To assess the influence of gray-level discretization on inter- and intra-observer reproducibility of texture radiomics features on clinical MR images.We studied two independent MRI datasets of 74 lacrymal gland tumors and 30 breast lesions from two different centers. Two pairs of readers performed three two-dimensional delineations for each dataset. Texture features were extracted using two radiomics softwares (Pyradiomics and an in-house software). Reproducible features were selected using a combination of intra-class correlation coefficient (ICC) and concordance and coherence coefficient (CCC) with 0.8 and 0.9 as thresholds, respectively. We tested six absolute and eight relative gray-level discretization methods and analyzed the distribution and highest number of reproducible features obtained for each discretization. We also analyzed the number of reproducible features extracted from computer simulated delineations representative of inter-observer variability.The gray-level discretization method had a direct impact on texture feature reproducibility, independent of observers, software or method of delineation (simulated vs. human). The absolute discretization consistently provided statistically significantly more reproducible features than the relative discretization. Varying the bin number of relative discretization led to statistically significantly more variable results than varying the bin size of absolute discretization.When considering inter-observer reproducible results of MRI texture radiomics features, an absolute discretization should be favored to allow the extraction of the highest number of potential candidates for new imaging biomarkers. Whichever the chosen method, it should be systematically documented to allow replicability of results. Medical imaging is progressively shifting from conventional visual image analysis to quantitative personalized medicine thanks to the recent development of data-driven analysis methods like radiomics . RadiomiRadiomics texture features are calculated from gray-level co-occurrence matrices and from other derived matrices that are computed on images after a gray-level discretization. The gray-level discretization consists in clustering pixels according to intensity values to facilitate the calculation of texture features . Two appIn this context, the purpose of this work was to investigate how pre-processing of images, and specifically gray-level discretization methods, could impact the number and type of reproducible texture features extracted from clinical MR images.This study was performed on two datasets: the DATASET 1 was prospectively acquired in a tertiary referral center specializing in ophthalmic diseases with the approval of the Institutional Research Ethics Board, of the Ile-De-France Ethics Committee (France) and adhered to the tenants of the Declaration of Helsinki ; signed informed consent was obtained from all subjects. The DATASET 2 was retrospectively built in a tertiary referral center specializing in breast lesions with the approval of the local Ethics Committee of Tenon Hospital  and the Institutional Review Board (IRB “blinded”), which waived the necessity for informed consent. Information was given to all patients.Two datasets of medical MR images were acquired in two distinct centers:The DATASET 1 consisted in multi-parametric MR images of 74 lacrymal gland lesions prospectively collected from December 2015 to April 2017 in a tertiary referral center specializing in ophthalmic diseases, using a 3 Tesla Philips INGENIA device with a 32-channel head coil . The MRI protocol, which included 6 MR sequences, is detailed in The DATASET 2 consisted in MR images of 30 breast lesions prospectively collected from November 2017 to April 2018 on a 1.5 Tesla General Electric MR scanner using a phased array dedicated breast coil covering both breasts. Patients were imaged in the prone position. The sequence of interest was an ultrafast dual-echo 3D spoiled gradient recalled acquisition sequence called DISCO , with pseudorandom variable density k-space segmentation, with an elliptical model where the central k-space regions were acquired more frequently than the peripheral region and a view-sharing reconstruction. The MRI acquisition parameters are detailed in Two readers reviewed the whole imaging DATASET 1, a senior neuro-radiologist specialized in orbital imaging with 8 years of experience (*blinded*) and a junior radiologist with 6 months of experience (*blinded*), blinded to patient ID, medical history, lab results and pathological results. Each reader independently performed a 2D manual delineation of the two lacrimal glands of each patient on each MR sequence. The slice of delineation was independently chosen by each reader, considered as the slice where the lesion had the largest diameter. The delineation included both the tumor and the normal glands. A total of 444 ROIs were drawn by each reader . One reader (*blinded*) performed a second segmentation session to assess the intra-observer reproducibility. The segmentations were performed using an in-house software developed on Matlab  and dedicated to the radiomics analysis .Two junior radiologists reviewed the whole imaging DATASET 2  blinded to patient ID, medical history, lab results and pathological results. Each reader independently performed a 2D manual delineation of the breast lesions on the DISCO sequence. The slice of delineation was independently chosen by each reader, considered as the slice where the lesion had the largest diameter. One reader (*blinded*) performed a second segmentation session to assess the intra-observer reproducibility. The segmentations were performed using the ITK-SNAP software  .To overcome the potential bias of the small number of readers that may not represent the true inter- and intra-observer segmentation variability, we developed a computer simulation of a high number of delineations (5000) based on morphological binary openings, closings and elastic transforms applied to a manual reference delineation for each ROI of DATASET 1. The Dice coefficient, which is a measurement of similarity between two segmentations, was calculated between each simulated ROI and the human reference one. Then, one simulated segmentation per Dice coefficient interval of 0.05 from 1.0 (no transformation) to 0.65 (maximum tested transformation) was randomly selected. This interval from 1.0 to 0.65 was chosen to encompass the whole variability different manual segmentations may provide. This led to 7 randomly selected simulated ROIs for each associated human reference ROI with Dice coefficients ranging from 0.65 to 0.95. The segmentations simulations were visually checked to make sure it was consistent with a potential manual delineation variability. An example of computer simulations is given in Two methods of discretization were used:An absolute discretization with fixed bin size (FBS method), where a new bin is assigned to pixel intensities each BS gray level, starting from 0, according to the following BS(x) the discretized gray-level of voxel x. The term [min(I(x)/BS) + 1] ensures that the bin count starts at 1. We tested six different bin sizes: 1, 5, 10, 20, 25 and 50.Where I(x) is the intensity of voxel x, BW the bin size and IA relative discretization with fixed bin number (FBN method), starting from the minimum intensity value of the segmented area and defined as follows:BN(x) the discretized gray-level of the voxel x. We tested eight different fixed bin numbers: 8, 16, 32, 64, 128, 256, 512 and 1024.Where I(x) is the intensity of voxel x, BN the bin number and IWe performed the feature extraction for each discretization using two radiomics-dedicated softwares:Griethuysen et al., version 1.3.0) [Pyradiomics open-source software (n 1.3.0)  was usedAn in-house software developed on Matlab  and dedicated to the radiomics analysis  was alsoTexture features extracted are listed in In order to select features that are reproducible between observer delineations, we performed a 2-way random intraclass correlation coefficient (ICC)  on pair combinations of readings  and a Lin’s concordance correlation coefficient (CCC) on the intra-observer pair. A feature was considered as highly reproducible and therefore selected if all three ICC values were above 0.8 and the CCC value was above 0.9. All other features were eliminated.For each discretization method and each MR sequence of DATASET 1, we analyzed the highest number of reproducible texture features obtained from the Pyradiomics software with FBS versus FBN discretization methods, using an exact Fisher test. We also assessed if there was a difference in reproducible feature distributions among chosen bin sizes for FBS or among chosen bin numbers for FBN, using Chi-square tests.For each discretization method and each MR sequence, we repeated the same analysis on the data extracted from the DATASET 1 using our in-house feature extraction software.For each discretization method, we repeated the same analysis using the Pyradiomics software on DATASET 2.Feature Extraction paragraph, using the Pyradiomics software.Texture features were extracted from each Dice coefficient associated ROI as described in the We focused on the simulated ROIs associated with a Dice coefficient of 0.85 because the mean Dice coefficient of manual delineations of pooled sequences of DATASET 1 (Experiment 1) was 0.84. We performed feature reduction according to the delineation variability by selecting features for which the ICC between the reference human ROI (Dice 1) and the simulated ROI (Dice 0.85) was above 0.8. We then performed the same analysis as described in previous sections.We also analyzed the variation of the highest number of reproducible features according to the Dice coefficient for FBS methods versus FBN methods.The flowchart of the study is detailed in All feature extractions and discretization methods were implemented using Python language for Pyradiomics software and Matlab language for our in-house feature extraction software. All statistical analyses of extracted data were performed using R-3.3.3  . P -valuThe results of the experiments 1, 2, 3 and 4 are detailed in the Tables In each of the above experiments, the gray-level discretization method impacted both the number and the distribution of reproducible features observed. Overall, the highest number of reproducible features was consistently obtained using FBS methods. The difference between FBS and FBN methods was significant in most cases. An illustration of the highest number of reproducible texture features for the FBS and FBN methods in each experiment is given in The highest number of reproducible features extracted from computer simulated delineations