A new class of deformation is presented for a planar loop structure made up of slender elastic bodies and joints. In demonstrating the circumferential shortening of the multi-jointed elastic loop, diverse three-dimensional (3D) deformations emerge through piecewise deflections and discrete rotations. These 3D morphologies correspond to conformations of molecular ring systems. Through image processing, the 3D reconstructions of the deformed structures are characterized by number, geometry, and initial imperfections of the body segments. We elucidate from measurements that the conformational deformation without self-stress results from a cyclical assembly of compressive bending of elastic bodies with high shear rigidity. The mechanical insights gained may apply in controlling the polymorphism exhibited by the cyclical structures across scales. They are found everywhere at different length scales from sub-oceanic cables to human-sized rods and ropes, to plant and animal microstructural fabrics, to molecular pillars and chains such as carbon nanotubes and double-stranded deoxyribonucleic acids (DNAs). Their unique large deformability have received attention from the scientific community including the field of theoretical and applied mechanics, and even at present the problems associated with the intricate deformation into three-dimensional (3D) configurations are being tackled in areas such as the compressive buckling from 2D into 3D architecture14. Depending on the elastic rod, there are four types of deformations, i.e., stretching/compression, shearing, bending, and twisting, which are paired with the axial and shear forces and the bending and torsion moments induced inside the rod. Hockling of stretched and twisted cables is a typical example of large 3D deformations of inextensible elastic rods without shearing, collectively named Kirchhoff’s rods. The terminal torques and low tensions applied to an initially straight configuration induce helical buckling, whereby the deformation mode transitions from torsion to 3D deflection17. After a helix begins to form locally, more and more twisting yields a loop of helices as a self-contacting formation perpendicular to the longitudinal axis. This writhing leads to a final phase called a plectoneme; e.g., a plectonemic phase emerges in supercoiled helixes of DNA21.The deformation of a slender elastic body is concisely described by the elastic rod model represented by a single arc-length parameter prescribing the center linesolenoid22, which involves shortest-wavelength writhing in the longitudinal direction. The uniform helical formation and the post-buckling localization, including the plectonemic or solenoid phase, can be simulated using elastic rod theory and its extension. The extended theory describes the local manner of deformation through axial extension and shear as well as that of bending and torsion24. Although understanding this hockling phenomena is of significance from the viewpoint of the 3D deformation of higher-order structures, the resulting coiled morphology is ill-reproduced, in general, given the specific control parameters such as the external action of terminal torsion and stretching.Non-negligible stretching and shearing cause, after uniform helical buckling, another type of deformation that features self-contact, known as a tangle model25. The tangle model comprises a number of rigid elbowed bodies, linked in a loop by revolute hinges. The possible morphology corresponds to the conformation of a molecular ring system, which is described by the Dreiding stereo model or through conformational analyses27.A control of the diverse structural morphologies undergoing large deformation is of interest to the various fields as illustrated previously. In this context, we focus on modeling that reduces the limited degrees of freedom in deformation to achieve a controllable system for the 3D morphology. We present another concept of a slender structure subjected to conventional contraction forces that is capable of being deformed into countable 3D patterns. The concept concerns the tangle mobility of interconnected pivotal segments, collectively referred to as a 30.Our main structural idea is to replace all the rigid equilateral components in a tangle model with soft tubular bodies. These elastic bodies are intersected via joints in a loop configuration, and tuning of the number of bodies and joints can control the degrees of freedom for each deformation. In the design of our proposed structure, we select bellowed tubing for the geometry of the elastic body. Bellows are an ancient mechanical component that have been used where compression and/or flexion is required. Their fundamental mechanical properties have been established from modelling their elasto-plasticity to beam modelling using finite-element methodsWith deformable loops made of a finite number of these elastic bodies and joints, we examined the circumferential shortening of each loop, which is equivalent to applying resultant forces directed towards the center. Following an analysis of the conformational deformations accompanying the piecewise deflections and discrete rotations, we report the manner of the various morphologies, which depends on structural factors, specifically, the number of bodies, the stiffness of stretching, bending, and twisting, and initial imperfections.pseudo-rotation.The proposed structure forms a loop, assembled alternately with equilateral elastic bodies and joints. Figure If the line inside the structure is shortened, the multi-jointed loop is compressed circumferentially. The resultant of the corresponding compressive force vectors acting on a joint is a force concentrated toward the center and all the point-symmetric forces on the circle are regarded as a cohesive attraction. The number of joints equals the number of point forces; the joints play a role in allocating points of application on the midline of the loop.single-traction/double-traction tests. In the deformed configuration with bending, the direction of each force is shifted toward that of the local radius of curvature.This loading can be applied in a traction test in which one reels off the line at a single side or both sides using a mechanical device such as a winch; hereafter, such tests are referred to as n is the number of bodies and 24.Figure mentclass2pt{minimmentclass2pt{minimTo investigate how the 3D deformation is affected by the geometry of elastic bodies, we performed double-traction tests for two different shapes, specifically, bellows and straight tubes. For the additional tests, we prepared small loops with entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaIn the unloading test , we performed image processing for deformed structures with To investigate the conformational deformation dependent on the number of bodies  from multiple-angle views of the deformed structure within X, Y) of the displaced joints during deformation. The convex hull yields the area enveloped by the conformation projected onto the XY-plane, which we denote by S. The curve of S(R) provides a criterion for the onset of conformational deformation as demonstrated in the next section for later discussion.To characterize the conformational deformations observed above, we assess the surface area of the convex hull derived from a point set of the positions of joints from the front view of the structure. We first performed a 3D reconstruction of the joint positions in world coordinates (S to R for S(R), there is a considerable decrease after the onset of the conformation , which is superposed on that in Fig. S begins with the near-initial value of Figure =24 Fig. b show diX, Y, Z), with the rotational axis of the actuator aligning with the Y-axis and the XY-plane being parallel to the camera image at Z-directional depth positions of joints , the structures softened moderately until the onset of the conformation at T(R) reduces once but the structures gain resistance. When T drops instantaneously around S(R) in Fig. The three measured forces Fig. d exhibitFrom a mechanical aspect, we next discuss the conformational deformation observed in the double-traction tests. The in-plane deformation up to the onset of conformational deformation can be represented as an even in-plane deformation of a curved beam element that substitutes for the bent bellowed tubes undergoing compression and bending. The details of modeling are described in Supplementary Information In the initial configuration of a circular loop, a uniform distribution of self-stress induced by its pure bending moment exists along the entire structure. This self-stress is released in response to a traction force during the double-traction test. We consider the state of the bending moment vanishing on all the joints together as the onset of conformational deformation so that the balance of each joint could be satisfied at any configuration of the conformation. Specifically, this hypothesis is a necessary condition for conformational deformation and is adopted latter in performing the convex-hull analyses.R to the actual compression displacement u of a loop structure. Based on the mechanics of materials, we are led to a linear relation for the traction force T with u and then derive an equation for T(R) as in Eq. (T(R) are about two times higher than those predicted, which means that in experiments the friction along a line on the joints and the attachment apparatus is non-negligible; see Fig. In observing the traction line to be extensible, we set a correction factor s in Eq. . To comps in Eq. d, the meS(R), when considering the isometric shrinkage of the corresponding polygon. Figure The pre-conformational model enables us to readily formulate a change in the convex-hull area, The inconsistency for  it Fig. c1. In otIn summary, we proposed a multi-jointed loop structure comprising a finite number of elastic tubular bodies, alternately connected with revolute joints. Subjected to concentrated forces acting toward the center, the structure is capable of forming 3D conformations through the coupling of piecewise continuum deflections and discrete revolute rotations.n), and the initial imperfections of the structure that lead to phenomena such as buckling. Note that n also corresponds to the number of point forces acting on the joints.Conformational deformations were demonstrated in traction tests of the traction line passing through the loop structure, which depends on the type of traction (single/double traction), the number of bodies  quantifies the conformational deformation projected onto the plane coordinates in front view. There are two inflection points on a single curve of S(R), the first indicating the onset of the conformational deformation and the second signifying the folding deformation, which does not appear necessarily.By applying image processing techniques, deformation analyses were performed on 3D reconstructions of the displaced joints. The convex-hull area The three structures for Last, we predicted the pre-conformational deformation using the linear analyses with a curved beam element and discussed the differences between predictions and measurements. Both the changes in normalized convex-hull area are consistent at the early stage of deformation and also the onsets of 3D deformation are well estimated given the condition of zero moment acting on joints. Moment-free-joints indicate that self-stresses never exist in the deformed configuration. From energy considerations, therefore, a series of conformations arises as a consequence of exploring the lowest energy configuration of a loop without self-stress, which is the driving mechanism underlying conformational deformation. For a comprehensive understanding of the deformation mechanics with a large set of test pieces, we first need to identify and reduce unexpected frictional forces inside the structure to reconsider the integrated design of the materials and experimental system.25. We therefore expect that mechanical insights into the new deformation phenomena will provide unique perspectives on more complex conformation states of molecular chains and protein architectures38. Although these deformations behave passively in our experiments, active conformational deformation is also possible from the multi-jointed elastic loop by controlling the unidirectional bending of soft actuators40. In this manner, friction-free planar rotations work preferentially on joints instead of through disallowed pseudo-rotations.The mechanical stress field in our developed loop structures cannot be translated directly into the real complicated intermolecular forces. However, the measured deformation morphologies correspond to the conformations of the tangle model, which are linked to the conformations of cyclic molecular systemsSupplementary methods, materials, figures, tables and discussions are presented in detail in Supplementary Movie S1.Supplementary Movie S2.Supplementary Movie S3.Supplementary Movie S4.Supplementary Movie S5.Supplementary Information 6.


