In our estimates, we include deposits that have been neglected in global carbonate budgets: Holocene Halimeda bioherms located on the shelf, and postglacial pre-Holocene (now) drowned coral reefs located on the shelf edge. Our results show that in the Great Barrier Reef alone, these drowned reef deposits represent ca. 135 Gt CaCO3, comparatively representing 16–20% of the younger Holocene reef deposits. Globally, under plausible assumptions, we estimate the presence of ca. 8100 Gt CaCO3 of Holocene reef deposits, ca. 1500 Gt CaCO3 of drowned reef deposits and ca. 590 Gt CaCO3 of Halimeda shelf bioherms. Significantly, we found that in our scenarios the periods of pronounced reefal mass accumulation broadly encompass the occurrence of the Younger Dryas and periods of CO2 surge  observed in Antarctic ice cores. Our estimations are consistent with reef accretion episodes inferred from previous global carbon cycle models and with the chronology from reef cores from the shelf edge of the Great Barrier Reef.More accurate global volumetric estimations of shallow-water reef deposits are needed to better inform climate and carbon cycle models. Using recently acquired datasets and International Ocean Discovery Program (IODP) Expedition 325 cores, we calculated shallow-water CaCO The authors assigned a medium degree of confidence to the current estimates of atm-CO2 contributions from coral reef accretion and carbonate compensation depth changes. This uncertainty derives not just from the use of proxy data and their limited availability, but from the complex relationships between the carbon and other biogeochemical cycles. Such uncertainty is reflected in the range of the contributions to postglacial atm-CO2 attributed to shallow-water reefs in global carbon models (− 9 to 30 ppm5).The role of calcium carbonate deposits in the carbon cycle, and the influence on climate change during the late-Quaternary is poorly constrained. The fifth assessment report of the Intergovernmental Panel on Climate Changecoral reef hypothesis 7. This hypothesis proposes that the increase of the atm-CO2 is at least partly due to the enhanced shallow-water CaCO3 accretion by corals. This hypothesis relies on the availability of new areas of marine flooded shelf during the last transgression and on the consequent increase in coral reef development. This would have changed the alkalinity balance, at least locally, and ultimately increasing the transfer of CO2 to the atmosphere8. Because of the reduced marine shelf area during glacial times and the subsequent increase in reef area from glacial to Holocene times, it is assumed that the coral reefs acted as secondary amplifiers—not precursors—of a climatic change that had already initiated10 with early atm-CO2 rise generally preceding global surface temperature increase11.The role that coral reefs may have played in this process has been termed the coral reef hypothesis is supported by the extensive coral reefs of Holocene age worldwide16. Moreover, a possible lower contribution from terrestrial sources in this same period17 argues in favour of alternative carbon sources, such as that represented by reef accretion. Simplified box models7 have suggested that the activity of the coral reefs can explain a significant rise of the atm-CO2 during postglacial times. However, the dissolution ratios, accretion rates and calcite saturation depth informing these models are poorly constrained and they possibly overestimate the total carbon derived from corals.The 219. Notably, Ridgwell et al.5 inferred two possible minor episodes of global reef growth from 17.0 to 13.8 ka BP and from 12.3 to 11.2 ka BP. With no conclusive evidence available at that time for such global reef growth episodes20, they attributed the increase in CO2 to changes in the biogeochemical properties of the Southern Ocean surface.More complex models have considered the effect of coral reefs in postglacial atm-CO22). Globally, drowned reefs may constitute an important fraction of postglacial carbonate24 and an alternative earlier source of postglacial carbon. Halimeda bioherms are another contributing component, with recent investigations from the GBR suggesting they are volumetrically relevant in postglacial carbonate budgets27.Interestingly, recent evidence in Tahiti and the Great Barrier Reef (GBR) has revealed glacial and early-postglacial (30 to 10 ka BP) coral reef episodes in line with those inferred intervals (IODP Expeditions 310 & 32528 and applying parameters (e.g. reef area) with large associated uncertainties. New datasets are, however, providing valuable constraints to these parameters within the GBR region. For example, new GIS datasets of reef boundaries29 have the potential to improve the estimations of reef area. Additionally, the recent surveys and investigations on the drowned shelf edge reefs of the GBR represent the most complete dataset investigating drowned reefs: IODP Exp. 325 well-dated fossil reef cores31, seismic lines33, multibeam bathymetry34, and surface sediment and rock dredge samples36. New detailed bathymetry and interpretations are also providing new constraints on the spatial distribution and volume of Halimeda bioherms in the northern GBR27.Estimations of global and regional reef carbonate area and volume have been attempted using a range of assumptionsHalimeda deposits on regional  and global postglacial shallow-water CaCO3 budgets. Our scientific objectives are to: (1) estimate the volume, mass and timing of postglacial shallow calcium carbonate deposition across the entire GBR using the most recent GIS datasets, data from two IODP Exp. 325 control zones from the shelf-edge reef system and the regional volume of the Halimeda bioherms; (2) extend the resulting volumetric and mass estimates globally based on assumptions ground-truthed in the GBR; and (3) compare our results with past regional and global volumetric and mass estimates.In this paper we investigate the impact of drowned coral reefs and 38 controlled by major glacial-interglacial sea-level fluctuations. The last glacial-interglacial fluctuation initiated during the LGM30 when sea level was approaching minimum levels (120–130 m below present)39.The GBR shelf along the northeastern coast of Australia Fig.  accommod40. These reefs currently lie between 40 and 130 m below present sea level and extend for more than 2000 km from the northern to the southern central GBR and possibly farther42  layer of carbonate sands and mud, dominated by Halimeda fragments, foraminifera, mollusks and bryozoans47.As the glaciation ended and sea level rose during the postglacial, extensive fringing- and barrier-reef structures developed along the shelf edge of the GBR until ca. 10 ka BP48. Both Pleistocene and Holocene reef deposits in the GBR display local and regional variations that are the expression of broader physiographic trends and of the physical processes linked to the postglacial marine flooding49.The shallower structures of the modern GBR were colonized after ca. 9 ka BP following the demise of the shelf-edge reefs. Since then, extensive reef development has occurred along the whole GBR from the vicinity of Fraser Island in the south, to Cape York in the north50, and excluding continental islands and shelf-edge reefs. These features were sliced into latitudinal 50 km wide slices to assess latitudinal variations. We assumed that the reef area polygons represented the main reef and bioclastic deposits directly related to Holocene reef growth. However, unaccounted fore- and back-reef aprons may constitute a significant portion of the reefal carbonate volume51.We estimated the areal trends of the Holocene reefs in the GBR. Reef area was estimated from GIS layers containing polygons representing the outline of the Holocene reefs Figs.  and 2. T3 deposits by multiplying the area derived from the interpreted GIS layer by the thickness derived from historical reef cores that have drilled through the Holocene . The volumetric and mass calculations were also performed at a sub-regional level in 50 km wide latitudinal slices that allowed reconstruction of latitudinal trends  between 14° and 21° S  than our best estimate of 751 Gt CaCO3. However, Rees13 include reefs in the Australian region that are not part of the GBR system sensu stricto. The Kinsey and Hopley55 estimated reef area value is more in agreement with our figure: ca. 20,000 km2. A more recent estimate places the GBR’s shallow-water reef area at 16,110 km250, but they do not include sediment wedges associated with these reefs, which are found at greater depths and constitute up to the double of the reef framework volume51. If we calculate the mass areal accumulation (MAA) using Rees13 estimates (Table −2) than even the highest MAA in Hydrographers Passage , vertical accretion rate, the maximum cumulative thickness and petrophysical parameters. A regional bathymetric dataset57 provided the basis for the reconstruction for the postglacial marine flooded area in the GBR .The volume and mass of the shelf edge deposits at a regional scale were ground-truthed in two control zones along the shelf edge of the GBR: Hydrographers Passage and Noggin Passage Fig. . Here, eflooded area and the accumulation represented by the MAA, scaled back by the proportion of bathymetric surface covered by reefs (reef area ratio) without considering any temporal evolution. The latter, the postglacial-thickness method, attempts to reconstruct the temporal evolution of the reef accretion by considering the change in flooded area since the LGM. It is layer-based, with each layer corresponding to a 5 m sea-level step in which data-derived thickness constraints  the mass areal accumulation method and (2) the postglacial thickness method. The former is a bulk calculation of volume based on nts Fig.  are applnts