decreased according to the Dice coefficient from 1 to 0.65. The number of reproducible features obtained from the FBS method was much less impacted by variability of ROI delineation than the number obtained by the FBN method, and the difference between FBS and FBN methods increased with the decrease of the Dice coefficient. The results for the water DIXON-T2-WI are illustrated in Whatever the discretization method, the DATASET and the MR sequence, the three most reproducible features were: GLRLM Gray-Level Non-Uniformity, GLDM Gray-Level Non-Uniformity, GLDM Dependence Non-Uniformity. The three least reproducible features were: GLDM Large Dependence Low Gray-Level Emphasis, GLCM Cluster Shade and GLRLM Long Run Low Gray-Level Emphasis.We assessed the influence of gray-level discretization methods on radiomics texture feature inter- and intra-observer reproducibility in two independent clinical MR datasets. Texture feature reproducibility was shown to substantially depend on the gray-level discretization method. The absolute discretization (FBS) method was found to provide a higher number of reproducible features than the relative discretization (FBN) method. The relative discretization was also found to give more different results when varying the bin numbers than the absolute discretization when varying the bin sizes.Although the gray-level discretization has been assessed in PET and CT studies –8, few sWe assessed the influence of gray-level discretization on the inter and intra-observer reproducibility of texture features because most of the radiomics studies currently integrate the inter and intra-observer reproducibility as a data reduction step to select robust features and exclude all others ,15–17. TOur results may be explained by the calculation process used to perform relative and absolute discretization methods. When using relative discretization (FBN), the intensity range of the segmented image will impact the bin size, which in turn determines the intensity histogram, the co-occurrence and other derived matrices used to compute texture features. Most of the inter and intra-observer segmentation variability is related to slightly different contours providing intensity outliers, thereby changing the intensity range of the region of interest. Conversely, the absolute discretization (FBS) method is independent from the intensity range of the segmented image and so may be less sensitive to inter- and intra-observer delineation variability.We used two independent and very different datasets for external validation, two pairs of readers independently delineating the datasets, computer simulations of manual delineation variability, two delineation softwares to get rid of the delineation software confounding factor, and two radiomics softwares for which the calculation method of gray-level co-occurrence matrices and other derived matrices was slightly different. The purpose was to determine whether our results applied to different data, quantification methods, or organs. The fixed bin size method of gray-level discretization consistently provided less variable results. There are currently no specific guidelines to address the choice of the best bin width or bin number. Available studies use bin numbers varying from 8 to 1000, as suggested by the IBSI ,14, or bThis study has some limitations. We did not normalize signal intensity on the MR images before extracting texture features. The IBSI recommends to use the normalization on raw MR data because it is based on arbitrary units. The normalization may impact texture feature values, especially when dealing with multiple MR machines and when comparing feature values among patients ,19,20. HThis study showed that the gray-level discretization of MR images influences the number and distribution of inter and intra-observer reproducible texture features. We showed that using a fixed bin size method (absolute discretization) allowed preserving a higher number of reproducible features which may be potential imaging biomarkers candidates. Considering the impact of the discretization method on results, the precise method used should be clearly documented in each radiomics study to improve the replicability of the results, as recommended in the recent evaluation criteria and reporting guidelines published to improve reliability, comparability and generalizability of radiomics-based studies . The staS1 TableThe DATASET 1 included 6 MR sequences: T1 Weighted Images (WI); Apparent Diffusion Coefficient (ADC) maps calculated voxel-wise as the linear slope of signal decrease between b0 and b1000 of Diffusion Weighted Imaging (DWI) acquisitions; In-Phase and Water DIXON-T2-WI ; Post-contrast In-Phase and Water DIXON-T1-WI , obtained after administration of intravenous contrast injection of a single bolus (0.1 mmol/kg) of Gadobutrol . The DATASET 2 included 1 MR sequence called DISCO .(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableReproducible features using the Pyradiomics software and manual delineations on DATASET 1 according to the gray-level discretization. The intensity normalization was performed as follows: images were centered by their mean and scaled by their standard deviation; intensity values below or above three standard deviations from the mean were excluded; intensity values were shifted to get only positive values; intensities were then scaled to the initial common range . The normalization was not applied to the parametric ADC map.(DOCX)Click here for additional data file.S5 TablePyradiomics computes one matrix per distance and/or angle (according to the texture feature), normalizes the matrix, calculates one feature value per normalized matrix and averages the results to obtain the final texture value. Our in-house software computes a weighted sum of all non-normalized matrices obtained with the different distances/angles , then directly calculates the final feature value based on this unique matrix. These two methods are described in the IBSI guidelines. There is no current recommendation to favor one method over another.(DOCX)Click here for additional data file.


The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The  vice versa. We discovered that psbA mRNA, which encodes a protein that is particularly prone to photodamage, exhibits dynamic changes in ribosome occupancy in response to light and that it is unique in this regard. Ribosome occupancy on other chloroplast mRNAs is static even after many hours in the dark; however, these mRNAs are nonetheless translated at reduced rates in the dark due to a reduced rate of ribosome movement. Resolution of these superimposed effects clarifies the phenomenology of light-regulated chloroplast translation and provides a basis for exploring underlying mechanisms.Our experiments address the effects of light on protein synthesis within chloroplasts, whose ~80 genes are essential for photosynthesis and account for a large fraction of the protein synthesis in leaf tissue. Light is necessary for photosynthesis but it also triggers photo-oxidative damage. It is known that light-induced changes in chloroplast translation aid photosystem repair and prioritize energy consumption in the dark. However, prior studies have been limited in scope and offer conflicting views of the nature of these responses. In this study, we used new methods to generate a comprehensive description of chloroplast translation in maize at various time scales after shifting plants from dark to light and  In an elaborate repair cycle, damaged D1 is degraded and then replaced with newly synthesized D1 [psbA translation has been intensively studied due to its central role in maintaining photosynthetic homeostasis and the ease of monitoring D1 synthesis. However, light-regulated translation in chloroplasts is not limited to the psbA mRNA [rbcL mRNA, whose translation initiation and elongation rates have been shown to change in response to light [Energy from sunlight fuels life on earth through the process of photosynthesis. Light is both an essential resource and a source of stress for photosynthetic organisms, as it damages cellular structures through photo-oxidative processes and the production of reactive oxygen species. Photosynthesis is compromised when excess light damages the Photosystem II (PSII) reaction center or when excitation of Photosystem I (PSI) and PSII is unbalanced , 2. A kesized D1 , 4. To csized D1 . The phesbA mRNA . This hato light –12.in vivo.Despite a large body of literature on light-regulated chloroplast translation, major gaps remain in the characterization of the basic phenomenology: i.e. which genes respond to light at the translational level, with what kinetics, and at which step in translation. Studies have been limited to the few most rapidly synthesized proteins, so no information is available for the majority of the chloroplast’s ~80 protein-coding genes. In addition, many studies examined light responses in plants that had been grown for extended periods in the absence of light, at which point effects on chlorophyll synthesis and energy supply confound data interpretation. For example, an influential series of reports employed a de-etiolation regime in which barley seedlings were germinated and grown in the absence of light for many days. Illumination of these plants triggered the incorporation of radiolabeled amino acids into chlorophyll binding proteins in isolated chloroplasts, a result that was initially interpreted as light-induced translation . Howevervice versa. We distinguished effects of light on translation initiation and elongation rates by following the kinetics of ribosome occupancy after introducing lincomycin, which specifically inhibits ribosomes at the first few codons in an open reading frame (ORF). Our results show that transition from dark to light is accompanied by a global increase in the rate of translation elongation in chloroplasts, and that the rapid recruitment of ribosomes to the psbA mRNA is superimposed on this global response. Surprisingly, the abundance and positions of ribosomes on all chloroplast mRNAs other than psbA are maintained largely unchanged even after 12 hours in the dark, implying a balanced decrease in rates of initiation and elongation. This comprehensive analysis clarifies the phenomenology of light-regulated chloroplast translation and provides a basis for mechanistic hypotheses to be tested in future studies.In this study, we revisit the phenomenon of light-regulated chloroplast translation using ribosome profiling , a metho-2s-1) to minimize photodamage. Three biological replicates were performed for each comparison.We grew maize seedlings for 8 days in light-dark cycles. Plants at this stage are photosynthetically competent but have