Emerging polymer 3D-printing technologies are enabling the design and fabrication of mechanically efficient lattice structures with intricate microscale structures. During fabrication, manufacturing inconsistencies can affect mechanical efficiency, thereby driving a need to investigate how design and processing strategies influence outcomes. Here, mechanical testing is conducted for 3D-printed lattice structures while altering topology, relative density, and exposure time per layer using digital light processing (DLP). Experiments compared a Cube topology with 800 µm beams and Body-Centered Cube (BCC) topologies with 500 or 800 µm beams, all designed with 40% relative density. Cube lattices had the lowest mean measured relative density of ~42%, while the 500 µm BCC lattice had the highest relative density of ~55%. Elastic modulus, yield strength, and ultimate strength had a positive correlation with measured relative density when considering measurement distributions for thirty samples of each design. BCC lattices designed with 50%, 40%, and 30% relative densities were then fabricated with exposure-per-layer times of 1500 and 1750 ms. Increasing exposure time per layer resulted in higher scaling of mechanical properties to relative density compared to design alteration strategies. These results reveal how design and fabrication strategies affect mechanical performance of lattices suitable for diverse engineering applications. Emerging 3D-printing technologies are enabling the design and fabrication of complex, mechanically efficient lattice structures from polymer materials ,2,3. 3D-There are numerous 3D-printing processes capable of producing lattice structures, including extrusion, resin, and powder-based printing ,16. EachAlthough many types of lattices have been experimentally and theoretically characterized ,29,30, iTherefore, this study focuses on the mechanical and dimensional characterization of polymer lattices fabricated with varied design and processing strategies using DLP printing. Design alterations include topology, beam diameter, and unit cell length to create lattices of different relative densities within the constraints of DLP polymer printing technologies. Cube and BCC unit cells were selected to form lattices with 40% relative density for benchmarking statistical distributions of print dimensions and mechanics for 30 samples each. Mechanical characterization includes unconfined compression testing to determine elastic modulus, yield strength, and ultimate strength to benchmark lattice performance. A second experiment was conducted by varying lattice’s relative density from 50% to 30% with 1500 and 1750 ms layer exposure times to compare how different design and processing strategies affect lattice manufacturing and mechanics. The study provides novelty through comparing design and processing strategies for polymer 3D printing in relation to actual measured outcomes that may differ from ideal cases for a variety of conditions. Outcomes are expected to highlight which design and printing strategies are most advantageous for maximizing performance of lattice structures for specified applications.Lattices were designed using a Python script to automate lattice construction from beams organized as unit cells using macros with Abaqus software  . Beams wThe first lattice had a Cube unit cell, 800 µm beam diameter, and 1.8 mm unit cell length and is referred to as the Cube-800ø design. The second lattice had a BCC unit cell, 800 µm beam diameter, and 3.1 mm unit cell length and is referred to as the BCC-800ø design. The last lattice had a BCC unit cell, 500 µm beam diameter, and unit cell length of 1.9 mm and is referred to as the BCC-500ø design. The design of these three lattices provides a basis for comparison with one design parameter controlled each in relation to the BCC-800ø design, which is only different from the Cube-800ø design by unit cell topology and the BCC-500ø design based on its beam diameter design. Designs of lattices were determined based on constraints in the build process such that lattice beam diameters were large enough for printing based on native resolution of the printing system, while ensuring lattices were small enough for printing an entire batch of thirty lattices at once.Lattices were fabricated using a DLP printing approach with an Envision One cDLM printer  using E-RigidForm Amber resin  that has an elastic modulus of approximately 3000 MPa. The Envision One printing system has a native xy-resolution of 93 µm, z-resolution of 50 to 150 µm, an ultraviolet light-emitting diode (UV-LED) light source at 385 nm wavelength, and build envelope of 180 by 101 by 175 mm. Prints commenced with manufacturer layer thickness of 50 µm, support thickness of 0.8 mm, support spacing of 1.75 mm, upright orientation, ambient temperature, initial burn-in time of 17,500 ms, waiting time before exposure of 1000 ms, separation velocity of 300 µm per second, and exposure time of 1500 ms or 1750 ms per layer. Support material size was determined through preliminary testing to minimize support material to ensure its removal without destroying the surrounding structure while ensuring support material was printed large enough to facilitate structural stability during printing. Preliminary work and past studies informed these parameters as suitable limits for high-quality prints according to hardware constraints .Envision One Rapid Prototype software  was used to prepare digital files from STLs and add support material structure. All samples were printed with an orientation indicated by Samples were removed from the build platform using a metal spatula, rinsed by hand with isopropyl alcohol, then placed in a bath of isopropyl alcohol with stirring to remove excess uncured resin. Support material was removed from each sample using a metal blade. Samples were placed in a PCA 2000 ultraviolet curing chamber (EnvisionTec) for two-hours for postprocessing. Thirty samples were fabricated for each of the 3, and input for Equation (1) [The height tion (1) :(1)ρr=wAn Olympus DSX-HRSU digital microscope  with 5× objective was used to produce stitched images of lattice faces to enable measurement of beam diameters. Images were stacked to produce an in-focus image that accounted for the depth of diagonal beams being at a different focal plane than orthogonal beams. Beam diameters were measured for unit cells on the side face of lattices, where side faces refer to those that are perpendicular to the build platform during fabrication. Mean beam diameters were measured for three representative samples of each design and categorized according to the beam’s orientation relative to the build direction. Beams that were aligned with the build direction were labelled as 0°, those that were diagonal (for BCC topologies only) were labelled as 45°, and those that were perpendicular to the build direction were labelled as 90°. For each lattice face, the unit cells making up the two middle columns of the face were used to collect measurements, as indicated in Mechanical testing was conducted using an Instron 5966 universal testing machine  and protocols according to the ISO-13314 standard  that areMechanical testing results were analyzed using a Python script to calculate the mechanical properties of lattices, including elastic modulus, yield stress, and ultimate stress. Elastic modulus (tion (2) :(2)Em=σ2The yield strength , then the BCC-800ø design (0.46), and least accurate for the BCC-500ø design (0.54). The differences in relative density accuracy may occur due to the smaller spacing between beams in the BCC-500ø design compared to the other two designs, which leads to a high accumulation of resin curing in void regions of the lattice. These findings are in agreement with past studies for resin printing that have demonstrated greater resin accumulation and fusion toward the center of lattice structures . The resThe fabricated designs in The microstructures of lattices were imaged for three samples of each design, with representative images provided in Beam diameters were measured and aggregated for the mid-point of beams on the vertical column of unit cells for each microscope image, which resulted in the measurement of forty-two 0° beam diameter measurements and fifty-four 90° beam diameter measurements for each design, with seventy-two 45° beam diameter measurements for each BCC design, according to methods described in The thirty fabricated samples for each The mean mechanical properties for elastic modulus, yield strength, and ultimate strength are plotted for each design in The mechanical properties of lattices were then investigated by considering the coupled measurement between each sample’s relative density and resulting mechanics to determine how the variation in relative density due to fabrication inconsistencies affects mechanics. Mechanical properties were fit to a linear regression model to determine correlations, according to Equation (3) :(3)p∗=mIn Equation (3), Regression results demonstrate a positive slope for all relationships, which indicates that an increase in relative density results in an increase in mechanical properties. The magnitude differs across designs, with a generally steeper slope for BCC-800ø and BCC-500ø mechanical properties. The correlational coefficient is generally higher for BCC designs than the Cube design, thereby indicating less expected variation when considering how increases in relative density will affect the targeted properties. These relationships are all plotted with individual measured data points for each sample in BCC topologies were designed with 500 µm beam diameters and relative densities of 50%, 40%, and 30%  to deterFabrication accuracy was further assessed using microscopy to determine consistency of beam diameters and pores on lattice surfaces . ResultsThe mean beam diameters for each design and exposure time were measured according to When comparing designs of different relative densities, the beam diameters remained consistent. For instance, for the 50%, 40%, and 30% relative density designs of 1750 ms exposure time per layer, the 0° mean beam diameters were 480 µm, 460 µm, and 480 µm, respectively. These results suggest that exposure timer per layer has a greater effect on mean beam diameter accuracy compared to altering unit cell length.Mechanical results for elastic modulus, yield stress, and ultimate strength were plotted when considering all fifteen samples printed and tested for a given layer exposure time as a single data set . ClusterAll data was fit with regression models according to Equation (3) when considering the relative density of all printed designs based on the set exposure time to quantify how relative density scales with mechanical properties for each condition . Each reRegression results demonstrate a positive correlation with relative density for each mechanical property for all designs, which is illustrated with Overall, these results demonstrate that lattices of the same relative density can have different scaling with mechanical properties depending on each lattice’s design configuration and process parameters. Previous studies have demonstrated such phenomena for printing processes, such as fused deposition modeling ,47, but Mechanical and dimensional characterization was conducted for sets of lattices with different topology, beam diameter, and relative density designs in addition to altered processing by varying exposure time per layer using DLP printing. Printing demonstrated that lattices were qualitatively printed accurately with a distribution of measured relative densities that primarily varied due to resin accumulation around pores. In comparison to fused deposition modeling, PolyJet, and sintered lattices, the DLP printed lattices generally have a high fidelity in reproducing microscale features. For instance, fused deposition modeling/fused filament fabrication beams tend to have globules of material that create large discrepancies in diameters ,49, PolyMechanics were investigated for differently designed lattices for two sets of experiments. Both sets of experiments contained a control design referred to as BCC-500ø or BCC-0.4When comparing the BCC-500ø and BCC-0.4Limitations exist from the extensive time required for experiments, imaging techniques, and mechanical characterization that could be investigated further in future investigations. Future work could investigate further trade-offs in topology, relative density, and beam diameter mechanics using design for experiments, in particular, by conducting experiments to determine how mechanical efficiency scales with varied topology, design, and processing strategies. Further processing experiments could consider higher or lower exposure times. However, lower exposure time per layer may result in no printed structure forming, while higher exposure leads to further fusion of beams that further deviate from the mechanical efficiency provided by lattice structures. Microscopy imaging only considered one face of each lattice at a time to infer the inner structure density based on relative density measurements, but future studies could investigate beams throughout the inner volume of lattices, which is possible with microCT analyses ,51. FurtThis study investigated the mechanics of lattices configured with two different topologies (Cube/BCC), three different designed relative densities , and two different exposure times per layer (1500 and 1750 ms) using DLP printing. Initially, thirty samples each of Cube and BCC unit cell topologies with beam diameters of 500 µm or 800 µm were printed with 40% relative density. The mean measured relative density was higher than intended for all designs and ranged from 42% for the Cube-800ø design to 54% for the BCC-500ø design. These higher relative densities are attributed to regions of solid fusions around pores, especially in the smaller spacing of the BCC-500ø design. Elastic modulus, yield strength, and ultimate strength were tested and plotted against relative density for each sample, which demonstrated a positive correlation between mechanical properties and relative density due to manufacturing deviations for all designs. A second experiment was conducted to compare strategies for redesigning BCC-500ø designs with 50% or 30% relative density and altering the exposure time per layer. When comparing printed designs of similar relative density measurements, those that were processed at higher exposure times had greater mechanical efficiency .Key conclusions of the study are: (1) manufacturing variations in lattices affect relative density distributions; (2) higher relative density from manufacturing variations results in higher relative mechanical properties; (3) scaling of mechanical properties with relative density varies based on design and processing strategies; and (4) increasing exposure time per layer results in greater improvements in mechanical efficiency than investigated design alteration strategies. The findings highlight a need for considering and evaluating diverse polymer processing strategies to promote high mechanical efficiency of 3D-printed polymer lattices. Future work could further investigate how topology, materials, and processing each uniquely affect manufacturing and mechanics to find optimal strategies for lattice construction that benefit diverse engineering applications.


