggest csaw for differential peak analysis, MEME suite for motif detection and enrichment, ChIPseeker for annotation and visualization, HMMRATAC for nucleosome detection, and HINT-ATAC for footprint analysis. If RNA-seq data is available, regulatory networks can be reconstructed using PECA method. However, researchers can always refer to this review for alternative tools for each step and we recommend selecting the tool based on the context of the experimental system and the data collected.We envisage that this review will encourage researchers to appreciate the complexity and current major hurdles in ATAC-seq data analysis. New ATAC-seq-specific tools and comprehensive benchmarking studies would enable the answering of more biological questions with ATAC-seq in the near future.Additional file 1.Review history. (DOCX 21 kb)


The objectives of this cross-sectional study were to: (i) determine whether there are differences in self-esteem and self-efficacy for healthy lifestyle choices between children living in food secure and food insecure households; and (ii) determine whether the association between household food insecurity (HFI), self-esteem and self-efficacy differs by gender. Survey responses of 5281 fifth-grade students (10 and 11 years of age) participating in the Canadian Children’s Lifestyle and School Performance Study II were analyzed using logistic and linear regression. HFI status was determined by the six-item short-form Household Food Security Survey Module (HFSSM). Students from food insecure households had significantly higher odds of low self-esteem, and significantly lower scores for global self-efficacy to make healthy choices, compared to students from food secure households. These associations were stronger for girls than for boys and appeared independent of parental educational attainment. Household income appeared to be the essential underlying determinant of the associations of food insecurity with self-esteem and self-efficacy. Upstream social policies such as improving the household income of low-income residents will reduce food insecurity and potentially improve self-esteem and self-efficacy for healthy choices among children. This may improve health and learning, and in the long term, job opportunities and household earnings. In Canada and the United States, household food insecurity (HFI) as measured on national surveys refers to self-reports of uncertain or insufficient food access, due to limited financial resources. Recent national prevalence data of HFI in Canada and the United States were reported as 12% , which cSuch consequences resulting from HFI include multiple poor health outcomes, higher health care utilization and costs ,6, higheAs HFI experienced in childhood is a stressor and precursor to unfavourable cognitive outcomes in childhood and adulthood , it is iIn order to prevent mental health issues, greater understanding of how FI influences self-esteem and self-efficacy among children is required. Further, the limited evidence of the association between household FS status and a child’s perceived self-efficacy to make healthy lifestyle choices, such as PA and HE needs to be extended. Therefore, the aim of this paper is to examine the relationship between household FS and self-esteem and self-efficacy for healthy lifestyle choices among grade five children living in Nova Scotia, Canada, where 22.8% of children lived in FI households in 2015–16 . The relIn this cross-sectional study, we used data from the Children’s Lifestyle and School Performance Study II (CLASS II) conducted in 2011 in Nova Scotia, Canada. Nova Scotia is a province on the eastern coast of Canada, consisting of a peninsula and offshore islands with a population just under 1 million people . CLASS IThe student survey consisted of questions on eating behaviours at school and home, physical activity, mental wellbeing, self-esteem, and self-efficacy. Parent(s)/guardian(s)’ survey contained questions about the home environment, household FI experience, and sociodemographic factors. Specifically, the home survey contained the US Department of Agriculture 6-item short-form Household Food Security Survey Module (HFSSM), a tool that has been validated in the US and is recommended when there is the need to reduce respondent burden or when asking questions about children’ FI is deemed too sensitive . A scoreOutcomes included global self-esteem, assessed using a series of ten questions with a 3-point Likert scale response options similar to the Emotional Functioning and Social Functioning items on the PedsQL . The folOutcomes also included self-efficacy for healthy lifestyle choices, assessed using 9 items, 4 for physical activity and 5 for healthy