Fig. b. It rel.31 Fig.  enabled The estimates obtained applying the mass areal accumulation method are lower than those estimated by applying the postglacial thickness method but are within the same order of magnitude. Not surprisingly, the application of both methods results in similar latitudinal trends Fig. e becausereef area ratio = ca. 20%). This proportion is almost twice the reef area ratio estimated at a regional scale for the shallower, Holocene reefs when considering the whole of the GBR shelf.In the control zones, Noggin Passage and Hydrographers Passage Fig. , ca. 20%−2, whilst inter-reef areas display MAA values that are an order of magnitude lower (7767–8406 kg m−2). The southern control zone (Hydrographers Passage) has a higher average MAA  than the northern site (Noggin Passage) in all the geomorphic areas assessed . This is consistent with a thinner reef veneer (< 10 m) in the terrace and outer geomorphic areas and less distinguishable barrier structures found at Noggin Passage40.In both control zones Table  we found40. On the contrary, in the southern-central GBR (e.g. Hydrographers Passage control zone) wider and a more gentle gradient provides more substrate availability34.Volumetrically, the carbonate deposits of the shelf edge wane in the northern GBR when compared to the central and southern GBR Fig. d, e. In 2  and 48% (maximum case) of the area occupied by banks with no Holocene cover in that same study. Kleypas58 inferred that the global area available for reef growth during the LGM lowstand was approximately 20% of that available today. In the GBR, that figure is at least within the same order of magnitude of our estimations: a maximum shelf edge flooded area of ca. 29,523 km2 .It is useful to establish some comparisons to appreciate the magnitude of the shelf edge deposits. At the shelf edge, our estimates suggest that the area covered by reef formations is between 3000 and 11,000 kmHalimeda bioherms are a significant component of the region’s postglacial carbonate budget. A comparison of the most up-to-date morphometric data27 shows that the postglacial Halimeda deposits are equivalent in mass to 5.5–10.5% of the Holocene reef mass on the GBR shelf . Despite these new constraints, questions remain about the extent of pre-Holocene Halimeda deposits, particularly at the shelf edge.3 deposits for the GBR to the entire globe using the reef area estimate of Spalding et al.56. Using this area, we applied the parameters ground-truthed in the GBR dataset. The global reef area (RAGLOBAL) was multiplied by average thickness and petrophysical parameters  to obtain global postglacial CaCO3 deposits. We accounted for the drowned postglacial reefs by applying two factors to the global Holocene estimates: a factor based in the ratio of shelf edge-to-Holocene reef area  and a factor based in the mass ratio  (Table We extrapolated the estimates and trends of CaCOM) Table .Table 3Greef area ratio at the shelf edge (ca. 20%) is twice the reef area ratio estimated for the whole GBR shelf  and shelf area in low latitudes  as reported in Kleypas58, suggesting a global reef area ratio of 5 to 6%. This percentage would be even lower if we apply the global reef areas of 300 × 103 km261, 255 × 103 km216 or 284 × 103 km256. The lower value of the global reef area ratio (5–6%) compared to the GBR values of this study (ca. 10%) could be partly explained by uncertainties in the topographic/bathymetric datasets used by Kleypas58 , and by difficulties in predicting reef habitat using the ReefHab model58, or by the inclusion of shelf areas that are not potential reef habitats. It is also possible that the GBR had more favourable regional conditions for reef development compared to other global locations.We found that the 2 by Spalding et al.56 is lower than estimates from other authors  ranging from 4073 Gt CaCO364 to a maximum of 54,550 Gt CaCO365 , we obtain a global value of 1460–1785 Gt CaCO3 accumulated in the drowned reefs from 19 to 10 ka BP. These results are modest compared to the combined Holocene reef deposits (ca. 8100 Gt CaCO3). However, given the direct evidence on large-scale pre-Holocene shelf edge reef systems in the GBR  and past evidence of other large-scale drowned reefs in global locations24, the question of the impact of drowned reefs on the LGM-to-postglacial global CaCO3 budgets remains relevant.If we extend the shelf edge-to-Holocene reef ratios estimated in the GBR to global scales  to a maximum of 3.2 Gt CaCO3 y−1 (using area in Copper65). However, if we split the averages between Holocene and pre-Holocene, we find the differences are of one order of magnitude . These were periods of high substrate availability and favourable environmental conditions for reef growth, as influenced by shelf physiography and sea-level rise49.The timing of the CaCO3 mass accumulation at regional level, but it can provide a broad temporal trend. The periods of higher CaCO3 accumulation in the shelf edge  . These findings are consistent with a more recent analysis of all available postglacial vertical reef accretion data (including IODP Exp. 325), which shows rapid accretion rates (9.6 to more than 20 mm yr−1) during these periods of higher CaCO3 accumulation .The postglacial thickness method cannot establish a precise chronology for CaCO3) and their global extrapolations (1500 Gt CaCO3) suggest that pre-Holocene reef accretion is likely to be more relevant to the global CaCO3 budgets  than currently recognized. The impact of these deposits in the atm-CO2 should be assessed by global process-based carbon models that reflect the full complexity of the atmosphere–ocean–land biogeochemical cycles . The latitudinal distribution of the shelf edge reefs is also strongly correlated to shelf availability.The shelf-edge reefs of the GBR constitute an important portion of the postglacial shallow reef deposits: these (now) drowned reefs occupy an area of between 3000 and 12,000 km3 from Holocene reefs (best estimate), which is consistent with previously published estimates. Following from recently published results in the GBR, a minimum of ca. 590 Gt CaCO3 from Halimeda deposits should be added to the global Holocene CaCO3 mass, representing a 4–8% increase in the budget.By globally extrapolating the GBR constraints, we estimate a total accumulation of 8100 Gt CaCO3 represent 16–20% more mass in the global postglacial budget than if considering the Holocene reef deposits alone. Their inclusion in global carbon models could provide new constraints on postglacial global atmospheric and climate models.Global extrapolations supported by recent surveys suggest that a significant proportion of postglacial, pre-Holocene shallow-water carbonate deposits can be attributed to now drowned postglacial reefs. These deposits of ca. 1500 Gt CaCO3 deposition in the GBR is broadly coeval with two distinct episodes of postglacial atm-CO2 increase (ca. 14.9–14.4 ka and ca. 13.0–11.5 ka BP67). Any causal relationship must be confirmed by more complex, process-based models of the global carbon and other biogeochemical cycles and global surveys of drowned reefs and reef areas.Our results support a more prominent role in the postglacial carbon cycle for pre-Holocene shallow-water coral reefs. Significantly, the timing of higher CaCOWe estimated the carbonate volume in the GBR for: (1) early postglacial reef deposits (21–10 ka); and (2) Holocene reef deposits. We also attempted a global extrapolation of these carbonate deposits based on these new and well-constrained regional GBR estimates.57, GIS layers with reef locations and extensions50; previous GBR Holocene drill cores (see Appendix 68 for data sources). We distinguished the areas at the shelf edge (defined here between the modern outer GBR reef front and the 130 m isobath) from the other areas of the shelf and extracted the corresponding bathymetric subset.Data were available across the whole GBR: bathymetry at ~ 100 m resolution47; (2) extensive and precise chronologic database (> 580 published U-Th and 14C ages), lithological and petrophysical properties directly measured from the IODP Exp. 325 drill holes and cores31; (3) high-resolution (5 m) bathymetric coverage in these sites34; and (4) extensive knowledge on the development history and age structure of the shelf-edge reef system21.More locally, data were available for two densely surveyed control zones at the shelf edge  = 2930 kg m−3Aragonite density (ρR) = 35%30.Reef formation porosity  as obtained from the seismic interpretations47. The three different velocity scenarios (1700–3300 m s−1) to convert from seismic time to true depth47 were considered to obtain a minimum, best estimate, and maximum values for each set of maps.Mass areal accumulation (MAA) [kg m−2] at the shelf-edge control zones: these values were calculated for each control zone by transforming each formation volume estimate to carbonate mass (CaCO3 mass = formation volume × ρA × (1—ΦR)) and subsequently dividing the mass by the total shelf edge area of the control zones (MAA = mass · [shelf edge area]−1). The calculations were also applied for each of the main geomorphic zones47: inner barrier, outer barrier, terrace and shelf break combined, and inner and outer platforms combined. Three values were applied to account for plausible minimum, best and maximum scenarios: the average MAA value of all reef locations, and the minimum and the maximum MAA value in any reef location (Table AA) kg m− at the sVertical accretion rate [m ky−1] at the shelf-edge control zones: the glacial-postglacial boundary recognized in the cores and the radiometric ages measured in the Exp. 325 core samples21 allowed the estimation of the vertical accretion gradients (m ky−1) for the entire postglacial Pleistocene time period (21–10 ka) , this became a necessary constraint