Vitis vinifera cv. Malbec and cv. Syrah grafted on 110 Richter rootstock. Fifty-one metabolites were detected and unequivocally annotated employing UPLC-MS based metabolite profiling. Integrating the data using hierarchical clustering showed a significant effect of late pruning treatments on must and wine metabolites. Syrah metabolite profiles were characterized by a general trend of higher metabolite content in the late shoot pruning treatments, while Malbec profiles did not show a consistent trend. In summary, late shoot pruning exerts a significant effect, though varietal specific, on must and wine quality-related metabolites, possibly related to enhanced photosynthetic efficiency, which should be taken into consideration when planning mitigating strategies in warm climates.Continually increasing global temperature could severely affect grape berry metabolite accumulation and ultimately wine polyphenol concentration and color intensity. To explore the effect of late shoot pruning on grape berry and wine metabolite composition, field trials were carried out on  ClimateA possibility for adapting viticulture to impending climate changes is to improve photosynthetic efficiency during grape ripening processes, e.g., by postponing winter pruning from dormancy to post-dormancy. Other field practices capable of modifying vine phenology include post-veraison leaf removal, post-bud-break pruning, and severe trimming or forcing vine regrowth . Late shThe goal of this work was to investigate the effect of incremental late shoot pruning on the chemical composition of must and wine from Syrah and Malbec varieties. Earlier works showed phenological syncing between the treatments, but lower yield and higher quality in the late shoot pruned vines . Using a22.12/vine) , 186 m above sea level. The experimental layout was a randomized complete block design with five treatments, and it was replicated four times. Row orientation was east/west, with a slight tendency to the south and vine and row spacing 1.5 and 3 m, respectively , LSP2 (pruning after 2 weeks of bud break), LSP3 (pruning after 3 weeks of bud break), WP+T (standard winter pruning and cluster thinning as control), and WP (standard winter pruning as control). These pruning treatments were randomly assigned to blocks, so there were 11*4 vines for each treatment and treatments were applied to the same vines in both years.The winter pruning and late shoot pruning treatments were performed exactly as described by  on the v2.3Each year, berries from all the treatments were harvested at a more or less, similar TSS (°Brix) value. Must samples were collected from bulk unfermented juice after settling for an hour and then decanting the liquid. The wine samples were obtained from the micro vinification process of the corresponding treatments . Must an2.3.1https://www.labogene.com). While wine samples were directly lyophilized in polypropylene tubes without pulverization. Metabolites were then extracted by adding 1ml pre-chilled methanol: chloroform: water extraction solution (2.5:1:1 v/v), following the protocol described by (http://www.elma-ultrasonic.com/) and centrifuged at 14000rpm for 10 min . The supernatant was then decanted into new tubes, mixed with 300μl of chloroform and 300μl of MiliQ water , vortexed for 10s and then centrifuged again for 5min at 14000rpm. The water/methanol phase, obtained from the extraction protocol (around 1ml), was collected, and filtered using 0.22 μm  and stored in vials for UPLC analysis.For the metabolite analysis, must samples were pulverized by mortar and pestle while keeping the material frozen by pouring liquid nitrogen. Approximately 200 mg of must powder was weighed and lyophilized in ScanVac CoolSafe™  system operating in both positive and negative ion modes, alternatively (https://www.waters.com/) version 4.1 was used for UPLC system control and data acquisition. The acquired raw data were processed using the MarkerLynx application manager  as described by www.chemspider.com).Each sample was analyzed twice in an Ultra Performance Liquid Chromatography coupled with a Quadrupole Time-of-Flight Mass-Spectrometer . We used two different internal standard references as analytes have different ionizing efficiency in positive and negative ion modes, e.g., some analytes are not ionized in negative mode, but very well ionized in positive mode. We annotated metabolites uniquely in both negative and positive ion modes. We used corticosterone as an internal standard reference to normalize data of metabolites annotated in positive ion mode, and due to poor ionization of corticosterone in negative mode, we use another internal standard ampicillin as a reference to normalize data of metabolites annotated in negative mode to minimizes the variability in sample preparation as well as variability generated by the instrument such as injection volume. Further accuracy was acquired by normalizing samples’ values to the relative dry weight (post-lyophilization). Therefore, data refer to the relative metabolite abundance based on ion counts. For hierarchical clustering representation, mean values were used, and metabolite values were ln (x+1) transformed. In addition, unit variant scaling was applied for this analysis.The two datasets (wine and must) were utilized for statistical analysis based on i) hierarchical clustering as a multivariate approach to the study; ii) analysis of variance for each detected metabolite  considering the effect of three factors , the effect of their interactions ; iii) Pearson correlation analysis for investigating on the putative relationships among metabolites both as must and wine autocorrelations and must-to-wine bipartite correlation. Statistical analysis and data visualizations were performed in the R environment  using R 33.1Standard winter pruning was conducted according to common agricultural practice in mid-February. In examining the seasonal patterns of phenological development throughout the three seasons, previous findings showed that late pruning treatments re-started the phenological process at a similar pace . A one-wpost hoc Tukey’s test revealed a significant difference between cultivars  and between vintages (2017 vs 2018) , and strong positive correlations among flavanols in Malbec (Sheet2) as well as strong positive correlations of astilbin, coutarate, caff-tart, and trans-piceid with the group of flavanol. Similar relations were also observed in the wine samples. When comparing the two cultivars separately, the Malbec correlation matrix reported higher indices in the number of correlations and their strength (Sheet1 and Sheet2). On the contrary, must and wine anthocyanins did not correlate well except for the wine delph-3-glu, delph-3-acet and peo-3-acet with all the must anthocyanins (Sheet1 and Sheet2).To study the coordination of metabolic processes concerning the pruning treatments, a correlation analysis using Pearson correlation was performed. The correlation analysis of must samples revealed high positive correlations among anthocyanins and flavanols. Vitisin_A showed a negative correlation with most of the metabolite classes except anthocyanins and stilbenes in must samples of Syrah and Malbec. Cultivar differences included a high number of positive correlations of gallocatechin and quercetin with anthocyanins in Syrah (4The wine quality is reflected by the amount and composition of a large number of primary and secondary metabolites that shape its sensorial experience. Considering this view, it is likely that optimizing the environmental conditions during the grape ripening period will be required to produce good quality wine. These conditions can be achieved by modifying the viticulture practices in which late shoot pruning is the most important and economical way. Late shoot pruning can delay the phenology towards a cooler environment, thus able to minimize the effect of elevated temperature. The current study aimed to dissect the effect of late shoot pruning on differential metabolite accumulation in two red grapevine varieties, Malbec and Syrah.In the LC-MS based metabolite profiles, amino acids did not follow a general trend, but their concentration decreased in late pruning, likely because these aromatic amino acids are precursors to the biosynthesis of secondary metabolites , and plaAmong stilbenes, our study showed that resveratrol and viniferin were significantly modulated by the pruning treatments, with Syrah showing higher resveratrol content and Malbec in viniferin content. These results are consistent with previous studies showing the high plasticity of these phytoalexins  and theiOur study shows a clear varietal specificity with respect to the effect of pruning treatments on the metabolite profile. For example, the role of the genotype is evident in anthocyanin’s pattern of change across the experimental setup, which follows a general trend of accumulation towards the later pruning treatments in Syrah. These results are supported by the findings that anthocyanin content changes in relation to climate conditions , and theThe effectiveness of the time-dependent late pruning technique for improving grape composition was confirmed in both Syrah and Malbec. Grapes from the late shoot pruning treatments had a higher concentration of polyphenols in the must, which is in line with earlier observations of late pruning to decouple the ripening dynamics of sugars and phenolic substances .Wine metabolite profiling also confirmed the ameliorating effect of pruning, which is consistent with previous findings . StatistLate shoot pruning can be effective when environmental conditions may limit the achievement of the desired grape ripeness, which is supported by earlier studies performed by  as well 5The late shoot pruning shifted grapevine phenology and perturbed its metabolism. Nonetheless, a combination of a highly regulated berry phenological syncing, renewed leaf development with improved carbon assimilation capacity and a lower cluster load, led to improved metabolic features such as more stilbenes and flavonoids. Having that said, varietal-specific qualitative and quantitative metabolic alterations of the berry metabolism should be considered, and similar studies should be conducted before upscaling conclusions.The original contributions presented in the study are included in the article/CP: Conceptualization, Data curation, Methodology, Investigation. PV: Data curation, Methodology, Investigation, Writing – original draft, Writing – review & editing. GH: Formal analysis, Methodology, Investigation. YS: Formal analysis, Methodology, Investigation. MH: Formal analysis, Methodology, Investigation. DF-M: Formal analysis, Methodology, Investigation. ED: Conceptualization, Investigation, Methodology, Resources. YN: Conceptualization, Investigation, Methodology, Supervision, Resources, Project administration, Writing – original draft, Writing – review & editing. AF: Conceptualization, Investigation, Methodology, Supervision, Resources, Project administration, Writing – original draft, Writing – review & editing. All authors contributed to the article and approved the submitted version.