eating. Students were asked “If you wanted to, how confident are you that you could (i) be physically active no matter how tired you may be?; (ii) be physically active even if you have a lot of homework?; (iii) ask your parent or other adult to play a physical activity or sport with you?; (iv) be physically active for at least 60 min on 5 or more days per week?; (v) eat healthy food at school?; (vi) choose a healthy snack between school and dinner time?; (vii) eat healthy food if you are alone at home?; (viii) choose a healthy snack when you are bored?; (ix) choose a healthy snack when you are sad?” Response choices included ‘not at all confident’, ‘a little bit confident’, ‘quite confident’, and ‘very confident’. The responses were scored as 1, 2, 3, 4, with the highest score representing higher self-efficacy. The scores were summed for global self-efficacy , physical activity, and healthy eating. These scores were then transformed to a scale of 1 to 100 to make them comparable across the three measures on self-efficacy. The items for self-efficacy have each demonstrated good internal consistency . The intwww.nsclass.ca.Covariates used in analyses included gender, region of residence , parental educational attainment, household income, and bodyweight status  . As such, gender-stratified multivariable models, adjusted for the same covariates (except gender), were used to examine whether the associations were distinct for girls and boys.For self-esteem items, the responses were ordinal and the sum scores were not normally distributed. Self-esteem was therefore dichotomized whereby sum scores lower than the 15th percentile were defined as low self-esteem, which is similar to the parametric concept of one standard deviation below the mean, an approach commonly applied in self-esteem research . Univarip = 0.251). For consistency with self-esteem models, gender-stratified analyses was conducted using a multivariable model, adjusting for the same covariates (except gender).For the associations of self-efficacy with FI status, univariate linear regression models were conducted for global, PA, and HE self-efficacy scores. A multivariable linear regression model with robust standard errors was used to account for clustering of students and adjusted for gender, region of residence, and bodyweight status. This model was further adjusted for parental education and household income. Models were tested for gender-HFI interaction, but it was not found to be significant . Similar statistically significant gradients were observed for household income and parental education, with lower household income and parental education associated with lower self-esteem and self-efficacy (data not shown).Of students with low self-esteem, 9.4% were from households experiencing severe FI whereas among students with normal self-esteem only 6.6% came from FI households. Likewise, among students in the lowest self-efficacy tertile, a higher percentage (8.3%) were from severe FI households relative to students in the highest self-efficacy tertile (5.7%). These differences were statistically significant (p = 0.006). In the gender-stratified Model 1, the association with lower self-esteem was only significant for girls from moderate FI households and boys from households with severe FI . This association was not significant among girls living in marginal or severe FI households or among boys living in marginal FI or moderate FI. When this model was further adjusted for parent educational attainment (Model 2), the association between HFI and low self-esteem remained significant only among students (girls and boys combined) and girls living in moderate FI households. They had respectively 40% and 2 times higher odds of low self-esteem when compared to their FS counterpart. Model 3 was further adjusted for household income. It showed that girls from moderate FI households had an associated 67% higher odds of low self-esteem when compared to their FS counterpart. This association was no longer significant for girls and boys combined.p = 0.251). The associations of FI with global self-efficacy score remained statistically significant after adjusting for gender, bodyweight status, and region of residence, while they remained statistically significant only for girls in the gender-stratified Model 1. When these models were further adjusted for parent educational attainment, associations remained significant only for students (girls and boys combined) and for girls from moderate FI households (Model 2). The association between FI and global self-efficacy was no longer significant after further adjusting for household income.The individual associations of FI with self-efficacy for physical activity and