not exhausted seed reserves. We subjected these plants to one of three light-shift regimes : (i) PlaLeaf tissue was processed for ribosome profiling (Ribo-seq) and RNA-seq analysis as described previously . Ribo-sepsbA RNA is highly dynamic in response to light, increasing approximately 6-fold after 15 minutes of light at dawn, decreasing approximately 8-fold after 1 hour in the dark at midday, and increasing approximately 8-fold after 15 minutes of reillumination at midday . Strikingly, the psbA mRNA was the only chloroplast mRNA to behave in this manner: ribosome footprint abundance on all other chloroplast ORFs changed less than two-fold in each comparison. The abundance of psbA mRNA did not vary , indicating that the changes in ribosome footprint abundance are due solely to effects on translation. Several other mRNAs showed small changes in ribosome density (ratio of Ribo-seq to RNA-seq reads) in response to light, but the change exceeded 2-fold only for ndhJ at midday (psbA changes very little even after extended times in light or dark.The results are presented in t midday  bottom. psbA maintained similar ribosome density (Ribo-seq/RNA-seq) at all time points. The Ribo-seq RPKM from psbA after 1 or 12 hours in the dark was similar to that from other genes encoding photosystem subunits. Illuminating plants for 15 minutes restored psbA ribosome occupancy to the same level as that in plants that had been maintained in the light for 7 hours.Normalized read counts for each condition are plotted separately in psbA mRNA shifted toward lower molecular weight fractions shortly after shifting to the dark (and vice versa), whereas the sedimentation profiles of the rbcL and atpB/E mRNAs were indistinguishable even when comparing plants that had been in the dark or light for many hours  or intact plants (rbcL) to the dark [in vivo pulse-labelling assays to examine how the rates of RbcL and PsbA synthesis change over time after shifting plants to the dark or light  under any single condition , 23, thethe dark , 24–27. or light . Pulse-lor light , precedipsbA changes very little following shifts from light-to-dark or dark-to-light, even after many hours in the new condition. This is consistent with two scenarios: either light has minimal effect on their translation or it triggers concerted changes in rates of initiation and elongation such that the average number of ribosomes associated with each mRNA is maintained. The data shown in psbA and rbcL mRNAs does decrease soon after a shift to the dark. However, it is not known whether other chloroplast mRNAs are similarly affected.Results above show that ribosome occupancy on all chloroplast ORFs other than To provide a plastome-wide view of the effects of light on translation elongation, we performed ribosome profiling over a time course following treatment of seedlings with the peptidyl-transferase inhibitor lincomycin . LincomypsbA mRNA is reduced to its dark steady-state level  and ribosome density (Ribo-seq/RNA-seq) following one hour in the dark at midday, and restoration to the original level after 15 minutes of reillumination (psbA and was not accompanied by a change in ribosome distribution (ndhJ and ndhK). In general, RNA levels were more dynamic in Arabidopsis than in maize over the conditions sampled. In particular, RNAs encoding many ribosomal proteins and NDH subunits increased after one hour in the dark and even more at the end of the night. For most ribosomal genes, this increase in RNA was reflected by an increase in ribosome footprint abundance such that ribosome density (Ribo-seq/RNA-seq) remained fairly constant. However, for most ndh genes, ribosome footprint abundance did not increase proportionally to RNA abundance in the dark, resulting in reduced ribosome density .We repeated a subset of experiments with Arabidopsis to determine whether light affects chloroplast translation similarly in a C4 monocot (maize) and a C3 dicot (Arabidopsis) . Similarmination . Also asribution , implyinpsbB, psbH, psbK, ndhC, rps11). Thus, although ribosomes remained associated with all RNAs through the night in both species, there was generally more clearing of ribosomes over the night in Arabidopsis.The only striking difference between the data for Arabidopsis and maize involved ribosome density at the end of the night  also decreased following the dark shift and increased after reillumination, albeit with less dynamic range than D1 synthesis. Decreased synthesis of these proteins in the dark was not reflected by reduced ribosome footprint abundance  ribosomes are gained and lost on the psbA mRNA shortly after shifting plants to the light or dark, respectively; (ii) the rate of translation elongation decreases globally following a shift to the dark. The superposition of a gene-specific recruitment of ribosomes to psbA on the global increase in translation elongation upon shifting from dark to light programs a massive increase in D1 synthesis. Ribosome occupancy and distribution on all other chloroplast ORFs changes very little, whether comparing short or long times (e.g. 15 minutes versus 7 hours) after the shift from one condition to another. That ribosome occupancy remains constant despite the global change in elongation rate suggests that initiation rates change roughly in concert with elongation rates on all chloroplast ORFs except psbA.Results presented here provide a plastome-wide accounting of ribosome occupancy, distribution, and elongation rate at various time scales following the transfer of maize seedlings with developed chloroplasts from dark to light, and psbA translation [psbA) had been assayed, (ii) psbA-specific effects were rarely resolved from plastome-wide effects, (iii) in organello translation assays generally monitored only the elongation phase and employed ATP and reducing agents, which may have obscured light-induced effects that rely on fluctuations in these signals. By providing a comprehensive view of which chloroplast ORFs respond to light in plants harboring developed chloroplasts, over what time scales, and at what step in translation, our results provide a broad and relatively physiological context in which to interpret this large body of prior work. Our findings validate some prior views, expand on others, and suggest that some commonly held assumptions are incorrect.Most prior studies of the effects of light on chloroplast translation involved assays in isolated chloroplasts, and/or illumination of plants that had been germinated and grown in the absence of light and therefore lacked chlorophyll. These approaches provided important insights into the role of chlorophyll in stabilizing nascent chlorophyll binding proteins –32 and tnslation , 33–35. nslation , 36, 37.psbA translation in chloroplasts [psbA ribosome occupancy changes rapidly when green plants are shifted between dark and light may appear to be nothing more than confirmatory. However, prior experiments involved illumination of undeveloped chloroplasts (de-etiolation) and/or failed to address whether light increases the rate of psbA translation initiation over and above any global stimulation. To our knowledge, the only prior studies to examine the effect of light on psbA ribosome association in plants involved the illumination of etiolated barley seedlings [psbA and rbcL mRNAs were recruited to polysomes following illumination, but this was mirrored by a global increase in chloroplast polysome content. A second set of experiments cited as evidence for light-induced psbA translation initiation employed a reporter gene fused to the psbA 5’UTR in tobacco plastids [It is well known that light stimulates roplasts , 38, 39,eedlings , 40, 41;plastids , 43. HowpsbA translation by light has been intensively studied in the single-celled alga Chlamydomonas reinhardtii. The prevailing view from this body of work is that psbA translation is regulated at different steps in the contexts of PSII biogenesis and repair; the former is believed to involve regulated initiation and the latter regulated elongation [psbA translation initiation we observed is a homeostatic repair mechanism: the response takes place in the context of developed chloroplasts, it is triggered within minutes of the shift to light, and it results in a substantial over-production of D1 with respect to other PSII subunits. Thus, our results add an important piece to this understanding by showing that light induces a rapid increase in the rate of translation initiation on the psbA RNA over and above any global increase in developed plant chloroplasts, and that this does not require excessive light intensities. The physiological and environmental contexts of light-regulated psbA translation are entirely different in land plants and Chlamydomonas: the former are sessile, multicellular, and land-dwelling whereas the latter are motile, single-celled, and aquatic. It would not be surprising if distinct mechanisms have evolved to maintain PSII homeostasis in these different contexts. That said, psbA mRNA in Chlamyomonas is lost from polysomes after one hour in the dark and regained within ~15 minutes of illumination at a moderate light intensity [psbA, they do suggest that light may regulate psbA translation at the initiation step in the context of PSII repair (and not just de novo biogenesis) in Chlamydomonas, as in plants.The regulation of ongation , 38, 44.ntensity , much aspsbA, whose translation elongation rate decreased and increased when isolated chloroplasts were shifted to the dark and light, respectively [rbcL, which maintained polysome association after shifting Amaranth seedlings to the dark despite reduced RbcL synthesis [psbA translation elongation previously detected in isolated chloroplasts occurs also in vivo. Second, we showed that the effects on both rbcL and psbA are not gene-specific, but rather reflect a plastome-wide change in translation elongation rate. Our results leave open the possibility that the magnitude of this effect varies among ORFs, but additional experiments would be required to make firm conclusions in this regard.Classic studies demonstrated that the rate of translation elongation on several chloroplast mRNAs in plants changes in response to light. The two primary examples focused on (i) ectively –27, 46, ynthesis . By usinpsbA RNA in barley chloroplasts [psbA translation by altering pausing at specific sites. A subsequent study showed that toe-print patterns change many hours after illuminating etiolated barley seedlings [psbA mRNA in mature maize and Arabidopsis chloroplasts over the time scales we examined. Similar results were obtained for all other chloroplast ORFs. Thus, our results provide strong evidence that light does not have any major effects on ribosome