Ipomoea batatas L.) with a strain of Lactobacillus plantarum leads to an increase in its exopolysaccharides (EPS) content which is useful for enhancing the functional properties of flour. The objective of this study was to develop healthy and nutritious meal-based biscuits from fermented sweet potato (FSP) flour supplemented with mackerel flour. Eleven formulations containing wheat flour, FSP flour, nonfermented sweet potato (NFSP) flour, and mackerel flour at different proportions defined following a mixture design were used to prepare biscuits. Physicochemical, microbiological, and sensory analyses of the different biscuits were performed. Biscuits prepared with FSP at 100% scored the lowest lipid (10.83 ± 0.97 g/100 g DM) and the highest sugar (67.43 ± 0.64 g/100 g DM) contents. The incorporation of mackerel flour in the formulation led to a significant (p < 0.05) increase in the protein and mineral contents of biscuits thus conferring an immune-boosting property to these latters. All the biscuits were of good microbiological quality independent of the formulation. The highest DPPH free radicals scavenging activity  were observed with biscuits prepared with FSP flour at 100%. The sensorial characteristics of biscuits prepared with equal proportions of wheat and FSP flours were highly appreciated by the panelists with scores close to the ones prepared with wheat flour at 100%. The results of this study demonstrate the potential of FSP flour as a substitute for wheat flour in biscuits preparation. It also suggests biscuits prepared with FSP flour supplemented with mackerel flour as a functional and immune-boosting food.The fermentation of sweet potato ( Nowadays, the demand for safe and nutritious diets satisfying energy needs and nutrition deficiency is constantly growing. To satisfy that demand, the development of new products has become the main concern of food industries . Hence, Ipomoea batatas L.) stands as an example of these roots and tubers due to its easy cultural conditions , its nutritional value, and its richness in bioactive compounds like flavonoids, anthocyanins, β-carotenes, phenolic compounds, and vitamin C [Lactobacillus plantarum. The swelling property of EPS reported in the literature improves the functional properties of flour [Sweet potato  due to its richness in minerals such as zinc, unsaturated fatty acids, and proteins [To improve the nutritional value of biscuits, they are generally supplemented. According to Ahmad and Ahmed , the supproteins . Hence, Ipomoea batatas L.) was used in this study. That variety was chosen because it is a β-carotenes fortified variety highly cultivated, prized, and consumed by the whole class of Cameroonians [Scomber scombrus) of approximately 500 g each were sampled in a fish shop, introduced in an icebox, and transported to the laboratory.The yellow variety of sweet potato , and rinsed with sterile distilled water. The tubers were peeled and cut manually with stainless steel in slices of 5 mm thickness. The slices were pooled, heated at 100°C for 5 min, and allowed to cool at room temperature (25 ± 2°C) in aseptic conditions. The precooked slices were fermented by the strain L. plantarum following the method described by Panyoo et al. [L. plantarum at 8 Log CFU/mL. The mixture was homogenized and incubated at 37°C for 33 h. After fermentation, the mixture was dried at 50°C for 72 h in a ventilated oven , ground and sieved (Ø = 500 μm). The flour of fermented sweet potato was packaged in a polyethylene bag and stored at 4°C for analyses. A sample of sweet potato treated in the same conditions but not inoculated with L. plantarum was used as a control to produce the nonfermented flour of sweet potato.Samples of yellow sweet potato were superficially cleaned with tap water, disinfected with chlorine . The mackerel flour was produced following the method described by Murali et al. [Ø = 500 μm) to obtain flour (821.62 g) which was packaged in a polyethylene bag and stored at 4°C for analyses.The fish species used in this study was i et al.  with somThe moisture and ash contents of flours were determined following the method AOAC . Total sThe method of Philips et al.  was usedμm) and added to the creamy paste. The mixture was thoroughly homogenized with a spatula. The dough obtained was cut and shaped using a biscuit mould (0.5 cm thick discs of 6 cm diameter). The shaped doughs were deposited on a clean stainless steel tray previously greased with margarine. The trays containing the shaped dough were introduced into a Memmert electric oven  and baked at 180°C for 15–20 min. The biscuits were removed from the oven and cooled at room temperature (25 ± 2°C) before being packaged in polyethylene bags. To determine the quantities of wheat flour, fermented sweet potato flour, and mackerel flour leading to a biscuit with the best microbiological, physicochemical, and sensorial qualities, eleven formulations with variable quantities of these ingredients were generated using Minitab version 18.1 . The different formulations are presented in The method described by Eyenga et al.  with somFor the proximal composition of the different samples of biscuits, moisture and ash were determined following the method AOAC . Total s−1 to 10−6). The total aerobic mesophilic flora was enumerated on Plate Count Agar  following the pour plate method ISO 4833-1 [Staphylococcus spp. while total coliforms and E. coli were enumerated following the method ISO4832 [Samples were prepared following the normalized method ISO 6887-2 . For eacO 4833-1 . Yeast aO 4833-1 . The metO 4833-1  was used ISO4832 . FollowiThe sensory characteristics of the 11 biscuits produced were evaluated by 20 trained panelists (12 women and 8 men) following the method described by Eyenga et al.  with somμg/mL), and 1 mL of each solution was mixed with 4 mL of DPPH solution (0.2 mM in methanol). The mixture was homogenized and stored in darkness at room temperature (25 ± 1°C) for 30 min. Then, the optical density was read at 536 nm against the blank (solvent). The DPPH radical scavenging activity was calculated using the formula:control refers to the optical density of the solution free of extract, and ODsample refers to the optical density of the solution containing extract at a given concentration.The antioxidant activity of biscuits produced in this study was assessed through their ability to scavenge free radicals. For this, the DPPH (2.2-diphenyl-1-picrylhydrazyl) free radical scavenging method described by Uddin et al.  was used50) was calculated from a graph representing the DPPH inhibition percentage as a function of the biscuit concentrations. It was used to compare the antioxidant activity of the different biscuit samples. Ascorbic acid (vitamin C) was used as standard.The concentration providing 50% of DPPH inhibition .μm. These particles' sizes were higher than that of wheat flour which varies between 50 and 200 μm. This result suggests that the use of fermented sweet potato flour in biscuit production might lead to a more granular biscuit. A similar observation was made by Eyenga et al. [Dacryodes edulis) powder.Flour production yields of 75.88, 66.66, and 64.61% were obtained from fermented sweet potato, nonfermented sweet potato, and mackerel, respectively. A granulometric distribution of fermented sweet potato flour revealed that its particles' size was between 200 and 500 a et al.  as the ap < 0.05) in water content observed between the flours from fermented (12.75 ± 0.27%) and nonfermented (8.08 ± 0.40%) sweet potato could be ascribed to the presence of exopolysaccharides (EPS) produced by the strain L. plantarum during the fermentation [p > 0.05), thus, demonstrating that the fermentation of sweet potato by L. plantarum did not influence its mineral content. The ash content of wheat flour (0.87 ± 0.04 g/100 g DM) was significantly (p < 0.05) lower than that of sweet potato flour. This difference arises from their physiologic growth conditions and their mineral composition.Globally, the flour from FSP scored the highest water content (12.75 ± 0.27%) while the least one was recorded with wheat flour (6.70 ± 0.22%). The significant difference  flour was noticed by Wijanarka et al. [Some functional characteristics of sweet potato flours were assessed, and the results are presented in s et al. . The vals et al.  with swea et al. . It was a et al. . Hence, p > 0.05) difference was noticed between the water solubility index of flours from fermented (54.53 ± 0.15%) and nonfermented (56.36 ± 1.61%) sweet potato. The high values of the water solubility index obtained with sweet potato flours could be explained by the difference in starch structure. Indeed, during the preheating treatment applied to sweet potato, the structure of starch granules changed leading to an increase in their water solubility [The water solubility index of sweet potato flours was also assessed. While wheat flour showed the least value of water solubility index (25.43 ± 1.84%), no significant (lubility .p < 0.05) decrease in the oil holding capacity of sweet potato