healthy eating are presented in the In terms of self-efficacy for healthy eating, students from moderately FI households had significantly lower scores than students from food secure households before and after adjusting for gender, bodyweight status, and region of residence. In gender-stratified models, this difference was significant for girls from moderate and severe FI households, but not for boys. The association with lower self-efficacy for healthy eating remained significant only among students (girls and boys combined) and girls living in moderate FI households after further considering parental education. When this model was further adjusted for household income, the association remained significant only for girls from moderately FI households .The objectives of this study were to: (i) to determine whether there are differences in self-esteem and self-efficacy for healthy lifestyle choices between students living in households with FS and FI; and (ii) to determine whether there are gender differences within the association between FI, self-esteem and self-efficacy among children. Our findings revealed that one quarter (25.2%) of children who participated in the CLASS II study were living in food insecure households. We demonstrated that these children living in households with FI were more likely to have low global self-esteem and low self-efficacy for healthy lifestyle choices. These associations were generally more pronounced for girls than for boys and independent of region of residence, bodyweight status and parental educational attainment. Household income appeared to be the essential underlying determinant of the associations of FI with self-esteem and self-efficacy, to some extent lesser in the association in girls. Although 7.1% of children lived in households with severe FI, we did not observe a gradient of severity of HFI with likelihood of low self-esteem and self-efficacy for physical activity and healthy eating.Our observed FI prevalence (25.2%) was relatively consistent with provincial estimates of HFI for the same year (2011) in that 23% of Nova Scotian households with children were food insecure . FindingGiven we found self-esteem and self-efficacy were compromised among children from food insecure households, especially among girls, upstream policy actions targeting the structural determinants of HFI  should bFurther research demonstrating the relationship between HFI and low self-esteem and self-efficacy is warranted, given that low self-esteem contributes to depression in youth ,53. ThisStrengths of this study include the large population-based sample, increasing the study’s representativeness. Sampling accounted for urban and rural residents, with all public schools in the province invited to participate. The relative high participation rate was another strength. To our knowledge, no other studies have investigated the gender differences in children between HFI modelled as three levels of severity and self-esteem and self-efficacy, reinforcing the importance of our contribution to the scarce evidence base. However, there were limitations associated with this study. This research was cross-sectional in nature, and therefore we could not establish causation. A review of longitudinal studies investigating associations between FI and mental health in adults suggested a bidirectional relationship existed (for example FI increasing depression and depression increasing FI) . TherefoVarious studies examined the association between HFI and poor mental health in high-income countries such as Canada. These studies generally concluded that HFI is associated with poor mental health outcomes. In this study, we examined in children if self-esteem and self-efficacy were associated with HFI at three increasing levels of severity. Given there is a hypothetical cyclical relationship between FI and self-esteem and self-efficacy, interventions that address both child poverty, low-self-esteem and low self-efficacy are required to break the cycle of intergenerational HFI. Such actions could improve physical and cognitive health among children now and throughout their entire lifespan. This is critically important, given the link between mental health problems in childhood and in later life .