pausing on chloroplast mRNAs. Although ribosome dwell time can be influenced by RNA structure, the sequence of the nascent peptide, and other features [Our results also clarified the effects of light on ribosome pausing. Pioneering ribosome toe-printing assays had revealed that ribosomes pause at specific sites on the roplasts , leadingeedlings . By contfeatures , 47, thepsbA was the only ORF to experience a substantial increase in ribosome occupancy following a transfer to light was unexpected because light has been reported to activate translation of several other chloroplast mRNAs via effects on proteins that bind their 5’-UTRs [psbA was maintained almost unchanged even after twelve hours in the dark was also unexpected. Although there is prior evidence that ribosomes remain bound to several chloroplast mRNAs in the dark [psbA), and that it applies to no more than a handful of RNAs in Arabidopsis. Although we did not directly measure initiation rates, our measurements of ribosome occupancy and elongation rates suggest that ribosome occupancy is maintained through a concerted reduction in the translation elongation and initiation rates. The maintenance of ribosome association even after many hours in the dark may have evolved as a means to protect mRNAs from degradation, and to allow a rapid resumption of translation upon exposure to light.Our finding that  5’-UTRs . That rithe dark , 36, it the dark . Our respsbA-specific manner. The light-induced signals that trigger psbA-specific ribosome recruitment, the plastome-wide increase in elongation rate, and the plastome-wide increase in initiation rate may be shared or distinct. Studies involving isolated chloroplasts treated with inhibitors of photosynthetic electron transport [trans-thylakoid proton gradient have all been invoked as potential triggers of light-induced effects on chloroplast translation [psbA-specific response from the global responses, and that assess the effects of disrupting specific steps in photosynthesis in intact plants will be required to pinpoint the photosynthesis-dependent signals that regulate chloroplast translation.Our results provide a foundation on which to delve into the mechanisms underlying each layer of light regulation, including the nature of the light-induced signal(s), the proteins whose activities are modulated by those signals, and the mechanisms by which these stimulate or inhibit translation in either a global or ransport , 35, 37 vice versa), strongly suggesting that the effects are mediated by post-translational modifications of pre-existing proteins. Candidates for proteins that regulate psbA-specific ribosome recruitment include HCF173 and HCF244, which are required specifically for psbA translation initiation in Arabidopsis [Changes in translation can be observed within minutes of shifting plants from light to dark  seedlings were grown on agar plates , 0.3%(w/v) Phytogel, 1% (w/v) sucrose, pH 5.8). Seeds were planted with 1-cm spacing to minimize shading. Plants were grown at 22°C under light/dark cycles of 16 h/8 h for 14 days for ribosome profiling, or for 10 days for pulse labeling. Light intensity was approximately 100 μmol m-2s-1, consistent with typical Arabidopsis growth light intensities and much lower than high-light treatments [2 and stored at -80°C. The treatment and harvest of maize and Arabidopsis in the dark were performed under a dim green light.Arabidopsis  and seedlings remained intact. Cotton threads (DMC crochet thread size 5) soaked in 1 mg/ml lincomycin (Sigma) were sewn four times through the stem of each seedling beneath the first (lowest) leaf. With 2 threads in each sewing, each wick consisted of 8 threads. The threads were cut at ~5 cm and the ends were placed in a 1.5 ml tube containing lincomycin. The apical half of leaves two and three were processed for ribosome profiling.35S Protein Labeling Mix  in 10 μL was added to the wounds. After 15-min of labeling, a 3-cm tissue section spanning the wounds was harvested and frozen in liquid N2. Plants were illuminated with a light intensity of 250 μmol m-2s-1 or maintained in the dark, as indicated. The tissue was homogenized in protein homogenization buffer . Lysates were fractionated by SDS-PAGE and transferred to nitrocellulose. Radiolabeled proteins were then detected with a Storm phosphorimager. Because the labeling efficiency of the leaf scratch method is variable, we used radiolabeled cytosolic proteins to normalize sample loading. This precluded the use of cycloheximide, which limited the number of chloroplast gene products we could resolve.For maize, the labeling was performed as in . In brie35S Protein Labeling Mix. Labeling was performed for 20 min under a light intensity of approximately 100 μmol m-2s-1 . After labeling, leaves were washed once in the labeling buffer (lacking 35S) and then frozen in liquid N2. The tissue was homogenized as for maize. The membrane fraction was collected by centrifugation at 13,000 x g for 5 min and washed once with the homogenization buffer. Samples were fractionated in SDS-PAGE gels containing 6M urea (11% polyacrylamide), with loading normalized on the basis of equal chlorophyll , 1% (w/v) sucrose, pH 5.8). Labeling was then initiated by the addition of 15 μl of EasyTag Express ed as in ). ProteiWith the exception of the first replicate of the experiment in Ribo-seq libraries were prepared using the NEXTflex Small RNA Sequencing Kit v2 or v3 (Bioo Scientific) with additional steps described previously . rDNA warps12, where exon 2 was used. The rationale for these choices was discussed previously [Read processing, alignment and analysis were performed according to the procedures described previously . In brieeviously .For the lincomycin assays, the ribosome run-off time course was determined from the RPKM values in the ORF body (from codon 8 to the stop codon). Because maize chloroplast ribosome footprints translating the same codon share a similar 3’-end position regardless of footprint size , the norRead mapping statistics and chloroplast RPKM values from all the experiments are provided in psbA, atpB/E, rbcL and ndhJ probes used for RNA gel blots correspond to maize chloroplast genome nucleotide positions 295–1074, 54590–55790, 57036–57607 and 50535–51014, respectively.Polysome analyses were performed as described previously . The psbmaizegdb.org) was used for other genomes. The gene set from maize genome annotation 6a (phytozome.jgi.doe.gov) was reduced to the gene set annotated in 5b+  (gramene.org). For Arabidopsis, we used TAIR10 genome and annotation (arabidopsis.org).Illumina read sequences were deposited at the NCBI Sequence Read Archive with accession number SRP133508. Alignments of reads to the maize chloroplast genome used Genbank accession X86563. B73 RefGen_v3 assembly (S1 Table(XLSX)Click here for additional data file.S1 FigA) Size distribution of ribosome footprints. Values are the mean ± SEM from all fifteen samples analyzed in (B). Three-nucleotide periodicity of Ribo-seq data. The frame placements were inferred from the locations of the 5’ ends of the different footprint sizes at the start and stop codons. Values are the mean ± SEM, from the same samples described in (A).atpI-atpH-atpF-atpA transcription unit. The lower panels show Ribo-seq reads mapping to the same genes.(C) Confinement of Ribo-seq reads to ORFs in chloroplasts. The upper panel shows RNA-seq read coverage across the chloroplast (D) Metagene analysis of reads mapping near all cytosolic start and stop codons. Read coverage is quantified from the same datasets summarized in (A).(E) Pearson correlation coefficients between each sample pair combination were calculated using RPKM values for each protein coding gene in the chloroplast genome. The correlation coefficients were used as the input for hierarchical clustering. The number following each sample name refers to the replicate.(F) Histogram showing depth of sequencing data. The plot shows the frequency distribution of chloroplast genes according to read count in the indicated maize Ribo-seq (top) or RNA-seq (bottom) light-shift datasets (mean of 3 replicates).(TIF)Click here for additional data file.S2 FigThe data are the same as those in (TIF)Click here for additional data file.S3 FigThe experiment was performed as described in (TIF)Click here for additional data file.S4 FigpsbZ and psbH locate in the stop codon and near sequences encoding the first transmembrane segment, respectively.Ratios of normalized read coverage for each of the light-shift comparisons diagrammed in (TIF)Click here for additional data file.S5 FigSize distribution of chloroplast ribosome footprints at start codon regions (first 7 codons), in ORF bodies (codon 8 to stop codon), and at non-start codon regions that captured ribosomes following lincomycin treatment. The mean ± SEM are shown from 2 replicates. The build-up of ribosomes at start codons over the lincomycin time course reflects the fact that lincomycin does not inhibit initiation but does inhibit clearance from start codons. For the analysis of ribosome build-up at non-start codon regions (bottom), we selected sites at which ribosome occupancy increased >5-fold in the light after 30 min of lincomycin treatment (87 sites).(TIF)Click here for additional data file.S6 Fig(A) Distribution of ribosomes along the indicated chloroplast ORFs following LIN treatment in the dark or light. The plots show the normalized abundance of ribosome footprints with 3’ ends at each position. Plots for each of two replicates are shown in separate graphs to illustrate reproducibility (replicate shown in parentheses). The region occupied by initiating ribosomes (first 7 codons) is shaded in gray.(B) Examples of ORFs that accumulate ribosomes at specific sites during the LIN time course. The non-start codon positions at which ribosomes accumulated more than 5-fold after 30 min LIN treatment in light are marked with vertical dashed lines.(C) Correlation plot showing the ratio of the normalized RPKM as in  in dark psbA) was slower in Replicate 1. This difference correlates with the regions of the leaf at which these genes are preferentially expressed: ribosomal protein genes peak in translational output in the basal region, photosynthesis genes peak in the apical region, and psbA is one of just a few genes whose output increases all the way to the leaf tip [(D) Correlation plot showing the normalized RPKM in dark after 30 minutes of LIN treatment. Each symbol represents the data for one ORF. A higher value indicates slower ribosome clearance from the ORF body. The clearance of ribosomes from ribosomal protein genes was similar in the two replicates, whereas the clearance from photosynthesis genes Click here for additional data file.