to avoid unrealistic cumulative reef thickness as sea-level rise progressed .Postglacial shelf margin .49 Fig. . The relTo estimate the postglacial Pleistocene carbonate deposits, we first produced the following input for both of the estimation methods we applied:3 accumulation was calculated using the following parameters:Reef area [m2] from the Queensland coast to the outer GBR: the reef area was estimated from GIS layers containing polygons representing the outline of the Holocene reefs  and their interpretation, which could result in an overestimation of the reef area in the northern GBR, the under or overestimation of the extent of the bioclastic cover, or the misrepresentation of some locations as Holocene reefs when they might be older Pleistocene outcrops50.ef area m from thens Figs. , 3e. We Reef thickness from Holocene GBR drill cores [m]: a dataset of Holocene reef thicknesses was used to estimate minimum, average and maximum thickness values for the Holocene reef thickness68  was multiplied by the reef area ratio (RAR) and mass areal accumulation (MAA) values to obtain CaCO3 accumulation values in the GBR shelf margin  was converted to an equivalent rate relative to past sea-level steps. This allowed us to associate an incremental reef thickness to each of the 5 m sea-level steps considered. Conversion from geological age to equivalent sea level was performed using a simplified relative sea-level curve based on Lambeck et al.39 for ages more recent than 10 ka BP, and based on Webster et al.21, Yokoyama et al.31 for ages before 10 ka BP  by each sea-level step (5 m). Subsequently, the product of this thickness and the flooded area at each flooding stage (FASL) gave us the formation volume for each one of the thirty-three latitudinal zones and for each sea-level step. The volume was scaled down by the reef area ratio (RAR) values  and density (ρA):To obtain an estimate of reef volume at each sea-level step, we first estimated reef thickness by multiplying the converted postglacial ess Fig. . The CaCPleistocene CaCO3 mass for each sea level (SL) between 130 and 0 m:Incremental reef volume = VASL × 5 m × FASL × RARIncremental CaCO3 mass = Incremental reef volume × ρA × (1—ΦR)Cumulative CaCO3 mass = Σ CaCO3 mass of Holocene carbonate in the GBR, the reef thickness (RT) values were multiplied by the reef area (RA) to obtain cumulative Holocene carbonate volumes for the GBR as a whole and for each one of the thirty-three latitudinal zones  and density (ρA) (Table Holocene reef volume = RA × RTHolocene reef CaCO3 mass = Holocene reef volume × ρA × (1 − ΦR)To obtain values of total nes Fig. . Mass vaA) Table , as summ3 budgets. We extrapolated the estimates and trends of CaCO3 deposits for the GBR to the entire globe. We used published estimates of global reef area and parameters ground-truthed by the GBR dataset. The global reef area (RAGLOBAL) was multiplied by average thickness (RT) and petrophysical parameters  to obtain global postglacial CaCO3 deposits.We consider the contribution of drowned postglacial reefs in global CaCOA),Assumption 1: on average, the proportion of postglacial drowned reefs areas corresponding to a given Holocene reef area is similar across all reef provinces. We expressed this assumption as a ratio of shelf margin area to total Holocene reef area, the area adjustment factor .Assumption 2: on average, the postglacial drowned reefs mass corresponding to a given Holocene reef mass is similar across all reef provinces. We expressed this assumption as a ratio of shelf margin CaCOWe accounted for the drowned Pleistocene reefs by applying two assumptions in our calculations to obtain two equivalent results.RAglobal) have a large range of uncertainty as demonstrated by the range of values proposed by different authors  have a large associated uncertainty: despite the evidence for drowned reefs in other geographical locations, the exact global extension and morphology of drowned reefs is not well constrained. Other carbonate provinces might differ in morphology, in accretion trends and in the proportion of Pleistocene reefs present along their margins compared to the more recent Holocene deposits of those provinces.Global postglacial CaCO3 accumulation = RAglobal × RTGBR × ρA × (1—ΦR)Assumption 1:Area-adjusted global postglacial CaCO3 mass = RAglobal × RTGBR × ρA × (1—ΦR) × (1 + AFA)Assumption 2:Mass-adjusted global postglacial CaCO3 mass = RAglobal × RTGBR × ρA × (1—ΦR) × (1 + AFM).The values for global reef area  varying according to temperature, salinity and pressure70.The chemical equilibrium of the shallow ocean is complex, with the concentration of the main inorganic carbon species (CO71, reefal CaCO3 accretion provides CO2 to the environment by increasing the concentration of CO2 in the ocean water. The equivalent CO2 and C mass based on the stoichiometry of the chemical reaction: Ca2+  + 2HCO3− = => CaCO3 + CO2 + H2O66 gives an incomplete picture because the exact proportion would depend on physical conditions that would have varied during the postglacial period. Experimentally, it has been demonstrated that for each mole of CaCO3 precipitated in seawater only a fraction of a mole of CO2 is fed to the surrounding waters due to the buffering effect of marine water72. It is then unclear how much of this carbon is released into the atmosphere, especially at centennial timescales72. The pathways for the released CO2 molecules are varied—they can be absorbed into inorganic or organic marine carbon cycles, or they can also be transferred into the atmosphere by the balancing of the partial pressure of CO2. We have not attempted to quantify the corresponding postglacial CO2 contribution to the surrounding waters and to the atmosphere. This would require more complex carbon models which are beyond the scope of this study.According to the coral reef hypothesisSupplementary Information.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.


So far, the engine has served over a million queries and the source code has been downloaded several thousand times.With the growth of protein structure data, the analysis of molecular interactions between ligands and their target molecules is gaining importance. PLIP, the protein–ligand interaction profiler, detects and visualises these interactions and provides data in formats suitable for further processing. PLIP has proven very successful in applications ranging from the characterisation of docking experiments to the assessment of novel ligand–protein complexes. Besides ligand–protein interactions, interactions with DNA and RNA play a vital role in many applications, such as drugs targeting DNA or RNA-binding proteins. To date, over 7% of all 3D structures in the Protein Data Bank include DNA or RNA. Therefore, we extended PLIP to encompass these important molecules. We demonstrate the power of this extension with examples of a cancer drug binding to a DNA target, and an RNA–protein complex central to a neurological disease. PLIP is available online at   The Protein Data Bank (PDB)  is contiPLIP, the protein–ligand interaction profiler , addresset al. run an inverse docking screen to identify targets of curcumin, a natural compound  contains DNA and RNA (Figure Studying the interactions of nucleic acids to ligands and to proteins is important. DNA/RNA can act, for instance, as a target for novel cancer drugs such as the drug XR5944  and as aet al. have solved the structure of FUS bound to RNA (FUS is a nuclear RNA-binding protein involved in RNA metabolism. It can undergo liquid–liquid phase separation, forming membraneless organelles . Aberrand to RNA . We appld to RNA . PLIP deet al. published the structure of two XR5944 molecules bound to the naturally occurring DNA TFF1-ERE (The cancer drug candidate XR5944 is a DNA bis-intercalator that strongly binds DNA in a sequence-specific manner ,16–18. BTFF1-ERE . PLIP idet al. obtained the X-ray structure revealing the transcribing complex in the ‘post-translocation’ state with the nucleotide added to the RNA transcript (Transcription, the synthesis of RNA from a DNA template, is one of the most important steps in the control of cell growth and differentiation ,25. Durianscript . Figure PLIP uses four steps to detect and report the relevant interactions of a complex: structure preparation, functional characterisation and rule-based matching and filtering of interactions. In the preparation step, the input structure is hydrogenated and the ligands are extracted along with their binding sites. If the advanced option for analysing DNA or RNA as receptor is selected, PLIP will automatically detect the nucleic acid residues as part of the receptor and exclude them from the ligand molecules. The RNA and DNA residues to be considered are detected by their name U, A, C, G and DT, DA, DC, DG, respectively. After the correct detection of receptor and ligands, PLIP continues with the subsequent interaction detection and visualisation as previously described in .There are a number of tools  analysinInteractions between nucleic acids and small molecules play a crucial role in various biological processes and have a strong impact on drug discovery. In that regard, the PLIP web tool has been adapted to recognise nucleic acid molecules as receptors and provide a comprehensive analysis and visualisation of non-covalent interactions with a one-click loading of structures. Moreover, its novel design displays a user-friendly platform, which facilitates the intuitive use of the tool’s features and exploration of the results for a better understanding of the biological processes behind the binding mechanism. Furthermore, the availability of PLIP source code enables local batch processing, customisation of the algorithm for special applications as well as active development of the tool in the community.


Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.The Gram-negative rod-shaped bacterium  Pseudomonas aeruginosa is a Gram-negative environmental gammaproteobacterium and a critical life-threatening pathogen in humans with compromised immune defense , the genome of P. aeruginosa is one of the largest among prokaryotes -6S RNA complex. We describe in-gradient distribution for thousands of mRNAs and sRNAs and predict hundreds of protein complexes involving metabolic and signal transduction proteins. In addition to performing Grad-seq on naive bacteria, we apply the approach for the first time to visualize transcript associations of an invading phage (ΦKZ) and to identify phage-encoded regulatory RNAs. These data sets are provided along with an online browser that helps to visualize the obtained sedimentation profiles in a searchable fashion and allows for them to be compared with Grad-seq data from other Gram-negative and Gram-positive species.Despite its importance as a human pathogen and model bacterium, global data informing on RNA/protein complexes are lacking for P. aeruginosa (see workflow in 600] of 0.3) in a rich culture medium to allow comparisons to previously established analyses under standard growth conditions. In addition, the exponential phase is optimal for phage infection and replication of ΦKZ  forms a ribonucleoprotein particle (RNP) with the SmpB protein to rescue stalled ribosome by a trans-translation mechanism  A total of 2,182 proteins were selected above the threshold from panel A; 614 proteins were discarded. (C) Normalization of protein levels by human albumin (UPS2) spike-in. (D) Dose-response curve for the RNA-seq analysis based on the ERCC spike-in. The linear dynamic range of detection was valid down to log10 sum of RNA reads of 1.5. (E) A total of 8,297 annotations were detected above the threshold of >100 reads/gradient, 2,971 annotations were excluded. (F) Normalization of RNA levels by the ERCC-130 spike-in. Download FIG S1, PDF file, 0.2 MB.Analysis and normalization of Grad-seq. (A) Dose-response curve for the MS analysis, plotted as relative concentration in UPS2 spike-in versus recovered MS counts. The linear dynamic range of detection is valid down to logCopyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the 10.1128/mBio.03454-20.2FIG S2FIG S2, PDF file, 0.4 MB.Sedimentation profiles of complexes that peak in fractions 8 to 20. (A) Approximately 30 uncharacterized proteins sedimented in HMW fractions and are named here by UniProt identifier. (B) Selected abundant protein bands in the Grad-seq experiment were selected for identification. (C) Protein entries were filtered by selected fraction where the protein was mostly abundant in panel B and a range of molecular weights based on the molecular size marker. The resulting table was ranked by abundance resulting in candidates. The abundance (iBAQ) was evaluated in contrast to the second-best hit. (D) Digital sedimentation profiles were correlated with the sedimentation profiles in panel B. Download Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the Viewing the RNA-seq results from individual fractions , we obseGrad-seq primarily recover cytosolic proteins, as it is performed on a cleared lysate. Indeed, membrane-associated proteins were underrepresented among the ∼2,200 detected proteins . For a qE. coli. Many metabolic processes involve multienzyme complexes, some of which are giant. Several such large complexes can readily be discerned from our Grad-seq data , covering 73.6% of the transcriptome . QuantitE. coli ). Note try phase .E. coli, S. enterica, and S. pneumoniae in which mRNAs generally cosedimented with ribosomes  of the same mRNA  constitute a potentially large class of bacterial riboregulators –50. Rece–51–ame mRNA . These iame mRNA , which iame mRNA . Obviousame mRNA . With rehlI gene . While tractions , supportTo validate the Grad-seq-based detection of UTR-derived fragments, we chose to probe the 3′ UTR of PA2758 mRNA on a Northern blot. We readily detected a distinct ∼50-nt RNA species with the predicted sedimentation in fractions 1 to 4, distinct from the full-length PA2758 mRNA . This 3′In silico target prediction using the CopraRNA algorithm  and modulate translation initiation. (C) Predicted interaction regions around stop codons. (D) A second example covers an interaction in the 5' UTR of the universal stress protein A family protein uspK. Download FIG S3, PDF file, 0.2 MB.CopraRNA analysis of RNA-RNA interaction of the PA2758 3′ UTR fragment. (A) Predicted interaction region around start codons were determined being in the first stem-loop that may represent the seed sequence. (B) An example is the intergenic region between Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the P. aeruginosa is known to possess two large posttranscriptional networks that depend on globally acting RBPs: one governed by Hfq and the other by two CsrA-like Rsm proteins. The mRNA and ncRNA ligands of these RBPs have been mapped in a transcriptome-wide fashion  data  data  provide eq) data .10.1128/mBio.03454-20.4FIG S4https://github.com/RickGelhausen/HRIBO]) supports the possibility of a short translated fragment starting at a putative start codon that is out of frame with ptrB. In addition, an inverted repeat in-between may serve as a Rho-independent terminator, because no stop-codon is reached in the reading frame. Download FIG S4, PDF file, 0.4 MB.ncRNA OsiS may harbor a hidden small open reading frame that may drive sedimentation in 30S fractions. Terminator 5′phosphate-dependent exonuclease (TEX) treatment indicates a TEX-unaffected 5′ end downstream of the annotated TSS . SequencCopyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the P. aeruginosa under optimal growth conditions, characterized by a rich supply of nutrients and rapid growth. To determine how this molecular organization is perturbed by a biotic stress, we subjected P. aeruginosa to infection by a phage, specifically, the lytic giant phage ΦKZ, which is a well-established model phage of pseudomonads and known to usurp many cellular functions of its host  The absorption profile and apparent proteome are not affected upon infection. (B) The global picture of the RNome is consistent with that shown in FIG S5, PDF file, 1.2 MB.Gradient fractionation of ΦKZ-infected Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the While altered abundance of host transcripts is one consequence of phage predation, we were particularly interested in the possibility that the phage infection might also perturb host transcriptome organization, altering the association of host transcripts with cellular factors to promote viral replication. Such changes, which should be visible as shifts in the gradient, could point out stress response mechanisms and pathways that remain invisible in standard RNA-seq experiments.liuR, coding for the transcriptional regulator of the of metabolic liu genes  are expressed  annotated phage genes , 51NC_00xpressed F and G. Global inspection of the Grad-seq data reinforced the notion that phage mRNAs are prioritized for translation. That is, in contrast with host mRNAs which were primarily detected in fractions 3 to 8 , the phain silico prediction, the ncRNA forms two stem-loops, the second of which might constitute the Rho-independent transcriptional terminator of the PHIKZ298 gene   and D. TWe carefully checked both UTR-derived RNA species for potential small ORFs, including alternative start codons, but found none that would be preceded by a strong Shine-Dalgarno sequence. We therefore consider both as ncRNAs and take them as illustrative examples how the extra information provided by Grad-seq, compared to that by simple RNA-seq, may help to discover overlooked transcripts in phage genomes.P. aeruginosa from a commonly studied growth condition, (ii) to assess the quality of these high-throughput results by independent methods, (iii) to illustrate how the technique might be utilized in the future to study bacteriophage-host interactions, and (iv) to organize the data such that they can be accessed and interrogated by nonspecialists. To facilitate the latter, we have set up an online browser  to provide the interested microbiologist with previously unavailable Grad-seq profiles of cellular transcripts and proteins in the model bacterium  browser , which i species , 28, 29. species , which iP. aeruginosa Grad-seq data cover only one growth condition, we believe that they already offer a huge discovery space. Given the bacterium’s impressive metabolic capabilities, the mass spectrometry (MS) profiles of all detected proteins should give new insights into metabolic multienzyme complexes. Obviously, not all these complexes are sufficiently stable to survive the long (overnight) ultracentrifugation procedure during which cellular complexes separate according to size and shape in a glycerol gradient. For example, the PDH complex dissociated into stable smaller subcomplexes  in ractions . In the  ligands  and B. Woli ProQ . The pseria ProQ , lack th10.1128/mBio.03454-20.6FIG S6Pseudomonas harbors important residues of a FinO domain. (A) Genomic locus of ProQ in PAO1. (B) Conserved residues in a FinO-domain (PF04352) are represented in red. Important residues in ProQ for RNA binding (E. coli. (C) ProQ proteins in Pseudomonas represent the condensed representatives in the diverse class of FinO domain proteins that can have C- or N-terminally associated domains. Download FIG S6, PDF file, 0.2 MB.ProQ in  binding  are indiCopyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the P. aeruginosa. The two partners show in an almost ideal form both narrow peaks and exceptionally high correlation in the gradient, primarily in fractions 5 and 6  Overrepresentation analysis for biological processes revealed that 5′ UTR transcripts that are shifted by >0.5 relative fractions are enriched for transcription factors. 