Mesenchymal stem cells (MSCs) are adult stromal cells with the capacity to differentiate into multiple types of cells. MSCs represent an attractive option in regenerative medicine due to their multifaceted abilities for tissue repair, immunosuppression, and anti-inflammation. Recent studies demonstrate that MSCs exert their effects via paracrine activity, which is at least partially mediated by extracellular vesicles (EVs). MSC-derived EVs (MSC-EVs) could mimic the function of parental MSCs by transferring their components such as DNA, proteins/peptides, mRNA, microRNA (miRNA), lipids, and organelles to recipient cells. In this review, we aim to summarize the mechanism and role of miRNA transfer in mediating the effects of MSC-EVs in the models of human diseases. The first three sections of the review discuss the sorting of miRNAs into EVs, uptake of EVs by target cells, and functional transfer of miRNAs via EVs. Then, we describe the composition of miRNAs in MSC-EVs. Next, we provide the existing evidence that MSC-EVs affect the outcomes of renal, liver, heart, and brain diseases by transferring their miRNA contents. In conclusion, EV-mediated miRNA transfer plays an important role in disease-modulating capacity of MSCs. Mesenchymal stem  cells (MSCs) are a heterogeneous population of progenitor cells which possess angiogenic, anti-apoptotic, and immunomodulatory properties. Many clinical trials including the work from our group  have tesEVs are membrane-contained vesicles released by most types of cells including MSCs. A crucial criterion of EVs is that they must be harvested from extracellular fluids such as tissue culture or bodily compartment through ultracentrifugation based on the position statement from the International Society for Extracellular Vesicles (ISEV) . EVs canThe majority of total RNA content in EVs includes tRNAs, mRNAs, miRNAs, and long non-coding RNAs. Sequence analysis showed that approximately 13% of the total RNA content of EVs from milk is miRNAs . miRNAs Many studies have shown that miRNAs are selectively sorted in EVs. Such sorting of miRNAs was demonstrated in PC-3 prostate cancer cells in which miRNAs with a low number in their names were found to be underrepresented in EVs . In dendlet-7a, miR-100, and miR-320a into the EVs [The secretory mechanism of miRNAs has been postulated and is regulated by a ceramide-dependent pathway. Ceramide is synthesized by neutral sphingomyelinase 2 (nSMase2) and controls the secretion of EVs. Decreased synthesis of ceramide via nSMase2 inhibition led to reduced secretion of miRNAs. Overexpression of nSMase2 elevated the extracellular miRNA levels . However the EVs . Alix, w the EVs . However the EVs . Finally the EVs .Table 1MThe contents of EVs may gain entry into target cells via two mechanisms: endocytosis and fusion. The primary entry mechanism of EVs is endocytosis, including clathrin-dependent endocytosis and clathrin-independent pathways which are mediated by caveolin, macropinocytosis, phagocytosis, and lipid raft . A populMechanisms for uptake of MSC-EVs by target cells have also been investigated in several studies. In a model of myocardial infarction, Arslan et al. reported that MSC-EVs were internalized by cardiomyocytes at the infarct site via endocytosis or phagocytosis. They also demonstrated that homogenized EVs failed to reduce the infarct size . Vonk etCXCL11/ITAC [Many reports have documented that miRNAs packaged in EVs can affect the function of the recipient cells. Valadi et al. were the first to report that EVs contained transferable and functional mRNAs and miRNAs, showing that new mouse proteins were discovered in the recipient cells after transferring the content of mouse EVs to human mast cells . Pegtel L11/ITAC . In anotL11/ITAC . In addiL11/ITAC . TransfeL11/ITAC .Several studies have compared the contents of miRNAs between MSC-EVs and their parental MSCs. Shao et al. reported that the expression of miR-21 and miR-15, which inhibits cardiac functions, was significantly lower in MSC-EVs compared to that of MSCs . miRNA aThere is a lack of a consensus miRNA signature among MSC-EVs from different sources. Table Cantaluppi et al. were the first to show that EVs from endothelial progenitor cells alleviated kidney injury from ischemia-reperfusion via transfer of miRNAs to resident renal cells . Since tIn a mouse model of myocardial infarction, MSC-EVs alleviated cardiac injury. In vitro, EVs enriched with miR-22 were produced by MSCs after ischemic precondition, transferred to cardiomyocytes, and reduced ischemia-induced cardiomyocyte apoptosis. The protective effect of miR-22 was facilitated by target protein methyl-CpG-binding protein 2 . In a raSimilar findings have also been documented in liver diseases. In a study of hematoma cells, Lou et al. reported that miR-122 was effectively packaged into EVs of MSCs with miR-122 overexpression, transferred into hepatoma cells, and reduced the expression of target genes, such as cyclin G1 and insulin-like growth factor receptor 1 in the cancer cells. In vivo, the MSC-EVs sensitized hepatoma cells to chemotherapeutic agent sorafenib . The samXin et al. reported that MSC