Pembrolizumab therapy was discontinued and a complete remission of PNP was obtained using oral steroids. Reintroduction of pembrolizumab resulted in flare of PNP. Given the close temporal relation between pembrolizumab initiation and the subsequent clinical expression of a widespread PNP, the patient was diagnosed with pre-existing subclinical PNP exacerbated by PD-1 inhibitor. The extreme rarity of PNP in the setting of cutaneous SCC and the effects of challenge, dechallenge, and rechallenge of pembrolizumab argue in favor of a checkpoint inhibitor related adverse effect. Our case is the first PNP associated with anti-PD-1 therapy and serological follow-up suggest that one infusion of pembrolizumab is sufficient to allow clinical expression of underlying pemphigus auto-immunity.A 64-year-old patient developed a widespread autoimmune mucocutaneous blistering disease 3 weeks after the initiation of the anti-programmed death-1 (anti-PD-1) pembrolizumab therapy administered for a locally advanced cutaneous squamous cell carcinoma (SCC) of the buttocks arising from hidradenitis suppurativa. A diagnosis of paraneoplastic pemphigus (PNP) was made based on the presence of a suprabasal acantholysis associated with intercellular deposits of immunoglobulin G and C3 on basement membrane zone. Analysis of the patient's sera was positive on monkey bladder and detected circulating antibodies against desmoglein 3 and desmoplakin I prior to the initiation of pembrolizumab. At that time, the patient had few localized blisters limited to the peri-tumoral skin of the buttocks with acantholysis but without  The immune system has developed sophisticated negative regulatory mechanisms to contain the development of autoimmunity. These regulatory mechanisms can be diverted by cancer cells to limit antitumor immunity. By counteracting these inhibitory signals, cancer immunotherapy aims to enhance immune responses to fight against tumors. Among several immunotherapeutic strategies tested these recent years, immune checkpoint inhibitors have come to the forefront in cancer treatment, showing remarkable benefit in the treatment of a wide range of cancer. By blocking negative regulators of lymphocyte activation, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or its ligand, programmed cell death ligand 1 (PD-L1), immune checkpoint inhibitors restore the function of effector T cells to target and destroy cancer cells. The drawback of inducing an effective immunity targeting cancers is the potential for autoimmune side effects, which are commonly termed immune-related adverse events (irAEs) . A majorCutaneous side effects are among the most frequent checkpoint inhibitor irAEs. Skin related toxicity of any grade may be seen in up to 25% of patients treated with antibodies targeting CTLA-4  and arouA 64-year-old man of Algerian origin presented with a 20-year history of sinuses and abscesses of the buttocks that had been treated with several minor surgical procedures over the past 10 years. A family history was noted because the patient's son had similar early-stage lesions. Clinical examination revealed a diffuse involvement of the buttocks, perineal, and perianal regions, with multiple interconnecting tracts and abscesses across entire areas . He was Physical examination also revealed few small intact bullae on the buttocks and painful erosions of the buccal mucosa. There were no vesicular or bullous lesions on the upper trunk, arms, legs or extremities, and no history of a prior bullous eruption. Mouth swabs were positive for herpes simplex virus 1 (HSV-1) DNA. Histopathological examination of a buttock blister revealed intraepidermal blistering and suprabasal acantholysis. Direct immunofluorescence (DIF) was negative for deposits of immunoglobulin (Ig) G, IgA, IgM, and C3. A diagnosis of pemphigus could therefore not be confirmed. The patient was treated with oral valaciclovir, which resulted in the almost complete healing of the oral and cutaneous lesions.Due to the poor performance status  and high-risk of sepsis-related complications, cytotoxic chemotherapy was contraindicated for SCC treatment, and a treatment by anti-programmed cell death protein 1 (anti PD-1) was decided. One week after the first pembrolizumab administration (2 mg per kg), an intense inflammatory swelling affecting the HS areas was noted, followed by a relapse of the bullous cutaneous eruption . Three wSeveral series of skin punch biopsies were performed for histopathologic examination and DIF studies . Histolode novo autoimmune disease. Therefore, we performed immunoblot analysis on the patient's serum collected 7 days before initiation of pembrolizumab therapy. Immunoblotting indicated the presence of circulating antibodies against the desmosomal components desmoplakin I and desmoglein 3. Consistently, IIF performed on monkey bladder epithelium using the same serum revealed a positive staining. Together, these results supported the presence of a subclinical PNP before the initiation of pembrolizumab  between pembrolizumab initiation and the widespread mucocutaneous eruption, we suspected that PD-1 blockage was associated with the aggravation of a preexisting condition, rather than the development of a olizumab .The pembrolizumab therapy was withheld and oral prednisone at 1 mg per kg daily was started. After 3 weeks of steroid treatment, complete clinical remission of the PNP was obtained and anti-desmoplakin I antibodies were no more detectable by immunoblotting . Given tA wide range of inflammatory skin disorders has been observed in patients treated with checkpoints inhibitors, including autoimmune blistering diseases. Current anti-PD1/PD-L1 therapy-associated autoimmune blistering diseases reported in the literature (including our patient) are presented in PNP may have developed independently of pembrolizumab therapy. However, several data are suggestive of a checkpoint inhibitor related adverse effect in our patient: (1) PNP has never been reported previously in the setting of a cutaneous SCC, making the combination of PNP and cutaneous SCC an extremely rare phenomenon; (2) the close temporal relation between pembrolizumab initiation and the subsequent development of PNP clinical lesions ; (3) the favorable outcome after pembrolizumab interruption and corticosteroids treatment ; (4) the relapse after pembrolizumab reintroduction . Using the Begaud scoring system , we asseSeveral pathophysiologic models for PNP have been proposed. First, neoplasms arising from immune cell expansion have been shown to dysregulate B-cell homeostasis resulting in the subsequent production of autoantibodies targeting desmosome components. This is best illustrated by the identification of B-cell tumor clones producing antibodies targeting desmosomal plakin proteins in resected Castleman tumors , thymomaIn summary, we reported a patient with extensive skin SCC who developed PNP after one infusion of pembrolizumab. The presence of autoantibodies directed against desmoplakin I and desmoglein 3 was demonstrated before the anti-PD-1 therapy. PNP in this patient was clearly induced since rechallenge was positive. So PNP should be added to the list of immune-related adverse events associated with checkpoint blockade such as BP.The datasets generated for this study are available on request to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AY, GB, and FC designed the study. SG-M conducted the immunological analyses. CP-S and AM performed the pathological studies. MA and CL collected clinical data. AY wrote the first draft of the manuscript. GB did modifications. FC and EM corrected the final version. All authors contributed to manuscript revision, read and approved the submitted version.FC is a consultant for Principia Biopharma. EM is consultant for MSD and has received support for congress from BMS and MSD. The remaining 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.