3′ UTR transcripts that are shifted by more than 1 relative fraction towards the top are enriched for factors of aerobic respiration and proton transmembrane transport. (B) Forty-nine transcripts encoding oxidative phosphorylation factors are significantly more shifted towards the top than transcripts coding for metabolic factors. At the same time, the relative position in the reference condition was, for these transcripts, significantly higher and matches the trend in panel A. (C) Averaged sedimentation profile of the oxidative phosphorylation pathway pae00190 CDS and selected examples coding for ATP synthase, NADH-oxidoreductase, and cytochrome Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the Yet, we also observe the reciprocal behavior, i.e., host mRNAs that move into ribosome-associated fractions. A particularly noteworthy case is the mRNA of protein PA4441, which carries a predicted DUF1043 domain that is also found in cyclic oligonucleotide-based anti-phage signaling system (CBASS) effectors . Put simP. aeruginosa strain PA14, this sRNA may promote adaptive immunity against bacteriophages through suppression of Rho-dependent termination of early CRISPR transcripts  The 5′ UTR appeared in two fragments, of which the larger one was allocated to RNAP fractions and strongly diminished upon ΦKZ infection. Download FIG S8, PDF file, 1.8 MB.5S rRNA indicates processing effects and its major RNase E is altered in the 5′ UTR. (A) 5S rRNA sedimentation was not affected upon infection, but it become smeary in Northern probing that may indicate processing defects that could lead to diminished reads in sequencing. (B) The read coverage of the 5′ UTR of Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the rne-II motif  disappeits mRNA .P. aeruginosa by constructing a proteinaceous nucleus-like compartment   of 15 to ensure immediate infection of the entire culture. To ensure that infected cell culture was in the early infection phase, the infected culture was mixed and incubated for 5 min at room temperature without shaking, followed by incubation at 37°C for 5 min with shaking, as described previously .eviously . The cul10.1128/mBio.03454-20.10TABLE S2Table S2, XLSX file, 0.01 MB.STAR methods. Download Copyright © 2021 Gerovac et al.2021Gerovac et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This is an open-access article distributed under the terms of the 2, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.2% [vol/vol] Triton X-100, 20 U/ml DNase I [Thermo Fisher Scientific], 200 U/ml SUPERase-IN [Life Technologies]) and one volume of 0.1-mm glass beads. Lysis was obtained by vortexing this mix for 30 s, followed by 15 s of cooling on ice. This process was repeated 10 times, and the lysate was cleared at 12,000 × g for 10 min at 4°C. The supernatant was used for SDS-PAGE analysis and RNA preparation by TRIzol (Thermo Fischer Scientific). Aliquots of 180 μl supernatant were layered on a linear 10% to 40% (wt/vol) glycerol gradient and were sedimented in a SW40Ti rotor at 100,000 × g for 17 h at 4°C. The gradient was fractionated in 590-μl fractions, and the absorption was determined at 260 nm. Protein samples for SDS-PAGE were prepared for each individual fraction and analyzed as previously described . This mix was used as an external RNA control for read normalization and to determine the dynamic range in sequencing, as suggested in best practice guidelines by the External RNA Controls Consortium . RNA-seqThe nanoscale liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis was performed as previously described . Protein18 Stage tips (18 Empore SPE disks (3M) in a 200-μl pipette tip. Peptides were eluted with 60% acetonitrile-0.3% formic acid, lyophilized in a laboratory freeze-dryer (Christ), and stored at −20°C. Prior to nanoLC-MS/MS, the peptides were dissolved in 2% acetonitrile-0.1% formic acid.Peptides were purified through Cage tips . Each St18-AQ, 1.9 μm (Dr. Maisch), and separated with a 140-min linear gradient from 3% to 40% acetonitrile and 0.1% formic acid at a flow rate of 500 nl/min.NanoLC-MS/MS analysis was performed as previously described  with an Both MS and MS/MS scans were acquired in the Orbitrap analyzer with a resolution of 60,000 for MS scans and 15,000 for MS/MS scans. High-energy collisional dissociation (HCD) fragmentation with 35% normalized collision energy was applied. A Top Speed data-dependent MS/MS method with a fixed cycle time of 3 s was used. Dynamic exclusion was applied with a repeat count of 1 and an exclusion duration of 60 s. Precursors that were singly charged were excluded from selection. Minimum signal threshold for precursor selection was set to 50,000. Predictive automatic gain control (AGC) was used with a target value of 2 × 105 for MS scans and 5 × 104 for MS/MS scans. EASY-IC was used for internal calibration.Pseudomonas and ΦKZ reference sequences  using the READemption align function . Approxn 0.4.3, ) based oPseudomonas aeruginosa UP000002438 (strain PAO1), the ΦKZ proteome UP000002098 . The Grad-seq browser is online, accessible at https://helmholtz-hiri.de/en/datasets/gradseqpao1. READemption 0.4.5 is deposited at Zenodo 1134354 (https://zenodo.org/record/1134354). The READemption analysis folder is deposited at Zenodo 4024332 (https://zenodo.org/record/4024332).MS data are accessible at the ProteomeXchange consortium  via the


Libraries were prepared by selecting polyadenylated mRNAs and RNA Sequencing (RNASeq) was performed using Illumina HiSeq2000 platform. RNASeq data presented in this article were deposited in the NCBI sequence read archive (SRA) under the accession number SRP144764 and links to these data were also indicated in the Data INRAE repository (https://doi.org/10.15454/JJZ3QQ). Transcriptome analyses of these data were published in Hérault et al. (2019) and Liu et al. (2020).Duck species are known to have different ability to fatty liver production in response to overfeeding and gene expression analyses can help to characterize mechanisms involved in these differences. This data article reports the sequencing of RNAs extracted from the liver of Pekin and Muscovy duck species and of their reciprocal hybrids, Mule and Hinny ducks fed  Specifications TableValue of the Data •ad libitum or overfed and can be used to analyze responses to overfeeding and differences between genetic types.These data represent hepatic transcriptomes from 4 different duck genetic types (“pure” species and hybrids) fed •Any researchers involved in liver gene expression and metabolism can benefit from these data and process raw FASTQ files.•These data can be included in meta-analyses to characterize responses to feeding in different duck breeds.1ad libitum (n = 10) or overfed (n = 10), Muscovy ducks fed ad libitum (n = 9) or overfed (n = 10), Mule duck hybrids fed ad libitum (n = 10) or overfed (n = 10) and Hinny duck hybrids fed ad libitum (n = 10) or overfed (n = 10). A liver weight increase was observed in overfed ducks when compared to ducks fed ad libitum ad libitum. This increase was more or less significant depending on the genetic type  for direct access to FASTQ files and number of reads are indicated in 22.1ad libitum (free access to the diet). From 4 to 6 weeks of age, they were fed ad libitum with the growing diet. From 6 to 12 weeks of age, they were fed with the growing diet at restricted levels appropriate for each genetic type (ranging from 200 to 250 g per duck at the beginning to 360–380 g at the end of the period). At 12 weeks of age, ducks were either fed ad libitum with the growing diet or overfed 14 days with high carbohydrate overfeeding diet containing corn and corn meal, respectively indicated as ‘Ad libitum’ and ‘Overfed’ in As described previously  male ducFourteen hours after the last meal, ducks were rendered unconscious and unable to feel pain by electronarcosis and were slaughtered by neck sectioning and bleeding. Immediately after bleeding, liver were weighted , and sam2.2Total RNA were extracted from liver samples using NucleoSpin® RNA L kit  including guanidinium thiocyanate, silica membrane and on-column RNase-free DNase digestion according to the manufacturer's instructions without modification. RNA concentration was determined using a NanoDrop ND-1000 Spectrophotometer . Quality and integrity of RNA were checked using Lab-on-a-Chip Eukaryote Total RNA Nano chip and Bionalyzer 2100 device . RNA with absorbance ratio λ260 nm/λ280 nm and λ260 nm/λ230 nm >1.8 and RNA integrity number (RIN) > 7.4 were selected (resulting in 9–10 RNA samples per genetic type and per diet).http://get.genotoul.fr/en/). RNA libraries were prepared according to Illumina's protocols without modification using the Illumina TruSeq RNA Sample Prep Kit v2 . PolyA+ mRNA were first isolated using oligo(dT) beads. Then, mRNA were fragmented and reverse transcribed into double stranded cDNA. Adapters and hexamer tags (Libraries preparation and sequencing experiments were performed at the Genotoul genomics facility GeT-PlaGe (mer tags  were lighttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/).The reads were of good quality  as controlled using FastQC (Liver samples were collected in a previous study Frédéric Hérault: Conceptualization, Methodology, Formal analysis, Resources. Elisabeth Baéza: Conceptualization, Resources. Christian Diot: Conceptualization, Methodology, Supervision, Project administration, Writing- Original draft and Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene  Oryza sativa) is one of the most important global cereal crops and feeds roughly half of the world’s population. Rice is cultivated in hot and humid climates, an environment also suitable for the propagation of viruses. The first major outbreak of rice viruses was caused by the Rice dwarf virus (RDV) in Japan in 1897 [Nilaparvata lugens Stål, BPH) is the most destructive pest of rice and a substantial threat to rice production, causing billions of dollars in crop losses annually [1F) was completed by selecting and cultivating Rby1 and Rby2 carrying Bph3. Both of these