treatment of rats with stroke elevated miR-133b level in the ipsilateral hemisphere. In vitro, miR-133b was transferred from MSC-EVs to primary cultured neurons and astrocytes. Cultured neurons treated with EVs from MSCs exposed to brain extracts of middle cerebral artery occlusion significantly induced the length branch number and length . The gromiRNA transfer also mediates the effects of MSC-EVs in brain tumors. Figueroa et al. found that EVs from glioma-associated MSCs elevated proliferation and tumorigenicity of glioma stem-like cells in vivo. miRNA profile analysis identified that miR-1587 mediated the effects of MSC-EVs on glioma stem-like cells, in part via downregulation of the nuclear hormone receptor corepressor-1 . In anotOver the past decade, MSC-EV-based therapies have shown promising results in animal models for a myriad of diseases. MSC-EVs exert their effects mainly via immunomodulation, tissue regeneration, anti-apoptosis, and regulation of tumor progression. MSC-EVs are cell-free products and reduce the risks associated with the use of native or engineered MSCs. The effects of MSC-EVs are of equal potency to those observed with whole MSCs. Therefore, MSC-EVs may be a promising alternative to MSC therapy. As with other cell types, the main functional RNA components in MSC-EVs are miRNAs, which are selectively sorted into EVs and protected from degradation. The release of MSC-EVs allows cells to communicate with other adjacent or distant cells by transferring miRNAs and other biologically active molecules into recipient cells via endocytosis and/or fusion. The effects of MSC-EVs are dependent on the profiles of their miRNAs, which regulate the expression of multiple target genes and participate in various cell signaling processes. However, there is a wide knowledge gap between miRNAs in MSC-EVs and altered gene expression in the target cells. Foremost, it remains unknown what the miRNA signatures of MSC-EVs are. There has not been any individual miRNA or miRNA panel that has been reported as a consensus miRNA profile of MSC-EVs. The lack of miRNA signatures may result from small sample sizes and heterogeneity in methodology, culture condition, cell origin, and cell status. It remains unclear how diverse miRNA profiles in different MSC-EVs exert similar protective function in animal models. Secondly, the sorting mechanisms of miRNAs into MSC-EVs have not been determined. The existing sorting pathways proposed in the literature apply to only a limited number of miRNAs and to some cell types. A general mechanism of miRNA sorting into EVs or MSC-EVs is still unavailable. Lastly, the principle target genes of miRNAs in MSC-EVs also remain unspecified. Based on the bioinformatics tools, dozens or hundreds of genes are potential targets of a single miRNA. Many of the target genes could work independently, synergistically, or even antagonistically. It would be difficult to attribute an observed miRNA-dependent phenotype to a single target as reported in most literature. Further studies may pave the way to develop MSC-EVs therapeutics based on miRNA delivery.


Gastro-oesophageal reflux is common in children, especially in the first year of life, and it may be regarded as physiological. Good functioning of the lower oesophageal sphincter depends largely on the anatomical relationships between oesophagus, stomach and diaphragm hiatus. Relative immaturity of these structures in newborn babies and young children is a risk factor in reflux disease, which may result in a wide variety of typical and/or atypical symptoms and, sometimes, serious complications such as oesophagitis and stenosis.Reflux disease may be diagnosed and studied, basing on morphological and functional aspects and, since the advent of pH-metry, it is possible to personalise the therapeutic approach to children with reflux.Surgical treatment of reflux disease in children has recently been improved due to a mini-invasive surgical approach. Absolute indications are recurrent pneumonia, intractable pain due to oesophagitis and retarded growth, often in association with neurological impairment.In the last three years, 18 children with reflux disease underwent videolaparoscopic surgery in our department, 14 by the Nissen and 4 by the Toupet technique.Post-operative pH-metry always showed a reduction in exposure of the distal oesophagus to acid  and an improvement in oesophageal clearance (short refluxes percentage) indicative of good functioning of the gastro-oesophageal junction.PH-metry proved to be an invaluable technique for planning therapeutic strategy. In follow-up evaluations, it enabled us to monitor functioning of the gastro-oesophageal junction and to avoid other more difficult