Patients undergoing major cancer surgery frequently require post-acute care for complications and adverse effects. Enhanced recovery after surgery programmes mean that patients are increasingly discharged home earlier. Symptom/complication detection post-discharge is sub-optimal. Systematic patient monitoring post-discharge following surgery may be optimally achieved through routine electronic patient-reported outcome (ePRO) data capture. ePRO systems that employ clinical algorithms to guide management of patients and automatically alert clinicians of clinically-concerning symptoms can improve patient outcomes and decrease hospital admissions. ePRO systems that provide individually-tailored self-management advice and integrate live ePRO data into electronic health records (EHR) may also advance personalised health and patient-centred care. This study aims to develop a hospital EHR-integrated ePRO system to improve detection and management of complications post-discharge following cancer-related surgery.The ePRO system was developed in two phases: (1) Development of a web-based ePRO symptom-report from validated European Organisation for Research and Treatment of Cancer (EORTC) questionnaires, clinical opinion and patient interviews, followed by hospital EHR integration; (2) Development of clinical algorithms triggering symptom severity-dependent patient advice and clinician alerts from: (i) prospectively-collected patient-completed ePRO symptom-report data; (ii) stakeholder meetings; (iii) patient interviews. Patient advice was developed from: (i) clinician-patient telephone consultations and patient interviews; (ii) review of hospital patient information leaflets (PIL) and patient support websites.n = 66) refined advice/alert accuracy. 15 telephone consultations, 7 patient interviews and review of 28 PILs and 3 patient support websites identified 4 themes to inform self-management advice. Comparisons between ePRO symptom-report data, telephone consultations and clinical events/outcomes (n = 27 patients) further refined clinical algorithms.Phase 1, including interviews with 18 patients, identified 35 symptom-report items. In phase 2, 130/300 (43%) screened patients were eligible. 61 (47%) consented to participate and 59 (97%) provided 444 complete self-reports. Stakeholder meetings  and patient interviews (A hospital EHR-integrated ePRO system that alerts clinicians and provides patient self-management advice has been developed to improve the detection and management of problems and complications after discharge following surgery. An ongoing pilot study will inform a multicentre randomised trial to evaluate the effectiveness of the ePRO system compared to usual care. Surgery may cure or alleviate cancer but is associated with significant complications and adverse effects (AEs). Up to 30% of patients undergoing major abdominal surgery for oesophageal cancer experience acute or long-term complications, such as infection or the need for further intervention , and as Prompt identification of complications and AEs after patients are discharged from hospital following surgery for cancer is critical to improve patient safety, outcomes and experiences and enable healthcare professionals (HCP) to plan appropriate care –17. TeleSpecific features of ePRO systems may provide added benefit. Firstly, the use of clinical algorithms to evaluate patient-reported symptom data and alert healthcare providers when severity reaches a pre-determined threshold can help improve patient outcomes , 19. BasPatient safety and care may be further enhanced if ePRO are embedded within systems with the functionality to provide patients with individually-tailored advice to support at-home symptom self-management . In a muSeveral of the ePRO systems developed to date are intended for the oncology setting –28. MostThe platform used to develop the ePRO system was adapted from the model developed for the eRAPID (Electronic patient self-Reporting of Adverse-events: Patient Information and aDvice) study . The eRAThe surgical ePRO system was developed between February 2014 and August 2017 in two phases  questionnaires. EORTC items were selected for their relevance to symptoms and complications experienced by patients after cancer-related major abdominal surgery, including oesophageal, gastric and hepato-pancreato biliary  cancer. In this context, UGI is used as a fully inclusive term, to include hepato-pancreato biliary cancer. EORTC items were also selected based on the familiarity of the measures to the clinical and study teams and their established routine use in international trials of PROMs to improve patient outcomes and care . CognitiAnalysis of the care pathway for patients undergoing major UGI surgery at a single UK hospital trust  was undertaken by study researchers  and the cancer nurse specialist team  to determine relevant clinician points of contact and appropriate timepoints for ePRO self-report completion by patients during their recovery post-discharge following surgery.To facilitate the integration of the ePRO system into the existing hospital EHR system at UHBT , several meetings were held between the study team, the University of Leeds IT team and the UHBT IT team between March 2015 and January 2016. Analysis of IT system performance and integration (e.g. downtime) was undertaken to monitor any issues related to integration.Phase 2 of the study was conducted in three steps.Informed by previous research undertaken to develop the eRAPID systems in Leeds and Manchester ; 31], i, i31], iClinical algorithms to guide severity-specific tailored patient self-management advice based on patient-reported symptoms were developed using four data sources Fig. . BetweenFollowing discharge, participants were requested to prospectively complete the ePRO self-report at discharge (baseline), twice in the first week (day 2 and day 7 approximately) and weekly thereafter for 8 weeks. One email and/or text message reminder with a direct link to the ePRO website was sent to participants on the days they were due to complete the ePRO self-report. However, patients could choose to log in and complete the self-report at any time during this period that they felt unwell or wanted advice. Participants were also asked to participate in a weekly telephone interview with a study researcher/research nurse, in which they were asked to reflect on the nature and severity of any symptoms they had experienced during the previous week. In addition, two stakeholder meetings with a patient and public representative and clinicians responsible for the clinical care of patients undergoing major UGI surgery were conducted. Finally, end of study interviews with a subset of approximately 10% of study participants were conducted approximately 10 weeks post-discharge. Purposive sampling was used to obtain a sample of participants with a diverse range of post-operative experiences and who had used the ePRO system to varying extents. Targeted verbatim transcription of interview audio-recordings was undertaken , wherebyData from the completed patient-completed ePRO self-reports, weekly patient interviews, stakeholder meetings and end of study patient interviews were considered together. Data were prospectively analysed in an iterative, cyclical manner as data collection and analyses proceeded to develop and refine the symptom severity thresholds that would subsequently inform the development of the clinical algorithms to trigger ePRO system actions. The purpose of this was to identify and agree upon symptoms and symptom severities associated with a ‘typical’ recovery following surgery and those that would be considered clinically concerning and to identify symptoms (and associated timepoints) for which advice or reassurance would have been beneficial. These data were used to further refine the selection of items included in the developmental version of the ePRO self-report questionnaire (described above).Patient self-management advice was developed using three data sources. Data were collated from, firstly, audio-recordings of end-of-study interviews (described above) and, secondly, telephone-based clinical consultations undertaken in the first week post-discharge  between a UHBT cancer nurse specialist (CNS) and participants as part of usual care. These consultations were audio-recorded to explore any advice and/or reassurance offered by the CNS to patients. Targeted verbatim transcription of audio-recordings was undertaken, and data analysed to identify themes relating to post-operative advice and reassurance and consider appropriate phrasing and terminology to inform the development of the ePRO system self-management advice. Between September 2016 and January 2017, a scoping search of online patient information/advice and literature provided by or recommended by the UK National Health Service (NHS) Trusts in England (e.g. on NHS Trust websites or NHS Trust recommended websites such as Macmillan Cancer Support) was undertaken. Search terms specific to patient information and advice (e.g. ‘patient information leaflet’) and relevant surgical procedures (e.g. ‘oesophagectomy’) were used. Information/literature was excluded if it did not relate to relevant procedures or was not from an NHS-approved source. Relevant themes and terminology were extracted from included information/literature using a content analysis approach and used, alongside data from the telephone consultations (described above), to draft the ePRO system patient self-management advice, in accordance with the three pre-determined symptom severity levels described above (Table Data previously generated from a subset of participants’ earlier in Phase 2 of the study (described above) who reported clinically-significant symptoms were reviewed and used to test and refine the clinical algorithms. Participants included in the subset were those who had reported symptoms of a severity that would trigger actions by the ePRO system . Actions triggered by the ePRO system and the content of any patient self-management advice provided as part of those actions were compared, firstly, with the patient advice provided by the CNS or study research nurse during the routine telephone consultations and weekly telephone interviews and, secondly, with any subsequent clinical events or outcomes of participants . The latter were identified from hospital readmission alerts, hospital EHR, and patients’ reports of accessing healthcare services reported during weekly follow-up telephone interviews. Any discrepancies between actions triggered by the ePRO system and advice provided by the nurses were discussed within the study team. Where considered necessary, the clinical algorithms were refined and tested further in an iterative, cyclical manner until no further refinements were required.Sociodemographic and clinical details of participants taking part in Phase 1 and 2 of the study are shown in Table n = 18, 16 men, mean age 66 – Table Ninety-five items from 7 validated EORTC questionnaires were identified. Cognitive interviews with patients . The integrated ePRO system was launched in April 2016. Between April 2016 and August 2017 there were four instances of downtime/integration loss totalling 145 days. This was due to an EHR software update resulting in a loss of EHR integration and clinicians being unable to access ePRO results through the EHR. A temporary workaround solution was developed to allow researchers and clinicians to access ePRO results independently of EHR during periods of downtime. This workaround required clinicians to access the ‘administrator view’ of the live ePRO system hosted on a secure server until an additional software update was issued to resolve the issue.n = 59 participants) also indicated that, for some items, patients tended to report symptoms that were already being appropriately managed or had resolved. To overcome this retrospective reporting of managed or resolved symptoms, an additional sub-item was added to 8 items. This sub-item asked patients if the reported symptom was a current issue, and the response incorporated into the clinical algorithm to trigger the appropriate level of action by the ePRO system in order to guide patient management appropriately.Of 300 patients screened, 130 (43%) patients were considered eligible and invited to participate, and 61 (47%) patients consented  indicated that, to account for natural