rice lines grow as healthy rice plants following BPH infestation, but become sterile at a mature stage. We speculated that the observed sterile symptoms in BC1F are due to BPH-transmitted viruses and subsequent viral infections. To test this hypothesis, we first tried to isolate the virus from BPH-infested sterile rice plants. However, we did not find any virus. Then, we used deep metatranscriptomic sequencing to detect viral genomes in the sterile rice plants, and virus-derived small interfering RNAs (vsiRNAs) were also identified through small RNA sequencing. Our analyses reveal multiple known and many novel RNA viruses as well as abundant vsiRNAs. Together, the results provide insights into the diversity of viruses in rice plants, and our findings indicate that multiple virus infections occur in sterile rice plants.Rice . Ms55, a high-yield cultured elite Brown planthopper (BPH) was collected from a rice field at China National Rice Research Institute, Hangzhou (where the BPHs are mixed biotypes and mainly biotype 2) and maintained on the susceptible cultivar Ms55 under greenhouse conditions at China National Rice Research Institute.Bioassays were conducted following the method described by Liu with minor modification . Line seTotal RNA were isolated from rice samples Rby1-21 and Rby2-45. To isolate total RNA, the samples were homogenized in 700 μL of lysis buffer by TissueRuptor . Total RNA was then extracted by using an RNeasy Plus mini kit according to the manufacture’s protocol (Qiagen). The quality of the resultant RNA was evaluated using an Agilent 2100 bioanalyzer  before library construction. rRNA was removed by using a Ribo-Zero Magnetic kit (Plant Leaf) , the remaining RNA was fragmented, reverse-transcribed into cDNA, ends repaired and adaptor ligated by using an Illumina Truseq Total RNA Library Preparation Kit. Finally, ligated RNA was amplified to generate a cDNA library. Paired-end (150 bp) sequencing of each library was then performed on the HiSeq 2500 platform (Illumina). Library preparation and sequencing were carried out by Novogene Co., Ltd. . Sequencing reads were assembled de novo by using Trinity. The assembled contigs were first compared against the database of all reference RNA virus proteins downloaded from Genbank by using BLASTX with an E value cutoff at 1E-5 to maximize sensitivity while minimizing false-positive results. The resultant contigs were then compared to a nonredundant nucleotide (nt) and protein (nr) database to remove non-viral sequences. We also performed domain-based BLAST to detect highly divergent viruses. The assembled contigs were compared to the Conserved Domain Database (CDD) version 3.16 with an expected value threshold of 1E-2. The quality-filter virus contigs with unassembled overlaps were then merged using SeqMan implemented in the Lasergene software package v7.1 (DNAStar). To confirm the assembly results, reads were mapped back to the virus genomes with Bowtie2 and inspected using an integrated genomics viewer (IGV) for any assembly errors. The final sequences of the virus genomes were obtained from the majority consensus of the mapping assembly. Samtools were used to determine the sequencing depth and coverage [The potential ORFs of the newly identified virus genomes were annotated based on predicted amino acid sequences and conserved positions in the genome compared to the closest related virus genome available in GenBank. Functional domains within each ORF were identified using BLAST against the Conserved Domain Database (CDD) with an expected value threshold of 1E-5. For viruses with multiple RNA segments, we used various strategies to search for viral genome segments, as described previously . Non-RdRWe used RT-PCR and Sanger sequencing to verify the presence of each putative virus in the samples. Primers were designed from the contigs assembled from next-generation sequencing. Genome walking and RT-PCR were used to fill in sequence gaps for putative viral contigs that contained incomplete sequences. To exclude the possibility that putative viral contigs expressed endogenous virus elements (EVEs), DNA isolated from the corresponding samples was examined by PCR and Sanger sequencing. Genome termini were determined using 5′/3′ RACE kits (TaKaRa).To determine the abundance of RNA transcripts, we mapped total reads to the assembled genes or genomes using Bowtie . The reaTo determine the phylogenetic relationships of the newly identified RNA viruses, the amino acid sequences of the viral RNA-dependent RNA polymerase (RdRp)  of theseOne microgram of total RNA isolated from each rice sample was used to generate a library of small RNAs using a TruSeq Small RNA Sample Preparation Kit (Illumina) according to the sample-preparation instructions. Briefly, small RNA (<40 nt) was ligated with a single-stranded 3′-adapter and a bar-coded 5′-adapter. Ligated small RNA was reverse transcribed and amplified by PCR to generate individual DNA colony template library. Both libraries of Rby1-21 and Rby2-45 were used for 50 bp single-end sequencing by the Illumina HiSeq 2000 platform in two lanes.Bioinformatic analysis of small RNA data was performed using the CLC Genomic Workbench software package (Qiagen). Briefly, small RNA reads were quality-checked; low-quality reads and adapter sequences were first removed from the raw small RNA data set. Trimmed small RNA sequences shorter than 15 nucleotides were discarded. The remaining reads were mapped to the rice genome to remove host-related reads. The unmapped reads were subsequently mapped to putative viruses with the same stringency settings.indica rice variety resistant to BPH, Ms55 was used as a recurrent parent to backcross with the TZ21 line that harbors a BPH resistance gene, Bph3. These crosses resulted in BC1F population that may be resistant to BPH  and rice ragged stunt virus (RRSV) were present in both Rby1-21 and Rby2-45, while Rice Picorna-like virus 1 (RPiV1), Rice Toti-like virus (RtoV), and a BPH virus, Nilaparvata lugens reovirus (NLRV), were present only in Rby2-45 . We alsoUsing a BLAST-based method for viral genomes in GenBank, we identified eight novel viruses in Rby1-21 and Rby2-45, including three negative-sense RNA viruses and five positive-sense RNA viruses . Fuyang Mononegavirales and Bunyavirales orders (family of Phasmaviridae and Peribunyaviridae)  . For allviridae) . In the avirales . Howeveres order .Tombusviridae, Picornaviridae, and Nodaviridae families  sequences or surface contamination, we mapped raw reads from Rby1-21 and Rby2-45 genomes to our set of BLAST-derived candidate viruses . We founRice plants utilize small RNA pathways for viral defense . When vi, RRSV  and RpeV (3639 reads). To map these resulting small RNA reads to putative viruses, we first removed rice-specific small RNA reads and then aligned the remaining reads to viral genomes . Small RTo test whether the presence of these small RNA sequences was due to active virus infections, we next analyzed all 18–30 nt small RNAs from the identified viruses in BPH-infested Rby1-21 and Rby2-45. VsiRNAs detected in plants infected with RNA viruses are typically 21 nucleotides long and produced by Dicer-like 4 (DCL4), whereas 22-nucleotide vsiRNAs are produced by Dicer-like 2 (DCL2) ,17. We oSogatella furcifera [Viruses are a major threat to global rice production . Here, wurcifera ,21. HereTraditionally, virus discovery has focused on viruses that are pathogenic to their hosts and can be isolated. These viruses cause severe diseases and induce obvious symptoms in their plant or animal hosts. However, viral infections can also be asymptomatic or induce inconspicuous symptoms in their hosts. These viruses may accumulate in relatively low titers in their host organisms or become latent such that virus production ceases. Consequently, these mild viruses are not easily isolated or cultured from their hosts using traditional methods. Metatranscriptomics has emerged as a powerful approach to uncover hidden viruses in many organisms, including humans, arthropods, and plants ,25,26,27Cymbidium ringspot virus (CymRSV), a member of the family Tombusviridae [Tombusviridae family and had a strong sense strand bias in Rby1-21 and Rby2-45. RRSV is a member of the Reoviridae family and contains a double-stranded RNA genome. RRSV vsiRNAs also showed a strong sense strand bias in Rby1-21 and Rby2-45, suggesting RRSV-derived vsiRNA originated from highly structured single-stranded viral RNA. Similar results were observed in singly (RRSV) or doubly (RRSV and SRBSDV) virus-infected rice plants [RNA interference (RNAi) in plants can function as an antiviral defense mechanism against invading viruses. To counteract RNA silencing, many plant viruses have evolved viral suppressors of RNA silencing (VSR) that target various components of the plant RNAi machinery. Different plants show different symptoms and produce different vsiRNA profiles, likely due to differences in plant RNAi components . vsiRNA sviridae . In our e plants . Taken tindica rice variety, with the Bph3-gene containing TZ21 line to produce a BPH-resistant BC1F line. Interestingly, we determined that BC1F lines are sterile only after BPH infestation. Using deep metatranscriptomics sequencing and vsiRNA analysis, we determined this sterility is likely due to infection by BPH-derived viruses. Our data suggest that the BPH-infested Rby1-21 line harbor active RTV1 and RRSV viral infections. Similarly, we find that the BPH-infested Rby2-45 line harbors active RTV1, RRSV, and RPeV viral infections. Notably, our sequencing failed to identify DNA polymerase sequences indicative of DNA viruses. We identified numerous RNA virus sequences that lacked sequence similarity to rice reference sequences. Interestingly, these RNA virus sequences displayed signatures of DCL2, DCL3, or DCL4 processing (high levels of 21–24 nt vsiRNAs), suggesting they may also be of a viral origin [1F rice may be caused by BPH-transmitted RNA viruses. Further studies are required to test this specific hypothesis. Additionally, our results showed that rice plants did not display obvious disease symptoms when they were co-infected by multiple viruses. Viruses in mixed infections can interact with each other in different ways [Breeding pest-resistant rice cultivars is an important approach for maintaining crop yields ,32,33. Hl origin ,35. BPH ent ways ,37. In t