and invasive tests in patients with severe neurological impairment. Gastro-oesophageal reflux disease is quite common in children. It has multifaceted aetiology and manifests in a variety of forms, depending on its amount, the chemical and physical properties of the refluxed material, oesophageal clearance, mucosal alterations, and reflex or mechanical effects of refluxed material on the airways.Until recently, diagnosis was based solely on symptoms and upper digestive contrast X-ray. Lately, however, endoscopy and 24-hour pH-metry have been employed as new basic device. Analysis of 24-hour pH curve, mainly with respect to new generation parameters, has increased the accuracy of diagnosis, especially in cases in which X-ray and endoscopy are negative, and makes it possible to identify patients not responding to pharmacological therapy and requiring surgery.The aim of this study was to evaluate 24-hour pH curve as a direct indicator of therapeutic efficacy in the follow-up of patients undergoing laparoscopic surgery.Eighteen children with reflux disease underwent videolaparoscopic surgery at the Section of Paediatric Surgery, Department of Paediatrics, of the University of Siena - Italy in the period 1998 – 2001. Indications for surgery were unsuccessful medical therapy and worsening of clinical symptoms and reflux. Six patients were excluded from the study as their post-operative follow up was not completed. The other twelve patients had a mean age of 6.08 years (age range 17 months to 16 years) at the time of the operation. Gender distribution did not show any significant statistical bias . Eleven patients had brain damage, with neurological and psychomotor impairment. These patients often had atypical symptoms, with recurrent airway infections, pneumonia and acute life-threatening events.Pre-operatively, gastro-oesophageal transit, morphological alterations and gastro-oesophageal reflux were evaluated by upper digestive tract contrast X-ray. Gastroscopy was then performed to identify any malformations and assess the macroscopic and microscopic condition of serial oesophageal mucosa biopsy specimens.Twenty-four-hour monitoring of gastro-oesophageal pH was used to evaluate reflux qualitatively and quantitatively. This method was originally standardised at our digestive pathophysiology unit. A computerised system (“pH-informer” - Deltron Electronics - Milan) was used with 1 second pH sampling, which is essential for bidimensional parameters evaluation . In nine cases (75.0%) videolaparoscopic Nissen fundoplication was performed; in four of these cases (44.4%) a temporary gastrostomy was necessary. In three cases (25.0%) a videolaparoscopic Toupet plasty was performed. Post-operative recovery was normal in all cases and no patient suffered from dysphagia.All patients underwent follow-up, consisting of clinical examination, upper alimentary X-ray and pH-metry, within six months of the operation. Patients subsequently underwent clinical follow-up for a mean period of 17.7 months (range 7–30 months). Pre and post-operative pH data, expressed in medians , were statistically compared by the Mann-Whitney test for repeated measurements .Upper digestive tract X-ray showed abundant reflux in 7 cases (58.3%), and a cardio-tuberositary malpositioning in 3 cases (42.8%). Gastroscopy showed reflux in nine cases (75.0%), three  with hiatal hernia and oesophagitis. PH-metry was positive in all cases with reflux, and traditional and new generation parameters had high values.At follow-up, X-rays did not show any alteration in gastro-oesophageal transit and, from a functional point of view, distal oesophageal acid exposure and oesophageal clearance were normal . Table 3In patients with severe cerebral handicap, the first sign of reflux disease is often severe oesophagitis, which can be avoided if early pH-metric assessment is performed. This functional test provides an exact picture of the function of the gastro-oesophageal junction, through evaluation of distal true acid exposure. This is expressed by hydrogen ions concentration (mmol/l) and by oesophageal clearing capacity, expressed by the number of short refluxes (less than 0.5 seconds in duration) in the 24-hour period. In these patients reflux disease often manifests as an increase in convulsive crises, or respiratory symptoms unresponsive to specific therapy but responsive to anti-reflux therapy, or in severe cases, to surgical therapy.PH-metry proved to be an invaluable technique for deciding therapeutic strategy. In follow-up evaluations, it enabled us to monitor functioning of the gastro-oesophageal junction and to avoid other more difficult and invasive tests in patients with severe neurological impairment.