improvements in symptoms during recovery, length of time since hospital discharge should be considered alongside symptom severity in the development of the clinical algorithms. For example, a high level of pain is expected at discharge but would be concerning and potentially indicative of an AE if experienced 6 weeks later. The clinical algorithms were refined to incorporate time since hospital discharge accordingly so that threshold scores for relevant symptoms were altered after 3 weeks post discharge to reflect the changes in symptoms experienced during typical recovery. Items included in the final ePRO self-report questionnaire are detailed in Table n = 7, 4 men, mean age 58 years) confirmed the acceptability and relevance of self-management advice and reassurance themes. These themes then formed the basis for developing the content of the self-management advice.Data from 15 routine care telephone consultations conducted by a CNS with 8 participants  in the first week post-discharge were analysed. Four main themes relating to post-operative advice and reassurance sought by participants and/or provided by the CNS were identified, including pain, other physical symptoms, diet and nutrition and managing recovery  were sourced. Using methods of content analysis, these data were combined to produce first drafts of patient self-management advice. These first drafts were reviewed and revised according to input from stakeholder meetings , resulting in the development of self-management advice for 22 symptoms. It was recognised through these iterative processes that some symptom advice differed depending on type of surgical procedure; this was made clear in the advice content to ensure patients followed the advice relevant to their procedure.Data previously generated from 27 participants’  earlier in Phase 2 of the study (described above) who reported clinically-significant symptoms were reviewed and used to test and refine the clinical algorithms. Comparisons were made between sets of data  from these 27 patients, and any discrepancies identified and discussed with the study team. Over an 8-month period, refinements to the ePRO self-report questionnaire and clinical algorithms were made (Table An electronic system for the routine capture of PRO data has been developed to improve the detection and management of complications after hospital discharge following surgery. Unique features of the ePRO system include its full integration into hospital EHR and the function to apply clinical algorithms based on patient-reported symptom-severity to guide the management of patients, and automatically and instantly alert clinicians of atypical symptoms. The ePRO system also accounts for patient-level characteristics to provide individually-tailored self-management advice necessary to provide the wide range of support required by different patients during their recovery at home , with paUntil now, few ePRO systems have been developed specifically to support patients after discharge from hospital following surgery. Of 24 controlled trials identified in a 2014 systematic review examining the effects of including PROMs in routine clinical practice , 29. OnlThis study has several strengths. Multiple data sources, including analyses of interviews and patient-clinician consultations, have directly informed the selection and refinement of ePRO self-report items, content of patient self-management advice and development of the clinical algorithms. The ePRO system has also been developed in close collaboration with multiple key stakeholder groups such as patients, patient representatives, nurses, dieticians and surgeons. This approach has ensured that the ePRO system actions are relevant and meaningful to both patients and clinicians. In addition, the ePRO system uses established, validated measures to assess patients’ HRQL, with the EORTC modules the most frequently used PROMs in oncology studies.This study does, however, have some limitations. While patient participants with a broad range of sociodemographic characteristics were included, the study was conducted in a single centre in the example context of cancer-related major abdominal surgery and it is possible that the characteristics of the sample do not typify those of the wider population of patients undergoing surgery. There is also a difference in the characteristics of patients who participated in phase 1 and phase 2 of the study, with more patients undergoing oesophago-gastric surgery in phase 1 compared to more undergoing hepatobiliary surgery in phase 2. Further work to develop and test the system in a wider setting and in a broader sample of patients is necessary to evaluate its application as a useful adjunct to patient post-operative care more generally. Depending on the surgical context, the use of alternative disease-specific patient-reported questionnaires may be considered to adapt the ePRO system self-report and algorithms to other patient groups. Integration of the ePRO system with the hospital EPR was lost for a total of 145 days over the 17-month study period due to an unsupported EHR software update. While it was possible to implement a temporary workaround, this incident highlights the potential impact IT issues can have on the widespread use of ePRO systems within EHR and indicates that successful use in routine clinical care would require full integration into hospital EHR and ongoing maintenance by hospital IT departments.Surgery is associated with significant complications and AEs, many of which occur after patients are discharged home and require further clinical intervention. Self-management of symptoms in the absence of adequate medical knowledge to distinguish between expected and concerning symptoms can be worrying and burdensome for patients and delay necessary treatment –9. An ePEmbedding prospectively-collected ePRO data into routine clinical practice has the potential to bring wider benefits to patients and healthcare systems through standardising practice , streaml


Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides . Additionally, their high stability, easily modifiable surface, and enormous diversity in shape and size, distinguish viruses from synthetic nanocarriers used for drug delivery. Indeed, several plant and bacterial viruses  have been investigated and applied as drug carriers. The ability to remove the genetic material within the capsids of some plant viruses and phages produces empty viral-like particles that are replication-deficient and can be loaded with therapeutic agents. This review summarizes the current applications of plant viruses and phages in drug discovery and as drug delivery systems and includes a discussion of the present status of virus-based materials in clinical research, alongside the observed challenges and opportunities. In the 18th century, the term “virus” was defined as a morbid principle or poisonous substance produced in the body as the result of some disease . Owing t11 plant viruses or phage to mice showed no sign of toxicity, the same dose of animal viruses caused severe hepatotoxicity . . 223]. IIn addition to doxorubicin, other anticancer pro-drug/drug molecules have also been successfully loaded inside the viral nanoparticles and their drug delivery efficiencies in cancer cells have been evaluated. Proflavine, mostly known as a bacteriostatic, is one such compound that has shown antiproliferative activity in cancer cells and tumors due to its intercalation into DNA . Loading3)2Cl2]) is another anticancer drug that has been efficiently delivered to cancer cells via viral particles. TMV particles have been decorated with mannose and lactose moieties to specifically target the galectin-rich human breast cancer cell line MCF-7 and asialoglycoprotein receptor (ASGPR) over-expressing hepatocellular carcinoma cell line HepG2, respectively. For this purpose, alkyne modified TMV particles were modified with azido sugar derivatives via the copper(I)-catalyzed azide–alkyne cycloaddition. The interior capsid surface of the TMV particles was covalently modified with cisplatin molecules through a stable chelate structure with the carboxyl groups of glutamate residues, and the drug later slowly released and resulted in enhanced apoptosis efficiency in specific targeted cell lines [Cisplatin (cis-[Pt(NHll lines . A similll lines .The plant hormone indole-3-acetic acid (IAA) is a prodrug used in a virus-based drug carrier to treat human prostate cancer cells. IAA generates a radical upon reaction with horseradish peroxidase and produces radical-dependent cytotoxicity as well as cell death. The viral drug carrier was designed by engineering M13 phage to display a short peptide to enhance prostate cancer cell recognition/penetration. Moreover, a NeutrAvidin–horseradish peroxidase conjugate was attached to the p9 phage coat protein of M13 fused with a biotinylated peptide . The treStaphylococcus aureus with the low potency antibiotic chloramphenicol, well-known for its toxicity to blood cells, conjugated to the fd phage retarded the growth of bacterial cells ~20-fold more efficiently than free chloramphenicol [Antibiotics are another group of drugs that have been loaded in viral-based drug carriers. It has been reported that the conjugation of many copies of the drug molecule onto the phage’s major coat proteins increased potency by creating a microenvironment around bacterial cell with a locally high drug concentration. Treatment of gram positive pathogenic bacteria phenicol .2)-5-iodoacetoamino-1,10-phenathroline(phen-IA). An antibody specific for Staphylococcus aureus, was chemically conjugated to the lysine residues of the coat proteins [S. aureus cells compared to free photosensitizer due to the enhanced cell targeting ability. On the other hand, the cell killing efficiency of the photosensitizer-labelled CCMV particles was approximately the same as that achieved using and anti-S.aureus antibody-photosensitizer conjugate. While the number of photosensitizers per binding event for the CCMV platform (~70) was significantly higher than the antibody-photosensitizer conjugate (~2), it was suggested that the large size of the CCMV particles significantly reduced the proximity of photosensitizer to the cell surface, which is an important factor in efficacy due to the very short diffusion length of singlet oxygen. The encapsulation of photosensitizers within the capsid cavity of CCMV particles has been another approach for PDT. Water-soluble zinc phthalocyanine (ZnPc) was encapsulated inside CCMV by two different routes: i) self-assembly of coat proteins dimers around aggregated ZnPc molecules at neutral pH; and ii) diffusion of ZnPc molecules into empty CCMV particles through capsid pores at acidic pH [1+), dicationic (Zn-Por2+), tricationic (Zn-Por3+), and tetracationic (Zn-Por4+)) were employed [+3 loaded TMV-F3 particles accumulated on the cell membrane and showed a fivefold increase in cell killing efficacy compared to the free drug. The results are explained with some possible mechanisms which are the cell toxicity through cell membrane disruption by light activation, the release of the photosensitizers at the cell surface and favored cell uptake of the Zn-Por molecules due to their positive charge. The self-assembly of drug-loaded liposomes on M13 phage has been another PDT delivery approach using phage as a nanocarrier [Efficient delivery of drug molecules to targeted cells is also important for photodynamic therapy (PDT) and photothermal therapy (PTT). PDT relies on the activation of a photosensitizer by light in the presence of oxygen, which produces reactive oxygen species (ROS). As cell damage occurs due to the reaction of ROS with cellular components, it is important to deliver the photosensitizers to the targeted cells and avoid their nonspecific delivery to the healthy cells. The nonspecific dispersal of the photosensitizer throughout the body creates sensitivity in patients. For instance, sunlight must be avoided for several weeks following treatment. Moreover, the insolubility of many photosensitizers in physiological solutions is another problem encountered in PDT-related drug delivery systems and it is necessary to develop new platforms to address this challenge. For this purpose, icosahedral CCMV particles have been explored for PDT by dual labelling with both cell-targeting moieties and photosensitizers. The surface coat protein