In clinical trials and meta-analysis, atherosclerotic vascular events (AVEs) during treatment with immune-checkpoint inhibitors (ICIs) have been reported with low incidence. However, preclinical data suggest that these drugs can promote atherosclerosis inflammation and progression of atherosclerosis plaques, and there is now growing and convincing evidence from retrospective studies that ICIs increase the risk of atherosclerotic vascular events including arterial thrombosis, myocardial infarction and ischemic stroke. Prospective studies are needed to increase knowledge on long-term effect of ICIs or their combinations with other cardio-toxic drugs, but in the meantime a careful assessment and optimization of cardiovascular risk factors among patients treated with ICIs is advisable. Immune checkpoint inhibitors (ICIs) have extended survival across many tumor types and their use in cancer treatment has been increasing over time . ICIs arInitially, atherosclerotic vascular events (AVEs) such as arterial thrombosis, coronary artery disease (CAD), acute coronary syndrome (ACS), myocardial infarction (MI) and ischemic stroke were not specifically recognized as irAEs and therefore not usually considered as a possible toxicity of ICIs. However, there is now growing preclinical and clinical evidence suggesting a possible correlation between ICIs and AVEs. In the present review we summarize and discuss the available literature on this topic.*” OR “immune checkpoint inhibitor*” OR “anti-CTLA-4” OR “anti-PD-1” OR “anti-PD-L1” OR “ipilimumab” OR “tremelimumab” OR “nivolumab” OR “pembrolizumab” OR “atezolizumab” OR “durvalumab” OR “cemiplimab”) AND .For the present review, the PubMed database was searched from the inception to 31st January, 2021, using the following terms: -cholesterol, that may undergo oxidative modification leading to the formation of oxidide LDL (oxLDL). The accumulation of oxLDL may elicit an innate inflammatory response with the recruitment of circulating monocytes that, infiltrating arterial walls, differentiate into macrophages and at a later stage transform into foam cells that eventually die creating a core area in the plaque that consists of necrotic cells and cholesterol crystals .As the atherosclerotic plaque grows, accumulation of immune cells and particularly T cells occurs at the shoulder regions of the lesion. In this context of chronic inflammation, adaptive immune response plays a crucial role, and T-cells that recognize autoantigenic components of LDL regulate plaque development . ParticuT cell functions are finely regulated by immune checkpoints, including CTLA-4 and PD-1 that represent now targets for cancer immunotherapy. CTLA-4 is mainly involved in the priming phase of T cell activation, whereas PD-1 is involved in the effector phase . When thin vivo atherosclerosis experimental model based on hypercholesterolemic, low-density lipoprotein receptor (LDL-R) knock-out mice (ldlr−/− mice) , 1211, 1in vivo study showed that ldlr−/− mice receiving high-cholesterol diet for 10 weeks had increased PD-L1 and B7-1 expression in dendritic cells (DCs) from the iliac lymph nodes, and increased PD-L1 and PD-L2 expression in peritoneal macrophages, compared with mice receiving control-diet (pdl−/−ldlr−/− mice) developed a significant increase in the aortic atherosclerotic burden after 10 weeks of high-cholesterol diet, with a 2-fold increase of plaques in aortic root and a 3-fold increase of plaques in aortic arch and descending aorta, when compared with the control group (ldlr−/− mice). In comparison with the control group, pdl−/−ldlr−/− mice had also increased smooth muscle cells and collagen deposition in the plaques, increased CD4+ and CD8+ T-cells and macrophages in the intima, increased CD4+ T-cells with activation phenotype (CD25+CD62Llo) in the iliac lymph nodes, and increased serum TNF-a levels. Furthermore, macrophages and DCs taken from pd1−/−ldlr−/− mice led to increased CD4+ T cell proliferation in vitro as compared with those taken from control mice . Among the 31 patients with an early acute vascular event, 90% had ≥ 2 cardiovascular risk factors  and 55% had ≥3 risk factors. A multivariable analysis identified non-small cell lung cancer (NSCLC), history of acute vascular events and dyslipidemia as significant risk factors for AVEs during treatment with ICIs (p < 0.0001), and in 25% of cases death occurred within 1 month from the vascular event.Only few retrospective studies have investigated the association between ICIs and AVEs . In partith ICIs . Not unep < 0.001), in a multivariable model that included known cardiovascular risk factors . Particularly, the use of ICIs was associated with a higher risk for MI , coronary revascularization , and ischemic stroke  (p < 0.001). Interestingly, in an imaging sub-study on 40 melanoma patients treated with ICIs, there was a >3-fold increase in the rate of atherosclerotic plaque progression after ICIs initiation (from 2.1% per year pre- to 6.7% per year post-ICI). As compared with non-statin users, patients receiving statins had lower yearly rates of plaque progression of total aortic plaque volume  and non-calcific plaque   .In contrast with these results, a smaller retrospective study reported an improvement of atherosclerosis with nivolumab . Among 3Data from prospective studies and meta-analysis suggested that AVEs are a rare event during treatment with ICIs. In fact, ICI-related AVEs have been only sporadically reported in prospective clinical trials. Particularly, few cases of MI were described in patients treated with atezolizumab for urothelial cancer  and withIn a meta-analysis evaluating the incidence of cardiovascular irAEs in cancer patients treated with ICIs, the incidence of MI was as low as 0.4% (95% CI 0.0–0.07%), although this result could be an under-estimation given that the 26 studies included were not specifically designed to evaluate the incidence of cardiovascular toxicity and only 6 out of 26 reported the incidence of MI as an irAE . Similarpost-hoc analysis of a prospective observational study reported high incidence of arterial thromboembolic events among 217 NSCLC patients treated with ICIs  (p = 0.015) and a trend toward better OS   and 2.0% of stroke  . Consists, 6.5%) . Interes = 0.14) .In prospective clinical trials and meta-analysis, the incidence of AVEs during treatment with ICIs was relatively low . HoweverPatients enrolled in clinical trials are usually a highly selected population, and elderly patients who may have subclinical atherosclerosis, as well as those with high cardiovascular risk or history of cardiovascular disease, have been often excluded or under-represented in clinical trials investigating ICIs , 23. ThiIn a recently published, well-designed matched cohort retrospective study, treatment with ICIs significantly increased the risk for AVEs and the atherosclerotic plaques volume. This finding is consistent with preclinical data showing that CTLA-4 and PD-1 blockade accelerates the progression of atherosclerotic plaques , 24. HowThe research on the correlation between ICIs and AVEs is now particularly important, since several combinations of ICIs with other drugs such as anti-angiogenesis agents, that potentially increase the risk for arterial thrombosis and acute vascular events, have been recently introduced in clinical practice . AlthougSome prospective studies designed with the aim to collect data on AVEs and other cardiovascular toxicities among cancer patients receiving ICIs are currently ongoing , and their results will probably provide better knowledge on the correlation between ICIs and AVEs. However, research efforts should be also directed to translational studies aiming to identify novel circulating biomarkers or possibly immunogenomic factors that may predict for cardiovascular toxicity of ICIs  . BaselinThis approach will require ever closer cooperation between oncologists and cardiologist in the near future.AI wrote the draft. All authors critically revised and approved the manuscript.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 transcriptomes encompass a large number of functional non-coding RNAs (ncRNAs), only some of which have protein-coding capacity. Since their initial discovery, ncRNAs have been classified into two broad categories based on their biogenesis and mechanisms of action, housekeeping ncRNAs and regulatory ncRNAs. With advances in RNA sequencing technology and computational methods, bioinformatics resources continue to emerge and update rapidly, including workflow for in silico ncRNA analysis, up-to-date platforms, databases, and tools dedicated to ncRNA identification and functional annotation. In this review, we aim to describe the biogenesis, biological functions, and interactions with DNA, RNA, protein, and microorganism of five major regulatory ncRNAs  in plants. Then, we systematically summarize tools for analysis and prediction of plant ncRNAs, as well as databases. Furthermore, we discuss the silico analysis process of these ncRNAs and present a protocol for step-by-step computational analysis of ncRNAs. In general, this review will help researchers better understand the world of ncRNAs at multiple levels. In the last few years, a number of non-coding RNAs (ncRNAs) have been described in plants involved in several processes, ranging from RNA maturation, splicing, regulation of transcription, post-transcriptional RNA modifications, and nucleosome remodeling. Therefore, it is unquestionable that ncRNAs play a significant role in gene regulatory network ,2,3,4. WCurrent studies have revealed that