flour. This could be ascribed to the reduction of nonpolar compounds  during the fermentation of sweet potato. Adams et al. [Another important functional property of flours assessed in this study was their oil holding capacity since oils can improve the mouth feel of products as well as their retention of flavors . Regardis et al.  also foup < 0.05). The incorporation of mackerel flour in the biscuit formulation has not shown a significant (p < 0.05) effect on their water contents. The highest water content (13.98 ± 1.33%) was recorded with biscuit BS6 prepared only with FSP flour. Its counterpart biscuit BS10 prepared only with NFSP flour scored a water content of 11.05 ± 0.26%. This could be attributed to the high water holding capacity of the flour from FSP as shown previously. According to FAO [g to FAO , the watp < 0.05) increase in lipid contents of biscuits was noticed. This can be justified by the low oil holding capacity of flour from FSP. Hence, during the baking process, lipids are not held in the dough leading to a reduction of lipids' contents in the biscuits. A similar observation was noticed by Nguyen and Nguyen [Low lipid contents were recorded with biscuits BS4 (10.83 g/100 g DM), BS6 (11.46 g/100 g DM), and BS7 (11.98 g/100 g DM). These biscuits were characterized by their high proportions of flours from FSP. However, as the proportion of FSP flour decreases, a significant (d Nguyen . The higd Nguyen  with bisp < 0.05) from one biscuit to another. Globally, the protein content of biscuits was found to increase with the proportion of mackerel flour and decrease when the proportion of FSP flour decreases. This observation can be attributed to the highest protein content of mackerel flour as reported in the literature [L. plantarum through metabolic activity leading to the production of EPS during the fermentation of sweet potato could explain this result. However, it is important to notice that biscuits BS7 prepared with 74.38% of FSP flour, 1.25% of mackerel flour, and only 24.37% of wheat flour scored a high protein content of 10.91 g/100 g DM. This observation demonstrates the suitability of FSP flour as a substitute for wheat flour in the production of biscuits that could be consumed by persons independent of their age to fight against malnutrition. The protein contents of biscuits prepared with flour from FSP were generally higher than 10.13 ± 0.80 g/100 g DM reported by Eyenga et al. [The protein contents of the different samples vary significantly (terature . The higp < 0.05) from 47.17 g/100 g DM (BS5) to 66.43 g/100 g DM (BS4). This high proportion of total carbohydrates in biscuits could result from the chemical composition of flours. The results of the present study demonstrate the high energetic potential of the different biscuits and suggest their suitability for children. Indeed, the quality of food formulated with as target adults or children for whom the energy requirements are high is generally assessed through their energy content [Sugars were present in all biscuits at levels that range significantly  compared to wheat flour (0.87 g/100 g DM).p < 0.05) from 1.33 ± 0.03 to 3.00 ± 0.01 Log CFU/g. The most contaminated sample was biscuit BS1 (3 Log CFU/g). Globally, there was no association between the proportions of flour from FSP and the TAMF loads. Compared to the norms for which the TAMF count of biscuits should not exceed 3 Log CFU/g [E. coli and total coliforms were absent in all samples (Staphylococcus spp. were detected in biscuits BS1 (0.77 ± 0.10 Log CFU/g), BS2 (0.53 ± 0.08 Log CFU/g), BS3 (0.81 ± 0.05 Log CFU/g), and BS7 (0.87 ± 0.04 Log CFU/g), their loads were all lower than the threshold value recommended by the European Commission regulation (1 Log CFU/g) [The results of the microbiological analysis of the different samples of biscuits are presented in og CFU/g , all bis samples . Althougg CFU/g) . Yeasts g CFU/g) , the loaFor newly developed food products like biscuits prepared with FSP and mackerel flours, the descriptive sensory analysis appears as a powerful tool to predict its acceptance by consumers. The biscuits prepared with FSP flour at different proportions are depicted in For the development of new products intended for human consumption, color is generally used as the criteria that mostly influences the purchasing decision. In this study, the color of biscuits was assessed by the panelists, and biscuits BS5 and BS9 show the highest scores of 7.90 ± 1.16 and 7.55 ± 0.99, respectively . These bBesides color, one of the desirable textural criteria of biscuits generally appreciated by consumers is their crispness . In thisp > 0.05), the odor scores increase as the proportion of FSP flour decreases. The highest scores were recorded with biscuits prepared with wheat flour and free of sweet potato flour (BS5 and BS9). This can be explained by the low oil retention ability of the FSP flour as it is well known that lipids contribute to improving the retention of flavors. Hence, wheat flour with its high oil retention capacity will contain more flavors developed during the baking process. Another explanation for the fact that panelists have mostly appreciated the biscuit BS5 prepared with wheat and mackerel flours could be the specific aromatic compounds brought by the mackerel flour.Odor of the biscuits was also evaluated by the panelists. Globally, although nonsignificant , the taste globally decreases as its proportion in the biscuits decreases. This result suggests that the incorporation of the mackerel flour in the dough formulation might bring an improvement in the final taste of biscuits.With regards to taste, the most appreciated biscuit was BS9 (7.90) prepared with wheat flour at 100%. Surprisingly, biscuit BS4 prepared with 100% FSP flour also showed a high taste score of 7.80. This appreciation of their taste by the panelists could be attributed to the high sugar content of these biscuits as starch was hydrolyzed into reducing sugars during the fermentation . MoreoveRegarding the overall acceptability, the least appreciated biscuit was BS7 with a score of 5.36. Biscuits prepared with FSP flour at 100% (BS4) showed a weak overall acceptability score. The highest overall acceptability score (8.10) was recorded with biscuit BS9 prepared with 100% of wheat. Srivastava et al.  also repL. plantarum in sweet potato paste during fermentation were exploited as a swollen baking agent to confer the same characteristics as wheat flour to sweet potato flour. Besides this baking ability, the antioxidant activity of the aqueous, ethanolic, and methanolic extracts of the different biscuits containing these EPS was assessed through their ability to scavenge DPPH free radicals. This test was chosen because free radicals are the main cause of the imbalance of antioxidants leading to oxidative stress in the human body. DPPH free radical was reported to act as an extensively proven tool for evaluating the ability of antioxidants to scavenge free radicals [p < 0.05) with the biscuits and the solvents. The IC50  of the different extracts were determined and the results are presented in 50 values of the different extracts ranged from 1.90 ± 0.02 to 9.95 ± 0.07 μg/mL and were all higher than that of the standard ascorbic acid (0.021 ± 0.001 μg/mL).In this study, the properties of EPS produced by radicals . All the50 values. This can be attributed to their high contents in EPS. Several studies have demonstrated the antioxidant activity of EPS [β-carotene [With regards to the biscuits, the antioxidant activity generally increases when the proportion of FSP flour in the formulation increases. The extracts from biscuit BS4 (100% FSP flour) were more active as they scored the lowest ICy of EPS . Indeed,y of EPS . Besidescarotene . This rep < 0.05) in IC50 values were noticed (50 values recorded with aqueous extracts compared to the methanolic ones also justify the fact that polar compounds of biscuits such as polysaccharides could be mostly involved in the antioxidant activity.Considering the extraction solvents, ethanolic extracts were more active than aqueous and methanolic extracts independent of the biscuits as significant differences ( noticed . This caThis study demonstrated the ability of FSP flour to be used as a swollen baking agent in biscuit preparation and suggests its potential as a substitute for wheat flour. Besides their nutritional values, biscuits prepared with FSP flour deserve functional properties and can be suitable for the management of chronic diseases. The incorporation of mackerel flour in the formulation has led to a significant improvement in the protein and mineral contents of the biscuits thus conferring an immune-boosting property to these latters. Biscuits formulated with equal proportions of wheat and FSP flours were highly appreciated by the panelists. However, given that some sensorial characteristics of biscuits prepared with the FSP flour at 100% were not highly appreciated by the panelists, further studies should be performed to optimize the formulation and characterize the EPS.


Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function. Aminoacyl-tRNA synthetases possess unique domains. In this study the structure of the vertebrate IARS1 and EARS1 complex reveals that vertebrate IARS1 protects the DNA repair factor BRCA1 from proteolytic degradation via its UBX-fold domain. In addition to their catalytic role in translation, vertebrate ARSs are involved in the regulation of various types of cell signaling, which function in cell cycle regulation, tumor suppression, cytokine activity, RNA splicing, cell adhesion, and angiogenesis3. These non-catalytic orthogonal functions are primarily achieved through various unique domains (UNEs) of ARSs acquired during evolution4. Spliceosome analyses identified a number of ARS variants with null catalytic activities that retain important cellular functions through UNEs, demonstrating the significance of the non-translational functions of UNEs5. ARSs assemble into a multi-synthetase complex (MSC), which is comprised of nine ARSs (arginyl- (RARS1), aspartyl- (DARS1), glutaminyl- (QARS1), glutamyl-proryl- (EPRS1), isoleucyl- (IARS1), leucyl- (LARS1), lysyl- (KARS1), and methionyl-tRNA synthetase 1 (MARS1)) and three auxiliary proteins , AIMP2 (p38), and AIMP3 (p18)) in vertebrates7. Within the MSC, each ARS communicates with other ARSs through interactions between the UNEs and between the UNEs and catalytic  domains, and through the three auxiliary proteins. Although the exact role of the MSC remains elusive, it has been proposed to provide a channel for the efficient delivery of charged tRNAs to ribosomes9. A recent study suggests that the MSC does not affect global translation, but is critical for the nuclear localization of ARSs, and ARSs released from the MSC contribute to various cellular events15. The MSC undergoes dynamic structural changes under different conditions, and various forms of the MSC are expected to be present18.Aminoacyl-tRNA synthetases (ARSs) catalyze the ligation of each amino acid to the 3’ hydroxyl group of the cognate tRNA and thereby establish the genetic code for protein synthesis19. Systematic depletion and yeast two-hybrid (Y2H) analyses suggest that IARS1 binds to the WHEP domain of EPRS1 through its UNE-I domain21. However, crosslinking and mass spectrometry (XL-MS) analyses revealed that IARS1 interacts with RARS1, LARS1, and MARS1 in the MSC18. These results suggest the dynamic and multi-valent interactions of IARS1 with the components of MSC. Furthermore, little is known about the orthogonal function of IARS1 UNE-I. Upregulation of the gene encoding IARS1 increases p38 MAPK pathway activation and reduces PI3K pathway activation, leading to phenotypic switching and apoptosis in aorta vascular smooth muscle cells22. However, it is unknown whether these functions are correlated with the UNE-I domain.IARS1 is one of the least characterized ARSs in the MSC. It is a class 1a ARS and has an UNE-I domain at its C-terminal end that is formed by tandem ~90 residue repeatsIn this study, we determined the structure of the chicken EARS1–IARS1 complex. We show that UNE-I is formed by tandem repeats of ubiquitin regulatory X (UBX) domains and that IARS1 interacts with a distinct hairpin loop in the EARS1 anticodon-binding domain via its tandem UBX domains. We also show that phosphomimetic mutation of one or two serine residues in the EARS1 hairpin loop releases IARS1 from the complex. IARS1 UBX domains interact with the heterodimeric RING domains of BRCA1-BARD1 and the BRCT domain of BRCA1 and protect the complex from ubiquitin (Ub)-mediated degradation. We show that the IARS1 UNE-I domain participates in DNA repair via maintaining the stability of the BRCA1-BARD1 complex.21; therefore, we performed mass spectrometry analysis using cells expressing wild-type IARS1, C-terminal-deleted IARS1 , and the IARS1 UNE-I domain (residues 942–1262) alone. While wild-type IARS1 most extensively captured EPRS1, it also interacted with LARS1, QARS1, RARS1, and DARS1. However, IARS1ΔC failed to interact with other ARSs except LARS1  directly associated with full-length IARS1, and removal of the three WHEP motifs did not affect complex formation  complexed with the IARS1 C-terminal domain (residues 965–1265) containing UNE-I, which diffracted at no better than 3.5–4 Å resolution. While we could not improve the size and quality of the crystals, we determined the structure of the GgEARS1–IARS1 complex at 2.4 Å resolution using the Pohang Advanced Light Source X-ray free electron laser  structure at 2.5 Å resolution  and C-terminal anticodon-binding domain  and distal β-barrel (Dβ) domains)  or human QARS124  Fig. . The helin Figs. . This loGgEARS1 structure is the first eukaryotic EARS1 structure, we briefly compared it with those of other related ARSs. Overall, each domain of GgEARS1 is very similar to the corresponding domain of QARS1 or archaeal EARS (PDB 3AII), except the Dβ domain, in which significant differences are observed between GgEARS1 and archaeal EARS25  of EARS1  from the hairpin loop and examined binding between EARS1 and IARS1 Fig. . Hairpin84–694 fr29. To investigate whether phosphorylation of these residues leads to release of IARS1 from EARS1, we generated single or double phosphomimetic Ser-to-Glu mutants. While mutation of Ser688 or Ser691 to glutamate retained the interaction of EARS1 with IARS1, simultaneous mutation of both residues dissociated IARS1 from EARS1  and one Tyr (Tyr690) residues are present. Although Ser691 is not involved in the direct interaction with the UBX domains, Ser688 makes contact with Glu1216 of UBX2, whereas Tyr690 interacts with Leu1198 and Asn1206 Fig. . In the RS1 Fig. . To furtRS1 Fig. .30. Therefore, we hypothesized that IARS1 interacts with various cellular proteins through its UBX domains and protects these proteins from E3 ligase-mediated degradation -RING (residues 26–142) domains of the BRCA1-BARD1 complex and the BRCT domain (residues 1645–1863) of BRCA1 interacted with IARS1, the BRCT domain (residues 566–777) of BARD1 did not  of active IARS1 localizes to the nucleus and that IARS1 is primarily located in the cytoplasmWe next examined the interaction between endogenous IARS1 and BRCA1 in the nucleus. We first isolated the nuclear fractions of HeLa cells. Co-IP analysis of BRCA1 using a BRCA1-specific antibody in the nuclear extract revealed that BRCA1 captured endogenous IARS1, which further supports the interaction between BRCA1 and IARS1 Fig. .IARS1#1 to #3) and one targeted the 3’ untranslated region (3’UTR). All siRNAs significantly reduced the levels of BRCA1 protein and the IARS1-binding protein LARS1  analysis of BRCA1. siRNAIARS1#3 and 3’UTR-specific siRNAIARS1 did not affect the mRNA level of BRCA1  and non-homologous end joining (NHEJ) in cells treated with siRNAIARS1#1 and #2. We have used siRNAIARS1#1 and #2 because they most significantly reduced the level of BRCA1 protein among the four siRNAs we examined  exposure, consistent with the previous finding that reduction of BRCA1 impairs RAD51 recruitment to DSBs44  at DSB sites using the ER-tIP Fig. . Inactiv50. Chromosomes from IARS1-depleted cells had a higher incidence of end-to-end fusions, inversions, and aneuploidy than chromosomes from scrambled siRNA-treated cells; IARS1 depletion increased the frequency of sister chromatid exchange (SCE) by approximately 10% compared with control cells  and prevents their ubiquitylation. When anchored on the BRCA1–BARD1 RING domains and/or the BRCT domain of BRCA1, the UBX domains can competitively interact at or near the ubiquitination site of BRCA1 and, together with the unbound catalytic domain, may hinder access of Ub-conjugating enzymes or proteases  and disable their orthogonal functions. For example, AIMP3 (p18) affects the activities of ATM and ATR during the DNA damage response53. Thus, removal of IARS1 might affect ATM activation and associated metabolism during DNA damage signaling.Although the cellular function of the UBX domain is not clearly understood, its structural similarity with Ub suggests that it interacts with cellular proteins and protects them from Ub-mediated degradationes Figs. . Associa20 and several MSC components were reported to interact with BRCA132, depletion of IARS1 could disrupt the assembly of the MSC and/or decreases the stability of the MSC components, which would subsequently affect the stability of BRCA1. Interestingly, the depletion of two MSC components, AIMP3 and EPRS1 did not affect the BRCA1 level  column, and size exclusion chromatography (SEC). To purify the LARS1 UNE-L domain (residues 1062−1176), the corresponding gene was inserted into the pGEX6P1 vector and expressed in Escherichia coli Rosetta (DE3) cells. The UNE-L domain was initially purified by GST affinity chromatography. After treatment with PreScission protease , the protein was further purified by IEX and SEC.For biochemical analyses, genes encoding full-length human IARS1 (residues 1−1262) and C-terminal-truncated IARS1 (residues 1−1150 and 1−1081) were amplified by PCR and inserted into the pFASTbacHTb vector with a His6 tag at the N-terminus. All constructs were expressed in Sf9 cells (Life Technologies) at 27 °C for 72 h. The proteins were purified using a Nieprs1 constructs, three versions of genes  were amplified, inserted into the pGEX4T3 vector, expressed in E. coli Rosetta (DE3) cells, and purified using a GST column followed by gel filtration. For the pull-down assay, GgEARS1 catalytic domain mutants  were inserted into the pET28a vector, expressed in E. coli Rosetta (DE3) cells, and purified by His-affinity chromatography, IEX, and SEC.To generate human GgEARS1 phosphomimetic mutant  or the hairpin loop-deleted mutant was mixed with the GST-UNE-I domain at a ratio of 1:1.5, respectively, and incubated for 30 min at 4 °C. The protein mixtures were pulled down as in the IARS1-EARS1 pull-down assay. For GST pull-down analysis, samples eluted from the Ni-sepharose beads with or without GgIARS1 were diluted 2-fold in His A buffer and then incubated with GST-sepharose beads for 2 hr at 4 °C. The beads were washed three times with His A buffer and eluted with a buffer containing 10 mM L-glutathione. All eluted proteins were analyzed by SDS-PAGE and stained with Coomassie Blue.Human His-IARS1 or IARS1ΔUBX2 protein was mixed with human GST-EARS1 or EARS1Δhairpin (residues 1−1015 and Δ684−694) proteins at a ratio of 1:1.5, respectively, and incubated for 30 min at 4 °C. The protein mixtures were loaded onto Ni-sepharose beads for 2 hr at 4 °C and eluted with 250 mM imidazole prepared in His A buffer  for further analysis. A His-tagged GgEARS1 catalytic domain (residues 176−706) and UNE-I domains of GgIARS1 (residues 965−1265) were fused by a 12 residue linker (SGSLVPRGSSGS), which contained a thrombin cleavage site, inserted into the pET28a vector (Novagen), and expressed in E. coli Rosetta (DE3) cells. The EARS1–UNE-I chimera was initially purified using a Ni2+ column, and the intervening linker was cleaved by the thrombin protease during dialysis. The EARS1–IARS1 complex protein was trimmed by subtilisin and further purified by IEX and SEC. The final purified complex protein was concentrated to 30 mg/mL and used for crystallization. GgEARS1–IARS1 complex microcrystals were obtained using the batch crystallization method. A solution containing GgEARS1–IARS1 complex protein and crystallization solution  were mixed at a ratio of 1:2, respectively, and incubated for 1 week at 20 °C.For crystallization, genes encoding the GgEARS1–IARS1 microcrystals were collected using the fixed-target serial femtosecond crystallography method with the PAL-XFEL61. Data collection and processing are described in GgEARS1–IARS1 complex was determined by the molecular replacement method. The crystal structure of GgEARS1 alone was initially determined at 2.5 Å resolution and used to locate the catalytic domains of GgEARS1 molecules in the GgEARS1–IARS1 complex  were employed to build the complete model. The final refined model of the GgEARS1-IARS1 complex at 2.4 Å resolution (Rwork/Rfree of 19.3%/24.3%) contained 94.4% of residues in its most favored region and 0.6% residues in the disallowed region  or co-IP buffer  supplemented with 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Roche). The soluble fraction of the cell lysate containing Flag-tagged EPRS1 proteins was isolated by centrifugation and incubated with anti-Flag M2 agarose affinity beads (Sigma) for 2 h at 4 °C. The beads were washed three times with IP buffer and eluted with IP buffer containing 0.4 mg/mL Flag peptide. The isolated soluble fraction of the cell lysate containing Flag-tagged IARS1 proteins was incubated with anti-Flag G1 resin (Genscript) overnight at 4 °C. The beads were washed three times with co-IP buffer and proteins were eluted with 2 × SDS loading buffer. Co-immunoprecipitated proteins were analyzed by immunoblotting.For BRCA1 immunoprecipitation, an anti-BRCA1 antibody  was mixed with the cell lysate overnight at 4 °C with constant rotation. Protein G beads  were added and incubated for 2 hr. After four washes with 1/2 buffer X, precipitates were dissolved in 2 × SDS loading buffer and separated by SDS-PAGE. Cytoplasmic and nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). The isolated nuclear fraction was incubated with agarose conjugated with an anti-BRCA1 antibody  overnight at 4 °C with constant rotation. After five washes with 1/2 buffer X, precipitates were dissolved in 2× SDS loading buffer and separated by SDS-PAGE.HEK293T cells transiently expressing Flag-tagged IARS1 were lysed with buffer X, and the crude lysates were incubated with Flag-M2 beads. Bead-bound proteins were washed and eluted with 0.4 mg/mL Flag peptide in buffer X. Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. For liquid chromatography-tandem mass spectrometry analyses, gel lanes were sliced into bands and processed as follows. Briefly, the protein bands were divided into 10-mm sections and in-gel digestion was performed with trypsin. Trypsin digest products were separated by online reversed-phase chromatography using a Thermo Scientific EASY-nLC 1200 ultra-high performance liquid chromatography system equipped with an autosampler, an Acclaim PepMap™ 100 reversed-phase peptide trap (75 μm inner diameter and 2 cm length), and a PepMap™ RSLC C18 reversed-phase analytical column . Samples were then subjected to electrospray ionization at a flow rate of 300 nL/min. The chromatography system was coupled in-line with an Orbitrap Fusion Lumos Mass Spectrometer. Obtained spectra were screened against the UniProt human database using Proteome Discoverer Sorcerer 2.1 software with a SEQUEST-based search algorithm. The comparative analysis of proteins identified in this study was performed using Scaffold 4 Q + S.4 cells per well in 2 mL. Cells were treated with olaparib (PARP inhibitor) at 37 °C for 14 days. For continuous exposure to the inhibitor, cells were re-fed with fresh medium containing the inhibitor every 4 days. Cells were stained with 0.5% crystal violet before colonies were counted. Clonogenic survival was determined by dividing the number of colonies on each treated plate by the number of colonies on the untreated plate.HeLa cells were transfected with scrambled or IARS1-targeting siRNA using Lipofectamine RNAiMAX. At 24 hr after transfection, cells were trypsinized and re-plated in 6-well plates at a density of 1 × 105 cells per well after 2 days. The following day, cells were co-transfected with 0.5 µg of the I-SceI expression vector or empty vector and 0.1 µg of the dsRED vector (used as a transfection control) in 0.1 mL Opti-MEM containing 3 µL Lipofectamine 3000 (Invitrogen). After 6 hr, the medium was removed and replaced with growth medium. At 2 days after I-SceI transfection, the percentage of GFP + cells was analyzed using a Becton Dickinson FACSVerse flow cytometer. The DNA repair efficiencies were determined as described previously65. Experiments were repeated at least three times, and the average values were used.To measure DNA repair efficiencies, transfected cells were plated in a 12-well plate at a density of 1 × 1066. Briefly, for each sample, a Ct was calculated by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample. ssDNA% was calculated using the following equation: ssDNA% = 1/(2^(4Ct-1)+0.5)*10067.The level of resection adjacent to specific DSBs was measured by quantitative polymerase chain reaction (qPCR). The sequences of qPCR primers and probes are shown in Supplementary Table Cells were cultured in medium containing BrdU at a final concentration of 25 µg/mL for 48 hr. Colcemid (0.2 µg/mL) was added for the final 4 hr. Then, metaphase cells were harvested by trypsinization, swollen in 0.075 M KCl for 15 min at 37 °C, fixed twice with methanol:acetic acid (3:1), dropped onto glass microscope slides, and stained with 5% Giemsa. Images were acquired using a fluorescence microscope . At least 35 metaphase cells were randomly analyzed in each condition.The following materials and methods are described in Supplementary Information: Crystallization of EARS1 and data collection, Determination of the EARS1 structure, Data collection using XFELs, SFX data processing, Cell culture, cDNAs, siRNAs, Antibodies, Transfection of cell lines, Immunoblot analysis, RNA extraction and cDNA synthesis, Quantitative real-time PCR, Immunofluorescence microscopy, and Laser microirradiation.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Diabetic eye screening programmes have been developed worldwide based on evidence that early detection and treatment of diabetic retinopathy are crucial to preventing sight loss. However, little is known about the decision-making processes and training needs of diabetic retinal graders, particularly in low- and middle-income countries.To provide data for improving evidence-based diabetic retinopathy training to help novice graders process fundus images more like experts.n = 18) and expert graders in Northern Ireland (n = 5) were selected through a purposive sampling technique. Data were collected from 21st February to 3rd September 2019. The interviewer used neutral prompts during think-aloud sessions to encourage participants to verbalise their thought processes while grading fundus images from anonymised patients, followed by semi-structured interviews. Thematic framework analysis was used to identify themes, supported by illustrative quotes from interviews. Mann–Whitney U tests were used to compare graders’ performance.This is a mixed-methods qualitative study conducted in southern Vietnam and Northern Ireland. Novice diabetic retinal graders in Vietnam (Expert graders used a more systematic approach when grading images, considered all four images per patient and used available software tools such as red-free filters prior to making a decision on management. The most challenging features for novice graders were intra-retinal microvascular abnormalities and new vessels, which were more accurately identified by experts.Taking more time to grade fundus images and adopting a protocol-driven “checklist” approach may help novice graders to function more like experts. Diabetes (DM) is one of the largest global health challenges of this century, affecting ~463 million adults aged 20–79 years old worldwide , 2. AccoDiabetic eye screening programmes (DESPs) have been developed worldwide in response to evidence showing that early detection and treatment of DR can prevent over 90% of sight loss –9. In EnImplementing DESPs in low- and middle-income countries (LMICs) can be challenging, and requires careful planning and evaluation . In partA key question, when training personnel to review fundus photos is how well novice graders carry out this important task. It would be useful to know how novice versus expert graders process information while grading images when developing training programmes for novice graders. Prior studies suggest that expert clinical decisions are typically based on pattern recognition, or the quick and efficient recall of previous cases, resulting in high diagnostic accuracy –23. In cThis study seeks to evaluate the clinical decision-making processes of expert and novice diabetic retinal graders in Vietnam and NI, and to use the findings to develop recommendations for more effective DR grader training programmes in Vietnam and other LMICs.This mixed-methods study used multiple qualitative methods to collect data from a structured (simulation-based) interview with Likert-scale ratings during think-aloud (TA) sessions and from semi-structured interviews. The TA method entails systematically capturing participants’ statements about their thoughts while completing a task in order to evaluate their decision-making processes . TA studParticipants  were recruited from DESPs in five locations in southern Vietnam, supported by Orbis International, Queen’s University Belfast (QUB) and health care providers from the DESP in NI. The sample includes 14 novice graders  and four experts  from Vietnam, plus five experts from NI . All invited graders participated in the study.Two 45° field images using the Canon CR-2 digital fundus camera were taken per eye, one image centred on the optic disc and the other centred on the macula (image size 2736 × 1824). These were chosen to align with the UKs’ NHS DESP guidelines. Twenty-four macular and disc-centred images from six consenting patients in Vietnam  were selected by a research optometrist from QUB (KC) from the database of the Orbis-supported DESP. The selected images represent a wide range of DR and maculopathy grades and warranted a range of responses ranging from annual re-screen to routine and urgent referrals. Interview guides were developed by researchers at QUB  based on their clinical and research experience. They were then translated from English to Vietnamese by bilingual Vietnamese-English speakers  before pilot