of the virus was genetically-modified to display a cysteine residue, used to attach the photosensitizer Ru, so-called hyperthermal therapy, employs gold nanoparticles as a heat source for inducing cell damage as a result of a light-to-heat conversion process. As gold nanoparticles generate local heating under light illumination, efficient delivery of gold nanoparticles to targeted cells is desirable for selective cell killing. Clusters of gold nanoparticles on T7 phage particles have been fabricated as a PTT delivery platform to treat prostate cancer cells in vitro . The assPolymeric materials are widely used as drug delivery systems because of their ability to be use as a matrix that protects drug molecules and controls drug release via e.g., its rate of degradation or swelling profile. Moreover, in some cases, the high water content and soft structures of polymer matrices make them similar to natural extracellular matrices, which contributes to minimizing tissue irritation and cell adhesion and makes them promising drug delivery systems. However, an initial burst, or very fast release of drug molecules remains a challenge that can limit their use to certain types of drugs. The incorporation of viruses into polymeric matrices has been proposed as a solution to overcome this limitation and to better control the release of the drugs molecules. The affinity-based polymeric drug release system is one such platforms in which M13 phage are embedded inside a polymer matrix to suppress the release of drug proteins due to their specific interactions with phage particles. For this purpose, the p3 coat protein of M13 phage was genetically modified to display peptides that bind to antibodies and mixed with a gelatin solution containing antibodies to form hybrid hydrogels . While tco- phenylboronic acid (PBA)) onto M13 surface through amino groups of the coat proteins [co-PBA) chains were conjugated per phage particle. Due to the temperature-responsive gelation behavior of the polymer, the polymer–M13 phage bioconjugate was mixed with insulin at 4 ºC and could then be converted into hydrogels by injecting into PBS buffer at 37 ºC. The insulin-encapsulated hydrogels demonstrated glucose-responsive release behavior, which can be an interesting strategy in the regulation of diabetes. During hydrogel formation, the virus particles interact with poly(NIPAMco-PBA) polymer, which is in a collapsed hydrophobic state. However, in the presence of glucose, the boronic acid moieties of poly(NIPAMco-PBA) couple to glucose to yield hydrophilic boronates that are substantially more hydrophilic. This changes the structure of hydrogel matrix and enables faster release of encapsulated insulin molecules. While 70% of insulin was released over 24 h, the release rate of insulin increased in the presence of glucose with a peak value of 90% at 10 h. Similar carbohydrate-responsive polymers have also been prepared by cross-linking phenylboronic acid (PBA)–M13 bioconjugates with poly, resulting in hydrogels with excellent injectability and self-healing behavior  concentrations and pH enables their use as an additional release mechanism in drug delivery systems. Doxorubicin-loaded RCNMV particles could be incorporated into two different polymeric matrices (poly(lactic acid)(PLA) and poly(lactic acid):polyethylene oxide (PLA:PEO)) by electrospinning into fibers [RCNMV particles has been another virus incorporated into polymeric drug delivery systems. The ability to trigger the opening and closing of RCNMV capsid pores by changing divalent ion ; and caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC) . On the vimentin .After their administration into the body, viral particles, like many other nanoparticles, are recognized as foreign agents by the cells of the host immune system and are eliminated from the blood. B-cells are one type of these immune cells and B-cell dependent immunoglubins have been shown to play an important role in rapid neutralization of T7 phage in murine blood, resulting in a short half-life (<5 min) . The retThe studies above suggest that the fast blood clearance of viral particles are at least partially due to the host immune response and highlight a need for a coating to tailor the properties of the viral particles for enhanced blood circulation times. For this, the modification of the viral capsid with passivating agents, such as polymers or proteins, has been investigated. For instance, the modification of the CPMV capsid with poly(ethylene glycol) (PEG) effectively blocked CPMV–cell interactions and shielded the viral particles from inducing a primary antibody response ,249. TheOverall, a unique feature of viruses highlighted in this review is that they are perfectly-defined nanomaterials that can be conveniently modified, either at the genetic level or using bioconjugate chemistry, to yield interesting tools for drug discovery or drug delivery. Indeed, the structural diversity of viruses as well as the connection between genotype and phenotype has provided several complementary platforms for the discovery of different classes of therapeutic polypeptides by phage display, some of which have made it to the clinic. While in vitro selection procedures by phage display are the most commonplace, a better understanding of the health risks posed by plant viruses and bacteriophages to Humans may unbridle more complex in vivo selection procedures towards targets that cannot be emulated in vitro. Indeed, the available yet limited number of studies examining the application of in vivo phage screening in human patients have shown promising results ,254. In


Simulated chromatin structures recapitulate known features of genome organization, including the formation of chromatin loops, topologically associating domains (TADs) and compartments, and are in quantitative agreement with chromosome conformation capture experiments and super-resolution microscopy measurements. Detailed characterization of the predicted structural ensemble reveals the dynamical flexibility of chromatin loops and the presence of cross-talk among neighboring TADs. Analysis of the model’s energy function uncovers distinct mechanisms for chromatin folding at various length scales and suggests a need to go beyond simple A/B compartment types to predict specific contacts between regulatory elements using polymer simulations.We introduce a computational model to simulate chromatin structure and dynamics. Starting from one-dimensional genomics and epigenomics data that are available for hundreds of cell types, this model enables  de novo prediction of genome organization for hundreds of cell types with available epigenomics but not Hi-C data.Three-dimensional genome organization is expected to play crucial roles in regulating gene expression and establishing cell fate, and has inspired the development of numerous innovative experimental techniques for its characterization. Though significant progress has been made, it remains challenging to construct chromosome structures at high resolution. Following the maximum entropy approach pioneered by Zhang and Wolynes, we developed a predictive model and parameterized a force field to study chromatin structure and dynamics using genome-wide chromosome conformation capture data (Hi-C). Starting from one-dimensional sequence information that includes histone modification profiles and CTCF binding sites, this model predicts chromosome structure at a 5kb resolution, thus establishing a sequence-structure relationship for the genome. A significant advantage of this model over comparable approaches is its ability to study long-range specific contacts between promoters and enhancers, in addition to building high-resolution structures for loops, TADs and compartments. Furthermore, the model is shown to be transferable across chromosomes and cell types, thus opens up the opportunity to carry out  The human genome contains about 2 meters of DNA that is packaged as chromatin inside a nucleus of only 10 micrometers in diameter . A detaiThe importance of chromatin organization has inspired the development of a variety of experimental techniques for its characterization. For example, using a combination of nuclear proximity ligation and high-throughput sequencing, chromosome conformation capture and related methods quantify the interaction frequency in three-dimensional space between pairs of genomic loci ,6, and hPolymer modeling has played a critical role in our understanding of the genome organization and in interpreting features of Hi-C contact maps . In partIn parallel, bioinformatics studies have provided powerful tools in addressing potential biases in Hi-C data –48, and The difficulty in predicting contacts between specific regulatory elements using polymer models is at least twofold. First, existing phase separation models based on A/B compartments or six subcompartments are inadequate for such purposes, despite their success in recapitulating the long-range block-wise patterns observed in Hi-C. As chromosome compartments are defined based on contact patterns revealed by Hi-C at a coarse resolution from 50kb to 1 Mb, they tend to group many regulatory elements together as one “active” type and fail to capture the distinction among them ,7,47. Thde novo prediction of the structural ensemble for any given chromatin segment using only one-dimensional sequencing data that is available for hundreds of cell types.In this paper, we report the development of a predictive and transferable polymer model to simulate the structure and dynamics of chromosomes at five kilo base resolution. This model takes combinatorial patterns of epigenetic marks and genomic location and orientation of CTCF binding sites as input, and can be parameterized from Hi-C data with a robust and efficient maximum entropy approach ,65. A keWe introduce a predictive model to study cell-type specific 3D chromatin folding. This model takes a sequence of chromatin states derived from genome-wide histone modification profiles and a list of CTCF binding sites as input. We selected these genomic features due to their known roles in organizing the chromatin at various length scales . At the Methods.As shown in r is a sum of three components, and UChrom(r) = U(r) + UCS(r) + UCTCF(r). U(r) is a generic polymer potential that is included to ensure the continuity of the chromatin, and to enforce excluded volume effect among genomic loci. UCS(r) is a key innovation of the chromatin model, and is crucial to capture the formation of TADs and compartments. It quantifies the chromatin state specific interaction energies between pairs of loci. As detailed in Section: Physical principles of chromatin organization and Methods, we used a general form for UCS(r) to capture its dependence on genomic separation. UCTCF(r) is inspired by the loop extrusion model  and plotted in Figure O in Experimental contact maps at 5kb resolution from Ref. were downloaded using the Gene Expression Omnibus (GEO) accession number GSE63525 (see Extended Data Sheet). We used the combined contact matrices constructed from all read pairs that map to the genome with a MAPQ> = 30. The raw matrices were then normalized with the KR method using the normalization vector provided in the same dataset. To convert the contact matrices into probabilities, we further divided each matrix element with the diagonal value A key input of the computational model is the sequence of chromatin states that captures the variation of epigenetic modifications along the genome sequence. Following Ref. , we defiTo capture the formation of chromatin loops, we compiled a list of CTCF-binding sites along the chromatin of interest using cell-type specific ChIP-Seq data.Starting from the peak profile downloaded from ENCODE (see Extended data sheet), we identified the center of binding for each peak of both CTCF and cohesin subunit Rad21. As both CTCF and cohesin molecules are found at the boundaries of most chromatin loops, we selected loop forming CTCF binding sites as those that have at least one Rad21 molecule located within 50bp of their genomic locations.We then determined the orientation of each CTCF-binding site as follows. We first attempted to align the binding sites to the set of CTCF motifs compiled in Refs.  and 96]96] Click here for additional data file.S1 TableSource for experimental data used in the study.(XLSX)Click here for additional data file.