Methotrexate-associated lymphoproliferative disorder (MTX-LPD) can present as a benign lymphoid proliferation or a malignant lymphoma in patients taking MTX. Almost 50% of MTX-LPD cases show spontaneous remission after withdrawal of MTX treatment. Studies have suggested that the hyper-immune state of rheumatoid arthritis, the immunosuppressive state associated with MTX, and the carcinogenicity of the Epstein-Barr virus might contribute to MTX-LPD development. Although most cases of MTX-LPD occur at extranodal sites, few cases of MTX-LPD affecting the stomach and duodenum have been reported. To our knowledge, no other study has reported on the endoscopic observations of dramatic withdrawal and appearance of multiple digestive tract lesions in a short period of time. Herein, we report the clinical course and imaging findings of our case, which may be useful for understanding the pathological condition of MTX-LPD.We describe the case of a 70-year-old woman with MTX-LPD of the stomach and duodenum. Disease regression was temporarily achieved after cessation of MTX treatment; however, it subsequently recurred, and complete response was only achieved after six cycles of rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisolone (R-CHOP) chemotherapy.The first-choice therapy for patients taking MTX who develop suspected MTX-LPD should be the withdrawal of MTX treatment. Even after remission is achieved, patients should be kept under careful observation, and if the disease recurs, chemotherapy should be commenced promptly. Methotrexate-associated lymphoproliferative disorder (MTX-LPD) can present as a benign lymphoid proliferation or a malignant lymphoma in patients taking MTX. In 1991, Ellman and colleagues reported the first case of lymphomas in a patient with rheumatoid arthritis (RA) treated with MTX . ApproxiHere, we describe a case of MTX-LPD in the stomach and duodenum that temporarily resolved after the cessation of MTX treatment. In order to achieve complete response after recurrence of disease, prompt chemotherapy was required. To our knowledge, there are no other reports on the endoscopic observations of dramatic withdrawal and the appearance of multiple digestive tract lesions within a short period. The imaging findings along with the patient’s clinical course in this case may prove useful for understanding the pathological condition of MTX-LPD.Helicobacter pylori infection, which was resolved five years prior; and RA, for which she had been taking MTX (6 mg per week) for the past 6 months. Her symptoms were investigated with esophagogastroduodenoscopy (EGD), which initially revealed no abnormality apart from atrophic gastritis. Following a two-month course of acid-suppressing drugs, she remained symptomatic; therefore, a repeat EGD was conducted, which revealed the emergence of multiple elevated lesions. As a result, she was referred to our hospital.A 70-year-old woman presented to the clinic with a history of epigastric distress. Her medical history was significant for Physical examination at that time revealed the abdomen to be soft and flat, with no hepatosplenomegaly or lymphadenopathy. Laboratory tests showed elevated levels of lactate dehydrogenase  and soluble interleukin-2 receptor (sIL-2R) . The lymphocyte count was 2375/μl .1 diffuse large B-cell lymphoma (DLBCL) (Lugano classification).EGD performed at the time of admission to our hospital revealed multiple “dish-like” lesions in the stomach and duodenum Fig. a, d. IndInitial management consisted of the discontinuation of MTX, which resulted in symptom improvement and reduction of sIL-2R level. Two weeks after the withdrawal of MTX, the lymphocyte count increased from 2375/μl to 5616/μl (52%). EGD conducted 1 month after discontinuation revealed a reduction in the number of lesions with some scarring  and a DLBCL histology type are predictive factors of shorter survival 0 years a. CD5-posFew reports have focused on the endoscopic features of gastric or duodenal lesions in MTX-LPD. Ikeda et al.  reportedOur case was classified as carrying a low rate of spontaneous remission and aggressive characteristics because of the patient’s advanced age, DLBCL histology type, CD5-positivity and EBV-negativity. In only a month after cessation of MTX treatment, her symptoms improved, and EGD showed that most lesions had disappeared. Subsequently, however, the symptoms exacerbated and the sIL-2R level increased. Repeat EGD revealed regrowth of lesions, and she was started on R-CHOP chemotherapy, which resulted in complete response.Various new drugs with high efficacy, such as biological preparations, have been developed for treating RA. However, these drugs are very expensive, and it is highly likely that MTX—which is inexpensive and highly effective—will remain the first-line drug. Hence, future studies must examine the mechanism underlying the development of MTX-LPD, and the elucidation of this mechanism will help in preventing the occurrence of MTX-LPD.In lymphoma patients treated with MTX, the first-choice therapy is the withdrawal of MTX treatment if MTX-LPD is suspected. Even after the remission of MTX-LPD, careful observation is important, and if the disease recurs, chemotherapy should be commenced promptly.