testing with a Vietnamese grader. Minimal changes were made to the interview guide based on the feedback.Data were collected from 21st February to 3rd September 2019. After providing written informed consent, participants were given standardised written and oral instructions with non-ocular examples of what occurs during a TA interview, followed by a practice session that involved solving simple mathematics problems. All interviews were conducted in Vietnamese at the graders’ usual hospital settings by an English-speaking optometrist (KC) assisted by bilingual Vietnamese team members . For each of the six sets of images, graders gave a DR and maculopathy grade; rated their difficulty in making that assessment on a 5-point Likert scale (1 = very difficult to 5 = very easy); made a recommendation as to appropriate follow-up care ; and rated their confidence in providing the recommendation on another 5-point Likert scale . At the end of the TA sessions, semi-structured interviews were conducted by the same researchers to collect information on demographic variables, training experience, job roles and recommendations to improve future training. All interviews were recorded using a digital voice recorder , the English portions transcribed verbatim by the interviewer (KC), and then reviewed by an expert qualitative researcher (LL) to ensure accuracy.After creating verbatim written transcripts of the translated audio files from interviews, a series of tables was created to compile quotes from each transcript related to a specific topic based on the interview guide , 30. TwoU test was used to compare differences between DR graders in NI versus Vietnam in terms of (a) time taken to grade six image sets, (b) self-reported difficulty and (c) self-reported confidence. Data were entered into an Excel spreadsheet and then analysed  using SPSS Statistics version 25.0 software .Descriptive statistics were used to characterise the participants by age, sex and work experience. We assessed the data for normality using histograms, since the data were not normally distributed and the sample size was small (<30), data are presented as median and interquartile range (IQR). The non-parametric Mann–Whitney To ensure the study was rigorous, a clear protocol was followed, and all steps were clearly documented. Prolonged visits to Vietnam and continuous communications with experts in the field were made. Triangulation of methods and of researchers were also used .This research adhered to the tenets of the Declaration of Helsinki. Ethical approval was granted by the Hanoi Medical University Institutional Review Board in Vietnam and the Research Ethics Committee at QUB. Permission was also obtained from hospital directors at tertiary , provincial  and district  level hospitals in Vietnam, where Vietnamese participants worked. Written informed consent was obtained from participants prior to recruitment.To promote readability of statements by study participants, quotes appear in italics; minor editing is shown with square brackets around added words and an ellipse to show where words have been removed. Participants were identified by source using a unique ID number, indicated by grader type, country and interview number e.g. nurse, Vietnam, GR1.As expected, length of grading experience was greater among experts than novice graders, although the experts in NI grading images for five times longer than the experts in Vietnam (11.4 ± 10.9 years vs. 2.13 ± 1.93 years). In total, 21/23 (91.3%) of the graders in the study were female (Table p = 0.004). Table Experts in NI spent longer time  grading all six image sets compared to graders in Vietnam  accurately graded and recommended an annual re-screen for the patient in image set #4, which displayed no signs of DR Fig. . HoweverDifferent heuristic approaches to the task of grading fundus images between novice and expert graders were identified from the TA interview data. Experts tended to take a more systematic approach when grading images, examining the macular and disc images of both eyes before assigning a grade for the patient. In contrast, novice graders frequently graded the macular and disc images of each eye separately. Also, the novice graders tended to not comment on blood vessel appearance whereas expert graders, particularly those in NI, discussed the retinal vasculature in detail. Experts, but not novice graders, typically used all available software grading tools such as the red-free filter to reach their decision. As one expert grader from Vietnam explained, ‘The [red-free] filter [lets me] see the images more clearly’ . In contrast, an expert grader from NI reported using the filter for a specific purpose ‘[I will use] the red-free filter to look for signs of IRMA [intra-retinal microvascular abnormalities (IRMA)]’, .The TA interviews also provided data showing that all graders, regardless of level of experience, could accurately detect specific signs of DR such as microaneurysms, haemorrhages and exudates. However, the DR terminology used by experts was much more precise than what novice graders reported. As one novice grader explained, ‘There are white spots, but I can’t think of the name’ . In contrast, an expert grader from Vietnam reported, ‘[In this image] we can see cotton wool spots’ . Experts in Vietnam and NI were much better at identifying IRMA, fibrosis and new vessels compared to novice graders. Recommended management plans varied accordingly. An expert grader from Vietnam reported, ‘I can see some fibrovascular membrane, which is a sign of R3’  and an expert from NI stated, ‘There is fibrosis so [the DR] is quite severe’ .It is…never…as easy to grade normal as abnormal [images] because you never really quite know if you missed something. And [so] usually I take twice as long to grade normal as abnormal [images]!’‘Data collected from the TA interviews also underscored the potential challenges, even for experts, of grading normal fundus image sets. As one senior grader from NI  reported:Graders in Vietnam reported being satisfied with their DR training course content. The main strength of the training for novice graders was their ability to develop new skills and better identify eye disease early, ‘It is really good that I can see damage to the eyes’, ; ‘To detect it [DR] early is better for patients so the patient can seek early treatment’, . Novice graders clearly indicated that additional training would be beneficial, perhaps by being mentored by more experienced graders at the workplace ). Non-medical graders in NI complete a mandatory, highly structured training programme and noted that there is value in learning to grade images oneself as for online courses ).To our knowledge, this is the first study to explore the clinical decision-making thought processes of DR graders in Vietnam. Expert graders in NI took more time to grade than other participants, and experts in both countries used a more systematic approach when grading images compared to novice graders. The systematic use of tools such as red-free filters was noted to be a crucial part of experts’ ability to detect more challenging findings such as IRMA, which was frequently missed by novice graders.Contrary to our findings, previous studies have found that experts spend less time completing clinical tasks compared to novice graders –36. ExpeAccording to the Early Treatment Diabetic Retinopathy Study, seven-field colour fundus photography is the gold standard for fundus imaging; however, this is time-consuming and potentially impractical for large-scale screening programmes. Imaging in single or multiple fields varies across countries, although most programmes use two field 45° field images, which is quicker and more convenient for patients. This protocol has been adopted by the UKs’ NHS DESP and was subsequently used in our study. The challenges of inconsistent DR programmes exist globally, but the concepts of basic DR grader training remain the same. Trainees should be reminded to examine fundus images systematically, review all of a patient’s photos and carefully assess retinal vasculature in each image before classifying DR disease. Zoom features and red-free filters are particularly useful to detect and confirm DR signs; therefore, consistent use of such grading tools is recommended. These insights are useful because training highly competent cadres of non-physician graders is particularly important in low-resource settings such as Vietnam, to allow ophthalmologists to focus more fully on delivering treatment.Artificial intelligence (AI) and deep learning algorithms may significantly change future approaches to DR grading and are beginning to do so already in settings such as Scotland , 38. HowStrengths of the study include the enrolment of multiple cadres at different levels of training in two distinct settings, collecting data during TA interviews conducted in real-life workplace settings, following a carefully designed study protocol, involving an experienced qualitative researcher on the team and as a co-analyst, achieving thematic saturation from the interview data, and having selected relevant images of anonymous patients from an on-going DESP.Limitations must also be acknowledged. The manner in which both NI and Vietnam participants graded fundus images in the current study may not completely reflect their typical work patterns: we did not use their usual grading software because it provides certain prompts. Instead, our goal was to document graders’ spontaneous approaches in order to understand their actual thought processes. Furthermore, application of these results outside of southern Vietnam and NI must be made with caution because the workplace context and culture may vary considerably from those in the present study.In conclusion, this study provides insights into DR graders’ decision-making processes that highlight ways to improve their training. Training graders to achieve high sensitivity and high specificity is crucial to develop sustainable DESPs and prevent blindness in a population. Practical and interactive sessions were favourable among graders in Vietnam and evaluations of these approaches are more likely to lead to positive changes in grading processes and patient outcomes. Adopting a TA approach to train graders either in person or via online learning may be beneficial for the future , 39. SinNo previous TA studies have been conducted previously to assess diabetic retinal graders’ decision-making processes.Non-ophthalmologists have successfully been trained in DR screening and grading in high-income settings.Expert graders in Northern Ireland take longer to grade images for DR severity compared to diabetic retinal graders in Vietnam, ensuring that no DR features are missed.Expert graders use a more systematic approach to grade fundus images for DR compared to novices.Findings from this study have been incorporated into existing diabetic eye screening training programmes in Vietnam.