ncRNAs can be transcribed from DNA sequences in protein-coding genes, intergenic or intronic regions . In termSo far, the biogenesis of some regulatory ncRNAs has been clearly described ,19,20; hWith the advancement of high-throughput RNA-seq technologies, the diversity of ncRNAs world has been unveiled. To date, numerous studies have applied RNA-seq technology to discover known and novel classes of ncRNAs in diverse tissues and developmental stages . These pIn this review, we mainly focus on five regulatory ncRNAs, including microRNA (miRNA), small interfering RNA (siRNA), tRNA-derived small RNA (tsRNA), circular RNA (circRNA), and long ncRNA (lncRNA) in plants. Here, we will show a regulator ncRNAs biogenesis landscape and give a hypothesis of tsRNAs biogenesis in plants based on previous studies. Besides, we will discuss the ncRNAs interaction with DNA, RNA, protein, and microorganism for helping our understanding of the dynamic inter-molecular networks within plant cells. Furthermore, we will summarize the algorithms, databases, and RNA-seq-based analysis pipelines of the regulatory ncRNAs in the field of plants to incentivize researchers to make greater use of RNA-seq technologies and bioinformatics approaches to discover the individual, as well diverse, regulatory ncRNAs in the exceedingly complicated landscape of ncRNAs.Currently, massive endogenous ncRNAs with various regulatory potentials have been discovered in various plant species ,23. BasemiRNA biogenesis is a multistep process involving transcription, processing, modification, and assembly of the RNA-induced silencing complex (RISC) A 34,35,,35,36. FMYB genes and is down-regulated in salt-stress responses among Arabidopsis ,55TAS ge6 (RDR6) . FinallyAS genes . In Araba-siRNAs . The TASolerance .cis-NAT-siRNAs and trans-NAT-siRNAs. However, only cis-NAT-siRNAs have been described in plants. trans-NAT-siRNAs remain only a hypothetical possibility. Therefore, in this review, cis-NAT-siRNAs are collectively referred to as nat-siRNAs. Previously, nat-siRNAs were thought to be generated by the hybridization of separately transcribed complementary RNAs. However, to date, many of the nat-siRNAs investigated depend on RDR for their accumulation . T. T89]. Tstresses . Besidesstresses , Arabidobidopsis , and tombidopsis .Recent studies have demonstrated that ncRNAs are involved in the maintenance of plant homeostasis through ncRNA-associated interaction with DNA, RNA, protein, and microorganism, respectively, and have important implications for plant growth, development, differentiation, and reproduction ,101. TypA series of experimental methods, such as PAR-CLIP , HITS-CLHomo sapiens [Furthermore, the interactions among congener and isotypic ncRNAs can influence several biological processes, including epigenetic modifications and translation. For example, miRNA response elements (MRE), such as circRNAs, lncRNAs, and eRNAs, act as competing endogenous RNAs (ceRNAs) with rich implications for gene regulation in various physiological and pathophysiological processes at the post-transcriptional level . ncRNAs  sapiens ,117,118,So far, numerous ncRNAs have been reported to be involved in the regulation of gene expression in the nucleus as direct regulators ,121,122.Furthermore, some lncRNAs have been demonstrated to be able to divide the nuclear region into distinct compartments and participate in the organization of multi-chromosomal regions ,125,126.ncRNA–protein interactions play a crucial role in regulating cell metabolism. It is widely known that numerous RNA binding proteins (RBPs) can change the fate or function of the bound RNAs in the nucleus region. To date, the RNA–protein regulatory relationship can be detected by CLIP-seq . TherefoGenerally, ncRNAs achieve and regulate various functions by forming various ribonucleoprotein (RNP) complexes with proteins. For example, snRNPs, which are composed of snRNAs and proteins, can direct both canonical splicing and alternative splicing. Besides, numerous nucleotides in pre-rRNA, pre-snRNA, and pre-tRNA undergo post-transcriptional modification by nucleolar RNP particles . Small nALTERNATIVE SPLICING COMPETITOR (ASCO) lncRNAs have been reported to be regulators of alternative splicing in Arabidopsis through interactions with splicing factors [As for circRNA, research has shown that they can serve as protein sponges to transport proteins into specific subcellular compartments. On the other hand, lncRNA can achieve their function via recruitment, inhibition, and acting indirectly through genome organization and transcription . ALTERNA factors . ApparenPlants have an animal-like innate immune system ,140. WheMetarhizium anisopliae and Sclerotinia sclerotiorum, respectively, in conidia formation, and sclerotia are differentially expressed [Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small ncRNAs [Currently known microorganisms regulate their pathogenic capacity based on two modes of small ncRNA regulation. (1) The first way is to regulate their own toxicity through sRNAs derived from microorganisms, such as the sRNA of the entomopathogenic fungi xpressed ,143. (2)l ncRNAs .Arabidopsis miR863-3p fine tunes plant immune responses during infection by sequentially silencing negative and positive regulators of plant immunity [Arabidopsis AGO that is highly induced by bacterial infection [rhizobia-legume symbiosis, AGO7 of Lotus japonicas is required for TAS3 ta-siRNA biogenesis, and it is important for the development of nitrogen-fixing nodules in plant roots [In addition to the innate immune system, plants have evolved two ways of regulating small ncRNAs in response to infection by pathogenic microorganisms. The first way is that plant endogenous sRNAs are involved in the regulation of immune responses. For example, immunity . Anotherimmunity . The secimmunity ,148,149.nfection . In rhiznt roots ,152.In the past two decades, substantial research efforts have been devoted to discovering non-coding regulatory RNAs and studying their functions, including miRNAs, lncRNAs, and circRNAs. With the development of next-generation sequencing (NGS), high-throughput sequencing has been widely used to characterize the ncRNA transcriptomes under various conditions, which provides an unprecedented opportunity to discover ncRNAs and identify differentially expressed ncRNA transcripts. However, rapidly growing sequencing data have created challenges for the identification, annotation, and storage of ncRNAs. Here, we systematically summarize the prediction tools, database resources, and integration workflows of various ncRNAs in plants.Next-generation sequencing offers unprecedented opportunities to discover and quantify various ncRNAs. To date, numerous computational methods have been developed for single ncRNA category, such as miRDeep-P2, miR-island, phasiRNAClassifier, NATpare, tsRFinder, RNAplonc, FEELnc, Circle-Map, and CircMarker ,160,161.With the decreasing cost of NGS sequencing and the advent of various tools for prediction and characterization of ncRNAs, the number of annotated ncRNAs has grown exponentially. Therefore, relevant databases of ncRNAs are emerging rapidly. There are lots of ncRNA-related databases in plants, most of which focus on a single-type ncRNA, such as PmiREN2.0 , MepmiRDp, FEELnc, Circle-Map, CPAT, and many other pipelines [Currently, the research on various ncRNA functions using RNA-Seq and sRNA-seq technology has achieved great success. However, only a few tools and platforms are available for the analysis and prediction of most ncRNAs in plants, such as mirTools 2.0 and sRNAtools ,162. Mosipelines ,172,245.In this workflow, we present a protocol for step-by-step computational analysis of ncRNAs, which mainly consists of three parts: (1) raw fastq data preprocessing, including read quality filtering and 3′ adapter trimming; (2) alignment and annotation; (3) sequence feature analysis, including ncRNA data alignment and novel ncRNA prediction.Although identification and characterization of biogenesis and fundamental functions of regulator ncRNAs are necessary, efforts to understand the depth and diversity of ncRNAs are the way forward. In the past decade, with the advancement of high-throughput sequencing technology, many bioinformatics methods and tools have emerged, which has enabled the discovery and research of many ncRNAs. Studies have found that ncRNAs mainly include housekeeping ncRNAs, such as tRNA, rRNA, etc., which are necessary to maintain plant life activities, and regulatory ncRNAs, such as miRNA, lncRNA, circRNA, etc., which play a role in regulating the special life process of plants. The interactions between these ncRNAs and the regulation of the expression of other encoded genes constitute a complex regulatory network of ncRNAs in plants.Arabidopsis has been revealed [Compared with numerous studies, such as miRNA and lncRNA, the functional characterization of circRNA is still in its infancy. Even in humans and animals, there are only a few reports on the functional study of circRNAs. Although many plants circRNAs have been identified from plant circRNA databases or sequencing analyses, the functional mechanism of only one circRNA from revealed . Due to At present, the research of these ncRNAs is more focused on their regulatory mechanisms during the growth and development of plant organs or tissues and in special environments. However, the expression and changes of ncRNAs in different cell types remain unclear . In receIn this review, we systematically summarized the functions and regulatory relationships of major classes of ncRNAs in plants. Besides, we reviewed and summarized the computational methods, tools, and knowledge bases of ncRNA in plants in detail. Notably, we proposed a general protocol for step-by-step computational analysis of different types of ncRNAs to help researchers choose appropriate ncRNA analysis tools and platforms. We hope this work contributes to a better understanding of the complex ncRNA world.